Although many driver mutations are thought to promote carcinogenesis via abnormal splicing, the landscape of splicing-associated variants (SAVs) remains unknown due to the complexity of splicing abnormalities. Here, we developed a statistical framework to systematically identify SAVs disrupting or newly creating splice site motifs and applied it to matched whole-exome and transcriptome sequencing data from 8976 samples across 31 cancer types, generating a catalog of 14,438 SAVs. Such a large collection of SAVs enabled us to characterize their genomic features, underlying mutational processes, and influence on cancer driver genes. In fact, ∼50% of SAVs identified were those disrupting noncanonical splice sites (non-GT-AG dinucleotides), including the third and fifth intronic bases of donor sites, or newly creating splice sites. Mutation signature analysis revealed that tobacco smoking is more strongly associated with SAVs, whereas ultraviolet exposure has less impact. SAVs showed remarkable enrichment of cancer-related genes, and as many as 14.7% of samples harbored at least one SAVs affecting them, particularly in tumor suppressors. In addition to intron retention, whose association with tumor suppressor inactivation has been previously reported, exon skipping and alternative splice site usage caused by SAVs frequently affected tumor suppressors. Finally, we described high-resolution distributions of SAVs along the gene and their splicing outcomes in commonly disrupted genes, including ,, , and, which offers genetic clues for understanding their functional properties. Collectively, our findings delineate a comprehensive portrait of SAVs, novel insights into transcriptional de-regulation in cancer.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive neoplasm immunophenotypically resembling regulatory T cells, associated with human T-cell leukemia virus type-1. Here we performed whole-genome sequencing (WGS) of 150 ATL cases to reveal the overarching landscape of genetic alterations in ATL. We discovered frequent (33%) loss-of-function alterations preferentially targeting the CIC long isoform, which were overlooked by previous exome-centric studies of various cancer types. Long but not short isoform-specific inactivation of Cic selectively increased CD4+CD25+Foxp3+ T cells in vivo. We also found recurrent (13%) 3′-truncations of REL, which induce transcriptional upregulation and generate gain-of-function proteins. More importantly, REL truncations are also common in diffuse large B-cell lymphoma, especially in germinal center B-cell-like subtype (12%). In the non-coding genome, we identified recurrent mutations in regulatory elements, particularly splice sites, of several driver genes. In addition, we characterized the different mutational processes operative in clustered hypermutation sites within and outside immunoglobulin/T-cell receptor genes and identified the mutational enrichment at the binding sites of host and viral transcription factors suggesting their activities in ATL. By combining the analyses for coding and non-coding mutations, structural variations, and copy number alterations, we discovered 56 recurrently altered driver genes, including 11 novel ones. Finally, ATL cases were classified into two molecular groups with distinct clinical and genetic characteristics based on the driver alteration profile. Our findings not only help to improve diagnostic and therapeutic strategies in ATL, but also provide insights into T-cell biology and have implications for genome-wide cancer driver discovery.

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Although many driver mutations are thought to promote carcinogenesis via abnormal splicing, the landscape of these splicing-associated variants (SAVs) remains unknown due to the complexity of splicing abnormalities. Here we developed a statistical framework to identify SAVs disrupting or newly creating splice site motifs and applied it to sequencing data from 8,976 samples across 31 cancer types. We constructed a catalog of 14,438 SAVs, approximately 50% of which consist of SAVs disrupting non-canonical splice sites (including the 3rd and 5th intronic bases of donor sites) or newly creating splice sites. Smoking-related signature substantially contributes to SAV generation. As many as 14.7% of samples harbor at least one SAVs in cancer-related genes, particularly in tumor suppressors. Importantly, in addition to previously reported intron retention, exon skipping or alternative splice site usage more frequently affected these genes. Our findings delineate a comprehensive portrait of SAVs, providing a basis for cancer precision medicine.

Loading the bacterial replicative helicase DnaB onto DNA requires a specific loader protein, DnaC/DnaI, which creates the loading-competent state by opening the DnaB hexameric ring. To understand the molecular mechanism by which DnaC/DnaI opens the DnaB ring, we solved 3.1-Å co-crystal structure of the interaction domains of Escherichia coli DnaB–DnaC. The structure reveals that one N-terminal domain (NTD) of DnaC interacts with both the linker helix of a DnaB molecule and the C-terminal domain (CTD) of the adjacent DnaB molecule by forming a three α-helix bundle, which fixes the relative orientation of the two adjacent DnaB CTDs. The importance of the intermolecular interface in the crystal structure was supported by the mutational data of DnaB and DnaC. Based on the crystal structure and other available information on DnaB–DnaC structures, we constructed a molecular model of the hexameric DnaB CTDs bound by six DnaC NTDs. This model suggested that the binding of a DnaC would cause a distortion in the hexameric ring of DnaB. This distortion of the DnaB ring might accumulate by the binding of up to six DnaC molecules, resulting in the DnaB ring to open.

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In acute myeloid leukemia (AML), MLL (KMT2A) rearrangements are among the most frequent chromosomal abnormalities; however, knowledge of the genetic landscape of MLL-rearranged AML is limited. In this study, we performed whole-exome sequencing (n = 9) and targeted sequencing (n = 56) of samples from pediatric MLL-rearranged AML patients enrolled in the Japanese Pediatric Leukemia/Lymphoma Study Group AML-05 study. Additionally, we analyzed 105 pediatric t(8;21) AML samples and 30 adult MLL-rearranged AML samples. RNA-sequencing data from 31 patients published in a previous study were also reanalyzed. As a result, we identified 115 mutations in pediatric MLL-rearranged AML patients (2.1 mutations/patient), with mutations in signaling pathway genes being the most frequently detected (60.7%). Mutations in genes associated with epigenetic regulation (21.4%), transcription factors (16.1%), and the cohesin complex (8.9%) were also commonly detected. Novel CCND3 mutations were identified in 5 pediatric MLL-rearranged AML patients (8.9%) and 2 adult MLL-rearranged AML patients (3.3%). Recurrent mutations of CCND1 (n = 3, 2.9%) and CCND2 (n = 8, 7.6%) were found in pediatric t(8;21) AML patients, whereas no CCND3 mutations were found, suggesting that D-type cyclins exhibit a subtype-specific mutation pattern in AML. Treatment of MLL-rearranged AML cell lines with CDK4/6 inhibitors (abemaciclib and palbociclib) blocked G1 to S phase cell-cycle progression and impaired proliferation. Pediatric MLL-MLLT3–rearranged AML patients with coexisting mutations (n = 16) had significantly reduced relapse-free survival and overall survival compared with those without coexisting mutations (n = 9) (P = .048 and .046, respectively). These data provide insights into the genetics of MLL-rearranged AML and suggest therapeutic strategies.

Background Chronic myeloid leukemia (CML) is characterized by the BCR-ABL1 fusion gene. Despite the dramatic improvement of its prognosis in recent years by the development of tyrosine kinase inhibitors (TKIs), a minority of chronic phase (CP) CML patients fail to respond to TKI therapies and progress to blast crisis (BC), showing dismal clinical outcomes. While acquired mutations in ABL1 kinase have been identified as a common mechanism for TKI resistance, recent genetic studies have revealed that patients with BC frequently harbor one or more genetic alterations implicated in myeloid malignancies, suggesting additional mutations other than ABL1 mutations might drive disease progression. However, our knowledge about the mechanism of TKI resistance and progression to BC is largely limited by the scarcity of matched CP and BC samples, which were investigated for genetic alterations in relatively small number of genes. Here, we performed comprehensive genomic studies of CML-BC using paired CP and BC samples to investigate the mutation profiles associated with BC. Method We performed whole-exome sequencing of 53 patients with CML-BC, including 40 myeloid and 13 lymphoid crisis cases, as well as corresponding CP controls to investigate acquired mutations during disease progression from CP to BC. We also performed targeted-capture sequencing of known and putative driver genes in an additional 15 CML-BC samples. Combined, a total of 68 CML-BC samples were analyzed for somatic mutations, copy number abnormalities, and structural variations. Results Commonly affecting ASXL1, GATA2, and IKZF1, mutations were found only in a minority of CP cases (10/53 [19%]). However, most cases acquired somatic mutations during disease evolution from CP to BC; in whole-exome sequencing, an average of 17 additional non-synonymous mutations were newly acquired per case during evolution from CP to BC. Mutations in CML-BC frequently involved known driver genes, such as ASXL1, RUNX1, ABL1, TP53, BCOR/BCORL1, and WT1. In addition, we identified novel targets of recurrent mutations, including UBE2A, NBEAL2 and KLC2. Of note, most these driver mutations were not detected in corresponding CP samples and newly acquired, whereas ASXL1 mutations were often found in corresponding CP samples in a minor population, suggesting that ASXL1 mutations at CP might play an important role in the disease progression to BC. Mutational profiles were similar between cases with and without a history of TKI therapy before BC, except for frequent ABL1 mutations among TKI-treated cases, mostly affecting the kinase domain. Compared with lymphoid BC, myeloid BC showed a higher number of somatic mutations, which was more prominent for ASXL1, TP53, and WT1 mutations. Copy number abnormalities were rarely found in CML-CP cases (8/53), but were common and newly acquired in 29 (55%) cases with CML-BC, 18 of which showed complex karyotype-like (≥3) abnormalities. Amplification of chromosome 6 and/or 8 were characteristics of myeloid BC, while deletion of chromosome 7 was more characteristic of lymphoid BC. In some cases, structural variations other than BCR-ABL1 translocation were newly acquired in CML-BC, which frequently involved genes implicated in myeloid malignancies such as RUNX1, CBFB, and MECOM. When mutations, copy number abnormalities, and structural variants were combined, most BC cases had at least one driver alterations, which might be involved in CML-BC. Conclusion Through a comprehensive sequencing analysis using paired samples of CP and BC, we demonstrate a role of additional driver events during the clonal evolution to BC. Additional mutations were common even in CML-CP, some of which might contribute to the progression to BC. Disclosures Takaori-Kondo: Celgene: Honoraria, Research Funding; Novartis: Honoraria; Janssen Pharmaceuticals: Honoraria; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria.