EGFR mutations are present in approximately 15–50% of non-small cell lung cancer (NSCLC), which are predictive of anti-EGFR therapies. At variance, NSCLC patients harboring KRAS mutations are resistant to those anti-EGFR approaches. Afatinib and allitinib are second-generation pan-EGFR drugs, yet no predictive biomarkers are known in the NSCLC context. In the present study, we evaluated the efficacy of pan-EGFR inhibitors in a panel of 15 lung cancer cell lines associated with the KRAS mutations phenotype. Methods: KRAS wild-type sensitive NCI-H292 cell line was further transfected with KRAS mutations (p.G12D and p.G12S). The pan-EGFR inhibitors’ activity and biologic effect of KRAS mutations were evaluated by cytotoxicity, MAPK phospho-protein array, colony formation, migration, invasion, and adhesion. In addition, in vivo chicken chorioallantoic membrane assay was performed in KRAS mutant cell lines. The gene expression profile was evaluated by NanoString. Lastly, everolimus and pan-EGFR combinations were performed to determine the combination index. Results: The GI50 score classified two cell lines treated with afatinib and seven treated with allitinib as high-sensitive phenotypes. All KRAS mutant cell lines demonstrated a resistant profile for both therapies (GI50 < 30%). The protein array of KRAS edited cells indicated a significant increase in AKT, CREB, HSP27, JNK, and, importantly, mTOR protein levels compared with KRAS wild-type cells. The colony formation, migration, invasion, adhesion, tumor perimeter, and mesenchymal phenotype were increased in the H292 KRAS mutated cells. Gene expression analysis showed 18 dysregulated genes associated with the focal adhesion-PI3K-Akt-mTOR-signaling correlated in KRAS mutant cell lines. Moreover, mTOR overexpression in KRAS mutant H292 cells was inhibited after everolimus exposure, and sensitivity to afatinib and allitinib was restored. Conclusions: Our results indicate that allitinib was more effective than afatinib in NSCLC cell lines. KRAS mutations increased aggressive behavior through upregulation of the focal adhesion-PI3K-Akt-mTOR-signaling in NSCLC cells. Significantly, everolimus restored sensibility and improved cytotoxicity of EGFR inhibitors in the KRAS mutant NSCLC cell lines.Background:  Lung cancer remains among the most common tumor worldwide, with the highest mortality, approaching 1.7–1.8 million deaths per year . Only 20According to the histological context, lung cancer can be mainly classified into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), with incidences of 15% and 85%, respectively. In addition, NSCLC receives histologically two subclasses: adenocarcinomas, comprising about 40% of the NSCLC cases, and squamous cell carcinomas. Complete surgical resection is amenable in 70% of adenocarcinoma , 25 to 3EGFR, KRAS gene mutation, and ALK translocation are some of the most common mechanisms of carcinogenesis in NSCLC [EGFR mutated (L858R and or exon del 19) patients [EGFR mutant T790M/L858R and exhibits antitumor activity in pre-clinical and clinical trials [Genetic alterations are associated with NSCLC tumorigenesis, and currently guide the therapeutic strategies . EGFR, Kin NSCLC . EGFR alpatients . The secpatients . Less exl trials . Cytotoxl trials .EGFR mutation-positive advanced NSCLC [EGFR T790M mutation relapsed or refractory to first-line EGFR TKI therapy, thus definitively establishing this third-generation TKI as current standard treatment [The emergence of a third generation of EGFR-TKI is based on the efficacy and safety study of osimertinib, which showed excellent performance in second-line treatment and offered more survival benefits for patients with the T790M mutation . Osimerted NSCLC . In addireatment . Other treatment .KRAS mutations are mostly located in codons 12 and 13 and can occur between 10 to 25% in European patients, approximately 10% in East Asians [KRAS G12C-mutated locally advanced or metastatic NSCLC [KRAS G12C-mutated tumors, is currently being evaluated in phase 1/2 with results resembling the efficacy of sotorasib [KRAS G12C mutant [KRAS mutant NSCLC patients is ongoing [t Asians , and in t Asians . The oncic NSCLC . Moreoveotorasib . A recenC mutant , and a c ongoing .KRAS G12C mutation is still a therapeutic challenge, and the high cost of these anti-KRAS inhibitors constitutes an important barrier in low and middle-income countries. Interestingly, it has been shown that KRAS mutant patients can exhibit high EGFR expression levels [KRAS mutations in a panel of lung cancer cell lines.Despite these developments, NSCLC patients’ platinum-refractory with non-n levels . Therefo50) values. Of the cell lines evaluated, three harbor EGFR mutations  and four KRAS mutations   compared to erlotinib and lapatinib inhibitors (30.4 ± 20.3 and 22.8 ± 23.6). Allitinib was the most potent EGFR inhibitor in the cell line panel, with an IC50 value of 1.8 ± 1.9 µM, while afatinib showed values of 2.4 ± 2.3 µM  . The afa 0.07 µM . Notably± 2.3 µM .KRAS mutant cell line (A549), and shows cytotoxic activity to H292 KRAS wild-type cell line (KRAS mutant cell lines (A549), wild-type (H292), and EGFR mutant cells (H1975 and PC9) to a fixed concentration of both inhibitors (0.2 µM) for 5 days. As shown in KRAS mutant cell line A549. However, a significant proliferation curves inhibition in EGFR mutant cell lines (H1975 and PC9) and EGFR wild-type (H292) was observed. We also investigated the intracellular pathway changes by Western blot to determine the biological effect of irreversible EGFR inhibitors (KRAS mutant cell lines (A549 and SKLU-1)  to afatinib, and eight presented a resistant phenotype. Meanwhile, ten cell lines were classified as sensitive to allitinib and five cell lines were classified as resistant . Alternaell line . To evalhibitors C. Cell lhibitors C. Finall SKLU-1) C.KRAS mutations in NSCLC, gene-edited lung cancer cell lines H292-WT, H292-G12D, and H292-G12S were used. Primarily, adhesion ligand proteins were analyzed in both mutant cells, and we found a significant increase in cell–protein adhesion in KRAS mutant cells compared to wild-type H292 cells  assay. Basal protein expression levels were altered, due to KRAS mutation insertion, and the main phospho-protein targets altered were AKT isoforms, ERK1 and 2, JNK2, HSP27, panJNK, P38δ, RSK1, RSK2, and mTOR  and H292 KRAS-G12S (32 genes) compared to KRAS wild-type cells  revealed a tight network for the mTOR-mediated signaling pathway with a PPI (protein-protein interaction) enrichment of p-value: <1.0 × 10−16 . Kaplan–Meier curve , and we found a significant correlation between mTOR (mRNA) overexpression with low overall survival rates in patients with lung adenocarcinoma  and afatinib or allitinib (pan-EGFR inhibitors) drugs . As depicted in KRAS mutant cell lines after exposure to everolimus alone (Since the mTOR pathway was a major pathway upregulated by s) drugs A–C. H292bination . Similarbination . We furtus alone .KRAS mutations in a panel of lung cancer cell lines.In the present study, we compared the in vitro efficacy of EGFR inhibitors  and conducted a comprehensive analysis of the molecular mechanisms triggered by EGFR mutations (T790M). Additionally, allitinib showed greater GI levels than afatinib, and superior proliferation inhibition in EGFR mutant cells. A large-scale study conducted by our group previously reported similar cytotoxic effects to other tumor types, such as melanoma, pancreas esophagus cell lines, where the majority (70%) of the KRAS mutant cell lines were classified as allitinib resistant [KRAS mutations in clinical trials (NCT04671303) [It was observed that allitinib showed a superior cytotoxicity effect in a panel of NSCLC cell lines, including those harboring secondary esistant . These f4671303) .EGFR and KRAS wild-type cell lines in the presence of both pan-EGFR inhibitors. On the contrary, these pathways are not inhibited in the KRAS mutant cells. It is well known that the ERK-MAPK pathway plays a central role in oncogenic KRAS-driven malignant phenotypes of NSCLC [KRAS oncogenic desensitizes cells to EGF at the level of the EGFR. In contrast, a recent study reported that cells (A549 gene-editing) with concomitant KRAS-EGFR mutation exhibited higher sensitivity to EGFR-tyrosine kinase inhibitors (TKIs). Indeed, EGFR phosphorylation is strongly compromised in the presence of oncogenic KRAS [KRAS mutant cells.In addition, we showed complete inhibition of the phospho-ERK and phospho-AKT activity only in of NSCLC . Therefonic KRAS , as obseKRAS mutation promoted an aggressive phenotype in the H292 gene-edited cell line, as well as an increase in cell–protein adhesion, superior clonogenicity profile, and higher invasiveness rates, and stronger metastatic abilities. KRAS mutant cells showed an increased cell-to-extracellular matrix interaction and concomitant integrin subunit beta 8 gene (ITGB8) overexpression, which plays an important role in cell adhesion to protein matrix components and has been associated as a diagnostic biomarker for lung NSCLC patients [KRAS mutant patients. FAK may be surrogate markers of aberrant KRAS signaling found in aggressive phenotype in lung cancer [KRAS-mutated adenocarcinoma cell line model (A549-FL), used to examine the metastasis mechanisms that identified epithelial–mesenchymal transition (EMT) markers [KRAS mutant cell lines, with downregulation of E-cadherin, and upregulation of N-cadherin, vimentin, and SLUG. Importantly, SLUG overexpression has been identified as the key factor responsible for resistance to MEK1/2 inhibition and increased metastasis in other cancer types [In our study, patients . Furtherpatients . Our fin markers . We founer types . Additioer types  mainly kKRAS mutation (G12D) led to an increase in tumor number and size [KRAS mutant cells showed an increase in TCF1/TCF7 transcription factors, which regulate important genes such as c-myc, that is responsible for the proliferation stimulus and apoptosis decision and matrix metalloproteinase-7 expression [KRAS mutant gene in H292 edited cells.The crosstalk of oncogenic pathways is present in NSCLC tumorigenesis. A recent study found that the activation of Wnt/β-catenin alone does not promote tumor development but concomitant activation of Wnt/β-catenin signaling and and size . A similpression . Based oKRAS mutant cells, as a differentiated pattern of MAPK pathway proteins. Both KRAS mutation cells display strong phosphorylation of several proteins, such as AKT, ERK1/2, JNK, HSP27, and predominantly mTOR protein, compared to wild-type cells. The PI3K/AKT/mTOR axis is involved in tumor survival, proliferation, and distant metastasis, supporting the development of target therapies [Molecular analyses showed a critical difference between wild-type and herapies . A recenherapies . Anotherherapies ,38.KRAS mutant cell lines (G12D and G12S). Superior fold change was detected in secreted phosphoprotein 1 (SPP1) in KRAS mutant cell lines. The SPP1 overexpression is associated with aggressive phenotypes of lung cancer [SPP1 was deleterious for mice harboring KRAS G12D during the early stages of carcinogenesis [EFNA2 gene, also overexpressed in both KRAS mutant cell lines, is considered a ligand of Ephrin family receptors, and it is involved in the WNT/ß-catenin signaling pathway by TCF/LEF 1 transcriptional complex activation [KRAS mutant cells.In the comparative analysis of the gene expression profile, we found 18 genes simultaneously differentially expressed in both g cancer  and receg cancer . Moreoveogenesis . In additivation , which wKRAS mutant cells presented a significantly ITGB4 and ITGA6 downregulation. These genes are members of the integrin family of the adhesion receptors that play a major role in epithelial organization and function and display a very high adhesive strength for laminin matrices regulating metastasis development [ITGB4/ITGA6 genes may be related to the aggressiveness, increased invasion, and changes in adhesion found in mutated KRAS cells. We also found a significant loss in IL2RA and TNC expression in KRAS mutant cells, and little is known about the role of these two genes in lung carcinogenesis.elopment . Based oKRAS mutant cells, and the in-silico analyses indicated a significant association between mTOR overexpression and worse overall survival in NSCLC patients. Likewise, a recent study confirmed that phosphorylated-mTOR protein is associated with metastasis and poor prognosis of NSCLC patients after surgical resection [KRAS mutant cells. Similar dual inhibition encouraging results are reported in other cancer types, such as triple-negative breast cancers [KRAS mutations.Importantly, we observed that the mTOR pathway was significantly enriched in esection . Therapeesection . Moreoveesection . However cancers . NeverthKRAS mutant H292-G12D and H292-G12S previously developed by our group [2 at 37 °C. Cell culture reagents were purchased from Sigma-Aldrich , and to avoid the misidentified and/or cross-contamination, cell lines were authenticated by STR analysis [TM PLUS Mycoplasma Detection Kit .Fifteen lung cancer cell lines from different anatomic sites were used in this study, namely NCI-1975; COR-L23; COR-L105; LUDLU-1; NCI-H322; NCI-H358; NCI-H727, NCI-H2228 NCI-H827; PC9; SK-MES-1; SK-LU-1; A549, H292, and Calu-3 . We alsour group . The epianalysis . Culturev/v) in all experiments.Afatinib (Cat. No. S1011), allitinib (Cat. No. S2185), lapatinib (Cat. No. S2111), erlotinib (Cat. No. S1023), and everolimus (Cat. No. S1078) were purchased from Selleck Chemicals . All drugs were diluted in DMSO at 10 mM and stored at −20 °C for future use. Everolimus was dissolved in distilled water at 10 mM. In all experiments, DMSO or water were used as control vehicles at a final concentration of 1%  as previously reported [3 cells were plated in 96-well plates in DMEM or RPMI-10%, Fetal bovine serum (FBS) allowed to adherence overnight in a CO2 atmosphere. Then, cells were treated with increased concentrations of afatinib, allitinib, and everolimus  in DMEM 0.5% FBS. Absorbance was measured at 490 nm using the Varioskan Flash multimode reader . Results were normalized with DMSO control values. GraphPad Prism software  was used to calculate calculated IC50 values, using a nonlinear regression analysis. The cytotoxicity was measured by ApoToxiGLo kit , according to the manufacturer’s instructions. In this assay, cell lines were plated in 96-well plates in DMEM or RPMI 10% allowed adherence overnight in a CO2 atmosphere and treated with fixed concentrations  for 72 h. Mean growth inhibition (GI) values were calculated at a fixed concentration of 1 µM . The A431 cell line was used to determine the cutoff sensibility value due to high EGFR expression levels and our previous allitinib sensitivity results [According to the manufacturer’s instructions, cell viability was determined 72 h after drug treatments, using the colorimetric CellTiter 96reported . To asse results . Cell li results ,47. All  results .® (1:10 in PBS). After 24 h, the excess liquid was removed, and cell plates were incubated with 100 μL/well of 0.1% BSA for 2 h and washed with PBS. Wild-type and KRAS mutant cell lines were seeded in duplicate and incubated at 37 °C in a 5% CO2 humidified atmosphere for 2 h. Non-adherent cells were rinsed with PBS solution. Adhered cells were fixed with 10% of trichloroacetic acid (TCA) and at last, stained with crystal violet solution (0.5%). Additionally, crystal violet dye was dissolved in 10% of acetic acid solution and absorbance was obtained at 590 nm using a Varioskan Flash multimode reader . Absorbance values of the samples were plotted in GraphPad Prism software to create relative colony growth. All experiments were performed in triplicates at least three times.To assess the cellular adhesion attachment event, we used the CAM assay as previously described . The 96-KRAS mutant cell lines  were seeded in duplicate into 24-well plates (0.7 × 103 cells per well) and allowed to adhere overnight in media with 10% FBS. Subsequently, cells were treated with fixed concentrations of afatinib and allitinib (0.2 µM) for 21 days. After, cell lines were fixed with methanol for 10 min. The colonies were stained with crystal violet solution (0.5%) and the colony images were obtained using an optical system, Olympus SZX7. Moreover, crystal violet dye was dissolved in 10% of acetic acid solution and absorbance was obtained at 590 nm using Varioskan Flash multimode reader . Absorbance values of the samples were plotted in GraphPad Prism software to create relative colony growth. All experiments were performed in triplicates at least three times.Wild-type and EGFR mutant cell lines (PC9 and H1975), KRAS wild-type cell line (H292), and KRAS mutant cell lines (A549 and SKLU-1). Cell lines were plated in 6-well plates (5.0 × 105) in DMEM or RPMI; 10% FBS allows for adherence overnight in a CO2 atmosphere. After 24 h, cell lines were rinsed in ice-cold PBS, then scraped and lysed in lysis buffer . In total, 30 μg of total protein was resolved by 10% SDS-PAGE and immediately transferred to nitrocellulose membranes in TransBlot Turbo transfer . Primary antibody was purchased from Cell Signaling Technology : human total EGF Receptor , pEGFR-Tyr1068 , p44/42 MAPK ; p.p44/42 MAPK-Thr202/Tyr204 ; AKT(pan) ; pAKT-Ser473 ; total PARP ; AKT1; AKT2 ; AKT3 ; phospho-mTOR ; c-MYC ; c-JUN ; CD44 ; Ciclin D1 ; Met ; TCF1/TCF7 ; Snail ; Slug ; E-caderin ; N-Caderin ; α-Smoth (Cat. No. 19245 RRID: AB_2734735); Vimentin  and β-tubulin (Cat. No. 2128 RRID: AB_823664) as endogenous control. Primary antibodies were diluted in TBS-T solution at 1:1000 and incubated overnight at 5 °C. Then, membranes were incubated with anti-rabbit secondary antibody Antirabbit  at dilution 1:5000. Immune detection was performed using Pierce™ ECL Western , in automatic ImageQuant mini LAS4000 chemiluminescence systems . Western blot experiments were performed three times. A total of the 750 μg of fresh protein lysates was used to perform Human Phospho-Mitogen-activated Protein Kinase (MAPK) Antibody Array . The protein content of each cell lines was incubated overnight at 4 °C with nitrocellulose membranes each containing 24 different kinases antibodies spotted in duplicate on a nitrocellulose membrane. Then, phospho-MAPKs membranes were incubated with a pan anti–MAPKs-HRP antibody for 2 h at room temperature. Blot detection was carried out by chemiluminescence reagent in ImageQuant LAS 4000 mini . Densitometric analyses of dot or bands were measured using ImageJ software  [EGFR inhibition and intracellular pathways signaling were evaluated in KRAS mutant cell lines tumor proliferation, we used the CAM in vivo assay as previously described [6 cells were suspended and immersed in Matrigel solution, then incubated inside CAM on day 9 of development. After 4 days, the 3D tumor model was observed, and daily growth was monitored throughout the development. At the end of the assay, the tumor was photographed in ovo (14 and 17 days of development) and ex ovo (17 day of development) using a stereomicroscope (Olympus SZX7), and the perimeter of the tumors was measured using ImageJ software (version 9.0). The results were expressed as the mean perimeter ± SD.To assess in vivo escribed . Fertili® DNA Polymerase system (Promega) according to the manufacturer’s instructions. Real-time PCR was performed using a Step One Plus instrument . PCR conditions were 95 °C for 2 min to activate DNA polymerase, followed by 40 cycles at 94 °C for 15 s and specific annealing temperature of each primer  was used to determine gene expression in the KRAS mutated cells.Real-time quantitative PCR (RT-qPCR) was performed for specific genes linked to epithelial–mesenchymal transition, migration, and invasion using the GoTaqh primer . The difKRAS-G12D, and H292-KRAS-G12S were performed using the NanoString nCounter PanCancer Pathways panel (770 gene transcripts) according to the manufacturer’s instruction and as described (manuscript accepted in Scientific Reports) . This panel assesses 13 canonical pathways . Briefly, 100 ng aliquots of RNA quantified by Qubit 2.0 System  were hybridized with capture and reporter probes and hybridization buffer in a total volume of 15 μL, incubated at 65 °C for 20 h. After codeset hybridization, the samples were purified, and RNA-probe complexes were immobilized to a cartridge using the NanoString nCounter Prep Station  for 4 h. Finally, the cartridges containing immobilized and aligned RNA-probes complexes were scanned in the nCounter Digital Analyzer (NanoString Technologies) using 280 FOVs (Fields of View), and image data were subsequently generated using the high-resolution setting. Raw data quality control assessments were performed with nSolver Analysis Software version 4.0 with the default settings . Quantile normalization and differential expression were performed within NanoStringNorm package  [® platform, available at https://rosalind.onramp.bio accessed on 15 October 2021. Genes with fold change (FC) ≥ ±2 and p < 0.05 were considered significant.Gene expression analysis on H292-WT, H292-WA, USA)  in the RmTOR gene in adenocarcinomas, the PrognoScan database was used. This database is an extensive collection of publicly available cancer microarray datasets with clinical annotation and the biological relationship between gene expression and prognosis (http://www.prognoscan.org/ accessed on 15 October 2021) [p-value < 0.05 was considered statistically significant.For meta-analysis of the prognostic value of the er 2021) . This dat-test, and differences between groups were tested using a two-way analysis of variance. Statistical analysis was carried out using GraphPad Prism version 9. The level of significance in all the statistical analyses was set at p < 0.05.Single comparisons between the different conditions studied were made using Student’s KRAS mutant status. We elucidate the phenotypic and molecular mechanisms activated by KRAS mutations and identified novel genes, such as SPP1, EFNA2, ITGA6, ITGB8, ITGB4, IL2RB, and TNC, associated with PI3K/AKT/mTOR pathway activation. Our findings provide preclinical evidence supporting the rationale for combining the mTOR inhibitor with afatinib or allitinib for NSCLC treatment in KRAS mutant patients.In summary, we showed a higher cytotoxicity effect of allitinib in a representative panel of lung cancer, and the sensibility was associated with


DNMT3A and DNMT3B) and maintenance (DNMT1) methylation. Mutations in DNMT3A are observed in approximately 22% of acute myeloid leukemia (AML). We hypothesized that DNMT1 or DNMT3B could function as a synthetic lethal therapeutic strategy for DNMT3A-mutant AML. CRISPR-Cas9 tiling screens were performed to identify functional domains within DNMT1/DNMT3B that exhibited greater dependencies in DNMT3A mutant versus wild-type cell lines. Although increased sensitivity to DNMT1 mutation was observed in some DNMT3A mutant cellular models tested, the subtlety of these results prevents us from basing any conclusions on a synthetic lethal relationship between DNMT1 and DNMT3A. Our data suggests that a therapeutic window for DNMT1 methyltransferase inhibition in DNMT3A-driven AML may exist, but validation in more biologically relevant models is required.Aberrant DNA methylation patterns are a prominent feature of cancer. Methylation of DNA is mediated by the DNA methyltransferase (DNMT) protein family, which regulates de novo ( DNMT3A mutation.The methyltransferase domain of DNA methyltransferase protein 1 is an essential functional domain in acute myeloid leukemia, which is independent of  The most observed epigenetic mark in the mammalian genome is DNA methylation at the 5-position of cytosines in symmetric CpG dinucleotides. DNA methylation is essential for mammalian development and plays critical roles in several biological processes, including regulation of gene expression, genomic imprinting, X-chromosome inactivation, stem cell regulation, and transposon silencing3. Both DNMT3A and DNMT3B comprise Pro-Trp-Trp-Pro (PWWP) and Atrx-Dnmt3-Dnmt3l (ADD) domains that are reported to bind to DNA and interact with histone tails, as well as a C-terminal catalytic methyltransferase (MTase) domain4. Once methylation has been established by the de novo methyltransferases, these marks are maintained in subsequent cell divisions by the maintenance DNA methyltransferase, DNMT15. Although DNMT1 contains a MTase domain in the C-terminus of the protein similar to the de novo methyltransferases, it contains functional domains different from DNMT3A and DNMT3B upstream of its catalytic domain.DNA methylation of cytosines in CpG dinucleotide pairs is mediated by the DNA methyltransferase (DNMT) protein family. To date, there are five members of the DNMT protein family, of which three are known to exhibit enzymatic activity on mammalian DNA. The initiating DNA methylation mark on cytosines is mediated by the de novo DNA methyltransferases, DNMT3A and DNMT3BDNMT family members in cancer. DNMT1, DNMT3A, and DNMT3B are overexpressed across several types of cancer lineages, including acute myeloid leukemia (AML), melanoma, breast cancer, colorectal cancer, prostate cancer, and stomach cancer11. Mutations in DNMT3A are observed in patients with myelodysplastic syndromes  and AML (~22%)13. Of the DNMT3A mutations observed in AML patients, over half encompass a missense mutation at the R882 amino acid position13. DNMT3A mutations are associated with poor prognosis in MDS and AML patients, and increased risk of MDS transitioning to AML17. Therefore, a therapeutic strategy to target DNMT3A mutant MDS/AML is of high interest.Previous reports have examined the expression and mutation of DNMT3A, DNMT3B, and DNMT118. We hypothesized that DNMT1 or DNMT3B could be used as a synthetic lethal therapeutic strategy for DNMT3A-mutant AML. CRISPR-Cas9 tiling-single guide RNA (sgRNA) knockout screens have been shown as an efficient screening paradigm to identify essential protein domains within a protein-of-interest21. Using a custom pooled lentiviral library with sgRNAs tiling the coding region of DNMT1 and DNMT3B, we performed CRISPR-Cas9 knockout tiling screens across a panel of AML cell lines either wild-type or mutant for DNTM3A. While knockout mutations of DNMT3B had minimal effects on cell proliferation across the AML cell lines screened, we identified sgRNAs spanning DNMT1 that resulted in decreased cell proliferation. Independent analyses using STARS22 and ProTiler19 identified the catalytic MTase domain of DNMT1 as the most essential functional domain for viability. While DNMT1 knockout mutations did exhibit stronger effects in some AML cell lines screened compared to others, we were unable to determine if DNMT1 is synthetic lethal with DNMT3A mutational status in these cell lines. We engineered paired isogenic cell lines that were either wild-type or mutant for DNMT3A to help minimize background genetic influence to focus on the hypothesized genetic interaction. CRISPR-Cas9 tiling screens across some isogenic cell lines revealed higher sensitivity to DNMT1 knockout mutations in the cell line mutant for DNMT3A compared to the isogenic wild-type cell line, with sgRNAs targeting the MTase domain exhibiting the most consistent results. However, the subtlety in these results prevents us from making conclusions on a synthetic lethal relationship between DNMT1 and DNMT3A. Our studies suggest that a therapeutic window for DNMT1 methyltransferase inhibition in DNMT3A-driven AML may exist, but validation in more biologically relevant models will be required.A previous study identified a putative trigenic negative interaction between DNMT3A is mutated in more than 22% of AML, we wanted to determine if either of the other catalytically active DNMT protein family members, DNMT1 or DNMT3B, can exhibit a synthetic lethal relationship with DNMT3A mutation. The Cancer Dependency Map (DepMap) provides publicly available data from genome-wide pooled shRNA and CRISPR screens across a large panel of cancer cell lines24. We first looked at the effects of shRNA knockdown and CRISPR knockout of DNMT1 and DNMT3B across a panel of AML cell lines screened in Project Achilles. DepMap categorizes DNMT1 as a common essential gene (a gene that ranks within the topmost depleting genes in at least 90% of cancer cell lines tested) based on the gene effects observed across a large number of cell lines screened using the shRNA and CRISPR libraries26. This trend is observed when looking at DNMT1 shRNA and CRISPR data across the AML cell line panel  and THP-1 cells (DNMT3A wild-type), which were very sensitive to DNMT1 mutations, and OCI-M1 cells (DNMT3A wild-type), which were less sensitive to DNMT1 mutations. We assessed the mutational spectrum across three functional domains within DNMT1 – the CXXC domain, which was not essential for proliferation in any cell line tested, and the RFTS and MTase domains, in which we observed certain sgRNAs inhibiting proliferation in some AML cell lines screened.Although the As expected, each individual sgRNA that was tested with NGS analyses exhibited different percentages of out-of-frame and in-frame insertions and deletions (indels)  in DNMT1, we utilized 2 different computational approaches. First, we used ProTiler, a computational method to map CRISPR knockout hyper-sensitive (CKHS) regions, which correlates with functional domains associated with a stronger sgRNA depletion effect across the coding region of the protein-of-interestMT1 Fig.  or DNMT3T3B Fig.  in AML clls Fig. . In contned Fig. . ProTileMT1 Fig. . As expeMT1 Fig. .Fig. 5Mu22. CRISPR sgRNAs along the span of both DNMT1 and DNMT3B were grouped into bins of 11 neighbors to act as “pseudo-genes” for the purpose of the STARS algorithm. STARS was run across DNMT1  to identify significantly depleted regionsMT1 Fig.  and DNMTned Fig. . The 2 mDNMT3A wild-type and mutant cell lines does not clearly suggest that DNMT1 is synthetic lethal with DNMT3A mutation. Our shRNA experiments found that AML cell lines are sensitive to DNMT1 knockdown regardless of DNMT3A mutational status. Additionally, published and internal CRISPR genome-wide screens found that both DNMT3A wild-type and mutant AML cell lines are sensitive to DNMT1 knock-out mutations28. A caveat of these experiments is that in addition to the mutational status of DNMT3A, AML cell lines have many differences within their genetic backgrounds, making it difficult to make any conclusions on any potential DNMT1/DNMT3A genetic interaction. To better understand the relationship between these two genes, we generated isogenic AML cell line pairs, in which each cell line in a pair is only different with respect to their DNMT3A mutational status. A clinically relevant R882C mutation in DNMT3A was knocked into one allele in OCI-M1 and NOMO-1 cells, which are normally wild-type for DNMT3A. Conversely, we reverted DNMT3A mutations in OCI-AML2 and SIG-M5  cells back to the wild-type sequence  and isogenic (DNMT3A wildtype) screens  occurring in the very end of the MTase domain, as well as early in the protein sequence. However, a larger portion of the MTase domain, and BAH1/BAH2 domains exhibit significant and negative residuals, indicating these regions are more lethal in the DNMT3A mutant clone  occurring in the very end of the MTase domain, as well as early in the protein sequence. Some significant and negative residual values  occur in the BAH domains and early in the MTase domain  and reference parental cell lines by fitting data from the pair to a non-linear function and determining the resulting residual of each sgRNA. Neighboring sgRNAs again were binned in groups of 11 to determine an average residual and significance value along the one Fig. . In the ain Fig. . In the ain Fig. . In addi21. Using the CRISPR/Cas9 tiling screen methodology, we identified the MTase domain of DNMT1 as a potential dependency in AML. Mutations within the DNMT1 methyltransferase resulted in proliferative disadvantages across the AML cell lines screened, and consistently scored regardless of analysis method utilized. These findings are not surprising, as the MTase domain is essential for the catalytic function of DNMT130.CRISPR/Cas9 tiling screens have proven a powerful tool for drug target discovery. Tiling sgRNAs across the coding region of a gene-of-interest allows for the identification of individual functional domains as cancer dependencies and could guide more efficacious drug targets for pharmacological inhibition. The findings from previously published tiling screens led to the hypothesis that in-frame mutations generated in essential functional domains can uncover regional vulnerabilities within proteins-of-interest by disrupting catalytic components or important protein:protein interactions, whereas in-frame mutations in non-essential functional domains would have minimal effect on protein function32, and can also function as an auto-inhibitory domain of DNMT133. BAH domains can bind to histone tails in a modification-dependent manner and recently, the BAH domains in DNMT1 were reported to be essential for the localization of DNMT1 to replication foci34. These domains could be considered as therapeutic approaches in lieu of the MTase domain, as these domains are unique to DNMT1 with respect to catalytically active DNMT family members.While sgRNAs targeting the MTase domain had the most impact on proliferation across AML cell lines screened, sgRNAs targeting other functional domains in DNMT1, specifically the RFTS and BAH domains, also exhibited proliferative dependencies, albeit to a lesser extent. The RFTS domain of DNMT1 is essential for replication-dependent DNA methylation by recruiting DNMT1 to DNA methylation sitesDNMT3A, as well as the generation of isogenic DNMT3A WT/mutant cell line pairs, allowed us to better examine the potential synthetic lethal interaction between DNMT3A and other DNMT family members DNMT1 and DNMT3B. Although studies have reported that DNMT3B is essential for cancer cell growth and survival37, we did not identify an essential role for the de novo methyltransferase across any AML cell lines with respect to viability in our CRISPR-based screens. Conversely, we did find AML cell lines dependent on DNMT1 expression at varying degrees. Knockout mutations of DNMT1 exhibited proliferative effects across all AML cell lines screened independent of the mutational status of DNMT3A. However, it is difficult to interpret these data due to the variability in genetic background across the AML cell lines screened. DNMT3A wildtype/mutant isogenic AML cell line pairs allowed us to study the effects on DNMT1 knockout mutations in AML cell lines in which the only difference in their genetic background is the mutational status of DNMT3A. We found that the isogenic cell line that is mutant for DNMT3A sometimes exhibited the strongest proliferative effects with knockout mutations in DNMT1. This, in addition to the DepMap CRISPR/shRNA data that shows that DNMT3A mutant AML cell lines were the most dependent on DNMT1 expression26, suggests a potential increased dependency between DNMT1 and DNMT3A that could be utilized as a therapeutic treatment for AML.The use of AML cell lines wild-type or mutant for 38. Reduction of Dnmt1 expression in a murine adrenocortical tumor cell line induces DNA demethylation and inhibits tumorigenesis39. In an ApcMin/+, Trp53-/- acinar cell pancreatic cancer model, decreased Dnmt1 expression reduces tumor burden, but not tumor size40. Reduction of Dnmt1 in an ApcMin/+ colorectal cancer mouse model completely suppresses polyp formation and a reduction in hypermethylation41. The role of the maintenance methyltransferase has also been well-studied in breast cancer models. Dnmt1 is essential for mammary and cancer stem cell maintenance and tumorigenesis7. Furthermore, DNMT1 promotes tumorigenesis in breast stromal fibroblasts, and reduction of DNMT1 expression suppresses these properties42. These studies support our findings that DNMT1 is required for cancer cell survival and makes it an interesting therapeutic target.The role of DNMT1 in tumorigenesis has been well-studied across several cancer lineages. A conditional knockout of Dnmt1 has been shown to block the development of leukemia, and haploinsufficiency of Dnmt1 delays the progression of leukemogenesis and impair LSC self-renewal43. While effective, there remains a need for more potent and selective inhibitors that can improve outcomes for these patients. A few studies have reported the generation of selective DNMT1 and DNMT3B inhibitors46. The question that remains is whether a therapeutic window exists for DNMT3A mutated AML with a selective DNMT1 inhibitor.To date, the most widely used epigenetic modulators as a therapeutic agent are pan DNMT inhibitors 5-azacitidine (azacitidine) and 5-aza-2′-deoxycytidine (decitabine). Azacitidine and decitabine are pyrimidine analogs that are modified in position 5 of the pyrimidine ring. They incorporate into RNA and DNA and covalently trap DNMT1, leading to protein degradation and reduced DNA methylation. Azacitidine and decitabine are approved globally and are treatments for MDS or AML as single agents or in combinationR882C/+ mutation into the DNMT3A locus. The DNMT3A mutations in OCI-AML2 and SIG-M5 cells were also engineered back to the wild-type sequence by Synthego. Clonal expansions of NOMO-1 and SIG-M5 parental pools obtained from Synthego were generated with a limited dilution series in 384-well plates. All cell lines used in this study were authenticated and confirmed mycoplasma negative at Genetica/LabCorp. Cas9-expressing OCI-M1 cells (OCI-M1_311 cas9) were obtained from the Broad Institute. For the remaining cell lines used in this study, Cas9-expressing cell lines were generated by lentiviral transduction using one of the following: pXPR_BRD101 (Broad Institute), pLEX_311-Cas9v2 (Broad Institute), pLV-ENO-Cas9 (Vector Builder), pLV-TPT1-Cas9 (Vector Builder), pLV-TUBA1B-Cas9 (Vector Builder), or pLV-TMSB4X-Cas9 (Vector Builder), which express Cas9 from either the EFS, EF1a, TPT1, TUBA1B, ENO, or TMSB4X promoters, respectively, as well as the blasticidin resistance gene. Cells were selected for 12–14 days post-selection on blasticidin. Cas9 activity for each Cas9-expressing cell line was tested using pXPR_011 or pRDA_047 (Broad Institute), lentiviral GFP reporters that co-expresses a sgRNA guide targeting GFP47. A Cas9 cell line with a reduction in GFP expression by at least 50% 10–12 days post-transduction was considered usable for pooled screens.The parental cell lines used in this study are the following: OCI-AML2 , OCI-AML3  SIG-M5 , THP-1 , NOMO-1 , and OCI-M1 . OCI-M1 and NOMO-1 cells were sent to Synthego  for knock-in of the clinically relevant DNMT1#1: TRCN0000364186; shDNMT1#2: TRCN0000232751; shDNMT3B#1: TRCN0000424360; shDNMT3B#2: TRCN0000414235; shControl: SHC007. THP-1 and OCI-AML2 cells transduced with both shRNA constructs were seeded in 200 µL media per well in a 96-well round bottom, ultra-low attachment plates (Costar). Cell seeding numbers were chosen based on growth curves to ensure linear growth throughout the experiment . Spheroid-like growth was monitored in real-time by live-cell imaging  acquiring one image every four hours for 8 days. Confluence (%) as a measure of spheroid-like cell confluence size was determined using an integrated analysis tool (Phase Confluence % tool available in the IncuCyte software). Percent confluence at the beginning of the experiment (t = 0) was used as baseline.The Incucyte® Live-Cell Analysis System (Sartorius) is an automated image acquisition system placed in a standard tissue culture incubator with an integrated analysis software that captures and analyzes images of cells over time. THP-1 and OCI-AML2 cells were transduced with shRNA lentivirus of one construct followed by antibiotic selection 24 h post-transduction. Cells stably expressing the shRNA were transduced with the second shRNA lentivirus 1 week later, with antibiotic selection 24 h post-transduction. The following shRNAs were obtained from Sigma Aldrich (MISSION® pLKO.1): shDNMT1 and DNMT3B mRNA expression upon shRNA transduction, RT-qPCR was performed. RNA was isolated from transduced cells 10 days post-transduction using the Qiagen RNeasy Mini kit (Qiagen). cDNA was generated using the Invitrogen SuperScript IV Reverse Transcriptase kit (Thermo Fisher). DNMT1 and DNMT3B TaqMan primers were purchased from Thermo Fisher (TaqMan Gene Expression Assay) and the specific Ct values were normalized to the GusB Ct values (housekeeping gene).To evaluate the effects on 22. This library contains 77,441 sgRNAs total, comprising of approximately 4 sgRNAs per gene and 1000 non-targeting control sgRNAs. A custom pooled DNMT1/3B lentiviral library consisting of 1493 sgRNAs was created by the Broad Institute. This library contains 777 sgRNAs spanning the coding region of DNMT1 and 421 sgRNAs targeting the coding region of DNMT3B. Non-targeting sgRNAs (250 sgRNAs) and 45 sgRNAs targeting common essential genes were also included in the library as negative and positive controls, respectively. See Supplementary Data The Brunello library (CP0041) was used for genome-wide CRISPR/Cas9 pooled screens (Broad Institute)DNMT1/3B tiling screens were performed at approximately 5000x representation and 3 replicates per screen. Cells were transduced at an MOI between 0.3 and 0.5 to ensure that each cell received at most 1 sgRNA. The volume of virus used to achieve optimal transduction rates was predetermined for each cell line. Cells were spun in 12-well plates at 2000 rpm for 2 h at 30 °C. Cells were grown with puromycin selection beginning at 24 h post-transduction. Cells were passaged every 3–4 days for up to 3 weeks. Cells were collected at days 5 and 23 of the screen, pelleted, resuspended in PBS, and stored at −20 °C. Genomic DNA (gDNA) isolation was performed using the QIAamp DNA Blood Midi Kit or QIAamp DNA Blood Maxi Kit (Qiagen) using manufacturer’s instructions. Purified gDNA was sent to the Broad Institute for NGS and deconvolution22. Readcounts were normalized to reads per million and log 2 transformed, then a log2 fold change (L2FC) from the plasmid DNA (pDNA) was calculated per sgRNA. Standardized log fold change values were calculated (described below) and used to plot the data as a relative comparison across screens. The raw count values from NGS on all DNMT1/3B tiling screens presented in this study are included in Supplementary Data Genome-wide CRISPR pooled screens were performed at a representation of 500× and 3 replicates per screen. 22 were performed for the DNMT1/3B tiling screens by taking the log2 fold changes score of each sgRNA. The probability mass function of a binomial distribution was then used to calculate a score for every sgRNA that scores in the top 10% of all sgRNAs for an experiment. Instead of mapping sgRNAs to genes, a mapping file was created for STARS where neighboring sgRNAs were binned in groups of 11 across the coding region of both DNMT1 and DNMT3B. At least 2 sgRNAs in the top 10% were required for a bin of sgRNAs to score, and permutation testing was used to generate the null distribution, allowing for the calculation of p-values and FDRs per bin. The ProTiler algorithm19 was run on a standardized log2 fold change score, where the median of the non-targeting guides across all screens was set to 0 and the median of the positive ‘common essential’ controls across all screens was set to −1. The residual analysis48 uses a natural cubic spline with three degrees of freedom for fitting. The residuals from the fit of the L2FC for the clone(s) compared to the L2FC of the reference parental line, across all sgRNAs in the DNMT1/DNMT3B custom library, were averaged across the same bins (instead of by gene) as used for the STARS analysis above. Z-scores p-values were calculated, and FDRs were calculated using the Benjamini & Hochberg method.STARS analysesR2 values in Supplementary Fig. A Dunnet’s multiple comparison test was performed on QPCR data shown in Fig. g 48 h post-transfection, and resuspended in 50 µL DirectPCR Cell Lysis Buffer (Viagen Biotech) containing 2 µL Proteinase K (Thermo Fisher Scientific). Lysates were incubated at 68 °C for 15 min, followed by 10 min at 98 °C. Regions of DNMT1 were PCR amplified and purified using the QIAquick PCR Purification Kit using the manufacturer’s instructions (Qiagen). Purified PCR product was sent to Genewiz  for Amplicon-EZ Next Generation Sequencing and analyses. The following primers were used for PCR amplification: RFTS-F 5′-GAGCAGCTGTAGGCCAAGTC-3′; RFTS-R 5′-AGGCTACCCCAACTGAACCT-3′; CXXC-F 5′-TGGTGGTGTGATCTTGGCTA-3′; CXXC-R 5′-CTGACCTACCTCCGCTCTTG-3′; BAH1-F 5′-GTGGGGGACTGTGTCTCTGT-3′; BAH1-R 5′-TGAAAGCTGCATGTCCTCAC-3′; BAH2-F 5′-GGGGGAGTCTACCTTGCAGT-3′; BAH2-R 5′-CGAGGAAGTAGAAGCGGTTG-3′; SAM-H-F 5′-GTTGCAGTGAGCCAAGATCA-3′; SAM-H-R 5′-TGACGGTTGTGCTGAAGAAG-3′; SAM-L-H 5′-TCACTTGAGCCTCTGGGTCT-3′; SAM-L-R 5′-ATCAGTGCATGTTGGGGATT-3′.Individual sgRNAs targeting either the RFTS, CXXC, BAH1, BAH2, or MTase domains were ordered from IDT (see Supplementary Data Further information on research design is available in the Peer Review FileSupplementary MaterialDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Reporting Summary


Poultry breeding programs are established to ameliorate the genetic potential of domestic birds through a selection process and crossbreeding programs. In this context, crossbreeding has been considered as an essential tool to produce favorable genotypes, which are affected by different genetic resources and non-genetic factors. Thus, the aim was to evaluate the impact of crossing white and brown quail lines on growth performance, carcass traits, and the relative expression of growth-related genes. The crossbred WBQ quail line  exhibited a significantly greater dressing percentage and better feed conversion ratio (FCR), as well as higher mRNA expression of growth hormone receptor (GHR) and insulin-like growth factor-1 (IGF-1) genes.p < 0.001). Meanwhile, both crossbred quail lines (WBQ and BWQ) showed significantly lower FCR compared with both purebred quail lines (p = 0.001). Both crossbred quail lines showed greater dressing percentages compared with both purebred quail lines (p = 0.038). The brown quail line showed significantly (p = 0.05) higher levels of serum triglycerides and VLDL compared with the white and BWQ lines. The WBQ crossbred line exhibited significantly higher mRNA expression of GHR and IGF-1 genes compared with other quail lines (p < 0.001). Both crossbred lines (WBQ and BWQ) exhibited negative heterosis percentages for body weight  and feed intake . Meanwhile, heterosis percentages for FCR  and dressing percentage  were improved in both crossbred lines. The WBQ line showed high heterosis percentages for the expression of GHR and IGF-1 genes . In conclusion, the WBQ line exhibited significantly greater dressing percentage and better FCR, as well as higher mRNA expression of GHR and IGF-1 genes. These results may be helpful to improve breeding programs and to develop commercial lines of meat-type Japanese quail.The aim was to evaluate the performance, blood lipid profile, and the relative expression of growth-related genes in purebred white and brown quail lines and their crossbred lines. A total of 240 one-day-old Japanese quail chicks of white and brown line, their crossbred line , and reciprocal crossbred line  were divided into four equal groups (60 birds each). The white quail line showed significantly higher final body weight, daily gain, and feed intake compared with the other quail lines ( The Japanese quail belongs to the order Galliformes, family Phasianidae, genus Coturnix, and species japonica . FurtherPoultry breeding programs are established to ameliorate the genetic potential of domestic birds through a selection process and crossbreeding programs . In thisIt is widely accepted that growth rate is greatly influenced by heterosis, which has been utilized in the breeding strategies of domestic fowls . In commThe experiment was conducted at the Experimental Animal Research Center, Faculty of Veterinary Medicine, Benha University, Egypt. The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Animal Ethics Committee of Banha University, Egypt .The parent Japanese quail lines were obtained from the Quail Production Unit, Agricultural Experiment and Research Unit, Faculty of Agriculture, Ain Shams University, Egypt. Both lines mainly differ in the colors of their feathers (white and brown). Furthermore, both lines were developed from the same random-bred population (non-selected pedigreed meat-type population), and the mating system was completely random. The random-bred base population was developed through selection for bodyweight at 28 days. A total of 240 one-day-old male Japanese quail chicks of white and brown line, their crossbred line , and reciprocal crossbred line  were the materials of this study. In addition, an excess number of sexed chicks were kept on hand to avoid any sexing problems. All chicks were of homogeneous size, and the initial body weight was 7.35 ± 0.45 g. Each genetic group was divided into 4 replicates (15 birds in each replicate). All quail chicks were reared under the same managerial and climatic conditions. All chicks were managed in an experimental room, which was supported with digital heaters to meet the required temperature and humidity. The brooding temperature was set at 35 °C for the first four days, then dropped to 32 °C at the end of the first week, and subsequently kept at 26–29 °C. The relative humidity was maintained at the recommended value (65 ± 5.0%). Feed was given ad libitum, and manual watering devices were used. Additionally, all birds were fed on the same basal diet .On a weekly basis, the body weight, average daily feed intake (ADFI), and the feed conversion ratio (FCR) were recorded for each genetic group. The average daily gain was calculated as final BW-initial BW/age (days). Moreover, the overall FCR of quails from each replicate was calculated as feed intake (g)/weight gain (g). At the end of experiment (6 weeks of age), 12 birds were chosen from each genetic group (3 birds/replicate), fastened for 12 h, and then slaughtered according to the standard pre-stunning technique. Consequently, the evisceration process and removal of the internal organs were performed. Following the effective drain for 5 min, the carcasses were chilled at 2 °C for 30 min. The dressing percentage was estimated as the carcass weight relative to the live body weight . ThereafAt the end of trial, three birds were chosen from each replicate to obtain 2 ml blood samples in plain tubes, which were then centrifuged at 3000 rpm for 10 min to separate serum portion. The obtained serum samples were stored at −20 °C until analysis. Total cholesterol , triglycerides , and serum high-density lipoprotein  were measured using Centronic kits . According to the formula of Friedewald et al. , serum lFor RNA analysis, three liver samples were collected from each replicate. Using the TRIZOL reagent, the RNA material was extracted . The cDNA was synthesized from RNA , and then samples of cDNA were stored at −20 °C.For each gene, the real time-PCR products were utilized to determine the expression of these genes by using one pair of primer . The PCRThe SPSS software program  was used to analyze the obtained data through the application of ANOVA procedures. The possible variations between the quail lines were evaluated by the Duncan multiple range test. The outputs are illustrated as means and the standard error of mean (SE). The applied model comprised the following effects:ij = the dependent variable.Yμ = the population mean.i = the fixed effect of genetic types .Gij = random error.eThe unit of heterosis was calculated as [(F1 − M)/M × 100%], in which M is the parental means and F1 is the crossbred means .p < 0.001). Meanwhile, both crossbred quail lines (WBQ and BWQ) showed significantly lower FCR compared with both purebred quail lines (p = 0.001).The effects of crossing white and brown quail lines on growth performance traits are illustrated in p = 0.04). Meanwhile, both purebred quail lines showed higher gizzard percentages compared with both crossbred quail lines (p = 0.01). The current trial did not record significant differences in the heart, liver, spleen, and intestine percentages between the experimental groups.The effects of crossing white and brown quail lines on carcass traits are described in p = 0.05) higher levels of serum triglycerides and VLDL compared with the white line and BWQ crossbred line. Meanwhile, the current study did not report significant differences in the levels of serum cholesterol, HDL and LDL between the experimental groups.The effects of crossing white and brown quail lines on blood lipid profile are described in GHR and IGF-1 genes are illustrated in GHR and IGF-1 genes compared with both purebred lines and the BWQ line (p < 0.001).The effect of crossing white and brown quail lines on the relative expression of Heterosis percentages of growth performance and carcass traits are described in Crossbreeding has been considered as a valuable tool to produce favorable genotypes, which is usually influenced by different genetic resources and non-genetic factors. Hence, the present work was conducted to elucidate the impact of crossing white and brown quail lines on growth performance, carcass traits, and the expression of some growth-related genes. In the current study, the white quail line showed significantly higher body weight, ADG, and ADFI compared with other quail lines. Consistent with these findings, Abou Khadiga et al.  suggesteIn the current study, both crossbred quail lines (WBQ and BWQ) showed significantly lower FCR compared with both purebred quail lines. This may be attributed to the lower feed intake and averaged ADG of the crossbred lines. Additionally, Varkoohi et al.  suggesteBoth crossbred quail lines, herein, showed greater dressing percentages compared with both purebred quail lines. In accordance with these findings, Ali et al.  demonstrp < 0.05) in the level of serum cholesterol between imported and local Japanese quail flocks. Similarly, the plasma cholesterol levels were significantly differed among the selected lines of Japanese quails [In the present study, the brown quail line exhibited significantly higher levels of serum triglycerides and VLDL compared with the white and BWQ quail lines. Meanwhile, other parameters of lipogram did not differ among the quail lines. Jatoi et al.  reportede quails . On the e quails  reportedHormonal regulation of growth involves a complex series of interactions among different hormones including the somatotropic axis . Furthermore, growth hormone can affect growth directly, but the important effects are mostly mediated through the IGF-1 activity . Herein,It is believed that IGF-1 plays a crucial role in the growth rate and feed efficiency of the birds. Moreover, the lower the IGF-1 level is, the slower is the growth . AdditioAlthough both crossbred lines (WBQ and BWQ) exhibited negative heterosis percentages for body weight and ADG, the heterosis percentages of FCR were improved. Moreover, both crossbred lines exhibited positive heterosis percentages of dressing percentage. Moritsu et al.  conducteIn the present study, the WBQ quail line showed high and positive heterosis percentages for GHR and IGF-1 gene expressions compared with the BWQ line. Consistent with these findings, Wang et al.  have repIt can be concluded that the crossbred quail lines (WBQ and BWQ) showed greater dressing percentages and better FCR compared with the purebred brown and white quail lines. Furthermore, the WBQ line exhibited a significantly higher mRNA expression of GHR and IGF-1 genes compared with the other quail lines. The WBQ exhibited positive heterosis percentages for dressing percentage and the expression of GHR and IGF-1 genes. The current study may be helpful to improve breeding programs and to develop commercial lines of meat-type Japanese quail.


AZD0156 is an oral inhibitor of ATM, a serine threonine kinase that plays a key role in DNA damage response (DDR) associated with double-strand breaks. Topoisomerase-I inhibitor irinotecan is used clinically to treat colorectal cancer (CRC), often in combination with 5-fluorouracil (5FU). AZD0156 in combination with irinotecan and 5FU was evaluated in preclinical models of CRC to determine whether low doses of AZD0156 enhance the cytotoxicity of irinotecan in chemotherapy regimens used in the clinic.in vitro and in vivo models.Anti-proliferative effects of single-agent AZD0156, the active metabolite of irinotecan (SN38), and combination therapy were evaluated in 12 CRC cell lines. Additional assessment with clonogenic assay, cell cycle analysis, and immunoblotting were performed in 4 selected cell lines. Four colorectal cancer patient derived xenograft (PDX) models were treated with AZD0156, irinotecan, or 5FU alone and in combination for assessment of tumor growth inhibition (TGI). Immunofluorescence was performed on tumor tissues. The DDR mutation profile was compared across Enhanced effects on cellular proliferation and regrowth were observed with the combination of AZD0156 and SN38 in select models. In cell cycle analysis of these models, increased G2/M arrest was observed with combination treatment over either single agent. Immunoblotting results suggest an increase in DDR associated with irinotecan therapy, with a reduced effect noted when combined with AZD0156, which is more pronounced in some models. Increased TGI was observed with the combination of AZD0156 and irinotecan as compared to single-agent therapy in some PDX models. The DDR mutation profile was variable across models.AZD0156 and irinotecan provide a rational and active combination in preclinical colorectal cancer models. Variability across in vivo and in vitro results may be related to the variable DDR mutation profiles of the models evaluated. Further understanding of the implications of individual DDR mutation profiles may help better identify patients more likely to benefit from treatment with the combination of AZD0156 and irinotecan in the clinical setting.The online version contains supplementary material available at 10.1186/s12885-022-10084-7. Drugs targeting DNA damage response (DDR) pathways are of great interest in oncology drug development. This interest stems from the ability of these drugs to target the DDR induced by cytotoxic chemotherapy and radiation therapy, which may serve as a mechanism of resistance to these treatments. The addition of a DDR inhibitor to a standard cytotoxic regimen thus provides a rational approach to improve standard therapies already known to benefit patients . Such agAtaxia-telangiectasia mutated (ATM) targeted agents are among the DDR inhibitors currently undergoing evaluation for cancer treatment. ATM is a serine threonine kinase that plays a key role in DNA double-strand break repair through both the homologous recombination repair and nonhomologous end joining pathways. ATM interacts with multiple other DDR factors, including the MRE11-NBS1-RAD50 (MRN) complex, as well as H2AX, MDM2, and CHK2 downstream .50 value of 0.00058 μM. The IC50 values of related targets further demonstrate this selectivity: ATR 6.2 μM (cell), mTOR 0.61 μM (cell), PIK3Ca 1.4 μM (cell), DNAPK 0.14 μM (enzyme). Though no single-agent effect of AZD0156 was observed in an SW620 colorectal cancer xenograft mouse model, tumor regression was observed when combined with irinotecan. Combination effects were also observed with the addition of AZD0156 to olaparib in HBCx-10 patient derived triple-negative breast cancer (TNBC) xenografts harboring BRCA2 and TP53 mutations  × 100). Specific growth rate was also assessed using GraphPad Prism 9.0. Mice were euthanized with cervical dislocation following anesthesia under isoflurane to ensure tumors were oxygenated. Animal studies were performed in a facility accredited by the American Association for Accreditation of Laboratory Animal Care in accordance with NIH guidelines and approved by the University of Colorado Institutional Animal Care and Use Committee prior to initiation (IACUC number 021).Patient derived xenograft models were generated as previously described . Brieflyior work . For ranSlides were fixed and blocked with 1% BSA and 1X PBS for 1 h in a humidified chamber at room temperature, then incubated overnight with γH2AX and P-RAD50 primary antibodies and washed with 1X PBS. Next slides were incubated with secondary antibodies AlexaFluor 488 and AlexaFluor 555 (Life Technologies) for 1 h in a humidified chamber at room temperature and washed in PBS. Counterstaining with DAPI in 1X PBS was performed for 30 min, followed by additional PBS wash. Slides were mounted with Fluoromount-G mounting media (SouthernBiotech), and imaged using Olympus FV-1000 confocal microscope at 20X magnification as described previously .For the 12 cell lines evaluated, prototypical DDR mutations were evaluated based on data provided in the Cancer Cell Line Encyclopedia . For evaP = 0.05 was used as the cut-off for statistical significance. Error bars represent standard error of the mean.GraphPad Prism software version 9.0 was used to perform statistical analyses. One-way ANOVA with Tukey’s multiple comparisons test was used for all analyses. 50 across 4 CRC cell lines evaluated was close to 5 uM, though at these high doses there is concern for off-target effects of closely related kinases [The ICP < 0.0001, RKO P = 0.0005, LOVO P < 0.0001, HT29 P = 0.0002). When compared to single-agent SN38, the decrease in proliferation of the combination was not statistically significant, though a trend was noted in the HCT8 and LOVO cell lines  were selected for further in vitro assessments. The combination of AZD0156 (100 nM) and SN38 (10 nM) resulted in decreased proliferation as compared to single-agent AZD0156 across all cell lines (HCT8 P = 0.0001), RKO (P < 0.0001) and HT29 (P < 0.0001) cell lines. There was a statistically significant decrease in percent confluence following treatment with this same combination when compared to SN38 in the HCT8 (P < 0.0001) and HT29 (P < 0.0001) cell lines. Similar patterns were seen at the 100 nM dose of AZD0156 in combination with SN38 when compared to single-agent AZD0156 , and single-agent SN38  , with findings more consistent with an additive effect of the AZD0156 and SN38 combination . This corresponded to increased G2/M arrest, with statistically significant differences observed in the RKO (P = 0.0005) and HT29 (P = 0.0192) cell lines. When AZD0156 was added to SN38, further decreases in G1 phase cells were observed in cell lines with AZD0156 at 50 nM  and 100 nM  when compared to AZD0156 at respective doses as a single agent. The proportion of cells in G2/M phase increased accordingly with the combination of SN38 and AZD0156 at 50 nM  and 100 nM   was observed, with evidence of tumor regression. Both single-agent irinotecan and the triplet of AZD0156, irinotecan, and 5FU were associated with significant tumor growth inhibition as well (P < 0.0001 for both). Interestingly, the addition of 5FU to the combination did not further enhance this effect, though tumor growth inhibition was not statistically significantly greater in the AZD0156/irinotecan arm as compared to the triplet arm. A trend toward tumor growth reduction was noted when comparing the combination of AZD0156 and irinotecan to single-agent irinotecan, though the difference was not statistically significant. Also of note in this model, a non-statistically significant increase in tumor volume was noted in the single-agent AZD0156 arm as compared to vehicle control (P = 0.1021). This pattern was not observed across other PDX models , doses of irinotecan and 5FU were decreased from 15 mg/kg to 7.5 mg/kg and 60 mg/kg to 30 mg/kg, respectively, on day 11 of treatment due to the sensitivity of the model to these single-agents. Following these dose adjustments, a clear effect of the combination of AZD0156 and irinotecan as compared to single-agent AZD0156  and AZD0156/irinotecan/5FU triple combination (P = 0.0090) were again associated with a statistically significant reduction in tumor volume when compared to single-agent AZD0156. A trend toward tumor growth reduction was noted in the AZD0156/irinotecan doublet (P = 0.0358) and AZD0156/irinotecan/5FU triplet as compared to single-agent irinotecan, but this was not statistically significant , the AZD0156/irinotecan doublet (P < 0.0001), as well as the triple combination arm (P = 0.0005) and single-agent irinotecan arm (P = 0.0007). When the doublet and triplet combination were compared to single-agent irinotecan, the differences were not significant. In the CRC102 model, no statistically significant differences in specific growth rate were noted.Specific growth rate (SGR) analysis was performed for both models  Fig. 5BB5B. In tIn two additional PDX models, CRC001 (KRAS G12D MT) and CRC042 (KRAS G13D MT), less pronounced effects on tumor growth inhibiton were observed with the addition of AZD0156 to chemotherapy  or irinotecan (P = 0.0005), consistent with increased accumulation of double-strand breaks in the setting of ATM inhibition  or irinotecan (P = 0.0265) as single-agents in the CRC102 model , is associated with greater effect in both CRC cell lines as well as in PDX models. Though a trend toward enhancement in the cytotoxicity of SN38 and irinotecan was observed with the addition of low doses of AZD0156 in these models, these trends were not statistically significant.SN38 has been previously shown to induce a G2/M cell cycle arrest , which wIn addition to indicating cycle cycle arrest, these observed changes in P-CHK2 by immunoblotting suggest suppression of ATM signaling and DNA damage response induced by AZD0156. This is further supported by a similar pattern of P-CHK2 expression in prior work with the combination of AZD0156 and olaparib [in vivo. Though in most of the PDX models evaluated, the triplet combination of AZD0156/irinotecan/5FU was associated with similar tumor growth inhibition as the doublet combinations, the finding of tumor regression in the AZD0156/irinotecan treated arm of the CRC026 model is notable. This is also the model in which the doses of both irinotecan and 5FU were adjusted at day 15 due to significant single-agent sensitivity, and it is possible that both the sensitivity of the model and the adjustment could have impacted these findings. Overall, the addition of 5FU to the AZD0156/irinotecan combination is that it did not induce a negative effect on the doublet therapy. However, the lack of added benefit of this additional cytotoxic agent may provide an opportunity to minimize the toxicity of a dual-cytotoxic regimen for the treatment of CRC through substitution of a targeted agent.Though 5FU as an antimetabolite chemotherapeutic agent does not induce DNA double-strand breaks, given the frequent clinical use of the combination as FOLFIRI for the treatment of CRC this compound was tested in combination with AZD0156 and irinotecan in vitro and in vivo results described here may be in part related to these variable molecular profiles. Prior work in other ATM inhibitors has evaluated this relationship, though without clear correlation. For example, in colorectal cancer cell lines, enhancement of the effect of radiation and topoisomerase I and II agents with ATM inhibitor KU59403 was not affected by p53 mutation status [Given the diverse DDR alterations noted across models evaluated in this work, it is possible that variability in both n status . Howevern status . Improven status .We have previously evaluated the combination of early-generation ATM inhibitor AZ31 with SN38 / irinotecan. In this work, a combination effect of AZ31 and SN38 is documented, and determined to be related to cell cycle arrest, rather than apoptosis . In addiHowever, there are some differences in our data as compared to prior work with AZ31 and other ATM inhibitors. For example, an improved combination effect of AZ31 and irinotecan was observed in the setting of irinotecan resistance , while iin vivo and in vitro results described above. Though we believe the varied DDR alterations present across the models evaluated in this work likely contribute to this variability, a clear relationship was not identified in this work, and will require further assessment in the future.The key limitation of this work are the variations in vitro and in vivo models indicate that the effect of the combination in colorectal cancer may be influenced by the underlying DDR phenotype of the individual tumor. Further understanding of this relationship in future studies will be important in identifying a subpopulation of colorectal cancer patients most likely to benefit from treatment with this combination.Our results suggest that the addition of AZD0156 to irinotecan enhances the effect of AZD0156, which is otherwise limited in CRC models. However, despite suggestive trends, a significant enhancement in the effect of irinotecan was not demonstrated. Variability in results across Additional file 1: Supplemental Table 1. Prototypical DNA Damage Repair Mutations in CRC Models Utilized in this Study. Supplemental Figure 1. Effect of AZD0156 and SN38 on proliferation in CRC cell lines. AZD0156 dose 50nM, SN38 dose 10nM. Percent confluence relative to vehicle at 120 hours as assessed by Incucyte ZOOM. Supplemental Figure 2. Effect of AZD0156 and SN38 on proliferation in additional CRC cell lines. Percent confluence relative to vehicle as measured by IncuCyte ZOOM™ over 120 hours in an additional 8 CRC cell lines treated with AZD0156 50 nM and 100 nM and SN38 10 nM, alone and in combination. Supplemental Figure 3. Statistical evaluation of synergy based on Bliss independence. Comparison of single agent and combination effect as assessed by Incucyte ZOOM™ to Bliss independence, derived from individual groups by computing survival fraction under the assumption that treatments act independently, as per Demidenko E, et al. [Supplemental Figure 4.In vivo effects of AZD0156 and chemotherapy in CRC PDX models. (A) Effect of AZD0156, irinotecan, and 5FU, alone and in combination in the CRC026 and CRC102 PDX models. Additional treatment groups added to data provided in Figure 5 of the main text. Doses of irinotecan and 5FU were reduced in single-agent and combination arms at day 11 in the CRC026 model due to its sensitivity to these single agents. (B) Specific Growth Rates of CRC026 and CRC102. Supplemental Figure 5. (A) Effect of AZD0156, irinotecan, and 5FU, alone and in combination in additional CRC001 and CRC042 PDX models. (B) Specific growth rates of CRC001 and CRC042.Additional file 2. Original, full-length images from immunoblotting, corresponding to cropped images shown in Fig. Additional file 3. ARRIVE guidelines checklist referencing details of animal experiments as outlined in the manuscript.