Intact Immune System Research Articles

Elucidating the direct effects of the novel HDAC inhibitor bocodepsin (OKI-179) on T cells to rationally design regimens for combining with immunotherapy.

Histone deacetylase inhibitors (HDACi) are currently being explored for the treatment of both solid and hematological malignancies. Although originally thought to exert cytotoxic responses through tumor-intrinsic mechanisms by increasing expression of tumor suppressor genes, several studies have demonstrated that therapeutic responses depend on an intact adaptive immune system: particularly CD8 T cells. It is therefore critical to understand how HDACi directly affects T cells in order to rationally design regimens for combining with immunotherapy. In this study, we evaluated T cell responses to a novel class-selective HDACi (OKI-179, bocodepsin) by assessing histone acetylation levels, which revealed rapid responsiveness accompanied by an increase in CD4 and CD8 T cell frequencies in the blood. However, these rapid responses were transient, as histone acetylation and frequencies waned within 24 hours. This contrasts with in vitro models where high acetylation was sustained and continuous exposure to HDACi suppressed cytokine production. In vivo comparisons demonstrated that stopping OKI-179 treatment during PD-1 blockade was superior to continuous treatment. These findings provide novel insight into the direct effects of HDAC inhibitors on T cells and that treatment schedules that take into account acute T cell effects should be considered when combined with immunotherapies in order to fully harness the tumor-specific T cell responses in patients.

Advances in glioma models using in vivo electroporation to highjack neurodevelopmental processes.

Glioma is the most prevalent type of neurological malignancies. Despite decades of efforts in neurosurgery, chemotherapy and radiation therapy, glioma remains one of the most treatment-resistant brain tumors with unfavorable outcomes. Recent progresses in genomic and epigenetic profiling have revealed new concepts of genetic events involved in the etiology of gliomas in humans, meanwhile, revolutionary technologies in gene editing and delivery allows to code these genetic "events" in animals to genetically engineer glioma models. This approach models the initiation and progression of gliomas in a natural microenvironment with an intact immune system and facilitates probing therapeutic strategies. In this review, we focus on recent advances in in vivo electroporation-based glioma modeling and outline the established genetically engineered glioma models (GEGMs).

A Network Landscape of HPVOPC Reveals Methylation Alterations as Significant Drivers of Gene Expression via an Immune-Mediated GPCR Signal.

HPV-associated oropharynx carcinoma (HPVOPC) tumors have a relatively low mutational burden. Elucidating the relative contributions of other tumor alterations, such as DNA methylation alterations, alternative splicing events (ASE), and copy number variation (CNV), could provide a deeper understanding of carcinogenesis drivers in this disease. We applied network propagation analysis to multiple classes of tumor alterations in a discovery cohort of 46 primary HPVOPC tumors and 25 cancer-unaffected controls and validated our findings with TCGA data. We identified significant overlap between differential gene expression networks and all alteration classes, and this association was highest for methylation and lowest for CNV. Significant overlap was seen for gene clusters of G protein-coupled receptor (GPCR) pathways. HPV16-human protein interaction analysis identified an enriched cluster defined by an immune-mediated GPCR signal, including CXCR3 cytokines CXCL9, CXCL10, and CXCL11. CXCR3 was found to be expressed in primary HPVOPC, and scRNA-seq analysis demonstrated CXCR3 ligands to be highly expressed in M2 macrophages. In vivo models demonstrated decreased tumor growth with antagonism of the CXCR3 receptor in immunodeficient but not immunocompetent mice, suggesting that the CXCR3 axis can drive tumor proliferation in an autocrine fashion, but the effect is tempered by an intact immune system. In conclusion, methylation, ASE, and SNV alterations are highly associated with network gene expression changes in HPVOPC, suggesting that ASE and methylation alterations have an important role in driving the oncogenic phenotype. Network analysis identifies GPCR networks, specifically the CXCR3 chemokine axis, as modulators of tumor-immune interactions that may have proliferative effects on primary tumors as well as a role for immunosurveillance; however, CXCR3 inhibition should be used with caution, as these agents may both inhibit and stimulate tumor growth considering the competing effects of this cytokine axis. Further investigation is needed to explore opportunities for targeted therapy in this setting.

Methionine restriction-induced sulfur deficiency impairs antitumour immunity partially through gut microbiota

Restriction of methionine (MR), a sulfur-containing essential amino acid, has been reported to repress cancer growth and improve therapeutic responses in several preclinical settings. However, how MR impacts cancer progression in the context of the intact immune system is unknown. Here we report that while inhibiting cancer growth in immunocompromised mice, MR reduces T cell abundance, exacerbates tumour growth and impairs tumour response to immunotherapy in immunocompetent male and female mice. Mechanistically, MR reduces microbial production of hydrogen sulfide, which is critical for immune cell survival/activation. Dietary supplementation of a hydrogen sulfide donor or a precursor, or methionine, stimulates antitumour immunity and suppresses tumour progression. Our findings reveal an unexpected negative interaction between MR, sulfur deficiency and antitumour immunity and further uncover a vital role of gut microbiota in mediating this interaction. Our study suggests that any possible anticancer benefits of MR require careful consideration of both the microbiota and the immune system.

O02 Neoantigens from actinic keratosis are predicted to be more immunogenic than those from cutaneous squamous cell carcinoma – a strategy for immune escape?

Abstract Actinic keratosis (AK) is a precursor of cutaneous squamous cell carcinoma (cSCC). More than 70% of cSCC arise from AKs, but fewer than 0.6% of AKs progress to cSCC. We have shown that the molecular landscapes of AK and cSCC are similar in terms of tumour mutational burden (TMB), copy number burden, mutational signatures and driver gene mutations but differ in levels of intrasample heterogeneity. We have also demonstrated that alterations in signalling pathways, including transforming growth factor-β, may be important for AK-to-SCC progression. However, the pathomechanisms underlying progression are not well characterized and it remains unclear why the majority of AKs do not progress, and most will regress. There is some evidence that differences in the neoantigen landscapes of AK and cSCC may play a role, and a previous study examining neoantigens in AK and cSCC from nine patients was the first to suggest that there is downregulation of the expression of highly immunogenic neoantigens in the immune escape of cSCC. In this study we further explore, in a larger data set, the hypothesis that the neoantigen recognition potential of AK vs. cSCC is a key determinant of AK-to-cSCC progression. We generated exome and transcriptome data from AK (n = 5) and cSCC (n = 15) with matched normal exome data (from blood). Human leucocyte antigen typing was performed using the OptiType algorithm and neoantigens restricted to major histocompatibility complex (MHC) class I were predicted using the NeoPredPipe workflow. Fitness of neoantigens, defined as the overall effectiveness of a neoantigen at stimulating an immune response, was determined by combining measures of MHC binding affinity and T-cell receptor recognition potential for the predicted neoantigen, and an overall neoantigen recognition potential score was derived for each neoantigen. The difference in median total neoantigen burden for AK and cSCC was not statistically significant [11 224 (range 30–17 330) and 1680 (range 3159–67 357), respectively (P = 0.07)]. Neoantigen burden was strongly correlated with TMB in both AK and cSCC. We found that, compared with cSCC, AK had predicted neoantigens with significantly higher mean MHC-binding affinity (P = 0.049), mean T-cell receptor recognition potential (P = 0.0093) and overall neoantigen recognition potential (P = 0.02). Taken together, our results confirm that neoantigens in AKs are predicted to be more effective at eliciting an immune response than those in cSCC. This suggests there may be a selection pressure in AKs for mutations that result in fewer immunogenic neoantigens resulting in immune escape and progression to cSCC. Finally, our results underscore the critical requirement for an intact immune system in prevention of AK-to-cSCC progression.

Intracellular Acidification in a Rat C6 Glioma Model following Cariporide Injection Investigated by CEST-MRI.

Acidification of cancerous tissue induced pharmacologically may slow tumor growth and can be detected using magnetic resonance imaging. Numerous studies have shown that pharmacologically inhibiting specific transporters, such as the Na+/H+ exchanger 1 (NHE1), can alter glycolitic metabolism and affect tumor acidosis. The sodium proton exchanger inhibitor Cariporide can acidify U87MG gliomas in mice. This study aimed to determine whether Cariporide could acidify C6 glioma tumors in rats with an intact immune system. C6 glioma cells were implanted in the right brain hemisphere of ten rats. Chemical exchange saturation transfer (CEST) MRI (9.4T) was acquired on days 7-8 and 14-15 after implantation to measure in vivo tissue intracellular pH (pHi) within the tumors and on the contralateral side. pHi was basic relative to contralateral tissue at both time points assessed using the amine and amide concentration-independent detection (AACID) value. On day 14-15, measurements were made before and up to 160 min after Cariporide injection (N = 6). Twenty minutes after drug injection, the average AACID value in the tumor significantly increased by ∼6.4% compared to pre-injection, corresponding to 0.31 ± 0.20 lower pHi, while in contralateral tissue, AACID value increased significantly by ∼4.3% compared to pre-injection, corresponding to 0.22 ± 0.19 lower pHi. Control rats without tumors showed no changes following injection of Cariporide dissolved in 10% or 1% DMSO and diluted in PBS. This study demonstrates the sensitivity of CEST-based pH-weighted imaging for monitoring the response of tumors to pharmacologically induced acidification.

Companion Animals as a Key to Success for Translating Radiation Therapy Research into the Clinic.

Many successful preclinical findings fail to be replicated during translation to human studies. This leads to significant resources being spent on large clinical trials, and in some cases, promising therapeutics not being pursued due to the high costs of clinical translation. These translational failures emphasize the need for improved preclinical models of human cancer so that there is a higher probability of successful clinical translation. Companion-animal cancers offer a potential solution. These cancers are more similar to human cancer than other preclinical models, with a natural evolution over time, genetic alterations, intact immune system, and a permanent adaptation to the microenvironment. These advantages have led pioneers in veterinary radiation oncology to aid human medicine by elucidating basic principles of radiation biology. More recently, the veterinary and human radiation oncology fields have increasingly collaborated to achieve advancements in education, radiotherapy techniques, and trial networks. This review describes these advancements, including significant prior research findings and the evolution of the veterinary radiation oncology discipline. It concludes by describing how companion-animal models can help shape the future of human radiotherapy. Taken as a whole, this review suggests companion-animal cancers may become widely used for preclinical radiotherapy research.

Discovery of (S)-N-(2-Amino-4-fluorophenyl)-4-(1-(3-(4-((dimethylamino)methyl)phenyl)-6-oxopyridazin-1(6H)-yl)ethyl)benzamide as Potent Class I Selective HDAC Inhibitor for Oral Anticancer Drug Candidate.

A novel series of benzamide derivatives were successively designed and synthesized prepared from the pyridazinone scaffold. Among them, (S)-17b, demonstrated potent inhibitory activity in vitro toward human class I HDAC isoforms and human myelodysplastic syndrome (SKM-1) cell line. Also, (S)-17b strongly increased the intracellular level of acetyl-histone H3 and P21 simultaneously and effectively induced G1 cell cycle arrest and apoptosis. Through oral dosing in SKM-1 xenograft models, (S)-17b exhibited excellent in vivo antitumor activity. In addition, compound (S)-17b showed better antitumor efficacy on mouse models with intact immune system than those with thymus deficiencies. Furthermore, this compound displayed a favorable pharmacokinetic profile in ICR mice and SD rat, respectively, minimal metabolic property differences among hepatocytes from five species, and a low inhibition upon the human ether-a-go-go (hERG) channel with an IC50 value of 34.6 μΜ. This novel compound (S)-17b may serve as a new drug candidate for further investigation.

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.

Host and microbe adaptation underlying true fungal commensalism

Abstract Fungi are found ubiquitously in the mammalian gut however the role of commensal fungi in the host is poorly understood. Laboratory mice are used to model host-fungi interactions however due to ultra-clean housing they do not harbor fungi. The human commensal fungus Candida albicans is widely studied to dissect host-commensal fungi interactions however prior dysbiosis of commensal bacteria is required to colonize laboratory mice. Insights from such forced host-fungal interactions are unsuitable for extrapolating mechanisms underlying fungal commensalism and its breakdown. To discover the natural fungal commensal of mice, we used pet-store “dirty” mice. We found that pet-store mice have a high fungal burden in their feces and their gut is predominantly colonized by single species of fungus, Kazachstania pintolopesii. Furthermore, we found that K. pintolopesii isolated from pet-store mice, stably colonized laboratory mice to high levels in the presence of an intact immune system and commensal bacteria. Laboratory mice colonized with K. pintolopesii exhibited increased neutrophil frequency in blood but did not show any pathological symptoms. Surprisingly, even a systemic K. pintolopesii challenge did not show any pathology in laboratory mice indicating unique adaptations of this fungus to the murine host. Remarkably, we found that laboratory mice stably colonized with K. pintolopesii are significantly protected from systemic C. albicans challenge suggesting that commensal fungi promote systemic antifungal responses in their host. In summary, our work established a novel unmanipulated murine model of fungal commensalism and provide unique insight into cellular and molecular mechanisms of mammalian host and fungal mutualism. This research was supported by grants from NIH (R01 DK113265), Searle Scholars program, and AAI Careers in Immunology Fellowship.

Harnessing the Immunological Effects of Radiation to Improve Immunotherapies in Cancer.

Ionizing radiation (IR) is used to treat 50% of cancers. While the cytotoxic effects related to DNA damage with IR have been known since the early 20th century, the role of the immune system in the treatment response is still yet to be fully determined. IR can induce immunogenic cell death (ICD), which activates innate and adaptive immunity against the cancer. It has also been widely reported that an intact immune system is essential to IR efficacy. However, this response is typically transient, and wound healing processes also become upregulated, dampening early immunological efforts to overcome the disease. This immune suppression involves many complex cellular and molecular mechanisms that ultimately result in the generation of radioresistance in many cases. Understanding the mechanisms behind these responses is challenging as the effects are extensive and often occur simultaneously within the tumor. Here, we describe the effects of IR on the immune landscape of tumors. ICD, along with myeloid and lymphoid responses to IR, are discussed, with the hope of shedding light on the complex immune stimulatory and immunosuppressive responses involved with this cornerstone cancer treatment. Leveraging these immunological effects can provide a platform for improving immunotherapy efficacy in the future.

Isogenic Mammary Models of Intraductal Carcinoma Reveal Progression to Invasiveness in the Absence of a Non-Obligatory In Situ Stage.

In breast cancer, progression to invasive ductal carcinoma (IDC) involves interactions between immune, myoepithelial, and tumor cells. Development of IDC can proceed through ductal carcinoma in situ (DCIS), a non-obligate, non-invasive stage, or IDC can develop without evidence of DCIS and these cases associate with poorer prognosis. Tractable, immune-competent mouse models are needed to help delineate distinct mechanisms of local tumor cell invasion and prognostic implications. To address these gaps, we delivered murine mammary carcinoma cell lines directly into the main mammary lactiferous duct of immune-competent mice. Using two strains of immune-competent mice (BALB/c, C57BL/6), one immune-compromised (severe combined immunodeficiency; SCID) C57BL/6 strain, and six different murine mammary cancer cell lines (D2.OR, D2A1, 4T1, EMT6, EO771, Py230), we found early loss of ductal myoepithelial cell differentiation markers p63, α-smooth muscle actin, and calponin, and rapid formation of IDC in the absence of DCIS. Rapid IDC formation also occurred in the absence of adaptive immunity. Combined, these studies demonstrate that loss of myoepithelial barrier function does not require an intact immune system, and suggest that these isogenic murine models may prove a useful tool to study IDC in the absence of a non-obligatory DCIS stage-an under-investigated subset of poor prognostic human breast cancer.

Abstract 1726: Loss of NF1 drives hormone dependent mammary carcinogenesis in a rat model with intact immune system

Abstract Loss of NF1 plays a major role as an oncogenic driver in up to 33% of all breast cancers (BC). Loss of NF1 is also a prognostic indicator for increased cancer risk at an earlier age, poorer outcomes, and therapeutic resistance. NF1 has recently been shown to bind the estrogen receptor alpha (ER) and act as a transcriptional corepressor. This helps explain findings in ER+ BC. Changes to NF1 that abrogate ER signaling thereby lead to Ras driven tumor resistance to endocrine therapy as cells are able to grow in low levels of E2 (and tamoxifen). Hence, NF1-mutant tumors represent a distinct molecular class in need of new therapeutics targeting both Ras and ER pathways. We have generated preclinical mammalian models of NF1 loss and BC that can be used to evaluate the role of NF1/ER transcriptional signaling in BC, the role of immune cells in BC, and therapeutics. Our models include a pathogenic patient missense allele c.3827G>A, p.R1276Q (knockin or KI) as well as a 14 base pair deletion c.3661_3674del, p.P1220fs*1223 (knockout or KO) model. Heterozygous (het) Nf1 female rats develop mammary gland adenocarcinoma spontaneously, but het KO rats develop multiple tumors with earlier onset. Tumors are generally Grade 2 and do not differ by genotype. By 16 weeks, 70% of KO females have developed at least one tumor whereas only 20% of KI rats and 2% of WT rats have developed tumors. This impacts survival as 76% of KI and 58% of KO females survive to one year. By 2 years, both alleles have 54% survival. The divergence in phenotype between KI and KO alleles may be due to residual function of R1276Q missense NF1 protein. A more in-depth analysis indicates that mammary tumor formation likely begins relatively early, as we find evidence of aberrant morphology and hyperplasia prior to the formation of palpable tumors. Interestingly, we find histological evidence of lung metastases and expression of breast markers GCDFP15, MGA, and CEA in the lung. Again, we see allele-specific effects in that KO rats develop lung tumors earlier than KI rats. Using single nuclei RNASeq to characterize the transcriptional profile of the mammary tumors, we find allele-specific effects beyond repression of Ras activity that drive aggressive tumor development. We identified different tumor cell populations (2 epithelial cell populations, Myeloid cells, B cells, T cells, Basal mammary cells, and WT specific cells) and identified different pathways altered due to the loss of Nf1 including Ephrin B signaling, Cyclin and Cell Cycle signaling, and Glycolysis signaling. Our overall goal is to characterize the phenotype of these rat models in terms of histopathology, Ras signaling, hormone signaling, immune components, and targeted drug response; which can then be compared with what is known regarding patients with somatic or germline inactivation of NF1 and breast cancer. Ultimately, this will provide better prognostic predictions for patients and better therapeutic options. Citation Format: Deeann Wallis, Christian Fay, Kelley Bradley, Erik Westin, William Bradley, Hui Liu, David Crossman, Jeremy B. Foote, Robert Kesterson. Loss of NF1 drives hormone dependent mammary carcinogenesis in a rat model with intact immune system [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 1726.

Abstract 2509: Similar variant histology and immune cell infiltration in canine and human muscle-invasive bladder cancer

Abstract Introduction: Muscle-Invasive Bladder Cancer (MIBC) is associated with a poor overall survival rate. One important factor that contributes to poor treatment outcomes is the histologic heterogeneity of MIBC tumors, which limits the translational value of some rodent models. As opposed to mice, dogs with spontaneously occurring bladder cancer have been shown to present with similar clinical behavior as human MIBC. In addition, canines have an intact immune system, which could provide for an ideal pre-clinical study population to investigate the efficacy of immunotherapy. In this study, we evaluated canine MIBC tumor biopsies for their similarities to human MIBC with regards to histology and immune cell infiltration. Materials & Methods: Nine canine MIBC tumor samples were provided by the ISU pathology service and evaluated by a human uropathologist. Canine MIBC tumors were phenotypically characterized using either immunofluorescence (IF) or RNA in situ hybridization (RNA-ISH), including CD44, KRT7 and FOXA1, as well as specific markers characterizing the immune cell infiltrate, such as CD163 (tumor-associated macrophages), CD8a (cytotoxic T-lymphocytes), PD-L1 (immune-checkpoint protein) and TGFβ2 (cancer-associated fibroblasts). Results: Histologically, all canine MIBCs were classified as invasive, high-grade urothelial carcinomas (UC) with identical morphology to human MIBC. Interestingly, previously described histological variants of human MIBC tumors were also observed within the canine MIBC samples, such as glandular differentiation in 3/9 canine MIBC samples; epithelial-mesenchymal transition with sarcomatoid differentiation 1/9, squamous differentiation in 2/9 and micropapillary carcinoma in 1/9, all of which have been associated with poor clinical outcomes in human patients. IF was strongly positive for KRT7, FOXA1 and CD44, and immune cell markers CD163 and TGFβ2. However, CD8a was only weakly expressed (5/9) or negative, and PD-L1 was only positive in 4/9 canine MIBC biopsies. Conclusions: Canine MIBC tumors showed significant heterogeneity with highly similar histological variant morphologies to those reported in humans with MIBC. Furthermore, phenotypic characterization of immune cell infiltrates and checkpoint proteins suggest that the tumors were typically immunologically “cold”. These data further establish the utility of the spontaneous canine model of MIBC for pre-clinical investigation of novel therapeutic drug candidates, including immunotherapies. Citation Format: Karin Allenspach, John Cheville, Hannah Wickham, Christopher Zdyrski, Vojtech Gabriel, Basant Ahmed, Mei-Jyun Ciou, Fabrice Lucien, Igor Frank, Olufemi Fasina, Pablo Pineyro, Johnathan P. Mochel. Similar variant histology and immune cell infiltration in canine and human muscle-invasive bladder 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 2509.

Abstract 5723: An in vivo pharmacogenomics platform replicates and extends biomarkers of therapy response identified via causal inference analysis of clinical data

Abstract Recent development of therapies targeting oncogenes have dramatically improved cancer care for subsets of patients and generated a wave of interest in cancer precision medicine. However, effective targeted therapies are scarce as we have a limited understanding of how drug responses are modulated by tumor genotype. Here, we utilize both human data and in vivo models to identify genetic drivers of therapy response in KRAS-driven non-small cell lung cancer patients treated with chemotherapy. We first present an integrative causal inference analysis of three clinical data resources: (1) the recently released Genomics Evidence Neoplasia Information Exchange, Biopharma Collaboration dataset (GENIE-BPC; n=197), (2) a selected set of patients from the Tempus Clinico-genomic Database (n=330), and (3) an additional cohort from Memorial Sloan Kettering Cancer Center (n=218). Each dataset was first analyzed separately using a causal inference pipeline. Doubly robust estimators for each variant were inferred within a counting process survival analysis model accounting for time-varying treatments and immortal time bias. Meta-analysis of the results from all three cohorts identified three commonly mutated and highly replicable genes with a significant effect on overall survival: KEAP1, SMARCA4, and CDKN2A. As further validation, we used our murine in vivo pharmacogenomics (PGx) platform that can quantify the effects of therapies across thousands of tumors of diverse genotypes. These tumors are initiated de novo in the native microenvironmental context in mice with an intact adaptive immune system. We tested a chemotherapy combination of carboplatin and pemetrexed in mice with KrasG12D-driven lung tumors and inactivation of each of 60 putative tumor suppressors. Treatment led to >75% reduction in tumor sizes relative to tumor suppressor inactivated (matched) vehicle-treated controls. These models identified causal effects for two out of the three candidates above: KEAP1 (resistance) and CDKN2A (sensitive). Moreover, our PGx platform identified additional candidate genes beyond those found using the clinical data, which had insufficient sample size for these rarely mutated genes. Together, we demonstrate how leveraging our PGx platform together with human data within a causal inference framework may improve the stratification of patients by their clinical outcomes, profoundly advancing the promise of precision medicine. Citation Format: David Amar, Erick Scott, Ian P. Winters, Gregory D. Wall, Dmitri A. Petrov, Monte M. Winslow, Joseph Juan, Ian K. Lai, Lafia Sebastian, Edwin A. Apilado, Gabriel Grenot, Vy B. Tran, Charles Rudin, Michael J. Rosen. An in vivo pharmacogenomics platform replicates and extends biomarkers of therapy response identified via causal inference analysis of clinical data. [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 5723.

Abstract 2339: Exploration of multiple immunotherapy modalities in ostesarcoma

Abstract Osteosarcoma (OS) is the most common primary bone malignancy affecting long bones in children and the third most frequent in adults in the United States. It is highly invasive and metastatic. The survival rates over the last 20 years are unchanged. Therefore, it is essential to develop novel, targeted therapeutic approaches to treat OS. In pursuit of that goal, novel models are needed. Due to the high similarity in cancer genetics, the spontaneous occurrence of tumors, and the presence of an intact immune system, dogs are deemed an excellent cancer immunotherapy model. OS is also the most common malignant bone tumor (80%) in dogs. Monotherapies like oncolytic viruses and immune checkpoint inhibitors have been shown to be insufficient for effective cancer treatment. Therefore, we propose a combination of immune checkpoint inhibition and oncolytic virotherapy to target osteosarcoma tumor cells. In this combined therapy, the oncolytic virus will serve to both stimulate an inflammatory response and transmit the checkpoint inhibitor, which in turn will block PD-1-directed down-regulation of T cells, allowing the adaptive immune system to respond better to tumors. In order to evaluate this approach in the canine model, we have developed an oncolytic virus (CAV2-AU-M2) based on canine adenovirus type 2 and armed with an immune checkpoint inhibitor, an anti-canine PD1 single domain camelid antibody (sdAb). Four canine osteosarcoma cell lines, D17, D22, MCKOS, and CF11, were cultured in two-dimensional (2D) and three-dimensional (3D) cultures and were infected with CAV2-AU-M2 to analyze the infective and lytic effects of the virus. CAV2-AU-M2 infected and lysed the cells in 2D. The virus also produced anti-PD1 sdAb upon infecting the cells. Similarly, results were evident in 3D cultures. The virus infected the spheroids in the outer layers on Day 4 and slowly diffused towards the core in 7 days. The spheroids infected started showing some cell death on day 4, which progressed significantly by Day 7. This approach to oncolytic virotherapy makes the tumor more visible to the immune system and abrogates tumor-induced immune suppression. Citation Format: Aliaa A. Ismail, Daniel J. Patton, Theresa A. Higgins, Maninder Sandey, Bruce F. Smith, Payal Agarwal. Exploration of multiple immunotherapy modalities in ostesarcoma [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 2339.

Abstract 2464: Novel models of breast cancer metastasis with different metastatic competence

Abstract Metastasis is responsible for poor overall survival of cancer patients and a better understanding of the molecular mechanisms that drive metastasis is crucial for improving disease outcome. In mouse models, gene expression profiling of cancer cells isolated from metastases and from their matched primary tumors is an approach that has been successfully employed to identify biomarkers and drivers of metastasis. However, only few such tumor pairs have been derived from immunocompetent mouse models, limiting our ability to compare the metastatic potentials of these pairs in the context of an intact immune system. Moreover, most of the metastatic cancer cell lines currently available on the C57BL/6 background have a relatively low metastatic potential, complicating the evaluation of therapeutic interventions that may inhibit metastasis. To address these critical limitations, we developed novel cell lines from distinct spontaneous lung metastases and corresponding primary breast tumors from C57BL/6 mice. Specifically, C57BL/6 mice were orthotopically injected with copGFP-expressing E0771 breast cancer cells and the tumors were allowed to grow for 28 days. Tumor cells at different stages of the metastatic cascade - primary tumor evolution, transit through the blood circulation (circulating tumor cells), survival at secondary site, dormancy, and metastatic outgrowth, were captured by collecting and culturing copGFP-positive cancer cell lines from the primary tumor, blood circulation, and metastatic lungs at the experimental endpoint. These cells were reintroduced orthotopically (mammary fat pad injection) and intravenously (tail-vein injection) to evaluate tumor growth and metastasis in vivo. Interestingly, cell lines derived from overt metastases displayed a wide array of metastatic competences, indicating that some metastases retain that ability to further metastasize while others do not. RNA sequencing of these cell lines identified genes and pathways that are significantly upregulated in highly metastatic vs. poorly metastatic cancer cell lines. Future studies will investigate whether modulation of these pathways in the primary tumor and CTCs can be used to predict metastatic recurrence and serve as a basis for developing novel biomarker-based therapeutic approaches for the treatment of metastatic disease. We also envision that the cell lines we generated will be useful for studies of the influence of host-tumor cells interactions on metastasis on the immunocompetent C57BL/6 background. Citation Format: Akshita B. Bhatt, Ruishan Wang, Hong Jia, Myriam Labelle. Novel models of breast cancer metastasis with different metastatic competence [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 2464.

Use of multi-color flow cytometry for canine immune cell characterization in cancer.

Although immunotherapy is becoming a standard approach of human cancer treatment, only a small but critical fraction of patients responds to the therapy. It is therefore required to determine the sub-populations of patients who will respond to immunotherapies along with developing novel strategies to improve efficacy of anti-tumor immune reactions. Current development of novel immunotherapies relies heavily on mouse models of cancer. These models are important for better understanding of mechanisms behind tumor immune escape and investigation of novel strategies to overcome it. Nevertheless, the murine models do not necessarily represent the complexity of spontaneously occurring cancers in humans. Dogs spontaneously develop a wide range of cancer types with an intact immune system under similar environment and exposure to humans, which can serve as translational models in cancer immunotherapy research. To date though, there is still a relatively limited amount of information regarding immune cell profiles in canine cancers. One possible reason could be that there are hardly any established methods to isolate and simultaneously detect a range of immune cell types in neoplastic tissues. To date only a single manuscript describes characterization of immune cells in canine tumour tissues, concentrating solely on T-cells. Here we describe a protocol for multi-color flow cytometry to distinguish immune cell types in blood, lymph nodes, and neoplastic tissues from dogs with cancer. Our results demonstrate that a 9-color flow cytometry panel enables characterization of different cell subpopulations including myeloid cells. We also show that the panel allows detection of minor/aberrant subsets within a mixed population of cells in various neoplastic samples including blood, lymph node and solid tumors. To our knowledge, this is the first simultaneous immune cell detection panel applicable for solid tumors in dogs. This multi-color flow cytometry panel has the potential to inform future basic research focusing on immune cell functions in translational canine cancer models.

Immunological and tumor-intrinsic mechanisms mediate the synergistic growth suppression of experimental glioblastoma by radiotherapy and MET inhibition

The hepatocyte growth factor (HGF)/MET signaling pathway has been proposed to be involved in the resistance to radiotherapy of glioblastoma via proinvasive and DNA damage response pathways.Here we assessed the role of the MET pathway in the response to radiotherapy in vitro and in vivo in syngeneic mouse glioma models. We find that the murine glioma cell lines GL-261, SMA-497, SMA-540 and SMA-560 express HGF and its receptor MET and respond to exogenous HGF with MET phosphorylation. Glioma cell viability or proliferation are unaffected by genetic or pharmacological MET inhibition using tepotinib or CRISPR/Cas9-engineered Met gene knockout and MET inhibition fails to sensitize glioma cells to irradiation in vitro. In contrast, the combination of tepotinib with radiotherapy prolongs survival of orthotopic SMA-560 or GL-261 glioma-bearing mice compared with radiotherapy or tepotinib treatment alone. Synergy is lost when such experiments are conducted in immunodeficient Rag1−/− mice, and, importantly, also when Met gene expression is disrupted in the tumor cells. Combination therapy suppresses a set of pro-inflammatory mediators including matrix metalloproteases that are upregulated by radiotherapy alone and that have been linked to poor outcome in glioblastoma. Several of these mediators are positively regulated by transforming growth factor (TGF)-β, and pSMAD2 levels as a surrogate marker of TGF-β pathway activity are suppressed by combination treatment. We conclude that synergistic suppression of experimental syngeneic glioma growth by irradiation and MET inhibition requires MET expression in the tumor as well as an intact immune system. Clinical evaluation of this combined strategy in newly diagnosed glioblastoma is warranted.

Abstract P3-08-01: Loss of NF1 drives hormone dependent mammary carcinogenesis in a rat model with intact immune system

Abstract Loss of NF1 plays a major role as an oncogenic driver in many cancer types and can be found in up to 33% of all breast cancers (BC). Loss of NF1 is also a prognostic indicator for increased cancer risk at an earlier age, poorer outcomes, and therapeutic resistance. In addition, certain NF1 genotypes may increase cancer risks, while others do not. NF1 is largely perceived as a classic Ras-opathy syndrome due to inactivating mutations in neurofibromin affecting RAS-MAPK signaling. However, recently it has been shown that NF1 binds estrogen receptor alpha (ER) and acts as a transcriptional corepressor. This helps explain some BC findings specifically in ER+ BC. In this model, specific changes to NF1 that abrogate ER signaling lead to Ras driven tumor resistance to endocrine therapy as cells are able to grow in low levels of E2 (and tamoxifen). Hence, NF1-mutant tumors represent a distinct molecular class in need of new therapeutics. A preclinical mammalian model of NF1 loss and BC would be helpful in both evaluating the role of NF1/ER transcriptional signaling in BC, evaluating the role of immune cells in BC, and testing therapeutics. Novel Nf1 rat models have a very robust ER+ BC phenotype, therefore more closely recapitulating clinical tumors compared to other preclinical models. Our models include a pathogenic patient missense allele c.3827G>A, p.R1276Q (knockin or KI), associated in humans with familial spinal NF1 and malignancy, as well as a 14 base pair deletion c.3661_3674del, p.P1220fs*1223 (knockout or KO) model. Heterozygous (het) Nf1 females develop mammary gland adenocarcinoma spontaneously, but het KO rats develop multiple tumors with earlier onset while het KI rats tend to develop fewer tumors with later onset. Tumors are generally Grade 2 and do not differ by genotype. By 16 weeks, 70% of KO females have developed at least one tumor whereas only 20% of KI rats and 2% of WT rats have developed tumors. This impacts survival as by 1 year 76% of KI females survive yet only 58% of KO females survive. However, by 2 years, both alleles have 54% survival. The divergence in phenotype between patient and null alleles may be due to residual function of R1276Q missense NF1 protein. A more in-depth analysis indicates that mammary tumor formation likely begins relatively early, as we find evidence of aberrant morphology and hyperplasia prior to the formation of palpable tumors. Interestingly, we find histological evidence of lung metastases and expression of breast markers GCDFP15, MGA, and CEA in the lung. Again, we see allele-specific effects in that KO rats develop lung tumors earlier than KI rats. While het male rats also develop mammary tumors at low rates, they experience longer survival times (76% of KO males survive 1 year and 98% of KI males survive 1 year) and males also develop tumors in other locations. Using single nuclei RNASeq to characterize the transcriptional profile of the mammary tumors, we find allele-specific effects beyond repression of Ras activity that drive aggressive tumor development. We identified different tumor cell populations (2 epithelial cell populations, Myeloid cells, B cells, T cells, Basal mammary cells, and WT specific cells) and identified different pathways altered due to the loss of Nf1 including Ephrin B signaling, Cyclin and Cell Cycle signaling, and Glycolysis signaling. Our overall goal is to characterize the phenotype of these rat models in terms of histopathology, Ras signaling, hormone signaling, immune components, and targeted drug response and compare/contrast them with what is known regarding patients with somatic or germline inactivation of NF1 and breast cancer. Ultimately, this will provide better prognostic predictions for patients and better therapeutic options for treatment. Citation Format: Deeann Wallis, Christian Fay, Kelley Bradley, Erik Westin, William Bradley, Hui Liu, Laura Lambert, David Crossman, Jeremy Foote, Robert Kesterson. Loss of NF1 drives hormone dependent mammary carcinogenesis in a rat model with intact immune system [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-08-01.