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Atypical retinoids (AR) or retinoid-related molecules (RRMs) represent a promising class of antitumor compounds. Among AR, E-3-(3'-adamantan-1-yl-4'-hydroxybiphenyl-4-yl)acrylic acid (adarotene), has been extensively investigated. In the present work we report the results of our efforts to develop new adarotene-related atypical retinoids endowed also with POLA1 inhibitory activity. The effects of the synthesized compounds on cell growth were determined on a panel of human and hematological cancer cell lines. The most promising compounds showed antitumor activity against several tumor histotypes and increased cytotoxic activity against an adarotene-resistant cell line, compared to the parent molecule. The antitumor activity of a selected compound was evaluated on HT-29 human colon carcinoma and human mesothelioma (MM487) xenografts. Particularly significant was the in vivo activity of the compound as a single agent compared to adarotene and cisplatin, against pleural mesothelioma MM487. No reduction of mice body weight was observed, thus suggesting a higher tolerability with respect to the parent compound adarotene.

Hybrid molecules targeting simultaneously DNA polymerase α (POLA1) and histone deacetylases (HDACs) were designed and synthesized to exploit a potential synergy of action. Among a library of screened molecules, MIR002 and GEM144 showed antiproliferative activity at nanomolar concentrations on a panel of human solid and haematological cancer cell lines. In vitro functional assays confirmed that these molecules inhibited POLA1 primer extension activity, as well as HDAC11. Molecular docking studies also supported these findings. Mechanistically, MIR002 and GEM144 induced acetylation of p53, activation of p21, G1/S cell cycle arrest, and apoptosis. Oral administration of these inhibitors confirmed their antitumor activity in in vivo models. In human non-small cancer cell (H460) xenografted in nude mice MIR002 at 50 mg/kg, Bid (qd × 5 × 3w) inhibited tumor growth (TGI = 61%). More interestingly, in POLA1 inhibitor resistant cells (H460-R9A), the in vivo combination of MIR002 with cisplatin showed an additive antitumor effect with complete disappearance of tumor masses in two animals at the end of the treatment. Moreover, in two human orthotopic malignant pleural mesothelioma xenografts (MM473 and MM487), oral treatments with MIR002 and GEM144 confirmed their significant antitumor activity (TGI = 72–77%). Consistently with recent results that have shown an inverse correlation between POLA1 expression and type I interferon levels, MIR002 significantly upregulated interferon-α in immunocompetent mice.

Nowadays, the increasing of multidrug-resistant pathogenic bacteria represents a serious threat to public health, and the lack of new antibiotics is becoming a global emergency. Therefore, research in antibacterial fields is urgently needed to expand the currently available arsenal of drugs. We have recently reported an alkyl-guanidine derivative (2), characterized by a symmetrical dimeric structure, as a good candidate for further developments, with a high antibacterial activity against both Gram-positive and Gram-negative strains. In this study, starting from its chemical scaffold, we synthesized a small library of analogues. Moreover, biological and in vitro pharmacokinetic characterizations were conducted on some selected derivatives, revealing notable properties: broad-spectrum profile, activity against resistant clinical isolates, and appreciable aqueous solubility. Interestingly, 2 seems neither to select for resistant strains nor to macroscopically alter the membranes, but further studies are required to determine the mode of action.

Small molecule modulators of chromatin modifying enzymes became the focus of drug discovery efforts, because of their direct antitumor effects, such as apoptosis and cell-cycle arrest, in addition to their indirect immunomodulation properties (Peedicayil, 2012; West and Johnstone, 2014; Falkenberg and Johnstone, 2014). Recent studies have shown that inhibition of HDACs or POLA1 activity exerts anti-proliferative effects and also potentiates the immune response by activating type I Interferon (Medon et al., 2017; Starokadomskyy et al, 2016). To exploit a potential synergy, hybrid molecules targeting simultaneously POLA1 and HDACs, were designed. Here, we present in vitro and in vivo data concerning the antitumor activity of new dual POLA1-HDAC11 inhibitors. Among a library of screened molecules, MIR002 and MIR144 showed an antiproliferative activity at nanomolar concentrations on a panel of human tumor cell lines. In vitro functional assays revealed that these molecules potently inhibit POLA1 and specifically act on HDAC11 that recently emerged as regulator of several immune cells differentiation, pointing out its immunomodulatory role. Mechanistically, MIR002 and MIR144 induce acetylation of Histone H4 and alpha-Tubulin, canonical targets of HDACs, and p53-K382, as well. Activation of p53 leads to p21 increase and cell cycle arrest in the G1/S boundary. These compounds could be even more effective in treating human cancers when they are appropriately combined with other chemotherapeutics, such as Cisplatin. Indeed, the combination of MIR002 or MIR144 with Cisplatin, revealed a synergistic interaction (Combination Index < 1) on NCI-H460 and H460-R9A (ST1926-resistant sub-line) cell growth. The in vivo oral administration of MIR002 or MIR144 showed a potent antitumor activity, with a Tumor Growth Inhibition comprised between 60 and 100% in a series of human and murine solid and hematological tumors (human MM473 and MM432 Pleural Mesothelioma, H460 and H460R9A NSCLC, A2780-Dx and A2780-DDP ovarian carcinomas, NB-4 Acute Promyelocitic leukemia and Murine EL-4 Lymphoma) xenografted in nude and immunocompetent mice. The strongest antitumor effect of MIR144 was observed in immunocompetent mice rather than in nude mice suggesting that an immunomodulatory effect, along with the direct antiproliferative activity, contributes to tumor inhibition. Further analysis confirmed that MIR144 antitumor activity involves the induction/activation of several components of the host immune system, both at humoral and cellular levels. The large spectrum of antitumor activity, together with the high tolerability observed, open the possibility for their clinical investigation in different population of cancer patients. Citation Format: Claudio Pisano, Lucio Merlini, Sergio Penco, Raffaella Cincinelli, Nadine Darwiche, Mario B. Guglielmi, Ilaria La Porta, Maddalena Pizzulo, Federica Prosperi, Giacomo Signorino, Fabiana Colelli, Francesco Cardile, Laura Focareta, Alessandra Fucci, Egildo L. D'Andrea, Assunta Riccio, Sabrina Dallavalle. Host antitumor immune response activated by Dual POLA1-HDAC11 inhibitors endowed with a large spectrum of antitumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3096.

The anti-proliferative and pro-apoptotic effects of Retinoid-Related Molecules (RRMs) are described as independent from Retinoids' receptors-mediated transcriptional activity. Prototypes of this class are CD437 and its more potent analogue ST1926, which have a strong antitumor activity by targeting DNA polymerase 1 alpha (POLA1) (Han et al. Nat Chem Biol. 2016; Abdel-Samad et al. AJCR, in press). With aim to identify new RRMs with an improved pharmacological profile, we synthetized and screened a library of RRMs for their antitumor properties and inhibitory activity on POLA1. From this screening, four molecules, MIR002, MIR020, MIR072 and MIR075, were selected. All exert a potent anti-proliferative effect, with G1/S arrest and apoptosis, in more than 50 cancer cell lines derived from human hematological and solid tumors. From a mechanistic point of view, these RRMs modulate, at different extent, POLA1 activity and/or expression. Notably, NSCLC cells harboring POLA1-L764F mutation (H460-R9A), are resistant to both CD437 and ST1926 (IC50>50 higher than the one of wild type H460 cells), while they are sensitive to MIR002, showing for this RRM-derivative an improved/different pharmacological profile with respect to CD437 and ST1926. Hints on the possibility of these new RRMs to be orally absorbed were obtained using cancer cells overexpressing or not P-glycoprotein (Pgp), known as the major player limiting the oral absorption of several chemotherapeutic agents. Results from these experiments revealed that the Tested compounds are not Pgp substrate thus suggesting the possibility for their absorption via oral route MIR002 in vivo activity was assessed in tumors from Malignant Mesothelioma derived cells (MM487), and lung cancer cells (H460 and H460-R9A). In all the evaluated models, MIR002 induced a strong Tumor Growth Inhibition either alone or in combination with cisplatin (TGI>61% and TGI>80-100%, respectively). The treatment of orthotopic models of malignant mesothelioma (MM487) with MIR072 in combination with Cisplatin, resulted in a impressive synergic antitumor activity if compared to Cisplatin monotherapy (TGI 95% vs 55%). Tests on orthotopic transplants of hepatocellular carcinoma cells (HepG2), showed that MIR020 has significant antitumor effects (TGI 72%). Finally, the activity of MIR075 was evaluated on glioblastoma luciferase-expressing cells (U-87MG) intracranically injected. Also this compound displayed a significant tumor growth inhibition (TGI 72%), as measured by IVIS imaging system. Taken together, the results from in vitro and in vivo experiments indicate that this new class of RRMs, including MIR002, MIR072, MIR020, and MIR075, modulate POLA1 functions and activate pro-apoptotic pathways. The large spectrum of antitumor activity, together with the high tolerability observed, opens the possibility for their clinical investigation in different population of cancer patients. Citation Format: Claudio Pisano, Lucio Merlini, Sergio Penco, Raffaella Cincinelli, Nadine Darwiche, Mario B. Guglielmi, Ilaria La Porta, Giacomo Signorino, Gabriele De Rubis, Fabiana Colelli, Francesco Cardile, Alessandra Fucci, Egildo L. D'Andrea, Sabrina Dallavalle. Preclinical antitumor activity of novel DNA polymerase 1 (POLA1) inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4848.

Hepatocyte growth factor (HGF) and macrophage-stimulating protein (MSP) have an intrinsic dual nature: they are trophic cytokines preventing apoptosis on one side and scatter factors promoting invasion on the other. For therapeutic use, their anti-apoptotic activity must be separated from their pro-invasive activity. To this end, we engineered chimeric factors containing selected functional domains of HGF and/or MSP in different combinations, and tested their biological activity. Here we present a chimeric cytokine derived from the alpha-chains of HGF and MSP, named Metron factor 1 for its ability to concomitantly activate the HGF receptor (Met) and the MSP receptor (Ron). We provide evidence that Metron factor 1 prevents apoptosis and stimulates cell proliferation at nanomolar concentrations, but is devoid of any pro-invasive activity. In an in vivo murine model of drug-induced nephrotoxicity, intravenous injection of recombinant Metron factor 1 prevented renal damage and preserved tubular integrity.

A recent work from Han et al. disclosed DNA polymerase 1 alpha (POLA1) as the key target for the anti-cancer effects of CD437, a molecule belonging to the class of Retinoid Related Molecules (RRMs). Before this evidence, this family of compounds, of which CD437 and ST1926 represent the prototypes, has been characterized for its antiproliferative, antitumor and pro-apoptotic activity. With aim to identify a new RRM with an improved pharmacological profile, we recently selected MIR002 as a novel compound endowed with a potent antitumor activity and a peculiar pharmacological profile. In vitro treatment with MIR002 leads to a G1/S arrest and exerts a potent anti-tumor/proapoptotic activity in a wide range of cancer cell lines, including H460-R9A cells, which are cross resistant to ST1926 and CD437 (IC50>70 fold of H460-R9A vs H460). Moreover, in H460-R9A cells, we identified the L764F mutation in POLA1, already described as a mutation conferring resistance to CD437. We also demonstrated that POLA1 is the molecular target of both MIR002 and ST1926 and that, interestingly, the expression of the L764F mutant, although associated with the resistance to RRM’s, only marginally reduces the cytotoxic effects of MIR002. In order to investigate the activity of MIR002 against tumor growth, we examined a panel of in vivo xenograft tumor models, including those originating from human Non-Small Cell Lung Cancer (NSCLC) and Malignant Pleural Mesothelioma (MPM). In these models, using a q2dx5x3w schedule in doses ranging from 50 to 70 mg/Kg administered orally, we observed a strong tumor growth inhibition (TGI>70%). Interestingly, the combination of MIR002 with Cisplatin (4mg/Kg; qd7x3w; i.v.) in MPM models showed a synergistic antitumor efficacy, reaching a TGI>90%, and cured animals. Interestingly, we previously identified POLA1 as a key pro-proliferative player in 9 primary MPM samples. The ex-vivo analyses indicated that POLA1 expression was strongly modulated upon MIR002 treatment. Furthermore, in the same models, we also observed that the expression/secretion of multiple circulating factors is affected by MIR002 and we identified some of them as potential biomarkers of tumor responsiveness to MIR002. Taken together, our results identify MIR002 as a novel anti-cancer compound and suggest POLA1 as a target for new antitumor strategies to be investigated in Clinical Trials. Citation Format: Claudio Pisano, Lucio Merlini, Sergio Penco, Raffaella Cincinelli, Nadine Darwiche, Mario B. Guglielmi, Antonietta Esposito, Ilaria La Porta, Giacomo Signorino, Gabriele De Rubis, Fabiana Colelli, Francesco Cardile, Alessandra Fucci, Egildo L. D'Andrea, Sabrina Dallavalle. MIR002: a new POLA1 inhibitor endowed with a large spectrum of antitumor activity [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 4195. doi:10.1158/1538-7445.AM2017-4195

In order to isolate, characterize, and establish culture cell lines with different diagnostic and prognostic significance, derived from multiclonal neoplasms, a ductal infiltrating mammary tumor was induced in rats by 7,12-dimethylbenz[a]anthracene. Clones with different DNA/protein content, being the DI of 1.16, 1.30, and 1.60, respectively, were observed in the primary tumor. Biparametric flow cytometry suggested that the clone at 1.30 is made up of two subpopulations with different protein and slightly different DNA contents. The culture, after a few passages, exhibited the presence of aneuploid cells and the absence of diploid components, demonstrating that only tumor cells survived. The limiting dilution method gave rise to four lines with DI of 1.16, 1.25, 1.30, and 1.50; a mean chromosome number of 45, 46, 47, and 88, respectively; and different morphological and ultrastructural features. These characteristics were stable during the experimental procedure, that is, for about 20 passages. Conversely, the detection of cytoskeletal proteins indicated that the tumor epithelial cells underwent early dedifferentiation into sarcoma-like cells showing markers of stromal cell type and thus exhibiting phenotypic instability in vitro, a feature reported in many advanced human breast cancers in vivo. In conclusion, this cellular model represents the in vivo situation and appears suitable for in vitro studies of tumor cell characteristics and might be used to predict clinical behavior.

The possibility that a significant fraction of cardiac myocyte loss in various disease states occurs through apoptosis has elicited considerable attention in recent years. Evidence from human studies as well as in vitro and animal models of disease has shown that cardiac myocyte apoptosis can be induced by a variety of stimuli and in a number of disease states, including hypoxia, ischemia–reperfusion, myocardial infarction, mechanical stretch, aortic constriction, and heart failure. Because adult cardiac myocytes are terminally differentiated cells, the effects of such loss can never be fully compensated. Interest in cardiomyocyte apoptosis has been fueled by the possibility that once the proximal and distal signals were defined that initiate this pathway of cell removal, it would be possible to develop stategies to selectively interfere with such signaling and prevent the loss of cardiac function. This article examines the evidence for possible proximal stimuli of apoptosis in the heart, including ligand-dependent activation of the membrane receptors for Fas ligand and tumor necrosis factor-α, and, in particular, activation of the tumor suppressor gene p53. It relates what is known about the mechanism by which these stimuli in other cells induce apoptosis and discusses possible strategies for inhibiting apoptosis in the heart.

A systematic study of medicinal chemistry aimed at identifying a new generation of HDAC inhibitors, through the introduction of a thiol zinc-binding group (ZBG) and of an amide-lactam in the ω-position of the polyethylene chain of the vorinostat scaffold, allowed the selection of a new class of potent pan-HDAC inhibitors (pan-HDACis). Simple, highly versatile, and efficient synthetic approaches were used to synthesize a library of these new derivatives, which were then submitted to a screening for HDAC inhibition as well as to a preliminary in vitro assessment of their antiproliferative activity. Molecular docking into HDAC crystal structures suggested a binding mode for these thiol derivatives consistent with the stereoselectivity observed upon insertion of amide-lactam substituents in the ω-position. ST7612AA1 (117), selected as a drug candidate for further development, showed an in vitro activity in the nanomolar range associated with a remarkable in vivo antitumor activity, highly competitive with the most potent HDAC inhibitors, currently under clinical trials. A preliminary study of PK and metabolism is also illustrated.

Objectives:To analyse the role of growth factors (platelet derived growth factor, PDGF; basic fibroblast growth factor, bFGF; interleukin 1, IL-1) in the genesis of myointimal hyperplasia in arterial allografts.Materials:Two groups of experiments were performed: isografts and allografts. The isograft group consisted of 15 inbred Lewis rats in which a 1 cm long segment of aorta was inserted as an abdominal aortic interposition graft. The aortic segments were obtained from syngenic Lewis rats. The allograft group consisted of 15 inbred Lewis rats, in which a 1 cm long segment of aorta was interposed at the abdominal aorta level. The aortic segments were obtained from allogenic Brown-Norway rats.Chief outcome measures:The animals were killed 4 weeks after surgery and were analysed by morphometric analysis (n = 3 for each group). In addition, production of PDGF, bFGF and IL-1 by aortic segments (n = 12 for each group) in organ culture was assessed.Main results:Allografts had more myointimal hyperplasia, than isografts (p < 0.05). PDGF and bFGF production, generally considered to be the cause of myointimal hyperplasia, was not increased in allografts. IL-1 production was higher in allografts (p < 0.001).Main conclusions:Myointimal hyperplasia in aortic allografts is dependent on growth factors produced by the graft itself. These growth factors are different from PDGF and bFGF that generally have been implicated in the genesis of naturally occurring myointimal hyperplasia and atherosclerosis. IL-1 may have a principal role in the genesis of myointimal hyperplasia in arterial allografts.

Poly(ADP-ribose)polymerase-I (PARP-1) enzyme is involved in maintaining DNA integrity and programmed cell death. A virtual screening of commercial libraries led to the identification of five novel scaffolds with inhibitory profile in the low nanomolar range. A hit-to-lead optimization led to the identification of a group of new potent PARP-1 inhibitors, acyl-piperazinylamides of 3-(4-oxo-3,4-dihydro-quinazolin-2-yl)-propionic acid. Molecular modeling studies highlighted the preponderant role of the propanoyl side chain.

Malignant pleural mesothelioma (MPM) development is mainly correlated with exposure to asbestos and appears to be a trend toward an increase in its incidence in the years to come. Although novel therapeutic strategies are under investigation, alone or in combination with conventional therapy, MPM remains a cancer with high medical need. With the aim to identify putative novel therapeutic targets/biomarkers expressed in specific MPM histotypes we investigated 9 different primary MPM cell lines at molecular, biochemical and pharmacological levels. A transcriptomic analysis has been conducted comparing the gene expression in epitheliod (E-MM473), sarcomatoid (S-MM432) and biphasic (B-MM487) primary cell lines. We found more than 1000 genes differentially regulated and statistically validated (p< = 0.05). The hierarchical clustering of the top 100 expressed genes revealed an histotype specific expression while few of them have a similar expression among the histotypes. 14 out of 100 genes were chosen based on their role in cell survival (hk1; gdf15; ifnar1/2; bmpr1/2; tgfrb; cdca8; birc5; plk1; srsf6; pin1; nox1; wt1). Their expression was confirmed via RT-PCR in all the 9 MPM cell lines (E-MM473, E-MM288, E-MM317, E-MM404, E-MM481; S-MM432, S-MM472; B-MM487, B-MM491) vs normal mesothelium cells (MET5A). Among the validated genes, gdf15 and hk1 expression strongly correlated with the epithelioid and bifasic histotype, respectively. The tumor relevance of these genes has been confirmed by siRNA interference in which hk1 and gdf15 transcript reduction significantly affected S-MM432 (>90%) and E-MM473/B-MM487 cell survival (>50%). GDF15 belongs to the TGF-β superfamily and its role in different pathologies including cancer is controversial (Breit et al., 2011). Its overexpression has been associated to tumor chemoresistance (Chung-Ying et al.,2007) including Cisplatin (Cis) (Li et al., 2015; Yang et al. 2014). To this end, we investigated if GDF15 protein levels in MPM cells exposed to Cis correlate with its citotoxicity. Interestingly GDF15 protein level, which is high in E-MM473, medium in B-M M432 and low in S-MM487, is inversely correlated with the cells sensitivity to the treatment (i.e. E-MM473 are 2-5 times more resistant) and GDF15 silencing increases Cis-induced cytotoxicity. To assess the relationship between GDF15 levels/ Cis resistance, efficacy of Cis (4mg/kg; q7dx3w;i.v.) on stably expressing luciferase E-MM473(highGDF15) and B-MM487 (lowGDF15), orthotopically xenografted in nude mice, were investigated. While, E-MM473 showed a poor response to the Cis (TGI 58%), an impressive tumor response (TGI 95%) was observed in treated B-MM487. Our results suggest and support the need of further clinical investigation to correlate GDF15 plasma level to the chemotherapy response in MPM patients. Citation Format: Claudio Pisano, Gianluca Carletta, Fulvio D’Angelo, Antonietta Esposito, Erminia Bianchino, Assunta Riccio, Michela Festa, Francesco Cardile, Emanuele Carchia, Walter D’Acunto, Giacomo Signorino, Mario B. Guglielmi, Pasquale De Luca. Molecular profiling of primary malignant pleuralmesothelioma histotypes cell lines reveals relationship between GDF15overexpression and cisplatin resistance both in in vitro and in vivo models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2936.

Adarotene belongs to the so-called class of atypical retinoids. The presence of the phenolic hydroxyl group on Adarotene structure allows a rapid O-glucuronidation as a major mechanism of elimination of the drug, favoring a fast excretion of its glucuronide metabolite in the urines. A series of ether, carbamate and ester derivatives was synthesized. All of them were studied and evaluated for their stability at different pH. The cytotoxic activity in vitro on NCI-H460 non-small cell lung carcinoma and A2780 ovarian tumor cell lines was also tested. A potential back-up of Adarotene has been selected to be evaluated in tumor models. (C) 2012 Elsevier Ltd. All rights reserved.

A structural investigation on the isoxazole scaffold led to the discovery of 3,4-isoxazolediamide compounds endowed with potent Hsp90 inhibitory properties. We have found that compounds possessing a nitrogen atom directly attached to the C-4 heterocycle ring possess in vitro Hsp90 inhibitory properties at least comparable to those of the structurally related 4,S-diarylisoxazole derivatives. A group of compounds from this series of diamides combine potent binding affinity and cell growth inhibitory activity in both series of alkyl- and aryl- or heteroarylarnides, with IC50 in the low nanomolar range. The 3,4-isoxazolediamides were also very effective in causing dramatic depletion of the examined client proteins and, as expected for the Hsp90 inhibitors, always induced a very strong increase in the expression levels of the chaperone Hsp70. In vivo studies against human epidermoid carcinoma A431 showed an antitumor effect of morpholine derivative 73 comparable to that induced by the reference compound 10.

Purpose: Acetyl-l-carnitine (ALC) plays a relevant role in energy metabolism and stress response because of its function in the complex metabolic system regulating the acetyl-CoA levels that provide a source of acetyl groups for metabolic and acetylation-regulated processes. Because acetylation may influence p53 activity/stability and, therefore, the response to platinum compounds, this study was designed to investigate the effect of ALC in combination with platinum compounds. Experimental Design: The antiproliferative and antitumor activity studies were done in a panel of human tumor cell lines with functional or defective p53. The antimetastatic drug efficacy was investigated in the s.c. growing H460/M tumor subline, which is able to generate lung metastases. Results: ALC enhanced the sensitivity to cisplatin of tumor cells with functional p53. The sensitization by ALC was reflected in an improved in vivo antitumor efficacy of the combination over cisplatin alone in wild-type p53 lung tumors. ALC did not increase the cisplatin efficacy in the p53-mutant SW620 tumor. ALC exhibited a significant antimetastatic activity, and this effect was better exploited in combination with the histone deacetylase inhibitor, ST3595. The in vivo ALC/cisplatin combination caused the activation of p53, associated with protein acetylation and induction of target genes. Conclusions: ALC was effective in enhancing the antitumor potential of platinum compounds in wild-type p53 tumors. ALC, alone and in combination with a histone deacetylase inhibitor, exhibited an outstanding antimetastatic activity. Both effects, likely mediated by protein acetylation, may have implications for platinum-based therapy and combinations with histone deacetylase inhibitors. Clin Cancer Res; 16(15); 3944–53. ©2010 AACR.

ST1968 is a novel hydrophilic camptothecin (CPT) derivative of the 7-oxyiminomethyl series. Because ST1968 retained ability to form remarkably stable cleavable complexes, this study was done to investigate its preclinical profile of antitumor activity in a large panel of human tumor models, including irinotecan-resistant tumors. Although less potent than SN38 in vitro, i.v. administered ST1968 caused a marked tumor inhibition, superior to that of irinotecan, in most tested models. ST1968 exhibited an impressive activity against several tumors including models of ovarian and colon carcinoma in which a high rate of cures was observed. In the most responsive tumors, complete and persistent tumor regressions were achieved even with low suboptimal doses. Even tumors derived from intrinsically resistant cells exhibited a significant responsiveness. Histologic analysis of treated tumors supports a contribution of both proapoptotic and antiangiogenic effects to ST1968 antitumor efficacy. A study done in yeast cells transformed with CPT-resistant mutant forms of topoisomerase I documented that, in contrast to other tested CPT, ST1968 was active against yeasts expressing the mutant K720E enzyme. Based on its outstanding efficacy superior to that of irinotecan and of its good therapeutic index, ST1968 has been selected for clinical development. [Mol Cancer Ther 2008;7(7):2051–9]

With the aim of understanding the influence of fluorine on the double bond of the cis-stilbene moiety of combretastatin derivatives and encouraged by a preliminary molecular modeling study showing a different biological environment on the interaction site with tubulin, we prepared, through various synthetic approaches, a small library of compounds in which one or both of the olefinic hydrogens were replaced with fluorine. X-ray analysis on the difluoro-CA-4 analogue demonstrated that the spatial arrangement of the molecule was not modified, compared to its nonfluorinated counterpart. SAR analysis confirmed the importance of the cis-stereochemistry of the stilbene scaffold. Nevertheless, some unpredicted results were observed on a few trans-fluorinated derivatives. The position of a fluorine atom on the double bond may affect the inhibition of tubulin polymerization and cytotoxic activity of these compounds.

We studied the anticancer activity of a series of new combretastatin derivatives with B-ring modifications. The structure-activity relationship (SAR) information confirmed the importance of cis-stereochemistry and of a phenolic moiety in B-ring. We selected the benzo[b]thiophene and benzofuran combretastatin analogues 11 (ST2151) and 13 (ST2179) and their phosphate prodrugs (29 and 30) for their high antitumor activity in in vitro and in vivo models. Cell exposure to IC50 of 11, 13, and CA-4 led to the arrest of various cell types in the G2/M phase of the cell cycle and induction of apoptosis. Mainly, 11 and 13 induced the formation of multinucleated cells with abnormal chromatin distribution, with only a minimal effect on the microtubule organization, with respect to CA-4. Interestingly, both the pharmacokinetic profile of 29 and its in vivo antitumor effect and those of 30, active even after oral administration, suggest additional pharmacological differences between these compounds and CA-4P.

library were cultured for 13 days, after which shRNAs were recovered by PCR. Deep sequencing was applied to determine the relative abundance of each shRNA in BRAF (V600E) cells as compared to WT2 CC cells. Results and Conclusions: Based on the results of the pooled shRNA screen, we were able to identify six candidate synthetic lethal genes in BRAF mutant CC cell lines. In particular, further validation showed RANBP2 gene knock-down to be synthetic lethal with BRAFV600E and BRAFlikeness in CC. Experiments addressing the identification and the biological characterization of RANBP2 will be presented.

Mesothelioma, sometimes referred to as asbestos cancer, is known to be among the most aggressive and difficult to treat tumours. The disease attacks the mesothelium, a protective two-layered membrane that covers the internal organs of the body including the lungs, heart and abdominal organs. Mesothelioma can affect any of these layers, but is usually seen in the pleural (lung) or peritoneal (abdomen) mesothelium. The most commonly diagnosed form of this cancer is pleural mesothelioma, caused primarily by the inhalation of asbestos fibers. The prevalence of this cancer is estimated at less than 1% of all cancers, however its incidence is increasing, with an expected peak in the next 10-20 years. Mesotheliomas are tumours that have a poor prognosis due to limited treatment options. The most aggressive form is the sarcomatoid histotype, followed by the biphasic and the epithelioid histotypes.In order to setup in vivo models of human mesothelioma, translational related to human pathology in terms of histology, antigen expression and pharmacological response to therapy, we have established three mesothelioma in vivo models using primary cells derived from patients having different mesothelioma histotypes.We stably transfected MM-432 (sarcomatoid), MM-473 (epithelioid) and MM-487 (biphasic) mesothelioma cell lines with a luciferase expression vector. The selected high luc-espressing clones were inoculated intrapleurally in immunodeficient nude mice, and they successfully invaded and proliferated within the murine host. In particular, the epithelioid histotype presented tumour growth 100% of the time, after a short latency period. The biphasic histotype failed to present tumour growth in 30% of the cases, however was far more aggressive than the epithelial histotype upon establishment of tumour growth. Moreover, the ex-vivo Immunocytochemistry (i.e for CEA, EMA, Mesothelin, Podoplanin, calretinin) and biochemical characterization (i.e.EGFR, VEGF, Top1, TKs pathways) of the three histotypes, revealed histotype-related differences. Finally, the cytotoxic responses to a panel of antitumor drugs (i.e . Doxorubicin , Topotecan , Ciclofosfamide, Dacarbazina, Gemcitabine, Temozolomide, Bortezomib, 5-azacitidina, Paclitaxel, Etoposide, 5-FU, Cisplatin, and AZD-2281) revealed that the three original cell lines, compared to the corresponding luc-expressing clones, had similar sensitivities to drugs depending upon the tumor histotypes. Further, in vivo characterizations focused on chemotherapeutic responses of these models, are currently in progress. Citation Format: Claudio Pisano, Alison G. Cole, Marcella Barbarino, Erminia Bianchino, Mario Guglielmi, Carmela Melito, Grazia Mercadante, Alfonsina Porciello, Assunta Riccio, Ilaria La Porta, Sara Orecchia, Roberta Libener, Laura Mazzucco, Simonetta Andrea Licandro, Pasquale De Luca. Mice models of three human mesothelioma histotypes derived from primary patient tumors. [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 5556. doi:10.1158/1538-7445.AM2014-5556

Histone deacetylase inhibitors (HDACi) are one of the last frontiers in pharmaceutical research. Two HDACi are already part of armamentarium of the anticancer drugs of the oncologists: Vorinostat, Zolinza® (SAHA) and Romidepsin, Istodax® (depsipeptide; FK-228), both licensed by the FDA for the treatment of cutaneous T-cell lymphoma (CTCL). Beyond them, more than twenty new drugs are currently under pre-clinical and clinical investigation as single agent and in combination therapies against different cancers and in other several novel therapeutic indications. Mainly they are hydroxamic acids and benzamide derivatives; however none has emerged superior to SAHA in terms of potency. In our search, we have selected a new generation of HDACi: thiol-based SAHA bearing a lactam amide in ω-position. This new generation was highly competitive compared to competing drugs, displaying sub-micromolar to low nanomolar inhibitor activity on HDACs, being especially powerful on HDAC6 isoform. They exhibited higher anti-proliferative activity than SAHA on different human cell lines. In vivo they were orally administered and showed a higher potency than SAHA, with a negligible toxicity. The overall profile of this new class of HDAC inhibitors, including synthesis and a comprehensive pharmacological characterization, will be presented. These encouraging results prompted us to select a drug candidate which is currently in a phase of pre-clinical evaluation. Citation Format: Giuseppe Giannini, Gianfranco Battistuzzi, Davide Vignola, Loredana Vesci, Ferdinando Maria Milazzo, Mario Berardino Guglielmi, Marcella Barbarino, Claudio Pisano, Walter Cabri. New thiol-ω(γ-lactam amide) SAHA analogues as potent histone deacetylase (HDAC) inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1010. doi:10.1158/1538-7445.AM2013-1010

Synthetic lethality (SL) is a cellular condition in which two or more non-allelic and non-essential mutations, which are not lethal on their own, become deadly when present within the same cell. PARP inhibitors (PARPi) represent a paradigmatic example of therapeutic application of the SL approaches. In cancer cells with BRCA1 or BRCA2 loss of function, inhibition of PARP1 activity leads to an accumulation of single strand breaks converted to double strand breaks but cannot be repaired by homologous recombination. However, preclinical and preliminary clinical evidences suggest a potentially broader scope for PARPi. Currently, several PARPi are under different phases of clinical investigation as monotherapy or in combination with DNA damaging agents. Although PARPi in monotherapy are quite safe, their combination with cytotoxic agents leads to exacerbation of the typical cytotoxic-related side effects, thus reducing the potentiality to improve the therapeutic index (T.I.) of these combinations. The study aim was to assess if the combination of PARPi and DNA-damaging agents with the widely used antimalarial agent DHA could improve both their antitumor activity and T.I. We have preliminary observed, in different cancer cells, that DHA was an inducer of endoplasmic reticulum (ER) stress (increased expression of typical ER stress-related genes), activation of checkpoint kinases and transcriptional up-regulation of different p53-target genes and of mitochondrial membrane depolarization. A consequence is PARP activation that, in absence of other factors, leads the cells to recover from damage and to their rescue. On the contrary, we observed that the simultaneous use of DHA with a PARPi or cytotoxic agents causes a massive cell death. More specifically a strong synergism was observed on tumor cells, when DHA was combined with different drugs: doxorubicin and platinum compounds on NCI-H460, temozolomide (TMZ) on MDA-MB436 and CAPAN-1. In addition, a synergistic interaction was identified between DHA and the PARPi (ABT-888 or AZD-2281) on various tumor cells (e.g., A431, HCT116, SW620, MDA-MB436, NCI-H460, A2780/DDP, CAPAN-1). In vivo, the combination of DHA (delivered at 200 mg/kg p.o. according to the schedule qdx5/w) with ABT-888 or AZD-2281, resulted in a significantly higher tumor growth inhibition than with monotherapy in NCI-H460 NSCLC, HCT116 colon ca. and MDA-MB436 triple-negative breast cancer BRCA1 negative xenografted in nude mice. DHA was also able to synergize in vivo with the alkylating agent TMZ against MDA-MB436 tumor model. No toxic effects of administrations of DHA in combination with chemotherapeutics in terms of body weight recordings were found throughout the experiments.Taken together, these results clearly pinpoint the clinical potential offered by DHA in the SL approaches in developing less toxic and more efficacious therapies to treat cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1774. doi:1538-7445.AM2012-1774

Hsp90 (heat shock protein 90) is a component of a molecular chaperone complex, involved in the folding, maturation and stabilisation of key signalling proteins which control cell proliferation, survival and transformation. It works by modulating a set of cancer-associated proteins, that are often over-expressed and/or mutated in tumor cells, collectively referred as ‘‘clients’’. Inhibition of Hsp90 causes destabilization and eventual degradation of client proteins involved in cell cycle, tumor growth, angiogenesis and apoptosis. Therefore, their modulation results in suppression of tumor growth by multiple parallel mechanisms. Consequently, inhibition of Hsp90 is believed to be a potential target for cancer therapy, also supported by a number of molecules currently under investigation in different phase of clinical trials. We recently synthesized 1,4,5-trisubstituted-1,2,3-triazole derivatives and surprisingly found that these new molecules demonstrated to be extremely active both on the biological target and on human tumor cell lines, even at nanomolar concentration. 3D QSAR analysis was also performed in order to rationalize HSP90 binding data. The overall profile of this new class of 1,2,3-triazoles, including stereoselective synthesis and a comprehensive pharmacological in vitro characterization, will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3896. doi:1538-7445.AM2012-3896

The novel hydroxamic acid-based compound ST3595, was identified within an in vitro screening for its inhibitory activity on histone deacetylase's (HDAC's) (Pisano C. et al., Clin Cancer Res 2010, 16 (15): 3944-53; Dalla Valle S et al., Eur J Med Chem 2009, 44 (5): 1900-12). With aim to define its antitumor potential, we investigated the in vivo activity of ST3595 in a variety of tumor models. In addition, we performed a comparative study of ST3595 and vorinostat (SAHA) to better understand the cellular/molecular basis of the antiproliferative activity. In in vivo experiments, ST3595, delivered orally or i.p. at well-tolerated doses (100 mg/kg) induced a significant tumor growth inhibition (ranging from 57 to 87%) in a variety of human tumor xenografts (H460 non-small cell lung, HCT116 colon and A2780 ovarian carcinoma). In in vitro studies, ST3595 exhibited arrest of H460 cells (wt p53) in S phase associated with a large extent of apoptosis. Under conditions producing comparable antiproliferative effects, treatment with vorinostat resulted in a G2/M arrest with a marginal induction of apoptosis. In HT29 cells (mutant p53) ST3595 caused arrest in S and G2/M phases with apoptosis induction, whereas vorinostat induced G1 accumulation without evidence of apoptosis, thus suggesting only antiproliferative effects. To explore the basis of the different cellular effects, we investigated the modulation of various factors implicated in DNA damage response and repair, cell cycle control and regulation of apoptosis. In H460 cells, we found that, in contrast to vorinostat, ST3595 was an effective inducer of p53 acetylation, supporting a role of this p53 modification in the proapoptotic activity of ST3595. In addition, in contrast to vorinostat, ST3595 strongly affected the expression of critical proteins involved in cell cycle control and DNA damage response. In particular, ST3595 induced activation and phosphorylation of H2AX, and of the checkpoint kinases chk1 and chk2, thus supporting an interference of the HDAC inhibitor in the processes controlling DNA integrity. In the light of the use of broad-specificity HDAC inhibitors in cancer therapy and in combination with other antitumor agents, the ability of ST3595 to modulate DNA damage response and it proapoptotic efficacy may have relevant implications in enhancing antitumor activity of well-established cytotoxic agents. Preclinical studies of ST3595 with genotoxic agents support this interpretation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2623. doi:10.1158/1538-7445.AM2011-2623