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.

OCT4 and SOX2 Work as Transcriptional Activators in Reprogramming Human Fibroblasts.

SOX2 and OCT4, in conjunction with KLF4 and cMYC, are sufficient to reprogram human fibroblasts to induced pluripotent stem cells (iPSCs), but it is unclear if they function as transcriptional activators or as repressors. We now show that, like OCT4, SOX2 functions as a transcriptional activator. We substituted SOX2-VP16 (a strong activator) for wild-type (WT) SOX2, and we saw an increase in the efficiency and rate of reprogramming, whereas the SOX2-HP1 fusion (a strong repressor) eliminated reprogramming. We report that, at an early stage of reprogramming, virtually all DNA-bound OCT4, SOX2, and SOX2-VP16 were embedded in putative enhancers, about half of which were created de novo. Those associated with SOX2-VP16 were, on average, stronger than those bearing WT SOX2. Many newly created putative enhancers were transient, and many transcription factor locations on DNA changed as reprogramming progressed. These results are consistent with the idea that, during reprogramming, there is an intermediate state that is distinct from both parental cells and iPSCs.

Polycomb Responds to Low Levels of Transcription.

How is Polycomb (Pc), a eukaryotic negative regulator of transcription, targeted to specific mammalian genes? Our genome-wide analysis of the Pc mark H3K27me3 in murine cells revealed that Pc is preferentially associated with CpG island promoters of genes that are transcribed at a low level and less so with promoters of genes that are either silent or more highly expressed. Studies of the CpG island promoter of the Kit gene demonstrate that Pc is largely absent when the gene is silent in myeloid cells, as well as when the gene is highly expressed in mast cells. Manipulations that increase transcription in the former case, and reduce it in the latter, increase Pc occupancy. The average negative effect of Pc, we infer, is about 2-fold. We suggest possible biological roles for such negative effects and propose a mechanism by which Pc might be recruited to weakly transcribed genes.

Regulation of a mammalian gene bearing a CpG island promoter and a distal enhancer.

A quantitative nucleosome occupancy assay revealed rules for nucleosome disposition in yeast and showed how disposition affects regulation of the GAL genes. Here, we show how those findings apply to the control of Kit, a mammalian gene. The Kit promoter lies in a CpG island, and its enhancer (active in mast cells) lies some 150 kb upstream. Nucleosomes form with especially high avidities at the Kit promoter, a reaction that, we surmise, ensures extremely low basal expression. In mast cells, transcriptional activators displace nucleosomes that are less tightly formed at the Kit enhancer. In turn, the active enhancer replaces a single Kit promoter nucleosome with the transcriptional machinery, thereby inducing transcription over 1,000-fold. As at the yeast GAL genes, the inhibitory effects of nucleosomes facilitate high factors of induction by mammalian activators working in the absence of specific repressors.

A RSC/nucleosome complex determines chromatin architecture and facilitates activator binding.

How is chromatin architecture established and what role does it play in transcription? We show that the yeast regulatory locus UASg bears, in addition to binding sites for the activator Gal4, sites bound by the RSC complex. RSC positions a nucleosome, evidently partially unwound, in a structure that facilitates Gal4 binding to its sites. The complex comprises a barrier that imposes characteristic features of chromatin architecture. In the absence of RSC, ordinary nucleosomes encroach over the UASg and compete with Gal4 for binding. Taken with our previous work, the results show that both prior to and following induction, specific DNA-binding proteins are the predominant determinants of chromatin architecture at the GAL1/10 genes. RSC/nucleosome complexes are also found scattered around the yeast genome. Higher eukaryotic RSC lacks the specific DNA-binding determinants found on yeast RSC, and evidently Gal4 works in those organisms despite whatever obstacle broadly positioned nucleosomes present.

A distant upstream locus control region is critical for expression of the Kit receptor gene in mast cells.

The Kit receptor tyrosine kinase functions in hematopoiesis, melanogenesis, and gametogenesis and in interstitial cells of Cajal. We previously identified two upstream hypersensitive site (HS) clusters in mast cells and melanocytes. Here we investigated the roles of these 5' HS sequences in Kit expression using transgenic mice carrying Kit-GFP reporter constructs. In these mice there is close correspondence between Kit-GFP reporter and endogenous Kit gene expression in most tissues analyzed. Deletion analysis defined the 5' upstream HS cluster region as critical for Kit expression in mast cells. Furthermore, chromatin immunoprecipitation analysis in mast cells showed that H3 and H4 histone hyperacetylation and RNA polymerase II recruitment within the Kit promoter and in the 5' HS region were associated with Kit expression. Therefore, the 5' upstream hypersensitivity sites appear to be critical components of locus control region-mediated Kit gene activation in mast cells.

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.