ARID5B Regulates Leukemia Sensitivity to Antimetabolites in Children with Acute Lymphoblastic Leukemia Via Effects on Cell Cycle Progression

Acute lymphoblastic leukemia (ALL) in children is a prototype of cancer that can be cured by chemotherapy alone. However, the molecular mechanisms for anti-leukemic drug sensitivity and genetic basis of inter-patient variability in treatment response are not fully understood. Taking a genome-wide approach, we recently identified genetic variants in the ARID5B gene that strongly predispose children to developing ALL and also a high risk of relapse following therapy (J Clin Oncol 2012 30:751, Nat Genet 2009 41:1001). To understand the mechanisms by which ARID5B is linked to treatment outcome in childhood ALL, we sought to 1) characterize ARID5B expression in different genetic subtypes of ALL, 2) determine the effects of ARID5B expression on cytotoxicity of chemotherapeutic agents commonly used in ALL therapy, and 3) describe molecular pathways linking ARID5B to anti-leukemic drug sensitivity. In 567 children with newly diagnosed ALL treated at St. Jude Children’s Research Hospital (GSE33315), ARID5B expression was highest in cases with hyperdiploid karyotype (>50 chromosomes) and lowest in T-cell ALL and cases with MLL rearrangements. This pattern was validated in an independent cohort of 106 children from the Dutch Childhood Oncology Group (GSE13351). In 59 patients treated on the Children’s Oncology Group (COG) CCG1961 trial, lower ARID5B expression was associated with higher rates of relapse (P=0.01, GSE7440). Importantly, when we compared matched newly-diagnosed vs. relapsed ALL blasts from a cohort of 60 patients enrolled in COG trials (GSE28460), ARID5B expression was further downregulated at disease recurrence (P=0.0009). shRNA-mediated ARID5B knockdown in 3 ALL cell lines (Nalm6, SEM, and UOCB-1) substantially increased resistance to antimetabolites (an average of 5.16 and 35.3-fold increase in IC50 for methotrexate [MTX] and 6-mercaptopurine [6MP], respectively), with minimal effects on glucocorticoids, vincristine, asparaginase, and daunorubicin. Because cytotoxic effects of MTX and 6MP are highly dependent on the rate of cell proliferation, we postulate that ARID5B directly influences cell cycle entry. In all 3 cell lines, ARID5B knockdown led to significant blockade at the G1/S checkpoint, increasing the percent of cells in G0/G1 phase. At the molecular level, downregulation of ARID5B resulted in higher levels of p21 and reduction in phosphorylated Rb, consistent with the retention at G0/G1 phase. ARID5B expression was restricted to nucleus but affected both nuclear and cytoplasmic p21 expression in a time-dependent fashion. Interestingly, there was a highly significant negative correlation between p21 expression and MTX- and 6MP-induced apoptosis in all 3 ALL cell lines. Taken together, we hypothesize that lower expression of ARID5B impairs ALL cell cycling by upregulating p21, contributing to resistance to MTX and 6MP and eventually leukemia relapse. Finally, we compared global gene expression in ARID5B knockdown vs. control ALL cells, and via the Connectivity Map analysis we identified histone deacetylase (HDAC) inhibitors as promising agents for overcoming ARID5B-related drug resistance. Indeed, ARID5B knockdown cells were significantly more sensitive to panobinostat than controls, suggesting HDAC inhibitors as potential therapeutic options for patients with ARID5B-deficient and drug resistant ALL.