Academic literature on the topic 'CSNK1A1'

Abstract Abstract 209 In order to identify novel approaches to the targeting of acute myeloid leukemia (AML), we performed a pooled in vivo shRNA screen on murine leukemic stem cells (LSCs) targeting factors related to Wnt-signaling. We found that silencing of casein kinase 1 alpha (Csnk1a1), a serine-threonine kinase and a critical negative regulator of beta catenin, dramatically depleted murine LSCs in vivo. This is a surprising result since beta catenin is essential for MLL-AF9 AML. Validation experiments with shRNA vectors co-expressing GFP recapitulated the result from the pooled screen and confirmed efficient knockdown of both the Csnk1a1 transcript and protein. To rule out off-target effects of the Csnk1a1 shRNAs, we co-expressed the shRNAs with a Csnk1a1 cDNA mutated at the shRNA binding sites, and observed a complete rescue of the proliferative defect. Additionally, we demonstrated that a kinase dead form of Csnk1a1(D136N) failed to rescue this proliferation defect. These results indicate the specific effect of these hairpins on Csnk1a1 function in leukemia cells. The role of Csnk1a1 in normal hematopoietic stem and progenitor cells (HSPCs) is not known. We introduced the Csnk1a1 shRNA vectors into HSPCs and followed GFP over time in a bone marrow transplantation setting. Over a 24-week period, we observed a 3–4 fold depletion of GFP positive donor cells with two independent Csnk1a1 shRNAs compared to control. In contrast, the same shRNAs resulted in a 20–25 fold depletion of leukemia cells in vivo over a 2-week time period, suggesting that leukemia cells are selectively dependent on Csnk1a1. To more rigorously study Csnk1a1 in hematopoiesis, we generated a Csnk1a1 conditional knockout mouse (loxP sites flanking critical exon 3) and crossed it with the Mx1-cre mouse, allowing for hematopoietic specific inducible Csnk1a1 excision. In competitive bone marrow transplantations, Csnk1a1(−/−) donor cells exhibited a severe competitive disadvantage resulting in a 20-fold depletion of donor cells over a 12-week period. Interestingly, Csnk1a1(−/−) donor cells were devoid of myeloid lineage cells, suggesting that Csnk1a1 is particularly important for the generation or survival of myeloid cells. Moreover, in line with our shRNA results, we found that Csnk1a1(−/−) cells were resistant to MLL-AF9 mediated transformation, demonstrating that Csnk1a1 is essential also for leukemia initiation. To identify critical targets of Csnk1a1, we performed gene expression profiling of Csnk1a1 silenced cells. We identified enrichment of a p53 signature using Gene Set Enrichment Analysis (FDR= 0.001). Induction of p53 and its target p21 was confirmed by western blots in both Csnk1a1 silenced leukemia cells and in Csnk1a1(−/−) bone marrow cells. Furthermore, we demonstrated that p53(−/−) leukemia cells are resistant to the proliferative defect induced by Csnk1a1 silencing. We next tested whether D4476, a small molecule casein kinase inhibitor, would exhibit selective anti-leukemic effects. Whereas treatment of LSCs with D4476 inhibited their proliferation (IC50: 7μM), concentrations up to 40μM had minimal effects on normal HSPCs. Confirming the specificity of the compound, we found that cells carrying Csnk1a1 shRNAs were sensitized to D4476 in a dose dependent manner. In contrast, overexpression of Csnk1a1 desensitized leukemia cells for D4476 treatment, suggesting that D4476 kills leukemia cells in a Csnk1a1 dependent manner. Finally, we mixed 10,000 HSPCs with 10,000 LSCs and treated them ex vivo with either D4476 or DMSO control for 48 hours followed by injection into lethally irradiated mice. Whereas exposure to the drug caused prolonged latency of disease with some recipients never developing leukemia, there was no significant effect on HSPC donor cell chimerism at 8 weeks post transplantation compared control, indicating limited toxicity from the drug. In summary, these findings identify Csnk1a1 as critical for maintaining both normal HSCs and LSCs via modulation of p53 activity. Importantly, LSCs were significantly more sensitive to small molecule inhibition of Csnk1a1, suggesting that Csnk1a1 may be an attractive new drug target in AML. Disclosures: No relevant conflicts of interest to declare.

Abstract The immunomodulatory (IMiD) drug lenalidomide is a highly effective treatment for multiple myeloma and myelodysplastic syndrome (MDS) with deletion of chromosome 5q (del(5q)). Recently, we and others demonstrated that lenalidomide activates the CRBN-CRL4 E3 ubiquitin ligase to ubiquitinate IKZF1 and IKZF3. Degradation of these lymphoid transcription factors explains lenalidomide’s growth inhibition of multiple myeloma cells and increased IL-2 release from T cells. However, it is unlikely that degradation of IKZF1 and IKZF3 accounts for lenalidomide’s activity in MDS with del(5q). Instead, we hypothesized that ubiquitination of a distinct CRBN substrate in myeloid cells explains the efficacy of lenalidomide in del(5q) MDS. Applying quantitative proteomics in the myeloid cell line KG-1, we identified a novel target, casein kinase 1A1 (CSNK1A1), that had increased ubiquitination and decreased protein abundance following lenalidomide treatment. CSNK1A1 is encoded in the del(5q) commonly deleted region and is thus a potential lenalidomide target in del(5q) MDS. Previous studies have demonstrated that Csnk1a1 is a therapeutic target in a murine model of acute myeloid leukemia. We validated that lenalidomide treatment decreased CSNK1A1 protein levels in multiple human cell lines in a dose-dependent manner without altering CSNK1A1 mRNA levels. Moreover, lenalidomide treatment increased ubiquitination of CSNK1A1 in cell lines. The decrease in CSNK1A1 protein levels in response to lenalidomide was abrogated by treatment with the proteasome inhibitor MG132 and by Cullin-RING ubiquitin ligase inhibition with MLN4924. CSNK1A1 co-immunoprecipitated with CRBN in the presence of lenalidomide, demonstrating direct interaction of CSNK1A1 with the substrate adaptor for the ubiquitin ligase. Homozygous genetic inactivation of the CRBN gene by CRISPR/Cas9 genome editing in 293T cells eliminated lenalidomide-induced degradation of CSNK1A1. In aggregate, these experiments demonstrate that CSNK1A1 is a CRBN-CRL4 substrate that is ubiquitinated and degraded in the presence of lenalidomide. We next explored how degradation of CSNK1A1 might explain the specificity of lenalidomide for cells with del(5q). ShRNA-mediated knockdown of CSNK1A1 sensitized primary human CD34+ cells to lenalidomide treatment, indicating that haploinsufficiency for CSNK1A1 might increase lenalidomide sensitivity in del(5q) hematopoietic cells. We sought to further validate this finding in a genetically defined Csnk1a1 conditional knockout mouse model. While murine cells are resistant to the effects of IMiDs, murine Ba/F3 cells overexpressing human CRBN (hCRBN), but not murine CRBN, degraded CSNK1A1 in response to lenalidomide. To examine the effect of Csnk1a1 haploinsufficiency on lenalidomide sensitivity, we isolated hematopoietic stem and progenitor cells from Csnk1a1+/- and Csnk1a1+/+ mice and transduced them with a retroviral vector expressing hCRBN. When treated with lenalidmide, Csnk1a1+/- cells expressing hCRBN were depleted over time relative to wild-type controls. The enhanced sensitivity of Csnk1a1+/- cells to lenalidomide was associated with induction of p21 and was rescued by heterozygous deletion of p53, demonstrating a critical downstream role for p53 consistent with clinical observations that TP53 mutations confer lenalidomide resistance. In aggregate, these studies demonstrate that lenalidomide induces the ubiquitination and consequent degradation of CSNK1A1 by the CRBN-CRL4 E3 ubiquitin ligase. del(5q) cells have only one copy of CSNK1A1, so they are selectively depleted over wild-type cells, explaining lenalidomide’s clinical efficacy in del(5q) MDS. Although the idea that heterozygous deletions could be cancer vulnerabilities was first proposed 20 years ago, lenalidomide provides the first example of an FDA-approved and clinically effective drug that derives its therapeutic window from specifically targeting a haploinsufficient gene. Disclosures Ebert: Celgene: Research Funding; Genoptix: Consultancy.

Hematopoietic Stem/Progenitor cells (HSPCs) with 5q haploinsufficiency in del(5q) myelodysplastic syndrome (MDS) acquire a clonal advantage in the bone marrow and out-compete normal hematopoiesis. A critical, yet unsolved question remains: how does genetic haploinsufficiency in del(5q) cells contribute to the clonal advantage of HSPCs? We investigated the role of haploinsufficiency for three candidate genes in the common deleted region on chromosome 5 (Csnk1a1, Egr1 and Apc) in direct competition with each other and wild-type (wt) cells on a single cell level by employing a novel lentiviral genetic barcoding strategy. We introduced genotype and cell-specific barcodes into HSPCs from murine models haploinsufficient for Csnk1a1, Egr1 or Apc. Barcoded HSPCs were sort-purified, genotypes mixed and subsequently competitively transplanted into lethally irradiated mice and re-transplanted after 16 weeks in a secondary transplant. The barcoded progeny was reliably recovered from peripheral blood and relative contribution of the barcoded clones to differentiated blood lineages was followed over 32 weeks. Despite heterogeneity in clonal evolution among the mice, all haploinsufficient clones had the potential to outcompete wt clones (3 of 5 mice in the primary transplant, 3 out of 4 mice in the secondary transplant). Csnk1a1 haploinsufficient clones showed the largest clonal abundance and clonal persistence. Expansion of oligoclonal Csnk1a1 haploinsufficient HSPCs was further enhanced in the secondary transplant in all mice. Egr1 haploinsufficient clones showed potential for prominent oligoclonal expansion in one mouse, but decreased in abundance in all secondary transplants. Apc haploinsufficient clones showed persistence but not expansion in 3 out of 5 mice. These results were validated by conventional competitive transplants, which demonstrated that Csnk1a1 and Egr1 haploinsufficient cells achieved the highest advantage over wt hematopoiesis in the primary transplant and more enhanced in the secondary transplant. Since Csnk1a1 regulates β-catenin protein stability, we hypothesized that the clonal expansion of Csnk1a1 haploinsufficient HSPCs is dependent on β-catenin levels. We performed a second genetic barcoding competitive transplant, comparing Csnk1a1-/+ HSPC directly to double haploinsufficient Csnk1a1-/+/Ctnnb1-/+ (β-catenin encoding) HSPCs. We included additional double haploinsufficient mutants Csnk1a1-/+/Apc-/+ and Csnk1a1-/+/Egr1-/+. Results showed pronounced expansion of Csnk1a1-/+ clones, while Csnk1a1-/+/Ctnnb1-/+ clones were outcompeted over time, suggesting that the advantage of Csnk1a1-/+ clones is β-catenin dependent. Csnk1a1-/+/Egr1-/+ and Csnk1a1-/+/Apc-/+ clones were less advantageous than Csnk1a1-/+ clones. To further investigate the mechanism of clonal fitness in Csnk1a1-/+ haploinsufficient HSPCs, we performed droplet based single cell RNA sequencing of Csnk1a1-/+ and wt Lin-Sca1+cKit+ (LSK) HSPCs. Csnk1a1 -/+ LSK were characterized by a higher fraction of cells expressing cell cycle genes compared to wt cells. In line, transcriptional alterations in the most primitive HSCs suggest that the clonal advantage is conveyed by canonical Wnt signaling activating downstream targets such E2F proteins. Csnk1a1-/+ haploinsufficient multipotent progenitors and myeloid/lymphoid primed progenitors expressed marked upregulation of metabolic pathways, mitochondrial respiration, cell cycle and differentiation, ubiquitination/proteasome system and deregulation of ribosome biogenesis. In conclusion, we demonstrate using a novel genetic barcoding approach in a competitive transplant setting that Csnk1a1-/+ haploinsufficient HSPCs have the potential for oligoclonal expansion and clonal persistence. Wnt/β-catenin signaling plays a central role in the clonal expansion. Interestingly, in Csnk1a1 haploinsufficiency the HSC state is preserved and the increased proliferation and metabolic activation are hallmark features of differentiating progenitor cells at MPP stage, increasing with cell cycle activation, thus ensuring clonal stability and preventing HSC exhaustion over time. Disclosures Brümmendorf: University Hospital of the RWTH Aachen: Employment; Janssen: Consultancy; Pfizer: Consultancy, Research Funding; Merck: Consultancy; Novartis: Consultancy, Research Funding; Ariad: Consultancy. Ebert:Celgene: Research Funding; Deerfield: Research Funding; Broad Institute: Other: Contributor to a patent filing on this technology that is held by the Broad Institute..

Despite extensive insights into the underlying genetics and biology of acute myeloid leukemia (AML), overall survival remains poor and new therapies are needed. We found that casein kinase 1 α (Csnk1a1), a serine-threonine kinase, is essential for AML cell survival in vivo. Normal hematopoietic stem and progenitor cells (HSPCs) were relatively less affected by shRNA-mediated knockdown of Csnk1a1. To identify downstream mediators of Csnk1a1 critical for leukemia cells, we performed an in vivo pooled shRNA screen and gene expression profiling. We found that Csnk1a1 knockdown results in decreased Rps6 phosphorylation, increased p53 activity, and myeloid differentiation. Consistent with these observations, p53-null leukemias were insensitive to Csnk1a1 knockdown. We further evaluated whether D4476, a casein kinase 1 inhibitor, would exhibit selective antileukemic effects. Treatment of leukemia stem cells (LSCs) with D4476 showed highly selective killing of LSCs over normal HSPCs. In summary, these findings demonstrate that Csnk1a1 inhibition causes reduced Rps6 phosphorylation and activation of p53, resulting in selective elimination of leukemia cells, revealing Csnk1a1 as a potential therapeutic target for the treatment of AML.

Abstract Background and Aim: Deletion of 5q is the most frequent cytogenetic aberration in MDS and is associated with distinct clinical characteristics, disease course and sensitivity to lenalidomide. The serine-threonine kinase CSNK1A1 is located in the commonly deleted region at 5q32 and has been described as a tumor-suppressor gene in colon cancer and acute myeloid leukemia through regulation of ß-catenin and p53. Recently, missense mutations in CSNK1A1 have been described in individual patients with del(5q) MDS. The aim of our study was to characterize the frequency and potential prognostic impact of CSNK1A1mutations in MDS and AML following MDS. Methods: 192 patients with MDS or AML following MDS (sAML) and deletion of chromosome 5q and 406 patients with MDS/sAML without deletion of chromosome 5q were included in the current analysis (n=598 in total). Patients with MDS (n=442) or sAML (n=156) were cytogenetically characterized by chromosome banding analysis and molecularly analyzed for mutations in exon 3 and 4 of CSNK1A1, the region critical for the kinase function, by Sanger sequencing. Patients with mutated CSNK1A1 were also analyzed for mutations in TP53 by next-generation or Sanger sequencing. Results: CSNK1A1 mutations were found in 17 (8.9%) of 192 MDS patients with del(5q). The mutation frequency was similar between patients with isolated del(5q) (n=153) and patients with concurrent cytogenetic aberrations or missing additional cytogenetic information (n=39)(9.2% vs 7.7%, P=.7). No mutation of CSNK1A1 was found in any of 406 MDS/sAML patients without del(5q). Thirteen patients (76%) had missense mutations affecting amino acid E98 in exon 3 of CSNK1A1. Of these, the glutamic acid to lysin substitution was the most frequent amino acid substitution (n=7). All mutations of glutamic acid 98 had a high probability to be damaging to the protein based on PolyPhen2 predictions (scores 0.922 to1). One patient had an Asn86Tyr mutation concurrently with the Glu98Ala mutation. Four patients (24%) had missense mutations affecting aspartic acid 140 in exon 4 of CSNK1A1. These mutations had moderate PolyPhen2 prediction scores (0.558-0.798). Three of the 17 CSNK1A1 mutated patients had additional cytogenetic aberrations besides del(5q), i.e. one trisomy 8, one trisomy 11, and one monosomy 7. None of the CSNK1A1 patients had a concurrent TP53 mutation. Del(5q) patients with wildtype or mutated CSNK1A1 had a similar median age (73.3 vs 77.5 years, P=.15). 70% and 59% of wildtype and mutated CSNK1A1 patients had female sex, respectively (P=.33). The WBC count was similar between wildtype and mutated CSNK1A1patients (3.9 vs 4.6, P=.47). Survival information was available for 155 patients with del(5q) (81%) including 16 patients (94%) with mutated CSNK1A1. Median follow-up from the time of sample harvest was 2.02 years. The probability of survival at 2 years was 41% for CSNK1A1 mutated and 72% for CSNK1A1wildtype patients (P=.059, log-rank test), suggesting a potential negative prognostic impact of CSNK1A1 mutations in del(5q) MDS patients. Conclusion: CSNK1A1 mutations are highly specific for MDS patients with del(5q) and are one of the most frequent recurrent genetic aberrations in these patients. Our survival analysis suggests that CSNK1A1 mutations have an unfavorable prognostic effect in patients with MDS and del(5q); however, the prognostic impact has to be confirmed in additional patients. Mutation analysis of exon 3 and 4 of CSNK1A1 should be included in the routine workup of MDS patients with deletion of 5q. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Kobbe:Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Medac: Other; Astellas: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Neovii: Other. Haferlach:MLL: Equity Ownership.

Abstract Heterozygous deletion of RPS14 occurs in del(5q) MDS and has been linked to impaired erythropoiesis, characteristic of this disease subtype. We previously generated a mouse model with conditional inactivation of Rps14 and demonstrated a p53-dependent erythroid differentiation defect with apoptosis at the transition from polychromatic to orthochromatic erythroblasts resulting in age-dependent progressive anemia, megakaryocyte dysplasia, and loss of hematopoietic stem cell (HSC) quiescence. We now sought to determine the mechanistic basis for the anemia using unbiased, quantitative mass spectrometry in erythroid progenitor cells. We found powerful induction of proteins involved in innate immune signaling, particularly the danger associated molecular pattern (DAMP) heterodimeric S100A8/S100A9 proteins. We found significantly increased S100a8 in the erythroid progenitor populations affected by the differentiation block (RIII-RIV population) and in monocytes and macrophages of Rps14 haploinsufficient bone marrows, all representing cells of the erythroblastic niche. Recombinant S100A8 was sufficient to impair erythropoiesis in wild-type cells. We rescued the erythroid differentiation defect in Rps14 haploinsufficient HSCs by genetic inactivation of S100a8 expression using CRISPR/Cas-mediated gene inactivation in primary mouse Rps14 haploinsufficient HSPC. We validated the association between induction of S100A8 and a severe erythroid phenotype in bone marrow samples of patients with del(5q) MDS. To examine whether ribosomal haploinsufficiency also leads to activation of S100A8 in patients with del(5q) MDS, we measured S100A8 expression using immunofluorescence in bone marrow biopsies from MDS patients with and without del(5q). In del(5q) MDS, the frequency of S100A8-positive cells was associated with disease severity, as reflected by transfusion burden. RPS14, CSNK1A1 and miR-145 are universally co-deleted in the 5q- syndrome and each represent different clinical features of del(5q) MDS in murine models. Haploinsufficiency of miR-145 or -146a also induces inappropriate activation of innate immune signaling. To analyze the combinatorial effect of haploinsufficiency Rps14, Csnk1a1 and miRNA-145, we transduced hematopoietic stem and progenitor cells (HSPC) from compound haploinsufficient Rps14 and Csnk1a1 mice and stably knocked down both miR-145/miR-146a by retrovirus-mediated overexpression of respective target sequences. Compound haploinsufficiency of Rps14, Csnk1a1 and miR-145/146a led to a progressive anemia comparable to Rps14 haploinsufficiency with splenomegaly and an erythroid differentiation defect at the RIII/RIV population, indicating that the anemia is mainly driven by Rps14 haploinsufficiency. Bone marrow histology demonstrated the typical 5q-phenoytpe of megakaryocytes, in line with significant thrombocytosis. At 10 months of age, hematopoietic stem and progenitor cells were significantly increased (lineagelow ckit+ Sca1+; LSK), in particular multipotent progenitor cells (MPPs; lineagelow ckit+ Sca1+ CD48- CD150+) to significantly higher extents than in solely Rps14 or Csnk1a1 haploinsufficient cells. We next asked if compound haploinsufficiency of the three 5q-genes has combinatorial or synergistic effects on S100a8 expression. Compound haploinsufficiency of Csnk1a1, Rps14 and miR-145/146a induced the highest expression of S100a8 in monocytes, while haploinsufficiency of Rps14 alone induced the highest expression of S100a8 in the RIII erythroid population, suggesting that cell-type specific induction mediates the phenotype. Our data indicate an unexpected link between haploinsufficiency for a ribosomal gene, Rps14, activation of S100A8, and inhibition of erythropoiesis. We demonstrate that compound haploinsufficiency for Csnk1a1 and miR145/146a with Rps14 haploinsuffciency increases the expression of S100a8, mainly in monocytes, and recapitulates the phenotype of del(5q) MDS by cooperating, cell-type specific effects. Disclosures Platzbecker: Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Boehringer: Research Funding.

Glioblastoma (GBM) is the malignant form of glioma, and the interplay of different pathways working in concert in GBM development and progression needs to be fully understood. Wnt signaling and sonic hedgehog (SHH) signaling pathways, having basic similarities, are among the major pathways aberrantly activated in GBM, and hence, need to be targeted. It becomes imperative, therefore, to explore the functioning of these pathways in context of each other in GBM. An integrative approach may help provide new biological insights, as well as solve the problem of identifying common drug targets for simultaneous targeting of these pathways. The beauty of this approach is that it can recapitulate several known facts, as well as decipher new emerging patterns, identifying those targets that could be missed when relying on one type of data at a time. This approach can be easily extended to other systems to discover key patterns in the functioning of signaling molecules. Studies were designed to assess the relationship between significant differential expression of genes of the Wnt (Wnt/β-catenin canonical and Wnt non-canonical) and SHH signaling pathways and their connectivity patterns in interaction and signaling networks. Further, the aim was to decipher underlying mechanistic patterns that may be involved in a more specific way and to generate a ranked list of genes that can be used as markers or drug targets. These studies predict that Wnt pathway plays a relatively more pro-active role than the SHH pathway in GBM. Further, CTNNB1, CSNK1A1, and Gli2 proteins may act as key drug targets common to these pathways. While CTNNB1 is a widely studied molecule in the context of GBM, the likely roles of CSNK1A1 and Gli2 are found to be relatively novel. It is surmised that Gli2 may be antagonistic to CSNK1A1, preventing the phosphorylation of CTNNB1 and SMO proteins in Wnt and SHH signaling pathway, respectively, by CSNK1A1, and thereby, aberrant activation. New insights into the possible behavior of these pathway molecules relative to each other in GBM reveal some key interesting patterns.

Abstract The immunomodulatory drug lenalidomide (LEN) is the treatment of choice for del(5q) MDS patients. LEN has been shown to trigger the specific degradation of CSNK1A1 and IKZF1 proteins after binding the E3-ligase substrate adaptor CRBN. When brought below a certain expression threshold, CSNK1A1 deficiency activates a p53-dependent apoptotic response. Thus, the unique sensitivity of del(5q) cells to LEN is explained by CSNK1A1 haploinsufficiency in del(5q) MDS patients. Despite its efficacy, 50% of LEN-treated patients eventually relapse within an interval of 2-3 years after treatment. Treatment failure is associated to low platelet counts and occurrence of additional mutations, such as TP53. To identify novel genetic determinants of LEN resistance, we have compared whole genome sequencing data of paired samples from six del(5q) patients who have been treated with LEN and eventually became resistant to the treatment. We identified 2 patients with mutations in TP53. The remaining four presented RUNX1 alterations: two patients had protein coding mutations in RUNX1 and two had a significant reduction in RUNX1, but not TP53, transcript levels. As a model of sensitivity, we studied the response to LEN in two human del(5q) cell lines, MDS-L and KG-1a. RUNX1 protein levels are postranscriptionally upregulated upon exposure to LEN, accompanied by increased levels of RUNX1 activity. Deletion of CRBN expression cancelled these effects. RUNX1 overexpression inhibited clonogenic growth and induced apoptosis. We then generated RUNX1 knock-out (KO) clones derived from MDS-L cells using CRISPR/Cas9 system. RUNX1 KO cells presented increased proliferation, increased colony growth and reduced apoptosis in the presence of LEN compared to wild-type (WT) control clones. These results were validated with different shRNAs against RUNX1. Genetic rescue experiments showed that RUNX1 mutants were unable to restore sensitivity to the drug compared to RUNX1 WT. Finally, modeling RUNX1 loss-of-function (LOF) in CSNK1A-depleted human CD34+ cells abrogated the effects of LEN on colony forming cell assays. Thus, RUNX1 function is required for the elimination of del(5q) cells by LEN. To understand the molecular mechanisms underlying the resistant phenotype, we performed RNA-seq on MDS-L cells treated with LEN for 24h. We observed a significant upregulation of Platelet specific genes (ITGB3, ITGA2B, VWF, THBD, SELP, TREML1, GATA1) coupled to downregulation of Cell Cycle genes (E2F2, E2F1, MCM5, CDKN1A), suggesting that LEN induces differentiation in to the Megakaryocytic (Meg) lineage. We found a significant upregulation of CD41+/CD61+ double positive cells after LEN exposure in vitro and in vivo, associated to the appearance of multinucleated cells. Importantly, the apoptotic response was associated to the emergence of the differentiating population. At the molecular level, CRBN is required for LEN-induced differentiation. Further downstream we identified IKZF1 degradation as key trigger, as IKZF1 overexpression restrained Meg differentiation and a IKZF1 dominant negative isoform enhanced it. In contrast, CSNK1A overexpression did not alter differentiation after LEN, but did reduce apoptotic induction. Moreover, we identified GATA2 targets enriched in LEN-regulated genes and showed that GATA2 overexpression or downregulation using shRNAs significantly increased or reduced LEN induced differentiation respectively. Finally, gene expression analysis after LEN exposure showed that Meg signatures were not enriched in resistant RUNX1 KO cells compared to WT control. Accordingly, RUNX1 KO cells did not undergo differentiation upon LEN exposure. RUNX1 LOF in CSNK1A-depleted primary human CD34+ cells blocked CFU-Mk growth in LEN treated cells. GATA2 overexpression was unable to restore LEN effects in RUNX1 deficient cells, suggesting a cooperative mechanism between both transcription factors. Luciferase assays using the human CD41 promoter showed that RUNX1 mutants reduced promoter transactivation compared to RUNX1 WT. Remarkably, we observed a similar phenotype for LEN-resistant TP53 KO cells. As a conclusion, our results suggest that GATA2, RUNX1 and TP53 cooperate to drive Meg differentiation after LEN-mediated degradation of IKZF1 protein. Loss of function mutations affecting RUNX1 or TP53 alter the activity of GATA2 transcriptional complex, rendering del(5q) cells unresponsive to LEN. Disclosures Platzbecker: Celgene: Research Funding.

Hintergrund und Zielsetzung: DMD ist die häufigste Form der Muskeldystrophie im Kindesalter und bis heute unheilbar. Sie wird durch das Fehlen des Proteins Dystrophin verursacht, welches verschiedene Signaltransduktionswege beeinflusst. Das Anliegen der Arbeit ist die Untersuchung und Modulation von Signaltransduktionswegen, die als alternative Therapiestrategie den Verlust von Dystrophin kompensieren könnten. Experimentelle Strategie: Für die Charakterisierung von Dystrophin nachgeschalteten Prozessen wurden mRNA-Expressionsanalysen in Muskelgeweben von DMD-Patienten und einem DMD-Brüderpaar mit einem infrafamiliär unterschiedlichen Verlauf der DMD durchgeführt. Aus diesen Expressionsdaten wurde erstmalig ein Petri-Netz entwickelt, welches Dystrophin mit in diesem Zusammenhang bisher unbekannten Signaltransduktionswegen verknüpft. Das Petri-Netz wurde auf Netzwerkintegrität und –verhalten mittels Invarianten- (INA) und theoretischen Knockout- (Mauritius Maps) Analysen untersucht. Durch beide Methoden läßt sich der maßgebliche Teilsignalweg bestimmen. In diesem Signalweg wurden die Proteinaktivität und die Genexpression durch siRNA, Vektor-DNA und chemische Substanzen in humanen SkMCs moduliert. Anschließend wurden die Proliferation und die Vitalität der Zellen sowie auch die Expression auf mRNA- und Protein-Niveau untersucht. Ergebnisse: RAP2B und CSNK1A1 waren in dem DMD-Brüderpaar differentiell exprimiert und konnten erstmalig in einem neuen, komplexen Signalweg in Zusammenhang mit Dystrophin nachgeschalteten Prozessen dargestellt werden. Mittelpunkt dieses Signalweges ist die De- und Aktivierung des Transkriptionsfaktors NFATc. Seine Zielgene umfassen neben anderen den negativen Proliferationsfaktor p21, das Dystrophin homologe UTRN und den Differenzierungsfaktor MYF5. Folglich würde ein Anstieg von UTRN eine unerwünschte Reduktion der Proliferationsrate von Myoblasten implizieren. Letzteres konnte bereits nachgewiesen werden und stellte das Motiv für weitere Studien dar. Jedoch zeigten siRNA- und Vektor-DNA-Experimente, daß NFATc nicht der ausschlaggebende Faktor für diese Zielgene ist. Die Substanzen Deflazacort (DFZ) und Cyclosporin A (CsA) wurden dagegen beschrieben, die Aktivierung von NFATc zu beeinflussen. Die Ergebnisse zeigten, daß beide Substanzen die Proliferation von Myoblasten erhöhen können. Die gleichzeitige Applikation von DFZ und CsA führte zu einem Anstieg der UTRN-Expression. Schlußfolgerung: Die Modulation der Proliferation und UTRN-Expression ist unabhängig von einander möglich. Entsprechend der Grundidee der Arbeit zeichnet sich eine neue Therapiestrategie ab, welche Dystrophin nachgeschaltete Prozesse einbezieht.
Background and aim: DMD is the most common muscular dystrophy in childhood and incurable to date. It is caused by the absence of dystrophin, what influences several signal transduction pathways. The thesis is interested in the investigation and modulation of signal transduction pathways that may compensate the lack of dystrophin as an alternative therapy strategy. Experimental strategy: To study Dystrophin downstream pathways the mRNA expression of DMD patients and two DMD siblings with an intra-familially different course of DMD were analysed in muscle tissue. On the basis of these expression data a Petri net was first developed implicating signal transduction pathways and Dystrophin downstream cascades. Invariant (INA) and theoretical knockout (Mauritius Maps) analyses were applied for studying network integrity and behaviour. Both methods provide information about the most relevant part of the network. In this part modulation of protein activity and of gene expression using siRNA, vector-DNA, and chemical substances were performed on human SkMCs. Subsequently, the cells were studied by proliferation and vitality tests as well as expression analyses at mRNA and protein level. Results: RAP2B and CSNK1A1 were differently expressed in two DMD siblings, and first are part of a signal transduction pathway implicating Dystrophin downstream processes. The central point of this pathway is the de- and activation of the transcription factor NFATc. Its target genes are, among others, the negative proliferation factor p21, the Dystrophin homologue UTRN, and the differentiation factor MYF5. Consequently, an increase in UTRN implicates an undesirably reduced myoblast proliferation rate. Latter was found in DMD patients and was target for further studies. But, siRNA and vector DNA experiments showed that NFATc is not the decisive factor for the target genes. Deflazacort and cyclosporin A are known to influence the activation of NFATc. The results first showed that both substances do induce myoblast proliferation. The use of deflazacort in combination with cyclosporin A resulted in an increase of UTRN expression. Conclusion: The modulation of proliferation and UTRN-expression independently of each other is possible. According to the basic idea of this study, a new therapeutic strategy becomes apparent, which considers Dystrophin downstream processes.

Glioblastoma is the most common and lethal malignant brain tumor with a survival rate of 14.6 months and a tumor recurrence rate of ninety percent. Two key causes for glioblastomas grim outcome derive from the lack of applicable prognostic markers and effective therapeutic targets. By employing a loss of function RNAi screen in glioblastoma cells we found a list of 20 kinases that can be considered glioblastoma survival kinases. These survival kinases which we term as survival kinase genes, (SKGs) were investigated to find prognostic markers as well as therapeutic targets for glioblastoma. Analyzing these survival kinases in The Cancer Genome Atlas patient database, we found that CDCP1, CDKL5, CSNK1𝜀, IRAK3, LATS2, PRKAA1, STK3, TBRG4, and ULK4 genes could be used as prognostic markers for glioblastoma with or without temozolomide chemotherapeutic treatment as a covariate. For the first time, we found that patients with increased levels of NEK9 and PIK3CB mRNA expression had a higher probability of recurrent tumors. We also discovered that expression of CDCP1, IGF2R, IRAK3, LATS2, PIK3CB, ULK4, or VRK1 in primary glioblastoma tumors was associated with tumor recurrence prognosis. To note, of these recurrent prognostic candidates, PIK3CB expression in recurrent tumor tissue had much higher expression compared to primary tissue. Further investigation in the PI3K pathway showed a strong correlation with recurrence rate, days to recurrence and survival emphasizing the role of PIK3CB in tumor recurrence in glioblastoma. In efforts to find effective therapeutic targets for glioblastoma we used SKGs as potential candidates. We chose the serine/threonine kinase, Casein Kinase 1 Epsilon (CSNK1𝜀) as a target for glioblastoma because multiple shRNAs targeted this gene in our loss of function screen and multiple commercially available inhibitors of this gene are available. Casein kinase 1 epsilon protein and mRNA expression were investigated using computational tools. It was revealed that CSNK1𝜀 expression has higher expression in glioblastoma than normal tissue. To further examine this gene we knocked down (KD) or inhibited CSNK1𝜀 in glioblastoma cells lines and noticed a significant increase in cell death without any significant effect on normal cell lines. KD and inhibition of CSNK1𝜀 in cancer stem cells, a culprit of tumor recurrence, also revealed limited self-renewal and proliferation in cancer stem cells and a significant decrease in cell survival without affecting normal stem cells. Further analysis of downstream effects of CSNK1𝜀 knockdown and inhibition indicate a significant increase in the protein expression of β-catenin (CTNNB1). We found that CSNK1𝜀 KD activated β-catenin, which increased GBM cell death, but can be rescued using CTNNB1 shRNA. Our survival kinase screen, computational analyses, patient database analyses and experimental methods contributed to the discovery of novel prognostic markers and therapeutic targets for glioblastoma.
Ph. D.

Myelodysplasia syndromes (MDS) are defined by a heterogeneous group of myeloid malignancies characterized by peripheral blood cytopenia and dishematopoiesis and frequently progress to acute myeloid leukemia. Conventional karyotype has a crucial role in myelodysplastic syndrome (MDS) and is one of items of the International Prognostic Scoring System (IPSS) for patient risk stratification and treatment selection. Approximately 50–60% of cases of MDS present chromosomal abnormalities, like the deletions of chromosome 5q and 7q, trisomy 8, and complex karyotypes. New genomic technologies have been developted, like single-nucleotide polymorphism array and next-generation sequencing. They can identify the heterozygous deletions wich result in haplo-insufficient gene expression (e.g., CSNK1A1, DDX41 on chromosome 5, CUX1, LUC7L2, EZH2 on chromosome 7) involved in the pathogenesis of myelodysplasia syndromes. Genetic abnormalities are multiple, the most recurrent one are involved in the RNA splicing like SF3B1, SRSF2, U2AF1, ZRSR2, LUC7L2, and DDX41. Epigenetic modifications are also identified, such as histone modification as ASXL1, EZH2. Finally, it can be DNA methylation (e.g., TET2, DNMT3A, IDH1/IDH2). On this review we will summarize the most recent progress in molecular pathogenesis of MDS, and try to better understand the pathogenesis of the specific subgroups of MDS patients and applications of discovery of new genetic mutation in the development of new therapeutic.