Direct engagement of the PI3K pathway by mutant KIT dominates oncogenic signaling in gastrointestinal stromal tumor.

Gastrointestinal stromal tumors (GISTs) predominantly harbor activating mutations in the receptor tyrosine kinase KIT. To genetically dissect in vivo the requirement of different signal transduction pathways emanating from KIT for tumorigenesis, the oncogenic KitV558Δ mutation was combined with point mutations abrogating specific phosphorylation sites on KIT. Compared with single-mutant KitV558Δ/+ mice, double-mutant KitV558Δ;Y567F/Y567F knock-in mice lacking the SRC family kinase-binding site on KIT (pY567) exhibited attenuated MAPK signaling and tumor growth. Surprisingly, abrogation of the PI3K-binding site (pY719) in KitV558Δ;Y719F/Y719F mice prevented GIST development, although the interstitial cells of Cajal (ICC), the cells of origin of GIST, were normal. Pharmacologic inhibition of the PI3K pathway in tumor-bearing KitV558Δ/+ mice with the dual PI3K/mTOR inhibitor voxtalisib, the pan-PI3K inhibitor pilaralisib, and the PI3K-alpha-restricted inhibitor alpelisib each diminished tumor proliferation. The addition of the MEK inhibitor PD-325901 or binimetinib further decreased downstream KIT signaling. Moreover, combining PI3K and MEK inhibition was effective against imatinib-resistant KitV558Δ;T669I/+ tumors.

Oncogenic Kit signaling and therapeutic intervention in a mouse model of gastrointestinal stromal tumor.

Kit receptor-activating mutations are critical in the pathogenesis of gastrointestinal stromal tumors (GIST). We investigated mechanisms of oncogenic Kit signaling and the consequences of therapeutic intervention in a mouse model of human GIST. Treatment of GIST mice with imatinib decreased cell proliferation and increased apoptosis in the tumor. Analysis of tumor tissue from imatinib-treated mice showed diminished phosphatidylinositol 3-kinase (PI3-kinase) and mammalian target of rapamycin (mTOR) signaling suggesting that oncogenic Kit signaling critically contributes to the translational response in GIST. Treatment with RAD001 (everolimus), an mTOR inhibitor, diminished the translational response and cell proliferation in tumor lesions, pointing to mTOR inhibition as a therapeutic approach for imatinib-resistant GIST. Analysis of RNA expression profiles in GIST lesions with and without imatinib treatment showed changes in expression of IFN-inducible genes and cell cycle regulators. These results convincingly show that KitV558Delta/+ mice represent a unique faithful mouse model of human familial GIST, and they demonstrate the utility of these mice for preclinical investigations and to elucidate oncogenic signaling mechanisms by using genetic approaches and targeted pharmacological intervention.

Functional characterization of the interaction between human La and hepatitis B virus RNA.

The La protein is a multifunctional RNA-binding protein and has also been suggested to be involved in the stabilization of hepatitis B virus (HBV) RNA. Here we demonstrate that antibodies against the human La protein specifically precipitate HBV RNA from HBV ribonucleoprotein-containing mammalian cell extracts, providing evidence for the association between human La and HBV RNA. Moreover, we report that the turnover of HBV RNA depends on structural features and less on the primary sequence of the La-binding site on the viral RNA. In addition we show that the interaction between human La and HBV RNA in vitro is modulated by accessory factor(s) in a phosphorylation-dependent manner. Taken together these data indicate that both structural features, the composition of La/HBV ribonucleoprotein particles as well as interacting cellular factors, are critical determinants in the regulation of the stability of the HBV RNA.

Critical role for Kit-mediated Src kinase but not PI 3-kinase signaling in pro T and pro B cell development.

The Kit receptor functions in hematopoiesis, lymphocyte development, gastrointestinal tract motility, melanogenesis, and gametogenesis. To investigate the roles of different Kit signaling pathways in vivo, we have generated knock-in mice in which docking sites for PI 3-kinase (KitY719) or Src kinase (KitY567) have been mutated. Whereas steady-state hematopoiesis is normal in KitY719F/Y719F and KitY567F/Y567F mice, lymphopoiesis is affected differentially. The KitY567F mutation, but not the KitY719F mutation, blocks pro T cell and pro B cell development in an age-dependent manner. Thus, the Src family kinase, but not the PI 3-kinase docking site in Kit, mediates a critical signal for lymphocyte development. In agreement with these results, treatment of normal mice with the Kit tyrosine kinase inhibitor imatinib (Gleevec) leads to deficits in pro T and pro B cell development, similar to those seen in KitY567F/Y567F and KitW/W mice. The two mutations do not affect embryonic gametogenesis but the KitY719F mutation blocks spermatogenesis at the spermatogonial stages and in contrast the KitY567F mutation does not affect this process. Therefore, Kit-mediated PI 3-kinase signaling and Src kinase family signaling is highly specific for different cellular contexts in vivo.

Gastrointestinal stromal tumors in a mouse model by targeted mutation of the Kit receptor tyrosine kinase.

Oncogenic Kit mutations are found in somatic gastrointestinal (GI) stromal tumors (GISTs) and mastocytosis. A mouse model for the study of constitutive activation of Kit in oncogenesis has been produced by a knock-in strategy introducing a Kit exon 11-activating mutation into the mouse genome based on a mutation found in a case of human familial GIST syndrome. Heterozygous mutant KitV558Delta/+ mice develop symptoms of disease and eventually die from pathology in the GI tract. Patchy hyperplasia of Kit-positive cells is evident within the myenteric plexus of the entire GI tract. Neoplastic lesions indistinguishable from human GISTs were observed in the cecum of the mutant mice with high penetrance. In addition, mast cell numbers in the dorsal skin were increased. Therefore KitV558Delta/+ mice reproduce human familial GISTs, and they may be used as a model for the study of the role and mechanisms of Kit in neoplasia. Importantly, these results demonstrate that constitutive Kit signaling is critical and sufficient for induction of GIST and hyperplasia of interstitial cells of Cajal.