Venetoclax Resistance Research Articles

Venetoclax triggers sublethal apoptotic signaling in venetoclax-resistant acute myeloid leukemia cells and induces vulnerability to PARP inhibition and azacitidine

Venetoclax plus azacitidine treatment is clinically beneficial for elderly and unfit acute myeloid leukemia (AML) patients. However, the treatment is rarely curative, and relapse due to resistant disease eventually emerges. Since no current clinically feasible treatments are known to be effective at the state of acquired venetoclax resistance, this is becoming a major challenge in AML treatment. Studying venetoclax-resistant AML cell lines, we observed that venetoclax induced sublethal apoptotic signaling and DNA damage even though cell survival and growth were unaffected. This effect could be due to venetoclax inducing a sublethal degree of mitochondrial outer membrane permeabilization. Based on these results, we hypothesized that the sublethal apoptotic signaling induced by venetoclax could constitute a vulnerability in venetoclax-resistant AML cells. This was supported by screens with a broad collection of drugs, where we observed a synergistic effect between venetoclax and PARP inhibition in venetoclax-resistant cells. Additionally, the venetoclax-PARP inhibitor combination prevented the acquisition of venetoclax resistance in treatment naïve AML cell lines. Furthermore, the addition of azacitidine to the venetoclax-PARP inhibitor combination enhanced venetoclax induced DNA damage and exhibited exceptional sensitivity and long-term responses in the venetoclax-resistant AML cell lines and samples from AML patients that had clinically relapsed under venetoclax-azacitidine therapy. In conclusion, we mechanistically identify a new vulnerability in acquired venetoclax-resistant AML cells and identify PARP inhibition as a potential therapeutic approach to overcome acquired venetoclax resistance in AML.

Acquired BCL2 variants associated with venetoclax resistance in acute myeloid leukemia

We report and characterize three venetoclax-resistant BCL2 variants arising during venetoclax/azacitidine therapy in acute myeloid leukemia (AML). Our results indicate the potential for on-target venetoclax resistance in patients with AML at relapse.

KS18, a Mcl-1 inhibitor, improves the effectiveness of bortezomib and overcomes resistance in refractory multiple myeloma by triggering intrinsic apoptosis.

Despite a record number of clinical studies investigating various anti-myeloma treatments, the 5-year survival rate for multiple myeloma (MM) patients in the US is only 55%, and almost all patients relapse. Poor patient outcomes demonstrate that myeloma cells are "born to survive" which means they can adapt and evolve following treatment. Thus, new therapeutic approaches to combat survival mechanisms and target treatment resistance are required. Importantly, Mcl-1, anti-apoptotic protein, is required for the development of MM and treatment resistance. This study looks at the possibility of KS18, a selective Mcl-1 inhibitor, to treat MM and overcome resistance. Our investigation demonstrates that KS18 effectively induces cell death in MM by dual regulatory mechanisms targeting the Mcl-1 protein at both transcriptional and post-translational levels. Specifically, KS18 suppresses Mcl-1 activation via STAT-3 pathway and promotes Mcl-1 phosphorylation/ubiquitination/proteasome-dependent protein degradation (UPS). Significantly, KS18 triggered caspase-dependent apoptosis in MM patient samples and bortezomib-resistant cells, synergizing with venetoclax to boost apoptosis. KS18 promises to overcome bortezomib and venetoclax resistance and re-sensitize myeloma cells to chemotherapy. Furthermore, the study shows the tremendous impact of KS18 in inhibiting colony formation in bortezomib-resistant cells and demonstrates significant tumor shrinkage in KS18-treated NSG mice without notable toxicity signs after 4weeks of therapy with a single acceptable dose each week, indicating its powerful anti-neoplastic and anti-resistance characteristics. This study strongly implies that KS18 may treat MM and provide new hope to patients who are experiencing recurrence or resistance.

Anti-CLL1 liposome loaded with miR-497-5p and venetoclax as a novel therapeutic strategy in acute myeloid leukemia

Acute myeloid leukemia (AML) is a lethal hematologic malignancy. Chemotherapy resistance results in a dismal survival of 1-2 years in older adults with AML. Therefore, novel therapies are urgently required. In this context, microRNA (miRNA)-based treatments remain an untapped strategy in AML. Using patient-derived specimens, we found increased inflammatory cytokines including IL-6 in serum of older adults with AML, and decreased miR-497-5p in CD34+ leukemic blasts. Target prediction revealed that miR-497-5p could directly target mitogen-activated protein kinase-1 (MAP2K1) mRNA, to indirectly target cytokines and JAK/STAT signaling pathway through p38-MAPK signaling pathway, potentially inhibiting leukemic growth, and overcoming chemoresistance from venetoclax. To improve miRNA delivery and minimize off-target effects, which represent key barriers to clinical translation, we developed liposomes for co-delivery of miR-497-5p and venetoclax. We decorated our liposomes with a peptide targeting CLL1, which is present on 92% leukemia blasts while being absent in normal hematopoietic cells. This targeted approach demonstrated high efficacy in inhibiting AML growth in mice with minimal toxicity, as well as reduced exposure to chemoresistance. Our findings suggested that anti-CLL1-decorated, miR-497-5p and venetoclax-loaded liposomes represent a promising novel miRNA-based therapeutic, which should be further investigated as a strategy to reduce venetoclax resistance in AML.

AML-444 A Covalent CDK7 Inhibitor Overcomes Venetoclax Resistance and Induces Apoptosis via C-Myc/p53 Axis in Acute Myeloid Leukemia

AML-444 A Covalent CDK7 Inhibitor Overcomes Venetoclax Resistance and Induces Apoptosis via C-Myc/p53 Axis in Acute Myeloid Leukemia

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

A Covalent CDK7 Inhibitor Overcomes Venetoclax Resistance and Induces Apoptosis via C-Myc/p53 Axis in Acute Myeloid Leukemia

A Covalent CDK7 Inhibitor Overcomes Venetoclax Resistance and Induces Apoptosis via C-Myc/p53 Axis in Acute Myeloid Leukemia

Targeting Acute Myeloid Leukemia Stem Cells through Perturbation of Mitochondrial Calcium.

Acute myeloid leukemia stem cells (LSCs) are uniquely reliant on oxidative phosphorylation (OXPHOS) for survival. Moreover, maintenance of OXPHOS is dependent on BCL-2, creating a therapeutic opportunity to target LSCs using the BCL-2 inhibitor venetoclax. Although venetoclax-based regimens have shown promising clinical activity, the emergence of drug resistance is prevalent. Thus, in the present study, we investigated how mitochondrial properties may influence venetoclax responsiveness. Our data show that utilization of mitochondrial calcium is fundamentally different between drug-responsive and nonresponsive LSCs. By comparison, venetoclax-resistant LSCs demonstrate an active metabolic (i.e., OXPHOS) status with relatively high levels of calcium. Consequently, we tested genetic and pharmacological approaches to target the mitochondrial calcium uniporter. We demonstrate that inhibition of calcium uptake reduces OXPHOS and leads to eradication of venetoclax-resistant LSCs. These findings demonstrate a central role for calcium signaling in LSCs and provide an avenue for clinical management of venetoclax resistance. Significance: We identify increased utilization of mitochondrial calcium as a distinct metabolic requirement of venetoclax-resistant LSCs and demonstrate the potential of targeting mitochondrial calcium uptake as a therapeutic strategy.

Design, synthesis, and biological activity evaluation of novel HDAC3 selective inhibitors for combination with Venetoclax against acute myeloid leukemia

Histone deacetylases (HDACs) are highly attractive targets in the drug development process, and the development of subtype-selective HDAC inhibitors is the research direction for HDAC inhibitors. As an important member of the HDAC family, HDAC3 has been found to be closely related to the pathological progression of many diseases due to its abnormal expression. In previous studies, we discovered compound 13a, which has potent inhibitory activity against HDAC1, 2, and 3. In this work, we improved the HDAC3 isotype selectivity of 13a, and obtained compound 9c through rational drug design. 9c shows a selectivity of 71 fold for HDAC3 over HDAC1 and can significantly inhibit the proliferation activity of MV4-11 cells in vitro. Furthermore, when combined with Venetoclax, 9c can effectively induce apoptosis in MV4-11 cells in vitro and reduce the expression of anti-apoptotic proteins, the development of HDAC3 selective inhibitors may serve as a potential lead compound to reverse Venetoclax resistance.

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.

Venetoclax resistance in acute lymphoblastic leukemia is characterized by increased mitochondrial activity and can be overcome by co-targeting oxidative phosphorylation

Deregulated apoptosis signaling is characteristic for many cancers and contributes to leukemogenesis and treatment failure in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Apoptosis is controlled by different pro- and anti-apoptotic molecules. Inhibition of anti-apoptotic molecules like B-cell lymphoma 2 (BCL-2) has been developed as therapeutic strategy. Venetoclax (VEN), a selective BCL-2 inhibitor has shown clinical activity in different lymphoid malignancies and is currently evaluated in first clinical trials in BCP-ALL. However, insensitivity to VEN has been described constituting a major clinical concern. Here, we addressed and modeled VEN-resistance in BCP-ALL, investigated the underlying mechanisms in cell lines and patient-derived xenograft (PDX) samples and identified potential strategies to overcome VEN-insensitivity. Leukemia lines with VEN-specific resistance were generated in vitro and further characterized using RNA-seq analysis. Interestingly, gene sets annotated to the citric/tricarboxylic acid cycle and the respiratory electron transport chain were significantly enriched and upregulated, indicating increased mitochondrial metabolism in VEN-resistant ALL. Metabolic profiling showed sustained high mitochondrial metabolism in VEN-resistant lines as compared to control lines. Accordingly, primary PDX-ALL samples with intrinsic VEN-insensitivity showed higher oxygen consumption and ATP production rates, further highlighting that increased mitochondrial activity is a characteristic feature of VEN-resistant ALL. VEN-resistant PDX-ALL showed significant higher mitochondrial DNA content and differed in mitochondria morphology with significantly larger and elongated structures, further corroborating our finding of augmented mitochondrial metabolism upon VEN-resistance. Using Oligomycin, an inhibitor of the complex V/ATPase subunit, we found synergistic activity and apoptosis induction in VEN-resistant BCP-ALL cell lines and PDX samples, demonstrating that acquired and intrinsic VEN-insensitivity can be overcome by co-targeting BCL-2 and the OxPhos pathway. These findings of reprogrammed, high mitochondrial metabolism in VEN-resistance and synergistic activity upon co-targeting BCL-2 and oxidative phosphorylation strongly suggest further preclinical and potential clinical evaluation in VEN-resistant BCP-ALL.

Discovery of a Covalent Inhibitor That Overcame Resistance to Venetoclax in AML Cells Overexpressing BFL-1.

Clinical and biological studies have shown that overexpression of BFL-1 is one contributing factor to venetoclax resistance. The resistance might be overcome by a potent BFL-1 inhibitor, but such an inhibitor is rare. In this study, we show that 56, featuring an acrylamide moiety, inhibited the BFL-1/BID interaction with a Ki value of 105 nM. More interestingly, 56 formed an irreversible conjugation adduct at the C55 residue of BFL-1. 56 was a selective BFL-1 inhibitor, and its MCL-1 binding affinity was 10-fold weaker, while it did not bind BCL-2 and BCL-xL. Mechanistic studies showed that 56 overcame venetoclax resistance in isogenic AML cell lines MOLM-13-OE and MV4-11-OE, which both overexpressed BFL-1. More importantly, 56 and venetoclax combination promoted stronger apoptosis induction than either single agent. Collectively, our data show that 56 overcame resistance to venetoclax in AML cells overexpressing BFL-1. These attributes make 56 a promising candidate for future optimization.

Venetoclax resistance leads to broad resistance to standard-of-care anti-MM agents, but not to immunotherapies

To our knowledge, venetoclax is the first example of personalized medicine for multiple myeloma (MM), with meaningful clinical activity as a monotherapy and in combination in patients with myeloma harboring the t(11:14) translocation. However, despite the high response rates and prolonged progression-free survival, a significant proportion of patients eventually relapse. Here, we aim to study adaptive molecular responses after the acquisition of venetoclax resistance in sensitive t(11:14) MM cell models. We therefore generated single-cell venetoclax-resistant t(11:14) MM cell lines and investigated the mechanisms contributing to resistance as well as the cells' sensitivity to other treatments. Our data suggest that acquired resistance to venetoclax is characterized by reduced mitochondrial priming and changes in B-cell lymphoma-2 (BCL-2) family proteins' expression in MM cells, conferring broad resistance to standard-of-care antimyeloma drugs. However, our results show that the resistant cells are still sensitive to immunotherapeutic treatments, highlighting the need to consider appropriate sequencing of these treatments after venetoclax-based regimens.

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.

Synergistic effects of RPT1G, a first-in-class small molecule NAMPT inhibitor, with venetoclax and olaparib in hematological malignancies.

e18512 Background: MultipleBCL-2 and PARP inhibitors are currently in clinical trials, or have been approved for use, in hematological malignancies and solid tumors. Venetoclax, an inhibitor of the apoptosis regulator B-cell lymphoma-2 (BCL-2), has activity in patients with chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). PARP inhibitors, such as Olaparib, exploit synthetic lethality in homologous recombination-deficient tumors, notably in breast and ovarian cancers. However, patients treated with BCL-2 or PARP inhibitors often face relapse and refractory disease, requiring alternative strategies to counter adaptive responses. Nicotinamide Phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the nicotinamide adenine dinucleotide (NAD+) salvage pathway, is upregulated in malignancies and plays a key role in cancer metabolism (e.g., Warburg effect). NAMPT makes nicotinamide mononucleotide which is then converted to NAD+, while PARP enzymes consume NAD+, placing NAMPT directly upstream of PARP. NAMPT overexpression is associated with Venetoclax resistance in leukemia patients, highlighting the link between NAD+, mitochondrial metabolism, and BCL-2 inhibition. Consequently, NAMPT inhibition may synergize with BCL-2 or PARP inhibitors, potentially enhancing 1st-line of treatment outcomes and re-sensitizing refractory cancer cells to BCL-2 or PARP inhibition. Methods: In this study, we investigated the combination effects of Venetoclax and Olaparib with RPT1G, a novel hyperbolic NAMPT inhibitor that simultaneously inhibits and stabilizes NAMPT in a catalytically active state. RPT1G disrupts NAMPT activity without ever turning it off completely avoiding the severe on-target toxicities seen with complete NAMPT inhibitors. Several cell lines, including OCI-AML3 and MV4;11 (AML), RS4;11 (ALL), and RL (diffuse large B-cell lymphoma) were treated with RPT1G and either Venetoclax or Olaparib to determine the degree of drug interactions. In a follow up in vivo study, we treated mice in a human xenograft model using semi-resistant MV4;11 cells with RPT1G alone or in combination with Venetoclax to measure the ability of RPT1G to enhance the effects of Venetoclax. Results: RPT1G achieves significant single-agent efficacy in leukemia and lymphoma mouse xenograft models. When RTP1G is used in combination with either Venetoclax or Olaparib significant synergy is seen across a broad range of concentrations in vitro. Consistent with in vitro synergy results, in a xenograft model of human AML, mice treated with a combination of RPT1G and Venetoclax show enhanced efficacy as compared to Venetoclax alone. Conclusions: Combination of RPT1G with either Venetoclax or Olaparib synergistically enhances killing of leukemic cells and presents as an effective strategy to improve treatment outcomes for patients with diverse malignancies.

A transcriptomic based deconvolution framework for assessing differentiation stages and drug responses of AML

The diagnostic spectrum for AML patients is increasingly based on genetic abnormalities due to their prognostic and predictive value. However, information on the AML blast phenotype regarding their maturational arrest has started to regain importance due to its predictive power for drug responses. Here, we deconvolute 1350 bulk RNA-seq samples from five independent AML cohorts on a single-cell healthy BM reference and demonstrate that the morphological differentiation stages (FAB) could be faithfully reconstituted using estimated cell compositions (ECCs). Moreover, we show that the ECCs reliably predict ex-vivo drug resistances as demonstrated for Venetoclax, a BCL-2 inhibitor, resistance specifically in AML with CD14+ monocyte phenotype. We validate these predictions using LUMC proteomics data by showing that BCL-2 protein abundance is split into two distinct clusters for NPM1-mutated AML at the extremes of CD14+ monocyte percentages, which could be crucial for the Venetoclax dosing patients. Our results suggest that Venetoclax resistance predictions can also be extended to AML without recurrent genetic abnormalities and possibly to MDS-related and secondary AML. Lastly, we show that CD14+ monocytic dominated Ven/Aza treated patients have significantly lower overall survival. Collectively, we propose a framework for allowing a joint mutation and maturation stage modeling that could be used as a blueprint for testing sensitivity for new agents across the various subtypes of AML.

PICALM::MLLT10 may indicate a new subgroup of acute leukemias with miscellaneous immunophenotype and poor initial treatment response but showing sensitivity to venetoclax.

The PICALM::MLLT10 fusion gene is a rare but recurrent event in acute leukemia (AL) associated with poor prognosis. It is still confused whether PICALM::MLLT10 can solely correspond to acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL) or acute leukemias of ambiguous lineage (ALAL). Here, we reported a series of PICALM::MLLT10 positive AL patients with miscellaneous immunophenotype including T-ALL, ALAL, AML, and B-ALL, complex karyotype, half of extramedullary disease (EMD), frequently concomitant PHF6 mutation, and poor initial treatment response to standard chemotherapy aiming to different immunophenotype, but showing sensitivity to combining chemotherapy especially integrated with venetoclax, suggesting this fusion gene may indicate a new subgroup of AL. Eighteen PICALM::MLLT10 positive patients of 533 AL patients (18/533, 3.4%) were identified by RNA sequencing in our center. We found PICALM::MLLT10 positive AL showing miscellaneous immunophenotype, higher expression of leukemic stemness genes and lower expression of biomarkers of venetoclax resistance, more extramedullary involvement, and especially poor response to conventional induction chemotherapy, but may benefit from venetoclax as well as low-dose Ara-C, granulocyte colony-stimulating factor (G-CSF), and anthracyclines combination chemotherapy. Sequential hematopoietic stem cell transplantation (HSCT) after chemotherapy combined with venetoclax may further improve long-term survival in AL patients with complete remission (CR) even measurable residual disease (MRD) positive.

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.

Blockage of BCL-XL overcomes venetoclax resistance across BCL2+ lymphoid malignancies irrespective of BIM status

AbstractVenetoclax (VEN), a B-cell lymphoma 2 (BCL2) inhibitor, has a promising single-agent activity in mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), and large BCLs, but remissions were generally short, which call for rational drug combinations. Using a panel of 21 lymphoma and leukemia cell lines and 28 primary samples, we demonstrated strong synergy between VEN and A1155463, a BCL-XL inhibitor. Immunoprecipitation experiments and studies on clones with knockout of expression or transgenic expression of BCL-XL confirmed its key role in mediating inherent and acquired VEN resistance. Of note, the VEN and A1155463 combination was synthetically lethal even in the cell lines with lack of expression of the proapoptotic BCL2L11/BIM and in the derived clones with genetic knockout of BCL2L11/BIM. This is clinically important because BCL2L11/BIM deletion, downregulation, or sequestration results in VEN resistance. Immunoprecipitation experiments further suggested that the proapoptotic effector BAX belongs to principal mediators of the VEN and A1155463 mode of action in the BIM-deficient cells. Lastly, the efficacy of the new proapoptotic combination was confirmed in vivo on a panel of 9 patient–derived lymphoma xenografts models including MCL (n = 3), B-ALL (n = 2), T-ALL (n = 1), and diffuse large BCL (n = 3). Because continuous inhibition of BCL-XL causes thrombocytopenia, we proposed and tested an interrupted 4 days on/3 days off treatment regimen, which retained the desired antitumor synergy with manageable platelet toxicity. The proposed VEN and A1155463 combination represents an innovative chemotherapy-free regimen with significant preclinical activity across diverse BCL2+ hematologic malignancies irrespective of the BCL2L11/BIM status.