Yvonne Chekaluk

Regulable neural progenitor-specific Tsc1 loss yields giant cells with organellar dysfunction in a model of tuberous sclerosis complex

Tuberous sclerosis complex (TSC) is a multiorgan genetic disease in which brain involvement causes epilepsy, intellectual disability, and autism. The hallmark pathological finding in TSC is the cerebral cortical tuber and its unique constituent, giant cells. However, an animal model that replicates giant cells has not yet been described. Here, we report that mosaic induction of Tsc1 loss in neural progenitor cells in Tsc1cc Nestin-rtTA+ TetOp-cre+ embryos by doxycycline leads to multiple neurological symptoms, including severe epilepsy and premature death. Strikingly, Tsc1-null neural progenitor cells develop into highly enlarged giant cells with enlarged vacuoles. We found that the vacuolated giant cells had multiple signs of organelle dysfunction, including markedly increased mitochondria, aberrant lysosomes, and elevated cellular stress. We found similar vacuolated giant cells in human tuber specimens. Postnatal rapamycin treatment completely reversed these phenotypes and rescued the mutants from epilepsy and premature death, despite prenatal onset of Tsc1 loss and mTOR complex 1 activation in the developing brain. This TSC brain model provides insights into the pathogenesis and organelle dysfunction of giant cells, as well as epilepsy control in patients with TSC.

Mosaic and Intronic Mutations in TSC1/TSC2 Explain the Majority of TSC Patients with No Mutation Identified by Conventional Testing

Tuberous sclerosis complex (TSC) is an autosomal dominant tumor suppressor gene syndrome due to germline mutations in either TSC1 or TSC2. 10–15% of TSC individuals have no mutation identified (NMI) after thorough conventional molecular diagnostic assessment. 53 TSC subjects who were NMI were studied using next generation sequencing to search for mutations in these genes. Blood/saliva DNA including parental samples were available from all subjects, and skin tumor biopsy DNA was available from six subjects. We identified mutations in 45 of 53 subjects (85%). Mosaicism was observed in the majority (26 of 45, 58%), and intronic mutations were also unusually common, seen in 18 of 45 subjects (40%). Seventeen (38%) mutations were seen at an allele frequency < 5%, five at an allele frequency < 1%, and two were identified in skin tumor biopsies only, and were not seen at appreciable frequency in blood or saliva DNA. These findings illuminate the extent of mosaicism in TSC, indicate the importance of full gene coverage and next generation sequencing for mutation detection, show that analysis of TSC-related tumors can increase the mutation detection rate, indicate that it is not likely that a third TSC gene exists, and enable provision of genetic counseling to the substantial population of TSC individuals who are currently NMI.

TSC1 involvement in bladder cancer: diverse effects and therapeutic implications

TSC1 is often mutated in bladder cancer. However the importance of this event in disease pathogenesis and its implications for therapy are uncertain. We used genomic sequencing to examine the involvement of TSC1 in bladder cancer, and signalling pathway analysis and small‐molecule screening to identify targeted therapeutic strategies in TSC1 mutant bladder cancer cell lines. TSC1 loss of heterozygosity was seen in 54% of bladder cancers. Two (4.9%) of these 41 bladder cancers had TSC1 mutations by exon‐based sequencing. Analysis of 27 bladder cancer cell lines demonstrated inactivating TSC1 mutations in three: RT‐4, HCV29, 97–1. Interestingly, only RT‐4 showed classic feedback inhibition of AKT, and was highly sensitive to treatment with mTOR inhibitors rapamycin and Torin1. 97–1 cells showed constitutive EGFR activation, and were highly sensitive to combined treatment with the mTOR inhibitor Torin1 and EGFR inhibitors Lapatinib or Afatinib. A BRAF missense mutation G469V was found in HCV29 cells, and AKT activation was dependent on BRAF, but independent of ERK. A kinase inhibitor screen of HCV29 cells showed strong growth inhibition by the Hsp90 inhibitor NVP‐AUY922, and we then found synergistic inhibitory effects of NVP‐AUY922 combined with either Torin1 or rapamycin on cell survival for both HCV29 and 97–1 cells. In aggregate, these findings indicate that there are highly variable mutation profiles and signalling pathway activation in TSC1‐mutant bladder cancer. Furthermore, combined Hsp90/mTOR inhibition is a promising therapeutic approach for TSC1 mutant bladder cancer. Copyright

Identification of Nine Genomic Regions of Amplification in Urothelial Carcinoma, Correlation with Stage, and Potential Prognostic and Therapeutic Value

We performed a genome wide analysis of 164 urothelial carcinoma samples and 27 bladder cancer cell lines to identify copy number changes associated with disease characteristics, and examined the association of amplification events with stage and grade of disease. Multiplex inversion probe (MIP) analysis, a recently developed genomic technique, was used to study 80 urothelial carcinomas to identify mutations and copy number changes. Selected amplification events were then analyzed in a validation cohort of 84 bladder cancers by multiplex ligation-dependent probe assay (MLPA). In the MIP analysis, 44 regions of significant copy number change were identified using GISTIC. Nine gene-containing regions of amplification were selected for validation in the second cohort by MLPA. Amplification events at these 9 genomic regions were found to correlate strongly with stage, being seen in only 2 of 23 (9%) Ta grade 1 or 1–2 cancers, in contrast to 31 of 61 (51%) Ta grade 3 and T2 grade 2 cancers, p<0.001. These observations suggest that analysis of genomic amplification of these 9 regions might help distinguish non-invasive from invasive urothelial carcinoma, although further study is required. Both MIP and MLPA methods perform well on formalin-fixed paraffin-embedded DNA, enhancing their potential clinical use. Furthermore several of the amplified genes identified here (ERBB2, MDM2, CCND1) are potential therapeutic targets.

Integrative analysis of 1q23.3 copy-number gain in metastatic urothelial carcinoma

Purpose: Metastatic urothelial carcinoma of the bladder is associated with multiple somatic copy-number alterations (SCNAs). We evaluated SCNAs to identify predictors of poor survival in patients with metastatic urothelial carcinoma treated with platinum-based chemotherapy. Experimental Design: We obtained overall survival (OS) and array DNA copy-number data from patients with metastatic urothelial carcinoma in two cohorts. Associations between recurrent SCNAs and OS were determined by a Cox proportional hazard model adjusting for performance status and visceral disease. mRNA expression was evaluated for potential candidate genes by NanoString nCounter to identify transcripts from the region that are associated with copy-number gain. In addition, expression data from an independent cohort were used to identify candidate genes. Results: Multiple areas of recurrent significant gains and losses were identified. Gain of 1q23.3 was independently associated with a shortened OS in both cohorts [adjusted HR, 2.96; 95% confidence interval (CI), 1.35–6.48; P = 0.01 and adjusted HR, 5.03; 95% CI, 1.43–17.73; P < 0.001]. The F11R, PFDN2, PPOX, USP21, and DEDD genes, all located on 1q23.3, were closely associated with poor outcome. Conclusions: 1q23.3 copy-number gain displayed association with poor survival in two cohorts of metastatic urothelial carcinoma. The identification of the target of this copy-number gain is ongoing, and exploration of this finding in other disease states may be useful for the early identification of patients with poor-risk urothelial carcinoma. Prospective validation of the survival association is necessary to demonstrate clinical relevance. Clin Cancer Res; 20(7); 1873–83. ©2014 AACR.

Comprehensive detection of germline variants by MSK-IMPACT, a clinical diagnostic platform for solid tumor molecular oncology and concurrent cancer predisposition testing

BackgroundThe growing number of Next Generation Sequencing (NGS) tests is transforming the routine clinical diagnosis of hereditary cancers. Identifying whether a cancer is the result of an underlying disease-causing mutation in a cancer predisposition gene is not only diagnostic for a cancer predisposition syndrome, but also has significant clinical implications in the clinical management of patients and their families.MethodsHere, we evaluated the performance of MSK-IMPACT (Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets) in detecting genetic alterations in 76 genes implicated in cancer predisposition syndromes. Output from hybridization-based capture was sequenced on an Illumina HiSeq 2500. A custom analysis pipeline was used to detect single nucleotide variants (SNVs), small insertions/deletions (indels) and copy number variants (CNVs).ResultsMSK-IMPACT detected all germline variants in a set of 233 unique patient DNA samples, previously confirmed by previous single gene testing. Reproducibility of variant calls was demonstrated using inter- and intra- run replicates. Moreover, in 16 samples, we identified additional pathogenic mutations other than those previously identified through a traditional gene-by-gene approach, including founder mutations in BRCA1, BRCA2, CHEK2 and APC, and truncating mutations in TP53, TSC2, ATM and VHL.ConclusionsThis study highlights the importance of the NGS-based gene panel testing approach in comprehensively identifying germline variants contributing to cancer predisposition and simultaneous detection of somatic and germline alterations.

Targeted deletion of Tsc1 causes fatal cardiomyocyte hyperplasia independently of afterload

Despite high expression levels, the role of Tsc1 in cardiovascular tissue is ill defined. We launched this study to examine the role of Tsc1 in cardiac physiology and pathology. Mice in which Tsc1 was deleted in cardiac tissue and vascular smooth muscle (Tsc1c/cSM22cre(+/-)), developed progressive cardiomegaly and hypertension and died early. Hearts of Tsc1c/cSM22cre(+/-) mice displayed a progressive increase in cardiomyocyte number, and to a lesser extent, size between the ages of 1 and 6 weeks. In addition, compared to control hearts, proliferation markers (phospho-histone 3 and PCNA) were elevated in Tsc1c/cSM22cre(+/-) cardiomyocytes at 0-4 weeks, suggesting that cardiomyocyte proliferation was the predominant mechanism underlying cardiomegaly in Tsc1c/cSM22cre(+/-) mice. To examine the contribution of Tsc1 deletion in peripheral vascular smooth muscle to the cardiac phenotype, Tsc1c/cSM22cre(+/-) mice were treated with the antihypertensive, hydralazine. Prevention of hypertension had no effect on survival, cardiac size, or cardiomyocyte number in these mice. We furthermore generated mice in which Tsc1 was deleted only in vascular smooth muscle but not in cardiac tissue (Tsc1c/cSMAcre-ER(T2+/-)). The Tsc1c/cSMAcre-ER(T2+/-) mice also developed hypertension. However, their survival was normal and no cardiac abnormalities were observed. Our results suggest that loss of Tsc1 in the heart causes cardiomegaly, which is driven by increased cardiomyocyte proliferation that also appears to confer relative resistance to afterload reduction. These findings support a critical role for the Tsc1 gene as gatekeeper in the protection against uncontrolled cardiac growth.

Abstract 1571: Novel microfluidic technology and biology based approach for isolation and characterization of circulating cells from the peripheral blood of patients with Lymphangioleiomyomatosis

The rare multisystem disease Lymphangioleiomyomatosis (LAM) that effects almost exclusively women is characterized by the aberrant proliferation of smooth muscle-like cells (LAM cells). Although the specific molecular determinants are unknown, metastasis seems to be the mechanism by which the LAM cells are disseminated. In fact the multisystem manifestations of LAM have been correlated with a metastatic dissemination of the LAM cells, which were identified in donor lungs transplanted to LAM patients. Furthermore, LAM cells have been detected in blood, chyle, and urine. However, there is still a significant gap between the detection of circulating cells and their use as a biomarker for diagnosis, monitoring of response to a therapy, and prognosis. It is well established that the LAM is characterized by cystic lung destruction caused by LAM cells (smooth-muscle-like-cells) that have mutations in tumor suppressor genes, tuberous sclerosis complex (TSC) 1 or 2, and have the capacity to metastasize. Given the limitations of the sensitivity of the current technologies, the genotyping of the circulating LAM cells (CLCs) has not been established. Hence we applied a novel microfluidic technology (CTC-chip) for the reliable detection of circulating LAM cells from blood. An integrated technology and biology based approach is employed to address the efficient and specific isolation and applications of CLCs. Since LAM cells isolated from LAM patient lungs expresses CD44 and CD44v6 and additionally CD44v6 positive cells show loss heterozygosity (LOH) at the TSC2 locus, we evaluated CD44 as a capture antibody and Cd44v6 together with Cd45 as a negative marker as detection antibodies. Furthermore, to enhance the sensitivity and specificity of isolation and identification of CLCs, we extensively tested various LAM specific, neural crest and melanocytic markers. Specifically, to increase the specificity of the capture we explored several neural crest markers. Since, stem cell precursors that can give rise to smooth muscle and melanocytes have shown to express neural stem cell marker Ng2, we optimized the antibodies against Ng2 as a capture moiety. To enhance the specificity of the detection, we stained CLCs for gp100, a melanocyte-related protein immunorecognized by antibody HMB45. Using this strategy, blood samples from 10 healthy controls and 25 samples from LAM patients are analyzed. The results indicate a clear difference in the levels of CLCs between healthy and patient population. Using this novel biology and technology approach, we were able to detect and identify CLCs was with high specificity and sensitivity. We hypothesize that with the presented strategies, it is feasible to use CLCs as a blood “biopsy”, which may lead not only to elucidating cellular and molecular mechanisms, but also may pave the way to finding cure for LAM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1571. doi:10.1158/1538-7445.AM2011-1571