ON-TARGETplus siRNA pools for gene expression knockdown.

Metastatic castration-resistant prostate cancer (mCRPC) remains the primary cause of prostate cancer-related mortality. Despite the availability of treatments, the molecular mechanisms underlying tumor invasion and metastasis are not fully understood, highlighting the need for novel therapeutic strategies. In this study, we developed fully human monoclonal antibodies (mAbs) that prevent the proteolytic cleavage of the transforming growth factor-beta (TGFβ) type I receptor (TβRI) by steric hindrance. This cleavage, mediated by the metalloprotease ADAM17 (a disintegrin and metalloprotease domain 17; also known as TACE), results in the generation of a soluble intracellular domain (TβRI-ICD) that is translocated to the nucleus of castration-resistant prostate cancer (CRPC) cells and promotes epithelial-to-mesenchymal transition (EMT), invasion, and metastasis. High levels of TGFBR1 correlated with poor survival in two independent clinical cohorts of patients with mCRPC, and a strong positive correlation between TGFBR1 and ADAM17 expression was observed. In a preclinical human orthotopic mCRPC mouse model, treatment with therapeutic mAbs effectively prevented the nuclear accumulation of TβRI-ICD, inhibited EMT, and suppressed tumor growth, invasion, and metastasis. Notably, the therapeutic effect was comparable to that of docetaxel, a current standard-of-care chemotherapy, without noticeable side effects on body weight, proximal aorta or heart function detected in immune-deficient mice. These findings suggest that targeting TβRI cleavage using specific mAbs is a novel precision medicine approach for the treatment of mCRPC. By selectively blocking the prometastatic activity of TβRI-ICD without disrupting physiological TGFβ signaling, this strategy may provide a safer and more effective alternative to existing therapies for advanced prostate cancer.

List of the 44 3D structures of HSC70 (HSPA8) in complex with the BAG domain of BAG-1 used in comparative structural analyses.

Antibodies used for western blot analyses.

Abstract Background: Transcription factor (TF) activity can be determined by nucleosome footprints in low-pass whole genome sequencing (lpWGS) of plasma cell-free DNA (cfDNA). cfDNA wrapped around nucleosomes is protected from enzymatic digestion, and TFs induce phased nucleosome positioning around their binding sites, which results in oscillatory sequencing coverage and preferential depletion at open chromatin regions. Herein, we quantify nucleosome occupancy at binding sites for hundreds of TFs, evaluating the clinical utility of these functional readouts in plasma taken from subjects treated on the CARD prospective randomized trial of cabazitaxel (CAB) vs second androgen receptor pathway inhibitor (ARPI). Methods: Plasma cfDNA lpWGS (median coverage ∼1.6x) was used to infer nucleosome footprints at TF binding sites (TFBS) of 682 TFs. Overall, 217 CARD trial subjects comprised a Test cohort and 174 patients receiving a taxane on the FIRSTANA and PROSELICA prospective trials comprised a validation set. cfDNA lpWGS of a cohort of 104 healthy participants was used as a control. Hazard ratios (HR) were computed using Cox proportional hazards models. Odds ratios (OR) were computed using logistic regression. Longitudinal changes between baseline and subsequent timepoints were investigated using linear mixed effect models. All analyses were adjusted for tumor fraction. Results: Out of 682 TFs with available binding site annotations, 357 consistently yielded signal-to-noise ratios of at least 2:1 and were further analyzed. Analytical validation utilizing technical replicates (different samples; same time point) and biological replicates (different samples, taken at baseline and screening, weeks apart) was pursued, determining the dynamic range for these TFs at 1000 TFBS each. Assay limits of detection (LOD) and limits of quantification (LOQ) for sequencing coverage and tumor fraction were determined for each TF. Of 357 TFs, 244 were significantly mCRPC associated (Wilcoxon test, adjusted p-value < 0.05) relative to healthy controls, including AR, NKX3-1, E2F1, MYC, MYCN, and MAZ. Predictive analysis in CARD showed accessibility at UBP1 binding sites at baseline was associated with taxane sensitivity over ARSI (OR 1.44, 95% 1.07-1.95, p-value < 0.05). Furthermore, average FOXP1 accessibility scores increased at progression (average increase 1.76, SE 0.49, p-value 0.02), alongside AR (average increase 1.16, SE 0.49, p-value 0.02) and GRHL2 scores (average increase 1.33, SE 0.53, p-value 0.01). These plasma-derived data suggest patients progress with increased AR signaling. Conclusions: Evaluating nucleosome footprints at TFBS using lpWGS is a robust approach that identifies valuable functional biomarkers of drug sensitivity. Such studies offer the opportunity to interrogate disease progression through serial clinical samples, where phenotype profiling is otherwise unfeasible. Citation Format: Denisa Bogdan, Jan Rekowski, George Seed, Claudia Bertan, Jane Goodall, Gemma Fowler, Penelope Flohr, Christine Geffriaud-Ricouard, Mustapha Chadjaa, Sandrine Mace, Isaac Lazzeri, Ellen Heitzer, Suzanne Carreira, Wei Yuan, Johann S. de Bono. Plasma cell-free DNA nucleosome footprints in metastatic castration-resistant prostate cancer (mCRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2585.

Thio-2 reduces genome-wide androgen receptor binding in LNCaP prostate cancer cells.

Synthego BAG-1 sgRNA for BAG-1 CRISPR knockout clones.

Development and characterization of BAG-1 knockout mice.

Development of castration resistant prostate cancer patient derived xenograft organoids.

Mouse organ hematoxylin and eosin, and BAG-1 immunohistochemistry, in BAG-1 knockout mice.

Abstract Purpose: Activation of the phosphoinositide 3-kinase/AKT (PI3K/AKT) signaling pathway promotes tumor immune evasion by suppressing effector T-cell infiltration and enhancing regulatory T-cell (Treg) activity, contributing to resistance to immune checkpoint inhibitors (ICI). Preclinical studies have demonstrated that inhibition of this pathway can restore antitumor immunity and synergize with PD-1/PD-L1 blockade. We explore the synergistic clinical potential of targeting the PI3K/AKT pathway in combination with atezolizumab to overcome immunotherapy resistance in recurrent glioblastoma (GBM) and advanced solid tumors. Patients and Methods: Phase Ib, investigator-initiated, open-label study (NCT03673787) composed of a proof-of-concept dose escalation part A of ipatasertib plus atezolizumab in a 3 + 3 design. Adult patients with treatment-refractory advanced cancers were enrolled into cohort A1 and recurrent GBM into cohort A2. Part B enrolled patients into six exploratory cohorts. The study aims to evaluate the safety, immune-modulatory effects, and preliminary efficacy of the combination of ipatasertib with atezolizumab. Results: The combination was well tolerated, with no dose-limiting toxicities at the recommended phase II dose of ipatasertib 400 mg daily plus atezolizumab 1,200 mg every 3 weeks. Pharmacodynamic analysis demonstrated depletion of FOXP3+ Tregs and increased infiltration of CD8+ effector T cells within the tumor microenvironment (TME). Durable exceptional responses were seen in some patients with treatment-refractory or recurrent GBM. Conclusions: This is the first report in clinical samples showing that ipatasertib efficiently depletes FOXP3+ Tregs and results in increased infiltration of effector CD8+ T cells in the TME. This was associated with preliminary efficacy in a subset of patients with treatment-refractory GBM.

Despite recent therapeutic advances, advanced prostate cancer (PCa) remains lethal as tumors develop resistance to current treatments. Novel and more effective therapeutic strategies to induce cell death in these tumors are urgently needed. Our group recently reported that NXP800, a drug in clinical development, drives unfolded protein response (UPR) and targets AR and E2F, decreasing the growth of castration-resistant PCa (CRPC) models in vitro and in vivo. BH3 mimetics are small molecules that inhibit antiapoptotic BCL-2 family proteins, thereby promoting apoptosis, and have shown particular promise in hematological malignancies. However, their efficacy in CRPC has been limited, likely due to functional redundancies among antiapoptotic proteins such as MCL1, BCLXL, and BCL2. We investigated the potential of combining NXP800 with BH3 mimetics targeting MCL1 (S63845) or BCLXL (A-1331852) to drive cell death by inducing the intrinsic apoptosis pathway in CRPC models. Cell viability and caspase 3/7 activity were assessed by luminescence assays, while additional apoptosis markers were evaluated by western blot following treatment with NXP800, S63845, and A-1331852, as single agents or in combination. To identify key mediators of the synergistic effects, an siRNA screen targeting BH3-only proteins was performed in CRPC cells before treatment with the single agents or their combination. To assess the molecular consequences of NXP800 treatment in vivo, RNA-seq was performed on tumors from CRPC-bearing mice treated with NXP800 (35 mg/kg daily for 5 days), with particular focus on genes involved in the intrinsic apoptosis pathway. NXP800 synergized with MCL1 and BCLXL inhibitors in CRPC cells, inducing apoptosis as evidenced by caspase 3/7 activation and PARP cleavage. Co-silencing of the mitochondrial pore–forming proteins BAX and BAK, as well as treatment with the pan-caspase inhibitor Q-VD-OPh, prevented cell death induced by NXP800 in combination with BH3 mimetics, indicating that the effect is caspase-dependent and involves activation of the intrinsic apoptosis pathway. Blocking NXP800-induced eIF2α phosphorylation using ISRIB abolished the synergistic effect observed with BH3 mimetics. Thapsigargin, which induces the unfolded protein response via SERCA inhibition, recapitulated the synergy and triggered apoptosis in combination with BH3 mimetics. RNA-seq analysis of LNCaP95 xenograft tumors treated with NXP800 revealed induction of specific BH3-only proteins whose silencing (in vitro) prevented caspase 3/7 activation and abolished the synergistic cell death observed with NXP800 in combination with MCL1 or BCLXL inhibition. NXP800 sensitizes CRPC cells to BH3 mimetics by inducing UPR and dysregulating BH3-only proteins. These findings highlight the potential of combining UPR-inducing agents with BH3 mimetics as a therapeutic strategy in CRPC. Juan M. Jiménez-Vacas, Jonathan Welti, Denisa Bogdan, Ines Figueiredo, Bora Gurel, Wanting Zeng, Tomas Goldsmith, Souvik Das, Joe Taylor, Nicholas Waldron, Claudia Bertan, Suzanne Carreira, Wei Yuan, Paul Workman, Steven P. Balk, Johann de Bono, Adam Sharp. Induction of the unfolded protein response unveils a vulnerability of advanced prostate cancer cells to BH3 mimetics [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Prostate Cancer Research and Treatment; 2026 Jan 20-22; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(2_Suppl):Abstract nr B032.

New androgen receptor (AR) pathway inhibitors (ARPIs) in clinical development, including AR degraders and CYP11A inhibitors, largely target ligand-dependent AR activation and have reported antitumor activity in metastatic castration-resistant prostate cancer (mCRPC) resistant to established ARPIs, predominately against tumors with AR mutations. We hypothesized that AR-mutated mCRPC exhibits lower AR splice variant 7 (AR-V7) expression and remains full-length-AR (FL-AR) driven, explaining, in part, the antitumor activity of these AR ligand-binding domain (LBD) targeting drugs. The data herein demonstrate that mCRPC tissue biopsies with detectable AR mutations express significantly lower levels of AR-V7 protein and associate with better overall survival and enhanced sensitivity to ARPIs. This is independent of differences in the total number of global splicing events but may be related to differences in splicing factor expression between AR-mutated and nonmutated mCRPC. In conclusion, AR-mutated mCRPC frequently exhibits low AR-V7 expression, arguably explaining the enhanced sensitivity to ARPIs observed in these cancers. Consequently, AR mutation status may serve as a biomarker to predict response to AR-directed therapies.

Mouse organ hematoxylin and eosin, and BAG-1 immunohistochemistry, in BAG-1 knockout mice.

TaqMan probes for qRT-PCR analyses.

Treatment of locally advanced and metastatic prostate cancer (PC) with androgen receptor-targeting (AR-targeting) therapies has limited durability, with disease eventually progressing to castrate-resistant PC (CRPC). Constitutively active AR splice variants (AR-Vs), such as AR-V7, play a key role in driving treatment resistance and disease progression. Importantly, the failure to attenuate AR-V function represents a major unmet clinical need, and as such, defining how AR-Vs are generated is likely to yield new therapeutic targets. Our knowledge of factors that mediate splicing of AR-V-encoding mRNAs remains limited. Here, we have employed an RNA-targeting CasRx approach to identify selective protein interactors of AR-V7 mRNA in PC. TRA2B and its ortholog, TRA2A, were identified as splicing regulators of AR transcripts that facilitate AR-V synthesis at the expense of full-length AR isoforms. TRA2B expression correlated with AR-V7 transcript in CRPC and attenuation of TRA2-mediated splicing diminished PC cell growth. Exploiting TRA2B function may therefore provide new therapeutic opportunities in advanced disease.

Thio-2 downregulates C-MYC expression through a BAG-1 independent mechanism.