Effect of chronic exposure to acidic pH on the MOLM-13 acute myeloid leukemia cell line

Acidification of the extracellular microenvironment (pHe ≈ 6.4-6.8) constitutes a major metabolic stress in cancers, promoting immune evasion, tumor progression, and therapy resistance. In our laboratory, we previously demonstrated the pivotal role of calcium signaling in the resistance mechanisms of leukemic cells. Acute myeloid leukemia (AML), a malignant hematologic disorder with a high relapse rate, exemplifies this issue through the persistence of leukemic stem cells, resistance to apoptosis, and enhanced efflux pump activity. Furthermore, the Warburg effect drives extracellular acidosis, favouring the emergence of aggressive, therapy-resistant phenotypes and altering intracellular metabolic and signaling pathways that support survival and proliferation. This study aimed to determine whether prolonged exposure to acidic extracellular pH modifies the calcium signature of leukemic cells and contributes to adaptive resistance mechanisms in AML. Experiments were conducted on the MOLM-13 cell line, derived from a relapse case harboring an FLT3 tandem duplication. Cells were cultured for three weeks at acidic (pHe ≈ 6.5) or neutral (pHe ≈ 7.4) pH conditions to induce phenotypic adaptation. Flow cytometry analyses assessed viability and apoptosis (Annexin V/IP), cell-cycle distribution (Ki67/IP), and the leukemic stem cell fraction (CD34⁺/CD38⁻). Intracellular calcium dynamics and intracellular pH were monitored in real time using specific fluorescent probes, while efflux pump activity was quantified by rhodamine exclusion. Gene expression related to calcium homeostasis and pH regulation was evaluated by qPCR to assess transcriptional adaptations to chronic acid stress. Preliminary results indicate that chronic exposure to acidic pH does not impair viability nor induce apoptosis or necrosis. Cell-cycle analysis shows increased G₀ and S phases with G₁ predominance, accompanied by a reduction in the stem-like fraction. Intracellular pH, calcium stores, and capacitive calcium influx are reduced, whereas efflux pump activity declines by approximately 10%. At the transcriptional level, NFATc1, STIM2, and PMCA4 are downregulated, whereas ORAI1 is overexpressed, suggesting the activation of adaptive or resistance mechanisms in response to sustained acidosis, which may support the survival of residual leukemic cells under metabolic stress. Although preliminary, these findings highlight the significant impact of chronic extracellular acidification on calcium signaling and the adaptive capacities of leukemic cells. Further investigations, including precise intracellular pH quantification, extended acid exposure, and analyses of primary AML samples from diverse mutational backgrounds, will refine these observations and may ultimately facilitate the identification of novel calcium-related functional biomarkers and potential therapeutic targets to overcome resistance in AML.