Astrocytomas are the most common type of brain tumors in children. Activated BRAF protein kinase mutations are characteristic of pediatric astrocytomas with KIAA1549-BRAF fusion genes typifying low-grade astrocytomas and V600E BRAF alterations characterizing distinct or higher-grade tumors. Recently, BRAF-targeted therapies, such as vemurafenib, have shown great promise in treating V600E-dependent melanomas. Like V600E BRAF, BRAF fusion kinases activate MAPK signaling and are sufficient for malignant transformation; however, here we characterized the distinct mechanisms of action of KIAA1549-BRAF and its differential responsiveness to PLX4720, a first-generation BRAF inhibitor and research analog of vemurafenib. We found that in cells expressing KIAA1549-BRAF, the fusion kinase functions as a homodimer that is resistant to PLX4720 and accordingly is associated with CRAF-independent paradoxical activation of MAPK signaling. Mutagenesis studies demonstrated that KIAA1549-BRAF fusion-mediated signaling is diminished with disruption of the BRAF kinase dimer interface. In addition, the KIAA1549-BRAF fusion displays increased binding affinity to kinase suppressor of RAS (KSR), an RAF relative recently demonstrated to facilitate MEK phosphorylation by BRAF. Despite its resistance to PLX4720, the KIAA1549-BRAF fusion is responsive to a second-generation selective BRAF inhibitor that, unlike vemurafenib, does not induce activation of wild-type BRAF. Our data support the development of targeted treatment paradigms for BRAF-altered pediatric astrocytomas and also demonstrate that therapies must be tailored to the specific mutational context and distinct mechanisms of action of the mutant kinase.

Angiocentric gliomas are pediatric low-grade gliomas (PLGGs) without known recurrent genetic drivers. We performed genomic analysis of new and published data from 249 PLGGs including 19 Angiocentric Gliomas. We identified MYB-QKI fusions as a specific and single candidate driver event in Angiocentric Gliomas. In vitro and in vivo functional studies show MYB-QKI rearrangements promote tumorigenesis through three mechanisms: MYB activation by truncation, enhancer translocation driving aberrant MYB-QKI expression, and hemizygous loss of the tumor suppressor QKI. This represents the first example of a single driver rearrangement simultaneously transforming cells via three genetic and epigenetic mechanisms in a tumor.

The second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ), formed by the p110 family of PI3-kinases, promotes cellular growth, proliferation, and survival, in large part by activating the protein kinase Akt/PKB. We show that inositol polyphosphate multikinase (IPMK) physiologically generates PIP 3 as well as water soluble inositol phosphates. IPMK deletion reduces growth factor-elicited Akt signaling and cell proliferation caused uniquely by loss of its PI3-kinase activity. Inhibition of p110 PI3-kinases by wortmannin prevents IPMK phosphorylation and activation. Thus, growth factor stimulation of Akt signaling involves PIP 3 generation through the sequential activations of the p110 PI3-kinases and IPMK. As inositol phosphates inhibit Akt signaling, IPMK appears to act as a molecular switch, inhibiting or stimulating Akt via its inositol phosphate kinase or PI3-kinase activities, respectively. Drugs regulating IPMK may have therapeutic relevance in influencing cell proliferation.signal transduction | cancer A large family of inositol phosphates serves multiple functions, with inositol 1,4,5-trisphosphate (IP 3 ) being well known as a second messenger releasing intracellular calcium (1). Inositol diphosphates, incorporating an energetic pyrophosphate bond, display numerous physiological roles, including pyrophosphorylation of a variety of protein targets (2-4). These inositol pyrophosphates are synthesized by a family of IP 6 kinase enzymes (5). Recently, novel isomers of inositol pyrophosphates have been described that are synthesized by a distinct inositol phosphate kinase enzyme designated Vip1 (6, 7).Inositol polyphosphate multikinase (IPMK) is a member of the IP 6 kinase family of enzymes but is not primarily associated with the formation of inositol pyrophosphates. Instead it generates several inositol phosphates, converting IP 3 to IP 4 and IP 4 to IP 5 , with its primary physiologic role in this pathway being to form the bulk of IP 5 in cells (5,(8)(9)(10)(11). IPMK also possesses phosphatidylinositol 3-kinase (PI3K) activity in vitro (12), specifically phosphorylating phosphatidylinositol(4,5)-bisphosphate (PIP 2 ) to generate phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ), a second messenger known to promote cellular growth, proliferation, survival, and migration (13). The physiologic role of this activity has not heretofore been established. The principal PI3K activity in cells has been attributed to a family of enzymes identified by Cantley and associates (reviewed in ref. 14), whose catalytic subunits are designated p110. PIP 3 generated by p110 in response to extracellular stimuli, such as growth factors, is a principal stimulus of the Akt/mammalian target of rapamycin (mTOR) signaling pathway, which in turn regulates protein synthesis and plays a role in some cancers (15)(16)(17).We wondered whether the PI3K activity of IPMK contributes to the generation of PIP 3 under physiologic conditions to influence Akt signaling and cell growth. There is good reason to assume that IPMK is ...