Small Molecule Inhibitor Of Bcl-2 Research Articles

Proapoptotic Bcl-2 inhibitor as host directed therapy for pulmonary tuberculosis.

Mycobacterium tuberculosis establishes within host cells by inducing anti-apoptotic Bcl-2 family proteins, triggering necrosis, inflammation, and fibrosis. Here, we demonstrate that navitoclax, an orally bioavailable, small-molecule Bcl-2 inhibitor, significantly improves pulmonary tuberculosis (TB) treatments as a host-directed therapy. Addition of navitoclax to standard TB treatments at human equipotent dosing in mouse models of TB, inhibits Bcl-2 expression, leading to improved bacterial clearance, reduced tissue damage / fibrosis and decreased extrapulmonary bacterial dissemination. Using immunohistochemistry and flow cytometry, we show that navitoclax induces apoptosis in several immune cells, including CD68 + and CD11b + cells. Finally, positron emission tomography (PET) in live animals using novel, clinically translatable biomarkers for apoptosis ( 18 F-ICMT-11) and fibrosis ( 18 F-FAPI-74) demonstrates that navitoclax significantly increases apoptosis and reduces fibrosis in pulmonary tissues, which are confirmed using post-mortem studies. Our studies suggest that proapoptotic drugs such as navitoclax can improve pulmonary TB treatments, and should be evaluated in clinical trials.

Paired comparisons of venetoclax concentration in cerebrospinal fluid, bone marrow, and plasma in acute leukemia patients.

Venetoclax, a small molecule inhibitor of BCL-2, has demonstrated efficacy in treating acute leukemias and has been recommended as one of the first-line anti-leukemia therapies. Although venetoclax has been suggested to probably possess the ability to penetrate the central nervous system (CNS), current data to elucidate the characteristics of venetoclax in cerebrospinal fluid (CSF), bone marrow (BM), and plasma are still lacking. This study investigated the real-world characteristics of venetoclax concentrations in CSF, BM, and plasma in acute leukemia patients. Thirteen acute leukemia patients treated with venetoclax were included, with paired samples of CSF, BM, and plasma collected and venetoclax concentrations measured using LC-MS/MS. With the results, the median venetoclax concentrations were 2030 ng/mL in plasma, 16.7 ng/mL in CSF, and 1390 ng/mL in BM. The percentages of CSF/plasma and BM/plasma were 0.74% and 70.37%, respectively. While no direct correlation was observed between CSF and plasma venetoclax levels, there was a trend toward an improved CSF/plasma percentage over time following the last administration of venetoclax. In contrast, a strong correlation was found between BM and plasma levels. This study demonstrated that venetoclax could reach its effective concentration in most patients, suggesting its potential clinical utility in the management of CNS involvement in acute leukemia.

Venetoclax Induces BCL-2-Dependent Treg to TH17 Plasticity to Enhance the Antitumor Efficacy of Anti-PD-1 Checkpoint Blockade.

The specific BCL-2 small molecule inhibitor venetoclax induces apoptosis in a wide range of malignancies, which has led to rapid clinical expansion in its use alone and in combination with chemotherapy and immune-based therapies against a myriad of cancer types. While lymphocytes, and T cells in particular, rely heavily on BCL-2 for survival and function, the effects of small molecule blockade of the BCL-2 family on surviving immune cells is not fully understood. We aimed to better understand the effect of systemic treatment with venetoclax on regulatory T cells (Treg), which are relatively resistant to cell death induced by specific drugging of BCL-2 compared to other T cells. We found that BCL-2 blockade altered Treg transcriptional profiles and mediated Treg plasticity toward a TH17-like Treg phenotype, resulting in increased IL17A production in lymphoid organs and within the tumor microenvironment. Aligned with previously described augmented antitumor effects observed when combining venetoclax with anti-PD-1 checkpoint inhibition, we also demonstrated that Treg-specific genetic BCL-2 knockout combined with anti-PD-1 induced tumor regression and conferred overlapping genetic changes with venetoclax-treated Tregs. As long-term combination therapies using venetoclax gain more traction in the clinic, an improved understanding of the immune-modulatory effects caused by venetoclax may allow expansion of its use against malignancies and immune-related diseases.

A retrospective observational study of rapid titration of venetoclax (VEN) in patients with mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL).

e19018 Background: CLL and MCL can present with rapid disease progression that requires quick control. VEN is an oral, small-molecule inhibitor of BCL-2 that requires careful dose titration to avoid TLS. However, standard outpatient titration of VEN takes 29 days to reach max dose, with the low doses achieving little disease control. A common practice at our site is to rapidly titrate VEN to 100mg within 1 week with hospitalization for careful TLS monitoring and management. We present the safety and outcomes of 39 identified patients treated as such. Methods: The rapid VEN titration plan was: VEN 20mg x 2 days, 50mg x 2 days, then 100mg daily with q6-q8 hour monitoring of TLS labs plus ongoing IV hydration and allopurinol with or without rasburicase per clinical judgment. Most patients were treated in such manner if TLS did not occur. Clinically stable patients were typically discharged after 2 days of VEN 100mg, then continued weekly outpatient dose escalation to the 200mg and then the 400mg dose levels. If a patient was progressing on a Bruton's Tyrosine Kinase inhibitor, the inhibitor was often given concurrently with the early doses of VEN. Results: Patient characteristics are listed in the table. 23% (5 of 22) of patients with CLL and 35% (6 of 17) of patients with MCL had laboratory TLS (Howard SC, NEJM2011). In the patients with CLL, 2/5 were low risk for TLS, and in the patients with MCL, 3/6 were low risk for TLS. Only 4.5% (1 of 22) of CLL patients and no MCL patients had clinical TLS (Grade 2 creatinine elevation). In that specific patient, considered low risk for TLS, the elevated creatinine did not recover but was subsequently deemed secondary to Richter’s transformation involving the kidneys. No unexpected toxicity was observed outside of those typically associated with VEN and anti-CD20 antibody, when used. The mean number of days to reach 100mg was 6.5 days (quartile [Q] 1:5d; Q3: 10d), and the mean number of days to reach the planned VEN target dose was 18.8 with range (Q1:14d; Q3: 25d). Sixty-four percent of patients achieved the 400mg dose level. Conclusions: This single-institution retrospective analysis highlights the low rate of clinical TLS when using VEN with rapid titration in a controlled environment for high-risk patients needing rapid disease control. However, lab TLS occurred in low-risk patients treated in this manner, necessitating hospitalization despite low risk. No unexpected or additional toxicity was observed. [Table: see text]

The future paradigm of HMA + VEN or targeted inhibitor approaches: sequencing or triplet combinations in AML therapy.

The routine use of next-generation sequencing methods has underscored the genetic and clonal heterogeneity of acute myeloid leukemia (AML), subsequently ushering in an era of precision medicine-based targeted therapies exemplified by the small-molecule inhibitors of FLT3, IDH1/IDH2, and BCL2. This advent of targeted drugs in AML has broadened the spectrum of antileukemic therapies, and the approval of venetoclax in combination with a hypomethylating agent has been a welcome addition to our AML patients unable to tolerate intensive chemotherapy. Mounting evidence demonstrates that molecularly targeted agents combined with epigenetic therapies exhibit synergistic augmented leukemic cell kill compared to single-agent therapy. With such great power comes greater responsibility in determining the appropriate frontline AML treatment regimen in a molecularly defined subset and identifying safe and effective combination therapies with different mechanisms of action to outmaneuver primary and secondary resistance mechanisms in AML.

Targeting CREB-Binding Protein (CREBBP) Overcomes Resistance to Azacitidine and Venetoclax Therapy in Acute Myeloid Leukemia (AML)

Acute myeloid leukemia (AML) is a clonal myeloid malignancy arising from hematopoietic stem or progenitor cells (HSPC) within the bone marrow (BM). Despite advancements in our understanding of AML pathogenesis, patients continue to face grim outcomes, with a five-year relative survival rate of approximately 20%. This highlights the pressing need to identify molecular features of AML that are amenable to therapeutic intervention. The ideal molecular targets would be those broadly dysregulated and integral to multiple signaling pathways implicated in AML pathogenesis. Recent therapeutic regimens have incorporated venetoclax, a small molecule inhibitor of BCL2, in combination with hypomethylating agents, showing moderate success. Venetoclax (VEN) acts as a BH3 mimetic, specifically inhibiting BCL2. While clinical outcomes of VEN combined with hypomethylating agents (decitabine or azacitidine - AZA) have been promising, a substantial subset of patients remains refractory or experiences relapse following treatment with these regimens. Due to the molecular and cellular heterogeneity of AML, identifying ideal mechanisms to circumvent resistance remains elusive. In our investigations, we have observed that patient-derived cells from AML exhibit a higher frequency of dysregulated immune and inflammatory pathways than previously appreciated, referred to as “oncogenic immune signaling states.” Furthermore, immune and inflammatory-related pathways are preferentially expressed in AML patients who are resistant/refractory to VEN/AZA compared to those who respond favorably. To identify critical immune and inflammatory-related pathways contributing to VEN/AZA responses, we developed an oncogenic immune signaling-targeted CRISPR-library, aimed at identifying potential therapeutic targets involved in VEN/AZA resistance in AML. Utilizing this targeted CRISPR-screen approach, we successfully identified CREB-binding protein (CREBBP) as the top candidate linked to resistance to VEN/AZA therapy. Notably, CREBBP alterations have been associated with a poor prognosis in de novo AML, making it a compelling target for further investigation. To validate CREBBP's role in VEN/AZA resistance, we treated a VEN/AZA-resistant AML cell line, THP1 (FAB subtype M5, KMT2A-r), with commercially available pharmacologic inhibitors of CREBBP, A485, and CCS1477, and assessed the response. Remarkably, inhibition of CREBBP with either A485 or CCS1477 significantly decreased cell proliferative ability and clonogenic potential. Importantly, co-treatment with either A485 or CCS1477 and VEN/AZA synergistically abrogated cell proliferation and clonogenic potential of THP1 cells, effectively overcoming resistance (Figure 1). Our ongoing studies include validating CREBBP as a potential therapeutic target for overcoming VEN/AZA resistance in AML. We have created CREBBP-knockdown cell lines through genetic manipulation (shRNA) and will utilize these to confirm the specificity of CREBBP in modifying resistance to VEN/AZA. In addition we will utilize patient-derived xenograft studies in immunocompromised (NRG) mouse models. By investigating CREBBP's role in mediating resistance, we aim to unlock new avenues for enhancing the efficacy of VEN/AZA therapy in AML patients, particularly those facing resistance to current therapies. In conclusion, our research highlights the critical involvement of CREBBP in VEN/AZA resistance in AML. Targeting this molecular pathway holds the promise of offering a potential therapeutic approach to improve treatment outcomes for AML patients, particularly those facing resistance to current therapies. Understanding and harnessing CREBBP's influence could pave the way for developing personalized and effective strategies to combat this challenging hematological malignancy.

Preclinical Evaluation of T-Cell Prolymphocytic Leukemia Demonstrates Heterogeneous BCL2-Family Dependency That May be Effectively Targeted with Small Molecule Inhibitors

T-cell prolymphocytic leukemia (T-PLL) is a rare, aggressive, mature T-cell leukemia with very few treatment options and adverse prognosis. There are currently no approved therapies for patients with relapsed T-PLL, where the median OS is less than 6 months. There are limited reports suggesting clinical activity of the BCL2 inhibitor venetoclax in patients with T-PLL. We aimed to characterize the dependencies of T-PLL on members of the BCL2-family of antiapoptotic proteins using BH3 profiling, a functional assay to assess the propensity of a cell to undergo apoptosis (priming) and the relative dependence of a cell on different antiapoptotic proteins. We then sought to experimentally exploit these dependencies with targeted therapies alone, and in combination, designed to inhibit the antiapoptotic proteins towards a therapeutic goal. Cell lines of mature T cell lymphomas/leukemias (TCL/L) including SUPT11 (T-cell leukemia with rearranged TCL1) (n=10) and bone marrow and peripheral blood samples from untreated patients with T-PLL (n=7) were analyzed by BH3 profiling. We also used a relapsed/refractory T-PLL patient sample that were able to engraft and expand in a PDX model. Finally, scRNA/ATAC seq has also been performed in a set of naïve-treated T-PLL patients. In contrast to TCL/L that demonstrated heterogeneity on BCL2 family member dependency, T-PLL samples were primed and consistently exhibited dependency on BCL2/BCL-xL and MCL1. Integration of scRNA/ATAC seq confirmed high expression of BCL2 and MCL1 in T-PLL cells from four patients supporting the premise that BCL2 and MCL1 could potentially be targeted for therapeutic benefit. Therefore, SUPT11 cells (used as a model of T-PLL) were exposed to venetoclax (25 and 50nM; BCL-2inh), fadraciclib (25 and 50nM; CDK2/9inh - indirect inhibitor of MCL-1 as it decreases mRNAs with high decay rates including MCL1 and MYC) or a combination of venetoclax and fadraciclib. We demonstrate that exposure to fadraciclib caused a rapid decline in the levels of phosphorylation on Serine 2 of RNA polymerase II C-terminal domain, as well as decline in the levels of MCL1 within 4 hours and completely depleted these proteins by 8-12 h of exposure. We also show that there was a synergistic increase both in mitochondrial dysfunction as measured by cytochrome C release by 18h and in cell death in cells exposed to the combination at 24h. These results suggest that combining venetoclax and fadraciclib might represent a potential therapeutic approach for T-PLL. In conclusion, preclinical evaluation of a T-PLL cell line and primary patient samples demonstrate BCL2-family dependency that can be effectively targeted with small molecule inhibitors of BCL2 and CDK2/9. In vivo studies in PDX models of T-PLL are underway to form the basis for clinical trials to study these combinations.

2722. Risk of Infectious Adverse Events of Venetoclax Therapy in the Treatment of Hematologic Malignancies: A Systematic Review and Bayesian Meta-Analysis of Randomized Controlled Trials

Abstract Background Venetoclax is a small molecule inhibitor of BCL-2 used in the treatment of acute myelogenous leukemia and chronic lymphocytic leukemia. Recent post-marketing studies on venetoclax suggested that it may predispose to opportunistic infections (OIs). We sought to systematically review the randomized controlled trial (RCT) evidence on venetoclax to assess whether it predisposes patients to infectious adverse events (IAEs) and neutropenia. Methods We systematically reviewed the RCTs comparing venetoclax therapy to active (i.e., a regimen with additional anti-cancer agents) or placebo controls in patients with hematologic malignancies, irrespective of treatment line. Data on IAEs and neutropenia were pooled by Bayesian meta-analysis and we computed the probability of any increased risk in infectious or neutropenic complications (Risk Ratio [RR] >1). Subgroup analyses were conducted based on treatment line, use of an inactive comparator, malignancy type, and prophylaxis strategy. Additionally, we pooled the incidence rates of fatal OIs between venetoclax and comparator arms and compared them by univariate meta-regression. Results Seven RCTs were included comprising 2067 patients. In venetoclax versus comparator regimens there was a 81.1% probability of any increase in IAEs (RR=1.14, 95% Confidence Interval [95%CI]=0.76-1.66). The probabilities of venetoclax increasing the risk of grade 3-5 and fatal IAEs were 86.6% (RR=1.16, 95%CI=0.86-1.55) and 81.1% (RR=1.40, 95%CI=0.66-2.94), respectively. There was a 97.2% likelihood that venetoclax increased the risk of grade 3-5 neutropenia (RR=1.44, 95%CI=1.01-2.10). Venetoclax treatment did not increase the incidence of fatal OIs (P=0.55). Conclusion Our results suggest that venetoclax likely increases the risk of high-grade neutropenia and IAEs overall, but not fatal OIs. However, our analyses did not identify any specific IAEs that would benefit from routine anti-infective prophylaxis or pre-emptive testing. Disclosures Michael Sebag, MD, PhD, Amgen: Advisor/Consultant|BMS: Advisor/Consultant|Focus Therapeutics: Advisor/Consultant|Gilead: Advisor/Consultant|Janssen: Advisor/Consultant|Novartis: Advisor/Consultant|Pfizer: Advisor/Consultant|Sanofi: Advisor/Consultant Matthew Cheng, MD, Amplyx Pharmaceuticals: Grant/Research Support|AstraZeneca: Advisor/Consultant|AstraZeneca: Honoraria|Cidara Therapeutics: Grant/Research Support|GEn1E lifesciences: Advisor/Consultant|GEn1E lifesciences: Stocks/Bonds|Kanvas Biosciences, Inc.: Board Member|Kanvas Biosciences, Inc.: Pending patents|Kanvas Biosciences, Inc.: Ownership Interest|Merck: Honoraria|nomic bio: Advisor/Consultant|nomic bio: Stocks/Bonds|Pfizer: Honoraria|Scynexis Inc.: Grant/Research Support|Takeda: Advisor/Consultant|Takeda: Honoraria

Tolerability in Transplant-Ineligible Patients with Newly Diagnosed Acute Myeloid Leukemia Treated with Venetoclax Based Combination Therapy: A Systematic Review and Meta-Analysis of Phase III Randomized Controlled Trials

Introduction Therapeutic challenge remains in older patients with acute myeloid leukemia (AML) with significant comorbidities who are not fit for induction chemotherapy and haemopoietic stem cell transplantation. Leukemic cells depend on BCL-2 protein (anti-apoptotic protein) for their survival. Venetoclax is a selective small-molecule BCL-2 inhibitor which binds to BCL-2 resulting in apoptosis of leukemic cells. In recent years, venetoclax has been approved in combination with azacitidine or low dose cytarabine in transplant-ineligible patients with newly diagnosed AML (ND AML). However, there are notable toxicities. The purpose of our meta-analysis is to determine tolerability, rates of treatment emergent serious adverse events (SAE), treatment discontinuation (TD) and treatment interruption (TI), dose reduction and 30-day mortality in transplant-ineligible patients with ND AML treated with venetoclax based combination therapy. Methods We systematically conducted a comprehensive literature search using MEDLINE, EMBASE databases and meeting abstracts from inception through June 30 th, 2023. Phase III RCTs utilizing venetoclax in transplant-ineligible patients with ND AML that mention tolerability, rate of treatment discontinuation and interruption, dose reduction, treatment emergent serious adverse events, 30-day mortality were incorporated in the analysis. Mantel-Haenszel (MH) method was used to calculate the estimated pooled risk ratio (RR) with 95% confidence interval (CI). Heterogeneity was assessed with Cochran's Q- statistic. Fixed effects model was applied. Results A total of 637 transplant-ineligible patients with ND AML from 2 phase III RCTs (VIALE-A and VIALE-V) were eligible. VIALE-A and VIALE-V compared azacitidine + venetoclax vs azacitidine + placebo, and venetoclax + low dose cytarabine vs placebo + low dose cytarabine respectively. The randomization ratio was 2:1 in both studies.TD due to treatment emergent adverse events (TEAE) was noted in 104 (24.5%) of venetoclax group vs 45 (21.2%) in control group with RR of 1.15 (95% CI: 0.85 -1.57; P = 0.37).TI due to TEAE was higher in the venetoclax group (68.9%) compared to control group (55.6%) with RR of 1.24 (95% CI: 1.08 -1.42; P = 0.002). 21 (4.9%) of patients in venetoclax group and 10 (4.7%) in the control group required dose reduction of treatment due to TEAE, RR 1.03 (95% CI: 0.50 -2.16; P = 0.93).Treatment emergent SAEs were reported in 77.4% in the venetoclax group vs 60% in control group (RR, 1.30; 95% CI: 1.15-1.46; P <0.0001). 30-day mortality was reported in 39 (9.1%) of patients in the venetoclax group and 20 (9.4%) in the control group with RR of 0.96 (95% CI: 0.58 -1.60; P = 0.89). Conclusions Our meta-analysis showed that there was no significant difference in the rate of treatment discontinuation and dose reduction due to adverse events in transplant-ineligible patients with ND AML treated with venetoclax combination regimens, compared to control arm although rates of treatment emergent SAEs and treatment interruption were considerably higher in the venetoclax-treated arm. However, the 30-day mortality rate was similar between the two groups. Longer follow up and timely supportive care are required to improve treatment emergent SAEs and treatment interruption.

A new cyclin-dependent kinase-9 inhibitor A09-003 induces apoptosis in acute myeloid leukemia cells with reduction of myeloid cell leukemia sequence-1 protein

Acute myeloid leukemia (AML) is the most common type of hematological disease in adults, and has a very poor outcome [1]. Based on its wide range of efficacy in AML models, a small-molecule inhibitor of the anti-apoptotic protein BCL-2, venetoclax (ABT-199/GDC-0199), was developed for clinical trials. However, venetoclax showed limited monotherapy activity [2]. The overexpression of myeloid cell leukemia sequence-1 protein (Mcl-1)—due to mutations in Fms-like tyrosine kinase 3 internal tandem duplication (FLT-3 ITD)—was considered to be the main reason for low efficacy of venetoclax in clinical trials [3-5]. To achieve venetoclax sensitization in AML, targeting CDK-9 with venetoclax is a promising therapeutic strategy.In this study, we developed A09-003 as a potent inhibitor of CDK-9, with an IC50 value of 16 nM. A09-003 inhibited cell proliferation in various leukemia cell lines. In particular, the proliferation inhibitory effect of A09-003 was most potent in MV4-11 and Molm-14 cells, harboring the FLT-3 ITD mutation with a high expression profile of Mcl-1. Marker analysis revealed that A09-003 reduced CDK-9 phosphorylation and reduced RNA polymerase II activity with decreased Mcl-1 expression. Finally, combining A09-003 with venetoclax induced apoptotic cell death in a synergistic manner. In summary, this study shows the potential of A09-003 in AML therapy.

The roles of protocadherin-7 in colorectal cancer cells on cell proliferation and its chemoresistance.

Despite the high mutation frequencies of KRAS, NRAS, and BRAF in colorectal cancer (CRC), there are no effective and reliable inhibitors for these biomarkers. Protocadherin-7 (PCDH7) is regarded as a potentially targetable surface molecule in cancer cells and plays an important role in their proliferation, metastasis, and drug resistance. However, the roles and underlying mechanisms of PCDH7 in CRC remain unclear. In the current study, we found that different colorectal cancer cells expressed PCDH7 over a wide range. The levels of PCDH7 expression were positively associated with cell proliferation and drug resistance in CRC cells but negatively correlated with the potential for cell migration and invasion. Our data indicated that PCDH7 mediated the resistance of CRC cells to ABT-263 (a small-molecule Bcl-2 inhibitor that induces apoptosis) by inhibiting cell apoptosis, which was supported by the downregulation of caspase-3, caspase-9, and PARP cleavage. We found that PCDH7 effectively promoted Mcl-1 expression at both mRNA and protein levels. Furthermore, PCDH7 activated the Wnt signaling pathway, which was confirmed by the increase in β-catenin and c-Myc expression. Finally, and notably, S63845, a novel Mcl-1 inhibitor, not only effectively attenuated the inhibitory effect of PCDH7 on cell apoptosis induced by ABT-263 in vitro but also sensitized PCDH7-overexpressed CRC cell-derived xenografts to ABT-263 in vivo. Taken together, although PCDH7 inhibited the migration and invasion of CRC cells, it could facilitate the development of drug resistance in colorectal cancer cells by positively modulating Mcl-1 expression. The application of the Mcl-1 inhibitor S63845 could be a potential strategy for CRC chemotherapy, especially in CRC with high levels of PCDH7.

A real-world pharmacovigilance study of FDA Adverse Event Reporting System (FAERS) events for venetoclax.

Venetoclax (VEN) is the first selective small molecule Bcl-2 inhibitor approved by FDA and used in adult chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL) and some acute myeloid leukemia (AML). However, the long-term safety of VEN in large sample population was unknown. This study evaluated the adverse events (AEs) of VEN from FDA Adverse Event Reporting System (FAERS) since its approval in 2016 by data mining. The disproportionality analyses, including four algorithms of reporting odd ratio (ROR), proportional reporting ratio (PRR), bayesian configuration promotion neural network (BCPNN), and multi item gamma poisson shrinker (MGPS), were employed to quantify the signals of VEN-associated AEs. From the FAERS database, a total of 8,379,682 reports were collected during the study period. After removing the duplication, the number of reports with VEN as the primary suspect (PS) was 19,107. The 19,107 cases of AEs involved 27 organ systems, 256 significant PTs which conforming to the four algorithms. Unexpected serious AEs, such as pleural effusion, splenic infarction, atrial fibrillation, skin squamous cell carcinoma, etc., have signals. The median time of occurrence of AEs related to VEN was 31 days (inter quartile range [IQR] 7-131 days), and half of the reported AEs occurred within 1 month after administration. Our research has found new significant AEs signals of VEN, which improved its safety information in real-world after marketing approval, and contributed to its risk control of use in clinic.

Azacitidine plus venetoclax in patients with high-risk myelodysplastic syndromes or chronic myelomonocytic leukaemia: phase 1 results of a single-centre, dose-escalation, dose-expansion, phase 1–2 study

Therapies beyond hypomethylating agents such as azacitidine are needed in high-risk myelodysplastic syndromes. Venetoclax is an orally bioavailable small molecule BCL-2 inhibitor that is synergistic with hypomethylating agents. We therefore aimed to evaluate the safety, tolerability, and preliminary activity of azacitidine combined with venetoclax for treatment-naive and relapsed or refractory high-risk myelodysplastic syndromes or chronic myelomonocytic leukaemia. We did a single centre, dose-escalation, dose-expansion, phase 1-2 trial at the University of Texas MD Anderson Cancer Center (Houston, TX, USA). This Article details the phase 1 results. We enrolled patients (≥18 years) with treatment-naive or relapsed or refractory high-risk myelodysplastic syndromes or chronic myelomonocytic leukaemia and bone marrow blasts of more than 5%. No specific Eastern Cooperative Oncology Group status restriction was used. Patients were treated with intravenous or subcutaneous azacitidine (75 mg/m2) for 5 days and oral venetoclax (100-400 mg) for 7-14 days. The primary outcome was safety and tolerability as well as determination of the maximum tolerated dose and recommended phase 2 dose of the azacitidine and venetoclax combination using a 3 + 3 study design. All patients who received one dose of study drug were included in the analyses. This study is registered with ClinicalTrials.gov, number NCT04160052. The phase 2 dose-expansion part of the trial is ongoing. Between Nov 12, 2019, and Dec 17, 2021, a total of 23 patients were enrolled in the phase 1 portion of this study (17 [74%] hypomethylating agent naive and six [26%] post-hypomethylating agent failure). 18 (78%) patients were male and five (22%) were female; 21 (91%) were white and two (9%) were Asian. Median follow-up was 13·2 months (IQR 6·8-18·3). The maximum tolerated dose was not reached and the recommended phase 2 dose was established as azacitidine 75 mg/m2 for 5 days plus venetoclax 400 mg for 14 days. The most common grade 3-4 treatment-emergent adverse events were neutropenia (nine [39%] of 23), thrombocytopenia (nine [39%]), lung infection (seven [30%]), and febrile neutropenia (four [17%]). Three deaths due to sepsis, which were not deemed treatment-related, occurred on the study drugs. The overall response rate was 87% (95% CI 66-97; 20 of 23 patients). Azacitidine with venetoclax is safe and shows encouraging activity in patients with high-risk myelodysplastic syndromes or chronic myelomonocytic leukaemia. MD Anderson Cancer Center.

P499: THE PHASE 1B OMNIVERSE TRIAL OF ORAL AZACITIDINE IN COMBINATION WITH VENETOCLAX FOR TREATMENT OF RELAPSED/REFRACTORY OR NEWLY DIAGNOSED ACUTE MYELOID LEUKEMIA (TRIAL IN PROGRESS)

Background: For older patients (pts) with acute myeloid leukemia (AML) who are ineligible for standard intensive chemotherapy (IC) regimens, traditional lower-intensity AML treatment (Tx) options include low-dose cytarabine (LDAC) or hypomethylating agents (HMAs; azacitidine [AZA] and decitabine), which are generally well tolerated but associated with suboptimal outcomes vs IC [Vey 2020]. Therefore, there remains a need for less toxic and more efficacious lower-intensity AML Tx regimens, especially agents that can be administered in the outpatient setting, which may increase convenience and reduce resource utilization. Venetoclax (VEN) is an oral, selective, small-molecule BCL2 inhibitor that has demonstrated considerable activity when combined with LDAC or HMAs in older pts with AML. In a recent phase 3 trial, VEN + injectable AZA significantly improved response and survival vs AZA alone in IC-ineligible pts with newly diagnosed (ND) AML [DiNardo 2020]. In the USA, VEN is approved in combination with an HMA or LDAC for Tx of pts with ND AML ≥ 75 years (y) of age or who cannot use IC due to comorbidities. Oral-AZA (CC-486) is approved for pts with AML in first remission (CR1) after IC who are ineligible for transplant or other curative therapies. In the randomized, phase 3 QUAZAR AML-001 trial of Oral-AZA in pts with AML in CR1 after IC, Oral-AZA 300 mg QD for 14 days (d)/28d Tx cycle was generally well tolerated and associated with significantly improved overall survival vs placebo. Gastrointestinal symptoms were the most common adverse events reported with Oral-AZA [Wei 2020]. Oral-AZA has also shown clinical activity in pts with active AML [Savona 2015]. As incorporation of AZA into DNA is S-phase-restricted, extended Oral-AZA dosing regimens (> 7d/28d Tx cycle) increase drug incorporation into cycling tumor cells to prolong epigenetic activity throughout the Tx cycle [Laille 2015]. An all-oral VEN + Oral-AZA combination regimen allows for outpatient administration and thus improves pt adherence [Eek 2016]. Aims: Describe the study design and objectives of the OMNIVERSE trial of Oral-AZA + VEN in older pts with ND AML or pts with AML relapsed/refractory (R/R) to prior Tx. Methods: OMNIVERSE (NCT04887857) is a multicenter, open-label, 2-part phase 1b trial (Figure). The key objectives of the trial are to assess safety and determine the maximum tolerated dose of Oral-AZA + VEN in pts with R/R AML (WHO 2016 criteria) ineligible to receive further IC (part I), and then in pts with ND AML ≥ 75 y of age, or those ≥ 18–74 y of age ineligible for IC or HSCT due to comorbidities (part 2). Main eligibility criteria include an ECOG performance status of 0–2 (ECOG 3 is allowed for pts ≥ 18–74 y of age with comorbidities) and an unfavorable cytogenetic risk profile for pts with ND AML. The initial dose of Oral-AZA is 300 mg QD × 14d/28d cycle, with de-escalation to 200 mg × 14d/28d cycle allowed for dose-limiting toxicities. VEN 400 mg is taken orally QD in continuous 28d cycles (or for 21d/cycle for dose level –2). A modified toxicity probability interval-2 design is used to evaluate the planned dose levels. The sample size is dependent on the dose levels included in the study (≤ 18 pts/part). Results: N/A Image:Summary/Conclusion: Study enrollment began in 2021. The trial is ongoing at clinical sites in the United States and Australia.

P757: RESULTS OF A PHASE 1 STUDY OF AZACITIDINE COMBINED WITH VENETOCLAX FOR TREATMENT-NAIVE AND RELAPSED HIGH-RISK MYELODYSPLASTIC SYNDROME AND CHRONIC MYELOMONOCYTIC LEUKEMIA

Background: Patients (pts) with higher-risk myelodysplastic syndromes (HR-MDS) who are ineligible for allogeneic stem cell transplant (SCT) have limited treatment options beyond the hypomethylating agents (HMAs) azacitidine (AZA) and decitabine (DAC). Responses to HMA monotherapy are time-limited and prognosis after HMA-failure is dismal. Venetoclax (VEN) is an oral small molecule BCL-2 inhibitor with the potential to prolong/deepen responses when combined with an HMA. Aims: To evaluate the safety/tolerability, recommended phase 2 dose (RP2D), and preliminary efficacy of VEN in combination with AZA for the treatment of HR-MDS/CMML. Methods: This was a single-center dose-escalation phase 1 study with “3 + 3” design. Key inclusion criteria were a diagnosis of MDS or CMML by WHO 2016, age ≥ 18 yrs, int-2 or high risk by IPSS, and bone marrow (BM) blasts of 5-19%. Pts could be treatment-naïve or relapsed/refractory, defined as failure after 4+ cycles of HMA. Prior VEN exposure was not permitted. Treatment consisted of AZA 75 mg/m2 on D1-5 and escalating doses of VEN on D1-7 or 1-14 (Table 1) every 28D. VEN dosing was adjusted for pts on concomitant CYP3A inhibitors. The primary objective was safety/tolerability and determination of the RP2D. Secondary objectives were response rates, overall survival (OS), and progression-free survival (PFS). Adverse events (AEs) were graded as per the CTCAE v5.0 and responses assessed using the modified IWG 2006 criteria. All pts provided informed consent. The study was registered on ClinicalTrials.gov (NCT04160052). Results: Data cutoff was February 19th, 2022. 23 pts (13 HMA-naïve MDS, 4 HMA-naïve CMML, 4 HMA-failure MDS, 2 HMA-failure CMML) were enrolled. The median age was 68 years (range 58-84) and median BM blasts 11% (range 6-19%). IPSS was int-2 in 78% of pts and high in 22% of pts. 8/23 (35%) had complex cytogenetics, 6/23 (26%) had TP53 mutations, and 5/23 (22%) had therapy-related MDS/CMML. A single dose-limiting toxicity (DLT) occurred at dose level +2 (BM aplasia and delayed count recovery). The maximum tolerated dose (MTD) was not reached. The most common grade 3/4 AEs were neutropenia (39%), thrombocytopenia (39%), anemia (13%), and infections: pneumonia (30%), neutropenic fever (17%), diverticulitis (9%), sepsis (9%), cellulitis (4%), and splenic abscess (4%). The RP2D was established at AZA 75 mg/m2 on D1-5 plus VEN 400 mg on D1-14. 3 deaths occurred on study, all from sepsis. 30 and 60-day mortality were 4% and 9%, respectively. Other reasons for discontinuing therapy included progression of MDS/CMML (n=6), transformation to AML (n=4), SCT (n=4), and social reasons (n=1). The ORR by intention-to-treat (ITT) analysis was 82% (14/17; 3 CR, 5 mCR+HI, 6 mCR) in the HMA-naïve cohort and 100% (6/6; 6 mCR) in the HMA-failure cohort. A median of 3 cycles (range 1-11) were given. Responses occurred after a median of 1 cycle (range 1-2) and lasted a median of 4.5m (range 1.2m to 21.2m). At a median follow-up of 13.2m, median OS was not reached (1-year OS 68.6%) in the HMA-naïve cohort and 8.3m (1-year OS 22%) in the HMA-failure cohort. Median PFS was 13.1m (1-year PFS 51.5%) in the HMA-naïve cohort and 6.8m (1-year PFS 0%) in the HMA-failure cohort. Image:Summary/Conclusion: AZA combined with VEN is tolerable and yields a high ORR in HR-MDS/CMML, with or without prior HMA. Rates of myelosuppression and infection are high and careful monitoring is required. The RP2D is AZA 75 mg/m2 on D1-5 plus VEN 400 mg on D1-14, every 28D. The phase 2 dose expansion component of this study is ongoing.

Navitoclax plus ruxolitinib in JAK inhibitor-naive patients with myelofibrosis: Preliminary safety and efficacy in a multicenter, open-label phase 2 study.

7015 Background: Ruxolitinib (RUX), a Janus kinase (JAK) 1/2 inhibitor, is the current standard of care for patients (pts) with myelofibrosis (MF) that improves splenomegaly and disease symptoms with limited impact on disease biology. Many pts lose response over time, highlighting an unmet need for novel therapies. Navitoclax (NAV) is an oral, small-molecule inhibitor of BCL-XL and BCL-2 that has a synergistic effect when used in combination with JAK inhibitors to enhance apoptosis. This ongoing, open-label, multicenter, phase 2 trial (NCT03222609) is evaluating the efficacy and safety of NAV with/without RUX in pts with MF. Here, we report results from JAK inhibitor-naïve pts treated with NAV+RUX. Methods: Enrolled pts had primary or secondary MF with splenomegaly (DIPSS ≥INT-1) and did not receive prior JAK-2 therapy or bromodomain and extraterminal motif (BET) inhibitors. Pts initiated NAV at 100 mg QD or 200 mg QD if baseline (BL) platelet count was ≤150 × 109/L or >150 × 109/L, respectively. RUX was given BID with starting dose based on BL platelet count per local label. The primary endpoint was spleen volume reduction of ≥35% (SVR35) from BL at wk 24. Key secondary endpoints were ≥50% reduction in total symptom score (TSS50), bone marrow (BM) fibrosis reduction, and anemia response. Adverse events (AEs) were monitored throughout the study. Results: As of Oct 04, 2021, 32 pts received NAV+RUX. Median duration of f/u was 6.1 (range, 1.9 ─ 18.6) mos. 28 (88%) pts received NAV 200 mg and 4 (13%) received 100 mg OD. Median age was 69 (44 ─ 83) yrs, and median spleen volume was 1889.08 cm3 (645.6 ─ 7339.6). Median NAV and RUX exposures were 24.1 (5.1 ─ 80.9) and 20.1 (0.1 ─ 80.1) wks, respectively. 31 (97%) pts reported ≥1 AE (Grade ≥3 AEs, 25 [78%]; serious AEs, 6 [19%]). Most common Grade ≥3 AEs were anemia (34%), thrombocytopenia (31%), and neutropenia (19%). 3 (9%) and 2 (6%) pts reported an AE leading to NAV and RUX discontinuation, respectively, and 2 (6%; 1 PD, 1 cardiac disorder unrelated to NAV) AEs led to death ≤30 days after last NAV dose. SVR35 was achieved by 52% of evaluable pts at wk 24 (SVR35 in INT-2, 50%; HR, 33%) and by 76% at any time on treatment (Table). Median time to first SVR35 was 12.1 (11 ─ 47) wks. Conclusions: The combination of NAV+RUX was well tolerated and demonstrated early and robust reductions in spleen volume, anemia, and BM fibrosis in pts without prior JAK-2 inhibitor exposure. SVR35, TSS50, and BM fibrosis improved over time. Clinical trial information: NCT03222609. [Table: see text]

Efficacy and safety of enasidenib and azacitidine combination in patients with IDH2 mutated acute myeloid leukemia and not eligible for intensive chemotherapy

Preclinically, enasidenib and azacitidine (ENA + AZA) synergistically enhance cell differentiation, and venetoclax (VEN), a small molecule Bcl2 inhibitor (i) is particularly effective in IDH2 mutated acute myeloid leukemia (IDH2mutAML). This open label phase II trial enrolled patients (pts) with documented IDH2mutAML. All patients received AZA 75 mg/m2/d x 7 d/cycle and ENA 100 mg QD continuously. Concomitant Bcl2i and FLT3i were allowed (NCT03683433).Twenty-six pts received ENA + AZA (median 68 years, range, 24–88); 7 newly diagnosed (ND) and 19 relapsed/refractory (R/R). In R/R AML patients, three had received prior ENA and none had received prior VEN. The composite complete remission rate (CRc) [complete remission (CR) or complete remission with incomplete hematologic recovery (CRi)] was 100% in ND AML, and 58% in R/R AML. Median OS was not reached in ND AML with median follow-up of 13.1 months (mo); Pts treated in first relapse had improved OS than those with ≥2 relapse (median OS not reached vs 5.2 mo; HR 0.24, 95% CI 0.07–0.79, p = 0.04). Two patients received ENA + AZA with a concomitant FLT3i, one responding ND AML patient and one nonresponding R/R AML patient. Seven R/R AML pts received ENA + AZA + VEN triplet, and with median follow up of 11.2 mo, median OS was not reached and 6-mo OS was 70%. The most frequent treatment-emergent adverse events include febrile neutropenia (23%). Adverse events of special interest included all-grade IDH differentiation syndrome (8%) and indirect hyperbilirubinemia (35%). ENA + AZA was a well-tolerated, and effective therapy for elderly pts with IDH2mut ND AML as well as pts with R/R AML. The addition of VEN to ENA + AZA appears to improve outcomes in R/R IDH2mutAML.Clinical trial registration information: https://clinicaltrials.gov/.NCT03683433

LOR-253 Overcomes Resistance to ABT-199 by Targeting MTF1 in AML

Introduction: Chemoresistance is one of the major challenges for the acute myeloid leukemia (AML) treatment. Venetoclax (ABT-199), a selective small molecule BCL-2 inhibitor, is being clinically vetted and is an effective therapy for some B-cell lymphomas, yet many patients who initially respond to ABT-199 develop resistance. Thus, enhancing the sensitivity of resistant cells to chemotherapy is a great interest to clinical trial. Method: The resistant cell lines were generated by culturing in the medium containing ABT-199. CCK8 analysis was used to detect the cell viability. Flowcytometric analysis with Annexin-V/PI was used to test the apoptosis. CRISPR/Cas9 by lentivirus delivering well-validated shRNAs in pLKO.1 vector was used to knockout the expression of MTF1. Western blot with the antibodies was used to determine the expression of the molecules. Clonogenic growth assay was used to determine the growth of parental and DTEP cells. Results: Here we report that resistance to the BCL-2 targeting drug ABT-199 in AML cell lines evolves from outgrowth of persister clones harbor BCL2. Furthermore, persister status is generated via adaptive super-enhancer remodeling that reprograms transcription and offers opportunities for overcoming ABT-199 resistance. Notably, pharmacogenomic screens revealed that persisters are vulnerable to inhibition of metal regulatory transcription factor 1 (MTF1), which is essential for the transcriptional reprogramming that drives and sustains ABT-199 resistance. Conclusion: LOR-253, which is a MTF1-targeting agent, add novel insights to overcome ABT-199-resistance in B-cell lymphomas.

Characterization of LP-118, a Novel Small Molecule Inhibitor of Bcl-2 and Bcl-Xl in Chronic Lymphocytic Leukemia Resistant to Venetoclax

Chronic lymphocytic leukemia (CLL) is the most common form of adult leukemia, which is characterized by the accumulation of mature CD19+CD5+ B cells that evade apoptosis by upregulating anti-apoptotic BH3 protein, B cell lymphoma protein 2 (Bcl-2). Venetoclax, a first-in-class Bcl-2 inhibitor has transformed therapy for CLL. However, with continuous administration of venetoclax, patients often acquire resistance via mutations at or near the drug binding pocket at G101 in Bcl-2 (Blombery et al, Cancer Discov 2019, Lucas et al, Blood, 2020). In some patients, the acquisition of a mutation in tumor suppressor protein TP53 or the upregulation of anti-apoptotic family members including Bcl-xL has been shown to drive resistance to venetoclax. In particular, a switch to Bcl-xL dependency provides the rationale for dual inhibition of Bcl-2/-xL.We performed BH3 profiling on treatment naïve and venetoclax relapsed/refractory (R/R) primary CLL cells to investigate the dependency of several anti-apoptotic proteins and their contributions to cell survival. Primary CLL cells were incubated with Bcl-xL specific HRK peptides, Mcl-1 specific MS-1 peptides, Bcl-2 interacting BAD peptides, or BIM peptides which interact with all anti-apoptotic members. We observed that the majority of these cells responded to both BIM and BAD peptides indicating the dependency on anti-apoptotic protein Bcl-2. However, in some samples, including venetoclax R/R primary CLL cells, showed dependency on other anti-apoptotic proteins including Bcl-xL as indicated by a depolarization from the HRK peptide.We subsequently performed pre-clinical investigations of LP-118, a novel small molecule inhibitor of Bcl-2, more potent than venetoclax and with added advantage of selectively binding to Bcl-xL while minimizing the platelet toxicity. LP-118 was rationally designed to have an enzymatic IC 50 for Bcl-xL at 10.1 nM which was in-between venetoclax (62.2 nM) and navitoclax (2.9 nM) to prevent the on-target effects of platelet toxicity. Since most venetoclax- R/R patients have mutation in Bcl-2 and/or have increased dependency on Bcl-xL, we hypothesized that these patients would benefit from the additional inhibition of Bcl-xL.To test this hypothesis, treatment naïve and venetoclax R/R primary CLL cells were incubated with LP-118, venetoclax, or navitoclax continuously for 18 hours at concentrations ranging from 0.1 nM to 10 nM. Apoptosis was measured through Annexin V/Tetramethylrhodamine methyl ester perchlorate (TMRM) staining followed by flow cytometry. We found that treatment naive CLL cells were more sensitive to LP-118 than venetoclax and navitoclax with IC 50 values at 0.5nM, 10 nM, and >10 nM, respectively. Venetoclax R/R CLL cells were 50% killed by 1 nM LP-118 and were not sensitive to either venetoclax or navitoclax. To investigate the mechanism of LP-118 induced cell death, we used intracellular flow cytometry and observed pore-forming pro-apoptotic protein Bak transforming to an active conformation as early as 8 hours after treatment in venetoclax R/R and treatment naive CLL cells. Furthermore, after 12 hours of treatment we observed a release of cytochrome c indicating that cells were going through apoptosis.To determine whether cells harboring the Bcl-2 Gly101Val mutation respond to LP-118, we transfected RS4; 11 cells with plasmids containing the mutation. We treated these mutant cells for 72 hours and found that while these mutant cells were not sensitive to venetoclax, they responded to LP-118 with IC 50 of 20 nM. Furthermore, LP-118 induced quicker and earlier bak transformation and cytochrome C release in these mutant cells than venetoclax. To further evaluate the efficacy of LP-118, we tested this compound in a RS4; 11 xenograft model. We treated mice daily via oral gavage and observe that 100 mpk of LP-118 significantly decreases overall tumor growth and increases survival compared to 100 mpk of venetoclax (p=0.0004).Collectively, our in vitro studies demonstrated preclinical efficacies of LP-118 in primary CLL samples and Gly101Val mutant cells. LP-118 is highly potent in venetoclax resistant patient samples and cell lines. Additional in vivo work has further confirmed the efficacy of LP-118 in mutant cell lines. This work justifies continued preclinical and clinical work with this agent, and a phase 1 first in human study will begin soon in patients with relapsed hematologic malignancies (NCT04771572). DisclosuresTan: Guangzhou Lupeng Pharmaceutical Company Ltd.,: Current Employment. Chen: Newave Pharmaceutical Inc.: Current Employment. Anthony: Newave Pharmaceutical Inc.: Current Employment. Chen: Newave Pharmaceutical Inc.: Current Employment. Shen: Guangzhou Lupeng Pharmaceutical Company Co. Ltd.,: Current Employment. Byrd: Newave: Membership on an entity's Board of Directors or advisory committees; Vincerx Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Novartis, Trillium, Astellas, AstraZeneca, Pharmacyclics, Syndax: Consultancy, Honoraria. Woyach: AbbVie Inc, ArQule Inc, AstraZeneca Pharmaceuticals LP, Janssen Biotech Inc, Pharmacyclics LLC, an AbbVie Company,: Consultancy; AbbVie Inc, ArQule Inc, Janssen Biotech Inc, AstraZeneca, Beigene: Other: Advisory Committee; AbbVie Inc, Loxo Oncology Inc, a wholly owned subsidiary of Eli Lilly & Company: Research Funding; Gilead Sciences Inc: Other: Data & Safety.

Identifying Mechanisms Associated with Venetoclax Resistance in Multiple Myeloma (MM)

Venetoclax (VTX) is a selective small-molecule inhibitor of BCL-2 that exhibits antitumoral activity against MM cells presenting lymphoid features and those with translocation t(11;14). Despite its impressive clinical activity, VTX therapy for a prolonged duration can lead to drug resistance. Therefore, it is important to understand the underlying mechanisms of resistance in order to develop strategies to prevent or overcome resistance. In the present study, we established four VTX resistant human myeloma cell lines (HMCLs) from four sensitive HMCLs, including three with t(11;14), in culture with a stepwise increase in treatment dose with VTX. To identify the molecular basis of acquired VTX resistance, whole exon sequencing (WES), mRNA-sequencing (mRNAseq), and protein expression assays were performed in the four isogenic VTX-sensitive/resistant HMCLs and three MM patients with samples collected before VTX administration and after clinical resistance to the drug. Compared with sensitive cell lines and patient samples collected before VTX administration, mRNAseq analysis identified downregulation of BIM and upregulation of BCLXL in both resistant cell lines and MM cells from relapse patients. Other transcriptional changes detected included upregulation of AURKA, BIRC3, BIRC5, and IL32. Enrichment analysis of differentially expressed genes suggested involvement of PI3K and MAPK signaling, likely associated with cytokines, growth factors (EGF, FGF and IGF family members), and receptor tyrosine kinase (EGF and FGF). Western blot analysis was performed to compare BCL2 family expression in resistant cell lines versus sensitive cell lines and it showed upregulation of BCL2 survival members (such as MCL-1 and BCLXL), and downregulation of pro-apoptotic BH3 members (such as BIM and PUMA). BIM expression was completely lost in one resistant cell line, and introduction of exogenous BIM into this cell line enhanced VTX sensitivity. Interestingly, BCL2 was upregulated in some resistant cell lines generated after a long-term treatment with VTX, suggesting BCL2 expression level may not be suitable as a marker of VTX sensitivity for acquired resistance. Unlike in CLL, BCL2 mutations were not identified through WES in any resistant cell lines or primary patient sample harvested after relapse. While 8 genes were mutated in two resistant samples , no clear mutational pattern emerged .Based on the above, we further tested some specific inhibitors in in vitro or ex vivo cell models to help understanding resistant mechanism and identify strategies to overcome VTX resistance. We found that inhibition of MCL-1, with the compound S68345, substantially enhanced VTX sensitivity in three resistant HMCLs and in primary cells from one relapsed MM patient. A BCLXL inhibitor (A155463) only significantly enhanced VTX sensitivity in one resistant cell line after co-treatment with VTX. Co-treatment of the other three resistant cell lines with VTX, S68345 and A155463 resulted in the most synergistic anti-myeloma activity, suggesting those cell lines are co-dependent on MCL-1, BCLXL, and BCL2 for survival, although they are more dependent on MCL-1. We also found that inhibition of PI3K signaling, IGF1, RTK (EGF and FGF) and AURKA significantly increased VTX sensitivity, partially through downregulation of MCL-1, and BCLXL, and upregulation of BIM. Conventional anti-MM drugs such as dexamethasone, bortezomib and lenalidomide, were shown to have little activity on augmenting VTX sensitivity in most resistant cell lines.In summary, we find that acquired resistance to VTX in MM is largely associated with BCL2 family regulation, including upregulation of survival members such as MCL-1, BCLXL, BCL2, and downregulation of pro-apoptotic members, especially BIM. Our study also indicates that upstream signaling involved in BCL2 family regulation during acquired resistance is likely related to cytokine, growth factor, and/or RTK-induced cell signaling such as PI3K. Co-inhibition of MCL-1, or BCLXL, as well as the upstream PI3K, RTK (FGF and EGF), IGF-1 mediated signaling were effective in overcoming VTX resistance. DisclosuresFonseca: Mayo Clinic in Arizona: Current Employment; Amgen: Consultancy; BMS: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Bayer: Consultancy; Janssen: Consultancy; Novartis: Consultancy; Pharmacyclics: Consultancy; Sanofi: Consultancy; Merck: Consultancy; Juno: Consultancy; Kite: Consultancy; Aduro: Consultancy; OncoTracker: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy; AbbVie: Consultancy; Patent: Prognosticaton of myeloma via FISH: Patents & Royalties; Scientific Advisory Board: Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Caris Life Sciences: Membership on an entity's Board of Directors or advisory committees.