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Mutations in splicing factors (SF) are the predominant class of mutations in myelodysplastic syndrome (MDS), but convergent downstream disease drivers remain elusive. To identify common direct targets of missplicing by mutant U2AF1 and SRSF2, we performed RNA sequencing and enhanced version of the cross-linking and immunoprecipitation assay in human hematopoietic stem/progenitor cells derived from isogenic induced pluripotent stem cell (iPSC) models. Integrative analyses of alternative splicing and differential binding converged on a long isoform of GNAS (GNAS-L), promoted by both mutant factors. MDS population genetics, functional and biochemical analyses support that GNAS-L is a driver of MDS and encodes a hyperactive long form of the stimulatory G protein alpha subunit, Gαs-L, that activates ERK/MAPK signaling. SF-mutant MDS cells have activated ERK signaling and consequently are sensitive to MEK inhibitors. Our findings highlight an unexpected and unifying mechanism by which SRSF2 and U2AF1 mutations drive oncogenesis with potential therapeutic implications for MDS and other SF-mutant neoplasms.Significance:SF mutations are disease-defining in MDS, but their critical effectors remain unknown. We discover the first direct target of convergent missplicing by mutant U2AF1 and SRSF2, a long GNAS isoform, which activates G protein and ERK/MAPK signaling, thereby driving MDS and rendering mutant cells sensitive to MEK inhibition.This article is highlighted in the In This Issue feature, p. 587

Supplementary table from Integrative RNA-omics Discovers GNAS Alternative Splicing as a Phenotypic Driver of Splicing Factor–Mutant Neoplasms

Supplementary Data from Integrative RNA-omics Discovers GNAS Alternative Splicing as a Phenotypic Driver of Splicing Factor–Mutant Neoplasms

Supplementary Figure 2 PDF file - 182K, This Figure shows the activation status of RhoA and Rac1 in mouse lungs and validation of RhoA-GTP antibody on mouse lung tissue by IHC

Supplementary Figure 6 PDF file - 141K, This Figure shows that FAK is co-expressed with RHOA in lung adenocarcinomas. In addition, we provide the validation of RHOA-GTP antibody on human specimens by IHC

Supplementary Figure 1 PDF file - 311K, This Figure shows that the KrasG12D;Ink4a/Arf -/- mice show increased tumor burden, increased Ki-67, Cyclin D1 expression and decreased survival compared to KrasG12D;Ink4a/Arf +/+ mice

Supplementary Figure 7 PDF file - 205K, This Figure shows that pharmacological inhibition of FAK results in suppression of NSCLC in vivo

Supplementary Methods PDF file - 123K, This file contains the Supplementary materials and methods linked to the manuscript. In addition the references associated with the materials are reported

Supplementary Figure 4 PDF file - 169K, This Figure shows that RHOA activation is essential for the survival of NSCLC expressing mutant KRAS in association with INK4a/ARF or p53 deficiency

Supplementary Figure 1. Pro-inflammatory cytokines are elevated in MF mice and reversed with JAK inhibitor treatment. Supplementary Figure 2. Principal component analysis of MF cells. Supplementary Figure 3. Single cell analysis of immunophenotypically-defined subpopulations from MF and control mice. Supplementary Figure 4. Elevated IL8 expression in MF patients. Supplementary Figure 5. Complete removal of Il6 from the mouse system shows only minor effects on MPLW515L disease. Supplementary Figure 6. Peripheral blood and cytometric flow analysis of MPLW515L transplanted mice with and without Stat3 deletion. Supplementary Figure 7. Constitutive activation of STAT3 in the BM of MPLW515L mice and primary MF patients. Supplementary Figure 8. Schematic illustration of bone marrow transplantation experiments using Stat3-deficient mice or littermate control mice as donors. Supplementary Figure 9. Excision of Stat3 post engraftment prolongs survival and reduces leukocytosis. Supplementary Figure 10. Mutant-restricted deletion of Stat3 does not affect disease severity in vivo. Supplementary Figure 11. Single cell analysis of populations from MF and control mice. Supplementary Figure 12. Mutant and non-mutant hematopoietic cells feature a higher percentage of cytokine secreting cells. Supplementary Figure 13. Single cell analysis of populations from Jak2V617F MF and control mice. Supplementary Figure 14. Mutant and non-mutant hematopoietic cells feature a higher percentage of cytokine secreting cells. Supplementary Figure 15. JAK1/2 inhibition reduces cytokine expression from CD45.1-positive wild-type cells and CD45.2-positive mutant cell population. Supplementary Table 1. Primary myelofibrosis patient characteristics. Supplementary Table 2. Genetic alterations of patient PMF#11.

Supplementary Figure 5 PDF file - 199K, This Figure shows that FAK is a critical target of RHOA in mutant KRAS;INK4a/ARF deficient NSCLC cells

Supplementary Figure 3 PDF file - 76K, This Figure shows the knockdown efficiency of shRNAs against KRAS and that MEK1/2 inhibition suppresses RHOA

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Excess-over-BLISS (EOB) scores for drug combinations

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The ubiquitin-like SUMO proteins covalently modify protein substrates and regulate their functional properties. In a broad spectrum of cancers, the tumor suppressor PML undergoes ubiquitin-mediated degradation primed by CK2 phosphorylation. Here, we report that the SUMO E3-ligase inhibitor PIAS1 regulates oncogenic signaling through its ability to sumoylate PML and the PML-RARA oncoprotein of acute promyelocytic leukemia (APL). PIAS1-mediated SUMOylation of PML promoted CK2 interaction and ubiquitin/proteasome-mediated degradation of PML, attenuating its tumor suppressor functions. In addition, PIAS1-mediated SUMOylation of PML-RARA was essential for induction of its degradation by arsenic trioxide, an effective APL treatment. Moreover, PIAS1 suppression abrogated the ability of arsenic trioxide to trigger apoptosis in APL cells. Lastly, PIAS1 was also essential for PML degradation in non–small cell lung carcinoma (NSCLC) cells, and PML and PIAS1 were inversely correlated in NSCLC cell lines and primary specimens. Together, our findings reveal novel roles for PIAS1 and the SUMOylation machinery in regulating oncogenic networks and the response to leukemia therapy. Cancer Res; 72(9); 2275–84. ©2012 AACR.

Follicular lymphoma, the most common indolent subtype of non-Hodgkin lymphoma, is associated with a relatively long overall survival rate ranging from 6 to 10 years from the time of diagnosis. However, in 20% to 60% of follicular lymphoma patients, transformation to aggressive diffuse large B-cell lymphoma (DLBCL) reduces median survival to only 1.2 years. The specific functional and genetic determinants of follicular lymphoma transformation remain elusive, and genomic alterations underlying disease advancement have only been identified for a subset of cases. Therefore, to identify candidate drivers of follicular lymphoma transformation, we performed systematic analysis of a B-cell–specific regulatory model exhibiting follicular lymphoma transformation signatures using the Master Regulator Inference algorithm (MARINa). This analysis revealed FOXM1, TFDP1, ATF5, HMGA1, and NFYB to be candidate master regulators (MR) contributing to disease progression. Accordingly, validation was achieved through synthetic lethality assays in which RNAi-mediated silencing of MRs individually or in combination reduced the viability of (14;18)-positive DLBCL (t-DLBCL) cells. Furthermore, specific combinations of small-molecule compounds targeting synergistic MR pairs induced loss of viability in t-DLBCL cells. Collectively, our findings indicate that MR analysis is a valuable method for identifying bona fide contributors to follicular lymphoma transformation and may therefore guide the selection of compounds to be used in combinatorial treatment strategies. Cancer Res; 76(3); 664–74. ©2015 AACR.

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Supplementary figure legends for S1-S4

B-cell maturation antigen (BCMA) is a prominent tumor-associated target for chimeric antigen receptor (CAR)-T cell therapy in multiple myeloma (MM). Here, we describe the case of a patient with MM who was enrolled in the CARTITUDE-1 trial ( NCT03548207 ) and who developed a progressive movement disorder with features of parkinsonism approximately 3 months after ciltacabtagene autoleucel BCMA-targeted CAR-T cell infusion, associated with CAR-T cell persistence in the blood and cerebrospinal fluid, and basal ganglia lymphocytic infiltration. We show BCMA expression on neurons and astrocytes in the patient's basal ganglia. Public transcriptomic datasets further confirm BCMA RNA expression in the caudate of normal human brains, suggesting that this might be an on-target effect of anti-BCMA therapy. Given reports of three patients with grade 3 or higher parkinsonism on the phase 2 ciltacabtagene autoleucel trial and of grade 3 parkinsonism in the idecabtagene vicleucel package insert, our findings support close neurological monitoring of patients on BCMA-targeted T cell therapies.

Background & aims Tufting enteropathy (TE) is a rare congenital disorder often caused by mutations in the gene encoding epithelial cell adhesion molecule (EpCam). The disease leads to diarrhea, intestinal failure and dependence on total parenteral nutrition (TPN). These patients often have liver impairments, but the pathology and mechanism of the damage are not well understood. We evaluated liver biopsies from TE patients to understand the pathophysiology. Methods We identified three patients with TE who underwent liver biopsy. Two normal controls and 45 patients on TPN secondary to short gut syndrome were selected for comparison (5 were age- and TPN duration-matched to the TE patients). Results We found that all TE patients showed a complete loss of EpCam expression in enterocytes and biliary epithelial cells, while the normal and TPN groups show basolateral expression. Histologically TE patients showed ductopenia, which was not seen in control groups. E-cadherin and β-catenin are normally located along the lateral membrane of biliary epithelial cells. However, they were relocated to the apical membrane in TE patients, indicating a defect in the apical-basal polarity of cholangiocytes. We examined hepatic reparative cells and found near absence of hepatic progenitor cells and intermediate hepatobiliary cells with mild reactive ductular cells in TE patients. Conclusion Our findings show that TE is associated with disrupted polarity of cholangiocyte and ductopenia. We demonstrate for the first time a role of EpCam in the maintenance of integrity of biliary epithelium. We also provided evidence for a disrupted development of hepatic reparative cells.

Genetics has played an important role in risk stratification for plasma cell myeloma patients, providing therapeutic guidance. In this study, we investigated the correlation of bone marrow morphologic features and genetic aberrations, including gene expression profiles, translocations, and gene mutations. For the first time we show that high plasma cell volume, diffuse sheet growth pattern, immature cell morphology, high mitotic index, and increased reticulin fibrosis, significantly correlates with high risk disease determined by MyPRS gene expression profiles. Furthermore, we show the association between MyPRS risk stratification and chromosomal alterations and specific gene mutations. We also demonstrate the combinational effect of TP53 mutation and 17p loss on the histological changes in bone marrow.

Myelodysplastic syndrome (MDS) is characterized by ineffective hematopoiesis with morphologic dysplasia and a propensity to transform into overt acute myeloid leukemia (AML). Our analysis of two cohorts of 20 MDS and 49 AML with multi-lineage dysplasia patients shows a reduction in Nucleophosmin 1 (NPM1) expression in 70% and 90% of cases, respectively. A mouse model of Npm1 conditional knockout (cKO) in hematopoietic cells reveals that Npm1 loss causes premature aging of hematopoietic stem cells (HSCs). Mitochondrial activation in Npm1-deficient HSCs leads to aberrant activation of the NLRP3 inflammasome, which correlates with a developing MDS-like phenotype. Npm1 cKO mice exhibit shortened survival times, and expansion of both the intra- and extra-medullary myeloid populations, while evoking a p53-dependent response. After transfer into a p53 mutant background, the resulting Npm1/p53 double KO mice develop fatal leukemia within 6 months. Our findings identify NPM1 as a regulator of HSC aging and inflammation and highlight the role of p53 in MDS progression to leukemia.

We report a case of a relapsed hemophagocytic intramedullary plasmacytoma, previously non-phagocytic, in conjunction with development of a new clone with different cytogenetic abnormalities forming a solitary plasmacytoma.

Classifying diffuse large B-cell lymphoma (DLBCL) according to the cell-of-origin (COO) was first proposed using gene expression profiling; accordingly, DLBCL is classified into germinal-center B-cell type and activated B-cell type. Immunohistochemistry (IHC)-based classification using different algorithms is used widely due to the ability to use formalin-fixed paraffin-embedded tissue. Recently, newer techniques using RNA expression from formalin-fixed paraffin-embedded were introduced including the nCounter NanoString platform assay. In this brief report, we study the degree of concordance between the NanoString assay and 6 commonly utilized IHC-based algorithms to classify DLBCL cases by COO. Stains for CD10, BCL2, BCL6, FOXP-1, MUM-1, and LOM2 were used to classify a cohort of DLBCL by COO according to the respective IHC-algorithms. Then, RNA was extracted from the same cases for NanoString assay classification. The degree of concordance was calculated between the NanoString classification and each IHC-algorithm as well as among the different IHC-algorithm themselves. The concordance in COO classification of DLBCL between NanonoString assay and IHC-based algorithms is variable depending on the used IHC-algorithm; the highest concordance is seen with the Visco algorithm (κ=0.69; P=0.001). Therefore, discrepancies between the recently introduced NanoString assay and the commonly utilized IHC-algorithms are expected to some extent and should be taken into consideration when interpreting conflicting results.

Myocyte enhancer-binding factor 2B (MEF2B) has been implicated as a transcriptional regulator for BCL6. However, details about the interaction between MEF2B and BCL6 during expression, as well as the relationship of MEF2B to the expression of other germinal center (GC) markers, have not yet been fully explained. Using germinal center B-cell-like diffuse large B-cell lymphoma (GC-DLBCL) and activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL) cell lines, we analyzed the expression of MEF2B and its associations with BCL6, CD10, and ERK. Furthermore, small interfering RNA (siRNA) was used to study the possible effects of MEF2B knockdown on these proteins and cell growth. Analysis of the BCL6 transcriptional complex was performed using electrophoretic mobility shift assay. The correlation between MEF2B expression and the genetic type of DLBCL was assessed using immunohistochemistry on 111 patient samples, and via in silico analysis of publicly available microarray (Gene Expression Omnibus (GEO)) datasets. Our results indicate that the expression of MEF2B protein is important for the growth of GC-DLBCL cells, as evidenced by MEF2B knockdown inhibition of cell growth and the subsequent suppression of BCL6, CD10, and ERK phosphorylation. Analysis of BCL6 transcription factors in nuclear extracts of MEF2-expressing DLBCL cells showed involvement of MEF2B with AP-2α and BCL6 proteins in the formation of the BCL6 gene transcriptional complex. Indeed, differential expression of MEF2B in the GC-DLBCL is statistically significant compared to the ABC-DLBCL in the GEO datasets, as well as in tissue microarray, as indicated via immunohistochemistry (Visco-Young algorithm). Our findings indicate that MEF2B is an essential component of the BCL6 gene transcriptional complex for the regulation of DLBCL growth via the promotion of BCL6 expression. Beyond its regulatory role in DLBCL growth, MEF2B expression correlated positively with BCL6 and CD10 expression, and was preferentially expressed in the GBC-DLBCL group.

Significance Fifty percent of diffuse large B cell lymphoma (DLBCL) evade immune-surveillance via somatic genetic lesions abrogating the expression of the class I major histocompatibility complex (MHC-I) complex on the cell surface, thus preventing the presentation of tumor neoantigens to the immune system. The results herein significantly extend these findings by showing that an additional 40% of DLBCL cases, despite expressing MHC-I, carry monoallelic HLA-I genetic alterations that limit the repertoire of neoantigens for presentation to immune cells. Both MHC-INEG and MHC-IPOS/monoallelically disrupted cases have significantly higher mutational load. Notably, homozygosis of HLA-I loci is significantly and preferentially enriched in the germline of DLBCL patients, suggesting a stepwise process by which limited neoantigen presentation is selected during DLBCL development., Fifty percent of diffuse large B cell lymphoma (DLBCL) cases lack cell-surface expression of the class I major histocompatibility complex (MHC-I), thus escaping recognition by cytotoxic T cells. Here we show that, across B cell lymphomas, loss of MHC-I, but not MHC-II, is preferentially restricted to DLBCL. To identify the involved mechanisms, we performed whole exome and targeted HLA deep-sequencing in 74 DLBCL samples, and found somatic inactivation of B2M and the HLA-I loci in 80% (34 of 42) of MHC-INEG tumors. Furthermore, 70% (22 of 32) of MHC-IPOS DLBCLs harbored monoallelic HLA-I genetic alterations (MHC-IPOS/mono), indicating allele-specific inactivation. MHC-INEG and MHC-IPOS/mono cases harbored significantly higher mutational burden and inferred neoantigen load, suggesting potential coselection of HLA-I loss and sustained neoantigen production. Notably, the analysis of >500,000 individuals across different cancer types revealed common germline HLA-I homozygosity, preferentially in DLBCL. In mice, germinal-center B cells lacking HLA-I expression did not progress to lymphoma and were counterselected in the context of oncogene-driven lymphomagenesis, suggesting that additional events are needed to license immune evasion. These results suggest a multistep process of HLA-I loss in DLBCL development including both germline and somatic events, and have direct implications for the pathogenesis and immunotherapeutic targeting of this disease.

Mutations in splicing factors (SF) are the predominant class of mutations in myelodysplastic syndrome (MDS), but convergent downstream disease drivers remain elusive. To identify common direct targets of missplicing by mutant U2AF1 and SRSF2, we performed RNA sequencing and enhanced version of the cross-linking and immunoprecipitation assay in human hematopoietic stem/progenitor cells derived from isogenic induced pluripotent stem cell (iPSC) models. Integrative analyses of alternative splicing and differential binding converged on a long isoform of GNAS (GNAS-L), promoted by both mutant factors. MDS population genetics, functional and biochemical analyses support that GNAS-L is a driver of MDS and encodes a hyperactive long form of the stimulatory G protein alpha subunit, Gαs-L, that activates ERK/MAPK signaling. SF-mutant MDS cells have activated ERK signaling and consequently are sensitive to MEK inhibitors. Our findings highlight an unexpected and unifying mechanism by which SRSF2 and U2AF1 mutations drive oncogenesis with potential therapeutic implications for MDS and other SF-mutant neoplasms. Significance: SF mutations are disease-defining in MDS, but their critical effectors remain unknown. We discover the first direct target of convergent missplicing by mutant U2AF1 and SRSF2, a long GNAS isoform, which activates G protein and ERK/MAPK signaling, thereby driving MDS and rendering mutant cells sensitive to MEK inhibition. This article is highlighted in the In This Issue feature, p. 587