Deficiency of Phox2b protein due to either MO knockdown or overexpression of <i>PHOX2B</i> variants leads to increased <i>phox2b</i> and <i>ascl1</i> RNA expression in the SCG.

<p>(A–D) Dorsal views (cranial to the left) of 4-dpf embryos expressing a <i>phox2b</i> ATG MO (B,D) or mismatched control MO (A,C), showing expression of <i>phox2b</i> (A, B) and <i>ascl1</i> (C, D) as determined by whole-mount ISH. Insets depict an enlarged view of the area of the SCG. (E–I) Area of the SCG is shown in 4-dpf embryos (lateral view, cranial to the left) in which capped mRNA (100 ng/µl) for wild-type (WT) human <i>PHOX2B</i> (F) and the indicated variants (G–I) was injected and ISH performed for <i>ascl1</i>. CT, control, water injected. (J, K) Whole-mount ISH in control vs. <i>phox2b</i> MO-injected embryos double labeled with <i>phox2a (blue)</i> and <i>th</i> (red) riboprobes. Arrows point to the SCG.</p

Schematic representation of the effect of aberrant Phox2b on sympathetic neuronal development in the zebrafish model.

<p>Phox2b is the master regulator of a differentiation cascade involving <i>Hand2</i>, <i>Gata2/3</i> and <i>Tfap2a</i> that ultimately leads to terminal differentiation of neuron progenitors, marked by <i>dbh</i> and <i>th</i> expression. Phox2b regulates itself as well as <i>ascl1a</i> and can directly activate <i>dbh</i>. A decreased dosage of the <i>phox2b</i> gene, either by allelic deletion (Phox2b KD) or by dominant-negative mutations (<i>676delG</i> or <i>K155X</i>) can compromise normal Phox2b function so that the cells are unable to progress through the various developmental stages and instead remain in an undifferentiated state.</p

<i>phox2b-</i>deficient embryos show impaired differentiation of sympathetic neurons in the SCG.

<p>(A–F) Lateral/oblique views of 4-dpf embryos after whole-mount ISH for <i>th</i> (A–C) and <i>dbh</i> (D–F) in control and <i>phox2b</i>–deficient embryos. Arrows indicate the SCG. Knockdown of <i>phox2b</i> by injection of a splice MO (4 ng) (MO^splice) inhibits the expression of <i>th</i> and <i>dbh</i> (B, E) which is rescued by coexpression of human <i>PHOX2B</i> mRNA (10 ng/µl) (C, F). Relative intensity levels of <i>th</i> (G) and <i>dbh</i> (H) expression in embryos injected with <i>phox2b</i> MOs that inhibit translation (MO^ATG) or splicing (MO^splice). Mismatched control MO (MO^mm) and <i>PHOX2B</i> mRNA-rescue (MO^splice/<i>PHOX2B</i>) are also shown. Data are presented as means ± SD (^***P<0.001; ^**P<0.01; n = 15 for each group).</p

<i>phox2b</i>, but not <i>ascl1</i>, is indispensable for sympathetic neuronal differentiation.

<p>(A–F) Whole-mount ISH of 3-dpf embryos for <i>th</i> and <i>dbh</i> following <i>ascl1</i> MO knockdown. Lateral views depict a minimal decrease in expression of <i>th</i> (A,B) and <i>dbh</i> (D,E) in <i>ascl1</i> MO-injected embryos compared to control MO-injected (A,D) embryos. Simultaneous knockdown of both <i>ascl1</i> and <i>phox2b</i> led to a significant decrease in expression of <i>th</i> and <i>dbh</i> in the SCG (C,F). (G) Relative intensity levels of <i>dbh</i>-staining in the SCG of embryos expressing <i>ascl1</i> MO, <i>phox2b</i> MO or the combination of the two. Data are presented as means ± SEM (^***P<0.001; n = 15 per group). (H–M) Whole-mount ISH for expression of <i>ascl</i> (H–J) and <i>phox2b</i> (K–M) in embryos in which <i>ascl1</i> expression was abrogated by MO knockdown, either singly (I, L) or in combination with <i>phox2b</i> (J, M). (N,O) Whole-mount ISH for <i>phox2a</i> expression in 4-dpf embryos in which <i>ascl1</i> expression was abrogated by MO knockdown. Arrows point to region of SCG.</p

Financial vs. Non-financial Stocks: Time-varying Correlations and Risks

We analyze the time-varying co-movements of both financial and non-financial stock returns across countries to analyze the conditional correlation exhibited by cross-country pairs during the recent financial crisis. Using an asymmetric bivariate GARCH model, the analysis is conducted for a number of developed and developing countries. Given the origins of this current crisis, we expect increased correlation between financial sectors. However, recent correlations are not excessively large when compared to those earlier in this decade. Principal components analysis reveals one common driver of these pairwise correlations which may be related to U.S. returns and market liquidity

The neuroblastoma-associated <i>676delG</i> and <i>K155X PHOX2B</i> variants cause decreased terminal differentiation in the SCG.

<p>Whole-mount ISH for <i>th</i> (A–F) and <i>dbh</i> (G–L) expression in 3-dpf embryos in which human neuroblastoma-derived mutations were overexpressed (lateral views are shown). The area encompassing the SCG (boxed) is shown enlarged to the right of each panel. Capped mRNA (100 ng/µl) for wild-type (WT) human <i>PHOX2B</i> and the <i>R100L</i>, <i>676delG</i>, <i>K155X</i> mutations were injected into one-cell embryos. CT, control water-injected. Relative intensity levels of <i>th</i> (M) and <i>dbh</i> (N) expression in the embryos depicted in panels A–F and G–L respectively. Data are presented as means ± SD (*P<0.01 vs. control-injected embryos; n = 6 per group). Whole-mount ISH for <i>th</i> (O) and <i>dbh</i> (P) in 3-dpf embryos expressing the <i>phox2b</i> MO and <i>PHOX2B K155X</i> mutant mRNA (P2BMO+<i>K155X</i>). Arrow indicates the region of the SCG.</p

<i>phox2b</i> is expressed in the SCG, a marker of the peripheral sympathetic nervous system in the zebrafish.

<p>(A–D) Whole-mount <i>in situ</i> hybridization (ISH) for <i>phox2b</i> expression in wild-type embryos at the indicated time points. <i>phox2b</i> expression is seen in cells of the prospective superior cervical ganglion (SCG; white arrows, boxed) at 36 hpf (A), which start to extend caudally to form the sympathetic chain. These cells are identified as sympathetic neuronal cells by their expression of <i>dbh</i> and <i>th</i> (E, F). Expression is also seen in the brachial arches (black asterisk) and hindbrain (white asterisk). By 50 hpf, <i>phox2b</i> expression is seen in the enteric precursors (B–D, black arrow). Expression continues in two parallel rows caudally, encompassing the sympathetic chain until 4 dpf. (E) Lateral view (cranial to the left) of a 4-dpf zebrafish embryo analyzed by whole-mount ISH for <i>dbh</i> expression, which is seen in the SCG (black square), the arch associated complex (AAC) also derived from the neural crest, and the hindbrain (HB). Inset shows enlarged dorsal view of the SCG. (F) Whole-mount ISH of 4-dpf embryo analyzed for <i>th</i> expression. Aggregates of cells expressing <i>th</i> are seen in the SCG (arrow) as well as in the midbrain (MB), HB and retina (R).</p

Impaired differentiation in the SCG due to <i>phox2b</i> deficiency is not rescued by retinoic acid.

<p>(A–F) Dorsal views of 3-dpf embryos injected with <i>phox2b</i> MO (D–F) or mismatched control MO (A–C) and treated with increasing concentrations of 13–<i>cis</i> retinoic acid (RA). (G) Relative intensity measurements of <i>th</i> expression in the SCG of embryos injected with various MOs and treated with different concentrations of RA. ATGK1, translation-blocking <i>phox2b</i> MO; P2BT2, splice blocking <i>phox2b</i> MO. (H–O) Whole-mount ISH of 3-dpf embryos in which the specified RNAs were overexpressed and analyzed for <i>th</i> expression following exposure to RA. Capped mRNA (100 ng/µl) for wild-type (WT) human <i>PHOX2B</i>, and the <i>R100L</i>, <i>676delG</i>, <i>K155X</i> mutations were injected into embryos at the one-cell stage. (P) Quantification of the relative intensity of <i>th</i> staining in the embryos depicted in H–O. Data are presented as means ± SD (^*P<0.05; n = 10 per group).</p

Phox2b deficiency causes arrest of SCG cells at an undifferentiated stage.

<p>(A,B) Dorsal views of ISH with FITC-labeled <i>th</i> (red) and digoxigenin-labeled <i>phox2b</i> (blue) riboprobes on 4-dpf embryos in which <i>phox2b</i> expression was abrogated by MO knockdown. Arrows point to the SCG. Insets show enlarged views of the SCG. (C, D) Lateral views of ISH with FITC-labeled <i>phox2b</i> (red) and digoxigenin-labeled <i>dbh</i> (blue) riboprobes in MO-injected (D) and control (C) embryos. (E) Quantitative real time-PCR analysis comparing <i>phox2b</i> mRNA expression levels in control vs. <i>phox2b</i> MO-injected (P2BATG MO) embryos. Data are presented as means ± SD (^****P<0.0001, ^***P<0.001; n = 15 per group). (F,G) Whole-mount ISH for <i>elavl3</i> in the area of the SCG (arrow) in <i>phox2b</i> MO-injected (G) compared to control MO-injected embryos at 4-dpf (F). (H–K) Lateral views of the SCG in 4-dpf embryos overexpressing the indicated RNAs analyzed for <i>elavl3</i> expression by whole-mount ISH.</p

Table_3_Synergistic Anti-Tumor Effect of Combining Selective CDK7 and BRD4 Inhibition in Neuroblastoma.xlsx

PurposeCyclin-dependent kinases (CDKs) that have critical roles in RNA polymerase II (Pol II)-mediated gene transcription are emerging as therapeutic targets in cancer. We have previously shown that THZ1, a covalent inhibitor of CDKs 7/12/13, leads to cytotoxicity in MYCN-amplified neuroblastoma through the downregulation of super-enhancer-associated transcriptional upregulation. Here we determined the effects of YKL-5-124, a novel covalent inhibitor with greater selectivity for CDK7 in neuroblastoma cells.Experimental DesignWe tested YKL-5-124 in MYCN-amplified and nonamplified neuroblastoma cells individually and in combination with other inhibitors in cell line and animal models. Cell viability, target validation, effects on cell cycle and transcription were analyzed.ResultsCDK7 inhibition with YKL-5-124 did not lead to significant cell death, but resulted in aberrant cell cycle progression especially in MYCN-amplified cells. Unlike THZ1, YKL-5-124 had minimal effects on Pol II C-terminal domain phosphorylation, but significantly inhibited that of the CDK1 and CDK2 cell cycle kinases. Combining YKL-5-124 with the BRD4 inhibitor JQ1 resulted in synergistic cytotoxicity. A distinct MYCN-gene expression signature associated with resistance to BRD4 inhibition was suppressed with the combination. The synergy between YKL-5-124 and JQ1 translated into significant tumor regression in cell line and patient-derived xenograft mouse models of neuroblastoma.ConclusionsThe combination of CDK7 and BRD4 inhibition provides a therapeutic option for neuroblastoma and suggests that the addition of YKL-5-124 could improve the therapeutic efficacy of JQ1 and delay resistance to BRD4 inhibition.</p