Cell cycle arrest and p53 prevent ON-target megabase-scale rearrangements induced by CRISPR-Cas9.

The CRISPR-Cas9 system has revolutionized our ability to precisely modify the genome and has led to gene editing in clinical applications. Comprehensive analysis of gene editing products at the targeted cut-site has revealed a complex spectrum of outcomes. ON-target genotoxicity is underestimated with standard PCR-based methods and necessitates appropriate and more sensitive detection methods. Here, we present two complementary Fluorescence-Assisted Megabase-scale Rearrangements Detection (FAMReD) systems that enable the detection, quantification, and cell sorting of edited cells with megabase-scale loss of heterozygosity (LOH). These tools reveal rare complex chromosomal rearrangements caused by Cas9-nuclease and show that LOH frequency depends on cell division rate during editing and p53 status. Cell cycle arrest during editing suppresses the occurrence of LOH without compromising editing. These data are confirmed in human stem/progenitor cells, suggesting that clinical trials should consider p53 status and cell proliferation rate during editing to limit this risk by designing safer protocols.

CRISPR-Cas9 globin editing can induce megabase-scale copy-neutral losses of heterozygosity in hematopoietic cells.

CRISPR-Cas9 is a promising technology for gene therapy. However, the ON-target genotoxicity of CRISPR-Cas9 nuclease due to DNA double-strand breaks has received little attention and is probably underestimated. Here we report that genome editing targeting globin genes induces megabase-scale losses of heterozygosity (LOH) from the globin CRISPR-Cas9 cut-site to the telomere (5.2 Mb). In established lines, CRISPR-Cas9 nuclease induces frequent terminal chromosome 11p truncations and rare copy-neutral LOH. In primary hematopoietic progenitor/stem cells, we detect 1.1% of clones (7/648) with acquired megabase LOH induced by CRISPR-Cas9. In-depth analysis by SNP-array reveals the presence of copy-neutral LOH. This leads to 11p15.5 partial uniparental disomy, comprising two Chr11p15.5 imprinting centers (H19/IGF2:IG-DMR/IC1 and KCNQ1OT1:TSS-DMR/IC2) and impacting H19 and IGF2 expression. While this genotoxicity is a safety concern for CRISPR clinical trials, it is also an opportunity to model copy-neutral-LOH for genetic diseases and cancers.

Spleen route accelerates engraftment of human hematopoietic stem cells.

Intravenous injections of human hematopoietic stem cells (hHSCs) is routinely used in clinic and for modeling hematopoiesis in mice. However, unspecific dilution in vascular system and non-hematopoietic organs challenges engraftment efficiency. Although spleen is capable of extra medullar hematopoiesis, its ability to support human HSC transplantation has never been evaluated. We demonstrate that intra-splenic injection results in high and sustained engraftment of hHSCs into immune-deficient mice, with higher chimerisms than with intravenous or intra-femoral injections. Our results support that spleen microenvironment provides a niche for HSCs amplification and offers a new route for efficient HSC transplantation.

Effect of tyrosine kinase inhibitors on stemness in normal and chronic myeloid leukemia cells.

Although tyrosine kinase inhibitors (TKIs) efficiently cure chronic myeloid leukemia (CML), they can fail to eradicate CML stem cells (CML-SCs). The mechanisms responsible for CML-SC survival need to be understood for designing therapies. Several previous studies suggest that TKIs could modulate CML-SC quiescence. Unfortunately, CML-SCs are insufficiently available. Induced pluripotent stem cells (iPSCs) offer a promising alternative. In this work, we used iPSCs derived from CML patients (Ph+). Ph+ iPSC clones expressed lower levels of stemness markers than normal iPSCs. BCR-ABL1 was found to be involved in stemness regulation and ERK1/2 to have a key role in the signaling pathway. TKIs unexpectedly promoted stemness marker expression in Ph+ iPSC clones. Imatinib also retained quiescence and induced stemness gene expression in CML-SCs. Our results suggest that TKIs might have a role in residual disease and confirm the need for a targeted therapy different from TKIs that could overcome the stemness-promoting effect caused by TKIs. Interestingly, a similar pro-stemness effect was observed in normal iPSCs and hematopoietic SCs. These findings could help to explain CML resistance mechanisms and the teratogenic side-effects of TKIs in embryonic cells.

Lack of beta-catenin in early life induces abnormal glucose homeostasis in mice.

AIMS/HYPOTHESIS: Wingless and iNT-1 (WNT) pathway members are critical for pancreatic development and exocrine tissue formation. Recently, much attention has focused on delineating the roles of beta-catenin in pancreatic organogenesis. However, little is known about the involvement of beta-catenin in the endocrine or exocrine function of the mature pancreas. We report for the first time the impact of beta-catenin deletion in the pancreatic beta cells. METHODS: We targeted the deletion of the beta-catenin gene in pancreatic beta cells by crossing a floxed beta-catenin mouse strain with a RIP-Cre mouse strain. RESULTS: Surprisingly, the majority of the mutant mice died shortly after birth and had deregulated glucose and insulin levels. The newborn mutant pancreases demonstrated increased insulin content, reflecting a defect in insulin release confirmed in vitro. Moreover, there was a reduction in total endocrine tissue at birth, while cellularity in islets was greater, suggesting that lack of beta-catenin affects beta cell size. Some newborns survived beta-catenin deletion and showed a milder phenotype during adulthood. CONCLUSIONS/INTERPRETATION: The deletion of beta-catenin in the maturing beta cells negatively impacts on islet morphology and function. This work reveals that lack of beta-catenin in early life is related to severe deregulation of glucose homeostasis.

Growth factor-induced signaling of the pancreatic epithelium.

Activated signaling proteins regulate diverse processes, including the differentiation of the pancreatic islet cells during ontogeny. Here we uncover the in vivo phosphorylation status of major growth factor-activated signaling proteins in normal adult mice and during pancreatic islet regeneration. We report elevated phospho-mitogen-activated protein kinase (phospho-MAPK), phospho-c-Jun-NH2-terminal kinase (phospho-JNK), and phospho-p38 MAPK expression during pancreatic regeneration. Immunoblotting experiments demonstrated elevated phosphorylation of p52 Src-homology/collagen (SHC) in the ductal network as well, substantiating the activation of this pathway. Furthermore, protein kinase B (PKB/Akt), a key signaling protein in the anti-apoptotic pathway, was phosphorylated to a greater extent in the ductal network from regenerating pancreas. We observed fibroblast growht factor (FGF)10 and platelet-derived growth factor (PDGF)AA expression in embryonic as well as regenerating adult pancreas. Epidermal growth factor (EGF) and PDGFAA stimulated MAPK and Akt phosphorylation, while FGF10 stimulated MAPK but not Akt phosphorylation in a time-dependent manner in freshly isolated cells from the adult ductal network. These data suggest that a heightened level of expression and stimulation of key signaling proteins underlie the expansion and differentiation processes that support pancreatic ontogeny and regeneration.

Differential expression of the nm23 genes in the developing human trophoblast.

NDP kinases are the non-specific enzymes which catalyse the synthesis of the NTPs through a transfer reaction using ATP as phosphoryl donor. In addition to their enzymatic activity, they display other not yet explained functions related to cell growth, differentiation and apoptosis, embryonic development, tumour progression and metastasis. In this study, the expression patterns of the three highly related NDP kinases A, B and C isoforms were investigated in the developing human trophoblast. Both NDP kinase A and B were found to be primarily present in the villous and extravillous cytotrophoblasts, while NDP kinase C was found almost exclusively in the syncytiotrophoblast layer. This suggests that NDP kinase A and B could be a marker for the mononuclear stage of differentiation of villous trophoblasts, while NDP kinase C could be a marker of the syncytiotrophoblast layer.

Knockout mice as model systems for studying nm23/NDP kinase gene functions. Application to the nm23-M1 gene.

Mice carrying a homozygous germ-line mutation in the nm23-M1 gene that eliminates its protein expression and drives expression of beta-galactosidase by nm23-M1 promoter have been generated. nm23-M1 gene inactivation is not teratogenic and the pups can grow to adult age without apparent health problems. However, they undergo a growth retardation and knocked out females cannot feed their pups. Both effects are background dependent. Beta-galactosidase mapping of nm23-M1 promoter activation during embryogenesis shows that the nm23-M1 gene is principally expressed in epithelial layer of tissues which require inductive epithelial-mesenchymal interactions for their formation. In conclusion, invalidated mice could be interesting models to analyze the role of nm23-M1 on signal transduction pathway regulation, or cancer induction and proliferation.

Characterization of the nm23-M2, nm23-M3 and nm23-M4 mouse genes: comparison with their human orthologs.

The nm23 gene family is thought to be involved in physiopathological processes such as growth, differentiation and cancer promotion, progression or metastasis. We report here the mouse nm23-M3 and nm23-M4 complementary DNA sequences and the genomic cloning, characterization and tissue expression pattern of the nm23-M2, nm23-M3 and nm23-M4 genes, in comparison with their human and rat orthologs and with the human nm23-H1 and mouse nm23-M1 genes. The organization and structure of the members of this gene family are remarkably similar in human and rodents. Accordingly, the striking similarities between the human and mouse nm23 genes enable the use of mouse transgenic and knock-out models for studying the role of nucleoside diphosphate kinase isoforms in human physiopathology.

Organization and expression of mouse nm23-M1 gene. Comparison with nm23-M2 expression.

Nm23 is a gene family encoding different isoforms of the nucleotide diphosphate kinase (NDPK), an enzyme involved in the synthesis of nucleoside triphosphates. In the present study, the organization and expression of the nm23-M1 gene encoding the mouse NDPKA isoform are described. This gene is about 10kb long and composed of five exons. The organization and the exon-intron boundaries are strictly conserved as compared to the human and rat related genes. The gene promoter region did not exhibit any consensus TATA box, SP1 binding element or Inr sequence. By contrast, TCF-1/LEF-1 binding elements and Pit-1 consensus sequence were present. Northern blotting and in situ hybridization methods were carried out in adult and 18.5 days post-coitum (dpc) mouse embryo, respectively. They showed tissue-specific expression of nm23-M1 transcripts, despite housekeeping gene promoter features. The strongest signals were detected in the nervous system, sensory organs and embryonic thymus. In contrast nm23-M2 mRNA was shown to be more widely expressed.The relationship between nm23-M1 gene tissue-specific expression and the putative binding element of the promoter region is discussed.

Catalytic mechanism of nucleoside diphosphate kinase investigated using nucleotide analogues, viscosity effects, and X-ray crystallography.

Nucleoside diphosphate (NDP) kinases display low specificity with respect to the base moiety of the nucleotides and to the 2'-position of the ribose, but the 3'-hydroxyl is found to be important for catalysis. We report in this paper the enzymatic analysis of a series of derivatives of thymidine diphosphate (TDP) where the 3'-OH group was removed or replaced by fluorine, azido, and amino groups. With Dictyostelium NDP kinase, kcat decreases 15-200-fold from 1100 s-1 with TDP, and (kcat/Km)NDP decreases from 12 x 10(6) to 10(3) to 5 x 10(4) M-1 s-1, depending on the substrate. The poorest substrates are 3'-deoxyTDP and 3'-azido-3'-deoxyTDP, while the best modified substrates are 2',3'-dehydro-3'-deoxyTDP and 3'-fluoro-3'-deoxyTDP. In a similar way, 3'-fluoro-2',3'-dideoxyUDP was found to be a better substrate than 2',3'-dideoxyUDP, but a much poorer substrate than 2'-deoxyUDP. (kcat/Km)NDP is sensitive to the viscosity of the solution with TDP as the substrate but not with the modified substrates. To understand the poor catalytic efficiency of the modified nucleotides at a structural level, we determined the crystal structure of Dictyostelium NDP kinase complexed to 3'-fluoro-2',3'-dideoxyUDP at 2.7 A resolution. Significant differences are noted as compared to the TDP complex. Substrate-assisted catalysis by the 3'-OH, which is effective in the NDP kinase reaction, cannot occur with the modified substrate. With TDP, the beta-phosphate, which is the leaving group when a gamma-phosphate is transferred to His122, hydrogen bonds to the 3'-hydroxyl group of the sugar; with 3'-fluoro-2',3'-dideoxyUDP, the beta-phosphate hydrogen bonds to Asn119 and moves away from the attacking Ndelta of the catalytic His122. Since all anti-AIDS nucleoside drugs are modified at the 3'-position, these results are relevant to the role of NDP kinase in their cellular metabolism.

Cloning of a second nm23-M1 cDNA: expression in the central nervous system of adult mouse and comparison with nm23-M2 mRNA distribution.

Nm23 has been identified as a gene family encoding different isoforms of the nucleoside diphosphate kinase. This protein is a key enzyme in the control of cellular concentrations of nucleoside triphosphates. Moreover, it has been shown to play important roles in various cellular functions such as differentiation and metastasis. In the present study, a second cDNA for nucleoside diphosphate kinase A (Nm23-M1) was isolated from a cDNA library of mouse embryonic stem cells. This clone encodes the same putative 152 aminoacids long protein as an already published cDNA but is longer in both its 5' and 3' untranslated regions. Tissue and cellular distribution of nm23-M1 mRNA was investigated by using Northern blot analysis and in situ hybridization. Nm23-M1 transcripts were found to be widely distributed throughout the mouse central nervous system with prominent expression in several restricted areas. No differences were noticed between the distribution of long and short transcripts. Furthermore, a similar pattern of expression was described in the central nervous system for nm23-M2 mRNA, encoding a second isoform of the nucleoside diphosphate kinase. However, the transcript of this isoform displayed a wider distribution and was expressed in all organs analysed by northern blotting. The possible involvement of nm23-M1 in differentiation of mouse nervous system is further discussed.