Jinyang Li

Supplementary Table 1: List of oligonucleotides used for qPCR

S1. Frequency of Pan-IN lesion types in KC versus KCC mice. S2. Re-expression of Ch25h in PDAC cell lines. S3. Presence of exogenous lipids diminishes effect of Ch25h re-expression in PDAC. S4. Re-expression of Ch25h does not affect Ras localization. S5. CH25H decreases autophagic flux in PDAC cells. S6. Ch25h slows PDAC tumor growth.

Cholesterol dependence is an essential characteristic of pancreatic ductal adenocarcinoma (PDAC). Cholesterol 25-hydroxylase (CH25H) catalyzes monooxygenation of cholesterol into 25-hydroxycholesterol, which is implicated in inhibiting cholesterol biosynthesis and in cholesterol depletion. Here, we show that, within PDAC cells, accumulation of cholesterol was facilitated by the loss of CH25H. Methylation of the CH25H gene and decreased levels of CH25H expression occurred in human pancreatic cancers and was associated with poor prognosis. Knockout of Ch25h in mice accelerated progression of Kras-driven pancreatic intraepithelial neoplasia. Conversely, restoration of CH25H expression in human and mouse PDAC cells decreased their viability under conditions of cholesterol deficit, and decelerated tumor growth in immune competent hosts. Mechanistically, the loss of CH25H promoted autophagy resulting in downregulation of MHC-I and decreased CD8+ T-cell tumor infiltration. Re-expression of CH25H in PDAC cells combined with immune checkpoint inhibitors notably inhibited tumor growth. We discuss additional benefits that PDAC cells might gain from inactivation of CH25H and the potential translational importance of these findings for therapeutic approaches to PDAC.Implications:Loss of CH25H by pancreatic cancer cells may stimulate tumor progression and interfere with immunotherapies.

Supplementary Tables 1, 2, and 3

Supplementary Figures 1-7 with legends

Fragility of regulatory T (Treg) cells manifested by the loss of neuropilin-1 (NRP1) and expression of IFNγ undermines the immune suppressive functions of Treg cells and contributes to the success of immune therapies against cancers. Intratumoral Treg cells somehow avoid fragility; however, the mechanisms by which Treg cells are protected from fragility in the tumor microenvironment are not well understood. Here, we demonstrate that the IFNAR1 chain of the type I IFN (IFN1) receptor was downregulated on intratumoral Treg cells. Downregulation of IFNAR1 mediated by p38α kinase protected Treg cells from fragility and maintained NRP1 levels, which were decreased in response to IFN1. Genetic or pharmacologic inactivation of p38α and stabilization of IFNAR1 in Treg cells induced fragility and inhibited their immune suppressive and protumorigenic activities. The inhibitor of sumoylation TAK981 (Subasumstat) upregulated IFNAR1, eliciting Treg fragility and inhibiting tumor growth in an IFNAR1-dependent manner. These findings describe a mechanism by which intratumoral Treg cells retain immunosuppressive activities and suggest therapeutic approaches for inducing Treg fragility and increasing the efficacy of immunotherapies.

Fragility of regulatory T (Treg) cells manifested by the loss of neuropilin-1 (NRP1) and expression of IFNγ undermines the immune suppressive functions of Treg cells and contributes to the success of immune therapies against cancers. Intratumoral Treg cells somehow avoid fragility; however, the mechanisms by which Treg cells are protected from fragility in the tumor microenvironment are not well understood. Here, we demonstrate that the IFNAR1 chain of the type I IFN (IFN1) receptor was downregulated on intratumoral Treg cells. Downregulation of IFNAR1 mediated by p38α kinase protected Treg cells from fragility and maintained NRP1 levels, which were decreased in response to IFN1. Genetic or pharmacologic inactivation of p38α and stabilization of IFNAR1 in Treg cells induced fragility and inhibited their immune suppressive and protumorigenic activities. The inhibitor of sumoylation TAK981 (Subasumstat) upregulated IFNAR1, eliciting Treg fragility and inhibiting tumor growth in an IFNAR1-dependent manner. These findings describe a mechanism by which intratumoral Treg cells retain immunosuppressive activities and suggest therapeutic approaches for inducing Treg fragility and increasing the efficacy of immunotherapies.

Supplementary Data from KAT6A and ENL Form an Epigenetic Transcriptional Control Module to Drive Critical Leukemogenic Gene-Expression Programs

The degree of metastatic disease varies widely among patients with cancer and affects clinical outcomes. However, the biological and functional differences that drive the extent of metastasis are poorly understood. We analyzed primary tumors and paired metastases using a multifluorescent lineage-labeled mouse model of pancreatic ductal adenocarcinoma (PDAC)—a tumor type in which most patients present with metastases. Genomic and transcriptomic analysis revealed an association between metastatic burden and gene amplification or transcriptional upregulation of MYC and its downstream targets. Functional experiments showed that MYC promotes metastasis by recruiting tumor-associated macrophages, leading to greater bloodstream intravasation. Consistent with these findings, metastatic progression in human PDAC was associated with activation of MYC signaling pathways and enrichment for MYC amplifications specifically in metastatic patients. Collectively, these results implicate MYC activity as a major determinant of metastatic burden in advanced PDAC.Significance:Here, we investigate metastatic variation seen clinically in patients with PDAC and murine PDAC tumors and identify MYC as a major driver of this heterogeneity.This article is highlighted in the In This Issue feature, p. 275

Supplementary Data from KAT6A and ENL Form an Epigenetic Transcriptional Control Module to Drive Critical Leukemogenic Gene-Expression Programs