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1.  Therapeutic Potential of HSP90 Inhibition for Neurofibromatosis Type 2 
Purpose
The growth and survival of neurofibromatosis type 2 (NF2)–deficient cells are enhanced by the activation of multiple signaling pathways including ErbBs/IGF-1R/Met, PI3K/Akt, and Ras/Raf/Mek/Erk1/2. The chaperone protein HSP90 is essential for the stabilization of these signaling molecules. The aim of the study was to characterize the effect of HSP90 inhibition in various NF2-deficient models.
Experimental Design
We tested efficacy of the small-molecule NXD30001, which has been shown to be a potent HSP90 inhibitor. The antiproliferative activity of NXD30001 was tested in NF2-deficient cell lines and in human primary schwannoma and meningioma cultures in vitro. The antitumor efficacy of HSP90 inhibition in vivo was verified in two allograft models and in one NF2 transgenic model. The underlying molecular alteration was further characterized by a global transcriptome approach.
Results
NXD30001 induced degradation of client proteins in and suppressed proliferation of NF2-deficient cells. Differential expression analysis identified subsets of genes implicated in cell proliferation, cell survival, vascularization, and Schwann cell differentiation whose expression was altered by NXD30001 treatment. The results showed that NXD30001 in NF2-deficient schwannoma suppressed multiple pathways necessary for tumorigenesis.
Conclusions
HSP90 inhibition showing significant antitumor activity against NF2-related tumor cells in vitro and in vivo represents a promising option for novel NF2 therapies.
doi:10.1158/1078-0432.CCR-12-3167
PMCID: PMC4331126  PMID: 23714726
2.  LIM Domain Kinases as Potential Therapeutic Targets for Neurofibromatosis Type 2 
Oncogene  2013;33(27):3571-3582.
Neurofibromatosis Type 2 (NF2) is caused by mutations in the neurofibromatosis 2 (NF2) gene that encodes a tumor suppressor protein called merlin. NF2 is characterized by formation of multiple schwannomas, meningiomas and ependymomas. Merlin loss of function is associated with increased activity of Rac and p21-activated kinases (PAK) and deregulation of cytoskeletal organization. LIM domain kinases (LIMK1 and 2) are substrate for Cdc42/Rac-PAK, and modulate actin dynamics by phosphorylating cofilin at serine-3. This modification inactivates cofilin’s actin severing and depolymerizing activity. LIMKs also translocate into the nucleus and regulate cell cycle progression. Significantly, LIMKs are overexpressed in several tumor types, including skin, breast, lung, liver and prostate. Here we report that mouse Schwann cells (MSCs) in which merlin function is lost as a result of Nf2 exon2 deletion (Nf2ΔEx2) exhibited increased levels of LIMK1, LIMK2, and active phospho-Thr508/505-LIMK1/2, as well as phospho-Ser3-cofilin, compared to wild-type normal MSCs. Similarly, levels of LIMK1 and 2 total protein and active phosphorylated forms were elevated in human vestibular schwannomas compared to normal human Schwann cells (SCs). Reintroduction of wild-type NF2 into Nf2ΔEx2 MSC reduced LIMK1 and LIMK2 levels. We show that pharmacological inhibition of LIMK with BMS-5, decreased the viability of Nf2ΔEx2 MSCs in a dose-dependent manner, but did not affect viability of control MSCs. Similarly, LIMK knockdown decreased viability of Nf2ΔEx2 MSCs. The decreased viability of Nf2ΔEx2 MSCs was not due to caspase-dependent or -independent apoptosis, but rather, to inhibition of cell cycle progression as evidenced by accumulation of cells in G2/M phase. Inhibition of LIMKs arrest cells in early mitosis by decreasing Aurora A activation. Our results suggest that LIMKs are potential drug targets for NF2 and tumors associated with merlin deficiency.
doi:10.1038/onc.2013.320
PMCID: PMC4016185  PMID: 23934191
LIMK; cell proliferation; Neurofibromatosis; schwannomas; cytoskeleton dynamics; cell cycle
4.  CTF Meeting 2012: Translation of the Basic Understanding of the Biology and Genetics of NF1, NF2, and Schwannomatosis Toward the Development of Effective Therapies 
The neurofibromatoses (NF) are autosomal dominant genetic disorders that encompass the rare diseases NF1, NF2, and schwannomatosis. The NFs affect more people worldwide than Duchenne muscular dystrophy and Huntington's disease combined. NF1 and NF2 are caused by mutations of known tumor suppressor genes (NF1 and NF2, respectively). For schwannomatosis, although mutations in SMARCB1 were identified in a subpopulation of schwannomatosis patients, additional causative gene mutations are still to be discovered. Individuals with NF1 may demonstrate manifestations in multiple organ systems, including tumors of the nervous system, learning disabilities, and physical disfigurement. NF2 ultimately can cause deafness, cranial nerve deficits, and additional severe morbidities caused by tumors of the nervous system. Unmanageable pain is a key finding in patients with schwannomatosis. Although today there is no marketed treatment for NF-related tumors, a significant number of clinical trials have become available. In addition, significant preclinical efforts have led to a more rational selection of potential drug candidates for NF trials. An important element in fueling this progress is the sharing of knowledge. For over 20 years the Children's Tumor Foundation has convened an annual NF Conference, bringing together NF professionals to share novel findings, ideas, and build collaborations. The 2012 NF Conference held in New Orleans hosted over 350 NF researchers and clinicians. This article provides a synthesis of the highlights presented at the conference and as such, is a “state-of-the-field” for NF research in 2012.
doi:10.1002/ajmg.a.36312
PMCID: PMC4150212  PMID: 24443315
neurofibromatosis type 1; neurofibromatosis type 2; NF1; NF2; schwannomatosis; tumor suppressor; SMARCB1; merlin neurofibromin; preclinical models
5.  Update From the 2011 International Schwannomatosis Workshop: From Genetics to Diagnostic Criteria 
Schwannomatosis is the third major form of neurofibromatosis and is characterized by the development of multiple schwannomas in the absence of bilateral vestibular schwannomas. The 2011 Schwannomatosis Update was organized by the Children’s Tumor Foundation (www.ctf.org) and held in Los Angeles, CA, from June 5–8, 2011. This article summarizes the highlights presented at the Conference and represents the “state-of-the-field” in 2011. Genetic studies indicate that constitutional mutations in the SMARCB1 tumor suppressor gene occur in 40–50% of familial cases and in 8–10% of sporadic cases of schwannomatosis. Tumorigenesis is thought to occur through a four-hit, three-step model, beginning with a germline mutation in SMARCB1 (hit 1), followed by loss of a portion of chromosome 22 that contains the second SMARCB1 allele and one NF2 allele (hits 2 and 3), followed by mutation of the remaining wild-type NF2 allele (hit 4). Insights from research on HIV and pediatric rhabdoid tumors have shed light on potential molecular pathways that are dysregulated in schwannomatosis-related schwannomas. Mouse models of schwannomatosis have been developed and promise to further expand our understanding of tumorigenesis and the tumor microenvironment. Clinical reports have described the occurrence of intracranial meningiomas in schwannomatosis patients and in families with germline SMARCB1 mutations. The authors propose updated diagnostic criteria to incorporate new clinical and genetic findings since 2005. In the next 5 years, the authors expect that advances in basic research in the pathogenesis of schwannomatosis will lead toward clinical investigations of potential drug therapies.
doi:10.1002/ajmg.a.35760
PMCID: PMC4020435  PMID: 23401320
neurofibromatoses; schwannoma; rhabdoid tumor
9.  Azacitidine in Chronic Myelomonocytic Leukemia: An Effective and Manageable Approach 
Chronic myelomonocytic leukemia (CMML) is an uncommon neoplastic hematological disorder, typically affecting the elderly, and characterized by a marked clinical heterogeneity and a remarkable propensity for transformation into acute myeloid leukemia. Hypomethylating agents represent the most innovative management approach in this difficult setting. At our institution, between 2010 and 2012, we have treated with azacitidine 10 CMML patients with a median age of 75 (62–86) years. The overall response rate of 70% was achieved without remarkable toxicities; in particular, most therapy-induced side effects were managed on outpatient basis. With a median follow-up of 12,5 (2–27) months, 6 patients are alive, and 4 of them continue to receive the treatment; the median survival from the start of therapy was not reached. In conclusion, also in the light of our encouraging experience, azacitidine can offer new chances of treatment also in the difficult setting of elderly CMML.
doi:10.4084/MJHID.2014.020
PMCID: PMC3965718  PMID: 24678397
13.  Managing myelodysplastic syndromes in very old patients: a teaching case report 
The introduction of hypomethylating agents in the treatment of myelodysplastic syndromes (MDS) has significantly changed the clinical scenario of these diseases, which afflict predominantly older individuals. However, some concerns regarding the optimal application of these innovative and costly agents in the treatment of geriatric high-risk MDS remain. We report here the case of a nonagenarian treated with hypomethylating agents achieving a long-lasting clinical response and a significant improvement in her functional status. Our case confirmed that functional status and biological status, rather than the chronological age alone, can substantially guide the plan of an appropriate treatment strategy in high-risk MDS patients; moreover, the current case emphasizes the need for targeted studies in the field of geriatric MDS in order to formulate guidelines on the appropriate use of these costly agents, so that candidate patients can receive adequate treatment to preserve their quality of life and life expectancy, but at the same time avoiding unnecessary costs deriving from the use of high-cost drugs for those in whom a significant therapeutic result cannot be reasonably expected.
doi:10.2147/CIA.S39536
PMCID: PMC3628529  PMID: 23610516
myelodysplastic syndromes; azacitidine; older patients
14.  Consensus Recommendations for Current Treatments and Accelerating Clinical Trials for Patients with Neurofibromatosis Type 2 
Neurofibromatosis type 2 (NF2) is a tumor suppressor syndrome characterized by bilateral vestibular schwannomas (VS) which often result in deafness despite aggressive management. Meningiomas, ependymomas and other cranial nerve and peripheral schwannomas are also commonly found in NF2 and collectively lead to major neurologic morbidity and mortality. Traditionally, the overall survival rate in patients with NF2 is estimated to be 38% at 20 years from diagnosis. Hence, there is a desperate need for new, effective therapies. Recent progress in understanding the molecular basis of NF2 related tumors has aided in the identification of potential therapeutic targets and emerging clinical therapies. In June 2010, representatives of the international NF2 research and clinical community convened under the leadership of Drs. D. Gareth Evans (University of Manchester) and Marco Giovannini (House Research Institute) to review the state of NF2 treatment and clinical trials. This manuscript summarizes the expert opinions about current treatments for NF2 associated tumors and recommendations for advancing therapies emerging from that meeting. The development of effective therapies for NF2 associated tumors has the potential for significant clinical advancement not only for patients with NF2 but for thousands of neuro-oncology patients afflicted with these tumors.
doi:10.1002/ajmg.a.34359
PMCID: PMC3319201  PMID: 22140088
Neurofibromatosis type 2; meningioma; schwannoma; vestibular schwannoma; ependymoma; radiotherapy; molecular therapy; surgery
16.  MEK inhibition exhibits efficacy in human and mouse neurofibromatosis tumors 
Neurofibromatosis type 1 (NF1) patients develop benign neurofibromas and malignant peripheral nerve sheath tumors (MPNST). These incurable peripheral nerve tumors result from loss of NF1 tumor suppressor gene function, causing hyperactive Ras signaling. Activated Ras controls numerous downstream effectors, but specific pathways mediating the effects of hyperactive Ras in NF1 tumors are unknown. We performed cross-species transcriptome analyses of mouse and human neurofibromas and MPNSTs and identified global negative feedback of genes that regulate Ras/Raf/MEK/ERK signaling in both species. Nonetheless, ERK activation was sustained in mouse and human neurofibromas and MPNST. We used a highly selective pharmacological inhibitor of MEK, PD0325901, to test whether sustained Ras/Raf/MEK/ERK signaling contributes to neurofibroma growth in a neurofibromatosis mouse model (Nf1fl/fl;Dhh-Cre) or in NF1 patient MPNST cell xenografts. PD0325901 treatment reduced aberrantly proliferating cells in neurofibroma and MPNST, prolonged survival of mice implanted with human MPNST cells, and shrank neurofibromas in more than 80% of mice tested. Our data demonstrate that deregulated Ras/ERK signaling is critical for the growth of NF1 peripheral nerve tumors and provide a strong rationale for testing MEK inhibitors in NF1 clinical trials.
doi:10.1172/JCI60578
PMCID: PMC3533264  PMID: 23221341
19.  Back to the Future: Proceedings From the 2010 NF Conference 
The neurofibromatoses (NF) encompass the rare diseases NF1, NF2, and schwannomatosis. The NFs affect 100,000 Americans; over 2 million persons worldwide; and are caused by mutation of tumor suppressor genes. Individuals with NF1 in particular may develop tumors anywhere in the nervous system; additional manifestations can include learning disabilities, bone dysplasia, cardiovascular defects, unmanageable pain, and physical disfigurement. Ultimately, the NFs can cause blindness, deafness, severe morbidity, and increased mortality and NF1 includes a risk of malignant cancer. Today there is no treatment for the NFs (other than symptomatic); however, research efforts to understand these genetic conditions have made tremendous strides in the past few years. Progress is being made on all fronts, from discovery studies—understanding the molecular signaling deficits that cause the manifestations of NF—to the growth of preclinical drug screening initiatives and the emergence of a number of clinical trials. An important element in fuelling this progress is the sharing of knowledge, and to this end, for over 20 years the Children’s Tumor Foundation has convened an annual NF Conference, bringing together NF professionals to share ideas and build collaborations. The 2010 NF Conference held in Baltimore, MD June 5–8,2010 hosted over 300 NF researchers and clinicians. This paper provides a synthesis of the highlights presented at the Conference and as such, is a “state-of-the-field” for NF research in 2010.
doi:10.1002/ajmg.a.33804
PMCID: PMC3079924  PMID: 21271647
neurofibromatosis; schwannomatosis; NF1; NF2; neurofibroma; learning disabilities; bone dysplasia; MPNST
20.  Characterization of the axon initial segment (AIS) of motor neurons and identification of a para-AIS and a juxtapara-AIS, organized by protein 4.1B 
BMC Biology  2011;9:66.
Background
The axon initial segment (AIS) plays a crucial role: it is the site where neurons initiate their electrical outputs. Its composition in terms of voltage-gated sodium (Nav) and voltage-gated potassium (Kv) channels, as well as its length and localization determine the neuron's spiking properties. Some neurons are able to modulate their AIS length or distance from the soma in order to adapt their excitability properties to their activity level. It is therefore crucial to characterize all these parameters and determine where the myelin sheath begins in order to assess a neuron's excitability properties and ability to display such plasticity mechanisms. If the myelin sheath starts immediately after the AIS, another question then arises as to how would the axon be organized at its first myelin attachment site; since AISs are different from nodes of Ranvier, would this particular axonal region resemble a hemi-node of Ranvier?
Results
We have characterized the AIS of mouse somatic motor neurons. In addition to constant determinants of excitability properties, we found heterogeneities, in terms of AIS localization and Nav composition. We also identified in all α motor neurons a hemi-node-type organization, with a contactin-associated protein (Caspr)+ paranode-type, as well as a Caspr2+ and Kv1+ juxtaparanode-type compartment, referred to as a para-AIS and a juxtapara (JXP)-AIS, adjacent to the AIS, where the myelin sheath begins. We found that Kv1 channels appear in the AIS, para-AIS and JXP-AIS concomitantly with myelination and are progressively excluded from the para-AIS. Their expression in the AIS and JXP-AIS is independent from transient axonal glycoprotein-1 (TAG-1)/Caspr2, in contrast to juxtaparanodes, and independent from PSD-93. Data from mice lacking the cytoskeletal linker protein 4.1B show that this protein is necessary to form the Caspr+ para-AIS barrier, ensuring the compartmentalization of Kv1 channels and the segregation of the AIS, para-AIS and JXP-AIS.
Conclusions
α Motor neurons have heterogeneous AISs, which underlie different spiking properties. However, they all have a para-AIS and a JXP-AIS contiguous to their AIS, where the myelin sheath begins, which might limit some AIS plasticity. Protein 4.1B plays a key role in ensuring the proper molecular compartmentalization of this hemi-node-type region.
doi:10.1186/1741-7007-9-66
PMCID: PMC3198992  PMID: 21958379
21.  Protein 4.1B Contributes to the Organization of Peripheral Myelinated Axons 
PLoS ONE  2011;6(9):e25043.
Neurons are characterized by extremely long axons. This exceptional cell shape is likely to depend on multiple factors including interactions between the cytoskeleton and membrane proteins. In many cell types, members of the protein 4.1 family play an important role in tethering the cortical actin-spectrin cytoskeleton to the plasma membrane. Protein 4.1B is localized in myelinated axons, enriched in paranodal and juxtaparanodal regions, and also all along the internodes, but not at nodes of Ranvier where are localized the voltage-dependent sodium channels responsible for action potential propagation. To shed light on the role of protein 4.1B in the general organization of myelinated peripheral axons, we studied 4.1B knockout mice. These mice displayed a mildly impaired gait and motility. Whereas nodes were unaffected, the distribution of Caspr/paranodin, which anchors 4.1B to the membrane, was disorganized in paranodal regions and its levels were decreased. In juxtaparanodes, the enrichment of Caspr2, which also interacts with 4.1B, and of the associated TAG-1 and Kv1.1, was absent in mutant mice, whereas their levels were unaltered. Ultrastructural abnormalities were observed both at paranodes and juxtaparanodes. Axon calibers were slightly diminished in phrenic nerves and preterminal motor axons were dysmorphic in skeletal muscle. βII spectrin enrichment was decreased along the axolemma. Electrophysiological recordings at 3 post-natal weeks showed the occurrence of spontaneous and evoked repetitive activity indicating neuronal hyperexcitability, without change in conduction velocity. Thus, our results show that in myelinated axons 4.1B contributes to the stabilization of membrane proteins at paranodes, to the clustering of juxtaparanodal proteins, and to the regulation of the internodal axon caliber.
doi:10.1371/journal.pone.0025043
PMCID: PMC3180372  PMID: 21966409
22.  Familial Adenomatous Polyposis-Associated Desmoids Display Significantly More Genetic Changes than Sporadic Desmoids 
PLoS ONE  2011;6(9):e24354.
Desmoid tumours (also called deep or aggressive fibromatoses) are potentially life-threatening fibromatous lesions. Hereditary desmoid tumours arise in individuals affected by either familial adenomatous polyposis (FAP) or hereditary desmoid disease (HDD) carrying germline mutations in APC. Most sporadic desmoids carry somatic mutations in CTNNB1. Previous studies identified losses on 5q and 6q, and gains on 8q and 20q as recurrent genetic changes in desmoids. However, virtually all genetic changes were derived from sporadic tumours. To investigate the somatic alterations in FAP-associated desmoids and to compare them with changes occurring in sporadic tumours, we analysed 17 FAP-associated and 38 sporadic desmoids by array comparative genomic hybridisation and multiple ligation-dependent probe amplification. Overall, the desmoids displayed only a limited number of genetic changes, occurring in 44% of cases. Recurrent gains at 8q (7%) and 20q (5%) were almost exclusively found in sporadic tumours. Recurrent losses were observed for a 700 kb region at 5q22.2, comprising the APC gene (11%), a 2 Mb region at 6p21.2-p21.1 (15%), and a relatively large region at 6q15-q23.3 (20%). The FAP-associated desmoids displayed a significantly higher frequency of copy number abnormalities (59%) than the sporadic tumours (37%). As predicted by the APC germline mutations among these patients, a high percentage (29%) of FAP-associated desmoids showed loss of the APC region at 5q22.2, which was infrequently (3%) seen among sporadic tumours. Our data suggest that loss of region 6q15-q16.2 is an important event in FAP-associated as well as sporadic desmoids, most likely of relevance for desmoid tumour progression.
doi:10.1371/journal.pone.0024354
PMCID: PMC3170296  PMID: 21931686
23.  Pain in Blood Cancers 
Indian Journal of Palliative Care  2011;17(3):175-183.
Patients with blood-related cancers (BRC) suffer from a substantial symptom burden, including several pain syndromes sustained by different causes and pathogenetic mechanisms. So, with regard to pain, a multifaceted clinical scenario may be observed in this setting. Indeed, pain may be correlated to disease itself, to disease-associated complications, to iatrogenic causes or may be due to unrelated clinical conditions. A close diagnostic procedure for the assessment of the underlying causes of the pain and of its pathogenetic mechanisms may direct the treatment approach which should be based on a multidisciplinary management and requires the integration of etiology-targeted interventions and painkilling drugs. The World Health Organization's three-step analgesic ladder for cancer pain relief can provide adequate pain control using oral drugs in most patients with BRC on pain, although more complex interventions may be necessary for many difficult-to-treat pain syndromes which are not infrequently encountered in this setting.
doi:10.4103/0973-1075.92333
PMCID: PMC3276813  PMID: 22346041
Blood-related cancers; Hematological malignancies; Pain

Results 1-25 (42)