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- ABCB1 (2)
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Resistance formation after initial therapy response (acquired resistance) is common in high-risk neuroblastoma patients. YM155 is a drug candidate that was introduced as a survivin suppressant. This mechanism was later challenged, and DNA damage induction and Mcl-1 depletion were suggested instead. Here we investigated the efficacy and mechanism of action of YM155 in neuroblastoma cells with acquired drug resistance. The efficacy of YM155 was determined in neuroblastoma cell lines and their sublines with acquired resistance to clinically relevant drugs. Survivin levels, Mcl-1 levels, and DNA damage formation were determined in response to YM155. RNAi-mediated depletion of survivin, Mcl-1, and p53 was performed to investigate their roles during YM155 treatment. Clinical YM155 concentrations affected the viability of drug-resistant neuroblastoma cells through survivin depletion and p53 activation. MDM2 inhibitor-induced p53 activation further enhanced YM155 activity. Loss of p53 function generally affected anti-neuroblastoma approaches targeting survivin. Upregulation of ABCB1 (causes YM155 efflux) and downregulation of SLC35F2 (causes YM155 uptake) mediated YM155-specific resistance. YM155-adapted cells displayed increased ABCB1 levels, decreased SLC35F2 levels, and a p53 mutation. YM155-adapted neuroblastoma cells were also characterized by decreased sensitivity to RNAi-mediated survivin depletion, further confirming survivin as a critical YM155 target in neuroblastoma. In conclusion, YM155 targets survivin in neuroblastoma. Furthermore, survivin is a promising therapeutic target for p53 wild-type neuroblastomas after resistance acquisition (neuroblastomas are rarely p53-mutated), potentially in combination with p53 activators. In addition, we show that the adaptation of cancer cells to molecular-targeted anticancer drugs is an effective strategy to elucidate a drug’s mechanism of action.
The formation of acquired drug resistance is a major reason for the failure of anti-cancer therapies after initial response. Here, we introduce a novel model of acquired oxaliplatin resistance, a sub-line of the non-MYCN-amplified neuroblastoma cell line SK-N-AS that was adapted to growth in the presence of 4000 ng/mL oxaliplatin (SK-N-ASrOXALI4000). SK-N-ASrOXALI4000 cells displayed enhanced chromosomal aberrations compared to SK-N-AS, as indicated by 24-chromosome fluorescence in situ hybridisation. Moreover, SK-N-ASrOXALI4000 cells were resistant not only to oxaliplatin but also to the two other commonly used anti-cancer platinum agents cisplatin and carboplatin. SK-N-ASrOXALI4000 cells exhibited a stable resistance phenotype that was not affected by culturing the cells for 10 weeks in the absence of oxaliplatin. Interestingly, SK-N-ASrOXALI4000 cells showed no cross resistance to gemcitabine and increased sensitivity to doxorubicin and UVC radiation, alternative treatments that like platinum drugs target DNA integrity. Notably, UVC-induced DNA damage is thought to be predominantly repaired by nucleotide excision repair and nucleotide excision repair has been described as the main oxaliplatin-induced DNA damage repair system. SK-N-ASrOXALI4000 cells were also more sensitive to lysis by influenza A virus, a candidate for oncolytic therapy, than SK-N-AS cells. In conclusion, we introduce a novel oxaliplatin resistance model. The oxaliplatin resistance mechanisms in SK-N-ASrOXALI4000 cells appear to be complex and not to directly depend on enhanced DNA repair capacity. Models of oxaliplatin resistance are of particular relevance since research on platinum drugs has so far predominantly focused on cisplatin and carboplatin.
Background: SAMHD1 mediates resistance to anti-cancer nucleoside analogues, including cytarabine, decitabine, and nelarabine that are commonly used for the treatment of leukaemia, through cleavage of their triphosphorylated forms. Hence, SAMHD1 inhibitors are promising candidates for the sensitisation of leukaemia cells to nucleoside analogue-based therapy. Here, we investigated the effects of the cytosine analogue CNDAC, which has been proposed to be a SAMHD1 inhibitor, in the context of SAMHD1. Methods: CNDAC was tested in 13 acute myeloid leukaemia (AML) cell lines, in 26 acute lymphoblastic leukaemia (ALL) cell lines, ten AML sublines adapted to various antileukaemic drugs, 24 single cell-derived clonal AML sublines, and primary leukaemic blasts from 24 AML patients. Moreover, 24 CNDAC-resistant sublines of the AML cell lines HL-60 and PL-21 were established. The SAMHD1 gene was disrupted using CRISPR/Cas9 and SAMHD1 depleted using RNAi, and the viral Vpx protein. Forced DCK expression was achieved by lentiviral transduction. SAMHD1 promoter methylation was determined by PCR after treatment of genomic DNA with the methylation-sensitive HpaII endonuclease. Nucleoside (analogue) triphosphate levels were determined by LC-MS/MS. CNDAC interaction with SAMHD1 was analysed by an enzymatic assay and by crystallisation. Results: Although the cytosine analogue CNDAC was anticipated to inhibit SAMHD1, SAMHD1 mediated intrinsic CNDAC resistance in leukaemia cells. Accordingly, SAMHD1 depletion increased CNDAC triphosphate (CNDAC-TP) levels and CNDAC toxicity. Enzymatic assays and crystallisation studies confirmed CNDAC-TP to be a SAMHD1 substrate. In 24 CNDAC-adapted acute myeloid leukaemia (AML) sublines, resistance was driven by DCK (catalyses initial nucleoside phosphorylation) loss. CNDAC-adapted sublines displayed cross-resistance only to other DCK substrates (e.g. cytarabine, decitabine). Cell lines adapted to drugs not affected by DCK or SAMHD1 remained CNDAC sensitive. In cytarabine-adapted AML cells, increased SAMHD1 and reduced DCK levels contributed to cytarabine and CNDAC resistance. Conclusion: Intrinsic and acquired resistance to CNDAC and related nucleoside analogues are driven by different mechanisms. The lack of cross-resistance between SAMHD1/ DCK substrates and non-substrates provides scope for next-line therapies after treatment failure.
The nucleoside analogue nelarabine, the prodrug of arabinosylguanine (AraG), is effective against T-cell acute lymphoblastic leukaemia (T-ALL) but not against B-cell ALL (B-ALL). The underlying mechanisms have remained elusive. Here, data from pharmacogenomics studies and a panel of ALL cell lines reveal an inverse correlation between nelarabine sensitivity and the expression of SAMHD1, which can hydrolyse and inactivate triphosphorylated nucleoside analogues. Lower SAMHD1 abundance is detected in T-ALL than in B-ALL in cell lines and patient-derived leukaemic blasts. Mechanistically, T-ALL cells display increased SAMHD1 promoter methylation without increased global DNA methylation. SAMHD1 depletion sensitises B-ALL cells to AraG, while ectopic SAMHD1 expression in SAMHD1-null T-ALL cells induces AraG resistance. SAMHD1 has a larger impact on nelarabine/AraG than on cytarabine in ALL cells. Opposite effects are observed in acute myeloid leukaemia cells, indicating entity-specific differences. In conclusion, SAMHD1 promoter methylation and, in turn, SAMHD1 expression levels determine ALL cell response to nelarabine.
The nucleoside analogue nelarabine, the prodrug of arabinosylguanine (AraG), has been known for decades to be effective against acute lymphoblastic leukaemias of T-cell (T-ALL), but not of B-cell (B-ALL) origin. The mechanisms underlying this lineage-specific drug sensitivity have remained elusive. Data from pharmacogenomics studies and from a panel of ALL cell lines revealed an inverse correlation of SAMHD1 expression and nelarabine sensitivity. SAMHD1 can hydrolyse and thus inactivate triphosphorylated nucleoside analogues. Transcriptomic and protein expression profiling of cell lines and patient-derived leukaemic blasts revealed lower SAMHD1 abundance in T-ALL than in B-ALL. Mechanistically, SAMHD1 promoter methylation strongly correlated with suppressed SAMHD1 expression, while T-ALL cells did not display increased global DNA methylation. Targeted SAMHD1 degradation using virus-like particles containing Vpx sensitised B-ALL cells to AraG, while ectopic SAMHD1 expression in SAMHD1-null T-ALL cells induced AraG resistance. SAMHD1 had a larger impact on cytarabine activity than on nelarabine/ AraG activity in acute myeloid leukaemia (AML) cells, but more strongly affected nelarabine/ AraG activity in ALL cells. This indicates a critical role of the cancer entity. In conclusion, lineage-specific differences in SAMHD1 promoter methylation and, in turn, SAMHD1 expression levels determine ALL cell response to nelarabine. SAMHD1 is a potential biomarker for the identification of ALL patients likely to benefit from nelarabine therapy and a therapeutic target to overcome nelarabine resistance.
Intact-cell maldi-tof mass spectrometry for the authentication of drug-adapted cancer cell lines
(2019)
The use of cell lines in research can be affected by cell line misidentification. Short tandem repeat (STR) analysis is an effective method, and the gold standard, for the identification of the genetic origin of a cell line, but methods that allow the discrimination between cell lines of the same genetic origin are lacking. Here, we use intact cell MALDI-ToF mass spectrometry analysis, routinely used for the identification of bacteria in clinical diagnostic procedures, for the authentication of a set of cell lines consisting of three parental neuroblastoma cell lines (IMR-5, IMR-32 and UKF-NB-3) and eleven drug-adapted sublines. Principal component analysis (PCA) of intact-cell MALDI-ToF mass spectrometry data revealed clear differences between most, but not all, of the investigated cell lines. Mass spectrometry whole-cell fingerprints enabled the separation of IMR-32 and its clonal subline IMR-5. Sublines that had been adapted to closely related drugs, for example, the cisplatin- and oxaliplatin-resistant UKF-NB-3 sublines and the vincristine- and vinblastine-adapted IMR-5 sublines, also displayed clearly distinctive patterns. In conclusion, intact whole-cell MALDI-ToF mass spectrometry has the potential to be further developed into an authentication method for mammalian cells of a common genetic origin.
Survivin is a drug target and the survivin suppressant YM155 a drug candidate for high-risk neuroblastoma. Findings from one YM155-adapted subline of the neuroblastoma cell line UKF-NB-3 had suggested that increased ABCB1 (mediates YM155 efflux) levels, decreased SLC35F2 (mediates YM155 uptake) levels, decreased survivin levels, and TP53 mutations indicate YM155 resistance. Here, the investigation of ten additional YM155-adapted UKF-NB-3 sublines only confirmed the roles of ABCB1 and SLC35F2. However, cellular ABCB1 and SLC35F2 levels did not indicate YM155 sensitivity in YM155-naïve cells, as indicated by drug response data derived from the Cancer Therapeutics Response Portal (CTRP) and the Genomics of Drug Sensitivity in Cancer (GDSC) databases. Moreover, the resistant sublines were characterised by a remarkable heterogeneity. Only seven sublines developed on-target resistance as indicated by resistance to RNAi-mediated survivin depletion. The sublines also varied in their response to other anti-cancer drugs. In conclusion, cancer cell populations of limited intrinsic heterogeneity can develop various resistance phenotypes in response to treatment. Therefore, individualised therapies will require monitoring of cancer cell evolution in response to treatment. Moreover, biomarkers can indicate resistance formation in the acquired resistance setting, even when they are not predictive in the intrinsic resistance setting.
Survivin is a drug target and its suppressant YM155 a drug candidate mainly investigated for high-risk neuroblastoma. Findings from one YM155-adapted subline of the neuroblastoma cell line UKF-NB-3 had suggested that increased ABCB1 (mediates YM155 efflux) levels, decreased SLC35F2 (mediates YM155 uptake) levels, decreased survivin levels, and TP53 mutations indicate YM155 resistance. Here, the investigation of 10 additional YM155-adapted UKF-NB-3 sublines only confirmed the roles of ABCB1 and SLC35F2. However, cellular ABCB1 and SLC35F2 levels did not indicate YM155 sensitivity in YM155-naïve cells, as indicated by drug response data derived from the Cancer Therapeutics Response Portal (CTRP) and the Genomics of Drug Sensitivity in Cancer (GDSC) databases. Moreover, the resistant sublines were characterized by a remarkable heterogeneity. Only seven sublines developed on-target resistance as indicated by resistance to RNAi-mediated survivin depletion. The sublines also varied in their response to other anti-cancer drugs. In conclusion, cancer cell populations of limited intrinsic heterogeneity can develop various resistance phenotypes in response to treatment. Therefore, individualized therapies will require monitoring of cancer cell evolution in response to treatment. Moreover, biomarkers can indicate resistance formation in the acquired resistance setting, even when they are not predictive in the intrinsic resistance setting.
The survivin suppressant YM155 is a drug candidate for neuroblastoma. Here, we tested YM155 in 101 neuroblastoma cell lines (19 parental cell lines, 82 drug-adapted sublines). 77 cell lines displayed YM155 IC50s in the range of clinical YM155 concentrations. ABCB1 was an important determinant of YM155 resistance. The activity of the ABCB1 inhibitor zosuquidar ranged from being similar to that of the structurally different ABCB1 inhibitor verapamil to being 65-fold higher. ABCB1 sequence variations may be responsible for this, suggesting that the design of variant-specific ABCB1 inhibitors may be possible. Further, we showed that ABCC1 confers YM155 resistance. Previously, p53 depletion had resulted in decreased YM155 sensitivity. However, TP53-mutant cells were not generally less sensitive to YM155 than TP53 wild-type cells in this study. Finally, YM155 cross-resistance profiles differed between cells adapted to drugs as similar as cisplatin and carboplatin. In conclusion, the large cell line panel was necessary to reveal an unanticipated complexity of the YM155 response in neuroblastoma cell lines with acquired drug resistance. Novel findings include that ABCC1 mediates YM155 resistance and that YM155 cross-resistance profiles differ between cell lines adapted to drugs as similar as cisplatin and carboplatin.
The survivin suppressant YM155 is a drug candidate for neuroblastoma. Here, we tested YM155 in 101 neuroblastoma cell lines (19 parental cell lines, 82 drug-adapted sublines). Seventy seven (77) cell lines displayed YM155 IC50s in the range of clinical YM155 concentrations. ABCB1 was an important determinant of YM155 resistance. The activity of the ABCB1 inhibitor zosuquidar ranged from being similar to that of the structurally different ABCB1 inhibitor verapamil to being 65-fold higher. ABCB1 sequence variations may be responsible for this, suggesting that the design of variant-specific ABCB1 inhibitors may be possible. Further, we showed that ABCC1 confers YM155 resistance. Previously, p53 depletion had resulted in decreased YM155 sensitivity. However, TP53-mutant cells were not generally less sensitive to YM155 than TP53 wild-type cells in this study. Finally, YM155 cross-resistance profiles differed between cells adapted to drugs as similar as cisplatin and carboplatin. In conclusion, the large cell line panel was necessary to reveal an unanticipated complexity of the YM155 response in neuroblastoma cell lines with acquired drug resistance. Novel findings include that ABCC1 mediates YM155 resistance and that YM155 cross-resistance profiles differ between cell lines adapted to drugs as similar as cisplatin and carboplatin.