Understanding the networks encoding simple differentiation programs could give us insight into complex pluripotent stem-cell behavior.

The intracellular mechanisms that allow stem and stem-like cells to differentiate into specialized cell types remain poorly understood. Thus a strong impetus exists to investigate the mechanistic basis of differentiation induced by growth factors, cytokines and hormones. In collaboration with the lab of Dr. Andrew Yen (Department of Biomedical Sciences), this project is centered on understanding the mechanisms that promote differentiation in retinoic acid-induced HL60 myeloblastic leukemia cells. The HL60 cell line has been a durable experimental model since the late 1970s. These bipotent, leukemic progenitor cells differentiate into granulocytes in response to the morphogenic compound retinoic acid (RA). In short, the immortal HL60 leukemia cell line provides a model system for studying the intracellular signaling networks that allow precursor stem-like cells to become committed to terminal differentiation. In my project, both wild-type and RA-resistant HL60 cells are used in concert to establish mechanistic anomalies and identify critical differences between RA-induced signaling in differentiation-capable and differentiation-resistant HL60 cells. 

There exists a repertoire of phenotypic markers that may be used to disambiguate the hematopoietic maturity of induced HL60. In these cells, retinoic acid (RA) treatment results in G1/G0 cell cycle arrest as the cells become committed to differentiation. Also, RA-induced HL60 cells begin to produce reactive oxygen species (ROS), a function of mature granulocytes. Many surface proteins are upregulated in RA-treated HL60, including CD38, BLR1, and CD11b. However, HL60 cells that are RA-resistant fail to arrest in the G1/G0 phase of cell cycle after RA treatment and fail to produce ROS. Sustained activation of the Raf/MEK/ERK proteins (the MAPK signaling axis) is a long-standing feature of RA-treated HL60. Interestingly, inhibition of c-Raf activity is known inhibit ERK activation but induce growth arrest and CD11b expression in RA-treated HL60. In addition to promoting MEK/ERK activation, c-Raf is capable of interacting with other proteins that are upregulated during the RA-induced differentiation of HL60, such as the hematopoietic proteins Vav and Lyn, each of which binds to the surface protein CD38. Another protein known to interact with both Vav and CD38 is Cbl, a protein whose binding to CD38 is necessary for RA-induced differentiation of HL60. 

Elucidation of core differentiation mechanisms becomes less and less tractable as the pooled knowledge of RA-induced interactions and signaling cascades becomes more unwieldy. However, computational methods can be used to model a network of biochemical reactions and interactions that accurately describe a complex biomolecular system. The previously developed HL60 model contains over 700 species and 1300 interactions (Tasseff et al. Integr Biol 2011 3(5):578–591). However, the model excludes many noteworthy factors, like the hematopoietic-specific Vav protein. Much work remains to be done to incorporate interactions and pathways that were omitted previously.