Previous studies of ours suggest that TKI-dependent combination therapy likely represents a potentially useful approach to counteracting both intrinsic and stroma-associated drug resistance in leukemia patients. With the recent discovery of numerous FLT3 inhibitor-responsive serine/threonine and tyrosine phosphorylation sites uncovered in primary AML patient bone marrow R112 biological activity samples, identification of protein kinase inhibitors that are able to enhance the potency of FLT3 inhibitors makes intuitive sense. Here, selective inhibitors targeting kinases involved in PI3K/ Akt and Ras/MEK/MAPK FD&C Blue No. 1 signaling were identified in a chemical screen as synergizing with PKC412 against mutant FLT3- expressing cells in the presence of adherent stroma. Akt inhibitors synergized with FLT3 inhibitors in the presence of either SCM or adherent stroma, as compared to p38 MAPK inhibitors, which synergized with FLT3 inhibitors only in the presence of adherent stroma. One possibility for this may be traced to the nature of stromal protection by SCM, characterized by highly concentrated levels of stromal-derived cytokines. Of relevance, studies have implicated Akt- and MAPKmediated signaling in stromal enhancement of leukemia cell viability. For instance, co-culture of leukemia cells and bone marrow-derived stroma has been shown to lead to activation of the MAPK/ERK pathway and integrin-linked kinase, which phosphorylates Akt. ILK/Akt is likely critical for leukemia cell survival in bone marrow, and thus inhibitors of ILK have been proposed as an approach to simultaneously target both leukemia cells and leukemia-activated stromal cells. Additionally, p38 MAPK activation has been found to play a role in stromadependent survival of B-CLL cells and ALL cells. In addition, continuous FLT3 inhibitor treatment leads to the development of drug-resistant cells characterized by constitutive activation of parallel downstream PI3K/Akt and/or Ras/MEK/ MAPK signaling pathways, which is believed to compensate for the loss of FLT3 activity in terms of survival and growth. In support of this, constitutive activation of ERK/Akt/STAT pathways has been observed in AML despite small molecule inhibition of FLT3-ITD activity, suggesting that optimal treatment of AML may require FLT3 inh
