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The Figure shows the long term behavior of sensitive and resistant cells (in absence of enzalutamide

Single file with supplemental figures, methods, tables, legends: Figure S1: Effects of FGF blockade in MM; Figure S2: FGF inhibition induces oxidative stress-mediated apoptosis in MM cells; Figure S3: Mitochondrial oxidative stress is responsible for the antitumor activity exerted in vivo by FGF inhibition; Figure S4: Oxidative stress-induced apoptosis following FGF inhibition is c-Myc-dependent; Figure S5: Western blot analysis for caspase 3 activation in PBMCs compared to NSC12-sensitive patient-derived MM cells treated with NSC12 for 12 hours; Figure S6: Effects of FGF inhibition in BTZ-resistant KMS-11 cells; Figure S7: High doses of BTZ hamper basal and NSC12-induced c-Myc degradation; Table S1: List of Log2 2-fold down- or upregulated genes in KMS-11 cells after 6 and 12 hours of NSC12 6μM treatment; Table S2: Patient-derived MM cells treated with NSC12; Reagent table.

Supplementary Tables with parameters

Additional Material and Methods

Multiple myeloma, the second most common hematologic malignancy, frequently relapses because of chemotherapeutic resistance. Fibroblast growth factors (FGF) act as proangiogenic and mitogenic cytokines in multiple myeloma. Here, we demonstrate that the autocrine FGF/FGFR axis is essential for multiple myeloma cell survival and progression by protecting multiple myeloma cells from oxidative stress–induced apoptosis. In keeping with the hypothesis that the intracellular redox status can be a target for cancer therapy, FGF/FGFR blockade by FGF trapping or tyrosine kinase inhibitor impaired the growth and dissemination of multiple myeloma cells by inducing mitochondrial oxidative stress, DNA damage, and apoptotic cell death that were prevented by the antioxidant vitamin E or mitochondrial catalase overexpression. In addition, mitochondrial oxidative stress occurred as a consequence of proteasomal degradation of the c-Myc oncoprotein that led to glutathione depletion. Accordingly, expression of a proteasome-nondegradable c-Myc protein mutant was sufficient to avoid glutathione depletion and rescue the proapoptotic effects due to FGF blockade. These findings were confirmed on bortezomib-resistant multiple myeloma cells as well as on bone marrow–derived primary multiple myeloma cells from newly diagnosed and relapsed/refractory patients, including plasma cells bearing the t(4;14) translocation obtained from patients with high-risk multiple myeloma. Altogether, these findings dissect the mechanism by which the FGF/FGFR system plays a nonredundant role in multiple myeloma cell survival and disease progression, and indicate that FGF targeting may represent a therapeutic approach for patients with multiple myeloma with poor prognosis and advanced disease stage.Significance:This study provides new insights into the mechanisms by which FGF antagonists promote multiple myeloma cell death.

Supplemantary informations for the model parameterization

Enzalutamide (MDV3100) is a potent second-generation androgen receptor antagonist approved for the treatment of castration-resistant prostate cancer (CRPC) in chemotherapy-naïve as well as in patients previously exposed to chemotherapy. However, resistance to enzalutamide and enzalutamide withdrawal syndrome have been reported. Thus, reliable and integrated preclinical models are required to elucidate the mechanisms of resistance and to assess therapeutic settings that may delay or prevent the onset of resistance. In this study, the prostate cancer multistage murine model TRAMP and TRAMP-derived cells have been used to extensively characterize in vitro and in vivo the response and resistance to enzalutamide. The therapeutic profile as well as the resistance onset were characterized and a multiscale stochastic mathematical model was proposed to link the in vitro and in vivo evolution of prostate cancer. The model showed that all therapeutic strategies that use enzalutamide result in the onset of resistance. The model also showed that combination therapies can delay the onset of resistance to enzalutamide, and in the best scenario, can eliminate the disease. These results set the basis for the exploitation of this “TRAMP-based platform” to test novel therapeutic approaches and build further mathematical models of combination therapies to treat prostate cancer and CRPC.Significance: Merging mathematical modeling with experimental data, this study presents the “TRAMP-based platform” as a novel experimental tool to study the in vitro and in vivo evolution of prostate cancer resistance to enzalutamide.

Enzalutamide (MDV3100) is a potent second-generation androgen receptor antagonist approved for the treatment of castration-resistant prostate cancer (CRPC) in chemotherapy-naïve as well as in patients previously exposed to chemotherapy. However, resistance to enzalutamide and enzalutamide withdrawal syndrome have been reported. Thus, reliable and integrated preclinical models are required to elucidate the mechanisms of resistance and to assess therapeutic settings that may delay or prevent the onset of resistance. In this study, the prostate cancer multistage murine model TRAMP and TRAMP-derived cells have been used to extensively characterize in vitro and in vivo the response and resistance to enzalutamide. The therapeutic profile as well as the resistance onset were characterized and a multiscale stochastic mathematical model was proposed to link the in vitro and in vivo evolution of prostate cancer. The model showed that all therapeutic strategies that use enzalutamide result in the onset of resistance. The model also showed that combination therapies can delay the onset of resistance to enzalutamide, and in the best scenario, can eliminate the disease. These results set the basis for the exploitation of this “TRAMP-based platform” to test novel therapeutic approaches and build further mathematical models of combination therapies to treat prostate cancer and CRPC. Significance: Merging mathematical modeling with experimental data, this study presents the “TRAMP-based platform” as a novel experimental tool to study the in vitro and in vivo evolution of prostate cancer resistance to enzalutamide.

Bladder tumors are a diffuse type of cancer with a relatively benign prognosis when treated at early stage/low-grade stages, but with poor outcome in the muscle invasive (MIBC) form or at recurrence. Long Pentraxin-3 (PTX3) is a component of the innate immunity with pleiotropic functions in the regulation of immune response, tissue remodeling and cancer progression. PTX3 may act as an oncosuppressor in different contexts, functioning as an antagonist of the FGF/FGFR system, rewiring the immune-microenvironment or acting through mechanisms not yet fully clarified. In this study, we report for the first time that PTX3 is differentially expressed in bladder cancer (BC) biopsies and tumor cell lines during the different stages of BC progression. Taking advantage of BC cell lines representative of different tumor grades, we demonstrate that PTX3 production by tumor cells decreases along the progression from low-grade to high grade/MIBC because of promoter methylation. In vitro and in vivo data reveal that PTX3 modulation in BC cells has a significant impact on different biological features of BC growth, including cell proliferation, motility, metabolism, stemness and drug resistance. In conclusion, PTX3 exerts an oncosuppressive effect on BC progression and may represent a potential functional biomarker in BC evolution. Citation Format: Sara Matarazzo, Laura Melocchi, Sara Rezzola, Elisabetta Grillo, Federica Maccarinelli, Arianna Giacomini, Sara Taranto, Luca Zammataro, Marianna Cerasuolo, Mattia Bugatti, William Vermi, Marco Presta, Roberto Ronca. Long Pentraxin-3 modulates bladder cancer progression [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C121. doi:10.1158/1535-7163.TARG-19-C121

Multiple myeloma (MM) remains incurable because of chemotherapeutic resistance. Fibroblast growth factors (FGF) play a pivotal role in MM by acting as an autocrine/paracrine mitogen on plasma cells, bone marrow-derived endothelial cells and fibroblasts. Also, a recurrent chromosomal translocation t(4;14) is associated with FGFR3 upregulation in MM patients. Thus, agents able to hamper FGF signaling may represent a novel approach for MM treatment. Recently we have identified the PTX3-derived small molecule NSC12 as the first orally active antitumor pan-FGF trap. Here, the role of FGF/FGFR system in MM cell survival and the therapeutic potential of NSC12 were investigated. In vitro effects of NSC12 were tested on four human MM cell lines harboring (KMS-11 and OPM-2 cells) or not (U-266 and RPMI8226 cells) the t(4:14) translocation and on patient-derived MM cells (N = 26). In vivo effects of FGF blockade were investigated using subcutaneous (KMS-11 and RPMI8226 cells) and systemic (MM-1S cells) xenografts grown in NOD/SCID mice. In addition, gene expression profiling (GEP) and gene set enrichment analyses (GSEA) were performed in NSC12-treated MM cells. NSC12 blocked the proliferation of all human MM cell lines tested, causing inhibition of FGFR signaling, downregulation of the anti-apoptotic protein mcl-1 and caspase 3 activation. Cytofluorimetric analysis revealed a significant increase of apoptotic cells as early as 6 hrs after NSC12 treatment, with 100% of cell death after 24 hrs even when MM cells were co-cultured with patient-derived bone marrow stromal cells. In vivo, NSC12 blocked FGFR activation and reduced the growth of subcutaneous xenografts and MM cell dissemination in the BM. GEP and GSEA analyses of NSC12-treated cells revealed the upregulation of oxidative stress-induced genes and the downmodulation of c-Myc targets. Accordingly, NSC12 caused the rapid proteasomal degradation of c-Myc paralleled by GSH depletion, mitochondrial ROS production and depolarization, DNA damage and finally apoptosis in KMS-11 cells but not in KMS-11 cells expressing the undegradable mutated (T58A) form of c-Myc. Interestingly, these findings were confirmed on Bortezomib-resistant MM cells as well as on bone marrow-derived primary MM cells from newly diagnosed and relapsed/refractory patients, including plasma cells bearing the t(4;14) translocation obtained from high-risk MM patients. Our data reveal that FGF blockade triggers MM mitochondrial oxidative stress, DNA damage and apoptotic cell death via the proteasomal degradation of the c-Myc oncoprotein. Altogether, our findings dissect for the first time the mechanism by which the FGF/FGFR system plays a non-redundant role in MM cell survival and disease progression, and indicate that FGF targeting may represent a therapeutic approach for MM patients with poor prognosis and advanced disease stage. Citation Format: Roberto Ronca, Gaia Cristina Ghedini, Federica Maccarinelli, Antonio Sacco, Silvia Laura Locatelli, Eleonora Foglio, Sara Taranto, Elisabetta Grillo, Sara Matarazzo, Riccardo Castelli, Vanessa Desantis, Nadia Cattane, Annamaria Cattaneo, Marco Mor, Carmelo Carlo-Stella, Aldo Maria Roccaro, Marco Presta, Arianna Giacomini. FGF trapping impairs multiple myeloma growth through c-Myc degradation-induced mitochondrial oxidative stress [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C052. doi:10.1158/1535-7163.TARG-19-C052

Bladder cancer is one of the most common cancers in the world. Although most of urothelial carcinomas (UCs) are low-grade papillary tumors, their propensity to recur and to progress to become invasive with a poor survival rate represents a major challenge. Different members of the Fibroblast growth factor (FGF) family are expressed by human bladder cancer cells, and aberrant expression of FGF receptors (FGFRs) is a typical feature of bladder cancer compared to normal urothelium. High expression levels of FGFR1 are associated with poor survival and FGFR3 is frequently dysregulated in UC. Thus, the FGF/FGFR system may represent a promising therapeutic target in invasive and noninvasive bladder cancer. Long-pentraxin 3 (PTX3) acts as a natural FGF trap by binding FGFs and hampering their biologic activity in various tumor models. Preliminary observations have shown that low-grade UCs express PTX3 while high-grade/invasive UCs lose PTX3 expression. Accordingly, bladder cancer cell lines share an overall presence of FGFRs and FGFs but express different levels of PTX3 in vitro and in vivo. Of note, high-grade/invasive cells express very low or undetectable levels of PTX3, whereas low-grade, papilloma-like cells express high levels of PTX3. Indeed, invasive bladder cancer cell xenografts grow faster in nude mice and express much lower levels of PTX3 than low grade-derived lesions, thus confirming an inverse correlation between UC grade and PTX3 expression. This hypothesis is reinforced by a preliminary case study performed on a small cohort of biopsies from UC patients confirming that the presence of PTX3 is a common feature of low-grade samples while PTX3 is poorly expressed or absent in aggressive/invasive cases. These data point to PTX3 as a natural FGF trap that could play a role in modulating bladder cancer progression and invasiveness and indicate that inhibition of the FGF/FGFR system by natural and synthetic FGF traps may represent a promising target for the therapy of UCs. Citation Format: Sara Matarazzo, Federica Maccarinelli, Gaia Cristina Ghedini, Laura Melocchi, Mattia Bugatti, William Vermi, Marco Presta, Roberto Ronca. FGF/PTX3 crosstalk in bladder cancer: novel prognostic and therapeutic implications [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A039.

Despite advances in systemic and supportive therapies, multiple myeloma (MM) remains incurable because of chemotherapeutic resistance. Fibroblast growth factor-2 (FGF2) plays a pivotal role in MM by acting as an autocrine/paracrine mitogen on plasma cells, bone marrow-derived endothelial cells, and fibroblasts. In keeping with the pivotal role of the FGF system in MM, a recurrent chromosomal translocation t(4;14) is associated with upregulation of FGF receptor-3 (FGFR3) in MM patients. Agents able to hamper FGF signaling are therefore of interest as a novel approach for the treatment of MM. The soluble pattern recognition receptor long pentraxin-3 (PTX3) owns a unique FGF-binding N-terminal domain. Structural/functional characterization of this domain led to the characterization of the pentapeptide ARPCA as a potent FGF antagonist in vitro and in vivo. Subsequently, molecular modeling based on ARPCA structure and small-molecule library fishing have allowed the identification of a novel, orally active 480 Da FGF trap (NSC12) able to hamper tumor growth and vascularization in FGF-driven human lung and prostate tumor models. Here, the therapeutic potential of NSC12 for MM treatment was tested on four human MM cell lines harboring (KMS-11 and OPM-2 cells) or not (U-266 and RPMI8226 cells) the t(4:14) translocation. NSC12 blocked the proliferation of all human MM cell lines with an IC50 ≅ 3 µM. Western blot analysis showed that NSC12 inhibited the activation of FGFR3 signaling and strongly downregulated the antiapoptotic protein mcl-1, leading to the activation of caspase 3. Cytofluorimetric analysis revealed a significant increase of annexin-V+ apoptotic cells as early as 6 hrs after NSC12 treatment, with 100% of cell death after 24 hrs of treatment. Interestingly, apoptosis was observed even when MM cells were cocultured with patient-derived bone marrow stromal cells. In vivo, oral delivery of NSC12 significantly blocked the growth of KMS-11 (286.9 ± 22.4 vs 459.8 ± 27.4 mm3, p Citation Format: Arianna Giacomini, Gaia Cristina Ghedini, Federica Maccarinelli, Silvia Laura Locatelli, Antonio Sacco, Riccardo Castelli, Vanessa Desantis, Aldo Maria Roccaro, Marco Mor, Carmelo Carlo-Stella, Roberto Ronca, Marco Presta. Inhibition of the fibroblast growth factor system by a new FGF trap induces oxidative stress and mitochondrial apoptosis in multiple myeloma cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B134.