Citations

Abstract Androgen receptor (AR) inhibition by androgen deprivation and/or antiandrogen administration is the mainstay therapy for advanced prostate cancer. However, most prostate cancers ultimately become resistant to these therapies, indicating the importance of identifying mechanisms driving resistance to improve patient outcomes. Here we demonstrated that acute treatment with the antiandrogen enzalutamide (ENZ) decreased glutathione (GSH) production, increased lipid peroxidation, and induced ferroptosis in prostate cancer cells. Consistently, meta-analysis of transcriptomic data linked the androgen-AR axis to metabolism-related biological processes, including lipid metabolism. The cystine transporter gene SLC7A11 was a key AR target, and full-length AR (AR-FL) transactivated SLC7A11 transcription by directly occupying the SLC7A11 promoter and putative enhancer regions. AR variants (AR-V) preferentially bound the SLC7A11 enhancer and upregulated SLC7A11 expression, thereby conferring resistance to ferroptosis induced by ENZ treatment. However, this effect was abolished following downregulation of AR-Vs using the dual CBP/p300 and BET inhibitor NEO2734. These findings reveal ferroptosis induction as an anticancer mechanism of antiandrogens and SLC7A11 as a direct target gene of AR-FL and AR-Vs. AR-V-mediated SLC7A11 expression represents a mechanism coupling ferroptosis resistance to prostate cancer progression. Significance: Upregulation of SLC7A11 can be induced by androgen receptor variants to inhibit antiandrogen-induced prostate cancer cell ferroptosis and to drive castration resistance in prostate cancer.

Histone‐lysine N‐methyltransferase SETD2 (SETD2), the sole histone methyltransferase that catalyzes trimethylation of lysine 36 on histone H3 (H3K36me3), is often mutated in clear cell renal cell carcinoma (ccRCC). SETD2 mutation and/or loss of H3K36me3 is linked to metastasis and poor outcome in ccRCC patients. Epithelial‐to‐mesenchymal transition (EMT) is a major pathway that drives invasion and metastasis in various cancer types. Here, using novel kidney epithelial cell lines isogenic for SETD2, we discovered that SETD2 inactivation drives EMT and promotes migration, invasion, and stemness in a transforming growth factor‐beta‐independent manner. This newly identified EMT program is triggered in part through secreted factors, including cytokines and growth factors, and through transcriptional reprogramming. RNA‐seq and assay for transposase‐accessible chromatin sequencing uncovered key transcription factors upregulated upon SETD2 loss, including SOX2, POU2F2 (OCT2), and PRRX1, that could individually drive EMT and stemness phenotypes in SETD2 wild‐type (WT) cells. Public expression data from SETD2 WT/mutant ccRCC support the EMT transcriptional signatures derived from cell line models. In summary, our studies reveal that SETD2 is a key regulator of EMT phenotypes through cell‐intrinsic and cell‐extrinsic mechanisms that help explain the association between SETD2 loss and ccRCC metastasis.

Abstract Purpose: Men with metastatic castration-resistant prostate cancer (mCRPC) frequently develop resistance to androgen receptor signaling inhibitor (ARSI) treatment; therefore, new therapies are needed. Trophoblastic cell-surface antigen (TROP-2) is a transmembrane protein identified in prostate cancer and overexpressed in multiple malignancies. TROP-2 is a therapeutic target for antibody–drug conjugates (ADC). Experimental Design: TROP-2 gene (TACSTD2) expression and markers of treatment resistance from prostate biopsies were analyzed using data from four previously curated cohorts of mCRPC (n = 634) and the PROMOTE study (dbGaP accession phs001141.v1.p1, n = 88). EPCAM or TROP-2–positive circulating tumor cells (CTC) were captured from peripheral blood for comparison of protein (n = 15) and gene expression signatures of treatment resistance (n = 40). We assessed the efficacy of TROP-2–targeting agents in a mouse xenograft model generated from prostate cancer cell lines. Results: We demonstrated that TACSTD2 is expressed in mCRPC from luminal and basal tumors but at lower levels in patients with neuroendocrine prostate cancer. Patients previously treated with ARSI showed no significant difference in TACSTD2 expression, whereas patients with detectable AR-V7 expression showed increased expression. We observed that TROP-2 can serve as a cell surface target for isolating CTCs, which may serve as a predictive biomarker for ADCs. We also demonstrated that prostate cancer cell line xenografts can be targeted specifically by labeled anti–TROP-2 agents in vivo. Conclusions: These results support further studies on TROP-2 as a therapeutic and diagnostic target for mCRPC.

Establishing causal links between inherited polymorphisms and cancer risk is challenging. Here, we focus on the single-nucleotide polymorphism rs55705857, which confers a sixfold greater risk of isocitrate dehydrogenase ( IDH) –mutant low-grade glioma (LGG). We reveal that rs55705857 itself is the causal variant and is associated with molecular pathways that drive LGG. Mechanistically, we show that rs55705857 resides within a brain-specific enhancer, where the risk allele disrupts OCT2/4 binding, allowing increased interaction with the Myc promoter and increased Myc expression. Mutating the orthologous mouse rs55705857 locus accelerated tumor development in an Idh1 R132H -driven LGG mouse model from 472 to 172 days and increased penetrance from 30% to 75%. Our work reveals mechanisms of the heritable predisposition to lethal glioma in ~40% of LGG patients.

Androgen receptor (AR) messenger RNA (mRNA) alternative splicing variants (AR-Vs) are implicated in castration-resistant progression of prostate cancer (PCa), although the molecular mechanism underlying the genesis of AR-Vs remains poorly understood. The CDK12 gene is often deleted or mutated in PCa and CDK12 deficiency is known to cause homologous recombination repair gene alteration or BRCAness via alternative polyadenylation (APA). Here, we demonstrate that pharmacological inhibition or genetic inactivation of CDK12 induces AR gene intronic (intron 3) polyadenylation (IPA) usage, AR-V expression, and PCa cell resistance to the antiandrogen enzalutamide (ENZ). We further show that AR binds to the CCNK gene promoter and up-regulates CYCLIN K expression. In contrast, ENZ decreases AR occupancy at the CCNK gene promoter and suppresses CYCLIN K expression. Similar to the effect of the CDK12 inhibitor, CYCLIN K degrader or ENZ treatment promotes AR gene IPA usage, AR-V expression, and ENZ-resistant growth of PCa cells. Importantly, we show that targeting BRCAness induced by CYCLIN K down-regulation with the PARP inhibitor overcomes ENZ resistance. Our findings identify CYCLIN K down-regulation as a key driver of IPA usage, hormonal therapy–induced AR-V expression, and castration resistance in PCa. These results suggest that hormonal therapy–induced AR-V expression and therapy resistance are vulnerable to PARP inhibitor treatment.

Abstract Introduction: Pediatric rhabdomyosarcoma (RMS) is the most common soft tissue tumor in children, with nearly 20% of children presenting with locally aggressive and/or metastatic disease. Despite aggressive chemotherapy and radiotherapy, the 5-year overall survival rate is still less than 40%. In addition, resistance to chemotherapy remains the major cause of cancer-related mortality. Therefore, there is an unmet need to develop more effective and tolerable therapeutic strategies for the treatment of RMS. Our overall objective is to leverage transcriptomic and proteomic profiling methods to identify RMS-specific surface proteins targetable with immune-based therapies. Methods: To identify therapeutic targets, we performed cell-surface capture and mass spectrometry on fusion-positive (FP), fusion-negative (FN) RMS and normal skeletal muscle. Transcriptomic datasets of RMS tumor specimens and normal tissue were employed to identify high-confidence RMS-specific cell-surface proteins. Expression of immune checkpoint molecules was confirmed by flow cytometry, western blot and PCR. Paraffin-embedded tissue sections were used to determine protein expression and prognostic value in RMS patients. We developed an in vitro T-cell killing assay to assess the functional role of immune checkpoint molecules on the antitumor immune response. Results: Surfaceome profiling of RMS cells revealed unique protein signatures specific for each tumor type and normal tissue. We have identified 5061 cell-surface proteins in RMS and 31.6% of proteins were correctly located at the plasma membrane. By integrating proteomic and transcriptomic data, we have identified 30 and 38 proteins significantly overexpressed in fusion-negative and fusion-positive RMS respectively compared to normal muscle. We have identified the immune checkpoint molecule B7-H3 as cell-surface protein overexpressed in both RMS subtypes. Strong expression of B7-H3 was also observed in patient’s tumor specimens. Genetic deletion of B7-H3 gene in RMS cells was associated with higher antitumor immune response in tumor cells co-cultured with CD8+-T cells. No difference was observed in tumor cell proliferation and radioresistance. Conclusions: Through integrated transcriptomic and proteomic profiling, we have discovered B7-H3 as a key regulator of tumor immune-evasion in RMS. B7-H3 is a suitable immunotherapy target for the treatment of RMS. Further studies are warranted to elucidate underlying mechanisms of B7-H3 function in RMS. Citation Format: Roxane R. Lavoie, Fabrice Lucien, Patricio C. Gargollo, Mohamed E. Ahmed, Yohan Kim, Emily Baer, Doris Phelps, Cristine M. Charlesworth, Benjamin J. Madden, Liguo Wang, Peter J. Hougthon, John Cheville, Haidong Dong, Candace F. Granberg. Surfaceome profiling of rhabdomyosarcoma reveals B7-H3 as a mediator of immune evasion [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 A036.

Abstract BACKGROUND Determination of the causation of germline single nucleotide polymorphisms (SNPs) located in non-coding regions of the genome is challenging. The genomic region of 8q24 has been identified as important in many kinds of cancer, linked to a topologically associated domain (TAD) encompassing MYC; this TAD contains a GWAS SNP (rs55705857) associated with IDH-mutant glioma. METHODS Germline genotyping data from 622 IDH-mutant glioma and 668 controls were used to fine map the rs55705857 locus by detailed haplotype analysis. Chromatin immunoprecipitation sequencing (ChIP-seq) of histone markers H3K4me1, H3K4me3, H3K27ac and H3K36me3 was performed on normal brain samples (n=8) and human glioma samples (n=11 IDH-wt and 52 IDH-mut). RNAseq from 9 normal and 83 brain tumors (n=26 IDH-wt and 55 IDH-mut) were used to assess differential gene expression. RESULTS Fine-mapping identified rs55705857 SNP as the most likely causative allele (OR=8.69; p<0.001) within 8q24 for the development of IDH-mutant glioma. At rs55705857, both H3K27ac and H3K4me1 in IDH-mutant vs IDH-wt tumors were increased 3.05- and 1.58-fold, respectively (DiffBind; p=5.81×10-7 and p=2.31×10-3). ChromHMM analysis of the marks indicated that promoter and enhancer functions were significantly increased, and the activity broadened at rs55705857 in IDH-mut gliomas compared to IDH-wt tumors and normal brain samples. This enhancement correlated with significant increased MYC expression in IDH-mut gliomas (p=3.1×10-13), as well as alterations of Myc signaling targets. Publicly available ATACseq, ChIPseq and long-range DNA interaction data demonstrated that the rs55705857 locus is open and interacts with the MYC promoter. CONCLUSIONS Fine-mapping of the 8q24 locus provided strong evidence that rs55705857 is the causative 8q24 locus associated with IDH-mut glioma. Functional experiments suggest that IDH mutation facilitates rs55705857 interaction with MYC to alter downstream MYC targets.

Novel therapeutic strategies are needed for the treatment of rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in children. By using a combination of cell surface proteomics and transcriptomic profiling of RMS and normal muscle, we generated a catalog of targetable cell surface proteins enriched in RMS tumors. Among the top candidates, we identified B7-H3 as the major immunoregulatory molecule expressed by RMS tumors. By using a large cohort of tissue specimens, we demonstrated that B7-H3 is expressed in a majority of RMS tumors while not detected in normal human tissues. Through a deconvolution analysis of the RMS tumor RNA-seq data, we showed that B7-H3-rich tumors are enriched in macrophages M1, NK cells, and depleted in CD8+-T cells. Furthermore, in vitro functional assays showed that B7-H3 knockout in RMS tumor cells increases T-cell mediated cytotoxicity. Altogether, our study uncovers new potential targets for the treatment of RMS and provides the first biological insights into the role of B7-H3 in RMS biology, paving the way for the development of next-generation immunotherapies.

Abstract Introduction: Pediatric rhabdomyosarcoma (RMS) is the most common soft tissue tumor in children, with nearly 20% of children presenting with locally aggressive and/or metastatic disease. Despite aggressive chemotherapy and radiotherapy, the 5-year overall survival rate is still less than 40%. In addition, resistance to chemotherapy remains the major cause of cancer-related mortality. Therefore, there is an unmet need to develop more effective and tolerable therapeutic strategies for the treatment of RMS. Our overall objective is to leverage transcriptomic and proteomic profiling methods to identify RMS-specific surface proteins targetable with immune-based therapies. Methods: To identify therapeutic targets, we performed cell-surface capture and mass spectrometry on fusion-positive (FP), fusion-negative (FN) RMS and normal skeletal muscle. Transcriptomic datasets of RMS tumor specimens and normal tissue were employed to identify high-confidence RMS-specific cell-surface proteins. Expression of immune checkpoint molecules was confirmed by flow cytometry, western blot and PCR. Paraffin-embedded tissue sections were used to determine protein expression and prognostic value in RMS patients. We developed an in vitro T-cell killing assay to assess the functional role of immune checkpoint molecules on the antitumor immune response. Results: Surfaceome profiling of RMS cells revealed unique protein signatures specific for each tumor type and normal tissue. We have identified 5061 cell-surface proteins in RMS and 31.6% of proteins were correctly located at the plasma membrane. By integrating proteomic and transcriptomic data, we have identified 30 and 38 proteins significantly overexpressed in fusion-negative and fusion-positive RMS respectively compared to normal muscle. We have identified the immune checkpoint molecule B7-H3 as cell-surface protein overexpressed in both RMS subtypes. Strong expression of B7-H3 was also observed in patient's tumor specimens. Genetic deletion of B7-H3 gene in RMS cells was associated with higher antitumor immune response in tumor cells co-cultured with CD8+-T cells. No difference was observed in tumor cell proliferation and radioresistance. Conclusion: Through integrated transcriptomic and proteomic profiling, we have discovered B7-H3 as a key regulator of tumor immune-evasion in RMS. B7-H3 is a suitable immunotherapy target for the treatment of RMS. Further studies are warranted to elucidate underlying mechanisms of B7-H3 function in RMS. Citation Format: Roxane R. Lavoie, Yohan Kim, Mohamed E. Ahmed, Doris Phelps, Emily Baer, Cristine Charlesworth, Benjamin Madden, Liguo Wang, Haidong Dong, Peter Houghton, John Cheville, Patricio Gargollo, Candace Granberg, Fabrice Lucien. Surfaceome profiling identifies the immune checkpoint molecule B7-H3 as a key player of immune-evasion in rhabdomyosarcoma [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 3048.

Abstract Androgen receptor (AR) is a major survival factor for prostate cancer. Inflammation is implicated in many cancer types, including prostate cancer. Activation of MAP3K7 (also termed TAK1) and downstream IκB kinase β (IKKβ) by proinflammatory cytokines such as TNFα stimulates NF-κB survival pathways. Paradoxically, MAP3K7 is often deleted in human prostate cancer. Here, we demonstrate that AR protein expression is lower in inflammatory tumor areas compared with non-inflammatory tissues in patients with prostate cancer. Map3k7 knockout increased AR protein levels and activity in the mouse prostate, and MAP3K7 and AR protein levels were inversely correlated in prostate cancer patient specimens. TNFα treatment increased AR protein ubiquitination and proteasomal degradation. Mechanistically, activation of IKKβ by TNFα induced phosphorylation and TRCP1/2 E3 ligase–mediated polyubiquitination and degradation of AR protein. TNFα suppressed prostate cancer proliferation, which could be rescued by blockade of AR degradation. These findings reveal a previously unrecognized tumor suppressive function of the inflammation-activated MAP3K7–IKKβ axis in degrading AR protein. Moreover, they suggest that aberrant elevation of AR protein could be a prognostic biomarker and therapeutic target for MAP3K7-deficient prostate cancer. Significance: This study identifies that MAP3K7–IKKβ signaling plays a tumor-suppressive role in prostate cancer by degrading AR, revealing potential prognostic and therapeutic strategies for MAP3K7-deficient tumors.