Selective and Potent PROTAC Degraders of c-Src Kinase.

Using dasatinib linked to E3 ligase ligands, we identified a potent and selective dual Csk/c-Src PROTAC degrader. We then replaced dasatinib, the c-Src-directed ligand, with a conformation-selective analogue that stabilizes the αC-helix-out conformation of c-Src. Using the αC-helix-out ligand, we identified a PROTAC that is potent and selective for c-Src. We demonstrated a high degree of catalysis with our c-Src PROTACs. Using our c-Src PROTACs, we identified pharmacological advantages of c-Src degradation compared to inhibition with respect to cancer cell proliferation.

Reply to Correspondence on "Synergy and Antagonism between Allosteric and Active-Site Inhibitors of Abl Tyrosine Kinase".

Manley and co-workers provide data demonstrating that, at super-pharmacological concentrations (300 µM), a ternary complex between Abl, asciminib, and ATP-competitive inhibitors is possible. The work in our manuscript concerns the interplay of asciminib (and GNF-2) with ATP-competitive inhibitors at pharmacologically relevant concentrations (Cmax =1.6-3.7 µM for asciminib). Manley and co-workers do not question any of the studies that we reported, nor do they provide explanations for how our work fits into their preferred model. Herein, we consider the data presented by Manley and co-workers. In addition, we provide new data supporting the findings in our Communication. Asciminib and ATP-competitive inhibitors do not simultaneously bind Abl at pharmacologically relevant concentrations unless the conformation selectivity for both ligands is matched.

Synergy and Antagonism between Allosteric and Active-Site Inhibitors of Abl Tyrosine Kinase.

Allosteric inhibitors of Abl kinase are being explored in the clinic, often in combination with ATP-site inhibitors of Abl kinase. However, there are conflicting data on whether both ATP-competitive inhibitors and myristoyl-site allosteric inhibitors can simultaneously bind Abl kinase. Here, we determine whether there is synergy or antagonism between ATP-competitive inhibitors and allosteric inhibitors of Abl. We observe that clinical ATP-competitive inhibitors are not synergistic with allosteric ABL inhibitors, however, conformation-selective ATP-site inhibitors that modulate the global conformation of Abl can afford synergy. We demonstrate that kinase conformation is the key driver to simultaneously bind two compounds to Abl kinase. Finally, we explore the interaction of allosteric and conformation selective ATP-competitive inhibitors in a series of biochemical and cellular assays.

Regulation of androgen-responsive transcription by the chromatin remodeling factor CHD8.

The androgen receptor (AR) mediates the effect of androgens through its transcriptional function during both normal prostate development and in the emergence and progression of prostate cancer. AR is known to assemble coactivator complexes at target promoters to facilitate transcriptional activation in response to androgens. Here we identify the ATP-dependent chromatin remodeling factor chromodomain helicase DNA-binding protein 8 (CHD8) as a novel coregulator of androgen-responsive transcription. We demonstrate that CHD8 directly associates with AR and that CHD8 and AR simultaneously localize to the TMPRSS2 enhancer after androgen treatment. In the LNCaP cell line, reduction of CHD8 levels by small interfering RNA treatment severely diminishes androgen-dependent activation of the TMPRSS2 gene. We demonstrate that the recruitment of AR to the TMPRSS2 promoter in response to androgen treatment requires CHD8. Finally, CHD8 facilitates androgen-stimulated proliferation of LNCaP cells, emphasizing the physiological importance of CHD8. Taken together, we present evidence of a functional role for CHD8 in AR-mediated transcriptional regulation of target genes.

Regulation of HOXA2 gene expression by the ATP-dependent chromatin remodeling enzyme CHD8.

Chromodomain, helicase, DNA-binding protein 8 (CHD8) is an ATP-dependent chromatin remodeling enzyme that has been demonstrated to exist within a large protein complex which includes WDR5, Ash2L, and RbBP5, members of the Mixed Lineage Leukemia (MLL) histone modifying complexes. Here we show that CHD8 relocalizes to the promoter of the MLL regulated gene HOXA2 upon gene activation. Depletion of CHD8 enhances HOXA2 expression under activating conditions. Furthermore, depletion of CHD8 results in a loss of the WDR5/Ash2L/RbBP5 subcomplex, and consequently H3K4 trimethylation, at the HOXA2 promoter. These studies suggest that CHD8 alters HOXA2 gene expression and regulates the recruitment of chromatin modifying enzymes.

CHD8 is an ATP-dependent chromatin remodeling factor that regulates beta-catenin target genes.

ATP-dependent chromatin remodeling by the CHD family of proteins plays an important role in the regulation of gene transcription. Here we report that full-length CHD8 interacts directly with beta-catenin and that CHD8 is also recruited specifically to the promoter regions of several beta-catenin-responsive genes. Our results indicate that CHD8 negatively regulates beta-catenin-targeted gene expression, since short hairpin RNA against CHD8 results in the activation of several beta-catenin target genes. This regulation is also conserved through evolution; RNA interference against kismet, the apparent Drosophila ortholog of CHD8, results in a similar activation of beta-catenin target genes. We also report the first demonstration of chromatin remodeling activity for a member of the CHD6-9 family of proteins, suggesting that CHD8 functions in transcription through the ATP-dependent modulation of chromatin structure.

Regulation of tryptophan hydroxylase-2 gene expression by a bipartite RE-1 silencer of transcription/neuron restrictive silencing factor (REST/NRSF) binding motif.

Tryptophan hydroxylase-2 (TPH2) is the rate-limiting enzyme in raphe serotonin biosynthesis, and polymorphisms of TPH2 are implicated in vulnerability to psychiatric disorders. Dynamic transcription regulation of TPH2 may underlie differences in vulnerability. We identified a transcription element in the TPH2 promoter that resembles the binding motif for RE-1 silencer of transcription (REST; also known as NRSF) transcription factor. REST limits tissue expression of non-neuronal genes through a canonical 21-bp motif called the NRSE (neuron-restrictive silencing element). The NRSE in TPH2 is a novel bipartite variant interrupted by a 6-base insertion. We confirmed that this bipartite NRSE permits transcriptional repression by REST identical to canonical NRSE in rat C6-glioma cells. Synthetic permutations of the motif revealed considerable flexibility in the juxtaposition of the two halves of bipartite NRSE. Computational analysis revealed many bipartite NRSE variants conserved between mouse and human genomes. A subgroup of these was found to bind REST by chromatin immunoprecipitation. Messenger RNAs for TPH2 and potassium channel H6, another gene with a bipartite NRSE, were up-regulated by dominant-negative REST in C6-glioma cells. These findings, which indicate that TPH2 expression is part of the developmental program regulated by REST and suggest that many previously unrecognized genes may be regulated by REST through the novel motif, have implications for the mechanism of REST action.

Functional interplay between histone demethylase and deacetylase enzymes.

Histone deacetylase (HDAC) inhibitors are a promising class of anticancer agents for the treatment of solid and hematological malignancies. The precise mechanism by which HDAC inhibitors mediate their effects on tumor cell growth, differentiation, and/or apoptosis is the subject of intense research. Previously we described a family of multiprotein complexes that contain histone deacetylase 1/2 (HDAC1/2) and the histone demethylase BHC110 (LSD1). Here we show that HDAC inhibitors diminish histone H3 lysine 4 (H3K4) demethylation by BHC110 in vitro. In vivo analysis revealed an increased H3K4 methylation concomitant with inhibition of nucleosomal deacetylation by HDAC inhibitors. Reconstitution of recombinant complexes revealed a functional connection between HDAC1 and BHC110 only when nucleosomal substrates were used. Importantly, while the enzymatic activity of BHC110 is required to achieve optimal deacetylation in vitro, in vivo analysis following ectopic expression of an enzymatically dead mutant of BHC110 (K661A) confirmed the functional cross talk between the demethylase and deacetylase enzymes. Our studies not only reveal an intimate link between the histone demethylase and deacetylase enzymes but also identify histone demethylation as a secondary target of HDAC inhibitors.

53BP1 oligomerization is independent of its methylation by PRMT1.

p53 binding protein 1 (53BP1) participates in the repair of DNA double stranded breaks (DSBs) where it is recruited to or near sites of DNA damage. Although little is known about the biochemical functions of 53BP1, the protein possesses several motifs that are likely important for its role as a DNA damage response element. This includes two BRCA1 C-terminal repeats, tandem Tudor domains, and a variety of phosphorylation sites. Here we show that a glycine-arginine rich (GAR) stretch of 53BP1 lying upstream of the Tudor motifs is methylated. We demonstrate that arginine residues within this region are important for asymmetric methylation by the PRMT1 methyltransferase. We further show that sequences upstream of the Tudor domains that do not include the GAR stretch are sufficient for 53BP1 oligomerization in vivo. Thus, although Tudor domains bind methylated proteins, 53BP1 homo-oligomerization occurs independently of Tudor function. Lastly, we find that deficiencies in 53BP1 generate a "hyper-rec" phenotype. Collectively, these data provide new insight into 53BP1, an important component in maintaining genomic stability.

A chromatin remodelling complex that loads cohesin onto human chromosomes.

Nucleosomal DNA is arranged in a higher-order structure that presents a barrier to most cellular processes involving protein DNA interactions. The cellular machinery involved in sister chromatid cohesion, the cohesin complex, also requires access to the nucleosomal DNA to perform its function in chromosome segregation. The machineries that provide this accessibility are termed chromatin remodelling factors. Here, we report the isolation of a human ISWI (SNF2h)-containing chromatin remodelling complex that encompasses components of the cohesin and NuRD complexes. We show that the hRAD21 subunit of the cohesin complex directly interacts with the ATPase subunit SNF2h. Mapping of hRAD21, SNF2h and Mi2 binding sites by chromatin immunoprecipitation experiments reveals the specific association of these three proteins with human DNA elements containing Alu sequences. We find a correlation between modification of histone tails and association of the SNF2h/cohesin complex with chromatin. Moreover, we show that the association of the cohesin complex with chromatin can be regulated by the state of DNA methylation. Finally, we present evidence pointing to a role for the ATPase activity of SNF2h in the loading of hRAD21 on chromatin.

A core-BRAF35 complex containing histone deacetylase mediates repression of neuronal-specific genes.

BRAF35, a structural DNA-binding protein, initially was identified as a component of a large BRCA2-containing complex. Biochemical analysis revealed the presence of a smaller core-BRAF35 complex devoid of BRCA2. Here we report the isolation of a six-subunit core-BRAF35 complex with the capacity to deacetylate histones, termed the BRAF-histone deacetylase complex (BHC), from human cells. BHC contains polypeptides reminiscent of the chromatin-remodeling complexes SWI/SNF and NuRD (nucleosome remodeling and deacetylating). Similar to NuRD, BHC contains an Mi2-like subunit, BHC80, and a PHD zinc-finger subunit as well as histone deacetylases 1/2 and an MTA-like subunit, the transcriptional corepressor CoREST. We show that BHC mediates repression of neuron-specific genes through the cis-regulatory element known as the repressor element 1 or neural restrictive silencer (RE1/NRS). Chromatin-immunoprecipitation experiments demonstrate the recruitment of BHC by the neuronal repressor REST. Expression of BRAF35 containing a single point mutation in the HMG domain of the protein abrogated REST-mediated transcriptional repression. These results demonstrate a role for core-BRAF35-containing complex in the regulation of neuron-specific genes through modulation of the chromatin structure.