In this study, we uncovered a critical role for STK10 in erythropoiesis: knockdown of STK10 resulted in inhibition of ribosome biogenesis and reduced ribosome levels. On the other hand, we have shown that the p53 signaling pathway is activated and cell apoptosis is increased by STK10 silencing. Together, our work demonstrates that STK10 emerges as a key gene for erythropoiesis regulation in acquired PRCA.
After carefully ruling out the secondary ones, we finally detected 30 patients with acquired PRCA to provide a relatively comprehensive view of gene mutations in this disease. For the selection of potential driver genes, a stringent strategy was used to filter a large number of susceptibility genes. Finally, mutations in STK10, MKI67 and SEC22B, which met the deleterious mutation criteria were chosen for further analysis. In our study, ex vivo studies showed that only STK10 silenced K562 cell lines had significantly reduced hemin stimulated benzidine-positive cells and CD235a expression compared with nontransfected cells, however, the mutation did not affect the differentiation of megakaryocytes. The other two genes did not show effects on either erythroid or megakaryocyte hematopoiesis.
Patients with STK10 mutation did not have any difference in clinical manifestations compared with those without, however, mutated patients did have a lower level of STK10 mRNA levels and they had a poorer response to IST and 2/3 of patients died within the follow-up period, whereas only one patient died for the rest of 27 patients. It seems that STK10 mutation not only affects erythropoiesis but may lead to a worse outcome for patients.
RNA sequencing in STK10 silenced K562 cell lines showed that ribosome biogenesis and hematopoiesis were significantly affected. Subsequent research revealed that STK10 gene mutation could affect the ribosome biosynthesis pathway and downregulated the ribosome protein levels, contributing to abnormal erythropoiesis. Ribosome biogenesis is an essential cellular process, and its regulatory role in erythropoiesis has been implied by the pathological states in macrocytic anemia. In pathological conditions leading to macrocytic anemia caused by point mutations or deletions in ribosomal protein genes such as congenital Diamond-Blackfan anemia (DBA) or acquired 5q- syndrome,[10–12] ribosome levels in erythroid cells are reduced, suggesting that ribosomal levels regulate differentiation.[13] However, whether ribosome biogenesis is impaired in acquired PRCA is still unknown. This unexpected discovery in our research demonstrated that ribosome biogenesis also played a critical role in acquired PRCA, which has a similar pathogenesis to DBA. Recently Liu and colleagues performed single-cell RNA sequencing in five acquired PRCA patients, and the results also revealed defective ribosome biogenesis in these patients. They demonstrated that a similar mechanism may underlie acquired PRCA and DBA, which coincides with our study.[14]
STK10 is a serine/threonine kinase in the Ste20 family that is highly expressed in the hematopoietic tissue. It plays an essential role in the growth, maturation, and apoptosis of cells.[15] The effect of STK10 on erythropoiesis remains unclear. It has been demonstrated that p53 plays an important negative role in erythroid differentiation, and inhibition of p53 could improve erythropoiesis to some extent.[16, 17] The stress caused by impaired ribosome biogenesis induces stabilization and activation of p53 driving cell cycle arrest and apoptosis of erythroblasts.[18] In our study, P53 activation and cell apoptosis were observed following the knockdown of STK10, which may lead to the impairment of erythropoiesis and contribute to the pathogenesis of acquired PRCA. Similar results from other genes have been shown previously: ribosomal proteins L5, L11 and L23 could inhibit MDM2-mediated p53 ubiquitination and degradation, and result in the upregulation of p53.[19, 20] In RPS19 deficient Mdm2 knock-in mice, Mdm2 knock-in reversed the p53 response and improved the growth of hematopoietic progenitors.[21] TP53 mutations were also observed in other bone marrow failure disorders, such as MDS, and Fanconi anemia. These mutations damage the proliferation of hematopoietic stem and progenitor cells, and are correlated with shorter overall survival in MDS patients.[22, 23] Our results were closely consistent with these reports: PRCA patients with STK10 mutations may upregulate the p53 signaling pathway and have an inferior outcome.
For the first time, we attempted to address the genes that may be related to the erythroid hematopoiesis deficiency in patients with acquired PRCA. We can draw a new picture of the pathogenesis of patients with PRCA (Fig. 6). Apart from the immune-mediated attack, some patients with STK10 mutations can downregulate ribosome biogenesis and upregulate the p53 signaling pathway, and further damage erythropoiesis. Because it was difficult to obtain enough fresh peripheral blood specimens from these patients with severe anemia in the follow-up period, we confirmed in the K562 cell lines that the STK10 mutation could upregulate the expression of TP53 protein. Mutation in the STK10 gene may result in a worse reaction to regular IST and an unfavorable outcome, which may also correlate with the abnormal expression of TP53.
Thirty patients are a relatively small number for the gene mutation profile analysis, and the long survival of the PRCA patients makes it difficult to draw a survival curve in a short follow-up period. We can still draw some conclusions that some patients with acquired PRCA may have gene mutations that contribute to the damage of hematopoiesis. These mutations, although not very common, may even suggest a poor prognosis. Further studies with a larger patient cohort and longer follow-up time are needed to verify this conclusion.