Acute lymphoblastic leukemia (ALL) is an aggressive hematological tumor resulting from the malignant transformation of lymphoid progenitors. Despite intensive chemotherapy, 20% of pediatric and over 50% of adult ALL patients fail to achieve a complete remission or relapse after intensified chemotherapy, making disease relapse and resistance to therapy the most significant challenge in the treatment of this disease1,2. Using whole exome sequencing, here we identify mutations in the cytosolic 5'-nucleotidase II gene (NT5C2), which encodes a 5'-nucleotidase enzyme responsible for inactivation of nucleoside analog chemotherapy drugs, in 20/103 (19%) relapse T-ALLs and in 1/35 (3%) relapse B-precursor ALLs analyzed. NT5C2 mutant proteins show increased nucleotidase activity in vitro and conferred resistance to chemotherapy with 6-mercaptopurine and 6-thioguanine when expressed in ALL lymphoblasts. These results support a prominent role for activating mutations in NT5C2 and increased nucleoside analog metabolism in disease progression and chemotherapy resistance in ALL.

Key Points• RAS pathway mutations are prevalent in relapsed childhood ALL, and KRAS mutations are associated with a poorer overall survival.• RAS pathway mutations confer sensitivity to mitogenactivated protein kinase kinase inhibitors.For most children who relapse with acute lymphoblastic leukemia (ALL), the prognosis is poor, and there is a need for novel therapies to improve outcome. We screened samples from children with B-lineage ALL entered into the ALL-REZ BFM 2002 clinical trial (www. clinicaltrials.gov, #NCT00114348) for somatic mutations activating the Ras pathway (KRAS, NRAS, FLT3, and PTPN11) and showed mutation to be highly prevalent (76 from 206). Clinically, they were associated with high-risk features including early relapse, central nervous system (CNS) involvement, and specifically for NRAS/KRAS mutations, chemoresistance. KRAS mutations were associated with a reduced overall survival. Mutation screening of the matched diagnostic samples found many to be wild type (WT); however, by using more sensitive allelic-specific assays, low-level mutated subpopulations were found in many cases, suggesting that they survived up-front therapy and subsequently emerged at relapse. Preclinical evaluation of the mitogen-activated protein kinase kinase 1/2 inhibitor selumetinib (AZD6244, ARRY-142886) showed significant differential sensitivity in Ras pathway-mutated ALL compared with WT cells both in vitro and in an orthotopic xenograft model engrafted with primary ALL; in the latter, reduced RAS-mutated CNS leukemia. Given these data, clinical evaluation of selumetinib may be warranted for Ras pathway-mutated relapsed ALL. (Blood. 2014;124(23):3420-3430)

Alterations of the TP53 gene are of particular importance in the relapse stage of childhood ALL, in which they independently predict high risk of treatment failure in a significant number of patients. Therefore, they will aid in future risk assessment of children with ALL relapse.