Cell lines and cell culture
FaDu (male, hypopharyngeal squamous cell carcinoma, grade II) and Detroit 562 cells (female, oropharyngeal squamous cell carcinoma, metastatic) were purchased from the ATCC. The HNSCC cell lines UT-SCC38 (male, laryngeal squamous cell carcinoma, T2N0M0, primary) and UT-SCC42B (male, laryngeal squamous cell carcinoma, T4N3M0, metastatic) derived from laryngeal squamous carcinomas were kindly provided by R. Grenman (Department of Otolaryngology, University Central Hospital, Turku, Finland). HCA-LSC1 cell line was established in our laboratory from a male patient, primary laryngeal squamous carcinoma resistant to chemotherapy.
Cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 200 mg/mL streptomycin, 2 mmol/L L-glutamine, 20 mmol/L HEPES (pH 7.3), and 100 mmol/L non-essential amino acids. All the cells derived from HPV-negative primary HNSCC. All cell lines were periodically tested for mycoplasma contamination by PCR using the Biotools Detection kit (Madrid, Spain) specifically amplifying a conserved region of the mycoplasma 16S RNA gene. Cell line authentication was carried out by DNA (STR) profiling at the SCT Core Facilities (University of Oviedo, Spain).
Cas9-expressing HNSCC cell lines were generated by lentiviral transduction using pKLV2-EF1a-Bsd2Cas9-W (Addgene, #67978). Blasticidin selection was initiated 3 days after transduction at 20 µg/mL and maintained for at least 14 days.
CRISPR/Cas9 KO screen
For each HNSCC cell line, a total of 1.0 x 108 cells were transduced with a predetermined volume of the genome-wide gRNA lentiviral supernatant that gave rise to 30% transduction efficiency. Two days after transduction, cells were selected with puromycin for 5 days and further cultured, always keeping the total population above 3.0 x 107. After 25 days of culturing, at least 3.0 x 107 cells were collected as the final time point.
Illumina sequencing of gRNAs and statistical analysis
Genomic DNA extraction and Illumina sequencing of gRNAs were conducted as described previously (23). The numbers of reads for each guide were counted with an in-house script. Enrichment and depletion of guides and genes were analyzed using MAGeCK statistical package (30) by comparing read counts from each cell line with counts from matching plasmid as the initial population and used for DNA extraction and gRNA sequencing.
Generation of CDK7 KO cell lines
HNSCC cells lines expressing Cas9 were transduced with two specific CDK7 gRNAs (KO1 or KO2) by subcloning each gRNA targeting sequence (CDK7_KO1 guide RNA: TTCCATAAAATCAAAGACA; CDK7_KO2 guide RNA: TAAAAACCTTACCCTATGT) into the expression vector pKLV2-U6gRNA5(BbsI)-PKGpuro2AZsG-W (Addgene #67975) or with the empty vector as control. For lentiviral production, pLP1 (Addgene #209988), pLP2 (Addgene #20989), and the envelope plasmid pLP/VSV-G (Life Technologies) were transfected in HEK293T cells The resulting lentiviral particles were used to transduce HNSCC cells, which were then selected in puromycin (2–3 µg/mL) containing medium for six days.
Competitive proliferation assay
Cas9-expressing HNSCC cells were transduced at 50% efficiency with lentiviral particles containing specific gRNAs targeting CDK7 (either CDK7 KO1 or KO2) or an empty vector as a control were used. The percentage of green fluorescent protein (ZsG)-positive cells was measured by flow cytometry between days 6 and 13 post-transduction and normalized to the percentage of ZsG-positive cells at day 4. Data are represented as the relative number of (ZsG)-positive cells in each well.
Drugs
YKL-5-124 and samuraciclib (CT7001) were obtained from MedChem Express. Palbociclib was obtained from Selleckchem. For in vitro studies, stock solutions of both compounds were prepared at a concentration of 10 mM in sterile dimethyl sulfoxide (DMSO) and stored at − 80°C. Prior to each experiment, the drugs were thawed and diluted to the desired final concentrations. DMSO was used as the vehicle control condition.
For in vivo studies, YKL-5-124 and samuraciclib were prepared in a vehicle of 10% DMSO, 40% PEG-300, 5% Tween-80, and 45% saline at a stock concentration of 20 mg/mL or 100mg/mL, respectively, and stored at -80ºC. Usage concentration was prepared daily before administration. YKL-5-124 was administered intraperitoneally at 10 mg/kg, five days a week, with a corresponding intraperitoneal vehicle control group. Samuraciclib was given orally at 50 mg/kg daily, with a separate oral vehicle control group.
Cell viability assays
HNSCC cells were seeded into 96-well culture plates at a density of 2,000–4,000 cells per well and incubated overnight. Drugs were serially diluted in medium over a range of concentrations and added to the cells. After 5 days of treatment, cell viability was measured in triplicates or quadruplicates using MTS assay (CellTiter 96 Aqueous One Solution Cell Proliferation Assay from Promega, Madison, WI, USA) reading absorbance at 490 nm using a Synergy HT plate reader (BioTek, Winooski, VT, USA). For the IC50 studies, the number of viable cells upon each drug treatment was normalized to the number of vehicle (DMSO)-treated cells at day 5 and the IC50 values were calculated using Graphpad Prism10 as the [inhibitor] vs. normalized response – Variable slope function.
Proliferation assays
HNSCC cells were seeded into 6-well culture plates at a density of 3,000–5,000 cells per well and incubated overnight. YKL-5-124 or samuraciclib were serially diluted in medium over a range of concentrations and added to the cells. Treatment was renewed every 3 days. After 14 days of culture, cells were fixed with methanol and stained with crystal violet 0.1% w/v. Colonies were, then, scanned with GS-800 Calibrated Imagen Densitometer (Bio-Rad, 170–7980) and images were analyzed with Fiji software to measure the number of colonies and surface per well. Data were normalized to the DMSO-treated control condition.
Cell cycle analysis
HNSCC cells were plated in 6-well plates in complete cell culture medium. After 24 hours, cells were treated with either YKL-5-124, samuraciclib or vehicle in growth media for 72 hours. Then, cells were collected and fixed in cold 70% ethanol for at least 24 hours at -20ºC. Cell cycle analysis was performed using FxCycle™ PI/RNase Staining Solution (Life Technologies, #F10797) to measure DNA content by flow cytometry, according to the manufacturer’s instructions. Cell percentage in each cell cycle phase was determined using FlowJo software’s Cell cycle algorithm. Only significant accumulation of cells in a cell cycle phase is indicated
Apoptosis assay
HNSCC cells were plated in 6-well plates, incubated for 24 hours, and then treated with either YKL-5-124, samuraciclib or vehicle for 48 hours. Apoptotic cells were quantified through Annexin V and Propidium Iodide (PI) staining, using Dead Cell Apoptosis Kit with Annexin V FITC/Alexa Fluor™ 488 & Propidium Iodide for Flow Cytometry (Invitrogen, #V13242 and #V13241) according to manufacturer’s instructions.
Tumorsphere formation assays
HNSCC-derived cells lines were plated at a density of 500 cells/mL in 6-well tissue culture plates treated with a sterile solution of polyHEMA (10 g/L in 95% ethanol) (Sigma) to prevent cell attachment. Cells were grown in DMEM-F12 (GE Healthcare) supplemented with 1% Glutamax and 2% B27 Supplement (Life Technologies), 10 ng/mL human bFGF and 20 ng/mL human EGF (PeproTech) and 100 U/mL penicillin and 200 mg/mL streptomycin (Thermo Scientific). In addition, fresh aliquots of EGF and bFGF were added every three days. After 7 days, tumorspheres were treated for 5 days with different concentrations of either YKL-5-124, samuraciclib, or DMSO as vehicle condition. Tumorsphere viability was measured using the CellTiter-Glo 3D assay (Promega, #G9681) and luminescence quantification using a Synergy HT plate reader (BioTek).
Western blot analysis
Cells were lysed in RIPA buffer (Thermo Scientific, 89900) supplemented with protease and phosphatase inhibitors (Sigma Aldrich, 78430). Protein concentration was determined by BCA assay (Thermo Scientific, 23225), samples were subjected to SDS-PAGE by using NuPAGE™ 4 to 12% Bis-Tris gels (Life Technologies) in MOPS running buffer and blotted on Trans-Blot® Turbo™ Midi Nitrocellulose membranes (Bio-Rad Laboratories). Membranes were blocked using 5% BSA in TBS-T for 1 hour at room temperature. Membranes were then incubated with primary antibodies (Supplementary Table S1) overnight at 4ºC, then washed in TBS-T and incubated with secondary antibodies goat anti-Rabbit IRDye 800CW or anti-Mouse IRDye 680RD (IRDye, LICOR, at 1:10,000 dilution) for 1 hour at room temperature. Fluorescence was measured using Odyssey® Fc Imager (LICOR Biosciences) and analyzed with Image Studio Lite software (LICOR Biosciences).
RNA sequencing (RNA-seq) and bioinformatics analysis
Total RNA was isolated from preconfluent FaDu cells using GeneJET RNA Purification Kit (Thermo Scientific). For all the experimental conditions, RNA extraction and sequencing was done in triplicates. Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non-directional library.
Sequencing and initial bioinformatics analyses were performed by Novogene, Inc (Cambridge, UK). Briefly, paired-end clean reads were aligned to the GRCh38 reference genome using Hisat2 v2.0.5. GENCODE database was used to transcriptome annotations. Differential expression analysis of CDK7-inhibitor treatment conditions versus control (three biological replicates per condition) was performed using the DESeq2 R package (1.20.0).
Gene Set Enrichment Analysis for Hallmarks Gene Sets was performed by GSEA_4.1.0 on normalized gene counts lists. Only the Hallmark pathways that are significant (FDR < 0.25, GSEA weighted Kolmogorov-Smirnov test, p < 0.05) positively or negatively enriched by normalized enrichment score (NES) are shown. GSEA for Reactome pathways was performed for commonly downregulated or upregulated genes upon each treatment on GSEA web. Only statistically significant changes (FDR < 0.05) are shown.
Differential expression of common essential genes upon CDK7 inhibition in the panel of five HNSCC cell lines was represented as z-score of log2 (CPM).
Mouse xenografts
All experimental protocols were performed in accordance with the institutional guidelines of the University of Oviedo and CIEMAT, and approved by the corresponding Animal Research Ethical Committee prior to the study (date of approval 1st August 2019; approval number PROAE 46/2019; date of approval 8th March 2024; approval number PROAE 03/2024; date of approval 12th February 2021; approval number PROEX 045.8/21).
Female AthymicNude-Fox1nu mice of 5–6 weeks old (ENVIGO RMS) were subcutaneously (s.c.) inoculated in the flanks with 1.5x106 FaDu or 2x106 HCA-LSC1 cells in culture medium mixed 1:2 with VitroGel® Hydrogel Matrix (The Well Bioscience, #VHM01). Once tumors reached a measurable size (between 100 and 200 mm3), mice were randomized into four treatment groups (six mice per group): (i) Intraperitoneal vehicle; (ii) Oral vehicle; (iii) YKL-5-124 (10 mg/kg, 5 doses/week, intraperitoneal) and (iv) samuraciclib (50 mg/kg, 7 doses/week, orally). Mice were monitored daily for signs of toxicity and tumor size was measured with a caliper 2–3 times a week.
Tumor volume was determined using the equation (D × d2)/6 × 3.14, where D is the maximum diameter, and d is the minimum diameter. Tumor volumes for all mice in each xenograft-treatment group were averaged to obtain the mean tumor volume for the corresponding group. Animals were sacrificed by CO2 asphyxiation or cervical dislocation when the tumors of the control group reached approximately 1,000 mm3. Tumors were resected from the flanks, fixed in formol and embedded in paraffin for histological evaluation.
Patient-derived organoids (PDOs)
HNSCC PDOs were generated from fresh primary tumor biopsies from HNSCC patients surgically treated at the Hospital Universitario Central de Asturias (HUCA), following institutional review board guidelines, and approved by the Regional Ethics Committee from the Principality of Asturias (CEImPA) (date of approval 25th January 2021; approval number 2021.002, for the project PID2020-117236RB-100). Informed consent was obtained from all patients. Tissue samples were obtained through the Biobank of Principado de Asturias (PT23/00077, part of the ISCIII Platform of Biobanks and Biomodels) and processed as described by Driehuis and colleagues (31).
PDOs were grown in Matrigel droplets (Corning #356231) in expansion medium, consisting of basal medium (Advanced DMEM/F12 supplemented with 1% penicillin/streptomycin, 1% GlutaMAX, and 10 mM Hepes), supplemented with 1X B27 (Gibco #17504044), 10 mM Nicotinamide (Sigma #N0636), 1.25 mM N-Acetylcysteine (Sigma #A9165), 500 nM A83-01 (Tocris #2939), 1 µM Prostaglandin E2 (Tocris #2296), 0.3 µM CHIR99021 (Sigma #SML1046), 1 µM Forskolin (Tocris #1099), 50 ng/mL hEGF (Peprotech #AF-100-15), 10 ng/mL hFGF10 (Peprotech #AF-100-26), 5 ng/mL hFGF2 (Peprotech #AF-100-18B), 100 ng/mL human Noggin (Peprotech #120-10C), and 200 ng/mL human R-spondin-1 (hRspo) (Peprotech #120 − 38).
For drug response assays, organoids that had been cultured for two days were harvested using 1 mg/mL of Dispase (Sigma, #D4693-1G) to remove Matrigel. Organoids were subsequently washed and filtered using a 70 µM strainer to ensure uniform size, reducing variability in the assay. The filtered organoids were counted and seeded at a density of 2,000 organoids per well into 96-well plates (Nunc, Thermo, #236105) using 5 µL of 95% Matrigel per well. After three days, organoids were treated with increasing concentrations of the specified drugs prepared in the expansion media using DMSO as vehicle control. Five days post-treatment, the viability of the PDOs was assessed using the CellTiter-Glo 3D assay (Promega, #G9681) by measuring luminescence using a Synergy HT plate reader (BioTek).
Statistical significance
Statistical analysis was performed using GraphPad Prism version 6.0 (Graphpad Software Inc, La Jolla, CA, USA). Data is presented as the mean standard deviation (SD) of at least three independent experiments unless otherwise stated. Statistical significance was determined either using a Student’s unpaired t-test with two-tailed distribution for comparison across two groups or Two-Way ANOVA for comparing multiple samples/variables. In comparisons with control groups, the values of p < 0.05 were considered statistically significant (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).