Htoo Zarni Oo

GUNS (NCT04812366) is a multi-centre adaptive umbrella phase-II neoadjuvant trial evaluating 24 weeks of biomarker-directed therapy on major pathologic response (MPR) and residual cancer burden (RCB) in men with high-risk localized prostate cancer (HR-LPC) undergoing radical prostatectomy. MPR was defined by ≤ 5 mm longest diameter of the dominant focus or viable RCB ≤ 1 mm ² . Diagnostic biopsies underwent Tempus’ 648-gene DNA and whole-transcriptome RNA sequencing (seq). After 8 weeks of androgen receptor pathway inhibitor (ARPI) doublet therapy with androgen deprivation (ADT) plus apalutamide (APA), men were assigned to sub-protocols (SPs) defined by genomic alterations ^(alt) for an additional 16 weeks of an ARPI-doublet with or without additional therapy. SP-1 randomized men with AR-associated (ETS fusions, FOXA1 , SPOP ) or otherwise favourable genomic alterations to continue ARPI-doublet (SP-1A) versus triplet therapy that includes another ARPI, abiraterone (SP-1B). SP-2 randomized men with aggressive genomic alterations ( RB1 , PTEN , TP53 , AKT1 ) to ARPI-doublet (SP-2A) versus triplet therapy with docetaxel chemotherapy (SP-2B). This analysis reports genomic and pathologic response data for 100 evaluable men enrolled in SP-1 and 48 in SP-2, out of 322 participants enrolled in GUNS. DNA-seq revealed a genomic landscape dominated by ETS gene fusions (41%), FOXA1 (22%), TP53 (19%), PTEN (15%), and SPOP (12%) alterations. In SP-1, MPR was significantly higher in patients treated with triplet (SP-1B) versus doublet (SP-1A) therapy (58% vs. 32%, p = 0.0045) and overall RCB was lower (median 0.73 vs. 2.79 mm ² , p = 0.012). In SP-2, MPR was significantly higher with ARPI-docetaxel triplet (SP-2B) versus doublet (SP-2A) therapy (25% vs. 0%, p = 0.011) with a 4.6-fold reduction in median RCB (7.6 vs. 34.6 mm ² , p = 0.083). Secondary endpoints included ypN1 rates, which trended lower in SP-1B vs. SP-1A (14% vs. 28%) and SP-2B vs. SP-2A (29% vs. 46%). ERG fusions tended to have higher RCB, and SPOP ^alt and FOXA1 ^alt lower RCB, in SP-1, while PTEN-deficient tumours with a concurrent ETS fusion had the worst pathologic outcomes in SP-2. Transcriptomically, baseline AR signalling was strongly associated with lower RCB following ARPI-only combinations, while immunological signatures were unfavourable, associations that were not seen with docetaxel in SP-2B. In conclusion, genomic-biomarker-directed ARPI-triplet therapies improve MPR rates and RCB distributions in men with HR-LPC, supporting further validation in trials to evolve selection of treatment intensification strategies.

Tumors must adapt to high levels of endoplasmic reticulum (ER) stress to sustain tumor growth and metastases. The chaperone GRP78 (BiP/HSPA5) is a key component of the unfolded protein response (UPR) and essential for ER stress management and adaptive signaling supporting pro-survival UPR activities. Here, we report that oncofetal chondroitin sulfate (CS) glycosaminoglycans are required for ER stress adaptation in osteosarcoma. When osteosarcoma cells encounter ER stress, they upregulate 4- O -sulfated CS at the expense of other glycosaminoglycans leading to a reconfiguration of the glycocalyx in favor of an oncofetal CS subtype. Genetic ablation of the CS synthesis pathway impairs the UPR by preventing osteosarcoma cells from mounting GRP78 expression in response to ER stress. CS deficiency makes osteosarcoma cells hypersensitive to inhibition of GRP78 under both ambient and ER stress conditions, and acute ER stress drives CS-defective osteosarcoma cells into an apoptotic cell death that can be rescued by re-instating CS 4- O -sulfation capacity. This has direct implications for the metastatic progression of osteosarcoma, whereby oncofetal CS protects dissociated osteosarcoma cells from anoikis to allow pulmonary colonization in mice. Our data identify CS glycosaminoglycans as a critical component of the UPR that permits osteosarcoma cells to manage ER stress.

Table S2. Biological characteristics and genetic background of PDX lines

Fig. S4. Stroma accumulation and ECM remodeling coincided with VTCN1 upregulation in response to reduced AR activity in post-NHT patients with prostate cancer.

Fig. S3. Lack of correlation between B7-H4 expression and immune activity in post-NHT prostate cancer.

Fig. S1. scRNA-seq analysis of human prostate cancer cells from the LTL331 PDX model showing VTCN1 expression across all 17 clusters.

Table S1. Baseline patient characteristics of TMA samples

Fig. S2. VTCN1 (mouse B7-H4) expression enriched in post-castration prostate tissue in a normal mouse prostate tissue castration model.