Key Nodes In FLT3 Dependent Signaling Determine Growth and Survival Of Childhood Acute Lymphoblastic Leukemia

The balanced regulation of complex signaling networks plays an important role in cell proliferation, survival and apoptosis. Receptor tyrosine kinase (RTK) dependent signal transduction has been implicated in the pathogenesis of many malignancies including acute lymphoblastic leukemia (ALL) of childhood. Moreover, deregulated RTK activity was observed following targeted inhibition of constitutively active kinases or kinase-dependent pathways in a variety of malignancies, which conferred pharmacological resistance. In ALL, fms-like-tyrosine kinase 3 (FLT3) and platelet-derived growth factor receptor beta (PDGFRβ) are targeted for mutation (Roberts et al., 2012). However, in primary ALL the RTK dependent signaling state is poorly defined and the occurrence of relapse in the context of genotype-directed monotherapy regimens targeting RTKs, such as FLT3, underlines the need for an activity-based approach to RTK signaling in leukemia comprising the identification of critical downstream target proteins.

To select for driver RTKs in ALL, we combined the analyses of RTK expression in primary ALL (n=102), ALL cell lines and normal hematopoietic cells with the characterization of ligand dependent cell proliferation as well as shRNA mediated RTK repression in vitro and in vivo. We observed aberrant RTK expression patterns in ALL cells compared to normal lymphoid progenitor and stem cell populations as well as mature T- and B-lymphocytes. RNA interference mediated repression of growth promoting RTKs FLT3 and PDGFRβ in primary ALL led to a loss or reduction of the affected cell population in vivo. To identify critical signaling nodes we performed a phosphoproteomic characterization by iTRAQ (isobaric tags for relative and absolute quantification)-based mass spectrometry of the signal transduction of selected driver RTKs in the corresponding primary ALL samples. Primary ALL cells were propagated in NSG mice after xenotransplantation, and regulated phosphoproteins were identified after ligand stimulation. Our network-directed approach to RTK signaling in ALL thus allowed for the identification of downstream signaling nodes implicated in aberrant RTK activity.

We identified a total of 2241 phosphoproteins and observed a striking diversity of RTK driven signaling processes in primary ALL exhibiting only a marginal overlap between phosphoregulated proteins which illustrates the inter-individual heterogeneity and the challenge for non-combinatorial therapies. Despite a predominant receptor and cell type specific composition of potentiated signaling networks our phosphoproteomic analyses identified p21-activated protein kinase PAK2 as a novel key nodal point in FLT3 dependent signaling in ALL. The importance of PAK protein family members in the regulation of cell proliferation and survival and the emerging role of PAK proteins in the pathogenesis of a broad range of tumors suggests a hitherto unanticipated function in the malignant transformation of ALL and the signal transduction of FLT3 (Ye and Field, 2012).

As a kinase PAK2 represents a druggable target and may be suited for combinatorial intervention strategies targeting FLT3 signaling in order to induce synthetic lethality. Inhibition of group I PAK kinases (PAK1, 2, 3) using the allosteric inhibitor IPA-3 and RNA interference mediated repression of PAK2 led to the loss of ALL cells due to an impaired cell proliferation and an increased apoptosis. Notably, PAK2 depleted ALL cells showed an elevated sensitivity towards pharmacological FLT3 inhibition which underlines the potential role of PAK2 as a novel target in ALL and the need for novel small molecule PAK inhibitors with higher specificity and improved applicability in vivo.