Discovery of selective small-molecule HDAC6 inhibitor for overcoming proteasome inhibitor resistance in multiple myeloma

Article abstract of DOI:10.1073/pnas.1608067113

Multiple myeloma (MM) has proven clinically susceptible to modulation of pathways of protein homeostasis. Blockade of proteasomal degradation of polyubiquitinated misfolded proteins by the proteasome inhibitor bortezomib (BTZ) achieves responses and prolongs survival in MM, but long-term treatment with BTZ leads to drug-resistant relapse in most patients. In a proof-of-concept study, we previously demonstrated that blocking aggresomal breakdown of polyubiquitinated misfolded proteins with the histone deacetylase 6 (HDAC6) inhibitor tubacin enhances BTZ-induced cytotoxicity in MM cells in vitro. However, these foundational studies were limited by the pharmacologic liabilities of tubacin as a chemical probe with only in vitro utility. Emerging from a focused library synthesis, a potent, selective, and bioavailable HDAC6 inhibitor, WT161, was created to study the mechanism of action of HDAC6 inhibition in MM alone and in combination with BTZ. WT161 in combination with BTZ triggers significant accumulation of polyubiquitinated proteins and cell stress, followed by caspase activation and apoptosis. More importantly, this combination treatment was effective in BTZ-resistant cells and in the presence of bone marrow stromal cells, which have been shown to mediate MM cell drug resistance. The activity of WT161 was confirmed in our human MM cell xenograft mouse model and established the framework for clinical trials of the combination treatment to improve patient outcomes in MM.

Full text of DOI:10.1073/pnas.1608067113

Discovery of selective small-molecule HDAC6 inhibitor
for overcoming proteasome inhibitor resistance in
multiple myeloma
Teru Hideshimaa,1, Jun Qia,1, Ronald M. Paranala,1, Weiping Tangb,c, Edward Greenberga, Nathan Westa,
Meaghan E. Collinga, Guillermina Estiud,2, Ralph Mazitschekc,e, Jennifer A. Perrya, Hiroto Ohguchia, Francesca Cottinia,
Naoya Mimuraa, Güllü Görgüna, Yu-Tzu Taia, Paul G. Richardsona, Ruben D. Carrascoa, Olaf Wiestd,f, Stuart L. Schreiberc,
Kenneth C. Andersona,3,4, and James E. Bradnera,3,4
aDepartment of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02215; bSchool of Pharmacology, University of
Wisconsin-Madison, Madison, WI 53705; cBroad Institute of Harvard and MIT, Cambridge, MA 02142; dDepartment of Chemistry and Biochemistry,
University of Notre Dame, Notre Dame, IN 46556; eCenter for Systems Biology, Massachusetts General Hospital, Boston, MA 02142; and fLaboratory of
Computational Chemistry and Drug Discovery, Laboratory of Chemical Genomics, Shenzhen Graduate School, Peking University, Shenzhen 518055, China
Edited by Rabinder Prinjha, Epinova Epigenetics, Birmingham, United Kingdom, and accepted by Editorial Board Member Dinshaw J. Patel September 29,
2016 (received for review June 16, 2016)
alternative system to proteasomal degradation of ubiquitinated
misfolded/unfolded proteins that ultimately induces autophagic
clearance of proteins by lysosomal degradation (8). Histone deace-
tylase 6 (HDAC6) plays a central role in autophagic protein degra-
dation by recruiting ubiquitinated protein cargo for transport to
aggresomes. HDAC6 is a member of the class IIb family of HDAC
enzymes. HDAC6 possesses two functional deacetylase domains and
a zinc finger motif. HDAC6 was initially described as a tubulin
deacetylase; however, the literature defines additional substrates,
including Hsp90 and p300 (9). HDAC6 modulates cell morphology,
adhesion and migration, immune-mediated cell–cell interactions, and
Multiple myeloma (MM) has proven clinically susceptible to modulation
of pathways of protein homeostasis. Blockade of proteasomal degra-
dation of polyubiquitinated misfolded proteins by the proteasome
inhibitor bortezomib (BTZ) achieves responses and prolongs survival in
MM, but long-term treatment with BTZ leads to drug-resistant relapse
in most patients. In a proof-of-concept study, we previously demon-
strated that blocking aggresomal breakdown of polyubiquitinated
misfolded proteins with the histone deacetylase 6 (HDAC6) inhibitor
tubacin enhances BTZ-induced cytotoxicity in MM cells in vitro.
However, these foundational studies were limited by the pharmaco-
logic liabilities of tubacin as a chemical probe with only in vitro utility.
Emerging from a focused library synthesis, a potent, selective, and
bioavailable HDAC6 inhibitor, WT161, was created to study the
mechanism of action of HDAC6 inhibition in MM alone and in
combination with BTZ. WT161 in combination with BTZ triggers
significant accumulation of polyubiquitinated proteins and cell
stress, followed by caspase activation and apoptosis. More impor-
tantly, this combination treatment was effective in BTZ-resistant
cells and in the presence of bone marrow stromal cells, which have
been shown to mediate MM cell drug resistance. The activity of
WT161 was confirmed in our human MM cell xenograft mouse
model and established the framework for clinical trials of the
combination treatment to improve patient outcomes in MM.
Significance
Proteasome inhibitors show remarkable anti-multiple myeloma
(MM) activity in preclinical and clinical studies. However, re-
sistance develops in the majority of patients, and novel treat-
ments are urgently needed. Histone deacetylase 6 (HDAC6) has
been shown to mediate aggresomal protein degradation and
could be a potential target for combination treatment to
overcome drug resistance. Here we designed and developed an
HDAC6-selective small molecule inhibitor, WT161, and used this
compound to define mechanisms of anti-MM activity, both
alone and in combination with proteasome inhibitors in in vitro
and in vivo studies. This study has established the framework
for combination treatment of HDAC6 inhibitors with protea-
some inhibitors in MM and validates an in vivo quality chemical
probe for broad use by the research community.
histone deacetylase 6 proteasome inhibitor multiple myeloma
|
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bortezomib-resistance WT161
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ultiple myeloma (MM) is a B-cell malignancy character-
ized by the proliferation of bone marrow (BM) plasma cells in
M
association with high Ig production (1). Although treatable, MM is
considered incurable and has a 5-y overall survival rate of only 45%.
The treatment of MM has been transformed in the last decade be-
cause of the development of novel therapeutic agents that target MM
cells in the BM microenvironment and that can overcome conven-
tional drug resistance (1). Namely, the proteasome inhibitor borte-
zomib (BTZ), which blocks the degradation of polyubiquitinated
misfolded proteins, induces ER stress, and triggers apoptosis of MM
cells, has rapidly translated to clinical trials demonstrating remark-
able clinical efficacy. The second-generation proteasome inhibitors
carfilzomib (CFZ) (2), ixazomib (3), and marizomib (4) also are
showing improved pharmacological properties and promising re-
sponses. Nonetheless, MM cells develop resistance to BTZ, leading
to relapse of disease in most patients (5, 6).
Author contributions: T.H., J.Q., W.T., R.M., S.L.S., K.C.A., and J.E.B. designed research; T.H., J.Q.,
R.M.P., W.T., E.G., N.W., M.E.C., G.E., R.M., H.O., N.M., Y.-T.T., and R.D.C. performed research;
T.H., J.Q., W.T., R.M., S.L.S., and J.E.B. contributed new reagents/analytic tools; T.H., J.Q., R.M.P.,
E.G., N.W., M.E.C., G.E., R.M., J.A.P., F.C., G.G., P.G.R., O.W., K.C.A., and J.E.B. analyzed data; and
T.H., J.Q., R.M.P., J.A.P., O.W., K.C.A., and J.E.B. wrote the paper.
Conflict of interest statement: T.H. is a consultant for Acetylon Pharmaceuticals. R.M. has
financial interests in SHAPE Pharmaceuticals and Acetylon Pharmaceuticals and is the inventor
on intellectual property licensed to these two entities. K.C.A. is an advisor for Celgene, Mil-
lennium Pharmaceuticals, and Gilead Sciences and is a Scientific Founder of OncoPep, Acety-
lon Pharmaceuticals, and C4 Therapeutics. J.E.B. is a Scientific Founder of SHAPE
Pharmaceuticals, Acetylon Pharmaceuticals, Tensha Therapeutics, and C4 Therapeutics and is
the inventor on intellectual property licensed to these entities. As of January 1, 2016, J.E.B. is
an employee of the Novartis Institutes of Biomedical Research.
This article is a PNAS Direct Submission. R.P. is a Guest Editor invited by the Editorial Board.
1T.H., J.Q., and R.M.P. contributed equally to this work.
2Deceased May 9, 2014.
3K.C.A. and J.E.B. contributed equally to this work.
4To whom correspondence may be addressed. Email: james_bradner@dfci.harvard.edu or
kenneth_anderson@dfci.harvard.edu.
The proteasome serves an important cellular function in clearing
abnormal proteins in the cell. Tumor cells, and particularly MM
cells that produce high levels of Ig, are more heavily dependent on
this clearance mechanism and thus are sensitive to proteasome
inhibition (7). The aggresomal protein degradation pathway is an
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1608067113/-/DCSupplemental.
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tubacin has limited in vivo bioavailability, and its chemical syn-
thesis is complex, limiting its availability and utility as a chemical
probe (12). Therefore the development of a highly potent and
selective HDAC6 inhibitor with good bioavailability that can be
synthesized in large quantities is highly desired. More importantly,
access to a selective HDAC6 inhibitor would enable us to study
the combination effects of HDAC6 inhibition with proteasome
inhibitors in preclinical in vivo models of MM for translation to
clinical trials.
In this study, we minimized the scaffold of tubacin via synthesis of
a drug-like library of 400 hydrazones biased for deacetylase inhi-
bition as hydroxamates, which we then tested for HDAC substrate
specificity and cellular activity. From these efforts, we identified a
potent and selective HDAC6 inhibitor, WT161. The structural basis
for its HDAC6 inhibition and selectivity is delineated. Treatment of
MM cell lines with WT161 triggers the accumulation of acetylated
tubulin and cell death in MM cell lines more potently than tubacin.
Additionally, WT161 in combination with BTZ induces synergistic
cytotoxicity and overcomes BTZ resistance in vitro. The anti-MM
activity of WT161 in combination with BTZ is validated in our
murine xenograft model of human MM. The synergistic effect in-
dicates that dual blockade of proteasomal and aggresomal protein
degradation could be a potential therapeutic strategy for the
treatment of MM and for overcoming proteasome inhibitor
resistance in MM.
A
B
O
O
H
N
H
N
R1
2
O
H
+
HN
H
O
H2N
N
H
R2
O
O
O
1
DMSO, 75oC
SAHA
O
R2
O
H
N
H
N
O
H
R1
N
N
H
HN
O
H
O
O
O
C
E
H
N
O
O
S
O
H
N
N
H
O
O
N
HO
Tubacin
WT161
WT161
D
WT161
100
50
0
100
50
0
Tubulin
Lysine
1
-2
-1
0
-1
-8
Log[M]
-6
Log[mM]
Tubacin
Tubacin
100
50
0
100
50
0
Results
Discovery of the Potent and Selective HDAC6 Inhibitor WT161. Be-
cause the class I, II, and IV HDAC enzymes are zinc-dependent
hydrolases, the dominant pharmacophore model of HDAC inhibi-
tors features three key structural elements: a metal-binding domain,
a surface recognition element, and a linker bridging these two
chemical moieties (13). Improved ligand specificity can be achieved
via modulation of the structure of the chelator or via the structure
or conformation of the capping feature (14, 15). For example, the
1,3-dioxane–appending group in tubacin is responsible for HDAC6
selectivity compared with the pan-inhibitor SAHA, which has a
much smaller aryl surface-binding feature (Fig. 1A) (16).
Tubulin
Lysine
1
-2
-1
0
-1
-8
Log[M]
-6
Log[mM]
SAHA
SAHA
100
50
0
100
50
0
Tubulin
Lysine
We undertook a chemical strategy to identify more drug-like
HDAC6 inhibitors using a highly parallel approach to biased
chemical library synthesis (17). We designed a hydroxamic acid
building block, compound 1, possessing a reactive hydrazide sepa-
rated by a six-carbon linear aliphatic linker that mimics the structure
of SAHA and tubacin (Fig. 1B). Direct coupling of compound 1
with a library of 400 aldehydes and ketones produced 400 hydrazones
in 96-well plate format, which were directly profiled in biochem-
ical and cellular assays without further purification. From the
resulting library, we identified compound WT161 as a potent and
selective inhibitor of HDAC6 (Fig. 1 C and D and Table S1) (14).
Although some potency for HDAC6 was sacrificed as compared
to SAHA (SAHA IC50 = 0.03 nM; WT161 IC50 = 0.40 nM),
WT161 is still very potent and is more selective against HDAC6
1
-2
-1
0
-1
-8
Log[M]
-6
Log[mM]
Fig. 1. Development of the selective HDAC6 inhibitor WT161. (A) Chemical
structures of SAHA and tubacin. The hydroxamic acid is highlighted in blue.
(B) The synthesis scheme used to create a hydrazine library that was screened to
identify the HDAC6-selective inhibitor WT161. (C) Chemical structure of WT161
(hydroxamic acid is highlighted in blue). (D) Inhibitory activity of WT161,
tubacin, and SAHA against HDAC1–9 assessed in a biochemical activity assay.
The activity of HDAC6 (red) and HDAC3 (black) are highlighted. The activity
curves of the other HDACs are shown in gray. (E) Cells were incubated with
increasing concentrations of WT161, tubacin, or SAHA for 4 h before fixation
and staining with the antibodies for Ac-α-tubulin and acetylated lysine.
Measurements of the cellular effects of WT161, tubacin, and SAHA on tubulin
(red) and lysine (black) acetylation were quantified via high-content imaging.
than against the other family members (HDAC3: SAHA IC50
=
0.21 nM; WT161 IC50 = 51.61 nM; tubacin IC50 = 130.90 nM).
Biochemically, WT161 is more potent than tubacin and is equiva-
lently selective for HDAC6 (tubacin IC50 = 1.62 nM) and has a
dramatically simplified synthesis (three steps, 40% overall yield).
The activity and selectivity of WT161 in cells was confirmed
further using a miniaturized assay system we developed to monitor
the simultaneous effects on HDAC6 (α-tubulin acetylation) and
class I nuclear deacetylases (lysine acetylation), using high-content
imaging (15, 18). WT161 selectively inhibits HDAC6 and dra-
matically increases levels of acetylated α-tubulin (Ac-α-tubulin)
with little effect on global lysine acetylation (Fig. S1A). Further-
more, when compared directly with tubacin and SAHA, WT161
was found to increase Ac-α-tubulin in cells more effectively (Fig.
1E). Thus, we identified and validated the small molecule WT161
tumor cell invasion/metastasis as well as misfolded/unfolded protein
degradation and stress response. Inhibition of HDAC6 with nonse-
lective HDAC inhibitors, such as vorinostat (suberoylanilide
hydroxamic acid, SAHA) or panobinostat (LBH589), blocks lyso-
somal protein degradation. To date preclinical and clinical studies
have used pan-HDAC inhibitors together with BTZ to inhibit both
proteasomal and aggresomal protein degradation and to overcome
clinical BTZ resistance (10, 11). However, the side-effect profile of
the broad HDAC inhibitors with BTZ, including fatigue, diarrhea,
and thrombocytopenia, limits the clinical utility of this combined
treatment (10). Thus, targeting HDAC6 selectively could be a po-
tential approach to overcome drug resistance in MM while reducing
the overall toxicity seen with the use of less selective HDAC inhibitors.
We previously have shown that proteasomal blockade of protein as a potential chemical probe for HDAC6 inhibition.
degradation with BTZ, combined with aggresomal blockade of
protein degradation with a prototype HDAC6 inhibitor, tubacin,
Molecular Recognition of HDAC6 by WT161. As we have reported
triggers significant cytotoxicity in MM cells in vitro (4). However, previously, the shape of the protein surface around the active site
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Hideshima et al.

A
HDAC2
HDAC3
HDAC6
SAHA
B
WT161
Fig. 2. Interactions of SAHA and WT161 in the active site pocket of HDAC2, HDAC3, and HDAC6. (A) Snapshots from 30-ns MD simulation are superimposed
for SAHA with HDAC2 (Left), HDAC3 (Center), and HDAC6 (Right) with multiple orientations stabilized. (B) Snapshots from 30-ns MD simulation are
superimposed for WT161 with HDAC2 (Left) and HDAC3 (Center), with multiple orientations stabilized. Only a single orientation is found for the HDAC6-
WT161 complex (Right). The ligand is shown in different colors in the different snapshots.
WT161 Induces Accumulation of Acetylated Tubulin and Cytotoxicity
in MM Cells. To confirm the activity of WT161 in MM cells, we
measured the accumulation of Ac-α-tubulin. Both WT161 and
tubacin significantly induce Ac-α-tubulin in a dose-dependent
fashion without increasing histone H3K9 acetylation (Fig. 3A
and Fig. S2A). WT161 can induce the accumulation of Ac-
α-tubulin in MM cell lines in as little as 2 h, and this induction is
reversible (Fig. S2 B and C). WT161 similarly induced accumu-
lation of Ac-α-tubulin in a panel of five additional MM cell lines
and in patient-derived MM cells that were purified from BM
aspirates from two MM patients (Fig. 3 B and C).
We previously have shown that HDAC6 inhibition by either
tubacin or siRNA triggers growth inhibition in MM cells (4).
WT161 inhibited cell growth more potently than tubacin (Fig. 3D
and Fig. S2D). The IC50 values of WT161 and tubacin in MM1.S
cells were calculated as 3.6 μM and 9.7 μM, respectively. WT161
induced significant toxicity in all MM cell lines tested, with IC50s
between 1.5 and 4.7 μM (Fig. S2E). Overall, these data demonstrate
that WT161 is a more potent inhibitor of HDAC6 than tubacin.
of each HDAC enzyme influences ligand-binding preferences
(16). HDAC6 forms a lipophilic pocket (the yellow loop in Fig. 2,
Right) containing Phe200 and Phe201 held in place by a cation–π
interaction of Phe200 with Arg194. This stable pocket is not
conserved at the sequence or structural level in HDAC2 or
HDAC3 because the flexibility introduced by the insertion of a
tyrosine and the alteration of Arg194 to a lysine reduce the li-
pophilic contacts (Fig. S1B).
To study the putative determinants of HDAC inhibitor selectivity,
we performed molecular dynamic (MD) simulations of HDAC2, -3,
and -6 in the presence of SAHA and WT161. The snapshots of the
MD simulations of SAHA in HDAC2, -3, and -6 show that SAHA
binds deeply in the active site channel and engages in multiple,
nonspecific interactions with all loops surrounding the active site
mouth with many possible orientations in all three isoforms (Fig.
2A). This finding indicates that SAHA is not sufficiently lipophilic
to engage a specific HDAC isoform in stable interactions, thus
explaining its observed lack of selectivity.
In contrast to SAHA and in analogy to tubacin, WT161 ex-
hibits good shape complementarity to HDAC6 via the Y-shaped
structure of the lipophilic cap. The cap region fits into the hy-
drophobic pocket formed by Phe200, Phe201, and Leu270 in
HDAC6 and forms a cation–π interaction with the top of the
linker and Arg194 (Fig. S1C). Thus, only a single orientation of
WT161 bound to HDAC6 is found over the course of the MD
simulation (Fig. 2B). These interactions are observed in HDAC6
but not in HDAC2/3, which have a conserved Arg260 that binds
more weakly to the lipophilic cap of WT161. Thus, the MD
simulation provides the structural basis for the HDAC6 selec-
tivity of WT161.
WT161 Enhances Proteasome Inhibitor-Induced Cytotoxicity in MM
cells. To investigate the cytotoxic effect of WT161 combined with
the first- and second-generation proteasome inhibitors BTZ and
CFZ, we first examined the effect of selective HDAC6 knockdown
using lentiviral HDAC6 shRNA. HDAC6 knockdown alone
inhibited cell growth, and the inhibition was significantly enhanced
by both BTZ and CFZ treatments (Fig. S3A). If HDAC6 knock-
down can enhance BTZ and CFZ cytotoxicity, we would expect
WT161 to exhibit similar effects. Indeed, both BTZ and CFZ trig-
gered growth inhibition that was further enhanced with increasing
MM.1S
161
Pat #1
161
Pat #2
Tubacin
0.3 0.1
WT161
C
D
A
C
T
C
T
C
T
161
μM
3
1
0
0.1 0.3 1 3 Pan
HDAC6
Ac-α-tub
α-tub
Ac-α-tub
Fig. 3. WT161 is a selective and reversible HDAC6 in-
hibitor and inhibits MM cell growth. (A) MM.1S cells
were cultured with tubacin, WT161, or panobinostat
(50 nM) for 6 h. Whole-cell lysates were analyzed by
immunoblotting with indicated antibodies. (B) MM cell
lines were cultured with or without 1 μM WT161 for
6 h. (C) MM.1S and patient-derived MM cells were cul-
tured with DMSO (control; C), 1 μM tubacin (T), or 1 μM
WT161 (161) for 6 h. Whole-cell lysates were subjected
to immunoblotting with the indicated antibodies.
(D) The relative viability of MM.1S cells cultured with
increasing concentrations of tubacin or WT161 for 48
h was determined by 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl tetrazolium bromide (MTT) assay.
α-tub
Ac-H3K9
120
120
Tubacin
WT161
100
100
Histone H3
80
80
B
60
60
MM.1S H929 RPMI U266 OPM2 KMS11
WT161
HDAC6
-
+
-
+
-
+
-
+
-
+
-
+
40
40
20
20
0
0
0
2
4
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8
10
10
0
2
4
6
8
Concentration (μM)
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annexin V positivity (Fig. 5A and Fig. S6C) (20). Together,
these results demonstrate that inhibition of proteasomal and
aggresomal protein degradation with BTZ and WT161, re-
spectively, effectively activates cell-stress signaling, resulting
in apoptotic cell death in MM cell lines and in patient-
derived cells.
A
B
WT161(μM)
120
100
80
60
40
20
0
120
100
80
60
40
20
0
0
0.625
1.25
2.5
Overcoming BM Microenvironment and BTZ-Induced Resistance. The
BM microenvironment promotes MM cell proliferation and confers
resistance to apoptosis (1). For example, dexamethasone-induced
apoptosis in MM cells is completely abrogated by coculture with
BM stromal cells (BMSCs). In MM–BMSC cocultures, BTZ alone
inhibited cell growth, and the inhibition was further enhanced with
WT161 (Fig. 6A and Fig. S7A).
Although BTZ has achieved responses and markedly improved
outcomes in MM, drug resistance develops leading to relapse of
disease in most patients. We therefore examined whether the com-
bination of BTZ with WT161 can overcome acquired BTZ
resistance. The BTZ-sensitive ANBL-6 cell line and the subline
ANBL-6-V5R, which exhibits decreased response to BTZ, were
cultured with BTZ, in the presence or absence of WT161.
ANBL-6-V5R cells showed resistance to BTZ treatment com-
pared with parental ANBL-6 BTZ-sensitive cells; importantly,
this resistance was completely abrogated with increasing con-
centrations of WT161 (Fig. 6B). Additionally, WT161 was able
to enhance cytotoxicity in patient-derived cells from three indi-
viduals who had developed resistance to BTZ treatment (Fig.
S7B). Therefore, WT161 supplementation to BTZ treatment may
abrogate resistance conferred by the microenvironment and BTZ-
acquired resistance.
0
3
4
5
6
7
0
5
7.5
10 12.5 15
Carfilzomib (nM)
Bortezomib (nM)
120
BTZ (nM)
100
80
60
40
20
0
0
2.5
5
0
0.3
1
3
0
0.3
1
3
Tubacin (μM)
WT161 (μM)
Fig. 4. HDAC6 inhibition enhances BTZ-induced cytotoxicity in MM cells.
(A) RPMI8226 cells were cultured for 48 h with BTZ (Left) or CFZ (Right) in the
absence or presence of WT161; n = 3. (B) MM.1S cells were cultured for 24 h with
tubacin or WT161 in the absence or presence of BTZ. All data represent mean SD.
concentrations of WT161 (Fig. 4A). To demonstrate the synergy
between dual proteasomal and aggresomal inhibition further, we
tested BTZ and CFZ in combination with an additional HDAC6-
selective inhibitor, tubastatin A (Fig. S3B). Although tubastatin A
was slightly less potent than WT161, it displays synergism with both
BTZ and CFZ, as calculated by isobologram analysis with a com-
bination index (CI) less than 1 (Fig. S3C).
Accordingly, as shown in Fig. 4B and Fig. S4A, WT161 enhanced
BTZ-induced cytotoxicity more strongly than tubacin. Moreover,
WT161 enhanced the cytotoxicity induced by other classes of
proteasome inhibitors (MG132 and lactacystin) more potently
than tubacin (Fig. S4B). Last, WT161 enhanced BTZ-induced
cytotoxicity in patient MM cells (Fig. S5 A and B). Importantly,
this combination treatment did not induce toxicity in periph-
eral blood mononuclear cells (PBMCs) derived from healthy
volunteers, thus suggesting a favorable therapeutic index
(Fig. S5C).
We next examined the molecular mechanisms whereby WT161
and BTZ induce synergistic cytotoxicity in MM cells. Consistent
with our previous studies (4), treatment of MM.1S cells with BTZ
and WT161 triggered increased accumulation of polyubiquitinated
proteins and activation of the stress-activated protein kinase JNK
relative to treatment with either drug alone (Fig. 5A). Accumula-
tion of polyubiquitinated proteins induces endoplasmic reticulum
(ER) stress and the unfolded protein response (UPR) to restore
normal ER function. As evident in MM1.S cells and in cells from
MM patients treated ex vivo, treatment with both BTZ and WT161
triggered increased expression of CCAAT/enhancer-binding pro-
tein (C/EBP) homologous protein (CHOP), a proapoptotic protein
induced by the UPR (Fig. 5 A and B and Fig. S6A). Activating
transcription factor 4 (ATF4) directly activates CHOP, and it, too,
was markedly up-regulated after treatment of BTZ with WT161
(Fig. 5C) (19). If the ER stress is prolonged, apoptotic cell death
ensues (19). We observed down-regulation of ER stress sensor
proteins inositol-requiring enzyme 1α (IRE1α) and protein kinase
R (PRKR)-like ER kinase (PERK) with WT161, both alone and
with BTZ, in patient MM cells (Fig. 5D), suggesting that WT161
treatment impairs the UPR to augment further ER stress-induced
death signaling. Finally, the antiapoptotic protein X-linked inhibi-
tor of apoptosis (XIAP) was down-regulated with WT161 treat-
ment (Fig. S6B), and combination treatment induced apoptosis as
determined by the cleavage of caspases 3, 8, and 9 and poly
(ADP-ribose) polymerase (PARP) and as confirmed by
In Vivo Anti-MM Activity of WT161 with BTZ. Finally, the relatively
simple synthesis of WT161 provided sufficient material to ex-
amine the effect of WT161 with BTZ treatment in vivo. First, we
evaluated the pharmacokinetic (PK) properties of WT161 (Fig.
S8A). With reasonable half-life in mice (1.4 h) and drug exposure
[maximum concentration (Cmax) = 18 mg/L], WT161 was for-
mulated for i.p. administration. The i.p. dose, 50–100 mg/kg,
was empirically determined based on the in vitro activity of
WT161 in MM cell lines (IC50 = 1.5–4.7 μM) and drug exposure
0 nM BTZ 3 nM BTZ
A
C
4h
8h
WT161
Ub
0
1
3
0
1
3 μM
ATF4
Ac-α-tub
p-JNK
GAPDH
CHOP
Casp-8
Cleaved Casp-9
D
Casp-3
BTZ
WT161
-
-
-
+
+
-
+
+
PARP
GAPDH
p-IRE1α
B
IRE1α
PERK
0 nM BTZ
161 C
5 nM BTZ
C
T
T 161
CHOP
Actin
Ac-α-tub
α-tub
Fig. 5. WT161 enhances BTZ-induced ER stress and apoptotic cell death.
(A) MM.1S cells were cultured with increasing concentrations of WT161 for
16 h in the presence or absence of BTZ. (B) Patient-derived MM cells were
treated with DMSO (control; C), 2 μM tubacin (T), or 2 μM WT161 (161) in the
absence or presence of BTZ for 24 h. (C) MM.1S cells were cultured with
vehicle control, WT161 (3 μM), BTZ (3 nM), or BTZ+WT161 for 4 h and 8 h.
Whole-cell lysates were subjected to immunoblotting with the indicated
antibodies. (D) Patient-derived MM cells were treated with 5 nM BTZ 2 μM
WT161 for 24 h. Whole-cell lysates were subjected to SDS/PAGE and immu-
noblotting with the indicated antibodies.
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adjacent to the binding site. The HDAC6 protein contains a
large lipophilic pocket adjacent to the active site that is unique to
this protein. The large pendent unit of tubacin fills this lipophilic
pocket but precludes it from binding to alternate HDAC family
proteins. Based on this design principle, we developed WT161 to
have a zinc-binding motif and a hydroxymate head to bind the
enzyme’s active site connected by a linker to a large triphenyl amine
motif designed to bind with the lipophilic pocket of HDAC6. The
simplified triphenyl amine structure has successfully achieved better
shape complementarity than tubacin and avoids the complicated
synthesis of this pendent unit. WT161, which has no stereocenters,
can be synthesized in large quantities for further in-depth in vitro
and in vivo experimentation.
A
C
Vehicle
WT161
20
15
10
5
MM.1S
BTZ
12
BTZ + WT161
8
4
0
BTZ
0
0.6 1.25 2.5
0 μM WT161
0
0.6 1.25 2.5
0
1 μM WT161
1
3 5 8 10 12 151719222426
Days from the first day of treatment
B
120
D
Ac-α-tub
BTZ (nM)
0
5
10
20
80
Resistance mechanisms are the largest hurdle to treating and ef-
fectively curing MM and can persist initially or emerge in the course
of treatment. Thus, to examine the synergistic effects of proteasomal
and aggresomal inhibition in MM, we tested WT161 with the pro-
teasome inhibitors BTZ and CFZ. Excitingly, WT161 was able to
enhance both BTZ and CFZ cytotoxic effects in MM cell lines and
patient samples, with no effect on PBMCs. With this combinatorial
treatment, we observed the accumulation of ubiquitinated proteins,
ER stress and induction of the UPR, activation of stress signaling
(JNK activation), and cleavage of caspases followed by apoptotic cell
death. Interestingly, WT161 as a single agent does not induce ER
stress, the UPR, or ER stress-mediated apoptosis. We did observe
down-regulation of the antiapoptotic protein XIAP and the ER
stress-sensor proteins PERK and IRE1α with WT161 after 24-h
treatment. This down-regulation is also observed after 24 h of ex-
posure of MM cells to CFZ (22). At first glance, this effect seems
counterintuitive, but activation of PERK and IRE1α not only pro-
motes cellular adaptation to/survival of ER stress but also actively
inhibits the ER stress-induced apoptotic program. Thus, we believe
down-regulation is required to impair the UPR and to augment ER
stress-induced apoptosis. Many candidate proteins are involved in
orchestrating the switch from the protective UPR signaling to
40
0
WT161 0 0.3
1
3
0
0.3
1
3
Control
WT161
Sensitive
Resistant
Fig. 6. Anti-MM activity of WT161 overcomes resistance and decreases
tumor burden in vivo. (A) MM.1S cells were cocultured with BMSCs WT161
with increasing concentrations of BTZ (measured in nanomolars) for 24 h. Cell
proliferation was assessed by [3H]-thymidine uptake; n = 3. CPM, counts per
minute. (B) ANBL-6 (BTZ-sensitive) and ANBL-6-V5R (BTZ-resistant) cells were
cultured with increasing concentrations of WT161 for 24 h in the absence or
presence of increasing concentrations of BTZ. Cell growth was assessed by MTT
assay; n = 3. (C) SCID mice were injected s.c. with 5 × 106 MM.1S cells and were
treated with vehicle, WT161 (50 mg/kg, i.p.), BTZ (0.5 mg/kg, i.v.), or BTZ +
WT161 for 26 d. Tumor volume (100 × mm3) was measured and calculated
versus time (days); n = 9 mice per group. All data represent mean
(D) Immunostaining of Ac-α-tubulin in xenograft tissue from two control and
two WT161-treated mice. Representative results are shown.
SD.
information obtained from the PK study (21). We observed toxicity
with WT161 at 100 mg·kg−1·d−1 i.p., but at 50 mg·kg−1·d−1 i.p.
WT161 was well tolerated as a single agent and in combination with
BTZ. Next, we tested WT161 in combination with BTZ in a murine
xenograft model of human MM (MM.1S). Tumor-bearing mice
were randomized into four treatment groups: control, BTZ, WT161,
or BTZ + WT161. Although there was no significant difference in
tumor growth between the control and BTZ-treated (P = 0.07) or
WT161-treated (P = 0.095) cohorts, BTZ combined with WT161
demonstrated a significant antitumor effect (P = 0.0078) (Fig. 6C).
Moreover, there was no significant difference between BTZ or
WT161 treatment and the combinatorial treatment (P = 0.327 and
P = 0.079, respectively). The on-target activity of WT161 in vivo was
confirmed by assessing Ac-α-tubulin levels in resected tumor samples
(Fig. 6D and Fig. S8B). In conclusion, these data demonstrate that
simultaneous inhibition of the proteasome and aggresome by BTZ
and WT161, respectively, triggers significant anti-MM activities both
in vitro and in vivo and represents a viable therapeutic option for the
treatment of MM, especially proteasome inhibitor-resistant MM.
proapoptotic signaling. Some of these proteins, such as P58IPK
,
GADD34, and TRB3, are involved in shutting down the PERK-
mediated pathway (19). In the combination treatment, BTZ induces
ER stress and the UPR, and WT161 down-regulates antiapoptotic
proteins and the proteins required for adaptation and survival of ER
stress. These distinct activities combine to tip the balance toward
apoptosis more strongly than when either agent is used alone.
Additionally, components of the BM microenvironment are well
known to play critical roles in MM cell survival and environment-
mediated drug resistance (1). Using a cellular assay to mimic MM in
its microenvironment, we observed that the WT161–BTZ combi-
nation treatment could overcome the resistance conferred by
BMSCs. Finally, the combination of BTZ and WT161 was effective
against BTZ-resistant MM cell lines and samples derived from pa-
tients who had developed BTZ resistance. The molecular mecha-
nisms conferring BTZ resistance in MM have not yet been fully
elucidated, but our results suggest that HDAC6 inhibitors in com-
bination with BTZ could overcome clinical BTZ resistance in MM.
The easy and cost-effective synthesis of WT161 enabled us to
study HDAC6 inhibition in combination with proteasome inhibition
in a preclinical mouse model of human MM. We observed a sig-
nificant inhibitory effect on tumor growth with the WT161–BTZ
combinatorial treatment. Our studies provide the framework for
clinical evaluation of combined targeted therapy to achieve dual
blockade of proteasomal and aggresomal protein degradation and
to improve patient outcomes in MM. Importantly, WT161 has been
further refined to develop ACY-1215 (ricolinostat), a hydroxamic
acid HDAC6 inhibitor with similar anti-MM activities, which has
Discussion
For target validation of HDAC6 in MM and for broader use by
the biological community, we endeavored to create a potent, se-
lective, and bioavailable HDAC6 inhibitor. WT161 was identified
via biochemical and cellular screening from a hydrazone library
containing ∼400 molecules. Biochemically, WT161 is most selec-
tive against HDAC6, and in cells WT161 effectively demonstrated
the predicted cellular effects of HDAC6 inhibition, namely the
accumulation of acetylation on α-tubulin but not histones. Our
proof-of-concept work demonstrated that HDAC6 inhibition by
either siRNA knockdown or tubacin was cytotoxic to MM cells, so moved rapidly from preclinical to promising phase I/II clinical trials
we tested WT161 for similar effects. Notably, we determined that in relapsed/refractory MM (23, 24). Unlike preclinical studies, sin-
WT161 can induce acetylation of α-tubulin and cell death effi- gle-agent activity of ricolinostat in clinical trials is possibly restrained
ciently in MM cell lines and in patient samples earlier and at lower
doses than tubacin (4).
The structural origin of the HDAC6 selectivity of WT161 was
traced to the differences in the shape of the protein surface
by its limited PK properties. Specifically, the Cmax of ricolinostat in
serum at a dose of 160 mg reached 1.1 μM, which may not be
sufficient to induce direct anti-MM activities. A single dose of
WT161 at 5 mg/kg i.v. reaches a Cmax in serum of ∼40 μM.
Hideshima et al.
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Reagents and General Synthetic Procedure. Tubacin was synthesized in the J.E.B.
laboratory (32). BTZ, CFZ, tubastatin A, and panobinostat were purchased
from Selleck Chemicals. Antibodies used in this study were purchased di-
rectly from the vendors listed in SI Materials and Methods. Detailed synthetic
methods can be found in SI Materials and Methods and as previously
reported by Tang et al. (33). All reactions were performed and monitored by
LCMS. The intermediates and final product were fully characterized with pro-
ton and carbon-13 NMR (1H NMR and 13C NMR) spectra and high-resolution
mass spectra (HRMS). Compounds were biochemically profiled against
HDAC1–9 as previously reported (14).
HDAC6/aggresome inhibition may be more broadly applicable
to cancers other than MM. Malignant cells generally have higher
protein synthesis rates than their normal counterparts, making
them more prone to protein aggregation and perhaps more
sensitive to proteasome inhibitor-induced apoptosis. Inhibition
of proteasome activity has been demonstrated to induce proa-
poptotic ER stress in pancreatic carcinoma (25), head and neck
cancer (26), and nonsmall cell lung carcinoma (27).
Mechanistic and translational biology has moved in vivo, and
so must the field of chemical biology. The availability of tubacin
has provided significant insights into HDAC6 biology. Since the
development of tubacin, additional HDAC6 inhibitors have been
described, such as tubastatin A and, more recently, N-hydroxy-4-
{[N-(2-hydroxyethyl)-2-phenylacetamido]methyl} benzamide (HPB)
and an aminopyrrolidinone HDAC6 inhibitor (28–30). When these
molecules are validated in vivo, we anticipate an informative and
structurally diverse set of chemical probes of HDAC6 to drive further
mechanistic exploration and definitive clinical translation in cancer
and nonmalignant diseases.
Cell Lines. MM.1S, NCI-H929, RPMI8226, and U266 cells were obtained
from American Type Culture Collection (ATCC). The KMS11 cell line was
obtained from the Japanese Collection of Research Bioresources (JCRB)
Cell Bank. OPM-2 cells were purchased from Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH (German Collection of Microor-
ganisms and Cell Cultures). ANBL-6 and ANBL-6-VR5 cell lines were kindly
provided by Robert Orlowski, MD Anderson Cancer Center, Houston, TX.
Statistical Analysis. The statistical significance of differences observed in drug-
treated versus control cultures was determined using the Wilcoxon signed-
ranks test.
Materials and Methods
ACKNOWLEDGMENTS. This study was supported by NIH Grants SPORE
CA10070, P01 78378, and R01 CA50947 (to K.C.A.), R01 CA178264 (to T.H. and
K.C.A.), R01 GM38627 (to S.L.S.), K08 CA128972 (to J.E.B.), T32 HL07623-18
(to J.E.B.), and R01 CA152314 (to J.E.B. and O.W.); a Burroughs-Wellcome Foun-
dation Career Award for Medical Scientists (to J.E.B.); the Doris Duke Charitable
Foundation (J.E.B.); and National Science Foundation Grant TG-CHE090124 (to
O.W.). K.C.A. is an American Cancer Society Clinical Research Professor.
All experiments with patient samples were performed according to a protocol
approved by the Institutional Review Board of Dana-Farber Cancer Institute.
All animal studies were conducted according to protocols approved by the
Animal Ethics Committee of the Dana-Farber Cancer Institute. Mice used in
this study were handled in compliance with the NIH Guide for the Care and
Use of Laboratory Animals (31).
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