Relapse after chemotherapy treatment depends on the cancer initiating cells (CICs). PEDF (Pigmented Epithelium Derived Factor) is an anti-angiogenic, neurotrophic and self-renewal regulator molecule, also involved in CICs biology. Acute and chronic exposition of colon cancer cell lines to CT/CTE PEDF-derived peptides decreased drug-resistance to conventional colorectal cancer treatments, such as oxaliplatin or irinotecan. We confirmed a reduction in the irinotecan and oxaliplatin IC50 doses for all tested tumour cell lines. After xenograft transplantation, CT/CTE treatments also produced a reduction in resistance to conventional chemotherapy treatments as in culture-assays. Metastatic capacity of these treated cell lines was also depleted. The PEDF signaling pathway could be a future therapeutic tool for use as an adjuvant therapy that decreases IC50 dosis, adverse effects and treatment costs. This pathway could also be involved in an increase of the time relapse in patients, decreased tumourigenicity, and decreased capacity to produce metastasis. There are different hypotheses about the origin of tumours, none of which has yet been able to explain all cases. One such hypothesis is based on the high susceptibility of stem cells to accumulate mutations, together with the existence of epigenetic changes that are essential for the initiation and development of tumourgenic behaviour. For this reason tumour stem cells display some same general characteristics as healthy stem cells (SCs) such as quiescence, self-renewal, asymmetric cell division, cell regulation via Wnt, Notch, Sonic Hedgehog signaling (Shh), and resistance to toxic substances and drugs. All of these characteristics were postulated more than fifteen years ago –4. ThereSelf-renewal can be defined as the capacity of the cell to, at a minimum, originate a daughter cell with identical characteristics to the previous one. Consequently, stem cells can undergo multiple cell divisions maintaining their cell de-differentiation. Self-renewal is also produced by asymmetrical cell divisions of stem cells. This ability is essential for maintaining stem cells throughout the organism′s lifetime. Every stem cell must maintain an equilibrium between self- renewal and cell differentiation .PEDF (Pigment Epithelium-Derived Factor) protein has been related to the modulation of self-renewal mechanisms in stem cells , 7. PEDFPEDF exhibits various collaborative effects against tumour progression. PEDF may induce cellular differentiation and induce apoptosis in tumour cells –13. Alsoin vivo and in vitro effects of the C-terminal part of PEDF on tumoural cell lines’ growth and tumourogenicity. We have measured the IC50 of different chemotherapeutic treatments in combination with two fragments of the PEDF protein: the CT and CTE peptides . Also, we have measured the resistant population at the end of the treatment, which is an important date for comparing the effectivity of those treatments. We have used three different colorectal cancer cell lines, with different genetic expression hallmarks . A fourth cell line, HT29 was also used for comparison in some of the experiments due to the high oncogenicity of those cells. PEDF derived peptides have been used in combined treatment with conventional chemotherapy, oxaliplatin and irinotecan, both in first and second line chemotherapy for colorectal cancer patients. These two PEDF derived peptides are designed from the carboxi-terminal part of the PEDF protein. CTE is the same molecule as CT, from the C-terminal part of PEDF protein, but with a glutamic acid instead of the phosphorylable serine of this small molecule. The treatments employed in this paper were acute treatments, lasting two hours, and chronic treatments, lasting 6 weeks, with the cell culture medium. In both cases the optimum concentration was 8 nM, optimized in a previous work-group in murine neural models [Here we present the We have selected three different colorectal cancer cell lines in order to study the generic effect in multiple source colorectal tumours, and the chemotherapy used will be oxaliplatin and irinotecan, which are the most common first line chemotherapy agents used for colorectal cancer patients.The resistance to chemotherapy decreased in the cell lines  treated with PEDF derived peptides (CT and CTE). All the cell lines showed statistically significant reduction of IC50, oscillating between 20 and 70% depending on every cell line in both acute and chronic treatments.SW-480 and SW-620 cell lines showed a significant reduction, between 30 to 50% in the variables studied: IC50 and resistant population. All these parameters and the survival curves with PEDF-derived peptides are always under control survival curves Figure .In the SW-480 cell line there is a sharp 50% decrease of oxaliplatin and irinotecan IC50 value when they are combined with CT and CTE chronic or acute treatments Table . A reducp < 0.5 and n ≥ 3 for every condition)  Table . This den) Table  (Supplemn) Table . Resistap < 0.5 and n ≥ 3 for every condition)  Table . Resistan) Table .HT-29 also showed a decrease in the IC50 of the chemotherapy combined with PEDF-derived peptides. A significant response was observed in acute CT/CTE combined with oxaliplatin treatment, obtaining a decrease of 40% in the oxaliplatin IC50. Reduction of IC0 was also visible  and the In vivo xenograft assays were started with the injection of 500 and 5,000 tumoral cells in nude mice to follow the tumour growth in the mice flank. Those cells were previously exposed to chronic treatment with CT and CTE peptides during six passages/2 weeks. Controls are xenografts with the same number of untreated injected cells. The assays were performed in parallel with the three colorectal cell lines. In this type of assays we compared the growth of the tumour in the flank between control untreated cells and CT or CTE previously treated cells.In vivo assays showed a significant decrease in the tumourigenicity of tumour cells after treatment with PEDF derived peptides in DLD-1, SW-480 and SW-620 cells lines. Consequently, the observed behaviour varied depending on the origin and the molecular characteristics of every cell line. CT treatment showed less effectivity than CTE-PEDF in vivo. CTE peptide is more stable due to the negative charge in its glutamic acid (instead of the serine found in the CT peptide).Notably, SW-480 cell line assays confirmed a 25% reduction in the number of tumours after CTE treatment, and similar results with CT treatment Figure  right.SW-620 and DLD-1 cell lines exposed to PEDF-derived peptides in chronic treatment, resulted in a reduction of the developed tumour number in the same way as SW-480. We observed 50% fewer tumours as compared to the untreated cells.When taken together, the colorectal cell lines results from injections within 35 mice showed that the number of tumours was significantly lower after treatment than of the control tumours. Comparing the data of all tumour lines used, 90% of the tumours of untreated cells are observed at four weeks and only 50% of the treated xenografts developed tumours.Concerning the volume of the tumours, untreated SW-480 cells reached four times larger tumour sizes after four weeks, than those treated did in eight weeks. After 6 weeks, treated tumours were 94% smaller in size than untreated control ones. In addition, the beginning of the tumours was drastically delayed in treated cells when compared to the control’s: In the untreated control group, 90% of tumours appeared in the fourth week, while in treated groups this percentage was not reached until the eighth week Figure . Similarp < 0.05 and n ≥ 3 every tumour groups  that corresponds temporally with chemotherapy doses application, even oxaliplatin or irinotecan. This way, we investigated the mechanisms involved in the interaction between the chemotherapeutic compounds and the PEDF derived peptides. The second one, a chronic 6 week treatment, with 8 nM CT and CTE peptides, was carried out to study the effect of TICs self-renewal inhibition in metastatic transformation.The peptide concentration (8 nM) was selected based on the team’s previous experience with the effect of this peptide on stem cell populations , 7. An aRegarding the acute treatment approach, we observed a decrease from 50% to 75% in the resistance to chemotherapy. This decrease was observed in the four studied cell lines and with both chemotherapeutic agents, irinotecan and oxaliplatin. Even though acute treatments demonstrated lower efficacy than the chronic ones, the improvement of the acute treatment effects remains an essential task concerning possible future applications. In this sense, we have obtained important IC50 reductions in some of the treatments and cell lines. This promising fact would present potential advantages such as easier application, a less tedious application procedure for patients, and would be economically advantageous for the health system , 24. In Additionally, we observed a great improvement in the chronic treatments: tumour cell lines that were previously treated for 6 weeks with a constant 8 nM amount of CT and CTE peptides. The treatment application time was coordinated with the day of the cells passing process to obtain a more homogeneous response. During this assay, the main objective was to identify the role that these PEDF-derived peptides might be playing as regards cell division or signaling pathway activation related to stress induced by toxic substances , 20, 36.Here, we report the decreased resistance to chemotherapeutic agents, oxaliplatin and irinotecan, in four colorectal cancer cell lines when such chemotherapy is combined with a dose of PEDF-derived peptides (CT or CTE). Interestingly, this resistance varied depending on the chemotherapeutic agent used. Thus, we observed a major synergy between irinotecan and PEDF-derived peptides. This fact is possibly due to differences in the action mechanism of both drugs. As chemotherapeutics, oxaliplatin and irinotecan are cytotoxic compounds that affect cell division. In this sense, both irinotecan and oxaliplatin target different cellular mechanisms . TherefoConsidering the effect between PEDF peptides and the chemotherapeutic molecules used in this study, it is not possible to speak about synergism. We have tried to calculate the Combination Index to show if it was CI = 1 (additive effect) CI > 1 (antagonism) or CI < 1 (synergy). We have also performed a bibliographical study and concluded that the best methodology for this is based on the Median-Effect Equation (Chou) and the Combination Index Theorem  ,  and the In vivo tumourigenicity of colorectal cell lines decreased with the application of a dose of PEDF-derived peptides. According to the previously presented results, PEDF-derived peptides, CT and CTE, showed to be notably effective against one of the most aggressive colorectal tumour cell lines, SW480, and also against their metastatic SW620 cell line, both derived from the same patient. In this sense, we observed the effect on the chemotherapy combined treatment, showing a decrease in the resistance of cells derived from xenografts previously treated with CT/CTE . The most significant effect was observed after re-injection of treated xenograft cells. 70% of xenograft cells treated with CTE were not able to produce a new tumour (metastasis-like model). Regarding CT treatment, the effects were not so drastic, and we observed a 40% reduction in tumour growth. This is the first time that a treatment is able to modify the behaviour of cancer; modified initiating cells hindered the tumour′s re-growth, and therefore that of the metastasis animal model.The difficulty of finding an experimental model of the metastasis inhibition complicates the study of the treatments’ effects. There are few models of e assays , 46. HerTo sum up, this data suggests that PEDF-derived peptides play a potentially therapeutic role in colorectal cancer. In light of the findings reported here, it would be necessary to confirm the low toxicity and high efficacy of these peptides with additional pre-clinical and clinical trials. Moreover, it would be essential that these trials be carried out with purified CT/CTE peptides instead of conditioned medium. Nonetheless, preliminary studies indicate a similar effect in all of the cell lines when conditioned medium is replaced with purified peptides (data not shown). Furthermore, it would be interesting to evaluate the effect of PEDF-derived peptides on other types of cancer and also perform combinatorial analyses for a larger ratio of doses. The group is currently focused on elucidating the molecular mechanisms that underlay these observed effects. Tumour initiating cells are long-retaining labeling cells with asymmetric divisions . We post2 at 37° C in a humidified incubator.The human colon cancer cells DLD-1, HT-29, SW-480 and SW-620 were obtained from Mario Fraga′s laboratory and maintained in DMEN F12  supplemented with 10% fetal bovine serum , 200 nM L-glutamine  and 1% penicillin/streptomycin (Biowhittaker) in an atmosphere of 5% COn1nu, at 1 month of age and of indistinct gender were obtained from Charles River International Laboratories. Animals were housed and bred at 20–25° C, humidity of 50–60% and a 12 hours light-dark cycle. All of the animals were treated in accordance with the approval of the local ethics committee (University of Castilla–La Mancha).FOXC-terminal (CT) fragment of PEDF protein was cloned in the mammalian expression vector pcDNA 3.1 (-)-myc-His as we describe previously . The recColon cancer cell lines were treated with PEDF derived peptides (CT or CTE) at a final concentration of 8 nM (200 ng/ml) in the medium. Cells were treated in two ways. On the one hand, acute treatments were performed, that is, a single treatment with the peptides. On the other hand, chronic treatments were carried out consisting of a total of 6 treatments with the peptides for a total of fourteen days. Peptide treatment was added with culture medium in the passage time. Timing for the treatments was: two hours with 8 nM peptide exposition before chemotherapy treatment for acute treatments and six weeks with 8 nM peptide added to the culture medium for chronic ones.5,000 cells were seeded per well in 24-well plates, in a volume of 250 uL. The next day the cells were treated with increasing doses of chemotherapeutic agents and left in a humidified incubator at 37° C and 5% CO2 for 4 days. Subsequently the cells were fixed with 0.5% glutaraldehyde (Sigma) for 10 minutes. Next, the cells were stained with 0.1% crystal violet for 20 minutes. After several washes, the staining was solubilized with 10% acetic acid. Finally, a spectrophotometric reading was performed at an onsa length of 590 nm with a plate spectrophotometer . The IC50 was determined (the dose of drug necessary to eliminate 50% of the cell population), while the IC0 (the dose of drug necessary to eliminate 100% of the population) was obtained through logarithmic regressions made with the program DE.0 plus v1.0.™ Basement Membrane Matrix, BD) with a 25 gauge-needle. The growth of the tumours was monitored weekly by means of a caliper. The final tumour volume was calculated using the formula V= 2 × L1 × L2 × π/6. Tumours were mechanically and chemically disintegrated with EDTA at 37° C. Later they were washed with PBS and seeded in DMEN F12 medium  supplemented with 200 nM L-glutamine , 1% penicillin/streptomycin (Biowhittaker), 0,5% EGF and 0,04% FGF in an atmosphere of 5% CO2 at 37° C in a humidified incubator.Untreated cells (control) and cells treated with peptides derived from PEDF suspended in PBS were injected subcutaneously on both flanks of immunocompromised mice. The injections were of 5000 or 500 cells, in a final volume of 200 ul of a 1: 1 dilution of matrigel (BD Matrigelhttp://www.combosyn.com/) from Combosyn Inc. company. Also we have analyzed the results with the Combenefit software, also indicated to analyze synergy between molecules (https://www.cruk.cam.ac.uk/research-groups/jodrell-group/combenefit).To analyze the interaction between molecules we have used the software Drug Combination Computer Simulation CompuSyn (U test or the Chi-square test when necessary. The data is expressed as the mean plus the standard error (+SE). The obtained results are considered statistically significant when p < 0.05(*), p < 0.01(**) and p < 0.005(***).The statistical analysis was carried out using a Mann–whitney


The title compounds were less toxic to PBMC, since IC50 was 1.5 to eighteen times higher (IC50 = 5.51 to 17.61 µg·mL−1) than values shown by tumour cell lines. The mechanism of cell growth inhibition and structure–activity relationships remains as a target for future investigations.The cytotoxicity of a series of aminonaphthoquinones resulting from the reaction of suitable aminoacids with 1,4-naphthoquinone was assayed against SF-295 (glioblastoma), MDAMB-435 (breast), HCT-8 (colon), HCT-116 (colon), HL-60 (leukemia), OVCAR-8 (ovarian), NCI-H358M  and PC3-M (prostate) cancer cells and also against PBMC . The results demonstrated that all the synthetic aminonaphthoquinones had relevant cytotoxic activity against all human cancer lines used in this experiment. Five of the compounds showed high cytotoxicity and selectivity against all cancer cell lines tested (IC Approximately 518,510 new cases of cancer were estimated for Brazil between 2012 and 2013, including cases of non-melanoma skin cancer, which is the most common type for both men and women , followed by prostate , female breast , colon and rectum , lung , stomach  and cervix   propanoate (9). Obtained as an orange solid in 64% yield from ethyl L-alanine ester after 2 h reaction, mp 65–68 °C  8.01 , 7.99 , 7.69 , 7.59 , 6.30 , 5.62 , 4.18 , 4.04 , 1.50 , 1.22 ; 13C-NMR  183.1, 181.3, 171.5, 146.5, 134.6, 133.1, 132.1, 130.3, 126.3, 126.0, 101.8, 61.9, 50.4, 17.4, 14.0. Anal. Calcd. for C15H15NO4: C, 65.92; H, 5.53; N, 5.13. Found: C, 65.32; H, 5.80; N, 4.89.ot found ); IR butanoate (10). Obtained as an orange solid in 64% yield from ethyl 4-aminobutanoate ester after 2 h reaction, mp 120–122 °C  8.05 , 8.01 , 7.69 , 7.59 , 6.08 , 5.73 , 4.12 , 3.24 , 2.41 , 1.99 , 1.23 ; 13C-NMR  182.8, 181.6, 172.8, 148.0, 134.7, 133.4, 132.0, 130.3, 126.3, 126.1, 100.7, 60.7, 42.0, 31.6, 17.4, 23.1, 14.1. Anal. Calcd. for C15H15NO4: C, 65.92; H, 5.53; N, 5.13. Found: C, 65.71; H, 6.24; N, 4.77.ot found ); IR butanoic acid (11). To a mixture of LiOH (30 mg), THF (2 mL) and water (0.5 mL) was added the ester 10a (100 mg). The mixture was stirred at room temperature and inspected by TLC until complete disappearance of the ester 10 (after 2 h reaction). The mixture was filtered under vacuum, acidified with 10% HCl and an amorphous orange solid filtered off in 80% yield, mp 210–212 ° C  12.17 , 7.95 , 7.93 , 7.80 , 7.67 , 5.7 , 3.20 , 2.30 , 1.78 ; 13C-NMR : 181.5, 181.3, 174.3, 148.6, 134.8, 133.2, 132.1, 130.4, 125.8, 125.3, 99.3, 41.2, 30.6, 22.6. Anal. Calcd. for C16H17NO4 × H2O: C, 62.94; H, 6.27; N, 4.59. Found: C, 61.14; H, 5.63; N, 4.73.0–211 °C ,26); IR in vitro. This analysis is part of an initial screening for determination of the potential antitumor of these samples. The human cancer cell strains used were SF-295 (glioblastoma-human), MDAMB-435 (melanoma), HCT-8 (colon), HCT-116 (colon), HL-60 (leukemia), OVCAR-8 (ovarian cancer), NCI-H358M  and PC3-M (highly metastatic prostate cancer cell line). All cancer cells were provided by the National Cancer Institute  and were cultured in RPMI 1640 supplemented with 10% fetal bovine serum and 1% antibiotics, maintained at 37 °C and atmosphere containing 5% CO2. The peripheral blood mononuclear cells (PBMC) were isolated by gradient of density (Ficoll-Hypaque) and cultivated in RPMI 1640 medium, supplemented with bovine foetal serum (20%), antibiotic (1%) and phytohemagglutinin (4%). The cells were maintained at 37 °C and atmosphere containing 5% CO2.This experiment evaluated the cytotoxicity of aminonaphthoquinones (ANPQs) against human cancer cells, −1. In order to determine the IC50 the substances were tested at increasing concentrations in serial dilutions (5–0.010 µg·mL−1). Doxorubicin (5–0.0010 µg·mL−1) was used as positive control only for the determination of IC50.The different samples of ANPQs were diluted in pure sterile DMSO. The substances were tested at a concentration of 5 µg·mLAnalysis of cytotoxicity by the MTT method has been used in the screening program at the National Cancer Institute of the United States (NCI), which tests over 10,000 samples per year . It is a6 cell mL−1 to OVCAR-8, NCI-H358M, PC3-M, SF-295 and MDA-MB435 strains, 0.7 × 105 cell mL−1 to HCT-8 and HCT-116 strains, 0.3 × 106 to HL-60 strain, 1.0 × 106 cell mL−1 to PBMC. The plates were incubated for 72 h at 5% CO2 at 37 °C. After, they were centrifuged and the supernatant removed. Then the solution of MTT  was added, and the plates were incubated for 3 h. The absorbance was read after dissolving the precipitate with 150 µL of pure DMSO in a spectrophotometer plate at 595 nm.The cells were plated at a concentration of 0.1 × 10The experiments were analyzed by mean ± standard deviation of the mean (SDM) of the percentage of cell growth inhibition using GraphPad Prism. 2, 9, 6, 5 and 7) showed high cytotoxic activity and selectivity against the eight human cancer cell lines tested. Furthermore, the aminonaphthoquinones in this study were less toxic to peripheral blood mononuclear cells, since the IC50s were 1.5 to eighteen times higher than values shown by tumor cell lines. The mechanism of cell growth inhibition and structure–activity relationship of the compounds remains the target of future investigations.Our findings have demonstrated the cytotoxic potential against human cancer lines of a series of synthetic aminoacids conjugated with a naphthoquinone nucleus. As shown by various tumor cell lines, only five samples (


Personalized cancer therapy relies on identifying patient subsets that benefit from a therapeutic intervention and suggest alternative regimens for those who don’t. A new data integrative approach, based on graphical models, was applied on our multi-modal –omics, and clinical data cohort of metastatic melanoma patients. We found that response to chemotherapy is directly linked to ten gene expression, four methylation variables and PARP1 SNP rs1805407. PARP1 is a DNA repair gene critical for chemotherapy response and for which FDA-approved inhibitors are clinically available (olaparib). We demonstrated that two PARP inhibitors (ABT-888 and olaparib) make SNP carrier cancer cells of various histologic subtypes more sensitive to alkylating agents, but they have no effect in wild-type cells. Furthermore, PARP1 inhibitors act synergistically with chemotherapy in SNP carrier cells , but they are additive at best in wild-type cancer cells. Taken together, our results suggest that the combination of chemotherapy and PARP1 inhibition may benefit the carriers of rs1805407 in the future and may be used in personalized therapy strategies to select patients that are more likely to respond to PARP inhibitors. Advances in cancer management have improved the overall outlook of patients with metastatic malignancies but chemotherapy remains a mainstay of treatment for most common cancers. Virtually all patients develop resistance to chemotherapy after prolonged exposure given the first order kinetics of cytotoxics that generally cannot eradicate cancer. Understanding the mechanisms of this resistance presents new opportunities to improve the therapeutic index of cytotoxic agents and identify novel drug targets.1. Given the critical role of PARP1 in base excision repair, PARP inhibition emerged as a therapeutic target and early studies demonstrated dramatic potentiation of chemotherapeutic agents in the presence of PARP inhibition3. Recent evidence indicates that, in addition to the catalytic inhibition of PARP activity, PARP inhibitors (PARPi) induce cytotoxic PARP-DNA complexes through PARP “trapping” that augment the cytotoxicity of alkylating agents. It is therefore of utmost importance to identify molecular features that act not only as biomarkers for patient stratification but also offer insights into the mechanisms of resistance to chemotherapy. Metastatic melanoma remains an excellent model for chemotherapy resistance given its refractory nature, despite the fact that current management of metastatic melanoma is mostly based on non-chemotherapy based strategies .A large proportion of cytotoxic agents exert their effect through DNA damage. Thus, DNA repair pathways constitute cells’ main resistance mechanisms and potential drug targets. Base excision repair, a predominant pathway for single strand break (SSB) damage repair, utilizes a family of related enzymes termed poly-(ADP-ribose) polymerases (PARP), which are activated by DNA damageCausalMGM6, in high-throughput data from a cohort of metastatic melanoma patients on chemotherapy. We identified a number of features directly linked to chemotherapy response, including a SNP in the PARP1 gene that is highly predictive of resistance to chemotherapy. Subsequently, in vitro studies investigated the impact of this PARP1 variant on PARPi sensitivity and demonstrated its utility as a predictive biomarker. Given the role of PARP1 in DNA repair, we propose this SNP as a characteristic biomarker for PARPi sensitivity to guide patient selection for chemotherapy treatment alone or in combination with PARPi.In this study, we used a probabilistic graphical method we have developed, Using a retrospective cohort study design Table , we eval7.Total RNA was isolated from melanoma tissues using the PerfectPure RNA Tissue kit . RNA was quantified using Ribogreen RNA quantitation Kit  and its quality was evaluated by RNA Integrity Number using the Agilent Bioanalyzer. Whole genome gene expression analysis was carried out using the Illumina HT-12 Expression BeadChip  which targets > 25,000 genes  derived from the RefSeq  and UniGene databasesDNA was isolated from melanoma tissues using ArchivePure DNA Cell/Tissue kit . DNA samples (0.5 μg) were treated with sodium bisulphite using the EZ DNA methylation Gold kit , and bisulphite-treated DNA was applied to an Illumina Infinium HumanMethylation27 BeadChip  for DNA methylation profiling. This microarray permits the quantitative measurement of DNA methylation for 27,578 CpG dinucleotides spanning 14,495 genes. Methylation status of the interrogated CpG sites was determined by comparing β-values, the ratio of the fluorescent signal from the methylated allele to the sum from the fluorescent signals of both methylated and unmethylated alleles.2 ≥ 0.8). We selected ~6 SNPs per gene (where possible) to complete the 384 SNP Illumina design  minor allele frequency (MAF) <10%; (2) screening using the Illumina Assay Design Tool  to ensure SNPs can be incorporated in an optimal custom array design. The remaining SNPs of each gene were in strong LD . Each reaction contained 12.5 µL TaqMan Universal PCR Master Mix, 0.625 µL TaqMan SNP Genotyping Assay, 10.875 µL distilled water and 1 µL gDNA (10 ng/µL), with a final reaction volume of 25 µL. Positive control samples for the three possible genotypes were included with each genotyping reaction. In addition, at least two no template (negative) controls were included.Genomic DNA was extracted from the melanoma tumor or the full panel of cancer cell lines using QIAamp DNA Mini kit  and subjected to SNP Genotyping using a Taqman SNP Genotyping assay  , colon , breast , ovarian  and lung . HT-29, HCT-116 and H-522 cell lines were generously provided by Dr. John Schmitz ; Dr. Jian Yu (LoVo and SW620); Dr. Vera Levina ; Nupur Gangopadhyay ; Dr. Lisa Butterfield ; Dr. John Kirkwood (FEMX); Dr. Anda Vlad (Magee Woman Cancer Institute) (A2780); Dr. Hassane Zarour (SK-Mel-2); WM-115, A375, WM-852 and SK-MEL-31 were purchased from American Type Culture Collection (ATCC). All cell lines were cultured according to their recommendations (in RPMI-1640 or DMEM medium supplemented with 10% FBS) without antibiotics and routinely tested for Mycoplasma. Cell lines were grown in monolayer at 37 °C in a humidified atmosphere containing 5% CO50 values were defined as the concentrations of drug(s) that inhibited growth by 50% relative to controls.Cytotoxicity was assessed using the MTT colorimetric dye reduction assay, according to the manufacturer’s instructions (TACS® MTT Cell Proliferation Assay (MTT-CPA) from Trevigen, Inc.). Briefly, cells were seeded in triplicates in 96 wells at appropriate cell density , in their respective regular culture medium. After 24h, medium was aspirated and cells were incubated with increasing concentrations of test agents for 72h at 37 °C. The MTT Reagent was subsequently added to each well and the plates were incubated for another 4h at 37 °C in the dark. In each well, 100 µl of Detergent Reagent was then added and incubated at 37 °C until complete solubilization of formazan crystals. Cell viability was measured by reading the absorbance at 570 nm using a Safire 2 microplate reader . All values were normalized to vehicle treated control wells. The values obtained for each of the triplicates were averaged, and ICRPMI 1640 and Dulbecco’s modified Eagle medium (DMEM) with L-Glutamine, cell culture media were purchased from Cellgro . Heat-inactivated Foundation Fetal bovine serum (FBS) was purchased from Gemini bio-products. The PARPi veliparib  used in the cell proliferation assays was generously provided by Dr. Jan Beumer . The ABT-888 used in the drug interaction assays was purchased from Selleck Chemicals LLC . ABT-888 and olaparib were dissolved in dimethyl sulfoxide (DMSO) to a stock solution of 100 mM. Working solutions were then prepared in PBS for subsequent serial dilutions. The final concentration of DMSO in drug-treated cultures was always less than 0.5% (v/v) and did not contribute to cytotoxicity (data not shown). MMS, an alkylating agent that results in DNA damage at the same bases as dacarbazine and temozolomide and that is used extensively as an alternative in the laboratory setting, was purchased from Sigma.50) values of MMS alone or in combination with PARPi were calculated by curve fitting in Graph Pad Prism . IC50 values from three independent experiments were averaged and expressed as mean ± standard deviation (S.D.). A chemopotentiation factor was defined as the ratio between IC50s of MMS in the presence or absence of PARPi. Cells were classified as “resistant” if their potentiation factor (ratio) was less than 1, and “sensitive” if the ratio was >2. For each cell line, p-values were calculated using the Student t-test  to compare the IC50 means of each treatment group .The cytotoxic effects of the alkylating agent MMS, alone or in combination with PARP inhibitors veliparib (ABT-888) or olaparib were assessed by MTT assay (see above). Cells were treated with increasing concentrations of MMS either alone (0 to 1 mM) or in the presence of a fixed concentration of PARPi  for 72h at 37 °C. Data were normalized to vehicle alone (0.5% DMSO) for MMS treatments, or 10 nM ABT-888 or 5 nM olaparib, for combination treatments. Half-maximal inhibitory concentration  and fixed ratio combinations thereof based on equipotency of the two test agents. Five two-fold dilutions ranging from one fourth of the IC50 to four times the IC50 of each drug in combination plus a control were tested in three independent experiments with duplicate samples. MTT data were then analyzed using the Compusyn Software , which provides a convenient tool to perform median effect analysis as described by Chou and Talalay8. The program calculates a combination index (CI) for each actual data point from the combination curves that is used to identify synergistic, additive, and antagonistic drug interactions. The program also calculates predicted CI values for every effect level (affected fraction or Fa), which when plotted as a function of Fa serve to visualize and quantify the effects of drug combinations over the entire dose range. CI values of 1, <1, and >1 represent additivity, synergism, or antagonism, respectively. Predicted CI values of three independent experiments were averaged and analyzed by one-sample Student’s t-test for significant deviation from additivity .Interactions between PARPi (ABT-888 or olaparib) and MMS were investigated by the Chou and Talalay median effect principleMixed Graphical Models (MGM) we refer to graphical models that are learned over variables of mixed type, i.e., continuous and discrete variables. We used CausalMGM, an algorithm we recently developed6 and used for biomarker discovery9, to learn a directed graph over the variables in our dataset, which consisted of continuous  and discrete (single nucleotide variants (SNPs), response to TMZ treatment) variables. The resulting directed graph selected those variables that are directly connected to response to TMZ treatment. We used subsampling to identify stable edges as we describe previously4. This is standard procedure, since the number of independence tests required is exponential to the number of variables.With the term response” clinical variable, a binary variable indicating response/no response to TMZ treatment dichotomized at presence of a response or stability of disease at 4 months of therapy. The 1,000 most correlated features in the input dataset included 557 mRNA expression probes, 425 methylation probes, 14 miRNA probes and 4 SNPs. BRAF mutation status was also included in the input variables to see if it had an effect on any of the features linked to response, although its direct correlation with it in this dataset was poor (R2 = 0.025). The largest interconnected output network included 20 features directly connected to the response variable in our initial (undirected) learned network  correlated features with response, as other (indirect) association yield higher pairwise correlation values. ID2 expression is also directly linked to response. ID2 is known to induce growth and proliferation in squamous cell carcinoma13. No miRNA was found to be directly linked to TMZ response in our dataset.Our dataset consisted of gene and microRNA expression, DNA methylation, SNP data from a selected panel and the clinical variable “response to TMZ”. We used CausalMGM to learn a network over the top 1000 features most correlated (pairwise) with “ork Fig. . We emph it Fig. , but alsresponse. We found this SNP to be an excellent predictor of worse outcome since all 21 patients that had the SNP (C/T or C/C) showed no response to TMZ treatment . Because of the strong direct association, the role that PARP1 plays in repairing TMZ mediated DNA damage, and the availability of PARP inhibitors, we decided to further investigate this finding. rs1805407 is located on the 2nd intron of PARP1, ~4 Kbp downstream of the PARP1 transcription start site and 35 bp downstream of the 3’ splice site of exon 2. Since this SNP does not appear to have any functional or regulatory role, we used SNAP14 to find other SNPs in strong linkage disequilibrium (LD) with it. The CEU population panel of 1000 Genomes pilot 1 contained 51 variants in perfect LD (R2 = 1)  and is annotated as a ‘Splice region variant’ because it is located 5 bp upstream from the splice site between intron 2 and exon 3. Finally, we found a strong dependence between rs1805407 and two other SNPs that have been previously associated with melanoma susceptibility: rs3219090 22. Due to high LD values with rs1805407 none of the above SNPs was included in our dataset.Another SNP (rs1805405) is also in perfect LD with rs1805407  vs WT (T/T). The Affymetrix 500k SNP arrays used by Cell Miner did not include rs1805407, so we used the k-nearest neighbors method with three of the 51 perfectly correlated variants with probes in the array  to infer the rs1805407 genotype in each cell line. Analysis of the IC50 values in cell lines predicted to have at least one allele of rs1805407  vs wild type  showed statistically significant resistance or sensitivity to four compounds, three of which are alkylating agents with action similar to TMZ (TMZ is not included in the NCI-60 dataset)  were resistant .Literature search identified 15 cell lines (Supplementary Table ). MMS was used as alkylating agent and ABT-888 was used to inhibit PARP activity. All four SNP cell lines were found to be significantly more sensitive to the combination treatment in agreement with our hypothesis; while in all five WT cell lines the combination treatment had no potentiation effect : five WT (T/T) and four C/T for SNP rs1805407  µM and for MMS + olaparib was 39.22 (±8.80) µM . By comparison, the corresponding values for SW620 (WT) cells were not significant: 342.71 (±68.67) μM with MMS only and 357 (±55.57) μM with MMS + olaparib (5 nM).  (Supplementary Table We extended those results to olaparib treatment of two cell lines: A2780 (SNP) and SW620 (WT); and we observed similarly significant potentiation of MMS cytotoxicity with the addition of olaparib in A2780 Fig. . Specifi50 value (drug combination treatment). Cell survival was assessed by MTT assay. We then determined the Chou-Talalay combination index (CI)8 of ABT-888 with MMS in four established cell lines: two with the variant C/T  and two WT T/T . Although CI is not a statistical measure, it nevertheless provides insight into whether the effect of both agents is additive (CI = 1), synergistic (CI < 1), or antagonistic (CI > 1). Both SNP carrier cell lines  exhibited strong and moderate effect, respectively across different concentrations  and PARPi (ABT-888) on cell lines with different rs1805407 genotypes. Exponentially dividing cells were exposed for 72h to increasing concentrations of ABT-888 (0–500 µM) or MMS (0–1 mM) (single drug treatment) or combined at a fixed ratio based on their corresponding ICns Table . In WT cns Table .Table 2AApplication of different therapies in well-defined subgroups of patients is an important step towards cancer precision medicine. A probabilistic graphical analytic framework identified ten gene expression and four DNA methylation variables that were directly linked to TMZ response that provided useful information about potential mechanisms. We also discovered a novel biomarker (PARP1 SNP rs1805407) of potentially high impact in stratifying patients in chemotherapy.50 values, two of which were DNA damage agents. We found the WT cell lines to be more sensitive to these drugs. Follow-up in vitro experiments in cells from different cancer histologies showed that the sensitivity of SNP carriers to alkylating agent is increased after PARP inhibition. Furthermore, we showed that in SNP cell lines the combination treatment was synergistic, while in WT cell lines was at best additive or even antagonistic. We also note that this effect was independent of BRCA1 mutation since none of the tested cell lines carried this mutation. Since this SNP has high prevalence  it has the potential of transforming the current chemotherapeutic strategies. SNP rs1805407 can be potentially used in the future as companion diagnostic marker for selecting the patients that are more likely to benefit from chemotherapy. Adequately powered future validation studies will test the clinical application of this SNP as response biomarker.All patients carrying this mutation responded poorly to chemotherapy in our cohort. Since chemotherapy induces DNA damage and PARP1 is a key factor in DNA repair, the previous results led to the hypothesis that rs1805407 may be associated with improved DNA repair, which results in poor efficiency of the drug. Comparison of drug sensitivity values in WT and SNP carrier cell lines (NCI-60) showed four drugs with statistically significant differences in IC35. Extensive autoPARylation of PARP1 results in dissociation from DNA, which is required for DNA repair completion36. It has been proposed that PARP1 inhibition acts on the autoPARylation stage, thus preventing PARP1 dissociation from the DNA (“PARP1-trapping”)38. PARP-DNA complexes are themselves cytotoxic and may underlie the differential efficacy of clinically relevant PARPi’s, which differ markedly in their PARP trapping potency24. Based on our findings we postulated that SNP rs1805407 affects PARP1 activity during treatment with alkylating agents. We found that SNP cell lines were sensitive to combination therapy with ABT-888, while WT in general were not. We subsequently conducted experiments with the FDA approved PARPi olaparib in A2780 (SNP carrier) and SW6220 (WT) cells and the results were more profound than with ABT-888. These are very important results as SNP rs1805407 can be potentially used in the future to decide whether a patient will receive a combination therapy or not.The mechanism of action of this SNP is currently unknown and is beyond the scope of this paper. However, one can postulate a possible scenario, based on publicly available data and what is known in the literature about PARP1 action and inhibition. PARP1 acts as a “molecular sensor” to identify DNA single strand breaks. Upon binding, PARP-1 undergoes autoPARylation and promotes PARylation of chromatin proteins, which recruit DNA repair factorsIn summary, we showed that (1) SNP carrying metastatic melanoma patients have poor response to chemotherapy; and (2) SNP carrying cells have increased sensitivity to combination treatment . Both these results are consistent with the theory that the identified SNP rs1805407 is associated with higher PARP1 expression. In this case, cancer cells in SNP carrying patients will repair the DNA damage, caused by chemotherapy, more efficiently. At the same time, inhibition of PARP-1 in SNP carrying cells can lead to higher number of PARP-DNA complexes, which will increase cytotoxicity. Supportive of the hypothesis of increased PARP1 expression in SNP carrying cells is the fact that rs1805407 is in perfect linkage disequilibrium with two PARP1 promoter SNPs (rs2077197 and rs6665208), which overlap ChIP peaks of cancer transcription factors. Based on GTEx data, rs1805407 is indeed an eQTL for PARP1.41. However, its overexpression can lead to inhibition of DNA repair42. Also, the short isoform uniquely maintains the auto-modification domain, which is most critical to PARP trapping. Experiments on measuring PARP1 trapping cannot be conducted in the context of this study, because cell lines have drastically different baseline response to alkylating agents  and -in addition- PARP1 is induced by alkylating agents (presumably at different levels in different cell lines). A future detailed analysis in cell lines or mouse models will shed more light in the mechanisms in which SNP rs1805407 may affect chemotherapy response.In addition, we note that SNP rs1805405, which is in perfect linkage disequilibrium with our rs1805407, causes a mis-splicing event leading to increased expression of the shorter PARP1 form, PARP1-003. PARP1-003 includes only exons 1 and 2, thus coding only for the first zinc finger (ZF1) of PARP1. In PARP1 protein, ZF1 is responsible for recognition of DNA lesions and for initiation of DNA repairin vitro and in a clinical setting.We also found that patients with one or two alleles of rs1805407 in TCGA have significantly increased relative abundance of isoform PARP1-003 vs PARP1-001 (the full length mRNA), but the difference was small in absolute numbers. However, TCGA data are generated from pre-treatment samples, generally from primary tumors, and in melanoma predominantly from metastatic sites . PARP1 expression is increased by DNA damage (treatment). The presence of SNP rs1805405 may affect the splicing rates and relative abundances of PARP1 isoforms, thus altering their relative protein levels. This is relevant in patients, especially knowing that TMZ is utilized in regimens that span either 5 days or 21 days out of 28-day cycles. Since the focus of this paper is in the discovery of an important biomarker for chemotherapy, the exact mechanism of action was not fully elucidated. The above stated hypotheses can guide future experiments The accurate prediction of therapy outcomes based on the molecular characteristics of tumors may alter the current landscape of cancer therapy given that immunotherapy results in substantial objective responses in some patients only. One can therefore expect that the accurate patient stratification into therapy-response categories may allow the overall percentage of disease control to increase through the current therapeutic armamentarium. In addition, insights into the molecular mechanism that characterizes a therapy-resistant phenotype may usher in new strategies to overcome therapy resistance.Supplementary Information


SIRT2 expression increases in acute myeloid leukemia blasts. Downregulation of SIRT2 using siRNA causes apoptosis of HeLa cells. Therefore, selective inhibitors of SIRT2 are candidate therapeutic agents for cancer. Adult T-cell leukemia/lymphoma (ATL) is a T-cell malignancy that has a poor prognosis and develops after long-term infection with human T-cell leukemia virus (HTLV)-1. Sirtuin 1 inhibition has been shown to induce apoptosis and autophagy in HTLV-1-infected cell lines, whereas the effects of SIRT2 inhibition alone have not been elucidated.Sirtuin 2 (SIRT2) is a member of the sirtuin family, nicotinamide adenine dinucleotideWe assessed the efficacy of our small molecule selective SIRT2 inhibitors NCO-90/141 to induce leukemic cell death. Cell viability was examined using the cell proliferation reagent Cell Count Reagent SF. Apoptotic cells were detected by annexin V-FITC and terminal deoxynucleotidyl transferase dUTP nick end labeling assays by flow cytometry. Caspase activity was detected using an APOPCYTO Intracellular Caspase Activity Detection Kit. The presence of autophagic vacuoles was assessed using a Cyto-ID Autophagy Detection Kit.Our novel small molecule SIRT2-specific inhibitors NCO-90/141 inhibited cell growth of leukemic cell lines including HTLV-1-transformed T-cells. NCO-90/141 induced apoptosis via caspase activation and mitochondrial superoxide generation in leukemic cell lines. However, a caspase inhibitor did not prevent this caspase-associated cell death. Interestingly, NCO-90/141 increased the LC3-II level together with autophagosome accumulation, indicating autophagic cell death. Thus, NCO-90/141 simultaneously caused apoptosis and autophagy.These results suggest that NCO-90/141 are highly effective against leukemic cells in caspase-dependent or -independent manners via autophagy, and they may have a novel therapeutic potential for treatment of leukemias including ATL.The online version of this article (10.1186/s12885-018-4710-1) contains supplementary material, which is available to authorized users. SIRT2 hunctions . Howeverunctions . SIRT2 munctions . High exunctions , 9.+ T-cells and has a poor prognosis, which develops after long-term infection with human T-cell leukemia virus (HTLV)-1 [ATL is a T-cell malignancy derived from mature CD4(HTLV)-1 –12. Alth(HTLV)-1 –16. Ther(HTLV)-1 . Despite(HTLV)-1 –21. TherWe previously reported that NCO-01 and NCO-04 inhibit both SIRT1 and SIRT2 activities in enzyme assays and induce apoptotic cell death , 22. SIR+ T-cell line derived from an ATL patient; kindly provided by Dr. Naomichi Arima, Kagoshima University), [Cell lines S1T (HTLV-1-infected CD4ersity),  MT-2 (HTersity),  Jurkat (ersity), .The novel SIRT2 inhibitors evaluated in this study, NCO-90 and NCO-141, have been described in a previous report . ChemicaWe purchased primary antibodies against β-actin, histone H3, p53, acetyl-p53K382 (ac-p53), and SIRT2 from Cell Signaling Technology , a primary antibody against acetyl-histone H4K16 (ac-H4) from Abcam , and a primary antibody against LC3 from MBL. Horseradish peroxidase-conjugated secondary antibodies were purchased from Vector Laboratories .Cell lysates were obtained using RIPA Lysis Buffer RIPA Lysis Buffer . Nuclear extracts were obtained using NE-PER Nuclear and Cytoplasmic Extraction Reagents , according to the manufacturer’s protocol. Western blotting was performed as described previously . Images 50) was calculated using Grafit .Cell viability was examined using the cell proliferation reagent Cell Count Reagent SF  as described previously . The halApoptotic cells were stained with annexin V-FITC (MBL) and 7-amino-actinomycin D , and analyzed by flow cytometry using a Cell Analyzer EC800  as described previously , 28. TheDNA fragmentation was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays using a MEBSTAIN Apoptosis Kit Direct (MBL) .Measurements of mitochondrial superoxide generation were performed using MitoSOX Red  as described previously .Caspase activity was detected using an APOPCYTO Intracellular Caspase-3 or − 8 Activity Detection Kit (MBL) and CaspGLOW Fluorescein Active Caspase-9 Staining Kit  as described previously [The presence of autophagic vacuoles was assessed using a Cyto-ID Autophagy Detection Kit , according to the manufacturer’s instructions , 30. Autt-tests and Wilcoxon matched-pairs tests were performed using Excel 2010  and Statcel2 software . In all tests, values of P < 0.05 were considered as statistically significant.Data are expressed as means ± SD. For data analyses, two-tailed Student’s 50 values of 1.0 and 0.5 μM for NCO-90 and NCO-141, respectively, whereas NCO-90/141 did not inhibit SIRT1 at up to 300 μM. NCO-90/141 inhibited the growth of all four cell lines in a dose-dependent manner. NCO-90 showed potent activities with GI50 values of 38.3, 48.5, 48.2, and 40.2 μM in S1T, MT-2, Jurkat, and HL60 cells, respectively. NCO-141 showed potent activities with GI50 values of 34.9, 26.4, 36.7, and 12.1 μM in S1T, MT-2, Jurkat, and HL60 cells, respectively. In contrast, commercially available SIRT2 inhibitor AKG2 had no GI50 values in leukemic cell lines


The cytotoxic impacts of the novel tubugi-1–NPY peptide–toxin conjugate, as well as of free tubugi-1, and tubugi-1 bearing the thiol spacer (liberated from tubugi-1–NPY conjugate), and native tubulysin A as reference were investigated by in vitro cell viability and proliferation screenings. The tumor cell lines HT-29, Colo320 (both colon cancer), PC-3 (prostate cancer), and in conjunction with RT-qPCR analyses of the hY1R expression, the cell lines SK-N-MC (Ewing`s sarcoma), MDA-MB-468, MDA-MB-231 (both breast cancer) and 184B5  were investigated. As hoped, the toxicity of tubugi-1 was masked, with IC50 values decreased by ca. 1,000-fold compared to the free toxin. Due to intracellular linker cleavage, the cytotoxic potency of the liberated tubugi-1 that, however, still bears the thiol spacer (tubugi-1-SH) was restored and up to 10-fold higher compared to the entire peptide–toxin conjugate. The conjugate shows toxic selectivity to tumor cell lines overexpressing the hY1R receptor subtype like, e.g., the hard to treat triple-negative breast cancer MDA-MB-468 cells.Tubugi-1 is a small cytotoxic peptide with picomolar cytotoxicity. To improve its cancer cell targeting, it was conjugated using a universal, modular disulfide derivative. This allowed conjugation to a neuropeptide-Y (NPY)-inspired peptide [K Until recently, the medication of tumor diseases was primarily based on more or less unspecific chemotherapeutics and the corresponding combination therapies –5. HowevMore recently, peptide–drug conjugates (PDCs) have been recommended as targeted therapeutics –15. WhilGenerally, a useful PDC must exhibit at least four major skills that are all required for the selective and potent treatment of, for instance, cancer cells: (1) a sufficient in vivo half-life, ideally hours to days, to reach the diseased cells with a high portion of intact PDC; (2) a selective conjugate binding to a specific target molecule, e.g., a cell-surface receptor, that is characteristic for the diseased cells; (3) a fast and efficient but target-dependent binding, or better internalization, of the PDC into targeted cells; and (4) the efficient cleavage of the linker structure and, thereby, efficient liberation of the drug molecule from the conjugate at or within the diseased cell, resulting in an efficient intracellular drug dose, ideally killing the tumor cells.2 receptor subtype (hY2R) -pNPY, which is comparable to the human NPY analogue -hNPY – has been identified and claimed to be especially selective for the NPY Y1 receptor subtype in comparison to the other, very closely related NPY receptor subtypes hY2R, hY4R and hY5R -pNPY as well as a peptide–tubulysin A conjugate -pNPY – representing a comparable PDC – compared to wildtype pNPY for their binding affinities at the NPY Y1 receptor subtype. While -pNPY (IC50 = 1.3 nM) showed a comparable binding affinity as pNPY (IC50 = 1.8 nM), the Y1 receptor binding of the peptide–tubulysin A conjugate -pNPY (IC50 = 47.6 nM) was detected to be slightly reduced. However, when testing the functional receptor activation – using an second messenger (IP) accumulation assay, Ahrens and co-workers found all three peptides and PDC, respectively, in the same EC50 range (1.7 to 2.6 nM) at the NPY Y1 receptor. Interestingly, at the NPY Y2 receptor subtype the EC50 value of the subtype-unspecific wildtype pNPY was found in the same range, but the EC50 values of -pNPY and -pNPY were detected with higher than 100 nM, i.e., around two magnitudes higher than at the Y1 receptor subtype. Furthermore the authors illustrated the Y1 receptor subtype-specific endocytotic internalization of the aforementioned peptides -pNPY, several approaches of peptide conjugates have been published with diagnostic indications -pNPY -pNPY -pNPY-based conjugates proved a convincing in vivo efficacy. To further improve the general setting of peptide–drug conjugates, major efforts have been made to enhance target affinity and specificity as well as metabolic stability of the peptide moiety, and to identify novel PDC payloads permitting superior PDC efficacies.Meanwhile, based on this hY1R-prefering peptide [Fications –34. In 234]-pNPY . This st34]-pNPY –37. Howe34]-pNPY . More re34]-pNPY ,39–40. H50 like in taxanes or epothilones) .–46.44–46464(C-βA-),F7,L17,P34]-hNPY was conjugated with tubugi-1 (2) as therapeutic payload, using a linker that promises a more general use than just for the present case. The study furthermore comprises the investigation of the PDCs’ in vitro efficacies on the viability and proliferation of colon and prostate as well as several breast cancer and Ewing`s sarcoma cell lines. Thereby the correlation with the hY1R expression levels of the latter three cell lines was determined as proof for targeted delivery.The aim of this work was to prepare a novel branched NPY Y1-receptor-selective peptide–toxin conjugate version with a tubugi toxin. Therefore, the NPY analogue -hNPY (8), was synthesized by reacting the tubugi-1-SSPy (3) with the free thiol function of a β-alanine–cysteine dipeptide (βAC) linked to the side chain of Lys4 of the targeting peptide. For this purpose, 1 mol equiv of the tubugi-1-SSPy building block 3 and one molar equivalent of the targeting peptide, -hNPY, were reacted for 60 min in an air- and moisture-free atmosphere  with DTT  healthy cells.2), tubugi-1–NPY-derived conjugate 8, as well as its reduced linker product 9 were initially tested against HT-29, PC-3 and Colo320 tumor cells. Contrarily to, for instance, SK-N-MC cells shown in 2 and 8, respectively, as shown in To assess the impact of the chemical modifications due to the linker-assisted peptide–toxin conjugation and toxin liberation on the tubugi-1 toxin’s, in vitro antitumor efficiency of free tubugi-1 (1), used for comparison, and the synthetic analogue tubugi-1 (2) expressed similar cytotoxic activities against the selected cancer cell lines in medium pM concentrations. Both compounds, 1 and 2, are able to penetrate the cells’ membrane by unspecific, receptor-independent pathways, not discriminating between normal and transformed cells. For that reason it is very difficult to adjust a practicable therapeutic window for these toxins. All the more, it is important to mask the high toxicity of tubulysin A and tubugi-1 until the toxins are delivered to the cells targeted. Indeed, in its conjugated form, represented by 8, attached to the peptide moiety designed to target the NPY Y1 receptor, the toxicity of tubugi-1 was found to be masked, with IC50 values increased ≈1,000-fold compared to the free toxin. The restoration of the tubugi-1 toxicity presupposes the intracellular cleavage of the disulfide linker within the reducing environment of the endo-lysosomal compartments of the addressed tumor cells, what should be simulated by testing tubugi-1-SH (9). As shown in 8. The only slight increase of cytotoxic activity of compound 9 compared to the complete conjugate 8 in Colo320 cells is most likely caused by a generally weak responsiveness of Colo320 cells towards tubugi-1-SH and the entire conjugate tubugi-1–SS–NPY. When compared with HT-29 and PC-3 cells, the IC50 value of tubugi-1-SH is by factor 10 higher in Colo320. Since the membrane passage of tubugi-1-SH is not depending on a NPY receptor, there have to be other explanations for the reduced cytotoxic impact of tubugi-1 and corresponding derivatives in Colo320, rather than the NPY Y1 receptor expression level.The antiproliferative activities of the investigated compounds and conjugates are summarized in 8 permits in the systemic situation before reaching the target cells much lower toxicity than the cytotoxic compound tubugi-1 alone, thus opening a feasible therapeutic window for the class of tubugi toxins. In that context, a loss of tubugi-1 activity is expectable due to its chemical modification caused by the linker-based conjugation, and after linker cleavage the intracellular activities of 8, i.e., the activities of the linker cleavage product 9, are within an acceptable range, and are comparable or higher than that of some commercially used anticancer compounds . Further in vitro cell proliferation and viability assays were conducted to investigate the impact of various durations of incubation of 8, and for the correlation of its potency with the hY1R expression levels of the cells.A significant aspect of the present concept of a hY1R-targeting peptide–toxin conjugate is the fact that intact tubugi-1–NPY conjugate 8 in two treatment regimens. One regime considered a pulsed setting, i.e., initial treatment with the drug for 6 h, washing and subsequently culturing without the PDC to reach 72 h , both tested at even higher concentration of the PDC than for the Y1 cell lines. This points out good selectivity not only between tumor cell lines with the different hY1R expression levels but also good discrimination against normal (non-cancerous) cells.For that reason, a collection of tumor cell lines was used that represents a wide range of cellular hY1R expression levels, i.e., highly hY1R-overexpressing Ewing`s sarcoma SK-N-MC cells, the triple-negative breast cancer cell lines MDA-MB-468 and MDA-MB-321 which are moderately and weakly expressing, respectively, as well as the 184B5 cell line, representing a normal, but chemically immortalized mammary gland epithelium with very weak hY1R expression. These cell lines were incubated with PDC ach 72 h . In the ach 72 h . As to b RT-qPCR . Both th8) with a reductively cleavable disulfide linker. The tubugi-1–NPY conjugate has a strongly masked antitumor activity against HT-29, PC-3 and Colo320 cells in comparison to the active compound alone, but the activity is restored to a sufficient extent upon linker cleavage (tubugi-1–SS–NPY → tubugi-1-SH). Most importantly, the cytotoxic potential of tubugi-1–SS–NPY correlates very well with the hY1R expression levels of a panel of tumor cell lines. For instance, the hY1R-overexpressing Ewing`s sarcoma cell line SK-N-MC was much more affected by the PDC than the normal  cell line 184B5 with weak hY1R expression. However, further efforts should be made to improve activity after internalization of the PDC.The highly active cytotoxin tubugi-1 was successfully conjugated to a truncated and modified neuropeptide-Y mimetic to form a new peptide–toxin conjugate (PDC Overall, the investigations carried out up to this point provide a biological validation of the developed conjugate. The principally modular conjugation protocol for tubugis bears promise for further cancer targeting conjugates.File 1Complete experimental procedures and characterization data.


Eleven CRC cell lines were classified into molecular subtypes and tested for their response to pan-ERBB, MEK, and ERK inhibitors as single agents and in combination. All six inflammatory or TA cell lines were exquisitely sensitive to the combination of MEK and neratinib whereas all five stem-like cell lines were resistant. Growth inhibition in sensitive cell lines was greater with the combination than with either drug alone even in cell lines with KRAS mutations. The combination inhibited pERK in inflammatory cell lines but not in four out of five stem-like cell lines. MEK162 plus neratinib were synergistic in cell culture and xenograft models in inflammatory cell lines. The ERK inhibitor, SCH772984, down-regulated pERK, decreased cell viability, and was synergistic with neratinib in both inflammatory and stem-like subtypes. These results suggest that inhibition of pERK is a critical node in decreasing cell viability of stem-like CRC tumors. Our results also suggest that CRC molecular subtypes may yield predictive information and may help to identify patients who may respond to targeted inhibitors.Molecular subtypes of colorectal tumors are associated with prognosis and prediction for treatment benefit from chemotherapy. The purpose of this study was two-fold: 1) to determine the association of colorectal (CRC) molecular subtypes with response to  The current standard of care for stage II/III colon cancer is adjuvant chemotherapy with 5-fluorouracil + leucovorin (FULV) or FULV plus oxaliplatin. The addition of targeted therapies in adjuvant setting has not been shown to reduce recurrences. We examined the association of subtypes with prognosis and for prediction of oxaliplatin benefit, when added to FU/LV by subtyping tumors from patients enrolled into NSABP C-07 N = 1729), a clinical trial in which patients were randomly assigned to FU/LV with or without oxaliplatin. In agreement with other investigators 729, a cl, we showKRAS mutations. Anti-epidermal growth factor receptor (EGFR) monoclonal antibodies, panitumumab and cetuximab, have been shown to improve overall survival, progression-free survival, and overall response rates in patients with metastatic, KRAS WT tumors [KRAS WT tumors respond, and even for those who do, the response is limited [Unlike stage II/III disease, the standard of care for colorectal cancer patients with metastatic disease is driven by the presence or absence of T tumors , 6. Howe limited , 8 by re limited –11.KRAS mutants with intrinsic resistance to anti-EGFR antibodies should be sensitive to inhibition of downstream signaling elements. Preclinical models tested this hypothesis with agents targeting pathways downstream of KRAS, however, single-agent inhibitors were disappointing in both PDX models and patients [in vivo were successfully inhibited.Preclinical studies showed that resistance to an EGFR blockade consistently displayed persistent activation of mitogen-activated protein kinase (MEK) and extracellular signal-regulated kinase (ERK) irrespective of the upstream genetic alterations . Theoretpatients –14. Intepatients . Recentlpatients  have shoDifferences among CRC subtypes with respect to general biology, association with prognosis, and the important clinical observation that oxaliplatin benefit was limited to the enterocyte CRC subtype, led us to hypothesize that subtypes may show a differential response to targeted therapies . To expl2. Cells were cultured in DMEM medium with 1X penicillin/streptomycin, and 10% fetal bovine serum. Selumetinib, gefitinib, afatinib, neratinib, MEK162, and SCH772984, were purchased from Selleckchembio .All cell lines used in this study were purchased from American Type Culture Collection (ATCC) . The CRC cell lines NCI-H747, SW837, SW1116, SW1463, SNU-C1, NCIH508, SW480, SW620, C2BBE1, HS675.T and HCT116 were cultured at 37°C in a humidified atmosphere in the presence of 5% CO3/well) in 100 μl of growth medium were plated into each 96 microtiter well  plates. Cells were allowed to attach for 14 to 18 hours, then were exposed to different doses of inhibitors for 72 hours. The number of viable cells was determined by using the CellTiter-Blue cell viability assay . The CellTiter-Blue reagent, an indicator dye, was added to cells, incubated for 1 hour, then fluorescence was measured (560(20) Ex/ 590(10) Em). The release of fluorescence as a result of cellular metabolism indicates living cells. The percent of living cells was calculated by normalizing the absorbance of treated cells to untreated cells. Cells were treated with drug over a range of concentrations. Dimethyl sulfoxide (DMSO) (0.01%) was used as the control treatment. Results are shown as triplicates of mean ± SD.CRC cells . Intensities of spots were quantified using myImage analysis software .CRC cells were harvested after 48 hours of culture in the presence of drugs or control vehicle, lysed by using 1X cell lysis buffer containing 1mM PMSF. Cell lysates were subjected to PathScan RTK Signaling Antibody Array Kit  (Chemiluminescent Readout) as per manufacturer's protocol. Briefly, the PathScan RTK Signaling Antibody Array Kit is a slide-based antibody array founded upon the sandwich immunoassay principle. The array kit allows for the simultaneous detection of 28 receptor tyrosine kinases and 11 important signaling nodes when phosphorylated at tyrosine or other residues. Target-specific capture antibodies using biotinylated protein as positive control, and nonspecific IgG as negative control, were spotted in duplicate onto nitrocellulose-coated glass slides. Cell lysates were then incubated on the slide followed by a biotinylated detection antibody cocktail. Streptavidin-conjugated HRP and LumiGLO reagent were then used to visualize the bound detection antibody by chemiluminescence. An image of the slide was captured with myECLProtein was extracted from cells using 1X RIPA buffer containing a Halt Protease and Phosphatase Inhibitor Cocktail (ThermoFisher Scientific). Cell lysate proteins (30 μg) were separated on 4–12% NuPAGE Bis-Tris precast gel electrophoresis and transferred to iBlot 2 polyvinyl difluoride membranes . The blots were incubated with the appropriate antibodies to detect the protein level of interest, and the immune complexes were visualized by GE Healthcare Amersham ECL WB detection system (ThermoFisher Scientific). Western blots were probed with antibodies against phosphor-ERBB2/HER-2 (Tyr1248) , ERBB2 , phosphor-ERBB3 (Tyr1197) , ERBB3 , phosphor-ERK1/2 , phosphor-p44/p42 MAPK (diluted 1:1000), total p44/42 MAPK, (diluted 1:1000), total ERK1/2 , phosphor-AKT S473 , AKT , phosphor-EGFR , phosphor-p90RSK , and Actin .3 and were randomly assigned to different treatment groups and a vehicle control (0.5% carboxymethylcellulose) (seven mice/group). An initial study was performed for dose tolerability. Efficacy studies were subsequently undertaken with 10 mice in each of 4 cohorts: vehicle, MEK162 alone (3 mg/kg); neratinib alone (10 mg/kg); and MEK162 (3mg/kg) plus neratinib (10mg/kg). Each drug was suspended in 0.5% carboxymethylcellulose and orally administered q.d. Mice were treated daily for 4 weeks. Tumor volume  where L is the longest and W is the shortest axis) and body weight were measured twice weekly. Mice were euthanized by brief isofluorane anesthesia followed by cervical dislocation, two hours following the last dose of drug. Tumors were harvested and snap frozen in a dry ice/ethanol bath for immunoblot analysis. Mice exhibiting any signs of distress including weight loss >20%, tumor size >10% body weight, or tumor growth hindering mouse mobility and ability to eat drink/eat normally, were euthanized.The animal study protocol was approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh (IACUC protocol #18022278) and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Five million cells per mouse were inoculated subcutaneously in female athymic nude mice  at the axillary region using human CRC cell lines NCI-H747 (inflammatory subtype) or SW480 (stem-like subtype). Tumor xenografts were allowed to grow to an average size of 100–200 mmt test. Results were considered statistically significant with p values <0.05. The RTK data was quantitated with a digital imaging system using the myImage Analysis software (ThermoFisher Scientific). Raw data quantification of all figures is shown in Five replicate measurements were made for all cellular proliferation and apoptotic assays and these data are presented as a mean plus or minus on standard deviation (SD). Statistical differences were determined by Student KRAS mutation status and their molecular subtype. Three of the five CRCA subtypes  were included in this study and represent approximately 80% of stage II and III colon cancer based on our analysis of C-07 [Cell lines were selected for the testing of targeted agents based on  of C-07 .One of the goals of this study was to identify novel treatments for patients resistant to chemotherapy. As noted previously, the stem-like subtype has de novo resistance to chemotherapy and bodes for a poor outcome. Although TA and inflammatory subtypes (approximately 50% of CRC) have a more favorable prognosis when treated in early-stage disease (stage II/III), if disease recurs, these patients have limited options.Table 1shows the mutation status of BRAF, KRAS, PIK3CA, and NRAS, as well as the subtype designations of these cell lines based on the results of two other studies [ studies . Fig, pan-ERBB inhibitors (afatinib and neratinib)  Fig, and the EGFR inhibitor, gefitinib,  as single agents in five stem-like cell lines , two inflammatory subtypes , and four TA subtypes . The MEK inhibitors (MEK162 and selumetinib) were the most effective agents in decreasing cell viability of inflammatory and TA cell lines  Figs. The inflammatory cell lines had IC50 values below 1μM, and one TA cell line had an IC50 value at 1μM (MEK162), whereas the other four TA cell lines had IC50 values above 2μM (MEK162), however, IC50 values in stem-like cell lines were all above 2μM, and for a majority of stem-like cell lines, IC50 values were not achieved at concentrations of 5μM . The two inflammatory cell lines, although KRAS mutant, were sensitive to the MEK inhibitors. The pan-ERBB inhibitor, neratinib, had an IC50 above 4μM in both inflammatory, stem-like , and TA cell lines except NCI-H508 TA cell line, which had an IC50 at 1μM (neratinib) . One TA cell line, NCI-H508, had an IC50 value at 1μM (MEK162); all other TA cell lines had IC50 values from 2μM to 5μM (MEK162) . Afatinib and gefitinib had little effect on cell viability at concentrations between 125nM to 4μM  Figs.Recently published data has shown combining pan-ERBB and MEK inhibitors demonstrated strong synergistic inhibition of CRC cell proliferation . Therefo. For all inflammatory and TA cell lines, the combination of neratinib (0.5 μM) with varying doses of MEK162 (0.062 μM -1μM) treatment was significantly more effective in reducing the cell viability . The combination of MEK162 at 0.062μM and neratinib at 0.5μM inhibited approximately 70% of inflammatory cell lines and 60% of TA cell lines, but more than 80% of the stem-like cell lines remained viable even at 1μM MEK162 and neratinib at 0.5μM. The resistance of the stem-like cell lines was independent of KRAS mutation status. We observed the same pattern of sensitivity and resistance with a constant dose of MEK162 (0.5 μM) with variable doses of neratinib (0.062μM-1 μM) .To improve the efficacy of these agents, we combined two different classes of drugs: the MEK inhibitor, MEK162, and the pan-ERBB inhibitor, neratinib. This combination dramatically reduced the cell viability of inflammatory and TA cell lines compared to single-agent treatment V600E-mutated metastatic melanoma. The same pattern of sensitivity was seen when the MEK inhibitor, trametinib, was combined with neratinib . Stem-like cell lines were resistant to this combination. More than 80% of the stem-like cells remained viable after treatment with 6 nM trametinib and 62 nM neratinib. In contrast, at these same drug concentrations, the viability of inflammatory and TA cell lines ranged between 20–60% . Our data suggest that although intrinsic resistance to EGFR-targeted therapy could be overcome by combination targeted therapies in inflammatory or TA subtypes, this approach would not work for stem-like subtypes.We used the combination of neratinib and another MEK inhibitor, trametinib, which has US FDA approval as a single agent for the treatment of patients with BRAFKRAS mutations (n = 4) or KRAS wild-type (n = 2) was demonstrated by performing a CompuSyn analysis, which showed a combination index (CI) of ≤1  ) or unchanged  . More specifically, inhibition of pERK after treatment with the combination of MEK162 plus neratinib was demonstrated in inflammatory cells lines NCI-H747 (0.50-fold vs. control)  and SW837 (0.20-fold vs. control) . Conversely, pERK levels were either increased or unchanged after exposure to the MEK162 plus neratinib combination compared to the control in the stem-like cell lines SW480 (4.2-fold vs. control), C2BBE1 (no change vs. control), and HCT-116 .To determine the targets that might be responsible for the resistance in cell lines to the combination of MEK162 and neratinib, we used the PathScan RTK Signaling Array Kit. Both inflammatory (n = 2) and stem-like (n = 5) cell lines were incubated with either no drug (control) or with the combination of MEK162 plus neratinib for 48 hours. Thirty-nine different anti-phosph-tyrosine antibodies were interrogated using the PathScan RTK Signaling Antibody arrays. These receptor tyrosine kinases signal primarily through tyrosine phosphorylation events involving a wide range of downstream signaling cascades including the PI3K/Akt, MAPK, and JAK/Stat pathways, which are important for cell division, growth, metabolism, differentiation, migration, and survival. The most dramatic single difference in the phosphorylation levels between the stem-like cell lines (C2BBE1 and SW480) and the inflammatory cell lines (NCI-H747 and SW837), after treatment with the combination of MEK162 plus neratinib, was the phosphorylated protein p44/42 MAPK . In inflammatory cell lines the levels of pERK1/2 were reduced after treatment with MEK162 plus neratinib . Conversely, the MEK162 plus neratinib combination failed to inhibit pERK levels in four out of five stem-like cells lines . Only one stem-like cell line, SW620, showed down-regulation of pERK after exposure to the MEK162 plus neratinib combination . These results suggest that continued activation of pERK might result in resistance in most stem-like cell lines to the combination of MEK162 plus neratinib.To validate the PathScan RTK array results, we examined the RAF-MEK-ERK downstream targets using western blots. Cells were incubated with neratinib or MEK162, singly or in combination for 48 hours. Results were similar to those of the PathScan RTK array. The combination of MEK162 plus neratinib resulted in substantial reduction of pERK levels after 48 hours of treatment in inflammatory cell lines (NCI-H747 and SW837) Fig 4D). This suggests that the decrease of cell viability by the MEK162 plus neratinib combination in inflammatory cell lines may be due to the induction of caspase 3/7 activity, an essential event during apoptosis.To determine whether apoptosis was responsible for the decreasing cell viability in cell lines when treated with the combination of MEK162 plus neratinib, we assessed apoptosis activity in resistant and sensitive cell lines. Both inflammatory  and stem-like  cell lines were treated with the varying doses of neratinib in combination with a constant dose of MEK162 (0.5 μM) for 72 hours followed by evaluation of caspase 3/7 activity. Only the inflammatory cell lines, NCI-H747 and SW837, showed induction of apoptosis after treatment with the combination of MEK162 (0.5 μM) plus neratinib (0.062 μM- 1 μM), whereas stem-like cell lines, SW480,C2BBE1, HCT-116, and Hs.675.T, showed no sign of apoptosis after treatment (p≤0.002) and single-agent neratinib (p≤0.0001) inhibited growth in the inflammatory cell line xenograft. However, the combination resulted in greater tumor inhibition  in the inflammatory cell line than either agent alone . In tumor xenografts, with a stem-like subtype (SW480), MEK162 alone significantly inhibited tumor growth compared to the vehicle control (p≤0.006), however, neratinib alone did not (p = 0.145) . Furthermore, the combination of MEK162 plus neratinib had no additive effects on the inhibition of tumor growth compared to the MEK162 alone (p = 0.57) in the stem-like xenograft models . No effects on the body weight of the treated xenograft mice were seen , indicating that there were no toxic effects of treatments. Table 2shows the percent of tumor growth inhibition (TGI) in both inflammatory (NCI-H747) and stem-like (SW480) cell line xenograft models in three cohorts: neratinib alone, MEK162 alone, and the combination of MEK162 plus neratinib compared to control.We examined the effect of neratinib and MEK162 as single agents and in combination on an inflammatory (NCI-H747) and a stem-like (SW480) cell line in xenograft models. Both single-agent MEK162 (p≤0.0001) in inflammatory subtype tumors Fig . There was no inhibition of pERK levels by single-agent neratinib. p-ERK levels were significantly downregulated with the combination of neratinib and MEK162 compared to either drug alone (p≤0.001 vs. MEK162 only) (p≤2.8511E-10 vs. neratinib only) or control vehicle only (p≤1.060E-05) . In stem-like xenograft models, MEK162 significantly inhibited pERK as a single agent (p≤1.696E-06 vs. control), whereas neratinib showed no effect on pERK as a single agent Fig . pERK levels were not statistically different between MEK162 alone and the combination (p = 0.09 vs. combination) in the stem-like xenograft tissue . The inability to inhibit pERK and tumor growth with neratinib alone or in combination with MEK162 was correlated.Examination of the phosphorylation status from inflammatory and stem-like tumors was carried out on all seven mice from each of four cohorts . Single-agent MEK162 significantly reduced the pERK protein levels compared to the control (. The combination of neratinib (0.125 μM) and SCH772984 (1 μM) was more effective at decreasing cell viability in both inflammatory and stem-like cell lines than either drug alone . Moreover, inhibition of pERK was seen with single-agent SCH772984 in both inflammatory and stem-like cell lines and correlated with loss of cell viability . CompuSyn analysis showed that the combination of SCH772984 plus neratinib were highly synergistic CI ~0.19) in both inflammatory and stem-like subtypes .Most of our cell culture and xenograft data show that inhibition of pERK is associated with drug sensitivity. This observation suggests that pERK is a critical downstream target for signaling of the RAS/RAF pathway and led us to test the ERK inhibitor, SCH772984. The SCH772984 is a potent, ATP-competitive and non-competitive inhibitor of ERK1/2 with additional allosteric properties, which inhibits ERK activation/phosphorylation , 18. SCHIn this study, we demonstrated that the combination of MEK162 plus neratinib effectively inhibited cell viability in inflammatory or TA subtypes but not in the stem-like subtype. Tumor growth of an inflammatory cell line in mouse-xenograft models was inhibited by MEK162 and neratinib as single agents, however, the combination of MEK162 plus neratinib was more efficacious, synergistic, and independent of KRAS status. The finding that sensitivity appeared independent of KRAS status is a particularly important observation as currently there are no specific therapies for KRAS MT tumors . In contS2 File). Moreover, in resistant stem-like cell lines, we did not observe any change in ERBB3 expression levels with MEK, neratinib, or the combination . One plausible explanation for the difference in our findings and those of Sun et al., is that we used different pan-ERBB and MEK inhibitors, which may affect the amount of ERBB-RAS-RAF-ERK targets downregulation and assessment.Sun et al. , who tesOther investigators , 20 havePatients with stem-like tumors represent approximately 25% to 30% of the CRC population and have a very poor prognosis regardless of treatment  and thusMolecular subtyping of colorectal cancer with gene expression represents a relatively new approach to predicting treatment response. The development of a validated assay may be a valuable tool not only in determining prognosis but also by guiding more precise therapies.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.S7 Fig(TIF)Click here for additional data file.S8 Fig(TIF)Click here for additional data file.S9 Fig(TIF)Click here for additional data file.S10 Fig(TIF)Click here for additional data file.S11 Fig(TIF)Click here for additional data file.S12 Fig(TIF)Click here for additional data file.S1 File(XLSX)Click here for additional data file.S2 File(PDF)Click here for additional data file.