Cervical cancer is a major cause of cancer-related mortality in women in the developing world. Cancer Stem cells (CSC) have been implicated in treatment resistance and metastases development; hence understanding their significance is important.Primary culture from tissue biopsies of invasive cervical cancer and serial passaging was performed for establishing cell lines. Variable Number Tandem Repeat (VNTR) assay was performed for comparison of cell lines with their parental tissue. Tumorsphere and Aldefluor assays enabled isolation of cancer stem cells (CSC); immunofluorescence and flow cytometry were performed for their surface phenotypic expression in cell lines and in 28 tissue samples. Quantitative real-time PCR for stemness and epithelial-mesenchymal transition (EMT) markers, MTT cytotoxicity assay, cell cycle analysis and cell kinetic studies were performed.+ phenotype. In tissue samples of untreated invasive cervical cancer, CD133+ CSCs ranged from 1.3–23% of the total population which increased 2.8-fold in radiation-resistant cases. Comparison of CD133+ with CD133− bulk population cells revealed increased tumorsphere formation and upregulation of stemness and epithelial-mesenchymal transition (EMT) markers with no significant difference in cisplatin sensitivity.Four low-passage novel cell lines designated RSBS-9, − 14 and − 23 from squamous cell carcinoma and RSBS-43 from adenocarcinoma of the uterine cervix were established. All were HPV16+. VNTR assay confirmed their uniqueness and derivation from respective parental tissue. CSC isolated from these cell lines showed CD133+ phenotype and are increased in relapsed cases and hence should be targeted for achieving remission.Low-passage cell lines developed would serve as models for studying tumor biology. Cancer Stem Cells in cervical cancer display CD133The online version of this article (10.1186/s12885-018-4237-5) contains supplementary material, which is available to authorized users. Cancer of cervix is the fourth most common cancer among women and the third most common cause of cancer related mortality worldwide . Develop+) [CSC have been demonstrated in several solid cancers. In cancer cervix, cancer stem-like cells were first identified to exist by Feng et al.  and subs+) , 6, Anne+)  and CD49+)  have all+) –8. HenceThe study was approved by the Institute’s Ethics Committee (vide letter No.IC-CCRT/07/DTM-3226 dated 16.11.2009). All patients were enrolled after obtaining a voluntary written informed consent and the study was conducted as per the Helsinki declaration (2000). Tissue samples obtained from untreated patients of invasive cervical cancer comprising of 3 radical hysterectomies in FIGO stage IIA and 30 cervical biopsies from FIGO stage IIb/III were subjected to primary culture. The sample was collected directly in Dulbecco’s Modified Eagle Medium/ Nutrient Mixture F-12 Ham  (Invitrogen). The rest of the specimen was subjected to formalin fixation and paraffin-embedded sections were evaluated light microscopically for histopathological diagnosis.2) and 95% air. 17 cases showed contamination and in 16 cases successful primary cultures could be established. In 7 of these 16 cases, long-term primary cultures got established; and 4 were pursued further. The morphology of the adherent cells was observed under an indirect microscope by phase-contrast and morphology documented.The specimen collected was immediately mechanically disaggregated with a sterile surgical blade, washed 3 times with sterile phosphate buffered saline (PBS), digested by collagenase II by incubation for 1 h at 37 °C , filtered through a 70 μm mesh to get rid of stromal fragments and obtain a cell suspension. The tumour cells were placed in 6 well plates (Corning) containing DMEM/ F12 supplemented with 10% fetal bovine serum , penicillin (100 U/mL), and streptomycin (100 μg/mL) and placed in a humidified incubator at 37 °C containing 5% carbon dioxide  equipped with XR81M-B Camera .Approximately 5 × 10Karyotyping was performed on metaphase spreads of RSBS-9, RSBS-14, RSBS-23 and RSBS-43 cells at passage 32 by standard G-banding.Cultured cells were also placed under anchorage-independent/stem-cell conditions in Ultra Low Attachment plates  in serum-free DMEM/F12 supplemented with 5 μg/mL insulin (Sigma), 20 ng/mL human recombinant epidermal growth factor , 10 ng/mL basic fibroblast growth factor , and 0.4% bovine serum albumin . Floating aggregates or tumorspheres were observed microscopically and their numbers per well documented. Efficiency of tumorsphere formation ability was calculated based on manual counting of number of tumorspheres after plating equal numbers of sorted cells wherever required.high and ALDHlow cells  as per manufacturer’s instructions detailed in Additional file 6 cells derived from tumorspheres and corresponding adherent cells were screened for surface markers by flow Cytometry using fluorescent  labelled antibodies against CD117, CD90, CD44, CD49f and CD71. Isolation of the CD133+ cells was performed using a FACS Aria flow cytometer  and analyzed further  and in 6 patients who had been treated with chemoradiation therapy 46 Gy/23 fraction external beam radiation administered concurrently with 40 mg/m2 cisplatin weekly dose followed by intracavitary brachytherapy and who had subsequently relapsed. The levels of CD133, CD49f and CD44 was evaluated in each case after gating for CD45 negative cells  was performed on the biopsies of patients with untreated invasive cervical cancer  were used to plot the growth curve by using the MTT 3--2,5-diphenyl tetrazolium bromide assay detailed in Additional file Cell cycle analysis was performed after labelling the cellular deoxyribonucleic acid (DNA) with propidium iodide (PI) . The cells were then subjected to cell cycle analysis by FACS (BD FACS Aria II). Each experiment was analysed in duplicate and four independent experiments were performed  were used to prove and validate that the cervical cancer cell lines were derived from primary cervical cancer biopsies. Amplification of VNTR loci D1S80 (range of amplification product 387 to 723 bps) was achieved using the primers  for variable duration . Cytotoxicity was determined by MTT assay All experiments were performed twice in triplicates to determine the ICOCT-4, SNAIL, SLUG, VIMENTIN, ABCG2, N-CDHERIN, NANOG, TIWST and E-CADHERIN using gene specific primers  per the manufacturer’s instructions. 1 μg RNA was reverse-transcribed into cDNA using specific primers and per the manufacturer’s instructions . 1 μL of cDNA was used as template for qRT-PCR for Successful long-term primary cultures could be established in 7/33 or 21.2% cases; four of these were pursued and 4 permanent cell lines were derived. They were designated as RSBS-9, RSBS-14, RSBS-23 and RSBS-43 respectively with the ages of the patients being 49, 34, 45 years and 63 years respectively. All 4 cell lines were derived from cervical biopsy specimen and from patients with FIGO stage III disease. All the cell lines established were regularly checked for mycoplasma contamination.The histology of the primary tumour corresponding to the RSBS-9 cell line was a moderately differentiated keratinizing squamous cell carcinoma, for RSBS-14 and RSBS-23 cell lines were non-keratinizing squamous cell carcinoma, moderately and poorly differentiated respectively. RSBS-43 cell line was derived from a moderately differentiated adenocarcinoma. The parental tissue biopsies and the respective adherent cell lines derived are shown in Fig. Electron microscopy of the cell lines derived from squamous cell carcinoma  showed high nucleo-cytoplasmic ratio with keratin filaments in the cytoplasm and cell junctions in the form of hemi-desmosomes. RSBS-43 cell line derived from adenocarcinoma cervix showed a few irregular microvilli, prominent nucleolus and prominent rough endoplasmic reticulum and occasional cell junctions. Representative images from RSBS-14 and RSBS-43 are shown in Fig. Karyotyping was done on all the four cell lines at passage 32. All 4 cell lines displayed aneuploidy. The number of chromosomes ranged from 47 to 53 in the RSBS-9, from 44 to 47 in RSBS-14, from 47 to 57 in RSBS-23 and from 47 to 53 in the RSBS-43 cell lines. The modal chromosome number was 53, 47, 56 and 51 in the RSBS-9, 14, − 23 and − 43 respectively [Additional file The VNTR assay uses multiple primer sets read as informative or non-informative. This established that the cell lines were indeed derived from the corresponding tissue sample and are unique Fig. .Fig. 3VaHPV DNA typing for HPV-16, − 18, − 31, and − 45, the most common HPV types in cervical cancer, revealed positivity only for HPV-16 sequence  [Additional file Cell cycle analysis showed 9–15% cells in the S-phase and 0.05–7.6% in subG1 phase [Additional file Cisplatin is commonly used in cervical cancer as part of the chemotherapy protocol and hence cisplatin sensitivity was determined in these cell lines [Additional file Tumorsphere formation could be demonstrated from cells derived from the primary cultures obtained from biopsy specimen of invasive cervical carcinoma, from the four novel cell lines established and from HeLa and SiHa cell lines. In these cases, tumor cells were dissociated and grown under anchorage-independent or ‘stem-cell’ conditions Floating aggregates of cells formed small spheroids on day 7 and representative images (RSBS-14 and RSBS-43) are shown in Additional file high whereas rest were ALDHlow in Hela, SiHa and four novel cell lines. A representative plot is shown in Additional file high and ALDHlow cells. Equal numbers of cells  of ALDHhigh and ALDHlow cells were cultured and tumorspheres was assessed at day 10. ALDHhigh positive cells showed higher capacity for spheroid formation [Additional file Adherent cultures from the four novel cell lines as well as SiHa and HeLa cell lines were tested by the ALDEFLUOR assay which identifies cancer stem cell population based on the high aldehyde dehydrogenase activity (ALDH) activity of cells in the presence or absence of the ALDH inhibitor DEAB. Further, these cells were sorted by flow cytometry based on high / low ALDH activity. Compared with control cells treated by the DEAB inhibitor, we observed 2.9% to 6% cells to be ALDHNext, we wished to identify the surface phenotypic expression of putative stem cell markers in the tumorspheres enriched for cancer stem cells. Initially,the expression of known stem cell markers including CD44, CD49f, CD133 and CD71, CD90, CD105 was tested by flow cytometry in the tumorspheres and adherent bulk cells in each of the 4 cell lines RSBS-9, − 14, − 23 and − 43 and also HeLa and SiHa cell lines. CD71, CD90 and CD105 did not show any differential expression and so were not pursued further. There was a differential expression of CD44, CD49f and CD133. CD44 levels were higher in adherent cells as compared to tumorshpere cells. Adherent cells showed CD49f expression in the range of 45.7–89% whereas in tumorspheres it ranged from 61.1–90.7%. Similarly CD133 positivity ranged from 1.4 to 19% percentage in adherent cells whereas it was 12.5–63.6% in corresponding tumorspheres. A representative plot in RSBS-9 cell line is shown [Additional file + is a surface phenotypic marker of cervical cancer stem cells.Direct immunofluorescence was carried out on adherent cells and tumorspheres derived from all four novel cell lines and their corresponding tissue sections. Similar results were observed in all cell lines tested and a representative image panel from RSBS-14 cell line is shown in Fig. n = 22), CD133 showed 1.3–50.6% positivity (median 10.4%); CD49f showed 3.2–91.8% positivity (median 58.4%), CD44 showed positivity in 1.2–58.1% (median 14.3%). In 6 cases who had received chemoradiation as per standard protocol but had relapsed cases within 1 year and hence represent radiation resistance or treatment failure, there was a trend towards downregulation of CD44 (median 6.2%) and upregulation of CD133 (median 28.2%). CD49f expression was very variable and no trend was observed.Expression of CD44, CD49f and CD133 in patient samples by flow cytometry are shown in Fig. + cancer stem cells were sorted from adherent cultures of RSBS 9, − 14, − 23 and − 43 cells tumorosphere formation assessed. CD133+ cells formed more and larger spheroids as compared to CD133− cells [Fig. + cancer stem cells to cisplatin determined by MTT assay. There was no appreciable difference in the CD133+ versus the CD133− cells in the response to cisplatin in all 4 cell lines tested [Fig. CD133lls Fig. . Furtherted Fig. .Fig. 6a OCT4, NANOG) and EMT markers  in CD133+ sorted cells in relation to unsorted cells and after normalization to β-ACTIN housekeeping gene was evaluated and results shown in Fig. + RSBS-9 cells showed marked upregulation of OCT-4, NANOG, ABCG-2, SLUG, SNAIL, TWIST, N CADHERIN, and VIMENTIN; however, E-CADHERIN was also upregulated. CD133+RSBS-14 cells showed upregulation of OCT-4 and NANOG, upregulated E-CADHERIN with little change in the other markers. CD133+ RSBS-23 cells showed upregulation of VIMENTIN, TWIST, SNAIL, SLUG AND N-CADHERIN, nil expression of E-CADHERIN with no change in the other markers. CD133+ RSBS-43 cells showed upregulation of OCT-4, TWIST and SNAIL and downregulation of E-CADHERIN with no appreciable change in the other transcripts. Taken together, our results indicate a variable albeit an overall upregulation of stemness and EMT markers in the CD133+ sorted cancer stem cells in all the four cell lines evaluated.Relative expression of stemness markers Additional file 2:Figure S1. Representative karyotypes with modal chromosome numbers of RSBS-9 (A), RSBS-14 (B), RSBS-23 (C) and RSBS-43 (D) respectively. (TIFF 127 kb)Additional file 3:Figure S2. A. PCR for HPV-16 in cell lines and corresponding parental tissues showing positivity for all cell lines and the parental tissues. B. Cell cycle analysis. C. Cell growth curve and estimation of doubling time showing similar doubling time of 48 h approximately. D. Cisplatin sensitivity assay showing variable chemosensitivity in the cell lines. (TIFF 192 kb)Additional file 4:Figure S3. A. Representative figure of tumorspheres from RSBS-14 and RSBS-43 cell lines on day 7. B. Aldefluor assay: Representative plots of RSBS-14 cell line showing 3.2% cells with high ALDH levels. Cell sorted into ALDH low (−) and high (+) levels and tumorsphere assay performed showing spheres in ALDHhigh sorted cells. (TIFF 223 kb)Additional file 5:Figure S4. CD133 and CD49f expression in adherent (ADH) vs Tumorspheres (SPH) in RSBS-9 cell line by Flow Cytometric Immunophenotyping. Representative plots of CD49f and CD133 expression and data represented in histograms. Both markers showed increased expression in tumorspheres as compared to adherent cells. (TIFF 123 kb)Additional file 6:Figure S5. CD49f expression in adherent cells and tumorspheres. Representative images panel of RSBS-14 cell line showing similar and moderate levels in adherent cells and in tumorspheres. (TIFF 2512 kb)


The objective of this study was to evaluate the effect of platinum-based drugs on nuclear-factor erythroid2 like 2 (NRF2) signaling in non-small cell lung cancer cell lines with or without Kelch-like ECH-associated protein 1 (KEAP1) mutations and to determine the role of NRF2 and KEAP1 on platinum-based drug treatment.We used real-time PCR to assess relative mRNA expression and used western blotting and immunofluorescence assays to assess protein expression. Small interfering RNA and shuttle plasmids were used to modulate the expression of NRF2, wild-type KEAP1, and mutant KEAP1. Drug sensitivity to platinum-based drugs was evaluated with Cell Count Kit-8.We found that platinum-based therapies modified the NRF2 signaling pathway differently in KEAP1-mutated non-small cell lung cancer (NSCLC) cell lines compared with wild-type KEAP1 cell lines. The reactive degree of NRF2 signaling also varies between nedaplatin and cisplatin. The modification of NRF2 or KEAP1 expression in NSCLC cell lines disrupted downstream gene expression and cell sensitivity to platinum-based drugs. Finally, gene expression data retrieved from The Cancer Genome Atlas (TCGA) consortium indicated that KEAP1 mutation significantly affects NRF2 signaling activity in patients with NSCLC.Our findings suggest that NRF2 signaling plays an indispensable role in NSCLC cell sensitivity to platinum-based treatments and provides a rationale for using NRF2 as a specific biomarker for predicting which patients will be most likely to benefit from platinum-based treatment. Non-small cell lung cancer (NSCLC) is associated with considerably high mortality worldwide . SurgeryNuclear-factor (NF)-E2-related factor 2 (NRF2) is a core transcription factor involved in the cellular response to oxidative stress . Under bIn urethane-induced lung carcinogenesis model, NRF2 activation suppresses tumorigenesis, but somatic activation of NRF2 in cancer cells enhanced growth of tumors . StudiesUnless otherwise stated, all chemicals were purchased from Sigma-Aldrich . Antibodies against NRF2 (ab62352) were purchased from Abcam; antibodies against HO1 (#5853) and NQO1 (#3187) were purchased from Cell Signaling Technology; and antibodies against KEAP1 (10503-2-AP), GCLM  (14241-1-AP), and β-actin were purchased from Proteintech.The A549 (human lung adenocarcinoma), H292 (human mucoepidermoid carcinoma), H460 (human large cell lung cancer), and SKMES-1 (human squamous lung cancer) cell lines were obtained from the Cell Resource Center, Peking Union Medical College. The cells were cultured in RPMI-1640, with the exception of SKMES-1 cells, which were cultured in MEM. All media were supplemented with 10 % fetal bovine serum . No antibiotics were added to the media.G333C-forward, TGATCTACACCGCGGGCTGCTACTTCCGACAGTCG333C-reverse, AGCCCGCGGTGTAGATCAGGCGGCCCACCD236H-forward, AGCCCGCGGTGTAGATCAGGCGGCCCACCD236H-reverse, GGTCCCGGCTGATGAGGGTCACCAGTTGGThe small interfering RNAs used to silence NRF2 were:Sense, GGUUGAGACUACCAUGGUUTTAntisense, AACCAUGGUAGUCUCAACCTTThe wild-type KEAP1 coding sequence (CDS) was cloned into the pEnter shuttle plasmid. The Fast Mutagenesis system  was used to introduce the G333C and D236H mutations. The primers used were:Transfection was conducted with Lipofectamine 2000 (Invitrogen) according to a previously published method .2O. Cycling conditions were as follows: 95 °C for 10 min and 40 cycles of 95 °C for 15 s and 60 °C for 60 s. Cycle thresholds were calculated as the expression fold-changes according to the 2−△△CT algorithm. The primers used for real-time PCR are:NRF2, forward, TCCAGTCAGAAACCAGTGGATReverse, GAATGTCTGCGCCAAAAGCTGHO1, forward, AAGATTGCCCAGAAAGCCCTGGACReverse, AACTGTCGCCACCAGAAAGCTGAGNQO1, forward, GAAGAGCACTGATCGTACTGGCReverse, GGATACTGAAAGTTCGCAGGGGCLM, forward, TGTCTTGGAATGCACTGTATCTCReverse, CCCAGTAAGGCTGTAAATGCTCTotal messenger RNA (mRNA) was extracted from cells using TRIzol according to the manufacturer’s protocol . Seven hundred fifty-nanogram total mRNA was used to synthesize first strand complementary DNA (cDNA) using the cDNA synthesis kit (ThermoFisher Scientific). Quantification reactions were performed with the ABI 7900 HT platform. Each reaction consisted of 5 μl SYBR Green/ROX qPCR Master Mix (ThermoFisher Scientific), 1 μl cDNA, 1 μl 10 μM forward and reverse primer mix, and 3 μl ddHFragment 1, forward AGAGGTGGTGGTGTTGCTTATReverse TGGAGATGGAGGCCGTGTAFragment 2, forward CAGGTCAAGTACCAGGATGReverse GATGAGGGTCACCAGTTGFragment 3, forward ATCGGCATCGCCAACTTCReverse AGGTAGCTGAGCGACTGTFragment 4, forward CAGAAGTGCGAGATCCTGReverse GCTCTGGCTCATACCTCTFragment 5, forward GCCCTGGACTGTTACAACReverse GTCTCTGTTTCCACATCGTAFragment 6, forward GCTGTCCTCAATCGTCTCReverse AGTTCTGCTGGTCAATCTGNRF2 exon2, forward TCGTGATGGACTTGGAGCTGReverse AGCATCTGATTTGGGAATGTGTotal RNA was extracted and used to synthesize the first chain cDNA according to the procedure described in the real-time PCR analysis description. cDNA was used as a template to synthesize six segments of the KEAP1 gene using the following cycling conditions: initial denaturing step at 94 °C for 5 min followed by 30 cycles at 94 °C for 40 s, 60 °C for 40 s, and 72 °C for 55 s. Products were separated on 1 % agarose gels, and bands were visualized with ethidium bromide. The products were sequenced using the ABI3730 XL DNA Analyzer  and analyzed with Chromas 2.4.3 software. Primers used to amplify the KEAP1 gene fragments were:Sections were spliced together after manual inspection and compared with the reference sequence with BLAST to identify potential mutations.Cytoplasm and nuclear protein were extracted by using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (ThermoFisher Scientific). For total protein extraction, we used standard protocols for protein isolation and antibody detection, as previously described , 25. CelImmunofluorescence was performed as previously reported with some modifications . Cells wv/v) CCK-8 was added to each well. Absorbance at 490 nm was measured after a 2-h incubation at 37 °C. Cell survival was calculated as the ratio between the treated group and control groups. Ratios were graphed in line charts with Graphpad Prism 6 software.Cellular proliferation was evaluated using a Cell Count Kit-8 (CCK-8) with some modifications . Lung cahttp://www.compgenome.org/TCGA-Assembler/) in an R program. KEAP1 mutation status was defined as previously described [Expression data from The Cancer Genome Atlas (TCGA) squamous lung cancer patients was retrieved using the package TCGA-Assembler  Fig.  and thatp < 0.01, **p < 0.05) and H460 , nedaplatin induced a weak but significant increase in three downstream gene expression. Cisplatin treatment, however, significantly enhanced HO1, NQO1, and GCLM expression in both A549 and H460 cell lines after 24 h of treatment.To profile the expression of NRF2 signaling pathway components in response to platinum treatment, the four NSCLC cell lines  were treated with 30 μM nedaplatin or 30 μM cisplatin for 24 h. Total RNA from each group was extracted, and HO1, NQO1, and GCLM gene expression levels were assessed with real-time PCR. In the wild-type KEAP1 cell lines H292 Fig.  and SKMETo evaluate protein levels, the four cell lines were treated with 30 μM nedaplatin or 30 μM cisplatin for 3 or 24 h. Total proteins were extracted, and HO1, NQO1, GCLM protein expression was evaluated by using western blot analysis. In the wild-type KEAP1 H292 and SKMES-1 cell lines Fig. , a mild The results of immunofluorescence analysis of HO1 protein expression were consistent with the results of the western blot analysis. Over a 24-h treatment period, 30 μM nedaplatin or cisplatin induced a mild increase in HO1 protein expression in H292 and SKMES-1 cells Fig. . In the p < 0.001, **p < 0.05) and H460 , constitutive expression of wild-type KEAP1 over a 72-h period reduced the expression of the NRF2 downstream target genes HO1, NQO1, and GCLM at the mRNA and protein levels. Overexpression of wild-type KEAP1 sensitized A549 cells  and H460 cells  to nedaplatin and cisplatin over a 24-h treatment period.To investigate the impact of KEAP1 mutations status on NRF2 signaling, the KEAP1 CDS was cloned into the pEnter vector and transfected into the NSCLC cell lines. In the mutant KEAP1 cell lines A549  and SKMES-1 cells  transfected with the mutant KEAP1 construct. An identical trend was observed at the protein level  and SKMES-1  treated with 60 μM cisplatin or nedaplatin.Next, we introduced the G333C and D236H mutations into the KEAP1 pEnter construct. The mRNA expression levels of the NRF2-associated genes HO1, NQO1, and GCLM were significantly elevated in H292 cells  and SKMES-1 , siRNA transfection over a 72-h period reduced the expression of NRF2 and its downstream targets HO1, NQO1, and GCLM at the mRNA and protein levels. Pretreatment with siRNA targeting NRF2 increased sensitivity to nedaplatin and cisplatin in H292 cells  and SKMES-1  over a 24-h treatment period.To assess the effects of NRF2 expression on NSCLC cells exposed to platinum-based drugs, we designed a small interfering RNA (siRNA) construct targeting NRF2 mRNA to inhibit NRF2 activity. In the wild-type KEAP1 cell lines H292  and SKMES-1 , administration of the NRF2 activator over a 24-h period significantly increased the expression of the NRF2 downstream genes HO1, NQO1, and GCLM at the mRNA and protein levels. Pretreatment with 60 μm t-BHQ conferred chemo-resistance to nedaplatin and cisplatin in H292 cells  and in SKMES-1 cells  over a 24-h treatment period.In addition, NSCLC cell lines were treated with t-BHQ, a specific activator of NRF2. In the wild-type KEAP1 NSCLC cell lines H292  and KEAP1  were observed between the mutated patients with mutant KEAP1 cases and with wild-type KEAP1. However, expression levels of the NRF2 downstream target genes HO1 , NQO1 , and GCLM  were significantly enhanced in patients with mutant KEAP1 cases compared with those with wild-type KEAP1.A total of 100 individuals diagnosed with NSCLC were selected from the TCGA group for analysis. KEAP1 mutation status was defined as previously described . We founA large genomic study has demonstrated that multiple oncogenic and tumor suppressor pathways are involved in the initiation and progression of lung cancer . In addiFunctional KEAP1 represses NRF2 activity by recruiting the NRF2 protein to the actin cytoskeleton . SequencAfter exposure to cisplatin, the KEAP1 mutant cell lines A549 and H460 exhibited considerably increased levels of NRF2 signaling, whereas when the KEAP1 wild-type cell lines H292 and SKMES-1 exhibited no changes or only mild changes. Cisplatin is a weak inducer of ARE in the MCF-7 human breast cancer cell line (without KEAP1 mutation ) and indHowever, nedaplatin induces only weak activation or no activation of NRF2 signaling. The antineoplastic activity of nedaplatin, a cisplatin derivate developed in 1983 , might bAn early study by Devling et al.  has revet test p value 0.0116). This effect was also observed in Fig. t test p value 0.0218). We speculated that under siRNA intervention or activator treatment, some other pathways such as NF-kappa B and BACH1 are compensatorily involved in the process [NRF2 activity is involved in chemosensitivity in breast cancer  and ovar process . Our worTo confirm the significance of KEAP1 mutation in vivo, we evaluated public gene expression data from the publically available TCGA consortium. As expected, KEAP1 mutation did not significantly change alter the mRNA expression of NRF2 and KEAP1. However, mRNA levels of NRF2 downstream target genes were significantly higher in patients with KEAP1 mutations compared with patients with wild-type KEAP1, suggesting that KEAP1 and NRF2 interact primarily at the protein level and that KEAP1 mutations strongly affect NRF2 signaling Fig. . The assIn summary, the mRNA and protein expression profile of NRF2 and components of the NRF2-associated genes in four NSCLC cell lines were characterized and their responses to platinum-based therapies were analyzed. A causative effect of KEAP1-NRF2 signaling on platinum-based treatment was defined by engineered expressing either wild-type or mutant KEAP1 and knocking-down or activating NRF2. It is reasonable to hypothesize that KEAP1/NRF2 plays a key role in the cellular response to platinum chemotherapy in NSCLC and that KEAP1 could be explored as a specific biomarker for predicting a patient’s response in personalized therapy.


Bacterial L-asparaginase (ASNase), hydrolyzing L-asparagine (Asn), is an important drug for treating patients with acute lymphoblastic leukaemia (ALL) and natural killer (NK) cell lymphoma. Although different native or pegylated ASNase-based chemotherapy are efficient, disease relapse is frequently observed, especially in adult patients. The neo-synthesis of Asn by asparagine synthetase (AsnS) following ASNase treatment, which involves the amino acid response and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathways, is believed to be the basis of ASNase-resistance mechanisms. However, AsnS expression has not emerged as an accurate predictive factor for ASNase susceptibility. The aim of this study was to identify possible ASNase sensitivity/resistance-related genes or pathways using a new asparaginase, namely a pegylated r-crisantaspase, with a focus on classic Asn-compensatory responses and cell death under conditions of Asn/L-glutamine limitation. We show that, for B-ALL cell lines, changes in the expression of apoptosis-regulatory genes  are associated with ASNase susceptibility. The response of malignant NK cell lines to ASNase may depend on Asn-compensatory mechanisms and other cellular processes such as cleavage of BCL2A1, a prosurvival member of the Bcl-2 protein family. These results suggest that according to cellular context, factors other than AsnS can influence ASNase susceptibility. Bacterial L-asparaginase (ASNase) is an important component of anticancer regimens for acute lymphoblastic leukemia (ALL)134268910121314AsnS mRNA and protein expression; 2) a decrease in Asn efflux through Na+-independent exchange system; 3) a production of aspartic acid, the substrate for Asn synthesis by AsnS, via transamination; 4) a higher Gln synthetase (GlnS) activity through post-transcriptional regulation; 5) an activation of the Gln transporter91011161713141920Despite the high survival rate of patients treated with chemotherapeutic drugs including ASNase, about 20% of children and more than 50% of adults with ALL relapse, with leukemic cells becoming resistant to treatmentAsnS transcription for further neo-synthesis of Asn10222324252627293032Moreover, at the level of signaling transduction, the amino acid response (AAR) pathway, a well-known pathway that senses and responses to a deficiency of amino acids, as well as the survival-related mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin complex 1 (mTORC1) pathways are believed to be involved in ASNase resistance through supplying Asn and limiting its waste. Indeed, upon amino acid depletion, uncharged tRNAs activate general control nonderepressible 2 kinase (GCN2) of AAR pathway, which phosphorylates subsequently eukaryotic initiation factor 2 alpha (elF2α), leading to translational up-regulation of a transcription factor ATF4. Enriched ATF4 proteins promote activation of AsnS at baseline or after exposure to ASNase353637AsnS in ALL patient samples are generally so low that AsnS expression cannot be used as a predictive factor for ASNase sensitivity or resistanceAsnS has not been identified as a discriminating gene for ASNase sensitivity in the study of gene-expression patterns in sensitive versus resistant children ALL samplesAlthough AsnS expression levels are believed to be a key factor in these mechanisms of resistance to ASNase, several studies using clinical samples from children and adult ALL fail to show any correlations between the sensitivity to ASNase and the mRNA expression of In the present work, we sought to identify discriminating factors for ASNase susceptibility using two pairs of B-ALL and malignant NK cell lines with high or low sensitivity to ASNase. We demonstrate that the classical resistance-mechanisms involving the MEK/ERK pathway as well as increase in GlnS expression are associated with resistance to ASNase in the KHYG1 NK leukemia cell line. Furthermore, we show that ASNase induced cell death by apoptosis and that the induction of apoptosis after exposure to ASNase is delayed in resistant cell lines. Analysis of gene-expression indicates that activation of the NFκB pathway is associated with higher sensitivity of the RS B-ALL cell line, while cleavage of the prosurvival Bcl-2 family member BCL2A1 was observed in the sensitive MEC04 NK T-cell lymphoma cell line. These data point to different mechanisms involved in ASNase-susceptibility according to disease type.50 < 5 × 10−5), whereas Nalm-6 and KHYG1 were much less sensitive to ASNases (EC50 > 1 × 10−2)In our previous study, we have defined using MTT assay the susceptibility of different leukemic cell lines  and Nalm-6), non-Hodgkin lymphoma cell lines as well as malignant NK cell lines (including the NK T-cell lymphoma cell line MEC04 and the NK cell leukemia cell line KHYG1), to ASNase treatmentWe first analyzed the classic compensatory AAR and MER/ERK pathways in the four cell lines . WithoutThese results indicate that the increased activity of the MEK/ERK pathway and the moderate up-regulation of AsnS and GlnS expression, favoring the neo-synthesis of Asn to compensate for Asn limitation, are associated to the ASNase-resistance of KHYG1 cells.To determine whether differences in mTORC1 activity exist between sensitive and resistant cell lines, the phosphorylation of 4E-BP1 on T70 was analyzed . For malThese observations show that in response to ASNase treatment the expression of phosphorylated 4E-BP1 was reduced in both the sensitive and the resistant cell lines and that the activity of mTORC1 pathway does not represent a discriminating factor for ASNase susceptibility in these cell lines.−5/ml). In contrast, the induction of apoptosis (~23%) and late apoptosis, (~18%) in resistant Nalm-6 cells was observed only 48 h after ASNase treatment and only at high doses of ASNase (e.g. ≥ 5 × 10−1 U/ml)  through which ASNase-treated cells were dying, and the difference in the induction of cell death between sensitive and resistant cell lines . For B-A−1 U/ml) .We obser−1 U/ml) . In all These results indicate that B-ALL and malignant NK cells predominantly die by apoptosis in response to ASNase treatment and that, expectedly, the induction of apoptosis is delayed and less marked in resistant versus sensitive cell lines.In order to determine which apoptosis-related factors may influence the susceptibility of cells to ASNase, the mRNA expression of 93 apoptosis-related genes was analyzed using TaqMan® Human Apoptosis Array in cells treated or not with ASNase. According to their prominent function in the regulation or execution of apoptosis, the studied genes were classified as pro-apoptotic n = 72) or anti-apoptotic (n = 21) and were divided into four sub-groups: Bcl-2 family, caspases, NFκB and TNF/Fas pathways. We compared the baseline mRNA expression of these genes in sensitive versus resistant cell lines from the same disease type as well as between treated versus non-treated cells. The fold change of each gene at baseline (ratio sensitive/resistant) or 6 h after ASNase treatment (ratio treated/non-treated) were summarized in  or anti-CASP4, PYCARD, RELB, NFκB2, BCL3, TNFSF10, FAS; 2 anti-apoptotic: BCL-2, BCL2A1) were more expressed at baseline and 10 NFκB target genes  were increased in ASNase-treated cells. Among these genes, four  were both more expressed at baseline and induced after ASNase treatment  were more expressed in sensitive RS cells at baseline and 9 genes (7 pro and 2 anti-apoptotic) were less expressed, compared to resistant Nalm-6 cells. In ASNase-treated sensitive RS cells, the expression of 21 genes (16 pro- and 5 anti-apoptotic ) were induced compared to non-treated cells, whereas in ASNase-treated resistant Nalm-6 cells, only 5 pro-apoptotic genes were induced. These data indicate that the expression pattern of apoptosis genes is consistent with the phenotype of ASNase sensitivity/resistance of the analyzed cell lines. Moreover, in sensitive RS cells, 9 NFκB target genes  were less expressed in sensitive MEC04 cells at baseline, and 5 genes (4 pro- and 1 anti-apoptotic) were more expressed, compared to resistant KHYG1 cells. In response to ASNase, only the expression of 1 pro-apoptotic gene was increased in sensitive MEC04 cells, whereas 2 pro-apoptotic genes were decreased. In resistant KHYG1 cells, 4 genes (2 pro-apoptotic and 2 anti-apoptotic) were induced, and 5 pro-apoptotic genes displayed reduced expression levels after ASNase treatment . ContrarBCL2A1 , was ~80 times more expressed at the transcript level in sensitive MEC04 cells, compared to resistant KHYG1 cells. Because the prosurvival BCL2A1 protein was recently reported to be converted into a pro-apoptotic factor after proteolytic cleavageBCL2A1 could represent a discriminating factor for ASNase susceptibility in these cell lines. As shown in We noticed that a specific anti-apoptotic member of the Bcl-2 family, ASNase is an important drug for ALL treatment. Although this molecule is efficient, some patients, especially adults, relapse, and refractory ALL cases are also reported. It is therefore essential to identify possible ASNase resistance determinants in leukemic cells. In this work, we sought to exploit the differential ASNase susceptibility of two pairs of B-ALL and malignant NK cell lines, in order to identify ASNase sensitivity/resistance-related genes or pathways, with a focus on classic compensatory responses and cell death under conditions of Asn/Gln limitation.AsnS at baseline and after ASNase exposure, albeit showing high GlnS expression. Given that the phosphorylation of elF-2α was not induced (and even decreased) after ASNase exposure, AAR pathway was inactivated in both cell lines, which could be an explanation for the absence of induction of AsnS protein expression following ASNase treatment. Those observations are in line with previous findings showing absence of AsnS mRNA expression before and after ASNase treatment in sensitive RS cells36AsnS gene is silenced and the promoter region of AsnS gene is highly methylated42AsnS expression might be associated with specific epigenetic processes such as AsnS gene silencing.The survival of both highly ASNase-sensitive RS and MEC04 cell lines seems strictly dependent on extracellular Asn, because these two cell lines had undetectable protein expression of AsnS by binding to its promoter, resulting in an increase in the protein expression of AsnS. The authors propose that tumor-derived p53 mutants induce oncogenesis by transactivating growth-promoting genes such as AsnS and hTERT genes45In resistant KHYG1 cells, the high baseline expression of AsnS as well as the increase in both AsnS and GlnS expression following ASNase treatment could compensate for the deficiency in Asn and Gln by promoting the synthesis of both amino acids, explaining the low susceptibility of this cell line to ASNase. Yet, intriguingly, in ASNase-treated cells, the phosphorylation of Erk1/2 was greatly induced with a slight increase in AsnS expression, while phosphorylation of elF-2α was reduced to undetectable levels. These data are in contradiction with previous published data suggesting the existence of a positive regulatory loop between the AAR and MEK/ERK pathways as well as the involvement of the activated AAR pathway in the activation of the MEK/ERK pathway and in the induction of AsnS expression in response to amino acid restriction2745We demonstrated that ASNase-induced cell death was mediated by apoptosis, in agreement with previous reports4710In conclusion, our data provide evidence that, for B-ALL cell lines, changes in the expression of apoptosis-regulatory genes, especially NFκB-related genes, rather than AsnS-regulatory pathways could be crucial for the susceptibility of cells to ASNase. Regarding malignant NK cell lines, AsnS-regulatory pathways appear to be the main determinant implicated in the susceptibility of cells to ASNase, BCL2LA1 cleavage representing another potential mechanism. Anomalies making cells resistant to ASNase could be multiple and variable from one patient sample to another according to the type of disease and cellular context. Thus, our study provided a piece of puzzle for the understanding of ASNase-resistance mechanisms and for further development of more adaptive and personalized combinatory treatment for ALL (e.g. including inhibitors of Erk1/2 or modulators of the NFκB pathway).2. Malignant NK cell lines, MEC04  and KHYG1 (aggressive NK cell leukemia), were cultured under the same condition in the presence of 10 ng/ml interleukin-2 (IL-2) .The B-ALL cell lines RS (ATCC CRL-1873) and Nalm-6 (DSMZ ACC128) were cultured in RPMI 1640 medium , containing 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 U penicillin and 100 μg streptomycin, at 37°C in a humidified atmosphere with 5% COWestern blot was carried out as previously described6 cells/ml in round-bottom 96-well plates and treated with increasing doses of pegylated r-crisantaspase−5 U/ml to 5 U/ml for 24, 48 and 72 h. Non-treated cells were used as control. All samples were then subjected to Annexin V/PI apoptosis assay. Cells were incubated in 100 μl of Annexin V-binding buffer containing 10 μg/ml of PI (Sigma-Aldrich) and 2.5 μl Annexin V  for 15 min at room temperature before analysis by flow cytometry .Cells were seeded at 0.2 × 106 cells/ml and treated with or without 5 U/ml of pegylated r-crisantaspase for 6 h. Total RNA was extracted using Trizol solution and RNA quality was assessed by Bioanalyser . The reverse transcription was performed using High Capacity RNA-to-cDNA Master Mix  with 1 μg of total RNA in a volume of 20 μl. The product of reverse transcription diluted in TaqMan® Universal PCR Master Mix (Life Technologies) was then loaded into TaqMan® Human Apoptosis Array (Life Technologies) and quantitative PCR was performed with the Applied Biosystems 7900 Sequence Detection system (Life Technologies) according to the manufacturer's instructions. Relative quantification (RQ) was obtained using the equation RQ = 2−ΔΔCt after normalization of the Ct data to that of the reference gene, where Ct is the threshold cycle of fluorescence detection. Results were the combined data of two independent experiments. The reference gene for baseline expression analysis was 18S. The mean and standard deviation (S.D.) were Ct = 9.8 ± 0.4 for non-treated B-ALL samples and Ct = 10.2 ± 0.5 for non-treated malignant NK samples. 18S, GADPH and β-actin were the reference genes for normalizing data of ASNase-treated samples to that of non-treated samples. RS cells: 18S Ct = 8.7 ± 0.1; GADPH Ct = 17.5 ± 0.1; β-actin Ct = 17.9 ± 0.1. Nalm-6 cells: 18S Ct = 8.4 ± 0.2; GADPH Ct = 17.9 ± 0.1; β-actin Ct = 18.4 ± 0.1. MEC04: 18S Ct = 8.6 ± 0.2; GADPH Ct = 16.7 ± 0.2; β-actin Ct = 18.9 ± 0.2. KHYG1: 18S Ct = 9.9 ± 0.3; GADPH Ct = 17.4 ± 0.1; β-actin Ct = 17.3 ± 0.2.Cells were seeded at 0.2 × 10


Genomic analysis of ovarian cancer cell lines has revealed a panel that best represents the most common ovarian cancer subtype, high-grade serous ovarian cancer (HGSOC). However, these HGSOC-like cell lines have not been extensively applied by ovarian cancer researchers to date, and the most commonly used cell lines in the ovarian cancer field do not genetically resemble the major clinical type of the disease. For the HGSOC-like lines to serve as suitable models, they need to be characterized for common functional assays. To achieve that objective, we systematically studied a panel of HGSOC cells CAOV3, COV362, Kuramochi, OVCAR4, OVCAR5, OVCAR8, OVSAHO and SNU119 for migration, invasion, proliferation, clonogenicity, EMT phenotype and cisplatin resistance. They exhibited a range of efficacies and OVCAR5, OVCAR8 and Kuramochi were the most aggressive. SNU119 and OVSAHO cells demonstrated the lowest functional activities. Wide differences in expression of EMT markers were observed between cell lines. SNU119 were the most epithelial and OVCAR8 had the most mesenchymal phenotype. COV362 was the most resistant to cisplatin while CAOV3 was the most sensitive. Taken together, our systematic characterization represents a valuable resource to help guide the application of HGSOC cells by the cancer research community. Ovarian cancer (OC) is the most lethal gynecological malignancy and is the 5th leading cause of cancer related deaths among women in the United States of America . While mUntil recently, a major issue with identifying good experimental models has been a lack of knowledge regarding the molecular and mutational profiles that are most characteristic of OC . High-grThe Cancer Genome Atlas Research Network (TCGA) study  revealedThe realization that the most commonly used cell lines in OC research are not representative of HGSOC will steadily lead to a shift towards using the more HGSOC-like cell lines. In order to be useful, these HGSOC cell lines need to be well characterized and should accurately represent the disease . Since mRecent studies have implicated EMT as a driver of OC metastasis –13. An ain vitro functional assays, their sensitivity to cisplatin and their expression of epithelial and mesenchymal markers. The absence of published reports of such consolidated data hampers effective transition to the use of these HGSOC cell line models for ovarian cancer research. We believe that our data will be very beneficial to the field and will serve as a guide to optimize assay and treatment conditions for various mechanistic, drug development and screening studies. It will enable researchers to extensively use these to more accurately model OC.We report the characterization of a panel of these HGSOC cell lines for commonly used in vitro to study the role of genes or effect of treatments on metastasis [p < 0.0001). OVCAR5 and OVCAR4 were not different from each other but were different from all other cell lines. OVCAR8, CAOV3, COV362, and HeyA8 were not different from each other (with the exception of HeyA8 being different from OVCAR8), but were different from all other cell lines. Kuramochi was significantly different from all other cell lines. SNU119 and OVSAHO were not different from each other but were significantly different from all other cell lines. Since each cell line had a different propensity to migrate, the number of cells seeded per insert had to be varied between cell lines in order to obtain quantifiable migrated cell numbers. The migration was then normalized to the number of cells seeded and ranked accordingly . OVCAR5 and HeyA8 were not different from each other but were different from all other cell lines. OVCAR8 was different from all other cell lines, Kuramochi was not different from OVCAR4 but was different from all other cell lines. OVCAR4, COV362, and CAOV3 were not different but were different from all other cell lines. The independent experiments with each cell line are presented in In addition to the ability to migrate towards chemoattractants, cancer cells need to have the ability to invade through the extracellular matrix surrounding cells and the basement membrane. Therefore, the ability of these cells to invade through matrigel was studied by using transwell invasion assay , 17. Thep < 0.0001). HeyA8 was different from all other cell lines. OVCAR5, OVCAR8 and CAOV3 were not different from each other but different from all other cell lines. COV362, Kuramochi, OVCAR4 and SNU119 were not different from each other (with the exception of COV362 being different from SNU119) but were different from all other cell lines.Having studied the ability of the OC cells to migrate and invade in response to chemoattractants, we proceeded to test their proliferation and clonogenicity. Both these traits are essential for tumor progression and are extensively used to study the effects of therapies, role of specific regulators or genes in OC , 19. Forp < 0.0001). Both OVCAR8 and OVSAHO were different from all other cell lines. Kuramochi and OVCAR4 were not different from each other but were different from all other cell lines. OVCAR5, HeyA8, COV362, SNU119, and CAOV3 were not different from each other . The time required for forming colonies ranged from 7 days for HeyA8 cells to 4 weeks for OVSAHO cells  and CAOV3 was the most sensitive (IC50 dose of 3.14 μM) to cisplatin  were much less potent in the functional assays. These in vitro functional assay results mirrored their ability to form mouse xenograft tumors reported previously [The present study is the first to systematically compare the eviously . HeyA8 ceviously . Similareviously , 18, 23.eviously . These neviously . This shThere was no clear correlation between genetic resemblances to HGSOC and the EMT phenotype. All the cell line models were found to be relatively sensitive to cisplatin, which agreed with the previous report on COV362 and CAOV3 . Since mIn conclusion, these HGSOC cell line models can be used as better representatives of the most common form of the disease. However, there are significant variations in their functional abilities, their EMT status as well as their sensitivity to cisplatin. Therefore, it is imperative that the assay conditions be adapted to the fact that some of these are less aggressive in the common functional assays tested compared to the OC cell lines that have been predominantly used. The standardized conditions reported herein would be useful as an appropriate starting point.Dulbecco's Modified Eagle's Medium (DMEM) , MEM vitamins , MEM nonessential amino acids , Penicillin-Streptomycin  and trypsin  were purchased from Corning. FBS was purchased from Gibco . Giemsa stain  and crystal violet  were obtained from Sigma Aldrich. Thiazolyl blue tetrazolium bromide was from Acros Organics  and 4% paraformaldehyde solution was purchased from Fisher Scientific (NC9245948).All cell lines were grown in DMEM with 10% FBS along with 1% MEM vitamins, MEM nonessential amino acids and Penicillin-Streptomycin. CAOV3, HeyA8, OVCAR5 and OVCAR8 were obtained from Ernst Lengyel, University of Chicago and OVCAR4 from Joanna Burdette, University of Illinois at Chicago. OVSAHO was obtained from the Japanese Collection of Research Bioresources and SNU119 was from the Korean Cell Line Bank. COV362 (Sigma), and Kuramochi (Japanese Collection of Research Bioresources). All cell lines used were genetically validated and also confirmed to be mycoplasma free using respective services from Idexx BioResearch . The genetic validation was done using the CellCheck 16 (16 Marker STR Profile and Inter-species Contamination Test) and mycoplasma testing was done using Stat-Myco.2 incubator (5% CO2) and then seeded in the upper chamber in 500 μl of serum free DMEM and allowed to migrate at 37°C. The numbers of cells seeded per insert and migration times are included in Table Transwell migration assays were conducted using 8 μm pore size inserts . OC cells were trypsinized, recovered for 30 min at 37°C in the CON = 200,000/number of cells seeded for the cell lineSimilarly, the baseline migration time was set at 8 h and the migration time adjustment factor (T) was calculated as follows:T = 8/migration time for the cell lineThe final adjusted cell count took into account the cell number adjustment factor (N) and the migration time adjustment factor (T) and was determined by:Adjusted cell count = average number of cells per field × N × T2 incubator and then seeded in the transwell insert in 500 μl serum-free DMEM. The number of cells seeded per insert are included in Table Cellular invasion through the extracellular matrix proteins was assayed using growth factor reduced matrigel coated transwell inserts  in 24-well plates. OC cells were trypsinized, recovered for 30 min at 37°C in the COAdjusted cell count = average number of cells per field × NWhere N = 200,000/number of cells seeded for the cell line.OC cells were seeded in 96-well plates in 8 replicates and allowed to grow for 5 days. To identify the optimal number of cells for comparison between cell lines, 1000, 2000, 5000 and 10000 cells of each cell line were seeded per well. On the fourth day MTT -2,5-diphenyltetrazolium bromide) Assay was conducted to measure proliferation of the OC cells. 20 μl of MTT solution (5 mg/ml in sterile PBS) was added per well, mixed and incubated at 37°C. The incubation times for the OC cell lines are listed in Table OC cells were seeded in 6-well plates  and allowed to form colonies. Medium was changed every fifth day. Once visible colonies were formed  reaction was done for E-cadherin and vimentin using TaqMan gene expression assays as described previously . BrieflyWestern blotting was done as described previously . BrieflyCell diameter was analyzed using the Z2 coulter Particle Count and Size Analyzer  using a 100 μm aperture size. Briefly, cells were diluted 1:10 in diluent (Isoton II) and 0.5 ml was loaded into the counter. All particles over 12 um were counted and measured. Between sample readings, the counter was flushed with the diluent.p-value from the overall F-test is reported. Tukey's method was used for pair-wise comparisons. The overall error rate per outcome was controlled at 5%.ANOVA was used to compare mean migration, invasion, proliferation, and colony formation by cell line. The


We also investigated the effects of mitogenic agents, and found that they can markedly influence the metabolome of the melanocyte cell line, resembling the pattern of most proliferative cell lines.Melanoma is the most aggressive type of skin cancer and efforts to improve the diagnosis of this neoplasia are largely based on the use of cell lines. Metabolomics is currently undergoing great advancements towards its use to screening for disease biomarkers. Although NMR metabolomics includes both 1D and 2D methodologies, there is a lack of data in the literature regarding heteronuclear 2D NMR assignments of the metabolome from eukaryotic cell lines. The present study applied NMR-based metabolomics strategies to characterize aqueous and lipid extracts from murine melanocytes and melanoma cell lines with distinct tumorigenic potential, successfully obtaining fingerprints of the metabolites from the extracts of the cell lines by means of 2D NMR HSQC correlation maps. Relative amounts of the identified metabolites were compared between the 4 cell lines. Multivariate analysis of  Another melanoma model includes the Tm1 and Tm5 melanoma cell lines, which were established by submitting the melanocyte cell line Melan-a to sequential cycles of substrate-adhesion impedimentin vivo and a lower doubling time in vitro101H NMR spectroscopy allied to multivariate analysis. We also investigated the effect of mitogenic agents on the metabolic profile of the melanocyte cell line.Melanoma presents a major challenge in medical practice, due to difficulty in diagnosis, poor response to available therapies and high incidence in western populations61H NMR spectra of the aqueous extracts obtained from the four cell lines showed many overlapping signals 1H NMR spectra obtained from the cell extracts. The 4 cell lines were separated into 4 distinct groups on the scores plot  with PMA present in the media during the entire time (MA), 2) with the cell line cultivated in a medium without PMA for 24 hours (MA-24), and 3) where the cell line was cultivated for 72 h without the presence of PMA in the medium (MA-72). ¹H NMR and principal component analyses were then carried out on the lipid extracts of the cells. The first principal component explained 45% of the variance, the second 72%, and the third 84%, cumulatively. Even the explained variance in the first PC not being ideal, the 3 conditions were well discriminated on the scores plot of both PC1 vs PC2 , and the Melan-a cell line is no exception. Stranzl In summary, the present study successfully applied NMR techniques in order to identify and determine the relative amounts of aqueous and lipid-soluble metabolites of both melanocyte and melanoma murine cell lines. Through PCA analysis it was possible to identify the main contributors which differentiate the 4 cell lines. The results showed that the metabolite which most contributes to differentiation in the aqueous extract obtained from the 4 cell lines on both PC1 and PC2 was creatine, as confirmed by gene expression assays. Therefore, we can state that there is an imbalance in creatine metabolism , such asFurthermore, this study demonstrated that the metabolomic profile of the melanocyte cell line is influenced by the use of mitogenic agents, such as PMA. Although not so evident as the concentration changes observed for aqueous metabolites, lipid metabolism changes between the Melan-a cell line grown with and without PMA added on culture media could be revealed by the use of multivariate analysis techniques, showing that the main alterations were related to the metabolic pathway of cholesterol. The 2D multiplicity-edited HSQC NMR fingerprints proved to be a useful tool for identifying and measuring different expression patterns of metabolites in both water soluble and lipid extracts, enabling us to identify potential progression biomarkers and thus contribute to the development of methods for the prognosis of melanoma.2 tissue-culture flasks and maintained in RPMI medium supplemented with 5% (v/v) fetal bovine serum (FBS), both from Gibco , 200 nM 12-O-tetradecanoyl phorbol-13-acetate - PMA , 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco) in a humidified atmosphere containing 5% CO2 at 37 °C. Melanoma cell line B16-F10 was cultured under the same conditions, without PMA. The cells were grown until 75–80% density and then collected using 0.1% trypsin in PBSA containing 1 mM EDTA and centrifuged at 4000 rpm for 20 minutes (4 °C). The cell pellet was washed thrice with phosphate buffer, the supernatant discarded and the pellet subjected to the extraction process. We standardized a quantity of 15 mg for each cell line, which corresponded to approximately 4 × 75 cm2 tissue-culture flasks.Mouse melanoma cell lines  and the immortalized murine melanocyte cell line Melan-a were kindly provided by Dr. Roger Chammas . Melan-a, Tm1 and Tm5 cells were grown in 75 cm3:MeOH 3:1 (v/v), being vortexed for 1 minute and then kept for 30 min at room temperature. After this, it was centrifuged at 10000 rpm for 20 min, the supernatant collected and the solvent dried under a stream of nitrogenThe cell pellets were subjected to lysis, being frozen with liquid nitrogen and returned to a 37 °C water bath several times. After being freeze-dried overnight, 15 mg of each cell line, was weighted and transferred to a glass tube with screw cap and Teflon (volume of 4 mL). The pellet was extracted using acetonitrile:water 1:1 (1 mL), vortexed for 1 minute and then kept for 10 min at room temperature (this procedure was repeated thrice), and then centrifuged at 10000 rpm for 6 min. The supernatant was collected and lyophilized. The pellet from the previous aqueous extraction procedure was then subjected to lipid extraction using CHCl3, Deuterium oxide  and 3-trimethylsilyl-2H4-propionic acid sodium salt (TSP) were purchased from Cambridge Isotope Laboratories, Inc.  and from Sigma-Aldrich . The samples obtained from the aqueous extraction were deuterium-exchanged by repeated dissolution in D2O, and the samples obtained from the lipid extraction were deuterium exchanged by repeated dissolution in MeOD-D2O (2:1). Both were freeze-dried overnight. Their spectra were obtained from solutions in D2O (580 μL plus 20 μL of TSP 1 mg/mL) or CDCl3-MeOD  at 30 °C, using TMS (Tetramethylsilane) or TSP as a reference (δ = 0.00 ppm). Spectra were obtained on a Bruker 400 MHz AVANCE III NMR spectrometer with a 5 mm BBI inverse probe and a Bruker 600 MHz ASCEND equipped with a QXI probe . 1D 1H-NMR was carried out using enough scans to give a Signal/Noise (S/N) ratio of at least 2000/1 . Experiments were performed without tube rotation and with the TMS or TSP signal at a medium width ranging from 0.8–1.0 Hz. Water suppression was obtained by presaturation, using the 1D zgpr pulse sequence, applying a low power continued wave irradiation before the first 90° pulse on channel 1, using a power level for presaturation of 44.98 -dBW (3.17 × 10−5 Watts). For multivariate analysis and estimation of metabolites concentration, the spectra were acquired using the noesygppr1d.2 pulse sequence, with 128 transients and a delay of 10.0 s . 2D NMR experiments were carried out using multiplicity-edited 1H-13C HSQC, heteronuclear correlation via double inept transfer with decoupling during acquisition, using trim pulses in inept transfer with multiplicity editing during the selection step (hsqcedetgpsp.3), TOCSY, total homonuclear correlation via Hartman-Hahn transfer using MLEV17 sequence for mixing with a mixing time of 0.06 s (mlevphpr.2), and 1H-13C/1H-31P HMBC, heteronuclear correlation via zero and double quantum coherence optimized on long range couplings (hmbcgplpndqf and hmbcgpndqf) pulse sequences. The 2D experiments were recorded for quadrature detection in the indirect dimension, HSQC spectra were acquired using 128 scans per series of 2 K × 400 W data points with zero filling in F1 (4 K) prior to Fourier transformationMeOD, CDCl18 primers, according to manufacturer’s instructions. RT-qPCR reactions were performed in a StepOne Plus  thermal cycler, using SYBR Green PCR Master Mix (Applied Biosystems) and specific primers for the genes of interest. The equipment’s default cycling program was used (15 s at 95 °C and 1 min at 60 °C for 40 cycles). Amplification efficiency and data normalization were performed as previously describedGAMT (Guanidine-Acetate Methyl Transferase - EC 2.1.1.2) mRNA expression levels were quantified in murine cell lines Melan-a and B16-F10. For this, cDNA were synthesized using the ImProm II Reverse Transcription Kit (Promega), from 1 μg of total RNA, and using Oligo d(T)Melan-a cell line were grown in RPMI medium containing 200 nM of PMA during four passages. The medium was then removed and replaced with a medium which did not contain PMA. The cells were left for a further 24 and 72 hours of growth.2OD resonances (δ 3.33–3.38) was removed from all data sets prior to normalization and multivariate data analysis in order to remove deviations due to water suppression efficiency or solvent multiplicity inhomogeneities. Following a preliminary PCA analysis, the region corresponding to (CH2)n (δ 1.20–1.40) and N+ (CH3)3 (δ 3.18–3.25) resonances was also removed from all datasets, mainly because minor peak shape deformations in this region interfered with PCA analysis, due to the high intensity of signals. All remaining frequency regions of the spectra were analyzed without scaling, in order to preserve natural differences in intensities from the datasetsMultivariate analyses were performed on the spectra obtained from both aqueous and lipid extractions. Spectra obtained from 3 to 5 independent experiments, each one with 3 or more technical replicates were assembled to build the bucket tables. For spectra obtained from aqueous extractions, the spectral width considered was from 0.50 to 10.00 ppm. After NMR spectral phase and baseline correction, each spectrum was data-reduced to 1183 regions of equal width (0.005 ppm) using the AMIX  software package, version 3.8 . The confidence level was 95.00%. For spectra obtained from lipid extractions, the spectral width considered was from 0.50 to 6.00 ppm. Each spectrum was data-reduced to 453 regions of equal width (0.005 ppm). The spectral region close to CHDHow to cite this article: Santana-Filho, A. P. d. et al. NMR metabolic fingerprints of murine melanocyte and melanoma cell lines: application to biomarker discovery. Sci. Rep.7, 42324; doi: 10.1038/srep42324 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The Maternal Embryonic Leucine Zipper Kinase (MELK) has been reported to be a genetic dependency in several cancer types. MELK RNAi and small-molecule inhibitors of MELK block the proliferation of various cancer cell lines, and MELK knockdown has been described as particularly effective against the highly-aggressive basal/triple-negative subtype of breast cancer. Based on these preclinical results, the MELK inhibitor OTS167 is currently being tested as a novel chemotherapy agent in several clinical trials. Here, we report that mutagenizing MELK with CRISPR/Cas9 has no effect on the fitness of basal breast cancer cell lines or cell lines from six other cancer types. Cells that harbor null mutations in MELK exhibit wild-type doubling times, cytokinesis, and anchorage-independent growth. Furthermore, MELK-knockout lines remain sensitive to OTS167, suggesting that this drug blocks cell division through an off-target mechanism. In total, our results undermine the rationale for a series of current clinical trials and provide an experimental approach for the use of CRISPR/Cas9 in preclinical target validation that can be broadly applied.DOI:http://dx.doi.org/10.7554/eLife.24179.001 Like a person who is dependent on coffee to be productive, cancer cells are dependent on the products of certain genes in order to dominate their environment and grow. Cancer cells will stop growing and die when the activity of these gene products is blocked. These genes are known as cancer dependencies or “addictions”. As a result, researchers are constantly looking for cancer dependencies and developing drugs to block their activity.It was previously believed that a gene called MELK was an addiction in certain types of breast cancer. In fact, pharmaceutical companies had developed a drug to block the activity of MELK, and this drug is currently being tested in human patients. However, Lin, Giuliano et al. have now taken a second look at the role of MELK in breast cancer, and have come to a different conclusion.Using a gene editing technology called CRISPR/Cas9, Lin, Giuliano et al. removed MELK activity from several cancer cell lines. This did not stop cancer cells from multiplying, suggesting that MELK is not actually a cancer addiction.Additionally, when breast cancer cells that do not produce MELK were exposed to the drug that is supposed to block MELK activity, the drug still stopped cell growth. Since the drug works when MELK is not present in the cell, the drug must be binding to other proteins. This suggests that MELK is not the actual target of the drug.Lin, Giuliano et al. suggest that, in the future, CRISPR/Cas9 technology could be used to better identify cancer dependencies and drug targets before cancer drugs are given to human patients.DOI:http://dx.doi.org/10.7554/eLife.24179.002 Tumors of the breast can be divided into five distinct subtypes based on characteristic gene expression patterns. These breast cancer subtypes are referred to as Luminal A, Luminal B, Her2-enriched, normal-like, and basal . Basal bIn recent years, significant progress has been made in the treatment of certain malignancies by targeting cancer cell ‘addictions’, or genetic dependencies that encode proteins required for the growth of specific cancer types . Drugs tThe Maternal Embryonic Leucine Zipper Kinase (MELK) has received substantial attention as a potential cancer cell addiction and promising target for drug development. MELK was first identified as an AMPK family member expressed in the mouse pre-implantation embryo , and hasAs part of a project in our lab to characterize genes whose expression is associated with patient prognosis in cancer , we idenOver a dozen previous publications have reported that MELK is a cancer dependency, as blocking MELK with RNAi or small molecules inhibited the proliferation of cell lines derived from multiple tumor types . HoweverTo assess the efficacy of our CRISPR system, we purified GFP+ populations from cell lines harboring each individual guide RNA, and then we analyzed the targeted loci by Sanger sequencing. TIDE analysis, which decomposes raw sequencing traces into linear combinations of indel mutations , revealeWe next set out to determine whether MELK was required for cancer cell fitness. To test this, we measured cell proliferation over 15 days in culture in the 30 independent lines of A375, Cal51, and MDA-MB-231 that we had generated . SurprisIn order to assess whether a wider range of cancer cell lines were dependent on MELK for viability, we performed individual GFP dropout experiments in 13 Cas9-expressing cancer cell lines . In thesAs negative controls in this experiment, we utilized three gRNA’s targeting Rosa26, and as positive controls we designed six gRNA’s targeting the essential replication genes RPA3 and PCNA . We firsTo extend these observations, we repeated the GFP dropout experiments in six Cas9-expressing cell lines  from other cancer types previously suggested to require MELK expression. Consistent with our observations in the triple-negative breast cancer cells, guides targeting Rosa26 exhibited minimal dropout over five passages, while guides targeting RPA3 or PCNA were depleted up to 90-fold . HoweverSeveral laboratories have conducted genome-wide or kinase-focused screens to identify novel cancer addictions. If these unbiased screens indicated that cancer cell lines required MELK expression to proliferate, then that would bolster the contention that MELK could be a therapeutic target in cancer. We therefore examined data from four recent screens: a kinome-wide siRNA screen in 117 cancer cell lines , a genomIf MELK is not a cancer cell dependency, then drugs that inhibit MELK must either be ineffective at stopping cancer cell division or they must also act on other cellular targets. We therefore assessed the efficacy of the MELK inhibitor OTS167 , a therapeutic agent being tested in several clinical trials. We treated a variety of cancer cell lines with 7-point serial dilutions of OTS167, and we observed that OTS167 did in fact impede cell proliferation at nanomolar concentrations . As OTS167 was able to inhibit growth despite the non-essentially of MELK, we considered the possibility that OTS167 acted through an off-target effect. To test this, we set out to determine whether MELK expression was actually required for OTS167 sensitivity. We calculated the GI50 value of OTS167 in A375, Cal51, and MDA-MB-231 cells that harbored gRNA’s targeting either Rosa26 or MELK, and we found that cell populations with wild-type or mutant MELK displayed equivalent sensitivity to the drug . For insTo further explore this observation, we tested the efficacy of OTS167 against a panel of triple-negative or receptor-positive breast cancer cell lines. MELK is significantly up-regulated in triple-negative tumors relative to receptor-positive tumors , and oneLastly, to confirm that OTS167 treatment fails to phenocopy MELK mutations, we examined their effects on cell cycle progression. We found that treatment with OTS167 blocked cytokinesis in a dose-dependent manner, resulting in populations harboring 13% to 60% multinucleate cells. In contrast, cells transduced with either Rosa26 or MELK gRNAs progressed through the cell cycle without gross mitotic defects, and exhibited no significant difference in the frequency of multinucleate cells . These rTo unambiguously demonstrate that MELK is dispensable for the proliferation of certain cancer cell lines, we used CRISPR to generate clonal cell lines that lack MELK protein. We transduced the MDA-MB-231 triple-negative breast cancer cell line with sets of 2 guide RNAs and then expanded clonal populations from single cells . RecombiThe MDA-MB-231and Cal51 MELK-KO clones exhibited robust proliferation, demonstrating that MELK is fully dispensable for the growth of these cancer cell lines . In factAs a mitotic kinase highly expressed in many cancer types, MELK has been identified as a promising target for therapeutic intervention. However, through the use of CRISPR/Cas9-mediated mutagenesis, we have demonstrated that MELK is dispensable for growth in 13 out of 13 cancer cell lines tested, and that a MELK inhibitor currently in clinical trials blocks cell division by inhibiting another target. We believe that our results highlight the importance of using CRISPR/Cas9 technology to study and validate preclinical targets in cancer drug development.RAD51 and MAD2 are unusually sensitive to off-target RNAi inhibition  that failed to reveal an effect of MELK loss on cell fitness, further strengthening our conclusion that MELK is dispensable for cancer cell proliferation.CRISPR mutagenesis can also assist in the pharmacological study of potential drugs. Several lines of evidence indicate that OTS167 does indeed inhibit MELK: for instance, a crystal structure of OTS167 binding to the MELK kinase domain has been reported . HoweverOur results leave open the question of what role, if any, MELK plays in mammalian biology and cell cycle progression. While MELK is up-regulated in diverse tumor types, it is also expressed in several normal cell lineages, including embryonic cells, hematopoietic cells, and neural progenitor cells . MELK ma2. Cell counting was performed using the Cellometer Auto T4 system .The identity of every human cell line utilized in this paper was authenticated using STR profiling . Cell lines were also confirmed to be negative for mycoplasma contamination using the MycoAlert Detection Kit . Cell lines HCC70, HCC1937, MDA-MB-453, MDA-MB-468, and ZR-75–1 were grown in RPMI 1640 supplemented with 10% fetal bovine (FBS), 2 mM glutamine, 1% Nonessential Amino Acids , and 100 U/ml penicillin and streptomycin. HCC1143 and NCI-H1299 were grown in RPMI 1640 supplemented with 10% FBS, 2 mM glutamine, and 100 U/ml penicillin and streptomycin. T24 was grown in McCoy’s 5A media supplemented with 10% FBS, 2 mM glutamine, and 100 U/ml penicillin and streptomycin. Cal51, A375, MDA-MB-231, HCT116, Cama1, JIMT-1, and U118-MG cells were grown in DMEM supplemented with 10% FBS, 2 mM glutamine, and 100 U/ml penicillin and streptomycin. T47D cells were grown in RPMI supplemented with 10% FBS, 6.94 μg/ml insulin , 2 mM glutamine, and 100 U/ml penicillin and streptomycin. MCF7 cells were grown in DMEM supplemented with 10% FBS, 0.01 mg/ml insulin, 2 mM glutamine, and 100 U/ml penicillin and streptomycin. Cell lines were kindly provided by the individuals thanked in the acknowledgments. All cell lines were maintained in a humidified environment at 37°C and 5% COGuide RNAs targeting protein domains in MELK, PCNA, and RPA3 were designed with assistance from Osama El Demerdash (manuscript in preparation). Oligonucleotides were ordered from IDT and then cloned into the LRG 2.1 vector [a gift from Jun-Wei Shi (University of Pennsylvania) and Chris Vakoc (Cold Spring Harbor Laboratory)] using a BsmBI digestion . PlasmidHEK293T cells were transfected using the calcium-phosphate method . SupernaTo measure cell proliferation, 100,000 cells of each strain were plated on a six well plate and then allowed to grow for 72 hr. Cells were then trypsinized, counted, and 100,000 cells were re-plated in fresh media. Cells were passaged five times, and cumulative population doublings and doubling times were calculated at each passage.Solutions of 1.0% and 0.7% Difco Agar Noble in sterile water were autoclaved and then allowed to equilibrate at 42°C or 37.5°C, respectively. The 1% agar solution was then mixed 1:1 with 2 ml of the appropriate media, supplemented with 2X the concentration of serum, glutamine, and penicillin/streptomycin. 1 mL of this mixture was then plated in one well of a six well plate and allowed to solidify at room temperature for 30 min, resulting in a 0.5% agar base layer. Cells of interest were then trypsinized and re-suspended in their appropriate media. 20,000 cells were diluted in 1 ml of 2X media and then mixed with 1 ml of the 0.7% agar solution. 500 μl of this mixture was then plated on the solidified base layer, resulting in 5000 cells in a 0.375% agar suspension. The agar was allowed to solidify at room temperature for one hour before being transferred to an incubator. Fresh 1X media was added to the surface of each well 24 hr after plating, and then were re-fed every 3 days. After 21 days of growth, colonies were scored under 10x magnification. All experiments were plated in triplicate and performed twice.Cells were lysed with RIPA buffer . Lysates were quantified using the Pierce BCA Kit (Thermo Scientific), and equal amounts of protein were denatured and loaded onto an 8% SDS-PAGE gel. The protein was transferred onto a polyvinylidene difluoride membrane using the Trans-Blot Turbo Transfer System . The membrane was blocked in 5% non-fat milk-TBST and then incubated with anti-MELK  at a 1:3000 dilution, or blocked in 10% BSA-TBST and then incubated with anti-MELK  at a 1:1000 dilution. Anti-alpha-tubulin  was used as a loading control at a 1:3000 dilution. All primary antibody incubations were performed overnight at 4°C. Following incubation, the membranes were washed and then incubated in either anti-rabbit secondary  at 1:50000 for MELK or anti-mouse secondary  at 1:10000 for Tubulin for 1 hr at room temperature.Genomic DNA was extracted from transduced cell lines using the QIAmp DNA Mini kit . Loci targeted by guide RNAs were amplified using the primers listed in For every cell line of interest, 10,000 cells were plated in 100 μl of media in an 8 × 4 matrix on a flat-bottomed 96-well plate. Cells were allowed to attach for 24 hr, at which point the media in every well was changed. 500 nM of OTSSP167  was added to one row of cells, and then 6 3-fold serial dilutions were performed. After 72 hr of growth in the presence of the drug, cells were trypsinized and counted using a MacsQuant Analyzer 10 . The fraction of cells recovered at every drug concentration, relative to a row of untreated cells, was determined. GI50 values were calculated using a four-parameter inhibition vs. concentration model in Prism 7 . Sensitivity experiments in Cells were transduced on day 0 with sgRNA lentiviral supernatant, which was then replaced with fresh media on day 1. On day 3, the baseline percentage of GFP+ cells was measured using a MacsQuant Analyzer 10 (Milltenyi Biotec). Cells were then passaged every 3 or 4 days, according to their growth rate and confluence, and the percentage of GFP+ cells was measured at every split. Dropout values represent the fold decrease in GFP+ cells at each passage, relative to the GFP+ percentage on day 3. In preliminary experiments with A375 and MDA-MB-231, replicate dropout assays were highly reproducible across independent replicates. For that reason, GFP dropout experiments in the 13 tested cell lines were performed once.10,000  or 20,000 (MDA-MB-231) cells of interest were plated in 250 μl of media in a flat-bottomed 24-well plate and allowed to attach for 24 hr. Then the media was replaced, and OTS167 or Cytochalasin B  were added to control wells. Following an additional 24 hr period of growth, cells were stained with 2.5 μg/ml of Hoechst dye  for 30 min and imaged using appropriate filters. DNA staining experiments were performed twice. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by Tony Hunter (Senior Editor) and two reviewers, one of whom is a member of our Board of Reviewing Editors. The reviewers have opted to remain anonymous.Thank you for submitting your article "CRISPR/Cas9 mutagenesis invalidates a genetic target of clinical trials in cancer" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Lin and colleagues report their CRISPR-based analysis of the candidate oncology drug target, MELK, a kinase that has been previously implicated in cancer in numerous publications, and for which a pharmacologic inhibitor is currently being evaluated in early clinical trials. There has been particular interest in its role in triple-negative breast cancer. In short, they made use of CRISPR cutting technology to generate MELK mutant alleles in a panel of cancer cell lines, including several in which MELK dependency has previously been reported. In cell culture assays including competitive cell growth, proliferative potential, and anchorage-independent growth, they used these cells to demonstrate that, in contrast to previous claims, MELK disruption did not seem to significantly affect the oncogenic potential of any of the tested cell lines. Furthermore, after generating single cell-derived MELK-deficient clones of one of these lines, they showed that the clinical small molecule MELK inhibitor retained its growth inhibitory properties, suggesting that this inhibitor displays off-target activities that are responsible for the observed effects on cell growth. They also examined published datasets associated with independently performed CRISPR and RNAi screens that included MELK-targeted agents and concluded that MELK dependency was not observed in any of those unbiased screens. Based on these findings, the authors conclude that previously reported MELK dependency in cancer is likely to reflect an experimental artifact, and that the MELK inhibitor that is currently being evaluated in the clinic may be functioning through a distinct, currently unknown mechanism.Overall, this is a well-executed and well documented study. Although these are essentially "negative results", the fact that others are still pursuing MELK as a candidate therapeutic target, and that clinical trials focused on MELK inhibition are underway, justifies publication of the findings.Essential revisions:1) The authors need to demonstrate that the MELK protein is substantially depleted in cell lines following CRISPR engineering. They have shown this for one cell line by western blot, but it should be shown for a few of the other lines as well to rule out the possibility that in some (or even many) of these lines, a small amount of "residual", non-mutated, expressed MELK is sufficient to maintain normal proliferation.2) The authors have not mentioned one of the most likely possibilities for the MOA of OTS167, which is polypharmacology. It could even be that OTS167 inhibition of MELK + Target X explains how it kills cells. Most kinase inhibitors, including the prominent approved drugs, Gleevec (bcr-abl + ckit), dasatinib (Bcr-abl + src family kinases), Sutent (many) work in this manner. The single gene knockout studies here would not show differences in this case. This should be acknowledged explicitly.3) The authors should also address other possible explanations of the difference between genetic perturbation (compensation could take place by upregulation of other kinases during the time it takes to deplete the protein after gene editing) and pharmacology . These differences can be acute in the setting of oncogene addiction/cancer cell signaling. Essential revisions:1) The authors need to demonstrate that the MELK protein is substantially depleted in cell lines following CRISPR engineering. They have shown this for one cell line by western blot, but it should be shown for a few of the other lines as well to rule out the possibility that in some (or even many) of these lines, a small amount of "residual", non-mutated, expressed MELK is sufficient to maintain normal proliferation.To address the reviewers’ concern that residual MELK protein may remain in our cell lines, we derived a second set of MELK-knockout clones, this time from the Cal51 triple-negative breast cancer cell line . We confWe also conducted additional western blot analyses on sorted populations of A375 and Cal51 cells transduced with MELK gRNAs . We obseIn total, our manuscript presents functional evidence that our CRISPR system is active (dropout of gRNAs targeting PCNA and RPA3), sequencing evidence that our MELK gRNAs efficiently induce indel mutations in the MELK gene, immunoblot evidence that these indel mutations result in the loss of MELK protein expression in multiple independent cell lines, and we confirm that no residual MELK protein is left in six MELK-knockout clonal lines.2) The authors have not mentioned one of the most likely possibilities for the MOA of OTS167, which is polypharmacology. It could even be that OTS167 inhibition of MELK + Target X explains how it kills cells. Most kinase inhibitors, including the prominent approved drugs, Gleevec (bcr-abl + ckit), dasatinib (Bcr-abl + src family kinases), Sutent (many) work in this manner. The single gene knockout studies here would not show differences in this case. This should be acknowledged explicitly.In light of the apparent promiscuity of OTS167 , we agree with the reviewers that polypharmacology is a likely MOA of OTS167. We have added a discussion of this possibility in the fourth paragraph of the Discussion section.3) The authors should also address other possible explanations of the difference between genetic perturbation (compensation could take place by upregulation of other kinases during the time it takes to deplete the protein after gene editing) and pharmacology . These differences can be acute in the setting of oncogene addiction/cancer cell signaling.We agree that one potential drawback of CRISPR is the lag time between the introduction of a gRNA, the loss of the targeted protein, and the ability to analyze a resulting phenotype. In particular, during the periods of growth required to generate and analyze pure GFP+ populations or MELK-knockout clones, it is possible that cells could “adapt” to the loss of MELK. However, we believe that the cell competition assays that we also performed give us insight into the more immediate consequences of MELK loss, as cells are under observation from the time in which the gRNA is introduced onward. A transient loss of cell fitness, followed by adaptation, would be detectable as a decrease in GFP+ cells in passage 2 or 3 of this assay, followed by stabilization of the GFP+/GFP- ratio. In total, we conducted 91 independent cell competition assays (7 MELK gRNAs in 13 cell lines) that could have revealed a transient fitness defect, but we failed to detect any evidence that this was occurring. It’s true that we cannot rule out the possibility that immediate adaptation is taking place, e.g. cells are able to compensate for MELK loss in such a way that not even a transient fitness defect is evident, but we believe that our experiments have given us a good chance to detect anything short of perfect compensation. To clarify these points, we have added a discussion of this issue in our manuscript .


Even in Ph-negative ALL cell lines, IKZF1 deletion and bilallelic loss of CDKN2A were independently associated with higher BTZ sensitivity. BTZ showed only marginal cross-resistance to four representative chemotherapeutic agents  in B-cell precursor-ALL cell lines. To improve the efficacy and safety of proteasome inhibitor combination chemotherapy, we also analyzed the anti-leukemic activity of carfilzomib (CFZ), a second-generation proteasome inhibitor, as a substitute for BTZ. CFZ showed significantly higher activity than BTZ in the majority of ALL cell lines except for the P-glycoprotein-positive t ALL cell lines, and IKZF1 deletion was also associated with a favorable response to CFZ treatment. P-glycoprotein inhibitors effectively restored the sensitivity to CFZ, but not BTZ, in P-glycoprotein-positive t ALL cell lines. P-glycoprotein overexpressing ALL cell line showed a CFZ-specific resistance, while knockout of P-glycoprotein by genome editing with a CRISPR/Cas9 system sensitized P-glycoprotein-positive t ALL cell line to CFZ. These observations suggested that IKZF1 deletion could be a useful biomarker to predict good sensitivity to CFZ and BTZ, and that CFZ combination chemotherapy may be a new therapeutic option with higher anti-leukemic activity for refractory ALL that contain P-glycoprotein-negative leukemia cells.Prognosis of childhood acute lymphoblastic leukemia (ALL) has been dramatically improved. However, prognosis of the cases refractory to primary therapy is still poor. Recent phase 2 study on the efficacy of combination chemotherapy with bortezomib (BTZ), a proteasome inhibitor, for refractory childhood ALL demonstrated favorable clinical outcomes. However, septic death was observed in over 10% of patients, indicating the necessity of biomarkers that could predict BTZ sensitivity. We investigated  In vitro anti-leukemic activity of BTZ against ALL was firstly reported in 2000 [in vitro when combined with standard chemotherapeutic agents [Bortezomib (BTZ) is a proteasome inhibitor approved for the treatment of multiple myeloma (MM) . Recentl in 2000 . Subsequ in 2000 , and, in in 2000 . However in 2000 . In contc agents . Based oc agents , and a fc agents : 16 (73%Carfilzomib (CFZ), a second-generation proteasome inhibitor, demonstrated more potent and more specific proteasome inhibition against the chymotrypsin-like activity of the 20S proteasome in a stable and irreversible fashion –12. CFZ in vitro BTZ sensitivity and possible cross-resistance of BTZ with conventional chemotherapeutic agents using a large panel of ALL cell lines. We also tested the in vitro anti-leukemic activity of CFZ in ALL cell lines as a possible substitute for BTZ in BTZ combination chemotherapy for refractory ALL.In the present study, we investigated the association of cytogenetic abnormalities with 2 at 37°C.Seventy-nine BCP-ALL cell lines, nine T-ALL cell lines, and two MM cell lines listed in 5 cells were plated into a 96-well flat-bottom plate and assays performed in triplicate in the presence or absence of seven concentrations of each drug. The cells were cultured for 44 h for sensitivities to BTZ, CFZ, DNR, and VCR, or 68 h for sensitivities to Dex and L-Asp. After a 6 h additional incubation with alamarBlue, absorbance at 570 nm were monitored by a microplate spectrophotometer using 600 nm as a reference wavelength. Cell survival was calculated by expressing the ratio of the optical density of treated wells to that of untreated wells as a percentage. The concentration of agent required to reduce the viability of treated cells to 50% of untreated cells was calculated, and the median of three independent assays was determined as IC50 for each cell line.To determine the 50% inhibitory concentrations (IC50s) of BTZ, CFZ, Dex, daunorubicin (DNR), VCR, and L-Asp, an alamarBlue cell viability assay  was performed. 1–4 x 10To detect apoptosis, cells were cultured in the absence or presence of BTZ or CFZ for 17–18 h, stained with a fluorescein isothiocyanate (FITC)-conjugated Annexin-V and 7AAD . To verify combination of P-glycoprotein inhibitors, cells were pretreated with verapamil or nilotinib for 2 h, and subsequently cultured in the absence or presence of BTZ or CFZ for 17–18 h. Cell surface expression of P-glycoprotein was analyzed with a FITC-conjugated anti-P-glycoprotein antibody . For the functional assay of P-glycoprotein-mediated efflux of calcein-AM , cells were incubated with 0.25 mM of CAM for 10 min at 37°C in the presence or absence of verapamil or nilotinib. To detect apoptosis in P-glycoprotein knockout HALO1 cells, cells were stained with a FITC-conjugated anti-P-glycoprotein antibody and a phycoerythrin (PE)-conjugated Annexin-V . The stained cells were analyzed by flow cytometry .3, 1 mM phenylmethylsulfonyl fluoride, 100 μM sodium vanadate). The lysates were separated on a SDS-polyacrylamide gel under reducing conditions and then transferred to a nitrocellulose membrane. The membrane was incubated with the primary antibody at 4°C overnight, and then with horseradish peroxidase-labeled secondary antibody at room temperature for 1 hour. The bands were developed using an enhanced chemiluminescence detection (ECL) kit . Anti-CHOP  and anti-NOXA  mouse monoclonal antibodies were purchased from Cell Signaling Technology  and abcam , respectively. Horseradish peroxidase-conjugated anti-mouse IgG  was purchased from MBL .Cells were solubilized in lysis buffer . The relative copy number, obtained after normalization against controls, was used to determine genomic copy number of each gene. Values between 0.75 and 1.3 were considered to be within normal range. Values below 0.75 or 0.25 indicated monoallelic or biallelic loss, respectively.SALSA multiplex ligation-dependent probe amplification (MLPA) kit P335-A4 was used according to the manufacturer’s instructions  . The kitABCB1 gene using the CRISPR design tool . We selected 5’-tttggctgccatcatccatgg-3’, which showed the highest off-target hit score, and the synthesized oligomers were cloned into CRISPR/Cas9 vector . Three days after electroporation of the ABCB1-targeting CRSIPR/Cas9 vector using Neon electroporation transfection system (Thermo Fisher Scientific) into HALO1 cells, CD4-positive cells were selected using CD4-microbeads  and expanded for further analyses.To knock out P-glycoprotein  expression with CRISPR/Cas9 system, we screened exon 3 of the IKZF1 deletion and biallelic loss of CDKN2A in 65 Ph-negative BCP-ALL cell lines using Excel software.We applied Mann-Whitney test for comparison of drug sensitivities and chi-square test for comparison of incidence in gene deletion. We applied a paired t-test for comparison of two means that were from the same objects and Pearson’s correlation analysis for correlation between paired data. All analyses were performed using Excel software. A multivariate analysis of log IC50 value of BTZ was performed to evaluate the effects of in vitro sensitivity to BTZ using 79 BCP-ALL, 9 T-ALL, and 2 MM cell lines. Based on dose-response curves determined by the alamarBlue cell viability assay using seven concentrations of BTZ that ranged from 1.25 nM to 80 nM -ALL, 4 t-ALL, and 3 t-ALL cell lines  and t-ALL cell lines . No statistically significant difference was observed between Ph+ALL cell lines and t-ALL cell lines . Further, t-ALL cell lines tended to be more sensitive to BTZ than t-ALL cell lines (p = 0.098).We compared the sensitivity of 79 BCP-ALL cell lines with different types of translocations to BTZ, which included 14 Ph+ALL, 11 ll lines . The remIKZF1 [CDKN2A/CDKN2B [IKZF1, CDKN2A, and CDKN2B, while 11 (16.9%), 36 (55.4%), and 40 (61.5%) out of 65 Ph-negative BCP-ALL cell lines carried deletions of IKZF1, CDKN2A, and CDKN2B, respectively (IKZF1 (p<0.00001 by chi-square test), CDKN2A (p = 0.0014), or CDKN2B (p = 0.0035) was significantly more common in Ph+ALL cell lines than in Ph-negative BCP-ALL cell lines. Regarding deletion of CDKN2A/CDKN2B genes, biallelic loss of CDKN2A was significantly more common in Ph+ALL cell lines (64.3%) than in Ph-negative BCP−ALL cell lines , while there was no statistically significant difference in the incidence of biallelic loss of CDKN2B between Ph+ALL cell lines (42.9%) and Ph-negative BCP-ALL cell lines   .IKZF1 was significantly associated with higher BTZ sensitivity  was significantly lower than that of 54 BCP-ALL cell lines not carrying the deletion  , respectively, were almost similar, and those of 54 and 25 BCP-ALL cell lines with or without CDKN2B deletion , respectively, were identical  was significantly lower than that of 57 BCP-ALL cell lines with monoallelic or no loss of CDKN2A   was almost similar to that of 57 BCP-ALL cell lines with monoallelic or no loss of CDKN2B  (data not shown).Among the nine genes or region tested, only deletion of sitivity . The IC5ey test) . Althougdentical . When we= 0.012) , while tIKZF1 deletion and/or biallelic loss of CDKN2A. Among 65 Ph-negative BCP-ALL cell lines, 11 cell lines carrying IKZF1 deletion  were significantly more sensitive to BTZ than 54 cell lines without the deletion   were significantly more sensitive than 52 cell lines with monoallelic or no loss of CDKN2A  (p = 0.032)  and biallelic loss of CDKN2A (p = 0.007) were independently associated with higher BTZ sensitivity.The above observations suggested that higher BTZ sensitivity of Ph+ALL cell lines may be associated with ey test) , and 13 = 0.032) . Among 1f CDKN2A , suggestll lines . Of noteTo investigate cross-resistance of BTZ to the chemotherapeutic agents that were used in the BTZ combination therapy in the TACL study , 9, we ein vitro anti-leukemic activity of CFZ, a second-generation proteasome inhibitor with irreversible activity [We next investigated the activity . InductiMLL+ALL , t-ALL , t-ALL , and B-other ALL cell lines . Although deletion of IKZF1 was not associated with higher CFZ sensitivity in 79 BCP-ALL cell lines including Ph+ALL cell lines, 11 Ph-negative BCP-ALL cell lines carrying IKZF1 deletion  were significantly more sensitive to CFZ than 54 Ph-negative BCP-ALL cell lines without the deletion  . Finally, we examined the cross-resistance of CFZ to the four drugs that were used in the TACL study regimen using 79 BCP-ALL cell lines  . With rell lines . There wIn general, the anti-leukemic activity of CFZ was higher than that of BTZ, and the IC50 of CFZ was significantly lower than that of BTZ in 79 BCP-ALL cell lines and 9 T-ALL cell lines (p<0.000001 by paired t-test) . The medTo further verify a possible involvement of P-gp expression in specific resistance to CFZ, we examined the sensitivities to CFZ and BTZ in the presence or absence of verapamil  or nilotTo directly confirm an association of P-gp expression with specific resistance to CFZ, we first analyzed P-gp-positive subline of 697 cell line  that wasABCB1-specific CRISPR/Cas9 vector that contains cDNA of Cas9 fused to human CD4 cDNA via a 2A peptide sequence was electroporated into HALO1 cells. 3 days after electroporation, CD4-positive cells were harvested using anti-CD4 antibody and subsequently expanded for further analysis. Flow cytometric analysis of P-gp expression Click here for additional data file.S2 FigShade indicates fluorescence intensity of anti-P-glycoprotein antibody, and line indicates that of control antibody. Relative fluorescence intensity of each cell line is indicated in the middle of each panel, and ratio of bortezomib IC50 value to carfilzomib IC50 value of each cell line is indicated at the top of each panel.(TIF)Click here for additional data file.S3 Fig(A) Flow cytometric analysis of CAM staining in P-glycoprotein-positive HALO1 cells (left panels) and P-glycoprotein-negative RS4;11 cells (right panels) cultured in the presence or absence of P-glycoprotein (P-gp) inhibitors (0.8 μM of nilotinib or 5 μM of verapamil). Geometric mean (GeoMean) of CAM staining is indicated in each panel. (B) Effect of P-glycoprotein (P-gp) inhibitors on CAM staining of P-glycoprotein-positive HALO1 cells (left panel) and P-glycoprotein-negative RS4;11 cells (right panel). The vertical axis indicates GeoMean of CAM staining. Mean ± SD of triplicated experiments are indicated. Asterisks indicate significance (**p<0.01) in a paired t-test.(TIF)Click here for additional data file.S4 Fig(A) Flow cytometric analysis of CAM staining in parental 697 cells (left panels) and 697R cells (right panels) cultured in the presence or absence of P-glycoprotein (P-gp) inhibitors (0.8 μM of nilotinib or 5 μM of verapamil). Geometric mean (GeoMean) of CAM staining is indicated in each panel. (B) Effect of P-glycoprotein (P-gp) inhibitors on CAM staining of parental 697 cells (left panels) and 697R cells (right panels). The vertical axis indicates GeoMean of CAM staining. Mean ± SD of triplicated experiments are indicated. Asterisks indicate significance  in a paired t-test.(TIF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.S2 Table(TIF)Click here for additional data file.S3 Table(TIF)Click here for additional data file.S4 Table(TIF)Click here for additional data file.