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Cancer is increasingly becoming a rising cause of mortality worldwide. Microbiota, both within the gut and tumors, has emerged as a significant player influencing tumor growth and responses to therapies. The tumor niche provides a privileged microenvironment for microbial colonization. Recent evidence links microbiota and pancreatic tumorigenesis. Pancreatic ductal adenocarcinoma (PDAC), is an aggressive cancer surrounded by a highly immuno-suppressive tumor microenvironment (TME) which limits efficacy of most available therapies. We have previously reported that rare long-term survival in pancreatic cancer is associated with an intratumoral microbial signature which correlates with enhanced TME immunoactivation, suggestive of microbial mediated immune cell recruitment. A gut-to-tumor microbial crosstalk was also found in a human-to-mouse Fecal Microbiota Transplant (FMT) tumor model. While we have identified the vital role of microbiota in affecting tumor immunity, there is still a gap of knowledge about local microbial interactions within TME. Deeper understanding of the microbial mediated events that are triggered and follow PDAC development, which act to induce and support tumor growth, would provide us with potential novel targets that could be blocked to reverse PDAC immunosuppression and tumor growth. To interrogate the features of the pancreatic tumor microbial niche, we performed spatial co-detection of microbial and host targets to identify the cellular compartment which interacts with microbes inside the TME in clinical samples. We developed novel qualitative and quantitative imaging methodologies for evaluating crosstalk between the host and microbes. We also evaluated the effect of microbial burden on transcriptomic changes in host cells through multiple spatial sequencing approaches. Overall, we dissect the functional role of microbes in orchestrating TME organization and their effect on tumor signaling. Citation Format: Vidhi Chandra, Hajar Rajaei, Seyda Baydogan, Javier Gomez, Sammy Ferri-Borgogno, Anirban Maitra, Erick Riquelme, Ismet Sahin, Jared Burks, Michael Kim, Florencia McAllister. Interrogating host-intratumoral microbial interactions in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5918.

Purpose: National guidelines recommend universal germline genetic testing (GT) for patients with Pancreatic Ductal Adenocarcinoma (PDAC), but rates of testing remain low. Given the aggressiveness of PDAC, the window of opportunity for GT is short and often overshadowed by treatment initiation and other clinical milestones. Thus, there is an unmet need for a model that streamlines GT and makes it available to a wider audience in a rapid fashion. Moreover, in pandemic times, video-based alternatives for medical care are increasingly relevant. Methods: We implemented a novel care delivery model in which a seven-minute educational video describing the benefits, risks, and implications of GT was shown to PDAC patients. The video was shown in lieu of an initial consult with a genetic counselor. Only patients who had not undergone GT or previously met with a genetic counselor were included. After watching the video, patients could elect to pursue GT and get tested on-side or remotely (at home). Genetic counselors disclosed results and provided post-test counseling by phone. Clinical and germline data were collected through medical records on a cohort of PDAC patients seen at the Gastrointestinal Center-MD Anderson during a 2-year enrollment period (May 2019-July 2021), which included the COVID-19 pandemic period. Results: A total of 286 PDAC patients watched the educational video. From 175 patients that watched the video pre-pandemic, 12 declined testing, whereas in the post-pandemic period, none of the 111 patients declined testing (6.9% vs 0%; p Conclusions: GT can have tremendously beneficial effects, such as qualifying for targeted treatment options and facilitating cancer prevention in probands’ at-risk family members. Comparing uptake of GT pre- versus post-pandemic suggests that patients were more willing to trust information from a video platform, likely due to the global effect of living in a “virtual“ society as a result of the pandemic. We suggest an approach in which every PDAC patient is shown a genetics educational video and given the choice to undergo GT and post-result counseling, greatly reducing the burden on genetic counselors. We report here the feasibility of implementing video-based germline testing in PDAC patients which resulted in unexpectedly high uptake levels, particularly post-pandemic. Further investigations are needed to explore the feasibility of a fully remote GT model in diverse populations to assess additional barriers to universal GT. Citation Format: Saumya Kasliwal, Seyda Baydogan, Devon Harrison, Mark Hurd, Maureen Mork, Anirban Maitra, Florencia McAllister. Video-based germline testing for individuals with pancreatic ductal adenocarcinoma: Influence of COVID-19 pandemic [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 2244.

Pancreatic ductal adenocarcinoma (PDAC) continues to be an aggressive disease with an immunosuppressive tumor microenvironment, which is recalcitrant to most therapies. Interleukin-17 (IL-17), a potent pro-inflammatory cytokine, has been implicated in initiation of pancreatic premalignant lesions but its role in invasive PDAC has not been widely explored. We hypothesized that IL-17 triggers and sustains immunosuppression during PDAC. We employed pharmacological and genetic strategies to inhibit IL-17/IL-17RA signaling axis and characterized the tumor microenvironment with RNA sequencing, mass & flow cytometry and quantitative multiplex immunofluorescence. Our analysis revealed that IL-17 signaled through tumor cells to recruit neutrophils into the tumor microenvironment and spatially exclude cytotoxic CD8 T cells. Furthermore, IL-17 signaling in tumor cells triggered neutrophils to form extracellular traps (NETs), a primary mechanism mediating PDAC immunosuppression. Genetic or pharmacological inhibition of IL-17 overcame resistance to immune checkpoint blockade (PD-1, CTLA-4), in a CD8 T cell dependent manner. Inhibition of neutrophils or Padi4-dependent NETosis phenocopied IL-17 neutralization. Higher expression of IL-17 and PADI4 in human PDAC corresponded with poorer prognosis, according to TCGA data. Serum from PDAC patients had a higher capacity for NET formation and diminished ability for NET degradation in control neutrophils, in comparison to serum from healthy donors. In conclusion, the cross-talk of IL-17 with pancreatic cancer cells orchestrates neutrophil recruitment and activation of Padi4 dependent NETosis, ultimately resulting in spatial remodeling of the stroma and immunosuppression. IL-17 inhibition overcomes resistance to checkpoint blockade in a CD8 T cell dependent fashion. Clinical studies with IL-17 inhibitors and checkpoint blockade represent a promising novel combinatorial therapy for this lethal disease. Citation Format: Vidhi Chandra, Yu Zhang, Erick Riquelme, Prasanta Dutta, Pompeyo R. Quesada, Amanda Rakoski, Michelle Zoltan, Nivedita Arora, Seyda Baydogan, William Horne, Jared Burks, Hanwen Xu, Perwez Hussain, Huamin Wang, Sonal Gupta, Anirban Maitra, Jennifer M. Bailey, Seyed J. Moghaddam, Sulagna Banerjee, Ismet Sahin, Pratip Bhattacharya, Florencia McAllister. Interleukin 17 induced- neutrophil extracellular traps (NETs) mediate resistance to checkpoint blockade in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-014.

Autophagy is a highly complex lysosomal degradation process. Recently we demonstrated that autophagy genes encoding Beclin1, ATG7, LC3 promotes cell proliferation, survival, and invasion by promoting Cyclin D1 and Integrin β1/ Src signaling in triple negative breast cancer (TNBC). Studies showed that increased basal autophagy is linked to development of chemoresistance, relapses and metastasis and poor prognosis in TNBC. However, the major molecular mechanisms and the integrated global regulators controlling the autophagic machinery still remains unknown. Here, we identified miRNA-873 as a p53-driven tumor suppressor that is associated with favorable patient survival (TCGA database) and the key factor for regulating the major autophagy genes, including BCN1, LC3, ATG7, ATG16L1 and ATG13 in TNBC. Using in silico prediction algorithms we demonstrated that miR-873 has binding sites on the 3’-untranslated region (3’-UTR) of these genes and directly binds and suppresses their expression using by gene reporter assays. Introduction of mutations to the miR-873 binding sites on 3-UTR of these genes reversed the inhibitory effect of miR-873on these genes. Furthermore, knockdown of Beclin1, LC3 and ATG7 genes significantly suppressed Eukaryotic Elongation Factor-2 kinase (eEF2K), an unusual alpha kinase, which is highly overexpressed in TNBC patients and associated with poor prognosis. We also found that miR-873 also binds to the 3’-UTR of eEF2K mRNA and regulates its expression and inhibits starvation induced autophagy. Through knockdown and overexpression studies we also demonstrated that eEF2K regulates expression of abovementioned autophagic proteins. Interestingly, we found that BCN1, LC3, and ATG7 also regulates expression of EF2K, suggesting an existence of a novel negative feed-back loop. Lastly, we demonstrated that miR-873 expression is suppressed in TNBC patients and cell lines and restoration of its expression in vivo in MDA-MB-231 and MDA-MB-436 orthotopic xenograft models by systemic injection (I.V, tail vein once a week, 0.15 mg/kg) of miR-873 mimic molecules incorporated in novel single lipid (SLNP)-nanoparticles suppressed tumor growth. Furthermore, in vivo treatment of mice with SLNP-Beclin1, ATG7 or ATG8 siRNAs also completely suppressed TNBC tumor growth. In conclusion, our data suggest that p53/miR-873/eEF2K axis is a novel post-transcriptional regulator of autophagy and miR-873 functions as a master regulator of the post-transcriptional regulation of the major autophagy genes directly and indirectly through eEF2K dependent dual-suppressor mechanism and modulation of this axis could be used as a potential therapy for TNBC. Citation Format: Hamada Ahmed Mokhlis, Nermin Kahraman, Seyda Baydogan, Abdel-Aziz Hamed Abdel-Aziz, Ahmed Ashour, Cristina Ivan, Gabriel Lopez-Berestein, Bulent Ozpolat. MiR-873 is the master regulator of autophagy genes through a novel negative feedback mechanism mediated by Elongation factor 2 kinase (eEF-2K) and suppresses tumor growth and progression of triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4262.

Ovarian cancer, especially high-grade serous ovarian cancer (HGSOC), is the deadliest gynecological cancer with 50-70% 5-year mortality rates. Every year more than 22,000 new cases of ovarian cancer and 15,000 deaths are anticipated within the United States only. Recent clinical and experimental evidence indicates that tumor-associated macrophages (TAMs), the most abundant cells in the tumor microenvironment, play a significant role in tumor growth and progression by contributing to angiogenesis, invasion, metastasis, and drug resistance, leading to poor clinical outcomes and significantly shorter patient survival in HGSOC. More than 50% of cells in the peritoneal tumor microenvironment and malign ascites consist of TAMs in ovarian cancer (OC) patients. Especially, M2 macrophages have been shown to support tumor proliferation and promote tumor progression, angiogenesis, and drug resistance. But the mechanisms of these oncogenic effects are still not clear. The goal of our study to investigate the role of TAM-derived exosomes, which are 30-100nm microvesicles released from cells and are key factors in communication between cancer cells and the tumor microenvironment. To this end, we evaluated differentially expressed miRNAs in high-grade ovarian cancer cells (OVCAR3, OVCAR 432 and OVCAR5) after treatment with exosomes-derived from TAMs (M2 phenotype) using the Affymetrix Gene Chip miRNA 4.0 microarrays. We identified several miRNAs, including miR-6068 that we validated by qPCR and found it to be significantly upregulated in both HGSOC cells and their exosomes. We demonstrated that transfection of HGSOC cells with miR-6068 significantly increased proliferation, migration and invasion capacities of the cells in vitro, suggesting that this miR-6068 act as an oncogenic miR (oncomiR). Using in silico prediction algorithms we found that miR-6068 has binding sites on the 3’-untranslated region (3’-UTR) of PTPN4 gene encoding a phosphatase and demonstrated that it miR-6068 suppresses PTPN4 expression by Western blot and qPCR. Inhibition of PTPN4 by siRNA significantly induced cell proliferation in OC cells, suggesting that PTPN4 acts as a tumor suppressor. In conclusion, our results suggest that miR-6068 has an oncogenic role in ovarian cancer progression by targeting PTPN4 and may be a novel effective therapeutic target for ovarian cancer. Citation Format: Seyda Baydogan, Jianting Sheng, Nermin Kahraman, Pinar Kanlikilicer, Hamada Ahmed Mokhlis, Sayra Dilmac, Stephen T. C. Wong, Bulent Ozpolat. Exosomal transfer of tumor-associated macrophage derived miR-6068 promote ovarian cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3563.