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Zebrafish screen identifies novel compound with selective toxicity against leukemia.

ABSTRACT: To detect targeted antileukemia agents we have designed a novel, high-content in vivo screen using genetically engineered, T-cell reporting zebrafish. We exploited the developmental similarities between normal and malignant T lymphoblasts to screen a small molecule library for activity against immature T cells with a simple visual readout in zebrafish larvae. After screening 26 400 molecules, we identified Lenaldekar (LDK), a compound that eliminates immature T cells in developing zebrafish without affecting the cell cycle in other cell types. LDK is well tolerated in vertebrates and induces long-term remission in adult zebrafish with cMYC-induced T-cell acute lymphoblastic leukemia (T-ALL). LDK causes dephosphorylation of members of the PI3 kinase/AKT/mTOR pathway and delays sensitive cells in late mitosis. Among human cancers, LDK selectively affects survival of hematopoietic malignancy lines and primary leukemias, including therapy-refractory B-ALL and chronic myelogenous leukemia samples, and inhibits growth of human T-ALL xenografts. This work demonstrates the utility of our method using zebrafish for antineoplastic candidate drug identification and suggests a new approach for targeted leukemia therapy. Although our efforts focused on leukemia therapy, this screening approach has broad implications as it can be translated to other cancer types involving malignant degeneration of developmentally arrested cells.

SUBMITTER: Ridges S

PROVIDER: S-EPMC3382926 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Purpose: BCR-ABL-positive acute lymphoid leukemia (ALL) has a dire prognosis. Despite improved disease-free survival due to implementation of TKIs in front-line therapy, patient survival remains unsatisfactory and there exists a need for new treatments. Therefore, we performed a drug screen based on competition between untransformed cells and BCR-Abl-transformed cells, and identified several compounds that selectively reduce the competitive fitness of BCR-Abl-transformed cells. Using a systems approach we discovered that one of these compounds, DJ34, induces potent activation of p53 and rapid degradation of c-Myc, which is frequently deregulated in leukemia. DJ34 treatment induced apoptosis, cell cycle arrest and cell differentiation and inhibited survival of primary AML cells, both ex vivo and in a zebrafish PDX model. The goals of this study is to check the effect of DJ34 on transcriptome profile of BCR-Abl expressing BaF3 cells. Methods: mRNA profiles of BaF3 cells expressing BCR-Abl treated with DMSO or 20µM DJ34 for 6hrs were generated by Illumina Hiseq 4000 SE50, in triplicate. Then, clean reads (as provided by BGI) were mapped to the GRCm38.85 genome assembly using Tophat2 (v. 2.1.1) [1] using default options BAM files were indexed using SAMtools [2]. Differential expression data was produced using cuffquant and cuffdiff with default options, from the cufflinks suite (v 2.2.1 / SVN rev 4237) [3]. Reference genome for the primary assembly and gene annotations used are revision 85 of GRCm38, from ensembl.org. The .Bam and .tdf files were used for quantification and visualization in IGV respectively. [1] Kim, Daehwan, et al. "TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions." Genome biology 14.4 (2013): R36. [2] Li, Heng, et al. "The sequence alignment/map format and SAMtools." Bioinformatics 25.16 (2009): 2078-2079. [3] Trapnell, Cole, et al. "Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks." Nature protocols 7.3 (2012): 562. Results: Using data analysis workflow described above, we identified differentially expressed genes in the control and DJ34 treated BaF3 cells expressing BCR-Abl cells. Conclusions: Competition based drug screening has identified DJ34 that selectively erradicates BCR-Abl expressing BaF3 cells. Our RNa-seq basaed transcriptome analysis shows that DJ34 induces potent activation of p53 and rapid degradation of c-Myc, which is frequently deregulated in leukemia.

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Zebrafish screen identifies novel compound with selective toxicity against leukemia.

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