Inhibition of Lysine Demethylase 4A (KDM4A) Rescues 3p - PowerPoint PPT Presentation

Inhibition of Lysine Demethylase 4A (KDM4A) Rescues 3p Haploinsufficiency Ruhee Dere, Ph.D. 14 th International VHL Medical Symposium Oct 29, 2020

Ren Renal al Cel ell C Car arcinoma a – Los oss of of 3p 3p Tumor mor S Suppr ppressors Co-deletion of multiple tumor suppressors on chromosome 3p contribute to renal cell carcinoma (RCC) 90% of clear cell renal cell carcinoma (ccRCC) have mono-allelic loss of Hsieh J et al., JCO, 2018 the epigenetic regulator SETD2

Histone C e Code e Vs. T Tubulin C n Code His istone Code Vs Vs. . Tubulin in C Code Courtesy Dr. Verhey Laurence et al., Trends in Genetics, 2016

SETD2 2 – A Dual al C Chromat atin & & Tubulin M Methyltransf sferas ase α -tub α -tubK40me3 DAPI Merge Zoom Li F. et al., Cell, 2013 Park IY. et al., Cell, 2016 • SETD2 writes the tri-methyl mark on histone H3 SETD2 SETD2 SETD2 lysine 36 (H3K36me3) GST GST GST • In vitro methylation assays show SETD2 can α -Tubulin methylate α -tubulin • Mass spec identified K40 of α -tubulin as the site for trimethylation Histone • Methyl lysine antibody specific for K40me3 recognized a methyl mark on spindle microtubules polyMT depolyMT Histones • Loss of Setd2 results in multiple mitotic defects and Park IY. et al., Cell, 2016 genomic instability

Existence o of f Du Dual-Func nction n Epigen enetic Reg Regulat ators Nucleus Cytoplasm Erasers Writers Erasers KDM4A Altered cytoskeleton ?? Writers Cell proliferation Gene Expression SETD2 Genomic stability DNA repair ?? Migration & metastasis Chromatin structure me me meK40 meK40 Readers PBRM1 ?? Readers

KDM4A i is s a a Cyt ytoplasm asmic P Protei ein hTERT RPE-1 HKC MEFs CD44 HA-KDM4A WCE N C WCE N C N C N C KDM4A KDM4A KDM4A Lamin A/C Lamin A/C Lamin A/C LDH LDH LDH • KDM4A is the corresponding demethylase that Li. GM, Cancer Res, 2013 erases the methyl mark laid by SETD2 on chromatin (H3K36me3) KDM4A is a dioxygenase enzyme requiring Fe, α - • ketoglutarate as co-factors for its activity • KDM4A localizes in the cytoplasm

KD KDM4A B Bin inds Methylated T Tubulin in A B C RPE1 hTERT RPE-1 D HEK293T cells • KDM4A binds α -tubulin WCE • KDM4A binds methylated IP: KDM4A tubulin Fold enrichment • Tubulin 1A (TUB1A) is highly over IgG control Mass Spec Analysis enriched in KDM4A immunoprecipitates

KDM DM4A De Demethyla lates Tubuli lin • Recombinant KDM4A A B Substrate: Substrate: demethylates recombinant Purified Tubulin Recombinant H3K36me3 (bovine) Recombinant microtubules Recombinant - + - + KDM4A KDM4A • Overexpression of a catalytically H3K36me3 K40me3 dead KDM4A (H188A mutant) fails Tubulin (input) H3 (input) to decrease methylation on microtubules D C Loss of KDM4A increases K40me3 • signal at the mitotic spindle E

KDM4A L Local alizes at s at the e MTOCs • KDM4A localizes at the poles of the mitotic spindle • KDM4A is a centrosome-associated protein that colocalizes with γ - tubulin at the MTOCs during mitosis • Localization of KDM4A at MTOCs is independent of SETD2 methylation on microtubules

KDM4A 4A Oppos pposes S SET ETD2- Me Mediated Me Methylation on n MT MTs If KDM4A opposes the methyl mark on MTs laid by SETD2, then inhibition of KDM4A should rescue genomic defects associated with SETD2 haploinsufficiency. Chiang et al., Cancer Res, 2018

Loss o ss of K KDM4A Resc Rescues G es Gen enomic Instab ability y Asso ssociated ed wi with Mono-allel elic L Loss ss of Se Setd2

Summa mmary • KDM4A is a cytoplasmic protein that binds α -tubulin • KDM4A demethylates Erasers microtubules KDM4A Altered cytoskeleton ?? Writers • KDM4A localizes to the MTOCs Cell proliferation SETD2 Genomic stability of the mitotic spindle ?? Migration & metastasis meK40 meK40 Readers PBRM1 ??

Summa mmary KDM4A activity can be inhibited using the small molecule JIB-04 JIB-04 disrupts O 2 binding in the catalytic domain of JIB-04 KDM4A Cascella B., et al., 2017, Chem. Commun.

Ack cknow owledgements University of Michigan Kristen Verhey, Ph.D. U.T. Southwestern Elisabeth Martinez, Ph.D. Pratim Chowdhury Dr. Rahul Jangid Vanderbilt University Research Associate Post-doctoral Fellow W. Kimryn Rathmell, M.D., Ph.D. Frank Mason, Ph.D. Baylor College of Medicine Cheryl L. Walker, Ph.D. Nicolas Young, Ph.D. Funding Sung Jung, Ph.D. VHL Alliance Gilson Longenbaugh Foundation