Targeting Cholesterol Homeostasis Pathway to Overcome Acquired Cytarabine Resistance in Acute Myeloid Leukemia

Abstract

The current backbone of Acute Myeloid Leukemia (AML)therapy is treatment with the cytidine analogue Ara-C and the anthracycline daunorubicin. Despite a high rate of initial remissions, a substantial fraction of patients with AML relapse and acquire resistance to Ara-C. In acquired resistance, there is an initial therapy response, but resistance develops over time. To uncover the acquired drug resistant mechanisms, we utilized two cytarabine (Ara-C) resistant cell lines - THP1 and U937 developed from the parental respective cell lines (kind gift from Dr. Martin Michaelis, University of Kent). In-vitro cytotoxicity assay confirmed the cytarabine resistance in THP1 cell line (THP1 Ara-C R IC50: 1457µM; THP1 parental IC50: 56 µM) and U937 cell line (U937 Ara-C R IC50: 1578.3 µM and U937 parental IC50: 0.14 µM). THP1-AraC-R was marginally cross resistant to both DNR (IC50 Parent -0.19µM, AraC 0.26µM) and ATO (IC50 Parent -2.1µM, AraC >4µM). To identify the factors associated with Ara-C drug resistance, we subjected both the cell lines along with their parental counterparts for transcriptomic profiling. Several protein coding genes were differentially expressed in both the cell lines. A total of 845 genes were upregulated and 268 genes were downregulated in THP1 Ara-C R cell line while 344 genes were upregulated, and 647 genes were downregulated in U937 Ara-C R cell line. To unravel the pathways associated with Ara-C resistance, the differentially expressed genes were subjected to Gene Set Enrichment Analysis (GSEA). The pathway enrichment showed Cholesterol Homeostasis pathway to be highly enriched in THP1 (NES: 2.17 & FDR q-value: 0) and U937 (NES:1.25 & FDR q-value:0.13) Ara-C resistant cell lines in comparison to the respective parental cell lines. To address the role of Cholesterol Homeostasis pathway in mediating chemoresistance in these two Ara-C resistant AML cell lines (THP1, U937) we screened few selected pharmacological inhibitors targeting different genes in the Cholesterol Homeostasis pathway [HMG Co-A reductase (HMGCR) the rate limiting step in Cholesterol metabolism, Squalene synthase (SQS), an intermediate enzyme in the pathway of Cholesterol synthesis and HMG Co-A synthase (HMGCS1) catalyzes the condensation of acetyl Co-A with aceto-acetyl Co-A to form HMGCo-A ]. Among all the inhibitors screened, Hymeglusin, an inhibitor of HMGCS1, re-sensitized only the THP1 Ara-C resistant cell with good synergy (combinatorial Index for THP1 Ara-C cell line 0.68 and 0.2 with Ara-C). These results demonstrate the dependence of leukemic cells on the Cholesterol Homeostasis pathway to mediate cytarabine resistance and pharmacological inhibition of this pathway can result in the reversal of cytarabine resistance.