Acute myeloid leukemia (AML) is a clonal malignant disease characterized by a blockage in the differentiation of myeloid cells resulting in the accumulation of highly proliferating immature blast cells. With the success of All Trans Retinoic acid (ATRA) in acute promyelocytic leukemia (AML3), differentiation therapy has become a very attractive treatment option. Ligation of CD44 (a cell surface antigen) with anti-CD44 monoclonal antibodies (mAbs) is reported to reverse the blockage of differentiation and suppress the proliferation of blasts derived from most AML subtypes. However, the molecular mechanisms underlying this apparent ‘normalization’ (reversal) of AML cells induced by CD44 have not been fully elucidated. To expand our understanding of the cellular regulation and circuitry involved, we aimed to apply a quantitative phosphoproteomic approach using Stable Isotope Labeling with Amino acids in Cell culture (SILAC) to monitor dynamic changes of phosphorylation states in HL60 cells following treatment with CD44-mAbs.
Phosphoproteomic analysis identified differentially phosphorylated proteins among CD44-mAb treated and control HL60 cells that are involved in a number of major signaling pathways as determined by the Ingenuity Pathway analysis (IPA®) platform. Among others, Rho signaling emerged as a major pathway significantly changed by CD44-mAb treatment.
Rho GTPases are well-recognized regulators of the actin cytoskeleton but have also been implicated in diverse cellular events such as cell polarity, microtubule dynamics, membrane trafficking, transcriptional regulation, cell growth control and development. An interesting Rho family member, PAK2 was identified in our search. PAK2 is a ubiquitously expressed serine/threonine protein kinase, which is a direct target for small GTPases and has been identified as a switch between cell survival and cell death signaling depending on its mode of activation. Western-blot analyses of cell lysates of CD44-mAb treated and control HL60 cells confirmed that the phosphorylation of PAK2 ,as well as protein level,were altered as early as 5 minutes following treatment. PAK2 knockdown decreased the effect of CD44-mAb induction of proliferation and inhibition of proliferation proving its importance for mediating it’s signaling transduction. PAK1, a structural homologue of PAK2 had the opposite effect of augmenting CD44-mAb effects suggesting a different mechanism involved. This specificity is attributed to the specific mode of activation that PAK2 exhibits which is not shared with the rest of PAK group I members. Caspase-mediated cleavage of PAK2 producing pro-apoptotic fragments is hypothesized to be the signaling transduction mediated by CD44-mAb. In-Vivo experiments show that PAK2 is essential for leukemic cell migration to the spleen. Additionally, it proved essential for CD44-mAb inhibition of leukemic cells migration to the spleen. Further validation and characterization of PAK2’s activation mode, phosphorylation dynamics, subcellular localization as well as its role in invivo migration are essential in understanding its role in AML.
|Date of Award||May 2019|
|Original language||English (US)|
- Biological, Environmental Science and Engineering
|Supervisor||Jasmeen Merzaban (Supervisor)|