KPC Model Research Articles

Abstract B062: Low dose dual epigenetic therapy utilizing hypomethylating agents and HDAC inhibitors prolong survival in an orthotopic PDAC model and alter the tumor microenvironment

Abstract Background: We have previously studied the effect of hypomethylating agent Decitabine (5- aza-dC; DAC) on pancreatic cancer using the KPC model (KrasG12D/+;Trp53R172H/+;Pdx1- Cre) and observed increased CD4+/CD8+ TILs, greater PD1 expression and slowed tumor growth with increased tumor necrosis. Subsequently we showed a significant survival benefit with DAC+αPD1 compared to either single agent. However, we identified a specific myeloid cell population initially identified by Chi3l3 expression that may be responsible for treatment resistance or escape. Based on prior observations of synergy specifically affecting tumor infiltrating myeloid and lymphoid cells, we focused on identifying a dual epigenetic strategy (a combination of DAC plus histone deacetylase inhibitors (HDACi)) to enhance immunological synergy and test their treatment efficacy with αPD1. Methods: We used a combination primary KPC-derived cell lines, bone marrow (BMDM) and peritoneum derived (PM) myeloid cells and T cells in co-culture systems to assess cytotoxic synergy (Bliss/Lowe consensus) and immune effects (cytokine release, myeloid cell polarization and T cell activation) to evaluate combinations of DAC+HDACi. The KPC orthotopic model relying on US monitoring of tumor size for standardized initiation of therapy was used for survival analysis and profiling in treatment combinations of DAC+HDACi+αPD1. Results: We showed that hypomethylation therapy exerts a primarily paracrine effect on myeloid cells and this polarization results in a decrease in DAC–induced T cell activation. We therefore selected HDAC inhibitors that in combination with DAC alter this paracrine effect. We selected specific HDAC inhibitors based on induction of class of HDAC expression following hypomethylation therapy, highest Bliss synergy scores at low doses and reversal of cytokine release, M2 polarization and T cell inactivation. Three HDAC inhibitors were chosen for in vivo testing, Domatinostat, Pracinostat and Entinostat. Using the treatment paradigm of ultra-low dose DAC+ HDACi (mainly to minimize toxicity) combined with αPD1 in the KPC orthotopic model we show that the triple combination (DAC + Entinostat + αPD1) is well-tolerated and results in a significant survival benefit compared to single and dual agent combinations. In addition, we also demonstrate a reduction in the Chi3l3 expressing myeloid cell population. Conclusions: Our results show that the combination of hypomethylating agent plus HDAC inhibitor has several beneficial effects on the tumor immune response in PDAC. HDAC inhibitors specifically reduce the suppressive myeloid cell population we previously identified after DAC therapy. The combination of dual low dose epigenetic therapy using a hypomethylating agent plus HDAC inhibitor followed by αPD1 results in significant survival benefit in the orthotopic KPC model. Citation Format: Stephen Muzyka, Arturo Orlacchio, Amanda Ackermann, Yasmine Chinda, Yoona Shim, Igor Dolgalev, Tamas Gonda. Low dose dual epigenetic therapy utilizing hypomethylating agents and HDAC inhibitors prolong survival in an orthotopic PDAC model and alter the tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B062.

Temporally resolved proteomics identifies nidogen-2 as a cotarget in pancreatic cancer that modulates fibrosis and therapy response.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by increasing fibrosis, which can enhance tumor progression and spread. Here, we undertook an unbiased temporal assessment of the matrisome of the highly metastatic KPC (Pdx1-Cre, LSL-KrasG12D/+, LSL-Trp53R172H/+) and poorly metastatic KPflC (Pdx1-Cre, LSL-KrasG12D/+, Trp53fl/+) genetically engineered mouse models of pancreatic cancer using mass spectrometry proteomics. Our assessment at early-, mid-, and late-stage disease reveals an increased abundance of nidogen-2 (NID2) in the KPC model compared to KPflC, with further validation showing that NID2 is primarily expressed by cancer-associated fibroblasts (CAFs). Using biomechanical assessments, second harmonic generation imaging, and birefringence analysis, we show that NID2 reduction by CRISPR interference (CRISPRi) in CAFs reduces stiffness and matrix remodeling in three-dimensional models, leading to impaired cancer cell invasion. Intravital imaging revealed improved vascular patency in live NID2-depleted tumors, with enhanced response to gemcitabine/Abraxane. In orthotopic models, NID2 CRISPRi tumors had less liver metastasis and increased survival, highlighting NID2 as a potential PDAC cotarget.

Ras-dependent activation of BMAL2 regulates hypoxic metabolism in pancreatic cancer.

To identify drivers of malignancy in human pancreatic ductal adenocarcinoma (PDAC), we performed regulatory network analysis on a large collection of expression profiles from laser capture microdissected samples of PDAC and benign precursors. We discovered that BMAL2 plays a role in the initiation, progression, post resection survival, and KRAS activity in PDAC. Functional analysis of BMAL2 target genes led us to hypothesize that it plays a role in regulating the response to hypoxia, a critical but poorly understood feature of PDAC physiology. Knockout of BMAL2 in multiple human PDAC cell lines revealed effects on viability and invasion, particularly under hypoxic conditions. Loss of BMAL2 also affected glycolysis and other metabolic processes. We found that BMAL2 directly regulates hypoxia-responsive target genes. We also found that BMAL2 is necessary for the stabilization of HIF1A upon exposure to hypoxia, but destabilizes HIF2A under hypoxia. These data demonstrate that BMAL2 is a master transcriptional regulator of hypoxia responses in PDAC and may serve as a long-sought molecular switch that distinguishes HIF1A- and HIF2A-dependent modes of hypoxic metabolism.

Abstract 467: Genetic ablation of microRNA-10b improves treatment outcomes in mouse models of pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a recalcitrant and lethal disease with an overall 5-year survival rate of less than 12%. PDAC is a heterogeneous disease characterized by highly aggressive cancer cells, extensive desmoplastic reaction, and hypovascularity. These unique features endow PDAC tumors with an array of innate resistance mechanisms against standard-of-care (SOC) chemotherapy: gemcitabine/nab-paclitaxel or FOLFIRINOX. Thus, there is clinical need to develop novel targets that enhance treatment efficacy of SOC chemotherapy. microRNA-10b (miR-10b) expression is frequently upregulated in PDAC and correlates with poor outcome. High levels of miR-10b expression within cancer cells correlates with poor treatment response to gemcitabine-containing neoadjuvant chemotherapy and poor overall clinical outcome. We and others reported that miR-10b exerts cancer cell-intrinsic pro-survival, anti-apoptotic, and pro-metastatic functions via downregulation of key target genes (i.e., BIM, PTEN, TIAM1). We hypothesize that upregulation of miR-10b expression is a chemoresistance mechanism to increase cell survival and inhibit apoptosis in PDAC. To test this hypothesis, we generated a KPC model (KrasG12D-driven, p53-deleted by Pdx1-Cre) with global Mir-10b gene knockout (Mir-10bKO). Animals were recruited when their tumor volume reached 200 mm3 by multi-mouse MR imaging and were randomly assigned to control or treatment groups (100 mg/kg of gemcitabine q4d). Tumor growth was monitored by weekly MR imaging sessions throughout the duration of the treatment. Untreated KPC;Mir-10bKO animals survived several weeks longer than untreated KPC animals (21.7 vs. 18 weeks, p = 0.056), suggesting a weak but detectable tumor promoting role of miR-10b. Remarkably, loss of miR-10b activity enhances treatment response to gemcitabine and significantly prolongs survival in this KPC model. Treated KPC animals lived only 5 weeks longer than untreated KPC (23 vs 18 weeks, p = 0.015), whereas KPC;Mir-10bKO lived 9 weeks longer (27 vs 18 weeks, p = 0.002). Notably, we observed a more durable response in treated female KPC;Mir-10bKO animals (n = 4) compared to any other experimental group (n > 5). We are currently performing tissue and molecular analyses to identify direct target genes involved in chemoresistance and sex-specific modulation of treatment response. Citation Format: Katarzyna Kempinska, Sudhakar Samuel, Saiprashanth Rajesh, Christiane Mallett, Lorenzo Sempere. Genetic ablation of microRNA-10b improves treatment outcomes in mouse models of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 467.

Abstract 6831: Single-cell-based epigenetic and transcriptomic analysis in the KPC pancreatic cancer mouse model revealed dynamiccharacteristic subtypes of cancer-associated fibroblasts

Abstract Pancreatic cancer features robust fibrosis, with 40-80% fibrotic tissue and a dense stroma, highlighting the crucial need to grasp cellular interactions in the cancer microenvironment. In this study, we used single-cell RNA sequencing (scRNA-seq) to analyze pancreatic tissues from 60-day-old normal mouse (n=1) (GSE125588) and KPC (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre) cancer model with tumor diameter of 1-2 cm (n=2) (GSE129455). By examining fibroblast clusters, we identified distinct subtypes of cancer-associated fibroblasts (CAFs), and revealed four clusters: normal fibroblasts cluster, inflammatory CAFs (iCAFs) cluster marked by increased expression of IL6 and other cytokines, myofibroblast CAFs (myCAFs) cluster solely in the KPC model, and antigen-presenting CAFs (apCAFs) cluster expressing Mesothelin. We found that expression pattern of iCAFs in the KPC appeared similar to normal cells than myCAFs, suggesting a potential role in inhibiting cancer. it is reported that mesothelial cells might transition to apCAFs under the influence of IL6 and TGFβ due to tissue damage and inflammatory effects. This transition was visualized in our apCAF cluster, suggesting a potential pathway for the transformation of mesothelial cells into apCAFs. Next, in order to analyze epigenetic profiles of fibroblasts, we conducted single-cell Assay for Transposase-Accessible Chromatin with high-throughput sequencing (scATAC-seq) using 90-day-old KPC mice (n=2) and a wild-type mouse (n=1).scATAC-seq showed that there are mainly two clusters of fibroblasts, one cluster corresponded to myCAF, enriched with PDPN, while the other represented iCAF cluster, enriched with COL1A2. These data suggest that in terms of the epigenetic state, myCAF contain several different CAFs and have the potential to differentiate into either myCAFs or other type of based on certain stimulation. Ultimately, CAF differentiated into three distinct subtypes as revealed by scRNA-seq. This highlights that despite similar epigenetic states, various transcription factors characterize the functionality of these fibroblast subtypes. Further exploration of the underlying mechanism that lead to different CAF subtypes might help in controlling the microenvironment of pancreatic cancer. Citation Format: Keita Sasaki, Keiko Shinjo, Xingxing Wang, Kohei Kumegawa, Reo Raruyama, Shinji Mii, Yoshiteru Murofushi, Miho Suzuki, Atsushi Enomoto, Yutaka Kondo. Single-cell-based epigenetic and transcriptomic analysis in the KPC pancreatic cancer mouse model revealed dynamiccharacteristic subtypes of cancer-associated fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6831.

Abstract 6816: Prevention of cancer cachexia by inhibition of soluble epoxide hydrolase

Abstract Background: Cancer cachexia is a devastating syndrome characterized by progressive muscle wasting and hyperinflammation. The underlying mechanisms remain poorly understood with no treatment options available. Cachexia is driven by systemic inflammation, pro-inflammatory cytokines, and apoptotic cell death (debris). The resolution of inflammation as an active biochemical process is regulated by novel pro-resolving lipid mediators. Arachidonic acid-derived epoxyeicosatrienoic acids (EETs) are lipid mediators stimulating inflammation resolution. EETs are rapidly metabolized by the enzyme soluble epoxide hydrolase (sEH). sEH is a critical cause of inflammation and a biomarker in several chronic inflammatory diseases. sEH inhibitors promote tissue regeneration and counter-regulate pro-inflammatory cytokines. We hypothesized that pharmacological inhibition of the sEH may prevent cachexia. Methods: We investigated murine cancer cachexia models using genetically engineered pancreatic (KPC) and prostate (TRAMP C1) tumor cell lines, and “TRAMP” mice (transgenic adenocarcinoma of the mouse prostate). We performed LC-MS/MS-based profiling of bioactive lipids in plasma from KPC and colon cancer (CT26)-induced cachectic mice. Results: KPC cells injected intraperitoneally induced 19.7% and 54.1% reduction in muscle weight in the tibialis anterior and soleus, respectively, in immunocompetent C57BL/6 mice compared to non-tumor bearing (NTB) mice. LC-MS/MS revealed KPC and CT26 cachexia induced a pro-inflammatory eicosanoid-driven cytokine storm. The human sEH inhibitor EC5026 delayed the onset of cachexia, leading to sustained survival over 250 days post-injection in the KPC model (n=15 mice/group). sEH expression was increased in the gastrocnemius of KPC mice by day 22 post-tumor cell injection vs. NTB. EC5026 counter-regulated sEH expression compared to vehicle-treated. In the TRAMP model, 5/5 of mice treated with EC5026 survived 230 days post-treatment compared to no survival of vehicle-treated mice (n=5). The transplanted KPC and TRAMP showed reduced CD4+ and CD8+ T lymphocytes and B lymphocytes in the tibialis anterior and gastrocnemius vs. NTB after flow cytometry analysis. EC5026 stimulated a general increase in CD3+ T lymphocytes and counter-regulated an eicosanoid-driven cytokine storm as shown by cytokine assays. EC5026 treatment increased tibialis anterior and gastrocnemius muscle weights and body weights compared to vehicle-treated KPC and TRAMP mice. Conclusions: sEH inhibition may be a novel therapeutic approach to cachexia by targeting the immune response via stimulation of resolution of inflammation without toxicity or immunosuppression. Since sEH inhibitors have proven safe and effective in clinical trials for controlling multiple inflammatory diseases, this study provides a basis for the rapid clinical translation of sEH inhibitors to prevent/reverse cachexia in cancer patients. Citation Format: Rachel L. Bayer, Sarina Virani, Michael Gillespie, Jacqueline Capuano, Keira Smith, Katherine Quinlivan, Kimberly Vasquez, Jun Yang, Haixia Yang, Nicholas Mitsiades, Bruce D. Hammock, Dipak Panigrahy. Prevention of cancer cachexia by inhibition of soluble epoxide hydrolase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6816.

Abstract 2957: Senescence of stromal, endothelial, and myeloid cells in the tumor microenvironment has distinct effects on cancer progression

Abstract Accumulation of senescent cells, undermining the function of vital organs, is a culprit of aging. The effects of senescence in different types of cells on tumor growth and metastasis remain incompletely characterized. Cell senescence has been shown to result from inactivation of Telomerase (TERT), an enzyme that maintains telomere length and has obscure roles in regulating gene expression and supporting mitochondrial function. As we reported previously, knockout (KO) of Tert gene in mesenchymal stromal cells (MSC) of Pdgfra+ or Pdgfrb+ lineages leads to premature replicative stromal cell senescence in adipose tissue. To assess the effect of senescence in endothelial and myeloid cells, we also created mice with Tert KO driven by the Tie2e promoter or the LysM promoter, respectively. Here, to investigate the effect of these three KOs on cancer, we used syngeneic graft models of breast, prostate, and pancreatic carcinoma. Overall, Tert KO in cells of Pdgfra+, Pdgfrb+, Tie2e+, or LysM+ lineage had a suppressive effect on tumor growth. The strongest tumor suppression effect of Tert KO was observed in female mice orthotopically grafted with E0771 breast cancer cells. Intermediate suppressive effects of Tert KO were observed in mice orthotopically grafted with KPC pancreatic ductal adenocarcinoma cells, whereas the effect on RM1 prostate cancer cells grafts in male mice was the least significant. MSC Tert KO and myeloid Tert KO also suppressed the growth of breast and prostate cells grafts. Importantly, spontaneous metastases to the liver were induced by Tie2e-Tert KO in all mice with pancreatic KPC grafts. Analysis of adipose tissue in this endothelial senescence model revealed vasculature abnormality, leakiness, and tissue hypoxia, which provides a mechanistic explanation for increased tumor dissemination. In contrast, LysM-Tert KO did not promote KPC metastases, consistent with the lack of contribution of myeloid cells to the vasculature. Pdgfrb-Tert KO in the KPC model resulted in liver metastases observed for some but not all mice, consistent with the role of MSC in supporting vascular integrity. To test an independent model of replicative MSC senescence, we exhausted their pool by several cycles of ganciclovir treatment in mice expressing thymidine kinase under the control of Pdgfrb promoter. Upon KPC grafting in this model, reduced primary pancreatic tumor growth was also observed and liver metastases were also induced. We conclude that senescence of distinct cells in the tumor microenvironment affects early and late stages of cancer progression differently. Specifically, our data indicate that vascular cell senescence suppresses tumor growth but promotes metastases. These findings are important to consider as new approaches designed to mitigate cell senescence are investigated in the context of cancer. Citation Format: Joseph Rupert, Zhanguo Gao, Yongmei Yu, Mikhail Kolonin. Senescence of stromal, endothelial, and myeloid cells in the tumor microenvironment has distinct effects on cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2957.

Abstract 716: Depletion of stromal cells remodels tumor microenvironment and potentiates immune checkpoint blockade in pancreatic cancer

Abstract During carcinoma progression, mesenchymal stromal cells (MSCs) become recruited to tumors and contribute to the pool of cancer-associated fibroblasts (CAFs). Sub-populations of CAFs, have diverse and incompletely understood effects on disease progression and resistance to therapy. Adipose stromal cells (ASCs), the MSCs from fat tissue increasingly recruited by carcinomas in the context of obesity, have been shown to support cancer progression. Here, the roles of perivascular CAFs expressing platelet-derived growth factor receptor beta (Pdgfrb) and of CAFs expressing non-glycanated decorin (ngDCN), a marker of ASCs, were analyzed. We used mice orthotopically grafted with pancreatic ductal adenocarcinoma (KPC) cells. Ablation of cells expressing thymidine kinase under the control of Pdgfrb promoter with ganciclovir resulted in suppression of primary pancreatic tumor growth. However, ablation Pdgfrb+ cells induced spontaneous metastases to the liver. For depletion of ngDCN+ cells, we used a hunter-killer peptide D-CAN, which has been previously reported to induce apoptosis of ASCs and CAFs in mouse models. Here, D-CAN also had a negative effect on pancreatic tumor growth, however, there was no significant effect on metastatic dissemination. Single cell RNA sequencing of tumors demonstrated that targeting of Pdgfrb+ and ngDCN+ stromal cells had distinct effects on sub-populations of CAFs. Endothelial cell and cancer cell survival was decreased by depletion of either Pdgfrb+ or ngDCN+ stroma. However, pathway analysis revealed that depletion of Pdgfrb+ and ngDCN+ stromal cells has different effects on the markers of cancer cell aggressiveness. D-CAN treatment, and to a less extent Pdgfrb+ cell ablation, suppressed macrophage M2 polarization and increased infiltration of cytotoxic T-lymphocytes. This observation, confirmed in the MyCap graft model of prostate cancer, suggested that ablation of ngDCN+ stroma should synergize with immune checkpoint inhibitors. We tested the effect of D-CAN on the efficacy of anti-PDL1 antibody in the orthotopic KPC model. Compared to D-CAN and anti-PDL1 antibody alone, combination treatment had a synergistic effect on tumor growth. Importantly, liver metastases were suppressed by the D-CAN/anti-PDL1 antibody combination, but not by individual agents. We conclude that improved approaches to target mesenchymal stroma in tumors may be effective in combination with immunotherapy. Citation Format: Joseph Rupert, Alexes Daquinag, Lingyi Cai, Yulin Dai, Zhongming Zhao, Dimitris Anastassiou, Mikhail Kolonin. Depletion of stromal cells remodels tumor microenvironment and potentiates immune checkpoint blockade in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 716.

Abstract 5932: Immunomodulatory and anticancer effects of cyclical gemcitabine/nab-paclitaxel with continuous dosing of tinodasertib (AUM001) in preclinical KPC model

Abstract Background: PDAC has a poor 5-year survival outcome, and its aggressiveness limits the treatment efficacy. This is often driven by dysregulated mRNA translation of pro-tumorigenic genes that contribute to the proliferative and metastatic nature of PDAC. Synergizing chemotherapy with inhibition of oncogenic mRNA translation via MNK-coupled phosphorylation of eIF4E could potentially be viable. To support this hypothesis, we combined AUM001, a MNK inhibitor, with gemcitabine (GEM)-nab-paclitaxel (nPTX) with the aim of modulating tumor growth kinetics and maintaining it in a stable state. Methods: PDAC KPC cells were subcutaneously inoculated into BL6 mice and randomized to five treatment groups. Chemotherapy involved administering GEM-nPTX intraperitoneally on days 1, 4, and 8. AUM001 was given orally daily for 8 days for both mono- and combination therapies and was further administered for an additional 3 weeks in continuous dosing group. IHC staining for Ki67, MVD, and stromal markers was conducted on FFPE sections as well as Western blot analysis of tumor tissues. Apoptosis was assessed using Annexin V. Immune profiling were evaluated by flow cytometric assay and GeoMx spatial transcriptomics analysis. Results: The administration of GEM-nPTX and AUM001 (GPA) or GEM-nPTX alone (GP) led to an average 38% reduction in net tumor growth with no observed toxicity. Notably, continuous dosing of AUM001 (GPAc) following the cessation of chemotherapy exhibited a 45% decrease in net tumor growth (P=0.056) when compared to GPA. EIF4E phosphorylation and Myc expression were markedly inhibited in GPAc with lower recovery rate for MVD as compared to the GPA group at EOO (P < 0.05). A higher fraction of apoptotic cells was detected in the GPAc group at EOO than GPA group in reference to GP group (P=NS). GPA also resulted in a lower frequency of NK cells which was partially lifted in GPAc during observation phase. Macrophages which are known to have a pro-tumor role initially increased in frequency upon addition of AUM during chemotherapy, but subsequently decreased during observation. These modulations in infiltrating immune cells correlated with the differential expression of several immune related genes in the tumor associated CD45+ immune cells, revealed by the GeoMx analysis. Conclusions: This study showed that sustained administration of AUM001 alongside cyclical GEM-nPTX is well-tolerated with good anti-tumor outcomes. Additionally, the maintenance of AUM001 may enhance the decrease in pro-oncogenic protein expression and remodel the tumor immune microenvironment. In conclusion, continuous dosing of AUM001 following the cessation of chemotherapy could stabilize the tumor state, potentiating its application in clinical settings as maintenance therapy for PDAC. Citation Format: Wai Fook Leong, Sze Sing Lee, Jyue Yuan Lim, Siwen Sylvialin Chen, Fara Faye Desacola Regis, Romana Vidergar, Subhra K. Biswas, Balram Chowbay. Immunomodulatory and anticancer effects of cyclical gemcitabine/nab-paclitaxel with continuous dosing of tinodasertib (AUM001) in preclinical KPC model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5932.

Abstract 5275: Celecoxib and chloroquine enhance the antitumor effect of immune checkpoint blockade therapy, in a syngeneic mouse model for pancreatic cancer

Abstract Immune checkpoint inhibitors (ICIs) have shown impressive clinical activities in some cancer types, but they have yet to demonstrate any meaningful clinical benefit in patients with pancreatic ductal adenocarcinoma (PDAC) except those with microsatellite instability high (MSI-H) tumors. Several recent studies showed that concomitant use of NSAIDs such as celecoxib by patients with certain cancers could improve the survival benefit of ICI treatment (e.g., Sebastian et al, Clin Lung Cancer, 2023;). Furthermore, autophagy inhibitors such as chloroquine were also reported to sensitize pancreatic tumors to the treatment of ICIs in mouse models for PDAC (Yamamoto, et al, Nature, 2020). We recently reported that the combination of chemotherapy (triple combination of nab-paclitaxel + gemcitabine + cisplatin) and a mouse surrogate of the IFc-enhanced anti-CTLA-4 antibody botensilimab, showed significantly improved antitumor activity in immune competent KPC syngeneic mouse model for PDAC [Tanne, et al, Cancer Res (13_Supplement), 2020]. In this study, we sought to determine whether or not the addition of celecoxib and/or chloroquine further improve the antitumor activity of triple chemotherapy and ICI in the syngeneic KPC model. We first found that celecoxib (30mg/kg P.O. QDx21) significantly enhanced the tumor growth inhibition when added to triple chemotherapy (Gemcitabine 70mg/kg I.P., Cisplatin 4mg/kg I.P., Nab-paclitaxel 25mg/kg I.V. days 1,4 and 7) plus the murine equivalents of botensilimab (100ug I.P. BIW for three weeks) and balstilimab (200ug I.P. BIW for three weeks) (p=0.004, N=10). We then performed a pilot study to assess if adding chloroquine could further improve the activity. While adding chloroquine (60mg/kg I.P. QDx21) alone did not significantly affect tumor growth the addition of both chloroquine and celecoxib greatly improve the tumor growth inhibition compared to just the triple chemotherapy and ICIs (p=0.009, N=5). Overall, our data showed that celecoxib or the combination of celecoxib and chloroquine could substantially sensitize the mouse pancreatic tumors to the treatment of chemotherapy and ICIs. (This work was supported in part by the Seena Magowitz Foundation and the Purple Pansies) Citation Format: Jaeger Moore, Dhan Chand, Daniel Levey, Daniel Von Hoff, Haiyong Han. Celecoxib and chloroquine enhance the antitumor effect of immune checkpoint blockade therapy, in a syngeneic mouse model for pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5275.

Abstract A078: Response and resistance to KRAS inhibition in PDAC mouse models

Abstract Pancreatic ductal adenocarcinoma (PDAC), among the most lethal cancers with a 5-year survival rate of 11%, is a KRAS-driven disease. Studies have shown that a single glycine (G) to aspartic acid (D) substitution on codon 12 of KRAS protein (known as KRASG12D mutation) appears in 41% of all PDAC cases, highlighting the necessity of targeting KRASG12D as a therapeutic approach. MRTX1133, a recently developed potent, non-covalent, direct, and selective KRASG12D small molecule inhibitor demonstrates a high-affinity interaction with KRASG12D. Previous studies have shown striking efficacy of MRTX1133 on growth inhibition of mouse/human PDAC tumor cells carrying KRASG12D mutation in various in vivo and in vitro models, during short-term treatment. Here in our study, we investigated the efficacy of continuous long-term treatment with MRTX1133 in two different PDAC mouse models: KPC (a genetically engineered mouse model harboring a KRASG12D mutation) and its syngeneic orthotopic model. In both models, tumor sizes were detected by ultrasound imaging and after reaching 100-300 mm3 in volume, mice were enrolled in either MRTX1133 or vehicle treatment (30mg/kg, IP, BID). Tumor sizes were monitored by ultrasound regularly and the treatment continued until mice reached clinical endpoint. At endpoint, mice were sacrificed, and tumors were harvested for a series of downstream experiments. Our results showed that while, in both models, vehicle treated tumors grew aggressively and mice reached endpoint in average 6 days post treatment, drug treated tumors regressed rapidly and survival rate increased significantly to as late as 222 days post treatment in KPC model. However, in both models, drug treated tumors ultimately relapsed, developed resistance to the drug, and resulted in death. Further analysis of KPC mice ultrasound images evidenced that the relapsed tumors occurred at the same sites of the primary tumors. This suggests the possibility of relapsed tumors arising from the regrowth of the dormant residual cells from the primary tumors. Re-implantation of the resistant tumors either in the flank or pancreata of the new wildtype mice resulted in the formation of drug-resistant tumors in hosts. In addition, histopathology analysis of the tumor sections demonstrated a significant increase in the ratio of invasive PDAC to PanIN areas in the drug resistant tumors. Furthermore, multiplex immunofluorescence of both MRTX1133 resistant and vehicle treated tumors from KPC mice demonstrated co-expression of markers representing basal-like and classical PDAC subtypes. Moreover, flow cytometry analysis indicated an increase in the myeloid population of the resistant tumors comparing to the vehicle treated group. In the future directions, through applying a broad range of cellular and molecular experiments we will characterize the kinetics of KRAS inhibitor drug resistance and define possible combination therapy startegies to overcome drug resistance mechanisms. Citation Format: Laleh Abbassi, Jullien Dilly, Annan Yang, Giselle Uribe, Branden Parent, Connor Hennessey, Kevin Kapner, Alexander Jordan, Shatavisha Dasgupta, Micaela Morgado, Taimour Baslan, Li Qiang, Peter Winter, Seema Chugh, Alex Shalek, Ben Stanger, Srivastan Raghavan, Scott Lowe, Brian Wolpin, Stephanie Dougan, Jonathan Nowak, Andrew Aguirre. Response and resistance to KRAS inhibition in PDAC mouse models [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 A078.

Abstract C097: KRASG12C exhibits allele-specific biology in pancreatic cancer and targeting CD24 sensitizes KRASG12C-driven tumors to sotorasib treatment

Abstract Pancreatic adenocarcinoma (PDAC) is predominantly driven by KRAS oncogene. While all oncogenic KRAS mutations result in its hyperactivation, the allele-specific biology of KRAS mutations remain poorly understood. Addressing such issues has become particularly important with the recent advancements in the development of allele-specific and pan-KRAS inhibitors. Among the KRAS inhibitors, KRASG12C inhibitors (G12Ci), Sotorasib and Adagrasib, have been approved for treating KRASG12C-mutant lung cancer. Sotorasib also exhibited promising anticancer activity in human PDAC, with 21% response rate and over 70% patients experiencing certain level of tumor control. However, targeting KRAS oncogenes faces the major challenge of therapy resistance, which severely limited its survival benefit. While multiple studies have elucidated potential resistance mechanisms to G12Ci in lung cancer, the molecular mechanisms underlying its resistance in PDAC remains elusive, largely owing to the scarcity of relevant and faithful preclinical models. Here, we report the generation of a new autochthonous KRASG12C-driven PDAC model (p48Cre; LSL-KrasG12C; p53L/+, KCPC). Compared to the KRASG12D-driven KPC model (p48Cre; LSL-KrasG12D; p53L/+), tumor development in KCPC model is significantly delayed accompanied with corresponding decrease in both KRAS signaling and proliferation in premalignant lesions. As a result, the overall survival of the KCPC model is significantly prolonged compared to the KPC model, indicating allele-specific discrepancy of KRAS mutations during PDAC initiation. However, upon malignant progression, KRASG12C-driven mouse PDAC exhibit similar histopathological and molecular features as KRASG12D-driven tumors. Furthermore, similar to the clinical observation, KCPC tumors exhibit only partial response to Sotorasib treatment. CyTOF analysis indicates that such tumor persistence following G12Ci treatment occurs even in the presence of conversion from a ‘cold’ pro-tumor immune microenvironment to a ‘hot’ anti-tumor immune environment, as evidenced by the prominent increase in both cytotoxic T cells and M1-like macrophages. Interestingly, we discovered that the expression of CD24, which was recently identified as a new ‘do not eat me’ signal, is significantly upregulated on the surface of persist tumor cells, which suppresses the function of anti-tumor macrophages. Treatment with anti-CD24 blocking mAb enhanced macrophage-mediated phagocytosis in vitro and significantly sensitizes KCPC-derived xenograft tumors to Sotorasib treatment in vivo in a macrophage-dependent manner. Similar upregulation of CD24 following KRAS inhibition and therapeutic benefit of co-targeting CD24 and KRAS were also observed in KRASG12D-driven mouse and human PDAC, implicating the potential of such combination in a broader spectrum of KRAS-driven PDAC. Together, our findings revealed the allele-specific function of KRASG12C in the development of PDAC and identified a novel immune combination to improve the outcome of KRAS targeted therapy in PDAC. Citation Format: Yongkun Wei, Minghui Liu, Phuoc T. Nguyen, Dean N. Pan, Wantong Yao, Haoqiang Ying. KRASG12C exhibits allele-specific biology in pancreatic cancer and targeting CD24 sensitizes KRASG12C-driven tumors to sotorasib treatment [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 C097.

Abstract A089: Quantitative MRI metrics and single cell transcriptome reveal effects of stroma-directed drug and chemotherapy in a GEM model of pancreatic cancer

Abstract Hypothesis: The dense stroma present in pancreatic ductal adenocarcinoma (PDA) harbors a unique tumor microenvironment (TME) that is immune suppressive and underlies the chemo-resistance of PDA. We have evaluated synthetic vitamin D combined with chemoimmunotherapy in a pilot clinical study (NCT03519308). To elucidate the underlying mechanisms of this treatment, we designed a matched study in a genetically engineered mouse model of PDA (KPC mice) to test the hypothesis that synthetic vitamin D enhances the effect of chemotherapy with or without immune checkpoint inhibition. To detect changes in TME, we have integrated diffusion weighted (DW) and dynamic contrast enhanced (DCE) MRI with immunohistochemistry (IHC) and single cell RNA sequencing (scRNAseq). Methods: KPC mice were randomly assigned to one of four groups: 1) Control (untreated); 2) Nab-paclitaxel + gemcitabine + cisplatin (Chemo); 3) Chemo + synthetic vitamin D (calcipotriol, Cal); 4) Chemo + Cal + PD-L1. The treatment lasted for 14 days followed by euthanasia and tumor collection for IHC and scRNAseq. Respiratory motion-robust DW- and DCE-MRI were performed on treatment days 0, 7 and 14. Results: Compared to consistent tumor growth (measured by MRI) in the Control group, both progression and regression were observed in groups 2) - 4), suggesting that the KPC model captured the varied treatment responses observed in the clinic. Capillary perfusion/permeability of the tumor measured by Ktrans (a DCE-MRI metric) was reduced at day 7 in the Chemo group (P<0.05), whereas Chemo+Cal reversed the reduction of Ktrans. Chemo-induced Ktrans reduction was corroborated with a significant reduction of microvascular density by IHC of CD31 compared to the Control. Addition of PD-L1 did not appear to enhance the tumor growth delay by Chemo+Cal. scRNAseq analysis revealed that while Chemo significantly delayed the tumor growth compared to the Control, it increased the portion of mesenchymal cells in the tumor and upregulated epithelial-to-mesenchymal transition (EMT) genes known to resist chemotherapy. In contrast, Chemo+Cal increased the fraction of epithelial cells and downregulated EMT genes compared to Chemo. Trasncriptome analysis of the tumor cells demonstrated that Chemo treatment increased the expression of Fibronectin 1 (FN1), a gene associated with tumor invasiveness, EMT, and metastasis, while lowering the expression of Trefoil Factor Family1 (Tff1), which is related to tumor-suppression and chemosensitivity. Chemo+Cal treatment reversed the expression of both FN1 and Tff1 gene compared to Chemo treatment. Conclusion: Single cell transcriptome analysis provided valuable insights into treatment-induced molecular subtype changes, which may predict future resistance and invasiveness. Our study revealed important mechanisms of calcipotriol that enhance the efficacy of chemotherapy by improving microvascular perfusion and permeability and by reducing chemo-resistance by enriching epithelial cells in PDA tumors. Citation Format: Hoon Choi, Mamta Gupta, Thomas Karasic, Miguel Joaquim, Emma Furth, Stephen Pickup, Cynthia Clendenin, Hee Kwon Song, Yong Fan, Jeffrey Duda, James Gee, Mark Rosen, Peter O’Dwyer, Rong Zhou. Quantitative MRI metrics and single cell transcriptome reveal effects of stroma-directed drug and chemotherapy in a GEM model of pancreatic cancer [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 A089.

Abstract A029: Re-purposing non-oncology agent itraconazole to target the dynamic cellular ecosystem of pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival of only 10% and persists as the third most common cause of cancer-related death in Western societies. Precision medicine is a likely future for all cancer treatment, but may have its greatest impact in less common, high-mortality and molecularly heterogeneous cancers, including pancreatic cancer. Fortunately, in the era of Big Data, bioinformatic analyses of genomic datasets have helped us gain new insights of how novel molecular-guided therapies can modulate signalling within tumors and their ecosystem (Vennin et al. 2017; Waddell, N, Pajic, M, Patch, AM. et al. 2015; Chou A. et al. 2018; Lau, MT. et al 2020). In this study, we delineate, at single-cell resolution, the complex stromal mechanisms by which a clinically-used, orally available anti-fungal agent itraconazole improves the overall anti-tumor response in PDAC. Single-cell RNAseq analysis of the genetically-engineered KPC model (LSL- KrasG12D;LSL-Trp53 R172H/+) revealed significant downregulation of the pro-tumorigenic CD105+ cancer-associated fibroblast (CAF) signature post-itraconazole therapy in vivo. Moreover, single-cell trajectory inference of CAFs subsets revealed a marked effect of treatment on myofibroblasts (myCAFs), which produce the fibrotic PDAC desmoplasia. In support of these findings, immunofluorescence analyses further demonstrated decreased deposition of collagen and altered matrix remodelling within itraconazole-treated tumors. Within the antigen-presenting apCAF and inflammatory iCAF populations, itraconazole treatment led to decreased pro-tumorigenic Igf1-Itga6:Itgb4 signalling to specific tumor cell subsets, which was functionally associated with reduced metastatic colonisation in vivo. apCAFs also showed decreased expression of H2-Aa and CD74, major antigens expressed by apCAFs which act as decoys for corresponding receptors on CD4+/CD8+ T cells, thus leading to their impaired activation (Lakins et al, 2018). These potentially promising effects on the immune environment of PDAC were supported by significant positive changes within macrophage populations, including enrichment of M1-like pro-inflammatory macrophage signatures (confirmed via immunofluorescence), and further associated with positive signalling via Cxcl9-Cxcr3 and Tnf1rs-1a/1b ligand-receptors. scRNAseq revealed dampening of regulatory T cell homing and functional potency signals within the pancreatic tumor microenvironment was associated with increased CD8+ T-cell infiltration post-itraconazole treatment, and importantly, significantly improved response to immune checkpoint blockade in an advanced in vivo model of pancreatic cancer. In summary, these integrated bioinformatic and molecular studies provide scientific rationale for the development of itraconazole and immunotherapy combination in pancreatic cancer. Citation Format: Diego Chacon-Fajardo, Sean Porazinski, Jennifer Man, Howard Yim, Emad El-Omar, Anthony Joshua, Marina Pajic. Re-purposing non-oncology agent itraconazole to target the dynamic cellular ecosystem of pancreatic cancer [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 A029.

Abstract A061: A novel genetically engineered mouse model of Myc-driven pancreatic cancer recapitulates phenotypic heterogeneity, metastasis, and therapy resistance seen in clinical populations

Abstract One of the most aggressive cancers in the US is pancreatic ductal adenocarcinoma (PDAc), whose incidence is expected to rise in the coming years. The standard-of-care treatments have improved over the past few decades, but they still primarily involve harsh chemotherapy cocktails and have only marginally increased overall survival, to a 5-year survival rate of 12 percent. c-MYC (MYC), a potent transcription factor, is frequently dysregulated in PDAc. In PDAc and other cancers, MYC plays a significant role in tumor progression, and its dysregulation has been linked to tumor aggressiveness and therapeutic resistance. In various mouse models of cancer, it has been demonstrated that oncogenic MYC expression controls components of the tumor microenvironment (TME). MYC expression in PDAc has been associated with a desmoplastic immune suppressive TME, though the precise mechanism is still unknown. In this study, we introduce a novel genetically modified mouse model (GEMM) of PDAc that can be used to simulate the disease's heterogeneity and investigate how MYC controls the tumor’s immune and stromal microenvironments. In the pancreas of our KMCERT2 model, both mutant Kras and lowly deregulated Myc are expressed through inducible Cre-driven mechanisms. Our research demonstrates how deregulated MYC cooperates with oncogenic KRASG12D in the adult pancreas to promote PDAc, faithfully reproducing the inter- and intra-tumoral heterogeneity observed in clinical PDAc populations. Currently, an embryonic KrasG12D- and p53 loss/mutant-driven PDAc model (KPC) is used for the majority of murine studies of PDAc. To learn more about the mechanisms underlying observed phenotypes in our KMCERT2 model, RNA- and DNA sequencing were performed on microdissected autochthonous tumor specimens as well as KMCERT2 tumor-derived cell lines. In contrast to the KPC model, our inducible KMCERT2 model of PDAc exhibits genetic alterations, such as CDKN2A loss, comparable to human disease. A distinct loss of nuclear p53 expression is also observed. Intriguingly, multiplexed immunohistochemistry analysis of immune cell composition showed that MYC-driven tumors have a higher density of antigen-presenting cells (APCs) when compared to spontaneous KPC tumors. Standard-of-care medications like gemcitabine and FOLFIRINOX are frequently ineffective against human PDAc. Orthotopic therapeutic studies using our KMCERT2 tumor-derived cell lines show a variety of responses to these therapies, with both resistant and sensitive tumor models, allowing us to use this model to better understand the mechanisms causing therapeutic resistance and to test new therapies. Additionally, we observe consistent liver metastasis in both spontaneous and orthotopic transplant settings. Together, these studies investigate the function of dysregulated MYC expression in murine PDAc metastatic behavior, immune phenotypes, and therapeutic response. It also offers mouse models of PDAc that are both spontaneous and orthotopic, recapitulating the complex and highly metastatic nature of the human disease. Citation Format: Isabel A. English, Patrick J. Worth, Kevin A. MacPherson, Michael B. Heskett, Vidhi Shah, Jackie L. Phipps, Courtney Betts, Shamilene Sivagnanam, Carl Pelz, Amy S. Farrell, Brittany L. Allen-Petersen, Meghan M. Joly, Motoyuki Tsuda, Colin J. Daniel, Jenny Eng, Xiaoyan Wang, Ashley L. Kiemen, Laura D. Wood, Terry K. Morgan, Mary C. Thoma, Jennifer M. Chu, Paul T. Spellman, Lisa M. Coussens, Ellen M. Langer, Rosalie C. Sears. A novel genetically engineered mouse model of Myc-driven pancreatic cancer recapitulates phenotypic heterogeneity, metastasis, and therapy resistance seen in clinical populations [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 A061.

Intracellular Salmonella delivery of an exogenous immunization antigen refocuses CD8 T cells against cancer cells, eliminates pancreatic tumors and forms antitumor immunity.

Immunotherapies have shown great promise, but are not effective for all tumors types and are effective in less than 3% of patients with pancreatic ductal adenocarcinomas (PDAC). To make an immune treatment that is effective for more cancer patients and those with PDAC specifically, we genetically engineered Salmonella to deliver exogenous antigens directly into the cytoplasm of tumor cells. We hypothesized that intracellular delivery of an exogenous immunization antigen would activate antigen-specific CD8 T cells and reduce tumors in immunized mice. To test this hypothesis, we administered intracellular delivering (ID) Salmonella that deliver ovalbumin as a model antigen into tumor-bearing, ovalbumin-vaccinated mice. ID Salmonella delivers antigens by autonomously lysing in cells after the induction of cell invasion. We showed that the delivered ovalbumin disperses throughout the cytoplasm of cells in culture and in tumors. This delivery into the cytoplasm is essential for antigen cross-presentation. We showed that co-culture of ovalbumin-recipient cancer cells with ovalbumin-specific CD8 T cells triggered a cytotoxic T cell response. After the adoptive transfer of OT-I CD8 T cells, intracellular delivery of ovalbumin reduced tumor growth and eliminated tumors. This effect was dependent on the presence of the ovalbumin-specific T cells. Following vaccination with the exogenous antigen in mice, intracellular delivery of the antigen cleared 43% of established KPC pancreatic tumors, increased survival, and prevented tumor re-implantation. This response in the immunosuppressive KPC model demonstrates the potential to treat tumors that do not respond to checkpoint inhibitors, and the response to re-challenge indicates that new immunity was established against intrinsic tumor antigens. In the clinic, ID Salmonella could be used to deliver a protein antigen from a childhood immunization to refocus pre-existing T cell immunity against tumors. As an off-the-shelf immunotherapy, this bacterial system has the potential to be effective in a broad range of cancer patients.

A first-in-class pan-lysyl oxidase inhibitor impairs stromal remodeling and enhances gemcitabine response and survival in pancreatic cancer.

The lysyl oxidase family represents a promising target in stromal targeting of solid tumors due to the importance of this family in crosslinking and stabilizing fibrillar collagens and its known role in tumor desmoplasia. Using small-molecule drug-design approaches, we generated and validated PXS-5505, a first-in-class highly selective and potent pan-lysyl oxidase inhibitor. We demonstrate in vitro and in vivo that pan-lysyl oxidase inhibition decreases chemotherapy-induced pancreatic tumor desmoplasia and stiffness, reduces cancer cell invasion and metastasis, improves tumor perfusion and enhances the efficacy of chemotherapy in the autochthonous genetically engineered KPC model, while also demonstrating antifibrotic effects in human patient-derived xenograft models of pancreatic cancer. PXS-5505 is orally bioavailable, safe and effective at inhibiting lysyl oxidase activity in tissues. Our findings present the rationale for progression of a pan-lysyl oxidase inhibitor aimed at eliciting a reduction in stromal matrix to potentiate chemotherapy in pancreatic ductal adenocarcinoma.

Induction of cancer neoantigens facilitates development of clinically relevant models for the study of pancreatic cancer immunobiology

Neoantigen burden and CD8 T cell infiltrate are associated with clinical outcome in pancreatic ductal adenocarcinoma (PDAC). A shortcoming of many genetic models of PDAC is the lack of neoantigen burden and limited T cell infiltrate. The goal of the present study was to develop clinically relevant models of PDAC by inducing cancer neoantigens in KP2, a cell line derived from the KPC model of PDAC. KP2 was treated with oxaliplatin and olaparib (OXPARPi), and a resistant cell line was subsequently cloned to generate multiple genetically distinct cell lines (KP2-OXPARPi clones). Clones A and E are sensitive to immune checkpoint inhibition (ICI), exhibit relatively high T cell infiltration, and have significant upregulation of genes involved in antigen presentation, T cell differentiation, and chemokine signaling pathways. Clone B is resistant to ICI and is similar to the parental KP2 cell line in terms of relatively low T cell infiltration and no upregulation of genes involved in the pathways noted above. Tumor/normal exome sequencing and in silico neoantigen prediction confirms successful generation of cancer neoantigens in the KP2-OXPARPi clones and the relative lack of cancer neoantigens in the parental KP2 cell line. Neoantigen vaccine experiments demonstrate that a subset of candidate neoantigens are immunogenic and neoantigen synthetic long peptide vaccines can restrain Clone E tumor growth. Compared to existing models, the KP2-OXPARPi clones better capture the diverse immunobiology of human PDAC and may serve as models for future investigations in cancer immunotherapies and strategies targeting cancer neoantigens in PDAC.

Effects of S-pindolol in mouse pancreatic and lung cancer cachexia models.

It is known that S-pindolol attenuates muscle loss in animal models of cancer cachexia and sarcopenia. In cancer cachexia, it also significantly reduced mortality and improved cardiac function, which is strongly compromised in cachectic animals. Here, we tested 3mg/kg/day of S-pindolol in two murine cancer cachexia models: pancreatic cancer cachexia (KPC) and Lewis lung carcinoma (LLC). Treatment of mice with 3mg/kg/day of S-pindolol in KPC or LLC cancer cachexia models significantly attenuated the loss of body weight, including lean mass and muscle weights, leading to improved grip strength compared with placebo-treated mice. In the KPC model, treated mice lost less than half of the total weight lost by placebo (-0.9±1.0 vs. -2.2±1.4g for S-pindolol and placebo, respectively, P<0.05) and around a third of the lean mass lost by tumour-bearing controls (-0.4±1.0 vs. -1.5±1.5g for S-pindolol and placebo, respectively, P<0.05), whereas loss of fat mass was similar. In the LLC model, the gastrocnemius weight was higher in sham (108±16mg) and S-pindolol tumour-bearing (94±15mg) mice than that in placebo (83±12mg), whereas the soleus weight was only significantly higher in the S-pindolol-treated group (7.9±1.7mg) than that in placebo (6.5±0.9). Grip strength was significantly improved by S-pindolol treatment (110.8±16.2 vs. 93.9±17.1g for S-pindolol and placebo, respectively). A higher grip strength was observed in all groups; whereas S-pindolol-treated mice improved by 32.7±18.5g, tumour-bearing mice only show minimal improvements (7.3±19.4g, P<0.01). S-pindolol is an important candidate for clinical development in the treatment of cancer cachexia that strongly attenuates loss of body weight and lean body mass. This was also seen in the weight of individual muscles and resulted in higher grip strength.

Abstract B003: Combined inhibition of the MAPK and Hippo pathways drives efficacious tumor suppression in a faithful model of mutant Kras (KPC) PDAC

Abstract Oncogenic mutations in the KRAS oncogene hyperactivate the MAPK pathway, a key driver of oncogenesis in the majority of human malignancies. KRAS-driven tumors are aggressive and highly refractory to standard-of-care treatments, thus intense efforts have been devoted to developing inhibitors of its activity. However, pioneering studies in engineered conditional mKras driven mouse models of pancreatic ductal adenocarcinoma (KPC) have demonstrated that while mKras is necessary for tumor maintenance, the anti-neoplastic effects of mKras extinction can be counteracted by activation of the Hippo pathway. These data strongly suggest that the full realization of MAPK pathway (including KRAS) inhibitors’ clinical potential requires the concomitant suppression of the function of YAP/TEAD, the effectors of Hippo pathway. Here, we directly test this hypothesis utilizing Sporos BioDiscovery’s next generation TEAD inhibitor (SPR1) - optimized to maximize anti-neoplastic activity and minimize toxicity through fine-tuning of TEAD paralog specificity. Using mKras murine (KrasG12D mutant, p53 null, PDAC) and human cell lines - we demonstrated strong interaction between SPR1 TEAD inhibitors and the clinical MEK inhibitors (since no G12D inhibitor is yet approved) Trametinib/Binimetinib in vitro. Functional TEAD inhibition was also shown by the reduced expression of the YAP/TEAD transcriptional targets CTGF and CYR61. Further, using a well-established murine orthotopic model, KPC (KrasG12D;p53-/-;p48Cre), that faithfully mimics human PDAC, we demonstrated robust combined efficacy of Trametinib and SPR1 TEAD inhibitors in vivo in suppressing tumor growth and strikingly doubling the survival time of the tumor-bearing mice. To our knowledge, this is the first preclinical demonstration of the therapeutic potential of a MAPK + TEAD inhibitor combination in a mKras G12D-driven orthotopic PDAC; given the high barrier for efficacy of this KPC model –we believe these results favorably presage generalization to MAPK/TEAD inhibitor combinations in other cancer setting and translatability to the clinic. Citation Format: Deepavali Chakravarti, Florian Muller, Jeno Gyuris, Erkun Baloglu, Selvi Kunnimalayiaan, Jill Olson, Sharon Shacham. Combined inhibition of the MAPK and Hippo pathways drives efficacious tumor suppression in a faithful model of mutant Kras (KPC) PDAC [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B003.