Molecular and biologic characterization and drug sensitivity of pan-histone deacetylase inhibitor–resistant acute myeloid leukemia cells by Warren Fiskus, Rekha Rao, Pravina Fernandez, Bryan Herger, Yonghua Yang, Jianguang Chen, Ravindra Kolhe, Aditya Mandawat, Yongchao Wang, Rajeshree Joshi, Kelly Eaton, Pearl Lee, Peter Atadja, Stephen Peiper, and Kapil Bhalla Blood Volume 112(7):2896-2905 October 1, 2008 ©2008 by American Society of Hematology

Compared with HL-60, HL-60/LR cells are highly resistant to apoptosis induced by LAQ824, SAHA, and LBH589. Compared with HL-60, HL-60/LR cells are highly resistant to apoptosis induced by LAQ824, SAHA, and LBH589. HL-60 (A) or HL-60/LR cells (B) were treated with the indicated concentrations of LAQ824 (top panel), SAHA (middle panel), and LBH589 (bottom panel) for 48 hours. After this, the percentage of annexin V–stained apoptotic cells was determined by flow cytometry. Values on the curves represent the mean of 2 experiments performed in duplicate. IC50 values were determined and indicated in the top left of each graph. Error bars indicate SE. (C) After treatment of cells with 500 nM LAQ824 for 24 hours, cells were Wright stained and the morphology of the untreated and LAQ824-treated cells was determined by light microscopy with a Nikon Eclipse 50i microscope (Nikon, Melville, NY) using a PLAN 40×/0.65 NA objective and a Nikon CCD camera. (D) HL-60 and HL-60/LR cells were treated with 500 nM LAQ824 for 24 hours. After treatment, CD11b expression on the surface of the cells was measured by flow cytometry. Warren Fiskus et al. Blood 2008;112:2896-2905 ©2008 by American Society of Hematology

Compared with HL-60, a higher percentage of HL-60/LR cells are in S phase of the cell cycle, have shorter doubling time, and are resistant to LAQ824-mediated growth inhibition. Compared with HL-60, a higher percentage of HL-60/LR cells are in S phase of the cell cycle, have shorter doubling time, and are resistant to LAQ824-mediated growth inhibition. (A) Untreated and LAQ824-treated HL-60 versus HL-60/LR cells (at the indicated concentrations of LAQ824 for 24 hours) were counted, and the percentage of growth inhibition of the treated cells was calculated and depicted as the curves in the panel. Values on the curves represent the mean of 2 experiments performed in duplicate. (B) Equal numbers of the log-phase HL-60 versus HL-60/LR cells were started in culture, and aliquots of cells were withdrawn at the indicated intervals and counted. The values on the curves represent the mean numbers (of 2 experiments performed in duplicate) of cells at the indicated time interval. Error bars indicate SD. (Inset) The table in the panel shows the percentage of cells in the various phases of cell cycle, as determined by PI staining and DNA content analysis by flow cytometry. The values represent the means plus or minus SE of 3 experiments. (C) Groups of 6 NOD/SCID mice were injected with HL-60 or HL-60/LR cells and monitored. Survival of the mice was plotted, and median survival time was calculated. (D) HL-60 and HL-60/LR cells were stained with isotype controls or specific antibodies for MDR1, MRP1, LRP/MVP, and BCRP1 and then analyzed by flow cytometry. HL-60/VCR cells that express MDR1, LRP/MVP, and BCRP1 were used as a positive control. Open black-lined histograms represent the isotype control; filled gray histograms, specific antibody staining. Warren Fiskus et al. Blood 2008;112:2896-2905 ©2008 by American Society of Hematology

Treatment with LAQ824 treatment failed to induce Bak, Bim, and Bax, but attenuated Bcl-xL and XIAP levels in HL-60/LR versus HL-60 cells. Treatment with LAQ824 treatment failed to induce Bak, Bim, and Bax, but attenuated Bcl-xL and XIAP levels in HL-60/LR versus HL-60 cells. (A) After treatment of HL-60 and HL-60/LR cells with the indicated concentrations of LAQ824 for 8 and 24 hours, total cell lysates were harvested and immunoblot analyses were done for Bcl-2, Bcl-xL, XIAP, Mcl-1, Bak, Bax, and Bim. β-actin expression served as the control for protein loading. (B) HL-60 and HL-60/LR cells were treated with the indicated concentrations of Apo-2L/TRAIL for 24 hours. After this, cell lysates were obtained and immunoblot analyses were done for DR4, DR5, caspase-8, c-FLIP, and FADD. β-actin expression in the lysates served as the loading control. (C) HL-60 and HL-60/LR cells were treated with the indicated concentrations of LAQ824 for 24 hours. Then, immunoblot analyses were done for p-AKT, AKT, p-GSK3β, p-ERK1/2, ERK1/2, c-Raf, cyclin D1, p-STAT5, STAT5, TBP-2, and TRX. The levels of β-actin expression in the lysates served as the loading control. (D) HL-60 and HL-60/LR cells were treated for 16 hours with 250 nM LAQ824. Then cells were stained with HPF and ROS levels were determined by flow cytometry. Values represent the means plus or minus SE of 3 experiments. Warren Fiskus et al. Blood 2008;112:2896-2905 ©2008 by American Society of Hematology

Compared with HL-60, HL-60/LR cells lack HDAC6 expression and LAQ824 induces acetylation of histones H3 and H4 in both HL-60 as well as HL-60/LR cells. Compared with HL-60, HL-60/LR cells lack HDAC6 expression and LAQ824 induces acetylation of histones H3 and H4 in both HL-60 as well as HL-60/LR cells. (A) After treatment of HL-60 and HL-60/LR cells with the indicated concentrations of LAQ824 for 24 hours, histones were isolated and immunoblot analyses of acetylated histones H3 and H4 were performed. Ponceau staining was used to compare equal protein loading in each lane. (B) Total cell lysates from HL-60 and HL-60/LR cells were used to perform Western blot analyses with specific antibodies against HDAC1, HDAC2, HDAC3, HDAC4, HDAC6, HDAC10, and acetylated α-tubulin. The levels of β-actin served as the loading control. (C) Total RNA from HL-60 and HL-60/LR cells was analyzed by reverse-transcription (RT)–PCR using HDAC6 and β-actin primers. (D) HL-60 and HL-60/LR were fixed on slides, stained with FITC-conjugated anti-HDAC6 antibody (green) and/or DAPI (blue), and imaged with a Zeiss LSM510 confocal microscope with a 63×/1.2 NA water-immersion objective lens. (E) HL-60 and HL-60/LR cells were treated with decitabine and/or LAQ824 as indicated. Then, immunoblot analyses were done for p15 and HDAC6 on the total cell lysates. The levels of β-actin in the lysates served as the loading control. Warren Fiskus et al. Blood 2008;112:2896-2905 ©2008 by American Society of Hematology

Compared with HL-60, HL-60/LR cells lack p-HSF1 and hsp70, and LAQ824 treatment did not increase acetylation of hsp90 or induce p21 levels in HL-60/LR cells. Compared with HL-60, HL-60/LR cells lack p-HSF1 and hsp70, and LAQ824 treatment did not increase acetylation of hsp90 or induce p21 levels in HL-60/LR cells. (A) HL-60 and HL-60/LR cells were treated with the indicated concentrations of LAQ824 for 8 hours. After this, Western blot analyses were done for hsp70, hsp90, and HSF-1. (B) HL-60 and HL-60/LR cells were treated with the indicated concentrations of LAQ824 for 24 hours. Then total cell lysates were harvested and immunoblot analyses were done for p21, p15, CEBPα, and acetylated α-tubulin. The levels of β-actin expression in the lysates served as the loading control. (C) After treatment with the indicated concentrations of LAQ824, immunoprecipitates of hsp90 were immunoblotted with anti–acetyl lysine or anti-hsp90 antibody. (D) HL-60 and HL-60/LR cells were treated with 250 nM LAQ824 for 8 hours. Then, cell lysates were harvested and immunoblot analyses were done with anti–acetylated lysine 69 hsp90 or anti-hsp90 antibody. The levels of β-actin in the lysates served as the loading control. (E) HL-60 and HL-60/LR cells were treated with 250 nM LAQ824 for 8 hours. Then, cells were cytospun and fixed on slides, and stained with FITC-conjugated anti-acetylated lysine 69 hsp90 antibody (green) and/or DAPI (blue), and imaged with a Zeiss LSM510 confocal microscope with a 63×/1.2 NA water-immersion objective lens. Warren Fiskus et al. Blood 2008;112:2896-2905 ©2008 by American Society of Hematology

HL-60/LR cells are collaterally sensitive to 17-AAG. HL-60/LR cells are collaterally sensitive to 17-AAG. (A) HL-60 and HL-60/LR cells were treated with the indicated concentrations of 17-AAG for 48 hours. After this, the cells were stained with trypan blue and the percentage of positively stained nonviable cells was determined by light microscopy. Values on the curves represent the mean plus or minus SE of 2 experiments performed in duplicate. (B) HL-60 and HL-60/LR cells were treated with the indicated concentrations of 17-AAG for 24 hours. After this Western blot analyses were done for p-AKT, AKT, p-ERK1/2, ERK1/2, c-Raf, and hsp70 on the cell lysates. The levels of β-actin expression served as the loading control. (C) Reduced binding of ATP and p23 to hyperacetylated hsp90 in HL-60/LR versus HL-60 cells. After incubation of cell lysates containing hsp90 with ATP-sepharose, ATP-sepharose was pelleted and Western blot analysis was done for hsp90. Alternatively, immunoprecipitates were probed with anti-p23 and anti-hsp90 antibody. (D) After treatment of HL-60 and HL-60/LR cells with 17-AAG, lysates were immunoprecipitated with anti-Bax (6A7) antibody and immunoblotted with anti-BclXL or anti–total Bax antibody. Warren Fiskus et al. Blood 2008;112:2896-2905 ©2008 by American Society of Hematology