Atypical Teratoid Rhabdoid Tumor Cell Lines Research Articles

A kinome drug screen identifies multi-TKI synergies and ERBB2 signaling as a therapeutic vulnerability in MYC/TYR subgroup atypical teratoid rhabdoid tumors.

Atypical teratoid rhabdoid tumor (ATRT) is a rare, devastating, and largely incurable pediatric brain tumor. Although recent studies have uncovered 3 molecular subgroups of ATRTs with distinct disease patterns, and signaling features, the therapeutic profiles of ATRT subgroups remain incompletely elucidated. We examined the effect of 465 kinase inhibitors on a panel of ATRT subgroup-specific cell lines. We then applied multiomics analyses to investigate the underlying molecular mechanism of kinase inhibitor efficacy in ATRT subgroups. We observed that ATRT cell lines are broadly sensitive to inhibitors of the PI3K and MAPK signaling pathways, as well as CDKs, AURKA/B kinases, and polo-like kinase 1. We identified 2 classes of multikinase inhibitors predominantly targeting receptor tyrosine kinases including PDGFR and EGFR/ERBB2 in MYC/TYR ATRT cells. The PDGFRB inhibitor, Dasatinib, synergistically affected MYC/TYR ATRT cell growth when combined with broad-acting PI3K and MAPK pathway inhibitors, including Rapamycin and Trametinib. We observed that MYC/TYR ATRT cells were also distinctly sensitive to various inhibitors of ERBB2 signaling. Transcriptional, H3K27Ac ChIPSeq, ATACSeq, and HiChIP analyses of primary MYC/TYR ATRTs revealed ERBB2 expression, which correlated with differential methylation and activation of a distinct enhancer element by DNA looping. Significantly, we show the brain penetrant EGFR/ERBB2 inhibitor, Afatinib, specifically inhibited in vitro and in vivo growth of MYC/TYR ATRT cells. Taken together, our studies suggest combined treatments with PDGFR and ERBB2-directed TKIs with inhibitors of the PI3K and MAPK pathways as an important new therapeutic strategy for the MYC/TYR subgroup of ATRTs.

OTHR-16. HYPERACTIVATION OF THE INTEGRATED STRESS RESPONSE VIA PROTEASOME INHIBITION KILLS DMG AND ATRT WHILE SPARING NORMAL IPSC BRAIN ORGANOIDS

Abstract BACKGROUND H3K27-altered diffuse midline glioma (DMG) and atypical teratoid/rhabdoid tumor (ATRT) have increased baseline activation of the integrated stress response (ISR), an evolutionarily conserved system that enables cells to tolerate stress. This activation is manifested by elevated levels of ATF4. Transient or low-level expression protects cells from stress, while persistent, high-level activation leads to cell death. Ixazomib is an orally bioavailable proteasome inhibitor that causes endoplasmic reticulum stress, which is a major upstream activator of the ISR. We hypothesized that the high baseline level of ATF4 in DMG and ATRT would make these tumors susceptible to ISR activators such as ixazomib. METHODS After determining the IC50, DMG and ATRT cell lines were treated with increasing concentrations of ixazomib. To evaluate if ixazomib selectively kills DMG and ATRT, we treated iPSC brain organoids in parallel with tumor cells. Combination treatment of ixazomib and idarubicin, a brain-penetrant anthracycline, was conducted to assess synergy. RESULTS Ixazomib significantly induced apoptosis and suppressed proliferation in multiple cell lines, as shown by western blots for cleaved PARP and phospho-Rb expression as well as cleaved caspase-3 and BrdU immunofluorescence (DMSO vs. 25nM p<0.0001 by ANOVA). Additionally, western blots detected elevated ATF4 and CHOP expression in ixazomib treated cells, indicating activation of the ISR. CC3 immunofluorescence and western blot for cleaved PARP showed significantly increased apoptosis in DMG and ATRT cell lines treated with ixazomib (JHHDIPG1 and CHLA06 – DMSO vs. 25nM p<0.0001 by ANOVA), while iPSC brain organoids were not affected. Synergy testing of ixazomib and idarubicin showed an overall zero-interaction-potency (ZIP) synergy score of 23.464 in CHLA06 and 19.084 in HSJD007 (scores of 10 or above indicate synergy). CONCLUSIONS By over-activating the ISR, ixazomib kills DMG and ATRT cells without impacting normal iPSC brain organoids. This effect may potentially be enhanced with the addition of idarubicin.

ATRT-17. INHIBITING THE MAP KINASE PATHWAY WITH MEK INHIBITOR MIRDAMETINIB SUPPRESSES CANCER GROWTH IN ATYPICAL TERATOID/RHABDOID TUMORS

Abstract BACKGROUND Atypical teratoid/rhabdoid tumors (ATRT) are poor-prognosis infantile brain tumors. Our prior studies examining ATRT primary tissues have shown an increased expression of phospho-ERK, a downstream effector of MEK in the mitogen-activated protein kinase (MAPK) pathway. The elevated MAPK pathway potentially contributes to drug resistance, aggressive cancer growth, and poor treatment outcomes. Mirdametinib is a highly specific MEK1/2 inhibitor reported to have superior blood-brain-barrier penetration compared to other MEK inhibitors. We hypothesized that mirdametinib can suppress ATRT tumor growth and improve overall survival. METHODS Mirdametinib effects were assessed through western blot for MAPK markers. Western blot analysis, Muse flow cytometer assays, and immunofluorescence were used for proliferation and apoptosis. Mirdametinib impact on survival was determined in orthotopic xenograft models of ATRT. RESULTS Mirdametinib decreased MAPK pathway activity in five ATRT cell lines (western blot: phospho-ERK). Cell lines treated with mirdametinib exhibited lower rates of proliferation (western blot: phospho-Rb; BrdU immunofluorescence staining: CHLA05 p=0.015, CHLA06 p<0.0001, BT37 p<0.0001 compared to DMSO control by t-test). Mirdametinib induced apoptosis in ATRT cells (western blot: cPARP; Muse Annexin V Assay: CHLA04, CHLA05, CHLA06, and BT37, p<0.0001, CHLA266, p<0.01 compared to DMSO control by t-test; cleaved caspase-3 immunofluorescence staining: CHLA05, CHLA06, and BT37, p<0.0001 compared to DMSO control by t-test). Daily oral delivery of mirdametinib treatment significantly extended the survival of mice bearing the very aggressive orthotopic CHLA06 tumors (17 to 24 days, log-rank test p=0.038 compared to vehicle control) and decreased the expression of phospho-ERK (western blot). Testing against additional ATRT orthotopic xenografts is currently underway. CONCLUSIONS Mirdametinib inhibits the MAPK pathway both in vitro and in vivo, suppressing proliferation and inducing apoptosis in ATRT. Our studies suggest that pharmacologically inhibiting the MAPK pathway with mirdametinib may be an effective strategy to target ATRT and help extend survival in this deadly disease.

ATRT-15. COMBINING THE PI3K INHIBITOR PAXALISIB WITH NUCLEOSIDE ANALOG GEMCITABINE TO IMPROVE SURVIVAL OF ATYPICAL TERATOID/RHABDOID TUMORS

Abstract BACKGROUND Atypical teratoid/rhabdoid tumors (ATRT) have a dismal overall survival. Targeting the mTORC1/2 pathway kills ATRT tumor cells and increases the survival of mice bearing orthotopic xenografts. Paxalisib is a brain penetrant PI3K inhibitor that is in pediatric clinical trials for diffuse midline glioma. Paxalisib activates the integrated stress response (ISR) in ATRT. Consistent high-level activation of the ISR leads to cell death. We hypothesize paxalisib will synergize with the DNA-damaging agent gemcitabine, which also triggers the ISR, to over-activate this pathway and drive ATRT apoptosis. METHODS We analyzed ISR gene expression following mTOR inhibition via RNA-seq. Children’s Brain Tumor Network (CBTN) provided comparative data on ISR genes in ATRT and other pediatric brain tumors. Paxalisib/gemcitabine effects were evaluated through cell-based assays, western blot for ISR markers, and survival in three murine models. RESULTS ATRT have high baseline expression of ISR genes (CBTN: ATF4, eIF2α), and mTOR inhibition leads to activation of ISR genes (ATF4, CHOP, PPP1R15A, p<0.05). Paxalisib monotherapy significantly extends median survival in murine models compared to vehicle controls (CHLA-06: 40 to 54 days, p=0.001; BT-12: 21 to 35 days, p=0.05). Paxalisib/gemcitabine therapy synergized to slow cell growth and induce cell death (SynergyFinder BLISS score in five ATRT cell lines). Combination therapy leads to ISR activation (western blot: p-eIF2α, ATF4, CHOP). Combination slowed tumor growth (bioluminescent imaging) and led to significant extension of survival in orthotopic xenograft models (CHLA-06: 22 to 82.5 days; BT-37 56-day vehicle median survival while undefined combination survival). A third model is ongoing and showing similar results. CONCLUSIONS Paxalisib/gemcitabine therapy shows potential for targeting ATRT by over-activating the ISR. Our findings prompted the Pediatric Neuro-Oncology Consortium to consider this combination for inclusion in an upcoming clinical trial targeting relapsed/refractory ATRT, offering a novel approach to treat this aggressive tumor.

ATRT-19. CELL SURFACE PROTEOME ANALYSIS OF ATRT IDENTIFIES CD44V6 AS A CAR TARGET

Abstract Atypical teratoid rhabdoid tumor (ATRT) is an uncommon, aggressive tumor affecting the brain and spinal cord primarily in children. Despite significant strides in understanding cancer biology through DNA and RNA sequencing, nearly 90% of ATRTs exhibit a crucial deletion that triggers rampant tumor growth. The absence of easily targetable genetic mutations in ATRT complicates the development of effective treatments, necessitating innovative strategies for therapy. Evading immune system detection is a key feature of cancer cells. In our study, we conducted an exhaustive, unbiased examination of 350 surface receptors through high-throughput multicolor flow cytometry on samples from ATRT surgeries, cell lines, and xenograft models derived from patients. By using a combination of antibodies targeting CD31, CD45, CD11b, CCR2, Cx3CR1, CD4, and CD8, we could identify immune cell populations within the tumors. Our findings revealed a heightened presence of CD44, CD146, CD59, CD151, and CD276 on the surface. Using qPCR we identified CD44V6 a variant isoform of the CD44 molecule, which is a cell surface glycoprotein involved in cell-cell interactions, cell adhesion, and migration. CD44 has multiple isoforms generated through alternative splicing, with CD44V6 being one of these variants. This specific isoform includes an additional variable exon (exon v6) in its extracellular domain. CD44v6-directed CAR-T cells effectively controlled tumor growth in multiple myeloma and solid tumors such as HNSCC, lung and ovarian adenocarcinomas. We developed CD44V6-directed CAR-T cells and investigated the capacity of Anti-CD44v6 CAR-T cells to execute cytotoxicity against ATRT tumor cells. We see significant cell killing by Anti-CD44v6 CAR-T cells in vitro and a significant increase in the survival of ATRT tumor-bearing mice.

ATRT-07. PRC1/BMI1 ACTIVITY ALTERS CHROMATIN ACCESSIBILITY TO REGULATE DIFFERENTIATION IN ATYPICAL TERATOID RHABDOID TUMORS

Abstract BACKGROUND SMARCB1 is a member of the SWI/SNF chromatin remodeling complex that is responsible for determining cellular pluripotency and lineage commitment. Atypical Teratoid Rhabdoid Tumor (ATRT) is a highly aggressive pediatric brain tumor and characterized by SMARCB1 deletion. METHODS Genome wide RNAi screen, RNA-seq and ChIP-Seq analysis, colony formation assay, cell cycle, western blotting, immunoprecipitation, mass spectrometry analysis, mouse xenograft model, IVIS and MRI imaging. RESULTS We performed an RNAi-based functional genomic screen to identify key epigenetic regulators that co-operate with SMARCB1 loss and contribute to tumorigenesis in ATRT. 406 genes associated with epigenetic regulation in patient-derived ATRT cell lines were targeted. This screen identified BMI1, a component of the Polycomb Repressive Complex 1 (PRC1), as one of the top targets essential for ATRT cell growth. Using RNA-seq and ChIP-Seq analysis we demonstrated that BMI1 co-operates with SMARCB1 deletion to suppress transcription of pro-differentiation pathways and promote self-renewal of tumor stem cells. In SMARCB1 deficient cells BMI1 forms a partial PRC1 complex devoid of DNA binding components. We found that genetic inactivation of BMI1 suppresses ATRT cell clonogenicity and neurosphere formation, leads to S-phase arrest and induces ATRT cell differentiation in vitro. In vivo BMI1 inhibition induces apoptosis, leading to significantly increasing survival of treated mice. By Doxycycline-inducible SMARCB1 re-expression system we revealed activation of proapoptotic proteins p16 and p21. With immunoprecipitation for BMI1, followed by mass spectrometry analysis we demonstrated that re-expression of SMARCB1 activates two PRC1 chromatin localizing components CBX4 and CBX8. Moreover, SMARCB1 re-expression significantly prolongs mice survival and decreases tumor size as evaluated by IVIS imaging system and T2-MRI. CONCLUSION Thus, we demonstrated that SMARCB1 deletion results in reprograming of BMI1 chromatin occupancy away from lineage specification by altering the components of the PRC1 complex and that PRC1/BMI1 inhibition is a novel therapeutic approach in ATRT.

Modeling human brain rhabdoid tumor by inactivating tumor suppressor genes in induced pluripotent stem cells

Atypical teratoid/rhabdoid tumor (ATRT) is a rare childhood malignancy that originates in the central nervous system. Over ninety-five percent of ATRT patients have biallelic inactivation of the tumor suppressor gene SMARCB1. ATRT has no standard treatment, and a major limiting factor in therapeutic development is the lack of reliable ATRT models. We employed CRISPR/Cas9 gene-editing technology to knock out SMARCB1 and TP53 genes in human episomal induced pluripotent stem cells (Epi-iPSCs), followed by brief neural induction, to generate an ATRT-like model. The dual knockout Epi-iPSCs retained their stemness with the capacity to differentiate into three germ layers. High expression of OCT4 and NANOG in neurally induced knockout spheroids was comparable to that in two ATRT cell lines. Beta-catenin protein expression was higher in SMARCB1-deficient cells and spheroids than in normal Epi-iPSC-derived spheroids. Nucleophosmin, Osteopontin, and Ki-67 proteins were also expressed by the SMARCB1-deficient spheroids. In summary, the tumor model resembled embryonal features of ATRT and expressed ATRT biomarkers at mRNA and protein levels. Ribociclib, PTC-209, and the combination of clofilium tosylate and pazopanib decreased the viability of the ATRT-like cells. This disease modeling scheme may enable the establishment of individualized tumor models with patient-specific mutations and facilitate high-throughput drug testing.

SYST-12 LP-184, A NOVEL ACYLFULVENE-DERIVED TUMOR SITE ACTIVATED SMALL MOLECULE INHIBITS ADULT AND PEDIATRIC CNS TUMOR CELL GROWTH

Abstract Blood-brain barrier (BBB) permeable agents effective against recurrent, chemotherapy-resistant central nervous system (CNS) tumors are urgently needed, particularly in Glioblastoma multiforme (GBM) and atypical teratoid/rhabdoid tumors (ATRT). These represent extremely aggressive and lethal types of CNS malignancies. LP-184 is a next-generation acylfulvene class drug candidate and has received FDA orphan drug designations for the treatment of malignant gliomas and ATRT. LP-184 creates covalent DNA adducts that are selectively repaired via nucleotide excision repair (NER). Repair of LP-184-induced DNA damage is mediated by ERCC complexes. Consistent with this, we observed 3-6X decreased cancer cell viability in U87, M1123 and Mayo39 GBM cells treated with LP-184 in combination with the ERCC3 degrader Spironolactone (SP) relative to LP-184 alone. Mice bearing pre-established subcutaneous U87 xenografts were treated with SP alone, LP-184 alone or their combination. Tumors recurred after initial regression in 5/5 animals treated with LP-184 alone whereas tumors showed durable complete regression without recurrence in 4/5 animals treated with LP-184 + SP. Thus, LP-184 likely functions as a synthetically lethal agent in the presence of DNA repair deficiencies. LP-184 inhibited cell growth in CHLA06 and BT37 ATRT cell lines, with IC50s in the 20-25nM range. LP-184 induced apoptosis 96 hours after treatment in these ATRT cell lines as measured by immunofluorescence for cleaved caspase-3 and Western blot for c-PARP. LP-184 intravenous treatment at 4 mg/kg twice weekly for 2 weeks showed tumor regression in mice implanted with CHLA06 ATRT subcutaneous xenografts, with 112% tumor growth inhibition and 2/10 treated mice being tumor-free at study termination. LP-184 further showed favorable intracranial tumor bioavailability along with in vivo BBB permeability comparable to other CNS cancer drugs. These findings identify LP-184 as a promising new agent and support its further development for treating CNS tumors in adult and pediatric populations.

ATRT-17. CAR T CELL THERAPY TARGETING CLAUDIN-6 IS EFFECTIVE IN CELLULAR AND XENOGRAFT MODELS OF ATYPICAL TERATOID/RHABDOID TUMOR

Abstract Novel therapies are needed to for the treatment of atypical teratoid/rhabdoid tumor (ATRT), an aggressive brain tumor that predominantly affects young children and has an average 5-year survival under 50%. Claudin-6 (CLDN6) is a tight junction protein present during development and is expressed in up to 70% of ATRT specimens but not in normal tissue, making it a promising immunotherapeutic target. CLDN6-targeted chimeric antigen receptor (CAR) T cells in combination with a CAR T cell-amplifying mRNA vaccine have demonstrated antitumor activity against other CLDN6-expressing cancers in pre-clinical and a phase I adult trial (NCT04503278; Haanen J et al AACR, 2022). To assess the effectiveness of CLDN6-targeted CAR T cells against ATRT, we utilized a second-generation mRNA CAR with a 4-1BB costimulatory domain and single-chain variable fragment against CLDN6 (Reinhard et al, 2020). CLDN6 expression in patient-derived ATRT specimens was profiled by RNAseq (mean FPKM 11.4) and immunohistochemistry (positive staining in 53% of specimens). Tumor-derived cell lines were assessed for CLDN6 expression by flow cytometry. Co-culture of CLDN6-directed mRNA CAR T cells resulted in tumor-specific cytotoxicity compared to CD19-directed control CAR T cells in CLDN6-positive ATRT cell lines 7316-2187 (92% versus 15% at 10:1, p < 0.0001; 86% versus 0% at 5:1, p<0.0001) and 7316-2141 (75% versus 7% at 10:1, p<0.0001; 53% versus 0% at 5:1, p< 0.0001). Patient-derived, orthotopic xenograft ATRT models were created through intracranial injection of tumor cells into the cerebellum of NSG mice. Following engraftment with 7316-2141, repeated intratumoral administration of mRNA CLDN6 CAR T cells resulted in significant tumor regression (-1.9x108 vs. +2.4x109 p/sec/cm2/sr, p<0.001) and improved survival compared to mRNA CD19 CAR T cells (median OS not reached vs. 13 days, p=0.002). This work highlights the potential for targeting CLDN6 via CAR T cell therapy in patients with ATRT as a novel therapeutic strategy.

Abstract 3933: RRM2 inhibition by COH29 is a potential therapeutic strategy for atypical teratoid rhabdoid tumors

Abstract Introduction: Atypical teratoid rhabdoid tumors (ATRT) are a rare but aggressive malignancy in central nervous system, commonly occurring in early childhood. ATRT patients face a dismal prognosis despite aggressive therapy. Development of both effective and protective targeted therapy is an emerging necessity. It has been reported that RRM2 is a master driver of aggressiveness and a poor prognosis biomarker in several cancers. However, little is known about RRM2 function in ATRT. The aim of this study is to assess the roles of RRM2 in ATRT and elucidate the therapeutic potential of RRM2 to cure the ATRT. Methods: Expression of RRM2 was evaluated by molecular profiling analysis and was confirmed by IHC in both ATRT patients and PDX tissues. Follow-up in vitro studies used 3 different cell lines to elucidate the oncogenic role of RRM2 in ATRT cells. The efficacy of COH29, an RRM2 inhibitor, was assessed in vitro. ATRT model of SCID mice orthotopically injected with ATRT cells were used to test the efficiency of COH29 for in vivo. Western blot and RNA-sequencing were used to determine the mechanisms of RRM2 transcriptional activation in ATRT. Results: Molecular profiling results revealed that RRM2 was relatively highly expressed in ATRT tissues and cell lines. Clinical relevance analysis results showed that the RRM2 expression level was associated with poor prognosis among ATRT patients. Knockdown of RRM2 by shRNAs significantly reduced ATRT cells colonies formation, proliferation and migration. A similar but more pronounced inhibitory effect was observed when COH29 was used to target RMM2. Inhibition of RRM2 by shRNAs or COH29 significantly induced cell death via DNA damage and/or apoptosis pathways. In vivo results demonstrated that COH29 could suppressed ATRT tumor growth and extend mice overall survival. Conclusion: Our study identified RRM2 as a potential mediator of oncogenic cellular functions in ATRT cell lines. We propose that highly expressed RRM2 is associated with poor patient outcomes, and inhibition of RRM2 by COH29 may present a significant therapeutic value in ATRT. Citation Format: Le Hien Giang, Tai-Tong Wong, Che- Chang. RRM2 inhibition by COH29 is a potential therapeutic strategy for 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 3933.

Abstract 4875: The anti-tumor effects of GSK-3β inhibitor (9-1NG-41) in ETMR and ATRT pediatric brain tumors

Abstract Purpose Embryonal brain tumors (EBTs): ETMR (embryonal tumor with multilayered rosettes) and ATRT (atypical teratoid rhabdoid tumor) are aggressive malignancies with poor prognosis necessitating alternative strategies. GSK-3β is a serine threonine kinase often overexpressed in solid tumors and a positive regulator of NF-κB which promotes tumor growth and chemotherapy resistance. 9-ING-41 is a maleimide-based small molecule that crosses the blood brain barrier and selectively inhibits GSK-3β. Previously we demonstrated that 9-ING-41 decreases ETMR and ATRT cell viability through apoptosis and increases p53 signaling, however the exact mechanism leading to its activation is unknown. This study aims to determine the optimal dosing for 9-ING-41 and describe the phenotypic response of these cell lines to 9-ING-41 using cell viability assay, western blotting and neurosphere assay. This information will help further elucidate the mechanism of action for this drug. Methods ATRT cell lines ATRT 2141, ATRT-787199, ATRT 803499, LCH-091-07 and the ETMR cell line BT-183 were grown for 24hrs prior to 9-ING-41 treatment in DMEM and Neurocult medium, respectively. Cell viability was assessed after 72hrs of treatment using Cell Titer Glo 2.0. Western blots were analyzed for XIAP, cleaved caspase-3, p53, MDM2 after 24 and 48 hours at 200nM, 400nM and 600nM. Neurosphere assays were done by seeding two cells per well in a 96-well plate followed by treatment with 9-ING-41. Neurosphere frequency and size was monitored weekly for up to 4 weeks using Incucyte® S3 software. Pearson’s correlation graph was generated to determine the relationship between GSK-3β expression and sensitivity to 9-ING-41. Graph Pad Prism was used for statistical analysis and level of significance set to p=0.05. Results The IC50s of 9-ING-41 in cell lines ATRT 2141, ATRT-787199, ATRT 803499 and LCH-091-07 were 194nM, 558nM, 730nM & 5353nM, respectively. We observed decreased expression of NF-κB mediated anti-apoptotic marker XIAP and an increase in expression of apoptotic marker cleaved caspase-3 and tumor suppressor p53 in response to 9-ING-41. MDM2, a negative regulator of p53 had a significant increase in expression indicating it is not responsible for p53 increase which may suggest ATM pathway could be responsible for regulating p53 increase. GSK-3β expression in vitro, correlated with sensitivity to 9-ING-41. There was a dose dependent decrease in frequency and size of neurospheres in response to 9-ING-41. Conclusion 9-ING-41 decreases cell viability and neurosphere formation in ETMR and ATRT cell lines within clinically relevant doses. p53 stabilization is not mediated by MDM2 and may be downstream of the ATM pathway. Further studies are underway to demonstrate its efficacy in mouse models. Citation Format: Divya Gandra, Lauren Difabio, Kaitlyn H. Smith, Kimberly Q. McKinney, Giselle S. Sholler. The anti-tumor effects of GSK-3β inhibitor (9-1NG-41) in ETMR and ATRT pediatric brain 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 4875.

351 Claudin 6 Expression in Atypical Teratoid/Rhabdoid Tumors is a Target for CAR-T Cell Therapy

INTRODUCTION: Novel therapies for atypical teratoid/rhabdoid tumor (ATRT) are desperately needed. Claudin-6 (CLDN6) is a tight junction protein present during early development and in some tumors, but not at appreciable levels in normal tissue. CLDN6-targeted chimeric antigen receptor (CAR) T cells with a CAR-T cell–amplifying RNA vaccine have demonstrated antitumor activity against CLDN6-expressing cancers in pre-clinical and clinical trials (NCT04503278; Haanen J et al AACR, 2022). METHODS: CLDN6 expression was assessed on primary ATRT specimens by RNAseq and immunohistochemistry and on tumor-derived cell lines by flow cytometry. CLDN6-positive and negative cell lines were co-cultured with CLDN6-targeting CAR-T cells and killing efficacy was measured. A second-generation mRNA CAR with a 4-1BB costimulatory domain and single-chain variable fragment directed against CLDN6 was used (Reinhard et al, 2020). Cell lines were modified with reporter assays and intracranial xenografts were created in NSG mice. RESULTS: 50% of specimens had CLDN6 expression greater than 2 FPKM (N = 28). Immunohistochemistry was positive in 53% of specimens (N = 15). Multiple CLDN6-positive ATRT cell lines were identified by flow cytometry. Co-culture of CLDN6-directed mRNA CAR-T cells with ATRT line 7316-2187 resulted in tumor-specific cytotoxicity versus CD19-directed control CAR-T cells (92% versus 15% at 10:1, p < 0.0001; 86% versus 0% at 5:1, p < 0.0001). Similar results were seen with an additional CLDN6-positive ATRT cell line 7316-2141 (75% versus 7% at 10:1, p < 0.0001; 53% versus 0% at 5:1, p < 0.0001). Both CLDN6- and CD19-directed CAR-T cells showed no cytotoxicity against a CLDN6-negative cell line, 7316-4149. Xenografts were successfully created, and ongoing work is evaluating locoregional administration of CLDN6-directed CAR-T cells. CONCLUSIONS: This work highlights the potential of targeting CLDN6 with CAR-T cell therapy as a novel treatment strategy for patients with ATRT.

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.

ATRT-01. Reconstitution of cGAS/ STING pathway via epigenetic reprogramming leads to anti-viral inflammatory signaling in Atypical teratoid rhabdoid tumors (ATRTs)

BACKGROUND: Atypical teratoid rhabdoid tumors (ATRTs) are highly aggressive brain tumors that affect young children characterized by biallelic inactivation of the SMARCB1 gene. Though patients benefit from multimodal therapy, there is no improvement in overall survival which necessitates the exploration of alternative approaches. Innate-based immune and epigenetic therapies have shown benefits in several cancers. The role of innate immune signaling has not been investigated in ATRTs. Our previous data from several ATRT cell lines showed loss of expression of key innate signaling components, like cGAS and STING that are needed for sensing extracellular dsDNA. Additionally, ATRT cell lines do not respond to STING agonists, like cGAMP or ISD. RESULTS: Co-treatment of ATRT cell lines, BT-12 and BT-16 with two epigenetic modulators, panobinostat and decitabine, leads to re-expression of cGAS and STING in a time-dependent manner. Furthermore, treatment with decitabine alone leads to demethylation of several CpG sites on the STING promoter and increased expression of STING mRNA. Panobinostat and decitabine co-treatment reconstitute STING-mediated innate signaling, as measured by IRF-3 and STAT1 phosphorylation and production of ISG-15 and IFIT-1 after treatment with cGAMP, a STING agonist. Co-treatment with panobinostat and decitabine also induced expression of antiviral pro-inflammatory chemokines/cytokines in ATRT cell lines, including type III IFN, IL-6, IL-8, IL-28, and IL-29. CONCLUSION: Our data suggest that ATRT cell lines are unresponsive to innate agonists possibly due to the loss of expression of key innate immune components. However, the cGAS/STING pathway is reactivated by epigenetic drugs, specifically the combination of panobinostat and decitabine. This is further potentiated by treating with STING agonists like cGAMP. Combination treatment of ATRT cell lines with panobinostat and decitabine also induced antiviral inflammatory signaling. This response could be a potential treatment modality to inhibit tumor growth and/or mediate cancer immunotherapy in these aggressive tumors.

ATRT-19. Functional genomics reveal distinct modulators of response to CDK4/6 inhibitors in ATRTs

Brain tumors are the leading cause of cancer-related deaths in children, and atypical teratoid rhabdoid tumors (ATRTs) are among the most common aggressive brain tumors in infants. With no standard-of-care treatment so far, ATRTs continue to have relatively low survival estimates, illustrating the urgent need for more efficacious treatment options. We have previously used genome-wide CRISPR/Cas9 knockout screens in combination with small-molecule drug assays to identify targetable vulnerabilities in ATRTs. CDK4/6 inhibitors, among the most promising drugs in our study with direct translational potential, are capable of inhibiting tumor growth due to mutual exclusive dependency of ATRTs on either CDK4 or CDK6. We here used genome-wide loss-of-function and gain-of-function strategies to identify modulators of response to CDK4/6 inhibition in ATRTs. Of note, while some well-known resistance mechanisms such as loss of RB1 or FBXW7 are shared by ATRT cell lines, we have also identified modulators of response to CDK4/6 inhibition with opposing effects across ATRT cell lines. As such, loss of AMBRA1, a recently described master regulator of D type cyclins, can either oppose the effects of or synergize with CDK4/6 inhibitors based on the cellular background. We are currently using a proteomics approach to further delineate the mechanism driving this functional heterogeneity of AMBRA1 in ATRTs. Our study will therefore provide deeper insights into the response of ATRTs to CDK4/6 inhibitors, which represent one of the most promising class of targeted agents for the treatment of ATRTs.

ATRT-13. An integrative analysis of the ATRT proteome unravels novel drug targets and refines molecular subgrouping

INTRODUCTION: Atypical teratoid/rhabdoid tumors (ATRT) represent frequent brain tumors in infants. In recent years, large-scale landscaping efforts on the epigenome and transcriptome of these tumors have unravelled a high degree of heterogeneity and three major molecular subgroups, termed ATRT-TYR, ATRT-SHH,ATRT-MYC, have been identified. The ATRT-proteome, in turn, still represents a largely unchartered territory. METHODS: We have performed a peptide-based screening approach to characterize the proteome of 40 ATRTs and six ATRT cell-lines. All of these samples had matching methylation data available and 28 also corresponding gene expression data. RESULTS: Unsupervised clustering recapitulated the previously described ATRT groups, revealing also a clear split of the SHH-subgroup in a supratentorial (SHH_1) and an infratentorial subgroup (SHH_2). Overall, we identified 7265 proteins, of which 1320 were differentially expressed between the groups, with an enrichment of spliceosome associated terms in SHH_1 and integrins/cell adhesions molecules in SHH_2. ATRT-MYC displayed an overrepresentation of immune cell markers and the TYR subgroup an enrichment of PI3K- as well as mTOR-signaling. Particularly, genes that have previously been described as signature genes for the ATRT-groups such as FABP7 in ATRT-SHH and OTX2 and MITF in ATRT-TYR were among the highly correlating genes that were both expressed in the proteome and the gene expression datasets. On top of this, our analysis revealed highly differentially expressed drug targets such as the tyrosine-kinase MARCKS (overexpressed in ATRT-TYR) not previously identified in ATRT transcriptome data, which warrant investigation by in vitro drug tests. CONCLUSION: Our data reveal the importance of previously described regulatory hubs in the ATRT subgroups, but additionally highlight novel drug targets that merit further exploration. Currently, drug treatment experiments in ATRT cell lines are ongoing to validate these proteins as drug targets, ultimately aiming to establish new therapeutic strategies in this deadly disease.

Targeting the TP53/MDM2 axis enhances radiation sensitivity in atypical teratoid rhabdoid tumors.

Atypical teratoid rhabdoid tumor (ATRT) is a highly aggressive pediatric brain tumor. Despite radiation, aggressive chemotherapy and autologous stem cell rescue, children usually have a poor survival time. In the present study, the role of TP53/MDM2 interaction in ATRT was investigated. A functional genomic screen identified the TP53/MDM2 axis as a therapeutic target in the central nervous system (CNS) ATRT. Gene expression analysis revealed that all ATRT sub-groups expressed high levels of MDM2, which is a negative regulator of TP53. Using cell viability, colony formation and methylcellulose assays it was found that genetic MDM2 inhibition with short hairpin RNA or chemical MDM2 inhibition with small molecule inhibitors, Nutlin3 and idasanutlin (RG7388) decreased the growth of ATRT cell lines. Furthermore, idasanutlin significantly decreased the growth of intracranial orthotopic ATRT brain tumors, as evaluated using T2 MRI, and prolonged survival time relative to control animals. MRI of intracranial tumors showed that diffusion coefficient, an effective marker for successful treatment, significantly increased with idasanutlin treatment showing tumor necrosis/apoptosis. Immunohistochemistry revealed an increased number of caspase-3-positive cells in the idasanutlin treatment group, confirming the induction of apoptosis in vivo. Using flow cytometry and western blot analysis we show that inhibition of MDM2 enhanced radiation sensitivity in vitro by potentiating DNA damage via the induction of the TP53/Bax/Puma proapoptotic axis. Furthermore, DNA damage was associated with increased mitochondrial reactive oxygen species accumulation. The present study demonstrated that MDM2 expression level was increased in ATRT patient samples and MDM2 inhibition suppressed ATRT cell growth in vitro, and leads to apoptosis in vivo. MDM2 inhibition potentiates DNA damage and sensitizes ATRT cells to radiation. These findings highlight the TP53/MDM2 axis as a rational therapeutic target in CNS ATRT.

Targeting of cyclin-dependent kinases in atypical teratoid rhabdoid tumors with multikinase inhibitor TG02.

Atypical teratoid rhabdoid tumors (ATRTs) are aggressive pediatric brain tumors with no current standard of care and an estimated median patient survival of 12 to 18 months. Previous genetic analyses have implicated cyclin D1 and enhancer of zeste homolog 2 (EZH2), a histone methyltransferase that is implicated in many cancers, as key drivers of tumorigenicity in ATRTs. Since the effects of EZH2 and cyclin D1 are facilitated by a host of cyclin-dependent kinases (CDKs), the authors sought to investigate the potential therapeutic effects of targeting CDKs in ATRTs with the multi-CDK inhibitor, TG02. Human ATRT cell lines BT12, BT37, CHLA05, and CHLA06 were selected for investigation. The effects of TG02 on cell viability, proliferation, clonogenicity, and apoptosis were assessed via Cell Counting Kit-8 assays, cell counting, clonogenic assays, and flow cytometry, respectively. Similar methods were used to determine the effects of TG02 combined with radiation therapy (RT) or cisplatin. Synergism indices for TG02-cisplatin combination therapy were calculated using CompuSyn software. TG02 was observed to significantly impair ATRT cell growth in vitro by limiting cell proliferation and clonogenicity, and by inducing apoptosis. TG02 inhibited ATRT cell proliferation and decreased cell viability in a dose-dependent manner with nanomolar half maximal effective concentration (EC50) values (BT12, 207.0 nM; BT37, 127.8 nM; CHLA05, 29.7 nM; CHLA06, 18.7 nM). TG02 (150 nM) dramatically increased the proportion of apoptotic ATRT cells 72 hours posttreatment (TG02 8.50% vs control 1.52% apoptotic cells in BT12, p < 0.0001; TG02 70.07% vs control 15.36%, p < 0.0001). Combination therapy studies revealed that TG02 acted as a potent radiosensitizer in ATRT cells (BT12 surviving fraction, RT 51.2% vs RT + TG02 21.7%). Finally, CompuSyn analysis demonstrated that TG02 acted synergistically with cisplatin against ATRT cells at virtually all therapeutic doses. These findings were consistent in cell lines that cover all three molecular subgroups of ATRTs. The results of this investigation have established that TG02 is an effective therapeutic against ATRTs in vitro. Given the lack of standard therapy for ATRTs, these findings help fill an unmet need and support further study of TG02 as a potential therapeutic option for patients with this deadly disease.

Abstract 4808: Preclinical studies of the combination of ONC201, radiotherapy and Temozolomide against GBM, DIPG and ATRT cell lines

Abstract The American Cancer Society predicts, in 2018, there will be ~23,880 new cases and 16,830 deaths caused by primary cancer of the brain and spinal cord, in the US. CNS tumors are the most common cause of cancer-related deaths in adolescents and young adults. First-in-class small-molecule imipridone ONC201 can act as a dual inhibitor of ERK/AKT and can induce an integrated stress response (ISR), pro-apoptotic TRAIL receptor DR5 activation, cancer stem cell depletion, and growth arrest. ONC201 crosses the blood brain barrier and has demonstrated clinical benefits in glioblastoma patients. Radiation therapy is used alone or in combination with surgery and/or chemotherapy such as Temozolomide in the treatment of primary or metastatic brain tumors. We hypothesized that ONC201 may synergize with radiotherapy and Temozolomide in brain tumor treatment. Glioblastoma (GBM), diffuse intrinsic pontine glioma (DIPG) and atypical teratoid rhabdoid tumor (ATRT) cell lines were tested in this study. Cell viability and colony formation assays were performed with ONC201 alone up to 20 μM or in combination with radiotherapy up to 8 Gy and Temozolomide up to 100 μM. Western blots were used to document apoptosis of treated cells. We observed synergy between ONC201 and radiation and between ONC201 and Temozolomide with the best combination indices of 0.51 and 0.21 respectively. Further studies are evaluating the role of dopamine receptors engaged by ONC201, the ISR and TRAIL pathway in the synergistic effect that would support further development of the triple combination therapy in brain tumors. Citation Format: Lanlan Zhou, Wafik S. El-Deiry. Preclinical studies of the combination of ONC201, radiotherapy and Temozolomide against GBM, DIPG and ATRT cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4808.

Comprehensive Analysis of Chromatin States in Atypical Teratoid/Rhabdoid Tumor Identifies Diverging Roles for SWI/SNF and Polycomb in Gene Regulation

Biallelic inactivation of SMARCB1, encoding a member of the SWI/SNF chromatin remodeling complex, is the hallmark genetic aberration of atypical teratoid rhabdoid tumors (ATRT). Here, we report how loss of SMARCB1 affects the epigenome in these tumors. Using chromatin immunoprecipitation sequencing (ChIP-seq) on primary tumors for a series of active and repressive histone marks, we identified the chromatin states differentially represented in ATRTs compared with other brain tumors and non-neoplastic brain. Re-expression of SMARCB1 in ATRT cell lines enabled confirmation of our genome-wide findings for the chromatin states. Additional generation of ChIP-seq data for SWI/SNF and Polycomb group proteins and the transcriptional repressor protein REST determined differential dependencies of SWI/SNF and Polycomb complexes in regulation of diverse gene sets in ATRTs.