Pharmacologic modulation of RNA splicing enhances anti-tumor immunity.

Although mutations in DNA are the best-studied source of neoantigens that determine response to immune checkpoint blockade, alterations in RNA splicing within cancer cells could similarly result in neoepitope production. However, the endogenous antigenicity and clinical potential of such splicing-derived epitopes have not been tested. Here, we demonstrate that pharmacologic modulation of splicing via specific drug classes generates bona fide neoantigens and elicits anti-tumor immunity, augmenting checkpoint immunotherapy. Splicing modulation inhibited tumor growth and enhanced checkpoint blockade in a manner dependent on host T cells and peptides presented on tumor MHC class I. Splicing modulation induced stereotyped splicing changes across tumor types, altering the MHC I-bound immunopeptidome to yield splicing-derived neoepitopes that trigger an anti-tumor T cell response in vivo. These data definitively identify splicing modulation as an untapped source of immunogenic peptides and provide a means to enhance response to checkpoint blockade that is readily translatable to the clinic.

SnapShot: Splicing Alterations in Cancer.

RNA splicing, the spliceosome-catalyzed process by which pre-messenger RNA (pre-mRNA) is processed to mature mRNA, is altered in a number of ways in cancer. Tumor-specific splicing alterations are created by mutations that disrupt splicing-regulatory elements within genes and impair splicing recognition or by altering the RNA-binding preferences of individual splicing factors. This SnapShot summarizes our current understanding of splicing-factor alterations in cancers. To view this SnapShot, open or download the PDF.

NUDT21 limits CD19 levels through alternative mRNA polyadenylation in B cell acute lymphoblastic leukemia.

B cell progenitor acute lymphoblastic leukemia (B-ALL) treatment has been revolutionized by T cell-based immunotherapies-including chimeric antigen receptor T cell therapy (CAR-T) and the bispecific T cell engager therapeutic, blinatumomab-targeting surface glycoprotein CD19. Unfortunately, many patients with B-ALL will fail immunotherapy due to 'antigen escape'-the loss or absence of leukemic CD19 targeted by anti-leukemic T cells. In the present study, we utilized a genome-wide CRISPR-Cas9 screening approach to identify modulators of CD19 abundance on human B-ALL blasts. These studies identified a critical role for the transcriptional activator ZNF143 in CD19 promoter activation. Conversely, the RNA-binding protein, NUDT21, limited expression of CD19 by regulating CD19 messenger RNA polyadenylation and stability. NUDT21 deletion in B-ALL cells increased the expression of CD19 and the sensitivity to CD19-specific CAR-T and blinatumomab. In human B-ALL patients treated with CAR-T and blinatumomab, upregulation of NUDT21 mRNA coincided with CD19 loss at disease relapse. Together, these studies identify new CD19 modulators in human B-ALL.

Restoration of TET2 Function Blocks Aberrant Self-Renewal and Leukemia Progression.

Loss-of-function mutations in TET2 occur frequently in patients with clonal hematopoiesis, myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML) and are associated with a DNA hypermethylation phenotype. To determine the role of TET2 deficiency in leukemia stem cell maintenance, we generated a reversible transgenic RNAi mouse to model restoration of endogenous Tet2 expression. Tet2 restoration reverses aberrant hematopoietic stem and progenitor cell (HSPC) self-renewal in vitro and in vivo. Treatment with vitamin C, a co-factor of Fe2+ and α-KG-dependent dioxygenases, mimics TET2 restoration by enhancing 5-hydroxymethylcytosine formation in Tet2-deficient mouse HSPCs and suppresses human leukemic colony formation and leukemia progression of primary human leukemia PDXs. Vitamin C also drives DNA hypomethylation and expression of a TET2-dependent gene signature in human leukemia cell lines. Furthermore, TET-mediated DNA oxidation induced by vitamin C treatment in leukemia cells enhances their sensitivity to PARP inhibition and could provide a safe and effective combination strategy to selectively target TET deficiency in cancer. PAPERCLIP.

Splicing regulation of DNA repair via CCAR1.

In this issue of Molecular Cell, Harada et al.1 and Karasu et al.2 identify CCAR1 as a novel regulator of the Fanconi anemia/BRCA DNA repair pathway via modulating the splicing of the mRNA encoding FANCA.

Molecular impact of mutations in RNA splicing factors in cancer.

Somatic mutations in genes encoding components of the RNA splicing machinery occur frequently in multiple forms of cancer. The most frequently mutated RNA splicing factors in cancer impact intronic branch site and 3' splice site recognition. These include mutations in the core RNA splicing factor SF3B1 as well as mutations in the U2AF1/2 heterodimeric complex, which recruits the SF3b complex to the 3' splice site. Additionally, mutations in splicing regulatory proteins SRSF2 and RBM10 are frequent in cancer, and there has been a recent suggestion that variant forms of small nuclear RNAs (snRNAs) may contribute to splicing dysregulation in cancer. Here, we describe molecular mechanisms by which mutations in these factors alter splice site recognition and how studies of this process have yielded new insights into cancer pathogenesis and the molecular regulation of splicing. We also discuss data linking mutant RNA splicing factors to RNA metabolism beyond splicing.

GPATCH8 modulates mutant SF3B1 mis-splicing and pathogenicity in hematologic malignancies.

Mutations in the RNA splicing factor gene SF3B1 are common across hematologic and solid cancers and result in widespread alterations in splicing, yet there is currently no therapeutic means to correct this mis-splicing. Here, we utilize synthetic introns uniquely responsive to mutant SF3B1 to identify trans factors required for aberrant mutant SF3B1 splicing activity. This revealed the G-patch domain-containing protein GPATCH8 as required for mutant SF3B1-induced splicing alterations and impaired hematopoiesis. GPATCH8 is involved in quality control of branchpoint selection, interacts with the RNA helicase DHX15, and functionally opposes SURP and G-patch domain containing 1 (SUGP1), a G-patch protein recently implicated in SF3B1-mutant diseases. Silencing of GPATCH8 corrected one-third of mutant SF3B1-dependent splicing defects and was sufficient to improve dysfunctional hematopoiesis in SF3B1-mutant mice and primary human progenitors. These data identify GPATCH8 as a novel splicing factor required for mis-splicing by mutant SF3B1 and highlight the therapeutic impact of correcting aberrant splicing in SF3B1-mutant cancers.

RBM33: A new regulator of N-6-methyladenosine (m6A) methylation.

In this issue of Molecular Cell, Yu et al.1 identify RBM33 as a previously unrecognized m6A (N-6-methyladenosine) RNA binding protein that plays a critical role in ALKBH5-mediated m6A demethylation of a subset of mRNA transcripts by forming a complex with ALKBH5.

Proteomic discovery of chemical probes that perturb protein complexes in human cells.

Most human proteins lack chemical probes, and several large-scale and generalizable small-molecule binding assays have been introduced to address this problem. How compounds discovered in such "binding-first" assays affect protein function, nonetheless, often remains unclear. Here, we describe a "function-first" proteomic strategy that uses size exclusion chromatography (SEC) to assess the global impact of electrophilic compounds on protein complexes in human cells. Integrating the SEC data with cysteine-directed activity-based protein profiling identifies changes in protein-protein interactions that are caused by site-specific liganding events, including the stereoselective engagement of cysteines in PSME1 and SF3B1 that disrupt the PA28 proteasome regulatory complex and stabilize a dynamic state of the spliceosome, respectively. Our findings thus show how multidimensional proteomic analysis of focused libraries of electrophilic compounds can expedite the discovery of chemical probes with site-specific functional effects on protein complexes in human cells.

SRSF2 plays an unexpected role as reader of m5C on mRNA, linking epitranscriptomics to cancer.

A common mRNA modification is 5-methylcytosine (m5C), whose role in gene-transcript processing and cancer remains unclear. Here, we identify serine/arginine-rich splicing factor 2 (SRSF2) as a reader of m5C and impaired SRSF2 m5C binding as a potential contributor to leukemogenesis. Structurally, we identify residues involved in m5C recognition and the impact of the prevalent leukemia-associated mutation SRSF2P95H. We show that SRSF2 binding and m5C colocalize within transcripts. Furthermore, knocking down the m5C writer NSUN2 decreases mRNA m5C, reduces SRSF2 binding, and alters RNA splicing. We also show that the SRSF2P95H mutation impairs the ability of the protein to read m5C-marked mRNA, notably reducing its binding to key leukemia-related transcripts in leukemic cells. In leukemia patients, low NSUN2 expression leads to mRNA m5C hypomethylation and, combined with SRSF2P95H, predicts poor outcomes. Altogether, we highlight an unrecognized mechanistic link between epitranscriptomics and a key oncogenesis driver.

Mapping genotypes to chromatin accessibility profiles in single cells.

In somatic tissue differentiation, chromatin accessibility changes govern priming and precursor commitment towards cellular fates1-3. Therefore, somatic mutations are likely to alter chromatin accessibility patterns, as they disrupt differentiation topologies leading to abnormal clonal outgrowth. However, defining the impact of somatic mutations on the epigenome in human samples is challenging due to admixed mutated and wild-type cells. Here, to chart how somatic mutations disrupt epigenetic landscapes in human clonal outgrowths, we developed genotyping of targeted loci with single-cell chromatin accessibility (GoT-ChA). This high-throughput platform links genotypes to chromatin accessibility at single-cell resolution across thousands of cells within a single assay. We applied GoT-ChA to CD34+ cells from patients with myeloproliferative neoplasms with JAK2V617F-mutated haematopoiesis. Differential accessibility analysis between wild-type and JAK2V617F-mutant progenitors revealed both cell-intrinsic and cell-state-specific shifts within mutant haematopoietic precursors, including cell-intrinsic pro-inflammatory signatures in haematopoietic stem cells, and a distinct profibrotic inflammatory chromatin landscape in megakaryocytic progenitors. Integration of mitochondrial genome profiling and cell-surface protein expression measurement allowed expansion of genotyping onto DOGMA-seq through imputation, enabling single-cell capture of genotypes, chromatin accessibility, RNA expression and cell-surface protein expression. Collectively, we show that the JAK2V617F mutation leads to epigenetic rewiring in a cell-intrinsic and cell type-specific manner, influencing inflammation states and differentiation trajectories. We envision that GoT-ChA will empower broad future investigations of the critical link between somatic mutations and epigenetic alterations across clonal populations in malignant and non-malignant contexts.

ARAF protein kinase activates RAS by antagonizing its binding to RASGAP NF1.

RAF protein kinases are effectors of the GTP-bound form of small guanosine triphosphatase RAS and function by phosphorylating MEK. We showed here that the expression of ARAF activated RAS in a kinase-independent manner. Binding of ARAF to RAS displaced the GTPase-activating protein NF1 and antagonized NF1-mediated inhibition of RAS. This reduced ERK-dependent inhibition of RAS and increased RAS-GTP. By this mechanism, ARAF regulated the duration and consequences of RTK-induced RAS activation and supported the RAS output of RTK-dependent tumor cells. In human lung cancers with EGFR mutation, amplification of ARAF was associated with acquired resistance to EGFR inhibitors, which was overcome by combining EGFR inhibitors with an inhibitor of the protein tyrosine phosphatase SHP2 to enhance inhibition of nucleotide exchange and RAS activation.

Translating recent advances in the pathogenesis of acute myeloid leukemia to the clinic.

Despite FDA approval of nine new drugs for patients with acute myeloid leukemia (AML) in the United States over the last 4 years, AML remains a major area of unmet medical need among hematologic malignancies. In this review, we discuss the development of promising new molecular targeted approaches for AML, including menin inhibition, novel IDH1/2 inhibitors, and preclinical means to target TET2, ASXL1, and RNA splicing factor mutations. In addition, we review progress in immune targeting of AML through anti-CD47, anti-SIRPα, and anti-TIM-3 antibodies; bispecific and trispecific antibodies; and new cellular therapies in development for AML.

Targeting histone acetylation dynamics and oncogenic transcription by catalytic P300/CBP inhibition.

To separate causal effects of histone acetylation on chromatin accessibility and transcriptional output, we used integrated epigenomic and transcriptomic analyses following acute inhibition of major cellular lysine acetyltransferases P300 and CBP in hematological malignancies. We found that catalytic P300/CBP inhibition dynamically perturbs steady-state acetylation kinetics and suppresses oncogenic transcriptional networks in the absence of changes to chromatin accessibility. CRISPR-Cas9 screening identified NCOR1 and HDAC3 transcriptional co-repressors as the principal antagonists of P300/CBP by counteracting acetylation turnover kinetics. Finally, deacetylation of H3K27 provides nucleation sites for reciprocal methylation switching, a feature that can be exploited therapeutically by concomitant KDM6A and P300/CBP inhibition. Overall, this study indicates that the steady-state histone acetylation-methylation equilibrium functions as a molecular rheostat governing cellular transcription that is amenable to therapeutic exploitation as an anti-cancer regimen.

Emerging concepts of epigenetic dysregulation in hematological malignancies.

The past decade brought a revolution in understanding of the structure, topology and disease-inducing lesions of RNA and DNA, fueled by unprecedented progress in next-generation sequencing. This technological revolution has also affected understanding of the epigenome and has provided unique opportunities for the analysis of DNA and histone modifications, as well as the first map of the non-protein-coding genome and three-dimensional (3D) chromosomal interactions. Overall, these advances have facilitated studies that combine genetic, transcriptomics and epigenomics data to address a wide range of issues ranging from understanding the role of the epigenome in development to targeting the transcription of noncoding genes in human cancer. Here we describe recent insights into epigenetic dysregulation characteristic of the malignant differentiation of blood stem cells based on studies of alterations that affect epigenetic complexes, enhancers, chromatin, long noncoding RNAs (lncRNAs), RNA splicing, nuclear topology and the 3D conformation of chromatin.

Recurrent SRSF2 mutations in MDS affect both splicing and NMD.

Oncogenic mutations in the RNA splicing factors SRSF2, SF3B1, and U2AF1 are the most frequent class of mutations in myelodysplastic syndromes and are also common in clonal hematopoiesis, acute myeloid leukemia, chronic lymphocytic leukemia, and a variety of solid tumors. They cause genome-wide splicing alterations that affect important regulators of hematopoiesis. Several mRNA isoforms promoted by the various splicing factor mutants comprise a premature termination codon (PTC) and are therefore potential targets of nonsense-mediated mRNA decay (NMD). In light of the mechanistic relationship between splicing and NMD, we sought evidence for a specific role of mutant SRSF2 in NMD. We show that SRSF2 Pro95 hot spot mutations elicit enhanced mRNA decay, which is dependent on sequence-specific RNA binding and splicing. SRSF2 mutants enhance the deposition of exon junction complexes (EJCs) downstream from the PTC through RNA-mediated molecular interactions. This architecture then favors the association of key NMD factors to elicit mRNA decay. Gene-specific blocking of EJC deposition by antisense oligonucleotides circumvents aberrant NMD promoted by mutant SRSF2, restoring the expression of PTC-containing transcript. Our study uncovered critical effects of SRSF2 mutants in hematologic malignancies, reflecting the regulation at multiple levels of RNA metabolism, from splicing to decay.

Structural basis of cytokine-mediated activation of ALK family receptors.

Anaplastic lymphoma kinase (ALK)1 and the related leukocyte tyrosine kinase (LTK)2 are recently deorphanized receptor tyrosine kinases3. Together with their activating cytokines, ALKAL1 and ALKAL24-6 (also called FAM150A and FAM150B or AUGß and AUGα, respectively), they are involved in neural development7, cancer7-9 and autoimmune diseases10. Furthermore, mammalian ALK recently emerged as a key regulator of energy expenditure and weight gain11, consistent with a metabolic role for Drosophila ALK12. Despite such functional pleiotropy and growing therapeutic relevance13,14, structural insights into ALK and LTK and their complexes with cognate cytokines have remained scarce. Here we show that the cytokine-binding segments of human ALK and LTK comprise a novel architectural chimera of a permuted TNF-like module that braces a glycine-rich subdomain featuring a hexagonal lattice of long polyglycine type II helices. The cognate cytokines ALKAL1 and ALKAL2 are monomeric three-helix bundles, yet their binding to ALK and LTK elicits similar dimeric assemblies with two-fold symmetry, that tent a single cytokine molecule proximal to the cell membrane. We show that the membrane-proximal EGF-like domain dictates the apparent cytokine preference of ALK. Assisted by these diverse structure-function findings, we propose a structural and mechanistic blueprint for complexes of ALK family receptors, and thereby extend the repertoire of ligand-mediated dimerization mechanisms adopted by receptor tyrosine kinases.

Disruption of cotranscriptional splicing suggests that RBM39 is a therapeutic target in acute lymphoblastic leukemia.

There are few options for patients with relapse/refractory B-cell acute lymphoblastic leukemia (B-ALL), thus this is a major area of unmet medical need. Here, we reveal that inclusion of a poison exon in RBM39, which could be induced both by CDK9 or CDK9 independent CMGC (cyclin-dependent kinases, mitogen-activated protein kinases, glycogen synthase kinases, CDC-like kinases) kinase inhibition, is recognized by the nonsense-mediated mRNA decay (NMD) pathway for degradation. Targeting this poison exon in RBM39 with CMGC inhibitors lead to protein downregulation and inhibition of ALL growth, particularly in relapse/refractory B-ALL. Mechanistically, disruption of co-transcriptional splicing by inhibition of CMGC kinases including DYRK1A, or inhibition of CDK9, which phosphorylate the C-terminal domain of RNA polymerase II (Pol II), results in alteration of SF3B1 and Pol II association. Disruption of SF3B1 and transcriptional elongation complex alters Pol II pausing, which promotes the inclusion of a poison exon in RBM39. Moreover, RBM39 ablation suppresses the growth of human B-ALL, and targeting RBM39 with sulfonamides, which degrade RBM39 protein, showed strong anti-tumor activity in preclinical models. Our data reveal that relapse/refractory B-ALL is susceptible to pharmacologic and genetic inhibition of RBM39 and provide two potential strategies to target this axis.

The Augmented R-Loop Is a Unifying Mechanism for Myelodysplastic Syndromes Induced by High-Risk Splicing Factor Mutations.

Mutations in several general pre-mRNA splicing factors have been linked to myelodysplastic syndromes (MDSs) and solid tumors. These mutations have generally been assumed to cause disease by the resultant splicing defects, but different mutations appear to induce distinct splicing defects, raising the possibility that an alternative common mechanism is involved. Here we report a chain of events triggered by multiple splicing factor mutations, especially high-risk alleles in SRSF2 and U2AF1, including elevated R-loops, replication stress, and activation of the ataxia telangiectasia and Rad3-related protein (ATR)-Chk1 pathway. We further demonstrate that enhanced R-loops, opposite to the expectation from gained RNA binding with mutant SRSF2, result from impaired transcription pause release because the mutant protein loses its ability to extract the RNA polymerase II (Pol II) C-terminal domain (CTD) kinase-the positive transcription elongation factor complex (P-TEFb)-from the 7SK complex. Enhanced R-loops are linked to compromised proliferation of bone-marrow-derived blood progenitors, which can be partially rescued by RNase H overexpression, suggesting a direct contribution of augmented R-loops to the MDS phenotype.

The Dietary Supplement Chondroitin-4-Sulfate Exhibits Oncogene-Specific Pro-tumor Effects on BRAF V600E Melanoma Cells.

Dietary supplements such as vitamins and minerals are widely used in the hope of improving health but may have unidentified risks and side effects. In particular, a pathogenic link between dietary supplements and specific oncogenes remains unknown. Here we report that chondroitin-4-sulfate (CHSA), a natural glycosaminoglycan approved as a dietary supplement used for osteoarthritis, selectively promotes the tumor growth potential of BRAF V600E-expressing human melanoma cells in patient- and cell line-derived xenograft mice and confers resistance to BRAF inhibitors. Mechanistically, chondroitin sulfate glucuronyltransferase (CSGlcA-T) signals through its product CHSA to enhance casein kinase 2 (CK2)-PTEN binding and consequent phosphorylation and inhibition of PTEN, which requires CHSA chains and is essential to sustain AKT activation in BRAF V600E-expressing melanoma cells. However, this CHSA-dependent PTEN inhibition is dispensable in cancer cells expressing mutant NRAS or PI3KCA, which directly activate the PI3K-AKT pathway. These results suggest that dietary supplements may exhibit oncogene-dependent pro-tumor effects.