Abstract
Acute lymphoblastic leukemia is the most common malignancy diagnosed in children. High-risk leukemia patients have poor outcomes despite advancements in chemotherapy treatments. Due to current therapy dose limitations, toxicities to normal cells, long term side effects and leukemia cells developing resistance to chemotherapy, there is great need for the development of innovative therapies. Leukemia stem cells (LSCs) are thought to be the precursors to leukemia cells and are suspected to be the cause of leukemia proliferation, metastasis, and relapse. Therefore, it is critical to develop novel therapies to target LSCs specifically. However, due to the lack of ability to identify LSCs, no LSC targeted therapies have been developed. The goal of this project is to identify LSCs in childhood precursor B acute lymphoblastic leukemia (ALL), in order to target these LSCs and spare normal cells, by using a combination of flow cytometry, Raman spectroscopy, and peptide screening. Since recent reports have correlated CD9 with LSCs in acute lymphoblastic leukemia cell lines, primary human ALL cells obtained from the University of California, Davis Medical Center were sorted by flow cytometry based on expression levels of the cell surface marker CD9. Sorted populations were engrafted into immunodeficient mice to analyze their ability to re-initiate leukemia. Sorted candidates subsequently were characterized using the laser-based technique, Raman spectroscopy, in order to determine whether the intrinsic biochemical signatures obtained by Raman spectroscopy could further identify LSCs. Lastly, LSCs and primary ALL samples were screened through one-bead one-compound (OBOC) combinatorial libraries to investigate peptides that will specifically target cell surface marker of LSCs. The OBOC peptides identified will be used to selectively target LSCs using drug loaded nanoparticles. After establishing a mouse model that could successfully engraft human ALL, three primary ALL samples were chosen for our study. When injected with sorted cells, 25%of the CD9 positive injected mice and 14% of the CD9 negative injected mice, developed leukemia. Furthermore, the Raman analysis of these sorted cells showed CD9 positive and CD9 negative cells have very similar intracellular signatures. Together this indicates that CD9 is not a good marker for LSCs in childhood ALL. As for OBOC screening, unsorted primary ALL samples were screened against OBOC peptide libraries and 33 leukemia-specific ligand sequences were identified. Eleven of the most common sequences were synthesized into biotinylated ligands to test for their binding affinity to leukemia cells. Although the LSC population was not identified by CD9 sorting or Raman signatures of ALL patient samples, OBOC screening of the unsorted cancer cells did result in the identification of leukemia-specific ligands which have the potential to be used to coat drug loaded nanoparticles. These nanoparticles could then be used for direct targeting of leukemia cells which should have minimal toxicity to all other normal cells since the ligands are specific to leukemia. Furthermore, this OBOC technique could be applied to other childhood malignancies and lead to new cancer therapies.