Journal articles: 'BaF3'

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Kim, Julie, Sofya Rodov, Jacqueline Barrientos, Ravitharan Krishnadasan, and K. Gary J. Vanasse. "Bcl2 Associated Induction of the Suppressor of Cytokine Signaling-3 (SOCS3) Gene Involves Activation of the p44/42 MAPK Pathway." Blood 106, no. 11 (November 16, 2005): 2286. http://dx.doi.org/10.1182/blood.v106.11.2286.2286.

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Abstract SOCS3 has been shown to be an essential regulator of IL-6 signaling in hepatocytes and macrophages, as well as a negative regulator of G-CSF signaling in myeloid cells, and SOCS3 expression appears to be STAT3-dependent. However, the underlying cellular pathways which regulate SOCS3 expression in B cells remain to be clearly defined. We previously showed that polyclonal CD19+ B cells isolated from Bcl2 overexpressing Eμ-Bcl2 transgenic mice overexpress SOCS3 protein and that exogenous expression of Bcl2 in hematopoietic and fibroblast cell lines induces SOCS3 protein expression. We also found that a distinct subset of follicular lymphomas (FL) exhibit combined overexpression of Bcl2 and SOCS3. We hypothesized that Bcl2-mediated induction of SOCS3 in B cells may occur indirectly via deregulation of Bcl2-associated cell signaling pathways. In the current study, IL-3-dependent BaF3 pro-B cells were stably transduced with either a 717bp human Bcl2α cDNA (BaF3/Bcl2) or vector only control (Baf3Δ). Cell lines were initially grown in the presence of IL-3, with IL-3 removed 48 hours prior to protein measurements. Western analysis using Bcl2 antisera revealed high level Bcl2 expression in transduced cells (BaF3/Bcl2) and absence of Bcl2 in BaF3Δ control cells. When probed with SOCS3 antisera, BaF3/Bcl2 cells exhibited marked overexpression of SOCS3 protein whereas BaF3Δ control cells did not express SOCS3. To assess whether Bcl2-associated induction of SOCS3 is STAT3-dependent, we measured phospho-STAT3 levels relative to STAT3 in BaF3/Bcl2 cells. Interestingly, when probed with phospho-STAT3 and STAT3 antisera, BaF3/Bcl2 and BaF3Δ cells did not reveal phospho-STAT3 protein but revealed equivalent STAT3 protein levels. Since we had previously noted overexpression of p44/42 Map kinase (MAPK) in CD19+ B cells from Eμ-Bcl2 transgenic mice relative to littermate controls, we next wanted to determine whether p44/42MAPK signaling played a role in Bcl2-mediated SOCS3 induction. Western analysis using p44/42MAPK antisera revealed overexpression of p44/42MAPK in BaF3/Bcl2 cells relative to BaF3Δ control cells. BaF3/Bcl2 and BaF3Δ cells were then grown in the presence or absence of a specific p44/42MAPK pathway inhibitor (MEK 1/2 inhibitor U0126), and whole cell lysates were analyzed for SOCS3 expression by Western analysis. When probed with SOCS3 antisera, BaF3/Bcl2 cells selectively grown in the presence of the p44/42MAPK inhibitor revealed complete abrogation of SOCS3 protein expression while those grown in the absence of inhibitor continued to harbor SOCS3 protein. As expected, ERK1/2 protein levels were selectively decreased in response to treatment with the p44/42MAPK inhibitor, indicating specific inhibition of the p44/42MAPK pathway. Bcl2 protein levels in BaF3/Bcl2 cells remained unchanged in the presence or absence of the p44/42MAPK inhibitor. As controls, inhibitors of p38MAPK (SB203580), AKT (Triciribine), and PI3K (LY294002) were evaluated and none were found to affect SOCS3 protein levels in BaF3/Bcl2 cells. These data indicate that Bcl2-associated induction of SOCS3 in B cells involves activation of the p44/42MAPK cell signaling pathway and is independent of STAT3 activation. These studies illustrate a novel and previously unappreciated Bcl2-associated signaling pathway involving SOCS3 induction in B cells that may play an important role in B cell biology and in Bcl2-associated hematologic malignancies.

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ROCH, Anne-Marie, Gerard QUASH, Yvonne MICHAL, Jacqueline CHANTEPIE, Bernard CHANTEGREL, Christian DESHAYES, Alain DOUTHEAU, and Jacqueline MARVEL. "Altered methional homoeostasis is associated with decreased apoptosis in BAF3 bcl2 murine lymphoid cells." Biochemical Journal 313, no. 3 (February 1, 1996): 973–81. http://dx.doi.org/10.1042/bj3130973.

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Methional is a potent inducer of apoptosis in an interleukin 3-dependent murine lymphoid cell line BAF3 b0 when it is added to the culture medium. In these cells transfected with the bcl2 gene, BAF3 bcl2, the apoptotic-inducing activity of methional is dramatically reduced. The addition of disulfiram (an inhibitor of aldehyde dehydrogenase) in order to reduce methional oxidation brought about an increase in apoptosis in BAF3 b0 but not in BAF3 bcl2 cells. In contrast, the addition of quercetin (an inhibitor of aldehyde reductase) in an attempt to diminish methional reduction increased apoptosis in both BAF3 b0 and BAF3 bcl2 cells. The extent of DNA fragmentation in BAF3 bcl2 cells approached that in BAF3 b0 cells in the presence of quercetin and exogenous methional, suggesting a defect in methional biosynthesis in BAF3 bcl2 cells. Direct evidence for this was obtained by measuring labelled methional in cells incubated with the sodium salt of [U-14C]4-methylthio-2-oxobutanoic acid (MTOB), the precursor of methional. The 80% decrease in labelled methional in BAF3 bcl2 compared with BAF3 b0 cells was accompanied by a concomitant rise in the transamination of [14C]MTOB to [14C]methionine in BAF3 bcl2 cells. Inhibition of the transaminase, however, by a synthetic transition-state-type compound, pyridoxal-L-methionine ethyl ester, induced apoptosis in BAF3 b0 but not in BAF3 bcl2 cells, confirming that the defect in BAF3 bcl2 cells was not in the transaminase itself but rather in the oxidative decarboxylation step MTOB →methional. In addition, no evidence was obtained for the synthesis of [14C]malondialdehyde from [14C]methional in BAF3 bcl2 cells. As these cells show no deficiency in their content of reactive oxygen species compared with that of BAF3 b0 cells, they may possess some other defect in the β-hydroxylase enzyme system itself.

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Shibayama, Hirohiko, Naoyuki Anzai, Stephen E. Braun, Seiji Fukuda, Charlie Mantel, and Hal E. Broxmeyer. "H-Ras Is Involved in the Inside-out Signaling Pathway of Interleukin-3–Induced Integrin Activation." Blood 93, no. 5 (March 1, 1999): 1540–48. http://dx.doi.org/10.1182/blood.v93.5.1540.

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Abstract The proto-oncogene product, p21ras, has been implicated in the cellular mechanism of adhesion, although its precise role has been controversial. Numerous cytokines and growth-factors activate Ras, which is an important component of their growth-promoting signaling pathways. On the other hand, the role of Ras in cytokine-induced adhesion has not been elucidated. We therefore investigated the function of H-Ras in the inside-out signaling pathway of interleukin-3 (IL-3)–induced integrin activation in the murine Baf3 cell line after transfection of cells with either constitutively active, dominant-negative, or wild-type H-Ras cDNAs. Adhesion of Baf3 cells to fibronectin was induced by IL-3 in a dose-dependent manner via very late antigen-4 (VLA-4; 4β1 integrins) and VLA-5 (5β1 integrins) activation. On the other hand, IL-4 did not induce the adhesion of Baf3 cells to fibronectin, although IL-4 did stimulate the cell proliferation of Baf3 cells. Constitutively active H-Ras–transfected Baf3 cells adhered to fibronectin without IL-3 stimulation through VLA-4 and VLA-5, whereas dominant-negative H-Ras–transfected Baf3 cells showed significantly less adhesion induced by IL-3 compared with wild-type and constitutively active H-Ras–transfected Baf3 cells. Anti-β1 integrin antibody (clone; 9EG7), which is known to change integrin conformation and activate integrins, induced the adhesion of dominant-negative H-Ras–transfected Baf3 cells as much as the other types of H-Ras–transfected Baf3 cells. 8-Br-cAMP, Dibutyryl-cAMP, Ras-Raf-1 pathway inhibitors, and PD98059, a MAPK kinase inhibitor, suppressed proliferation and phosphorylation of MAPK detected by Western blotting with anti–phospho-MAPK antibody, but not adhesion of any type of H-Ras–transfected Baf3 cells, whereas U-73122, a phospholipase C (PLC) inhibitor, suppressed adhesion of these cells completely. These data indicate that H-Ras and PLC, but not Raf-1, MAPK kinase, or the MAPK pathway, are involved in the inside-out signaling pathway of IL-3–induced VLA-4 and VLA-5 activation in Baf3 cells.

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Shibayama, Hirohiko, Naoyuki Anzai, Stephen E. Braun, Seiji Fukuda, Charlie Mantel, and Hal E. Broxmeyer. "H-Ras Is Involved in the Inside-out Signaling Pathway of Interleukin-3–Induced Integrin Activation." Blood 93, no. 5 (March 1, 1999): 1540–48. http://dx.doi.org/10.1182/blood.v93.5.1540.405k10_1540_1548.

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The proto-oncogene product, p21ras, has been implicated in the cellular mechanism of adhesion, although its precise role has been controversial. Numerous cytokines and growth-factors activate Ras, which is an important component of their growth-promoting signaling pathways. On the other hand, the role of Ras in cytokine-induced adhesion has not been elucidated. We therefore investigated the function of H-Ras in the inside-out signaling pathway of interleukin-3 (IL-3)–induced integrin activation in the murine Baf3 cell line after transfection of cells with either constitutively active, dominant-negative, or wild-type H-Ras cDNAs. Adhesion of Baf3 cells to fibronectin was induced by IL-3 in a dose-dependent manner via very late antigen-4 (VLA-4; 4β1 integrins) and VLA-5 (5β1 integrins) activation. On the other hand, IL-4 did not induce the adhesion of Baf3 cells to fibronectin, although IL-4 did stimulate the cell proliferation of Baf3 cells. Constitutively active H-Ras–transfected Baf3 cells adhered to fibronectin without IL-3 stimulation through VLA-4 and VLA-5, whereas dominant-negative H-Ras–transfected Baf3 cells showed significantly less adhesion induced by IL-3 compared with wild-type and constitutively active H-Ras–transfected Baf3 cells. Anti-β1 integrin antibody (clone; 9EG7), which is known to change integrin conformation and activate integrins, induced the adhesion of dominant-negative H-Ras–transfected Baf3 cells as much as the other types of H-Ras–transfected Baf3 cells. 8-Br-cAMP, Dibutyryl-cAMP, Ras-Raf-1 pathway inhibitors, and PD98059, a MAPK kinase inhibitor, suppressed proliferation and phosphorylation of MAPK detected by Western blotting with anti–phospho-MAPK antibody, but not adhesion of any type of H-Ras–transfected Baf3 cells, whereas U-73122, a phospholipase C (PLC) inhibitor, suppressed adhesion of these cells completely. These data indicate that H-Ras and PLC, but not Raf-1, MAPK kinase, or the MAPK pathway, are involved in the inside-out signaling pathway of IL-3–induced VLA-4 and VLA-5 activation in Baf3 cells.

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5

Barrientos, Jacqueline C., Sofya Rodov, Arthur W. Zieske, and K. Gary J. Vanasse. "The Suppressor of Cytokine Signaling-3 Gene Is Overexpressed in Human De Novo Follicular Lymphomas and Promotes IL-3-Independent Proliferation of the BaF3 Pro-B Cell Line." Blood 104, no. 11 (November 16, 2004): 1107. http://dx.doi.org/10.1182/blood.v104.11.1107.1107.

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Abstract The recent generation of mice lacking functional SOCS3 in hepatocytes, macrophages, and neutrophils reveals SOCS3 to be an essential regulator of IL-6 signaling via mediation of gp130-related cellular complexes, as well as a negative regulator of G-CSF signaling in myeloid cells. Although SOCS3 would appear to be a critical physiologic regulator of inflammatory responses, its possible role in hematologic malignancies and the underlying mechanisms which regulate its expression in B cells remain to be clearly defined. We previously showed that CD19+ B cells isolated from Eμ-Bcl-2 transgenic mice express high levels of SOCS3 in addition to overexpression of Bcl-2. Moreover, hematopoietic cell lines transduced to stably overexpress Bcl-2 exhibited marked induction of SOCS3 compared to controls, suggesting Bcl-2-associated pathways may play a role in the induction of SOCS3. In the current study, we describe SOCS3 overexpression limited to neoplastic follicular lymphoma (FL) cells in Bcl-2-associated human de novo FL and show that overexpression of SOCS3 is capable of stimulating cytokine-independent cellular proliferation of the BaF3 pro-B cell line. We measured SOCS3 protein levels by immunohistochemistry in paraffin-embedded biopsies from twelve patients diagnosed with de novo, untreated histologic grade I or II FL which harbored t(14;18) and Bcl-2 overexpression. In 9/12 de novo FL cases examined, immunostaining with two distinct antibodies to SOCS3 revealed marked overexpression of SOCS3 protein that, within the follicular center cell region, was limited to neoplastic FL cells and co-localized with Bcl-2 primarily in the nucleus of positive cells. In contrast, SOCS3 protein was not detected by immunostaining in germinal center follicular B cells from benign hyperplastic tonsil tissue. To further evaluate the role of SOCS3 in B cell biology, the IL-3-dependent BaF3 pro-B cell line was stably transduced with either a retroviral expression construct containing a 675bp human SOCS3 cDNA (BaF3SOCS3) or with vector only control (BaF3Δ). Whereas no SOCS3 protein was detected in control cells, high level expression of SOCS3 in transduced BaF3SOCS3 cells was confirmed by Western analysis using SOCS3 anti-sera. Furthermore, Bcl-2 protein was not detected in either BaF3SOCS3 or control cell lines. 2 x 105 BaF3SOCS3, BaF3Δ, and non-transduced BaF3 cell lines were initially grown in the presence 10% fetal bovine serum (FBS) and 5% WEHI 3B cell-conditioned medium as a source of IL-3. IL-3 was then removed by washing with DMEM/10% FBS. Cell viability was then measured by recording absorbance at 490nm using incorporation of the MTS tetrazolium compound. Interestingly, BaF3SOCS3 cells overexpressing SOCS3 did not undergo apoptosis but were able to proliferate in the absence of IL-3, with percent viable cells approaching 400% at > 96 hours, which represented the final time-point measured. In contrast, BaF3Δ and non-transduced BaF3 cells underwent apoptotic cell death between 8 and 36 hours in response to IL-3 withdrawal. Thus, SOCS3 overexpression confers IL-3-independent cell proliferation to the BaF3 cell line. These data indicate that unlike its negative regulatory effect on G-CSF signaling in myeloid cells, overexpression of SOCS3 in B cells may promote B cell proliferation rather than growth suppression and may play an important role in the pathogenesis of de novo FL in humans.

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Kakumitsu, Haruko, Kazuya Shimoda, Takashi Haro, Kotaro Shide, Takashi Kumano, Seido Oku, Kenjirou Kamezaki, Akihiko Numata, Katsuto Takenaka, and Mine Harada. "Tyrosine Kinase 2 (Tyk2) Interacts with and Phosphorylates Siva-1, and Auguments the Apoptotic Effect Induced by Siva-1." Blood 108, no. 11 (November 16, 2006): 1726. http://dx.doi.org/10.1182/blood.v108.11.1726.1726.

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Abstract Jak-Stat pathway is essential to transduce cytokine signalings. We previously reported that tyrosine kinase 2 (Tyk2), one of the Jak kinases, was essential but Stat1 was dispensable in IFN-alpha-induced B lymphocyte apoptosis. This indicates the existence of other signaling molecules besides Stat1 downstream of activated Tyk2. Therefore, in order to find Tyk2-activated signaling molecules which transduce IFN-alpha signal and induce B lymphocyte apoptosis, we performed a yeast two-hybrid screen for proteins that interact with Tyk2, and identified Siva-1, which had been originally cloned as a CD27 binding protein. Siva-1 has a death domain (DD) homology region and induces apoptosis in various cells through binding CD27, which belongs to the tumor necrosis factor receptor (TNFR) family. We found that Tyk2 interacts with and phosphorylates Siva-1. Two regions of Siva-1, the N-terminus and the middle portion of the protein containing a death domain homology region, contributed to binding to Tyk2, and two tyrosines of Siva-1, Tyr53 and Tyr162, were phosphorylated by Tyk2. Because Siva-1 is so far thought to be a proapoptotic protein, we assessed whether Tyk2 had any effects on Siva-1-mediated apoptosis through the Tyk2-Siva-1 interaction. First, we established BaF3 cell (IL-3 dependent murine proB cell) lines stably expressing either wild-type Tyk2 (BaF3/WT Tyk2) or constitutively activated Tyk2 which Valine678 was replaced by Phenylalanine (BaF3/V678F Tyk2). In BaF3/V678F Tyk2 cells, Tyk2 and Stats are constitutively activated, and they grow in the absence of IL-3. We transiently transfected either GFP-Siva-1 or control GFP vector into BaF3/parent cells, BaF3/WT Tyk2 cells or BaF3/V678F Tyk2 cells. Thirty-six hours after transfection, we measured annexin V positive cells by flow cytometry on GFP-positive cells. Approximately 24% of both BaF3/parent cells and BaF3/WT Tyk2 cells transfected with GFP-Siva-1 fell into apoptosis, whereas only less than 7% of the cells transfected control vector showed apoptosis. The frequency of this Siva-1-induced apoptosis was increased in BaF3/V678F Tyk2 cells (over 43% vs <10% of expressing control vector), suggesting that activated Tyk2 augmented the apoptotic function of Siva-1. In conclusion, we show that Tyk2 associates with and phosphorylates Siva-1 and that this Tyk2-Siva-1 interaction enhances the apoptotic effect induced by Siva-1. This indicates that Siva-1 might be a probable key molecule downstream of Tyk2 which is activated in response to IFN-alpha.

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Kimura, Shinya, Hidekazu Segawa, Junya Kuroda, Takeshi Yuasa, and Taira Maekawa. "CNS-9, a Novel Specific Inhibitor of ABL Tyrosine Kinase Overcomes Resistance Mechanism of Imatinib." Blood 104, no. 11 (November 16, 2004): 761. http://dx.doi.org/10.1182/blood.v104.11.761.761.

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Abstract Imatinib mesylate (also known as STI-571 and Gleevec) has drastically changed the treatment of Philadelphia chromosome positive (Ph+) leukemias. However, the resistance to imatinib has frequently been reported, particularly in patients with advanced-stage disease. A novel orally bioavailable inhibitor of the ABL tyrosine kinase (TK) named CNS-9 was developed from the 2-(phenylamino)pyrimidine class to overcome resistance mechanisms of imatinib. Inhibition of TK phosphorylation (IC50) on wild type (wt) BCR/ABL in 293T cell line by CNS-9 was 22nM, which was 2-log more potent than imatinib. Importantly, CNS-9 inhibited TK phosphorylation of E255K mutant BCR/ABL with IC50 of 98nM, while imatinib could not inhibit it with clinically relevant concentration. The T315I mutant BCR/ABL protein was resistant to CNS-9 and imatinib. CNS-9 also inhibited TK phosphorylation of platelet-derived growth factor receptor (PDGFR) or c-Kit pathways at the very similar observed IC50s when compared with imatinib, in spite of significant higher potency against ABL. The ability of CNS-9 in vitro to inhibit 101 TK molecules was assayed by KinaseProfilerTM (Upstate), showing also more specific inhibitory activity against ABL than imatinib. The growth of BCR/ABL-positive cell lines K562, KU812, BaF3 harboring wt BCR/ABL (BaF3/wt) and E255K (BaF3/E255K) was inhibited by CNS-9 with IC50 of 5, 3, 17, and 110nM, respectively (Table 1). Generally, CNS-9 was 20 to 30-fold more potent on the growth inhibition than imatinib in these same cell lines. We next investigated the in vivo effect on the leukemic growth inhibition of CNS-9. Nude mice were injected subcutaneously with 3x107 KU812 (wt BCR/ABL) on Day 0. CNS-9 or imatinib were orally administrated twice a day from Day 7 to Day 18. The dosages of CNS-9 and imatinib, which inhibited completely tumor growth were 20mg/kg/day and 200mg/kg/day, respectively, indicating that CNS-9 is 10-fold potent than imatinib in vivo. To examine the in vivo effect of CNS-9 against mutant BCR/ABL, BaF3/wt, BaF3/E255K or BaF3/T315I were engrafted to nude mice and treated with CNS-9 or imatinib. CNS-9 was also 10-fold potent than imatinib against BaF3/wt. Intriguingly, mice harboring BaF3/wt or BaF3/E255K showed significantly prolonged survival when treated with CNS-9. Consistent with in vitro assay, CNS-9 had no effect on T315I, and imatinib was not effective against both E255K and T315I. In conclusion, CNS-9 is substantially more inhibitory and more specifically than imatinib toward BCR/ABL-dependent cell growth both in vitro and in vivo Moreover, CNS-9 may be effective for leukemia patients whose leukemic cells harbor E255K mutant. The efficacy and safety of CNS-9 for Ph+ leukemias should be verified in early phase clinical trials. The IC50s values of leukemic cell lines for CNS-9 and imatinib CNS-9 (nM) imatinib (nM) K562 p210 wt BCR/ABL 5 130 KU812 p210 wt BCR/ABL 3 67 U937 BCR/ABL (−) >1000 >1000 BaF3 p190 wt BCR/ABL 17 360 BaF3 p190 E255K BCR/ABL 110 >1000 BaF3 p190 T315I BCR/ABL >1000 >1000

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Berg, Stephanie, Manuel O. Diaz, Sucha Nand, and Jiwang Zhang. "Germline Mutations Predispose to Familial Myeloproliferative Neoplasms." Blood 134, Supplement_1 (November 13, 2019): 2969. http://dx.doi.org/10.1182/blood-2019-124805.

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Background: Myeloproliferative neoplasms (MPNs), commonly polycythemia vera (PV), primary myelofibrosis (PMF) and essential thrombocythemia (ET), are a group of malignant hematologic disorders characterized by proliferation and accumulation of terminally differentiated blood cells of erythroid, myeloid and megakaryocytic origin (Cazzola, Blood 2014).Somatic driver mutations, frequently JAK2V617F, MPLW515L/K or mutant CALR in sporadic MPNs affect signaling by the thrombopoietin (THPO) and/or the erythropoietin (EPO) receptor (Pickman, PLoS 2016). MPL is the receptor of THPO and JAK2 is the key downstream mediator of THPO/MPL signaling (Nangalia NEJM 2013). In familial MPNs (families with more than 1 member developing a MPN) a germline mutation predisposes to disease development. These mutations can reveal pathways or processes involved in the pathogenesis of the disease, independent of, or cooperating with, the somatic driver mutations (Rumi, JCO 2007). We identified 3 putative predisposing germline mutations in 1 family, 3 affected generations developed MPN (PMF, ET and PV): RBSN frameshift,JAK2R340Q & PRKAA2Q425P missense variants. RBSN encodes Rabenosyn-5 protein (RBSN) which plays a role in early growth-factor receptor clathrin-dependent endocytosis (Nielsen, J Cell Biol 2000) (Fig.1).We discovered that wild type (WT) Rabenosyn-5 regulates cell surface MPL levels in hematopoietic progenitor cells and predict that MPL levels on the cell surface are negatively associated with WT Rabenosyn-5 levels. We hypothesize that the RBSN mutation predisposes to MPN development in this family, leading to abnormal activation of THPO signaling by reducing MPL turnover. Methods: Peripheral blood DNA from patient samples were analyzed for potential putative mutations using Agilent SureSelect Human ALL Exon V5+UTRs exome capture kit followed by parallel sequencing with Illumina HiSeq 2000. In vitro studies involved using the BaF3 system and CRISPR-Cas9 gene editing technique. We generated BaF3-MPL (BaF3 cells transduced with human CD4-MPL fusion) cell lines that rely on IL3 or human THPO cytokines for growth & proliferation (Fig.2&3). We transduced RBSN into the BaF3-MPL cell lines and produced RBSN heterozygous (HT) and homozygous (HM) mutants. We first generated LentiCRISPRV2GFP-mRBSN vector by inserting RBSN specific guide RNA into the LentiCRISPRV2GFP vector and generated lentivirus expressing both Cas9 and guide RNA by co-transfecting 293T cells with LentiCRISPRV2GFP vector and viral packaging vectors. The final virus was used to infect BaF3-MPL cells. Transduced cells were purified by FACS to select GFP+ cells and individual cells were grown in culture. RBSN gene mutations were examined using PCR and sequenced accordingly. To study whether the RBSN mutation alters the surface MPL levels, we stained BaF3-MPL, HT-RBSN-BaF3 and HM-RBSN-BaF3 cells with APC-CD4 antibody and the surface levels of MPL were detected by FACS by comparing the mean fluorescence intensity (MFI) of APC. Results: We identified several colonies with either HT-RBSN-BaF3 or HM-RBSN-BaF3. The guide RNA we designed specifically targeted the 12th exon of the RBSN gene which best recapitulates the mutations observed in our patients, thus, the frameshift mutations in HT-RBSN-BaF3 and HM-RBSN-BaF3 cell lines we predict will make truncated forms of RBSN which will be verified by Western Blotting for future experiments. Surface levels of MPL in the HT-RBSN-BaF3 and HM-RBSN-BaF3 cells are greater than that in BaF3-MPL cells, suggesting RBSN negatively regulates THPO-MPL signaling by regulating the cell surface MPL levels (Fig. 4). Conclusions: RBSN is a regulator of receptor trafficking, which mediates the fusion of the endocytosed receptor to the early endosome and then lysosome for degradation. We propose that genetic inactivation of RBSN might prevent the endocytosed MPL from endosome-degradation and enhance the reuse of MPL by recycling. Future experiments are planned to include in vivo studies to study whether inactivation of RBSN promoted MPN-like disease development into NSG mice and transducing our established cell lines to label individual cell compartments to study THPO-stimulated signaling. Our findings, if confirmed, will further clarify aspects of familial and somatic MPN pathogenesis and may inform efforts to devise new therapeutic and diagnostic strategies for this disease complex. Disclosures No relevant conflicts of interest to declare.

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Yamamoto, Yukiya, Sachiko Iba, Akihiro Abe, and Nobuhiko Emi. "Elongation of MPL Transmembrane Domain Is a Novel Activating-Mutation in Essential Thrombocythemia." Blood 126, no. 23 (December 3, 2015): 1628. http://dx.doi.org/10.1182/blood.v126.23.1628.1628.

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Abstract Essential thrombocythemia (ET) is a clonal disease of hematopoietic stem cell. Somatic gene mutations including JAK2 or CALR are hallmark of diagnosis and molecular targets for developing novel therapies. However, non-mutated ET cases within JAK2 and CALR still exist. The third mutated gene of ET is MPL, which is known as encoding thrombopoietin receptor. MPL W515 and S505 are hot spot for missense mutations leading to constitutive active MPL signaling. These missense mutations are located within transmembrane or juxtamembrane domain of MPL. Here, we present a novel activating-mutation of MPL in an ET patient. The patient was a 54-year-old woman with occasional headache. A full blood count showed a hemoglobin level of 12.2 g/dL, a white blood cell count of 11.2 x 103/mL and a platelet count of 164 x 104/mL. A bone marrow sample was hypercellular, containing increased megakaryocytes. Chromosomal analysis showed normal karyotype. Further genetic analysis did not detect JAK2 V617F or CALR mutation. Finally, we directly sequenced MPL exon 10. The result showed MPL c.1496-1497AT>TGGGCCTCAGCTGGGCG (Figure 1). This mutation has been considered as an in-frame mutation, indicating MPL p.H499LGLSWA (reference: NP_005364). The amino acids insertion in transmembrane domain of MPL belongs to hydrophobic family, suggesting that MPL H499 mutation (H499ins) might construct stable structure. To investigate whether MPL H499ins is functionally active, we established stable BaF3/MPL H499ins cell lines. In contrast to BaF3/MPL wild-type, BaF3/MPL H499ins cells proliferate without WEHI3-conditioned medium as well as BaF3/MPL W515L or S505N. Western blot analysis showed BaF3/MPL H499ins cells constitutionally activate downstream signaling including JAK-STAT, MAPK and AKT. Furthermore, we established stable BaF3 cell lines with MPL H499 LGLSWALGLSWA (H499 insx2), MPL H499del and others. In contrast to BaF3/H499del, H499L, H499LG and H499insx3, BaF3/MPL H499insx2 cells proliferate without WEHI3-conditioned medium. This result suggests that elongation of MPL transmembrane domain is a novel oncogenic mechanism leading to constitutive active MPL signaling. Phosphorylation of MPL Y626 has significant role to transduce MPL signaling. To explore if constitutive activation of MPL H499ins depends on phosphorylation of MPL Y626, we established stable BaF3 cell lines with MPL H499insY626F. The BaF3 cells could not proliferate without WEHI3-conditioned medium. This result clearly shows phosphorylation of MPL Y626 has a pivotal role for constitutive activation of MPL H499ins. Finally, we examined potential effect to inhibit constitutive MPL signaling with JAK1/2 inhibitor, Ruxolitinib. In contrast to K562, growth of BaF3/MPL H499ins cells were inhibited with Ruxolitinib at half maximal effective concentration 113 nM as well as MPL W515L or S505N (Figure 2). In summary, elongation of MPL transmembrane domain is a novel oncogenic mechanism in ET patients. Ruxolitinib is also a potent inhibitor against MPL activating mutations as well as JAK2 V617F-associated myeloproliferative neoplasm. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

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Persico, Justin, Imawati Budjahardo, and Kenneth Kaushansky. "The Homodimeric Hematopoietic Cytokine Receptor C-Mpl Requires Cross Phosphorylation in Order to Signal Cell Proliferation." Blood 112, no. 11 (November 16, 2008): 3734. http://dx.doi.org/10.1182/blood.v112.11.3734.3734.

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Abstract Objectives: The three primary regulators of hematopoiesis, erythropoietin, granulocyte colony-stimulating factor and thrombopoietin, bind to homodimeric members of the cytokine receptor superfamily and utilize Janus Kinase (JAK) 2 to initiate signaling. Recently, mutations in JAK2, particularly JAK2V617F, were found to contribute to the pathogenesis of the myeloproliferative disorders. In vitro studies have determined that only homodimeric cytokine receptors can support JAK2-mediated cytokine hypersensitivity. As part of a strategy to identify novel approaches to inhibit mutant JAK2 function we tested whether the homodimeric receptors initiate signaling by JAK2 mediated receptor trans-phosphorylation, and whether JAK2V617F escapes this requirement. Methods: We introduced the engineered receptor Myr/FKBPF36V/c-Mplcyto into hematopoietic cell lines containing either wild-type JAK2 or JAK2V617F, a receptor designed to adopt either a monomeric or dimeric state depending on the absence or presence, respectively, of the chemical dimerizer AP20187. To evaluate the effects of receptor dimerization on the growth of wild-type and V617F mutant JAK2 cell lines we measured reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). If either of the cell lines were able to support cell proliferation in absence of receptor dimerization it would indicate that signaling is initiated by ipsilateral receptor phosphorylation, as opposed to the trans-phosphorylation employed by subfamilies of heterodimeric receptors, such as that for IL-2. Results: The Myr/FKBPF36V/c-Mplcyto receptor construct was subcloned into a retroviral vector, transduced into Baf3, Baf3/JAK2 and Baf3/JAK2V617F cells and spontaneous, IL-3- and AP20187-induced cell proliferation was assessed. Equal expression of the receptor construct in each cell line was confirmed by western blotting. Both Baf3/JAK2- and Baf3/JAK2V617F-derived cell lines transduced with wild type c-Mpl served as controls, and quantitative western blotting was used to verify that equal levels of the two receptor constructs were introduced into the cell lines. Growth factor dependence was confirmed in the control cell lines with both thrombopoietin and IL-3 and was confirmed with IL-3 in the experimental cell lines. There was an increased sensitivity to growth factors in the control cell line containing the JAK2 V617F mutant, consistent with a myeloproliferative phenotype. When Myr/FKBPF36V/Mplcyto was introduced into either Baf3/JAK2 or Baf3/JAK2V617F cells, the cells remained dependent on either IL-3 or AP20187, although maximal rates of cell growth were significantly greater in the Baf3/JAK2V617F/Myr/FKBPF36V/Mplcyto cells than in Baf3/JAK2/Myr/FKBPF36V/Mplcyto cells. The maximal rate of growth of Baf3/JAK2V617F/Myr/FKBPF36V/Mplcyto cells also significantly exceeded that of the Baf3 parental cell line. Furthermore, we found that in the absence of chemically induced dimerization neither Myr/FKBPF36V/c-Mplcyto/JAK2 nor Myr/FKBPF36V/c-Mplcyto JAK2V617F cells proliferated. Conclusions: These results argue that JAK2 induces signaling by trans-phosphorylation of the cytoplasmic domains of c-Mpl and that the kinase hyperactivity displayed by JAK2V617F cannot overcome this requirement. Therefore it may be possible to alter or inhibit trans-phosphorylation and attenuate JAK2V617F-mediated myeloproliferation.

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Lu, Lihui, Arup Ghose, Matt Quail, Bruce Ruggeri, and Mangeng Cheng. "NPM-ALK Mutants in Kinase Domain Exhibit Altered Kinase Activity and Various Sensitivity to ALK Inhibitors." Blood 112, no. 11 (November 16, 2008): 1603. http://dx.doi.org/10.1182/blood.v112.11.1603.1603.

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Abstract Abnormal expression of constitutively active anaplastic lymphoma kinase chimeric proteins (NPM-ALK) in the pathogenesis of anaplastic large-cell lymphoma (ALCL) is well defined. Recently, small molecule inhibitors have been reported that provide solid proof-of-concept validation that inhibition of ALK is sufficient to attenuate the growth and proliferation of ALK+ALCL cells. Although robust clinical response of ALCL patients to the ALK inhibitors is expected, some of those patients are also anticipated to develop resistance to an ALK inhibitor, most likely associated with single point mutations in the kinase domain of ALK. In this study, the nonsense mutants of NPM-ALK on the phosphate anchor region and the gatekeeper region were generated by site-directed mutagenesis and their kinase activity was measured in cells. NPM-ALK/L182M, L182V and L256M mutants displayed comparable or higher kinase activity in cells relative to NPM-ALK wt, and were able to render BaF3 cells into growth factor-independent growth, while NPM-ALK/L182R, L256R, L256V, L256P and L256Q displayed much weaker or little kinase activity in cells and failed to transform BaF3 cells. While NPM-ALK/L182M and L182V displayed comparable sensitivity to a fused pyrrolocarbazole (FP)-derived ALK inhibitor as NPM-ALK wt, they were >30-fold less sensitive to a diaminopyrimidine (DAP)-derived ALK inhibitor. On the other hand, NPM-ALK/L256M displayed >30-fold less sensitivity to both the FP-derived and the DAP-derived ALK inhibitors. Consistent with NPM-ALK autophosphorylation inhibition, the FP-ALK inhibitor induced growth inhibition and cytotoxicity of BaF3/NPM-ALK/L182M and L182V cells but not L256M cells, and the DAP ALK inhibitor failed to induce growth inhibition and cytotoxicity of all three BaF3/NPM-ALK mutant cell lines in culture. In the absence of an ALK inhibitor, BaF3 cells harboring NPM-ALK mutants did not display growth advantage in culture with 10% serum and in mice over the NPM-ALK wt cells. However, the BaF3/NPM-ALK mutants displayed significant growth advantage in culture with low serum (2%) over the BaF3/NPM-ALKwt cells, suggesting the BAF3/NPM-ALK mutant cells may proliferate better in the stressed conditions. Analyses of binding of ALK inhibitors to ALK wt and mutants in ALK homology models are in progress and the results will be discussed.

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Schwall, R. H., L. Y. Chang, P. J. Godowski, D. W. Kahn, K. J. Hillan, K. D. Bauer, and T. F. Zioncheck. "Heparin induces dimerization and confers proliferative activity onto the hepatocyte growth factor antagonists NK1 and NK2." Journal of Cell Biology 133, no. 3 (May 1, 1996): 709–18. http://dx.doi.org/10.1083/jcb.133.3.709.

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Hepatocyte growth factor (HGF) is a potent epithelial mitogen whose actions are mediated through its receptor, the proto-oncogene c-Met. Two truncated variants of HGF known as NK1 and NK2 have been reported to be competitive inhibitors of HGF binding to c-Met, and to function as HGF antagonists (Lokker, N.A., and P.J. Godowski. 1993. J. Biol. Chem. 268: 17145-17150; Chan, A.M., J.S. Rubin, D.P. Bottaro, D.W. Hirschfield, M. Chedid, and S.A. Aaronson. 1991. Science (Wash. DC). 254:1382-1387). We show here, however, that NK1 acts as a partial agonist in mink lung cells. Interestingly, NK1, which is an HGF antagonist in hepatocytes in normal conditions, was converted to a partial agonist by adding heparin to the culture medium. The interaction of NK1 and heparin was further studied in BaF3 cells, which express little or no cell surface heparan sulfate proteoglycans. In BaF3 cells transfected with a plasmid encoding human c-Met, heparin and NK1 synergized to stimulate DNA synthesis and cell proliferation. There was no effect of heparin on the IL-3 sensitivity of BaF3-hMet cells, and no effect of NK1 plus heparin in control BaF3 cells, indicating that the response was specific and mediated through c-Met. The naturally occurring HGF splice variant NK2 also stimulated DNA synthesis in mink lung cells and exerted a heparin-dependent effect on BaF3-hMet cells, but not on BaF3-neo cells. The activating effect of heparin was mimicked by a variety of sulfated glycosaminoglycans. Mechanistic studies revealed that heparin increased the binding of NK1 to BaF3-hMet cells, stabilized NK1, and induced dimerization of NK1. Based on these studies, we propose that the normal agonist activity of NK1 and NK2 in mink lung cells is due to an activating interaction with an endogenous glycosaminoglycan. Consistent with that model, a large portion of the NK1 binding to mink lung cells could be blocked by heparin. Moreover, a preparation of glycosaminoglycans from the surface of mink lung cells induced dimerization of NK1. These data show that the activity of NK1 and NK2 can be modulated by heparin and other related glycosaminoglycans to induce proliferation in cells expressing c-Met.

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Boyapalle, Sandhya, Kathy Rocha, Fei Guo, Purva Bali, Maria Balasis, Warren Fiskus, Sandhya Kumaraswamy, Michael Pranpat, and Kapil Bhalla. "Increased Levels of the Heat Shock Protein (hsp) 70 Contribute as a Pro-Survival and Pro-Growth Mechanism in the Transformation of Bone Marrow Progenitor Cells." Blood 106, no. 11 (November 16, 2005): 1374. http://dx.doi.org/10.1182/blood.v106.11.1374.1374.

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Abstract Hsp70 is an ATP-dependent molecular chaperone that assists in the folding of native proteins into active conformation and prevents aggregation of misfolded and mutated abnormal proteins. In normal non-transformed cells, the expression of hsp70 is low and largely stress-inducible due to misfolded and denatured proteins. Recent studies in our laboratory have demonstrated that human acute leukemia cells abundantly express hsp70, which exerts strong antiapoptotic effects upstream and downstream of the mitochondria. Additionally, as compared to the controls, the mouse myeloid 32D or BaF3 cells transformed by Bcr-Abl or FLT-3 also show increased expression of hsp70. To further elucidate the pro-survival and pro-growth effects of hsp70 and its role in the leukemia transformation, we created stable hsp70 transfectants of the IL-3-dependent 32D and BaF3 (normally maintained in culture in IL-3 containing 10% WEHI medium), i.e., 32D/hsp70 and BaF3/hsp70 cells. These cells displayed 3 to 5 fold higher levels of hsp70, as compared to the control 32D or BaF3 cells. Both 32D/hsp70 and BaF3/hsp70 cells showed significantly improved growth and survival supported by 10% WEHI medium. Following culture in 0%, and less so in 1%, WEHI medium for 24 hours, 32D and BaF3 cells undergo cell cycle G1 phase accumulation, with corresponding decline in the % of cells in the S phase. Following this exposure, they also show markedly increased apoptosis and loss of clonogenic survival, as determined by the colony growth assays in methylcellulose. In contrast, under similar conditions of exposure to reduced % of WEHI conditioned medium, 32D/hsp70 and BaF3/hsp70 cells displayed significantly less accumulation in G1 phase, as well as reduced loss of clonogenic survival and apoptosis (p<0.05). This was also associated with reduced loss of mitochondrial membrane potential and increased accumulation of reactive oxygen species. Notably, following IL-3 withdrawal, exposure to 10 ng/ml of G-CSF for 72 hours induced significantly less differentiation of 32D/hsp70 versus 32D cells, as determined by increase in the % of cells expressing CD11b and GR1 (determined by specific antibody staining and flow cytometry) or by evaluation of the morphologic features of differentiation (p<0.05). Western blot analyses demonstrated that both 32D/hsp70 and BaF3/hsp70 cells, compared to their controls, possessed significantly higher expression of IL-3β receptor (R) and pSTAT5. Importantly, the supernatants of hsp70 overexpressing cells, compared to their controls, also showed higher levels of IL-3, as detected by an ELISA. In addition, BaF3/hsp70, versus the control cells, showed increased DNA binding activity and transactivation by the AP1 transcription factor, utilizing a protein/DNA binding array assay (Panomics, Redwood City, CA) and AP-1-luciferase cis-reporting analysis (Stratagene, La Jolla, CA), respectively. These findings strongly suggest that increased hsp70 levels confer a growth and survival advantage through an IL3- IL-3βR-STAT5-dependent mechanism in the marrow progenitor cells, which may contribute to the transformation induced by leukemia associated oncoproteins.

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Gbadamosi, Mohammed, Tiffany A. Hylkema, Soheil Meshinchi, and Jatinder K. Lamba. "Novel CD33 Antibodies Unravel Localization, Biology, and Therapeutic Implications of CD33 Isoforms." Blood 134, Supplement_1 (November 13, 2019): 908. http://dx.doi.org/10.1182/blood-2019-122596.

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Background: Recent promising results from the CD33-directed immunoconjugate, gemtuzumab ozogamicin (GO), has transformed the realm of acute myeloid leukemia (AML) treatment strategies and has opened the door for several other CD33-directed immunotherapeutic strategies to be tested in clinical settings. Starting from accelerated approval in 2000, GO was discontinued in 2010 due to high levels of death in a major phase 3 clinical trial until follow-up efficacy studies allowed for its re-approval in 2017. Despite the complex path of GO as a therapeutic, its impact in the field has been unprecedented and thus ongoing studies seek to improve treatment strategies using GO. Among the most prevalent challenges to this goal is the variation in structure and expression levels of CD33. In particular, previous studies from our group reported rs12459419 C>T, a splicing SNP, resulting in formation of a shorter CD33 isoform (CD33D2) that lacks the critical immunogenic IgV domain (Lamba, 2017). Given that GO recognizes the IgV domain, its therapeutic effect is compromised in patients expressing CD33D2 isoform (CT and TT genotypes). The lack of benefit observed in rs12459419 heterozygous patients is perplexing and warrants in depth investigations to understand biology of CD33D2 isoform. However, the lack of a specific antibody that can recognize the CD33D2 isoform is a significant roadblock to advancing the: (1) understanding of CD33D2 biology; (2) evaluation of CD33D2 isoform as a novel drug target; and (3) explanation regarding why heterozygous patients have compromised response to GO, despite having intermediate levels of CD33FL isoform. Methods: To overcome this roadblock, we focused on developing novel CD33 antibodies specifically targeted to the shared IgC domain between both isoforms (Figure 1). In brief, peptides spanning IgC domain of CD33 were used as immunogens in either Balb/c or C57BL/6 mice for antibody generation. The novel antibodies, named 5C11-2 and HL2541, were used to assess surface-level, intracellular expression, and intracellular localization of CD33 isoforms. The newly developed IgC targeting antibodies and the well-established IgV targeting P67.6 antibody were tested by flow cytometry and western blotting on the CD33 negative BaF3 cell line and BaF3 cells engineered to express CD33FL (BaF3-CD33FLGFP) and CD33D2 (BaF3-CD33D2GFP), as well as AML cell lines representing different rs1245419 genotypes. Results: As expected, naïve Baf3 cells showed no GFP fluorescence signal whereas BaF3-CD33FLGFP, BaF3-CD33D2GFP were positive for GFP confirming expression of CD33 isoforms (Figure 2A). Subsequent staining with P67.6 only produced a positive signal in BaF3-CD33FLGFP cells while BaF3-CD33D2GFP, which lack the IgV domain, were negative (Figure 2B). Interestingly both novel IgC targeting antibodies, 5C11-2 and HL2541, only stained positive for BaF3-CD33D2GFP cells and were negative for BaF3-CD33FL cells (Figure 2C). Western blotting confirmed the recognition of both CD33FL and CD33D2 isoforms by the IgC targeting novel antibody 5C11-2 (Figure 3). These results indicate a potential steric hindrance caused the bulky nature of the clustered extracellular Ig-folds that may have impacted recognition of native CD33FL by IgC targeting antibodies. Cytometry analysis using 5C11-2 on MV4-11 cells, which are heterozygous for the rs12459419 SNP, showed fluorescence signal when stained IgV specific P67.6 but were negative for the IgC specific novel antibody (Figure 4). These results warrant further investigation to establish the exact localization of CD33D2 in context of different cell types and primary AML cells from patients. Nonetheless, our results report successful development of novel CD33 antibodies that recognizes CD33D2 isoform, which holds promise in establishing CD33 biology and understanding the therapeutic implications such as mechanisms contributing to compromised response in rs12459419 heterozygous patients. Disclosures No relevant conflicts of interest to declare.

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Barge, RM, JP de Koning, K. Pouwels, F. Dong, B. Lowenberg, and IP Touw. "Tryptophan 650 of human granulocyte colony-stimulating factor (G-CSF) receptor, implicated in the activation of JAK2, is also required for G- CSF-mediated activation of signaling complexes of the p21ras route." Blood 87, no. 6 (March 15, 1996): 2148–53. http://dx.doi.org/10.1182/blood.v87.6.2148.bloodjournal8762148.

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Granulocyte colony-stimulating factor (G-CSF) induces rapid phosphorylation of JAK kinases as well as activation of the p21ras route through interaction with its specific receptor (G-CSF-R). The cytoplasmic membrane-proximal region of G-CSF-R (amino acids 631 to 684) is necessary for proliferation induction and activation of JAK2. In contrast, activation of Shc and Syp, signaling molecules implicated in the p21ras signaling route, depends on the phosphorylation of tyrosine residues located in the membrane-distal region (amino acids 685 to 813) of G-CSF-R. We investigated whether G-CSF-induced activation of signaling complexes of the p21ras route depends on the function of the membrane-proximal cytoplasmic region of G-CSF-R. A G- CSF-R mutant was constructed in which tryptophan 650 was replaced by arginine and expressed in BAF3 cells (BAF/W650R). In contrast to BAF3 cell transfectants expressing wild-type G-CSF-R, BAF/W650-R cells did not proliferate and did not show activation of JAK2, STAT1, or STAT3 in response to G-CSF. Immunoprecipitations with anti-Shc and anti-Grb2 antisera showed that mutant W650R also failed to activate Syp and Shc. These data indicate that the membrane-proximal cytoplasmic domain of G- CSF-R is not only crucial for proliferative signaling and activation of JAK2 and STATs, but is also required for activation of the p21ras route, which occurs via the membrane-distal region of G-CSF-R.

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Tabayashi, Takayuki, Sigal Gery, Maya Koren-Michowitz, and H. Phillip Koeffler. "Adaptor Protein Lnk Negatively Regulates Bcr-Abl-Induced Cell Proliferation through Inhibition of the Stat5 Signaling Pathway." Blood 116, no. 21 (November 19, 2010): 3406. http://dx.doi.org/10.1182/blood.v116.21.3406.3406.

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Abstract Abstract 3406 Adaptor protein Lnk negatively regulates not only several hematopoietic cytokine receptors including MPL, EpoR and c-Kit, but also non-receptor tyrosine kinases such as JAK2 and Src. Our previous studies demonstrated that Lnk, when expressed in hematopoietic cell lines, binds and regulates the mutant proteins, JAK2V617F and MPLW515L. Recent in vivo studies have shown that Lnk has an important role in the development of myeloproliferative neoplasms. These data suggest that Lnk may have the ability to inhibit constitutively activated signaling pathways in hematopoietic malignancies. However, how Lnk can attenuate the activity of Bcr-Abl is unclear. In the present study, we tested the hypothesis that Lnk might play a role in regulating Bcr-Abl function. In order to assess if Lnk can inhibit the proliferation of Bcr-Abl-positive hematopoietic cells, Bcr-Abl-expressing BaF3 cells were stably transfected with either Lnk (BaF3/Bcr-Abl/Lnk) or vector only (BaF3/ Bcr-Abl). Colony-formation assays revealed that Lnk significantly inhibited the proliferation of Bcr-Abl-expressing BaF3 cells. Similarly, overexpression of Lnk inhibited growth in the human CML cell line, K562. To determine the cause of growth inhibition by Lnk, assays for apoptosis were performed. Annexin V staining demonstrated that Lnk overexpression induced apoptosis in Bcr-Abl-expressing BaF3 cells. Western blotting analysis of protein lysates from BaF3/ Bcr-Abl /Lnk cells and BaF3/ Bcr-Abl cells found that Lnk-mediated growth inhibition was associated with downregulation of the Stat5 signaling pathway, but not associated with MAPK and PI3K signaling pathways. In addition, experiments in 293T cells expressing Bcr-Abl and Stat5 with either wild-type Lnk or SH2 mutant Lnk revealed that wild-type Lnk, but not SH2 mutant Lnk, inhibited phosphorylation of Stat5. Interestingly, Lnk inhibited Bcr-Abl-induced Stat5 phosphorylation in a dose-dependent manner. These data suggest that the SH2 domain of Lnk is essential for Lnk–mediated downregulation of the Stat5 signaling pathway in Bcr-Abl-positive cells. Taken together, our data suggest that Lnk inhibits Bcr-Abl-induced cell proliferation by attenuating the Stat5 signal transduction and may become a therapeutic target for Bcr-Abl-positive leukemias such as chronic myeloid leukemia and Philadelphia chromosome positive acute lymphoblastic leukemia. Disclosures: No relevant conflicts of interest to declare.

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Yano, Mio, Toshihiko Imamura, Kenichi Sakamoto, Daisuke Asai, Gen Kano, Hajime Hosoi, Takao Deguchi, et al. "Clinical Significance of LNK (SH2B3) Expression in Pediatric B Cell Precursor Acute Lymphoblastic Leukemia." Blood 124, no. 21 (December 6, 2014): 3772. http://dx.doi.org/10.1182/blood.v124.21.3772.3772.

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Abstract Background: In pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL), aberrant JAK-STAT signaling is one of the key leukemogenic mechanisms. Although activating mutations of JAK2 are found in high-risk BCP-ALL patients in western countries, such mutations are rare in Japanese cohorts, led to speculation of other factors contributing abnormal activation of JAK-STAT pathway. The adaptor protein LNK (SH2B3) is one of the negative regulator of JAK-STAT signaling, and its loss of function mutations have been identified in myeloproliferative neoplasms and high-risk BCP-ALL. In addition, the loss of function of LNK has been demonstrated to increase proliferation of B cells in vivo. Based on these findings, we conducted genetic analysis to determine the prognostic impact of LNKin BCP-ALL, and functional analysis to investigate its possible mechanisms. Methods: For genetic analysis, we evaluated diagnostic bone marrow or peripheral blood samples of 164 pediatric BCP-ALL patients treated with the Japan Association of Childhood Leukemia Study (JACLS) ALL02 protocol along with peripheral blood samples from 9 healthy volunteers. The LNK expression level was determined by qRT-PCR. Deletion of IKZF1 was determined by MLPA, and direct sequencing was employed to detect LNK mutations in patients with IKZF1 deletion. For functional analysis, thrombopoietin (TPO)-dependent BaF3-MPL cells expressing LNK (BaF3-MPL-LNK) was established by retroviral transduction to investigate if LNKexpression levels impact on drug sensitivity for prednisolone (PSL), doxorubicin (DOX) or vincristine (VCR). For growth assay, BaF3-MPL cells with or without LNK were cultured for four days in the presence of TPO and the viable cell number was counted. For drug sensitivity test, BaF3-MPL cells with or without LNK were treated with each drug for 48 hours in the presence of TPO, then the IC50 was calculated. Phospho-specific flow cytometory (Phos-Flow) was performed to measure JAK-STAT activation. Results: The LNK expression levels in pediatric BCP-ALL patients’ samples were significantly lower than in samples from healthy donors (P < 0.01). When analyzing all 164 cases, the expression level of LNK was decreased in relapsed patients but there was no statistical significance (P = 0.067). IKZF1 deletion was found in 25 (15% of all) patients, and in these patients, LNK expression level dose not relate to relapse (P = 0.39). Intriguingly, when patients with known high-risk factor (i.e., IKZF1 deletion or poor response to PSL) were excluded, the expression level of LNK was significantly higher in non-relapsed patients (P < 0.05). In functional assay, we observed inhibition of TPO dependent growth of BaF3-MPL cells by expression of LNK (P < 0.01), consistent with previous reports. Phos-Flow analysis revealed that LNK expression suppressed TPO–induced phosphorylation of STAT5 in BaF3-MPL cells. In drug sensitivity test, we found that IC50 of PSL and DOX were substantially lower in BaF3-MPL-LNK cells from in BaF3-MPL-mock cells (0.70 vs 3.93 nM, P < 0.01 and 0.61 vs 1.14 nM, P < 0.05, respectively). Decline in IC50 of VCR by LNK expression was not statistically significant (1.38 vs 2.45 nM, P = 0.056). We next compared the impact of LNK with Ruxolitinib (RUX), a potent synthesized JAK2 inhibitor. The diminution in IC50 of PSL in BaF3-MPL-mock cells treated with RUX (50 nM) was comparable of that in BaF3-MPL-LNK cells (0.31 nM, combination index (CI) = 0.39), consistent with our hypothesis that LNK is working as a JAK2 inhibitor. Since we identified two amino-acid substitutions in N-terminal proline-rich dimerization domain (R139H) and PH domain (P242S), we also examined their function. Transductions of these genes in BaF3-MPL cells, however, did not alter cell growth, suggesting they are single nucleotide variants. Discussions: Our findings that high LNK expression is associated with low relapse rate in intermediate risk (IKZF1 intact, good PSL response) patients indicate potential of LNK to restrain relapse in such patients, presumably by suppressing JAK-STAT signaling. Since we proved the impact of LNK expression to improve sensitivity of PSL in vitro which was comparable to RUX, RUX could compensate lack of internal LNK expression to induce cell death of BCP-ALL cells. Collectively, targeting JAK-STAT pathway will be promising therapeutic option for intermediate risk BCP-ALL patients with low expression level of LNK. Disclosures No relevant conflicts of interest to declare.

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Zhang, Weiguo, Marina Konopleva, Yuexi Shi, Xiaoyang Ling, and Michael Andreeff. "Combination of Sorafenib and Arsenic Trioxide Exerts Synergistic Anti-Leukemia Effects in Sorafenib-Resistant FLT3-ITD-Harboring AML Cells." Blood 112, no. 11 (November 16, 2008): 2656. http://dx.doi.org/10.1182/blood.v112.11.2656.2656.

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Abstract Sorafenib, originally developed as a small molecular Raf inhibitor, has been reported by us to exert directly effect by targeting internal tandem duplication (ITD) mutations of Fms-like tyrosine kinase 3 (FLT3) gene and resulted in an impressive reduction of leukemia blasts in the peripheral blood and bone marrow in AML patients [Zhang W., et al: (2008) J Natl Cancer Inst. 100:184–98]. However, complete remission was not achieved by single agent therapy. Long term treatment with sorafenib seems inducible of the chemoresistance in AML cells lines and animal models although the MAK-ERK activity still could be profoundly inhibited by sorafenib. On the other hand, arsenic trioxide (ATO) has shown great promise in the treatment of patients with acute promyelocytic leukemia (APL). However, ATO efficacy as a single agent in clinical trials to treat non-APL AML was limited and associated with activation of MEK-ERK signaling [Bonati A., et al. (2006) Curr Pharm Biotechnol. 7:397–405]. Therefore, we hypothesized that a combination therapy with sorafenib and ATO could abrogate the ATO-induced upregulation of pro-survival signaling of MEK-ERK and lead to improvement efficacy against hematological malignancies. We first established sorafenib-resistant leukemia cells by long-term culture of murine Baf3-FLT3-ITD cells in vitro with low doses of sorafenib; or by isolation of resistant cells from SCID mice which survived following prolonged treatment with low doses of sorafenib (named Baf3-ITD-Re and Baf3-ITD-Rm cells, respectively). Anti-leukemic activity of sorafenib and/or ATO was investigated in the resistant cells and parental cells (Baf3-ITD) by determination of annexin V positivity and ERK phosphorylation after treatment with sorafenib and/or ATO. In vivo effects were investigated in a leukemic murine model by injecting Baf3-ITD-Rm into SCID mice via tail vein, and tumor progression was determined by monitoring the body bioluminescence of xenograft-bearing mice and by comparing spleen and liver size. The infiltration with leukemia cells was analyzed by histology. Our results showed that sorafenib-induced resistant cells Baf3-ITD-Re and Baf3-ITD-Rm have 1000-fold resistance to sorafenib-mediated cell apoptosis compared with parent cells Baf3-ITD. ATO treatment upregulated the level of phosphorylated ERK in these sorafenib-resistant cells. Combined sorafenib and ATO showed synergistic effect in the resistant cells, and was even more effective in stroma cell co-culture systems (CIs were 0.81 ± 0.12 vs. 0.26 ± 0.12). In a mouse leukemia model of sorafenib-resistance, combined administration with sorafenib and ATO for two weeks synergized anti-leukemia efficacy, significantly decreased the leukemia burden and effectively decreased spleen and liver size. Furthermore, histological analyses showed profoundly reduced leukemia cell infiltration in spleen, liver and bone marrow in the combination therapy. In summary, the combination of sorafenib with ATO shows a synergistic effects in a sorafenib resistant leukemia model, which may suggest a potential therapeutic strategy in sorafenib-resistant leukemia patients.

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Kim, Kyu-Tae, Obdulio Piloto, and Donald Small. "FLT3 Inhibitor Resistant Cells Show Different Downstream Signaling Pathway Changes." Blood 106, no. 11 (November 16, 2005): 4381. http://dx.doi.org/10.1182/blood.v106.11.4381.4381.

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Abstract Receptor tyrosine kinase FLT3 plays an important role in leukemogenesis, especially in acute myeloid leukemia (AML). Tyrosine kinase inhibitors (TKI) targeting wild-type and mutant FLT3 have been developed and shown to have activity in clinical trials. However, as seen with Gleevac in CML, prolonged incubation with TKIs can select for resistant clones that may contribute to disease progression. To study resistance to TKIs against FLT3 we developed FLT3 inhibitor resistant cell lines by co-culturing MOLM14 and BaF3/ITD cells, expressing FLT3/ITD mutants with increasing concentrations of the FLT3 inhibitor CEP-701. The resulting cell lines, MOLM14(R) and BaF3/ITD(R) are resistant to CEP-701 induced cytotoxicity. MOLM14(R) is also resistant to other selective FLT3 TKIs including CEP-5214 and PKC412. In contrast, BaF3/ITD(R) cells were still sensitive to CEP5214 and PKC412. Western blot analysis reveals that CEP-701, CEP-5214 and PKC412 all still inhibit FLT3 in MOLM14(R) cells implying selection of a clone no longer dependent on FLT3 signaling. FLT3 phosphorylation is not inhibited by CEP-701 in BaF3/ITD(R) cells but is still inhibited by CEP-5214 and PKC412. Thus the BaF3/ITD(R) cells appear to remain FLT3-dependent. Sequencing of FLT3 from the resistant clones showed that the resistance was not the result of drug resistance mutations in FLT3/ITD. To investigate possible mechanisms of resistance in FLT3-dependent and FLT3-independent FLT3 inhibitor resistant cells, we examined pathways downstream of FLT3. Previously, we and others reported that constitutive FLT3 activation results in specific changes in gene expression in myeloid leukemic cells. As expected for cells with continued FLT3/ITD activation, Western blot analysis of BaF3/ITD(R) cells treated with CEP-701 show that they maintain activation of Erk/MAPK, Akt, and STAT5 pathways and induction of FLT3 dependent genes including Pim-1 and cMyc. In the apparently FLT3-independent MOLM-14(R) clones, inhibition of FLT3 activity resulted in decreased phosphorylation of downstream Akt and Stat5. However, we found Erk/MAPK phosphorylation and cMyc expression were not decreased in response to FLT3 TKI. This implies that whatever pathway has been selected for the ability to grow in this inhibitor is still feeding into this part of the downstream signaling pathway normally activated by FLT3/ITD. Thus, BaF3/ITD(R) FLT3-dependent and MOLM-14(R) FLT3 independent cells differ in response to several FLT3 inhibitors that results from the differences in their mechanisms of resistance.

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20

Hartley, C., G. Dyas, I. Archibeque, A. Al-Assaad, B. Sun, P. Lincoln, J. Zhan, T. Arvedson, and G. Molineux. "A comparison of potency of and intracellular signaling events provoked by, small molecule and protein-based mpl agonists." Journal of Clinical Oncology 27, no. 15_suppl (May 20, 2009): e14624-e14624. http://dx.doi.org/10.1200/jco.2009.27.15_suppl.e14624.

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e14624 Background: Thrombopoietin (TPO) interacts with its receptor (mpl) to initiate signal transduction and increase platelet production. Other mpl agonists have been developed for clinical use: MDGF (a recombinant protein) and romiplostim (a peptibody) are protein-based mpl agonists; eltrombopag (a small molecule) is a nonpeptide mpl agonist. Methods: Mpl agonists were compared for their ability to induce cellular responses and activate relevant signaling pathways in mpl-expressing cells. Proliferation was assessed by ATP bioluminescence in Baf3/mpl cells treated with mpl agonists for 2 days. Differentiation was assessed by flow cytometric-detection of CD41 and CD61 expression on human CD34+ cells cultured with mpl agonists for 7 days. Activation of signaling pathways was determined by Western blot analysis of proteins from serum-starved Baf3/mpl cells treated with mpl agonists for 20 minutes. Experiments were repeated 3 or more times. Results: The TPO concentration that produced a half-maximal (EC50) proliferative response of Baf3/mpl cells was comparable with that of romiplostim (Table). In contrast, a 30-fold greater concentration of MGDF and a 2,000-fold greater concentration of small molecule were required to produce the same degree of proliferation. In addition, a 1,000,000-fold greater concentration of small molecule compared with romiplostim was required to produce an EC50 for differentiation of CD34+ cells. Mpl agonists activated identical signaling pathways in Baf3/mpl cells: a similar dose-response for phosphorylation of JAK2, Stat3, Stat5, Erk1/2, and AKT was observed at their respective EC10, EC50, and EC100 concentrations. Conclusions: In Baf3/mpl cells, the small molecule was between 1,000- and 1,000,000-fold less potent than protein-based mpl agonists when corrected for molecular mass differences. Small molecule and protein-based mpl agonists were indistinguishable when mpl intracellular signaling was compared at similarly effective concentrations. [Table: see text] [Table: see text]

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Collins, M. K., J. Marvel, P. Malde, and A. Lopez-Rivas. "Interleukin 3 protects murine bone marrow cells from apoptosis induced by DNA damaging agents." Journal of Experimental Medicine 176, no. 4 (October 1, 1992): 1043–51. http://dx.doi.org/10.1084/jem.176.4.1043.

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Murine bone marrow-derived cells, dependent on interleukin 3 (IL-3) for their growth in culture, undergo programmed cell, or apoptosis, upon cytokine withdrawal. Here it is reported that a variety of DNA damaging agents cause a more rapid onset of apoptosis in a factor-dependent cell line, BAF3, deprived of IL-3. In contrast, when cultured in the presence of IL-3, or other growth promoting factors, BAF3 cells are highly resistant to X-irradiation and the cytotoxic drugs etoposide and cisplatin. Overexpression of the bcl2 gene product also protects BAF3 cells from DNA damage. The presence of IL-3 is not required during the initial events of DNA damage or its repair. In the absence of IL-3, cells still complete the repair of DNA breaks within 15 min, and continue to cycle for 5 h. At this time, IL-3 is necessary to prevent the accelerated onset of DNA cleavage from a G2 arrest point.

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22

Johnson, Jan, Manish Ghandi, Jennifer N. Minear, and Brian J. Lannutti. "PRV-1 Interacts with the Cytoplasmic Domain of Mpl and Interferes with TPO-Induced Signaling Cascades." Blood 108, no. 11 (November 16, 2006): 1160. http://dx.doi.org/10.1182/blood.v108.11.1160.1160.

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Abstract Polycythemia rubra vera-1 (PRV-1) is a member of the uPAR/CD59/Ly6 family of cell surface receptors. An increase in the expression level of PRV-1 mRNA has been shown in patients with polycythemia vera and essential thrombocythemia, however, the ligand and function of PRV-1 remains unclear. Here, we show for the first time that PRV-1 expression alters the TPO-mediated Mpl receptor-signaling pathway. We also report a novel interaction between PRV-1 and the cytoplasmic region of Mpl (121 amino acids) using a yeast two-hybrid screen. Further characterization of this interaction in yeast containing truncated versions of the Mpl receptor cytoplasmic region indicates that the binding of PRV-1 to Mpl is regulated at the distal end of the cytoplasmic region. Deletion analysis utilizing Mpl receptors truncated after 53 (T53) or 69 (T69) cytoplasmic amino acid residues failed to show an association with PRV-1. Further mapping of the cytoplasmic domain of Mpl showed that truncations equal or greater than 111 cytoplasmic residues (T111) restored the interaction with PRV-1. To examine the physiological relevance of this interaction, we co-expressed Mpl and PRV-1 in BaF3 cells. Western blot analysis with an anti-phosphotyrosine antibody on TPO-stimulated BaF3/Mpl/PRV-1 whole cell lysates demonstrated a noticeable change in intracellular phosphorylation when compared to BaF3/Mpl. We found no affect on Jak2 and Mpl tyrosine phosphorylation. In contrast, there were higher levels of tyrosine-phosphorylated STAT5 and STAT3 in BaF3/Mpl cells expressing PRV-1. We then further investigated the effect of PRV-1 expression on MAPK signaling. We found that TPO-stimulation of BaF3/Mpl/PRV-1 cells resulted in a reduction of MAPK phosphorylation compared with cells expressing Mpl alone. These results indicate a novel function for PRV-1 as a signaling molecule in cytokine signaling cascades and may lead to a greater understanding of the mechanism of overexpression of PRV-1 in myeloproliferative disorders.

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Schmidt-Tanguy, Aline, Annette Romanski, Mathilde Hunault-Berger, and Oliver G. Ottmann. "Different Roles of Two Autotaxin Isoforms in Proliferation, Migration and Adhesion in the Non-Mutational Tyrosine Kinase Inhibitor Resistant Acute Lymphoblastic Leukemia Cell Line SupB15." Blood 112, no. 11 (November 16, 2008): 1915. http://dx.doi.org/10.1182/blood.v112.11.1915.1915.

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Abstract The Bcr-Abl oncogene is present in 30–40% of adult patients with acute lymphoblastic leukemia (ALL). The Abl kinase inhibitor imatinib-based therapy has become standard for this subset ALL. Acquired resistance to imatinib occurs frequently and is associated with mutations in the tyrosine kinase domain (TKD) approximately in about 80% of patients. In contrast, TKD mutations are uncommon in primary imatinib resistance which appears to be multifactorial, although the underlying mechanisms have been incompletely elucidated. We have established a Ph+ cell line for the analysis of non-mutational resistance mechanisms of imatinib resistance: SupB15RT, a Bcr-Abl expressing lymphoblastic cell line derived from SupB15WT cell line by gradually increasing the exposure to imatinib. SupB15RT shows cross-resistance to the second generation Abl kinase inhibitors Nilotinib and Dasatinib. We have shown that several commonly implicated mechanisms of imatinib resistance do not play a role in conferring the imatinib resistance in SupB15RT cells. By comparative gene expression analysis of SupB15WT vs. SupB15RT cells using Affymetrix- Microarrays, we identified 29 differentially regulated genes. Autotaxin (ATX) is one of the most highly up-regulated genes in imatinib resistant SupB15RT cells, and suggested a contribution to imatinib resistance. ATX is an exo-enzyme (pyrophosphophatase/phosphodiesterase). It plays a role in tumor progression and migration as a tumor cell autocrine motilty factor in various solid tumor cell types. ATX is involved in the synthesis of the signaling molecule, lysophosphatidic acid (LPA) which promotes survival and motility. It was the aim of this study to determine whereas ATX plays a functional role for imatinib resistance in Ph+ ALL. Using RT-PCR we demonstrated that 2 isoforms of ATX are expressed in SupB15RT cells: ATXshort and ATXlong. ATXlong (863 aa) contains highly basic insertion in the catalytic domain (52 residues). We retroviraly transfected BaF3 cells with p185 and/or ATXshort or ATXlong to analyze its influence on growth, adhesion and migration in mouse cell model. In comparison to wild type BaF3 cells the proliferation of BaF3 cells expressing ATXshort is enhanced (1,5-fold), whereas ATXlong expressing BaF3 cells showed no difference in proliferation in comparison to Mock infected cells. The proliferation of p185 expressing BaF3 cells co-expressing ATXshort or ATXlong is not inhibited by the treatment with 1μM imatinib after 3 days in contrast to p185 expressing BaF3 cells. In adhesion experiments, BaF3 cells expressing ATXshort showed a higher attachment independent of p185 expression. We also performed migration experiments using transwell assays. These assays showed more migration with cells co-expressing p185 and ATXlong compared to p185 alone. This is in agreement with our results for SupB15RT vs. SupB15WT with a 3-fold migration increase of SupB15RT. Application of 10% fetal calf serum (FCS) in migration experiments resulted in a 1,5-fold higher migration of the ATXlong expressing BaF3 cells compared to culture without FCS. One explanation for this finding may be that FCS contains lysophosphatidic choline (LPC) which is converted to LPA by ATX. Although expression of both 2 isoforms of ATX is important for the increased proliferation, it seems that the 2 isoforms have different cellular functions in Ph+ lymphoblastic cells. ATXshort seems to enhance adhesion whereas ATXlong plays an important role in motility. Taken together our results indicate a role for ATX in TK- inhibitor resistant SupB15RT cells through LPA signaling via LPA receptors. The ratio between ATXshort and ATXlong probably is important for the intracellular signaling and has to be explored.

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van der Kuip, Heiko, Alexander W. Goetz, Cornelius Miething, Justus Duyster, and Walter E. Aulitzky. "Adhesion to fibronectin selectively protects Bcr-Abl+cells from DNA damage–induced apoptosis." Blood 98, no. 5 (September 1, 2001): 1532–41. http://dx.doi.org/10.1182/blood.v98.5.1532.

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The phenotype of Bcr-Abl–transformed cells is characterized by a growth factor-independent survival and a reduced susceptibility to apoptosis. Furthermore, Bcr-Abl kinase alters adhesion features by phosphorylating cytoskeletal and/or signaling proteins important for integrin function. Integrin-mediated adhesion to extracellular matrix molecules is critical for the regulation of growth and apoptosis. However, effects of integrin signaling on regulation of apoptosis in cells expressing Bcr-Abl are largely unknown. The influence of adhesion on survival and apoptosis in Bcr-Abl+ and Bcr-Abl− BaF3 cells was investigated. p185bcr-abl–transfected BaF3 cells preadhered to immobilized fibronectin had a significant survival advantage and reduced susceptibility to apoptosis following γ-irradiation when compared with the same cells grown on laminin, on polylysin, or in suspension. Both inhibition of Bcr-Abl kinase by STI571 and inhibition of specific adhesion reversed the fibronectin-mediated antiapoptotic effect in BaF3p185. The DNA damage response of Bcr-Abl−BaF3 cells was not affected by adhesion to fibronectin. In contrast to parental BaF3 cells, BaF3p185 adherent to fibronectin did not release cytochrome c to the cytosol following irradiation. The fibronectin-mediated antiapoptotic mechanism in Bcr-Abl–active cells was not mediated by overexpression of Bcl-XL or Bcl-2 but required an active phosphatidylinositol 3-kinase (PI-3K). Kinase-active Bcr-Abl in combination with fibronectin-induced integrin signaling led to a hyperphosphorylation of AKT. Thus, cooperative activation of PI-3K/AKT by Bcr-Abl and integrins causes synergistic protection of Bcr-Abl+ cells from DNA damage–induced apoptosis.

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25

Zhou, Jie, Aibin Liang, Shaoguang Li, Wenjun Zhang, and Jianfei FU. "Activation of JAK2/STAT5 Pathway Reduces Expression Level of DNMT3a in MPN Cell Line." Blood 134, Supplement_1 (November 13, 2019): 5394. http://dx.doi.org/10.1182/blood-2019-125434.

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Introduction: Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell (HSC) disorders characterized by overproduction of mature blood cells and increased risk of transformation to acute myeloid leukemia (AML), and JAK2V167F is the most frequent MPN driving mutation detected in >95% of PV and 50-60% ET and PMF. DNMT3A is a de novo DNA methyltransferase that catalyzes the addition of methyl groups into active chromatin in CpG-rich regions leading to gene inactivation. Dnmt3a-/- HSC have enhanced self-renewal and a block in differentiation in vivo. Previous study showed that JAK2V617F and Dnmt3a loss cooperate to induce myelofibrosis through activated enhancer-driven inflammation, while whether JAK2V617F regulates DNMT3a still remains unclear. AZ960 is a potent and selective ATP competitive inhibitor of the JAK2 kinase, and previous studies reported that AZ960 possessed the activity selectively against JAK2. LY2784544 has been identified as a selective inhibitor of JAK2V617F and has undergone clinical trials for the treatment of several myeloproliferative disorders. Methods: Empty vector (control) and mutant JAK2V617F were transduced into BaF3 cells using a lentivirus system. JAK2V617F-expressing BaF3 cells grow IL-3 independent and were selected by fluorescence-activated cell sorting (FACS) for GFP expression. The protein expression levels of p-STAT5 and DNMT3a were detected by western blotting. JAK2V617F-expressing and control BaF3 cells were incubated with gradient concentration of LY2784544 or AZ960 to inhibit JAK2/STAT5 pathway. Results: The expression levels of p-STAT5 were obviously up-regulated in the JAK2V617F-expressing BaF3 cells, and DNMT3a was down-regulated. After 1-hour incubation in the serial diluted LY2784544, p-STAT5 were reduced in JAK2V617F-expressing BaF3 cells, with expression of DNMT3a elevated. To further confirm the correlation between JAK2/STAT5 pathway and expression of DNMT3a, another JAK2 inhibitor AZ960 was tested similar to LY2784544. With p-STAT5 expression suppressed, protein level of DNMT3a showed significantly promotion. Conclusion: We observed that JAK2V167F mutation suppresses protein expression levels of DNMT3a in MPN cell lines. JAK2 inhibition by AZ960 and LY2784544 significantly improved expression levels of DNMT3a. The activation of JAK2/STAT5 pathway reduces expression level of DNMT3a in MPN cell line, and the specific mechanism still needs to be explored. Figure Disclosures No relevant conflicts of interest to declare.

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Nabinger, Sarah C., Seiji Fukuda, Reuben Kapur, and Rebecca Chan. "The Role of Protein Tyrosine Phosphatase, Shp2, in FLT3-ITD-Induced Leukemogenesis." Blood 112, no. 11 (November 16, 2008): 1804. http://dx.doi.org/10.1182/blood.v112.11.1804.1804.

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Abstract Internal tandem duplications of the FMS-like receptor tyrosine kinase (FLT3-ITDs), an in-fame insertion of several amino acids within the juxtamembrane domain, are present in 25% of acute myeloid leukemia (AML) patients and confer a poor prognosis. FLT3-ITDs induce FLT3 ligand (FL)-independent hyperactivation of Erk and promiscuous activation of STAT5; however, the molecular mechanisms underlying aberrant activation of these signaling molecules is largely unknown. Tyrosine 599 (Y599) of WT FLT3 recruits the protein tyrosine phosphatase, Shp2, upon stimulation with FL, resulting Erk activation. In several FLT3-ITDs, including N51-FLT3 and N73-FLT3, Y599 is duplicated. These findings led us to hypothesize that increased recruitment of Shp2 to N51-FLT3 or N73- FLT3, via Y599, results in enhanced Shp2 activation and contributes to N51-FLT3- and N73-FLT3-induced cellular hyperproliferation, Erk hyperactivation, and promiscuous STAT5 activation. Using Baf3 cells stably expressing WT FLT3, N51-FLT3, or N73- FLT3, co-immunoprecipitation assays demonstrated that Shp2 is phosphorylated and associates with WT FLT3 in a FL-dependent manner. However, in contrast, Shp2 is constitutively hyperphosphorylated and associated with FLT3-N51 and FLT3-N73 independent of FL stimulation. To investigate the functional role of Shp2 in Flt3-ITD-induced leukemogenesis, Baf3 cells expressing WT FLT3, N51-FLT3, or N73-FLT3 were transfected with a mammalian expression vector encoding a U6 polymerase III– directed Shp2-specific short-hairpin RNA (shRNA) or a scrambled shRNA and selected in puromycin. Western blot analysis revealed significant reduction of Shp2 expression by the Shp2-specific shRNA and no change in Shp2 expression by the scrambled shRNA in all cell lines. Upon knock-down of Shp2 in Baf3/WT-FLT3 cells, proliferation was minimally reduced based on thymidine incorporation assays; however, knock-down of Shp2 in Baf3/N51-FLT3 and Baf3/N73-FLT3 cells significantly reduced proliferation, both at baseline and in response to FL stimulation. Collectively, these data suggest that constitutive recruitment of Shp2 to N51-FLT3 and N73-FLT3 contributes to the FLT3- ITD-induced hyperproliferative phenotype and imply that inhibition of Shp2 function may provide a novel therapeutic approach to FLT3-ITD-bearing leukemias.

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Goh, Kee C., Wai C. Ong, Changyong Hu, Hannes Hentze, Ai L. Liang, Walter Stunkel, Yong C. Tan, et al. "SB1518: A Potent and Orally Active JAK2 Inhibitor for the Treatment of Myeloproliferative Disorders." Blood 110, no. 11 (November 16, 2007): 538. http://dx.doi.org/10.1182/blood.v110.11.538.538.

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Abstract JAK2 is the most common mutated gene in bcr-abl-negative chronic myeloproliferative disorders (MPDs) making it an attractive target for drug discovery. Pre-clinical studies have shown that inhibition of JAK2 signaling pathways confer therapeutic benefits in animal models of MPD. We describe here SB1518, a potent, selective and orally active inhibitor of JAK2 with therapeutic potential for the treatment of MPDs. SB1518 is a potent ATP-competitive inhibitor of both JAK2 kinase (IC50 = 22 nM) and its JAK2V617F mutant (IC50 = 19 nM) without significant activity against 40 other representative kinases across the human kinome. SB1518 selectively inhibits the proliferation of cell lines driven by JAK2 or its mutants (e.g. IC50 = 81 nM for a murine BaF3 cell line stably transfected to express erythropoietin receptor and GFP-labeled JAK2V617F). It reduces basal phosphorylation of JAK2 and STAT5 in BaF3-JAK2V617F cells in a concentration-dependent manner. The excellent physicochemical, metabolic and pharmacokinetic properties of SB1518 render it amenable to oral dosing. SB1518 was investigated in a model of advanced MPD established by intravenous injection of BaF3-JAK2V617F cells in nude mice. In several studies using different initial tumor burden and/or drug regimen, we observed dose-dependent and statistically significant therapeutic effects including normalization of elevated white blood cell count and reduction of GFP-labeled BaF3 cells in the peripheral blood, resolution of hepatosplenomegaly, reduction of phospho-STAT5 in diseased organs, prolonged survival and alleviation of terminal-stage anemia and thrombocytopenia. These therapeutic effects were observed at doses well tolerated by the animals. In conclusion, our data demonstrate the therapeutic potential of SB1518 for the treatment of myeloproliferative disorders caused by aberrant JAK2 signaling.

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28

Dong, F., C. van Buitenen, K. Pouwels, L. H. Hoefsloot, B. Löwenberg, and I. P. Touw. "Distinct cytoplasmic regions of the human granulocyte colony-stimulating factor receptor involved in induction of proliferation and maturation." Molecular and Cellular Biology 13, no. 12 (December 1993): 7774–81. http://dx.doi.org/10.1128/mcb.13.12.7774.

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The granulocyte colony-stimulating factor receptor (G-CSF-R) transduces signals important for the proliferation and maturation of myeloid progenitor cells. To identify functionally important regions in the cytoplasmic domain of the G-CSF-R, we compared the actions of the wild-type receptor, two mutants, and a natural splice variant in transfectants of the mouse pro-B cell line BAF3 and two myeloid cell lines, 32D and L-GM. A region of 55 amino acids adjacent to the transmembrane domain was found to be sufficient for generating a growth signal. The immediate downstream sequence of 30 amino acids substantially enhanced the growth signaling in the three cell lines. In contrast, the carboxy-terminal part of 98 amino acids strongly inhibited growth signaling in the two myeloid cell lines but not in BAF3 cells. Truncation of this region lead to an inability of the G-CSF-R to transduce maturation signals in L-GM cells. An alternative carboxy tail present in a splice variant of the G-CSF-R also inhibited growth signaling, notably in both the myeloid cells and BAF3 cells, but appeared not to be involved in maturation.

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29

De Grandis, Maria, Marie Cambot, Marie-Paule Wautier, Bruno Cassinat, Christine Chomienne, Yves Colin, Jean-Luc Wautier, Caroline Le Van Kim, and Wassim El Nemer. "JAK2V617F activates Lu/BCAM-mediated red cell adhesion in polycythemia vera through an EpoR-independent Rap1/Akt pathway." Blood 121, no. 4 (January 24, 2013): 658–65. http://dx.doi.org/10.1182/blood-2012-07-440487.

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Abstract Polycythemia vera (PV) is characterized by an increased RBC mass, spontaneous erythroid colony formation, and the JAK2V617F mutation. PV is associated with a high risk of mesenteric and cerebral thrombosis. PV RBC adhesion to endothelial laminin is increased and mediated by phosphorylated erythroid Lu/BCAM. In the present work, we investigated the mechanism responsible for Lu/BCAM phosphorylation in the presence of JAK2V617F using HEL and BaF3 cell lines as well as RBCs from patients with PV. High levels of Rap1-GTP were found in HEL and BaF3 cells expressing JAK2V617F compared with BaF3 cells with wild-type JAK2. This finding was associated with increased Akt activity, Lu/BCAM phosphorylation, and cell adhesion to laminin that were inhibited by the dominant-negative Rap1S17N or by the specific Rap1 inhibitor GGTI-298. Surprisingly, knocking-down EpoR in HEL cells did not alter Akt activity or cell adhesion to laminin. Our findings reveal a novel EpoR-independent Rap1/Akt signaling pathway that is activated by JAK2V617F in circulating PV RBCs and responsible for Lu/BCAM activation. This new characteristic of JAK2V617F could play a critical role in initiating abnormal interactions among circulating and endothelial cells in patients with PV.

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30

Dong, F., C. van Buitenen, K. Pouwels, L. H. Hoefsloot, B. Löwenberg, and I. P. Touw. "Distinct cytoplasmic regions of the human granulocyte colony-stimulating factor receptor involved in induction of proliferation and maturation." Molecular and Cellular Biology 13, no. 12 (December 1993): 7774–81. http://dx.doi.org/10.1128/mcb.13.12.7774-7781.1993.

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The granulocyte colony-stimulating factor receptor (G-CSF-R) transduces signals important for the proliferation and maturation of myeloid progenitor cells. To identify functionally important regions in the cytoplasmic domain of the G-CSF-R, we compared the actions of the wild-type receptor, two mutants, and a natural splice variant in transfectants of the mouse pro-B cell line BAF3 and two myeloid cell lines, 32D and L-GM. A region of 55 amino acids adjacent to the transmembrane domain was found to be sufficient for generating a growth signal. The immediate downstream sequence of 30 amino acids substantially enhanced the growth signaling in the three cell lines. In contrast, the carboxy-terminal part of 98 amino acids strongly inhibited growth signaling in the two myeloid cell lines but not in BAF3 cells. Truncation of this region lead to an inability of the G-CSF-R to transduce maturation signals in L-GM cells. An alternative carboxy tail present in a splice variant of the G-CSF-R also inhibited growth signaling, notably in both the myeloid cells and BAF3 cells, but appeared not to be involved in maturation.

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31

Collins, M. K., I. J. Furlong, P. Malde, R. Ascaso, J. Oliver, and A. Lopez Rivas. "An apoptotic endonuclease activated either by decreasing pH or by increasing calcium." Journal of Cell Science 109, no. 9 (September 1, 1996): 2393–99. http://dx.doi.org/10.1242/jcs.109.9.2393.

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DNA fragmentation in isolated nuclei from the murine IL3-dependent bone marrow cell line BAF3 could be stimulated either by decreasing pH below 6.5 or by adding microM calcium at neutral pH. An endonuclease which could also be stimulated either by a decrease in pH, to 6.5, or by the presence of microM calcium at neutral pH, was purified 10(4)-fold from nuclei of BAF3 cells. Digestion of DNA with the purified enzyme resulted in 5′-terminal hydroxyl and 3′-terminal phosphate ends. These characteristics are distinct from those described for other mammalian endonucleases. The possible role of this enzyme in genome digestion during apoptosis is discussed.

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32

Mirza, Humayun, Valentina A. Schmidt, Claudia K. Derian, Jolyon Jesty, and Wadie F. Bahou. "Mitogenic Responses Mediated Through the Proteinase-Activated Receptor-2 Are Induced by Expressed Forms of Mast Cell α- or β-Tryptases." Blood 90, no. 10 (November 15, 1997): 3914–22. http://dx.doi.org/10.1182/blood.v90.10.3914.

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Abstract The proteinase-activated receptor-2 (PAR-2) is the second member of a putative larger class of proteolytically activated receptors that mediate cell activation events by receptor cleavage or synthetic peptidomimetics corresponding to the newly generated N-terminus. To further study the previously identified mitogenic effects of PAR-2, we used the interleukin-3 (IL-3)–dependent murine lymphoid cell line, BaF3, for generation of stable cell lines expressing PAR-2 (BaF3/PAR-2) or the noncleavable PAR-2 mutant PAR-2Arg36 → Ala36. Only BaF3 cells expressing either wild-type or mutated receptor exhibited mitogenic responses when grown in IL-3–deficient media supplemented with PAR-2 activating peptide (SLIGRL, PAR39-44). This effect was dose dependent with an EC50 of ∼80 μmol/L, sustained at 24, 48, and 72 hours, and was also demonstrable using thrombin receptor peptide TR42-47. Because tryptase shares ∼70% homology with trypsin (previously shown to activate PAR-2), we studied recombinantly expressed forms of α- and β-tryptases as candidate protease agonists for PAR-2. Hydrolytic activity of the chromogenic substrate tosyl-glycyl-prolyl-argly-4-nitroanilide acetate was present as a sharp peak at Mr ∼130, confirming the presence of secretable and functionally active homotetrameric α- and β-tryptases in transfected COS-1 cells. Dose-dependent proliferative responses were evident using either secreted form of tryptase with maximal responses seen at ∼3 pmol/L (0.1 U/L). Receptor proteolysis was necessary and sufficient for mitogenesis because active site-blocked tryptase failed to induce this response, and proliferative responses were abrogated in BaF3 cells expressing PAR-2Arg36 → Ala36. These results specifically identify both forms of mast cell tryptases as serine protease agonists for PAR-2 and have implications for elucidating molecular mechanisms regulating cellular activation events mediated by proteases generated during inflammatory, fibrinolytic, or hemostatic-regulated pathways.

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Shankar, Deepa B., Jenny C. Chang, Bertrand Parcells, Salemiz Sandoval, Junling Li, Ru-Qi Wei, Paul Tapang, et al. "The Multi-Targeted Receptor Tyrosine Kinase Inhibitor, ABT-869, Induces Apoptosis of AML Cells Both In Vitro and In Vivo." Blood 106, no. 11 (November 16, 2005): 616. http://dx.doi.org/10.1182/blood.v106.11.616.616.

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Abstract Children with acute myeloid leukemia (AML) have less than 60% overall survival despite aggressive chemotherapy and bone marrow transplantation. Only one third of the adult patients diagnosed with AML will be cured. AML blast cells from up to 30% of patients express a constitutively active receptor tyrosine kinase, FLT3-ITD, which contains an internal tandem duplication in the juxtamembrane domain. Patients with FLT3-ITD have a worse prognosis. ABT-869 is a novel multi-targeted small molecule inhibitor of receptor tyrosine kinases and is a potent inhibitor of FLT3, c-Kit, and all members of the VEGF and PDGF receptor families. To determine the effects of ABT-896 on AML cells, we treated AML cell lines, primary cells, and tumors in xenograft models with varying concentrations of the drug. In vitro viability assays showed that ABT-869 inhibited the growth of two different cell lines, MV-4-11 (human AML cell line that expresses FLT3-ITD) and BAF3-ITD (murine B-cell line stably transfected with the FLT3-ITD) at an IC50 of 10nM. ABT-869 was also effective against another mutation of FLT3, D835V, but at higher concentrations (IC50 of 100nM). Phosphorylation of FLT3 and activation of downstream signaling molecules, STAT5 and ERK, were inhibited by ABT-869 in a concentration-dependent manner. Cells were also stained with Annexin V-FITC and Propidium Iodide, and analyzed using FACS. ABT-869 induced apoptosis, caspase-3 activation, and PARP cleavage after 48 hours. To examine the in vitro effects of ABT-869 on normal hematopoietic progenitor cells, we performed methylcellulose-based colony assays with human bone marrow. No significant difference was observed in the number and type of colonies formed using BM cells treated with ABT-869 or control, up to a concentration of 1 micromolar. These results suggest that ABT-869 is not toxic to normal bone marrow progenitor cells at concentrations that are effective against AML cells. To examine the effects of ABT-869 in vivo, we treated SCID mice injected with MV-4-11, Baf3-ITD, Baf3-D835V, or Baf3-WT cells, with oral preparations of ABT-869. Complete regression of MV-4-11 tumors was observed in mice treated with ABT-869 at 20 and 40 mg/kg/day. No adverse effects were detected in the peripheral blood counts, bone marrow, spleen or liver. Histology of the tumors from the control-treated group showed a high degree of proliferation by Ki-67 staining, increased mitotic figures, and a well-defined tumor mass. In contrast, the tumors from mice treated with ABT-869 showed a number of apoptotic bodies by TUNEL staining and the presence of reactive, inflammatory cells. Interestingly, we also observed that mice that received ABT-869 the day after injection of AML cells remained tumor-free for over 2 months in contrast to the mice receiving the vehicle alone. Inhibition of FLT3 phosphorylation was demonstrated in the tumors from mice treated with ABT-869. We are evaluating the activity of ABT-869 treatment of SCID mice injected with Baf3-ITD, Baf3-D835V, or Baf3-WT cells. NOD-SCID mouse models are currently being used to analyze the effects of ABT-869 on primary AML cells in vivo. Our preclinical studies demonstrate that ABT-869 is effective and nontoxic, and provide rationale for the treatment and prevention of relapse in AML patients.

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Page, Elyse C., Susan L. Heatley, David T. Yeung, Paul Q. Thomas, and Deborah L. White. "A Novel Role for HMGN1 in Down Syndrome Acute Lymphoblastic Leukemia." Blood 134, Supplement_1 (November 13, 2019): 1462. http://dx.doi.org/10.1182/blood-2019-126244.

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Background Down Syndrome (DS) Acute Lymphoblastic Leukemia (ALL) patients have extremely poor outcomes with mortality rates four times greater than non-DS ALL patients within their first two years of diagnosis. They are more suspectible to treatment related toxicities and experience higher relapse rates compared to other ALL patients. Approximately 60% of DS-ALL patients harbor rearrangement of cytokine receptor like factor 2 (CRLF2r), specifically P2RY8-CRLF2, and/or the CRLF2 F232C activating mutation. These lesions are considered poor risk and currently no targeted therapy exist. How increased chromosome 21 gene dosage affect disease phenotype is not yet fully elucidated. However, the high mobility group nucleosome-binding domain-containing protein 1 (HMGN1) on chromosome 21, which competes with histone H1 to bind the nucleosome and results in gene activation may be a candidate for targeted therapy in DS-ALL. Methods We aimed to determine the role of HMGN1 in CRLF2r DS-ALL. To model CRLF2r DS-ALL, the trisomy 21 cell line, SET-2, was transduced with a retroviral vector encoding the CRLF2 F232C activating mutation. Gene knockdown of HMGN1 using CRISPR/Cas9 was performed in the SET-2 CRLF2r line and the non-trisomy-21, non-CRLF2 expressing Jurkat line. Individual knockdowns of another two genes on chromosome 21, DYRK1A and ERG were also performed. Knockdown of JAK2 was used as a control as it is critical for CRLF2 signaling. CellTiter-Glo was used to investigate proliferation of knockdown lines to test the hypothesis that HMGN1 is essential for CRLF2r DS-ALL cell proliferation. Lentiviral vectors encoding the P2RY8-CRLF2 fusion gene, CRLF2 F232C activating mutation or an overexpression construct of HMGN1 were transduced into BaF3 cells individually or in combination to test the hypothesis that overexpressing HMGN1 is associated with activation of CRLF2. Quantitative PCR (qRTPCR) for CRLF2 and flow cytometry for the CRLF2/IL7Rα receptor (TSLPR) were used to determine the effect of increased HMGN1 on CRLF2 expression. AnnexinV/7-AAD cell death assays were performed to determine if the effects of HMGN1 could be reduced by the demethylase inhibitor GSK-J4. Results Knockdown of HMGN1 resulted in an 80-90% decrease in HMGN1 protein expression in SET-2 CRLF2 and Jurkat lines compared to the Cas9 controls. While knockdowns of DYRK1A and ERG did not impair the proliferation of SET-2 CRLF2 cells, HMGN1 and JAK2 knockdowns led to a complete proliferation arrest over a period of 120hrs (p=<0.001, n=3), demonstrating their effect on cell division. However, no change in proliferation was observed in the Jurkat knockdown lines. Overexpression of either HMGN1 or P2RY8-CRLF2 alone in BaF3 cells did not result in cytokine independent transformation. However, cytokine independence was triggered in BaF3 cells when HMGN1 and P2RY8-CRLF2 were co-expressed (p=<0.001, n=3); demonstrating a role for HMGN1 in leukemic transformation. Importantly, the overexpression of HMGN1 in the BaF3 P2RY8-CRLF2 line increased the mRNA expression of CRLF2 by 5.8-fold compared to the BaF3 P2RY8-CRLF2 line without HMGN1 (p=0.034, n=3) and increased the mean fluorescence intensity of TSLPR by flow cytometry from 42 to 308 (p=0.008, n=3) (figure 1.a-c) indicating a novel role for HMGN1 in P2RY8-CRLF2 activation. While there are no pharmacological inhibitors for HMGN1, Lane et al. (2014) have shown that the restoration of H3K27 methylation using the demethylase inhibitor GSK-J4 was able to prevent DS-ALL cells from repassaging. Therefore, we have employed the inhibitor GSK-J4 to determine if it can reduce cell survival in HMGN1 overexpressed BaF3 cells. Specific inhibition of BaF3 P2RY8-CRLF2 HMGN1 cells was evident by decreased cell viability at a concentration of 3.8µM compared to BaF3 P2RY8-CRLF2 or BaF3 HMGN1 lines (p=<0.001, n=3) (figure 4.d). Thus, demonstrating a role for HMGN1 in the modification of the P2RY8-CRLF2 methylome and suggesting HMGN1 as a potential therapeutic target. Conclusion These data support the hypotheses that HMGN1 has a significant role in DS-ALL cell proliferation and that overexpression of HMGN1 results in activation of P2RY8-CRLF2. This is the first report of a novel role for HMGN1 in P2RY8-CRLF2 activation and leukemic transformation in CRLF2r DS-ALL. Additionally, we show that HMGN1 is a potential candidate for the development of a pharmacological inhibitor for CRLF2r DS-ALL. Disclosures Yeung: Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Pfizer: Honoraria; Amgen: Honoraria. White:BMS: Honoraria, Research Funding; AMGEN: Honoraria, Speakers Bureau.

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Liu, Pengpeng, Yu Liu, Chong Chen, Ting Liu, and Ting Niu. "Alox15b Gene Contributes to Lymphoma Tumorigenesis Via PI3K/AKT/mTOR Pathway Activation and Has a Synergistic Effect with Alox5 Gene." Blood 132, Supplement 1 (November 29, 2018): 4112. http://dx.doi.org/10.1182/blood-2018-99-120130.

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Abstract Deletion of the short arm of chromosome 17(del17p) is associated with resistance to standard treatment and the very poor clinical outcome in lymphocytic malignancy. Alongside with TP53 gene, ALOX15B gene is also lost in del(17p) patients. In our pervious study, partially knockdown of Alox15b in mice could accelerate lymphoma tumorigenesis with or without TP53 loss. Accumulation of arachidonic acid (AA) and reduction of apoptosis was observed. But how Alox15b gene and AA effect lymphoma tumorigenesis is not fully understood. Here we show that PI3K/AKT/mTOR pathway is activated in Alox15b-knockdown lymphocytes and contributes to the drug resistance. Besides, we also find that Alox15bgene and Alox5gene play a synergistic role lymphoma tumorigenesis in mice model. Experiments were performed using murine preB cell line, baf3 cell. We introduced shRNAs targeting Alox15b at 2 different loci (shAlox15b.1252 and shAlox15b.2865) to create 2 separated Alox15b-deficient cell lines. Intracellular AA level was tested by liquid chromatograph-mass spectrometer(LC-MS). And Alox15b-deficient lymphocytes showed an accumulation of intracellular AA. Then the activation status of PI3K/AKT/mTOR pathway was detected by examining the phosohor-S6 (pS6) protein level using Western Blot assay in wild-type baf3 cells with exogenous-added AA and Alox15b-deficient baf3 cell lines respectively. The results showed that exogenous-added AA activated PI3K/AKT/mTOR pathway in a dose-dependent manner in wild-type baf3 cells. Also, Alox15b-dificient baf3 cell lines showed an increase in pS6 protein level than control, suggesting that in Alox15b-deficient lymphocytes, PI3K/AKT/mTOR pathways is activated via accumulation of AA In addition, the cell viability test was involved to test how Alox15b gene knockdown affected lymphocyte's reaction to rapamycin(RAPA), a mTOR inhibitor. The results showed Alox15b-dificient baf3 cells are more resistant to RAPA, which indicated that mTOR inhibitors may not be a suitable choice in del17p patients. At Last, we collected primary murine lymphoma cells induced by shAlox15b-shTP53/shREN-shTP53 and blood sample from del17p/non-del17p CLL patients to verify our assumption. In mice samples, as AA accumulation was already observed in previous study, we only test pS6 level. And one sample from each experimental group (shAlox15b-shTP53) showed an increase in pS6 level. In human samples, we can see a slightly increase in AA in del17p patient. But due to individual variation, the result isn't statistical significant. Besides, we constructed a plasmid, namely eMLM-shAlox15b-shAlox5, with 2 shRNA targeting Alox5 and Alox15b gene at the same time, introduced the plasmid into primary murine preB cells and transplanted back to recipient mice. After verified by flow cytometry and pathological staining, survival analysis revealed the fact that silencing Alox5 and Alox15b gene at the same time can accelerate lymphoma formation, suggesting that Alox5 and Alox15b gene have a synergistic effect in lymphoma tumorigenesis. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

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Robinson, Lisa J., Jia Xue, and Seth J. Corey. "The Src Related Tyrosine Kinase Lyn Mediates Proliferative Signals from Leukemia-Related Flt3 Mutants." Blood 104, no. 11 (November 16, 2004): 2562. http://dx.doi.org/10.1182/blood.v104.11.2562.2562.

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Abstract Fms-like tyrosine kinase-3 (Flt3) is a hematopoietic growth factor receptor that regulates the survival and proliferation of myeloid and B-cell precursors. Mutations of Flt3 resulting in constitutive activation are the most common known molecular abnormalities in acute myeloid leukemia. Cells expressing activated Flt3 mutants show enhanced survival and proliferation, and leukemia-associated Flt3 mutations produced a fatal myeloproliferative disorder, in a mouse model (Kelly et al. Blood 2002). Nevertheless, the signaling pathways mediating Flt3 effects on proliferation are incompletely understood. Src kinases, such as Lyn, have also been linked to myeloproliferative disease, but the participation of Src-related kinases in Flt3 signaling has not been established. We therefore determined whether Src-family kinases were activated by Flt3 in human leukemic cell lines that express either wild-type or mutant Flt3. Flt3-ligand stimulation of THP1 cells, which express wild-type Flt3, significantly increased the phosphorylation of Lyn, the principal Src-family kinase found in these cells. In MV4;11 cells, which express a constitutively-active tandem duplication mutant of Flt3, Lyn phosphorylation is constitutively elevated. In both cases, a Lyn phosphorylation state specific antibody confirmed phosphorylation at tyrosine 397 of human Lyn; phosphorylation at this site is indicative of Lyn kinase activation. To determine whether the increase in Lyn phosphorylation seen in MV4;11 cells could be attributed specifically to effects of mutant Flt3, we examined Lyn phosphorylation in the murine leukemic Baf3 cells transduced with either wild-type or mutant human Flt3. Lyn phosphorylation and activation in Baf3 cells expressing the Flt3 mutant was significantly increased, compared to basal Lyn phosphorylation in Baf3 cells expressing wild-type Flt3 or vector alone. This result suggested that the constitutive Lyn phosphorylation seen in MV4;11 cells could be caused by mutant Flt3. In Baf3 cells expressing wild-type Flt3, Lyn phosphorylation was stimulated by Flt3-ligand treatment as expected. The Baf3 cell line is growth factor dependent, requiring IL-3 for survival and proliferation. However, as previously reported, expression of activated Flt3 mutants render the cells growth factor independent. We found that the Src-family specific inhibitors PP1 and PP2, but not the inactive analog PP3, inhibited the growth factor-independent proliferation of Baf3 cells expressing mutant Flt3. Moreover, anti-sense oligonucleotides that specifically reduced Lyn expression also inhibited proliferation of cells expressing Flt3 mutants. Previous studies identified Stat5 and Erk as mediators of the proliferation signal from mutant Flt3. We therefore examined the effects of Src kinase inhibitors on the activation of these signaling proteins by mutant Flt3. No change in Stat5 phosphorylation was detected, but inhibition of Lyn did inhibit Erk phosphorylation. These results suggest that Lyn is upstream from Erk in a Flt3 signaling pathway important for proliferation. Flt3 activation of Lyn also raises the possibility that Src kinase inhibitors could have therapeutic efficacy in Flt3-related acute leukemias.

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Tauchi, Tetsuzo, Daigo Akahane, Seiichi Okabe, Eishi Ashihara, Shinya Kimura, Taira Maekawa, and Kazuma Ohyashiki. "Combined Effects of Novel Heat Shock Protein 90 Inhibitor NVP-AUY922 and Nilotinib against Mutant Forms of BCR-ABL." Blood 112, no. 11 (November 16, 2008): 724. http://dx.doi.org/10.1182/blood.v112.11.724.724.

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Abstract NVP-AUY922 (Novartis) is a novel 4,5-diaryloxazole ATP-binding site heat shock protein 90 (HSP90) inhibitor, which has been shown to inhibit the chaperone function of HSP90 and deplete the levels of HSP90 client protein. Combining AUY922 with ABL kinase inhibitors may provide several advantages, such as enhanced efficacy and reducing the potential emergence of new resistant mutations. Treatment with AUY922 has been shown to exert greater potency against BCR-ABL mutants compared with wild type (wt) BCR-ABL. In the present study, we investigated the combined effects of AUY922 and Nilotinib on mutant forms of BCR-ABL-expressing cells. Co-treatment with AUY922 and Nilotinib resulted in significantly more inhibition of growth than treatment with either agent alone in BaF3 cells expressing wt-BCR-ABL and BCR-ABL mutants (M244V, G250E, Q252H, Y253F, E255K, T315A, T315I, F317L, F317V, M351T, H396P). The observed data from the isobologram indicated the synergistic effect of simultaneous exposure to AUY922 and Nilotinib even in BaF3 cells expressing T315I. In contrast, we did not observe enhanced effects of AUY922 and imatinib in T315I BCR-ABL-expressing cells. Co-treatment with 500nM of Nilotinib significantly increased AUY922-induced apoptosis in BaF3 cells expressing T315I. Combined treatment with AUY922 and Nilotinib in BaF3 T315I also associated with more PARP cleavage, resulting from increased activation of caspase-3 and -9 during apoptosis. Following co-treatment of BaF3 T315I with AUY922 and Nilotinib caused more attenuation of phospho-T315I BCR-ABL levels and the downstream signal transducer, including phosho-CrkL, phospho-Stat5, and phospho-Akt. These results demonstrate that Nilotinib appears to inhibit T315I BCR-ABL kinase activity in BCR-ABL structurally compromised by loss of HSP90 chaperone activity. To assess the in vivo efficacy of AUY922 and Nilotinib, athymic nude mice were injected i.v. with mixture of BaF3 cells expressing wild type BCR-ABL and mutant forms of BCR-ABL(M244V, G250E, Q252H, Y253F, E255K, T315A, T315I, F317L, F317V, M351T, H396P). 24 hrs after injection, the mice were divided four groups (5 mice per group), with each group receiving either vehicle, AUY922 (50mg/kg; i.p. two times per week), Nilotinib (30mg/kg; p.o. once every day), AUY922 (50mg/kg; i.p. two times per week) + Nilotinib (30mg/kg; p.o. once every day). Animals treated with either vehicle or Nilotinib-alone died of a condition resembling acute leukemia by 28 days; animals treated with AUY922 alone survived more than 40 days, and those treated with the combination of AUY922 + Nilotinib survived more than 60 days. Histopathologic analysis of vehicle or Nilotinib-alone treated mice revealed infiltration of the spleen and bone marrow with leukemic blasts. In contrast, histopathologic analysis of organs from AUY922 plus Nilotinib-treated mice demonstrated normal tissue architecture and no evidence of residual leukemia. Taken together, these preclinical studies show that the combination of AUY922 and Nilotinib exhibits a desirable therapeutic index that can reduce the in vivo growth of mutant forms of BCR-ABL-expressing cells, including T315I, in an efficacious manner.

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Casolari, Debora A., Diana G. Iarossi, Carolyn M. Butcher, Sarah C. Bray, Wendy T. Parker, Chris N. Hahn, Susan Branford, et al. "Aberrant Activation of Epidermal Growth Factor Receptor in MPN May Respond to the Kinase Inhibitor Gefitinib." Blood 124, no. 21 (December 6, 2014): 1882. http://dx.doi.org/10.1182/blood.v124.21.1882.1882.

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Abstract Epidermal growth factor receptor (EGFR) expression is elevated in peripheral blood (PB) cells of polycythemia vera (PV) patients (Skov et al, Eur J Haematol 2011;87:54-60) and EGFR inhibitors (AEE788, erlotinib) inhibit erythroid burst-forming units (BFUE) from PV patients but not normal donors. The mechanism underlying the effect of EGFR inhibitors on MPN progenitor growth has not been established but could be due to an off-target effect on JAK2 activity (Li et al, J Biol Chem 2007;282:3428-32; Gaikwad et al, Exp Hematol 2007;35:1647-56). Therefore, we investigated the growth of BFUE in the presence and absence of the EGFR inhibitor gefitinib (10µM), which does not inhibit JAK2, and observed inhibition of growth of both erythropoietin (Epo)-dependent and -independent colonies from PB mononuclear cells (PBMNC) from PV patients but not from normal individuals. These results suggest a potential role for EGFR signalling in supporting growth and/or survival of PV progenitors. Therefore, to evaluate the possibility of somatic genetic abnormalities leading to EGFR hypersensitivity in MPN, we performed targeted exon capture and massively parallel sequencing, and sensitive mass array screening of 155 MPN patient samples. We identified a low-frequency, recurrent somatic variant of EGFR (p. C329R) in 3/155 MPN patients. The human EGFR C329 residue is homologous to the residue C359 of the C. elegans gene let-23, target of a known gain-of-function mutation (Katz et al, Mol Cell Biol 1996;16(2):529-37); it also aligns with the cysteine residue affected in the highly-transforming mutant ErbB2 C334S found in lung cancer (Greulich et al, PNAS 2012; 109:14476–14481); and it lies within the extracellular cysteine-rich region of EGFR that is the target of frequent somatic mutations in glioma. To confirm the hypothesis that the EGFR C329R mutant leads to altered cytokine response, we transduced Ba/F3 cells with empty vector, EGFR wild type (WT) or mutant constructs (BaF3/MIG, BaF3/EGFR and BaF3/EGFRC329R, respectively). Both WT and mutant receptors showed constitutive activation and transforming ability when expressed at high levels. However, BaF3/EGFRC329R cells display increased levels of STAT activation associated with a slight proliferation advantage when compared to BaF3/EGFR. Given that gefitinib inhibited the growth of both BaF3/EGFR and BaF3/EGFRC329R but did not affect BaF3/MIG cells grown in IL-3, we next compared the effect of gefitinib (10µM) on the growth of BFUE from PV patient samples with and without the EGFR C329R mutation. We observed significant inhibition of Epo-independent BFUE from all PV samples but not of Epo-dependent BFUE from normal controls (Figure A). Furthermore, genotyping of JAK2 and EGFR from the individual colonies obtained in BFUE assay (treated or not with gefitinib, 10µM) for a PV patient that is positive for EGFR C329R showed that drug treatment significantly reduced the proportion of JAK2 V617F heterozygous BFUE compared to the vehicle-treated control (chi-squared test = 0.0002, Figure B). This suggests that signalling from EGFR contributes to proliferation and/or survival in JAK2 V617F heterozygous BFUE from this patient. The results presented here are consistent with an EGFR signalling role in supporting growth of PV progenitors, particularly in the context of a heterozygous JAK2V617F mutation. STAT5 signalling is essential for PV (Walz et al, Blood 2012;119:3550-3560; Yan et al, Blood 2012;199:3539-3549) and JAK2-independent activation of STAT5 through EGFR (Quesnelle et al, J. Cell. Biochem. 2007; 102:311–319) via various mechanisms may contribute to the level of STAT5 activation required for the PV phenotype. A recent study demonstrating a role for EGFR in hematopoietic stem cells (Doan et al, Nat Med 2013;19:295-304) also highlights the potential of aberrant EGFR signalling to contribute to altered properties of MPN stem cells. Finally, given that gefitinib is currently in clinical use for treatment of solid tumors, these findings raise the possibility that gefitinib may have clinical utility in the context of MPN. Figure 1 Figure 1. Disclosures Branford: Novartis: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Otsuka: Honoraria, Research Funding.

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Imbert, Véronique, Nadia Lounnas, Catherine Frelin, Nadège Gonthier, Emmanuel Griessinger, Nicolas Sirvent, Michael Hummelberger, et al. "Pharmacological Blockade of NF-kB Targets Both Imatinib-Sensitive or -Resistant Chronic Myeloid Leukemia Cells for Cell Death." Blood 108, no. 11 (November 16, 2006): 4793. http://dx.doi.org/10.1182/blood.v108.11.4793.4793.

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Abstract The Bcr-Abl inhibitor imatinib is now the first line therapy for all newly diagnosed CML patients in chronic phase. Nevertheless resistance to the drug emerges as CML progresses to an acute deadly phase. Additional cellular targets should thus be identified to develop alternative therapeutic strategies. The transcription factor NF-kB is a pro-survival factor, found abnormally active in numerous hematologic malignancies. In the present study we show that the constitutive and abnormal activation of NF-kB in Bcr-Abl transformed BaF3 cells and in the LAMA84 CML line could be downregulated after inhibition of Bcr-Abl. Pharmacological blockade of NF-kB by the IKK2 inhibitor AS602868 (Serono International S.A.) prevented proliferation of BaF3/Bcr-Abl, LAMA84 and primary CML cells. Importantly, AS602868 led to apoptosis of an imatinib resistant variant of LAMA84 and of BaF3 clones expressing mutated form of Bcr-Abl derived from imatinib resistant patients. Moreover, NF-kB inhibition affected proliferation and hematopoietic colony formation of primary imatinib resistant CML cells. Finally, the IKK2 inhibitor prolonged survival of mice intravenously injected with the imatinib resistant clone LAMA84-r. Our data strongly suggest that NF-kB mediates important survival functions in CML cells for bcr-abl and that targeting NF-kB with the IKK2 inhibitor AS602868 may represent a new promising therapeutic strategy for CML treatment.

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Hu, Mickey C. T., and Shu L. Chien. "The Cytoplasmic Domain of Stem Cell Antigen CD34 Is Essential for Cytoadhesion Signaling But Not Sufficient for Proliferation Signaling." Blood 91, no. 4 (February 15, 1998): 1152–62. http://dx.doi.org/10.1182/blood.v91.4.1152.

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Abstract CD34 is widely used as a marker in the identification and purification of human hematopoietic stem and progenitor cells; however, its function within hematopoiesis is largely unknown. We have investigated the contribution of cytoplasmic domain of CD34 in cytoadhesion signaling and proliferation signaling in hematopoietic cells. Engagement of particular determinants of CD34 by monoclonal antibodies leads to homotypic adhesiveness of the full-length CD34-transfected BaF3 cells. However, this homotypic adhesiveness is abrogated in BaF3 cells transfected with the truncated CD34 lacking the cytoplasmic domain. Cytoadhesion signaling through the cytoplasmic domain of CD34 cannot be restored through that of erythropoietin receptor (EPOR) or granulocyte colony-stimulating factor receptor (G-CSFR), suggesting that the cytoplasmic domain of CD34 is required for its signal transduction of cellular adhesion. In constrast, we show that replacing the cytoplasmic domain of EPOR or G-CSFR with that of CD34 abolished growth signal transduction in response to EPO or G-CSF in the chimeric receptor-transfected BaF3, 32D, and FDCP1 cells, whereas the wild-type EPOR- or G-CSFR-transfected cells responded to EPO or G-CSF growth signaling well. These results suggest that the cytoplasmic portion of CD34 may not contain the elements necessary to transduce a proliferative signal in hematopoietic cells. Thus, the function of CD34 in hematopoiesis is primarily on hematopoietic cell adhesion.

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Williams, Allen B., Aviv Gazit, Bao Nguyen, Shimon Slavin, and Don Small. "Inhibition of Mutant FLT3 Activation by the Novel Tyrphostin, AGS324." Blood 106, no. 11 (November 16, 2005): 3371. http://dx.doi.org/10.1182/blood.v106.11.3371.3371.

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Abstract FLT3 is a member of the class III tyrosine kinase family, consisting of five immunoglobin-like extracellular domains, a single transmembrane domain followed by a short juxtamembrane region and an interrupted kinase domain. Constitutive activation of FLT3 through mutation occurs in about 30% of AML cases. Studies show that targeted inhibition of FLT3 by small molecule tyrosine kinase inhibitors results in clinical responses in some patients. In light of the finding of resistant mutants selected by Gleevec treatment in CML, it is likely that similar mutants will arise in FLT3. Thus, the need for development of additional FLT3 inhibitors will be important to overcome this resistance. Here, we investigate a series of compounds derived from AG1295 (a FLT3 inhibitor with an IC50 of 300–500 nM) for activity against FLT3. Of these, AGS324 exhibited the most potent activity against FLT3 ITD in transfected BaF3 cells. FLT3 ITD phosphorylation was completely inhibited by treatment with AGS324 with an IC50 of ~40 nM. Likewise, downstream STAT5 and MAP kinase phosphorylation were reduced in parallel to inhibition of FLT3 activation. AGS324 inhibited proliferation of BaF3 FLT3 ITD expressing cells with an IC50 of 60 nM. IL-3 could completely rescue BaF3 ITD cells from AGS324 mediated cytotoxicity even at 10 uM of inhibitor, indicating no interference with IL-3 signaling pathways. There was also no activity against proliferation of BaF3 parental cells. Like AG1295, it showed greatly reduced activity against the D835Y point mutant for inhibition of FLT3 phosphorylation (IC50 >5 uM) and proliferation (IC50 > 10 uM). Not all cells expressing kinase domain point mutants were resistant to inhibition by AGS324. Cells expressing the D835G mutation were inhibited in a similar fashion to BaF3 ITD cells. In addition cells expressing a FLT3 Y842E activating mutation were also inhibited by AGS324. Proliferation of MV4-11 cells, derived from an AML patient, which express a FLT3 ITD mutation, was also inhibited with a similar IC50 (~80 nM). Interestingly, SEM-K2 cells, which express amplified wild-type FLT3 were relatively resistant to inhibition of proliferation and activation of FLT3 by AGS324. Several other compounds that resulted from modifications to AG1295 showed varying degrees of potency against FLT3. Taken together, these findings indicate that AGS324 is a potent inhibitor of FLT3 ITD and some kinase domain point mutations but has reduced activity against wtFLT3. Some structural modifications of AG1295 greatly increase potency against FLT3 ITD as well as some kinase domain mutations. We present some SAR data for this group of tyrphostins, which may be useful for more specific targeting of mutant FLT3.

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Wang, Yongchao, Warren Fiskus, Kavita Natarajan, Anand Jillella, Cornelia Quadt, Peter Ataja, Ross Levine, and Kapil N. Bhalla. "Co-Treatment with JAK2 Inhibitor TG101209 and Panobinostat or hsp90 Inhibitor AUY922 Attenuates Mutant JAK2-V617F Levels and Activity in Human Myeloproliferative Disorder Cells." Blood 112, no. 11 (November 16, 2008): 3739. http://dx.doi.org/10.1182/blood.v112.11.3739.3739.

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Abstract The mutant JAK2-V617F is present and constitutively active in myeloproferative disorders (MPD), including polycythemia vera (PV), essential thrombocytosis and primary myelofibrosis (MF). JAK2-V617F activates the signal transduction through the STAT, RAS/MAPK and PI3K/AKT pathways, conferring proliferative and survival advantage in the MPD hematopoietic progenitor cells (HPCs). We have previously reported that the panhistone deacetylase (HDAC) inhibitors e.g., panobinostat (Novartis Pharmaceuticals Inc), induces growth arrest and apoptosis of neoplastic HPCs. Panobinostat (PS), by inhibiting HDAC6, induces hyperacetylation of heat shock protein (hsp) 90, which attenuates the ATP binding and chaperone function of hsp90. This is associated with polyubiquitylation and degradation of hsp90 client protein signaling kinases, e.g, FLT-3, Bcr-Abl, AKT and c-RAF. Additionally, the mutant versions of signaling oncoprotein kinases, e.g., FLT-3, Bcr-Abl, B-RAF and c-KIT, may be even more dependent on hsp90 chaperone function for maintaining their proper folding and active conformation. This makes the mutant oncoprotein kinase even more susceptible to misfolding and proteasomal degradation upon inhibition of hsp90 function. In the present studies we determined the effects of PS or the non-geldanamycin analogue hsp90 inhibitor (AUY922, Novartis) and/or the JAK-2 inhibitor TG101209 (TG) (TargeGen, San Diego, CA) in the cultured (human erythroleukemia HEL and mouse BaF3 cells expressing JAK2-V617F) and primary human MPD cells (from two patients with PV and MF. Treatment with PS (10 to 30 nM) dose-dependently increased % of cells in G1 and decreased % of cells in S phase of cell cycle, as well as induced apoptosis of HEL and BaF3/JAK2-V617F cells. This was associated with attenuation in the levels of JAK2-V617F, p-JAK2, p-STAT3, p-STAT5, p-AKT (serine 473), AKT, p-ERK1/2, p-GATA1 (serine 310), GATA1 and Bcl-xL in HEL cells. PS induced lysine acetylation of hsp90 but not of JAK2. Similar effects were observed, following treatment with AUY922 (AUY). Both PS and AUY induced significantly less apoptosis of BaF3/hEpoR versus BaF3/JAK2-V617F cells. PS and AUY also reduced the chaperone association of JAK2-V617F with hsp90. Treatment with TG (0.2 to 2.0 uM) also dose-dependently attenuated the levels of p-STAT3, p-STAT5, p-AKT (serine 473), AKT, p-ERK1/2, p-GATA1 (serine 310) and Bcl-xL, which was associated with apoptosis of HEL and BaF3/JAK2-V617F cells. Co-treatment with PS and TG induced more apoptosis of the cultured and primary MPD cells than treatment with either agent alone. AUY also increased apoptosis of HEL cells induced by TG. Co-treatment with PS and AUY synergistically induced apoptosis of HEL cells (combination indices < 1.0). Co-treatment with 20 nM of PS or 15 nM of AUY augmented TG mediated attenuation of p-STAT3, p-STAT5 and Bcl-xL levels in HEL and BaF3/JAK2-V617F cells. These findings demonstrate for the first time that pan-HDAC inhibitor PS and hsp90 inhibitor AUY attenuate the levels and signaling of JAK2-V617F and enhance the anti-JAK2-V617F activity of TG in human MPD cells. Our findings also support the rationale to determine the in vivo activity of PS or AUY in combination with JAK2 inhibitors against human MPD.

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Gloc, Ewa, Mariusz Warszawski, Wojciech Młynarski, Małgorzata Stolarska, Grazyna Hoser, Tomasz Skorski, and Janusz Błasiak. "TEL/JAK2 tyrosine kinase inhibits DNA repair in the presence of amifostine." Acta Biochimica Polonica 49, no. 1 (March 31, 2002): 121–28. http://dx.doi.org/10.18388/abp.2002_3828.

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The TEL/JAK2 chromosomal translocation (t(9;12)(p24;p13)) is associated with T cell childhood acute lymphoblastic leukemia. The TEL/JAK2 fusion protein contains the JAK2 catalytic domain and the TEL-specific oligomerization domain. TEL-mediated oligomerization of the TEL/JAK2 proteins results in the constitutive activation of the tyrosine kinase activity. Leukemia cells expressing TEL/JAK2 tyrosine kinase become resistant to anti-neoplastic drugs. Amifostine is a pro-drug which can selectively protect normal tissues against the toxicity of anticancer drugs and radiation. We investigated the effects of amifostine on idarubicin-induced DNA damage and repair in murine pro-B lymphoid BaF3 cells and BaF3-TEL/JAK2-transformed cells using alkaline single cell gel electrophoresis (comet assay). Idarubicin induced DNA damage in both cell types but amifostine reduced its extent in control non-transformed BaF3 cells and enhanced it in TEL/JAK2-transformed cells. The transformed cells did not show measurable DNA repair after exposure to amifostine and idarubicin, but cells treated only with idarubicin were able to recover within a 60-min incubation. Because TEL/JAK2-transformed cells can be considered as model cells for certain human leukemias and lymphomas we anticipate an enhancement of idarubicin cytotoxicity by amifostine in these diseases. Moreover, TEL/JAK2 tyrosine kinase might be involved in cellular response to DNA damage. Amifostine could promote apoptosis or lower the threshold for apoptosis induction dependent on TEL/JAK2 activation.

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Wang, J. K., G. Gao, and M. Goldfarb. "Fibroblast growth factor receptors have different signaling and mitogenic potentials." Molecular and Cellular Biology 14, no. 1 (January 1994): 181–88. http://dx.doi.org/10.1128/mcb.14.1.181.

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Fibroblast growth factor (FGF) receptors (FGFRs) are structurally related receptor protein tyrosine kinases encoded by four distinct genes. Activation of FGFR-1, -2, and -3 by FGFs induces mitogenic responses in various cell types, but the mitogenic potential of FGFR-4 has not been previously explored. We have compared the properties of BaF3 murine lymphoid cells and L6 rat myoblast cells engineered to express FGFR-1 or FGFR-4. Acidic FGF binds with high affinity to and elicits tyrosine phosphorylation of FGFR-1 or FGFR-4 receptors displayed on BaF3 cells, but only FGFR-1 activation leads to cell survival and growth. FGFR-4 activation also fails to elicit detectable signals characteristic of the FGFR-1 response: tyrosine phosphorylation of SHC and extracellular signal-related kinase (ERK) proteins and induction of fos and tis11 RNA expression. The only detected response to FGFR-4 activation was weak phosphorylation of phospholipase C gamma. A chimeric receptor containing the extracellular domain of FGFR-4 and the intracellular domain of FGFR-1 confers FGF-dependent growth upon transfected BaF3 cells, demonstrating that the intracellular domains of the receptors dictate their functional capacity. Activation of FGFR-1 in transfected L6 myoblasts induced far stronger phosphorylation of phospholipase C gamma, SHC, and ERK proteins than could activation of FGFR-4 in L6 cells, and only FGFR-1 activation induced tyrosine phosphorylation of a characteristic 80-kD protein. Hence, the signaling and biological responses elicited by different FGF receptors substantially differ.

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Wang, J. K., G. Gao, and M. Goldfarb. "Fibroblast growth factor receptors have different signaling and mitogenic potentials." Molecular and Cellular Biology 14, no. 1 (January 1994): 181–88. http://dx.doi.org/10.1128/mcb.14.1.181-188.1994.

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Fibroblast growth factor (FGF) receptors (FGFRs) are structurally related receptor protein tyrosine kinases encoded by four distinct genes. Activation of FGFR-1, -2, and -3 by FGFs induces mitogenic responses in various cell types, but the mitogenic potential of FGFR-4 has not been previously explored. We have compared the properties of BaF3 murine lymphoid cells and L6 rat myoblast cells engineered to express FGFR-1 or FGFR-4. Acidic FGF binds with high affinity to and elicits tyrosine phosphorylation of FGFR-1 or FGFR-4 receptors displayed on BaF3 cells, but only FGFR-1 activation leads to cell survival and growth. FGFR-4 activation also fails to elicit detectable signals characteristic of the FGFR-1 response: tyrosine phosphorylation of SHC and extracellular signal-related kinase (ERK) proteins and induction of fos and tis11 RNA expression. The only detected response to FGFR-4 activation was weak phosphorylation of phospholipase C gamma. A chimeric receptor containing the extracellular domain of FGFR-4 and the intracellular domain of FGFR-1 confers FGF-dependent growth upon transfected BaF3 cells, demonstrating that the intracellular domains of the receptors dictate their functional capacity. Activation of FGFR-1 in transfected L6 myoblasts induced far stronger phosphorylation of phospholipase C gamma, SHC, and ERK proteins than could activation of FGFR-4 in L6 cells, and only FGFR-1 activation induced tyrosine phosphorylation of a characteristic 80-kD protein. Hence, the signaling and biological responses elicited by different FGF receptors substantially differ.

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Bartalucci, Niccolò, Costanza Bogani, Serena Martinelli, Carmela Mannarelli, Jean-Luc Villeval, and Alessandro M. Vannucchi. "The PI3K Specific Inhibitor BKM120 Results Effective and Synergizes With Ruxolitinib In Preclinical Models Of Myeloproliferative Neoplasms." Blood 122, no. 21 (November 15, 2013): 1599. http://dx.doi.org/10.1182/blood.v122.21.1599.1599.

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Abstract Background and Aims A gain-of-function mutation in Janus kinase 2 (JAK2V617F) is at the basis of the majority of chronic myeloproliferative neoplasms (MPN). The dual JAK1/JAK2 inhibitor ruxolitinib (ruxo) determined rapid and sustained responses in splenomegaly and symptomatic improvement in patients with myelofibrosis (MF), supporting the central role of dysregulated JAK2 signaling. Enhanced activation of other downstream pathways including the PI3K/mTOR pathway has been documented as well. We previously reported (Bogani et al, PlosOne 2013;8:54828) that targeting mTOR by the allosteric inhibitor RAD001 resulted in inhibition of JAK2VF mutated cells and produced clinical benefits in a phase I/II trial (Guglielmelli et al, Blood 2011;118:2069). In this study we evaluated the effects of BKM120, a specific PI3K inhibitor, alone and in combination with ruxolitinib, in in-vitro and in-vivo MPN models. Methods To evaluate cell proliferation, colony formation, apoptosis, cell cycle and protein phosphorylation status we used mouse BaF3 and BaF3-EPOR cells expressing wild type (WT) or VF mutated JAK2, the human VF-mutated HEL and SET2 cell lines, and primary MPN CD34+ cells from patients with MF or polycythemia vera (PV). Effect of drug combination was analyzed according to Chou and Talalay calculating the combination index (CI); a CI <1 indicates synergistic activity. For in vivo studies we used two mouse models: (1) SCID mice receiving iv BaF3-EPOR VF-luciferase (luc) cells (gift of T. Radimerski) were randomized on day 6 to different treatment groups based on baseline luminescence. (2) C57Bl6/J JAK2 VF Knock-in mice were generated by insertion of the reversed JAK2V617F exon 13 sequence; mating with Vav-Cre transgenic mice activates the VF allele producing a MPN phenotype in progenies with VF heterozygous expression (Hasan et al, Blood 2013;Epub). Mice were treated for 15 days, then blood, spleen and bone marrow cells were analyzed. Results We found that BKM120 preferential inhibited BAF3 VF and BaF3-EpoR VF cells (IC50: 364±200nM and 1100±207nM, respectively) compared to their respective WT counterpart (5300±800nM and 3122±1000nM: p<.05). HEL and SET2 cells resulted also sensitive to BKM120 (2000±500nM and 1000±300nM). Interestingly we found that BKM120 significantly increased G2/M phase and decreased S phase of cell cycle (p<.01) and induced apoptosis (IC50, SET2=10µM, BaF3-EPOR VF=1.8 µM). Western blot analysis showed marked reduction of phospho-mTOR and its target phospho-4EBP1 as well as downregulation of phospho-STAT5 at 6 and 24h of treatment. BKM120 impaired colony formation from MF and PV CD34+ cells at doses 2 to 8-fold lower than healthy controls (p<.01). BKM120 strongly inhibited EEC colony growth from PV pts (IC50, 9±4nM). Co-treatment of BKM120+ruxo resulted in synergistic inhibition of proliferation of SET2 (median CI=0.45) and BaF3-EPOR VF (median CI=0.8) cells. Triple combinations including BKM120/ruxo plus either RAD001 (Torc1 inhibitor) or PP242 (Torc1/2 inhibitor) resulted highly synergistic (median CI=0.27 and 0.52) to indicate the importance of complete mTOR inhibition. BKM120 at 45mpk and 60mpk increased mean lifespan of BaF3 VF luc mouse model from 21d in control mice to 27.2d and 28d in BMK120 treated mice. In KI mice, co-treatment with 60mpk BKM120 + 60mpk ruxo resulted in improvement of splenomegaly (median spleen weight: 1.4, 0.82, 0.8 and 0.6 g respectively for controls, 60mpk BKM120, 60mpk ruxo and 60mpk BKM120+60mpk ruxo) and reduction of leukocytosis and reticulocyte count. The level of phosho-STAT5 and -4EBP1 in the spleen was significantly reduced in mice receiving BKM120+ruxo as compared to single drug treatment. We finally analyzed the effects of BKM120+/-ruxo on the in-vitro clonogenic growth of BM cells from VF and WT KI mice mixed in a 1:1 ratio. The proportion of VF-positive colonies resulted reduced in a dose dependent manner by 19%, 33% and 44% (p<.03) compared to controls with 50nM, 100nM and 300nM BKM120 respectively. A 25% and 39% of VF-positive colonies reduction was achieved with 50nM and 100nM ruxolitinib. The combined treatment with 100nM BKM120 + 50nM ruxo resulted in a 50% decrease of the number of mutated colonies (p<.02). Conclusions Inhibition of PI3K by BKM120 alone and combined with JAK2 inhibitor ruxolitinib resulted in enhanced activity in preclinical models of MPN, providing a rationale for the ongoing combination clinical trial. Disclosures: Vannucchi: Novartis: Membership on an entity’s Board of Directors or advisory committees.

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Jiang, Xuejie, Po Yee Mak, Hong Mu, Weiguo Zhang, Duncan Mak, Peter Ruvolo, Teresa McQueen, et al. "Concurrent Inhibition of Beta-Catenin/CBP and FLT3 Strongly Suppresses c-Myc and Synergistically Induces Apoptosis in FLT3 Mutant AML Cells." Blood 126, no. 23 (December 3, 2015): 3685. http://dx.doi.org/10.1182/blood.v126.23.3685.3685.

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Abstract Introduction. The FLT3-ITD mutations are frequently observed in acute myeloid leukemia (AML) and correlate with poor prognosis. FLT3-ITD signaling activates MAPK/ERK, JAK/STAT5, and PI3K/AKT pathways, and stabilizes and increases the nuclear localization and transcriptional activity of β-catenin. The effectiveness of FLT3 tyrosine kinase inhibitors (TKIs) is often limited because of additional mutations in the FLT3 gene, TKI-induced alternative signaling activation, and lack of TKIs against AML stem cells (LSCs). Interestingly, the novel β-catenin/cAMP response-element binding protein (CBP) modulator C82 binds to CBP and inhibits its interaction with β-catenin, and disrupts Wnt/β-catenin mediated cell proliferation, self-renewal and survival, which are essential for LSCs/progenitor cells. Consequently, we evaluated the effects of C82 in combination with TKIs in AML cells with FLT3 mutations. Methods. BaF3 cells harboring various FLT3 mutations, Molm13 and MV4-11 cells with FLT3-ITD mutation, as well as FLT3-wild type BaF3 and OCI-AML3 cells were treated with C82 or TKI (sorafenib or quizartinib) alone, or in combination. Apoptosis and cell cycle distribution were determined by flow cytometry, synergistic cytotoxicity (CI) of the combination treatment was calculated by isobologram analysis, and cellular localization of β-catenin was determined by confocal imaging. Protein expression was measured by western blotting and RNA expression by qRT-PCR. Results. Combination of C82 with sorafenib synergistically induced apoptosis in BaF3 FLT3-ITD, BaF3 FLT3-D835G, BaF3 FLT3-ITD plus 676, Molm13, and MV4-11 cells, and in sorafenib resistant BaF3 FLT3-ITD plus 842 cells, even when they were co-cultured with human bone marrow-derived mesenchymal stem cells (MSCs) to mimic the AML microenvironment (CI < 1). Similar results were obtained with Molm13 and MV4-11 cells treated with C82 plus quizartinib. Cell cycle analysis showed that the combination of C82 and sorafenib suppressed cell cycle progression and increased the proportion of cells in subG1 and G1 phases. Furthermore, the combination of C82 and sorafenib inhibited nuclear localization of β-catenin and reduced CD44 levels. Pathway PCR array analysis revealed that C82 and sorafenib, either alone or in combination, suppressed the expression of survivin, cyclin D1, and c-Myc, all downstream targets of β-catenin and several frizzled family receptors/co-receptor and ligands, such as FZD1, FZD3, FZD5, FZD6, FZD7, low-density lipoprotein receptor, WNT10B, and WNT3 (≥2 fold). Western blot analysis confirmed that C82 and TKI (sorafenib or quizatinib) either alone or in combination suppressed c-Myc, survivin, and CD44. Selective inhibition of FLT3-ITD downstream signaling indicated that TKI-induced c-Myc inhibition is primarily mediated through the FLT3-ITD/STAT5/c-Myc signaling cascade. Conclusion: These results demonstrate that combined inhibition of β-catenin and FLT3 tyrosine kinase exerts synergistic cytotoxicity in AML cells with FLT3 mutations. These findings provide a rationale for developing clinical protocols employing β-catenin/CBP modulators and TKIs to overcome FLT3 inhibitor resistance and to determine whether treatment outcomes in patients with FLT3 mutant AML can be improved. Disclosures Zhang: Karyopharm: Research Funding. Cortes:BMS: Consultancy, Research Funding; Teva: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; ARIAD Pharmaceuticals Inc.: Consultancy, Research Funding. Andreeff:Oncoceutics, Inc.: Membership on an entity's Board of Directors or advisory committees. Carter:PrismBiolab: Research Funding.

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Metais, Jean-Yves, Ashley E. Dunfee, Rodrigo T. Calado, and Cynthia E. Dunbar. "BCL2A1 Is A Survival and Immortalization Factor for Primitive Myeloid Hematopoietic Cells." Blood 110, no. 11 (November 16, 2007): 3365. http://dx.doi.org/10.1182/blood.v110.11.3365.3365.

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Abstract We recently reported development of an acute myeloid leukemia in a rhesus macaque transplanted with autologous CD34+ cells transduced with a murine stem cell virus-derived replication defective retrovirus vector expressing only marker genes under control of the strong MCSV LTR. This animal had an unusual clonal reconstitution pattern the first year following transplant, with a single transduced myeloid progenitor cell clone accounting for up to 80% of then normal myelopoiesis (Kelly, 2005). The same vector-containing clone then transformed to AML five years following transplantation, and each tumor cell was shown to contain two vector insertions, one localized 20 kb upstream the CDw92 gene on chromosome 9, and the second localized in the first intron of BCL2A1 on chromosome 15 (Seggewiss, 2006), a gene in the anti-apoptotic BCL2 family not previously linked to myeloid leukemia. BCL2A1 was highly expressed in the tumor cells. This tumor was the first hematopoietic malignancy reported in a recipient of primitive cells transduced with a replication-incompetent vector containing only marker genes, and suggested that BCL2A1 could have potent effects on myeloid cell behavior. To investigate the impact of the BCL2A1 gene product on hematopoietic cells, we cloned the murine and human HA-tagged BCL2A1 cDNAs into lentivirus vectors and transduced the murine BaF3 hematopoietic cell line as a model to study the impact of expression of these proteins on hematopoiesis. We confirmed overexpression of the proteins in the producer cell line as well as in transduced cells by western blot using an anti-HA monoclonal antibody. BaF3 cell proliferation and survival are dependant on IL-3, and under IL-3 replete conditions overexpression of murine or human BCL2A1 did alter proliferation compared with untransduced cells or cells transduced with an empty vector. Removal of IL-3 from the cell culture media leads to rapid apoptosis of BaF3 cells, with cell cycle arrest in the G1 and an apoptotic subpopulation appearing within 24 hours of IL-3 removal. 45% untransduced or empty vector cells were apoptotic, and this fraction decreased to 30% and 15% respectively for BaF3 cells expressing murine or human BCL2A1. These results were confirmed by direct analysis of apoptosis. Only BaF3 cells over-expressing human BCL2A1 were still alive and arrested in G1 after 3 days of culture without IL-3. The murine BCL2A1 had similar but less striking effects. Gene expression analyses on the BaF3 cell populations are ongoing, to identify potential downstream targets of the BCL2A1 protein. The BCL2A1 and empty vectors were also utilized in murine bone marrow cell immortalization assay, previously utilized to identify genes impacting on the survival and expansion of primary myeloid progenitor cells (Du, 2005). In an initial set of experiments, clonal clonal expansion was obtained with marrow cells expressing murine (4 clones) and human (5 clones) BCL2A1 but not for empty vector or untransduced murine marrow. Mice have also been transplanted with primary bone marrow cells transduced with the BCL2A1 and control vectors, and are being followed for in vivo expansion of transduced clones and development of leukemia. In conclusion, we have confirmed the role of BCL2A1 as an anti-apoptotic protein, now in myeloid hematopoietic cells, and will continue to investigate the role of this gene product in hematopoiesis and leukemogenesis.

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Tauchi, Tetsuzo, Seiichi Okabe, Eishi Ashihara, Shinya Kimura, Taira Maekawa, and Kazuma Ohyashiki. "Combined Effects of the Hedgehog Pathway Inhibitor LDE225 and Nilotinib In a Random Mutagenesis Screen." Blood 116, no. 21 (November 19, 2010): 595. http://dx.doi.org/10.1182/blood.v116.21.595.595.

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Abstract Abstract 595 The hedgehog signaling pathway is a developmental pathway that has been shown to play a role in primitive hematopoiesis. Recent studies have demonstrated that hedgehog pathway is activated in chronic myeloid leukemia (CML) stem cells via up-regulation of Smoothened (Smo), a seven transmembrane domain receptor protein. LDE225 is a small molecule Smo antagonist which has entered Phase I clinical evaluation in patients with solid tumors. In the present study, we investigated the combined effects of LDE225 and nilotinib in a random mutagenesis study for BCR-ABL mutation (Blood, 109; 5011, 2007). We performed a comprehensive drug combination experiment using a broader range of concentrations for LDE225 and nilotinib. Compared with single agents, the combination of LDE225 and nilotinib was more effective at reducing the outgrowth of resistant cell clones. No outgrowth was observed in the presence of 2 μM nilotinib plus 20 μM LDE225. Also co-treatment with LDE225 and nilotinib resulted in significantly more inhibition of growth than treatment with either agent alone in BaF3 cells expressing wt-BCR-ABL and BCR-ABL mutants (M244V, G250E, Q252H, Y253F, E255K, T315A, T315I, F317L, F317V, M351T, H396P). The observed data from the isobologram indicated the synergistic effect of simultaneous exposure to LDE225 and nilotinib even in BaF3 cells expressing T315I. Co-treatment with 2μM of nilotinib significantly increased LDE225-induced apoptosis in BaF3 cells expressing T315I. Combined treatment with LDE225 and nilotinib in BaF3 T315I also associated with more PARP cleavage, resulting from increased activation of caspase-3 and -9 during apoptosis. To assess the mechanism of combination effect between LDE225 and nilotinib on T315I BCR-ABL-expressing cells, we used RNA interference to determine whether reduction of nilotinib off-target affected the growth inhibition. BaF3 cells expressing T315I BCR-ABL pretreated with DDR1 siRNA showed enhanced growth inhibition with LDE225. These results demonstrate that the enhanced growth inhibition by LDE225 and nilotinib in T315I-expressing cells may be mediated by DDR1. To assess the in vivo efficacy of LDE225 and nilotinib, athymic nude mice were injected s.c. with BaF3 cells expressing random mutagenesis for BCR-ABL mutation. 7 days after injection (average tumor volume, 100 mm3), the mice were randomised into four groups (5 mice per group), with each group receiving either vehicle, LDE225 (20mg/kg; p.o. once every day), nilotinib (30mg/kg; p.o. once every day), LDE225 (20mg/kg; p.o. once every day) + nilotinib (30mg/kg; p.o. once every day). The LDE225 and nilotinib combination more effectively inhibited tumor growth in mice compared to either vehicle- or nilotinib- or LDE225-treated mice. Histopathologic analysis of tumor tissue from LDE225 plus nilotinib-treated mice demonstrated an increased number of apoptotic cells detected by TUNEL staining. To investigate combined effects of LDE225 and nilotinib on primary Ph-positive acute lymphocytic leukemia (ALL) cells, NOD/SCID mice were injected i.v. with bone marrow mononuclear cells from a Ph positive ALL patient. Treatment with LDE225 and nilotinib demonstrated a marked segregation of apoptotic cells in both the central bone-marrow cavity and the endosteal surface. These results suggest that the combination with a Smo inhibitor and ABL TKIs may help to eliminate the Ph positive ALL cells. Taken together, the present study shows that the combination of LDE225 and nilotinib exhibits a desirable therapeutic index that can reduce the in vivo growth of mutant forms of BCR-ABL-expressing cells. Disclosures: Tauchi: Novartis Pharma KK: Research Funding.

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Andradottir, Silja D., and Magnus K. Magnusson. "Regulatory Role of the Sprouty Gene Family on PDGFβR Fusion Oncogenes." Blood 106, no. 11 (November 16, 2005): 3509. http://dx.doi.org/10.1182/blood.v106.11.3509.3509.

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Abstract Constitutively activated fusion tyrosine kinases of the Platelet-dervied growth factor β receptor (PDGFβR) family have been described in patients with chronic myelomonocytic leukemia (CMML). Like other tyrosine kinase driven myeloproliferative syndromes, CMML is characterized by progression leading to highly aggressive acute leukemia. In order to understand the progression of these malignancies we are studying tyrosine kinase pathway regulatory genes. In this study we focus on the functional role of the sprouty gene family in the regulation of PDGFβR fusion oncogenes. Sprouty (Spry) has recently been identified as a repressor of receptor tyrosine kinases signaling in vertebrates and invertebrates. The studies of sprouty in the mammalian system have thus far mostly focused on the regulation of the epidermal and fibroblast growth factor receptor, while nothing is known about the possible regulation of PDGF receptors by sprouty proteins and nothing is known about regulation of mutationally activated tyrosine kinases. Expression plasmids containing human sprouty wildtype genes (Spry1-3 WT) were constructed, along with a series of plasmids containing dominant negative variants by site-direct mutagenesis in critically conserved domains [Spry1(Y53F), Spry2(Y55F), Spry3(Y27F)]. Stable cell lines containing these plasmids have been generated in the BaF3 background with or without the constitutively activated Rabaptin-5/PDGFβR (R/P) fusion oncoprotein. Effects on cell growth and downstream signaling events were studied. Spry1 WT and Spry3 WT signifcantly inhibit growth of R/P transformed BaF3 cell lines. This inhibition was much more pronounced in IL3 depleted media indicating that the inhibition is mediated through PDGFβR tyrosine kinase inhibition. The dominant negative forms, Spry1(Y53F) and Spry3(Y27F) stimulated growth of the the same BaF3 cell lines. Results from [3H]thymidine uptake studies in these cell lines showed decreased uptake in Spry1 WT and Spry3 WT transduced cells and increased uptake in the dominant negative forms, indicating that the effects are through increased proliferation rather than decreased apoptosis. Interestingly, R/P transformed BaF3 cell lines transfected with plasmid containing Spry2 WT and Spry2(Y55F) showed inverse results, Spry2 WT stimulated growth while Spry2(Y55F) inhibited growth. A possible explanation for stimulatory effects of Spry2 is that this Spry variant contains a Cbl binding domain previously shown to prevent Cbl mediated ubiquitylation and degradation of RTKs. We are currently studying the downstream targets of the Spry regulation of PDGFβR focusing on Ras and MAPkinase pathways. In conclusion, we have shown that Spry1 and Spry3 inhibits growth of PDGFβR transformed BaF3 cell lines, while Spry2 stimulates growth. This is the first evidence for regulatory role of Sprouty genes in activated fusion tyrosine kinase. This conserved family of tyrosine kinase regulatory genes is an ideal target for studies of disease progression in tyrosine kinase driven malignancies.

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Journal articles on the topic 'BaF3'

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