Recurrent mutations in the spliceosome are observed in several human cancers, but their functional and therapeutic significance remains elusive. SF3B1, the most frequently mutated component of the ...spliceosome in cancer, is involved in the recognition of the branch point sequence (BPS) during selection of the 3′ splice site (ss) in RNA splicing. Here, we report that common and tumor-specific splicing aberrations are induced by SF3B1 mutations and establish aberrant 3′ ss selection as the most frequent splicing defect. Strikingly, mutant SF3B1 utilizes a BPS that differs from that used by wild-type SF3B1 and requires the canonical 3′ ss to enable aberrant splicing during the second step. Approximately 50% of the aberrantly spliced mRNAs are subjected to nonsense-mediated decay resulting in downregulation of gene and protein expression. These findings ascribe functional significance to the consequences of SF3B1 mutations in cancer.
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•SF3B1 hotspot mutations are neomorphic and induce aberrant 3′ splice site selection•Mutant SF3B1 utilizes a different branch point than that used by wild-type SF3B1•SF3B1 mutants require the canonical 3′ splice site to induce aberrant splicing•∼50% of aberrant mRNAs undergo NMD leading to downregulation of canonical isoforms
Darman et al. report that SF3B1 mutations found in cancer induce aberrant 3′ splice site selection. To induce aberrant splicing, mutant SF3B1 requires canonical 3′ splice site but utilizes a different branch point than wild-type SF3B1. Approximately 50% of the aberrant mRNAs undergo NMD resulting in downregulation of canonical transcripts.
The transcription factor SOX2 is an essential regulator of pluripotent stem cells and promotes development and maintenance of squamous epithelia. We previously reported that SOX2 is an oncogene and ...subject to highly recurrent genomic amplification in squamous cell carcinomas (SCCs). Here, we have further characterized the function of SOX2 in SCC. Using ChIP-seq analysis, we compared SOX2-regulated gene profiles in multiple SCC cell lines to ES cell profiles and determined that SOX2 binds to distinct genomic loci in SCCs. In SCCs, SOX2 preferentially interacts with the transcription factor p63, as opposed to the transcription factor OCT4, which is the preferred SOX2 binding partner in ES cells. SOX2 and p63 exhibited overlapping genomic occupancy at a large number of loci in SCCs; however, coordinate binding of SOX2 and p63 was absent in ES cells. We further demonstrated that SOX2 and p63 jointly regulate gene expression, including the oncogene ETV4, which was essential for SOX2-amplified SCC cell survival. Together, these findings demonstrate that the action of SOX2 in SCC differs substantially from its role in pluripotency. The identification of the SCC-associated interaction between SOX2 and p63 will enable deeper characterization the downstream targets of this interaction in SCC and normal squamous epithelial physiology.
KRAS mutations in codons 12 and 13 are established predictive biomarkers for anti-EGFR therapy in colorectal cancer. Previous studies suggest that KRAS codon 61 and 146 mutations may also predict ...resistance to anti-EGFR therapy in colorectal cancer. However, clinicopathological, molecular, and prognostic features of colorectal carcinoma with KRAS codon 61 or 146 mutation remain unclear.
We utilized a molecular pathological epidemiology database of 1267 colon and rectal cancers in the Nurse's Health Study and the Health Professionals Follow-up Study. We examined KRAS mutations in codons 12, 13, 61 and 146 (assessed by pyrosequencing), in relation to clinicopathological features, and tumor molecular markers, including BRAF and PIK3CA mutations, CpG island methylator phenotype (CIMP), LINE-1 methylation, and microsatellite instability (MSI). Survival analyses were performed in 1067 BRAF-wild-type cancers to avoid confounding by BRAF mutation. Cox proportional hazards models were used to compute mortality hazard ratio, adjusting for potential confounders, including disease stage, PIK3CA mutation, CIMP, LINE-1 hypomethylation, and MSI.
KRAS codon 61 mutations were detected in 19 cases (1.5%), and codon 146 mutations in 40 cases (3.2%). Overall KRAS mutation prevalence in colorectal cancers was 40% (=505/1267). Of interest, compared to KRAS-wild-type, overall, KRAS-mutated cancers more frequently exhibited cecal location (24% vs. 12% in KRAS-wild-type; P < 0.0001), CIMP-low (49% vs. 32% in KRAS-wild-type; P < 0.0001), and PIK3CA mutations (24% vs. 11% in KRAS-wild-type; P < 0.0001). These trends were evident irrespective of mutated codon, though statistical power was limited for codon 61 mutants. Neither KRAS codon 61 nor codon 146 mutation was significantly associated with clinical outcome or prognosis in univariate or multivariate analysis colorectal cancer-specific mortality hazard ratio (HR) = 0.81, 95% confidence interval (CI) = 0.29-2.26 for codon 61 mutation; colorectal cancer-specific mortality HR = 0.86, 95% CI = 0.42-1.78 for codon 146 mutation.
Tumors with KRAS mutations in codons 61 and 146 account for an appreciable proportion (approximately 5%) of colorectal cancers, and their clinicopathological and molecular features appear generally similar to KRAS codon 12 or 13 mutated cancers. To further assess clinical utility of KRAS codon 61 and 146 testing, large-scale trials are warranted.
Carbamoyl phosphate synthetase 1 (CPS1) catalyzes the first step in the ammonia-detoxifying urea cycle, converting ammonia to carbamoyl phosphate under physiologic conditions. In cancer, CPS1 ...overexpression supports pyrimidine synthesis to promote tumor growth in some cancer types, while in others CPS1 activity prevents the buildup of toxic levels of intratumoral ammonia to allow for sustained tumor growth. Targeted CPS1 inhibitors may, therefore, provide a therapeutic benefit for cancer patients with tumors overexpressing CPS1. Herein, we describe the discovery of small-molecule CPS1 inhibitors that bind to a previously unknown allosteric pocket to block ATP hydrolysis in the first step of carbamoyl phosphate synthesis. CPS1 inhibitors are active in cellular assays, blocking both urea synthesis and CPS1 support of the pyrimidine biosynthetic pathway, while having no activity against CPS2. These newly discovered CPS1 inhibitors are a first step toward providing researchers with valuable tools for probing CPS1 cancer biology.
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•A high-throughput screen identifies a small-molecule inhibitor of CPS1, H3B-120•H3B-120 binds to an allosteric pocket to achieve highly selective inhibition of CPS1•Analogs of H3B-120 block CPS1 cellular activity in primary human hepatocytes
CPS1 is a proposed oncoprotein of interest for drug development. Yao et al. describe the discovery of small-molecule inhibitors of CPS1 and discuss their proposed impact for studying CPS1 cancer biology.
The Drosophila melanogaster male-specific lethal (MSL) complex binds the single male X chromosome to upregulate gene expression to equal that from the two female X chromosomes. However, it has been ...puzzling that approximately 25% of transcribed genes on the X chromosome do not stably recruit MSL complex. Here we find that almost all active genes on the X chromosome are associated with robust H4 Lys16 acetylation (H4K16ac), the histone modification catalyzed by the MSL complex. The distribution of H4K16ac is much broader than that of the MSL complex, and our results favor the idea that chromosome-wide H4K16ac reflects transient association of the MSL complex, occurring through spreading or chromosomal looping. Our results parallel those of localized Polycomb repressive complex and its more broadly distributed chromatin mark, trimethylated histone H3 Lys27 (H3K27me3), suggesting a common principle for the establishment of active and silenced chromatin domains.
Mutations affecting RNA splicing factors are commonly found in leukemias and mutations in SF3B1, SRSF2, and U2AF1 impart specific changes to global splicing. We analyzed RNA-seq data from acute ...myeloid leukemia (AML) patients from The Cancer Genome Atlas (TCGA) to evaluate for spliceosomal alterations characteristic of mutant SRSF2, SF3B1, and U2AF1 . Although only 1 patient with an SRSF2 mutation was reported in the TCGA AML publication, we identified 19 additional patients to have the characteristic splicing changes of mutant SRSF2 . Mutational analysis of the RNA-seq data identified the SRSF2 proline 95 hotspot mutation in each of these 19 patients (19/178, 11%), making SRSF2 amongst the most commonly mutated genes in the AML TCGA dataset. Interestingly, 47% of SRSF2 mutant patients also had a co-existing IDH2 mutation (and conversely, 50% of IDH2 mutant patients had a co-existing SRSF2 mutation). These data are similar to recent observation that SRSF2 mutations were the most common accompanying mutation in IDH2 mutant myeloid leukemia patients treated on the phase I clinical trial of AG-221.
We next sought to understand the functional contribution of SRSF2 mutations to co-existing IDH2 mutations. Overexpression of IDH2 R140Q and IDH2 R172K in Vav -cre Srsf2 P95H or Vav- cre Srsf2 wildtype(WT) backgrounds revealed a clear collaborative effect of mutant IDH2 and mutant Srsf2 (Figure A-B). As early as 4 weeks post-transplant, the peripheral blood of Srsf2 mutant mice transplanted with IDH2 R140Q or IDH2 R172K forms had a substantially greater percentage of GFP+ cells than seen in an Srsf2 WT background. Moreover, these mice exhibited significant myeloid skewing, macrocytic anemia, and thrombocytopenia of greater magnitude than seen with mutant IDH2 expressed in the SRSF2 WT background. IDH2 / Srsf2 double-mutant mice developed a lethal, mixed myelodysplastic/myeloproliferative neoplasm that was serially transplantable (Figure B). Effects from overexpression of mutant IDH2 were recapitulated in mice expressing Idh2 R140Q and Srsf2 P95H from endogenous loci(Mx1 -cre Idh2R140Q/+Srsf2P95H/+ double knock-in mice) (Figure C), providing the first model where spliceosomal gene mutations promote leukemogenesis in vivo .
Given prior data identifying inhibition of TET2 as a mediator of IDH2 -mutant leukemogenesis, we evaluated if loss of TET2 might promote transformation of SRSF2 mutant cells. However, deletion of TET2 in an SRSF2 mutant background (Mx1 -cre Tet2fl/flSrsf2P95H/+) was insufficient to promote transformation (Figure D). We next analyzed RNA-seq data from the TCGA, Leucegene, and our own unpublished AML cohort using a novel analysis method to detect unannotated as well as complex splicing events that might explain the basis for the collaboration of mutant IDH2 and SRSF2 . This revealed that IDH2/SRSF2 double-mutant AML had increased aberrant splicing events compared to SRSF2 single-mutant AML and also that IDH2 -mutations are associated with a small but reproducible change in RNA splicing (Figure E). The vast majority of splicing changes affected by SRSF2 and/or IDH2 mutations were a change in cassette exon splicing with SRSF2 mutations promoting inclusion of C-rich containing mRNA sequences, regardless of the presence of concomitant IDH2 mutations (Figure F). One of the most robust mis-splicing events in IDH2/SRSF2 mutant AML was an intron retention event in INTS3 resulting in its reduced mRNA and protein expression (Figure G-H). INTS3 encodes a component of the integrator complex that participates in small nuclear RNA (snRNA) processing. Consistent with this, IDH2 / SRSF2 double-mutant cells exhibited alterations in processing of U2 and U4 snRNAs similar to that seen with direct INTS3 downregulation. Biologically, attenuation of INTS3 expression caused a blockade of myeloid differentiation in HL-60 cells upon ATRA treatment, an effect which was further enhanced in an IDH2 mutant background.
These data uncover an important role for spliceosomal gene mutations in the pathogenesis of IDH2 mutant leukemogenesis and identify perturbations in the integrator complex as a novel mediator driving the transformation of IDH2 and SRSF2 mutant cells. The frequent coexistence of IDH2 and SRSF2 mutations underscores the enormous therapeutic potential for modulation of splicing in the ~50% of IDH2 -mutant leukemia patients who also harbor a spliceosomal gene mutation.
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De Botton:Servier: Honoraria; Agios: Honoraria, Research Funding; Celgene: Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Stein:Pfizer: Consultancy, Other: Travel expenses; Novartis: Consultancy, Research Funding; Agios Pharmaceuticals, Inc.: Consultancy, Research Funding; Celgene Corporation: Consultancy, Other: Travel expenses, Research Funding; Seattle Genetics: Research Funding; GSK: Other: Advisory Board, Research Funding; Constellation Pharma: Research Funding. Peng:H3 Biomedicine Inc.: Employment. Buonamici:H3 Biomedicine Inc.: Employment. Palacino:H3 Biomedicine Inc.: Employment. Smith:H3 Biomedicine Inc.: Employment. Thompson:Merck: Membership on an entity's Board of Directors or advisory committees; Charles River Laboratories: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Other: Founder. Levine:Roche: Research Funding; Roche: Research Funding; Qiagen: Equity Ownership; Qiagen: Equity Ownership; Celgene: Research Funding; Celgene: Research Funding.
Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders characterized by dysplastic hematopoiesis and peripheral blood cytopenias. Recently, somatic mutations affecting ...components of the spliceosomal machinery have been discovered in the majority of MDS patients. SF3B1 mutations are most frequent and strongly correlate with the presence of bone marrow ring sideroblasts and a favorable prognosis. SF3B1 mutations, including the K700E substitution which accounts for more than 50% of all mutations, are missense, heterozygous and cluster in a hotspot within the heat domain of the protein suggesting that they are gain-of-function variants.
The molecular effects of SF3B1 mutations and the mechanisms through which they drive clonal expansion and dyserythropoiesis remain obscure. Therefore, to assess their molecular and phenotypic consequences, we generated a mouse model carrying a conditional floxed knock-in allele (Sf3b1flox-K700E/+) by homologous recombination of JM8 murine embryonic stem cells. To induce expression of Sf3b1 K700E in adult hematopoietic stem and progenitor cells, Sf3b1flox-K700E/+/Mx1-Cre+ were injected with pIpC from 4-8 weeks of age. Here we report the initial characterization of these animals.
Monthly peripheral blood counts from mutants and wild-type (WT) littermates starting one month post-pIpC injection showed a reduction in hemoglobin levels (at 8 weeks WT=17g/dl mut=14.5g/dl, p<0.03). Additionally, flow cytometric analysis of bone marrow samples demonstrated a modest but consistent decrease in late erythroid progenitor cells (Ter119+ and CD71-/low). The myeloid compartment showed relative expansion of Gr1+/Mac1+ and Mac1+ cells whereas analysis of hematopoietic stem and progenitor cells (HSPCs) revealed a decrease in HSCs (% of total events WT=0.04%; Sf3b1flox-K700E/+=0.01%) in mutant mice. In competitive transplantation experiments into sub-lethally irradiated syngeneic recipients we observed a lower engraftment potential of Sf3b1flox-K700E Lin-ve HSPCs (CD45.2) compared to wild-type cells (CD45.1). Flow cytometric analysis of peripheral blood of recipient animals showed that Sf3b1flox-K700E cells contributed more to the myeloid lineage than wild-type cells (Sf3b1flox-K700E Mac1+/Gr1+ 8.95%; Mac1+ 15% vs WT Mac1+/Gr1+ 4.08%; Mac1+ 5.57%). At a median follow-up of 56 weeks, mutant animals did not show decreased survival or signs of illness as compared to WT controls.
Finally, as Sfb31 mutations are predicted to affect splicing of pre-mRNA and consequently alter the gene expression, we performed RNAseq analysis in unselected and Lin-ve bone-marrow cells from mutant and controls animals. Comparison between wt and mutant samples showed deregulated expression of genes implicated in human MDS (Mmp9, Puma, Bcl2l1). We then looked at the pattern of aberrant splicing promoted by Sf3b1flox-K700E, and found that mutant animals have an increased use of cryptic 3'' splice sites (ss) throughout their genome. We showed that the majority of these alternative 3' ss are novel and we characterized them as being located 15 to 24 nucleotides upstream from the canonical 3' ss and associated with sequence features including a shorter polypyrimidine tract and an enrichment of adenines -8 to -18 bases upstream of the cryptic 3' ss. Interestingly, similar features have been reported in human cancers with SF3B1 hotspot mutations. We predict that ~33% of the mRNAs affected by aberrant splicing will include an aberrant premature termination codon, promoting RNA degradation through nonsense-mediated decay.
In conclusion, our conditional Sf3b1K700E knock-in mouse is a faithful molecular model of the consequences of these mutations in the mouse hematopoietic system. The mild phenotype we observe in comparison to SF3B1-mutant human MDS may be explained by the requirement for additional mutations to progress to overt MDS and is more reminiscent of SF3B1-associated clonal hemopoiesis, relatively common phenomenon in elderly humans without overt hematological abnormalities.
Additionally, our initial characterization of novel splice sites preferentially recognised by the mutant Sf3b1 protein suggests that transcriptional consequences of the mutation may differ between species, dependant on the degree of conservation of the relevant intronic regions.
Seiler:H3 Biomedicine: Employment. Peng:H3 Biomedicine: Employment. Buonamici:H3 Biomedicine: Employment. Campbell:14M genomics: Other: Co-founder and consultant.
The Drosophila MSL complex mediates dosage compensation by increasing transcription of the single X chromosome in males approximately two-fold. This is accomplished through recognition of the X ...chromosome and subsequent acetylation of histone H4K16 on X-linked genes. Initial binding to the X is thought to occur at "entry sites" that contain a consensus sequence motif ("MSL recognition element" or MRE). However, this motif is only ∼2 fold enriched on X, and only a fraction of the motifs on X are initially targeted. Here we ask whether chromatin context could distinguish between utilized and non-utilized copies of the motif, by comparing their relative enrichment for histone modifications and chromosomal proteins mapped in the modENCODE project. Through a comparative analysis of the chromatin features in male S2 cells (which contain MSL complex) and female Kc cells (which lack the complex), we find that the presence of active chromatin modifications, together with an elevated local GC content in the surrounding sequences, has strong predictive value for functional MSL entry sites, independent of MSL binding. We tested these sites for function in Kc cells by RNAi knockdown of Sxl, resulting in induction of MSL complex. We show that ectopic MSL expression in Kc cells leads to H4K16 acetylation around these sites and a relative increase in X chromosome transcription. Collectively, our results support a model in which a pre-existing active chromatin environment, coincident with H3K36me3, contributes to MSL entry site selection. The consequences of MSL targeting of the male X chromosome include increase in nucleosome lability, enrichment for H4K16 acetylation and JIL-1 kinase, and depletion of linker histone H1 on active X-linked genes. Our analysis can serve as a model for identifying chromatin and local sequence features that may contribute to selection of functional protein binding sites in the genome.
Heterozygous mutations in several core members of the spliceosome complex have been reported in Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML). In particular high frequency SF3B1 ...hotspot mutations, a component of the U2 complex involved in the interaction with the branch point (BP) and recognition of the 3' splice sites (ss) during splicing, have been identified in Refractory Anemia with Ringed Sideroblasts (RARS) a subtype of MDS. Using computational analyses of RNAseq from several cancer types including RARS, we identified that SF3B1 hotspot mutations induce aberrant 3'ss selection by recognizing a cryptic AG located between 15 to 24 nucleotides upstream of the canonical AG. Experimental confirmation of these motif features was performed using minigenes in SF3B1 mutant cells. Furthermore, we discovered that SF3B1 mutant utilized a different BP from that used by SF3B1 wild-type providing novel mechanistic insights into changes in function induced by the hotspot mutations. The induction of aberrant splicing can introduce premature termination codons thus targeting mRNA for degradation by Nonsense Mediated Decay (NMD). We predicted that close to 50% of the aberrantly spliced genes would be subject to NMD and showed (using isogenic Nalm-6 cells engineered by AAV homology to express SF3B1K700E or SF3B1K700K) that several of these genes were downregulated at the transcript and protein levels. These downregulated genes/proteins might be involved in the pathogenesis of SF3B1 mutant cancers. Interestingly, pathway analysis of genes differentially expressed or aberrantly spliced in SF3B1 mutant compared to wild-type in RARS samples identified cell differentiation and epigenetics as the primary misregulated pathways. To study the impact of SF3B1 mutations on differentiation, we used the TF-1 differentiation cell model where erythroid differentiation is induced by treatment with erythropoietin (EPO). EPO treatment, as expected, induced erythroid differentiation in TF-1 cells transduced with SF3B1WT, but a block in erythroid differentiation was observed in TF-1 cells transduced with SF3B1K700E (the most common mutation in MDS and chronic lymphocytic leukemia (CLL)). Intriguingly, SF3B1G742D, which is found mutated in CLL but not MDS, did not block differentiation in this myeloid differentiation model, implying that specific SF3B1 mutations and splicing aberrations have important context dependent effects. Pathway analysis comparing SF3B1K700E vs. SF3B1WT or SF3B1G742D identified several genes involved in heme biosynthesis or downstream of GATA1 to be downregulated (such as, AHSP, ALAS2, CCL5, CD36, EPOR, GP1BB, HBB, HBE1, HBG1, PRG2) in SF3B1K700E cells only. This is consistent with the role of SF3B1K700E in RARS. In our analyses, we also identified that ABCB7 is aberrantly spliced and that the aberrant transcript is subject to NMD, causing downregulation of the canonical transcript and protein. ABCB7 is a mitochondrial transporter important in cellular iron metabolism and in heme production; moreover, partial loss of function mutation in ABCB7 has been identified in X-linked sideroblastic anemia and ataxia, demonstrating an iron overload phenotype in cells with defective ABCB7. Interestingly, when ABCB7 was knocked down in TF-1 cells we observed block in differentiation similar to that observed in SF3B1K700E cells suggesting a link between SF3B1 mutation and ABCB7 levels and impaired differentiation. Taken together, these data suggest that SF3B1 mutations induce aberrant splicing and as a consequence downregulation of several genes that contribute to the block in erythroid differentiation, one of the key biological defects observed in MDS.
Buonamici:H3 Biomedicine: Employment. Darman:H3 Biomedicine: Employment. Perino:H3 Biomedicine: Employment. Agrawal:H3 Biomedicine: Employment. Peng:H3 Biomedicine: Employment. Seiler:H3 Biomedicine: Employment. Feala:H3 Biomedicine: Employment. Bailey:H3 Biomedicine: Employment. Chan:H3 Biomedicine: Employment. Fekkes:H3 Biomedicine: Employment. Keaney:H3 Biomedicine: Employment. Kumar:H3 Biomedicine: Employment. Kunii:H3 Biomedicine: Employment. Lee:H3 Biomedicine: Employment. Mackenzie:Eisai: Employment. Matijevic:Eisai: Employment. Mizui:H3 Biomedicine: Employment. Myint:Eisai: Employment. Park:H3 Biomedicine: Employment. Pazolli:H3 Biomedicine: Employment. Thomas:H3 Biomedicine: Employment. Wang:H3 Biomedicine: Employment. Warmuth:H3 Biomedicine: Employment. Yu:H3 Biomedicine: Employment. Zhu:H3 Biomedicine: Employment. Furman:Acerta Pharma BV: Research Funding; Gilead: Consultancy; Pharmacyclics LLC, an AbbVie Company: Consultancy, Honoraria, Speakers Bureau. Ebert:Celgene: Consultancy; H3 Biomedicine: Consultancy; Genoptix: Consultancy, Patents & Royalties. Smith:H3 Biomedicine: Employment.
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