A hallmark of pancreatic ductal adenocarcinoma (PDAC) is an exuberant stroma comprised of diverse cell types that enable or suppress tumor progression. Here, we explored the role of oncogenic KRAS in protumorigenic signaling interactions between cancer cells and host cells. We show that KRAS mutation (KRAS*) drives cell-autonomous expression of type I cytokine receptor complexes (IL2r γ -IL4r α and IL2r γ -IL13r α 1) in cancer cells that in turn are capable of receiving cytokine growth signals (IL4 or IL13) provided by invading Th2 cells in the microenvironment. Early neoplastic lesions show close proximity of cancer cells harboring KRAS* and Th2 cells producing IL4 and IL13. Activated IL2r γ -IL4r α and IL2r γ -IL13r α 1 receptors signal primarily via JAK1-STAT6. Integrated transcriptomic, chromatin occupancy, and metabolomic studies identifi ed MYC as a direct target of activated STAT6 and that MYC drives glycolysis. Thus, paracrine signaling in the tumor microenvironment plays a key role in the KRAS*-driven metabolic reprogramming of PDAC. SIGNIFICANCE:Type II cytokines, secreted by Th2 cells in the tumor microenvironment, can stimulate cancer cell-intrinsic MYC transcriptional upregulation to drive glycolysis. This KRAS*-driven heterotypic signaling circuit in the early and advanced tumor microenvironment enables cooperative protumorigenic interactions, providing candidate therapeutic targets in the KRAS* pathway for this intractable disease.

Immunotherapies targeting programmed cell death protein 1 (PD-1) and programmed cell death ligand 1 (PD-L1) immune checkpoints represent a major breakthrough in cancer treatment.PD-1 is an inhibitory receptor expressed on the surface of activated T-cells that dampens T-cell receptor (TCR)/CD28 signaling by engaging with its ligand PD-L1 expressed on cancer cells.Despite the clinical success of PD-1 blockade using monoclonal antibodies, most patients do not respond to the treatment, and the underlying regulatory mechanisms of PD-1 remain incompletely defined. Here we show that PD-1 is extensively N-glycosylated in T cells and the intensities of its specific glycoforms are altered upon TCR activation. Glycosylation was critical for maintaining PD-1 protein stability and cell surface localization. Glycosylation of PD-1, especially at the N58 site, was essential for mediating its interaction with PD-L1. The monoclonal antibody STM418 specifically targeted glycosylated PD-1, exhibiting higher binding affinity to PD-1 than FDAapproved PD-1 antibodies, potently inhibiting PD-L1/PD-1 binding, and enhancing anti-tumor immunity. Together these findings provide novel insights into the functional significance of PD-1 glycosylation and offer a rationale for targeting glycosylated PD-1 as a potential strategy for immunotherapy. SignificanceFindings demonstrate that glycosylation of PD-1 is functionally significant and targeting glycosylated PD-1 may serve as a means to improve immunotherapy response.

GATA4, an essential cardiogenic transcription factor, provides a model for dominant transcription factor mutations in human disease. Dominant GATA4 mutations cause congenital heart disease (CHD), specifically atrial and atrioventricular septal defects (ASDs and AVSDs). We found that second heart field (SHF)-specific Gata4 heterozygote embryos recapitulated the AVSDs observed in germline Gata4 heterozygote embryos. A proliferation defect of SHF atrial septum progenitors and hypoplasia of the dorsal mesenchymal protrusion, rather than anlage of the atrioventricular septum, were observed in this model. Knockdown of the cell-cycle repressor phosphatase and tensin homolog (Pten) restored cell-cycle progression and rescued the AVSDs. Gata4 mutants also demonstrated Hedgehog (Hh) signaling defects. Gata4 acts directly upstream of Hh components: Gata4 activated a cisregulatory element at Gli1 in vitro and occupied the element in vivo. Remarkably, SHF-specific constitutive Hh signaling activation rescued AVSDs in Gata4 SHF-specific heterozygous knockout embryos. Pten expression was unchanged in Smoothened mutants, and Hh pathway genes were unchanged in Pten mutants, suggesting pathway independence. Thus, both the cell-cycle and Hh-signaling defects caused by dominant Gata4 mutations were required for CHD pathogenesis, suggesting a combinatorial model of disease causation by transcription factor haploinsufficiency.Gata4 | ASDs | Hedgehog signaling | second heart field | cell cycle

Rationale Mutations of TBX5 cause Holt–Oram syndrome (HOS) in humans, a disease characterized by atrial or occasionally ventricular septal defects in the heart and skeletal abnormalities of the upper extremity. Previous studies have demonstrated that Tbx5 regulates Osr1 expression in the second heart field (SHF) of E9.5 mouse embryos. However, it is unknown whether and how Tbx5 and Osr1 interact in atrial septation. Objective To determine if and how Tbx5 and Osr1 interact in the posterior SHF for cardiac septation. Methods and Results In the present study, genetic inducible fate mapping showed that Osr1-expressing cells contribute to atrial septum progenitors between E8.0 and E11.0. Osr1 expression in the pSHF was dependent on the level of Tbx5 at E8.5 and E9.5 but not E10.5, suggesting that the embryo stage before E10.5 is critical for Tbx5 interacting with Osr1 in atrial septation. Significantly more atrioventricular septal defects (AVSDs) were observed in embryos with compound haploinsufficiency for Tbx5 and Osr1. Conditional compound haploinsufficiency for Tbx5 and Osr1 resulted in a significant cell proliferation defect in the SHF, which was associated with fewer cells in the G2 and M phases and a decreased level of Cdk6 expression. Remarkably, genetically targeted disruption of Pten expression in atrial septum progenitors rescued AVSDs caused by Tbx5 and Osr1 compound haploinsufficiency. There was a significant decrease in Smo expression, which is a Hedgehog (Hh) signaling pathway modulator, in the pSHF of Osr1 knockout embryos at E9.5, implying a role for Osr1 in regulating Hh signaling. Conclusions Tbx5 and Osr1 interact to regulate posterior SHF cell cycle progression for cardiac septation.

Atrial septal defects (ASDs) are a common human congenital heart disease (CHD) that can be induced by genetic abnormalities. Our previous studies have demonstrated a genetic interaction between Tbx5 and Osr1 in the second heart field (SHF) for atrial septation. We hypothesized that Osr1 and Tbx5 share a common signaling networking and downstream targets for atrial septation. To identify this molecular networks, we acquired the RNA-Seq transcriptome data from the posterior SHF of wild-type, Tbx5(+/) (-), Osr1(+/-), Osr1(-/-) and Tbx5(+/-)/Osr1(+/-) mutant embryos. Gene set analysis was used to identify the Kyoto Encyclopedia of Genes and Genomes pathways that were affected by the doses of Tbx5 and Osr1. A gene network module involving Tbx5 and Osr1 was identified using a non-parametric distance metric, distance correlation. A subset of 10 core genes and gene-gene interactions in the network module were validated by gene expression alterations in posterior second heart field (pSHF) of Tbx5 and Osr1 transgenic mouse embryos, a time-course gene expression change during P19CL6 cell differentiation. Pcsk6 was one of the network module genes that were linked to Tbx5. We validated the direct regulation of Tbx5 on Pcsk6 using immunohistochemical staining of pSHF, ChIP-quantitative polymerase chain reaction and luciferase reporter assay. Importantly, we identified Pcsk6 as a novel gene associated with ASD via a human genotyping study of an ASD family. In summary, our study implicated a gene network involving Tbx5, Osr1 and Pcsk6 interaction in SHF for atrial septation, providing a molecular framework for understanding the role of Tbx5 in CHD ontogeny.

Dominant mutations of Gata4, an essential cardiogenic transcription factor (TF), were known to cause outflow tract (OFT) defects in both human and mouse, but the underlying molecular mechanism was not clear. In this study, Gata4 haploinsufficiency in mice was found to result in OFT defects including double outlet right ventricle (DORV) and ventricular septum defects (VSDs). Gata4 was shown to be required for Hedgehog (Hh)-receiving progenitors within the second heart field (SHF) for normal OFT alignment. Restored cell proliferation in the SHF by knocking-down Pten failed to rescue OFT defects, suggesting that additional cell events under Gata4 regulation is important. SHF Hh-receiving cells failed to migrate properly into the proximal OFT cushion, which is associated with abnormal EMT and cell proliferation in Gata4 haploinsufficiency. The genetic interaction of Hh signaling and Gata4 is further demonstrated to be important for OFT development. Gata4 and Smo double heterozygotes displayed more severe OFT abnormalities including persistent truncus arteriosus (PTA). Restoration of Hedgehog signaling renormalized SHF cell proliferation and migration, and rescued OFT defects in Gata4 haploinsufficiency. In addition, there was enhanced Gata6 expression in the SHF of the Gata4 heterozygotes. The Gata4-responsive repressive sites were identified within 1kbp upstream of the transcription start site of Gata6 by both ChIP-qPCR and luciferase reporter assay. These results suggested a SHF regulatory network comprising of Gata4, Gata6 and Hh-signaling for OFT development.

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<p>Supplementary Figures 1-6</p>