Brain Metastasis Model Research Articles

Ferritin nanoconjugates guide trastuzumab brain delivery to promote an antitumor response in murine HER2 + breast cancer brain metastasis

Brain metastasis (BM) represents a clinical challenge for patients with advanced HER2 + breast cancer (BC). The monoclonal anti-HER2 antibody trastuzumab (TZ) improves survival of BC patients, but it has low central nervous system penetrance, being ineffective in treating BM. Previous studies showed that ferritin nanoparticles (HFn) may cross the blood brain barrier (BBB) through binding to the transferrin receptor 1 (TfR1). However, whether this has efficacy in promoting the trans-BBB delivery of TZ and combating BC BM was not studied yet. Here, we investigated the potential of HFn to drive TZ brain delivery and promote a targeted antitumor response in a murine model of BC BM established by stereotaxic injection of engineered BC cells overexpressing human HER2. HFn were covalently conjugated with TZ to obtain a nanoconjugate endowed with HER2 and TfR1 targeting specificity (H-TZ). H-TZ efficiently achieved TZ brain delivery upon intraperitoneal injection and triggered stable targeting of cancer cells. Treatment with H-TZ plus docetaxel significantly reduced tumor growth and shaped a protective brain microenvironment by engaging macrophage activation toward cancer cells. H-TZ-based treatment also avoided TZ-associated cardiotoxicity by preventing drug accumulation in the heart and did not induce any other major side effects when combined with docetaxel. These results provided in vivo demonstration of the pharmacological potential of H-TZ, able to tackle BC BM in combination with docetaxel. Indeed, upon systemic administration, the nanoconjugate guides TZ brain accumulation, reduces BM growth and limits side effects in off-target organs, thus showing promise for the management of HER2 + BC metastatic to the brain.

Overexpression of CD2/CD27 could inhibit the activation of nitrogen metabolism pathways and suppress M2 polarization of macrophages, thereby preventing brain metastasis of breast cancer

ObjectiveOur study aimed to reveal the possible molecular mechanisms of CD2 and CD27 in influencing the tumor microenvironment of breast cancer (BC) brain metastasis based on the TCGA (The Cancer Genome Atlas) and SRA (Sequence Read Archive) databases. MethodsWe calculated the proportions of tumor-infiltrating immune cells and the immune and stromal cell scores in 1222 BC samples from the TCGA-BRCA dataset, followed by identification of candidate DEGs. We further screened for BC brain metastasis-related DEGs in the BC brain metastasis dataset SUB12911144 from the SRA database. Finally, we established a mouse breast cancer brain metastasis model for in vivo validation. ResultsWe further screened two immune-regulatory DEGs (CD2 and CD27). GSEA analysis showed that the downregulation of CD2 and CD27 expression was closely related to the activation of nitrogen metabolism pathways. CIBERSORT algorithm analysis showed a correlation between the expression of 16 types of tumor-infiltrating immune cells and CD2 and 19 types of tumor-infiltrating immune cells and CD27. In addition, CD2 and CD27 expression were negatively associated with the proportion of M2 macrophages. In vivo experimental results demonstrated that overexpression of CD2/CD27 could suppress the M2 polarization of macrophages and inhibit breast cancer brain metastasis. ConclusionIn the tumor microenvironment, overexpression of CD2/CD27 inhibited the activation of nitrogen metabolism pathways and suppressed M2 polarization of macrophages, thereby preventing brain metastasis of breast cancer.

Machine learning identifies experimental brain metastasis subtypes based on their influence on neural circuits

A high percentage of patients with brain metastases frequently develop neurocognitive symptoms; however, understanding how brain metastasis co-opts the function of neuronal circuits beyond a tumor mass effect remains unknown. We report a comprehensive multidimensional modeling of brain functional analyses in the context of brain metastasis. By testing different preclinical models of brain metastasis from various primary sources and oncogenic profiles, we dissociated the heterogeneous impact on local field potential oscillatory activity from cortical and hippocampal areas that we detected from the homogeneous inter-model tumor size or glial response. In contrast, we report a potential underlying molecular program responsible for impairing neuronal crosstalk by scoring the transcriptomic and mutational profiles in a model-specific manner. Additionally, measurement of various brain activity readouts matched with machine learning strategies confirmed model-specific alterations that could help predict the presence and subtype of metastasis.

CDK4/6 Inhibition Sensitizes Intracranial Tumors to PD-1 Blockade in Preclinical Models of Brain Metastasis.

Brain metastases are associated with high morbidity and are often resistant to immune checkpoint inhibitors. We evaluated whether CDK4/6 inhibitor (CDKi) abemaciclib can sensitize intracranial tumors to programmed cell death protein 1 (PD-1) inhibition in mouse models of melanoma and breast cancer brain metastasis. Treatment response was evaluated in vivo using immunocompetent mouse models of brain metastasis bearing concurrent intracranial and extracranial tumors. Treatment effect on intracranial and extracranial tumor-immune microenvironments (TIME) was evaluated using immunofluorescence, multiplex immunoassays, high-parameter flow cytometry, and T-cell receptor profiling. Mice with humanized immune systems were evaluated using flow cytometry to study the effect of CDKi on human T-cell development. We found that combining abemaciclib with PD-1 inhibition reduced tumor burden and improved overall survival in mice. The TIME, which differed on the basis of anatomic location of tumors, was altered with CDKi and PD-1 inhibition in an organ-specific manner. Combination abemaciclib and anti-PD-1 treatment increased recruitment and expansion of CD8+ effector T-cell subsets, depleted CD4+ regulatory T (Treg) cells, and reduced levels of immunosuppressive cytokines in intracranial tumors. In immunodeficient mice engrafted with human immune systems, abemaciclib treatment supported development and maintenance of CD8+ T cells and depleted Treg cells. Our results highlight the distinct properties of intracranial and extracranial tumors and support clinical investigation of combination CDK4/6 and PD-1 inhibition in patients with brain metastases. See related commentary by Margolin, p. 257.

Warburg effect enhanced by AKR1B10 promotes acquired resistance to pemetrexed in lung cancer-derived brain metastasis

BackgroundResistance to pemetrexed (PEM), a rare chemotherapeutic agent that can efficiently cross the blood-brain barrier, limits the therapeutic efficacy for patients with lung cancer brain metastasis (BM). Aldo-keto reductase family 1 B10 (AKR1B10) was recently found to be elevated in lung cancer BM. The link between AKR1B10 and BM-acquired PEM is unknown.MethodsPEM drug-sensitivity was assessed in the preclinical BM model of PC9 lung adenocarcinoma cells and the BM cells with or without AKR1B10 interference in vitro and in vivo. Metabolic reprogramming of BM attributed to AKR1B10 was identified by chromatography-mass spectrometry (GC-MS) metabolomics, and the mechanism of how AKR1B10 mediates PEM chemoresistance via a way of modified metabolism was revealed by RNA sequencing as well as further molecular biology experimental approaches.ResultsThe lung cancer brain metastatic subpopulation cells (PC9-BrM3) exhibited significant resistance to PEM and silencing AKR1B10 in PC9-BrM3 increased the PEM sensitivity in vitro and in vivo. Metabolic profiling revealed that AKR1B10 prominently facilitated the Warburg metabolism characterized by the overproduction of lactate. Glycolysis regulated by AKR1B10 is vital for the resistance to PEM. In mechanism, AKR1B10 promoted glycolysis by regulating the expression of lactate dehydrogenase (LDHA) and the increased lactate, acts as a precursor that stimulates histone lactylation (H4K12la), activated the transcription of CCNB1 and accelerated the DNA replication and cell cycle.ConclusionsOur finding demonstrates that AKR1B10/glycolysis/H4K12la/CCNB1 promotes acquired PEM chemoresistance in lung cancer BM, providing novel strategies to sensitize PEM response in the treatment of lung cancer patients suffering from BM.

SYST-28 FUNCTIONAL MAPPING REVEALS WIDESPREAD REMODELLING AND A NOVEL TARGETABLE PTP4A2-ROBO1 SIGNALING AXIS AT GLIOBLASTOMA RECURRENCE

Abstract Resistance to genotoxic therapies and tumor recurrence are hallmarks of glioblastoma (GBM), an aggressive brain tumor. Here, we explore the functional drivers of post-treatment recurrent GBM. By conducting genome-wide CRISPR-Cas9 screens in patient-derived GBM models, we uncover distinct genetic dependencies in recurrent tumor cells that were absent in their patient-matched primary predecessors, accompanied by increased mutational burden and differential transcript and protein expression. These analyses map a multilayered genetic response to drive tumor recurrence, identifying protein tyrosine phosphatase 4A2 (PTP4A2) as a novel modulator of self-renewal, proliferation and tumorigenicity at GBM recurrence. Mechanistically, genetic perturbation or small molecule inhibition of PTP4A2 represses axon guidance activity through a dephosphorylation axis with roundabout guidance receptor 1 (ROBO1), exploiting a functional dependency on ROBO signaling. Roundabout guidance receptor 1 (ROBO1) protein is involved in axonal guidance during neurodevelopment and aberrant ROBO1 signaling axis is associated with higher tumorigenicity in GBM. ROBO1 was highly expressed on the surface of malignant and treatment-refractory brain tumor initiating cells (BTICs) in rGBM, brain metastasis (BM) and medulloblastoma (MB). Importantly, engineered anti-ROBO1 single-domain antibodies also mimic the effects of PTP4A2 inhibition. We therefore developed and validated second-generation CAR-T cells against ROBO1, which demonstrated upregulation of activation markers, enhanced cytokine release, markedly increased proliferation, and induction of potent and specific tumor cell death compared to untransduced cells in all the three brain cancers. These findings were further validated in vivo in rGBM, MB and BM models, where ROBO1 CAR-T cells showed significant reduction in tumor burden and increase in survival of treated mice. We conclude that functional reprogramming drives tumorigenicity and dependence on a multi-targetable PTP4A2-ROBO1 signaling axis at GBM recurrence.

BSBM-10 CHARACTERIZATION OF A RADIATION-RESISTANT MODEL OF LUNG CANCER BRAIN METASTASIS

Abstract Lung cancer is the most common cancer to metastasize to the brain, with 50% of all brain metastases originating from lung primary tumors. Traditional therapy for lung cancer brain metastases includes surgical resection, radiation, and chemotherapy. Due to the presence of the blood-tumor barrier, penetration of therapeutics is significantly hindered. Even when used in combination, tumor recurrence and regrowth after initial chemoradiotherapy remain a significant issue. To recapitulate recurrent disease, we aim to characterize a radio-resistant preclinical model of brain metastasis to investigate its effect on growth, chemosensitivity, and drug permeation across the blood-tumor barrier. Herein, we have generated a human NSCLC cell line bearing EGFR exon 19 deletion (PC9) that has been serial passaged in mice to colonize brain (PC9-Br). PC9-Br cells were serially irradiated and allowed to proliferate for 10-14 days prior to re-irradiation. The total dose administered to PC9-Br cells was 60 Gy to generate a radioresistant population (PC9-Br-RR). Clonogenic, MTT, and SRB analysis indicates a significant increase in survival percentage of PC9-Br-RR compared to PC9-Br irradiation at 2, 4, 6, and 8 Gy. While PC9-Br-RR cells are more resistant to cisplatin than PC9-Br (IC50: 2.5 µM and 1.5 µM, respectively at 72 h), they are more sensitive to osimertinib in vitro (IC50: 6.9 nM and 33 nM, respectively at 72 h). In vivo, PC9-Br-RR tumors developed more rapidly in mice than PC9-Br and brain-to-body tumor burden analysis showed PC9-Br-RR cells have increased brain tropism. Collectively, this data supports the characterization of a radio-resistant brain metastasis model to investigate the role of acquired radio-resistance at the blood-tumor barrier.

BSBM-21 MACHINE LEARNING IDENTIFIES EXPERIMENTAL BRAIN METASTASIS SUBTYPES BASED ON THEIR INFLUENCE ON NEURAL CIRCUITS

Abstract A high percentage of patients with brain metastases frequently develop neurocognitive symptoms, however understanding how brain metastasis co-opt the function of neuronal circuits beyond a mass effect remains unknown. We report a comprehensive multidimensional modelling of brain functional analysis in the context of brain metastasis. By testing different pre-clinical models of brain metastasis from various primary sources and oncogenic profiles we dissociated the heterogeneous impact on brain function that we detected from the homogeneous inter-model tumor size or glial response. In contrast we report a potential underlying molecular program responsible for impairing neuronal crosstalk in a model-specific manner. Additionally, measurement of various brain activity readouts matched with machine learning strategies confirmed model-specific alterations that could help to predict the presence and subtype of metastasis. We envision that our findings not only increase our knowledge on the molecular basis of neurocognitive impairment associated with brain metastases but they are also the first step towards new therapeutic strategies to prevent or stop the decline in quality of life associated with these symptoms. In addition, our computational findings exploiting electrophysiological profiles suggest the possibility to exploit them as novel biomarkers.

BSBM-11 COMBINING IMMUNOTHERAPY AND WHOLE-BRAIN RADIATION THERAPY IN A NOVEL SYNGENEIC LUNG CANCER BRAIN METASTASIS PRECLINICAL MODEL: DOES TIMING OF ADMINISTRATION MATTER?

Abstract Approximately 50% of all brain metastasis cases originate from primary lung tumors. Whole-brain radiation therapy (WBRT) is a common treatment for patients with multiple brain metastases, but patients rarely survive longer than one year after diagnosis. Recent studies demonstrate improved outcomes with combinatorial radiotherapy and immunotherapy, but the optimal timing of immunotherapy administration is unclear. Eliciting a robust immune response post-immunotherapy is vital for therapeutic efficacy. Our work shows that the BBB is disrupted and proinflammatory cytokines are increased in the brain 12 hours post-WBRT in immunocompetent but not immunocompromised mice. Additionally, efflux transporter activity is decreased at this time point. This study utilizes a novel Lewis Lung Carcinoma cell line, LLC-Br, which was developed in our laboratory to preferentially metastasize to the brain. Mice were treated with immune checkpoint inhibitors (ICIs) before or after WBRT then we evaluated tumor progression, survival, and blood-tumor barrier (BTB) permeability. We hypothesized administration of ICI following WBRT would increase survival, decrease tumor burden, and increase BTB permeability in our preclinical syngeneic lung cancer brain metastasis model.

Circulating hsa_circ_0072309, acting via the miR-100/ACKR3 pathway, maybe a potential biomarker for the diagnosis, prognosis, and treatment of brain metastasis from non-small-cell lung cancer.

One of the main causes of lung cancer-related death is brain metastasis (BM). Finding early indicators of BM derived from lung cancer is crucial. Therefore, this study was designed to determine if serum hsa_circ_0072309 may be employed as a potential biomarker for BM induced by non-small-cell lung cancer (NSCLC) and to understand its possible underlying mechanism. Primary lung cancer and healthy neighboring tissues were obtained from all patients, while BM tissues were taken from BM+ patients. Serum specimens were collected from all patients and healthy volunteers. Hsa_circ_001653, miR-100, and ACKR3 RNA expressions were analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and atypical chemokine receptor 3 (ACKR3) protein expression by western blotting (WB), immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA). In order to examine the effect of serum hsa_circ_0072309 and its relevant mechanism on BM development, an NSCLC-associated BM model in mice was established. According to the results, miR-100 expression was down-regulated in primary lung cancer tissues compared to healthy lung tissues in all NSCLC patients, and circ_0072309 and ACKR3 expression were up-regulated. In BM tissues compared with primary lung tumors of BM+ patients, in serum samples from all patients compared to healthy volunteers, and in lung tumors of BM+ patients compared to those from BM- patients. Patients' serum exhibits the same level of hsa_circ_0072309/miR-100/ACKR3 expression as in BM samples. Advanced tumor-node-metastasis (TNM) stage, higher BM, shorter post-operative overall survival (OS), and progression-free survival (PFS) are all substantially associated with increased serum circ_0072309 levels in BM+ patients. In animal models, serum owning hsa_circ_0072309 from BM+ patients facilitates BM formation by regulating the miR-100/ACKR3 pathway. The current preliminary research reveals serum hsa_circ_0072309 as a possible biomarker and target for early diagnosis, prognosis, and therapy of NSCLC-derived BM and suggests a substantial role for the hsa_circ_0072309/miR-100/ACKR3 axis in the formation of BM from NSCLC.

Tumor-suppressive action of miR-30a-5p in lung adenocarcinoma correlates with ABL2 inhibition and PI3K/AKT pathway inactivation.

ABL2 contributes to the oncogenic potential of cancers, pointing to its inhibition as a possible strategy against malignant diseases. Bioinformatics prediction of upstream effector miR-30a-5p for ABL2 allowed us to hypothesize and then validate mechanistic actions of miR-30a-5p in lung adenocarcinoma (LUAD). The ABL2 expression in LUAD was analyzed in the TCGA data, clinical samples, and cell lines. The shRNA-mediated silencing of ABL2 was introduced to illustrate its effect on malignant phenotypes of LUAD cells. The binding affinity between ABL2 and miR-30a-5p was verified by luciferase activity and RNA pull-down assay. Ectopic expression, knockdown methods, and PI3K inhibitor LY294002 were used to investigate their effects on in vitro biological characteristics and in vivo tumor growth of LUAD cells. Using nude mouse lung adenocarcinoma in situ and brain metastasis models to validate the inhibitory effect of miR-30a-5p on LUAD by regulating the ABL2/PI3K/AKT signaling axis. High expression of ABL2 and poor expression of miR-30a-5p were noticed in LUAD tissues and cell lines. Importantly, miR-30a-5p was demonstrated to target and downregulate ABL2, subsequently inactivating the PI3K/AKT pathway. miR-30a-5p inhibited the malignant phenotypes of LUAD cells by inhibiting ABL2 expression and inactivating the PI3K/AKT pathway. For in vivo experiments, miR-30a-5p was substantiated to thwart tumor tumorigenesis by regulating the ABL2/PI3K/AKT axis. In addition, miR-30a-5p suppresses the occurrence and development of in situ lung cancer and brain metastasis via the ABL2/PI3K/AKT signaling pathway. This study underscores the inhibitory role of miR-30a-5p in LUAD through the ABL2/PI3K/AKT axis, which may be a viable target for LUAD treatment.

Biglycan and reduced glycolysis are associated with breast cancer cell dormancy in the brain.

Exit of quiescent disseminated cancer cells from dormancy is thought to be responsible for metastatic relapse and a better understanding of dormancy could pave the way for novel therapeutic approaches. We used an in vivo model of triple negative breast cancer brain metastasis to identify differences in transcriptional profiles between dormant and proliferating cancer cells in the brain. BGN gene, encoding a small proteoglycan biglycan, was strongly upregulated in dormant cancer cells in vivo. BGN expression was significantly downregulated in patient brain metastases as compared to the matched primary breast tumors and BGN overexpression in cancer cells inhibited their growth in vitro and in vivo. Dormant cancer cells were further characterized by a reduced expression of glycolysis genes in vivo, and inhibition of glycolysis in vitro resulted in a reversible growth arrest reminiscent of dormancy. Our study identified mechanisms that could be targeted to induce/maintain cancer dormancy and thereby prevent metastatic relapse.

Insights into the regulation of brain metastasis by natural killer cells using single-cell RNA sequencing and imaging mass cytometry

Abstract Metastatic brain tumors are incurable, rapidly fatal, and five times more common than primary glial or meningeal tumors of the central nervous system (CNS). Natural Killer (NK) cells are cytotoxic lymphocytes with strong in vivo anti-tumor activity, a native ability to cross the blood-brain barrier (BBB), and a demonstrated role in the regulation of metastasis in peripheral organs. NK cells perform critical roles in anti-tumor immunosurveillance via the expression of germline-encoded activation and inhibition receptors on the cell membrane. These receptors recognize ligands on both normal and transformed cells, and ultimately determine whether an NK cell will be stimulated to kill a neoplastic cell or inhibited from killing a healthy cell. Notably, NK cells are under investigation in clinical trials as both drug targets and adoptive cell therapies. We are working to characterize NK cell-mediated regulation of brain metastasis, which appears to be dysfunctional relative to the robust NK cell anti-metastatic activity observed in peripheral tissues. We seek to elucidate a molecular mechanism for this NK cell dysfunction using in vivo models of brain metastasis in conjunction with flow cytometry, single-cell RNA sequencing (scRNA-seq), and imaging mass cytometry (IMC). An improved understanding of the functional status and transcriptional programs of NK cells in brain metastasis will inform the development of neuroprotective anti-metastatic therapies aimed at modulating and potentiating the anti-tumor action of both native and adoptively-transferred NK cells. Supported by grants from the National Cancer Institute (R01CA237376-01A1), the UCI Stem Cell Research Center, and the UCI Medical Scientist Training Program

Short-term topiramate treatment prevents radiation-induced cytotoxic edema in preclinical models of breast-cancer brain metastasis.

Brain edema is a common complication of brain metastases (BM) and associated treatment. The extent to which cytotoxic edema, the first step in the sequence that leads to ionic edema, vasogenic edema, and brain swelling, contributes to radiation-induced brain edema during BM remains unknown. This study aimed to determine whether radiation-associated treatment of BM induces cytotoxic edema and the consequences of blocking the edema in preclinical models of breast-cancer brain metastases (BCBM). Using in vitro and in vivo models, we measured astrocytic swelling, trans-electric resistance (TEER), and aquaporin 4 (AQP4) expression following radiation. Genetic and pharmacological inhibition of AQP4 in astrocytes and cancer cells was used to assess the role of AQP4 in astrocytic swelling and brain water intake. An anti-epileptic drug that blocks AQP4 function (topiramate) was used to prevent cytotoxic edema in models of BM. Radiation-induced astrocytic swelling and transient upregulation of AQP4 occurred within the first 24 hours following radiation. Topiramate decreased radiation-induced astrocytic swelling and loss of TEER in astrocytes in vitro, and acute short-term treatment (but not continuous administration), prevented radiation-induced increase in brain water content without pro-tumorigenic effects in multiple preclinical models of BCBM. AQP4 was expressed in clinical BM and breast-cancer cell lines, but AQP4 targeting had limited direct pro-tumorigenic or radioprotective effects in cancer cells that could impact its clinical translation. Patients with BM could find additional benefits from acute and temporary preventive treatment of radiation-induced cytotoxic edema using anti-epileptic drugs able to block AQP4 function.

Abstract 3582: Differentiation of niraparib and olaparib brain penetration in a mouse brain metastatic tumor model

Abstract Purpose: There remains an unmet need to provide effective treatment therapy for patients with primary and metastatic brain tumors; lack of drug penetration across the blood brain barrier (BBB) is a key factor. Synthetic lethality remains an attractive mechanism in treating brain tumors post radiotherapy. Here, we evaluated brain penetration and distribution of niraparib and olaparib in a mouse brain tumor model. Experimental: Female mice (CrTac:NCr-Foxn1nu; 6 w/o) received 2.5E5 luciferase transfected human breast cancer line (MDA 231-BRM2-831) via intracardiac injection. Mice were imaged twice/week using bioluminescence imaging (BLI) to monitor tumor growth. On day 35, mice with brain metastases (BM) were treated via oral gavage once daily for 5 days with either niraparib (35 mg/kg, n=4 BM, n=3 control), olaparib (50 mg/kg, n=3 BM, n=3 control), or vehicle (n=3 control). Terminal blood samples and brains were collected 2 hr post final dose. Serial tissue sections were collected for MALDI-IMS, H&E and IHC staining from 5 distinct horizontal planes in the brain. Tissue collected between each imaging plane was homogenized for LC-MS bioanalysis. Summary of Data: In vivo BLI imaging was used to identify mice with BM and tumor presence was confirmed ex vivo using IHC. Quantitative MALDI IMS of coronal brain sections collected from mice administered niraparib showed consistent concentrations distributed throughout the brain parenchyma with locally higher concentrations detected from tumor regions. Table 1 summarizes the LC-MS bioanalysis concentrations in plasma and bulk brain homogenates and brain section concentrations detected by MALDI IMS. The estimated mean unbound brain-to-plasma partition coefficient (Kp,uu,brain) was 3.0x and 5.6x higher for niraparib compared to olaparib in control and BM mice, respectively. Conclusions: Herein, we demonstrated that niraparib has a higher brain penetration and distribution compared to olaparib in both control mice and mice with BM. Summary of Quantification Results Displayed as Total Conc. with Adjusted Free Conc. in Parentheses Compound Group Terminal Plasma Mean (ng/mL) LC-MS Bulk Brain Homogenate Mean (ng/g) MALDI IMS Brain Section Mean (ng/g) Kp,uu Brain Niraparib Control 3847 (769) 658 (111) 532 (89) 0.18 Niraparib BM 2535 (507) 543 (91) 473 (80) 0.14 Niraparib Vehicle BLQ BLQ Olaparib Control 226 (68) 6 (2) BLQ 0.06 Olaparib BM 119 (36) 5 (2) BLQ 0.03 Olaparib Vehicle BLQ BLQ Citation Format: Mark Reid Groseclose, Jeremy Barry, Tinamarie Skedzielewski, Gerald McDermott, Chakravarthi Balabhadrapatruni, Bill Benson, Yongle Pang, David K. Lim, Hoang Tran, Mike Ringenberg, Keyur Gada, Amine Aziez, Elaine Paul, Hasan Alsaid. Differentiation of niraparib and olaparib brain penetration in a mouse brain metastatic tumor model. [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 3582.

Abstract 4008: TAS2940, a novel brain-penetrable pan-ERBB inhibitor, exhibits tumor regression and prolongs survival in intracranial models bearing ERBB aberrations

Abstract Background: Primary and metastatic brain tumors have significant unmet medical needs as few drugs can penetrate the blood-brain barrier (BBB). Brain metastasis is more common in non-small cell lung cancer (NSCLC) and breast cancer than in other cancers. Genetic alterations in ERBB family genes are frequently observed in those cancers, with about half of HER2/EGFR aberrations metastasizing in the brain. EGFR aberrations have been also observed in glioblastoma. Several pan-ERBB inhibitors are clinically available or under clinical development, such as afatinib, neratinib, and poziotinib. However, none are highly brain-penetrable as they are pumped out via the BBB. Here, we report TAS2940 as a novel potent pan-ERBB inhibitor that elicits brain penetrability and anti-tumor effects in brain metastasis models. Methods: The kinase selectivity of TAS2940 was evaluated in a panel of 257 kinases. Autophosphorylation of wild-type and mutant ERBB in stably expressing cell lines was evaluated using a quantitative immunofluorescence assay. The growth inhibitory effect on cell lines expressing HER2/EGFR aberrations was evaluated based on quantitation of the cellular ATP concentration. Cell lines were transplanted subcutaneously or intracranially in BALB/cAJcl-nu/nu mice. HER2 tyrosine kinase inhibitors, including TAS2940, were then orally administered once daily followed by evaluation of tumor volume and body weight. Results: TAS2940 showed potent and selective inhibition against ERBB family proteins over more than 250 kinases on enzymatic assay. TAS2940 also showed preferential cytotoxicity in cells harboring HER2/EGFR genetic alterations in vitro, while sparing cells without ERBB genetic aberrations. TAS2940 showed potent anti-proliferative activity in cells containing HER2 genetic alterations or EGFR exon 20 insertions, commonly found in NSCLC or breast cancer, as well as EGFRvIII alterations, frequently observed in glioblastoma. In vivo xenograft tumor models with various EGFR genetic alterations showed that TAS2940 exerted anti-tumor effects, such as growth retardation or regression. We further examined brain penetration by assessing the brain to blood concentration of TAS2940 in mice with intact BBBs, showing higher brain penetrability compared to other ERBB inhibitors. Evaluation of TAS2940 in intracranial brain metastasis mouse models resulted in tumor regression and prolonged survival. Conclusion: TAS2940 is a novel potent and selective pan-ERBB inhibitor with high brain penetrability in vivo. These results support the clinical development of TAS2940 in primary and metastatic brain tumors driven by ERBB genetic aberrations. Citation Format: Kei Oguchi, Hikari Araki, Shingo Tsuji, Masayuki Nakamura, Akihiro Miura, Kaoru Funabashi, Akiko Osada, Sakiho Tanaka, Takamasa Suzuki, Shinji Mizuarai. TAS2940, a novel brain-penetrable pan-ERBB inhibitor, exhibits tumor regression and prolongs survival in intracranial models bearing ERBB aberrations. [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 4008.

Abstract 6276: SKL27969, an oral selective PRMT5 inhibitor, sensitizes the effect of DNA-damaging agents in preclinical models of multiple solid tumors

Abstract Background: Protein arginine methyltransferase 5 (PRMT5) is overexpressed in various human malignancies and induces epigenetic and post-translational changes, which have a significant impact on cell growth, proliferation, apoptosis, and DNA damage repair. SKL27969 is a potent and selective brain-penetrating PRMT5 inhibitor demonstrating antitumor activity in the preclinical models of glioblastoma (GBM) and non-small cell lung cancer (NSCLC) and triple-negative breast cancer (TNBC) brain metastasis models. In this study, we evaluated the antitumor activity of SKL27969 alone or in combination with DNA-damaging agents in various cancer models. Methods: Cell cycle analysis, apoptosis, proliferation, and invasion assays were conducted to determine in vitro profiles of SKL27969. In vivo efficacy of SKL27969 was evaluated in human cancer cell line-derived xenograft (CDX) or patient-derived xenograft (PDX) models. Immunohistochemistry and RNA-sequencing analysis in tumor samples from animal models were conducted to confirm the in vivo mechanism of action (MoA). In vitro synergistic effects of SKL27969 with combination partner drugs were analyzed using Combenefit software. Results: SKL27969 exhibits strong anti-proliferative effect by inducing cell cycle arrest and apoptosis and also inhibits invasion of cancer cells. An evaluation of SKL27969 in a panel of over 100 cancer cell lines revealed a broad spectrum of anti-proliferative activity in various cancer types, and an especially more sensitive response in cell lines harboring genetic alterations in the mitotic DNA damage checkpoint, DNA repair, or RNA processing pathways. Administration of SKL27969 demonstrated significant tumor growth inhibition, with a decrease in tumor symmetric dimethylarginine (SDMA) in NSCLC and TNBC subcutaneous xenograft models. Transcriptome and immunohistochemical analysis of SKL27969-treated tumors revealed that the cell cycle checkpoint pathways and DNA damage repair genes were downregulated, supporting the rationale for combination treatment with DNA-damaging agents such as chemotherapeutic agents. Robust antitumor effects were observed after treatment with SKL27969 in combination with paclitaxel or gemcitabine in NSCLC PDX or pancreatic cancer CDX models. Further ex vivo studies to confirm the in vivo MoA of this synergism, and in vivo combination studies of SKL27969 with standard chemotherapeutic agents in other solid cancer CDX models, are ongoing. Conclusion: This is the first preclinical study that suggests the therapeutic potential of SKL27969 in combination with currently approved DNA-damaging agents, and supports further clinical investigation of SKL27969’s role in improving the therapeutic benefit of standard therapies. Citation Format: Mijin Moon, Yongje Shin, Soyoung Ki, Jungtae Na, Ho Yeon Lee, Ilkyoo Koh, Beomjin Hong, Jiyeon Yun, Janice Laramy, Vijaykumar Vashi, Sook-Kyung Park. SKL27969, an oral selective PRMT5 inhibitor, sensitizes the effect of DNA-damaging agents in preclinical models of multiple solid 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 6276.

Abstract 1146: Tepotinib treatment inhibits tumor growth and affects tumor permeability in an intracranial PDX model of MET-amplified NSCLC brain metastasis

Abstract Background: Tepotinib is approved to treat patients with NSCLC harboring MET exon 14 skipping. We previously provided preclinical proof of concept for tepotinib to treat NSCLC metastasized to the brain (Friese-Hamim M, et al. Lung Cancer 2022;163:77-86). Here, we report a detailed analysis of the effects of high-dose tepotinib in preclinical models of MET-amplified NSCLC brain metastases. Experimental methods: Using the previously reported intracranial MET-amplified patient-derived xenograft (PDX) models (LU5349 and LU5406), we measured the effects of tepotinib at the clinically relevant dose of 125 mg/kg QD and above (300 mg/kg QD) or a vehicle control on plasma concentrations, tumor growth (by T2-weighted MRI), intra-tumoral compound distribution (by MALDI MSI) and tumor vasculature (by DCE-MRI for Ktrans, a measure of tumor permeability), as well as biomarkers in tumor tissue (pMET, pAKT, Ki67, endoglin, caspase-3, and MoIgG). For model LU5349, animals were imaged on the day before euthanasia at Day (D) 0, 6 and 16 of treatment (n=10 per time point). Results: Plasma concentrations of tepotinib 2 hours after the final administration at each time point were not proportional by dose, indicating a limited absorption at the higher dose. By D16, tumor measurements (±SD) in tepotinib-treated mice (5.5 ± 3.4 mm3) were approximately 20-fold smaller than those in control mice (109.3 ± 50.1 mm3). At the lower dosing level, concentrations of tepotinib within the tumor regions decreased from 12 ± 6.4 µM (D0) to 3.2 ± 2.7 µM (D6) and then increased to 4.6 ± 1.2 µM (D16). In the higher dose group, concentrations of tepotinib in tumor regions decreased from 15.1 ± 8.7 µM (D0) to 6.8 ± 2.9 µM (D6) and then to 4.5 ± 3.6 µM (D16). Concentrations of tepotinib were below the limit of detection in non-tumor regions. Ktrans of the tumor was also affected by tepotinib treatment. While there was an apparent decline in mean Ktrans over time in control tumors (7.4 ± 3.0 × 10−2 s−1 [D0], 6.4 ± 2.8 × 10−2 s−1 [D6], 5.6 ± 3.5 × 10−2 s−1 [D16]), tepotinib treatment caused a further reduction at both the lower (7.8 ± 3.2 × 10−2 s−1 [D0], 2.9 ± 1.3 × 10−2 s−1 [D6], 2.7 ± 1.4 × 10−2 s−1 [D16]) and higher dose (8.0 ± 3.0 × 10−2 s−1 [D0], 2.7 ± 1.1 × 10−2 s−1 [D6], 2.9 ± 1.2 × 10−2 s−1 [D16]), indicating potential anti-vascular effects in the tumor. Conclusions: These preclinical analyses of tepotinib treatment of MET-amplified NSCLC brain metastases provide new insights, considering the observed tumor regression, the compound’s distribution to tumor versus non-tumor brain tissue and anti-vascular effects (via DCE-MRI), which may provide a predictive biomarker of response and patient survival. A possible causal association between reduced vascular permeability and the restricted distribution of tepotinib to tumor regions may exist. Analyses of the second PDX model and tumor biomarkers are ongoing and will be presented. Citation Format: Anderson Clark, Joachim Albers, Patrick McConville, Sylwia A. Stopka, Michael S. Regan, Marina DiPiazza, Nathalie Y.R. Agar. Tepotinib treatment inhibits tumor growth and affects tumor permeability in an intracranial PDX model of MET-amplified NSCLC brain metastasis [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 1146.

Abstract 4680: Intracranial breast cancer brain metastases (BCBM) models - stereotactic vs. intracarotid inoculation

Abstract Breast cancer is the most prevalent cancer in women worldwide. 30% of breast cancer patients develops metastases at distant organs, with 10-20% of these patients developing brain metastases. The median survival time for patients with brain metastases is only 7.2 months and there is an urgent need for effective therapies. Development of novel therapies relies on good translational animal models. Subcutaneous tumors are technically easy and highly reproducible but lacks the natural tumor environment and the impact of the blood brain barrier. Breast cancer brain metastasis (BCBM) models are most often established by stereotactic intracranial inoculation. However, this leads to formation of single tumors and does not take the extravasation and seeding part of the metastatic cascade into account and resembles primary tumors to a larger extend. Intracarotid inoculation incorporates extravasation and seeding of micro metastases making it a better suited translational animal model for BCBM. A panel of intracranial BCBM was established through stereotactic and intracarotid inoculation of breast cancer cell lines with high survival and take rates (>85%). The panel included the ST941 PDX model, an estrogen receptor positive model harbouring the ESR1-Y537S mutation and exhibiting resistance to fulvestrant. The panel also included the triple negative breast cancer cell line MDA-MB-231 as well as HER2+ models. Tumor establishment and progression were evaluated using bioluminescence and magnetic resonance imaging (MRI). Whole brain and tumors were isolated, and paraffin embedded for histological evaluation of tumor establishment, invasiveness, and infiltration. Intracranial tumor establishment was evident early after inoculation using bioluminescence imaging and tumor growth was later confirmed by MRI. Tumor growth rate were dependent on inoculation method with rapid tumor growth observed with stereotactic inoculation. While stereotactic inoculation led to single tumor establishment with defined tumor borders, intracarotid inoculation led to micro seeding with multiple foci established throughout the brain. At early timepoints, the metastases consisted of <150 cells per metastases, but at later timepoints larger metastases were formed. Tumor infiltration was more diffused in brains with multiple metastases and the blood brain barrier integrity was different when comparing the models and inoculation methods. A panel of BCBM was successfully established using both stereotactic and intracarotid inoculation. While bioluminescence allowed the earliest tumor imaging, MRI provided additional information on tumor establishment such as location, numbers, and blood brain barrier permeability. The models showed varying tumor establishment, infiltration, and marker expression. The established panel can be used as a platform for testing novel anti-cancer therapies targeting BCBM. Citation Format: Sigrid Cold, Moritz Braig, Trine Bjoernbo Engel, Maria Zeiler Alfsen, Maria Nikoline Kristiansen, Lotte Kellemann Kristensen, Andreas Kjaer, Carsten Haagen Nielsen. Intracranial breast cancer brain metastases (BCBM) models - stereotactic vs. intracarotid inoculation. [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 4680.

Abstract 63: Machine learning identifies experimental brain metastasis subtypes based on their influence on neural circuits

Abstract A high percentage of patients with brain metastases frequently develop neurocognitive symptoms, however understanding how brain metastasis co-opt the function of neuronal circuits beyond a mass effect remains unknown. We report a comprehensive multidimensional modelling of brain functional analysis in the context of brain metastasis. By testing different pre-clinical models of brain metastasis from various primary sources and oncogenic profiles we dissociated the heterogeneous impact on brain function that we detected from the homogeneous inter-model tumor size or glial response. In contrast we report a potential underlying molecular program responsible for impairing neuronal crosstalk in a model-specific manner. Additionally, measurement of various brain activity readouts matched with machine learning strategies confirmed model-specific alterations that could help to predict the presence and subtype of metastasis. Citation Format: Mariam Masmudi-Martín, A Sanchez-Aguilera, A Navas-Olive, P Baena, C Hernández-Oliver, S Martínez, M Lafarga, Renacer Red Nacional de Metástasis Cerebral, MZ Lin, L Menendez de la Prida, M Valiente. Machine learning identifies experimental brain metastasis subtypes based on their influence on neural circuits [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 63.