Under normal conditions our intestines are inhabited by trillions of diverse microorganisms composing the intestinal microbiota, which are mostly non-pathogenic anaerobic commensal bacteria vital for the maintenance of immune homeostasis. The composition and diversity of the intestinal microbiota can be disturbed by various factors including diet, antibiotics, and exposure to intestinal pathogens. Alterations of the intestinal microbiota contributes to many diseases including graft-vs.-host disease (GVHD), a life threatening complication that occurs after allogeneic hematopoietic cell transplantation  caused by an allogeneic reaction of donor T cells against recipient target tissues. Intestinal GVHD is most difficult to treat and connected to a high mortality. Due to recent advances in high-throughput sequencing technology, composition of the microbiome during allo-HCT has been characterized, and some common patterns have been identified. Metabolites produced by intestinal bacteria were shown to promote intestinal tissue homeostasis and immune tolerance post-allo-HCT. In this review, we discuss the role of the intestinal microbiota and metabolites in the context of acute GVHD. Moreover, novel therapeutic approaches that aim at protecting or regenerating intestinal cell populations will be highlighted. Allogeneic hematopoietic cell transplantation  is the only curative therapy option for most acute leukemias. Prior allo-HCT patients undergo a conditioning regimen, consisting of chemotherapy with or without radiotherapy to decrease the number of malignant cells, and prevent rejection of the transplanted hematopoietic donor cells. Subsequently, hematopoietic stem cells (HSCs) from the bone marrow or peripheral blood of an allogeneic donor are infused. In addition to the HSCs, the graft contains allogeneic donor T cells, which can on the one hand attack residual malignant cells, known as graft-vs.-leukemia (GvL) effect, but on the other hand may attack healthy host tissues, resulting in graft-vs.-host disease (GVHD). Although the understanding of the disease pathophysiology has improved significantly in the last decades, acute GVHD is still a major cause of non-relapse morbidity, and mortality post-allo-HCT . In partThe important influence of intestinal microbiota on immune responses, including post-allo-HCT, becomes more and more recognized and the gut is one of the main targets of acute GVHD. Severe gastrointestinal (GI) GVHD remains a major issue after allo-HCT, since it is difficult to treat and involvement of the GI tract is reported in almost all fatal acute GVHD cases . In addiThe first hint that the intestinal microbiota affect GVHD development dates back to the early 1970s, when first studies in murine models showed that GVHD was reduced and survival was prolonged in antibiotics-treated or germ-free mice undergoing allo-HCT –13.Consequently, efforts were made to translate these insights into the human setting. Clinical trials investigated gut decontamination using antibiotics or laminar-airflow isolation rooms for patients undergoing allo-HCT, in order to minimize contact to microbiota –19. HoweHowever, interest in the interplay between the microbiome and inflammatory conditions has been growing again in recent years. Major advances in modern high-throughput sequencing technology made it possible to investigate changes in the composition of the intestinal microflora after allo-HCT in depth and to analyze which bacteria might be detrimental and which may be beneficial with respect to immune tolerance post-allo-HCT.Firmicutes and Bacteroidetes phyla  preceding the development of GVHD in pediatric allo-HCT patients , also known as stool transplantation, has been used to successfully treat recurrent patients . Very repatients , 45. A prelieved . AnotherMicrobial metabolites comprise a multitude of different intermediate products and end products of intestinal microbiota metabolism with various functions, yet their role in GVHD pathophysiology is merely starting to be discovered. Very recent publications revealed that one of the mechanisms, how intestinal bacteria can influence immune tolerance post-allo-HCT, is the production of metabolites Figure . Table 2Il22 transcription and mucosal immune homeostasis  . Daily iFocusing more on the receptors of bacterial metabolites, the group of Dr. Reddy recently revealed an important regulatory role for GPR43, a G-protein-coupled receptor on IECs recognizing the microbiota-derived short-chain fatty acids, butyrate, and propionate, during GVHD development. Sensing of the microbial metabolites by GPR43 reduced GVHD severity and mortality in murine models by activating the NLRP3 inflammasome in recipient non-hematopoietic cells via ERK phosphorylation. Importantly, GPR43 did not seem to play a role for host and donor hematopoietic cells, since absence of GPR43 did not lead to systemic activation of donor T cells or inflammation . While pIn a recently published study, Varelias et al. demonstrated that recipient mucosal-associated invariant T (MAIT) cells are present in acute GVHD target organs in murine models, including the intestinal lamina propria . These cSevere GI GVHD remains a major issue after allo-HCT, since it is difficult to treat and involvement of the GI tract is reported in almost all fatal acute GVHD cases . TherefoIntestinal stem cells (ISCs) represent important players for both physiological renewal of intestinal cells and tissue regeneration after injury. In a murine model, it was shown that ISCs are damaged during the conditioning regimen and the development of GVHD . This coIL-22 is a member of the IL-10 cytokine family that plays opposing roles in different autoimmune diseases, being able to drive or regulate disease –73. Simi+ ISCs (in vivo treatment approaches and showed the feasibility of both recombinant IL-22 and a recombinant human IL-22-dimer/Fc-fusion protein (F-652) for the treatment of murine GVHD. Based on the results from these preclinical studies, a Phase IIa clinical trial investigating F-652 in combination with systemic corticosteroids in patients with grade II-IV lower GI acute GVHD is currently recruiting (NCT0240665).In contrast, Hanash et al. demonstrated that recipient innate lymphoid cell (ILC)-derived IL-22 was able to enhance regeneration of IL-22R-expressing ISCs in a more acute major mismatch GVHD model . Host de+ ISCs . MoreoveThe described conflicting data of both a regulatory and inflammatory role for IL-22 in GVHD might result from many factors differing between the studies, including kinetics of the model, source of IL-22, presence or absence of other cytokines and pathophysiological differences between acute and chronic GVHD models.Reg3γ deficient mice this protection was completely abrogated, emphasizing the important role of REG3γ for gastrointestinal crypt homeostasis  to regulate GI acute GVHD . REG3α beostasis .REG3A SNPs were not associated with acute GVHD severity in this retrospective study including 350 patients  in the genes encoding two of the most abundant Paneth cell antimicrobial peptides REG3α and HD5 were studied . Interespatients .Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor family, regulating differentiation, and proliferation of epithelial cells, including IECs, as well as intestinal stem cells , 84. AlmIn the last 10 years it has become evident that a loss of diversity of the intestinal microbiota flora due to reduced food intake, chemotherapy-related damage, and antibiotics promotes the development of GVHD, which changed the dogma that antibiotics-based decontamination of the intestinal tract improves outcome post-allo-HCT. With the discovery that indoles and butyrate promote intestinal homeostasis the connection between antibiotics treatment and unfavorable outcome post-allo-HCT could be explained. Beyond bacterial species that were connected to GVHD, recent studies have also identified fungi and viruses that occurred more frequently in patients with severe GVHD. Based on this improved understanding on how the microbiome and the intestinal tract interact, novel strategies have been developed such as FMT that hold promise to overcome acute GVHD in a majority of patients. Ongoing clinical studies will provide important information on the role of FMT and regenerative strategies.NK and RZ collected literature, discussed the studies, and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


CCNE1 was the only significantly up-regulated gene among E2F targets at resistance with CCNE1 genomic amplification being observed in two resistant models. Rb was downregulated in all resistant models; a reduction of RB1 copy number was observed in three resistant cell lines. In silico analyses showed that CCNE1/RB1 ratio correlated with palbociclib IC50 in different datasets of both breast and non-breast cancer cell lines, performing better than CCNE1 or RB1 taken separately. Finally, the CCNE1/RB1 ratio was shown to be an adverse prognostic factor in patients with ER+ BC and to be able to discriminate palbociclib-sensitive versus resistant among patients enrolled in the NeoPalAna trial, a neoadjuvant trial testing palbociclib, performing better than CCNE1 or RB1 alone. Our data suggest that the CCNE1/RB1 ratio may be a viable biomarker of palbociclib resistance, warranting further clinical validation.CDK4/6 inhibitors represent a new treatment standard for hormone receptor-positive (HR+), HER2-negative advanced breast cancer (BC) patients. Although efficacious, resistance to these agents is universal. Here, we profiled a large panel of HR+ BC cell lines with conditioned resistance to the CDK4/6 inhibitor palbociclib, and analyzed cell cycle-related markers by gene expression profiles (GEP) and western blot (WB). GEP showed high molecular heterogeneity among the models, with E2F targets being significantly enriched both during treatment and at the time of resistance. By both WB and GEP, a common molecular feature at the time of palbociclib resistance was the concomitant overexpression of cyclin E1 and down-regulation of Rb.  CCNE1, and reduced expression of another, called Rb. The ratio of CCNE1:Rb expression proved to be an accurate prognostic factor among patients enrolled in a clinical trial of palbociclib, performing better than either CCNE1 or Rb levels on their own.The expression levels of two genes involved in controlling the cell cycle offers a promising predictive indicator of whether women with hormone receptor–positive breast cancer are likely to develop resistance to the drug palbociclib. A team led by Luca Malorni and Ilenia Migliaccio from the Hospital of Prato, Italy, analyzed gene and protein profiles in a panel of breast cancer cell lines with differential sensitivity to palbociclib. The researchers found that, compared to palbociclib-sensitive cell lines, those that were drug-resistant tended to have elevated expression of one cell-cycle–related gene, called  However, acquired resistance to these agents is universal, and results from clinical trials indicate that approximately 10 to 15% of patients may exhibit de novo resistance.4Palbociclib is a highly specific, orally active CDK4/6 inhibitor currently approved for the treatment of hormone receptor-positive, HER2-negative (HR+/HER2neg) advanced breast cancer (BC) in combination with the endocrine agents letrozole or fulvestrant.5 The primary target of CDK4/6 action is the retinoblastoma susceptibility gene product (Rb) and other Rb family members (such as p107 and p130).6 Phosphorylation of Rb (pRb) by active cyclin-CDK complexes leads to release of transcription factors of the E2F family, and transcription of genes required for S-phase entry.6 Constraint upon CDK activity and G1 progression is provided by the universal CDK inhibitors of the Cip-Kip family, including p21 and p27, and the specific CDK4/6 inhibitors of the Ink family typified by p16.7CDK4 and CDK6 are kinases activated by binding to D-type cyclins, bearing a crucial role in cell proliferation through the regulation of cell cycle entry.10 However, these events have been observed only in a limited number of in vitro and in vivo models, and these data have not been fully validated in the clinical setting. Therefore, the aim of our study is to further investigate if common cell cycle alterations, especially in the Rb/E2F pathway, could be observed in palbociclib-resistant models using a large panel of HR+ BC cell lines.Preclinical data have already shown that deregulation of cell cycle genes and proteins is associated with resistance to palbociclib, including overexpression of cyclin E1 and loss of Rb.We challenged eight BC cell lines with palbociclib including T47D, ZR75-1, MCF7, MCF7 Estrogen Deprivation Resistant (MCF7 EDR), MCF7 Tamoxifen Resistant (MCF7 TamR), CAMA1, MDA MB 361, and BT474. We developed PDR derivatives for all BC cell lines with the exception of CAMA1, which failed to restore growth with 350 nM palbociclib over two separate attempts. The time to develop resistance for all other models ranged from 10 to 27 weeks.P-value < 0.05), while PDR derivatives did not show significant differences in cell growth when treated with palbociclib 1 µM compared to vehicle-treated cells  lower proportions of β-galactosidase positive cells during palbociclib treatment compared to their PDS counterparts, demonstrating the ability to revert the palbociclib-induced senescence-like phenotype  or with palbociclib starting treatment concentration (STC) for 3 days (PDS treated) and from PDR cells continuously receiving palbociclib 1 µM.CCNE1 and RB1 were consistently deregulated in all PDR models versus PDS treated cell lines  ; similarly, genes down-regulated at the time of resistance were also significantly enriched in E2F targets indicating that the pathway remains inhibited by palbociclib at the time of resistance  Fig. . Interes05) Fig. , while t05) Fig. .CCNE1 expression being a common event.Overall these data suggest that palbociclib effectively inhibits cell growth of HR+ BC cells through inhibition of the Rb/E2F pathway. However, at the time of resistance, signaling is only partially restored, with increased To validate the role of cell cycle-related proteins during palbociclib treatment and at the time of resistance, we performed western blot (WB) analysis for selected markers in PDS cell lines treated with palbociclib for 3 and 6 days at the STCs, and in PDR models continuously receiving palbociclib 1 μM.14 were consistently downregulated by palbociclib. Conversely, cyclin D1 and cyclin E1 levels tended to increase during treatment. All other markers were modulated heterogeneously, or were not modulated by the treatment , these markers significantly correlated with higher IC50 values for palbociclib  and among all drugs tested in the dataset20 palbociclib showed the strongest association with CCNE1/RB1 ratio . In addition, analyzing GE data from BC cell lines of the Iorio et al. dataset,20 but taking into account the IC50 values for palbociclib obtained from a previous BC-focused study,19 we were able to demonstrate that CCNE1, RB1, and CCNE1 /RB1 ratio correlated with higher IC50 also in BC cell lines  was found in patients with a higher CCNE1/RB1 ratio  showing a poorer survival compared to those with a ratio <1 , before starting palbociclib (C1D1) and after 2 weeks of palbociclib (C1D15), and surgical samples were analyzed for Ki67, GE, and mutation profiles.23 We found that CCNE1/RB1 was significantly higher in patients resistant to palbociclib at C0D1, C1D1, C1D15 (p(WMW) = 0.0048; p = 0.013; p = 0.032, respectively, Fig. CCNE1/RB1 performed better than CCNE1 and RB1 alone in discriminating palbociclib-resistant versus sensitive patients at C0D1 (AUC(CCNE1/RB1) = 0.92; AUC(CCNE1) = 0.83; AUC(RB1) = 0.69), and at C1D1 (AUC(CCNE1/RB1) = 0.92; AUC(CCNE1) = 0.75; AUC(RB1) = 0.83), while showing equivalent performance compared to CCNE1 at C1D15 (AUC(CCNE1/RB1) = 0.9; AUC(CCNE1) = 0.9; AUC(RB1) = 0.55)  Fig. .26 We obtained reliable models of acquired resistance to palbociclib, as revealed by functional analyses investigating proliferation, IC50 and β-galactosidase expression, for all cell lines except CAMA1. Yang et al. succeeded in the development of models of CAMA1 resistant to abemaciclib,10 probably due to the different approach used10 and the different inhibitory profile of abemaciclib.27In this study, we developed a panel of HR+ BC cell lines with acquired resistance to palbociclib by chronically exposing cells to increasing doses of the drug. To account for the heterogeneity of luminal BC, we selected eight BC cell lines with different initial degrees of sensitivity to palbociclib. We deliberately challenged cell lines to palbociclib alone, rather than combined long-term estrogen deprivation (LTED) and palbociclib in order to identify markers specifically related to palbociclib resistance, thus avoiding the confounding effect of endocrine treatment. We acknowledge that the resultant models might not fully recapitulate the clinical scenario wherein CDK4/6 inhibitor resistance occurs in the setting of dual hormonal therapy and palbociclib. However, we did include two models previously resistant to hormonal therapy in order to account for the effect of combined palbociclib and endocrine resistance. Two HR+/HER2+ cell lines were also included to search for strong common mechanisms of resistance that might occur in HR+ BC despite HER2 status, given that palbociclib has also shown efficacy in HER2+ BC models.29 As expected, and in line with previous reports,31 we observed an inhibition of this pathway in response to palbociclib. We observed a marked reduction of the RBsig, a gene-signature of loss of RB1 function12 and found an enrichment in E2F targets within genes downregulated by palbociclib, consistent with previous observations.30 The activity of palbociclib on the Rb/E2F pathway was confirmed by WB, as revealed by decreased levels of total Rb coupled with decreased phosphorylation of Rb on S807/811 after 3 and 6 days of treatment. Together with Rb, we also observed a general decrease in p107 levels in all cell lines. As already reported by previous studies with cell lines sensitive to CDK4/6 inhibitors,31 our PDS models displayed cyclin D1 accumulation, reduction of E2F-regulated proteins (such as cyclins A1 and E2 and CDK2), and p130 stabilization when receiving palbociclib.As a first step, we explored the effects of palbociclib on the Rb/E2F pathway, which is the target of CDK4/6 inhibitors.9 Our PDS models showed a persistent inhibition of the Rb/E2F pathway after 3 and 6 days of treatment. However, we did not analyze earlier time points, and therefore cannot exclude a partial recovery of the pathway at 72 h as suggested by Herrera-Abreu et al.9It has been demonstrated that BC cells show adaptation following CDK4/6 inhibition as early as 72 h. This has been shown to be mediated by non-canonical activation of cyclin D1/CDK2 complexes, which in turn induce a recovery of Rb phosphorylation and S-phase entry despite CDK4/6 inhibition.33 In particular, cyclin E overexpression and Rb loss have been previously indicated as possible mechanisms of acquired resistance to CDK4/6 inhibitors in some BC cell lines.10 Our GE and WB data showed that both cyclin E1 overexpression and Rb underexpression occur across different and heterogeneous BC cellular models, including HER2+ cells, which has not been shown previously. We also demonstrated that CCNE1 amplification and RB1 deletion might explain only partially these findings, suggesting that other mechanisms could be implicated. Intriguingly, these genomic alterations occurred in both HER2+ and HER2neg cell lines, again supporting the hypothesis that strong mechanisms of acquired resistance to palbociclib might occur in HR+ BC independent of HER2 status. This is of potential clinical relevance as CDK4/6 inhibitors are currently under investigation for patients with HR+ /HER2+ BC.34It has already been shown that alterations in cell cycle genes and proteins are associated with acquired resistance to palbociclib.32At the time of resistance, we observed a slight reactivation of the Rb/E2F pathway as demonstrated by the increased RBsig expression in PDR cells, and by the modulation of some of the E2F targets analyzed. However, the pathway remained significantly inhibited compared to the PDS untreated cells, indicating that palbociclib treatment may still be inhibiting the pathway. This is consistent with previously published data in ovarian cancer, wherein cells resistant to palbociclib did not fully restore E2F target gene expression.31 Intriguingly, phosphorylation status of p27 has also been suggested as a mechanism of resistance to CDK4/6 inhibitors.35Another common marker that we found at the time of acquired resistance is the reduced expression of p27. In a previous study, Dean et al. similarly suggested that loss of CDK inhibitors p21 and p27 may foster the ability of cells to escape from palbociclib-induced cell cycle arrest.36 Loss of RB1 has been already suggested as a mechanism of de novo resistance to these drugs.19 Here, we identified CCNE1 as an additional potential marker of de novo resistance in a large panel of pan-cancer and BC cell lines. To date, the role of cyclin E1 as a marker of de novo resistance has been demonstrated in other tumor types such as ovarian and gastric cancer38 but only suggested in BC.39 Our data suggest that both CCNE1 and the CCNE1/RB1 ratio, which evaluates both CCNE1 and RB1, might serve as “pan-cancer” markers of de novo resistance to palbociclib and that the CCNE1/RB1 ratio might serve as better marker compared to the single biomarkers CCNE1 and RB1 used separately, both in BC and in other cancer types. In BC, this is evident for palbociclib IC50 values from Finn et al,19 but not when values from Iorio et al.20 were considered  upon which the patient had experienced previous progression as a prior line of therapy for ER+/HER2neg metastatic BC. This might be a potential additional setting to validate the role of the CCNE1/RB1 as a marker of de novo resistance to palbociclib.One limitation of our study is the lack of clinical validation of our findings on the role of 41 and to palbociclib in vitro.42 Similarly, overexpression of LMW cyclin E detected by immunohistochemistry has been associated with a lesser benefit from treatment with palbociclib and endocrine therapy in patients with metastatic BC.42 In our WB studies of the PDR models, LMW isoforms of cyclin E1 were not detected, suggesting that the full length is predominantly overexpressed in our models of acquired resistance. However, our in silico analyses could not exclude the possibility that LMW cyclin E may contribute to the effect on de novo resistance we described for cyclin E1, as both RNA and RPPA data cannot distinguish between the full length and the LMW isoforms.Recent data have shown that expression of the cytoplasmic low-molecular-weight isoforms (LMW) of cyclin E is associated with resistance to aromatase inhibitorsCCNE1/RB1 ratio have poorer OS compared to patients with a ratio <1, suggesting that this may represent a population of patients with an adverse outcome, which might mandate tailored therapeutic strategies. However, more detailed studies are necessary to dissect the prognostic versus predictive value of this biomarker in early BC, which would be of particular interest since CDK4/6 inhibitors are currently being tested in the adjuvant setting.43We demonstrated that patients with early ER+ BC with a high CCNE1 overexpression/amplification and reduced expression/loss of RB1 as common events that occur at the time of acquired resistance in luminal BC cell lines, independent of HER2 status. Furthermore, we have identified the CCNE1/RB1 ratio as a potential marker of de novo resistance to palbociclib both in vitro and in clinical samples.The strengths of our study include the size of the employed cell lines panel: to the best of our knowledge, this is the largest panel of HR+ BC cell lines ever developed to investigate acquired resistance to palbociclib. In turn, we have expanded previous knowledge on acquired resistance to palbociclib by confirming Results of our study, if further confirmed and tested in clinical trials, might lead to the identification of new biomarkers of de novo and acquired resistance to palbociclib, which would assist to better personalize treatment of patients with HR+ BC.44 MDA MB 361 were purchased from Sigma-Aldrich and BT474 from Interlab Cell Line Collection, Genova, Italy.T47D and ZR75-1 cell lines were obtained from Dr Livia Malorni, CNR Avellino, Italy; CAMA1 were obtained by American Type Culture Collection; MCF7 parental cells, MCF7 EDR and MCF7 TamR were previously described.Cells were cultured at 37 °C and 5% CO2. T47D, ZR75-1, MCF7, CAMA1, MDA MB 361, and BT474 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/glucose and L-glutamine (Lonza) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Hyclone) and, 10,000 U penicillin and 10 mg steptomycin/mL solution (P/S) (Sigma-Aldrich). MCF7 EDR and MCF7 TamR were grown in DMEM with 4,5 g/glucose and without L-glutamine and phenol red (Lonza) supplemented with 10% charcoal-stripped FBS (CS-FBS) (GIBCO), and P/S. MCF7 TamR cell medium was supplemented with 100 nM final concentration of (Z)−4-Hydroxytamoxifen (Sigma-Aldrich) dissolved in 100% ethanol.PDR were generated by long term culturing of cells with increasing concentrations of palbociclib from the STC up to 1 μM. STC was preliminary assessed on each model and defined as the concentration that, after 9 days of treatment, inhibited cells growth rate between 40 and 60%. Cells were considered resistant to palbociclib when they restored the ability to regularly grow in the presence of palbociclib 1 μM.Palbociclib STC was 50 nM for T47D, ZR75-1, MCF7 EDR, and MCF7 TamR cell lines, 100 nM for CAMA1 and 350 nM for MCF7, MDA MB 361 and BT474 cell lines. PDR cells were then continuously cultured in their original media with the addition of palbociclib 1 μM. Palbociclib was provided by Pfizer and dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich).In January 2016, all cell lines and their PDR derivatives have been authenticated by short tandem repeat DNA profiling analysis. This analysis was performed in service by BMR Genomics, Padova, Italy.For proliferation and IC50 assays a total of 3000 cells/well of PDS or PDR cell lines, cultured in their individual media, were plated in 96-well plates in triplicate, 24 h before beginning treatments. These consisted of palbociclib 1 µM, palbociclib STC or 0.01% DMS0 (vehicle) for 6 days for proliferation assays or doubling dilutions of palbociclib, from 64 µM to 0.97 nM for 9 days for the IC50 assays. Media were replaced every 72 h. Cells were fixed with 4% glutaraldehyde grade II (Sigma-Aldrich) and stained with 0.05% methylene blue (Sigma-Aldrich). The dye was subsequently extracted with 3% HCl (Carlo Erba) and absorbance measured at 655 nm. Three biological replicates for each PDS or PDR cell line were performed.PDS and PDR cell lines were plated in 12-well plates in duplicate, in a range of 10,000–20,000 cells/well. Cells were plated in their individual medium which was replaced after 24 h with medium containing palbociclib 1 µM or 0.01% DMSO as a control. Beta-galactosidase activity was assessed at day 6 by Senescence detection kit (BioVision) according to the manufacturer’s instructions. Positive and negative cells were counted in six randomly selected 20X fields and the percentage of positive cells was calculated. Three biological replicates for each PDS or PDR line were performed.TM MiniPrep kit (Zymo Research), according to the manufacturer’s instructions. DNA concentrations and quality were checked by spectrophotometer.DNA was isolated from PDS and PDR cells grown in their individual media supplemented with 0.01% DMSO or palbociclib 1 µM, respectively. DNA extraction was performed with the Quick-gDNATM RNA MiniPrep kit (Zymo Research) according to the manufacturer’s instructions. RNA quantification and quality analysis were performed on a Bioanalyzer 2100 System (Agilent), using the RNA 6000 nano Kit (Agilent). cDNA synthesis and biotin-labeled cRNA generation were performed using the Illumina TotalPrep RNA Amplification Kit (Ambion), according to the manufacturer’s protocol using 500 ng of total RNA. Quality assessment and quantification of cRNAs were performed with Agilent RNA kits on the Bioanalyzer 2100 System. Hybridization of cRNAs (750 ng) was carried out using Illumina Human 48k gene chips (Human HT-12 v4 BeadChip). Array washing was performed using Illumina High Temp Wash Buffer for 10′ at 55 °C, followed by staining using streptavidin-Cy3 dyes (Amersham Biosciences). Probe intensity data were obtained using the Illumina GenomeStudio software.RNA was isolated from PDS cells treated with 0.01% DMSO, as a control, or with palbociclib at the STC for 3 days, and from PDR cells at early (before or at passage 15) or late (after passage 15) stage of resistance, grown in their individual media supplemented with palbociclib 1 µM. The number of passages of PDR cells was calculated starting from the moment they restored the ability to regularly proliferate in the presence of palbociclib 1 µM. RNA was extracted from cells with the Direct-zolP-value < 0.05 in at least one sample were considered for downstream analysis (n = 31016). In case of multiple probes matching to a single gene, the probe with the highest signal-to-noise ratio was taken into consideration. To mitigate the effect of cell line specific expression profiles, the log2-ratio values of normalized expression of treated PDS and PDR to the corresponding untreated PDS GE profiles were computed. . The list KEGG_CELL_CYCLE (http://software.broadinstitute.org/gsea/msigdb/cards/KEGG_CELL_CYCLE) was used to identify cell cycle pathway genes. Top variable cell cycle genes  was performed by cubic spline algorithm implemented in the Illumina GenomeStudio software version 1.9.0. Only probes showing a detection nes Fig.  were defhttp://software.broadinstitute.org/gsea/msigdb/cards/HALLMARK_E2F_TARGETS) as gene set. Differential expression between log2-ratio expression values was tested by Wilcoxon–Mann–Whitney (WMW) test.E2F gene set enrichment was computed with Gene Set Enrichment Analysis (GSEA) version 2-2.2.3 by using log2-ratio values of averaged (mean) FC across PDS treated and PDR cell line models as ranking parameters and HALLMARK_E2F_TARGETS . A volume of 15 μg of proteins were separated under denaturing conditions by electrophoresis on 10% or 7.5% polyacrylamide gels  and transferred to nitrocellulose membranes  by electroblotting . The blots were checked using the ChemiDoc MP Imaging System and the Image Lab software (Bio-Rad) to confirm uniform transfer of all samples. Blots were then blocked with Tris-buffered saline (Sigma-Aldrich) containing 0.1% Tween 20 solution (Sigma-Aldrich) and 5% non-fat dry milk  or 5% BSA (Santa Cruz Biotechnology) for 1 h at room temperature (RT) and then incubated with primary and secondary antibodies.PDS cells treated with 0.01% DMSO, as a control, or with the STC of palbociclib for 3 or 6 days and PDR cells grown in their individual media supplemented with palbociclib 1 µM were lysed with RIPA buffer , supplemented with 1% Protease Inhibitor Cocktail (Sigma-Aldrich) and 1% HaltTM software v 5.2.1 (Bio-rad). WB were repeated in two biological replicates. A list of primary and secondary antibodies with their working conditions is provided in Supplementary Table Analysis of WB bands and normalization to total proteins were performed using the Image LabRB1 and CCNE1 between PDR and PDS cells were assessed with RB1, CCNE1 and the reference gene EIF2C1 CNV Assays on a QX200TM droplet digital PCR (ddPCR) system (Bio-Rad). PCR reactions were prepared with approximately 10 ng of DNA and ddPCR supermix for probes (Bio-Rad). RB1 and CCNE1 probes (Bio-Rad) were stained in FAM while EIF2C1 probe (Bio-Rad) was stained in HEX. The ratios CCNE1/EIF2C1and RB1/EIF2C1 were calculated using Quantasoft software (Bio-Rad). At least one negative control with no DNA was included in each run. Experiments were performed three times in duplicate.Copy number alterations of P-values < 0.05 were considered significant.For proliferation assays, growth fold changes were computed as ratio, dividing growth rate of treated cells by their controls treated with DMSO. Mean value of growth fold change of all experiments ±standard error of the mean (SEM) was plotted. Two–way ANOVA with Sidak’s multiple comparisons test was performed with GraphPad Prism version 7.03 and yp, were computed as: yp = (y − T0)/(DMSO − T0), where y are the observed values. The model performance was estimated by a weighted standard error, and a weighted goodness-of-fit. Once the model was fitted, the drug 50% inhibitory concentration (IC50) could be estimated from inhibition rates by inverting the function . Mean value of IC50 of all experiments ±standard error (SE) was calculated. Student’s t tests were performed and P-values < 0.05 were considered significant. Mean value of senescence percentages of all experiments ±SE was plotted. Student’s t tests were performed and P-values < 0.05 were considered significant. Calculations and plots for IC50 and senescence were performed by R (version 3.3.1).For IC50, the signals were converted to proportions adjusting on time zero (T0) and on untreated DMEM control values and fitted using the 4-parameter logistic regression (R package nplr version 0.1–4). The proportion values, For GE data, the two-sided Wilcoxon–Whitney test was used to check for significant differences between two distributions. The distributions of PFS and OS were estimated using the Kaplan–Meier method and compared with the log-rank test.All codes used to analyze the data and generate the figures are available upon request.Supplementary material


The anti-proliferative activity of the synthesised compounds was evaluated against two cell lines, namely; colon cancer HT-29 and triple negative breast cancer MDA-MB-468 cell lines. Also, their inhibitory activity against the cell surface expression of CD133 was examined. In particular, compound 4b emerged as a promising hit molecule as it manifested good antineoplastic potency against both tested cell lines , beside its ability to inhibit the cell surface expression of CD133 by 50% suggesting a promising potential of effectively controlling the tumour by eradicating the tumour bulk and inhibiting the proliferation of the CSCs. Moreover, compounds 4a and 4c showed moderate activity against HT-29  and MDA-MB-468  cell lines, while they inhibited the CD133 expression by 14% and 48%, respectively. Finally, a single crystal X-ray diffraction was recorded for compound 4d.Cancer stem cells (CSCs) have been objects of intensive study since their identification in 1994. Adopting a structural rigidification approach, a novel series of 3-phenylthiazolo[3,2- Indeed, three main key properties of CSCs render them highly distinguishablePertaining to their close similarity to normal cells, it is problematic to segregate CSCs from non-CSCs within a tumour. But for the presence of surface cell antigens, the identification and separation of tumour initiating cells from more differentiated tumour cells would not have been possiblea]benzimidazoles  thio)-1-arylethan-1-ones . Chemical shifts are expressed in δ values (ppm) using the solvent peak as internal standard. All coupling constant (J) values are given in hertz. The abbreviations used are as follows: s, singlet; d, doublet; m, multiplet. Elemental analyses were carried out at the Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt. Analytical thin layer chromatography (TLC) on silica gel plates containing UV indicator was employed routinely to follow the course of reactions and to check the purity of products. All reagents and solvents were purified and dried by standard techniques.Melting points were determined using a Gallenkamp melting point apparatus and are uncorrected. Infrared (IR) Spectra were recorded as KBr disks using the Perkin Elmer FT-IR (Fourier transform infrared) Spectrum BX apparatus. Mass spectra were measured on an Agilent Triple Quadrupole 6410 QQQ LC/MS (Liquid chromatography/Mass spectroscopy) equipped with an ESI (electrospray ionisation). NMR spectra were recorded on a Bruker NMR spectrometer. 1a–d (5 mmol) in glacial acetic acid (10 ml), 2-mercaptobenzimidazole 2  and conc. sulphuric acid (50 mmol) were added. The reaction mixture was heated under reflux for 2 h. The solid product obtained upon cooling was filtered off, washed with cold water then with petroleum ether, and recrystallised from ethanol to afford the corresponding sulphate salts 3a–d, respectively.To a solution of the appropriate acetophenone 3a–d (2 mmol) in water (10 ml), an aqueous solution of sodium bicarbonate was added. The reaction mixture was stirred at room temperature for 2 h. The solid formed was collected by filtration, washed with water, dried, and crystallised from ethanol to afford compounds 4a–d, respectively.To a suspension of the appropriate sulphate salts 1H NMR (DMSO-d6) δ ppm: 3.84 , 7.13 , 7.20–7.32 , 7.54 , 7.71 ; 13C NMR (DMSO-d6) δ ppm: 55.86 (OCH3), 109.23, 112.01, 114.68, 116.44, 119.23, 120.86, 121.47, 123.61, 130.61, 130.69, 133.66, 148.64, 157.03, 159.92; Anal. Calcd. for C16H12N2OS: C, 68.55; H, 4.31; N, 9.99; Found C, 68.73; H, 4.28; N, 10.12.White crystals (yield 80%), m.p. 173–175 °C; 1H NMR (DMSO-d6) δ ppm: 3.82 , 6.99 , 7.09 , 7.12 , 7.27–7.31 , 7.69 , 9.67 ; 13C NMR (DMSO-d6) δ ppm: 56.23 (OCH3), 107.63, 112.07, 113.40, 116.14, 119.11, 120.06, 120.72, 122.38, 123.48, 130.22, 134.22, 148.17, 148.67, 148.77, 156.94; Anal. Calcd. for C16H12N2O2S: C, 64.85; H, 4.08; N, 9.45; Found C, 65.14; H, 4.02; N, 9.34.White crystals (yield 78%), m.p. 190–193 °C; 1H NMR (DMSO-d6) δ ppm: 3.79 , 3.83 , 7.07 , 7.22–7.38 , 7.71 ; 13C NMR (DMSO-d6) δ ppm: 56.62 (OCH3), 60.71 (OCH3), 106.95, 108.79, 112.23, 119.19, 120.89, 123.58, 124.64, 130.21, 133.83, 139.20, 148.68, 153.59, 156.93; Anal. Calcd. for C18H16N2O3S: C, 63.51; H, 4.74; N, 8.23; Found C, 63.69; H, 4.69; N, 8.11.White crystals (yield 83%), m.p. 177–179 °C; 1H NMR (DMSO-d6) δ ppm: 5.28 , 6.73 , 7.22–7.38 , 7.59 , 7.68 ; Anal. Calcd. for C15H11N3S: C, 67.90; H, 4.18; N, 15.84; Found C, 68.15; H, 4.14; N, 15.73.White crystals (yield 75%), m.p. 185–186 °C; 4d were performed on a Bruker SMART APEX II D8 Venture diffractometer with graphite-monochromated Mo Kα radiation (λ = 0.71073 Å) at 100 K. The structure was solved by direct method and refined with SHELXTL. E-maps provided the positions of all the non-H-atoms. The full-matrix least-squares refinement was carried out on F2’s using anisotropic temperature factors for all non-H-atoms. Crystallographic data for the structure reported in this paper have been deposited at the Cambridge Crystallographic Data Centre and allocated with the deposition number: CCDC 1429525.The measurements of the crystal of compound a]benzimidazoles 4a–d was evaluated at Stem Cell Therapy and Tissue Reengineering Program, King Faisal Specialized Hospital and Research Center, Riyadh, Saudi Arabia. In vitro anti-proliferative activity was measured by the cell growth inhibition assay. This assay was conducted by use WST-1(water soluble tetrazolium-1) reagent50 for each compound. HT-29 colon cancer cell line and MDA-MB-468 triple negative breast cancer cell line were purchased from the American Type Culture Collection. Cells were maintained in RPMI 1640 , supplemented with 10% FBS  , 100 IU/mL penicillin, 100 mg/mL streptomycin, and 2 mmol/L L-glutamine (Sigma). Cells were seeded into 96-well plates at 0.4 * 104/well and incubated overnight. The medium was replaced with fresh one containing the desired concentrations of the test compounds. After 48 h, 10 μl of the WST-1 reagent were added to each well and the plates were re-incubated for 4 h at 37 °C. The amount of formazan was quantified using ELISA (Enzyme Linked Immunosorbent Assay) reader at 450 nm.Anti-proliferative activity of the synthesised 3-phenylthiazolobenzimidazoles 4a–d, with 75–83% yield . The crystallographic data and hydrogen-bond geometry of 4d are presented in Supplementary data.Characterisation of compound a]benzimidazoles 4a–d was evaluated against human colon cancer cell line HT-29 and triple negative breast cancer cell line MDA-MB-468 using the WST-1 assay as described by Ngamwongsatit et al50) values. Among the tested compounds, compound 4b bearing a terminal phenyl ring substituted with two electron-donating groups proved to be the most potent against both cell lines; HT-29 and MDA-MB-468 with IC50 values of 9 and 12 μM, respectively. Compared to the positive control 5-FU , these results proclaimed compound 4b to be superior to the reference drug. Moreover, compounds 4a and 4c showed moderate activity against HT-29 cell line with IC50 values of 21 and 29 μM, respectively, which are comparable to that of 5-FU (IC50 = 15 μM). Luckily, both compounds evinced good antineoplastic potency against MDA-MB-468 cell line relative to 5-FU . Unfortunately, compound 4d bearing a p-amino phenyl group did not manifest any significant anti-proliferative activity against the cancer colon cell line HT-29 while it demonstrated moderate activity against triple negative breast cancer cell line MDA-MB-468 (53 μM)  .a]benzimidazoles 4a–d on cell surface expression of CD133 was evaluated at 10 μM via flow cytometry. The analysis was performed on a BD LSR II™ (BD Biosciences). The results expressed as a side population inhibition (%). Scrutinizing the obtained results disclosed that compound 4a bearing a m-methoy phenyl group only limitedly inhibited the CD133 by 13%, whereas compound 4b that possesses an extra electron donating group; an additional p-hydroxy group significantly inhibited the CD133 expression by 50%. Coherent to these findings, compound 4c that has three methoxy groups grafted on its terminal phenyl ring produced a comparable inhibition of CD133 (48%). These findings are in accordance with the previous conclusion that outlined the importance of incorporation of a lipophilic electron-donating group represented by a terminal phenyl ring substituted with a methoxy or a hydroxyl group. Regrettably, compound 4d containing a p-amino phenyl ring did not display any marked activity against the cell surface expression of CD133  based on structural rigidification of a series of 2-((benzimidazol-2-yl) thio)-1-arylethan-1-ones (Series 1) that proved to have antineoplastic activity and inhibitory activity of cell surface expression of CD133. The anti-proliferative activity of the synthesised compounds was evaluated against two cell lines; colon cancer cell line HT-29 and triple negative breast cancer cell line MDA-MB-468. Moreover, their inhibitory activity against the cell surface expression of CD133 was determined in an attempt to explore their potential to eradicate CSCs as well as the tumour bulk cells. Compound 4b emerged as a promising hit molecule as it manifested excellent antineoplastic potency against both tested cell lines  beside its ability to inhibit the cell surface expression of CD133 by 50% suggesting a promising potential of effectively controlling the tumour by eradicating the tumour bulk and inhibiting the proliferation of the CSCs. Moreover, compounds 4a and 4d exhibited good anti-proliferative activity against both cell lines and also significant inhibition potential of cell surface expression of CD133. On the other hand, structure of compound 4d was further substantiated via X-ray single crystal analysis.In conclusion, we designed and synthesised a novel series of 3-arylthiazolo[3,2-


These results suggest that combination targeted therapy may be an effective strategy against colorectal cancer.Treatment of colorectal cancer mostly relies on traditional therapeutic approaches, such as surgery and chemotherapy. Limited options of targeted therapy for colorectal cancer narrowly focus on blocking cancer-generic targets VEGFR and EGFR. Identifying the oncogenic drivers, understanding their contribution to proliferation, and finding inhibitors to block such drivers are the keys to developing targeted therapy for colorectal cancer. In this study, ten colorectal cancer cell lines were screened against a panel of protein kinase inhibitors blocking key oncogenic signaling pathways. The results show that four of the 10 cell lines did not respond to any kinase inhibitors significantly, the other six were mildly inhibited by AZD-6244, BMS-754807, and/or dasatinib. Mechanistic analyses demonstrate that these inhibitors independently block the MAP kinase pathway, IR/IGF-1R/AKT pathway, and Src kinases, suggesting a multi-driver nature of proliferative signaling in these cells. Most of these cell lines were potently and synergistically inhibited by pair-wise combinations of these drugs. Furthermore, seven of the 10 cell lines were inhibited by the triple combination of AZD-6244/BMS-754807/dasatinib with IC Colorectal cancer (CRC) is the third most commonly diagnosed cancer, and the third leading cause of cancer-related deaths in the US and worldwide . In the Cell proliferation is sustained by signal transduction pathways that relay external growth signals to regulate cellular metabolism and gene expression. Cancer cells often rely on constitutively activated signaling pathways that short-circuit normal signaling and regulatory processes. The most prominent oncogenic signaling proteins are soluble and receptor protein tyrosine kinases (PTKs) , moleculTargeted therapy for CRC has largely relied on monoclonal antibodies against EGFR and VEGFR ,6. The aIn this study, we evaluate the responses of 10 CRC cell lines to the treatment of a panel of kinase inhibitors. Six of the 10 cell lines are inhibited by multiple inhibitors blocking independent signaling pathways, including the Mek inhibitor AZD-6244, the insulin receptor (IR)/insulin-like growth factor 1 receptor (IGF-1R) inhibitor BMS-754807, and the Src kinase inhibitor dasatinib. Mechanistic analysis and drug combination studies indicate that the proliferation of these cells are supported by multiple pathways, and can only be effectively blocked by combination of drugs that respectively block these pathways. This study provides a mechanistic explanation for why targeted therapy has not worked well for CRC, and a rational strategy for combination targeted therapy. d values below 20 nM are shown in http://lincs.hms.harvard.edu/kinomescan/). Two of the PTK inhibitors, BMS-754807, specific for the kinases in the IR family and Met . Be. Be28]. The responses by most of these CRC cells to multiple kinase inhibitors are intriguing. For effective targeted therapy by a single agent, the cancer cell needs to be addicted to a single oncogene or a network ,31. Yet Even though AZD-6244 is a specific Mek inhibitor, it strongly activated Mek phosphorylation (Ser217/Ser221) in all three cell lines, but inhibited the phosphorylation of Erk (Thr202/Tyr204), a direct substrate for Mek. The fact the AZD-6244 blocked Erk activation and inhibited the proliferation of all three cells indicated that the function of Mek plays an important role for the proliferation of these cells. This is consistent with the fact all these cells contain KRAS mutations. AZD-6244 activation of Mek phosphorylation was somewhat surprising, but it could be a feedback mechanism. Mek phosphorylation on Ser217/Ser221 is catalyzed by Raf, blocking Mek activity by AZD-6244 appears to result in hyper-phosphorylation of Mek, perhaps as an attempt to compensate for the loss of Mek function. This is most likely a feedback mechanism in these cells compensating for the blockade of function by AZD-6244. Such a feedback mechanism has been observed for Akt inhibitor, GSK-690693 in other systems . NeverthWe attempted to determine the protein levels and the phosphorylation status of IR and IGF-1R in all three cells under treatments. While the IR and IGF-1R proteins could be readily determined B, WesterDasatinib inhibited Src autophosphorylation on Tyr416 and Src phosphorylation on Tyr527, catalyzed by another PTK, Csk, in all three cell lines. This result indicated that dasatinib did block Src kinase activity as well as Csk kinase activity, consistent with its reported specificity. Although normally down-regulation of Tyr527 phosphorylation correlates to activation of Src kinase activity, dasatinib also directly inhibited Src activity, thus the net effect of dasatinib on Src was likely inhibitory. A comparison of the activation levels of Src among the three cell lines suggests that LS-174T contains the highest level of phosphor-Tyr416 in control and under AZD-6244 and BMS-754807 treatments, suggesting that Src activation level was higher in LS-174T than in the other two cell lines. This is consistent with the fact that LS-174T is the only cell line among the three that is sensitive to inhibition by dasatinib. Although phosphor-Tyr416 in LS-1034 and LS-513 was clearly detectable and it was clearly inhibited by desatinib, Src apparently did not play a major role in the proliferation of these two cell lines, as dasatinib did not significantly inhibit the proliferation of these two cells. The above results together demonstrate that each of these drugs independently blocks a specific pathway. Thus, they support that hypothesis that these CRC cancer cells are indeed multi-driver/multi-pathway dependent cancer cells. The fact that multiple independent pathways are contributing to the proliferation of these cells explains the observations that these cells are sensitive to multiple inhibitors, but the inhibition by any one of them is only mild.The results from the previous sections suggested that CRC cell proliferation was driven simultaneously by IR/IGF-1R/AKT pathway, the MAP kinase pathway and/or the Src kinases. One logical prediction of the multi-driver proliferation model is that complete inhibition would require a combination of drugs that collectively block all the activated pathways in a given cell line. We tested this prediction by determining the responses of selected cells to combinations of drugs that were individually only mildly effective. 50’s were mostly in the high nM to low μM range. We determined if these cells would be more effectively inhibited by the combination (1:1 mixture) of the two drugs. As shown in 50’s were summarized visually in 50 for the two-drug combination ranged from 46 nM for NCI-H747 to 153 nM for LS-1034. These nM level IC50 values indicate that these CRC cell lines are extremely sensitive to the combination of AZD-6244 and BMS-754807. As shown in 60 and IC70 values  as a function of percentage of inhibition . Synergy0 values B for the50’s of 1.41 μM and 0.61 μM, respectively, but the two drugs combined had an IC50 of 102 nM. The DRI value also increased dramatically with the increase in the percentage of inhibition, reaching 17-fold at 70% inhibition. The benefit was even higher at 80% inhibition, however, the DRI could not be calculated because AZD-6244 did not reach 80% inhibition up to 20 μM. These results are consistent with the mechanistic understanding that both Src kinase activity and the MAP kinase pathway independently contribute to the proliferation of LS-174T, and further suggest that a cancer based on this mechanism would be subject to combination targeted therapy with dasatinib and AZD-6244.While inhibition by AZD-6244 and BMS-754807 seems to be a common feature of CRC cells, LS-174T, NCI-H747 and SK-CO-1 displayed sensitivity to dasatinib . Interes50 of 40 nM, marginally better than that of the AZD-6244 and BMS-754807 combination, at 52 nM. This could be due to the fact the dasatinib was a much weaker inhibitor than AZD-6244 and BMS-754807 for this cell line.Based on the results in the previous sections, most of the CRC cell lines were inhibited by AZD-6244, BMS-754807 and/or dasatinib, and two cell lines, SK-CO-1 and NCI-H747 were inhibited by all three. The synergistic effects in pair-wise combinations led us to determine if the three-drug combination would be even more potent than the two-drug combinations. To test this proposition, we tested SK-CO-1 against the 1:1:1 mixture of the three drugs at a series of concentrations A. The tr50s ranging from 10 nM for NCI-H747 to 1061 nM for LS-123, with seven of the 10 cell lines inhibited by the triple combination with IC50’s below 84 nM. The extremely low IC50 of 10 nM for NCI-H747 is consistent with the observation that it is more sensitive to each drug alone than the other cell lines  of imatinib inhibiting BCR-Abl dependent CML cell lines [50’s of the double and triple combinations are also shown to Although different cell lines were more potently inhibited by one combination or another, we thought that the triple combination may be a one-size-fits-all formulation for most CRC cells . We tested all 10 cell lines against the triple combination (1:1:1 ratio) B. The cell lines . Interesll lines . FurtherThe molecular mechanisms driving the proliferation of CRC cells have been extensively studied ,39. The 50, suggesting that receptor PTKs in the PDGFR, EGFR, FGFR, MET, and soluble PTKs in the Abl families do not play major roles in driving the proliferation of CRC cells. This is largely consistent with the conclusion from genetic analysis that receptor PTKs are rarely mutated and are unlikely to play any major roles in driving CRC proliferation [It is also noteworthy what kinase inhibitors did not inhibit the proliferation of the CRC cells. PTK inhibitors sunitinib, BGJ398, crizotinib, and imatinib did not inhibit any of the cell lines significantly, while gefitinib only inhibited one cell line, LS-513, with a modest ICferation . It is sferation . The preFour of the cell lines did not respond to the individual kinase inhibitors, and interestingly three of them contained hypermutated genomes. At least two scenarios are plausible. Because these cells’ genomes contain 10–20 times as many mutations as other cancer cell lines, it is possible that many signaling pathways are affected. About 8% all cancers contain hypermutated genomes. A recent study determined that normal cancers contain an average of four driver mutations/tumor, and hypermutated genomes likely contain many times more . It is pDespite the success of targeted therapy in treating some cancers, it has not reached most cancers. One of the most basic challenges targeted therapy faces is that it has worked effectively only for cancers that are addicted to a driver oncogene or pathway. Successful applications of Abl inhibitor for BCR-Abl-dependent chronic myeloid leukemia, EGFR inhibitors for EGFR mutation dependent non-small cell lung cancer, and ErbB2 inhibitors for ErbB2-dependnet breast cancer are all examples of both the power of targeted therapy and the challenge it faces. However, most cancers contain multiple driver genes that may independently contribute to the proliferative advantage of a cancer cell. A good strategy for dealing with this has not been worked out. Most of the CRC cells we studied here appear to be multi-driver cancer cells. None of the 10 CRC cell lines displayed an exclusive reliance on any one gene or pathway, as most of them contain activated MAP kinase, IR/IGF-1R/AKT, and/or the Src kinase pathways.Even though there is a certain level of heterogeneity among the tested cells, the level of similarity is also surprising. While it is not surprising that all six sensitive cells responded to AZD-6244, due to the mutation of BRAF and KRAS in these cells, it was surprising that five of the six sensitive cells responded to BMS-754807, because none of these cells contains a mutation in IR or IGF-1R. However, the relationship between IR/IGF-1R signaling is complicated by cros-talks and feedback loops ,41,42,43The inhibition of several cell lines by dasatinib suggests that Src is playing a role in driving the proliferation of these cells. Western blot analyses confirmed that dasatinib indeed blocked Src activation by autophosphorylation, and thus Src kinase activity. Indeed Src has been reported to be associated with some CRC cell lines ,45,46. TThe multi-driver nature of CRC cell proliferation could explain why CRC cannot be effectively treated by individual kinase inhibitors, even though these inhibitors clearly block their intended targets, and their intended targets clearly play important roles in CRC cell proliferation. The multi-driver nature of CRC cells also dictates that effective targeted therapy against CRC would require the combination of several inhibitors, each blocking one activated pathway. Our results confirmed this hypothesis. If the multi-driver mechanism applies to other solid tumors, then the combination of kinase inhibitors may be an effective and necessary approach of targeted therapy. With the realization of the limits of mono-agent targeted therapy ,48, combA key feature of the AZD-6244/BMS-754807 or AZD-6244/dasatinib drug combination is that the synergy increases dramatically with the level of inhibition. While the synergy at 10–20% inhibition is often minimal, it increases dramatically as the level of inhibition increases. At 60–70% inhibition, the dose reduction index often reached higher than 10, and as high as more than 30. This feature is fully consistent with the multi-driver proliferation mechanism, and would be particularly beneficial for combination targeted therapy. To inhibit the proliferation of these cells to a significant level, for example 70–80%, with an individual drug would require concentrations of 20 μM or higher even for the most potent inhibitors. Signaling inhibitors at such high concentrations would likely cause off-target effects and significant toxicity ,51. This50 values below 100 nM. As a benchmark for targeted therapy, CML cells dependent on BCR-Abl for proliferation are inhibited by Abl inhibitor imatinib with IC50 values in the range of 80–600 nM (38). Projecting these results from this study into patient treatment provides at least two treatment approaches that are worthy of further investigation. First, if a patient can be pre-tested for the drug sensitivity to decide which drug combination the patient would respond to, then an effective drug combination can be selected for the patient. This is the ideal scenario of targeted therapy or precision therapy. Second, if a drug sensitivity pre-test is not feasible, the triple combination of AZD-6244, BMS-754807 and dasatinib may be an attractive treatment option because majority of CRC cell lines can be strongly inhibited by the combination (IC50 ≤ 84 nM). The treatment with a triple combination may have another benefit. Increasing evidence demonstrates significant intra-tumor genetic heterogeneity, where a solid tumor may contain heterogeneous sub-population of cancer cells driven by different genetic mutations [The study demonstrated that double or triple combinations of AZD-6244, BMS-754807 and dasatinib are particularly effective in blocking the proliferation of most CRC cell lines, with many ICutations ,53. If t2. Kinase inhibitors were purchased from Selleckchem , LC Laboratories  or AdooQ Bioscience . All CRC cell lines were purchased from ATCC , and were used within 6 months of their purchase. ATCC authenticates all its cell lines by short tandem repeat profiling, cell morphology monitoring, karyotyping, and cytochrome C oxidase I testing. The cells were grown in ATCC-formulated McCoy’s 5a Medium Modified , RPMI-1640 Medium , or Eagle’s Minimum Essential Medium , containing 10% FBS and 1% penicillin-streptomycin . All cells were cultured at 37 °C in humid atmosphere containing 5% COCells were plated into 96-well plates at 25,000 cells per well, and were incubated with drugs at indicated concentrations in 100 μL medium containing 1% DMSO for 72 h. For adherent cells , at the end of the incubation, 10 μL of staining solution containing 5 mg/mL MTT dye -2,5-diphenyltetrazolium bromide) (Thermo Fisher Scientific) was added into each well. After incubation at 37 °C for another 4 h, the medium in the wells was removed by aspiration. DMSO (100 μL) was added into each well, and when purple formazon was dissolved, absorbance at 490 and 750 nm for each well was determined using a VersaMax Tunable Microplate Reader with SOFTmax Pro v5 . The A490-A750 values were taken as indicator of cell viability. For semi-suspension cells (NCI-H747), cell viability was determined by staining the cells with Biolog Redox Dye Mix MB . At the end of 72 h drug treatment, 10 μL Biolog Redox Dye Mix MB was added into each well, and the plates were incubated at 37 °C for 2 h before the absorbance of the water soluble formazan product at 590 and 750 nm was determined using the microplate reader. The A590-A750 values were taken as indicator of cell viability. All cell growth and drug inhibition experiments were performed in triplicates at least twice. Statistical analysis was performed on six repeat data sets for each assay.50 of the cell line for the drug was also determined using a 16-concentration 2x dilution series of the drug, ranging from 0.6 nM to 20 μM. The IC50 values were estimated from the dose response curves as the concentrations that resulted in 50% inhibition of the cell’s viability. To determine if the proliferation of a cell line is sensitive to inhibition by the kinase inhibitors, each cell was screened against a panel of ten kinase inhibitors  at four To determine the effects of the kinase inhibitors on the signaling network in the CRC cell lines, the cell lines were cultured to 70% confluency, and then treated by the indicated drugs. The culture medium was then replaced by fresh medium containing kinase inhibitors at designated concentrations. The cells were incubated for 2 h under treatment. After the treatment, the culture medium was aspirated, and the cells were then resuspended in the SDS-PAGE loading buffer containing a protease inhibitor cocktail for mammalian cell and tissue extracts  and protein phosphatase inhibitors  and lysed in a 95 °C heat bath immediately. The lysates were then cleared by centrifugation and the supernatants were fractionated by SDS-PAGE, and immunoblotted by various antibodies recognizing selected signaling proteins and their specific phosphorylated forms. The loading was normalized based on the β-actin expression levels in these lysates determined by an antibody specific for this protein. All antibodies were purchased from Cell Signaling Technology .x) was determined manually from the graphs of drug dose response curves. The combination index (CI) was calculated for a given percentage of viability inhibition according to Chou [40, IC50, IC60, etc., were determined from the dose response curves, and the CI at a given level of inhibition (X%) was calculated by the following equation: CI = ICxab/ICxa + ICxab/ICxb, where ICxab is the concentration of the drug combination that resulted in X% inhibition, and ICxa and ICxb are the concentrations of drug a and drug b, respectively, that caused X% inhibition to cell proliferation. The dose reduction index (DRI), a measure of how many folds the combination concentration may be reduced, for a given percentage of inhibition, compared with the doses of each drug alone, is calculated as: DRI = 1/CI [To determine if two drugs were synergistic in their inhibition of a cell line’s proliferation, the cell line viability was determined by incubating the cells in the presence of each drug alone or both drugs (1:1 ratio) at 16 concentrations ranging from 0.6 nM to 20 μM. The concentration of a drug or drug combination that inhibited cell proliferation at X%  using Excel with the assumption of two tailed distribution and equal variance. The P values are reported in the appropriate figure legends.We performed three types of statistical analysis to evaluate the data. First, the IChttps://cancer.sanger.ac.uk/cosmic) [Genomic data for the CRC cell lines were collected from the COSMIC database, the Catalogue of Somatic Mutations in Cancer (/cosmic) .


Bacopa monnieri. Previously, we showed that bac II reduced endothelial cell migration and tube formation and induced apoptosis in colorectal cancer cell lines. The aim of the current study was to examine the effects of treatment with combined doses of bac I and bac II using four cell lines representative of the breast cancer subtypes: triple negative (MDA-MB-231), estrogen receptor positive (T47D and MCF7) and human epidermal growth factor receptor 2 (HER2) positive (BT-474). Drug treatment outcome measures included cell viability, proliferation, cell cycle, apoptosis, migration, and invasion assays. Relationships were analysed by one- and two-way analysis of variance with Bonferroni post-hoc analysis. Combined doses of bac I and bac II, each below their half maximal inhibitory concentration (IC50), were synergistic and reduced the viability and proliferation of the four breast cancer cell lines. Cell loss occurred at the highest dose combinations and was associated with G2/M arrest and apoptosis. Migration in the scratch wound assay was significantly reduced at apoptosis-inducing combinations, but also at non-cytotoxic combinations, for MDA-MB-231 and T47D (p < 0.0001) and BT-474 (p = 0.0003). Non-cytotoxic combinations also significantly reduced spheroid invasion of MDA-MB-231 cells by up to 97% (p < 0.0001). Combining bac I and II below their IC50 reduced the viability, proliferation, and migration and invasiveness of breast cancer cell lines, suggesting synergy between bac I and II.Bacopaside (bac) I and II are triterpene saponins purified from the medicinal herb  Breast cancer remains the second most common cancer in women worldwide and the fourth leading cause of cancer death . Breast Bacopa monnieri [Terpenoids are a diverse family of natural products found in a variety of fruits, vegetables and medicinal plants . Classifmonnieri ,12,13,14monnieri ,15, withmonnieri ). We theTo further support the use of bac I and bac II in cancer, we previously showed that these drugs block the functional activity of the transmembrane protein aquaporin (AQP) 1 . AQP1 isTogether, these studies provide a strong rationale to investigate the effects of bac I and II in breast cancer. We therefore tested the anti-cancer activity of the drugs individually and combined on breast cancer cell lines from the major molecular subtypes, MDA-MB-231 (TNBC), T47D (ER+/PR+), MCF7 (ER+/PR−), and BT-474 (HER2+), and looked for potential synergy between the two drugs.50s) shown in 50 values for bac I for MDA-MB-231, T47D and MCF7 were 99 μM  91–109), 89 μM (95% CI 73–109), and 83 μM (95% CI 79–87), respectively, whilst BT-474 was more sensitive at 59 μM (95% CI 56–63) , 19 μM (95% CI 17–20) and 16 μM (95% CI 15–17), respectively, whilst T47D was less sensitive at 29 μM (95% CI 9–96) (A viability assay (MTS) was performed on the four breast cancer cell lines treated with varying concentrations of either bac I or bac II. The cell lines showed a dose response effect to both drugs with half maximal inhibitory concentrations (ICI 56–63) a. The ICCI 9–96) b.50 5.5-fold lower than for bac I for MDA-MB-231, we used a constant dose of bac II well below the IC50—specifically, 2.5 μM—whilst the concentration of bac I was varied; this was done to determine the effect of bac II, at a dose that did not reduce viability, on the IC50 of bac I. For MDA-MB-231 cells, the IC50 of the combinations was determined as 13 μM bac I (95% CI 10–16 μM) + 2.5 μM bac II. Bac II shifted the IC50 for Bac I from 99 μM (95% CI 91–109) to 13 μM (95% CI 10–16)  of 0.270 (95% CI 0.240–0.301)  c, indica0–0.301) d. Isobolp < 0.0001 relative to vehicle at day 3). Relative to day 0, a decrease in absorbance occurred at day 1 with this dose for all cell lines (p ˂ 0.0001), as well as with 10 μM bac I + 2.5 μM bac II (p < 0.0001) for MDA-MB-231 and BT-474 (p = 0.0002) for BT-474 , and by 13% and 20% with 10 μM bac I + 5 μM bac II for T47D (p < 0.0001). Sub-G1 events, suggestive of dead cells and/or debris, increased with increasing combinations of bac I and bac II, most notably for MDA-MB-231 and BT-474.Based on the cell loss induced with combination doses, cell cycle analysis was performed, comparing vehicle and drug-treated cells. For each breast cancer cell line, there was an increase in the percentage of cells in G2/M with increasing doses of bac I and bac II. The dose combinations at which this occurred were cell-line dependent. Compared to the vehicle, with a dose of 5 μM bac I + 5 μM bac II, the proportion of cells in G2/M increased by 17% and 9% for MDA-MB-231 a,b, and for T47D c,d, and for T47D e,f, respThe morphology of breast cancer cell lines treated with each drug alone and in combination was observed microscopically after three days in culture. Bac I and bac II, each at 5 μM, and bac I, at 10 μM, had no observable effects on morphology compared to the vehicle control, while 10 μM bac II induced small vacuoles in MDA-MB-231 only; therefore, the highest doses of each drug selected for use in combination were doses of ≤10 μM bac I and ≤5 μM bac II . Combinep < 0.001, p < 0.0001 respectively) (Cells were treated with a vehicle at a dose that slowed proliferation but did not cause cell loss and one or more doses that induced cell loss. ctively) a,b,g,h. p < 0.0001). MCF7 cells were more resistant (p < 0.0001). For BT-474 (p < 0.0001). Representative images of wound closure assays are shown in We assessed the effects of bac I and bac II on breast cancer cell migration, since AQP1 is known to be important in this process and these drugs target AQP1 function. esistant c: an inhr BT-474 d, at dosp < 0.0001). Furthermore, the doses of 2.5 μM bac I + 2.5 μM bac II and 5 μM bac I + 2.5 μM bac II significantly reduced invasion, by 68% and 97%, respectively (p < 0.0001)—doses that did not induce apoptosis , being very low in T47D and BT-474, and undetectable in MCF7 (data not shown). Cell lines expressing AQP1 transcript were treated for 24 h with a non-cytotoxic combination of bac I and bac II that reduced migration: specifically, 5 μM bac I + 2.5 μM bac II for MDA-MB-231 and BT-474, and 10 μM bac I + 2.5 μM bac II for T47D. Cells were also treated with the 5 μM bac I + 5 μM bac II combination that induced apoptosis. Compared to vehicle, AQP1 transcript expression in MDA-MB-231 cells was reduced with the non-cytotoxic dose (p = 0.004) and by 2.5-fold with the cytotoxic dose (p < 0.0001) (AQP1 transcript expression was significantly higher in MDA-MB-231 relative to the other cell lines ( 0.0001) a. For T4 0.0001) b,c. ThesThe cell membrane permeabilisation effects of high concentrations of saponins lead to hemolysis and cytotoxicity. At lower doses, however, anti-cancer properties such as the inhibition of proliferation, induction of apoptosis and attenuation of invasiveness have been demonstrated (reviewed in ). Here, B. monnieri containing approximately 20% bac II w/w [B. monnieri extract was cytotoxic to human cancer cell lines of the colon, lung, cervix, and breast [B. monnieri extract in skin carcinogenesis [There are no studies investigating the anti-cancer effects of combining bac I and II. Previous studies have focused on the anti-cancer effects of either the whole plant extract, bacoside A (a mixture of saponins from c II w/w ), or bacd breast ,30,31; bd breast  and gliod breast  cell lind breast . Similard breast  and in ed breast . We haved breast . In vivod breast  and of Bogenesis ,36, as wogenesis , and Ehrogenesis , respectThese studies provide strong evidence to investigate the anti-cancer efficacy of bac I and bac II combined in breast cancer cell lines from each of three main molecular subtypes, as performed here for the first time. We found that all cell lines were sensitive to the synergistic effects of bac I and bac II combined, but particularly MDA-MB-231 and BT474, representative of the more aggressive subtypes of breast cancer of TNBC and HER2+, respectively. We also found that certain dose levels and combinations were cytotoxic, consistent with previous studies using the whole extract and its derivatives.B. monnieri extract [A novel aspect of our study, however, was the finding that lower doses of bac I and II combined, at doses that did not induce apoptosis, also had potent anti-cancer activity in vitro. Previously, we showed that non-cytotoxic concentrations of bac II alone inhibited endothelial cell tube formation—an in vitro measure of angiogenesis —but othe extract . This pr extract .B. monnieri extract containing bac I and bac II at levels higher than these. A large single oral dose of 5000 mg/kg of whole extract, containing 51.5 mg/kg bac I and 91 mg/kg bac II, caused no acute toxicity in Sprague-Dawley rats; in addition, rats receiving up to 1500 mg/kg of whole extract daily for 270 days, containing 15.5 mg/kg bac I and 27.3 mg/kg bac II, showed no evidence of chronic toxicity [B. monnieri whole extract at too low a dose to be comparable to the amounts of bac I and bac II used here [In terms of safety, the highest doses used in our in vitro study to induce apoptosis were 10 μM bac I and 5 μM bac II, equivalent in mice to approximately 7 mg/kg and 4 mg/kg, respectively, while the human equivalent dose  would betoxicity . Others toxicity  and clintoxicity  and micetoxicity —5 to 8 tsed here ,45,46,47sed here , the estThe rationale for combining the two drugs was based on their ability to block AQP1 ion and water channel function. Both bac I and bac II block the water channel function of AQP1, but only bac I blocks the ion channel function . We therB. monnieri and bacoside A, the latter of which contains bac II but not bac I. Both induced the death of human glioblastoma cell lines in vitro through macropinocytosis, a process whereby cell membrane buckling leads to the accumulation of extracellular fluid inside large vacuoles within the cell [There have been reports of other suggested mechanisms of anti-cancer activity for extracts of the cell . This buthe cell . They althe cell . The potbacopa monnieri, were obtained from ChromaDex , solubilised in methanol at 10 mM and 1.5 mM stock solutions, respectively, and stored at −20 °C. Cell lines MDA-MB-231, T47D, MCF7 and BT-474 were purchased from the American Type Culture Collection . Cells were maintained in complete medium, either in Dulbecco’s modified Eagle’s medium  for MDA-MB-231, MCF7 and BT-474 or Roswell Park Memorial Institute (RPMI) 1640 medium  for T47D, containing 10% foetal bovine serum (FBS) , 200 U/mL of penicillin, 200 μg/mL of streptomycin  and 2 mM L-alanyl-L-glutamine dipeptide . Cells were grown under standard culture conditions at 37° C with 5% CO2 in air and used within 4 passages.The analytical standards bacopaside I  and bacopaside II , derived from the medicinal herb 4 cells per well of a 96 well plate and incubated under standard culture conditions overnight. Next, cells were treated with either vehicle (1% methanol), bac I, bac II or combinations of bac I and bac II, for 24 h. MTS assay was performed using the CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay  according to the manufacturer’s instructions. Absorbance was read at 492 nm and results were calculated as the mean absorbance normalized to the vehicle control. The half maximal inhibitory concentration (IC50) for each drug alone and in combination was determined by non-linear regression analysis.The effect of bac I and bac II, alone and in combination, on cell viability was determined by MTS assay as described previously ,21. Cell50 of bac I and bac II combined, bac II was used at a constant concentration of 2.5 μM whilst the concentration of bac I was varied. The isobologram method [50s for bac I and bac II alone on the y- and x-axis respectively. The IC50 combination of bac I and bac II was also plotted on the same graph. Its position would indicate whether the IC50 combination was antagonistic, additive, or synergistic, depending on whether the point was above, on or below the theoretical line respectively. This was also reflected by the combination index, calculated by the formula, CI = d1/Dx1 + d2/Dx2, where Dx1 was the IC50 of bac I, Dx2 was the IC50 of bac II, and d1 and d2 were the doses of bac I and bac II respectively that combined gave an IC50. The combination was antagonistic, additive or synergistic if the combination index was greater than, equal to or less than one [To determine the ICm method  was used3 (MDA-MB-231 and MCF7), 3 × 103 (T47D) or 4 × 103 (BT-474) cells per well of a 96 well plate followed by overnight incubation. Cells were then treated with the vehicle or bac I and/or bac II, and crystal violet absorbance at 595 nm was measured on days 0, 1 and 3 of treatment.The crystal violet assay was used to measure cell proliferation and was performed as described previously . Briefly5 cells were seeded per well of a six-well plate, incubated overnight, then treated with either the vehicle or combinations of bac I and bac II for 24 h. Cells were resuspended in ice-cold Dulbecco’s phosphate buffered saline (DPBS) with drop-wise addition of 100% ice cold ethanol to a final concentration of 70% ethanol. Cells were centrifuged at 300× g for 5 min and cell pellets resuspended in 0.25% Triton X-100 in DPBS. Cells were stained for 2 h in darkness with 25 μg/mL propidium iodide  and 40 μg/mL bovine pancreas ribonuclease A  in DPBS and analysed on the BD FACSCanto II cell analyser . Doublet populations were excluded and 50,000 single cell events were captured per sample. Data was analysed using FlowJo software v10.4.0 , specifically by applying the Watson model to determine the percentage of cells in each stage of the cell cycle.Cell cycle analysis was performed as previously described ,52. Brie5 per well of a six well plate, cultured overnight then treated with either vehicle or bac I and bac II for 24 h. Cells were harvested and stained with annexin-V and propidium iodide for 15 min in darkness at room temperature then run on a BD FACSCanto II cell analyser . Compensation was performed to account for the spectral overlap between fluorochromes. Cells were gated to exclude debris and doublets and at least 10,000 single cell events were acquired per sample. Data was analysed using FlowJo software v10.4.0 .Apoptosis was determined by annexin-V and propidium iodide (PI) staining, using the Annexin-V-FLUOS staining kit , as previously described . Briefly4 (T47D and MCF7), 4 × 104 (MDA-MB-231), or 5 × 104 (BT-474) cells per well of a 96 well plate, incubated under standard conditions until 80–90% confluent, then serum starved overnight. A circular wound was made with a p10 pipette tip. Cells were resuspended in complete medium supplemented with 1 μg/mL mitomycin C  to inhibit cell proliferation, and either vehicle or bac I and/or bac II. The area of wound closure was measured using NIS Elements software .The scratch wound assay was performed as described previously ,53. Cell® 3D culture spheroid cell invasion assay  was set up as per the manufacturer’s instructions using the kit contents, including spheroid formation extracellular matrix (ECM) and invasion matrix. MDA-MB-231 cells were seeded in the spheroid formation ECM at 3000 cells per well of a Costar® 96-well ultra-low attachment plate  followed by incubation for 72 h. Spheroids were subsequently embedded in invasion matrix and vehicle or drug treatments were added. The area of invasion on day 3 was measured using NIS elements software  and was expressed normalised to the mean invasion of the vehicle control.The Cultrex5 cells per well in six-well plates and incubated until 70 to 80% confluent. RNA was isolated with the PureLink RNA mini kit  at time 0 and following 24 h treatment with vehicle or combinations of bac I and bac II. The iScript cDNA Synthesis Kit  was used to reverse transcribe 200 ng of RNA. Transcript expression was determined as previously described [−ΔCt relative quantification method, normalizing to CCSER2 reference gene.Breast cancer cell lines were seeded at 3 × 10escribed , using mData were analysed using GraphPad Prism version 7.0c for Mac OS X  and is presented as means ± standard deviation (SD). Statistical analysis was done by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test unless otherwise stated.This study is the first of its kind to test the anti-cancer potential of bac I and bac II combined on breast cancer cell lines from each of the three molecular subtypes. Bac I and bac II combined behaved synergistically, the combination was more potent than either drug alone, and all subtypes were susceptible to their effects; however, the degree of susceptibility was cell line-specific. In particular, the more aggressive TNBC (MDA-MB-231) and HER2+ (BT-474) cell lines were the most sensitive. Further studies will explore the toxicity and pharmacokinetics of the two drugs in mouse models of breast cancer and elucidate the exact mechanisms underlying the observed effects.