Small molecule p300/catenin antagonist enhances hematopoietic recovery after radiation

<div><p>There is currently no FDA approved therapeutic agent for ARS mitigation post radiation exposure. Here we report that the small molecule YH250, which specifically antagonizes p300/catenin interaction, stimulates hematopoiesis in lethally or sublethally irradiated mice. A single administration of YH250 24 hours post irradiation can significantly stimulate HSC proliferation, improve survival and accelerate peripheral blood count recovery. Our studies suggest that promotion of the expansion of the remaining HSC population via stimulation of symmetric non-differentiative proliferation is at least part of the mechanism of action.</p></div

Edited P19 cells exhibited higher β-catenin/TCF transcription, as well as increased survivin/BIRC5 expression.

<p>A. Edited P19 cells exhibited significantly higher β-catenin/TCF transcription compared with wild type (WT) cells under basal conditions, as assessed by Topflash/Fopflash (TOP/FOP) activity. n = 3, *p < 0.05. B and C. In both wild type and edited P19 cells, Wnt3a significantly activated the Survivin/luciferase reporter construct (compared with empty vehicle). This activation was effectively inhibited by specific, direct small molecule CBP/catenin antagonist ICG-001 in wild type cells (B). However, in the P19 edited cells, although Wnt3a increased Survivin/luciferase activity, ICG-001 did not reduce the activity (C). Representative of n = 3, *p < 0.05, **p < 0.01. D. Expression of survivin/BIRC5 mRNA, which is highly dependent on CBP, was enhanced by Wnt3a in wild type (WT) and to a lesser extent in p300 edited P19 cells. Whereas treatment with ICG-001 in WT cells significantly decreased survivin/BIRC5 message, there was essentially no effect of ICG-001on survivin/BIRC5 expression in edited cells. n = 3, **p < 0.01. Vehicle/control data (set to 1) as indicated with a red, dashed horizontal.</p

Nuclear receptor/Wnt beta-catenin interactions are regulated via differential CBP/p300 coactivator usage

<div><p>Over 400 million years ago, the evolution of vertebrates gave rise to a life cycle in which the organism began to live longer particularly as an adult. To accommodate such a longer lifespan, the organism underwent adaptation, developing a mechanism for long-lived cellular homeostasis. This adaptation required a population of long-lived relatively quiescent somatic stem cells (SSCs) along with a more proliferative differentiated daughter cell population, and was necessary to safeguard the genetic attributes with which SSCs were endowed. Intriguingly, cAMP response element binding protein (CREB)-binding protein (CBP) and E1A-binding protein, 300 kDa (p300), the highly homologous Kat3 coactivators had diverged, through duplication of ancestral Kat3, immediately preceding the evolution of vertebrates, given that both CBP and p300 have been detected in nearly all vertebrates versus non-vertebrates. We now demonstrate that a relatively small, highly evolutionarily conserved, amino terminal 9 amino acid deletion in CBP versus p300, plays a critical role in allowing for both robust maintenance of genomic integrity in stem cells and the initiation of a feed-forward differentiation mechanism by tightly controlling the interaction of the nuclear receptor family with the Wnt signaling cascade in either an antagonistic or synergistic manner.</p></div

Absence of “p300 insertion” sequence sterically inhibits nuclear receptor and β-catenin from simultaneously binding to CBP; however, nuclear receptor may bind concurrently to p300 (due to lack of steric inhibition) and thereby antagonize CBP/β-catenin signaling.

<p>A. Sequence alignment of the distal N-termini of p300 (P1) and CBP (C1), depicting conserved sites for binding of β-catenin (DELI motif) and for binding of nuclear receptor (LXXLL motif), as well as the 9aa “p300 insertion” sequence which is deleted/absent from CBP. (Note: Sequences depicted are for human.) B. Absence of “p300 insertion” sequence prevents nuclear receptor and β-catenin from simultaneously binding to CBP due to steric inhibition (yellow triangle), whereas presence of the sequence allows concurrent binding and synergy of β-catenin and nuclear receptor due to abrogation of steric hindrance (lavender crescent). C. Three-dimensional structural modeling of the N-terminus of CBP/p300 and its putative interactions with β-catenin and nuclear receptor ligand binding domain of RXR alpha are shown in a ribbon representation. (Left) Interaction between the N-terminus of CBP (CBP-NT) (pink color) and β-catenin (white color) is shown. The nuclear receptor (NR) binding motif (LXXLL) is highlighted in red. (Right) Interaction between the N-terminus of p300 (p300-NT) and β-catenin (white color) and nuclear receptor (RXR alpha) (green color). The nuclear receptor binding motif in p300 (LXXLL) is highlighted in yellow color.</p

More β-catenin is associated with p300 in edited P19 cells than in wild type (WT) cells, as demonstrated by immunoblot for β-catenin using samples of nuclear protein subjected to immunoprecipitation with CBP, p300, or (control) rabbit IgG antibody.

<p>n = 3. (5% input/control immunoblotted for β-catenin is depicted on the far left).</p

p300 edited P19 cells do not undergo neuronal differentiation.

<p>A. Expression of ephrin B1, a gene critical for neuronal migration and the maintenance of neuronal progenitors, was induced by Wnt and all-trans retinoic acid (ATRA) in an additive manner in wild type P19 cells, as assessed by realtime RTPCR. Although expression of ephrin B1 was induced by Wnt3a and ATRA, individually, there was antagonism between the two treatments in the edited cells. n = 3, *p < 0.05, **p < 0.01. Vehicle/control data (set to 1) as indicated with a red, dashed horizontal. B and C. Wild type (WT) and edited P19 cells were subjected to an established neuronal differentiation protocol (as detailed in the Experimental Procedures). As demonstrated with light microscopy, WT cells stopped proliferating and neurite outgrowth ensued, whereas edited cells continued to proliferate and neurite outgrowth was not observed (20X magnification) (B). By immuno-fluorescence microscopy, ephrin B1 was clearly expressed after subjected to the differentiation protocol in WT but not edited P19 cells (20X magnification) (C).</p

Schematic of CBP and p300 showing identity over various regions.

<p>CBP and p300 have molecular weights of approximately 300kDa and are encoded over 33 and 31 exons and consist of 2441 and 2414 amino acids (aa), respectively. β-catenin, with direct small molecule CBP/catenin (PRI-724/ICG-001) antagonist, competitively binds to CBP’s distal N-terminus, the least conserved region within these two Kat3 coactivators. CBP, cAMP response element binding protein (CREB)-binding protein; p300, E1A-binding protein, 300 kDa; Br, Bromodomain; CH, Cysteine/histidine; KIX, kinase-inducible domain interacting domain.</p