Abstract: The chromosomal translocation t(2;5)(p23;q35) involving the ALK tyrosine kinase gene results in expression of the NPM-ALK fusion protein which represents the driving force for survival and proliferation of a subset of Anaplastic Large Cell Lymphoma. More recently, a distinct chromosomal rearrangement of the ALK gene leading to a new fusion variant EML4-ALK, has been identified as a low frequency event, mutually exclusive with respect to EGFR and K-ras mutation, in Non Small Cell Lung cancer patients. As previously found for NPM-ALK, this new fusion variant has constitutively active ALK kinase and was demonstrated to have strong oncogenic potential. Taken together these findings support the hypothesis that ALK represents an innovative and valuable target for cancer therapy both in ALCL and NSCLC patients whose tumors harbor translocated ALK. Here we further describe the preclinical characterization of NMS-E628, an orally available small-molecule inhibitor of ALK kinase activity. Proliferation profiling on a wide panel of human tumor cell lines demonstrated that the compound selectively blocks proliferation of ALK-dependent cell lines and potently inhibits ALK-dependent signaling. In vivo, NMS-E628 induced complete tumor regression when administered orally for ten consecutive days to SCID mice bearing Karpas-299 or SR-786 xenografts, with ex vivo analyses demonstrating dose-dependent target modulation that was maintained for up to 18 hours after single treatment. NMS-E628 was also highly efficacious in a transgenic mouse leukemia model in which human NPM-ALK expression was targeted to T cells. In this latter model, which faithfully recapitulates pathological features of human ALCL, treatment of NPM-ALK transgenic mice with NMS-E628 for as little as 3 consecutive days induced complete regression of tumor masses observed in the thymus and in lymph nodes. NMS-E628 was also highly efficacious in inhibiting the in vitro and in vivo growth of the NSCLC cell line NCI-H2228, which bears the EML4-ALK rearrangement. Complete regressions were also achieved in this model, and prolonged inhibition of ALK phosphorylation and downstream effector activation were observed at active doses. NMS-E628 has favorable pharmacokinetic and toxicological properties and biodistribution analysis revealed that it is able to cross the blood-brain barrier in different animal species. To confirm that therapeutic doses are reached in the brain, NCI-H2228 cells were injected intracranially in nude mice and NMS-E628 was administered orally with different schedules. Dose-dependent increase in survival, together with inhibition of tumor growth as assessed by MRI, confirmed that NMS-E628 does indeed possess antitumor activity in this setting, an important finding considering that a significant proportion of NSCLC patients develop brain metastases. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A244.

Abstract: FLT3 (FMS-like tyrosine kinase 3) and KIT are both members of the class III receptor tyrosine kinase family characterized by an autoinhibitory juxtamembrane (JM) domain that docks with the kinase domain to stabilize a catalytically inactive conformation. Therefore, mutations or deletions in this or in adjacent regions cause constitutive activation of these kinases as observed in 30% of AML patients for FLT3 and in 70% of adult GIST and a subset of melanoma patients for KIT. NMS-P088 is a member of a novel indazole sub-series, which selectively targets both FLT3 and KIT kinases, with different mutations, both primary and secondary, some of which involved in resistance to inhibitors clinically used to target these kinases, such as quizartinib for FLT3 and imatinib for KIT. The compound is able to arrest in vitro growth of human cell lines (AMLs and GISTs) which bear constitutively activated FLT3 or KIT, with IC50s in the low nanomolar range and with high selectivity towards cell lines, including AMLs, which are not dependent on these kinases. When tested against a panel of BA-F3 cells engineered to be driven by different FLT3 or KIT mutants, NMS-P088 compared favorably with reference compounds, both in vitro and in vivo. For example, comparative in vivo tumor inhibition values against BA-F3_FLT3-ITD(F691L) model were 85% for 15 mg/kg NMS-P088 vs. 14% for 40 mg/kg quizartinib. In vivo studies conducted on nude mice bearing the human MOLM-13 AML model revealed that repeated oral administration of NMS-P088 was able to significantly inhibit tumor growth and to increase survival time and that strong inhibition of FLT3 signaling is sustained for at least 48 h after a single treatment. Preclinical profiling of the compound showed a good oral bioavailability in all species, indicated a good therapeutic window in 7-day repeated toxicological studies in rat and in dog and excellent BBB penetration. The low myelotoxicity observed in exploratory toxicological studies suggests that NMS-P088 is potentially well-suited to combination with myelotoxic chemotherapeutic agents and this point is currently subject of preclinical evaluation. Furthermore, no adverse effects on cardiac parameters were observed in a canine telemetry study in which animals were exposed to Cmax levels several fold higher than those required for efficacy in the mouse. NMS-P088 is currently undergoing advanced preclinical development activities. Citation Format: Marina Ciomei, Nadia Amboldi, Sabrina Cribioli, Daniele Casero, Angela Greco, Antonella Isacchi, Enrico A. Pesenti, Daniele Donati, Arturo Galvani, Andrea Lombardi Borgia. NMS-P088, a dual FLT3-KIT inhibitor active also on gatekeeper mutations and devoid of QTc prolongation. [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 798. doi:10.1158/1538-7445.AM2015-798

Abstract: Background: NMS-03592088 is a novel, potent inhibitor of the FLT3, CSF1R and KIT receptor tyrosine kinases (KD 〈 1 nM for all three targets). The compound demonstrated high preclinical efficacy following oral administration in all tested target-dependent tumor models, including those harboring kinase domain secondary resistance mutations, such us the FLT3 residue 691 gatekeeper mutation and the KIT residue 670 and exon 17 mutations. In a FLT3-ITD model of disseminated AML, efficacy observed following single agent treatment with NMS-03592088 was further significantly increased when administered in combination with cytarabine, with excellent tolerability. In preclinical studies conducted in non-human primates, a dose-related increase of circulating CSF1 levels was observed in association with the administration of NMS-03592088, consistent with in vivo inhibition of CSF1R by the compound, thus providing the opportunity for the use of CSF1 levels as a potential pharmacodynamic biomarker of CSF1R modulation in the clinical setting. All three targets of NMS-03592088 are relevant in different settings of hematologic malignancies and solid tumors. In particular, FLT3 mutations occur in approximately 30% of acute myeloid leukemia patients (AML), and are associated with a poor prognosis; KIT mutations are reported in patients with the core-binding factor (CBF) subtype of AML and the CSF1 and/or CSF1R genes are frequently expressed in AML blasts. Recent experimental evidence suggests a potential therapeutic rationale for CSF1R blockade in AML, possibly due to interference with microenvironmental support [Edwards DK et al, Blood, 2019, 133: 588]. Furthermore, chronic myelomonocytic leukemia (CMML) blasts express high levels of CSF1R and NMS-03592088 was able to effectively inhibit their proliferation, concomitant with the suppression of intracellular CSF1R dependent signalling. A clinical trial exploring safety, tolerability and efficacy of NMS-03592088 in patients with AML and CMML is therefore warranted. Trial design: This first-in-human study (EudraCT Number: 2018-002793-47) is designed as an open-label multicenter Phase I/II trial including patients with relapsed or refractory AML or CMML who have exhausted standard treatment options, or for whom standard therapy is considered unsuitable. The study is designed to characterize the safety, tolerability, pharmacokinetics, pharmacodynamics and to explore the preliminary anticancer activity of NMS-03592088 administered orally as single agent once daily for 21 consecutive days, followed by a 7-day break within a 28 day cycle. The study includes an initial conventional phase I part with an accelerated dose titration design in subsequent cohorts of 3+3 patients aimed at defining the maximal tolerated dose (MTD) and the recommended phase 2 dose (RP2D), followed by a limited dose expansion to confirm the RP2D. Once the RP2D is confirmed, a single-stage exploratory Phase II part will start comprising two parallel cohorts, one cohort will consist of AML FLT3 mutated patients and one of patients with CMML. Patients previously treated with FLT3 inhibitors are allowed to participate. The primary endpoint of the Phase II portion of the study is Overall Response Rate. Efficacy will be assessed according to standard criteria [Döhner H et al, Blood 2017, 129: 424; Savona MR et al., Blood, 2015, 125: 1857]. Exploratory endpoints are included to evaluate the potential effects of treatment with NMS-03592088 on circulating levels of CSF1 in plasma, the potential correlation of cellular CSF1R expression levels with clinical outcome in both AML and CMML, and the mutational status of a panel of leukemia-related genes, not limited to FLT3. The Phase I part started in Italy in March, 2019 and is currently ongoing. Disclosures Ciomei: NMS: Employment. Zanetta:Clioss: Employment. Fiorentini:Accelera: Employment. Bosotti:NMS: Employment. Ardini:NMS: Employment. Lombardi Borgia:NMS: Employment. Pulci:Accelera: Employment. Gatto:Clioss: Employment. Di Sanzo:Clioss: Employment. Colajori:Clioss: Consultancy. Davite:Clioss: Employment. Galvani:NMS: Employment. Gan:NMS: Employment. Rossi:Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Mundipharma: Honoraria; BMS: Honoraria; Sandoz: Honoraria; Daiichi-Sankyo: Consultancy; Roche: Membership on an entity's Board of Directors or advisory committees. Rambaldi:Jazz: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau, travel support; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Research Funding, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Research Funding, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Italfarmaco: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Research Funding, Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Speakers Bureau; Omeros: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Isacchi:NMS: Employment.