OTS167 blocks FLT3 translation and synergizes with FLT3 inhibitors in FLT3 mutant acute myeloid leukemia.

Internal tandem duplication (-ITD) mutations of Fms-like tyrosine kinase 3 (FLT3) provide growth and pro-survival signals in the context of established driver mutations in FLT3 mutant acute myeloid leukemia (AML). Maternal embryonic leucine zipper kinase (MELK) is an aberrantly expressed gene identified as a target in AML. The MELK inhibitor OTS167 induces cell death in AML including cells with FLT3 mutations, yet the role of MELK and mechanisms of OTS167 function are not understood. OTS167 alone or in combination with tyrosine kinase inhibitors (TKIs) were used to investigate the effect of OTS167 on FLT3 signaling and expression in human FLT3 mutant AML cell lines and primary cells. We describe a mechanism whereby OTS167 blocks FLT3 expression by blocking FLT3 translation and inhibiting phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and eukaryotic translation initiation factor 4B (eIF4B). OTS167 in combination with TKIs results in synergistic induction of FLT3 mutant cell death in FLT3 mutant cell lines and prolonged survival in a FLT3 mutant AML xenograft mouse model. Our findings suggest signaling through MELK is necessary for the translation and expression of FLT3-ITD, and blocking MELK with OTS167 represents a viable therapeutic strategy for patients with FLT3 mutant AML.

LFA-1 co-stimulation inhibits T(h)2 differentiation by down-modulating IL-4 responsiveness.

Initial T cell activation in the context of different co-stimulatory receptors can influence subsequent lineage commitment into T(h) effector cell subtypes. Specifically, CD28 co-stimulation promotes T(h)2 differentiation, whereas leukocyte function-associated antigen-1 (LFA-1) co-stimulation promotes T(h)1 differentiation and inhibits T(h)2 differentiation. In this report, we have addressed the mechanism of LFA-1-mediated inhibition of T(h)2 responses. We show that co-stimulation through LFA-1 does not decrease early IL-4 secretion, but rather induces a loss in IL-4 responsiveness. T cells primed in the context of LFA-1 co-stimulation require a 5-fold increase in the concentration of IL-4 required to drive T(h)2 differentiation, which is not mediated by a loss in IL-4R expression. To determine whether LFA-1 co-stimulation impacts on proximal signaling from the IL-4R, we first identified a kinetic window where we could separate IL-4-driven T(h)2 differentiation from initial T cell priming. T cells were primed for 2 days under different co-stimulation conditions and re-cultured in the presence of IL-4. Subsequent T(h)2 differentiation was absolutely dependent on addition of IL-4. Proximal IL-4R signaling, as evidenced by tyrosine phosphorylation of signal transducer and activator of transcription-6 (STAT6), was not inhibited by initial co-stimulation through LFA-1, yet these T cells still required higher amounts of IL-4 and corresponding higher levels of STAT6 activation to up-regulate GATA-3 and induce T(h)2 differentiation. Thus, LFA-1 co-stimulation appears to interfere with GATA-3 expression downstream of STAT6. These results suggest that LFA-1 co-stimulation functions as a threshold modulator of T(h)2 differentiation, increasing the effective concentration of IL-4 required to drive T(h)2 responses.