Ramiro Vázquez

Coumarins: old compounds with novel promising therapeutic perspectives.

Natural as well as synthetic coumarins have recently drawn much attention due to its broad pharmacological activities. Many coumarins and their derivatives exert anti-coagulant, anti-tumor, anti-viral, anti-inflammatory and anti-oxidant effects, as well as anti-microbial and enzyme inhibition properties. The recognition of key structural features within coumarin family is crucial for the design and development of new analogues with improved activity and for the characterization of their mechanism of action and potential side effects. The different substituents in the coumarin nucleus strongly influence the biological activity of the resulting derivatives. Although some coumarins have been already characterized to evoke a particular biological activity, the challenge would be the design and synthesis of new derivatives with high specific activity for other pharmacological targets and define their mechanism of action to achieve new therapeutic drugs. The present review highlights the current progress in the development of coumarin scaffolds for drug discovery as novel anti-cancer agents. The major challenges about coumarins include the translation of current knowledge into new potential lead compounds and the repositioning of known compounds for the treatment of cancer.

Toward establishing structure-activity relationships for oxygenated coumarins as differentiation inducers of promonocytic leukemic cells

The presumption that some coumarins might be lead compounds in the search for new differentiation agents against leukemia is based on the fact that natural coumarins, 5-(3-methyl-2-butenyloxy)-6,7-methylenedioxycoumarin (C-2) and 5-methoxy-6,7-methylenedioxycoumarin (C-1) inhibit proliferation and induce differentiation in U-937 cells [Riveiro, M. E.; Shayo, C.; Monczor, F.; Fernandez, N.; Baldi, A.; De Kimpe, N.; Rossi, J.; Debenedetti, S.; Davio, C. Cancer Lett.2004, 210, 179-188]. These promising findings prompted us to investigate the anti-leukemia activity of a broader range of related polyoxygenated coumarins. Twenty related natural or synthetically prepared coumarins, including a range of 5-substituted ayapin derivatives which have become easy accessible via newly developed synthesis methods, were evaluated, where treatments with 5-(2,3-dihydroxy-3-methylbutoxy)-6,7-methylenedioxycoumarin (D-3) and 5-(2-hydroxy-3-methoxy-3-methylbutoxy)-6,7-methylenedioxycoumarin (D-2) were able to inhibit the cell growth and induce the differentiation of U-937 cells after 48 h treatment. These results provide insight into the correlation between some structural properties of polyoxygenated coumarins and their in vitro leukemic differentiation activity.

Synthesis, structural characterization, and pro-apoptotic activity of 1-indanone thiosemicarbazone platinum(II) and palladium(II) complexes: potential as antileukemic agents.

In the search for alternative chemotherapeutic strategies against leukemia, various 1‐indanone thiosemicarbazones, as well as eight novel platinum(II) and palladium(II) complexes, with the formula [MCl2(HL)] and [M(HL)(L)]Cl, derived from two 1‐indanone thiosemicarbazones were synthesized and tested for antiproliferative activity against the human leukemia U937 cell line. The crystal structure of [Pt(HL1)(L1)]Cl.2MeOH, where L1=1‐indanone thiosemicarbazone, was solved by X‐ray diffraction. Free thiosemicarbazone ligands showed no antiproliferative effect, but the corresponding platinum(II) and palladium(II) complexes inhibited cell proliferation and induced apoptosis. Platinum(II) complexes also displayed selective apoptotic activity in U937 cells but not in peripheral blood monocytes or the human hepatocellular carcinoma HepG2 cell line used to screen for potential hepatotoxicity. Present findings show that, in U937 cells, 1‐indanone thiosemicarbazones coordinated to palladium(II) were more cytotoxic than those complexed with platinum(II), although the latter were found to be more selective for leukemic cells suggesting that they are promising compounds with potential therapeutic application against hematological malignancies.

The bromodomain inhibitor OTX015 (MK-8628) exerts anti-tumor activity in triple-negative breast cancer models as single agent and in combination with everolimus

Triple-negative breast cancer (TNBC) is an aggressive and heterogeneous subgroup of breast tumors clinically defined by the lack of estrogen, progesterone and HER2 receptors, limiting the use of the targeted therapies employed in other breast malignancies. Recent evidence indicates that c-MYC is a key driver of TNBC. The BET-bromodomain inhibitor OTX015 (MK-8628) has potent antiproliferative activity accompanied by c-MYC down-regulation in several tumor types, and has demonstrated synergism with the mTOR inhibitor everolimus in different models. The aim of this study was to evaluate the anti-tumor activity of OTX015 as single agent and in combination with everolimus in TNBC models. OTX015 was assayed in three human TNBC-derived cell lines, HCC1937, MDA-MB-231 and MDA-MB-468, all showing antiproliferative activity after 72 h (GI50 = 75–650 nM). This was accompanied by cell cycle arrest and decreased expression of cancer stem cells markers. However, c-MYC protein and mRNA levels were only down-regulated in MDA-MB-468 cells. Gene set enrichment analysis showed up-regulation of genes involved in epigenetic control of transcription, chromatin and the cell cycle, and down-regulation of stemness-related genes. In vitro, combination with everolimus was additive in HCC1937 and MDA-MB-231 cells, but antagonistic in MDA-MB-468 cells. In MDA-MB-231 murine xenografts, tumor mass was significantly (p < 0.05) reduced by OTX015 with respect to vehicle-treated animals (best T/C = 40.7%). Although everolimus alone was not active, the combination was more effective than OTX015 alone (best T/C = 20.7%). This work supports current clinical trials with OTX015 in TNBC (NCT02259114).

OTX015 (MK-8628), a novel BET inhibitor, exhibits antitumor activity in non-small cell and small cell lung cancer models harboring different oncogenic mutations

Inhibitors targeting epigenetic control points of oncogenes offer a potential mean of blocking tumor progression in small cell and non-small cell lung carcinomas (SCLC, NSCLC). OTX015 (MK-8628) is a BET inhibitor selectively blocking BRD2/3/4. OTX015 was evaluated in a panel of NSCLC or SCLC models harboring different oncogenic mutations. Cell proliferation inhibition and cell cycle arrest were seen in sensitive NSCLC cells. MYC and MYCN were downregulated at both the mRNA and protein levels. In addition, OTX015-treatment significantly downregulated various stemness cell markers, including NANOG, Musashi-1, CD113 and EpCAM in H3122-tumors in vivo. Conversely, in SCLC models, weak antitumor activity was observed with OTX015, both in vitro and in vivo. No predictive biomarkers of OTX015 activity were identified in a large panel of candidate genes known to be affected by BET inhibition. In NSCLC models, OTX015 was equally active in both EML4-ALK positive and negative cell lines, whereas in SCLC models the presence of functional RB1 protein, which controls cell progression at G1, may be related to the final biological outcome of OTX015. Gene expression profiling in NSCLC and SCLC cell lines showed that OTX015 affects important genes and pathways with a very high overlapping between both sensitive and resistant cell lines. These data support the rationale for the OTX015 Phase Ib (NCT02259114) in solid tumors, where NSCLC patients with rearranged ALK gene or KRAS-positive mutations are currently being treated.

Promising in vivo efficacy of the BET bromodomain inhibitor OTX015/MK-8628 in malignant pleural mesothelioma xenografts.

It has recently been reported that a large proportion of human malignant pleural mesothelioma (MPM) cell lines and patient tissue samples present high expression of the c‐MYC oncogene. This gene drives several tumorigenic processes and is overexpressed in many cancers. Although c‐MYC is a strategic target to restrain cancer processes, no drugs acting as c‐MYC inhibitors are available. The novel thienotriazolodiazepine small‐molecule bromodomain inhibitor OTX015/MK‐8628 has shown potent antiproliferative activity accompanied by c‐MYC downregulation in several tumor types. This study was designed to evaluate the growth inhibitory effect of OTX015 on patient‐derived MPM473, MPM487 and MPM60 mesothelioma cell lines and its antitumor activity in three patient‐derived xenograft models, MPM473, MPM487 and MPM484, comparing it with cisplatin, gemcitabine and pemetrexed, three agents which are currently used to treat MPM in the clinic. OTX015 caused a significant delay in cell growth both in vitro and in vivo. It was the most effective drug in MPM473 xenografts and showed a similar level of activity as the most efficient treatment in the other two MPM models (gemcitabine in MPM487 and cisplatin in MPM484). In vitro studies showed that OTX015 downregulated c‐MYC protein levels in both MPM473 and MPM487 cell lines. Our findings represent the first evidence of promising therapeutic activity of OTX015 in mesothelioma.

Abstract 3530: Gene expression profile of OTX015, a BET bromodomain inhibitor, in preclinical models of non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC) models

Background. BET bromodomain proteins recognize chromatin modifications and act as epigenetic readers contributing to gene transcription. OTX015, a novel BET bromodomain inhibitor currently in clinical Phase Ib studies in hematologic malignancies and solid tumors, including lung cancer. We have shown in vitro and in vivo activity of OTX015 in NSCLC and SCLC models (Riveiro et al; EORTC 2014), however the mechanism of action and relevant affected genes are not fully characterized. This study aimed to elucidate pathways and genes affecting response/resistance to OTX015 in lung models.Methods. OTX015 (OncoEthix SA, Switzerland), GI50 values were determined with the MTT assay after 72h exposure in 5 NSCLC cell lines (H2228, H3122, A549, HOP62, HOP92) and 4 SCLC cell lines (H82, H69, DMS79, DMS114). For cell cycle analysis, cells were stained with propidium iodide and analyzed with a FACScan flow cytometer. RNA was isolated after treatment with DMSO or OTX015 (500 nM) for 2, 4, 8 or 12 and 24h and gene expression profiles (GEP) were obtained in all NSCLC and 2 SCLC (H82 and DMS114) cell lines with Illumina HumanHT-12 Expression BeadChips. Results. OTX015 displayed antiproliferative effects in 4 of the 5 NSCLC cell lines with GI50 values from 110 to 940 nM, whereas A549 cells (harboring mutations in KRAS and LKB1 genes) were resistant to OTX015. Among SLCL models, DMS114 cells showed dose-dependent sensitivity to OTX015 [GI50 = 120nM], while H82, H69, and DMS79 cells were resistant [GI50 >6 μM], despite overexpressing CMYC and/or NMYC. In sensitive-NSCLC models we observed an increase in cells in the S phase after OTX015 treatment, whereas an increase in the G1 phase was observed in the sensitive DMS114 cell line. GEP showed that changes in expression levels of EFR3B and genes related to RNA biogenesis were higher in SCLC cells with reduced sensitivity to OTX015 than in sensitive cells. On the other hand, genes related to histones and chromatin structure were the most differentially expressed after OTX015 exposure in both sensitive and resistant cell lines. In NSCLC models, EFR3B and FOS gene up-regulation, IL7R and IL6-related gene down-regulation were associated with higher OTX015 sensitivity Expression changes more prominent in OTX015-resistant cells affected the transcription factor FOXD1 and genes related to RNA processing. Conclusions. Our data contribute to elucidate the mechanisms associated with OTX015 sensitivity in several lung cancer in vitro models, supporting the rationale for the ongoing clinical Phase Ib study in solid tumors including NSCLC patients (KRAS+; ALK+). Citation Format: Maria Eugenia Riveiro, Ivo Kwee, Lucile Astorgues-Xerri, Mohamed Bekradda, Ramiro Vazquez, Andrea Rinaldi, Esteban Cvitkovic, Francesco Bertoni. Gene expression profile of OTX015, a BET bromodomain inhibitor, in preclinical models of non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC) models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3530. doi:10.1158/1538-7445.AM2015-3530

Abstract 5530: OTX015, a novel pan BET-BRD inhibitor is active in non-small-cell lung cancer (NSCLC) cell lines bearing the fusion protein EML4-ALK

Background: Various inhibitors targeting the activity of BET proteins have been recently developed and have shown potent anti-proliferative effects in several tumors, including NSCLC. The fusion of the echinoderm microtubule-associated protein-like 4 (EML4) and the anaplastic lymphoma kinase (ALK) genes results in the chimeric oncogene EML4-ALK, identified as a distinct entity of NSCLC patients that enables effective ALK-targeted therapy. However, most of these patients invariably acquire resistance within few months. Herein, we report preclinical findings obtained with a novel oral pan-BET-BRD inhibitor, OTX015, in a panel of NSCLC cell lines, some of which bear the fusion protein EML4-ALK. Materials and Methods: Five established NSCLC cell lines (i.e. H2228, H3122, A549, HOP62 and HOP92) were exposed to increasing concentrations of OTX015 (OncoEthix SA, Switzerland). In each case, the effect on cell viability was determined by the MTT assay after 72 h of exposure. GI50 values, representing 50% growth inhibition concentration, were calculated using GraphPad Prism 5.0 software. Protein levels were determined by Western blot using commercial antibodies. RNA was extracted with the Qiagen RNAEasy kit and reverse-transcribed using the Superscript First-Strand Synthesis System for RT-PCR kit following manufacturer9s instructions. RT-PCR was performed using Fast SYBR Green Master Mix on a StepOnePlus Real-Time PCR System. Results: OTX015 displayed anti-proliferative activity in EML4-ALK-positive H2228 and H3122 cells after a 72-h treatment, with GI50 values of 629 and 627nM, respectively. The corresponding GI50 values for the benchmark compound, JQ-1, were 2161 and 1265 nM. Interestingly, OTX015 was also active in the EML4-ALK-negative A549 cell line, with a GI50 of 432 nM. BRD4/3/2, c-MYC, BCL-2, p21 and CyclinD1 were detected at protein and mRNA levels in all cell lines. Both OTX015-sensitive and -resistant cells exhibited similar basal expression levels for the aforementioned proteins. EML4-ALK variants 1 and 3 were identified in H3122 and H2228 cells, respectively. Assessment of the signaling pathways involved in the anti-proliferative activity of OTX015 in these cells showed a transient up-regulation of STAT3, followed by a down-regulation after 24 h and up to 72 h of exposure. These results indicate that the key downstream effector of OTX015 is STAT3, frequently found to be overexpressed in crizotinib-resistant cell lines. Interestingly, c-MYC protein and mRNA levels were not altered by OTX015. EML4-ALK-positive H3122 cells showed down-regulation of n-MYC mRNA levels after OTX015 treatment. Conclusion: Our results indicate that NSCLC cell lines with genomic ALK alterations are sensitive to BET-BRD inhibition by OTX015, suggesting its potential clinical use as anticancer agent in EML4-ALK positive NSCLC patients. Citation Format: Ramiro Vazquez, Lucile Astorgues-Xerri, Mohamed Bekradda, Esteban Cvitkovic, Patrice Herait, Michela Boi, Giorgio Inghirami, Maurizio D9Incalci, Maria E. Riveiro, Eric Raymond. OTX015, a novel pan BET-BRD inhibitor is active in non-small-cell lung cancer (NSCLC) cell lines bearing the fusion protein EML4-ALK. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5530. doi:10.1158/1538-7445.AM2014-5530

Abstract 5080: BET inhibitors INCB054329 and INCB057643 display significant activity in androgen-independent prostate cancer models

Prostate cancer is a leading cause of cancer death worldwide. Deregulated transcriptional factors and epigenetic effectors contribute to prostate cancer progression and castration resistance providing ideal targets for developing new therapeutic strategies. Bromodomain and extra-terminal (BET) proteins act transcriptional co-activators interacting with multiple co-regulatory molecules at gene promoters and enhancers. BET inhibitors (BETi) disrupt transcriptional regulatory complexes and have broad anticancer activity. INCB054329 and INCB057643 are BETi with proven pre-clinical activity in hematological and solid tumors. Both compounds are currently in clinical trials (NCT02431260 and NCT02711137) in advanced cancer patients. In this study we explored for the first time the activity of these two BETi in prostate cancer, comparing their effects in androgen-dependent and independent models in vitro and in vivo. We assessed the effect of INCB054329 and INCB057643 on cell proliferation, colony formation and tumor-sphere assays in androgen-dependent (LNCaP and VCaP) and androgen-independent (DU145, PC3, 22Rv1) cells. The in vivo antitumor activity was evaluated in mice with 22Rv1 derived subcutaneous xenografts with drugs administered orally (BID, daily for 3 weeks). INCB054329 and INCB057643 inhibited proliferation of prostate cancer cell lines. In short-term cell proliferation assays the BETi appeared more effective against androgen-dependent (VCaP and LNCaP) than androgen-independent (DU145 and PC3) cells (GI50 of ≤100 nM and ≥500 nM, respectively). 22Rv1 cells, which express androgen-independent AR splice variants, exhibited an intermediate level of sensitivity to both compounds (GI50: 150-250 nM). Interestingly, in colony and tumor-sphere forming assays all the cell lines showed substantially greater sensitivity to the BETi than in short-term assays. Notably, androgen-independent 22Rv1 cells were highly responsive with GI50 values ≤100 nM similar to those seen in androgen-sensitive LNCaP cells. Treatment of mice bearing 22Rv1 tumor xenografts with INCB054329 (50 mg/kg) and INCB057643 (3 mg/kg) led to significant inhibition of tumor growth (T/C%: 42 and 45%, respectively) and consistent reduction of tumor weight relative to vehicle-treated mice. These results provide evidence of activity of INCB054329 and INCB057643 in prostate cancer cell lines. Although short term assays suggest a preferential anti-proliferative effect in androgen sensitive prostate cancer cells, both compounds also exhibit significant activity in an androgen-independent model both in vitro and in vivo suggesting that they might represent a valid therapeutic option for treatment of castration-resistant prostate cancer. Citation Format: Ramiro Vazquez, Gianluca Civenni, Martina Marchetti, Sabrina Zadic, Phillip Liu, Bruce Ruggeri, Giuseppina M. Carbone, Carlo V. Catapano. BET inhibitors INCB054329 and INCB057643 display significant activity in androgen-independent prostate cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5080. doi:10.1158/1538-7445.AM2017-5080

Abstract 3526: OTX015 effects in triple-negative breast cancer (TNBC) models are independent of hypoxia conditions and synergistic with other anticancer agents

Background: TNBC is an aggressive and heterogeneous subtype group of breast cancers clinically defined by the lack of estrogen and progesterone receptors, as well as the human epidermal growth factor receptor 2 (HER2). Few therapeutic options have shown clinical benefit beyond cytotoxic chemotherapy. Clinical studies have demonstrated that more than 50% of human breast cancers present a much lower median O2 partial pressure than normal breast tissue, correlating with chemo- and radio-resistance. We previously reported the in vivo effects of the BET-bromodomain inhibitor OTX015 (OncoEthix SA, Switzerland) in TNBC models (Vazquez et al.; EORTC-ENA 2014; #580). The aim of this work was to evaluate the in vitro antitumor activity of OTX015 both in normoxic and hypoxic environments, as well as in combination with antitumor agents. Materials and Methods: OTX015 growth inhibition concentrations 50% (GI50) values were determined in HCC197, MDA-MB-231 and MDA-MB-468 human-derived TNBC cell lines after 48 and 72 h in normoxia and hypoxia (0.1% atmospheric O2), employing the MTT assay and cell counting. RT-PCR was performed with Fast SYBR Green Master Mix on a StepOnePlus Real-Time PCR System at baseline, and after 24, 48 and 72 h of OTX015 treatments. OTX015 was combined with docetaxel or the mTOR inhibitor, everolimus, and the combination index (CI) determined by the Chou-Talalay method (CI 1.1, antagonism). Results: OTX015 showed antiproliferative activity in the 3 cell lines after 48- and 72-h treatments in normoxia and hypoxia. MDA-MB-231 was the most sensitive in both conditions. Hypoxia significantly (p Citation Format: Ramiro Vazquez, Lucile Astorgues-Xerri, Maria E. Riveiro, Luca Beltrame, Sergio Marchini, Francesco Bertoni, Ivo Kwee, Mohamed Mohamed, Esteban Cvitkovic, Roberta Frapolli, Maurizio D9Incalci. OTX015 effects in triple-negative breast cancer (TNBC) models are independent of hypoxia conditions and synergistic with other anticancer agents. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3526. doi:10.1158/1538-7445.AM2015-3526