Autochthonous Model Research Articles

Abstract 918: Development of a platform for programmable in vivo oncogene activation and screening using CRISPRa technology

Abstract Conventional genetically engineered mouse models (GEMMs) are time consuming, laborious and offer limited spatio-temporal control. We have developed a streamlined platform for in vivo gene activation using CRISPR activation (CRISPRa) technology. Our model system allows for flexible, sustained and timed activation of one or more target genes, in vitro or in vivo, using single or pooled lentiviral guides. Using Myc and Yap1 as model oncogenes, we implemented this platform to study the effect of oncogene activation on the tumorigenic potential of primary pancreatic organoids, as well as in an autochthonous model of lung adenocarcinoma. We found that Myc-activation in pancreatic organoids increased their tumorigenic potential and resulted in significantly shorter survival relative to controls when transplanted orthotopically. In vivo Myc activation in the lung accelerated tumor progression and resulted in significantly shorter overall survival relative to non-targeted tumor controls. Furthermore, we found that Myc-activation drives the acquisition of an immune suppressive “cold” tumor microenvironment. Through cross-species validation of our results, we were able to link MYC to a previously described, immunosuppressive transcriptomic subtype in patient tumors, thus identifying a patient cohort that may benefit from combined MYC/immune-targeted therapies. Our work demonstrates how CRISPRa can be used for rapid functional validation of putative oncogenes and may allow for the identification and evaluation of potential metastatic and oncogenic drivers through competitive screening. Citation Format: Fredrik Ivar Thege, Dhwani N. Rupani, Bhargavi B. Barathi, Anirban Maitra, Andrew D. Rhim, Sonja M. Wörmann. Development of a platform for programmable in vivo oncogene activation and screening using CRISPRa technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 918.

Abstract 2194: Rapid interrogation of colon cancer driver genes in vivo using split Cre and CRISPR-Cas9

Abstract Colorectal cancer (CRC) is the second leading cause of cancer-related deaths worldwide, with a five-year survival rate of only 63% with current treatment regimens. While genetically engineered mouse models (GEMMs) have contributed widely to our understanding of the underlying genetics and molecular mechanisms of the disease, major limitations exist. Most models of CRC lack flexibility to initiate focal adenocarcinomas with different genetic perturbations within the native microenvironment of the distal colon. Specifically, no autochthonous model of metastatic CRC compatible with in vivo CRISPR-Cas9 genome engineering has been described. Therefore, we developed a novel split Cre-recombinase system restricting activation of oncogenic KrasG12D and deletion of the tumor suppressor gene (TSG) Trp53 to Lgr5+ stem cells of the colon crypts. Importantly, these events are not only critical drivers of CRC progression and metastasis in mice and humans but can also lead to non-specific transformation of the surrounding colonic stroma. By expressing one half of Cre from the endogenous Lgr5 locus and other half via colonoscopy-guided submucosal injection of lentivirus, we restricted Cre-mediated recombination to enterocytes. Lentivirus was also engineered to express a single guide RNA (sgRNA) against Apc, facilitating tumor initiation via deletion of this critical CRC TSG in mice expressing Cas9. A second sgRNA was included to facilitate rapid interrogation of putative cancer driver genes. As proof-of-principle, we targeted Smad4, a critical suppressor of metastasis in CRC, and observed adenocarcinoma formation and liver metastases with complete recombination and editing of all targeted genes. To prioritize candidate TSGs for future in vivo interrogation, we applied MutSigCV to large human sequencing datasets and identified genes significantly co-mutated with APC and KRAS in CRC. Altogether, this faithful and tractable model of CRC can be used for rapid functional interrogation of TSGs and should accelerate downstream mechanistic and preclinical studies. Citation Format: Daniel Zhang, Peter Westcott, Santiago Naranjo, Haley Hauck, William Rideout, Nate Sacks, Arjun Bhutkar, Justin Patten, Norihiro Goto, Joseph Sedlak, Tyler Jacks. Rapid interrogation of colon cancer driver genes in vivo using split Cre and CRISPR-Cas9 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2194.

A Programmable In Vivo CRISPR Activation Model Elucidates the Oncogenic and Immunosuppressive Functions of MYC in Lung Adenocarcinoma.

A streamlined platform for programmable CRISPR gene activation enables rapid evaluation and functional validation of putative oncogenes in vivo.

Synergy of a STING agonist and an IL-2 superkine in cancer immunotherapy against MHC I–deficient and MHC I+ tumors

Cyclic dinucleotides (CDN) and Toll-like receptor (TLR) ligands mobilize antitumor responses by natural killer (NK) cells and T cells, potentially serving as complementary therapies to immune checkpoint therapy. In the clinic thus far, however, CDN therapy targeting stimulator of interferon genes (STING) protein has yielded mixed results, perhaps because it initiates responses potently but does not provide signals to sustain activation and proliferation of activated cytotoxic lymphocytes. To improve efficacy, we combined CDN with a half life-extended interleukin-2 (IL-2) superkine, H9-MSA (mouse serum albumin). CDN/H9-MSA therapy induced dramatic long-term remissions of the most difficult to treat major histocompatibility complex class I (MHC I)–deficient and MHC I+ tumor transplant models. H9-MSA combined with CpG oligonucleotide also induced potent responses. Mechanistically, tumor elimination required CD8 T cells and not NK cells in the case of MHC I+ tumors and NK cells but not CD8 T cells in the case of MHC-deficient tumors. Furthermore, combination therapy resulted in more prolonged and more intense NK cell activation, cytotoxicity, and expression of cytotoxic effector molecules in comparison with monotherapy. Remarkably, in a primary autochthonous sarcoma model that is refractory to PD-1 checkpoint therapy, the combination of CDN/H9-MSA with checkpoint therapy yielded long-term remissions in the majority of the animals, mediated by T cells and NK cells. This combination therapy has the potential to activate responses in tumors resistant to current therapies and prevent MHC I loss accompanying acquired resistance of tumors to checkpoint therapy.

Investigating Cutaneous Squamous Cell Carcinoma in vitro and in vivo: Novel 3D Tools and Animal Models.

Cutaneous Squamous Cell Carcinoma (cSCC) represents the second most common type of skin cancer, which incidence is continuously increasing worldwide. Given its high frequency, cSCC represents a major public health problem. Therefore, to provide the best patients’ care, it is necessary having a detailed understanding of the molecular processes underlying cSCC development, progression, and invasion. Extensive efforts have been made in developing new models allowing to study the molecular pathogenesis of solid tumors, including cSCC tumors. Traditionally, in vitro studies were performed with cells grown in a two-dimensional context, which, however, does not represent the complexity of tumor in vivo. In the recent years, new in vitro models have been developed aiming to mimic the three-dimensionality (3D) of the tumor, allowing the evaluation of tumor cell-cell and tumor-microenvironment interaction in an in vivo-like setting. These models include spheroids, organotypic cultures, skin reconstructs and organoids. Although 3D models demonstrate high potential to enhance the overall knowledge in cancer research, they lack systemic components which may be solved only by using animal models. Zebrafish is emerging as an alternative xenotransplant model in cancer research, offering a high-throughput approach for drug screening and real-time in vivo imaging to study cell invasion. Moreover, several categories of mouse models were developed for pre-clinical purpose, including xeno- and syngeneic transplantation models, autochthonous models of chemically or UV-induced skin squamous carcinogenesis, and genetically engineered mouse models (GEMMs) of cSCC. These models have been instrumental in examining the molecular mechanisms of cSCC and drug response in an in vivo setting. The present review proposes an overview of in vitro, particularly 3D, and in vivo models and their application in cutaneous SCC research.

β-Hydroxybutyrate suppresses colorectal cancer.

Colorectal cancer (CRC) is among the most frequent forms of cancer, and new strategies for its prevention and therapy are urgently needed1. Here we identify a metabolite signalling pathway that provides actionable insights towards this goal. We perform a dietary screen in autochthonous animal models of CRC and find that ketogenic diets exhibit a strong tumour-inhibitory effect. These properties of ketogenic diets are recapitulated by the ketone body β-hydroxybutyrate (BHB), which reduces the proliferation of colonic crypt cells and potently suppresses intestinal tumour growth. We find that BHB acts through the surface receptor Hcar2and induces the transcriptional regulator Hopx, thereby altering gene expression and inhibiting cell proliferation. Cancer organoid assays and single-cell RNA sequencing of biopsies from patients with CRC provide evidence that elevated BHB levels and active HOPX are associated with reduced intestinal epithelial proliferation in humans. This study thus identifies a BHB-triggered pathway regulating intestinal tumorigenesis and indicates that oral or systemic interventions with a single metabolite may complement current prevention and treatment strategies for CRC.

Mucin 5AC–Mediated CD44/ITGB1 Clustering Mobilizes Adipose-Derived Mesenchymal Stem Cells to Modulate Pancreatic Cancer Stromal Heterogeneity

Secreted mucin 5AC (MUC5AC) promotes pancreatic cancer (PC) progression and chemoresistance, suggesting its clinical association with poor prognosis. RNA sequencing analysis from the autochthonous pancreatic tumors showed a significant stromal alteration on genetic ablation of Muc5ac. Previously, depletion or targeting the stromal fibroblasts showed an ambiguous effect on PC pathogenesis. Hence, identifying the molecular players and mechanisms driving fibroblast heterogeneity is critical for improved clinical outcomes. Autochthonous murine models of PC (KrasG12D, Pdx1-Cre [KC] and KrasG12D, Pdx1-Cre, Muc5ac-/- [KCM]) and co-implanted allografts of murine PC cell lines (Muc5ac wild-type and CRISPR/Cas knockout) with adipose-derived mesenchymal stem cells (AD-MSCs) were used to assess the role of Muc5ac in stromal heterogeneity. Proliferation, migration, and surface expression of cell-adhesion markers on AD-MSCs were measured using live-cell imaging and flow cytometry. MUC5AC-interactome was investigated using mass-spectrometry and enzyme-linked immunosorbent assay. The KCM tumors showed a significant decrease in the expression of α-smooth muscle actin and fibronectin compared with histology-matched KC tumors. Our study showed that MUC5AC, carrying tumor secretome, gets enriched in the adipose tissues of tumor-bearing mice and patients with PC, promoting CD44/CD29 (integrin-β1) clustering that leads to Rac1 activation and migration of AD-MSCs. Furthermore, treatment with KC-derived serum enhanced proliferation and migration of AD-MSCs, which was abolished on Muc5ac-depletion or pharmacologic inhibition of CXCR2 and Rac1, respectively. The AD-MSCs significantly contribute toward α-smooth muscle actin-positive cancer-associated fibroblasts population in Muc5ac-dependent manner, as suggested by autochthonous tumors, co-implantation xenografts, and patient tumors. MUC5AC, secreted during PC progression, enriches in adipose and enhances the mobilization of AD-MSCs. On recruitment to pancreatic tumors, AD-MSCs proliferate and contribute towards stromal heterogeneity.

Safety and efficacy of prophylactic and therapeutic vaccine based on live-attenuated Listeria monocytogenes in hepatobiliary cancers

Primary liver cancer (PLC) comprising hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) represents the third deadliest cancer worldwide with still insufficient treatment options. We have previously found that CD4 T helper 1 (Th1) response is indispensable for the protection against PLC. In the present research, we aimed to test the potent inducers of Th1 responses, live-attenuated Listeria monocytogenes ∆actA/∆inlB strain as preventive/therapeutic vaccine candidate in liver fibrosis, HCC, and CCA. Studies were performed using autochthonous models of HCC and CCA, highly reflecting human disease. L. monocytogenes ∆actA/∆inlB demonstrated strong safety/efficacy in premalignant and malignant liver diseases. The protective mechanism relied on the induction of strong tumor-specific immune responses that keep the development of hepatobiliary cancers under control. Combination therapy, comprising Listeria vaccination and a checkpoint inhibitor blockade significantly extended the survival of HCC-bearing mice even at the advanced stages of the disease. This is the first report on the safety and efficacy of Listeria-based vaccine in liver fibrosis, as well as the first proof of principle study on Listeria-based vaccines in CCA. Our study paves the way for the use of live-attenuated Listeria as safe and efficient vaccine and a potent inducer of protective immune responses in liver fibrosis and hepatobiliary malignancies.

Loss of ASAP1 in the MMTV-PyMT model of luminal breast cancer activates AKT, accelerates tumorigenesis, and promotes metastasis

ASAP1 is a multi-domain adaptor protein that regulates cytoskeletal dynamics, receptor recycling and intracellular vesicle trafficking. Its expression is associated with poor prognosis in a variety of cancers, and can promote cell migration, invasion and metastasis. Although amplification and expression of ASAP1 has been associated with poor survival in breast cancer, we found that in the autochthonous MMTV-PyMT model of luminal breast cancer, ablation of ASAP1 resulted in an earlier onset of tumor initiation and increased metastasis. This was due to tumor cell-intrinsic effects of ASAP1 deletion, as ASAP1 deficiency in tumor, but not in stromal cells was sufficient to replicate the enhanced tumorigenicity and metastasis observed in the ASAP1-null MMTV-PyMT mice. Loss of ASAP1 in MMTV-PyMT mice had no effect on proliferation, apoptosis, angiogenesis or immune cell infiltration, but enhanced mammary gland hyperplasia and tumor cell invasion, indicating that ASAP1 can accelerate tumor initiation and promote dissemination. Mechanistically, these effects were associated with a potent activation of AKT. Importantly, lower ASAP1 levels correlated with poor prognosis and enhanced AKT activation in human ER+/luminal breast tumors, validating our findings in the MMTV-PyMT mouse model for this subtype of breast cancer. Taken together, our findings reveal that ASAP1 can have distinct functions in different tumor types and demonstrate a tumor suppressive activity for ASAP1 in luminal breast cancer.

Chemotherapy-induced adenosine A2B receptor expression mediates epigenetic regulation of pluripotency factors and promotes breast cancer stemness.

Rationale: Triple-negative breast cancer (TNBC) is characterized by its unique molecular profile, aggressive nature and lack of targeted therapy. Chemotherapy induces expression of pluripotency factors and mediates an active induction of breast cancer stem cells (BCSCs) in TNBC, which potentiates the risk of tumor recurrence and metastasis and increases patient mortality. Adenosine receptor 2B (A2BR) expression and activation of its downstream signaling pathway has been implied to promote breast cancer metastasis. This study is to investigate the role of A2BR in the regulation of chemotherapy-induced BCSC enrichment.Methods: We generated shRNA-mediated A2BR knockdown subclones in TNBC cell lines and evaluated the effect on the BCSC phenotype by Aldefluor and mammosphere assays in vitro. We performed chromatin immunoprecipitation (ChIP) assay to investigate recruitment of transcription factor FOXO3 and histone modification enzymes KDM6A and p300 to the regulatory regions of pluripotency factors, as well as levels of histone modification marks H3K27ac and H3K27me3 on these regions. We employed both xenograft model and genetically engineered, autochthonous breast cancer model to evaluate the effect of A2BR on chemotherapy-induced BCSC enrichment in vivo.Results: We demonstrated that chemotherapy increased protein level of A2BR, which contributed to chemotherapy-induced pluripotency factor expression and BCSC enrichment in TNBC. A2BR mediated activation of p38 MAPK and nuclear translocation of chromatin remodeling factor SMARCD3, which interacted and recruited histone demethylase KDM6A and histone acetyltransferase p300 specifically to the pluripotency factor genes NANOG, SOX2 and KLF4. Recruitment of KDM6A and p300 decreased histone H3K27me3 and increases H3K27ac marks, and increased transcription factor FOXO3 binding to NANOG, SOX2 and KLF4 genes, leading to transcriptional activation of these genes and BCSC specification. Genetic or pharmacological inhibition of A2BR blocked chemotherapy-mediated epigenetic activation of pluripotency factor genes and BCSC enrichment in vitro and in vivo, and delayed tumor recurrence after chemotherapy was discontinued.Conclusion: Chemotherapy-induced A2BR expression mediates epigenetic activation of pluripotency factors and promotes breast cancer stemness. Targeting A2BR in combination with chemotherapy may block BCSC enrichment and improve outcome in TNBC.

Loss of Rnf43 Accelerates Kras-Mediated Neoplasia and Remodels the Tumor Immune Microenvironment in Pancreatic Adenocarcinoma

RNF43 is an E3 ubiquitin ligase that is recurrently mutated in pancreatic ductal adenocarcinoma (PDAC) and precursor cystic neoplasms of the pancreas. The impact of RNF43 mutations on PDAC is poorly understood and autochthonous models have not been characterized sufficiently. In this study, we describe a genetically engineered mouse model (GEMM) of PDAC with conditional expression of oncogenic Kras and deletion of the catalytic domain of Rnf43 in exocrine cells. We generated Ptf1a-Cre;LSL-KrasG12D;Rnf43flox/flox (KRC) and Ptf1a-Cre; LSL-KrasG12D (KC) mice and animal survival was assessed. KRC mice were sacrificed at 2 months, 4 months, and at moribund status followed by analysis of pancreata by single-cell RNA sequencing. Comparative analyses between moribund KRC and a moribund Kras/Tp53-driven PDAC GEMM (KPC) was performed. Cell lines were isolated from KRC and KC tumors and interrogated by cytokine array analyses, ATAC sequencing, and invitro drug assays. KRC GEMMs were also treated with an anti-CTLA4 neutralizing antibody with treatment response measured by magnetic response imaging. We demonstrate that KRC mice display a marked increase in incidence of high-grade cystic lesions of the pancreas and PDAC compared with KC. Importantly, KRC mice have a significantly decreased survival compared with KC mice. Using single-cell RNA sequencing, we demonstrated that KRC tumor progression is accompanied by a decrease in macrophages, as well as an increase in T and B lymphocytes, with evidence of increased immune checkpoint molecule expression and affinity maturation, respectively. This was in stark contrast to the tumor immune microenvironment observed in the KPC PDAC GEMM. Furthermore, expression of the chemokine CXCL5 was found to be specifically decreased in KRC cancer cells by means of epigenetic regulation and emerged as a putative candidate for mediating the unique KRC immune landscape. The KRC GEMM establishes RNF43 as a bona fide tumor suppressor gene in PDAC. This GEMM features a markedly different immune microenvironment compared with previously reported PDAC GEMMs and puts forth a rationale for an immunotherapy approach in this subset of PDAC cases.

Anti-PD-1 Therapy with Adjuvant Ablative Fractional Laser Improves Anti-Tumor Response in Basal Cell Carcinomas.

Simple SummaryIn some mouse models, ablative fractional laser (AFL) enhances the efficacy of anti-programmed cell death1 therapy (aPD-1), which was recently approved for basal cell carcinoma (BCC). In this explorative study, we aimed to assess locally applied AFL as an adjuvant to systemic aPD-1 treatment in a clinically relevant BCC model. BCC-carrying mice received aPD-1 alone, AFL alone, aPD-1+AFL, or no treatment. Both aPD-1 and AFL alone significantly increased survival time relative to the untreated controls, while aPD-1 that had been complemented with AFL further promoted survival and improved tumor clearance and growth rates. The BCCs were poorly immune infiltrated, but aPD-1 with adjuvant AFL and AFL alone induced substantial immune cell infiltration in tumors and increased the levels of relevant immune cell subtypes. Thus, the anti-tumor response that was generated by aPD-1 with adjuvant AFL may potentially be promoted by increased immune activity in tumors. In conclusion, the use of a local AFL adjuvant to systemic aPD-1 therapy could hold substantial promise for BCC treatment.The efficacy of anti-programmedcelldeath1therapy (aPD-1), which was recently approved for basal cell carcinoma (BCC) treatment, can be enhanced by adjuvant ablative fractional laser (AFL) in syngeneic murine tumor models. In this explorative study, we aimed to assess locally applied AFL as an adjuvant to systemic aPD-1 treatment in a clinically relevant autochthonous BCC model. BCC tumors (n = 72) were induced in Ptch1+/−K14-CreER2p53fl/fl-mice (n = 34), and the mice subsequently received aPD-1 alone, AFL alone, aPD-1+AFL, or no treatment. The outcome measures included mouse survival time, tumor clearance, tumor growth rates, and tumor immune infiltration. Both aPD-1 and AFL alone significantly increased survival time relative to untreated controls (31 d and 34.5 d, respectively vs. 14 d, p = 0.0348–0.0392). Complementing aPD-1 with AFL further promoted survival (60 d, p = 0.0198 vs. aPD-1) and improved tumor clearance and growth rates. The BCCs were poorly immune infiltrated, but aPD-1 with adjuvant AFL and AFL alone induced substantial immune cell infiltration in the tumors. Similar to AFL alone, combined aPD-1 and AFL increased neutrophil counts (4-fold, p = 0.0242), the proportion of MHCII-positive neutrophils (p = 0.0121), and concordantly, CD4+ and CD8+ T-cell infiltration (p = 0.0061–0.0242). These descriptive results suggest that the anti-tumor response that is generated by aPD-1 with adjuvant AFL is potentially promoted by increased neutrophil and T-cell engraftment in tumors. In conclusion, local AFL shows substantial promise as an adjuvant to systemic aPD-1 therapy in a clinically relevant preclinical BCC model.

Development and immunohistochemical characterization of patient-derived xenograft models for muscle invasive bladder cancer.

Objective(s):Patient-derived xenograft (PDX) models have become a valuable tool to evaluate chemotherapeutics and investigate personalized cancer treatment options. The role of PDXs in the study of bladder cancer, especially for improvement of novel targeted therapies, continues to expand. In this study, we aimed to establish autochthonous PDX models of muscle-invasive bladder cancer (MIBC) to provide a useful tool to conduct research on personalized therapy. Materials and Methods:Tumors from MIBC patients undergoing radical cystectomy were subcutaneously transplanted into immunodeficient mice. The tumor size was measured by a caliper twice a week for up to five months. After the first growth in mice, they were serially passaged. Hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) of 11 markers (Ki67, P63, GATA3, KRT5/6, KRT20, E-cadherin, 34βE12, PD-L1, EGFR, Nectin4, and HER2) were used to evaluate phenotype maintenance of original tumors.Results:From 10 MIBC patients, two PDX models (P8X20 and P8X26) were successfully established (20% success rate). These models mostly retained primary tumor characteristics including histology, morphology, and molecular nature of the original cancer tissues. IHC analysis showed that the expression level of 7 markers for the model P8X20, and 8 markers for the model P8X26 was exactly similar between the patient tumor and the next generations.Conclusion:We developed the first autochthonous PDX models of MIBC in Iran. Our data suggested that the established MIBC PDX models reserved mostly histopathological characteristics of primary cancer and could provide a new tool to evaluate novel biomarkers, therapeutic targets, and drug combinations.

Quantitative Detection of Disseminated Melanoma Cells by Trp-1 Transcript Analysis Reveals Stochastic Distribution of Pulmonary Metastases.

A better understanding of the process of melanoma metastasis is required to underpin the development of novel therapies that will improve patient outcomes. The use of appropriate animal models is indispensable for investigating the mechanisms of melanoma metastasis. However, reliable and practicable quantification of metastases in experimental mice remains a challenge, particularly if the metastatic burden is low. Here, we describe a qRT-PCR-based protocol that employs the melanocytic marker Trp-1 for the sensitive quantification of melanoma metastases in the murine lung. Using this protocol, we were able to detect the presence of as few as 100 disseminated melanoma cells in lung tissue. This allowed us to quantify metastatic burden in a spontaneous syngeneic B16-F10 metastasis model, even in the absence of visible metastases, as well as in the autochthonous Tg(Grm1)/Cyld−/− melanoma model. Importantly, we also observed an uneven distribution of disseminated melanoma cells amongst the five lobes of the murine lung, which varied considerably from animal to animal. Together, our findings demonstrate that the qRT-PCR-based detection of Trp-1 allows the quantification of low pulmonary metastatic burden in both transplantable and autochthonous murine melanoma models, and show that the analysis of lung metastasis in such models needs to take into account the stochastic distribution of metastatic lesions amongst the lung lobes.

Oncogenic KRAS Requires Complete Loss of BAP1 Function for Development of Murine Intrahepatic Cholangiocarcinoma.

Simple SummaryIntrahepatic cholangiocarcinoma (ICC) is a primary liver cancer that currently has limited treatment options. As a result, patients with this disease generally have a poor prognosis. Previous studies have demonstrated that mutations in KRAS and loss of BRCA-1-associated protein 1 (BAP1) are frequently found in ICC. We developed a mouse model for ICC that incorporates KRAS and BAP1 mutations in a liver-dependent fashion to aid in the improvement of our understanding of this devastating disease. Our findings suggest that complete loss of BAP1 function combined with mutant KRAS appears to be a requirement for inducing ICC formation within the liver.Intrahepatic cholangiocarcinoma (ICC) is a primary biliary malignancy that harbors a dismal prognosis. Oncogenic mutations of KRAS and loss-of-function mutations of BRCA1-associated protein 1 (BAP1) have been identified as recurrent somatic alterations in ICC. However, an autochthonous genetically engineered mouse model of ICC that genocopies the co-occurrence of these mutations has never been developed. By crossing Albumin-Cre mice bearing conditional alleles of mutant Kras and/or floxed Bap1, Cre-mediated recombination within the liver was induced. Mice with hepatic expression of mutant KrasG12D alone (KA), bi-allelic loss of hepatic Bap1 (BhomoA), and heterozygous loss of Bap1 in conjunction with mutant KrasG12D expression (BhetKA) developed primary hepatocellular carcinoma (HCC), but no discernible ICC. In contrast, mice with homozygous loss of Bap1 in conjunction with mutant KrasG12D expression (BhomoKA) developed discrete foci of HCC and ICC. Further, the median survival of BhomoKA mice was significantly shorter at 24 weeks when compared to the median survival of ≥40 weeks in BhetKA mice and approximately 50 weeks in BhomoA and KA mice (p < 0.001). Microarray analysis performed on liver tissue from KA and BhomoKA mice identified differentially expressed genes in the setting of BAP1 loss and suggests that deregulation of ferroptosis might be one mechanism by which loss of BAP1 cooperates with oncogenic Ras in hepato-biliary carcinogenesis. Our autochthonous model provides an in vivo platform to further study this lethal class of neoplasm.

Abstract PO-063: Functional interrogation of immune escape in neoantigen-expressing pancreatic cancer identifies a critical role for the CD155/TIGIT axis

Abstract The CD155/TIGIT axis can be co-opted during immune evasion in chronic viral infections and cancer. While insights regarding mechanisms of immune escape have fueled tremendous clinical successes in a broad range of tumor types, microsatellite-stable pancreatic adenocarcinoma (PDAC), which represents greater than 98% of patients, is largely refractory to available immune checkpoint blockade. The recent recognition that a subset of pancreas cancer harbors potential neoantigens has intensified interest in defining the molecular and cellular mechanisms of immune evasion in PDAC to guide effective therapeutic strategies that leverage the adaptive immune system in this disease. However, difficulty in precisely defining the tumor-reactive T cell compartment has hampered prior efforts to delineate the full spectrum of mechanisms by which PDAC evades immune eradication. We recently developed multiple organoid-based and autochthonous preclinical models to delineate the molecular and cellular mechanisms of immune evasion in this subset of patients. Specifically, we utilized a high affinity MHC class I-restricted antigen (OVA257–264) or recently described missense-derived MHC class I-restricted neoantigens (mutations in the laminin 4 alpha subunit or in alpha-1,3-glucosyltransferase). While a subset of animals either successfully clear these tumors, or are arrested in a state of immune-mediated tumor dormancy, all three neoantigen models lead to a significant subset of tumors that acquire the ability to evade immune clearance. In these preclinical models, we demonstrate that intratumoral neoantigen-specific CD8+ T cells adopt multiple states of dysfunction, typified by T cell exhaustion (best marked by TIGIT+PD1+ co-positivity). Global profiling of the tumor-immune microenvironment in neoantigen-expressing murine PDAC compared to non-neoantigen-expressing PDAC identified an expected increase in CD8+ T cells, and specifically in neoantigen-specific CD8+ T cells, but also identified additional changes within the broader leukocyte contexture. Using flow cytometric and single-cell transcriptomic analyses, we demonstrate that human PDAC TILs express similar dysfunctional programs and are enriched in TIGIT+PD1+ TILs. Furthermore, we show that CD155, the high affinity TIGIT ligand, is highly expressed on the surface of murine and human PDAC tumor cells. Using genetic (CRISPR-activation) and/or pharmacologic modulation, we functionally interrogate the CD155/TIGIT axis and demonstrate that increased signaling through CD155 and/or TIGIT is sufficient to promote immune evasion in neoantigen-expressing PDAC. Lastly, we identified a combination immunotherapy (TIGIT/PD-1 co-blockade plus CD40 agonism) that elicits profound anti-tumor responses in these preclinical models. Using a suite of high-resolution analyses, we are dissecting the mechanisms of effectiveness and resistance to this combination immunotherapy, which is set to enter clinical trials in pancreatic cancer later this year. Citation Format: William Freed-Pastor, Laurens Lambert, Zackery Ely, Nimisha Pattada, Arjun Bhutkar, Alex Jaeger, George Eng, Kim Mercer, William Hwang, Tyler Jacks. Functional interrogation of immune escape in neoantigen-expressing pancreatic cancer identifies a critical role for the CD155/TIGIT axis [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-063.

Abstract PR-010: Collateral amplification of the KRAS linked gene PTHLH governs pancreatic cancer growth and metastasis and reveals a new therapeutic vulnerability

Abstract Purpose: Metastasis is the leading cause of cancer-related death in PDAC, yet very little is understood regarding the underlying biology. As a result, targeted therapies to inhibit metastasis are lacking. Whole-genome sequencing has established that the squamous/quasi-mesenchymal/basal-like PDAC subtype, which is characterized by its high metastatic proclivity, is annotated by KRAS gene amplification. Here, we report that the squamous lineage gene parathyroid hormone-related protein (PTHrP encoded by PTHLH) is located directly adjacent to KRAS and is co-amplified in metastatic PDAC patients. We hypothesize that this collateral amplification of PTHrP may exert its own oncogenic and pro-metastatic phenotype beyond KRAS and set out to determine if this will confer a novel therapeutic vulnerability. Methods: We generated a novel genetically engineered mouse model whereby we deleted the cytokine Pthlh in the autochthonous KPCY model. To functionally demonstrate the oncogenic and pro-metastatic roles of PTHrP, we further employed genetic deletion and pharmacological inhibition in orthotopic injection, tail vein metastasis assays, mouse hospital pre-clinical trials, and patient-derived 3D organoid models. Results: In silico analysis established that PTHLH is co-amplified along with KRAS in TCGA, is specifically enriched in metastatic patients from the COMPASS trial, and correlates with significantly decreased overall survival in both cohorts. Further examination revealed that PTHLH is a squamous/quasi-mesenchymal/basal-like lineage marker. We generated KPCY-PthlhCKO mice and showed that they have significantly reduced primary and metastatic tumor burden and dramatically increased overall survival relative to KPCY controls. In parallel experiments, we treated mice with an anti-PTHrP neutralizing monoclonal antibody, which similarly reduced primary and metastatic tumor growth. Finally, RNA-seq revealed a downstream mechanism whereby PTHrP is important for metastatic competency through induction of EMT, thus facilitating entry into the metastatic cascade. Loss of PTHrP reduced the ability of tumor cells to undergo EMT both in vivo and in vitro, resulting in a nearly complete elimination of disseminating cells in KPCY-PthlhCKO mice. Thus, KPCY-PthlhCKO tumors are locked in a well-differentiated epithelial state and are unable to initiate the metastatic process. Conclusions: This work has demonstrated the importance of the previously unappreciated role for PTHrP signaling in pancreatic cancer cell plasticity and metastasis, and future studies will look to translate anti-PTHrP therapy into clinical trials. In a broader sense, we establish a new paradigm of collateral amplification, where an assumed passenger gene (PTHLH) is co-amplified along with a known oncogene (KRAS) and endows the evolving tumor with its own oncogenic and pro-metastatic phenotype. Citation Format: Jason R. Pitarresi, Robert J. Norgard, Anna M. Chiarella, Kensuke Suzuki, Richard Kremer, Ben Z. Stanger, Anil K. Rustgi. Collateral amplification of the KRAS linked gene PTHLH governs pancreatic cancer growth and metastasis and reveals a new therapeutic vulnerability [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-010.

Bacterial cancer therapy in autochthonous colorectal cancer affects tumor growth and metabolic landscape.

Bacterial cancer therapy (BCT) shows great promise for treatment of solid tumors, yet basic mechanisms of bacterial-induced tumor suppression remain undefined. Attenuated strains of Salmonella enterica serovar Typhimurium (STm) have commonly been used in mouse models of BCT in xenograft and orthotopic transplant cancer models. We aimed to better understand the tumor epithelium–targeted mechanisms of BCT by using autochthonous mouse models of intestinal cancer and tumor organoid cultures to assess the effectiveness and consequences of oral treatment with aromatase A–deficient STm (STmΔaroA). STmΔaroA delivered by oral gavage significantly reduced tumor burden and tumor load in both a colitis-associated colorectal cancer (CAC) model and in a spontaneous Apcmin/+ intestinal cancer model. STmΔaroA colonization of tumors caused alterations in transcription of mRNAs associated with tumor stemness, epithelial-mesenchymal transition, and cell cycle. Metabolomic analysis of tumors demonstrated alteration in the metabolic environment of STmΔaroA-treated tumors, suggesting that STmΔaroA imposes metabolic competition on the tumor. Use of tumor organoid cultures in vitro recapitulated effects seen on tumor stemness, mesenchymal markers, and altered metabolome. Furthermore, live STmΔaroA was required, demonstrating active mechanisms including metabolite usage. We have demonstrated that oral BCT is efficacious in autochthonous intestinal cancer models, that BCT imposes metabolic competition, and that BCT has direct effects on the tumor epithelium affecting tumor stem cells.

Reprogramming of Neutrophils as Non-canonical Antigen Presenting Cells by Radiotherapy-Radiodynamic Therapy to Facilitate Immune-Mediated Tumor Regression.

Ineffective antigen cross-presentation in the tumor microenvironment compromises the generation of antitumor immune responses. Radiotherapy-radiodynamic therapy (RT-RDT) with nanoscale metal-organic frameworks (nMOFs) induces robust adaptive immune responses despite modest activation of canonical antigen presenting dendritic cells. Here, using transplantable and autochthonous murine tumor models, we demonstrate that RT-RDT induces antitumor immune responses via early neutrophil infiltration and reprogramming. Intravenous or intratumoral injection of nMOFs recruited peripheral CD11b+Ly6G+CD11c- neutrophils into tumors. The activation of nMOFs by low-dose X-rays significantly increased the population of CD11b+Ly6G+CD11c+ hybrid neutrophils with upregulated expression of the co-stimulatory molecules CD80 and CD86 as well as major histocompatibility complex class II molecules. Thus, nMOF-enabled RT-RDT reshapes a favorable tumor microenvironment for antitumor immune responses by reprogramming tumor-infiltrating neutrophils to function as non-canonical antigen presenting cells for effective cross-presentation of tumor antigens.

New Paleomagnetic Constraints on the Cretaceous Tectonic Framework of the Antarctic Peninsula

AbstractUnderstanding the tectonic framework of the Antarctic Peninsula is hindered by a paucity of paleomagnetic data from key locations. In this study, we present paleomagnetic data from the South Shetland Islands, to the northwest of the Antarctic Peninsula, which provides valuable paleoposition constraints on the Western domain of the Antarctic Peninsula. We report a key reliable paleopole (58.1°S, 354.3°E, A95 = 6.3°) from Livingston Island in the South Shetland Islands at ∼102 Ma. Plate reconstruction models from the Early Cretaceous attach the South Shetland Islands to the Pacific margin of southern Patagonia‐Fuegian Andes at ∼140 Ma. The South Shetland Islands then experienced southward translation to its current position to the northwest of the Antarctic Peninsula following counterclockwise rotation during ∼100–90 Ma. A similar counterclockwise rotation has also been identified from southern Patagonia‐Fuegian Andes but is absent in the Antarctic Peninsula, suggesting a direct affinity between the South Shetland Islands and southern Patagonia‐Fuegian Andes. However, the consistent, almost northward Cretaceous paleomagnetic declination in the Antarctic Peninsula, and the near‐synchronous tectonic‐magmatic history between the Antarctic Peninsula and the southern Patagonia‐Fuegian Andes support an autochthonous continental subduction model for most of the Antarctic Peninsula.