SF3B1 is the most commonly mutated RNA splicing factor in cancer
, but the mechanisms by which SF3B1 mutations promote malignancy are poorly understood. Here we integrated pan-cancer splicing ...analyses with a positive-enrichment CRISPR screen to prioritize splicing alterations that promote tumorigenesis. We report that diverse SF3B1 mutations converge on repression of BRD9, which is a core component of the recently described non-canonical BAF chromatin-remodelling complex that also contains GLTSCR1 and GLTSCR1L
. Mutant SF3B1 recognizes an aberrant, deep intronic branchpoint within BRD9 and thereby induces the inclusion of a poison exon that is derived from an endogenous retroviral element and subsequent degradation of BRD9 mRNA. Depletion of BRD9 causes the loss of non-canonical BAF at CTCF-associated loci and promotes melanomagenesis. BRD9 is a potent tumour suppressor in uveal melanoma, such that correcting mis-splicing of BRD9 in SF3B1-mutant cells using antisense oligonucleotides or CRISPR-directed mutagenesis suppresses tumour growth. Our results implicate the disruption of non-canonical BAF in the diverse cancer types that carry SF3B1 mutations and suggest a mechanism-based therapeutic approach for treating these malignancies.
Although mutations in the gene encoding the RNA splicing factor SF3B1 are frequent in multiple cancers, their functional effects and therapeutic dependencies are poorly understood. Here, we ...characterize 98 tumors and 12 isogenic cell lines harboring
hotspot mutations, identifying hundreds of cryptic 3' splice sites common and specific to different cancer types. Regulatory network analysis revealed that the most common
mutation activates MYC via effects conserved across human and mouse cells.
mutations promote decay of transcripts encoding the protein phosphatase 2A (PP2A) subunit PPP2R5A, increasing MYC S62 and BCL2 S70 phosphorylation which, in turn, promotes MYC protein stability and impair apoptosis, respectively. Genetic PPP2R5A restoration or pharmacologic PP2A activation impaired SF3B1-mutant tumorigenesis, elucidating a therapeutic approach to aberrant splicing by mutant SF3B1. SIGNIFICANCE: Here, we identify that mutations in
, the most commonly mutated splicing factor gene across cancers, alter splicing of a specific subunit of the PP2A serine/threonine phosphatase complex to confer post-translational MYC and BCL2 activation, which is therapeutically intervenable using an FDA-approved drug.
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RNA-binding proteins (RBPs) are essential modulators of transcription and translation frequently dysregulated in cancer. We systematically interrogated RBP dependencies in human cancers using a ...comprehensive CRISPR/Cas9 domain-focused screen targeting RNA-binding domains of 490 classical RBPs. This uncovered a network of physically interacting RBPs upregulated in acute myeloid leukemia (AML) and crucial for maintaining RNA splicing and AML survival. Genetic or pharmacologic targeting of one key member of this network, RBM39, repressed cassette exon inclusion and promoted intron retention within mRNAs encoding HOXA9 targets as well as in other RBPs preferentially required in AML. The effects of RBM39 loss on splicing further resulted in preferential lethality of spliceosomal mutant AML, providing a strategy for treatment of AML bearing RBP splicing mutations.
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•CRISPR/Cas9 domain screen reveals RBP dependencies in AML•RBM39 is required for AML maintenance through mis-splicing of HOXA9 target genes•Proteomic studies identify an essential RBP splicing network in AML•Pharmacologic RBM39 degradation leads to broad anti-leukemic effects
Using a CRISPR/Cas9 screen targeting RNA-binding domains of classical RNA-binding proteins (RBPs), Wang et al. uncover a network of interacting RBPs upregulated in acute myeloid leukemia (AML) and crucial for RNA splicing and AML survival, highlighting RBM39 as a central, targetable component of the RBP network.
Histiocytic neoplasms are a heterogeneous group of clonal haematopoietic disorders that are marked by diverse mutations in the mitogen-activated protein kinase (MAPK) pathway
. For the 50% of ...patients with histiocytosis who have BRAF
mutations
, RAF inhibition is highly efficacious and has markedly altered the natural history of the disease
. However, no standard therapy exists for the remaining 50% of patients who lack BRAF
mutations. Although ERK dependence has been hypothesized to be a consistent feature across histiocytic neoplasms, this remains clinically unproven and many of the kinase mutations that are found in patients who lack BRAF
mutations have not previously been biologically characterized. Here we show ERK dependency in histiocytoses through a proof-of-concept clinical trial of cobimetinib, an oral inhibitor of MEK1 and MEK2, in patients with histiocytoses. Patients were enrolled regardless of their tumour genotype. In parallel, MAPK alterations that were identified in treated patients were characterized for their ability to activate ERK. In the 18 patients that we treated, the overall response rate was 89% (90% confidence interval of 73-100). Responses were durable, with no acquired resistance to date. At one year, 100% of responses were ongoing and 94% of patients remained progression-free. Cobimetinib treatment was efficacious regardless of genotype, and responses were observed in patients with ARAF, BRAF, RAF1, NRAS, KRAS, MEK1 (also known as MAP2K1) and MEK2 (also known as MAP2K2) mutations. Consistent with the observed responses, the characterization of the mutations that we identified in these patients confirmed that the MAPK-pathway mutations were activating. Collectively, these data demonstrate that histiocytic neoplasms are characterized by a notable dependence on MAPK signalling-and that they are consequently responsive to MEK inhibition. These results extend the benefits of molecularly targeted therapy to the entire spectrum of patients with histiocytosis.
Adult stem and progenitor cells are uniquely capable of self-renewal, and targeting this process represents a potential therapeutic opportunity. The early erythroid progenitor, burst-forming unit ...erythroid (BFU-E), has substantial self-renewal potential and serves as a key cell type for the treatment of anemias. However, our understanding of mechanisms underlying BFU-E self-renewal is extremely limited. Here, we found that the muscarinic acetylcholine receptor, cholinergic receptor, muscarinic 4 (CHRM4), pathway regulates BFU-E self-renewal and that pharmacological inhibition of CHRM4 corrects anemias of myelodysplastic syndrome (MDS), aging, and hemolysis. Genetic down-regulation of CHRM4 or pharmacologic inhibition of CHRM4 using the selective antagonist PD102807 promoted BFU-E self-renewal, whereas deletion of
increased erythroid cell production under stress conditions in vivo. Moreover, muscarinic acetylcholine receptor antagonists corrected anemias in mouse models of MDS, aging, and hemolysis in vivo, extending the survival of mice with MDS relative to that of controls. The effects of muscarinic receptor antagonism on promoting expansion of BFU-Es were mediated by cyclic AMP induction of the transcription factor CREB, whose targets up-regulated key regulators of BFU-E self-renewal. On the basis of these data, we propose a model of hematopoietic progenitor self-renewal through a cholinergic-mediated "hematopoietic reflex" and identify muscarinic acetylcholine receptor antagonists as potential therapies for anemias associated with MDS, aging, and hemolysis.
Drug relapse after periods of abstinence is a common feature of substance abuse. Moreover, anxiety and other mood disorders are often co-morbid with substance abuse. Cholinergic receptors in the ...ventral tegmental area (VTA) are known to mediate drug-seeking and anxiety-related behavior in rodent models. However, it is unclear if overlapping VTA cholinergic mechanisms mediate drug relapse and anxiety-related behaviors associated with drug abstinence. We examined the effects of VTA cholinergic receptor blockade on cue-induced cocaine seeking and anxiety during cocaine abstinence. Male Sprague–Dawley rats were trained to self-administer intravenous cocaine (~0.5 mg/kg/infusion, FR1 schedule) for 10 days, followed by 14 days of forced abstinence. VTA infusion of the non-selective nicotinic acetylcholine receptor antagonist mecamylamine (0, 10, and 30 μg/side) or the non-selective muscarinic receptor antagonist scopolamine (0, 2.4 and 24 μg /side) significantly decreased cue-induced cocaine seeking. In cocaine naïve rats, VTA mecamylamine or scopolamine also led to dose-dependent increases in open arm time in the elevated plus maze (EPM). In contrast, rats that received I.V. cocaine, compared to received I.V. saline rats, displayed an anxiogenic response on day 14 of abstinence as reflected by decreased open arm time in the EPM. Furthermore, low doses of VTA mecamylamine (10 μg /side) or scopolamine (2.4 μg /side), that did not alter EPM behavior in cocaine naive rats, were sufficient to reverse the anxiogenic effects of cocaine abstinence. Together, these data point to an overlapping role of VTA cholinergic mechanisms to regulate relapse and mood disorder-related responses during cocaine abstinence.
•VTA mAChR and nAChR blockade attenuates cue-induced cocaine seeking on forced abstinence day 14.•VTA mAChR and nAChR blockade produces an anxiolytic-like effect on the elevated plus maze in cocaine naive rats.•I.V. cocaine self-administration and forced abstinence produces an anxiogenic-like effect on the elevated plus maze.•VTA mAChR and nAChR blockade attenuates the cocaine-induced anxiogenic-like effect on the elevated plus maze.
Altered expression of XPO1, the main nuclear export receptor in eukaryotic cells, has been observed in cancer, and XPO1 has been a focus of anticancer drug development. However, mechanistic evidence ...for cancer-specific alterations in XPO1 function is lacking. Here, genomic analysis of 42,793 cancers identified recurrent and previously unrecognized mutational hotspots in
XPO1 mutations exhibited striking lineage specificity, with enrichment in a variety of B-cell malignancies, and introduction of single amino acid substitutions in XPO1 initiated clonal, B-cell malignancy
. Proteomic characterization identified that mutant XPO1 altered the nucleocytoplasmic distribution of hundreds of proteins in a sequence-specific manner that promoted oncogenesis. XPO1 mutations preferentially sensitized cells to inhibitors of nuclear export, providing a biomarker of response to this family of drugs. These data reveal a new class of oncogenic alteration based on change-of-function mutations in nuclear export signal recognition and identify therapeutic targets based on altered nucleocytoplasmic trafficking. SIGNIFICANCE: Here, we identify that heterozygous mutations in the main nuclear exporter in eukaryotic cells, XPO1, are positively selected in cancer and promote the initiation of clonal B-cell malignancies. XPO1 mutations alter nuclear export signal recognition in a sequence-specific manner and sensitize cells to compounds in clinical development inhibiting XPO1 function.
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Rearrangements involving the neurotrophic receptor kinase genes (NTRK1, NTRK2, and NTRK3; hereafter referred to as TRK) produce oncogenic fusions in a wide variety of cancers in adults and children. ...Although TRK fusions occur in fewer than 1% of all solid tumors, inhibition of TRK results in profound therapeutic responses, resulting in Breakthrough Therapy FDA approval of the TRK inhibitor larotrectinib for adult and pediatric patients with solid tumors, regardless of histology. In contrast to solid tumors, the frequency of TRK fusions and the clinical effects of targeting TRK in hematologic malignancies are unknown. Here, through an evaluation for TRK fusions across more than 7,000 patients with hematologic malignancies, we identified TRK fusions in acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), histiocytosis, multiple myeloma, and dendritic cell neoplasms. Although TRK fusions occurred in only 0.1% of patients (8 of 7,311 patients), they conferred responsiveness to TRK inhibition in vitro and in vivo in a patient-derived xenograft and a corresponding AML patient with ETV6-NTRK2 fusion. These data identify that despite their individual rarity, collectively, TRK fusions are present in a wide variety of hematologic malignancies and predict clinically significant therapeutic responses to TRK inhibition.
Inversions between chromosome 3q21 and 3q26 (“inv(3)/t(3;3)”) mark an aggressive, poor prognosis form of AML with limited treatment options and also occasionally occur in MDS and CML. Inv(3)/t(3;3) ...repositions a distal GATA2enhancer from 3q21 to the EVI1locus at 3q26 inducing ectopic EVI1 expression and reducing GATA2 expression. At the same time, additional genomic alterations exist in inv(3)/t(3;3) leukemias but the contributions of these events to inv(3)/t(3;3) leukemia are not well understood.
Here we evaluated genomic alterations in 63 patients with inv(3)/t(3;3) leukemia. Mutations in NRAS and the core RNA splicing factor SF3B1 were the most common individual alterations (each occurring in 27% of patients; Fig.A). We next quantified gene expression and splicing in AML samples with and without inv(3)/t(3;3) abnormalities and SF3B1 mutations. Amongst inv(3)/t(3;3) AML, the majority of gene expression changes were driven by the inv(3)/t(3;3) rearrangement while the majority of splicing changes were driven by mutant SF3B1. The most abundant category of splicing change in inv(3)/SF3B1 co-mutant cells was alteration in 3‘ splice site usage (Fig.B). Intriguingly, one of the most robust changes in splicing in inv3/SF3B1 co-mutant cells was aberrant splicing of EVI1itself such that SF3B1 mutant cells promoted expression of a novel isoform of EVI1using an intron proximal 3‘ splice site (Fig.C; the official gene name “MECOM” corresponds to the genes MDS1and EVI1at this locus).
While several mRNA isoforms of EVI1have been previously described, these all result in loss of EVI1 functional domains. In contrast, the unique EVI1 isoform in SF3B1 mutant/inv(3) AMLs contains in an in-frame insertion of six amino acids in the second zinc finger domain (ZF2) of EVI1 (a region of EVI1 known to be affected by germline mutations in leukemia predisposition syndromes). These data identify that nearly one-third of inv(3) AML patients express a heretofore undescribed isoform of EVI1. Of note, this unannotated EVI1isoform is also present in EVI1expressing/SF3B1K700Emutant leukemias lacking inv(3)).
The above findings highlight a novel model where inv(3)/t(3;3) AML is driven by ectopic expression of distinct oncogenic isoforms of EVI1. To test this model and understand the contribution of SF3B1K700Eto inv(3)/t(3;3) AML, we crossed transgenic mice bearing the entire human inv(3)(q21;q26) locus whereby the GATA2enhancer misdirects human EVI1expression (“inv3 mice”; Yamazaki et al. Cancer Cell2014) to Sf3b1K700Econditional knockin mice (Mx1-cre Sf3b1K700E/WT).Given that the human MECOMlocus (coding and noncoding regions) was recapitulated in this mouse model, the concordant novel EVI1isoform was expressed in inv3/Sf3b1K700Emice as in patients. While Mx1-cre Sf3b1K700Emice develop an MDS-like disorder, inv3 mice develop myeloid and lymphoid leukemias with lethality ~300 days after birth. However, expression of the Sf3b1K700E/WTmutation in inv3 hematopoietic cells resulted in a highly penetrant MDS, which transformed to a lethal AML by a median of 241 days (Fig.D;p=0.0021). In the first 6 months following transplant, Mx1-cre inv3 Sf3b1K700Emice had leukopenia, macrocytic anemia, and morphologic dysplasia (Fig.E-F) that eventually transformed to a disease with a high WBC count and large numbers of immature cells around time of death. In competitive reconstitution assays, Mx1-cre inv3 Sf3b1K700Ehematopoietic stem cells (HSCs) failed to differentiate into mature peripheral blood cells despite having a competitive advantage at the level of HSCs (Fig.G-H).
RNA-seq of hematopoietic precursors from the above models identified (i) a substantial change in splicing in inv3/Sf3b1K700Emutant leukemias versus those driven by inv3 alone, and (ii) alterations in a host of RNA binding proteins in inv3/Sf3b1K700Emutant leukemias (Fig.I).
These data highlight a high occurrence of SF3B1 mutations in inv(3)/t(3;3) leukemias, present a new genetically accurate model for inv(3) AML, and uncover a novel oncogenic isoform of EVI1 expressed in a large proportion of inv(3)/t(3;3) patients. Ongoing work focused on identifying the mechanistic effect of the SF3B1-mutant induced aberrant EVI1isoform may provide novel insight into the role of EVI1 in promoting leukemogenesis and engender development of therapeutic opportunities targeting EVI1splicing.
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No relevant conflicts of interest to declare.
Mutations in the RNA splicing factor SF3B1 are recurrent in CLL and myeloid neoplasms but their functional role in promoting tumorigenesis remain poorly understood. While SF3B1 mutations have been ...identified as promoting use of aberrant 3' splice sites (3'ss), consistent identification of mis-spliced transcripts and pathways that functionally link mutant SF3B1 to transformation remains elusive. Moreover, large-scale analyses of the impact of mutant SF3B1 on gene expression and gene regulatory networks, which may be distinct from aberrant splicing changes, remain to be performed. We therefore sought to elucidate the effects of SF3B1 mutations across hematopoietic malignancies and cancer lineages at the level of both mRNA splicing and expression. To this end, we collected RNA-seq data from 79 tumors and 12 isogenic cell lines harboring SF3B1 hotspot mutations. The most frequent hotspot, K700E, was the most common mutation in CLL and breast cancers while mutations at position R625 were restricted to melanomas (Figure A, B).
Regulatory network analysis of differentially expressed genes in SF3B1 mutated CLL identified MYC as the top master regulator (Figure C). MYC activation in SF3B1 mutated CLL was also verified by differential expression analyses (Figure D) and was common to SF3B1K700E mutant cancers while absent in cancers with mutations affecting R625. Taken together, these observations suggested that tumors harboring SF3B1K700E mutations activate the MYC transcriptional program.
We next sought to verify the effects of c-Myc activation by mutant Sf3b1 in the B-cell lineage in vivo. We crossed Cd19-cre Sf3b1K700E/+ mice with Eμ-Myc transgenic mice to generate Cd19-cre+ control, Sf3b1K700E/+, Eμ-MycTg/+, and Sf3b1K700E/+Eμ-MycTg/+ double-mutant mice. While control or single mutant primary mice did not develop disease over one year, double-mutant mice developed a lethal B-cell malignancy. This effect was consistent in serial transplantation, where mice transplanted with double-mutant cells had shorter survival compared to single-mutant controls (Figure E). These data provide the first evidence that SF3B1 mutations contribute to tumorigenesis in vivo.
To understand the molecular mechanism for MYC activation across SF3B1 mutant human and mouse cells, we analyzed RNA-seq data from CLL patients, isogenic Nalm-6 cells, and splenic B-cells from the mouse models. This revealed a significant overlap in aberrant (3'ss) events across SF3B1 mutant samples. Interestingly, mis-spliced events across mouse and human SF3B1K700E mutant samples identified aberrant 3'ss usage and decay of PPP2R5A (Figure F), a gene whose product has previously been shown to regulate c-MYC protein stability and the only gene whose aberrant splicing was most prominent in K700E compared with R625 mutant SF3B1. PPP2R5A is a subunit of the PP2A phosphatase complex that dephosphorylates Serine 62 (S62) of c-MYC, resulting in an unstable form of c-MYC that is a substrate for proteasomal degradation. Consistent with this, SF3B1K700E mutant cells exhibited dramatic increase in S62-phosphorylated c-MYC and increased stability of c-MYC protein. MYC expression, stability, and S62 phosphorylation could be abrogated in SF3B1 mutant cells by restoring PPP25RA expression. In addition to c-MYC S62 phosphorylation, PPP2R5A-containing PP2A reduced S70 phosphorylation of BCL2 (a modification important for apoptosis induction) in SF3B1 mutant cells. To functionally evaluate the importance of impaired PP2A enzymatic activity in SF3B1 mutant cells further, we assessed the therapeutic potential of the FDA-approved oral PP2A activator, FTY-720. SF3B1 mutant cells were more sensitive to FTY-720 treatment than SF3B1 WT counterparts, experiencing growth arrest at lower concentration (Figure G). Moreover, both S62-phosphorylated c-MYC and S70-phosphorylated BCL2 decreased in a dose-dependent manner upon treatment with FTY-720 (Figure H).
Here through combined evaluation of the effects of the SF3B1 mutation on splicing, gene expression, and transcriptional networks across cancer types, we identify a novel mechanism by which mutant SF3B1-mediated alterations in RNA splicing contribute to activation of oncogenic MYC through effects on MYC proteolysis. Moreover, these data highlight a novel therapeutic approach targeting the impact of mutant SF3B1 on post-translational modification of MYC.
Mato:Janssen: Consultancy, Honoraria; Celgene: Consultancy; Prime Oncology: Speakers Bureau; TG Therapeutics: Research Funding; Regeneron: Research Funding; Abbvie: Consultancy; Sunesis: Honoraria, Research Funding; Acerta: Research Funding; AstraZeneca: Consultancy; Pharmacyclics: Consultancy, Honoraria, Research Funding.
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