Malignant Rhabdoid Tumor Cell Lines Research Articles

HER2 expression-independent antitumor effect of trastuzumab deruxtecan (T-DXd) on pediatric solid tumors.

e15016 Background: Treatment of pediatric solid tumors shows limited improvement even with intensification of conventional chemotherapy, suggesting novel approaches such as targeted therapies are needed. Trastuzumab Deruxtecan (T-DXd), a HER2-targeting antibody–drug conjugate, exhibits significant anti-cancer activity in breast cancers with a broad range of HER2 expression. T-DXd might be a promising agent for refractory or relapsed pediatric solid tumors, but its efficacy in pediatric tumors needs to be studied. Methods: Anti-cancer efficacy of T-DXd and its payload DXd were assessed in vitro by utilizing our in-house cancer cell line models. Cytotoxicity assays were performed for 60 pediatric cancer cell lines, including Neuroblastoma (NB) = 25, Ewing sarcoma / Ewing sarcoma family of tumors (EWS / EWSFT) = 17, Rhabdomyosarcoma (RMS) = 10, Others (Osteosarcoma, Brain tumor, Wilms tumor, Hepatoblastoma, and Malignant rhabdoid tumor (MRT) = 8, and 2 breast cancer cell lines expressing HER2 high or low (IHC score 2+ or 0) as positive or low control, respectively. We analyzed HER2 expression on the cell surface by flow cytometry and evaluated relative mean fluorescence intensity (MFI) values. Results: In the flow cytometric assessment, pediatric cancer cell lines showed smaller median MFI of 1.40 (0.67 - 2.71) compared with breast cancer control with HER2 high expression (33.77). The median MFI of each cell lines were 0.94 in NB, 1.76 in EWS / EWSFT, 1.42 in RMS, 1.52 in other pediatric cancers. Although low or no expression of HER2, certain inhibitory activities to the cell growth were observed for T-DXd against a part of NB, EWS, RMS and MRT cell lines, with the minimum IC50 values of 14.5 nM, 6.5 nM, 10.2 nM, 8.1 nM, respectively. In NB, EWS and RMS, a wide range of sensitivity to T-DXd were seen among different cell lines in the same disease. Most of cell lines showed high sensitivity to DXd (Median IC50 value: 0.90 nM [0.08 - 6.46 nM]), but, among them, cell lines with lower sensitivity to DXd tend to show less sensitivity to T-DXd. There was no clear correlation between sensitivity to T-DXd and HER2 expression among pediatric cancer cell lines, indicating that other mechanism such as intrinsic sensitivity to payload might affect antitumor activity of T-DXd. Conclusions: T-DXd exhibited antitumor efficacy against pediatric solid tumors in vitro irrespective of HER2 expression. Our results suggest that T-DXd might be an alternative therapy for pediatric solid tumor cases for whom conventional chemotherapies and other targeting therapies are ineffective. This study supports further investigation for T-DXd in this population.

Validation of a non-oncogene encoded vulnerability to exportin 1 inhibition in pediatric renal tumors

Malignant rhabdoid tumors (MRTs) and Wilms' tumors (WTs) are rare and aggressive renal tumors of infants and young children comprising ∼5% of all pediatric cancers. MRTs are among the most genomically stable cancers, and although WTs are genomically heterogeneous, both generally lack therapeutically targetable genetic mutations. Comparative protein activity analysis of MRTs (n= 68) and WTs (n= 132) across TCGA and TARGET cohorts, using metaVIPER, revealed elevated exportin 1 (XPO1) inferred activity. Invitro studies were performed on a panel of MRT and WT cell lines to evaluate effects on proliferation and cell-cycle progression following treatment with the selective XPO1 inhibitor selinexor. Invivo anti-tumor activity was assessed in patient-derived xenograft (PDX) models of MRTs and WTs. metaVIPER analysis identified markedly aberrant activation of XPO1 in MRTs and WTs compared with other tumor types. All MRT and most WT cell lines demonstrated baseline, aberrant XPO1 activity with invitro sensitivity to selinexor via cell-cycle arrest and induction of apoptosis. Invivo, XPO1 inhibitors significantly abrogated tumor growth in PDX models, inducing effective disease control with sustained treatment. Corroborating human relevance, we present a case report of a child with multiply relapsed WTs with prolonged disease control on selinexor. We report on a novel systems-biology-based comparative framework to identify non-genetically encoded vulnerabilities in genomically quiescent pediatric cancers. These results have provided preclinical rationale for investigation of XPO1 inhibitors in an upcoming investigator-initiated clinical trial of selinexor in children with MRTs and WTs and offer opportunities for exploration of inferred XPO1 activity as a potential predictive biomarker for response. This work was funded by CureSearch for Children's Cancer, Alan B. Slifka Foundation, NIH (U01 CA217858, S10 OD012351, and S10 OD021764), Michael's Miracle Cure, Hyundai Hope on Wheels, Cannonball Kids Cancer, Conquer Cancer the ASCO Foundation, Cycle for Survival, Paulie Strong Foundation, and the Grayson Fund.

Establishment and characterization of NCC-MRT1-C1: a novel cell line of malignant rhabdoid tumor.

Malignant rhabdoid tumor (MRT) is a sarcoma histologically characterized by rhabdoid cells and genetically characterized by loss of function of the chromatin remodeling complex SWI/SNF induced by SMARCB1 gene deficiency. MRT mainly occurs in children, may arise in various locations, but is predominantly in the central nervous system (CNS) and kidney. Although MRT exhibits poor prognosis, standard treatment has not yet been established due to its extreme rarity. Patient-derived cancer cell lines are critical tools for basic and pre-clinical research in the development of chemotherapy. However, none of the MRT cell lines was derived from adult patients, and only one cell line was derived from the MRT of a soft tissue, despite the clinical behavior of MRT varying according to patient age and anatomic site. Herein, we reported the first cell line of MRT isolated from the soft tissue of an adult patient and named it NCC-MRT1-C1. NCC-MRT1-C1 cells showed a biallelic loss of the SMARCB1 gene. NCC-MRT1-C1 cells demonstrated rapid proliferation, spheroid formation, invasion capability in vitro, and tumorigenesis in nude mice. Screening of antitumor agents in NCC-MRT1-C1 cells resulted in the identification of six effective drugs. In conclusion, we report the first MRT cell line from the soft tissue of an adult patient. We believe that NCC-MRT1-C1 is a useful tool for developing novel chemotherapies for MRT.

Human Malignant Rhabdoid Tumor Antigens as Biomarkers and Potential Therapeutic Targets.

Simple SummaryAtypical teratoid rhabdoid tumor (ATRT) is a deadly type of human pediatric brain cancer without effective treatments. ATRT is mainly linked to the inactivation of a tumor suppressor gene, SMARCB1; however, additional biomarkers remain to be identified to develop novel therapeutic strategies. Therefore, different tumor antigens and extracellular matrix modulators were investigated in two human ATRT and one kidney malignant rhabdoid tumor cell lines and compared with the nonmalignant HEK293 cell line. Alpha-fetoprotein (AFP), mucin-16 (MUC16 or cancer antigen 125/CA125), osteopontin (OPN), and mesothelin (MSLN) are highly expressed in these human malignant rhabdoid cancer cell lines. Inhibiting MMPs using a small-molecule inhibitor decreased cell survival. This biomarker discovery process may lead to the identification of novel diagnostic and therapeutic strategies, such as the development of targeted and immunotherapies against cancer biomarkers, to treat cancer patients.Introduction: Atypical teratoid rhabdoid tumor (ATRT) is a lethal type of malignant rhabdoid tumor in the brain, seen mostly in children under two years old. ATRT is mainly linked to the biallelic inactivation of the SMARCB1 gene. To understand the deadly characteristics of ATRT and develop novel diagnostic and immunotherapy strategies for the treatment of ATRT, this study investigated tumor antigens, such as alpha-fetoprotein (AFP), mucin-16 (MUC16/CA125), and osteopontin (OPN), and extracellular matrix modulators, such as matrix metalloproteinases (MMPs), in different human malignant rhabdoid tumor cell lines. In addition, the roles of MMPs were also examined. Materials and methods: Five human cell lines were chosen for this study, including two ATRT cell lines, CHLA-02-ATRT and CHLA-05-ATRT; a kidney malignant rhabdoid tumor cell line, G401; and two control cell lines, human embryonic kidney HEK293 and HEK293T. Both ATRT cell lines were treated with a broad-spectrum MMP inhibitor, GM6001, to investigate the effect of MMPs on cell proliferation, viability, and expression of tumor antigens and biomarkers. Gene expression was examined using a reverse transcription polymerase chain reaction (RT-PCR), and protein expression was characterized by immunocytochemistry and flow cytometry. Results: All the rhabdoid tumor cell lines tested had high gene expression levels of MUC16, OPN, AFP, and MSLN. Low expression levels of neuron-specific enolase (ENO2) by the two ATRT cell lines demonstrated their lack of neuronal genotype. Membrane-type 1 matrix metalloproteinase (MT1-MMP/MMP-14) and tissue inhibitor of metalloproteinases-2 (TIMP-2) were highly expressed in these malignant rhabdoid tumor cells, indicating their invasive phenotypes. GM6001 significantly decreased ATRT cell proliferation and the gene expression of MSLN, OPN, and several mesenchymal markers, suggesting that inhibition of MMPs may reduce the aggressiveness of rhabdoid cancer cells. Conclusion: The results obtained from this study may advance our knowledge of the molecular landscapes of human malignant rhabdoid tumors and their biomarkers for effective diagnosis and treatment. This work analyzed the expression of human malignant rhabdoid tumor antigens that may serve as biomarkers for the development of novel therapeutic strategies, such as cancer vaccines and targeted and immunotherapies targeting osteopontin and mesothelin, for the treatment of patients with ATRT and other malignant rhabdoid tumors.

Abstract 283: Target cell-derived, G401-CLENs for selective delivery of model therapeutics to rhabdoid tumors

Abstract One of the rarest and most aggressive pediatric cancers to date is the malignant rhabdoid tumor (MRT), maintaining a survival rate of 16%. Considered a renal cancer, approximately 20-25 new cases are diagnosed in the USA each year, with the average age of diagnosis being about 11 months old. Conventional strategies for treating MRT are limited due to several factors including off-target associated toxicities, patient population, age, metastasis to brain tissue, and diminished survival rates. CLENs (cell membrane lipid-extracted nanoliposomes), a novel drug delivery system, was previously developed and evaluated for selective delivery of cytotoxic drug agents to breast cancer cells and compared to more conventional liposomes. The purpose of this investigation was to optimize and characterize G401-CLENs for selective targeting and delivery of model payloads to a cellular model of rhabdoid tumors. The MRT cell line (G-401 [G401] (ATCC® CRL-1441™) was cultured in McCoy's 5A Medium (ATCC® 30-2007™), supplemented by 10% FBS. The G401 cellular lipid materials (otherwise known as lipid extracts (LE)) were derived from G401 cells. Follow up studies include the incorporation of G401-LE in liposomes to form G401-CLENs. For development, special consideration was given to distinct determinants of targeting (i.e., particle size and zeta potential) and cellular uptake by G401-CLENs. Other analyses include a comparison of delivery of model and functional siRNA (BRD9 Silencer Select Pre-designed, siRNA ID s35295, Ambion) to G401 target cells using G401-CLENs, and conventional nano-preparations in vitro. G401-LE cell membrane components were extracted from rhabdoid G401 cells. On-going physiochemical characterization studies of G401-CLENs and functional in vitro and fluorescence microscopic analyses are currently underway. Citation Format: Lindsay Bourdeau, Carlos Cruz, Taylor Williams. Target cell-derived, G401-CLENs for selective delivery of model therapeutics to rhabdoid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 283.

The forkhead box M1 (FOXM1) expression and antitumor effect of FOXM1 inhibition in malignant rhabdoid tumor.

Malignant rhabdoid tumor (MRT) is a rare, highly aggressive sarcoma with an uncertain cell of origin. Despite the existing standard of intensive multimodal therapy, the prognosis of patients with MRT is very poor. Novel antitumor agents are needed for MRT patients. Forkhead box transcription factor 1 (FOXM1) is overexpressed and is correlated with the pathogenesis in several human malignancies. In this study, we identified the clinicopathological and prognostic values of the expression of FOXM1 and its roles in the progression of MRT. We investigated the FOXM1 expression levels and their clinical significance in 23 MRT specimens using immunohistochemistry and performed clinicopathologic and prognostic analyses. We also demonstrated correlations between the downregulation of FOXM1 and oncological characteristics using small interfering RNA (siRNA) and FOXM1 inhibitor in MRT cell lines. Histopathological analyses revealed that primary renal MRTs showed significantly low FOXM1 protein expression levels (p = 0.032); however, there were no significant differences in other clinicopathological characteristics or the survival rate. FOXM1 siRNA and FOXM1 inhibitor (thiostrepton) successfully downregulated the mRNA and protein expression of FOXM1 in vitro and the downregulation of FOXM1 inhibited cell proliferation, drug resistance to chemotherapeutic agents, migration, invasion, and caused the cell cycle arrest and apoptosis of MRT cell lines. A cDNA microarray analysis showed that FOXM1 regulated FANCD2 and NBS1, which are key genes for DNA damage repair. This study demonstrates that FOXM1 may serve as a promising therapeutic target for MRT.

MDM2 and MDM4 Are Therapeutic Vulnerabilities in Malignant Rhabdoid Tumors.

Malignant rhabdoid tumors (MRT) are highly aggressive pediatric cancers that respond poorly to current therapies. In this study, we screened several MRT cell lines with large-scale RNAi, CRISPR-Cas9, and small-molecule libraries to identify potential drug targets specific for these cancers. We discovered MDM2 and MDM4, the canonical negative regulators of p53, as significant vulnerabilities. Using two compounds currently in clinical development, idasanutlin (MDM2-specific) and ATSP-7041 (MDM2/4-dual), we show that MRT cells were more sensitive than other p53 wild-type cancer cell lines to inhibition of MDM2 alone as well as dual inhibition of MDM2/4. These compounds caused significant upregulation of the p53 pathway in MRT cells, and sensitivity was ablated by CRISPR-Cas9-mediated inactivation of TP53. We show that loss of SMARCB1, a subunit of the SWI/SNF (BAF) complex mutated in nearly all MRTs, sensitized cells to MDM2 and MDM2/4 inhibition by enhancing p53-mediated apoptosis. Both MDM2 and MDM2/4 inhibition slowed MRT xenograft growth in vivo, with a 5-day idasanutlin pulse causing marked regression of all xenografts, including durable complete responses in 50% of mice. Together, these studies identify a genetic connection between mutations in the SWI/SNF chromatin-remodeling complex and the tumor suppressor gene TP53 and provide preclinical evidence to support the targeting of MDM2 and MDM4 in this often-fatal pediatric cancer. SIGNIFICANCE: This study identifies two targets, MDM2 and MDM4, as vulnerabilities in a deadly pediatric cancer and provides preclinical evidence that compounds inhibiting these proteins have therapeutic potential.

Redirecting T Cells to Glypican-3 with 4-1BB Zeta Chimeric Antigen Receptors Results in Th1 Polarization and Potent Antitumor Activity.

T cells engineered to express CD19-specific chimeric antigen receptors (CARs) have shown breakthrough clinical successes in patients with B-cell lymphoid malignancies. However, similar therapeutic efficacy of CAR T cells in solid tumors is yet to be achieved. In this study we systematically evaluated a series of CAR constructs targeting glypican-3 (GPC3), which is selectively expressed on several solid tumors. We compared GPC3-specific CARs that encoded CD3ζ (Gz) alone or with costimulatory domains derived from CD28 (G28z), 4-1BB (GBBz), or CD28 and 4-1BB (G28BBz). All GPC3-CARs rendered T cells highly cytotoxic to GPC3-positive hepatocellular carcinoma, hepatoblastoma, and malignant rhabdoid tumor cell lines in vitro. GBBz induced the preferential production of Th1 cytokines (interferon γ/granulocyte macrophage colony-stimulating factor) while G28z preferentially induced Th2 cytokines (interleukin-4/interleukin-10). Inclusion of 4-1BB in G28BBz could only partially ameliorate the Th2-polarizing effect of CD28. 4-1BB induced superior expansion of CAR T cells in vitro and in vivo. T cells expressing GPC3-CARs incorporating CD28, 4-1BB, or both induced sustained tumor regressions in two xenogeneic tumor models. Thus, GBBz CAR endows T cells with superior proliferative potential, potent antitumor activity, and a Th1-biased cytokine profile, justifying further clinical development of GBBz CAR for immunotherapy of GPC3-positive solid tumors.

CD146 is a novel marker for highly tumorigenic cells and a potential therapeutic target in malignant rhabdoid tumor.

Malignant rhabdoid tumor (MRT) is a rare, highly aggressive pediatric malignancy that primarily develops during infancy and early childhood. Despite the existing standard of intensive multimodal therapy, the prognosis of patients with MRT is dismal; therefore, a greater understanding of the biology of this disease is required to establish novel therapies. In this study, we identified a highly tumorigenic sub-population in MRT, based on the expression of CD146 (also known as melanoma cell adhesion molecule), a cell adhesion molecule expressed by neural crest cells and various derivatives. CD146+ cells isolated from four MRT cell lines by cell sorting exhibited enhanced self-renewal and invasive potential in vitro. In a xenograft model using immunodeficient NOD/Shi-scid IL-2Rγ-null mice, purified CD146+ cells obtained from MRT cell lines or a primary tumor exhibited the exclusive ability to form tumors in vivo. Blocking of CD146-related mechanisms, either by short hairpin RNA knockdown or treatment with a polyclonal antibody against CD146, effectively suppressed tumor growth of MRT cells both in vitro and in vivo via induction of apoptosis by inactivating Akt. Furthermore, CD146 positivity in immunohistological analysis of 11 MRT patient samples was associated with poor patient outcomes. These results suggest that CD146 defines a distinct sub-population in MRT with high tumorigenic capacity and that this marker represents a promising therapeutic target.

A NOXA/MCL-1 Imbalance Underlies Chemoresistance of Malignant Rhabdoid Tumor Cells.

Malignant rhabdoid tumor (MRT) is a rare aggressive pediatric cancer characterized by inactivation of SNF5, a core subunit of SWI/SNF complexes. Previously, we showed that SNF5 contributes to transcriptional activation of NOXA, a pro-apoptotic protein that binds and inhibits the anti-apoptotic protein MCL-1. In this study, we found that NOXA expression was downregulated in MRT cell lines as well as in clinical MRT samples and that ectopically expressed NOXA bound MCL-1 and increased the sensitivity of MRT cell lines to doxorubicin (DOX) by promoting apoptosis. Consistent with this finding, knockdown of MCL-1 in MRT cell lines induced apoptosis and increased DOX sensitivity in MRT cells, and the MCL-1 inhibitor TW-37 synergized with DOX to induce MRT cell death. Our results suggest that modulation of the NOXA/MCL-1 pathway may be a potential strategy for the treatment of patients with MRT. J. Cell. Physiol. 231: 1932-1940, 2016. © 2015 Wiley Periodicals, Inc.

Abstract C162: A medium-throughput single cell CRISPR-Cas9 assay to assess gene essentiality

Abstract Target selection for oncology and other indications is a critical step in the successful development of therapeutics, however it remains one of the most challenging areas of drug discovery. In fact, up to two-thirds of oncology relevant targets reported in literature have not been confirmed on follow-up studies, indicating that target validation in oncology is especially challenging. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene editing of specific loci offers an alternative method to RNA interference and complements small molecule inhibitors for determining whether or not a cell line is dependent on a specific gene product for proliferation and/or survival. Importantly, CRISPR-Cas9 may be advantageous for some studies as it offers efficient and specific gene knock-out leading to complete loss of protein function. This may be especially useful for some target classes, including epigenetic targets, which appear to require near complete loss of protein function to observe phenotypes. In our initial studies using CRISPR-Cas9 to verify the essential nature of EZH2 (Enhancer of Zest 2) expression for the proliferation of SMARCB1/SNF5/INI1 mutant malignant rhabdoid tumor cell lines, we observed that the initial reduction in proliferation was lost over time. We hypothesized that in the few cells that retain proliferative capacity at least one allele of EZH2 remains functional, and this hypothesis suggests that carrying out CRISPR-Cas9 studies for individual target genes without analyzing single cell clones could produce misleading results. To verify this, we developed a medium throughput assay to analyze 10s-100s of single cell clones for target gene disruption using CRISPR-Cas9 gene knockout, followed by a restriction digest and fluorescent undigested fragment length analysis to successfully assess EZH2 allele status. Significantly, these data support our hypothesis that retention of one functional copy of EZH2 is required for the proliferation of EZH2-dependent cell lines and that this can be rapidly assessed by our assay. Thus, the assessment of zygosity of the gene of interest can be evaluated in a medium-throughput manner in single cell clones. The assay is thereby able to unambiguously indicate whether or not a specific gene is essential for survival and/or proliferation in a given cell line, and offers a unique approach for target validation using gene editing. Importantly, this approach should be applicable to any target of interest that is expected to affect cell proliferation or survival. Such data can aid in the development of more robust cancer therapeutics by increasing confidence in target selection. Citation Format: Alexandra R. Grassian, Tim Scales, Sarah K. Knutson, Nicola J. McCarthy, Chris E. Lowe, Jon D. Moore, Robert A. Copeland, Heike Keilhack, Jesse J. Smith, Julie A. Wickendon, Scott Ribich. A medium-throughput single cell CRISPR-Cas9 assay to assess gene essentiality. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C162.

Abstract 489: Loss of NOXA expression by INI1/SNF5 loss impaired sensitivity to chemotherapeutic agents in malignant rhabdoid tumor in vitro and iv vivo

Abstract Background: We previously showed that INI1/SNF5 reexpression increased transcriptional activity at the NOXA locus in malignant rhabdoid tumor (MRT) cell lines resulting in NOXA protein production. In other cancer cells, the binding between NOXA and MCL-1 induces apoptosis after exposure to chemotherapeutic agents. Therefore, we hypothesized that loss of NOXA alters the apoptotic pathway in MRTs and contributes to their resistance to chemotherapeutic agents. Methods: The TTC549 and KP-MRT-RY MRT cell lines were transfected with the pcDNA 3.1(+)-wild type hNOXA plasmid, a kind gift from Dr. Kelekar, University of Minnesota, using Lipofectamine reagent and then selected with G418. To generate MCL-1 knockdown MRT cell lines, we transfected a small interfering (si) RNA targeting MCL-1 using Lipofectamine reagent. Cells were plated in growth medium for 24 hrs and then treated with serial dilutions of doxorubicin (DOX) and/or TW-37. To generate mice xenografts for both TTC549 MCL-1 stable knockdown (KD) and control cell lines, we established two independently derived MCL-1 stable knockdown TTC549 cell lines using lentiviral vectors encoding a shRNA targeting MCL-1 and a negative control cell line (pLKO.1). We injected either 5 × 106 TTC549 MCL-1 KD or control cell lines s.c. into athymic mice. One week after injection, mice were treated once daily with DOX (15mg/kg) or vehicle alone for five days. Results: In both NOXA expressing and MCL-1 KD MRT cells, we observed a dramatic reduction of the IC50 values of DOX treatment. Moreover, apoptosis was also observed at 48 hrs after DOX treatment at concentrations less than the IC50 values for control cells. Immunoprecipitation of MCL-1 showed that expressed NOXA associated with MCL-1 in MRT cells. Furthermore, we observed that mean tumor volume in the DOX-treated MCL-1 KD group was significantly smaller than that in the vehicle-treated MCL-1 knockdown group. Next, we assessed the effects of TW-37, an inhibitor that binds to the Bcl-2 homology domain 3 (BH3) of proteins including MCL-1, in combination with DOX. Using the combination index, we found that DOX and TW-37 displayed modest synergy in both MRT cell lines (CI<1) in vitro and in vivo. Discussion: Ectopic expression of NOXA or knockdown of MCL-1 increased DOX sensitivity in MRT cell lines. These results indicate that loss of INI1/SNF5-mediated regulation of NOXA expression impairs an apoptotic pathway leading to repression of MCL-1 followed by the resistance to chemotherapeutic agents. We also found that TW-37, a BH3 only protein mimetic, has a synergistic effect with DOX on MRT cell lines in vitro and in vivo, providing a potential foundation for molecular targeting therapy for MRT. Citation Format: Kazutaka Ouchi, Yasumichi Kuwahara, Tomoko Iehara, Eiichi Konishi, Hajime Hosoi. Loss of NOXA expression by INI1/SNF5 loss impaired sensitivity to chemotherapeutic agents in malignant rhabdoid tumor in vitro and iv vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 489. doi:10.1158/1538-7445.AM2015-489

SNF5/INI1 deficiency redefines chromatin remodeling complex composition during tumor development.

Malignant rhabdoid tumors (MRT), a pediatric cancer that most frequently appears in the kidney and brain, generally lack SNF5 (SMARCB1/INI1), a subunit of the SWI/SNF chromatin-remodeling complex. Recent studies have established that multiple SWI/SNF complexes exist due to the presence or absence of different complex members. Therefore, the effect of SNF5 loss upon SWI/SNF complex formation was investigated in human MRT cells. MRT cells and primary human tumors exhibited reduced levels of many complex proteins. Furthermore, reexpression of SNF5 increased SWI/SNF complex protein levels without concomitant increases in mRNA. Proteomic analysis, using mass spectrometry, of MRT cells before and after SNF5 reexpression indicated the recruitment of different components into the complex along with the expulsion of others. IP-Western blotting confirmed these results and demonstrated similar changes in other MRT cell lines. Finally, reduced expression of SNF5 in normal human fibroblasts led to altered levels of these same complex members. These data establish that SNF5 loss during MRT development alters the repertoire of available SWI/SNF complexes, generally disrupting those associated with cellular differentiation. These findings support a model where SNF5 inactivation blocks the conversion of growth-promoting SWI/SNF complexes to differentiation-inducing ones. Therefore, restoration of these complexes in tumors cells provides an attractive approach for the treatment of MRTs. SNF5 loss dramatically alters SWI/SNF complex composition and prevents formation of complexes required for cellular differentiation.

Abstract 1001: CD146 is a novel marker of highly tumorigenic populations and a therapeutic target in malignant rhabdoid tumor

Abstract Background: Malignant rhabdoid tumor (MRT) is a rare, highly aggressive pediatric malignancy, particularly develops during infancy and early childhood. In contrast to significant advances in multimodality therapy for other pediatric malignancies, effective therapies for MRT have not been established. The origin of tumor cells in MRT has remained undetermined: however, MRT arises from various parts of body with some preference, eading to the hypothesis that MRT derives from neural crest or its derivatives. CD146 is a cell surface glycoprotein expressed by various neural crest-derived tissues, which has been expected to be a therapeutic target for immunotherapy against various CD146-expressing malignancies, such as melanoma and osteosarcoma. Objective: In the current study, we investigated (1) the expression of CD146 in MRT, (2) tumorigenic potential of CD146-expressing cells in MRT, (3) in vitro and in vivo effect of anti-CD146 neutralizing antibody against MRT, and (4) underlying molecular mechanisms involved in CD146 expression. Material and Method: The expression levels of CD146 in MRT cell lines and clinical samples were investigated by fluorescence-activated cell sorting (FACS) or immunohistochemical analyses. Tumor cells were then sorted by FACS according to the expression of CD146, and purified CD146+ and CD146- cells were characterized by colony-formation, sphere-formation, and invasion assays. In vivo tumorigenic potential was assessed by subcutaneous transplantation of the purified CD146+ and CD146- cells into NOD/Shi-scid, IL-2Rγnull (NOG) mice. We developed rabbit anti-CD146 neutralizing antibody to investigate the effect of blocking CD146 both in vitro and in vivo. DNA microarray was performed by using purified CD146+ and CD146- cells, and the association of candidate pathways was validated by shRNA silencing or treatment of the neutralizing antibody against CD146. Results: The expression of CD146 was observed in all MRT cell lines and clinical samples. Compared to CD146- cells, CD146+ cells in MRT exhibited significantly higher colony-, and sphere-forming potential, and more invasive capacity. The data of xeno-transplant demonstrated that stable engraftment of the purified tumor cells as well as successful engraftment after serial transplantation was exclusively observed in CD146+ cell-injected mice. Furthermore, our anti-CD146 neutralized antibody effectively inhibited both in vitro proliferation and in vivo tumor formation. Microarray analysis showed that various molecular pathways were engaged in higher tumorigeneity in CD146+ cells. Details of underlying mechanisms are now under investigation. Conclusion: Our data shows that CD146 defines a distinct subpopulation with highly tumorigenic and self-renewal capacity in MRT. Notable anti-tumor effect of anti-CD146 neutralizing antibody will facilitate a novel immunotherapy for MRT. Citation Format: Seishiro Nodomi, Katsutsugu Umeda, Satoshi Saida, Yasumichi Kuwahara, Takayuki Hamabata, Tomoo Daifu, Itaru Kato, Hidefumi Hiramatsu, Ken-ichiro Watanabe, Souichi Adachi, Eiichi Konishi, Hajime Hosoi, Toshio Heike. CD146 is a novel marker of highly tumorigenic populations and a therapeutic target in malignant rhabdoid tumor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1001. doi:10.1158/1538-7445.AM2014-1001

Abstract 2245: Mechanism of replicative senescence induced by SNF5 loss

Abstract Cellular senescence was initially described as the phenomenon by which normal diploid cells cease to divide after reaching the Hayflick limit. However, with the identification of replicative senescence, other inducers of this process have appeared in the literature including oncogene induced senescence (OIS). Of interest, different oncogenes can initiate OIS through alternative pathways e.g. HRAS (G12V) and BRAF (V600E) that effect senescence through p16 dependent and p16 independent pathways, respectively. Alternatively, loss of tumor suppressor gene function can also induce senescence. In malignant rhabdoid tumors (MRTs), SNF5 behaves as a tumor suppressor and its re-expression in MRT cell lines results in growth arrest via the RB pathway. Paradoxically, when we knocked out SNF5 in normal human fibroblasts (NHF-1), we also observed senescence. We therefore hypothesize that perturbation of the SWI/SNF complex (e.g. knocking down SNF5) results in cellular senescence in most normal tissues. This suggests an epigenetic mechanism of senescence occurring through a p16 independent pathway, as previous publications have demonstrated a critical role for SNF5 in p16 expression. Additionally, in BRAF induced senescence, changes in levels of CD44, phospho-MEK1/2, and p16 are observed. However, we do not find these changes during SNF5 loss. Our findings suggest a relationship between SWI/SNF's chromatin remodeling activity and the regulation of cellular senescence. Importantly, our studies will identify the key signaling pathways that allow MRTs to escape cellular senescence after SNF5 loss. These results will further elucidate the role of SNF5 loss in MRT development and provide new targets for clinical intervention. Acknowledgement: We thank the members of the Weissman lab for their help, advice, and support and R01CA91048 (B.E.W.) , T32ES007126 (D.W.), UNC Dissertation Completion Fellowship (D.W.) for financial support. Citation Format: Darmood Wei, HoYoon Chung, Dennis A. Simpson, William K. Kaufmann, Bernard E. Weissman. Mechanism of replicative senescence induced by SNF5 loss. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2245. doi:10.1158/1538-7445.AM2014-2245

Effect of telomerase inhibition on preclinical models of malignant rhabdoid tumor

Novel treatment approaches are desperately needed for malignant rhabdoid tumor (MRT). Telomerase is an attractive therapeutic target because it is specific to cancer and critical for cancer cell immortality. We evaluated the effect of the telomerase inhibitor imetelstat in preclinical models of MRT. Three MRT cell lines, BT-12, G401, and RT-peri, were treated with the telomerase inhibitor imetelstat. The effects of imetelstat on telomere length, DNA damage response, and cell proliferation were assessed. The efficacy of imetelstat invivo was evaluated in subcutaneous xenografts derived from each of the cell lines. Treatment with imetelstat resulted in inhibition of telomerase activity, marked telomere shortening, and activation of the DNA damage response pathway, as measured by formation of γ-H2AX nuclear foci, phosphorylation of ATM, and phosphorylation of TP53. Imetelstat-treated G401 cells underwent complete growth arrest after 16 passages. The other two cell lines exhibited growth inhibition. Imetelstat resulted in 40-50% growth inhibition compared to placebo-treated controls in all three xenograft models. The activity of imetelstat as a single agent suggests that further studies of telomerase inhibitors in combination with other agents may be warranted.

Fibroblast Growth Factor Receptors as Novel Therapeutic Targets in SNF5-Deleted Malignant Rhabdoid Tumors

Malignant rhabdoid tumors (MRTs) are aggressive pediatric cancers arising in brain, kidney and soft tissues, which are characterized by loss of the tumor suppressor SNF5/SMARCB1. MRTs are poorly responsive to chemotherapy and thus a high unmet clinical need exists for novel therapies for MRT patients. SNF5 is a core subunit of the SWI/SNF chromatin remodeling complex which affects gene expression by nucleosome remodeling. Here, we report that loss of SNF5 function correlates with increased expression of fibroblast growth factor receptors (FGFRs) in MRT cell lines and primary tumors and that re-expression of SNF5 in MRT cells causes a marked repression of FGFR expression. Conversely, siRNA-mediated impairment of SWI/SNF function leads to elevated levels of FGFR2 in human fibroblasts. In vivo, treatment with NVP-BGJ398, a selective FGFR inhibitor, blocks progression of a murine MRT model. Hence, we identify FGFR signaling as an aberrantly activated oncogenic pathway in MRTs and propose pharmacological inhibition of FGFRs as a potential novel clinical therapy for MRTs.

Study of the gene expression and microRNA expression profiles of malignant rhabdoid tumors originated in the brain (AT/RT) and in the kidney (RTK)

Malignant rhabdoid tumors (MRT) can occur in a variety of anatomical sites. The most frequent locations are the brain, where they are named atypical teratoid/rhabdoid tumors (AT/RT), and the kidney, where they are named rhabdoid tumors of the kidney (RTK). MRTs at all sites are recognized as the same entity due to their similar morphology, aggressive behavior, and a common genetic abnormality, an inactivating mutation of the SMARCB1/INI-1/hSNF5/BAF47 gene. We aim to investigate potential molecular differences between AT/RT and RTK. We analyzed the microRNA (miRNA) and gene expression (GE) profiles of 10 RTK, 13 AT/RT, and 2 human MRT cell lines (G401-RTK and MON-AT/RT). Illumina V2 MicroRNA Chips (Illumina, Inc., CA, USA) were used for miRNA analysis, and Illumina HT-12 whole genome expression arrays were used for GE analysis. The distribution of p values from GE showed a significant difference between RTK and AT/RT, with 20% of the genes having p values ≤0.05 and the principal component analysis of the GE data showed separation between RTK and AT/RT. However, the miRNA expression failed to identify the different tumor groups. Among the 122 genes significantly differentially expressed between AT/RT and RTK, we found both genes related to brain development (i.e., FABP7, 22-fold increase in AT/RT) and genes related to kidney development (i.e., TCF21, sixfold increase in RTK). Based on our results, we hypothesized that although MRT are indeed the same tumor, independent of the site of origin, the GE differences reflect the influence of microenvironment over tumor development.

Establishment and characterization of MRT cell lines from genetically engineered mouse models and the influence of genetic background on their development

Malignant rhabdoid tumors (MRTs) are rare, aggressive cancers occuring in young children primarily through inactivation of the SNF5(INI1, SMARCB1) tumor suppressor gene. We and others have demonstrated that mice heterozygous for a Snf5 null allele develop MRTs with partial penetrance. We have also shown that Snf5(+/-) mice that lack expression of the pRb family, due to TgT121 transgene expression, develop MRTs with increased penetrance and decreased latency. Here, we report that altering the genetic background has substantial effects upon MRT development in Snf5(+/--) and TgT121 ;Snf5(+/-) mice, with a mixed F1 background resulting in increased latency and the appearance of brain tumors. We also report the establishment of the first mouse MRT cell lines that recapitulate many features of their human counterparts. Our studies provide further insight into the genetic influences on MRT development as well as provide valuable new cell culture and genetically engineered mouse models for the study of CNS-MRT etiology.

Abstract 1359: Establishment and characterization of mouse malignant rhabdoid tumor cell lines

Abstract Malignant rhabdoid tumors (MRTs) are rare, aggressive cancers occuring in young children primarily through inactivation of the SNF5 tumor suppressor gene. Espetially, rhabdoid tumor arised from brain is usually called AT/RT. We and others have demonstrated that mice heterozygous for a Snf5 null allele develop MRTs with partial penetrance. We have also shown that Snf5+/− mice that lacked expression of the pRb family, due to TgT121 transgene expression, developed MRTs with increased penetrance and decreased latency. In this current study, we observed that the genetic background altered MRT development in Snf5+/− and TgT121;Snf5+/− mice, resulting in increased latency and the appearance of significant frequency of brain tumors. We also report the establishment of the first mouse MRT cell lines that recapitulate many features of their human counterparts including lack of genomic instability and expression of smooth muscle and neuronal differentiation markers. The cell lines also form aggressive tumors upon inoculation into immunocompromised mice that resemble the tumors of origin. Our studies provide further insight into the genetic influences on MRT development as well as provide valuable new cell culture and genetically engineered mouse models for the study of AT/RT etiology. 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 1359. doi:1538-7445.AM2012-1359