Abstract
We have recently shown that small molecule inhibitors of Aurora A kinase (AURKA) including dimethyfasudil and MLN827 induce polyploidization and differentiation of normal and malignant megakaryocytes, as evidenced by increased DNA content and upregulation of the cell surface markers CD41 and CD42. Furthermore, our pre-clinical studies demonstrate that MLN8237 shows potent anti-leukemia activity and anti-myelofibrotic activity in MPNs by promoting polyploidization and terminal differentiation of abnormal megakaryocytes. To determine the mechanism by which AURKA inhibitors ameliorate the leukemic and myelofibrotic phenotypes, we have examined the functional requirement for Aurka in adult hematopoiesis. To circumvent the embryonic lethality of the germline knockout and study the requirement for AURKA in adult hematopoiesis, we induced deletion in Aurka floxed mice with Mx1-Cre. Complete loss of AURKA caused a rapid and profound defect in hematopoiesis, with the mice developing pancytopenia and marked hypocellularity of the bone marrow. Notably, we observe an increase in the proportion of CD41 and CD42-positive megakaryocytes in contrast to the deficiency of myeloid and lymphoid cells in the bone marrow. To determine whether the observed defects are cell autonomous, we transplanted Aurkafl/fl, Mx1-Cre bone marrow cells to lethally irradiated recipients, confirmed engraftment, and induced deletion with pIpC. Upon AURKA deletion, the transplanted mice develop an identical phenotype to the one observed in the conditional knockout mice, demonstrating a cell autonomous requirement for AURKA during hematopoiesis. Moreover, in competitive transplantation experiments, Aurka-/- cells are selectively lost and fail to contribute to hematopoiesis. Mechanistically, loss of AURKA led to a significant degree of apoptosis in hematopoietic cells, likely due to mitotic catastrophe resulting from impaired chromosome segregation. To bypass the requirement for AURKA in progenitor cells and examine AURKA function in the megakaryocyte lineage, we deleted AURKA in megakaryocytes ex vivo by infecting Aurka floxed bone marrow with MSCV-Cre. We found that deletion of AURKA resulted in increased CD41 and CD42 expression as well as increased ploidy of the megakaryocyte fraction. Together these results are consistent with a selective differentiation effect of AURKA deficiency on megakaryocytes.
To investigate whether AURKA modulates differentiation of megakaryocytes through interactions with lineage specific transcription factors, we performed co-immunoprecipitation experiments between AURKA and megakaryocyte transcription factors containing consensus AURKA phosphorylation sites in primary megakaryocytes. Results confirmed a robust interaction between AURKA and p45 NF-E2. Additionally, we found by an in vitro kinase assay that AURKA phosphorylates p45 NF-E2 on the S170 residue, as the S170A mutant of NF-E2 cannot be phosphorylated by AURKA. To explore the functional importance of NF-E2 phosphorylation at S170, we created a phosphomimetic mutant, S170D, and assayed its ability to induce megakaryocyte differentiation in primary cultures. While overexpression of wild-type NF-E2 significantly increased the expression of the megakaryocyte differentiation markers CD41 and CD42, overexpression of the S170D mutant was markedly less effective in promoting megakaryocyte differentiation, indicating that the loss of phosphorylation promotes NF-E2 activity and that NF-E2 activity may be suppressed by AURKA phosphorylation. Finally, to determine if AURKA inhibitors promote megakaryocyte differentiation by allowing activation of NF-E2, we knocked-down down NF-E2 in megakaryocytic cell lines and subsequently treated with diMF or MLN8237. Strikingly, cells with NF-E2 knocked-down displayed significantly less CD41 and CD42 expression in response to AURKA inhibitor treatment compared to control knockdowns. This experiment demonstrates that NF-E2 is required for the differentiation effect caused by AURKA inhibition. Taken together, our data show that Aurora A kinase is required for adult hematopoiesis and that Aurora A regulates NF-E2 function during megakaryocyte differentiation. Our results also reveal that inhibition of AURKA kinase activity couples polyploidization and terminal differentiation of megakaryocytes.
Disclosures:
Crispino: Sanofi: Research Funding.
Interaction and Feedback Regulation between STK15/BTAK/Aurora-A Kinase and Protein Phosphatase 1 through Mitotic Cell Division Cycle
