Abstract: We report that the TPM3‐NTRK1 rearrangement is a low‐frequency recurring event in CRC. A validated IHC method for the identification of TRKA‐positive patients is provided. Pharmacological inhibition of TRKA is proposed as a novel therapeutic strategy for CRC.

Abstract: FLT3, KIT and CSF1R are members of the class III receptor tyrosine kinase family, characterized by an autoinhibitory juxtamembrane (JM) domain that docks within the kinase domain to stabilize a catalytically inactive conformation. Activating rearrangements of the JM domain of FLT3 (FLT3-ITD) occur in 20-25% of AML and represent a driver of disease and a negative prognostic factor. Another 5-7% of AML harbours an activating D835 mutation in the activation loop of the kinase domain. Several FLT3 inhibitors, including quizartinib, crenolanib and gilteritinib, are currently in advanced clinical testing. Moreover midostaurin, a multikinase inhibitor with activity on FLT3, has been recently approved in FLT3 mut AML in combination with standard chemotherapy. Unfortunately, the clinical response to FLT3 inhibitors is shortened by the emergence of resistance mutations at critical residues such as D835 and F691, the so called “gatekeeper” residue. F961L mutations have been described to induce resistance to quizartinib, crenolanib and gilteritinib, making F691L an unmet medical need in AML. NMS-P088 is a potent and selective inhibitor of FLT3 and KIT kinases, including variants with both primary and secondary resistance mutations, as well as of CSF1R, with exquisite cellular selectivity for cell lines dependent on these targets. NMS-P088 has remarkable (subnanomolar) activity against the MOLM-13 and MV4-11 AML cell lines harbouring the FLT3-ITD rearrangement. On a panel of BA/F3 cells harbouring FLT3-ITD and its mutant forms NMS-P088 showed high potency and very significant activity on F691L, superior to that of key comparators tested in parallel. In the disseminated MOLM-13 AML model, repeated oral administration of NMS-P088 as single agent was able to significantly increase survival time, and showed synergy with cytarabine. The outstanding in vitro activity of NMS-P088 on BA/F3_FLT3-ITD harbouring the F691L mutation was also confirmed in vivo, with high TGI for NMS-P088, while quizartinib tested in parallel resulted not active. NMS-P088 has activity on CSF1-dependent macrophages both in vitro and in vivo and showed single agent efficacy in a syngeneic tumor model done in immunocompetent mice, with robust reduction of CSF-1R positive intratumoral macrophages, providing a rationale for testing in clinical setting as a modulator of host vs tumor response. GLP toxicity studies revealed good tolerability at efficacious exposures, with no cardiac effects and excellent BBB penetration. Thus NMS-P088, a potent FLT3 inhibitor with activity on the gatekeeper mutation, is a preclinical candidate with potential to address an unmet medical need in AML, both as single agent and in combination, as well as for testing in different solid tumors sensitive to immunomodulation. Citation Format: Marina Ciomei, Elena Ardini, Gemma Texido, Rachele Alzani, Wilma Pastori, Dario Ballinari, Sabrina Cribioli, Fabio Gasparri, Nilla Avanzi, Daniele Casero, Daniele Donati, Arturo Galvani, Andrea Lombardi Borgia, Antonella Isacchi. NMS-P088, a FLT3-KIT-CSF-1R inhibitor with activity on FLT3 F691L as a novel agent in AML [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 805.

Abstract: FMS-like tyrosine kinase 3 (FLT3) 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 deletion 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-P948 is an alkoxy-indazole derivative potent dual inhibitor of FLT3 and KIT (IC50 26 and 89 nM, respectively), active also against FLT3 and KIT mutations that confer secondary resistance, such as the gatekeeper mutation T670I of KIT. It is a very selective compound, resulting, when tested on a panel of more than 150 human tumor cell lines, strongly active only on cell lines whose proliferation is driven by one of these two kinases (for FLT3: Eol-1, IC50= 8 nM; MV-4–11, IC50= 8 nM; MOLM-13, IC50= 18 nM and for KIT: GIST-430 IC50= 7 nM and GIST-882 IC50= 99 nM). Mechanism of action is demonstrated on both kinases, with activating and resistance mutations, at low nanomolar concentrations. NMS-P948 has good oral bioavailability in rodents. Antitumor efficacy is observed at as low as 10 mg/kg in xenograft models and no toxicity in terms of body weight loss was observed up to 60 mg/kg. In disseminated human AML MOLM-13 model (FLT3-ITD) NMS-P948 produces a very prolonged survival of treated mice (T/C= 390 at 60 mg/kg OS daily × 10) and this antitumor efficacy results superior to that of quizartinib administered with the same schedule at the MTD (T/C= 207 at 40 mg/kg). In conclusion, NMS-P948 is a promising dual inhibitor of mutated FLT3 and KIT with good in vitro and in vivo pharmacological and pharmacokinetic characteristics and with the potential to act also on patients that developed resistance to first line therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B230.

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.

Abstract: NMS-P088 is a potent and selective inhibitor of FLT3 and KIT kinases, including variants with both primary as well as secondary resistance mutations, in particular retaining potent activity against residue 691 gatekeeper mutation which still represents an unmet medical need. Different form most clinically advanced selective FLT3 inhibitors, NMS-P088 was also shown to strongly inhibit CSF1R in biochemical assays (Ki= 4.5 nM; Kd 0.88nM). FLT3, KIT and CSF1R are members of the class III receptor tyrosine kinase family. Activating rearrangements of the JM domain of FLT3 (FLT3 ITD) occur in 20-25% of Acute Myeloid Leukemia (AML) and represent a driver of disease and a negative prognostic factor. Another 5-7% of AML cases harbor an activating D835 mutation in the activation loop of the kinase domain. KIT is also found mutated in circa 8.0% of AML, primarily in core binding factor (CBF) AML. CSF1R has been shown to polarize macrophages towards an immunosuppressive and tumor-promoting phenotype. CSF1 and/or CSF1R genes are expressed in AML blasts and CSF1R mediates supportive interactions between AML and stromal cells in the AML microenvironment. CSF1R has also been found to be highly expressed in blast samples from chronic myelomonocytic leukemia (CMML), a clonal hematopoietic stem cell disorder with poor survival rates post- blast transformation. Hypomethylating agents or standard induction with chemotherapy are the most commonly used therapeutic intervention for CMML and no targeted therapy is currently approved. We found that in vitro NMS-P088 has potent activity on CSF1-dependent macrophages, inhibiting CSF1-stimulated proliferation and cell signalling. In mice NMS-P088 efficiently and dose-dependently decreased tissue infiltration of CSF1R expressing macrophages in liver, consistent with potent in vivo inhibition of this kinase. Furthermore, it showed single agent efficacy in a syngeneic mouse tumor model, with robust reduction of CSF-1R positive intratumoral macrophages. Importantly, we observed high expression of CSF1R in blast samples derived from CMML patients, and the compound was able to inhibit their proliferation and CSF1R signalling. It has been reported that treatment with CSF1R inhibitors induces upregulation of circulating CSF1 ligand as compensatory feedback modulation. Accordingly, during preclinical studies of NMS-088 conducted in non-human primates, a dose-related increase of circulating CSF1 levels was consistently observed. These data confirm in vivo CSF1R inhibition by NMS-P088 and support the opportunity to monitor CSF1 levels as a pharmacodynamic biomarker of CSF1R modulation in the clinical setting. Based on its original kinase targets profile, including FLT3 and KIT gatekeeper resistance mutations as well as CSF1R, preclinical efficacy and safety NMS-P088 was selected to initiate in a clinical trial to potentially address unmet medical needs in AML and CMML. Citation Format: Marina Ciomei, Elena Ardini, Laura Gianellini, GianMaria Borleri, Gemma Texido Romero, Roberta Ceruti, Wilma Pastori, Nilla Avanzi, Daniele Casero, Paola Gnocchi, Andrea Lombardi Borgia, Federico Lussana, Alessandro Rambaldi, Arturo Galvani, Antonella Isacchi. NMS-P088, a novel FLT3, KIT and CSF1R inhibitor, is a promising clinical candidate for AML and CMML treatment [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 1324.

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.