Abstract: Altered expression and activity of cyclin-dependent kinase (CDK) and tropomyosin receptor kinase (TRK) families are observed in a wide variety of tumors. In those malignancies with aberrant CDK activation, the retinoblastoma protein (pRb) pathway is deregulated, leading to uncontrolled cell proliferation. Constitutive activation of TRKs is instead linked to cancer cell survival and dissemination. Here, we show that the novel small-molecule PHA-848125, a potent dual inhibitor of CDKs and TRKs, possesses significant antitumor activity. The compound inhibits cell proliferation of a wide panel of tumoral cell lines with submicromolar IC50. PHA-848125–treated cells show cell cycle arrest in G1 and reduced DNA synthesis, accompanied by inhibition of pRb phosphorylation and modulation of other CDK-dependent markers. The compound additionally inhibits phosphorylation of TRKA and its substrates in cells, which functionally express this receptor. Following oral administration, PHA-848125 has significant antitumor activity in various human xenografts and carcinogen-induced tumors as well as in disseminated primary leukemia models, with plasma concentrations in rodents in the same range as those found active in inhibiting cancer cell proliferation. Mechanism of action was also confirmed in vivo as assessed in tumor biopsies from treated mice. These results show that the dual CDK-TRK inhibitor PHA-848125 has the potential for being a novel and efficacious targeted drug for cancer treatment. Mol Cancer Ther; 9(8); 2243–54. ©2010 AACR.

Abstract: Most of the widely used anticancer drugs target the elongation step of DNA synthesis, either directly, for example by restricting the pool of dNTPs or by acting as chain terminators following incorporation into the nascent DNA strands, or indirectly, for example by targeting enzymes that facilitate replication fork progression, or by intercalating DNA and thereby creating a physical block to fork progression. The arrest of replication forks induced by these agents invariably results in DNA strand breakage, which, while being potentially toxic to normal proliferating tissues, is also susceptible to escape by tumor cells via activation of the ATR/ATM dependent S-phase checkpoint pathway and subsequent repair of the damaged lesion. Targeting the initiation step of DNA replication, rather than elongation, might represent a strategy to overcome activation of the S-phase checkpoint, and in this context a potentially attractive molecular target is the serine/threonine kinase Cdc-7. Cdc7 promotes DNA replication initiation via phosphorylation of one or more subunits of the MCM DNA helicase complex, thus facilitating unwinding of double-stranded DNA at replication origins. Phosphorylation of Mcm2 at Ser40 and Ser53 is uniquely dependent upon Cdc7 and is essential for initiation of DNA replication and cell growth. The altered expression of proteins involved in the initiation of DNA replication closely correlates with aggressive tumor phenotypes and is a powerful marker of clinical outcome in a variety of malignancies. In particular, Cdc7 protein levels and Cdc7 dependent activity are increased in many cancer cell lines, and in tumors compared to matched normal tissue, where correlation with negative prognosis has been observed for ovarian, breast, colon and uterine carcinomas, diffuse large B cell non-Hodgkin's and Hodgkin's lymphomas. Somatic mutations of the CDC7 gene have also been identified in colorectal and gastric carcinomas. Depletion of Cdc7 by RNA interference results in p53 independent apoptosis of tumor cells, whereas normal cells respond with reversible arrest of cell cycle progression, perhaps corresponding to a physiological checkpoint. Sustained inhibition of Cdc7 in the presence of drugs affecting the elongation step of DNA replication such as topoisomerase inhibitors or DNA intercalating agents, increases cell death. Taken together, these findings suggest Cdc7 kinase may represent a promising novel cancer target. NMS-1116354 is a potent (low nM) ATP competitive small molecule inhibitor of Cdc7 kinase activity. Tested on a panel of more than 50 kinases representative of the human kinome, NMS-1116354 was found to be highly selective, with cross-reactivity observed only for CDK9. NMS-1116354 was tested for anti-proliferative activity against a panel of 171 human cancer cell lines of both solid and hematological origin. Cancer cell proliferation was inhibited with IC50 values & lt; 1 μM in 99 cell lines. Lymphoma, multiple myeloma and breast cancer cell lines, in particular triple negative, were especially sensitive to NMS-1116354. Activity of NMS-1116354 was independent of p53 status, and was maintained in cell lines resistant to 5-FU, cisplatin, gemcitabine and doxorubicin. NMS-1116354 induced rapid (within 3 hours of treatment) and massive cell apoptosis as assessed by induction of active caspase 3 and sub-G1 accumulation. Thus, in vitro, NMS-1116354 has potent, broad and p53-independent anti-cancer activity. In mechanism of action studies in cells, NMS-1116354 inhibited pSer40-Mcm2 (a phosphorylation event which we have previously described as being a specific biomarker of Cdc7 kinase activity) at drug concentrations consistent with those required for cell proliferation inhibition and apoptosis induction. Unlike DNA elongation inhibitors, such as hydroxyurea (HU), NMS-1116354 did not induce the DNA damage checkpoint response as assessed by Chk1 and Chk2 phosphorylation. Thus, effects observed following treatment of cells with NMS-1116354 essentially recapitulated those observed after genetic ablation of the protein using anti-Cdc7 siRNA. In cells, NMS-1116354 was also found to down-regulate the pro-survival protein Mcl-1, consistent with inhibition of Cdk9, the only other tested kinase against which the compound has significant activity. Downregulation of Mcl-1 protein may thus potentially contribute to anti-tumor activity of the compound, particularly in contexts which are dependent on the anti-apoptotic activity of this protein. NMS-1116354 has excellent oral biovailalability and is well tolerated in mice after prolonged treatment. Administration of NMS-1116354 to tumor-bearing animals induced potent tumor growth inhibition, including tumor regression, in xenograft models of human breast, colon, and ovarian cancer, as well as in the transgenic TRAMP model of prostate carcinoma. Tumor regression in 7 out of 10 treated animals was also observed in the rat DMBA carcinogen induced mammary tumor model. NMS-1116354 treatment also increased survival time and induced tumor regressions in AML and multiple myeloma animal models. Ex-vivo analysis of mouse xenografts treated with NMS-1116354 showed dose dependent inhibition of Mcm2 phosphorylation and modulation of the expression of a specific set of Cdc7 regulated genes in skin and tumors after both single and repeated administration. Additionally, Mcl-1 expression was found to be down-regulated in white blood cells of treated mice, as well as in tumor cells in the case of hematological cancer models, where interestingly, down-regulation correlated with antitumor activity. Thus, NMS-1116354 has potent anti-cancer activity in vivo on several different solid and hematological cancer models, in which the compound inhibits Cdc7 kinase activity and reduces Mcl-1 protein levels. These findings confirm the dual mechanism of action of the compound in vivo and support the use of Mcm2 phosphorylation, Mcl-1 down-regulation and a specifically identified gene signature as biomarkers of target modulation in clinical trials, both in tumors and in surrogate tissues (i.e. skin and blood). In drug combination studies, NMS-1116354 exhibited synergistic effects when combined with Irinotecan, Docetaxel, anti-metabolites (Gemcitabine and 5-FU) and Bortezomib in vitro and in vivo, opening a possible path for its clinical development in combination with approved drugs. Phase I clinical trials to evaluate the safety of NMS-1116354 in cancer patients were initiated in 2009. The Phase I program currently ongoing includes clinical studies with NMS-1116354 administered orally as single agent exploring different schedules in patients with solid tumors and hematological malignancies. In conclusion, NMS-1116354 is a novel Cdc7 and Cdk9 kinase inhibitor with a unique mechanism of action which involves inhibition of initiation of DNA replication and down-regulation of the pro-survival protein Mcl-1. The compound is endowed with potent single agent antitumor activity in solid and hematological cancer models, is synergistic in combination with approved drugs, and is presently in Phase I clinical studies. Selected References Montagnoli A. et al., Drf1, a novel regulatory subunit for human Cdc7 kinase. EMBO J. 2002; 21: 3171-3181. Montagnoli A. et al., Cdc7 inhibition reveals a p53-dependent replication checkpoint that is defective in cancer cells. Cancer Res. 2004; 64: 7110-7116. Montagnoli A. et al., Identification of Mcm2 Phosphorylation Sites by S-phase-regulating Kinases. J Biol. Chem. 2006; 281: 10281-10290. Tenca P. et al., Cdc7 is an active kinase in human cancer cells undergoing replication stress. J Biol. Chem. 2007; 282: 208-215. Montagnoli A. et al., A Cdc7 kinase inhibitor restricts initiation of DNA replication and has antitumor activity. Nature Chem. Biol. 2008; 4: 357-365. Vanotti E. et al., Cdc7 kinase inhibitors: pyrrolopyridinones as potential antitumor agents. Synthesis and structure-activity relationships. J. Med. Chem. 2008; 51: 487-501. Menichincheri et al., First Cdc7 Kinase Inhibitors: Pyrrolopyridinones as Potent and Orally Active Antitumor Agents. 2. Lead Discovery. J. Med. Chem. 2009, 22; 293-307. Ermoli et al. Cell division cycle 7 kinase inhibitors: 1H-pyrrolopyridines, synthesis and structure-activity relationships. J. Med. Chem. 2009, 23; 4380-90. Warr MR, Shore GC., Unique biology of Mcl-1: therapeutic opportunities in cancer. Current Molecular Medicine 2008; 8: 138-147. Citation Format: Francesco Colotta, Jurgen Moll, Barbara Valsasina, Ermes Vanotti, Sonia Rainoldi, Francesco Sola, Vanessa Marchesi, Maria Menichincheri, Antonella Ciavolella, Veronica Patton, Clara Albanese, Daniele Volpi, Nilla Avanzi, Dario Ballinari, Francesco Fiorentini, Antonella Isacchi, Enrico Pesenti, Arturo Galvani, Corrado Santocanale, Alessia Montagnoli. NMS-1116354: More than an inhibitor of Cdc 7 kinase in S-phase [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr DD01-01