Supplementary Table 2. Representativeness of Study Participants

Supplementary Table 3. Genetic variant detected in the custom gene panel through whole exome sequencing.

and Objectives: Metastatic castration-sensitive prostate cancer (mCSPC) is a clinically heterogeneous disease in which genetic mutations and tumor burden influence patient outcomes. Mutations in some DNA repair genes (e.g. BRCA1/2 and ATM) have been related to prostate cancer development and progression and may serve as therapeutic targets. The GENPRO12 study aims to evaluate the mutational profiles in non-mCSPC and mCSPC patients and to explore its clinical associations. This preliminary report includes 109 mCSPC patients, stratified by synchronous/metachronous disease. Each group was further classified into risk/volume (high/low) categories based on CHAARTED and LATITUDE criteria. Tumor samples were collected and analyzed using next-generation sequencing (NGS) ACHILLES test (Olmos et al. Ann Oncol 2024) to identify mutations in 40 genes, including BRCA1/2, ATM, and other prostate cancer-related genes. Clinicopathological data were extracted from medical records. Median age at diagnosis of mHSPC was 72 (range 47-92) with a median PSA 46.9 (0.3-3956). 90 patients had synchronous metastatic disease, 69 were CHAARTED high-volume, 76 were LATITUDE high-risk, and 25 had visceral metastases (17 in the lung only). All but 11 patients had at least 1 alteration in the genes analyzed. The most relevant altered genes are listed in the table. BRCA2, PTEN, RB1 Loss of Function (LoF) and MYC gain were more frequent in de novo and/or high-volume cases. Genetic alterations with prognostic and/or predictive value can be detected using prostate-specific NGS assays on archived diagnostic FFPE samples. Their prevalence may vary based on mCSPC clinical presentation, reflecting distinct disease biologies. Updated results, including outcomes and treatment responses, will be presented at the meeting. María Ovejero-Sánchez, Ana Jambrina, Ignacio González-Ginel, Daniel Tello, Manuel Balongo, Ana Gutierrez-Pecharroman, Pedro P López-Casas, Alfredo Rodríguez-Antolín, Elena Castro, David Olmos. Mutation prevalence in metastatic castration-sensitive prostate cancer based on metastasis timing and risk stratification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5787.

Neuroendocrine prostate cancer (NEPC) is a highly aggressive variant of prostate cancer with poor clinical outcomes and limited treatment options, highlighting the urgent need for therapies targeting its unique molecular profile of ecubectedin, a novel inhibitor of transcription from the ecteinascidin’s family, in both in vitro and in vivo models of NEPC. Methodology: Human NEPC cells, NCI-H660, along with eight NE-SCLC cell lines (as the well-known NE model) were treated with ecubectedin for 72 hours. Cell viability was assessed using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) short-term assay. Treatment-induced changes in gene expression profiles were analyzed by bulk RNA sequencing. In vivo antitumoral activity of ecubectedin was evaluated in in two human-derived NEPC models: H660 (cell-derived xenograft or CDX) and CP-IBI-034 (a transformed NEPC patient-derived xenograft or PDX obtained from a liver metastasis, confirmed histologically). Both H660 and CPI-IBI-034 were implanted subcutaneously (s.c.) in athymic nu/nu mice. Ecubectedin/placebo was intravenously (i.v.) administered (at 0.75 mg/kg) on days 0, 7 and 14 in n=6-8 mice per arm and model. Results: Ecubectedin markedly reduced NCI-H660 cell viability in similar proportions to NE-SCLC, with an average IC50 of 2.3 nM. At a transcriptional level, ecubectedin induced significant downregulation of key NE regulators, including ASCL1, NEUROD1, POU3F2, MYCN, EZH2 and SOX2, in both NEPC and NE-SCLC. In CP-IBI-034 tumor bearing mice, ecubectedin significantly reduced (vs placebo) the tumor volume during the experimental phase (up to Day 35) with a 22% of ecubectedin-treated mice resulting in complete tumor remissions. In H660 xenografts, a significantly antitumor activity (vs placebo) was also observed with complete tumor remissions in 100% of mice that lasted until the end of the experiment (Day 91). Conclusions: Ecubectedin may impact transcriptional networks driving the cell plasticity and NE transformation in NEPC tumors. More importantly, a significant in vitro and in vivo antitumor activity of ecubectedin was observed in NEPC models, suggesting a significant potential as a promising novel therapeutic for this indication associated to very poor outcomes. Laura Almalé, Maria José Guillen, Paula Diaz, Ismael Fernández-Miranda, Paula Iglesias, Marcelo Lima Ribeiro, Maria Ovejero-Sánchez, Elena Castro, Pedro P. López-Casas, Carmen Cuevas, Pablo Avilés, David Olmos. Ecubectedin's role in targeting transcriptional regulators in neuroendocrine prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1771.

Supplementary Table 4. Preclinical model recommendation

PURPOSE:Pancreatic ductal adenocarcinoma (PDAC) has limited treatment options. We compared the efficacy of comprehensive precision medicine against that of the conventional treatment in PDAC. PATIENTS AND METHODS:We report a phase III trial of advanced PDAC in which patients were randomized (1:2) to a conventional treatment treated at physician's discretion (arm A) or to precision medicine (arm B). Subjects randomized to arm B underwent a tumor biopsy for whole-exome sequencing and to generate avatar mouse models and patient-derived organoids for phenotypic drug screening, with final treatment recommended by the molecular tumor board. The primary objective was median overall survival (OS). RESULTS:A total of 137 patients were enrolled with 125 randomized, 44 to arm A and 81 to arm B. Whole-exome sequencing was performed in 80.3% (65/81) patients of arm B, with potentially actionable mutations detected in 21.5% (14/65). Experimental models were generated in 16/81 patients (19.8%). Second-line treatment was administered to 39 patients in the experimental arm, but only four (10.2%) received personalized treatment, whereas 35 could not receive matched therapy because of rapid clinical deterioration, delays in obtaining study results, or the absence of actionable targets. The median OS was 8.7 and 8.6 months (P = 0.849) and the median progression-free survival was 3.8 and 4.3 months (P = 0.563) for the conventional and experimental arms, respectively. Notably, the four patients who received personalized treatment had a median OS of 19.3 months. CONCLUSIONS:Personalized medicine was challenging to implement in most patients with PDAC, limiting the interpretation of intention-to-treat analysis. Survival was improved in the subset of patients who did receive matched therapy.

Supplementary Table 5. Complete Efficacy Data

Supplementary Table 1. Custom virtual gene panel included in the whole exome sequencing analysis

Supplemental Figure 1. Glesatinib is a potent and selective MET inhibitor. Supplemental Figure 2. Waterfall plot of glesatinib activity across pre-clinical tumor models. Supplemental Figure 3. SNU-638 Criz-res line is homogeneous and harbors the F1200L and Y1230H resistance mutations on the same allele. Supplemental Figure 4. Long term treatment of a METex14 del mutant and amplified PDX model (PULM-039) leads to capmatinib and crizotinib-resistant tumors but not glesatinib-resistant tumors. Supplemental Figure 5. Molecular basis for resistance that arises from the G1163R mutation. Supplemental Figure 6. Glesatinib is active against type I MET inhibitor-resistance mutations in a spheroid growth assay.

Supplemental Figure 3. A) iRGD-TAMRA (red) binding after 15 min at 4 degree in normal cells (Low integrins-NRP1) and in human and mice PDAC cells (High integrins-NRP1). Scale bars 200μm B) qRT-PCR of miR-21-5p expression after treatment of PANC1, BxPC3 and PL-45 cells with TPN-21 at the dose of 100nM after 48h. C) Representation of 3D-model treatment course with TPN-21. For qPCR each miRNA sample was normalized on the basis of its 18s content. Error bars, mean {plus minus} s.d *P = 0.01-0.05; **P = 0.001-0.01; ***P < 0.001; ****P < 0.0001 N.S., not significant, two-tailed t-test; n = 3 biological replicates.

Abstract Purpose: Since drug responses vary between patients, it is crucial to develop pre-clinical or co-clinical strategies that forecast patient response. In this study, we tested whether RNA-based therapeutics were suitable for personalized medicine by using patient-derived-organoid (PDO) and patient-derived-xenograft (PDX) models. Experimental Design: We performed microRNA (miRNA) profiling of PDX samples to determine the status of miRNA deregulation in individual pancreatic ductal adenocarcinoma (PDAC) patients. To deliver personalized RNA-based-therapy targeting oncogenic miRNAs that form part of this common PDAC miRNA over-expression signature, we packaged antimiR oligonucleotides against one of these miRNAs in tumor-penetrating nanocomplexes (TPN) targeting cell surface proteins on PDAC tumors. Results: As a validation for our pre-clinical strategy, the therapeutic potential of one of our nano-drugs, TPN-21, was first shown to decrease tumor cell growth and survival in PDO avatars for individual patients, then in their PDX avatars. Conclusions: This general approach appears suitable for co-clinical validation of personalized RNA medicine and paves the way to prospectively identify patients with eligible miRNA profiles for personalized RNA-based therapy. Clin Cancer Res; 24(7); 1734–47. ©2018 AACR.

Supplemental Figure 2. A) Relative expression Left panel: Mean of fluorescence and right panel: qPCR validation of miR-21-5p expression level in the set of cell lines, 3D-models and organoid compared to normal controls. B) qPCR analysis of PDCD4 and PTEN expression levels in the PANC1 stable cell lines (Lenti-21) inhibiting miR- 21 activity (pLenti-III-miR-off). C) qRT-PCR of miR-21-5p expression after lipofectamine transfection of PANC1, BxPC3 and PL-45 cells with anti-miR-21 inhibitor (mirVana ®) at the dose of 50nM after 48h. For qPCR each miRNA sample was normalized on the basis of it's 18s content and on the basis of GAPDH for mRNAs.

5003 Background: Understanding of the association between HRR mutations and outcomes in mCRPC pts is limited. This analysis investigated the prevalence and outcomes of pts with/out HRR mutations (somatic and/or germline), stratified as BRCA, non-BRCA, or HRR non-BRCA, who initiated 1L mCRPC treatment with novel hormonal therapy (NHT) or taxane. Methods: Eligible pts from PROREPAIR-B (NCT03075735), PROSENZA (NCT02922218), PROSTAC (NCT02362620), and PROSABI (NCT02787837) studies underwent paired somatic/germline DNA analyses using a custom NGS panel that included ATM, BRCA1, BRCA2, BRIP1, CDK12, CHEK2, FANCA, HDAC2, PALB2, RAD51B, and RAD54L. Those with pathogenic (likely) mutations in ≥1 allele of ≥1 gene were considered deficient (HRR). rPFS, PFS2, and OS were reported for BRCA, non-BRCA, and HRR non-BRCA subgroups; associations between mutations and outcomes were assessed using inverse probability weighted Cox models, which balanced the baseline (BL) characteristics between subgroups. Hazard ratios (HRs) with 95% confidence intervals (CIs) and p values are presented. Results: Of 729 pts, 223 (30.6%) were HRR, including 96 (13.2%) BRCA. 60.4% of pts were treated with NHT and 39.6% with taxane in 1L; 80.7% received at least second-line treatment. Median age at BL was 72.2 years, 63.5% had Gleason >7, 53.1% had ECOG ≥1, and 13.3% presented with visceral metastases. BL characteristics after adjustment were similar (standard mean difference: <0.1 in HRR and maximum 0.11 for all pts). BRCA pts had significantly worse rPFS, PFS2, and OS than non-BRCA pts; BRCA pts also had significantly worse PFS2 and OS than HRR non-BRCA pts. There were no significant differences between the outcomes of somatic and germline BRCA pts. Conclusions: BRCA pts, regardless of somatic/germline origin, had significantly worse rPFS, PFS2, and OS than the total non-BRCA population and significantly worse PFS2 and OS than non-BRCA pts in the HRR subgroup. It is crucial to screen early for HRR mutations, particularly in BRCA1/2, to begin timely, targeted mCRPC treatment and improve prognosis.[Table: see text]

Table S1. Baseline Characteristics in the Stromal SPARC-Evaluable Population

PDF file - 38KB, Protocol for Avatar Generation.

Supplemental Figure 1. A) Left panel: Visualization of the miRNA profile of normal pancreas samples (n=3 control). Right panel: Visualization of the miRNA profile of PDAC PDX tumor samples (n=27 cases). Relative expression is shown as mean of fluorescence (MFI). miRNAs profiling was done through Firefly Circulating miRNA Assay and normalized using two miRNAs that are not significantly deregulated between all samples miR-22-3p and miR-30b-5p defined by the geNorm-like 37 algorithm. B) Visualization of miRNA profiles of PDAC cell lines (n=4), PDAC 3Dmodel (n=2) and Patient-derived-organoid (PDO 286 and PDO 281) compared to normal pancreatic cell and organoid (n=2 control). miRNAs profiling was done via Firefly Circulating miRNA Assay and normalized using miR-181b-5p, miR-103-3p, miR-30b-5p defined by the geNorm-like algorithm. Normalized miRNA signal intensities are presented as fold-change (log10 of the ratio between a probe value to the average of all the other samples for that probe). Green represents highly expressed miRNAs and red represents lowly expressed miRNAs.

Supplemental Figure 4. A) qPCR analysis of miR-21-5p expression level after 4 treatments with TPN-21 in PDO 286. B) Relative tumor burden after (4 I.V injection of PBS (n=6) TPN-control n=6 or TPN-21 n=5 (5mg/kg). For qPCR each miRNA sample was normalized on the basis of its 18s content.

Toxicology analysis. Liver transaminases aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured after Antartina{trade mark, serif} treatment and H&E stained tissues from BALB/c mice untreated (n= 5) or treated with 5 mg/kg (n=5) and 50 mg/kg (n=5) of Antartina{trade mark, serif}

Figure S1 . Representative SPARC staining. (A) Representative SPARC staining in stroma. (B) Representative SPARC staining in tumor epithelia.