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Supp Tables S1-S6 (including variant classification, bioinformatics predictions versus experimental data, primer sequences, summary of biological, clinical, tumoral, familial and multifactorial data, and description of statistical analysis approaches)

BRCA2 is a clinically actionable gene implicated in breast and ovarian cancer predisposition that has become a high priority target for improving the classification of variants of unknown significance (VUS). Among all BRCA2 VUS, those causing partial/leaky splicing defects are the most challenging to classify because the minimal level of full-length (FL) transcripts required for normal function remains to be established. Here, we explored BRCA2 exon 3 (BRCA2e3) as a model for calibrating variant-induced spliceogenicity and estimating thresholds for BRCA2 haploinsufficiency. In silico predictions, minigene splicing assays, patients' RNA analyses, a mouse embryonic stem cell (mESC) complementation assay and retrieval of patient-related information were combined to determine the minimal requirement of FL BRCA2 transcripts. Of 100 BRCA2e3 variants tested in the minigene assay, 64 were found to be spliceogenic, causing mild to severe RNA defects. Splicing defects were also confirmed in patients' RNA when available. Analysis of a neutral leaky variant (c.231T>G) showed that a reduction of approximately 60% of FL BRCA2 transcripts from a mutant allele does not cause any increase in cancer risk. Moreover, data obtained from mESCs suggest that variants causing a decline in FL BRCA2 with approximately 30% of wild-type are not pathogenic, given that mESCs are fully viable and resistant to DNA-damaging agents in those conditions. In contrast, mESCs producing lower relative amounts of FL BRCA2 exhibited either null or hypomorphic phenotypes. Overall, our findings are likely to have broader implications on the interpretation of BRCA2 variants affecting the splicing pattern of other essential exons.Significance:These findings demonstrate that BRCA2 tumor suppressor function tolerates substantial reduction in full-length transcripts, helping to determine the pathogenicity of BRCA2 leaky splicing variants, some of which may not increase cancer risk.

Sequencing-based genetic tests to identify individuals at increased risk of hereditary breast and ovarian cancers have resulted in the identification of more than 40,000 sequence variants of BRCA1 and BRCA2. A majority of these variants are considered to be variants of uncertain significance (VUS) because their impact on disease risk remains unknown, largely due to lack of sufficient familial linkage and epidemiological data. Several assays have been developed to examine the effect of VUS on protein function, which can be used to assess their impact on cancer susceptibility. In this study, we report the functional characterization of 88 BRCA2 variants, including several previously uncharacterized variants, using a well-established mouse embryonic stem cell (mESC)-based assay. We have examined their ability to rescue the lethality of Brca2 null mESC as well as sensitivity to six DNA damaging agents including ionizing radiation and a PARP inhibitor. We have also examined the impact of BRCA2 variants on splicing. In addition, we have developed a computational model to determine the probability of impact on function of the variants that can be used for risk assessment. In contrast to the previous VarCall models that are based on a single functional assay, we have developed a new platform to analyze the data from multiple functional assays separately and in combination. We have validated our VarCall models using 12 known pathogenic and 10 neutral variants and demonstrated their usefulness in determining the pathogenicity of BRCA2 variants that are listed as VUS or as variants with conflicting functional interpretation.

Stenotrophomonas maltophilia is a Gram-negative bacterium known to cause respiratory tract infections and other diseases in humans. Here, we describe the isolation and genome annotation of S. maltophilia siphophage Suzuki. Its 56,042-bp genome has 83 predicted protein-coding genes and demonstrates similarity with Xylella phages Sano and Salvo.

BRCA2 is a clinically actionable gene implicated in breast and ovarian cancer predisposition that has become a high priority target for improving the classification of variants of unknown significance (VUS). Among all BRCA2 VUS, those causing partial/leaky splicing defects are the most challenging to classify because the minimal level of full-length (FL) transcripts required for normal function remains to be established. Here, we explored BRCA2 exon 3 (BRCA2e3) as a model for calibrating variant-induced spliceogenicity and estimating thresholds for BRCA2 haploinsufficiency. In silico predictions, minigene splicing assays, patients' RNA analyses, a mouse embryonic stem cell (mESC) complementation assay and retrieval of patient-related information were combined to determine the minimal requirement of FL BRCA2 transcripts. Of 100 BRCA2e3 variants tested in the minigene assay, 64 were found to be spliceogenic, causing mild to severe RNA defects. Splicing defects were also confirmed in patients' RNA when available. Analysis of a neutral leaky variant (c.231T>G) showed that a reduction of approximately 60% of FL BRCA2 transcripts from a mutant allele does not cause any increase in cancer risk. Moreover, data obtained from mESCs suggest that variants causing a decline in FL BRCA2 with approximately 30% of wild-type are not pathogenic, given that mESCs are fully viable and resistant to DNA-damaging agents in those conditions. In contrast, mESCs producing lower relative amounts of FL BRCA2 exhibited either null or hypomorphic phenotypes. Overall, our findings are likely to have broader implications on the interpretation of BRCA2 variants affecting the splicing pattern of other essential exons. Significance: These findings demonstrate that BRCA2 tumor suppressor function tolerates substantial reduction in full-length transcripts, helping to determine the pathogenicity of BRCA2 leaky splicing variants, some of which may not increase cancer risk.

Next-generation sequencing of Sri Lankan families with inherited cancer syndromes resulted in the identification of five BRCA2 variants of unknown clinical significance. Interpreting such variants poses significant challenges for both clinicians and patients. Using a mouse embryonic stem cell-based functional assay, we found I785V, N830D, and K2077N to be functionally indistinguishable from wild-type BRCA2. Specific but mild sensitivity to olaparib and reduction in homologous recombination (HR) efficiency suggest partial loss of function of the A262T variant. This variant is located in the N-terminal DNA binding domain of BRCA2 that can facilitate HR by binding to dsDNA/ssDNA junctions. P3039P is clearly pathogenic because of premature protein truncation caused by exon 23 skipping. These findings highlight the value of mouse embryonic stem cell-based assays for determining the functional significance of variants of unknown clinical significance and provide valuable information regarding risk estimation and genetic counseling of families carrying these BRCA2 variants.

Poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) olaparib has been approved for treatment of advanced ovarian cancer associated with BRCA1 and BRCA2 mutations. BRCA1- and BRCA2-mutated cells, which are homologous recombination (HR) deficient, are hypersensitive to PARPi through the mechanism of synthetic lethality. Here we examine the effect of PARPi on HR-proficient cells. Olaparib pretreatment, PARP1 knockdown or Parp1 heterozygosity of Brca2cko/ko mouse embryonic stem cells (mESCs), carrying a null (ko) and a conditional (cko) allele of Brca2, results in viable Brca2ko/ko cells. PARP1 deficiency does not restore HR in Brca2ko/ko cells, but protects stalled replication forks from MRE11-mediated degradation through its impaired recruitment. The functional consequence of Parp1 heterozygosity on BRCA2 loss is demonstrated by a significant increase in tumorigenesis in Brca2cko/cko mice. Thus, while olaparib efficiently kills BRCA2-deficient cells, we demonstrate that it can also contribute to the synthetic viability if PARP is inhibited before BRCA2 loss., The PARP inhibitor olaparib is an approved treatment method for women with BRCA1 and BRCA2 mutation associated cancers. Here the authors show that olaparib can contribute to synthetic viability of cells if PARP1 is inhibited before BRCA2 loss.

Topical anesthetics are found in a variety of prescription and non-prescription preparations, from teething gels to hemorrhoid creams. In 2003, there were 8576 exposures to local/topical anesthetics reported to the American Association of Poison Control Centers, with 67% of cases in the age group younger than 6 years old. This report reviews the available literature involving topical anesthetic exposures in children younger than 6 years old, including the National Library of Medicine's Pub Med database (limited to English language) and data from POISINDEX. Additionally, we reviewed the American Association of Poison Control Centers' annual reports from 1983 to 2003. There were 7 deaths in this age range from topical anesthetics. Although the number of deaths is low, the fact that there have been deaths reveals the serious nature of the toxicity that can result from these readily available non-prescription analgesics. Toxicity may result from topical absorption, ingestion, or aspiration. Additionally, toxicity can result from unintentional as well as therapeutic mishaps. Although the number of cases is limited, these medications can be toxic at low doses-which, in children younger than 6 years of age, may amount to as little as a teaspoon.

Integral proteins of the nuclear envelope inner membrane have been proposed to reach their sites by diffusion after their co-translational insertion in the rough endoplasmic reticulum. They are then retained in the inner nuclear membrane by binding to nuclear structures. One such structure is the nuclear lamina, an intermediate filament meshwork composed of A-type and B-type lamin proteins. Emerin, MAN1, and LBR are three integral inner nuclear membrane proteins. We expressed these proteins fused to green fluorescent protein in embryonic fibroblasts from wild-type mice and Lmna -/- mice, which lack A-type lamins. We then studied the diffusional mobilities of emerin, MAN1, and LBR using fluorescence recovery after photobleaching. We show that emerin and MAN1, but not LBR, are more mobile in the inner nuclear membrane of cells from Lmna -/- mice than in cells from wild-type mice. In cells from Lmna -/- mice expressing exogenous lamin A, the protein mobilities were similar to those in cells from wild-type mice. This supports a model where emerin and MAN1 are at least partly retained in the inner nuclear membrane by binding to A-type lamins, while LBR depends on other binding partners for its retention.

Membrane blebbing during the apoptotic execution phase results from caspase-mediated cleavage and activation of ROCK I. Here, we show that ROCK activity, myosin light chain (MLC) phosphorylation, MLC ATPase activity, and an intact actin cytoskeleton, but not microtubular cytoskeleton, are required for disruption of nuclear integrity during apoptosis. Inhibition of ROCK or MLC ATPase activity, which protect apoptotic nuclear integrity, does not affect caspase-mediated degradation of nuclear proteins such as lamins A, B1, or C. The conditional activation of ROCK I was sufficient to tear apart nuclei in lamin A/C null fibroblasts, but not in wild-type fibroblasts. Thus, apoptotic nuclear disintegration requires actin-myosin contractile force and lamin proteolysis, making apoptosis analogous to, but distinct from, mitosis where nuclear disintegration results from microtubule-based forces and from lamin phosphorylation and depolymerization.

Mutations in the lamin A/C gene (LMNA) cause a variety of human diseases including Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy, and Hutchinson-Gilford progeria syndrome. The tissue-specific effects of lamin mutations are unclear, in part because the function of lamin A/C is incompletely defined, but the many muscle-specific phenotypes suggest that defective lamin A/C could increase cellular mechanical sensitivity. To investigate the role of lamin A/C in mechanotransduction, we subjected lamin A/C–deficient mouse embryo fibroblasts to mechanical strain and measured nuclear mechanical properties and strain-induced signaling. We found that Lmna–/– cells have increased nuclear deformation, defective mechanotransduction, and impaired viability under mechanical strain. NF-κB–regulated transcription in response to mechanical or cytokine stimulation was attenuated in Lmna–/– cells despite increased transcription factor binding. Lamin A/C deficiency is thus associated with both defective nuclear mechanics and impaired mechanically activated gene transcription. These findings suggest that the tissue-specific effects of lamin A/C mutations observed in the laminopathies may arise from varying degrees of impaired nuclear mechanics and transcriptional activation.

Patients with pituitary tumors frequently complain of quality of life problems despite normal hormonal blood levels. The goals of the study were to determine whether the sickness-related quality of life reported by these patients was poorer than that expected by the general population and whether some of these complaints and areas of dysfunction were more troublesome than others. Forty-three patients from a southeast Michigan pituitary disorders support group volunteered to complete a survey focusing on demographic, disease data and common complaints plus the Sickness Impact Profile (SIP). The results indicated that mental and physical fatigue were the most troublesome symptoms with sleep and libidinal complaints also of significant concern. The SIP indicated significant discontent among patients with regard to overall quality of life. Although this observational investigation seemed to underscore the fact that patients with pituitary tumors have a decreased quality of life despite apparently normal hormonal status, further subgroup analysis and subsequent surveys will be required to determine the actual significance of these findings.

Thymic epithelial cells (TEC), as part of thymic stroma, provide essential growth factors/cytokines and self-antigens to support T cell development and selection. Deletion of Rb family proteins in adult thymic stroma leads to T cell hyperplasia in vivo. To determine whether deletion of Rb specifically in keratin (K) 18 positive TEC was sufficient for thymocyte hyperplasia, we conditionally inactivated Rb and its family members p107 and p130 in K18+ TEC in genetically engineered mice (TgK18GT121; K18 mice). We found that thymocyte hyperproliferation was induced in mice with Rb inactivation in K18+ TEC, while normal T cell development was maintained; suggesting that inactivation of Rb specifically in K18+ TEC was sufficient and responsible for the phenotype. Transplantation of wild type bone marrow cells into mice with Rb inactivation in K18+ TEC resulted in donor T lymphocyte hyperplasia confirming the non-cell autonomous requirement for Rb proteins in K18+ TEC in regulating T cell proliferation. Our data suggests that thymic epithelial cells play an important role in regulating lymphoid proliferation and thymus size.

The nuclear lamina is a protein meshwork lining the nucleoplasmic face of the inner nuclear membrane and represents an important determinant of interphase nuclear architecture. Its major components are the A- and B-type lamins. Whereas B-type lamins are found in all mammalian cells, A-type lamin expression is developmentally regulated. In the mouse, A-type lamins do not appear until midway through embryonic development, suggesting that these proteins may be involved in the regulation of terminal differentiation. Here we show that mice lacking A-type lamins develop to term with no overt abnormalities. However, their postnatal growth is severely retarded and is characterized by the appearance of muscular dystrophy. This phenotype is associated with ultrastructural perturbations to the nuclear envelope. These include the mislocalization of emerin, an inner nuclear membrane protein, defects in which are implicated in Emery-Dreifuss muscular dystrophy (EDMD), one of the three major X-linked dystrophies. Mice lacking the A-type lamins exhibit tissue-specific alterations to their nuclear envelope integrity and emerin distribution. In skeletal and cardiac muscles, this is manifest as a dystrophic condition related to EDMD.

A-type lamins are intermediate filament proteins that provide a scaffold for protein complexes regulating nuclear structure and function. Mutations in the LMNA gene are linked to a variety of degenerative disorders termed laminopathies, whereas changes in the expression of lamins are associated with tumourigenesis. The molecular pathways affected by alterations of A-type lamins and how they contribute to disease are poorly understood. Here, we show that A-type lamins have a key role in the maintenance of telomere structure, length and function, and in the stabilization of 53BP1, a component of the DNA damage response (DDR) pathway. Loss of A-type lamins alters the nuclear distribution of telomeres and results in telomere shortening, defects in telomeric heterochromatin, and increased genomic instability. In addition, A-type lamins are necessary for the processing of dysfunctional telomeres by non-homologous end joining, putatively through stabilization of 53BP1. This study shows new functions for A-type lamins in the maintenance of genomic integrity, and suggests that alterations of telomere biology and defects in DDR contribute to the pathogenesis of lamin-related diseases.

Lamins A/C, the major constituent of the nuclear lamina, confer mechanical stability to nuclei. We examined the myonuclei of LMNA null mice at the myotendinous junctions (MTJ), the site of longitudinal force transmission from contractile proteins to extracellular proteins. The right soleus and rectus femoris muscles of five null mutants aged 5-7 weeks and two wild-type animals aged 5 weeks and 6 months were examined by electron microscopy. The myofibers merging into the tendons were assessed for nuclear disintegration and cytoplasmic degeneration. The myofibers of the wild-type rectus femoris and soleus muscles revealed 19-27 nuclei/50 myofibers and 5-8/20, respectively, with no signs of degeneration. The rectus femoris muscle fibers of the null mice contained 75-117 myonuclei/50 myofibers, the soleus muscle, 13-36 nuclei/20 myofibers. Eleven to twenty-one per 50 myonuclei of the rectus femoris myonuclei showed chromatin clumping, nuclear fragmentation, nuclear inclusions and invaginations, and intranuclear filaments. The values were 12-19/50 for the soleus myonuclei. Moreover, 5-12/50 rectus femoris myofibers and 5-14/20, of the soleus myofibers showed cytoplasmic degeneration. None of these changes was found distal to the MTJ. These results favor the notion that myonuclei lacking a functional lamina are susceptible to mechanical stress in vivo. These alterations may contribute to the development of early joint contractures, a feature of ADEDMD.

Nuclear lamins are major architectural elements of the mammalian cell nucleus, and they have been implicated in the functional organization of the nuclear interior, possibly by providing structural support for nuclear compartments. Colocalization studies have suggested a structural role for lamins in the formation and maintenance of pre-mRNA splicing factor compartments. Here, we have directly tested this hypothesis by analysis of embryonic fibroblasts from knock-out mice lacking A- and C-type lamins. We show that the morphology and cellular properties of splicing factor compartments are independent of A- and C-type lamins. Genetic loss of lamins A/C has no effect on the cellular distribution of several pre-mRNA splicing factors and does not affect the compartment morphology as examined by light and electron microscopy. The association of splicing factors with the nuclear matrix fraction persists in the absence of lamins A/C. Live cell microscopy demonstrates that the intranuclear positional stability of splicing factor compartments is maintained and that the exchange dynamics of SF2/ASF between the compartments and the nucleoplasm is not affected by loss of lamin A/C. Our results demonstrate that formation and maintenance of intranuclear splicing factor compartments is independent of lamins A/C, and they argue against an essential structural role of lamins A/C in splicing factor compartment morphology.

Numerous studies of the underlying causes of ageing have been attempted by examining diseases associated with premature ageing, such as Werner's syndrome and Hutchinson–Gilford progeria syndrome (HGPS). HGPS is a rare genetic disorder resulting in phenotypes suggestive of accelerated ageing, including shortened stature, craniofacial disproportion, very thin skin, alopecia and osteoporosis, with death in the early teens predominantly due to atherosclerosis1. However, recent reports suggest that developmental abnormalities may also be important in HGPS1,2. Here we describe the derivation of mice carrying an autosomal recessive mutation in the lamin A gene (Lmna) encoding A-type lamins, major components of the nuclear lamina3. Homozygous mice display defects consistent with HGPS, including a marked reduction in growth rate and death by 4 weeks of age. Pathologies in bone, muscle and skin are also consistent with progeria. The Lmna mutation resulted in nuclear morphology defects and decreased lifespan of homozygous fibroblasts, suggesting premature cell death. Here we present a mouse model for progeria that may elucidate mechanisms of ageing and development in certain tissue types, especially those developing from the mesenchymal cell lineage.

Dunnigan's Familial Partial Lipodystrophy (FPLD) is an autosomal dominant disease characterized by regional fat loss and insulin resistance. FPLD is caused by mutations in the LMNA gene, which encodes intermediate filaments of the nuclear lamina. Different LMNA mutations cause Emery-Dreifuss muscular dystrophy and/or a dilated cardiomyopathy. It is not known how LMNA mutations cause any of the disease phenotypes. Here we measure physical and metabolic characteristics of Lmna-/- and +/- mice to determine their usefulness as models for FPLD. Lmna-/- mice, which die prematurely of muscular dystrophy, have little fat, but do not show the insulin resistance characteristic of FPLD. Lmna+/- mice, despite treatment with a high fat diet, do not have decreased fat stores or metabolic features of FPLD. We also show, in mice, that Lmna transcripts are expressed at high levels in muscle and adipose tissue, but do not vary by body region or sex. In conclusion, Lmna+/- and -/- mice do not mimic Dunnigan's FPLD, and differential expression of lamins A and C does not appear to contribute to sex- or tissue-specific LMNA phenotypes.

AUR is a commonly seen genitourinary emergency. It has many etiologies, including obstructive, neurogenic, pharmacologic, and extraurinary causes. Treatment is immediate bladder decompression by transurethral catheterization and treatment of the provoking etiology. Urinary incontinence is less commonly seen as a presenting complaint in the ED. For the emergency physician, the key lies in recognizing its underlying cause. Neurologic and pharmacologic causes need to be considered in all patients. Urinary incontinence that is not caused by a neurologic emergency can be referred for further outpatient evaluation.

Local and distant invasion is a hallmark property of glioblastoma (GBM) that prevents curative surgical resection, and may be the key reason for GBM recurrence. Although cancer stem cells (CSCs) have been suggested to cause tumor recurrence based on their resistance to radiotherapy and chemotherapy, it is not clear whether CSCs are also involved in GBM invasiveness. We hypothesize that the invading cells in GBM have stem-like properties. To test our hypothesis, we propose to characterize cells at the invasive front of GBM using a syngeneic intracranial transplant tumor model. Three core regulatory networks are dysregulated in most human GBMs, including the RTK/RAS/PI3K/PTEN, INK/Cyclin D/CDK/RB, and ARF/p53 pathways. We have previously generated a genetically engineered mouse (GEM) model for GBM development via adult-inducible astrocyte-specific perturbation of RB, K-RAS and PTEN. These mice develop astrocytomas that progress to high grade GBM, and faithfully recapitulate the histopathology and molecular characteristics of most human GBMs. We have isolated primary tumor cells from these mice that are maintained in serum-free neural stem cell culture medium and expanded as neurospheres. These primary and low-passage cultures contain a mixed population of cancer stem cells and progenitor cells, and are highly invasive in vitro by matrigel invasion assay. With > 90% penetrance, intracranial injection of 25,000 cells leads to invasive GBM formation and a 32-day median survival in syngeneic host mice. Thus, the model provides an efficient approach for dissecting GBM cell invasion mechanisms in vivo. We will further trace the migration/invasion of GBM cells in the host brain after labeling with lentiviral-expressed mCherry. Host blood vessels are labeled with YFP via Tie2-Cre endothelial expression of a Rosa26-YFP allele. A time course analysis of stem cell marker expression will be used to assess properties of invading cells at the diffusive tumor margin. Laser capture microdissection will be used for invasive rim-cell and non-invasive core-cell isolation, and RNA samples will be compared by unbiased transcriptome studies. Results will provide hypotheses for invasion mechanisms and may identify potential biomarkers for invasive cells. Citation Format: Nailing Zhang, Yurong Song, Sanaz Jansen, Amit Adhikari, Sophie Wang, Lucy Lu, Teresa Sullivan, Terry Van Dyke. Characterization of the invading cells in glioblastoma using a syngeneic intracranial transplant tumor model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2626. doi:10.1158/1538-7445.AM2013-2626

Astrocytomas are the most common malignant brain tumors which maintain a poor survival rate despite decades of research effort. Understanding the initiation process and identifying astrocytoma cell(s) of origin have been of immense interest. Genetically engineered mouse models (GEMMs) are ideal for studying the initiation process compared to evolved human tumor cells. Our lab has developed an adult inducible GEMM that alters key human glioblastoma (Grade IV) pathways. Inactivation of Rb tumor suppression (TS) by expressing N-terminal 121aa of large T antigen (T121) under GFAP promoter is sufficient to initiate grade II astrocytoma. Addition of mutant KrasG12D leads to grade III and further PTEN deletion to glioblastomas. KrasG12D activation and PTEN deletion alone or in combination failed to initiate astrocytomas, suggesting that Rb TS inactivation is essential for its initiation. Studies from several cancers have identified tumor-initiating cells, a subset of tumor cells with stem cell-like properties responsible for generating the entire tumor mass. Their origin remains a mystery. We hypothesize that Rb TS inactivation reprograms astrocytes into stem/progenitor-like state generating potential cell(s) of origin for astrocytoma. Immunostaining of progenitor and proliferation markers (e.g. nestin and Ki-67) showed their co-expression in the cortex with T121, suggesting a possible reprogramming of mature astrocytes. Moreover, astrocyte differentiation marker S100beta was downregulated in T121 cells. To rule out the possibility of migration resulting in the observed cortical progenitor cells, focal induction of T121 in cortex using lenti-cre injection was performed. We found expression of Ki-67 and nestin and reduced expression of S100beta, suggesting that the reprogramming was independent of the germinal zones in the brain. Furthermore, cortical T121 expressing astrocytes were able to generate neurospheres but astrocytes from wildtype cortex failed to do so. These cells also showed self-renewability and multilineage ability, emphasizing their dedifferentiated status upon Rb-TS inactivation. We were able to achieve in-vitro reprogramming of mature astrocytes upon T121 expression suggesting that the reprogramming does not require brain microenvironment. Sphere assays using cortical cells from mice where individual Rb family member was inactivated suggests that loss of individual Rb family members alone is insufficient in reprogramming the mature astrocytes. With the Rb pathway known to be altered in more than 75% of glioblastomas, our results are a step in the direction of unraveling the process of tumor initiation for this deadly disease. Citation Format: Amit S. Adhikari, Teresa Sullivan, Yurong Song, Xiuyun Lu, Norene O'Sullivan, Terry Van Dyke. Reprogramming of mature astrocytes by abrogation of Rb tumor suppression leads to astrocytoma initiation in GEMMs. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5014. doi:10.1158/1538-7445.AM2013-5014

Astrocytomas are the most common primary human malignant brain tumors. Despite standard-of-care surgery, radiation, and chemotherapy, no curative treatment for these devastating diseases is currently available. Tumor-initiating cells (TICs), a small population of tumor cells is shown to be responsible for astrocytoma initiation and progression. Though their presence has been convincingly shown, there is considerable debate about their origin. By using genetically engineered mice, we hypothesis that loss of Rb family, induces dedifferentiation of mature astrocytes which are capable of evolving into TICs. Our lab has developed an adult mouse model where inactivation of retinoblastoma (Rb) family proteins by expressing N-terminal 121aa of large T antigen (T121), initiates grade II astrocytoma. pRb inactivation and mutant KrasG12D activation leads to grade III astrocytoma and further PTEN inactivation results in Grade IV glioblastomas. We have utilized the Rb family inactivation model to investigate the dedifferentiation of mature astrocytes. We have performed IHC using progenitor markers, Sox2 and nestin and a proliferation marker Ki67 and observe the presence of these markers coinciding with T121 expression suggesting a possible dedifferentiation of mature astrocytes. Quantitative analysis of the overlap between Sox2 and Ki-67 with T121 indicates that the marker presence is specific to T121 expression. In addition to these markers, we also show that S100α an astrocyte differentiation marker is downregulated in pRb inactivated cells further supporting the hypothesis of possible dedifferentiation of mature astrocytes. Since T121 expression is induced in all GFAP expressing cells, there is a possibility that the GFAP expressing cells in the germinal areas such as SVZ and dentate gyrus could be migrating into the cortex and form the observed progenitor cells. To address this issue we used lenti-Cre to focally induce T121 expression in the cortex. Sox2 expression, along with the reduced expression of differentiation marker S100α in these cells, suggests that the dedifferentiation events are independent of the germinal zones in the adult brain. As a functional assay to identify progenitor/ stem cells we performed neurosphere assay. In our assay conditions, wildtype adult cortex was unable to generate any spheres compared to T121 expressing cells. We found that the longer the post induction time the higher the sphere forming ability of T121 cells. The multilineage ability of these T spheres cells was confirmed by inducing differentiation into different neural lineages. To test the tumor forming ability of the sphere cells, we have performed subcutaneous injections and awaiting the results. Thus we conclude that in our system inactivation of Rb family members induced dedifferentiation in mature astrocytes. 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 3346. doi:1538-7445.AM2012-3346

The discovery of high-risk breast cancer susceptibility genes, such as Breast cancer associated gene 1 (BRCA1) and Breast cancer associated gene 2 (BRCA2) has led to accurate identification of individuals for risk management and targeted therapy. The rapid decline in sequencing costs has tremendously increased the number of individuals who are undergoing genetic testing world-wide. However, given the significant differences in population-specific variants, interpreting the results of these tests can be challenging especially for novel genetic variants in understudied populations. Here we report the characterization of novel variants in the Malaysian and Singaporean population that consist of different ethnic groups (Malays, Chinese, Indian, and other indigenous groups). We have evaluated the functional significance of 14 BRCA2 variants of uncertain clinical significance by using multiple in silico prediction tools and examined their frequency in a cohort of 7840 breast cancer cases and 7928 healthy controls. In addition, we have used a mouse embryonic stem cell (mESC)-based functional assay to assess the impact of these variants on BRCA2 function. We found these variants to be functionally indistinguishable from wild-type BRCA2. These variants could fully rescue the lethality of Brca2-null mESCs and exhibited no sensitivity to six different DNA damaging agents including a poly ADP ribose polymerase inhibitor. Our findings strongly suggest that all 14 evaluated variants are functionally neutral. Our findings should be valuable in risk assessment of individuals carrying these variants.

Nuclear lamin A and C alleles that are linked to three distinct human diseases have been expressed both in HeLa cells and in fibroblasts derived from Lmna null mice. Point mutations that cause dilated cardiomyopathy (L85R and N195K) and autosomal dominant Emery-Dreifuss muscular dystrophy (L530P) modify the assembly properties of lamins A and C and cause partial mislocalization of emerin, an inner nuclear membrane protein, in HeLa cells. At the same time, these mutant lamins interfere with the targeting and assembly of endogenous lamins and in this way may cause significant changes in the molecular organization of the nuclear periphery. By contrast, lamin A and C molecules harboring a point mutation (R482W), which gives rise to a dominant form of familial partial lipodystrophy, behave in a manner that is indistinguishable from wild-type lamins A and C, at least with respect to targeting and assembly within the nuclear lamina. Taken together, these results suggest that nuclear structural defects could contribute to the etiology of both dilated cardiomyopathy and autosomal dominant Emery-Dreifuss muscular dystrophy.