Currently, there are few data concerning β3-AR in myeloid malignancies. Here, we evaluated β3-AR in myeloid leukemia cell lines and the effect of β3-AR antagonist SR59230A. In addition, we investigated the potential role of β3-AR blockade in doxorubicin resistance. Using flow cytometry, we assessed cell death in different in vitro myeloid leukemia cell lines  treated with SR59230A in hypoxia and normoxia; furthermore, we analyzed β3-AR expression. We used healthy bone marrow cells (BMCs), peripheral blood mononuclear cells (PBMCs) and cord blood as control samples. Finally, we evaluated the effect of SR59230A plus doxorubicin on K562 and K562/DOX cell lines; K562/DOX cells are resistant to doxorubicin and show P-glycoprotein (P-gp) overexpression. We found that SR59230A increased cancer cell lines apoptosis especially in hypoxia, resulting in selective activity for cancer cells; moreover, β3-AR expression was higher in malignancies, particularly under hypoxic condition. Finally, we observed that SR59230A plus doxorubicin increased doxorubicin resistance reversion mainly in hypoxia, probably acting on P-gp. Together, these data point to β3-AR as a new target and β3-AR blockade as a potential approach in myeloid leukemias. Leukemia is the term used to identify a group of different cancers involving blood and bone marrow (BM). Myeloid malignancies are clonal disorders associated with uncontrolled proliferation and altered differentiation of hematopoietic stem cells (HSCs) with a consequent increase in immature myeloid cells ,2. In recent years, the development of new technologies and the discovery of novel molecular findings, have improved leukemia diagnosis. An important challenge in the treatment of myeloid malignancies is represented by the phenomenon of resistance to antineoplastic drugs. Understanding the cellular and molecular mechanisms associated with chemoresistance is crucial in order to improve cancer patients’ outcome and survival. There are two main types of chemoresistance: primary drug resistance and acquired drug resistance. In patients with primary resistance, tumor cells appear resistant before chemotherapy; while in patients with acquired resistance, tumor cells become resistant after antineoplastic treatment . In geneMDR1) gene. In particular, it is a 170-kDa efflux pump, which using ATP hydrolysis, plays an important role in the extrusion of different compounds out of cells, including drugs and xenobiotics, with a consequent decrease in intracellular substances accumulation. P-gp is expressed in healthy tissues but also in different types of cancer [MDR1 expression was an independent prognostic factor for induction therapy outcome and overall survival in AML patients [It is known that an abnormal expression of efflux proteins, and especially of P-glycoprotein (P-gp), is associated with chemoresistance in myeloid neoplasms ,5,6. P-gf cancer ,5, enhanf cancer . Moreovepatients . βeta-adrenergic receptors (β-ARs) are G-protein-coupled receptors involved in catecholamines-activated signal transduction pathways. Three types of β-ARs are known: beta1-adrenoreceptors (β1-ARs), beta2-adrenoreceptors (β2-ARs) and beta3-adrenoreceptors (β3-Ars). These receptors are localized and expressed in distinct and specific tissues. β1-ARs are expressed abundantly in cardiac tissue, kidney and adipose tissue; β2-ARs are localized in gastrointestinal tract, bronchi, skeletal muscle, liver, immune and non- immune cells; finally, β3-ARs are mainly present in intestine, adipose tissue and endothelium, moreover they are expressed in the smooth muscle cells of the detrusor muscle in the urinary bladder . Interesβ-ARs play a key role in different biological processes that are crucial in cancer biology and they promote tumor progression . In partFurthermore, β-ARs expression has been showed not only in cancer cells, but also in tumor microenvironment cells, including cancer associated fibroblasts, macrophages, and endothelial cells ,13. Finally, different studies suggest that β-AR blocker drugs are associated with reduction of cancer cell proliferation, progression and metastasis improving outcome and survival ,11. For In this study, we investigated the effect of β3-AR antagonist SR59230A, belonging to the class of aryloxypropanolaminotetralins, on different in vitro models of myeloid leukemias. Moreover, we analyzed the potential involvement of β3-AR in the phenomenon of chemoresistance, which generally represents a crucial challenge in cancer treatment. Indeed, chemoresistance influences patients’ clinical outcome promoting recurrence and metastasis and increasing mortality risk.Here, we demonstrate in in vitro models that β3-AR is highly expressed in myeloid malignancies and could be involved in cancer cell lines survival in particular under hypoxic conditions; in addition, we show that SR59230A treatment in combination with doxorubicin could reduce resistance to doxorubicin, especially in hypoxia.In order to investigate the effect of β3-ARs blockade on apoptosis in leukemic malignancies, four different cell lines of myeloid leukemia were treated with β3-ARs antagonist SR59230A . SR59230As reported in It is known that β3-AR is highly expressed in different tumor tissues, including hematologic malignancy . TherefoNotably, data revealed an increase in β3-AR expression in K562, HL60 and KCL22 cell lines after 48 h of hypoxic exposure A, while To exclude drug toxicity, we performed an analysis of SR59230A effect on bone marrow cells (BMCs) samples of healthy donors; in particular, BMCs were treated under normoxia and hypoxia conditions for 48 h. As shown in To clarify the ineffectiveness on healthy samples we analyzed the expression of β3-AR in healthy cells such as BMCs and peripheral blood mononuclear cells (PBMCs). As reported in 50) values which for K562 and K562/DOX cell lines were 15.3 and 13.1 µM, respectively. Therefore, the two lines had the same sensitivity towards the compound. These results led us to continue the studies with two concentrations, 3 µM and 5 µM, which had an intrinsic toxicity of around 20% .As shown in A preliminary study was conducted on the K562 cell line to investigate a putative role of the β3-AR in multidrug resistance (MDR). According to literature , the hypAccording to Calvani et al., UCP-2 is strongly expressed in various cancer types, among which leukemia and pancreatic cancer. Moreover, UCP-2 overexpression is regulated by β3-ARs in stem cells . FurtherTherefore, in this report, we treated K562 both sensitive and resistant to doxorubicin with SR59230A. Results showed that, in K562 resistant cell line, SR59230A inhibited the upregulation of UCP2 observed in hypoxia compared to normoxia conditions E. Moreover, we observed also that HIF-1a protein expression induced in hypoxia was reverted by treatment with SR59230A E, confirLiterature data showed that β-ARs blockade could be involved in prevention and treatment of different types of tumor . PreclinIn order to better understand the role of β3-AR in myeloid malignancies, we focused our attention on the effect of the β3-AR antagonist SR59230A on this type of tumor; in particular, we used K562, KCL22, HEL and HL60 myeloid leukemia cell lines and we demonstrated that β3-AR blockade results in reduced cancer cell lines survival by increasing apoptosis, especially under hypoxic conditions.The finding that SR59230A enhances cell lines death supports the idea that β3-AR could influence biological processes involved in the regulation of the balance between cell growth and apoptosis. To confirm that β3-AR could play a crucial role in disease condition, and especially in cancer, we analyzed BM and PBMCs samples from healthy donors as control samples. SR59230A was selective for myeloid leukemia malignancies and non-toxic for normal cells, demonstrating that β3-AR could be a possible marker for malignancies. Literature data have already shown the β3-AR upregulation under hypoxia in melanoma ; here weSince β3-AR was slightly induced under hypoxic conditions in normal cells and hypoxia is a feature of different pathologies, this receptor could be a putative selective marker for cancer therapy and other pathologies involving hypoxic environment.Furthermore, in this study we focused our attention on the potential effect of the β3-AR antagonist SR59230A on myeloid leukemia’s resistance to doxorubicin. Indeed, cancer MDR represents a significant clinical problem for cancer treatment, including hematological malignancies. Doxorubicin is an antineoplastic compound frequently used in different tumor types ; unfortuMDR1 gene expression in various tumors, including colon and liver cancer [MDR1 expression in tumor cells [Several studies evidenced the involvement of HIF1α in the regulation of r cancer . In partor cells ; for insor cells , while Dor cells . Moreoveor cells . Concernor cells .In this work we showed that the combination of β3-AR antagonist SR59230A with doxorubicin reduced resistance to doxorubicin in K562/DOX cell line, which represents a model of a doxorubicin-resistant cell line with higher expression of P-gp in comparison with the K562 cell line. MDR1 expression in the K562 cell line maintained in hypoxia, consistent with the finding that hypoxia is involved in MDR [We showed an increase in d in MDR ; we thenMDR1 gene is hypoxia responsive: indeed, hypoxia promotes transcriptional induction of MDR1 and consequently P-gp expression [According to Comerford et al., pression . Finally2 humidified atmosphere incubator with 21% O2 for normoxia, and 1% O2 for hypoxia condition. Four different human myeloid leukemia cell lines were cultured in our study: K562 and KCL22 (chronic myelogenous leukemia cell lines), HEL (erythroleukemia cell line), HL60 (Acute Promyelocytic Leukemia cell line). Cell lines were obtained from ATCC. Cell lines were cultured in RPMI medium (Gibco) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine (200 mmol/L), 100 U/mL penicillin–streptomycin. All cell lines were maintained at 37 °C in a 5% CO2. To maintain the resistance, every month, the resistant cell line was cultured for three days with 400 nM doxorubicin. K562/DOX cell line overexpresses almost exclusively the membrane glycoprotein P-gp. The P-gp expressing K562/DOX cell line was obtained from Prof. J. P. Marie . This cell line was cultured in RPMI 1640 medium (GIBCO) supplemented with 10% fetal calf serum (FCS) (GIBCO) at 37 °C in a humidified incubator with 5% COAll cell lines were routinely tested for mycoplasma contamination.10 M to 104 M for 72 h in a humidified atmosphere with 5% CO2. The MTT working solution was then added and plates were further incubated for 3 h. Following incubation cells, formazan crystals were inspected microscopically. The supernatant was then carefully removed by slow aspiration and the formazan crystals were dissolved in 150 μL of Dimethyl Sulfoxide (DMSO). The absorbance of the solution was then read on an automated plate reader at a wavelength of 570 nm. The percentage of growth compared to the untreated control was transformed into dose–response curves with the GraphPad Prism 5 program and calculated the IC50 values. Toxicity test was repeated three times.The intrinsic toxicity of the SR59230A compound was determined through MTT assay after the exposure of parental cell line, K562, and resistant cell line, K562/DOX, to the compound in a concentration range of 104 cells/well) were seeded, in exponential growth phase, and solutions of doxorubicin, or a solution of doxorubicin in combination with the compound, were added to the wells repeated in quadruplicate. Then the plates were incubated at 37 °C for 72 h in a humidified atmosphere with 5% CO2. The MTT test was applied as described for intrinsic toxicity and the ability of the SR59230A compound to increase cytotoxicity of doxorubicin was expressed by the RF values obtained as the ratio between the doxorubicin IC50 values on K562/DOX cell line in the absence and in the presence of the compound. The same procedure was adopted for the evaluation after 48 h of the reversal activity of resistance in hypoxic environment with 1% O2. The cell line was adapted to O2 decrease for 24 h. At 48 h, a sample of control cells maintained at 21% O2 or 1% O2 was collected for the molecular analysis by PCR. Toxicity test was repeated three times.The doxorubicin toxicity was evaluated in absence and in the presence of the SR59230A compound tested at 3 µM and 5 µM concentrations. Cells  using a RotorGene 3000  instrument. Primers were purchased from IDT (Germany). An amount of 500 ng of total RNA was retro-transcribed using iScript  and amplified with specific primers: MDR1, Fw CAGCTATTCGAAGAGTGGGCACAAAC and Rv GCCTCTGCATCAGCTGGACTGTTG. PCR amplification was carried out by SsoAdvancedTM Universal SYBR® Green Supermix  according to manual instruction. In the present analysis 18s rRNA was confirmed to be stable and was used as the normalizer Fw CGGCTACCACATCCAAGGAA and Rv GTTATTTTTCGTCACTACCTCCCCGGG. The RT-qPCR was performed using the following procedure: 98 °C for 2 min, 40 cycles of 98 °C for 5 s, 60 °C for 10 s. The program was set to reveal the melting curve of each amplicon from 60 to 95 °C with a read every 0.5 °C. For the ADRB3 gene the following primers were used: RefSeq Accession No. NC_000008.10, NG_011936.1, NT_167187.1; unique assay ID qHsaCED0047996 (Biorad).The expression levels of The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Pediatric Ethics Committee of Regione Toscana (Project identification code “Beta 3 2019 #235/2019”). 2) at 21% O2 or 1% O2. Peripheral Blood (PB) samples of BM blood were collected according to clinical management. BM aspirates were collected from health donors that provided a written informed consent. BMCs were isolated and expanded using α MEM supplemented with 10% heat inactivated FBS, penicillin-streptomycin, 4-(2-hydro-xyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium pyruvate , and 5 ng/mL basic fibroblast growth factor . BMCs were incubated under standard conditions  in a hypoxic or normoxic incubator. In order to evaluate SR59230A toxicity, all cell lines were treated with increasing concentration of SR59230A  for 24 h and 48 h.SR59230A  effects on apoptosis were evaluated on BMCs cultured in 24-well plates  in hypoxic (1%) or normoxic (21%) conditions. For the evaluation of β1-, β2-, and β3-adrenoreceptor expression level, K562, HEL and HL60 cell lines were cultured under normoxic and hypoxic conditions for 24 h, and then stained with the anti-β1 (ORB 129489 (PE)), anti-β2 (ORB 15065 (FITC))  and anti-β3-AR antibodies. The anti-β3-AR antibody (ab140713) used for cytofluorimetric analysis was obtained from ABCAM and conjugated with the R-Phycoerythrin Conjugation Kit . Expression levels were obtained by using a MACSQuant Analyzer 10 flow cytometry. 3 times.Apoptosis was analyzed using FITC Annexin V Apoptosis Detection Kit with Propidium Iodide according to the manufacture’s protocol. The cell lines were washed twice with cold BioLegend’s Cell Staining Buffer and were resuspend in Annexin V Binding Buffer. FITC Annexin V and Propidium Iodide solution were added to cell suspension and further incubated for 15 min at room temperature in the dark; afterwards Annexin V Binding Buffer was added, the cell lines were analyzed by flow cytometry and data were analyzed with Flowlogic Software. Flow cytometry experiments were repeated Cord blood cells were obtained from health donors. For the colony formation assay, 100,000/mL of mononucleated cells was plated in p35 mm dishes in Methocult  and treated with SR59230A . After 14 days, the number of BFU-E and CFU-GM were counted. After cells lysis and quantification, 20 μg of total proteins was loaded on SDS-PAGE followed by WB analysis. PVDF membranes were treated with blocking solution for 1 h at room temperature and then they were incubated overnight at 4 °C with gentle shaking with the following primary antibodies: anti-human HIF-1a #610959 (BD Transduction Laboratories), anti-MDR-1 (E1Y7S) #13978 , anti-UCP2 (G-6) sc-390189 and anti-β-Actin (C4) sc-47778 (Santa Cruz Biotechnology). The next day membranes were incubated with specific secondary antibodies for 1 h at room temperature. Chemiluminescent protein revelation was performed using Clarity Western ECL Substrate (Biorad) and the images were acquired through the Chemidoc Imaging System (Biorad®). The Western blot experiments were repeated three times and to verify the application of equal amounts of protein, the intensity of the corresponding protein bands of interest was normalized based on that of the β-actin band for each sample.P < 0.05 were considered significant.Statistical analysis was performed using the GraphPad Prism 6.0 . Values are presented as mean ± SD. Differences with To assess normal distribution and homoscedasticity for each quantitative outcome in each group Kolmogorov–Smirnov’s test and Bartlett’s Test was used, respectively. In order to evaluate difference in quantitative outcomes between groups, according to normality and homoscedasticity tests results, ANOVA and posthoc t-test with Bonferroni correction for multiple comparison were used. Posthoc test was performed only if ANOVA analyses were statistically significant.In summary, this work offers a new interesting perspective on myeloid leukemias treatment; in particular our data highlight β3-AR as an attractive target to reduce cancer cell survival in myeloid malignancies . Moreove


FAM50A/FAM50B, two genes of unknown function. Silencing of FAM50B occurs across a range of tumour types and in this context disruption of FAM50A reduces cellular fitness whilst promoting micronucleus formation and extensive perturbation of transcriptional programmes. Our studies reveal the fitness effects of FAM50A/FAM50B in cancer cells.Genetic redundancy has evolved as a way for human cells to survive the loss of genes that are single copy and essential in other organisms, but also allows tumours to survive despite having highly rearranged genomes. In this study we CRISPR screen 1191 gene pairs, including paralogues and known and predicted synthetic lethal interactions to identify 105 gene combinations whose co-disruption results in a loss of cellular fitness. 27 pairs influence fitness across multiple cell lines including the paralogues  Fam50a/Fam50b synthetic lethality was in isogenic cell models as well as in an in vivo setting.Systematic screens for synthetic lethal gene pairs represent a powerful approach to define interactions that may be exploited in the clinic. Here, the authors use a dual-guide CRISPR screening approach to screen a curated selection of gene pairs across three cell lines and validate the  Drosophila genetics communities who realised that the disruption of multiple genes simultaneously could elicit cell death in situations where disruption of such genes singly did not1. In more recent times, synthetic lethality has been exploited as an approach to treat cancers, the most notable example being the development of PARP inhibitors to treat patients with BRCA1/2 mutant tumours4. Although substantial efforts have been made to identify synthetic lethal (SL) interactions in human cancer cells, we are still a significant way from having a genome-wide map of cancer dependencies, with many established SL interactions appearing to be highly context dependent5. Thus, systematic screens for SL gene pairs represent a powerful approach to define interactions that may be exploited in the clinic and to understand the context dependencies in which they operate.A major precept of cancer genetics is that normal cells acquire somatic mutations that provide a fitness advantage and drive tumour evolution and growth. Since these alterations are generally not found in normal cells, they may be exploited therapeutically, either by direct pathway inhibition, for example, in the context of activated oncogenes, or via synthetic lethality. The concept of synthetic lethality was pioneered by the yeast and 9. As above, several theories have been proposed for the creation of paralogues, one being that paralogues have developed to create functional redundancy, presumably as a result of selective pressure. Examples of essential paralogues include RPL22L1 and RPL22, which are ribosomal proteins, and YAP1/WWTR1(TAZ) in the Hippo pathway9. Importantly, although several paralogue dependencies have been established, we are currently unable to accurately predict which paralogue pairs may be essential or functionally related by their sequence alone. Intriguingly, many essential paralogue pairs are part of the same protein complex8. A good example of this are components of the BAF/PBAF complexes, such as ARID1A/ARID1B and SMARCA2/SMARCA4, which are required for a range of processes such as transcription and chromatin regulation11. Disruption of these gene pairs results in growth arrest and cell death; phenotypes which appear to be influenced by cell lineage and differentiation status12.The mammalian genome has evolved to carry parallel pathways or in some cases multiple redundant genes on which cells rely for survival—the principal reason for these gene sets seems to be to buffer and protect cells from the adverse consequences of gene loss, allowing cells to operate with higher fidelity and/or plasticity, even under stressful conditions. One set of these genes are the paralogues; genes derived from a common ancestral gene that now reside in different regions of the genome13. Generally, these screens have been performed using either compounds/targeted-agents, shRNA/siRNAs, or more recently with CRISPR17. Many of these screens have been performed in the context of specific genetic changes, such as in panels of cells with defined genetic alterations, or in isogenic cell lines, so that genetic interactions can be readily identified18. More recently, it has become possible to use paired gRNAs, also known as combinatorial or multiplex CRISPR screening, to identify essential gene pairs24. This approach to exploring genetic epistasis facilitates the identification of gene combinations that are SL by screening at scale. Here, we deployed CRISPR screening to interrogate 1191 gene pairs including 645 paralogues, 447 mutually exclusive genetic interactions defined using mutual exclusivity modelling of cancer data, and a set of 95 literature curated SL pairs. Our screening of two melanoma cell lines (A375 and MeWo) and a retinal epithelial line (RPE-1) identified 27 SL pairs occurring in ≥2 cell lines. This included the poorly characterised Family with sequence similarity 50 Member A & B (FAM50A/FAM50B) gene pair, whose disruption precipitates a loss of cellular fitness associated with apoptosis and widespread dysregulation of transcription. FAM50A/FAM50B are particularly notable among our collection of genetic interactions because ~4% of cancers profiled by the TCGA show loss of FAM50B expression (0–10% across tumour categories), thus highlighting the FAM50A/FAM50B axis as a potential therapeutic target.As noted above, cancer cell line screens are powerful tools for the identification of SL interactions because they can be used to systematically and comprehensively screen the genome without making any prior assumptions about which genes interact26, where pairs of genes had been identified as ‘co-lost’ less frequently than expected by chance. We intersected the gene sets from these studies, resulting in 447 overlapping candidate pairs for which gRNAs could be designed. The second gene set consisted of the 95 highest scoring gene pair interactions for which gRNAs could be designed as defined by a curated database of SL interactions (SynLethDB)27. Notably, this set of genes includes pairs derived from a vast array of biological contexts and tumour types, allowing us to assess if these interactions were essential in our system and in the cell lines we screened. Our library also included gRNAs for four gene pairs to test interactions between PARG1/XRCC1, KDM6A/UTY, KDM6A/KDM6B and KDM6B/UTY, implicated from genome sequencing studies28. The final gene set consisted of paralogous pairs. To define these genes, we built a computational pipeline to identify paralogue pairs  with >20% DNA sequence homology/similarity and filtered this collection to identify genes where there was a single common orthologue in either Caenorhabditis elegans  or Drosophila melanogaster  and where disruption of this gene resulted in death of the organism. In this way, we identified 701 gene pairs, 645 of which were amenable to targeting by CRISPR (see Methods). In order to assess the performance of our library we also included a panel of established essential and non-essential genes30. A complete list of all gene pairs is provided in Supplementary Data Gene pair sets were chosen to be included in our library based on three distinct biological rationales Fig. . The fir31. We designed 3–5 guides for each gene and placed guides together as pairs and also paired each of them with a non-targeting/control guide, to allow assessment of both guide–guide and single-guide activity  and was assembled using Gibson cloning33 and BAGEL29 to compare the profile of gene essentiality by comparison of the screen results to established essential and non-essential genes, which were included in the library as controls  at day 14 (to select genes whose loss resulted in a cytotoxic rather than cytostatic effect), or an independent screen performed on each respective cell line using a whole-genome single gRNA CRISPR library34 , and all of the nine interactions common to all three cell lines were paralogues ; Fig. C. elegans/D. melanogaster) 29 and calculated the residual, representing a baseline neutral genetic interaction in each cell line , selected based on their recurrence or biological interest, and proceeded to validate these pairs using competitive cell growth assays. To do this, we placed a gRNA targeting one of the genes in the pair in a mCherry-expressing vector, and another, targeting the other gene, in a BFP-expressing vector (see Methods). We transduced cells with both vectors to create four populations (see below), and measured population dynamics at timepoints between day 4 and 14 Fig. . These p8, revealing 167/234 overlapping gene pairs were in agreement with 22 of these pairs ‘hits’ in both studies. These pairs included EAF1/EAF2, DDX39A/DDX39B and CHMP1A/CHMP1B. In the same way, we compared our data set to a study by Gonatopoulos-Pournatzis et al.22 Noting that RPE-1 was the only cell line screened in both studies, 94/170 overlapping genes pairs were concordant as either hits or non-hits. Of these, 17 were defined as SL interactions in both studies. These interactions included CNOT7/CNOT8, TTC7A/TTC7B and SAR1A/SAR1B, all of which are paralogous gene pairs. The gene pairs ARID1A/ARID1B, SEC23A/SEC23B, SLC25A28/SLC25A37, SMARCA2/SMARCA4, TTC7A/TTC7B and UAP1/UAP1L1 were hits in all three data sets. Collectively, this analysis orthogonally validates nearly 40 SL interactions and also the quality of our screen.Across the cell lines screened in this study we identified a rich collection of SL interactions, several of which were validated as described above. To extend this analysis we compared our data to other previously generated SL data sets   rescued the lethal phenotype associated with FAM50A disruption  Fig. . We nextion Fig. . To furtion Fig.  in RKO cFAM50A alone  associated with germline hypomorphic alleles of FAM50A where patients developed to adulthood with phenotypes including developmental delay39. This observation suggests a therapeutic window for FAM50A inhibition. Notably, although we observed the interaction of FAM50A/FAM50B in all cell lines screened, many genetic interactions are highly context dependent and influenced by both the genetics of the cell line and the growth environment. Thus, further studies will be required to establish all contexts in which the FAM50A/FAM50B interaction is operative.Of note, in our CRISPR screens we observed that inactivation of FAM50A in tumours that have lost FAM50B expression we used the TOV21G cell line carrying the Dox-inducible FAM50A gRNA construct mentioned above (see Methods). As shown in Fig. FAM50A in TOV21G cells precipitated a profound reduction in cellular fitness. Mice xenografted with the Dox-inducible line and fed a Dox-containing diet  exhibited a significant decrease in tumour growth compared with controls on a Dox-free diet  showed CRISPR editing at the gRNA cut site. These edits included in-frame events, which presumably were not disruptive of FAM50A but altered the cut site such that it was no longer a substrate for the gRNA, non-disruptive missense mutations and null alleles (nonsense and frameshift mutations). Wildtype traces, representing unedited alleles, were <1% of sequence reads  or a CRISPR engineered FAM50B null A375 clone (A375-F50B−), we introduced a lentiviral vector carrying a gRNA to disrupt either FAM50A or a non-essential gene (AIPL1) and cultured the cells for 8 days prior to transcriptome sequencing. In both cell lines, gene set enrichment analysis revealed that disruption of FAM50A on a FAM50B null background resulted in statistically significant (padj < 0.05) transcriptional changes that included genes of the TP53 and TNFα/NFκβ pathways, and apoptosis regulators  and FAM50B (F50B−) knockout A375 cells relative to parental A375 cells revealing some pathway overlaps  , where loss of FAM50A/FAM50B also caused an induction of apoptotic cell death  and PARP inhibitors , each of the 27 gene pairs we identified could be considered as targets for therapy development if follow-up studies can identify selectivity for cancer cells. Another way of using essential gene pair data is to identify situations where one member of the pair is lost somatically, thus exposing the other member of the pair as a candidate drug target and the data we have generated here could be mined for this purpose.FAM50B was silenced in a significant proportion of tumours across a range of histologies; 0–10% in each tumour type examined. Further, we showed that co-disruption of FAM50A and FAM50B both in vitro and in vivo precipitates a profound reduction in tumour cell fitness, a phenotype that could be partially rescued by reintroduction of FAM50B. Importantly, we observed dysregulation of a range of cell regulatory transcriptional programmes and the formation of extensive micronuclei together with apoptotic cell death, suggesting a specific role for FAM50A/FAM50B in cellular survival via maintenance of genome stability. In agreement with our study, Dede et al.45 recently identified FAM50A/FAM50B as a candidate SL gene pair in cancer cell lines. Our screen is one of the first to profile gene paralogues as potential drug targets at scale and thus represents a blueprint for endeavours to prioritise all genes for screening.Of note, using TCGA expression data and referencing our collection of 27 recurrent SL gene pairs, we determined that 31. A detailed protocol is available at Protocols.io (dx.doi.org/10.17504/protocols.io.bpqhmmt6). The gRNAs used are provided in Supplementary Data 46. Subsequently, using the ENSEMBL Perl API v84, we identified those gRNAs whose cutting sites were located within a protein domain as reported by Pfam and then discarded sequences containing BbsI restriction sites. From the resulting set of gRNAs we selected 3–5 per gene to be included in the library. Each of these gRNAs were paired with each of the 3–5 gRNAs designed for the other gene in the pair. gRNAs were also individually paired with a non-targeting Fluc_gRNA control (GTGTTGGGCGCGTTATTTATCGG) from the Firefly  luciferase gene to allow assessment of single gene lethality. Single-targeting guides were always under the sU6 promoter (with the non-targeting gRNA under the hU6 promoter). Combinatorial guides were in a single orientation (eg hU6 guide_A + sU6 guide_B). The final library contained 41,838 different combinations of gRNAs including gRNAs targeting a total of 1191 gene pairs, 12,803 gRNAs combined with the Fluc_gRNA control and gRNAs against essential/non-essential genes to aid in statistical analysis. To make virus, 20 × 106 293 T cells were transfected with 11.25 μg pMD2.G (Addgene #12259), 17 μg psPAX2 (Addgene #12260) and 22.5 μg of dual gRNA library plasmid. Media was changed 12 h post transduction and virus was harvested 36 h later and stored at −80 °C.The CRISPR gRNA library was constructed using the method previously described by Vidigal and Ventura2 in media as specified by ATCC. Cell line identity was confirmed by STR profiling and all cells were screened and found negative for mycoplasma. Cas9-expressing cell line generation was performed using a lentivirus produced with the pKLV2EF1a-Cas9Bsd-W plasmid . The activity was confirmed with a BFP/GFP reporter assay32. All lines had ≥90% Cas9 activity prior to screening . For each replicate, 48× PCRs containing 3 µg genomic DNA were performed using KAPA HiFi Polymerase (ThermoFisher), using the primers listed in Supplementary Data 35 to define synergy between gRNA pairs using the equation:2FC of the pair observed in the screen and log2FC expected by the model. We performed both the control and experimental replicates in triplicate, therefore comparing each experimental replicate to each control resulted in nine comparisons per construct. For each of these nine comparisons, we plotted observed vs predicted effects, and using Loess regression, the behaviour of the population was modelled. Population modelling in this way accounts for the effect of CRISPR-mediated cutting of the genome and the potential effect of single vs double cutting. For each paired gRNA construct, a residual  was interpolated. The variance of residuals was found to be heteroscedastic, with greater variance observed when the expected effect of the guide pair was more lethal. To correct for this heteroscedasticity, variance smoothing was performed19. This was done by ranking each g1g2 construct by the expected fold change; the residuals were then put into bins of 200 (arbitrary number) and the variance of each bin calculated. The value of each residual was then divided by the variance of the bin that it belonged to so as to create a variance adjusted residual, resulting in equal variance across the model. For each gene pair, we then generated up to 225 (25 × 9) independent residuals from each of the gRNA paired constructs.We used the Bliss independence modelt test and the robust ranking algorithm (RRA). Gene pairs typically detected in only the RRA analysis often had a single guide producing a large residual, with the remaining constructs having minimal biological effect and a mean effect close to zero. Given that in these cases, the significance of the pair was often driven by a single guide, possibly driven by off-target effects, we did not wish to consider these pairs as hits. Gene pairs typically detected in only the t test had a minor global negative shift, with few guide pairs ranking in the bottom 10% of residuals. Given that we were interested in pairs which had a large biological effect (favouring the RRA), but did not want to select genes where a single guide was causing outliers (favouring the t test) we chose to take the overlap between the two analyses to ensure size and consistency of effect.To derive ‘hits’ from the screen we required significance in two independent statistical tests; a t test the equation used was:33 using the bottom 10% of residual values. For each test (t test or RRA) we used a Bonferroni test to correct for multiple testing and counted as significant pairs with an FDR < 0.1.For the 30 analysis of the single gene targeting constructs , when put under the hU6 promoter in a gene pair, were more likely to produce a significant residual . This is likely due to enhanced gRNA activity in this context. In view of this, we did not consider any pairs containing genes lethal in isolation as screen hits. Genes were considered lethal if BAGEL or MAGeCK detected them as lethal in our screen at day 14, or in an independent screen34 (using an FDR < 0.1 for MAGeCK and a PPV < 5% for BAGEL).We noted that lethal gRNAs defined by either MAGeCK or BAGEL19. In total, eight pairs underwent low-throughput validation, with three additional non-interacting pairs used as controls . Guides showing activity across all cell lines were chosen for the validation  and overexpression of FAM50B confirmed by western blotting . To create inducible knockouts, a gRNA against FAM50A (Supplementary Data Two cell lines (RKO-Cas9 and TOV21G-Cas9) lacking Cells were seeded into six-well plates at 1000 cells/well. 24 h post seeding, media was changed to either contain doxycycline (0.1 µg/ml) or an equivalent volume of dimethyl sulfoxide (DMSO). The assay was terminated as cells in the control (DMSO) wells approached confluence. Cells were fixed with 100% ice-cold methanol and stained with 1% crystal violet solution.The CaspGlow assay (ThermoFisher) was used to assess apoptosis at day 7/8 after viral transduction as per the manufacturer’s instructions.FAM50A and FAM50B, A375 FAM50B cells (A375-F50B−) and TOV21G cells were seeded at 500,000 cells per well in six-well plates with 8 µg/ml polybrene, and transduced with either the BFP-F50A_gRNA lentivirus or the BFP-AIPL1_gRNA lentivirus to infect >90% of cells. Media was changed at 24 h and cells were maintained in culture until harvest (day 7–8). The experiment was performed as three independent transductions per condition. RNA extraction was performed with the Direct-zol RNA microprep  according to manufacturers’ instructions. Library preparation was performed using the KAPA stranded RNAseq kit using RiboErase. Samples were multiplexed and sequenced on a HiSeq 2500 with 75 bp paired-end reads. Sequences were pseudo-aligned to the Homo Sapiens transcriptome and quantified using the Kallisto quantifier47 (v 0.44.0). We ran DESeq248 on the gene-level counts, first removing all genes with low-level counts. Gene set enrichment analysis was performed with fgsea (https://bioconductor.org/packages/release/bioc/html/fgsea.html).To assess the transcriptional effect of co-disruption of 6 TOV21G- iF50A clone 9 cells  in the right flank. The mice were fed Mouse Breeders Diet  and one week after dosing were randomly assigned into two cohorts with one being fed a Doxycycline diet  and the other remaining on the Mouse Breeders Diet for the remainder of the study. The developing tumours were measured every second day until they reached 1.2 cm2 , at which point animals were humanely sacrificed and the mass excised and stored at −80 °C. The care of mice in this study and all experimental procedures was in accordance with Home Office guidelines (P6B8058BO). Procedures were further approved by the Animal Welfare Ethical Review Body (AWERB) of the Welcome Trust Sanger Institute. Housing and husbandry conditions were exactly as detailed previously49. The experiment was independently replicated twice. The first cohort were female mice and the second cohort were male.For examination of primary tumour growth, NOD-SCID mice at 6–8 weeks of age were subcutaneously administered 2.5 × 10To count micronuclei, cells were grown on coverslips, then at the time of the assay, coverslips were washed once with PBS then fixed with three parts ethanol/1 part acetic acid for one minute. Coverslips were then washed twice with PBS before mounting onto microscope slides with Vectashield DAPI mounting medium (Vector Labs). Micronuclei were counted on a Leica microscope, selecting random fields and counting all nuclei and micronuclei in each field until >500 nuclei had been counted.All mouse experiments were performed under Home Office Project Licence P6B8058BO with ethics board approval.Further information on research design is available in the Supplementary FiguresPeer Review FileDescription of Additional Supplementary FilesSupplementary Data_1Supplementary Data_2Supplementary Data_3Supplementary Data_4Supplementary Data_5Supplementary Data_6Supplementary Data_7Supplementary Data_8Reporting Summary


To identify the aberrantly expressed long non‐coding RNAs (lncRNAs) in ovarian high‐grade serous carcinoma (HGSC).Total RNA was isolated in HGSC cell lines, ovarian surface epithelial cells, and normal ovaries. Aberrantly expressed lncRNAs in HGSC were identified by PCR array, which analyzes 84 kinds of lncRNAs. To infer their functions, HGSC cell lines with different levels of expression of the identified lncRNAs were established, and then, activities of proliferation, migration, and apoptosis were examined. Expression levels of the identified lncRNAs were also examined in multiple ovarian HGSC tissues.Ten aberrantly expressed lncRNAs, six upregulated and four downregulated, were identified in the HGSC cell lines. The authors established four HGSC cell lines: in two of the cell lines, one of the upregulated lncRNAs was knocked down, and in two other cell lines, one of the downregulated lncRNAs (MEG3 and POU5F1P5) was overexpressed. Migration activities were inhibited in the HGSC cell lines overexpressing MEG3 or POU5F1P5 while there were no differences in proliferation and apoptosis between the established and control cell lines. The four lncRNAs downregulated in the HGSC cell lines were also observed to be downregulated in ovarian HGSC tissues.The authors identified four downregulated lncRNAs in ovarian HGSC. Based on histopathology and molecular genetics, epithelial ovarian cancers (EOCs) are mainly classified into endometrioid, clear cell, mucinous, low‐grade serous, and high‐grade serous carcinomas (HGSC).In recent years, with the advances in large‐scale sequencing technology, it has been widely accepted that more than 98% of the human genome is transcribed into non‐coding RNAs that do not encode obvious proteins.In this study, we focused on HGSCs, which are the most frequently occurring and aggressive subtypes among ovarian cancers, and aimed to identify lncRNAs involved in the progression and malignant behaviors of HGSC. We compared lncRNA expression profiles between HGSC cell lines and normal ovarian cells and ovarian tissue samples, and identified the aberrantly expressed lncRNAs specific to the HGSC cell lines. In addition, some of the lncRNAs downregulated in the HGSC cell lines were observed to be downregulated in ovarian HGSC tissues. Our results also suggest that some of the identified lncRNAs have roles in malignant behaviors.22.1Human ovarian cancer cell lines, KURAMOCHI and TYK‐nu, were used as HGSC cell models. Both cell lines were purchased from Japanese Collection of Research Bioresources Cell Bank. KURAMOCHI and TYK‐nu cells were cultured in RPMI‐1600 (Wako) and eagle’s minimal essential medium (Sigma‐Aldrich) supplemented with 10% fetal bovine serum, respectively. Human ovarian surface epithelial cells (HOSE) were purchased from Cosmo‐bio and cultured in ovarian surface epithelial cell medium (ScienCell Research Laboratories).Twenty‐two cases of HGSC specimens were obtained from the patients who underwent operation at Shimane University hospital. Ten cases of normal ovarian tissues were obtained from the patients who underwent oophorectomy by a reason different from HGSC at Yamaguchi University hospital. Dissected specimens were immediately immersed in liquid nitrogen and stored at −80°C until RNA isolation.2.2Total RNA was isolated from the cell lines using an RNeasy mini kit (Qiagen) according to the manufacturers’ instructions. Total RNA was also isolated from the tissue specimens by ISOGEN reagent (Nippon Gene), followed by chloroform extraction and 2‐propanol precipitation.GAPDH as a reference gene.First‐strand cDNA was synthesized from 1 μg of total RNA by random hexamers using a QuantiTect Reverse Transcription Kit (Qiagen) as previously reported.2.32 First Strand Kit (Qiagen) according to the manufacturers’ instructions.2 lncRNA PCR Array Human Cancer Pathway Finder (Qiagen)). Real‐time PCR was carried out with the RT2 SYBR Green Master Mix (Qiagen) under the cycling conditions . The relative expression levels were calculated with the delta‐delta Ct method using ACTB as a reference gene. Significant changes in the lncRNA expression were defined as at least twofold upregulation or downregulation of ones in both two HGSC cell lines compared to those in HOSE and normal ovaries.cDNA synthesis was performed using the RT2.44 cells in 6‐well plates and, at 50% confluence, were transfected with the 20 nmol/L siRNAs by RNAi MAX (Invitrogen).For knockdown of LINC00152 and LINC01234, siRNAs for each lncRNA (Lincode Human lncRNAs siRNA SMART pool) and non‐targeting control (Lincode Non‐Targeting Pool siRNA) were purchased from Dharmacon. TYK‐nu cells were plated at approximately 5 × 104 cells in 6‐well plates. The stable cell lines were established by sorting with 2 μg/mL blasticidin S for a month.For overexpression of MEG3 and POU5F1P5, firstly the expression vectors of each lncRNA were constructed. The full length of MEG3 and POU5F1P5 cDNA was amplified by RT‐PCR using a HOSE cDNA as a template, primers shown in Table 2.54 cells in 6‐well plates, respectively.TYK‐nu lines, in which the lncRNA expression was altered, and the control lines were plated at approximately 6 × 102.66 cells/mL in 0.25 mg/mL RNase in PBS. Resultant single cell suspension was stained with 7‐aminoactinomycin D (7‐AAD) (Bio‐Rad Laboratories) and assayed using NovoCyte Flow Cytometer (ACEA Biosciences). The percentage of cells in each cell cycle stage  was evaluated with a NovoExpress software (ACEA Biosciences).For cell cycle assay, TYK‐nu lines overexpressing MEG3 and POU5F1P5 and the control lines were trypsinized, fixed for 30 minutes in cold 70% ethanol, and then adjusted to a concentration of 1 × 10For apoptosis assay, an Annexin V‐FITC Apoptosis Detection Kit (Affymetrix) was used according to the manufacturer's protocol. In brief, the cells of TYK‐nu lines overexpressing MEG3 or POU5F1P5 and the control lines were trypsinized into single cells and were stained with annexin V‐FITC and 7‐AAD. Ten thousand cells were counted, and annexin V‐positive cells were measured by NovoCyte Flow Cytometer. Cells that were annexin V‐positive and 7‐AAD‐negative, and double‐positive, were considered to be early and late apoptotic cells, respectively.2.75 cells in 6‐well plates and cultured until they reached confluence. Linear scratch wounds were created on the cell layer in the center of each well with a 1000‐µL sterile pipette tip. After 72 hours, images were taken to evaluate the wounds at the same fields under the microscope, and the separation distances between wound sides were quantified as previously reported.Cells of TYK‐nu lines overexpressing MEG3 or POU5F1P5 and the control lines were plated at approximately 2 × 102.84 cells with serum‐free medium in insert chambers on the well (12‐well Transwell system) (Corning Life Science). As a chemo‐attractant, the medium containing 10% FBS was filled under the insert chamber. After 24‐hours culture, the cells on the upper surface of the insert chambers were completely removed by wiping with cotton swabs. The membranes of the insert chambers were fixed, and the migrated cells were stained with Diff‐Quick (Sysmex). The stained cells were counted at 200× magnification in five randomized field views, and the mean was calculated. Three independent experiments in triplicate were performed.Cell migration assay was performed with a BioCoat Matrigel Invasion Chamber (Corning Life Science) according to the manufacturer's protocol as previously reported.2.9t test and Wilcoxon test. A probability value of P < .05 was considered to be significant. All the statistical analyses were performed by using the SPSS 5.0 J for Windows software package (SAS Institute).The significance of the difference between the two groups was analyzed by Student's 33.1To identify aberrantly expressed lncRNAs in HGSC, we used HGSC cell lines because using cell lines generally yields more uniform data than using tissue specimens that reflect individual differences. To analyze lncRNAs, we used a PCR array that can comprehensively analyze the expression of 84 kinds of lncRNAs associated with various cancer types. The expression levels of the lncRNAs in the two HGSC cell lines, KURAMOCHI and TYK‐nu, were compared to those in human ovarian surface epithelial cells (HOSE) and normal ovarian tissues. Of the 84 lncRNAs analyzed, six upregulated lncRNAs  and four downregulated lncRNAs  were identified as aberrantly expressed lncRNAs in the HGSC cell lines  and four downregulated lncRNAs  were examined in 22 specimens of ovarian HGSC tissues and 10 specimens of normal ovarian tissues by real‐time RT‐PCR. The expression levels of the four downregulated lncRNAs  were significantly lower in ovarian HGSC tissues compared to normal ovarian tissues, confirming that the expression profiles of these four lncRNAs were consistent between the HGSC cell lines and ovarian HGSC tissues Figure . However4In this study, based on a comparison of the expression profiles of HGSC cell lines with human ovarian surface epithelial cells (HOSE) and normal ovarian tissues, we identified ten aberrantly expressed lncRNAs in the HGSC cell lines and showed that among them four aberrantly downregulated lncRNAs  were similarly downregulated in ovarian HGSC tissues. The expression levels of ADAMTS9‐AS2, MEG3, and XIST were especially low in ovarian HGSC tissues compared with normal ovaries. Therefore, they are possible candidates of HGSC‐specific lncRNAs for molecular targeting therapy in future directions. These lncRNAs offer therapeutic applications by restoration of the lncRNAs that suppress malignant behaviors in a highly cancer‐specific manner like a drug delivery system.Some of the HGSC‐specific lncRNAs may have roles in malignant behaviors. Among four aberrantly downregulated lncRNAs , MEG3 and POU5F1P5 inhibited the cell migration when they were overexpressed. This is the first report of an inhibitory effect of MEG3 and POU5F1P5 on cell migration in ovarian HGSC. However, it is unclear why MEG3 and POU5F1P5 did not inhibit cell proliferation.POU5F1P5 has been reported to be upregulated in various cancer tissues and cancer cell lines,MEG3 inhibits malignant behaviors in a variety of cancers.Downregulation of ADAMTS9‐AS2 or XIST is associated with progression of cell proliferation and malignant behaviors in ovarian cancer.About the four upregulated lncRNAs , knockdown using siRNAs did not show any inhibitory effects and their expression levels were not different between ovarian HGSC tissues and normal ovarian tissues. We cannot clearly explain the difference in actions and expressions between upregulated and downregulated lncRNAs. We speculate as follows; the lncRNAs in this study were originally identified in HGSC cell lines. Although the HGSC cell lines used in this study are derived from ovarian HGSC cells, ovarian HGSC tissues reflect individual differences, indicating the heterogeneity. Therefore, the character of the HGSC cell lines is not always consistent with the character of the ovarian HGSC tissues.In conclusion, we identified four new lncRNAs that are downregulated in ovarian HGSC. These four lncRNAs may have uses in the diagnosis and treatment of HGSC.The authors declare that there are no conflicts of interest.All of the experiments handling human tissues were conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Institutional Review Board of Yamaguchi University Graduate School of Medicine. Informed consent was obtained from the patients before the collection of any samples.


Programmed death 1/programmed death ligand 1 (PD-1/PD-L1) targeted immunotherapy affords clinical benefit in ~20% of unselected patients with lung cancer. The factor(s) that determine whether a tumor responds or fails to respond to immunotherapy remains an active area of investigation. We have previously defined divergent responsiveness of two KRAS-mutant cell lines to PD-1/PD-L1 blockade using an orthotopic, immunocompetent mouse model. Responsiveness to PD-1/PD-L1 checkpoint blockade correlates with an interferon gamma (IFNγ)-inducible gene signature and major histocompatibility complex class II (MHC II) expression by cancer cells. In the current study, we aim to identify therapeutic targets that can be manipulated in order to enhance cancer-cell-specific MHC II expression.Responsiveness to IFNγ and induction of MHC II expression was assessed after various treatment conditions in mouse and human non-small cell lung cancer (NSCLC) cell lines using mass cytometric and flow cytometric analysis.Single-cell analysis using mass and flow cytometry demonstrated that IFNγ consistently induced PD-L1 and MHC class I (MHC I) across multiple murine and human NSCLC cell lines. In contrast, MHC II showed highly variable induction following IFNγ treatment both between lines and within lines. In mouse models of NSCLC, MHC II induction was inversely correlated with basal levels of phosphorylated extracellular signal-regulated kinase (ERK) 1/2, suggesting potential mitogen-activated protein (MAP) kinase-dependent antagonism of MHC II expression. To test this, cell lines were subjected to varying levels of stimulation with IFNγ, and assessed for MHC II expression in the presence or absence of mitogen-activated protein kinase kinase (MEK) inhibitors. IFNγ treatment in the presence of MEK inhibitors significantly enhanced MHC II induction across multiple lung cancer lines, with minimal impact on expression of either PD-L1 or MHC I. Inhibition of histone deacetylases (HDACs) also enhanced MHC II expression to a more modest extent. Combined MEK and HDAC inhibition led to greater MHC II expression than either treatment alone.These studies emphasize the active inhibitory role that epigenetic and ERK signaling cascades have in restricting cancer cell-intrinsic MHC II expression in NSCLC, and suggest that combinatorial blockade of these pathways may engender new responsiveness to checkpoint therapies. In the USA, lung cancer has an incidence of 225 000 patients every year leading to approximately 160 000 deaths.Our group has previously analyzed determinants of response to immune checkpoint blockade, using orthotopic implantation of KRAS mutant NSCLC lines into syngeneic hosts.In this study, we aimed to characterize the determinants of divergent IFNγ responsiveness between these tumor lines in vitro in order to gain mechanistic insights that may lead to novel approaches to enhance IFNγ sensitivity and thus make more tumors susceptible to PD-1/PD-L1 checkpoint blockade, with an emphasis on MHC II regulation. Our studies point to a critical requirement for epigenetic and post-translational-dependent mechanisms that actively restrict IFNγ induction of the MHC class II machinery in NSCLC.Murine cell lines: CMT167 and LLC cells stably expressing firefly luciferase were used in this study.2. Cells were regularly analyzed for morphology and functional characteristics to assure that the lines maintained consistent phenotypic characteristics for the duration of the study. To avoid cross-contamination, all cell lines used were not maintained in long-term cultures but rather maintained as frozen stocks and only kept in live culture for 2–4 weeks period. The EA1 cell line was derived from C57BL/6 mice treated with adenovirus containing Cas9 and guide RNAs leading to generation of tumors containing an EML4-ALK fusion as previously described.2. We followed similar procedures for maintaining the integrity of our cell lines as our group has previously reported.Human cell lines. Human NSCLC cell lines included: H460 , A549 cells (KRASG12S mutation) and SW1573 cells (KRASG12C mutation). Cells were periodically tested for mycoplasma infection, maintained as frozen stocks and cultured for only 2–4 weeks before use in experiments. Human lung adenocarcinoma cell lines were maintained in RPMI 1640  containing 10% FBS at 37°C in a humidified 5% CO2 atmosphere.CMT167, LLC cells, EA1 and EA2 cells were subjected to various treatments. After described treatment period, cells were trypsinized, collected and washed. For surface stains, cells were resuspended in flow cytometry staining bufferCMT167 and LLC cells were cultured for 48 hours under basal conditions or with 100 ng/mL IFNγ. Cells were then collected with trypsin, washed with PBS and resuspended in cisplatin (Fluidigm). Cells were incubated in cisplatin for 5 min, with the staining reaction quenched with MaxPar cell staining buffer (Fluidigm). Cells were subjected to barcoding using the Fluidigm Cell-ID 20 Plex Palladium barcoding kit according to manufacturer’s protocol (Fluidigm cat #201060). After barcoding, all samples were combined, and stained with a cocktail of metal-conjugated and fluorescently conjugated antibodies to detect cell surface proteins for 30 min at room temperature (RT) . Cells were then washed and incubated with secondary antibodies  for 30 min at RT. Cells were permeablized with FoxP3 Fix/Perm buffer overnight at 4°C. Cells were then washed and incubated with antibodies to detect intracellular antigens for 2 hours at 4°C. Cells were washed, resuspended in intercalator solution, washed and resuspended with equilibration beads prior to collection on a Helios mass cytometer (Fluidigm).https://github.com/nolanlab) (as in191Iridium (Ir)+, 193Ir+, 195Platinum (Pt)-) events. All viable nucleated events were imported into the Cytofkit analysis pipeline.Samples were normalized using NormalizerR2013b_MacOSX and debarcoded using SingleCellDebarcoderR2013b_MacOSX, with both programmes downloaded from the Nolan laboratory GitHub page (ab) as in. Normali10.1136/jitc-2019-000441.supp1Supplementary dataRecombinant mouse IFNγ  and human IFNγ  were used respectively to stimulate murine and human cell lines. Cells were further treated with various inhibitors including IκB kinase (IKK)-16 , ruxolitinib , cobimetinib , trametinib  and trichostatin A (TSA) . Compounds were diluted in a manner consistent with manufacturer recommendations.For quantitative real-time-PCR analysis, treated cells were harvested and homogenized in 350 µL of Buffer RLT . Total RNA was isolated using an RNAeasy Kit (Qiagen) and reverse transcription was performed on 1 µg of total RNA using qScript cDNA SuperMix (Quanta BioSciences 95 048–025). Real-time PCR analysis was conducted in triplicate in a C1000 Touch Thermal Cycler (Bio-Rad) using Power SYBR Green PCR Master Mix (Applied Biosystems 4367659). The relative message levels of each target gene were normalized to GAPDH.https://github.com/JinmiaoChenLab/cytofkit; Release 3.6), Excel (V.15.13), Adobe Illustrator.Flow cytometry data were analyzed using Kaluza software V.2.1.1 (Beckman Coulter) and GraphPad Prism software V.8. Mass cytometry data were analyzed using FlowJo V.10.2.4, R studio (V.1.0.136), the Cytofkit package  and EML4-ALK fusion  cell lines.10.1136/jitc-2019-000441.supp2Supplementary dataIFNγ induction of cell surface proteins was also evaluated in a panel of human NSCLC cell lines. Three of three human NSCLC cell lines showed IFNγ-dependent induction of PD-L1 and MHC class I , with cells uniformly inducing these proteins . In contWe next sought to understand mechanisms that restrain MHC II induction within and between NSCLC lines. Curiously, basal pERK1/2 levels were inversely correlated with high MHC II inducibility when we compared oncogene-matched NSCLC cell lines that had high vs low MHC II induction   trametinib . As expeWe next analyzed the impact of MEKi inhibition in LLC cells, a cell line that failed to induce MHC II expression after IFNγ stimulation. Treatment of LLC cells with trametinib in the absence of IFNγ induce a marked morphologic change in LLC cells , but faiTrametinib was further tested for its ability to modulate IFNγ responsiveness in human NSCLC cell lines. H1299 cells treated with IFNγ and trametinib had increased PD-L1 and MHC I expression relative to cells treated with IFNγ alone, though these cells failed to express MHC II under any conditions . In contHistone deacetylase inhibitors (HDACi) are another class of molecules that have been reported to promote MHC II expression in cancer cells.Given that combinatorial treatment with an HDACi and MEKi only induced MHC II in a subset of LLC cells, we tested the impact of STAT3 and CDK4/6 inhibitors as additional candidate regulators of MHC II expression. Notably, both STAT3We next explored the mechanisms promoting IFNγ responsiveness in either CMT167 cells treated with IFNγ, or with LLC cells treated with IFNγ in the presence or absence of trametinib. For these studies, we focused on the role of the janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway  of MHC II regulation in NSCLC cell lines, with a focus on MEK-ERK and HDAC-dependent pathways.The MEK-ERK pathway has previously been implicated in regulating the expression of MHC I and MHC II.A primary focus of our current studies has been on the expression of MHC II, given its divergent regulation between cell lines regardless of oncogene driver. It is important to note that MHC II expression, and the broader MHC II processing and presentation pathway, is coordinately regulated by the MHC class II transactivator, CIITA,Based on the enhanced induction of MHC II following treatment with MEKi and HDACi, we postulate that cell-to-cell variation in these pathways may contribute to the variable induction of MHC II following IFNγ stimulation. Further, we postulate that differential responsiveness to IFNγ stimulation observed between NSCLC cell lines may result from inter-cell line variability in: (1) the baseline activation of MEK/ERK signaling pathways and (2) locus-specific variation in chromatin accessibility . Consistent with this model, MHC II is uniformly induced in CMT167 cells following IFNγ and MEKi cotreatment. In contrast, the failure to uniformly induce MHC II in LLC cells following simultaneous treatment with IFNγ, MEKi and HDACi strongly suggests that MEK-ERK signaling and HDACs actively restrain IFNγ-dependent signaling in some, but not all, NSCLC cell lines, and that there are additional, unidentified modulators of this pathway.Our studies emphasize an important link between MEK and HDAC inhibitors and potential sensitivity of NSCLC cell lines to mechanisms of immune detection by regulating cancer cell-specific MHC II expression. It has previously been shown in triple negative breast cancer that alterations in RAS/MAPK signaling is associated with reduced tumor infiltrating lymphocytes. MEK inhibition led to increased MHC expression and was synergistic with anti-PD-1 immunotherapy in syngeneic models.In total, our studies demonstrate that IFNγ signaling in NSCLC cell lines is actively restrained by HDAC-dependent epigenetic modifications and by MEK-ERK signaling. How these two pathways regulate MHC I and MHC II expression at the molecular level, and whether these pathways are mechanistically linked, remains an ongoing subject of investigation. Given that both HDACs and MEK-ERK signaling can be therapeutically targeted, our data emphasize the potential utility of HDAC and/or MEK inhibition in individuals with an IFNγ rich TME. The potential utility of these combinatorial treatments is further exemplified by recent studies showing the benefit of either HDAC inhibition or MEK inhibition in the context of PD-1/PD-L1 targeted immunotherapy.


General characteristics of each cell line including doubling time, in vitro morphology, mutational profiles, and protein expressions of CRC-related genes were described. Whole exome sequencing was performed on each cell line to configure mutational profiles. Single nucleotide variation, frame shift, in-frame deletions and insertions, start codon deletion, and splice stop codon mutation of various genes were found and classified based on their pathogenicity reports. In addition, cell viability was assayed to measure their sensitivities to 24 anti-cancer drugs including anti-metabolites, kinase inhibitors, histone deacetylase inhibitors, alkylating inhibitors, and topoisomerase inhibitors, all widely used for various cancers. On testing, five CRC cell lines showed MSI, of which MLH1 or MSH6 gene was mutated. These newly established CRC cell lines can be used to investigate biological characteristics of CRC, particularly for investigating gene alterations associated with CRC.Colorectal cancer (CRC) represents the third most frequently diagnosed malignancy worldwide and is the second most common cause of tumor-associated mortalities in Korea. Due to the disease’s aggressive behavior, the 5-year survival rate for CRC patients remains unpromising. Well-characterized cell lines have been used as a biological model for studying the biology of cancer and developing novel therapeutics. To assist  It remains the second most perpetual type of tumor in both genders , and the number of CRC cases continues to increase. Approximately 25% of CRC cases are diagnosed in stage IV, and recurrence with distance metastasis follows after primary resection in nearly 50% of CRC patients3. Due to its aggressive behavior, the 5-year relative survival rate remains disapproving4. Neoadjuvent therapy is generally performed before surgical resection as single- or multi-agent chemotherapy to improve prognosis5. While roughly 50% of CRC patients respond to customary chemotherapy, the majority develop drug resistance through the course of treatment, and relapse or distance metastasis often follows. In recent years, novel anti-cancer agents that target surface growth factor receptors have been developed as adjuvant therapy to decrease the risk of the cancer recurrence6.Colorectal cancer (CRC) represents the third most frequently diagnosed tumor worldwide and is the second most common cause of tumor-associated mortalities in Korea7. However, most of the widely used CRC cell lines were derived from Caucasian and African American populations. Accordingly, inter-heterogeneity from ethnic diversity has been biased toward Western countries. To address this need, we established and characterized 18 novel CRC cell cultures  from 18 Korean CRC patients. Characterization includes cellular phenotypes, growth rates, mutations of CRC-driver genes, and sensitivities to 24 anti-colorectal cancer drugs that are approved by the National Cancer Institute. The 24 drugs are categorized as anti-metabolites , kinase inhibitors , histone deacetylase inhibitors , alkylating inhibitors , topoisomerase inhibitors (Irinotecan), growth factor receptor inhibitors , natural compounds , and miscellaneous . These newly established 18 cell lines can be used to study the molecular biology of CRC, specifically to investigate genomic alterations related to CRC.Well-characterized cell lines have been used as models for studying the biology of cancer and developing novel therapeutics8. These novel 18 CRC cell lines were deposited at Korean Cell Line Bank .Eighteen human CRC cell lines  were established from pathologically proven colorectal tumor tissues acquired from Korean CRC patients. Each participant was given informed consent before cell line establishment and experiment. The detailed procedure was described previouslyTM kit 9. Samples were arranged in the kit, including positive and negative controls for mycoplasma contamination. Mycoplasma control DNA served as a positive control and sterilized distilled water was used as the negative control. PCR amplification was performed under the following conditions: denaturation, 94 °C; annealing, 58 °C; and extension, 75°C. To confirm the specificity of contamination, PCR products were analyzed with gel electrophoresis using 2% agarose gel. PCR amplification and contaminated products had sizes of 570 bp and 260 bp, respectively.Mycoplasma contamination test was performed as described previously using e-Myco9. For growth properties, cells were seeded into 96-well plates at a density of 2.0 × 103 cells/well and were treated with EZ-cytox , a water-soluble tetrazolium salt solution that could be reduced by succinate-tetrazolium reductase to produce formazan dye. After incubating at 37 °C for 2 h, optical density (OD) was assessed at 450 nm using a Multiskan™ GO Microplate Spectrophotometer . The number of cells was analyzed in triplicate at 24-hour intervals for at least 7 days. The doubling time of the cells was calculated from the growth phase. Growth curve and growth properties were drawn and calculated using GraphPad Prism software with normalized OD values. Cell morphology was assessed using an Axiovert 100 microscope at 100× magnification.Cell growth rate was measured with same method described previously10. Briefly, total DNA was isolated from cell pellet by using QIAamp DNA Mini Kit  according to manufacturer’s protocol. Quantified and diluted gDNA solution was added to reaction mixture consisted of Amp FISTR PCR reaction mix, Taq DNA polymerase, and Amp FISTR identifier primer set . DNA was amplified using a GeneAmp PCR System 9700 (Applied Biosystem) with annealing temperature set to 59°C. Gene Scan-500 Rox standard (0.05 μl) and 9 μl oHi-Di Formamide (Applied Biosystem) were added to 1 μl of PCR product of each cell line and denatured at 95 °C for 2 min. The mixture was then analyzed with a 3500 xL Genetic Analyzer (Applied Biosystems).DNA fingerprinting analysis was performed as decreased before5 cells/well, tumor cells were seeded into a 96-well plate. Optimal concentrations of anti-cancer drugs were then used to treat 18 CRCs. These concentrations were: 100 μg/ml of TAS-102, 100 μg/ml of Regorafenib, 1000 μg/ml of Leucovorin calcium, 1000 μg/ml of Capecitabine, 50 μg/ml of Apitolisib, 100 μg/ml of Belinostat, 50 μg/ml of Trametinib, 50 μg/ml of Cyclopamine, 100 μg/ml of ICG-001, 100 μg/ml of Buparlisib, 50 μg/ml of SAHA, 50 μg/ml of Afatinib, 5 μg/ml of AZD2014, 100 μg/ml of MK-5108, 50 μg/ml of Olaparib, 100 μg/ml of Irinotecan, 50000 μg/ml of 5-FU, 100 μg/ml of Oxaliplatin, 100 μg/ml of Baicalein, 100 μg/ml of Curcumin, 100 μg/ml of Genistein, 200 μg/ml of Resveratrol, 1000 μg/ml of Cetuximab, and 1000 μg/ml of Bevacizumab. The 96-well plate containing anti-cancer drugs was incubated for 72 h at 37 °C. After incubation, 10 ul EZ-Cytox solution was applied to each well. After the plate was incubated for 2 h at 37 °C, optical density value was assessed at 450 nm with a Multiskan™ GO Microplate Spectrophotometer (Thermo Fisher Scientific).At density of 2 × 109. Cells were harvested with a cell scraper after washing with cold PBS. Whole protein was extracted with EzRIPA buffer  supplied with 1% protease inhibitor and 1% phosphatase inhibitor in accordance with the cell viability assay time frame. The volume of lysis buffer was adjusted to the number of cells collected in each vial. The protein concentration was determined by SMARTTM micro BCA protein assay kit . Proteins in equal amounts were loaded on a 4-12% Bis-Tris gel (Invitrogen) and run at 50 volts for 2 h. Proteins on gel were then transferred to a PVDF membrane (Invitrogen) by electro-blotting with constant current of 80 mA at 4 °C overnight. Proteins on transferred membrane were blocked by incubating with 1.5% to 2.0% skim milk in 0.05% Tween 20-TBS buffer including 1 mM MgCl2 at room temperature for an hour. The membrane was then incubated with primary antibodies against EGFR  (1:2000), HER2  (1:1000), MLH1  (1:500), MSH2  (1:500), EpCAM  (1:1000), E-cadherin  (1:1000), vimentin  (1:2000), and β-actin  (1:100) followed by incubation with mouse or rabbit IgG 2nd antibody  (1:5000) conjugated with peroxidase that matched with the primary antibody used. Chemiluminescent working solution WESTZOLTM (Intron biotechnology) was then used to treat the membrane which was then exposed to Fuji RX film  for 1-5 minutes.Detailed procedure was described previously9. SureSelect sequencing libraries were prepared using SureSelect Human All Exon 50 Mb Kit (Agilent) according to manufacturer’s instructions using a Bravo automated liquid handler. Three micrograms of genomic DNA were fragmented to a median size of 150 bp using a Covaris-S2 instrument . Adapter ligated DNA was amplified by PCR. PCR product quality was then assessed by capillary electrophoresis. Hybridization buffer and DNA blocker mix were incubated at 95 °C for 5 minutes and 65 °C for 10 min in a thermal cycler. The hybridization mixture was then added to a bead suspension and incubated at RT for 30 min while mixing. These beads were washed and DNA was eluted from beads with 50 ml SureSelect elution buffer (Agilent). The flow cell was then loaded on a HiSeq. 2500 sequencing system (Illumina).Detailed procedure was described previously11. For microsatellite instability (MSI) analysis, BAT25 and BAT26 (two mononucleotide microsatellite markers) were evaluated using a capillary-based sequencing analysis8. PCR was performed as described above except that forward primers were labeled with a fluorescent dye. Labeled samples were run on an ABI 3730 genetic analyzer (Applied Biosystems). GeneMapper software v4.0 (Applied Biosystems) was used to calculate the size of each fluorescent PCR product. For gel-based MSI analysis, desired fragments were amplified in the presence of [a-P32] deoxycytidine triphosphate. PCR products were denatured and separated on 6 M urea/7%polyacrylamide gels run at 60 W.Detailed procedure was described previouslyThe study protocol was approved by the Institutional Review Board of Seoul National University Hospital (IRB No. H-1102-098-357). The study was performed in accordance with the Declaration of Helsinki.In vitro and in vivo characteristics of newly established 18 CRC cell lines are summarized in Tables Human specimens were obtained from CRC patients who underwent surgeries at Seoul National University (SNU) Hospital from 1999 to 2008. Eighteen colorectal carcinoma cell lines  were established in RPMI 1640 medium supplemented with 10% FBS. in vitro cultivation, sixteen CRC cell lines  grew as monolayers of substrate-adherent cells. SNU-NCC-61cell line showed spindle morphology while other cell lines showed polygonal morphology. SNU-2359 and SNU-2493 grew as floating clumps. SNU-NCC-376 cell line formed floating and adherent aggregates  had pathogenic mutations in MLH1 and the protein expression was exclusively low accordingly. Two cell lines (SNU-2359 and SNU-2493) harbored benign mutation in MLH1 (c.655 A > G/p.Ile219Va), and protein structure was not affected. Although no pathogenic MSH2 mutation was present in the newly established CRC cell lines, the protein expression of MSH2 was varying, which implicated the protein expression of MSH2 was determined by RNA splicing or epigenetical alternations , we determined pathogenic mutations. Results are summarized in Fig. TP53 (83%) and APC (67%). KRAS and SMAD4 mutations were also prevalent in the sample sets at 44%. The most hyper-mutated cell line was SNU-2621B (10 mutations). Genes that are related to DNA repair such as POLD1, MSH6, and PMS2 were mutated in the SNU-2621B cell line. Similarly, SNU-1983 was also hyper-mutated (9 mutations) and DNA repair genes such as MLH1 and POLD1 were mutated. The truncation mutations of MLH1 and MSH6 genes in SNU-1566, SNU-1983 and SNU-2621B cell lines were confirmed with Sanger sequencing (Table Fifteen genes in developing CRC were screened in the 18 newly established CRC cell lines. Using Clinvar database (Areas under curve (AUCs) of 18 CRC cell lines in response to NIH approved 24 anti-cancer drugs, including anti-metabolite , kinase inhibitor , histone deacetylase inhibitor , alkylating inhibitor , topoisomerase inhibitor (Irinotecan), growth factor receptor inhibitor , natural compounds , and miscellaneous , were estimated Fig. . CRC cel12. Although there has been much research on inventing novel therapeutics, the molecular basis of drug response and aggressive behavior remains obscure due to its genetic intricacy, and more comprehensive analysis is called for to refine regimes for treatment and prevention13.New CRC cases continue to increase. At the time of detection, many CRC cases have already progressed to stage IV, which makes surgical resection unfeasible, and nearly 50% of CRC cases have shown recurrence or distance metastasis after primary resection14. Clinical correlation between original human materials and cancer cell lines can decrease due to the accumulation of genetic aberrations with increasing subculture numbers19. Therefore, novel CRC cell lines can deliver suitable biological models for investigating a broader spectrum of molecular characteristics of CRC. J. H. Oh et al. established 12 human CRC cell lines from 6 primary and 6 metastatic tumors of 11 Korean CRC patients10. In addition, J. L. Ku et al. established 13 CRC cell lines from 10 primary and 3 metastatic tumors of 13 Korean patients8. In this study, we established 18 novel CRC cell lines from 13 primary and 5 metastatic tumors of Korean patients who underwent surgical resection from 1999 to 2008 in SNU Hospital. Novel cell lines established through this study will be deposited to the Korean Cell Line Bank at various passages.The importance of human CRC cell lines lies in their similarity to original tissues and their renewability, which facilitate the study of human CRC. Several CRC cell lines such as HCT‐116, LoVo, SW‐480, and WiDr have accelerated the CRC research. Nevertheless, those accessible CRC cell lines are somewhat obsolete and possibly acquire genetic alternations as passagingMLH1, MSH2, and MSH620. Hereditary non-polyposis CRC, which accounts for 2–5% of all CRC cases is also concurrent with germline mutations in MMR genes. Nearly 90% of reported mutations in MMR genes were harbored in MLH1 and MSH222. In this study, five cell lines harbored pathogenic mutations in MMR genes. MLH1 was mutated in SNU-1983, SNU-2359, SNU-2434, SNU-2493, and SNU-3030. Among these cell lines, three  had pathogenic mutations in MLH1, and the protein expression was exclusively low accordingly. Interestingly, we found no pathogenic MSH2 mutation in the newly established CRC cell lines. Although Wei et al. reported that there were different patterns of MSH2 and MLH1 mutations between Asian and Caucasian population23, the prevalence of MLH1 mutation in comparison with MSH2 mutation in an Asian population has not been reported. Although we found no pathogenic MSH2 mutation, the protein expression of MSH2 varied, which implied that the protein expression of MSH2 was determined by RNA splicing or epigenetic alternations. Two  of these five cell lines were derived from patients with hereditary non-polyposis CRC.Nearly 15% of sporadic CRC cases show the MSI phenotype, which is prompted by inactivation of mismatch repair (MMR) genes such as APC, KRAS, and tp53 are frequently abberant genes in CRCs15, and these three genes were mostly mutated in the CRC cell lines characterized in this study as well. Most of the identified APC germline alternations are nonsense mutations or frameshift mutations near the 5’ end of the gene, which truncated the protein structure24. We considered APC mutations pathogenic when they were reported in Clinvar (https://www.ncbi.nlm.nih.gov/clinvar), and the types of pathogenic APC mutations we identified in this study were also nonsense or frameshift.KRAS serves as a fundamental mediator in the transduction of several growth or differentiation factor stimuli25. Most aberrations in KRAS harbor codons 12, 13, 59, and 6126. In this study, KRAS mutations gene were harbored in 8 of 18 cell lines (44%). Two cell lines (SNU-1566 and SNU-2423) had a mutation at codon 13, and six lines had a mutation at codon 12. Mutation types were G to A or G to T transitions.tp5327. In this study, mutations of tp53 were present in 16 (88%) of the 18 cell lines. All tp53 mutations were at codons 72 (n = 12), 74 (n = 1), 176 (n = 1), 196 (n = 1), 213 (n = 1), 245 (n = 1), 337 (n = 1), 342 (n = 1), 800 (n = 1), and c.376-1 G > A in our study. Interestingly, pPro72Arg mutation is commonly found in gastric cancer28. This mutated codon is associated with colorectal cancer29. SMAD4 serves as the fundamental component of TGF-β signaling, and it is reported to be inactivated in many types of tumor including pancreas, stomach, and colon30. SMAD4 mutation has been found in 10~35% of CRC34. Similarly, we found mutation of SMAD4 in 2 (10.5%) of 18 cell lines. SMAD4 mutations were at codons 386 (n = 1) and 442 (n = 1). PTEN mutations are known to occur in 5–14% of CRC37. PTEN serves as an anti-oncogene. Over-activation of PI3K/AKT pathway is mainly associated with loss of PTEN38. In this study, we found mutation of PTEN in only 1 of the 18 lines (5.2%), SNU-1983. SNU-1983 had KRAS mutation without BRAF or PIK3CA mutation. Mutations in BRAF, specifically valine-to-glutamate change at residue 600 (V600E), account for approximately 10% of CRC cases39. The present study showed BRAF mutation with V600E in 4 (21%) of 18 lines. STK11 regulates cell polarity and is a tumor suppressor. This gene is mainly related to Peutz-Jeghers syndrome40. Cadherin-1 (CDH1), in the classical cadherin superfamily, is associated with cancer proliferation and invasiveness41. SNU-2621B had mutation in STK11 and CDH1, but no other cell lines had these mutations. SNU-2621B had one STK11 mutation (pGly163) and two CDH1 mutations (pArg74* and Arg800fs). Two CDH1 mutations (pArg74* and Arg800fs) usually occur in gastric cancer42.Nearly 50% of CRC cases have several genetic alternations in All 18 CRC cell lines were sensitive to Apitolisib, Trametinib, Belinostat, 5-FU, and Buparlisib. Interestingly, SNU-2465 was resistant to Apitolisib, whereas all other lines were susceptible. All 18 CRC cell lines were resistant to Cetuximab, Bevacizumab, Leucovorin calcium, Olapraib, Cyclopamine, and Resveratrol. These novel cell lines will be deposited at the Korean Cell Line Bank and distributed worldwide for those who study colorectal cancer. These lines can be used as valuable materials to investigate biological properties of heterogeneous CRC.Supplementary Information.