(247c) Role of Lysine Deacetylases in Megakaryocyte Differentiation
Low platelet count, or thrombocytopenia, is an important clinical issue for cancer patients either as a direct symptom of leukemia or as a side effect of chemotherapy. It is desirable to produce platelets in vitro for transfusions rather than relying solely on platelet donations. However, for this process to be feasible as a clinical option, significant advances must be made to increase the yield of platelets collected per input stem cell. Our lab previously found that nicotinamide (NIC), a form of vitamin B3, enhances the platelet-producing potential of megakaryocytes (Mks) produced in culture1. One role of NIC is to inhibit sirtuins, an NAD+-dependent class of lysine deacetylase enzymes (KDACs). Since select inhibitors of sirtuins elicit similar enhancements in Mk maturation, we hypothesized that NIC mediates its effects by inhibiting sirtuins.
In the last decade, post-translational acetylation of proteins has been recognized as a prevalent modification that regulates protein function in a variety of signaling contexts2. Acetylation, which is regulated by KDACs and acetyl transfereases, has been demonstrated to be important for stem cell maintenance and the differentiation of many cell types including oligodendrocytes, cardiomyocytes and mesenchymal stem cells3. Additionally, two KDAC isoforms, KDAC1 and KDAC2, have been shown to directly impact hematopoietic stem cell differentiation4. We have been investigating the molecular regulation of KDACs in megakaryopoiesis, with a focus on the roles of sirtuins throughout the course of Mk differentiation. A better mechanistic understanding could then be exploited in vitro for platelet production or in vivoto provide the basis for developing better treatments for patients with megakaryocytic leukemias.
Gaining an understanding of sirtuin (SIRT) activity, rather than protein expression levels alone, is important because the SIRT proteins can be post-translationally modified to alter their endogenous activity, and these alterations would not be revealed through protein levels alone. We have developed a high-throughput system utilizing mass spectrometry to profile endogenous deacetylase activity in megakaryocytic cell lines. We validated that we can discriminate between activity of the Zn2+-dependent KDACs (class I and class II) and the SIRTs (class III) using class-specific inhibitors. Results using a small panel of peptide substrates suggested that in the CHRF-288-11 (CHRF) Mk cell line, SIRT activity decreased throughout the course of PMA-mediated Mk differentiation. This trend was retained upon expansion to a library of 361 acetylated peptide substrates. We are extending our analysis to primary Mk cultures where we can profile KDAC activity in various culture populations that include mature Mks, immature Mks, and non-Mks. In CHRF cell lysates, SIRT1, 2, and 7 protein levels decreased after PMA stimulation, while SIRT3 protein levels actually increased. This highlights the importance of analyzing enzyme activity in addition to protein levels, and also the need for SIRT isoform-specific peptide substrate fingerprints. We aim to identify SIRT isoform-specific peptide fingerprints using Mk cell lines with SIRT overexpression/knockdown. Development of this assay system will be widely useful as tool to understand the roles of SIRTs in a variety of contexts. Currently, there is evidence in the literature that, in addition to differentiation, SIRTs play a role in tumorigenesis, regulation of metabolism, cardiovascular disease, and several other processes, but there is a lack of reliable tools to examine activity of these enzymes, which limits discovery of molecular mechanisms and isoform-selective inhibitors.
Additionally, we have utilized mouse models with the genes for specific sirtuin proteins (SIRT3 and SIRT7) knocked out of their genome to help us to understand the importance of these sirtuin proteins for in vivo hematopoiesis. We have found that SIRT3 and SIRT7 seem to be dispensable for in vivomegakaryopoiesis and platelet production based on steady state platelet counts, reticulated platelets, bone marrow Mk ploidy, bone marrow histology, and recovery after induced thrombocytopenia. This suggests that these two KDAC isoforms are not promising targets for enhancing platelet production.
1 Giammona, L. M. et al. Mechanistic studies on the effects of nicotinamide on megakaryocytic polyploidization and the roles of NAD+ levels and SIRT inhibition. Experimental Hematology 37, 1340-1352.e1343, doi:10.1016/j.exphem.2009.08.004 (2009).
2 Choudhary, C. et al. Lysine Acetylation Targets Protein Complexes and Co-Regulates Major Cellular Functions. Science 325, 834-840, doi:10.1126/science.1175371 (2009).
3 Kretsovali, A., Hadjimichael, C. & Charmpilas, N. Histone deacetylase inhibitors in cell pluripotency, differentiation, and reprogramming. Stem Cells Int 184154, 8 (2012).
4 Wada, T. et al. Expression Levels of Histone Deacetylases Determine the Cell Fate of Hematopoietic Progenitors. Journal of Biological Chemistry 284, 30673-30683, doi:10.1074/jbc.M109.042242 (2009).