1. Lineage-inappropriate PAX5 expression in t(8;21) acute myeloid leukemia requires signaling-mediated abrogation of polycomb repression
by Debleena Ray, So Yeon Kwon, Hiromi Tagoh, Olaf Heidenreich, Anetta Ptasinska, and Constanze Bonifer
Blood
Volume 122(5):
August 1, 2013
©2013 by American Society of Hematology

2. PAX5 is organized in an open chromatin conformation in t(8;21) AML and non-t(8;21) myeloid precursors.
PAX5 is organized in an open chromatin conformation in t(8;21) AML and non-t(8;21) myeloid precursors. (A) Quantitative real-time PCR analysis showing PAX5 expression relative to TBP expression in Ramos (B-cell line), Nalm-6 (pre-B cell line), Kasumi-1, and SKNO-1 (t(8;21) AML cell lines), primary cells from 2 t(8;21) AML patients [t(8;21)#1-CD34+ cells from the peripheral blood of a t(8;21) patient, t(8;21)#2-CD34+ cells from the bone marrow of a t(8;21) AML patient after relapse], KG1, HL-60 [myeloid cell lines without t(8;21) translocation], HL-60+PMA (HL-60 cell line differentiated to macrophage like cells by treatment with PMA) and HeLa (epithelial cell line). The y-axis represents average relative PAX5 expression from 3 independent experiments; the error bars represent SD between 3 independent experiments. For the patient samples, the error bars represent variability between qPCR measurements. (B-C) Schematic diagram showing the mammalian sequence conservation from the UCSC genome browser. The black bars represent the positions of the DHSs identified at the (B) PAX5 promoter region and (C) enhancer, respectively, in Nalm-6, Kasumi-1, SKNO-1, HL-60, KG-1, and HeLa cell lines. (D-E) DNase I footprinting experiments examining the distal promoter element HS 8 showing that the transcription factor binding pattern and the chromatin fine structure at HS 8 is identical in Nalm-6, t(8;21) AML cells and HL-60. In panel D, samples from the different cell lines digested with different concentrations of DNase I were separated by electrophoresis on the same gel; thereafter, the samples showing equal DNAse I digestion (as shown by the digestion pattern at the TBP locus) were digitally cut and realigned to prepare the figure. PMA, phorbol myristate acetate.
Debleena Ray et al. Blood 2013;122:
©2013 by American Society of Hematology

3. PAX5 is poised for transcription in non-t(8;21) myeloid precursors and is repressed by polycomb-repressive complexes.
PAX5 is poised for transcription in non-t(8;21) myeloid precursors and is repressed by polycomb-repressive complexes. ChIP-qPCR experiments measuring relative enrichment of (A) H3K4me3, (B) H3K9ac, (C) total RNA Pol II, (D) elongating form of RNA Pol II, (E) H3K27me3, (F) H3K27ac, and (G) EZH2 at the PAX5 promoter hypersensitive sites. Relative enrichment is calculated over the inactive IVL locus in panels A, B, C, and D and relative to input in E and F and relative to TBP in panel G. Each bar graph is representative of at least 2 independent experiments analyzed in duplicate. The error bars represent variability in qPCR measurements.
Debleena Ray et al. Blood 2013;122:
©2013 by American Society of Hematology

4. PAX5 is neither a direct nor an indirect target of RUNX1/ETO.
PAX5 is neither a direct nor an indirect target of RUNX1/ETO. (A) qRT-PCR showing (A,C) RUNX1/ETO and (B,D) PAX5 expression after transfecting (left) a primary t(8;21) sample and (right) Kasumi-1 cell line with siRNA mismatch (siRNA MM) and siRNA RUNX1/ETO (siRNA RE) to knock-down RUNX/ETO. Materials were from a previous study.5 qRT-PCR, quantitative reverse transcription PCR.
Debleena Ray et al. Blood 2013;122:
©2013 by American Society of Hematology

5. Aberrant signaling is required for PAX5 deregulation in t(8;21) AML
Aberrant signaling is required for PAX5 deregulation in t(8;21) AML. (A) Schematic diagram showing chronic AKT, JNK, JAK/STAT, and MAP kinase signaling in t(8;21) AML downstream of an activating mutation (indicated by a star) in a tyrosine kinase (growth fa...
Aberrant signaling is required for PAX5 deregulation in t(8;21) AML. (A) Schematic diagram showing chronic AKT, JNK, JAK/STAT, and MAP kinase signaling in t(8;21) AML downstream of an activating mutation (indicated by a star) in a tyrosine kinase (growth factor) receptor or activated rat sarcoma (RAS) signaling. The circles indicate the signaling components targeted by small-molecule inhibitors. (B) Table listing the signaling components targeted, small-molecule inhibitors used, and the effect of the inhibitors on PAX5 expression in Kasumi-1 cells. For detailed data, see supplemental Figure 4A. (C-F) Quantitative reverse transcription PCR experiment measuring expression of PAX5, INK4/ARF, TBP, and RPL13A, respectively, after simultaneous treatment with JNK, MEK, and p38 inhibitors. Each bar graph is representative of 3 independent experiments. The error bars represent the variability in qPCR measurements.
Debleena Ray et al. Blood 2013;122:
©2013 by American Society of Hematology

6. Inhibition of chronic signaling re-recruits polycomb to the PAX5 promoter.
Inhibition of chronic signaling re-recruits polycomb to the PAX5 promoter. (A-E) ChIP-qPCR experiments measuring relative enrichment of the elongating form of the RNA Pol II complex as well as H3K27ac, H3K27me3, EZH2, and H2Aub1 at the PAX5 promoter hypersensitive sites in untreated Kasumi-1, DMSO-treated Kasumi-1, JNK, MEK, and p38 inhibitor-treated Kasumi-1 and HL-60 cells. (A,B) Relative enrichment shown is over the inactive IVL locus. (C-E) Enrichment is relative to (C) input, (D) TBP, and (E) Chr18. Each bar graph is a representative of 2 independent experiments and the error bars show variability between qPCR measurements. (F) Western blot showing BMI1 and phospho-BMI1 expression in untreated Kasumi-1, DMSO-treated Kasumi-1, JNK, MEK, and P38-treated Kasumi-1 cells and HL-60 cells. GAPDH expression in each of the above cell lines was used as a loading control. (G) The HDAC inhibitor TSA rescues the inhibition of signaling mediated downregulation of PAX5 expression. qRT-PCR experiment showing PAX5 expression in untreated, DMSO-treated, ethanol-treated, ethanol + DMSO treated, TSA-treated, JNK + MEK + P38 inhibitor–treated, and TSA + JNK + MEK + p38 inhibitor–treated Kasumi-1 cells. The y-axis shows PAX5 expression relative to GAPDH expression. The bar graph shows average values from 2 independent experiments measured in duplicate; error bars show the variability in qPCR measurements between the 2 experiments.
Debleena Ray et al. Blood 2013;122:
©2013 by American Society of Hematology

7. siRNA-mediated depletion of MAPKAPK3 and KIT signaling molecules downregulates PAX5 expression in Kasumi-1 cells.
siRNA-mediated depletion of MAPKAPK3 and KIT signaling molecules downregulates PAX5 expression in Kasumi-1 cells. (A-B) Immunostaining showing KIT and MAPKAPK3 proteins (red), respectively, in Kasumi-1 cells after siRNA-mediated depletion. (siNEG) cells transfected with a control siRNA. (C) qRT-PCR showing expression of PAX5, RPL13A, and TBP relative to GAPDH in Kasumi-1 cells transfected with control siRNA (siNEG), MAPKAPK3 siRNA, and KIT siRNA. Values obtained with siNEG were set as one. The bar graph is representative of 2 independent experiments measured in duplicate; the error bars show variability in qPCR measurements.
Debleena Ray et al. Blood 2013;122:
©2013 by American Society of Hematology

8. Signaling-mediated abrogation of polycomb silencing activates PAX5 expression in t(8;21) AML. In B cells, the PAX5 promoter is active and robust transcription occurs.
Signaling-mediated abrogation of polycomb silencing activates PAX5 expression in t(8;21) AML. In B cells, the PAX5 promoter is active and robust transcription occurs. In myeloid precursors, the gene is not expressed, but is poised for transcription and repressed by polycomb-repressive complexes. In t(8;21) AML, this polycomb repression is relieved via activated MAP kinase signaling, potentially cooperating with so far uncharacterized transcription factors (TF). This leads to the chronic activation of MAPKAPK3, which in turn leads to the dissociation of polycomb complexes from the PAX5 promoter region, effective transcriptional elongation, and aberrant expression of PAX5.
Debleena Ray et al. Blood 2013;122:
©2013 by American Society of Hematology