Anne F. Buckley

Transcription factor LKLF is sufficient to program T cell quiescence via a c-Myc-dependent pathway.

T lymphocytes circulate in a quiescent state until they encounter cognate antigen bound to the surface of an antigen-presenting cell. The molecular pathways that regulate T cell quiescence remain largely unknown. Here we show that forced expression of the lung Krüppel-like transcription factor (LKLF) in Jurkat T cells is sufficient to program a quiescent phenotype characterized by decreased proliferation, reduced cell size and protein synthesis and decreased surface expression of activation markers. Conversely, LKLF-deficient peripheral T cells produced by gene targeting showed increased proliferation, increased cell size and enhanced expression of surface activation markers in vivo. LKLF appeared to function, at least in part, by decreasing expression of the proto-oncogene encoding c-Myc. Forced expression of LKLF was associated with markedly decreased c-Myc expression. In addition, many effects of LKLF expression were mimicked by expression of the dominant-negative MadMyc protein and rescued by overexpression of c-Myc. Thus, LKLF is both necessary and sufficient to program quiescence in T cells and functions, in part, by negatively regulating a c-Myc–dependent pathway.

An Animal Model of MYC-Driven Medulloblastoma

Medulloblastoma (MB) is the most common malignant brain tumor in children. Patients whose tumors exhibit overexpression or amplification of the MYC oncogene (c-MYC) usually have an extremely poor prognosis, but there are no animal models of this subtype of the disease. Here, we show that cerebellar stem cells expressing Myc and mutant Trp53 (p53) generate aggressive tumors following orthotopic transplantation. These tumors consist of large, pleiomorphic cells and resemble human MYC-driven MB at a molecular level. Notably, antagonists of PI3K/mTOR signaling, but not Hedgehog signaling, inhibit growth of tumor cells. These findings suggest that cerebellar stem cells can give rise to MYC-driven MB and identify a novel model that can be used to test therapies for this devastating disease.

Epidermal growth factor receptor expression and gene copy number in conventional hepatocellular carcinoma.

Epidermal growth factor receptor (EGFR) is frequently overexpressed in hepatocellular carcinoma, but its relationship with EGFR gene copy number has not been studied. This study examined EGFR expression and gene copy number in hepatocellular carcinoma and evaluated their relationship to clinicopathologic features in 76 tumors. Moderate to strong expression of EGFR was observed by immunohistochemical analysis in 50 (66%) of 76 hepatocellular carcinomas. Fluorescence in situ hybridization (FISH) showed extra EGFR gene copies in 17 (45%) of 38 tumors. This was accompanied by gains of chromosome 7, indicating that this was the result of balanced polysomy rather than gene amplification. There was no correlation between EGFR expression by immunohistochemical analysis and gene copy number by FISH. EGFR expression showed borderline association with cirrhosis but not with other clinicopathologic parameters examined. EGFR overexpression is present in a majority of hepatocellular carcinomas, suggesting a role for EGFR antagonists in therapy. The increased expression does not correlate with an increase in the EGFR gene copy number.

Type 1 angiotensin receptors on macrophages ameliorate IL-1 receptor–mediated kidney fibrosis

In a wide array of kidney diseases, type 1 angiotensin (AT1) receptors are present on the immune cells that infiltrate the renal interstitium. Here, we examined the actions of AT1 receptors on macrophages in progressive renal fibrosis and found that macrophage-specific AT1 receptor deficiency exacerbates kidney fibrosis induced by unilateral ureteral obstruction (UUO). Macrophages isolated from obstructed kidneys of mice lacking AT1 receptors solely on macrophages had heightened expression of proinflammatory M1 cytokines, including IL-1. Evaluation of isolated AT1 receptor-deficient macrophages confirmed the propensity of these cells to produce exaggerated levels of M1 cytokines, which led to more severe renal epithelial cell damage via IL-1 receptor activation in coculture compared with WT macrophages. A murine kidney crosstransplantation concomitant with UUO model revealed that augmentation of renal fibrosis instigated by AT1 receptor-deficient macrophages is mediated by IL-1 receptor stimulation in the kidney. This study indicates that a key role of AT1 receptors on macrophages is to protect the kidney from fibrosis by limiting activation of IL-1 receptors in the kidney.

Radioprotection of the brain white matter by Mn(III) N-butoxyethylpyridylporphyrin-based superoxide dismutase mimic, MnTnBuOE-2-PyP5+

Cranial irradiation is a standard therapy for primary and metastatic brain tumors. A major drawback of radiotherapy (RT), however, is long-term cognitive loss that affects quality of life. Radiation-induced oxidative stress in normal brain tissue is thought to contribute to cognitive decline. We evaluated the effectiveness of a novel mimic of superoxide dismutase enzyme (SOD), MnTnBuOE-2-PyP5+(Mn(III) meso-tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin), to provide long-term neuroprotection following 8 Gy of whole brain irradiation. Long-term RT damage can only be assessed by brain imaging and neurocognitive studies. C57BL/6J mice were treated with MnTnBuOE-2-PyP5+ before and after RT and evaluated three months later. At this time point, drug concentration in the brain was 25 nmol/L. Mice treated with MnTnBuOE-2-PyP5+/RT exhibited MRI evidence for myelin preservation in the corpus callosum compared with saline/RT treatment. Corpus callosum histology demonstrated a significant loss of axons in the saline/RT group that was rescued in the MnTnBuOE-2-PyP5+/RT group. In addition, the saline/RT groups exhibited deficits in motor proficiency as assessed by the rotorod test and running wheel tests. These deficits were ameliorated in groups treated with MnTnBuOE-2-PyP5+/RT. Our data demonstrate that MnTnBuOE-2-PyP5+ is neuroprotective for oxidative stress damage caused by radiation exposure. In addition, glioblastoma cells were not protected by MnTnBuOE-2-PyP5+ combination with radiation in vitro. Likewise, the combination of MnTnBuOE-2-PyP5+ with radiation inhibited tumor growth more than RT alone in flank tumors. In summary, MnTnBuOE-2-PyP5+ has dual activity as a neuroprotector and a tumor radiosensitizer. Thus, it is an attractive candidate for adjuvant therapy with RT in future studies with patients with brain cancer. Mol Cancer Ther; 14(1); 70–79. ©2014 AACR.

Gq signaling causes glomerular injury by activating TRPC6

Familial forms of focal segmental glomerulosclerosis (FSGS) have been linked to gain-of-function mutations in the gene encoding the transient receptor potential channel C6 (TRPC6). GPCRs coupled to Gq signaling activate TRPC6, suggesting that Gq-dependent TRPC6 activation underlies glomerular diseases. Here, we developed a murine model in which a constitutively active Gq α subunit (Gq(Q209L), referred to herein as GqQ>L) is specifically expressed in podocytes and examined the effects of this mutation in response to puromycin aminonucleoside (PAN) nephrosis. We found that compared with control animals, animals expressing GqQ>L exhibited robust albuminuria, structural features of FSGS, and reduced numbers of glomerular podocytes. Gq activation stimulated calcineurin (CN) activity, resulting in CN-dependent upregulation of TRPC6 in murine kidneys. Deletion of TRPC6 in GqQ>L-expressing mice prevented FSGS development and inhibited both tubular damage and podocyte loss induced by PAN nephrosis. Similarly, administration of the CN inhibitor FK506 reduced proteinuria and tubular injury but had more modest effects on glomerular pathology and podocyte numbers in animals with constitutive Gq activation. Moreover, these Gq-dependent effects on podocyte injury were generalizable to diabetic kidney disease, as expression of GqQ>L promoted albuminuria, mesangial expansion, and increased glomerular basement membrane width in diabetic mice. Together, these results suggest that targeting Gq/TRPC6 signaling may have therapeutic benefits for the treatment of glomerular diseases.

Fluctuating response of a cystic vestibular schwannoma to radiosurgery: case report.

OBJECTIVE A vestibular schwannoma (VS) is a benign tumor of the VIIIth cranial nerve that can often be treated by microsurgery or radiosurgery and demonstrates high tumor control rates. Radiosurgery is typically performed as gamma knife surgery (GKS), although other modalities are being applied with increasing frequency. A differentiating feature in responsiveness to microsurgery or GKS is whether the VS is cystic or solid. A cystic VS is less responsive to GKS than a solid VS, representing a challenging clinical problem. GKS treatment of a cystic VS usually results in sustained expansion, sustained regression, or transient expansion followed by sustained regression. In this article, we report an atypical fluctuating course of a cystic VS after GKS, ultimately requiring surgical intervention. CLINICAL PRESENTATION A 66-year-old woman presented with asymmetric hearing loss and tinnitus. Magnetic resonance imaging revealed a 2.0-cm unilateral cystic VS within the cerebellopontine angle. INTERVENTION After GKS with a 12-Gy dose to the 50% isodose line, the tumor expanded transiently to 3.2 cm and then regressed to 1.0 cm over the next 2 years. She presented several months later with new-onset dizziness, ataxia, and facial numbness. Magnetic resonance imaging revealed a 3.2-cm multicystic VS that compressed the brainstem. After microsurgical tumor excision, the patients symptoms abated. CONCLUSION Our case report is a novel demonstration that a cystic VS that has regressed after GKS is still at risk for expansion. The mechanisms responsible for radiation-induced cystic tumor expansion have not been thoroughly elucidated. The risk of unpredictable tumor enlargement should be discussed with patients when considering GKS for cystic tumors.

Radioimmunotargeting of Malignant Glioma by Monoclonal Antibody D2C7 Reactive Against Both Wild-Type and Variant III Mutant Epidermal Growth Factor Receptors

INTRODUCTION Malignant glioma remains a significant therapeutic challenge, and immunotherapeutics might be a beneficial approach for these patients. A monoclonal antibody (MAb) specific for multiple molecular targets could expand the treatable patient population and the fraction of tumor cells targeted, with potentially increased efficacy. This motivated the generation of MAb D2C7, which recognizes both wild-type epidermal growth factor receptor (EGFRwt) and a tumor-specific mutant, EGFRvIII. METHODS D2C7 binding affinity was determined by surface plasmon resonance and its specificity characterized through comparison to EGFRwt-specific EGFR.1 and EGFRvIII-specific L8A4 MAbs by flow cytometry and immunohistochemical analysis. The three MAbs were labeled with (125)I or (131)I using Iodogen, and paired-label internalization assays and biodistribution experiments in athymic mice with human tumor xenografts were performed. RESULTS The affinity of D2C7 for EGFRwt and EGFRvIII was 5.2×10(9) M(-1) and 3.6×10(9) M(-1), and cell-surface reactivity with both receptors was documented by flow cytometry. Immunohistochemical analyses revealed D2C7 reactivity with malignant glioma tissue from 90 of 101 patients. Internalization assays performed on EGFRwt-expressing WTT cells and EGFRvIII-expressing NR6M cells indicated a threefold lower degradation of (125)I-labeled D2C7 compared with (131)I-labeled EGFR.1. Uptake of (125)I-labeled D2C7 in NR6M xenografts (52.45±13.97 %ID g(-1) on Day 3) was more than twice that of (131)I-labeled L8A4; a threefold to fivefold tumor delivery advantage was seen when compared to (131)I-labeled EGFR.1 in mice with WTT xenografts. CONCLUSIONS These results suggest that D2C7 warrants further evaluation for the development of MAb-based therapeutics against cancers expressing EGFRwt and EGFRvIII.

Adjunctive albuterol enhances the response to enzyme replacement therapy in late-onset Pompe disease

Effective dosages for enzyme replacement therapy (ERT) in Pompe disease are much higher than for other lysosomal storage disorders, which has been attributed to low cation‐independent mannose‐6‐phosphate receptor (CI‐MPR) in skeletal muscle. We have previously demonstrated the benefit of increased CI‐MPR‐mediated uptake of recombinant human acid‐α‐glucosidase during ERT in mice with Pompe disease following addition of albuterol therapy. Currently we have completed a pilot study of albuterol in patients with late‐onset Pompe disease already on ERT for >2 yr, who were not improving further. The 6‐min walk test (6MWT) distance increased in all 7 subjects at wk 6 (30±13 m; P=0.002), wk 12 (34±14 m; P=0.004), and wk 24 (42±37 m; P=0.02), in comparison with baseline. Grip strength was improved significantly for both hands at wk 12. Furthermore, individual subjects reported benefits; e.g., a female patient could stand up from sitting on the floor much more easily (time for supine to standing position decreased from 30 to 11 s), and a male patient could readily swing his legs out of his van seat (hip abduction increased from 1 to 2+ on manual muscle testing). Finally, analysis of the quadriceps biopsies suggested increased CI‐MPR at wk 12 (P=0.08), compared with baseline. With the exception of 1 patient who succumbed to respiratory complications of Pompe disease in the first week, only mild adverse events have been reported, including tremor, transient difficulty falling asleep, and mild urinary retention (requiring early morning voiding). Therefore, this pilot study revealed initial safety and efficacy in an open label study of adjunctive albuterol therapy in patients with late‐onset Pompe disease who had been stable on ERT with no improvements noted over the previous several years.—Koeberl, D. D., Austin, S., Case, L. E., Smith, E. C., Buckley, A. F., Young, S. P., Bali, D., Kishnani, P. S. Adjunctive albuterol enhances the response to enzyme replacement therapy in late‐onset Pompe disease. FASEB J. 28, 2171–2176 (2014). www.fasebj.org

Comparison of the Dako EGFR pharmDx kit and Zymed EGFR antibody for assessment of EGFR status in colorectal adenocarcinoma.

Immunohistochemistry is widely used to assess epidermal growth factor receptor (EGFR) expression on colorectal carcinomas to select patients for treatment with cetuximab, an anti-EGFR antibody. The data comparing different commercial EGFR antibodies is limited, and no cost comparisons have been made. We analyzed 65 advanced colorectal cancers from 36 patients using the EGFR pharmDx kit (DakoCytomation) and Clone 31G7 (Zymed Laboratories, Inc). EGFR expression was seen in 35 (53%) tumors (21 primary, 14 metastatic) with the Dako pharmDx kit. The Zymed antibody showed positive results in 41 (63%) tumors (25 primary, 16 metastatic). The cost per test was