Overcome Venetoclax Resistance Research Articles

Using the Iron Chelator Deferasirox to Overcome Venetoclax Resistance in Mantle Cell Lymphoma

Using the Iron Chelator Deferasirox to Overcome Venetoclax Resistance in Mantle Cell Lymphoma

MCL-229 Using the Iron Chelator Deferasirox to Overcome Venetoclax Resistance in Mantle Cell Lymphoma

MCL-229 Using the Iron Chelator Deferasirox to Overcome Venetoclax Resistance in Mantle Cell Lymphoma

Boanmycin induces apoptosis and overcomes venetoclax resistance in acute myeloid leukemia

Abstract Objectives This study aimed to investigate the efficacy of boanmycin, a clinical drug used for head and neck cancers, in the treatment of acute myeloid leukemia (AML), particularly in venetoclax-resistant AML cells. Methods The cell viability assay was conducted to measure the inhibitory effects of boanmycin on the AML cell lines and patient primary cells using the CCK8 reagent. The colony formation assay was performed to evaluate the colony formation ability of HL60 and venetoclax-resistant HL60 (HL60-res) cells with or without boanmycin treatment. Flow cytometry was performed to detect cell apoptosis level, and Western blot was used to assess changes in apoptosis-related proteins. Results Our findings reveal that boanmycin significantly inhibits AML cell proliferation and colony formation, and induces apoptosis. Importantly, boanmycin exhibits substantial inhibitory effects on venetoclax-resistant cells, and suppresses the proliferation of peripheral blood mononuclear cells (PBMCs) or bone marrow mononuclear cells (BMMCs) derived from newly diagnosed and relapsed AML patients. Conclusions Boanmycin may overcome venetoclax resistance and offer therapeutic benefits for patients with venetoclax-resistant AML.

Identifying Targetable Vulnerabilities to Circumvent or Overcome Venetoclax Resistance in Diffuse Large B-Cell Lymphoma.

Clinical trials with single-agent venetoclax/ABT-199 (anti-apoptotic BCL2 inhibitor) revealed that diffuse large B-cell lymphoma (DLBCL) is not solely dependent on BCL2 for survival. Gaining insight into pathways/proteins that increase venetoclax sensitivity or unique vulnerabilities in venetoclax-resistant DLBCL would provide new potential treatment avenues. Therefore, we generated acquired venetoclax-resistant DLBCL cells and evaluated these together with intrinsically venetoclax-resistant and -sensitive DLBCL lines. We identified resistance mechanisms, including alterations in BCL2 family members that differed between intrinsic and acquired venetoclax resistance and increased dependencies on specific pathways. Although combination treatments with BCL2 family member inhibitors may overcome venetoclax resistance, RNA-sequencing and drug/compound screens revealed that venetoclax-resistant DLBCL cells, including those with TP53 mutation, had a preferential dependency on oxidative phosphorylation. Mitochondrial electron transport chain complex I inhibition induced venetoclax-resistant, but not venetoclax-sensitive, DLBCL cell death. Inhibition of IDH2 (mitochondrial redox regulator) synergistically overcame venetoclax resistance. Additionally, both acquired and intrinsic venetoclax-resistant DLBCL cells were similarly sensitive to inhibitors of transcription, B-cell receptor signaling, and class I histone deacetylases. These approaches were also effective in DLBCL, follicular, and marginal zone lymphoma patient samples. Our results reveal there are multiple ways to circumvent or overcome the diverse venetoclax resistance mechanisms in DLBCL and other B-cell lymphomas and identify critical targetable pathways for future clinical investigations.

Selective eradication of venetoclax-resistant monocytic acute myeloid leukemia with iron oxide nanozymes

The clinical treatment of human acute myeloid leukemia (AML) is rapidly progressing from chemotherapy to targeted therapies led by the BCL-2 inhibitor venetoclax (VEN). Despite its unprecedented success, VEN still encounters clinical resistance. Thus, uncovering the biological vulnerability of VEN-resistant AML disease and identifying effective therapies to treat them are urgently needed. We have previously demonstrated that iron oxide nanozymes (IONE) are capable of overcoming chemoresistance in AML. The current study reports a new activity of IONE in overcoming VEN resistance. Specifically, we revealed an aberrant redox balance with excessive intracellular reactive oxygen species (ROS) in VEN-resistant monocytic AML. Treatment with IONE potently induced ROS-dependent cell death in monocytic AML in both cell lines and primary AML models. In primary AML with developmental heterogeneity containing primitive and monocytic subpopulations, IONE selectively eradicated the VEN-resistant ROS-high monocytic subpopulation, successfully resolving the challenge of developmental heterogeneity faced by VEN. Overall, our study revealed an aberrant redox balance as a therapeutic target for monocytic AML and identified a candidate IONE that could selectively and potently eradicate VEN-resistant monocytic disease.

Abstract 7089: Proteomic evaluation of combination data in acute myeloid leukemia to inform mechanism of action and new targets

Abstract Background: Acute myeloid leukemia (AML) is a prevalent adult leukemia. In the US, around 20,380 individuals were diagnosed with AML in 2023. Despite standard therapies, relapse and resistance persist. Cyclin-dependent kinase 9 (CDK9) has been recognized an actionable therapeutic target in AML. The potent inhibitor, AZD4573, induces durable apoptosis and suppresses tumor growth. Our study reveals that combining AZD4573 with standard-of-care (SoC) treatments deepens cell death responses in multiple AML cell models. We also used proteomics tools to understand the mechanisms underlying the vulnerability and resistance in SoC and CDK9 combinations. Methods: In the drug combination study, three SoC agents (cytarabine, decitabine, and venetoclax) were evaluated in combination using a 6 × 6 dose-response matrix in four AML cell models (MOLM13, MOLM16, MV411, and OCI-AML2). The HSA synergy score and Emax drug activity were used to assess the benefits of the drug combinations. In the proteomics evaluation study, MOLM13 cells treated for 7 days under different conditions - (1) vehicle control; (2) cytarabine alone; (3) decitabine alone; (4) venetoclax alone; (5) AZD4573 alone; (6) AZD4573+cytarabine; (7) AZD4573+decitabine; (8) AZD4573+venetoclax - were collected for proteomics analysis using LC-MS/MS. Differential expression and missing pattern analyses were used to identify dysregulated proteins. Functional enrichment analysis was employed to identify dysregulated biological processes and pathways in response to different treatments. Results: Our drug combination screens demonstrated the efficacy of AZD4573 in combination with cytarabine, decitabine, and venetoclax in 3 of 4 AML cell models. In our proteomic analysis, we took two approaches. First, we compared the proteomes of AML cells treated with SoC agents, identifying shared downregulated pathways such as serine biosynthesis, notably pointing to the rate-limiting enzyme PHGDH as a potential therapeutic target in AML. Cytarabine and decitabine induced an inflammatory response, while venetoclax increased the expression of cell division regulators like Aurora kinase B (AURKB). Interestingly, targeting AURKB with AZD2811 has been shown to overcome venetoclax resistance in AML. Second, we compared proteome changes induced by various AZD4573 combinations. We identified IRF2BP2 as a shared vulnerable protein across the three AZD4573 combinations. POUF2F2 expression was significantly induced by cytarabine but suppressed when combined with AZD4573. Given that POUF2F2 activates the PDK1-Akt pathway, our findings suggest that AZD4573-induced POUF2F2 downregulation suppresses the PDK1-AKT pathway, inhibiting tumor progression. Conclusions: Our proteomics analysis identifies vulnerable and resistant pathways and proteins, shedding light on the mechanism of action and revealing potential new targets for AML. Citation Format: Lori Chan, Zuleyha Ozen, Anthony Iannetta, Eric Miele, Funda Kar, Andrew Jarnuczak, Geoff Nelson, Aleksandra Markovets, Justine Roderick-Richardson, Jelena Urosevic, Lisa Drew, Jerome Mettetal. Proteomic evaluation of combination data in acute myeloid leukemia to inform mechanism of action and new targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7089.

Abstract 5991: Improved venetoclax therapy by a novel conjugate with artemisinin by NOXA-mediated reduction of Mcl-1 and cyclin D1 protein in myeloid leukemia cells

Abstract Evasion of apoptosis is crucial for the growth, survival and chemoresistance of myeloid leukemia. The Bcl-2 selective inhibitor venetoclax is the only apoptosis inducer approved for clinical use. Venetoclax in combination with hypomethylating agents or low-dose cytarabine has now become the standard care for elderly AML patients. However, a sizeable fraction of patients are either refractory to venetoclax combination therapy or ultimately relapse. High or induced expression of Mcl-1 and Bcl-xL mediates venetoclax resistance. Previously we reported that artemisinin enhances venetoclax apoptosis induction by degrading Mcl-1 through NOXA induction. We designed a novel conjugate A1 of venetoclax with dihydroartemisinin. A1 shows dual functions of inhibiting Bcl-2 and inducing NOXA. A1 treatment releases Bim from both Bcl-2 and Mcl-1, further degrades Mcl-1 protein, and presents ten-fold stronger apoptotic induction ability in venetoclax insensitive cells. Acquired increase of Mcl-1 protein was observed in venetoclax resistant MOLM-13/VEN cells, which are sensitive to A1-induced apoptosis. In Bcl-xL expressing venetoclax refractory leukemia cells A1 obtains a new mechanism of inhibiting cell cycling by downregulating cyclin D1 through a new NOXA/cyclin D1 cascade, mediated through the endoperoxide moiety of artemisinin and intracellular iron, independent of Bcl-2 inhibition. We reveal a new compound A1 overcoming venetoclax resistance by NOXA-mediated degradation of Mcl-1 and cyclin D1 protein. Citation Format: Jingyi Zhang, Zhenwei Zhang, Samuel Waxman, Linxiang Zhao, Yongkui Jing. Improved venetoclax therapy by a novel conjugate with artemisinin by NOXA-mediated reduction of Mcl-1 and cyclin D1 protein in myeloid leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5991.

Venetoclax Resistance in Acute Myeloid Leukemia.

Venetoclax is a BH3-mimetics agent interacting with the anti-apoptotic protein BCL2, facilitating cytochrome c release from mitochondria, subsequent caspases activation, and cell death. Venetoclax combined with azacitidine (VEN-AZA) has become a new standard treatment for AML patients unfit for intensive chemotherapy. In the phase III VIALE-A study, VEN-AZA showed a 65% overall response rate and 14.7 months overall survival in comparison with 22% and 8 months in the azacitidine monotherapy control arm. Despite these promising results, relapses and primary resistance to venetoclax are frequent and remain an unmet clinical need. Clinical and preclinical studies have been conducted to identify factors driving resistance. Among them, the most documented are molecular alterations including IDH, FLT3, TP53, and the newly described BAX mutations. Several non-genetic factors are also described such as metabolic plasticity, changes in anti-apoptotic protein expression, and dependencies, as well as monocytic differentiation status. Strategies to overcome venetoclax resistance are being developed in clinical trials, including triplet therapies with targeted agents targeting IDH, FLT3, as well as the recently developed menin inhibitors or immunotherapies such as antibody-drug conjugated or monoclonal antibodies. A better understanding of the molecular factors driving venetoclax resistance by single-cell analyses will help the discovery of new therapeutic strategies in the future.

Comprehensive characterization of IFNγ signaling in acute myeloid leukemia reveals prognostic and therapeutic strategies

Interferon gamma (IFNγ) is a critical cytokine known for its diverse roles in immune regulation, inflammation, and tumor surveillance. However, while IFNγ levels were elevated in sera of most newly diagnosed acute myeloid leukemia (AML) patients, its complex interplay in AML remains insufficiently understood. We aim to characterize these complex interactions through comprehensive bulk and single-cell approaches in bone marrow of newly diagnosed AML patients. We identify monocytic AML as having a unique microenvironment characterized by IFNγ producing T and NK cells, high IFNγ signaling, and immunosuppressive features. IFNγ signaling score strongly correlates with venetoclax resistance in primary AML patient cells. Additionally, IFNγ treatment of primary AML patient cells increased venetoclax resistance. Lastly, a parsimonious 47-gene IFNγ score demonstrates robust prognostic value. In summary, our findings suggest that inhibiting IFNγ is a potential treatment strategy to overcoming venetoclax resistance and immune evasion in AML patients.

Novel therapies upon failure of HMA plus venetoclax.

The efficacy and tolerability of the combination of hypomethylating agents with venetoclax (HMA-VEN) in patients with newly diagnosed acute myeloid leukemia has been a practice-changing milestone in the field. However, treatment failure and relapse remain major barriers to prolonged survival. TP53 mutation is a predictor of primary induction failure and portends especially poor outcomes. Prelinical data suggest that VEN resistance stems from these genetic changes, which lead to increases in antiapoptotic proteins such as MCL-1 and BCLXL. For patients who discontinue HMA-VEN for reasons other than disease progression, such as post allotransplantation, infection, and personal preference, rechallenge with HMA-VEN at the time of relapse may be considered. For those who progress on HMA-VEN, clinical trials with novel agents or rational drug combinations are preferred if available. If no trial option is available, fit patients may benefit from intensive chemotherapy. Emerging therapies aim to overcome venetoclax resistance, target interactions that promote leukemogenesis, and harness the immune system to irradicate leukemic blasts and stem cells.

Abstract LB_A20: Overcoming cancer drug resistance with AI-designed RNA epigenetic inhibitors

Abstract Background: Drug resistance is a major obstacle to successful treatment of cancer and leukemia. The role of RNA epigenetics in cancer drug resistance is largely unknown. Our previous studies demonstrated that specific RNA cytosine methyltransferases (RCMTs), namely NSUN1 (aka NOL1, NPO2) and NSUN2, mediate the formation of drug-resistant active chromatin structures (ACS) at nascent RNAs to regulate drug resistance in leukemia cells (Cheng, J.X. et al. Nat Commun, 2018). The Cancer Genome Atlas Program (TCGA) data demonstrate that elevated expression levels of NSUN1 and NSUN2 are correlated with significantly shorter patients’ survival in almost all cancer subtypes (https://www.proteinatlas.org/ENSG00000111641-NOP2/pathology). However, due to the lack of NSUN1 and NSUN2 proteins’ crystal structures, no NSUN1/2-targeting therapeutics have been developed yet. Methods: 1. Argonne artificial intelligence (AI)/supercomputer design of NSUN1/2-targeting small-molecule compound libraries, simulation of compound-protein interactions and analysis of structure activity relationship (SAR). 2. Novel RNA epigenetic technologies, including NSUN1/2-targeting fluorescence releasing assay (FRA), 5-ethynyl uridine click chemistry (EC) and proximity ligation rolling cycle amplification (PL-RCA)-coupled confocal microscopy (CM), flow cytometry (FCM) and RNA sequencing (RNA-seq). 3. Functional drug screening, H&E histology and immunohistochemistry (IHC). 4. Enforced NSUN1/2 expression and knockout (KO) cell lines and syngeneic AML mouse models. Results:1. Identification of the ligand-binding modules/surfaces in NSUN1 and NSUN2 proteins and generation of NSUN1/2-targeting small-molecule libraries by AI/supercomputing. 2. Identification of several NSUN1/2 inhibitors that effectively inhibit growth of the venetoclax-resistant human monocytic leukemia (SC), erythroid leukemia (K562) and lung cancer (H82) cells. 3. Demonstration of the lead inhibitors execute their effects through disruption of NSUN1/2-mediated (ACS) and translational complexes (TLC). 4. Characterization of the lead inhibitors’ binding modules with AI/supercomputing SAR analysis. 5. In vivo validation of the efficacies of the identified lead inhibitors using syngeneic AML mouse models. Conclusion:By leveraging the power of the Argonne AI/supercomputer and our functional assays, we identified several small-molecule inhibitors that can disrupt the NSUN1/2-mediated ACS and TLC to overcome venetoclax resistance in AML cell lines and syngeneic mice. This study has not only illustrated the importance of NSUN1 and NSUN2 in oncogenic bioprocesses and drug (venetoclax) resistance, but also paved the way to the development of novel RNA epigenetics-driven therapeutics for leukemia and other cancer subtypes. Citation Format: Jason X Cheng, Shaun Wood, Derrick Tang, Linda Degenstein, Andrzej Joachimiak, Rick Stevens. Overcoming cancer drug resistance with AI-designed RNA epigenetic inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_A20.

Overcoming Venetoclax Resistance in Leukemia with AI-Designed RNA Epigenetic Inhibitors

Background : Venetoclax resistance is a major obstacle to successful treatment of acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Its underlying mechanisms are multifaceted and have been linked to monocytic differentiation, reprograming mitochondrial metabolism, and increased expression of BRD4 and other BCL2-like proteins, etc. Our previous studies demonstrated that specific RNA cytosine methyltransferase, namely NSUN1 (aka NOL1, NPO2) and NSUN2, mediate the formation of drug-resistant active chromatin structures (ACS) at nascent RNAs to regulate drug resistance in leukemia cells (Cheng, J.X. et al. Nat Commun, 2018) ( Figure 1A). The Cancer Genome Atlas Program (TCGA) data demonstrated that elevated expression of NSUN1 and NSUN2 is correlated with significantly shorter patients' survival in almost all cancer subtypes. However, due to the lack of the crystal structures of NSUN1 and NSUN2 proteins, no NSUN1/2-targeting therapeutics have been developed yet. Methods: AI/supercomputing design of NSUN1/2-targeting drug libraries and analysis of structure activity relationship (SAR). Newly developed functional technologies, including NSUN1/2-targeting fluorescence releasing assay (FRA), 5-ethynyl uridine click chemistry (EC) and proximity ligation rolling cycle amplification (PL-RCA)-coupled confocal microscopy (CM), flow cytometry (FCM) and RNA sequencing (RNA-seq). NSUN1/2-targeting functional drug screening (NSUN1/2-FDS) assay and drug growth inhibition (MTT) assay. H&E histology and immunohistochemistry (IHC). Enforced NSUN1/2 expression and knockout (KO) cell lines and syngeneic AML mouse models. Results: Identification of several ligan-binding modules/surfaces in NSUN1 and NSUN2 proteins and generation of small-molecule libraries to targe these ligand-binding surfaces/modules with AI/supercomputing structural prediction and ligand-receptor interaction simulation. Identification of several effective small-molecule inhibitors of NSUN1 and NSUN2 by functional screening of the AI-designed drug libraries in venetoclax-resistant human monocytic leukemia (SC), erythroid leukemia (K562) and lung cancer (H82) cells. Defining the binding modules of the identified NSUN1/2 inhibitors and selecting lead inhibitors through supercomputer/AI-directed SAR analysis. Elucidation of the action mechanisms of NSUN1/2 lead inhibitors thorough disruption of NSUN1/2-mediated ACS in the nucleus and translational complexes (TLC) in the ribosomes and mitochondria ( Figure 1A). In vivo validation of the efficacies of the identified lead inhibitors using a syngeneic AML mouse model. As shown in Figure 1B, NSUN1-17, one of the lead inhibitors, efficiently eliminated the AML cells/blasts from the blood vessels and parenchyma of the lungs of the syngeneic AML mice. In addition, our siRNA knock-down experiments demonstrated that downregulation of NSUN1/2 expression re-sensitizes venetoclax-resistant monocytic leukemia cells to a low dose of venetoclax. Our NIH/3T3 soft-agarose assay confirmed the oncogenic transformation capability of NSUN1 and NSUN2. We have also successfully established the homozygous ( Nsun2-/-) mouse model with markedly reduced body weight, but a normal lifespan, compared to the heterozygous mice ( Nsun2+/-). Conclusion: By leveraging the Argonne AI/supercomputer and our functional assays, we identified several small-molecule inhibitors that can disrupt the NSUN1/2-mediated ACS and TLC to overcome venetoclax resistance in AML cell lines and syngeneic mice. This study has not only illustrated the importance of NSUN1 and NSUN2 in maintaining oncogenic transcription and translation as well as venetoclax resistance in AML, but also paved the way to the development of novel RNA epigenetics-driven therapeutics for leukemia and other cancers.

Mitochondrial STAT3 Plays a Critical Role in Survival of AML Cells

Introduction: Signal transducer and activator of transcription 3 (STAT3) has been shown to be critical to the survival of acute myeloid leukemia (AML) cells. It has been previously shown to mediate oxidative phosphorylation (OXPHOS) in acute myeloid leukemia bulk and stem cells via its transcriptional regulation of the amino acid transporter SLC1A5 and thereby glutamine uptake to AML cells (Amaya et al. Blood 2022). Further, STAT3 has been shown to regulate the transcription of important mitochondrial genes such as MCL-1 (Shastri et al. JCI 2018). Although STAT3 has been previously shown to also translocate to the mitochondria of myeloid malignant cells, its role in the mitochondria is less well defined in this cell population. In this study, we sought to understand the role of mitochondrial STAT3 in AML bulk leukemia and leukemia stem cell population. Methods: Human primary AML samples and MOLM-13 cells were utilized for experiments. To assess STAT3 localization to the mitochondria, mitochondrial fractions were isolated followed by Western Blot analysis. Flow cytometry-based imaging (ImageStream) was also utilized to quantify localization of STAT3 to the mitochondria. Mitochondrial mass was assessed using flow cytometry based MitoTracker assays. Metabolomic assays were performed by mass spectrometry at the University of Colorado metabolomics core. Viability assays were performed with flow cytometry using Annexin V and Dapi stains. Patient derived xenograft models were performed as previously described (Amaya et al. Blood 2022) in NSG-S mouse with Busulfan conditioning. Results: In this study, we show that primary human AML cells have constitutive STAT3 activation via phosphorylation of S727, which has been shown to signal mitochondrial localization. Further, we show that STAT3 is localized in the mitochondria of leukemia cells in primary patient samples and the cell line MOLM-13. Using genetic (siRNA) and pharmacologic (Stattic) inhibition of STAT3, we show a decrease in pSTAT3 at S727, with subsequent decreased translocation of STAT3 to the mitochondria. This leads to mitochondrial dysfunction including decreased TCA cycle metabolites, as well as decreased intracellular ATP upon STAT3 inhibition. STAT3 inhibition also leads to decreased mitochondrial mass suggesting autophagy upon mitochondrial STAT3 (mitoSTAT3) inhibition. To assess whether mitoSTAT3 is functionally important, we performed viability assays in MOLM-13, as well as leukemia stem cells isolated from primary AML cells. Inhibition of mitochondrial STAT3 leads to decreased viability in MOLM-13 and primary AML cells, and reduced engraftment in PDX mouse models using primary AML samples (Figure 1A), suggesting a decrease in leukemia stem cell potential. Interestingly, STAT3 inhibition with Stattic is able to overcome venetoclax resistance for primary AML patient samples in vitro (Figure 1B). Conclusions: In this study, we show mitoSTAT3 plays a key role in mitochondrial function of AML cells. mitoSTAT3 inhibition leads to mitochondrial dysfunction, a decrease in mitochondrial mass and eventually cell death. We show that Stattic is an effective inhibitor of mitoSTAT3 in AML, leading to cell death and decreased leukemia stem cell function. Further studies are needed to assess the mechanistic function of STAT3 in the mitochondria.

ERK1/2 Inhibition Overcomes Resistance to Venetoclax in AML By Inhibiting Drp1 Dependent Mitochondrial Fission

Background: Primary or secondary resistance to venetoclax is frequently associated with mutational/non-mutational activation of MAPK pathways. ERK1/2 are terminal kinases in MAPK pathway and may be an appropriate target regardless of the upstream mechanisms that activate the pathway. ERK1/2 mediated phosphorylation of Drp1 promotes mitochondrial fission and MAPK-driven tumor growth in RAS driven solid cancers (Kashatus et al., Mol Cell. 2015). However, the role of Drp1 dependent mitochondrial dynamics in therapeutic resistance in AML is unexplored. Methods: ERK1/2 was inhibited using Compound 27 (ERKi, Heightman et al., J Med Chem. 2018), an analog of ASTX029 (Munck et al., Mol Cancer Ther. 2021) in vitro and using ASTX029 in vivo. Preclinical models of venetoclax resistance and primary patient samples (n=8) were used to assess the synergy of concomitant Bcl2 and ERK1/2 inhibition (ERKi). In addition, a comprehensive analysis of alteration of signaling pathways, apoptotic signatures and DNA damage responses in response to ERKi+/-venetoclax were analyzed by mass cytometry based proteomic analysis (CyTOF) and immunoblotting. The potential clinical relevance of ERK1/2 inhibition to overcome venetoclax resistance was confirmed in a PDX model of AML (established from an AML patient who relapsed after venetoclax/decitabine treatment). Mitochondrial images were acquired using super-resolution imaging with OMX-Blaze followed by quantification with Imaris. Results : We previously reported the synergy of ERK1/2 inhibition using Compound 27 with venetoclax at inducing apoptosis in RAS mutated and/or venetoclax resistant AML cells including venetoclax resistant isogenic lines (Sharma et al., Blood 140; Supplement 1, 2022). Venetoclax+ERKi depleted leukemia progenitor cells in primary AML samples (CI:0.03-0.23) and impaired clonogenic growth of NRAS mutant PDX cells. In a PDX mouse model of post venetoclax/decitabine-relapsed AML, ASTX029+venetoclax treatment improved survival compared to vehicle (median survival 76.5 days vs. 50 days, p=0.0006) and venetoclax alone (median survival 76.5 days vs. 51.5 days, p=0.0065) (Figure 1) with corresponding reduction in leukemia burden in bone marrow (p<0.0001) and spleen (p<0.0001). CyTOF analysis using PDX bone marrow showed decreased expression of Mcl-1 and pMcl-1-T163 and an increased expression of BIM in response to ERKi+/-venetoclax (Figure 1). To maintain stemness in AML, mitochondrial ROS mitigation and Drp1-mediated mitochondrial fission are crucial (Schimmer et al., Cell Stem Cell. 2018). The inhibition of ERK1/2 resulted in decreased pDrp1-Ser616, along with an increase in mitochondrial length (p<0.001) suggesting impaired mitochondrial fission. This was accompanied by a decrease in mitochondrial membrane potential (p<0.0001) and an increase in mitochondrial ROS (p<0.0001). Overexpression (OE) of a phospho-mimetic i.e. Drp1-Ser616Glu-Ser637Ala led to shorter mitochondrial length (Figure 2), suggesting enhanced fission, a distinct metabolic phenotype with decreased ROS production and decreased mitochondrial depolarization with venetoclax+/- ERKi. Finally, Drp1 phospho-mimetic OE reversed apoptosis induction by venetoclax +/- ERKi (Figure 2) as compared to the wild-type and phospho-null (Ser616Ala) Drp1 (p<0.001) expressing cells, supporting the role of mitochondrial fission in resistance to venetoclax. Conclusion: The increased mitochondrial fission driven by ERK1/2 mediated phosphorylation of Drp1 contributes to venetoclax resistance in AML and inhibiting ERK1/2/Drp1 axis overcomes resistance to venetoclax by inhibiting mitochondrial fission (Figure 2). These data provide a strong rationale for the combination of ERK1/2 and Bcl-2 inhibitors in the treatment of AML.

Venetoclax Resistance Results in Broad Resistance to Majority of Anti-MM Agents Due to the Suppression of Apoptosis but Can be Overcome By BCMA-Targeted Immunotherapy

Venetoclax represents the first example of personalized medicine in multiple myeloma (MM), as it has meaningful clinical activity in patients harboring the t(11;14) translocation. This subgroup of patients generally displays upregulated BCL-2 expression and/or a higher BCL-2/BCL-X L ratio, reflecting an imbalance between cell survival (promoted by BCL-2) and cell death (promoted by BCL-X L). The Bellini study demonstrated significantly superior response rate and progression-free survival (PFS) with combination of Venetoclax and Bortezomib in relapsed myeloma, however, the overall survival in the arm with venetoclax was inferior. Part of the decrease in overall survival (OS) was considered to be related with increased incidence of infections, however, OS is also significantly impacted by response to subsequent therapies. Here, we investigated whether acquired venetoclax resistance could lead to global resistance to multiple subsequent anti-MM therapies and in turn can explain the decreased OS. We exposed venetoclax-sensitive myeloma cells (KMS27 and KMS12PE) to high-dose venetoclax treatment and generated monoclonal drug-tolerant expanded persister (DTEP) clones from single cell with a three- to ten-fold increase in IC50 compared to the parental cells (4 single cell clones for each cell line). Interestingly, the venetoclax resistance evolved without genomic alterations, as the parental and resistant cells did not show marked differences in mutational frequencies or copy number variations, including the absence of genomic alterations in the BCL-2 gene, such as the Gly101Val mutation, 1q21 gain or 8p loss. Consistent with previous literature in other disease models, these resistant clones also exhibited reduced mitochondrial priming and had increased protein expression of anti-apoptotic regulators, including MCL-1, BCL-X L, and BCL-W, thus overcoming the venetoclax-induced inhibition of the anti-apoptotic regulator BCL-2, resulting in the sequestration of the pro-apoptotic ligand BIM. We next evaluated if the altered mitochondrial priming observed in the resistant cells was also responsible for a broader resistance to anti-cancer agents. We observed that venetoclax-resistant clones were resistant to most standard-of-care anti-MM agents including alkylating agents (Melphalan, cyclophosphamide, bendamustine), proteasome inhibitors (bortezomib and carfilzomib) or immunomodulatory drugs (lenalidomide and pomalidomide) than parental cells, suggesting a broad resistance to anti-cancer agents possibly due to reduced apoptotic signaling. This indicates that switching to these agents after the acquisition of venetoclax resistance may not be efficacious. Since these data suggested a functional substitution between BCL-2 family members, we next evaluated if MCL1 or BCL-XL represent a co-dependency in resistant cells that can be therapeutically targeted to overcome resistance. Simultaneous inhibition of MCL1 (via S63845) or BCL-XL (via A155463) and BCL2 (via venetoclax) increased BIM release and enhanced cell death in the resistant clones compared to single agents, with combination index (CI) values < 0.3 in all doses tested. We further investigated the efficacy of immunotherapeutic approaches to overcome venetoclax resistance as these immunotherapies do not rely exclusively on traditional apoptotic signaling to mediate cell death. We observed that both antibody-dependent cellular cytotoxicity (ADCC) induced by Daratumumab and BCMA targeting CAR-T cells induced comparable cell death in venetoclax-resistant clones and parental cells. Moreover, myeloma cells from a patient with t(11;14) MM progressing on venetoclax, showed significant cytotoxicity to anti-BCMA CAR T cells in vitro and achieved a deep response subsequently when treated with Cilta-cel. In conclusion, we report that resistance to venetoclax confers broad resistance to standard-of-care myeloma drugs and that immunotherapies, especially CAR-T cells and CD38 monoclonal antibody, may remain effective, thus conferring potential benefits in managing acquired resistance to venetoclax.

High Expression Level of MMP7 and Mir-489-3p/MMP7 Axis Associates with Venetoclax Resistance in Acute Myelogenous Leukemia

The BCL2 inhibitor venetoclax used to treat acute myelogenous leukemia (AML). Although this has been a promising therapeutic option for patients with AML, many of these patients develop resistance and relapsed disease. Matrix metalloproteinases (MMPs) were demonstrated to have important implications in the progression and invasiveness of many malignant disorders. In contrast, the biological significance of these molecules in AML is not clear. We determined the levels of MMP-7 in the bone marrow of 76 patients with AML. 70 bone marrow donors served as normal controls. The level of MMP-7 was significantly higher in the AML than in the normal controls. MMP-7 levels were significantly lower in the AML patients who achieved a complete remission (CR) than in those who did not. Through in vitro studies, we observed that MMP7 levels were elevated in AML cell lines (U937, THP-1) and in Venetoclax-resistant AML cell lines (U937R, THP-1R) and that downregulation of MMP7 could enhance Venetoclax efficacy in U937R and THP-1R cells. Our in vitro and in vivo investigations show that miR-489-3p was downregulated in Venetoclax-resistant AML cell lines and promoting level of miR-489-3p expression can directly suppressing MMP-7 expression. Pharmacological inhibition of miR-489-3p markedly reversed the effects of Venetoclax-resistant upon AML proliferation and metastasis. Thus, our study suggests that MMP-7 is an essential factor in AML, and regulator the miR-489-3p/MMP7 axis may be a novel treatment strategy for overcoming Venetoclax resistance in AML.

Abstract A35: Overcoming Venetoclax Resistance in Acute Myeloid Leukemia (AML) via the Novel Imipridone ONC213

Abstract Older adults make up most of the AML patient population and have an especially dismal prognosis due to limited therapeutic options. The Bcl-2 inhibitor, venetoclax (VEN), was approved for use in combination with low-dose cytarabine or a hypomethylating agent [azacitidine (AZA) or decitabine] in AML patients who are ≥75 years old or ineligible for intensive chemotherapy (IC). Due to superior survival data and tolerability, the combination of VEN and AZA has become a new standard of care for older patients and those unfit for IC. Though this was a promising therapeutic option for these patients, resistance to this therapy results in a 1-year overall survival rate of only 30-40% and the median overall survival following treatment failure is less than three months, highlighting the urgent need of new therapies to address this. Previous studies have found VEN resistance can be overcome by targeting mitochondria and Mcl-1. Our studies on ONC213, a novel imipridone, have found that it has potent antileukemic activity against both AML cell lines and primary patient samples. It reduces Mcl-1 and targets mitochondria of AML cells – and was thus a promising agent to overcome VEN resistance. When used in combination, ONC213 and VEN are highly synergistic in both AML cell lines and primary patient samples, including those intrinsically resistant to VEN. The combination also targets AML progenitor cells determined by colony formation assays and shows in vivo efficacy in a cell line derived xenograft mouse model (median survival of 57 days vs 37 days in control mice, p=0.002). To determine if ONC213 could overcome acquired resistance to VEN and AZA, we developed VEN and AZA double resistant ML-2 and MV4-11 AML cells (designated ML-2/VEN+AZA-R and MV4-11/VEN+AZA-R) by stepwise increasing concentrations of VEN and AZA at 1:3 fixed ratio over an extended period. ML-2/VEN+AZA-R and MV4-11/VEN+AZA-R AML cells demonstrate 437- and 1156-fold, respectively, increase in VEN+AZA (1:3 ratio) IC50 compared to parental cells (5,285.3 nM vs 12.1 nM and 6,704.3 nM vs 5.8 nM, respectively). ONC213 can resensitize resistant cells to VEN and the combination of ONC213 and VEN is highly synergistic in both ML-2/VEN+AZA-R and MV4-11/VEN+AZA-R cells as evident by a combination index (CI) value of < 0.05. ONC213 alone and in combination with VEN suppress oxidative phosphorylation (OXPHOS) and Mcl-1 in AML cells, including those with inherent resistance to VEN or with acquired resistance to VEN+AZA. Overexpression of Mcl-1 can partially rescue cells from combination therapy. These results suggest that ONC213 suppresses OXPHOS and Mcl-1 to overcome VEN resistance. Here we demonstrate that VEN resistance can be addressed through dual targeting of mitochondria and Mcl-1 using the novel imipridone, ONC213. ONC213 resensitizes VEN + AZA resistant cells to VEN and offers a new approach to combat VEN+AZA resistance in AML. Citation Format: Jenn L. Carter, Yongwei Su, Holly Edwards, Lisa Polin, Joshua E. Allen, Varun V. Prabhu, Jay Yang, Maik Huettermann, Yubin Ge. Overcoming Venetoclax Resistance in Acute Myeloid Leukemia (AML) via the Novel Imipridone ONC213 [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 A35.

Abstract 3860: ERK1/2 inhibition overcomes resistance to venetoclax in AML by altering mitochondrial metabolism

Abstract Background: Primary or secondary resistance to venetoclax is pervasively associated with mutational/non-mutational activation of RAS/RAF/ERK pathway which confers metabolic alterations as a compensatory adaptation. Preclinical models of venetoclax resistance and primary patient samples (n=8) were used to assess the synergy of concomitant Bcl2 and ERK1/2 inhibition followed by RNAseq analysis to identify molecular mechanisms responsible for overcoming resistance to venetoclax. Results: ERK1/2 inhibition using Compound 27 (ERKi, Heightman et al., J Med Chem. 2018), an analog of ASTX029, was highly synergistic with venetoclax at inducing apoptosis in AML. NRAS-mutant OCI-AML3 and THP1 cells, that are inherently resistant to venetoclax, were sensitized to venetoclax when combined with ERKi with ~90% apoptosis compared to 20% by venetoclax alone (Combination Index = 0.008). Synergy was also confirmed using isogenic OCI-AML2 cells with lab-generated acquired resistance to venetoclax, resulting in 75% apoptosis versus 22% by venetoclax (CI=0.6). Leukemia progenitor cells in primary AML samples were also depleted by the ven+ERKi combination (CI:0.03-0.23). In line with our in-vitro findings, treatment with ASTX029 as a single agent or in combination with venetoclax significantly inhibited leukemia burden and increased survival in an OCIAML3 xenograft model (p<0.05). Single agent ERKi and combination treatment using an NRAS-mutant PDX resulted in significantly impaired clonogenic potential and decreased expression of cMyc and CD44 in CD34+CD38- population as demonstrated by CyTOF, indicating depletion of progenitors. Transcriptomic profiling of OCIAML3 cells revealed upregulation of 6 pathways in response to venetoclax including oxidative phosphorylation (OXPHOS), electron transport chain, Myc targets, E2F targets, epithelial mesenchymal transition and KRAS signaling. Interestingly, these pathways were downregulated after ERKi+venetoclax treatment along with decrease in glycolysis and fatty acid metabolism (FDR<0.05). Since, venetoclax treatment significantly enriched for OXPHOS, we examined the metabolic effect of ERKi to overcome venetoclax resistance. Seahorse analysis showed increased basal and maximal respiration, and ATP production in OCIAML2 venetoclax resistant versus parental cells. Combining ERKi with venetoclax reduced OCR and ATP production in OCIAML3 and OCIAML2-venetoclax resistant cells. This was accompanied by an increase in membrane depolarization (p=0.001) as well as enhanced mitochondrial ROS (p<0.001). Conclusion: The inhibition of ERK1/2 alters mitochondrial metabolism and functional integrity to overcome resistance to venetoclax by causing apoptosis in venetoclax resistant AML cells. These data provide a strong rationale for the combination of ERK1/2 and Bcl-2 inhibitors in treatment of AML and warrant further investigation in the clinic. Citation Format: Priyanka Sharma, Lauren Ostermann, Sujan Piya, Natalia Baran, Anudishi Tyagi, Christopher Hindley, Kim-Hien Dao, Martin Sims, V. Lokesh Battula, Michael Andreeff, Gautam Borthakur. ERK1/2 inhibition overcomes resistance to venetoclax in AML by altering mitochondrial metabolism. [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 3860.

Dual targeting of BCL-2 and MCL-1 in the presence of BAX breaks venetoclax resistance in human small cell lung cancer.

No targeted drugs are currently available against small cell lung cancer (SCLC). BCL-2 family members are involved in apoptosis regulation and represent therapeutic targets in many malignancies. Expression of BCL-2 family members in 27 SCLC cell lines representing all known four SCLC molecular subtypes was assessed by qPCR, Western blot and mass spectrometry-based proteomics. BCL-2 and MCL-1 inhibition (venetoclax and S63845, respectively) was assessed by MTT assay and flow cytometry and in mice bearing human SCLC tumours. Drug interactions were calculated using the Combenefit software. Ectopic BAX overexpression was achieved by expression plasmids. The highest BCL-2 expression levels were detected in ASCL1- and POU2F3-driven SCLC cells. Although sensitivity to venetoclax was reflected by BCL-2 levels, not all cell lines responded consistently despite their high BCL-2 expression. MCL-1 overexpression and low BAX levels were both characteristic for venetoclax resistance in SCLC, whereas the expression of other BCL-2 family members did not affect therapeutic efficacy. Combination of venetoclax and S63845 resulted in significant, synergistic in vitro and in vivo anti-tumour activity and apoptosis induction in double-resistant cells; however, this was seen only in a subset with detectable BAX. In non-responding cells, ectopic BAX overexpression sensitised to venetoclax and S63845 and, furthermore, induced synergistic drug interaction. The current study reveals the subtype specificity of BCL-2expression and sheds light on the mechanism of venetoclax resistance in SCLC. Additionally, we provide preclinical evidence that combined BCL-2 and MCL-1 targeting is an effective approach to overcome venetoclax resistance in high BCL-2-expressing SCLCs with intact BAX.

Augmenting Venetoclax Activity Through Signal Transduction in AML.

Venetoclax, a small-molecule B-cell lymphoma 2 (BCL-2) inhibitor, selectively eradicates leukemic stem cells (LSCs). While venetoclax has revolutionized the treatment of acute myeloid leukemia (AML), treatment failure and disease relapse are common. Mechanisms underlying venetoclax resistance are surprisingly heterogeneous. Venetoclax resistance encompasses a spectrum of genetic and epigenetic changes, with numerous pathways contributing to the upregulation of additional anti-apoptotic proteins. In this review, we address the mechanisms of venetoclax resistance in the context of signal transduction. We emphasize how aberrant cell signaling impairs apoptosis and predisposes to venetoclax failure. Commonly activated pathways, such as FLT3, PI3K/AKT/mTOR, and RAS, contribute to upregulated anti-apoptotic mediators and are frequently responsible for refractory disease or disease relapse. We highlight novel combination strategies aimed at disabling constitutively active signal transduction to augment response and overcome venetoclax resistance.