Sarah B. Gitto

Constitutively Active Akt1 Cooperates with KRasG12D to Accelerate In Vivo Pancreatic Tumor Onset and Progression

BACKGROUND AND AIMS: Pancreatic adenocarcinoma is a deadly disease characterized by metastatic progression and resistance to conventional therapeutics. Mutation of KRAS is the most frequent early event in pancreatic tumor progression. AKT isoforms are frequently activated in pancreatic cancer, and reports have implicated hyperactivation of AKT1, as well as AKT2, in pancreatic tumor formation. The objective here is to delineate the role of AKT in facilitating in vivo pancreatic tumor progression in the context of KRAS mutation and predisposition to pancreatic cancer. METHODS: Mice with Akt1 and KRas mutant alleles expressed using the pancreas Pdx promoter were mated to characterize the incidence and frequency of histologic and genetic alterations known to occur commonly in human pancreatic ductal adenocarcinoma. RESULTS: Active Akt1 (Akt1Myr, containing a myristoylation sequence) cooperated with active mutant KRasG12D to accelerate pancreatic carcinoma onset and progression and increase phosphorylation of downstream effectors in the Akt pathway. Mucin and smooth muscle actin expression was found in and around pancreatic intraepithelial neoplasms (PanINs), and accelerated time to metastasis was found in Akt1Myr/KRasG12D mice. CONCLUSIONS: In contrast to prior reports of pancreatic KRas mutant mice mated with mice deficient for various tumor suppressor genes, which resulted in aggressive disease within a few months of age, Akt1Myr/KRasG12D mice enabled the study of PanINs and spontaneous pancreatic transformation more characteristic of human pancreatic progression in elderly individuals. The Akt1Myr/KRasG12D model holds promise for delineating the tumor biology and biomarkers critical for understanding their cooperation in cancer oncogenesis and future targeting in therapeutic strategies.

Anti-ovarian tumor response of donor peripheral blood mononuclear cells is due to infiltrating cytotoxic NK cells.

Treatment of ovarian cancer, a leading cause of gynecological malignancy, has good initial efficacy with surgery and platinum/taxane-based chemotherapy, but poor long-term survival in patients. Inferior long-term prognosis is attributed to intraperitoneal spreading, relapse and ineffective alternate therapies. Adoptive cell therapy is promising for tumor remission, although logistical concerns impede widespread implementation. In this study, healthy PBMCs were used to examine the immune response in a mouse model with human ovarian cancer, where natural killer (NK) cells were found to be the effector cells that elicited an anti-tumor response. Presence of tumor was found to stimulate NK cell expansion in mice treated intraperitoneally with PBMC+Interleukin-2 (IL-2), as compared to no expansion in non-tumor-bearing mice given the same treatment. PBMC+IL-2 treated mice exhibiting NK cell expansion had complete tumor remission. To validate NK cell mediated anti-tumor response, the intratumoral presence of NK cells and their cytotoxicity was confirmed by immunohistochemistry and granzyme activity of NK cells recovered from the tumor. Collectively, this study highlights the significance of NK cell-cytotoxic response to tumor, which may be attributed to interacting immune cell types in the PBMC population, as opposed to clinically used isolated NK cells showing lack of anti-tumor efficacy in ovarian cancer patients.

Difluoromethylornithine Combined with a Polyamine Transport Inhibitor Is Effective against Gemcitabine Resistant Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is highly chemo-resistant and has an extremely poor patient prognosis, with a survival rate at five years of <8%. There remains an urgent need for innovative treatments. Targeting polyamine biosynthesis through inhibition of ornithine decarboxylase with difluoromethylornithine (DFMO) has had mixed clinical success due to tumor escape via an undefined transport system, which imports exogenous polyamines and sustains intracellular polyamine pools. Here, we tested DFMO in combination with a polyamine transport inhibitor (PTI), Trimer44NMe, against Gemcitabine-resistant PDAC cells. DFMO alone and with Trimer44NMe significantly reduced PDAC cell viability by inducing apoptosis or diminishing proliferation. DFMO alone and with Trimer44NMe also inhibited in vivo orthotopic PDAC growth and resulted in decreased c-Myc expression, a readout of polyamine pathway dysfunction. Moreover, dual inhibition significantly prolonged survival of tumor-bearing mice. Collectively, these studies demonstrate that targeting polyamine biosynthesis and import pathways in PDAC can lead to increased survival in pancreatic cancer.

Recent insights into the pathophysiology of mTOR pathway dysregulation

Mechanistic target of rapamycin (mTOR) dysregulation is present in a variety of human pathologies including neurological disease, cancer, diabetes, and cardiac disease. Hyperactivation leads to increased protein synthesis and cell growth, which are essential for growth, development, and cancer. Inhibition of mTOR results in induction of autophagy, a cell survival mechanism thought to be deficient in neurodegeneration. Counteracting the balance of mTOR signaling with target specific inhibitors is of interest in pathological conditions where mTOR signaling is upregulated. The US Food and Drug Administration (FDA) has approved the use of rapamycin for treatment of renal cell, pancreatic neuroendocrine, and hormone positive breast cancer. Many clinical trials are underway to determine the efficacy of mTOR inhibitors in other pathologies as monotherapies or combinational therapies with chemotherapeutics, tyrosine kinase inhibitors, molecular targeted therapies, and vascular endothelial growth factor (VEGF) inhibitors. Collectively, this review is an overview of the current practices and outcomes of pharmaceutically targeting this highly studied mediator of normal and aberrant cell function.

PD-L1 blockade enhances anti-tumor efficacy of NK cells

ABSTRACT Anti-PD-1/anti-PD-L1 therapies have shown success in cancer treatment but responses are limited to ~ 15% of patients with lymphocyte infiltrated, PD-L1 positive tumors. Hence, strategies that increase PD-L1 expression and tumor infiltration should make more patients eligible for PD-1/PD-L1 blockade therapy, thus improving overall outcomes. PD-L1 expression on tumors is induced by IFNγ, a cytokine secreted by NK cells. Therefore, we tested if PM21-particle expanded NK cells (PM21-NK cells) induced expression of PD-L1 on tumors and if anti-PD-L1 treatment enhanced NK cell anti-tumor efficacy in an ovarian cancer model. Studies here showed that PM21-NK cells secrete high amounts of IFNγ and that adoptively transferred PM21-NK cells induce PD-L1 expression on SKOV-3 cells in vivo. The induction of PD-L1 expression on SKOV-3 cells coincided with the presence of regulatory T cells (Tregs) in the abdominal cavity and within tumors. In in vitro experiments, anti-PD-L1 treatment had no direct effect on cytotoxicity or cytokine secretion by predominantly PD-1 negative PM21-NK cells in response to PD-L1+ targets. However, significant improvement of NK cell anti-tumor efficacy was observed in vivo when combined with anti-PD-L1. PD-L1 blockade also resulted in increased in vivo NK cell persistence and retention of their cytotoxic phenotype. These results support the use of anti-PD-L1 in combination with NK cell therapy regardless of initial tumor PD-L1 status and indicate that NK cell therapy would likely augment the applicability of anti-PD-L1 treatment.

Abstract A30: Chronic inflammation induces severe stromal damage and early pancreatic tumors in mice with activated Akt1 and KRas

Inflammation and the immune environment is implicated as a risk for pancreatic ductal adenocarcinoma (PDAC), especially in chronic conditions. However, a mechanistic link is unknown. Recently, radiotherapy followed by immune checkpoint inhibitors are being tested and show promising results, thereby introducing immune modulation as a new investigational area for the treatment of pancreatic cancer. A potential barrier is that pancreatic fibrosis and desmoplastic reactivity are common characteristics of PDAC and makes therapeutic delivery difficult. To define how inflammation and desmoplasia effects the immune-pancreatic stromal environment and progression of PDAC, we have chronically injected caerulein into genetically engineered mice that we previously showed spontaneously develop PDAC through the cooperation of mutant active Akt1 and KRas. We show that injected Akt1Myr/KRasG12D mice have increased tissue remodeling, stellate cell activation, collagen production, and mucin production when compared to KRasG12D mice. They also have increased ductal proliferation, accelerated tumor formation, and increased immune cell infiltration in the pancreas ducts and stroma compared to control PBS- and caerulein-injected KRasG12D mice. Pro-inflammatory cytokines and macrophages likely activate stellate cells, which in turn facilitate additional cytokines to enhance a pro-tumor immune environment. Other than macrophages, the role that innate immune cells contribute to pancreatic cancer has not been well studied. Ongoing studies will delineate the immune cell infiltration, cytokine levels, and the effect on tumor formation in double mutant Akt1Myr/KRasG12D mice. Understanding the mechanisms by which inflammation affects tumor formation can lead to strategies to inhibit pro-tumor changes, impede PDAC development and improve overall patient survival. Citation Format: Sarah B. Gitto, Kathryn A. Cline, Amr S. Khaled, Deborah A. Altomare.{Authors}. Chronic inflammation induces severe stromal damage and early pancreatic tumors in mice with activated Akt1 and KRas. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A30.