Anne Reiman

Immunodeficiency in ataxia telangiectasia is correlated strongly with the presence of two null mutations in the ataxia telangiectasia mutated gene

Immunodeficiency affects over half of all patients with ataxia telangiectasia (A‐T) and when present can contribute significantly to morbidity and mortality. A retrospective review of clinical history, immunological findings, ataxia telangiectasia mutated (ATM) enzyme activity and ATM mutation type was conducted on 80 consecutive patients attending the National Clinic for Ataxia Telangiectasia, Nottingham, UK between 1994 and 2006. The aim was to characterize the immunodeficiency in A‐T and determine its relationship to the ATM mutations present. Sixty‐one patients had mutations resulting in complete loss of ATM kinase activity (group A) and 19 patients had leaky splice or missense mutations resulting in residual kinase activity (group B). There was a significantly higher proportion of patients with recurrent sinopulmonary infections in group A compared with group B (31 of 61 versus four of 19 P = 0·03) and a greater need for prophylactic antibiotics (30 of 61 versus one of 19 P = 0·001). Comparing group A with group B patients, 25 of 46 had undetectable/low immunoglobulin A (IgA) levels compared with none of 19; T cell lymphopenia was found in 28 of 56 compared with one of 18 and B cell lymphopenia in 35 of 55 compared with four of 18 patients (P = 0·00004, 0·001 and 0·003 respectively). Low IgG2 subclass levels and low levels of antibodies to pneumococcal polysaccharide were more common in group A than group B (16 of 27 versus one of 11 P = 0·01; 34/43 versus six of 17 P = 0·002) patients. Ig replacement therapy was required in 10 (12·5%) of the whole cohort, all in group A. In conclusion, A‐T patients with no ATM kinase activity had a markedly more severe immunological phenotype than those expressing low levels of ATM activity.

Lymphoid tumours and breast cancer in ataxia telangiectasia; substantial protective effect of residual ATM kinase activity against childhood tumours

Background:Immunodeficiency in ataxia telangiectasia (A-T) is less severe in patients expressing some mutant or normal ATM kinase activity. We, therefore, determined whether expression of residual ATM kinase activity also protected against tumour development in A-T.Methods:From a total of 296 consecutive genetically confirmed A-T patients from the British Isles and the Netherlands, we identified 66 patients who developed a malignant tumour; 47 lymphoid tumours and 19 non-lymphoid tumours were diagnosed. We determined their ATM mutations, and whether cells from these patients expressed any ATM with residual ATM kinase activity.Results:In childhood, total absence of ATM kinase activity was associated, almost exclusively, with development of lymphoid tumours. There was an overwhelming preponderance of tumours in patients <16 years without kinase activity compared with those with some residual activity, consistent with a substantial protective effect of residual ATM kinase activity against tumour development in childhood. In addition, the presence of eight breast cancers in A-T patients, a 30-fold increased risk, establishes breast cancer as part of the A-T phenotype.Conclusion:Overall, a spectrum of tumour types is associated with A-T, consistent with involvement of ATM in different mechanisms of tumour formation. Tumour type was influenced by ATM allelic heterogeneity, residual ATM kinase activity and age.

Modeling ATM mutant proteins from missense changes confirms retained kinase activity

Ataxia‐telangiectasia mutated (ATM) is the gene mutated in the cancer‐predisposing disorder ataxia‐telangiectasia (A‐T). We modeled ATM sequence variants identified in UK A‐T patients to determine the stability and kinase activity of the resulting proteins as well as the distribution of these mutations across the coding region. Of 20 missense changes modeled, 10 proteins showed ATM kinase activity and 10 showed none. In the majority of cases the mutant ATM protein was unstable, although this was variable. Reduction in ATM kinase activity can result either from the presence of low levels of unstable mutant protein with relatively normal specific kinase activity or from stable mutant protein with deficient ATM kinase activation. Indeed, ATM mutant proteins without kinase activity toward downstream targets were still able to autophosphorylate on serine 1981, although in a much less efficient manner, suggesting that this was not sufficient for ATM activation. In terms of function, green fluorescent protein (GFP)‐tagged kinase inactive ATM proteins could form ionizing radiation (IR)‐induced foci (IRIF), at least temporarily, which colocalized with the DNA double‐strand break (DSB) marker γH2AX. Consistent with this, both kinase active and inactive mutant ATM proteins were able to interfere with phosphorylation of targets by endogenous ATM. Since the majority of missense mutations occurred C‐terminal to aa1966, including all 10 mutations with absence of kinase activity, the implication was that mutations N‐terminal to this, with exceptions, are less likely to result in loss of kinase activity and therefore, are less likely to be identified in A‐T patients. Hum Mutat 30:1–9, 2009.

Folliculin interacts with p0071 (plakophilin-4) and deficiency is associated with disordered RhoA signalling, epithelial polarization and cytokinesis

Inherited mutations in the folliculin (FLCN) gene cause the Birt-Hogg-Dubé syndrome of familial hair follicle tumours (fibrofolliculomas), lung cysts and kidney tumours. Though folliculin has features of a tumour suppressor, the precise function of the FLCN gene product is not well characterized. We identified plakophilin-4 (p0071) as a potential novel folliculin interacting protein by yeast two-hybrid analysis. We confirmed the interaction of folliculin with p0071 by co-immunoprecipitation studies and, in view of previous studies linking p0071 to the regulation of rho-signalling, cytokinesis and intercellular junction formation, we investigated the effect of cell folliculin status on p0071-related functions. Folliculin and p0071 partially co-localized at cell junctions and in mitotic cells, at the midbody during cytokinesis. Previously, p0071 has been reported to regulate RhoA signalling during cytokinesis and we found that folliculin deficiency was associated with increased expression and activity of RhoA and evidence of disordered cytokinesis. Treatment of folliculin-deficient cells with a downstream inhibitor of RhoA signalling (the ROCK inhibitor Y-27632) reversed the increased cell migration phenotype observed in folliculin-deficient cells. Deficiency of folliculin and of p0071 resulted in tight junction defects and mislocalization of E-cadherin in mouse inner medullary collecting duct-3 renal tubular cells. These findings suggest that aspects of folliculin tumour suppressor function are linked to interaction with p0071 and the regulation of RhoA signalling.

Birt Hogg-Dubé syndrome-associated FLCN mutations disrupt protein stability.

Germline mutations in the FLCN gene cause Birt‐Hogg‐Dubé syndrome, familial spontaneous pneumothorax, or apparently nonsyndromic inherited RCC. The vast majority of reported FLCN mutations are predicted to result in a truncated/absent gene product and so infrequent missense and inframe‐deletion (IFD) FLCN mutations might indicate critical functional domains. To investigate this hypothesis we (1) undertook an in silico evolutionary analysis of the FLCN sequence and (2) investigated in vitro the functional effects of naturally occurring FLCN missense/IFD mutations. The folliculin protein sequence evolved more slowly and was under stronger purifying selection than the average gene, most notably at a region between codons 100 and 230. Pathogenic missense and IFD FLCN mutations that impaired folliculin tumor suppressor function significantly disrupted the stability of the FLCN gene product but two missense substitutions initially considered to be putative mutations did not impair protein stability, growth suppression activity, or intracellular localization of folliculin. These findings are consistent with the distribution of FLCN mutations throughout the coding sequence, and suggest that multiple protein domains contribute to folliculin stability and tumor suppressor activity. In vitro assessment of protein stability and tumor suppressor activity provides a practical strategy for assessing the pathogenicity of potential FLCN mutations. Hum Mutat 32:1–9, 2011.

Therapeutic Targeting the Loss of the Birt-Hogg-Dubé Suppressor Gene

Brit-Hogg-Dubé (BHD) syndrome, an autosomal dominant familial cancer, is associated with increased risk of kidney cancer. BHD syndrome is caused by loss-of-function mutations in the folliculin (FLCN) protein. To develop therapeutic approaches for renal cell carcinoma (RCC) in BHD syndrome, we adopted a strategy to identify tumor-selective growth inhibition in a RCC cell line with FLCN inactivation. The COMPARE algorithm was used to identify candidate anticancer drugs tested against the NCI-60 cell lines that showed preferential toxicity to low FLCN expressing cell lines. Fifteen compounds were selected and detailed growth inhibition (SRB) assays were done in paired BHD RCC cell lines (UOK257 derived from a patient with BHD). Selective sensitivity of FLCN-null over FLCN-wt UOK257 cells was observed in seven compounds. The most selective growth-inhibitory sensitivity was induced by mithramycin, which showed an approximately 10-fold difference in GI50 values between FLCN-null (64.2 ± 7.9 nmol/L, n = 3) and FLCN-wt UOK257 cells (634.3 ± 147.9 nmol/L, n = 4). Differential ability to induce caspase 3/7 activity by mithramycin was also detected in a dose-dependent manner. Clonogenic survival studies showed mithramycin to be approximately 10-fold more cytotoxic to FLCN-null than FLCN-wt UOK257 cells (200 nmol/L). Following mithramycin exposure, UOK257-FLCN-null cells were mainly arrested and blocked in S and G2-M phases of the cell cycle and low dose of rapamycin (1 nmol/L) potentiated mithramycin sensitivity (1.5-fold in G2-M population and 2-fold in G2-M period time, 2xGI50, 48 hours). These results provide a basis for further evaluation of mithramycin as a potential therapeutic drug for RCC associated with BHD. Mol Cancer Ther; 10(1); 80–9. ©2011 AACR.

Pathogenic ATM mutations occur rarely in a subset of multiple myeloma patients.

Ataxia Telangiectasia (A‐T) patients have biallelic inactivation of the ATM gene and exhibit a 200‐fold‐increased frequency of lymphoid tumours. ATM mutations have been found in a number of adult lymphoid malignancies but there is no data on the occurrence of ATM mutations in multiple myeloma tumours. The purpose of our work was to investigate the occurrence of ATM mutations in multiple myeloma and to this end we screened 45 sporadic cases for ATM mutations using denaturing high‐performance liquid chromatography analysis and DNA sequencing. Pathogenic ATM mutations were identified in 2/45 of the myelomas compared with a published estimate of ATM mutant allele frequency in the UK population of 2/521 (P = 0·033). One was the missense mutation 7181C>T which was then modelled in an expression system and the S2394L protein shown to have no ATM kinase activity. The second myeloma had the pathogenic ATM splice site mutation IVS40‐1G>C leading to loss of exon 41. We also report a 48‐year‐old ataxia telangiectasia patient who developed multiple myeloma. Taken together our study suggests that ATM mutation may play a role in the pathogenesis of a subset of multiple myelomas.

Knockdown of Slingshot 2 (SSH2) serine phosphatase induces Caspase3 activation in human carcinoma cell lines with the loss of the Birt–Hogg–Dubé tumour suppressor gene ( FLCN )

Birt–Hogg–Dubé (BHD) syndrome, is a dominantly inherited familial cancer syndrome associated with susceptibility to renal cell carcinoma (RCC) caused by inactivating mutations in the folliculin (FLCN) gene. The precise functions of the FLCN gene product are still under investigation but RCC from BHD patients show loss of the wild-type allele consistent with a tumor suppressor gene function. In a search for potential synthetic-lethal targets for FLCN using a phosphatase siRNA library screening approach, we found that knockdown of SSH2 serine phosphatase (one of the three members of Slingshot family and previously implicated in actin reorganization) specifically induced Caspase3/7 activity in a dose-dependent manner (up to six-fold increase, 10 nM, 72 h) in two human FLCN-deficient cell lines (BHD-origin renal cell carcinoma UOK257 and thyroid carcinoma FTC133) but not in their folliculin expressing isogenic cell lines. SSH2 siRNA-induced knockdown was accompanied by increased expression of SSH1 and SSH3 (suggesting a compensatory regulatory mechanism among members of SSH family). FLCN-null cells exhibited evidence of dysregulated cofilin de/phosphorylation pathways. Knockdown of SSH2 in FLCN-null cells was associated with an alteration in cell cycle kinetics (20% increase in G1, 30% and 40% decrease in S and G2M, respectively). Combination treatment of multiple SSH family (SSH2 plus SSH1 and/or SSH3) siRNAs potentiated induction of Caspase3/7 activity and changes in the cell cycle kinetics. These data indicate that: (a) apoptotic cell death in FLCN-null cells can be triggered by SSH2 knockdown through cell cycle arrest; (b) SSH2 represents a potential therapeutic target for the development of agents for the treatment of BHD syndrome and, possibly, related tumors.

Abstract LB-145: Folliculin interacts with p0071 (Plakophilin-4) and deficiency is associated with disordered RhoA signalling, epithelial polarization and cytokinesis

Inherited mutations in the folliculin gene (FLCN) cause the Birt-Hogg-Dube syndrome of familial hair follicle tumours (fibrofolliculomas), lung cysts and kidney tumours. Though folliculin has features of a tumour suppressor, the precise function of the FLCN gene product is not well characterised. We identified plakophilin-4 (PKP4, p0071) as a potential novel folliculin interacting protein by yeast-2-hybrid analysis. We confirmed the interaction of folliculin with p0071 by co-immunoprecipitation studies and, in view of previous studies linking p0071 to regulation of rho-signalling, cytokinesis and intercellular junction formation, we investigated the effect of cell folliculin status on p0071-related functions. Folliculin and p0071 partially colocalised at cell junctions and in mitotic cells, at the midbody during cytokinesis. Previously, p0071 has been reported to regulate RhoA signalling during cytokinesis and we found that folliculin deficiency was associated with increased expression and activity of RhoA and evidence of disordered cytokinesis. Treatment of folliculin deficient cells with a downstream inhibitor of RhoA signalling (the ROCK inhibitor Y-27632) reversed the increased cell migration phenotype observed in folliculin deficient cells. Deficiency of folliculin and of p0071 resulted in tight junction defects and mislocalisation of E-cadherin in IMCD-3 renal tubular cells. These findings suggest that aspects of folliculin tumour suppressor function are linked to interaction with p0071 and regulation of RhoA signalling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-145. doi:1538-7445.AM2012-LB-145

Abstract LB-259: Knockdown of slingshot2 serine phosphatase induces caspase3 activation in human carcinoma cell lines with the loss of the Birt-Hogg-Dubé tumor suppressor gene

Birt-Hogg-Dube (BHD) Syndrome, is a dominantly familial cancer syndrome associated with susceptibility to renal cancer carcinoma (RCC) caused by inactivating mutations in the folliculin (FLCN) gene. The precise functions of the FLCN gene product are still under investigation but RCC from BHD patients show loss of the wild type allele consistent with a tumour suppressor gene function. In search for potential synthetic-lethal targets for FLCN using a phosphatase siRNA library screen approach, we found that knockdown of SSH2 serine phosphatase (one of the three members of Slingshot family and actively involved in Actin reorganization) specifically induced caspase 3/7 activity in a dose-dependent manner (up to 6 fold increase, 10nM, 72hrs) in two human cell lines (BHD-origin renal cell carcinoma UOK-257 and thyroid carcinoma FTC-133) with loss of FLCN expression, but not in their folliculin expressing isogenic cell lines. SSH2 siRNA-induced knockdown was verified by real-time PCR (up to 60% reduction in SSH2 transcripts, 10nM siRNA, 48hrs), but SSH1 and SSH3 transcripts were increased up to 3 fold (suggesting a compensatory regulatory mechanism among members of SSH family). Further investigation of the underlining mechanism of caspase induction by SSH2 knockdown in FLCN-null cells exhibited that there was a dose-dependent increase in G1 cell population (up to 20% increase, 20nM SSH2 siRNA, 72hrs). Combination treatment of SSH2 + SSH1 siRNAs, but not SSH2 + SSH3 siRNAs potentiated induction of caspase 3 activity and G1 cell cycle arrest. These data indicate that apoptotic cell death in FLCN-null cells can be triggered by SSH2 knockdown through G1 arrest and provide a tool to develop potential therapeutic agents for BHD patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-259. doi:1538-7445.AM2012-LB-259