Aberrant cell cycle regulation and osteoblastic differentiation in diamond-blackfan anemia (DBA) mesenchymal stem cells Free

Diamond-Blackfan anemia (DBA) is a rare hematological disorder characterized by red blood cell aplasia. Advances in genomic studies have identified mutations in ribosomal protein genes (such as RPS19, RPL11, RPS26) and non-ribosomal genes (GATA1, EPO, ADA2, TSR2) as the underlying causes of DBA. In addition to anemia, DBA patients often present with bone development-associated defects, including short stature, thumb or craniofacial anomalies, and an increased risk of developing osteosarcoma. These findings suggest a dysregulated non-hematopoietic bone marrow microenvironment (BME) in DBA patients, which is not well understood. Mesenchymal stem cells (MSCs) are a crucial component of the BME and can differentiate into osteoblasts, adipocytes, and chondrocytes. In this study, we determined the molecular defects of DBA MSCs using both mouse and human RPL11 haploinsufficient MSCs. Mouse DBA MSCs were derived from RPL11^+/fl carrying Cre-ERt2 and Mx1-Cre, in which the Rpl11 gene is deleted upon injecting tamoxifen or polyinosinic:polycytidylic acid (poly(I:C)), respectively. Additionally, we used cell-permeable Cre in vitro to delete the gene and observe the immediate effect of gene deletion. For human MSCs, we either knocked out the RPL11 gene using the CRISPR/Cas9 system or used DBA patient-derived induced pluripotent stem cells (iPSCs)-derived mesenchymal stem cells.

Using these models, we identified a decrease in the proliferation of MSCs from RPL11 mutant mice and a DBA patient compared to healthy MSCs (7.04x10⁴±1.01 and 3.62x10⁴±1.00 in WT and DBA, respectively). The reduced proliferation observed in DBA MSCs is due to G2/M phase cell cycle arrest. Interestingly, DBA cells did not show elevated p53 compared to WT MSCs, which is often observed in DBA patients. DBA cells showed a significant reduction of G2/M phase-associated mRNA and proteins such as cyclin A, cyclin B, and CDK1. DBA MSCs showed an increased accumulation of binucleated cells (27.01%±7.501) compared to healthy MSCs (4.995%±1.596), suggesting cytokinesis failure as the underlying cause for G2/M phase arrest.

Cytokinesis failure is known to activate the Hippo pathway in cells. When the Hippo pathway is activated, a core kinase called LATS1/2 (large tumor suppressor kinases 1/2) phosphorylates a downstream target called YAP (yes-associated protein), a transcription co-activator that mediates cell proliferation, survival, cytoskeleton arrangement, and osteogenic differentiation in MSCs. Phosphorylation of YAP inhibits the nuclear translocation of YAP through cytoplasmic retention or protein degradation. Western blot analysis and fluorescence microscopy showed a 30% increase in cytoplasmic YAP and a reduction in nuclear YAP in DBA MSCs compared to normal MSCs. As a result of YAP inactivation, actin remodeling and osteogenic differentiation potential were significantly reduced in DBA MSCs. Both mouse and human DBA MSCs showed increased cortical actin accumulation and loss of lamellipodia and stellate morphology. Furthermore, DBA MSCs expressed low RUNX2 (Runt-related transcription factor 2), which is an essential transcription factor for osteoblast differentiation. As expected, both mouse and human DBA MSCs show reduced osteogenic differentiation potential compared to healthy MSCs.

Based on these results, we hypothesize that reactivating YAP nuclear translocation increases proliferation in DBA MSCs. To test this hypothesis, we treated mouse DBA and healthy MSCs with a LATS inhibitor at 10nM, 100nM, 1µM, 5 µM, and 10 µM. After 7 days of treatment, DBA MSCs partially restored proliferation compared to control MSCs, with statistical significance starting from 1 µM (p-value < 0.0001 compared to the DMSO control). Among other drugs tested, such as corticosteroids and L-leucine, the LATS inhibitor was the only effective drug that restored proliferation. In addition, the LATS inhibitor restored the lamellipodia formation and stellate morphology of DBA MSCs in a dose-dependent manner.In conclusion, we characterized and identified the impaired YAP signaling pathway of bone marrow mesenchymal stem cells in DBA. Results from this study will lay the groundwork for understanding the bone marrow microenvironment of DBA and identifying potential targets for more effective treatment in DBA.