ALBERT

Description: Chronic lymphocytic leukaemia (CLL) is the most common form of adult leukaemia worldwide. Patients display a highly variable clinical course, with some requiring immediate therapeutic intervention while others can remain untreated for years. We have previously reported that DNA methylation of the homeobox A4 (HOXA4) promoter can serve as part of a three gene prognostic signature with CD38 and BTG4 to predict time to first treatment (TTT) in Stage A patients. HOXA4 encodes a transcription factor that is expressed in haematopoietic progenitor cells and is involved in embryonic development and B-cell differentiation, and its aberrant epigenetic regulation has been identified in multiple forms of leukaemia. In this study we have sought to elucidate the role of HOXA4in the progression of CLL and determine the functional consequences of its expression. We analysed DNA methylation of the HOXA4 promoter by pyrosequencing in a heterogeneous cohort of 163 CLL patients (median age: 70; median follow-up: 10 years), of whom 60% were Binet Stage A, 16% Stage B and 24% Stage C. Data was collected regarding treatment history, TTT and overall survival, as well as cytogenetic abnormalities and IGVH mutation status. HOXA4 methylation increased with disease progression and was significantly higher in Stage C patients (median 74%) than those with Stage A (62%; p = 0.03) and Stage B disease (65%; p 〈 0.05). HOXA4 methylation was positively correlated with IGVH sequence homology (r = 0.34, p 〈 0.0001) and negatively associated with TTT among patients who have started chemotherapy (p = 0.04) and with overall survival (p = 0.04). No associations were observed between HOXA4 methylation and 11q, 13q or 17p deletions, or TP53 and ATMmutations. To investigate the role of HOXA4 in the evolution of the disease, we analysed samples taken at multiple timepoints from 42 patients, of whom 29 were undergoing treatment and 13 remained untreated. HOXA4methylation significantly increased in patients undergoing treatment (p = 0.01), but did not differ in untreated patients (p = 0.19). We hypothesised that silencing of HOXA4 may be selected for during treatment due to its expression conferring increased sensitivity to chemotherapy. Using a lentiviral system, we observed that re-expression of HOXA4increased drug sensitivity in a malignant differentiated B-cell line (Raji). Significantly higher apoptosis was identified after treatment with 3 μM and 10 μM fludarabine (both p 〈 0.001) and 1 μM and 10 μM ibrutinib (p 〈 0.01 and p 〈 0.001), but not 1 μM and 10 μM idelalisib. To confirm the translational relevance our observations, we overexpressed HOXA4 in primary CLL cells derived from four patients and confirmed increased apoptosis in response to 3 μM and 10 μM fludarabine treatment in comparison to control cells (p = 0.02 and p 〈 0.01). Further work is underway in primary CLL cells to elucidate the pathways under the control of HOXA4 that may confer this drug sensitivity. Our ongoing work may indicate that HOXA4 is also implicated in the progression of CLL through directing malignant cells to the protective bone marrow niche, thereby further reducing sensitivity to antimetabolites. In cell lines HOXA4 up-regulates the expression of RGS2 and RGS16, which are negative regulators of the CXCR4-CXCL12 signalling axis, and we have identified selection for biallelic HOXA4methylation in primary acute lymphoblastic leukaemia cells following engraftment in mice (median in primary cells: 80%; engrafted cells: 92%; p 〈 0.0001). To determine the origins of HOXA4 dysregulation during the course of the disease, we analysed prospective blood samples from the European Prospective Investigation into Cancer and Nutrition (EPIC) from 20 individuals diagnosed with CLL up to 17 years after blood draw (median: 7 years) and 20 age-matched controls who remained free of cancer. We observed that HOXA4methylation was significantly higher among future CLL patients (median: 49% vs 42%; p = 0.01) and was inversely correlated with time to diagnosis, but did not reach statistical significance (r = -0.39, p = 0.09). Together, our findings suggest that silencing of the HOXA4 gene is an early event in CLL which is selected for during the course of disease through reduced sensitivity to chemotherapeutic agents. Our ongoing work will identify downstream targets that may be implicated in conferring sensitivity, and which may serve as biomarkers to predict prognosis and inform treatment strategies. Disclosures Junge: AstraZeneca: Other: Salary, Research Funding.

Description: The stress-inducible transcription factor, NF-κB, plays an important in role in B-cell malignancies, regulating expression of a plethora of genes including those that drive proliferation, apoptosis and survival. Non-canonical ('alternative') NF-κB signalling, resulting in processing of the p100 subunit to p52 and dimerization with RelB, is less well-studied than the canonical pathway. Previous studies on NF-κB signalling, particularly in CLL, showed that the RelA (canonical) subunit is highly expressed and associated with poor outcome. In multiple myeloma and lymphoma, mutations in genes that alter NF-κB activity (NOTCH1, BIRC3, MYD88, TRAF2/3) can promote cell survival, by increasing activation of the CD40 receptor or NIK (NF-κB-inducing kinase), resulting in constitutive non-canonical NF-κB signalling. More recently, a comprehensive study showed that recurrent mutations in TRAF3, BIRC3 and NFKB2, all of which regulate non-canonical NF-κB signalling, also occur in CLL (Puente XS et al, Nature 2015). Taken together, these data suggest that non-canonical NF-κB signalling is important in CLL and that this pathway represents a novel therapeutic target. We hypothesised that receptor-activated proliferation in CLL cells is reliant on non-canonical NF-κB-regulated gene transcription, and that targeting this pathway would decrease CLL cell survival. Since the IKKα kinase phosphorylates p100, which in turn leads to active p52/RelB dimers in the nucleus, it is a key driver of the non-canonical signalling pathway. We used cell line models and patient-derived CLL cells to evaluate a series of selective, first-in-class IKKα inhibitors. Initially, we assessed 20 novel IKKα inhibitors in MEC-1 (CLL-like, BIRC2/3 and TP53 deleted) and Maver-1 (mantle cell lymphoma, TRAF3 deleted) cell lines, as these are representative of B-cell malignancies with constitutive activation of non-canonical NF-κB. Compounds S and U were found to be potent (cell-free IC50 values 〈 50nM) and selective (e.g. 〉50-fold over IKKβ). In growth inhibition studies, compound S inhibited cell proliferation with GI50 values of 4.3 μM (MEC-1) and 2.7 μM (Maver-1) whereas compound U gave GI50 values of 0.47 μM (MEC-1) and 0.5 μM (Maver-1). We then examined selected cases from our CLL cohort. Western blotting of nuclear extracts showed that expression of NF-κB subunits in patient-derived CLL cells was heterogeneous: RelA was constitutively expressed in most cases, and although expression of the non-canonical subunits, p52 and RelB, varied, high levels of p52 were associated with high RelB expression, indicating that non-canonical signalling is active in CLL. The panel of 20 IKKα inhibitors were also tested in viability and apoptosis assays in patient-derived CLL cells, revealing LC50 values ranging from 0.5 μM (Compound U) - 5μM (48 hr exposure). Decreasing cell survival correlated with increased caspase 3/7 activation. To model the tumour microenvironment, we used CD40L-expressing fibroblasts in co-culture with primary CLL cells to stimulate NF-κB signalling and CLL cell proliferation. CD40L stimulation led to increased levels of nuclear RelB and p52 levels after 2-8 hrs co-culture, concomitant with activation of IKKα, as demonstrated by rapid phosphorylation of p100 subunit. Compound S was evaluated in CD40L-stimulated CLL cells where it was shown to inhibit p100 processing and decrease levels of nuclear RelB and p52. Compound S significantly reduced CD40L-stimulated proliferation of CLL cells following 7 day treatment (1μM), in a similar fashion to the clinically-used PI3Kδ inhibitor, Idelalisib, and was also effective at reducing proliferation of TP53-mutated patient-derived CLL cells. Compound S had little or no effect on accumulation of nuclear RelA (p65), indicating selectivity of these inhibitors for IKKα over IKKβ and suggesting that targeting receptor-stimulated IKKα in B cells may avoid 'global' toxicity anticipated by use of IKKβ-targeting therapies. Future studies will examine the gene expression profile following IKKα inhibition in CLL, and also interrogate the underlying genotypes among patients that may drive NF-κB signaling. These data provide proof of concept that targeting non-canonical NF-κB signaling is a valid therapeutic strategy in CLL. Disclosures No relevant conflicts of interest to declare.

Description: The ataxia telangiectasia and RAD3-related (ATR) protein kinase is a component of the cellular DNA damage response pathway and promotes cell survival by signalling repair of collapsed replication forks generated by replication stress. We hypothesised that inhibition of ATR potentiates the anti-leukaemic activity of chain terminating nucleoside analogues used in the treatment of acute myeloid leukaemia (AML). We used VE-821 and its derivative VX-970 (Vertex Pharmaceuticals, Abingdon, UK) as potent and specific inhibitors of ATR kinase activity to examine the effects of ATR inhibition in AML cell lines, primary AML cells and AML xenografts. Co-treatment with 1mM VE-821 did not consistently potentiate the anti-proliferative effects of cytarabine, clofarabine or fludarabine in a panel of AML cell lines. However, there was consistent potentiation of hydroxyurea and gemcitabine in all 7 AML cell lines tested. Treatment with hydroxyurea, which induces replication stress via depletion of dNTPs, resulted in phosphorylation of CHK1, a downstream target of ATR. CHK1 phosphorylation was attenuated when 1mM VE-821 was co-administered with hydroxyurea. Exposure of cells to gemcitabine or hydroxyurea slowed transit through S phase, which was pronounced in combination with VE-821. HL-60 AML cell clones expressing either a constitutively active or inducible shRNA construct targeting ATR had reduced ATR protein expression compared to control cells and were significantly more sensitive to the anti-proliferative effects of gemcitabine and hydroxyurea, but not to cytarabine, clofarabine or fludarabine. The growth inhibitory effects of hydroxyurea and gemcitabine were also significantly potentiated by VE-821 in primary AML patient samples, which included three adult patients with de novo AML and a paediatric patient with therapy-related AML. In contrast, ATR inhibition did not potentiate the inhibitory effects of hydroxyurea or gemcitabine in primary bone marrow cells from healthy donors ex vivo. We next sought to determine whether ATR inhibition potentiated hydroxyurea and gemcitabine in an orthotopic mouse model of AML. MV4-11 AML cells engineered to express firefly luciferase (MV4-11 pSLIEW) were intrafemorally transplanted into immunodeficient Rag2-/- gc-/- mice. Bioluminescent imaging via IVIS Spectrum (PerkinElmer, Buckinghamshire, UK) demonstrated localised femoral engraftment first detectable 4-5 days post-injection, with luciferase signal developing in other parts of the body (liver, ovaries) between days 15 and 18 in untreated mice. Treatment was initiated 7 days post-injection when disease was localised to the femur and prior to emergence of disseminated luciferase signal. Single agent hydroxyurea (250mg per kg, IP days 0-4 and 7-11) conferred some early disease control compared to controls as determined by luciferase total body flux measured on day 14, but this was not statistically significant (p=0.18) and did not affect overall survival (mean 35 days for controls and 37 days for hydroxyurea, p=0.47). Monotherapy with VX-970 (60 mg per kg, orally on days 0-4 and 7-11) also conferred early disease control compared to vehicle-treated mice (p=0.18), and resulted in significantly longer overall survival (mean 40 days, p=0.017). Combination treatment with hydroxyurea and VX-970 did not result in more effective early disease control or improved overall survival compared to monotherapy with either agent. Treatment with gemcitabine monotherapy (100 mg per kg, intraperitoneal injection on days 0, 3, 7 and 10) conferred significant early disease control (p=0.002) and significantly improved overall survival compared to controls (mean survival 73 days, p

Description: Introduction. Current treatment protocols for chronic lymphocytic leukemia (CLL), including FCR (fludarabine, cyclophosphamide and rituximab), have dramatically improved response rates. However, since none of these therapies is curative, continued preclinical studies on innovative therapeutic strategies are warranted. In particular, the identification of new agents that interfere with the survival of CLL cells by promoting their apoptosis is one approach to improve therapeutic outcome. Taking into account that p53 deletions and/or mutations in CLL are restricted to 10%-15% of patients at diagnosis, we have investigated the efficacy of the second-generation MDM2 antagonist RG7388, in primary CLL samples, examining its ability to disrupt the p53-MDM2 interaction, resulting in expression of p53 transcriptional targets that trigger apoptosis. Methods. Heparinized peripheral blood samples were obtained from CLL patients (n=42) with informed consent, in accordance with institutional guidelines and the Declaration of Helsinki. Mononuclear cells were purified by Lymphoprep density-gradient centrifugation. Cells were resuspended in RPMI 1640 medium supplemented with 10% FCS and 1% penicillin/streptomycin. Cells were treated with increasing concentrations of RG7388 and incubated at 37°C, 5% CO2. Ex vivo cytotoxicity was assessed in all samples 48 hours after treatment using an XTT assay. A pilot time-course experiment identified 6 hours after treatment as the most informative time point for gene expression analysis. Therefore, in 8 samples cells were treated for 6 hours and RNA was extracted using standard methods. qRT-PCR on a panel of nine p53 transcriptional targets (CDKN1A, MDM2, PUMA, BAX, FAS, FDXR, ZMAT3, TP53INP1, DR5) was performed using SYBR Green chemistry. The functional integrity of p53 was assessed by western blot following treatment with MDM2 antagonists in 20 samples. Apoptosis was assessed using a Caspase 3/7 luminescent assay. Results. Treatment with RG7388 significantly decreased cell viability (LC50 〈 1µM) in 28/42 CLL samples (mean LC50 = 0.40 ± 0.05 µM). As expected, the 8 CLL samples that displayed non functional p53 on western blot and/or mutated TP53 by sequencing were more drug-resistant (mean LC50 = 5.22 ± 1.13 µM) than those sensitive responders (LC50 〈 1µM) with a functional p53 (mean LC50 = 0.39 ± 0.05 µM, n=12). Interestingly, among p53 functional samples, we identified a small subset that showed intermediate response (1 µM 10 µM, n=3) to RG7388 treatment. RG7388 significantly upregulated the mRNA expression of MDM2 and of p53 transcriptional targets involved in the intrinsic pathway of apoptosis (PUMA and BAX), and the extrinsic pathway of apoptosis (death receptor 5 (DR5)) (Figure 1A, n=5 samples). Interestingly, only a slight upregulation of CDKN1A was observed and upregulation of pro-apoptotic genes dominated. Overall, in p53 functional CLL samples, exposure to 3 µM RG7388 for 6 hours led to a 7.8-fold increase in MDM2, a 7-fold increase in PUMA, a 4.9-fold increase in DR5, a 4.5-fold increase in BAX, and a 3.3-fold increase in CDKN1A (Figure 1A). Conversely, no significant upregulation of p53 target genes was seen in p53 non-functionalCLL samples (Figure 1B, n=3 samples). Treatment with RG7388 led to a concentration-dependent increase in caspase 3/7 activity in p53 functional CLL samples (Figure 1C). No significant difference in caspase 3/7 activity was observed in p53 non-functional samples after RG7388 treatment (Figure 1D). Interestingly, when we analysed samples taken two years apart from a fludarabine- and chlorambucil-resistant CLL patient, we found that treatment with RG7388 decreased cell viability, induced p53 accumulation and upregulation of p53-target genes, suggesting that inhibiting the p53-MDM2 interaction might be a promising treatment strategy in chemo-resistant CLL patients that show a functional p53. Conclusion. Taken together, our data indicate that RG7388 induces a characteristic dominantly pro-apoptotic gene expression profile of p53-target genes in primary p53-functional CLL samples. RG7388 showed a consequent potent apoptotic effect on CLL cells, which was dependent on the presence of a functional p53. Identification of patients in whom functional p53 activation drives apoptosis may translate into improved outcomes for patients treated with non-genotoxic MDM2 antagonists. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: Acute myeloid leukemia (AML) is a hematological malignancy with an undefined heritable risk. Here we perform a meta-analysis of three genome-wide association studies, with replication in a fourth study, incorporating a total of 4018 AML cases and 10488 controls. We identify a genome-wide significant risk locus for AML at 11q13.2 (rs4930561; P = 2.15 × 10−8; KMT5B). We also identify a genome-wide significant risk locus for the cytogenetically normal AML sub-group (N = 1287) at 6p21.32 (rs3916765; P = 1.51 × 10−10; HLA). Our results inform on AML etiology and identify putative functional genes operating in histone methylation (KMT5B) and immune function (HLA).