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Supplemental Figure S1. FACS analysis of Aldefluor stained SU-DIPG XIII cells. Supplemental Figure S2: Downstream signaling changes by inhibition of MAPK and PI3K/mTOR in DIPG cells. Supplemental Figure S3. Bru-sequencing transcriptome analysis identifies gene sets regulated by MAPK and PI3K/mTOR inhibition in ALDH positive SU-DIPG XIII cells. Supplemental Figure S4. A-C. Heatmaps of Bru-seq RPKM values in single agent treated (901 or GSK) ALDH+ SU-DIPG XII cells highlight treatment effects on "stemness" and DDR genes. Supplemental Figure S5: A.-B. Illustrative result of BOILED-Egg predictive model for GSK-458 and GDC-0084, respectively. Supplemental Figure S6: A-C. SU-DIPG-XIII cells were Aldefluor stained and sorted into an ALDH+ and ALDH- population. Supplemental Figure S7: A. Aldefluor stain and FACS of SU-DIPG XIII cells. B. ALDH+ cells were treated with 50 nM 901+GSK each and 24 hrs later Aldefluor re-stained and analyzed by flow cytometry. Table S1. Characteristics of Patient-Derived Diffuse Intrinsic Pontine Glioma (DIPG) Cells. Table S2. Selected Genes Upregulated in ALDH+ DIPG cells Table S3. Selected Metabolome Genes Upregulated in ALDH+ DIPG Cells. Table S4. RT-qPCR primer sequences.

Understanding the cancer stem cell (CSC) landscape in diffuse intrinsic pontine glioma (DIPG) is desperately needed to address treatment resistance and identify novel therapeutic approaches. Patient-derived DIPG cells demonstrated heterogeneous expression of aldehyde dehydrogenase (ALDH) and CD133 by flow cytometry. Transcriptome-level characterization identified elevated mRNA levels of MYC, E2F, DNA damage repair (DDR) genes, glycolytic metabolism, and mTOR signaling in ALDH+ compared with ALDH−, supporting a stem-like phenotype and indicating a druggable target. ALDH+ cells demonstrated increased proliferation, neurosphere formation, and initiated tumors that resulted in decreased survival when orthotopically implanted. Pharmacologic MAPK/PI3K/mTOR targeting downregulated MYC, E2F, and DDR mRNAs and reduced glycolytic metabolism. In vivo PI3K/mTOR targeting inhibited tumor growth in both flank and an ALDH+ orthotopic tumor model likely by reducing cancer stemness. In summary, we describe existence of ALDH+ DIPGs with proliferative properties due to increased metabolism, which may be regulated by the microenvironment and likely contributing to drug resistance and tumor recurrence.Implications:Characterization of ALDH+ DIPGs coupled with targeting MAPK/PI3K/mTOR signaling provides an impetus for molecularly targeted therapy aimed at addressing the CSC phenotype in DIPG.

Combined pathway analyses of sequencing data

Regulatory T cells (Treg) are abundant in human and mouse pancreatic cancer. To understand the contribution to the immunosuppressive microenvironment, we depleted Tregs in a mouse model of pancreatic cancer. Contrary to our expectations, Treg depletion failed to relieve immunosuppression and led to accelerated tumor progression. We show that Tregs are a key source of TGFβ ligands and, accordingly, their depletion reprogramed the fibroblast population, with loss of tumor-restraining, smooth muscle actin–expressing fibroblasts. Conversely, we observed an increase in chemokines Ccl3, Ccl6, and Ccl8 leading to increased myeloid cell recruitment, restoration of immune suppression, and promotion of carcinogenesis, an effect that was inhibited by blockade of the common CCL3/6/8 receptor CCR1. Further, Treg depletion unleashed pathologic CD4+ T-cell responses. Our data point to new mechanisms regulating fibroblast differentiation in pancreatic cancer and support the notion that fibroblasts are a heterogeneous population with different and opposing functions in pancreatic carcinogenesis.Significance:Here, we describe an unexpected cross-talk between Tregs and fibroblasts in pancreatic cancer. Treg depletion resulted in differentiation of inflammatory fibroblast subsets, in turn driving infiltration of myeloid cells through CCR1, thus uncovering a potentially new therapeutic approach to relieve immunosuppression in pancreatic cancer.See related commentary by Aykut et al., p. 345.This article is highlighted in the In This Issue feature, p. 327

Pancreatic ductal adenocarcinoma (PDA) is characterized by an immune-suppressive tumor microenvironment that renders it largely refractory to immunotherapy. We implemented a multimodal analysis approach to elucidate the immune landscape in PDA. Using a combination of CyTOF, single-cell RNA sequencing and multiplex immunohistochemistry on patient tumors, matched blood and non-malignant samples, we uncovered a complex network of immune-suppressive cellular interactions. These experiments revealed heterogeneous expression of immune checkpoint receptors in individual patients’ T cells and increased markers of CD8+ T cell dysfunction in the advanced disease stage. Tumor-infiltrating CD8+ T cells had an increased proportion of cells expressing an exhausted expression profile that included upregulation of the immune checkpoint TIGIT, a finding that we validated at the protein level. Our findings point to a profound alteration of the immune landscape of tumors, and to patient-specific immune changes that should be taken into account as combination immunotherapy becomes available for pancreatic cancer. Using single-cell RNA sequencing, CyTOF and multiplex immunohistochemistry, Steele et al. survey the immune landscape in pancreatic cancers, adjacent tissue and blood, observing heterogeneous immune checkpoint receptor expression within patients.

Oncogenic KRAS is the hallmark mutation of human pancreatic cancer and a driver of tumorigenesis in genetically engineered mouse models of the disease. While the tumor cell-intrinsic effects of oncogenic Kras expression have been widely studied, its role in regulating the extensive pancreatic tumor microenvironment is less understood. Using a genetically engineered mouse model of inducible and reversible oncogenic Kras expression and a combination of approaches that include mass cytometry and single cell RNA sequencing, we have discovered that non-cell autonomous (i.e., extrinsic) oncogenic KRAS signaling reprograms pancreatic fibroblasts, activating an inflammatory gene expression program. As a result, fibroblasts become a hub of extracellular signaling, mediating the polarization and function of pro-tumorigenic myeloid cells while also preventing tissue repair. Our study provides fundamental new knowledge on the mechanisms underlying the formation of the fibroinflammatory stroma in pancreatic cancer and highlights stromal pathways with the potential to be exploited therapeutically.

Ewing sarcoma (ES) is driven by the tumor initiating fusion EWS::FLI1 that causes widespread transcriptional dysregulation via epigenetic reprogramming. ES cells are highly plastic and heterogenous and have features of both neural crest and mesenchymal lineages. Epigenetic plasticity is well established as a mediator of disease progression and drug resistance, leading to the creation of heterogeneous tumor cell subpopulations that can initiate metastasis and relapse. Successful therapeutic strategies for metastatic or relapsed ES patients are limited and unchanged in the last three decades. Identification of new therapeutic strategies, and defining the effect of new agents on ES biology, is critical to improving patient outcomes. Given the epigenetic dependencies of ES, epigenetic modifying agents are an important class of drugs to investigate. The class of bromodomain and extra-terminal domain protein inhibitors (BETi) have shown preclinical efficacy in ES models but drug combinations will be needed. We are testing the hypothesis that transcriptional rewiring downstream of BETi will create new dependencies in ES cells that could be therapeutically exploited with biologically-defined combinations. Our studies show that the BETi, BMS-986158, the first BETi to be tested in pediatric patients, has profound cytostatic effects in preclinical ES models in vitro and in vivo. Our data further reveal that long term exposure to BMS-986158 creates a persister population of cells that reacquires proliferative capacity. We performed RNA-seq studies on parent, acutely treated (72 hours), and persister populations from three ES cell lines to define the effects of short and long term BET inhibition. Exposure to BMS-986158 led to strong inhibition of the EWS::FLI1-activated gene signature that was evident both acutely and in persister cells. Thus, despite continued inhibition of EWS::FLI1 activity, persister cells restored their ability to survive and proliferate. Gene Set Enrichment Analysis of transcripts that were significantly altered in persister cells identified gene programs involved in development and lineage commitment, including polycomb targets, suggesting that persister cells may have been rewired to more stem-like states. In addition, in two of the three cell lines, persister cells demonstrated upregulation of MYC target genes and cell cycle programs that were initially repressed by BET inhibition. Ongoing studies are investigating the molecular mechanisms by which MYC programs are reactivated in persister cells. We anticipate that epigenetic reprogramming mediates the transcriptional rewiring of MYC programs in persister cells, thereby escaping the cytostatic effects of continued BET inihbition. By defining these mechanisms we aim to identify new vulnerabilities that can be exploited in the context of BETi combination trials in the future. Citation Format: Shireen S. Ganapathi, Nicolas M. Garcia, Veerin R. Sirihorachai, Elizabeth R. Lawlor. Emergence of persister cells following bromodomain inhibition in Ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A011.

This study uses biomaterial scaffolds to identify a macrophage gene signature defined by high expression of C1qa, C1qb, and Trem2 that is elevated systemically in mouse and human pancreatic cancer., Pancreatic ductal adenocarcinoma (PDA) is accompanied by reprogramming of the local microenvironment, but changes at distal sites are poorly understood. We implanted biomaterial scaffolds, which act as an artificial premetastatic niche, into immunocompetent tumor-bearing and control mice, and identified a unique tumor-specific gene expression signature that includes high expression of C1qa, C1qb, Trem2, and Chil3. Single-cell RNA sequencing mapped these genes to two distinct macrophage populations in the scaffolds, one marked by elevated C1qa, C1qb, and Trem2, the other with high Chil3, Ly6c2 and Plac8. In mice, expression of these genes in the corresponding populations was elevated in tumor-associated macrophages compared with macrophages in the normal pancreas. We then analyzed single-cell RNA sequencing from patient samples, and determined expression of C1QA, C1QB, and TREM2 is elevated in human macrophages in primary tumors and liver metastases. Single-cell sequencing analysis of patient blood revealed a substantial enrichment of the same gene signature in monocytes. Taken together, our study identifies two distinct tumor-associated macrophage and monocyte populations that reflects systemic immune changes in pancreatic ductal adenocarcinoma patients.

Pancreatic Ductal Adenocarcinoma (PDA) has an exceedingly poor prognosis with only 10% 5-year survival rate. Kras mutations are found in over 90% of cases of pancreatic cancer and drive the formation of pancreatic intraepithelial neoplasia (PanIN), precursor lesions to PDA. Both PanIN and PDA are characterized by dense stroma, containing fibroblasts and immune cells. The infiltrating immune cells have a suppressive phenotype and prevent anti-tumor immunity by cytotoxic T cells. The mechanisms underlying the immunosuppression in pancreatic cancer are only partially understood. Our goal was to unravel the non-cell autonomous role of oncogenic Kras (Kras*) expressing epithelial cells in driving the formation of a complex, tumor promoting microenvironment during the onset of pancreatic cancer. Our laboratory has described a mouse model (iKras*) of inducible and reversible expression of Kras* in the pancreas. Taking advantage of the reversibility of Kras* expression in this model, we conducted a thorough characterization of the immune infiltration and function upon modulation of Kras* at different stages of pancreatic carcinogenesis. iKras* mice and wild type littermates were enrolled in experiments at the age of 8-12 weeks. Kras* expression was activated, then we induced acute pancreatitis to promote the formation of pre-neoplastic lesions. After 3 weeks, a time when widespread low-grade lesions and fibrosis are observed, mice were either harvested or Kras* expression was inactivated and 3 days or 1 week later the pancreas was harvested. We performed flow cytometry, immunohistochemistry and cytometry time of flight (CyTOF) in the pancreas to analyze myeloid cell populations, as well as functional markers (Arg1, iNOS, IFN). Myeloid cells and T cells infiltrated the pancreas in presence of active Kras*. Inactivation of Kras* resulted in a relatively modest decrease in infiltrating myeloid cells. However, analysis of the functional marker Arg1, a putative immune suppressive molecule expressed in myeloid cells, indicated that its expression depends on Kras*-expressing cells. When we further studied the interactions among cell types using single cell RNA sequencing, we discovered that non-cell autonomous (extrinsic) oncogenic KRAS signaling reprograms pancreatic fibroblasts, activating an inflammatory gene expression program. As a result, fibroblasts become a hub of extracellular signaling, mediating the polarization and function of pro-tumorigenic myeloid cells while also preventing tissue repair. Citation Format: Ashley Velez-Delgado, Katelyn L. Donahue, Kristee L. Brown, Wenting Du, Valerie Irizarry-Negron, Rosa E. Menjivar, Emily L. Lasse-Opsahl, Nina G. Steele, Stephanie The, Jenny Lazarus, Veerin R. Sirihorachai, Filip Bednar, Timothy L. Frankel, Yaqing Zhang, Marina Pasca di Magliano. Extrinsic KRAS signaling shapes the pancreatic microenvironment through fibroblast reprogramming [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-016.

Summary Oncogenic mutations in BRAF are believed to initiate serrated colorectal cancers; however, the mechanisms of BRAF-driven colon cancer are unclear. We find that oncogenic BRAF paradoxically suppresses stem cell renewal and instead promotes differentiation. Correspondingly, tumor formation is inefficient in BRAF-driven mouse models of colon cancer. By reducing levels of differentiation via genetic manipulation of either of two distinct differentiation-promoting factors ( Smad4 or Cdx2 ), stem cell activity is restored in BRAF V600E intestines, and the oncogenic capacity of BRAF V600E is amplified. In human patients, we observe that reduced levels of differentiation in normal tissue is associated with increased susceptibility to serrated colon tumors. Together, these findings help resolve the conditions necessary for BRAF-driven colon cancer initiation. Additionally, our results predict that genetic and/or environmental factors that reduce tissue differentiation will increase susceptibility to serrated colon cancer. These findings offer an opportunity to identify susceptible individuals by assessing their tissue-differentiation status.