Upregulation of MAPK/MCL-1 Maintaining Mitochondrial Oxidative Phosphorylation Confers Acquired Resistance to BCL-2 Inhibitor Venetoclax in AML
Authors: Zhang Qi , Han Lina , Shi Ce , Pan Rongqing , MA Man Chun John , Ryan Jeremy , Kurtz Stephen E , Mu Hong , Ruvolo Vivian , Ma Helen , Andreeff Michael , Mi Yingchang , Tyner Jeffrey W. , Leverson Joel , Letai Anthony , Davis Richard Eric , Konopleva Marina 
Published: November 2019

Abstract
ABT-199 (venetoclax), a selective small-molecule antagonist of the anti-apoptotic protein BCL-2, enables the activation of pro-apoptotic proteins and the induction of cancer cell death. Our previous studies found that AML is a BCL-2 dependent disease and responds robustly to venetoclax by induction of apoptotic cell death (Pan et al., Cancer Discovery 2014). Despite initial responses to single agent venetoclax in a Phase II trial of relapsed AML, patients ultimately developed resistance and progressed (Konopleva et al., Cancer Discovery 2016). In this study we investigated mechanisms of acquired resistance to venetoclax in preclinical AML models.
First, we generated 5 AML cell lines resistant to ³1µM venetoclax. No BAX (exon5 and 6) or BCL2 (exon2) mutations were found in resistant cells. Immunoblotting analysis demonstrated increased expression of anti-apoptotic proteins MCL-1, BCL-2 A1, and BCL-XL, and a decrease of pro-apoptotic PUMA protein in selected resistant cell lines. To probe the functional interactions between the pro- and anti-apoptotic proteins, we next performed co-immunoprecipitation (co-IP) studies. The anti-BIM and anti-MCL-1 co-IPs revealed reduced levels of BIM:BCL-2 complexes and increased BIM:MCL-1 complexes in resistant cells compared to their parental counterparts (Fig 1B). The BH3 profiling technique examines mitochondrial sensitivity to different BH3 mimetic peptides, and has proven to be a useful tool to determine cell dependence on anti-apoptotic BCL-2 family proteins. BH3 profiling demonstrated that resistant cells had increased responses to NOXA, MS1 and HRK peptides, indicating increased dependence on MCL-1 and/or BCL-XL (Fig 1C). To characterize the functional role of MCL-1 in resistance to venetoclax, we co-treated parental and resistant cells with selective BCL-XL or MCL-1 inhibitors A-1155463 (Leverson et al. Science Transl Med 2015) and A-1210477 (Leverson et al., Cell Death Dis 2015). The combination of venetoclax with either A-1155463 or A-1210477 showed synergistic growth inhibition in all 5 parental cell lines. Notably, 4 of the 5 resistant cell lines (OCI-AML2, Kasumi, MV4-11, MOLM13) became more sensitive to an MCL-1 inhibitor but not to a BCL-XL inhibitor (Fig 1E). However, no further sensitization was seen in combination with venetoclax in resistant cells.
To characterize additional mechanisms of resistance to venetoclax in AML cells, we conducted RNA sequencing of single cell clones (2 clones/cell line) isolated from paired isogenic cells (OCI-AML2, MV4-11, MOLM13). Analysis of RNA expression patterns by gene set enrichment analysis (GSEA) revealed elevated expression of genes in the RAS/MAPK pathway (Fig 1F), consistent with increased p-ERK and p-p90-RSK protein levels (Fig 1G). Inhibition of MAPK with MEK inhibitor GDC-0973 reduced MCL-1 expression in parental but not in resistant cells, indicating that MAPK activation partially contributed to high MCL-1 levels (Fig 1G).
GSEA of RNAseq data further uncovered altered expression of genes involved in mitochondrial oxidative phosphorylation (OxPhos) in 3 resistant cell lines with high MCL-1 expression (OCI-AML2, MV4-11 and MOLM-13). Notably, BCL-2 was reported to sustain AML stem cell survival through maintenance of the mitochondrial activity of OxPhos (Lagadinou etal., Cell Stem Cell, 2013). Analysis of mitochondrial respiration using a Seahorse Bioanalyzer demonstrated similar levels of oxygen consumption rate (OCR) in parental and resistant cells. Inhibition of BCL-2 with 100nM venetoclax for only 2 hrs. fully blocked baseline and maximal respiratory activity in parental but not in resistant cells. In turn, inhibition of MCL-1 with A-1210477 inhibited respiration in both parental and resistant cells, indicating a role for MCL-1 in sustaining mitochondrial activity in venetoclax-resistant AML cells, which can maintain unperturbed mitochondrial function.
In summary, we identified a novel mechanism of resistance to targeted BCL-2 inhibition through upregulation of MAPK leading to increased levels of anti-apoptotic MCL-1 that binds and neutralizes BIM and maintains the mitochondrial OxPhos pathway in AML cells. Concomitant inhibition of BCL-2 and MCL-1, or of BCL-2 and OxPhos could induce synergistic cell death in AML and conceivably prevent the emergence of venetoclax resistance.

Disclosures
Tyner: Constellation Pharmaceuticals: Research Funding; Janssen Research & Development: Research Funding; Agios Pharmaceuticals: Research Funding; Genentech: Research Funding; Array Biopharma: Research Funding; Inctye: Research Funding; Seattle Genetics: Research Funding; Aptose Biosciences: Research Funding; AstraZeneca: Research Funding; Takeda Pharmaceuticals: Research Funding; Leap Oncology: Consultancy. Leverson:AbbVie: Employment, Other: Shareholder in AbbVie. Letai:Astra-Zeneca: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding. Konopleva:Calithera: Research Funding; Cellectis: Research Funding.