Self‐renewal of Bcr‐Abl+ chronic myeloid leukemia (CML) cells is sustained by a nuclear activated serine/threonine‐(S/T) unphosphorylated β‐catenin. Although β‐catenin can be tyrosine (Y)‐phosphorylated, the occurrence and biological relevance of this covalent modification in Bcr‐Abl‐associated leukemogenesis is unknown. Here we show that Bcr‐Abl levels control the degree of β‐catenin protein stabilization by affecting its Y/S/T‐phospho content in CML cells. Bcr‐Abl physically interacts with β‐catenin, and its oncogenic tyrosine kinase activity is required to phosphorylate β‐catenin at Y86 and Y654 residues. This Y‐phospho β‐catenin binds to the TCF4 transcription factor, thus representing a transcriptionally active pool. Imatinib, a Bcr‐Abl antagonist, impairs the β‐catenin/TCF‐related transcription causing a rapid cytosolic retention of Y‐unphosphorylated β‐catenin, which presents an increased binding affinity for the Axin/GSK3β complex. Although Bcr‐Abl does not affect GSK3β autophosphorylation, it prevents, through its effect on β‐catenin Y phosphorylation, Axin/GSK3β binding to β‐catenin and its subsequent S/T phosphorylation. Silencing of β‐catenin by small interfering RNA inhibited proliferation and clonogenicity of Bcr‐Abl+ CML cells, in synergism with Imatinib. These findings indicate the Bcr‐Abl triggered Y phosphorylation of β‐catenin as a new mechanism responsible for its protein stabilization and nuclear signalling activation in CML.

The bacterial CRISPR/Cas system has proven to be an efficient tool for genetic manipulation in various organisms. Here we show the application of CRISPR-Cas9 technology to edit the human Rhodopsin (RHO) gene in a mouse model for autosomal dominant Retinitis Pigmentosa. We designed single or double sgRNAs to knock-down mutant RHO expression by targeting exon 1 of the RHO gene carrying the P23H dominant mutation. By delivering Cas9 and sgRNAs in a single plasmid we induced an efficient gene editing in vitro, in HeLa cells engineered to constitutively express the P23H mutant RHO allele. Similarly, after subretinal electroporation of the CRISPR/Cas9 plasmid expressing two sgRNAs into P23H RHO transgenic mice, we scored specific gene editing as well as significant reduction of the mutant RHO protein. Successful in vivo application of the CRISPR/Cas9 system confirms its efficacy as a genetic engineering tool in photoreceptor cells.

Inhibition of deregulated protein tyrosine kinases represents an attractive strategy for controlling cancer growth. However, target specificity is an essential aim of this strategy. In this report, pp60(c-Src) kinase and β-catenin were found physically associated and constitutively activated on tyrosine residues in human colorectal cancer cells. The use of specific small-interfering RNAs (siRNA) validated pp60(c-Src) as the major kinase responsible for β-catenin tyrosine phosphorylation in colorectal cancer. Src-dependent activation of β-catenin was prevented by SKI-606, a novel Src family kinase inhibitor, which also abrogated β-catenin nuclear function by impairing its binding to the TCF4 transcription factor and its trans-activating ability in colorectal cancer cells. These effects were seemingly specific, as cyclin D1, a crucial β-catenin/TCF4 target gene, was also down-regulated by SKI-606 in a dose-dependent manner accounting, at least in part, for the reduced growth (IC₅₀, 1.5-2.4 μmol/L) and clonogenic potential of colorectal cancer cells. Protein levels of β-catenin remained substantially unchanged by SKI-606, which promoted instead a cytosolic/membranous retention of β-catenin as judged by immunoblotting analysis of cytosolic/nuclear extracts and cell immunofluorescence staining. The SKI-606-mediated relocalization of β-catenin increased its binding affinity to E-cadherin and adhesion of colorectal cancer cells, with ensuing reduced motility in a wound healing assay. Interestingly, the siRNA-driven knockdown of β-catenin removed the effect of SKI-606 on cell-to-cell adhesion, which was associated with prolonged stability of E-cadherin protein in a pulse-chase experiment. Thus, our results show that SKI-606 operates a switch between the transcriptional and adhesive function of β-catenin by inhibiting its pp60(c-Src)–dependent tyrosine phosphorylation; this could constitute a new therapeutic target in colorectal cancer. (Cancer Res 2006; 66(4): 2279-86)

The rare patients who are able to spontaneously control HIV replication in the absence of therapy show signs of a particularly efficient cellular immune response. To identify the molecular determinants that underlie this response, we characterized the T cell receptor (TCR) repertoire directed at Gag293, the most immunoprevalent CD4 epitope in the HIV-1 capsid. HIV controllers from the ANRS CODEX cohort showed a highly skewed TCR repertoire that was characterized by a predominance of TRAV24 and TRBV2 variable genes, shared CDR3 motifs, and a high frequency of public clonotypes. The most prevalent public clonotypes generated TCRs with affinities at the higher end of values reported for naturally occurring TCRs. The high-affinity Gag293-specific TCRs were cross-restricted by up to 5 distinct HLA-DR alleles, accounting for the expression of these TCRs in HIV controllers of diverse genetic backgrounds. Transfer of these TCRs to healthy donor CD4⁺ T cells conferred high antigen sensitivity and polyfunctionality, thus recapitulating key features of the controller CD4 response. Transfer of a high-affinity Gag293-specific TCR also redirected CD8⁺ T cells to target HIV-1 capsid via nonconventional MHC II restriction. Together, these findings indicate that TCR clonotypes with superior functions are associated with HIV control. Amplification or transfer of such clonotypes may contribute to immunotherapeutic approaches aiming at a functional HIV cure.

HIV controllers are rare individuals who spontaneously control HIV replication in the absence of antiretroviral therapy. To identify parameters of the CD4 response that may contribute to viral control rather than merely reflect a persistently low viremia, we compared the T helper profiles in two groups of patients with more than 10 years of viral suppression: HIV controllers from the Agence Nationale de Recherche sur le SIDA et les Hépatites Virales (ANRS) CO18 cohort ( n = 26) and efficiently treated patients ( n = 16). Cells specific for immunodominant Gag and cytomegalovirus (CMV) peptides were evaluated for the production of 10 cytokines and cytotoxicity markers and were also directly quantified ex vivo by major histocompatibility complex (MHC) class II tetramer staining. HIV controller CD4 ⁺ T cells were characterized by a higher frequency of gamma interferon (IFN-γ) production, perforin ⁺ /CD107a ⁺ expression, and polyfunctionality in response to Gag peptides. While interleukin 4 (IL-4), IL-17, and IL-21 production did not differ between groups, the cells of treated patients produced more IL-10 in response to Gag and CMV peptides, pointing to persistent negative immunoregulation after long-term antiretroviral therapy. Gag293 tetramer-positive cells were detected at a high frequency (0.12%) and correlated positively with IFN-γ-producing CD4 ⁺ T cells in the controller group ( R = 0.73; P = 0.003). Tetramer-positive cells were fewer in the highly active antiretroviral therapy (HAART) group (0.04%) and did not correlate with IFN-γ production, supporting the notion of a persistent immune dysfunction in HIV-specific CD4 ⁺ T cells of treated patients. In conclusion, HIV controllers maintained a population of highly efficient Th1 effectors directed against Gag in spite of a persistently low antigenemia, while patients treated in the long term showed a loss of CD4 effector functions.

Statins are widely used hypocholesterolemic drugs that inhibit 3‐hydroxy‐3‐methyl‐glutaryl‐coenzyme A reductase, a rate‐limiting enzyme of the mevalonate pathway whose biosynthetic endproduct is cholesterol. As a result of this activity, statins may perturb the composition of cell membranes, resulting in lipid raft disruption. Furthermore, by inhibiting protein prenylation, a process also dependent on mevalonate, statins block membrane targeting and activity of small GTPases. Antigen uptake, processing and presentation involve the interplay of Rab and Rho family GTPases. Furthermore, lipid rafts have been implicated both in antigen internalization by the BCR and in MHC class II clustering at the immunological synapse. Here we have addressed the effects of simvastatin on antigen processing and presentation by human B cells and dendritic cells. The results show that simvastatin potently suppresses tetanus toxoid processing and presentation to CD4⁺ T cells by HLA‐DR by inhibiting protein antigen uptake through both receptor‐mediated endocytosis and macropinocytosis. This effect can be largely accounted for by defective prenylation of Rho and Rab GTPases in the absence of any measurable perturbation of lipid rafts. In addition, simvastatin was found to preferentially affect the invariant chain‐dependent MHC class II pathway, thereby identifying this route of antigen processing and presentation as a selective target of statins.