Billie M. Moats-Staats

Treatment of childhood kaposiform hemangioendothelioma with sirolimus

Sirolimus (Rapamune), a mammalian target of Rapamycin (mTOR) inhibitor, which has been used extensively in children following solid organ transplantation, has been demonstrated to have anti‐angiogenic activity in pre‐clinical models. Limited experience suggests that it may have application to the treatment of vascular lesions. We describe our experience with a 1‐year‐old female with a kaposiform hemangioendothelioma and Kasabach–Merritt phenomenon who had rapid and dramatic response to sirolimus (0.1 mg/kg/day). This case provides further rationale for clinical trials of sirolimus in the treatment of vascular lesions. Pediatr Blood Cancer. 2010;55:1396–1398.

Tissue-Specific Developmental Regulation of the Messenger Ribonucleic Acids Encoding the Growth Hormone Receptor and the Growth Hormone Binding Protein in Rat Fetal and Postnatal Tissues

ABSTRACT: Tissue responsiveness to growth hormone is likely to be regulated by local concentrations and availability of the membrane-bound growth hormone receptor (GHR) and perhaps by the actions of the soluble growth hormone binding protein (GHBP). To determine whether the developmental regulation of the GHR and GHBP might vary among tissues, we have measured the relative abundance of the 4.3-kb GFR and 1.3-kb GHBP mRNA in rat fetal and postnatal liver, kidney, lung, and ileum by Northern hybridization of polyadenylated RNA with a 32P-labeled antisense riboprobe prepared from a rat GHR cDNA. The GHR and GHBP mRNA were both present in the four tissues studied at fetal age 19 d (E19). In postnatal liver, both transcripts increased in abundance 3− to 4-fold after 14 d to mature levels at 42 d (p = 0.0001). Similar changes were seen in postnatal kidney for GHR mRNA abundance; however, GHBP mRNA abundance increased only 2− to 3-fold to mature levels by 28 d (kidney GHR versus GHBP mRNA profile, p = 0.0001). In lung, a 2-fold linear increase in GHR mRNA abundance was observed (p = 0.0019), but the GHBP mRNA did not change (GHR versus GHBP mRNA profile, p = 0.0006). Both transcripts decreased in abundance by 2− to 3-fold from E19 to 42 d in ileum (p < 0.05). The abundance of both transcripts was three to 10 times greater in 60-d liver than in the other three tissues at 60 d. The variation in abundance and in the developmental profiles of the GHR and GHBP mRNA observed in these fetal and postnatal tissues suggests that the GHR and GHBP could mediate differences within and between tissues in the responsiveness to growth hormone. The differential regulation of the two transcripts evident in kidney and lung supports the emerging evidence that the GHBP may have a funciton distinct from that of the GHR.

Novel mutation in the SLC19A2 gene in an African-American female with thiamine-responsive megaloblastic anemia syndrome

Thiamine‐responsive megaloblastic anemia (TRMA) syndrome is an autosomal recessive disorder characterized by diabetes mellitus (DM), progressive sensorineural deafness, and thiamine‐responsive anemia. Mutations in the SLC19A2 gene encoding a high‐affinity thiamine transporter protein THTR‐1 are responsible for the clinical features associated with TRMA syndrome. We report an African‐American female with TRMA‐syndrome associated with thyroid disease and retinitis pigmentosa caused by a novel mutation in the SLC19A2 gene. The patient presented at 12 months of age with paroxysmal atrial tachycardia and hepatosplenomegaly. One month later, she developed DM requiring intermittent insulin therapy. At 2‐1/2 years of age, profound sensorineural hearing loss was discovered. By 4 years of age, daily insulin therapy (0.5 U/kg/day) was instituted and her insulin requirement gradually increased to 1.0 U/kg/day by 9 years of age. She developed optic atrophy, retinitis pigmentosa, and visual impairment by 12 years of age with severe restriction of peripheral vision by 16 years. At age 19, a thiamine‐responsive normocytic anemia was discovered. She was diagnosed with autoimmune thyroiditis at 20 years and she experienced a psychotic episode associated with a mood disorder at age 21. With oral thiamine therapy, her insulin requirement decreased by 30% over a 20 month period. Molecular analysis revealed that the patient is homozygous for a missense mutation (C152T) in exon 1 of the SLC19A2 gene.

Expression of insulin-like growth factor system genes during the early postnatal neurogenesis in the mouse hippocampus

Insulin‐like growth factor‐1 (IGF‐1) is essential to hippocampal neurogenesis and the neuronal response to hypoxia/ischemia injury. IGF (IGF‐1 and ‐2) signaling is mediated primarily by the type 1 IGF receptor (IGF‐1R) and modulated by six high‐affinity binding proteins (IGFBP) and the type 2 IGF receptor (IGF‐2R), collectively termed IGF system proteins. Defining the precise cells that express each is essential to understanding their roles. With the exception of IGFBP‐1, we found that mouse hippocampus expresses mRNA for each of these proteins during the first 2 weeks of postnatal life. Compared to postnatal day 14 (P14), mRNA abundance at P5 was higher for IGF‐1, IGFBP‐2, ‐3, and ‐5 (by 71%, 108%, 100%, and 98%, respectively), lower for IGF‐2, IGF‐2R, and IGFBP‐6 (by 65%, 78%, and 44%, respectively), and unchanged for IGF‐1R and IGFBP‐4. Using laser capture microdissection (LCM), we found that granule neurons and pyramidal neurons exhibited identical patterns of expression of IGF‐1, IGF‐1R, IGF‐2R, IGFBP‐2, and ‐4, but did not express other IGF system genes. We then compared IGF system expression in mature granule neurons and their progenitors. Progenitors exhibited higher mRNA levels of IGF‐1 and IGF‐1R (by 130% and 86%, respectively), lower levels of IGF‐2R (by 72%), and similar levels of IGFBP‐4. Our data support a role for IGF in hippocampal neurogenesis and provide evidence that IGF actions are regulated within a defined in vivo milieu.

A non-transformed oligodendrocyte precursor cell line, OL-1, facilitates studies of insulin-like growth factor-I signaling during oligodendrocyte development

The process by which oligodendrocyte progenitors differentiate into mature oligodendrocytes is complex and incompletely understood in part because of the paucity of oligodendrocyte precursors cell lines that can be studied in culture. We have developed a non‐immortalized rat oligodendrocyte precursor line, called OL‐1, which behaves in a fashion consistent with developing oligodendrocytes in vivo. This OL‐1 line provides a model for the study of oligodendrocyte development and offers an alternative to the CG‐4 cell line. When OL‐1 cells are propagated in conditioned growth media, they have morphology consistent with immature oligodendrocytes and exhibit A2B5 antigen positive and myelin basic protein‐negative immunoreactivity. Withdrawal of conditioned growth media and culture in serum‐free medium results in OL‐1 cell maturation, manifested by a shift to myelin basic protein‐positive immunoreactivity, A2B5 antigen‐negative immunoreactivity, decreased NG2 mRNA expression, increased expression of proteolipid protein mRNA, and increased expression of CNP protein. In addition, the expression of proteolipid protein and its splicing variant DM‐20 exhibit a pattern that is similar to brain proteolipid protein expression during development. When OL‐1 cells are exposed to Insulin‐like growth factor‐I, there are significant increases in proteolipid protein mRNA expression (p < 0.05), the number of cell processes (p < 0.05), and cell number (p < 0.05). Treatment with the caspase inhibitors Z‐DEVD‐FMK and Z‐VAD‐FMK (inhibitors of caspases 3, 6, 7, 8, 10 and 1, 3, 4, respectively), Insulin‐like growth factor‐I, or both, results in a similar increase in cell number. Because Insulin‐like growth factor‐I does not substantially increase the BrdU labeling of OL‐1 cells, these data collectively indicate that Insulin‐like growth factor‐I increases OL‐1 cell number predominately by promoting survival, rather than stimulating proliferation. This non‐immortalized oligodendrocyte precursor cell line, therefore, exhibits behavior consistent with the in vivo development of oligodendrocytes and provides an excellent model for the study of developing oligodendrocytes.

A novel mutation (E767K) in the second extracellular loop of the calcium sensing receptor in a family with autosomal dominant hypocalcemia

Autosomal dominant hypocalcemia resulting from gain‐of‐function mutations of the calcium sensing receptor (CASR) is a rare familial disorder that can become evident at any age. We report a novel mutation (E767K) of the CASR in a family with autosomal dominant hypocalcemia. Ten members of the family had a history of hypocalcemia. The index case exhibited marked hypocalcemia and seizures in the newborn period, while her father who also has hypocalcemia, was largely asymptomatic except for a myocardial infarction‐like event at 21 years of age, a new presentation of the disorder. The E767K mutation, which resides in the second extracellular loop adjacent to the fifth transmembrane domain, co‐segregated with hypocalcemia in these two individuals. Both subjects are heterozygous for the mutation. The proband is also heterozygous for the previously reported CASR polymorphism of G990R in the intracellular domain, while her father is homozygous. The co‐segregation of this naturally occurring mutation with autosomal dominant hypocalcemia supports the previously reported experimental model in which it was proposed that the three acidic residues (767, 758, and 759) in exo‐loop 2 in CASR help maintain an inactive conformation of the receptor.

PTEN deficiency mediates a reciprocal response to IGFI and mTOR inhibition.

Recent evidence implicates the insulin-like growth factor (IGF) pathway in development of Ewing sarcoma, a highly malignant bone and soft-tissue tumor that primarily affects children and young adults. Despite promising results from preclinical studies of therapies that target this pathway, early-phase clinical trials have shown that a significant fraction of patients do not benefit, suggesting that cellular factors determine tumor sensitivity. Using FAIRE-seq, a chromosomal deletion of the PTEN locus in a Ewing sarcoma cell line was identified. In primary tumors, PTEN deficiency was observed in a large subset of cases, although not mediated by large chromosomal deletions. PTEN loss resulted in hyperactivation of the AKT signaling pathway. PTEN rescue led to decreased proliferation, inhibition of colony formation, and increased apoptosis. Strikingly, PTEN loss decreased sensitivity to IGF1R inhibitors but increased responsiveness to temsirolimus, a potent mTOR inhibitor, as marked by induction of autophagy. These results suggest that PTEN is lost in a significant fraction of primary tumors, and this deficiency may have therapeutic consequences by concurrently attenuating responsiveness to IGF1R inhibition while increasing activity of mTOR inhibitors. The identification of PTEN status in the tumors of patients with recurrent disease could help guide the selection of therapies. Implications: PTEN status in Ewing sarcoma affects cellular responses to IGFI and mTOR-directed therapy, thus justifying its consideration as a biomarker in future clinical trials. Mol Cancer Res; 12(11); 1610–20. ©2014 AACR.

Dexamethasone changes the composition of insulin-like growth factor binding proteins in the newborn mouse ileum.

Background Early postnatal glucocorticoid exposure accelerates the maturation of the bowel mucosa but results in bowel wall thinning in the newborn mouse ileum and increases the risk of focal ileal perforation in extremely premature infants. We have previously demonstrated a redistribution of insulin-like growth factor-I (IGF-I) from the submucosa in control animals to the distal villi of those treated with early postnatal dexamethasone, implicating IGF-I as an important mediator of dexamethasones capacity to alter tissue growth. To investigate the possibility that IGF binding proteins (IGFBPs) might contribute to this process, we characterized the localization and abundance of IGFBP peptides and mRNAs in the same model. Methods Newborn mice received daily intraperitoneal injections of dexamethasone (l &mgr;g/g) or phosphate-buffered saline and then were euthanized on day 3 of life. Their ileums were harvested and prepared for microscopy. Tissue sections of ileum from both treatment conditions were processed in parallel for immunolocalization of each of the six IGFBP peptides and for in situ hybridization of each of the six IGFBP transcripts. Results Transcripts for IGFBP-1, -2, and -3 were highly abundant and ubiquitous the ileal mucosa, whereas transcripts for IGFBP-4, -5, and -6 were less abundant in epithelial cells. There were no differences in abundance between control and dexamethasone-treated ileum with regard to mRNA localization or abundance for IGFBP-1, -2, -3, and -6. In contrast, mRNA transcripts for IGFBP-4 and -5 were modestly increased with dexamethasone treatment (although only IGFBP-4 was significant). Strikingly different patterns of IGFBP immunolocalization were observed between control and dexamethasone-treated animals. IGFBP-1, -2, -3, and -5 were not detected in control ileum, whereas IGFBP-4 and -6 were both present in the mucosa. In contrast, dexamethasone treatment resulted in dramatic mucosal increases in IGFBP-2, -3, -4, and -5, paralleling the changing distribution of IGF-I that we previously reported. Conclusion Taken together, these findings further implicate the IGF system as an important participant in dexamethasone-induced maturation in the newborn mouse ileum.

Expression of the Insulin-Like Growth Factor System in Postpneumonectomy Lung Growth

The insulin-like growth factors (IGF-I and IGF-II) may play an important role in postpneumonectomy compensatory lung growth by translating hormonal inputs and mechanical forces into cellular proliferation signals. We examined the mRNA abundance of IGF-I, IGF-II, and IGF binding proteins (IGFBPs) in lungs of rats on postoperative days 1, 2, 3, 5, and 7 following left pneumonectomy (PNX) or shamoperation (SC) and in normal animals (CON). There was no difference in the abundance of lung IGF-I mRNA (measured by Northern analysis) or serum IGF-I (measured by radioimmunoassay (RIA)) between SC and PNX animals. IGF-II mRNA abundance was initially decreased following PNX (73% decrease compared to SC animals on day 1, p < .05) and then rose to approach SC group values on subsequent days. Transcripts for IGFBP-2, -3, -4, -5, and -6 were decreased in both the SC and PNX groups compared to CON animals on the day following pneumonectomy, then rose back to baseline by postoperative day 2-3. Tissue IGFBPs, measured by ligand blot analyses, were not different in either the SC or PNX groups. In contrast, all serum IGFBP bands were increased on postoperative day 1 following either sham or PNX surgery. In addition, serum IGFBP-4 was increased in PNX animals compared to the SC group on days 1 and 2 (increase of 38% and 78%, respectively, p < .05). We conclude that the changes observed in lung IGF and IGFBP expression following pneumonectomy do not represent major.

Valproic acid reduces the tolerability of temsirolimus in children and adolescents with solid tumors

A pediatric study has established a maximum tolerated dose (MTD) for temsirolimus (Tem) of more than 150 mg/m2 intravenously/week. A phase I trial was conducted to establish the MTD for Tem in combination with valproic acid (VPA) in children and adolescents with refractory solid tumors. The secondary aims included expression of mammalian target of rapamycin (mTOR) markers on archival tumor tissue; Tem pharmacokinetics; assessment of histone acetylation (HA); and tumor response. Patients were treated with VPA (5 mg/kg orally three times daily) with a target serum level of 75–100 mcg/ml. Tem was started at an initial dose of 60 mg/m2/week. Pharmacokinetics and HA measurements were performed during weeks 1 and 5. Two of the first three patients experienced dose-limiting toxicity (grade 3 mucositis). Tem at 35 mg/m2/week was found to be tolerable. Peak Tem concentrations were higher in all patients compared with those in previously published reports of single agent Tem. Increases in HA are correlated with VPA levels. All tumor samples expressed mTORC1 and mTORC2. An objective response was observed in one patient (melanoma), whereas transient stable disease was observed in four other patients (spinal cord ependymoma, alveolar soft part sarcoma, medullary thyroid carcinoma, and hepatocellular carcinoma). The MTD of Tem when administered with VPA is considerably lower than when used as a single agent, with mucositis the major dose-limiting toxicity. The combination merits further study and may have activity in melanoma. Attention to drug–drug interactions will be important in future multiagent trials including Tem.