Calcium channel blockers potentiate gemcitabine chemotherapy in pancreatic cancer.

There is currently no effective treatment for pancreatic ductal adenocarcinoma (PDAC). While palliative chemotherapy offers a survival benefit to most patients, nearly all will eventually progress on treatment and long-term survivability remains poor. Given the lack of subsequent line treatment options, in this study, we sought to identify novel strategies to prevent, delay, or overcome resistance to gemcitabine, one of the most widely used medications in PDAC. Using a combination of single-cell RNA sequencing and high-throughput proteomic analysis, we identified a subset of gemcitabine-resistant tumor cells enriched for calcium/calmodulin signaling. Pharmacologic inhibition of calcium-dependent calmodulin activation led to the rapid loss of drug-resistant phenotypes in vitro, which additional single-cell RNA sequencing identified was due to impaired activation of the RAS/ERK signaling pathway. Consistent with these observations, calcium chelation or depletion of calcium in the culture media also impaired ERK activation in gemcitabine-resistant cells, and restored therapeutic responses to gemcitabine in vitro. We observed similar results using calcium channel blockers (CCBs) such as amlodipine, which inhibited prosurvival ERK signaling in vitro and markedly enhanced therapeutic responses to gemcitabine in both orthotopic xenografts and transgenic models of PDAC. Combined, these results offer insight into a potential means of gemcitabine resistance and suggest that select CCBs may provide a clinical benefit to PDAC patients receiving gemcitabine-based chemotherapy.

XP-524 is a dual-BET/EP300 inhibitor that represses oncogenic KRAS and potentiates immune checkpoint inhibition in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is associated with extensive dysregulation of the epigenome and epigenetic regulators, such as bromodomain and extraterminal motif (BET) proteins, have been suggested as potential targets for therapy. However, single-agent BET inhibition has shown poor efficacy in clinical trials, and no epigenetic approaches are currently used in PDAC. To circumvent the limitations of the current generation of BET inhibitors, we developed the compound XP-524 as an inhibitor of the BET protein BRD4 and the histone acetyltransferase EP300/CBP, both of which are ubiquitously expressed in PDAC tissues and cooperate to enhance tumorigenesis. XP-524 showed increased potency and superior tumoricidal activity than the benchmark BET inhibitor JQ-1 in vitro, with comparable efficacy to higher-dose JQ-1 combined with the EP300/CBP inhibitor SGC-CBP30. We determined that this is in part due to the epigenetic silencing of KRAS in vitro, with similar results observed using ex vivo slice cultures of human PDAC tumors. Accordingly, XP-524 prevented KRAS-induced, neoplastic transformation in vivo and extended survival in two transgenic mouse models of aggressive PDAC. In addition to the inhibition of KRAS/MAPK signaling, XP-524 also enhanced the presentation of self-peptide and tumor recruitment of cytotoxic T lymphocytes, though these lymphocytes remained refractory from full activation. We, therefore, combined XP-524 with an anti-PD-1 antibody in vivo, which reactivated the cytotoxic immune program and extended survival well beyond XP-524 in monotherapy. Pending a comprehensive safety evaluation, these results suggest that XP-524 may benefit PDAC patients and warrant further exploration, particularly in combination with immune checkpoint inhibition.

p110γ deficiency protects against pancreatic carcinogenesis yet predisposes to diet-induced hepatotoxicity.

Pancreatic ductal adenocarcinoma (PDAC) is notorious for its poor survival and resistance to conventional therapies. PI3K signaling is implicated in both disease initiation and progression, and specific inhibitors of selected PI3K p110 isoforms for managing solid tumors are emerging. We demonstrate that increased activation of PI3K signals cooperates with oncogenic Kras to promote aggressive PDAC in vivo. The p110γ isoform is overexpressed in tumor tissue and promotes carcinogenesis via canonical AKT signaling. Its selective blockade sensitizes tumor cells to gemcitabine in vitro, and genetic ablation of p110γ protects against Kras-induced tumorigenesis. Diet/obesity was identified as a crucial means of p110 subunit up-regulation, and in the setting of a high-fat diet, p110γ ablation failed to protect against tumor development, showing increased activation of pAKT and hepatic damage. These observations suggest that a careful and judicious approach should be considered when targeting p110γ for therapy, particularly in obese patients.

Metastatic Thymoma Harboring a Deleterious BRCA2 Mutation Derives Durable Clinical Benefit from Olaparib.

Thymomas comprise a group of rare epithelial neoplasms of the anterior mediastinum. Whereas localized disease carries a favorable prognosis, the majority of patients with metastatic thymomas experience progression or recurrence over a 10-year period. Although targeted therapies have become standard of care in many malignancies, no clinically actionable mutations have consistently been identified in metastatic thymomas. Here, we describe a patient with an aggressive thymoma complicated by extensive pleural metastases. Over a 16-year period, she progressed on multiple treatment regimens. To identify additional treatment options, tissue from a pleural metastasis was sent for next-generation sequencing, revealing mutations in BRCA2, tyrosine kinase 2, and SET domain containing 2. Based on supporting evidence for poly (ADP-ribose) polymerase (PARP) inhibition in other BRCA-mutated tumors, the patient was started on the PARP inhibitor olaparib. She derived significant clinical benefit from treatment, with imaging showing overall stabilization of her disease. Here, we review the genotyping results of her tumor and discuss the functional and clinical significance of the mutations in her cancer as well as implications for managing patients with advanced BRCA-mutant thymomas. KEY POINTS: Targeted therapy has yet to enter the standard clinical management of metastatic thymomas. Patients with BRCA2-mutant thymomas may benefit from poly (ADP-ribose) polymerase inhibition.

Quercetin Enhances the Anti-Tumor Effects of BET Inhibitors by Suppressing hnRNPA1.

Bromodomain and extraterminal domain (BET) proteins, which are important epigenetic readers, are often dysregulated in cancer. While a number of BET inhibitors are currently in early phase clinical trials, BET inhibitors show limited single-agent activity. The purpose of this study is to determine if Quercetin, a naturally occurring polyphenolic flavonoid often found abundant in fruits and vegetables, can enhance the anti-tumor effects of BET inhibitors. The efficacy of the combination was evaluated in vitro and in a xenograft model of pancreatic cancer. Co-treatment with BET inhibitors and Quercetin promoted apoptosis, decreased sphere-forming ability by cancer cells, and decreased cell proliferation. We found that hnRNPA1, a nuclear protein known to control mRNA export and mRNA translation of anti-apoptotic proteins, mediates some anti-tumor effects by Quercetin. Additionally, we show that combining BET inhibitors with Quercetin or hnRNPA1 knockdown decreased the anti-apoptotic protein Survivin. Significantly, Quercetin decreased hnRNPA1 in vivo and enhanced the effects of BET inhibitors at suppressing tumor growth. Together, these results demonstrate that Quercetin enhances the efficacy of BET inhibitors by suppressing hnRNPA1, and identify combination therapy with Quercetin and BET inhibitors for the treatment of cancer patients.

Cancer Cell Invasion in Three-dimensional Collagen Is Regulated Differentially by Gα13 Protein and Discoidin Domain Receptor 1-Par3 Protein Signaling.

Cancer cells can invade in three-dimensional collagen as single cells or as a cohesive group of cells that require coordination of cell-cell junctions and the actin cytoskeleton. To examine the role of Gα13, a G12 family heterotrimeric G protein, in regulating cellular invasion in three-dimensional collagen, we established a novel method to track cell invasion by membrane type 1 matrix metalloproteinase-expressing cancer cells. We show that knockdown of Gα13 decreased membrane type 1 matrix metalloproteinase-driven proteolytic invasion in three-dimensional collagen and enhanced E-cadherin-mediated cell-cell adhesion. E-cadherin knockdown reversed Gα13 siRNA-induced cell-cell adhesion but failed to reverse the effect of Gα13 siRNA on proteolytic invasion. Instead, concurrent knockdown of E-cadherin and Gα13 led to an increased number of single cells rather than groups of cells. Significantly, knockdown of discoidin domain receptor 1 (DDR1), a collagen-binding protein that also co-localizes to cell-cell junctions, reversed the effects of Gα13 knockdown on cell-cell adhesion and proteolytic invasion in three-dimensional collagen. Knockdown of the polarity protein Par3, which can function downstream of DDR1, also reversed the effects of Gα13 knockdown on cell-cell adhesion and proteolytic invasion in three-dimensional collagen. Overall, we show that Gα13 and DDR1-Par3 differentially regulate cell-cell junctions and the actin cytoskeleton to mediate invasion in three-dimensional collagen.

Ablation of 5-lipoxygenase mitigates pancreatic lesion development.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC), which continues to have a dismal prognosis, is associated with a pronounced fibroinflammatory response. Inflammation in vivo can be mediated by 5-lipoxygenase (5LO), an enzyme that converts omega-6 fatty acids (FA) to eicosanoids, including leukotriene B4 (LTB4). We have previously shown that diets rich in omega-6 FA increase pancreatic lesions and mast cell infiltration in EL-Kras mice. In this study, we evaluated the role of 5LO in generating higher levels of LTB4 from human cells and in mediating lesion development and mast cell infiltration in EL-Kras mice. MATERIALS AND METHODS: Human pancreatic ductal epithelial and cancer cells were treated with omega-6 FA in vitro. EL-Kras mice lacking 5LO (EL-Kras/5LO(-/-)) mice were generated and fed standard chow or omega-6 FA diets. Pancreatic lesion frequency and mast cell infiltration were compared with EL-Kras/5LO(+/+) mice. Human PDAC tumors were evaluated for 5LO expression and mast cells. RESULTS: Human pancreatic ductal epithelial and pancreatic cancer cells treated with omega-6 FA generated increased LTB4 levels in vitro. EL-Kras/5LO(-/-) mice developed fewer pancreatic lesions and had decreased mast cell infiltration when compared with EL-Kras/5LO(+/+) mice. Human PDAC tumors with increased 5LO expression demonstrate increased mast cell infiltration. Additionally, diets rich in omega-6 FA failed to increase pancreatic lesion development and mast cell infiltration in EL-Kras/5LO(-/-) mice. CONCLUSIONS: The expansion of mutant Kras-induced lesions via omega-6 FA is dependent on 5LO, and 5LO functions downstream of mutant Kras to mediate inflammation, suggesting that 5LO may be a potential chemopreventive and therapeutic target in pancreatic cancer.

Comparative effectiveness in esophagogastric cancer.

Cancer of the esophagus and the gastroesophageal junction (GEJ) continues to have a dismal prognosis, with the incidence of esophageal cancer increasing in the United States. Although radical resection was initially the primary treatment for this disease process, systemic chemotherapy and radiation have been shown to play a role in prolonging survival in most patient populations. This chapter explores the evidence that guides treatment for esophageal and GEJ cancer today. Chemotherapy and radiation therapy were introduced as treatment modalities for esophageal and GEJ cancers when it became evident that surgical therapy alone provided poor long-term survival rates. A variety of treatment strategies have been explored including preoperative (neoadjuvant) and postoperative (adjuvant) chemotherapy, with and without radiation. The evidence suggests that neoadjuvant chemotherapy or chemoradiotherapy provides better outcomes compared to surgery alone for esophageal, GEJ, and gastric cancers. Studies indicate a trend towards improved survival when neoadjuvant chemoradiotherapy is compared to chemotherapy alone. When patients have undergone resection with node-positive disease without receiving neoadjuvant therapy, some form of adjuvant treatment is recommended. This chapter also explores the surgical management of esophageal, GEJ, and gastric cancers including the extent of the gastric lymph node dissection. It also includes a discussion about adherence to national guidelines in terms of gastric cancer treatment and esophageal and gastric lymph node examinations.

Interplay between ß1-integrin and Rho signaling regulates differential scattering and motility of pancreatic cancer cells by snail and Slug proteins.

The Snail family of transcription factors has been implicated in pancreatic cancer progression. We recently showed that Snail (Snai1) promotes membrane-type 1 matrix metalloproteinase (MT1-MMP)- and ERK1/2-dependent scattering of pancreatic cancer cells in three-dimensional type I collagen. In this study, we examine the role of Slug (Snai2) in regulating pancreatic cancer cell scattering in three-dimensional type I collagen. Although Slug increased MT1-MMP expression and ERK1/2 activity, Slug-expressing cells failed to scatter in three-dimensional collagen. Moreover, in contrast to Snail-expressing cells, Slug-expressing cells did not demonstrate increased collagen I binding, collagen I-driven motility, or α2ß1-integrin expression. Significantly, inhibiting ß1-integrin function decreased migration and scattering of Snail-expressing cells in three-dimensional collagen. As Rho GTPases have been implicated in invasion and migration, we also analyzed the contribution of Rac1 and Rho signaling to the differential migration and scattering of pancreatic cancer cells. Snail-induced migration and scattering were attenuated by Rac1 inhibition. In contrast, inhibiting Rho-associated kinase ROCK1/2 increased migration and scattering of Slug-expressing cells in three-dimensional collagen and thus phenocopied the effects of Snail in pancreatic cancer cells. Additionally, the increased migration and scattering in three-dimensional collagen of Slug-expressing cells following ROCK1/2 inhibition was dependent on ß1-integrin function. Overall, these results demonstrate differential effects of Snail and Slug in pancreatic cancer and identify the interplay between Rho signaling and ß1-integrin that functions to regulate the differential scattering and migration of Snail- and Slug-expressing pancreatic cancer cells.

Biochemical role of the collagen-rich tumour microenvironment in pancreatic cancer progression.

PDAC (pancreatic ductal adenocarcinoma) is among the most deadly of human malignances. A hallmark of the disease is a pronounced collagen-rich fibrotic extracellular matrix known as the desmoplastic reaction. Intriguingly, it is precisely these areas of fibrosis in which human PDAC tumours demonstrate increased expression of a key collagenase, MT1-MMP [membrane-type 1 MMP (matrix metalloproteinase); also known as MMP-14]. Furthermore, a cytokine known to mediate fibrosis in vivo, TGF-ß1 (transforming growth factor-ß1), is up-regulated in human PDAC tumours and can promote MT1-MMP expression. In the present review, we examine the regulation of PDAC progression through the interplay between type I collagen (the most common extracellular matrix present in human PDAC tumours), MT1-MMP and TGF-ß1. Specifically, we examine the way in which signalling events through these pathways mediates invasion, regulates microRNAs and contributes to chemoresistance.

Concurrent PEDF deficiency and Kras mutation induce invasive pancreatic cancer and adipose-rich stroma in mice.

BACKGROUND AND AIMS: Pigment epithelium-derived factor (PEDF), a non-inhibitory SERPIN with potent antiangiogenic activity, has been recently implicated in metabolism and adipogenesis, both of which are known to influence pancreatic cancer progression. Increased pancreatic fat in human pancreatic tumour correlates with greater tumour dissemination while PEDF deficiency in mice promotes pancreatic hyperplasia and visceral obesity. Oncogenic Ras, the most common mutation in pancreatic ductal adenocarcinoma (PDAC), has similarly been shown to promote adipogenesis and premalignant lesions. METHODS: In order to determine whether concurrent loss of PEDF is sufficient to promote adipogenesis and tumorigenesis in the pancreas, the authors ablated PEDF in an EL-Kras(G12D) mouse model of non-invasive cystic papillary neoplasms. RESULTS: EL-Kras(G12D)/PEDF deficient mice developed invasive PDAC associated with enhanced matrix metalloproteinase (MMP)-2 and MMP-9 expression and increased peripancreatic fat with adipocyte hypertrophy and intrapancreatic adipocyte infiltration (pancreatic steatosis). In support of increased adipogenesis, the stroma of the pancreas of EL-Kras(G12D)/PEDF deficient mice demonstrated higher tissue levels of two lipid droplet associated proteins, tail-interacting protein 47 (TIP47, perilipin 3) and adipose differentiation-related protein (ADRP, Pperilipin 2), while adipose triglyceride lipase, a key factor in lipolysis, was decreased. In patients with PDAC, both tissue and serum levels of PEDF were decreased, stromal TIP47 expression was higher and the tissue VEGF to PEDF ratio was increased (p<0.05). CONCLUSIONS: These data highlight the importance of lipid metabolism in the tumour microenvironment and identify PEDF as a critical negative regulator of both adiposity and tumour invasion in the pancreas.

Targeting BET Proteins Decreases Hyaluronidase-1 in Pancreatic Cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is characterized by the presence of dense stroma that is enriched in hyaluronan (HA), with increased HA levels associated with more aggressive disease. Increased levels of the HA-degrading enzymes hyaluronidases (HYALs) are also associated with tumor progression. In this study, we evaluate the regulation of HYALs in PDAC. METHODS: Using siRNA and small molecule inhibitors, we evaluated the regulation of HYALs using quantitative real-time PCR (qRT-PCR), Western blot analysis, and ELISA. The binding of BRD2 protein on the HYAL1 promoter was evaluated by chromatin immunoprecipitation (ChIP) assay. Proliferation was evaluated by WST-1 assay. Mice with xenograft tumors were treated with BET inhibitors. The expression of HYALs in tumors was analyzed by immunohistochemistry and by qRT-PCR. RESULTS: We show that HYAL1, HYAL2, and HYAL3 are expressed in PDAC tumors and in PDAC and pancreatic stellate cell lines. We demonstrate that inhibitors targeting bromodomain and extra-terminal domain (BET) proteins, which are readers of histone acetylation marks, primarily decrease HYAL1 expression. We show that the BET family protein BRD2 regulates HYAL1 expression by binding to its promoter region and that HYAL1 downregulation decreases proliferation and enhances apoptosis of PDAC and stellate cell lines. Notably, BET inhibitors decrease the levels of HYAL1 expression in vivo without affecting the levels of HYAL2 or HYAL3. CONCLUSIONS: Our results demonstrate the pro-tumorigenic role of HYAL1 and identify the role of BRD2 in the regulation of HYAL1 in PDAC. Overall, these data enhance our understanding of the role and regulation of HYAL1 and provide the rationale for targeting HYAL1 in PDAC.

BET inhibition rescues ciliogenesis and ameliorates pancreatitis-driven phenotypic changes in mice with Par3 loss.

The apical-basal polarity of pancreatic acinar cells is essential for maintaining tissue architecture. However, the mechanisms by which polarity proteins regulate acinar pancreas tissue homeostasis are poorly understood. Here, we evaluate the role of Par3 in acinar pancreas injury and homeostasis. While Par3 loss in the mouse pancreas disrupts tight junctions, Par3 loss is dispensable for pancreatogenesis. However, with aging, Par3 loss results in low-grade inflammation, acinar degeneration, and pancreatic lipomatosis. Par3 loss also exacerbates pancreatitis-induced acinar cell loss, resulting in pronounced pancreatic lipomatosis and failure to regenerate. Moreover, Par3 loss in mice harboring mutant Kras causes extensive pancreatic intraepithelial neoplastic (PanIN) lesions and large pancreatic cysts. We also show that Par3 loss restricts injury-induced primary ciliogenesis. Significantly, targeting BET proteins enhances primary ciliogenesis during pancreatitis-induced injury and, in mice with Par3 loss, limits pancreatitis-induced acinar loss and facilitates acinar cell regeneration. Combined, this study demonstrates how Par3 restrains pancreatitis- and Kras-induced changes in the pancreas and identifies a potential role for BET inhibitors to attenuate pancreas injury and facilitate pancreas tissue regeneration.

Pancreatic cancer cells respond to type I collagen by inducing snail expression to promote membrane type 1 matrix metalloproteinase-dependent collagen invasion.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by pronounced fibrotic reaction composed primarily of type I collagen. Although type I collagen functions as a barrier to invasion, pancreatic cancer cells have been shown to respond to type I collagen by becoming more motile and invasive. Because epithelial-mesenchymal transition is also associated with cancer invasion, we examined the extent to which collagen modulated the expression of Snail, a well known regulator of epithelial-mesenchymal transition. Relative to cells grown on tissue culture plastic, PDAC cells grown in three-dimensional collagen gels induced Snail. Inhibiting the activity or expression of the TGF-ß type I receptor abrogated collagen-induced Snail. Downstream of the receptor, we showed that Smad3 and Smad4 were critical for the induction of Snail by collagen. In contrast, Smad2 or ERK1/2 was not involved in collagen-mediated Snail expression. Overexpression of Snail in PDAC cells resulted in a robust membrane type 1-matrix metalloproteinase (MT1-MMP, MMP-14)-dependent invasion through collagen-coated transwell chambers. Snail-expressing PDAC cells also demonstrated MT1-MMP-dependent scattering in three-dimensional collagen gels. Mechanistically, Snail increased the expression of MT1-MMP through activation of ERK-MAPK signaling, and inhibiting ERK signaling in Snail-expressing cells blocked two-dimensional collagen invasion and attenuated scattering in three-dimensional collagen. To provide in vivo support for our findings that Snail can regulate MT1-MMP, we examined the expression of Snail and MT1-MMP in human PDAC tumors and found a statistically significant positive correlation between MT1-MMP and Snail in these tumors. Overall, our data demonstrate that pancreatic cancer cells increase Snail on encountering collagen-rich milieu and suggest that the desmoplastic reaction actively contributes to PDAC progression.

Contribution of epithelial-to-mesenchymal transition and cancer stem cells to pancreatic cancer progression.

Pancreatic adenocarcinoma remains among the most lethal of human malignancies. Overall 5-y survival is less than 5%, and only 20% of patients presenting with localized disease amenable to surgical resection. Even in patients who undergo resection, long-term survival remains extremely poor. A major contributor to the aggressiveness of multiple cancers, and pancreatic cancer in particular, is the process of epithelial-to-mesenchymal transition (EMT). This review highlights the growing evidence of EMT in pancreatic cancer progression, focusing on the contribution of EMT to the development of cancer stem cells and on interaction of EMT with other pathways central to cancer progression, such as Hedgehog signaling, the K-ras oncogene, and transforming growth factor-beta (TGF-ß). We will also discuss EMT-targeting agents currently in development and in clinical trials that may help to reduce the morbidity and mortality associated with pancreatic cancer.

The difficulty in translating the preclinical success of combined TGFß and immune checkpoint inhibition to clinical trial.

Immune checkpoint inhibitors (ICIs) have transformed the treatment paradigm for solid tumors. However, even in cancers generally considered ICI-sensitive, responses can vary significantly. Thus, there is an ever-increasing interest in identifying novel means of improving therapeutic responses, both for cancers in which ICIs are indicated and those for which they have yet to show significant anti-tumor activity. To this end, Transforming Growth Factor ß (TGFß) signaling is emerging as an important barrier to the efficacy of ICIs. Accordingly, several preclinical studies now support the use of combined TGFß and immune checkpoint blockade, with near-uniform positive results across a wide range of tumor types. However, as these approaches have started to emerge in clinical trials, the addition of TGFß inhibitors has often failed to show a meaningful benefit beyond the current generation of ICIs alone. Here, we summarize landmark clinical studies exploring combined TGFß and immune checkpoint blockade. These studies not only reinforce the difficulty in translating results from rodents to clinical trials in immune-oncology but also underscore the need to re-evaluate the design of trials exploring this approach, incorporating both mechanism-driven combination strategies and novel, predictive biomarkers to identify the patients most likely to derive clinical benefit.

The immune modifying effects of chemotherapy and advances in chemo-immunotherapy.

Immune checkpoint inhibitors (ICIs) have transformed the treatment paradigm for several malignancies. While the use of single-agent or combined ICIs has achieved acceptable disease control rates in a variety of solid tumors, such approaches have yet to show substantial therapeutic efficacy in select difficult-to-treat cancer types. Recently, select chemotherapy regimens are emerging as extensive modifiers of the tumor microenvironment, leading to the reprogramming of local immune responses. Accordingly, data is now emerging to suggest that certain anti-neoplastic agents modulate various immune cell processes, most notably the cross-presentation of tumor antigens, leukocyte trafficking, and cytokine biosynthesis. As such, the combination of ICIs and cytotoxic chemotherapy are beginning to show promise in many cancers that have long been considered poorly responsive to ICI-based immunotherapy. Here, we discuss past and present attempts to advance chemo-immunotherapy in these difficult-to-treat cancer histologies, mechanisms through which select chemotherapies modify tumor immunogenicity, as well as important considerations when designing such approaches to maximize efficacy and improve therapeutic response rates.

Gα13 loss in Kras/Tp53 mouse model of pancreatic tumorigenesis promotes tumors susceptible to rapamycin.

Gα13 transduces signals from G-protein-coupled receptors. While Gα13 functions as a tumor suppressor in lymphomas, it is not known whether Gα13 is pro-tumorigenic or tumor suppressive in genetically engineered mouse (GEM) models of epithelial cancers. Here, we show that loss of Gα13 in the Kras/Tp53 (KPC) GEM model promotes well-differentiated tumors and reduces survival. Mechanistically, tumors developing in KPC mice with Gα13 loss exhibit increased E-cadherin expression and mTOR signaling. Importantly, human pancreatic ductal adenocarcinoma (PDAC) tumors with low Gα13 expression also exhibit increased E-cadherin expression and mTOR signaling. Treatment with the mTOR inhibitor rapamycin decreases the growth of syngeneic KPC tumors with Gα13 loss by promoting cell death. This work establishes a tumor-suppressive role of Gα13 in pancreatic tumorigenesis in the KPC GEM model and suggests targeting mTOR in human PDAC tumors with Gα13 loss.

Loss of SMAD4 Is Associated With Poor Tumor Immunogenicity and Reduced PD-L1 Expression in Pancreatic Cancer.

Transforming Growth Factor ß (TGFß) is a key mediator of immune evasion in pancreatic ductal adenocarcinoma (PDAC), and the addition of TGFß inhibitors in select immunotherapy regimens shows early promise. Though the TGFß target SMAD4 is deleted in approximately 55% of PDAC tumors, the effects of SMAD4 loss on tumor immunity have yet to be fully explored. Using a combination of genomic databases and PDAC specimens, we found that tumors with loss of SMAD4 have a comparatively poor T-cell infiltrate. SMAD4 loss was also associated with a reduction in several chemokines with known roles in T-cell recruitment, which was recapitulated using knockdown of SMAD4 in PDAC cell lines. Accordingly, JURKAT T-cells were poorly attracted to conditioned media from PDAC cells with knockdown of SMAD4 and lost their ability to produce IFNγ. However, while exogenous TGFß modestly reduced PD-L1 expression in SMAD4-intact cell lines, SMAD4 and PD-L1 positively correlated in human PDAC samples. PD-L1 status was closely related to tumor-infiltrating lymphocytes, particularly IFNγ-producing T-cells, which were more abundant in SMAD4-expressing tumors. Low concentrations of IFNγ upregulated PD-L1 in tumor cells in vitro, even when administered alongside high concentrations of TGFß. Hence, while SMAD4 may have a modest inhibitory effect on PD-L1 in tumor cells, SMAD4 indirectly promotes PD-L1 expression in the pancreatic tumor microenvironment by enhancing T-cell infiltration and IFNγ biosynthesis. These data suggest that pancreatic cancers with loss of SMAD4 represent a poorly immunogenic disease subtype, and SMAD4 status warrants further exploration as a predictive biomarker for cancer immunotherapy.

Inhibition of MNKs promotes macrophage immunosuppressive phenotype to limit CD8+ T cell antitumor immunity.

To elicit effective antitumor responses, CD8+ T cells need to infiltrate tumors and sustain their effector function within the immunosuppressive tumor microenvironment (TME). Here, we evaluate the role of MNK activity in regulating CD8+ T cell infiltration and antitumor activity in pancreatic and thyroid tumors. We first show that human pancreatic and thyroid tumors with increased MNK activity are associated with decreased infiltration by CD8+ T cells. We then show that, while MNK inhibitors increase CD8+ T cells in these tumors, they induce a T cell exhaustion phenotype in the tumor microenvironment. Mechanistically, we show that the exhaustion phenotype is not caused by upregulation of programmed cell death ligand 1 (PD-L1) but is caused by tumor-associated macrophages (TAMs) becoming more immunosuppressive following MNK inhibitor treatment. Reversal of CD8+ T cell exhaustion by an anti-PD-1 antibody or TAM depletion synergizes with MNK inhibitors to control tumor growth and prolong animal survival. Importantly, we show in ex vivo human pancreatic tumor slice cultures that MNK inhibitors increase the expression of markers associated with immunosuppressive TAMs. Together, these findings demonstrate a role of MNKs modulating a protumoral phenotype in macrophages and identify combination regimens involving MNK inhibitors to enhance antitumor immune responses.