Functionally-instructed modifiers of response to ATR inhibition in experimental glioma.

BACKGROUND: The DNA damage response (DDR) is a physiological network preventing malignant transformation, e.g. by halting cell cycle progression upon DNA damage detection and promoting DNA repair. Glioblastoma are incurable primary tumors of the nervous system and DDR dysregulation contributes to acquired treatment resistance. Therefore, DDR targeting is a promising therapeutic anti-glioma strategy. Here, we investigated Ataxia telangiectasia and Rad3 related (ATR) inhibition (ATRi) and functionally-instructed combination therapies involving ATRi in experimental glioma. METHODS: We used acute cytotoxicity to identify treatment efficacy as well as RNAseq and DigiWest protein profiling to characterize ATRi-induced modulations within the molecular network in glioma cells. Genome-wide CRISPR/Cas9 functional genomic screens and subsequent validation with functionally-instructed compounds and selected shRNA-based silencing were employed to discover and investigate molecular targets modifying response to ATRi in glioma cell lines in vitro, in primary cultures ex vivo and in zebrafish and murine models in vivo. RESULTS: ATRi monotherapy displays anti-glioma efficacy in vitro and ex vivo and modulates the molecular network. We discovered molecular targets by genome-wide CRISPR/Cas9 loss-of-function and activation screens that enhance therapeutic ATRi effects. We validated selected druggable targets by a customized drug library and functional assays in vitro, ex vivo and in vivo. CONCLUSION: In conclusion, our study leads to the identification of novel combination therapies involving ATRi that could inform future preclinical studies and early phase clinical trials.

Augmenting MEK inhibitor efficacy in BRAF wild-type melanoma: synergistic effects of disulfiram combination therapy.

BACKGROUND: MEK inhibitors (MEKi) were shown to be clinically insufficiently effective in patients suffering from BRAF wild-type (BRAF WT) melanoma, even if the MAPK pathway was constitutively activated due to mutations in NRAS or NF-1. Thus, novel combinations are needed to increase the efficacy and duration of response to MEKi in BRAF WT melanoma. Disulfiram and its metabolite diethyldithiocarbamate are known to have antitumor effects related to cellular stress, and induction of endoplasmic reticulum (ER) stress was found to synergize with MEK inhibitors in NRAS-mutated melanoma cells. Therefore, we investigated the combination of both therapeutics to test their effects on BRAF-WT melanoma cells and compared them with monotherapy using the MEKi trametinib. METHODS: The effects of combined therapy with disulfiram or its metabolite diethyldithiocarbamate and the MEKi trametinib were evaluated in a series of BRAF-WT melanoma cell lines by measuring cell viability and apoptosis induction. Cytotoxicity was additionally assessed in 3D spheroids, ex vivo melanoma slice cultures, and in vivo xenograft mouse models. The response of melanoma cells to treatment was studied at the RNA and protein levels to decipher the mode of action. Intracellular and intratumoral copper measurements were performed to investigate the role of copper ions in the antitumor cytotoxicity of disulfiram and its combination with the MEKi. RESULTS: Diethyldithiocarbamate enhanced trametinib-induced cytotoxicity and apoptosis induction in 2D and 3D melanoma culture models. Mechanistically, copper-dependent induction of oxidative stress and ER stress led to Janus kinase (JNK)-mediated apoptosis in melanoma cells. This mechanism was also detectable in patient-derived xenograft melanoma models and resulted in a significantly improved therapeutic effect compared to monotherapy with the MEKi trametinib. CONCLUSIONS: Disulfiram and its metabolite represent an attractive pharmaceutical approach to induce ER stress in melanoma cells that potentiates the antitumor effect of MEK inhibition and may be an interesting candidate for combination therapy of BRAF WT melanoma.

Generation and characterization of three induced pluripotent stem cells lines from an 86-year old female individual diagnosed with an invasive lobular mammary carcinoma.

Invasive lobular carcinoma (ILC) is a distinct type of breast cancer and is accounting up to 10-15 % of all mammary carcinomas showing a pronounced increase in incidence rates over the last two decades. We generated three induced pluripotent stem cell (iPSC) lines from CD34+ progenitor cells isolated from a mammary carcinoma patient diagnosed with ILC. Here, we describe the characterization of the iPSCs by array-based comparative genomic hybridization (array CGH), immunocytochemistry, flow cytometry, reverse transcriptase polymerase chain reaction and directed in vitro differentiation. The iPSC lines will find application in the field of breast cancer research.

Therapeutic Efficacy of Pharmacological Ascorbate on Braf Inhibitor Resistant Melanoma Cells In Vitro and In Vivo.

High-dose ascorbate paradoxically acts as a pro-oxidant causing the formation of hydrogen peroxide in an oxygen dependent manner. Tumor cells (in particular melanoma cells) show an increased vulnerability to ascorbate induced reactive oxygen species (ROS). Therefore, high-dose ascorbate is a promising pharmacological approach to treating refractory melanomas, e.g., with secondary resistance to targeted BRAF inhibitor therapy. BRAF mutated melanoma cells were treated with ascorbate alone or in combination with the BRAF inhibitor vemurafenib. Viability, cell cycle, ROS production, and the protein levels of phospho-ERK1/2, GLUT-1 and HIF-1α were analyzed. To investigate the treatment in vivo, C57BL/6NCrl mice were subcutaneously injected with D4M.3A (BrafV600E) melanoma cells and treated with intraperitoneal injections of ascorbate with or without vemurafenib. BRAF mutated melanoma cell lines either sensitive or resistant to vemurafenib were susceptible to the induction of cell death by pharmacological ascorbate. Treatment of BrafV600E melanoma bearing mice with ascorbate resulted in plasma levels in the pharmacologically active range and significantly improved the therapeutic effect of vemurafenib. We conclude that intravenous high-dose ascorbate will be beneficial for melanoma patients by interfering with the tumor's energy metabolism and can be safely combined with standard melanoma therapies such as BRAF inhibitors without pharmacological interference.

Disulfiram as a Therapeutic Agent for Metastatic Malignant Melanoma-Old Myth or New Logos?

New therapeutic concepts such as anti-PD-1-based immunotherapy or targeted therapy with BRAF and MEK inhibitors have significantly improved the survival of melanoma patients. However, about 20% of patients with targeted therapy and up to 50% with immunotherapies do not respond to their first-line treatment or rapidly develop resistance. In addition, there is no approved targeted therapy for certain subgroups, namely BRAF wild-type melanomas, although they often bear aggressive tumor biology. A repurposing of already approved drugs is a promising strategy to fill this gap, as it will result in comparatively low costs, lower risks and time savings. Disulfiram (DSF), the first drug to treat alcoholism, which received approval from the US Food and Drug Administration more than 60 years ago, is such a drug candidate. There is growing evidence that DSF has great potential for the treatment of various human cancers, including melanoma. Several mechanisms of its antitumor activity have been identified, amongst them the inhibition of the ubiquitin-proteasome system, the induction of reactive oxygen species and various death signaling pathways. This article provides an overview of the application of DSF in humans, its molecular mechanisms and targets in cancer therapy with a focus on melanoma. The results of clinical studies and experimental combination approaches of DSF with various cancer therapies are discussed, with the aim of exploring the potential of DSF in melanoma therapy.