Abstract Pancreatic ductal adenocarcinoma (PDAC) is the deadliest malignancy of the pancreas, with an overall five-year survival rate of only 12%. Despite therapeutic advances, effective therapies are still limited. It is now known that many of the DNA damage repair (DDR) genes that are mutated in PDAC are crucial for the proper functioning of the homologous recombination repair (HRR) pathway. Loss-of-function alterations in DDR genes such as BRCA1/2 mutations render PDAC cells vulnerable to certain DNA damage agents, for example, poly (ADP-ribose) polymerase inhibitors (PARPi) and platinum-based agents. Since this treatment strategy is currently limited to only 5-10% of PDAC patients, it underscores a dire need to find new therapeutic avenues and expand the PDAC patient population that could benefit from these therapies. We have recently published that BARD1 (BRCA1- Associated- Ring- Domain- 1), an obligate binding partner of BRCA1 in the process of HRR, is upregulated in PDAC cells exposed to PARPi/platinums. PDAC tissue microarray stained for BARD1 also shows higher BARD1 expression in PDAC tissues compared to normal pancreas. Here, we explored the effects of targeting BARD1 on PDAC cells. For this, we created genetic models of BARD1 inhibition and utilized these model systems to study if loss of BARD1 slows PDAC growth, invasion, and chemo-sensitizes PDAC cells to PARPis and DNA damage agents. Doxycycline inducible shRNA and BARD1 CRISPR KO models were created to inhibit gene expression of BARD1 in PDAC cells. Loss of BARD1 protein and mRNA expression was confirmed by western blot and qRT-PCR analyses. Using colony formation assays, Pico green cell survival assays, and Boyden matrigel invasion assays, we found that inhibition of BARD1 resulted in slower growth and decrease in invasion of pancreatic cancer cells in vitro. The expression levels of EMT markers such as zeb-1 and vimentin, were also decreased. These studies were recapitulated in a mouse xenograft subcutaneous model, where BARD1 CRISPR KO (+/- and -/-) and WT PDAC cells were injected subcutaneously on flanks of mice and tumor growth was monitored over time. Kaplan Meier curves were calculated and plotted using Log-rank test. We found that inhibiting BARD1 significantly (P=0.026) delayed PDAC tumor growth in vivo and extended survival (P<0.001). CRISPR KO of BARD1 also enhanced the efficacies of PARP inhibitors like olaparib, rucaparib and veliparib, and lowered their IC50s (fold changes >10). Our work indicates that targeting BARD1 affects key hallmarks of cancer (growth and invasion), such that the window of therapeutic opportunity is extended, which further allows chemo-sensitization of PDAC cells to DNA damaging agents. Our ongoing studies will explore if BARD1 loss is a predictive marker of PDAC sensitivity to other DNA-damaging agents besides PARPi and allow the development of therapeutic strategies to target BARD1 in PDAC. Citation Format: Aditi Jain, Rutuj P. Kusurkar, Eleanor Jenkins, Avinoam Nevler, Wei Jiang, Charles J. Yeo. Targeting BARD1 in human pancreatic ductal adenocarcinoma: How to expand the target population of vulnerable tumors? [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B006.

Abstract Background: Clinically and histologically identical pancreatic ductal adenocarcinomas (PDACs) differ in response to treatment, disease progression, and survival. Molecular subtyping defines patient populations that differ in terms of prognosis and response to gemcitabine and 5-FU-based chemotherapy. Our goal has been to develop biomarker-based Immunohistochemical protocols that are more accurate than molecular subtyping to guide individualized therapeutic intervention. We focused on two biomarkers highly associated with the basal and classical subtypes, respectively keratin 17 (K17), which promotes tumor growth and chemoresistance, and GATA6, a transcription factor involved in normal pancreatic development that is correlated with longer-term survival in PDAC. Methods: Formalin-fixed, paraffin-embedded (FFPE) tissue specimens (n=120) were selected from the archival collections of Stony Brook University Hospital and Thomas Jefferson University Hospital. Dual localization of K17 and GATA6 was performed by indirect immunoperoxidase methods utilizing Vina Green chromogen for K17 and diaminobenzidine (DAB) for GATA6. The detection of K17 staining in ≥ 10% of tumor cells was scored as high for K17 while the detection of moderate to low intensity staining for GATA6 was scored as low, as described (K. Duan et al. Sci Repts, 2021, 14951). Results: The analysis of IHC-stained PDAC revealed that K17 and GATA6 were heterogeneously expressed, with the strongest GATA6 expression in glandular components and the strongest K17 expression in single cells that diffusely infiltrated the stroma and peripancreatic soft tissue. Furthermore, K17 and GATA6 expression were not mutually exclusive (>50% of cases had high expression of both markers). Correlation of IHC test results in the context of tissue histomorphology revealed that PDAC is highly plastic, with the capacity to transition from well-differentiated to poorly differentiated states that are highlighted by changes in K17 and GATA6 expression. Furthermore, testing for both markers provided greater accuracy in prognostic stratification than testing either marker alone (K17: HR=1.670, p=0.0241; GATA6: HR=0.6755, p=0.0491; K17+GATA6: HR=2.008, p=0.0092). Additionally, high K17 expression, correlated with chemoresistance to gemcitabine, in both low and high-GATA6 cases, indicating that K17 has a dominant negative effect on treatment response. Conclusions: K17 and GATA6 are heterogeneously expressed but not mutually exclusive and highlight the plasticity of diffusely infiltrative versus glandular components of PDAC. Dual color IHC to score both markers provides enhanced prognostic accuracy compared to independent testing for K17 and GATA6. Moreover, high K17 expression overrides GATA6 status to identify the patients that are unlikely to benefit from gemcitabine based chemotherapies, suggesting that K17 may serve as a predictive biomarker to inform treatment selection. Citation Format: Michael Horowitz, Lyanne Oblein, Jaymie Oentoro, Anika Porwal, Wei Jiang, Natalia Marchenko, Luisa Escobar-Hoyos, Kenneth Shroyer. Keratin 17 and GATA6 expression in pancreatic ductal adenocarcinoma: A subtyping stratification approach [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B066.

Abstract Background: Two chemotherapies based on either gemcitabine- or 5-fluorouracil (5-FU) remain standard-of-care for pancreatic ductal adenocarcinomas (PDACs). Patients, however, differ in response and thus many clinical trials for experimental drugs have failed. Therefore, there is an urgent need to establish clinically deployable and rapid biomarkers to decide the best chemotherapeutic regimen for subgroups of patients and prevent toxic side effects. We previously reported that keratin 17 (K17) expression, detected at either the level of messenger RNA or protein by immunohistochemistry (IHC), is as accurate as molecular subtyping to identify subgroups of PDAC patients with shortest survival. In this work, we determined the predictive value of K17 testing for both chemotherapeutic approaches to treat PDAC. Methods: We profiled keratin 17 (K17) expression, a hallmark biomarker of the basal molecular subtype of PDAC, using immunohistochemistry in two cohorts of formalin-fixed paraffin-embedded PDACs (total n=305). These cases were collected through the Know Your Tumor® initiative and three high-volume treating institutions in the U.S. We performed multivariate predictive analyses and determined the threshold to define positive vs negative test. Results: Patients with advance stage disease, low K17, and treated with 5-FU- chemotherapy had three-times longer survival rates compared to counterparts treated with gemcitabine-based chemotherapy. These results contrast to those found in cases with high K17, where no differences were seen with either regimen. Based on RECIST, 72% of low K17 cases responded to treatment compared to 24% of high K17 cases. Predictive value of K17 was independent of mutation status and clinical variables. Conclusions: Our findings indicate that 5-FU-, rather than gemcitabine- based therapies are more likely to extend survival for patient's tumors expressing low rather than high K17. A K17 IHC test rapid test in PDACs has been patented (US11,092,603) and it is currently being tested in prospective phase II clinical trials (NCT04469556, NCT02047474 and NCT03991962). Results from these trials will test the presented findings. Citation Format: Lyanne Oblein, Lucia Roa, Sruthi Babu, Michael Horowitz, Emanuel Petricoin, Lynn M. Matrisian, Edik Blais, Natalia Marchenko, Felicia Allard, Ali Alkalin, Wei Jiang, Brent Larson, Andrew Hendifar, Vincent Picozzi, Minsig Choi, Luisa Escobar-Hoyos, Kenneth Shroyer. Keratin 17 as a predictor of chemotherapy response in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C063.

Abstract Background. Keratin 17 (K17) is a negative prognostic and predictive biomarker, overexpressed in the biologically most aggressive forms of pancreatic ductal adenocarcinoma (PDAC). In other anatomic sites and disease processes, K17 expression impacts the inflammatory microenvironment, correlating with changes in the expression of inflammatory cytokines and the infiltration of CD8+ T cells. Thus, we hypothesized that K17 expression also impacts the immune response in PDAC. Methods. We aimed to determine the relationship between intratumoral and peritumoral immune cell infiltrates versus K17 expression in tumor cells, using brightfield multiplexed immunohistochemistry (mIHC) and our suite of deep learning tools to quantitatively evaluate the expression of four biomarkers of T-cells and macrophages. The study included a multi-institutional cohort of 235 PDAC cases from Stony Brook University Hospital (n=54), Thomas Jefferson University (n=67), Cedars Sinai Medical Center (n=7) and Know Your Tumor PanCAN-Perthera (n=107). Peritumoral immune cells were scored in zones defined as stromal tissue within ≤25 µm of the closest tumor cell. Intratumor immune cells were defined as those that physically touched or overlapped with tumor cells. Immune cell counts in peritumoral and intratumoral K17-negatvie areas of the tumor were normalized to immune cell counts in K17-positive areas. Results. The density of CD8+ T cells in K17-negative peritumoral zones was greater than in K17-positive peritumoral zones in 82.6% of PDACs. Similarly, the density of intratumoral CD8+ T cells in K17-negative areas was greater than in K17-positive areas in 93.2% of cases. Less striking inverse correlations for immune cell density relative to K17 expression were found for CD4+ peritumor lymphocytes and both intratumoral and peritumor CD16+ macrophages. By contrast, CD163+ (tumor-promoting) macrophages were more numerous in K17+ peritumoral and intratumoral zones. All differences were statistically significant and independent of tumor grade, stage, histologic subtype, and neoadjuvant treatment history (p< 0.0001). Conclusions. Correlations between K17 and inflammatory cells suggests K17 expression negatively influences the number and infiltration of T-cells and contributes to the marked immunosuppressive effect of PDAC. These findings suggest that the biologic impact of K17 goes beyond its role as a negative prognostic biomarker that impacts tumor growth, metastasis, and chemoresistance, to suppress the immune response to PDAC. Understanding how K17 influences immune cell function may inform future immunotherapeutic opportunities for PDAC treatment. Citation Format: Lyanne Oblein, Sruthi Babu, Mariana Torrente-Goncalves, Lucia Roa, Michael Horowitz, Mahmudul Hasan, Danielle Fassler, Jaymie Oentoro, Emanuel Petricoin, Lynn M. Matrisian, Natalia Marchenko, Edik Blais, Felicia Allard, Ali Alkalin, Wei Jiang, Brent Larson, Andrew Hendifar, Chao Chen, Shahira Abousamra, Dimitris Samaras, Tahsin Kurc, Joel Saltz, Luisa Escobar-Hoyos, Kenneth Shroyer. Keratin 17 excludes CD8-positive T cells and recruits CD163-positive macrophages in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B041.

7039 Background: Disease relapse and infection cause significant morbidity and mortality after allogeneic HSCT for acute myeloid leukemia (AML) and myelodysplasia (MDS). Wilms’ tumour 1 (WT1) and preferentially expressed antigen in melanoma (PRAME) are both commonly overexpressed in these conditions. We assessed the safety of a novel combination of tumour associated antigen (TAA) and multipathogen (MP) specific T cells administered prophylactically after HSCT in a phase 1 trial. Methods: Patients with overexpression of WT1 or PRAME by ddPCR on diagnostic tumour samples were eligible. TAA and MP specific T cells were ex vivo expanded from stem cell donors by stimulating apheresis derived mononuclear cells with tumour, viral or fungal peptides. T cells specific for CMV, EBV, Adenovirus (AdV) and Aspergillus (Asp) antigens were produced separately and pooled in equal parts into a MP product. Patients received 1 infusion of MP specific and up to 4 infusions of TAA specific T cells at 4-weekly intervals from 28 days post HSCT (cell dose 2x107/m2 per infusion). Results: Ten HSCT recipients have received a total of 38 infusions. Median age was 48 years (17-67), disease AML (n = 6) or high risk MDS (n = 4), DRI intermediate (n = 4) or high (n = 6), conditioning myeloablative (n = 8) or reduced intensity (n = 2), donor source sibling (n = 7) or matched unrelated (n = 3). Median expression of WT1 on diagnostic bone marrow was 1442 copies/104 copies ABL (0-3870), PRAME 131 copies/104 copies ABL (4-12300). Patients received WT1 (n = 4), PRAME (n = 5) or both WT1 and PRAME specific T cells (n = 1). Mean tumour antigen specificity in the TAA product was 1.4% and 13.3% of CD3+ cells for WT1 and PRAME respectively. Mean total pathogen specificity in the MP product was approximately 44% (CMV = 14.0%, EBV = 14.8% and AdV = 11.6% of CD3+ cells, Asp = 14.8% of CD4+ cells). All patients received MP specific T cells. No immediate infusion related adverse events were reported. At a median 540 days post transplant (80-1265), 8 of 10 patients remain alive and in complete disease remission. Two patients did not proceed after completing 3 of 5 infusions due to the development of graft versus host disease (GVHD); both are in remission. There were 2 deaths; one patient with progressive disease who had persistent high risk MDS pre and post HSCT, and one with multiorgan failure who had multiple post transplant complications (venoocclusive disease, sepsis, GVHD), both prior to infusion. Low level viral reactivations occurred (CMV n = 5, EBV n = 7, BKV n = 3, HHV6 n = 4, AdV n = 1), however none required treatment and there were no cases of viral tissue disease or EBV PTLD. There were no invasive fungal infections. Conclusions: Prophylactic infusions of donor derived WT1/PRAME and multipathogen specific T cells post HSCT are well tolerated and associated with low rates of infection and relapse. Clinical trial information: NCT02895412.

We designed a trial to simultaneously address the problems of graft versus host disease (GVHD), infection, and recurrence of malignancy after allogeneic stem cell transplantation. CD34(+) stem cell isolation was used to minimize the development of acute and chronic GVHD. Two prophylactic infusions, one combining donor-derived cytomegalovirus, Epstein-Barr virus, and Aspergillus fumigatus specific T-cells and the other comprising donor-derived CD19 directed chimeric antigen receptor (CAR) bearing T-cells, were given 21-28 days after transplant. Two patients were transplanted for acute lymphoblastic leukemia from HLA identical siblings using standard doses of cyclophosphamide and total body irradiation without antilymphocyte globulin. Patients received no post-transplant immune suppression and were given no pre-CAR T-cell lymphodepletion. Neutrophil and platelet engraftment was prompt. Following adoptive T-cell infusions, there was rapid appearance of antigen-experienced CD8(+) and to a lesser extent CD4(+) T-cells. Tetramer-positive T-cells targeting CMV and EBV appeared rapidly after T-cell infusion and persisted for at least 1 year. CAR T-cell expansion occurred and persisted for up to 3 months. T-cell receptor tracking confirmed the presence of product-derived T-cell clones in blood targeting all three pathogens. Both patients are alive over 3 years post-transplant without evidence of GVHD or disease recurrence. Combining robust donor T-cell depletion with directed T-cell adoptive immunotherapy targeting infectious and malignant antigens permits independent modulation of GVHD, infection, and disease recurrence. The combination may separate GVHD from the graft versus tumor effect, accelerate immune reconstitution, and improve transplant tolerability.

INTRODUCTION:The myelodysplastic syndromes (MDSs) are heterogeneous myeloid malignancies, conventionally diagnosed by cytomorphology and cytogenetics, with an emerging role for flow cytometry. This study compared the performance of a 4-parameter flow cytometry scoring system, the Ogata Score, with other modalities in the diagnosis of MDS.METHODS:Bone marrow aspirate and trephine biopsies from 238 patients performed to assess for possible MDS were analysed, and the flow cytometry score was retrospectively applied. The sensitivity and specificity of the flow cytometry score, the aspirate microscopy, the trephine microscopy with immunohistochemistry, and cytogenetic and molecular results were determined relative to the final diagnosis.RESULTS:The medical records of the 238 patients were reviewed to determine the final clinical diagnosis made at the time of the bone marrow examination. This final diagnosis of MDS, possible MDS or not MDS, was based on clinical features and laboratory tests, including all parameters of the bone marrow investigation, except for the flow cytometry score, which was only determined for this study. The flow cytometry score was 67.4% sensitive and 93.8% specific. Aspirate microscopy had higher sensitivity (83.7%) and similar specificity (92.0%), whereas trephine microscopy had similar sensitivity (66.3%) and specificity (89.4%) to flow cytometry. Although the flow cytometry score had a lower sensitivity than aspirate microscopy, in 18 patients (7.6% of the total) the flow cytometry score was positive for MDS, whereas aspirate microscopy was negative or inconclusive.CONCLUSION:The flow cytometry score and trephine microscopy exhibited reasonable sensitivity and high specificity, and complement aspirate microscopy in the assessment of MDS.

High-throughput single-cell technologies hold the promise of discovering novel cellular relationships with disease. However, analytical workflows constructed for these technologies to associate cell proportions with disease often employ unsupervised clustering techniques that overlook the valuable hierarchical structures that have been used to define cell types. We present treekoR, a framework that empirically recapitulates these structures, facilitating multiple quantifications and comparisons of cell type proportions. Our results from twelve case studies reinforce the importance of quantifying proportions relative to parent populations in the analyses of cytometry data - as failing to do so can lead to missing important biological insights.

Infectious diseases contribute significantly to morbidity and mortality in recipients of allogeneic haematopoietic stem cell transplantation (aHSCT), particularly in the era of highly immunosuppressive transplant regimens and alternate donor transplants. Delayed cellular immune recovery is a major mechanism for the increased risk in these patients. Adoptive cell therapy with ex vivo manipulated pathogen-specific T cells (PSTs) is increasingly taking its place as a treatment strategy using donor-derived or third party–banked cells. The majority of clinical trial data in the form of early-phase studies has been in the prophylaxis or treatment of cytomegalovirus (CMV), Epstein-Barr virus (EBV) and adenovirus (AdV). Advancements in methods to select and enrich PSTs offer the opportunity to target the less common viral pathogens as well as fungi with this technology. Early clinical studies of PSTs targeting polyomaviruses (BK virus and JC virus), human herpesvirus 6 (HHV6), varicella zoster virus (VZV) and Aspergillus spp. have shown promising results in small numbers of patients. Other potential targets include herpes simplex virus (HSV), respiratory viruses and other invasive fungal species. In this review, we describe the burden of disease of this wider spectrum of pathogens, the progress in the development of manufacturing capability, early clinical results and the opportunities and challenges for implementation in the clinic.

Background Administration of partially HLA-matched third party virus-specific T cells (VST) from a cryopreserved cell bank is safe and effective after failure of standard antiviral therapy to resolve viral infection occurring after allogeneic stem cell transplantation (HSCT). Aim In this phase I trial, we assessed the safety and efficacy of administering partially HLA-matched third party VST at the time of initial antiviral therapy following HSCT rather than waiting for failure of at least two weeks of standard antiviral treatment. Methods A cryopreserved cell bank of VST directed at cytomegalovirus (CMV), Epstein Barr virus (EBV) or adenovirus (Adv) was established using G-CSF mobilised peripheral blood from healthy stem cell donors. After stimulation with peptide mixes, VST were selected by expression of CD137+ cells and cultured with cytokines. HSCT recipients were treated with up to 4 doses of 2x107 of VST/m2, the first commencing within 7 days of initial antiviral treatment for viral reactivation. Results A total of 188 doses of VST were manufactured from 7 donors with 12 product manufacturing runs (CMV n=3, EBV n=4 and Adv n=5). Median virus specificity was 75% for CMV, 83% for EBV and 37% for Adv. Thirty HSCT recipients were treated with VST a median of 55 days post-transplant. Data from 25 patients treated for initial viral reactivation were available for analysis (CMV n=22, EBV n=2, Adv n=1). Median age was 58 years (0-71). Patients underwent transplant for myeloid malignancies (n=16), lymphoid malignancies (n=5) and non-malignant conditions (n=4). Patients with malignant disease were transplanted in CR1 (n=8), CR2 (n=3), >CR2 (n=3) or with active disease (n=7). Conditioning was myeloablative in 11 patients and reduced intensity in 14 patients. Donors were matched unrelated (n=20), haploidentical (n=4) or siblings (n=1). 21 patients received some form of T cell depletion (most commonly pre-transplant thymoglobuline in vivo). All patients received VST within 7 days of commencing initial antiviral therapy. 18 patients received a single VST infusion, 6 received 2 and 1 received 4 VST infusions. There were 3 mild infusion related adverse events (vomiting, hypertension, fever). 3 patients had aGVHD pre-infusion (2 grade 1 skin, 1 grade 3 GI). Two patients died of acute GVHD (1 patient with resolved grade 3 GI GVHD pre-VST infusion developed grade 4 GI GVHD 89 days post- infusion as immunosuppression was weaned; the other patient developed de novo liver and GI GVHD 30 days post infusion in the context of a rapid wean in immunosuppression for severe BK virus haemorrhagic cystitis). 2 patients developed de novo grade 1 GVHD post-infusion. 2 patients developed mild limited cGVHD and 1 patient developed extensive cGVHD after VST infusion. 23/25 patients (92%) had complete viral clearance of the infection for which VST were given, 2 had a partial viral response. Median time to best viral response was 20 days. There were 5 deaths (refractory aGVHD in 2 patients, pulmonary VOD/CMV pneumonitis, disease relapse, and sepsis/aspiration pneumonia). 4 of 25 patients died within 12 months of transplant for a 1 year NRM of 12%. At a median follow up of 431 days (112-1391) post-transplant, 20 of 25 patients (80%) remain alive (Figure 1). Conclusion Infusion of third party partially HLA-matched donor-derived VST at the time of first antiviral treatment for CMV, EBV and Adv post HSCT is associated with minimal infusion toxicity, a low rate of moderate to severe GVHD and complete viral clearance in 92% of recipients. Overall survival in this group of high-risk patients requiring treatment for viral reactivation after HSCT is high. A randomised trial will be performed to determine whether administration of third-party VST in addition to standard anti-viral treatment improves transplant outcomes. Figure 1 Disclosures Gottlieb: Haemalogix P/L: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy; Gilead: Consultancy; AbbVie: Consultancy; University of Sydney: Employment; Merck: Consultancy. Ritchie:Amgen: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy; BMS: Research Funding; Takeda: Research Funding; Beigene: Research Funding; Imago: Research Funding; Novartis: Honoraria; Sanofi: Honoraria.

Previously, it has been shown that pancreatic ductal adenocarcinoma (PDA) tumors exhibit high levels of hypoxia, characterized by low oxygen pressure (pO2) and decreased O2 intracellular perfusion. Chronic hypoxia is strongly associated with resistance to cytotoxic chemotherapy and chemoradiation in an understudied phenomenon known as hypoxia-induced chemoresistance. The hypoxia-inducible, pro-oncogenic, serine-threonine kinase PIM1 (Proviral Integration site for Moloney murine leukemia virus 1) has emerged as a key regulator of hypoxia-induced chemoresistance in PDA and other cancers. Although its role in therapeutic resistance has been described previously, the molecular mechanism behind PIM1 overexpression in PDA is unknown. Here, we demonstrate that cis-acting AU-rich elements (ARE) present within a 38-base pair region of the PIM1 mRNA 3'-untranslated region mediate a regulatory interaction with the mRNA stability factor HuR (Hu antigen R) in the context of tumor hypoxia. Predominantly expressed in the nucleus in PDA cells, HuR translocates to the cytoplasm in response to hypoxic stress and stabilizes the PIM1 mRNA transcript, resulting in PIM1 protein overexpression. A reverse-phase protein array revealed that HuR-mediated regulation of PIM1 protects cells from hypoxic stress through phosphorylation and inactivation of the apoptotic effector BAD and activation of MEK1/2. Importantly, pharmacological inhibition of HuR by MS-444 inhibits HuR homodimerization and its cytoplasmic translocation, abrogates hypoxia-induced PIM1 overexpression and markedly enhances PDA cell sensitivity to oxaliplatin and 5-fluorouracil under physiologic low oxygen conditions. Taken together, these results support the notion that HuR has prosurvival properties in PDA cells by enabling them with growth advantages in stressful tumor microenvironment niches. Accordingly, these studies provide evidence that therapeutic disruption of HuR's regulation of PIM1 may be a key strategy in breaking an elusive chemotherapeutic resistance mechanism acquired by PDA cells that reside in hypoxic PDA microenvironments.