Yuliya Pylayeva-Gupta

RAS oncogenes: weaving a tumorigenic web

RAS proteins are essential components of signalling pathways that emanate from cell surface receptors. Oncogenic activation of these proteins owing to missense mutations is frequently detected in several types of cancer. A wealth of biochemical and genetic studies indicates that RAS proteins control a complex molecular circuitry that consists of a wide array of interconnecting pathways. In this Review, we describe how RAS oncogenes exploit their extensive signalling reach to affect multiple cellular processes that drive tumorigenesis.

MyD88 inhibition amplifies dendritic cell capacity to promote pancreatic carcinogenesis via Th2 cells

MyD88 blockade exaggerates the ability of dendritic cells to promote the transition from chronic pancreatitis to pancreatic cancer.

Wild-type H- and N-Ras promote mutant K-Ras driven tumorigenesis by modulating the DNA damage response

Mutations in KRAS are prevalent in human cancers and universally predictive of resistance to anticancer therapeutics. Although it is widely accepted that acquisition of an activating mutation endows RAS genes with functional autonomy, recent studies suggest that the wild-type forms of Ras may contribute to mutant Ras-driven tumorigenesis. Here, we show that downregulation of wild-type H-Ras or N-Ras in mutant K-Ras cancer cells leads to hyperactivation of the Erk/p90RSK and PI3K/Akt pathways and, consequently, the phosphorylation of Chk1 at an inhibitory site, Ser 280. The resulting inhibition of ATR/Chk1 signaling abrogates the activation of the G2 DNA damage checkpoint and confers specific sensitization of mutant K-Ras cancer cells to DNA damage chemotherapeutic agents in vitro and in vivo.

EZH2 couples pancreatic regeneration to neoplastic progression

Although the polycomb group protein Enhancer of Zeste Homolog 2 (EZH2) is well recognized for its role as a key regulator of cell differentiation, its involvement in tissue regeneration is largely unknown. Here we show that EZH2 is up-regulated following cerulein-induced pancreatic injury and is required for tissue repair by promoting the regenerative proliferation of progenitor cells. Loss of EZH2 results in impaired pancreatic regeneration and accelerates KRas(G12D)-driven neoplasia. Our findings implicate EZH2 in constraining neoplastic progression through homeostatic mechanisms that control pancreatic regeneration and provide insights into the documented link between chronic pancreatic injury and an increased risk for pancreatic cancer.

IL35-Producing B Cells Promote the Development of Pancreatic Neoplasia

UNLABELLED A salient feature of pancreatic ductal adenocarcinoma (PDAC) is an abundant fibroinflammatory response characterized by the recruitment of immune and mesenchymal cells and the consequent establishment of a protumorigenic microenvironment. Here, we report the prominent presence of B cells in human pancreatic intraepithelial neoplasia and PDAC lesions as well as in oncogenic Kras-driven pancreatic neoplasms in the mouse. The growth of orthotopic pancreatic neoplasms harboring oncogenic Kras was significantly compromised in B-cell-deficient mice (μMT), and this growth deficiency could be rescued by the reconstitution of a CD1d(hi)CD5(+) B-cell subset. The protumorigenic effect of B cells was mediated by their expression of IL35 through a mechanism involving IL35-mediated stimulation of tumor cell proliferation. Our results identify a previously unrecognized role for IL35-producing CD1d(hi)CD5(+) B cells in the pathogenesis of pancreatic cancer and underscore the potential significance of a B-cell/IL35 axis as a therapeutic target. SIGNIFICANCE This study identifies a B-cell subpopulation that accumulates in the pancreatic parenchyma during early neoplasia and is required to support tumor cell growth. Our findings provide a rationale for exploring B-cell-based targeting approaches for the treatment of pancreatic cancer.

Microdissection and culture of murine pancreatic ductal epithelial cells.

Given the complexity of morphological presentation and variability in clinical outcomes observed in -epithelial cancers, it is important to understand how genomic perturbations and resultant molecular aberrations lead to acquisition of tumorigenic phenotypes. Complex 3D epithelial culture systems provide investigators with the ability to propagate and manipulate primary cells in an appropriate physical setting in order to deconstruct the contribution of a given genetic lesion(s) to the process of cellular transformation. Pancreatic ductal epithelial cells (PDEC) can give rise to pancreatic intraepithelial neoplasia-precursor lesions that precede pancreatic ductal adenocarcinoma (PDA). In this chapter, we describe a series of methods for derivation and culture of primary PDEC, which can be used to elucidate the mechanistic contribution of oncogenic insults to the initiation and progression of pancreatic tumorigenesis.

Abstract B62: The role of B regulatory cells in pancreatic cancer

Background: Formation of pancreatic ductal adenocarcinoma (PDA) is accompanied by pronounced changes in stromal responses and immune surveillance programs, which are now recognized as some of the major drivers in PDA tumor evolution, and likely contribute to its notorious resistance to therapy. However, the cellular and molecular mechanisms that underlie immune response modulation in the context of pancreatic tumorigenesis remain poorly understood. We have observed that B lymphocytes accumulate at sites of pancreatic neoplasia and infiltrate lesion-adjacent stroma. The role of B-cell-mediated immune regulation in solid cancers has only recently begun to be appreciated, and virtually nothing is known about B cell function in pancreatic tumorigenesis. Our studies test the role of B cells in the initiation of pancreatic cancer and provide novel insight into functions of B regulatory cells in cancer pathogenesis. Experimental Design and Methods: In this study, we orthotopically grafted KRasG12D- pancreatic ductal epithelial cells (PDEC) into pancreata of recipient mice with normal or selectively immunocompromised immune responses. In particular, we investigated the role of B lymphocytes in pancreatic neoplasia by implanting KRasG12D-PDEC into the B cell deficient μMT strain, and evaluated the role of specific B cell subsets using adoptive lymphocyte transfer. Results: We demonstrate that B lymphocytes are specifically expanded in pancreatic neoplasia. We find that rather than being restricted to peri-pancreatic lymph node tissue, B cells infiltrate human PanIN lesions, as well as both LSL-KRasG12D; p48-Cre (KC) and pancreata orthotopically grafted with KRasG12D-PDEC. Such infiltration is closely correlated with expression of the B cell chemoattractant CXCL13 in the stromal compartment of the pancreas. Transplantation of KRasG12D-PDEC into pancreata of syngeneic B cell deficient μMT mice resulted in reduced growth of the cancer cells, indicating that B cells exert a tumor promoting effect. This defect in growth of KRasG12D-PDEC was accompanied by robust infiltration of macrophages. We found that pancreata-associated macrophages in μMT mice with KRasG12D-PDEC lesions expressed less of TAM-like CD206 and, correspondingly, more of M1-like CD86 on their cell surface. This observation suggested that absence of B cells prevents (or reverses) pro-tumorigenic macrophage polarization in pancreatic cancer towards a more anti-tumorigenic macrophage phenotype. After a more detailed analysis of B cell subtypes, we found that a regulatory subtype of B cells (Bregs, as defined by CD19+CD1dhighCD5+ surface phenotype and IL-10 expression) is specifically expanded in pancreata of KC and orthotopically grafted animals. Correspondingly, we find that a proportion of CD20+ B cells that infiltrate human PanIN lesions express IL-10. Reconstitution of μMT mice with B regulatory cells rescued the in vivo KRasG12D-PDEC growth defect. Conclusion: These results identify a novel mode of immunomodulation in pancreatic neoplasia and suggest that regulatory B cells may have an important role in promoting pro-tumorigenic immune responses. Citation Format: Yuliya Pylayeva-Gupta, Jesse S. Handler, Cristina Hajdu, Dafna Bar-Sagi. The role of B regulatory cells in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B62.

Abstract PR03: Selective sensitization of mutant K-Ras cancer cells to DNA damage based therapies by targeting wild type H- and N-Ras.

Mutations in Kras are highly prevalent in human cancers and are strongly predictive of resistance to chemotherapy. As the constitutive activation of K-Ras has been believed to confer functional autonomy and dominance over the wild type Ras forms, it has been thought that wild type Ras forms are dispensable to the mutant K-Ras driven tumorigenic phenotype. Here we demonstrate a functional dependence of mutant K-Ras driven tumors on wild type Ras for the activation of the DNA damage response, and that this dependence can be exploited to specifically sensitize K-Ras cancers to DNA[[Unable to Display Character: –]]damage-inducing agents. We show that in mutant K-Ras cancer cells, wild type H-Ras and N-Ras are required for the activation of Chk1, the G2 DNA damage checkpoint, and the cellular response to DNA-damage-inducing chemotherapy. Specifically, we show that targeted depletion of wild type H- and N-Ras in mutant K-Ras cancer cells leads to the hyperactivation of the Erk/p90RSK and PI3K/Akt pathways, and as a consequence, the phosphorylation of Chk1 at an inhibitory site, Ser 280. The resulting inhibition of ATR/Chk1 signaling abrogates the activation of the G2 DNA damage checkpoint, and confers a specific sensitization of mutant K-Ras cancer cells to DNA damage therapeutic agents both in vitro and in vivo. Our results demonstrate a previously unappreciated role for wild type Ras in the DNA damage response of mutant K-Ras cancer cells and reveal a promising approach for targeting K-Ras-driven tumors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PR03. Citation Format: Elda Grabocka, Yuliya Pylayeva-Gupta, Eyoel Yemanaberhan, Veronica Lubkov, Laura Taylor, Dafna Bar-Sagi. Selective sensitization of mutant K-Ras cancer cells to DNA damage based therapies by targeting wild type H- and N-Ras. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr PR03.

Abstract A66: KRas-mediated modulation of the immune response in pancreatic neoplasia.

It is becoming increasingly clear that stromal responses elicited by early-stage neoplastic lesions can promote tumor progression towards a more invasive and potentially metastatic state. However, the molecular mechanisms that underlie the early recruitment of stroma, and in particular immune cells, to sites of neoplasia remain poorly understood. To address this question in the context of pancreatic adenocarcinoma, we make use of orthotopic pancreatic grafts of primary murine pancreatic ductal epithelial cells (PDEC) harboring oncogenic KRas (KRasG12D). We find that engrafted KRasG12D-expressing PDEC can elicit a significant immunological response. The nature and composition of immune infiltrates was assessed using FACS sorting as well as immunohistochemical techniques and was directly compared to those derived from p48Cre;LSL-KRasG12D mice. Our observations to date indicate that the expression of KRasG12D promotes the recruitment of immunosuppressive leukocytes in the absence of a notable increase in the cytotoxic T cell population. These results support the hypothesis that effective tumor immunity mediated by an adaptive T cell response is actively suppressed at the very early stage of pancreatic neoplasia. We are currently investigating the molecular mechanisms by which oncogenic KRas modulates the intratumoral immune response. Citation Format: Yuliya Pylayeva-Gupta, Kyoung Eun Lee, George Miller, Dafna Bar-Sagi. KRas-mediated modulation of the immune response in pancreatic neoplasia. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A66.

Abstract A102: Dendritic cells contribute to pancreatic fibroinflammatory disease and the transition to neoplasia.

Introduction: Chronic pancreatitis is the most prevalent risk factor for the development of pancreatic cancer. Dendritic cells (DC) are known to augment inflammation in a range of clinically significant contexts, yet their role in chronic pancreatitis and pancreatic cancer development has not been intensely studied. We postulated a primary role for DC in pancreatic inflammation and the transition to carcinogenesis. Methods: Chronic pancreatitis was induced using caerulein (50µg/kg, 3 weeks) in C57BL/6 mice. The p48Cre;Kras G12D model was used to study pancreatic carcinogenesis. Pancreatic DC populations were expanded by i.p. adoptive transfer of bone marrow-derived DC (1x106 cells, 3X/week) or using Flt3L (10µg/day x 10 days). MyD88 was inhibited in DC using CD11c-Cre MyD88 Floxed +/+ bone marrow chimeric p48Cre;Kras G12D mice. Results: DC accounted for 1-3% of CD45 + leukocytes in normal pancreata, but increased to 10-15% during chronic pancreatitis. Moreover, the absolute number of pancreatic DC increased 100-fold. The number of DC was similarly increased in the pancreata of p48Cre;Kras G12D mice compared with aged matched WT or p48Cre controls. Furthermore, the surface phenotype of DC infiltrating p48Cre;Kras G12D pancreata differed from controls in that they were highly mature, expressing elevated CD40 and CD86. DC adoptive transfer to mice experiencing chronic pancreatitis resulted in severely exacerbated fibro-inflammatory disease, including greater than 60% reduction in acinar cell volume and a marked reduction in islet cell mass. Further, mice treated with Flt3L and simultaneous challenge with caerulein showed exacerbated acinar destruction, fibrosis, and inflammation. In addition to the fibroinflammatory changes, DC-overexpansion in pancreatitis led to widespread development of early PanIN lesions. Overall, approximately 40% of ducts were classified as PanINs in mice adoptively transferred with DC. Moreover, DC markedly accelerated malignant transformation when transferred for four weeks to p48Cre;Kras G12D mice as evidenced by a two-fold increase in tumor weight. Protein analysis revealed that DC transfer resulted in altered pancreatic expression of numerous cell cycle regulatory and tumor suppressor genes including upregulated expression of p21, p27, p-p27, p53, and Rb. DC effects in pancreatic fibroinflammation and carcinogenesis were mediated by induction of Th2-deviated CD4 + T cells. The number of intra-pancreatic CD4 + T cells and CD4:CD8 T cell ratio was markedly increased in mice adoptively transferred with DC. Additionally, intra-pancreatic CD4 + T cells in DC-treated mice exhibited a strong Th2 differentiation. Accentuation of the DCTh2 axis by inhibiting MyD88 signaling in DC further accelerated carcinogenesis in p48Cre;Kras G12D mice. CD4 + T cell depletion, protected p48Cre;Kras G12D mice adoptively transferred with DC from developing accelerated carcinogenesis. Further, adoptive transfer of Th2-deviated CD4 + T cells from pancreatitic mice that had previously been transferred with DC markedly accelerated carcinogenesis. Conclusion: DC expand in chronic pancreatitis and pancreatic cancer. DC over-expansion in chronic pancreatitis exacerbates endocrine and exocrine destruction, induces marked inflammation, and early PanIN lesions. DC expansion in p48Cre;Kras G12D results in marked acceleration of tumor growth. DC effects on pancreatic fibroinflammation and neoplasia are mediated by induction of pancreatic-antigen-restricted Th2 cells. Citation Format: Saman Zarbakhsh, Harry Mushlin, Raghavendra Rao, Sanna Badar, Mohsin Jamal, Dafna Bar-Sagi, George Miller, Adeel Rehman, Christopher Graffeo, Atsuo Ochi, Rocky Barilla, Constantinos Zambirinis, Nina Fallon, Cristina Hajdu, Yuliya Pylayeva-Gupta. Dendritic cells contribute to pancreatic fibroinflammatory disease and the transition to neoplasia. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A102.