1. Cancer Biology
  2. Cell Biology
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MELK is not necessary for the proliferation of basal-like breast cancer cells

  1. Hai-Tsang Huang
  2. Hyuk-Soo Seo
  3. Tinghu Zhang
  4. Yubao Wang
  5. Baishan Jiang
  6. Qing Li
  7. Dennis L Buckley
  8. Behnam Nabet
  9. Justin M Roberts
  10. Joshiawa Paulk
  11. Shiva Dastjerdi
  12. Georg E Winter
  13. Hilary McLauchlan
  14. Jennifer Moran
  15. James E Bradner
  16. Michael J Eck
  17. Sirano Dhe-Paganon
  18. Jean J Zhao  Is a corresponding author
  19. Nathanael S Gray  Is a corresponding author
  1. Dana-Farber Cancer Institute, United States
  2. Harvard Medical School, United States
  3. University of Dundee, United Kingdom
  4. Novartis Institutes for Biomedical Research, United States
Research Article
Cite this article as: eLife 2017;6:e26693 doi: 10.7554/eLife.26693
16 figures, 4 tables, 2 data sets and 3 additional files

Figures

Figure 1 with 1 supplement
Biochemical characterization of HTH-01-091 and other MELK inhibitors.

(A) The chemical structures of all MELK inhibitors used in this study. (B) Overlaid histograms comparing the percent remaining kinase activities of 140 kinases (ICKP panel) when treated with OTSSP167 (1 μM) versus HTH-01-091 (1 μM). The kinases are ranked and therefore in different orders between the two compounds. See also Figure 1—figure supplement 1, Figure 1—source data 1 and Table 2 for more inhibitor selectivity and potency data. (C) Crystal structure of MELK(2-333) in complex with HTH-01-091. (PDB accession code: 5TWL). (D) Crystal structure of MELK(1-340) in complex with MRT199665. (PDB accession code: 5TX3).

https://doi.org/10.7554/eLife.26693.002
Figure 1—source data 1

Kinase profiling of OTSSP167 and HTH-01-091 by the International Center for Kinase Profiling (ICKP).

https://doi.org/10.7554/eLife.26693.004
Figure 1—figure supplement 1
KINOMEscan TREEspot analysis of MELK inhibitors.

(A) Kinase profile of JW-7-25-1 at 10 μM (KINOMEscan, 353 kinases). (B) Kinase profile of HTH-01-091 at 1 μM (KINOMEscan, 456 kinases). (C) Kinase profile of OTSSP167 at 10 μM (KINOMEscan, 456 kinases). The circle that represents MELK is in blue while all other bound kinases are in red. The S(35) score indicates the percentage of kinases within the panel that demonstrate <35% retention on the solid support after inhibitor treatment.

https://doi.org/10.7554/eLife.26693.003
Figure 2 with 1 supplement
HTH-01-091 is cell permeable, causes MELK degradation, but demonstrates poor antiproliferative effects in basal-like breast cancer cell lines.

(A) Immunoblots analyzing the pull-down levels of MELK and ERK1/2 by ATP-biotin probes and streptavidin beads from MDA-MB-468 cells treated with different doses of HTH-01-091 and 10 mM MG132 for 1 hour. (B) Immunoblots for MELK and α-tubulin after treatment of MDA-MB-468 cells with 1 μM of OTSSP167, MRT199665 or 10 μM of HTH-01-091, MELK-T1 and NVS-MELK8a for specified amount of time. See also Figure 2—figure supplement 1 for rescue experiments using carfilzomib and MLN4924. (C) 3-day antiproliferation assays of HTH-01-091 and other MELK inhibitors tested in MDA-MB-468 (basal) and ZR-75-1 (luminal) cells. Values represent two independent experimental mean ±SD. Error bars shorter than the height of the symbol are not drawn. Potency (MELK): ***IC50 <1 nM, **IC50 <5 nM, *IC50 <20 nM, measured by Z’LYTE biochemical assay. See also Tables 3 and 4 for a summary of antiproliferative IC50 values in a panel of breast cancer cell lines at 3 and 7 days post treatment, respectively.

https://doi.org/10.7554/eLife.26693.007
Figure 2—figure supplement 1
MELK inhibitor-induced MELK degradation is dependent on the proteosome, but not the cullin-RING ubiquitin ligase family.

Immunoblots for MELK and GAPDH after pretreatment of MDA-MB-468 cells with carfilzomib (400 nM) or MLN4924 (1 μM) for 4 hours, followed by treatment of 1 μM of OTSSP167, MRT199665 or 10 μM of HTH-01-091 for 4 hours.

https://doi.org/10.7554/eLife.26693.008
Figure 3 with 2 supplements
WT and MELK−/− MDA-MB-468 cells have similar growth rates, cell cycle distribution, and sensitivity to MELK inhibitors.

(A) Immunoblots for MELK and GAPDH in WT (clone E9) and MELK−/− (clone C7) clones isolated from MDA-MB-468 cells transfected with sgMELK-3 and Cas9. See also Figure 3—figure supplements 1 and 2, and Figure 3—source data 1 for the details of the generation of MELK−/− clones. (B) 5-day proliferation curves comparing the growth rates of parental, WT, and MELK−/− MDA-MB-468 cells. Values represent two independent experimental mean ±SD. (C) Cell cycle analysis comparing WT and MELK−/− MDA-MB-468 cells. Values represent triplicate mean ±SD. (D) 3-day proliferation assays comparing the sensitivity of WT and MELK−/− MDA-MB-468 cells to OTSSP167, HTH-01-091 and NVS-MELK8a. Values represent duplicate mean ±SD. Error bars shorter than the height of the symbol are not drawn.

https://doi.org/10.7554/eLife.26693.011
Figure 3—source data 1

Genotype analysis of single cell clones selected from MDA-MB-468 cells transfected with Cas9/sgMELK-3.

https://doi.org/10.7554/eLife.26693.014
Figure 3—figure supplement 1
CRISPR/Cas9-mediated knockout of MELK in MDA-MB-468 cells.

(A) Tracking of Indels by Decomposition (TIDE) analysis (Brinkman et al., 2014) of MDA-MB-468 cells transfected with Cas9/sgMELK-3 and enriched by the transfection marker OFP. (B) Immunoblots for MELK and VINC in MDA-MB-468 cells transfected with PX458 or Cas9/sgMELK-3 and enriched by the transfection marker GFP or OFP, respectively. (C) Immunoblots for MELK and VINC in ten MDA-MB-468 clones selected from MDA-MB-468 cells transfected with Cas9/sgMELK-3. Clone E9 is the WT clone, and clone C7 is the MELK−/− clone shown in Figure 3. For the genotyping results, see also Figure 3—source data 1.

https://doi.org/10.7554/eLife.26693.012
Figure 3—figure supplement 2
Genotype analysis of cell clones selected from MDA-MB-468 cells transfected with Cas9/sgMELK-3.

Trace images of the sequence around exon 5 of MELK and the actual sequences of the PCR amplicons from three clones isolated from MDA-MB-468 cells transfected with Cas9/sgMELK-3, including clone (A) E9, (B) C7 and (C) C9.

https://doi.org/10.7554/eLife.26693.013
Figure 4 with 3 supplements
MELK−/− MDA-MB-468-FKBP12F36V-MELK(sg3R) cells grow normally in response to pharmacologically induced FKBP12F36V-MELK degradation.

(A) Chemical structure of heterobifunctional dTAG molecule dTAG-47. See also Figure 4—figure supplement 3 for the chemical structures of dTAG-7, dTAG-13 and dTAG-36. (B) Immunoblots for MELK and GAPDH after treatment of dTAG-7, 13, 36, and 47 at indicated concentrations in MELK−/− MDA-MB-468-FKBP12F36V-MELK(sg3R) cells for 4 hours. See Figure 4—figure supplement 1 for details of the generation of MELK−/− MDA-MB-468-FKBP12F36V-MELK(sg3R) clones. (C) Same as in 4B, but with treatment at 500 nM and extended treatment times for 14, 24, 48, and 72 hours. (D) Changes in abundance of 7270 proteins (peptide count ≥2) comparing MELK−/− MDA-MB-468-FKBP12F36V-MELK cells treated with dTAG-7 (250 nM) or DMSO for 1 hour, versus p-value (dTAG-7: triplicate, DMSO: duplicate, limma moderated t-test). See also Figure 4—source datas 1 and 2 for the original and the processed data. (E) Crystal violet staining image showing parental MDA-MB-468 cells and MELK−/− MDA-MB-468-FKBP12F36V-MELK(sg3R) cells after treatment with DMSO or 500 nM of dTAG-47 for 9 days. Immunoblots showing MELK and GAPDH from a duplicate plate on Day 9 confirmed sustained depletion of FKBP12F36V-MELK(sg3R). These are representative data from one of two independent experiments.

https://doi.org/10.7554/eLife.26693.015
Figure 4—source data 1

Raw integrated ion intensities from quantitative multiplexed proteomics.

https://doi.org/10.7554/eLife.26693.019
Figure 4—source data 2

Processed Data and Statistical Analysis by limma (topTable).

https://doi.org/10.7554/eLife.26693.020
Figure 4—figure supplement 1
Single clones selected from MDA-MB-468-FKBP12F36V-MELK(sg3R) cells subject to CRISPR/Cas9-mediated knockout of endogenous MELK.

Immunoblots for MELK and GAPDH in single cell clones selected from MDA-MB-468-FKBP12F36V-MELK(sg3R) transfected with Cas9/sgMELK-3 to knockout endogenous MELK. Clone 1D4 is clone 1 and clone 1A3 is clone 2 in Figure 4. Clone 1D4 was confirmed with heterozygous −1 and +1 indels; clone 1A3 was confirmed with homozygous −1 deletions.

https://doi.org/10.7554/eLife.26693.016
Figure 4—figure supplement 2
FKBP12F36V-MELK(sg3R) recapitulates two phenotypes of endogenous MELK to suggest preserved functions.

(A) Immunoblots for MELK, cyclin B1 and GAPDH in asynchronous versus M-phase arrested MDA-MB-468 cells and MDA-MB-468-FKBP12F36V-MELK(sg3R) cells (clone 1). (B) Immunoblots for MELK and GAPDH in MDA-MB-468 cells and MDA-MB-468-FKBP12F36V-MELK(sg3R) cells (clone 1) treated with MRT199665 (1 μM) for the indicated amount of time.

https://doi.org/10.7554/eLife.26693.017
Figure 4—figure supplement 3
Chemical structures of dTAG molecules.

FKBP12F36V-binding ligands conjugated by variable linkers to thalidomide or pomalidomide, including (A) dTAG-7, (B) dTAG-13 and (C) dTAG-36 are capable of inducing degradation of FKBP12F36V-tagged proteins of interest.

https://doi.org/10.7554/eLife.26693.018
Figure 5 with 2 supplements
MELK-targeting shRNAs may induce antiproliferation of MDA-MB-468 cells through off-target activities while CRISPRi-mediated MELK knockdown does not affect proliferation.

(A) Immunoblots for MELK and GAPDH after 2-day treatment with or without doxycycline (100 ng/mL) in MDA-MB-468 cells transduced with doxycycline-inducible shRNA constructs. NT-1 represents a non-targeting control. Hairpins shMELK-1, 2, and 5 target three different regions within MELK’s coding region and 3’-UTR. See also Figure 5—figure supplement 1. (B) A crystal violet staining image of MDA-MB-468 cells transduced with doxycycline-inducible shRNA constructs after treatment with or without doxycycline (100 ng/mL) for 9 days with an initial seeding of 10,000 cells in a 12-well plate. (C) A crystal violet staining image of MELK−/− MDA-MB-468 cells (clone C7) transduced with doxycycline-inducible shRNA constructs after treatment with or without doxycycline (100 ng/mL) for 9 days with an initial seeding of 10,000 cells in a 12-well plate. See also Figure 5—figure supplement 1E. (D) A bar graph quantifying the intensity of crystal violet staining of cells treated as described in B and C. Values from the doxycycline treated groups are normalized to the untreated. Values represent mean ±SD from two independent experiments (*p≤0.05; **p≤0.01; ***p≤0.001, two-tailed Student’s t-Test). (E) A scheme that depicts the guide sequences that target the transcription start site of MELK and were tested in our study. See also Figure 5—figure supplement 2A. (F) An H1-based doxycycline-inducible sgRNA construct was modified from tet-pLKO-puro, which constitutively coexpresses tetR (tet repressor protein). TRE, tet response element. (G) Immunoblots for MELK and GAPDH after 2-day treatment with or without doxycycline (100 ng/mL) in MDA-MB-468-KRAB-dCas9 cells transduced with doxycycline-inducible sgRNA constructs. See also Figure 5—figure supplement 2B. (H) A crystal violet staining image of MDA-MB-468-KRAB-dCas9 cells transduced with doxycycline-inducible sgRNA constructs after treatment with or without doxycycline (100 ng/mL) for 9 days with an initial seeding of 10,000 cells in a 12-well plate. See also Figure 5—figure supplement 2C. (I) A bar graph quantifying the intensity of crystal violet staining of cells treated as described in H. Values from the doxycycline treated groups are normalized to the untreated. Values represent mean ±SD from two independent experiments (for n.s., p>0.05, two-tailed Student’s t-Test).

https://doi.org/10.7554/eLife.26693.021
Figure 5—figure supplement 1
Validating MELK-targeting short hairpins.

(A) Relative expression of MELK mRNA levels quantified by RT-qPCR in MDA-MB-468 cells after 2-day doxycycline-induced (100 ng/mL) shRNA expression. Values represent triplicate means from a single experiment. NT-1 is a non-targeting control. Hairpins shMELK-1, 2, 3, 4, and 5 (abbreviated as sh1, 2, 3, 4, and 5) target five different regions within the coding region and 3’-UTR of MELK. (B) Immunoblots for MELK and GAPDH after 2-day treatment with or without doxycycline (100 ng/mL) in MDA-MB-468 cells transduced with doxycycline-inducible shRNAs. NT-1 and NT-2 represent two non-targeting controls. (C) Crystal violet staining image of MDA-MB-468 cells transduced with doxycycline-inducible shRNA constructs after treatment with or without doxycycline (100 ng/mL) for 8 days. The bar graph represents quantification of the staining intensities in the doxycycline treated groups relative to the untreated groups. Values came from a single experiment. (D) Relative expression of MELK mRNA levels quantified by RT-qPCR in MDA-MB-468 cells after 8-day doxycycline-induced (100 ng/mL) shRNA expression. After 8-day induced expression of shMELK-1, 2 or 4, MDA-MB-468 cells were mostly dead and no data are shown. Values represent triplicate means from a single experiment. (E) Immunoblots for MELK and GAPDH after 2-day treatment with or without doxycycline (100 ng/mL) in WT and MELK−/− MDA-MB-468 cells transduced with doxycycline-inducible shRNAs.

https://doi.org/10.7554/eLife.26693.022
Figure 5—figure supplement 2
Validating MELK knockdown mediated by CRISPR interference.

(A) Relative expression of MELK mRNA levels quantified by RT-qPCR in MDA-MB-468 cells stably expressing KRAB-dCas9 and MELK transcription start site-targeting sgRNAs. Values represent duplicate means from two independent experiments. Short-guide RNA sgNT2 is a non-targeting control that targets EGFP. Primer pair 1 and 2 for RT-qPCR are listed in Supplementary file 2. (B) Relative expression of MELK mRNA levels quantified by RT-qPCR in MDA-MB-468-KRAB-dCas9 cells after 2-day doxycycline-induced (100 ng/mL) sgRNA expression. Values represent duplicate means from two independent experiments. (C) Relative expression of MELK mRNA levels quantified by RT-qPCR in MDA-MB-468-KRAB-dCas9 cells after 9-day doxycycline-induced (100 ng/mL) sgRNA expression. Values represent duplicate means from two independent experiments.

https://doi.org/10.7554/eLife.26693.023
Chemical structure 1
Ethyl 6-chloro-4-((trans-4-((dimethylamino)methyl)cyclohexyl)amino)quinoline-3-carboxylate.
https://doi.org/10.7554/eLife.26693.024
Chemical structure 2
Preparation of (6-chloro-4-((trans-4-((dimethylamino)methyl)cyclohexyl)amino)quinolin-3-yl)methanol.
https://doi.org/10.7554/eLife.26693.025
Chemical structure 3
6-chloro-3-(((2,4-dimethoxybenzyl)amino)methyl)-N-(trans-4-((dimethylamino)methyl)cyclohexyl)quinolin-4-amine.
https://doi.org/10.7554/eLife.26693.026
Chemical structure 4
Preparation of 9-chloro-3-(2,4-dimethoxybenzyl)−1-(trans-4-((dimethylamino)methyl)cyclohexyl)−3,4-dihydropyrimido[5,4 c]quinolin-2(1H)-one.
https://doi.org/10.7554/eLife.26693.027
Chemical structure 5
Preparation of 9-(3,5-dichloro-4-hydroxyphenyl)−1-(trans-4-((dimethylamino)methyl)cyclohexyl)−3,4-dihydropyrimido[5,4 c]quinolin-2(1H)-one (HTH-01-091).
https://doi.org/10.7554/eLife.26693.028
Chemical structure 6
tert-butyl (8-((2-(2,6-dioxopiperidin-3-yl)−1,3-dioxoisoindolin-4-yl)amino)octyl)carbamate.
https://doi.org/10.7554/eLife.26693.029
Chemical structure 7
4-((8-aminooctyl)amino)−2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione.
https://doi.org/10.7554/eLife.26693.030
Chemical structure 8
(2S)-(1R)−3-(3,4-dimethoxyphenyl)−1-(2-(2-((8-((2-(2,6-dioxopiperidin-3-yl)−1,3-dioxoisoindolin-4-yl)amino)octyl)amino)−2-oxoethoxy)phenyl)propyl 1-((S)−2-(3,4,5-trimethoxyphenyl)butanoyl)piperidine-2-carboxylate (dTAG-36).
https://doi.org/10.7554/eLife.26693.031
Chemical structure 9
tert-butyl (8-((2-(2,6-dioxopiperidin-3-yl)−1,3-dioxoisoindolin-5-yl)amino)octyl)carbamate.
https://doi.org/10.7554/eLife.26693.032
Chemical structure 10
5-((8-aminooctyl)amino)−2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione trifluoroacetate.
https://doi.org/10.7554/eLife.26693.033
Chemical structure 11
(2S)-(1R)−3-(3,4-dimethoxyphenyl)−1-(2-(2-((8-((2-(2,6-dioxopiperidin-3-yl)−1,3-dioxoisoindolin-5-yl)amino)octyl)amino)−2-oxoethoxy)phenyl)propyl 1-((S)−2-(3,4,5-trimethoxyphenyl)butanoyl)piperidine-2-carboxylate (dTAG-47).
https://doi.org/10.7554/eLife.26693.034

Tables

Table 1
Biochemical IC50 values of MELK inhibitors.
https://doi.org/10.7554/eLife.26693.005
Biochemical IC50 (nM)
MELK*PIK3CAmTOR*GSK3A*CDK7
JW-7-25-15.05.512.363.4
HTH-01-09110.596263217401230
OTSSP1670.566.535.71.649.1
MRT1996651.4–3.3
NVS-MELK8a11.9
MELK-T113.5
  1. *Kinase activity measured by Z’-LYTE assay at [ATP]=apparent Km, in accordance with Z’-LYTE Screening Protocol and Assay Conditions provided by Life Technologies.

    Kinase activity measured by Adapta assay at [ATP]=apparent Km, in accordance with Adapta Screening Protocol and Assay Conditions provided by Life Technologies.

Table 2
Enzymatic IC50 values of the main targets of HTH-01-091 measured by the International Center for Kinase Profiling (ICKP).
https://doi.org/10.7554/eLife.26693.006
Kinase% activity remaining (1 µM)Enzymatic IC50* (nM)
PIM31
PIM1160.6
PIM22
RIPK2342.5
DYRK3541.8
SmMLCK8108.6
CLK211
MELK1315.3
HIPK213
DAPK115
  1. *Radioactive filter-binding assay provided by the ICKP.

Table 3
3-day antiproliferative activities of MELK inhibitors in a panel of breast cancer cell lines*.
https://doi.org/10.7554/eLife.26693.009
3-day antiproliferative IC50 (μM)Note
SubtypeBasal-likeLuminal
Cell lineMDA-MB-468BT-549HCC70ZR-75-1MCF7T-47DMELK IC50 (nM)Selectivity
HTH-01-0914.006.168.80>108.753.8710.5Good
OTSSP1670.0140.0210.0340.0550.0350.1060.5Poor
MRT1996650.580.400.394.830.445.891.4–3.3Poor
NVS-MELK8a5.418.055.99>106.06>1011.9Good
MELK-T1>10>1013.5Good
  1. *IC50 values were estimated based on ‘log(inhibitor) vs. normalized response -- Variable slope’ using GraphPad Prism 7. Experiments were performed in duplicates.

Table 4
7-day antiproliferative activities of MELK inhibitors in a panel of breast cancer cell lines*.
https://doi.org/10.7554/eLife.26693.010
7-day antiproliferative IC50 (μM)
Basal-likeLuminal
Cell lineMDA-MB-468MDA-MB-468
(second source)
BT-549HCC70MCF7T-47D
HTH-01-0912.7110.72.822.434.130.78
OTSSP1670.0120.0330.0090.0210.0270.009
MRT1996650.160.910.310.0750.110.62
NVS-MELK8a2.968.44.984.172.814.90
  1. *IC50 values were estimated based on ‘log(inhibitor) vs. normalized response -- Variable slope’ using GraphPad Prism 7. Experiments were performed in duplicates.

Data availability

The following data sets were generated
  1. 1
  2. 2
    Structure of Maternal Embryonic Leucine Zipper Kinase
    1. Hyuk-Soo Seo
    2. Hai-Tsang Huang
    3. Nathanael S Gray
    4. Sirano Dhe-Paganon
    (2017)
    Publicly available at the RCSB Protein Data Bank (accession no. 5TWL).

Additional files

Supplementary file 1

Crystallization conditions and data collection and refinement statistics for crystal structures.

https://doi.org/10.7554/eLife.26693.035
Supplementary file 2

Oligonucleotides and primers.

The oligos and primers necessary for all cloning, genotyping and RT-qPCR work in this study are listed.

https://doi.org/10.7554/eLife.26693.036
Transparent reporting form
https://doi.org/10.7554/eLife.26693.037

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