Alterations in EGFR, KRAS, and ALK are oncogenic drivers in lung cancer, but how oncogenic signaling influences immunity in the tumor microenvironment is just beginning to be understood. Immunosuppression likely contributes to lung cancer, because drugs that inhibit immune checkpoints like PD-1 and PD-L1 have clinical benefit. Here, we show that activation of the AKT-mTOR pathway tightly regulates PD-L1 expression in vitro and in vivo. Both oncogenic and IFNg-mediated induction of PD-L1 was dependent on mTOR. In human lung adenocarcinomas and squamous cell carcinomas, membranous expression of PD-L1 was significantly associated with mTOR activation. These data suggest that oncogenic activation of the AKT-mTOR pathway promotes immune escape by driving expression of PD-L1, which was confirmed in syngeneic and genetically engineered mouse models of lung cancer where an mTOR inhibitor combined with a PD-1 antibody decreased tumor growth, increased tumor-infiltrating T cells, and decreased regulatory T cells.

Loss of glycogen synthase kinase 3␤ (GSK-3␤) in mice results in embryonic lethality via hepatocyte apoptosis. Consistent with this result, cells from these mice have diminished nuclear factor B (NF-B) activity, implying a functional role for GSK-3␤ in regulating NF-B. Here, we have explored mechanisms by which GSK-3␤ may control NF-B function. We show that cytokine-induced IB kinase activity and subsequent phosphorylation of IB␣, p105, and p65 are not affected by the absence of GSK-3␤ activity. Furthermore, nuclear accumulation of p65 following tumor necrosis factor treatment is unaffected by the loss of GSK-3␤. However, NF-B DNA binding activity is reduced in GSK-3␤ null cells and in cells treated with a pharmacological inhibitor of GSK-3. Expression of certain NF-B-regulated genes, such as IB␣ and macrophage inflammatory protein 2, is minimally affected by the absence of GSK-3␤. Conversely, we have identified a subset of NF-B-regulated genes, including those for interleukin-6 and monocyte chemoattractant protein 1, that require GSK-3␤ for efficient expression. We show that efficient localization of p65 to the promoter regions of the interleukin-6 and monocyte chemoattractant protein 1 genes following tumor necrosis factor alpha treatment requires GSK-3␤. Therefore, GSK-3␤ has profound effects on transcription in a gene-specific manner through a mechanism involving control of promoter-specific recruitment of NF-B.

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Constitutive nuclear factor KB (NF-KB) activation is among the many deregulated signaling pathways that are proposed to drive pancreatic cancer cell growth and survival. Recent reports suggest that glycogen synthase kinase-3B (GSK-3B) plays a key role in maintaining basal NF-KB target gene expression and cell survival in pancreatic cancer cell lines. However, the mechanism by which GSK-3B facilitates constitutive NF-KB signaling in pancreatic cancer remains unclear.In this report, we analyze the contributions of both GSK-3 isoforms (GSK-3A and GSK-3B) in regulating NF-KB activation and cell proliferation in pancreatic cancer cell lines (Panc-1 and MiaPaCa-2). We show that GSK-3 isoforms are differentially required to maintain basal NF-KB DNA binding activity, transcriptional activity, and cell proliferation in Panc-1 and MiaPaCa-2 cells. Our data also indicate that IKB kinase (IKK) subunits are not equally required to regulate pancreatic cancer-associated NF-KB activity and cell growth. Importantly, we provide the first evidence that GSK-3 maintains constitutive NF-KB signaling in pancreatic cancer by regulating IKK activity. These data provide new insight into GSK-3-dependent NF-KB regulation and further establish GSK-3 and IKK as potential therapeutic targets for pancreatic cancer.

Constitutive activation of the KRAS oncogene in human malignancies is associated with aggressive tumor growth and poor prognosis. Similar to other oncogenes, KRAS acts in a cell-intrinsic manner to affect tumor growth or survival. However, we describe here a different, cell-extrinsic, mechanism through which mutant KRAS contributes to tumor development. Tumor cells carrying mutated KRAS induced highly suppressive T cells, and silencing KRAS reversed this effect. Overexpression of the mutant KRASG12V gene in wild-type KRAS tumor cells led to Treg induction. We also demonstrate that mutant KRAS induces the secretion of interleukin-10 and transforming growth factor-β1 (both required for Treg induction) by tumor cells through the activation of the MEK-ERK-AP1 pathway. Finally, we report that inhibition of KRAS reduces the infiltration of Tregs in KRAS-driven lung tumorigenesis even before tumor formation. This cell-extrinsic mechanism allows tumor cells harboring a mutant KRAS oncogene to escape immune recognition. Thus, an oncogene can promote tumor progression independent of its transforming activity by increasing the number and function of Tregs. This has a significant clinical potential, in which targeting KRAS and its downstream signaling pathways could be used as powerful immune modulators in cancer immunotherapy.

Mutations in KRAS drive the oncogenic phenotype in a variety of tumors of epithelial origin. The NF-κB transcription factor pathway is important for oncogenic RAS to transform cells and to drive tumorigenesis in animal models. Recently TAK1, an upstream regulator of IKK, which controls canonical NF-κB, was shown to be important for chemoresistance in pancreatic cancer and for regulating KRAS+ colorectal cancer cell growth and survival. Here we show that KRAS+ upregulates GSK-3α leading to its interaction with TAK1 to stabilize the TAK1/TAB complex to promote IKK activity. Additionally, GSK-3α is required for promoting critical non-canonical NF-κB signaling in pancreatic cancer cells. Pharmacologic inhibition of GSK-3 suppresses growth of human pancreatic tumor explants, consistent with the loss of expression of oncogenic genes such as c-myc and TERT. These data identify GSK-3α as a key downstream effector of oncogenic KRAS via its ability to coordinately regulate distinct NF-κB signaling pathways.

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We previously reported that an Nkx2-5-GFP bacterial artificial chromosome in transgenic mice recapitulated the endogenous gene activity in the heart. Here, we identified three additional previously uncharacterized distal enhancer modules of Nkx2-5: UH6, which directed transgene expression in the right ventricle, interventricular septum, and atrial ventricular canal; UH5, which directed expression in both atria; and UH4, which directed transgene expression in tongue muscle. Nkx2-5 enhancers drive cardiogenic gene activity from the earliest progenitors to the late-stage embryonic heart, reside within its 27 kb of 5 flanking sequences, organized in a tandem array. Nkx2-5 enhancers involved with stomach-, tongue-, and chamber-restricted expression displayed lacZ transgene activity and chromatin histone acetylation patterns consistent with tissue-specific expression. An examination of Nkx2-5 gene activity in murine embryonic stem cells converted to beating embryoid bodies showed that only the proximal active region 2 and GATA-Smad enhancers were chromatin-remodeled. Chromatin remodeling of active region 2 and GATA-Smad enhancers were blunted by noggin coexpression, which indicated dependence on bone morphogenetic protein signaling for their chromatin activation during activation of Nkx2-5 expression.enhancer modules ͉ heart development ͉ Nkx2-5 gene regulation