Cancer Cell-intrinsic Mechanisms Research Articles

Abstract 6819: Exploring the impact of IDH1-mutations on antitumor immunity in intrahepatic cholangiocarcinoma

Abstract Intrahepatic cholangiocarcinoma (CCA) is an aggressive biliary tract cancer that carries unfavorable prognosis. 10-20% of intrahepatic CCAs harbor a mutation in IDH1 that can be targeted with mutant IDH1-specific inhibitors. However, objective and durable responses to treatment with mutant IDH1 inhibitors are rare in CCA. Mutant IDH1 has neomorphic enzymatic activity that produces the oncometabolite D-2-hydroxyglutarate (D-2-HG). D-2-HG promotes biliary tumor formation through cancer cell-intrinsic mechanisms but is also a paracrine factor in the tumor microenvironment (TME). Our group and others have identified that IDH1-mutant CCAs are not responsive to immunotherapy treatment approaches in part due to decreased CD8+ T cell infiltration and an increase in immunosuppressive macrophage cells in the TME. To better understand the immunosuppressive features of the IDH1-mutant CCA TME, we generated an isogenic cell line panel of IDH1-mutant (MUT) and IDH1-wild type (WT) mouse cholangiocarcinoma cells in the SB1 cell line background through a CRISPR homology directed repair approach. Compared to IDH1-WT cells, IDH1-MUT cells produce high levels of D-2-HG and both cell line panels readily form tumors in immunocompetent C57BL/6 mice. Tumors formed from IDH1-mut cells have reduced CD8+ T cell infiltration compared to IDH1-WT tumors, a finding that correlates with analysis of human CCA samples. Using cytometry by time of flight (CyTOF), we identified that CD8+ T cells in IDH1-MUT tumors have decreased expression of key regulators of the antitumor immune response including EOMES, TBET, and GZMB compared to those isolated from IDH1-WT tumors. Analysis of secreted factors from these IDH1-MUT and IDH1-WT cells identified that IDH1-MUT cells release increased levels of CCL2, a chemokine shown to diminish the antitumor immune response across multiple cancer types, compared to IDH1-WT cells. Further, IDH1-MUT tumors grown in syngeneic, immunocompetent mice have increased CCL2 staining by IHC compared to IDH1-WT tumors. CCL2 expression is also increased in a preliminary analysis of IDH1-mutant human CCA primary tumors. Treatment of mice harboring orthotopic liver tumors formed from IDH1-MUT cells with a CCL2 neutralizing antibody resulted in decreased tumor size compared to isotype control. Together, our data suggest that CCL2 is a mediator of immunosuppression in the IDH1-mutant CCA TME and that modulation of CCL2 activity may improve outcomes in this rare disease subtype. Further work to understand the mechanisms through which IDH1 mutations regulate CCL2 levels in these tumors is underway. Citation Format: Emma Kartalia, James M. Leatherman, Jae W. Lee, Kiyoko Yoshima, Daniel J. Zabransky, Mark Yarchoan. Exploring the impact of IDH1-mutations on antitumor immunity in intrahepatic cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6819.

Abstract B019: The bone microenvironment's impact on the heterogeneity of resistance to proteasome inhibitors in multiple myeloma

Abstract Multiple myeloma (MM) is an osteolytic plasma cell malignancy that, despite being responsive to therapies such as proteasome inhibitors, frequently relapses. Understanding the mechanism and the niches where resistant disease evolves remains of major clinical importance. Cancer cell intrinsic mechanisms and bone ecosystem factors are known contributors to the evolution of resistant MM but the exact contribution of each is difficult to define with current in vitro and in vivo models. Using a novel bmathematical model that incorporates key cellular species of the bone ecosystem that control normal bone remodeling and, in MM, yields a protective environment we studied how, under therapy, the bone ecosystem contributes to the evolutionary dynamics of resistant MM under control and proteasome inhibitor treatment. Our results show that Environmentally-Mediated Drug Resistance (EMDR) might not be sufficient to explain resistant disease. But our results highlight the importance of EMDR in facilitating the emergence of resistance, and more importantly facilitating the survival of tolerant cells, allowing the tumor multiple routes for resistant disease. Our results also provide evidence that intervention with therapies targetting the bone ecosystem would significantly improve the ability of treatment to increase the time to progression for patients with multiple myeloma and, potentially, other diseases where EMDR plays a role. Citation Format: David Basanta, Ryan Bishop, Anna Miller, Conor C. Lynch, Matthew Froid, Ariosto S. Silva, Kenneth H. Shain. The bone microenvironment's impact on the heterogeneity of resistance to proteasome inhibitors in multiple myeloma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr B019.

Deciphering the molecular biology of inflammatory breast cancer through molecular characterization of patient samples and preclinical models.

Inflammatory breast cancer is an aggressive subtype of breast cancer with dismal patient prognosis and a unique clinical presentation. In the past two decades, molecular profiling technologies have been used in order to gain insight into the molecular biology of IBC and to search for possible targets for treatment. Although a gene signature that accurately discriminates between IBC and nIBC patient samples and preclinical models was identified, the overall genomic and transcriptomic differences are small and ambiguous, mainly due to the limited sample sizes of the evaluated patient series and the failure to correct for confounding effects of the molecular subtypes. Nevertheless, data collected over the past 20 years by independent research groups increasingly support the existence of several IBC-specific biological characteristics. In this review, these features are classified as established, emerging and conceptual hallmarks based on the level of evidence reported in the literature. In addition, a synoptic model is proposed that integrates all hallmarks and that can explain how cancer cell intrinsic mechanisms (i.e. NF-κB activation, genomic instability, MYC-addiction, TGF-β resistance, adaptive stress response, chromatin remodeling, epithelial-to-mesenchymal transition) can contribute to the establishment of the dynamic immune microenvironment associated with IBC. It stands to reason that future research projects are needed to further refine (parts of) this model and to investigate its clinical translatability.

The Role of the Toll-like Receptor 2 and the cGAS-STING Pathways in Breast Cancer: Friends or Foes?

Breast cancer stands as a primary malignancy among women, ranking second in global cancer-related deaths. Despite treatment advancements, many patients progress to metastatic stages, posing a significant therapeutic challenge. Current therapies primarily target cancer cells, overlooking their intricate interactions with the tumor microenvironment (TME) that fuel progression and treatment resistance. Dysregulated innate immunity in breast cancer triggers chronic inflammation, fostering cancer development and therapy resistance. Innate immune pattern recognition receptors (PRRs) have emerged as crucial regulators of the immune response as well as of several immune-mediated or cancer cell-intrinsic mechanisms that either inhibit or promote tumor progression. In particular, several studies showed that the Toll-like receptor 2 (TLR2) and the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathways play a central role in breast cancer progression. In this review, we present a comprehensive overview of the role of TLR2 and STING in breast cancer, and we explore the potential to target these PRRs for drug development. This information will significantly impact the scientific discussion on the use of PRR agonists or inhibitors in cancer therapy, opening up new and promising avenues for breast cancer treatment.

Abstract IA019: Metabolic adaptations in renal cell carcinoma

Abstract Clear cell renal cell carcinoma (ccRCC) is characterized by deregulated hypoxic signaling, metabolic defects, and a complex tumor microenvironment (TME) highly enriched in both lymphoid and myeloid immune subsets. Loss of the oxygen sensing gene, von Hippel Lindau (VHL), is a critical early event in ccRCC pathogenesis and promotes stabilization of hypoxia inducible (transcription) factors (HIF) that upregulate pro-growth signaling pathways, including angiogenesis and aerobic glycolysis. However, whether Vhl loss in cancer cells impacts the frequency, energetic requirements, or other functional properties of immune subsets in the TME remains unclear. Here, using Vhl WT and Vhl KO in vivo murine kidney cancer models, we found that Vhl KO tumors across multiple clones were slower growing and promoted increased immune cell infiltration in the TME. Enhanced proinflammatory signatures in tumor associated macrophage (TAM) subsets from Vhl deficient tumors were also observed, and these features were rescued when Vhl was reintroduced to the cancer cells. Moreover, in vivo quantification of the PET tracer and glucose analog, [18F]fluorodeoxyglucose (FDG), revealed that both myeloid derived suppressor cells (MDSC) and TAM populations from Vhl KO tumors consume more glucose than myeloid subsets from Vhl intact tumors. Notably, Vhl loss did not confer increased glucose uptake in cancer cells or lymphoid subsets in vivo. Overall, these data are consistent with enhanced metabolism and proinflammatory properties observed in the myeloid compartment of human ccRCC and highlight cancer cell intrinsic mechanisms driving environmental reprograming as a means of shaping the immune landscape in ccRCC. Citation Format: W. Kimryn Rathmell. Metabolic adaptations in renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr IA019.

Effect of stimulator of interferon genes (STING) signaling on radiation-induced chemokine expression in human osteosarcoma cells.

Cancer cell-intrinsic mechanisms affecting radiation immunomodulation could be exploited to optimize systemic effects of localized radiation. Radiation-induced DNA damage is sensed by cyclic GMP-AMP synthase (cGAS), which ultimately activates stimulator of interferon (IFN) genes (STING). Resultant expression of soluble mediators such as CCL5 and CXCL10 can facilitate recruitment of dendritic cells and immune effector cells into the tumor. The primary objectives of this study were to determine the baseline expression levels of cGAS and STING in OSA cells and evaluate the dependence of OSA cells on STING signaling for eliciting radiation-induced expression of CCL5 and CXCL10. cGAS and STING expression, and CCL5/CXCL10 expression in control cells, STING-agonist treated cells, and cells treated with 5 Gy ionizing radiation were assessed utilizing RTqPCR, Western blot, and ELISA. U2OS and SAOS-2 OSA cells were deficient in STING relative to human osteoblasts (hObs), while SAOS-2-LM6 and MG63 OSA cells expressed equivalent amounts of STING compared to hObs. A dependence on baseline or induced STING expression was observed for STING-agonist, and radiation-induced, expression of CCL5 and CXCL10. This finding was confirmed by performing siRNA knockdown of STING in MG63 cells. These results show that STING signaling is necessary for radiation-induced expression of CCL5 and CXCL10 in OSA cells. Additional studies are necessary to determine whether STING expression in OSA cells in vivo alters immune cell infiltrates after radiation exposure. These data may also have implications for other potentially STING-dependent characteristics such as resistance to oncolytic virus cytotoxicity.

Abstract 4117: Molecular insights into resistance mechanisms to therapy with T cell bispecific antibodies

Abstract T Cell Bispecific Antibodies (TCBs) are potent molecules that induce durable complete remissions with a favorable safety profile in Relapsed Refractory Non Hodgkin Lymphoma patients. Despite the promising clinical efficacy, a proportion of patients escape under treatment due to the occurrence of primary and acquired resistance mechanisms that may involve the modulation of target antigen expression, cancer cell intrinsic mechanisms, T cell functionality or treatment-induced changes in the tumor microenvironment (TME). Therefore, a better understanding of TCB-induced resistance mechanisms is of great importance to enable the design of novel therapeutic approaches and/or combination strategies to overcome resistance. We present the results of exploratory preclinical analysis using an aggressive lymphoma model in humanized mice and a syngeneic melanoma model in immunocompetent mice, focusing on the assessment of T cell functionality, TME reprogramming, and cancer cell intrinsic changes upon longitudinal TCB treatments, encompassing tumor regression and escape. Flow cytometry, IHC, scRNAseq and ex vivo functional analysis of T cell cytotoxicity and cytokine release demonstrate that intra-tumor T cell functionality is preserved over serial TCB treatments, including tumor escape. On the other hand, TCB treatment induced a reprogramming of the TME consisting of rapid myeloid cell infiltration, followed by the expansion of intra-tumor T cells at a later time point (favoring higher CD8:CD4 ratio). Cumulative evidence from in vitro and in vivo scRNAseq experiments provided insights into macrophage reaction to TCB-mediated T cell inflammation, consisting of a rapid (as early as 2 h) activation and upregulation of several pro-inflammatory molecules (IL-1b, CXCL8, CXCL9, CXCL10, CXCL1, CCL2, CCL3, CCL4, CCL5, IFN-g-induced genes: GBP2 -8 and BATF2). This was followed by a shift towards a more suppressive phenotype at later time points (20-96 h) as evidenced by the expression of PD-L1, INHBA, IDO, FLT1 genes. The analysis of cancer cells showed upregulation of Wnt/beta catenin, p53 and Hedgehog signaling pathways at tumor escape. In contrast, proliferation signatures including MYC, E2F target genes, MTORC signaling, and G2M checkpoint, along with metabolic pathways of oxidative phosphorylation and glycolysis were enriched in cancer cells at regression. Taken together, our data show that T cell cytotoxicity and functional states are preserved during tumor regression and tumor escape phases, suggesting that T cell dysfunctionality is unlikely to be the sole cause of TCB resistance. TME reprogramming, consisting of a rapid myeloid cell infiltration and their acquisition of a suppressive phenotype over time, along with a selection of cancer cell clones that escape the TCB-mediated cytotoxicity and upregulate Wnt, p53 and Hedgehog pathways could contribute to TCB-mediated resistance, leading to tumor escape. Citation Format: Johannes Sam, Llucia Alberti Servera, Sina Nassiri, Atul Sethi, Alison Ribeiro, Gabrielle Leclercq-Cohen, Petra Schwalie, Tamara Hüsser, Emilio Yangueez, Sylvia Herter, Christian Klein, Pablo Umana, Marina Bacac. Molecular insights into resistance mechanisms to therapy with T cell bispecific antibodies. [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 4117.

A novel cell line panel reveals non-genetic mediators of platinum resistance and phenotypic diversity in high grade serous ovarian cancer

ObjectivesResistance to cancer therapy is an enduring challenge and accurate and reliable preclinical models are lacking. We interrogated this unmet need using high grade serous ovarian cancer (HGSC) as a disease model. MethodsWe created five in vitro and two in vivo platinum-resistant HGSC models and characterised the entire cell panel via whole genome sequencing, RNASeq and creation of intraperitoneal models. ResultsMutational signature analysis indicated that platinum-resistant cell lines evolved from a pre-existing ancestral clone but a unifying mutational cause for drug resistance was not identified. However, cisplatin-resistant and carboplatin-resistant cells evolved recurrent changes in gene expression that significantly overlapped with independent samples obtained from multiple patients with relapsed HGSC. Gene Ontology Biological Pathways (GOBP) related to the tumour microenvironment, particularly the extracellular matrix, were repeatedly enriched in cisplatin-resistant cells, carboplatin-resistant cells and also in human resistant/refractory samples. The majority of significantly over-represented GOBP however, evolved uniquely in either cisplatin- or carboplatin-resistant cell lines resulting in diverse intraperitoneal behaviours that reflect different clinical manifestations of relapsed human HGSC. ConclusionsOur clinically relevant and usable models reveal a key role for non-genetic factors in the evolution of chemotherapy resistance. Biological pathways relevant to the extracellular matrix were repeatedly expressed by resistant cancer cells in multiple settings. This suggests that recurrent gene expression changes provide a fitness advantage during platinum therapy and also that cancer cell-intrinsic mechanisms influence the tumour microenvironment during the evolution of drug resistance. Candidate genes and pathways identified here could reveal therapeutic opportunities in platinum-resistant HGSC.

Mutant p53 drives an immune cold tumor immune microenvironment in oral squamous cell carcinoma

The critical role of the tumor immune microenvironment (TIME) in determining response to immune checkpoint inhibitor (ICI) therapy underscores the importance of understanding cancer cell–intrinsic mechanisms driving immune-excluded (“cold”) TIMEs. One such cold tumor is oral cavity squamous cell carcinoma (OSCC), a tobacco-associated cancer with mutations in the TP53 gene which responds poorly to ICI therapy. Because altered TP53 function promotes tumor progression and plays a potential role in TIME modulation, here we developed a syngeneic OSCC models with defined Trp53 (p53) mutations and characterized their TIMEs and degree of ICI responsiveness. We observed that a carcinogen-induced p53 mutation promoted a cold TIME enriched with immunosuppressive M2 macrophages highly resistant to ICI therapy. p53-mutated cold tumors failed to respond to combination ICI treatment; however, the combination of a programmed cell death protein 1 (PD-1) inhibitor and stimulator of interferon genes (STING) agonist restored responsiveness. These syngeneic OSCC models can be used to gain insights into tumor cell–intrinsic drivers of immune resistance and to develop effective immunotherapeutic approaches for OSCC and other ICI-resistant solid tumors.

Abstract 655: Macrophage mediated resistance to TKI therapy in ALK fusion positive non-small cell lung cancer

Abstract Patients with lung cancer bearing driver oncogenes such as epidermal growth factor receptor (EGFR) mutations or anaplastic lymphoma kinase (ALK) fusions, often experience long term disease control on targeted therapy. Despite excellent initial responses, ultimately these tumors develop resistance to targeted therapy. While several cancer cell intrinsic mechanisms of targeted therapy resistance are well known, they do not account for all cases of resistance. Based on our previous observations, we hypothesized components of the tumor microenvironment, specifically monocytes and macrophages, also contribute to the development of targeted therapy resistance. We generated bone marrow derived macrophages from C57BL/6 mice and polarized them to an M0, M1, or M2 phenotype over the course of 6 days. At day 7, cells were plated in fresh media and after 2 days, the conditioned media (CM) was collected. Macrophage polarization was verified via qPCR. Murine cell lines, EA-1, EA-2, and EA-3 are unique, murine lung adenocarcinomas bearing an oncogenic ALK fusion derived from C57BL/6 mice. These murine cell lines were then cultured in the macrophage CM prior to exposure to ALK fusion specific tyrosine kinase inhibitors (TKI), alectinib or crizotinib. We observed that culture in M2-CM resulted in at least a 10-fold increase in alectinib resistance across all 3 murine cell lines when compared to the same cell lines cultured in M1 or M0-CM. We next tested for the activity of known, clinically relevant bypass signaling pathways as mediator of M2-CM protection. Aftatinib, an inhibitor of EGFR and human epidermal growth factor receptor 2 (HER2) family of proteins, did not alter M2-CM protection in the presence of alectinib. M2-CM did not impart TKI resistance to the murine cell lines in the presence of crizotinib, a TKI which inhibits ALK fusion activity and MET receptor tyrosine kinase activity, suggesting bypass signaling through MET drives the M2-CM resistance. Ongoing animal model studies demonstrate a clear role for innate immune cells in decreasing tumor responsiveness to TKI. In summary, our work demonstrates TKI therapy may be modulated by the presence of and interaction with specific immune cells within the tumor microenvironment. These data support exploring macrophage targeting therapies to improve TKI responsiveness. Citation Format: Melanie Mandell, Katherine Priest, Anh Le, Lynn Heasley, Raphael Nemenoff, Erin L. Schenk. Macrophage mediated resistance to TKI therapy in ALK fusion positive non-small cell lung cancer [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 655.

Abstract SP037: New insights to cancer-associated systemic inflammation

Abstract The occurrence of distant metastases accounts for over 90% of breast cancer deaths. Despite these devastating effects, metastatic disease is poorly understood and incurable. Clearly, there is an urgent need for novel therapeutic strategies that prevent and/or cure metastatic disease. For successful dissemination and metastasis, cancer cells must evade detection and destruction by the immune system and adapt to a foreign tissue environment. This process is enabled by factors secreted by the primary tumor that shape both the intratumoral microenvironment and the systemic immune landscape. By manipulating systemic immune responses and creating conditions of chronic inflammation, tumors can have a long-distance reach and transform distant tissues that are intrinsically hostile to metastatic colonization into niches that are more hospitable for metastatic lesions to grow out. Key feature of this systemic process - often referred to as pre-metastatic niche formation- is the mobilization and polarization of (immature) myeloid immune cells from the bone marrow to the circulation, tumor and distant organs. We and others have used preclinical tumor models to demonstrate that these tumor-mobilized myeloid cells, including neutrophils and macrophages, contribute to a systemic immunosuppressive environment that inhibits anti-tumor immune responses and fosters metastasis formation. The pro-metastatic function of systemic inflammation is supported by clinical observations demonstrating that neutrophil accumulation in blood of cancer patients is strongly associated with poor survival and increased risk for metastasis. Targeting pro-metastatic inflammation represents an attractive strategy to prevent or treat metastatic disease. Importantly, the oncology field can benefit from compounds that have already been developed for the treatment of inflammatory diseases. In this Educational Session talk, I will discuss current insights into how tumors engage the immune system systemically, how systemic inflammation impacts disease progression and therapy response and how we can utilize these insights for the design of novel therapeutic opportunities. A great challenge in the field of immuno-oncology is the unexplained inter-patient heterogeneity in the intratumoral and systemic composition and functional state of the immune system. I will share some of our recent insights into how cancer cell-intrinsic mechanisms lie at the basis of this heterogeneity in the immune milieu of breast cancer, which may set the stage for personalized immune-intervention strategies. Citation Format: KE de Visser. New insights to cancer-associated systemic inflammation [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr SP037.

Metabolic Codependencies in the Tumor Microenvironment.

Metabolic reprogramming enables cancer cell growth, proliferation, and survival. This reprogramming is driven by the combined actions of oncogenic alterations in cancer cells and host cell factors acting on cancer cells in the tumor microenvironment. Cancer cell-intrinsic mechanisms activate signal transduction components that either directly enhance metabolic enzyme activity or upregulate transcription factors that in turn increase expression of metabolic regulators. Extrinsic signaling mechanisms involve host-derived factors that further promote and amplify metabolic reprogramming in cancer cells. This review describes intrinsic and extrinsic mechanisms driving cancer metabolism in the tumor microenvironment and how such mechanisms may be targeted therapeutically. SIGNIFICANCE: Cancer cell metabolic reprogramming is a consequence of the converging signals originating from both intrinsic and extrinsic factors. Intrinsic signaling maintains the baseline metabolic state, whereas extrinsic signals fine-tune the metabolic processes based on the availability of metabolites and the requirements of the cells. Therefore, successful targeting of metabolic pathways will require a nuanced approach based on the cancer's genotype, tumor microenvironment composition, and tissue location.

Abstract B21: The gastrointestinal microbiota controls cancer cell intrinsic mechanisms to promote the progression of acute lymphoblastic leukemia

Abstract Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer and the second most common acute leukemia in adults. Despite best existing therapy, older children and elderly ALL patients have poor prognoses. Thus, there is a need to identify alternative therapeutics for these patients. There has been controversy about the function of the gut microbiota in cancer, with some reports showing that it supports tumor growth and others showing tumor growth inhibition. Recently, Dr. Melvyn Greaves proposed that ALL development may have a microbial trigger; however, gut microbiota studies in ALL are scarce. Little is known about the role of the gut microbiota in ALL progression and in reshaping cancer cell-intrinsic pathways that are as important as immune responses in the outcome of ALL. Recently, we found that gut microbiota depletion in immunocompromised mice reduces ALL growth by inducing apoptosis and inhibiting proliferation. We show that in immunocompromised microbiome-depleted mice, the rate of ALL tumor growth is reduced compared to microbiome-competent mice. Molecularly, we found that microbiome ablation results in changes in cancer cell-intrinsic pathways of apoptosis and proliferation in these tumors. To further understand how the gut microbiota affects these cancer cell-intrinsic pathways, we have performed RNA sequencing on tumors from microbiome-competent and microbiome-depleted mice accompanied by 16s rRNA sequencing from stool samples and mass spectrometry of blood serum to identify metabolites secreted by the gut microbiota that reach the tumor site, affecting cancer cell growth. Our goal is to define bacterial species and metabolites involved in ALL progression and to mechanistically determine how these bacterial species and metabolites regulate ALL growth via regulation of cancer cell-intrinsic pathways. These data suggest that the gut microbiota may regulate ALL progression in an immune-independent manner via transcriptional changes of cancer cell-autonomous pathways. We envision that the gut microbiota can be reshaped in ALL patients who are elderly, chemo-refractive, and/or immunocompromised to alter cancer cell-intrinsic pathways to treat ALL. The clinical impact of these findings is significant given that ALL patients who are immunocompromised could still benefit from therapeutics aiming at modulating the gut microbiota. Citation Format: Wadie Mahauad-Fernandez, Soumaya Zlitni, Ami Bhatt, Dean Felsher. The gastrointestinal microbiota controls cancer cell intrinsic mechanisms to promote the progression of acute lymphoblastic leukemia [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr B21.

Abstract PR08: Intratumoral T-cell distribution in murine and patient pancreatic cancer correlates with tissue heterogeneity and survival

Abstract The complex microenvironment characteristic of pancreatic ductal adenocarcinoma (PDAC) has been implicated as a critical mediator of tumor progression and therapeutic response. Spatial relationships of individual cellular and acellular components within tumors may offer novel insights into the dynamic and complex functions of PDAC. Using eight-color multiplex immunohistochemistry and novel spatial analysis algorithms, we report a heterogeneous and increased population of infiltrating T lymphocytes in the tumors compared to uninvolved pancreatic tissues of treatment-naïve PDAC patients. Spatial distribution of cytotoxic T cells in proximity to cancer cells independently correlates with increased overall patient survival. Collagen-I and aSMA-positive fibroblasts do not correlate with a paucity of T-cell accumulation around cancer cells, suggesting that PDAC desmoplasia may not be a simple physical barrier. However, T-cell infiltration was reduced in proximity to specific cancer cell subpopulations, supporting cancer-cell intrinsic mechanisms for immune avoidance. Further expansion of this technology using early and late primary tumors and metastatic tissue of genetically engineered mouse models (KrasLSL-G12D; p53R172H; PDX1-Cre; EYFPLSL) demonstrates similar T-cell heterogeneity and spatial associations. These data support the validity of multiplex and spatial analysis platforms for exploring the structure-function relationships in the PDAC microenvironment and suggest differential spatial relationships between cancer subtypes and the surrounding microenvironment may be a determining factor in PDAC progression. This abstract is also being presented as Poster A04. Citation Format: Julienne L. Carstens, Pedro Correa de Sampaio, Soutik Barua, Huamin Wang, Jared K. Burks, Arvind Rao, Valerie S. LeBleu, Raghu Kalluri. Intratumoral T-cell distribution in murine and patient pancreatic cancer correlates with tissue heterogeneity and survival [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr PR08.

PAK4 inhibition improves PD-1 blockade immunotherapy.

Lack of tumor infiltration by immune cells is the main mechanism of primary resistance to programmed cell death protein 1 (PD-1) blockade therapies for cancer. It has been postulated that cancer cell-intrinsic mechanisms may actively exclude T cells from tumors, suggesting that the finding of actionable molecules that could be inhibited to increase T cell infiltration may synergize with checkpoint inhibitor immunotherapy. Here, we show that p21-activated kinase 4 (PAK4) is enriched in non-responding tumor biopsies with low T cell and dendritic cell infiltration. In mouse models, genetic deletion of PAK4 increased T cell infiltration and reversed resistance to PD-1 blockade in a CD8 T cell-dependent manner. Furthermore, combination of anti-PD-1 with the PAK4 inhibitor KPT-9274 improved anti-tumor response compared with anti-PD-1 alone. Therefore, high PAK4 expression is correlated with low T cell and dendritic cell infiltration and a lack of response to PD-1 blockade, which could be reversed with PAK4 inhibition.

Abstract 1485: Hepatic cell cycle-related kinase shapes a metastatic-prone liver microenvironment via crosstalk between myeloid-derived suppressor cell and natural killer T cell

Abstract Metastasis is a prominent cause of cancer-related death governed by both cancer cell-intrinsic mechanisms and extrinsic microenvironment. Clinical observations demonstrated liver as a common metastatic site for various cancers, which may be due to its immune tolerant environment. Myeloid-derived suppressor cell (MDSC) is a heterogeneous cell population of immature myeloid cells that contribute to the formation of a favorable metastatic environment partially via suppression of immune effector cells. However, the underlying mechanisms in liver tropism of tumor metastasis remain poorly understood. We have previously discovered that cell cycle-related kinase (CCRK) can promote primary hepatocellular carcinoma (HCC) development via MDSCs (Gut 2018; Nat Commun accepted). Here we hypothesize that the accumulation of hepatic MDSCs induced by CCRK may contribute to the formation of a favorable metastatic liver microenvironment. By the construction of a liver-specific CCRK inducible transgenic (TG) mouse model by a Cre/loxP system, induction of CCRK expression by tamoxifen injection could increase CD11b+Gr-1+Ly6G+Ly6Clow polymorphonuclear (PMN)-MDSCs liver accumulation specifically in male mice with upregulated Cxcl1 and Gcsf expression. Intrahepatic injection of a mouse hepatoma cell line Hep1-6 in male TG mice developed larger tumors compared to control and positively associated with increased PMN-MDSCs levels in liver. Moreover, the tumorigenicity was abolished by PMN-MDSC depletion. Notably, intrasplenic injection of a mouse melanoma cell line B16F10 exhibited increased level of liver-infiltrating PMN-MDSCs and enhanced liver metastasis in male TG mice compared to control mice. Moreover, depletion of PMN-MDSCs suppressed metastasis in liver. Mechanistically, anti-tumor NKT cells, rather than NK cells and CD8+T cells, were negatively correlated with tumor weight and MDSC proportion, indicating the potential crosstalk between MDSC and NKT in liver metastasis. Our findings suggest that hepatic CCRK expression create a tumor growth- and metastasis-supportive liver microenvironment via enhancing immunosuppression. Additional colorectal cancer metastasis models will be evaluated in our established TG mice. Moreover, the roles and underlying mechanisms of CCRK-PMN-MDSC interactions in the establishment of liver metastasis await further investigation. This project is supported by Collaborative Research Fund C4017-14G and the Focused Innovations Scheme 1907309. Citation Format: Xuezhen ZENG, Jingying Zhou, Zhewen Xiong, Hanyong Sun, Weiqin Yang, Wenshu Tang, Yu Feng, Alfred Sze-Lok Cheng. Hepatic cell cycle-related kinase shapes a metastatic-prone liver microenvironment via crosstalk between myeloid-derived suppressor cell and natural killer T cell [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1485.

Abstract ES2-3: Exosome-mediated stimulation of metastasis

Abstract Although cancer cell intrinsic mechanisms are important in driving aggressive features of cancer, stromal cells in the tumor microenvironment also serve critical roles. In particular, heterotypic interaction between cancer cells and stromal fibroblasts can generate signals for cancer progression, therapy resistance, and inflammatory responses. Among the signals resulting from cellular cross-talk include anti-viral pathways that induce interferon-stimulated genes (ISGs). These observations suggest 1) the existence of signals in the tumor microenvironment that mimic a viral infection, 2) resulting activation of anti-viral signaling may contribute to how stromal cells promote aggressive features of cancer, and 3) mechanisms must exist to prevent this anti-viral signaling under non-pathological conditions. We have demonstrated that exosomes and their RNA produced by stromal fibroblasts represents one such “virus mimic” by engaging RNA pattern recognition receptors (PRRs) such as RIG-I. Triggering of stromal NOTCH-MYC by breast cancer cells results in a POL3-driven increase in RN7SL1, an endogenous non-coding RNA normally shielded from PRR recognition by RNA binding proteins SRP9/14. This increase in RN7SL1 alters its stoichiometry with SRP9/14 and generates unshielded RN7SL1 that is deployed in stromal exosomes. After these stromal exosomes are transferred to immune cells, the unshielded RN7SL1 can drive an inflammatory response. Upon exosome transfer to breast cancer cells, unshielded RN7SL1 activates the RIG-I to enhance tumor growth, metastasis, and therapy resistance. The significance of this “virus mimicry” and failure to shield endogenous RN7SL1 from PRR activation is corroborated by evidence from patient tumors and blood. In total, these results demonstrate how exosomes, unshielding of endogenous RNAs, and engagement of pattern recognition receptors couple stromal activation with aggressive features of breast cancer. Citation Format: Minn A. Exosome-mediated stimulation of metastasis [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr ES2-3.

Immune Checkpoint Inhibition for Pancreatic Ductal Adenocarcinoma: Current Limitations and Future Options.

Pancreatic ductal adenocarcinoma (PDAC), as the most frequent form of pancreatic malignancy, still is associated with a dismal prognosis. Due to its late detection, most patients are ineligible for surgery, and chemotherapeutic options are limited. Tumor heterogeneity and a characteristic structure with crosstalk between the cancer/malignant cells and an abundant tumor microenvironment (TME) make PDAC a very challenging puzzle to solve. Thus far, targeted therapies have failed to substantially improve the overall survival of PDAC patients. Immune checkpoint inhibition, as an emerging therapeutic option in cancer treatment, shows promising results in different solid tumor types and hematological malignancies. However, PDAC does not respond well to immune checkpoint inhibitors anti-programmed cell death protein 1 (PD-1) or anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) alone or in combination. PDAC with its immune-privileged nature, starting from the early pre-neoplastic state, appears to escape from the antitumor immune response unlike other neoplastic entities. Different mechanisms how cancer cells achieve immune-privileged status have been hypothesized. Among them are decreased antigenicity and impaired immunogenicity via both cancer cell-intrinsic mechanisms and an augmented immunosuppressive TME. Here, we seek to shed light on the recent advances in both bench and bedside investigation of immunotherapeutic options for PDAC. Furthermore, we aim to compile recent data about how PDAC adopts immune escape mechanisms, and how these mechanisms might be exploited therapeutically in combination with immune checkpoint inhibitors, such as PD-1 or CTLA-4 antibodies.

Abstract 5062: CAFs in breast cancer circulation: The influence of cancer cell intrinsic mechanisms

Abstract Background: Metastatic disease is the primary cause of breast cancer (BC) mortality. Tumor-stromal cell interactions play a pivotal in tumor initiation, progression and metastasis. Cancer-associated fibroblasts (CAFs) are the majority of stroma in BC and are critical to BC tumorigenicity and malignancy. Metastasis occurs due to the transport of circulating tumor cells (CTC) and clusters of CTCs through the vasculature. We recently identified circulating CAFs (cCAFs) as a novel circulating biomarker associated with metastatic BC. cCAFs not only circulate individually, in BC patient blood and in blood from both spontaneous and xenograft murine BC models, but cCAFs are also found in clusters with CTCs. In this study, we examine the egress of CAFs and follow them through to metastatic sites, and evaluate the ability of BC cell subtype/metastatic propensity to influence cCAF and cCAF/CTC cluster egress. Methods: We used NSG mice with orthotopic xenograft implantation of BC cells, primary CAF cell lines, or co-implantation of BC and CAF cell lines. We used two different BC cell lines, the nonmetastatic BC cell line, MCF-7, and the highly metastatic primary BC cell line, DT28. We also employed the MMTV-PyMT spontaneous model of BC metastasis to evaluate cCAFs and CTCs in a preclinical model. Mice were sacrificed at specific time points, and cardiac blood was collected to ascertain the temporal dynamics of cCAF and CTC presence. Blood was filtered using the faCTChecker microfluidic filtration instrument (Circulogix). Filters were enumerated by IF for cCAFs, CTCs, and cCAF/CTC co-clusters. To explore BC-intrinsic factors that influence cCAF and cCAF/CTC cluster egress, we modeled egress from the primary tumor in vitro using transendothelial cell migration assays, where the chemoattractant for CAF cells was conditioned media from the different BC cells lines. Results: In both spontaneous and orthotopic xenograft models of BC, cCAFs, CTCs, and cCAF/CTCs appear early in tumor development. cCAF/CTC clusters increase in correlation with tumor burden and metastasis. CAFs injected alone in orthotopic xenografts are able to egress independently of BC cells and cCAFs and cCAF clusters were seen; however, co-inoculation with BC cells resulted in substantially higher numbers of both individual cCAFs and cCAF clusters, and now co-clusters were seen. cCAFs appear about 4 days post-injection, and precede appearance of CTCs and CAF/CTC clusters. CAFs co-injected with BC cells into the MFP appear at metastatic sites. In in vitro transendothelial assays, BC secreted factors that potentiate CAF egress are identified as well as the if cCAF/CTC clusters egress as clusters or cluster in the circulation. Conclusion: Although CAFs are highly motile and cCAFs precede CTCs into the circulation, BC subtype influences the ability of CAFs to egress. Targeting cCAF egress and/or cCAF/BC cell clusters provides novel avenues to prevent or treat BC metastasis. Citation Format: Utsav Sharma, Philip Miller, Kelsie Medina Saenz, Angela Spartz, Marc Lippman, Dorraya El-Ashry. CAFs in breast cancer circulation: The influence of cancer cell intrinsic mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5062.

Abstract 4755: Role of PAK4 in cancer immune cell exclusion

Abstract Lack of T-cell infiltration is the main mechanism of primary resistance to checkpoint blockade therapies. Here, we performed a transcriptomic analysis of metastatic melanoma biopsies taken from patients treated with anti-PD1 (n=23) with biopsies pre- (n=17) and during treatment (n=22), and investigated cancer cell intrinsic mechanisms of immune evasion. We classified our samples based on their T-cell infiltration status using a validated set of immune genes for T-cell infiltration (Spranger et al., 2015), normalized within gene and scored the samples from 0 (least infiltrated) to 1 (most infiltrated). In order to identify genes that anti-correlate with infiltration, we performed differential gene expression analysis between non-infiltrated (n= 18) and infiltrated tumors (n=21) regardless clinical response and condition. The analysis resulted in total of 1904 genes enriched in the non-infiltrated group (log2foldchange > 1, q-value < 0.05 and infiltration threshold = 0.5). Since Wnt signaling pathway has been previously suggested as a potential mechanism of immune exclusion, we aimed to select targetable proteins implicated in β-catenin signaling. Interestingly, p21 (RAC1) Activated Kinase 4 (PAK4) was significantly over-expressed in the non-infiltrated group (log2folchange = 1.04, q-value = 1.08e-05). PAK4 is a kinase involved in cell migration, proliferation and apoptosis and it is known that it can directly phosphorylate β-catenin to promote Wnt signaling. In addition, PAK4 was found to be also significantly enriched in on-treatment samples of non-responders (n=7) compared to responders (n=7) (mean of non-responder = 3.68, mean of responders = 2.69, p-value = 0.007). We next aimed to determine genes negatively associated with infiltration using Pearson correlation coefficient. PAK4 was on the top 5 of genes negatively associated with CD8A (pcc = -0.56, p-value 1.8e-04), HLA-DMB (pcc = -0.64, p-value 8.4e-06), TNF (pcc = -0.66, p-value 4.8e-06) among other immune genes. Altogether, this data suggests that PAK4 have an active role in T-cell exclusion. In order to validate our finding, we analyzed 472 melanoma samples from the TCGA. We first performed RNA-seq deconvolution to estimate the infiltration status of each sample and then determined genes that anti-correlated with the different immune cell subtypes. In line with the hypothesis that PAK4 was causatively excluding T-cell from tumors, we found that PAK4 performed the strongest anti-correlation with dendritic cells (pcc= -0.45, p-value = 2.2e-16) and CD8+ T-cells (pcc = -0.22, p-value = 1.12e-06). PAK4 expression was enriched in TCGA non-infiltrated samples (p-value = 4.92e-09, infiltration threshold = 0.5). In summary, this data suggests that PAK4 might be driving T-cell exclusion and that inhibition of PAK4 kinase activity with small molecules could facilitate T-cell infiltration and synergize with current checkpoint blockade therapies. Citation Format: Gabriel Abril-Rodriguez, Catherine S. Grasso, Jesse M. Zaretsky, Beata Berent-Maoz, Siwen Hu-Lieskovan, Antoni Ribas. Role of PAK4 in cancer immune cell exclusion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4755.