Discovery of novel FGF trap small molecules endowed with anti-myeloma activity.

Fibroblast growth factors (FGFs) act as proangiogenic and mitogenic cytokines in several cancers, including multiple myeloma (MM). Indeed, corrupted FGF autocrine and paracrine secretion induces an aberrant activation of the FGF receptor (FGFR) signaling sustaining cancer cell spreading and resistance to pharmacological treatments. Thus, FGF traps may represent a promising anti-cancer strategy to hamper the ligand-dependent activation of the FGF/FGFR system. We previously identified NSC12 as the first orally available small molecule FGF trap able to inhibit the growth and progression of several FGF-dependent tumor models. NSC12 is a pregnenolone derivative carrying a 1,1-bis-trifluoromethyl-1,3-propanediol chain in position 17 of the steroid nucleus. Investigation of structure-activity relationships (SARs) provided more potent and specific NSC12 steroid derivatives and highlighted that the C17-side chain is pivotal for the FGF trap activity. Here, a scaffold hopping approach allowed to obtain two FGF trap compounds (22 and 57) devoid of the steroid nucleus and able to efficiently bind FGF2 and to inhibit FGFR activation in MM cells. Accordingly, these compounds exert a potent anti-tumor activity on MM cell lines both in vitro and in vivo and on MM patient-derived primary cells, strongly affecting the survival of both proteasome-inhibitor sensitive and resistant MM cells. These results propose a new therapeutic option for relapsed/refractory MM patients and set the bases for the development of novel FGF traps prone to chemical diversification to be used in the clinic for the treatment of those tumors in which the FGF/FGFR system plays a pivotal role, including MM.

Proteomic Analysis Highlights the Impact of the Sphingolipid Metabolizing Enzyme ß-Galactosylceramidase on Mitochondrial Plasticity in Human Melanoma.

Mitochondrial plasticity, marked by a dynamism between glycolysis and oxidative phosphorylation due to adaptation to genetic and microenvironmental alterations, represents a characteristic feature of melanoma progression. Sphingolipids play a significant role in various aspects of cancer cell biology, including metabolic reprogramming. Previous observations have shown that the lysosomal sphingolipid-metabolizing enzyme ß-galactosylceramidase (GALC) exerts pro-oncogenic functions in melanoma. Here, mining the cBioPortal for a Cancer Genomics data base identified the top 200 nuclear-encoded genes whose expression is negatively correlated with GALC expression in human melanoma. Their categorization indicated a significant enrichment in Gene Ontology terms and KEGG pathways related to mitochondrial proteins and function. In parallel, proteomic analysis by LC-MS/MS of two GALC overexpressing human melanoma cell lines identified 98 downregulated proteins when compared to control mock cells. Such downregulation was confirmed at a transcriptional level by a Gene Set Enrichment Analysis of the genome-wide expression profiling data obtained from the same cells. Among the GALC downregulated proteins, we identified a cluster of 42 proteins significantly associated with GO and KEGG categorizations related to mitochondrion and energetic metabolism. Overall, our data indicate that changes in GALC expression may exert a significant impact on mitochondrial plasticity in human melanoma cells.

The PTX3/TLR4 autocrine loop as a novel therapeutic target in triple negative breast cancer.

BACKGROUND: The pattern recognition receptor long pentraxin-3 (PTX3) plays conflicting roles in cancer by acting as an oncosuppressor or as a pro-tumor mediator depending on tumor context. Triple negative breast cancer (TNBC) represents the most aggressive histotype of breast cancer, characterized by the lack of efficacious therapeutic targets/approaches and poor prognosis. Thus, the characterization of new molecular pathways and/or alternative druggable targets is of great interest in TNBC. METHODS: The expression of PTX3 in BC tumor samples and in BC cell lines has been analyzed using the Gene Expression-Based Outcome for Breast Cancer Online (GOBO), qPCR, Western blot and ELISA assay. The contribution of tumor and stromal cells to PTX3 production in TNBC was assessed by analyzing single cell RNA sequencing data and RNAscope performed on TNBC tumor samples. In order to investigate the effects of PTX3 in TNBC, different cell lines were engineered to knock-down (MDA-MB-231 and BT549 cells) or overexpress (MDA-MB-468 and E0771 cells) PTX3. Finally, using these engineered cells, in vitro (including gene expression profiling and gene set enrichment analyses) and in vivo (orthotopic tumor models in immune-compromised and immune competent mice) analyses were performed to assess the role and the molecular mechanism(s) exerted by PTX3 in TNBC. RESULTS: In silico and experimental data indicate that PTX3 is mainly produced by tumor cells in TNBC and that its expression levels correlate with tumor stage. Accordingly, gene expression and in vitro results demonstrate that PTX3 overexpression confers a high aggressive/proliferative phenotype and fosters stem-like features in TNBC cells. Also, PTX3 expression induces a more tumorigenic potential when TNBC cells are grafted orthotopically in vivo. Conversely, PTX3 downregulation results in a less aggressive behavior of TNBC cells. Mechanistically, our data reveal that PTX3 drives the activation of the pro-tumorigenic Toll-like receptor 4 (TLR4) signaling pathway in TNBC, demonstrating for the first time that the PTX3/TLR4 autocrine stimulation loop contributes to TNBC aggressiveness and that TLR4 inhibition significantly impacts the growth of PTX3-producing TNBC cells. CONCLUSION: Altogether, these data shed light on the role of tumor-produced PTX3 in TNBC and uncover the importance of the PTX3/TLR4 axis for therapeutic and prognostic exploitation in TNBC.

The Pro-Oncogenic Sphingolipid-Metabolizing Enzyme ß-Galactosylceramidase Modulates the Proteomic Landscape in BRAF(V600E)-Mutated Human Melanoma Cells.

ß-Galactosylceramidase (GALC) is a lysosomal enzyme involved in sphingolipid metabolism by removing ß-galactosyl moieties from ß-galactosylceramide and ß-galactosylsphingosine. Previous observations have shown that GALC may exert pro-oncogenic functions in melanoma and Galc silencing, leading to decreased oncogenic activity in murine B16 melanoma cells. The tumor-driving BRAF(V600E) mutation is present in approximately 50% of human melanomas and represents a major therapeutic target. However, such mutation is missing in melanoma B16 cells. Thus, to assess the impact of GALC in human melanoma in a more relevant BRAF-mutated background, we investigated the effect of GALC overexpression on the proteomic landscape of A2058 and A375 human melanoma cells harboring the BRAF(V600E) mutation. The results obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS) demonstrate that significant differences exist in the protein landscape expressed under identical cell culture conditions by A2058 and A375 human melanoma cells, both harboring the same BRAF(V600E)-activating mutation. GALC overexpression resulted in a stronger impact on the proteomic profile of A375 cells when compared to A2058 cells (261 upregulated and 184 downregulated proteins versus 36 and 14 proteins for the two cell types, respectively). Among them, 25 proteins appeared to be upregulated in both A2058-upGALC and A375-upGALC cells, whereas two proteins were significantly downregulated in both GALC-overexpressing cell types. These proteins appear to be involved in melanoma biology, tumor invasion and metastatic dissemination, tumor immune escape, mitochondrial antioxidant activity, endoplasmic reticulum stress responses, autophagy, and/or apoptosis. Notably, analysis of the expression of the corresponding genes in human skin cutaneous melanoma samples (TCGA, Firehose Legacy) using the cBioPortal for Cancer Genomics platform demonstrated a positive correlation between GALC expression and the expression levels of 14 out of the 27 genes investigated, thus supporting the proteomic findings. Overall, these data indicate for the first time that the expression of the lysosomal sphingolipid-metabolizing enzyme GALC may exert a pro-oncogenic impact on the proteomic landscape in BRAF-mutated human melanoma.

Iron supplementation enhances RSL3-induced ferroptosis to treat naïve and prevent castration-resistant prostate cancer.

Prostate cancer (PCa) is a leading cause of death in the male population commonly treated with androgen deprivation therapy that often relapses as androgen-independent and aggressive castration-resistant prostate cancer (CRPC). Ferroptosis is a recently described form of cell death that requires abundant cytosolic labile iron to promote membrane lipid peroxidation and which can be induced by agents that inhibit the glutathione peroxidase-4 activity such as RSL3. Exploiting in vitro and in vivo human and murine PCa models and the multistage transgenic TRAMP model of PCa we show that RSL3 induces ferroptosis in PCa cells and demonstrate for the first time that iron supplementation significantly increases the effect of RSL3 triggering lipid peroxidation, enhanced intracellular stress and leading to cancer cell death. Moreover, the combination with the second generation anti-androgen drug enzalutamide potentiates the effect of the RSL3 + iron combination leading to superior inhibition of PCa and preventing the onset of CRPC in the TRAMP mouse model. These data open new perspectives in the use of pro-ferroptotic approaches alone or in combination with enzalutamide for the treatment of PCa.

The natural FGF-trap long pentraxin 3 inhibits lymphangiogenesis and lymphatic dissemination.

The lymphatic vascular system represents a major route for dissemination of several solid tumors, including melanoma. Even though the members of the Vascular Endothelial Growth Factor family VEGF-C and VEGF-A have been shown to drive tumor lymphangiogenesis, experimental evidence indicates that also the pro-angiogenic factor Fibroblast Growth Factor-2 (FGF2) may play a role in the lymphangiogenic switch by triggering the activation of lymphatic endothelial cells (LECs) in cooperation with VEGFs.The soluble pattern recognition receptor Long Pentraxin 3 (PTX3) acts as a natural FGF trap, thus exerting an oncosuppressive role in FGF-dependent tumors. Here, the capacity of PTX3 to modulate lymphangiogenesis was assessed in vitro and in vivo. The results demonstrate that recombinant human PTX3 inhibits the lymphangiogenic activity exerted by the VEGF-A/FGF2/sphingosine-1-phosphate (VFS) cocktail on human and murine LECs. In keeping with in vitro data, a reduced lymphangiogenic response was observed in a lymphangiogenic Matrigel plug assay following the subcutaneous injection of the VFS cocktail in PTX3-overexpressing transgenic TgN(Tie2-hPTX3) mice when compared to wild-type or Ptx3 null animals. Accordingly, the capacity of B16F10-VEGFC-luc melanoma cells to colonize the primary tumor-draining lymph node after grafting into the foot pad was dramatically impaired in PTX3-overexpressing mice.Together with the observation that both the VFS cocktail and melanoma cell conditioned media caused a significant downregulation of PTX3 expression in LECs, these data indicate that the FGF trap activity of PTX3 may exert a key effect in the modulation of lymphangiogenesis and tumor metastatic dissemination.

Production and Biochemical Characterization of Dimeric Recombinant Gremlin-1.

Gremlin-1 is a secreted cystine-knot protein that acts as an antagonist of bone morphogenetic proteins (BMPs), and as a ligand of heparin and the vascular endothelial growth factor receptor 2 (VEGFR2), thus regulating several physiological and pathological processes, including embryonic development, tissue fibrosis and cancer. Gremlin-1 exerts all these biological activities only in its homodimeric form. Here, we propose a multi-step approach for the expression and purification of homodimeric, fully active, histidine-tagged recombinant gremlin-1, using mammalian HEK293T cells. Ion metal affinity chromatography (IMAC) of crude supernatant followed by heparin-affinity chromatography enables obtaining a highly pure recombinant dimeric gremlin-1 protein, exhibiting both BMP antagonist and potent VEGFR2 agonist activities.

Oncosuppressive and oncogenic activity of the sphingolipid-metabolizing enzyme ß-galactosylceramidase.

ß-galactosylceramidase (GALC) is a lysosomal enzyme that removes ß-galactose from ß-galactosylceramide, leading to the formation of the oncosuppressor metabolite ceramide. Recent observations have shown that GALC may exert opposite effects on tumor growth by acting as an oncosuppressive or oncogenic enzyme depending on the different experimental approaches, in vitro versus in vivo observations, preclinical versus clinical findings, and tumor type investigated. This review will recapitulate and discuss the contrasting experimental evidence related to the impact of GALC on the biological behavior of cancer and stromal cells and its contribution to tumor progression.

FGFR blockade by pemigatinib treats naïve and castration resistant prostate cancer.

Prostate cancer (PCa) is a leading cause of cancer mortality in the male population commonly treated with androgen deprivation therapy (ADT) and relapsing as aggressive and androgen-independent castration-resistant prostate cancer (CRPC). In PCa the FGF/FGFR family of growth factors and receptors represents a relevant mediator of cancer growth, tumor-stroma interaction, and a driver of resistance and relapse to ADT. In the present work, we validate the therapeutic efficacy the FDA-approved FGFR inhibitor pemigatinib, in an integrated platform consisting of human and murine PCa cells, and the transgenic multistage TRAMP model of PCa that recapitulates both androgen-dependent and CRPC settings. Our results show for the first time that pemigatinib causes intracellular stress and cell death in PCa cells and prevents tumor growth in vivo and in the multistage model. In addition, the combination of pemigatinib with enzalutamide resulted in long-lasting tumor inhibition and prevention of CRPC relapse in TRAMP mice. These data are confirmed by the implementation of a stochastic mathematical model and in silico simulation. Pemigatinib represents a promising FDA-approved FGFR inhibitor for the treatment of PCa and CRPC alone and in combination with enzalutamide.

ß-Galactosylceramidase in cancer: friend or foe?

Lysosomal ß-galactosylceramidase (GALC) removes ß-galactose from ß-galactosylceramide, thus generating the oncosuppressor metabolite ceramide. Recent observations have shown that GALC may exert opposite effects on tumor growth and differentiation, questioning its contribution to the sphingolipid metabolism in cancer cells and its role in tumor progression.

Caffeine Inhibits Direct and Indirect Angiogenesis in Zebrafish Embryos.

In this study, we report the effects of caffeine on angiogenesis in zebrafish embryos both during normal development and after exposure to Fibroblast Growth Factor 2 (FGF2). As markers of angiogenesis, we measured the length and width of intersegmental vessels (ISVs), performed whole-mount in situ hybridization with fli1 and cadh5 vascular markers, and counted the number of interconnecting vessels (ICVs) in sub-intestinal venous plexus (SIVP). In addition, we measured angiogenesis after performing zebrafish yolk membrane (ZFYM) assay with microinjection of fibroblast growth factor 2 (FGF2) and perivitelline tumor xenograft assay with microinjection of tumorigenic FGF2-overexpressing endothelial (FGF2-T-MAE) cells. The results showed that caffeine treatment causes a shortening and thinning of ISVs along with a decreased expression of the vascular marker genes and a decrease in the number of ICVs in the SIVP. Caffeine was also able to block angiogenesis induced by exogenous FGF2 or FGF2-producing cells. Overall, our results are suggestive of the inhibitory effect of caffeine in both direct and indirect angiogenesis.

Pentraxin 3 Inhibits the Angiogenic Potential of Multiple Myeloma Cells.

During multiple myeloma (MM) progression the activation of the angiogenic process represents a key step for the formation of the vascular niche, where different stromal components and neoplastic cells collaborate and foster tumor growth. Among the different pro-angiogenic players, Fibroblast Growth Factor 2 (FGF2) plays a pivotal role in BM vascularization occurring during MM progression. Long Pentraxin 3 (PTX3), a natural FGF antagonist, is able to reduce the activation of stromal components promoted by FGF2 in various in vitro models. An increased FGF/PTX3 ratio has also been found to occur during MM evolution, suggesting that restoring the "physiological" FGF/PTX3 ratio in plasma cells and BM stromal cells (BMSCs) might impact MM. In this work, taking advantage of PTX3-inducible human MM models, we show that PTX3 produced by tumor cells is able to restore a balanced FGF/PTX3 ratio sufficient to prevent the activation of the FGF/FGFR system in endothelial cells and to reduce the angiogenic capacity of MM cells in different in vivo models. As a result of this anti-angiogenic activity, PTX3 overexpression causes a significant reduction of the tumor burden in both subcutaneously grafted and systemic MM models. These data pave the way for the exploitation of PTX3-derived anti-angiogenic approaches in MM.

Specific targeting of the KRAS mutational landscape in myeloma as a tool to unveil the elicited antitumor activity.

Alterations in KRAS have been identified as the most recurring somatic variants in the multiple myeloma (MM) mutational landscape. Combining DNA and RNA sequencing, we studied 756 patients and observed KRAS as the most frequently mutated gene in patients at diagnosis; in addition, we demonstrated the persistence or de novo occurrence of the KRAS aberration at disease relapse. Small-molecule inhibitors targeting KRAS have been developed; however, they are selective for tumors carrying the KRASG12C mutation. Therefore, there is still a need to develop novel therapeutic approaches to target the KRAS mutational events found in other tumor types, including MM. We used AZD4785, a potent and selective antisense oligonucleotide that selectively targets and downregulates all KRAS isoforms, as a tool to dissect the functional sequelae secondary to KRAS silencing in MM within the context of the bone marrow niche and demonstrated its ability to significantly silence KRAS, leading to inhibition of MM tumor growth, both in vitro and in vivo, and confirming KRAS as a driver and therapeutic target in MM.

Chemical modification of NSC12 leads to a specific FGF-trap with antitumor activity in multiple myeloma.

Inhibition of FGF/FGFR signaling is a promising strategy for the treatment of malignances dependent from FGF stimulation, including multiple myeloma (MM). The steroidal derivative NSC12 (compound 1) is a pan-FGF trap endowed with antitumor activity in vivo. Chemical modifications of compound 1 were explored to investigate structure-activity relationships, focusing on the role of the bis(trifluoromethyl)1,3-propanediol chain, the stereochemistry at C20 and functionalization of C3 position. Our studies unveiled compound 25b, the pregnane 3-keto 20R derivative of compound 1 as an effective agent, blocking the proliferation of MM cells in vitro by inhibiting FGF-dependent receptor activation and slowing MM growth in vivo. Importantly, the absence of the hydroxyl group at C3 prevents binding to estrogen receptors, which might concur to the antitumor activity observed for compound 1, leading to a specific FGF/FGFR system inhibitor, and further supporting the role of FGFR in anticancer therapy in MM.

Expression of activated VEGFR2 by R1051Q mutation alters the energy metabolism of Sk-Mel-31 melanoma cells by increasing glutamine dependence.

Vascular endothelial growth factor receptor 2 (VEGFR2) activating mutations are emerging as important oncogenic driver events. Understanding the biological implications of such mutations may help to pinpoint novel therapeutic targets. Here we show that activated VEGFR2 via the pro-oncogenic R1051Q mutation induces relevant metabolic changes in melanoma cells. The expression of VEGFR2R1051Q leads to higher energy metabolism and ATP production compared to control cells expressing VEGFR2WT. Furthermore, activated VEGFR2R1051Q augments the dependence on glutamine (Gln) of melanoma cells, thus increasing Gln uptake and their sensitivity to Gln deprivation and to inhibitors of glutaminase, the enzyme initiating Gln metabolism by cells. Overall, these results highlight Gln addiction as a metabolic vulnerability of tumors harboring the activating VEGFR2R1051Q mutation and suggest novel therapeutic approaches for those patients harboring activating mutations of VEGFR2.

VEGF-Independent Activation of Müller Cells by the Vitreous from Proliferative Diabetic Retinopathy Patients.

Proliferative diabetic retinopathy (PDR), a major complication of diabetes mellitus, results from an inflammation-sustained interplay among endothelial cells, neurons, and glia. Even though anti-vascular endothelial growth factor (VEGF) interventions represent the therapeutic option for PDR, they are only partially efficacious. In PDR, Müller cells undergo reactive gliosis, produce inflammatory cytokines/chemokines, and contribute to scar formation and retinal neovascularization. However, the impact of anti-VEGF interventions on Müller cell activation has not been fully elucidated. Here, we show that treatment of MIO-M1 Müller cells with vitreous obtained from PDR patients stimulates cell proliferation and motility, and activates various intracellular signaling pathways. This leads to cytokine/chemokine upregulation, a response that was not mimicked by treatment with recombinant VEGF nor inhibited by the anti-VEGF drug ranibizumab. In contrast, fibroblast growth factor-2 (FGF2) induced a significant overexpression of various cytokines/chemokines in MIO-M1 cells. In addition, the FGF receptor tyrosine kinase inhibitor BGJ398, the pan-FGF trap NSC12, the heparin-binding protein antagonist N-tert-butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe Boc2, and the anti-inflammatory hydrocortisone all inhibited Müller cell activation mediated by PDR vitreous. These findings point to a role for various modulators beside VEGF in Müller cell activation and pave the way to the search for novel therapeutic strategies in PDR.

Avian Reovirus P17 Suppresses Angiogenesis by Promoting DPP4 Secretion.

Avian reovirus p17 (ARV p17) is a non-structural protein known to activate autophagy, interfere with gene transcription and induce a significant tumor cell growth inhibition in vitro and in vivo. In this study, we show that ARV p17 is capable of exerting potent antiangiogenic properties. The viral protein significantly inhibited the physiological angiogenesis of human endothelial cells (ECs) by affecting migration, capillary-like structure and new vessel formation. ARV p17 was not only able to suppress the EC physiological angiogenesis but also rendered ECs insensitive to two different potent proangiogenic inducers, such as VEGF-A and FGF-2 in the three-dimensional (3D) Matrigel and spheroid assay. ARV p17 was found to exert its antiangiogenic activity by upregulating transcription and release of the well-known tumor suppressor molecule dipeptidyl peptidase 4 (DPP4). The ability of ARV p17 to impact on angiogenesis is completely new and highlights the "two compartments" activity of the viral protein that is expected to hamper the tumor parenchymal/stromal crosstalk. The complex antitumor activities of ARV p17 open the way to a new promising field of research aimed to develop new therapeutic approaches for treating tumor and cancer metastasis.

Simultaneously characterization of tumoral angiogenesis and vasculogenesis in stem cell-derived teratomas.

Tumor neovascularization may occur via both angiogenic and vasculogenic events. In order to investigate the vessel formation during tumor growth, we developed a novel experimental model that takes into account the differentiative and tumorigenic properties of Embryonic Stem cells (ESCs). Leukemia Inhibitory Factor-deprived murine ESCs were grafted on the top of the chick embryo chorionallantoic membrane (CAM) in ovo. Cell grafts progressively grew, forming a vascularized mass within 10 days. At this stage, the grafts are formed by cells with differentiative features representative of all three germ layers, thus originating teratomas, a germinal cell tumor. In addition, ESC supports neovascular events by recruiting host capillaries from surrounding tissue that infiltrates the tumor mass. Moreover, immunofluorescence studies demonstrate that perfused active blood vessels within the tumor are of both avian and murine origin because of the simultaneous occurrence of angiogenic and vasculogenic events. In conclusion, the chick embryo ESC/CAM-derived teratoma model may represent a useful approach to investigate both vasculogenic and angiogenic events during tumor growth and for the study of natural and synthetic modulators of the two processes.

A novel variant of VEGFR2 identified by a pan-cancer screening of recurrent somatic mutations in the catalytic domain of tyrosine kinase receptors enhances tumor growth and metastasis.

In cancer genomics, recurrence of mutations in gene families that share homologous domains has recently emerged as a reliable indicator of functional impact and can be exploited to reveal the pro-oncogenic effect of previously uncharacterized variants. Pan-cancer analyses of mutation hotspots in the catalytic domain of a subset of tyrosine kinase receptors revealed that two infrequent mutations of VEGFR2 (R1051Q and D1052N) recur in analogous proteins and correlate with reduced patient survival. Functional validation showed that both R1051Q and D1052N mutations increase the enzymatic activity of VEGFR2. The expression of VEGFR2R1051Q potentiates the PI3K/Akt signaling axis in cancer cells, increasing their tumorigenic potential in vitro and in vivo. In addition, it confers to cancer cells an increased sensitivity to the VEGFR2-targeted tyrosine kinase inhibitor Linifanib. In the context of an efficacious application of anti-cancer targeted therapies, these findings indicate that the screening for uncharacterized mutations, like VEGFR2R1051Q, may help to predict patient prognosis and drug response, with significant clinical implications.