SMARCB1 Deficiency Research Articles

Primary adenocarcinoma of the spermatic cord: a case report and review of the literature

BackgroundPrimary malignant neoplasms of the spermatic cord are extremely rare, with most reported cases being sarcomas or metastatic carcinomas. However, primary adenocarcinoma of the spermatic cord has not been previously reported.Case presentationA 34-year-old male with a solid mass in the right spermatic cord, was eventually diagnosed with primary adenocarcinoma. Histological examination revealed a moderately-to-poorly differentiated adenocarcinoma exhibiting glandular, cribriform, or nested growth patterns, characterized by medium to large-sized cells and focal extracellular mucus. Immunohistochemical analysis demonstrated positive staining for CK (AE1/AE3), CK8/18, CK19, MOC31 (EP-CAM), and Ber-EP4, while negative staining was observed for CK7, D2-40, WT-1, MC, PAX-8, NKX3.1, PSA, CEA, TTF-1, and NapsinA. Furthermore, a complete loss of INI-1 expression and consistent BRG1 expression were noted in all tumor cells. Next-generation sequencing revealed SMARCB1 deletion, low tumor mutation burden (TMB-L), and microsatellite stability (MSS).ConclusionWe reported the first case of primary adenocarcinoma of the spermatic cord with SMARCB1 (INI-1) deficiency. This case contributes to the expanding understanding of rare neoplasms and underscores the importance of further research into therapeutic strategies targeting SMARCB1-deficient tumors.

Targeting dependency on a paralog pair of CBP/p300 against de-repression of KREMEN2 in SMARCB1-deficient cancers

SMARCB1, a subunit of the SWI/SNF chromatin remodeling complex, is the causative gene of rhabdoid tumors and epithelioid sarcomas. Here, we identify a paralog pair of CBP and p300 as a synthetic lethal target in SMARCB1-deficient cancers by using a dual siRNA screening method based on the “simultaneous inhibition of a paralog pair” concept. Treatment with CBP/p300 dual inhibitors suppresses growth of cell lines and tumor xenografts derived from SMARCB1-deficient cells but not from SMARCB1-proficient cells. SMARCB1-containing SWI/SNF complexes localize with H3K27me3 and its methyltransferase EZH2 at the promotor region of the KREMEN2 locus, resulting in transcriptional downregulation of KREMEN2. By contrast, SMARCB1 deficiency leads to localization of H3K27ac, and recruitment of its acetyltransferases CBP and p300, at the KREMEN2 locus, resulting in transcriptional upregulation of KREMEN2, which cooperates with the SMARCA1 chromatin remodeling complex. Simultaneous inhibition of CBP/p300 leads to transcriptional downregulation of KREMEN2, followed by apoptosis induction via monomerization of KREMEN1 due to a failure to interact with KREMEN2, which suppresses anti-apoptotic signaling pathways. Taken together, our findings indicate that simultaneous inhibitors of CBP/p300 could be promising therapeutic agents for SMARCB1-deficient cancers.

The IL6/JAK/STAT3 signaling axis is a therapeutic vulnerability in SMARCB1-deficient bladder cancer

SMARCB1 loss has long been observed in many solid tumors. However, there is a need to elucidate targetable pathways driving growth and metastasis in SMARCB1-deficient tumors. Here, we demonstrate that SMARCB1 deficiency, defined as genomic SMARCB1 copy number loss associated with reduced mRNA, drives disease progression in patients with bladder cancer by engaging STAT3. SMARCB1 loss increases the chromatin accessibility of the STAT3 locus in vitro. Orthotopically implanted SMARCB1 knockout (KO) cell lines exhibit increased tumor growth and metastasis. SMARCB1-deficient tumors show an increased IL6/JAK/STAT3 signaling axis in in vivo models and patients. Furthermore, a pSTAT3 selective inhibitor, TTI-101, reduces tumor growth in SMARCB1 KO orthotopic cell line-derived xenografts and a SMARCB1-deficient patient derived xenograft model. We have identified a gene signature generated from SMARCB1 KO tumors that predicts SMARCB1 deficiency in patients. Overall, these findings support the clinical evaluation of STAT3 inhibitors for the treatment of SMARCB1-deficient bladder cancer.

10175-GGE-17 MOLECULAR CHARACTERIZATION OF LOW-GRADE DIFFUSELY INFILTRATIVE TUMOR WITH INI1 DEFICIENCY

Abstract Central nervous system low-grade diffusely infiltrative tumor with INI1 deficiency (CNS LGDIT-INI1) is a pathologically low-grade tumor characterized by the infiltrative growth of INI1-deficient tumor cells. Although CNS LGDIT-INI1 is known to progress to an atypical teratoid/rhabdoid (AT/RT)-like tumor, little is yet known about the molecular characteristics distinguishing CNS LGDIT-INI1 from AT/RT. To elucidate the molecular profile of CNS LGDIT-INI1, we performed whole-genome sequencing (WGS), RNA-seq, and DNA methylation array analysis on three cases (1 child and 2 adults) with CNS LGDIT-INI1, including a paired primary-recurrent case. Additionally, 44 publicly available AT/RT cases were analyzed. All the three cases demonstrate homozygous deletions of the SMARCB1 gene, supporting its fundamental role in the tumorigenesis of CNS LGDIT-INI1. The median number of somatic mutations is 1.3/Mb (1.0-2.8) predominantly comprising age-related C-to-T transitions at the CpG site, which is slightly higher than 0.82/Mb in AT/RT, potentially suggesting an association with advanced age of onset. CNS LGDIT-INI1 exhibits a relatively stable genome where broad copy number variations are extremely rare except for the SMARCB1 region, whereas the AT/RT genome shows multiple copy number alterations. Gene set enrichment analysis based on gene expression reveals that CNS LGDIT-INI1 exhibits relatively lower activation in the cell cycle and protein translational activity compared to AT/RT, which is consistent with the low cell proliferation in CNS LGDIT-INI1. The DNA methylation profile of LGDIT resembles that of AT/RT-MYC, suggesting a common cell of origin between CNS LGDIT-INI1 and AT/RT-MYC. In conclusion, CNS LGDIT-INI1 grows based on the loss of SMARCB1, exhibiting a stable genome and low proliferation activity. Considering the similarity of the DNA methylation profile and SMARCB1 deficiency, it is suggested that CNS LGDIT-INI1 may be a precursor of AT/RT.

Primary cilia contribute to the aggressiveness of atypical teratoid/rhabdoid tumors

Atypical teratoid/rhabdoid tumor (AT/RT) is a highly malignant brain tumor in infants that is characterized by loss of nuclear expression of SMARCB1 or SMARCA4 proteins. Recent studies show that AT/RTs comprise three molecular subgroups, namely AT/RT-TYR, AT/RT-MYC and AT/RT-SHH. The subgroups show distinct expression patterns of genes involved in ciliogenesis, however, little is known about the functional roles of primary cilia in the biology of AT/RT. Here, we show that primary cilia are present across all AT/RT subgroups with specific enrichment in AT/RT-TYR patient samples. Furthermore, we demonstrate that primary ciliogenesis contributes to AT/RT biology in vitro and in vivo. Specifically, we observed a significant decrease in proliferation and clonogenicity following disruption of primary ciliogenesis in AT/RT cell line models. Additionally, apoptosis was significantly increased via the induction of STAT1 and DR5 signaling, as detected by proteogenomic profiling. In a Drosophila model of SMARCB1 deficiency, concomitant knockdown of several cilia-associated genes resulted in a substantial shift of the lethal phenotype with more than 20% of flies reaching adulthood. We also found significantly extended survival in an orthotopic xenograft mouse model of AT/RT upon disruption of primary ciliogenesis. Taken together, our findings indicate that primary ciliogenesis or its downstream signaling contributes to the aggressiveness of AT/RT and, therefore, may constitute a novel therapeutic target.

P53 Pathway Inactivation Drives SMARCB1-deficient p53-wildtype Epithelioid Sarcoma Onset Indicating Therapeutic Vulnerability Through MDM2 Inhibition.

Loss of the gene SMARCB1 drives the development of malignant rhabdoid tumors, epithelioid sarcomas, and other malignancies. The SMARCB1 protein is a core component of the SWI/SNF (SWItch/Sucrose Non-Fermentable) family of chromatin remodeling complexes, which are important regulators of gene expression and cell differentiation. Here, we use CRISPR-Cas9 to create germline smarcb1 loss of function in zebrafish. We demonstrate that the combination of smarcb1 deficiency with mutant p53 results in the development of epithelioid sarcomas, angiosarcomas, and carcinomas of the thyroid and colon. Although human epithelioid sarcomas do not frequently harbor p53 mutations, smarcb1-deficient tumors in zebrafish were only observed following disruption of p53, indicating that p53 signaling in human tumors might be attenuated through alternative mechanisms, such as MDM2-mediated proteasomal degradation of p53. To leverage this possibility for the treatment of human epithelioid sarcoma, we tested small molecule-mediated disruption of the p53-MDM2 interaction, which stabilized p53 protein leading to p53-pathway reactivation, cell-cycle arrest, and increased apoptosis. Moreover, we found that MDM2 inhibition and the topoisomerase II inhibitor doxorubicin synergize in targeting epithelioid sarcoma cell viability. This could be especially relevant for patients with epithelioid sarcoma because doxorubicin represents the current gold standard for their clinical treatment. Our results therefore warrant reactivating p53 protein in SMARCB1-deficient, p53-wildtype epithelioid sarcomas using combined doxorubicin and MDM2 inhibitor therapy.

Malignant rhabdoid tumor of the liver in a middle-aged woman: a case report and literature review

BackgroundExtrarenal malignant rhabdoid tumor (EMRT) is a rare and high-mortality malignant tumor, which is more common in infants and rarely seen in adults. We firstly report a case of liver malignant rhabdoid tumor (MRT) with a loss of SMARCB1 gene (alias INI1, SNF5, BAF47) expression in a middle-aged woman, and preliminarily summarize the clinical characteristics and discuss its potential treatment of liver MRT by reviewing 55 cases reported in the past.Case presentationWe report a 40-year-old woman who was admitted to our hospital for right epigastric pain. Previously, the patient was treated with liver hematoma in another hospital until she came to our hospital for abdominal pain again. In our hospital, we performed surgical treatment on her and the pathology diagnosed EMRT with negative expression of SMARCB1. After surgery, the patient underwent genetic testing, but failed to screen for sensitive targeted or conventional chemotherapy drugs, and she did not receive further treatment. Due to lack of timely diagnosis and effective chemotherapy drugs, tumor recurrence and metastasis occurred one year after surgery. Then the patient chose traditional Chinese medicine for treatment. And the metastatic tumors had still progressed after one year of treatment, but the patient didn’t have obvious discomfort symptoms.ConclusionsLiver MRT is a highly aggressive tumor with high metastatic potential and poor prognosis. It lacks specific symptoms and signs and is easy to be ignored and misdiagnosed. The mortality rate is extremely high as there is no effective treatment. But most tumors are accompanied by SMARCB1 deficiency, which may offer new research directions for cancer therapeutics. For the present, early detection, early diagnosis and early resection remain the key to improve the prognosis of patients.

Cytomorphologic Spectrum of SMARCB1-Deficient Soft Tissue Neoplasms.

The SWI/SNF complex core subunit SMARCB1 is inactivated in a variety of neoplasms that share characteristic "rhabdoid" cytomorphology. The aim of this study was to evaluate SMARCB1-deficient soft tissue neoplasms on cytology to identify diagnostic clues. Eleven SMARCB1-deficient tumors, including six epithelioid sarcomas, three malignant rhabdoid tumors, one epithelioid malignant peripheral nerve sheath tumor (MPNST), and one poorly differentiated chordoma with fine-needle aspiration (FNA), serous effusion, or touch prep (TP) from two institutions, were included. Targeted next-generation sequencing (NGS) was performed in two cases. Evaluation of FNA (n = 4), effusion (n = 4), and TP (n = 3) in nine adult and two pediatric patients demonstrated cellular samples (n = 11), epithelioid cells with rhabdoid morphology (n = 9), eccentrically located nuclei with prominent nucleoli (n = 7), and cytoplasmic bodies (n = 4); two patients were diagnosed on FNA with cell block. Immunohistochemistry (IHC) demonstrated SMARCB1 loss in all cases and keratin and/or EMA expression in all but the epithelioid MPNST; NGS identified SMARCB1 inactivation in both cases. SMARCB1-deficient soft tissue neoplasms comprise a variety of tumors with epithelioid morphology and frequent expression of keratin and/or EMA. Recognition of characteristic rhabdoid morphology on cytology can prompt IHC and/or NGS testing for SMARCB1 deficiency and help establish the diagnosis.

Nucleoporin 210 Serves a Key Scaffold for SMARCB1 in Liver Cancer.

The roles of chromatin remodelers and their underlying mechanisms of action in cancer remain unclear. In this study, SMARCB1, known initially as a bona fide tumor suppressor gene, was investigated in liver cancer. SMARCB1 was highly upregulated in patients with liver cancer and was associated with poor prognosis. Loss- and gain-of-function studies in liver cells revealed that SMARCB1 loss led to reduced cell proliferation, wound healing capacity, and tumor growth in vivo. Although upregulated SMARCB1 appeared to contribute to switch/sucrose nonfermentable (SWI/SNF) complex stability and integrity, it did not act using its known pathways antagonism with EZH2 or association between TP53 or AMPK. SMARCB1 knockdown induced a mild reduction in global H3K27 acetylation, and chromatin immunoprecipitation sequencing of SMARCB1 and acetylated histone H3K27 antibodies before and after SMARCB1 loss identified Nucleoporin210 (NUP210) as a critical target of SMARCB1, which bound its enhancer and changed H3K27Ac enrichment and downstream gene expression, particularly cholesterol homeostasis and xenobiotic metabolism. Notably, NUP210 was not only a putative tumor supporter involved in liver cancer but also acted as a key scaffold for SMARCB1 and P300 to chromatin. Furthermore, SMARCB1 deficiency conferred sensitivity to doxorubicin and P300 inhibitor in liver cancer cells. These findings provide insights into mechanisms underlying dysregulation of chromatin remodelers and show novel associations between nucleoporins and chromatin remodelers in cancer. SIGNIFICANCE: This study reveals a novel protumorigenic role for SMARCB1 and describes valuable links between nucleoporins and chromatin remodelers in cancer by identifying NUP210 as a critical coregulator of SMARCB1 chromatin remodeling activity.

SMARCB1-deficient carcinomas of the head and neck region: a cytopathologic characterization

SMARCB1 encodes for a component of the SWI/SNF complex and is widely implicated in carcinogenesis. In the head and neck, SMARCB1-deficient carcinomas typically arise in the sinonasal tract but can be found at other sites. EZH2 inhibitors have emerged as potential targeted therapy against SWI/SNF-deficient tumors. We sought to characterize the cytomorphology of head and neck carcinomas with SMARCB1 deficiencies to identify potential candidates for targeted therapy. Head and neck carcinomas with SMARCB1 mutations were retrospectively identified and confirmed to be SMARCB1-deficient by both molecular (fluorescent in-situ hybridization or next generation sequencing) and immunohistochemical means. Cases with positive cytology were reviewed and their cytologic features cataloged. A total of 19 specimens from 13 patients were reviewed, including 8 specimens from 7 sinonasal carcinomas, 4 specimens from 3 thyroid carcinomas, 3 specimens from 2 skin carcinomas, and 4 specimens from 1 carcinoma of unknown primary origin. High-grade features were common, including mitoses (11 of 19) necrosis (13 of 19) and multinucleation (16 of 19). Tumors showed either dense cytoplasm with distinct cell borders (10 of 19) or delicate cytoplasm with indistinct cell borders (9 of 19). Most tumors showed no distinct epithelial differentiation (12 of 19), while some (7 of 19) showed glandular or signet ring features. A minor cohort demonstrated rhabdoid cells (4 of 19). Head and neck carcinomas with SMARCB1 deficiencies have a wide array of morphologies and tend to demonstrate high-grade features. Only a minor cohort demonstrate rhabdoid-type cells. Evaluation of SMARCB1 deficiency for potential targeted therapy should not be limited to tumors with rhabdoid morphology.

SWI/SNF complex-deficient soft tissue neoplasms: An update.

The SWItch Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complex is a large multi-subunit protein assembly that orchestrates chromatin compaction and accessibility for gene transcription in an ATP-dependent manner. As a key epigenetic regulator, the SWI/SNF complex coordinates gene expression, cell proliferation and differentiation, and its biologic functions, in part, antagonize the polycomb repressive complex 2. The mammalian SWI/SNF complex consists of 15 subunits encoded by 29 genes, some of which are recurrently mutated in human cancers, in the germline or sporadic setting. Most SWI/SNF-deficient tumors share common "rhabdoid" cytomorphology. SMARCB1 (INI1) is the subunit most frequently inactivated in soft tissue neoplasms. Specifically, SMARCB1 deficiency is observed as the genetic hallmark in virtually all malignant rhabdoid tumors, and most cases of epithelioid sarcoma and poorly differentiated chordoma. In addition, subsets of myoepithelial carcinoma (10-40%), extraskeletal myxoid chondrosarcoma (20%), epithelioid schwannoma (40%), and epithelioid malignant peripheral nerve sheath tumor (70%) demonstrate SMARCB1 loss. The gene encoding the SS18 subunit is involved in the SS18-SSX rearrangement, which is pathognomonic of synovial sarcoma and indirectly inactivates SMARCB1. Finally, undifferentiated SMARCA4-deficient thoracic sarcomas are defined by SMARCA4 subunit inactivation, leading to SMARCA4 and SMARCA2 loss. Rarely, inactivation of alternate but biologically equivalent key regulators can substitute for canonical subunit deficiency, such as SMARCA4 inactivation in cases of SMARCB1-retained epithelioid sarcoma. This review briefly highlights SWI/SNF complex biologic functions and its roles in human cancer and provides a detailed update on recent advances in soft tissue neoplasms with canonical SWI/SNF complex deficiency, correlating morphologic, genomic, and immunohistochemical findings.

SMARCB1 Deficiency Integrates Epigenetic Signals to Oncogenic Gene Expression Program Maintenance in Human Acute Myeloid Leukemia.

SWI/SNF is an evolutionarily conserved multi-subunit chromatin remodeling complex that regulates epigenetic architecture and cellular identity. Although SWI/SNF genes are altered in approximately 25% of human malignancies, evidences showing their involvement in tumor cell-autonomous chromatin regulation and transcriptional plasticity are limiting. This study demonstrates that human primary acute myeloid leukemia (AML) cells exhibit near complete loss of SMARCB1 (BAF47 or SNF5/INI1) and SMARCD2 (BAF60B) associated with nucleation of SWI/SNFΔ SMARCC1 (BAF155), an intact core component of SWI/SNFΔ, colocalized with H3K27Ac to target oncogenic loci in primary AML cells. Interestingly, gene ontology (GO) term and pathway analysis suggested that SMARCC1 occupancy was enriched on genes regulating Rac GTPase activation, cell trafficking, and AML-associated transcriptional dysregulation. Transcriptome profiling revealed that expression of these genes is upregulated in primary AML blasts, and loss-of-function studies confirmed transcriptional regulation of Rac GTPase guanine nucleotide exchange factors (GEF) by SMARCB1. Mechanistically, loss of SMARCB1 increased recruitment of SWI/SNFΔ and associated histone acetyltransferases (HAT) to target loci, thereby promoting H3K27Ac and gene expression. Together, SMARCB1 deficiency induced GEFs for Rac GTPase activation and augmented AML cell migration and survival. Collectively, these findings highlight tumor suppressor role of SMARCB1 and illustrate SWI/SNFΔ function in maintaining an oncogenic gene expression program in AML.Implications: Loss of SMARCB1 in AML associates with SWI/SNFΔ nucleation, which in turn promotes Rac GTPase GEF expression, Rac activation, migration, and survival of AML cells, highlighting SWI/SNFΔ downstream signaling as important molecular regulator in AML. Mol Cancer Res; 16(5); 791-804. ©2018 AACR.

Effect of SMARCB1 deficiency in renal medullary carcinoma (RMC) on genes associated with nucleosome assembly and telomere organization.

614 Background: RMC is a highly aggressive tumor with close to universal fatality despite therapy. It is almost exclusively found in young African-Americans with sickle cell trait, and is characterized by complete loss of expression of SMARCB1, a major chromatin remodeler involved in regulation of gene expression. We investigated the effects of SMARCB1 loss on mutation frequency, gene expression, and cell growth in RMC. Methods: Whole exome sequencing (WES) and RNA sequencing (RNA-seq) were performed in RMC tissues from 15 and 11 patients respectively, each with matched adjacent normal kidney tissue controls. In vitro experiments were performed in a cell line (RMC2C) we established from a patient with RMC. SMARCB1 was conditionally re-expressed using a tetracycline-inducible lentivector. Gene ontology (GO) analysis was performed using DAVID. Results: WES showed that RMC harbors a low number (median of < 25/tumor sample) of non-synonymous exomic single nucleotide variants (SNVs) or small indels. GO analysis revealed that the most significant pathways upregulated in RMC compared with normal tissue were those associated with nucleosome assembly and telomere organization (p values < 0.0001). Re-expression of SMARCB1 at near-endogenous levels suppressed the growth rate of RMC2C cells. Subsequent silencing of SMARCB1 expression restored the growth rate of these cells. RNA-seq of RMC2C cells expressing SMARCB1 demonstrated that the most significant downregulated pathways compared with SMARCB1-negative RMC2C cells were those associated with nucleosome assembly and telomere organization (p values < 0.0001). Conclusions: RMC harbors a remarkably simple genome, as evidenced by our WES analysis. Therefore, consistently detected alterations, such as SMARCB1 loss, are likely to serve as drivers for this disease. Indeed, in vitro restoration of SMARCB1 expression suppressed the growth of RMC cells and repressed genes associated with nucleosome assembly and telomere organization, identifying for the first time a causal link between loss of SMARCB1 and dysregulation of these genes. These results provide the basis for future therapeutic strategies targeting SMARCB1 loss in RMC.

Claudin-4 expression distinguishes SWI/SNF complex-deficient undifferentiated carcinomas from sarcomas

Inactivation of SWI/SNF (switch/sucrose non-fermentable) chromatin remodeling complex subunits is a feature shared by select carcinomas and sarcomas with epithelioid morphology and variable keratin expression, making the distinction between carcinoma and sarcoma challenging in some cases. The tight junction-associated protein claudin-4 is a marker of epithelial differentiation that is expressed in nearly all carcinomas. Claudin-4 expression has been reported in the glandular component of biphasic synovial sarcoma but has not been systematically evaluated in other sarcoma types. In this study we assessed claudin-4 expression in SWI/SNF complex-deficient neoplasms showing loss of SMARCB1 (INI1), SMARCA4 (BRG1), or ARID1A and other sarcomas with epithelioid morphology. Immunohistochemistry for claudin-4 was performed on 130 neoplasms, including 90 soft tissue tumors with epithelioid morphology and/or SMARCB1 deficiency (20 epithelioid sarcomas (10 conventional, 10 proximal-type); 10 epithelioid angiosarcomas; 10 epithelioid hemangioendotheliomas; 15 epithelioid malignant peripheral nerve sheath tumors; 10 malignant rhabdoid tumors; 15 myoepithelial carcinomas; 10 biphasic synovial sarcomas), 10 ovarian clear cell carcinomas, 10 ovarian small cell carcinomas of hypercalcemic type, and 20 SWI/SNF complex-deficient undifferentiated carcinomas (14 SMARCB1 deficient and 6 SMARCA4 deficient, including rhabdoid carcinomas of various sites and sinonasal carcinomas). Membranous expression of claudin-4 (≥5% of cells) was observed in all biphasic synovial sarcomas (epithelial component only), all ovarian clear cell carcinomas, and 16 (80%) SWI/SNF complex-deficient undifferentiated carcinomas. All other soft tissue tumors were negative for claudin-4, with the exception of two myoepithelial carcinomas and one malignant rhabdoid tumor. Interestingly, none of the ovarian small cell carcinomas of hypercalcemic type expressed claudin-4. In summary, expression of claudin-4 is highly specific for true epithelial differentiation and may be useful to distinguish SWI/SNF complex-deficient undifferentiated carcinomas from sarcomas with epithelioid morphology. The lack of claudin-4 expression in ovarian small cell carcinomas of hypercalcemic type suggests that these tumors may be better regarded as sarcomas rather than carcinomas.

SMAD dependent signaling plays a detrimental role in a fly model of SMARCB1-deficiency and the biology of atypical teratoid/rhabdoid tumors.

Atypical teratoid/rhabdoid tumors (ATRT) are highly malignant brain tumors arising in young children. The majority of ATRT is characterized by inactivation of the chromatin remodeling complex member SMARCB1 (INI1/hSNF5). Little is known, however, on downstream pathways involved in the detrimental effects of SMARCB1 deficiency which might also represent targets for treatment. Using Drosophila melanogaster and the Gal4-UAS system, modifier screens were performed in order to identify the role of SMAD dependent signaling in the lethal phenotype associated with knockdown of snr1, the fly homolog of SMARCB1. Expression and functional role of human homologs was next investigated in ATRT tumor samples and SMARCB1-deficient rhabdoid tumor cells. The lethal phenotype associated with snr1 knockdown in Drosophila melanogaster could be shifted to later stages of development upon additional knockdown of several decapentaplegic pathway members including Smox, and Med. Similarly, the transforming growth factor beta (TGFbeta) receptor type I kinase inhibitor SB431542 ameliorated the detrimental effect of snr1 knockdown in the fruit fly. Examination of homologs of candidate decapentaplegic pathway members in human SMARCB1-deficent ATRT samples revealed SMAD3 and SMAD6 to be over-expressed. In SMARCB1-deficent rhabdoid tumor cells, siRNA-mediated silencing of SMAD3 or SMAD6 expression reduced TGFbeta signaling activity and resulted in decreased proliferation. Similar results were obtained upon pharmacological inhibition of TGFbeta signaling using SB431542. Our data suggest that SMAD dependent signaling is involved in the detrimental effects of SMARCB1-deficiency and provide a rationale for the investigation of TGFbeta targeted treatments in ATRT.

Abstract A29: Balanced translocations disrupting SMARCB1 are hallmark recurrent genetic alterations in renal medullary carcinomas

Abstract Renal medullary carcinoma (RMC) is a rare and highly aggressive neoplasm that most often occurs in young patients or children with sickle cell trait or disease (SCD). Although BAF47 staining is constantly negative in these tumors, the genetic mechanism underlying this phenotype remains unknown, since all cases reported so far show SMARCB1 hemizygous deletion only. By investigating a series of five RMC by gene expression profiling, array-CGH, and RNA and whole-exome sequencing, we demonstrated that SMARCB1 inactivation is related to an original mechanism of inter-chromosomal balanced translocations that disrupt SMARCB1 sequence and thus contribute to its inactivation. RMC were also characterised by a simple genome, with a low mutation rate and a lack of recurrent molecular alterations other than SMARCB1 deficiency, emphasizing its potency as single genetic event to drive oncogenesis. Finally, gene expression profiling revealed that RMC share common oncogenic pathways with pediatric malignant rhabdoid tumors, another lethal tumor subtype characterised by SMARCB1 deficiency Citation Format: Julien Calderaro, Julien Masliah-Planchon, Wilfrid Richer, Laetitia Planque, Pascale Maille, Alexandre de la Taille, Helene Boussion, Olivier Delattre, Yves Allory, Franck Bourdeaut. Balanced translocations disrupting SMARCB1 are hallmark recurrent genetic alterations in renal medullary carcinomas. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A29.

Balanced Translocations Disrupting SMARCB1 Are Hallmark Recurrent Genetic Alterations in Renal Medullary Carcinomas

BackgroundRenal medullary carcinoma (RMC) is a rare and highly aggressive neoplasm that most often occurs in the setting of sickle cell trait or sickle cell disease (SCD). Most patients present with metastatic disease resistant to conventional chemotherapy, and therefore there is an urgent need for molecular insight to propose new therapies. ObjectiveTo determine the molecular alterations and oncogenic pathways that drive RMC development. Design, setting, and participantsA series of five frozen samples of patients with RMC was investigated by means of gene expression profiling, array comparative genomic hybridization, and RNA and whole exome sequencing (WES). Outcome measurements and statistical analysisRNA and DNA sequencing read data were analyzed to detect gene fusions and somatic mutations. Gene fusions mutations were validated by real-time polymerase chain reaction and fluorescence in situ hybridization. Gene expression profiling was analyzed by unsupervised hierarchical clustering and Gene Set Enrichment Analysis (Broad Institute, Cambridge, MA, USA). Results and limitationsWe observed inactivation of the tumor suppressor gene SMARCB1 in all tumors. In all four cases developed in patients with SCD, we identified an original mechanism of interchromosomal balanced translocations that disrupt the SMARCB1 sequence and thus contribute to its inactivation. Gene expression profiling revealed that RMC shares common oncogenic pathways with pediatric malignant rhabdoid tumors, another tumor subtype characterized by SMARCB1 deficiency. ConclusionsRMCs are characterized by an original mechanism of interchromosomal balanced translocations that disrupt the SMARCB1 sequence. WES reveals that RMCs show no other recurrent genetic alteration and an overall stable genome, underscoring the oncogenic potency of SMARCB1 inactivation. Patient summaryOur comprehensive molecular study supports a pivotal role of the tumor suppressor gene SMARCB1 in the development of renal medullary carcinoma. The use of therapeutic strategies based on the biologic effects of its inactivation should now open new perspectives for this typically lethal malignancy.

Identification of genes involved in the biology of atypical teratoid/rhabdoid tumours using Drosophila melanogaster

Atypical teratoid/rhabdoid tumours (AT/RT) are malignant brain tumours. Unlike most other human brain tumours, AT/RT are characterized by inactivation of one single gene, SMARCB1. SMARCB1 is a member of the evolutionarily conserved SWI/SNF chromatin remodelling complex, which has an important role in the control of cell differentiation and proliferation. Little is known, however, about the pathways involved in the oncogenic effects of SMARCB1 inactivation, which might also represent targets for treatment. Here we report a comprehensive genetic screen in the fruit fly that revealed several genes not yet associated with loss of snr1, the Drosophila homologue of SMARCB1. We confirm the functional role of identified genes (including merlin, kibra and expanded, known to regulate hippo signalling pathway activity) in human rhabdoid tumour cell lines and AT/RT tumour samples. These results demonstrate that fly models can be employed for the identification of clinically relevant pathways in human cancer.

SMARCB1 Deficiency in Tumors From the Peripheral Nervous System

Inactivation of SMARCB1 tumor-suppressor gene was originally described as highly specific for rhabdoid tumors (RTs). Nevertheless, recent reports have illustrated that SMARCB1 alterations also characterize other tumors; in particular, some familial schwannomatosis and epithelioid malignant peripheral nerve sheath tumors, both from peripheral nervous system (PNS) origin, lack BAF47 expression. To document the putative role of SMARCB1 in PNS, we reviewed PNS tumors referred to our institution for a molecular analysis of SMARCB1 because of histologic features compatible with RT. Clinicopathologic, radiologic, and molecular characteristics were detailed for the 12 cases showing loss of expression and/or biallelic inactivation of SMARCB1. The status of the NF2 gene, likely to synergize with SMARCB1 in PNS tumors, was also analyzed. Patients' age ranged from 0 to 45 years (median age, 6.6 y). Neurological symptoms were observed in 7/12 cases with radiologic features evoking a neuroblastic tumor in 6 cases and a peripheral nerve tumor in 4 cases. The mean delay before diagnosis was 3 months. Histologic examination revealed rhabdoid features in 11/12 tumors. All tumors showed a complete loss of SMARCB1 expression. Interestingly, adjacent nervous proliferation resembling neurofibromas were observed in 3 cases, suggesting a multistep transformation. Three tumors harbored a hemizygous deletion at the NF2 locus, but all NF2 sequences were normal. We report the first series of PNS RT. In patients with aggressive PNS tumors, RT should be suspected, and anti-SMARCB1 immunohistochemical analysis should be performed. SMARCB1 inactivation, occasionally associated with NF2 deletion, might have oncogenic effects in peripheral nerves.