FDA-approved disulfiram as a novel treatment for aggressive leukemia.

Acute leukemia continues to be a major cause of death from disease worldwide and current chemotherapeutic agents are associated with significant morbidity in survivors. While better and safer treatments for acute leukemia are urgently needed, standard drug development pipelines are lengthy and drug repurposing therefore provides a promising approach. Our previous evaluation of FDA-approved drugs for their antileukemic activity identified disulfiram, used for the treatment of alcoholism, as a candidate hit compound. This study assessed the biological effects of disulfiram on leukemia cells and evaluated its potential as a treatment strategy. We found that disulfiram inhibits the viability of a diverse panel of acute lymphoblastic and myeloid leukemia cell lines (n = 16) and patient-derived xenograft cells from patients with poor outcome and treatment-resistant disease (n = 15). The drug induced oxidative stress and apoptosis in leukemia cells within hours of treatment and was able to potentiate the effects of daunorubicin, etoposide, topotecan, cytarabine, and mitoxantrone chemotherapy. Upon combining disulfiram with auranofin, a drug approved for the treatment of rheumatoid arthritis that was previously shown to exert antileukemic effects, strong and consistent synergy was observed across a diverse panel of acute leukemia cell lines, the mechanism of which was based on enhanced ROS induction. Acute leukemia cells were more sensitive to the cytotoxic activity of disulfiram than solid cancer cell lines and non-malignant cells. While disulfiram is currently under investigation in clinical trials for solid cancers, this study provides evidence for the potential of disulfiram for acute leukemia treatment. KEY MESSAGES: Disulfiram induces rapid apoptosis in leukemia cells by boosting oxidative stress. Disulfiram inhibits leukemia cell growth more potently than solid cancer cell growth. Disulfiram can enhance the antileukemic efficacy of chemotherapies. Disulfiram strongly synergises with auranofin in killing acute leukemia cells by ROS induction. We propose testing of disulfiram in clinical trial for patients with acute leukemia.

Combination drug screen targeting glioblastoma core vulnerabilities reveals pharmacological synergisms.

BACKGROUND: Pharmacological synergisms are an attractive anticancer strategy. However, with more than 5000 approved-drugs and compounds in clinical development, identifying synergistic treatments represents a major challenge. METHODS: High-throughput screening was combined with target deconvolution and functional genomics to reveal targetable vulnerabilities in glioblastoma. The role of the top gene hit was investigated by RNA interference, transcriptomics and immunohistochemistry in glioblastoma patient samples. Drug combination screen using a custom-made library of 88 compounds in association with six inhibitors of the identified glioblastoma vulnerabilities was performed to unveil pharmacological synergisms. Glioblastoma 3D spheroid, organotypic ex vivo and syngeneic orthotopic mouse models were used to validate synergistic treatments. FINDINGS: Nine targetable vulnerabilities were identified in glioblastoma and the top gene hit RRM1 was validated as an independent prognostic factor. The associations of CHK1/MEK and AURKA/BET inhibitors were identified as the most potent amongst 528 tested pairwise drug combinations and their efficacy was validated in 3D spheroid models. The high synergism of AURKA/BET dual inhibition was confirmed in ex vivo and in vivo glioblastoma models, without detectable toxicity. INTERPRETATION: Our work provides strong pre-clinical evidence of the efficacy of AURKA/BET inhibitor combination in glioblastoma and opens new therapeutic avenues for this unmet medical need. Besides, we established the proof-of-concept of a stepwise approach aiming at exploiting drug poly-pharmacology to unveil druggable cancer vulnerabilities and to fast-track the identification of synergistic combinations against refractory cancers. FUNDING: This study was funded by institutional grants and charities.

In vitro and in vivo drug screens of tumor cells identify novel therapies for high-risk child cancer.

Biomarkers which better match anticancer drugs with cancer driver genes hold the promise of improved clinical responses and cure rates. We developed a precision medicine platform of rapid high-throughput drug screening (HTS) and patient-derived xenografting (PDX) of primary tumor tissue, and evaluated its potential for treatment identification among 56 consecutively enrolled high-risk pediatric cancer patients, compared with conventional molecular genomics and transcriptomics. Drug hits were seen in the majority of HTS and PDX screens, which identified therapeutic options for 10 patients for whom no targetable molecular lesions could be found. Screens also provided orthogonal proof of drug efficacy suggested by molecular analyses and negative results for some molecular findings. We identified treatment options across the whole testing platform for 70% of patients. Only molecular therapeutic recommendations were provided to treating oncologists and led to a change in therapy in 53% of patients, of whom 29% had clinical benefit. These data indicate that in vitro and in vivo drug screening of tumor cells could increase therapeutic options and improve clinical outcomes for high-risk pediatric cancer patients.

High-Content Imaging of Unbiased Chemical Perturbations Reveals that the Phenotypic Plasticity of the Actin Cytoskeleton Is Constrained.

Although F-actin has a large number of binding partners and regulators, the number of phenotypic states available to the actin cytoskeleton is unknown. Here, we quantified 74 features defining filamentous actin (F-actin) and cellular morphology in >25 million cells after treatment with a library of 114,400 structurally diverse compounds. After reducing the dimensionality of these data, only ∼25 recurrent F-actin phenotypes emerged, each defined by distinct quantitative features that could be machine learned. We identified 2,003 unknown compounds as inducers of actin-related phenotypes, including two that directly bind the focal adhesion protein, talin. Moreover, we observed that compounds with distinct molecular mechanisms could induce equivalent phenotypes and that initially divergent cellular responses could converge over time. These findings suggest a conceptual parallel between the actin cytoskeleton and gene regulatory networks, where the theoretical plasticity of interactions is nearly infinite, yet phenotypes in vivo are constrained into a limited subset of practicable configurations.

A novel small molecule that kills a subset of MLL-rearranged leukemia cells by inducing mitochondrial dysfunction.

Survival rates for pediatric patients suffering from mixed lineage leukemia (MLL)-rearranged leukemia remain below 50% and more targeted, less toxic therapies are urgently needed. A screening method optimized to discover cytotoxic compounds selective for MLL-rearranged leukemia identified CCI-006 as a novel inhibitor of MLL-rearranged and CALM-AF10 translocated leukemias that share common leukemogenic pathways. CCI-006 inhibited mitochondrial respiration and induced mitochondrial membrane depolarization and apoptosis in a subset (7/11, 64%) of MLL-rearranged leukemia cell lines within a few hours of treatment. The unresponsive MLL-rearranged leukemia cells did not undergo mitochondrial membrane depolarization or apoptosis despite a similar attenuation of mitochondrial respiration by the compound. In comparison to the sensitive cells, the unresponsive MLL-rearranged leukemia cells were characterized by a more glycolytic metabolic phenotype, exemplified by a more pronounced sensitivity to glycolysis inhibitors and elevated HIF1α expression. Silencing of HIF1α expression sensitized an intrinsically unresponsive MLL-rearranged leukemia cell to CCI-006, indicating that this pathway plays a role in determining sensitivity to the compound. In addition, unresponsive MLL-rearranged leukemia cells expressed increased levels of MEIS1, an important leukemogenic MLL target gene that plays a role in regulating metabolic phenotype through HIF1α. MEIS1 expression was also variable in a pediatric MLL-rearranged ALL patient dataset, highlighting the existence of a previously undescribed metabolic variability in MLL-rearranged leukemia that may contribute to the heterogeneity of the disease. This study thus identified a novel small molecule that rapidly kills MLL-rearranged leukemia cells by targeting a metabolic vulnerability in a subset of low HIF1α/low MEIS1-expressing MLL-rearranged leukemia cells.

Identification of new MRP4 inhibitors from a library of FDA approved drugs using a high-throughput bioluminescence screen.

Multidrug resistance protein 4 (MRP4) effluxes a wide variety of drugs and endogenous signaling molecules from cells and has been proposed as an attractive therapeutic target in several solid tumors, including neuroblastoma and colorectal cancer. MRP4 also regulates the pharmacokinetics of its drug substrates and its absence can increase their tissue penetration. We observed that MRP4 can efflux the bioluminescence substrate d-luciferin, and exploited this phenomenon to develop a robust, high throughput, live cell-based bioluminescent screen to identify new MRP4 inhibitors. We applied this screen to a combined library of 3600 compounds, all of which were either FDA-approved drugs or bioactive compounds with defined mechanisms of action. From the primary screen, 36 compounds effectively inhibited MRP4 (>4-fold increase in bioluminescence), with inhibitors of receptor tyrosine kinases and phosphodiesterases highly over-represented. Selected compounds were tested for their ability to sensitize MRP4-overexpressing cell lines to the MRP4 substrate drugs 6-mercaptopurine and SN-38, with sensitization up to 6.5-fold with the ryanodine receptor antagonist dantrolene. These newly identified MRP4 inhibitors are readily available and are either established drugs or well-characterized bioactive compounds. As such, they should be immediately useful as investigative tools, and suitable for testing both in vitro and in vivo.