Background: Mutational/non-mutational activation of MAPK pathway is a pervasive mechanism of resistance to venetoclax (ABT199) in AML. Our work with ASTX029 and Compound 27, inhibitors of ERK1/2, linked suppressed mitochondrial fission as a mechanism of overcoming MAPK driven resistance to ABT199 in AML. We demonstrated that ERK1/2 inhibition decreased phosphorylation of DRP1, a dynamin like GTPase that drives mitochondrial fission, induced apoptosis in combination with ABT199 in vitro and extended the survival of PDX mouse model of post-ABT199/decitabine relapsed AML (Sharma et al., Blood. 2023). ABT199 resistant AML cells exhibit tighter mitochondrial cristae, resulting in lower cytochrome C release, and enhanced oxidative phosphorylation (OXPHOS). Additionally, gene silencing of BAX and mitochondrial metalloprotease OMA1 were identified as the two top hits that lead to ABT199 resistance (Qi et al., Signal Transduct Target Ther. 2022). Bax activates OMA1 (Jiang et al., PNAS. 2014) and OMA1 cleaves membrane bound long (L-OPA1) to short (S-OPA1) isoform. Steady-state balance of long and short OPA1 isoforms is important for functioning as a molecular staple at cristae junctions. L-Opa1 and S-opa1 oligomerize at cristae junctions to prevent cytochrome C mobilization and release (Varanita et al., Cell Metabolism. 2015). By maintaining tight mitochondrial cristae, Opa1 stabilizes respiratory chain super complexes and inhibits ROS generation. Herein, we investigated the functional aspect of ERK1/2 inhibition on mitochondrial cristae remodeling and metabolism in context of overcoming resistance to ABT199 in AML. Methods: ERK1/2 was inhibited in vitro using Compound 27 (ERKi, Heightman et al., J Med Chem. 2018) and in vivo using ASTX029 (Munck et al., Mol Cancer Ther. 2021), which is currently under clinical trial in solid tumors (NCT03520075). RNAseq, gene set enrichment analysis (GSEA) and CyTOF were performed to identify the mechanism and validated by immunoblotting and flowcytometry. Oxygen consumption rate (OCR) was measured using Seahorse based Mito Stress test. Mitochondrial images were acquired using transmission electron microscopy (TEM) followed by quantification with Imaris. Results: ERKi synergized strongly with ABT199 at inducing apoptosis in RAS mutated and/or ABT199 resistant AML cells and primary samples (p<0.05). Transcriptome profiling revealed that ABT199 upregulated 6 pathways in AML cells: OXPHOS, Myc targets, E2F targets, G2M checkpoint, epithelial mesenchymal transition and KRAS signaling. Interestingly, these pathways were downregulated in response to ERKi+/-ABT199 treatment (FDR<0.05). OPA1, one of the core enrichment proteins for OXPHOS gene set, maintains mitochondrial cristae and contributes to resistance to ABT199 (Chen et al., Cancer Discov. 2019). ERKi in combination with ABT199 induced proteolysis of OPA1 and increased the S-Opa1/L-Opa1 ratio in ABT199 resistant OCIAML2 cells and NRAS mutated OCIAML3 cells. Single cell proteomics using CyTOF showed increased expression of Bax in response to ASTX029+ABT199 in OCIAML3 xenograft. Knockdown of Bax inhibited the proteolysis of Opa1 as well as apoptosis mediated by ERKi+ABT199. Structurally, ERK1/2 inhibition in combination with ABT199 increased cristae width (p<0.0001) with simultaneous release of cytochrome C (p<0.001) as observed by TEM and flowcytometry respectively. Since, ABT199 treatment significantly enriched for OXPHOS, we examined the metabolic effect of ERKi in overcoming ABT199 resistance. Mito Stress test showed increased basal (p<0.0001) and maximal OCR (p<0.0001) and ATP production (p<0.0001) in OCIAML2 ABT199 resistant versus parental cells. Combining ERKi with ABT199 reduced OCR and ATP production in OCIAML3 and OCIAML2 ABT199 resistant cells (p<0.0001). ERKi alone or in combination with ABT199 reduced the activity of electron transport chain complex I (p<0.01). This was accompanied by an increase in mitochondrial membrane depolarization by ERKi+ABT199 (p<0.0001), suggesting mitochondrial dysfunction. Conclusion: Inhibition of ERK1/2, results in mitochondrial dysfunction and mediates Bax dependent proteolysis of Opa1 in AML, which alters mitochondrial cristae width and releases cytochrome C to overcome ABT199 resistance. The findings in this study provide a strong rationale for combining Bcl-2 and ERK1/2 inhibitors as part of an AML treatment protocol.