Herling, Marco

Abstract Background:Recurrent mutations in ATM, JAK1/3 and STAT5B genes have been identified in T-cell prolymphocytic leukemia (T-PLL), a mature T cell leukemia with poor survival. However, much remains to be understood regarding the dysregulated pathways involved in the pathogenesis of this aggressive disease. Here, we integrated the analyses of whole exome sequencing (WES) and transcriptomes of a cohort of 12 T-PLL samples to gain further insights into molecular mechanisms of this malignancy. Methods: Leukemic cells were isolated from peripheral blood or bone marrow samples of T-PLL patients. Tumor DNA and RNA, and germline DNA were extracted from purified leukemic T cells and bone marrow derived stromal cells, respectively. WES (n=12) and transcriptome analysis (n=10) were performed and data analyzed with published standard methods (Robinson et al. 2010 and DePristo et al. 2011) Results: 7 paired tumor and germline DNA and 5 tumor-only DNA samples were used for WES analysis. Mutations in ATM and TET2 were detected in 4 (33%) and 1 (8%) patients, respectively. JAK3 (p.M511I, p.Q501H and p.Q503H) or STAT5B (2 p.T628S and 1 p.N642H) mutations each occurred in 3 (25%) cases and were mutually exclusive, consistent with prior reports. Importantly, we found several novel mutations in genes with regulatory roles in the NOTCH signaling pathway. Specifically, 2 somatic mutations p.A3V and p.A3S in NOTCH2 and 2 other mutations (p.S244L and p.A1414V) in NOTCH4 were identified in 4 (33%) patients. One mutation was in the EGF- like and another one in the NODP domain critical for NOTCH-mediated cellular differentiation. Two (16.7%) novel missense mutations (p.G174D and p.V836I) were found in MAML2. Three missense mutations (2 p.S583N and 1 p.R190H) occurred in MAML1. Both MAML2 and MAML1 are essential cofactors of the NOTCH signaling pathway. In addition, a somatic mutation in the NOTCH signal regulator DTX2 (p.G268D) was detected. Altogether 7 (58.3%) patients harbored mutations in genes with a regulatory role in the NOTCH pathway. Among the epigenetic modifiers, we found deletions and point mutations in KDM6B and KDM6A in 4 (30%) patients including 2 codon deletions in KDM6B (p.X751Val) and 1 missense mutation each in KDM6B (p.R1016Q) and KDM6A (p.V510G). KDM6A and KDM6B are histone H3 lysine 27 demethylases known to be involved in regulation of DNA repair and play an important role in embryonic differentiation. Transcriptome analyses of 10 T-PLL cases versus normal CD3+ T cells from 5 healthy donors revealed 706 up-regulated and 655 down-regulated genes. Consistent with prior data, TCL1A and TCL1B were significantly overexpressed (23.7 and 13.1 fold, respectively). Using Ingenuity Pathway Analysis, we found PTEN Signaling, glutamate receptor, and axonal guidance signaling to be among the top 3 upregulated pathways. The top upstream regulators implicated for the up-regulated signals were FGF, Wnt/β-catenin and TNF. The top 3 down-regulated pathways were granulocyte adhesion and diapedesis, IL-17A in psoriasis and IL-10 signaling. The top regulators for the down-regulated pathways were LPS, TNF and IFNg. Wnt/β-catenin and Human embryonic stem cell pluripotency signaling were among the top 20 up-regulated pathways. Many genes involved in the regulation of Wnt signal pathway were included in the above pathways, including Wnt ligands (Wnt9A, Wnt4), transmembrane receptors (CDH1, LRP5, FZD6, FZD7, SMO), cytoplasmic factor (AXIN2), and nuclear factors (MYC, SOX12, SOX8, SOX18, CCND1). Summary: Our study identified recurrent mutations in the NOTCH signaling pathway as well as dysregulated embryonic developmental pathway, e.g. Wnt/NOTCH as newly implicated molecular mechanisms in T-PLL pathogenesis for further functional studies. Additionally, novel (KDM6A and KDM6B) and known mutations (ATM, TET2) in chromatin regulatory genes underscore the importance of epigenetic dysregulation in the tumorigenesis of T-PLL. Disclosures Parikh: Pharmacyclics: Honoraria, Research Funding. Mustjoki:Ariad: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Ding:Merck: Research Funding.

Abstract T-cell prolymphocytic leukemia (T-PLL) is a rare disease with an aggressive clinical course and a median overall survival of less than three years. Although almost 75% of T-PLL patients are reported to harbor translocations causing the activation of the proto-oncogene TCL1A, T-PLL is genetically heterogenous: most T-PLL patients also have mutations or deletions in the ATM gene and the genes involved in the JAK-STAT pathway are mutated in 76% of cases. There is an urgent need for more rational based therapies, but clinical trials are difficult to perform due to the rareness of the disease. Here, we systematically explored the diversity of drug responses in T-PLL patient samples ex vivo using a drug sensitivity and resistance testing (DSRT) system including 306 oncology drugs (approved or investigational). We also aimed to determine any associations between the genetic aberrations and drug sensitivities in T-PLL patients. Primary mononuclear cells were gathered from 30 T-PLL patients for drug testing. Cells were plated in 384-well plates and subjected to the 306 substances using a 10,000-fold concentration range. After 72 hours, cell viabilities were measured, the results were depicted as dose-response curves for each compound, and differential drug sensitivity scores (sDSS), representing leukemia-specific responses, were computed by comparing patient samples to healthy donors. Drug response profiles across patients were clustered and visualized by hierarchical clustering. The subgroups resulting from the clustering were statistically compared using a two-sample t-test to understand which drug classes were driving the grouping. To delineate heterogeneous pathway dependencies, drug sensitivities were correlated with somatic genetic variants and recurrent chromosomal aberrations. Genetic characterization was performed by targeted amplicon sequencing of tumor cells to profile known recurrent genetic variants (STAT5b, IL2RG, JAK1, JAK3, ATM). Information on chromosomal aberrations (TCL1A translocations, ATM deletions) was derived from parallel clinicopathologic databases. Amplicon sequencing revealed that 70% of T-PLL patients (21/30) harbored a mutation in genes involved in the JAK-STAT pathway (JAK1, JAK3, STAT5b or IL2RG). The most prevalent mutation led to an M511I amino acid exchange in the JAK3 protein (26% of patients). Interestingly, the STAT5b mutations (5/30) did not coexist with any of the JAK mutations in our cohort. Based on DSRT analysis, all T-PLL samples were sensitive to the CDK-inhibitor SNS-032 and the anti-cancer antibiotic actinomycin D. Next, we clustered patients using sDSS values for all 306 different substances, and this showed that patient samples could be divided into 3 main groups, based on their drug responses (Figure). According to two-sample t-test, the grouping was driven by the selective sensitivities of Groups II and III to HDAC inhibitors (belinostat, panobinostat, quisinostat, CUDC-101 and vorinostat) and the selective sensitivity of Group III to PI3K/AKT/mTOR inhibitors (AZD-8055, MK-2206, apitolisib, dactolisib, PF-04691502, ZSTK474, and omipalisib), HSP90 inhibitors (BIIB021, luminespib, alvespimycin, and tanespimycin) as well as JAK inhibitors (ruxolitinib, momelotinib, tofacitinib, gandotinib). Group I samples were on the other hand relatively resistant to these classes of drugs. Surprisingly, despite the prevalence of the signature event of activation of TCL1 (the established AKT coactivator) in nearly all cases, only a subset of cases (group III) responded to PI3K/AKT/mTOR inhibitors. Strikingly, the grouping of selective responses to HDAC, JAK, PI3K/mTOR/Akt and HSP90 inhibitors did not link to the presence of JAK/STAT mutations, TCL1A translocations, or ATM deletion status. Ex vivo drug screening of primary T-PLL samples revealed heterogenous selective drug responses in specific drug classes (such as HDAC-, JAK-, HSP90- and PI3K/Akt/mTOR-inhibitors). Surprisingly, the drug response patterns did not correlate with known recurrent genetic aberrations suggesting that sequencing for recurrent genetic biomarkers cannot easily be turned into effective therapeutic strategies in T-PLL, and that further elucidation of the biological pathways driving T-PLL is needed. Figure 1. Mutation status and clustering of HDAC-, PI3K/mTOR/Akt-, HSP90-, and JAK-inhibitor responses in PLL patients based on sDSS values Figure 1. Mutation status and clustering of HDAC-, PI3K/mTOR/Akt-, HSP90-, and JAK-inhibitor responses in PLL patients based on sDSS values Disclosures Koschmieder: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel reimbursement for scientific conferences, Research Funding; Novartis Foundation: Research Funding; Baxalta/CTI: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Janssen Cilag: Other: Travel reimbursement for scientific conferences ; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel reimbursement for scientific conferences. Wennerberg:Pfizer: Honoraria, Research Funding. Ding:Merek: Research Funding. Mustjoki:Pfizer: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.