Molecular and phenotypical mechanisms driving immune evasion in myeloid neoplasms

• Grant number: 24/00286-6
• Support Opportunities: Regular Research Grants
• Start date: June 01, 2024
• End date: May 31, 2026
• Field of knowledge: Health Sciences - Medicine - Medical Clinics
• Mobility Program: SPRINT - Projetos de pesquisa - Mobilidade
• Principal Investigator: Lorena Lôbo de Figueiredo Pontes
• Grantee: Lorena Lôbo de Figueiredo Pontes
• Principal researcher abroad: Robert Samuel Welner
• Institution abroad: University of Alabama at Birmingham (UAB), United States
• Host Institution: Hemocentro de Ribeirão Preto. Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da USP (HCMRP). Secretaria da Saúde (São Paulo - Estado). Ribeirão Preto , SP, Brazil
• Associated research grant: 21/06841-3 - Molecular and phenotypical mechanisms driving immune evasion in myeloid malignancies, AP.JP2

Abstract

Background: Natural Killer (NK) cells, the major subset among innate lymphoid cells, can affect frequency and self-renewal of normal hematopoietic stem cells (Figueiredo-Pontes et al, Stem Cell Reports, 2021). In agreement, NK maturation and dysfunction may contribute to leukemogenesis in myeloproliferative neoplasms (Bianco & Arantes et al, Frontiers in Immunology, 2021) and Acute Myeloid Leukemia (Amanda F. O. Costa, et al, PhD Thesis, FMRP-USP, 2022, Manuscript in preparation). Our preliminary data suggest a shift of NK cells from highly cytotoxic toward immature cytokine-producing that favor survival of Leukemia Initiating Cells (LICs). Therefore, we hypothesized that, in myeloid neoplasms, exposure to the leukemic cytokine milieu may prevent NK functional maturation and anti-leukemic activity. The mechanisms driving such defective immune phenotype are not known. The following research plan aims to elucidate the molecular variation of the immune cytotoxic cells (NK and T cells) and their immune evasion mechanisms that contribute to disease burden and resistance in myeloid neoplasms.Methods and preliminary data: We are conducting experiments of Single-cell RNA sequencing (sc-RNA-seq) of bone marrow samples from patients with AML with two different selections: patients of different molecular risk categories and patients with similar molecular profiles that relapsed or not after consolidation therapy. Briefly, sc-RNA-seq was performed using the Chromium Single Cell Gene Expression (10x Genomics). Pre-selection for directed sequencing was done using the CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) strategy. ADT-labeled cells are encapsulated within a droplet as single cells with DNA-barcoded microbeads, and when lysed, release both bound ADTs and mRNA that are separated based on size during sequencing. Thus, proteomics and transcriptomics data can be obtained from a single sequencing run. We used 53 TotalSeq antibodies (https://www.biolegend.com/enus/ totalseq) to target the cells of interest including NK cells, T cells, macrophages and myeloid progenitors. The first experiments were carried out in Prof. Welner's laboratory at UAB, USA, and addressed NK subsets only from AML samples categorized as favorable (N=4, normal karyotype and NPM1 mutation), intermediate (N=16, normal karyotype with both NPM1 and FLT3-ITD mutations) and adverse risk (N=4, normal karyotype with FLT3-ITD mutation). Four healthy bone marrow samples were processed as reference. The data revealed differential signaling pathways in NK subsets from different genetic risk AMLs and are currently under analysis. More recently, in our lab at USP, Brazil, the post-doctoral fellow Camila Araújo has selected 24 diagnostic samples from AML patients distributed in three molecular subgroups: NMP1-mutated, NPM1 and FLT3-ITD-mutated and FLT3-ITD-mutated aiming to compare those who relapsed within the first 12 months after consolidation and those that did not. We wonder if the immune dysfunction has a role in leukemia persistence that outweighs the molecular profile. Our focus is to investigate potential differences in antitumoral immune cells and their ligands at the LIC level that justify the different clinical outcomes, especially in the double-mutated group considered to be of intermediate genetic risk and for whom consolidation therapy decisions are not well established. (AU)