The Loss ss of KDM6A Accelerates s the Deve velopment of Multiple Mye yeloma Daphné Dupéré-Richer Licht Lab The University of Florida The University of Florida
Epigenetic Regulator Gene Anomalies in MM Function Gene Diag Mut (%) Relapse Mut (%) CNV CREBBP 0.8 4 Loss 13% Histone Ac p300 1.3 2 Loss 13% NSD1 0 2.6 Gain 44% NSD2 15% t(4;14) Histone MT SETD2 1.3 2.6 Gain 33% KMT2C 1.5 6.4 Gain 50% Histone DM KDM6A 1.3 0.6 Loss 25% SMARCA4 0.2 2,6 Gain 50% Chromatin ARID4A 1 - Loss 23% Remodeling CHD2 1.5 0.6 Gain 50%
KD KDM6A - JmjC Histone Demethylase Targeting H3K27me3 and me2 - X-linked gene - Tumor Suppressor in Many Cancers - Mutations Encompassing the Whole Locus, Frequent Copy Loss Suggests Tumor Suppressor KDM6A mutations in MM K1071fs P1159fs W1245* P1207_ K1345_ G102D Q172P D114fs Y116fs Q218_ Q147* Q191* Q539* Q607* T807fr E278* R563* V91fs A17S 0 200 400 600 800 1000 1200 1401 TPR - Tetratricopeptide repeat domain JmjC domain
KDM6 KD M6A A is s a Centra ral Pro rotein of the COMP MPAS ASS-like ke En Enhancer C Compl plex Activation Repression SWI/SNF ARID1A/2/4A CBP /5B PRC2 p300 SMARCA4 EED KDM6A EZH2 KMT2C/D H3K27me OFF ON H3Kac H3K4me Data generated by The CoMMpass ℠ study from MMRF
Gain of Function Model Shows That KDM6A is a In Vitro Tumor Suppressor in MM ARP-1 ARD KDM6A null KDM6A wt ARD pKDM6A +Dox ARD ARP1 (ng/ml) 6 100 12 25 50 KDM6A H4 Ezponda et al Cell Reports 2017
Gain of Function Model Shows That KDM6A is a In Vitro Tumor Suppressor in MM 1400 ARD Cumulative cell 1200 Add back counts (x10 6 ) 1000 800 600 400 200 0 ARP-1 ARD 0 d 3 d 6 d 9 d 12 d KDM6A null KDM6A wt Time ARD pKDM6A +Dox ARD ARP1 (ng/ml) 6 100 12 25 50 KDM6A H4 Ezponda et al Cell Reports 2017
New mo model of KD KDM6 M6A A loss ss in MM MM Cell Line In vitro by y Ac Acute Gene Editing Tetracoid Repeats KDM6A gene jmjC KDM6A exon 4 exon 6 exon 24 250 kb 1401 a.a TPR JmjC Exon 4 Exon 6 gRNA gRNA Cas9
In vitro KDM6 KD M6A A Disru sruption Ad Adva vantages s Gro rowth of MM MM Cells Mutant allele frequency Mutant allele frequency KDM6A mutant allele 100 KDM6A mutant allele 100 frequency (%) frequency (%) 80 Arp-1 90 Mutant Alleles Karpas-620 Increase Over Time 60 80 Exon 4 MM1S Exon 6 40 70 20 60 0 5 10 0 10 Day Day
In vitro KDM6 KD M6A A Disru sruption Ad Adva vantages s Gro rowth of MM MM Cells Mutant allele frequency Mutant allele frequency KDM6A mutant allele 100 KDM6A mutant allele 100 frequency (%) frequency (%) 80 Arp-1 90 Mutant Alleles Karpas-620 Increase Over Time 60 80 Exon 4 MM1S Exon 6 40 70 20 60 0 5 10 0 10 Day Day Karpas-620 ARP-1 MM1S WT E4 E6 ARD WT E4 E6 WT E4 E6 KDM6A KD Pools of KDM6A KO cells HDAC2 HDA C2
A Ra A Rapid Mo Mouse se Mo Model for r Mu Multiple Mye Myeloma ma In vivo LTR promoter 8 Gy L-GP130 EGFP IRES fetal liver cells Kdm6af/f Cre+/- Cd19 Wild type Recipient E.14 Donor Method adapted from Dechow et el JCI, 2014
A Ra A Rapid Mo Mouse se Mo Model for r Mu Multiple Mye Myeloma ma In vivo Sanguineous Splenomegaly Tumor Tumor ascites LTR promoter 8 Gy L-GP130 EGFP IRES Control BM Spleen Tumor 0.10 2.08 82.8 1.02 6.02 43.0 GFP+ fetal liver cells Kdm6af/f Cre+/- Cd19 Wild type 0 t n 2 95,6 u 2 o Recipient B E.14 Donor C CD138 GFP GFP+ CD138+ Tumor Cells Method adapted from Dechow et el JCI, 2014
KDM6A is a tumor suppressor in vitro and in vivo What Molecular Function of KDM6A is Critical? Histone Demethylation? Where in the Genome Does KDM6A Bind in MM Cells? What Genes are Affected?
Jmj JmjC Deme methyl ylation Ac Activi vity y is s Disp spensa sable for r KD KDM6 M6A A Gro rowth Suppre ressi ssion H1146A JmjC Inactive Mutant E1148A JmjC TPR
JmjC Deme Jmj methyl ylation Ac Activi vity y is s Disp spensa sable for r KD KDM6 M6A A Gro rowth Suppre ressi ssion H1146A JmjC Inactive Mutant E1148A JmjC TPR pKDM6A pKDM6A empty vector JmjC-dead Cumulative cell count (x106) 16 16 16 n o d o x no dox no dox dox dox 12 12 dox 12 8 8 8 4 4 4 0 0 0 D0 D3 D6 D9 D12 D0 D3 D6 D9 D12 D0 D3 D6 D9 D12 Expression of wt or JmjC Dead KDM6A Causes Similar Growth Suppression
Jmj JmjC Deme methyl ylation Ac Activi vity y is s Disp spensa sable for r KD KDM6 M6A A Gro rowth Suppre ressi ssion H1146A JmjC Inactive Mutant E1148A JmjC TPR RNA-Seq After KDM6A Add-Back pKDM6A pKDM6A empty vector JmjC-dead Cumulative cell count (x106) 16 16 16 n o d o x no dox no dox dox dox 12 12 dox 12 8 8 8 4 4 4 - Membrane receptor activity 0 0 - Cell activation 0 D0 D3 D6 D9 D12 D0 D3 D6 D9 D12 D0 D3 D6 D9 D12 - Cell-cell adhesion Expression of wt or JmjC Dead KDM6A Causes Large Overlap of Genes Affected with Add-Back Similar Growth Suppression of Wild-Type and JmjC Inactive KDM6A
Tagging the Endogenous s KD KDM6 M6A A Locus s for r Detection of Binding Sites Bi KDM6A 1401 a.a JmjC HA TPR Stop codon ARP1 gRNA + ARD WT HA +/+ ssODN HA HA-tag KDM6A HDAC2
Va Valida dati tion o of H f HA T Taggi gging o g of K f KDM6A KDM6A edited/HA-tagged 800 HA-tag 0 HA 800 KDM6A wt untag 0 800 WT KDM6A 0 800 KO 0 0 2200 RARRES3 HRASLS2 HA and KDM6A Antibodies Detect the Same Peaks KDM6A Peaks Disappear in KDM6A KO cells
KD KDM6 M6A A binding si sites s in MM MM Cells 2000 WT Gene Expression Drops 0 RNA 2000 KO KDM6A Binding 0 800 HA-tag 0 HA 800 untag 0 800 WT KDM6A 0 800 KO Binding lost in CRISPR-KO Cells 0 2200 WT H3K27Ac 0 2200 KO 0 210 WT 0 H3K27me3 210 KO 0 RARRES3 HRASLS2
Conclusi sions 1- KDM6A is a Growth/Tumor Suppressor in MM Cell Lines 2- KDM6A Shows Tumor Suppressive Activity in a Mouse Model of MM Decreases Latency, Associated with Invasive Phenotype
Conclusi sions 1- KDM6A is a Growth/Tumor Suppressor in MM Cell Lines 2- KDM6A Shows Tumor Suppressive Activity in a Mouse Model of MM Decreases Latency, Associated with Invasive Phenotype 3- KDM6A Binds and Affects Adhesion/Invasion Genes Overrepresentation of STAT, ETS, CTCF binding Sites
Conclusi sions 1- KDM6A is a Growth/Tumor Suppressor in MM Cell Lines 2- KDM6A Shows Tumor Suppressive Activity in a Mouse Model of MM Decreases Latency, Associated with Invasive Phenotype 3- KDM6A Binds and Affects Adhesion/Invasion Genes Overrepresentation of STAT, ETS, CTCF binding Sites 4- KDM6A Loss can Lead to Loss of Gene Expression Correlates with H3K27Ac Loss at Enhancers H3K27me3 Changes Not Evident at Many Genes
KD KDM6A A - Deme methyl ylase se Ac Activi vity y Ap Appears rs Disp spensa sable at Ma Many y Genes s Possible Functions SWI/SNF Assist/Cooperate in Binding of Specific TFs ARID1A/2/4A CBP /5B p300 SMARCA4 Recruitment of Enhancer Active HMTs KDM6A KMT2C, D KMT2C/D ETS Recruitment of Histone acetyl transferases Recruitment of remodelers OFF ON
Open Questions SWI/SNF KDM6A Loss - Partial Loss of Enhancer Marks ARID1A/2/4A CBP /5B What is the Basis Of Redundancy? p300 SMARCA4 KDM6A Which KDM6A Target Genes/Pathways Lead KMT2C/D to Accelerated Growth? How Can KDM6A Loss be Overcome? Synthetic Lethal Screens Prior Work-EZH2i OFF ON KDM6A/COMPASS Complex
Acknowledgments Licht Lab Jianping Li Crissandra Jarrett Washington University Sayantan Maji Lucas Wartman Teresa Ezponda Timothy Ley Richard Bennett Sharon Norton Xiaoxiao Huang Darby Monagle Amin Sobh Gabriel Prado UFHCC Bioinformatics Alberto Riva
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