Studies Towards Broadening The Substrate Profile And Regulation Of Histone Deacetylase 1

Aberrant expression of histone deacetylase 1 (HDAC1) is implicated in multiple diseases, including cancer. As a consequence, HDAC1 has emerged as an important therapeutic target for drug development. HDAC1 regulates key cellular processes, such as cell proliferation, apoptosis, and cell survival, by deacetylating both histone and non-histone substrates. Due to the lack of simple tools to identify physiological substrates of HDAC1, the full spectrum of HDAC1 activities in the cell remains unclear. Here, we employed a substrate trapping strategy to identify cellular substrates of HDAC1. Using this approach, we identified mitosis-related protein Eg5 as a substrate. HDAC1 colocalizes with Eg5 during mitosis, suggesting a role for HDAC1 in the mitotic defects observed with HDAC inhibitor drugs. By extending substrate trapping strategy to HEK293 cells, we identified Lysine Specific Demethylase 1 (LSD1) as an HDAC1 substrate. Significantly, LSD1 is overexpressed in multiple cancers and has emerged as a potential anti-cancer drug target. LSD1 is typically found in association with another epigenetic enzyme, histone deacetylase (HDAC). HDAC and LSD1 inhibitor compounds have been tested as combination anti-cancer agents. However, the functional link between LSD1 and HDAC has yet to be understood in detail. Here we uncovered that HDAC1 mediated deacetylation of LSD1 at K374 in the substrate binding lobe, which affected the histone 3 binding and gene expression activity of LSD1. The mechanistic link between HDAC1 and LSD1 established here suggests that HDAC inhibitors influence LSD1 activity, which will ultimately guide drug design targeting epigenetic enzymes. Discovery of novel substrates using trapping mutants will reveal the full activities of HDAC1 in both physiological and pathological conditions, which will lead to a better understanding of HDAC inhibitor mechanism of action. My second project focused on studying the effect of Single nucleotide polymorphisms (SNPs) of HDAC1. HDAC1 is upregulated in multiple diseases, and has emerged as an important therapeutic target for drug development. SNPs in multiple genes are often linked to the diseases, such as cancer. Here, we used the Hypothesis driven SNP search (HyDn-SNP-S) program to identify a HDAC1 SNP-F437C. The presence of SNP-F437C on HDAC1 affected acetylation at K432 and phosphorylation at S393, which ultimately altered enzymatic activity. These studies shed insights into the altered posttranslational modifications caused by HDAC1 exonic SNP. The study also revealed the significance of studying SNPs of HDAC in understanding the mechanisms leading to HDAC deregulation in cancer

LSD1 Substrate Binding and Gene Expression Are Affected by HDAC1-Mediated Deacetylation

Lysine Specific Demethylase 1 (LSD1) catalyzes the demethylation of histone 3 to regulate gene expression. With a fundamental role in gene regulation, LSD1 is involved in multiple cellular processes, including embryonic development, cell proliferation, and metastasis. Significantly, LSD1 is overexpressed in multiple cancers and has emerged as a potential anticancer drug target. LSD1 is typically found in association with another epigenetic enzyme, histone deacetylase (HDAC). HDAC and LSD1 inhibitor compounds have been tested as combination anticancer agents. However, the functional link between LSD1 and HDAC has yet to be understood in detail. Here, we used a substrate trapping strategy to identify cellular substrates of HDAC1. Using inactive HDAC1 mutants, we identified LSD1 as an HDAC1 substrate. HDAC1 mediated deacetylation of LSD1 at K374 in the substrate binding lobe, which affected the histone 3 binding and gene expression activity of LSD1. The mechanistic link between HDAC1 and LSD1 established here suggests that HDAC inhibitors influence LSD1 activity, which will ultimately guide drug design targeting epigenetic enzymes

Targeted Degradation of Oncogenic KRASG12C by VHL-recruiting PROTACs

We report the development of LC-2, the first PROTAC capable of degrading endogenous KRASG12C. LC-2 covalently binds KRASG12C with a MRTX849 warhead and recruits the E3 ligase VHL, inducing rapid and sustained KRASG12C degradation leading to suppression of MAPK signaling in both homozygous and heterozygous KRASG12C cell lines. LC-2 demonstrates that PROTAC-mediated degradation is a viable option for attenuating oncogenic KRAS levels and downstream signaling in cancer cells.</p

Mutagenesis Studies of the 14 Å Internal Cavity of Histone Deacetylase 1: Insights toward the Acetate-Escape Hypothesis and Selective Inhibitor Design

Histone deacetylase (HDAC) proteins are promising targets for cancer treatment, as shown by the approval of two HDAC inhibitors for the treatment of cutaneous T-cell lymphoma. HDAC1 in particular has been linked to cell growth and cell cycle regulation and is therefore an attractive target for anticancer drugs. The HDAC1 active site contains a hydrophobic 11 Å active-site channel, with a 14 Å internal cavity at the bottom of the active site. Several computational and biochemical studies have proposed an acetate-escape hypothesis where the acetate byproduct of the deacetylation reaction escapes via the 14 Å internal cavity. Selective HDAC inhibitors that bind to the 14 Å cavity have also been created. To understand the influence of amino acids lining the HDAC1 14 Å cavity in acetate escape and inhibitor binding, we used mutagenesis coupled with acetate competition assays. The results indicate that amino acids lining the 14 Å cavity are critical for catalytic activity and acetate competition, confirming the role of the cavity in acetate escape. In addition, these mutagenesis studies will aid in HDAC1-inhibitor design that exploits the 14 Å cavity