Deletion 6q Drives T-cell Leukemia Progression by Ribosome Modulation.

Deletion of chromosome 6q is a well-recognized abnormality found in poor-prognosis T-cell acute lymphoblastic leukemia (T-ALL). Using integrated genomic approaches, we identified two candidate haploinsufficient genes contiguous at 6q14, SYNCRIP (encoding hnRNP-Q) and SNHG5 (that hosts snoRNAs), both involved in regulating RNA maturation and translation. Combined silencing of both genes, but not of either gene alone, accelerated leukemogeneis in a Tal1/Lmo1/Notch1-driven mouse model, demonstrating the tumor-suppressive nature of the two-gene region. Proteomic and translational profiling of cells in which we engineered a short 6q deletion by CRISPR/Cas9 genome editing indicated decreased ribosome and mitochondrial activities, suggesting that the resulting metabolic changes may regulate tumor progression. Indeed, xenograft experiments showed an increased leukemia-initiating cell activity of primary human leukemic cells upon coextinction of SYNCRIP and SNHG5. Our findings not only elucidate the nature of 6q deletion but also highlight the role of ribosomes and mitochondria in T-ALL tumor progression. SIGNIFICANCE: The oncogenic role of 6q deletion in T-ALL has remained elusive since this chromosomal abnormality was first identified more than 40 years ago. We combined genomic analysis and functional models to show that the codeletion of two contiguous genes at 6q14 enhances malignancy through deregulation of a ribosome-mitochondria axis, suggesting the potential for therapeutic intervention.This article is highlighted in the In This Issue feature, p. 1494.

ABT-199 mediated inhibition of BCL-2 as a novel therapeutic strategy in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk subtype of acute lymphoblastic leukemia (ALL) with gradually improved survival through introduction of intensified chemotherapy. However, therapy-resistant or refractory T-ALL remains a major clinical challenge. Here, we evaluated B-cell lymphoma (BCL)-2 inhibition by the BH3 mimetic ABT-199 as a new therapeutic strategy in human T-ALL. The T-ALL cell line LOUCY, which shows a transcriptional program related to immature T-ALL, exhibited high in vitro and in vivo sensitivity for ABT-199 in correspondence with high levels of BCL-2. In addition, ABT-199 showed synergistic therapeutic effects with different chemotherapeutic agents including doxorubicin, l-asparaginase, and dexamethasone. Furthermore, in vitro analysis of primary patient samples indicated that some immature, TLX3- or HOXA-positive primary T-ALLs are highly sensitive to BCL-2 inhibition, whereas TAL1 driven tumors mostly showed poor ABT-199 responses. Because BCL-2 shows high expression in early T-cell precursors and gradually decreases during normal T-cell differentiation, differences in ABT-199 sensitivity could partially be mediated by distinct stages of differentiation arrest between different molecular genetic subtypes of human T-ALL. In conclusion, our study highlights BCL-2 as an attractive molecular target in specific subtypes of human T-ALL that could be exploited by ABT-199.

Spontaneous abrogation of the G2DNA damage checkpoint has clinical benefits but promotes leukemogenesis in Fanconi anemia patients.

DNA damage checkpoints in the cell cycle may be important barriers against cancer progression in human cells. Fanconi anemia (FA) is an inherited DNA instability disorder that is associated with bone marrow failure and a strong predisposition to cancer. Although FA cells experience constitutive chromosomal breaks, cell cycle arrest at the G2 DNA damage checkpoint, and an excess of cell death, some patients do become clinically stable, and the mechanisms underlying this, other than spontaneous reversion of the disease-causing mutation, are not well understood. Here we have defined a clonal phenotype, termed attenuation, in which FA patients acquire an abrogation of the G2 checkpoint arrest. Attenuated cells expressed lower levels of CHK1 (also known as CHEK1) and p53. The attenuation could be recapitulated by modulating the ATR/CHK1 pathway, and CHK1 inhibition protected FA cells from cell death. FA patients who expressed the attenuated phenotype had mild bone marrow deficiency and reached adulthood, but several of them eventually developed myelodysplasia or leukemia. Better understanding of attenuation might help predict a patient's clinical course and guide choice of treatment. Our results also highlight the importance of evaluating the cellular DNA damage checkpoint and repair pathways in cancer therapies in general.

Aberrant activation of stress-response pathways leads to TNF-alpha oversecretion in Fanconi anemia.

Fanconi anemia (FA), an inherited syndrome that associates bone marrow failure, cancer predisposition, and genetic instability, is characterized by an overproduction of the myelosuppressive cytokine TNF-alpha through unknown mechanisms. We demonstrate here that FANC pathway loss-of-function results in the aberrant activation of 2 major stress-signaling pathways: NF-kappaB and MAPKs. These responses are independent on TNF-alpha expression. On the contrary, inhibition of the MAPK pathways normalizes TNF-alpha oversecretion in FA. Moreover, our data show that the overexpression of the matrix metalloproteinase MMP-7 is the key event directly responsible for the high rate of TNF-alpha shedding and release from the cytoplasmic membrane in FA. TNF-alpha overproduction is, indeed, normalized by MMP-7 inhibition. Finally, MAPK inhibition impacts on MMP-7 overexpression. Evidence is provided of the existence of a linear pathway in which FANC mutations activate MAPK signaling that induces MMP-7 overexpression leading, in fine, to TNF-alpha oversecretion. TNF-alpha may, in turn, sustain or amplify both MAPKs and NF-kappaB activation. Aberrant expression or activity of NF-kappaB and/or MAPKs has been already involved in bone marrow failure and leukemia, and their inhibition offered clinical benefit for patients. In conclusion, our data provide a strong rationale for new clinical trials on FA patients.

Fanconi anemia C gene product regulates expression of genes involved in differentiation and inflammation.

Loss of Fanconi anemia (FA) proteins activity by recessive inherited mutations in one of the FA genes leads to a disease characterized by bone marrow failure, myeloid leukemia and DNA damage hypersensitivity. The aim of this work was to improve our understanding of the FA syndrome defining the transcription profile of the FA complementation group C (FANCC)-deficient cells in comparison to their ectopically corrected counterpart using oligonucleotide microarrays. In this way, 49 RNAs have been isolated, which showed a consistent differential pattern of expression among FANCC mutated and corrected cells. The observed specific changes in gene expression suggest that FANCC regulates specifically myeloid differentiation and unmasks a previously unsuspected anti-inflammatory role for the FA proteins. In spite of the DNA damage hypersensitivity of the syndrome, no gene coding for a protein directly involved in DNA repair/damage response was found to be deregulated in our analysis. This observation suggests that FANCC does not directly control genes involved in DNA repair at the transcriptional level, but does not exclude a regulation at the translational or post-translational level, or by protein/protein interactions. The potential role of the differentially expressed genes in FA phenotype as well as a functional- and cellular-based clustering of the identified genes are presented and discussed.

Recombinant adeno-associated virus serotype 4 mediates unique and exclusive long-term transduction of retinal pigmented epithelium in rat, dog, and nonhuman primate after subretinal delivery.

We previously described chimeric recombinant adeno-associated virus (rAAV) vectors 2/4 and 2/5 as the most efficient vectors in rat retina. We now characterize these two vectors carrying the CMV.gfp genome following subretinal injection in the Wistar rat, beagle dog, and cynomolgus macaque. Both serotypes displayed stable GFP expression for the duration of the experiment (6 months) in all three animal models. Similar to the AAV-2 serotype, AAV-2/5 transduced both RPE and photoreceptor cells, with higher level of transduction in photoreceptors, whereas rAAV-2/4 transduction was unambiguously restricted to RPE cells. This unique specificity found conserved among all three species makes AAV-2/4-derived vectors attractive for retinal diseases originating in RPE such as Leber congenital amaurosis (RPE65) or retinitis pigmentosa due to a mutated mertk gene. To provide further important preclinical data, vector shedding was monitored by PCR in various biological fluids for 2 months post-rAAV administration. Following rAAV-2/4 and -5 subretinal delivery in dogs (n = 6) and in nonhuman primates (n = 2), vector genome was found in lacrymal and nasal fluids for up to 3-4 days and in the serum for up to 15-20 days. Overall, these findings will have a practical impact on the development of future gene therapy trials of retinal diseases.

Sustained tetracycline-regulated transgene expression in vivo in rat retinal ganglion cells using a single type 2 adeno-associated viral vector.

BACKGROUND: Viral vector delivery of neurotrophic-expressing transgenes in the retina may retard or prevent the onset of blindness associated with photoreceptor degeneration. A key safety issue is to achieve regulated expression of these genes in the retina. The purpose of our study was to evaluate whether a single recombinant AAV-2 (rAAV) encoding for a tetracycline (Tet)-regulated destabilized reporter gene could provide quantitative profiles of gene regulation targeted to the rat neuroretina. METHODS: A rAAV vector carrying a destabilized green fluorescent protein (dgfp) under a tet-regulatable promoter and the tetracycline-repressed transactivator (tTA) was generated (rAAVtetoff.dgfp) and administered intravitreally in nine Wistar rats. Retinas were monitored for 6 months using noninvasive fluorescence imaging and the animals were subjected to two cycles of doxycycline (Dox), a tetracycline analog. Eyes were ultimately examined by histology. RESULTS: Intravitreal injection of rAAVtetoff.dgfp resulted in effective transduction of ganglion cells. Following full expression of the transgene in the absence of Dox, 95% of the GFP signal was shut down 48 h post Dox administration and the signal was undetectable 7 days later. Initial levels of GFP expression were restored 21 days after Dox administration ceased. This pattern of expression was repeated twice over a period of 6 months. CONCLUSIONS: This report demonstrates that rAAVtetoff.dgfp intravitreally injected rats displayed tight and sustained long-term regulation of the reporter gene in ganglion cells. These findings may have important implications regarding rAAV-mediated gene therapy using neuroprotective approaches for retinitis pigmentosa and glaucoma.

The Fanconi anemia pathway and the DNA interstrand cross-links repair.

Fanconi anemia (FA) is a genetic cancer-predisposition syndrome characterized by bone marrow failure and cellular and chromosomal hypersensitivity to DNA cross-linking agents. Seven FA genes have been isolated and their products associate to form a pathway that interacts functionally or physically with several DNA-damage response proteins involved in cell cycle checkpoints and/or DNA repair. These proteins include BLM, ATM, BRCA1, XPF and the MRE11/RAD50/NBS1 complex. In spite of several recent striking progresses in the biochemistry and the molecular biology of the disorder, the precise function(s) of the FA proteins remain(s) poorly determined. However, several recent data indicate that the FA pathway could be involved in the coordination of both cell cycle checkpoints and DNA repair.