Combined inhibition of receptor tyrosine and p21-activated kinases as a therapeutic strategy in childhood ALL.

Receptor tyrosine kinase (RTK)-dependent signaling has been implicated in the pathogenesis of acute lymphoblastic leukemia (ALL) of childhood. However, the RTK-dependent signaling state and its interpretation with regard to biological behavior are often elusive. To decipher signaling circuits that link RTK activity with biological output in vivo, we established patient-derived xenograft ALL (PDX-ALL) models with dependencies on fms-like tyrosine kinase 3 (FLT3) and platelet-derived growth factor receptor ß (PDGFRB), which were interrogated by phosphoproteomics using iTRAQ mass spectrometry. Signaling circuits were determined by receptor type and cellular context with few generic features, among which we identified group I p21-activated kinases (PAKs) as potential therapeutic targets. Growth factor stimulation markedly increased catalytic activities of PAK1 and PAK2. RNA interference (RNAi)-mediated or pharmacological inhibition of PAKs using allosteric or adenosine triphosphate (ATP)-competitive compounds attenuated cell growth and increased apoptosis in vitro. Notably, PAK1- or PAK2-directed RNAi enhanced the antiproliferative effects of the type III RTK and protein kinase C inhibitor midostaurin. Treatment of FLT3- or PDGFRB-dependent ALLs with ATP-competitive PAK inhibitors markedly decreased catalytic activities of both PAK isoforms. In FLT3-driven ALL, this effect was augmented by coadministration of midostaurin resulting in synergistic effects on growth inhibition and apoptosis. Finally, combined treatment of FLT3 D835H PDX-ALL with the ATP-competitive group I PAK inhibitor FRAX486 and midostaurin in vivo significantly prolonged leukemia progression-free survival compared with midostaurin monotherapy or control. Our study establishes PAKs as potential downstream targets in RTK-dependent ALL of childhood, the inhibition of which might help prevent the selection or acquisition of resistance mutations toward tyrosine kinase inhibitors.

Identification of novel parasitophorous vacuole proteins in P. falciparum parasites using BioID.

Malaria blood stage parasites develop within red blood cells where they are contained in a vacuolar compartment known as the parasitophorous vacuole (PV). This compartment holds a key role in the interaction of the parasite with its host cell. However, the proteome of this compartment has so far not been comprehensively analysed. Here we used BioID in asexual blood stages of the most virulent human malaria parasite Plasmodium falciparum to identify new proteins of the PV. The resulting proteome contained many of the already known PV proteins and validation by GFP-knock-in of 10 previously in P. falciparum uncharacterised hits revealed 5 new PV proteins and two with a partial PV localisation. This included proteins peripherally attached to the inner face of the PV membrane as well as proteins anchored in the parasite plasma membrane that protrude into the PV. Using selectable targeted gene disruption we generated mutants for 2 of the 10 candidates. In contrast we could not select parasites with disruptions for another 3 candidates, strongly suggesting that they are important for parasite growth. Interestingly, one of these included the orthologue of UIS2, a protein previously proposed to regulate protein translation in the parasite cytoplasm but here shown to be an essential PV protein. This work extends the number of known PV proteins and provides a starting point for further functional analyses of this compartment.