Loss of mice before the end of the experiment and reason of sacrification. CRS: CIC-rearranged sarcoma; DDLPS: dedifferentiated liposarcoma; LMS: leiomyosarcoma; SynSa: synovial sarcoma.

Abstract Background: PM54, a synthetic ecteinascidin analog derived from lurbinectedin, binds GC-rich promoter regions to block transcription, induce DNA double-strand breaks, and trigger S-phase arrest and apoptosis. We aimed to define PM54 transcriptional mechanisms across tumor types and to identify transcriptomic features associated with heightened sensitivity. Methods: Comprehensive transcriptomic profiling (RNA-seq) was performed following acute PM54 exposure (50 nM, 6 h) in a panel of 32 cancer cell lines representing six tumor types: 12 small-cell lung cancers (SCLC), 8 gastric cancers, 6 prostate cancers, 4 breast cancers, 1 ovarian cancer, and 1 melanoma. Hierarchical clustering, differential expression, GSEA, and GO analyses were used to define temporal transcriptional response clusters. In vivo validation was conducted in four CDX models (SCLC DMS-53, ovarian A2780, melanoma WM-266-4, and TNBC MDA-MB-231) after a single PM54 dose, with RNA-seq performed on day 7. Results: PM54 induced a rapid, broad transcriptional repression in all models, with >2,000 downregulated genes per line and a conserved core of 1,170 commonly repressed genes (versus 226 commonly induced). Repressed programs included cell cycle, RNA Pol II transcription, chromatin organization, DNA repair and MYC targets; early upregulated signatures implicated metabolism, PARP signaling and stress responses. Unsupervised clustering based on PM54 transcriptional effect identified a “primed” responder cluster (characterized by high baseline transcription and rapid transcriptional shutdown after treatment) and an “adaptive” responder cluster. Curiously, all three POU2F3 SCLC lines and prostate cancer models were into the primed responder cluster. Consistently, in vivo transcriptomic analyses recapitulated these clusters, demonstrating enhanced transcriptional repression, MYC/mTOR signaling suppression, and markedly improved survival outcomes in primed (+192%, +345% and +412% in ovarian, melanoma and SCLC, respectively) compared to adaptive responders (+185% in breast cancer and +240% in gastric cancer). Conclusions: PM54 rapidly suppresses proliferative/MYC-driven programs and elicits adaptive metabolic/immune responses. Baseline transcriptional state predicts response and may guide patient selection and combination strategies. Citation Format: Ismael Fernández-Miranda, Javier Robles, Maria José Guillen, Pablo Avilés, Marcelo L. Ribeiro, Carmen Cuevas. PM54 targets oncogenic transcriptional networks across multiple cancer types [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 2981.

Average number of apoptotic cells evaluated on (A) H&E and (B) cPARP IHC staining. C, Double-strand break formation shown by γH2AX IHC staining. The results show the mean ± standard deviation. Statistical significance was calculated using the Mann–Whitney U test, *P < 0.05. ns, not significant.

Representative dot plots of BrdU and propidium iodide (PI) staining or composite histograms of PI staining of MDA-MB-231, A2780 or 22Rv1 cell lines treated for 24-h with 10 nM of PM534 (blue) or vehicle (control, red) and pulsed with BrdU for 1-h. DNA content was stained with PI (2n for G1 and 4n for G2) while BrdU incorporation and staining reflects S phase.

A: HUVEC adhesion (% of adhesion compared to control) on an extracellular matrix composed of fibronectin and type I collagen quantified with Sulforhodamine B following 24 hours of incubation with PM534 or colchicine. Results are presented as the mean percentage (n=3). Dotted line indicates a 50% reduction of adhesion. B: Survival curves for HUVEC cells following the incubation with PM534 and colchicine, in which 70% of cell were alive at 10 nM of PM534.

Supplemental Figure 5: Overview of representative tumor sections of STS336SynSa on day 16, treated with A) vehicle, B) doxorubicin, C) trabectedin, D) lurbinectedin, E) ecubectedin, and F) PM54. Tumors treated with ecubectedin and PM54 showed alteration in the myxoid stroma after treatment, potentially explaining the absence of tumor volume shrinkage despite the increase in mitosis and decrease in apoptosis. SynSa: synovial sarcoma.

List of treatment groups, models, number of tumors included at the start of the experiment and the number of tumors included in the analysis. Tumors from mice that were sacrificed prior to the end of the experiment (e.g., due to unethical tumor volume >2000mm 3 or body weight loss), were not included in the analysis. Tumors excluded from analysis of phase 2 of the STS336SynSa experiment concerns tumors from mice designated to the group intended for follow-up at any point in the experiment (i.e., any moment between days 0 and 41). CRS: CIC-rearranged sarcoma; DDLPS: dedifferentiated liposarcoma; LMS: leiomyosarcoma; SynSa: synovial sarcoma

Abstract Trabectedin, prototype of the ecteinascidin class of drugs, is a known second- or later-line therapeutic option for advanced soft tissue sarcoma (STS). We evaluated the antitumor activity of two novel synthetic trabectedin derivatives, ecubectedin and PM54, in selected patient-derived xenograft (PDX) STS models. In total, 364 Naval Medical Research Institutenu/nu mice were transplanted bilaterally with two leiomyosarcoma (LMS), two dedifferentiated liposarcoma (DDLPS), one synovial sarcoma (SynSa), and one CIC-rearranged sarcoma (CRS) PDX models. Mice were randomized to six groups and treated via tail vein injection with (i) vehicle, (ii) doxorubicin, (iii) trabectedin, (iv) lurbinectedin, (v) ecubectedin, or (vi) PM54. Treatment was given on days 1, 8, and 15, and mice were sacrificed on day 16. The SynSa experiment included extra mice to investigate posttreatment xenograft evolution. Antitumor activity was assessed by tumor volume measurement, histopathologic, and immunohistochemical analyses. In all LMS and DDLPS models, ecubectedin and PM54 led to tumor growth delay compared with trabectedin. Histopathologic evaluation of treated tumors showed moderately increased antitumor activity of novel ecteinascidins compared with trabectedin. The CRS model showed tumor shrinkage in response to ecubectedin (63% regression from baseline) and PM54 (24% regression from baseline), while the SynSa model showed tumor volume stabilization. Both translocation-related models showed significant antitumor effect on histopathologic evaluation in response to the novel drugs compared with trabectedin. Ecubectedin and PM54 have modest antitumor activity in STS PDX models, with the strongest effects seen in the translocation-related sarcoma models, showing the potential of this class of drugs in the treatment of STS. Significance: Advanced STS remains without truly effective therapeutic options. Translocation-related sarcomas demonstrate higher vulnerability to next-generation ecteinascidin treatment compared with more complex sarcoma subtypes. This vulnerability builds upon known literature and could be exploited in future clinical trials using the next-generation ecteinascidins.

Protein corrected transport rate (pmol/min/pmol transporter protein) of PM534 in Abcb1KO-MDCKII-MDR1-LV cells. Data are expressed as mean (n=3) and 95% CI.

Supplemental Figure 2: Evolution of absolute tumor volume of individual tumors from day 1 to day 16 (A-F) or from day 1 to day 17 and to day 41 (G). Statistical significance indicate either tumor growth or shrinkage, whereas non-significance indicates tumor volume stabilization. * p<0.05, CRS: CIC-rearranged sarcoma; DDLPS: dedifferentiated liposarcoma; LMS: leiomyosarcoma; STS: soft tissue sarcoma; TV: tumor volume; ns: not significant; SynSa: synovial sarcoma.

Trabectedin is a marine-derived antitumor agent from the ecteinascidin family that is effective in treating Ewing sarcoma. It features a carbinolamine that forms reversible covalent bonds with N2 of guanine, distinguishing it from other structurally similar duplex DNA alkylating agents. Importantly, the ecteinascidins are structurally distinct from other carbinolamine alkylating drugs by inclusion of a C-subunit that widens the DNA minor groove and bends DNA into the major groove. Taking into account the sensitivity of soft tissue sarcomas to Trabectedin and the role of the Ewing sarcoma protein (EWS) in binding to G-quadruplexes (G4s), this research proposes a refined understanding of Trabectedin’s molecular target by exploring its potential interaction with G4 structures. Building on these insights, we designed experiments using Trabectedin in combination with G4s, including the wild-type MYCN G4s, to assess whether Trabectedin could form stable covalent bonds to a consensus sequence within a duplex stem loop potentially influenced by its association with a G4 structure. Using a combination of circular dichroism, quantitative Förster resonance energy transfer (FRET) melting assays, and LC-MS, we demonstrate that Trabectedin can engage and stabilize composite G4–duplex architectures, provided a guanine-containing covalent bonding site is present within the associated hairpin loop. However, an LC-MS affinity assay revealed that Trabectedin still retains a preference for duplex DNA over the G4-containing construct. Finally, we observed that Trabectedin treatment of cells significantly increased G4 frequency, suggesting a modulation of G4 dynamics. We propose a context-dependent recognition model wherein Trabectedin’s dominant reactivity still lies with duplex DNA, but its structural compatibility allows engagement with reactive motifs embedded within G4-associated stem-loop architectures, potentially contributing to its unique downstream biological effects. To explain why Trabectedin is more potent and clinically efficacious than other carbinolamine-containing antitumor antibiotics such as Saframycin A, we propose a unique “search and lock” mechanism in which Trabectedin preferentially covalently bonds to G4-associated stem loops due to context-dependent enhanced stability of its covalent adduct.

Tumor volume assessment in STS models after treatment.

Evaluation of mitotic and apoptotic activity Arrows indicate whether mitotic or apoptotic activity was significantly higher (↑), lower (↓) or not statistically different (=) compared to trabectedin. Statistically significant changes were calculated using the Mann-Whitney U test, compared to trabectedin. CRS: CIC-rearranged sarcoma; DDLPS: dedifferentiated liposarcoma; H&E: hematoxylin and eosin; LMS; leiomyosarcoma; SynSa; synovial sarcoma

Supplemental Figure 3: Relative change of individual tumor volumes from baseline (here indicated as 0), on day 16 (A-F) or day 41 (G), sorted per treatment for each experiment.

Average number of mitotic cells evaluated on (A) H&E and (B) mitotic activity shown by pHH3 IHC staining. C, Percentage of Ki-67–positive cells. The results show the mean ± standard deviation. Statistical significance was calculated using the Mann–Whitney U test, *P < 0.05. ns, not significant.

Supplemental Figure 4: Evolution of body weight during treatment, presented as relative body weight (% change from normalized baseline) ± standard deviation. Figure 2F depicts the first phase of the experiment of STS336SynSa, while Figure 2G depicts the complete experiment. Mice sacrificed prior to end of experiment are removed from data after date of scarification. 3x/4x: 3 or 4 mice of the same group were sacrificed on the same day; * p<0.05; CRS: CIC-rearranged sarcoma; DDLPS: dedifferentiated liposarcoma; LMS: leiomyosarcoma; ns: not significant; STS: soft tissue sarcoma; TV: tumor volume; SynSa: synovial sarcoma.

This study evaluates PM534, a novel colchicine-binding domain inhibitor, for its potential in cancer therapy. PM534 exhibited potent in vitro efficacy against a panel of 14 human cancer cell lines, including breast, ovarian, and prostate cancers, with concentration needed to reduce the growth of treated cells to half that of untreated cells values in the low nanomolar range. Both continuous (72 hours) and short-term (1-24 hours) exposure led to irreversible effects, inducing G2-M cell cycle arrest and multinucleation. Additionally, PM534 also impaired angiogenic process. It effectively inhibited HUVEC functions, including adhesion, with an IC50 of 2.3 nmol/L, markedly more potent than colchicine (IC50 = 1,800 nmol/L). At concentrations as low as 1.6 nmol/L, PM534 delayed wound closure in migration assays, completely inhibiting migration above 4 nmol/L. Additionally, PM534 abrogated invasion and disrupted capillary-like network formation at concentrations starting from 0.5 nmol/L without inducing cytotoxicity. In vivo PM534 demonstrated robust antitumor efficacy across six xenograft models, including ovarian (A2780 and ES-2), triple-negative breast (MDA-MB-231 and HCC1937), and prostate (VCaP and 22Rv1) tumors. This treatment also led to statistically significant increases in median survival times across all models, without inducing signs of systemic toxicity. Mechanistically, PM534 induced apoptosis, mitotic catastrophe, and necrosis in tumor tissues. Importantly, PM534 retained efficacy in models overexpressing multidrug resistance proteins P-glycoprotein or β-III tubulin, overcoming common resistance mechanisms that limit the effectiveness of other tubulin-binding agents. Collectively, these findings highlight PM534 as a promising antitumor agent with potent activity against diverse and treatment-resistant malignancies. A phase I clinical trial (NCT#5835609) is underway to assess the therapeutic potential of PM534 in patients with advanced solid tumors.

Cancer is the leading cause of disease-related deaths among children in high-income countries. Tumor heterogeneity and lack of mechanism-of-action-based therapeutic options are key challenges to overcome to improve pediatric cancer patient survival. To address these challenges, we formed the EU-IMI-2 funded public-private partnership "ITCC-Pediatric Preclinical Proof-of-Concept Platform" (ITCC-P4), which built a large repertoire of patient-derived xenograft (PDX) models representing all major high-risk solid pediatric cancer types for in vivo drug testing. A total of 353 PDX models were established from diagnostic and relapsed pediatric cancers and molecularly characterized, together with matched germline/tumor samples. Serial PDX models were also established, spanning diagnostic/posttreatment, primary/relapse, and metastasis-derived pairs. Proof-of-concept in vivo drug screening data in neuroblastoma and rhabdomyosarcoma models identified potential predictive biomarkers for targeted therapy. Molecular data from the PDX models, accessible at https://r2platform.com/itcc-p4, allowed the selection of models for preclinical testing based on oncogenic drivers and/or potential biomarkers. Operated by a non-profit entity, this sustainable platform aids academic and industrial researchers in developing and prioritizing innovative therapies for pediatric cancer.

Abstract Background: PM54, a next-generation synthetic ecteinascidin, combines enhanced DNA-minor-groove binding with dual activity: direct cytotoxicity and immune modulation through immunogenic cell death and innate signalling cascades. This study aimed to characterize the immunomodulatory effects of PM54 alone and in combination with PD-1/PD-L1 blockade in in vitro and humanized tumor models. Methods: PBMCs from healthy donor were pre-stimulated with anti-CD3/CD28 complex and coculture for 96h with MDA-MB-231 (high PD-L1) pre-treated with PM54 and IOs (anti-PD1/PD-L1). Number of resulting target cells was analysed by flow cytometry. NXG mice engrafted with human hematopoietic CD34+ cells (Hu-CD34+) were xenografted with MDA-MB-231 xenografts. Randomly allocated mice (n≥4/group) were treated with placebo, PM54 (0.6 mg/kg, i.v., Days 0, 14) ± atezolizumab (10 mg/kg, i.p., twice weekly). On day 28, animals were euthanized, and tumors weighed and analyzed by multiparametric flow cytometry and RNA-seq. Results: In vitro coculture experiments showed an increase in the immune mediated cytotoxicity of target cells upon combination of lower doses of PM54 with atezolizumab, sensitizing cells to immunotherapy. On day 28, treatments of Hu-CD34+ bearing MDA-MB-231 tumors induced a statistically significant tumor reduction (vs placebo: 988.4 mm3). The median of tumor volume (mm3) was 305.8 (p=0.0159) for PM54, 715.6 (p=0.0317) for atezolizumab and 199.7 (p=0.0286) for the combination. Of note, statistically significant tumor reduction was also recorded in the combination vs PM54 or atezolizumab (in both, p=0.0159). In humanized MDA-MB-231, PM54 + atezolizumab expanded CD3+ cells (32.2% vs 9.3%), increased CD8+ cells (44.7% vs 19.8%), reduced Tregs (10.0% vs 42.8%), modestly increased cytotoxic NK cells, and downregulated PD-L1 on tumor and immune compartments. Transcriptomics analysis demonstrated that the combination arm renders the highest number of deregulated genes (1404 up and 981 down genes vs 537 and 908 up genes or 278 and 552 down genes in PD-L1 o PM54 treatment, respectively). Among them, GSEA showed an increase upon treatment or combination in MHC genes, IFNg and IFNα response, and NK activity and TCR signaling, leading to an enhance inflammatory response. Moreover, CIBERSORT analysis of immune populations complemented cytometry evaluation with an increase in CD8 T cells, Tfh, activated DCs and NK cells and M1/M2 ratio increase upon treatment and combination. Conclusions: PM54 is a dual action immunochemotherapeutic that drives ICD, increases allogenic immune cytotoxicity of tumoral cells and, in vivo, augments effector CD8+ responses, diminishes Tregs, and reprograms suppressive myeloid cells. These immunologic effects synergize with PD-1/PD-L1 blockade, supporting development of PM54-based combinations for transcriptionally driven, immune-cold malignancies. Citation Format: Eugenio Bustos-Morán, Daniel Torralba, Ismael Fernández-Miranda, Maria José Guillen, Pablo Avilés, Marcelo L. Ribeiro, Carmen Cuevas, . PM54 reshapes the tumor microenvironment to potentiate checkpoint blockade [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 2841.

A–G, Evolution of tumor volume during treatment, presented as relative tumor volume (% change from normalized baseline) ± standard deviation. F, The first 16 days of the STS336SynSa experiment. G, The complete experiment, including the follow-up period after the first 16 days. Tumors from mice sacrificed prior to the end of the experiments were not included in statistical evaluation. Statistical significance is indicated for ecubectedin or PM54 compared with trabectedin at the end of the experiment (day 16 or 41). *, P < 0.05. 3×/4×: 3 or 4 mice of the same group were sacrificed on the same day. TV, tumor volume.