Response Of Pancreatic Ductal Adenocarcinoma Research Articles

IGFBP2 Drives Regulatory T Cell Differentiation through STAT3/IDO Signaling Pathway in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) represents one of the deadliest malignancies. Elevated regulatory T cell (Treg) infiltration has a potent immunosuppressive function in tumor biology, which contributes to low survival in PDAC. Nonetheless, the crosstalk between malignant cells and tumor-infiltrating Tregs in PDAC is not well understood. Here, clinical data demonstrates that the insulin-like growth factor binding protein 2 (IGFBP2) is associated with Treg accumulation in the microenvironment of PDAC in humans. Additionally, IGFBP2 increases Treg infiltration in the tumor microenvironment and promotes disease progression in mouse PDAC. Bioinformatic analysis and mechanistic assessment reveals IGFBP2 upregulated indoleamine 2, 3-dioxygenase (IDO) by activating signal transducer and activator of transcription 3 (STAT3) signaling in PDAC cells, thus inducing Treg differentiation and an immunosuppressive tumor microenvironment. These findings provide mechanistic insights into an important molecular pathway that promotes an immunosuppressive microenvironment, which suggests the IGFBP2 axis as a potential target for improved immune response in PDAC.

Differentiation between Pancreatic Ductal Adenocarcinoma and Normal Pancreatic Tissue for Treatment Response Assessment using Multi-Scale Texture Analysis of CT Images.

Pancreatic ductal adenocarcinoma (PDAC) is the most prevalent type of pancreas cancer with a high mortality rate and its staging is highly dependent on the extent of involvement between the tumor and surrounding vessels, facilitating treatment response assessment in PDAC. This study aims at detecting and visualizing the tumor region and the surrounding vessels in PDAC CT scan since, despite the tumors in other abdominal organs, clear detection of PDAC is highly difficult. This retrospective study consists of three stages: 1) a patch-based algorithm for differentiation between tumor region and healthy tissue using multi-scale texture analysis along with L1-SVM (Support Vector Machine) classifier, 2) a voting-based approach, developed on a standard logistic function, to mitigate false detections, and 3) 3D visualization of the tumor and the surrounding vessels using ITK-SNAP software. The results demonstrate that multi-scale texture analysis strikes a balance between recall and precision in tumor and healthy tissue differentiation with an overall accuracy of 0.78±0.12 and a sensitivity of 0.90±0.09 in PDAC. Multi-scale texture analysis using statistical and wavelet-based features along with L1-SVM can be employed to differentiate between healthy and pancreatic tissues. Besides, 3D visualization of the tumor region and surrounding vessels can facilitate the assessment of treatment response in PDAC. However, the 3D visualization software must be further developed for integrating with clinical applications.

Targeting the bicarbonate transporter SLC4A4 overcomes immunosuppression and immunotherapy resistance in pancreatic cancer

Solid tumors are generally characterized by an acidic tumor microenvironment (TME) that favors cancer progression, therapy resistance and immune evasion. By single-cell RNA-sequencing analysis in individuals with pancreatic ductal adenocarcinoma (PDAC), we reveal solute carrier family 4 member 4 (SLC4A4) as the most abundant bicarbonate transporter, predominantly expressed by epithelial ductal cells. Functionally, SLC4A4 inhibition in PDAC cancer cells mitigates the acidosis of the TME due to bicarbonate accumulation in the extracellular space and a decrease in lactate production by cancer cells as the result of reduced glycolysis. In PDAC-bearing mice, genetic or pharmacological SLC4A4 targeting improves T cell-mediated immune response and breaches macrophage-mediated immunosuppression, thus inhibiting tumor growth and metastases. In addition, Slc4a4 targeting in combination with immune checkpoint blockade is able to overcome immunotherapy resistance and prolong survival. Overall, our data propose SLC4A4 as a therapeutic target to unleash an antitumor immune response in PDAC.

Abstract B002: ARNTL2 is a Kras-dependent master regulator of hypoxic response in pancreatic ductal adenocarcinoma

Abstract We employed regulatory network analysis to identify master transcriptional regulators whose activity drives key pancreatic cancer phenotypes. Regulatory network analysis is a systems biology approach that computationally infers the activity of transcriptional regulators based on the integrated expression of their positive and negative target genes. To focus on epithelial cell biology, we performed laser capture microdissection and RNA sequencing on 200 human PDAC samples and 45 low-grade precursors (PanIN and IPMN), generating a rich dataset with clinical, epidemiological, and histopathological annotation. Using a transcriptional regulatory network derived from this dataset, we performed master regulator analysis to identify master regulators of four phenotypes: precursors vs. PDAC (initiation), low-grade vs. high grade histopathology (progression), survival post resection, and association with KRAS activity. Changes in the activity of multiple established PDAC drivers and suppressors were associated with tumor initiation and progression, providing a strong validation from existing biology. Integrating across these phenotypes, the top master regulator of PDAC malignancy was found to be ARNTL2, a member of the PAS family of bHLH transcription factors. The canonical function of ARNTL2 is as the heterodimeric binding partner of the circadian rhythm protein CLOCK. However, functional analysis ARNTL2 inferred transcriptional targets highlighted a potential role in hypoxia response and prior studies have demonstrated the ability of ARNTL2 to heterodimerize with HIF1A. Indeed, ARNTL2 was inferred to directly or indirectly regulate every single hypoxia-responsive protein in a previously published hypoxia signature. We therefore studied the role of ARNTL2 in both normoxic and hypoxic conditions and found that its knockdown in PDAC cells led to a selective decrease in viability and invasion under hypoxic conditions. ARNTL2 activity was induced in response to hypoxia and inhibited following treatment with multiple RAF, MEK, and ERK inhibitors, validating its computationally-ingerred association with RAS activity. Strikingly, we found that ARNTL2 was required in PDAC cells for the stabilization of HIF1A in response to hypoxia. Conversely, HIF2A was further stabilized in response to hypoxia upon ARNTL2 deletion, suggesting that ARNTL2 serves to control the balance between HIF1A- and HIF2A dependent metabolic programs in PDAC. Consistent with this, loss of ARNTL2 had sizeable impacts on profiles in PDAC cells and led to a significant reduction of lactate dehydrogenase A under hypoxic conditions. Together these data demonstrate that ARNTL2 is a previously underappreciated master regulator of hypoxia response in pancreatic ductal adenocarcinoma. Citation Format: H. Carlo Maurer, Alvaro Curiel-Garcia, Pasquale Laise, Carmine F. Palermo, Stephen A. Sastra, Gulam A. Manji, Alina Iuga, Kristen Johnson, Jeanine Genkinger, Costas A. Lyssiotis, Andrea Califano, Kenneth P. Olive. ARNTL2 is a Kras-dependent master regulator of hypoxic response in pancreatic ductal adenocarcinoma [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 B002.

Abstract B010: Investigating endoplasmic reticulum stress responses in pancreatic ductal adenocarcinoma under lipid imbalance

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer-related death with a five-year survival rate of 10%. The complex genetic makeup and heterogeneity of its microenvironment make PDAC difficult to treat. Desmoplasia, a major feature of PDAC’s TME, are dense fibrotic and inflammatory stromal regions. Desmoplastic reaction results in poor intratumoral perfusion and elevated interstitial fluid pressure, creating regions with severe oxygen and nutrient deprivation. One result is a deficiency in unsaturated fatty acids (uFAs) because the de novo synthesis of uFAs requires oxygen. Our results indicate that PDAC cells experience endoplasmic reticulum (ER) stress and trigger subsequent ER stress responses under uFA limited conditions in vitro. The ER stress responses are governed by three major stress sensors: Inositol-requiring enzyme 1𝛼 (IRE1𝛼), PKR-like ER kinase (PERK) and activating transcription factor 6𝛼 (ATF6𝛼). We hypothesize that ER stress responses are tumor promoting in PDAC and used as a survival strategy under uFA deprivation. In addition, previous studies showed that MYC-driven cancer cells are highly reliant on the ER stress response pathways. Therefore, we hypothesize that the two major PDAC transcriptional subtypes “Classical” and “Basal-like” have distinct dependencies on ER stress response pathways, with the basal-like subtype being more dependent due to an enrichment of MYC gene signatures. Our results suggest that opposite to cancer cell promoting, ER stress responses mediated by IRE1𝛼, PERK and ATF6𝛼 are cytotoxic in vitro under uFA deprived conditions. By analyzing the published bulk RNA-seq from patient samples, we found that the basal-like PDAC subtype is enriched with an ER stress gene signature. To avoid contamination from other cell populations, we are currently analyzing a published scRNA-seq dataset from 15 independent patient samples. We will investigate whether the ER stress signature is also enhanced in basal-like cells. Basal-like PDAC cells are usually more mesenchymal; we therefore created isogenic “epithelial” and “mesenchymal” lines to mimic PDAC subtypes. However, we did not see signaling preferences between the lines. One caveat is that the basal-like subtype have many more features than being mesenchymal, and our isogenic system might not be a faithful modeling of the subtypes. We are studying whether hypoxic PDAC cells experience ER stress in vivo due to a deprivation in uFAs using multiplex fluorescent imaging, stimulated Raman microscopy and/or mass spectrometry. We are evaluating the role of ER stress responses in vivo and see whether inhibiting ER stress response alone or in combination with other reagents gives rise to potential therapeutic opportunities. This project will provide a better understanding of the role ER stress responses play under uFA deprivation and will also enable us to further understand PDAC subtypes in terms of how they utilize ER stress responses. Citation Format: Yanqing (Christine) Jiang, Xu Han, Mai Wang, Raymond Ng, Pamela Burgess-Jones, John Tobias, Nathan Coffey, Andrew Hu, Brian Keith, Celeste Simon. Investigating endoplasmic reticulum stress responses in pancreatic ductal adenocarcinoma under lipid imbalance [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 B010.

Intra-tumoral infiltration of adipocyte facilitates the activation of antitumor immune response in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a highly fatal malignancy that is characterized by an immunosuppressive microenvironment. The immune suppression in PDAC is largely driven by heterogeneous stromal and tumor cells. However, how adipocyte in the tumor microenvironment (TME) is related to the immune cell infiltration in PDAC has rarely been published. We identified adipocytes by performing bioinformatics analyses, and explored the clinical outcomes and TME characters in PDAC with different levels of adipocyte infiltration. Interestingly, in contrast to adiposity, high adipocyte infiltration in the TME was related to significantly increased median overall survival and a lower total tumor mutational burden. Functionally, high adipocyte infiltration was associated with the immune response, particularly with the abundant cytokine infiltration in PDAC samples. Moreover, adipocyte infiltration in the TME was positively associated with anticancer signatures in the immune microenvironment. Immunohistochemistry and RT-PCR were performed with PDAC tissue samples from our center to study the expression of adipocytes in PDAC. The mature adipocytes were strongly associated with the immune composition and prognosis of patients with PDAC. Primary adipocytes were isolated from mice to construct a PDAC transplantation tumor model. In vivo experiments showed that adipocytes elicited increased CD8+ T cell infiltration and potent antitumor activity in tumor-bearing mice. Overall, we innovatively found that adipocytes facilitated the antitumor immune response in the TME by performing mouse experiments and analyzing PDAC samples. This study provides a new perspective on the activation of the immune microenvironment in PDAC.

IgG+ Extracellular Vesicles Measure Therapeutic Response in Advanced Pancreatic Cancer.

(1) Background: Pancreatic ductal adenocarcinoma (PDAC) is expected to be the second-leading cause of cancer deaths by 2030. Imaging techniques are the standard for monitoring the therapy response in PDAC, but these techniques have considerable limits, including delayed disease progression detection and difficulty in distinguishing benign from malignant lesions. Extracellular vesicle (EV) liquid biopsy is an emerging diagnosis modality. Nonetheless, the majority of research for EV-based diagnosis relies on point analyses of EVs at specified times, while longitudinal EV population studies before and during therapeutic interventions remain largely unexplored. (2) Methods: We analyzed plasma EV protein composition at diagnosis and throughout PDAC therapy. (3) Results: We found that IgG is linked with the diagnosis of PDAC and the patient’s response to therapy, and that the IgG+ EV population increases with disease progression and reduces with treatment response. Importantly, this covers PDAC patients devoid of the standard PDAC seric marker CA19.9 expression. We also observed that IgG is bound to EVs via the tumor antigen MAGE B1, and that this is independent of the patient’s inflammatory condition and IgG seric levels. (4) Conclusions: We here propose that a population analysis of IgG+ EVs in PDAC plasma represents a novel method to supplement the monitoring of the PDAC treatment response.

An m5C methylation regulator-associated signature predicts prognosis and therapy response in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is the most aggressive digestive malignancy due to frequent late-stage diagnosis, rapid progression and resistance to therapy. With increasing PDAC incidence worldwide, there is an urgent need for new prognostic biomarkers and therapy targets. Recently, RNA methylation has emerged as a new tumorigenic mechanism in different cancers. 5-methylcytosine (m5C) is one of the most frequent RNA modifications and occurs on a variety of RNA species including mRNA, thereby regulating gene expression. Here we investigated the prognostic role of m5C-regulator-associated transcriptional signature in PDAC. We evaluated m5C-regulator status and expression in 239 PDAC samples from publicly available datasets. We used unsupervised consensus clustering analyses to classify PDACs based on m5C-regulator expression. From the resulting signature of differentially expressed genes (DEGs), we selected prognosis-relevant DEGs to stratify patients and build a scoring signature (m5C-score) through LASSO and multivariate Cox regression analyses. The m5C-score represented a highly significant independent prognostic marker. A high m5C-score correlated with poor prognosis in different PDAC cohorts, and was associated with the squamous/basal subtype as well as activated cancer-related pathways including Ras, MAPK and PI3K pathways. Furthermore, the m5C-score correlated with sensitivity to pathway-specific inhibitors of PARP, EGFR, AKT, HER2 and mTOR. Tumors with high m5C-score were characterized by overall immune exclusion, low CD8+ T-cell infiltration, and higher PD-L1 expression. Overall, the m5C-score represented a robust predictor of prognosis and therapy response in PDAC, which was associated with unfavorable molecular subtypes and immune microenvironment.

Abstract 3507: β-Hydroxy-β-methylbutyrate (HMB) sensitizes murine pancreatic ductal adenocarcinoma (PDAC) to immunotherapy

Abstract Background: Pancreatic cancer is the fourth leading cause of cancer-related death in the United States. PDAC often presents at advanced tumor stages, resulting in poor survival rates. Immunotherapies such as inhibition of programmed death 1 (PD1) are effective in many cancers, but not in PDAC. HMB is a leucine metabolite that reduces PDAC progression and cachexia in association with enhanced antitumor immune responses. The purpose of this study was to determine if HMB treatment enhances immunotherapeutic response in PDAC. Methods: C57BL/6J mice bearing subcutaneously transplanted PANC02 tumors were fed either a control (AIN93G) or HMB (1% w/w) supplemented diet. Once tumors were palpable, mice were randomized for treatment with vehicle or anti-PD1 immunotherapy (n=12-13/group). Tumors were harvested and global transcriptomic analysis was performed. Results: Both HMB and anti-PD1 interventions alone were insufficient to reduce tumor growth. However, the combination of HMB and anti-PD1 resulted in significantly smaller tumors than the anti-PD1 alone group, indicating that HMB was able to stimulate PDAC response to ICI. Transcriptomic analysis revealed limited transcriptional effects attributable to immunosurveillance following treatment with either HMB or anti-PD1 alone; however, the combination produced a robust antitumor immune response. Digital cytometry analysis revealed altered immune cell composition in the tumor microenvironment. Conclusion: The combination of HMB with anti-PD1 treatment elicited significant antitumor responses in a murine model of PDAC progression. Given that immunotherapy trials in PDAC have not yet proven effective, even when combined with cytotoxic chemotherapy, our preclinical findings support clinical evaluation of combined ICI and HMB regimens as a therapeutic strategy against PDAC. Citation Format: Suhas K. Etigunta, Michael F. Coleman, Kristyn A. Liu, Alex J. Pfeil, Zhengrong Cui, Stephen D. Hursting. β-Hydroxy-β-methylbutyrate (HMB) sensitizes murine pancreatic ductal adenocarcinoma (PDAC) to immunotherapy [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 3507.

Abstract 1052: Augmentation of standard chemotherapy response by inhibition of Aurora kinase A in preclinical models of pancreatic cancer

Abstract Background: The prognosis of pancreatic ductal adenocarcinoma (PDAC) is very poor with a 5-year survival rate of 8%. Current standard chemotherapeutic treatment nab-paclitaxel (NPT) plus gemcitabine (Gem) has been able to give an average prognosis of 8.5 months after detection. Aurora Kinase A (AURKA), an enzyme responsible for the regulation of cytokinesis, is mutated in over 55% of PDAC. Increased expression of AURKA inhibits metabolic stress-induced autophagy, thus promoting cell survival. Additionally, amplification of AURKA has been shown to lead to taxane resistance. Through inhibition of AURKA signaling with a small-molecule inhibitor alisertib (MLN8237, ALS), we evaluated an enhancement of NPT+Gem chemotherapy response in PDAC. Methods: In vitro cell proliferation was evaluated in PDAC-associated cell lines by WST-1 assay. Protein expression was measured by Immunoblot analysis. Tumor growth and survival studies were performed using human PDAC cells AsPC-1 in NOD/SCID mice. Intratumoral mechanism of action was determined by IHC and Immunoblot analysis. Results: In AsPC-1 subcutaneous xenografts, chemotherapy regimen (NPT+Gem) and alisertib inhibited tumor growth while their combination exhibited an additive effect. The increase in tumor size was 255 mm3 in controls, 105 mm3 after NPT+Gem, 133 mm3 after ALS and 38 mm3 after NPT+Gem+ALS treatment. In peritoneal dissemination xenografts, animal survival was 23 days in controls that was increased by NPT+Gem (41 days, 78% increase), ALS (25 days, 9% increase) and NPT+Gem+ALS exhibited an additive effect (49 days, 113% increase). NPT+Gem and ALS effect on tumor cell proliferation and apoptosis corresponded with subcutaneous tumor growth data. In vitro proliferation analysis of PDAC epithelial cells (AsPC-1, Panc-1), stromal cells and endothelial cells demonstrated that NPT+Gem and ALS inhibited cell proliferation and their combination treatment had an additive effect. Immunoblot analysis of PDAC cells showed increased levels of cleaved caspase-3 and cleaved PARP-1 in combination therapy pointing to increased levels of apoptosis with NPT+Gem+ALS treatment in all cell lines tested. Conclusion: These findings demonstrate that nab-paclitaxel plus gemcitabine standard chemotherapy response can be enhanced through specific inhibition of aurora kinase A signaling by alisertib in PDAC. The data support the potential of this combinatorial therapeutic strategy for clinical PDAC therapy. Citation Format: David Kronenberger, Margaret A. Schwarz, Md Sazzad Hassan, Urs von Holzen, Roderich E. Schwarz, Niranjan Awasthi. Augmentation of standard chemotherapy response by inhibition of Aurora kinase A in preclinical models of pancreatic cancer [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 1052.

Abstract 1995: β-Hydroxy-β-methylbutyrate promotes immunotherapy response and pro-inflammatory macrophage polarization in a mouse model of pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) continues to represent a critical unmet therapeutic need in the US, and is exacerbated by obesity. Current chemotherapeutic regimens poorly control PDAC progression, and immunotherapy has yet to be widely successful against PDAC. Obesity promotes a potently protumor PDAC microenvironment including suppression of antitumor immune response. The purpose of our studies was to test whether dietary supplementation with β-hydroxy-β-methylbutyrate (HMB), shown to be safe and well tolerated in cancer patients, could offset obesity-mediated exacerbation of PDAC and promote antitumor immunosurveillance. Methods: Lean and obese C57BL/6 mice were treated with HMB alone or in combination with anti-PD1 immunotherapy following subcutaneous injection of Panc02 PDAC cells. Bone marrow-derived macrophages (BMDM) were treated with HMB in vitro with or without lipopolysaccharide (LPS). Tumor/macrophage transcriptomic analysis using Affymetrix arrays followed by gene set enrichment was performed, and tumor microenvironment immune cell composition was determined by mass cytometry. Results: a) HMB cooperates with obesity and immunotherapy to reduce PDAC progression, as indicated by reduced tumor growth with the combination regimen of HMB + anti-PD1 relative to obese or anti-PD1 immunotherapy treated mice without HMB supplementation; b) HMB mitigates PDAC immunosuppression and promotes tumor immune surveillance, as demonstrated by induction of an antitumor immune-related gene expression profile in response to HMB supplementation; c) HMB promotes pro-inflammatory polarization of tumor-associated macrophages and BMDM, as shown by increased CD38 positivity and reduced Arg1 positivity in tumor-associated macrophages from HMB supplemented mice relative to control. Further, BMDM transcriptional response to HMB alone or HMB in combination with LPS paralleled that of BMDM treated with LPS relative to untreated LPS. Conclusion: Overall, our findings suggest HMB is an adjuvant for antitumor immune responses in PDAC. Moreover, HMB promotes immune surveillance in PDAC, synergizing with immunotherapy. Hence, HMB-induced suppression of PDAC tumor growth and promotion of immune surveillance may offer significant synergy with chemotherapies or immunotherapies in PDAC. Funding: This work was supported by R35CA197627 to SDH. Citation Format: Michael F. Coleman, Kristyn A. Liu, Alexander J. Pfeil, Suhas K. Etigunta, Xiaohu Tang, Laura M. Lashinger, Zhengrong Cui, Stephen D. Hursting. β-Hydroxy-β-methylbutyrate promotes immunotherapy response and pro-inflammatory macrophage polarization in a mouse model of pancreatic cancer [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 1995.

A phase I study to evaluate the safety and tolerability of SX-682 in combination with PD-1 inhibitor as maintenance therapy for unresectable pancreatic adenocarcinoma.

TPS631 Background: Survival outcomes for advanced pancreatic ductal adenocarcinoma (PDAC) remain dismal despite improvements in systemic therapy regimens developed over the past decade. In addition, current first-line therapies result in cumulative cytopenias and neuropathy, highlighting the need for more effective, less toxic maintenance treatment strategies. There are currently no standard approved maintenance treatments for patients with PDAC not associated with BRCA or DNA-repair mutations. Pre-clinical data suggest a potential synergistic effect of combining blockade of CXC chemokine receptors (CXCR) with immunotherapy or chemotherapy in pancreatic cancer1,2. We are currently conducting a Phase I study (NCT04477343) evaluating SX-682, an oral CXCR1/2 inhibitor, and Nivolumab as maintenance treatment for advanced PDAC. Methods: This is an open-label, dose escalation Phase I clinical trial evaluating the combination of SX-682 and Nivolumab. Patients must have histologically confirmed unresectable PDAC and have completed at least 16 weeks of first-line chemotherapy with disease stability or treatment response at time of enrollment. Radiographically measurable disease must be present per iRECIST criteria. Patients receive a 3-week run-in phase of twice-daily dosing of SX-682, followed by combination of twice-daily dosed SX-682 and every 2-week Nivolumab (240 mg IV). Dose finding of SX-682 is performed using Bayesian optimal interval (BOIN) design to determine the maximum tolerated dose (MTD) when combined with Nivolumab. Pre-treatment and one on treatment (Day 28-35) biopsies are required for enrollment to evaluate change in tumor microenvironment immune cell composition by single cell-RNA sequencing, flow cytometry, RNA RT-qPCR, and IHC. The primary endpoint is to determine MTD; the key secondary endpoint is progression-free survival (PFS), defined as the time from enrollment to progression via iRECIST criteria or death. Nine of a planned 20 patients have been enrolled. Dose-level 1 (SX-682 50 mg BID) completed enrollment without dose-limiting toxicity (DLT). Dose-level 2, which commenced in June 2021, (SX-682 100 mg BID) is without DLTs, but has not completed enrollment at time of abstract submission. Nywening TM, Belt BA, Cullinan DR, et al. Targeting both tumour-associated CXCR2(+) neutrophils and CCR2(+) macrophages disrupts myeloid recruitment and improves chemotherapeutic responses in pancreatic ductal adenocarcinoma. 1) Gut. 2018;67(6):1112-1123. Steele CW, Karim SA, Leach JDG, et al. CXCR2 Inhibition Profoundly Suppresses Metastases and Augments Immunotherapy in Pancreatic Ductal Adenocarcinoma. 2) Cancer Cell. 2016;29(6):832-845. Clinical trial information: NCT04477343.

CD73 induces GM-CSF/MDSC-mediated suppression of T cells to accelerate pancreatic cancer pathogenesis.

Metabolic alterations regulate cancer aggressiveness and immune responses. Given the poor response of pancreatic ductal adenocarcinoma (PDAC) to conventional immunotherapies, we investigated the link between metabolic alterations and immunosuppression. Our metabolic enzyme screen indicated that elevated expression of CD73, an ecto-5’–nucleotidase that generates adenosine, correlates with increased aggressiveness. Correspondingly, we observed increased interstitial adenosine levels in tumors from spontaneous PDAC mouse models. Diminishing CD73 by genetic manipulations ablated in vivo tumor growth, and decreased myeloid-derived suppressor cells (MDSC) in orthotopic mouse models of PDAC. A high-throughput cytokine profiling demonstrated decreased GM-CSF in mice implanted with CD73 knockdowns. Furthermore, we noted increased IFN-γ expression by intratumoral CD4+ and CD8+ T cells in pancreatic tumors with CD73 knockdowns. Depletion of CD4+ T cells, but not CD8+ T cells abrogated the beneficial effects of decreased CD73. We also observed that splenic MDSCs from Nt5e knockdown tumor-bearing mice were incompetent in suppressing T cell activation in the ex vivo assays. Replenishing GM-CSF restored tumor growth in Nt5e knockout tumors, which was reverted by MDSC depletion. Finally, anti-CD73 antibody treatment significantly improved gemcitabine efficacy in orthotopic models. Thus, targeting the adenosine axis presents a novel therapeutic opportunity for improving the anti-tumoral immune response against PDAC.

Estimated tumor blood flow as a predictive imaging indicator of therapeutic response in pancreatic ductal adenocarcinoma: use of three-phase contrast-enhanced computed tomography.

Preoperative chemotherapy or chemoradiotherapy is a common strategy for treating pancreatic ductal adenocarcinoma (PDAC). This study aimed to assess the association between the therapeutic response in PDAC and tumor blood circulation. The medical records of patients who underwent chemotherapy or chemoradiotherapy prior to pancreatectomy for PDAC were reviewed. Of these, patient data that included three-phase contrast-enhanced computed tomography (CECT) findings before treatments were used in this study. We evaluated the estimated tumor blood flow (eTBF) using CECT. According to the therapeutic histopathological response defined by the Evans classification, patients were divided into poor (grade I/IIa) and good (grade IIb/III/IV) responder groups. The variables, including eTBF, were compared between the two groups. Thirty patients were enrolled in this study. Of these, 13 (43.3%) (grade IIB/III/IV: 8/4/1 patients) were categorized into the good responder group and 17 patients (56.7%) (grade I/IIA: 4/13 patients) were categorized into the poor responder group. eTBF was significantly higher in the good responder group (0.39s-1 vs. 0.32s-1, p = 0.007). An eTBF ≥ 0.36s-1 was found to be an independent predictive factor for the destruction of over 50% of tumor cells (p = 0.036; odds ratio, 9.71; 95% confidence interval, 1.16-81.30). eTBF can be used to predict the therapeutic histopathological response in PDAC prior to treatment.

Extracellular volume fraction with MRI: As an alternative predictive biomarker to dynamic contrast-enhanced MRI for chemotherapy response of pancreatic ductal adenocarcinoma

PurposeTo assess the feasibility of extracellular volume (ECV) fraction determined with equilibrium contrast-enhanced MRI for prediction of treatment response to chemotherapy in pancreatic ductal adenocarcinoma (PDAC) in comparison with dynamic contrast-enhanced MRI (DCE-MRI), and to clarify the association between ECV fraction and DCE-MRI-derived pharmacokinetic parameters. MethodsThis retrospective study included 58 consecutive patients with histologically confirmed PDAC who underwent DCE-MRI before systemic chemotherapy. Tumor pharmacokinetic parameters, including the volume transfer coefficient (Ktrans), rate constant (kep), and extracellular extravascular volume fraction (ve) of DCE-MRI, and ECV fraction determined with equilibrium contrast-enhanced MRI were compared between the response and non-response groups. The correlation of tumor ECV fraction with each DCE-MRI-derived pharmacokinetic parameter was examined using Spearman’s rank correlation coefficient. ResultsTumor Ktrans, ve, and ECV fraction were significantly higher in the response group than in the non-response group (all, P < 0.001), whereas no significant difference was found in kep (P = 0.119). Tumor ECV fraction showed the highest area under receiver operating characteristic curve of 0.918, with a sensitivity of 89.3%, specificity of 90.0%, and accuracy of 89.7% (cut off, >37.6%). The ECV fraction showed a significant positive correlation with Ktrans (Spearman’s coefficient = 0.66, P < 0.001) and ve (Spearman’s coefficient = 0.79, P < 0.001). ConclusionsECV fraction determined with equilibrium contrast-enhanced MRI was as useful as DCE-MRI-derived pharmacokinetic parameters for predicting treatment response to chemotherapy in patients with PDAC.

Abstract PO-027: Investigating unfolded protein responses in pancreatic ductal adenocarcinoma under lipid imbalance

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer-related death with a five-year survival rate of only 10%. A major feature of PDAC tumor microenvironment (TME) is desmoplasia, which are dense fibrotic and inflammatory stromal regions constituting the PDAC TME. This desmoplastic reaction results in poor intratumoral perfusion and elevated interstitial fluid pressure, creating regions with severe oxygen and nutrient deprivation. One result is a significant deficiency in unsaturated fatty acids (UFAs) because the de novo synthesis of UFAs requires molecular oxygen. Our data indicate that PDAC cells experience endoplasmic reticulum (ER) stress and trigger a subsequent unfolded protein response (UPR) under tumor-like stress, which can be mitigated by UFAs. The UPR regulates an intricate signaling network mediated by three major stress sensors: Inositol-requiring enzyme 1a (IRE1a), PKR-like ER kinase (PERK) and activating transcription factor 6a (ATF6a). UPR output can be either cytoprotective or cytotoxic depending on tumor types and the severity of stress. Our data suggest that B-I09, a selective IRE1 RNase inhibitor, impairs PDAC cell viability under stress conditions, indicating a pro-survival role for IRE1a. Previous studies showed that MYC-driven cancer cells are highly reliant on UPR pathways. Therefore, we hypothesize that the two major PDAC transcriptional subtypes “Classical” and “Basal-like” may have distinct dependencies on UPR pathways, with the Basal-like subtype being more dependent due to an enrichment of MYC gene signatures. Overall, we hypothesize that IRE1a, PERK and/or ATF6a play(s) an oncogenic role in PDAC tumorigenesis due to lipid saturation abnormalities, and the Basal-like PDAC subtype is more sensitive to inhibition of UPR pathway(s). We are evaluating the role of IRE1a, PERK and ATF6a using pharmacologic inhibition and genetic knockdown in vitro and in vivo under UFA deprived conditions. We will also evaluate PDAC intracellular and interstitial fluid lipidomics using LC-MS. In addition, we will assess whether hypoxic PDAC cells experience ER stress in vivo due to UFA deprivation using Stimulated Raman scattering (SRS) microscopy. To explore the subtype-specific dependency on the UPR pathways, we are querying a UPR gene signature in different tumor cell clusters using single cell RNA-seq datasets of patient samples. In addition, we will employ an isogenic system for PDAC subtypes and examine subtype-specific responses to UPR inhibition in vitro and in vivo. Our goal is to better understand UPR pathways in PDAC cells as a survival mechanism in response to the “lipotoxicity” induced by ER membrane saturation, with a consideration of intratumoral heterogeneity of PDAC cell subtype. Citation Format: Yanqing (Christine) Jiang, Xu Han, Michelle Burrows, Carson Poltorack, Celeste Simon, Brian Keith. Investigating unfolded protein responses in pancreatic ductal adenocarcinoma under lipid imbalance [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-027.

Abstract PO-072: Inhibiting vasoactive intestinal peptide receptor signaling elicits T cell dependent anti-tumor response of pancreatic ductal adenocarcinoma to immune checkpoint therapy

Abstract Pancreatic ductal adenocarcinoma (PDAC) is unresponsive to immune checkpoint therapy largely due to a paucity of T cells within the tumor microenvironment (TME) and abundant immunosuppressive signaling pathways. In this study we show that human PDAC tumors over-express vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide that suppresses T cell effector properties and promotes the generation of regulatory T cells (Tregs). Therefore, we treated tumor-bearing mice with VIP receptor peptide antagonists and measured T cell homing, activation, and anti-tumor responses in preclinical murine models of PDAC. Pharmacological inhibition of VIP receptor (VIP-R) signaling using daily subcutaneous injections of peptide antagonists had no discernable toxicity in healthy and tumor-bearing mice. Treatment with VIP-R antagonists in combination with anti-PD-1 checkpoint blockade significantly decreased tumor burden, and improved survival in subcutaneous and orthotopic murine PDAC models. Combination therapy significantly enhanced T cell activation and proliferation and decreased frequencies of Tregs within the TME. Anti-tumor responses were T cell dependent, as the combination therapy failed to improve survival in CD4 or CD8 deficient mice using knock-out strains and antibody depletion. Furthermore, combination therapy significantly increased frequencies of tumor specific T cells (measured with a tetramer reagent) and provided protective immunity against tumor rechallenge. Combination therapy led to significant increases in the infiltration of adoptively transferred GFP+ T cells into PDAC tumors and decreased CXCR4 expression levels on T cells. Encouragingly, peptide-based VIP-R antagonists enhanced the in vitro activation of human T cells isolated from peripheral blood of PDAC patients. Human T cells cultured with VIP-R antagonists had increased proliferation, activation, and decreased proportions of T regs and exhausted T cells co-expressing PD-1, Tim-3 and Lag-3. Taken together, our findings show that VIP is a targetable mechanism of immune escape in PDAC. Inhibiting VIP receptor signaling improves T cell effector properties and synergistically improves anti-tumor responses to checkpoint inhibitors in mouse PDAC models. Additionally, as the VIP sequence is identical between human and mice, and since VIP-R antagonists have similar effects on human and murine T cells in culture, clinical translation is highly feasible. Citation Format: Sruthi Ravindranathan, Passang Tenzin, Jian Ming Li, Rohan Dhamsania, Michael Ware, Mohammad Zaidi, Shuhua Wang, Jingru Zhu, Maria Cardenas, Yuan Liu, Gaurav Joshi, Sanjeev Gumber, Brian Robinson, Anish Sen-Majumdar, Shanmuganathan Chandrakasan, Haydn Kissick, Alan Frey, Susan Thomas, Bassel El-Rayes, Gregory Lesinski, Edmund K. Waller. Inhibiting vasoactive intestinal peptide receptor signaling elicits T cell dependent anti-tumor response of pancreatic ductal adenocarcinoma to immune checkpoint therapy [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-072.

FUS-induced circRHOBTB3 facilitates cell proliferation via miR-600/NACC1 mediated autophagy response in pancreatic ductal adenocarcinoma

BackgroundCircular RNAs (circRNAs) are becoming a unique member of non-coding RNAs (ncRNAs) with emerging evidence of their regulatory roles in various cancers. However, with regards to pancreatic ductal adenocarcinoma (PDAC), circRNAs biological functions remain largely unknown and worth investigation for potential therapeutic innovation.MethodsIn our previous study, next-generation sequencing was used to identify differentially expressed circRNAs in 3 pairs of PDAC and adjacent normal tissues. Further validation of circRHOBTB3 expression in PDAC tissues and cell lines and gain-and-loss function experiments verified the oncogenic role of circRHOBTB3. The mechanism of circRHOBTB3 regulatory role was validated by pull-down assays, RIP, luciferase reporter assays. The autophagy response of PANC-1 and MiaPaca-2 cells were detected by mCherry-GFP-LC3B labeling and confocal microscopy, transmission electron microscopy and protein levels of LC3B or p62 via Western blot.ResultscircRHOBTB3 is highly expressed in PDAC cell lines and tissues, which also promotes PDAC autophagy and then progression in vitro and in vivo. Mechanistically, circRHOBTB3 directly binds to miR-600 and subsequently acts as a miRNA-sponge to maintain the expression level of miR-600-targeted gene NACC1, which facilitates the autophagy response of PDAC cells for adaptation of proliferation via Akt/mTOR pathway. Moreover, the RNA-binding protein FUS (FUS) directly binds to pre-RHOBTB3 mRNA to mediate the biogenesis of circRHOBTB3. Clinically, circRHOBTB3, miR-600 and NACC1 expression levels are correlated with the prognosis of PDAC patients and serve as independent risk factors for PDAC patients.ConclusionsFUS-mediated circRHOBTB3 functions as a tumor activator to promote PDAC cell proliferation by modulating miR-600/NACC1/Akt/mTOR axis regulated autophagy.

TLR2 activation promotes tumour growth and associates with patient survival and chemotherapy response in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor prognosis, and is plagued by a paucity of targeted treatment options and tumour resistance to chemotherapeutics. The causal link between chronic inflammation and PDAC suggests that molecular regulators of the immune system promote disease pathogenesis and/or therapeutic resistance, yet their identity is unclear. Here, we couple endoscopic ultrasound-guided fine-needle aspiration, which captures tumour biopsies from all stages, with whole transcriptome profiling of PDAC patient primary tumours to reveal enrichment of the innate immune Toll-like receptor 2 (TLR2) molecular pathway. Augmented TLR2 expression associated with a 4-gene "TLR2 activation" signature, and was prognostic for survival and predictive for gemcitabine-based chemoresistance. Furthermore, antibody-mediated anti-TLR2 therapy suppressed the growth of human PDAC tumour xenografts, independent of a functional immune system. Our results support TLR2-based therapeutic targeting for precision medicine in PDAC, with further clinical utility that TLR2 activation is prognostic and predictive for chemoresponsiveness.

Abstract 1565: β-hydroxy-β-methylbutyrate enhances anti-PD1 immunotherapy in a mouse model of pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) has a five year survival rate in single digits.This is driven by a number of factors including limited effective therapeutic options for the management of PDAC. Immunotherapies such as inhibition of programmed death 1 (PD1) have provided dramatic benefit to cancers with historically poor treatment options such as melanoma. However, PDAC has thus far proven unresponsive to immunotherapies. It is well understood that the development of effective anti-tumor immunity is in part limited by the metabolically hostile tumor microenvironment. β-Hydroxy-β-Methylbutyrate (HMB) is a leucine metabolite which has been shown to reduce cancer cachexia and tumor progression. We sought to determine if HMB treatment would alter immunotherapy response in PDAC via reduction of metabolic competition. Methods: C57BL/6 mice were injected with PANC02 cells subcutaneously to generate PDAC tumors, then fed either a control (AIN93G) or HMB supplemented diet. Once tumors were palpable, mice were further randomized for treatment with anti-PD1 immunotherapy or a vehicle control. Tumors were collected, measured and weighed; RNA isolated; and cDNA synthesized for global transcriptomic analysis. Gene set enrichment analysis followed by enrichment mapping was used to determine the pathway activation induced by HMB, anti-PD1, or the combination. CIBERSORTx deconvolution was used to determine microenvironment immune cell composition. Results: As anticipated, neither HMB nor anti-PD1 therapies alone were sufficient to reduce tumor growth. The combination of HMB and anti-PD1; however, resulted in significantly smaller tumors than the anti-PD1 treated group, indicating that HMB was sufficient to enable PDAC response to immunotherapy. Transcriptomic analysis demonstrated only moderate immunomodulatory effects of HMB or anti-PD1 alone; however, the combination potently induced putatively anti-tumor immune response. Deconvolution analysis indicated that the combination of HMB and anti-PD1 increased the abundance of anti-tumor immune cell types. HMB treatment, as expected, was very well tolerated by all animals with no adverse effects detected. Overall, HMB promotes efficacy of anti-PD1 immunotherapy in a model of PDAC. Discussion: Here, we report that the combination of HMB with anti-PD1 elicited significant anti-tumor response, without the need for the prior induction of immunological cell death via chemotherapy or radiation. This is particularly remarkable as many PDAC immunotherapy trials with or without other treatments did not result in significant benefit. HMB has been well tolerated in numerous cancer cachexia clinical trials and is both cheaply and commonly available. Thus, this combination is a highly promising avenue for the advancement of immunotherapy in PDAC. This research was supported by R35CA197627 to S. Hursting. Citation Format: Suhas K. Etigunta, Michael F. Coleman, Alex J. Pfeil, Laura M. Lashinger, Zhengrong Cui, Stephen D. Hursting. β-hydroxy-β-methylbutyrate enhances anti-PD1 immunotherapy in a mouse model of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1565.