RPPA-Based Protein Profiling Reveals Enhanced PI3K/AKT/mTOR Signaling in ETV6/RUNX1-Positive Acute Lymphoblastic Leukemia Patients with Low CD200 Expression

Although our understanding of pediatric acute lymphoblastic leukemia (ALL) biology has improved through RNA expression profiling, DNA-based somatic mutation analysis and genome-wide DNA methylation assays, these methods are not able to provide insight into altered protein expression and impact on signaling pathways activity. Recent studies clearly indicated the need to target the physiological effects of deregulated pathways rather than their individual gene components, in order to improve therapeutic options.

Recently, we identified CD200 deletion as a recurrent genetic alteration in B-cell precursor ALL (BCP-ALL) with prognostic relevance in intermediate risk BCP-ALLs. However, the exact mechanisms by which loss of CD200 triggers precursor B-cell transformation as well as the signaling cascades that are activated downstream of CD200 remain to be elucidated.

Given this, we used reverse phase protein array (RPPA) technology in a cohort of 72 ETV6/RUNX1-positive BCP-ALL primary patient samples at diagnosis to study the impact of low CD200 expression on several key cellular signaling pathways. Interestingly, differential protein analysis between CD200-low and CD200-high BCP-ALLs suggested that reduced CD200 levels are associated with enhanced PI3K/AKT/mTOR signaling. More specifically, eIF4G, a scaffold protein that plays a role in initiation of translation, was found to be hyperphosphorylated in CD200-low cases (p = 0.015). Also AKT was found hyperphoshorylated at the activation site (p = 0.033). In addition, hyperphosphorylation of c-RAF (p = 0.026) and B-RAF (p = 0.035) at their activation site were also observed in CD200-low patients. Next to B-RAF, hyperphosphorylation of the p44/42 activation site was noted (p = 0.006), highlighting a global hyperactivation of the RAS/MAPK pathway. Interestingly, the expression of integrin α4/β1 was markedly decreased in CD200-low ETV6-RUNX1-positive patients (p = 0.001). These heterodimeric cell surface receptors play a pivotal role in cell adhesion and migration, as well as in growth and survival and a decreased expression of some subunits of integrin has been observed in several tumor entities. In leukemia, the α4/β1 dimer, also named VLA-4, has been demonstrated several times to contribute to drug resistance and relapse occurrence since its presence allows leukemia cells to adhere to the bone marrow niche and escape from chemotherapeutics action (Hsieh YT, Blood 2013). The relevance of our observation in this context however needs to be further elucidated.

Finally, these observations were validated using two different B-ALL cell lines REH and AT-2, both ETV6/RUNX1-positive, but REH is characterized by a CD200 deletion, whereas AT-2 displays no copy number aberrations at this particular genomic locus.

Altogether, these results revealed several targetable pathways in ETV6/RUNX1-positive BCP-ALL patients with low CD200 expression, and provide a framework for testing of interesting inhibiting compounds including PI-103, LY294002 and silvestrol.

BDM and PVV are shared last authors