Spatial multi-omics uncovers distinct transcriptional and metabolic responses to AR-NTD degradation in CRPC

Rationale: Castration-resistant prostate cancer (CRPC) remains incurable with current androgen deprivation therapies.Mutations in the Androgen Receptor (AR), splice variants such as AR-V7 that retain the N-terminal domain (NTD) of AR, along with significant intratumoral heterogeneity sustain AR signaling and drive therapeutic resistance.Consequently, the persistent AR⁺/PSA⁺ subpopulations keep supporting tumor growth and facilitates lineage plasticity.Therefore, therapies capable of degrading both full-length AR and AR variants while reprogramming the broader tumor ecosystem are critically needed.Methods: A potent UroA analog, ASR603 (3-hydroxy-8-methoxy-6H-dibenzo[b,d]pyran-6-one), was synthesized that bind and degraded the AR-NTD in CRPC cells.In vivo efficacy was evaluated in 22Rv1 xenografts in both castrated and noncastrated mice, as well as in a patient-derived xenograft (PDX) model treated orally with ASR603.Mechanistic investigations employed imaging mass cytometry (CyTOF), Visium-based spatial transcriptomics, mass spectrometry-based ubiquitinomics and spatially resolved mass spectrometry imaging (MSI) based metabolomics, as well as glycomics to characterize AR-dependent subpopulations, ubiquitination sites, and metabolic rewiring.Results: ASR603 selectively inhibited the viability and anchorage-independent growth of AR⁺ and AR-V7⁺ CRPC cells, including enzalutamide-resistant cells, while sparing nonmalignant RWPE-1 cells.ASR603 reduced the AR, AR-V7, and PSA expression confirming specific inhibition of the AR signaling.In both castrated and noncastrated 22Rv1 xenografts as well as PDX model, oral ASR603 significantly inhibited tumor growth and decreased AR, PSA, and Ki-67 expression, demonstrating potent antitumor activity independent of systemic androgen levels.Single-cell imaging mass cytometry of PDX tumors demonstrated selective depletion of AR⁺/PSA⁺ luminal clusters, with concomitant reduction in HOXB13 expression but retention of E-cadherin.AR-low regions were characterized by high Hsp70, low Ki-67, and morphological disorganization, consistent with a stress-adapted, low-proliferative tumor state.Spatial transcriptomics identified an AR-low cluster with broad loss of AR targets and proliferation genes, as well as enrichment of autophagy, lysosomal, mTOR, and ubiquitin-mediated proteolysis pathways Mechanistic analyses demonstrated that ASR603 promotes ubiquitinproteasome mediated degradation of AR and AR-V7 via N-terminal ubiquitination sites (K301 and K313).ASR603 treatment was also associated with metabolic rewiring characterized by altered tryptophan and taurine related metabolites that inversely correlated with tumor burden.These findings indicate metabolic dysfunction in AR-depleted tumor regions.Conclusion: ASR603, an AR degrader, targeted N-terminal ubiquitination sites to eliminate AR⁺/PSA⁺ and AR-V7⁺ subpopulations thereby disrupting the AR-dependent luminal programs.These alterations induce an integrated stress response that reprograms CRPC tumors into a stress-adapted, metabolically dysfunctional, and structurally compromised state.These findings support AR-NTD directed degradation as a promising theranostic strategy for reshaping the CRPC tumor ecosystem and for defining metabolic response signatures in advanced prostate cancer.