Doctor of Philosophy

dissertationAcute lymphoblastic leukemia (ALL) is the most common cancer of childhood, with approximately 2000 cases diagnosed annually in the US. Although cure rates for childhood ALL are currently ~80%, T-cell ALL (T-ALL) is still more difficult to treat than B-cell ALL, requiring harsher treatments with concomitant harsher side effects. The goal of this study was to identify more targeted therapies for treating T-ALL with the intent of reducing harsh treatment side effects, thus preserving both lives and long-term quality of life. To meet this goal, 26,400 compounds from the ChemBridge library were screened utilizing zebrafish larvae since they have the combined attributes of vertebrate physiology and small size. The transgenic lck:eGFP zebrafish line with T-cell specific GFP was chosen since compounds which eliminate immature T-cells in the thymus might also eliminate developmentally arrested leukemic blasts. The screen identified five "hit" compounds that cause reduction in GFP without sickening the larvae or causing general cell cycle effects. Of these five compounds, one compound, "Lenaldekar" (LDK), was effective in killing human Jurkat T-ALL without harming healthy lymphocytes. In vivo, LDK shows efficacy in treating leukemia in both zebrafish and mouse xenograft models of T-ALL without observable toxicity or endorgan damage. Furthermore, expanded leukemia testing showed that T-ALL, B-ALL, and CML are all largely LDK-sensitive, including most treatment-refractory relapsed Ph+ leukemias and primary patient samples. Moreover, some AML and multiple myeloma cell lines also show LDK sensitivity. Molecular characterization shows that LDK down-regulates the PI3K/AKT/mTOR (P/A/mT) pathway, which pathway is up-regulated in ~50% of T-ALL cases. Recent results suggest that LDK may achieve this effect via inactivation of the insulin-like growth factor 1 receptor (IGF1-R), which activates the P/A/mT pathway. In addition, LDK treatment elicits a second activity of G2/M arrest in most sensitive cell lines, which arrest appears to be independent of P/A/mT pathway inhibition. Future directions include identifying and modeling LDK's direct biochemical target(s) with the intent of utilizing structure-activity relationships to optimize LDK's chemical structure and efficacy. This study's ultimate goal is to bring LDK into clinical trials for the treatment of T-ALL in both monotherapy and combination therapy applications

Zebrafish screen identifies novel compound with selective toxicity against leukemia

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