Acute myeloid leukemia (AML) is a heterogeneous disease characterized by the accumulation of immature nonfunctional highly proliferative hematopoietic cells in the blood, due to a blockage in myeloid differentiation at various stages. Since the success of the differentiation agent, All-trans retinoic acid (ATRA), in the treatment of acute promyelocytic leukemia (APL), much effort has gone into trying to find agents that are able to differentiate AML cells and specifically the leukemic stem cell (LSC). CD44 is a cell surface receptor that is over-expressed on AML cells. When bound to anti-CD44 monoclonal antibodies (mAbs), this differentiation block is relieved in AML cells and their proliferation is reduced. The molecular mechanisms that AML cells undergo to achieve this reversal of their apparent phenotype is not fully understood. To this end, we designed a study using quantitative phosphoproteomics approaches aimed at identifying differences in phosphorylation found on proteins involved in signaling pathways post-treatment with CD44-mAbs. The Rho family of GTPases emerged as one of the most transformed pathways following the treatment with CD44-mAbs. The P21 activated kinase 2(PAK2), a target of the Rho family of GTPases, was found to be differentially phosphorylated in AML cells post-treatment with CD44-mAbs. This protein has been found to possess a role similar to that of a switch that determines whether the cell survives or undergoes apoptosis. Beyond confirming these results by various biochemical approaches, our study aimed to determine the effect of knock down of PAK2 on AML cell proliferation and differentiation. In addition, over-expression of PAK2 mutants using plasmid cloning was also explored to fully understand how levels of PAK2 as well as the alteration of specific phospohorylation sites could alter AML cell responses to CD44-mAbs. Results from this study will be important in determining whether PAK2 could be used as a potential therapeutic target for AML once its levels are altered.

Acute myeloid leukemia (AML) is a subset of leukemia that is characterized by the clonal expansion of cytogenetically and molecularly abnormal myeloid blasts. These blasts are highly proliferative accumulating in bone marrow and blood which leads to severe infections, anemia, and bone marrow failure. The poor prognosis of AML patients caused by the low tolerance to intensive chemotherapy has encouraged the pursuit of alternative therapeutic approaches. Differentiation therapy which involves the use of agents that can release the differentiation block in these leukemic blasts has emerged as a promising therapeutic approach. The use of All-trans retinoic acid (ATRA) represents a successful example of such an approach, nonetheless its efficacy is restricted to one subtype of AML. Efforts have been focused on finding differentiation agents which are effective for the other more common AML subtypes. Anti-CD44 targeted antibodies that activate the CD44 cell surface antigen are a promising candidate. Previous studies have shown that anti-CD44 treatment has been able to release the differentiation block in AML1 through AML5 subtypes. The exact mechanism by which anti-CD44 treatment is able to induce its effects has not been fully elucidated. Recent studies highlight the role that epigenetic mechanisms play during haematopoiesis and leukemogenesis and therefore, in this work we investigated the epigenetic mechanisms associated with anti-CD44 induced differentiation. Using AML cell lines from different subtypes, we demonstrated that anti-CD44-induced differentiation results in an extensive change of histone modification levels. We found that inhibiting enzymes responsible for the H3K9ac, H3K4me, H3K9me, and H3K27me modifications, attenuated the anti-proliferative and differentiation promoting effects of antic-CD44 treatment. Taken together, these data highlight the promising potential of using anti-CD44 as a therapeutic agent across multiple subtypes in AML

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