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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.

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.

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