Choline Kinase a Is Required for Alkylating Agent Resistance in T-Cell Acute Lymphoblastic Leukemia

Abstract

Conventional chemotherapy is responsible for most clinical cures achieved in oncology, but these drugs are toxic, and resistance is a major problem. Indeed,10-20% of children and >50% of adults with T-cell acute lymphoblastic leukemia (T-ALL) exhibit resistance to front-line therapy, leading to a dismal prognosis. Improved treatment options are needed for these patients. To identify mechanisms of alkylating agent resistance, we performed a genome-wide CRISPR screen in T-ALL cells treated with the alkylating agent nitrogen mustard, the active functional group of drugs such as cyclophosphamide and melphalan. The results revealed a number of internal positive controls, including genes implicated in the Fanconi and other DNA repair pathways. This screen also implicated a role for choline kinase A (CHKA) in nitrogen mustard resistance. CHKA is best-known for its ability to catalyze phosphocholine synthesis, a key step in phosphatidylcholine biosynthesis, and has no known role in DNA damage repair. We found that CHKA shRNA knockdown induces profound sensitivity to nitrogen mustard treatment in leukemia cells (p= 0.0012), but not in normal hematopoietic progenitor CD34+ cells. This effect was rescued by restoring expression of wild-type CHKA, but not by an ATPase-deficient CHKA point mutant. This indicates that CHKA kinase activity is required for resistance. We next assessed the role of CHKA in resistance to a panel of therapeutics with distinct mechanisms of action. We found that CHKA loss induced selective sensitivity to drugs that induce bulky DNA adducts. By contrast, CHKA had little effect on drugs that induce other forms of DNA damage, such as free radical damage or double-strand DNA breaks. Studies to elucidate the mechanism through which CHKA mediates resistance to these drugs are underway. To investigate the therapeutic potential of CHKA inhibition, we tested a small molecule CHKA inhibitor (CK2) in combination with alkylating agents in a panel of patient-derived T-ALL models. In vitro, we found that the pharmacologic inhibition of CHKA is synergistic with alkylating agent treatment with a bliss score of 16.683 for PDX9 and 21.982 for PDX CBAT (score >10 indicating strong synergy). We then tested the potential of this approach in these T-ALL PDX models when engrafted in vivo in NRG mice and treated with CK2 and cyclophosphamide alone or in combination. We found that the addition of CK2 further improved the survival benefit of cyclophosphamide in this model (p= 0.0260). Taken together, our findings support a new role of choline kinase A in alkylating agent resistance in T-ALL and provide a rationale to explore the possibility of increasing the efficacy of conventional chemotherapy in the context of drug-resistant leukemias by combining CHKA inhibition with alkylating agents.