AbstractAtypical Teratoid/rhabdoid Tumors Research Articles

Abstract 7555: Selective inhibition of the CoREST complex as a novel therapeutic target of atypical teratoid rhabdoid tumors

Abstract Atypical teratoid/rhabdoid tumors (AT/RT) are deadly infantile brain tumors, primarily arising from a single recurring biallelic loss-of-function mutation in SMARCB1 which is a member of the SWI-SNF chromatin remodeling complex. The partially functional SWI-SNF complex (without SMARCB1) is able to inhibit EZH2 at most locations of the genome except at gene promoter regions where EZH2 co-localizes with the REST complex. This results in hypermethylation and subsequent repression of genes coding for neuronal differentiation and tumor suppressors. CoREST is an important transcriptional repressor and consists of LSD1 and HDAC1/2 bound together by the scaffolding protein RCOR. Increased activity of EZH2 and the CoREST complex may help AT/RT cells maintain a stem cell-like state, driving its aggressive growth and therapy resistance. We hypothesize that functional inhibition of the CoREST complex by targeting LSD1 and HDAC1/2 might help restore the epigenetic balance lost in AT/RT cells.We selectively inhibited the CoREST complex in AT/RT cells (BT37, CHLA05 and CHLA06) by using a bi-functional LSD1 and HDAC1/2 inhibitor, Corin, which has previously been shown to be effective against melanoma and diffuse midline glioma. Administration of corin to AT/RT cells significantly increased H3K4me1, H3K9Ac and H3K27Ac histone marks (p < 0.001, Western blotting). Corin inhibited AT/RT cell growth and proliferation (MUSE Viacount p<0.05, BrdU immunofluorescence compared to control), and increased apoptosis (cPARP Western blotting, CC3 immunofluorescence, MUSE Annexin V assay, p<0.05 compared to control). Corin appears to be specific in targeting AT/RT as it did not affect brain organoids derived from iPSCs (cPARP Western blotting). Corin induced neuronal differentiation in AT/RT as seen by the increased expression of Beta-3-tubulin, MAP2 and Synaptophysin (Immunofluorescence). Enrichment analysis of differentially expressed genes after whole mRNA sequencing of AT/RT cells treated with corin for 24 h revealed upregulation of pathways related to neuronal differentiation and axonal development and downregulation of pathways related to proliferation and cell cycle (pFDR < 0.05, FC>2). In mice bearing orthotopic CHLA06 tumors, intratumoral delivery of a single dose of corin for 24 h increased H3K27me3 and H3K27ac, demonstrating that corin engaged its target in vivo, reduced growth and induced apoptosis (Ki67, cPARP). Survival studies show a trend toward single agent activity against the very aggressive CHLA06 orthotopic xenograft (increased in median survival from 14 to 35 days compared to control). These studies demonstrate that targeting the CoREST complex disrupts AT/RT epigenetic abnormalities, reversing their stem cell-like state, slowing tumor cell growth, and inducing apoptosis, that may translate into the clinical setting to help improve AT/RT’s dismal survival. Citation Format: Anupa Geethadevi, Marianne Collard, Tyler Findlay, Yiming Deng, Robert Fisher, Nikhil Vaidya, Charles Eberhart, Philip A. Cole, Rhoda Alani, Jeffrey Rubens, Eric Raabe. Selective inhibition of the CoREST complex as a novel therapeutic target of atypical teratoid rhabdoid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7555.

Abstract 6565: Improving survival of atypical teratoid/rhabdoid tumor orthotopic xenografts through the combination of PI3K inhibitor paxalisib and nucleoside analog gemcitabine

Abstract Atypical teratoid/rhabdoid tumors (AT/RT) are an aggressive infantile brain tumor with a dismal four-year event-free survival of 37%. We have previously identified high activation of mTORC1 and mTORC2 within AT/RT and shown that the highly brain penetrant PI3K inhibitor paxalisib is capable of simultaneously inhibiting mTORC1/2 by acting upstream of both complexes. In two orthotopic xenograft models, paxalisib slowed tumor growth and significantly extended survival (CHLA-06: 40 to 54 days, p=0.001; BT-12: 21 to 35 days, p=0.02). RNAseq analysis following mTOR inhibition in AT/RT cell lines showed an upregulation of genes involved in the activation of the integrated stress response (ISR) (ATF4, CHOP, PPP1R15A t-test p<0.05). Analysis of primary pediatric brain tumors through the Children’s Brain Tumor Network showed increased levels of ATF4 mRNA and eIF2α protein and mRNA in AT/RT compared to other indolent pediatric brain tumors. Increased expression of ISR components suggests this pathway may be a dependency in AT/RT. Activation of this pathway is known to be cytoprotective, however persistent over-activation drives cell death through induction of pro-apoptotic factors. Gemcitabine is a pyrimidine analog functioning to inhibit DNA synthesis and induce DNA damage; notably, recent literature has correlated DNA damage to activation of the ISR with downstream phosphorylation of eIF2α. We hypothesize that due to high baseline activation of ISR for AT/RT, paxalisib combined with gemcitabine will result in an over-activation of the ISR leading to AT/RT cell death. Western blot analysis of combination therapy compared to DMSO control showed induction of phospho-eIF2α, ATF4, and CHOP, denoting an activation of the ISR. Combination therapy in AT/RT cells induces cell death (Western blot analysis cPARP, MUSE Annexin V assay, SynergyFinder BLISS score [viability] CHLA-06: 21.50, CHLA-266: 22.60) and synergizes to decrease cell growth (SynergyFinder BLISS score [inhibition] CHLA-06: 16.80, BT-12: 14.30, CHLA-266: 13.90, BT-37: 11.40, CHLA-05: 10.40). Paxalisib/gemcitabine combination therapy in murine orthotopic xenograft models of AT/RT has slowed tumor growth (bioluminescent imaging) and led to significant extension of survival. Mice bearing BT-37 orthotopic xenografts and treated with vehicle control had a median survival of 56 days, while all of the paxalisib/gemcitabine treated mice were alive at 100 days of treatment (p<0.0001). In CHLA-06 orthotopic tumors combination therapy extended median survival from 22 days for control to 82.5 days (p<0.0001). Based on these findings, the Pacific Pediatric Neuro-Oncology Consortium (PNOC) is planning to include paxalisib/gemcitabine combination therapy in the next international clinical trial for patients with relapsed/recurrent AT/RT. Citation Format: Tyler Findlay, Kristen Malebranche, Anupa Geethadevi, Charles Eberhart, Jeffrey Rubens, Eric Raabe. Improving survival of atypical teratoid/rhabdoid tumor orthotopic xenografts through the combination of PI3K inhibitor paxalisib and nucleoside analog gemcitabine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6565.

Abstract 145: Unraveling SMARCB1 depletion in hiPSC-derived neural crest cells: Implications for ATRT cell of origin

Abstract Atypical teratoid rhabdoid tumors (ATRTs) are highly aggressive pediatric brain cancers that lack standardized treatment regimens and are associated with poor prognoses. Despite being driven by biallelic inactivation of a single gene, SMARCB1, ATRTs exhibit three epigenetic subgroups (SHH, TYR, and MYC), each with different clinical outcomes. Like many pediatric brain tumors, ATRTs share the fundamental underlying mechanism of stalled differentiation, suggesting that it may arise from neural progenitor cells that fail to terminally differentiate. However, unlike other brain tumors, ATRTs share molecular similarities with malignancies found outside of the CNS, especially in the kidney and soft tissues. This unique feature implies a putative cell of origin that is not restricted to the CNS: neural crest cells, a transient embryonic cell population that emerges from the neuroectoderm but then migrates throughout the embryo during development. To study SMARCB1 loss in a genetically defined neural progenitor cellular context with isogenic controls, the Furnari lab engineered a doxycycline inducible SMARCB1 knockdown system into human induced pluripotent stem cells (hiPSCs). After simultaneous SMARCB1 knockdown with directed neural differentiation, the resulting SMARCB1-depleted neural progenitor cells exhibited transcriptomic profiles that were similar to ATRTs, particularly to the SHH subgroup. Building upon these findings, the engineered hiPSCs were differentiated into neural crest cells using a modified version of a published protocol. Via RT-qPCR, these hiPSC-derived neural crest cells were confirmed to express typical neural crest marker genes and lose expression of SMARCB1 in the presence of doxycycline. Remarkably, ATP assays demonstrated that neural crest cells that were differentiated without SMARCB1 acquired enhanced proliferation in comparison to those differentiated with SMARCB1 intact. Further work aims to tease apart this proliferative phenotype in the neural crest cellular context via RNAseq, evaluate the impact of cell identity upon subgroup status, and to determine whether SMARCB1 depletion post-differentiation also enhances proliferation. Together, these findings feature the utility of hiPSC-derived models for investigating mechanisms underlying ATRTs to hopefully identify novel subgroup-specific targets. Citation Format: Clark Wang, Frank Furnari. Unraveling SMARCB1 depletion in hiPSC-derived neural crest cells: Implications for ATRT cell of origin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 145.

Abstract 6498: MEK inhibitor mirdametinib suppresses MAP kinase pathway activity and inhibits tumor growth in atypical teratoid/rhabdoid tumors

Abstract Atypical teratoid/rhabdoid tumors (AT/RT) are deadly infantile brain tumors currently lacking effective treatments. Our prior studies examining AT/RT primary tissues have shown an elevated expression of phospho-ERK, a downstream effector of MEK in the mitogen-activated protein (MAP) kinase pathway. The increased activity of this pathway potentially leads to more aggressive cancer growth, emergence of drug resistance, and poor treatment outcomes in AT/RT patients. Mirdametinib is a novel, highly specific MEK1/2 inhibitor that has been reported to have superior blood-brain-barrier penetration compared to other MEK inhibitors. It is currently in development as a monotherapy for low-grade glioma as well as NF1-related plexiform neurofibromas. We hypothesized that mirdametinib can inhibit AT/RT tumor growth and improve overall survival. We examined the in vitro effect of mirdametinib treatment on five AT/RT cell lines (CHLA04, CHLA05, CHLA06, CHLA266, and BT37). Mirdametinib decreased MAP kinase pathway activity in the tested cell lines (western blot, phospho-ERK). Cell lines treated with mirdametinib exhibited decreased viability at 72hr (Muse Cell Viability Assay, CHLA05 t-test p=0.011, CHLA06 t-test p<0.001, BT37 t-test p<0.01 compared to DMSO control) and lower rates of proliferation (western blot, phospho-RB; BrdU immunofluorescence staining, CHLA05 t-test p=0.015, CHLA06 t-test p<0.0001, BT37 t-test p<0.0001 compared to DMSO control). Furthermore, mirdametinib induced apoptosis in AT/RT cells (western blot, cPARP; Muse Annexin V Assay, CHLA04, CHLA05, CHLA06, and BT37, t-test p<0.0001, CHLA266, t-test p<0.01 compared to DMSO control; cleaved caspase-3 immunofluorescence staining, CHLA05, CHLA06, and BT37, t-test p<0.0001 compared to DMSO control). We then tested the efficacy of mirdametinib in vivo. We treated mice bearing orthotopic CHLA06 tumors with a daily oral delivery of the drug. Mirdametinib treatment significantly extended the survival of treated mice in this very aggressive model (17 to 24 days, log-rank test p=0.038 compared to vehicle control) and decreased the expression of phospho-ERK (western blot, phospho-ERK), demonstrating that mirdametinib engaged its target in vivo. Testing against additional AT/RT orthotopic xenografts is currently underway. Our studies suggest that pharmacologically inhibiting the MAP kinase pathway may be an effective strategy to target AT/RT. Mirdametinib can potentially be an effective treatment to help extend survival in this deadly disease. Citation Format: Yiming Deng, Kristen Malebranche, Tyler Findlay, Anupa Geethadevi, Charles Eberhart, Jeffrey Rubens, Eric Raabe. MEK inhibitor mirdametinib suppresses MAP kinase pathway activity and inhibits tumor growth in atypical teratoid/rhabdoid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6498.

SDPS-18 RHABDOID TUMOR PREDISPOSITION SYNDROME IN TWO INFANT SIBLINGS WITH ATYPICAL TERATOID RHABDOID TUMORS DUE TO A GERMLINE SMARCB1 MUTATION

Abstract Atypical teratoid rhabdoid tumors (ATRT) are aggressive, malignant tumors of the central nervous system (CNS) that develop due to a loss-of-function mutation in the SMARCB1 gene. Rhabdoid tumor predisposition syndrome (RTPS) is characterized by familial cases as a result of pathogenic germline variants of this gene and an increased risk of developing ATRT within the first 2 years of life, which carries a poor prognosis. Genetic penetrance is incomplete with variable expressivity but the patient's age at time of diagnosis remains the most significant indicator of survival. We present a case of RTPS in a sibship born to unaffected parents, one of which was a carrier. The first sibling was diagnosed at 8 months of age after presenting with vomiting, developmental regression, and CN VI palsy for 3 months. Imaging revealed a posterior fossa mass, acquired obstructive hydrocephalus, and spinal metastasis. Four years later, her 3-month-old sister was brought in for evaluation due to vomiting, irritability, and macrocephaly for 2 months, along with a new vertical gaze palsy. Imaging findings also showed a posterior fossa mass and acquired obstructive hydrocephalus but without metastasis. Both siblings underwent craniotomy with subtotal tumor resection and ventriculoperitoneal shunt placement. Chemotherapy was promptly initiated but despite active treatment, they had rapid disease progression and ultimately passed away. Whole exome sequencing of both patients revealed an in-frame deletion involving the SNF5 homology domain of SMARCB1, with consistent findings on whole transcriptome sequencing, and evidence of loss of a single copy of chromosome 22. In this case report, we present a family faced with the devastating outcomes of RTPS and emphasize the importance of genetic testing and counseling for these families after diagnosis in the first child. Families should also be streamlined to a fertility specialist prior to subsequent pregnancies as an opportunity for familial screening.

ATRT-04. TARGETING THE COREST COMPLEX HELPS REPROGRAM AT/RT’S ABNORMAL EPIGENETIC LANDSCAPE TOWARD A DIFFERENTIATION PHENOTYPE

Abstract Atypical teratoid/rhabdoid tumors (AT/RT) are deadly infantile brain tumors. Their aggressive phenotype results from a single recurring biallelic loss-of-function mutation in genes encoding components of the SWI/SNF chromatin-remodeling complex - mainly SMARCB1 and SMARCA4 in remaining tumors. Residual SWI/SNF activity continues to inhibit EZH2 methyltransferase except at promoter regions colocalized to REST, which associates with neuronal differentiation and tumor suppressor genes. At these sites, EZH2 increases repressive H3K27me3 marks, helping maintain AT/RT in a stem cell-like state, and driving its aggressive growth and therapy resistance. We study the consequences of disrupting SWI/SNF inhibition at sites of EZH2/REST co-localization to target AT/RT precisely. The repressive action of REST is carried out mainly by the CoREST complex, consisting of LSD1, HDAC1/2 and RCOR. Lentiviral knockdown of RCOR2 reversed AT/RT’s stem cell-like state and induced differentiation along the glial and neuronal lineage. A decrease in the H3K27Me3 further confirmed the absence of COREST complex assembly at these sites. We used a bifunctional LSD1 and HDAC1/2 inhibitor, Corin, to pharmacologically target the CoREST complex in AT/RT cells. Corin also induced neuronal differentiation in AT/RT with increased expression of neuronal differentiation markers and decreased expression of stem cell markers. Further, Corin reduced AT/RT cell viability and proliferation (p<0.05) and increased apoptosis (p<0.05). In mice bearing orthotopic CHLA06 tumors, convection-enhanced delivery of a single dose of Corin increased H3K27ac, demonstrating that Corin engaged its target in vivo and induced apoptosis (western blot, cPARP). These studies demonstrate that targeting the CoREST complex disrupts AT/RT epigenetic abnormalities, reversing their stem cell-like state, slowing tumor cell growth, and inducing apoptosis. Targeting the CoREST complex with Corin is a promising new strategy that may translate into the clinical setting to help improve AT/RT’s dismal survival.

ATRT-16. THE PI3K INHIBITOR PAXALISIB COMBINES SYNERGISTICALLY WITH THE MEK INHIBITOR MIRDAMETINIB TO TARGET ATYPICAL TERATOID/RHABDOID TUMORS

Abstract Atypical teratoid/rhabdoid tumors (AT/RT) are aggressive pediatric brain tumors and the most common malignant brain tumors of infancy. The four-year event-free survival rate is only 37%. There are limited treatment options for patients with relapsed AT/RT, but precision therapies may help improve survival for patients with this deadly disease. We have previously identified strong activation of the PI3K-AKT-mTOR (mTOR) signaling pathways in AT/RT. Paxalisib is a highly brain-penetrant PI3K inhibitor acting upstream of mTOR. We find that Paxalisib slows tumor growth in orthotopic xenograft models of AT/RT and extends median survival from 40 to 54 days (p=0.001, log-rank test). RNASeq after mTOR pathway inhibition identifies reflexive activation of the RAS-RAF-MEK-ERK (MAPK) pathway as a possible mechanism of therapy resistance (KEGG pathway analysis). The RAS-RAF-MEK-ERK (MAPK) pathway plays a pivotal role in regulating cell growth and survival and activation of the pathway which contributes to the aggressive growth of numerous cancers. Mirdametinib is a small, allosteric MEK inhibitor currently used in clinical trials for both pediatric low- and high-grade gliomas (PD0325901). We find that dual inhibition of both the mTOR and MAPK pathways by Paxalisib and Mirdametinib synergize to reduce AT/RT growth and viability (Bliss synergy score 16.77). Paxalisib also combines with Mirdametinib to induce high levels of apoptosis and cell senescence (as determined by western blot: cPARP, pRB, P21, P16). Naïve treatments and a current survival study in mice bearing AT/RT orthotopic tumors suggest that combination therapy decreases mTOR and MAPK pathway activation (as determined by western blot). These data support using Paxalisib and Mirdametinib combination therapy as a promising novel therapy to treat relapsed AT/RT.

ATRT-01. PARP INHIBITION AS A THERAPEUTIC TARGET IN ATRT

Abstract Atypical teratoid rhabdoid tumors (ATRTs) are highly aggressive pediatric brain tumors with poor survival. The available treatments rely on toxic chemotherapy and radiotherapy, which alone can cause poor outcomes for young patients. Poly (ADP-ribose) polymerases (PARP), multifunctional enzymes which play an important role in DNA damage repair and genome stability have emerged as a new target in cancer therapy. An FDA approved drug screen revealed that Rucaparib, a PARP inhibitor, is important for ATRT cell growth.In this study we investigated the effect of Rucaparib treatment in ATRT. Using MTS and colony formation assays we found that Rucaparib treatment decreased ATRT cell growth and inhibited clonogenic potential of ATRT cells. Flow cytometry and Western blot analysis showed that Rucaparib treatment led to cell cycle arrest and apoptosis. Moreover, Rucaparib treatment led to DNA damage accumulation as seen by increased expression of yH2AX by immunofluorescence. In vivo, Rucaparib treatment significantly decreased tumor growth and prolonged survival in orthotopic mouse models. Furthermore, Immunohistochemistry revealed an increased number of P53 and Caspase3positive cells in the Rucaparib treatment group, confirming the induction of apoptosis in vivo. Furthermore, Rucaparib pretreatment sensitizes ATRT cells to radiation and significantly increased survival of xenograft-bearing mice. Thus, we demonstrated that Rucaparib has potential to be a new therapeutic strategy for ATRT as shown by its ability to decrease ATRT tumor growth both in vitro and in vivo.

ATRT-15. ATRTS HIJACK EPIGENETIC MECHANISMS TO PROMOTE IMMUNOSUPPRESSION AND ESCAPE FROM NK CELL THERAPY

Abstract Atypical Teratoid/Rhabdoid Tumors (ATRTs) are malignant tumors of the central nervous system in children. They are almost universally characterized by genetic loss of SWI/SNF Related, Matrix Associated, Actin Dependent Regulator of Chromatin Subfamily member B1 (SMARCB1) - a core subunit of the ATP-dependent SWI/SNF chromatin remodeling complex, and dysregulation of lineage restricted enhancers. Here, we show that tumor subgroup-specific upregulation of a distant member of the transforming growth factor-β (TGF-β), bone morphogenic factor (BMF) family of proteins drives insensitivity to NK-mediated cytolysis in vitro and in vivo. Mechanistically, subgroup-specific loss of chromatin occupancy by the RE1 Silencing Transcription Factor (REST) to its consensus RE1 motif, allowed deposition of enhancer-specific histone H3 lysine4 (K4) mono-methylation and histone H3K27 acetylation marks to activate an enhancer-promoter interaction. Restoration of REST binding or BET-domain inhibition resulted in a significant reduction in the enhancer marks and enhancer-promoter interaction and involved histone H3K27me3 deposition at the REST binding site, to facilitate gene silencing and sensitization of ATRTs to NK-mediated cytolysis. Thus, our study has identified a novel function for REST as a modulator of tumor-intrinsic enhancer activity and tumor-extrinsic immune microenvironment. Of equal significance, we highlight a role of REST in recruiting the polycomb repressive complex-2 (PRC2) in gene silencing in ATRTs.

Abstract 1141: The CoREST complex as a novel target to disrupt AT/RT’s abnormal epigenetic landscape

Abstract Atypical teratoid/rhabdoid tumors (AT/RT) are deadly infantile brain tumors. Their aggressive phenotype results from a single recurring biallelic loss-of-function mutation in genes encoding components of the SWI/SNF chromatin-remodeling complex - SMARCB1 in the majority of tumors and SMARCA4 in remaining tumors. Residual SWI/SNF activity continues to inhibit EZH2 methyltransferase except at gene promoter regions where EZH2 co-localizes with the REST complex. At these sites, EZH2 increases repressive H3K27me3 marks. EZH2 co-localizes with REST on neuronal differentiation and tumor suppressor genes. As a result, this epigenetic abnormality helps maintain AT/RT in a stem cell-like state, driving its aggressive growth and therapy resistance. We study the consequences of disrupting SWI/SNF inhibition at sites of EZH2/REST co-localization to target AT/RT precisely. While there are no known interactions between the REST complex and SWI/SNF, there are interactions between REST’s primary co-factor CoREST and SWI/SNF. The CoREST complex consists of LSD1, HDAC1, and the scaffolding protein RCOR. We disrupted the CoREST complex with lentiviral knockdown of RCOR2 (kdRCOR2). KdRCOR2 reversed AT/RT’s stem cell-like state, driving neuronal differentiation and slowing AT/RT growth. Interestingly, kdRCOR1 had no impact on cell differentiation (microscopy and qRT-PCR) and had a modest impact on cell growth (MUSE cell viability assay). We next used Corin to pharmacologically target the CoREST complex in 3 AT/RT cell lines (CHLA05, CHLA06, and BT37). Corin is a bi-functional inhibitor of LSD1 and HDAC1/2, the component enzymes of the CoREST complex. Similar to kdRCOR2, Corin induced neuronal differentiation in AT/RT with increased adherence to plastic and new axonal development (microscopy). Cells also increased expression of neuronal differentiation markers identified by immunofluorescence (MAP2) and qRT-PCR (CEND1, and NEUROD2, t-test p<0.05), and decreased expression of stem cell markers (western blot SOX2 and LIN28). In addition, Corin reduced AT/RT cell viability and proliferation (MUSE Cell viability assay p<0.05; MUSE BrdU assay p<0.05) and increased apoptosis (MUSE Annexin V assay, p<0.05). In mice bearing orthotopic CHLA06 tumors, convection-enhanced delivery of a single dose of Corin increased H3K27me3 and H3K27ac, demonstrating that Corin engaged its target in vivo and induced apoptosis (western blot, PARP). These studies demonstrate that targeting the CoREST complex disrupts AT/RT epigenetic abnormalities, reversing their stem cell-like state, slowing tumor cell growth, and inducing apoptosis. Targeting the CoREST complex with Corin is a promising new strategy that may translate into the clinical setting to help improve AT/RT’s dismal survival. Citation Format: Anupa Geethadevi, Marianne Collard, Nikhil Vaidya, Tyler Findlay, Kristen Malebranche, Charles Eberhart, Eric Raabe, Philip Cole, Rhoda Alani, Jeffrey Rubens. The CoREST complex as a novel target to disrupt AT/RT’s abnormal epigenetic landscape [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1141.

Abstract 5509: The PI3K inhibitor Paxalisib combines synergistically with the MEK inhibitor Mirdametinib to target atypical teratoid/rhabdoid tumors

Abstract Atypical teratoid/rhabdoid tumors (AT/RT) are aggressive pediatric brain tumors and the most common malignant brain tumors of infancy. The four-year event-free survival rate is only 37%. For patients with relapsed AT/RT there are limited treatment options, but precision therapies may help improve survival for patients with this deadly disease. We have previously identified strong activation of the PI3K-AKT-mTOR (mTOR) signaling pathways in AT/RT. Paxalisib is a highly brain-penetrant PI3K inhibitor acting upstream of mTOR. We find that Paxalisib slows tumor growth in orthotopic xenograft models of AT/RT and extends median survival from 40 to 54 days (p=0.001, log-rank test). RNASeq after mTOR pathway inhibition identifies reflexive activation of the RAS-RAF-MEK-ERK (MAPK) pathway as a possible mechanism of therapy resistance (KEGG pathway analysis). The RAS-RAF-MEK-ERK (MAPK) pathway plays a pivotal role in regulating cell growth and survival and activation of the pathway contributes to the aggressive growth of numerous cancers. Mirdametinib is a small, allosteric MEK inhibitor currently used in clinical trials for both pediatric low- and high-grade gliomas (PD0325901). We find that dual inhibition of both the mTOR and MAPK pathways by Paxalisib and Mirdametinib synergize to reduce AT/RT growth and viability (Bliss synergy score 16.77). Paxalisib also combines with Mirdametinib to induce high levels of apoptosis and cell senescence (as determined by western blot: cPARP, pRB, P21, P16). Naïve treatments in mice bearing AT/RT orthotopic tumors suggests the combination therapy decrease mTOR and MAPK pathway activation (as determined by western blot). These data support the use of Paxalisib and Mirdametinib combination therapy as a promising novel combination therapy to treat relapsed AT/RT. Citation Format: Kristen J. Malebranche, Tyler Findlay, Anupa Geethadevi, Charles Eberhart, Eric Raabe, Jeffrey Rubens. The PI3K inhibitor Paxalisib combines synergistically with the MEK inhibitor Mirdametinib to target atypical teratoid/rhabdoid tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5509.

ATRT-24. CDK7 Inhibition in AT/RT

Atypical teratoid/rhabdoid tumors (AT/RT) are CNS tumors with a 5-year survival of ~35%. AT/RT is characterized by loss-of-function mutations in the SMARCB1 component of the SWI/SNF (SWItch/Sucrose Non-Fermentable) complex. Based on preliminary CRISPR-Cas9 gene essentiality screen results identifying AT/RT vulnerabilities, we hypothesized that interaction between CDK7 and the SWI/SNF complex via SMARCB1 provides a potential target to improve clinical survival of patients. CDK7 expression was identified by microarray in AT/RT, medulloblastoma, glioblastoma and normal brain. Established cell lines (BT12, BT16, CHLA06), patient derived lines (MAF-737, MAF-1298, MAF-1337), normal human astrocytes (NHA) and NIH3T3 mouse embryonic fibroblast cells were utilized for in vitro response to CDK7 inhibition. Murine cerebellar xenografts of MAF-737 were utilized to evaluate genetic and pharmacologic response to CDK7 inhibition. The NCI Approved Oncology Drugs (AOD-9) Panel was evaluated with an IC25 dose of CDK7 inhibitor THZ2 to identify potential synergistic combinations. CDK7 is up-regulated in AT/RT compared to other brain tumors or normal brain. In vitro, AT/RT cells are highly susceptible to CDK7 pharmacologic inhibition with nM IC50 levels. AT/RT cells with shRNA against CDK7 implanted in vivo show significantly reduced growth. Evaluation of in vivo tumors treated with THZ2 demonstrate decreased Ki-67 and reduced pRBP1 demonstrating effective inhibition of the target as well as a decrease in cell proliferation. Combination therapy of THZ2 with the AOD-9 Panel found significant synergy with antimetabolite therapies, specifically pemetrexed, pralatrexate, and methotrexate. There was no synergy with other standard chemotherapy. Our findings demonstrate that CDK7 is highly expressed in AT/RT and necessary for proliferation of AT/RT cells, suggesting it as a potential therapeutic target. Antimetabolites, which are currently used in several AT/RT protocols, synergized with CDK7 inhibition offers a potential future combination therapy for patients.

ATRT-12. LIN28A expression correlates with poor prognosis and the MYC subgroup in AT/RTs

Atypical teratoid/rhabdoid tumors (AT/RTs) are malignant embryonal tumors of the central nervous system, which mainly affect young children. These tumors are defined by loss of SMARCB1 (or SMARCA4 in rare cases) and can be categorized into three main DNA methylation subgroups (i.e. –MYC, -SHH, -TYR). AT/RTs commonly display heterogeneous expression of LIN28A, an RNA-binding protein, which regulates pluripotency and plays critical roles during embryonic development. The biological impact and clinical significance of LIN28A expression in AT/RTs remains unknown. In this study, we investigated 80 samples of molecularly and clinically characterized AT/RTs for LIN28A expression using immunohistochemistry. Staining signal of tumor tissue was assessed and scored via semi-automated digital image analysis. Global LIN28A expression intensity and heterogeneity were tested for correlation with DNA methylation subtype, tumor localization, as well as patient age, gender and overall survival. LIN28A was found with strongly varying staining patterns and intensities across our cohort of AT/RTs, with significantly elevated expression in the MYC subgroup. Moreover, we identified strong global and focal LIN28A expression as an independent negative prognostic factor in AT/RTs. In summary, we show that AT/RT-MYC tumors display significantly increased LIN28A expression in comparison to the other DNA methylation subgroups, suggesting a subgroup-specific intratumoral role of LIN28A. Furthermore, we demonstrate an impact of LIN28A expression on survival in AT/RTs. Further investigations on the functions of LIN28A in AT/RTs in vitro and in vivo are ongoing and aim to uncover potential therapeutic implications in these tumors.

ATRT-10. Single-cell transcriptional profiling of ATRTs reveals heterogeneous signatures of tumor and non-malignant cell populations

Atypical Teratoid/Rhabdoid Tumors (ATRTs) are known for exhibiting high inter-tumor heterogeneity, even though they are almost all characterized by a common loss of SMARCB1 (or rarely SMARCA4). Three subgroups have been identified at bulk methylome and transcriptome level: ATRT-TYR, ATRT-SHH, and ATRT-MYC. To better understand the biology underlying each subgroup and potentially unveil their (different) cell(s) of origin, we performed single-cell transcriptomic analyses in 22 ATRTs using fresh frozen samples and both 10X and Smartseq technology. All data, grouped by technology, underwent quality control and normalization, regressing out the biases introduced by each sample. Tumor microenvironment (TME) and tumor bulk (TB) clusters were characterized by a combination of copy number variant analyses, enrichment in literature lists of marker genes for specific cell populations, and in-depth analysis of differentially enriched (DE) genes. Non-negative Matrix Factorization (NMF) was applied to TB to reveal major transcriptional profiles, which were grouped into meta-signatures. A total of 71 gene lists were retrieved from NMF (TB) and DE analyses (TME + TB), that gathered into 11 signature groups by Jaccard similarity, with one extra group accounting for unique signatures. Three groups targeted TME, accounting for either microglia, fibroblasts and endothelial cells, or OPCs, oligodendrocytes, astrocytes and neurons. These signatures are enriched in specific clusters across technologies. The remaining eight groups divide into two types, either enriched in clusters predominantly formed by cells of one or two ATRT subgroups or signatures enriched for a particular phenotype, such as cilial, cycling, axonogenesis or EM transition. While the first type is enriched across clusters in a gradient fashion, the second shows enrichment for selected clusters across technologies. Further analyses on the integrated dataset and additional samples are ongoing to validate and refine these 11 signature groups in ATRTs to see how this may lead to new treatment approaches.

ATRT-03. Adapted Treatment Protocol: Synchronous Atypical Teratoid/Rhabdoid CNS Tumor and Extra CNS Disease

Abstract Atypical teratoid/rhabdoid tumors (AT/RTs) of the central nervous system (CNS) are rare, aggressive, early childhood tumors with unfavorable prognosis. There have been 31 cases reported of children with AT/RT of the CNS and extra CNS primary tumors. In addition to its aggressive tendencies, malignant rhabdoid tumors (MRTs) of the kidney have also shown a common genetic abnormality-inactivating mutation of SMARC B1/INI-gene. We report a 22-month-old male who presented at 15 months of age with metastatic AT/RT of the posterior fossa and synchronous malignant rhabdoid tumor of the left kidney. MRI of the brain demonstrated a midline posterior fossa mass and left renal mass was noted incidentally on imaging of spine. Pathology was consistent with MRT of the kidney and pathogenic variant was found in the tumor sample, specifically SMARCB1 homozygous/biallelic deletion. Patient underwent a subtotal resection of the posterior fossa tumor and subsequent radical resection of the mass on kidney rhabdoid tumor. He was treated as per ACNS033 protocol with 2 cycles of induction with high-dose methotrexate followed by vincristine, cyclophosphamide, cisplatin, and etoposide with complete response followed by three tandem stem cell transplants with thiotepa and carboplatin for which he has been tolerating and responding favorably. Focal radiation therapy to the brain and flank area is planned at end of therapy. In a large series of synchronous AT/RTs reported in 2017 only 3 of the 31 patients were considered long-term survivors. All received a combination of high dose intrathecal or intravenous chemotherapy, total resection of at least one of the tumors, focal radiation, and autologous peripheral blood stem cell transplant. We demonstrated a case with a favorable response with our treatment. Treatment continues to be challenging given the tumor’s rarity and mortality as there are no standardized protocols or randomized controlled trials.

ATRT-11. Anatomico-biological correlations define a new layer for ATRT molecular subgroups pointing to potential lineages of origin

Atypical teratoid rhabdoid tumors (ATRT) are divided in three molecular subgroups, the so-called MYC, TYR and SHH subgroups. This heterogeneity suggests some diversity in the cells of origin, which remain hypothetical thus far. A careful radiological review of 55 MRI at diagnosis was performed in parallel with a careful analysis of mouse tumor origin in the Rosa26-CreERT2::Smarcbflox/flox model. Methylation, bulk RNAseq and scRNAseq analyses were integrated to these anatomic information to highlight potential origin for each molecularly and anatomically defined subgroups. We demonstrated that mouse Myc-ATRTs derive from extra-parenchymal meningeal areas, a finding consistant with many human MYC ATRT being clearly of intra-cranial extra-axial origin. Although this finding could point to a neural crest origin, transcriptomic features fail to unravel any lineage-specific signature. We also defined a distinct supra-tentorial SHH ATRT subgroup, characterized both in mouse and Humans by neural features pointing to the ganglionic eminence progenitors as the candidate origin. Finally we identified a distinct infra-tentorial SHH ATRT subgroup, not observed in mice, with hindbrain/midbrain boundary progenitor signature. scRNAseq from human SHH infra-tentorial tumors consistently suggest a dedifferentiation process involving the Notch pathway in the oncogenic transformation of hindbrain/midbrain neural progenitors.

Dual mTORC1/2 inhibition compromises cell defenses against exogenous stress potentiating Obatoclax-induced cytotoxicity in atypical teratoid/rhabdoid tumors

Atypical teratoid/rhabdoid tumors (AT/RT) are the most common malignant brain tumors of infancy and have a dismal 4-year event-free survival (EFS) of 37%. We have previously shown that mTOR activation contributes to AT/RT’s aggressive growth and poor survival. Targeting the mTOR pathway with the dual mTORC1/2 inhibitor TAK-228 slows tumor growth and extends survival in mice bearing orthotopic xenografts. However, responses are primarily cytostatic with limited durability. The aim of this study is to understand the impact of mTOR inhibitors on AT/RT signaling pathways and design a rational combination therapy to drive a more durable response to this promising therapy. We performed RNASeq, gene expression studies, and protein analyses to identify pathways disrupted by TAK-228. We find that TAK-228 decreases the expression of the transcription factor NRF2 and compromises AT/RT cellular defenses against oxidative stress and apoptosis. The BH3 mimetic, Obatoclax, is a potent inducer of oxidative stress and apoptosis in AT/RT. These complementary mechanisms of action drive extensive synergies between TAK-228 and Obatoclax slowing AT/RT cell growth and inducing apoptosis and cell death. Combination therapy activates the integrative stress response as determined by increased expression of phosphorylated EIF2α, ATF4, and CHOP, and disrupts the protective NOXA.MCL-1.BIM axis, forcing stressed cells to undergo apoptosis. Combination therapy is well tolerated in mice bearing orthotopic xenografts of AT/RT, slows tumor growth, and extends median overall survival. This novel combination therapy could be added to standard upfront therapies or used as a salvage therapy for relapsed disease to improve outcomes in AT/RT.

ATRT-05. REPURPOSED ANTI-MALARIAL QUINACRINE ACTIVATES P53 AND INHIBITS ATRT TUMORIGENICITY

Atypical teratoid rhabdoid tumors (ATRTs) are fatal pediatric brain tumors that warrant improved therapies urgently. ATRTs are characterized by loss of INI1, a subunit of the SWI/SNF chromatin-remodeling complex. ATRTs grow aggressively despite majority of primary tumors expressing p53, suggesting inactivation of this tumor suppressor pathway. Reactivation of p53 could be a potential therapeutic strategy for inhibiting ATRT growth. Our laboratory specializes in researching mechanisms contributing to ATRT pathogenesis and potential therapies. In line with this, we investigated an anti-malarial drug called quinacrine that has been safely used in children for decades and can induce p53 in renal cell carcinoma. We used 5 patient-derived ATRT cell lines (BT-37, BT-12, CHLA-06, CHLA-266, CHLA-05) for our studies. We show that ATRT cell lines treated with quinacrine for 6 hours show increased expression of p53, suggesting its activation. Treatment of ATRT cell lines with increasing doses of quinacrine for 24 hours showed dose-dependent decrease in cell growth and proliferation (assessed by MTS assay and BrdU incorporation, P<0.05) and increase in apoptotic cell death (CC-3 and cleaved PARP expression). Nude mice harboring flank tumors of ATRT cell lines and treated with quinacrine for 3 weeks showed significant reduction in tumor growth compared to control animals (P<0.05). Since quinacrine is a substrate for the drug-efflux proteins P-gp/BCRP, we used quinacrine in combination with elacridar (Pgp/BCRP inhibitor) in our intracranial xenograft experiments to increase quinacrine’s retention in the brain. Mice harboring intracranial xenografts of ATRT cells showed increased survival when treated with quinacrine and elacridar (median survival 46 days) compared to control animals (median survival 25 days). These results suggest that quinacrine inhibits ATRT growth, partly by activating p53. Our studies are the first to show quinacrine’s effect on ATRTs and our current experiments include further investigation of quinacrine’s mechanism.

СОВРЕМЕННЫЕ МЕТОДЫ ДИАГНОСТИКИ И ЛЕЧЕНИЯ АТИПИЧНЫХ ТЕРАТОИД-РАБДОИДНЫХ ОПУХОЛЕЙ ЦНС У ДЕТЕЙ

Atypical teratoid rhabdoid tumors (ATRT) of the CNS in children are a group of aggressive malignant neoplasms with extremely poor prognosis, and therefore these tumors are classified as IV (highest) grade of malignancy. At the time of diagnosis, 20% of patients are found to have metastasized to the brain and spinal cord. The pathogenesis of ATRT is based on the biallelic mutation of the SMARCB1 gene, more rarely SMARCA4 genes. Despite the use of intensive multimodal therapy, the survival rate of patients with ATRT remains low. Recently, several groups of scientists, based on epigenetic and transcriptional studies, have identified 3 molecular groups of ATRT with different therapeutic sensitivity to chemoradiation therapy and, accordingly, have a different prognosis of the disease. This article presents the current literature data on epidemiology, pathogenesis, clinic, diagnosis and therapy of patients with ATRT, and describes the directions of treatment.

ATRT-18. SHH-SUBTYPE ATYPICAL TERATOID/RHABDOID TUMORS ARE SELECTIVELY SENSITIVE TO GEMCITABINE TREATMENT

Atypical Teratoid Rhabdoid Tumors (ATRT) are highly malignant embryonal tumors of the central nervous system with a dismal prognosis. ATRT can be divided into three molecular subgroups of which the Sonic Hedgehog (SHH) subgroup is most prevalent. In this study, we developed and validated a novel patient-derived ATRT model, which we used along a panel of other primary ATRT models for large scale drug discovery assays. We found that ATRTs are selectively sensitive to the nucleoside analogue gemcitabine, with SHH-subtype ATRTs being the most sensitive subgroup. Gene expression profiles and protein analysis indicated that gemcitabine treatment causes degradation of Sirtuin 1 (SIRT1), which causes ATRT specific cell-death through NF-kB and p53 activation. Furthermore, we found that this gemcitabine induced loss of SIRT1 results in a nucleus-to-cytoplasm shift of the SHH signaling activator Gli, explaining the additional gemcitabine sensitivity in SHH-subtype ATRT. Treatment of SHH-subgroup ATRT xenograft-bearing mice resulted in a >40% increase in median survival (p<0.01, log-rank test) and long-term survivors in two independent models. To prepare translation of our findings to the clinic, we investigated potential gemcitabine induced resistance mechanisms by conducting kinome-wide CRISPR/Cas9 knockout screens in primary ATRT cells. Through these experiments we found that low-dose gemcitabine treatment combined with inhibition of protein kinase C zeta (PKCζ) prevents regrowth of resistant ATRT subclones. Together, these findings show that ATRT are highly sensitive to gemcitabine treatment; and as such we suggest that gemcitabine may be rapidly incorporated into future treatment regimens for SHH-ATRT.