Assaying pharmacodynamic endpoints with targeted therapy: Flavopiridol and 17AAG induced dephosphorylation of histone H1.5 in acute myeloid leukemia

Abstract

Histone H1 is commonly used to assay kinase activity in vitro. As many promising targeted therapies affect kinase activity of specific enzymes involved in cancer transformation, H1 phosphorylation can serve as potential pharmacodynamic marker for drug activity within the cell. In this report we utilized a phosphoproteomic workflow to characterize histone H1 phosphorylation changes associated with two targeted therapies in the Kasumi-1 Acute Myeloid Leukemia (AML) cell line. The phosphoproteomic workflow was first validated with standard casein phosphoproteins and then applied to the direct analysis of histone H1 from Kasumi-1 nuclear lysates. Ten H1 phosphorylation sites were identified on the H1 variants, H1.2, H1.3, H1.4, H1.5 and H1.x. Liquid chromatography mass spectrometry profiling of intact H1s demonstrated global dephosphorylation of H1.5 associated with therapy by the cyclin dependent kinase inhibitor, flavopiridol, and the Hsp90 inhibitor, 17AAG (17-(Allylamino)-17-demethoxygeldanamycin). In contrast, independent treatments with a nucleotide analog, proteosome inhibitor and histone deacetylase inhibitor did not exhibit decreased H1.5 phosphorylation. The data presented herein demonstrate that potential of histones to assess the cellular response of reagents that have direct and indirect effects on kinase activity that alters histone phosphorylation. As such, this approach may be a highly informative marker for response to targeted therapies influencing histone phosphorylation.

Alterations of Histone H1 Phosphorylation During Bladder Carcinogenesis

“…The sites of phosphorylation of H1 histones in various human cell lines have been extensively characterized previously 23, 29, 32, 34-36 . There is some variability in the level and sites of phosphorylation depending on the cell line, however, in all publications that looked at mitotic or asynchronous cells, mitotic-specific CDK-dependent phosphorylation on T146 (T147 for H1.3) was reported for histones H1.2-H1,4.…”

“…Due to limited availability of normal human bladder epithelial cells and in order to have similar amount of protein for each replicate, we had to use all the extracted histones from normal human bladder epithelial cells for one replicate. Histones were extracted as described previously 32 . Briefly, cell pellets were resuspended in 1 mL of NP-40 lysis buffer [10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl 2 , 0.5% NP-40, 0.15 mM spermine, 0.5 mM spermidine, 1 mM PMSF, protease inhibitor cocktail (1:1000)] and incubated on ice for 5 min.…”

“…Techniques to specifically identify CDK-dependent linker histone phosphorylation have been reported and are suggested to have potential for cancer diagnosis and prognosis 19 . Protein phosphorylation is often aberrant in cancer states due to the dysregulation of cellular signaling pathways 31 , and its alterations can be used as a pharmacodynamic marker in patients treated with targeted kinase inhibitors 32 .…”

The impact of cruciferous vegetable isothiocyanates on histone acetylation and histone phosphorylation in bladder cancer

“…After treatment, the cells were harvested by scraping and then snap frozen. Histones were extracted as described previously [34]. Reagents for histone extraction were obtained from Sigma-Aldrich (St. Louis, MO).…”

Quantitative Mass Spectrometry Reveals that Intact Histone H1 Phosphorylations are Variant Specific and Exhibit Single Molecule Hierarchical Dependence

“…Our data suggest that cell cycle mediated T146 phosphorylation is variant-specific. Kinase inhibitors blocking the phosphorylation of H1 residues may also prove to be useful therapeutics (78). The specificity of such inhibitors is crucial.…”