Regulatory Phosphorylation of Ikaros by Bruton's Tyrosine Kinase

<div><p>Diminished Ikaros function has been implicated in the pathogenesis of acute lymphoblastic leukemia (ALL), the most common form of childhood cancer. Therefore, a stringent regulation of Ikaros is of paramount importance for normal lymphocyte ontogeny. Here we provide genetic and biochemical evidence for a previously unknown function of Bruton's tyrosine kinase (BTK) as a partner and posttranslational regulator of Ikaros, a zinc finger-containing DNA-binding protein that plays a pivotal role in immune homeostasis. We demonstrate that BTK phosphorylates Ikaros at unique phosphorylation sites S214 and S215 in the close vicinity of its zinc finger 4 (ZF4) within the DNA binding domain, thereby augmenting its nuclear localization and sequence-specific DNA binding activity. Our results further demonstrate that BTK-induced activating phosphorylation is critical for the optimal transcription factor function of Ikaros.</p></div

BTK Expression Levels Control DNA Binding Activity and Transcription Factor Function of Ikaros.

<p>[<b>A1</b>] BTK Western Blot Analysis of RAJI and Hek293T cells. RAJI cells expressed predominantly the 77 kDa isoform of BTK, whereas Hek293T cells expressed predominantly a 65 kDa isoform of BTK (labeled as BTKi). Both RAJI and Hek293T cells express SYK and IK1. [<b>A2</b>] <u>Upper panel</u>: BTK Western blot analysis of whole cell lysates from Hek293T cells treated with medium only (CON), BTK siRNA or Ku80 siRNA that was used as a control. Each siRNA was used at a 50 nM concentration. BTK siRNA (but not Ku80 siRNA) resulted in depletion of BTK protein without a decrease in the amount of IK protein. <u>Lower panel</u><b>:</b> Ku80 Western blot analysis of whole cell lysates from Hek293T cells treated with medium only (CON), BTK siRNA or Ku80 siRNA. BTK siRNA did not affect the expression level of the control protein Ku80. In contrast, Ku80 siRNA resulted in depletion of Ku80 protein. [<b>A3</b>] Additional Controls. SYK vs. IK Western blot analysis of whole cell lysates from 293T cells treated with medium only (CON), SYK siRNA or BTK siRNA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071302#pone.0071302-Uckun2" target="_blank">[13]</a>. BTK siRNA did not cause a decrease in the amount of SYK or IK proteins [modified from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071302#pone-0071302-g002" target="_blank">Figure 2</a> of our recent open access article published in PNAS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071302#pone.0071302-Uckun2" target="_blank">[13]</a>. [<b>A4</b>] EMSAs were performed on nuclear extracts (NE) from untreated control (CON) Hek293T cells as well as Hek293T cells treated for 72 h with BTK siRNA (50 nM) using biotin-labeled DNA probe IK-BS1. Both 0.4 µg (1×) and 4 µg (10×) amounts of NE were used. IK activity was measured by the electrophoretic mobility shifts of the biotin-labeled IK-BS1 probe, representing IK-containing nuclear complexes (indicated with arrow heads). The position of the probe is also indicated with an arrowhead at the bottom of the gel. The biotin-labeled DNA was detected using a streptavidin-horseradish peroxidase conjugate and a chemiluminescent substrate. The membrane was exposed to X-ray film and developed with a film processor. [<b>B</b>] IK binding sites of validated IK target genes. By cross-referencing IK-regulated gene set (GSE323211) with the archived CHiPseq data (GSM803110) the location of potential IK binding sites for validated IK target genes <i>Itga4</i> (NM_010576; chr2:79095583–79173271), <i>Eif4e3</i> (NM_025829; chr6:99575131–99616765), <i>Kif23</i> (NM_024245; chr9:61765085–61794606) and <i>Tnfaip8l2</i> (NM_027206; chr3:94943443–94946282) was visualized in the mouse mm9 reference database using the Integrative Genomics Browser. [<b>C</b>] RT-PCR was used to examine the expression levels of randomly selected IK target genes after 72 h treatment with medium alone (CON), 50 nM scrambled siRNA (sc-siRNA), <i>IKZF1</i> siRNA vs. <i>BTK</i> siRNA. <u>C1</u>: Expression levels of 4 randomly selected IK target genes were reduced by siRNA-mediated depletion of BTK, whereas treatment with sc-siRNA had no such effect. <u>C2</u>: Included as a positive control, <i>IKZF1</i> siRNA also abrogated or reduced the expression of all 4 IK target genes.</p

Co-localization and Physical Interactions of Native Ikaros and BTK Proteins in Human Cells.

<p>[A] Nuclear co-localization of Native IK and BTK. ALL-N1 cells were fixed and stained with polyclonal rabbit anti-IK1 (primary Ab)/Alexa Fluor 568 F(ab')₂ fragment of goat anti-rabbit IgG (secondary Ab) (red) and mouse anti-BTK MoAb (primary Ab)/Alexa Fluor 488 goat anti-mouse IgG (secondary Ab) (green) antibodies. Nuclei were stained with blue fluorescent dye 4′,6-diamidino-2-phenylindole (DAPI). MERGE panels depict the merge three-color confocal image showing co-localization of IK1 and BTK in DAPI-stained nucleus as magenta immunofluorescent foci (System magnification: 315×). Representative foci of colocalization are indicated with white arrowheads. [B] Co-immunoprecipitation of Native IK and BTK. B1 depicts the results of the BTK Western blot analysis of the IK and BTK immune complexes immunoprecipitated (IP) from ALL-N1 cells. B.2 depicts the results of the IK Western blot analysis of the BTK and IK immune complexes from the same cells. Controls included immunoprecipitations performed without using a primary (1⁰) antibody.</p

BTK Expression Levels Control Nuclear Localization of Ikaros.

<p>[<b>A</b>] Cells were stained with mouse monoclonal antibody (mAb) against human BTK and an anti-IK mouse mAb, which was raised against murine IK and which cross-reacts with human IK. TOTO-3 was used for nuclear staining. Depicted are the confocal two-color (green/blue: BTK/TOTO-3 and IK/TOTO-3) merge images of abundantly BTK⁺ (high BTK) leukemic cells from a pediatric B-precursor ALL case vs. leukemic cells with low BTK expression level from an infant B-precursor ALL case. Nuclear staining for IK was observed in the confocal fluorescence images of primary leukemic B-cell precursors with high BTK expression level. By contrast, leukemic cells with low BTK expression levels showed an aberrant, predominantly cytoplasmic localization of IK (System Magnification: 500×). [<b>B</b>] The DT-40 cell line and its subclones were stained with the rabbit polyclonal, H-100 (sc-13039) antibody against the N-terminus of IK. Depicted are the confocal two-color (red/blue) IK/DAPI merge images of wildtype (WT) DT40 cells, BTK-deficient DT40 cells, and BTK-deficient DT40 cells reconstituted with wildtype <i>btk</i>. The contour of the DAPI-stained nuclei (blue) was marked with a dotted line and shows a significant amount of IK protein (red) outside the nucleus of the BTK⁻ DT40 cells. [<b>C1</b>] Upper panel: Depicted are the confocal single-color and two-color merge images of control Hek293T cells stained with the secondary goat anti-mouse (GAM) antibody and DAPI. No false positive green fluorescence was detected (System Magnification: 630×). Lower panel: Depicted are the confocal single-color and two-color (green/blue: BTK/DAPI) merge images of test Hek293T cells stained with anti-BTK/goat-anti-mouse (GAM) antibody combination and DAPI. BTK (green fluorescence) was localized primarily in the cytoplasm of Hek293T cells around the DAPI-stained blue nucleus. [<b>C2</b>] Left panel: Merge confocal images of untreated control Hek293T cells two-color stained with anti-BTK/goat-anti-mouse (GAM) antibody combination and DAPI. Right panel: Merge confocal images of BTK-siRNA transfected Hek293T cells two-color stained with anti-BTK/goat-anti-mouse (GAM) antibody combination and DAPI 72 h post-transfection. (System Magnification: 630×). BTK depletion was confirmed by Western blot analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071302#pone-0071302-g003" target="_blank">Figure 3</a>) [<b>D</b>] Confocal images of Hek293T cells expressing the HA-tagged mutant IK proteins D₆ or A₁₂ stained with the mouse monoclonal anti-HA antibody (HA-probe F-7)(primary Ab)/Alexa Fluor 488 goat anti-mouse IgG (secondary Ab) (green) antibody and blue fluorescent DNA dye 4′, 6-diamidino-2-phenylindole (DAPI) following treatment with BTK siRNA (50 nM) vs. PBS (CON). (System Magnification: 630×).</p

BTK phosphorylation sites of Ikaros.

<p>[<b>A</b>] We identified Ser²¹⁴ (S214) and Ser²¹⁵ (S215) as two unique BTK phosphorylation sites within the mouse IK peptide ²¹⁴SSLEEHK²²⁰ corresponding to a consensus sequence encoded by Exon 5 and found in IK from mouse (NM_001025597, human (NM_006060.4), and chicken (NM_205088). [<b>B</b>] Alignment of mouse, human, and chicken IK protein segments containing the identified BTK phosphorylation sites. [<b>C</b>] Schematic diagram of BTK-IK (203–224) complex. BTK is shown as a molecular surface colored in gray, and IK is shown as secondary structure colored in green. Key residues in catalytic site of BTK, ATP and substrate residues (Ser214 and Ser215) in the peptide of IK are shown as a stick model. The model was built by Modeler <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071302#pone.0071302-Satterthwaite1" target="_blank">[1]</a> and shown with Pymol. [<b>D</b>] Loss of IK DNA binding activity by BTK-resistance mutations. EMSAs were performed on nuclear extracts (NE) from Hek293T cells transfected with expression vectors for wildtype (WT) or mutant IK proteins using the Thermo Scientific LightShift Chemiluminescent EMSA Kit and biotin-labeled DNA probe gamma-satellite A. IK activity was measured by the electrophoretic mobility shifts of the biotin-labeled probe, representing IK-containing nuclear complexes (indicated with arrow head). The biotin-labeled DNA was detected using streptavidin-horseradish peroxidase conjugate and a chemiluminescent substrate. The membrane was exposed to X-ray film and developed with a film processor. [E] Confocal two-color merge image of a representative Hek293T cell expressing FLAG-tagged wildtype IK protein (green) in the DAPI-stained (blue) nucleus. (System Magnification: 630×). [F] Confocal two-color merge image of representative Hek293T cells expressing FLAG-tagged mutant IK protein with alanine substitutions at BTK phosphorylation sites S214 and S215 (green) in the cytoplasm outside the DAPI-stained nucleus. (System Magnification: 630×). To detect the FLAG-tagged wildtype (in E) and BTK-phosphorylation site mutant IK proteins (in F), cells were stained with a monoclonal mouse anti-DDK antibody and a secondary goat anti-mouse antibody conjugated with Alexa Fluor 488. Fluorescent cells were imaged using the PerkinElmer Ultraviewer Confocal Dual Spinning Disc Scanner (Shelton, CT).</p

Transcript Levels of Ikaros Target Genes in Primary Leukemia Cells from Pediatric Ph^-/BCR-ABL^- and Ph ⁺ /BCR-ABL⁺ BPL Patients on the MILE Study.

<p>Expression levels of IK target genes were compared between primary leukemic cells (GSE13159) from 122 pediatric BPL patients with t(9;22) translocation (Ph ⁺ /BCR-ABL⁺) and 237 pediatric BPL patients without t(9;22) translocation (Ph^-/BCR-ABL^-). Heat map depicts up and down regulated transcripts ranging from red to green respectively for standardized expression values and clustered according to average distance metric (<b>A</b>). 25 transcripts representing 16 IK target genes were expressed at significantly higher levels in leukemia cells from Ph⁺ patients, no significant differences were observed for 10 transcripts representing 9 IK target genes and 7 transcripts representing 5 IK target genes were significantly down regulated (<b>Table S2</b> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080732#pone.0080732.s001" target="_blank">File <b>S1</b></a>). Hierarchical cluster analysis identified MDFIC (0.69 SD units, P = 8.8 x 10^-12), DHRS3 (0.6 SD units, P = 3.7 x 10^-11), <i>GSN</i> (0.49 SD units, P = 3.4 x 10^-10), <i>ITGA4</i> (0.53 SD units, P = 2.6 x 10^-9) and TSPAN13 (0.2 SD units, P = 1.2 x 10^-7) as the most significantly up-regulated genes in the 122 Ph⁺ patient samples. Rank ordered difference in standard deviation units for Ph⁺ samples compared to Ph^- samples for enrichment of IK target genes <b>(B1)</b> as well as lymphoid priming genes <b>(B2)</b> using a supervised approach implemented in GSEA2.08 (Broad institute). Enrichment scores were calculated for the ranked members of the gene sets and normalized to the gene set size (NES) for which the p-value was calculated using 1000 permutations of the pre-ranked gene list and the FDR corrected for testing 2 gene sets. There was a significant enrichment of IK target genes (NES = 2.04, P < 0.001) and lymphoid priming genes (NES = 1.62, P = 0.013) that included the leading edge subsets MDFIC<i>, EIF4E3</i>, DHRS3<i>, ITGA4</i>, PTK2<i>, GSN, S100A10, GRAMD3</i>, F13A1, CALCRL, ATP1B1, LAMC1, TES, ADD3<i>, TCTEX1D1, ATRNL1</i>, IL12RB1<i>, RNF125, MCOLN3</i>, RUNX2, PDLIM2<i>, TSGA10, TREML2</i> for IK targets and <i>IGJ</i>, CNN3<i>, CSF1R, PTGER2, LTB</i>, DNTT, CD52, MEF2C, and RUNX2 for lymphoid priming genes. There was a significant increase in the multivariate mean for 45 transcripts in the Ph⁺ subset of specimens (MANOVA, F_1,357 = 30.65, P<0.0001). The mean level of expression for each transcript in each BPL subset is illustrated in the heat map organized using a two-way hierarchical clustering method (average distance metric) to group expression profiles of transcripts and BPL subsets (<b>C</b>).</p

Dimeric Drug Polymeric Nanoparticles with Exceptionally High Drug Loading and Quantitative Loading Efficiency

Encapsulation of small-molecule drugs in hydrophobic polymers or amphiphilic copolymers has been extensively used for preparing polymeric nanoparticles (NPs). The loadings and loading efficiencies of a wide range of drugs in polymeric NPs, however, tend to be very low. In this Communication, we report a strategy to prepare polymeric NPs with exceptionally high drug loading (>50%) and quantitative loading efficiency. Specifically, a dimeric drug conjugate bearing a trigger-responsive domain was designed and used as the core-constructing unit of the NPs. Upon co-precipitation of the dimeric drug and methoxy­poly­(ethylene glycol)-<i>block</i>-poly­lactide (mPEG-PLA), NPs with a dimeric drug core and a polymer shell were formed. The high-drug-loading NPs showed excellent stability in physiological conditions. No premature drug or prodrug release was observed in PBS solution without triggering, while external triggering led to controlled release of drug in its authentic form

Exon-specific Detection of IKZF1 Transcripts in Normal Hematopoietic Cells and Primary Leukemic Cells from Pediatric Ph⁺ vs. Ph^- BPL Patients.

<p>IKZF1 transcript levels, as measured by 5 IKZF1 probe sets specific for IKZF1 Exons 1-4, were compared between primary leukemic cells from 123 pediatric Ph⁺ BPL patients, 327 pediatric Ph^- BPL patients and non-leukemic hematopoietic cells from 74 normal bone marrow specimens analyzed in 2 independent studies (viz.: GSE13159 and GSE13351). Depicted in (<b>A</b>) are bar charts of RMA-normalized transcript levels for the 5 IKZF1 probe sets, including 2 probe sets exhibiting Exon 4 specificity to test for reduction in signal due to an intragenic IKZF1 deletion involving a region within Exons 4-7 or Exons 2-7. No significant reduction in expression levels were observed for these 2 Exon 4 probesets (<b>B</b>) and significant increases in expression were observed for both comparisons of Ph⁺ with Normal and Ph^- samples for the two Exon 3 probesets. Heat map depicts up- and down-regulated transcripts ranging from red to green respectively for RMA-normalized IKZF1 transcript levels of leukemic cells mean centered to normal hematopoietic cells and clustered according to the average distance metric (<b>C</b>).</p

Pediatric Ph⁺ BPL is not Characterized by IK-deficiency.

<p><b>[A1-A4] Genomic PCR</b>. <b>Analysis of the Human <i>Ikaros</i>/<i>IKFZ1</i> Gene Exons E4-E7 in High-Risk BPL</b>. We performed exon-specific IKZF1 PCR with DNA sequencing on purified genomic DNA samples from 3 pediatric patients with Ph⁺ high-risk BPL. Exons E4-E7 and their intron-exon junctions were PCR amplified using the PCR primers in Table <b>S3</b> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080732#pone.0080732.s001" target="_blank">File <b>S1</b></a>. Depicted are agarose gels documenting that normal size PCR products (E4: 577-bp, E5: 646-bp, E6: 530-bp, E7:785-bp) for each of the 4 IKZF1 exons were obtained in each of the 3 patients, providing strong evidence against the existence of homozygous deletions of the entire IKZF1 locus or within. IKZF1 exons E4-E7. <b>[A5] Western Blot Analysis of Ikaros Expression in Ph⁺ High-Risk</b>. <b>BPL</b>. Depicted is an anti-IK Western blot of whole cell lysates of primary leukemia cells from 3. Ph⁺ BPL patients. Similar to RAJI cells and primary cells from an MLL-AF4⁺ ALL case that were included as controls, Ph⁺ ALL cells from each of the 3 cases expressed an intact 57 kDa IK1 protein. <b>[B] Nuclear Expression of Intact Ikaros Protein and Its Regulators in Ph⁺ and Ph^- High-Risk BPL</b>. Depicted are Western blots of nuclear protein extracts (NE) from primary leukemic cells of 8 high-risk pediatric BPL patients, including 4 Ph⁺ ALL cases. NE were examined for presence of IK (B1), Ku70 (B2), SYK (B3) and BTK (B4). See text for discussion. <b>[C & D] Assessment of Sequence-Specific DNA Binding Function of Nuclear Ikaros in Ph⁺ and Ph^- High-Risk BPL</b>. Electrophorectic mobility shift assays (EMSA) were performed on nuclear extracts from BPL cells using the Thermo Scientific LightShift Chemiluminescent EMSA Kit and the <i>IK-BS1</i> test probe containing a high-affinity IK1 binding site and the <i>IK</i>-<i>BS5</i> negative control probe that has a single base pair (G>A) substitution at position 3 within the core consensus and does not bind IK [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080732#B10" target="_blank">10</a>], [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080732#B12" target="_blank">12</a>], [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080732#B13" target="_blank">13</a>]. The nuclear protein extracts from each of the 4 Ph⁺ and 4 Ph^- BPL cases showed significant gel retardation of the IK1-specific IK-BS1 probe (but not the control IK-BS5 probe) (<b>C</b>). Supershift assays were performed in 3 Ph⁺ and 4 Ph^- BPL cases with an anti-IK monoclonal antibody (2 µg/sample) to confirm the presence of IK in the retarded DNA-binding protein complexes, as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080732#B12" target="_blank">12</a>] (<b>D</b>). Positions of the retarded and supershifted bands are indicated with arrowheads. In each case, the retarded complex of the IK-BS1 probe was supershifted with the anti-IK antibody.</p

Transcript Levels of Ikaros Target Genes in Primary Leukemia Cells from Pediatric Ph^- and Ph⁺ BPL Patients.

<p>Expression levels of IK target genes were compared for primary leukemic cells from 155 pediatric Ph^-/BCR-ABL^- BPL patients and 20 Ph ⁺ /BCR-ABL ⁺ /BPL patients on the Mullighan study (GSE12995). Transcript signal values were obtained from hybridization onto the Affymetrix Human Genome U133A genechip arrays. Heat map depicts up and down regulated transcripts ranging from red to green respectively for mean centered log₁₀ transformed expression values and clustered according to average distance metric (<b>A</b>). Rank ordered difference in standard deviation units for Ph⁺ samples (N=20) compared to other samples (N=155) in the Mullighan study (GSE12995) were processed for enrichment of IK target genes <b>(B1)</b> and IK-regulated lymphoid priming genes <b>(B2)</b> using a supervised approach implemented in GSEA2.08 (Broad institute). Enrichment scores for calculated for the ranked members of the gene sets and normalized to the gene set size (NES) for which the P-value was calculated using 1000 permutations of the pre-ranked gene list and the FDR corrected for testing 2 gene sets. There was a significant enrichment of IK target genes (NES = 1.43, P = 0.046) for Ph⁺ patients that included the leading edge subset comprised of TSPAN13<i>, GSN</i>, MDFIC<i>, ITGA4, TREML2, RNF125</i>, IQGAP2, LAMC1, TES, DHRS3<i>, S100A10</i>, IL12RB1, ADD3<i>, GRAMD3, ATRNL1</i>, RUNX2<i>, MCOLN3</i>, ATP1B1, and CALCRL. Likewise, there was a significant enrichment of lymphoid priming genes (NES = 1.51, P = 0.039) for Ph⁺ patients that included the leading edge subset comprised of <i>IGJ</i>, CNN3, CD52, DNTT<i>, CSF1R</i>, SATB1<i>, LTB, PTGER2</i>, MEF2C, RUNX2 (Figure <b>1B.2</b>). There was a significant increase in the multivariate mean for 45 transcripts in the Ph⁺ subset of specimens compared to the pooled mean of the other subsets (MANOVA, F_1,173 = 11.29, P=0.001). The mean level of expression for each transcript in each BPL subset is illustrated in the heat map organized using a two-way hierarchical clustering method (average distance metric) to group expression profiles of transcripts and specimen subsets (<b>C</b>).</p