Abstract 3973: Transcriptomic profiling of in-vitro modelled drug resistant AML cell lines unravel metabolic and stemness gene signatures
Authors: Illangeswaran Raveen Stephen , Jebanesan Daniel Zechariah , Sivakumar Kezia Kanimozhi , Vidhyadharan Rakhi Thalayattu , Mathews Vikram , Velayudhan Shaji Ramachandran , Balasubramanian Poonkuzhali 
Published: June 2022

Abstract
Disease recurrence is the major cause of treatment failure and mortality in acute myeloid leukemia (AML). A small fraction of leukemic cells that withstand chemotherapeutic insult can rewire cellular processes to re-emerge with lethal disease. Approaches to decipher drug resistance mechanisms in these drug persister cells can pave the way for newer therapies and improve survival in patients. To uncover drug resistant mechanisms that are clinically relevant, we generated in-house Cytarabine (AraC) and Arsenic trioxide (ATO) resistant cells from naïve MV4-11 AML cell line by incremental dose exposure method. Single cell clones were generated from AraC (MV411-AraC-R)/ATO (MV411-ATO-R) drug tolerant cells. Both the AraC-R and ATO-R clones were resistant to AraC (600-fold) and ATO (2-fold) respectively compared to the naïve cells. AraC-R cells were marginally cross resistant to daunorubicin (DNR) and ATO. Interestingly, the ATO-R cells were highly cross resistant to both AraC (600-fold) and DNR (4-fold) demonstrating altered drug resistance mechanism. We next subjected the AraC-R, ATO-R and the naïve cells to RNAseq analysis to identify transcriptomic changes post resistance development. 1218 transcripts in AraC-R cells and 7654 transcripts in ATO-R cells were differentially expressed compared to the naïve cells, suggesting ATO exposure alters the transcriptome drastically compared to AraC. We performed gene set enrichment analysis (GSEA) with these differentially expressed gene signatures. AraC-R gene signatures were highly enriched (NES-2.8 & 2.0 FDR-0.00) for oxidative phosphorylation and mitochondrial related signatures, consistent with previous report suggesting AraC exposed residual cells are highly enriched for oxidative phosphorylation and are not enriched for stemness gene signatures (Farge et al). ATO-R gene signatures were positively enriched (NES- 2.0 & 1.4 FDR 0.005 & 0.03) for Leukemic stem cell gene signatures (Eppert_LSC and Gentles_LSC) and relapse (NES- 2.0 & 1.5 FDR 0.0 & 0.01) gene signatures (CALCRL_RIC and Hackles_relapse) and negatively enriched for mitochondrial gene signatures showing reduced reliance on mitochondrial metabolism. Since AraC-R cells showed high dependency on mitochondrial metabolism, we treated these cells with venetoclax (VEN) a known oxphos inhibitor for 48 hours and assessed the viability. Ven markedly sensitized the AraC-R cells compared to naïve cells (IC50 13nM and 323nM), while ATO-R cells were markedly resistant to Ven (IC50 8049nM) corroborating our transcriptomic findings. These finding suggest transcriptome profiling of in-vitro modelled drug resistant cell lines can mimic clinically relevant drug resistant mechanism and could aide in identifying alternate therapies to circumvent disease relapse. Further metabolomic characterization and mechanistic studies are ongoing to substantiate these findings.
Citation Format: Raveen Stephen Illangeswaran, Daniel Zechariah Jebanesan, Kezia Kanimozhi Sivakumar, Rakhi Thalayattu Vidhyadharan, Vikram Mathews, Shaji Ramachandran Velayudhan, Poonkuzhali Balasubramanian. Transcriptomic profiling of in-vitro modelled drug resistant AML cell lines unravel metabolic and stemness gene signatures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3973.