ClinicalTrials.gov; No.: NCT01752114; URL: www.clinicaltrials.gov).

The development of arrays that can profile molecular activities in cells is important to understanding signaling pathways in normal and pathological settings. While oligonucleotide arrays are now routinely used to profile global gene expression, there is still a lack of tools for profiling enzyme activities in cell lysates. This paper describes the combination of peptide arrays formed on self-assembled monolayers and mass spectrometry to provide a label-free approach for identifying patterns of enzyme activities in cell lysates. The approach is demonstrated by profiling lysine deacetylase (KDAC) activities in cell lysates of the CHRF megakaryocytic (Mk) cell line. Class-specific deacetylase inhibitors were used to show that terminal Mk differentiation of CHRF cells is marked by a pronounced decrease in sirtuin activity and by little change in activity of KDACs 1-11. This work establishes a platform that can be used to identify changes in global activity profiles of cell lysates for a wide variety of enzymatic activities.

Inhibition of histone deacetylases (HDACi) has emerged as a promising approach in the treatment of many types of cancer, including leukemias. Among the HDACs, Class III HDACs, also known as sirtuins (SIRTs), are unique in that their function is directly related to the cell’s metabolic state through their dependency on the co-factor NAD+. In this study, we examined the relation between SIRTs and the growth, survival, and differentiation of K562 erythroleukemia cells. Using a mass spectrometry approach we previously developed, we show that SIRT expression and deacetylase activity in these cells changes greatly with differentiation state (undifferentiated vs. megakaryocytic differentiation vs. erythroid differentiation). Moreover, SIRT1 is crucially involved in regulating the differentiation state. Overexpression of wildtype (but not deacetylase mutant) SIRT1 resulted in upregulation of glycophorin A, ~2-fold increase in the mRNA levels of α, γ, ε, and ζ - globins, and spontaneous hemoglobinization. Hemin-induced differentiation was also enhanced by (and depended on) higher SIRT1 levels. Since K562 cells are bipotent, we also investigated whether SIRT1 modulation affected their ability to undergo megakaryocytic (MK) differentiation. SIRT1 was required for commitment to the MK lineage and subsequent maturation, but was not directly involved in polyploidization of either K562 cells or an already-MK-committed cell line, CHRF-288-11. The observed blockage in commitment to the MK lineage was associated with a dramatic decrease in the formation of autophagic vacuoles, which was previously shown to be required for K562 cell MK commitment. Autophagy-associated conversion of the protein LC3-I to LC3-II was greatly enhanced by overexpression of wildtype SIRT1, further suggesting a functional connection between SIRT1, autophagy, and MK differentiation. Based on its clear effects on autophagy, we also examined the effect of SIRT1 modulation on stress responses. Consistent with results of prior studies, we found that SIRT1 silencing modestly promoted drug-induced apoptosis, while overexpression was protective. Furthermore, pan-SIRT inhibition mediated by nicotinamide pre-treatment substantially increased imatinib-induced apoptosis. Altogether, our results suggest a complex role for SIRT1 in regulating many aspects of K562 cell state and stress response. These observations warrant further investigation using normal and leukemic primary cell models. We further suggest that, ultimately, a well-defined mapping of HDACs to their substrates and corresponding signaling pathways will be important for optimally designing HDACi-based therapeutic approaches.

Elucidating ways to enhance megakaryopoiesis in vivo would have therapeutic applications for thrombocytopenia and transfusion medicine. Nicotinamide has been shown to enhance endomitosis in megakaryocytes cultured in vitro, suggesting that it may be beneficial for the production of platelets in culture. We hypothesized that regular injections of nicotinamide in mice would also increase platelets in vivo. However, we found that platelet counts were reduced by about 25% with daily injections of nicotinamide. Altering the schedule, duration, or nicotinamide dose did not improve platelet production. Consistent with lower platelet levels, nicotinamide also tended to decrease megakaryocyte frequency in sternum and spleen sections, as well as colony formation in vitro by bone marrow progenitor cells. However, there was no effect on the fraction or ploidy of CD41+ cells harvested from bone marrow. Together, our results suggest that, although nicotinamide increases polyploidization of megakaryocytes in culture, it does not have translatable effects in vivo.

In vitro-derived platelets (PLTs), which could provide an alternative source of PLTs for patient transfusions, are formed from polyploid megakaryocytes (MKs) that extend long cytoplasmic projections, termed proplatelets (proPLTs). In this study, we compared polyploidization and proPLT formation (PPF) of MKs cultured on surfaces that either promote or inhibit protein adsorption and subsequent cell adhesion. A megakaryoblastic cell line exhibited increased polyploidization and arrested PPF on a low-attachment surface. Primary human MKs also showed low levels of PPF on the same surface, but no difference in ploidy. Importantly, both cell types exhibited accelerated PPF after transfer to a surface that supports attachment, suggesting that pre-culture on a non-adhesive surface may facilitate synchronization of PPF and PLT generation in culture.