Despite progress in the treatment of acute lymphoblastic leukemia (ALL), T-cell ALL (T-ALL) has limited treatment options, particularly in the setting of relapsed/refractory disease. Using an unbiased genome-scale CRISPR-Cas9 screen we sought to identify pathway dependencies for T-ALL which could be harnessed for therapy development. Disruption of the one-carbon folate, purine and pyrimidine pathways scored as the top metabolic pathways required for T-ALL proliferation. We used a recently developed inhibitor of SHMT1 and SHMT2, RZ-2994, to characterize the effect of inhibiting these enzymes of the one-carbon folate pathway in T-ALL and found that T-ALL cell lines were differentially sensitive to RZ-2994, with the drug inducing a S/G2 cell cycle arrest. The effects of SHMT1/2 inhibition were rescued by formate supplementation. Loss of both SHMT1 and SHMT2 was necessary for impaired growth and cell cycle arrest, with suppression of both SHMT1 and SHMT2 inhibiting leukemia progression in vivo. RZ-2994 also decreased leukemia burden in vivo and remained effective in the setting of methotrexate resistance in vitro. This study highlights the significance of the one-carbon folate pathway in T-ALL and supports further development of SHMT inhibitors for treatment of T-ALL and other cancers.

Despite a remarkable increase in the genomic profiling of cancer, integration of genomic discoveries into clinical care has lagged behind. We report the feasibility of rapid identification of targetable mutations in 153 pediatric patients with relapsed/refractory or high-risk leukemias enrolled on a prospective clinical trial conducted by the LEAP Consortium. Eighteen percent of patients had a high confidence Tier 1 or 2 recommendation. We describe clinical responses in the 14% of patients with relapsed/refractory leukemia who received the matched targeted therapy. Further, in order to inform future targeted therapy for patients, we validated variants of uncertain significance, performed ex vivo drug-sensitivity testing in patient leukemia samples, and identified new combinations of targeted therapies in cell lines and patient-derived xenograft models. These data and our collaborative approach should inform the design of future precision medicine trials. Significance: Patients with relapsed/refractory leukemias face limited treatment options. Systematic integration of precision medicine efforts can inform therapy. We report the feasibility of identifying targetable mutations in children with leukemia and describe correlative biology studies validating therapeutic hypotheses and novel mutations. See related commentary by Bornhauser and Bourquin, p. 1322. This article is highlighted in the In This Issue feature, p. 1307

Despite progress in the treatment of acute lymphoblastic leukemia (ALL), T-cell ALL (T-ALL) has limited treatment options particularly in the setting of relapsed/refractory disease. Using an unbiased genome-scale CRISPR-Cas9 screen we sought to identify pathway dependencies for T-ALL which could be harnessed for therapy development. Disruption of the one-carbon folate, purine and pyrimidine pathways scored as the top metabolic pathways required for T-ALL proliferation. We used a recently developed inhibitor of SHMT1 and SHMT2, RZ-2994, to characterize the effect of inhibiting these enzymes of the one-carbon folate pathway in T-ALL and found that T-ALL cell lines were differentially sensitive to RZ-2994, with a S/G2 cell cycle arrest. The effects of SHMT1/2 inhibition were rescued by formate supplementation. Loss of both SHMT1 and SHMT2 was necessary for impaired growth and cell cycle arrest, with suppression of both SHMT1 and SHMT2 impairing leukemia progression in vivo. RZ-2994 decreased leukemia burden in vivo and remained effective in the setting of methotrexate resistance in vitro. This study highlights the significance of the one-carbon folate pathway in T-ALL and supports further development of SHMT inhibitors for treatment of T-ALL and other cancers.