Molecular Modes of Action of an Aqueous Nerium oleander Extract in Cancer Cells In Vitro and In Vivo.

Cancer drug resistance remains a major obstacle in clinical oncology. As most anticancer drugs are of natural origin, we investigated the anticancer potential of a standardized cold-water leaf extract from Nerium oleander L., termed Breastin. The phytochemical characterization by nuclear magnetic resonance spectroscopy (NMR) and low- and high-resolution mass spectrometry revealed several monoglycosidic cardenolides as major constituents (adynerin, neritaloside, odoroside A, odoroside H, oleandrin, and vanderoside). Breastin inhibited the growth of 14 cell lines from hematopoietic tumors and 5 of 6 carcinomas. Remarkably, the cellular responsiveness of odoroside H and neritaloside was not correlated with all other classical drug resistance mechanisms, i.e., ATP-binding cassette transporters (ABCB1, ABCB5, ABCC1, ABCG2), oncogenes (EGFR, RAS), tumor suppressors (TP53, WT1), and others (GSTP1, HSP90, proliferation rate), in 59 tumor cell lines of the National Cancer Institute (NCI, USA), indicating that Breastin may indeed bypass drug resistance. COMPARE analyses with 153 anticancer agents in 74 tumor cell lines of the Oncotest panel revealed frequent correlations of Breastin with mitosis-inhibiting drugs. Using tubulin-GFP-transfected U2OS cells and confocal microscopy, it was found that the microtubule-disturbing effect of Breastin was comparable to that of the tubulin-depolymerizing drug paclitaxel. This result was verified by a tubulin polymerization assay in vitro and molecular docking in silico. Proteome profiling of 3171 proteins in the NCI panel revealed protein subsets whose expression significantly correlated with cellular responsiveness to odoroside H and neritaloside, indicating that protein expression profiles can be identified to predict the sensitivity or resistance of tumor cells to Breastin constituents. Breastin moderately inhibited breast cancer xenograft tumors in vivo. Remarkably, in contrast to what was observed with paclitaxel monotherapy, the combination of paclitaxel and Breastin prevented tumor relapse, indicating Breastin's potential for drug combination regimens.

The epigenetic modifier HDAC2 and the checkpoint kinase ATM determine the responses of microsatellite instable colorectal cancer cells to 5-fluorouracil.

The epigenetic modifier histone deacetylase-2 (HDAC2) is frequently dysregulated in colon cancer cells. Microsatellite instability (MSI), an unfaithful replication of DNA at nucleotide repeats, occurs in about 15% of human colon tumors. MSI promotes a genetic frameshift and consequently a loss of HDAC2 in up to 43% of these tumors. We show that long-term and short-term cultures of colorectal cancers with MSI contain subpopulations of cells lacking HDAC2. These can be isolated as single cell-derived, proliferating populations. Xenografted patient-derived colon cancer tissues with MSI also show variable patterns of HDAC2 expression in mice. HDAC2-positive and HDAC2-negative RKO cells respond similarly to pharmacological inhibitors of the class I HDACs HDAC1/HDAC2/HDAC3. In contrast to this similarity, HDAC2-negative and HDAC2-positive RKO cells undergo differential cell cycle arrest and apoptosis induction in response to the frequently used chemotherapeutic 5-fluorouracil, which becomes incorporated into and damages RNA and DNA. 5-fluorouracil causes an enrichment of HDAC2-negative RKO cells in vitro and in a subset of primary colorectal tumors in mice. 5-fluorouracil induces the phosphorylation of KAP1, a target of the checkpoint kinase ataxia-telangiectasia mutated (ATM), stronger in HDAC2-negative cells than in their HDAC2-positive counterparts. Pharmacological inhibition of ATM sensitizes RKO cells to cytotoxic effects of 5-fluorouracil. These findings demonstrate that HDAC2 and ATM modulate the responses of colorectal cancer cells towards 5-FU.

Cryo-EM structure of the Mre11-Rad50-Nbs1 complex reveals the molecular mechanism of scaffolding functions.

The DNA double-strand break repair complex Mre11-Rad50-Nbs1 (MRN) detects and nucleolytically processes DNA ends, activates the ATM kinase, and tethers DNA at break sites. How MRN can act both as nuclease and scaffold protein is not well understood. The cryo-EM structure of MRN from Chaetomium thermophilum reveals a 2:2:1 complex with a single Nbs1 wrapping around the autoinhibited Mre11 nuclease dimer. MRN has two DNA-binding modes, one ATP-dependent mode for loading onto DNA ends and one ATP-independent mode through Mre11's C terminus, suggesting how it may interact with DSBs and intact DNA. MRNs two 60-nm-long coiled-coil domains form a linear rod structure, the apex of which is assembled by the two joined zinc-hook motifs. Apices from two MRN complexes can further dimerize, forming 120-nm spanning MRN-MRN structures. Our results illustrate the architecture of MRN and suggest how it mechanistically integrates catalytic and tethering functions.

Assessing the Effect of Histone Deacetylase Inhibitors on DNA Double-Strand Break Repair by Nonhomologous End Joining.

This book chapter describes a plasmid-based reporter method, first described by Bennardo et al. (2008) that we use in our laboratory for determining the activity of the repair of DNA double-strand breaks by nonhomologous end joining. This method can be used to measure the impact of epigenetic modifiers of the histone deacetylase family on this DNA repair pathway by flow cytometry.

Class I HDAC overexpression promotes temozolomide resistance in glioma cells by regulating RAD18 expression.

Overexpression of histone deacetylases (HDACs) in cancer commonly causes resistance to genotoxic-based therapies. Here, we report on the novel mechanism whereby overexpressed class I HDACs increase the resistance of glioblastoma cells to the SN1 methylating agent temozolomide (TMZ). The chemotherapeutic TMZ triggers the activation of the DNA damage response (DDR) in resistant glioma cells, leading to DNA lesion bypass and cellular survival. Mass spectrometry analysis revealed that the catalytic activity of class I HDACs stimulates the expression of the E3 ubiquitin ligase RAD18. Furthermore, the data showed that RAD18 is part of the O6-methylguanine-induced DDR as TMZ induces the formation of RAD18 foci at sites of DNA damage. Downregulation of RAD18 by HDAC inhibition prevented glioma cells from activating the DDR upon TMZ exposure. Lastly, RAD18 or O6-methylguanine-DNA methyltransferase (MGMT) overexpression abolished the sensitization effect of HDAC inhibition on TMZ-exposed glioma cells. Our study describes a mechanism whereby class I HDAC overexpression in glioma cells causes resistance to TMZ treatment. HDACs accomplish this by promoting the bypass of O6-methylguanine DNA lesions via enhancing RAD18 expression. It also provides a treatment option with HDAC inhibition to undermine this mechanism.

Epigenetic Anti-Cancer Treatment With a Stabilized Carbocyclic Decitabine Analogue.

5-Aza-2'-deoxycytidine (Decitabine, AzadC) is a nucleoside analogue, which is in clinical use to treat patients with myelodysplastic syndrome or acute myeloid leukemia. Its mode of action is unusual because the compound is one of the few drugs that act at the epigenetic level of the genetic code. AzadC is incorporated as an antimetabolite into the genome and creates covalent, inhibitory links to DNA methyltransferases (DNMTs) that methylate 2'-deoxycytidine (dC) to 5-methyl-dC (mdC). Consequently, AzadC treatment leads to a global loss of mdC, which presumably results in a reactivation of silenced genes, among them tumor suppressor and DNA damage response genes. Because AzadC suffers from severe instability, which limits its use in the clinic, a more sophisticated AzadC derivative would be highly valuable. Here, we report that a recently developed carbocyclic AzadC analogue (cAzadC) blocks DNMT1 in the AML cell line MOLM-13 as efficient as AzadC. Moreover, cAzadC has a surprisingly strong anti-proliferative effect and leads to a significantly higher number of double strand breaks compared to AzadC, while showing less off-target toxicity. These results show that cAzadC triggers more deleterious repair and apoptotic pathways in cancer cells than AzadC, which makes cAzadC a promising next generation epigenetic drug.

In Vitro Assessment of the Genotoxic Hazard of Novel Hydroxamic Acid- and Benzamide-Type Histone Deacetylase Inhibitors (HDACi).

Histone deacetylase inhibitors (HDACi) are already approved for the therapy of leukemias. Since they are also emerging candidate compounds for the treatment of non-malignant diseases, HDACi with a wide therapeutic window and low hazard potential are desirable. Here, we investigated a panel of 12 novel hydroxamic acid- and benzamide-type HDACi employing non-malignant V79 hamster cells as toxicology guideline-conform in vitro model. HDACi causing a ≥10-fold preferential cytotoxicity in malignant neuroblastoma over non-malignant V79 cells were selected for further genotoxic hazard analysis, including vorinostat and entinostat for control. All HDACi selected, (i.e., KSK64, TOK77, DDK137 and MPK77) were clastogenic and evoked DNA strand breaks in non-malignant V79 cells as demonstrated by micronucleus and comet assays, histone H2AX foci formation analyses (γH2AX), DNA damage response (DDR) assays as well as employing DNA double-strand break (DSB) repair-defective VC8 hamster cells. Genetic instability induced by hydroxamic acid-type HDACi seems to be independent of bulky DNA adduct formation as concluded from the analysis of nucleotide excision repair (NER) deficient mutants. Summarizing, KSK64 revealed the highest genotoxic hazard and DDR stimulating potential, while TOK77 and MPK77 showed the lowest DNA damaging capacity. Therefore, these compounds are suggested as the most promising novel candidate HDACi for subsequent pre-clinical in vivo studies.

Anticancer drug and ionizing radiation-induced DNA damage differently influences transcription activity and DDR-related stress responses of an endothelial monolayer.

The endothelium contributes to the pathophysiology of adverse effects caused by conventional (genotoxic) anticancer therapeutics (cAT). The relevance of structurally different types of cAT-induced DNA lesions for eliciting selected endothelial stress responses is largely unknown. Here, we analyzed the cAT-induced formation of DNA double-strand breaks (DSB), transcription blockage and DNA damage response (DDR) in time kinetic analyses employing a monolayer of primary human endothelial cells (HUVEC). We observed that the degree of cAT-induced transcription blockage, the number of DSB and activation of DDR-related factors diverge. For instance, ionizing radiation caused the formation of numerous DSB and triggerd a substantial activation of ATM/Chk2 signaling, which however were not accompanied by a significant transcription inhibition. By contrast, the DNA cross-linking cAT cisplatin triggered a rapid and substantial blockage of transcription, which yet was not reflected by an appreciable number of DSB or increased levels of pATM/pChk2. In general, cAT-stimulated ATM-dependent phosphorylation of Kap1 (Ser824) and p53 (Ser15) reflected best cAT-induced transcription blockage. In conclusion, cAT-induced formation of DSB and profound activation of prototypical DDR factors is independent of the inhibition of RNA polymerase II-regulated transcription in an endothelial monolayer. We suggest that DSB formed directly or indirectly following cAT-treatment do not act as comprehensive triggers of superior signaling pathways shutting-down transcription while, at the same time, causing an appreciable stimulation of the DDR. Rather, it appears that distinct cAT-induced DNA lesions elicit diverging signaling pathways, which separately control transcription vs. DDR activity in the endothelium.

The SIAH1-HIPK2-p53ser46 Damage Response Pathway is Involved in Temozolomide-Induced Glioblastoma Cell Death.

Patients suffering from glioblastoma have a dismal prognosis, indicating the need for new therapeutic targets. Here we provide evidence that the DNA damage kinase HIPK2 and its negative regulatory E3-ubiquitin ligase SIAH1 are critical factors controlling temozolomide-induced cell death. We show that HIPK2 downregulation (HIPK2kd) significantly reduces the level of apoptosis. This was not the case in glioblastoma cells expressing the repair protein MGMT, suggesting that the primary DNA lesion responsible for triggering HIPK2-mediated apoptosis is O6 -methylguanine. Upon temozolomide treatment, p53 becomes phosphorylated whereby HIPK2kd had impact exclusively on ser46, but not ser15. Searching for the transcriptional target of p-p53ser46, we identified the death receptor FAS (CD95, APO-1) being involved. Thus, the expression of FAS was attenuated following HIPK2kd, supporting the conclusion that HIPK2 regulates temozolomide-induced apoptosis via p-p53ser46-driven FAS expression. This was substantiated in chromatin-immunoprecipitation experiments, in which p-p53ser46 binding to the Fas promotor was regulated by HIPK2. Other pro-apoptotic proteins such as PUMA, NOXA, BAX, and PTEN were not affected in HIPK2kd, and also double-strand breaks following temozolomide remained unaffected. We further show that downregulation of the HIPK2 inactivator SIAH1 significantly ameliorates temozolomide-induced apoptosis, suggesting that the ATM/ATR target SIAH1 together with HIPK2 plays a proapoptotic role in glioma cells exhibiting p53wt status. A database analysis revealed that SIAH1, but not SIAH2, is significantly overexpressed in glioblastomas. IMPLICATIONS: The identification of a novel apoptotic pathway triggered by the temozolomide-induced DNA damage O6 -methylguanine supports the role of p53 in the decision between survival and death and suggests SIAH1 and HIPK2 as new therapeutic targets.

Temozolomide Induces Senescence and Repression of DNA Repair Pathways in Glioblastoma Cells via Activation of ATR-CHK1, p21, and NF-κB.

The DNA-methylating drug temozolomide, which induces cell death through apoptosis, is used for the treatment of malignant glioma. Here, we investigate the mechanisms underlying the ability of temozolomide to induce senescence in glioblastoma cells. Temozolomide-induced senescence was triggered by the specific DNA lesion O6-methylguanine (O6MeG) and characterized by arrest of cells in the G2-M phase. Inhibitor experiments revealed that temozolomide-induced senescence was initiated by damage recognition through the MRN complex, activation of the ATR/CHK1 axis of the DNA damage response pathway, and mediated by degradation of CDC25c. Temozolomide-induced senescence required functional p53 and was dependent on sustained p21 induction. p53-deficient cells, not expressing p21, failed to induce senescence, but were still able to induce a G2-M arrest. p14 and p16, targets of p53, were silenced in our cell system and did not seem to play a role in temozolomide-induced senescence. In addition to p21, the NF-κB pathway was required for senescence, which was accompanied by induction of the senescence-associated secretory phenotype. Upon temozolomide exposure, we found a strong repression of the mismatch repair proteins MSH2, MSH6, and EXO1 as well as the homologous recombination protein RAD51, which was downregulated by disruption of the E2F1/DP1 complex. Repression of these repair factors was not observed in G2-M arrested p53-deficient cells and, therefore, it seems to represent a specific trait of temozolomide-induced senescence. SIGNIFICANCE: These findings reveal a mechanism by which the anticancer drug temozolomide induces senescence and downregulation of DNA repair pathways in glioma cells.

Multiple DNA damage-dependent and DNA damage-independent stress responses define the outcome of ATR/Chk1 targeting in medulloblastoma cells.

Targeting of oncogene-driven replicative stress as therapeutic option for high-risk medullobastoma was assessed using a panel of medulloblastoma cells differing in their c-Myc expression [i.e. group SHH (c-Myc low) vs. group 3 (c-Myc high)]. High c-Myc levels were associated with hypersensitivity to pharmacological Chk1 and ATR inhibition but not to CDK inhibition nor to conventional (genotoxic) anticancer therapeutics. The enhanced sensitivity of group 3 medulloblastoma cells to Chk1 inhibitors likely results from enhanced damage to intracellular organelles, elevated replicative stress and DNA damage and activation of apoptosis/necrosis. Furthermore, Chk1 inhibition differentially affected c-Myc expression and functions. In c-Myc high cells, Chk1 blockage decreased c-Myc and p-GSK3α protein and increased p21 and GADD45A mRNA expression. By contrast, c-Myc low cells revealed increased p-GSK3ß protein and CHOP and DUSP1 mRNA levels. Inhibition of Chk1 sensitized medulloblastoma cells to additional replication stress evoked by cisplatin independent of c-Myc. Importantly, Chk1 inhibition only caused minor toxicity in primary rat neurons in vitro. Collectively, targeting of ATR/Chk1 effectively triggers death in high-risk medulloblastoma, potentiates the anticancer efficacy of cisplatin and is well tolerated in non-cancerous neuronal cells.

XRCC3 contributes to temozolomide resistance of glioblastoma cells by promoting DNA double-strand break repair.

Glioblastoma is the most frequent and aggressive form of high-grade malignant glioma. Due to the dismal prognosis faced by patients suffering from this disease, there is a need for identifying new targets that might improve therapy. The aim of this study was to determine the contribution of the DNA double-strand break (DSB) repair protein X-ray repair cross-complementing 3 (XRCC3) to the resistance of glioma cells to the chemotherapeutic drug temozolomide. Analysis of a publicly available database, E-GEOD-4290, showed that gliomas overexpress XRCC3 (NM_005432) compared to normal brain tissue. Using an isogenic glioma cell system, in which XRCC3 was downregulated by interference RNA, we demonstrate that XRCC3 protects glioma cells against temozolomide-induced reproductive cell death, apoptosis and cell cycle inhibition. Furthermore, XRCC3 knockdown significantly reduced the rate of repair of DSBs following TMZ treatment, which results in increased drug sensitivity. This study confirms the importance of homologous recombination in the resistance of glioma cells to the methylating drug temozolomide and adds XRCC3 to the list of homology-directed DNA repair proteins as possible targets for therapeutic intervention.

Chloroethylating nitrosoureas in cancer therapy: DNA damage, repair and cell death signaling.

Chloroethylating nitrosoureas (CNU), such as lomustine, nimustine, semustine, carmustine and fotemustine are used for the treatment of malignant gliomas, brain metastases of different origin, melanomas and Hodgkin disease. They alkylate the DNA bases and give rise to the formation of monoadducts and subsequently interstrand crosslinks (ICL). ICL are critical cytotoxic DNA lesions that link the DNA strands covalently and block DNA replication and transcription. As a result, S phase progression is inhibited and cells are triggered to undergo apoptosis and necrosis, which both contribute to the effectiveness of CNU-based cancer therapy. However, tumor cells resist chemotherapy through the repair of CNU-induced DNA damage. The suicide enzyme O6-methylguanine-DNA methyltransferase (MGMT) removes the precursor DNA lesion O6-chloroethylguanine prior to its conversion into ICL. In cells lacking MGMT, the formed ICL evoke complex enzymatic networks to accomplish their removal. Here we discuss the mechanism of ICL repair as a survival strategy of healthy and cancer cells and DNA damage signaling as a mechanism contributing to CNU-induced cell death. We also discuss therapeutic implications and strategies based on sequential and simultaneous treatment with CNU and the methylating drug temozolomide.

Assessing the Effect of Class I Histone Deacetylase Activity on DNA Double-Strand Break Repair by Homologous Recombination.

Here we describe the method used in our laboratory for determining the activity of homologous recombination repair of DNA double-strand breaks in cell lines. This plasmid-based method, first published by Pierce et al. 1999 from Maria Jasin's laboratory, is used along with flow cytometry for demonstrating the positive regulation of class I histone deacetylases on the repair of DNA double-strand breaks by homologous recombination.

DNA damage and the balance between survival and death in cancer biology.

DNA is vulnerable to damage resulting from endogenous metabolites, environmental and dietary carcinogens, some anti-inflammatory drugs, and genotoxic cancer therapeutics. Cells respond to DNA damage by activating complex signalling networks that decide cell fate, promoting not only DNA repair and survival but also cell death. The decision between cell survival and death following DNA damage rests on factors that are involved in DNA damage recognition, and DNA repair and damage tolerance, as well as on factors involved in the activation of apoptosis, necrosis, autophagy and senescence. The pathways that dictate cell fate are entwined and have key roles in cancer initiation and progression. Furthermore, they determine the outcome of cancer therapy with genotoxic drugs. Understanding the molecular basis of these pathways is important not only for gaining insight into carcinogenesis, but also in promoting successful cancer therapy. In this Review, we describe key decision-making nodes in the complex interplay between cell survival and death following DNA damage.

The inducible E3 ubiquitin ligases SIAH1 and SIAH2 perform critical roles in breast and prostate cancers.

The ubiquitin-dependent proteasomal degradation of proteins controls signaling and cellular survival. An increasing body of evidence suggests that the E3 ubiquitin ligases SIAH1 and SIAH2 are able to dictate the growth, development, and chemo-/radiosensitivity of breast and prostate cancer cells. Here we review the current knowledge on the impact of SIAHs on breast and prostate tumorigenesis. Furthermore, we summarize how stress, hormones, and cytokines regulate SIAH1 and SIAH2 in transformed mammalian cells.

Cytolethal distending toxin (CDT) is a radiomimetic agent and induces persistent levels of DNA double-strand breaks in human fibroblasts.

Cytolethal distending toxin (CDT) is a unique genotoxin produced by several pathogenic bacteria. The tripartite protein toxin is internalized into mammalian cells via endocytosis followed by retrograde transport to the ER. Upon translocation into the nucleus, CDT catalyzes the formation of DNA double-strand breaks (DSBs) due to its intrinsic endonuclease activity. In the present study, we compared the DNA damage response (DDR) in human fibroblasts triggered by recombinant CDT to that of ionizing radiation (IR), a well-known DSB inducer. Furthermore, we dissected the pathways involved in the detection and repair of CDT-induced DNA lesions. qRT-PCR array-based mRNA and western blot analyses showed a partial overlap in the DDR pattern elicited by CDT and IR, with strong activation of both the ATM-Chk2 and the ATR-Chk1 axis. In line with its in vitro DNase I-like activity on plasmid DNA, neutral and alkaline Comet assay revealed predominant induction of DSBs in CDT-treated fibroblasts, whereas irradiation of cells generated higher amounts of SSBs and alkali-labile sites. Using confocal microscopy, the dynamics of the DSB surrogate marker γ-H2AX was monitored after pulse treatment with CDT or IR. In contrast to the fast induction and disappearance of γ-H2AX-foci observed in irradiated cells, the number of γ-H2AX-foci induced by CDT were formed with a delay and persisted. 53BP1 foci were also generated following CDT treatment and co-localized with γ-H2AX foci. We further demonstrated that ATM-deficient cells are very sensitive to CDT-induced DNA damage as reflected by increased cell death rates with concomitant cleavage of caspase-3 and PARP-1. Finally, we provided novel evidence that both homologous recombination (HR) and non-homologous end joining (NHEJ) protect against CDT-elicited DSBs. In conclusion, the findings suggest that CDT functions as a radiomimetic agent and, therefore, is an attractive tool for selectively inducing persistent levels of DSBs and unveiling the associated cellular responses.

Targeting components of the alternative NHEJ pathway sensitizes KRAS mutant leukemic cells to chemotherapy.

Activating KRAS mutations are detected in a substantial number of hematologic malignancies. In a murine T-cell acute lymphoblastic leukemia (T-ALL) model, we previously showed that expression of oncogenic Kras induced a premalignant state accompanied with an arrest in T-cell differentiation and acquisition of somatic Notch1 mutations. These findings prompted us to investigate whether the expression of oncogenic KRAS directly affects DNA damage repair. Applying divergent, but complementary, genetic approaches, we demonstrate that the expression of KRAS mutants is associated with increased expression of DNA ligase 3α, poly(ADP-ribose) polymerase 1 (PARP1), and X-ray repair cross-complementing protein 1 (XRCC1), all essential components of the error-prone, alternative nonhomologous end-joining (alt-NHEJ) pathway. Functional studies revealed delayed repair kinetics, increased misrepair of DNA double-strand breaks, and the preferential use of microhomologous DNA sequences for end joining. Similar effects were observed in primary murine T-ALL blasts. We further show that KRAS-mutated cells, but not KRAS wild-type cells, rely on the alt-NHEJ repair pathway on genotoxic stress. RNA interference-mediated knockdown of DNA ligase 3α abolished resistance to apoptotic cell death in KRAS-mutated cells. Our data indicate that targeting components of the alt-NHEJ pathway sensitizes KRAS-mutated leukemic cells to standard chemotherapeutics and represents a promising approach for inducing synthetic lethal vulnerability in cells harboring otherwise nondruggable KRAS mutations.

B-Raf inhibitor vemurafenib in combination with temozolomide and fotemustine in the killing response of malignant melanoma cells.

In the treatment of metastatic melanoma, a highly therapy-refractory cancer, alkylating agents are used and, for the subgroup of BRAFV600E cancers, the B-Raf inhibitor vemurafenib. Although vemurafenib is initially beneficial, development of drug resistance occurs leading to tumor relapse, which necessitates the requirement for combined or sequential therapy with other drugs, including genotoxic alkylating agents. This leads to the question whether vemurafenib and alkylating agents act synergistically and whether chronic vemurafenib treatment alters the melanoma cell response to alkylating agents. Here we show that a) BRAFV600E melanoma cells are killed by vemurafenib, driving apoptosis, b) BRAFV600E melanoma cells are neither more resistant nor sensitive to temozolomide/fotemustine than non-mutant cells, c) combined treatment with vemurafenib plus temozolomide or fotemustine has an additive effect on cell kill, d) acquired vemurafenib resistance of BRAFV600E melanoma cells does not affect MGMT, MSH2, MSH6, PMS2 and MLH1, nor does it affect the resistance to temozolomide and fotemustine, e) metastatic melanoma biopsies obtained from patients prior to and after vemurafenib treatment did not show a change in the MGMT promoter methylation status and MGMT expression level. The data suggest that consecutive treatment with vemurafenib and alkylating drugs is a reasonable strategy for metastatic melanoma treatment.