BackgroundChildren 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.MethodsFourteen 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.ResultsAra-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.ConclusionsWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-017-0493-5) contains supplementary material, which is available to authorized users.

Ewing sarcoma is the second most common cancer of bone and soft tissue arising in children and young adults. Although the survival rate has improved for patients treated for localized disease, the survival rate for patients with metastatic tumor remains lower than 30%. In order to identify novel therapeutic targets and to better understand the genes involved in growth and survival of Ewing sarcoma, we employed a functional genomics approach based on siRNA screening. Four Ewing sarcoma cell lines, TC-32, TC-71, SK-ES-1 and RD-ES, were transfected with a library of siRNA targeting 287 cancer-associated genes. The resulting siRNA screening data for each cell line were normalized and statistical cut-offs were determined. The results indicated that siRNAs targeting Fibroblast Growth Factor Receptor 4, FGFR4 were among the most effective in reducing cell viability in all four of the Ewing sarcoma cell lines. Validation of the siRNA screens showed that siRNAs to FGFR4, reduced viability much greater that those to FGFR1, FGFR2, or FGFR3. Furthermore, siRNA targeting FGFR4 were able to induce caspase 3 activity. FGFR4 protein is expressed on Ewing sarcoma cells as determined by western blot analysis, although expression levels were lower compared to FGFR4 expression on rhabdomyosarcoma cells. Targeting FGFR activity in Ewing sarcoma cells using a pan-FGFR inhibitors PD-173074 and BGJ-398 demonstrated that Ewing sarcoma cells were sensitive to FGFR inhibition. Furthermore, treatment of Ewing sarcoma cells with the selective FGFR4 inhibitor BLU9931 resulted in growth inhibition and decreased ERK signaling. These results indicate that FGFR4 may play an important role in growth and survival of Ewing sarcoma and could serve as a potential therapeutic target for this disease. Citation Format: Justin J. Montoya, Daniel H. Wai, David W. Lee, Peter A. Azorsa, Oliver B. Pepper, Robert J. Arceci, David O. Azorsa. A role for FGFR4 in growth and survival of Ewing sarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3338. doi:10.1158/1538-7445.AM2017-3338

Background: Acute myeloid leukemia (AML) continues to have an overall poor prognosis of about 50% in children. Treatment options for relapsed or refractory AML are limited. Bortezomib, a well-established proteasome inhibitor, is used in AML therapy. It binds to the beta 5 subunit (PSMB5) of the 26S proteasome and inhibits the chymotrypsin (CT)-like activity. Although patients develop resistance to bortezomib, the mechanism is not fully elucidated. Disulfiram (DS) is an aldehyde dehydrogenase (ALDH) inhibitor used to treat alcoholism without major side effects. DS has anticancer cytotoxicity that is in part copper (Cu2+)-dependent (DS/Cu2+). One of the reported mechanisms of action of DS is through proteasome inhibition. We previously demonstrated that both DS/Cu2+ and bortezomib are cytotoxic in a panel of AML cell lines. In the present study, we focused on two acute megakaryoblastic leukemia cell lines derived from patients with Down syndrome (CMY and CMK), one of the cell lines is resistant to cytarabine (CMY), a key drug used in AML treatment. Down syndrome children with leukemia are more prone to suffer from significant toxicity to chemotherapy; therefore, the need for novel treatment options for this category of patients is warranted. Purpose: The purpose of the present study was to develop an in vitro model of bortezomib resistance in CMY and CMK cell lines, to understand the mechanism of resistance, and to identify drugs that are cytotoxic in the bortezomib-resistant cell lines. Methods and Results: The bortezomib-resistant cell lines CMY and CMK were generated by exposure to stepwise increasing concentrations of bortezomib over a period of 9 months up to a concentration of 200 nM in CMY, and 100 nM in CMK cells. Viability was determined using Cell Titer Glo assay. CMY B200nM was 70 fold resistant to bortezomib with decreased inhibition of CT-like activity compared to the parent CMY cell line. Similar results were observed in the CMK cell line. An A362C mutation in PSMB5 causing a change from glutamine to proline was found in the bortezomib resistant CMY cell line. In order to confirm that this mutation caused bortezomib resistance, the PSMB5 (A362C) from CMY B200nM was cloned into the pcDNA3.1 vector and transfected into HEK 293A cells. This caused a two-fold higher bortezomib resistance than the corresponding control. The bortezomib-resistant AML cell lines were cross-resistant to the proteasome inhibitors carfilzomib and MG132 compared to the parent cell line, but were sensitive to DS/Cu2+. Moreover, the PSMB5 mRNA levels and the beta 5-subunit protein levels increased with bortezomib resistance , as evaluated by quantitative real-time RT-PCR and western blot, respectively. Although DS/Cu2+ induced ubiquitination similar to bortezomib, no comparable inhibition of CT-like activity was observed, suggesting a different mechanism of proteasome inhibition. Conclusions: The present results show that (1) the A362C mutation in the PSMB5 may underlie bortezomib-resistance in the cytarabine-resistant CMY cell line. This may provide an explanation for resistance to bortezomib in AML patients, which we hope to assess in the ongoing COG AML trial in which patients are randomized to receive bortezomib plus chemotherapy versus chemotherapy alone, and (2) DS/Cu2+ sensitivity may provide an effective and less toxic treatment approach for AML to be tested in the Down syndrome group of patients with poor outcome following relapse. Citation Format: Ranjan Bista, Oliver Pepper, David W. Lee, David A. Azorsa, Robert J. Arceci, Eiman Aleem. Bortezomib-resistant pediatric acute myeloid leukemia cell lines derived from Down syndrome patients are sensitive to disulfiram. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B02.