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Figures S1-S3

Table S2

Table S1

Despite significant recent advances in precision medicine, pancreatic ductal adenocarcinoma (PDAC) remains near uniformly lethal. Although immune-modulatory therapies hold promise to meaningfully improve outcomes for patients with PDAC, the development of such therapies requires an improved understanding of the immune evasion mechanisms that characterize the PDAC microenvironment. Here, we show that cancer cell–intrinsic glutamic-oxaloacetic transaminase 2 (GOT2) shapes the immune microenvironment to suppress antitumor immunity. Mechanistically, we find that GOT2 functions beyond its established role in the malate–aspartate shuttle and promotes the transcriptional activity of nuclear receptor peroxisome proliferator–activated receptor delta (PPARδ), facilitated by direct fatty acid binding. Although GOT2 is dispensable for cancer cell proliferation in vivo, the GOT2–PPARδ axis promotes spatial restriction of both CD4+ and CD8+ T cells from the tumor microenvironment. Our results demonstrate a noncanonical function for an established mitochondrial enzyme in transcriptional regulation of immune evasion, which may be exploitable to promote a productive antitumor immune response.Significance:Prior studies demonstrate the important moonlighting functions of metabolic enzymes in cancer. We find that the mitochondrial transaminase GOT2 binds directly to fatty acid ligands that regulate the nuclear receptor PPARδ, and this functional interaction critically regulates the immune microenvironment of pancreatic cancer to promote tumor progression.See related commentary by Nwosu and di Magliano, p. 2237..This article is highlighted in the In This Issue feature, p. 2221

Supplementary Figure from A Cancer Cell–Intrinsic GOT2–PPARδ Axis Suppresses Antitumor Immunity

Supplementary Methods

PCR scheme employed for comparing relative levels of spliced and unspliced species.

Intron retention is not a sequencing or amplification artifact.

Supplementary Table 1. Normalized RPKM expression values and normalized Affymetrix microarray intensity values for each CRPC sample used for correlation analysis. Supplementary Table 2. Complete listing of high probability, protein-coding somatic mutations detected by RNA-seq in CRPC samples. Mutations are classified as either a nonsense or missense single nucleotide variant (SNV) or as a >1 base insertion (INS) or deletion (DEL). The gene symbol and Genbank ID is provided for each predicted amino acid change given. Supplementary Table 3. High confidence putative fusions detected by ChimeraScan (at least one breakpoint-spanning read) and deFuse (greater than 50% probability score) for each CRPC sample. Supplementary Table 4. RPKM values for approximately 45,500 transcripts for all 8 CRPC samples. Supplementary Table 5. Top 100 expressed genes as measured by the mean RPKM across all 8 CRPC samples. Supplementary Table 6. Top 100 expressed genes as measured by the median RPKM across all 8 CRPC samples. Supplementary Table 7. Novel candidate lncRNAs observed CRPC. List is sorted by average FPKM for expression of the lncRNA. Supplementary Table 8. Transcripts, gene names, and gene description for the 1,465 transcripts with the widest range of expression used for unsupervised clustering of TCGA and CRPC samples. The average RPKM difference between CRPC and TCGA primary prostate cancers is shown. Supplementary Table 9. RNA-SeQC mapping statistics for 8 CRPC RNA-seq samples. For each sample, the Illumina instrument model, RIN score, type of run, and the number of total and uniquely-mapping reads is shown. In addition, it is indicated which percentage of quality-filtered reads mapped to exon, intron or intergenic coordinates, the number of Ensembl genes determined as expressed, and the percentage of reads mapping to ribosomal RNA. Supplementary Tables 10-11. Intronic RPB values for genes in CRPC 49 and 66. Supplementary Table 12. Global analysis of splicing in CRPC. For each CRPC sample, the number of sequence reads uniquely mapping to an exon-exon splice junction or overlapping an exon-intron splice boundary is shown. The percentage of unspliced out of the total is indicated. Supplementary Table 13. Primer sequences for the detection of correctly or incorrectly spliced portions of AR, KLK2, KLK3, STEAP2, CPSF6, and CDK19. Supplementary Table 14. Complete results for ChimeraScan on CRPC samples. Supplementary Table 15. Complete results for deFuse on CRPC samples.

Clustering and pathway analysis of CRPC samples.

Positive correlation between gene expression platforms in CRPC samples.

Supplementary Methods and Materials

Supplementary Figure Legends

PHF20L1:LRRC6 fusion transcript detected in CRPC 66.

Supplementary Figure Legends

Men with metastatic prostate cancer who are treated with androgen deprivation therapies (ADT) usually relapse within 2 to 3 years with disease that is termed castration-resistant prostate cancer (CRPC). To identify the mechanism that drives these advanced tumors, paired-end RNA-sequencing (RNA-seq) was performed on a panel of CRPC bone marrow biopsy specimens. From this genome-wide approach, mutations were found in a series of genes with prostate cancer relevance, including AR, NCOR1, KDM3A, KDM4A, CHD1, SETD5, SETD7, INPP4B, RASGRP3, RASA1, TP53BP1, and CDH1, and a novel SND1:BRAF gene fusion. Among the most highly expressed transcripts were 10 noncoding RNAs (ncRNAs), including MALAT1 and PABPC1, which are involved in RNA processing. Notably, a high percentage of sequence reads mapped to introns, which were determined to be the result of incomplete splicing at canonical splice junctions. Using quantitative PCR (qPCR), a series of genes (AR, KLK2, KLK3, STEAP2, CPSF6, and CDK19) were confirmed to have a greater proportion of unspliced RNA in CRPC specimens than in normal prostate epithelium, untreated primary prostate cancer, and cultured prostate cancer cells. This inefficient coupling of transcription and mRNA splicing suggests an overall increase in transcription or defect in splicing.Implications: Inefficient splicing in advanced prostate cancer provides a selective advantage through effects on microRNA networks but may render tumors vulnerable to agents that suppress rate-limiting steps in splicing. Mol Cancer Res; 13(1); 98–106. ©2014 AACR.

Extensive intronic coverage in a subset of genes.

Heatmap representation ERG expression in CRPC samples.

High-confidence fusion transcripts detected in CRPC.

Supplementary Table Legends

Positive correlation between intron and exon expression in CRPCsamples.

Supplementary Figure S3 from BET Bromodomain Inhibition Blocks an AR-Repressed, E2F1-Activated Treatment-Emergent Neuroendocrine Prostate Cancer Lineage Plasticity Program

ROS production in MCL cells

ROS scavengers do not abrogate AZD5991-induced apoptosis

Supplementary Figure S2 from BET Bromodomain Inhibition Blocks an AR-Repressed, E2F1-Activated Treatment-Emergent Neuroendocrine Prostate Cancer Lineage Plasticity Program

Supplementary Dataset S1 shows the list of copy number gain genes.

Supplementary Data from BET Bromodomain Inhibition Blocks an AR-Repressed, E2F1-Activated Treatment-Emergent Neuroendocrine Prostate Cancer Lineage Plasticity Program

Purpose: Most studies characterizing antitumor properties of invariant natural killer T (iNKT) cells have used the agonist, α-galactosylceramide (α-GalCer). However, α-GalCer induces strong, long-lasting anergy of iNKT cells, which could be a major detriment for clinical therapy. A novel iNKT cell agonist, β-mannosylceramide (β-ManCer), induces strong antitumor immunity through a mechanism distinct from that of α-GalCer. The objective of this study was to determine whether β-ManCer induces anergy of iNKT cells.Experimental Design: Induction of anergy was determined by ex vivo analysis of splenocytes from mice pretreated with iNKT cell agonists as well as in the CT26 lung metastasis in vivo tumor model.Results: β-ManCer activated iNKT cells without inducing long-term anergy. The transience of anergy induction correlated with a shortened duration of PD-1 upregulation on iNKT cells activated with β-ManCer, compared with α-GalCer. Moreover, whereas mice pretreated with α-GalCer were unable to respond to a second glycolipid stimulation to induce tumor protection for up to 2 months, mice pretreated with β-ManCer were protected from tumors by a second stimulation equivalently to vehicle-treated mice.Conclusions: The lack of long-term functional anergy induced by β-ManCer, which allows for a second dose to still give therapeutic benefit, suggests the strong potential for this iNKT cell agonist to succeed in settings where α-GalCer has failed. Clin Cancer Res; 19(16); 4404–11. ©2013 AACR.

Supplementary Figure S4 from BET Bromodomain Inhibition Blocks an AR-Repressed, E2F1-Activated Treatment-Emergent Neuroendocrine Prostate Cancer Lineage Plasticity Program

Supplementary Table S2 S4 S6 S7 from BET Bromodomain Inhibition Blocks an AR-Repressed, E2F1-Activated Treatment-Emergent Neuroendocrine Prostate Cancer Lineage Plasticity Program

BAX overexpression re-sensitizes OCI-:Y3 cells to AZD5991

PDF file, 680K, Mice were injected i.p. with 2.4, 0.48, 0.24 or 0.048 nmol of a-GalCer or vehicle control. After 1 month, splenocytes were harvested and restimulated in vitro with 100 nM a-GalCer or vehicle. IFN-g in culture supernatants was measured after 48 hours of stimulation. Representatives of at least 2 replicate experiments are shown. Bars represent mean plus-minus SD; *, statistically significantly different from vehicle control stimulation; #, statistically significant from corresponding stimulation in vehicle pretreated group p

Supplementary Table S3 from BET Bromodomain Inhibition Blocks an AR-Repressed, E2F1-Activated Treatment-Emergent Neuroendocrine Prostate Cancer Lineage Plasticity Program

Purpose:The purpose of this study was to measure genomic changes that emerge with enzalutamide treatment using analyses of whole-genome sequencing and RNA sequencing.Experimental Design:One hundred and one tumors from men with metastatic castration-resistant prostate cancer (mCRPC) who had not been treated with enzalutamide (n = 64) or who had enzalutamide-resistant mCRPC (n = 37) underwent whole genome sequencing. Ninety-nine of these tumors also underwent RNA sequencing. We analyzed the genomes and transcriptomes of these mCRPC tumors.Results:Copy number loss was more common than gain in enzalutamide-resistant tumors. Specially, we identified 124 protein-coding genes that were more commonly lost in enzalutamide-resistant samples. These 124 genes included eight putative tumor suppressors located at nine distinct genomic regions. We demonstrated that focal deletion of the 17q22 locus that includes RNF43 and SRSF1 was not present in any patient with enzalutamide-naïve mCRPC but was present in 16% (6/37) of patients with enzalutamide-resistant mCRPC. 17q22 loss was associated with lower RNF43 and SRSF1 expression and poor overall survival from time of biopsy [median overall survival of 19.3 months in 17q22 intact vs. 8.9 months in 17q22 loss, HR, 3.44 95% confidence interval (CI), 1.338–8.867, log-rank P = 0.006]. Finally, 17q22 loss was linked with activation of several targetable factors, including CDK1/2, Akt, and PLK1, demonstrating the potential therapeutic relevance of 17q22 loss in mCRPC.Conclusions:Copy number loss is common in enzalutamide-resistant tumors. Focal deletion of chromosome 17q22 defines a previously unappreciated molecular subset of enzalutamide-resistant mCRPC associated with poor clinical outcome.

Figure S3 shows the prognostic significance of copy number loss of 17q24, 2q24, and 4q35 containing putative tumor suppressor genes.

Supplementary Table S1 from BET Bromodomain Inhibition Blocks an AR-Repressed, E2F1-Activated Treatment-Emergent Neuroendocrine Prostate Cancer Lineage Plasticity Program

Supplementary Dataset S2 shows the list of copy number loss genes.

Caspase blockade does not abrogate mitochondrial effects of AZD5991

Supplementary Figure S6 from BET Bromodomain Inhibition Blocks an AR-Repressed, E2F1-Activated Treatment-Emergent Neuroendocrine Prostate Cancer Lineage Plasticity Program

Purpose:Lineage plasticity in prostate cancer—most commonly exemplified by loss of androgen receptor (AR) signaling and a switch from a luminal to alternate differentiation program—is now recognized as a treatment resistance mechanism. Lineage plasticity is a spectrum, but neuroendocrine prostate cancer (NEPC) is the most virulent example. Currently, there are limited treatments for NEPC. Moreover, the incidence of treatment-emergent NEPC (t-NEPC) is increasing in the era of novel AR inhibitors. In contradistinction to de novo NEPC, t-NEPC tumors often express the AR, but AR's functional role in t-NEPC is unknown. Furthermore, targetable factors that promote t-NEPC lineage plasticity are also unclear.Experimental Design:Using an integrative systems biology approach, we investigated enzalutamide-resistant t-NEPC cell lines and their parental, enzalutamide-sensitive adenocarcinoma cell lines. The AR is still expressed in these t-NEPC cells, enabling us to determine the role of the AR and other key factors in regulating t-NEPC lineage plasticity.Results:AR inhibition accentuates lineage plasticity in t-NEPC cells—an effect not observed in parental, enzalutamide-sensitive adenocarcinoma cells. Induction of an AR-repressed, lineage plasticity program is dependent on activation of the transcription factor E2F1 in concert with the BET bromodomain chromatin reader BRD4. BET inhibition (BETi) blocks this E2F1/BRD4-regulated program and decreases growth of t-NEPC tumor models and a subset of t-NEPC patient tumors with high activity of this program in a BETi clinical trial.Conclusions:E2F1 and BRD4 are critical for activating an AR-repressed, t-NEPC lineage plasticity program. BETi is a promising approach to block this program.

Effect of AZD5991 on Bcl-2 family members

Purpose:Bcl-2 has been effectively targeted in lymphoid malignancies. However, resistance is inevitable, and novel approaches to target mitochondrial apoptosis are necessary. AZD5991, a selective BH3-mimetic in clinical trials, inhibits Mcl-1 with high potency.Experimental Design:We explored the preclinical activity of AZD5991 in diffuse large B-cell lymphoma (DLBCL) and ibrutinib-resistant mantle cell lymphoma (MCL) cell lines, MCL patient samples, and mice bearing DLBCL and MCL xenografts using flow cytometry, immunoblotting, and Seahorse respirometry assay. Cas9 gene editing and ex vivo functional drug screen assays helped identify mechanisms of resistance to Mcl-1 inhibition.Results:Mcl-1 was expressed in DLBCL and MCL cell lines and primary tumors. Treatment with AZD5991 restricted growth of DLBCL cells independent of cell of origin and overcame ibrutinib resistance in MCL cells. Mcl-1 inhibition led to mitochondrial dysfunction as manifested by mitochondrial membrane depolarization, decreased mitochondrial mass, and induction of mitophagy. This was accompanied by impairment of oxidative phosphorylation. TP53 and BAX were essential for sensitivity to Mcl-1, and oxidative phosphorylation was implicated in resistance to Mcl-1 inhibition. Induction of prosurvival proteins (e.g., Bcl-xL) in stromal conditions that mimic the tumor microenvironment rendered protection of primary MCL cells from Mcl-1 inhibition, while BH3-mimetics targeting Bcl-2/xL sensitized lymphoid cells to AZD5991. Treatment with AZD5991 reduced tumor growth in murine lymphoma models and prolonged survival of MCL PDX mice.Conclusions:Selective targeting Mcl-1 is a promising therapeutic approach in lymphoid malignancies. TP53 apoptotic network and metabolic reprogramming underlie susceptibility to Mcl-1 inhibition.

PDF file, 1823K, Mice were injected i.p. with 2.4 nmol (2 ug) of a-GalCer, b-ManCer or vehicle control. Splenocytes were harvested and restimulated in vitro with 100 nM a-GalCer, 500 nM b-ManCer or vehicle. IFN-g in culture supernatants was measured after 48 hours of stimulation. The concentrations of IFN-g in the supernatants (upper panels) and fold change over vehicle stimulated control to account for the variable backgrounds among groups (lower panels) at indicated time points are shown. Representatives of at least 2 replicate experiments are shown. Bars represent mean plus-minus SD; *, statistically significantly different from vehicle control stimulation; #, statistically significant from corresponding stimulation in vehicle pretreated group p

PDF file, 791K, Mice were injected i.p. with 2.4 or 12 nmol of b-ManCer, or 2.4 nmol of a-GalCer or vehicle control. After 6 weeks, splenocytes were harvested and restimulated in vitro with 100 nM a-GalCer, 500 nM of b-ManCer or vehicle. IFN-g and IL-4 in culture supernatants was measured after 48 hours of stimulation. Representatives of at least 2 replicate experiments are shown. Bars represent mean�SD; *, statistically significantly different from vehicle control stimulation; #, statistically significant from corresponding stimulation in vehicle pretreated group p

Supplementary Table S5 from BET Bromodomain Inhibition Blocks an AR-Repressed, E2F1-Activated Treatment-Emergent Neuroendocrine Prostate Cancer Lineage Plasticity Program

Figure S1 shows that copy number alterations of well-described prostate cancer tumor suppressor genes are not different between enzalutamide-naïve and -resistant tumors.

In vivo safety of AZD5991

Figure S2 shows a heatmap illustrating copy number alteration of the 124 protein-coding genes in individual samples.

Supplementary Table S8-S12 from BET Bromodomain Inhibition Blocks an AR-Repressed, E2F1-Activated Treatment-Emergent Neuroendocrine Prostate Cancer Lineage Plasticity Program

Supplementary Figure Legend contains the description of supplementary Figure S1-S4 and supplementary Datasets S1-S2.