Autochthonous Model Research Articles

Cell microparticles loaded with tumor antigen and resiquimod reprogram tumor-associated macrophages and promote stem-like CD8+ T cells to boost anti-PD-1 therapy

The durable response rate to immune checkpoint blockade such as anti-programmed cell death-1 (PD-1) antibody remains relatively low in hepatocellular carcinoma (HCC), mainly depending on an immunosuppressive microenvironment with limited number of CD8+ T cells, especially stem-like CD8+ T cells, in tumor tissues. Here we develop engineered microparticles (MPs) derived from alpha-fetoprotein (AFP)-overexpressing macrophages to load resiquimod (R848@M2pep-MPsAFP) for enhanced anti-PD-1 therapy in HCC. R848@M2pep-MPsAFP target and reprogram immunosuppressive M2-like tumor-associated macrophages (TAMs) into M1-like phenotype. Meanwhile, R848@M2pep-MPsAFP-reprogrammed TAMs act as antigen-presenting cells, not only presenting AFP antigen to activate CD8+ T cell-mediated antitumor immunity, but also providing an intra-tumoral niche to maintain and differentiate stem-like CD8+ T cells. Combination immunotherapy with anti-PD-1 antibody generates strong antitumor immune memory and induces abundant stem-like CD8+ T cell proliferation and differentiation to terminally exhausted CD8+ T cells for long-term immune surveillance in orthotopic and autochthonous HCC preclinical models in male mice. We also show that the R848-loaded engineered MPs derived from macrophages overexpressing a model antigen ovalbumin (OVA) can improve anti-PD-1 therapy in melanoma B16-OVA tumor-bearing mice. Our work presents a facile and generic strategy for personalized cancer immunotherapy to boost anti-PD-1 therapy.

CUL4B functions as a tumor suppressor in KRAS-driven lung tumors by inhibiting the recruitment of myeloid-derived suppressor cells.

Lung cancer is the leading cause of cancer-related death worldwide. KRAS mutations are the most common oncogenic alterations found in lung cancer. Unfortunately, treating KRAS-mutant lung adenocarcinoma (ADC) remains a major oncotherapeutic challenge. Here, we used both autochthonous and transplantable KRAS-mutant tumor models to investigate the role of tumor-derived CUL4B in KRAS-driven lung cancers. We showed that knockout or knockdown of CUL4B promotes lung ADC growth and progression in both models. Mechanistically, CUL4B directly binds to the promoter of Cxcl2 and epigenetically represses its transcription. CUL4B deletion increases the expression of CXCL2, which binds to CXCR2 on myeloid-derived suppressor cells (MDSCs) and promotes their migration to the tumor microenvironment. Targeting of MDSCs significantly delayed the growth of CUL4B knockdown KRAS-mutant tumors. Collectively, our study provides mechanistic insights into the novel tumor suppressor-like functions of CUL4B in regulating KRAS-driven lung tumor development.

Ablative radiotherapy improves survival but does not cure autochthonous mouse models of prostate and colorectal cancer

BackgroundGenetically engineered mouse models (GEMMs) of cancer are powerful tools to study mechanisms of disease progression and therapy response, yet little is known about how these models respond to multimodality therapy used in patients. Radiation therapy (RT) is frequently used to treat localized cancers with curative intent, delay progression of oligometastases, and palliate symptoms of metastatic disease.MethodsHere we report the development, testing, and validation of a platform to immobilize and target tumors in mice with stereotactic ablative RT (SART). Xenograft and autochthonous tumor models were treated with hypofractionated ablative doses of radiotherapy.ResultsWe demonstrate that hypofractionated regimens used in clinical practice can be effectively delivered in mouse models. SART alters tumor stroma and the immune environment, improves survival in GEMMs of primary prostate and colorectal cancer, and synergizes with androgen deprivation in prostate cancer. Complete pathologic responses were achieved in xenograft models, but not in GEMMs.ConclusionsWhile SART is capable of fully ablating xenografts, it is unable to completely eradicate disease in GEMMs, arguing that resistance to potentially curative therapy can be modeled in GEMMs.

EPEN-01. ZFTA-RELA FUSION PROGRAMS CONVERGE ON YAP PATHWAY ACTIVATION IN EPENDYMOMA

Abstract Approximately two thirds of supratentorial ependymoma are driven by the ZFTA-RELA gene fusion. ZFTA is a recently described gene with a role in DNA binding and transcriptional regulation, while RELA (p65) is a known principal mediator of the canonical NFkB pathway, a pathway activated many human malignancies. The underlying mechanism of NFkB pathway protein interaction with the ZFTA-RELA fusion to drive tumorgenesis has not yet been described. In this study, we utilized our autochthonous mouse tumor model using in utero electroporation (IUE) of the embryonic mouse brain to explore these questions. Using rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) targeting HA-tagged ZFTA-RELA fusion in our IUE model, we demonstrate that both canonical and non-canonical NFkB proteins interact with ZFTA-RELA protein in ZFTA-RELA fusion ependymoma. Focusing on select key NFkB proteins, we performed chromatin immunoprecipitation sequencing (ChIP-Seq) targeting p50/NFkB1, a protein that partners with RELA/p65 in the canonical NFkB pathway, revealing that p50/NFkB1 regulatory programs define active genes that are both distinct from and shared with the ZFTA-RELA fusion protein. Functionally, we performed CRISPR-CAS9 knock out (KO) of p50/NFkB1 and identified no phenotypic effect, as KO lines demonstrated no growth impairment. Furthermore, NFkB targeting drugs did not impair tumor growth. Given these findings, suggesting redundant transcriptional programing, we identified that ZFTA and RELA intersect with TEAD transcription factor programs to activate YAP signaling and that p50 localizes to YAP-TEAD pathway members. Targeting TEAD 1 and 4 with ChIP-Seq, we found that TEAD 1 and 4 co-regulate p50/NFkB1 and ZFTA-RELA target genes. Finally, we demonstrate YAP pathway activation across all supratentorial ependymoma, including ZFTA-RELA ependymoma. Our pathway analysis consistently converged on YAP/TEAD pathways, suggesting collaboration of ZFTA-RELA fusion with YAP/TEAD in transcriptional regulation for these tumors. Targeting YAP/TEAD may therefore represent a promising therapeutic strategy for ZFTA-RELA fusion ependymoma.

High-Throughput Identification, Modeling, and Analysis of Cancer Driver Genes In Vivo.

The vast number of genomic and molecular alterations in cancer pose a substantial challenge to uncovering the mechanisms of tumorigenesis and identifying therapeutic targets. High-throughput functional genomic methods in genetically engineered mouse models allow for rapid and systematic investigation of cancer driver genes. In this review, we discuss the basic concepts and tools for multiplexed investigation of functionally important cancer genes in vivo using autochthonous cancer models. Furthermore, we highlight emerging technical advances in the field, potential opportunities for future investigation, and outline a vision for integrating multiplexed genetic perturbations with detailed molecular analyses to advance our understanding of the genetic and molecular basis of cancer.

Abstract B029: Foxf2 activates antitumor immunity to repress the progression of prostate cancer by repressing Fgl1

Abstract Targeting immune suppressive mechanisms in the tumor microenvironment has revolutionized cancer treatment. Although immune checkpoint inhibition (ICI) has yielded meaningful responses across many cancer types, the ICI efficacy is still limited for prostate cancer. We generated a mouse model with prostate epithelial specific expression of Foxf2, a Forkhead box transcription factor. Foxf2 overexpression suppressed tumor growth in two mouse models for prostate cancer with prostate specific Pten deletion or TRAMP. Foxf2 expression strongly induced tumor cell-intrinsic immune responses by recruiting and activating CD8+ T cells and switching the polarization of macrophages from an immunosuppressive M2-like phenotype to a more inflammatory M1-like state in both syngeneic and autochthonous mouse prostate cancer models in an immunocompetent context. Furthermore, we showed that overexpression of Foxf2 in mouse and human prostate cells repressed the expression of FGL1, a major ligand of LAG-3 that mediates T cell suppression. In this study, we take a combination of bioinformatic, molecular, cellular, and genetic approaches and reveal that Foxf2 overexpression in prostate cancer suppresses tumor progression by activating antitumor immunity. Citation Format: Deyong Jia, Zhicheng Zhou, OhJoon Kwon, Li Xin. Foxf2 activates antitumor immunity to repress the progression of prostate cancer by repressing Fgl1 [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B029.

SAT-219 - Capillarization of tumor endothelial cells within tumor associated with T cell exclusion in an autochthonous mouse HCC model

SAT-219 - Capillarization of tumor endothelial cells within tumor associated with T cell exclusion in an autochthonous mouse HCC model

Heart, Not Souls, Of Consciousness in Asabano Ethnopsychology

Ethnic cultural conceptualizations of consciousness often posit souls or other spirits, but these do not always address consciousness itself. This article describes an autochthonous model of consciousness that was current among the Asabano people of central New Guinea before first contact in the mid-twentieth century. In their conceptualization, one's own souls were not seen as essences of the self or agents of personal awareness. Rather, they merely inflected awareness, which was understood to occur in the heart. This autochthonous model of consciousness attributed selfish impulses and bodily strength to a 'little soul' and generous inclinations to a 'big soul'. The heart was the seat of thought and feeling and the recipient of the souls' proddings, and sensory perceptions originating in other body parts.

Single-Cell Evolutionary Analysis Reveals Drivers of Plasticity and Mediators of Chemoresistance in Small Cell Lung Cancer.

Understanding the evolutionary dynamics of SCLC can shed light on key mechanisms such as cellular plasticity, heterogeneity, and chemoresistance.

Targeted TLR9 Agonist Elicits Effective Antitumor Immunity against Spontaneously Arising Breast Tumors.

Spontaneous tumors that arise in genetically engineered mice recapitulate the natural tumor microenvironment and tumor-immune coevolution observed in human cancers, providing a more physiologically relevant preclinical model relative to implanted tumors. Similar to many cancer patients, oncogene-driven spontaneous tumors are often resistant to immunotherapy, and thus novel agents that can effectively promote antitumor immunity against these aggressive cancers show considerable promise for clinical translation, and their mechanistic assessment can broaden our understanding of tumor immunology. In this study, we performed extensive immune profiling experiments to investigate how tumor-targeted TLR9 stimulation remodels the microenvironment of spontaneously arising tumors during an effective antitumor immune response. To model the clinical scenario of multiple tumor sites, we used MMTV-PyMT transgenic mice, which spontaneously develop heterogeneous breast tumors throughout their 10 mammary glands. We found that i.v. administration of a tumor-targeting TLR9 agonist, referred to as PIP-CpG, induced a systemic T cell-mediated immune response that not only promoted regression of existing mammary tumors, but also elicited immune memory capable of delaying growth of independent newly arising tumors. Within the tumor microenvironment, PIP-CpG therapy initiated an inflammatory cascade that dramatically amplified chemokine and cytokine production, prompted robust infiltration and expansion of innate and adaptive immune cells, and led to diverse and unexpected changes in immune phenotypes. This study demonstrates that effective systemic treatment of an autochthonous multisite tumor model can be achieved using a tumor-targeted immunostimulant and provides immunological insights that will inform future therapeutic strategies.

Epigenetic plasticity cooperates with cell-cell interactions to direct pancreatic tumorigenesis.

The response to tumor-initiating inflammatory and genetic insults can vary among morphologically indistinguishable cells, suggesting as yet uncharacterized roles for epigenetic plasticity during early neoplasia. To investigate the origins and impact of such plasticity, we performed single-cell analyses on normal, inflamed, premalignant, and malignant tissues in autochthonous models of pancreatic cancer. We reproducibly identified heterogeneous cell states that are primed for diverse, late-emerging neoplastic fates and linked these to chromatin remodeling at cell-cell communication loci. Using an inference approach, we revealed signaling gene modules and tissue-level cross-talk, including a neoplasia-driving feedback loop between discrete epithelial and immune cell populations that was functionally validated in mice. Our results uncover a neoplasia-specific tissue-remodeling program that may be exploited for pancreatic cancer interception.

Melanocyte PDL1 promotes early tumor progression in a novel autochthonous NRAS-mutant melanoma model

Abstract Tumor-intrinsic PDL1 drives melanoma treatment resistance. Melanocytes (melanoma cell-of-origin) do not express PDL1, but PDL1 is expressed in malignant melanomas, suggesting melanocyte PDL1 promotes melanomagenesis. We developed a novel autochthonous mouse model that develops NRAS Q61R-mutant melanomas lacking PDL1 only in melanocytes (PDL1 KOTN Q61R) and littermate controls (PDL1 +/+TN Q61R). We induced mice with 4-OH tamoxifen ± UV exposure to accelerate melanoma development. Tumor latency significantly increased in PDL1 KOvs. PDL1 +/+TN Q61Rmice at 0 kJ/m 2and 2 kJ/m 2UV(p<0.02), suggesting melanocyte/tumor PDL1 promotes melanomagenesis. Latencies were similar at 4.5 kJ/m 2, suggesting immune contributions, e.g., from local immunosuppression. We derived transplantable cell lines from autochthonous tumors. PDL1 +/+TN Q61Rcell lines had increased mTORC1 and stemness, and reduced STING and reduced Chk1 inhibitor sensitivity in vitro and in vivo vs. PDL1 KOTN Q61R, phenocopied by PDL1 KOB16. Tumor PDL1 suppressed ERK signals (NRAS target) in TN Q61Rvs. PDL1 KOcell lines and promoted resistance to NRAS-targeting small molecule drugs, suggesting tumor PDL1 promotes NRAS signals. Transplanted 4.5 kJ/m 2UV-inducedPDL1 KOTN Q61Rtumors were resistant to αPD1, αPDL1, αPDL2, and CD122-biased IL2 in WT mice vs. B16 melanomas, which are sensitive to biased IL2. Our model allows studies of PDL1 signals in melanomagenesis and progression and distinguishes bona fide cell-intrinsic PDL1 signals. This research was funded by the NIH T32GM113896 (STX MSTP) Award (C.O.) and the NCI (CA204965, CA054515) supported Curiel.

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.

CRISPR-Cas-amplified urinary biomarkers for multiplexed and portable cancer diagnostics

Synthetic biomarkers, bioengineered sensors that generate molecular reporters in diseased microenvironments, represent an emerging paradigm in precision diagnostics. Despite the utility of DNA barcodes as a multiplexing tool, their susceptibility to nucleases in vivo has limited their utility. Here we exploit chemically stabilized nucleic acids to multiplex synthetic biomarkers and produce diagnostic signals in biofluids that can be ‘read out’ via CRISPR nucleases. The strategy relies on microenvironmental endopeptidase to trigger the release of nucleic acid barcodes and polymerase-amplification-free, CRISPR-Cas-mediated barcode detection in unprocessed urine. Our data suggest that DNA-encoded nanosensors can non-invasively detect and differentiate disease states in transplanted and autochthonous murine cancer models. We also demonstrate that CRISPR-Cas amplification can be harnessed to convert the readout to a point-of-care paper diagnostic tool. Finally, we employ a microfluidic platform for densely multiplexed, CRISPR-mediated DNA barcode readout that can potentially evaluate complex human diseases rapidly and guide therapeutic decisions.

Abstract 3: In vivo CRISPR activation screening identifies tissue-specific oncogene selection

Abstract Background: While the genetic landscapes of pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC) are relatively well described, the contribution of individual genes and transcriptomic programs to progression and metastasis remain incompletely understood. To quantify the tissue-specific selective advantage of transcriptomic drivers of tumor progression and metastasis, we have developed a platform for in vivo CRISPR activation (CRISPRa) oncogene screening. Material and Methods: Our platform consists of an autochthonous CRISPRa-competent mouse tumor model (PPKS; P53(F/F), Kras(LSL-G12D/+), R26(LSL-SAM), coupled with in vivo delivery of Cre/sgRNA lentiviral libraries. To evaluate tissue-specific differences in oncogene selection, we designed a CRISPRa library targeting 450 of the most frequently altered genes across human adenocarcinoma. We next generated lung and pancreas tumors through administration of controlled doses of Cre/sgRNA library in vivo. Tumors and metastatic lesions were isolated and guide enrichment was determined using NGS. We also performed CRISPRa screening on proto-oncogenic (PPKS) pancreatic organoids orthotopically transplanted into syngeneic or immunodeficient mice (NU/J). The results from our CRISPRa screening were confirmed using single-gene activation in vivo and in pancreatic organoids. Results: We found that pancreatic tumors displayed dramatically higher levels of guide selection relative to lung tumors. While, on average, 99.7% of guides were represented in primary lung tumors at survival endpoint, only 40.4% of guides were detected in pancreatic tumors. We identified very strong selection for Egfr-activation in primary lung tumors, followed by MafB, Sox2 and Myc. In contrast, we found near-complete dominance of Myc-activation in pancreatic tumors. Since EGFR and MYC represent two of the most frequently altered genes in NSCLC and PDAC, respectively, this indicates our ability to recapitulate clinically observed oncogene selection. To determine if the selection for Myc-activation in the pancreas was driven by immune response, we performed screening in pancreatic organoids transplanted into immune competent and incompetent mice and found that Myc was uniformly the most highly enriched target. To confirm our findings, we generated Myc-activated proto-oncogenic organoids and autochthonous tumors, and both displayed accelerated tumor formation relative to non-targeted controls. Conclusions: We present one of the first implementations of autochthonous in vivo CRISPR activation screening, allowing for direct observation and quantification of oncogene selection and competition in vivo. Our model faithfully recapitulated oncogene selection observed in patient tumors by identifying EGFR and MYC as dominating oncogenes in the context of KRAS mutant lung and pancreas tumors, respectively. Citation Format: Fredrik Ivar Thege, Sonja M. Woermann, Anirban Maitra. In vivo CRISPR activation screening identifies tissue-specific oncogene selection [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 3.

Abstract NG12: Altered calcium signaling in cancer: PTHrP-KRAS collateral amplification governs pancreatic cancer metastasis, cachexia, and immunosuppression

Abstract Purpose: Metastasis is the leading cause of cancer-related death in pancreatic ductal adenocarcinoma (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 the PTHLH gene) is located directly adjacent to KRAS and is co-amplified as part of the KRAS amplicon in metastatic PDAC patients. We hypothesize that this collateral amplification of PTHrP, and downstream activation of calcium signaling, exerts its own oncogenic and pro-metastatic phenotypes beyond KRAS and set out to determine if this will confer a novel therapeutic vulnerability. Finally, we postulate that PTHrP-mediated Calcium signaling may be responsible for driving other non-cell autonomous PDAC-associated phenotypes, including cancer cachexia and the organization of an immunosuppressive microenvironment. Methods: We generated a novel genetically engineered mouse model whereby we deleted the cytokine PTHrP in the autochthonous KPCY model of pancreatic cancer (mutant Kras and mutant p53 activated in the pancreatic epithelium). To functionally demonstrate the pro-metastatic and cachexia-inducing roles of PTHrP, we further employed genetic deletion and pharmacological inhibition in orthotopic injection and mouse hospital pre-clinical trials. Results: In silico analysis established that PTHLH (the gene encoding the PTHrP protein) is co-amplified along with KRAS in TCGA and correlates with significantly decreased overall survival. 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. Mechanistic studies revealed that intracellular calcium was responsible for turning on an epithelial-to-mesenchymal (EMT) program that could be impeded by inhibiting PTHrP, thus blocking tumor cells from entering the metastatic cascade. Additionally, both genetic deletion and pharmacological inhibition of PTHrP in vivo led to a profound reduction in cachexia-related adipose tissue wasting and muscle atrophy. Re-introduction of PTHrP into a PDAC cell line with low cachexia-inducing potential (and low baseline PTHrP) dramatically increased the degree of cachexia and metastasis observed upon orthotopic implantation. Thus, PTHrP is both necessary and sufficient to induce cachexia and metastasis. Additionally, mice implanted with PTHrP overexpressing tumor cells had a dramatic reduction in overall survival. Finally, we demonstrated that PTHrP is responsible for organizing a pro-tumor immunosuppressive microenvironment by recruiting Myeloid Derived Suppressive Cells (MDSCs). Blocking PTHrP signaling reduces the degree of local immune suppression allowing for the enhanced recruitment of CD8+ T cells. Thus, PTHrP is a pleiotropic molecule that governs many pancreatic cancer-associated disease phenotypes. Conclusions: This work has demonstrated the importance of the previously unappreciated roles of PTHrP signaling in driving diverse pancreatic cancer phenotypes from metastasis to cachexia to immunosuppression, and future studies will look to translate anti-PTHrP therapy into clinical trials. Citation Format: Jason Robert Pitarresi. Altered calcium signaling in cancer: PTHrP-KRAS collateral amplification governs pancreatic cancer metastasis, cachexia, and immunosuppression. [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 NG12.

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 SY15-01: Tumor agnostic dendritic cell vaccines

Abstract Dendritic cells (DCs) are specialized myeloid cells with the ability to uptake, process, and present antigens to T lymphocytes; they also generate cytokine and chemokine gradients that regulate immune cell trafficking, activation, and function. DCs pulsed with tumor antigens have been used as a platform for therapeutic vaccination in cancer. In a broadly tested clinical application called a DC vaccine, autologous monocyte-derived DCs (moDCs) are isolated from a patient with cancer, exposed to tumor antigens ex vivo (antigen loading), and matured before reinfusion to the patient. However, in spite of significant development and testing, DC vaccines based on antigen-loaded moDCs have delivered mixed clinical results. The suboptimal performance of DC vaccines may be due to the use of moDCs - a heterogenous population of non-professional antigen-presenting cells - and the artificial nature of the procedure used to "load" selected tumor antigens on moDCs ex vivo. To address both issues, we developed a DC vaccine that uses a population of mouse or human DC progenitors (DCPs) engineered to produce immunostimulatory cytokines. While not involving antigen loading, the new DC platform outperformed antigen-loaded moDCs in mouse models of cancer. Engineered DCPs produced professional cDC1 and cCD2 in mice, reprogrammed the tumor microenvironment to a form characterized by broad IFNg signaling, and inhibited tumors, including autochthonous models, in an antigen-agnostic fashion. In one application, engineered DCPs synergized with antigen-specific CAR-T cells to eradicate gliomas in mice. Citation Format: Michele De Palma. Tumor agnostic dendritic cell vaccines. [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 SY15-01.

Abstract 1152: NKTR-255, a polymer-conjugated IL-15, dramatically improves ROR1 CAR-T cell persistence and anti-tumor efficacy in an autochthonous model of ROR1+ lung cancer

Abstract CAR-T cells have mediated remarkable clinical responses against hematological malignancies, but this success has yet to extend to solid tumors, where many factors in the tumor microenvironment (TME) inhibit their persistence and function. In a phase 1 trial at our Center, CAR-T cells targeting the tumor-associated antigen ROR1 in patients with non-small cell lung cancer (NSCLC) and triple negative breast cancer rapidly became dysfunctional and persisted poorly at tumor sites, ultimately failing to mediate objective responses. To evaluate strategies to improve ROR1 CAR-T efficacy, we adapted the KrasLSL-G12D/+;p53fl/fl (KP) autochthonous model of lung adenocarcinoma to express ROR1 (KPROR1). This aggressive model mimics the initiation, progression, and suppressive TME of human NSCLC. ROR1 CAR-T cells in KPROR1 mice rapidly declined in number, lost the ability to produce pro-inflammatory cytokines, and failed to significantly enhance tumor control, recapitulating the major barriers to CAR-T efficacy we observed in patients. NKTR-255 is a novel immunotherapeutic consisting of a polymer conjugated recombinant human interleukin-15 (rhIL-15) that could enhance the activity of CAR-T cells. NKTR-255 binds to the IL-15 receptor and IL-2/IL-15 receptor, thus maintaining the full potential spectrum of IL-15 biology while exerting a sustained effect on CD8+ memory T cell and NK cell expansion and persistence. We hypothesized that the combination of CAR-T cells and NKTR-255 would be feasible and safe and may extend the duration of efficacy of ROR1 CAR-T cells in ROR1+ lung tumors by improving their expansion and persistence. To test this, we treated tumor-bearing KPROR1 mice with control or ROR1 CAR-T cells with or without weekly injections of NKTR-255. NKTR-255 treatment resulted in a robust expansion of ROR1 CAR-T cells in the peripheral blood relative to vehicle-treated mice, which correlated with increased CAR-T cell infiltration into lung tumors. CAR-T cells in NKTR-255-treated mice also showed reduced signs of T cell exhaustion, including reduced expression of inhibitory receptors and enhanced ability to produce effector cytokines upon ex vivo re-stimulation. Importantly, combination therapy with both NKTR-255 and ROR1 CAR-T cells resulted in significantly improved tumor control relative to either treatment alone. Together, our work shows that NKTR-255 dramatically improves the persistence, function, and anti-tumor activity of ROR1 CAR-T cells in an aggressive autochthonous model of ROR1+ lung cancer. Our findings provide rationale to combine NKTR-255 with CAR-T cell therapy as a strategy to enhance the anti-tumor efficacy of CAR-T cells in patients with solid tumors. Citation Format: W. Sam Nutt, Takahiro Miyazaki, Mario Marcondes, Stanley R. Riddell, Shivani Srivastava. NKTR-255, a polymer-conjugated IL-15, dramatically improves ROR1 CAR-T cell persistence and anti-tumor efficacy in an autochthonous model of ROR1+ lung 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 1152.

Phagocytosis increases an oxidative metabolic and immune suppressive signature in tumor macrophages.

Phagocytosis is a key macrophage function, but how phagocytosis shapes tumor-associated macrophage (TAM) phenotypes and heterogeneity in solid tumors remains unclear. Here, we utilized both syngeneic and novel autochthonous lung tumor models in which neoplastic cells express the fluorophore tdTomato (tdTom) to identify TAMs that have phagocytosed neoplastic cells in vivo. Phagocytic tdTompos TAMs upregulated antigen presentation and anti-inflammatory proteins, but downregulated classic proinflammatory effectors compared to tdTomneg TAMs. Single-cell transcriptomic profiling identified TAM subset-specific and common gene expression changes associated with phagocytosis. We uncover a phagocytic signature that is predominated by oxidative phosphorylation (OXPHOS), ribosomal, and metabolic genes, and this signature correlates with worse clinical outcome in human lung cancer. Expression of OXPHOS proteins, mitochondrial content, and functional utilization of OXPHOS were increased in tdTompos TAMs. tdTompos tumor dendritic cells also display similar metabolic changes. Our identification of phagocytic TAMs as a distinct myeloid cell state links phagocytosis of neoplastic cells in vivo with OXPHOS and tumor-promoting phenotypes.