SLIDE 1
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
SLIDE 2 Epigenetic Regulator Gene Anomalies in MM
Function Gene Diag Mut (%) Relapse Mut (%) CNV
Histone Ac CREBBP 0.8 4 Loss 13% p300 1.3 2 Loss 13% Histone MT NSD1 2.6 Gain 44% NSD2 15% t(4;14) SETD2 1.3 2.6 Gain 33% KMT2C 1.5 6.4 Gain 50% Histone DM KDM6A 1.3 0.6 Loss 25% Chromatin Remodeling SMARCA4 0.2 2,6 Gain 50% ARID4A 1
CHD2 1.5 0.6 Gain 50%
SLIDE 3 KD KDM6A
200 400 600 800 1000 1200 1401 TPR - Tetratricopeptide repeat domain JmjC domain
A17S V91fs G102D D114fs Y116fs Q147* Q172P Q191* Q218_ E278* Q539* R563* Q607* T807fr K1071fs P1159fs P1207_ W1245* K1345_
KDM6A mutations in MM
- 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
SLIDE 4 KD KDM6 M6A A is s a Centra ral Pro rotein of the COMP MPAS ASS-like ke En Enhancer C Compl plex
Data generated by The CoMMpass℠ study from MMRF
Activation Repression
ON OFF
H3K4me H3K27me H3Kac
SMARCA4 ARID1A/2/4A /5B
KMT2C/D CBP p300 KDM6A
SWI/SNF EZH2
PRC2
EED
SLIDE 5 ARP-1 ARD KDM6A wt KDM6A null
Gain of Function Model Shows That KDM6A is a Tumor Suppressor in MM
KDM6A H4 ARP1 ARD 6 12 25 50 ARD pKDM6A +Dox 100 (ng/ml)
In Vitro
Ezponda et al Cell Reports 2017
SLIDE 6 ARP-1 ARD KDM6A wt KDM6A null
Gain of Function Model Shows That KDM6A is a Tumor Suppressor in MM
200 400 600 800 1000 1200 1400
0 d 3 d 6 d 9 d 12 d ARD Add back
Cumulative cell counts (x106) Time
KDM6A H4 ARP1 ARD 6 12 25 50 ARD pKDM6A +Dox 100 (ng/ml)
In Vitro
Ezponda et al Cell Reports 2017
SLIDE 7 Exon 4 gRNA Exon 6 gRNA
KDM6A gene
New mo model of KD KDM6 M6A A loss ss in MM MM Cell Line by y Ac Acute Gene Editing
TPR JmjC 1401 a.a
KDM6A
exon 4 exon 6 exon 24
Tetracoid Repeats jmjC 250 kb
In vitro
Cas9
SLIDE 8 60 70 80 90 100
10
KDM6A mutant allele frequency (%)
Day Mutant allele frequency
Exon 4 Exon 6
KD KDM6 M6A A Disru sruption Ad Adva vantages s Gro rowth of MM MM Cells In vitro
Mutant Alleles Increase Over Time
20 40 60 80 100 5 10 KDM6A mutant allele frequency (%)
Day Mutant allele frequency
Arp-1 Karpas-620 MM1S
SLIDE 9 KD KDM6A
ARD WT E4 E6
ARP-1 HDA HDAC2 C2
WT E4 E6
Karpas-620
WT E4 E6
MM1S
60 70 80 90 100
10
KDM6A mutant allele frequency (%)
Day Mutant allele frequency
Exon 4 Exon 6
KD KDM6 M6A A Disru sruption Ad Adva vantages s Gro rowth of MM MM Cells In vitro
Mutant Alleles Increase Over Time Pools of KDM6A KO cells
20 40 60 80 100 5 10 KDM6A mutant allele frequency (%)
Day Mutant allele frequency
Arp-1 Karpas-620 MM1S
SLIDE 10 A A Ra Rapid Mo Mouse se Mo Model for r Mu Multiple Mye Myeloma ma
Method adapted from Dechow et el JCI, 2014
In vivo
fetal liver cells
Kdm6af/f Cd19 Cre+/- E.14 Donor
L-GP130 EGFP
IRES
LTR promoter 8 Gy
Wild type Recipient
SLIDE 11 A A Ra Rapid Mo Mouse se Mo Model for r Mu Multiple Mye Myeloma ma
Method adapted from Dechow et el JCI, 2014
In vivo
GFP+ CD138+ Tumor Cells
95,6 0.10
Sanguineous ascites Splenomegaly Tumor Tumor
2.08
GFP+
CD138 B 2 2 GFP C
n t
1.02 43.0 82.8 6.02
Tumor Spleen BM Control
fetal liver cells
Kdm6af/f Cd19 Cre+/- E.14 Donor
L-GP130 EGFP
IRES
LTR promoter 8 Gy
Wild type Recipient
SLIDE 12
What Molecular Function of KDM6A is Critical? Histone Demethylation? Where in the Genome Does KDM6A Bind in MM Cells? What Genes are Affected?
KDM6A is a tumor suppressor in vitro and in vivo
SLIDE 13 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
TPR JmjC
H1146A E1148A
JmjC Inactive Mutant
SLIDE 14 4 8 12 16
Cumulative cell count (x106)
pKDM6A
no dox dox 4 8 12 16
pKDM6A JmjC-dead
n
dox 4 8 12 16
D0 D3 D6 D9 D12 D0 D3 D6 D9 D12 D0 D3 D6 D9 D12
empty vector
no dox dox
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
TPR JmjC
H1146A E1148A
Expression of wt or JmjC Dead KDM6A Causes Similar Growth Suppression JmjC Inactive Mutant
SLIDE 15 4 8 12 16
Cumulative cell count (x106)
pKDM6A
no dox dox 4 8 12 16
pKDM6A JmjC-dead
n
dox 4 8 12 16
D0 D3 D6 D9 D12 D0 D3 D6 D9 D12 D0 D3 D6 D9 D12
empty vector
no dox dox
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
TPR JmjC
H1146A E1148A
Large Overlap of Genes Affected with Add-Back
- f Wild-Type and JmjC Inactive KDM6A
Expression of wt or JmjC Dead KDM6A Causes Similar Growth Suppression RNA-Seq After KDM6A Add-Back JmjC Inactive Mutant
- Membrane receptor activity
- Cell activation
- Cell-cell adhesion
SLIDE 16 Stop codon gRNA + ssODN HA-tag
KDM6A
Tagging the Endogenous s KD KDM6 M6A A Locus s for r Detection of Bi Binding Sites
TPR JmjC
HA
1401 a.a
HDAC2 KDM6A HA ARD WT HA+/+
ARP1
SLIDE 17 Va Valida dati tion o
f HA T Taggi gging o g of K f KDM6A
HA and KDM6A Antibodies Detect the Same Peaks KDM6A Peaks Disappear in KDM6A KO cells
KDM6A edited/HA-tagged KDM6A wt
800 800 800 800 2200
HA-tag WT KO untag
KDM6A HA
RARRES3
HRASLS2
SLIDE 18 KDM6A Binding Binding lost in CRISPR-KO Cells Gene Expression Drops
KD KDM6 M6A A binding si sites s in MM MM Cells
RARRES3
HRASLS2
800 800 800 800 2200 2200 210 210 2000 2000
HA-tag WT KO untag
H3K27Ac H3K27me3 KDM6A HA
WT KO WT KO WT KO
RNA
SLIDE 19
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
SLIDE 20
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
SLIDE 21
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
SLIDE 22 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
Assist/Cooperate in Binding of Specific TFs Recruitment of Enhancer Active HMTs KMT2C, D Recruitment of Histone acetyl transferases Recruitment of remodelers
ON OFF SMARCA4 ARID1A/2/4A /5B
KMT2C/D CBP p300 KDM6A SWI/SNF ETS
SLIDE 23 Open Questions
KDM6A Loss - Partial Loss of Enhancer Marks What is the Basis Of Redundancy? Which KDM6A Target Genes/Pathways Lead to Accelerated Growth? How Can KDM6A Loss be Overcome? Synthetic Lethal Screens Prior Work-EZH2i KDM6A/COMPASS Complex
ON OFF
SMARCA4 ARID1A/2/4A /5B
KMT2C/D CBP p300 KDM6A
SWI/SNF
SLIDE 24
Licht Lab Jianping Li Crissandra Jarrett Sayantan Maji Teresa Ezponda Richard Bennett Sharon Norton Xiaoxiao Huang Darby Monagle Amin Sobh Gabriel Prado
Acknowledgments
UFHCC Bioinformatics Alberto Riva Washington University Lucas Wartman Timothy Ley
