Multicenter phase 2 trial of sirolimus for tuberous sclerosis: kidney angiomyolipomas and other tumors regress and VEGF- D levels decrease

Background

Tuberous sclerosis (TSC) related tumors are characterized by constitutively activated mTOR signaling due to mutations in TSC1 or TSC2.

Methods

We completed a phase 2 multicenter trial to evaluate the efficacy and tolerability of the mTOR inhibitor, sirolimus, for the treatment of kidney angiomyolipomas.

Results

36 adults with TSC or TSC/LAM were enrolled and started on daily sirolimus. The overall response rate was 44.4% (95% confidence intervals [CI] 28 to 61); 16/36 had a partial response. The remainder had stable disease (47.2%, 17/36), or were unevaluable (8.3%, 3/36). The mean decrease in kidney tumor size (sum of the longest diameters [sum LD]) was 29.9% (95% CI, 22 to 37; n = 28 at week 52). Drug related grade 1–2 toxicities that occurred with a frequency of >20% included: stomatitis, hypertriglyceridemia, hypercholesterolemia, bone marrow suppression (anemia, mild neutropenia, leucopenia), proteinuria, and joint pain. There were three drug related grade 3 events: lymphopenia, headache, weight gain. Kidney angiomyolipomas regrew when sirolimus was discontinued but responses tended to persist if treatment was continued after week 52. We observed regression of brain tumors (SEGAs) in 7/11 cases (26% mean decrease in diameter), regression of liver angiomyolipomas in 4/5 cases (32.1% mean decrease in longest diameter), subjective improvement in facial angiofibromas in 57%, and stable lung function in women with TSC/LAM (n = 15). A correlative biomarker study showed that serum VEGF-D levels are elevated at baseline, decrease with sirolimus treatment, and correlate with kidney angiomyolipoma size (Spearman correlation coefficient 0.54, p = 0.001, at baseline).

Conclusions

Sirolimus treatment for 52 weeks induced regression of kidney angiomyolipomas, SEGAs, and liver angiomyolipomas. Serum VEGF-D may be a useful biomarker for monitoring kidney angiomyolipoma size. Future studies are needed to determine benefits and risks of longer duration treatment in adults and children with TSC.

Trial Registration

Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00126672","term_id":"NCT00126672"}}NCT00126672     

Nucleolin, defective for MPF phosphorylation, localizes normally during mitosis and nucleologenesis

To determine what effect maturation promoting factor (MPF, p34 ^cdc2 kinase/cyclin B) phosphorylation has on nucleolin’s distribution during mitotic nucleolar disassembly and reassembly, we altered Chinese hamster ovary (CHO) nucleolin (the N protein) such that it cannot be phosphorylated by p34 ^cdc2 . As expected, the transiently expressed epitope-tagged N protein showed no apparent defect in nucleolar localization in interphase CHO cells, even after hypotonic shock and recovery to quickly disassemble and then reassemble interphase nucleoli. In mitotic CHO cells, the N protein localized to the perichromosomal sheath and the cytoplasm, as is typical for nucleolin. Similar to epitope-tagged wild-type nucleolin, the N protein also maintained its association with persistent nucleoli characteristic of mitotic Chinese hamster lung (Dede) cells. In synchronized HeLa cells, the N protein again localized to the perichromosomal sheath and the cytoplasm as nucleoli disassembled during prophase. In HeLa cell telophase, the N protein localized normally to nucleolus-derived foci within the cytoplasm and prenucleolar bodies within reforming nuclei. The observations indicate that MPF phosphorylation is not essential for nucleolin’s localizations to the perichromosomal sheath and the cytoplasm during prophase and metaphase, and that functional MPF phosphorylation sites are not essential for nucleolin’s localizations during nucleologenesis. Accepted: 15 April 1999     

The carbonic anhydrase isoforms of Chlamydomonas reinhardtii: intracellular location, expression, and physiological roles

Aquatic photosynthetic organisms, such as the green alga Chlamydomonas reinhardtii, respond to low CO(2) conditions by inducing a CO(2) concentrating mechanism (CCM). Carbonic anhydrases (CAs) are important components of the CCM. CAs are zinc-containing metalloenzymes that catalyze the reversible interconversion of CO(2) and HCO(3)(-). In C. reinhardtii, there are at least 12 genes that encode CA isoforms, including three alpha, six beta, and three gamma or gamma-like CAs. The expression of the three alpha and six beta genes has been measured from cells grown on elevated CO(2) (having no active CCM) versus cells growing on low levels of CO(2) (with an active CCM) using northern blots, differential hybridization to DNA chips and quantitative RT-PCR. Recent RNA-seq profiles add to our knowledge of the expression of all of the CA genes. In addition, protein content for some of the CA isoforms was estimated using antibodies corresponding to the specific CA isoforms: CAH1/2, CAH3, CAH4/5, CAH6, and CAH7. The intracellular location of each of the CA isoforms was elucidated using immunolocalization and cell fractionation techniques. Combining these results with previous studies using CA mutant strains, we will discuss possible physiological roles of the CA isoforms concentrating on how these CAs might contribute to the acquisition and retention of CO(2) in C. reinhardtii.     

Development of Anncaliia algerae (Microsporidia) in Drosophila melanogaster

Representatives of the genus Anncaliia are known as natural parasites of dipteran and coleopteran insects, amphipod crustaceans, but also humans, primarily with immunodeficiency. Anncaliia algerae‐caused fatal myositis is considered as an emergent infectious disease in humans. A. (=Nosema, Brachiola) algerae, the best studied species of the genus, demonstrates the broadest among microsporidia range of natural and experimental hosts, but it has never been propagated in Drosophila. We present ultrastructural analysis of development of A. algerae in visceral muscles and adipocytes of Drosophila melanogaster 2 wk after per oral experimental infection. We observed typical to Anncaliia spp. features of ultrastructure and cell pathology including spore morphology, characteristic extensions of the plasma membrane, and presence of “ridges” and appendages of tubular material at proliferative stages. Anncaliia algerae development in D. melanogaster was particularly similar to one of A. algerae and A.(Brachiola) vesicularum in humans with acute myositis. Given D. melanogaster is currently the most established genetic model, with a fully sequenced genome and easily available transgenic forms and genomic markers, a novel host–parasite system might provide new genetic tools to investigate host–pathogen interactions of A. algerae, as well to test antimicrosporidia drugs.     

Differences in the Developmental Fate of Cultured and Noncultured Myoblasts When Transplanted into Embryonic Limbs

Myoblasts from embryonic, fetal, and adult quail and chick muscles were transplanted into limb buds of chick embryos to determine if myoblasts can form muscle fibers in heterochronic limbs and to define the conditions that affect the ability of transplanted cells to populate newly developing limb musculature. Myoblasts from each developmental stage were either freshly isolated and transplanted or were cultured prior to transplantation into limb buds of 4- to 5-day (ED4-5) chick embryos. Transplanted myoblasts, regardless of the age of the donor from which they were derived, formed muscle fibers within embryonic limb muscles. Transplanted cloned myoblasts formed muscle fibers, although there was little evidence that the number of transplanted myoblasts significantly increased following transplantation or that they migrated any distance from the site of injection. The fibers that formed from transplanted clonal myoblasts often did not persist in the host limb muscles until ED10. Diminished fiber formation from myoblasts transplanted into host limbs was observed whether myoblasts were cloned or cultured at high density. However, when freshly isolated myoblasts were transplanted, the fibers they formed were numerous, widely dispersed within the limb musculature, and persisted in the muscles until at least ED10. These results indicate that transplanted myoblasts of embryonic, fetal, and adult origin are capable of forming fibers during early limb muscle formation. They also indicate that even in an embryonic chick limb where proliferation of endogenous myoblasts and muscle fiber formation is rapidly progressing, myoblasts that are cultured in vitro do not substantially contribute to long-term muscle fiber formation after they are transplanted into developing limbs. However, when the same myoblasts are freshly isolated and transplanted without prior cell culture, substantial numbers of fibers form and persist after transplantation into developing limbs. Thus, these studies demonstrate that the extent to which transplanted myoblasts fuse to form fibers which persist in host musculature depends upon whether donor myoblasts are freshly isolated or maintained in vitro prior to injection.     

Helix formation in model peptides based on nucleolin TPAKK motifs

The structures formed by peptide models of the N-terminal domain of the nucleolar protein nucleolin were studied by CD and nmr. The sequences of the peptides are based on the putative nucleic acid binding sequence motif TPAKK: The peptides TP1 and TP2 have the sequence acetyl-G(ATPAKKAA)_nG-amide, with n = 1 and 2, respectively. CD measurements indicate structural changes in both peptides when the lysine side chains are uncharged by increasing the pH or acetylation of the side-chain amines. When trifluoroethanol (TFE) is added, more extensive structural changes are observed, resembling helical structure based on nmr nuclear Overhauser effect (NOE) and C^α proton chemical shift changes, and CD spectra. The structure formed in 0.5M NaClO₄ as observed by nmr is similar to that when the lysine side chains are acetylated, due presumably to interactions of perchlorate ion with side-chain charges on lysines. The helical structure observed in TPAKK motifs may be stabilized via N-capping interactions involving threonine. The structures observed in TFE suggest that the Thr-Pro sequence initiates short helical segments in TPAKK motifs, and these helical structures might interact with nucleic acids, presumably via interactions between lysines and threonines of nucleolin. © 1995 John Wiley & Sons, Inc.     

Developmentally regulated expression of three slow isoforms of myosin heavy chain: diversity among the first fibers to form in avian muscle.

At least three slow myosin heavy chain (MHC) isoforms were expressed in skeletal muscles of the developing chicken hindlimb, and differential expression of these slow MHC isoforms produced distinct fiber types from the outset of skeletal muscle myogenesis. Immunohistochemistry with isoform-specific monoclonal antibodies demonstrated differences in MHC content among the fibers of the dorsal and ventral premuscle masses and distinctions among fibers before splitting of the premuscle masses into individual muscles (Hamburger and Hamilton Stage 25). Immunoblot analyses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of myosin extracted from the hindlimb demonstrated the presence throughout development of different mobility classes of MHCs with epitopes associated with slow MHC isoforms. Immunopeptide mapping showed that one of the MHCs expressed in the embryonic limb was the same slow MHC isoform, slow MHC1 (SMHC1), that is expressed in adult slow muscles. SMHC1 was expressed in the dorsal and ventral premuscle masses, embryonic, fetal, and some neonatal and adult hindlimb muscles. In the embryo and fetus SMHC1 was expressed in future fast, as well as future slow muscles, whereas in the adult only the slow muscles retained expression of SMHC1. Those embryonic muscles destined in the adult to contain slow fibers or mixed fast/slow fibers not only expressed SMHC1, but also an additional slow MHC not previously described, designated as slow MHC3 (SMHC3). Slow MHC3 was shown by immunopeptide mapping to contain a slow MHC epitope (reactive with mAb S58) and to be structurally similar to a MHC expressed in the atria of the adult chicken heart. SMHC3 was designated as a slow MHC isoform because (i) it was expressed only in those muscles destined to be of the slow type in the adult, (ii) it was expressed only in primary fibers of muscles that subsequently are of the slow type, and (iii) it had an epitope demonstrated to be present on other slow, but not fast, isoforms of avian MHC. This study demonstrates that a difference in phenotype between fibers is established very early in the chicken embryo and is based on the fiber type-specific expression of three slow MHC isoforms.