Pancreatic cancer is one of the deadliest cancers, due to late diagnosis and very few available therapeutic treatments. The Kras gene is frequently mutated in pancreatic cancer, but previous clinical trials using RAS inhibitors have proven ineffective. Additionally, many drug treatments targeting the MAPK pathway have shown little success due to the inherent drug resistance of pancreatic cancer cells and to acquired resistance through activation of alternative proliferative pathways such as JAK-STAT and PI3K-AKT. Our pathway analysis after MEK inhibition in pancreatic cancer cells displayed sustained and/or upregulation of AKT activity in multiple cell lines. Therefore, we hypothesize that a combination treatment of Trametinib and Omipalisib would block two prominent mitogenic pathways (MEK and AKT, respectively) to suppress cancer cell proliferation and migration. Multiple analyses including Western blot, clonogenic, 5-Ethynyl-2’-Deoxyuridine (EDU), and scratch migration assays were employed to determine the augmentation of cellular function in murine and anthropic pancreatic cancer cell lines after single or combination treatment with Trametinib and/or Omipalisib. Results demonstrated that application of Omipalisib alone was successful at blocking pAKT but failed to suppress pERK. Conversely, Trametinib alone selectively inhibited pERK but did not affect pAKT levels. The combination treatment successfully suppressed both pathways in relatively low quantities, indicating their efficacy as a dual therapeutic. Dual therapy was further effective at inhibiting cell growth as evidenced by clonogenic and EdU assays. Recovery and closure from an in vitro scratch wound was also significantly inhibited with combination of Omipalisib and Trametinib. In vivo studies demonstrated that both the pancreatic xenograft mean tumor growth and final tumor size were significantly reduced in response to the combination treatment compared to vehicle. In conclusion, dual therapy with Omipalisib and Trametinib showed a greater anti-tumor efficacy than treatment with either drug alone. Currently, we are determining the prolonged effectiveness of this combination treatment program in genetically-engineered mouse models. If successful, a pharmacological application of Omipalisib and Trametinib has the potential to provide a beneficial therapeutic option to pancreatic cancer patients. Citation Format: Jarrid Jack, Alexandra Pierce, Bailey Bye, McKinnon Walsh, Prabhakar Chalise, Michael N. VanSaun. Dual MEK and AKT inhibition suppresses pancreatic cancer growth and migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4027.

While the process of antibody generation in response to pathogens and immunization is well understood, the normal antibody repertoire needs characterization to better understand the diversity of the antibody response. The antibody molecule is generated from multiple gene segments; variable (V), diversity (D), and joining (J). These segments rearrange (V, D, and J in the heavy chain, V and J in the light chain) and combine with a constant region, which determines the effector function within in the host. The V, D, and J gene segments are combined to form the complementary determining region 3 (CDR3), providing much of the variability within the B cell receptor repertoire. To address heavy chain rearrangement in the unstimulated antibody repertoire, we pooled and sequenced the total splenic RNA from three groups of normal C57BL/6J. V, D, J, and constant region usage and CDR3 formation were analyzed. We developed a bioinformatic workflow to analyze the most common usage and combinations of the V, D, and J gene segments and their pairings with constant regions. We examined length and composition of CDR3 along with the ontology of the observed antibodies. The preliminary screen identified over 33,000 sequences as possible antibodies; 5,743 were hypothesized to be productive sequences. The largest V-gene family represented was V1, followed by V2 and V6. The average CDR3 length was 11 amino acids and we identified a number of high-occurrence CDR3 amino acid sequences. These data provide a glimpse of the normal C57BL/6J heavy chain response and validate the bioinformatics needed for our future immunization studies. Supported by NASA grants NNX13AN34G and NNX15AB45G and NIH grant GM103418 and the Molecular Biology Core supported by CVM-KSU.

The 5-year overall survival for pancreatic ductal adenocarcinoma (PDAC) patients is an abysmal 12% with local disease and 3% for metastatic disease. Obesity is a major risk factor for PDAC showing a three-month decrease in overall survival when compared to lean PDAC patients. The causal relationship between obesity and cancer has driven seminal findings to support a pro-tumorigenic role of white adipose tissue (WAT). Our previous results agreed with wider based studies demonstrating obese WAT as highly inflamed and eliciting pro-tumorigenic effects, through secretion of adipose derived factors that increase tumor proliferation, migration, and xenograft size. While multiple studies have implicated obese WAT as eliciting pro-tumorigenic effects, the effects of brown adipose tissue (BAT) on PDAC have not been explored. Unlike WAT, the BAT is generally protective against obesity, regulates core body temperature, and supports an anti-inflammatory environment. While regulation of these metabolic effects by BAT are well established, the influence of BAT has on PDAC function remains to be determined. Considering its contradictory role to WAT, we hypothesized that BAT would exhibit anti-tumorigenic effects on pancreatic cancer. To test this hypothesis, we isolated and cultured adipose tissue from lean and obese mice in order to collect perigonadal WAT (pgWAT) and BAT conditioned media (CM). PDAC cells were cultured in the absence or presence of pgWAT-CM or BAT-CM and then assayed for alterations in proliferation. While pgWAT induced proliferation of PDAC cells, we detected a significant inhibition of proliferation by BAT-CM. In order to adapt to environmental demands, WAT can convert into BAT and vice versa. Taking account of this phenomenon, we hypothesized that induction of WAT browning would reduce tumor growth by limiting the release of energy-rich molecules from obese WAT and mimicking BAT. To test our hypothesis, 3T3-L1 pre-adipocytes were differentiated into mature white adipocytes and subsequently “browned” by utilizing Cl-316, 243, trametinib, or roscovitine. From these treatments, we validated that brown-like gene signatures were upregulated while white-like gene signatures were downregulated after the treatments. The adipose conditioned media (ACM) from these “browned” adipocytes was applied to a PDAC cell line and proliferation was measured. Our results demonstrated that the browned ACM inhibited PDAC proliferation when compared to non-treated white ACM. These results support the idea that efficient browning of adipose tissue could be therapeutically beneficial for obese PDAC patients. Currently, we are analyzing the ACM from brown adipose tissue and browned WAT to determine alterations in secreted cytokines. Identifying the adipose derived cytokines involved in promoting PDAC proliferation will potentially provide alternate targets for therapeutics. In conclusion, we have identified an anti-tumorigenic role for BAT that further suggests the predominant subtype of adipose is important in relation to PDAC progression. Citation Format: Austin Eades, Michael VanSaun, McKinnon R. Walsh, Bailey Bye, Appolinaire Olou, Joe Ambrose, Jarrid Jack. Opposing effects of white and brown adipose tissue on PDAC growth [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C081.

CXCR1/2 inhibitors are being implemented with immunotherapies in PDAC clinical trials. Cytokines responsible for stimulating these receptors include CXCL ligands, typically secreted by activated immune cells, fibroblasts, and even adipocytes. Obesity has been linked to poor patient outcome and altered anti-tumor immunity. Adipose-derived cytokines and chemokines have been implicated as potential drivers of tumor cell immune evasion, suggesting a possibility of susceptibility to targeting specifically in the context of obesity. RNA-sequencing of human PDAC cell lines was used to assess differential influences on the cancer cell transcriptome after treatment with conditioned media from peri-pancreatic adipose tissue of lean and obese PDAC patients. The adipose-induced secretome of PDAC cells was then assessed by cytokine arrays and ELISAs. Lentiviral transduction and CRISPR-Cas9 was used to knock out CXCL5 from a murine PDAC cell line for orthotopic tumor studies in diet-induced obese, syngeneic mice. Flow cytometry was used to define the immune profiles of tumors. Anti-PD-1 immune checkpoint blockade therapy was administered to alleviate T cell exhaustion and invoke an immune response, while the mice were monitored at endpoint for differences in tumor size. The chemokine CXCL5 was secreted in response to stimulation of PDAC cells with human adipose conditioned media (hAT-CM). PDAC CXCL5 secretion was induced by either IL-beta or TNF, but neutralization of both was required to limit secretion. Ablation of CXCL5 from tumors promoted an immune phenotype susceptible to PD-1 inhibitor therapy. While application of anti-PD-1 treatment to control tumors failed to alter tumor growth, knockout CXCL5 tumors were diminished. In summary, our findings show that known adipokines TNF and IL-beta can stimulate CXCL5 release from PDAC cells in vitro. In vivo, CXCL5 depletion alone is sufficient to promote T cell infiltration into tumors in an obese setting, but requires checkpoint blockade inhibition to alleviate tumor burden.