BCR-ABL+ Bp-ALL Exhibits Heightened Sensitivity to Simultaneous Induction of Energy and ER Stress Via Downregulation of Mcl-1 Expression
Authors: Leclerc Guy J. , DeSalvo Joanna , Leclerc Gilles M. , Lampidis Theodore J. , Barredo Julio C. 
Published: October 2019

Abstract
Tumor metabolism has emerged as a hallmark of cancer by which the oncogenic profile of cancer cells pairs energy availability with growth and survival. BCR-ABL+ acute lymphoblastic leukemia (BCR-ABL+ ALL) accounts for 25% of ALL and the BCR-ABL fusion protein has been correlated with alterations in glucose metabolism. The glucose analogue 2-deoxy-D-glucose (2DG), induces simultaneous energy and ER-stress by inhibiting glycolysis (energy stress) via blocking HK and PGI enzymes, and interfering with N-linked glycosylation through its incorporation in place of mannose into the lipid-linked oligosaccharide (LLO) chain leading to premature termination of LLO synthesis (ER stress). We recently demonstrated that inhibition of both aerobic glycolysis and N-linked glycosylation by 2DG led to apoptosis in ALL (Mol Cancer Res 10:969, 2012). Among ALL subtypes, BCR-ABL+ ALL cell lines SupB15 and TOM1 exhibited the highest sensitivity to 2DG suggesting that in ALL cells BCR-ABL expression may be linked to heightened sensitivity to energy and/or ER stress. To test this premise, we constructed NALM6 (Bp-ALL) stable cell lines expressing the BCR-ABL p190 fusion and found that expression of the BCR-ABL p190 fusion in NALM6 cells significantly increased 2DG-induced apoptosis compared to mock transfected controls. To investigate the contribution of each form of stress (energy and ER stress) in the increased sensitivity of BCR-ABL+ cells to 2DG, we examined the effects of agents known to preferentially induce energy stress (lowering ATP) such as 2-fluoro-2-deoxy-D-glucose and oxamate, and ER stress such as tunicamycin. We found no differences in cell death or ATP levels between BCR-ABL+ and control ALL cells treated with agents solely inducing energy or ER stress, indicating the increased susceptibility of BCR-ABL+ ALL cells to 2DG results from the simultaneous induction of both types of stress. Consistent with these data, Fluorophore-Assisted Carbohydrate Electrophoresis and immunoblotting demonstrated that 2DG induced equivalent inhibition of N-linked glycosylation and upregulation of the UPR signaling in BCR-ABL+ and control ALL cells. Similar findings were seen in primary ALL patient samples. Consequently, simultaneous energy and ER stress are required for the heightened vulnerability to these forms of stress conferred by BCR-ABL in ALL. Based on the known function of Mcl-1 promoting survival in BCR-ABL+ ALL, we investigated its role in the mechanism of cell death induced by energy and/or ER stress agents. We uncovered that BCR-ABL+ ALL cell lines exhibited the lowest levels of Mcl-1 expression compared to control BCR-ABL- ALL and CML cells, and this basal level of Mcl-1 expression directly correlated with their resistance to energy and/or ER stressing agents. Further, we found that Mcl-1 knock-down by shRNA sensitized ALL and CML cells to the dual induction of energy and ER stress by 2DG. We also found that Mcl-1 expression was further downregulated in BCR-ABL+ and - ALL cells treated with energy and/or ER stressors which correlated with the level of apoptosis. Using qRT-PCR or co-treatment with the pancaspase inhibitor qVD-OPH or the proteasome inhibitor bortezomib, we showed that Mcl-1 downregulation by energy and/or ER stressors is neither due to transcriptional, caspase dependent cleavage, or post-translational regulation mechanisms but likely due to translational control of the Mcl-1 protein via AMPK/mTOR and the UPR PERK/p-eIF2α. Finally, because TKIs lead to decrease ATP (energy stress) in BCR-ABL+ cells, we co-treated BCR-ABL+ ALL cells with TKIs plus ER stressors and found that the simultaneous induction of these forms of stress by these combinations significantly increased cell death compared to single drug. Further, we found that TKIs + ER stressors further downregulated Mcl-1 and GRP78 expression compared to single agent treatment. Taken together, our data demonstrate that simultaneous induction of energy and ER stress leads to significant induction of apoptosis in BCR-ABL+ ALL cells, and these heightened vulnerability to these forms of stress is likely mediated by downregulation of Mcl-1 expression via AMPK activation/mTOR inhibition and PERK/p-eIF2α. Our study demonstrates that strategies with agents that induce energy and/or ER stress plus BCR-ABL TKIs warrant further consideration as a potential approach for future clinical translation into trials for BCR-ABL+ ALL patients.

Disclosures
No relevant conflicts of interest to declare.