STK33 kinase inhibitor BRD-8899 has no effect on KRAS-dependent cancer cell viability

Article (PDF Available)inProceedings of the National Academy of Sciences 109(8):2860-5 · February 2012with44 Reads
DOI: 10.1073/pnas.1120589109 · Source: PubMed
Abstract
Approximately 30% of human cancers harbor oncogenic gain-of-function mutations in KRAS. Despite interest in KRAS as a therapeutic target, direct blockade of KRAS function with small molecules has yet to be demonstrated. Based on experiments that lower mRNA levels of protein kinases, KRAS-dependent cancer cells were proposed to have a unique requirement for the serine/threonine kinase STK33. Thus, it was suggested that small-molecule inhibitors of STK33 might have therapeutic benefit in these cancers. Here, we describe the development of selective, low nanomolar inhibitors of STK33's kinase activity. The most potent and selective of these, BRD8899, failed to kill KRAS-dependent cells. While several explanations for this result exist, our data are most consistent with the view that inhibition of STK33's kinase activity does not represent a promising anti-KRAS therapeutic strategy.

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STK33 kinase inhibitor BRD-8899 has no effect
on KRAS-dependent cancer cell viability
Tuoping Luoa,b,1, Kristina Massona,1, Jacob D. Jaffea, Whitney Silkwortha, Nathan T. Rossa,2, Christina A. Scherera,
Claudia Schollc, Stefan Fröhlingc, Steven A. Carra, Andrew M. Sterna, Stuart L. Schreibera,b,d,3, and Todd R. Goluba,d,e,3
aBroad Institute of Harvard and MIT, 7 Cambridge Center, Cambridge, MA 02142; bDepartment of Chemistry and Chemical Biology, Harvard University,
Cambridge, MA 02138; cDepartment of Internal Medicine III, University Hospital of Ulm, 89081 Ulm, Germany; dHoward Hughes Medical Institute,
Bethesda, MD 20817; and eDepartment of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02215
Contributed by Stuart L. Schreiber, December 14, 2011 (sent for review September 23, 2011)
Approximately 30% of human cancers harbor oncogenic gain-of-
function mutations in KRAS. Despite interest in KRAS as a therapeu-
tic target, direct blockade of KRAS function with small molecules has
yet to be demonstrated. Based on experiments that lower mRNA
levels of protein kinases, KRAS-dependent cancer cells were pro-
posed to have a unique requirement for the serine/threonine kinase
STK33. Thus, it was suggested that small-molecule inhibitors of
STK33 might have therapeutic benefit in these cancers. Here, we
describe the development of selective, low nanomolar inhibitors
of STK33s kinase activity. The most potent and selective of these,
BRD8899, failed to kill KRAS-dependent cells. While several explana-
tions for this result exist, our data are most consistent with the
view that inhibition of STK33s kinase activity does not represent
a promising anti-KRAS therapeutic strategy.
Oncogenic mutations in the RAS family member KRAS are
among the most common mutations in human cancer (1).
For example, KRAS-mutation frequencies in lung, colon, and
pancreas adenocarcinoma are 30%, 50%, and 90% respectively
(2). These observations, coupled with functional studies, suggest
that KRAS is a highly attractive therapeutic target for many
cancers. Unfortunately, small-molecule targeting of KRAS has
not yet been achieved, and no effective KRAS inhibitors have
been described. Blockade of prenylation of the KRAS C-terminal
membrane anchoring domain with farnesyltransferase inhibitors
has met with limited success, due at least in part to increased
expression of geranylgeranyl transferase (3). Similarly, targeting
other steps in the processing of the KRAS C-terminal region
through the inhibition of Ras-converting enzyme or isoprenyl-
cysteine-carboxymethyltransferase has yet to be clinically vali-
dated (2). Efforts to target the downstream effector pathways of
KRAS with MEK inhibitors alone or in combination with PI3K
inhibitors have shown promising preclinical results and are cur-
rently being evaluated in the clinic (4). Nevertheless, the therapeu-
tic targeting of KRAS remains one of the grand challenges in
cancer research.
Recently, an alternative approach to targeting KRAS has been
proposednamely, the RNA interference (RNAi)-based screen-
ing for synthetic lethal gene/RNA interactions that might then
suggest protein targets more druggablethan the targeted mRNA
or the KRAS protein itself (59). A recently reported RNAi screen
suggested the serine-threonine kinase STK33 as such a target
(9). Knock down of STK33 was reported to induce apoptosis in
KRAS-dependent AML cancer cell lines but spare KRAS wild-
type cells. While the normal function of the STK33 protein is un-
known, the results led the authors to propose that a small-molecule
inhibitor of STK33s protein kinase activity would selectively kill
KRAS-mutant cancer cells. As no such small-molecule inhibitors
exist, we set out to discover them, and to characterize their activity
as anti-KRAS agents.
Results
High Throughput Screening for STK33 Kinase Inhibitors. We first
established an assay suitable for screening for STK33 kinase
inhibitors. Using baculovirus-expressed full length human recom-
binant STK33 and the general kinase substrate myelin basic
protein (MBP), a biochemical assay was optimized that quantified
the STK33 kinase-dependent generation of ADP (see Materials
and Methods). Radiometric analysis and mass spectroscopy (MS)
(10, 11) were used to corroborate that the ADP generated was
quantitatively coupled to phosphorylation of MBP (SI Appendix,
Fig. S1). The reaction rate was linear with respect to time and
enzyme concentration and Michaelis-Menten constants were de-
termined (SI Appendix,Fig.S2). The relatively nonspecific kinase
inhibitor dimethyl fasudil (BRD7446) was identified in a pilot
screen as a low micromolar STK33 inhibitor; it therefore served as
a positive control for subsequent screening (SI Appendix,Fig.S2E).
A high throughput screen of 27,500 compounds from diverse
chemical collections (SI Appendix, Table S1) was performed in du-
plicate at 100 μM ATP (threefold above the KM;ATP). Coefficients
of variation (CVs) of 46% for the DMSO vehicle control and
corresponding Zfactor values for the positive control between
0.64 and 0.76 were obtained (12). The hit rate was 0.4% at an in-
hibition threshold of 20% inhibition where Zfactor equaled zero
(SI Appendix,Fig.S3). The IC50 values of the 102 primary hits were
measured at two ATP concentrations (100 and 25 μM) using both
the ADP-Glo and HTRF assays.
The STK33-inhibitory activity of 95/102 compounds (93%) was
verified in replicate experiments, and these compounds clustered
into 37 discrete chemotypes (SI Appendix, Fig. S4). Four groups
appeared to be ATP-noncompetitive inhibitors but were either
very weak or contained chemically reactive moieties, and were
therefore not prioritized. All other hits were ATP-competitive
inhibitors with IC50 values in the micromolar range, except the
chemotype of staurosporine (a notoriously nonselective kinase
inhibitor) that inhibited STK33 with low nanomolar potency.
Fasudil (BRD7868) (Table 1), a known inhibitor of Rho-asso-
ciated protein kinase (ROCK) (13) as well as seven of its analogs
yielded a structure-activity relationship for inhibiting STK33.
This series was therefore prioritized as the lead structure for
chemical optimization due to its low micromolar potency, known
selectivity for other kinases, physical properties, and synthetic
feasibility.
Author contributions: T.L., K.M., J.D.J., W.S., N.T.R., S.A.C., A.M.S., S.L.S., and T.R.G.
designed research; T.L., K.M., J.D.J., and W.S. performed research; C. Scholl and S.F.
contributed new reagents/analytic tools; T.L., K.M., J.D.J., N.T.R.,and C.A. Scherer analyzed
data; and T.L., K.M., A.M.S., S.L.S., and T.R.G. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
1T.L. and K.M. contributed equally to this work.
2Present address: Novartis Institutes for BioMedical Research, 250 Massachusetts
Avenue, Cambridge, MA 02142.
3To whom correspondence may be addressed. E-mail: stuart_schreiber@harvard.edu or
golub@broadinstitute.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/
doi:10.1073/pnas.1120589109/-/DCSupplemental.
28602865 PNAS February 21, 2012 vol. 109 no. 8 www.pnas.org/cgi/doi/10.1073/pnas.1120589109
STK33 Can Autophosphorylate In Vitro and Is Inhibited by a Fasudil
Analog. During the optimization of STK33 inhibitors, we sought
to confirm that the candidate inhibitors were truly functioning as
STK33 inhibitors, and were not simply an artifact of a screening
assay based on an artificial kinase substrate (MBP). To address this
concern, we assessed whether STK33, like many kinases, is autop-
hosphorylated, and if so, whether our candidate STK33-inhibitory
compounds blocked such autophosphorylation (SI Appendix,Fig
.S1A) (9, 14). Mass spectrometry revealed several sites of phos-
phorylation on STK33 (Fig. 1). Two sites (Thr440 and Ser441)
were found to be phosphorylated in the preparation of recombi-
nant STK33 itself and the remaining sites were due to bona fide
in vitro kinase activity of the enzyme. Among the sites observed
were the Thr491/Ser493/Thr496 cluster that had been previously
reported to be regulated during the cell cycle in HeLa cells (15).
We then verified that the lead scaffold that emerged from the
biochemical screen (represented in BRD3773, BRD3752, and
BRD3695, see structures in Table 1), indeed inhibited the autopho-
sphorylation of STK33 at these sites in a concentration-dependent
manner (Fig. 1). These results suggested that the scaffold was
a suitable starting point for further optimization with respect to
STK33 inhibition.
Chemical Optimization of STK33 Inhibitors. For chemical optimiza-
tion, 250 fasudil analogs were synthesized and tested in the
biochemical assay (SI Appendix, Schemes S1S5, Table S13,
and SI Materials and Methods). Structurally, fasudil comprises
two fragments: an isoquinoline ring and a homopiperazine ring,
connected by the sulfonyl functionality on the 5-position of the
isoquinoline. The sulfonamide functionality and nitrogen on
the isoquinoline ring are critical for the potency against STK33
(SI Appendix, Table S2). Replacing the basic amine nitrogen with
carbon or oxygen, or converting it to an amide, resulted in signif-
icantly reduced potency (BRD7657 vs. BRD2998 and BRD4220,
BRD7868 vs. BRD0188, BRD7647 vs. BRD6818, Table 1, SI
Appendix, Table S3). We observed that the two additional methyl
groups in dimethyl fasudil lead to a sevenfold increase in potency
compared to fasudil (BRD7446 vs. BRD7868). While one mono-
methyl fasudil isomer, namely BRD1869, was threefold less
potent than fasudil (BRD7868), the other monomethyl fasudil
isomer (BRD3773) was over 70-fold more potent against
STK33 compared to fasudil (BRD7868). Therefore we explored
the effect of monomethylation at several positions of the isoqui-
noline ring; only 4-methyl substitution significantly increased
the potency (SI Appendix, Table S4). An SAR study performed
on isoquinoline 4-substitution indicated that only a small alkyl
group, such as ethyl (BRD5930), is tolerated, while polar or bulky
groups resulted in decreased activity (SI Appendix, Table S5).
Further optimization on the amine fragment led to two more
nanomolar STK33 inhibitors (BRD3752 and BRD3695); their
enantiomers were 60100-fold less active (BRD0828 and
BRD9573) (Table 1, SI Appendix, Table S6).
Further chemical optimization was guided by measures of
STK33 selectivity. As measured by kinase activity profiling
against 241 kinases (SI Appendix, Table S7), BRD3773 exhibited
relative STK33 selectivity, inhibiting 36 kinases with estimated Ki
values within 10-fold of that of STK33. Compound BRD3695
showed further selectivity with respect to those 36 kinases (SI
Appendix, Fig. S5), with inhibition confined to the AGC subfamily
of kinases, and therefore became the lead for further chemical
optimization.
Modification of the pyrrolidine ring of BRD3695 (SI Appendix,
Table S8) yielded more potent, low nanomolar analogs
(BRD4980, BRD9949, and BRD8899). These compounds were
more potent than their corresponding (2R, 4S) diastereomers
(BRD1045, BRD7071, and BRD5749). The low nanomolar po-
tency of BRD8899 against STK33 as well as other selected inhi-
bitors was confirmed by competition binding assay (SI Appendix,
Table S9) (16). Furthermore, the specificity of BRD8899 was
retained, exhibiting significant off-target inhibition of a few other
kinases, including RIOK1 (97% inhibition), MST4 (96%), RSK4
(89%), ATK1(85%), KITD816V(85%), ROCK1 (84%), and FLT3
(81%), while STK33 was inhibited by 89% (SI Appendix,
Table S10). BRD8899 thus represents a 200-fold improvement
in potency compared to the initial screening hit (BRD7446), with
increased selectivity for STK33 (Fig. 2).
Characterization of BRD8899 in Cell-Based Assays. Having demon-
strated that BRD8899 is a potent and selective inhibitor of
STK33 kinase activity in biochemical assays, we next turned to
its characterization in cells. It has been previously reported that
STK33 knock down via RNAi results in the selective killing of
mutant KRAS-dependent cancer cells (9), a result that we con-
firmed using the same shRNAs in KRAS-mutant AML cell lines
(NOMO-1 and SKM-1 AML cells) compared to KRAS-wild-type
lines (THP-1 and U937) (SI Appendix, Fig. S7). We next asked
whether BRD8899 could recapitulate KRAS-associated pattern
of cell killing. We therefore tested BRD8899 at a range of doses
across 35 cancer cell lines, but observed no effect on cell viability
in any of the lines at concentrations as high as 20 μM (Fig. 3A,
SI Appendix, Fig. S6 and Table S12). (9) Because BRD8899 (our
most potent STK33 inhibitor) failed to kill KRAS-mutant cell
lines as predicted by the RNAi experiments, we tested additional
analogs for their ability to kill cancer cells. While some of these
compounds reduced cell viability, such cytotoxicity was uncorre-
lated with KRAS-mutation status across the panel of cell lines
Fig. 1. Proteomic assessment of STK33 autophosphorylation. Sites on STK33
determined to be autophosphorylated are shown on the left. Increasing con-
centrations of the three fasudil analogs shown were added to an in vitro
phosphorylation reaction in which the only substrate present was STK33 it-
self. The heatmap is colored according to how the level of phosphorylation at
a given site changed in response to addition of compound at the specified
concentration vs. DMSO control. Some compound concentrations were found
to increase phosphorylations at given sites, leading to values >100%.1am-
biguously localized phosphorylation within the span of bracketed amino
acids. 2ambiguously localized double phosphorylation within the span of
bracketed amino acids.
Luo et al. PNAS February 21, 2012 vol. 109 no. 8 2861
BIOCHEMISTRY
(Fig. 3B,SI Appendix, Fig. S6). We therefore conclude that the
observed cell death is most likely explained by off-target effects of
these less potent compounds, rather than by STK33-inhibitory
activity.
Establishing Bioactivity of BRD8899 in Cells. The experiments
described above suggest that treatment of KRAS-mutant cancer
cells with low nanomolar biochemical potency against STK33
does not result in cell death, arguing against the notion that
the enzymatic function of STK33 represents an attractive thera-
peutic target in KRAS-mutant cancers. However, it is possible
that while BRD8899 is highly potent in biochemical assays, it
fails to inhibit STK33 in cellseven at 1,000-fold higher concen-
trations. To address this possibility, we sought a biomarker of
BRD8899 activity in cells. We first looked for evidence of STK33
autophosphorylation in cells, given the observation that such
autophosphorylation occurs with recombinant STK33, and is in-
hibited by BRD8899. However, looking across multiple cell lines,
we found endogenous STK33 to be expressed at low levels, and
phosphorylation was not detectable by mass spectrometry (for
details, see Materials and Methods). We therefore turned to
the kinase profiling data for BRD8899, where we observed simi-
larly potent inhibition of the kinase MST4 in addition to STK33.
Treatment of NOMO-1 cells with BRD8899 resulted in decreased
phosphorylation of the MST4 substrate ezrin (Fig. 4A), but had
no effect on ERK phosphorylation, as predicted from the kinase
profiling data (Fig. 4B). This finding suggests that BRD8899
indeed gets into cells, inhibits the activity of a target (MST4)
predicted by kinase profiling experiments, and yet does not kill
KRAS-mutant cancer cells. These results are most consistent
with BRD8899 likely inhibiting STK33 in cells, but STK33 kinase
activity not serving as a compelling target for KRAS-mutant
tumors. Of course in the absence of a direct biomarker of
STK33 activity in cells, we cannot exclude that whereas BRD8899
inhibits MST4 (as an off-target effect), it does not effectively
inhibit STK33 in cells.
Discussion
Cancer genomes have served as powerful guides to the develop-
ment of cancer therapeutics that target oncogene dependencies.
Several examples point to the utility of targeting mutant, onco-
genic kinases with small-molecule inhibitors, with dramatic
clinical success being seen in chronic myeloid leukemia (17), gas-
trointestinal stromal tumors (18), lung cancer (19), and melano-
ma (20). However, the therapeutic path for nonkinase mutations
in cancer is less established. In particular, recurrent mutations
in KRAS have been known to exist for over a quarter century,
and yet KRAS has been considered undruggable.
A general solution to approaching cancer targets, however,
has recently been suggested. Borrowing from the concepts of
synthetic lethality first explored in yeast, nononcogene addic-
tionhas been proposed (8, 21). One avenue to identify non-
oncogene codependencies (proteins on which oncogenes are
dependent yet not themselves mutated) relies on genetic screens
Table 1. Biochemical activity of selected STK33 inhibitors
R1R2IC50 (μM) R1R2IC50 (μM) R1R2IC50 (μM)
H
NH
NH
2
BRD7657: H-9
14 H
NBRD0188
>186 Me
NH
NH
BRD9573
4.8
H
N
NH
BRD7868: Fasudil
14 H
N
NH
Me
BRD1869
50 Me
NH
NH
HO
BRD4980
0.037
Me
N
NH
Me
BRD7446: Dimethyl-fasudil
2.0 Me
N
NH
BRD3773
0.19 Me
NH
NH
HO
BRD1045
0.12
H
BRD7647: H-89
NH
N
H
Br 2.2 Et
N
NH
BRD5930
0.28 Me
NH
NH
H2N
BRD9949
0.020
HNH
Me
BRD2998
>186 Me
NNH
2
BRD3752
0.047 Me
NH
NH
H
2
N
BRD7071
0.42
H
NH
OH
BRD4220
110 Me
NNH2
BRD0828
3.7 Me
NH
NH
HN
Me
O
BRD8899
0.011
H
NH
N
H
Br
O
BRD6818
>186 Me
NH
NH
BRD3695
0.063 Me
NH
NH
HN
Me
O
BRD5749
0.52
Representative fasudil analogs, tested for their inhibition of STK33 kinase activity in vitro. The side groups for R1and R2are presented for each analog
(see the top structure for positions), along with the compound name and the IC50 at 25 μMATP.
2862 www.pnas.org/cgi/doi/10.1073/pnas.1120589109 Luo et al.
to infer potentially more readily druggable proteins whose inhibi-
tion is selectively lethal in the context of particular mutations.
Such approaches are highly attractive because they do not require
prior knowledge of the biological basis of the synthetic lethal
interactionrather, the relationship is revealed through an un-
biased screen such as an RNAi screen. On the other hand, while
this approach might establish a synthetic relationship between
a genetic feature of a cancer and the lowering of a target mRNA,
they do not establish such a relationship involving a function
of the encoded protein. Assuming RNAi-induced changes in
mRNA levels and small-molecule-modulated protein function
are equivalent, the report of STK33 essentiality in mutant KRAS-
dependent cancer cells (9) was encouraging because STK33, as a
serine/threonine kinase, was in principle an accessible target of
small molecules, and such compounds could form the basis of
anti-KRAS therapeutics. We therefore developed a biochemical
STK33 kinase assay to identify specific STK33 inhibitors that
could be evaluated in cellular proof-of-principle studies. A high
throughput screen identified a series of STK33 kinase-inhibitory
compounds, which were subsequently optimized for potency
(representing 200 1;000-fold increase in potency) while mini-
mizing off-target effects (inhibiting only three other kinases more
potently than STK33). Specifically, our lead compound,
BRD8899, exhibited an IC50 of 11 nM in an STK33 biochemical
assay. However, contrary to essentiality of STK33 predicted by
initial genetic studies of STK33, BRD8899 failed to kill KRAS-
mutant cells at concentrations as high as 20 μM.
There are a number of explanations of the failure of a potent
STK33 kinase inhibitor to kill KRAS-mutant cells, and several
are offered here. It is conceivable that while BRD8899 (and its
analogs) inhibit STK33 kinase activity in biochemical assays, it
does not do so in living cells. For example, it is possible that
BRD8899 does not penetrate cells. However, several pieces of
evidence argue against this possibility. Ideally, one would have
a direct measure of STK33 kinase activity in cells, but to date,
no such biomarker has been developed for STK33. We attempted
to identify such a direct biomarker, looking for evidence of the
STK33 autophosphorylation we observed using recombinant
STK33 protein. Unfortunately, owing to the low level of STK33
expression in the two cancer cell lines analyzed (NOMO-1 and
SKM-1), we were unable to document such phosphorylation.
We therefore turned to indirect measures of BRD8899 bioactivity,
using the predicted off-target effects of the compound suggested by
kinase specificity profiling. Indeed, we found that BRD8899 inhi-
bits the kinase MST4 in cells (resulting in subsequent decreased
phosphorylation of an MST4 substrate, ezrin). These biomarker
studies, while indirect, suggest that BRD8899 does in fact enter
cells, and can inhibit the activity of its kinase targets. While it is
conceivable that BRD8899 inhibits MST4 but not STK33, we con-
sider that scenario less likely. Although other explanations exist,
such as a cellular context dependency recently reported with small-
molecule inhibitors of PKC and PDK1 (23, 24), overall we surmise
that inhibiting STK33 kinase activity is not likely to be an effective
therapeutic strategy for KRAS-mutant cancer.
While inhibition of STK33 kinase activity may not result in
KRAS-related cell killing, it is possible that nonkinase activities
of STK33 may be responsible for its observed essentiality in
RNAi-based studies. For example, STK33 could have a distinct
function other than its kinase activity. It is conceivable that
STK33 has a more structural, scaffolding function that is required
for the proper function of a multiprotein complex. Whereas
RNAi-mediated knock down of STK33 might disrupt such puta-
tive scaffolding activity, a small-molecule kinase inhibition may
not. Such nonkinase activities of STK33 cannot be excluded by
our present studies. A similar conclusion was reached in a recent
independent study (22).
An alternative explanation for the failure of STK33 inhibitors
to recapitulate STK33 the knock-down phenotype is that the
shRNA reagents have off-target (non-STK33) effects. Arguing
against this alternative explanation is the observation that a domi-
nant-negative STK33 construct was shown to result in selective
lethality to KRAS-dependent cell lines (9). Nevertheless, until
clear rescue experiments using non-RNAi-inhibitable STK33 ex-
pression constructs demonstrate rescue of shRNA-mediated kill-
ing, an off-target mechanism must remain a formal possibility. We
note that a recent study using STK33 siRNA oligonucleotides tran-
siently transfected into AML cell lines failed to show a KRAS-spe-
cific killing effect (22). However, the transfection efficiency was
not reported and only partial STK33 knock down was achieved
in those experiments, making it difficult to interpret the results.
It has been recently suggested that while gain of function
mutations within oncogenes lead to increases in rate-determining
Fig. 2. Small molecule/kinase interaction maps for
BRD7446 and BRD8899. The kinase profiling (16)
was done at 5 μM for BRD7446 and 1 μMfor
BRD8899, resulting in equal inhibition of STK33 in
a biochemical assay. Kinases found to bind are
marked with red circles; larger circles indicate higher
affinity binding. STK33 is indicated by the red arrow.
Luo et al. PNAS February 21, 2012 vol. 109 no. 8 2863
BIOCHEMISTRY
steps in signaling, their synthetic lethal partners may operate
through relatively rapid signaling steps (8). Although these fast steps
might be modulated by irreversiblemaneuvers such as RNAi
knock down and dominant negative expression, achieving this same
level of target modulation with reversible small molecule competi-
tive inhibitors may be more difficult. The extent to which this phe-
nomenon explains our STK33 results remains to be determined.
The experiments described here used cell viability as the
readout of STK33 modulation. As such, these studies do not
preclude the possibility of an important role of STK33 in other
cancer phenotypes (e.g., migration, adhesion). However, our
study indicates that the pharmaceutical targeting of STK33 ki-
nase activity may not result in an effective strategy for patients
with KRAS-mutant cancers. Nevertheless, the potential of syn-
thetic-lethal RNAi screens should not be underestimated; they
represent a powerful strategy for identifying promising new
anticancer targets.
Materials and Methods
STK33 and Kinase Assays. Human full length STK33 containing an amino
terminal histidine tag expressed by baculovirus in Sf21 cells was purchased
from Millipore Corporation (catalog # 14-671-K, purity >93% by SDS-PAGE
and Coomassie blue staining). Myelin basic protein (bovine) was purchased
from Millipore Corporation (catalog # 13104). ATP, ADP, 4-Morpholine-
propanesulfonic acid (MOPS), MgCl2, Brij-35, glycerol, 2-mercaptoethanol,
and BSA were purchased from Sigma-Aldrich. The 384-well general plates
were purchased from VWR (Corning 3570); 384-well low volume plates
were purchased from Greiner Bio-One (384W SV, HiBase, PS, LUMITRAC
200, Medium Binding, 30 μLwell, catalog # 784075). CyBi®-Well vario
was purchased from CyBio AG. CyBi tips were purchased from CyBio AG
(CyBi-Tip Trays 384 standard; catalog # OL 3800-25-513-N). HTRF Transcreener
ADP assay was purchased from Cis-bio US (catalog # 62ADPPEC); ADP-Glo
assays were purchased from Promega Corporation (catalog # V9103). The
Envision 2012 multilabel reader was purchased from PerkinElmer, Inc. Multi-
drop Combi reagent dispenser and cassettes were purchased from Thermo
Fisher Scientific, Inc.
Kinase reactions were performed under 10 mM MOPS-NaOH (pH 7.0),
10 mM MgCl2, 0.3 mM EDTA, 0.001% Brij-35, 0.5% glycerol, 0.01% 2-mercap-
toethanol, and 0.1mgmL BSA. The enzyme concentration (STK33) and
substrate concentration (MBP and ATP) were varied depending on the
experiment. Reactions were initiated by the addition of ATP (final concentra-
tion 25 500 μM depending on experiments) and incubated at 30 °C or
room temperature for the indicated time. For additional details, see
SI Appendix,SI Materials and Methods.
Mass Spectrometric Assessment of Phosphorylation State of MBP and STK33
for In Vitro Assays. For MBP, assay conditions were as described in STK33
and Kinase Assaysand SI Appendix, Fig. S1. For STK33, 1 μg of total
STK33 was incubated with 100 μM ATP, in the absence or presence of
the concentration of fasudil analog indicated in Fig. 1. The reaction was
terminated by the addition of SDS-PAGE loading buffer containing 1% SDS
containing 10 mM DTT and heating to 95 °C for 5 min. Samples were reduced
at room temperature for 30 min and cysteines were subsequently alkylated
with 30 mM iodoacetamide for 30 min. SDS-PAGE separation and gel band
sample preparation was done as described (10). Liquid Chromatography
Mass spectrometry (LCMS) was performed on an Orbitrap mass spectrometer
(ThermoFisher Scientific) coupled to a nano-flow chromatography system
(Agilent 1100), as described (11). For each survey scan, the top 10 most abun-
dant peptidic ions were selected for collision-induced dissociation (MS/MS
sequencing), using intelligent sampling dynamic exclusion principles (21).
The data were analyzed using SpectrumMill Proteomics Workbench (Agilent)
as described (21). Extracted ion chromatograms corresponding to the phos-
phopeptides from either MBP or STK33 were generated using XCalibur (Ther-
moFisher Scientific) (7.5ppm window width). Area under these curves
was used as the basis for quantification of the effect of the fasudil analogs
on site-specific phosphorylation. All peak areas were normalized to total
protein amount for each condition, as determined by the peak area under
a peptide that could not be phosphorylated in the assay.
Analysis of STK33 Expression in Cells. NOMO-1 and SKM-1 cells were grown in
SILAC (stable isotope labeling with amino acids in culture) heavy medium.
Recombinant STK33 was added at known concentrations in SILAC light
medium and proteins within lysates were separated on gel electrophoresis
and then analyzed by MS. Based on the light standard, the levels of endo-
genous STK33 were estimated to approximately 10 ng1.5×108cells. Similar
experiments were done using NOMO-1 cells stably expressing Flag-STK33 in
a pLenti6.2 vector, and the levels of overexpressed protein were estimated
to be approximately 10-fold higher. For the phosphorylation studies by
MS, synthetic phospho-peptides for STK33 (based on known phosphorylation
sites from the in vitro kinase assay) were added into the digest of Flag-STK33
expressed in NOMO-1 cells to estimate the detectable amounts of phospho-
STK33 in cells.
Cell Lines and Culture Conditions. NOMO-1, SKM-1, NB4, THP-1, U937,
OCI-AML3, MM1S, and RPMI-8226, were kindly provided by Gary Gilliland
(Brigham and Womens Hospital, Boston). TF-1, P31/FUJ, MOLM-16, PL-21,
EOL-1, GDM-1, Colo-205, HeyA8, OvCar-3, CaOV3, OvCar-8, and KYSE-405
cells were provided by the Broad-Novartis Cancer Cell Line Encyclopedia.
The remaining cell lines were obtained from the American Type Culture
Collection and the German Collection of Microorganism and Cell Cultures.
A
B
Fig. 3. Cell viability assay for fasudil analogs BRD8899, BRD3773, BRD9573,
and BRD3695. (Aand B). Dose-response curves of KRAS and STK33 dependent
NOMO-1 and SKM-1 cells and KRAS and STK33 independent THP-1 and U937
cells (as previously confirmed by RNAi), using CellTiter Glo as a measurement
for viability. Cells were plated in 384-well plates and treated with compounds
for 72 h. The luminescence values of each compound treatment divided by
the median of all DMSO values for each individual cell line was taken as a
measurement of relative viability. Error bars represent the standard deviation
of three replicates. KRAS mutant and KRAS wild-type cell lines are depicted in
red or blue, respectively. For 31 additional cell lines screened, see SI Appendix,
Fig. S6.
2864 www.pnas.org/cgi/doi/10.1073/pnas.1120589109 Luo et al.
All cell lines were maintained under the manufacturers recommended
standard conditions.
STK33 and KRAS Knock Down. Generation of virus, transduction, and selection
of cells was done as previously described (9). In brief, cells were transduced
with pLKO.1puromycin lentiviral shRNA vectors from the TRC shRNA library,
and selected with 4μgmL puromycin for 48 h. Cell viability was measured
at timepoints 0 and 72 h after selection. Hairpins targeting the following
sequences in STK33 were used: GCAGTTCAAGTTTCACATCTA (2,078) GAACA-
CATCATACATCTGGAA (2,079) and CTTGCCATTAACTTGCTGCTA (2,081). Hair-
pins targeting the following sequences in KRAS were used: GCAGACGT-
ATATTGTATCATT (33,259) GAGGGCTTTCTTTGTGTATTT (33,260) and CCTAT-
GGTCCTAGTAGGAAAT (33,262).
Cell Viability Assays. Cells were plated in 384-well plates at optimized densi-
ties and incubated overnight prior to treatment. Compounds were added to
cells using a CyBio Well Vario after which cells were cultured under standard
condition for 72 h (unless stated otherwise). Cell viability was measured using
CellTiter Glo luminescence (Promega) and readout using an LJL Biosystems
Analyst microplate reader. Raw numbers were the normalized to the median
of the DMSO treated wells for each plate.
Protein Assays and Western Blotting. Whole-cell lysates were prepared using a
lysis buffer from Cell Signaling Technology (# 9803) and subjected to Western
blotting according to standard procedures. The membranes were developed
using a LI-COR Odyssey Infrared Imaging system, and the images were ana-
lyzed by Adobe Photoshop and ImageJ softwares. The following antibodies
were used: anti-MST4 (Cell Signaling, #3822), anti-ezrin (Cell Signaling,
#3245), anti-phospho-ezrin (Cell Signaling, #3141), anti-phospho-ERK (Cell
Signaling, #437) and anti-ERK (Santa Cruz, #135900).
ACKNOWLEDGMENTS. We thank members of the Broad Institute Chemical
Biology Platform for high throughput screening assistance. We also thank
D. Gary Gilliland at Merck Research Laboratories and Robert J. Gould at
Epizyme for helpful advice. This study was supported by a Starr Cancer Con-
sortium grant. In addition, T.L. was supported by a grant from the National
Institute of General Medical Sciences (GM38627 awarded to S.L.S.), K.M. was
supported by the Swedish Research Council (Vetenskapsrådet), and C.S.
was supported by an Emmy Noether Fellowship from the German Research
Foundation. Support for these studies was also provided by the National
Institutes of Health (NIH) Genomics Based Drug Discovery-Driving Medical
Projects Grant RL1-GM084437 and RL1-CA133834, administratively linked
to NIH grants RL1-HG004671 and UL1-DE019585.
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A
BFig. 4. MST4 and ERK kinase activity was used as surrogate
biomarker and negative control for BRD8899, respectively.
(Aand B.) NOMO-1 cells were treated with the indicated
concentrations of staurosporine, the farnesyl transferase in-
hibitor L744 (as positive controls) or the fasudil analog
BRD8899 for 24 h. Samples were lysed and subjected to im-
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Luo et al. PNAS February 21, 2012 vol. 109 no. 8 2865
BIOCHEMISTRY
    • "In 2009, Scholl et al. discovered from an RNAi screen that KRAS-driven cancers are dependent on a gene that encodes for a serine/threonine protein kinase STK33 [58]. In 2012, Luo et al. developed a potent and selective kinase inhibitor for STK33 which failed to reproduce the synthetic lethality observed in the original screen [107]. It can be argued that this is due to the fact that STK33 has other non-kinase functions that are critical to the viability of KRAS-driven cancer cells, for example protein scaffolding . "
    [Show abstract] [Hide abstract] ABSTRACT: Lung cancer is a heterogeneous disease consisting of multiple histological subtypes each driven by unique genetic alterations. Despite the development of targeted therapies that inhibit the oncogenic mutations driving a subset of lung cancer cases, there is a paucity of effective treatments for the majority of lung cancer patients and new strategies are urgently needed. In recent years, the concept of synthetic lethality has been established as an effective approach for discovering novel cancer-specific targets as well as a method to improve the efficacy of existing drugs which provide partial but insufficient benefits for patients. In this review, we discuss the concept of synthetic lethality, the various types of synthetic lethal interactions in the context of oncology and the approaches used to identify these interactions, including recent advances that have transformed the ability to discover novel synthetic lethal combinations on a global scale. Lastly, we describe the specific synthetic lethal interactions identified in lung cancer to date and explore the pharmacological challenges and considerations in translating these discoveries to the clinic.
    Full-text · Article · Dec 2016
    • "Additionally, transcription factors such as GATA2 [65,66] are largely considered undruggable. A potentially druggable hit, serine/threonine protein kinase 33 (STK33), was initially considered a tractable target [67]; however, follow-up studies have determined that both genetic depletion and pharmacological inhibition of STK33 has no effect on cell growth [68]. Likewise, genetic and pharmacological validation of the hit TBK1 [69] found no reproducible requirement for TBK1 in the growth of KRAS-mutant tumor cell lines in vitro [70]. "
    [Show abstract] [Hide abstract] ABSTRACT: Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest cancers with a dismal 7% 5-year survival rate and is projected to become the second leading cause of cancer-related deaths by 2020. KRAS is mutated in 95% of PDACs and is a well-validated driver of PDAC growth and maintenance. However, despite comprehensive efforts, an effective anti-RAS drug has yet to reach the clinic. Different paths to inhibiting RAS signaling are currently under investigation in the hope of finding a successful treatment. Recently, direct RAS binding molecules have been discovered, challenging the perception that RAS is an “undruggable” protein. Other strategies currently being pursued take an indirect approach, targeting proteins that facilitate RAS membrane association or downstream effector signaling. Unbiased genetic screens have identified synthetic lethal interactors of mutant RAS. Most recently, metabolic targets in pathways related to glycolytic signaling, glutamine utilization, autophagy, and macropinocytosis are also being explored. Harnessing the patient’s immune system to fight their cancer is an additional exciting route that is being considered. The “best” path to inhibiting KRAS has yet to be determined, with each having promise as well as potential pitfalls. We will summarize the state-of-the-art for each direction, focusing on efforts directed toward the development of therapeutics for pancreatic cancer patients with mutated KRAS.
    Full-text · Article · Apr 2016
    • "Our models raise the possibility that STK33 arose in the original knockdown screen and failed in the pharmacologic follow-up because of its mechanism of autoregulation (Luo et al., 2012; Scholl et al., 2009; Weı¨werWeı¨wer et al., 2012). Indeed, STK33 continues to autophosphorylate in vitro at inhibitor concentrations >50-fold higher than the halfmaximal inhibitory concentration (IC 50 ) for trans-substrate phosphorylation (Luo et al., 2012). An STK33-like knockdown/inhibitor discrepancy has also been described recently for ATM (Lee et al., 2015 ), a protein kinase whose activity is governed by autophosphorylation (Bakkenist and Kastan, 2003). "
    [Show abstract] [Hide abstract] ABSTRACT: Chemical inhibition and genetic knockdown of enzymes are not equivalent in cells, but network-level mechanisms that cause discrepancies between knockdown and inhibitor perturbations are not understood. Here we report that enzymes regulated by negative feedback are robust to knockdown but susceptible to inhibition. Using the Raf–MEK–ERK kinase cascade as a model system, we find that ERK activation is resistant to genetic knockdown of MEK but susceptible to a comparable degree of chemical MEK inhibition. We demonstrate that negative feedback from ERK to Raf causes this knockdown-versus-inhibitor discrepancy in vivo. Exhaustive mathematical modeling of three-tiered enzyme cascades suggests that this result is general: negative autoregulation or feedback favors inhibitor potency, whereas positive autoregulation or feedback favors knockdown potency. Our findings provide a rationale for selecting pharmacologic versus genetic perturbations in vivo and point out the dangers of using knockdown approaches in search of drug targets.
    Full-text · Article · Feb 2016
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