Abstract A05: Multiplexed CyTOF analysis of FLT3-ITD AML landscape identifies proteomic profiles associated with resistance to targeted therapies
Authors: Muftuoglu Muharrem , Yilmaz Musa , Basyal Mahesh , Li Li , Daver Naval , Andreeff Michael 
Published: May 2023

Abstract
Targeted therapies(TT) combining FLT3 inhibitors with hypomethylating agents (HMA) and Venetoclax (Ven) are highly effective against FLT3-ITD acute myeloid leukemia (AML) with high response rates. Signaling mutations along with emergence of rarer mutations are the key contributors to primary and secondary resistance to TT. We hypothesize that resistant leukemia clones have unique proteomic profiles that facilitate survival under therapy pressure(TP) and deciphering proteomic profiles at the single-cell level will delineate resistance mechanisms and adaptive responses driven by TP. We performed multiplexed single-cell proteomic analysis of serial PB and BM samples (n:162) from patients treated with FLT3i+HMA+/-VEN using CyTOF. Unsupervised analysis identified leukemia cells and non-malignant cellular elements of the leukemia compartment in an unbiased manner. Next, we interrogated the leukemia proteomic landscape to identify leukemia associated proteomic features. Since Ven targets BCL2 and FLT3i modulates the expression of anti-apoptotic molecules we assessed the expression of apoptosis regulators and found that leukemia cells with immature phenotype, including those with leukemia stem cell(LSC) phenotype, almost always expressed moderate-high levels of BCL2. Monocytic cells(MC) lacked BCL2 expression and had the highest levels of MCL1. Despite expressing moderate levels of MCL1 and BCL-XL, TT was effective in substantially eliminating CD34+ immature leukemia cells. We observed a relative enrichment of MCs, either benign or malignant, after TT. Since cells with MC phenotype were inherently resistant to TT we hypothesized that leukemia cell subsets having similar proteomic profiles to MCs will persist after TT. Phenotypic interrogation of leukemia cells (UMAP) revealed that LSCs generally clustered on the opposite pole distant from MCs and leukemia cells on poles facing MCs were more differentiated. Remarkably, FLT3-ITD mutation partners differentially altered the proteomic landscape. NPM1 mutant FLT3-ITD AML cells displayed a less diverse leukemia architectural organization with differentiation block. Contrarily, signaling mutations diversified the leukemia landscape into a diverse continuum of differentiation states. We also observed that RAS/MAPK mutations could overcome differentiation block and gave rise to a differentiation continuum encompassing less-differentiated, transitional and differentiated leukemia cells. The transitional CD34+ leukemia cells, mapped in close vicinity to MCs, and differentiated MCs that preferentially persisted at D28, had active signaling pathways and expressed CD36. The presence of CD34+ leukemia cells with persistent signaling at D28 was indicative of poor clinical outcomes. Conclusion: Multiplexed single-cell proteomic analysis identified a unique mode of resistance and proteomic features of surviving cells in with FLT3-ITD AML patients treated with TT, and elucidated how different mutation partners in FLT3-ITD AML differentially altered proteomic landscape to drive resistance to TT.
Citation Format: Muharrem Muftuoglu, Musa Yilmaz, Mahesh Basyal, Li Li, Naval Daver, Michael Andreeff. Multiplexed CyTOF analysis of FLT3-ITD AML landscape identifies proteomic profiles associated with resistance to targeted therapies [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A05.