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Current standard of care for patients with pediatric acute lymphoblastic leukemia (ALL) is mainly effective, with high remission rates after treatment. However, the genetic perturbations that give rise to this disease remain largely undefined, limiting the ability to address resistant tumors or develop less toxic targeted therapies. Here, we report the use of next-generation sequencing to interrogate the genetic and pathogenic mechanisms of 240 pediatric ALL cases with their matched remission samples. Commonly mutated genes fell into several categories, including RAS/receptor tyrosine kinases, epigenetic regulators, transcription factors involved in lineage commitment, and the p53/cell-cycle pathway. Unique recurrent mutational hotspots were observed in epigenetic regulators CREBBP (R1446C/H), WHSC1 (E1099K), and the tyrosine kinase FLT3 (K663R, N676K). The mutant WHSC1 was established as a gain-of-function oncogene, while the epigenetic regulator ARID1A and transcription factor CTCF were functionally identified as potential tumor suppressors. Analysis of 28 diagnosis/relapse trio patients plus 10 relapse cases revealed four evolutionary paths and uncovered the ordering of acquisition of mutations in these patients. This study provides a detailed mutational portrait of pediatric ALL and gives insights into the molecular pathogenesis of this disease. Cancer Res; 77(2); 390–400. ©2016 AACR.

Pediatric ALL is the most common childhood tumor and the leading cause of childhood cancer deaths. To gain a better understanding of the landscape of somatic mutations in ALL, we performed whole exome and targeted sequencing of 240 pediatric B-ALL patients with their matched remission samples. The significantly mutated genes fall into several common categories: RAS/receptor tyrosine kinases, epigenetic regulators, transcription factors involved in lineage commitment and p53/cell cycle pathway. RAS/receptor tyrosine kinases: the most frequently mutated genes were members of RAS signaling (NRAS, KRAS, FLT3, PTPN11). Besides the well know hotspot mutations [G12D/V/C (NRAS 13 cases, KRAS 13 cases), G13D (NRAS 14 cases, KRAS 11 cases) and Q61H/L/R/K (NRAS 15 cases, KRAS 1 case)], novel mutational sites were also identified for KRAS: A146T/P (3 cases), K117N/T (4 cases) and V14I (1 case). High frequency missense mutations of PTPN11 clustered in SH2 domain (included the canonical hotspot A72T (5 cases) and E76K/V (4 cases)) and tyrosine-phosphatase catalytic domain (G503R/V). For FLT3, well-appreciated activating hotspot mutations in the kinase domain (D835Y/Y842C) and several novel recurrent mutationswere identified. Epigenetic regulators: hotspot mutations were identified in histone H3K36 methyltransferase WHSC1. Mutation E1099K located in the SET domain, was identified in 10 patients as well as two of the 5 ALL cell lines that we sequenced (RS4;11, SEM). Stable silencing of E1099K mutant WHSC1 in RS4;11 cells by either lentiviral shRNA or CRISPR guide RNA (sgRNA) markedly reduced clonogenic growth both in vitro and in vivo, underscoring the critical role of WHSC1 in lymphoid malignancies. Two highly-related histone/non-histone acetyltransferases, CREBBP and EP300, were also prominently mutated in our cohort. Mutations of CREBBP predominantly occurred in the acetyltransferase domain, particularly in the hotspot R1446C/H. Mutations of chromatin remodeling genes (ARID1A and ARID2) have been identified in a number of cases. Silencing of ARID1A in ALL cell lines by lentiviral shRNA resulted in upregulation of the pro-growth regulator c-MYC, while forced expression of ARID1A reduced c-MYC luciferase reporter activity. In addition, silencing of ARID1A by either shRNA or CRISPR-sgRNA resulted in enhanced clonogenic growth, suggesting that ARID1A may be involved in the c-MYC pathway and modulates the ALL cell proliferation. Mutations of epigenetic regulators were also found in the polycomb complex (EZH2, EED, SUZ12), chromatin/nucleosome structure modifying proteins (CHD2, CHD3, CHD4), TET family proteins [TET1 (2 cases), TET2 (5 cases)] and histone modification proteins (HDAC1, SIRT1, BCOR, BRD8, lysine demethylase PHF2/KDM6A, histone acetyltransferase KAT6B). Transcription factors and p53/cell cycle pathway: a number of alterations of transcription factors essential for hematopoietic and lymphoid differentiation were noted including the lineage regulator PAX5 (5 missense, 3 indels) and ETV6 (6 cases, 3 were frameshift indel and 1 was a splice-site mutations). In addition, mutations were also found in other lineage transcription factors (IKZF2, IKZF3, EBF1), WT1 (6 cases, including 3 indels and 1 stop-gain mutations), RUNX family member [RUNX2 (7 cases), RUNX1 (1 case)], ERG1 (3 cases), GATA1/3 (1 case each) and CTCF. Somatic mutations of genes involved in the p53 pathway occurred in 18 patients, including TP53, ATM and the kinases that regulate p53 activities (HIPK1, HIPK2). Germline TP53 pathogenic variants were found in these 2 patients. Taken together, we extensively interrogated the mutational landscape of a large cohort of pediatric ALL samples by exome and targeted resequencing. This study provides a detailed mutational portrait of pediatric ALL and gives new insights into the molecular pathogenesis of this disease. Disclosures Kantarjian: Amgen: Research Funding; ARIAD: Research Funding; Bristol-Myers Squibb: Research Funding; Pfizer Inc: Research Funding; Delta-Fly Pharma: Research Funding; Novartis: Research Funding. Ogawa:Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding.