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Additional file 1: Supplementary Table S1. Pancreatic cancer cells sensitivity. Supplementary Table S2. Screening domatinostat in combination with chemotherapeutics on cell proliferation by SRB assay and Calcusyn software. Supplementary Table S3. Screening domatinostat in combination with chemotherapeutics on cell proliferation by SRB assay and Calcusyn software. Supplementary Table S4. Screening domatinostat in combination with chemotherapeutics on cell proliferation by SRB assay and Calcusyn software. Supplementary Table S5. Screening domatinostat in combination with chemotherapeutics on cell proliferation by SRB assay and Calcusyn software. Supplementary Table S6. Domatinostat modulates FOXM1 activity. Supplementary Table S7. Critical FOXM1 role in pancreatic cancer patients. Supplementary Figure S1. Chemotherapy effect on pancreatic cancer cells. Supplementary Figure S2. Domatinostat plus gemcitabine/taxol combination induces Annexin-V exposure. Supplementary Figure S3. Domatinostat plus gemcitabine/taxol combination induces cell cycle perturbation and a clear block in phase S. Supplementary Figure S4. Pancreatic cancer cells enriched in stem-cell features, culturing model validation. Supplementary Figure S5. Domatinostat effects on CSC population. Supplementary Figure S6. The effect of domatinostat on PDAC spheroid cultures. Supplementary Figure S7. Domatinostat effects on FOXM1 expression. Supplementary Figure S8. Domatinostat modulates FOXM1 activity. Supplementary Figure S9. A network analysis revealed FOXM1 as upstream regulator of significant domatinostat-modulated proteins involved in stemness, oxidative stress and DNA repair. Supplementary Figure S10. Critical FOXM1 role in pancreatic cancer patients. Supplementary Figure S11. FOXM1 expression in transfected cells. Supplementary Figure S12. Domatinostat modulates FOXM1 activity. Supplementary Figure S13. Domatinostat does not exert toxic effect alone and in combination in vivo. Supplementary Figure S14. Domatinostat synergistically improves overall survival of PANC1 mice affecting FOXM1 and OCT4 expression.

Key Points Question Does sequential scheduling of bevacizumab administration in combination with chemotherapy improve treatment efficacy in patients with metastatic colorectal cancer in keeping with the tumor vascular normalization hypothesis? Findings In this phase 3 randomized clinical trial of 230 patients, the primary end point objective response rate did not significantly differ between the sequential and the concomitant schedule of bevacizumab administration in combination with standard oxaliplatin-based regimens. However, the sequential schedule of bevacizumab administration was associated with longer overall survival, fewer adverse effects, and better health-related quality of life. Meaning Sequential bevacizumab scheduling plus chemotherapy might be relevant to optimize therapeutic efficacy and to explore antiangiogenic combination treatments with innovative perspectives., This randomized clinical trial compares outcomes of concomitant and sequential administration of bevacizumab in combination with standard oxaliplatin-based regimens in untreated or single line–treated patients with metastatic colorectal cancer., Importance Although bevacizumab is a standard of care in combination treatments for metastatic colorectal cancer (mCRC), its clinical benefit has been limited. Objective To determine whether sequential scheduling of bevacizumab administration in combination with chemotherapy improves treatment efficacy in patients with mCRC, in keeping with the tumor vascular normalization hypothesis. Design, Setting, and Participants This open-label, randomized clinical phase 3 trial was conducted from May 8, 2012, to December 9, 2015, at 3 Italian centers. Patients aged 18 to 75 years with unresectable, previously untreated, or single line–treated mCRC were recruited. Follow-up was completed December 31, 2019, and data were analyzed from February 26 to July 24, 2020. Interventions Patients received 12 biweekly cycles of standard oxaliplatin-based regimens (modified FOLFOX-6 [levo–folinic acid, fluorouracil, and oxaliplatin]/modified CAPOX [capecitabine and oxaliplatin]) plus bevacizumab administered either on the same day as chemotherapy (standard arm) or 4 days before chemotherapy (experimental arm). Main Outcomes and Measures The primary end point was the objective response rate (ORR) measured with Response Evaluation Criteria in Solid Tumors, version 1.1. Secondary end points included progression-free survival, overall survival, safety, and quality of life (QOL). Results Overall, 230 patients (136 men [59.1%]; median age, 62.3 [interquartile range, 53.3-67.6] years) were randomly assigned to the standard arm (n = 115) or the experimental arm (n = 115). The median duration of follow-up was 68.3 (95% CI, 61.0-70.0) months. No difference in ORR (57.4% [95% CI, 47.8%-66.6%] in the standard arm and 56.5% [95% CI, 47.0-65.7] in the experimental arm; P = .89) or progression-free survival (10.5 [95% CI, 9.1-12.3] months in the standard arm and 11.7 [95% CI, 9.9-12.9] months in the experimental arm; P = .15) was observed. However, the median overall survival was 29.8 (95% CI, 22.5-41.1) months in the experimental arm compared with 24.1 (95% CI, 18.6-29.8) months in the standard arm (adjusted hazard ratio, 0.73; 95% CI, 0.54-0.99; P = .04). Moreover, the experimental arm was associated with a significant reduction in the rate of severe diarrhea (6 [5.3%] vs 19 [16.5%]; P = .006) and nausea (2 [1.8%] vs 8 [7.0%]; P = .05) and improved physical functioning (mean [SD] change from baseline, 0.65 [1.96] vs −7.41 [2.95] at 24 weeks; P = .02), and constipation scores (mean [SD] change from baseline, −17.2 [3.73] vs −0.62 [4.44]; P = .003). Conclusions and Relevance In this randomized clinical trial, sequential administration of bevacizumab plus chemotherapy did not improve ORR, the primary end point. However, the overall survival advantage, fewer adverse effects, and better health-related QOL associated with sequential bevacizumab administration might provide the basis for exploring antiangiogenic combination treatments with innovative perspectives. Trial Registration EudraCT Identifier: 2011-004997-27; ClinicalTrials.gov Identifier: NCT01718873

INTRODUCTION: Metastatic pancreatic ductal adenocarcinoma (PDAC) represents an unmet clinical need because the lack of effective treatment strategies and a very poor prognosis. Here we propose a novel therapeutic strategy, based on the repurposing of valproic acid (VPA), a safe and generic drug with epigenetic modulating activity, and simvastatin (SIM), a widely used generic cholesterol lowering drug, in combination with the standard gemcitabine/nab-paclitaxel (GEM/NP) treatment in metastatic PDAC setting. METHODS: Synergistic anti-tumor effect of the combined treatment was assessed in PANC1, ASPC1, PANC28 and BxPC3 PDAC cell lines in vitro by evaluating combination index, apoptosis, clonogenic capability, tumor spheroids and fibroblasts/tumor cells microtissues growth. Antitumor effect was confirmed in vivo on heterotopic and orthotropic xenograft PANC1 models in athymic mice. Expression and functional role of TGF-β and downstream epithelial-to-mesenchymal-transition (EMT) markers were studied by Ingenuity pathway analysis (IPA), mRNA and protein expression and by cell migration scratch assay. RESULTS: We showed, both in vitro and in vivo, the ability of VPA/SIM combination, used at low dosages, to synergistically improve the anti-proliferative and pro-apoptotic effect of chemotherapy (GEM/NP). Mechanistically, VPA/SIM treatment, alone or in combination with chemotherapy, induced e-Cadherin and impaired vimentin and ZEB-1 expression, functionally linked to the synergistic inhibition of cell migration. In line, IPA highlighted a protein network connecting HDACs and HMGCR, the targets of VPA and SIM respectively, with the two main EMT markers. Notably, the most significantly cancer enriched features associated with this network were “migration of tumor cell lines”, “fibrosis” and “invasion of tumor cell lines”, and TGFβ emerged as a hierarchical dominant network- node. Indeed, VPA/SIM inhibited TGFβ transcription and TGFβ-regulated EMT gene expression in PDAC cells. Consistently we also observed a significant reduction of circulating TGFβ1 levels in mice treated with VPA/SIM in combination with GEM/NP, paralleled by a reduced fibrosis on PDAC xenograft tumor sections, confirming the involvement of TGFβ functional down-modulation in the mechanism of VPA/SIM antitumor synergistic interaction and chemo-sensitization. CONCLUSIONS: Overall, we proposed a novel combination strategy, based on two safe and generic drugs, able to sensitize a widely employed regimen in metastatic PDAC patients, that warrant further clinical evaluation. Citation Format: Maria Serena Roca, federica iannelli, maria rita milone, veronica barile, cristina testa, tania moccia, carlo vitagliano, fabiana tatangelo, di gennaro elena, antonio avallone, francesca bruzzese, alessandra leone, alfredo budillon. Repurposing of valproic acid and simvastatin in pancreatic cancer: in vitro and in vivo synergistic antitumor interaction and sensitization to gemcitabine/nab-paclitaxel via inhibition of TGFβ-EMT signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1840.