Heme-Sensing Pathway Modulates Susceptibility of Poor Prognosis B-Lineage Acute Leukemia to BH3-Mimetics

Abstract

211 Words Body: 5960 Words (not counting references nor supplementary material) Heme-sensing pathway dictates apoptotic response 2 Abstract:Anti-apoptotic MCL1 is one of the most frequently amplified genes in human cancers and elevated expression confers resistance to many therapeutics including the BH3-mimetic agents dihydroartemisinin (DHA) translationally represses MCL-1 and synergizes with BH3-mimetics. To explore how DHA represses MCL-1, a genome-wide CRISPR screen identified that loss of genes in the heme synthesis pathway renders mouse BCR-ABL + B-ALL cells resistant to DHAinduced death. Mechanistically, DHA disrupts the interaction between heme and the eIF2α kinase heme regulated inhibitor (HRI) triggering the integrated stress response. Genetic ablation ofEif2ak1, which encodes HRI, blocks MCL-1 repression in response to DHA treatment and represses the synergistic killing of DHA and BH3-mimetics compared to wild-type leukemia. Furthermore, BTdCPU, a small-molecule activator of HRI, similarly triggers MCL-1 repression and synergizes with BH3-mimetics in mouse and human leukemia including both Ph + and Ph-like B-ALL. Lastly, combinatorial treatment of leukemia bearing mice with both BTdCPU and a BH3-mimetic extended survival and repressed MCL-1 in vivo. These findings reveal for the first time that the HRI-dependent cellular heme-sensing pathway can modulate apoptosis in leukemic cells by repressing MCL-1 and increasing their responsiveness to BH3-mimetics. This signaling pathway could represent a generalizable mechanism for repressing MCL-1 expression in malignant cells and sensitizing them to available therapeutics. Heme-sensing pathway dictates apoptotic response Heme-sensing pathway dictates apoptotic response 6 Materials and Methods Cells and Cell Culture. Mouse p185 + Arf-null B-ALL (hereafter referred to as BCR-ABL + B-ALL) (10) and PAX5-JAK2, RCSD1-AB1, and RCSD1-ABL2 fusion-expressing Ph-like Ba/F3 cells (23) were grown in RPMI with 10% fetal bovine serum, 55 µM 2mercaptoethanol, 2 mM glutamine, penicillin, and streptomycin (Invitrogen). Navitoclax and Venetoclax were obtained from Selleckchem. DHA was obtained from AvaChem Scientific. BTdCPU was obtained from Millipore and synthesized by the The human Ph + leukemia cell lines OP-1, TOM-1, BV-173, and SUP-B15 were cultured in RPMI with 20% fetal bovine serum, 55 µM 2-mercaptoethanol, 2 mM glutamine, penicillin, and streptomycin. Genome-wide CRISPR Screen. Cas9-expressing p185 + B-ALL cells were transduced with the Brie CRISPR KO library (Addgene #73633) at a multiplicity of infection of 0.5with an sgRNA coverage of 400x (24). One day after transduction, puromycin was added (1 µg/mL) to select for infected cells. Cells were then cultured in either DMSO or 10 µM DHA containing media for 24 hours (h), followed by a 48h culture in drug free media.As previously described, genomic DNA was extracted from the surviving cells (Qiagen DNeasy Blood and Tissue kit) and amplified by PCR for Illumina sequencing of sgRNAs by Nextseq (24). MAGeCK was used to ...