Preclinical Activity of Selective SYK Inhibitors, Entospletinib and Lanraplenib, Alone or Combined with Targeted Agents in Ex Vivo AML Models with Diverse Mutational Backgrounds
Authors: Day Melinda A. L. , Sergeev Philipp , Heckman Caroline A. , Schinzel Anna , Obholzer Nikolaus D. , Lin Charles Y. , Kumar Pavan , DiMartino Jorge , Saffran Douglas C. 
Published: November 2021

Abstract
Spleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase that mediates integrin and Fc receptor signaling in myeloid cells. SYK has been implicated as an oncogenic driver in acute myeloid leukemia (AML) with aberrant expression of HOXA9 and MEIS1 and cooperates with FLT3 internal tandem duplication to drive leukemogenesis. The oral SYK inhibitor entospletinib (ENTO) has demonstrated clinical activity in HOXA9/MEIS1 driven AML and is currently being investigated in a phase 3 trial of previously untreated patients with nucleophosmin1-mutated (NPM1 mut) AML. Lanraplenib (LANRA) is a next generation oral SYK inhibitor with potency and selectivity comparable to ENTO. In healthy volunteers and patients with autoimmune disease, LANRA has shown pharmacokinetic properties that compare favorably with ENTO. To support the clinical development of LANRA for the treatment of AML, ex vivo treatment of patient-derived AML cells was used to compare its activity to that of ENTO, both as a single-agent and in combination with other AML therapies. First, ENTO and LANRA single-agent activities were evaluated in peripheral blood-derived blasts from 15 AML patients, representing different mutational backgrounds including NPM1, FLT3, PTPN11, and NRAS mutations. AML cells were seeded into 96 well plates and treated with ENTO and LANRA for 6 days. Comparable effects on viability were observed across the 15 models with the 2 compounds, and in 11 of the models, the half maximal inhibitory concentration (IC 50) values were within 2-fold of each other. ENTO had a slightly lower IC 50 value than LANRA in the FLT3-mutated models possibly due to the direct FLT3 inhibitory activity of ENTO. Next, we tested the activity of ENTO and LANRA ex vivo in bone marrow-derived AML blasts from 29 AML patients representing diverse mutational backgrounds, including NPM1, IDH1, FLT3, and RAS mutations as well as MLL rearrangements. The models were treated for 9 days with either ENTO or LANRA, and viability was assessed using Annexin V and 7-aminoactinomycin D staining. Again, ENTO and LANRA showed comparable effects on cell viability with no significant differences between the compounds when compared across the different mutational backgrounds. Both studies suggest the potential for anti-leukemic activity in several different genetically defined subsets of AML. Matrix combination assays were performed by combining ENTO or LANRA with either cytarabine (NPM1 mut), gilteritinib (FLT3 mut), or trametinib (RAS mut) with cell viability and death assessed after a 3-day incubation period. Increased cell death in an additive manner was observed in all combinations tested, with results for ENTO and LANRA being similar, indicating the utility of both compounds in combinatorial treatment paradigms. These results support the clinical evaluation of LANRA in genetically defined subsets of AML. A phase 1b/2 study of LANRA in combination with the selective FLT3 inhibitor gilteritinib, in patients with relapsed or refractory FLT3 mut AML is planned for the end of this year.

Disclosures
Day: Cyteir Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Heckman: Novartis: Research Funding; Orion Pharma: Research Funding; Celgene/BMS: Research Funding; Oncopeptides: Consultancy, Research Funding; Kronos Bio, Inc.: Research Funding. Schinzel: Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Obholzer: Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Lin: Kronos Bio, Inc.: Current Employment. Kumar: Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company. DiMartino: Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Saffran: Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company.