Down-regulation of RhoA inhibits YAP by activating Hippo signaling to promote macrophage M1 polarization and enhance its phagocytosis, thereby inhibiting AML progression in preclinical models.

BACKGROUND: Acute myeloid leukemia (AML) is a malignant tumor of the hematopoietic system. RhoA has been implicated in AML progression, but its role in regulating macrophage polarization and phagocytic function in the AML microenvironment remains unclear. METHODS: RhoA expression level in AML tissues was analyzed by GEPIA database. RhoA expressions in AML cells were detected using RT-qPCR and Western blot. AML cell malignant phenotype was detected by knocking down RhoA using lentivirus-mediated siRNA technology. THP-1 macrophage polarization model and Transwell co-culture system were constructed. ELISA, immunofluorescence and flow cytometry detected macrophage M1/M2 polarization markers, Hippo/YAP signaling pathway and CD47-SIRPα phagocytosis checkpoint molecules. RhoA knockdown's effects on tumor growth and macrophage function were evaluated by establishing subcutaneous tumor-bearing and systemic leukemia models in mice. RESULTS: RhoA was overexpressed in AML. RhoA knockdown suppressed the malignant phenotype of AML cells, reducing cell viability by 36.9% and 32.5% in MOLM-13 and HL-60 cells, respectively, while increasing apoptosis and inhibiting migration and invasion. In the co-culture system, knockdown of RhoA promoted M1 polarization of macrophages, up-regulated iNOS and CD86 expressions, and increased macrophage phagocytic capacity toward MOLM-13 cells from 7.75% to 33.38% and toward HL-60 cells from 15.85% to 51.80%. Mechanistically, RhoA Knockdown activated the Hippo signaling axis (increased p-LATS1/LATS1 ratio) and inhibited the YAP signaling axis (increased p-YAP level and decreased total YAP protein expression). In vivo, RhoA knockdown reduced subcutaneous tumor weight by 59.9%, prolonged median survival from 21.7 to 32.4 days (P = 0.0077), and increased macrophage phagocytosis from 6.12 ± 0.75% to 19.21 ± 1.37%. CONCLUSION: Down-regulation of RhoA by activating Hippo signaling and inhibiting YAP signaling promoted macrophage M1 polarization and enhanced its phagocytosis, thereby effectively inhibiting AML malignant phenotype. However, these findings were primarily derived from cell-line-based in vitro systems and murine AML models, and further validation using primary human macrophages and patient-derived AML samples is warranted.