Inhibition of ERK1/2 Reverses Venetoclax-Induced Autophagy to Overcome Resistance in Acute Myeloid Leukemia

Background: Activation of the RAS/MAPK pathway confers inherent/acquired resistance to the Bcl-2 inhibitor, venetoclax, in AML (Zhang et al., Signal Transduct Target Ther. 2022). Pro-survival Bcl-2 inhibits apoptosis as well as autophagy. Phosphorylation of Bcl-2 by ERK1/2 leads to dissociation of the autophagic protein Beclin 1 from Bcl-2 and promotes autophagy (Pattingre et al., Cell. 2005). Thus, while Bcl-2 inhibition can induce apoptosis, its inhibition leads to induction of autophagy (Pattingre et al., Cell. 2005) that can protect tumor cells from organelle damage and nutrient deprivation.

Objective: We hypothesized that terminal MAPK pathway inhibition using an ERK1/2 inhibitor would reverse venetoclax-induced autophagy and synergistically lead to cell death/apoptosis.

Methods: We inhibited ERK1/2 using Compound 27 (Heightman et al., J Med Chem. 2018), a close analog of ASTX029, which is a dual-mechanism ERK1/2 inhibitor currently under clinical investigation in solid tumors (NCT03520075). We studied the potential synergy of ERK1/2 and Bcl-2 inhibition in decreasing cell proliferation and inducing apoptosis and in overcoming venetoclax resistance in AML preclinical models and primary patient samples (n=8). We performed simultaneous analysis of cell proliferation, stress response and DNA damage using single cell proteomics analysis to understand the mechanism of synergy. Autophagy was measured by lipidated LC3 quantification (flow cytometry and immunoblotting). Colony formation assay using NRAS-mutant PDX was performed to analyze the effect on progenitor cells. For insights into the mechanisms of synergy, we conducted large-scale proteomics analyses by single cell mass cytometry (CyTOF) and simultaneously measured cell proliferation, stress response, DNA damage and Bcl-2 family proteins.

Results: Inhibition of ERK1/2 sensitized OCI-AML3 (NRAS-mutant) cells (Combination Index [CI]: 0.008) and venetoclax-resistant OCI-AML2 cells (CI: 0.05) to venetoclax and shifted the cytostatic effect of the single agents to a cytotoxic effect. Combination treatment induced apoptosis in leukemia initiating progenitor cells (LICs, CD34+CD38-) in primary AML samples with 54% apoptosis in combination versus 10% with venetoclax alone (CI = 0.03-0.23), while sparing normal CD34+CD38- cells.

Mechanistically, basal ERK1/2 phosphorylation and autophagy (LC3 lipidation), were higher in venetoclax-resistant OCI-AML2 cells versus parental cells (p=0.0008) and both were inhibited with ERK1/2 inhibition alone and in combination with venetoclax. Simultaneous analysis of cell proliferation, stress response and DNA damage showed downregulation of proliferation (Ki-67) and an increase in ATF4 in response to ERK1/2 inhibition. The combination of Compound 27 and venetoclax inhibited autophagy (lipidated LC3) and increased apoptosis (increased cleaved PARP and cleaved caspase 3, Fig 1a).

Venetoclax-induced autophagy was also confirmed in OCI-AML3 cells at 24 h post treatment (p=0.03). ERK1/2 inhibition reversed venetoclax-induced autophagy in OCI-AML3 (p=0.0003) and OCI-AML2-venetoclax-resistant cells (p=0.0007). CyTOF analysis showed decreased p-AMPK in response to ERK1/2 inhibition in OCI-AML3 cells. As AMPK/FIS1-mediated mitochondrial autophagy is required for self-renewal of AML stem cells (Pie et al., Cell Stem Cell. 2018), we confirmed decreased clonogenic potential of AML progenitor cells by reduced colony formation in response to ERK1/2 inhibition using an NRAS-mutant AML PDX model (p=0.0002). Single cell proteomics (CyTOF) showed decreased p-AMPK in response to single agent ERK1/2 inhibition or the combination with venetoclax in LIC populations (CD34+CD38-) in the NRAS-mutant AML PDX model. In addition to p-AMPK the expression of cMyc and CD44 was also reduced in the combination treatment, indicating reduced stemness (Fig 1b).

Conclusion: Mechanistically, our results indicate that pharmacological inhibition of ERK1/2 impairs autophagy and sensitizes venetoclax-resistant AML cells to apoptosis, thus overcoming primary and secondary resistance to venetoclax. In addition, combination treatment induced apoptosis of LICs and reduced clonogenic potential and markers of stem cell function. 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.

Hindley:Astex Pharmaceuticals: Current Employment. Dao:Astex Pharmaceuticals, Inc.: Current Employment. Sims:Astex Pharmaceuticals: Current Employment. Andreeff:Brooklyn ITX: Research Funding; Glycomimetics: Consultancy; Aptose: Consultancy, Membership on an entity's Board of Directors or advisory committees; Senti Bio: Consultancy, Research Funding; Syndax: Consultancy, Research Funding; Pinot Bio: Research Funding; Medicxi: Consultancy; Oxford Biomedical UK: Research Funding; Reata: Current holder of stock options in a privately-held company; AstraZeneca: Research Funding; Breast Cancer Research Foundation: Research Funding; Daiichi-Sankyo Inc.: Consultancy, Research Funding; Kintor Pharmaceutical: Research Funding; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; NCI: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; Oncolyze: Current holder of stock options in a privately-held company; Chimerix: Current holder of stock options in a privately-held company. Borthakur:Catamaran Bio, Abbvie, PPD Development, Protagonist Therapeutics, Janssen: Consultancy; Pacylex, Novartis, Cytomx, Bio Ascend: Membership on an entity's Board of Directors or advisory committees; Astex Pharmaceuticals, Ryvu, PTC Therapeutics: Research Funding.