Abstract B022: Reprogramming Apoptotic Resistance in PDAC Through Synthetic Oncolytic Immunotherapy

Pancreatic stellate cells (PSCs) are the main sources of cancer-associated fibroblasts in pancreatic ductal adenocarcinoma (PDAC), which account for at least 50% of PDAC’s total tumor volume and its dense desmoplastic stroma. In recent years, immune-stromal cell interactions have been the subject of research in a variety of diseases. Interestingly, natural killer (NK) cells are relatively understudied in PDAC and may be exploited to identify new treatment opportunities. We have explored how NK cell-PSC interactions are mediated by known NK cell ligands. Human PSC (hPSC, CRC-811) or PDAC (MIA-PaCa-2, PANC-1) cell lines were co-cultured with DiO stained NK cells (NK-92, donor NK cells) for 4 hours, then target cells were assessed for apoptosis by flow cytometry using AnnexinV and Sytox stains. We found that although NK cells lyse both PSCs and PDAC cells, there was more NK cell-mediated lysis against PSCs. Using flow cytometry, we found that PSCs express the well-known MICA/B ligand, which binds the activating NKG2D receptor on NK cells to initiate its cytotoxic activity. In co-culture, neutralizing MICA/B in vitro did not completely inhibit NK cell-mediated lysis of PSCs, suggesting other interactions need to be explored. To identify other molecular determinants, NK cell ligand and receptor expression was evaluated by quantitative PCR in human PSC, PDAC, NK, and other leukocytic (K562) cell lines. Cell surface validation of highly expressed NK cell ligands and receptors were confirmed by flow cytometry and/or Western blot. NK cell ligands that were both highly expressed and localized to the cell surface were: NKG2D ligands—MICA and ULBP2 (both activating), KIR2DS4 (CD158I) ligand—HLA-A (activating), and NKp44 ligands—NKp44L and PCNA (activating and inhibitory, respectively). Their corresponding receptors were also expressed and localized to the cell surface by NK cells. These results highlight novel NK cell-PSC interactions that may mediate immune suppression in PDAC. These findings warrant further investigation. Citation Format: Zoe X. Malchiodi, Allison Fitzgerald, Elena Galvano, Louis M. Weiner. Identification of molecules mediating natural killer cell-pancreatic stellate cell interactions [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 6169.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, primarily due to intrinsic or acquired resistance to chemotherapy, such as Gemcitabine (Gem). Naturally occurring botanicals, including Andrographis (Andro), can help enhance the anti-tumorigenic therapeutic efficacy of conventional chemotherapy through time-tested safety and cost-effectiveness. Accordingly, we hypothesized that Andro might reverse Gem resistance in PDAC. The critical regulatory pathways associated with Gem resistance in PDAC were identified by analyzing publicly available transcriptomic profiling and PDAC tissue specimens. A series of systematic in vitro experiments were performed using Gem-resistant (Gem-R) PDAC cells and patient-derived 3D-organoids to evaluate the Andro-mediated reversal of Gem resistance in PDAC. Transcriptomic profiling identified the calcium signaling pathway as a critical regulator of Gem-resistance (Fold enrichment: 2.8, p = 0.002). Within this pathway, high ERBB3 expression was significantly associated with poor prognosis in PDAC patients. The combination of Andro and Gem exhibited superior anti-cancer potential in Gem-R PDAC cells through potentiating cellular apoptosis. The combined treatment down-regulated ERBB3 and decreased intracellular calcium concentration in Gem-R PDAC cells. Finally, these findings were successfully interrogated in patient-derived 3D-organoids. In conclusion, we demonstrate novel evidence for Andro-mediated reversal of chemoresistance to Gem in PDAC cells through the regulation of ERBB3 and calcium signaling.

Emerging evidence has demonstrated that radiotherapy (RT) can not only cause direct damage to cancer cells but also lead to immunogenic cell death (ICD), which involves the activation of host antitumor immune response in tumor immune microenvironment (TIME). RT-induced ICD comprises the release of damage-associated molecular patterns (DAMPs) from dying cancer cells that result in the activation of tumor-specific immunity to elicit long-term antitumor efficacy in both original and abscopal tumor sites. Adenosine triphosphate (ATP), as an important DAMP released by irradiated cancer cells and an essential factor within purinergic pathway, can be further hydrolyzed to adenosine (ADO) by two key ectonucleotidases, CD39 and CD73, to further modulate the antitumor immunity in TIME through purinergic signaling via the interaction to its specific receptors such as adenosine 2A receptor (A2AR) and A2BR widely expressed on the surface of the components in TIME, including cancer cells and many immune effector cells. In this review, we first introduced key components in purinergic pathway including ATP, ADO, their receptors, and essential ectonucleotidases. Then we reviewed the regulation of ATP and ADO levels and their main mechanisms by which they promote tumor growth and broadly suppress antitumor immunity through inhibiting the pro-inflammatory response of dendritic cells, cytotoxic T lymphocytes, and natural killer cells, while improving the anti-inflammatory response of regulatory T cells, macrophages, and myeloid-derived suppressor cells in TIME, especially after irradiation. Finally, we presented an overview of dozens of promising therapeutics including pharmacological antagonists and specific antibodies targeting ADO receptors and ectonucleotidases CD39 or CD73 investigated in the clinic for cancer treatment, especially focusing on the preclinical studies and clinical trials being explored for blocking the purinergic signaling to enhance RT as a combination antitumor therapeutic strategy, which has a robust potential to be translated to the clinic in the future.

Pancreatic ductal adenocarcinoma (PDAC) is characterised by a dismal prognosis and insensitivity to immune checkpoint blockers (ICBs); however, the underlying mechanism remains elusive. This study aimed to identify tumour cell-intrinsic regulators that promote immune evasion and ICB resistance in PDAC. Multi-omics analysis and clinical cohort studies identified protein disulfide isomerase family A member 6 (PDIA6) as a regulator of the immune microenvironment. Flow cytometry, multiplex immunohistochemistry, electron microscopy and Glutathione S-Transferase (GST) pulldown assays confirmed that PDIA6 repressed PRKR-like endoplasmic reticulum kinase (PERK) activation and immunogenic cell death (ICD). Chromatin immunoprecipitation confirmed that KRASG12D and YY1 modulated PDIA6 transcription. LSL-Kras G12D/+ ;LSL-Trp53 R172H/+ ;Pdx-1-Cre (KPC) mouse models showed that PDIA6 inhibition improved ICB response. Multi-omics screening identified PDIA6 as a biomarker of CD8+ T-cell paucity and poor prognosis in patients with PDAC. High PDIA6 levels predicted poor ICB response in the PDAC cohorts. PDIA6 inhibition reprogrammed the immunosuppressive tumour microenvironment and hindered mouse PDAC growth in the presence of CD8+ T-cell, which is attributed to enhanced ICD. PDIA6 interacted with cysteine 453 of PERK, abrogating the disulphide bond-mediated dimerisation and activation of PERK, an ICD inducer. Oncogenic KRASG12D potently upregulated PDIA6 via YY1-mediated transcriptional activation. We identified a small-molecule inhibitor of PDIA6, PACMA31, and demonstrated that targeting PDIA6 with PACMA31 improved ICB efficacy in a PDAC mouse model with KRAS mutations. PDIA6, driven by KRASG12D, alleviates ICD and promotes immune evasion, functioning as a predictive biomarker to screen ICB-sensitive patients and a therapeutic target to improve ICB efficacy in PDAC with KRAS mutations.

Cancer is the major cause of death besides cardiovascular disease. Leukaemia represents the most prevalent malignancy in children with a frequency of 30 % and is one of the ten leading types of cancer in adults. Philadelphia Chromosome-positive B-ALL (Ph+ B-ALL) is driven by the cytogenetic aberration of the reciprocal chromosomal translocation t(9;22)(q34;q11) leading to the formation of the Philadelphia chromosome with a BCR-ABL1 fusion gene. This fusion gene encodes a BCR-ABL1 oncoprotein which is characterized by a constitutively enhanced tyrosine kinase activity promoting amplified proliferation, differentiation arrest and resistance to cell death. Ph+ B-ALL is considered the most aggressive ALL subtype with a long-term survival rate in the range of only 30 % despite intensive standard of care including chemotherapy in combination with a tyrosine kinase inhibitor (TKI) followed by allogeneic stem cell transplantation after remission for clinically fit patients. The efficacy of chemotherapy has long been mainly attributed to tumour cell toxicity while immune modulating effects have been overlooked, especially in light of known immunosuppressive properties. Accumulative evidence, however, emphasizes the ability of chemotherapeutic agents, including TKIs, to normalise or re-educate a dysfunctional tumour microenvironment (TME) resulting in enhanced anti-tumour immunity. One of the underlying mechanisms of immune modulation is the induction of immunogenic cell death (ICD). ICD is an anti-tumour agent-induced cell death modality determined by the capacity to convert cancer cells into anti-cancer vaccines. The induction of ICD relies on the release of damage-associated molecular patterns (DAMPs) from dying tumour cells succumbing to ICD. Translocation of CALR to the cell surface, extracellular secretion of ATP and release of HMGB1 from the nucleus are key hallmarks of ICD that mediate anti-tumour immunity upon binding to antigen presenting cells resulting in a tumour antigen-specific immune response. Besides these molecular determinants, ICD is functionally defined by the inhibition of tumour growth in a vaccination assay in which mice are injected with tumour cells exposed to the potential ICD inducer in-vitro and then re-challenged with live tumour cells of the same cancer type. Both molecular and functional criteria determine the gold standard approach to assess ICD. By increasing the immunogenicity of cancer cells, ICD contributes to the restoration of immunosurveillance as an essential feature of tumour rejection, which is clinically reflected by improved therapeutic efficacy and disease outcome in patients. Therefore, identifying novel ICD inducers is an objective of interest in the context of cancer therapy. In respect of these considerations, the aim addressed in the present work is the examination of the second-generation TKI Nilotinib for the ability to induce ICD. The thesis is set in the context of the group's research on the role of Gas6/TAM signalling within the TME regarding the pathogenesis of acute leukaemia. In in-vivo experiments of our research group it has been consistently observed that the use of Nilotinib enhances the anti-leukaemic immunity mediated by a deletion of Gas6. Against the background of increasing importance of chemotherapeutic agents as potent modulators of a dysregulated TME, it was hypothesized that Nilotinib may synergize with a Gas6-deficient environment by inducing ICD in Ph+ B-ALL cells. In growth inhibition and Annexin V/Propidium iodide cell death assays Nilotinib was shown to induce cell death in concentration-dependent manner that occurs bimodally in terms of cell death modality ranging between apoptosis and necrosis. By ICD marker analysis, comprising flow-cytometric detection of CALR exposure, chemoluminescence-based ATP measurement and immunoblotting for HMGB1, it was found that Nilotinib-induced cell death is not accompanied by CALR exposure and ATP secretion, but is associated with the release of HMGB1. In macrophages co-culture experiments with Nilotinib-treated leukaemic cells, no relevant shift in terms of macrophages activation and polarisation was observed in either a juxtacrine or paracrine setup. In consistency with the results obtained in the in-vitro experiments, Nilotinib was not potent to elicit a protective immune response in mice within a vaccination assay. Conclusively, Nilotinib was identified to not qualify as bona fide ICD inducer. The role of Nilotinib-induced cell death and HMGB1 release are proposed as objective for further investigation concerning the synergistic interplay between Nilotinib and a Gas6-deficient environment. Efforts addressing exploration and optimisation of the immunological potential of chemotherapeutic agents are a promising approach aimed at providing cancer patients with the best possible treatment in future.

Ferroptosis has emerged as a critical mechanism in overcoming drug resistance in various tumors; however, its role in gemcitabine-resistant pancreatic ductal adenocarcinoma (PDAC) has not been thoroughly explored. Additionally, the effectiveness of ferroptosis in many cancer therapies has been limited. In our study, we found that GPX4 is upregulated in gemcitabine-resistant (GR) PDAC patient tissues and cell lines. Notably, inhibiting GPX4 can reverse gemcitabine resistance in PDAC through the induction of ferroptosis, highlighting it as a potential therapeutic target. To address this, we developed a novel nanomedicine, GSH-depleted and ferroptosis-induced (FiN), designed to encapsulate and deliver the GPX4 inhibitor RSL3, resulting in a dual ferroptosis-induced nanomedicine (DFiN). In a PDAC patient-derived xenograft (PDX) mouse model exhibiting gemcitabine resistance, DFiN demonstrated significant accumulation in GR PDAC tumors, leading to a reduction in intracellular GSH levels and inhibition of GPX4. This dual action promoted lipid peroxidation and synergistically induced ferroptosis, effectively reversing gemcitabine resistance. Both in vitro and in vivo experiments confirmed that DFiN is effective in overcoming gemcitabine resistance in GR PDAC cells and PDX tumors, with minimal toxicity observed. This study presents a promising strategy for addressing drug resistance in cancer therapy.

Decision letter: Neutrophil-mediated fibroblast-tumor cell il-6/stat-3 signaling underlies the association between neutrophil-to-lymphocyte ratio dynamics and chemotherapy response in localized pancreatic cancer: A hybrid clinical-preclinical study

Resistance to standard therapy remains a challenge in treatment of pancreatic ductal adenocarcinoma (PDAC) resulting in need for novel therapeutic strategies. Anti-VEGF therapy with r84 delays PDAC progression; however, chronic hypoxia ultimately results in transition to a mesenchymal phenotype and rapid progression at a later timepoint. COX-2 inhibition with apricoxib, a novel antagonist in Phase II trials, reverses EMT in PDAC cells. We investigate the relationship between COX-2 and VEGF production in PDAC cell lines to evaluate the efficacy of a combination strategy in preclinical models of PDAC. In vitro, changes in VEGF production by PDAC cells following apricoxib were assessed by ELISA at baseline and following forced induction of EMT. The effect of r84, apricoxib, or combination on tumor growth and metastatic incidence was determined in SCID mice with established orthotopic pancreatic xenografts. Improvement of survival and reduction in tumor burden was assessed in a murine genetic model of PDAC using p48-Cre; KrasG12D;Cdkn2alox/lox mice, tissue was collected at 4 weeks of therapy with control antibody, r84 or apricoxib for analysis. PDAC cell lines grown in the presence of TGF-β and collagen demonstrated increased Zeb1 and decreased ECAD expression by Western blot. Treatment with clinically-achievable doses of apricoxib reversed this change in phenotype. High expression of COX-2 correlated with high levels of VEGF in conditioned media in human PDAC cell lines. VEGF production was initially sensitive to COX-2 inhibition, with complete depletion of VEGF in the first 6 hours, but VEGF levels returned to baseline by 16 hours and continued to rise throughout the first 72 hours post apricoxib. Upon repeat dosing at 24 hours, VEGF production had become independent of COX-2 and no changes were observed. Similar results were seen following induction of EMT, however, baseline VEGF production was dramatically increased, and while there was an initial decrease after apricoxib, production remained more than 2-fold higher than in cells under normal culture conditions. In vivo, apricoxib and r84 had minimal effect on primary tumor growth as single agents; however each agent resulted in a reduction of metastatic incidence. Combination therapy reduced primary tumor size and virtually eliminated metastases. Overall survival was significantly improved with apricoxib as a single agent. Apricoxib treated animals also demonstrated a delay in progression of disease and decreased collagen deposition in the microenvironment compared to r84, which increased collagen deposition.We conclude that COX-2 inhibition by apricoxib delays progression of PDAC and results in mesenchymal to epithelial transition. Combination with anti-VEGF therapy results in potent antitumor and antimetastatic effects in our models and warrants further evaluation as a strategy to augment chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3819. doi:1538-7445.AM2012-3819

Pancreatic cancer (PC) remains one of the most challenging cancers and has the worst prognosis. Tumor-associated MUC1 (tMUC1) is overexpressed and aberrantly glycosylated in over 80% of human pancreatic ductal adenocarcinoma (PDA). Chimeric antigen receptor (CAR) engineered T cells are an emerging cancer immunotherapy strategy and recently, we successfully engineered tMUC1-specific human and mouse CAR T cells and demonstrated their effectiveness as monotherapy against PDA in vitro and in vivo. In this study, we observed varying sensitivity among human PDA cell lines in response to tMUC1-targeted CAR T cell cytolysis. Notably, highly resistant HPAFII cells released greater amounts of interferon (IFN)-regulated ICAM-1, CXCL10, and CXCL11 compared to the more sensitive MiaPaCa-2 cells following CAR T cell challenge. Blocking IFN signaling using Ruxolitinib, a JAK1/2 inhibitor (JAKi), significantly reduced the upregulation of ICAM-1 and CXCL10. Western blot analysis revealed that both type I and type II IFN signaling pathways were elevated in PDA cells upon CAR T cell treatment. JAKi effectively suppressed this signaling response, with a more pronounced impact on the type I IFN pathway. Importantly, both IFN blockade and transient knockdown of IFN receptors significantly enhanced the sensitivity of PDA cells to CAR T cell-mediated cytolysis in vitro. Further mechanistic study revealed that CAR T cells partially lose their cytolytic potential after engaging with PDA cells. Treatment with CAR T cells triggered the up-regulation of immune checkpoint PD-L1 expression on PDA cells via tumor cell’ own IFN signaling. Thus, blocking PD-L1 in HPAFII enhanced its response to CAR T cells. Similarly, neutralizing CXCL10 enhanced CAR T cell killing of HPAFII cells suggested CXCL10’s involvement in resistance to CAR T cell cytolysis. RNA-seq data indicated higher expression of multiple genes along the IFN signaling pathway which were associated with poor prognosis in PDA patients. Taken together, tumor intrinsic IFN signaling may drive immune evasion in PDA cells against tMUC1-targeted T cell-mediated immunotherapy. This identifies tumor IFN signaling as a potential therapeutic target to improve CAR T cell efficacy in PDA treatment.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the sixth leading cause of death worldwide. PDAC carries a 5-y survival of less than 10%, as it is often diagnosed at a late stage and is widely refractory to available therapies. PDAC tumors are hypoperfused, resulting in poor nutrient delivery. To exist in this hostile microenvironment, PDAC cells rely on intracellular and extracellular scavenging pathways to acquire metabolic substrates for growth. Autophagy and other lysosome-dependent recycling pathways are aberrantly regulated in PDAC. Although autophagy modulation is an emerging therapeutic strategy for PDAC, the co-dependences induced by lysosomal inhibition have not been systematically explored. Cationic amphiphilic drugs (CADs) accumulate in lysosomes, destabilize their membranes, and can have anti-tumorigenic effects. CADs include hundreds of pharmacologic agents used to treat a broad spectrum of common diseases. The aim of this study was to investigate anti-PDAC activity of several clinically-approved and newly synthesized CADs. Material and methods: Imidazoline, quinoline, and chrysene derivatives were examined. Anti-tumor activity of CADs was evaluated in human PDAC cell lines in vitro and in Tg(fli1:EGFP) zebrafish model in vivo. Effects on mitochondria, lysosomes, and autophagy flux were examined by immunofluorescent microscopy. Levels of reactive oxygen species (ROS), changes in the mitochondrial membrane potential, and induction of apoptosis by the selected CADs were evaluated by flow cytometry. Results: The newly synthesized derivatives of quinoline and chrysene induced apoptosis in PDAC cells in vitro in the 2–10μM range, while the only FDA-approved imidazoline with apoptotic activity was rilmenidine, at doses higher than 100μM. Most of the tested compounds induced the expansion of the acidic compartment, as measured by acridine orange and LAMP1 staining, and modulated autophagy, as seen by LC3 staining intensity and distribution. Mitochondrial oxidative stress was induced by the tested CADs as well as the dissipation of the mitochondrial membrane potential (ΔΨm). Ultimately, both newly synthesized CADs and rilmenidine limited the growth and spread of PANC1 cancer cells in the Tg(fli1:EGFP) zebrafish model. Conclusion: Quinoline and chrysene CADs had the potential for a dual lysosome/mitochondrion targeting in PDAC cells and were inducing apoptosis almost in a nM range. While rilmenidine was less potent than newly synthetized CADs in inducing cell death in vitro, all the tested compounds had significant anti-tumor effects in the zebrafish model. These results imply that CADs have promising anti-PDAC effects and that effects on the other cells in the tumor microenvironment other than the cancer cells themselves have to be examined. No conflict of interest.

Advances in Immunogenic Cell Death for Cancer Immunotherapy

Immunogenic cell death (ICD) is a type of cancer cell death triggered by certain chemotherapeutic drugs, oncolytic viruses, physicochemical therapies, photodynamic therapy, and radiotherapy. It involves the activation of the immune system against cancer in immunocompetent hosts. ICD comprises the release of damage-associated molecular patterns (DAMPs) from dying tumor cells that result in the activation of tumor-specific immune responses, thus eliciting long-term efficacy of anticancer drugs by combining direct cancer cell killing and antitumor immunity. Remarkably, subcutaneous injection of dying tumor cells undergoing ICD has been shown to provoke anticancer vaccine effects invivo. DAMPs include the cell surface exposure of calreticulin (CRT) and heat-shock proteins (HSP70 and HSP90), extracellular release of adenosine triphosphate (ATP), high-mobility group box-1 (HMGB1), type I IFNs and members of the IL-1 cytokine family. In this review, we discuss the cell death modalities connected to ICD, the DAMPs exposed during ICD, and the mechanism by which they activate the immune system. Finally, we discuss the therapeutic potential and challenges of harnessing ICD in cancer immunotherapy.

<div>Abstract<p>A hallmark of pancreatic ductal adenocarcinoma (PDAC) is the presence of a dense, desmoplastic stroma and the consequent altered interactions between cancer cells and their surrounding tumor microenvironment (TME) that promote disease progression, metastasis, and chemoresistance. We have previously shown that IL6 secreted from pancreatic stellate cells (PSC) stimulates the activation of STAT3 signaling in tumor cells, an established mechanism of therapeutic resistance in PDAC. We have now identified the tumor cell–derived cytokine IL1α as an upstream mediator of IL6 release from PSCs that is involved in STAT3 activation within the TME. Herein, we show that IL1α is overexpressed in both murine and human PDAC tumors and engages with its cognate receptor IL1R1, which is strongly expressed on stromal cells. Further, we show that IL1R1 inhibition using anakinra (recombinant IL1 receptor antagonist) significantly reduces stromal-derived IL6, thereby suppressing IL6-dependent STAT3 activation in human PDAC cell lines. Anakinra treatment results in significant reduction in IL6 and activated STAT3 levels in pancreatic tumors from <i>Ptf1a<sup>Cre/+</sup>;LSL-Kras<sup>G12D/+</sup>; Tgfbr2<sup>flox/flox</sup></i> (PKT) mice. Additionally, the combination of anakinra with cytotoxic chemotherapy significantly extends overall survival compared with vehicle treatment or anakinra monotherapy in this aggressive genetic mouse model of PDAC. These data highlight the importance of IL1 in mediating tumor–stromal IL6/STAT3 cross-talk in the TME and provide a preclinical rationale for targeting IL1 signaling as a therapeutic strategy in PDAC.</p></div>

Introduction: We explored HuR's (an RNA binding protein) significance in the DNA damage response (DDR) in pancreatic ductal adenocarcinoma (PDA) cells. Experimental Procedures: We treated PDA cells with DNA damaging agent's mitomycin C (MMC), oxaliplatin, cisplatin, carboplatin, gemcitabine and a PARP inhibitor (ABT-888). HuR subcellular localization was assessed by immunoblotting. Additionally, we measured γH2AX foci, a marker of DNA damage, by immunofluorescence. Drug sensitivity assays were performed in isogenic PDA cells that differed in HuR expression (siRNA oligos against HuR, cDNAs encoding HuR for overexpression, and appropriate controls). A BrdU/PI pulse chase experiment monitored the cell cycle progression after treating cells with low (150nM) and high (1μM) doses of MMC. Ribonucleoprotein-immunoprecipitation (RIP) assays were used to isolate mRNAs bound to HuR to identify HuR targets in the DDR. Results: Enhanced-HuR cytoplasmic protein expression was observed in PDA cells exposed to all DNA damaging agents. Under high doses of certain DNA damaging agents (e.g., MMC) HuR was cleaved in a caspase-dependent manner. To assess HuR's role in the DDR, isogenic PDA cells lines manipulated with HuR levels were treated with MMC (150nM) for 2 hrs. γH2AX foci assay revealed a 3-fold increase in foci number with MMC treatment in the HuR depleted cells, as compared to the control cells, suggesting that DNA repair was delayed in the absence of HuR. Cell cycle analyses demonstrated that HuR-depleted PDA cells progressed through the G2/M checkpoint and into mitosis upon MMC treatment. A combinatorial approach with RIP-sequencing and an siRNA library screen for genes resistant to DNA damaging agents was performed, and WEE1, a mitotic inhibitor kinase, was identified as a novel HuR target. A separate RIP-qPCR experiment validated WEE1 as an HuR binder with 56-fold more expression upon MMC stress, compared to an IgG control-IP. WEE1 protein expression and Cdk1 (a target of the WEE1 kinase and G2/M phase regulator) phosphorylation was reduced in HuR depleted cells, validating HuR as an upstream post-transcriptional regulator of WEE1 and an indirect inhibitor of the cdk1-cyclin complex in the face of DNA damage. Conclusions: We demonstrate that HuR-deficiency allows PDA cells to enter the cell cycle more efficiently upon DNA damage. This event is dependent on HuR's regulation of WEE1 leading to activation of the CDK1-cyclin complex. These data support a model where HuR is integral to the G2/M checkpoint, forcing a cell cycle recovery response which is critical for PDA cells to avoid DNA damage. Therefore, HuR promotes survival of PDA cells through its acute activation of the DDR pathway. Disruption of HuR's molecular interaction with WEE1 represents a therapeutic opportunity to enhance clinically utilized chemotherapeutic DNA damaging agents for PDA. Citation Format: Shruti Lal, Richard A. Burkhart, Zoe Norris, Neil Beeharry, Vikram Bhattacharjee, Isidore Rigoutsos, Timothy Yen, Charles J. Yeo, Jordan M. Winter, Jonathan R. Brody. A novel chemoresistance mechanism: HuR post-trancriptionally regulates WEE1, the mitotic inhibitor, upon DNA damage in pancreatic adenocarcinoma cells. [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 628. doi:10.1158/1538-7445.AM2013-628

The function of tubulin acetylation in the context of cancer initiation and progression remains unknown. In studying the frequency of primary cilia in early pancreatic cancer, we discovered an increase in acetylated alpha-tubulin labeling (AAT) labeling in murine PanIN, even in the absence of primary cilia. We therefore hypothesized that human pancreatic ductal adenocarcinoma (PDAC) cells may upregulate expression of AAT as they progress from non-invasive to invasive and metastatic disease. Performing confocal microscopy on a human PanIN (n=23) and metastatic PDAC (n=10) tissue arrays, we have determined that in 15% of PanIN lesions, 13-30% of the cells have detectable levels of AAT, with a primarily cytoplasmic/cell membrane pattern of labeling. In contrast, over 80% of metastatic PDAC cells are AAT-positive. Surprisingly, high-resolution 3D confocal imaging of both fixed and unfixed human PDAC cell lines demonstrated immunoreactive AAT to be present on the cell surface of unfixed cells, as previously reported in human leukemia cells (Quillen, JCB 1985). To evaluate the functional significance of this finding, we developed a FACS-based strategy to isolate viable PDAC cells based on levels of cell surface AAT. Applying this strategy to four different human pancreatic cancer cell lines and three early passage human xenografts, we have determined that 5-10% of human pancreatic cancer cells have detectable levels of cell-surface AAT. PDAC cells positive for cell surface AAT are markedly enriched in cells also positive for either CD133 or CD24/CD44, representing putative pancreatic cancer stem cell populations. We have functionally analyzed the tumor-initiating capacity of cell surface AAT+ cells using in vitro tumor sphere experiments. In this assay, cell surface AAT+ cells show a statistically significant 100-fold increase in tumor sphere-forming capacity relative to cell surface AAT-negative cells. Tumor sphere-forming capacity was significantly reduced by neutralizing anti-AAT antibodies, as well as by an siRNA targeting α-tubulin K40 acetyltransferase (αTAT). We further evaluated the functional significance of cell surface AAT by analyzing the in vivo tumor initiating capacities of cell surface AAT-positive and AAT-negative cells from a human PDAC xenograft. Cell surface AAT-positive cells exhibited significantly increased tumor initiating frequency relative to the AAT-negative population (**p<0.0000539). AAT-positive cells had a tumor-initiating frequency similar to an Aldefluor-bright population, suggesting that the presence of cell surface acetylated alpha tubulin may mark a pancreatic cancer tumor initiating population. These data provide important new information regarding cellular heterogeneity in human pancreatic cancer, and suggest that novel strategies targeting cell surface tubulin may have therapeutic utility. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5338. doi:1538-7445.AM2012-5338