Bcl-2 Antagonist Research Articles

Combinatorial Therapy of CDK9 Inhibitor with CD19 CAR-T to Reciprocally Overcome Therapy Resistance and Enhance Treatment Efficacies Against Aggressive B-Cell Lymphomas

Diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) are subgroups of aggressive B-cell lymphoma associated with unacceptably high rates of refractoriness to intensive treatment and significantly shortened survival in this setting. In addition to standard therapies, modern lymphoma care has incorporated targeted agents including Bruton's tyrosine kinase inhibitors and BCL-2 antagonists. Despite achieving initial clinical responses, many patients eventually succumb to disease relapse following treatment with these novel agents. Meanwhile, chimeric-antigen receptor (CAR) T cell therapies have produced unprecedented initial responses in patients with relapsed or refractory DLBCL and MCL, yet at follow-up, only 40% of patients are able to sustain durable remissions. Thus, there is an urgent need to improve the duration of response and delay or prevent CAR-T therapy resistance. To this end, we previously performed mechanistic and translational studies aimed at developing CDK9 as a novel therapeutic target in DLBCL and MCL. This work involved the interrogation of gene expression and functional screening of a large panel of primary patient samples and lymphoma cell lines, ultimately revealing an essential transcription state characterized by increased RNA polymerase II-mediated transcription as the primary driver of therapy resistance evolution and fatal disease progression. Although clinical trials of CDK9 inhibitors (CDK9i) in B-cell lymphomas have shown promise in drug and CAR-T resistant lymphoma patients, mono-targeting of CDK9 has encountered clinical challenges, particularly the development of resistance in initial responders. Thus, understanding CDK9i resistance evolution mechanisms and identifying ways to target the key resistance-associated molecular determinants and subpopulation(s) of cells capable of giving rise to resistant disease holds great potential for advancing lymphoma therapy. Here, we extend our previous work with CDK9i to identify the underlying molecular determinants of CDK9i resistance and, ultimately, to design rational combination therapies to enhance and sustain CDK9i efficacy. In doing so, we are able to demonstrate that: 1) DLBCL and MCL are exquisitely sensitive to CDK9 inhibition (NVP2/AZD4573) regardless of genetic background or resistance status, 2) populations of CDK9i (NVP2/AZD4573) treatment-induced drug tolerant persister (DTP) cells are responsible for driving therapy resistance evolution, 3) CDK9i (NVP2/AZD4573) treatment induces an immunogenic response via activation of proinflammatory and IFN pathways ex vivo and in vivo, and 4) CDK9i and CD19 CAR-T therapies exhibit reciprocally enhanced efficacy in a manner that can overcome therapy resistance in DLBCL and MCL. The outcomes of this study have broad applicability across B-cell malignancies for eradicating both targeted and CAR-T therapy resistance, which we anticipate can be readily translated to the clinic and have immediate impact on DLBCL/MCL patient care.

Targeting the tumor microenvironment for treating double-refractory chronic lymphocytic leukemia

AbstractThe introduction of BTK inhibitors and BCL2 antagonists to the treatment of chronic lymphocytic leukemia (CLL) has revolutionized therapy and improved patient outcomes. These agents have replaced chemoimmunotherapy as standard of care. Despite this progress, a new group of patients is currently emerging, which has become refractory or intolerant to both classes of agents, creating an unmet medical need. Here, we propose that the targeted modulation of the tumor microenvironment provides new therapeutic options for this group of double-refractory patients. Furthermore, we outline a sequential strategy for tumor microenvironment-directed combination therapies in CLL that can be tested in clinical protocols.

Steroid-free combination of 5-azacytidine and venetoclax for the treatment of multiple myeloma.

Multiple myeloma (MM) is an incurable plasma cell malignancy that, despite an unprecedented increase in overall survival, lacks truly risk-adapted or targeted treatments. A proportion of patients with MM depend on BCL-2 for survival, and, recently, the BCL-2 antagonist venetoclax has shown clinical efficacy and safety in t(11;14) and BCL-2 overexpressing MM. However, only a small proportion of MM patients rely on BCL-2 (approx. 20%), and there is a need to broaden the patient population outside of t(11;14) that can be treated with venetoclax. Therefore, we took an unbiased screening approach and screened epigenetic modifiers to enhance venetoclax sensitivity in 2 non-BCL-2 dependent MM cell lines. The demethylase inhibitor 5-azacytidine was one of the lead hits from the screen, and the enhanced cell killing of the combination was confirmed in additional MM cell lines. Using dynamic BH3 profiling and immunoprecipitations, we identified the potential mechanism of synergy is due to increased NOXA expression, through the integrated stress response. Knockdown of PMAIP1 or PKR partially rescues cell death of the venetoclax and 5-azacytidine combination treatment. The addition of a steroid to the combination treatment did not enhance the cell death, and, interestingly, we found enhanced death of the immune cells with steroid addition, suggesting that a steroid-sparing regimen may be more beneficial in MM. Lastly, we show for the first time in primary MM patient samples that 5-azacytidine enhances the response to venetoclax ex vivo across diverse anti-apoptotic dependencies (BCL-2 or MCL-1) and diverse cytogenetic backgrounds. Overall, our data identify 5-azacytidine and venetoclax as an effective treatment combination, which could be a tolerable steroid-sparing regimen, particularly for elderly MM patients.

Venetoclax with decitabine or azacitidine in relapsed or refractory acute myeloid leukemia.

Relapsed or refractory acute myeloid leukemia (AML) is associated with poor outcomes and resistance to therapy. The addition of venetoclax, a BCL-2 antagonist, to lower-intensity therapies results in improved survival in the first-line setting compared to monotherapy with a hypomethylating agent or low-dose cytarabine. Despite this, much remains unknown about the performance of venetoclax with a hypomethylating agent following the first-line setting. Additionally, while the ELN 2022 guidelines appear to improve the prognostication of AML, clarification is needed to determine how the revision applies to lower-intensity strategies. To investigate this, we retrospectively analyzed the performance of venetoclax with decitabine or azacitidine in relapsed or refractory AML under the ELN 2022 guidelines. We demonstrated that the ELN 2022 revision is not optimized for lower-intensity venetoclax-based strategies. To refine the prognostication schema, we showed significantly improved response and survival benefits for patients with mutated NPM1 and IDH. Relatively, patients with mutated NRAS, KRAS, and FLT3-ITD were associated with inferior response and survival. Furthermore, there is an unmet clinical need for tools to improve the selection of lower-intensity therapy candidates with borderline functional status. Using an incremental survival computation method, we discovered that a CCI score threshold of 5 distinguishes patients at an elevated risk of death. Together, these novel findings highlight areas of refinement to improve survival in relapsed or refractory AML.

Waldenström Macroglobulinemia: Targeted Agents Taking Center Stage.

With the worldwide approval of the oral covalent Bruton tyrosine kinase (BTK) inhibitors ibrutinib and zanubrutinib for treating patients with Waldenström macroglobulinemia (WM), targeted agents have certainly taken center stage in the therapeutic landscape of WM. This review discusses the biological and clinical data supporting current and up-and-coming targeted agents in WM. Bruton tyrosine kinase inhibitors induce fast, deep, and durable responses in patients with WM, comparable to chemoimmunotherapy; however, there is a glaring absence of comparative studies between these regimens. The high response and progression-free survival rate and the ease of administration of BTK inhibitors must be balanced against their specific adverse-event profile with unique toxicity (e.g., bleeding and cardiac arrhythmia) and the indefinite duration of the therapy. Novel targeted agents of interest include BCL2 antagonists (e.g., venetoclax and sonrotoclax) and non-covalent BTK inhibitors (e.g., pirtobrutinib and nemtabrutinib), among others. The therapeutic landscape of patients with WM will benefit from the robust participation of patients in clinical trials.

Utilizing Chemotherapy to Enhance the Anti-Tumor Properties of Ex Vivo Expanded Gamma Delta (γδ) T Cells Against Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is the second most common type of blood cancer in children with a high rate of treatment failure and aggressive relapse. While allogeneic HSCT offers a cure, limitations arise in achieving remission prior to transplant. Immunotherapy-based approaches can induce remission, gaining interest in AML. Here, we explore the use of gamma delta (γδ) T cells as a promising tool to fulfill this unmet need. Unlike predominant alpha beta (αβ) T cells, γδ T cells do not require antigen presentation and identify their targets in a major histocompatibility complex (MHC) independent manner. Allogeneic γδ T cells thus have minimal risk of causing graft-versus-host disease (GvHD), creating ‘off-the-shelf’ cellular therapeutics. This would be ideal to target AML, especially given the aggressive nature of relapsed disease. Cytotoxic mechanisms used by γδ T cells include recognition of cellular stress molecules such as NKG2D and DNAM-1 receptor ligands on target cells, activation of death receptor pathway through Fas-FasL and TRAIL-R interactions, as well as γδ ΤCR mediated killing through recognition of butyrophilins (BTNs) on target cells. These stress molecules are expressed on AML cells and can be further upregulated through chemotherapeutic agents such as azacitidine (a hypomethylating agent) and venetoclax (BCL-2 antagonist), drugs currently in use to treat relapsed AML. Here, we tested these drugs in combination with γδ T cells to target AML. Methods Our studies explored four different AML cell lines (Kasumi-1, Nomo-1, MV4-11 and MOLM13). Each one was respectively treated at their corresponding inhibitory concentration 25 (IC 25) and 50 (IC 50) of azacitidine and/or venetoclax for 24 hours. After treatment, AML cells were assessed by flow cytometry for expression of NKG2D and DNAM-1 ligands, death receptor pathway molecules (CD95, TRAIL-R1/R2) as well as the butyrophillins (BTN2A and BTN3A1) to elucidate which pathways are over-expressed, aiding in γδ T cell targeting and cytotoxicity. Next, we co-cultured AML cells pre-treated with azacitidine and/or venetoclax with γδ T cells and measured cytotoxicity using a flow cytometry-based assay and a bioluminescence assay. Results: Each cell line expressed a unique pattern of markers at baseline. Cell lines treated with azacitidine had a robust expression of the NKG2D ligands ULBP1, ULBP2/5/6 and ULBP3. Azacitidine also increased CD155 and TRAIL-R2 in Nomo-1 while CD112 increased in MOLM13. Most venetoclax-treated AML cells over-expressed BTN2A and BTN3A1 along with CD95 in MOLM13. Cell lines were then treated with a combination of both drugs. Dual treatment of cells resulted in a significant increase in expression of most NKG2D markers as well the death receptor Fas (CD95). Additionally, increase in CD155 as well as BTNs was seen in several cell lines. Importantly, azacitidine and venetoclax had minimal effects on the viability of γδ Τ cells when treated for 24 hours. In vitro cytotoxicity studies indicate increased cell death across multiple AML cell lines when γδ T cells were co-cultured with cells pretreated with azacitidine and venetoclax. Conclusions: Azacitidine and venetoclax treatment resulted in upregulation of several markers involved in γδ Τ cell-mediated cytotoxicity. Ex vivo expanded γδ Τ cells combined with chemotherapy-treated AML cells resulted in increased overall cytotoxicity, which is likely both additive and synergistic. Our next steps include preclinical validation of this combinatorial approach in an in-vivo mouse study.

Targeting Mitochondrial Apoptotic Priming State to Personalize Therapy for Relapsed Acute Myeloid Leukemia

Despite of remarkable changes in therapeutic landscape of AML in past 5 years, relapsed AML patients still succumb to disease. In majority of patients, the common cause of death ultimately is the emergence multi-drug resistance at relapse. Using patient-derived xenograft (PDX) as our models, we investigated the basis of multi-drug resistance and methods for identifying drug sensitivity in relapsed AML. We hypothesized that there is differential activation of drug-induced mitochondrial apoptotic signaling between treatment-naïve and relapsed myeloblasts. To study mechanisms of drug resistance in AML, we developed PDX models (n=18 models) of in vivo acquired resistance to single agents (BCL-2/MCL-1 inhibitor, FLT3 inhibitor, SMAC mimetics and BRD-4 inhibitor) of distinct mechanisms, which caused board chemoresistance across all models. We tested whether multidrug resistance is associated with reduction in apoptotic signaling, using BH3 profiling, a method for measurement of mitochondrial sensitivity to apoptosis via exposure to synthetic BH3 domain peptides, on matched pre- and post-resistant PDXs. Using BH3 profiling, we found that a reduction in mitochondrial apoptotic priming consistently accompanied the acquisition of multidrug resistance irrespective of the mechanism of drug action and genetic status of PDX models. Next we created pharmacologic sensitivity landscape for 22 AML PDX models by exposing splenic myeloblasts isolated after confirmed engraftment (>70% circulating hCD45+ blasts) to drug a drug panel (n=40) for 14 hours. We first demonstrate that ex vivo drug-induced mitochondrial sensitivity singling measured via dynamic BH3 profiling (DBP) segregates PDXs according to prior treatment. By using unsupervised hierarchical clustering we found that AML PDXs developed from treatment naïve patients clustered together and showed greater apoptotic priming responses to a larger number of targeted agents than PDXs from R/R patients, which also formed discrete clusters. We replicated drug-response patterns from PDX models in primary and relapsed AML patients by analyzing ex vivo sensitivity responses to >500 drugs across relapse and diagnosis patients (Malani et al, 2021 and Tyner et al, 2018). We show that human AML patients at relapse also broadly lose sensitivity to agents in a similar pan-resistant manner as PDX models. Using unbiased whole transcriptomic analysis of naïve and resistant PDXs (n=78 samples), we found common pathways of resistance where positive enrichment was observed for cytokine-cytokine receptor interactions, xenobiotics biodegradation, metabolic pathways, Hippo signaling, and Ras signaling in resistant compared to treatment-naïve PDXs. Conversely, mechanisms relating to DNA repair and replication, such as base excision repair, mismatch repair, and homologous recombination, were negatively enriched. Moreover, pathways associated with specific drug resistance were also observed. One of the known multi-drug resistance mechanisms to chemotherapy drugs in AML is increased expression of efflux pumps from the superfamily of ATP binding cassette. We found enrichment in ABC transporter family pathway signatures in all 3 PDX models, DFAM-61786, DFAM-15354, and DFAM-61345, resistant to quizartinib, birinapant, and JQ-1 and validated higher drug-efflux activity in quizartinib and birinapant resistant model using calcein-AM efflux assay. We finally tested whether DBP could predict in vivo response of patient myeloblasts in PDX models. We tested in vivo sensitivity to 5 drugs of disparate mechanisms of action: birinapant and LCL-161 (SMAC mimetics), JQ-1 (BRD-4 inhibitor), venetoclax (BCL-2 antagonist), and quizartinib (FLT-3 inhibitor) in 4-9 different PDX models each. Using ex vivo DBP assay, we identified drugs that prime not only treatment naïve PDXs but also R/R PDXs. We identified persistent in vivo activity in a drug-specific manner, with BH3 mimetics and HDAC inhibitors showing overlapping activity across different drug-resistant models. Overall, we demonstrate that acquired resistance to targeted therapy in AML is accompanied by common mechanism of reduction in mitochondrial priming along with drug-specific resistance mechanisms. Further, we find that, even in the context of a multiply-resistant PDX model, DBP can still identify therapeutic vulnerabilities that can be efficaciously exploited in vivo.

Synergistic Growth Inhibition of NPM1 Mutant AML PDX By Combined Therapy with BCL-2 Inhibitor Venetoclax (ABT-199) and Menin Inhibitor DS-1594b In Vivo

Introduction: Mixed-lineage-leukemia (MLL) translocations are prevalent in hematologic malignancies, resulting in aggressive leukemias resistant to standard therapies and low 5-year survival rates. These translocations lead to the production of MLL fusion proteins that promote leukemia development by enhancing proliferation, inhibiting hematopoietic differentiation, and driving leukemogenesis. Acute myeloid leukemia (AML), an aggressive blood cancer with a grim prognosis, particularly in elderly patients, frequently involves mutations in the nucleophosmin (NPM1) gene. Importantly, the interaction between Menin, MLL, and MLL fusion proteins play a crucial role in the pathogenesis of AML, both with MLL rearrangements (MLLr) and in leukemias carrying NPM1 mutations. Inhibition of the Menin-MLL interaction using novel Menin inhibitors has demonstrated efficacy in pre-clinical and clinical studies for both AML subsets. In this study, we investigate the effectiveness of the novel Menin-MLL inhibitor DS-1594b, developed by Daiichi Sankyo Co. Ltd., in combination with the BCL-2 antagonist venetoclax (ABT-199), in a patient-derived xenograft (PDX) model of NPM1-mutated acute leukemia. We have previously reported synergistic lethality and differentiation-inducing effects of this drug combination in both AML cell lines and primary leukemia samples in vitro (Ciaurro et al., Blood 2022). Our findings shed light on the potential of this therapeutic combination for the targeted treatment of AML with NPM1 mutations. Methods: NSG mice (female, 8-10 weeks old) were purchased from Jackson Laboratory. Mice were inoculated via the tail vein with NPM1m PDX/luc/GFP (3×10 6 cells). Once leukemia engraftment was confirmed by bioluminescent imaging mice were randomized in 4 groups: vehicle, ABT199, DS-1594b alone, and combination. After 12 days drugs were administered as follows: DS1594b at a dosage of 50 mg/kg by oral gavage for 4 weeks; ABT-199 at a dosage of 50 mg/kg by oral gavage for 2 weeks and 100mg/kg for the other 2 weeks. NSG mice were euthanized by CO 2 inhalation at the endpoint of the study in accordance with IACUC protocols. BM and spleens were harvested in a sterile environment and flushed in HBSS with 1% FBS. For CyTOF data spleen cells were initially barcoded and stained with metal-conjugated antibodies according to Fluidigm's protocols and samples were analyzed by CyTOF (Helios) Mass Cytometer. Results: New in vitro data confirmed synergistic lethality on additional AML cell lines and demonstrated upregulation of PARP-C and Caspase3-C apoptosis pathways using western blot analysis. Further, additional 3 primary AML patient samples showed additive or synergistic growth inhibition when treated with combination of drugs. In the in vivo experiment with NPM1m PDX/luc/GFP AML PDX model, we found that co-treatment with DS-1594b and venetoclax for four weeks resulted in a greater reduction in AML burden compared to treatment with either agent alone or a vehicle control as shown by Bioluminescence imaging. Moreover, weight of mice in the combo group was not significantly affected, while mice lost weight in vehicle and in single agent cohort due to rapid disease progression. The survival curve clearly demonstrated the benefits of the combination treatment, with the DS-1594b group having modest anti-tumor efficacy, and the combination group showing the greatest efficacy (Fig. 1A). To study the effects on cell differentiation, we used CyTOF technique, which allowed us to analyze a complete panel of 30 markers. We found two populations of cells, which we named populations (pop) 3 and pop 4, that were enriched in the group treated with the combination of drugs compared to the control group (Fig 1B). These populations of cells had higher levels of expression of CD33 and CD38, which are well-known markers of cellular differentiation. Conclusions: In this study, we present the in vivo effectiveness of DS-1594b in combination with venetoclax in a PDX model of NPM1-mutated AML. Moreover, the combination treatment exhibits differentiation-inducing effects, which contribute to its therapeutic effectiveness, and was safe. Overall, our findings strongly indicate that the combination of DS-1594b and venetoclax exhibits promising anti-leukemic activity in vivo and holds potential as a therapeutic strategy for AML patients with mtNPM1 mutations.

Ibrutinib and Venetoclax in Symptomatic, Treatment-Naive Patients with Waldenström Macroglobulinemia

Background: BTK inhibitors and BCL2 antagonists as monotherapy are highly active and well tolerated in Waldenström macroglobulinemia (WM). We initiated a prospective study evaluating ibrutinib and venetoclax in treatment-naive WM. Study therapy was stopped on March 31, 2022, after four ventricular arrhythmia events, including two grade 5 events. Herein, we present follow-up data after stopping therapy to assess ongoing safety and response durability of this combination in WM. Methods: This was an investigator-initiated, multicenter, prospective phase II study in symptomatic treatment-naïve WM patients. Study therapy was given in 4-week cycles. Intended therapy consisted of ibrutinib at 420 mg/day on cycle 1. Venetoclax was added on cycle 2 at 100 mg/day for one week, 200 mg/day for one week, and 400 mg/day for two weeks. Ibrutinib 420 mg/day and venetoclax 400 mg/day were given together for cycles 3-24. All patients underwent baseline laboratory studies, a bone marrow biopsy with MYD88 and CXCR4 genotyping, and CT scans to evaluate extramedullary disease. Responses were assessed using modified IWWM6 criteria. The study was registered under clinicaltrials.gov (NCT04273139). Results: Between July 2020 and January 2022, 45 (30 males; 15 females) patients were enrolled. Baseline characteristics were: median age 67 (range 40-82 yrs); serum IgM 4297 (range 572-9211 mg/dL); hemoglobin 10.2 (range 7.8-15.3 g/dL); bone marrow involvement 60% (5-90%); presence of lymphadenopathy (>1.5 cm) 25 (56%); and splenomegaly (>15 cm) 13 (29%). MYD88 L265P was detected in all patients, and CXCR4 mutations in 17 (38%; 10 nonsense and 7 frameshift). Median time on treatment was 10.2 months (range 1.9-20.8) before treatment was halted. Overall response rate was 100%, and major response 96%. Best responses included very good partial response (VGPR; n=19, 42%); partial response (n=24, 53%); and minor response (n=2, 4%). CXCR4 mutations were associated with lower VGPR rates (29% v 50%; p=0.18). At a data cutoff date of May 31, 2023, the median study follow-up is 24.4 months (range 3-33.6). There were 12 progression events, including two treatment-emergent deaths and one transformation to DLBCL. Five patients started a new therapy. At 24 months, progression-free survival (PFS) rate was 76% (95% CI 59-86; Figure A), next treatment-free rate was 89% (95% CI 72-96), and overall survival (OS) rate was 96% (95% CI 84-99). CXCR4 mutations did not impact PFS (p=0.89; Figure B), time to next treatment (TTNT) (p=0.43), or OS (p=0.27). Median time after end of therapy (EOT) was 13.3 months (95% CI 12.9-13.5). There were 11 progression events after EOT, and the 12-month PFS rate after EOT was 79% (95% CI 63-88; Figure C). CXCR4 mutations (p=0.35) or time on therapy (<12 vs. ≥12 months; p=0.76) did not impact PFS after EOT. There was a trend towards longer PFS after EOT in patients who had attained VGPR (p=0.12; Figure D). No treatment-emergent events, especially arrhythmia, were observed after treatment stopped. Thirty-one patients underwent a bone marrow biopsy one year after EOT. No emergent mutations, especially in BTK and TP53, were detected. Conclusion: In symptomatic, treatment-naive patients with WM, the combination of ibrutinib and venetoclax was shown to be highly effective with a VGPR rate of 42% and a 24-month PFS rate of 76%, despite stopping therapy early due to unexplained ventricular arrhythmia. VGPR attainment might predict longer PFS after EOT, and PFS was similar in patients who received <12 vs. ≥12 months of therapy. No treatment-emergent toxicities, including arrhythmia, occurred after EOT. The combination of ibrutinib and venetoclax holds significant promise. However, given that the cause of the unexpected ventricular arrhythmia remains unclear, it is challenging to recommend the combination at the current dose and schedule. The potential for shorter treatment durations or safer combinations offer hope for maintaining efficacy while mitigating adverse effects.

The BCL2 inhibitor venetoclax mediates anticancer effects through dendritic cell activation.

BCL2 is an apoptosis-inhibitory oncoprotein that also possesses apoptosis-unrelated activities. Pharmacological BCL2 inhibitors have been developed with the scope of driving BCL2-dependent cancer cells into apoptosis, and one BCL2 antagonist, venetoclax, has been clinically approved for the treatment of specific leukemias and lymphomas. Nonetheless, it appears that venetoclax, as well as genetic BCL2 inhibition, can mediate anticancer effects through an indirect action. Such an indirect effect relies on the enhancement of the immunostimulatory function of dendritic cells, hence increasing tumor immunosurveillance. Mechanistically, BCL2 inhibition involves improved antigen presentation by conventional type-1 dendritic cells (cDC1s) due to the activation of an interferon response, leading to a T cell-mediated anticancer immune response that can be further enhanced by PD-1 blockade. These findings support the emerging hypothesis that successful antineoplastic drugs generally mediate their effects indirectly, through the immune system, rather via merely cell-autonomous effects on malignant cells.

Bcl-2 Antagonist Obatoclax Reactivates Latent HIV-1 via the NF-κB Pathway and Induces Latent Reservoir Cell Apoptosis in Latently Infected Cells.

The implementation of combined antiretroviral therapy (cART) has rendered HIV-1 infection clinically manageable and efficiently improves the quality of life for patients with AIDS. However, the persistence of a latent HIV-1 reservoir is a major obstacle to achieving a cure for AIDS. A "shock and kill" strategy aims to reactivate latent HIV and then kill it by the immune system or cART drugs. To date, none of the LRA candidates has yet demonstrated effectiveness in achieving a promising functional cure. Interestingly, the phosphorylation and activation of antiapoptotic Bcl-2 protein induce resistance to apoptosis during HIV-1 infection and the reactivation of HIV-1 latency in central memory CD4+ T cells from HIV-1-positive patients. Therefore, a Bcl-2 antagonist might be an effective LRA candidate for HIV-1 cure. In this study, we reported that a pan-Bcl-2 antagonist obatoclax induces HIV-1 reactivation in latently infected cell lines in vitro and in PBMCs/CD4+ T cells of HIV-infected individuals ex vivo. Obatoclax promotes HIV-1 transcriptional initiation and elongation by regulating the NF-κB pathway. Obatoclax activates caspase 8 and does not induce the phosphorylation of the antiapoptotic protein Bcl-2 in latent HIV-1 infected cell lines. More importantly, it preferentially induces apoptosis in latently infected cells. In addition, obatoclax exhibited potent anti-HIV-1 activity on target cells. The abilities to reactivate latent HIV-1 reservoirs, inhibit HIV-1 infection, and induce HIV-1 latent cell apoptosis make obatoclax worth investigating for development as an ideal LRA for use in the "shock and kill" approach.

FRI645 A Case Of Diabetic Ketoacidosis After Initiation Of Decitabine And Venetoclax Chemotherapy

Abstract Disclosure: R. Maini: None. J. Milosavljevic: None. S. Aleksic: None. Background: Decitabine, a pyrimidine nucleotide analogue, and venetoclax, a Bcl-2 inhibitor (ABT-199), are chemotherapy agents used for the treatment of myelodysplastic syndromes. Clinical trials have shown hyperglycemia as an adverse reaction for both of these medications, but DKA has not been reported. Clinical Case: A 71-year-old woman with a history of T2D and myelodysplastic syndrome (MDS) presented with nausea, vomiting, and diarrhea for five days. Initial tests revealed blood glucose 839 mg/dL, an anion gap of 19 mEq/L (n 7-16), serum bicarbonate 14 mmol/L (n 20-30), venous pH of 7.24, and hydroxybutyrate >4 mmol/L (n< 0.3), consistent with diabetic ketoacidosis. Her anti-glutamic acid decarboxylase autoantibodies were negative. Chest CT showed bibasilar pulmonary opacities, concerning for multifocal pneumonia. She was placed on an insulin drip and fluids according to the institution's protocol and her anion gap closed within a day. She was transitioned to subcutaneous insulin. The patient had well-controlled T2D, with an HbA1c of 5.6% two weeks before her presentation and an admission fructosamine level of 377 umol/L (n 200-285). Her BMI was 25. Her only T2D medication was semaglutide 0.25 mg weekly but had not taken a dose in three weeks due to the national shortage. Five weeks prior to this presentation, she began weekly chemotherapy infusions of decitabine and oral venetoclax as part of a clinical trial (2021-13466) for MDS treatment, with her last infusion one week before her presentation. Conclusion: This is the first case to demonstrate DKA precipitated by decitabine and venetoclax treatment. While both agents have been linked to severe hyperglycemia in safety clinical trials, DKA has not been previously reported. Data from animal studies have shown that prolonged exposure of mouse islets to Bcl-2 antagonists increase levels of reactive oxygen species and induce pancreatic β cell apoptosis (1). Further research is needed to assess the benefits and risks of this chemotherapy regimen in patients with diabetes.

Intracellular BAPTA directly inhibits PFKFB3, thereby impeding mTORC1-driven Mcl-1 translation and killing MCL-1-addicted cancer cells

Intracellular Ca2+ signals control several physiological and pathophysiological processes. The main tool to chelate intracellular Ca2+ is intracellular BAPTA (BAPTAi), usually introduced into cells as a membrane-permeant acetoxymethyl ester (BAPTA-AM). Previously, we demonstrated that BAPTAi enhanced apoptosis induced by venetoclax, a BCL-2 antagonist, in diffuse large B-cell lymphoma (DLBCL). This finding implied a novel interplay between intracellular Ca2+ signaling and anti-apoptotic BCL-2 function. Hence, we set out to identify the underlying mechanisms by which BAPTAi enhances cell death in B-cell cancers. In this study, we discovered that BAPTAi alone induced apoptosis in hematological cancer cell lines that were highly sensitive to S63845, an MCL-1 antagonist. BAPTAi provoked a rapid decline in MCL-1-protein levels by inhibiting mTORC1-driven Mcl-1 translation. These events were not a consequence of cell death, as BAX/BAK-deficient cancer cells exhibited similar downregulation of mTORC1 activity and MCL-1-protein levels. Next, we investigated how BAPTAi diminished mTORC1 activity and identified its ability to impair glycolysis by directly inhibiting 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) activity, a previously unknown effect of BAPTAi. Notably, these effects were also induced by a BAPTAi analog with low affinity for Ca2+. Consequently, our findings uncover PFKFB3 inhibition as an Ca2+-independent mechanism through which BAPTAi impairs cellular metabolism and ultimately compromises the survival of MCL-1-dependent cancer cells. These findings hold two important implications. Firstly, the direct inhibition of PFKFB3 emerges as a key regulator of mTORC1 activity and a promising target in MCL-1-dependent cancers. Secondly, cellular effects caused by BAPTAi are not necessarily related to Ca2+ signaling. Our data support the need for a reassessment of the role of Ca2+ in cellular processes when findings were based on the use of BAPTAi.

DNAi-peptide nanohybrid smart particles target BCL-2 oncogene and induce apoptosis in breast cancer cells

Genomic DNA sequences provide unique target sites, with high druggability value, for treatment of genetically-linked diseases like cancer. B-cell lymphoma protein-2 (BCL-2) prevents Bcl-2-associated X protein (BAX) and Bcl-2 antagonist killer 1 (BAK) oligomerization, which would otherwise lead to the release of several apoptogenic molecules from the mitochondrion. It is also known that BCL-2 binds to and inactivates BAX and other pro-apoptotic proteins, thereby inhibiting apoptosis. BCL-2 protein family, through its role in regulation of apoptotic pathways, is possibly related to chemo-resistance in almost half of all cancer types including breast cancer. Here for the first time, we have developed a nanohybrid using a peptide-based carrier and a Deoxyribonucleic acid inhibitor (DNAi) against BCL-2 oncogene to induce apoptosis in breast cancer cells. The genetically designed nanocarrier was functionalized with an internalizing RGD (iRGD) targeting motif and successfully produced by recombinant DNA technology. Gel retardation assay demonstrated that the peptide-based carrier binds single-stranded DNAi upon simple mixing. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) analyses further revealed the formation of nanohybrid particles with a size of 30 nm and a slightly positive charge. This hemocompatible nanohybrid efficiently delivered its contents into cancer cells using iRGD targeting moiety. Gene expression analysis demonstrated that the nanohybrids, which contained DNAi against BCL-2 proficiently suppressed the expression of this oncogene in a sequence specific manner. In addition, the nanohybrid, triggered release of cytochrome c (cyt c) and caspase3/7 activation with high efficiency. Although the DNAi and free nanocarrier were separately unable to affect the cell viability, the nanohybrid of 20 nM of DNAi showed outstanding antineoplastic potential, which was adjusted by the ratio of the MiRGD nanocarrier to DNAi. It should be noted that, the designed nanohybrid showed a suitable specificity profile and did not affect the viability of normal cells. The results suggest that this nanohybrid may be useful for robust breast cancer treatment through targeting the BCL-2 oncogene without any side effects.

Abstract A03: Mitochondrial dynamics alterations in BH3 mimetics resistance in acute myeloid leukemia

Abstract BH3 mimetics are used as an efficient strategy to promote mitochondrial-dependent cell death in malignancies, including acute myeloid leukemia (AML). Venetoclax, a potent BCL-2 antagonist, is used clinically in combination with hypomethylating agents for the treatment of AML, while various compounds targeting MCL-1 are in clinical trials. Yet, resistance to single or combinatorial BH3 mimetics therapies eventually ensues. Integration of multiple genome-wide CRISPR/Cas9 screens revealed that loss of mitophagy and mitochondrial dynamics regulators sensitizes AML cells to BH3 mimetics. One such regulator is Mitofusin-2 (MFN2), which is essential in AML, while its protein levels positively correlate with drug resistance in patients with AML. Resistance to BH3 mimetics is accompanied by alterations in mitochondrial morphology, enhanced mitochondria-endoplasmic reticulum interactions, and augmented mitophagy flux. Genetic or chemical targeting of MFN2, or global pharmacologic inhibition of autophagy, synergizes with BH3 mimetics and is a promising strategy to circumvent resistance in AML. Citation Format: Christina Glytsou, Xufeng Chen, Emmanouil Zacharioudakis, Wafa Al-Santli, Hua Zhou, Evripidis Gavathiotis, Iannis Aifantis. Mitochondrial dynamics alterations in BH3 mimetics resistance in acute myeloid leukemia [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A03.

A tumor microenvironment model of chronic lymphocytic leukemia enables drug sensitivity testing to guide precision medicine

The microenvironment of chronic lymphocytic leukemia (CLL) cells in lymph nodes, spleen, and bone marrow provides survival, proliferation, and drug resistance signals. Therapies need to be effective in these compartments, and pre-clinical models of CLL that are used to test drug sensitivity must mimic the tumor microenvironment to reflect clinical responses. Ex vivo models have been developed that capture individual or multiple aspects of the CLL microenvironment, but they are not necessarily compatible with high-throughput drug screens. Here, we report on a model that has reasonable associated costs, can be handled in a regularly equipped cell lab, and is compatible with ex vivo functional assays including drug sensitivity screens. The CLL cells are cultured with fibroblasts that express the ligands APRIL, BAFF and CD40L for 24 h. The transient co-culture was shown to support survival of primary CLL cells for at least 13 days, and mimic in vivo drug resistance signals. Ex vivo sensitivity and resistance to the Bcl-2 antagonist venetoclax correlated with in vivo responses. The assay was used to identify treatment vulnerabilities and guide precision medicine for a patient with relapsed CLL. Taken together, the presented CLL microenvironment model enables clinical implementation of functional precision medicine in CLL.

Abstract 2526: The Bcl-2 family proteins in CLL: Effects by the cytogenetics, microenvironments and therapy

Abstract The balance between the pro- and anti-apoptotic members of the Bcl-2 family proteins governs the fate of CLL cells and determines their response to therapeutics. Thus, we have developed an assay to quantify the Bcl-2 family proteins at the molecular level and express them as molecules/pg total protein. This set of proteins represents the 10 major Bcl-2 members in CLL: Bcl-2, Bcl-XL, Mcl-1, BIM, BID, BMF, BAX, BAK, PUMA and Noxa. This analysis enabled us to evaluation of the interplay of these proteins and how they respond to cytogenetic aberrations, drug treatment or environmental changes. In 36 CLL samples that represent 6 districted cytogenetic features: normal cytogenetics, del17p, del11q, del13q14, 11q/13q double deletion, and trisomy 12, we found that the major anti-apoptotic proteins in CLL is Bcl-2, that averaged >9,000 molecules/pg of total protein. Mcl-1 was expressed at less than one tenth of Bcl-2 and Bcl-XL was expressed less than one percent of Bcl-2 molecules. The major pro-apoptotic proteins were Bax and Puma which were expressed at similar levels to Bcl-2. Among the cytogenetic groups, there were significant increases of Bcl-2 in the 11q/13q group whereas Mcl-1 was increased in 11q and trisomy 12 groups compared to samples with normal cytogenetic characteristics. Next, we screened CLL response to different microenvironments by incubating primary CLL cells in vitro in media supplemented with either fetal bovine serum (FBS), patient autologous plasma, a layer of StromaNKtert cells representing the stroma cell support in the bone marrow, a B-cell activating media (BCA) that consisted of anti-CD40, IL-4 and anti-IgM to mimic the lymphoid microenvironment, or oligodeoxynucleotide (ODN) plus IL-15 to represent the T-cell independent stimulation in the lymph nodes. Compared to FBS, homologous patient plasma moderately promoted CLL viability. However, stroma cells, BCA media as well ODN+IL-15 greatly induced levels of Mcl-1 and Bcl-XL and were more effective in promoting CLL survival. For the pro-apoptotic proteins, BMF was induced by BCA media, and PUMA, NOXA and BID were greatly induced by ODN+IL-15. We also examined the changes in Bcl-2 family proteins in response to therapeutic agents with different mechanisms of action, such as the CDK9 inhibitor fadraciclib, the Bcl-2 antagonist venetoclax, the Mcl-1 inhibitors AZD5991 and S63845, the proteasome inhibitor carfilzomib, the BTK inhibitor ibrutinib and the splicing inhibitor herboxidiene. Fadraciclib and herboxidiene were effective in reducing Mcl-1 to induce apoptosis. While both the Mcl-1 inhibitors and carfilzomib increased the Mcl-1 level, and carfilzomib increased Noxa that counteracted its effect on Mcl-1. Puma was reduced by most of the drugs except ibrutinib. Thus, the systematic analysis of the Bcl-2 family proteins provide insights on how the interplay of members of this important family affects the destiny of the CLL cells. Citation Format: Rong Chen, Ping Xiong, Chaomei Liu, Yuling Chen, Yingjun Jiang, Varsha Gandhi, Nitin Jain, William Wierda, William Plunkett. The Bcl-2 family proteins in CLL: Effects by the cytogenetics, microenvironments and therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2526.

The super elongation complex drives transcriptional addiction in MYCN-amplified neuroblastoma.

MYCN amplification in neuroblastoma leads to aberrant expression of MYCN oncoprotein, which binds active genes promoting transcriptional amplification. Yet, how MYCN coordinates transcription elongation to meet productive transcriptional amplification and which elongation machinery represents MYCN-driven vulnerability remain to be identified. We conducted a targeted screen of transcription elongation factors and identified the super elongation complex (SEC) as a unique vulnerability in MYCN-amplified neuroblastomas. MYCN directly binds EAF1 and recruits SEC to enhance processive transcription elongation. Depletion of EAF1 or AFF1/AFF4, another core subunit of SEC, leads to a global reduction in transcription elongation and elicits selective apoptosis of MYCN-amplified neuroblastoma cells. A combination screen reveals SEC inhibition synergistically potentiates the therapeutic efficacies of FDA-approved BCL-2 antagonist ABT-199, in part due to suppression of MCL1 expression, both in MYCN-amplified neuroblastoma cells and in patient-derived xenografts. These findings identify disruption of the MYCN-SEC regulatory axis as a promising therapeutic strategy in neuroblastoma.

Synergistic Interactions between the Hypomethylating Agent Thio-Deoxycytidine and Venetoclax in Myelodysplastic Syndrome Cells

Interactions between the novel hypomethylating agent (HMA) thio-deoxycytidine (T-dCyd) and the BCL-2 antagonist ABT-199 (venetoclax) have been examined in human myelodysplastic syndrome (MDS) cells. The cells were exposed to agents alone or in combination, after which apoptosis was assessed, and a Western blot analysis was performed. Co-administration of T-dCyd and ABT-199 was associated with the down-regulation of DNA methyltransferase 1 (DNMT1) and synergistic interactions documented by a Median Dose Effect analysis in multiple MDS-derived lines (e.g., MOLM-13, SKM-1, and F-36P). Inducible BCL-2 knock-down significantly increased T-dCyd's lethality in MOLM-13 cells. Similar interactions were observed in the primary MDS cells, but not in the normal cord blood CD34+ cells. Enhanced killing by the T-dCyd/ABT-199 regimen was associated with increased reactive oxygen species (ROS) generation and the down-regulation of the anti-oxidant proteins Nrf2 and HO-1, as well as BCL-2. Moreover, ROS scavengers (e.g., NAC) reduced lethality. Collectively, these data suggest that combining T-dCyd with ABT-199 kills MDS cells through an ROS-dependent mechanism, and we argue that this strategy warrants consideration in MDS therapy.

Dual mTORC1/2 Inhibition Synergistically Enhances AML Cell Death in Combination with the BCL2 Antagonist Venetoclax.

Acute myelogenous leukemia (AML) is an aggressive disease with a poor outcome. We investigated mechanisms by which the anti-AML activity of ABT-199 (venetoclax) could be potentiated by dual mTORC1/TORC2 inhibition. Venetoclax/INK128 synergism was assessed in various AML cell lines and primary patient AML samples in vitro. AML cells overexpressing MCL-1, constitutively active AKT, BAK, and/or BAX knockout, and acquired venetoclax resistance were investigated to define mechanisms underlying interactions. The antileukemic efficacy of this regimen was also examined in xenograft and patient-derived xenograft (PDX) models. Combination treatment with venetoclax and INK128 (but not the mTORC1 inhibitor rapamycin) dramatically enhanced cell death in AML cell lines. Synergism was associated with p-AKT and p-4EBP1 downregulation and dependent upon MCL-1 downregulation and BAK/BAX upregulation as MCL-1 overexpression and BAX/BAK knockout abrogated cell death. Constitutive AKT activation opposed synergism between venetoclax and PI3K or AKT inhibitors, but not INK128. Combination treatment also synergistically induced cell death in venetoclax-resistant AML cells. Similar events occurred in primary patient-derived leukemia samples but not normal CD34+ cells. Finally, venetoclax and INK128 co-treatment displayed increased antileukemia effects in in vivo xenograft and PDX models. The venetoclax/INK128 regimen exerts significant antileukemic activity in various preclinical models through mechanisms involving MCL-1 downregulation and BAK/BAX activation, and offers potential advantages over PI3K or AKT inhibitors in cells with constitutive AKT activation. This regimen is active against primary and venetoclax-resistant AML cells, and in in vivo AML models. Further investigation of this strategy appears warranted.