Abstract
BACKGROUND: Acute myeloid leukemia (AML) remains a high-risk hematologic malignancy due to frequent relapse and therapeutic resistance. Although induction therapy can achieve cytological remission, a fraction of leukemic cells (minimal residual disease, MRD) persists within the protective bone marrow (BM) microenvironment. MRD is heterogeneous and may include subclones with intrinsic survival features present before therapy. Among these, rare BM-resident leukemic cells (BMresLC) may represent pre-adapted precursors of MRD, maintained in a low-proliferative (Ki67(low)) or quiescent state. We previously showed that calcium signaling through ORAI1-dependent store-operated calcium entry (SOCE) contributes not only to AML stemness and drug resistance but also to the regulation of the G0-G1 cell-cycle transition and the emergence of slow-cycling leukemic cells. With this study, we have characterized the stemness and calcium signature of BMresLC before any therapeutic intervention. Our results, beyond further characterizing a population of cells rarely studied, could thus pave the way to new therapeutic opportunities combining current treatments with the targeting of relevant pathways highlighted by our work. METHODS: A patient-derived xenograft (PDX) model in NSG (NOD/SCID/IL2Rγ(null)) mice was used to localize, isolate, and characterize human BMresLC. Whole-bone clearing and 3D-Imaris imaging enabled spatial localization of rare leukemic cells. Flow cytometry and qPCR assessed cell-cycle status, immunophenotype (CD34, CD38, TIM-3, PD-L1, Ki67), stemness, and calcium-signaling components (ORAI1-3, STIM1-2, NFATc1-4). SOCE was measured using Indo-1 assays. Comparative analyses were performed against diagnostic AML cells, public MRD RNA-seq datasets, and prognosis-stratified patient cohorts. RESULTS: BMresLC displayed an immune-evasive immunophenotype and contained a small fraction of Ki67(neg) quiescent cells, but were not enriched in fully quiescent cells. Instead, they predominantly exhibited a Ki67(low) slow-cycling profile, consistent with a low-proliferative persistent state. Transcriptional analysis revealed overexpression of stemness-associated genes and selective downregulation of calcium-signaling components ORAI1, ORAI2, STIM2, and NFATc1/c4, consistent with a SOCE-suppressed calcium signature. Functional assays confirmed reduced calcium influx. Compared with post-therapy MRD datasets, BMresLC showed some stemness and immune-evasion traits but displayed a distinct pre-therapeutic calcium signature, suggesting that it represents an early, persistent state preceding full MRD remodeling. Prognostic subgroup analysis further showed that BMresLC calcium and stemness profiles partially recapitulate features of adverse-risk AML, including differences in CD34, CD38, PD-L1, MMRN1, LAPTM4B, NFATc2, and STIM2 expression. CONCLUSIONS: Our findings identify a distinctive calcium- and stemness-based signature in BMresLC, representing a pre-MRD survival state characterized by slow cycling rather than enrichment in strict quiescence. This pre-therapeutic signature may contribute to MRD establishment and relapse risk in AML.