Daniel S. Wechsler

Characterization of Gene Expression Profiles Associated with Glioma Progression Using Oligonucleotide-Based Microarray Analysis and Real-Time Reverse Transcription-Polymerase Chain Reaction

Diffuse astrocytoma of World Health Organization (WHO) grade II has an inherent tendency to spontaneously progress to anaplastic astrocytoma (WHO grade III) and/or glioblastoma (WHO grade IV). The molecular basis of astrocytoma progression is still poorly understood, in particular with respect to the progression-associated changes at the mRNA level. Therefore, we compared the transcriptional profile of approximately 6800 genes in primary WHO grade II gliomas and corresponding recurrent high-grade (WHO grade III or IV) gliomas from eight patients using oligonucleotide-based microarray analysis. We identified 66 genes whose mRNA levels differed significantly (P < 0.01, > or =2-fold change) between the primary and recurrent tumors. The microarray data were corroborated by real-time reverse transcription-polymerase chain reaction analysis of 12 selected genes, including 7 genes with increased expression and 5 genes with reduced expression on progression. In addition, the expression of these 12 genes was determined in an independent series of 43 astrocytic gliomas (9 diffuse astrocytomas, 10 anaplastic astrocytomas, 17 primary, and 7 secondary glioblastomas). These analyses confirmed that the transcript levels of nine of the selected genes (COL4A2, FOXM1, MGP, TOP2A, CENPF, IGFBP4, VEGFA, ADD3, and CAMK2G) differed significantly in WHO grade II astrocytomas as compared to anaplastic astrocytomas and/or glioblastomas. Thus, we identified and validated a set of interesting candidate genes whose differential expression likely plays a role in astrocytoma progression.

Treatment with sirolimus results in complete responses in patients with autoimmune lymphoproliferative syndrome

We hypothesized that sirolimus, an mTOR inhibitor, may be effective in patients with autoimmune lymphoproliferative syndrome (ALPS) and treated patients who were intolerant to or failed other therapies. Four patients were treated for autoimmune cytopenias; all had a rapid complete or near complete response. Two patients were treated for autoimmune arthritis and colitis, demonstrating marked improvement. Three patients had complete resolution of lymphadenopathy and splenomegaly and all patients had a reduction in double negative T cells, a population hallmark of the disease. Based on these significant responses, we recommend that sirolimus be considered as second‐line therapy for patients with steroid‐refractory disease.

PICALM modulates autophagy activity and tau accumulation

Genome-wide association studies have identified several loci associated with Alzheimer’s disease (AD), including proteins involved in endocytic trafficking such as PICALM/CALM (phosphatidylinositol binding clathrin assembly protein). It is unclear how these loci may contribute to AD pathology. Here we show that CALM modulates autophagy and alters clearance of tau, a protein which is a known autophagy substrate and which is causatively linked to AD, both in vitro and in vivo. Furthermore, altered CALM expression exacerbates tau-mediated toxicity in zebrafish transgenic models. CALM influences autophagy by regulating the endocytosis of SNAREs, such as VAMP2, VAMP3 and VAMP8, which have diverse effects on different stages of the autophagy pathway, from autophagosome formation to autophagosome degradation. This study suggests that the AD genetic risk factor CALM modulates autophagy, and this may affect disease in a number of ways including modulation of tau turnover.

Iron deprivation in cancer--potential therapeutic implications.

Iron is essential for normal cellular function. It participates in a wide variety of cellular processes, including cellular respiration, DNA synthesis, and macromolecule biosynthesis. Iron is required for cell growth and proliferation, and changes in intracellular iron availability can have significant effects on cell cycle regulation, cellular metabolism, and cell division. Perhaps not surprisingly then, neoplastic cells have been found to have higher iron requirements than normal, non-malignant cells. Iron depletion through chelation has been explored as a possible therapeutic intervention in a variety of cancers. Here, we will review iron homeostasis in non-malignant and malignant cells, the widespread effects of iron depletion on the cell, the various iron chelators that have been explored in the treatment of cancer, and the tumor types that have been most commonly studied in the context of iron chelation.

Hemolysis Following Coil Embolization of a Patent Ductus Arteriosus

We describe the development of hemolysis from moderate residual shunting across a patent ductus arteriosus following coil embolization. The fall in hemoglobin levels from 11.6 to 6.0 gm/dl necessitated a second coil procedure which resulted in complete closure of the residual shunting and resolution of hemolysis. Therefore, appearance of anemia following coil embolization of patent ductus arteriosus should be monitored closely; however, repeat coil embolization with elimination of residual shunt will lead to prompt recovery of normal hemoglobin levels.

WHIM syndrome caused by a single amino acid substitution in the carboxy-tail of chemokine receptor CXCR4

WHIM syndrome is a rare, autosomal dominant, immunodeficiency disorder so-named because it is characterized by warts, hypogammaglobulinemia, infections, and myelokathexis (defective neutrophil egress from the BM). Gain-of-function mutations that truncate the C-terminus of the chemokine receptor CXCR4 by 10-19 amino acids cause WHIM syndrome. We have identified a family with autosomal dominant inheritance of WHIM syndrome that is caused by a missense mutation in CXCR4, E343K (1027G → A). This mutation is also located in the C-terminal domain, a region responsible for negative regulation of the receptor. Accordingly, like CXCR4(R334X), the most common truncation mutation in WHIM syndrome, CXCR4(E343K) mediated approximately 2-fold increased signaling in calcium flux and chemotaxis assays relative to wild-type CXCR4; however, CXCR4(E343K) had a reduced effect on blocking normal receptor down-regulation from the cell surface. Therefore, in addition to truncating mutations in the C-terminal domain of CXCR4, WHIM syndrome may be caused by a single charge-changing amino acid substitution in this domain, E343K, that results in increased receptor signaling.

A novel chromosomal inversion at 11q23 in infant acute myeloid leukemia fuses MLL to CALM, a gene that encodes a clathrin assembly protein

Rearrangements involving the MLL gene at chromosome band 11q23 are common in infant acute myeloid leukemias (AMLs). We recently encountered an infant patient with rapidly progressive AML whose leukemic cells harbored a previously undescribed MLL rearrangement involving an inversion of 11q [inv(11)(q14q23)]. We used panhandle PCR to determine that this rearrangement juxtaposed the MLL (Mixed‐Lineage Leukemia) gene to the CALM (Clathrin Assembly Lymphoid Myeloid leukemia) gene at 11q14–q21. The CALM protein participates in recruitment of clathrin to internal membrane surfaces, thereby regulating vesicle formation in both endocytosis and intracellular protein transport. Intriguingly, CALM has been identified in other cases of AML as a translocation partner for the AF10 gene, which has independently been found to be an MLL partner in AML. We identified the MLL‐CALM fusion transcript (but not the reciprocal CALM‐MLL transcript) in leukemia cell RNA by RT‐PCR. The predicted 1803 amino acid MLL‐CALM fusion protein includes amino‐terminal MLL domains involved in transcriptional repression, and carboxy‐terminal CALM‐derived clathrin‐binding domains. The genomic breakpoint in MLL is in the 7th intron (within the breakpoint cluster region); the corresponding CALM breakpoint is in the 7th CALM intron. In contrast, breakpoints in CALM‐AF10 translocations lie in the 17th–19th CALM introns (30 kb downstream); also, in these translocations, CALM provides the 5′ end of the fusion transcript. Together with its previously recognized association with AF10 in AML, the identification of CALM as an MLL fusion partner suggests that interference with clathrin‐mediated trafficking pathways may be an underappreciated mechanism in leukemogenesis.

Refractory Iron Deficiency Anemia as the Primary Clinical Manifestation of Celiac Disease

In the absence of dietary insufficiency, iron deficiency is usually caused by chronic blood loss or intestinal malabsorption. Celiac disease is one of the most common causes of intestinal malabsorption during childhood, and its association with insulin-dependent diabetes mellitus has been previously reported. Here the authors describe an otherwise asymptomatic diabetic adolescent boy with iron deficiency anemia that was not responsive to oral iron therapy. A diagnosis of celiac disease was made based on both anti-endomysial antibody titers and small intestinal biopsy. Institution of a gluten-free diet resulted in correction of the anemia. These observations emphasize the importance of considering a diagnosis of celiac disease in patients with nonresponsive iron deficiency anemia, particularly in the setting of insulin-dependent diabetes mellitus.

The PICALM Protein Plays a Key Role in Iron Homeostasis and Cell Proliferation

The ubiquitously expressed phosphatidylinositol binding clathrin assembly (PICALM) protein associates with the plasma membrane, binds clathrin, and plays a role in clathrin-mediated endocytosis. Alterations of the human PICALM gene are present in aggressive hematopoietic malignancies, and genome-wide association studies have recently linked the PICALM locus to late-onset Alzheimers disease. Inactivating and hypomorphic Picalm mutations in mice cause different degrees of severity of anemia, abnormal iron metabolism, growth retardation and shortened lifespan. To understand PICALM’s function, we studied the consequences of PICALM overexpression and characterized PICALM-deficient cells derived from mutant fit1 mice. Our results identify a role for PICALM in transferrin receptor (TfR) internalization and demonstrate that the C-terminal PICALM residues are critical for its association with clathrin and for the inhibitory effect of PICALM overexpression on TfR internalization. Murine embryonic fibroblasts (MEFs) that are deficient in PICALM display several characteristics of iron deficiency (increased surface TfR expression, decreased intracellular iron levels, and reduced cellular proliferation), all of which are rescued by retroviral PICALM expression. The proliferation defect of cells that lack PICALM results, at least in part, from insufficient iron uptake, since it can be corrected by iron supplementation. Moreover, PICALM-deficient cells are particularly sensitive to iron chelation. Taken together, these data reveal that PICALM plays a critical role in iron homeostasis, and offer new perspectives into the pathogenesis of PICALM-associated diseases.

High-risk surgically resected pediatric melanoma and adjuvant interferon therapy.

Pediatric patients with high‐risk surgically resected melanoma are at risk for relapse, yet little is known about these young patients and how they tolerate high‐dose interferon therapy.