Mohd Ishaq Dar

IIIM-CSIR
Selective Killing of Mixed Lineage Leukemia Cells by a
Potent Small-Molecule DOT1L Inhibitor
Cancer Cell; 20, 53–65, July 12, 2011
,
Scott R.Daigle, Edward J.Olhava, Carly A.Therkelsen, Christina R.Majer, Christopher J.
Sneeringer, Jeffrey Song, L. Danielle Johnston, Margaret Porter Scott, Jesse J. Smith, Yonghong
Xiao, Lei Jin, Kevin W. Kuntz, Richard Chesworth, Mikel P. Moyer, Kathrin M. Bernt, Jen-Chieh
Tseng, Andrew L. Kung, Scott A. Armstrong, Robert A. Copeland, Victoria M. Richon, Roy M. Pollock
Belfer Institute for Applied Cancer Science, Dana-Farber Cancer Institute, Boston, MA 02115, USA,
Division of Hematology/Oncology, Children’s Hospital and Department of Pediatric Oncology, DanaFarber Cancer Institute and Harvard Medical School, Boston, MA 02115, USA,
Harvard Stem Cell Institute, Boston, MA 02138, USA
Mohd Ishaq Dar
Junior Research Fellow
Cancer Pharmacology Division
Targeting the common S-adenosylmethionine(SAM) cofactorbinding site of PMTs is an attractive strategy
IIIM-CSIR
There are 60 PMTs encoded in the human genome, including 51 lysine methyltransferases (PKMTs) and 9
protein arginine methyltransferases (PRMTs) which methylate both histone and non-histone proteins in a
cell.
Sites for methylation
K4, K9, K27, K36, and K79
K20
H3
H4
K9, K20 and K27
Methylation
transcriptional repression,
K4, K36, and K79
Methylation
transcriptional activation
DOTIL methylates Histone H3 of core histone at K79.
+ s-adenosylhomocysteine
IIIM-CSIR
Dot IL is the only PKMT which does not contain SET domain hence
inhibitors to it will be more specific.
DOTIL plays critical roles in normal cell differentiation, initiation of MLL
as well as induction of pluripotency in stem cells.
DOT 1 (Disruptor of telomere silencing) was originally discovered in yeast
through a genetic screen to identify proteins whose overexpression leads to
impaired telomeric silencing . Mutation of putative methyl transferase domain
inactivates its silencing fuction in yeast.
Role of DOTIL in Leukemia
IIIM-CSIR
Mislocated enzymatic activity of DOT1L has been proposed as a driver of leukemogenesis in mixed
lineage leukemia (MLL).We can prevent Leukemia by blocking DOTIL. Meis1, like several Hox genes,
is a known marker of MLL fusion protein-induced leukemia .
EPZ004777 as inhibitor of Dot1
IIIM-CSIR
Exposure of leukemic cells to DOTIL inhibitor EPZ004777 results in selective killing of cells
bearing MLL gene translocation, with little effect on non-MLL-translocated cells.
EPZOO4777 was less effective in-vivo.
Mechanism of catalysis of DOTIL
IIIM-CSIR
Different Analogs of SAM having same nucleoside core
IIIM-CSIR
S-adenosylmethionine
EPZ004777
EPZ-5676
The chemical structure of these inhibitors retain the nucleoside
core of the SAM substrate , SAH product. As such they were
designed to bind to DOT IL within the same SAM binding
pocket.
IIIM-CSIR
Catalytic site remodelling of the DOT1L methyltransferase by
selective inhibitors.
The X-ray crystal structure of
inhibitors (FED1, EPZ004777, EPZ-5676) in complex with the
human DOT1L methyltransferase domain showed these compounds
occupy SAM
binding
pocket
Induce conformational changes in DOT1L leading to the opening of a hydrophobic pocket
beyond the amino acid portion of SAM.
This structural rearrangement creates additional surface for interaction that contribute to high
affinity and selectivity of these compounds.
IIIM-CSIR
Mixed lineage leukemia is a gentically distinct form of acute leukemia.
A universal hallmark of MLL disease is a chromosomal translocation .
Affecting the MLL gene on chromosome 11q23
Translocation involving MLL are found in 70% of infant leukemias.
Normally, the MLL gene encodes for a SET domain histone methyltransferase that
catalyzes the methylation of lysine 4 of histone H3 (H3K4) at specific gene loci.
In the disease- linked translocations , the catalytic
SET
domain is lost and the
remaining MLL protein is fused inframe to a variety of partners such as AF4,
AF9,AF10 and ENL.
Interaction of Dot1L in MLL
IIIM-CSIR
DOT1L interacts with six unique MLL fusion proteins created by chromosomal
translocations
These fusion products retain the gene specific recognition elements and are capable
of interacting directly, or indirectly with another histone methyl transferase , DOTIL.
They also gain the ability to recruit Dot 1L to the aberrant gene locations.
EPZ-5676 inhibitor of DOTIL
IIIM-CSIR
A phase 1 trial for Epizyme's DOT1L inhibitor EPZ-5676 was initiated in September 2012. EPZ-5676
is the first HMT inhibitor (HMTi) to enter human clinical development.
EPZ-5676 has shown low oral bioavailability but good bioavailibilty by intravenous mode in mouse
and rat model.
EPZ-5676
IIIM-CSIR
Selective Killing of Mixed Lineage Leukemia Cells by a Potent SmallMolecule DOT1L Inhibitor
Based on the chemical structure of SAM and SAH they have synthesized
EPZ004777
EPZ004777
Inhibition of recombinant human DOTIL by EPZ004777
IIIM-CSIR
a
Enzyme
IC50 (nM)
DOT1L
0.4 ± 0.1
CARM1
>50,000
EHMT2
>50,000
c
EZH1
c
>50,000
EZH2
>50,000
PRMT1
>50,000
PRMT5
521 ± 137
PRMT8
>50,000
SETD7
>50,000
WHSC1
>50,000
Inhibition of recombinant human DOTIL by EPZ004777. EPZ is a potent and
selective DOT 1L inhibitor by enzyme based assay.
Immunoblot assay of the extracted histones
IIIM-CSIR
Next they tested the ability of EPZ004777 to inhibit DOTIL in cells by immunoblot
assay of the extracted histones
Effect of EPZ004777 on expression of leukemia promoting genes
IIIM-CSIR
(C) Quantitative real-time PCR analysis of HOXA9 and MEIS1 mRNA levels in MV4-11 and MOLM-13 cells
following 6 day incubation with EPZ004777.Relative mRNA expression levels are plotted as a percentage
of those in vehicle-treated control cells.
(D) Time course of HOXA9, MEIS1 and TBP mRNA expression in MV4-11 and MOLM-13 cells over 8 days
of incubation with 3 μM EPZ004777 as measured by quantitative real-time PCR. Relative mRNA
expression levels are plotted as a percentage of those at day 0. Error bars: standard deviation (SD).This
efect was not due to the general inhibitory effect on gene expression since transcript levels of the
hosekeeping gene TBP were unaffected.
IIIM-CSIR
EPZ004777 treatment on proliferation of MLL rearranged and nonrearraged cells
Growth of MV4-11 (MLL-AF4),MOLM-13 (MLL-AF9), and Jurkat (non-MLL-rearranged) cells
during several days incubation with 3 μM EPZ004777. Viable cells were counted every 3 to
4 days in the presence of EPZ004777 (+) or DMSO vehicle control (−) and results plotted on a
logarithmic scale
Effect of EPZ004777 on the proliferation of leukemia cell lines bearing MLL-AF4, MLL-AF9,
and MLL-ENL fusions, or cell lines lacking an MLL rearrangement.
IC50 value of EPZ004777 IN MLL rearranged and non rearranged cells
IIIM-CSIR
MLL Gene
Fusion
MLL-AF4
MLL-AF4
MLL-AF4
MLL-AF9
MLL-AF9
MLL-ENL
Nonrearranged
Cell Type
IC50 (μM)
RS4;11
SEM
MV4-11
THP-1
MOLM-13
KOPN-8
REH
6.47
1.72
0.17
3.36
0.72
0.62
13.90
Nonrearranged
Kasumi-1
32.99
Nonrearranged
697
36.57
Nonrearranged
HL-60
>50
Nonrearranged
Jurkat
>50
Nonrearranged
U937
>50
a
Flow cytometryic Analysis of cell cycle & Annexin
IIIM-CSIR
In MV4-11 cells, a modest increase in G0/G1 phase, and a decrease in S-phase cells were
apparent after 4 days of incubation with 3 μM EPZ004777. This was followed by an
increase in sub-G1 and Annexin-positive cells over the next 6 days, consistent with
apoptotic cell death .Compound treatment also led to caspase activation
EPZ004777 Increases Caspase 3/7 Activation in
MV4‐11 Cells. MV4‐11 cells treated with 3 uM
EPZ004777 for up to 10 days were analyzed
using the Guava EasyCyte Plus instrument.
IIIM-CSIR
EPZ004777 monitered for cell surface expression of myeloid differentiation marker
CD14 by flow cytometry.
This provides further evidence that small-molecule inhibition of DOTIL promotes
some degree of differentiation prior to cell killing
IIIM-CSIR
Whether EPZ00477 reverses the MLL-Rearranged gene signature.
Gene Set Enrichment analysis of genes upregulated by EPZOO4777 treatment of MOLM-13
cells as compared with members of KEGG hematopoetic cell lneage gene set.
Down regulation of MLL-AF9 taget genes by EPZ004777
IIIM-CSIR
GSEA of genes downregulated
by EPZ004777 treatment of
MOLM-13 cells as compared
with genes bound by MLL-AF9
in
MLL-AF9-transformed
murine
hematopoietic
progenitors (Compared with
Bernt et al., 2011 ).
IIIM-CSIR
Down regulation of MLL-AF4 taget genes by
EPZ004777
GSEA
of
genes
downregulated
by
EPZ004777 treatment
of MV4-11 cells as
compared with genes
bound by MLL-AF4 in
human
SEM
cells
(Guenther et al., 2008).
IIIM-CSIR
Gene expression changes caused by EPZ004777 treatment of MOLM-13 cells
with those caused by genetic knockout of DOTIL in a mouse model of MLL-AF9
leukemia..
They found a significant overlap between these gene expression changes indicating
that EPZOO4777 treatment and genetic ablation of DOTIL cause cell killing of MLLrearranged cells through similar pathways
GSEA of genes downregulated by EPZ004777 treatment of MOLM-13 cells as compared with genes
downregulated following genetic knockout of Dot1l in a murine MLL-AF9 leukemia model ( Bernt et al.,
2011 [ Cancer Cell]).
Efficacy of EPZ004777 in Vivo
IIIM-CSIR
 Next they tested the efficacy of EPZ004777 in a mouse xenograft model of MLL.
To determine whether EPZOO4777 was capable of inhibiting DOTIL activity in vivo.
They have used a mouse MV4-11 subcutaneous xenograft model, Since tumours
can be readily harvested.
Due to poor pharmacokinetic properties of EPZ00477 ,compound was delivered
via subcutaneously implanted mini-osmotic pumps.
Pumps were loaded with 50 mg/ml of drug.
Next they tested the efficacy of EPZ004777 in a more therapeutically relevant model of
MLL in which MV4-11 cells were injected into the tail vein of immunodeficient mice
resulting in disseminated leukemic disease.
Female NSG mice were injected via tail vein with 1 106 MV4-11 cells stably expressing
the firefly luciferase gene. Leukemia engraftment was confirmed by bioluminescence
imaging 5 days after inoculation
Animals were dying of leukemic disease
EPZ004777 exposure caused a dose-dependent and statistically significant increase in
survival for the 150, 100, and 50 mg/ml dose groups,
In conclusion
They have designed and characterized a potent, selective DOTIL inhibitor as a
starting point towards the development of medicines for the treatment of patients
suffering from MLL.
The current results also provide validation for small-molecule inhibition of HMTS as
a therapeutic modality in cancer.
IIIM-CSIR
Thank You
The association of 11q23 rearrangements with ALL, AML, or MPAL is unique in
that most other chromosomal rearrangements tend to be associated with
leukemias of a particular hematopoietic lineage. These observations led to the
name Mixed-Lineage Leukemia (MLL) for the gene that resides on 11q23.
MLL-AF4 encodes a protein of 2304 amino acids, with the NH2-terminal 1439
amino acids derived from MLL on chromosome 11 and the COOH-terminal 865
amino acids from the AF4 gene on chromosome 4.
11q23 rearrangements are found in 70% of leukemias in infants 1 year of age
whether the immunophenotype is more consistent with acute lymphoblastic
leukemia (ALL) or acute myelogenous leukemia (AML).1
Whereas MLL translocations can be found in either ALL or AML, particular
translocations show lineage specificity, with the t(4;11) found most often in
ALL, the t(9;11)(p21;q23) found most often in AML, and the t(11;16) found
most often in myelodysplastic syndrome and secondary leukemia. A

Download Report