Most B cell precursor acute lymphoblastic leukemia (BCP ALL) can be classified into known major genetic subtypes, while a substantial proportion of BCP ALL remains poorly characterized in relation to its underlying genomic abnormalities. We therefore initiated a large-scale international study to reanalyze and delineate the transcriptome landscape of 1,223 BCP ALL cases using RNA sequencing. Fourteen BCP ALL gene expression subgroups (G1 to G14) were identified. Apart from extending eight previously described subgroups (G1 to G8 associated with MEF2D fusions, TCF3–PBX1 fusions, ETV6–RUNX1–positive/ETV6–RUNX1–like, DUX4 fusions, ZNF384 fusions, BCR–ABL1/Ph–like, high hyperdiploidy, and KMT2A fusions), we defined six additional gene expression subgroups: G9 was associated with both PAX5 and CRLF2 fusions; G10 and G11 with mutations in PAX5 (p.P80R) and IKZF1 (p.N159Y), respectively; G12 with IGH–CEBPE fusion and mutations in ZEB2 (p.H1038R); and G13 and G14 with TCF3/4–HLF and NUTM1 fusions, respectively. In pediatric BCP ALL, subgroups G2 to G5 and G7 (51 to 65/67 chromosomes) were associated with low-risk, G7 (with ≤50 chromosomes) and G9 were intermediate-risk, whereas G1, G6, and G8 were defined as high-risk subgroups. In adult BCP ALL, G1, G2, G6, and G8 were associated with high risk, while G4, G5, and G7 had relatively favorable outcomes. This large-scale transcriptome sequence analysis of BCP ALL revealed distinct molecular subgroups that reflect discrete pathways of BCP ALL, informing disease classification and prognostic stratification. The combined results strongly advocate that RNA sequencing be introduced into the clinical diagnostic workup of BCP ALL.

BackgroundHereditary predisposition is rarely suspected for childhood acute lymphoblastic leukemia (ALL). Recent studies identified germline ETV6 variations associated with marked familial clustering of hematologic malignancies, pointing to this gene as a potentially important genetic determinant for ALL susceptibility. The aims of the current study are to comprehensively identify ALL predisposition variants in ETV6 and to determine the extent to which they contribute to the overall risk of childhood ALL.MethodsWhole-exome sequencing of an index family with multiple cases of ALL was performed to identify causal variants for ALL predisposition. Targeted sequencing of ETV6 was done in 4,405 children from the Children's Oncology Group (COG) and St. Jude Children's Research Hospital frontline ALL trials. Patients were included in this study on the basis of their enrollment in these clinical trials and the availability of germline DNA. ETV6 variant genotypes were compared with non-ALL controls to define ALL-related germline risk variants. ETV6 variant function was characterized bioinformatically and correlated with clinical and demographic features in 2,021 children with ALL.FindingsWe identified a novel nonsense ETV6 variant (p.R359X) with a high penetrance of familial ALL. Subsequent targeted sequencing of ETV6 in 4,405 childhood ALL cases discovered 31 exonic variants (4 nonsense, 21 missense, 1 splice site, and 5 frame shift variants) that are potentially related to ALL risk in 35 cases (0.79%). Fifteen (48%) of the 31 ALL-related ETV6 variants clustered in the ETS domain and predicted to be highly deleterious. Children with ALL-related ETV6 variants were significantly older at leukemia diagnosis than others (10.2 years [IQR 5.3-13.8] vs 4.7 years [IQR 3.0-8.7], P=0.017). The hyperdiploid leukemia karyotype was strikingly overrepresented in ALL cases harboring germline ETV6 risk variants compared to the wildtype group (9 of 14 cases [64.3%] vs 538 of 2,007 cases [26.8%]; P=0.0050).InterpretationOur findings indicated germline ETV6 variations as the basis of a novel genetic syndrome associated with predisposition to childhood ALL.FundingThis study was supported by the National Institutes of Health and by the American Lebanese Syrian Associated Charities.

PML/RARalpha is of crucial importance in acute promyelocytic leukemia (APL) both pathologically and therapeutically. Using a genome-wide approach, we identified in vivo PML/RARalpha binding sites in a PML/RARalpha-inducible cell model. Of the 2979 targeted regions, >62% contained canonical PU.1 motifs and >84% of these PU.1 motifs coexisted with one or more RARE half (RAREh) sites in nearby regions. Promoters with such PU.1-RAREh binding sites were transactivated by PU.1. PU.1-mediated transactivation was repressed by PML/RARalpha and restored by the addition of all-trans retinoic acid (ATRA). Genes containing such promoters were significantly represented by genes transcriptionally suppressed in APL and/or reactivated upon treatment with ATRA. Thus, selective targeting of PU.1-regulated genes by PML/RARalpha is a critical mechanism for the pathogenesis of APL.

Chromosomal translocations are a genomic hallmark of many hematologic malignancies. Often as initiating events, these structural abnormalities result in fusion proteins involving transcription factors important for hematopoietic differentiation and/or signaling molecules regulating cell proliferation and cell cycle. In contrast, epigenetic regulator genes are more frequently targeted by somatic sequence mutations, possibly as secondary events to further potentiate leukemogenesis. Through comprehensive whole-transcriptome sequencing of 231 children with acute lymphoblastic leukemia (ALL), we identified 58 putative functional and predominant fusion genes in 54.1% of patients (n = 125), 31 of which have not been reported previously. In particular, we described a distinct ALL subtype with a characteristic gene expression signature predominantly driven by chromosomal rearrangements of the ZNF384 gene with histone acetyltransferases EP300 and CREBBP. ZNF384-rearranged ALL showed significant up-regulation of CLCF1 and BTLA expression, and ZNF384 fusion proteins consistently showed higher activity to promote transcription of these target genes relative to wild-type ZNF384 in vitro. Ectopic expression of EP300-ZNF384 and CREBBP-ZNF384 fusion altered differentiation of mouse hematopoietic stem and progenitor cells and also potentiated oncogenic transformation in vitro. EP300-and CREBBP-ZNF384 fusions resulted in loss of histone lysine acetyltransferase activity in a dominant-negative fashion, with concomitant global reduction of histone acetylation and increased sensitivity of leukemia cells to histone deacetylase inhibitors. In conclusion, our results indicate that gene fusion is a common class of genomic abnormalities in childhood ALL and that recurrent translocations involving EP300 and CREBBP may cause epigenetic deregulation with potential for therapeutic targeting.