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Purpose/Objective(s): Pancreatic ductal adenocarcinoma (PDAC) is characterized by an unusually high degree of hypoxia in the tumor microenvironment, which activates hypoxic signaling pathways orchestrated by hypoxia-inducible factor 1α (HIF1α). It has been shown previously that HIF1α significantly increases PDAC radioresistance and growth, though its mechanism of action is unknown. Here, we test the hypothesis that HIF1α confers these traits in PDAC through involvement in DNA damage repair and KRAS signaling in vitro. Materials/Methods: We performed studies with the KPC cell line (KrasLSL-G12D/+;TP53LSL-R172H/+). We used CRISPR-Cas9, a genome editing tool, to generate a HIF1α knockout (KO) cell line. Hypoxic microenvironments were induced using 100uM of the oxygen scavenger CoCl2. Radioresistance and double-strand DNA damage was evaluated and compared between wild-type (WT) and KO cells after treatment with increasing doses of ionizing radiation using neutral comet assays. Tumor signaling pathways were mapped and analyzed using western blot and co-immunoprecipitation. The KRASG12D inhibitor, BI-2852, was obtained from Boehringer-Ingelheim. Cancer cells were evaluated for growth and migration using cell proliferation assays and scratch wound assays following treatment with 50 uM BI-2852. Bioinformatic analysis was conducted using TCGA (The Cancer Genome Atlas) data; high and low expression were defined as samples with mRNA expression z-scores ≥ 1 and ≤ -1, respectively, Results: HIF1α KO cells demonstrated significantly increased radioresistance compared to WT cells following 8 and 10 Gy of radiation in hypoxic conditions (t-test, P Conclusion: Overall, this study demonstrates that HIF1α promotes DNA damage repair in PDAC and reveals a HIF1α/KRAS/p53 signaling axis. Future studies will test anti-HIF1α and anti-KRAS combination therapies in PDAC mouse models. Citation Format: Kevin J. Tu, Sanjit K. Roy, Amit Sawant, Hem D. Shukla. HIF1α is involved in radiation-induced DNA damage repair and anti-apoptotic pathways in pancreatic ductal adenocarcinoma. [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 4810.

3-Bromopyruvate (3 BP) is a promising anticancer drug against pancreatic cancer. It exerts potent anticancer effects by inhibiting hexokinase II enzyme of glycolysis pathway and ATP generation in cancer cells. In present investigation we have examined the effect of 3-BromoPyruvate employing panc-2 cancer cells, and syngeneic pancreatic cancer mouse model. The clonogenic survival assay demonstrated dose dependent inhibition of cell growth, and we observed 95% cell death at 15 uM conc. Further, ATP production was severely inhibited in cancer cells treated with same concentration of 3-BP. We also observed that 3-BP in combination with low doses of irradiation was more effective in killing cancer cells than 3-BP alone. Electron microscopy revealed that 3-BromoPyruvate severely damaged mitochondrial integrity which might have severely affected ATP generation in cancer cells. We further tested 3-BromoPyruvate (2.5-10.0 mg/kg) in syngeneic panc-2 tumor mouse model and observed that 3-BP at10.0 mg/kg dose significantly reduced tumor growth (volume) about 35-40%. Immunohistochemistry (IHC) data showed that 3-BP inhibits hexokinase II (HK2) and TGFbeta1 and enhanced active caspase-3 expression in tumor tissues as compared to untreated control. The disruption of the interaction between VDAC1 and HK2 due to 3BP treatment was confirmed by CO-IP. Interestingly, 3 BP at higher dose induces caspase-3 activation in tumor derived organoid without showing any effect on normal pancreatic organoid. Altogether, the results in this study demonstrated that 3 BP could be a promising anti-tumor drug against pancreatic cancer and its efficacy could be improved in combination with radiation therapy. Citation Format: Sanjit K. Roy, Tijana Dukic, Binny Bhandary, Aneesh Acharya, Kevin J. Tu, Young H. Ko, Hem D. Shukla. 3-Bromopyruvate in combination with radiation inhibits pancreatic cancer growth by dismantling mitochondria and ATP generation in a preclinical mouse model [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 5243.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth most lethal cancer in the United States with an estimated 60,430 new cases and 48,220 deaths in 2021. PDACs are characterized by extensive desmoplastic stroma and severe hypovascularity, resulting in an intra-tumoral hypoxic microenvironment. In response to hypoxic stress, cancer cells set off many adaptive responses including metabolism regulation, cell survival, and inflammation through the stabilization and activation of the hypoxia-inducible factor (HIF) family of transcription factors. In this study, we used the gene-editing tool CRISPR-cas9 to knock out HIF1A and investigate whether HIF1A affects the radioresistant and invasive characteristics of PDACs in a KPC cell line model. To first observe how HIF1A affects PDAC radioresistance, we performed a clonogenic survival assay with increasing doses of radiation on both wild-type and HIF1A knockout (KO) cells treated and untreated with CoCl2-induced hypoxia conditions (100 uM and 200 uM CoCl2). Our data showed that under hypoxia, KO cells exhibited significant cell death when treated with 6, 8, and 10 Gy of radiation as compared to wild-type KPC cells, emphasizing the role of HIF1A in radiation resistance. In addition, to understand the role of HIF1A in regulating the invasive behavior of PDACs, we performed a cell proliferation assay on wild-type and KO KPC cells. HIF1A KO cells treated with CoCl2 exhibited significantly reduced proliferation compared to wild-type cells also treated with CoCl2 (p0.05). Because western blot demonstrated increased HIF1A expression in wild-type cells following CoCl2 treatment, our results provided evidence for the role of HIF1A activation in promoting PDAC invasiveness. Through western blot, we also confirmed the association between HIF1A expression with p53 degradation in PDAC. We used COREMINE, a literature mining tool, to map a direct interaction between HIF1A with KRAS in PDACs (p=0.000016). We propose HIF1A as a switch to activate KRAS and degrade p53 under hypoxic conditions in PDAC proliferation. Thus, modulating the HIF1A switch may be an important mechanism to reduce the tumor-promoting microenvironment and inhibit cancer growth. Citation Format: Kevin J. Tu, Sanjit K. Roy, Binny Bhandary, Amit Sawant, Hem D. Shukla. Loss of HIF1A decreases resistance to radiation and invasiveness in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-126.

In the US, hepatocellular carcinoma (HCC) incidence has tripled over the past two decades. This trend indicates that social determinants, genetic and environmental factors are driving the epidemic at the population level. Furthermore, the disease has disproportionately affected minority and disadvantaged populations. SATB2 (special AT-rich binding protein-2), a transcription factor and epigenetic regulator, influences gene expression both by modulating chromatin architecture and by functioning as a transcriptional co-factor. SATB2 is highly expressed in embryonic stem cells and progenitor cells, whereas its expression is low or absent in human normal liver tissues. We have recently demonstrated that overexpression of SATB2 can transform normal epithelial cells to cancer stem-like cells in pancreatic, colorectal and breast cancer models, and the expression of SATB2 was significantly higher in cancer tissues compared to normal tissues. The purpose of this study was to examine the expression of SATB2 gene in HCC from AA and CA patients and assess its oncogenic potential by measuring cell viability, epithelial-mesenchymal transition (EMT), stem cell markers and pluripotency maintaining factors in cancer stem cells (CSCs). We compared the expression of SATB2 in primary hepatocytes, Hep3b (from AA), HepG2 (from CA) and HCC CSCs. Hep3b (higher), HepG2 (lower) and CSCs (highest) expressed SATB2 protein, whereas human primary normal hepatocytes did not express SATB2. Knockout of SATB2 in CSCs by Crispr/Cas9 technique significantly inhibited the expression of SATB2 gene, stem cell markers (CD24, CD44 and CD133), pluripotency maintaining factors (cMyc, KLF4, SOX2 and OCT4), and EMT compared to NTC control group. The expression of SATB2 was significantly higher in HCC tissues than adjacent normal controls. Furthermore, the expression of SATB2 was significantly higher in HCC tissues derived from AA compared to those in CA. These data suggest that SATB2 is an oncogenic factor and its expression, which is higher in in HCC tissues derived from AA, may explain the disparity in HCC outcomes among AA. This abstract is also being presented as Poster B049. Citation Format: S.K. Roy, Y. Ma, D.M. Danos, S. Shankar, L. Miele, R.A. Scribner, R.K. Srivastava. Higher expression of SATB2 gene in hepatocellular carcinoma of African American patients determines aggressiveness phenotypes than those in Caucasian Americans [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr PR08.

Colorectal cancer (CRC) mortality rates dropped by half in the past three decades, but CRC continues to demonstrate racial disparities in incidence and survival. It is believed that unfavorable socioeconomic conditions may lead to an inflammatory phenotype. Furthermore, epigenetic events play a major role in regulating inflammatory genes that have been linked to CRC health disparities. The objective of the study was to identify gene(s) that are dysregulated in CRC using The Cancer Genome Atlas (TCGA) database, and, furthermore, to characterize those genetic event(s) that are associated with CRC health disparities. The Engrailed-2 (En2) gene encodes for a homeobox-containing transcription factor regulating embryonic brain development. The data indicate that Nanog, IL-8, and EN2 were upregulated in colorectal cancer. EN2 was highly expressed in CRC stem cells and cell lines but its expression was very low or absent in normal cells. EN2 gene was highly expressed in CRC tissues derived from African American patients compared to those from Caucasian Americans. The expression of EN2 significantly increased with patients' age. There was no difference in EN2 expression between males and females of CRC patients. Higher expression of EN2 was correlated with poor survival of CRC patients with greater IL-8 expression. EN2 may act as a novel prognostic biomarker for CRC. Our data indicate that CRC disparities may be associated with epigenetic factors regulating the inflammatory environment. Citation Format: Y.M. Ma, S.K. Roy, R.K. Srivastava, S. Shankar. Epigenetics and inflammatory environment may contribute to racial disparities in colorectal cancer [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr C066.

Multiple lines of evidence suggest that the Sonic Hedgehog (Shh) signaling pathway is aberrantly reactivated in pancreatic cancer stem cells (CSCs). The objectives of this study were to examine the molecular mechanisms by which GANT-61 (Gli transcription factor inhibitor) regulates stem cell characteristics and tumor growth. Effects of GANT-61 on CSC's viability, spheroid formation, apoptosis, DNA-binding and transcriptional activities, and epithelial-mesenchymal transition (EMT) were measured. Humanized NOD/SCID/IL2Rgammanull mice were used to examine the effects of GANT-61 on CSC's tumor growth. GANT-61 inhibited cell viability, spheroid formation, and Gli-DNA binding and transcriptional activities, and induced apoptosis by activation of caspase-3 and cleavage of Poly-ADP ribose Polymerase (PARP). GANT-61 increased the expression of TRAIL-R1/DR4, TRAIL-R2/DR5 and Fas, and decreased expression of PDGFRα and Bcl-2. GANT-61 also suppressed EMT by up-regulating E-cadherin and inhibiting N-cadherin and transcription factors Snail, Slug and Zeb1. In addition, GANT-61 inhibited pluripotency maintaining factors Nanog, Oct4, Sox-2 and cMyc. Suppression of both Gli1 plus Gli2 by shRNA mimicked the changes in cell viability, spheroid formation, apoptosis and gene expression observed in GANT-61-treated pancreatic CSCs. Furthermore, GANT-61 inhibited CSC tumor growth which was associated with up-regulation of DR4 and DR5 expression, and suppression of Gli1, Gli2, Bcl-2, CCND2 and Zeb1 expression in tumor tissues derived from NOD/SCID IL2Rγ null mice. Our data highlight the importance of Shh pathway for self-renewal and metastasis of pancreatic CSCs, and also suggest Gli as a therapeutic target for pancreatic cancer in eliminating CSCs. Citation Format: Junsheng Fu, Mariana Rodova, Sanjit K. Roy, Sharmila Shankar, Rakesh K. Srivastava. GANT-61 inhibits pancreatic cancer stem cell growth in vitro and in NOD/SCID/IL2R gamma null mice xenograft. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4012. doi:10.1158/1538-7445.AM2013-4012

Our previous studies showed that B cell receptor (BCR) induced apoptosis in lymphoma was preceded by suppression of PI-3K [Carey GB, Scott DW (2001) J. Immunol, 166: 1618-1626]. PI-3K stimulates mTOR, a suppressor of autophagy and mitophagy. In addition, Bcl-XL is known to inhibit BCR-driven apoptosis in lymphoma, although the underlying mechanism is not clear. Therefore the present study examined autophagy in BCR induced apoptosis in lymphoma cells expressing high or low levels of Bcl-XL. WEHI-231 and WEHI-231 cells expressing Bcl-XL (WEHI-231/Bcl-XL) were treated with anti-IgM to cross-link the BCR and to determine the cellular processes leading to apoptosis. The results confirmed dramatic mitochondrial dysfunction followed by apoptosis in parental WEHI-231 compared to WEHI-231/BclXL. BCR-crosslinking resulted in a massive and rapid induction of the autophagic response in parental WEHI-231 compared to those expressing Bcl-XL. Furthermore, treatment with STF-62247, a small molecule inducer of autophagy, resulted in apoptosis in WEHI-231 but not WEHI-231/BclXL cells. These data suggest that BclXL prevents BCR-driven apoptosis in lymphoma via blocking autophagic and mitophagic responses. Hence, combinations of small molecule autophagy inducers and Bcl-XL antagonists may prove to be effective in addressing lymphoma. Supported by NCI Grant UH2CA158689 to G.B.C. Citation Format: Gregory B. Carey, Sanjit K. Roy, Alphius Sesay. Bcl-XL overexpression prevents B Cell receptor driven autophagy in IgM+ lymphoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1728. doi:10.1158/1538-7445.AM2013-1728