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Supplementary Figure 3: Association of chemokine expression with a T cell-inflamed phenotype was validated in a second independent PDAC patient cohort

Supplementary Table 1: Top 99 genes associating with CD8+ TIL counts in primary resected cases

Purpose:The molecular drivers of antitumor immunity in pancreatic ductal adenocarcinoma (PDAC) are poorly understood, posing a major obstacle for the identification of patients potentially amenable for immune-checkpoint blockade or other novel strategies. Here, we explore the association of chemokine expression with effector T-cell infiltration in PDAC.Experimental Design:Discovery cohorts comprised 113 primary resected PDAC and 107 PDAC liver metastases. Validation cohorts comprised 182 PDAC from The Cancer Genome Atlas and 92 PDACs from the Australian International Cancer Genome Consortium. We explored associations between immune cell counts by immunohistochemistry, chemokine expression, and transcriptional hallmarks of antitumor immunity by RNA sequencing (RNA-seq), and mutational burden by whole-genome sequencing.Results:Among all known human chemokines, a coregulated set of four (CCL4, CCL5, CXCL9, and CXCL10) was strongly associated with CD8+ T-cell infiltration (P < 0.001). Expression of this “4-chemokine signature” positively correlated with transcriptional metrics of T-cell activation (ZAP70, ITK, and IL2RB), cytolytic activity (GZMA and PRF1), and immunosuppression (PDL1, PD1, CTLA4, TIM3, TIGIT, LAG3, FASLG, and IDO1). Furthermore, the 4-chemokine signature marked tumors with increased T-cell activation scores (MHC I presentation, T-cell/APC costimulation) and elevated expression of innate immune sensing pathways involved in T-cell priming (STING and NLRP3 inflammasome pathways, BATF3-driven dendritic cells). Importantly, expression of this 4-chemokine signature was consistently indicative of a T-cell–inflamed phenotype across primary PDAC and PDAC liver metastases.Conclusions:A conserved 4-chemokine signature marks resectable and metastatic PDAC tumors with an active antitumor phenotype. This could have implications for the appropriate selection of PDAC patients in immunotherapy trials.

Recent advances in cancer metabolism suggest that targeting amino acid metabolism represents a promising strategy for the development of novel therapeutic agents. Tumor, stromal and myeloid-derived suppressor cells (MDSC) within the tumor microenvironment (TME) create a nutrient-poor environment that inhibit immune function and support tumor growth. GCN2 (general control nonderepressible 2), a stress response kinase, plays a key role in maintaining cellular homeostasis under a wide range of stressors. Phosphorylation of GCN2 (pGCN2) in response to stress leads to inhibition of global protein synthesis and subsequently leads to 1) T cell anergy and apoptosis, 2) enhanced MDSC-dependent immune suppression and 3) tumor cell survival. Collectively, these roles suggest that GCN2 inhibition could have both a direct anticancer and an immune-activating effect. Treating nutrient-deprived T cells in vitro with a RAPT GCN2 inhibitor (RPT-GCN2i) rescued CD4+ and CD8+ T cell proliferation and effector functions. The RPT-GCN2i also reversed T cell suppression mediated by MDSCs derived from healthy donors or cancer patients. Using syngeneic mouse tumor models, we demonstrated that translational induction of activating transcription factor 4 (ATF4) is a strong marker of GCN2 pathway activation in vivo. Oral administration of an RPT-GCN2i exhibited notable drug-target occupancy and potently inhibited GCN2 kinase and ATF4 in the TME. RPT-GCN2i as a single agent and in combination with checkpoint blockade or angiogenesis inhibitor (anti-VEGFR) led to delays in tumor growth rate in various syngeneic tumor models. In addition, GCN2 inhibition redirected MDSC within the TME from a suppressive to inflammatory phenotype through downregulation of Arg1 and iNOS. Our results show that inhibition of GCN2 is an attractive approach for enhancing antitumor immune response and therefore GCN2 is a promising therapeutic target for the treatment of cancer. Citation Format: Lisa Marshall, Buvana Ravishankar, Urvi Kolhatkar, Mengshu Xu, Lavanya Adusumilli, Deepa Pookot, Thant Zaw, Chandru Ramana, Raashi Sreenivasan, Mikhail Zibinsky, Jeffrey Jackson, Grant Shibuya, Paul Leger, Omar Robles, Anqi Ma, Andrew Ng, Anton Shakhmin, Justy Guagua, Scott Jacobson, Steve Wong, Delia Bradford, Tracy L. McGaha, M Teresa Ciudad, James E. Talmadge, Holly C. Britton, George Katibah, Gene Cutler, David Wustrow, Paul D. Kassner, Dirk G. Brockstedt. Targeting the stress response kinase GCN2 to restore immunity in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3153.

Pancreatic cancer continues to have the highest mortality rate of all solid cancers, with a 5-year overall survival of approximately 8%. Despite recent progress in other malignancies, immunotherapy remains ineffective against pancreatic cancer. Previous work from our group and others has shown that tumors with mismatch repair (MMR) and homologous repair (HR) deficiency have increased immune activity, likely attributable to the inherently increased mutational and neoantigen load in these patients. However, there remains a subset of pancreatic cancer patients with increased CD8+ T cell infiltration, despite lacking these specific mutational signatures. Furthermore, an increasing body of evidence has shown that mutational burden and neoantigen load only partially explain the T cell-inflamed phenotype seen in tumors, implicating the presence of additional mechanisms that drive T cell infiltration in cancer. Several recent studies have highlighted the importance of proper CD8+ T cell priming by antigen presenting cells (APCs), particularly Batf3+ dendritic cells (DCs), and subsequent tumor infiltration via chemokines for immunotherapy to be effective. In this study, we investigated the involvement of chemokines in cytotoxic T lymphocyte infiltration in pancreatic cancer. Using a bioinformatics-driven approach, we analyzed 78 treatment-naïve, primary pancreas cancer resections for associations between CD8+ tumour infiltrating lymphocytes (TILs) and chemokine expression by RNAseq. A panel of chemokines, including CCL4, CCL5, CXCL9, CXCL10, and CXCL11, was highly associated with CD8+ TILs (p < 0.001). Segregating 173 tumour-enriched patient RNAseq samples based on expression of CXCL9 and CXCL10, we found those with higher expression of these chemokines had increased immune activation signatures. Importantly, this included increased MHC I presentation, presence of Batf3+ DCs, and T cell/APC co-stimulation (p < 0.001), while we observed no differences in conventional predictors of CD8+ T cell infiltration such as SNV counts or neoantigens between groups. These results were consistent across ICGC and TCGA data sets. Moreover, these results were also recapitulated in 72 tumor-enriched liver metastases, suggesting an underlying immunobiology that may occur in both primary and metastatic sites. The cellular sources of these chemokines, as determined by immunohistochemical analysis, and the role of tumor-sensing innate immune pathways leading to CD8+ T cell priming, by pathway and differential gene expression analysis, are currently being investigated. Taken together, these results demonstrate a potential role for these chemokines in recruiting CD8+ T cells in pancreatic cancer. Understanding mediators driving cytotoxic T cell infiltration will help identify mechanisms leading to proper CD8+ T cell priming and homing into tumors, to stratify patients amenable for known and novel immunotherapies. Citation Format: Joan Miguel Romero, Barbara Grünwald, Ashton Connor, Gun Ho Jang, Prashant Bavi, Aaditeya Jhaveri, Mehdi Masoomian, Sandra Fischer, Amy Zhang, Robert E. Denroche, Tracy McGaha, Faiyaz Notta, Pamela Ohashi, Grainne O'Kane, Julie Wilson, Jennifer Knox, Steven Gallinger. Mediators of CD8+ cytotoxic T lymphocyte infiltration in pancreatic cancer [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 1500.

Background: Squamous cell carcinoma of the oral cavity (SCCOC) often presents at early stages but its prognosis remains guarded, with a 5-year survival rate of 60% despite curative-intent therapies. Preoperative window-of-opportunity (WOO) studies in resectable SCCOC enable pharmacodynamic evaluation of molecular endpoints without compromising curative-intent treatment. Preoperative nivolumab in SCCOC was safe and showed promising tumor responses in CheckMate-358 WOO study (Ferris et al. ESMO 2017, LBA46). Sitravatinib, a receptor tyrosine kinase inhibitor which potently inhibits Tyro, AXL, Mer, and VEGF family of receptors, has shown encouraging results when combined with nivolumab in non-small cell lung cancer patients who have progressed on anti-PD-1 agents (Leal et al. ESMO 2018, 1129O). We hypothesize that sitravatinib and nivolumab have synergistic antitumor and immunogenic effects by increasing tumor immune infiltration and by blocking oncogenic pathways implicated in disease progression and immune-checkpoint resistance. Methods: Trial design: SNOW is a single-center, non-randomized WOO study of preoperative sitravatinib and nivolumab in patients with resectable SCCOC. Sitravatinib 120 mg is given orally once daily from day 1 until 48h before surgery or for a maximum period of 28 days. Nivolumab 240mg is given intravenously on day 15 for one dose only. Surgery is planned between days 23-30 following study treatment initiation. Fresh tumor biopsies and serial blood samples for extensive immunophenotyping and evaluation of other pharmacodynamic biomarkers, as well as clinical photographs of the tumor, are collected at baseline, on day 15 prior to nivolumab and at the time of surgery. 18FAZA-PET scans are performed at baseline and before surgery. Key eligibility criteria: previously untreated and resectable SCCOC; T2-4a, N0-2 or T1 (greater than 1 cm)-N2; no history of tumor bleeding or invasion of major vessels; adequate organ function; no autoimmune disorders; no immunosuppressive therapy. Study objectives: primary objective is to evaluate the immune and pharmacodynamic effects of sitravatinib plus nivolumab. Secondary objectives are: safety and tolerability including toxicity, rate of surgery completion within the planned window and rate of postoperative complications; antitumor activity including rate of complete pathological response; pharmacokinetics of sitravatinib alone and in combination with nivolumab. Correlative studies: tumor and blood immunophenotyping, tumor genome and transcriptome analysis, changes in intratumoral hypoxia based on 18FAZA-PET testing. Sample size: SNOW is a proof-of-concept study with no specific statistical assumptions at trial onset. We plan to enroll 12-15 patients evaluable for correlative studies. Study activation: Aug 30th, 2018. Two patients enrolled as of Jan 10th2019. Clinical trial identification: NCT03575598. Citation Format: Marc Oliva Bernal, Douglas Chepeha, Amy Prawira, Douglass Vines, Anna Spreafico, Scott Bratman, John De Almeida, Aaron Hansen, David Goldstein, Ralph Gilbert, Patrick Gullane, Dale H. Brown, Ilan Weinreb, Bayardo Perez-Ordoñez, Pamela S. Ohashi, Tracy McGaha, Ben X. Wang, Jonathan Irish, Isan Chen, Lillian L. Siu. Sitravatinib and nivolumab in oral cavity cancer window of opportunity study (SNOW) [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 CT124.

This abstract is being presented as a short talk in the scientific program. A full abstract is printed in the Proffered Abstracts section (PR05) of the Conference Proceedings. Citation Format: Amy Paschall, Ruihua Zhang, Kankana Bardhan, Chen-Feng Qi, Liang Peng, Geming Lu, Jianjun Yang, Miriam Merad, Mary Zimmerman, Tracy McGaha, Gang Zhou, Andrew Mellor, Scott I. Abrams, Herbert Morse, Keiko Ozato, Huabao Xiong, Kebin Liu. IRF8 regulates GM-CSF expression in T cells and tumor cells to mediate myeloid-derived suppressor cell differentiation. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A84.

Myeloid cells are a heterogenous and abundant population of haematopoietic cells that are virtually present in all mammalian tissues, where they monitor local microenvironment to maintain homeostasis. All myeloid cells originate from the pluripotent hematopoietic stem cells that undergo progressive restriction in their lineage potential to give rise to mature granulocytes and macrophages. Lineage restriction and differentiation are regulated by timely activation of specific set of lineage-specific transcription factors in concert with down-regulation of other set(s) of transcription factors that are important for alternative cell lineage potential. Altered expression of these lineage-specific transcription factors often leads to deregulation of myelopoiesis and resultant hematopoietic disorders. Therefore, lineage-specific transcription factors are essential for myeloid cell lineage differentiation and maturation. Mice with a null mutation of irf8, the gene that encodes IFN regulatory factor 8 (IRF8), exhibit massive accumulation of CD11b+Gr1+ immature myeloid cells (IMCs). Therefore, IRF8 is a myeloid cell lineage-specific transcription factor that plays an essential function in the regulation of myelopoiesis. Particularly, IRF8 may determine differentiation, lineage commitment, and immune function of monocytes versus granulocytes under physiological conditions. A hallmark of cancer-bearing mice is the accumulation of CD11b+Gr1+ myeloid-derived suppressor cells (MDSCs). Interestingly, IRF8 is silenced in MDSCs from tumor-bearing mice. Therefore, IRF8 is apparently a key transcription factor that mediates MDSC differentiation. However, the molecular mechanism underlying IRF8 regulation of MDSCs is largely unknown. Because MDSCs is induced by inflammation, we therefore hypothesized that IRF8 may repress the expression of proinflammatory factors to mediate differentiation of MDSCs/IMCs under physiological and pathological conditions. To test this hypothesis, we made use of conventional IRF8 KO mice, mice with IRF8 deficiency only in myeloid cells, mice with IRF8 deficiency only in T cells, and tumor-bearing mouse models. Here we report an intriguing finding that although IRF8 conventional mice exhibit deregulated myeloid cell differentiation and resultant accumulation of CD11b+Gr1+ IMCs, surprisingly, mice with IRF8 deficiency only in myeloid cells exhibit normal myeloid cell lineage differentiation. Instead, mice with IRF8 deficiency only in T cells exhibited deregulated myeloid cell differentiation and IMC accumulation. We further demonstrated that IRF8-deficient T cells exhibit elevated GM-CSF expression and secretion. Treatment of mice with GM-CSF increased IMC accumulation, and adoptive transfer of IRF8-deficient T cells, but not GM-CSF-deficient T cells, increased IMC accumulation in the recipient chimera mice. Moreover, overexpression of IRF8 decreased GM-CSF expression in T cells. These data thus determine that IRF8 functions in T cells to repress GM-CSF expression to suppress IMCs. However, in tumor-bearing mice, IRF8 is silenced in MDSCs but not in T cells, suggesting a different mechanism of MDSC regulation by IRF8. We observed that silencing IRF8 using IRF8-specific siRNA dramatically increase GM-CSF expression in tumor cells. Therefore, IRF8 represses GM-CSF expression in tumor cells to mediate MDSC differentiation. In summary, we determine that IRF8 regulates GM-CSF expression in T cells and tumor cells, respectively, to mediate myelopoiesis under physiological and pathological conditions. This abstract is also presented as Poster A84. Citation Format: Amy Paschall, Ruihua Zhang, Kankana Bardhan, Chen-Feng Qi, Liang Peng, Geming Lu, Jianjun Yang, Miriam Merad, Mary Zimmerman, Tracy McGaha, Gang Zhou, Andrew Mellor, Scott I. Abrams, Herbert Morse, Keiko Ozato, Huabao Xiong, Kebin Liu. IRF8 regulates GM-CSF expression in T cells and tumor cells to mediate myeloid-derived suppressor cell differentiation. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr PR05.