Inhibition of Anaplastic Lymphoma Kinase (Alk) as Therapeutic Target to Improve Brain Function in Neurofibromatosis Type 1 (Nf1).

Neurofibromatosis type 1 (Nf1) is a neurodevelopmental disorder and tumor syndrome caused by loss of function mutations in the neurofibromin gene (Nf1) and is estimated to affect 100,000 people in the US. Behavioral alterations and cognitive deficits have been found in 50-70% of children with Nf1 and include specific problems with attention, visual perception, language, learning, attention, and executive function. These behavioral alterations and cognitive deficits are observed in the absence of tumors or macroscopic structural abnormalities in the central nervous system. No effective treatments for the behavioral and cognitive disabilities of Nf1 exist. Inhibition of the anaplastic lymphoma kinase (Alk), a kinase which is negatively regulated by neurofibromin, allows for testing the hypothesis that this inhibition may be therapeutically beneficial in Nf1. In this review, we discuss this area of research and directions for the development of alternative therapeutic strategies to inhibit Alk. Even if the incidence of adverse reactions of currently available Alk inhibitors was reduced to half the dose, we anticipate that a long-term treatment would pose challenges for efficacy, safety, and tolerability. Therefore, future efforts are warranted to investigate alternative, potentially less toxic and more specific strategies to inhibit Alk function.

Long-term effects of pharmacological inhibition of anaplastic lymphoma kinase in neurofibromatosis 1 mutant mice.

Neurofibromatosis type 1 (NF1) is associated with behavioral alterations and cognitive impairments. There is a genetic interaction between NF1 and the receptor tyrosine kinase Alk. Short-term pharmacological Alk inhibition, with a compound FDA-approved for cancer starting 10 days prior to cognitive testing, was shown to improve cognitive performance of NF1 heterozygous (HET) mice. However, effects of long-term Alk inhibition on behavioral cognitive performance are not known. Therefore, in the study described below we determine the effects of prolonged pharmacological Alk inhibition for 24 weeks on behavioral and cognitive performance of NF1 HET mice. As these studies have the ultimate objective of developing a treatment for humans with neurofibromatosis and acceptable side effects in the context of cancer are not acceptable in the context of long-term treatment of patients with neurofibromatosis, we included additional behavioral tests of anxiety-like and depressive-like behaviors as well. Long-term effects of Alk inhibition had genotype-dependent effects, consistent with a specific interaction between Alk and NF1. Beneficial effects of long-term Alk inhibition in NF1 HET mice included rescue of impairments in object recognition in NF1 HET males and females, and improved cognitive performance of NF1 HET males and females in the water maze test. In contrast, long-term Alk inhibition had detrimental effects in WT mice not seen after short-term treatments. As longer treatments are translationally more relevant for NF1 patients, these data highlight the important to assess long-term effects of drugs, especially of repurposed drugs used originally as part of cancer therapy.

Role of the parental NF1 carrier in effects of pharmacological inhibition of anaplastic lymphoma kinase in Neurofibromatosis 1 mutant mice.

Neurofibromatosis type 1 (NF1), a genetically determined neurodevelopmental disorder and tumor syndrome, is associated with cognitive impairments, including in executive function and sleep-related problems. Consistent with the human data, NF1 heterozygous (Het) mice show impaired spatial learning and memory in the water maze and extinction of contextual fear memory. It is not clear whether neurological phenotypes might depend on the parental carrier. In this study, we compared the behavioral and cognitive performance of NF1 Het and wild-type litter mates with either the father (PC) or the mother (MC) as the NF1 carrier on a F1 C57BL/66/129SvJ background. The behavioral and cognitive phenotypes and responsiveness to Alk inhibition in heterozygous NF1 offspring depended on whether the parental carrier was maternal or paternal. Alk inhibition (20 mg/kg) increased activity levels during the dark period in NF1 Het PC, but not MC, mice. In the water maze, NF1 Het PC, but not MC, mice showed reduced cognitive flexibility and impaired ability to locate the third hidden platform location, which was improved by Alk inhibition (3.6 mg/kg). Consistent with reduced cognitive flexibility, WT, but not NF1, mice showed better performance in the third than second water maze probe trial. Finally, Alk inhibition (10 mg/kg) increased baseline activity of NF1 MC, but not PC, mice during the fear conditioning test. Thus, the effective dose depends on the behavioral test and genotype but indicates that in NF1 patients cognitive flexibility might be particularly sensitive to Alk inhibition.

Pharmacological inhibition of Anaplastic Lymphoma Kinase rescues spatial memory impairments in Neurofibromatosis 1 mutant mice.

Heterozygous Neurofibromatosis 1 (NF1) loss of function mutations are found in 90% of patients with neurofibromatosis, a syndrome associated with disabling cognitive impairment. Drosophila studies have demonstrated a genetic interaction between Anaplastic Lymphoma Kinase (Alk) and NF1 in cognitive performance. In addition, pharmacologic inhibition of Alk improves cognitive performance in heterozygous NF1 mutant flies. In this study, we tested whether pharmacological inhibition of Alk in heterozygous NF1 mutant mice attenuates or rescues cognitive impairments. Cognitive impairment of spatial memory retention observed in heterozygous NF1 mutant mice was rescued by the Alk inhibitor. These data support the hypothesis that inhibition of Alk may cognitively benefit patients with Neurofibromatosis 1.

Genetic inhibition of Anaplastic Lymphoma Kinase rescues cognitive impairments in Neurofibromatosis 1 mutant mice.

Heterozygous Neurofibromatosis 1 (NF1) loss of function mutations occur in approximately 90% of patients with neurofibromatosis. A major, disabling phenotypic consequence of reduced NF1 function is cognitive impairment; a possibly related behavioral phenotype is impaired sleep. Recent results in Drosophila have demonstrated a genetic interaction between Anaplastic Lymphoma Kinase (Alk) and NF1 for both associative learning and sleep. Inhibition of Alk improves associative learning and sleep in heterozygous NF1 mutant flies. The results in Drosophila provide a strong motivation to investigate NF1/Alk genetic interactions in mice. In Drosophila, activation of Alk by its ligand, Jelly belly (Jeb), is the physiologically relevant target of negative regulation by NF1. Therefore, we tested whether genetic inhibition of Alk in heterozygous NF1 mutant mice attenuates or rescues cognitive impairments in mice. Our results are consistent with the hypothesis that NF1 functions in mice biochemically to inhibit signaling from Alk through Ras. The cognitive phenotypes observed in heterozygous NF1 mutant mice are rescued or ameliorated by genetic inhibition of Alk activity. In two tests of hippocampus-dependent learning, the Morris water maze and extinction of contextual fear, mutation of one or both alleles of Alk was sufficient to improve performance to wild type or near wild type levels in NF1-/+ mice. In addition, in NF1 mice genetic inhibition of Alk improves circadian activity levels. These data are intriguing in light of the circadian alterations seen in NF1 patients and indicate that inhibition of Alk activity may cognitively benefit patients with Neurofibromatosis 1.

Sudden cardiac death in the older athlete.

The overwhelming majority of sports-related sudden deaths occur among those older than 35 years of age. Because increasing numbers of older people are participating in organized endurance and competitive sporting events, the incidence of sports-related sudden death in older adults is expected to rise. Older athletes will approach clinical cardiologists for advice regarding their fitness for participation. It is important to recognize both that strenuous exercise is associated with a transient elevation in risk of sudden cardiac death and that appropriate training substantially reduces this risk. The approach to pre-participation screening for risk of sudden death in the older athlete is a complex issue and at present is largely focused on identifying inducible ischemia due to significant coronary disease. In this brief review, we summarize the current state of knowledge in this area with respect to epidemiology, mechanisms, and approaches to risk stratification, as viewed from the perspective of the consulting clinical cardiologist.

Organ allocation in adults with congenital heart disease listed for heart transplant: impact of ventricular assist devices.

BACKGROUND: Adults with congenital heart disease (CHD) listed for heart transplantation are rarely supported by ventricular assist devices (VADs). This may be a disadvantage to their priority for organ allocation. We sought to determine the relationship between VAD implantation and successful transplantation among patients listed for heart transplant. METHODS: Adults with CHD patients (N = 1,250) were identified from the United Network for Organ Sharing (UNOS) database from 1985 to 2010 and compared to patients without congenital etiology for heart failure (N = 59,606). VAD use at listing, listing status, status upgrades and reasons for upgrade prior to transplant were trended at 5-year intervals and appropriate statistical comparisons were made between groups. RESULTS: Since 1985, VAD use prior to transplant has increased significantly in patients without CHD, but not in CHD patients (17% vs 3% in 2006 to 2010, p < 0.0001). CHD patients were more likely to be listed as Status 2, compared to those without (66% vs 40%, p < 0.001 for 2006 to 2010), and less likely to be upgraded to Status 1 after listing (43% vs 55%, p = 0.03). Among those upgraded to Status 1, CHD patients were less likely to have a VAD at transplant than those without (3% vs 18%, p = 0.005). VAD use was more likely to result in death in CHD patients. CONCLUSIONS: VAD use is less common in CHD patients than in patients without CHD, both at the time of listing and transplantation. Reduced VAD use appears to contribute to lower listing status and organ allocation. These differences have grown more disparate over time. Separate criteria for organ allocation for CHD patients may be justified.

Jelly Belly trans-synaptic signaling to anaplastic lymphoma kinase regulates neurotransmission strength and synapse architecture.

In Drosophila, the secreted signaling molecule Jelly Belly (Jeb) activates anaplastic lymphoma kinase (Alk), a receptor tyrosine kinase, in multiple developmental and adult contexts. We have shown previously that Jeb and Alk are highly enriched at Drosophila synapses within the CNS neuropil and neuromuscular junction (NMJ) and postulated a conserved intercellular signaling function. At the embryonic and larval NMJ, Jeb is localized in the motor neuron presynaptic terminal whereas Alk is concentrated in the muscle postsynaptic domain surrounding boutons, consistent with anterograde trans-synaptic signaling. Here, we show that neurotransmission is regulated by Jeb secretion by functional inhibition of Jeb-Alk signaling. Jeb is a novel negative regulator of neuromuscular transmission. Reduction or inhibition of Alk function results in enhanced synaptic transmission. Activation of Alk conversely inhibits synaptic transmission. Restoration of wild-type postsynaptic Alk expression in Alk partial loss-of-function mutants rescues NMJ transmission phenotypes and confirms that postsynaptic Alk regulates NMJ transmission. The effects of impaired Alk signaling on neurotransmission are observed in the absence of associated changes in NMJ structure. Complete removal of Jeb in motor neurons, however, disrupts both presynaptic bouton architecture and postsynaptic differentiation. Nonphysiologic activation of Alk signaling also negatively regulates NMJ growth. Activation of Jeb-Alk signaling triggers the Ras-MAP kinase cascade in both pre- and postsynaptic compartments. These novel roles for Jeb-Alk signaling in the modulation of synaptic function and structure have potential implications for recently reported Alk functions in human addiction, retention of spatial memory, cognitive dysfunction in neurofibromatosis, and pathogenesis of amyotrophic lateral sclerosis.

Anaplastic lymphoma kinase and leukocyte tyrosine kinase: functions and genetic interactions in learning, memory and adult neurogenesis.

Anaplastic Lymphoma Kinase (Alk) is a receptor tyrosine kinase expressed throughout the adult mammalian hippocampus. Recent studies in Drosophila and prior studies in Caenorhabditis elegans have implicated Alk signaling in learning and neurogenesis. We have studied the roles of Alk and the closely related receptor Leukocyte Tyrosine Kinase (Ltk) in learning, behavior and neurogenesis. In the hippocampus, both receptors are expressed throughout the dentate gyrus, CA1 and CA3. To assess the functional roles of Alk and Ltk in the mammalian brain, we analyzed phenotypes in Alk mutant, Ltk mutant and Alk/Ltk double-mutant mice compared to wild-type littermates. Similar to Drosophila, we found enhanced performance in spatial memory in Alk mutant mice. Also similar to Drosophila, we observed reduced neurogenesis associated with loss of Alk function. We also report genetic interactions between Alk and Ltk with respect to neurogenesis and behavioral measures such as activity, anxiety levels, and retention of spatial memory.

Assessment of electronic health information system use and need in US adult congenital heart disease centers.

OBJECTIVES: Efforts to improve care for adult congenital heart disease (ACHD) patients necessitates collection of accurate, detailed, longitudinal data. We sought to document what electronic health record systems are currently available at ACHD centers and to assess national interest in a uniform ACHD-focused system. DESIGN: Directors of ACHD centers in the United States were invited to complete an online questionnaire regarding current health information systems at their institution both for general cardiology and for ACHD. Topics that were surveyed included utility and perceived limitations of currently available systems. The survey also assessed the level of interest in an ACHD-specific system, and its optimal functions. RESULTS: Thirty-four centers responded, representing both pediatric and adult institutions that care for patients with ACHD. Of these, 80% reported using a variety of commercially supported electronic medical record products, whereas only 50% employed an ACHD-specific noncommercial database to supplement their institutional system. Comparison of the two systems revealed that most clinical activities are pursued through the institutional electronic medical record system. Research and tracking of clinical activities were the primary uses of ACHD-specific systems, which have several noted limitations. Strong interest in an integrated ACHD-specific system was found among responders. CONCLUSIONS: There is both an unmet need and a strong interest in an ACHD-oriented electronic health record that could facilitate research, outcome tracking, quality assurance, and inter-institutional collaboration, all functions that are lacking in electronic health systems currently in use.

Lessons from development: A role for asymmetric stem cell division in cancer.

Asymmetric stem cell division has emerged as a major regulatory mechanism for physiologic control of stem cell numbers. Reinvigoration of the cancer stem cell theory suggests that tumorigenesis may be regulated by maintaining the balance between asymmetric and symmetric cell division. Therefore, mutations affecting this balance could result in aberrant expansion of stem cells. Although a number of molecules have been implicated in regulation of asymmetric stem cell division, here, we highlight known tumor suppressors with established roles in this process. While a subset of these tumor suppressors were originally defined in developmental contexts, recent investigations reveal they are also lost or mutated in human cancers. Mutations in tumor suppressors involved in asymmetric stem cell division provide mechanisms by which cancer stem cells can hyperproliferate and offer an intriguing new focus for understanding cancer biology. Our discussion of this emerging research area derives insight from a frontier area of basic science and links these discoveries to human tumorigenesis. This highlights an important new focus for understanding the mechanism underlying expansion of cancer stem cells in driving tumorigenesis.

Affinity Density: a novel genomic approach to the identification of transcription factor regulatory targets.

METHODS: A new method was developed for identifying novel transcription factor regulatory targets based on calculating Local Affinity Density. Techniques from the signal-processing field were used, in particular the Hann digital filter, to calculate the relative binding affinity of different regions based on previously published in vitro binding data. To illustrate this approach, the complete genomes of Drosophila melanogaster and D.pseudoobscura were analyzed for binding sites of the homeodomain proteinc Tinman, an essential heart development gene in both Drosophila and Mouse. The significant binding regions were identified relative to genomic background and assigned to putative target genes. Valid candidates common to both species of Drosophila were selected as a test of conservation. RESULTS: The new method was more sensitive than cluster searches for conserved binding motifs with respect to positive identification of known Tinman targets. Our Local Affinity Density method also identified a significantly greater proportion of Tinman-coexpressed genes than equivalent, optimized cluster searching. In addition, this new method predicted a significantly greater than expected number of genes with previously published RNAi phenotypes in the heart. AVAILABILITY: Algorithms were implemented in Python, LISP, R and maxima, using MySQL to access locally mirrored sequence data from Ensembl (D.melanogaster release 4.3) and flybase (D.pseudoobscura). All code is licensed under GPL and freely available at http://www.ohsu.edu/cellbio/dev_biol_prog/affinitydensity/.