David M. Langenau

I. Uncover the genetic programs underlying self-renewal of rhabdomyosarcoma cancer stem cells

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood and affects over 350 new patients each year in the United States. Embryonal RMS (ERMS), the most common subtype of pediatric RMS, is not only morphologically distinct from the other major pediatric subtype (alveolar rhabdomyosarcoma) but is also transformed by different molecular mechanisms. For example, our analysis of human ERMS suggests that the RAS pathway is activated in a majority of tumors by yet undiscovered mechanisms.

We have recently shown that expression of activated k-RAS in early muscle cells is sufficient to induce disease in transgenic zebrafish. These tumors are morphologically similar to human embryonal rhabdomyosarcoma and express the clinical diagnostic markers of this malignancy. Using fluorescence activated cell sorting and cell transplantation, we have demonstrated the existence of a cancer stem cell in this disease, a cell that is most similar to a non-transformed, activated muscle satellite cell. Using fluorescent transgenic zebrafish and cutting-edge live cell imaging, we are now visualizing ERMS development in real-time. Moreover, our fluorescent transgenic approaches allow for the identification of malignant cell sub-types and should afford unprecedented opportunities to visualize self-renewal within a tumor directly. Using conditional transgenic approaches and chemical genetics, we are currently interrogating which pathways modulate self-renewal, cancer stem cell number, and tumor growth in ERMS. Uncovering downstream molecular pathways that regulate self-renewal will be integral to identifying novel drugs for the treatment of this disease.

II. Identify the self-renewal programs in T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is a devastating disease of childhood, accounting for approximately 20% of pediatric ALLs and is associated with transformation of thymic precursor cells. The mechanisms governing self-renewal in this disease are largely unknown. Using a transgenic zebrafish model of T-ALL, we are currently using large-scale transplantation experiments to functionally assess self-renewal and tumor growth. Our results in Myc-induced T-ALL reveal that leukemia-initiating cell number is quite high in primary leukemias (0.45%-15.9%), signifying that self-renewal is a more common attribute in malignant T-ALL cells than previously suggested.

This observation was confirmed by engraftment of single cells into recipient animals with 7 of 44 recipient animals developing T-ALL. Additionally, serial passage of T-ALL cells into transplant recipients can lead to evolution of tumors with increased percentages of tumor-initiating cells. These experiments can be explained by two models: 1) either selection pressures favor clones with increased leukemia-initiating potential or 2) clones continue to amass genetic or epigenetic modifications that led to increased self-renewal. These two models are being tested experimentally in the laboratory. Together, these experiments establish the unprecedented opportunity to assess gene pathways involved in leukemia self-renewal that do not exist in more established vertebrate models of disease.