Genome-wide functional CRISPR screen reveals CDK7 as a targetable therapeutic vulnerability for head and neck cancer

Genome-wide functional CRISPR screen reveals CDK7 as a targetable therapeutic vulnerability for head and neck cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genome-wide functional CRISPR screen reveals CDK7 as a targetable therapeutic vulnerability for head and neck cancer María Otero-Rosales, Miguel Álvarez-González, Beatriz de Luxán-Delgado, and 15 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4679708/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Head and neck squamous cell carcinoma (HNSCC) remains a challenging prevalent lethal malignancy, with still scarce targeted therapies and rather limited clinical benefit. We conducted an optimized genome-wide functional CRISPR screen aimed at identifying actionable genetic vulnerabilities for rapid preclinical evaluation as novel targeted therapies. Cyclin-dependent kinases (CDKs) were prioritized as pivotal in cancer therapy. Methods: Whole-genome CRISPR KO screen was performed in a panel of five HNSCC cell lines. CDK7 was selected for further functional and molecular characterization. The effects of CRISPR CDK7 knockout (KO) and CDK7-selective inhibitors were thoroughly investigated in cellular models using viability, colony formation and apoptosis assays, cell cycle analysis, and global transcriptomics by RNAseq. CDK7 inhibition was also therapeutically evaluated in mouse xenografts and patient-derived organoids (PDOs). Results : CDK7 was identified as an essential gene across all five HNSCC cell lines screened. Genetic and pharmacological CDK7 inhibition significantly and consistently reduced tumor cell proliferation due to generalized cell cycle arrest and apoptosis induction. CDK7 KO, YKL-5-124 and samuraciclib also showed a potent antitumor activity effectively abrogating tumor growth in HNSCC PDOs and also in mouse xenograft models without significant toxicity. Mechanistically, CDK7 inhibition led to a broad downregulation of gene sets for cell cycle progression, DNA repair, and massively reduced the transcription of several essential genes and untargetable vulnerabilities identified by our CRISPR screen. Conclusions : CDK7 emerges as a promising targetable therapeutic vulnerability for HNSCC. Our study provides broad-based evidence for the robust antitumor activity of CDK7-selective inhibitors in disease-relevant preclinical models, strongly supporting patient testing. Head and neck squamous cell carcinoma CRISPR screen genetic vulnerability CDK7 essential gene cell cycle YKL-5-124 samuraciclib Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Head and neck squamous cell carcinoma (HNSCC) is the sixth most prevalent cancer globally, with significant morbidity and mortality rates. Despite advances in multimodal treatment strategies, including surgery, radiation, and chemotherapy, the five-year survival rate for HNSCC patients remains between 40–50% ( 1 ). The prognosis is particularly unfavorable for patients with recurrent or metastatic disease ( 2 ). Nowadays, precision medicine for HNSCC is restricted to the EGFR-specific inhibitor cetuximab and the immunotherapy agents nivolumab and pembrolizumab ( 3 – 5 ). Treatment with cetuximab benefits a minority of HNSCC patients despite harboring high EGFR gene amplification, and its effectiveness is often hindered by tumor resistance. Similarly, even though immunotherapeutic agents such as nivolumab and pembrolizumab have recently emerged as promising treatment options, their clinical benefit in HNSCC patients is rather low (20–30%). These challenges underline the need for identifying novel therapeutic targets to improve treatment outcomes in this disease. Cyclin-dependent kinases (CDKs) are pivotal in cancer therapy due to their role in cell cycle regulation and proliferation. CDK4/6 inhibitors such as palbociclib have demonstrated efficacy, particularly in hormone receptor-positive breast cancer ( 6 – 8 ). However, their therapeutic capability/application is often limited by the development of drug resistance and adverse side effects. In HNSCC, CDK inhibitors hold promising therapeutic potential and continue to be an active area of research. Recent studies suggest a potential synergy when combining palbociclib with cetuximab and radiotherapy ( 9 ). Among the CDKs, CDK7 has been identified as a crucial regulator in cancer biology. This kinase forms a trimeric complex with Cyclin H and MAT1, functioning as a Cdk-activating kinase (CAK) that regulates both cell cycle progression and global transcription. CDK7 controls the cell cycle at different levels by phosphorylating CDKs 1, 2, 4 and 6 in their T-loops promoting their activation ( 10 – 13 ). Additionally, the CAK complex is a key component of the transcription factor TFIIH involved in transcription initiation and elongation ( 14 ). CDK7 is aberrantly overexpressed in different types of cancer ( 15 – 17 ) and significant progress has been made in the development of selective CDK7 inhibitors, which have shown efficacy preclinically in multiple tumor types including breast ( 18 , 19 ), pancreatic ( 20 ), and lung cancer ( 21 ) among others. For the last decade, genome-wide CRISPR screens have revolutionized the landscape of target identification in biomedical research, offering a powerful tool to elucidate the functional relevance of genes across the entire genome ( 22 , 23 ). While being extensively utilized in the past several years to define the landscape of important biological processes, these screens continue to be developed and are indispensable in uncovering novel therapeutic targets and dissecting complex biological pathways in vitro and in vivo ( 24 – 29 ). Taking advantage of an optimized genome-wide functional CRISPR screen ( 27 ), we aimed to identify actionable genetic vulnerabilities that can be rapidly evaluated as potential targeted therapies in preclinical HNSCC models. As a result, our study led to the identification of CDK7 as a promising clinically targetable vulnerability in HNSCC. A detailed functional and molecular characterization of available CDK7-selective inhibitors further contributed to unprecedentedly demonstrate their robust antitumor activity in a broad range of disease-relevant cellular, patient-derived organoids (PDOs) and animal models. These findings encourage future clinical testing in HNSCC patients. Materials and Methods 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 10 8 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 10 7 . After 25 days of culturing, at least 3.0 x 10 7 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 1.5x10 6 FaDu or 2x10 6 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 mm 3 ), 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 CO 2 asphyxiation or cervical dislocation when the tumors of the control group reached approximately 1,000 mm 3 . 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). Results Genome-wide functional CRISPR-Cas9 screen revealed CDK7 as a vulnerability in HNSCC cells As an attempt to uncover essential genes with therapeutic potential, we conducted a CRISPR-Cas9 knockout screen across five different HNSCC cell lines: FaDu, UT-SCC38, HCA-LSC1, UT-SCC42B and Detroit 562. We used a comprehensive whole-genome library, targeting 18,010 genes with 90,709 different gRNAs ( 27 ). The HNSCC cell lines were transduced using a low multiplicity of infection (MOI) of 0.3 and subsequently selected with puromycin to ensure efficient integration of the gRNAs. Cells were cultured for 25 days, after which genomic DNA was extracted, and gRNAs were amplified and sequenced (Fig. 1 a). Guide-RNA depletion and enrichment was analyzed using MAGeCK software ( 30 ) to identify negatively and positively regulated genes, indicative of a role in cell viability. Cell-line-specific and commonly essential genes were determined based on their false discovery rate (FDR) score. Only genes with FDR < 0.10 were consider for further analysis, thus identifying between 500 and 1,500 essential genes per cell line (Fig. 1 b ). A focus on essential genes across multiple cell lines revealed 228 genes consistently essential in all five analyzed HNSCC cell lines (Fig. 1 c and Supplementary File S2) , including well-known oncogenes and drivers of HNSCC cell proliferation such as MYC and CCND1. Pathway analysis of the common essential genes showed a significant overlap in MYC and E2F targets and cell cycle checkpoint-related gene sets (Fig. 1 d). Given the translational potential of CDK inhibitors, we focused our attention on the essentiality of CDK proteins as targetable and druggable candidates. CDK1 and CDK7 emerged as the most crucial genes across all five HNSCC cell lines, while other CDKs such as CDK2 or CDK5 were found to be non-essential, or showed selectivity to certain cell lines, such as CDK4, CDK6 or CDK9 (Fig. 1 e). CDK1 is the only CDK in mammals that is essential for cell cycle progression ( 32 ). However, CDK1 inhibitors have either been associated with high toxicity or failed to demonstrate sufficient efficacy in patients. Interestingly, CDK7-selective inhibitors have been recently developed and are under current active testing in clinical trials ( 33 , 34 ). We therefore selected CDK7 for further investigation due to its critical role in cell cycle progression and transcription, as well as its function as an upstream regulator of other CDKs, including CDK1 itself. Single gRNA analysis revealed that every gRNA targeting CDK7 was depleted at the final time point in all HNSCC cell lines (Fig. 1 f). Genetic validation of CDK7 essentiality in HNSCC cells To validate the essentiality of CDK7 as a candidate gene in our screening, we first analyzed CDK7 protein levels in our HNSCC cell line panel ( Fig. 2 a ) . We next performed targeted CRISPR knockouts of CDK7 (CDK7 KO) using two distinct single gRNAs in three HNSCC cell lines: FaDu, UT-SCC38 and HCA-LSC1, which harbored different endogenous levels of CDK7. We confirmed that these two gRNAs robustly reduced CDK7 protein levels in all three HNSCC cell lines ( Fig. 2 b ) . Subsequently, we carried out competitive co-culture assays between control and KO cells to validate the results of our screen (Fig. 2 c). In these assays, CDK7 KO cells were significantly outcompeted by control cells in FaDu (Fig. 2 d), UT-SCC38 (Fig. 2 e) and HCA-LSC1 cells (Fig. 2 f ) , thus confirming a significant reduction in cell viability upon CDK7 depletion. These results highlight the critical role of CDK7 in maintaining cell proliferation/survival and validate our initial findings from the CRISPR screen. In addition, we assessed the effects of CDK7 KO on the phosphorylation of known direct CDK7 targets/substrates. Thus, we found that CDK7 depletion led to reduced phosphorylation levels of the Carboxy-terminal domain (CTD) of the RNA Polymerase II (Ser5), retinoblastoma protein (Rb) (Ser780), and CDK1 T-loop phosphorylation (Thr161) in the three HNSCC cell lines tested (Fig. 2 g). These data demonstrate that CDK7 depletion effectively abrogates HNSCC cell proliferation by targeting several key downstream effectors and regulators of cell cycle control and transcription. Therapeutic potential of CDK7 pharmacological inhibition The robust effects of genetic CDK7 KO prompted us to evaluate the therapeutic potential of available CDK7-selective inhibitors in preclinical HNSCC models. We selected two distinct compounds: YKL-5-124, a covalent selective CDK7 inhibitor ( 35 ), and samuraciclib (CT7001, ICEC0942), an orally bioavailable, ATP-competitive inhibitor ( 36 ) currently undergoing Phase I/II clinical trials in cancer patients ( 33 ). We first analyzed the effects of selective CDK7 inhibitors on the viability of a panel of five HNSCC cell lines. Both YKL-5-124 and samuraciclib (Fig. 3 a) significantly reduced HNSCC cell viability, although lower IC50 values were observed for YKL-5-124 (ranging from 35 to 100 nM) than samuraciclib (range 30 to 200 nM) after 5 days of treatment (Fig. 3 b). Remarkably, both CDK7-selective inhibitors were more effective in decreasing HNSCC cell viability than the CDK4/6 inhibitor palbociclib (> 10-fold higher IC50 values) (Fig. 3 a-b ). Analogous results were observed in colony formation assays performed along 14 days of treatment. Lower doses of YKL-5-124 and samuraciclib (ranging from 5 to 25 nM) completely abolished HNSCC cell proliferation, whereas considerably higher concentrations of palbociclib were required to achieve a comparable effect (Fig. 3 c). We next investigated the immediate molecular effects of CDK7 inhibition on the phosphorylation of downstream protein substrates. HNSCC cells were treated with 1 µM of either YKL-5-124 or samuraciclib for 24 hours to minimize potential compensatory mechanisms upon prolonged treatment. This resulted in decreased phosphorylation levels of CTD-RNA Polymerase II (Ser5), Rb (Ser780), and CDK1 (Thr161) in most HNSCC cell lines, thereby mirroring the changes previously observed by CDK7 KO. CDK7 protein levels remained unchanged or slightly increased upon treatment with CDK7 inhibitors. Noteworthy, a shift in CDK7 electrophoretic mobility was detected upon treatment with YKL-5-124 in all five HNSCC cell lines, likely due to the covalent binding of this compound (Fig. 3 d and Supplementary Figure S1 a). The functional consequences of CDK7 pharmacological inhibition were also investigated on cell cycle dynamics and apoptosis. YKL-5-124 treatment led to cell accumulation in S and/or G2/M phases depending on the cell line analyzed, and samuraciclib treatment led to cell accumulation in G2/M phase in all five HNSCC cell lines (Fig. 3 e and 3 f and Supplementary Figure S1 b and S1c ). This was also accompanied by a significant induction of apoptosis, especially after samuraciclib treatment, in all HNSCC cell lines tested ( Fig. 3 g and 3 h and Supplementary Figure S1 d) . Concordant to these findings, increased levels of cleaved PARP were also detected by Western blot upon YKL-5-124 and samuraciclib treatment (Fig. 3 d and Supplementary Figure S1 a ). Global transcriptional changes caused by CDK7 inhibition In order to delineate the global transcriptional changes caused by CDK7 inhibition, RNA-seq experiments were performed in FaDu cells treated with either YKL-5-124 or samuraciclib for 48 hours. As represented by Volcano plots, a total of 804 and 1,147 genes were found significantly up- and downregulated by YKL-5-124, respectively (Fig. 4 a), and 630 and 989 were significantly up- and downregulated genes upon samuraciclib treatment, respectively (Fig. 4 b). In addition, Venn diagrams depict the unique and overlapping changes by YKL-5-124 and samuraciclib, with a total of 645 common downregulated genes (Fig. 4 c) and 376 common upregulated genes (Fig. 4 d). GSEA using reactome pathways database of common downregulated genes showed a predominance of gene sets related to cell cycle and mitotic pathways, as well as upregulation of specific gene sets by both compounds (Fig. 4 e). GSEA of Hallmark gene sets further revealed a common downregulation of genes critical for cell cycle progression by both CDK7 inhibitors, including MYC and E2F targets, and G2M checkpoint genes (Fig. 4 f and Supplementary Figure S2 a ). In addition, Supplementary Figure S3 depicts gene expression changes across cell cycle pathway upon treatment with CDK7 inhibitors. Moreover, genes involved in DNA repair were also found significantly and commonly downregulated by both compounds, suggesting a broader impact on cellular regulatory mechanisms essential for cell division and genome stability (Fig. 4 f). Furthermore, YKL-5-124 treatment led to the downregulation of oncogenic pathways such as mTORC1, TNFα, and KRAS signaling, suggesting differential effects plausibly due to varying mechanisms of action between these two CDK7 inhibitors (Supplementary Figure S2 b) . Interestingly, a number of essential genes identified in our genome-wide CRISPR screen (most of them undruggable targets) were significantly downregulated by CDK7 inhibition (Fig. 4 g and Supplementary File S3 ), suggesting a common downregulated profile of genetic vulnerabilities. According to these findings, CDK7 inhibition emerges as a promising therapeutic strategy to effectively and broadly target genetic vulnerabilities in HNSCC. Impact of CDK7 inhibition on mice xenograft models We next examined the effects of both genetic and pharmacological inhibition of CDK7 on tumor growth in vivo. Deletion of CDK7 dramatically impaired the tumor growth of FaDu and HCA-LSC1 cell lines subcutaneously injected in immunodeficient mice ( Fig. 5 a ) , reaching final tumor volume reductions of 60 and 75% respectively ( Fig. 5 b ) . Noteworthy, treatment with YKL-5-124 and samuraciclib in a therapeutic setting ( Fig. 5 c-d ) also demonstrated to be effective at reducing the tumor volumes of both FaDu and HCA-LSC1 xenografts ( Fig. 5 e-f ) . Notably, YKL-5-124 exhibited a more potent antitumor activity, showing tumor reductions up to 74% after 12 days of treatment ( Fig. 5 g ) . By contrast, samuraciclib led to lower and delayed antitumor effects with tumor reductions by 40% after 12 days of treatment ( Fig. 5 g ) . Remarkably, none of these compounds caused any sign of adverse effect on the mice or body weight reduction (Supplementary Figure S4 ) . Impact of selective CDK7 inhibition on patient-derived organoids Finally, the therapeutic potential of CDK7 inhibitors was also tested in two HNSCC patient-derived organoid (PDO) models. Treatment of already formed PDOs with either YKL-5-124 or samuraciclib was effective to abrogate the growth of both HNSCC PDOs tested (Fig. 6 a-b ) , with IC50s ranging 329 to 397 nM for YKL-5-124 and 399 to 666 nM for samuraciclib (Fig. 6 c ). The effects of CDK7-selective inhibitors were compared to those of the CDK4/6 inhibitor palbociclib. Interestingly, PDO_84 showed resistance to palbociclib, while PDO_55 exhibited a lower sensitivity to palbociclib compared to both CDK7 inhibitors (IC50 of 2 µM) (Fig. 6 d-e ). These data are in line with our results on the viability of HNSCC cell lines. Also relevant from a therapeutic point of view, we explored the ability of CDK7-selective inhibitors to effectively target and eradicate cancer stem cells (CSC)-enriched HNSCC tumorsphere cultures. Clonal sphere-forming ability in non-adherent serum-free culture conditions is a hallmark of self-renewal and CSC-related phenotype. This is also the basis for the formation and growth of tumor organoids, which are 3D structures cultivated from patient-derived stem cells ( 37 ). Thus, we found that YKL-5-124 and samuraciclib were both effective in reducing the viability of CSC-enriched tumorsphere cultures in FaDu and UT-SCC38 cells, in a dose-dependent manner ( Supp Fig. S5 ). Discussion Despite advances in cancer treatment, the five-year survival rate for advanced HNSCC remains below 50%. Beyond conventional radio/chemotherapy, only anti-EGFR and anti-PD-1 therapies are currently approved for HNSCC treatment; however, their efficacy and clinical benefit are rather unsatisfactory. This prompted us to perform an unbiased genome-wide functional CRISPR screen across multiple HNSCC cell lines aimed at uncovering actionable genetic vulnerabilities. We took advantage of an optimized dropout CRISPR screen that allowed us to identify hundreds of essential genes for all five HNSCC cell lines screened. Special attention was focused on CDKs for their pivotal in cancer therapy as critical players in cell cycle regulation and proliferation. Our screen revealed CDK1 and CDK7 as common essentialities for the whole panel of HNSCC cell lines, thus emerging the best targetable candidates. The translational potential of CDK proteins for molecular targeted therapy is unquestionable. A growing number of CDK-selective inhibitors are being extensively evaluated in clinical trials. Notably, three CDK4/6 inhibitors: palbociclib (Pfizer), abemaciclib (Eli Lilly), and ribociclib (Novartis), have been long approved for clinical use in combination with endocrine therapy in breast cancer ( 6 – 8 ) and are under investigation for other solid tumors (NCT02465060, NCT03310879 and more). In particular, CDK4/6 inhibitors hold promise in HNSCC where CDKN2A/p16 deletion and/or amplification of CCND1 (Cyclin D1) frequently occur, ultimately causing sustained CDK4/6 activation. However, our screening results show that CDK4 and CDK6 are only essential in certain HNSCC cell lines, and initial clinical trials have not yielded encouraging results ( 38 ). Similarly, even though CDK1 has shown a broader regulatory/control effect on HNSCC cell proliferation, the clinical application of CDK1 inhibitors is hindered by their high toxicity and lack of efficacy in cancer patients ( 39 ). Interestingly, CDK7 has demonstrated widespread essentiality in all HNSCC cell lines screened and a remarkable progress has been made in the development of selective CDK7 inhibitors in recent years with promising data in other cancers. Given that CDK7 is an upstream regulator of other CDKs, CDK7 targeting may emerge an effective strategy to functionally target other critical/essential CDKs, such as CDK1. CDK7 is detected aberrantly overexpressed in different cancer types ( 15 , 16 ). Several CDK7-selective inhibitors have proved efficacy in various preclinical models of breast ( 18 , 19 ), pancreatic ( 20 , 40 ), and lung cancer ( 21 ) among others. Moreover, some CDK7 inhibitors have entered Phase I clinical trials for breast cancers ( 33 , 34 ). In the context of HNSCC, elevated levels of CDK7 have been associated with poor clinical outcomes ( 41 ). On this basis, a role has been suggested as a potential therapeutic target for HNSCC; however, this possibility has not yet been explored in preclinical and/or clinical settings. Noteworthy, the present study provides the first broad-based evidence to comprehensively demonstrate the robust antitumor activity of CDK7-selective inhibitors using a wide range of disease-relevant cellular and animal models. Therefore, our findings provides unprecedented preclinical rationale strongly supporting and encouraging selective pharmacologic CDK7 targeting as a novel therapeutic strategy to test in future clinical trials for HNSCC patients. Both genetic and pharmacologic CDK7 inhibition consistently demonstrated potent antiproliferative effects in five different HNSCC cell lines, showing approximately 10-fold lower IC50 than the CDK4/6 inhibitor palbociclib. Differences could be attributable to a distinct mechanism of action, since we found that CDK7-selective inhibitors caused both cell cycle arrest and apoptosis induction. Therefore, the dual action of YKL-5-124 and samuraciclib as cytostatic and cytotoxic agents plausibly explains the superior effectivity to other CDK inhibitors such as palbociclib being exclusively cytostatic. The therapeutic potential of CDK7 inhibitors was also tested in already formed HNSCC PDOs, which offer a more physiologically relevant patient-derived model that mimic the architectural and genetic heterogeneity of the primary tumors of origin. This led to concordant results further demonstrating that YKL-5-124 and samuraciclib were both highly effective at reducing the viability of HNSCC PDOs as compared to palbociclib treatment. These results also reflect the superior therapeutic efficacy of CDK7 inhibition. We also found some remarkable differences between the two CDK7-selective inhibitors tested in our preclinical HNSCC models. Thus, the covalent CDK7 inhibitor YKL-5-124 exhibited a more robust antitumor activity than samuraciclib (ATP-competitive inhibitor) in tumor xenografts and some HNSCC cellular models, although more apoptotic-inducing activity was detected for samuraciclib. Samuraciclib is currently undergoing Phase I/II clinical trials against various tumor types, in particular estrogen receptor-positive breast cancer where it showing acceptable safety profile with evidence of antitumor activity in combination with endocrine therapy ( 42 ). The effects of YKL-5-124 and samuraciclib were also further compared by global transcriptomics that revealed overlapping as well as specific changes. RNAseq analysis confirmed a common downregulation of cell cycle related signatures (i.e. MYC and E2F targets, and G2M checkpoint genes), as expected. In addition, DNA repair gene sets were also downregulated by both CDK7 inhibitors, which indicate a broader regulatory effect on cell division and genome stability. Among the specific transcription changes, YKL-5-124 led to downregulation of various oncogenic signaling pathways (i.e. mTORC1, TNFα, and KRAS), probably reflecting varying mechanisms of action between both inhibitors. Quite remarkably, CDK7-selective inhibitors significantly and massively reduced transcription of HNSCC essential genes identified in our genome-wide CRISPR screen, indicating that a broad spectrum of genetic vulnerabilities could be simultaneously inhibited by CDK7 targeting, including CDK1 and also numerous undruggable genes. Few essential genes were found to be upregulated by both CDK7 inhibitors, such as histone H2B that has been reported to be ubiquitinated by CDK7 ( 43 ). CDK7 inhibition could hence contribute to increase H2B expression. Altogether, these molecular insights may underlie the robust antitumor activity seen by both genetic and pharmacologic CDK7 in a wide range of HNSCC preclinical models, which pose the enormous therapeutic potential of CDK7 to design novel molecular-targeted treatments for HNSCC. Tumor heterogeneity is a well-known feature that greatly complicates cancer treatment and a long-lasting clinical effectivity. Research evidences indicate that heterogeneity develops through time as tumor-initiating stem cells, also known as cancer stem cells (CSCs) ( 44 ). CSC self-renewal, replication, and differentiation are postulated to produce a hierarchy of cells constituting the tumor mass ( 45 ). The same principle is applied to the generation and growth of tumor organoids that is based on CSC self-renewal features. Hence, complete eradication of tumors requires therapies able to effectively eliminate the CSC subpopulations responsible for treatment resistance, relapse and metastasis ( 46 , 47 ). On this basis, it is therefore a relevant finding that both YKL-5-124 and samuraciclib effectively reduced the viability of CSC-enriched tumorspheres as well as HNSCC PDOs, thereby suggesting that selective CDK7 targeting could also be an efficacious strategy to target and eradicate the CSC niche in HNSCC. Importantly, none of these CDK7 inhibitors led to any noticeable adverse effects in vivo, consistent with previous research demonstrating that CDK7 is non-essential in adult tissues with low proliferative rates and primarily impacts highly proliferative cells, a hallmark of cancer ( 48 ). It is also worth to mention that a recent study demonstrated CDK7 as a putative vulnerability to overcome CDK4/6 resistance in breast cancer ( 19 ). Concordant to these observations, we showed that CDK7-selective inhibitors effectively reduced the growth/viability of palbociclib-resistant HNSCC PDO models. Conclusions The present study uncovered a catalog of genetic vulnerabilities that could serve as the basis to design novel molecular-targeted treatments for HNSCC. As a proof-of-principle, CDK7 was revealed as a common essentiality in all HNSCC cell lines screened, and all concordantly displayed an exceptional dependency on CDK7 activity/function. Furthermore, genetic and pharmacologic CDK7 inhibition showed potent antitumor activity and efficacy in a wide range of disease-relevant HNSCC models. According to our findings, CDK7 inhibition emerges as a promising therapeutic strategy to effectively and broadly target CDK signaling and genetic vulnerabilities in HNSCC. All the herein preclinical results provide unprecedented support for future clinical testing of CDK7-selective inhibitors in HNSCC patients. Abbreviations AML Acute myeloid leukemia ATCC American Type Culture Collection BCA Bicinchoninic Acid Assay BSA Bovine Serum Albumin CAK Cdk-activating complex CDK Cycle-dependent kinases CPM Counts Per Million CRISPR Clustered Regularly Interspaced Short Palindromic Repeats DESeq2 Differential expression using Sequencing 2 DMEM Dulbeco’s Modified Eagle Medium DNA Deoxyribonucleic acid FBS Fetal Bovine Serum FDA Food and drugs administration FDR False Discovery Rate GSEA Gene Set Enrichment Analysis HEPES 4-(2-hidroxietil)-1-piperazinaetanosulfónico HNSCC Head and neck squamous cell carcinoma HPV Human Papillomavirus KO Knock-out MAGeCK Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout MOI Multiplicity of Infection MOPS 3-(N-Morpholino)propanesulfonic acid NES Normalized enrichment score PCR Polymerase Chain Reaction PDO Patient-derived organoid PI Propidium Iodide RIPA buffer Radio-Immunoprecipitation Assay buffer RNA Ribonucleic acid SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis STR Short Tandem repeat TBS-T Tris-Buffered Saline with Tween-20 TCGA The Cancer Genome Atlas Declarations Ethics approval and consent to participate This study was performed in accordance to the principles of the Declaration of Helsinki with the appropriate approval of the Ethical and Scientific Committees of the Hospital Universitario Central de Asturias and the Regional Ethics Committee from the Principality of Asturias (CEImPA) (date of approval 9 th March 2023; approval number: 2023.018, for the project PI22/00167). Patient informed consent was obtained for all the tissue samples collected by the Biobank of Principado de Asturias (PT20/00161 and PT23/00077). All the animal experimental procedures were performed in accordance with the institutional guidelines of the University of Oviedo and approved by the Animal Research Ethical Committee of the University of Oviedo (date of approval 1 st August 2019; approval number PROAE 46/2019; date of approval 8 th March 2024; approval number PROAE 03/2024). Data Availability RNA-sequencing data will be submitted to GEO and accession codes will be available before publication. Competing Interests K.T. is a shareholder of and has received research funding from Storm Therapeutics Ltd. All the other authors declare that they have no competing interests. Funding This study was supported by the Instituto de Salud Carlos III (ISCIII) through the project grants PI19/00560, PI21/00208, PI22/00167, and CIBERONC (CB16/12/00390 and CB16/12/00228) and was co-funded by the European Union, the Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Fundación Bancaria Cajastur-IUOPA, and Universidad de Oviedo. Additional funding was provided through the grant “Ayudas para Grupos de Investigación de Organismos del Principado de Asturias 2021–2023” (IDI/2021/000079), funded by Principado de Asturias through FICYT and the FEDER Funding Program from the European Union, and a grant from the MCIN/AEI (PID2020-117236RB-I00). M.O.-R. and E.P.-A are both recipients of a PFIS predoctoral fellowship from ISCIII (FI23/00037 and FI20/00064), and S.D.M. is supported by a grant from the Programa de Formación de Profesorado Universitario from the Spanish Ministry of Universities (FPU21/05639). S.A.-T. is a recipient of a Miguel Servet fellowship from ISCIII (CP23/00101) and co-funded by the European Union. K.T. is supported by Wellcome Trust (grants RG83195, G106133 and G127005), UKRI Medical Research Council (grant RG83195) and Leukaemia UK (grants G108148 and G117699). M.A.F was supported by the Asociación Española contra el Cáncer (AECC; 2019/INVES19001ALVA). M.A.G. is a recipient of a Severo Ochoa predoctoral fellowship from the Principado de Asturias (BP21-205), and F.H.-P. is funded by Fundación Alimerka and is a recipient of a Maria Zambrano postdoctoral fellowship at the University of Oviedo. Author’s contribution Conceptualization, F.H.-P. and J.M.G.-P.; Data curation, M.O.-R., F.H.-P., and J.M.G.-P.; Formal analysis, M.O.-R., F.H.-P., J.M.G.-P., M.A.-F., R.G.-E., C.L.; Funding acquisition, J.M.G.-P., J.P.R., M.A.-F., R.G.-E., C.L., K.T., G.V.; Investigation, M.O.-R., F.H.-P., M.A.-G., B.d.L.-D., S.D.M., E.P.-A., M.R.-S., A.L.-F., D.C., R.G.-D., K.T., R.G.-E.; Methodology, M.O.-R., F.H.-P., K.T., M.A.-G., B.d.L.-D., S.d.M, E.P.-A., M.R.-S., A.L.-F., C.L., D.C., R.G.-D., M.A.-F., S.A.-T.; Resources, J.M.G.-P., J.P.R., M.A.-F., R.G.-E., C.L.; Supervision, F.H.-P. and J.M.G.-P.; Visualization, M.O.-R. and F.H.-P.; Writing—original draft, M.O.-R., F.H.-P. and J.M.G.-P.; Writing—review & editing, M.O.-R., F.H.-P., J.M.G.-P., M.A.-F., R.G.-E., C.L., J.P.R., K.T., G.V. Acknowledgements We thank Dr. Reidar Grenman (Turku Univ., Finland) for kindly providing UT-SCC38 and UT-SCC42B. 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Supplementary Files SupplementaryFileS1withTableS1andFiguresS1S5.pdf SupplementaryFileS2HNSCC.common.dropouts.csv SupplementaryFileS3DE.essential.genes.after.treatment.csv SupplementaryfileUncroppedWBimages.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4679708","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":332478209,"identity":"51e52b3d-b5bc-43f3-bba1-c3478c802030","order_by":0,"name":"María Otero-Rosales","email":"","orcid":"","institution":"Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Instituto Universitario de Oncología del Principado de Asturias (IUOPA), University of Oviedo, Oviedo","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"","lastName":"Otero-Rosales","suffix":""},{"id":332478210,"identity":"b5b68dee-6847-4a2b-9267-280d41222b50","order_by":1,"name":"Miguel Álvarez-González","email":"","orcid":"","institution":"Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Instituto Universitario de Oncología del Principado de Asturias (IUOPA), University of Oviedo, Oviedo","correspondingAuthor":false,"prefix":"","firstName":"Miguel","middleName":"","lastName":"Álvarez-González","suffix":""},{"id":332478211,"identity":"cc6b1993-cc40-4644-9c70-7f33d8cf8dfd","order_by":2,"name":"Beatriz de Luxán-Delgado","email":"","orcid":"","institution":"Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Instituto Universitario de Oncología del Principado de Asturias (IUOPA), University of Oviedo, Oviedo","correspondingAuthor":false,"prefix":"","firstName":"Beatriz","middleName":"","lastName":"de Luxán-Delgado","suffix":""},{"id":332478212,"identity":"c6eb12be-ecb5-4618-a187-048e5c58dd3a","order_by":3,"name":"Sonia Del Marro","email":"","orcid":"","institution":"Molecular Oncology Unit, CIEMAT. 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(a)\u003c/strong\u003e Schematic diagram depicting the experimental workflow of the functional CRISPR/Cas9 KO screen performed in a panel of five different HNSCC cell lines. (\u003cstrong\u003eb)\u003c/strong\u003e Bar graph showing the number of essential genes identified in each HNSCC cell line, categorized by FDR. (\u003cstrong\u003ec)\u003c/strong\u003e Venn diagram illustrating the overlap of essential genes across all five HNSCC cell lines tested. (\u003cstrong\u003ed)\u003c/strong\u003e Pathway analysis of genes commonly essential across all HNSCC cells. (\u003cstrong\u003ee)\u003c/strong\u003e Heat map displaying the essentiality of genes coding for CDKs, based on FDR. (\u003cstrong\u003ef)\u003c/strong\u003e Line graph showing gRNAs counts of CDK7 gene at the initial (day 0) and final time points (day 25) of the screen for each cell line.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4679708/v1/222517e505b3e338ede38156.jpg"},{"id":61350664,"identity":"43cafcd2-2406-4d21-bb53-2a97654e7af5","added_by":"auto","created_at":"2024-07-29 18:43:43","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1230961,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenetic validation of CDK7 essentiality in HNSCC cells. (a)\u003c/strong\u003e Western Blot analysis displaying CDK7 protein levels across our panel of HNSCC cell lines, with β-actin serving as the loading control. (\u003cstrong\u003eb)\u003c/strong\u003e Western Blot analysis of CDK7 expression levels in FaDu, UT-SCC38, and HCA-LSC1 cell lines transduced with two independent sgRNAs to knockout CDK7 gene (KO1 and KO2) or control cells transduced with the empty vector, and β-actin protein as the loading control. (\u003cstrong\u003ec)\u003c/strong\u003e Schematic diagram depicting the experimental workflow for the in vitro competitive proliferation assays. (\u003cstrong\u003ed-f)\u003c/strong\u003e Bar graphs showing the percentage of CDK7 KO cells harboring ZsG protein over different time points in FaDu \u003cstrong\u003e(d)\u003c/strong\u003e, UT-SCC38 \u003cstrong\u003e(e)\u003c/strong\u003e, and HCA-LSC1 \u003cstrong\u003e(f)\u003c/strong\u003e cell lines (***, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001. Error bars, mean + SD of at least three replicates, Two-way ANOVA test). (\u003cstrong\u003eg)\u003c/strong\u003e Western blot analysis of the indicated CDK7 targets in empty vector- and CDK7 KO-transduced HNSCC cell lines, with β-actin protein serving as the loading control.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4679708/v1/f5bda5029a87ece7b0b55ab6.jpg"},{"id":61350130,"identity":"0123710a-bb2d-4c33-aa3f-ff223d6dd1cd","added_by":"auto","created_at":"2024-07-29 18:35:42","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2523382,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional characterization of CDK7 pharmacological inhibition in HNSCC cell lines. \u003c/strong\u003e(\u003cstrong\u003ea)\u003c/strong\u003e Measurement of cell viability in our panel of five different HNSCC cell lines treated with increasing concentrations of the CDK7-selective inhibitors YKL-5-124 and samuraciclib, and compared to the CDK4/6 inhibitor palbociclib. Four replicates per condition. Data shown as average + SD. (\u003cstrong\u003eb)\u003c/strong\u003e Table summarizing average IC50 values for each cell line treated with the aforementioned compounds (n \u0026gt;= 2). (\u003cstrong\u003ec)\u003c/strong\u003e Colony formation assays with increasing concentrations of YKL-5-124, samuraciclib, and palbociclib in the indicated HNSCC cell lines. (\u003cstrong\u003ed)\u003c/strong\u003e Western Blot analysis of protein changes in the indicated CDK7 targets and cleaved PARP (cPARP) in HNSCC cells treated with either vehicle (DMSO), YKL-5-124 (1μM), or samuraciclib (1μM) for 24 hours. β-actin protein was used as the loading control. (\u003cstrong\u003ee-f)\u003c/strong\u003e Flow cytometry analysis of cell cycle changes in HNSCC cell lines treated with the indicated doses of YKL-5-124 and samuraciclib for 72 hours. Two replicates per condition. Percentage of cells in each cell cycle phase are shown as a stacked barplot + SD. Only significant accumulation of cells in a cell cycle phase is indicated. Statistical significance was calculated using two-way ANOVA Dunnett’s multiple comparisons test. ***, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001.\u003cstrong\u003e (g-h)\u003c/strong\u003eFlow cytometry assessment of apoptosis and necrosis in HNSCC cell lines upon treatment for 48 hours with increasing concentrations of either YKL-5-124 or samuraciclib. Two replicates per condition. Statistical significance was calculated using two-way ANOVA Dunnett’s multiple comparisons test. ***, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4679708/v1/2a923a0d046e06e3a6e33357.jpg"},{"id":61350132,"identity":"e972ab2d-8068-4896-b116-be5448b85921","added_by":"auto","created_at":"2024-07-29 18:35:42","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1919333,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGlobal transcriptional changes and a common downregulated profile in FaDu cells caused by CDK7 inhibition.\u003c/strong\u003e \u003cstrong\u003e(a-b) \u003c/strong\u003eVolcano plots illustrating differentially expressed genes after 48 hours of treatment with either YKL-5-124\u003cstrong\u003e (a) \u003c/strong\u003eor samuraciclib\u003cstrong\u003e (b) \u003c/strong\u003eat 250 nM (padj \u0026lt;0.05, log2fc\u0026lt;-0.5 or log2fc\u0026gt;0.5). To enhance clarity, ribosomal coding genes were filtered out\u003cstrong\u003e. (c-d)\u003c/strong\u003e Venn diagram showing the unique and overlapping genes that were found downregulated \u003cstrong\u003e(c)\u003c/strong\u003e and upregulated \u003cstrong\u003e(d)\u003c/strong\u003e upon YKL-5-124 and samuraciclib treatment (padj\u0026lt;0.01, log2fc\u0026lt;-0.5 or log2fc\u0026gt;0.5). (\u003cstrong\u003ee)\u003c/strong\u003e Reactome pathway analysis of the common downregulated (\u003cem\u003eblue bars\u003c/em\u003e) and common upregulated (\u003cem\u003ered bars\u003c/em\u003e) genes by both CDK7 inhibitors. (\u003cstrong\u003ef)\u003c/strong\u003e Balloon plot displaying Gene Set Enrichment Analysis of genes downregulated (\u003cem\u003eblue color\u003c/em\u003e) or upregulated (\u003cem\u003ered color\u003c/em\u003e) by either YKL-5-124 or samuraciclib. (\u003cstrong\u003eg)\u003c/strong\u003e Heat map depicting the subsets of essential genes identified in FaDu cells by our functional CRISPR screen that were found commonly significantly upregulated (\u003cem\u003ein red\u003c/em\u003e) or downregulated (\u003cem\u003ein blue\u003c/em\u003e) by both CDK7 inhibitors, as compared to control DMSO-treated FaDu cells. Differential gene expression changes are shown as z-score of log2(CPM).\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4679708/v1/329394c15d4619277804f12b.jpg"},{"id":61350665,"identity":"de3eb1d4-6e46-4ad0-9a02-9d174956a4ee","added_by":"auto","created_at":"2024-07-29 18:43:43","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1448596,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of CDK7 inhibition on \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003exenograft models. (a) \u003c/strong\u003eTumor growth of FaDu and HCA-LSC1 cell lines with CDK7 KO (n= 12 tumors per group for FaDu xenografts, and n= 7 tumors for HCA-LSC1 xenografts). Data shown as mean \u003cu\u003e+\u003c/u\u003eSEM. Statistic two-way ANOVA, uncorrected Fisher’s LSD, ***, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001\u003cstrong\u003e (b)\u003c/strong\u003e Box plots representing final tumor volumes of CDK7 KO or empty vector groups. Unpaired t- test, ***, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001. (\u003cstrong\u003ec-d) \u003c/strong\u003eSchematic representation of YKL-5-124 \u003cstrong\u003e(c) \u003c/strong\u003eand samuraciclib \u003cstrong\u003e(d)\u003c/strong\u003etesting in vivo.\u003cstrong\u003e (e-f)\u003c/strong\u003e Tumor growth of FaDu and HCA-LSC1 cell lines in presence of vehicle/YKL-5-124 (n= 12 tumors per treatment condition) \u003cstrong\u003e(e)\u003c/strong\u003e or vehicle/samuraciclib (n= 12 tumors per condition) \u003cstrong\u003e(f)\u003c/strong\u003e treatment for 12 days. Data shown as mean \u003cu\u003e+\u003c/u\u003e SEM. Statistic two-way ANOVA, uncorrected Fisher’s LSD, ***, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001. \u003cstrong\u003e(g)\u003c/strong\u003e Box plots representing final tumor volumes among the different experimental groups within pharmacological CDK7 inhibition experiments. Unpaired t- test, ***, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4679708/v1/92bac3043647f66a5f76af3e.jpg"},{"id":61350134,"identity":"5bacb1ea-3f5e-4894-8022-b1812a1d9d6b","added_by":"auto","created_at":"2024-07-29 18:35:43","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1488351,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTherapeutic evaluation of CDK7-selective inhibitors in HNSCC PDOs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea) \u003c/strong\u003eRepresentative images of formed HNSCC PDOs treated with two doses of YKL-5-124 or samuraciclib for five days. (\u003cstrong\u003eb) \u003c/strong\u003eMeasurement of cell viability in HNSCC PDOs treated with increasing concentrations of YKL-5-124 and samuraciclib. Each condition was performed in quadruplicates. Data shown as mean \u003cu\u003e+\u003c/u\u003e SD. \u003cstrong\u003e(c) \u003c/strong\u003eTable summarizing IC50 values for each PDO treated with the aforementioned compounds. \u003cstrong\u003e(d)\u003c/strong\u003e Representative images of formed HNSCC PDOs treated with two doses of palbociclib for five days. \u003cstrong\u003e(e)\u003c/strong\u003e Measurement of cell viability in HNSCC PDOs treated with increasing concentrations of palbociclib. Each condition was performed in triplicates. Data shown as mean \u003cu\u003e+\u003c/u\u003e SD.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4679708/v1/c6094f4bda20eff7ee3489df.jpg"},{"id":61518210,"identity":"7f8758d8-cb09-4fce-8eb7-11e9a185565e","added_by":"auto","created_at":"2024-07-31 16:29:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11119027,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4679708/v1/91574abc-5be9-47f1-9599-18a85c6e3d89.pdf"},{"id":61350663,"identity":"5b1bde15-8fbd-4701-b777-3d95e1f931f4","added_by":"auto","created_at":"2024-07-29 18:43:42","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1653390,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFileS1withTableS1andFiguresS1S5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4679708/v1/b0c3b351004539c4f5640ba9.pdf"},{"id":61350131,"identity":"c3ffc8b9-35ea-4106-b51f-c68ac60de6cf","added_by":"auto","created_at":"2024-07-29 18:35:42","extension":"csv","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11833,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFileS2HNSCC.common.dropouts.csv","url":"https://assets-eu.researchsquare.com/files/rs-4679708/v1/ae534e45f71793d8f63bce99.csv"},{"id":61350135,"identity":"e7ffed8e-3090-4227-91a9-7de7a91a1b16","added_by":"auto","created_at":"2024-07-29 18:35:43","extension":"csv","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":4986,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFileS3DE.essential.genes.after.treatment.csv","url":"https://assets-eu.researchsquare.com/files/rs-4679708/v1/1e0d1e63dad3ce7ffa722b45.csv"},{"id":61350137,"identity":"7c25ce85-1233-40f7-abc1-4b785d016564","added_by":"auto","created_at":"2024-07-29 18:35:43","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":16311812,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryfileUncroppedWBimages.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4679708/v1/67bc108a4a115c047e398124.pdf"}],"financialInterests":"Competing interest reported. K.T. is a shareholder of and has received research funding from Storm Therapeutics Ltd. All the other authors declare that they have no competing interests.","formattedTitle":"Genome-wide functional CRISPR screen reveals CDK7 as a targetable therapeutic vulnerability for head and neck cancer","fulltext":[{"header":"Background","content":"\u003cp\u003eHead and neck squamous cell carcinoma (HNSCC) is the sixth most prevalent cancer globally, with significant morbidity and mortality rates. Despite advances in multimodal treatment strategies, including surgery, radiation, and chemotherapy, the five-year survival rate for HNSCC patients remains between 40\u0026ndash;50% (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). The prognosis is particularly unfavorable for patients with recurrent or metastatic disease (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNowadays, precision medicine for HNSCC is restricted to the EGFR-specific inhibitor cetuximab and the immunotherapy agents nivolumab and pembrolizumab (\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Treatment with cetuximab benefits a minority of HNSCC patients despite harboring high \u003cem\u003eEGFR\u003c/em\u003e gene amplification, and its effectiveness is often hindered by tumor resistance. Similarly, even though immunotherapeutic agents such as nivolumab and pembrolizumab have recently emerged as promising treatment options, their clinical benefit in HNSCC patients is rather low (20\u0026ndash;30%). These challenges underline the need for identifying novel therapeutic targets to improve treatment outcomes in this disease.\u003c/p\u003e \u003cp\u003eCyclin-dependent kinases (CDKs) are pivotal in cancer therapy due to their role in cell cycle regulation and proliferation. CDK4/6 inhibitors such as palbociclib have demonstrated efficacy, particularly in hormone receptor-positive breast cancer (\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). However, their therapeutic capability/application is often limited by the development of drug resistance and adverse side effects. In HNSCC, CDK inhibitors hold promising therapeutic potential and continue to be an active area of research. Recent studies suggest a potential synergy when combining palbociclib with cetuximab and radiotherapy (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmong the CDKs, CDK7 has been identified as a crucial regulator in cancer biology. This kinase forms a trimeric complex with Cyclin H and MAT1, functioning as a Cdk-activating kinase (CAK) that regulates both cell cycle progression and global transcription. CDK7 controls the cell cycle at different levels by phosphorylating CDKs 1, 2, 4 and 6 in their T-loops promoting their activation (\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Additionally, the CAK complex is a key component of the transcription factor TFIIH involved in transcription initiation and elongation (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). CDK7 is aberrantly overexpressed in different types of cancer (\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e) and significant progress has been made in the development of selective CDK7 inhibitors, which have shown efficacy preclinically in multiple tumor types including breast (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e), pancreatic (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e), and lung cancer (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e) among others.\u003c/p\u003e \u003cp\u003eFor the last decade, genome-wide CRISPR screens have revolutionized the landscape of target identification in biomedical research, offering a powerful tool to elucidate the functional relevance of genes across the entire genome (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). While being extensively utilized in the past several years to define the landscape of important biological processes, these screens continue to be developed and are indispensable in uncovering novel therapeutic targets and dissecting complex biological pathways \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e (\u003cspan additionalcitationids=\"CR25 CR26 CR27 CR28\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTaking advantage of an optimized genome-wide functional CRISPR screen (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e), we aimed to identify actionable genetic vulnerabilities that can be rapidly evaluated as potential targeted therapies in preclinical HNSCC models. As a result, our study led to the identification of CDK7 as a promising clinically targetable vulnerability in HNSCC. A detailed functional and molecular characterization of available CDK7-selective inhibitors further contributed to unprecedentedly demonstrate their robust antitumor activity in a broad range of disease-relevant cellular, patient-derived organoids (PDOs) and animal models. These findings encourage future clinical testing in HNSCC patients.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell lines and cell culture\u003c/h2\u003e \u003cp\u003eFaDu (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.\u003c/p\u003e \u003cp\u003eCells 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).\u003c/p\u003e \u003cp\u003eCas9-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 \u0026micro;g/mL and maintained for at least 14 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCRISPR/Cas9 KO screen\u003c/h2\u003e \u003cp\u003eFor each HNSCC cell line, a total of 1.0 x 10\u003csup\u003e8\u003c/sup\u003e 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 10\u003csup\u003e7\u003c/sup\u003e. After 25 days of culturing, at least 3.0 x 10\u003csup\u003e7\u003c/sup\u003e cells were collected as the final time point.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eIllumina sequencing of gRNAs and statistical analysis\u003c/h2\u003e \u003cp\u003eGenomic DNA extraction and Illumina sequencing of gRNAs were conducted as described previously (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). 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 (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e) 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eGeneration of CDK7 KO cell lines\u003c/h2\u003e \u003cp\u003eHNSCC 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\u0026ndash;3 \u0026micro;g/mL) containing medium for six days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCompetitive proliferation assay\u003c/h2\u003e \u003cp\u003eCas9-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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDrugs\u003c/h2\u003e \u003cp\u003eYKL-5-124 and samuraciclib (CT7001) were obtained from MedChem Express. Palbociclib was obtained from Selleckchem. For \u003cem\u003ein vitro\u003c/em\u003e studies, stock solutions of both compounds were prepared at a concentration of 10 mM in sterile dimethyl sulfoxide (DMSO) and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. Prior to each experiment, the drugs were thawed and diluted to the desired final concentrations. DMSO was used as the vehicle control condition.\u003c/p\u003e \u003cp\u003eFor \u003cem\u003ein vivo\u003c/em\u003e 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 -80\u0026ordm;C. 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCell viability assays\u003c/h2\u003e \u003cp\u003eHNSCC cells were seeded into 96-well culture plates at a density of 2,000\u0026ndash;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 \u0026ndash; Variable slope function.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eProliferation assays\u003c/h2\u003e \u003cp\u003eHNSCC cells were seeded into 6-well culture plates at a density of 3,000\u0026ndash;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\u0026ndash;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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell cycle analysis\u003c/h2\u003e \u003cp\u003eHNSCC 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 -20\u0026ordm;C. Cell cycle analysis was performed using FxCycle\u0026trade; PI/RNase Staining Solution (Life Technologies, #F10797) to measure DNA content by flow cytometry, according to the manufacturer\u0026rsquo;s instructions. Cell percentage in each cell cycle phase was determined using FlowJo software\u0026rsquo;s Cell cycle algorithm. Only significant accumulation of cells in a cell cycle phase is indicated\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eApoptosis assay\u003c/h2\u003e \u003cp\u003eHNSCC 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\u0026trade; 488 \u0026amp; Propidium Iodide for Flow Cytometry (Invitrogen, #V13242 and #V13241) according to manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTumorsphere formation assays\u003c/h2\u003e \u003cp\u003eHNSCC-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).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eCells 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\u0026trade; 4 to 12% Bis-Tris gels (Life Technologies) in MOPS running buffer and blotted on Trans-Blot\u0026reg; Turbo\u0026trade; 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 (\u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e) overnight at 4\u0026ordm;C, 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\u0026reg; Fc Imager (LICOR Biosciences) and analyzed with Image Studio Lite software (LICOR Biosciences).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRNA sequencing (RNA-seq) and bioinformatics analysis\u003c/h2\u003e \u003cp\u003eTotal 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.\u003c/p\u003e \u003cp\u003eSequencing 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).\u003c/p\u003e \u003cp\u003eGene 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\u0026thinsp;\u0026lt;\u0026thinsp;0.25, GSEA weighted Kolmogorov-Smirnov test, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;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\u0026thinsp;\u0026lt;\u0026thinsp;0.05) are shown.\u003c/p\u003e \u003cp\u003eDifferential expression of common essential genes upon CDK7 inhibition in the panel of five HNSCC cell lines was represented as z-score of log2 (CPM).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMouse xenografts\u003c/h2\u003e \u003cp\u003e 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).\u003c/p\u003e \u003cp\u003eFemale AthymicNude-Fox1nu mice of 5\u0026ndash;6 weeks old (ENVIGO RMS) were subcutaneously (s.c.) inoculated in the flanks with 1.5x10\u003csup\u003e6\u003c/sup\u003e FaDu or 2x10\u003csup\u003e6\u003c/sup\u003e HCA-LSC1 cells in culture medium mixed 1:2 with VitroGel\u0026reg; Hydrogel Matrix (The Well Bioscience, #VHM01). Once tumors reached a measurable size (between 100 and 200 mm\u003csup\u003e3\u003c/sup\u003e), 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\u0026ndash;3 times a week.\u003c/p\u003e \u003cp\u003eTumor volume was determined using the equation (D \u0026times; d2)/6 \u0026times; 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 CO\u003csub\u003e2\u003c/sub\u003e asphyxiation or cervical dislocation when the tumors of the control group reached approximately 1,000 mm\u003csup\u003e3\u003c/sup\u003e. Tumors were resected from the flanks, fixed in formol and embedded in paraffin for histological evaluation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePatient-derived organoids (PDOs)\u003c/h2\u003e \u003cp\u003e 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 (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePDOs 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 \u0026micro;M Prostaglandin E2 (Tocris #2296), 0.3 \u0026micro;M CHIR99021 (Sigma #SML1046), 1 \u0026micro;M 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\u0026thinsp;\u0026minus;\u0026thinsp;38).\u003c/p\u003e \u003cp\u003eFor 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 \u0026micro;M 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 \u0026micro;L 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).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eStatistical significance\u003c/h2\u003e \u003cp\u003eStatistical 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\u0026rsquo;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 \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant (* \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; ** \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; *** \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; **** \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eGenome-wide functional CRISPR-Cas9 screen revealed CDK7 as a vulnerability in HNSCC cells\u003c/h2\u003e \u003cp\u003eAs an attempt to uncover essential genes with therapeutic potential, we conducted a CRISPR-Cas9 knockout screen across five different HNSCC cell lines: FaDu, UT-SCC38, HCA-LSC1, UT-SCC42B and Detroit 562. We used a comprehensive whole-genome library, targeting 18,010 genes with 90,709 different gRNAs (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). The HNSCC cell lines were transduced using a low multiplicity of infection (MOI) of 0.3 and subsequently selected with puromycin to ensure efficient integration of the gRNAs. Cells were cultured for 25 days, after which genomic DNA was extracted, and gRNAs were amplified and sequenced (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Guide-RNA depletion and enrichment was analyzed using MAGeCK software (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e) to identify negatively and positively regulated genes, indicative of a role in cell viability.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCell-line-specific and commonly essential genes were determined based on their false discovery rate (FDR) score. Only genes with FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.10 were consider for further analysis, thus identifying between 500 and 1,500 essential genes per cell line (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb\u003cb\u003e).\u003c/b\u003e A focus on essential genes across multiple cell lines revealed 228 genes consistently essential in all five analyzed HNSCC cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec \u003cb\u003eand Supplementary File S2)\u003c/b\u003e, including well-known oncogenes and drivers of HNSCC cell proliferation such as MYC and CCND1. Pathway analysis of the common essential genes showed a significant overlap in MYC and E2F targets and cell cycle checkpoint-related gene sets (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eGiven the translational potential of CDK inhibitors, we focused our attention on the essentiality of CDK proteins as targetable and druggable candidates. CDK1 and CDK7 emerged as the most crucial genes across all five HNSCC cell lines, while other CDKs such as CDK2 or CDK5 were found to be non-essential, or showed selectivity to certain cell lines, such as CDK4, CDK6 or CDK9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). CDK1 is the only CDK in mammals that is essential for cell cycle progression (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). However, CDK1 inhibitors have either been associated with high toxicity or failed to demonstrate sufficient efficacy in patients. Interestingly, CDK7-selective inhibitors have been recently developed and are under current active testing in clinical trials (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). We therefore selected CDK7 for further investigation due to its critical role in cell cycle progression and transcription, as well as its function as an upstream regulator of other CDKs, including CDK1 itself. Single gRNA analysis revealed that every gRNA targeting CDK7 was depleted at the final time point in all HNSCC cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eGenetic validation of CDK7 essentiality in HNSCC cells\u003c/h2\u003e \u003cp\u003eTo validate the essentiality of CDK7 as a candidate gene in our screening, we first analyzed CDK7 protein levels in our HNSCC cell line panel \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. We next performed targeted CRISPR knockouts of CDK7 (CDK7 KO) using two distinct single gRNAs in three HNSCC cell lines: FaDu, UT-SCC38 and HCA-LSC1, which harbored different endogenous levels of CDK7. We confirmed that these two gRNAs robustly reduced CDK7 protein levels in all three HNSCC cell lines \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. Subsequently, we carried out competitive co-culture assays between control and KO cells to validate the results of our screen (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). In these assays, CDK7 KO cells were significantly outcompeted by control cells in FaDu (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed), UT-SCC38 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee) and HCA-LSC1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef\u003cb\u003e)\u003c/b\u003e, thus confirming a significant reduction in cell viability upon CDK7 depletion. These results highlight the critical role of CDK7 in maintaining cell proliferation/survival and validate our initial findings from the CRISPR screen.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, we assessed the effects of CDK7 KO on the phosphorylation of known direct CDK7 targets/substrates. Thus, we found that CDK7 depletion led to reduced phosphorylation levels of the Carboxy-terminal domain (CTD) of the RNA Polymerase II (Ser5), retinoblastoma protein (Rb) (Ser780), and CDK1 T-loop phosphorylation (Thr161) in the three HNSCC cell lines tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). These data demonstrate that CDK7 depletion effectively abrogates HNSCC cell proliferation by targeting several key downstream effectors and regulators of cell cycle control and transcription.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eTherapeutic potential of CDK7 pharmacological inhibition\u003c/h2\u003e \u003cp\u003eThe robust effects of genetic CDK7 KO prompted us to evaluate the therapeutic potential of available CDK7-selective inhibitors in preclinical HNSCC models. We selected two distinct compounds: YKL-5-124, a covalent selective CDK7 inhibitor (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e), and samuraciclib (CT7001, ICEC0942), an orally bioavailable, ATP-competitive inhibitor (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e) currently undergoing Phase I/II clinical trials in cancer patients (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). We first analyzed the effects of selective CDK7 inhibitors on the viability of a panel of five HNSCC cell lines. Both YKL-5-124 and samuraciclib (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) significantly reduced HNSCC cell viability, although lower IC50 values were observed for YKL-5-124 (ranging from 35 to 100 nM) than samuraciclib (range 30 to 200 nM) after 5 days of treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Remarkably, both CDK7-selective inhibitors were more effective in decreasing HNSCC cell viability than the CDK4/6 inhibitor palbociclib (\u0026gt;\u0026thinsp;10-fold higher IC50 values) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-b\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnalogous results were observed in colony formation assays performed along 14 days of treatment. Lower doses of YKL-5-124 and samuraciclib (ranging from 5 to 25 nM) completely abolished HNSCC cell proliferation, whereas considerably higher concentrations of palbociclib were required to achieve a comparable effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eWe next investigated the immediate molecular effects of CDK7 inhibition on the phosphorylation of downstream protein substrates. HNSCC cells were treated with 1 \u0026micro;M of either YKL-5-124 or samuraciclib for 24 hours to minimize potential compensatory mechanisms upon prolonged treatment. This resulted in decreased phosphorylation levels of CTD-RNA Polymerase II (Ser5), Rb (Ser780), and CDK1 (Thr161) in most HNSCC cell lines, thereby mirroring the changes previously observed by CDK7 KO. CDK7 protein levels remained unchanged or slightly increased upon treatment with CDK7 inhibitors. Noteworthy, a shift in CDK7 electrophoretic mobility was detected upon treatment with YKL-5-124 in all five HNSCC cell lines, likely due to the covalent binding of this compound (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed \u003cb\u003eand Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea).\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe functional consequences of CDK7 pharmacological inhibition were also investigated on cell cycle dynamics and apoptosis. YKL-5-124 treatment led to cell accumulation in S and/or G2/M phases depending on the cell line analyzed, and samuraciclib treatment led to cell accumulation in G2/M phase in all five HNSCC cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef \u003cb\u003eand Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb and S1c\u003c/b\u003e). This was also accompanied by a significant induction of apoptosis, especially after samuraciclib treatment, in all HNSCC cell lines tested \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh \u003cb\u003eand Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ed)\u003c/b\u003e. Concordant to these findings, increased levels of cleaved PARP were also detected by Western blot upon YKL-5-124 and samuraciclib treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed \u003cb\u003eand Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea\u003c/b\u003e).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eGlobal transcriptional changes caused by CDK7 inhibition\u003c/h2\u003e \u003cp\u003eIn order to delineate the global transcriptional changes caused by CDK7 inhibition, RNA-seq experiments were performed in FaDu cells treated with either YKL-5-124 or samuraciclib for 48 hours. As represented by Volcano plots, a total of 804 and 1,147 genes were found significantly up- and downregulated by YKL-5-124, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), and 630 and 989 were significantly up- and downregulated genes upon samuraciclib treatment, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). In addition, Venn diagrams depict the unique and overlapping changes by YKL-5-124 and samuraciclib, with a total of 645 common downregulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) and 376 common upregulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). GSEA using reactome pathways database of common downregulated genes showed a predominance of gene sets related to cell cycle and mitotic pathways, as well as upregulation of specific gene sets by both compounds (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). GSEA of Hallmark gene sets further revealed a common downregulation of genes critical for cell cycle progression by both CDK7 inhibitors, including MYC and E2F targets, and G2M checkpoint genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef \u003cb\u003eand Supplementary Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ea\u003c/b\u003e). In addition, \u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e\u003c/b\u003e depicts gene expression changes across cell cycle pathway upon treatment with CDK7 inhibitors. Moreover, genes involved in DNA repair were also found significantly and commonly downregulated by both compounds, suggesting a broader impact on cellular regulatory mechanisms essential for cell division and genome stability (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). Furthermore, YKL-5-124 treatment led to the downregulation of oncogenic pathways such as mTORC1, TNFα, and KRAS signaling, suggesting differential effects plausibly due to varying mechanisms of action between these two CDK7 inhibitors \u003cb\u003e(Supplementary Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eb)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInterestingly, a number of essential genes identified in our genome-wide CRISPR screen (most of them undruggable targets) were significantly downregulated by CDK7 inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg \u003cb\u003eand Supplementary File S3\u003c/b\u003e), suggesting a common downregulated profile of genetic vulnerabilities. According to these findings, CDK7 inhibition emerges as a promising therapeutic strategy to effectively and broadly target genetic vulnerabilities in HNSCC.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eImpact of CDK7 inhibition on mice xenograft models\u003c/h2\u003e \u003cp\u003eWe next examined the effects of both genetic and pharmacological inhibition of CDK7 on tumor growth in vivo. Deletion of CDK7 dramatically impaired the tumor growth of FaDu and HCA-LSC1 cell lines subcutaneously injected in immunodeficient mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e, reaching final tumor volume reductions of 60 and 75% respectively \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. Noteworthy, treatment with YKL-5-124 and samuraciclib in a therapeutic setting \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec-d\u003cb\u003e)\u003c/b\u003e also demonstrated to be effective at reducing the tumor volumes of both FaDu and HCA-LSC1 xenografts \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee-f\u003cb\u003e)\u003c/b\u003e. Notably, YKL-5-124 exhibited a more potent antitumor activity, showing tumor reductions up to 74% after 12 days of treatment \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg\u003cb\u003e)\u003c/b\u003e. By contrast, samuraciclib led to lower and delayed antitumor effects with tumor reductions by 40% after 12 days of treatment \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg\u003cb\u003e)\u003c/b\u003e. Remarkably, none of these compounds caused any sign of adverse effect on the mice or body weight reduction \u003cb\u003e(Supplementary Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eImpact of selective CDK7 inhibition on patient-derived organoids\u003c/h2\u003e \u003cp\u003eFinally, the therapeutic potential of CDK7 inhibitors was also tested in two HNSCC patient-derived organoid (PDO) models. Treatment of already formed PDOs with either YKL-5-124 or samuraciclib was effective to abrogate the growth of both HNSCC PDOs tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea-b\u003cb\u003e)\u003c/b\u003e, with IC50s ranging 329 to 397 nM for YKL-5-124 and 399 to 666 nM for samuraciclib (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec\u003cb\u003e).\u003c/b\u003e The effects of CDK7-selective inhibitors were compared to those of the CDK4/6 inhibitor palbociclib. Interestingly, PDO_84 showed resistance to palbociclib, while PDO_55 exhibited a lower sensitivity to palbociclib compared to both CDK7 inhibitors (IC50 of 2 \u0026micro;M) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed-e\u003cb\u003e).\u003c/b\u003e These data are in line with our results on the viability of HNSCC cell lines.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlso relevant from a therapeutic point of view, we explored the ability of CDK7-selective inhibitors to effectively target and eradicate cancer stem cells (CSC)-enriched HNSCC tumorsphere cultures. Clonal sphere-forming ability in non-adherent serum-free culture conditions is a hallmark of self-renewal and CSC-related phenotype. This is also the basis for the formation and growth of tumor organoids, which are 3D structures cultivated from patient-derived stem cells (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Thus, we found that YKL-5-124 and samuraciclib were both effective in reducing the viability of CSC-enriched tumorsphere cultures in FaDu and UT-SCC38 cells, in a dose-dependent manner (\u003cb\u003eSupp Fig. S5\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDespite advances in cancer treatment, the five-year survival rate for advanced HNSCC remains below 50%. Beyond conventional radio/chemotherapy, only anti-EGFR and anti-PD-1 therapies are currently approved for HNSCC treatment; however, their efficacy and clinical benefit are rather unsatisfactory. This prompted us to perform an unbiased genome-wide functional CRISPR screen across multiple HNSCC cell lines aimed at uncovering actionable genetic vulnerabilities. We took advantage of an optimized dropout CRISPR screen that allowed us to identify hundreds of essential genes for all five HNSCC cell lines screened.\u003c/p\u003e \u003cp\u003eSpecial attention was focused on CDKs for their pivotal in cancer therapy as critical players in cell cycle regulation and proliferation. Our screen revealed CDK1 and CDK7 as common essentialities for the whole panel of HNSCC cell lines, thus emerging the best targetable candidates. The translational potential of CDK proteins for molecular targeted therapy is unquestionable. A growing number of CDK-selective inhibitors are being extensively evaluated in clinical trials. Notably, three CDK4/6 inhibitors: palbociclib (Pfizer), abemaciclib (Eli Lilly), and ribociclib (Novartis), have been long approved for clinical use in combination with endocrine therapy in breast cancer (\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e) and are under investigation for other solid tumors (NCT02465060, NCT03310879 and more). In particular, CDK4/6 inhibitors hold promise in HNSCC where CDKN2A/p16 deletion and/or amplification of CCND1 (Cyclin D1) frequently occur, ultimately causing sustained CDK4/6 activation. However, our screening results show that CDK4 and CDK6 are only essential in certain HNSCC cell lines, and initial clinical trials have not yielded encouraging results (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Similarly, even though CDK1 has shown a broader regulatory/control effect on HNSCC cell proliferation, the clinical application of CDK1 inhibitors is hindered by their high toxicity and lack of efficacy in cancer patients (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInterestingly, CDK7 has demonstrated widespread essentiality in all HNSCC cell lines screened and a remarkable progress has been made in the development of selective CDK7 inhibitors in recent years with promising data in other cancers. Given that CDK7 is an upstream regulator of other CDKs, CDK7 targeting may emerge an effective strategy to functionally target other critical/essential CDKs, such as CDK1.\u003c/p\u003e \u003cp\u003eCDK7 is detected aberrantly overexpressed in different cancer types (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Several CDK7-selective inhibitors have proved efficacy in various preclinical models of breast (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e), pancreatic (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e), and lung cancer (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e) among others. Moreover, some CDK7 inhibitors have entered Phase I clinical trials for breast cancers (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). In the context of HNSCC, elevated levels of CDK7 have been associated with poor clinical outcomes (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). On this basis, a role has been suggested as a potential therapeutic target for HNSCC; however, this possibility has not yet been explored in preclinical and/or clinical settings. Noteworthy, the present study provides the first broad-based evidence to comprehensively demonstrate the robust antitumor activity of CDK7-selective inhibitors using a wide range of disease-relevant cellular and animal models. Therefore, our findings provides unprecedented preclinical rationale strongly supporting and encouraging selective pharmacologic CDK7 targeting as a novel therapeutic strategy to test in future clinical trials for HNSCC patients.\u003c/p\u003e \u003cp\u003eBoth genetic and pharmacologic CDK7 inhibition consistently demonstrated potent antiproliferative effects in five different HNSCC cell lines, showing approximately 10-fold lower IC50 than the CDK4/6 inhibitor palbociclib. Differences could be attributable to a distinct mechanism of action, since we found that CDK7-selective inhibitors caused both cell cycle arrest and apoptosis induction. Therefore, the dual action of YKL-5-124 and samuraciclib as cytostatic and cytotoxic agents plausibly explains the superior effectivity to other CDK inhibitors such as palbociclib being exclusively cytostatic. The therapeutic potential of CDK7 inhibitors was also tested in already formed HNSCC PDOs, which offer a more physiologically relevant patient-derived model that mimic the architectural and genetic heterogeneity of the primary tumors of origin. This led to concordant results further demonstrating that YKL-5-124 and samuraciclib were both highly effective at reducing the viability of HNSCC PDOs as compared to palbociclib treatment. These results also reflect the superior therapeutic efficacy of CDK7 inhibition.\u003c/p\u003e \u003cp\u003eWe also found some remarkable differences between the two CDK7-selective inhibitors tested in our preclinical HNSCC models. Thus, the covalent CDK7 inhibitor YKL-5-124 exhibited a more robust antitumor activity than samuraciclib (ATP-competitive inhibitor) in tumor xenografts and some HNSCC cellular models, although more apoptotic-inducing activity was detected for samuraciclib. Samuraciclib is currently undergoing Phase I/II clinical trials against various tumor types, in particular estrogen receptor-positive breast cancer where it showing acceptable safety profile with evidence of antitumor activity in combination with endocrine therapy (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe effects of YKL-5-124 and samuraciclib were also further compared by global transcriptomics that revealed overlapping as well as specific changes. RNAseq analysis confirmed a common downregulation of cell cycle related signatures (i.e. MYC and E2F targets, and G2M checkpoint genes), as expected. In addition, DNA repair gene sets were also downregulated by both CDK7 inhibitors, which indicate a broader regulatory effect on cell division and genome stability. Among the specific transcription changes, YKL-5-124 led to downregulation of various oncogenic signaling pathways (i.e. mTORC1, TNFα, and KRAS), probably reflecting varying mechanisms of action between both inhibitors. Quite remarkably, CDK7-selective inhibitors significantly and massively reduced transcription of HNSCC essential genes identified in our genome-wide CRISPR screen, indicating that a broad spectrum of genetic vulnerabilities could be simultaneously inhibited by CDK7 targeting, including CDK1 and also numerous undruggable genes. Few essential genes were found to be upregulated by both CDK7 inhibitors, such as histone H2B that has been reported to be ubiquitinated by CDK7 (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). CDK7 inhibition could hence contribute to increase H2B expression. Altogether, these molecular insights may underlie the robust antitumor activity seen by both genetic and pharmacologic CDK7 in a wide range of HNSCC preclinical models, which pose the enormous therapeutic potential of CDK7 to design novel molecular-targeted treatments for HNSCC.\u003c/p\u003e \u003cp\u003eTumor heterogeneity is a well-known feature that greatly complicates cancer treatment and a long-lasting clinical effectivity. Research evidences indicate that heterogeneity develops through time as tumor-initiating stem cells, also known as cancer stem cells (CSCs) (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). CSC self-renewal, replication, and differentiation are postulated to produce a hierarchy of cells constituting the tumor mass (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). The same principle is applied to the generation and growth of tumor organoids that is based on CSC self-renewal features. Hence, complete eradication of tumors requires therapies able to effectively eliminate the CSC subpopulations responsible for treatment resistance, relapse and metastasis (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). On this basis, it is therefore a relevant finding that both YKL-5-124 and samuraciclib effectively reduced the viability of CSC-enriched tumorspheres as well as HNSCC PDOs, thereby suggesting that selective CDK7 targeting could also be an efficacious strategy to target and eradicate the CSC niche in HNSCC. Importantly, none of these CDK7 inhibitors led to any noticeable adverse effects in vivo, consistent with previous research demonstrating that CDK7 is non-essential in adult tissues with low proliferative rates and primarily impacts highly proliferative cells, a hallmark of cancer (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). It is also worth to mention that a recent study demonstrated CDK7 as a putative vulnerability to overcome CDK4/6 resistance in breast cancer (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Concordant to these observations, we showed that CDK7-selective inhibitors effectively reduced the growth/viability of palbociclib-resistant HNSCC PDO models.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe present study uncovered a catalog of genetic vulnerabilities that could serve as the basis to design novel molecular-targeted treatments for HNSCC. As a proof-of-principle, CDK7 was revealed as a common essentiality in all HNSCC cell lines screened, and all concordantly displayed an exceptional dependency on CDK7 activity/function. Furthermore, genetic and pharmacologic CDK7 inhibition showed potent antitumor activity and efficacy in a wide range of disease-relevant HNSCC models. According to our findings, CDK7 inhibition emerges as a promising therapeutic strategy to effectively and broadly target CDK signaling and genetic vulnerabilities in HNSCC. All the herein preclinical results provide unprecedented support for future clinical testing of CDK7-selective inhibitors in HNSCC patients.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAML Acute myeloid leukemia\u003c/p\u003e\n\u003cp\u003eATCC American Type Culture Collection\u003c/p\u003e\n\u003cp\u003eBCA Bicinchoninic Acid Assay\u003c/p\u003e\n\u003cp\u003eBSA Bovine Serum Albumin\u003c/p\u003e\n\u003cp\u003eCAK Cdk-activating complex\u003c/p\u003e\n\u003cp\u003eCDK Cycle-dependent kinases\u003c/p\u003e\n\u003cp\u003eCPM Counts Per Million\u003c/p\u003e\n\u003cp\u003eCRISPR Clustered Regularly Interspaced Short Palindromic Repeats\u003c/p\u003e\n\u003cp\u003eDESeq2 Differential expression using Sequencing 2\u003c/p\u003e\n\u003cp\u003eDMEM Dulbeco\u0026rsquo;s Modified Eagle Medium\u003c/p\u003e\n\u003cp\u003eDNA Deoxyribonucleic acid\u003c/p\u003e\n\u003cp\u003eFBS Fetal Bovine Serum\u003c/p\u003e\n\u003cp\u003eFDA Food and drugs administration\u003c/p\u003e\n\u003cp\u003eFDR False Discovery Rate\u003c/p\u003e\n\u003cp\u003eGSEA Gene Set Enrichment Analysis\u003c/p\u003e\n\u003cp\u003eHEPES 4-(2-hidroxietil)-1-piperazinaetanosulf\u0026oacute;nico\u003c/p\u003e\n\u003cp\u003eHNSCC Head and neck squamous cell carcinoma\u003c/p\u003e\n\u003cp\u003eHPV Human Papillomavirus\u003c/p\u003e\n\u003cp\u003eKO Knock-out\u003c/p\u003e\n\u003cp\u003eMAGeCK Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout\u003c/p\u003e\n\u003cp\u003eMOI Multiplicity of Infection\u003c/p\u003e\n\u003cp\u003eMOPS 3-(N-Morpholino)propanesulfonic acid\u003c/p\u003e\n\u003cp\u003eNES Normalized enrichment score\u003c/p\u003e\n\u003cp\u003ePCR Polymerase Chain Reaction\u003c/p\u003e\n\u003cp\u003ePDO Patient-derived organoid\u003c/p\u003e\n\u003cp\u003ePI Propidium Iodide\u003c/p\u003e\n\u003cp\u003eRIPA buffer Radio-Immunoprecipitation Assay buffer\u003c/p\u003e\n\u003cp\u003eRNA Ribonucleic acid\u003c/p\u003e\n\u003cp\u003eSDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis\u003c/p\u003e\n\u003cp\u003eSTR Short Tandem repeat\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTBS-T Tris-Buffered Saline with Tween-20\u003c/p\u003e\n\u003cp\u003eTCGA The Cancer Genome Atlas\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was performed in accordance to the principles of the Declaration of Helsinki with the appropriate approval of the Ethical and Scientific Committees of the Hospital Universitario Central de Asturias and the Regional Ethics Committee from the Principality of Asturias (CEImPA) (date of approval 9\u003csup\u003eth\u003c/sup\u003e March 2023; approval number: 2023.018, for the project PI22/00167). Patient informed consent was obtained for all the tissue samples collected by the Biobank of Principado de Asturias (PT20/00161 and PT23/00077).\u0026nbsp;All the animal experimental procedures were performed in accordance with the institutional guidelines of the University of Oviedo and approved by the Animal Research Ethical Committee of the University of Oviedo (date of approval 1\u003csup\u003est\u003c/sup\u003e August 2019; approval number PROAE 46/2019; date of approval 8\u003csup\u003eth\u003c/sup\u003e March 2024; approval number PROAE 03/2024).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA-sequencing data will be submitted to GEO and accession codes will be available before publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eK.T. is a shareholder of and has received research funding from Storm Therapeutics Ltd. All the other authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Instituto de Salud Carlos III (ISCIII) through the project grants PI19/00560, PI21/00208, PI22/00167, and CIBERONC (CB16/12/00390 and CB16/12/00228) and was co-funded by the European Union, the Instituto de Investigaci\u0026oacute;n Sanitaria del Principado de Asturias (ISPA), Fundaci\u0026oacute;n Bancaria Cajastur-IUOPA, and Universidad de Oviedo. Additional funding was provided through the grant \u0026ldquo;Ayudas para Grupos de Investigaci\u0026oacute;n de Organismos del Principado de Asturias 2021\u0026ndash;2023\u0026rdquo; (IDI/2021/000079), funded by Principado de Asturias through FICYT and the FEDER Funding Program from the European Union, and a\u0026nbsp;grant from the\u0026nbsp;MCIN/AEI (PID2020-117236RB-I00). M.O.-R. and E.P.-A are both recipients of a PFIS predoctoral fellowship from ISCIII (FI23/00037 and FI20/00064), and S.D.M. is supported by a grant from the Programa de Formaci\u0026oacute;n de Profesorado Universitario from the Spanish Ministry of Universities (FPU21/05639). S.A.-T. is a recipient of a Miguel Servet fellowship from ISCIII\u0026nbsp;(CP23/00101) and co-funded by the European Union. K.T. is supported by Wellcome Trust (grants RG83195, G106133 and G127005), UKRI Medical Research Council (grant RG83195) and Leukaemia UK (grants G108148 and G117699).\u0026nbsp;M.A.F was supported by the Asociaci\u0026oacute;n Espa\u0026ntilde;ola contra el C\u0026aacute;ncer (AECC; 2019/INVES19001ALVA).\u0026nbsp;M.A.G. is a recipient of a Severo Ochoa predoctoral fellowship from the Principado de Asturias (BP21-205), and F.H.-P. is funded by Fundaci\u0026oacute;n Alimerka and is a recipient of a Maria Zambrano postdoctoral fellowship at the University of Oviedo.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthor\u0026rsquo;s contribution\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, F.H.-P. and J.M.G.-P.; Data curation, M.O.-R., F.H.-P., and J.M.G.-P.; Formal analysis, M.O.-R., F.H.-P., J.M.G.-P., M.A.-F., R.G.-E., C.L.; Funding acquisition, J.M.G.-P., J.P.R., M.A.-F., R.G.-E., C.L., K.T., G.V.; Investigation, M.O.-R., F.H.-P., M.A.-G., B.d.L.-D., S.D.M., E.P.-A., M.R.-S., A.L.-F., D.C., R.G.-D., K.T., R.G.-E.; Methodology, M.O.-R., F.H.-P., K.T., M.A.-G., B.d.L.-D., S.d.M, E.P.-A., M.R.-S., A.L.-F., C.L., D.C., R.G.-D., M.A.-F., S.A.-T.; Resources, J.M.G.-P., J.P.R., M.A.-F., R.G.-E., C.L.; Supervision, F.H.-P. and J.M.G.-P.; Visualization, M.O.-R. and F.H.-P.; Writing\u0026mdash;original draft, M.O.-R., F.H.-P. and J.M.G.-P.; Writing\u0026mdash;review \u0026amp; editing, M.O.-R., F.H.-P., J.M.G.-P., M.A.-F., R.G.-E., C.L., J.P.R., K.T., G.V.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. Reidar Grenman (Turku Univ., Finland) for kindly providing UT-SCC38 and UT-SCC42B. We also thank staff at the Molecular Histopathology Unit-IUOPA, Animal Facility and SCT Core Services from the University of Oviedo and Sanger Institute for their excellent technical support. Carmen Mart\u0026iacute;n Hern\u0026aacute;ndez and Cristina Herrero Igartua, members of the Molecular Oncology Unit at CIEMAT, for their valuable help. We want to particularly acknowledge for its collaboration the Principado de Asturias BioBank (PT20/00161 and PT23/00077), financed jointly by Servicio de Salud del Principado de Asturias, Instituto de Salud Carlos III and Fundaci\u0026oacute;n Bancaria Cajastur and integrated in the Spanish National Biobanks Network.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. 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EMBO J. 2012;31(11):2498-510.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Head and neck squamous cell carcinoma, CRISPR screen, genetic vulnerability, CDK7, essential gene, cell cycle, YKL-5-124, samuraciclib","lastPublishedDoi":"10.21203/rs.3.rs-4679708/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4679708/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Head and neck squamous cell carcinoma (HNSCC) remains a challenging prevalent lethal malignancy, with still scarce targeted therapies and rather limited clinical benefit. We conducted an optimized genome-wide functional CRISPR screen aimed at identifying actionable genetic vulnerabilities for rapid preclinical evaluation as novel targeted therapies. Cyclin-dependent kinases (CDKs) were prioritized as pivotal in cancer therapy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Whole-genome CRISPR KO screen was performed in a panel of five HNSCC cell lines. CDK7 was selected for further functional and molecular characterization. The effects of CRISPR CDK7 knockout (KO) and CDK7-selective inhibitors were thoroughly investigated in cellular models using viability, colony formation and apoptosis assays, cell cycle analysis, and global transcriptomics by RNAseq. CDK7 inhibition was also therapeutically evaluated in mouse xenografts and patient-derived organoids (PDOs).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: CDK7 was identified as an essential gene across all five HNSCC cell lines screened. Genetic and pharmacological CDK7 inhibition significantly and consistently reduced tumor cell proliferation due to generalized cell cycle arrest and apoptosis induction. CDK7 KO, YKL-5-124 and samuraciclib also showed a potent antitumor activity effectively abrogating tumor growth in HNSCC PDOs and also in mouse xenograft models without significant toxicity. Mechanistically, CDK7 inhibition led to a broad downregulation of gene sets for cell cycle progression, DNA repair, and massively reduced the transcription of several essential genes and untargetable vulnerabilities identified by our CRISPR screen.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: CDK7 emerges as a promising targetable therapeutic vulnerability for HNSCC. Our study provides broad-based evidence for the robust antitumor activity of CDK7-selective inhibitors in disease-relevant preclinical models, strongly supporting patient testing.\u003c/p\u003e","manuscriptTitle":"Genome-wide functional CRISPR screen reveals CDK7 as a targetable therapeutic vulnerability for head and neck cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-29 18:35:37","doi":"10.21203/rs.3.rs-4679708/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"52806ea2-99af-41e0-a522-da986ea8c55e","owner":[],"postedDate":"July 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-31T16:21:26+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-29 18:35:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4679708","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4679708","identity":"rs-4679708","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}