Sendai virus is robust and consistent in delivering genes into human pancreatic cancer cells.

Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly intratumorally heterogeneous disease that includes several subtypes and is highly plastic. Effective gene delivery to all PDAC cells is essential for modulating gene expression and identifying potential gene-based therapeutic targets in PDAC. Most current gene delivery systems for pancreatic cells are optimized for islet or acinar cells. Lentiviral vectors are the current main gene delivery vectors for PDAC, but their transduction efficiencies vary depending on pancreatic cell type, and are especially poor for the classical subtype of PDAC cells from both primary tumors and cell lines. Methods: We systemically compare transduction efficiencies of glycoprotein G of vesicular stomatitis virus (VSV-G)-pseudotyped lentiviral and Sendai viral vectors in human normal pancreatic ductal and PDAC cells. Results: We find that the Sendai viral vector gives the most robust gene delivery efficiency regardless of PDAC cell type. Therefore, we propose using Sendai viral vectors to transduce ectopic genes into PDAC cells.

MEK Inhibition Sensitizes Pancreatic Cancer to STING Agonism by Tumor Cell-intrinsic Amplification of Type I IFN Signaling.

PURPOSE: Stimulator of interferon genes (STING) agonists are currently in development for treatment of solid tumors, including pancreatic ductal adenocarcinoma (PDAC). Response rates to STING agonists alone have been promising yet modest, and combination therapies will likely be required to elicit their full potency. We sought to identify combination therapies and mechanisms that augment the tumor cell-intrinsic effect of therapeutically relevant STING agonists apart from their known effects on tumor immunity. EXPERIMENTAL DESIGN: We screened 430 kinase inhibitors to identify synergistic effectors of tumor cell death with diABZI, an intravenously administered and systemically available STING agonist. We deciphered the mechanisms of synergy with STING agonism that cause tumor cell death in vitro and tumor regression in vivo. RESULTS: We found that MEK inhibitors caused the greatest synergy with diABZI and that this effect was most pronounced in cells with high STING expression. MEK inhibition enhanced the ability of STING agonism to induce type I IFN-dependent cell death in vitro and tumor regression in vivo. We parsed NFκB-dependent and NFκB-independent mechanisms that mediate STING-driven type I IFN production and show that MEK signaling inhibits this effect by suppressing NFκB activation. CONCLUSIONS: Our results highlight the cytotoxic effects of STING agonism on PDAC cells that are independent of tumor immunity and that these therapeutic benefits of STING agonism can be synergistically enhanced by MEK inhibition.

T-cell Dysfunction upon Expression of MYC with Altered Phosphorylation at Threonine 58 and Serine 62.

As a transcription factor that promotes cell growth, proliferation, and apoptosis, c-MYC (MYC) expression in the cell is tightly controlled. Disruption of oncogenic signaling pathways in human cancers can increase MYC protein stability, due to altered phosphorylation ratios at two highly conserved sites, Threonine 58 (T58) and Serine 62 (S62). The T58 to Alanine mutant (T58A) of MYC mimics the stabilized, S62 phosphorylated, and highly oncogenic form of MYC. The S62A mutant is also stabilized, lacks phosphorylation at both Serine 62 and Threonine 58, and has been shown to be nontransforming in vitro. However, several regulatory proteins are reported to associate with MYC lacking phosphorylation at S62 and T58, and the role this form of MYC plays in MYC transcriptional output and in vivo oncogenic function is understudied. We generated conditional c-Myc knock-in mice in which the expression of wild-type MYC (MYCWT), the T58A mutant (MYCT58A), or the S62A mutant (MYCS62A) with or without expression of endogenous Myc is controlled by the T-cell-specific Lck-Cre recombinase. MYCT58A expressing mice developed clonal T-cell lymphomas with 100% penetrance and conditional knock-out of endogenous Myc accelerated this lymphomagenesis. In contrast, MYCS62A mice developed clonal T-cell lymphomas at a much lower penetrance, and the loss of endogenous MYC reduced the penetrance while increasing the appearance of a non-transgene driven B-cell lymphoma with splenomegaly. Together, our study highlights the importance of regulated phosphorylation of MYC at T58 and S62 for T-cell transformation. IMPLICATIONS: Dysregulation of phosphorylation at conserved T58 and S62 residues of MYC differentially affects T-cell development and lymphomagenesis.

Reprogramming of nucleotide metabolism by interferon confers dependence on the replication stress response pathway in pancreatic cancer cells.

We determine that type I interferon (IFN) response biomarkers are enriched in a subset of pancreatic ductal adenocarcinoma (PDAC) tumors; however, actionable vulnerabilities associated with IFN signaling have not been systematically defined. Integration of a phosphoproteomic analysis and a chemical genomics synergy screen reveals that IFN activates the replication stress response kinase ataxia telangiectasia and Rad3-related protein (ATR) in PDAC cells and sensitizes them to ATR inhibitors. IFN triggers cell-cycle arrest in S-phase, which is accompanied by nucleotide pool insufficiency and nucleoside efflux. In combination with IFN, ATR inhibitors induce lethal DNA damage and downregulate nucleotide biosynthesis. ATR inhibition limits the growth of PDAC tumors in which IFN signaling is driven by stimulator of interferon genes (STING). These results identify a cross talk between IFN, DNA replication stress response networks, and nucleotide metabolism while providing the rationale for targeted therapeutic interventions that leverage IFN signaling in tumors.

The activities of MYC, MNT and the MAX-interactome in lymphocyte proliferation and oncogenesis.

The MYC family of proteins plays essential roles in embryonic development and in oncogenesis. Efforts over the past 30 years to define the transcriptional activities of MYC and how MYC functions to promote proliferation have produced evolving models of MYC function. One picture that has emerged of MYC and its partner protein MAX is of a transcription factor complex with a seemingly unique ability to stimulate the transcription of genes that are epigenetically poised for transcription and to amplify the transcription of actively transcribed genes. During lymphocyte activation, MYC is upregulated and stimulates a pro-proliferative program in part through the upregulation of a wide variety of metabolic effector genes that facilitate cell growth and cell cycle progression. MYC upregulation simultaneously sensitizes cells to apoptosis and activated lymphocytes and lymphoma cells have pro-survival attributes that allow MYC-driven proliferation to prevail. For example, the MAX-interacting protein MNT is upregulated in activated lymphocytes and was found to protect lymphocytes from MYC-dependent apoptosis. Here we review the activities of MYC, MNT and other MAX interacting proteins in the setting of T and B cell activation and oncogenesis. This article is part of a Special Issue entitled: Myc proteins in cell biology and pathology.

A critical role for Mnt in Myc-driven T-cell proliferation and oncogenesis.

Mnt (Max's next tango) is a Max-interacting transcriptional repressor that can antagonize both the proproliferative and proapoptotic functions of Myc in vitro. To ascertain the physiologically relevant functions of Mnt and to help define the relationship between Mnt and Myc in vivo, we generated a series of mouse strains in which Mnt was deleted in T cells in the absence of endogenous c-Myc or in the presence of ectopic c-Myc. We found that apoptosis caused by loss of Mnt did not require Myc but that ectopic Myc expression dramatically decreased the survival of both Mnt-deficient T cells in vivo and Mnt-deficient MEFs in vitro. Consequently, Myc-driven proliferative expansion of T cells in vitro and thymoma formation in vivo were prevented by the absence of Mnt. Consistent with T-cell models, mouse embryo fibroblasts (MEFs) lacking Mnt were refractory to oncogenic transformation by Myc. Tumor suppression caused by loss of Mnt was linked to increased apoptosis mediated by reactive oxygen species (ROS). Thus, although theoretically and experimentally a Myc antagonist, the dominant physiological role of Mnt appears to be suppression of apoptosis. Our results redefine the physiological relationship between Mnt and Myc and requirements for Myc-driven oncogenesis.

The role of senescence and prosurvival signaling in controlling the oncogenic activity of FGFR2 mutants associated with cancer and birth defects.

Mutations in fibroblast growth factor receptors (FGFRs) cause human birth defect syndromes and are associated with a variety of cancers. Although forced expression of mutant activated FGFRs has been shown to oncogenically transform some immortal cell types, their activity in primary cells remains unclear. Here, we show that birth defect and cancer-associated FGFR2 mutants promote DNA-damage signaling and p53-dependent senescence in primary mouse and human cells. Senescence promoted by FGFR mutants was associated with downregulation of c-Myc and forced expression of c-Myc facilitated senescence escape. Whereas c-Myc expression facilitated senescence bypass, mutant FGFR2 signaling suppressed c-Myc-dependent apoptosis and led to oncogenic transformation. Cells transformed by coexpression of a constitutively activated FGFR2 mutant plus c-Myc appeared to be become highly addicted to FGFR-dependent prosurvival activities, as small molecule inhibition of FGFR signaling resulted in robust p53-dependent apoptosis. Our data suggest that senescence-promoting activities of mutant FGFRs may normally limit their oncogenic potential and may be relevant to their ability to disrupt morphogenesis and cause birth defects. Our results also raise the possibility that cancers originating through a combination of constitutive FGFR activation and deregulated Myc expression may be particularly sensitive to small molecule inhibitors of FGF receptors.