Sun, Rui

Immunotherapy has been effective in many cancer types but has failed in multiple clinical trials in prostate cancers, with the underlying mechanisms remaining largely unclear. Here, we demonstrate that androgen receptor pathway inhibitor (ARPI) plus irradiation (IR) triggered robust anticancer immunity in prostate cancers in both patients and mice. We show that androgen-activated AR suppressed innate immune signaling by inducing inhibitor of nuclear factor kappa-B kinase subunit epsilon ( IKBKE ) gene repression through HDAC2 interaction with an IKBKE enhancer RNA ( IKBKE eRNA, or IKBKE-e ). ARPI treatment caused IKBKE derepression and enhanced an IR-induced innate immune response via action of RIG-I and MDA5 dsRNA sensors. IKBKE-e ablation largely enhanced innate immunity in prostate cancer cells in culture and anticancer immunity in mice. Our results revealed AR, HDAC2, and IKBKE eRNA as critical intrinsic immune suppressors in prostate cancer cells, suggesting that rejuvenating inhibitor of nuclear factor kappa-B kinase subunit epsilon (IKKε) signaling by targeting IKBKE-e is an actionable strategy to elicit synthetic anticancer immunity in immunologically “cold” cancers such as prostate cancer.

Abstract CDK12 is frequently deleted and/or mutated in human cancers, and CDK12 inactivation is associated with aggressive phenotypes, including the tendency to metastasis. In this study, we showed that CDK12 deficiency induced intronic polyadenylation (IPA) usage in BRD4 to upregulate expression of the bromodomain-containing protein 4 (BRD4) short isoform (BRD4S). BRD4S demonstrated an enhanced capacity to form phase separation despite lacking the intrinsically disordered region located in the C-terminal of full-length BRD4. Oligomerization of BRD4S mediated phase separation through a base-interacting structural domain. CUT&Tag and RNA sequencing analyses revealed that BRD4S preferentially bound to the promoters of TGFβ signaling genes, including TGFB2 and LTBP1, driving their expression. Consistently, BRD4S drove cancer cell dissemination in vitro and metastasis in vivo, which could be blocked by BET or TGFβ signaling pathway inhibitors. Overall, this study identifies IPA usage at the BRD4 gene locus induced by CDK12 deficiency as a mechanism responsible for BRD4S genesis, which represents an important mechanism driving cancer metastasis and a potential target for therapeutic intervention in CDK12-deficient cancers. Significance: The CDK12–BRD4S–TGFβ signaling axis represents a key regulatory module that promotes metastatic progression and can be targeted by BET or TGFβ pathway inhibitors for treating CDK12-deficient cancers.

CDK12 primarily functions as a transcription regulatory cyclin-dependent kinase (CDK) that controls mRNA elongation, splicing, and polyadenylation. The CDK12 gene is implicated in human cancers since it is frequently mutated and/or deleted in prostate and ovarian cancer but paradoxically amplified in breast cancer. Here, we demonstrate that CDK12 promotes serine-933 phosphorylation of DNA2, a nuclease/helicase critical for replication fork stress regulation, and the phosphorylation subsequently facilitates DNA2 polyubiquitination and degradation mediated by the APC/C CDC20 E3 ubiquitin ligase. CDK12 inactivation induces but amplification suppresses genome-wide expression of interferon response and antigen processing and presentation machinery genes in ovarian and breast cancer cells, respectively. Besides causing aberrant DNA2 stabilization, replication stress, genomic instability, and cytosolic double-stranded DNA (dsDNA) accumulation, CDK12 loss also triggers cGAS–STING activation and innate immune response, which can be reversed by forced expression of replication protein A (RPA) subunits or DNA2 depletion. Our findings identify DNA2 as a phosphorylation substrate of CDK12, connecting CDK12 to cell cycle regulation. These data also reveal DNA2 protein destruction as a critical mechanism that dictates genomic instability, cGAS–STING signaling activation, and innate immune response in CDK12-deregulated cancers.

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.

Abstract Retinoblastoma (RB) protein can exert tumor suppressor functions even when it becomes phosphorylated. It is thus essential to understand how phosphorylated RB (p-RB) expression and function are regulated. Here, we demonstrated that RING finger domain protein TRIM28 bound and promoted ubiquitination and degradation of CDK4/6-phosphorylated RB protein. SETDB1, a known TRIM28 binding partner, protected p-RB from degradation through the binding of methylated RB by its Tudor domain independent of its methyltransferase activity. SETDB1 was found to be frequently overexpressed due to gene amplification and positively correlated with p-RB in prostate cancer patient specimens. Inhibition of SETDB1 expression using a gene-specific antisense oligonucleotide (ASO) reduced tumor growth but accelerated RB protein degradation, limiting the therapeutic efficacy. However, coadministration of the CDK4/6 inhibitor palbociclib blocked ASO-induced RB degradation and resulted in a much greater cancer-inhibitory effect than each inhibitor alone both in vitro and in vivo. This study identified CDK4/6-dependent, TRIM28-mediated proteasomal degradation as a mechanism of RB inactivation and reveals SETDB1 as a key inhibitor of this process. Our findings suggest that combined targeting of SETDB1 and CDK4/6 represents a viable approach for the treatment of cancers with SETDB1 gene amplification or overexpression. Significance: The identification of a role for TRIM28 and SETDB1 in regulating CDK4/6-phosphorylated RB stability uncovers a combination strategy using CDK4/6 and SETDB1 inhibition to decrease RB degradation and inhibit cancer growth.

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.

Significance The multiple-domain protein ZMYND8 is well known as a chromatin reader that recognizes methylated, acetylated, and/or mutated histones. Here we report that ZMYND8 is overexpressed in human ccRCC, and that it preferentially binds T487-phosphorylated EZH2. We show that in hypoxia-stimulated or von Hippel–Lindau–deficient cells, ZMYND8 binding of EZH2 is important for maintenance of EZH2 phosphorylation level, inhibition of PRC2 complex formation, and enhanced interaction between EZH2 and FOXM1. Our results reveal a reader function of ZMYND8 in recognizing a phosphorylation “code” on a nonhistone protein. Our findings also identify ZMYND8 binding as a facet of regulation and function of EZH2 phosphorylation in the Polycomb-dependent to -independent switch of EZH2/PRC2 activities.