Autochthonous Pancreatic Ductal Adenocarcinoma Research Articles

Abstract C101: Calcium signaling rewiring activates epithelial-to-mesenchymal transition

Abstract Purpose: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths in the United States and is often not diagnosed until it has already metastasized. Calcium and downstream signaling cascades contribute to epithelial-to-mesenchymal transition (EMT) and facilitate metastasis formation in PDAC. Ca2+/calmodulin-dependent kinase-II (CaMK2), a downstream effector kinase activated by Ca2+, has previously been shown in vitro to be an important mediator of a partial/hybrid EMT program that gives tumor cells high metastatic proclivity. We hypothesize that CaMK2 is a critical node in the partial EMT state and thus deleted Camk2b in vivo to study the effect of its loss on tumor progression and metastasis. Methods: We generated a genetically engineered mouse model where Camk2b was knocked out in the KPCY autochthonous PDAC mouse model (herein KPCY-Camk2bcKO). Isolated cell lines were treated with the EMT-inducing ligand TGF-b to study the effect of Camk2b on tumor cell plasticity. Results: Prior in vitro work indicated that CAMK2 is required for hybrid EMT and thus we expected KPCY-Camk2bcKO mice to have fewer metastases and live longer. Surprisingly, the genetic deletion of Camk2b accelerated tumor formation and reduced overall survival relative to KPCY controls. Despite dying sooner, there was no difference in overall tumor burden in KPCY-Camk2bcKO mice. Pathological and immunofluorescence analysis of KPCY-Camk2bcKO tumors showed an increased number of poorly differentiated regions and EMT as indicated by loss of the epithelial cell marker ECADHERIN (E-CAD) in YFP lineage-labeled tumor cells. In stark contrast to prior work, the loss of Camk2b increased tumor cell plasticity, potentially highlighting a rewiring of calcium signaling circuits in the Camk2b-null state. To this end, KPCY-Camk2bcKO tumor cell lines displayed a baseline mesenchymal morphology in vitro and had lower expression of Cdh1 (the gene encoding E-CAD) mRNA. Using a flow cytometry-based approach, we also found baseline E-CAD protein levels to be diminished relative to KPCY lines. Additionally, KPCY-Camk2bcKO tumor cells were more responsive to the EMT-inducing ligand TGF-b and downregulated their E-CAD levels more than KPCY cells. Notably, treatment with low dose TGF-b also dramatically reduced E-CAD expression in KPCY-Camk2bcKO but not KPCY cells, indicating that the knockout cells are easily pushed towards a mesenchymal state. Conclusions: While CAMK2 has been studied previously as a facilitator of the EMT program, this work paradoxically demonstrates that Camk2b loss enhances cellular plasticity within PDAC. In this context, we speculate that loss of Camk2b rewires calcium signaling to drive EMT. In future studies, we will investigate the effect of Camk2b loss on the metastatic cascade and uncover mechanisms through which Camk2b loss makes PDAC tumor cells more plastic. We will also begin to characterize the non-cell autonomous roles of CAMK2B in crosstalk with the tumor microenvironment. Citation Format: Jessica Peura, Yamini Ogoti, Jason Pitarresi. Calcium signaling rewiring activates epithelial-to-mesenchymal transition [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 C101.

Abstract B044: Targeting integrin alpha V beta 3 remodels the tumor microenvironment in pancreatic cancer

Abstract In pancreatic ductal adenocarcinoma (PDAC) the tumor microenvironment (TME) plays a crucial role in therapy resistance, metastasis, and recurrence. ProAgio is a novel therapeutic cytotoxin rationally designed to bind outside the RGD ligand pocket of the alphaV-beta3 integrin pair and then kill cells expressing this target. Pre-clinical studies demonstrated the safety and anti-tumor efficacy of ProAgio in animal models, and ProAgio is currently being tested in a Phase I clinical trial. The effect of ProAgio on TME cellular components has not been closely examined. We hypothesized that ProAgio remodels the PDAC TME to decrease stromal density and local immunosuppression by killing stromal cells thought to express the integrin pair, such as cancer associated fibroblasts (CAFs), proliferating endothelial cells, and tumor associated macrophages (TAMs). To identify potential cellular targets of ProAgio in the PDAC TME, we re-analyzed publicly available single cell RNA-seq datasets of human and mouse PDAC samples. We assessed integrin co-expression using flow cytometry, performed immunophenotyping and histopathological analysis to identify TME cellular and structural alterations induced by ProAgio. Integrins αV and β3 were both expressed at the RNA level in TAMs, myeloid cells, lymphocytes (T, B and NK), endothelial cells and CAFs. In the autochthonous KPC mouse model, we confirmed that granulocytes, macrophages, monocytes, CD4+ and CD8+T cells co-expressed integrin αVβ3 at the protein level. Amongst CAFs, inflammatory CAFs (iCAFs) had the highest percentage of cells co-expressing αV and β3 integrins. No anti-tumor effect was observed after 14 days of ProAgio treatment in a syngeneic KPC-derived PDAC orthotopic model, however, in the KPC autochthonous model the same treatment retarded tumor growth within 1 week. ProAgio treated autochthonous tumors displayed no changes in immune components or CAF subsets, but increased collagen area was observed. In addition, ProAgio treatment resulted in higher blood vessel area while integrin β3 protein levels remained stable. In conclusion, short-course ProAgio remodels the TME and delays tumor growth in an autochthonous PDAC model with mature stromal components, but does not deplete CAFs, TAMs or collagen area. Future studies will seek to refine our understanding of the cell subtypes affected by more prolonged ProAgio treatment and to determine the cellular and inflammatory mediators responsible for these changes. Citation Format: Mayrel Palestino Dominguez, Philip Homan, Xianyu Zhang, Sandra Navas Reyes, Theresa Guerin, Laura Bassel, Liu Zhi-ren, Serguei Kozlov, Christine Alewine. Targeting integrin alpha V beta 3 remodels the tumor microenvironment in 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 B044.

Abstract A014: Inhibition of KrasG12D with a novel small molecule inhibitor alters the pancreatic cancer microenvironment

Abstract Approximately 90% of pancreatic ductal adenocarcinoma (PDAC) patients harbor an oncogenic KRAS mutation, with Gly-to-Asp mutations (KrasG12D) being most common. PDAC is characterized by a robust, immunosuppressive tumor microenvironment that is rich in myeloid-derived suppressor cells (MDSCs) and has a paucity of T cells. The majority of therapeutic interventions in PDAC are ineffective, in part, due to this immunosuppressive TME. We aimed to examine the efficacy of a small molecule KrasG12D inhibitor, MRTX1133, in implantable and autochthonous PDAC models with an intact immune system. In vitro studies validated the specificity and potency of MRTX1133. In vivo, MRTX1133 prompted deep tumor regressions in all models tested, including complete or near-complete remissions after 14d. MRTX1133 treatment additionally reduced metastatic burden after 7d of treatment. An increase in tumor cell death was identified after only 36h of treatment, with concomitant loss of tumor cell proliferation. Inhibition of KrasG12D led to marked shifts in the TME composition. MRTX1133-treated tumors had an increase in fibroblasts, collagen deposition, and blood vessel density. There was a robust increase in M1-like macrophages after MRTX1133 treatment. Further, there was a loss of MDSCs, along with an increase in cytotoxic T cells. Proteome profiling on tumor cells treated with MRTX1133 identified a loss of tumor cell-derived GM-CSF (a MDSC recruiter), as well as an increase in CCL2 (a macrophage recruiter), providing evidence that MRTX1133 alters the TME through tumor secreted factors. T cells were necessary for MRTX1133’s full anti-tumor effect, and T cell depletion accelerated tumor regrowth after therapy. These results validate the specificity, potency, and efficacy of MRTX1133 in immunocompetent KrasG12D-mutant PDAC models, providing a rationale for clinical testing and a platform for further investigation of combination therapies. Citation Format: Samantha B. Kemp, Noah Cheng, Nune Markosyan, Rina Sor, Il-Kyu Kim, Jill Hallin, Jason Shoush, Liz Quinones, Natalie Brown, Jared B. Bassett, Nikhil Joshi, Salina Yuan, Molly Smith, William P. Vostrejs, Kia Z. Perez-Vale, Benjamin Kahn, Feiyan Mo, Timothy R. Donahue, Caius G. Radu, Cynthia Clendenin, James G. Christensen, Robert H. Vonderheide, Ben Z. Stanger. Inhibition of KrasG12D with a novel small molecule inhibitor alters the pancreatic cancer microenvironment [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 A014.

Cross-Dataset Single-Cell Analysis Identifies Temporal Alterations in Cell Populations of Primary Pancreatic Tumor and Liver Metastasis.

Pancreatic ductal adenocarcinoma (PDAC) has a unique tumor microenvironment composed of various cell populations such as cancer cells, cancer-associated fibroblasts (CAFs), immune cells, and endothelial cells. Recently, single-cell RNA-sequencing analysis (scRNA-seq) has systemically revealed the genomic profiles of these cell populations in PDAC. However, the direct comparison of cell population composition and genomic profile between primary tumors (at both early- and late-stage) and metastatic tumors of PDAC is still lacking. In this study, we combined and analyzed recent scRNA-seq datasets of transgenic KPC mouse models with autochthonous PDAC and matched liver metastasis, revealing the unique tumor ecosystem and cell composition of liver metastasis in contrast to primary PDAC. Metastatic PDAC tumors harbor distinct cancer cell subpopulations from primary tumors. Several unique markers, including HMGA1, were identified for metastasis-enriched cancer cell subpopulations. Furthermore, metastatic tumors reveal significantly enriched granulocytic myeloid-derived suppressor cells (G-MDSCs), mature neutrophils, and granulocyte-myeloid progenitors (GMPs). A common GMP population across primary tumors, liver metastases, and healthy bone marrow was identified as the putative cell origin of tumor-associated neutrophils/granulocytes.

Abstract 3870: TNF-MK2 signaling drives protective autophagy following MAPK pathway inhibition in pancreatic cancer

Abstract Targeting the oncogenic KRAS-RAF-MEK-ERK (MAPK) pathway has remain unsuccessful in the clinic for patients with pancreatic ductal adenocarcinoma (PDAC), a highly lethal cancer with few effective treatment options. To identify novel, effective therapeutic strategies, we investigated the impact of MEK and ERK inhibitors on production of inflammatory cytokines in PDAC cells. We found that both MEK and ERK inhibitors dramatically upregulated production of TNF, leading to activation of multiple TNF signaling pathways which include the pro-death and pro-survival NF-kB and p38.MK2 cascades. Silencing of TNF receptor 1 (TNFR1) abrogated the pro-apoptotic effect of MAPK inhibitors, suggesting that pro-death effect driven by TNF signaling is required for the therapeutic effect of MAPK inhibitors. However, targeting the NF-kB and MK2 pathways greatly augment the suppressive effect of MAPK inhibitors. Notably, the mechanisms via which MK2 promotes PDAC cell survival are mediated through phospho-activation of Heat shock protein 27 (Hsp27) and Beclin1, a critical mediator of autophagy. Intriguingly, silencing of TNFR1 abrogated induction of proactive autophagy following MAPK inhibition, strongly suggesting autocrine signaling as the inciting event of autophagy. Mechanistically, we showed that TNF signaling upregulate unfolded protein response (UPR) in PDAC cells, which is required for autophagy induction. Targeting MK2 abrogated transcription of key UPR genes and blocks subsequent autophagy. The combination of MK2 inhibitor ATI-450 and ERK inhibitor ulixertinib was more effective in curbing the growth of PDAC patient-derived xenograft in vivo and prolonged the survival of autochthonous PDAC mice (KPC model). Overall, our study provided novel insights on the mechanisms that drive protective autophagy following MAPK pathway inhibition and a rationale and feasible therapeutic combination that can be tested in clinical trials for PDAC patients. Citation Format: Iftikhar Ali Khawar, Qing Wei, Timothy Hung-Po Chen, Lin Lin, Patrick M. Grierson, Kian-Huat Lim. TNF-MK2 signaling drives protective autophagy following MAPK pathway inhibition in pancreatic cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3870.

Abstract 6394: Immune mechanisms underlie full therapeutic efficacy of a novel inhibitor of KrasG12D in mouse models of pancreatic ductal adenocarcinoma

Abstract The KRAS oncogene is mutated in greater than 90% of pancreatic ductal adenocarcinoma (PDAC) cases. MRTX1133 is a non-covalent inhibitor targeting the most common mutant KRAS in PDAC, KRASG12D, and has shown robust efficacy in various tumor models, including PDAC (Hallin et al., Nature Medicine, 2022; Kemp et al., Cancer Discovery, 2022). However, the interplay between MRTX1133 and the immune system has not been characterized. Using immunocompetent mouse models of PDAC, we aimed to elucidate whether the effects of MRTX1133 are immune dependent. Mice were injected subcutaneously with tumor cell clones derived from the autochthonous KPC mouse model of PDAC that reproducibly result in high or low levels of T cell infiltration. MRTX1133 suppressed tumor growth in both “T cell high” and “T cell low” tumor-bearing mice. However, complete regressions were observed only in mice bearing T cell high tumors (4/8 10d after treatment start), indicating that T cell abundance improves MRTX1133 efficacy. T cells were necessary for sustained tumor growth inhibition in both subcutaneous and autochthonous PDAC models, as mice depleted of CD4+ and CD8+ T cells demonstrated rapid tumor re-growth following cessation of MRTX1133. Batf3KO mice, which are deficient in cross-presenting dendritic cells, displayed reduced tumor control compared to wild type mice implanted with T cell high tumors. These data suggest that T cell priming is required to elicit MRTX1133’s complete anti-tumor activity. We also studied the impact of MRTX1133 on the tumor immune microenvironment. In the subcutaneous setting, T cell low tumor cells showed decreased proliferation (Ki-67+, P = 0.024) and increased apoptosis (CC3+, P = 0.022) after 36h of MRTX1133 treatment. T cell low tumor-bearing mice treated with MRTX1133 for 60h exhibited increased proportions of CD4+ (by 1.5-fold, P = 0.010) and CD8+ (by 2-fold, P = 0.004) T cells in the tumor. These tumor-infiltrating CD8+ T cells were more proliferative (Ki-67+, P < 0.001), activated (CD11a+, P = 0.006), and cytotoxic (GZMB+, P = 0.004) compared to corresponding T cells in vehicle-treated mice. Additionally, more CD8+ T cells in the tumor-draining lymph nodes of MRTX1133-treated mice were IFNγ+ (P < 0.001), illustrating a systemic immune effect from MRTX1133. Our findings indicate that T cells play an important role in MRTX1133’s demonstrated efficacy, suggesting a “vaccine effect” from mutant KRAS inhibition and offering a rationale for testing novel combinations of mutant KRAS inhibitors with cancer immunotherapies. Citation Format: Noah Cheng, Samantha B. Kemp, Nune Markosyan, Rina Sor, Il-Kyu Kim, Jill Hallin, Jason Shoush, Liz Quinones, Natalie V. Brown, Jared B. Bassett, Nikhil Joshi, Salina Yuan, Molly Smith, William P. Vostrejs, Kia Z. Perez-Vale, Benjamin Kahn, Feiyan Mo, Timothy R. Donahue, Caius G. Radu, Cynthia Clendenin, James G. Christensen, Robert H. Vonderheide, Ben Z. Stanger. Immune mechanisms underlie full therapeutic efficacy of a novel inhibitor of KrasG12D in mouse models of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6394.

Abstract 1725: Preclinical evaluation of RM-042, an orally bioavailable inhibitor of GTP-RAS, in models of pancreatic ductal adenocarcinoma

Abstract Approximately 95% of pancreatic ductal adenocarcinoma (PDAC) cases are driven by activating mutations in KRAS, accounting for over 45,000 deaths per year in the US. In KRAS-mutant cells, KRAS protein exists predominantly in an active, GTP-bound state, leading to excessive oncogenic signaling via RAF/MEK/ERK and other effector pathways. After decades of anticipation, the first therapeutic inhibitors of this oncoprotein target a specific mutant allele (KRASG12C) and have shown promising results in this rare subset of human PDAC patients (1-2% of cases). While these encouraging clinical results bolster support for targeting RAS in human PDAC, most patients harbor KRASG12D and KRASG12V mutations and remain unserved by targeted therapies. Here we report preclinical studies of RM-042, a first-in-class, potent, oral, small molecule inhibitor of GTP-bound RAS proteins, designed to treat cancers driven by a variety of RAS mutations. RMC-6236, a clinical candidate and RM-042, a tool compound, both act through non-covalent binding to an abundant intracellular chaperone protein, cyclophilin A (CypA), resulting in a binary complex that engages GTP-bound RAS to form a high-affinity, RAS-selective tri-complex that sterically inhibits RAS binding to effectors.We examined the anti-tumor activity of RM-042 in two complementary preclinical model systems of PDAC. Using primary human PDAC explant cultures, we found that RM-042 potently inhibits phospho-ERK expression (a marker of KRAS/RAF/MEK pathway activity), in a concentration-dependent manner and was associated with reduced viability and altered stromal content. Pilot short-term studies in the K-rasLSL.G12D/+, p53LSL.R172H/+, Pdx1Cretg/+ (KPC) mouse model of PDAC found that RM-042 induced initial tumor stabilizations and regressions associated with loss of pERK expression, in association with increased apoptosis and reduced proliferation, at tolerated doses. However, tumors collected at endpoints showed re-established pERK expression, suggesting activation of resistance/adaptation mechanisms. Ongoing studies will determine pharmacokinetic properties of RM-042 in the autochthonous PDAC mouse model, as well as the duration of tumor responses and evaluate a potential survival benefit provided by RM-042. Regulatory network analysis of single cell RNA sequencing performed on these tumors at multiple timepoints will provide a mechanistic understanding of the regulatory proteins that drive responses in each cell type in the tumors. Through this approach, we will assess the regulatory adaptations that occur as tumors initially respond and as they escape. Together, these studies provide complementary evidence from in vivo murine and ex vivo human models for promising anti-tumor activity in response to GTP-RAS inhibition, supporting the inclusion of PDAC patients in ongoing and future clinical trials of RMC-6236. Citation Format: Urszula N. Wasko, Stephen A. Sastra, Carmine F. Palermo, David Wildes, Mallika Singh, Kenneth P. Olive. Preclinical evaluation of RM-042, an orally bioavailable inhibitor of GTP-RAS, in models of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1725.

Abstract C013: Response to combination immunotherapy in a mouse model of PDAC is a function of baseline T cell content

Abstract Findings from the recently reported phase 2 PRINCE trial for patients with metastatic pancreatic cancer suggest potential clinical benefit with certain combinations of chemotherapy and immunotherapy. Overall survival following gemcitabine/nab-paclitaxel and either PD-1 blocking antibody or CD40 agonistic antibody (aCD40) correlated with the prevalence of distinct T cell subsets in circulation at baseline and with the extent of certain tumor infiltrating T cells (Padron et al., Nature Medicine, 2022). Here, we modeled the therapeutic effect of aCD40/PD-1/CTLA-4 therapy in the absence of chemotherapy in mice bearing tumors derived from the autochthonous pancreatic ductal adenocarcinoma (PDAC) KPC model and studied the dependency of tumor regression on the degree of tumor infiltrating T cells. We used a panel of KPC tumor cell clones that exhibit reproducible and transplantable levels of T cell infiltration ranging between high, intermediate, and low amounts. Mice were subcutaneously injected with clonal PDAC lines and treated 14-21 days later with either aCD40 alone or aCD40/PD-1/CTLA-4 (all i.p., n=5-8 mice per cohort of T cell high, intermediate and low clones). Systemic immune dynamics were surveyed by bleeding mice on day 0, 7, and 14 of treatment and analyzing for immune cell activation and exhaustion markers. In mice bearing T cell high and intermediate tumors, aCD40/PD-1/CTLA-4 induced higher rates of complete tumor regressions (71% for T cell high; 25% for T cell intermediate) and prolonged survival (P < 0.001 for T cell high; P = 0.013 for T cell intermediate) compared to untreated controls. Treatment with aCD40 alone was effective in mice with T cell intermediate tumors, leading to regressions in 29% of mice. In mice bearing T cell low tumors, however, tumor regressions after aCD40/PD-1/CTLA-4 were less frequent (12.5%), although survival was improved significantly following the treatment (P <0.001). aCD40 alone improved overall survival of T cell low bearing mice (P = 0.026) without inducing tumor regressions. Regardless of T cell high, intermediate, or low tumors, aCD40/PD-1/CTLA-4 increased the proportions of circulating activated and exhausted CD4+ and CD8+ T cells. aCD40/PD-1/CTLA-4 also increased T cell infiltration into T cell low tumors, but this enhanced infiltration did not correlate with complete regressions. Thus, using tumor clones derived from the same autochthonous genetic model, we show that response to combination immunotherapy is a function of baseline T cell content, with ‘hot’ tumors responding the best and ‘cold’ tumors responding the worst. Additional tumor cell-intrinsic factors such as those revealed in other mouse experiments and in the human samples from the PRINCE study likely restrict a robust anti-tumor immune response even when T cells have been recruited. Citation Format: Noah C. Cheng, Nune Markosyan, Robert H. Vonderheide. Response to combination immunotherapy in a mouse model of PDAC is a function of baseline T cell content [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 C013.

Syngeneic Mouse Orthotopic Allografts to Model Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is a very complex disease characterized by a heterogeneous tumor microenvironment made up of a diverse stroma, immune cells, vessels, nerves, and extracellular matrix components. Over the years, different mouse models of PDAC have been developed to address the challenges posed by its progression, metastatic potential, and phenotypic heterogeneity. Immunocompetent mouse orthotopic allografts of PDAC have shown good promise owing to their fast and reproducible tumor progression in comparison to genetically engineered mouse models. Moreover, combined with their ability to mimic the biological features observed in autochthonous PDAC, cell line-based orthotopic allograft mouse models enable large-scale in vivo experiments. Thus, these models are widely used in preclinical studies for rapid genotype-phenotype and drug-response analyses. The aim of this protocol is to provide a reproducible and robust approach to successfully inject primary mouse PDAC cell cultures into the pancreas of syngeneic recipient mice. In addition to the technical details, important information is given that must be considered before performing these experiments.

A Sampling of Highlights from the Literature

CRISPR-based screening can enhance understanding of T-cell biology (from marius walter via Wikimedia Commons)Using CRISPR-based screening, two groups have identified canonical BRG1/BRM-associated factor (cBAF) complex components as regulators of T-cell differentiation and function that are important for antitumor immunity. Guo et al. find cBAF levels influence the fate of activated CD8+ T cells: high levels promote effector T-cell differentiation and low levels promote memory T-cell differentiation. Belk et al. show cBAF complex components, including Arid1a, promote T-cell acquisition of epigenetic features of exhaustion and limit T-cell persistence. Reducing cBAF activity, either pharmacologically (Guo et al.) or genetically (Belk et al.), improves tumor control in mouse models. The two studies suggest modulating the cBAF complex may improve T cell–based cancer immunotherapy.Guo A, …, Green DR. Nature 2022 June 22;607:135–141.Belk JA, …, Satpathy AT. Cancer Cell 2022 July 11;40:768–86.E7.S. epidermis specific T-cell responses can drive skin irAEs (from NIAID via NIH Flickr)Increasing our understanding of the mechanisms underlying immune-related adverse events (irAEs) is important for optimizing the use of immune checkpoint inhibitors (ICI). Using a mouse model of skin irAEs, Hu et al. find that exposure to Staphylococcus epidermis at the time of anti-CTLA4 treatment induces an inflammatory response resembling skin irAEs in patients treated with ICIs. The response is driven by S. epidermis–specific T cells that produce IL17 and leads to the emergence of S. epidermis–specific memory T cells. Further analysis will determine whether modulating cross-talk between the host immune system and microbiota can alleviate skin irAEs.Hu ZI, …, Belkaid Y. PNAS 2022 June 21;119:e2200348119.Phase II clinical trial results testing chemoimmuno-therapy in PDAC (via Rawpixel)Immunotherapy has so far been ineffective in the treatment of pancreatic ductal adenocarcinoma (PDAC). Padrón et al. report results from a phase II clinical trial evaluating chemotherapy combined with the anti–PD-1 nivolumab or the CD40-agonist sotigalimab, or with both together. The only group of patients among whom the 1-year overall survival rate was significantly greater than a historical control rate of 35% was the group assigned nivolumab/chemotherapy. Analysis of circulating and tumor biomarkers identified distinct immune signatures of improved outcome for patients receiving nivolumab/chemotherapy or sotigalimab/chemotherapy that could be used for patient selection in future clinical trials.Padrón LJ, …, Vonderheide RH. Nat Med 2022 June 3;28:1167–77.B cells can promote or suppress tumors depending on the TME (Carsten Tolkmit via Flickr)The role of B cells in the suppression or promotion of tumors remains unclear. In colorectal cancer (CRC), Wang et al. discover a specific regulatory B-cell subset expressing leucine-tRNA-synthase-2 (LARS2) and TGFβ1 that contributes to CRC progression by altering metabolic processes in the tumor microenvironment (TME). In a second study, Sagiv-Barfi et al. demonstrate that B-cell activation and antigen presentation are key to inducing antitumor T-cell responses, and both B and T cells are needed for efficacy of combination therapy with IL12, anti-OX40, and CpG in breast cancer. The two studies highlight how B-cell responses in the TME not only depend on cancer type, but also on B-cell phenotype and regulatory function.Wang Z, …, Chu Y. Immunity 2022 June 14;55:1067–81.E8.Sagiv-Barfi I, …, Levy R. Sci Immunol 2022 May 27;7:eabn5859.Profiling the diversity of tumor antigens (Andy Mitchell via Wikimedia Commons)A greater understanding of in vivo cancer-specific patterns of peptide presentation by MHC class I complexes is needed to improve the development of antigen-specific immunotherapies. Jaeger et al. develop and use a new genetically engineered mouse model to profile the cancer immunopeptidome from autochthonous pancreatic ductal adenocarcinoma and lung adenocarcinomas. The authors find little correlation between mRNA abundance and peptide detection, suggesting significant posttranslational regulation of tumor-specific peptide presentation. Dendritic cell vaccines loaded with lung adenocarcinoma peptides uniquely identified by the assay induce antigen-specific T-cell responses ex vivo, highlighting the model’s ability to capture the cancer immunopeptidome.Jaeger AM, …, Jacks T. Nature 2022 June 15;607:149–55.A new map of human immune cell development (tzunghaor via openclipart)Understanding how and where the immune system develops can provide a better understanding of cancer origin and facilitate the development of novel immunotherapies. Through single-cell profiling and spatial transcriptomics of nine prenatal tissues, Suo et al. have generated a comprehensive atlas of the developing human immune system. Highlights of the findings include the discovery that human immune development occurs across multiple tissue sites, including sites not considered primary hematopoietic organs; the identification of a population of prenatal B1 cells; and the demonstration that unconventional T cells are selected on thymocytes. This road map provides a resource for advancing immune-based science and medicine.Suo C, …, Teichmann SA. Science 2022 May 12;376:eabo0510.

Deciphering the immunopeptidome in vivo reveals new tumour antigens.

Immunosurveillance of cancer requires the presentation of peptide antigens on major histocompatibility complex class I (MHC-I) molecules1-5. Current approaches to profiling of MHC-I-associated peptides, collectively known as the immunopeptidome, are limited to in vitro investigation or bulk tumour lysates, which limits our understanding of cancer-specific patterns of antigen presentation in vivo6. To overcome these limitations, we engineered an inducible affinity tag into the mouse MHC-I gene (H2-K1) and targeted this allele to the KrasLSL-G12D/+Trp53fl/fl mouse model (KP/KbStrep)7. This approach enabled us to precisely isolate MHC-I peptides from autochthonous pancreatic ductal adenocarcinoma and from lung adenocarcinoma (LUAD) in vivo. In addition, we profiled the LUAD immunopeptidome from the alveolar type 2 cell of origin up to late-stage disease. Differential peptide presentation in LUAD was not predictable by mRNA expression or translation efficiency and is probably driven by post-translational mechanisms. Vaccination with peptides presented by LUAD in vivo induced CD8+ T cell responses in naive mice and tumour-bearing mice. Many peptides specific to LUAD, including immunogenic peptides, exhibited minimal expression of the cognate mRNA, which prompts the reconsideration of antigen prediction pipelines that triage peptides according to transcript abundance8. Beyond cancer, the KbStrep allele is compatible with other Cre-driver lines to explore antigen presentation in vivo in the pursuit of understanding basic immunology, infectious disease and autoimmunity.

Insufficiency of compound immune checkpoint blockade to overcome engineered T cell exhaustion in pancreatic cancer

BackgroundAchieving robust responses with adoptive cell therapy for the treatment of the highly lethal pancreatic ductal adenocarcinoma (PDA) has been elusive. We previously showed that T cells engineered to express...

The MK2/Hsp27 axis is a major survival mechanism for pancreatic ductal adenocarcinoma under genotoxic stress.

Combination chemotherapies remain the cornerstone treatment for pancreatic ductal adenocarcinoma (PDAC), but de novo and acquired resistance is common. In this study, we aimed to identify and characterize resistance mechanisms to a FIRINOX chemotherapy regimen (a combination of 5-fluorouracil, irinotecan, and oxaliplatin) because it is the most aggressive regimen currently used clinically for patients with PDAC. Using an unbiased reverse-phase protein array, we detected phospho-activation of heat shock protein 27 (Hsp27) as the most up-regulated event after FIRINOX treatment in PDAC cells. Silencing HSP27 by RNA interference or by a small-molecule inhibitor enhanced apoptosis caused by FIRINOX in vitro. Mechanistically, FIRINOX up-regulated tumor necrosis factor–α (TNFα), causing autocrine phosphorylation and activation of transforming growth factor–β–activated kinase 1 (TAK1), MAPK activated protein kinase 2 (MAPKAPK2 or MK2), and, ultimately, Hsp27. Targeting MK2, the kinase that directly phosphorylates Hsp27, abrogated Hsp27 activation, sensitized PDAC cells to apoptosis, and suppressed SN-38–induced protective autophagy in vitro, in part by blocking phospho-activation of Beclin1. In an autochthonous PDAC mouse model, the MK2 inhibitor ATI-450 decreased PDAC development and progression. When combined with FIRINOX, ATI-450 eliminated most PDAC foci and marked prolonged mouse survival without causing additional toxicity. Last, we found that high phospho-MK2 expression in tumors was associated with poorer survival of patients with PDAC. Our study identified MK2 as a mediator of genotoxic stress–induced activation of prosurvival pathways and provides preclinical support for combining an MK2 inhibitor with FIRINOX-based chemotherapies to treat PDAC.

Abstract PO-017: Targeting NF-kB pathway through IRAK4 renders immune checkpoint blockade effective in pancreatic cancer

Abstract Effective immunotherapy in pancreatic ductal adenocarcinoma (PDAC) is impeded by multiple barriers in the tumor microenvironment. These include the dense extracellular matrix (ECM), excessive inhibitory myeloid cells, cytokines and chemokines, which collectively incapacitate anti-tumour T cells. Constitutive activation the NF-kB pathway is a mechanism that drives intrinsic survival of PDAC cells and stromal fibrosis, but its impact on anti-tumour immunity has not been investigated. Using The Cancer Genome Atlas database, we found that expression of RELA, a canonical NF-kB factor, in PDAC samples is associated with activated stroma and lower cytotoxic T cell signatures. In a PDAC tissue microarray, the staining intensity of activated IRAK4, the innate immune kinase that drives NF-kB signaling, negatively correlates with T cell abundance. Based on these findings, we investigated the immunological impact role of IRAK4 in PDAC. Transcriptomic analysis showed that ablation of IRAK4 in PDAC cells downregulates NF-kB and inflammatory signatures, and markedly decreases transcription of hyaluronan synthase 2 (HAS2). Accordingly, pharmacologic inhibition of IRAK4 significantly decreased intratumoral hyaluronan, as well as collagen, in autochthonous PDAC mice and potentiated standard chemotherapy. Furthermore, IRAK4 inhibition also significantly reduced production of several suppressive chemokines and checkpoint ligands PD-L1 and Nectin2, leading to revitalization of infiltrative CD4+ and CD8+ T cells. These effects were partly mediated through reduction of intratumoural hyaluronan, which we recapitulated with HAS inhibitor, 4-MU. Accordingly, combined IRAK4 inhibitors with immune checkpoint blockade (ICB) especially anti-CTLA4, were highly efficacious in abrogating tumour growth in autochthonous PDAC mice and doubling their survival. In summary, we showed that targeting the NF-kB pathway through IRAK4 renders ICB effective via multiple mechanisms and should be tested in clinical trials for PDAC patients. Citation Format: Daoxiang Zhang, Vikas Somani, Paarth B. Dodhiawala, Patrick M. Grierson, Lin Li, Kuljeet Seehra, Xiuting Liu, Brett L. Knolhoff, Marianna B. Ruzinova, David G. DeNardo, Kian-Huat Lim. Targeting NF-kB pathway through IRAK4 renders immune checkpoint blockade effective in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-017.

Dynamic Contrast-enhanced MRI Detects Responses to Stroma-directed Therapy in Mouse Models of Pancreatic Ductal Adenocarcinoma.

The dense stroma underlies the drug resistance of pancreatic ductal adenocarcinoma (PDA) and has motivated the development of stroma-directed drugs. Our objective is to test the concept that dynamic contrast-enhanced (DCE) MRI using FDA-approved contrast media, an imaging method sensitive to the tumor microenvironment, can detect early responses to stroma-directed drug. Imaging studies were performed in three mouse models exhibiting high desmoplastic reactions: the autochthonous PDA in genetically engineered mice (KPC), an orthotopic model in syngeneic mice, and a xenograft model of human PDA in athymic mice. An investigational drug, PEGPH20 (pegvorhyaluronidase alfa), which degrades hyaluronan (HA) in the stroma of PDA, was injected alone or in combination with gemcitabine. At 24 hours after a single injection of PEGPH20, Ktrans , a DCE-MRI-derived marker that measures how fast a unit volume of contrast media is transferred from capillaries to interstitial space, increased 56% and 50% from baseline in the orthotopic and xenograft tumors, respectively, compared with a 4% and 6% decrease in vehicle groups (both P < 0.05). Similarly, after three combined treatments, Ktrans in KPC mice increased 54%, whereas it decreased 4% in controls treated with gemcitabine alone (P < 0.05). Consistently, after a single injection of PEGPH20, tumor HA content assessed by IHC was reduced substantially in all three models while drug delivery (measured by paclitaxel accumulation in tumor) was increased by 2.6-fold. These data demonstrated a DCE-MRI marker, Ktrans , can detect early responses to stroma-directed drug and reveal the sustained effect of combination treatment (PEGPH20+ gemcitabine).

Tumor-Induced IL-6 Reprograms Host Metabolism to Suppress Anti-tumor Immunity

SummaryIn patients with cancer, the wasting syndrome, cachexia, is associated with caloric deficiency. Here, we describe tumor-induced alterations of the host metabolic response to caloric deficiency that cause intratumoral immune suppression. In pre-cachectic mice with transplanted colorectal cancer or autochthonous pancreatic ductal adenocarcinoma (PDA), we find that IL-6 reduces the hepatic ketogenic potential through suppression of PPARalpha, the transcriptional master regulator of ketogenesis. When these mice are challenged with caloric deficiency, the resulting relative hypoketonemia triggers a marked rise in glucocorticoid levels. Multiple intratumoral immune pathways are suppressed by this hormonal stress response. Moreover, administering corticosterone to elevate plasma corticosterone to a level that is lower than that occurring in cachectic mice abolishes the response of mouse PDA to an immunotherapy that has advanced to clinical trials. Therefore, tumor-induced IL-6 impairs the ketogenic response to reduced caloric intake, resulting in a systemic metabolic stress response that blocks anti-cancer immunotherapy.

NO• and Pancreatic Cancer: A Complex Interaction with Therapeutic Potential.

Pancreatic tumors express high level of nitric oxide synthases (NOSs) in particular inducible (iNOS/NOS2) and endothelial (eNOS/NOS3) forms. However, the role of nitric oxide (NO•) in the development and progression of pancreatic cancer is not clearly defined. Delineating the NO•-induced signaling in pancreatic cancer and its potential contribution in disease aggressiveness may provide therapeutic targets to improve survival in this lethal malignancy. Recent Advances: An increased expression of NOS2/iNOS in tumors is associated with poorer survival in early stage resected patients with pancreatic ductal adenocarcinoma (PDAC). Furthermore, genetic deletion of NOS2 enhanced survival in mice with autochthonous PDAC. Additionally, targeting NOS3/eNOS reduced the abundance of precursor lesions in mice, which trended toward improved survival. The extremely poor prognosis in pancreatic cancer is due to the late diagnosis and lack of effective therapy in advanced disease. One of the most critical issues is to decipher the underlying mechanism of disease aggressiveness and therapeutic resistance for identifying potential therapeutic target and effective treatment. Given the evidence of a strong association between inflammation and pancreatic cancer and clinical evidence, which suggests an association between NOS2 and disease aggressiveness, it is critical to define the role of NO• signaling in this lethal malignancy. Recent preclinical and clinical evidences indicate a potential therapeutic significance of targeting NO• signaling in pancreatic cancer. With the emergence of new preclinical models, including the patient-derived organoids, further preclinical evaluation using clinically tested NOS inhibitors is needed for designing future clinical investigation. Antioxid. Redox Signal. 26, 1000-1008.

Abstract IA21: The basis of immune suppression in murine pancreatic ductal adenocarcinoma

Abstract Immune therapy of human pancreatic ductal adenocarcinoma (PDAC) with the T cell checkpoint antagonists, anti-CTLA-4 and anti-PD-L1, has been unsuccessful. To determine the reason for this, we studied the KPC mouse model of autochthonous PDAC in which cancer cells express KrasG12D and p53R172H, and have lost the wild type p53 allele (LOH). These tumors resemble human PDAC in not responding to treatment with anti-CTLA-4 or anti-PD-L1. PDAC-bearing mice have CD8+ T cells specific for antigen(s) expressed by cancer cells. Thus, the ineffectiveness of T cell checkpoint antagonist immune therapy is not explained by the absence of an anti-tumor immune response, and immune suppression in the tumor microenvironment must occur mainly by another means. We have shown that this immune suppressive process depends on the stromal cell known as the “cancer-associated fibroblast” (CAF) that is identified by the membrane protein marker, “fibroblast activation protein” (FAP). Conditionally depleting the FAP+ CAF (Kraman M, et al., Science 2010) leads to T cell-mediated slowing of PDAC growth, and co-treatment with anti-PD-L1 enhances this effect (Feig C, et al. PNAS 2013). Three observations directed attention to the chemokine, CXCL12, as the mediator of immune suppression: the FAP+ CAF is the source of CXCL12 mRNA in PDACs; CXCL12 protein selectively “coats” the cancer cells; and T cells are excluded from the CXCL12-coated cancer cell-containing regions of the tumor. We treated PDAC-bearing mice for 24 hr with AMD3100/Plerixafor, a small molecule antagonist of the CXCL12 receptor, CXCR4, alone or together with anti-PD-L1. Tumors from AMD3100-treated mice exhibit frequent T cells within cancer cell-containing regions, and tumors from dual treated mice have even more T cells. Importantly, whereas tumor volume doubles in control mice in 6 days, AMD3100 alone arrests tumor growth, and AMD3100 + anti-PD-L1 treatment decreases tumor volume by 15%. Both therapies eliminate almost all cancer cells, leaving a residual mass containing only PanIN, inflammatory cells, and desmoplastic matrix. These findings may be relevant not only to human PDAC, but also to colorectal and ovarian carcinoma, all of which also contain FAP+ CAFs and CXCL12-coated cancer cells, demonstrate exclusion of T cells from the vicinity of cancer cells, and are resistant to therapy with anti-PD-1/anti-PD-L1. Citation Format: Douglas T. Fearon. The basis of immune suppression in murine pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr IA21.