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1
Pískala, Alois, Naeem B. Hanna, Milena Masojídková, Miroslav Otmar, Pavel Fiedler, and Karel Ubik. "Synthesis of N4-Alkyl-5-azacytidines and Their Base-Pairing with Carbamoylguanidines - A Contribution to Explanation of the Mutagenicity of 2'-Deoxy-5-azacytidine." Collection of Czechoslovak Chemical Communications 68, no. 4 (2003): 711–43. http://dx.doi.org/10.1135/cccc20030711.
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A series of N4-alkyl-5-azacytidines 3a-3h were prepared by treatment of the 4-methoxy analogue 4 with the respective amines. In the case of propyl-, butyl-, sec-butyl-, benzyl- or furfurylamine, aggregates of azacytidines 3a-3e with their hydrolytic products 5a-5e were isolated. Similar aggregates were obtained with 1-methyl-5-azacytosine (6) and 2-(methylcarbamoyl)guanidine (7). Compound 7 was prepared by the reaction of guanidine with methyl isocyanate; the reaction of 2 or 3 equivalents gave the di- or tricarbamoyl derivatives 11 and 12, respectively. Cyclization of 7 and 11 with DMF dimethyl acetal afforded azacytosines 6 and 13, respectively. Aggregates of guanosine with 5-azacytosine nucleosides 1, 2 and 15 or of 5-aza-5,6-dihydrocytosine nucleosides 16 and 17 with 5-azacytidine (1) and its 2'-deoxy congener 2 have been prepared by co-crystallization of the respective pairs of nucleosides. The anomers of (deoxyribosylcarbamoyl)guanidine 20a and 20b have been prepared by hydrolysis of the deoxy nucleoside 2. An aggregate of the picrate (8a) of (ribosylcarbamoyl)guanidine 8 with cytidine (9) has been obtained by co-crystallization of both components. Reaction of the methoxy nucleoside 4 with tert-butylamine gave, by contrast to the above mentioned amines, the α-anomer of O-methylribosylisobiuret 22, which was cyclized by DMF dimethyl acetal to the α-anomer of N4,N4-dimethyl-5-azacytidine 24. The connection of the base-pairing ability of carbamoylguanidines with the mutagenicity of 2'-deoxy-5-azacytidine (2) as well as the mechanism of inhibition of DNA methyltransferase by this nucleoside analogue is discussed. In contrast to the unsubstituted 5-azacytidine (1) or its N4-methyl derivatives, none of the N4-alkyl derivatives exhibited any antibacterial or antitumor activity at 100 μg/ml or 10 μmol/l concentrations, respectively.
2
Wang, C., and EA McCulloch. "Sensitivity to 5-azacytidine of blast progenitors in acute myeloblastic leukemia." Blood 69, no. 2 (February 1, 1987): 553–59. http://dx.doi.org/10.1182/blood.v69.2.553.553.
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Abstract In a previous study, we showed that the blast stem cells of acute myeloblastic leukemia (AML) were more sensitive to cytosine arabinoside (ara-C) when growing in suspension culture than during colony formation in methylcellulose. We suggested that the difference might be explained by considering the cellular mechanisms responsible for growth in suspension and colony formation. In the former, the clonogenic cells increase in number (self-renewal); in the latter, most of the divisions are terminal. The increased sensitivity to ara-C in suspension might then be attributed to its ability to inhibit self-renewal to a greater degree than cell division generally. A test of this hypothesis would be to compare the survival curves in suspension and in methylcellulose using a drug that spared or stimulated self-renewal. Such an agent is 5- azacytidine (5-aza) and has the additional advantage that its analogue, 6-azacytidine (6-aza) has no effect on self renewal. The data supported the hypothesis, since clonogenic AML blasts were much less sensitive to 5-aza in suspension than in methylcellulose. The effect of 6-aza, while qualitatively similar, was much less marked. Controls showed that the difference in survival curves could not be explained on a kinetic basis or by the secretion of growth factors by 5-aza-treated cells. We suggest that a comparison of the effects of drugs in suspension and in methylcellulose may be useful in preclinical screening of putative anti- AML compounds.
3
Wang, C., and EA McCulloch. "Sensitivity to 5-azacytidine of blast progenitors in acute myeloblastic leukemia." Blood 69, no. 2 (February 1, 1987): 553–59. http://dx.doi.org/10.1182/blood.v69.2.553.bloodjournal692553.
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In a previous study, we showed that the blast stem cells of acute myeloblastic leukemia (AML) were more sensitive to cytosine arabinoside (ara-C) when growing in suspension culture than during colony formation in methylcellulose. We suggested that the difference might be explained by considering the cellular mechanisms responsible for growth in suspension and colony formation. In the former, the clonogenic cells increase in number (self-renewal); in the latter, most of the divisions are terminal. The increased sensitivity to ara-C in suspension might then be attributed to its ability to inhibit self-renewal to a greater degree than cell division generally. A test of this hypothesis would be to compare the survival curves in suspension and in methylcellulose using a drug that spared or stimulated self-renewal. Such an agent is 5- azacytidine (5-aza) and has the additional advantage that its analogue, 6-azacytidine (6-aza) has no effect on self renewal. The data supported the hypothesis, since clonogenic AML blasts were much less sensitive to 5-aza in suspension than in methylcellulose. The effect of 6-aza, while qualitatively similar, was much less marked. Controls showed that the difference in survival curves could not be explained on a kinetic basis or by the secretion of growth factors by 5-aza-treated cells. We suggest that a comparison of the effects of drugs in suspension and in methylcellulose may be useful in preclinical screening of putative anti- AML compounds.
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Motoji, T., T. Hoang, D. Tritchler, and EA McCulloch. "The effect of 5-azacytidine and its analogues on blast cell renewal in acute myeloblastic leukemia." Blood 65, no. 4 (April 1, 1985): 894–901. http://dx.doi.org/10.1182/blood.v65.4.894.894.
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Abstract Blast cells from patients with acute myeloblastic leukemia were exposed to 5-azacytidine (5-aza) and its analogues 5-aza 2′-deoxycytidine (5- aza-dr) and 6-azacytidine (6-aza). Simple negative exponential survival curves were obtained for the three drugs, but the sensitivity varied; 5- aza-dr was most toxic, 6-aza was least toxic, and 5-aza was intermediate. Colonies surviving drug exposure were replated; 5-aza and 5-aza-dr were found to increase secondary plating efficiency, whereas 6- aza was inactive. The findings provide indirect evidence for a role for DNA methylation in the regulation of blast cell self-renewal.
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Motoji, T., T. Hoang, D. Tritchler, and EA McCulloch. "The effect of 5-azacytidine and its analogues on blast cell renewal in acute myeloblastic leukemia." Blood 65, no. 4 (April 1, 1985): 894–901. http://dx.doi.org/10.1182/blood.v65.4.894.bloodjournal654894.
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Blast cells from patients with acute myeloblastic leukemia were exposed to 5-azacytidine (5-aza) and its analogues 5-aza 2′-deoxycytidine (5- aza-dr) and 6-azacytidine (6-aza). Simple negative exponential survival curves were obtained for the three drugs, but the sensitivity varied; 5- aza-dr was most toxic, 6-aza was least toxic, and 5-aza was intermediate. Colonies surviving drug exposure were replated; 5-aza and 5-aza-dr were found to increase secondary plating efficiency, whereas 6- aza was inactive. The findings provide indirect evidence for a role for DNA methylation in the regulation of blast cell self-renewal.
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Potter, Victoria T., Victoria J. Tindell, Rachel Kesse-Adu, Janet Hayden, Patience Pasipanodya, Laura Reiff-Zall, Judith C. W. Marsh, et al. "Comparative Analysis of An Azacytidine Versus Azacytidine-HSCT Approach for the Treatment of Older Patients with AML/MDS." Blood 116, no. 21 (November 19, 2010): 2375. http://dx.doi.org/10.1182/blood.v116.21.2375.2375.
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Abstract Abstract 2375 While allogeneic HSCT remains the only curative option for patients with MDS, it is associated with considerable morbidity and mortality. 5-azacytidine (5-aza) has been shown to improve the overall survival (OS) of patients with int-2/high risk MDS when compared with best supportive care and conventional chemotherapy. The feasibility of 5-aza in the pre-transplant setting has been demonstrated allowing disease remission induction whilst minimising the toxicity of “induction” chemotherapy. However the additional benefit gained by patients receiving HSCT post 5-aza as compared to patients who receive continuous 5-aza therapy remains to be determined. We report on our single centre experience on the use of 5-aza in a cohort of 71 patients receiving 5-aza as de-novo therapy for high risk MDS/AML. The patients were classified into 2 cohorts. 41 patients received 5-aza in a non-intensive group (defined as patients with advanced age or significant co-morbidities precluding them from HSCT) and 30 patients received 5-aza induction therapy with the intention of proceeding to an allogeneic HSCT (intensive group). Patients in the non-intensive group were significantly older at start of 5-aza therapy than the intensive group (median age: 72 yrs vs 62 yrs, p=<0.01). There was no difference in the proportion of patients with advanced disease (int-2/high IPSS or AML) between groups (non-intensive: 80% vs intensive: 76%, p=0.76). Similarly, there was no significant difference in blast percentage at diagnosis or karyotype risk group between the non-intensive and intensive groups. In the intensive group, the median no. of cycles of 5-aza administered was 7(range 1–30). Out of 30 patients, 15(50%) patients did not proceed to transplant. 14 of these patients had disease progression while on 5-aza, out of which 11 patients received conventional chemotherapy. None of these 11 patients proceeded to HSCT primarily as a result of refractory MDS/AML or chemotherapy toxicity. 15(50%) patients attained a morphological remission (<5% bone marrow blasts) post-5-aza and eventually received a RIC HSCT (including two patients who received umbilical cord blood transplants). All transplanted patients had durable engraftment and the incidence of GVHD at 1 year post-transplant was 50%. At last follow-up, 6 patients who received HSCT were still alive. The actuarial OS at 1 yr post transplant was 35%+/−7%. Among the 41 patients in the non-intensive group, the median number of cycles of 5-aza received was 7 (range:1-46). At last follow-up, 8 patients (19%) were alive with 7 patients receiving ongoing 5 aza. The main reason for cessation of therapy was disease progression. In summary, only 50% of patients commencing 5-aza in the intensive group were subsequently able to receive an allogeneic HSCT. While the non-intensive group consisted of older patients, the median OS from start of 5-azacytidine was 22 months for both intensive and non-intensive groups. The 1 yr and 2 yr actuarial OS was 57%+/−8% and 24%+/−8% for the non-intensive group and 76%+/−8% and 17%+/−8% for the intensive group (p=0.76). While the development of RIC regimens has facilitated the expansion of allogeneic HSCT to older patients with AML/MDS, these findings suggest that in certain patients with co-morbidities, non-intensive novel therapies may be a preferable therapeutic option. Further studies in older AML/MDS patients to evaluate the benefit of novel agents such as 5-azacytidine when compared with HSCT are warranted. Disclosures: Mufti: Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau.
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Sripayap, Piyanuch, Tadashi Nagai, Mitsuyo Uesawa, Hiroyuki Kobayashi, Tomonori Tsukahara, Ken Ohmine, Kazuo Muroi, Gary Baley, and Keiya Ozawa. "Overcoming Resistance to 5-Azacytidine in Acute Myelogenous Leukemia." Blood 120, no. 21 (November 16, 2012): 1370. http://dx.doi.org/10.1182/blood.v120.21.1370.1370.
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Abstract Abstract 1370 Background: The DNA methylation inhibitor 5-azacytidine (AZA), which is approved for treatment of myelodysplastic syndrome, is also a potential agent for treatment of leukemia; however, drug resistance is an ongoing problem, and mechanisms underlying developing resistance to AZA are poorly understood. Therefore, clarifying the resistance mechanisms is central to establish effective countermeasures. Methods: To probe the mechanisms of resistance to AZA and to develop an effective method for overcoming them, we first generated two AZA-resistant cell lines, THP-1/AR and HL60/AR, from the human acute myelogenous leukemia cell lines THP-1 and HL60. We then studied variations between the parental and resistant lines. Results: AZA increased the percentages of sub-G1 and G2/M-phase cells in the AZA-sensitive parental cell lines; whereas, it had no similar effect in the resistant lines. Consistent with these results, the AZA-induced increases in the levels of cleaved forms of caspase 3, caspase 7, caspase 9, and PARP seen in sensitive cells were diminished in resistant cells. Furthermore, AZA markedly elevated the level of phospho JNK/SAPK in sensitive cells, but not in resistant cells. These results suggest that AZA induced apoptosis as well as G2/M arrest due to activation of JNK/SAPK signaling, and that induction of these changes was prevented in resistant cells. We also found that the activity as well as protein levels of DNA methyltransferases (DNMTs), which are the main target molecules of AZA, were suppressed by AZA in sensitive cells. However, in resistant cells, this effect was abrogated; and accordingly, AZA-induced up-regulation of p16 gene expression was also negated. These findings thus suggest that resistance was acquired by a DNMT-dependent mechanism. There was no remarkable difference between resistant cells and sensitive cells in the levels of uridine-cytidine kinase 2 (UCK2), which is a key enzyme for conversion of AZA to active form. However, several point mutations were found restrictedly in exon 4 of the UCK2 gene in both resistant cells. These results raised the possibility that the AZA activation process was perturbed due to reduction of UCK activity; and consequently, AZA failed to suppress DNMT in resistant cells. In addition, by microarray analysis, we identified eleven genes that were expressed at significantly different levels in resistant cells versus sensitive cells. Finally, we showed that the histone deacetylase inhibitor romidepsin induced p16 gene expression and increased the levels of apoptosis-related molecules, while suppressing growth in both sensitive and resistant cell lines. An isobologram analysis demonstrated that simultaneous administration of AZA and romidepsin resulted in an additive inhibitory effect on both AZA-sensitive and AZA-resistant cell growth. These results suggest that romidepsin can overcome AZA resistance; therefore, the combination of AZA and romidepsin not only augments the anti-leukemia effect but also prevents acquisition of resistance to AZA. Conclusions: Newly established 5-azacytidine-resistant cell lines THP-1/AR and HL60/AR are good models to analyze the mechanisms of drug resistance to 5-azacytidine. Using these cell lines, we revealed that acquisition of resistance is primarily caused by a DNMT-dependent mechanism, which can be surmounted with addition of romidepsin. It is likely that the combination of AZA and romidepsin can prevent patients from acquiring resistance to AZA while augmenting its anti-leukemia therapeutic effect. Disclosures: No relevant conflicts of interest to declare.
8
Yalamalle, V. R., D. M. Ithape, A. Kumar, K. Bhagat, S. Ghosh, and M. Singh. "Seed treatment with 5-azacytidine reduces ageing-induced damage in onion seeds." Seed Science and Technology 48, no. 3 (December 31, 2020): 407–12. http://dx.doi.org/10.15258/sst.2020.48.3.09.
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The effect of treating aged onion seeds with 5-azacytidine (5-aza) on germination and vigour was evaluated. Seeds of two onion varieties, 'Bhima Raj' (BRJ) and 'Bhima Red' (BRD) were treated with 0, 10, 25 or 50 μg mL–1 5-azacytidine (a DNA demethylating agent). In comparison with the control treatment (0 μg mL–1 5-azacytidine), treatment with 5-azacytidine enhanced seed germination, seedling length, seedling dry weight and seed vigour indices. 5-azacytidine treatment also increased the activity of superoxide dismutase (SOD) and total antioxidant capacity (TAC). Seed treatment with 5-azacytidine has the potential to enhance the viability and vigour of aged onion seeds. This study provides phenotypic and biochemical data for further exploring the role of DNA methylation in understanding the process of seed ageing.
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Bakanay, S. M., E. Karakiliç, S. Civriz-Bozdag, M. Arat, M. Ozcan, G. Gurman, O. Ilhan, M. Beksac, N. Konuk, and O. Arslan. "5-azacytidine treatment results in myelodysplastic syndrome." Journal of Clinical Oncology 27, no. 15_suppl (May 20, 2009): e18003-e18003. http://dx.doi.org/10.1200/jco.2009.27.15_suppl.e18003.
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e18003 Background: Myelodysplastic syndrome (MDS) is a clonal disease of hematopoiesis characterized by dysplasia in one or more series. 5-azacytidine (5-AZA) which is one of the methyl transferase inhibitors, targets the epigenetic changes in MDS and has been used for the last few years. Phase III studies which compare 5-AZA with supportive therapies report response rates up to 60%. Methods: In this study the aim was to retrospectively analyze the response rates of 26 MDS patients who were treated with 5-AZA between years 2002–2008. The patients were; median age 58 (21–84); male/female = 16/10; RAEB-I (7 patients); RAEB-II (18 patients); ve CMML (1 patient); secondary MDS (2 patients). According to an international prognostic scoring system, 6 patients were intermediate-1; 9 patients were intermediate-2; and 11 patients were high risk. Nine patients received 5-AZA as first-line therapy. The median leukocyte counts, absolute neutrophil counts (ANC), hemoglobin values, and thrombocyte counts at the begining of the cycles were; 2.4 (0.5–23)x10e9/L, 0.7 (0.1–16.5)x10e9/L, 8.6(5.3–11.4) gr/dl, 41(4–35)x10e9/L, respectively. The karyotype analysis revealed del5q in 3 patients; -7/del7q in 6 patients; trisomy 8 in 7 patients, del20q, del11q and complex karyotype (del7q+del5q+delY) in 1 patient each. The median cycles completed were 2 (1–6). Results: Seven patient were not elligible for response evaluation. Nine patients (47%) did not respond to the therapy and 8 of them were lost. Ten patients (53%) responded to the therapy with complete remission (n=4), partial remission (n = 2) and hematological improvement (n = 4). The responders and non-responders were similar in terms of median age, sex and pretreatment leukocyte, ANC and hemoglobin levels. However, there was statistically significant difference in terms of initial thrombocyte counts, total number of cycles received and serum ferritin levels. The patients who received 5-AZA as first line therapy had beter response than others (71% versus 29%). Conclusions: The observed response to 5-AZA therapy was similar to the reported rates in the literature. The most important factors for response were initial thrombocyte counts, total number of cycles, other therapies received before 5-AZA and the serum ferritin levels. No significant financial relationships to disclose.
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Locatelli, Silvia L., Roberto Papait, Giuseppa Careddu, Ada Koschorke, Giuliano G. Stirparo, Monica Balzarotti, Luca Castagna, Armando Santoro, and Carmelo Carlo-Stella. "Upregulation of Cereblon Expression By the DNA Methyltransferase Inhibitor Azacytidine Strongly Enhances Lenalidomide Cytotoxicity in Germinal Center B-Cell-like (GCB) and Activated B-Cell-like (ABC) Diffuse Large B-Cell Lymphoma (DLBCL)." Blood 124, no. 21 (December 6, 2014): 2253. http://dx.doi.org/10.1182/blood.v124.21.2253.2253.
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Abstract INTRODUCTION: Lenalidomide monotherapy exerts clinical activity in relapsed/refractory Diffuse Large B-cell Lymphoma (DLBCL) with better response rate and progression-free survival being recorded in activated B-cell-like (ABC) rather than germinal center B-cell-like (GCB)-DLBCL. Reasons for such a difference are likely due to different expression of key molecules involved in mediating activity of Lenalidomide, such as Interferon regulatory factor 4(IRF4) and cereblon (CRBN). Evidences supporting the key role of DNA methylation and histone modifications in regulating genome stability and gene expression in DLBCL prompted us to investigate the capacity of Azacytidine in modulating Lenalidomide activity, thereby sensitizing GCB-DLBCL to Lenalidomide and enhancing Lenalidomide efficacy in ABC-DLBCL. METHODS: DLBCL cell lines with ABC (U-2932, RIVA) or GCB (SU-DHL4, SU-DHL6) genotype were used to investigate the effects of Lenalidomide and Azacytidine on cell growth and cell death. Western blotting (WB) and immunofluorescence analysis were used to assess modulating effects of the two-drug combination on molecular determinants of Lenalidomide activity. Additionally, we studied CRBN, IRF4 and CRBN binding proteins expression, such as Ikaros and Aiolos (IKZF1 and IKZF3) by real time polymerase chain reaction (RT-PCR) in response to drug treatment. RESULTS: Graded concentrations of Lenalidomide (0.1-100 µM) inhibited cell proliferation by 20% to 40% and increased cell death up to 30% to 40% in ABC-DLBCL cell lines, whereas had minimal effects on GCB-DLBCL cell lines. Untreated ABC-DLBCL but not GCB-DLBCL consistently showed a high expression of CRBN and IRF4. Upon Lenalidomide treatment (3 days) CRBN was significantly upregulated and IRF4 downregulated in ABC-DLBCL, but not GCB-DLBCL cells. Since DNA methylation regulates gene expression in DLBCL cell lines, we next examined whether Azacytdine could modulate CRBN and IRF4 expression and in turn enhance responsiveness to Lenalidomide. Exposure of both ABC- and GCB-DLBCL cell lines to Azacytidine (up to 72 hours) induced a marked increase of CRBN and IRF4 transcripts; addition of Lenalidomide strongly increased Azacytidine-induced increase of CRBN and significantly downregulated IRF4 expression; the combined treatment induced a marked downregulation of Ikaros and Aiolos protein levels. At the cellular level, the concomitant Azacytidine (10 μM)/Lenalidomide (10 μM) treatment inhibited in a synergistic manner the mean (±SEM) cell growth of both ABC-DLBCL (Lena: -16 ± 4%; AZA: -22 ± 2%; AZA/Lena: -70 ± 1%, P<0.001) and GCB-DLBCL (Lena: -17 ± 3%; AZA: -40 ± 4%; AZA/Lena: -82 ± 2%, P<0.001). Additionally, the two drug exposure was associated with a 3-fold decrease of S phase cells(Lena: 28 ± 2%; AZA: 22 ± 0.8%; AZA/Lena: 9 ± 1%, P<0.001); a marked p21 overexpression, and a 3- to 4-fold cell death increase (P<0.001) in both ABC- and GCB-DLBCL. CONCLUSIONS: Our results indicate that Azacytidine sensitizes GCB-DLBCL to the cytotoxic effects of Lenalidomide and enhances Lenalidomide efficacy against ABC-DLBCL resulting in synergistic anti-proliferative and pro-apoptotic effects in both ABC- and GCB-DLBCL cell lines. Cytotoxicity of the two drug combination is mediated by signaling events involving CRBN upregulation and IRF4 downregulation leading to CRBN-binding proteins downregulation. Azacytidine-dependent activation of CRBN and IRF4 expression allow to hypothesize a methylation-driven regulation of these genes. These results might provide a rationale for clinical studies using Azacytidine and Lenalidomide combination in ABC- and GCB-DLBCL. Disclosures No relevant conflicts of interest to declare.
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Bogenberger, James M., William E. Pierceall, Ryan Lena, Reinhold Munker, Kaoru Tohyama, Lisa Sproat, Pierre Noel, et al. "BH3 Profiling Predicts 5-Azacytidine Response in Malignant Myeloid Cells." Blood 120, no. 21 (November 16, 2012): 1432. http://dx.doi.org/10.1182/blood.v120.21.1432.1432.
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Abstract Abstract 1432 Previously we demonstrated that inhibition of anti-apoptotic BCL-2 family members sensitize leukemic cells to 5-Azacytidine (5-Aza), using siRNA and pharmacological inhibition with the BH3-mimetic ABT-737, both in vitro and ex vivo (Bogenberger, JM., et. al. ASH Annual Abstracts 2011;118:Abstract 3513). Crucially, several anti-apoptotic BCL-2 members (e.g. BCL-2 and BCL-XL) required concurrent inhibition for potent and universal sensitization to 5-Aza. Anti-apoptotic BCL-2 proteins block the execution of programmed cell death (apoptosis) by binding to and counteracting two types of pro-apoptotic BCL-2 family proteins: the “BH3-only” proteins, including both activators (BIM and BID) and sensitizers (e.g. NOXA, PUMA, HRK), and the multi-domain effector proteins (BAX and BAK). Cells dependent on anti-apoptotic BCL-2 family members for survival have been defined as “primed for death” (Certo, M., et. al. Cancer Cell 2006 May;9(5):351-65). Importantly, the priming status reflects a dependence on anti-apoptotic BCL-2 family proteins and can be characterized with the BH3 profiling functional assay. This assay measures induction of mitochondrial outer membrane permeabilization (MOMP) in response to treatment with peptides derived from BH3-only proteins (Ni Chonghaile, T., et. al. Science 2011 Nov 25;334(6059);1129-33). Thus, the unique BH3 profile associated with a specific malignant cell population, is a function of the anti-apoptotic BCL-2 family member/s contextual expression in that cell population. Based on our observations of potent 5-Aza sensitization in combination with anti-apoptotic BCL-2 protein family inhibition, we hypothesized that BH3-profiling would predict response to 5-Aza. To address the potential utility of BH3 profiling in predicting response to 5-Aza in myeloid malignancies, we assayed a broad panel of AML-derived cell lines (N=13) by BH3 profiling and correlated BH3 metrics with 5-Aza response. Identical cell line passages were assayed by BH3 profiling and in 5-Aza drug dose response assays. The cell panel comprised lines derived from AML FAB subtypes M7, M6, M5, M4, M2 and M0, as well as diverse cytogenetics such as t(11;21), t(9;11), t(4;11), t(6;11), del 5q, del 7, and a broad spectrum of mutations such as FLT3, N-RAS, CDKN2A, NPM1 and DNMT3A. The panel included a normal karyotype AML cell line (CG-SH) (Munker, R., et. al. Leuk Res 2009 Oct:33(10):1405-8) and a blastic cell line derived from a patient with MDS (MDS-L) (Tohyama, K., et. al. Br J Haematol 1994 Jun;87(2):235-42). Partition modeling using several BH3 metrics discriminates 5-Aza response with statistical significance (N=13, Mann-Whitney p<0.01) between more sensitive (EC50<2uM) and less sensitive (EC50>2uM) AML lines. For instance, the Puma BH3 peptide alone distinguishes more sensitive from less sensitive cell lines (N=13, Mann-Whitney p=0.0046). Several more complex parameters, such as “Puma+Hrk”, “Puma+Noxa,” and “Bim+Puma+Noxa+Hrk,” also significantly discriminate 5-Aza response (Mann-Whitney p=0.0011). Using continuous variable analysis, a Puma+Hrk metric exhibited the strongest correlation with 5-Aza response (R2=0.85, p<0.0001), while an individual Puma metric yielded the second strongest correlation (R2=0.70, p=0.0004). In conclusion, BH3 profiling discriminates 5-Aza response and confirms a central role for anti-apoptotic BCL-2 members in 5-Aza response. Furthermore, HRK priming indicates that BCL-XL is an important, although not the sole, anti-apoptotic component determining 5-Aza response in myeloid cells. These BH3 profiling results are in agreement with our functional 5-Aza RNAi and BCL-XL/BCL-2 inhibitor data in combination with 5-Aza. Based on these results, we are currently evaluating BH3 profiles from 5-Aza responding and non-responding AML and MDS patients to validate BH3 profiling-derived metrics in predicting clinical outcomes to 5-Aza. Disclosures: Pierceall: Eutropics Pharmaceuticals: Employment, Equity Ownership. Lena:Eutropics Pharmaceuticals: Employment. Mesa:Sanofi: Research Funding; Incyte: Research Funding; Lilly: Research Funding; NS Pharma: Research Funding; YM Bioscience: Research Funding. Cardone:Eutropics Pharmaceuticals: Employment, Equity Ownership.
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Latagliata, Roberto, Pasquale Niscola, Maria Antonietta Aloe Spiriti, Ida Carmosino, Laura Cesini, Chiara Sarlo, Annalina Piccioni, et al. "Pulmonary Infections in Patients with Myelodysplastic Syndromes Receiving Azacytidine Treatment." Blood 128, no. 22 (December 2, 2016): 5544. http://dx.doi.org/10.1182/blood.v128.22.5544.5544.
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Abstract Azacytidine (AZA) is a demethylating agent widely used in the treatment of patients with high-risk Myelodysplastic Syndromes (MDS). Pulmonary infections are often reported in these patients during AZA treatment, however, their incidence, etiology and impact in the overall outcome are still unclear. A major problem is represented by the low level of clinical and microbiological documentation, as most of patients treated with AZA are managed on an outpatient basis. This study aims to evaluate the incidence and clinical impact of pulmonary infections in MDS patients treated with AZA in 5 hematological Institutes in Rome, with a relatively homogeneous infective diagnostic work-up in the presence of fever or other infective clinical signs. We retrospectively evaluated 146 MDS patients [M/F 93/53, median age 69.5 years, interquartile range (IQR) 65.0 - 75.6] treated with AZA at our Institutions from 04/2009 to 01/2016. All patients received AZA cycles at standard dosage (75 mg/m2 for 7 days every 28 days) as outpatients. In the event of febrile neutropenia or other infectious episodes patients underwent blood cultures, culture from other sites, and chest x-ray or (preferably) pulmonary CT-scan. Galactomannan assay from serum and from sputum/bronchoalveolar lavage was performed as indicated. The total number of AZA cycles was 1712, with a median per patient of 9 cycles (IQR 5 - 17 cycles). There were 75 episodes of lung infection (4.1% of AZA cycles), with 58 patients (39.7%) presenting at least 1 episode. Based on the above diagnostic work-up, pulmonary infiltrates were considered of fungal origin in 21 cases (28.0%), associated to bacteremia in 5 cases (6.7%) and of unknown origin in the remaining 49 cases (65.3%). As to the time of occurrence of lung infections, 29 episodes were documented in the first 4 cycles of AZA treatment (5.4% of 535 cycles) and the remaining 46 episodes beyond the 4th cycle of AZA treatment (3.9% of 1177 cycles). Overall, a pulmonary fungal disease was documented in 10 of 535 (1.9%) cycles 1-4 and in 11 of 1177 (0.9%) cycles beyond the 4th (p=0.001). Several clinical features (age, gender, Hb level, WBC and PLT counts, time from diagnosis) were evaluated as predictive factors for the occurrence of pulmonary infection, but none was significant. Out of 58 patients who developed at least one pulmonary infection, 39 (67.2%) definitively discontinued the AZA treatment within 3 months from the infectious episode due to deterioration of clinical conditions, hematologic disease progression and/or death. Attributable mortality rate of patients with pulmonary infection was 22.4% (13 of 58). The median Overall Survival (OS) of the whole cohort was 18.0 months (95%CI 14.4 - 21.5): the median OS of patients with pulmonary infection was 15.6 months (95%CI 13.1 - 18.0) compared with 21.5 months (95%CI 16.3 - 26.6) of patients without pulmonary infection (p=0.031). In conclusion, pulmonary infections are a common complication in MDS patients receiving AZA treatment, and are often associated to an interruption of AZA therapy. Pulmonary fungal infections are more frequently observed early during the first cycles of treatment. It should be defined if the poor outcome of patients who develop pulmonary infections during AZA therapy is an epiphenomenon of an immunologic deterioration associated to the hematologic disease progression or is independently related to the complication. If confirmed in other experiences, the results of our study raise the issue of the need of an antibacterial and/or antifungal prophylaxis particularly during the first months of AZA therapy. Disclosures Latagliata: Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Honoraria; Celgene: Honoraria; Janssen: Consultancy, Honoraria; Shire: Honoraria. Breccia:Celgene: Honoraria; Novartis: Consultancy, Honoraria; Ariad: Honoraria; Bristol Myers Squibb: Honoraria; Pfizer: Honoraria.
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Oran, Betul, Marcos de Lima, Guillermo Garcia-Manero, Peter F. Thall, Ruitao Lin, Amin M. Alousi, Chitra Hosing, et al. "Maintenance with 5-Azacytidine for Acute Myeloid Leukemia and Myelodysplastic Syndrome Patients." Blood 132, Supplement 1 (November 29, 2018): 971. http://dx.doi.org/10.1182/blood-2018-99-111582.
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Abstract Background: Relapse is the main cause of treatment failure after allogeneic stem cell transplant (SCT) in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Based on encouraging preliminary reports for S.C. 5-Azacitidine (AZA) in the post-transplant setting, we investigated whether AZA given as maintenance might improve relapse free survival (RFS). Methods: Between 2009 and 2017, > 900 AML/MDS aged 18-75 were screened for the randomized controlled study of post-transplant AZA if they were in complete remission after SCT. The randomization was planned with the treatment arm to receive AZA given as 32 mg/m2/day S.C. for 5 days in each of 12 cycles. The control arm (Ct.) had no intervention. The first cycle of AZA was started within 42-100 post SCT. Each cycle consisted of 28 days allowing next cycle to be started within 56 days following the previous one in the presence of toxicity. The primary outcome for treatment evaluation was RFS. The goal was to test the hypothesis that AZA, given for 12 cycles, provided at least a 50% improvement compared with Ct. in median RFS. An accrual rate of 6 pts. per month was assumed. The final analysis was to be done after 213 to 246 pts. were enrolled based on the number of events observed. However, the study was closed early due to slow accrual 8 years. Results: 187 pts were randomized to either AZA (n=93) Ct. (n=94). Of 93 patients, 87 started treatment with AZA; with a median of 62 days. There were no significant differences between the arms in age (median, 57y), race, cytogenetics, donor type, conditioning intensity, hematopoietic stem cell source, except disease status in AML and comorbidity (Table 1). The majority of the pts.in AZA arm (74.6%) did not receive the planned 12 cycles of treatment, with the number of cycles varying with a median of 4 (range, 1-12). The most common causes of discontinuation of AZA were relapse/death on AZA (46.9%), hematologic/non-hematologic toxicity (26.5%), or patient request/logistical reasons (26.5%). Median follow-up time was similar between arms; 4.6 years in AZA vs. 4.06 years in Ct. Median RFS was comparable, with 2.07 yrs. (AZA) vs. 1.28 yrs. (obs.) (p=0.43). Due to bias induced by the discontinuation of AZA, any conventional comparison analysis, such as a logrank test, would be biased. Therefore, to obtain unbiased comparisons of RFS, separate landmark analysis using logrank tests were conducted, with each including only the AZA pts. who were compliant up to the landmark time, and the comparable subset of Ct. pts. defined as those at risk of failure at the landmark time (Table 2). All landmark analysis showed no significant AZA effect on RFS. A Cox model regression analysis indicated that there was improvement in RFS with AZA beyond day 180, but this was not statistically significant. Additionally, three ad hoc comparisons of AZA to Ct. were done by stratifying AZA pts. into those who received: 1-4 cycles (group 1); , 5-8 cycles (group 2) ; ,or 9-12 cycles (group 3). Pts.in.Ct. were included in the comparative analysis for group 2 if they were at risk of failure at day 150, and for group 3 at day 270. Cox regression analyses showed improvement in RFS in AZA group if they received ≥9 cycles of AZA, but the effect was not significant (p=0.18). Overall, AZA maintenance was well-tolerated with only 1 grade 5 adverse event (AE) reported with AZA vs. 10 grade 5 AEs in the Ct . arm. Twenty grade 4 AEs were reported in AZA arm, 13 were attributed to AZA; all but one was bone marrow (BM) toxicity. All AZA attributable grade IV AEs resolved with a median of 23 days. In obs. arm, 8 grade 4 AEs were reported with 3 BM toxicity. Conclusion: The SC AZA vs. observation randomized trial conducted over 8 years demonstrated that a prospective trial in the post-transplant setting was feasible and safe but challenging. In this trial, although the aim was compare RFS between arms after 12 cycles of AZA, less than 30% of AZA pts. completed the planned 12 cycles for various reasons, including disease relapse, toxicity, compliance, and logistics for receiving the drug. Because accrual was slow, the study was terminated early. While RFS was comparable between the two arms, stratification by number of cycles of AZA received showed a trend towards improvement in RFS in AZA pts. receiving more cycles of therapy. This finding supports the notion that AZA given for 9-12 cycles as maintenance therapy warrants further study for prevention of relapse after allogeneic HSCT for AML and MDS. Figure Figure. Disclosures Oran: AROG pharmaceuticals: Research Funding; Celgene: Consultancy, Research Funding; ASTEX: Research Funding. Shpall:Affirmed GmbH: Research Funding. Rezvani:Affirmed GmbH: Research Funding. Champlin:Sanofi: Research Funding; Otsuka: Research Funding.
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Ohanian, Maro, Guillermo Garcia-Manero, Mark J. Levis, Elias Jabbour, Naval Guastad Daver, Gautam Borthakur, Tapan M. Kadia, et al. "Sorafenib plus 5-azacytidine (AZA) in older untreated FLT3-ITD mutated AML." Journal of Clinical Oncology 35, no. 15_suppl (May 20, 2017): 7029. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.7029.
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7029 Background: Sorafenib plus 5-azacytidine (AZA) is observed to be safe and effective in relapsed / refractory FLT3-ITD mutated acute myeloid leukemia (AML) patients (pts). Hypothesis: Combining sorafenib with AZA is safe and effective in older untreated FLT3-ITD mutated AML pts. Methods: Eligibility included: untreated FLT3-ITD mutated AML (≥10% mutation burden), age ≥60 yrs, adequate organ function, and ECOG performance status ≤ 2. The regimen was: AZA 75 mg/m2daily x 7 days and sorafenib 400 mg twice daily for 28 days. Results: 26 pts with untreated AML [median age 73 (61-86)] were enrolled: 16 (62%) pts had normal karyotype, 2 (8%) complex karyotype, 4 (15%) other miscellaneous abnormalities, and 4 (15%) with insufficient metaphases. Prior to the initiation of treatment, FLT3-ITD was detected in all pts with a median allelic ratio of 0.3735 (0.009-0.885). The overall response rate (ORR) in 25 evaluable pts was (76%) [7 (28%) with CR, 10 (40%) CRi/CRp, and 2 (8%) PR]. Pts underwent a median of 3 (1-35) treatment cycles. The median number of cycles to response was 2 (1-4), and the median time to achieve response, 1.77 months (mos) (0.689-4.271 mos). The median duration of CR/CRp/CRi is 14.5 mos (1.18—28.74). Three (18%) responding pts (CR, CRp, CRi) have proceeded to allogeneic stem cell transplant. With a median follow-up of 6.8 mos (0.2-18.8), 6 pts are alive, 3 in remission (CR/CRP/CRi). The median overall survival (OS) for the entire group is 8.3 mos; 9.2 mos in 17 responders. Evaluable pts treated with AZA + sorafenib (n = 25) were compared to a matched cohort of historical FLT3-ITD mutated pts > 60 yrs, but treated with hypomethylator-based (HMA) therapy without sorafenib (n = 20); the respective ORR (CR, CRp, CRi, PR) (76% vs. 70%, p = 0.653) and median OS (8.3 and 9.4 mos, p = 0.69) were similar. The remission duration for the responding pts treated with AZA+sorafenib was significantly longer (14.5 mos) than those on other HMA regimens without sorafenib (3.8 mos) (p = 0.01). Adverse events possibly attributable to the regimen included: grade (Gr) 1/2 nausea (n = 3), Gr 1/2 diarrhea (n = 2), Gr 1 dyspnea (n = 1), and Gr 1 breast pain (n = 1). Conclusions: The combination of AZA and Sorafenib is both well tolerated and effective in older untreated FLT3-ITD mutated AML. Clinical trial information: NCT02196857;NCT01254890.
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Daver, Naval Guastad, Guillermo Garcia-Manero, Jorge E. Cortes, Sreyashi Basu, Farhad Ravandi, Tapan M. Kadia, Gautam Borthakur, et al. "Phase IB/II study of lirilumab with azacytidine (AZA) in relapsed AML." Journal of Clinical Oncology 35, no. 15_suppl (May 20, 2017): e18505-e18505. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.e18505.
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e18505 Background: Lack of KIR-HLA class I interactions has been associated with NK-mediated antitumor efficacy in AML patients (pts) in remission upon KIR-mismatched haploidentical stem cell transplantation (SCT) (Ruggeri L et al., Science 2002). Blockade of KIR2DL1, 2 and 3 receptors induced augmented NK-cell mediated lysis of tumor cells (Romagne F et al., Blood 2009). Hypomethylating agents possess anti-leukemia activity, increase MHC II expression, and interferon-gamma signature. Methods: Pts with AML who failed prior therapy, have adequate performance status (ECOG ≤ 2) and organ function are eligible. AZA 75mg/m2 Days 1-7 was given with lirilumab on Day 8 at the dosage of 1 and 3 mg/kg in 2 consecutive cohorts of 6 pts each. Courses were repeated every 4-5 weeks. Lirilumab 3mg/kg established as recommended phase 2-dose with AZA. 13 additional pts treated at the RP2D. Responses evaluated at the end of 3 courses. Results: To date, 25 pts (16 de novo, 9 secondary AML), median age 64 years (range, 30 – 89), 52% adverse cytogenetics, median prior therapies 3 (range, 1-8), and prior alloSCT in 6 (28%) have been enrolled. All 25 pts had baseline next generation sequencing, common mutations included ASXL1 (n = 9), TP53 (n = 7), RAS (n = 4), TET2 (n = 4), and RUNX1 (n = 3). Two (8%) achieved CR/CRi (1 CR, 1 CRi) and 3 (12%) achieved hematologic improvement for an overall response rate of 20%. The median cycles to response was 3 (range, 1-11). The 8-week mortality is 12%, respectively. The median duration of response and overall survival were 2.0 months and 4.0 months, respectively (med f/u = 5.1 months). Grade 3/4 toxicities were similar to those seen with AZA based therapies in salvage including bloodstream infections in 15 (60%), 6 pneumonia, 1 UTI, 1 skin infection, 2 abdominal pain, and 1 mucositis. Immune mediated toxicities were observed in 4 (16%) pts (1 pneumonitis Grade 3, 2 colitis Grade 3, 1 colitis Grade 2). The immune mediated toxicities responded rapidly to steroids and 3 pts could be rechallenged safely with lirilumab. Six pts were postSCT and no Grade 3/4 GVHD flares were noted. Conclusions: Full doses of AZA and lirilumab were well tolerated in heavily pretreated pts with relapsed AML. The efficacy data are still preliminary and the study is ongoing. Clinical trial information: NCT02399917.
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Rohon, Peter, Jana Vondrakova, Anna Jonasova, Milena Holzerova, Marie Jarosova, and Karel Indrak. "Treatment of Chronic Myelomonocytic Leukemia with 5-Azacytidine: Case Reports." Case Reports in Hematology 2012 (2012): 1–4. http://dx.doi.org/10.1155/2012/369086.
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Epigenetic therapy with hypomethylating agent (5-azacytidine; AZA) is common in the management of specific subtypes of myelodysplastic syndrome (MDS), but there are only few studies in chronic myelomonocytic leukemia (CMML) patients. In this paper our experience with 3 CMML patients treated with AZA is described. In one patient transfusion independency was observed after 4 treatment cycles; in one case a partial response was recorded, but a progression to acute myeloid leukemia (AML) after 13 AZA cycles has appeared. In one patient, AZA in reduced dosage was administered as a bridging treatment before allogeneic stem cell transplantation (ASCT), but in the control bone marrow aspirate (before ASCT) a progression to AML was recorded. Future studies are mandatory for evaluation of new molecular and clinical features which could predict the efficiency of hypomethylating agents in CMML therapy with respect to overall survival, event-free survival, quality-adjusted life year, and pharmacoeconomy.
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Kim, Wontak, Katherine K. Soh, Jeremiah J. Bearss, Ye Sol Lee, Peter Peterson, Clifford J. Whatcott, Adam Siddiqui-Jain, et al. "Alvocidib Potentiates the Activity of Azacytidine in an MCL-1-Dependent Fashion." Blood 126, no. 23 (December 3, 2015): 1343. http://dx.doi.org/10.1182/blood.v126.23.1343.1343.
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Abstract Despite significant efforts, the clinical mechanism of action of hypomethylating agents such as 5-azacytidine (5-aza) is still poorly understood. 5-aza is currently indicated for the treatment of patients with myelodysplastic syndrome (MDS). While 5-aza has achieved good single-agent activity in acute myeloid leukemia (AML), complete response rates remain low when used as a single agent. In a recent report aimed at identifying rational therapeutic combinations with 5-aza, Bogenberger and colleagues identified multiple BCL-2 family member/BH3-containing therapeutic targets, which synergize with 5-aza when inhibited genetically or pharmacologically. The CDK9 inhibitor, alvocidib, has achieved significant improvement in complete response rates of newly diagnosed AML patients when administered before cytarabine and mitoxantrone (FLAM regimen) in a randomized multi-center Phase 2 trial when compared to 7+3 standard of care treatment. Recent reports suggest that the transcriptional repression of key anti-apoptotic proteins (eg., MCL-1) mediated by alvocidib's CDK9 inhibition, drive the pro-apoptotic activity of alvocidib in the FLAM regimen. We, therefore, hypothesized that alvocidib and 5-aza would synergize therapeutically in the treatment of AML by means of transcriptional repression of MCL-1 and sensitization to 5-aza. In this report, we demonstrate that treatment of AML cell lines with alvocidib inhibits both mRNA and protein expression of MCL-1 in a time and concentration-dependent fashion. Pre-treatment of cells with alvocidib, to repress MCL-1 expression prior to 5-aza treatment, reduced the 5-aza cell viability EC50 more than 2.5-fold, from 1.8 µM to 0.6 µM in MV4-11 cells. The alvocidib/5-aza combination also resulted in synergistic increases in caspase activity relative to either single agent within the combination, at multiple dose levels. Therefore, following reports suggesting inhibition of BCL-2 family members including MCL-1, sensitizes cells to 5-aza, our data suggest that the alvocidib/5-aza combination may constitute a viable therapeutic regimen. We also conclude that a CDK9 inhibitor/5-aza combination may be an effective clinical approach for the treatment of AML. Disclosures Kim: Tolero Pharmaceuticals: Employment. Soh:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment. Lee:Tolero Pharmaceuticals: Employment. Peterson:Tolero Pharmaceuticals: Employment. Whatcott:Tolero Pharmaceuticals: Employment. Siddiqui-Jain:Tolero Pharmaceuticals: Employment. Weitman:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment. Warner:Tolero Pharmaceuticals: Employment.
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Tibes, Raoul, Steven M. Kornblau, William E. Pierceall, Ryan Lena, Yi Hua Qiu, Michael Cardone, Leena Chaudhuri, et al. "BH3 Profiling As Predictor Of 5-Azacytidine and Decitabine Clinical Responses." Blood 122, no. 21 (November 15, 2013): 603. http://dx.doi.org/10.1182/blood.v122.21.603.603.
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Abstract Hypomethylating agents have changed treatment and outcomes in MDS and are active in AML patients. However a significant number of patients do not respond to 5-Azacytidine or Decitabine and there is no clinically validated assay that predicts response to 5-Azacytidine (5-Aza) or Decitabine (DAC). Clinical parameters and recently TET2 mutations have been identified as genetic predictors of response to 5-Aza. Several recent papers report on the possible predictive value of anti-apoptotic proteins, for example BCL2L10. In previous genome-scale RNA-interference (RNAi) screens anti-apoptotic BCL-2 family members, most potently BCL-XL, were identified as top targets whose down-regulation sensitize to 5-Aza. Results were confirmed with the BCL-XL and BCL-2 inhibitor ABT-737, as well as the BCL-2-specific inhibitor ABT-199, both of which sensitized myeloid cells to 5-Aza. Although it is expected that interfering with anti-apoptotic genes would sensitize to hypomethylating similar to cytotoxics, few of the other >900 genes from our RNAi sensitizer screens, including few of the 571 kinases, sensitized to 5-Aza as potently as inhibition of BCL-XL. Thus BCL-2 family members constitute an especially potent context to modulate the activity of 5-Aza. To assess if protein expression of the top targets BCL-XL, BCL-2 or MCL-1 correlate with and may explain a preferential sensitization, 577 primary AML samples were examined by Reverse Phase Protein Array (RPPA). There was substantial overlap of expression across and within FAB subgroups and cytogenetics, and expression of neither of these genes/proteins could explain the observed effects. This may be expected due to functional redundancies amongst the different anti-apoptotic BCL-2 family members, as well as distinct, yet overlapping binding affinities between anti- and pro-apoptotic BCL-2 proteins and BH3 peptides. Thus, we next aimed to functionally interrogate the overall balance of pro- and anti-apoptotic BCL-2 family members, also described as “primedness” by BH3 profiling. This assay uses peptides derived from pro-apoptotic BH3-only proteins to determine the capacity of cells to initiate apoptosis in response to pro-apoptotic BH3 molecules [anti-apoptotic BCL-2 molecules like BCL-XL and BCL-2 bind and inhibit BH3-only molecules]. This assay is a broad functional readout that incorporates many parameters including the consequences of varying BCL2L10 expression. To establish a proof-of-concept that the functional interrogation of specific apoptotic thresholds may be an optimal readout for 5-Aza response, we first assessed a broad panel of 13 AML-derived cell lines by BH3 profiling and in parallel 5-Aza drug dose response experiments. Several BH3 profiling metrics, including NOXA plus BIM, significantly correlated with 5-Aza sensitivity in vitro (Figure 1). Next, to correlate actual clinical responses to 5-Aza with BH3 profiling, specimens from 28 AML, MDS and MDS/MPN overlap patients treated with 5-Aza or DAC-based regimens and for whom clinical outcome was available, were assayed in BH3 assays. In the clinic, the best BH3 metrics were combined values from NOXA and BIM peptides similar to cell lines. NOXA plus BIM discriminated clinical responses defined as achieving any response (sensitive) versus a patient being resistance/refractory to 5-Aza/DAC based regimens with statistical significance (Mann-Whitney two-tailed p = 0.001). This was true for the entire group of patients (5-Aza- and DAC- regimens) with a Receiver Operating Characteristic (ROC) of an AUC=0.875, with a further increase for patients treated only with 5-Aza based regimens with an AUC=0.95.Figure 1BH3 metrics distinguish cell lines with higher EC 50 (> 2uM) from those with lower EC50 (generally < 1uM).Figure 1. BH3 metrics distinguish cell lines with higher EC 50 (> 2uM) from those with lower EC50 (generally < 1uM). In conclusion, due to significant expression overlap and functional redundancies of the BCL-2 family proteins, expression does not correlate with 5-Aza clinical or in vitro responses. However, BH3 profiling as a general functional readout of apoptotic primedness, significantly discriminated responses in patients as well as in vitro. Thus, BH3 profiling incorporates the entirety of pro- and anti-apoptotic molecules (including BCL2L10) and is promising to predict responses to hypomethylating agents. We propose to prospectively validate BH3 profiling as a predictive biomarker assay for 5-Aza or DAC based regimens. Disclosures: Pierceall: Eutropics: Employment, Equity Ownership. Lena:Eutropics: Employment, Equity Ownership. Cardone:Eutropics: Employment, Equity Ownership. Elashoff:Eutropics: Employment. Blake:Noel Blake: Employment, Equity Ownership.
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Rawson, Jonathan M. O., Michele B. Daly, Jiashu Xie, Christine L. Clouser, Sean R. Landman, Cavan S. Reilly, Laurent Bonnac, Baek Kim, Steven E. Patterson, and Louis M. Mansky. "5-Azacytidine Enhances the Mutagenesis of HIV-1 by Reduction to 5-Aza-2′-Deoxycytidine." Antimicrobial Agents and Chemotherapy 60, no. 4 (February 1, 2016): 2318–25. http://dx.doi.org/10.1128/aac.03084-15.
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ABSTRACT5-Azacytidine (5-aza-C) is a ribonucleoside analog that induces the lethal mutagenesis of human immunodeficiency virus type 1 (HIV-1) by causing predominantly G-to-C transversions during reverse transcription. 5-Aza-C could potentially act primarily as a ribonucleotide (5-aza-CTP) or as a deoxyribonucleotide (5-aza-2′-deoxycytidine triphosphate [5-aza-dCTP]) during reverse transcription. In order to determine the primary form of 5-aza-C that is active against HIV-1, Illumina sequencing was performed using proviral DNA from cells treated with 5-aza-C or 5-aza-dC. 5-Aza-C and 5-aza-dC were found to induce highly similar patterns of mutation in HIV-1 in terms of the types of mutations observed, the magnitudes of effects, and the distributions of mutations at individual sequence positions. Further, 5-aza-dCTP was detected by liquid chromatography–tandem mass spectrometry in cells treated with 5-aza-C, demonstrating that 5-aza-C was a substrate for ribonucleotide reductase. Notably, levels of 5-aza-dCTP were similar in cells treated with equivalent effective concentrations of 5-aza-C or 5-aza-dC. Lastly, HIV-1 reverse transcriptase was found to incorporate 5-aza-CTPin vitroat least 10,000-fold less efficiently than 5-aza-dCTP. Taken together, these data support the model that 5-aza-C enhances the mutagenesis of HIV-1 primarily after reduction to 5-aza-dC, which can then be incorporated during reverse transcription and lead to G-to-C hypermutation. These findings may have important implications for the design of new ribonucleoside analogs directed against retroviruses.
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Jilg, Stefanie, Johanna Kauschinger, Veronika Reidel, Catharina Müller-Thomas, Richard Hauch, Johannes Schauwecker, Burkhard Schmidt, et al. "Combination of 5-Azacytidine and ABT-199 Has a Synergistic Apoptotic Effect in High-Risk MDS/sAML after HMA Failure." Blood 128, no. 22 (December 2, 2016): 4297. http://dx.doi.org/10.1182/blood.v128.22.4297.4297.
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Abstract Introduction: Myelodysplastic syndromes (MDS) are one of the most common haematological disorders of the older patient. Allogeneic stem cell stransplantation (ASCT) is the sole curative treatment. The number of eligible patients is limited due to age and comorbidities and only approximately one-third of these patients are cured by ASCT. Further therapeutic strategies such as hypomethylating agents (HMA) have often a short response duration. We recently found that ABT-199 effectively induces apoptosis in the leukemic progenitor compartment of higher-risk MDS and sAML patients, whereas healthy controls and low-risk patients remain unaffected (Jilg et al., 2016). These data suggest that pro-apoptotic ABT-199 might harbor potential as a novel treatment modality in higher-risk MDS patients. However, it remains unclear whether ABT-199 is a powerful option in patients after HMA failure. Synergistic effects of ABT-199 and 5-azacytidine (5-AZA) were also not analyzed in primary material of MDS/sAML patients yet. Here we investigate the effect of ABT-199 as single treatment and in combination with 5-azacytdine in bone marrow samples of 28 high-risk MDS/sAML patients under HMA therapy. We measured induction of apoptosis after ABT-199/5-azacytidine treatment and analyzed the effect on colony forming capacity. Methods: Purified bone marrow mononuclear cells (BMMNC) were treated with 1μM ABT-199 or a soluble control (DMSO) and/or 5-AZA (from 0.25µM to 10µM) for 72h in vitro. Apoptosis was analyzed by flow cytometry after staining for 7-AAD, Annexin V, and CD34 as a progenitor marker. The long-term survival was investigated by colony formation assay. Control samples were obtained from human femoral heads discarded after implantation of total endoprosthesis of the hip joint from hematologically healthy age-matched donors. Combination indices were calculated using ComboSyn. Results: 28 primary samples of patients under HMA treatment (complete response n=5; stable disease n=10 and primary failure n=13) were treated with either ABT-199 single and/or combination therapy with ABT-199 and 5-AZA. Combination therapy showed a clear synergistic effect with the most favorable combination index (CI=0.1446) at ABT-199 (1µM) and 5-AZA (1µM). As expected ABT-199 only showed reduced activity in patients with complete remission. However BCL-2 inhibition effectively decreased the number of viable CD34+ cells in patients with stable disease under HMA treatment. Patients with HMA have a very poor prognosis. As expected 5-AZA (1µM) had only slight effects on MDS stem/progenitor cells of these patients in the ex vivo setting. ABT-199 monotherapy was still able to effectively induce apoptosis in a 72h read-out. Combination therapy (ABT-199 and 5-AZA at a concentration of 1µM each) efficiently induced apoptosis in the CD34+stem/progenitor cells as well as in the bulk of BMMNCs. When compared with single treatment, we found that induction of apoptosis was significantly increased in the stem/progenitor population from patients with HMA failure after combination treatment (ABT-199 mono vs combination p= 0.0089 and 5-AZA mono vs. combination p=0.0006). Conclusion: To identify novel treatment options in higher-risk MDS/sAML patients, we analyzed the apoptotic effect of ABT-199 single and ABT-199/5-azacytidine in combination in primary samples of patients under 5-azacytidine treatment. ABT-199 and 5-azacytidine showed strong synergistic effects with the most favorable combination index with 5-azacytidine (1µM) and ABT-199 (1µM). Monotherapy with ABT-199 was able to induce cell death ex vivo in high-risk MDS/sAML evenafter HMA failure. Combination therapy induced apoptosis very effectively and significantly reduced colony forming capacity. Age-matched healthy controls were only marginally effected. We therefore conclude that combination of low-dose 5-azacytidine with ABT-199 is more effective than single treatment in this pre-treated cohort of high-risk MDS/sAML patients. Since patients with HMA failure have a very poor prognosis with limited treatment options combination therapy of ABT-199 and 5-azacytidine seems to be a promising therapy option. Disclosures No relevant conflicts of interest to declare.
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Ades, L., A. Renneville, A. Marceau, M. Sebert, T. Braun, O. Nibourel, C. Preudhomme, and P. Fenaux. "142 MOLECULAR PROGNOSTIC FACTORS IN HIGHER RISK MDS RECEIVING FIRST LINE AZACYTIDINE (AZA)." Leukemia Research 39 (April 2015): S72. http://dx.doi.org/10.1016/s0145-2126(15)30143-0.
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Fabre, Claire, Claude Gardin, Rose-Marie Mbida, Bruno Quesnel, François Dreyfus, Françoise Isnard, Lionel Mannone, et al. "Treatment of AML with Azacytidine (AZA): Current Results of the French ATU Program." Blood 110, no. 11 (November 16, 2007): 1849. http://dx.doi.org/10.1182/blood.v110.11.1849.1849.
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Abstract Background: AZA is an approved drug for MDS by FDA, currently being investigated in AML. ATUs are patient named programs launched by French health authorities for yet unapproved but promising drugs, and one is ongoing for AZA in MDS and AML. Patients: 108 AML pts from 29 centres included from Sept 2004 to June 2007 in this program, and who had completed at least one course of AZA, are analyzed. They received AZA 75 mg/m2/d (d 1–7) (SC) every 4 weeks, planned for a minimum of 4 courses. Results: Median age was 75 y [range 43–91], M/F was 66/42. 51 pts had AML without and 57 pts with preceding MDS, p-AML and s-AML, respectively (resp). Pts had M0, 1, 2, 3, 4, 5, 6, and 7, in 8, 16, 30, 0, 21, 15, 6 and 4% of cases, resp. 42, 17 and 12 pts were resistant, in first or second relapse, resp, to previous cytotoxic chemotherapy (CT), including intensive anthr-AraC CT in 64 pts and LD AraC in 7, while 37 pts (all with s-AML) were untreated. Karyotype was normal, fav (t(8;21), inv 16), unfav (−7/7q- or complex), int (all others) and NA in 38, 0, 45, 18 pts and 7 pts, resp. Pts received a median of 4 cycles [range 2–16]. Response was assessed only after 4 cycles, unless pts progressed before. Thus, 15 pts were not yet evaluable. Of the remaining 93 pts, 12 (13%) achieved CR, 34 (36%) PR, (OR=49%) and 43 (46%) were considered failure. 4 pts died before end of cycle 2, without evidence of progression. 35 of the 46 responders received maintenance therapy after response was achieved. Allogeneic SCT was performed in 5 responders (2 CR+3 PR). Myelosuppression lead to dose reduction in 16% pts and hospitalization in 17% pts but no toxic death was seen. Other side effects included local reactions (reversible with local NSAID) (19%), grade I-II GI disorders (52%). OR was 34% (11% CR+23% PR) in p-AML and 60% in s-AML pts (12% CR+48% PR) (p<0.05). In p-AML, OR was 75%, 17%, 11% for normal, int and unfav karyotype (p<0.001), while in s-AML, OR was 73%, 57%, 48% for normal, int and unfav karyotype (p=NS). Considering previous treatments, OR in p-AML was 63% and 24% for pts in 1st relapse and > 1 relapse/refractory, resp (p<0.05). In s-AML, OR was 61% and 46%, in previously treated or not pts, resp (p=NS). Median actuarial response duration was 6 months (m) (range 1–22+) in s-AML, and 3 m (1–28+) in p-AML. Median actuarial survival was 9 m [3–30] and 5 m [2–31+[in s- and p-AML, resp. Conclusion: In this very high risk population (in relapse, refractory or post MDS), AZA gave 49% CR and PR, which were higher in s-AML. A prognostic value of cytogenetics and of prior treatment for response was seen in p-AML, but was only marginal in s-AML. An update will be presented.
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Stübig, Thomas, Anita Badbaran, Tim Luetkens, York Hildebrandt, Djordje Atanackovic, Thomas M. C. Binder, Boris Fehse, and Nicolaus Kröger. "5-Azacytidine Promotes an Inhibitory T-Cell Phenotype and Impairs Immune Mediated Antileukemic Activity." Mediators of Inflammation 2014 (2014): 1–12. http://dx.doi.org/10.1155/2014/418292.
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Demethylating agent, 5-Azacytidine (5-Aza), has been shown to be active in treatment of myeloid malignancies. 5-Aza enhances anticancer immunity, by increasing expression of tumor-associated antigens. However, the impact of 5-Aza immune responses remains poorly understood. Here, T-cell mediated tumor immunity effects of 5-Aza, are investigatedin vitroandin vivo. T-cells from healthy donors were treated with 5-Aza and analyzed by qRT-PCR and flow cytometry for changes in gene expression and phenotype. Functionality was assessed by a tumor lysis assay. Peripheral blood from patients treated with 5-Aza after alloSCT was monitored for changes in T-cell subpopulations. 5-Aza treatment resulted in a decrease in CD8+ T-cells, whereas CD4+ T-cells increased. Furthermore, numbers of IFN-γ+ T-helper 1 cells (Th1) were reduced, while Treg-cells showed substantial increase. Additionally, CD8+ T-cells exhibited limited killing capacity against leukemic target cells.In vivodata confirm the increase of Treg compartment, while CD8+ T-effector cell numbers were reduced. 5-Aza treatment results in a shift from cytotoxic to regulatory T-cells with a functional phenotype and a major reduction in proinflammatory Th1-cells, indicating a strong inhibition of tumor-specific T-cell immunity by 5-Aza.
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El Khawanky, Nadia, Amy Hughes, Wenbo Yu, Sanaz Taromi, Jade Clarson, Angel F. Lopez, Michael P. Brown, et al. "Azacytidine Sensitizes AML Cells for Effective Elimination By CD123 CAR T-Cells." Blood 134, Supplement_1 (November 13, 2019): 3904. http://dx.doi.org/10.1182/blood-2019-124684.
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Chimeric antigen receptor T-cells (CAR Tc) have yielded impressive remission rates in treatment-refractory B-cell malignancies (B-ALL and B-lymphomas) by targeting CD19, resulting in the first FDA approved CAR Tc therapies, Kymriah and Yescarta. However, the translation of these results for other cancer entities remains a challenge. Pre-clinical studies using second-generation CAR Tc against the interleukin-3 receptor alpha chain (CD123) engendered strong anti-leukemic activity. CD123 CAR Tc clinical studies resulted in transient responses, or complete remission but at the expense of on-target off-tumor toxicities. Our studies employing third-generation anti-CD123 CAR Tc demonstrate strong anti-leukemic activity with no adverse effects in vivo. However, the leukemia was not completely eradicated. Combining anti-CD123 CAR Tc with DNA hypomethylating (HMA) agents may enhance the anti-leukemic effect and survival. HMAs such as azacytidine (Aza) activate key epigenetically silenced pathways in AML cells, inhibiting cell proliferation while enhancing cell immunogenicity. We hypothesized that Aza will increase the expression of CD123 on AML cells resulting in long-term disease eradication by anti-CD123 CAR Tc. The anti-leukemic efficacy, survival advantage, safety and feasibility of the combination treatment with Aza and anti-CD123 CAR Tc were evaluated in vivo. HL-60 (CD123med), MLL-2 (CD123lo), MOLM-13 (CD123hi), primary de novo and relapsed/refractory (r/r) AML cells were cultured for 0-8 days in the presence of Aza (0µM-5µM) and analysed for their CD123 expression by flow cytometry, quantitative western blot and RNAseq. The anti-CD123 CAR was constructed with the humanized CSL362-based ScFv and the CD28-OX40-CD3ζ signaling domain, encoded in a third-generation lentiviral vector and expressed in CD3+ Tc from healthy donors. Rag2γc-/- mice (n=12-16/ group) were engrafted with 1x105 MOLM13/ffLuc AML cells and treated with PBS, 5x106 Non-transduced (NTD) Tc orCAR Tc, 4x 2.5mg/kg Aza, or 5x106 CAR Tc following 4x Aza (2.5mg/kg). Leukemic burden was assessed weekly by bioluminescence imaging. Tc activity and immunophenotyping was performed using flow cytometry at day 35 post engraftment, and survival was monitored. HL-60, MLL-2 and MOLM-13 cells showed significant increases in HLA-DR, PD-L1, STAT1 and IRF7 expression, as well as CD123 when exposed to Aza (Fig 1A,B). Interestingly, the increased effect was seen from day one regardless of concentration. This was similarly reflected in AML patient cells. Aza treatment also arrested cell proliferation and decreased viability in both cell lines and patient cells suggesting Aza can aid in the anti-leukemic effect. Rag2γc-/- mice engrafted with MOLM-13 and treated with Aza and CD123 CAR Tc demonstrated suppressed growth, and eradication of MOLM-13 cells compared to mice treated with CD123 CAR Tc or Aza alone. Additionally, a significant decrease in residual CD123+ cells in the bone marrow (BM) of dual treated mice was seen (Fig 1C). A higher frequency of residual CD8+ T-cells in the BM, and CD4+ Tc in the peripheral blood (PB) and BM of dual treated mice was observed compared to CAR Tc only treated mice. Most prominently, we found a significantly higher mean number of stem cell-like and central memory CD8+ Tc in the BM of dual treated mice (232 cells/µl and 208cells/µl, respectively) compared to the CAR Tc only group (55 cells/µl and 23 cells/µl, respectively). Assessment of immune checkpoint markers on residual CAR Tc of dual treated mice revealed significantly decreased levels of CTLA-4, PD-1 and TIM-3 in the BM, and CTLA-4 in the PB compared to the CAR Tc only group. While CAR Tc treatment alone demonstrated a survival advantage compared to PBS, NTD or Aza treated mice, Aza and CAR Tc treatment had a significantly higher survival rate compared to the CAR Tc only group (92% vs. 46% at day 50, p<.01). Our findings indicate that Aza increases immunogenicity and augments the cell surface expression of CD123 on AML cells, allowing enhanced recognition and elimination of malignant cells by CD123 CAR Tc. This is the first demonstration that HMAs and CAR Tc immunotherapy can be used synergistically to treat AML. Considering HMAs are currently under clinical investigation in AML, our data encourage further clinical evaluation of this dual treatment in r/r AML, including high-risk patients that are chemotherapy or allogeneic transplantation ineligible. Disclosures Hughes: Novartis, Bristol-Myers Squibb, Celgene: Research Funding; Novartis, Bristol-Myers Squibb: Consultancy, Other: Travel. White:BMS: Honoraria, Research Funding; AMGEN: Honoraria, Speakers Bureau. Yong:Novartis: Honoraria, Research Funding; Celgene: Research Funding; BMS: Honoraria, Research Funding.
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Balaian, Larisa, and Edward D. Ball. "5-Azacytidine Augments the Cytotoxicity of Mylotarg toward AML Blasts In Vitro and In Vivo." Blood 110, no. 11 (November 16, 2007): 1835. http://dx.doi.org/10.1182/blood.v110.11.1835.1835.
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Abstract Binding of CD33 on AML cells by monoclonal antibodies (mAb) mediates cytotoxicicity on AML cells modulated by the protein tyrosine kinase Syk and the phosphatase SHP-1. We have demonstrated that Syk- negative AML cells are relatively resistant to the effects CD33 ligation, but after exposure to the hypomethylating agent 5-azacytidine (5-aza) they become sensitive to the effects of both unconjugated and chemically-conjugated (gemtuzumab ozogamycin, GO) anti-CD33 mAb. Here we tested a panel of 40 primary AML samples for the expression of SHP-1. In 13% of the cases SHP-1 was undetectable. Anti-CD33 mAb and GO induced growth inhibition more effectively in AML cells that expressed SHP-1. Among SHP-1-positive samples, 69% demonstrated significant growth inhibition in response to CD33 ligation. In contrast, none of the SHP-1-negative samples responded to anti-CD33 mAb. These results show a correlation between SHP-1 expression and responsiveness of AML cells to CD33 ligation. However, 5-aza treatment restored SHP-1 expression and, therefore, increased the anti-proliferative effects of anti-CD33 mAb and GO. In 40% of SHP-1-negative samples, the AML cells were only marginally inhibited by 5-aza or anti-CD33 mAb alone, whereas the combination produced a more than additive effect in AML cells where 5-aza induced re-expression of SHP-1. The effect of GO was more than doubled by 5-aza in these cells. Total inhibition of DNA synthesis in the presence of 5-aza plus anti-CD33 mAb or GO reached 60–70% and was similar in SHP-1-positive and SHP-1-negative cells. Moreover, 5-aza significantly enhanced of GO-mediated cytotoxicity in AML progenitor cells. In a NOD/scid mouse model, which permits growth of human AML cells and allows measurement of in vivo therapeutic effects of therapeutic strategies for AML, we tested whether combined treatment of the mice with 5-aza would enhance the cytotoxicity of anti-CD33 mAb and GO. Suboptimal doses of 5-aza by itself, as well as treatment with murine anti-CD33 mAb alone did not cause significant cytoreduction. However, combined treatment of mice with anti-CD33 mAb and 5-aza, resulted in a significant response. Treatment with GO mediated up to 60% inhibition of AML cell proliferation. Combined treatment of mice with GO and 5-aza resulted in reduction of leukemia cells by >80%. These data show an interaction of 5-aza and anti-CD33/GO in an in vivo AML model. Based on these data, we hypothesize that the combination of 5-aza and GO may be a potent therapy for patients with AML. Moreover, Syk and SHP-1 may serve as biomarkers of leukemia cell response. Therefore, we initiated a clinical trial of 5-aza and GO combined therapy. Six patients with relapsed AML have been treated in a dose escalation of 5-aza preceeding GO (6 mg/m2 times two). All 6 were Syk-positive, while SHP-1 expression was detected in 4 samples and absent in two. 2 days of 5-aza treatment in vivo induced re-expression of SHP-1 in both previously SHP-1 negative patient cells. Moreover, significant increases were observed in the levels of Syk protein in one baseline positive sample and 1 SHP-baseline positive sample. Study of the effects of 5-aza alone ex vivo on the baseline patient cells showed no significant effect on leukemia cell proliferation. However, importantly, in all 6 samples 5-aza more than doubled the AML cells’ response to cytotoxic effects of GO and naked anti-CD33 mAb. These results suggest that 5-aza may augment the effects of anti-CD33 mAb therapy through demethylation of SYK, SHP-1, and possibly other genes. The clinical efficacy of the combined therapy requires further study.
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Koshy, Mabel, Louise Dorn, Linda Bressler, Robert Molokie, Donald Lavelle, Nasrin Talischy, Ronald Hoffman, Wendy van Overveld, and Joseph DeSimone. "2-deoxy 5-azacytidine and fetal hemoglobin induction in sickle cell anemia." Blood 96, no. 7 (October 1, 2000): 2379–84. http://dx.doi.org/10.1182/blood.v96.7.2379.
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Abstract Augmentation of the fetal hemoglobin (HbF) levels is of therapeutic benefit in patients with sickle cell anemia. Hydroxyurea (HU), by increasing HbF, lowers rates of pain crisis, episodes of acute chest syndrome, and requirements for blood transfusions. For patients with no HbF elevation after HU treatment, augmentation of HbF levels by 5-aza-2′-deoxycytidine (5-aza-CdR, decitabine) could serve as an alternate mode of treatment. Eight adult patients participated in a dose-escalating phase I/II study with 5-aza-CdR at doses ranging from 0.15 to 0.30 mg/kg given 5 days a week for 2 weeks. HbF, F cell, F/F cell, γ-globin synthesis ratio, complete blood count, and chemistry were measured. The average γ-globin synthesis relative to non-α-globin synthesis prior to therapy was 3.19% ± 1.43% and increased to 13.66% ± 4.35% after treatment. HbF increased from 3.55% ± 2.47% to 13.45% ± 3.69%. F cells increased from 21% ± 14.8% to 55% ± 13.5% and HbF/F cell increased from 17% to 24%. In the HU nonresponders HbF levels increased from 2.28% ± 1.61% to 2.6% ± 2.15% on HU, whereas on 5-aza-CdR HbF increased to 12.70% ± 1.81%. Total hemoglobin increased by 1 g/dL in 6 of 8 patients with only minor reversible toxicities, and all patients tolerated the drug. Maximum HbF was attained within 4 weeks of treatment and persisted for 2 weeks before falling below 90% of the maximum. Therefore 5-aza-CdR could be effective in increasing HbF in patients with sickle cell anemia who failed to increase HbF with HU. Demonstration of sustained F levels with additional treatment cycles without toxicity is currently being performed.
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Koshy, Mabel, Louise Dorn, Linda Bressler, Robert Molokie, Donald Lavelle, Nasrin Talischy, Ronald Hoffman, Wendy van Overveld, and Joseph DeSimone. "2-deoxy 5-azacytidine and fetal hemoglobin induction in sickle cell anemia." Blood 96, no. 7 (October 1, 2000): 2379–84. http://dx.doi.org/10.1182/blood.v96.7.2379.h8002379_2379_2384.
Full textAbstract:
Augmentation of the fetal hemoglobin (HbF) levels is of therapeutic benefit in patients with sickle cell anemia. Hydroxyurea (HU), by increasing HbF, lowers rates of pain crisis, episodes of acute chest syndrome, and requirements for blood transfusions. For patients with no HbF elevation after HU treatment, augmentation of HbF levels by 5-aza-2′-deoxycytidine (5-aza-CdR, decitabine) could serve as an alternate mode of treatment. Eight adult patients participated in a dose-escalating phase I/II study with 5-aza-CdR at doses ranging from 0.15 to 0.30 mg/kg given 5 days a week for 2 weeks. HbF, F cell, F/F cell, γ-globin synthesis ratio, complete blood count, and chemistry were measured. The average γ-globin synthesis relative to non-α-globin synthesis prior to therapy was 3.19% ± 1.43% and increased to 13.66% ± 4.35% after treatment. HbF increased from 3.55% ± 2.47% to 13.45% ± 3.69%. F cells increased from 21% ± 14.8% to 55% ± 13.5% and HbF/F cell increased from 17% to 24%. In the HU nonresponders HbF levels increased from 2.28% ± 1.61% to 2.6% ± 2.15% on HU, whereas on 5-aza-CdR HbF increased to 12.70% ± 1.81%. Total hemoglobin increased by 1 g/dL in 6 of 8 patients with only minor reversible toxicities, and all patients tolerated the drug. Maximum HbF was attained within 4 weeks of treatment and persisted for 2 weeks before falling below 90% of the maximum. Therefore 5-aza-CdR could be effective in increasing HbF in patients with sickle cell anemia who failed to increase HbF with HU. Demonstration of sustained F levels with additional treatment cycles without toxicity is currently being performed.
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Khong, Tiffany, Janelle Sharkey, and Andrew Spencer. "Azacytidine Suppressors Autocrine IL-6 Secretion and Demonstrates In Vitro and In Vivo Activity Against Multiple Myeloma." Blood 106, no. 11 (November 16, 2005): 1566. http://dx.doi.org/10.1182/blood.v106.11.1566.1566.
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Abstract Azacytidine (AZA), a DNA methyltransferase inhibitor, has been shown to inhibit cell growth and induce apoptosis in some cancer cells. We determined the impact of AZA on a panel of human myeloma cell lines (HMCL); KMS 12PE, KMS 18, LP-1, NCI-H929, OPM-2, RPMI-8226 and U266 and in an in vivo murine model of multiple myeloma (5T33 model). Dose responsiveness to AZA was determined via MTS assays with a range of AZA doses (1–10mM) for 72 hours. FACS and cell cycle analysis were used to evaluate the profile of the cells after exposure to AZA for 72 hours. MTS assays demonstrated a dose and time dependent AZA-induced inhibition of HMCL viability with effective concentrations of AZA ranging from 1–10 mM. This was associated with accumulation of cells in the Go/G1 phase with decreasing number of cells in the S and G2/M phases. Western Blot analysis using antibodies against caspases 3,8,10, PARP, phospho-ERK, ERK, Stat3 and phospho -Stat3 were performed to help characterize the mechanism(s) of cell killing. Cleavage of caspases 3,8,10 and PARP within 24 hours of AZA treatment confirmed early AZA-induced HMCL apoptosis. phospho-ERK which was absent in untreated U266 appeared after 48 hours exposure to 5mM AZA. Similarly inhibitors of caspases 3,8 and 9 were used to determine which apoptotic pathway was being preferentially activated by AZA. Inhibitors of both caspase 3 and 9 effectively abrogated AZA-induced apoptosis in U266 and NCI-H929. In contrast caspase 8 inhibitor was less effective which is consistent with AZA acting via the mitochondrial apoptotic pathway. Reactivation of p16 gene by AZA-induced hypomethylation was assessed with methylation specific PCR. MSP-PCR of the p16 gene indicated a loss of methylation and up-regulated transcription after 48 hours treatment with 5 mM AZA. The level of IL-6 in conditioned media from U266 cells treated with AZA was determined by ELISA assay and demonstrated a rapid fall in autocrine IL-6 production. RT-PCR demonstrated rapid AZA-induced cessation of IL-6 transcription temporarily associated with the disappearance of upstream phospho -Stat3. Addition of exogenous IL-6 did not rescue U266 from AZA-induced apoptosis. AZA was also administered to a 5T33 murine model of multiple myeloma at increasing concentrations (1, 3, 10 mg/kg). At 10 mg/kg the median survival of vehicle versus AZA treated mice was 28 days versus 30+ days (p=0.003). These findings justify further evaluation of AZA as a potential therapeutic agent for multiple myeloma.
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Wolfromm, W., I. Itzykson, T. Thepot, D. Dreyfus, S. Stamatoulas, D. de Botton, P. Prebet, et al. "P122 Treatment of CMML by azacytidine (AZA): a preliminary report on 23 patients (pts)." Leukemia Research 33 (May 2009): S130. http://dx.doi.org/10.1016/s0145-2126(09)70203-6.
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Thépot, Sylvain, Raphael Itzykson, Valerie Seegers, Christian Recher, Bruno Quesnel, Jacques Delaunay, Thomas Cluzeau, et al. "Azacytidine (AZA) as First Line Therapy in AML: Results of the French ATU Program." Blood 114, no. 22 (November 20, 2009): 843. http://dx.doi.org/10.1182/blood.v114.22.843.843.
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Abstract Abstract 843 Background: AZA prolongs survival in higher-risk MDS including patients (pts) with 20-29 % marrow blasts, now considered WHO-AML ( Lancet Onc, 2009). However, no large AML cohorts (especially with '30% marrow blasts) treated upfront with AZA have been reported. Methods: An AZA compassionate program (ATU) was initiated in France in Dec 2004 for higher risk MDS, and AML considered not candidates or refractory to intensive chemotherapy (IC). We retrospectively analyzed WHO AML pts having received at least 1 cycle of AZA in the 42 centers with complete pt reporting, excluding those previously treated by IC, allo SCT, low dose AraC or a hypomethylating agent. Results: 138 pts were included between Dec 2004 and Dec 2008; M/F: 86/52; median age 73 years (y) (range 31-87), 117 pts (85%) were > 65 y and 54 (40%) >75y. 65 pts (47%) had prior WHO MDS and 30 pts (22%) therapy related (tAML). 44 pts (32%) had 20-29% marrow blasts. Median WBC was 3.0 G/L [0.8-111.5]. Karyotype (MRC classification), was intermediate (int) in 60 pts,( including 38 normal (NK), and 7 isolated +8 ) adverse in 67 pts (including 42 -7/ del7q, 41 del5q/-5, 45 complex karyotype, two 3q26) and failed in 11 pts. With a median follow-up of 11.3 months, pts received a median of 4.5 AZA cycles (range 1-26). Treatment was according to FDA-EMEA approved schedule for MDS in 95 pts (69%) and a less intensive schedule (5d/4w, or <75 mg/m2/d) in 31% pts, 29 pts (21%) received concomitant valproic acid (VPA). First evaluation was made after 3 to 4 cycles. An overall AML response (ie according to AML-IWG criteria) was observed in 29 pts (21%) including 19 CR (14%), 3 CRp (2%) and 7 PR (5%) after a median of 3 cycles (1 – 11). An additional 25 pts (who had no CR, CRi or PR) achieved hematologic improvement (HI, according to MDS-IWG 2006 criteria). Neither any pretreatment characteristic including age, preceding MDS, tAML, karyotype, WBC, marrow blast %, combination with VPA were correlated with AML response. Median time to progression after AML response was 7.6 months In the 138 pts, 1 y-OS was 40%, 2 y OS 18% and median OS 10.2 months. In univariate analysis, pre-treatment characteristics negatively influencing OS were higher WBC, adverse cytogenetics, higher absolute PB blasts and diagnosis of tAML. In multivariate analysis: higher WBC (p=0.018), and adverse cytogenetics (p=0.0006) retained prognostic significance for OS. In particular, pts with WBC >10 G/L (32 pts in our cohort) carried poorer prognosis ( 1 y OS of 27% vs 44% ,p=0.01); NK had better OS (1-y OS: 66%) than adverse cytogenetics (1-y OS: 30%, p=0.01) but also other “intermediate-risk” abnormalities (1-y OS: 30%, p=0.03). Marrow blast % did not influence OS and survival, whatever the cut off chosen. In particular, pts with 20-29 % marrow blasts had 22% AML response and 1 y OS of 50%, compared to 21% and 1 y OS of 35%, respectively, in pts with >30% marrow blasts (p=NS and NS, respectively). Prior MDS also had no influence on survival. Overall, 33 pts required hospitalization during treatment, mainly for neutropenic fever. A landmark analysis at the time of evaluation showed that achievement of CR, CRi or PR was associated with improved OS (1y-OS 55% vs 31%,p=0.007). In pts with no AML-IWG response, however, achievement of HI also predicted better survival: 1 y-OS 55% vs 19 %, p=0.02. In the 54 pts older than 75 y (ie pts generally considered unfit for IC), 12 (22%) had AML response including CR in 9 (17%) and 3 PR (5%). 1y-OS was 41 % vs 38% for younger pts (p=NS). Hospitalisation was needed in 31% of them vs 32% in younger pts (p=NS). Conclusion: In this untreated cohort of generally older AML pts considered non candidates for intensive chemotherapy, response rate was 21% and 1 y OS 40%. Higher WBC counts and adverse karyotype were associated with poorer OS, but marrow blast %, whatever the threshold chosen, had no influence on outcome. Age above 75 y was associated with similar response and 1y OS. Finally, pts without AML IWG responses but with improved cytopenias also appeared to have improved survival. Disclosures: Off Label Use: Azacytidine is approved by FDA and EMEA in the treatment of high risk MDS and AML up to 30% of bone marrow blast.. Fenaux:CELGENE: Research Funding; AMGEN: Research Funding.
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Behnam, Y. T., and N. Maclean. "Effects of 5-azacytidine and 5-aza-2-deoxycytidine on alphafetoprotein levels in mice." Comparative Biochemistry and Physiology Part C: Comparative Pharmacology 97, no. 2 (January 1990): 357–61. http://dx.doi.org/10.1016/0742-8413(90)90154-2.
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Kuykendall, Jim R. "5-Azacytidine and Decitabine Monotherapies of Myelodysplastic Disorders." Annals of Pharmacotherapy 39, no. 10 (October 2005): 1700–1709. http://dx.doi.org/10.1345/aph.1e612.
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OBJECTIVE: To review and differentiate the pharmacology, toxicology, pharmacokinetics, and results of major clinical trials of 5-azacytidine (5-AzaC) and 5-aza-2'-deoxycytidine (decitabine) therapy of myelodysplastic disorders. DATA SOURCES: A PubMed/MEDLINE search was conducted (1966–October 2004) using the following terms: DNA methylation, myelodysplastic disorders, 5-azacytidine, and 5-aza-2'-deoxycytidine (decitabine). Additional data sources included bibliographies from identified articles and manufacturer information. STUDY SELECTION AND DATA EXTRACTION: Clinical trials for the treatment of various malignancies by hypomethylating agents were selected from data sources. All published, major clinical trials evaluating 5-AzaC or decitabine in myelodysplastic disorders and transformed myeloid leukemia treatment were included. DATA SYNTHESIS: Myelodysplastic disorders are a group of bone marrow stem cell hyperplasias and dysplasias that result in ineffective hematopoiesis. Myelodysplastic disorders and transformed leukemia have poor prognosis and minimal response to chemotherapy. DNA hypomethylating agents have been shown to improve overall response rates (increased neutrophil, leukocyte, and platelet counts), time to leukemic progression, and quality of life compared with supportive therapy. The incidence of the most common adverse effects (nausea, vomiting, myelosuppression) can be reduced by low-dose, continuous, or extended-interval infusion. CONCLUSIONS: Since appropriate dosing schedules of decitabine are being investigated, comparison of the clinical effectiveness of 5-AzaC and decitabine would be premature at this time. DNA hypomethylating agents show promise as monotherapies of myelodysplastic disorders and transformed leukemia and may be useful as a component of combination chemotherapy of various malignancies.
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Ayala, Rosa, Inmaculada Rapado, Esther Onecha, David Martínez-Cuadrón, Gonzalo Carreño-Tarragona, Juan Bergua, Susana Vives, et al. "The Mutational Landscape of Acute Myeloid Leukaemia Predicts Responses and Outcomes in Elderly Patients from the PETHEMA-FLUGAZA Phase 3 Clinical Trial." Cancers 13, no. 10 (May 18, 2021): 2458. http://dx.doi.org/10.3390/cancers13102458.
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We sought to predict treatment responses and outcomes in older patients with newly diagnosed acute myeloid leukemia (AML) from our FLUGAZA phase III clinical trial (PETHEMA group) based on mutational status, comparing azacytidine (AZA) with fludarabine plus low-dose cytarabine (FLUGA). Mutational profiling using a custom 43-gene next-generation sequencing panel revealed differences in profiles between older and younger patients, and several prognostic markers that were useful in young patients were ineffective in older patients. We examined the associations between variables and overall responses at the end of the third cycle. Patients with mutated DNMT3A or EZH2 were shown to benefit from azacytidine in the treatment-adjusted subgroup analysis. An analysis of the associations with tumor burden using variant allele frequency (VAF) quantification showed that a higher overall response was associated with an increase in TET2 VAF (odds ratio (OR), 1.014; p = 0.030) and lower TP53 VAF (OR, 0.981; p = 0.003). In the treatment-adjusted multivariate survival analyses, only the NRAS (hazard ratio (HR), 1.9, p = 0.005) and TP53 (HR, 2.6, p = 9.8 × 10−7) variants were associated with shorter overall survival (OS), whereas only mutated BCOR (HR, 3.6, p = 0.0003) was associated with a shorter relapse-free survival (RFS). Subgroup analyses of OS according to biological and genomic characteristics showed that patients with low–intermediate cytogenetic risk (HR, 1.51, p = 0.045) and mutated NRAS (HR, 3.66, p = 0.047) benefited from azacytidine therapy. In the subgroup analyses, patients with mutated TP53 (HR, 4.71, p = 0.009) showed a better RFS in the azacytidine arm. In conclusion, differential mutational profiling might anticipate the outcomes of first-line treatment choices (AZA or FLUGA) in older patients with AML. The study is registered at ClinicalTrials.gov as NCT02319135.
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Mercier, Cédric, Raphaelle Fanciullino, Cindy Serdjebi, Joseph Ciccolini, Bertrand Pourroy, Gerard Milano, L’Houcine Ouafik, and Régis T. Costello. "Cytidine Deaminase Status As a Predictive Marker Of Response and Toxicity In Patients With Haematological Malignancies Treated With Azacytidine Or Cytarabine." Blood 122, no. 21 (November 15, 2013): 3862. http://dx.doi.org/10.1182/blood.v122.21.3862.3862.
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Abstract Background Cytarabine and azacytidine are mainstays for treating haematological malignancies. As most nucleosidic analogs, both azacytidine and cytarabine are metabolized in the liver by an exclusive enzymatic step driven by cytidine deaminase (CDA). CDA expression polymorphism has been associated with clinical outcome in patients treated with gemcitabine. Methods We determined CDA levels in 39 adult patients (16F, 23M, mean age 77 years), treated mainly for AML and myelodysplastic syndromes with either aza-cytidine or a cytarabine-containing regimen. Response and treatment-related toxicities were monitored following current standards. In addition, impact of CDA status on azacytidine tolerance was evaluated in mice with or without CDA deficiency, as a proof of concept for the actual implication of metabolic deregulations in the toxicities observed in patients. Results In patients, mean CDA activity was 3.7 +/-2.8 U/mg (min: 1, max: 14.8 U/mg). Ten out of 39 patients (i.e., 25%) had low CDA activities and were considered as poor metabolizers (PM). Conversely, 8 patients (i.e., 20%) displayed high CDA activities (i.e., > 6U/mg) and were considered as high metabolizers (HM). PM showed severe toxicities, including two toxic-deaths. Conversely, HM showed little efficacy when treated with either azacytidine or cytarabine. In mice with CDA-deficiency, standard dose azacytidine led to profound and long-lasting neutropenia, as compared with normal mice. Drug monitoring confirmed that individuals with low CDA activity and toxicities showed higher concentrations of azacytidine as compared with normal individuals. Conclusions this study suggests that CDA status could be a relevant marker for predicting clinical outcome in patients treated with either azacytidine or cytarabine. CDA status could be further used as a covariate to tailor drug dosage so as to ensure an optimal efficacy/toxicity balance in patients with haematological malignancies. Disclosures: No relevant conflicts of interest to declare.
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Bories, Pierre, Sarah Bertoli, Françoise Huguet, Sophie Dobbelstein, Luc Fornecker, Odile Beyne Rauzy, Véronique Demas, Ana Berceanu, Eric Delabesse, and Christian Recher. "Efficacy of Frontline 5-Azacytidine in Older AML Patient Unfit for Chemotherapy." Blood 118, no. 21 (November 18, 2011): 2614. http://dx.doi.org/10.1182/blood.v118.21.2614.2614.
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Abstract Abstract 2614 Background: The efficacy of conventional treatment in older patients with acute myeloid leukemia remains unsatisfactory, with low remission rates and poor overall survival. Disappointing results of intensive chemotherapy in patient over 60y are explained by decreased bone marrow reserve and high chemoresistance rate. Novel treatment approaches for this age group are needed. Among these options, 5-azacytidine (AZA) have shown promising results, improving survival in high-risk myelodysplastic syndrome including patient with 20–29% marrow blast now considered WHO AML. Methods: AML patients (marrow blast≥20%) considered not candidates for ICT and treated frontline with only AZA in Toulouse University Hospital were retrospectively analyzed in this study. AZA 75 mg/m2/day was administered subcutaneously for 7 days every 4 weeks, which was defined as 1 cycle. Results: Ninety eight patients were registered between June 2007 and Dec 2010. In this period of time, a total of 470 AML patients older than 60 years were referred in our center. 234 received ICT, 98 AZA, and 138 LD-ARAC or BSC. Sex ratio M/F was 56/42 and median age was 76 (range 50–89). Eighty eight patients were >65y and 51 were >75y. Forty eight patients (48.9%) had de novo AML, 27 patients (27.5%) had prior history of WHO MDS, 5 patients (5.1%) had prior history of myeloproliferative disorder and 18 patients (18.3%) had therapy related AML. Median white blood cell (WBC) count was 2.2G/L (range 0.76–62) and median bone marrow (BM) blast was 35% (range 20–85). Karyotype (Medical Research Council classification) was intermediate in 48 patients (48.9%) including 36 normal and 6 trisomy 8, adverse in 44 patients (44.8%) including 24 -5/del5q, 28 -7/del7q, 35 complex karyotype and 21 monosomal karyotype, and failed in 6 patients (6.1%). Median follow up in survivors was 12.6 months. Patients received a median of 6 cycles of AZA (range 1–27). All patients received at least 1 cycle of AZA and were considered evaluable for response. Fifty patients (51%) presented a response to AZA. Overall response rate according to AML IWP criteria was documented in 24 patients (24.5%), including 13 CR, 5CRi (18CR/CRi 18.4%), and 6PR (6.1%). The median duration of remission for patients achieving CR or CRi was 9.5months. Among the 74 patients considered as failure according to AML-IWP criteria, 26 additional patients (26.5%) obtained hematological improvement HI (7 pts on 1 lineage, 13 pts on 2 lineages and 6 pts on the 3 lineages). Overall, 60 patients required hospitalization during treatment for neutropenic fever. The death rate during the first two months after AZA initiation was 18.3%, due to AML progression in 83% and to infection in 17% of cases. The unique prognosis factor that impacts the response rate in univariate and multivariate analysis was the cytogenetic risk group. We identified 18 CR/CRi/PR (37.5%) in the intermediate group and 6 CR/CRi/PR (13.6%) in the adverse group (p=0.02). Age, WBC count, BM blast, or secondary status of the AML were not statistically related to the response rate. However, it should be noted that only 14 patients had WBC>10G/L at diagnosis. In the entire cohort 1y OS was 50% and 2yOS was 28%. In univariate and multivariate analysis, intermediate karyotype was statistically linked to longer survival. Achievement of CR/CRi or PR translated in increased survival, and importantly in patient with no AML-IWP response, achievement of HI, even on 1 lineage only, translated in increased survival. Moreover when comparing patient treated with 1 or 2 courses, with patient receiving 3 or more courses, we noted a significant improve in survival while number of courses increased. Conclusion: AZA appears to be a valuable option for WHO AML patients considered unfit for chemotherapy and with a low WBC count. In this cohort of very old patients, response rates and more importantly survival rates with AZA are promising. Although, pretreatment prognosis factor such as high risk cytogenetic remains adverse factor, predictive factors of response are needed to guide therapeutic decision. Disclosures: Off Label Use: Azacytidine is approved by FDA and EMEA in the treatment of high risk MDS and AML up to 30% of bone marrow blast. In our study, Azacytidine use was supported by the published results of the French compasionate Azacytidine program in WHO AML patients with more than 30% blast. Recher:Celgene: Membership on an entity's Board of Directors or advisory committees.
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Cojocari, Dan, Sha Jin, Julie J. Purkal, Relja Popovic, Nari N. Talaty, Yu Xiao, Larry R. Solomon, Erwin Boghaert, Joel D. Leverson, and Darren C. Phillips. "5-Azacytidine Induces NOXA and PUMA Expression to Prime AML Cells for Venetoclax-Mediated Apoptosis." Blood 132, Supplement 1 (November 29, 2018): 2644. http://dx.doi.org/10.1182/blood-2018-99-111985.
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Abstract Acute myeloid leukemia (AML) is a clonal hematologic malignancy characterized by genomic heterogeneity and epigenetic changes, including aberrant DNA hypermethylation. Phase-Ib clinical data in relapsed/refractory AML patients indicate that combining venetoclax with the hypomethylating agents (HMAs) 5-azacitidine (5-Aza) or decitabine results in an overall response (OR) of 62% (DiNardo et al. 2018) compared to the historical OR of 28-29% with HMAs treatment alone (Kantarjian et al. 2013; Dombret et al. 2015). Subsequently, a randomized phase-III clinical trial was initiated to evaluate venetoclax activity in combination with 5-Aza in treatment-naïve AML patients ineligible for standard induction therapy (M15-656, NCT02993523). However, the underlying mechanism for the combinational activity observed between venetoclax and 5-Aza is unknown. In this study, we demonstrate that both chronic low-dose 5-Aza treatment, which induced global DNA demethylation, and acute treatment (24 hours, non-epigenetic effects) can drive combinational activity with venetoclax in AML through distinct mechanisms. Chronic culture with a low-dose 5-Aza for one week sensitized AML cell lines to venetoclax in vitro. In contrast, acute treatment with 5-Aza, activated the integrated stress response (ISR) pathway to induce expression of the BH3-only proteins NOXA (PMAIP1) and PUMA (BBC3) in human AML cell lines, independent of DNA methylation. This resulted in an increase in the amount of NOXA and/or PUMA in complex with anti-apoptotic proteins like BCL-2, BCL-XL and MCL-1, thereby "priming" AML cells for induction of apoptosis by venetoclax treatment. Priming for apoptosis resulted in significant synergistic cell death in a panel of AML cell lines treated with venetoclax and 5-Aza in vitro. In this panel of cell lines, the level of the PMAIP1, BBC3, and DDIT3 gene induction correlated with the synergy observed between venetoclax and 5-Aza. Importantly, subsequent PMAIP1 deletion significantly impacted the kinetics and depth of apoptosis induced by 5-Aza or venetoclax alone or in combination. In accordance with the in vitro combinational activity, the venetoclax/5-Aza combination provided added benefit over either agent alone in two xenograft models of AML. Together, these data provide a rationale for an ongoing randomized phase-III clinical trial evaluating venetoclax activity in combination with 5-Aza (M15-656, NCT02993523). Disclosures: DC, SJ, JP, RP, NT, YX, EB, JL, and DP are employees of AbbVie. LS is a former employee of AbbVie and was employed during the duration of this study. The design, study conduct, and financial support for this research were provided by AbbVie and Genentech. AbbVie participated in the interpretation of data, review, and approval of the publication. Disclosures Cojocari: AbbVie Inc: Employment. Jin:AbbVie Inc: Employment, Equity Ownership. Purkal:AbbVie Inc: Employment, Equity Ownership. Popovic:AbbVie Inc: Employment, Equity Ownership. Talaty:AbbVie Inc: Employment, Equity Ownership. Xiao:AbbVie Inc: Employment, Equity Ownership. Solomon:AbbVie Inc: Equity Ownership. Boghaert:AbbVie Inc: Employment, Equity Ownership. Leverson:AbbVie Inc: Employment, Equity Ownership, Patents & Royalties. Phillips:AbbVie Inc: Employment, Equity Ownership, Patents & Royalties.
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Cortvrindt, R., J. Bernheim, N. Buyssens, and K. Roobol. "5-azacytidine and 5-aza-2'-deoxycytidine behave as different antineoplastic agents in B16 melanoma." British Journal of Cancer 56, no. 3 (September 1987): 261–65. http://dx.doi.org/10.1038/bjc.1987.187.
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Daver, Naval Guastad, Sreyashi Basu, Guillermo Garcia-Manero, Jorge E. Cortes, Farhad Ravandi, Elias Jabbour, Stephany Hendrickson, et al. "Phase IB/II study of nivolumab with azacytidine (AZA) in patients (pts) with relapsed AML." Journal of Clinical Oncology 35, no. 15_suppl (May 20, 2017): 7026. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.7026.
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7026 Background: Blocking PD-1/PD-L1 pathways enhances anti-leukemia responses in murine AML (Zhang et al, Blood 2009). PD-1 positive CD8 T-cells are increased in bone marrow (BM) of pts with AML (Daver et al, ASH 2016). AZA up-regulates PD-1 in AML (Yang et al., Leukemia 2013). Methods: Pts were eligible if they had AML and failed prior therapy, had adequate performance status (ECOG ≤ 2), and organ function. AZA 75mg/m2Days 1-7 with nivolumab 3mg/kg on Day 1 and 14 was established as the recommended phase II dose. Courses were repeated every 4-5 weeks indefinitely. Responses were evaluated at the end of 3 courses. Results: 53 pts with med age 68 years (range, 44 – 90), secondary AML (43%), poor risk cytogenetics (43%), med prior regimens 2 (range, 1-7) have been enrolled. Common mutations included DNMT3A (n = 11), TP53 (n = 11), TET2 (N = 8), CEBPA (n = 8), ASXL1(n = 8). All 53 pts are evaluable for response: 11 (21%) achieved CR/CRi and 7 (14%) had hematologic improvement (HI) for an overall response rate of 35%. Additionally, 14 (26%) had ≥50% BM blast reduction, 3 (6%) had stable disease > 6 months, and 12 (23%) had progression. The CR/CRi have been durable with 9 of 11 (82%) pts with CR/CRi alive at 1 year, after censoring for SCT. Med survival for the 53 evaluable pts was 5.7 months (range, 0.9 – 16.2) and in the 27 salvage 1 pts was 9.3 months (range, 1.6 – 16.2). These compare favorably to historical survival with AZA-based salvage protocols at MDACC. Grade 3/4 and Grade 2 immune toxicities were observed in 7 (14%) and 6 (12%) pts, respectively. These responded rapidly to steroids and 12 of 13 pts were successfully rechallenged with nivolumab. Multicolor flow-cytometry data were available on pretherapy, end of cycle 1, and end of cycle 2 BM aspirates in 9 CR/CRi and 22 non-responders. Pts who achieved CR/CRi had higher pre-therapy total CD3 (P = 0.02) and higher CD8+ T-cells (P = 0.07) infiltrate in the BM. Responders demonstrated progressive increase in BM CD8+ and CD4+ infiltrate. Both responders and non-responders had increase in CTLA4+ CD8+ cells on therapy. Conclusions: Full dose AZA and nivolumab are tolerable and may produce durable responses in relapsed AML. Up-regulation of CTLA4 may be a mechanism of resistance to PD1 based therapies in AML. Clinical trial information: NCT02397720.
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Daver, Naval, Jorge Cortes, Lingsha Zhou, Sherry Pierce, Naveen Pemmaraju, Elias Jabbour, Gautam Borthakur, et al. "Ruxolitinib (RUX) in combination with 5-Azacytidine (AZA) as therapy for patients with myelofibrosis (MF)." Clinical Lymphoma Myeloma and Leukemia 15 (September 2015): S59. http://dx.doi.org/10.1016/j.clml.2015.07.120.
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Garrido, Ana, Miguel Ortín, Rodrigo Martino, Josep Nomdedeu, Ana Aventin, Montserrat Hoyos, Salut Brunet, and Jordi Sierra. "5-Azacytidine Before or After Stem Cell Transplantation in Acute Myeloid Leukaemia (AML) and Myelodysplastic Syndromes (MDS)." Blood 118, no. 21 (November 18, 2011): 4267. http://dx.doi.org/10.1182/blood.v118.21.4267.4267.
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Abstract Abstract 4267 The acceptable toxic profile of 5-aza-citidine (5-aza) allows its use in fragile or elderly patients in whom intensive chemotherapy should be avoided. Whether it is possible to take advantage of this low toxicity in patients awaiting for donor search and/or stem cell transplant (SCT) and in those experiencing leukemia recurrences after the procedure remains unknown. We analysed the clinical results of using 5-aza in these two settings to define the feasibility, safety and results of this approach. Patients and methods: From 2007 to 2011, 15 patients (11 males, 4 females) received 5-aza as last treatment prior to an allogeneic SCT (n=13) or as rescue after an early post-transplant relapse (n=2) at our centre. Diagnosis was MDS in 3 cases (median age 62; range 58–63) and AML in 12 cases (median age 58; range 37–67). Patients with MDS received a median of 6 courses of 5-aza (range 3–8) as the only treatment from diagnosis, except for one patient who had received panobinostat prior to 5-aza. Amongst patients with AML, 12 patients received 5-aza either as treatment for AML (2/12) or after remission (8/12) because of the high relapse risk while awaiting for a suitable donor to be found. Two patients with AML received 5-aza as treatment for early post-SCT relapse. AML patients treated with 5-aza before SCT received a median of 5 courses (range 1–19), whilst patients receiving treatment for relapse received 1 and 3 courses, respectively. Ten patients received a nonmyeloablative conditioning regimen, 1 received a conventional conditioning regimen, 2 patients are still in the process of donor search and the other 2 patients received 5-aza after an autologous stem cell transplantation relapse. RESULTS: All MDS patients engrafted and are in complete remission (CR) after a median of 696 days of follow-up (range 377–1227). One of those patients died because of aGvHD. Nine of 12 AML patients receiving 5-aza prior to SCT are alive after a median 373 days follow-up (133–995). One patient showing refractoriness to 3 different lines of treatment died from disease progression after 211 days. All patients receiving 5-aza as treatment for early relapse are dead, 41 and 401 days after starting treatment. Most interestingly, AML patients receiving 5-aza as maintenance of an already-achieved CR while awaiting transplantation did not experience disease progression despite the median time they remained on this treatment was prolonged (9 months). Graft-versus-host disease ≥ grade II was seen in 3 patients. No graft failures were seen and all patients who received an allogeneic stem cell transplantation remain in complete response. CONCLUSION: The use of 5-aza for maintaining or achieving a response in patients with AML who are awaiting SCT is a safe procedure and adds flexibility to schedule the treatment without the need to administer potentially toxic therapy. The use of 5-aza before transplant did not appear to interfere either with engraftment, incidence of GvHD or short-term relapse after transplant. Disclosures: No relevant conflicts of interest to declare.
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Roversi, Fernanda Marconi, Maura Lima Pereira Bueno, Cristiane Okuda Torello, Fernanda I. Della Via, Renata Giardini Rosa, Juares Ednaldo Romero Bianco, Adriana Silva Santos Duarte, et al. "A Novel Chemical Compound Inhibiting Hematopoietic Cell Kinase (iHCK) Has a Synergic Effect with Azacytidine (Aza) or Cytarabine (Ara-C) for Acute Myeloid Leukemia Treatment." Blood 134, Supplement_1 (November 13, 2019): 4650. http://dx.doi.org/10.1182/blood-2019-128959.
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Introduction. Hematopoietic cell kinase (HCK) belongs to the Src kinase family (SFK) involved in the oncogenic process and hematological malignancy. Some SFK inhibitors are currently under investigation in clinical trials for leukemia after demonstrating efficacy in patients with solid tumors. We have previously reported that HCK is overexpressed in leukemic cells and its inhibition by lentivirus resulted in reduction of cell growth and increased cell death (Roversi et al. BBA Mol Basis Dis. 2017, 1863(2):450-61). In light of the genomic and molecular diversity of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), the development of chemical compounds specific for new molecular targets is currently an important subject. Aims. To investigate the in vitro and in vivo effects of a new chemical compound targeting HCK inhibition (iHCK), alone or in combination with the most used drugs for treatment of MDS and AML (Azacytidine - Aza - or Cytarabine - Ara-C). Methods. After iHCK development, we tested its activity alone or in combination with Aza or Ara-C in CD34+ cells isolated from AML patients (n=5) as well as in a panel of myeloid leukemia cell lines (KG1, HL-60, HEL and K562). Additionally, we tested the iHCK in normal and malignant cells cultured in a 3D bioscaffold obtained by decellularization of bovine bone marrow (Bianco et al. Biomat Sci 2019, 7(4):1516-28), in order to mimic the bone marrow niche. After informed written consent and approval of the Ethical Committee of University of Campinas (CAAE 1000.0.146.00-11), in accordance to the Helsinki Declaration, CD34+ cells were isolated from bone marrows of healthy donors (HD), MDS and AML patients and were treated with iHCK or vehicle (DMSO) in liquid culture, for three days. Meanwhile, HS-5 mesenchymal cells were cultured into the 3D bioscaffold. iHCK or vehicle treated CD34+ cells were introduced into the 3D bioscaffold containing HS-5 and evaluated after 7 and 14 days, by light microscopy (hematoxilin and eosin regular staining) and immunohistochemistry (expression of CD34 and CD90 antigens). NOD.CB17-Prkdcscid/J mice received 2 Gy irradiation followed by transplantation with caudal intravenous injection of leukemia cells obtained from hCG-PML-RARα transgenic mice. After acute promyelocytic leukemia (APL) establishment, animals were treated or not with intraperitoneally iHCK and peripheral blood was collected for hematological analysis and protein was extracted from spleen and bone marrows for Western Blot analysis. ANOVA and Student's T-Test were used. Results.In leukemia cell lines and primary cells, the combinatory treatment of iHCK and Cytarabine (1μM) or 5-Azacitidine (1μM) demonstrated synergistic effects, compared to either drug alone, on the reduction of growth and induction of cell death (P<0.001; Figure 1). Further, Western blot revealed increased BAX expression and decreased BCL-XL expression. Moreover, iHCK treatment was able to reduce the activation of oncogenic pathways, MAPK/ERK and PI3K/AKT, leading to severe reduction of ERK, AKT and p70S6 phosphorylation. Treatment with iHCK reduced CD34+ MDS and AML cells proliferation cultured into the 3D bioscaffold but had no effect upon normal CD34+cells. In vivo analysis showed that APL mice treated with iHCK (5μM) for 48h had reduced leukocyte number compared to APL mice treated with vehicle (13.2±1.1 vs 49.4±18.8; P<0.001). No alterations in hemoglobin levels and platelet were found. Likewise, the in vivo iHCK (2.5μM, 5.0μM or 10.0μM) treatment decreased the phosphorylation of ERK, AKT and P70S6K proteins of leukemic cells (Figure 2). Conclusion.The iHCK pharmacological inhibitor has an antiproliferative activity in leukemic cells without altering cell death and survival rate of normal cells, demonstrating on-target malignant cell killing activity as a single agent or in combination with Azacytidine (Aza) or Cytarabine (Ara-C). Disclosures No relevant conflicts of interest to declare.
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Maurillo, Luca, Alessandra Spagnoli, Mariella Genuardi, Monia Lunghi, Nicola Di Renzo, Alfonso Maria D’Arco, Giuseppe Mele, et al. "5-Azacytidine for the Treatment of Acute Myeloid Leukemia: A Retrospective, Multicenter Study of 55 Patients." Blood 112, no. 11 (November 16, 2008): 1947. http://dx.doi.org/10.1182/blood.v112.11.1947.1947.
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Abstract 5-Azacytidine (AZA) is an hypomethylating agent approved in US for the treatment of all FAB subtypes of myelodysplastic syndromes (MDS). Some recent reports have raised the question of a possible efficacy of AZA in selected patients with acute myeloid leukaemia (AML). In September 2007, we started a retrospective study aiming to register and analyse all Italian patients with MDS or AML who had received AZA for the treatment of their disease outside of clinical trials, on the basis of a national patient named program. Among a total of 246 patients treated in 31 different Italian Institutions since 2005, 55 AML diagnosed according to WHO criteria were collected. Median age was 72 years (range 29–87) and 28 patients were male. Poor karyotype was present in 11 patients (20%), while 14 patients (25%) had secondary AML. Median time from diagnosis was 5 months (range 0–72). Eighteen patients (33%) received AZA as front-line treatment, as they were considered not eligible for intensive chemotherapy due to age, co-morbidities or poor performance status. Thirty-seven patients (67%) were pre-treated with growth factors (3 patients) or with one or more lines of chemotherapy (11 and 23 patients, respectively); most of the pre-treated patients (22 out of 34) had received high dose chemotherapy, including autologous or allogeneic stem cell transplantation. Low dose chemotherapy had been employed in the remaining cases. The median number of monthly AZA cycles administered was 4 (range 1–22). Thirty-nine patients (71%) received AZA at the fixed dose of 100 mg/d s.c., 16 patients (29%) received a dose of 75 mg/sqm/d s.c.. A seven-day per month schedule was employed in 43 patients (78%), while 11 patients (20%) received AZA for more than 7 days and one patient for 5 days. Twenty-nine patients (52.8%) received AZA alone, twenty-six (47.2%) in various combinations with growth factors (1), valproic acid +/− ATRA (21) or gentuzumab-ozogamycin (4).The most relevant toxicities observed (grade 3–4) were represented by further myelosuppression (15%), infections (24%: in particular, 1 fungal lung infection, 3 pneumonia and 1 septic shock) and gastrointestinal adverse events (20%). The overall response rate was 35.3% (18/51): 8 patients achieved a complete remission (CR) (15.7%), while a partial response (PR) was observed in 5 patients (9.8%). Five haematological improvements were also seen (9.8%). Response rate was significantly higher in untreated patients compared to pre-treated ones (p=0.02). A statistically significant difference (p=0.04) in response rate in favour of 75 mg/sqm/d versus 100 mg fixed dose was also observed. The actuarial probability of overall survival (OS) at 16 months was 45% for patients responding to AZA and 10% for those non responding (p=0.0027). In conclusion, our data show that: AZA can induce significant responses in about one third of AML patients; the “standard” dose of 75 mg/sqm/d seems to be more effective than 100 mg/d (one single vial) fixed dose; AZA is more effective in de novo as compared to pre-treated (refractory and/or relapsed) disease; AML patients responding to AZA have a significant survival advantage compared to non responders.
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Mercier, Cedric, Raphaelle Fanciullino, Cindy Serdjebi, Joseph Ciccolini, Bertrand Pourroy, Gerard Milano, Regis Costello, and L'houcine Ouafik. "Cytidine deaminase status as a predictive marker in patients with hematologic malignancies treated with azacytidine or cytarabine." Journal of Clinical Oncology 31, no. 15_suppl (May 20, 2013): 7117. http://dx.doi.org/10.1200/jco.2013.31.15_suppl.7117.
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7117 Background: Cytarabine and azacytidine are mainstays for treating haematological malignancies. As most nucleosidic analogs, both azacytidine and cytarabine are metabolized in the liver by an exclusive enzymatic step driven by cytidine deaminase (CDA). CDA is highly polymorphic and dysregulations have been repeatedly associated with poor clinical outcome with gemcitabine. Methods: We have used a test to determine, on a phenotype basis, CDA status in patients. This test was used prospectively in a subset of 39 adult patients (16F, 23M, mean age 77 years), all treated for various haematological malignancies (i.e., CML, AML, lymphomas, myelodysplastic syndromes) with either aza-cytidine or a cytarabine-containing regimen. Response and treatment-related toxicities were monitored following current standards. In addition, impact of CDA status on azacytidine tolerance was evaluated in mice with or without CDA deficiency, as a proof of concept for the actual implication of metabolic deregulations in the toxicities observed in patients. Results: In patients, mean CDA activity was 3.7 +/-2.8 U/mg (min: 1, max: 14.8 U/mg). Ten out of 39 patients (i.e., 25%) showed low CDA activities and were considered as PM. Conversely, 8 patients (i.e., 20%) displayed CDA activities particurlary elevated (i.e., > 6U/mg) and were considered as UM patients. Of note, PM patients all showed severe toxicities, including two toxic-deaths. Conversely, UM patients showed little efficacy when treated with either azacytidine or cytarabine. In mice with CDA-deficiency, standard azacytidine led to profound and long-lasting neutropenia, as compared with normal mice. Drug monitoring confirmed that individuals with low CDA activity and toxicities showed higher concentrations of azacytidine as compared with normal individuals. Conclusions: Overall this pilot study strongly suggests that CDA status could be a relevant marker for predicting clinical outcome in patients treated with either azacytidine or cytarabine. CDA status could be further used as a covariate to tailor drug dosage so as to ensure an optimal efficacy/toxicity balance in patients with haematological malignancies.
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Jawad, Mays, Nigel H. Russell, and Monica Pallis. "Azacytidine and Gemtuzumab Ozogamicin Co-Operate to Inhibit the Wnt Pathway and Increase Cytotoxicity in AML." Blood 112, no. 11 (November 16, 2008): 2991. http://dx.doi.org/10.1182/blood.v112.11.2991.2991.
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Abstract Thirty percent of acute myeloid leukaemia (AML) patients contain somatic mutations (internal tandem duplications-ITD) of FLT3 gene (FLT3/ITD) that are associated with poor prognosis. We have previously shown that Gemtuzumab Ozogamicin (Mylotarg), the CD33 calichaemicin-conjugate, targets the leukaemic stem and progenitor cells (LSPC) of FLT3/ITD samples better than WT samples (ASH 2007, Abstract #650). The Wnt signalling pathway has a role in the pathogenesis of AML, particularly in cells capable of self renewal, i.e. LSPC. Beta-catenin is one downstream effector of Wnt signalling, which upon stimulation will activate transcription of target genes, particularly oncogenes such as c-myc and cyclin D1, that influence cell proliferation, survival and cell fate. More recently, a synergistic effect on activation of Wnt signaling pathway in leukaemic transformation by FLT3/ITD has been shown, suggesting that beta-catenin induction may be one transforming event in AML achieved by FLT3/ITD activation. Therefore, identifying therapeutic agents that specifically target the Wnt pathway at various positions may be necessary for complete inhibition of this pathway in AML. In haemopoietic cells, treatment with the hypomethylating agent 5′-Azacytidine (Aza) results in progressive demethylation of previously methylated Wnt inhibitors and re-expression of transcripts and downregulation of Wnt signalling. The aim of this study is to investigate whether Aza targets the Wnt pathway in AML, whether the combination with Mylotarg further frustrates this pathway leading to cell death and whether this is more pronounced in FLT3/ITD samples. We examined cell survival in the presence of Aza, Mylotarg or the combination in FLT3/WT (HL60 and U937) and FLT3/ITD (MOLM13 and MV4-11) cell lines. Both single Aza and Mylotarg 48hr treatment resulted in significant cell kill (30–42% and 51–74%, respectively), whereas this effect was enhanced (additive) when the Aza/Mylotarg combination was used (69–85% cell kill). Although slightly more sensitive in FLT3/ITD cells (76–85% cell kill), our data indicate that the Aza/Mylotarg combination is effective even in FLT3/WT cells (69–71% cell kill). In order to assess possible mechanism of action, beta-catenin levels were measured in these treated cells. Reduction in beta-catenin levels was similar in Aza-treated FLT3/ITD and WT cells (7–23% and 9–13%, respectively). In contrast, Mylotarg and Aza/Mylotarg combination resulted in a significant beta-catenin reduction in FLT3/ITD cells compared to WT cells: 41–47% in FLT3/ITD and 0–16% in FLT3/WT for Mylotarg and 52–59% in FLT3/ITD and 0–17% in FLT3/WT for Aza/Mylotarg combination. The FLT3 inhibitor AG1296 also downregulated beta-catenin levels in MV4-11 cells, suggesting that Mylotarg may in fact be targeting the Wnt pathway by inhibiting FLT3 signalling. We have also compared cell survival in FLT3/ITD and compared them to FLT3/WT in LSPC from primary AMLs. CD34+CD38−CD123+ LSPC from FLT3/ITD samples were more sensitive to Aza compared to LSPC of FLT3/WT samples (15% vs 42% cell kill, respectively; n=3, 72hrs). The difference is enhanced when the Mylotarg/Aza combination is used (23% vs 61% cell kill in FLT3/WT and FLT3/ITD, respectively). We conclude that the Mylotarg/Aza combination effectively inhibits the Wnt pathway and targets LSPCs in AML.
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Singh, Anup Kumar, and Xiaochun Yu. "Novel Therapeutic Targeting of UHRF1 Restores 5'-Azacytidine Response in Resistant Acute Myeloid Leukemia." Blood 132, Supplement 1 (November 29, 2018): 1356. http://dx.doi.org/10.1182/blood-2018-99-115735.
Full textAbstract:
Abstract DNA hypermethylation plays a pivotal role in the pathogenesis of acute myeloid leukemia (AML). Most of the recurrent driver mutations and chromosomal translocations in AML involve genes encoding chromatin modifiers and DNA methylation relevant enzymes. Hypo-methylating drugs such as 5-Azacytidine (AZA) that target DNMTs prolong overall survival in AML patients. However, their long term treatments lead to emergence of acquired therapy resistance mostly through unknown mechanisms and hence there is an urgent need for alternate therapeutics to address AZA resistance in AML patients. Recently, it has been shown that AZA resistant leukemic cells are relatively quiescent with higher expression of many components of DNA methylation machinery that also includes UHRF1 (ubiquitin-like with PHD and ring finger domains 1). UHRF1 is a key epigenetic modulator that regulates DNA methylation and gene expression. It is a multi-domain nuclear protein with an SRA (SET-and-RING-associated) domain to recognize hemi-methylated DNA immediately after replication. It plays a crucial role in the maintenance of DNA methylation by recruiting DNMT1 to replication sites and facilitates methylation on newly synthesized DNA strand. UHRF1 is frequently overexpressed in multiple human neoplasms including AML and in the absence of UHRF1, hematopoietic stem cells undergo erythroid-biased differentiation at the expense of self-renewal capacity. Despite UHRF1 being key a therapeutic target against AML, specific, and cell-permeable inhibitors of UHRF1 have not been identified yet. In this study, we hypothesized that targeting UHRF1 using novel small molecule inhibitor will interfere with DNMT1-dependent DNA methylation at newly synthesized DNA strand, which may further synergize with antiproliferative effect of classical DNMT inhibitors in AML cells. In this study, we used in silico strategy to discover novel putative UHRF1 inhibitors by screening NCI compound database. For in vitro validation, we have first purified the SRA domain of UHRF1 followed by analysis of total DNA methylation levels using 5'-methyl cytosine (5mC) dot blot in the presence of each inhibitor. After a series of stringent in vitro and cell based assays we have identified lead compound 20 (C20) as a potent UHRF1 inhibitor which suppresses DNA methylation without affecting DNMTs in leukemic cells. Specificity of C20 against SRA domain was further established by isothermal titration calorimetry (ITC). We next found that C20 treatment significantly decreased UHRF1 and DNMT1 foci formation in the nucleus of mouse embryonic fibroblast and stem cells. Based on the its critical role in DNA methylation and enhanced expression in resistant cells, we assumed that AZA resistance in AML may be mediated by UHRF1 and C20 might restore AZA sensitivity by attenuating enhanced UHRF1 activity. To validate this, we pretreated AZA resistant leukemic cells (HL60R) with suboptimal dose of C20 followed by AZA treatment. Interestingly, we found a synergistic increase in antiproliferative effect by flow cytometry and colony formation assay. By analyzing the surface expression of myeloid differentiation markers, we found that C20 treatment promotes differentiation and decreases quiescent leukemic cell population. In conclusion, we report a novel UHRF1 inhibitor as a sensitizer of resistant AML cells towards AZA treatment potentially by promoting differentiation, suggesting a novel combination approach for future clinical evaluations. Disclosures No relevant conflicts of interest to declare.
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Gao, Qing, Maojuan Guo, Xijuan Jiang, Xiantong Hu, Yijing Wang, and Yingchang Fan. "A Cocktail Method for Promoting Cardiomyocyte Differentiation from Bone Marrow-Derived Mesenchymal Stem Cells." Stem Cells International 2014 (2014): 1–11. http://dx.doi.org/10.1155/2014/162024.
Full textAbstract:
A growing body of evidence supports the argument that bone marrow-derived mesenchymal stem cells (MSCs) can differentiate into cardiomyocyte-like cells in an appropriate cellular environment, but the differentiation rate is low. A cocktail method was designed: we investigated the role of 5-azacytidine (5-aza), salvianolic acid B (SalB), and cardiomyocyte lysis medium (CLM) in inducing MSCs to acquire the phenotypical characteristics of cardiomyocytes. The fourth-passage MSCs were treated with 5-aza, SalB, CLM, 5-aza+salB, 5-aza+CLM, SalB+CLM, and 5-aza+SalB+CLM for 2 weeks. Immunofluorescence results showed that cTnT expression in the 5-aza+salB+CLM group was stronger than other groups. Real-time qPCR and Western blotting analyses showed that cTnT, alpha-cardiac actin, mef-2c, Cx43, and GSK-3beta expression increased while beta-catenin expression decreased. The salB+5-aza+CLM group had the most evident effects. SalB combined with 5-aza and CLM improved cardiomyocyte differentiation from MSCs. In the MSCs differentiation process, the Wnt/beta-catenin signaling pathway had been inhibited.
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Mabaera, Rodwell, Christine Richardson, Sarah Conine, and Christopher H. Lowrey. "5-Azacytidine Induction of Human γ-Globin Gene Expression: Experimental Evaluation of Current Models." Blood 108, no. 11 (November 16, 2006): 1581. http://dx.doi.org/10.1182/blood.v108.11.1581.1581.
Full textAbstract:
Abstract 5-Azacytidine (5-Aza) was demonstrated to be a potent inducer of human fetal globin gene expression more than 20 years ago. More recently, 5-Aza-2-deoxycytidine has been shown to have similar properties. Since the 1980’s there have been two predominant hypotheses to explain the action of these agents. The first is based on the observation that these, and several other active inducing agents, are cytotoxic to differentiating erythroid cells and that drug treatment alters the kinetics of erythroid differentiation. This has been proposed to result in prolonged expression of the γ-globin genes which are normally expressed only early in differentiation. The second is based on the observation that both agents are DNA methyltransferase inhibitors and are presumed to cause demethylation of cellular DNA including the γ-globin gene promoters leading to activation of the genes. These two models lead to specific predictions that we have evaluated using an in vitro erythroid differentiation system. In this system, human adult CD34+ cells are cultured in SCF, Flt3 ligand and IL-3 for 7 days and then switched to Epo for 14 days. This results in an exponential expansion of erythroid cells. As has been described for normal human differentiation, these cells express small amounts of γ-globin mRNA early in differentiation followed by a much larger amount of β-globin mRNA. HPLC at the end of the culture period shows 99% HbA and 1% HbF. Treatment of cultures on a daily basis with 5-Aza starting on day 10 results in dose dependent increases in γ-globin mRNA, Gγ- and Aγ-chain production and HbF. The cytotoxicity model predicts that γ-globin expression will be prolonged to later in differentiation - and this is seen. However, a daily 5-Aza dose of 300 nM, which produces ~80% of the maximal response in γ-globin mRNA and HbF, has no effect on cell growth or differentiation kinetics. This argues against the toxicity model. We next examined the effect of 5-Aza on γ-globin promoter methylation using the bisulfite method. We studied CpGs at −344, −252, −162, −53, −50, +6, +19 and +50 relative to the start site. For untreated controls, all of the sites are nearly 100% methylated at day 1. By day 3, the upstream sites become ~50% methylated except the −53 CpG which was <20%. This pattern persisted at day 10. By day 14 the promoters had become largely remethylated. For cells treated with 5-Aza starting on day 10, there was no change in the levels of methylation seen on days 1,3 and 10, but at day 14 the low levels of upstream methylation persisted - just as γ-globin expression does. However, in both treated and untreated cells, down-stream CpG sites were highly methylated at all time points. This suggests that γ promoter demethylation may be due to a local and not a generalized effect of 5-Aza on cellular DNA methylation. We also made two unexpected observations. At a 300nM dose of 5-Aza, γ-globin mRNA is ~doubled while β-globin mRNA levels are ~halved - indicating that 5-Aza not only induces γ-globin expression also suppresses β-globin. Also despite only a doubling in γ-globin mRNA, there was an ~50-fold increase in HbF, from ~1% to more than 50%, while total per cell Hb levels were unchanged. Neither of these results are easily explained by current models of γ-globin gene induction. Our results raise the possibility that mechanisms beyond cytotoxicity and generalized DNA demethylation may be responsible for pharmacologic induction of γ-globin mRNA and HbF.
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Constantoulakis, P., T. Papayannopoulou, and G. Stamatoyannopoulos. "alpha-Amino-N-butyric acid stimulates fetal hemoglobin in the adult." Blood 72, no. 6 (December 1, 1988): 1961–67. http://dx.doi.org/10.1182/blood.v72.6.1961.bloodjournal7261961.
Full textAbstract:
The effect of alpha-amino-N-butyric acid (alpha ABA) on fetal hemoglobin production in the adult was examined in vivo after being administered to normal and anemic baboons and in erythroid progenitor cell cultures. Infusion of alpha ABA for five days resulted in four- to fivefold increases in the level of F reticulocytes of normal or chronically anemic baboons. The induction of HbF by alpha ABA was strikingly enhanced by the administration of 5-azacytidine. The addition of alpha ABA in culture produced a concentration-related increase of HbF in baboon CFUe and e-cluster colonies. In addition to the induction of HbF, alpha ABA stimulated the growth of all classes of erythroid progenitors in vivo or in culture. The activation of gamma- globin gene expression by alpha ABA is attributed to an interaction between regulatory sites of globin chromatin modified by alpha ABA and the immature intracellular environment of the expanding erythropoiesis. The combination of chromatin modification, DNA methylation, and the immature intracellular environment of rapid erythroid regeneration may explain the synergistic induction of HbF by alpha ABA and 5-azacytidine.
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Okoye-Okafor, Ujunwa Cynthia, Komal Kumar Javarappa, Dimitrios Tsallos, Joseph J. Saad, Lumie Benard, Victor Thiruthuvanathan, Sally Cole, et al. "Azacytidine Inhibits Megakaryopoiesis Via the Induction of Immunogenic RNA Species and Activation of Type-I Interferon Signaling." Blood 134, Supplement_1 (November 13, 2019): 1280. http://dx.doi.org/10.1182/blood-2019-131461.
Full textAbstract:
Management of hematologic disease- and therapy-related thrombocytopenia remains a serious clinical issue, especially in patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). The ribonucleoside and DNA-demethylating agent azacytidine (AZA), has proven useful for the treatment of patients with MDS and AML not eligible for stem cell transplantation. While low-dose AZA therapy induces clinical remissions in up to 50% of treated patients, it comes at the cost of aggravating pre-existing thrombocytopenia which is observed in a subset of patients; this can lead to increased bleeding and bleeding-associated mortality, and importantly, often requires dose modifications and delays of therapy. Thus, identification of strategies alleviating ineffective megakaryopoiesis will likely lead to increased therapeutic efficacy for patients with MDS/AML. Eltrombopag (EP), a second-generation small molecule thrombopoietin receptor (TPO-R) agonist was effective in raising platelet counts in patients with MDS as a single agent, as well as in combination with certain standard of care therapies. However, it failed to stimulate platelet production during the first four cycles of AZA treatment as uncovered by a recent phase III placebo-controlled clinical study (SUPPORT; NCT02158936). The goals of this study were to identify the cellular and molecular underpinnings of AZA-associated inhibition of megakaryopoiesis and to assess the ineffectiveness of EP in mitigating AZA treatment-associated thrombocytopenia. Our results demonstrate that at a clinically-equivalent and non-cytotoxic dose, AZA rapidly induces transient activation of interferon type I (IFN-I) signaling in various hematopoietic cell types, including stem and lineage-committed progenitor cells (HSPCs). We detected IFNα and IFNβ production and release using ELISA and intracellular flow cytometry on primary total mononuclear cell- and purified CD34-positive HSPC populations derived from cord blood, bone marrow from healthy volunteers or patients with MDS/AML. AZA-mediated activation of Type I IFNs in healthy control- and MDS/AML cells was preceded by an accumulation of double-stranded RNA (dsRNA) species and decreased total RNA cytosine methylation measured by immunocytochemistry and intracellular FACS analysis; this suggested that AZA triggered the accumulation of immunogenic RNA species which elicit an IFN-I response. In support, we found Toll like receptor 3 (TLR3) activation and phosphorylation of STAT1 in CD34+ HPSC, along with premature activation of Suppressor of Cytokine Signaling 1 (SOCS1), a well-known JAK/STAT-dependent signaling attenuator. This rapid AZA-induced viral mimicry response led to abrogation of thrombopoietin (TPO) or EP-stimulated TPO-R signaling and inhibition of ex vivo megakaryocyte progenitor proliferation quantified by colony formation in semi-solid medium. Importantly, inhibition of IFN-I signal activation using the JAK3 inhibitor decernotinib, the IFNα/β-blocking peptide, B18R, or RNA interference-mediated knock-down of SOCS1 counteracted the inhibitory effects of AZA on TPO-R stimulation and restored megakaryopoiesis. Given these observations, we pre-clinically tested a revised treatment protocol, in which primary cells were first exposed to AZA for four days followed by TPO-R stimulation using TPO or EP. This new treatment strategy alleviated AZA's inhibitory effects at the molecular and cellular levels, demonstrating that upon resolution of the AZA-mediated vial mimicry response, EP and TPO can effectively stimulate TPO-R signaling and megakaryopoiesis. Together, our data reveal a mechanistic basis of AZA-mediated inhibition of megakaryopoiesis in patients with MDS/AML. Additionally, we show that EP cannot overcome the megakaryopoiesis-inhibitory effects of acute IFN-I signaling activation upon AZA exposure. Findings of our study are consistent with and provide a molecular explanation for the observations made in the context of the SUPPORT study. In the future, it will be critical to better understand and potentially counteract the megakaryopoiesis-inhibitory effects by IFN-I pathway activation upon AZA therapy in patients with MDS/AML. Disclosures Okoye-Okafor: Novartis Pharmaceuticals: Research Funding. Pallaud:Novartis Pharmaceuticals: Employment. Marques Ramos:Novartis Pharmaceuticals: Employment. Verma:Janssen: Research Funding; BMS: Research Funding; Celgene: Honoraria; Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria. Heckman:Celgene: Research Funding; Novartis: Research Funding; Oncopeptides: Research Funding; Orion Pharma: Research Funding. Will:Novartis Pharmaceuticals: Research Funding.
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Chekhun, V. F., Yu V. Lozovska, L. A. Naleskina, T. V. Borikun, A. P. Burlaka, I. N. Todor, D. V. Demash, et al. "MODIFYING EFFECTS OF 5-AZACYTIDINE ON METAL-CONTAINING PROTEINS PROFILE IN GUERIN CARCINOMA WITH DIFFERENT SENSITIVITY TO CYTOSTATICS." Experimental Oncology 38, no. 4 (December 22, 2016): 283–87. http://dx.doi.org/10.31768/2312-8852.2016.38(4):283-287.
Full textAbstract:
Aim: To assess the influence of the treatment with 5-azacytidine (5-aza) on the profile of metal-containing proteins and factors of their regulation in Guerin carcinoma cells in vivo. Materials and Methods: The study was conducted on Wistar rats transplanted with wild-type Guerin carcinoma (Guerin/WT) and its strains resistant to cisplatin (Guerin/CP) or doxorubicin (Guerin/Dox). Animals were distributed in 6 groups treated with 5-aza and control animals without treatment. 5-Aza was injected by i.v. route (1 injection in 4 days at a dose of 2 mg/kg starting from the 4th day after tumor transplantation, 4 injections in total). Ferritin levels in blood serum and tumor tissue were measured by ELISA, transferrin and free iron complexes — by low-temperature EPR, miRNA-200b, -133a and -320a levels and promoter methylation — by real-time quantitative reverse transcription polymerase chain reaction. Results: The study has shown that 5-aza treatment caused demethylation of promoter regions of fth1 and tfr1 genes in all studied Guerin carcinoma strains. 5-Aza treatment resulted in a significant decrease of ferritin levels in tumor tissue (by 32.1% in Guerin/WT strain, by 29.8% in Guerin/Dox and by 69.1% in Guerin/CP). These events were accompanied by 3.5-fold and 2-fold increase of free iron complexes levels in tumor tissue of doxorubicin and cisplatin resistant strains, respectively. Also, 5-aza treatment resulted in significantly elevated levels of miR-200b, -133a, 320a expression in tumor tissue. After 5-aza treatment, ferritin levels in blood serum of animals with Guerin/Dox were increased by 23.9%, while in Guerin/Wt and Guerin/CP they were decreased by 17 and 16%, respectively. Conclusion: Alterations of epigenetic regulation upon in vivo treatment with 5-aza change the levels of metal-containing proteins due to DNA demethylation and altered miRNA expression profiles in Guerin carcinoma cells.