Abstract B001: Multiparameter flow cytometry based high throughput drug sensitivity assay for myelodysplastic syndrome permits inclusion of immune microenvironment, novel agents, and drug combinations and correlation with molecular/clinical features
Authors: Tanya Wu, Brian Ball, Guangrong Qin, Yueming Tang, Jacob Appelbaum, Stanley Lee, Sergei Doulatov, Timothy J Martins, Holly Yin, Zhe Wang, Timothy Monahan, Melinda Biernacki, Derek Stirewalt, Marie Bleakley, Guido Marcucci, Pamela S Becker
Journal: Cancer Research (2025)

Abstract Introduction. The current approved drugs for myelodysplastic syndrome (MDS) are limited, with a few targeted agents such as IDH and BCL-2 inhibitors, hypomethylating agents (HMAs), and growth factors. To identify new effective agents and combinations, evaluate individual patient responses and develop predictive models, we have developed a new multiparameter flow cytometry based high throughput drug screen (MFC-HTS) using the iQue 3 (Sartorius) for MDS with novel agents and immunotherapy and combinations. Methods. Blood and bone marrow samples were obtained from patients with MDS or secondary AML arising from MDS with informed consent on IRB approved protocols (Fred Hutchinson Cancer Center). High throughput drug sensitivity (HTS) assays were performed by conventional viability assay utilizing CellTiter-Glo, CLIA approved since 2014, at the University of Washington Quellos High Throughput Screening Core or by MFC-HTS assay in the Becker lab with drug additions performed at the High Throughput Drug Screening Core of the City of Hope Shared Resources. The flow cytometry based assay permits analysis of single cells using gating strategies for surface markers, permits assay within the immune microenvironment cells, and analysis for apoptosis, viability, cell populations including differentiated lineages using Forecyt software (Sartorius) with estimation of drug sensitivity scores, IC50s and area under the curve (AUC). The panel consists of about 60 drugs for assay on a single 384-well plate, including chemotherapy agents and targeted inhibitors, both FDA approved or investigational, as well as double (e.g.-venetoclax/HMA or venetoclax-ivosidenib) or triple drug combinations, antibodies/antibody drug conjugates or immune effector cells. The classes of drugs include degraders, IDH, PARP, nuclear export, kinase, DNMT, BET, PRMT5, IRAK, HDAC and BCL2 inhibitors, purine analogs, and other agents. We will use machine learning methods to analyze molecular and clinical features to develop a predictive model for optimizing therapy for individual MDS patients. We will stratify patient samples by IPSS-R, IPSS-M, and the Molecular Taxonomy of MDS classification (Bernard E et al Blood 2024). Results. The conventional viability assay distinguishes unique responses to venetoclax, navitoclax, cladribine, clofarabine, eprenetapopt, alvocidib, momelotinib, idarubicin, trametinib, mitoxantrone, cytarabine and combinations for MDS samples with normal or complex cytogenetics, and with different mutation backgrounds. The MFC-HTS demonstrates responses for blasts or LSCs or monocytes or lineages. Rapid analysis permits comparison of all the drugs of the same mechanism of action or subsets of agents. Unique responses were noted for samples with complex cytogenetics and TP53 mutation/deletion. Conclusions.This new robust high throughput drug sensitivity assay for MDS with rapid analysis of results by MFC and Forecyt® software permits inclusion of components of the marrow microenvironment and novel agents, including immunotherapy and cell therapy. Citation Format: Tanya Wu, Brian Ball, Guangrong Qin, Yueming Tang, Jacob Appelbaum, Stanley Lee, Sergei Doulatov, Timothy J Martins, Holly Yin, Zhe Wang, Timothy Monahan, Melinda Biernacki, Derek Stirewalt, Marie Bleakley, Guido Marcucci, Pamela S Becker. Multiparameter flow cytometry based high throughput drug sensitivity assay for myelodysplastic syndrome permits inclusion of immune microenvironment, novel agents, and drug combinations and correlation with molecular/clinical features [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Functional and Genomic Precision Medicine in Cancer: Different Perspectives, Common Goals; 2025 Mar 11-13; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(5 Suppl):Abstract nr B001.