Disulfiram overcomes bortezomib and cytarabine resistance in Down-syndrome-associated acute myeloid leukemia cells

Background Children with Down syndrome (DS) have increased risk for developing AML (DS-AMKL), and they usually experience severe therapy-related toxicities compared to non DS-AMKL. Refractory/relapsed disease has very poor outcome, and patients would benefit from novel, less toxic, therapeutic strategies that overcome resistance. Relapse/resistance are linked to cancer stem cells with high aldehyde dehydrogenase (ALDH) activity. The purpose of the present work was to study less toxic alternative therapeutic agents for relapsed/refractory DS-AMKL. Methods Fourteen AML cell lines including the DS-AMKL CMY and CMK from relapsed/refractory AML were used. Cytarabine (Ara-C), bortezomib (BTZ), disulfiram/copper (DSF/Cu2+) were evaluated for cytotoxicity, depletion of ALDH-positive cells, and resistance. BTZ-resistant CMY and CMK variants were generated by continuous BTZ treatment. Cell viability was assessed using CellTiter-Glo®, ALDH activity by ALDELUORTM, and proteasome inhibition by western blot of ubiquitinated proteins and the Proteasome-Glo™ Chymotrypsin-Like (CT-like) assay, apoptosis by Annexin V Fluos/Propidium iodide staining, and mutations were detected using PCR, cloning and sequencing. Results Ara-C-resistant AML cell lines were sensitive to BTZ and DSF/Cu2+. The Ara-C-resistant DS-AMKL CMY cells had a high percentage of ALDHbright “stem-like” populations that may underlie Ara-C resistance. One percent of these cells were still resistant to BTZ but sensitive to DSF/Cu2+. To understand the mechanism of BTZ resistance, BTZ resistant (CMY-BR) and (CMK-BR) were generated. A novel mutation PSMB5 Q62P underlied BTZ resistance, and was associated with an overexpression of the β5 proteasome subunit. BTZ-resistance conferred increased resistance to Ara-C due to G1 arrest in the CMY-BR cells, which protected the cells from S-phase damage by Ara-C. CMY-BR and CMK-BR cells were cross-resistant to CFZ and MG-132 but sensitive to DSF/Cu2+. In this setting, DSF/Cu2+ induced apoptosis and proteasome inhibition independent of CT-like activity inhibition. Conclusions We provide evidence that DSF/Cu2+ overcomes Ara-C and BTZ resistance in cell lines from DS-AMKL patients. A novel mutation underlying BTZ resistance was detected that may identify BTZ-resistant patients, who may not benefit from treatment with CFZ or Ara-C, but may be responsive to DSF/Cu2+. Our findings support the clinical development of DSF/Cu2+ as a less toxic efficacious treatment approach in patients with relapsed/refractory DS-AMKL

NCI60 Cancer Cell Line Panel Data and RNAi Analysis Help Identify EAF2 as a Modulator of Simvastatin and Lovastatin Response in HCT-116 Cells

Simvastatin and lovastatin are statins traditionally used for lowering serum cholesterol levels. However, there exists evidence indicating their potential chemotherapeutic characteristics in cancer. In this study, we used bioinformatic analysis of publicly available data in order to systematically identify the genes involved in resistance to cytotoxic effects of these two drugs in the NCI60 cell line panel. We used the pharmacological data available for all the NCI60 cell lines to classify simvastatin or lovastatin resistant and sensitive cell lines, respectively. Next, we performed whole-genome single marker case-control association tests for the lovastatin and simvastatin resistant and sensitive cells using their publicly available Affymetrix 125K SNP genomic data. The results were then evaluated using RNAi methodology. After correction of the p-values for multiple testing using False Discovery Rate, our results identified three genes (NRP1, COL13A1, MRPS31) and six genes (EAF2, ANK2, AKAP7, STEAP2, LPIN2, PARVB) associated with resistance to simvastatin and lovastatin, respectively. Functional validation using RNAi confirmed that silencing of EAF2 expression modulated the response of HCT-116 colon cancer cells to both statins. In summary, we have successfully utilized the publicly available data on the NCI60 cell lines to perform whole-genome association studies for simvastatin and lovastatin. Our results indicated genes involved in the cellular response to these statins and siRNA studies confirmed the role of the EAF2 in response to these drugs in HCT-116 colon cancer cells

Hepatocyte Permissiveness to Plasmodium Infection Is Conveyed by a Short and Structurally Conserved Region of the CD81 Large Extracellular Domain

Invasion of hepatocytes by Plasmodium sporozoites is a prerequisite for establishment of a malaria infection, and thus represents an attractive target for anti-malarial interventions. Still, the molecular mechanisms underlying sporozoite invasion are largely unknown. We have previously reported that the tetraspanin CD81, a known receptor for the hepatitis C virus (HCV), is required on hepatocytes for infection by sporozoites of several Plasmodium species. Here we have characterized CD81 molecular determinants required for infection of hepatocytic cells by P. yoelii sporozoites. Using CD9/CD81 chimeras, we have identified in CD81 a 21 amino acid stretch located in a domain structurally conserved in the large extracellular loop of tetraspanins, which is sufficient in an otherwise CD9 background to confer susceptibility to P. yoelii infection. By site-directed mutagenesis, we have demonstrated the key role of a solvent-exposed region around residue D137 within this domain. A mAb that requires this region for optimal binding did not block infection, in contrast to other CD81 mAbs. This study has uncovered a new functionally important region of CD81, independent of HCV E2 envelope protein binding domain, and further suggests that CD81 may not interact directly with a parasite ligand during Plasmodium infection, but instead may regulate the function of a yet unknown partner protein

CD9 participates in endothelial cell migration during in vitro wound repair

CD9, a widely expressed membrane protein of the tetraspanin family, has been implicated in diverse functions, such as signal transduction, cell adhesion, and cell motility. We tested the effects of an anti-CD9 monoclonal antibody (ALMA.1) on the migration and proliferation of human vascular endothelial cells (ECs) during repair of an in vitro mechanical wound mimicking angiogenic processes. ALMA.1 induced dose-dependent inhibition of wound repair with a 35±1.5% decrease at 20 μg/mL. Only cell migration was affected, because the rate of proliferation of ECs at the lesion margin was not modified and because the inhibition of repair was also observed for nonproliferating irradiated ECs. Monoclonal antibodies against CD63 tetraspanin (H5C6) and control mouse IgG (MOPC-21) were inactive. CD9, one of the most abundant proteins at the surface of ECs, colocalized with β1 or β3 integrins on EC membranes in double-labeling immunofluorescence experiments with ALMA.1 and an anti-β1 (4B4) or anti-β3 (SDF.3) monoclonal antibody. Moreover, ALMA.1 and 4B4 had additive inhibitory effects on lesion repair, whereas 4B4 alone also inhibited EC proliferation. In transmembrane Boyden-type assays, ALMA.1 induced dose-dependent inhibition of EC migration toward fibronectin and vitronectin with 45±6% and 31±10% inhibition, respectively, at 100 μg/mL. 4B4 inhibited migration toward fibronectin at 10 μg/mL but had no effect in the case of vitronectin. Adhesion of ECs to immobilized anti-CD9 monoclonal antibodies induced tyrosine-phosphorylated protein levels similar to those observed during interactions with β1 or β3 integrins. These results point to the involvement of CD9 in EC adhesion and migration during lesion repair and angiogenesis, probably through cooperation with integrins. As such, CD9 is a potential target to inhibit angiogenesis in metastatic and atherosclerotic processes

Clinical resistance associated with a novel MAP2K1 mutation in a patient with Langerhans cell histiocytosis

Patients with Langerhans cell histiocytosis (LCH) harbor BRAF V600E and activating mutations of MAP2K1/MEK1 in 50% and 25% of cases, respectively. We evaluated a patient with treatment‐refractory LCH for mutations in the RAS‐RAF‐MEK‐ERK pathway and identified a novel mutation in the MAP2K1 gene resulting in a p.L98_K104 > Q deletion and predicted to be auto‐activating. During treatment with the MEK inhibitor trametinib, the patient's disease showed significant progression. In vitro characterization of the MAP2K1 p.L98_K104 > Q deletion confirmed its effect on cellular activation of the ERK pathway and drug resistance