Identification of a Pharmacodynamic Biomarker for SEL24/MEN1703, a First-in-Class Dual PIM/FLT3 Kinase Inhibitor for Acute Myeloid Leukemia, and Its Implementation in the First-in-Human Study CLI24-001
Authors: Tomirotti Andrea , Merlino Giuseppe , Fiascarelli Alessio , Baldini Simone , Tagliavini Alessia , Borella Elisa , Mazan Milena , Brzózka Krzysztof , Bressan Alessandro , Pellacani Andrea , Salerno Massimiliano , Binaschi Monica , Bellarosa Daniela 
Published: November 2019

SEL24/MEN1703 is a first-in-class, orally available, dual PIM/FLT3 kinase inhibitor currently investigated in patients with Acute Myeloid Leukemia (AML) in the first-in-human study CLI24-001 (NCT03008187).
PIM and FLT3 kinases are considered to play an important role in AML and are inhibited by SEL24/MEN1703. Moreover, there is evidence that inhibition of PIM kinases might contribute to overcoming acquired resistance induced by approved FLT3 inhibitors1. In AML, different signal transducers in the FLT3 pathway are substrates of kinases. Therefore, their phosphorylation levels might be modulated by kinase inhibitors and may be exploited as a potential pharmacodynamic biomarker in clinical development. In particular, phosphorylation of S6, 4E-BP1, and STAT5 is regulated by both FLT3 and PIM1/2. Thus, the objective of this investigation was to identify the most promising pharmacodynamic biomarker/s for implementation in the clinical trials of SEL24/MEN1703.
Initially, we assessed the in vitro cytotoxic effect of SEL24/MEN1703 in a panel of 26 AML cell lines harboring different genetic mutations, to identify suitable cell lines for subsequent experiments. In the selected panel of AML cell lines, SEL24/MEN1703 resulted in the inhibition of phosphorylation of S6, 4-EBP1 and STAT5 as measured by immunoblotting. Notably, the reduction in phosphorylated S6 (pS6) in response to SEL24/MEN1703 was particularly evident. Since SEL24/MEN1703 displays a broad cytotoxic activity in AML cell lines, we clustered sensitive and resistant cell lines considering 0.5 μM as the IC50 cut-off value. Then, we investigated the relationship between SEL24/MEN1703 cytotoxic activity in AML cell lines and the inhibition of the above mentioned phosphorylated proteins in a 24-hour cytotoxic assay, showing a correlation between IC50 and the reduction of pS6 (Pearson correlation coefficient: -0.6905, R2= 0.477). To further confirm the in vitro data, SEL24/MEN1703 ability to modulate phosphoproteins was assessed also in xenograft mice bearing MOLM-16 cell line. The phosphorylation status of S6, 4E-BP1 and STAT5 was analyzed by immunoblot in tumor tissues from mice treated at 25 mg/kg of SEL24/MEN1703 at baseline and at 4, 8, and 16 hours after treatment. Results showed that also in vivo, SEL24/MEN1703 administration resulted in a decrease of pS6, with maximum reduction in this parameter observed 4 hours after the administration of the investigational compound.
Based on these results, pS6 was identified as the pharmacodynamic biomarker to be implemented in the CLI24-001 clinical trial. Among different available methods, flow cytometry was selected as the preferred platform to analyze patient samples, because of its ability to provide quantitative assessment of cellular events and pharmacodynamic evaluation in a selected, relevant cell subpopulation, such as the AML blast cells. The assessment of pS6 in the clinical trial is planned both at baseline and at cycle 1 day 14 for whole blood and bone marrow. In addition, pS6 levels will be measured in whole blood at additional time points during treatment cycles. We have implemented the measurement of pS6 in the CLI24-001 trial, and pS6 levels as well as their relationship with the main pharmacokinetic parameters in patients treated with SEL24/MEN703 at 100 and 125 mg will be presented.
1Green A.S. et al., Pim kinases modulate resistance to FLT3 tyrosine kinase inhibitors in FLT3-ITD acute myeloid leukemia, Sci Adv, 2015

Disclosures
Tomirotti: Menarini Ricerche S.p.A.: Employment. Merlino:Menarini Ricerche S.p.A.: Employment. Fiascarelli:Menarini Ricerche S.p.A.: Employment. Baldini:Menarini Ricerche S.p.A.: Employment. Tagliavini:Menarini Ricerche S.p.A: Employment. Borella:Menarini Ricerche S.p.A.: Employment. Mazan:Selvita S.A.: Employment. Brzózka:Selvita S.A.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Bressan:Menarini Ricerche S.p.A.: Employment. Pellacani:Menarini Ricerche S.p.A.: Employment; Amgen: Equity Ownership. Salerno:Menarini Ricerche S.p.A.: Employment. Binaschi:Menarini Ricerche S.p.A.: Employment. Bellarosa:Menarini Ricerche S.p.A.: Employment.