Abstract: PERK (PKR-like endoplasmic reticulum kinase) is a serine-threonine kinase associated to endoplasmic reticulum membrane. Together with ATF6 and IRE1, PERK is a key effector of the Unfolded Protein Response (UPR), a network of signaling pathways that ensures protein homeostasis in the endoplasmic reticulum. Multiple Myeloma (MM) and other protein-secreting tumors are highly dependent on UPR for survival, and inhibition of PERK may be an effective strategy to inhibit growth of these tumors. Here we describe the in vitro and in vivo properties of NMS-E194, a novel potent and selective ATP-competitive PERK inhibitor belonging to the arylsulfonamide chemical class. NMS-E194 inhibits PERK kinase with a Ki of ca 1.5 nM in biochemical assay and possesses high selectivity towards a panel of 54 kinases. When tested in cell proliferation assays, NMS-E194 is preferentially active on multiple myeloma (MM) and diffuse large B cell lymphoma cell lines. In mechanism of action studies, NMS-E194 shows a bi-phasic behaviour: at low nanomolar doses it activates PERK, causing ATF4 accumulation, while at & gt;200 nM it inhibits PERK and downstream pathway, inducing apoptosis. NMS-E194 possesses a favourable in vitro ADME profile and has promising PK properties in the mouse. NMS-E194 demonstrated strong anti-tumor activity following oral administration at 25 mg/kg daily to mice harbouring luciferized KMS-11 cells in a disseminated growth model of MM, with no overt toxicity. Overall, the data available so far warrant further development of NMS-E194 and support PERK inhibition as a novel therapeutic approach in MM and other PERK-dependent tumors. Citation Format: Claudia Perrera, Maurizio Pulici, Davide Carenzi, Ilaria Motto, Marina Fasolini, Elena Casale, Stefania Re Depaolini, Simona Rizzi, Daniela Asa, Fulvia Roletto, Simona Bindi, Daniele Casero, Patrizia Banfi, Elena Ardini, Nadia Amboldi, Dario Ballinari, Fabio Gasparri, Sabrina Cribioli, Laura Mancini, Marina Ciomei, Daniele Donati, Eduard Felder, Arturo Galvani, Antonella Isacchi, Barbara Valsasina. Characterization of NMS-E194, a selective and potent PERK inhibitor with efficacy in the KMS-11 multiple myeloma [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 5163. doi:10.1158/1538-7445.AM2017-5163

Abstract: Background: FLT3 mutations occur in approximately 30% of AML patients and are associated with aggressive disease. Despite the approval of midostaurin and gilteritinib, the prognosis for FLT3+ patients with relapsed or refractory disease is poor. NMS-088 is a novel, potent FLT3, KIT and CSF1R inhibitor with superior preclinical activity compared with approved FLT3 inhibitors in different FLT3-driven models. In addition, NMS-088 is active on FLT3 resistance mutation F691L. Dose escalation results from a Phase I/II study to establish safety, dose selection and preliminary clinical activity for NMS-088 in patients with R/R AML and CMML are described. Methods: In the Phase I 3+3 escalations, NMS-088 is administered daily for 21 of 28 days (schedule A) or continuously (schedule B). Patients must have R/R AML or CMML unsuitable for standard therapy. The primary objective is the MTD or MAD as assessed by DLTs. Secondary endpoints include safety, PK and ELN response. Results: as of January 26, 44 R/R AML or CMML patients were treated at doses from 20 to 360 mg/day in A or from 120 to 250 mg/day in B. Median age was 64 yrs, 41 pts had AML and 3 pts had CMML, median number of prior lines was 2 (range 1 to 10). FLT3 mutations were present in 24 out of 41 AML pts (20 FLT3-ITD, 2 FLT3 D835 and 2 FLT3-ITD and D835). The majority of pts with FLT3+ AML had received prior FLT3 inhibitors (86.4%). NMS-088 showed manageable safety with no MTD characterized. One pt had DLT (abnormal posture, decreased activity, dyspnea G3 and eyelid ptosis G1) at 360 mg in A (at day 21) and one pt had DLT (eyelid ptosis G3) at 180 mg in B (at day 29), both suggestive for myasthenic syndrome. Three additional pts experienced possible myasthenic syndrome at doses ≥ 180 mg. Overall the most frequent treatment emergent related adverse events (≥10%) were nausea (any grade 20.5%), vomiting (13.6%), asthenia (11.4%). Discontinuations due to related AEs were as follows: 2 DLT pts per protocol, 2 pts due to nausea (G1; day 161 at 270 mg) and myasthenia gravis (G3; day 39 at 300 mg in a pt with baseline AChR antibodies). There was a dose-dependent trend for response. A total of 5 out of 12 evaluable pts with FLT3+ AML treated at dose ≥ 300 mg achieved a response with 2 CRi, 1 CRi/MLFS and 2 MLFS. Remarkably, all these pts had received prior midostaurin and 2 pts received both midostaurin and gilteritinib. Two pts with response withdrew from treatment to receive HSCT (DoR 1.0+ mos each). For other responding pts DoR was 1.3, 2.8 and 7.9 mos. Conclusions: NMS-088 showed clinical efficacy in pts with FLT3+ R/R AML, including pts who have failed prior FLT3 inhibitors. Together with the manageable safety observed, these results warrant further development of this drug including potential as a novel valuable therapeutic option for pts who have exhausted available treatments. The trial is currently opened for enrollment (NTC03922100). Citation Format: Antonio Curti, Alessandro Rambaldi, Chiara Cattaneo, Roberto Cairoli, Matteo Della Porta, Patrizia Chiusolo, Federico Lussana, Marta Ubezio, Valentina Mancini, Isabel Cano, Carmen Besliu, Christian Hove Claussen, Rosalinda Gatto, Patrizia Crivori, Elena Colajori, Alessio Somaschini, Cristina Davite, Antonella Isacchi, Elena Ardini, Lisa Mahnke, Pau Montesinos. NMS-03592088, a novel, potent FLT3, KIT and CSF1R inhibitor with activity in FLT3 positive acute myeloid leukemia patients with prior FLT3 inhibitor experience [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT025.

Abstract: We previously demonstrated that natural product‐inspired 3,4‐dihydropyrrolo[1,2‐a]pyrazin‐1(2H)‐ones derivatives delivered potent and selective PIM kinases inhibitors however with non‐optimal ADME/PK properties and modest oral bioavailability. Herein, we describe a structure‐based scaffold decoration and a stereoselective approach to this chemical class. The synthesis, structure–activity relationship studies, chiral analysis, and pharmacokinetic data of compounds from this inhibitor class are presented herein. Compound 20c demonstrated excellent potency on PIM1 and PIM2 with exquisite kinases selectivity and PK properties that efficiently and dose‐dependently promoted c‐Myc degradation and appear to be promising lead compounds for further development.

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.

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 Meeting 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