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

Inhibition of Lysine Demethylase 4A (KDM4A) Rescues 3p Haploinsufficiency

Ruhee Dere, Ph.D. 14th International VHL Medical Symposium Oct 29, 2020

Hsieh J et al., JCO, 2018

Ren Renal al Cel ell C Car arcinoma a – Los

• ss of
• f 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 the epigenetic regulator SETD2

Histone C e Code e Vs. T Tubulin C n Code

His istone Code Vs Vs. . Tubulin in C Code

Laurence et al., Trends in Genetics, 2016

Courtesy Dr. Verhey

• SETD2 writes the tri-methyl mark on histone H3

lysine 36 (H3K36me3)

• In vitro methylation assays show SETD2 can

methylate α-tubulin

• Mass spec identified K40 of α-tubulin as the site for

trimethylation

• Methyl lysine antibody specific for K40me3

recognized a methyl mark on spindle microtubules

• Loss of Setd2 results in multiple mitotic defects and

genomic instability

SETD2 2 – A Dual al C Chromat atin & & Tubulin M Methyltransf sferas ase

Li F. et al., Cell, 2013

SETD2 GST SETD2 GST polyMT depolyMT Histones GST SETD2 α-Tubulin Histone

Park IY. et al., Cell, 2016 Park IY. et al., Cell, 2016

α-tub α-tubK40me3 DAPI Merge Zoom

Existence o

• f

f Du Dual-Func nction n Epigen enetic Reg Regulat ators

Writers Readers Erasers Gene Expression DNA repair Chromatin structure me me Nucleus Cytoplasm meK40 meK40 Writers SETD2 ?? Readers PBRM1 ?? Erasers KDM4A ?? Altered cytoskeleton Cell proliferation Genomic stability Migration & metastasis

KDM4A i is s a a Cyt ytoplasm asmic P Protei ein

• KDM4A is the corresponding demethylase that

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

WCE N C KDM4A Lamin A/C LDH MEFs WCE N C KDM4A Lamin A/C LDH HKC N C KDM4A Lamin A/C LDH hTERT RPE-1 N C CD44 HA-KDM4A

• Li. GM, Cancer Res, 2013

KD KDM4A B Bin inds Methylated T Tubulin in

• KDM4A binds α-tubulin
• KDM4A binds methylated

tubulin

• Tubulin 1A (TUB1A) is highly

enriched in KDM4A immunoprecipitates

B

RPE1

A

HEK293T cells WCE IP: KDM4A Mass Spec Analysis Fold enrichment

• ver IgG control

D

hTERT RPE-1

C

KDM DM4A De Demethyla lates Tubuli lin

H3K36me3 H3 (input)

• +

Substrate: Recombinant H3K36me3 Recombinant KDM4A Tubulin (input)

• +

K40me3 Substrate: Purified Tubulin (bovine) Recombinant KDM4A

A B C D E

• Recombinant KDM4A

demethylates recombinant microtubules

• Overexpression of a catalytically

dead KDM4A (H188A mutant) fails to decrease methylation on microtubules

• Loss of KDM4A increases K40me3

signal at the mitotic spindle

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

• n microtubules

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

KDM4A 4A Oppos pposes S SET ETD2- Me Mediated Me Methylation on n MT MTs

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

meK40 meK40 Writers SETD2 ?? Readers PBRM1 ?? Erasers KDM4A ?? Altered cytoskeleton Cell proliferation Genomic stability Migration & metastasis

• KDM4A is a cytoplasmic protein

that binds α-tubulin

• KDM4A demethylates

microtubules

• KDM4A localizes to the MTOCs
• f the mitotic spindle

KDM4A activity can be inhibited using the small molecule JIB-04 JIB-04 disrupts O2 binding in the catalytic domain of KDM4A

Cascella B., et al., 2017, Chem. Commun.

Summa mmary

JIB-04

Ack cknow

• wledgements

Pratim Chowdhury Research Associate

• Dr. Rahul Jangid

Post-doctoral Fellow Baylor College of Medicine Cheryl L. Walker, Ph.D. Nicolas Young, Ph.D. Sung Jung, Ph.D. Funding VHL Alliance Gilson Longenbaugh Foundation University of Michigan Kristen Verhey, Ph.D. U.T. Southwestern Elisabeth Martinez, Ph.D. Vanderbilt University

• W. Kimryn Rathmell, M.D., Ph.D.

Frank Mason, Ph.D.