Abstract: Activated ALK and ROS1 tyrosine kinases, resulting from chromosomal rearrangements, occur in a subset of non–small cell lung cancers (NSCLC) as well as other tumor types and their oncogenic relevance as actionable targets has been demonstrated by the efficacy of selective kinase inhibitors such as crizotinib, ceritinib, and alectinib. More recently, low-frequency rearrangements of TRK kinases have been described in NSCLC, colorectal carcinoma, glioblastoma, and Spitzoid melanoma. Entrectinib, whose discovery and preclinical characterization are reported herein, is a novel, potent inhibitor of ALK, ROS1, and, importantly, of TRK family kinases, which shows promise for therapy of tumors bearing oncogenic forms of these proteins. Proliferation profiling against over 200 human tumor cell lines revealed that entrectinib is exquisitely potent in vitro against lines that are dependent on the drug's pharmacologic targets. Oral administration of entrectinib to tumor-bearing mice induced regression in relevant human xenograft tumors, including the TRKA-dependent colorectal carcinoma KM12, ROS1-driven tumors, and several ALK-dependent models of different tissue origins, including a model of brain-localized lung cancer metastasis. Entrectinib is currently showing great promise in phase I/II clinical trials, including the first documented objective responses to a TRK inhibitor in colorectal carcinoma and in NSCLC. The drug is, thus, potentially suited to the therapy of several molecularly defined cancer settings, especially that of TRK-dependent tumors, for which no approved drugs are currently available. Mol Cancer Ther; 15(4); 628–39. ©2016 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

Abstract: The RET proto-oncogene encodes a receptor tyrosine kinase that is mainly expressed in neural crest-derived tissues where it plays an important role in cell differentiation, growth and survival. Germline activating point mutations of RET are associated with multiple endocrine neoplasia type 2 (MEN2), an inherited cancer syndrome characterized by development of medullary thyroid carcinoma (MTC), pheochromocytoma and parathyroid hyperplasia and are present in circa 50% of sporadic cases of MTC. More recently, a chromosomal rearrangement of the RET gene was identified in a subset of lung adenocarcinomas (1-2%) which is reported to result in expression of a fusion protein containing constitutively active RET kinase domain fused to the N-terminal portion of the kinesin KIF5B. We recently presented data on NMS-173, a very potent RET inhibitor characterized by an excellent in vivo activity profile upon i.v. administration. Here we describe the identification and the preclinical characterization of NMS-616, a novel analogue belonging to the same chemical class, suitable for oral administration. NMS-616 is a highly potent (IC50: 2 nM), ATP competitive RET inhibitor, characterized by high selectivity when tested against a panel of more than 50 kinases. NMS-616 potently blocked proliferation of MTC cell lines, such as the TT human cell line, which endogenously harbours constitutively activated RET, concomitant with abrogation of RET autophosphorylation and signaling pathway activation. The compound also inhibited the IL-3 independent proliferation of RET-driven Ba/F3 cells, with down-modulation of RET autophosphorylation and downstream signalling pathways. NMS-616 is characterized by a good in vitro ADME profile and in vivo pharmacokinetic parameters in the mouse, including oral bioavailability. When tested in vivo in a murine subcutaneous xenograft model employing TT cells, NMS-616 displayed dose-dependent tumor growth inhibition following daily oral administration at 50 and 100 mg/Kg, with tumor regression observed at both dose levels. In addition ex vivo analysis demonstrated dose-dependent target modulation following a single administration and the maintenance of RET phosphorylation inhibition for at least 24 hours. Citation Format: Elena Ardini, Patrizia Banfi, Francesca Quartieri, Paolo Polucci, Nilla Avanzi, Dario Ballinari, Laura Mancini, Edward Felder, Daniele Donati, Arturo Galvani, Enrico Pesenti, Antonella Isacchi, Maria Menichincheri. Identification of a highly potent, selective and orally available RET inhibitor with antitumor efficacy in RET-dependent tumor models. [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 2091. doi:10.1158/1538-7445.AM2013-2091

Abstract: RET, a receptor tyrosine kinase (RTK) expressed mainly in neural crest-derived tissues, plays a role in cell growth and differentiation and its physiological activation depends upon binding to the GDNF family. Genetic aberrations leading to constitutive RET activation are well-established as oncogenic events. Activating point mutations of RET, for example, are present in ca. 70% of medullary thyroid carcinoma patients including all hereditary cases, while RET gene rearrangements resulting in production of activated RET fusion proteins occur in approximately 10% of sporadic papillary thyroid carcinomas. More recently, recurring RET gene rearrangements have also been found in 1-2 % of lung adenocarcinomas and subsets of other solid tumors including colorectal and salivary gland carcinomas. Thus RET kinase represents an actionable therapeutic target in multiple clinical settings with high medical need. Consequently several small-molecule inhibitors targeting this kinase have been explored in clinical settings. A common feature of most advanced agents is their lack of selectivity and in particular their potent cross-reactivity against VEGFR2, an RTK whose inhibition is associated with serious, dose-limiting cardiovascular toxicity. Indeed, the high homology between the two kinases renders identification of ATP competitive compounds that selectively inhibit RET over VEGFR2 a highly challenging task. Here we describe the preclinical activity of NMS-E668, a potent and selective ATP-competitive RET inhibitor characterized by favorable activity, selectivity and ADME profiles. Biochemically, NMS-E668 has an excellent selectivity profile against a panel of & gt;50 kinases, notably including & gt;10-fold selectivity over VEGFR2. Selectivity of NMS-E668 for RET vs. VEGFR2 was confirmed in NIH-3T3 cells engineered to express activated forms of these kinases. NMS-E668 potently (IC50 circa 50 nM) and selectively inhibited proliferation of RET-dependent tumor cells, including TT medullary carcinoma cells harboring a RET C634W activating point mutation and LC2/ad lung carcinoma cells bearing the oncogenic fusion protein CCDC6-RET. NMS-E668 also potently inhibited IL3-independent growth of Ba/F3 cells expressing KIF5B-RET, the RET rearrangement that is most commonly found in lung adenocarcinomas. Cellular mechanism studies confirmed that NMS-E668 inhibits RET autophosphorylation and downstream signaling at doses consistent with antiproliferative activity. Tested in vivo against KIF5B-RET-driven Ba/F3 tumors, NMS-E668 displayed & gt;90% tumor growth inhibition accompanied by target modulation following oral administration at 10 and 20 mg/kg, with prolonged tumor regressions observed at the higher dose. Thus NMS-E668, a potent and VEGFR2-sparing RET inhibitor is an innovative and highly promising candidate for further development. Citation Format: Elena Ardini, Patrizia Banfi, Nilla Avanzi, Marina Ciomei, Paolo Polucci, Alessandra Cirla, Antonella Ermoli, Ilaria Motto, Elena Casale, Giulia Canevari, Cinzia Cristiani, Sonia Troiani, Federico Riccardi Sirtori, Nadia Amboldi, Dario Ballinari, Francesco Caprera, Eduard Felder, Arturo Galvani, Daniele Donati, Antonella Isacchi, Maria Menichincheri. NMS-E668, a potent and selective RET kinase inhibitor characterized by specificity towards VEGFR2 and high antitumor efficacy against RET-driven models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Me eting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2082. doi:10.1158/1538-7445.AM2017-2082

Abstract: Cdc7 is an essential serine/threonine protein kinase that licenses initiation of DNA synthesis at replication origins, promotes cell cycle checkpoint activation in response to replication stress and is involved in additional aspects of DNA metabolism, including repair mechanisms. Cdc7 is therefore a potential target for cancer therapy, with a distinct mechanism of action from known drugs that inhibit DNA replication. Overexpression of Cdc7 is found in various types of cancer, suggesting that its deregulated activity may promote survival of cancer cells, as well as tumor progression. In particular, its overexpression in breast cancer is linked to triple negative, HER2 positive subtypes, genomic instability, arrested tumor differentiation and enhanced survival. Depletion through RNA silencing, or inhibition of the kinase activity of Cdc7 induces S-phase arrest and apoptosis in cancer cell lines, while non-transformed epithelial cells arrest in G1, remain viable, and are able to resume cell proliferation on recovery of Cdc7 activity. We have previously reported the identification of NMS-P354, an orally bioavailable Cdc7 inhibitor which promotes tumor growth inhibition in preclinical cancer models and was very helpful in validating Cdc7 as potential target. Here, we describe the characterization of NMS-P862, a follow -on, novel, potent (IC50: 9 nM) and more selective Cdc7 inhibitor. Profiling of anti-proliferative activity against more than one hundred human tumor cell lines indicated relatively high activity (IC50 & lt; 0.5 μM) against triple negative or HER2 positive/double negative breast cancer lines, as well as against prostate cancer cell lines. In vivo, oral administration of NMS-P862 resulted in dose-dependent antitumor activity, including for example tumor regression in a carcinogen (7,12-dimethylbenz[α]antracene) induced rat breast cancer model, for which we observed 9/12 objective responses, assessed as per RECIST criteria. A favorable ADME profile with high oral bioavailability, together with permissive therapeutic safety margins in test species at efficacious exposures, indicate that NMS-P862 is suitable for further development, affording the possibility to target cancer via a novel mechanism which we believe is suited to both single agent and opportune drug combination approaches. Citation Format: Alessia Montagnoli, Maria Menichincheri, Nadia Amboldi, Dario Ballinari, Marina Ciomei, Francesco Fiorentini, Rosita Lupi, Daniele Pezzetta, Sonia Rainoldi, Daniele Pezzetta, Eduard Felder, Antonella Isacchi, Enrico Pesenti, Arturo Galvani. NMS-P862, a novel orally available selective small molecule Cdc7 inhibitor with antitumor efficacy in breast cancer. [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 4539. doi:10.1158/1538-7445.AM2014-4539

Abstract: RET chromosomal rearrangements initially identified in a subset of papillary thyroid cancers, as well as gain-of-function point mutations present in ca. 70% of medullary thyroid carcinomas, are well established as oncogenic events that induce constitutive RET activation. More recently, recurring activating RET gene rearrangements have also been found to be expressed in 1-2% of lung adenocarcinomas and subsets of other solid tumors, including colorectal and salivary gland carcinomas. RET kinase is therefore a validated actionable therapeutic target in multiple tumor types, and several small-molecule inhibitors targeting RET are being explored in clinical settings. A common feature of most advanced agents is their lack of selectivity and in particular their potent cross-reactivity against VEGFR2, a receptor tyrosine kinase whose inhibition has been described to be associated with dose-limiting cardiovascular toxicity. Indeed, the high homology between the two kinases renders identification of ATP competitive compounds that selectively inhibit RET over VEGFR2 a highly challenging task. Here we describe the preclinical activity of NMS-E668, a potent and selective ATP-competitive RET inhibitor characterized by favorable activity, selectivity and ADME profiles. NMS-E668 has low nM potency on RET and excellent biochemical selectivity when tested against a kinome panel, notably including circa 30-fold selectivity over VEGFR2. Importantly, 30-fold selectivity of NMS-E668 for RET vs. VEGFR2 was confirmed at the cellular level using NIH-3T3 cells engineered to express activated forms of the two receptors. NMS-E668 potently and selectively inhibited the proliferation of RET-dependent tumor cells, including TT medullary carcinoma cells harboring a RET C634W activating point mutation and LC-2/ad lung carcinoma cells bearing the oncogenic fusion protein CCDC6-RET. NMS-E668 also potently inhibited IL3-independent growth of Ba/F3 cells expressing KIF5B-RET, the RET rearrangement most commonly found in lung adenocarcinomas. Importantly, the proliferation of circa 100 non-RET-dependent tumor cell lines was not significantly affected by NMS-E668, confirming again its selectivity. Tested in vivo against the TT xenograft tumor model NMS-E668 displayed an excellent tumor growth inhibition with complete tumor regression achieved in all animals treated at the higher dose and with confirmed ex vivo target modulation. Good activity was also observed in additional RET-dependent models following oral administration of the compound. Thus NMS-E668, a potent and VEGFR2-sparing RET inhibitor, is an innovative and highly promising candidate for clinical development. Citation Format: Elena Ardini, Patrizia Banfi, Nilla Avanzi, Marina Ciomei, Paolo Polucci, Alessandra Cirla, Matteo D'Anello, Andrea Lombardi Borgia, Ilaria Motto, Cinzia Cristiani, Dario Ballinari, Eduard Felder, Daniele Donati, Arturo Galvani, Antonella Isacchi, Maria Menichincheri. NMS-E668, a highly potent orally available RET inhibitor with selectivity towards VEGFR2 and demonstrated antitumor efficacy in multiple RET-driven cancer models [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 4785.