Targeting the vulnerability to NAD+ depletion in B-cell acute lymphoblastic leukemia.

Although substantial progress has been made in the treatment of B-cell acute lymphoblastic leukemia (B-ALL), the prognosis of patients with either refractory or relapsed B-ALL remains dismal. Novel therapeutic strategies are needed to improve the outcome of these patients. KPT-9274 is a novel dual inhibitor of p21-activated kinase 4 (PAK4) and nicotinamide phosphoribosyltransferase (NAMPT). PAK4 is a serine/threonine kinase that regulates a variety of fundamental cellular processes. NAMPT is a rate-limiting enzyme in the salvage biosynthesis pathway of nicotinamide adenine dinucleotide (NAD) that plays a vital role in energy metabolism. Here, we show that KPT-9274 strongly inhibits B-ALL cell growth regardless of cytogenetic abnormalities. We also demonstrate the potent in vivo efficacy and tolerability of KPT-9274 in a patient-derived xenograft murine model of B-ALL. Interestingly, although KPT-9274 is a dual PAK4/NAMPT inhibitor, B-ALL cell growth inhibition by KPT-9274 was largely abolished with nicotinic acid supplementation, indicating that the inhibitory effects on B-ALL cells are mainly exerted by NAD+ depletion through NAMPT inhibition. Moreover, we have found that the extreme susceptibility of B-ALL cells to NAMPT inhibition is related to the reduced cellular NAD+ reserve. NAD+ depletion may be a promising alternative approach to treating patients with B-ALL.

BCOR regulates myeloid cell proliferation and differentiation.

BCOR is a component of a variant Polycomb group repressive complex 1 (PRC1). Recently, we and others reported recurrent somatic BCOR loss-of-function mutations in myelodysplastic syndrome and acute myelogenous leukemia (AML). However, the role of BCOR in normal hematopoiesis is largely unknown. Here, we explored the function of BCOR in myeloid cells using myeloid murine models with Bcor conditional loss-of-function or overexpression alleles. Bcor mutant bone marrow cells showed significantly higher proliferation and differentiation rates with upregulated expression of Hox genes. Mutation of Bcor reduced protein levels of RING1B, an H2A ubiquitin ligase subunit of PRC1 family complexes and reduced H2AK119ub upstream of upregulated HoxA genes. Global RNA expression profiling in murine cells and AML patient samples with BCOR loss-of-function mutation suggested that loss of BCOR expression is associated with enhanced cell proliferation and myeloid differentiation. Our results strongly suggest that BCOR plays an indispensable role in hematopoiesis by inhibiting myeloid cell proliferation and differentiation and offer a mechanistic explanation for how BCOR regulates gene expression such as Hox genes.

Uniquely conserved non-translated regions are involved in generation of the two major transcripts of protein phosphatase 2Cbeta.

Partial cDNAs of different isoforms of protein phosphatase 2Cbeta (PP2Cbeta or PPM1B) have been characterized in mammals. We disclose here the full cDNAs of two major PP2Cbeta isoforms from human, rat and mouse. These cDNAs (2.6 and 3.3 kb) are able to encode 53 kDa (PP2Cbetal) and 43 kDa (PP2Cbetas) polypeptides, respectively. The isoforms are co-expressed ubiquitously with the highest level in skeletal muscle, as assessed by Northern-blot analysis. Western and in situ analyses using monoclonal antibodies against PP2Cbeta confirmed the existence of two isoforms in the cytoplasm. Comparative sequence analysis revealed that both cDNAs consist of six exons with an alternate usage of the 3' exons that underlies the differences between them. The genomic structure of PP2Cbeta is similar to that of other PP2C paralogs and includes a non-coding first exon followed by a large intron and a large second exon that encoded most of the catalytic domain. Both variants of the ending exon include large non-coding regions. All non-translated regions (NTRs) are highly conserved between the orthologous genes, indicating their regulatory function. The 5'-NTR is long (379 bp), includes upstream start codons and is predicted to contain stable secondary structures. Such features inhibit translation initiation by the scanning mechanism. Introduction of this NTR element into a bi-luciferase expression-cassette enabled expression of the second cistron, suggesting that it might serve as an internal ribosome entry site, or it contains a cryptic promoter. Overexpression of PP2Cbeta under CMV-promoter in 293 cells led to cell-growth arrest or cell death.

LNK (SH2B3): paradoxical effects in ovarian cancer.

LNK (SH2B3) is an adaptor protein studied extensively in normal and malignant hematopoietic cells. In these cells, it downregulates activated tyrosine kinases at the cell surface resulting in an antiproliferative effect. To date, no studies have examined activities of LNK in solid tumors. In this study, we found by in silico analysis and staining tissue arrays that the levels of LNK expression were elevated in high-grade ovarian cancer. To test the functional importance of this observation, LNK was either overexpressed or silenced in several ovarian cancer cell lines. Remarkably, overexpression of LNK rendered the cells resistant to death induced by either serum starvation or nutrient deprivation, and generated larger tumors using a murine xenograft model. In contrast, silencing of LNK decreased ovarian cancer cell growth in vitro and in vivo. Western blot studies indicated that overexpression of LNK upregulated and extended the transduction of the mitogenic signal, whereas silencing of LNK produced the opposite effects. Furthermore, forced expression of LNK reduced cell size, inhibited cell migration and markedly enhanced cell adhesion. Liquid chromatography-mass spectroscopy identified 14-3-3 as one of the LNK-binding partners. Our results suggest that in contrast to the findings in hematologic malignancies, the adaptor protein LNK acts as a positive signal transduction modulator in ovarian cancers.

Use of magnetic resonance imaging for noninvasive characterization and follow-up of an experimental injury to normal mouse muscles.

A magnetic resonance imaging protocol was tested in a cardiotoxin-induced myonecrosis of hindlimb muscles of three normal mice to assess the usefulness of data provided by longitudinal follow-up of a few individuals. Magnetic resonance imaging examinations were performed sequentially at 4 T between days 1 and 11 post-injury. Axial T1-weighted images after injection of a paramagnetic contrast agent were used to determine the volume of lesions from regions of increased signal intensity. T2 measurements were performed from a single-slice ten-echo acquisition centered upon the largest section of lesion. Early after injury, a very large T2 increase was observed. As recovery proceeded, T2 values progressively decreased toward normal values. Similarly, the volumes of lesions decreased to virtually zero by days 10-11. The evolution of these indices followed the same time scheme observed in histological studies. The use of a volume probe allowed accurate measurement of T2 values, and the acquisition of volumetric data. Such magnetic resonance imaging follow-up should help gather valuable information using few animals.

Muscular transverse relaxation time measurement by magnetic resonance imaging at 4 Tesla in normal and dystrophic dy/dy and dy(2j)/dy(2j) mice.

Muscular transverse relaxation times values were measured in vivo in normal mice (strain C57BL6/J, n=14) and in murine models of human congenital muscular dystrophy (dy/dy, n=9; dy(2j)/dy(2j), n=8). A single-slice multi-echo sequence was used. Gastrocnemius/soleus complex, thigh and buttock muscles were studied. Muscular transverse relaxation times values were compared between different muscle groups in each type of animal and between animal groups. Differences were observed between normal and dy(2j)/dy(2j) mice from 3 to 12 weeks of age, and between normal and dy/dy mice at 6 weeks. In specific age ranges, the values of muscular transverse relaxation times in two dystrophic models are different from those in normal mice, and could thus be used as an index of modifications in dystrophic muscle to evaluate therapies.

An Escherichia coli gene divergently transcribed from a promoter overlapping the metA promoter.

The upstream region of the metA gene in Escherichia coli contains two promoters. We have identified by lacZ fusion an additional promoter in this region, and showed that it is transcribed in the opposite orientation from the metA gene. The putative translation product corresponds to a peptide of 147 amino acids-ORF19 by molecular mass. This peptide is probably not essential for growth, as an insertion mutant is viable.

Computed radiography.

In several medical centers computed radiography has almost completely replaced the use of conventional screen-film systems for general radiography. The aim of this paper is to explain the basic principles of the four most frequently numerical detectors used in the world, with emphasis on the phosphor plates, which are the most frequently used both in hospitals and by practitioners. The other two systems are based on a receptor with selenium. The fourth uses charged coupled device (CCD) detectors. The most important principles of digital processing are then described with concentration on unsharp mask filtering. In the future computed radiography will replace standard radiology and will create a system in medicine using the power of computers to archive--with more efficiency and less space--patient medical data. The transmission of data to workstations and the processing of this data is the topic of a new field in medicine.

[Techniques to improve the accuracy of CT-guided abdominal biopsy]. / Techniques permettant d'améliorer la rentabilité des ponctions abdominales sous scanner.

STUDY AIM: The aim of this paper is to present an overview of the various technical progresses made in the field of CT-guided abdominal biopsies. Recent improvements allowed to markedly increase the efficiency of biopsies and to decrease the number of complications. The main innovations concern the guidance technique itself with the availability of ultra-fast CT systems, the development of automated biopsy systems, which allow to improve the size and quality of tissue samples and numerous technical tricks, allowing an easier access to target lesions, either in patient positioning or in displacement of anatomical structures. A better management of tissue samples favored by a close collaboration with pathologists is also mandatory. The last section of the paper is an overview of the rare complications of CT-guided biopsies.

[Digital thoracic radiology: devices, image processing, limits]. / Radiographie thoracique numérisée: appareillages, traitements d'images, limites.

In a first part, the different techniques of digital thoracic radiography are described. Since computed radiography with phosphore plates are the most commercialized it is more emphasized. But the other detectors are also described, as the drum coated with selenium and the direct digital radiography with selenium detectors. The other detectors are also studied in particular indirect flat panels detectors and the system with four high resolution CCD cameras. In a second step the most important image processing are discussed: the gradation curves, the unsharp mask processing, the system MUSICA, the dynamic range compression or reduction, the soustraction with dual energy. In the last part the advantages and the drawbacks of computed thoracic radiography are emphasized. The most important are the almost constant good quality of the pictures and the possibilities of image processing.

[Role of MRI in the diagnosis of breast diseases]. / Place de l'IRM dans le diagnostic des affections mammaires.

UNLABELLED: ADVANTAGES AND LIMITATIONS: Magnetic resonance imaging of the breast is probably the most sensitive method for detecting or ruling out breast disease. It is however not as specific as expected. TECHNIQUE: All examinations are performed with and without gadolinium intravenous administration, excepted in the case of silicone implant reconstruction mammoplasty. Dynamic contrast-enhanced MRI sequences are necessary with a permanent balance between temporal resolution, spatial resolution, and signal. MRI of the breast is not indicated as a routine examination in a screening program, neither to improve the specificity of infra-clinic lesions, nor in simply dense breasts without any known risk factor or in circumscribed masses. INDICATIONS: MRI is best used to improved the sensitivity of mammography and sonography in selected patients. The selected indications are: evaluation of the volume and extension of breast lesions evaluation of the therapeutic response after chemotherapy, and detection or exclusion of the local recurrence in patients with breast conservation therapy. PERSPECTIVES: The future indications and perspectives of MRI include interventional breast radiology (MRI-guided core biopsy), and thermocoagulation therapy. It may be interesting for the evaluation of patients with contrast enhanced MRI lesions with normal mammography and sonography, and also in woman with a genetically defined high breast cancer risk.

Role of the adaptor protein LNK in normal and malignant hematopoiesis.

The signal transduction pathways, orchestrating the differentiation of hematopoietic stem and progenitor cells in response to cytokine stimulation, are strictly controlled by networks of feedback loops, highly selective protein interactions and finely tuned on/off switches. In hematological malignancies, the aberrant activation of signaling pathways is usually associated with mutations in tyrosine kinases. Recently, the role of negative signaling regulators is increasingly being recognized as an alternative mechanism involved in diseases such as leukemias and myeloproliferative neoplasms (MPNs). The adaptor protein LNK (Src homology 2 (SH2)B3) is a negative regulator of cytokine signaling that has an essential, nonredundant role in normal hematopoiesis. Indeed, LNK-deficient mice show marked expansion of early hematopoietic precursors, more mature myeloid and B-lineage lymphoid cells, as well as enhanced hematopoietic reconstitution. Murine models show that loss of LNK enhances the development of MPNs and may have a role in additional pathologies. LNK mutations were recently identified in patients with MPNs, and studies in animal models and hematopoietic cell lines suggest that LNK controls the aberrant signaling pathways induced by activated oncogenic kinases. In addition, genome-wide studies show that LNK is associated with autoimmune and cardiovascular disorders. These findings have implications for the future study of hematopoiesis, as well as for the development of novel stem cell and disease-specific therapies.

The role of circadian regulation in cancer.

Proper circadian regulation is essential for the well being of the organism, and disruption of circadian rhythms is associated with pathological conditions including cancer. In mammals, the core clock genes, Per1 and Per2, are key regulators of circadian rhythms both in the central clock in the hypothalamous and in peripheral tissues. Recent findings revealed molecular links between Per genes and cellular components that control fundamental cellular processes such as cell division and DNA damage. New data also shed light on mechanisms by which circadian oscillators operate in peripheral organs to influence tissue-dependent metabolic and hormonal pathways. Circadian cycles are linked to basic cellular functions, as well as to tissue-specific processes through the control of gene expression and protein interactions. By controlling global networks such as chromatin remolding and protein families, which themselves regulate a broad range of cellular functions, circadian regulation impinges upon almost all major physiological pathways. These molecular insights illustrate how disregulation of circadian rhythms might influence the susceptibility to cancer development and provide further support for the emerging role of circadian genes in tumor suppression.

The clock gene Per2 links the circadian system to the estrogen receptor.

Circadian rhythms regulate diverse physiological processes including homeostatic functions of steroid hormones and their receptors. Estrogen receptor-alpha (ERalpha) is essential for normal mammary gland physiology and is a prognostic marker for the treatment of breast cancer. We report that Per2, a core clock gene, links the circadian cycle to the ERalpha signaling network. Binding of enhances ERalpha degradation, while suppression of Per2 levels leads to ERalpha stabilization. In turn, Per2 itself is estrogen inducible in these cells, suggesting a feedback mechanism to attenuate stimulation by estrogen. In addition, overexpression of Per2 in breast cancer cells leads to significant growth inhibition, loss of clonogenic ability and apoptosis. Taken together, these results further support a critical role for peripheral circadian regulation in tissue homeostasis and suggest a novel role for clock genes in estrogen receptor-positive breast cancer.

Closed ruptures of the flexor digitorum tendons: MRI evaluation.

OBJECTIVE: To assess the MRI findings in cases of closed rupture of the flexor digitorum tendons (FDT). PATIENTS AND DESIGN: Ten patients with a clinical suspicion of rupture of FDT underwent MRI before surgery. None of the patients presented a skin injury. Fingers were imaged using axial T1-weighted SE sequences, three-dimensional GE images, and curved reconstructions. RESULTS: Twelve FDT had surgical confirmation of rupture. Flexor digitorum profundus (FDP) and flexor pollicis longus (FPL) tendons were more frequently ruptured (n=8) than flexor digitorum superficialis (FDS) tendons (n=4). MR images accurately depicted the level of the rupture. The gap between the tendon ends (mean 45 mm, range 21-70 mm) was assessed best with curved reconstructions and was well correlated with the surgical findings. The proximal end mainly retracted into the palm or the carpal tunnel (n=8), and less frequently into the digital canal (n=4). In two cases, the proximal end curled up in the palm, clinically simulating a rupture of a lumbrical muscle in one case. MRI also showed the appearance of the adjacent tendons. CONCLUSION: MRI accurately depicted the level of rupture and the gap between the tendon ends, which assisted the surgical choice between suture, graft or tendon transfer.