Targeting histone methylation for cancer therapy: enzymes, inhibitors, biological activity and perspectives
Cancer biology of MLL and LSD1
The biology of MLL has been extensively studies and reviewed 52]–54], 60]–62] and briefly summarized in the above section and here. The biological function of
MLL is essential for development: knockout of MLL in mice is embryonic lethal. MLL
has been found to associate with thousands of gene promoters and have a global role
in positive regulation of transcription of many important genes such as Hox families
of genes 81], 82]. Hox genes are transcriptional factors essential for the development of multiple
tissues including the hematopoietic system, while overexpression of certain members
(e.g., HoxA9 etc.) has been found to lead to leukemogenesis 83]. MLL gene translocations are frequently found in acute leukemia. Moreover, it is
noted that MLL-translocation occurs in one allele, with the wild-type (WT) MLL in
the other allele remaining intact. A recent study showed that MLL’s H3K4 methyltransferase
activity is essential for MLL-rearranged leukemia, suggesting inhibition of the SET
domain of MLL is a possibly viable approach to MLL leukemia treatment 84].
LSD1 plays an opposite role as a histone lysine demethylase 22], 23]. LSD1 contains four functional domains, including an N-terminal domain with a putative
nuclear localization peptide, a SWIRM, and an oxidase domain, inside which there is
a tower domain insert 85]. The last three domains are important for demethylation. In addition, the tower domain,
which is not present in a closely related enzyme LSD2, directly interacts with CoREST
(also known as RCOR1, repressor element-1 silencing transcription factor corepressor
1), through which LSD1 forms protein complexes that regulate histone lysine methylation
as well as gene expression. The biological function of LSD1 is crucial, as germline
LSD1 knockout in mice was found to be embryonic lethal and conditional knockout caused
increased H3K4me1/2, blocked hematopoiesis and pancytopenia (low in all blood cell
types) 86]. The primary substrates of LSD1 are H3K4me1 and me2, which are important histone
marks for active gene transcription. LSD1 was found to be part of an MLL transcription
complex 87]. A possible function of LSD1 is to counteract MLL and keep a balanced H3K4 methylation
(Fig. 4a). Of interest is that LSD1 has recently been found to be required for leukemia stem
cells transformed with MLL-AF9 88]. LSD1 knockdown abrogated the transforming ability of MLL-AF9, increased the H3K4me2
levels at MLL-AF9 target gene loci, and reduced the expression of HoxA9 and Meis1.
Presumably, LSD1 inhibition could counteract the loss of the SET domain in MLL-AF9
and restore a balanced H3K4 methylation. Pharmacological inhibition of LSD1 showed
similar activities against MLL-AF9 leukemia in vitro and in vivo 88]. Although there is a safety concern of LSD1 inhibition 89], 90] because of LSD1’s role in hematopoiesis 86], 91], a recent study showed after termination of LSD1 conditional knockout the impaired
hematopoiesis can be recovered in a mouse model 91]. These lines of evidence strongly support that LSD1 is a drug target for MLL leukemia.
In addition, H3K9 and other proteins have been found to be LSD1’s substrates 92]–96]. In the context of androgen receptor-mediated gene expression, histone H3 threonine
6 is phosphorylated by PKC?1, which prevents LSD1 from binding to methylated H3K4.
In complex with androgen receptor and PKC?1, LSD1 can change its substrate-specificity
and demethylate H3K9me1 and 2 92]. DNA methyltransferase 1 (DNMT1), which maintains the integrity of DNA methylation
as well as plays an important role in maintaining hematopoietic stem and progenitor
cells 97], is also a substrate of LSD1. DNMT1 is methylated in vivo and such methylation destabilizes
the protein. LSD1 can demethylate and therefore stabilize DNMT1. Therefore, LSD1 is
of importance in maintaining global DNA methylation 93]. In addition, LSD1 can demethylate other non-histone proteins, such as p53 94], MYPT1 95], and STAT3 96], and regulate gene expression mediated by these proteins.
Overexpression of LSD1 has been found in many types of cancer 98]–102], including AML (without an MLL-translocation), lung, breast, and prostate cancer.
These observations implicate that LSD1 is a potential drug target for these tumors.
It was recently found that a significant portion of cell lines of AML and small cell
lung cancer (SCLC) are highly sensitive to pharmacological inhibition of LSD1 103]. Although except for MLL-rearranged leukemia, the molecular mechanisms that link
LSD1 to these malignancies are not fully understood (likely because LSD1 has multiple
protein substrates), inhibition of LSD1 generally caused broad gene expression pattern
changes in these sensitive tumors, which could be responsible for the anti-proliferative
activity and other effects, e.g., inducing apoptosis and/or differentiation.
LSD1 inhibitors and their biological activities
A number of small molecule inhibitors of LSD1 have been discovered, developed, reported
in the journals and patents 103]–112] and reviewed recently 16], 113]. These compounds can be classified into reversible and irreversible inhibitors depending
upon their modes of action. We focus on the biological activities of the most potent
compounds. Figure 6 summarizes representative inhibitors of these two classes, together with their enzyme
and cellular activities. LSD1 belongs to a family of monoamine oxidases (MAO), using
FAD as the cofactor for the redox reaction (Fig. 2a). The common feature for irreversible LSD1 inhibitors is that upon oxidation, part
of the molecules is able to covalently bind to FAD and permanently deactivates the
enzyme 22]. However, for reversible inhibitors, there is no covalent interaction between the
inhibitor and the protein.
Fig. 6. Structures and activities of representative LSD1 inhibitors
The first inhibitors of LSD1 with a common cyclopropylamine core structure were derived
from Tranylcypromine (7, Fig. 6), an FDA-approved antidepression drug. Compound 7 is an inhibitor of MAO-A and -B,
enzymes that degrade neurotransmitters in the central nervous system. Compound 7 weakly
inhibits LSD1 with an IC
50
of ~15 ?M. More potent inhibitors have been developed based on the structure of 7.
Of importance for the inhibitor optimization is the introduction of a second amine-containing
N-substituent, such as those on the right side of cyclopropylamine moiety in highly
potent LSD1 inhibitors 8–10. These basic groups not only greatly increase the inhibitory
activity, but also render excellent LSD1 selectivity against MAO-A and -B 112]. Compound 8 (compound B in 88]) inhibited recombinant LSD1 in vitro with an IC
50
of 98 nM. It showed antitumor activities against MLL-AF9 transformed leukemic stem
cells. It inhibited colony-forming ability of MLL-AF9 containing leukemia cells with
EC
50
values as low as 50 nM. It also down-regulated expression of many leukemia-relevant
genes such as HoxA7, HoxA9 and Meis1. However, it exhibited somewhat severe toxicities
in a mouse model of MLL-AF9 leukemia, causing deaths of many experimental mice presumably
due to insufficient inhibitory potency and/or inappropriate dosages. Compound 9 (compound
1 in 111]) is a much more potent LSD1 inhibitor, almost quantitatively deactivating the enzyme
with an IC
50
of 9.8 nM. It exhibited potent anti-proliferative activity against MLL-rearranged
leukemia cell lines MV4-11 and Molm-13 with EC
50
values of 10 and 96 nM, while 9 is almost inactive against leukemia cells NB4 and
U937 without an MLL-translocation. The differential activities of compound 9 (as well
as several other LSD1 inhibitors) suggest that the LSD1 inhibitor is non-cytotoxic,
but LSD1 is essential for MLL-rearranged leukemia cells. In vivo antitumor studies
using a systemic murine model of MV4-11 leukemia showed that compound 9 did not exhibit
overt toxicities and was able to inhibit leukemia progression by 90 % and significantly
prolong survival of the experimental animals. Another potent LSD1 inhibitor GSK2879552
(10) was found to exhibit high anti-proliferative activity against 20 out of 29 AML
cell lines with EC
50
values ranging from ~3–100 nM 103]. In addition, anti-proliferation screening of compound 10 led to the finding that
a significant portion (9 out of 28) of small cell lung carcinoma (SCLC) cell lines
were susceptible to 10 with EC
50
s of ~2–240 nM. This compound also showed significant antitumor activity in a mouse
xenograft model of SCLC cancer. Similarly, compound 10 is also devoid of general cytotoxicity: it did not inhibit the growth of 100 cell
lines across a range of cancer types, showing a high selectivity of using LSD1 inhibitors
targeting cancer. Mechanistic studies showed that gene expression of TGF-? signaling,
which is dysregulated in SCLC, was significantly altered upon treatment with compound
10. This could be attributed to the antitumor activity of the LSD1 inhibitor. In addition,
DNA hypomethylation of a gene set was identified to be correlated with the sensitivity
of SCLC cells (including primary patient samples) to LSD1 inhibition 105]. This biomarker could be used as a major criterion for patient recruitment. Compound
10 has currently been in clinical trials for SCLC, while no clinical data have been
disclosed.
Several chemo-types of reversible LSD1 inhibitors have been disclosed, among which
compounds SP2509 (11) 107], GSK690 (12) 16], and 13 (compound 17 in 112]) possess low nM in vitro inhibitory activity. Compound 11 potently inhibited LSD1
with an IC
50
of 13 nM, showing a non-competitive mode of action. Treatment with 11 increased promoter-specific
H3K4 methylation, inhibited colony-formation, and induced differentiation and apoptosis
of several AML cell lines including MV4-11, Molm-13, and OCI-AML3. The combination
of 11 with an inhibitor of HDAC exhibited synergy and showed significantly improved
in vivo antileukemia activity in mouse models of AML 107]. Compounds 12 and 13 are quite similar, with the same 3-, 5-, 6-trisubstituted pyridine
core structure. Preliminary biological data of compound 12 were presented in the 2013
American Association of Cancer Research annual meeting, showing 90 nM IC
50
against LSD1, high enzyme selectivity, as well as low ?M cellular activity against
AML cells. Compound 13 showed an improved in vitro inhibitory activity (29 nM) against
LSD1 as well as good anti-proliferative activities (EC
50
: 0.36–3.6 ?M) against several sensitive cancer cells including MV4-11 with MLL-AF4
oncogene 112].
MLL inhibitors
In MLL-rearranged leukemia, the MLL gene translocation is heterozygous. The H3K4 methyltransferase
activity of the remaining copy of WT MLL was found to be essential for the malignancy
92]. Therefore, MLL inhibitors could be useful to treat MLL-rearranged leukemia. However,
compounds that directly inhibit the SET domain of MLL have not been reported. Alternatively,
the MLL SET domain alone was found to have extremely low methyltransferase activity
114], 115]. The optimal enzyme activity requires its complexation with three other proteins,
i.e., WDR5, ASH2L, and RbBP5. Among these, the interaction between WDR5 and MLL is
critical, which led to the finding of indirect MLL inhibitors, compounds that disrupt
the binding of WDR5 to MLL 116], 117]. Several compounds have recently been found to bind to WDR5 with Kd
values of 0.001–5.5 ?M. To date, the best compound MM-401 (14, Fig. 7), an extremely tight binder to WDR5 with a Kd
value of 1 nM, showed an IC
50
of 0.9 nM in disrupting the interaction between WDR5 and MLL. Indeed, compound 14
almost quantitatively inhibited the methyltransferase activity of the MLL complex
(0.5 ?M) with an IC
50
value of 0.32 ?M in vitro. Because the WDR5-MLL interaction is unique, compound 14
exhibited a high enzyme selectivity profile: it did not inhibit several closely related
SET domain methyltransferases including MLL2 – 4, SET1, and SET7/9. Consistent with
MLL’s role, it showed selective activity against MLL-rearranged leukemia cells. Compound
14 inhibited the proliferation of MLL-rearranged leukemia cells with EC
50
values of 12–30 ?M, while it had no effects on other non-MLL leukemia cells. The relatively
weak cellular activity might be due to the poor cell permeability of compound 14,
a cyclic peptidomimetic compound.
Fig. 7. Structure and activity of a compound that disrupts MLL:WDR5 interactions and thereby
inhibits MLL indirectly