Combination with Palbociclib Overcomes Venetoclax Resistance Mechanisms and Outperforms Single Agent Efficacy in Acute Myeloid Leukemia

Field: Pharmacology, Toxicology and Pharmaceutics (Life Sciences)

Introduction: Initial remission rates for acute myeloid leukemia (AML) patients treated with venetoclax (ven) plus azacytidine have been promising (Di Nardo et al, 2019), however, drug resistance and disease relapse continue to be a major hurdle mitigating long-term treatment and overall survival. Understanding the complex genetic and biological factors driving drug response is required to advance new therapeutic options for this heterogeneous disease. Recent studies from our lab identified novel drug combinations with ven, showing the CDK4/6 inhibitor, palbociclib (palbo) as one of the most effective partner agents (Eide et al, 2023). Investigation of the mechanisms of this drug combination may inform treatment strategies for patients with ven-resistant AML and provide better outcomes for patients. Methods: To identify drugs that may be more effective in combination with ven, we analyzed samples from a large cohort of AML patients treated ex vivo with ven, the partner single agent, or the combination. We used primary AML cells in patient derived xenograft (PDX) mouse models to evaluate the combination efficacy vs. single agents in reducing tumor burden. To elucidate the mechanism of this combination, we performed a genome-wide CRISPR screen to identify genes involved in resistance to palbo, ven, or the combination. Knockout (KO) of genes that led to drug resistance were generated in AML cell lines to validate screen hits and examine mechanisms of single agent and combination drug response and resistance. Results: The combination of ven+palbo showed greater efficacy than either single agent in ex vivo treated primary AML patient samples and was among the most efficacious of 25 ven inclusive combinations tested. Treatment with ven+palbo reduced tumor burden more than either single agent in PDX mouse models as assessed by WBC count and spleen weight. Western blot analysis of AML cell lines treated with ven+palbo showed increased PMAIP1 expression and decreased MCL1 expression compared to single agents. Treatment with palbo may sensitize cells to ven mediated apoptosis through the upregulation of PMAIP1, which binds to MCL1, since upregulation of MCL1 has been shown to cause ven resistance (Ramsey et al 2019). Genome wide CRISPR screens in OCI-AML2 cells identified several genes involved in resistance to single agents or the combination. Resistance screens in the presence of palbo showed loss of many genes involved in cell cycle regulation conferred palbo resistance, including Rb1, loss of which was previously reported to cause resistance to palbo in breast cancer patients (Condorelli et al 2018). Resistance screens in the presence of ven show loss of BAX and PMAIP previously reported to cause ven resistance (Nechiporuk et al 2019, Moujalled et al Blood 2023). Interestingly, loss of BAX led to enhanced sensitivity to palbo as well as abemaciclib, another FDA approved CDK4/6 inhibitor, indicating CDK4/6 inhibitors could be beneficial treatments for patients who have acquired ven resistance via BAX mutation. Resistance screens in the presence of ven+palbo revealed IKZF1 as a top hit. IKZF1 has a well-established role in the pathogenesis of acute lymphoblastic leukemia, and its role in AML is increasingly emerging (Wang et al 2023). RNA-seq analysis of IKZF1 KO cells showed a shift to a monocytic cell signature and increased expression of AXL RNA; a shift also seen at the protein level. Additionally, AXL transcript expression is significantly increased in IKZF1-mutated AML patient samples compared to WT. We found that IKZF1 KO cells are sensitive to several AXL inhibitors, offering a potential therapeutic approach for patients who develop resistance to the ven+palbo combination and/or patient subsets that harbor an IKZF1 mutation. Conclusion: Combination therapy of ven+palbo shows greater efficacy than either single agent in patient samples and PDX mouse models. A greater understanding of the ven+palbo efficacy and mechanisms of resistance has helped to inform the subsets of patients who may benefit from treatment with ven+palbo as well as identify additional therapies that may boost efficacy and mitigate resistance. This study provides a promising therapeutic strategy for improving the long-term treatment and survival of AML patients and highlights novel potential drug repurposing: AXL inhibitors to treat AML patients harboring an IKZF1 mutation; CDK4/6 inhibitors for AML with BAX mutation.