Mice lacking the epidermal growth factor receptor family member ErbB4 exhibit defects in cranial neural crest cell migration but die by embryonic day 11 because of defective heart development. To examine later phenotypes, we rescued the heart defects in ErbB4 mutant mice by expressing ErbB4 under a cardiac-specific myosin promoter. Rescued ErbB4 mutant mice reach adulthood and are fertile. However, during pregnancy, mammary lobuloalveoli fail to differentiate correctly and lactation is defective. Rescued mice also display aberrant cranial nerve architecture and increased numbers of large interneurons within the cerebellum.

The recruitment of the TATA box-binding protein (TBP) to promoters in vivo is often rate limiting in gene expression. We present evidence that TBP negatively autoregulates its accessibility to promoter DNA in yeast through dimerization. The crystal structure of TBP dimers was used to design point mutations in the dimer interface. These mutants are impaired for dimerization in vitro, and in vivo they generate large increases in activator-independent gene expression. Overexpression of wild-type TBP suppresses these mutants, possibly by heterodimerizing with them. In addition to loss of autorepression, dimerization-defective TBPs are rapidly degraded in vivo. Direct detection of TBP dimers in vivo was achieved through chemical cross-linking. Taken together, the data suggest that TBP dimerization prevents unregulated gene expression and its own degradation.

BackgroundDormant leukemia stem cells (LSC) promote therapeutic resistance and leukemic progression as a result of unbridled activation of stem cell gene expression programs. Thus, we hypothesized that 1) deregulation of the hedgehog (Hh) stem cell self-renewal and cell cycle regulatory pathway would promote dormant human LSC generation and 2) that PF-04449913, a clinical antagonist of the GLI2 transcriptional activator, smoothened (SMO), would enhance dormant human LSC eradication.MethodsTo test these postulates, whole transcriptome RNA sequencing (RNA-seq), microarray, qRT-PCR, stromal co-culture, confocal fluorescence microscopic, nanoproteomic, serial transplantation and cell cycle analyses were performed on FACS purified normal, chronic phase (CP) chronic myeloid leukemia (CML), blast crisis (BC) phase CML progenitors with or without PF-04449913 treatment.ResultsNotably, RNA-seq analyses revealed that Hh pathway and cell cycle regulatory gene overexpression correlated with leukemic progression. While lentivirally enforced GLI2 expression enhanced leukemic progenitor dormancy in stromal co-cultures, this was not observed with a mutant GLI2 lacking a transactivation domain, suggesting that GLI2 expression prevented cell cycle transit. Selective SMO inhibition with PF-04449913 in humanized stromal co-cultures and LSC xenografts reduced downstream GLI2 protein and cell cycle regulatory gene expression. Moreover, SMO inhibition enhanced cell cycle transit and sensitized BC LSC to tyrosine kinase inhibition in vivo at doses that spare normal HSC.ConclusionIn summary, while GLI2, forms part of a core HH pathway transcriptional regulatory network that promotes human myeloid leukemic progression and dormant LSC generation, selective inhibition with PF-04449913 reduces the dormant LSC burden thereby providing a strong rationale for clinical trials predicated on SMO inhibition in combination with TKIs or chemotherapeutic agents with the ultimate aim of obviating leukemic therapeutic resistance, persistence and progression.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0453-9) contains supplementary material, which is available to authorized users.

First generation EGFR TKIs (gefitinib, erlotinib) provide significant clinical benefit for NSCLC cancer patients with oncogenic EGFR mutations. Ultimately, these patients' disease progresses, often driven by a second-site mutation in the EGFR kinase domain (T790M). Another liability of the first generation drugs is severe adverse events driven by inhibition of WT EGFR. As such, our goal was to develop a highly potent irreversible inhibitor with the largest selectivity ratio between the drug-resistant double mutants (L858R/T790M, Del/T790M) and WT EGFR. A unique approach to develop covalent inhibitors, optimization of reversible binding affinity, served as a cornerstone of this effort. PF-06459988 was discovered as a novel, third generation irreversible inhibitor, which demonstrates (i) high potency and specificity to the T790M-containing double mutant EGFRs, (ii) minimal intrinsic chemical reactivity of the electrophilic warhead, (iii) greatly reduced proteome reactivity relative to earlier irreversible EGFR inhibitors, and (iv) minimal activity against WT EGFR.