Selective PKCδ Inhibitor B106 Elicits Uveal Melanoma Growth Inhibitory Effects Independent of Activated PKC Isoforms

Article abstract of DOI:10.1021/acschembio.8b00292

In uveal melanoma (UM) cells, the protein kinase C (pathway) is almost generally constitutively activated as a result of an activating mutation in either the GNAQ or the GNA11 G-protein. A pan-PKC inhibitor, sotrastaurin (also named AEB071), is in clinica

Full text of DOI:10.1021/acschembio.8b00292

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Correspondence/Rebuttal
Selective PKC# inhibitor B106 elicits uveal melanoma growth
inhibitory effects independent of activated PKC isoforms
Renier Heijkants, Amina Teunisse, Jelle de Vries, Huib Ovaa, and Aart Jochemsen
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00292 • Publication Date (Web): 07 Dec 2018
Downloaded from http://pubs.acs.org on December 12, 2018
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A
MEL202
MEL290
FM6
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Sotrastaurin
GFX
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GFX
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GFX
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B106
B106
B106
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μM
μM
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DMSO
1 µM Sotrastaurin
sG1: 4.9
1 µM B106
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sG1: 0.8
G1: 53.0
S: 17.1
sG1: 5.0
G1: 14.0
S: 6.6
G1: 91.4
S: 1.6
G2/M: 7.0
G2/M: 29.9
G2/M: 79.5
sG1: 0.9
G1: 66.3
S: 12.8
sG1: 0.7
G1: 66.2
S: 11.5
sG1: 1.3
G1: 22.1
S: 5.1
G2/M: 21.0
G2/M: 22.3
G2/M: 72.8
sG1: 1.5
G1: 40.8
S: 20.3
sG1: 10.7
G1: 5.4
S: 8.3
sG1: 1.6
G1: 39.7
S: 19.7
G2/M: 38.9
G2/M: 86.3
G2/M: 40.5
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C
MEL202
MEL290
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FM6
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p-PKCδ
p-PKCδθ
p-MARCKS
p-JNK
Vinculin
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Selective PKCδ inhibitor B106 elicits uveal melanoma growth inhibitory effects independent of
activated PKC isoforms
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Renier Heijkants, Amina Teunisse, Jelle de Vries, Huib Ovaa and Aart Jochemsen.
Department of Cell and Chemical Biology.
Leiden University Medical Center.
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Leiden, the Netherlands.
Abstract
In uveal melanoma (UM) cells the protein kinase C (pathway) is almost generally constitutively
activated as a result of an activating mutation in either the GNAQ or the GNA11 Gꢀprotein. A panꢀPKC
inhibitor, Sotrastaurin (also named AEB071), is in clinical trials for treatment of UM patients with
limited success and eliciting adverse effects. Interestingly, genetic interference with expression of just
one PKC isoform, e.g. PKCδ, is sufficient to reduce UM cell proliferation. Therefore, we tested the
effect of a recently described specific PKCδ inhibitor, B106, on growth and survival of UM cell lines.
Surprisingly, we found that B106 efficiently induced apoptosis in several cell lines, but apparently
independent of activated PKCδ.
Uveal melanoma (UM) is a lethal ocular malignancy, which is driven by oncogenic mutations in the αꢀ
subunits of Gꢀproteins GNAQ or GNA11 2. Activated GNAQ/11 are known to feed into various
signaling pathways, including the activation of protein kinase C (PKC) isoforms 3-6. Several studies
have shown that activated PKCs are essential for UM cell proliferation 7, 8. These results have spurred
the use of panꢀPKC inhibitors in the clinic to treat UM. Half of the patients show disease stabilization
1,
9
upon PKC inhibition, unfortunately all patients show adverse effects . It has been demonstrated by
genetic means that UM cells expression mutant GNAQ or GNA11 are, among other PKC isoforms,
dependent on expression PKCδ 3, 7, 8, 10. These results indicated that targeting of a single PKC isoform,
e.g. PKCδ, by a specific small molecule compound rather than the panꢀPKC inhibitor approach, could
yield a similar therapeutic effect with potentially less adverse effects.
We noted that Takashima et al. (2014) reported the development of a selective PKCδ inhibitor named
B106, which very efficiently induced apoptosis in cutaneous melanoma cell lines 11
.
We reasoned that this particular inhibitor could be regarded as an interesting therapeutic potential for
UM. To investigate this possibility, we tested the growth inhibitory effect of B106 on a panel of UM cell
lines in comparison with two panꢀPKC inhibitors, GF109203X (GFX) and the clinically used
Sotrastaurin. Three of the tested cell lines contain either a GNAQ mutation (MEL202, OMM2.3) or a
GNA11 mutation (MM66). The MEL285 and MEL290 cell lines do not contain a GNAQ or GNA11
mutation, although they are still regarded as uveal melanoma cell lines 12. The three UM cell lines
MEL202, OMM2.3 and MM66 were found to be growth inhibited by B106 treatment and even showed
a greater sensitivity for B106 compared to both panꢀPKC inhibitors (Fig 1A and Supplementary Fig
1A). In accordance with earlier observations, both MEL290 and MEL285 were resistant for panꢀPKC
inhibition correlating with the lack of PKC activity 13 8. Surprisingly, these latter UM cell lines were still
sensitive for B106 treatment, despite the absence of activated PKC isoforms and absence of
phosphorylated MARCKS, a hallmark of the presence of active PKC protein(s).
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As a comparison for B106 activity we made use of the NRAS mutated cell line FM6, which was also
investigated in the study by Takashima et al. In accordance with the original study the FM6 cells were
found to be very sensitive for B106, but were not growth inhibited by either Sotrastaurin or GFX,
suggesting that activated PKC is not relevant for the proliferation of these cells (Figure 1A). To assess
the effect of the compounds on cell cycle progression we performed flow cytometry analyses.
Treatment with 1 ꢁM Sotrastaurin for 24 hours induced G1 cell cycle arrest only in those cells
containing a GNAQ or GNA11 activating mutation (MEL202, OMM2.3 and MM66; Fig 1B and
Supplementary Fig 1B). Cell cycle profiles of FM6, MEL290 and MEL285 were hardly affected by
Sotrastaurin. B106, on the other hand, induced a clear G2/Mitosis arrest in all cell lines, regardless of
PKC activity (Fig 1B and Supplementary Fig 1B). These results suggested to us that the mode of
action of B106 is independent of presence of activated PKC isoforms.
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Takashima et al. showed that the activation of cꢀJun Nꢀterminal Kinases 1 and 2 (JNK), as assessed
by increased phosphorylation is, at least partly, essential for the B106ꢀinduced apoptosis in NRAS
mutated cutaneous melanoma cells 11. To investigate the effect of B106 in comparison with
Sotrastaurin on activation of JNK the cell lines were treated with 1 ꢁM of B106 and 4 ꢁM Sotrastaurin
for 2 hours. Interestingly, both B106 and Sotrastaurin treatment increased phosphorylated JNK in all
cell lines apart from MEL290 (Figure 1C and Supplementary Figure 1C). Whereas panꢀPKC inhibition
by Sotrastaurin reduced phosphorylation/activation of specific PKCδ and PKCδ/θ as well as the PKC
phosphorylation substrate MARCKS, incubation with B106 did not reduce PKCδꢀ or MARCKS
phosphorylation and in some cell lines phosphorylation of PKCδ was even found to be increased.
These data indicate that B106 is inhibiting cell growth and survival, but that this effect can be observed
independent on the presence of activated PKCδ in the cells and apparently also independent on the
activation of JNK, as illustrated by the MEL290 cell line.
Takashima et al. show that B106 is a selective PKCδ inhibitor when compared to PKCα in a cell free
system. However, it must be noted that in this in vitro kinase assay the IC50 for PKCδ is still 50 nM 11
which is relatively high compared to 2.1 nM for Sotrastaurin 14
,
.
To obtain more insight into the modeꢀofꢀaction of B106 as a kinase inhibitor, the compound was
provided to a company to perform a kinome analysis. The effect of B106 was determined on the
activity of 366 human kinases, at two concentrations, 0.1 ꢀM and 1.0 ꢀM. Surprisingly, hardly any
kinase was inhibited by B106. MEK2 activity was reduced the most, to 56% at 1.0 ꢀM while ROCK1
and PKCθ activities were reduced to 74% and 78%, respectively. PKCδ activity was not inhibited at all
under these conditions (Supplementary tables 1 and 2). Because MEK2, which is upstream of
ERK1/2, was identified as the most inhibited kinase in this assay, we determined the effect of B106 on
7,
8
ERK1/2 phosphorylation in comparison with Sotrastaurin. As reported before,
Sotrastaurin
decreased ERK1/2 phosphorylation but B106 actually slightly increased ERK1/2 phosphorylation (data
not shown).
The result of the kinome profiling actually supports our data presented here and deꢀsubstantiates the
claim that B106 is a PKCδ specific inhibitor.
We recognize the significance and potential high clinical impact of a selective PKCδ inhibitor.
Unfortunately, it appears that B106 does not live up to the promise. Our results indicate that very likely
a modeꢀofꢀaction of B106 exists, distinct from simple kinase inhibition, yet to be elucidated.
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How to explain the strong growth inhibitory and apoptosisꢀinducing activity of B106? Part of B106 is
still similar to Rottlerin, the structure of which has been one of the starting points in the development of
B106. Although Rottlerin was originally reported as a kinase inhibitor with some specificity for PKCδ,
more recent studies highly question this specificity and one report even indicates that PKCs are
completely insensitive to Rottlerin in a cell free assay 15, 16. Soltoff indicates that among the various
effects of Rottlerin it acts as a mitochondrial uncoupler, resulting in strongly reduced ATP levels in the
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cytoplasm, depolarisation of the mitochondrial membrane affecting ROS (Reactive Oxygen Species)
15,
levels and possibly inducing apoptosis
17. Although such effects have not been investigated for
B106, it is not excluded that increased ROS levels contribute to the apoptosis inducing effects of
B106, also because ROS do activate JNK via the ASK1ꢀMKK4 pathway 18, 19. Indeed, our preliminary
data indicate that the increase in phosphorylated JNK is attenuated by incubation with NꢀacetylꢀLꢀ
cysteine, a wellꢀknown ROS scavenger (data not shown).
We think it would be a possibility to employ similar techniques and approaches as Takashima et al.
have employed but with Sotrastaurin as a starting scaffold for chemical modifications to make it more
selective for PKCδ. Alternatively, recently a few publications describe the identification of isoformꢀ
20,
specific PKC inhibitors, i.e. PKCι and PKCζ
21; possibly similar approaches could be used to
develop PKCδꢀspecific inhibitor(s) to modify Sotrastaurin to make it more selective for PKCδ.
Methods
Cell culture
MEL202, MEL290, MEL285 and OMM2.3 were cultured in a mixture of RPMI and DMEMꢀF12 (1:1
ratio), supplemented with 10% foetal calf serum (FCS) and antibiotics 22. MM66 cells were cultured in
IMDM medium, supplemented with 15% FCS and antibiotics 6. FM6 were cultured in DMEM, 10% FCS
and antibiotics.
Growth assays
Cells were seeded in triplicate, in 96ꢀwell format and incubated for 3 days with drugs as indicated. Cell
survival was determined via the Cell TitreꢀBlue Cell Viability assay (Promega); fluorescence was
measured in a micro plate reader (Victor, Perkin Elmer).
Flow cytometry
Cells were incubated for 24 hours with 1 ꢁM B106, 1 ꢁM Sotrastaurin or vehicle (DMSO) and
harvested by trypsinization. PBS was used to wash the cells twice before fixing them in ice cold 70%
ethanol at ꢀ20 ºC for at least 16 hours. Subsequently, cells were washed in PBS containing 2% FCS
and stained for 30 minutes at 37 ºC using PBS containing 2% FCS, 50 ꢁg/ml RNAse and 50 ꢁg/ml
propidium iodide (PI). Analysis was performed on BD LSR II system (BD Biosciences).
Western blot analysis
After a 2ꢀhour incubation with 1 ꢁM B106, 4 ꢁM Sotrastaurin or vehicle (DMSO) cells were lysed in
Giordano buffer (50 mM TrisꢀHCl pH 7.4, 250 mM NaCl, 0.1% Triton Xꢀ100 and 5 mM EDTA;
supplemented with phosphataseꢀ and protease inhibitors). Proteins were separated using SDSꢀPAGE
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after which the proteins were blotted onto polyvinylidene fluoride transfer membranes (Millipore,
Darmstadt, Germany). Blocking of the membranes was achieved using TBST (10 mM TrisꢀHCl pH8.0,
150 mM NaCl, 0.2% Tween 20) containing 10% milk. Membranes were incubated with the following
primary antibodies: phosphoꢀJNK (Thr183/Tyr185; Cat. #4668), phosphoꢀMARCKS (Ser152/156; Cat.
#2741), phosphoꢀPKCδ (Thr505; Cat #9374), phosphoꢀPKCδ/θ (Ser643/676; Cat. #9376) all from Cell
Signaling Technology, and Vinculin (hVINꢀ1/V9131, SigmaꢀAldrich) and appropriate HRPꢀconjugated
secondary antibodies (Jackson Laboratories). Bands were visualized by exposure to Xꢀray film using
chemoluminescence.
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Kinome analysis
To determine the effect of B106 on activity of 366 human kinases, a stock of 10 mM of B106 dissolved
in DMSO was shipped to Eurofins Pharma Discovery Services UK Limited. Effect of B106 was
determined in the KinaseProfiler service.
Preparation of B106
Unless stated otherwise, all reagents were obtained from commercial suppliers and were used without
further purification. All air or moisture sensitive reactions were performed under an argon or nitrogen
atmosphere or under a positive flow of argon or nitrogen in heat gunꢀ or vacuum ovenꢀdried
glassware. Tetrahydrofuran (THF) was obtained from an Inert (Amesbury, MA) dry solvent system
(degassed solvents delivered through activated alumina columns, positive pressure of nitrogen).
Acetonitrile (ACN) and Toluene were dried over activated 4Å molecular sieves. Deionized and
degassed water (H2O) was used for reactions. Column chromatography was performed with
prepacked silica flash cartridges (GraceResolv, Grace Davison Discovery Science) on a Buchi
Sepacore purification system (SepacoreControl 1.2 Chromatography Software, Cꢀ620 Control Unit, Cꢀ
660 Fraction Collector, Cꢀ640 UV Photometer, Cꢀ605 Pump Modules, BÜCHI Labortechnik A). Nuclear
magnetic resonance (NMR) spectra were recorded on a Bruker UltrashieldTM 300 (75.00 MHz for 13C)
using the residual solvent as internal standard (1H: δ 7.26 ppm, 13C{1H}: δ 77.00 ppm for CDCl3),
Chemical shifts (δ) are given in ppm and coupling constants (J) are annotated in Hertz (Hz).
Resonances are described as s (singlet), d (doublet), t (triplet), q (quartet), br (broad singlet) and m
(multiplet) or combinations thereof. LCꢀMS measurements were performed on a system equipped with
a Waters 2795 Separation Module (Alliance HT), Waters 2996 Photodiode Array Detector (190ꢀ750
nm) Phenomenex Kinetex C18 (2.1x50, 2.6 ꢂm) and LCTTM ESI Mass Spectrometer. Electrospray
Ionization (ESI) highꢀresolution mass spectrometry was carried out using a Waters Xevo G2 XS QTOF
instrument in positive ion mode.
Synthesis of 5ꢀbromoꢀ2ꢀ((2ꢀmethylbutꢀ3ꢀynꢀ2ꢀyl)oxy)benzaldehyde:
To a thoroughly dried 100 mL round bottomed flask containing 2ꢀmethylbutꢀ3ꢀynꢀ2ꢀol (5.54 mL, 57.2
mmol, 1.15 equiv.) dissolved in dry ACN (50 mL) at 0° C was added 1,8ꢀDiazabicyclo[5.4.0]undecꢀ7ꢀ
ene (DBU, 11.1 mL, 74.6 mmol, 1.5 equiv.). To this mixture trifluoroacetic anhydride (8.08 mL, 57.2
mmol, 1.15 equiv.) was added dropwise over a 10 minute period. The reaction was stirred at 0° C for
45 minutes before being transferred via cannula to a 250 mL round bottomed flask containing 5ꢀ
bromoꢀ2ꢀhydroxybenzaldehyde (10.0 g, 49.7 mmol, 1 equiv.), DBU (9.65 mL, 64.6 mmol, 1.3 equiv.)
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and CuCl2.2H2O (8.5 mg, 0.050 mmol, 0.001 equiv.) in ACN at ꢀ5° C. After 1,5 hours cooling was
removed and the mixture was stirred at ambient temperature for 16 hours before being concentrated
under reduced pressure at 40 °C. The resulting residue was taken up in ethyl acetate (EtOAc),
washed once with H2O, once with 1 M HCl and once with brine before being dried using Na2SO4,
filtered and concentrated. This residue was adsorbed onto Celite and subjected to silica gel flash
chromatography eluting with 0% to 50% EtOAc in heptane to yield 8.42 g (63%) of the desired product
as a yellow solid.
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1HꢀNMR (300 MHz, CDCl3) δ: 10.31 (s, 1H), 7.93 (d, J = 2.6 Hz, 1H), 7.59 (dd, J = 8.9, 2.65 Hz, 1H),
7.42 (d, J = 8.9 Hz, 1H), 2.63 (s, 1H), 1.71 (s, 6H) (Supplementary Figure 2A). 13CꢀNMR (75 MHz,
CDCl3) δ: 188.7, 157.1, 137.3, 130.7, 130.0, 122.6, 115.9, 84.4, 75.9, 74.3, 29.4. ESI+ MS: 267.0,
269.0 [M+H]+ (data not shown).
Synthesis of 6ꢀbromoꢀ2,2ꢀdimethylꢀ2Hꢀchromeneꢀ8ꢀcarbaldehyde:
5ꢀBromoꢀ2ꢀ((2ꢀmethylbutꢀ3ꢀynꢀ2ꢀyl)oxy)benzaldehyde (1.0 g; 3.7 mmol; 1.0 equiv.) was taken up in
ACN (15 mL)in a 20 mL microwave vial. Butylated hydroxytoluene (BHT, 16 mg; 0.075 mmol; 0.02
equiv.) was added and the mixture was heated at 180 °C for 20 minutes. Two more identical runs with
additional fresh catalytic amounts of BHT were performed before the crude was adsorbed onto Celite
and subjected to silica gel flash chromatography eluting with 0% to 25% EtOAc in heptane to yield 488
mg (49%) of the desired product.
1HꢀNMR (300 MHz, CDCl3) δ: 10.35 (s, 1H), 7.71 (d, J = 2.5 Hz, 1H), 7.26 (d, J = 2.5 Hz, 1H), 6.28 (d,
J = 10.0 Hz, 1H), 5.75 (d, J = 10.0 Hz, 1H), 1.49 (s, 6H) (Supplementary Figure 2B). 13CꢀNMR (75
MHz, CDCl3) δ: 187.8, 155.1, 134.1, 132.6, 129.2, 125.4, 124.3, 120.5, 113.1, 78.1, 28.1. ESI+ MS:
267.0, 269.0 [M+H]+ (data not shown).
Synthesis of 9ꢀ(2ꢀ(trifluoroꢀλ4ꢀboraneyl)ethyl)ꢀ9Hꢀcarbazole, potassium salt:
In a thoroughly dried 100 mL roundꢀbottomed flask 2,5ꢀdimethylꢀhexaꢀ2,4ꢀdiene (4.11 mL; 27.4 mmol;
5.5 equiv.) was dissolved in THF (10 mL) and the mixture was cooled to 0 °C. A 1.0 M solution of
BH3.THF complex in THF (12.9 mL; 12.9 mmol; 2.5 equiv.) was added and the mixture was stirred at 0
°C for 3 hours. Then, a solution of 9ꢀvinylcarbazole (1.0 g; 5.17 mmol; 1.0 equiv.) in THF (1 mL) was
added. The reaction mixture was allowed to warm up to room temperature and stirred for an additional
3 hours before being cooled to 0 °C followed by addition of H2O (1.7 mL; 94 mmol; 18 equiv.). The
mixture was stirred at ambient temperature for 1,5 hours before a 37% aq. formaldehyde solution (4.3
mL; 58 mmol; 11 equiv.) was added and the mixture was stirred at ambient temperature overnight.
The reaction mixture was poured into brine (50 mL) and extracted with EtOAc (75 mL). The organic
layer was separated, dried (Na2SO4), filtered and concentrated to dryness under reduced pressure at
40 °C to give 4.4 g of a colourless oil. The resulting oil was dissolved in acetone (17 mL) and H2O (6.5
mL) before the addition of KHF2 (1.62 g; 20.7 mmol; 4 equiv.). The resulting mixture was stirred at
ambient temperature for 3.5 hours before being concentrated under reduced pressure at 40 °C to give
4.4 g of a pale yellow solid. The resulting residue was crystallized from acetone and Et2O. The
obtained white solids (168 mg) were used without further purification and characterization.
Synthesis of 6ꢀ(2ꢀ(9Hꢀcarbazolꢀ9ꢀyl)ethyl)ꢀ2,2ꢀdimethylꢀ2Hꢀchromeneꢀ8ꢀcarbaldehyde:
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6ꢀbromoꢀ2,2ꢀdimethylꢀ2Hꢀchromeneꢀ8ꢀcarbaldehyde (57 mg; 0.21 mmol; 1.0 equiv.), 9ꢀ(2ꢀ(trifluoroꢀλ4ꢀ
boraneyl)ethyl)ꢀ9Hꢀcarbazole potassium salt (64 mg; 0.21 mmol; 1.0 equiv.), PdCl2(dppf).DCM (8 mg;
0.011 mmol; 0.05 equiv.) and Cs2CO3 were taken up in 1,5 mL toluene. An amount of 0.5 mL H2O was
added and the mixture was stirred overnight at 80 °C. The mixture was allowed to cool to ambient
temperature and subsequently poured into brine (20 mL) and extracted with EtOAc (25 mL).
The organic layer was collected, dried (Na2SO4), filtered and concentrated to dryness in vacuo at 40
°C to give 74 mg of an orange oil. The obtained crude was adsorbed onto Celite and subjected to
silica gel flash chromatography eluting with 0% to 50% EtOAc in heptane to yield 29 mg of the desired
product as a colourless oil.
1HꢀNMR (300 MHz, CDCl3) δ: 10.44 (s, 1H), 8.13 – 8.07 (m, 2H), 7.56 (d, J = 2.3 Hz, 1H), 7.43 (ddd, J
= 8.3, 7.1, 1.2 Hz, 2H), 7.31 (d, J = 8.2 Hz, 2H), 7.23 (ddd, J = 8.0, 7.2, 1.0 Hz, 2H), 6.73 (d, J = 2.3
Hz, 1H), 6.14 (d, J = 9.9 Hz, 1H), 5.65 (d, J = 9.9 Hz, 1H), 4.49 (t, J = 7.4 Hz, 2H), 3.04 (t, J = 7.7 Hz,
2H), 1.47 (s, 6H) (Figure 1D and Supplementary Figure 2C). 13CꢀNMR (75 MHz, CDCl3) δ: 189.2,
155.1, 140.0, 132.7, 131.6, 130.8, 126.5, 125.6, 124.1, 122.9, 122.5, 121.2, 120.3, 118.9, 108.5, 77.6,
44.6, 34.1, 28.0. HRMS [ESI+.]: found 382.1817, calculated 382.1807 for C26H23NO2 [M+H]+ (data not
shown).
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Figure Legends
Figure 1. Differences in response upon panꢀPKC inhibition and B106 incubation in GNAQ/11 mutated
and wild type cells. (A) Cell viability assay in which uveal melanoma cell lines MEL202 and MEL290
and cutaneous melanoma cell line FM6 were incubated with panꢀPKC inhibitory drugs Sotrastaurin,
GFX or selective PKCδ inhibitor B106 for 3 days. (B) Effects of 1 ꢁM Sotrastaurin and 1 ꢁM B106 after
24ꢀhour incubation on the cell cycle profile of MEL202, MEL290 and FM6. (C) Western blot analysis of
effects induced by 4 ꢁM B106 (B) and 1 ꢁM Sotrastaurin (S) compared to solvent DMSO (D) after 2ꢀ
hour incubation on the amount of phosphorylatedꢀPKCδ, ꢀPKCδθ, ꢀMARCKS and ꢀJNK. Vinculin
1
expression was analysed to ensure equal loading. (D) HꢀNMR confirmed structure of synthesized
B106.
Acknowledgements
The authors like to thank G van der Heden for carefully reviewing the manuscript and thoughtful
discussions.
Supporting Information
The Supporting Information is available free of charge at http://pubs.acs.org
- data supporting the results of Figure 1 by extending to other cell lines
- 1H-NMR conformation of B106 synthesis
- full results of Kinome Profiling of B106
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