Abstract

Background/Objectives: Chronic lymphocytic leukemia (CLL) is characterized by clinical and biological heterogeneity shaped by intrinsic signaling programs and microenvironmental interactions. Established biomarkers, including IGHV mutation status and TP53 alterations, provide important clinical and molecular information, but do not fully capture the diversity of pathways that sustain leukemic cell fitness. Aberrant calcium signaling contributes to leukemic survival; however, the clinical relevance of Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2), a calcium-responsive kinase, has not been defined. This study evaluated CaMKK2 as a candidate marker associated with high-risk disease biology and a pathway of interest for further study. Methods: CaMKK2 expression was quantified in purified CD19+ CLL cells from a clinically annotated cohort balanced by immunoglobulin heavy chain variable region (IGHV) mutation status. Associations with time to treatment and overall survival were analyzed. Functional relevance was assessed by pharmacologic inhibition of CaMKK2 in primary CLL cells using metabolic (MTS) and apoptosis (Annexin V/PI) assays. Correlations between CaMKK2 expression and inhibitor sensitivity were determined. The impact of CaMKK2 inhibition on nurse-like cell (NLC) differentiation and macrophage-mediated leukemic support was evaluated in ex vivo culture systems. Results: Elevated CaMKK2 expression was enriched in IGHV-unmutated CLL and associated with shorter time to treatment and inferior overall survival. Pharmacological inhibition of CaMKK2 was associated with reduced primary CLL viability in a dose-dependent manner and increased Annexin V/PI-defined total cell death with sensitivity correlating with CaMKK2 expression levels. Inhibition also attenuated CD163+ macrophage polarization and impaired NLC-mediated support of leukemic cells. Conclusions: CaMKK2 expression is associated with IGHV-unmutated, high-risk CLL biology. Pharmacologic inhibition of CaMKK2 was associated with reduced leukemic cell viability and altered macrophage phenotypes in ex vivo systems. These findings are exploratory, derived from a limited cohort, and support further investigation into the role of CaMKK2 in CLL biology, but do not establish independent prognostic value or direct on-target causality.

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