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 longterm remission in adult zebrafish with cMYCinduced 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. (Blood. 2012;119(24):5621-5631) IntroductionThe yearly incidence in the US for all leukemia types, including acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and chronic myelogenous leukemia (CML), was estimated at more than 40 000 men and women in 2010, with a yearly death rate of 50%. 1 More than 2000 cases of ALL are diagnosed in US children every year, making it the most common childhood cancer. 2 T-cell ALL (T-ALL) represents approximately 15% and 25% of pediatric and adult ALL cases, respectively. 3 Although leukemia treatment has become increasingly efficient over the past 50 years, mortality from ALL is still 20% for children and more than 40% for adults, and T-ALL has been more difficult to treat than B-cell ALL (B-ALL). 4 Currently, research efforts are devoted to molecularbased risk stratification of patients and the development of targeted therapies to limit side effects [5][6][7] and to increase treatment efficacy.Development of targeted cancer therapies typically requires knowledge of the molecular target. 8 In the absence thereof, an alternative approach may use a robust readout designed to screen large numbers of compounds for specific effects 9 against the malignant cell type in question. More than 50% of patients with T-ALL have deregulated NOTCH1, 10 and in a recent study 47% had mutations in the PI3 kinase/AKT/mTOR (P/A/mT) pathway. 11 NOTCH1 signaling requires proteolytic cleavage by ␥-secretase and other proteases 12 to release the intracytoplasmic domain, providing severalpotential targets for therapeutic intervention. Targeted treatment approaches for T-ALL using...

Summary Children and adolescents with Burkitt Lymphoma (BL) and combined central nervous system (CNS) and bone marrow involvement still have a poor prognosis with chemotherapy alone. We therefore investigated in children and adolescents with bone marrow (≥25% blasts) and/or CNS-positive Burkitt lymphoma the chemoimmunotherapy combination of rituximab (375 mg/m2) and the standard chemotherapy arm of our previously reported French-American-British (FAB) Lymphome Malins de Burkitt (LMB) 96 trial. Central pathological and cytogenetic characterization was also performed. There were 40 evaluable patients with Burkitt histology (25 with leukaemia and 15 with CNS disease ± leukaemia). The chemoimmunotherapy regimen was well tolerated. The incidence of grade III/IV mucositis during induction cycles with combined chemotherapy and rituximab was 31% and 26%, respectively. The 3-year event-free survival (EFS)/overall survival (OS) was 90% (95% confidence interval [CI], 76–96%) in the entire cohort and 93% (95% CI, 61–99%) in patients with CNS disease. Based on the results of this trial, an international randomized study of FAB/LMB 96 chemotherapy ± rituximab for high-risk patients is currently under investigation.

T cell neoplasias are common in pediatric oncology, and include acute lymphoblastic leukemia (T-ALL) and lymphoblastic lymphoma (T-LBL). These cancers have worse prognoses than their B cell counterparts, and their treatments carry significant morbidity. While many pediatric malignancies have characteristic translocations, most T lymphocyte-derived diseases lack cytogenetic hallmarks. Lacking these informative lesions, insight into their molecular pathogenesis is less complete. Although dysregulation of the NOTCH1 pathway occurs in a substantial fraction of cases, many other genetic lesions of T cell malignancy have not yet been determined. To address this deficiency, we pioneered a phenotype-driven forward-genetic screen in zebrafish (Danio rerio). Using transgenic fish with T lymphocyte-specific expression of enhanced green fluorescent protein (EGFP), we performed chemical mutagenesis, screened animals for GFP+ tumors, and identified multiple lines with a heritable predisposition to T cell malignancy. In each line, patterns of infiltration and morphologic appearance resembled human T-ALL and T-LBL. T cell receptor analyses confirmed their clonality. Malignancies were transplantable and contained leukemia-initiating cells (LIC), like their human correlates. In summary, we have identified multiple zebrafish mutants that recapitulate human T cell neoplasia and show heritable transmission. These vertebrate models provide new genetic platforms for the study of these important human cancers.