Intracranial Xenograft Model Research Articles

Targeted Extracellular Vesicles Delivered Verrucarin A to Treat Glioblastoma.

Glioblastomas, accounting for approximately 50% of gliomas, comprise the most aggressive, highly heterogeneous, and malignant brain tumors. The objective of this study was to develop and evaluate a new targeted therapy, i.e., highly potent natural compound verrucarin A (Ver-A), delivered with monoclonal antibody-directed extracellular vesicle (mAb-EV). First, the high surface expression of epidermal growth factor receptor (EGFR) in glioblastoma patient tissue and cell lines was confirmed using immunohistochemistry staining, flow cytometry, and Western blotting. mAb-EV-Ver-A was constructed by packing Ver-A and tagging anti-EGFR mAb to EV generated from HEK293F culture. Confocal microscopy and the In Vivo Imaging System demonstrated that mAb-EV could penetrate the blood–brain barrier, target intracranial glioblastoma xenografts, and deliver drug intracellularly. The in vitro cytotoxicity study showed IC50 values of 2–12 nM of Ver-A. The hematoxylin and eosin staining of major organs in the tolerated dose study indicated minimal systemic toxicity of mAb-EV-Ver-A. Finally, the in vivo anti-tumor efficacy study in intracranial xenograft models demonstrated that EGFR mAb-EV-Ver-A effectively inhibited glioblastoma growth, but the combination with VEGF mAb did not improve the therapeutic efficacy. This study suggested that mAb-EV is an effective drug delivery vehicle and natural Ver-A has great potential to treat glioblastoma.

DDRE-50. INVESTIGATING THE ROLE OF LonP1 IN GLIOBLASTOMA TUMOR PROGRESSION

Abstract Glioblastoma (GBM), a WHO grade IV brain cancer, exhibits strong treatment resistance and a high rate of reoccurrence, which gives it a dismal prognosis, a 5% survival rate in the first 5 years. LonP1, a mitochondrial master regulator, can drive metabolic transformation, cytokine production, EMT, and treatment resistance in various cancer types, but its role in GBM remains unexplored. Our research group has previously shown that LonP1 is overexpressed in human malignant gliomas, particularly glioblastoma, and that this is associated with disease prognosis. Here, we present findings that demonstrate that LonP1 seems to drive enhanced tumor progression, invasiveness, angiogenesis in different high grade glioblastomas based on TCGA-subtype. Furthermore, in collaboration with Professor Bhaskar Das, we have validated a lead compound, BT317, with on-target inhibition of LonP1 protease activity. BT317 has enhanced activity against glioma stem cell lines (GSC) and has demonstrated low toxicity and efficacy in an intracranial xenograft model. This preliminary data highlights the potential of using combinatorial, pharmacological LonP1 and proteasome inhibition as a novel strategy for targeting specific subtypes of GBM.

CBIO-10. TARGETING HISTONE H4 Lys 20 MONOMETHYLATION IN THE TREATMENT OF GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma (GBM) is the most malignant primary tumor of the central nervous system, while the pathogenesis remains unclear. Protein lysine methyltransferase SETD8, which is responsible for the modification of histone H4K20me1, has been shown to play an important role in cellular transcriptional regulation and the development of a variety of tumors, yet its role in the malignant progression of GBM has not been elucidated. MATERIAL AND METHODS In the present study, we used primary GBM cell lines, intracranial xenograft model, transcriptome sequencing together with ChIP-sequencing, aiming to elucidate the molecular mechanism of SETD8-mediated H4K20me1 transcriptional regulation in promoting GBM progression. Furthermore, we evaluated the potential therapeutic significance in GBM using SETD8 small molecule inhibitor, UNC0379. RESULTS We found that SETD8 is aberrantly expressed in GBM tissues, accompanied by the dysregulation of H4K20me1 modification, which is associated with tumor pathology and prognosis. Using SETD8 inhibitor UNC0379 or knockdown of SETD8 significantly inhibited GBM cell proliferation in vitro and in vivo, and downregulated H4K20me1 modification level as well as transcriptome expression. CONCLUSION In summary, our work provide a novel insight into the role of SETD8/H4K20me1 axis. SETD8 overexpression mediated aberrant H4K20me1 modification act as a novel "transcriptional switch" in the malignant progression of glioma.

Nx-5948, a Selective Degrader of BTK with Activity in Preclinical Models of Hematologic and Brain Malignancies

Bruton's tyrosine kinase (BTK) plays a key role in cell survival in B cell malignancies, and covalent inhibitors of BTK, such as ibrutinib and acalabrutinib, have proven efficacious in chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and Waldenstrom's macroglobulinemia (WM). BTK inhibitors have also demonstrated clinical activity in small trials of patients with relapsed/refractory primary central nervous system lymphoma. The long-term efficacy of BTK inhibitors is limited by the emergence of resistance mutations, most commonly at C481 of BTK. These mutations preclude formation of a covalent bond with BTK and lead to diminished efficacy and disease progression. Several noncovalent BTK inhibitors, which do not require covalent binding to C481, are currently being investigated in clinical trials as potential therapies for patients with relapsed and refractory disease. However, other mutations have been shown to decrease the in vitro activity of these non-covalent BTK inhibitors, suggesting that mutations may ultimately limit the effectiveness of these compounds as well.Small molecule-induced protein degradation offers a unique approach to target BTK for the treatment of B-cell malignancies. Chimeric Targeting Molecules (CTMs) catalyze ubiquitylation and proteasomal degradation of target proteins and are comprised of a target binding element (“hook”), a linker, and ubiquitin ligase binding element (“harness”). NX-5948 is a CTM that contains a BTK hook linked to a cereblon (CRBN) harness. While some CRBN-binding drugs, such as lenalidomide and pomalidomide, promote the degradation of neo-substrate proteins, such as the transcription factors Aiolos and Ikaros, NX-5948 has been engineered to avoid Aiolos and Ikaros degradation and therefore does not possess IMiD activity.Preclinical studies have shown that NX-5948 catalyzes the degradation of 50% of cellular BTK (DC 50) at < 1 nM concentrations in lymphoma cell lines and peripheral blood mononuclear cells (PBMCs). NX-5948 impairs viability in the BTK-dependent ABC-DLBCL cell line, TMD8 (EC 50: < 10 nM after 72 hours). Importantly, NX-5948 induces degradation of the mutated BTK-C481S in cells and inhibits proliferation of BTK-C481S mutant TMD8 cells more effectively than ibrutinib (NX-5948 EC 50 values of < 10 nM versus > 1 μM for ibrutinib). NX-5948 also catalyzes selective degradation of BTK. In an unbiased proteomics experiment, NX-5948 treatment of TMD8 cells demonstrates robust BTK degradation without significant downregulation of other off-target proteins.Oral administration of NX-5948 in mice leads to dose-proportional exposure in plasma and BTK degradation to <10% of baseline levels in circulating and splenic B cells. In a TMD8 xenograft model in mice containing the BTK-C481S mutation, daily oral administration of NX-5948 resulted in superior tumor growth inhibition (TGI) as compared to ibrutinib. Following a single oral dose of NX-5948 in cynomolgus monkeys, BTK degradation to <10% of baseline levels is observed in the circulating B cells.In addition, NX-5948 penetrates the central nervous system (CNS) and demonstrates activity in a model of brain malignancies. Following oral dosing of NX-5948 to mice, NX-5948 was detectable in the cerebrospinal fluid at a comparable level to the unbound concentration of NX-5948 in mouse plasma. In an intracranial TMD8 xenograft model, treatment with NX-5948 resulted in degradation of BTK in intracranial TMD8 cells, decreased intracranial tumor burden and improved survival relative to treatment with vehicle.The potent BTK degradation activity of NX-5948 and the ability of this molecule to penetrate the CNS supports its development for the treatment of B-cell malignancies, including CNS lymphoma. DisclosuresRobbins: Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Noviski: Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Rountree: Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tan: Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Brathaban: Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Ingallinera: Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Karr: Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Kelly: Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Konst: Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Ma: Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tenn-McClellan: Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. McKinnell: Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Perez: Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Guiducci: Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Hansen: Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Sands: Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees.

CC-99282 is a Novel Cereblon (CRBN) E3 Ligase Modulator (CELMoD) Agent with Enhanced Tumoricidal Activity in Preclinical Models of Lymphoma

CC-99282 is a novel, oral CELMoD ® agent currently under investigation in phase 1 clinical studies in patients with relapsed or refractory (R/R) non-Hodgkin lymphomas (NHL) and chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). Mechanistically, CC-99282 interacts with the CRL4 CRBN E3 ubiquitin ligase substrate receptor CRBN to induce recruitment and ubiquitin-mediated proteasomal degradation of transcription factors Ikaros and Aiolos. The design intent for CC-99282 included efficient absorption, deep tissue distribution, and prolonged exposure to optimize activity in bulky lymphoma lesions. Recently, we reported that CC-99282 shows potent antitumor activity in different preclinical models of diffuse large B cell lymphoma (DLBCL; Lopez-Girona, et al. Hematol Oncol. 2021). Here, we provide an expanded analysis of CC-99282 activity as a monotherapy, as well as examine its synergistic activity with anti-CD20 antibody treatment, in preclinical models of NHL including DLBCL and follicular lymphoma (FL).Compared with existing agents targeting Ikaros/Aiolos that show activity in hematologic malignancies, such as lenalidomide, avadomide, and iberdomide (CC-220), CC-99282 induced a more rapid, deep, and sustained degradation of Ikaros/Aiolos, causing derepression of cyclin-dependent kinase (CDK) inhibitors and interferon-stimulated genes (IRF7, IFIT3, and DDX58), and the reduction of the highly critical oncogenic factors c-Myc and IRF4. These molecular changes were followed by potent, 10- to 100-fold enhanced, autonomous cell killing and induction of apoptosis (Figure). Our results show that these effects were independent of the cell of origin (activated B cell [ABC; TMD8 cell line], germinal center B cell [GCB; WSU-DLCL2 cell line], or primary mediastinal B cell lymphoma [PMBL] subtypes of DLBCL) or presence of high-risk chromosomal translocations (MYC, BCL2, and/or BCL6), as observed in a panel of 36 lymphoma cell lines that included DLBCL and FL cell lines. In vivo, CC-99282 demonstrated robust tissue distribution that favored target tissues and exhibited antitumor activity resulting in improved tumor regression and tumor-free animals in several lymphoma xenograft models, including an intracranial xenograft model. This strong antitumor activity was observed using various continuous and intermittent dosing paradigms.The potent, direct autonomous cell-killing activity of CC-99282 was augmented when CC-99282 was combined with the anti-CD20 antibody rituximab. In vitro combination studies of CC-99282 with rituximab in lymphoma cell lines demonstrated enhanced cell killing by human natural killer (NK) cells, macrophage-mediated phagocytosis, antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). In FL and DLBCL cell lines, we showed that the combination of CC-99282 with rituximab resulted in increases in both NK-mediated ADCC and macrophage-mediated ADCP of up to 20% compared with rituximab treatment alone. In vivo, combination treatment with CC-99282 and rituximab induced dose-dependent tumor growth inhibition in WSU-DLCL2 and RL (FL) xenograft models. In the WSU-DLCL2 model, CC-99282 (1 mg/kg) or rituximab (10 mg/kg) monotherapy resulted in modest tumor growth inhibition, whereas the combination of CC-99282 (1 mg/kg) and rituximab (10 mg/kg) resulted in tumor regression in 100% of animals. Similar results were obtained in FL xenograft models using the RL cell line, where combinations of CC-99282 (1 mg/kg) with rituximab (25 mg/kg) induced complete tumor regression in 100% of animals.In conclusion, CC-99282 is a novel CELMoD agent with an improved substrate degradation profile compared with existing Ikaros/Aiolos-degrading agents. CC-99282 demonstrated enhanced antiproliferative and apoptotic activities across a broad range of lymphoma cells and a robust distribution profile that favors target tissues such as lymphoid organs. In addition, CC-99282 acts synergistically in combination with anti-CD20 monoclonal antibody treatment. Collectively, these data support the clinical investigation of CC-99282 as monotherapy and in combination with rituximab in patients with R/R NHL. [Display omitted] DisclosuresCarrancio: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Groocock: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Janardhanan: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Jankeel: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Galasso: Ryan Galasso: Current Employment, Current equity holder in publicly-traded company. Guarinos: Bristol Myers Squibb: Current Employment. Narla: Bristol Myers Squibb: Current Employment. Groza: Bristol Myers Squibb: Current Employment. Leisten: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Pierce: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Rolfe: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Lopez-Girona: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company.

Histone chaperone FACT complex inhibitor CBL0137 interferes with DNA damage repair and enhances sensitivity of medulloblastoma to chemotherapy and radiation.

Medulloblastoma (MB) is a malignant pediatric brain tumor with a poor prognosis. Post-surgical radiation and cisplatin-based chemotherapy have been a mainstay of treatment, which often leads to substantial neurocognitive impairments and morbidity, highlighting the need for a novel therapeutic target to enhance the sensitivity of MB tumors to cytotoxic therapies. We performed a comprehensive study using a cohort of 71MB patients' samples and pediatric MB cell lines and found that MB tumors have elevated levels of nucleosome remodeling FACT (FAcilitates Chromatin Transcription) complex and DNA repair enzyme AP-endonuclease1 (APE1). FACT interacts with APE1 and facilitates recruitment and acetylation of APE1 to promote repair of radiation and cisplatin-induced DNA damage. Further, levels of FACT and acetylated APE1 both are correlate strongly with MB patients' survival. Targeting FACT complex with CBL0137 inhibits DNA repair and alters expression of a subset of genes, and significantly improves the potency of cisplatin and radiation in vitro and in MB xenograft. Notably, combination of CBL0137 and cisplatin significantly suppressed MB tumor growth in an intracranial orthotopic xenograft model. We conclude that FACT complex promotes chemo-radiation resistance in MB, and FACT inhibitor CBL0137 can be used as a chemo-radiation sensitizer to augment treatment efficacy and reduce therapy-related toxicity in high-risk pediatric patients.

USP7 inhibition induces apoptosis in glioblastoma by enhancing ubiquitination of ARF4

BackgroundGlioblastomas (GBMs) are grade IV central nervous system tumors characterized by a poor prognosis and a short median overall survival. Effective induction of GBM cell death is difficult because the GBM cell population is genetically unstable, resistant to chemotherapy and highly angiogenic. In recent studies, ubiquitin-specific protease 7 (USP7) is shown to scavenge ubiquitin from oncogenic protein substrates, so effective inhibition of USP7 may be a potential key treatment for GBM.MethodsImmunohistochemistry and western blotting were used to detect the expression of USP7 in GBM tissues. In vitro apoptosis assay of USP7 inhibition was performed by western blotting, immunofluorescence, and flow cytometry. Anti-apoptotic substrates of USP7 were defined by Co-IP and TMT proteomics. Western blotting and IP were used to verify the relationship between USP7 and its substrate. In an in vivo experiment using an intracranial xenograft model in nude mice was constructed to assess the therapeutic effect of target USP7.ResultsImmunohistochemistry and western blotting confirmed that USP7 was significantly upregulated in glioblastoma samples. In in vitro experiments, inhibition of USP7 in GBM induced significant apoptosis. Co-IP and TMT proteomics identified a key anti-apoptotic substrate of USP7, ADP-ribosylation factor 4 (ARF4). Western blotting and IP confirmed that USP7 interacted directly with ARF4 and catalyzed the removal of the K48-linked polyubiquitinated chain that binded to ARF4. In addition, in vivo experiments revealed that USP7 inhibition significantly suppressed tumor growth and promoted the expression of apoptotic genes.ConclusionsTargeted inhibition of USP7 enhances the ubiquitination of ARF4 and ultimately mediates the apoptosis of GBM cells. In a clinical sense, P5091 as a novel specific inhibitor of USP7 may be an effective approach for the treatment of GBM.

Interplay of m6 A and histone modifications contributes to temozolomide resistance in glioblastoma.

BackgroundDespite the development of new treatment protocols for glioblastoma (GBM), temozolomide (TMZ) resistance remains a primary hindrance. Previous studies, including our study, have shown that aberrant N6‐methyladenosine (m6A) modification is implicated in GBM pathobiology. However, the roles and precise mechanisms of m6A modification in the regulation of TMZ resistance in GBM remain unclear.Methodsm6A individual‐nucleotide‐resolution cross‐linking and immunoprecipitation sequencing (miCLIP‐seq) was performed to identify m6A modification of transcripts in TMZ‐resistant and ‐sensitive tumors. To explore the role of METTL3 in TMZ resistance, TMZ‐resistant GBM cells were transfected with METTL3 shRNA or overexpression lentivirus and then assessed by cell viability, tumor sphere formation, and apoptosis assays. An intracranial GBM xenograft model was developed to verify the effect of METTL3 depletion during TMZ treatment in vivo. ATAC‐seq, ChIP‐qPCR, and dual‐luciferase reporter assays were carried out to verify the role of SOX4/EZH2 in the modulation of METTL3 expression upon TMZ treatment.ResultsWe demonstrated that TMZ treatment upregulated the expression of the m6A methyltransferase METTL3, thereby increasing m6A modification of histone modification‐related gene transcripts. METTL3 is required to maintain the features of GBM stem cells. When combined with TMZ, METTL3 silencing suppressed orthotopic TMZ‐resistant xenograft growth in a cooperative manner. Mechanistically, TMZ induced a SOX4‐mediated increase in chromatin accessibility at the METTL3 locus by promoting H3K27ac levels and recruiting RNA polymerase II. Moreover, METTL3 depletion affected the deposition of m6A on histone modification‐related gene transcripts, such as EZH2, leading to nonsense‐mediated mRNA decay. We revealed an important role of EZH2 in the regulation of METTL3 expression, which was via an H3K27me3 modification‐independent manner.ConclusionsOur findings uncover the fundamental mechanisms underlying the interplay of m6A RNA modification and histone modification in TMZ resistance and emphasize the therapeutic potential of targeting the SOX4/EZH2/METTL3 axis in the treatment of TMZ‐resistant GBM.

The New PI3K/mTOR Inhibitor GNE-477 Inhibits the Malignant Behavior of Human Glioblastoma Cells.

The most common primary central nervous system tumor in adults is glioblastoma multiforme (GBM). The high invasiveness of GBM cells is an important factor leading to inevitable tumor recurrence and a poor prognosis of patients. GNE-477, a novel PI3K/mTOR inhibitor, has been reported to exert antiproliferative effects on other cancer cells. However, researchers have not clearly determined whether GNE-477 produces antitumor effects on GBM. In the present study, GNE-477 significantly inhibited the proliferation, migration and invasion of U87 and U251 cells. In addition, GNE-477 also induced apoptosis of GBM cells, arresting the cell cycle in G0/G1 phase. More importantly, GNE-477 also reduced the levels of AKT and mTOR phosphorylation in the AKT/mTOR signaling pathway in a concentration-dependent manner. An increase in AKT activity induced by SC79 rescued the GNE-477-mediated inhibition of GBM cell proliferation and apoptosis. The antitumor effects of GNE-477 and the regulatory effects on related molecules were further confirmed in vivo using a nude mouse intracranial xenograft model. In conclusion, our study indicated that GNE-477 exerted significant antitumor effects on GBM cells in vitro and in vivo by downregulating the AKT/mTOR pathway.

Abstract 1473: Nonclinical development of PF-07284890 (ARRY-461), a potent, brain-penetrant, small molecule inhibitor of BRAF V600-mutation-driven tumors in vitro and in vivo

Abstract Although approved BRAF inhibitors have transformed the treatment of patients with certain BRAF V600 mutant cancers, their long-term efficacy is thought to be limited by poor brain penetration. As a result, disease progression in the brain is a significant cause of morbidity and mortality. PF-07284890 (ARRY-461) is an orally bioavailable, brain penetrant, potent, small molecule inhibitor targeting BRAF V600 mutant tumors and is in clinical development in patients with BRAF V600 metastatic melanoma with progression to the brain. In biochemical in vitro studies, PF-07284890 inhibits BRAF and CRAF with IC50's of 5.8 and 4.1 nM, respectively. Additionally, PF-07284890 inhibits the clinically relevant kinase domain BRAF V600 mutants (V600E and V600K) (IC50 = 24-25 nM). In cell-based systems, PF-07284890 potently inhibits phosphorylation of ERK, a downstream marker of BRAF inhibition, and potently inhibits proliferation of BRAF V600E/K mutant melanoma cell lines (IC50 18-38 nM). PF-07284890 was designed to distribute to the brain and, as such, in vitro experiments indicate that PF-07284890 has high cellular membrane permeability and is not a substrate for human P glycoprotein (P-gp). After oral administration of PF-07284890 to mice and rats, PF-07284890 distributes to the brain where the free fraction adjusted exposure in the brain was approximately proportional to the free fraction adjusted exposure in plasma (i.e., Cbrain,u/Cplasma,u approaches 1.0). In vivo, PF-07284890 inhibits phosphorylation of ERK in A375 BRAF V600E tumors, achieving maximal target inhibitions at a dose of 10 mg/kg. PF-07284890 has been evaluated for its ability to control BRAF V600E cell line and patient-derived melanoma xenograft tumor growth in nude mice when implanted both subcutaneously and intracranially. Dose-related tumor growth inhibition was demonstrated at dose levels ranging from 1 to 30 mg/kg, BID in both subcutaneous and intracranial xenograft models. In all models, regardless of tumor location, maximal efficacy was observed in dose ranges of 10-30 mg/kg BID. In the intracranial A375-luc BRAF V600E melanoma xenograft model, significant and durable tumor regressions were seen at doses of 10 and 30 mg/kg/day which translated to a profound survival benefit in animals receiving PF-07284890 (median survival &amp;gt; 55 days post-implantation) compared to control animals (median survival = 15 days). GLP safety studies demonstrated a good safety profile for PF-07284890. PF-07284890 is fully brain penetrant, with the potential to address this key unmet medical need and thereby defines a new class of brain penetrant, potent and selective BRAF inhibitors. Citation Format: Karyn Bouhana, Deborah Anderson, Walter DeWolf, Suzy Brown, Lance Williams, Li Ren, David Moreno, Ross Wallace, Jay Brad Fell, Dylan Hartley, Patrice Lee. Nonclinical development of PF-07284890 (ARRY-461), a potent, brain-penetrant, small molecule inhibitor of BRAF V600-mutation-driven tumors in vitro and in vivo [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 1473.

Abstract 1070: SCR6106, an oral and brain penetrant SERD demonstrated anti-tumor activities in vitro and in vivo

Abstract Benefits of endocrine therapy in treating hormone receptor (HR)-positive (HR+) BC are well recorded in clinic. However, ~40% to 50% of HR+ patients eventually developed to endocrine resistance and disease relapsed. Fulvestrant, a selective estrogen receptor degrader (SERD) is approved to treat HR+ metastatic breast cancer in patients with disease progression after endocrine therapy. However, due to its poor bioavailability, it is given by intramuscular injection, which makes inconvenience in administration. Additionally, approximately 14% of hormone positive patients with advanced breast cancer are diagnosed with brain metastases, while, at present, there are no FDA-approved systemic therapies for the treatment of breast cancer brain metastases. Therefore, to develop a new generated oral and brain-penetrant SERD to overcome these challenges is believed to be needed. SCR6106, discovered and currently being developed by Simcere, is a novel, potent, orally delivered and non-steroidal SERD that both antagonizes and degrades ERα. SCR6106 showed high potency on inducing ERα protein degradation (DC50=0.29 nM) and anti-proliferation (IC50=0.43 nM) in MCF7 cells. In addition, SCR6106, showed high activities on ERα degradation and anti-proliferation in a panel of ER+ breast cancer cell lines. Meanwhile, ER target genes transcription were evaluated and SCR6106 exhibit strong suppressive phenotype in T-47D. SCR6106 demonstrated more potent than fulvestrant on anti-tumor growth in vivo. In MCF7 xenograft model, SCR6106 at 3 mpk QD induced 113% tumor growth inhibition (TGI), compared to fulvestrant 56.8% TGI at 250 mpk. Compared to fulvestrant, SCR6106 demonstrated stronger ERα degradation and inhibition of ER target genes transcription in tumors, which was consistent with anti-tumor activities. SCR6106 displayed good oral bioavailability in multiple pre-clinical species, which supports its oral administration in human. Significantly, SCR6106 possess a high BBB permeability, the B/P ratio is more than 4 in pre-clinical species. The exposure of SCR6106 observed in mouse brain is much higher than the IC90 value of anti-proliferation (MCF7), and SCR6106 demonstrated anti-tumor activities in a MCF-7 intracranial tumor xenograft model. In conclusion, SCR6106 is a novel, potent and oral SERD, which demonstrated anti-tumor activities in vitro and in vivo. It showed high potential ability to cross the blood-brain barrier, and could be used to treat HR+ patients with brain metastasis. Citation Format: Feng Zhou, Lei Liu, Guimei Yang, Peng Gu, Liting Xue, Wenqing Yang, Ping Chen, Renhong Tang. SCR6106, an oral and brain penetrant SERD demonstrated anti-tumor activities in vitro and in vivo [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 1070.

EMBR-06. EFFECTIVE INHIBITION OF MYC-AMPLIFIFIED GROUP 3 MEDULLOBLASTOMA BY FACT-TARGETED CURAXIN DRUG CBL0137

Medulloblastoma (MB) is the most common malignant pediatric brain tumor that can be categorized into four major molecular subgroups. Group 3 MB with MYC amplifification (MYCamp-G3-MB) has been shown to be highly aggressive and exhibited worst prognosis, indicating the need for novel effective therapy most urgently. A few epigenetic targeted therapeutic strategies have recently been proven to effectively treat preclinical models of MYCamp-G3-MB, including BET inhibition, HDAC inhibition and SETD8 inhibition, unveiling a promising direction for further investigation. In this study, we carried out systemic bioinformatic analyses of public-available MB datasets as well as functional genomic screening datasets of primary MYCamp-G3-MB lines to search for other potential therapeutic targets within epigenetic modulators. We identifified SSRP1, a subunit of histone-chaperone FACT complex, to be the top drug target candidate as it is highly cancer-dependent in whole-genome CRISPR-Cas9 screening across multiple MYCamp-G3-MB lines; signifificantly upregulated in MYCamp-G3-MB compared to normal cerebellum and most of the rest MB subtypes; its higher expression is correlated with worse prognosis; and it has a blood-brain-barrier penetrable targeted drug that has entered early phase human clinical trials already. Then we utilized RNA-interference approach to verify the cancer-dependency of SSRP1 in multiple MYCamp-G3-MB lines and further confifirmed the therapeutic effificacy of FACT-targeted curaxin drug CBL0137 on treating preclinical models of MYCamp-G3-MB in vitro and in vivo, including an orthotopic intracranial xenograft model. Mechanistically, transcriptome analyses showed CBL0137 preferentially suppressed cell-cycle and DNA-repair related biological processes. Moreover, it selectively disrupted transcription of MYC and NEUROD1, two critical oncogenic transcription factors of MYCamp-G3-MB, via depleting FACT complex from their promoter regions. In summary, our study demonstrates FACT-targeted CBL0137 works effectively on treating MYCamp-G3-MB, presenting another promising epigenetic-targeted therapeutic strategy against the most devastating form of MB.

RAB1A Regulates Glioma Cellular Proliferation and Invasion Via the mTOR Signaling Pathway and Epithelial–Mesenchymal Transition

Aim: We aimed at investigating the mechanism of RAB1A proliferation and invasion in gliomas. Materials & methods: Genome-wide expression profile data and immunohistochemistry were analyzed to assess RAB1A expression in gliomas. The Transwell assay, wound healing assay, brain slice coculture model, cellular fluorescenceand intracranial xenograft model of nude mice were used to determine the proliferation and invasion of glioma cells. Results & conclusion: RAB1A was highly expressed in gliomas compared with normal brain tissue. The overall survival time of gliomapatients with high RAB1A expressionwas significantly shortened. RAB1A regulated the activity of RAC1 by inhibiting the mTOR signaling pathway, affecting actin polymerization, cell morphology and cell polarity. RAB1A downregulation inhibited the epithelial-mesenchymal transition, proliferation andinvasion of glioma cells.

Abstract PD13-07: Subgroup analysis of patients with brain metastases from the phase 3 ASCENT study of sacituzumab govitecan versus chemotherapy in metastatic triple-negative breast cancer

Abstract Background: Patients (pts) with metastatic triple-negative breast cancer (mTNBC) who have brain metastases represent a poor prognosis cohort with a high unmet clinical need. Sacituzumab govitecan (SG) is an antibody-drug conjugate composed of an anti-Trop-2 antibody coupled to SN-38, an active metabolite of irinotecan, via a unique hydrolyzable linker that allows for SN-38 release intracellularly and in the tumor microenvironment (bystander effect). SN-38 can cross the blood-brain barrier and is a drug partner in central nervous system (CNS) disease regimens (PMID: 18784279; PMID: 26080460). Although antibody-based therapy raises concerns regarding CNS penetration, activity with SG has been seen in intracranial xenograft models (Brenner Neurooncol Adv 2019). In a subgroup analysis from ASCENT, the efficacy and safety of SG were evaluated in pts with stable brain metastases. Methods: In the phase 3 ASCENT study (NCT02574455), 529 pts with mTNBC refractory to or relapsing after at least 2 prior chemotherapies were randomized 1:1 to receive SG (10 mg/kg IV on days 1 and 8 every 21 days) or single-agent treatment of physician’s choice (TPC; capecitabine, eribulin, vinorelbine, or gemcitabine) until disease progression or unacceptable toxicity. Brain MRIs were required in pts with known brain metastases who were eligible if they had stable CNS disease for ≥4 wk by MRI. Per protocol, stable disease was defined as ≥2 wk from discontinuation of antiseizure medication and corticosteroid dose (≤20 mg prednisone equivalent) that was stable or decreasing for ≥2 wk before randomization. In these pts, brain MRIs were required throughout the study. The primary endpoint was progression-free survival (PFS) per independent central review (RECIST v1.1) in brain metastases-negative pts. Secondary endpoints included PFS per investigator assessment, PFS in the full population (in pts with/without brain metastases) by central review, objective response rate (ORR), overall survival (OS), and safety. Results: Overall, 61 of 529 (12%) enrolled pts had stable brain metastases at screening and were randomized to SG (n=32) or TPC (n=29). Median age was 53 y for SG and 51 y for TPC; all pts were female and had a median of 5 prior anticancer regimens. In this subset, median PFS was 2.8 mo (95% CI, 1.5-3.9) for SG vs 1.6 mo (95% CI, 1.3-2.9) for TPC by central review, and median OS was 6.8 mo (95% CI, 4.7-14.1) for SG vs 7.5 mo (95% CI, 4.7-11.1) for TPC. ORR for SG vs TPC, respectively, was 3% (1/32) vs 0% by central review, with a clinical benefit rate of 9.4% vs. 3.4%. Stable disease was achieved in 15 (47%) pts with SG vs 9 (31%) pts with TPC. In 53 pts who received at least 1 dose of treatment (SG, n=30; TPC, n=23), any-grade treatment-emergent adverse events (&amp;gt;20% with SG) for SG vs TPC were fatigue (63% vs 52%), diarrhea (50% vs 13%), neutropenia (43% vs 35%), nausea (43% vs 26%), decreased appetite (30% vs 17%), decreased neutrophil count (33% vs 22%), anemia (23% vs 35%), alopecia (23% vs 13%), and constipation (23% vs 22%). There were no treatment-related deaths. Two pts treated with SG are continuing study treatment for 16.2 and 6.3 mo as of the data cutoff date. Conclusions: Data interpretation in this population with poor prognosis is limited by the small sample size. In this exploratory analysis of pts with brain metastases from the phase 3 ASCENT study, SG was numerically better than TPC for tumor response and PFS but not OS. The safety profile was similar to that of the population without brain metastases for both study arms. SG is currently under clinical investigation for pts undergoing elective craniotomy for breast cancer with brain metastases or recurrent glioblastoma (NCT03995706) based on promising preclinical and intracranial clinical data. Citation Format: Véronique Diéras, Robert Weaver, Sara M. Tolaney, Aditya Bardia, Kevin Punie, Adam Brufsky, Hope S. Rugo, Kevin Kalinsky, Tiffany Traina, Leonard Klein, Delphine Loirat, Filipa Lynce, Brooke Daniel, Foluso Ademuyiwa, Sara A. Hurvitz, David M. Goldenberg, Quan Hong, Martin Olivo, Loretta M. Itri, Lisa Carey. Subgroup analysis of patients with brain metastases from the phase 3 ASCENT study of sacituzumab govitecan versus chemotherapy in metastatic triple-negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD13-07.

Inhibiting protein phosphatase 2A increases the antitumor effect of protein arginine methyltransferase 5 inhibition in models of glioblastoma.

Despite multi-model therapy of maximal surgical resection, radiation, chemotherapy, and tumor-treating fields, the median survival of glioblastoma (GBM) patients is less than 15 months. Protein arginine methyltransferase 5 (PRMT5) catalyzes the symmetric dimethylation of arginine residues and is overexpressed in GBM. Inhibition of PRMT5 causes senescence in stem-like GBM tumor cells. LB100, a first-in-class small molecular inhibitor of protein phosphatase 2A (PP2A), can sensitize therapy-resistant tumor cells. Here, we tested the anti-GBM effect of concurrent PRMT5 and PP2A inhibition. Patient-derived primary GBM neurospheres (GBMNS), transfected with PRMT5 target-specific siRNA, were treated with LB100 and subjected to in vitro assays including PP2A activity and western blot. The intracranial mouse xenograft model was used to test the in vivo antitumor efficacy of combination treatment. We found that PRMT5 depletion increased PP2A activity in GBMNS. LB100 treatment significantly reduced the viability of PRMT5-depleted GBMNS compared to PRMT5-intact GBMNS. LB100 enhanced G1 cell cycle arrest induced by PRMT5 depletion. Combination therapy also increased the expression of phospho-MLKL. Necrostatin-1 rescued PRMT5-depleted cells from the cytotoxic effects of LB100, indicating that necroptosis caused the enhanced cytotoxicity of combination therapy. In the in vivo mouse tumor xenograft model, LB100 treatment combined with transient depletion of PRMT5 significantly decreased tumor size and prolonged survival, while LB100 treatment alone had no survival benefit. Overall, combined PRMT5 and PP2A inhibition had significantly greater antitumor effects than PRMT5 inhibition alone.

Effective inhibition of MYC-amplified group 3 medulloblastoma by FACT-targeted curaxin drug CBL0137

Medulloblastoma (MB) is the most common malignant pediatric brain tumor that can be categorized into four major molecular subgroups. Group 3 MB with MYC amplification (MYCamp-G3-MB) has been shown to be highly aggressive and exhibited worst prognosis, indicating the need for novel effective therapy most urgently. A few epigenetic targeted therapeutic strategies have recently been proven to effectively treat preclinical models of MYCamp-G3-MB, including BET inhibition, HDAC inhibition and SETD8 inhibition, unveiling a promising direction for further investigation. In this study, we carried out systemic bioinformatic analyses of public-available MB datasets as well as functional genomic screening datasets of primary MYCamp-G3-MB lines to search for other potential therapeutic targets within epigenetic modulators. We identified SSRP1, a subunit of histone-chaperone FACT complex, to be the top drug target candidate as it is highly cancer-dependent in whole-genome CRISPR-Cas9 screening across multiple MYCamp-G3-MB lines; significantly upregulated in MYCamp-G3-MB compared to normal cerebellum and most of the rest MB subtypes; its higher expression is correlated with worse prognosis; and it has a blood-brain-barrier penetrable targeted drug that has entered early phase human clinical trials already. Then we utilized RNA-interference approach to verify the cancer-dependency of SSRP1 in multiple MYCamp-G3-MB lines and further confirmed the therapeutic efficacy of FACT-targeted curaxin drug CBL0137 on treating preclinical models of MYCamp-G3-MB in vitro and in vivo, including an orthotopic intracranial xenograft model. Mechanistically, transcriptome analyses showed CBL0137 preferentially suppressed cell-cycle and DNA-repair related biological processes. Moreover, it selectively disrupted transcription of MYC and NEUROD1, two critical oncogenic transcription factors of MYCamp-G3-MB, via depleting FACT complex from their promoter regions. In summary, our study demonstrates FACT-targeted CBL0137 works effectively on treating MYCamp-G3-MB, presenting another promising epigenetic-targeted therapeutic strategy against the most devastating form of MB.

STEM-23. INHIBITING PROTEIN PHOSPHATASE 2A INCREASES THE ANTITUMOR EFFECT OF PROTEIN ARGININE METHYLTRANSFERASE 5 INHIBITION IN MODELS OF GLIOBLASTOMA

Abstract BACKGROUND: Despite multi-model therapy of maximal surgical resection, radiation, chemotherapy, and tumor treating fields, median survival of Glioblastoma (GBM) patients is less than two years. Protein Arginine Methyltransferase 5 (PRMT5) catalyzes the symmetric di-methylation of arginine residues and is overexpressed in GBM. Inhibition of PRMT5 causes senescence of immature GBM tumor cells. LB100, first-in-class small molecule inhibitor of Protein Phosphatase 2A (PP2A) can sensitize therapy-resistant tumor cells. Here, we tested the anti-GBM effect of concurrent PRMT5 and PP2A inhibition. Methods: Patient-derived primary GBM neurospheres (GBMNS), transfected with PRMT5 target-specific siRNA were treated with LB100 and subjected to in vitro assays including PP2A activity and western blot. The intracranial mouse xenograft model was used to test the in vivo antitumor efficacy of combination treatment. Results: We found that PRMT5-depletion increased PP2A activity in GBMNS. LB100 treatment significantly reduced the viability of PRMT5-depleted GBMNS compared to PRMT5 intact GBMNS. LB100 enhanced the G1 cell cycle arrest induced by PRMT5-depletion. Combination therapy also increased the expression of phospho-MLKL. Necrostatin-1 rescued PRMT5-depleted cells from the cytotoxic effects of LB100, indicating that necroptosis caused the enhanced cytotoxicity of combination therapy. In the in vivo mouse tumor xenograft model, LB100 treatment combined with transient depletion of PRMT5 significantly decreased tumor size and prolonged mice survival, while LB100 treatment alone had no survival benefit. Conclusion: Overall, combined PRMT5 and PP2A inhibition had significantly greater antitumor effects than PRMT5 inhibition alone.

EXTH-75. IN VITRO AND IN VIVO EFFICACY OF COMBINATORIAL INHIBITION OF LSD1 AND HDACS IN PATIENT DERIVED GLIOBLASTOMA STEM CELL MODELS

Abstract Inhibitors of histone deacetylases (HDACi) have been tested in glioblastoma (GBM), however, single agent clinical efficacy has not been proven, prompting study of combinatorial approaches. A rational HDACi combination strategy is with inhibition of LSD1, lysine specific demethylase 1, a histone demethylase known to exist in complex with HDAC1/2. We previously showed in vivo efficacy of combining a brain permeant LSD1 inhibitor, tranylcypromine (TCP) together with vorinostat. More selective inhibitors of LSD1 have been developed and were tested in the current study together with the HDACi, vorinostat or panobinostat in a panel of patient derived glioblastoma stem cell (GSC) lines that have been characterized as radio-resistant or radiosensitive. To test in vitro sensitivity, dose response experiments were conducted in nine GSC cell lines using three LSD1 inhibitors or two HDACi as single agents or in combination. Two non-tumor lines were also tested to assess selective cytotoxicity for brain tumor lines. Cell viability and clonogenicity assays were performed. Several radio-resistant GSC lines showed sensitivity to single agent LSD1 inhibitors, whereas some radio-sensitive GSCs did not, indicating distinct molecular mechanisms of response. Intracranial orthotopic xenograft models were used to test in vivo efficacy using the radioresistant GSC20 line. Interestingly, LSD1 inhibition alone promoted tumor burden reduction and better survival rates than combination treatment. RNA-Seq guided biomarkers of sensitivity to the LSD1/HDAC inhibitor combination were assessed in vitro and in vivo. Both in vivo and in vitro data show that GSC20 treated with GSK LSD1 inhibitor had higher levels of HKDC1 gene with in vivo data also showing elevated levels of FTH1. In vivo mouse data for vorinostat and combination treatment groups showed that only the FTH1 gene level was elevated, providing a potential explanation for low in vivo efficacy. Collectively, our data suggests that LSD1 inhibition represents a viable strategy in GBM.

INNV-08. COMPARATIVE STUDY OF THE CANCER TREATMENT POTENTIAL EFFECT OF TUMOR-TREATING FIELDS AND COLD ATMOSPHERIC PLASMA

Abstract INTRODUCTION Cold Atmospheric Plasma (CAP) is ionized gas close to room temperature that generates reactive oxygen and nitrogen species, an electromagnetic field (EMF) and UV, which are selectively cytotoxic to cancer cells. Tumor Treating Fields (TTFields) is an FDA approved cancer treatment that utilizes “low intensity” and “intermediate frequency” alternating electric fields to produce an inhibitory effect on cancerous cells. OBJECTIVES In this study, we directly compared CAP and TTFields treatments using U87 glioblastoma tumor cells. We aimed to assess the relative merits of both technologies as potential approaches for brain cancer treatment. METHODS In this in vitro study, the cells were treated with either CAP or TTFields at room temperature. In both cases in order the optimize the treatment parameters, the energy we applied was varied for both methods by changing the power and time of the treatment. The resulting cell viability changes were measured 72 hours after treatment. RESULTS The results revealed that increased energy resulted in decreased cell viability of U87 glioblastoma tumor cells with both CAP and TTFields treatment. The rate of U87 glioblastoma tumor cell viability under CAP treatment was 80% of the original viability after using 4.8J of energy, compared with 15,752J of energy with TTFields treatment. Additionally, the CAP displayed significantly greater time-efficiency as compared to TTFields. For instance, the most effective TTFields treatment took 4 hours, while the CAP treatment displayed a comparable effect on tumor cell viability with a treatment time of 30 seconds. CONCLUSIONS This preliminary in vitro study indicates that CAP treatment is potentially a more favorable effective method of brain cancer therapy. This data indicates that CAP is significantly more energy-efficient and requires significantly less treatment time than TTFields. Future research looks to compare the 2 treatments in an intracranial mouse xenograft model.

Human macrophages engineered to secrete a bispecific T cell engager support antigen-dependent T cell responses to glioblastoma

BackgroundTargeted and effective treatment options are needed for solid tumors, including glioblastoma (GBM), where survival rates with standard treatments are typically less than 2 years from diagnosis. Solid tumors pose...