Faculty Bibliography

Regulatory T (Treg) cell infiltration constitutes a prominent feature of pancreatic ductal adenocarcinoma (PDA). However, the immunomodulatory function of Treg cells in PDA is poorly understood. Here, we demonstrate that Treg cell ablation is sufficient to evoke effective anti-tumor immune response in early and advanced pancreatic tumorigenesis in mice. This response is dependent on interferon-gamma (IFN-gamma)-producing cytotoxic CD8+ T cells. We show that Treg cells engage in extended interactions with tumor-associated CD11c+ dendritic cells (DCs) and restrain their immunogenic function by suppressing the expression of costimulatory ligands necessary for CD8+ T cell activation. Consequently, tumor-associated CD8+ T cells fail to display effector activities when Treg cell ablation is combined with DC depletion. We propose that tumor-infiltrating Treg cells can promote immune tolerance by suppressing tumor-associated DC immunogenicity. The therapeutic manipulation of this axis might provide an effective approach for the targeting of PDA.

Background: Mutant KRAS tumors show a dependency on WT-H/N-Ras for activation of ATR/Chk1-mediated G2 DNA damage response (Grabocka, Cell, 2015). We have shown in vitro that the Wee1 kinase inhibitor AZD1775, which acts to abrogate the G2 DNA damage checkpoint and induces replication stress during S-phase, selectively sensitizes RAS/RAF mutant cells to the DNA damaging agent irinotecan. Up to 65% of metastatic colorectal cancers harbor RAS or BRAF mutations and these patients have limited treatment options following first line therapy. Methods: This is an open label, single-arm, phase Ib study using a modified 3+3 dose-escalation schedule with expansion cohort. Primary objective is to determine the MTD of AZD1775 in combination with irinotecan as 2 -line therapy in patients with metastatic KRAS, NRAS or BRAF mutated colorectal cancer. Up to 18 patients will be enrolled in the dose escalation portion. Standard dose irinotecan is given on day 1 of every 2 week cycle. AZD1775 is administered PO twice daily for 3 to 5 days of each cycle, starting cycle 2. The maximum tolerated dose (MTD) is defined as the highest dose level at which <=1 of 6 patients experience a dose limiting toxicity. Once the MTD is reached and/or recommended dose for expansion is determined, a dose expansion cohort of 14 patients will be enrolled. Secondary endpoints include characterizing the safety profile at the MTD, obtaining a preliminary estimate of efficacy for the combination (measured by overall response rate, progressionfree and overall survival rates), and obtaining pharmacokinetic parameters. Pre- and on-treatment biopsies will be collected from the expansion cohort to determine: adequate target engagement of Wee1, changes in markers of DNA damage, TP53 mutation status, and changes in gene expression profiles in order to identify potential biomarkers of response. At February 2017, 2 patients have been enrolled on this study

Pancreatic cancer is a devastating disease that is largely refractory to currently available treatment strategies. Therapeutic resistance is partially attributed to the dense stromal reaction of PDAC tumors that includes a pervasive infiltration of immunosuppressive (M2) macrophages. Nab-paclitaxel (trade name Abraxane) is a nanoparticle albumin-bound formulation of paclitaxel that, in combination with gemcitabine, is currently the first line treatment for pancreatic cancer. Here, we show that macrophages internalized nab-paclitaxel via macropinocytosis. The macropinocytic uptake of nab-paclitaxel induced macrophage immunostimulatory (M1) cytokine expression and synergized with IFNgamma to promote inducible nitric oxide synthase (iNOS) expression in a TLR4-dependent manner. Nab-paclitaxel was internalized by tumor-associated macrophages in vivo, and therapeutic doses of nab-paclitaxel alone, and in combination with gemcitabine, increased the MHCII+CD80+CD86+ M1 macrophage population. These data revealed an unanticipated role for nab-paclitaxel in macrophage activation and rationalize its potential use to target immune evasion in pancreatic cancer.

Mammalian tissues rely on a variety of nutrients to support their physiological functions. It is known that altered metabolism is involved in the pathogenesis of cancer, but which nutrients support the inappropriate growth of intact malignant tumors is incompletely understood. Amino acids are essential nutrients for many cancer cells that can be obtained through the scavenging and catabolism of extracellular protein via macropinocytosis. In particular, macropinocytosis can be a nutrient source for pancreatic cancer cells, but it is not fully understood how the tumor environment influences metabolic phenotypes and whether macropinocytosis supports the maintenance of amino acid levels within pancreatic tumors. Here we utilize miniaturized plasma exchange to deliver labeled albumin to tissues in live mice, and we demonstrate that breakdown of albumin contributes to the supply of free amino acids in pancreatic tumors. We also deliver albumin directly into tumors using an implantable microdevice, which was adapted and modified from ref. 9. Following implantation, we directly observe protein catabolism and macropinocytosis in situ by pancreatic cancer cells, but not by adjacent, non-cancerous pancreatic tissue. In addition, we find that intratumoral inhibition of macropinocytosis decreases amino acid levels. Taken together, these data suggest that pancreatic cancer cells consume extracellular protein, including albumin, and that this consumption serves as an important source of amino acids for pancreatic cancer cells in vivo.

There is growing evidence that stress-coping mechanisms represent tumor cell vulnerabilities that may function as therapeutically beneficial targets. Recent work has delineated an integrated stress adaptation mechanism that is characterized by the formation of cytoplasmic mRNA and protein foci, termed stress granules (SGs). Here, we demonstrate that SGs are markedly elevated in mutant KRAS cells following exposure to stress-inducing stimuli. The upregulation of SGs by mutant KRAS is dependent on the production of the signaling lipid molecule 15-deoxy-delta 12,14 prostaglandin J2 (15-d-PGJ2) and confers cytoprotection against stress stimuli and chemotherapeutic agents. The secretion of 15-d-PGJ2 by mutant KRAS cells is sufficient to enhance SG formation and stress resistance in cancer cells that are wild-type for KRAS. Our findings identify a mutant KRAS-dependent cell non-autonomous mechanism that may afford the establishment of a stress-resistant niche that encompasses different tumor subclones. These results should inform the design of strategies to eradicate tumor cell communities.

Pancreatic ductal adenocarcinoma (PDA) is marked by an abundant fibroinflammatory microenvironment. Regulatory T (Treg) cell infiltration constitutes a prominent feature of PDA. However, the immunomodulatory function of Treg cells in PDA remains poorly understood. Using orthotopic and autochthonous mouse models of PDA we have found that Treg cell ablation is sufficient to evoke effective anti-tumor immune response in early and advanced pancreatic neoplasia. This response is dependent on IFN-gamma producing cytotoxic CD8 T cells. We show that Treg cells engage in extended interactions with tumor-associated CD11c dendritic cells (DCs) and restrain their immunogenic function by suppressing the expression of costimulatory ligands necessary for CD8 T cell activation. Consequently, tumor-associated CD8 T cells fail to display effector activities when Treg cell ablation is combined with DC depletion. We propose that tumor-infiltrating Treg cells promote immune-tolerance by suppressing DC immunogenicity. Therapeutic manipulation this axis might provide an effective approach for the targeting of PDA

Inflammation is paramount in pancreatic oncogenesis. We identified a uniquely activated gammadeltaT cell population, which constituted approximately 40% of tumor-infiltrating T cells in human pancreatic ductal adenocarcinoma (PDA). Recruitment and activation of gammadeltaT cells was contingent on diverse chemokine signals. Deletion, depletion, or blockade of gammadeltaT cell recruitment was protective against PDA and resulted in increased infiltration, activation, and Th1 polarization of alphabetaT cells. Although alphabetaT cells were dispensable to outcome in PDA, they became indispensable mediators of tumor protection upon gammadeltaT cell ablation. PDA-infiltrating gammadeltaT cells expressed high levels of exhaustion ligands and thereby negated adaptive anti-tumor immunity. Blockade of PD-L1 in gammadeltaT cells enhanced CD4+ and CD8+ T cell infiltration and immunogenicity and induced tumor protection suggesting that gammadeltaT cells are critical sources of immune-suppressive checkpoint ligands in PDA. We describe gammadeltaT cells as central regulators of effector T cell activation in cancer via novel cross-talk.

SOS is a key activator of the small GTPase Ras. In cells, SOS-Ras signaling is thought to be initiated predominantly by membrane recruitment of SOS via the adaptor Grb2 and balanced by rapidly reversible Grb2-SOS binding kinetics. However, SOS has multiple protein and lipid interactions that provide linkage to the membrane. In reconstituted-membrane experiments, these Grb2-independent interactions were sufficient to retain human SOS on the membrane for many minutes, during which a single SOS molecule could processively activate thousands of Ras molecules. These observations raised questions concerning how receptors maintain control of SOS in cells and how membrane-recruited SOS is ultimately released. We addressed these questions in quantitative assays of reconstituted SOS-deficient chicken B-cell signaling systems combined with single-molecule measurements in supported membranes. These studies revealed an essentially one-way trafficking process in which membrane-recruited SOS remains trapped on the membrane and continuously activates Ras until being actively removed via endocytosis.

A salient feature of pancreatic ductal adenocarcinoma (PDA) is an abundant fibroinflammatory response characterized by the recruitment of immune and mesenchymal cells and the consequent establishment of a pro-tumorigenic microenvironment. Here we report the prominent presence of B cells in human pancreatic intraepithelial neoplasia (PanIN) and PDA lesions as well as in oncogenic K-Ras-driven pancreatic neoplasms in the mouse. The growth of orthotopic pancreatic neoplasms harboring oncogenic K-Ras was significantly compromised in B cell-deficient mice (muMT), and this growth deficiency could be rescued by the reconstitution of a CD1dhighCD5+ B cell subset. The pro-tumorigenic effect of B cells was mediated by their expression of IL-35 through a mechanism involving IL-35-mediated stimulation of tumor cell proliferation. Our results identify a previously unrecognized role for IL-35-producing CD1dhighCD5+ B cells in the pathogenesis of pancreatic cancer and underscore the potential significance of a B cell/IL-35 axis as a therapeutic target.