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Randomized trials in acute myeloid leukemia (AML) have demonstrated improved survival by the BCL-2 inhibitor venetoclax combined with azacitidine in older patients, and clinical trials are actively exploring the role of venetoclax in combination with intensive chemotherapy in fitter patients with AML. As most patients still develop recurrent disease, improved understanding of relapse mechanisms is needed. We find that 17% of patients relapsing after venetoclax-based therapy for AML have acquired inactivating missense or frameshift/nonsense mutations in the apoptosis effector gene BAX. In contrast, such variants were rare after genotoxic chemotherapy. BAX variants arose within either leukemic or preleukemic compartments, with multiple mutations observed in some patients. In vitro, AML cells with mutated BAX were competitively selected during prolonged exposure to BCL-2 antagonists. In model systems, AML cells rendered deficient for BAX, but not its close relative BAK, displayed resistance to BCL-2 targeting, whereas sensitivity to conventional chemotherapy was variable. Acquired mutations in BAX during venetoclax-based therapy represent a novel mechanism of resistance to BH3-mimetics and a potential barrier to the long-term efficacy of drugs targeting BCL-2 in AML.

Selective targeting of BCL-2 with the BH3-mimetic venetoclax has been a transformative treatment for patients with various leukemias. TP-53 controls apoptosis upstream of where BCL-2 and its prosurvival relatives, such as MCL-1, act. Therefore, targeting these prosurvival proteins could trigger apoptosis across diverse blood cancers, irrespective of TP53 mutation status. Indeed, targeting BCL-2 has produced clinically relevant responses in blood cancers with aberrant TP-53. However, in our study, TP53-mutated or -deficient myeloid and lymphoid leukemias outcompeted isogenic controls with intact TP-53, unless sufficient concentrations of BH3-mimetics targeting BCL-2 or MCL-1 were applied. Strikingly, tumor cells with TP-53 dysfunction escaped and thrived over time if inhibition of BCL-2 or MCL-1 was sublethal, in part because of an increased threshold for BAX/BAK activation in these cells. Our study revealed the key role of TP-53 in shaping long-term responses to BH3-mimetic drugs and reconciled the disparate pattern of initial clinical response to venetoclax, followed by subsequent treatment failure among patients with TP53-mutant chronic lymphocytic leukemia or acute myeloid leukemia. In contrast to BH3-mimetics targeting just BCL-2 or MCL-1 at doses that are individually sublethal, a combined BH3-mimetic approach targeting both prosurvival proteins enhanced lethality and durably suppressed the leukemia burden, regardless of TP53 mutation status. Our findings highlight the importance of using sufficiently lethal treatment strategies to maximize outcomes of patients with TP53-mutant disease. In addition, our findings caution against use of sublethal BH3-mimetic drug regimens that may enhance the risk of disease progression driven by emergent TP53-mutant clones.

PURPOSE The B-cell lymphoma 2 (BCL-2) inhibitor venetoclax has an emerging role in acute myeloid leukemia (AML), with promising response rates in combination with hypomethylating agents or low-dose cytarabine in older patients. The tolerability and efficacy of venetoclax in combination with intensive chemotherapy in AML is unknown. PATIENTS AND METHODS Patients with AML who were ≥ 65 years (≥ 60 years if monosomal karyotype) and fit for intensive chemotherapy were allocated to venetoclax dose-escalation cohorts (range, 50-600 mg). Venetoclax was administered orally for 14 days each cycle. During induction, a 7-day prephase/dose ramp-up (days −6 to 0) was followed by an additional 7 days of venetoclax combined with infusional cytarabine 100 mg/m2 on days 1-5 and idarubicin 12 mg/m2 intravenously on days 2-3 (ie, 5 + 2). Consolidation (4 cycles) included 14 days of venetoclax (days −6 to 7) combined with cytarabine (days 1-2) and idarubicin (day 1). Maintenance venetoclax was permitted (7 cycles). The primary objective was to assess the optimal dose schedule of venetoclax with 5 + 2. RESULTS Fifty-one patients with a median age of 72 years (range, 63-80 years) were included. The maximum tolerated dose was not reached with venetoclax 600 mg/day. The main grade ≥ 3 nonhematologic toxicities during induction were febrile neutropenia (55%) and sepsis (35%). In contrast to induction, platelet recovery was notably delayed during consolidation cycles. The overall response rate (complete remission [CR]/CR with incomplete count recovery) was 72%; it was 97% in de novo AML and was 43% in secondary AML. During the venetoclax prephase, marrow blast reductions (≥ 50%) were noted in NPM1-, IDH2-, and SRSF2-mutant AML. CONCLUSION Venetoclax combined with 5 + 2 induction chemotherapy was safe and tolerable in fit older patients with AML. Although the optimal postremission therapy remains to be determined, the high remission rate in de novo AML warrants additional investigation (ANZ Clinical Trial Registry No. ACTRN12616000445471).

The BCL-2 inhibitor venetoclax combined with hypomethylating agents or low-dose cytarabine represents an important new therapy for older or unfit patients with acute myeloid leukemia (AML). We analyzed 81 patients receiving these venetoclax-based combinations to identify molecular correlates of durable remission, response followed by relapse (adaptive resistance), or refractory disease (primary resistance). High response rates and durable remissions were typically associated with NPM1 or IDH2 mutations, with prolonged molecular remissions prevalent for NPM1 mutations. Primary and adaptive resistance to venetoclax-based combinations was most commonly characterized by acquisition or enrichment of clones activating signaling pathways such as FLT3 or RAS or biallelically perturbing TP53. Single-cell studies highlighted the polyclonal nature of intratumoral resistance mechanisms in some cases. Among cases that were primary refractory, we identified heterogeneous and sometimes divergent interval changes in leukemic clones within a single cycle of therapy, highlighting the dynamic and rapid occurrence of therapeutic selection in AML. In functional studies, FLT3 internal tandem duplication gain or TP53 loss conferred cross-resistance to both venetoclax and cytotoxic-based therapies. Collectively, we highlight molecular determinants of outcome with clinical relevance to patients with AML receiving venetoclax-based combination therapies.

Introduction The Australasian Leukaemia and Lymphoma Group (ALLG) conducted a randomized trial (AMLM16) combining the FLT3 inhibitor sorafenib (SOR) or placebo (PBO) with intensive induction and consolidation chemotherapy, followed by 12 months of maintenance therapy in patients with FLT3-ITD mutant AML. The clinical results of the AMLM16 study are detailed in a companion abstract co-submitted to ASH 2020. We hypothesized that the optimal time to administer sorafenib would be d4-10 of induction in order to limit overlap with anthracyclines on d1-3 and to avoid high FLT3 ligand (FLT3L) levels beyond d10, which could abrogate FLT3 on-target activity and diminish the therapeutic index. Key objectives of the correlative studies were to correlate in vivo SOR activity on suppression of FLT3 phosphorylation (P-FLT3) using the plasma inhibitory assay (PIA) with relapse risk and to assess for the presence of FLT3-ITD measurable residual disease (MRD) after therapy. Methods Patients aged 18-65 years with newly diagnosed AML (excluding APML) were enrolled to a National Blood Cancer Registry; those with a FLT3-ITD mutant: wild-type allelic ratio (AR) ≥ 0.05 were eligible for enrolment to the AMLM16 study. Patients were randomized 2:1 to SOR or PBO 400mg orally bd on d4-10 of induction and each consolidation cycle in combination with intensive chemotherapy and for 12 months as maintenance monotherapy. Serum FLT3-L levels were measured by ELISA, SOR and its metabolites by mass spectrometry. SOR mediated inhibition of FLT3 was measured in serum samples using the PIA as previously described (Levis, Blood 2006) in which P-FLT3 inhibition to ≤15% baseline is defined as response. Quantitation of FLT3-ITD levels by NGS was performed at study screening and after each induction and consolidation chemotherapy cycle. Quantitation was by amplicon-based sequencing which detected ITDs >6 bp with a sensitivity of 0.001%: levels below and above this were termed measurable residual disease (MRD) negative and positive respectively. Prepared libraries were sequenced (Illumina) using 150 bp paired-end reads with a minimum coverage of 400,000 reads. Bioinformatics analysis was performed using getITD (Blätte, Leukemia, 2019). Results 98 evaluable patients were randomized to induction with either SOR (n=65) or PBO (n=33) with a FLT3-ITD AR ranging from 0.05 to 8.4. FLT3-ITD AR was ≥ 0.5 or ≥ 0.7 in 48% and 29%, respectively. During induction, FLT3-L levels increased by an average of 150-fold from d4 to d10 and a further 1.6-fold to d15, with a return to baseline by d28. Serial plasma samples to assess PIA were available for 63 patients (Fig A). Response to ≤15% on d10 (relative to d4) was observed in 88% of SOR patients, compared with 4.5% receiving PBO. The relapse risk was 32% and 62% among PIA responders and non-responders, respectively (Fig A). Among patients with PIA response to ≤15% by d10 (the last day of SOR/PBO), only 8.3% had evidence of a sustained PIA response by d15. Reduction of FLT3-ITD to undetectable levels (VAF We compared the relationship between inhibition of P-FLT3 on d10 and bone marrow FLT3-ITD MRD after induction with relapse risk (Fig D, E). In the SOR arm, if P-FLT3 was not reduced to ≤15%, all patients had persistent FLT3-ITD MRD after induction and the relapse risk was 80% (Fig D). In contrast, if P-FLT3 inhibition was ≤15% and FLT3-ITD MRD not detected, the relapse risk was only 20%. In the PBO arm after induction, persistent and undetectable FLT3-ITD MRD (VAF Conclusions These studies demonstrate that in vivo SOR inhibited P-FLT3 to ≤15% during induction in the majority of cases and was associated with reduced relapse risk. Persistent FLT3-ITD MRD post-induction was associated with a high relapse risk in both treatment arms. For patients receiving SOR, failure to reduce P-FLT3 response to ≤15% was associated with a high risk of persistent FLT3-ITD MRD and clinical relapse. Disclosures Anstee: Walter and Eliza Hall Institute: Patents & Royalties: milestone and royalty payments related to venetoclax.. Levis:Menarini: Honoraria; Amgen: Honoraria; FujiFilm: Honoraria, Research Funding; Daiichi-Sankyo: Honoraria; Astellas: Honoraria, Research Funding. Enjeti:Novartis: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Alexion: Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees; Bayer: Speakers Bureau; Sanofi: Speakers Bureau. D'Rozario:Abbvie: Membership on an entity's Board of Directors or advisory committees; BMS/ Celgene: Membership on an entity's Board of Directors or advisory committees. Marlton:AbbVie: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-La Roche Ltd: Membership on an entity's Board of Directors or advisory committees; Astra-Zeneca: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees. Roberts:Janssen: Research Funding; Servier: Research Funding; AbbVie: Research Funding; Genentech: Patents & Royalties: for venetoclax to one of my employers (Walter & Eliza Hall Institute); I receive a share of these royalties. Wei:Astra Zeneca: Honoraria, Research Funding; Novartis: Honoraria, Research Funding, Speakers Bureau; Abbvie: Honoraria, Research Funding, Speakers Bureau; Walter and Eliza Hall Institute of Medical Research: Patents & Royalties: AW is eligible for royalty payments related to venetoclax; Roche: Honoraria; Macrogenics: Honoraria; Servier: Consultancy, Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Pfizer: Honoraria; Bristol Myers Squibb: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria. OffLabel Disclosure: Sorafenib for FLT3-ITD AML

Improving outcomes in acute myeloid leukemia (AML) remains a major clinical challenge. Overexpression of pro-survival BCL-2 family members rendering transformed cells resistant to cytotoxic drugs is a common theme in cancer. Targeting BCL-2 with the BH3-mimetic venetoclax is active in AML when combined with low-dose chemotherapy or hypomethylating agents. We now report the pre-clinical anti-leukemic efficacy of a novel BCL-2 inhibitor S55746, which demonstrates synergistic pro-apoptotic activity in combination with the MCL1 inhibitor S63845. Activity of the combination was caspase and BAX/BAK dependent, superior to combination with standard cytotoxic AML drugs and active against a broad spectrum of poor risk genotypes, including primary samples from patients with chemoresistant AML. Co-targeting BCL-2 and MCL1 was more effective against leukemic, compared to normal hematopoietic progenitors, suggesting a therapeutic window of activity. Finally, S55746 combined with S63845 prolonged survival in xenograft models of AML and suppressed patient-derived leukemia but not normal hematopoietic cells in bone marrow of engrafted mice. In conclusion, a dual BH3-mimetic approach is feasible, highly synergistic, and active in diverse models of human AML. This approach has strong clinical potential to rapidly suppress leukemia, with reduced toxicity to normal hematopoietic precursors compared to chemotherapy.

Background: Recent randomized trials have demonstrated improvements in overall survival (OS) for the BCL-2 inhibitor venetoclax (VEN) in combination with azacitidine and low dose cytarabine in older unfit patients with AML. Pre-clinical studies identified BAX deficiency as a potential mechanism of VEN resistance in AML, but this has not been observed in patients to date (Chen, Cancer Disc 2019). Methods: Patient samples were derived from studies approved by the Alfred Ethics Committee. BAX sequencing was performed using targeted sequencing. In vivo studies used NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. S63845 and S55746 were obtained from Servier/Novartis, A1155463 from G. Lessene (WEHI), venetoclax and cytarabine from Selleckchem. OCI-AML cells were used for protein, viability and competition studies. Results: We used targeted sequencing to assess BAX among 44 patients relapsing after attaining initial remission on VEN-containing regimens for AML. BAX variants at relapse were identified in 6 patients (13.6%), with a variant allelic frequency (VAF) of 0.75% - 48% (Fig A). This included missense, frameshift (fs), nonsense and splice site mutations (Fig A). One variant, P168A, residing in the linker region prior to the α9 helix is known to reduce BAX translocation and activity. The emergence of BAX mutations at AML progression implicated its role in adaptive resistance (Fig B). In contrast, no BAX mutations were identified in a control cohort of 35 patients with AML relapsing after conventional chemotherapy (Fig A). To explore mechanisms of acquired resistance to BH3 mimetics, OCI-AML3 cells were exposed to increasing concentrations of BCL-2 and MCL1 inhibitors over 3-months. Acquired resistance (OCI-AML3-R) was associated with loss of BAX expression, with no changes observed in other BCL-2 family members (Fig C). In contrast, BAX expression was preserved in OCI-AML3 cells exposed to similar concentrations of DMSO (OCI-AML3 DMSO). Targeted BAX NGS revealed a BAX E41Gfs variant in exon 3 at baseline (VAF 46%). In OCI-AML3-R cells, this variant was detected at a VAF of 86%, with no copy number change detected on sequencing-based copy number assessment, consistent with development of loss of heterozygosity in OCI-AML3-R cells. OCI-AML3-R cells were also cross-resistant to an MCL1 inhibitor (S63845), combination of VEN with S63845 or triple combination with VEN, S63845 and a BCL-X inhibitor A1155463 (not shown). In a xenograft model of AML, cohorts transplanted with OCI-AML-R cells lacking wildtype BAX displayed reduced survival compared to mice transplanted with OCI-AML3-DMSO cells after combined treatment with VEN and S63845 (Fig D). As mitochondrial apoptosis is mediated by pro-apoptotic effectors BAX and BAK, we generated OCI-AML3 cells deficient in BAX or BAK by CRISPR/CAS9 gene editing. Only BAX-/- but not BAK-/- cells were resistant to cell death induced by inhibitors of BCL-2 and MCL1 (not shown). This specificity was confirmed in xenograft models of BAX-/- or BAK-/- AML, which confirmed resistance of BAX deficient cells to combined therapy with VEN and S63845 in vivo (Fig E). Finally, to determine if BAX deficiency confers generalized chemoresistance, a competition assay compared proportions of wildtype BAX expressing and BAX-/- OCI-AML3 cells during a 14-day exposure to either 1) VEN, 2) S63845, 3) combined VEN and S63845 or 4) Ara-C. VEN therapy led to expansion of BAX-/- deficient cells, which was enhanced in combination with S63845. In contrast, treatment with Ara-C depleted BAX-/- cells over 4 days, with death of both BAX and BAX-/- populations seen by day 7 (Fig F). Conclusion: We identified the presence of BAX mutations in AML samples from patients progressing on VEN-containing regimens. We show that BAX, but not BAK loss in an AML cell line is associated with resistance to BH3-mimetic drug combinations resulting in reduced survival in AML xenograft models. In contrast, BAX deficiency does not impede the cytotoxic actions of conventional chemotherapy. We conclude that selection of BAX deficient cells may represent a novel mechanism of resistance to BH3-mimetics in the treatment of AML and that the emergence of BAX variants should be considered in patients developing adaptive resistance to VEN-based therapies. Disclosures Moujalled: Walter and Eliza Hall Institute: Patents & Royalties: milestone and royalty payments related to venetoclax.; Servier: Research Funding. Brown:Servier: Research Funding. Anstee:Walter and Eliza Hall Institute: Patents & Royalties: milestone and royalty payments related to venetoclax.. Banquet:Servier: Current Employment. Chanrion:Servier: Current Employment. Maragno:Servier: Current Employment. Schoumacher:Servier: Current Employment. Lessene:Servier: Research Funding; Walter and Eliza Hall Institute: Patents & Royalties: milestone and royalty payments related to venetoclax.. Geneste:Servier: Current Employment. Huang:Genentech: Research Funding; Walter and Eliza Hall Institute: Patents & Royalties: milestone and royalty payments related to venetoclax.; Servier: Research Funding. Roberts:Servier: Research Funding; Janssen: Research Funding; Genentech: Patents & Royalties: for venetoclax to one of my employers (Walter & Eliza Hall Institute); I receive a share of these royalties; AbbVie: Research Funding. Blombery:Janssen: Honoraria; Invivoscribe: Honoraria; Amgen: Consultancy; Novartis: Consultancy. Wei:Genentech: Honoraria, Other: Advisory committee; Servier: Consultancy, Honoraria, Other: Advisory committee; Walter and Eliza Hall Institute: Patents & Royalties; Macrogenics: Honoraria, Other: Advisory committee; AMGEN: Honoraria, Other: Advisory committee, Research Funding; Astra-Zeneca: Honoraria, Other: Advisory committee, Research Funding; Janssen: Honoraria, Other; Abbvie: Honoraria, Other: Advisory committee, Research Funding, Speakers Bureau; Astellas: Honoraria, Other: Advisory committee; Pfizer: Honoraria, Other: Advisory committee; Celgene: Honoraria, Other: Advisory committee, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau.

Background:The small molecule BCL-2 inhibitor Venetoclax (VEN) has emerged as a promising frontline therapy in combination with low dose cytarabine (LDAC) or hypomethylating agents in older patients with acute myeloid leukemia (AML)(DiNardo et al, Lancet Oncol 2018, Blood 2019; Wei et al,JCO 2019). The anti-leukemic activity of single agent VEN as monotherapy in newly diagnosed AML is unknown. This information would be useful for studies considering VEN as a maintenance therapy. We have previously presented preliminary outcomes from a phase 1b/2 study: Chemotherapy and Venetoclax in Elderly AML Trial (CAVEAT), which incorporated a 7-day single agent VEN run-in phase prior to combination with chemotherapy (Wei et al, ASH 2018). The study procedures incorporated a bone marrow assessment performed at study screening and on day 8, prior to commencement of chemotherapy (Figure 1A). This allowed us to explore the single agent activity of venetoclax on bone marrow blasts in treatment naïve patients with AML and to assess molecular dynamics of response. Methods:The CAVEAT trial included 51 de novo or secondary patients with AML aged ≥65 years and considered fit for intensive chemotherapy. The study enrolled patients to 5 VEN dose cohorts (50-100-200-400-600mg). VEN was administered on days 1-14 during induction. Chemotherapy commenced on day 8, with a 7-day VEN monotherapy pre-phase. Molecular studies:a 54-gene myeloid NGS panel (Illumina TruSight) and MassARRAY MALDI-TOF mass spectrometry was performed on DNA extracted from bone marrow aspirates. FLT3-ITD testing was by fragment analysis, PCR and capillary electrophoresis. Minimal residual disease (MRD) testing was performed by digital droplet PCR for IDH mutations (assay sensitivity: 10-4-10-5). Results: A total of 46 patients had paired BM assessments at screening and on day 8 for comparison. A ≥50% relative reduction in BM blasts was recorded in 13/46 (28%) patients. Figure 1B shows the relative BM blast reduction stratified by molecular subgroups. Patients with NPM1(n=9) or IDH2 (n=8) mutation achieved greater BM blast reductions (median of 56% and 55%, respectively) than IDH1 (n=6), TP53 (n=10) or FLT3-ITD (n=5) mutant cases (median of 37%, 17% and 7%, respectively). Three NPM1 mutant cases with Clinically, complete remission (CR) and CR with incomplete count recovery (CRi) rates after chemotherapy were seen for the following mutations: NPM1(80%), IDH2 (100%), IDH1 (62%) and TP53 (36%) (Figure 1D). Despite all four response-evaluable patients with FLT3-ITD achieving CR/CRi, 3/4 relapsed with detectable FLT3-ITD (at 0.8, 1.5 and 12.3 months respectively). Median survival for each genomic sub-group were NPM1 (12.5m), IDH2 (not reached), IDH1 (6.1m),TP53 (3.8m), FLT3-ITD (5.5m). CyTOF analyses for changes in BCL-2 family expression are underway and results will be presented at the meeting. Conclusion: Treatment naïve NPM1 and IDH2 mutant AML blasts are highly sensitive to VEN alone and combination with cytarabine and anthracycline chemotherapy results in a high clinical response rate. TP53 and FLT3-ITD mutant cases were more resistant and outcomes were poor despite VEN combined with chemotherapy. Figure 1 Disclosures Reynolds: Alfred Health: Employment, Other: Biostatistician for trials funded by the Australian government and Abbvie, Amgen, Celgene, GSK, Janssen-Cilag, Merck, Novartis, Takeda, but sponsored by Alfred Health.; AUSTRALASIAN LEUKAEMIA & LYMPHOMA GROUP (ALLG): Consultancy; Novartis AG: Equity Ownership; Novartis Australia: Honoraria. Fong:Pfizer: Consultancy, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; Astellas: Consultancy; Novartis: Speakers Bureau. Ting:NHMRC: Research Funding. Fleming:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria; Pfizer: Honoraria; Ariad: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees. Moujalled:Walter and Eliza Hall Institute of Medical Research: Patents & Royalties: Share in venetoclax royalties . Ivey:Novartis: Honoraria. Roberts:AbbVie: Research Funding; Janssen: Research Funding; Walter and Eliza Hall Institute: Employment, Patents & Royalties: receives a fraction of its royalty stream related to venetoclax. Wei:AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: AHW is a former employee of the Walter and Eliza Hall Institute and receives a fraction of its royalty stream related to venetoclax, Research Funding, Speakers Bureau; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Macrogenics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra Zeneca: Honoraria, Research Funding; Janssen: Honoraria. OffLabel Disclosure: Venetoclax is a BCL-2 inhibitor that is FDA-approved in some indications. This presentation will focus on correlative study results in the CAVEAT trial which combines venetoclax with modified intensive chemotherapy in AML, which is not an approved indication.

Targeting BCL-2 with the BH3-mimetic venetoclax (VEN) has emerged as a highly efficacious treatment for elderly patients with acute myeloid leukaemia (AML), in combination with either low-dose Ara-C (Wei, JCO 2019) or hypomethylating agents (Di Nardo, Lancet Oncol 2018). Long-term survival outcomes remain poor among patients with adverse risk karyotype, who often harbour mutations in the TP53 gene. Therefore, identification of effective treatment options, with TP53 independent activity, represents an area of high unmet therapeutic need. We generated TP53-null AML cell lines using CRISPR-Cas9 gene editing and screened these cell lines against 48 compounds. The histone deacetylase inhibitors (HDACi) had substantial efficacy. Additional studies performed in factor dependent myeloid (FDM) cells demonstrated cell death from HDACi to be Bax/Bak dependent, but Bim and p53 independent. RNA-sequencing, following HDACi treatment, revealed a rapid upregulation of pro-apoptotic factors, including PMAIP1 (NOXA) and BBC3 (PUMA). These results indicate that treatment with HDACi upregulates pro-apoptotic factors, independently of P53, and led to our hypothesis that HDACi could sensitise tumor cells to BH3-mimetics. We then tested the activity of HDACi in combination with BH3-mimetics targeting BCL-2 against 61 primary AML patient samples. Overall, LC50 at 48h was reduced by >2 logs in 60% of cases. Importantly, TP53 mutant cases were sensitive to this combination. Patient-derived xenograft models confirmed that this combination was tolerable and able to suppress bone marrow AML in vivo. Our data supports the clinical use of combined HDAC and BCL-2 targeting for AML and represents a promising treatment option for patients with adverse risk genomic characteristics; in particular, those who have failed VEN treatment in combination with low dose chemotherapy, and cases defective in P53.

Zebrafish are increasingly used to study neurodegenerative conditions such as Parkinson's disease (PD). In rodents, the influence of the genetic background on important experimental parameters in PD research such as susceptibility to toxin exposure or motor behavior is well established. In contrast, little is known about the impact of the genetic background in commonly used zebrafish wild-type strains on these important experimental parameters. We determined the effect of the genetic background in five commonly used zebrafish wild-type strains on crucial, PD-related aspects, in particular the number of ascending dopaminergic neurons, their susceptibility to PD-related neurotoxins, and the expression levels of five genes involved in oxidative stress defense, protein degradation, cell death, and apoptosis. We also investigated whether the susceptibility to morpholino-mediated knockdown of the PD gene DJ-1 may have a varying effect on neuronal cell loss depending on the genetic background. Finally, we determined the influence of the genetic background on spontaneous motor behavior. There was remarkably little variation between the different wild-type strains for most parameters investigated. However, the susceptibility to the neurotoxin 1-methyl-4-phenylpyridinium differed between the five investigated strains and so did their spontaneous motor behavior.

Background: Venetoclax (VEN) is a potent small molecule BH3-mimetic drug that selectively targets the prosurvival protein BCL-2 and has an emerging role for treatment in Acute Myeloid Leukaemia (AML). This ongoing Phase 1b study aims to evaluate the optimal dose, safety and efficacy of VEN in combination with modified intensive chemotherapy in fit, elderly patients with AML who have not received prior induction chemotherapy. Here we present updated data including molecular correlates of response and treatment outcomes. Methods: Eligibility: Patients were enrolled with AML (excluding APL), age ≥65 years (≥60 years if monosomal karyotype), ECOG 0-1 and adequate organ function. Prior HMA/LDAC was permitted after a 14-day washout. Treatment: 1) Dose escalation cohorts comprised VEN 50mg (Cohort A), 100mg (B), 200mg (C), 400mg (D) or 600mg (E); 2) a 7-day VEN pre-phase incorporating dose ramp-up to minimise tumour lysis risk followed by a 7-day overlap with chemotherapy (day -6 to +7); 3) attenuated induction chemotherapy with cytarabine 100mg/m2/day IVI d1-5 staggered with idarubicin 12mg/m2 IV d2-3. For patients in remission, 4 cycles of consolidation were administered, comprising 14 days of VEN (day -7 to +7) with bolus cytarabine (100mg/m2/day IV d+1-2) and idarubicin (12mg/m2 IV d+1) followed by 7 cycles of VEN monotherapy maintenance; planned to commence q28d. Antifungal azoles were avoided during VEN exposure. The first patient was enrolled 17JUL2016. Molecular studies- Next generation sequencing using a 54-gene TruSight myeloid panel was performed on baseline bone marrow samples. FLT3-ITD testing was performed by capillary electrophoresis. RT-qPCR was used to detect minimal residual disease (MRD) in NPM1-mutated cases (assay sensitivity: 10-5-10-6). Results: The data cut-off date was 13JUL2018. 44 patients were enrolled with 3 patients still in the induction cycle. Median age was 72 years (range 63-80 years; 23% ≥75 years), 66% of patients were male, 41% had secondary AML and 38% prior hypomethylating agent (HMA) exposure. Main grade ≥3 non-haematological adverse events during induction were febrile neutropenia (56%), sepsis (32%), rapid atrial fibrillation (15%), diarrhoea (12%), nausea (10%) and localised infection (10%). No clinical TLS was observed. One hematologic dose-limiting toxicity was reported in cohort E (VEN 600mg), but a maximum tolerated dose has not been reached. We observed progressive delay in platelet count recovery as patients proceeded with each cycle of therapy (median time to platelets ≥50x109/L was 25d in induction, 37d in consolidation 1 and 57d in consolidation 4). Treatment-related mortality was 7% in induction. 18/38 (47%) had ≥30% relative reduction in bone marrow (BM) blasts after 7 days of pre-phase VEN monotherapy, with no correlation between VEN dosage and the degree of BM blast reduction. Overall CR/CRi rate was 71%. CR/CRi was achieved in 95% of patients with de novo AML (vs 42% in secondary/therapy-related AML). Response rates were 88% in intermediate vs 46% in adverse cytogenetic risk AML. Responses in patients with prior HMA therapy were 43% and 84% for previously untreated patients. Molecular data were available for 41 patients. 98% had ≥1 mutation detected. (Figure 1) Responses in response-evaluable patients were most common in NPM1 mutant AML (100%; n=7), followed by RUNX1 (90%; n=11), RAS (90%; n=10) and IDH (89%; n=9). Lowest responses were observed in TP53 mutant AML (33%; n=9). NPM1 MRD assessment was performed on 6 patients, of which 5 (83%) achieved NPM1 MRD negativity. (Figure 2) Updated response, response duration and survival data will be presented, along with molecular characteristics of relapse. Conclusion: To date, VEN up to 600mg in combination with 5+2 induction chemotherapy is tolerable in fit elderly patients with AML. Initial response rates >80% were observed in de novo AML, as well as NPM1, RUNX1, RAS and IDH mutant AML, whereas responses were lower in patients with prior HMA exposure, adverse karyotype, secondary and TP53 mutant AML. Molecular determinants of relapse require further study. Disclosures Wei: Servier: Consultancy, Honoraria, Other: Advisory committee, Research Funding; Amgen: Honoraria, Other: Advisory committee, Research Funding; Novartis: Honoraria, Other: Advisory committee, Research Funding, Speakers Bureau; Pfizer: Honoraria, Other: Advisory committee; Celgene: Honoraria, Other: Advisory committee, Research Funding; Abbvie: Honoraria, Other: Advisory board, Research Funding, Speakers Bureau. Fong:Celgene: Other: Advisory board; Novartis: Other: Advisory board; Amgen: Research Funding; Pfizer: Other: Speaker fees. Reynolds:Novartis: Equity Ownership, Other: former employee of Novartis AG and holds stock in the company. . Roberts:Janssen: Research Funding; AbbVie: Research Funding; Genentech: Research Funding; Walter and Eliza Hall: Employment, Patents & Royalties: Employee of Walter and Eliza Hall Institute of Medical Research which receives milestone and royalty payments related to venetoclax.

Background: Acute myeloid leukaemia (AML) arises from the transformation of hematopoietic stem and progenitor cells. Targeting BCL-2 with the BH3-mimetic venetoclax (VEN) has emerged as a highly efficacious treatment option for elderly patients with AML, in combination with either low-dose ara-C (LDAC) (Wei, ASH 2017) or hypomethylating agents (HMA) (Di Nardo, Lancet Oncol 2018). Long-term survival outcomes, however, remain poor among patients with adverse risk karyotype, who often harbour mutations in the TP53 gene. Identification of more effective treatment options with TP53 independent activity represents an area of high unmet therapeutic need in AML. Methods: Panobinostat (histone deacetylase inhibitor; HDACi) was obtained from Novartis, S55746 (BCL-2 inhibitor) was obtained from Servier, and venetoclax (BCL-2 inhibitor) was obtained from Selleck Chemicals. Primary AML samples were obtained from patients providing informed consent. For in vivo experiments, NOD/Rag-/-/Il2rgtm1Wjl Tg(IL3, CSF2, KITLG) (NRG-SG3) mice were used. Dosing for S55746 was 100mg/kg daily Mon-Fri and panobinostat 10mg/kg on Mon/ Wed/ Fri. Results: A panel of 22 drugs were screened for enhanced anti-leukaemic activity in combination with BH3-mimetics targeting either BCL-2 or MCL1 against 45 primary AML patient samples. This revealed the histone deacetylase inhibitor (HDACi) panobinostat to have substantially enhanced efficacy: LC50 at 48h was reduced by >2 logs when combined with VEN to target BCL-2 in 13/21 (62%) or S55746 in 19/24 (79%) cases. Targeted exome sequencing revealed adverse-risk AML, including TP53 mutant cases, to be sensitive to this combination. Studies performed in factor dependent myeloid (FDM) cells, derived from gene knockout mice, demonstrated cell death from panobinostat to be Bax/Bak dependent. CRISPR/Cas9 BIM (MV4;11) and TP53 (OCI-AML3) deleted AML cell lines confirmed panobinostat-VEN activity to be independent of these pathways. Comparison of panobinostat in combination with BCL-2 targeting showed the combination to be less affected by p53 deficiency than cytotoxic (idarubicin, ara-C) + BCL2 inhibitor combinations. Patient-derived xenograft (PDX) models confirmed that this combination was tolerable and able to rapidly suppress bone marrow AML in vivo. Exploratory CyTOF and Western blot studies in MV4;11 cells showed that panobinostat +/- BCL-2 targeting markedly suppressed levels of MYC, pSTAT5 and pERK. Conclusions: Combined targeting of HDAC and BCL-2 represents a promising, chemotherapy-free, treatment option for patients with AML, including sub-groups with adverse risk genomic characteristics. The combination may have particular relevance for patients with adverse risk AML, including cases defective in functioning TP53. Figure. Figure. Disclosures Teh: The Walter and Eliza Hall Institute of Medical Research: Other: Institutional funding for venetoclax including milestone and royalty payments.. Kraus-Berthier:servier: Employment. Kloos:Servier: Employment; Novartis: Other: Partnership. Schoumacher:Servier: Employment. Gray:The Walter and Eliza Hall Institute of Medical Research: Other: Institutional funding for venetoclax including milestone and royalty payments.. Wei:Celgene: Honoraria, Other: Advisory committee, Research Funding; Novartis: Honoraria, Other: Advisory committee, Research Funding, Speakers Bureau; Amgen: Honoraria, Other: Advisory committee, Research Funding; Pfizer: Honoraria, Other: Advisory committee; Servier: Consultancy, Honoraria, Other: Advisory committee, Research Funding; Abbvie: Honoraria, Other: Advisory board, Research Funding, Speakers Bureau.