T helper 9 (Th9) cells are specialized for the production of IL-9, promote allergic inflammation in mice, and are associated with allergic disease in humans. It has not been determined whether Th9 cells express a characteristic transcriptional signature. In this study, we performed microarray analysis to identify genes enriched in Th9 cells compared with other Th subsets. This analysis defined a transcriptional regulatory network required for the expression of a subset of Th9-enriched genes. The activator protein 1 (AP1) family transcription factor BATF (B cell, activating transcription factor-like) was among the genes enriched in Th9 cells and was required for the expression of IL-9 and other Th9-associated genes in both human and mouse T cells. The expression of BATF was increased in Th9 cultures derived from atopic infants compared with Th9 cultures from control infants. T cells deficient in BATF expression had a diminished capacity to promote allergic inflammation compared with wild-type controls. Moreover, mouse Th9 cells ectopically expressing BATF were more efficient at promoting allergic inflammation than control transduced cells. These data indicate that BATF is a central regulator of the Th9 phenotype and contributes to the development of allergic inflammation.

Spliceosome mutations are common in MDS and AML, yet the oncogenic changes due to these mutations have not been identified. A global analysis of exon usage in AML samples revealed distinct molecular subsets containing alternative spliced isoforms of inflammatory and immune genes. IRAK4 was the dominant alternatively spliced isoform in MDS/AML and is characterized by a longer isoform that retains exon 4, encoding a protein, IRAK4-Long (L) that assembles with the Myddosome, results in maximal activation of NF-κB, and is essential for leukemic cell function. Expression of IRAK4-L is mediated by mutant U2AF1 and is associated with oncogenic signaling in MDS/AML. Inhibition of IRAK4-L abrogates leukemic growth, particularly in AML cells with higher expression of the IRAK4-L isoform. Collectively, mutations in U2AF1 induce expression of therapeutically targetable “active” IRAK4 isoforms and provide a genetic link to activation of chronic innate immune signaling in MDS and AML.

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Libraries of transgenic Drosophila melanogaster carrying RNA interference (RNAi) constructs have been used extensively to perform large-scale functional genetic screens in vivo. For example, RNAi screens have facilitated the discovery of multiple components of the Hippo pathway, an evolutionarily conserved growth-regulatory network. Here we investigate an important technical limitation with the widely used VDRC KK RNAi collection. We find that approximately 25% of VDRC KK RNAi lines cause false-positive enhancement of the Hippo pathway, owing to ectopic expression of the Tiptop transcription factor. Of relevance to the broader Drosophila community, ectopic tiptop (tio) expression can also cause organ malformations and mask phenotypes such as organ overgrowth. To enhance the use of the VDRC KK RNAi library, we have generated a D. melanogaster strain that will allow researchers to test, in a single cross, whether their genetic screen of interest will be affected by ectopic tio expression.

Mutations in the cardiac splicing factor RBM20 lead to malignant dilated cardiomyopathy (DCM). To understand the mechanism of RBM20-associated DCM, we engineered isogenic iPSCs with DCM-associated missense mutations in RBM20 as well as RBM20 knockout (KO) iPSCs. iPSC-derived engineered heart tissues made from these cell lines recapitulate contractile dysfunction of RBM20-associated DCM and reveal greater dysfunction with missense mutations than KO. Analysis of RBM20 RNA binding by eCLIP reveals a gain-of-function preference of mutant RBM20 for 3′ UTR sequences that are shared with amyotrophic lateral sclerosis (ALS) and processing-body associated RNA binding proteins (FUS, DDX6). Deep RNA sequencing reveals that the RBM20 R636S mutant has unique gene, splicing, polyadenylation and circular RNA defects that differ from RBM20 KO. Super-resolution microscopy verifies that mutant RBM20 maintains very limited nuclear localization potential; rather, the mutant protein associates with cytoplasmic processing bodies (DDX6) under basal conditions, and with stress granules (G3BP1) following acute stress. Taken together, our results highlight a pathogenic mechanism in cardiac disease through splicing-dependent and -independent pathways.

PARP-14, a member of the poly ADP-ribose polymerase super family, promotes T helper cell 2 (Th2) differentiation by regulating interleukin-4 (IL-4) and STAT6-dependent transcription. Yet, whether PARP-14 globally impacts gene regulation has not been determined. In this report, using an RNA pol II ChIP-seq approach, we identify genes in Th2 cells that are regulated by PARP-14, and either dependent or independent of ADP-ribosyltransferase catalytic activity. Our data demonstrate that PARP-14 enhances the expression of Th2 genes as it represses the expression of Th1-associated genes. Among the relevant targets are Signal Transducer and Activator of Transcription genes required for polarizing Th1 and Th2 cells. To define a mechanism for PARP-14 function, we use an informatics approach to identify putative PARP-14 DNA binding sites. Two putative PARP-14 binding motifs are identified in multiple Th2 cytokine genes, and we demonstrate that PARP-14 interacts with each motif using in vitro binding assays. Taken together our results indicate that PARP-14 is an important factor for T helper cell differentiation and it binds to specific DNA sequences to mediate its function.

Despite growing awareness of the biologic features underlying MLL-rearranged leukemia, targeted therapies for this leukemia have remained elusive and clinical outcomes remain dismal. MBNL1, a protein involved in alternative splicing, is consistently overexpressed in MLL-rearranged leukemias. We found that MBNL1 loss significantly impairs propagation of murine and human MLL-rearranged leukemia in vitro and in vivo. Through transcriptomic profiling of our experimental systems, we show that in leukemic cells, MBNL1 regulates alternative splicing (predominantly intron exclusion) of several genes including those essential for MLL-rearranged leukemogenesis, such as DOT1L and SETD1A. We finally show that selective leukemic cell death is achievable with a small molecule inhibitor of MBNL1. These findings provide the basis for a new therapeutic target in MLL-rearranged leukemia and act as further validation of a burgeoning paradigm in targeted therapy, namely the disruption of cancer-specific splicing programs through the targeting of selectively essential RNA binding proteins.

BackgroundCancer outcomes for Medicaid enrollees may be affected by patients’ primary care (PC) utilization and complex Medicaid enrollment dynamics, which have recently changed for many states under the Affordable Care Act.MethodsWith New Jersey State Cancer Registry and linked Medicaid claims data, a retrospective cohort study was conducted for patients with incident breast, colorectal, or invasive cervical cancer (aged 21‐64 years) diagnosed in 2012‐2014. Associations of Medicaid enrollment factors and PC utilization with the stage at diagnosis and treatment delays were examined with multivariate logistic regression models.ResultsThe study included 19,209 total cancer cases and 3253 linked Medicaid cases. Medicaid cases were more likely to be diagnosed at a late stage and to experience treatment delays in comparison with non‐Medicaid cases. In adjusted analyses, Medicaid cases with 1 or more PC visits before the diagnosis had lower odds of a late‐stage diagnosis (odds ratio, 0.47; 95% confidence interval, 0.33‐0.67) in comparison with Medicaid cases with no outpatient visits. New enrollees (<6 months) and longer term enrollees in fee‐for‐service (FFS) Medicaid had greater odds of a late‐stage diagnosis and treatment delays in comparison with those in Medicaid managed care.ConclusionsMedicaid patients with cancer diagnosed just before and in the initial year of eligibility expansion had worse outcomes than non‐Medicaid cases. Poor outcomes were especially pronounced among new enrollees, those without outpatient visits before their diagnosis, and FFS enrollees. Targeted strategies to enhance care continuity, including access to PC providers before the diagnosis and a better understanding of pathways to cancer care upon Medicaid enrollment, are needed to improve outcomes in this population.