Type 2 diabetes (T2DM) is associated with aggregation of the human islet amyloid polypeptide (hIAPP) into cytotoxic amyloid species. Here we tested the effect of a diphenylpyrazole (DPP)-derived small molecule inhibitor, anle145c, on cytotoxicity and on aggregation properties of hIAPP. We demonstrate that incubation of hIAPP with the inhibitor yields ~10 nm-sized non-toxic oligomers, independent of the initial aggregation state of hIAPP. This suggests that anle145c has a special mode of action in which anle145c-stabilized oligomers act as a thermodynamic sink for the preferred aggregation state of hIAPP and anle145c. We also demonstrate that the inhibitor acts in a very efficient manner, with sub-stoichiometric concentrations of anle145c being sufficient to (i) inhibit hIAPP-induced death of INS-1E cells, (ii) prevent hIAPP fibril formation in solution, and (iii) convert preformed hIAPP fibrils into non-toxic oligomers. Together, these results indicate that anle145c is a promising candidate for inhibition of amyloid formation in T2DM. These so-called amyloid diseases in general have the hallmark of an age-related pathological effect. However, T2DM stands apart in that it is a common metabolic disease, linked to life style and obesity7, that increasingly affects people even at an early age9. Its prevalence around the world is beginning to take on epidemic proportions and T2DM thereby represents a major health problem in society10.Misfolding of proteins and aggregation into amyloid structures is associated with a number of diseases, including type 2 diabetes (T2DM), Parkinson’s disease and Alzheimer’s disease13. hIAPP is a hormone that is co-secreted with insulin in the β-cells in the islets of Langerhans in the pancreas. Under pathological conditions typical for T2DM, hIAPP is produced at relatively high concentrations, thereby promoting aggregation and contributing to pancreatic cell death15. The aggregation process furthermore can be affected by factors such as the presence of metal ions, insulin, sulfated glycans and membranes25, thereby complicating interpretation of in vivo studies on a molecular level. Nevertheless, an important role for hIAPP in T2DM is underscored by the observations that a high prevalence of hIAPP aggregates is found in humans with T2DM26 and that for species whose IAPP cannot form fibrils it has been reported that they do not develop T2DM characterized by islet amyloid deposits29.A prominent pathological event in T2DM is believed to be the formation of toxic amyloid species by a 37 amino acid protein known as human islet amyloid polypeptide (hIAPP)39. However, most of these inhibitors were found to act efficiently only at relatively high concentrations.Inhibition of hIAPP amyloid formation is considered to be an effective strategy to help combat T2DM. Several natural small molecule amyloid inhibitor compounds, such as epigallocatechin gallate (EGCG), resveratrol and curcumin, as well as a variety of synthetic inhibitors have been shown to be able to inhibit hIAPP fibrillation or to reduce the cytotoxic activity of hIAPP40. Notably, the lead compound “anle138b” was found to inhibit protein aggregation in case of prion disease, Parkinson’s disease (PD), Alzheimer’s disease (AD), Multiple System Atrophy (MSA) and Creutzfeldt-Jakob disease (CJD) in vitro and in vivo47. For none of the DPP compounds, the effect on hIAPP has been tested. Yet hIAPP is an interesting target, because it forms oligomers that have some features that are different from those of other well-studied amyloid forming proteins48.Recently, a family of di-phenyl pyrazole (DPP) amyloid inhibitors has been developed that act as promising oligomer modulators to combat amyloid diseases40. Therefore, artefacts due to aggregation of the inhibitor in the aqueous environment are avoided and the interaction of anle145c with hIAPP can easily and systematically be studied at concentrations that would not be feasible for anle138b.Here we set out to elucidate whether DPP compounds can be effective against aggregation of hIAPP. For this initial study, we selected the DPP-derived small-molecule inhibitor anle145c because it structurally resembles the very lipophilic lead compound anle138b and is likewise an efficient inhibitor of amyloid aggregation. Anle145c also has the advantage of being more water soluble than anle138b with a solubility between ~50 to 100 µM compared to ~0.2 µM for anle138bOur findings indicate that the molecule efficiently inhibits both aggregation and cytotoxicity by hIAPP at sub-stoichiometric concentrations and in addition is able to convert mature amyloid fibrils into non-toxic anle145c-stabilized oligomers by stabilizing non-toxic oligomers thermodynamically. The results suggest that anle145c is a promising candidate to inhibit protein aggregation in case of T2DM.In this study we investigated the interaction between hIAPP and the small-molecule inhibitor anle145c. Figure 15, most likely as a consequence of hIAPP aggregation.The ability of anle145c to protect living cells from hIAPP-induced cytotoxicity was tested using insulinoma (INS-1E) cells from a rat pancreatic beta cell line. These cells closely resemble the insulin-producing beta-cells in the pancreas that are known to become damaged under conditions of T2DMAs shown by the micrograph in Fig. To gain insight into the molecular nature of the interaction between hIAPP and anle145c, we next studied the aggregation behavior of hIAPP in the absence and presence of the inhibitor.49. As shown in Fig. 50.The aggregation kinetics of hIAPP were followed using a ThT binding assay. ThT is a widely used fluorescent dye that does not bind to monomeric hIAPP, but shows enhanced fluorescence upon binding to amyloid structuresTo discriminate between these possibilities, we performed negative stain TEM. As shown in Fig. 1H NMR on hIAPP in the absence and presence of anle145c as shown in Fig. 51. Quantification of the signal intensity shows that the kinetics of monomer and small oligomer depletion indeed are similar with a t1/2 value of 5.7 hours in both cases. The data also indicate that hIAPP monomers are consumed almost completely. For the aromatic region no discernable signals were left  of ~1 nm. After 16 h incubation, only very large particles are observed of ~2–10 µm, consistent with the formation of amyloid fibrils. When freshly dissolved hIAPP is incubated in the presence of the inhibitor at a sub-stoichiometric ratio of anle145c to hIAPP of 1/5, the initial RH is again ~1 nm. However, in this case after incubation only a relatively small increase in RH is observed to around 10 nm.The size of the particles was studied by dynamic light scattering (DLS). Figure The oligomeric species were further characterized by atomic force microscopy (AFM). As shown in Fig. Finally, the effect of anle145c on the conformational behavior of hIAPP was monitored using CD spectroscopy Fig. . CD specAn important question is whether anle145c is able to act on grown fibrils, and, if so, what kind of structures are formed. As shown in Fig. in vivo if they are non-toxic to cells. Thus, the effect of the oligomers was tested on INS-1E cell lines. The results, as illustrated in Fig. The anle145c-stabilized oligomers can only be relevant Together, the results obtained from addition of anle145c to hIAPP at different time-points of incubation indicate that anle145c-stabilized oligomers form a thermodynamic sink for the preferred aggregation state of hIAPP in the presence of anle145c and that these oligomers are non-cytotoxic.Here we investigated the interaction between the DPP-derived small molecule inhibitor anle145c and hIAPP in solution and we investigated the effect of the inhibitor on hIAPP-induced cytotoxicity. We will first discuss the situation in solution.48.We found that anle145c is a very efficient inhibitor of hIAPP aggregation in solution, with sub-stoichiometric concentrations of anle145c being sufficient for full inhibition of fibril formation. Instead of fibrils, oligomeric species were formed, which we call “anle145c-stabilized oligomers”. The size distributions as observed by DLS, EM and AFM experiments were consistent with oligomers of ~10 nm. The secondary structure of hIAPP in this oligomeric complex with anle145c was mostly random coil, similar to that of freshly dissolved monomeric hIAPP, but also similar to toxic as well as non-toxic oligomers1H NMR experiments indicated that anle145c does not recruit monomeric hIAPP species or small oligomers, since its presence does not seem to affect the half-time of monomer depletion. Moreover, recruitment of monomeric species would effectively decrease the hIAPP monomer concentration, leading to an increase in lag-time of fibril formation53, which was not observed by ThT measurements. Instead, our finding that the lag time for fibril formation is independent of the anle145c concentration suggests that anle145c acts on late oligomeric species that are present just prior to fibril formation. This would then prevent them from becoming part of the growing fibrils, but would not interfere with the process of fibril formation itself.i.e. a transition with a high energy barrier, from an oligomer in a random coil organization to an ordered β-sheet oligomer, which then can grow into fibrils54. Possibly, anle145c prevents this “activation” step by interacting with oligomeric species and allowing the formation of a thermodynamically more favorable state. This state then would consist of anle-stabilized hIAPP oligomers, in which hIAPP remains in a mostly random coil conformation.It has been proposed that hIAPP oligomers undergo “activation”, i.e. no matter what the initial population distribution of hIAPP aggregates is, anle145c-stabilized oligomers will be obtained with similar final size distributions.Consistent with being the thermodynamically preferred species, anle145c-stabilized oligomers could not only be obtained by incubation with monomeric hIAPP species, they also could be derived by incubation with fibrils. Both oligomer populations were found to behave very similar, in the sense that they have a similar size distribution and conformation and they are non-toxic to cells. Moreover, addition of anle145c to hIAPP populations at different times of incubation suggested that formation of anle145c-stabilized oligomers is controlled by thermodynamics, 56. In Fig. in vivo for α-synuclein with the related anle138b46. Both observations are consistent with the in vitro design principle of the DPP compounds, which is conversion of larger into smaller oligomers40. Nevertheless, the effect of anle145c on hIAPP is remarkable for two reasons.The exact pathways for fibril formation of hIAPP are not known but they are likely to involve many different potential oligomeric intermediatesFirst, we used the more water-soluble variant of the lead compound anle138b, which has not been extensively tested so far for its ability to inhibit amyloid formation. Rather surprisingly, the results in this study show that anle145c is perhaps the most effective inhibitor for hIAPP fibril formation reported thus far. Indeed, at substoichiometric concentrations of anle145c it was found that (i) fibril formation is prevented, (ii) hIAPP-induced cell death is inhibited and (iii) fibrils are converted into non-toxic anle145c-stabilized oligomers. To our knowledge this is the first time that such a high efficiency for an hIAPP inhibitor compound is reported for all three effects. Anle145c furthermore appears to act in an unprecedented way by trapping hIAPP in a thermodynamic sink in the form of non-toxic anle145c-stabilized oligomers.a priori one could not have made the assumption that DPP compounds would be effective against hIAPP toxic oligomers. This is because hIAPP oligomers have some features that are different from those of other well-studied amyloid forming proteins, such as α-synuclein57, i.e. they do not exhibit extensive secondary structure and they do not expose hydrophobic surfaces that allow them to bind hydrophobic molecules such as 1-anilinonaphtalene-8-sulphonic acid (ANS)48. The efficient inhibition of aggregation that was observed in the presence of anle145c, thus highlights the general efficiency of DPP compounds against amyloid aggregation.The second reason why the effects are remarkable, is that 59. Importantly, the higher efficiency of anle145c in cells further increases the potential of this compound as an inhibitor to prevent hIAPP islet amyloid formation and thereby combat T2DM.In cells, we observed efficient inhibition of the toxic effects of hIAPP at even smaller sub-stoichiometric ratios than those required for inhibition of hIAPP aggregation in solution. This is not uncommon, as for inhibitor compounds where comparisons were made, higher as well as lower concentrations were found to be required in cells than in solutionIn conclusion, this study adds a highly promising inhibitor, anle145c, to the existing array of potential inhibitors of hIAPP amyloid formation and it adds T2DM as one more amyloid disease that may be targeted by DPP compounds. The results thus highlight the potential of DPP-derived compounds as small molecule oligomer modulators for amyloid diseases in general.40.Human hIAPP (amino acid sequence: KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY including disulfide bond and amidated C-terminus) was obtained from Bachem AG (batch number 7001439). Thioflavin T (ThT) was obtained from Sigma Aldrich. Anle145c was supplied by the NMR-based structural biology department,  and was synthesized as described previously60. Briefly, hIAPP was dissolved in 1, 1, 1, 3, 3, 3-hexafluoro-2-propanol (HFIP) and incubated for 1 hour to eliminate any pre-existing supramolecular structures. HFIP was evaporated using nitrogen gas followed by 1 h in a vacuum desiccator. For the fluorescence, microscopy , DLS and cell viability assays, the resulting peptide film was dissolved in DMSO at 2 mM and incubated for one hour. If required, hIAPP was further diluted in DMSO to the desired concentration. For the NMR experiments, the peptide film was solubilized at a concentration of 50 µM in 10 mM phosphate buffer, pH 7.4 in D2O. For the CD experiments, the peptide film was solubilized at a concentration of 25 µM in 10 mM phosphate buffer, pH 7.4.hIAPP stock solutions were prepared as described previously6 for the NMR experiments.Stock solutions of anle145c were prepared as 10 mM solutions in DMSO, or in DMSO-d61. The kinetics of fibril formation were measured by monitoring the fluorescence intensity increase upon binding of the fluorescent dye Thioflavin T (ThT) to fibrils. A plate reader and standard 96-wells flat-bottom black microtiter plates in combination with a 430 nm excitation filter and a 535 nm emission filter were used. First 2.5 µL of an anle145c solution in DMSO at the desired concentration was added to 195 µL of a buffer containing 10 µM of ThT, 10 mM Tris-HCl, 100 mM NaCl at pH 7.4. The ThT assay was then started by adding 2.5 µL of a 0.4 mM hIAPP solution in DMSO. The microtiter plate was shaken for 10 s directly after addition of all components, but not during the measurement. All ThT assays were performed on triplicate samples on different days and using different hIAPP stock solutions. The resulting curves were analyzed using ORIGIN program and fitted to a Boltzmann sigmoidal equation, where Fi and Ft are the initial and final fluorescence values. This fitting allows the estimation of kinetic parameters such as the time for which the fluorescence reaches 50% of its maximal intensity (t1/2).Thioflavin-T binding fluorescence assays were performed using a Spectrafluor Tecan  plate reader as previously described61. hIAPP in solution was incubated in the absence and presence of anle145c under the same conditions as in the Thioflavin T assay. Aliquots (20 μL) of this mixture were placed onto glow-discharged 300-mesh carbon-coated copper grids for 2 min, then the grids were blotted and dried. Grids were negatively-stained, as described previously61, with saturated uranyl acetate (2% w/v) for 45 s, after which they were blotted and dried. Grids were examined using a Technai 12 electron microscope operating at 120 kV.TEM images were recorded using a Technai 12 electron microscope operating at 120 kV as previously described2.INS-1E rat insulinoma cells were cultured in RPMI 16% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, 2.5 mM glucose and 50 µM β-mercapto-ethanol. The human melanoma cell line MelJuSo (MJS) was cultured in RPMI 1640 (Invitrogen) containing 10% FCS (PAA laboratories), 100 U/ml penicillin, 100 µg/ml streptomycin and 2 mM L-glutamine (complete medium). All cell lines were cultivated at 37 °C and 5% COCells were seeded prior to incubation with hIAPP. hIAPP was added to the cells in the presence of varying concentrations of anle145c. The final concentration of hIAPP was 10 µM. After treatment the cells were thoroughly washed with PBS, trypsinized and transferred to 96-well plates. The cells were incubated for 10 min with DAPI (2 µg/ml) and washed three times to remove the staining solution. All samples were measured by flow cytometry using a FACS Canto II and analyzed using FlowJo V10 software.1H frequency of 500 MHz and equipped with a TCI cryoprobe (temperature 20 °C) as previously described51. Experiments were processed and analyzed with TopSpin program (Bruker). Measurements were carried out in 10 mM potassium phosphate buffer in D2O at pH 7.4. Peptide concentrations were 50 µM in the absence and in the presence of anle145c. The peptide film was dissolved in the phosphate buffer and immediately transferred in a Shigemi tube of 5 mm diameter, comprising a sample volume of 300 µL. The evolution in time of the peptide signal intensity was followed by recording one-dimensional 1H spectra until the peptide signal intensity decayed completely. Peptide 1H resonances  were then integrated and these values are plotted as a function of time, leading to a sigmoidal curve that can be fitted using R program by a Boltzmann sigmoidal equation.NMR experiments were recorded on a Bruker Avance III spectrometer operating at a DLS measurements of hIAPP in the absence and presence of varying concentrations of anle145c were performed at room temperature on a Zetasizer Nano ZS . Aliquots of 2.5 µL of an anle145c solution in DMSO at the desired concentration were added to 195 µL of a buffer containing 10 mM Tris-HCl, 100 mM NaCl at pH 7.4. Subsequently 2.5 µL of a 0.4 mM hIAPP solution in DMSO was added and the mixture was transferred to a low volume cuvette for measurements. Each measurement was an average of seven runs and took about 7 minutes to complete. The volume percentage was recorded, which describes the relative proportion of multiple components in the sample based on their mass or volume rather than based on their scattering (intensity). Processing of the data was automated through the Zetasizer software.62. hIAPP (50 µM) without and with anle145c (10 µM) was incubated for various time intervals in 10 mM Tris-HCl, 150 mM NaCl buffer solution and then deposited on freshly cleaved mica. The samples were dried with a gentle stream of nitrogen, rinsed with water, dried again with a stream of nitrogen and finally freeze-dried overnight, as described previously61. The dried samples were scanned in air with drive frequencies around 240 kHz and drive amplitudes between 15 and 379 mV. All height and amplitude images of sample regions were acquired with resolution of 5.1 Mpixels. For image analysis and processing, the software NanoScope version 5  was used.AFM measurements were performed at room temperature using scan rates between 1.0 and 1.5 Hz in the tapping mode on a MultiMode scanning probe microscope equipped with a Nano-Scope IIIa controller  using an E-scanner (scan size 15 mm-615 mm) and a MMMC cantilever holder  equipped with a silicon SPM sensor  as previously described61.CD spectra were measured on a Jasco 810 spectropolarimeter . Measurements were carried out in cells of 0.1 cm path length at room temperature in 10 mM phosphate buffer at pH 7.4 of hIAPP at different times of incubation in the presence and absence of anle145c. Measurements were taken every 0.2 nm at a scan rate of 20 nm/min. Each spectrum reported is the average of five scans. The peptide concentration was 25 µM. anle145c was added at a 5/1 molar ratio of hIAPP to anle145chIAPP, human islet amyloid polypeptide; ThT, thioflavin; RH, hydrodynamic radius; DLS, dynamic light scattering; AFM, atomic force microscopy; EM, electron microscopy. T2DM, type 2 diabetes; DPP, diphenylpyrazole.Supplementary Information


L, and MCL-1 inhibitors and discover that most cell lines depend on at least one combination for survival. We demonstrate that expression levels of BCL-2 genes predict single mimetic sensitivity, whereas EMT status predicts synergistic dependence on BCL-XL+MCL-1. Lastly, we use a CRISPR/Cas9 screen to discover that BFL-1 and BCL-w promote resistance to all tested combinations of BCL-2, BCL-XL, and MCL-1 inhibitors. Together, these results provide a roadmap for rationally targeting BCL-2 family dependencies in diverse human cancers and motivate the development of selective BFL-1 and BCL-w inhibitors to overcome intrinsic resistance to BH3 mimetics.While inhibitors of BCL-2 family proteins (BH3 mimetics) have shown promise as anti-cancer agents, the various dependencies or co-dependencies of diverse cancers on BCL-2 genes remain poorly understood. Here we develop a drug screening approach to define the sensitivity of cancer cells from ten tissue types to all possible combinations of selective BCL-2, BCL-X Dependency of diverse cancers on specific BCL-2 family members and their combinations is unknown. Here they perform drug screening and find most cell lines to be dependent on at least one combination of BCL-2 family members, and using a CRISPR screen find BCL-w and BFL-1 to mediate resistance to BH3 mimetics To date, the most well described of the anti-apoptotic BCL-2 genes are BCL-2, BCL-XL, and MCL-1, and recently, following over a decade of extensive research effort, potent and selective inhibitors of each of these proteins were developed. Much is known about the cancer types that respond well to selective BCL-2 inhibitors, and indeed the BCL-2 inhibitor venetoclax (ABT-199) is now FDA approved to treat certain leukemias such as chronic lymphocytic leukemia (CLL)3. In contrast, outside of a small number of studies in select cancer types, little is known regarding which cancers might respond well to single agent BCL-XL or MCL-1 inhibition7. Finally, to the best of our knowledge, no studies have systematically examined the dependencies of cancers on combinations of BCL-2 family proteins.The process of intrinsic apoptosis is tightly regulated by the BCL-2 family of proteins. In human cancers, the anti-apoptotic BCL-2 proteins play a critical role in protecting cells, which are often “primed” for apoptosis, from committing to irreversible cell deathL, MCL-1, and their combinations? What are the molecular features of tumors that drive these dependencies? Finally, which cancers fail to respond to BH3 mimetics, and how can this intrinsic resistance be overcome? To answer these questions, we developed a screening strategy to assess the sensitivity of cancer cell lines to all possible combinations of a selective BCL-2 inhibitor (ABT-199), a selective BCL-XL inhibitor (WEHI-539), and a selective MCL-1 inhibitor (A-1210477). Using this approach, we mapped cellular dependencies and co-dependencies on BCL-2, BCL-XL, and MCL-1 across a large number of primary and established cancer cell lines representing 10 distinct cancer types. These data provide new insights into the landscape of sensitivity to BH3 mimetics in human cancers, revealing molecular determinants of sensitivity and a role for a novel endoplasmic reticulum (ER) stress-epithelial-mesenchymal transition (EMT) axis in dictating the frequently observed synergy between BCL-XL and MCL-1 inhibitors in solid tumors. Collectively, these findings may help guide the use of BH3 mimetics as precision therapies in defined cancers.With these limitations in mind, we set out to address the following questions: What are the dependencies of diverse human cancers with respect to BCL-2, BCL-XL8. A drug panel consisting of all possible single, double, and triple agent combinations of these drugs, at these concentrations, was then constructed and assayed in cell lines after a 72 h treatment using a conventional viability assay (see Methods)  and MCL-1 (S63845) inhibitors, as well as a dual BCL-2/BCL-XL inhibitor (ABT-737), in cell lines exhibiting single agent BCL-2, BCL-XL, or MCL-1 inhibitor sensitivity, or a cell line resistant to the inhibition of all three genes  Fig. . To ensuies Fig.  6,9–11],9–11L8. 13. In both cases, data from primary cultures closely resembled those from established cell lines, suggesting that apoptotic regulatory mechanisms are largely maintained during culture. Further, the intermediate sensitivity of JH4.3 cells to BCL-XL inhibition, initially observed in vitro, was recapitulated in an in vivo xenograft model, suggesting correspondence between in vitro and in vivo sensitivities in these models , high-grade serous ovarian cancer (OV), colorectal adenocarcinoma (COAD), pancreatic ductal adenocarcinoma (PAAD), non-small cell lung cancer (LUAD), cutaneous melanoma (SKCM), liver hepatocellular carcinoma (LIHC), bladder cancer (BLCA), breast cancer (BRCA), and glioblastoma multiforme (GBM) (The Cancer Genome Atlas (TCGA) abbreviations used throughout; Fig. els Fig. .14, the majority of solid cancers did not. Exceptions to this observation were SKCM cell lines and one LIHC cell line (SNU 423), which exhibited a modest BCL-2 dependency. This finding likely explains why single agent BCL-2 inhibitors such as ABT-199 have been less efficacious in solid cancers compared to LAML. LAML cells did not depend on BCL-XL for survival but did exhibit a robust MCL-1 dependency, again consistent with recent evidence15. In solid cancer cell lines, a fraction were dependent on BCL-XL, MCL-1, or the combination of the two. In particular, we observed a specific dependence on BCL-XL in select tumor types, in particular in subsets of BLCA, SKCM, and PAAD lines. This finding was unexpected given that solid tumors have only rarely been associated with single agent sensitivity to BH3 mimetics and suggests the possibility of defining subsets of these tumors vulnerable to selective BCL-XL inhibition. We also observed MCL-1 dependence in subsets of BRCA, SKCM, and PAAD cell lines, with the sensitivity of BRCA lines to single agent MCL-1 inhibition being consistent with recent reports16. Finally, and surprisingly, we observed that 49 of 78 cell lines were sensitive (defined as a viability loss of >50%) to the combined inhibition of BCL-XL + MCL-1. Consistent with the lack of activity of single agent BCL-2 inhibition, the addition of ABT-199 to the three conditions above  did not cause a significant increase in viability loss, suggesting that BCL-2 does not significantly contribute to intrinsic BH3 mimetic resistance in solid cancers, and that BCL-2 selective inhibitors may be unlikely to improve the apoptotic responses of solid tumors to various drug therapies. Finally, we observed that cell lines from OV, COAD, and LUAD tumors were frequently insensitive to all combinations of BH3 mimetics, suggesting either lower overall degrees of apoptotic priming or different anti-apoptotic BCL-2 protein dependencies in these tumors. To distinguish between these two possibilities, we selected cell lines that were singly dependent on BCL-2, BCL-XL, or MCL-1 (single-gene), synergistically dependent on BCL-XL + MCL-1, or resistant to all tested BH3 mimetics. BH3 profiling was then performed on these cell lines using the BIM and PUMA peptides, which measure a cell’s ability to undergo apoptosis and overall BCL-2 priming, respectively  which are not inhibited by the available BH3 mimetics. In summary, the results of this large-scale profiling effort reveal that many solid tumor types are dependent upon some combination of BCL-2 family anti-apoptotic proteins, including a large subset of tumors with targetable dependencies on BCL-XL, MCL-1, or the combination, motivating the search for specific biomarkers of these vulnerabilities.Collectively, pan-cancer cell line response behaviors revealed a number of notable patterns. First, whereas many of the LAML cell lines tested exhibited some dependency on BCL-2, a finding that coheres with published literaturene SNU 42, which eL and MCL-1 dependency and synergy between BCL-XL and MCL-1. Two of the most dominant phenotypic drivers in a given cancer are oncogenic mutations (oncogene or tumor suppressor status) and tissue of origin. As such, we first sought to determine which of these two properties best predicted BCL-2 gene dependencies. To address this question, we filtered our list of 78 cell lines to only those that are in the Cancer Cell Line Encyclopedia (CCLE) and therefore can be linked to publicly available genomic annotation data. These cell lines were then grouped based on their predominant BCL-2 gene dependencies (with a 25% viability loss threshold): BCL-XL dependent, MCL-1 dependent, BCL-XL+MCL-1 co-dependent, or mostly resistant  and determined the status of each of these genes in all cell lines. To determine if tissue of origin or mutation status was a better predictor of dependency phenotype, we first performed a linear regression analysis , and the goodness-of-fit for each model  using a BCL-XL overexpression construct, then treated cells with A-1210477 or the combination of this drug with WEHI-539  were stably transduced with shRNA targeting NOXA. Knockdown of NOXA expression attenuated sensitivity to BCL-XL inhibition, protection that could be reversed with the addition of A-1210477.A key, distinguishing feature of cells from distinct tissues are their unique gene expression patterns. We and others have previously demonstrated that expression levels of BCL-2 genes often correlate, and at times even drive, dependencies on various BCL-2 genes. For example, a recent report demonstrated that expression of NOXA elicited a dependence on BCL-2 in a subset of neuroblastomasies Fig. . In agre539 Fig. . ConsistL and MCL-1 inhibitors is strongly associated with the expression levels of defined BCL-2 family members. To determine whether the expression levels of these genes have predictive value, we used a sliding scale analysis to identify threshold expression levels of BCL-XL and NOXA that best segregated tumor cell lines on the basis of MCL-1 or BCL-XL dependency, respectively. This analysis identified an expression threshold for NOXA that robustly segregated WEHI-539 responsive and non-responsive lines . Further, this analysis also identified a BCL-XL expression threshold that robustly segregated A-1210477 responsive and non-responsive lines . To independently validate this finding, we used data from a recent, large cell line chemogenomic profiling effort which determined the sensitivity of hundreds of cell lines to the dual BCL-2 and BCL-XL inhibitor ABT-263, among other agents18. The NOXA expression threshold identified above also effectively segregated sensitive and resistant lines (p < 0.0001), with lines expressing high levels of NOXA being more sensitive to ABT-263 than cell lines below the threshold, and sensitive lines exhibiting submicromolar median inhibitory concentration-50% (IC50) values  to some combination of these inhibitors. Approximately 50% of these responding lines exhibited single agent sensitivity to BCL-XL or MCL-1 inhibitors, while the remaining lines showed pronounced synergy between BCL-XL and MCL-1 inhibitors, with the combination being substantially more potent than expected on the basis of additivity alone  was performed . To further examine this relationship, we formulated “mesenchymal” and “epithelial” expression signatures (see Methods). Using these signatures, we observed that BCL-XL/MCL-1 inhibitor synergy is anti-correlated with the mesenchymal state and positively correlated with the epithelial state, both across individual cell lines  that might explain this finding.To functionally confirm these findings, we targeted E-cadherin with lentiviral shRNAs to induce EMT via the release of β-cateninrgy Fig. . Taken tL + MCL-1 inhibitor combination by either causing a decrease in sensitivity to the combination or by increasing sensitivity to the BCL-XL or MCL-1 inhibitor alone. In the cell line panel above, forcing EMT via shE-cadherin transduction failed to either decrease sensitivity to the drug combination or increase sensitivity the MCL-1 inhibitor  and in tissue-based groupings of cell lines , and probed for its expression via western blotting in 15 representative cell lines . Since BCL-XL and NOXA expression were the strongest correlates of BCL-XL/MCL-1 dependence, we first analyzed protein expression for these genes following induction of an EMT (via shE-cadherin). In PC-9 cells, E-cadherin knockdown did not affect BCL-XL protein expression, but caused a dramatic increase in NOXA protein expression , induction of ATF4 and ATF3, and ATF4/ATF3-dependent induction of NOXA expression24. Indeed, this pathway was induced in PC-9 cells transduced with multiple independent shE-cadherin hairpins, suggesting this might be the mechanism of NOXA induction  to every combination. This implies that there are additional mechanisms that protect these cell lines from apoptosis, even when BCL-2, BCL-XL, and MCL-1 are inhibited. To find additional molecular targets to sensitize these resistant cell lines, we employed a negative-selection CRISPR screen using a library of sgRNAs that target a variety of cancer-related genes, including BCL-2 genes. The innately resistant cell line TYKNU was transduced with the CRISPR library at a low multiplicity of infection of 0.3, which reduces the likelihood of a single cell receiving multiple knockouts. Screens were performed in the presence of combined BCL-XL and MCL-1 inhibition given that no additional toxicity was conferred by BCL-2 inhibition , a result which would be expected to lead to BCL-XL dependence via MCL-1 suppression. Similarly, PANC 03.27 cells are also exceptionally dependent on BCL-XL and express higher levels of NOXA compared to tissue average (PAAD). In each case, shRNA-mediated knockdown of NOXA was sufficient to reverse BCL-XL dependence, reverting these cells to a state more resembling the tissue average  and MCL-1  have recently been described27. Importantly A-1155463, A-1331852, and S63845 have all demonstrated safety and efficacy in early in vivo models, suggesting that these agents (or derivatives thereof) may someday make it to the clinic. These findings have culminated in the first FDA approved BCL-2 inhibitor (venetoclax), which has demonstrated clinical efficacy in hematological malignancies. However, analogous approaches targeting BCL-2 in solid malignancies have been far less successful, with only a handful of success stories28. Thus, there is a critical need to understand the contexts in which BH3 mimetics targeting BCL-2, BCL-XL, MCL-1, and other family members have activity, either as single agents or in defined combinations.The identification of ABT-737 as the first true BH3 mimetic initiated a paradigm shift in terms of how we think about targeting apoptotic machinery in cancer cells. In the first decade following this shift, great progress was made targeting BCL-2 in a variety of leukemias, ranging from CLL to LAML. Since the development of ABT-737, much research has focused on synthesizing new BH3 mimetics which can inhibit other BCL-2 family members, with a particular emphasis on BCL-XL dependence in the majority of tested BLCA cell lines as well as roughly 50% of PAAD cell lines. These findings demonstrate that, although rare, acute sensitivity to single agent BH3 mimetics can occur in solid cancers. Further, we discovered that the expression levels of the BCL-2 family proteins BCL-2, BCL-XL, and NOXA are associated with, and functionally regulate, sensitivity to single agent BCL-2, MCL-1, and BCL-XL inhibitors across tumors from diverse tissues of origin. These findings confirm and extend previous findings. For example, in LAML, it is appreciated that BCL-2 expression levels predict sensitivity to BCL-2 inhibition, and recent studies in breast cancer imply that BCL-XL function is critical for sensitivity to MCL-1 inhibition6. Together, the discovery that solid tumors often harbor exquisite sensitivities to single agent BH3 mimetics targeting MCL-1 or BCL-XL, and the discovery that these dependencies can be accurately predicted based on the expression of the BCL-2 family members BCL-XL and NOXA, respectively, may have direct translational implications.Here, addressing this need, we defined the landscape of BCL-2 gene dependencies in cancers derived from ten distinct tissues of origin. This effort identified both known and novel single-gene dependencies as well as combinatorial co-dependencies. Surprisingly, we observed unexpected cases of single gene dependencies across cell lines from a variety of tissues. For example, we identified a novel BCL-XL + MCL-1 co-inhibition across cell lines from multiple tissues of origin. Specifically, this combination was synergistically active in roughly 50% of solid tumor cell lines, a discovery that extends the results of recent reports identifying a BCL-XL/MCL-1 co-dependency in breast and small cell lung cancers7. To better understand the basis for this synergy, we performed GSEA analysis, coupled with functional validation experiments, ultimately identifying a signaling pathway linking EMT to PERK signaling which culminates in increased NOXA expression and functional dependence on BCL-XL. Thus, mesenchymal-like cells have increased BCL-XL dependence, whereas epithelial-like cells are characterized by synergistic co-dependence on BCL-XL and MCL-1. It is well established that EMT can promote resistance to a variety of chemotherapies and cellular stresses through the acquisition of stem cell-like properties, including resistance to apoptosis, and recent reports imply that BCL-XL may drive EMT-induced resistance to apoptosis29. Our results extend these findings by demonstrating that the process of EMT renders cells dependent on BCL-XL for their survival. As such, although cells that have undergone an EMT are more resistant to diverse chemotherapies, they exhibit increased sensitivity to BCL-XL inhibition. Finally, prior studies have established that the PERK-NOXA signaling pathway driving BCL-XL dependence can be induced by specific chemotherapies, suggesting that these agents may be broadly useful as potentiators of sensitivity to BCL-XL inhibitors30.Another striking feature of this dependency landscape is the pervasive and synergistic efficacy of BCL-XL, and MCL-1 has broadened the landscape of tumors amenable to targeting with BH3 mimetics, our findings demonstrate that some tumors remain insensitive to the combined inhibition of all three of these targets. Using an unbiased, negative-selection CRISPR screen, we identified and validated the BCL-2 family members BCL-w and BFL-1 as potent drivers of intrinsic resistance. As such, these data provide a clear rationale to develop selective, potent inhibitors of BCL-w and BFL-1 in order to further expand the applicability of BH3 mimetic therapies for solid tumors.Finally, although the growing armamentarium of drugs targeting BCL-2, BCL-X12 or at John Hopkins University31. All cell lines were grown at optimal confluency (no lower than 30% or higher than 80%) and were given fresh media every 2–3 days. For BH3 mimetics, ABT-199 (obtained from Selleckchem), WEHI-539 (obtained from ApexBio), and A-1210477 (obtained from Active Biochem) were used as BCL-2, BCL-XL, and MCL-1 inhibitors, respectively. The PERK inhibitor GSK2606414 was purchased from Tocris Bioscience and used at the indicated concentrations.Refer to Supplementary Table 32. A-1210477 is less potent than ABT-199 and WEHI-539 and thus a concentration of 10 µM was selected, which is consistent with previous reports4. A 500x stock of each of the following combinations of drugs was made at the selected concentrations: ABT-199, WEHI-539, A-1210477, ABT-199 + WEHI-539, ABT-199 + A-1210477, WEHI-539 + A-1210477, ABT-199 + WEHI-539 + A-1210477. All of these combinations yielded a final DMSO concentration of 0.2%, and therefore 0.2% DMSO was used as a vehicle control in the panel. Cell lines were then seeded in triplicate at 5000 cells per well in Greiner white-bottom 96 well plates, and allowed to adhere overnight. The next day, cells were incubated with the BH3 mimetic panel and viability was assessed via Cell Titer Glo (Promega) after a 72 h incubation in drug. This drug incubation period was selected based on previous optimization experiments in our lab. Percentage viability loss was calculated as follows: 100*[1.0 –  / )]. This turned viability loss into a positive signal ranging from 0 to 99 (with 0 indicating no apparent viability loss) and this signal was used in the various heatmap figures and bar graphs and is also referred to in the text as “dependence/dependency” scores. For calculating synergy of the BCL-XL + MCL-1 combination, we first divided all dependency scores by 100 to convert each value to a 0 to 1 scale, and used the Bliss formula: Synergy = (1- BCL-XL dependence) * (1-MCL-1 dependence) – (observed BCL-XL + MCL-1 co-dependence).Concentrations of ABT-199, WEHI-539, and A-1210477 were carefully selected to yield maximal inhibition (close to 100%) of their annotated targets without significant off-target effects. For ABT-199 and WEHI-539, 1 µM was used, and these concentrations are in line with those used by other groupsIn vivo experiment. All animal studies were performed at Duke University under an Institutional Animal Care and Use Committee (IACUC) approved protocol, and all studies adhered to the outlined guidelines on ethical usage of research animals. JH4.3 cells were dissociated in 0.25% typsin, washed twice in PBS, and then resuspended in PBS/Matrigel  solution at 2.5 × 107 cell/ml on ice. 100 µl of cell suspension were injected subcutaneously into the right flank of each 7-week-old male Athymic nu/nu mice (Duke Breeding Core). Tumors were measured twice a week using a Vernier caliper and volumes were calculated using the formula, L = longest diameter, W = shortest diameter)). When tumor size reach about 60–100 mm3, the mice were enrolled into A-1331852 treatment group or vehicle group at random. Each group contains 6–7 mice, a number expected to be sufficient to detect statistically significant differences using a one way ANOVA followed by the Student Newman Keuls test . A-1331852 was formulated in 60% Phosal 50PG, 27.5% PEG400, 10% ethanol, and 2.5% DMSO. Mice were administrated 100 µl of A-1331852 solution (prepared at 25 mg/kg) or vehicle by oral gavage every day. Endpoint of the study was determined using time to reach tumor volume of ~1000 mm3 or tumor ulceration. When tumors reached the endpoint, mice were euthanized under CO2 and tumors were harvested immediately.TP53, PIK3CA, PTEN, KRAS, EGFR, NF1, BRAF, RB1, ATM, BRCA2, and BRCA1. To investigate whether genetic mutation status or tissue of origin is a better predictor of drug effectiveness, we used the following linear modeling set up:To test if oncogene or tumor suppressor status correlated with BCL-2 gene dependency data, cell lines were first binned as either wild-type or mutant (including amplifications/deletions) for the following genes: y is an n-vector of drug sensitivity scores, X is an n × p design matrix jointly holding both cell line tissue of origin and mutation status of select oncogenes, β is the corresponding p-dimensional vector of additive effect sizes, and ɛ is normally distributed residual noise with variance σ2 and identity matrix I. For each single agent and drug combination, we compute a p-value denoting the significance of association for each tissue type and oncogene mutation of interest (See Supplementary Table R2) statistic, which assessed how well each data type fit model:1R2 statistic, the better the model fit.where https://portals.broadinstitute.org/ccle/home). We again used the linear model in Eq.  also predicts BCL-XL dependence in a larger data set, we mined NOXA expression and ABT-263  sensitivity data from a publicly available dataset18. Cell lines were split into two groups, above or below the NOXA expression threshold of 10.33, and ABT-263 IC50 values (LOG2) were plotted in box whisker plots. A t-test was performed to test for significance between the two groups (p < 0.0001)For cell lines included in the CCLE, mRNA expression data  was obtained from the CCLE download portal  version 6.0 compiled at the Broad Institute34. These collections included: (A) the H  gene sets, and (B) the C2 CP , BioCarta, KEGG, and Reactome gene sets. To identify significantly enriched gene sets, we first derived a self-curated signature of genes associated with BCL-2/BCL-XL/MCL-1 dependence using the Bayesian approximate kernel regression model35. This nonlinear framework identifies differentially expressed genes while simultaneously considering interaction effects between genes, as well as tissue specific effects between samples. In the context of the current study, we assessed drug synergy by regressing the RMA-normalized mRNA gene expression of each cell line36 onto the GI50 value of each inhibitor combination. Members of the self-curated gene signature were then determined by measuring the magnitude of the treatment effect onto the expression of each gene (posterior probabilities of association (PPA) > 0.5)37. These self-curated signature genes were passed through as input in the global test program. A gene set was differentially enriched if it had a Benjamini–Hochberg corrected p-value  below 0.05.To investigate gene set enrichment, we used the global test programL, we used a previously published V5-tagged BCL-XL ORF plasmid38. An ORF containing HcRED was used as a control. The shRNA and CRISPR sequences used are listed in Supplementary Table For overexpression of BCL-X6. Briefly, 5 µg of LCV2 vector was digested with ESP3I restriction enzyme at 37 C° for 2 h, resolved on a 1% agarose gel, and extracted using a Qiagen Gel extraction kit as per manufacturer’s protocol. sgRNAs were amplified using the NEB Phusion Hotstart Flex kit and the following PCR conditions: 98 C° for 30 s,  x 18 cycles, 72 C° for 3 min.Functional CRISPR/Cas9 constructs were generated via the Gibson assembly as describedThe following array primers were used:Array Forward = TAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGArray Reverse = ACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACAmplified inserts were then cleaned using the Axygen AxyPrep Mag PCR Clean-up kit as per manufacturer’s protocol. 100 ng of cut LCV2 vector were combined with 40 ng of each sgRNA and ligated using the Gibson Assembly protocol as per manufacturer’s protocol. The assembled sgRNA constructs were electroporated into e cloni bacteria, and spread over LB plates containing ampicillin (amp) overnight. Individual colonies were selected from these plates, inoculated in LB amp for 16 h, and the plasmid DNA was prepped using the Qiagen plasmid mini-prep protocol.6 cells were seeded in a 6 well plate and were allowed to attach for 24 h. Cells were then transduced using 0.2–0.5 mL of virus + RPMI 1640 (with 1% pen/strep and 10% FBS) to a final volume of 2 mL. Polybrene was added to a final concentration of 16 µg/mL. The plates were then centrifuged  for 1 h. Afterwards, the virus-containing media was replaced 2 mL of the cell line specific media. The next day, the media was replaced with fresh media containing 2 µg/mL puromycinmycin (puromycin) and the plates were incubated an additional 48 h in puromycin to kill non-transduced cells. A non-transduced well of cells was included to ensure adequate kill of non-transduced cells. For cells transduced was CRISPR/Cas9 constructs, the same procedure was done except that after 2 days in puromycin, cells were moved to 10 cm or 15 cm dishes and allowed to grow in puromycin for an additional 5 days. This ensures adequate time for CRISPR-mediated genetic ablation. For the experiments testing the effects of the BCL-XL ORF, NOXA shRNA, E-cadherin shRNA, BFL-1 CRISPR, BCL-w CRISPR; cells were plated in 96 well plates and treated with the BH3 mimetic panel as described in the above method section. Protein lysates were generated from each condition to provide examples of each genetic manipulation.Virus was produced using the same protocol for the ORF, shRNA, and CRISPR constructs. Briefly, 2.75 µg of plasmid construct DNA was mixed with the packaging plasmids PSPAX2 (2.75 µg) and VSVG (0.275 µg) in 17 µL of FuGene 6 and Opti-MEM (Gibco) up to a final volume of 280 µL. This mixture was incubated at room temperature for 30 min and then added drop-wise to a 10 cm dish containing 293 T cells grown to ~30% confluency. After 18 h, the media was replaced with 20 mL of harvest media (DMEM (Gibco) + 1% penicillin/streptomycin + 30% fetal bovine serum) and the harvest media was collected after 48 additional h and filtered (0.45 µM) via syringe. For transductions of ORF and shRNA virus, 0.1 × E6 6. Briefly, cell pellets were resuspended in lysis buffer, rotated at 4 C° for 10 min, and cleared of debris via 16,200 RCF centrifugation for 10 min at 4 C°. Lysates were then quantitated using a standard Bradford assay and denatured using 3x NuPage buffer. Westerns blots were run on a NuPage gradient gel. The following antibodies were used at 1:1000 dilutions, except NOXA and ATF3 which were used at 1:500: Vinculin , BCL-XL , NOXA (Santa Cruz Biotechnology #114C307), E-Cadherin , BCL-XL , PERK , p-eIF2α , ATF4 , ATF3 (Santa Cruz Biotechnology sc-188), β-actin , BCL-w , BFL-1 , SLUG . Rabbit or mouse HRP-conjugated secondary antibodies  were used at a dilution of 1:2000 and proteins were detected using standard ECL detection. To quantify relative expression of SLUG protein, we first used densitometry to quantify SLUG and actin levels from each tested cell line. Normalized SLUG expression was then compared amongst all cells lines and these data were used for the Pearson Correlation analysis.Whole-cell protein lysate generation, protein quantitation via Bradford assay, and western blotting was performed as described39. In brief, cells were digitonin permeabilized and incubated with fluorescent mitochondrial dye (JC-1) and fluorescence was analyzed over time in a 384 well plate format. Mitochondrial priming was measured based on changes in depolarization over time. Peptides from the BH3 domains of BIM, PUMA, BAD, NOXA, and HRK were used to determine the priming states of the analyzed cell lines.The BH3 profiling assay was performed as previously describedFor cell lines with mRNA expression data (CCLE cell lines), we quantified an epithelial and mesenchymal gene score for each cell line by summing the expression levels of either selected epithelial or mesenchymal markers. For epithelial markers, we used: MUC1, CDH1 (E-cadherin), EPCAM, and CLDN3. For the mesenchymal markers, we used: SNAIL, SLUG, TWIST1, TWIST2, VIM, and CDH2 (N-cadherin). These genes are all established, canonical markers of epithelial vs. mesenchymal states. These signatures were also used to calculate average signature scores for each tissue type used in this study.40. Briefly, 2 × 106 TYKNU cells were transduced with a pooled library of barcoded CRISPRs that target 398 genes important in cancer (5x sgRNA per gene plus 50 control sgRNAs). A multiplicity of infection (MOI) of 0.3 was used to reduce the chances of cells taking up more than one CRISPR construct. After selection in puromycin for ~10 days, cells were plated at >1 × 106 cells per condition (this ensures 1000x coverage of each CRISPR construct). The conditions were performed in duplicate and were either 0.2% DMSO or 1 µM WEHI-539+5 µM A-1210477. After 2 weeks of treatment, DNA was harvested from each condition and cleaned via the Axygen AxyPrep Mag PCR Clean-up kit. Two rounds of nested PCR were performed to amplify each CRISPR and to attach sample-unique barcodes. Ninety ng of DNA from each barcoded sample were pooled and sent for deep-sequencing to deconvolute the individual counts for each gene from each condition. The depletion metric (DM) for each gene was calculated based on the relative levels of the best 3 sgRNA (three-score) from each gene in the treated sample relative to the DMSO control sample. Relevant genes involved in apoptosis were highlighted as follows: red, BCL-w (BCL2L2) and green, BFL-1 (BCL2A1).Pooled screening was performed as describedSupplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Datas 6


The prognosis of stage III/IV Merkel cell carcinoma (MCC) is very poor. The Phosphatidylinositol 3-kinase p110δ specific inhibitor idelalisib has recently been reported to induce complete clinical remission in a stage IV MCC patient. Here we assessed the expression of p110δ in primary MCC and MCC cell lines including its functionality.50 compared to REH which was accompanied by a significant decrease of Akt phosphorylation. However, treating the MCC cells with the specific PI3K p110α subunit inhibitor BYL719 led to a more effective decrease of the cell viability compared to idelalisib: WaGa cells 30-fold, PeTa cells 15-fold and all other MCC cell lines 3-fold.Immunofluorescence microscopy revealed a specific cytoplasmic p110δ expression in 71.4% of the tested MCCs and in all tested MCC cell lines. Compared to the B cell leukemia cell line REH all MCC cell lines, except MKL-1, revealed a lower response towards the treatment with idelalisib. MKL-1 showed a 10-fold higher ICAlthough PI3K p110δ is expressed in the majority of MCCs and cell lines its inhibition by idelalisib alone does not suffice to effectively affect MCC cells viability. Merkel cell carcinoma (MCC) is a very aggressive virus associated non-melanoma human skin cancer –3. The Met al. [Despite its rarity the incidence of MCC has tripled in the USA and Europe . This anet al. .The activity of the PI3K-pathway has already been studied in MCC by inhibiting the p110α subunit , 13. YetHere we assessed the expression of p110δ in 21 MCC tissues and 7 MCC cell lines. The functionality of p110δ was analyzed by idelalisib treatment of MCC cell lines and compared to the p110α subunit by treatment with the p110α specific inhibitor BYL719.The expression of PI3K p110δ was analyzed in 18 primary and in three metastatic MCCs by immunofluorescence microscopy (IFM). A specific cytoplasmic staining could be observed in 13 of 18 primary MCC (72.2%) and in 2 out of 3 metastatic MCC (66.6%)  which is located downstream of PI3K. A significant decrease of AKT phosphorylation was restricted to REH and MKL-1 with increasing concentrations of idelalisib Figure .In addition, the MCC cell lines and the B-ALL cell line REH were treated with the specific PI3K p110α inhibitor BYL719. In general, all cell lines showed a higher sensitivity towards BYL719 treatment compared to the idelalisib treatment. Again, REH showed the strongest response compared to all other cell lines. The MCPyV-positive cell lines WaGa and PeTa were more sensitive for BYL719 compared to MKL-1 and MKL-2  cannot completely be resolved, because the patient described by Shiver et al. did also receive radiotherapy. Iyer and colleagues described single-fraction radiation therapy for MCCs as a convenient alternative for chemotherapy [To what extent the findings of our study add to the understanding of the recent case report of Shiver otherapy . So it motherapy .www.federa.org/sites/default/files/digital_version_first_part_code_of_conduct_in_uk_2011_12092012.pdf”). MCC have been defined by histology and immunohistochemistry for cytokeratin 20 (CK20), neural cell adhesion molecule (CD56), synaptophysin, chromogranin A and MCPyV  and have been reviewed by 2 experienced pathologists .Twenty-one formalin fixed and paraffin embedded (FFPE) MCC tissues, 18 primary and 3 metastases, were obtained from the archives of the Department of Pathology, Maastricht University Medical Center +, Maastricht, the Netherlands. All use of tissue and patient data is in agreement with the Dutch Code of Conduct for Observational Research with Personal Data (2004) and Tissue  with 10% fetal calf serum (FCS)  in an incubator at 37° C and 5% CO2.The MCPyV-positive MCC cell lines MKL-1, MKL-2, WaGa, PeTa; the MCPyV-negative MCC cell lines MCC13, MCC26 and the B-ALL cell line REH were used. All MCC cell lines were kindly provided by Jürgen Becker . REH was obtained from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures. The multiple myeloma cell line U266 was kindly provided by Department of Transplantation Immunology, Tissue Typing Laboratory from the Maastricht Medical Center, NL. As described by Ikeda and colleagues U266 is negative for the PI3K p110δ subunit , and thu2). The cells were harvested, pelleted, washed with cold PBS and resuspended in protein stabilizing buffer which consists of CHAPS Cell Extract Buffer  and phosphatase-protein inhibitor cocktail (Thermo Fisher Scientific). The proteins were isolated by freezing and thawing cycles by using liquid nitrogen. The protein concentration was determined by using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. 35–60 µg protein samples were separated by SDS-PAGE in 12% (w/v) polyacrylamide gels, transferred to nitrocellulose membranes according to the manufacturer’s instructions using Bio-Rad Mini Protean tetra systems. Followed by blocking with 5% BSA (Sigma), incubated with primary antibodies anti-PI3K p100δ A-8 1:300 (SANTA CRUZ BIOTECHNOLOGY); PI3K p100α antibody 1:1000 , AKT antibody 1:1000  and pAKT (Thr308) antibody 1:1000  and monoclonal anti-β-actin antibody clone AC-15 1:10000  diluted in blocking buffer and incubated over night at 4° C, washed and incubated with the secondary antibody Alkaline Phosphatase AffiniPure F(ab')2 Fragment Goat Anti-Mouse/Rabbit IgG + IgM (H + L) (Jackson ImmunoResearch). The blots were developed by NBT/BCIP (Thermo Fisher Scientific) incubation according to the manufacturer’s instructions. The blots were scanned using Canon CanoScan 9000F Mark II.The MCPyV-positive cell lines MKL-1, MKL-2, WaGa, PeTa and MCPyV-negative cell lines MCC13, MCC26 and the B-ALL cell line REH were grown in T25 flasks (25 cmThe expression of PI3K p110δ was assessed by immunofluorescence microscopy (IFM) in 18 primary and 3 metastatic MCCs, MCPyV-positive MCC cell lines MKL-1, MKL-2, WaGa, PeTa and MCPyV-negative MCC cell lines MCC13 and MCC26. Further the expression of PI3K p110δ was analyzed in the B cell acute lymphoblastic leukemia (B-ALL) cell line REH. All samples were formalin fixed and paraffin embedded. The FFPE sections were deparaffinized and rehydrated in xylene and ethanol baths. The antigen retrieval was performed by boiling the slides in 1 mM EDTA pH 8.0. The slides were blocked in 5% BSA TBST, incubated with the first antibody anti-PI3K p100δ antibody A-8 (SANTA CRUZ BIOTECHNOLOGY) at 4° C over-night, followed by washing and incubation with the secondary antibody Goat anti-Mouse IgG (H + L) DyLight 488 (Thermo Fischer Scientific).The nuclei of the cells were stained with DAPI mounting medium  according manufacturer’s instructions. The detection of the fluorescence stained cells was performed with the Leica microscope DM 5000 B (Leica).The cell lines MKL-1, MKL-2, WaGa, PeTa, MCC13, MCC26 and REH were treated with idelalisib  and BYL719 . Both agents were dissolved in DMSO. Following concentrations were used for the treatments: 10 nM, 100 nM, 500 nM, 1 µM, 12.5 µM, 25 µM, 50 µM and 100 µM.® RPMI 1640 medium (Life-science) with 10% fetal calf serum (FCS)  in an incubator at 37° C and 5% CO2. The effect of the drug on the cell viability was assessed by the XTT assay (Thermo Fisher Scientific) according to the protocol provided by the manufacturer. The read out of the XTT assay was done with the iMark™ Microplate Absorbance Reader (BIO-RAD).The cells were incubated in a 96 well plate  for 120 h, in GibcoTo determine the effect of idelalisib on the PI3K pathway the phosphorylation of AKT was assessed by Western blotting. Therefore, the cells were treated with 0.2% DMSO, 1 µM, 12.5 µM and 25 µM of idelalisib for 72 h.50) values were determined with Graphpad Prism 6. By using the One-Way ANOVA test the significance of the dose response curves was determined. The western blots were quantified via ImageJ [The half maximal inhibitory concentration (ICa ImageJ .The tested MCCs and MCC cell lines express PI3K p110δ. However, the MCC cell lines seem to be resistant towards treatment with the specific p110δ-inhibitor idelalisib. Our data indicate that idelalisib monotherapy will not suffice to treat MCC. In as much MCC patients might benefit of a combination of radiotherapy and idelalisib remains to be elucidated by clinical studies.


Epstein-Barr virus (EBV), which belongs to the gammaherpesvirus subfamily, is a ubiquitous tumor virus. EBV infects B cells via CD21 and is associated with a variety of B-cell neoplasms such as Burkitt lymphoma, EBV-positive diffuse large B-cell lymphoma, and posttransplant lymphoproliferative disorders [Chronic active EBV infection (CAEBV), which is defined as an EBV+ T/NK LPD in the 2017 WHO classification , is charOncotarget, Onozawa et al. report that signal transducers and activators of transcription (STAT) 3 is constitutively active in both EBV+ T/NK cell lines and peripheral blood mononuclear cells from CAEBV patients [In this issue of patients . They alet al. also demonstrate that ruxolitinib inhibits the phosphorylation of constitutively active STAT3 and induces apoptosis in an EBV-positive T-cell line. We recently reported that tofacinib, a JAK3-selective inhibitor, induces G1 cell-cycle arrest and inhibits the growth of EBV-positive T/NK lymphoma cells, in which the JAK/STAT pathway is activated [LMP1 gene, which is difficult, particularly in T/NK cell lines.STAT3 is an important signalling mediator and is constitutively activated in a variety of tumours, including lymphomas. Therefore, this mediator has been investigated as a target for the treatment of many types of cancer. Interestingly, STAT3 is activated in most EBV-associated hematological and epithelial malignancies. Latent membrane protein (LMP) 1 is an oncoprotein encoded by EBV, and constitutively activates the NF-κB, PI3K/AKT, and JNK pathways . Onozawactivated . Whetherex vivo and in vivo studies are necessary to clarify the efficacy of these drugs.Novel approaches involving molecular-targeted therapies are needed to establish the optimum treatment strategy for CAEBV. At present the only curative therapy is hematopoietic stem cell transplantation, but this has a high incidence of complications , 5. JAK


DXR) and paclitaxel (231PTR) were used. The Mammosphere-Forming Assay and aldehyde dehydrogenase (ALDH) enzyme activity assay showed an increase in BCSCs in the doxorubicin-resistant model. Moreover, the expression of some transcription factors involved in epithelial-mesenchymal transition (EMT), a process that confers BCSC characteristics, was upregulated after chemotherapy treatment. FASN inhibitors C75, (−)-Epigallocatechin 3-gallate (EGCG), and its synthetic derivatives G28, G56 and G37 were used to evaluate the effect of FASN inhibition on the BCSC-enriched population in our cell lines. G28 showed a noticeable antiproliferative effect in adherent conditions and, interestingly, a high mammosphere-forming inhibition capacity in all cell models. Our preliminary results highlight the importance of studying FASN inhibitors for the treatment of TNBC patients, especially those who progress after chemotherapy.Recent studies showed that Fatty Acid Synthase (FASN), a lipogenic enzyme overexpressed in several carcinomas, plays an important role in drug resistance. Furthermore, the enrichment of Breast Cancer Stem Cell (BCSC) features has been found in breast tumors that progressed after chemotherapy. Hence, we used the triple negative breast cancer (TNBC) cell line MDA-MB-231 (231) to evaluate the FASN and BCSC population role in resistance acquisition to chemotherapy. For this reason, parental cell line (231) and its derivatives resistant to doxorubicin (231 Additiooperties . Some trnd snail ,13.Moreover, it has recently been demonstrated that the regulation of lipid metabolism promotes BCSCs and cancer chemoresistance . Back in(−)-Epigallocatechin 3-gallate (EGCG) is a powerful antioxidant and the most abundant catechin in green tea. Its apoptotic effect leads to antiproliferative activity ,37,38,39DXR) and paclitaxel (231PTR), the most common drugs currently used in this BC subtype devoid of a validated targeted therapy.In the present study, we evaluated FASN and BCSC characteristics, i.e., mammosphere-forming capacity and ALDH1 activity, in the acquisition of chemoresistance in the TNBC model MDA-MB-231 (231). Moreover, we used the natural FASN inhibitor EGCG and its synthetic derivatives G28, G56, and G37 in comparison to C75  to targeFASN activity has demonstrated to play an important role in drug resistance through new phospholipid synthesis for membrane renovation and plasticity. It also decreases ceramide levels, inhibiting apoptosis via PARP activation ,48,49,50DXR) [PTR)  cells resistant to doxorubicin (231DXR)  and paclR) [PTR) . It has reatment A, while ent , comparable to those of 231 and 231PTR. Likewise, no morphological differences were found among the three cell models (data not shown). Since one of the BCSC population characteristics is its inherent chemoresistance [30) of cell viability for doxorubicin (70 nM) or paclitaxel (5 nM) at 48 h), and treatment duration were performed equally in both approaches. The 3--2,5-diphenyltetrazolium bromide (MTT) assay was carried out in adherent conditions to determine cell viability, whereas, in non-adherent culture, the Mammosphere-Forming Inhibition (MFIn) was used. The BCSC-enriched population showed inherent doxorubicin resistance when compared to adherent conditions for both sensitive and resistant cell lines (DXR). Nevertheless, MFIn values exhibited lower inhibition in both cell lines treated with doxorubicin. Furthermore, in non-adherent culture, a doxorubicin cytotoxic effect was significantly higher in parental cells (60.66% ± 1.17) compared to the resistant model . Similar results were found with paclitaxel treatment  and 31.24% ± 5.59 (231PTR).To determine whether there was an enrichment of this population in our resistant models, we used the Mammosphere-Forming Assay MFA; A in 231 d 231PTR B. 231 ansistance , we carrll lines C. In adhreatment D. The reTM assay. Obtained data revealed a significant increase of ALDH1 activity in the resistant counterpart 231DXR (26.52% ± 1.81) compared to parental  (br) cells from both sensitive and resistant cell lines were also increased, with no significance, after 12 and 24 h of doxorubicin treatment, reaching values of 42.15% ± 10.53 and 38.10% ± 6.71, respectively. 231DXR cells exhibited a non-significant increase of ALDH1 activity in comparison with 231 cells after 12 h treatment (36.87% ± 3.56 vs. 17.28% ± 5.68), showing similar levels at 24 h after treatment.We next measured the ALDH1 activity using the ALDEFLUOR < 0.05) E. Interebr population in paclitaxel-resistant 231 cell line, no significant differences were found between sensitive and resistant cell models (br population after 24 h of treatment in sensitive cells (p < 0.01), whereas ALDHbr population in 231PTR cells remained similar during treatment.Regarding an ALDHl models F. Paclitsnail, slug, twist, zeb1 and zeb2 was evaluated after doxorubicin or paclitaxel treatment. The basal levels of snail were 2-fold higher in 231DXR compared to 231 (p < 0.05). When doxorubicin was added, interestingly, a significant increase of snail was observed in the 231 cell line, while such a strong effect was not observed in 231DXR  but opposite results were observed in vimentin levels at the same time points  of cell viability at 48 h was used  ocompound .DXR and 231PTR), with values ranging from 45.26% ± 2.94 to 21.24% ± 5.18. EGCG cytotoxic effect was significantly diminished in non-adherent settings in all cell lines. EGCG reduced viability in adherent cells with percentages ranging from 81.54% ± 3.38 (231) to 85.43% ± 0.99 in 231PTR. Instead, MFIn values laid between 25.71% ± 4.42 (231) and 36% ± 14.70 in 231DXR.No differences were found with C75 treatment in adherent and non-adherent conditions in 231 or both resistant cell models (231DXR) and 83.50% ± 2.08 (231PTR) in adherent conditions, the same dose of G28 exhibited a significantly cytotoxic effect in suspension, with values of 98.10% ± 0.72 for 231, 94.70% ± 1.55 for 231DXR and 90.67% ± 1.60 for 231PTR.G28 displayed a noticeable cytotoxic effect in both adherent and suspension conditions. Although the reduction in viability ranged from 78.70% ± 2.50 (231) to 72.66% ± 6.76 (231DXR) and 84.66% ± 1.63 (231PTR). As a counterpart, G37 treatment resulted in similar values, concretely 75.24% ± 5.32 for sensitive 231 cells, 81.33% ± 4.04 for doxorubicin-resistant cells and 83.07% ± 1.93 for 231PTR cells. On the other hand, both compounds showed lower MFIn effect, especially G56, which exhibits decreased values of 27.56% ± 7.20 (231), 28.01% ± 3.42 (231DXR), and 28.47% ± 6.86 (231PTR), similar to those found in EGCG treatment.Regarding G56 and G37 compounds, both exhibited a strong effect in adherent conditions in all cell models. For instance, cells treated with G56 showed a reduction in viability of 88.56% ± 0.57 (231), 79.31% ± 5.18 , and exDXR and 231PTR showed increased ability to form mammospheres under drug treatment compared to 231 showing both, drug resistance and anchorage-independent growth. The ALDEFLUORTM assay indicated that ALDHbr cell population was significantly increased in the 231DXR cell line, whereas ALDHbr 231PTR cell percentage remained similar to that in the parental. Interestingly, the number of ALDHbr cells significantly increased in the parental cell line after doxorubicin treatment. Taken together, these results suggest that 231DXR cell line shows an increase in BCSC features, and that these features can be induced after doxorubicin treatment. On the other hand, despite the fact that we did not observe an obvious enrichment in BCSC characteristics in 231PTR compared to parental cells, the intrinsic BCSC features of this ML cell line were maintained.BCSCs are a low-represented population within tumors, primary and established cell lines; therefore, different methodologies have been settled to study the BCSC-enriched population with these cells in vitro. The 231 cell line is known to form vague mammospheres that can be passaged for several generations that can be passed on for several generations ,59,60,61DXR and 231PTR cell lines showed higher resistance to their respective chemotherapeutic agent in comparison with 231 in adherent or suspension condition. Additionally, and as expected, doxorubicin and paclitaxel showed more cytotoxic effects in adherent conditions compared to non-adherent ones. Interestingly, the ALDH1 activity was enhanced after doxorubicin treatment in both sensitive and resistant 231DXR cell models. This phenotype is associated with chemoresistance in patients [snail, an EMT inducer directly associated with tumor development and relapse [DXR compared to sensitive cells. Regarding paclitaxel resistance, no differences were observed in the ALDHbr population. The EMT-related transcription factor slug, linked to cancer progression and BCSC activity [PTR cells compared to 231 cell model with no changes in vimentin levels.Cell plasticity plays a key role in tumor progression, and the plastic CSC concept defines that bidirectional conversions between non-CSCs and CSCs may exist. Thus, the activation of the EMT process may lead to the CSC phenotype ,38,39. 2patients . Remarka relapse ,64,65, sactivity , was uprAlthough doxorubicin counts with undeniable advantages as an antitumor drug, it has been shown to promote stemness in murine cell lines ,68,69. RDXR cell model when treated with doxorubicin. It has been previously described in breast cancer that FASN protein levels increase as a mechanism to become resistant [FASN overexpression confers many advantages to tumor cells such as the ability to preserve a high proliferation rate, and it also plays a key role in drug resistance acquisition ,47,49. Wesistant . Gonzaleesistant . Additioesistant . Likewisesistant . FASN exesistant . In thisDXR and 231PTR (ML cell models) is not only a static drug-selected state, but also an acquired reversible phenotype that can be triggered during treatment. 231DXR presented a larger BCSC population compared to the doxorubicin sensitive 231 cell line, which was also enhanced with the presence of the chemotherapeutical agent. This might be explained by the activation of EMT induced through snail. Despite the preliminary nature of the obtained results, they provide a rationale to suppress FASN as a potential strategy for a highly proliferative neoplasia such as TNBC.In conclusion, the results presented in this work suggest that chemoresistance of 231l-glutamine (Gibco), and 1% sodium pyruvate (Gibco), 50 U/mL Pen/Strep (Linus). Cells were kept at 37 °C and 5% CO2 atmosphere. Doxorubicin-resistant cells MDA-MB-231 (231DXR) and paclitaxel-resistant cells (231PTR) were developed using a stepwise selection method. Increasing doses of doxorubicin  and paclitaxel  were added until their corresponding IC50 was reached. Briefly, cells were initially treated with a concentration of 0.1xIC50 of each drug. After 48 h, treatment medium was replaced for fresh medium. When the cells were capable of growing and reached appropriate confluence, they were treated with double the previous concentration for 48 h. The stepwise selection method was subsequently performed until the final concentration of drug was that of the parental IC50. It took around six months for each cell line. Resistance was confirmed by cell viability assay.MDA-MB-231 cell line was obtained from the American Type Culture Collection (ATCC) and was routinely grown in DMEM  supplemented with 10% FBS , 1% 50) of the MDA-MB-231 parental cells’ viability previously determined with MTT assay after 48 h [50 of paclitaxel was used , C75 (Sigma), EGCG-derivatives G28, G56 or G37 (dissolved in DMSO)) were added to each well. The synthesis of FASN inhibitors G28, G56, and G37 was performed as previously described [To elucidate inhibitory effects of chemotherapy drugs and FASN inhibitors, 231 and resistant counterparts were seeded in 96-well microplates at a cell density of 4 × 10escribed . Followiescribed . Graph p30 of parental cells calculated at 48 h) for five days. Finally, cell viability was also measured using a colorimetric MTT assay.On the other hand, cell viability inhibition experiments were performed seeding 5000 cells in adherent 6-well cell culture microplates with DMEM. Cells were incubated for 24 h to allow cell attachment and then chemotherapy drugs (doxorubicin or paclitaxel) or FASN inhibitors were added at a unique specific concentration  the Mammosphere-Forming Index (MFI) and (B) Mammosphere-Forming Inhibition (MFIn). For MFIn analysis, compounds were added at the moment of seeding. An IC30 of chemotherapy drugs and FASN inhibitors calculated at 48 h on MDA-MB-231 parental cells was used.The mammosphere-forming procedure was used to evaluate an CSC population, following previously described protocols ,56. AfteFormula: 50 and 3xIC50, respectively, obtained after 48 h of treatment. After 12 or 24 h treatment, cells were washed with PBS, and then Qiazol  was added. RNeasy mini kit (Qiagen) was used to isolate total RNA according to manufacturer instructions. RNA was reverse-transcribed into complementary DNA (cDNA) using High Capacity cDNA Archive Kit . Different gene expression levels were determined using LightCycler® 480 Real-time PCR System  with LightCycler® 480 SYBR Green I Master (Roche), following manufacturer instructions. Primers used are shown in Cells were treated with a concentration of doxorubicin or paclitaxel equal to the 0.5xICTM kit (STEMCELL Technologies) was used to determine ALDH enzyme activity, following company guidelines. Briefly, ALDH reagent (BAAA) freely diffuses into viable cells and ALDH enzyme catalyzes its conversion to BAA, which is negatively charged and retained into cells. Intracellular BAA increases fluorescence which can be analyzed through flow cytometry. As seen before for 12 and 24 h treatments, 0.5xIC50 and 3xIC50  for doxorubicin and paclitaxel, respectively, were used. After treatment, cells were detached and 2 × 105 cells per sample were collected. After washing with PBS, 500 µL of the kit buffer was added to each condition. Afterwards, 2.5 µL of the ALDH reagent was added and 250 µL of the suspension was immediately transferred to a new Eppendorf with 2.5 µL of the ALDH inhibitor N,N-diethylaminobenzaldehyde (DEAB), in order to consider background fluorescence (Aldefluorrescence . All samDXR) showed increased BCSC features in both sensitive and resistant cell models after chemotherapy treatment. We have previously recognized FASN as a possible co-target to resensitize chemoresistant cells to doxorubicin [DXR and 231PTR. However, these outcomes are still in initial states and more approaches should be performed. According to recent published research, FASN would not only play an important role in a highly proliferative neoplasia such as TNBC, but also in drug resistance acquisition and in sustaining malignancy in cancer [The study of the BCSC-enriched population in an ML TNBC cell line (MDA-MB-231) and its doxorubicin-resistant derivative (231orubicin . Moreoveorubicin . Resultsn cancer ,74,75, b


Benzothiazole (BTA) belongs to the heterocyclic class of bicyclic compounds. BTA derivatives possesses broad spectrum biological activities such as anticancer, antioxidant, anti-inflammatory, anti-tumour, antiviral, antibacterial, anti-proliferative, anti-diabetic, anti-convulsant, analgesic, anti-tubercular, antimalarial, anti-leishmanial, anti-histaminic and anti-fungal among others. The BTA scaffolds showed a crucial role in the inhibition of the metalloenzyme carbonic anhydrase (CA). In this review an extensive literature survey over the last decade discloses the role of BTA derivatives mainly as anticancer agents. Such compounds are effective against various types of cancer cell lines through a multitude of mechanisms, some of which are poorly studied or understood. The inhibition of tumour associated CAs by BTA derivatives is on the other hand better investigated and such compounds may serve as anticancer leads for the development of agents effective against hypoxic tumours. Five membered heterocyclic which incorporate oxygen, nitrogen and sulphur are found in broad spectrum therapeutic agents which have an enormous significance in drug discovery and drug development processesThe pharmacological profile of the drug used for the management of amyotrophic lateral sclerosis Riluzole  attracteThe BTA scaffold possesses a wide spectrum of biological activities such as anti-inflammatoryCancer is the most prominent, notably complex and lethal disease which became a serious concern of today’s medical science. It poses a great challenge to medical scientific community for development of drugs, medicines and procedures for safer treatment and cure of cancer disease2.2.1.1 (3–(5-fluorobenzo[d]thiazol-2-yl)phenol) and 2 (4–(5-fluorobenzo[d]thiazol-2-yl)phenol) having hydroxyl substituents on the third and fourth position of phenyl exhibited the best activity having GI50 values of 0.57 and 0.4 µM respectively against MCF-cell line as compared to BTA derivatives containing alkoxy, methyl sulphonyl and ethyl substituents on the benzothiazole (N-bis-benzothiazole and benzothiazolyl thiocarbamide derivatives and screened for cytotoxic activities against two human cell lines U-937 (human macrophage cell line), THP-1 (human leukaemia monocytic cell line) and B16-F10 (mouse melanoma cell line). The thiourea containing benzothiazole derivative 3 ), (4847.73 ± 2.39 µM)) and (34.58 ± 1.73 µM)) as compared to standard compound etoposide IC50 values (17.94 ± 0.89), (18.69 ± 0.94) and (2.16 ± 0.11 µM)) against U-937, B16-F10 and THP-1 cell lines respectivelyAiello et al. synthesised fluorinated 2-aryl benzothiazole derivatives and evaluate them for anti-tumour activities against cancer cell lines such as MDA-MB-468 (mammary gland/breast tissues derived from metastatic site) and MCF-7 cell line (human breast adenocarcinoma). The fluorinated BTA derivatives thiazole . Kumbharvative 3  demonstr4 , non-small cell lung cell lines , colon cancer cell lines , central nervous system cancer cell lines , melanoma cell lines , ovarian cancer cell lines , renal cancer cell lines , prostate cancer cell lines  and breast cancer cell lines  in comparison with reference drug 5-fluorouracil NSC 19893. The BTA scaffold 5 showed remarkable growth inhibitory activities against different human tumour cell lines such as leukaemia cell lines , non-small cell lung cell lines , colon cancer cell lines , central nervous system cancer cell lines , melanoma cell lines , ovarian cancer cell lines , renal cancer cell lines , prostate cancer cell lines  and breast cancer cell lines  as compared with reference drug 5-fluorouracil NSC 19893. The scaffolds 4 and 5 showed the best anticancer therapeutic potential due to presence of electron with drawing groups on para position of phenyl ringCaputo et al. afforded two types of five derivatives on the basis of an aryl amide and an aryl urea functionalities attached at C-2 of benzothiazole core and these scaffolds were screened against 60 human cancer cell lines. The urea moiety based fluorophenyl containing benzothiazole derivative 4  and cyan4  and A-498 (human kidney carcinoma cell line) with GI50 values of 0.542 µM and 1.02 µM respectively. This compound also possess efficacy against UO-31 and RXF 393 cell lines. The derivatives 7 and 9 because of 3,4-disubstitutedphenyl moiety, exhibited excellent anti-proliferative activities with low IG50 values of 1.85, 2.10 µM against RCC and ACHN cell lines respectively. The SAR study revealed the fact that sorafenib analogues possesses anti proliferative activity due to the presence of both urea spacer and phenyl disubstitution. Compound 8 demonstrated the highest CLogP value being the most lipophilic and potent derivative, in the low µM rangeEl-Damasy et al. synthesised the novel amide and urea based BTA series of 20 sorafenib analogues in which the pyridylamide privileged functionality was attached with an ether linkage at 6-position of the BTA ring. A selected group of 12 potent scaffolds were evaluated and appraised for anti-proliferative activities against sixty human cancer cell lines. These chlorotrifluoromethyl phenyl ureido picolinamide benzothiazoles 7 , bis-tri7 , MNK-45 (gastric cancer cell line), NCI-H226 (human lung cancer cell line), HT-29  and SK-N-SH (neuroblastoma cell line). The substituted 2-hydroxybenzylidene containing semicarbazide 10 -hydrazino moiety at the C-2 position which remarkably enhances the anti-tumour potential, whereas replacing the 4-hydroxy moiety with 4-methoxy decreased the activities against both cell linesGabr et al. obtained hydrazine derivatives by treating 2-amino-6-fluorobenzothiazole with hydrazine hydrate which was further treated with the suitable aldehydes to afford 27 different BTA Schiff base derivatives. These derivatives were screened for anti-tumour potential against Hela  and COS-7 (kidney fibroblast cancer) cell lines. The hydrazine based benzothiazole 11  exhibite12 , human lung cancer cell (H460), non-small cell lung cancer (A549) and human breast cancer (MDA-MB-231). Among these derivatives, the indole based hydrazine carboxamide scaffold 12  showed p2.2.13 (2–(1H-benzo[d]imidazol-2-ylthio)-N-(4-(benzo[d]thiazol-2-yl)-3-chlorophenyl) acetamide) (14 (N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(1-phenyl-1H-benzo[d]imidazol-2-yl-thio)-acetamide)  benzothiazole derivatives with reference to the heterocyclic substitution was benzimidazole Yurttas et al. obtained 2–(4-aminophenyl)BTA derivatives substituted with different heterocyclic rings and tested their antitumor potential against 60 human tumour cell lines. The BTA derivatives etamide)  and 14 (etamide)  showed r15 piperazin-1-yl)-2-oxoethyl)-4-chloro-benzenesulfonodithioamide) showed antiproliferative activity (CC50 = 8 ± 3 µM) against human derived DU-145 cell line (17 (N-(2–(4-(benzo[d]thiazol-2-yl) piperazin-1-yl)-2-oxoethyl)-2,5-dichloro benzenesulfonodithio-amide) demonstrated remarkable activities against several human derived cell lines such as HepG2 and DU-145  , DU-145 (human prostate cancer cell lines express androgen receptor), HepG-2 cell line (human liver cancer), WIL-2NS (Human splenic B-lymphoblastoid cells), MRC-5 (human lung fibroblast cell line), MCF-7 cell line (human breast adenocarcinoma), MT-4 (human T-cells containing an integrated HTLV-1 genome), SK-MES-1 cell line (human lung cancer) and SK-28 cell line (skin melanoma). Derivative ell line  whereas ctively) . Derivat50 values of these derivatives indicated that all compounds exhibited good potential against the aforementioned cell lines but the pyridine containing derivative 18 , MCF-7 (Breast) and HCT-116 . GIative 18  had a re2.4.in-vitro against different tumour cell lines. BTA derivatives 19 (N-(benzo[d]thiazol-2-yl)-2–(5-(1–(2-chlorophenoxy)propyl)-1,3,4-oxadiazol-2-ylthio)acetamide) and 20 (N-(benzo[d]thiazol-2-yl)-2–ethyl)-1,3,4-oxadiazol-2-ylthio) acetamide) exhibited remarkable activities against CCRF-CEM (leukaemia) cell lines. The CC50 values of compounds 19 and 20  indicated that the thiophene based acetamide benzothiazole derivatives 21 , morphol21 , HeLa, A549, HCT-116, HepG2 and SKRB-3 breast cancer cell line. The substituted bromopyridine acetamide benzothiazole derivative 29  showed p30 Kamal et al. reported the synthesis of novel phenyl pyridopyrimidinones based BTA derivatives which were examined against four different cancer cell lines such as ME-180, DU-145, MCF-7 and B-16. The pyridine containing pyrimidine benzothiazole 31  exhibite2.9.32  pathways, which regulate the equilibrium between apoptosis and cell proliferation. The SAR explained that the maximum cytotoxicity of derivative 34 against colon cancer cell line was due to the presence of methoxy group (–OCH3) in the phenylKambhare et al. synthesised the isoxazole pyrimidine based BTAs and evaluated them for anticancer activity by the MTT assay against different cell lines such as A549, Colo205, MCF-7 and U937 cell lines, in comparison with the standard drug etoposide. The pyridine containing pyrimidine derivative 34  demonstr35 , breast carcinoma (MCF-7), cervical carcinoma (HeLa), pancreatic carcinoma (MiaPaCa-2), colon carcinoma (SW 620), lung carcinoma (H 460), and diploid fibroblasts (WI 38). Among them, the pyrimidine based carbonitrile benzothiazole derivative 36  showed p2.11.37  cell line, human lung adenocarcinoma epithelial (A549) cell line, human breast adenocarcinoma (MCF-7) cell line, human colorectal adenocarcinoma (HT-29) cell line and mouse embryo fibroblast (NIH3T3) cell line. The piperidine based acetohydrazide derivative 37  showed m2.12.38  The sulphonamide scaffold based BTA 40  exhibite2.13.41 and the substituted chloromethylbenzamide benzothiazole 42 . The benzamide benzothiazole derivative azole 44  showed p45  phenyl]-2-aryloxyacetamide derivatives for cytotoxicity and anticancer activity against sixty human cancer cell lines derived from 9 neoplastic diseases, among which L (leukaemia), M (melanoma), RC  NSCLC  CC (colon cancer), OC (ovar ian cancer), BC (breast cancer), CNSC  and PC (prostate cancer). Derivatives 49 (N-(4-(benzo[d]thiazol-2-yl)-3-methoxyphenyl)-3–(4-chlorophenyl)propanamide) and 50 (N-(4-(benzo[d]thiazol-2-yl)-2-chlorophenyl)-2–(4-chlorophenylthio)acetamide)   exhibite51 . The SAR studies showed that the highest activity of compound 51 was due to the presence of three chlorine atoms in the compound as compared to other derivativesNoolvi et al. reported the synthesis of chloro substituted benzothiazole amines in order to produce isothiocyanates and thioureases. These BTA derivatives were tested for their anticancer activities. The dichlorophenyl containing chlorobenzothiazole 51  showed g52 (N-(4-methoxyphenyl)-acetamide substitutions on position 5 of the 4-thiazolidinones ring enhanced the anti-cancer potentialHavrylyuk et al. screened novel 4-thiazolidinone benzothiazole derivatives against ovarian, renal, prostate, leukaemia, melanoma, lung, colon, CNS and breast cancer cell lines. The thioxothiazolidine acetamide benzothiazole 52  showed t53  and liver cancer (HEPG2) cell lines. Among all these derivatives, nitrobenzylidene containing thiazolidine derivative 54  exhibite55  complexes containing benzothiazole derivatives and tested their anticancer activity against K-562 and KE-37 (human leukemic) cell lines. Among all these derivatives, the Ru(III) containing methylbenzothiazole 60  exhibite61 and derivative 62  cell line. Derivatives ative 62  exhibite63 and 64 benzothiazole-6-carbohydrazide 65  in Phase I/IIb clinical trials for the management of hypoxic metastatic tumours,Carbonic anhydrases  are zinc enzymes which catalyse the reversible interconversion between CO72, a CAI in clinical use for decades as lead molecule, a multitude of primary sulphonamides (e.g. compounds 73–75) as well as the secondary siulfonamide 76 were reported to act as highly efficient, frequently low nanomolar inhibitors against the tumour-associated isoforms CA IX and XIIex vivo or in vivo studies are available so far with these potent CA IX/XII inhibitors, but compounds belonging to other classes of sulphonamides were proved to possess significant antitumor effects in vivo when they act as potent inhibitors of these two CA isoforms,.Indeed, using the ethoxzolamide 4.Benzothiazole is a pharmacophore widely used in medicinal chemistry. This review points out to a growing interest in the development of lead or hybrid structures bearing the BTA moiety as antiproliferative and anticancer agents. The present work describes the potential of BTA scaffolds in the management of various types of cancers such as ovarian, prostate, central nervous system, renal, gastric, pancreatic, liver, breast and colon cancers. SAR studies reaveled that the anticancer activity of BTA scaffolds depends upon the nature of substituents present in these molecules, being multifactorial and not always easy to rationalise. The plethora of research on the anticancer profile of BTA derivatives mentioned in this review and their rationalisation based on the drug targets of these derivatives, when this was possible, may be useful for the development of novel such agents.