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5037 Combined Single Cell Flow Cytometry and Imaging Analyses Reveal Immunomodulatory Effects Exerted By Targeted Phospho-SYK Inhibitors with Elevated Sensitivity in NPM1 Mutated AML

Program: Oral and Poster Abstracts
Session: 803. Emerging Tools, Techniques and Artificial Intelligence in Hematology: Poster III
Hematology Disease Topics & Pathways:
Research, Biological therapies, Translational Research, drug development, Therapies, Immunotherapy, immunology, Biological Processes, Technology and Procedures, imaging
Monday, December 11, 2023, 6:00 PM-8:00 PM

Bridget A Robinson, PhD1*, Philipp Sergeev, MSc2*, Yoko Kosaka, PhD3*, Thomas Jacob, PhD1*, Andy Kaempf, MS4*, Evan F. Lind, PhD5, Cristina Tognon, PhD1*, Luis A. Carvajal, PhD6*, Jorge F. DiMartino, MD6, Caroline A. Heckman, PhD7 and Tania Q Vu, PhD1*

1Oregon Health & Science University, Portland, OR
2Institute for Molecular Medicine Finland (FIMM), Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland
3Department of Molecular Microbiology & Immunology, Oregon Health & Science University, Portland, OR
4Department of Hematology & Medical Oncology, Oregon Health & Science University, Portland, OR
5Department of Cell, Developmental & Cancer Biology, Oregon Health & Science University, Portland, OR
6Kronos Bio, Inc., San Mateo, CA
7Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki, Finland

Acute myeloid leukemia (AML) is driven by genetic lesions that can result in addiction to signaling pathways that lead to aberrant proliferation and survival. Several new targeted drugs are being assessed for activity in AML with some recently approved for the treatment of subgroups of patients with specific genetic features, such as mutations in FLT3, IDH1 or IDH2. Entospletinib (ENTO) and lanraplenib (LANRA) are inhibitors of spleen tyrosine kinase (SYK); the latter is a next-generation SYK inhibitor and is currently being evaluated in combination with gilteritinib (GILT) in patients with relapsed or refractory FLT3-mutated AML (NCT05028751). Although SYK is rarely mutated, it has been found to cooperate with FLT3, and is highly active especially in AML patients with mutated FLT3. In addition, SYK is important for immune cell signaling through antibody constant region Fc and B cell receptors.

To examine the multi-modulatory effect of pSYK inhibitors in AML, we used combined high parameter flow cytometry (HP-FC) and single cell miniaturized imaging (sc-MI) approaches to profile the immunologic, mutation, and functional protein pathway targets of ENTO and LANRA. Two different AML patient cohorts were assessed by ex vivo treatment with ENTO, LANRA and combinations of LANRA (trametinib, gilteritinib (GILT), aPD-1) over a range of time points (2h, 4h, 3d and 9d) and readouts for cellular phenotype (e.g., differentiation state, immune cell populations) and functional readouts (pSYK, viability) for each cohort were analyzed by HP-FC and sc-MI and associated with available mutation information (bulk gene and single cell RNA sequencing).

Differential analyses of data from flow cytometry (AML-HP-FC Cohort) and single cell imaging (AML-sc-MI Cohort) demonstrated: 1) sensitivity to ENTO and LANRA (³ 1uM) was higher in NPM1 and FLT3 mutated AML samples, as measured via cell viability and pSYK target inhibition; sensitivity was not drastically altered in the presence of other mutations (e.g. IDH1/2, MLL); 2) lower doses of ENTO and LANRA (<1uM) induced proliferation in specific cell types, including progenitor myeloid cells, and T and B lymphocytes, but induced more T cell proliferation in AML samples that have low baseline proliferation; analysis with next generation bulk gene and single cell RNA sequencing suggested downregulation of STAT1expression, downregulation of MYC- and mTOR-dependent pathways, and upregulation of IL-1 pathway genes; and 3) LANRA was more broadly effective at targeting pSYK inhibition than ENTO; however, both were effective in combinations (ENTO with trametinib and GILT, and LANRA with aPD-1).

In sum, analysis using collective flow and single cell imaging provides corroboratory and complementary information revealing new roles for ENTO and LANRA as immune modulating agents supporting B and T cell proliferation at lower dosages, and with demonstrated greater effect in individuals with NPM1 and FLT3 mutations. These support studies investigating the use of pSYK inhibitors in combination with other therapies to reduce tumor burden, and as an improved line of treatment for AML.

Acknowledgements: We are appreciative of the patients and families for their time and participation.

Disclosures: Lind: Kronos Bio: Research Funding. Tognon: Notable Labs: Research Funding. Carvajal: Kronos Bio: Current Employment, Current equity holder in publicly-traded company. DiMartino: Kronos Bio, Inc.: Current Employment. Heckman: WNTResearch: Research Funding; Novartis: Research Funding; Zentalis Pharmaceuticals: Research Funding; Autolus: Consultancy; Amgen: Honoraria; Oncopeptides: Research Funding; Kronos Bio: Research Funding. Vu: Kronos Bio: Research Funding.

*signifies non-member of ASH