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Purpose:Molecular targeted therapy using BRAF and/or MEK inhibitors has been applied to BRAFV600E-mutant high-grade gliomas (HGG); however, the therapeutic effect is limited by the emergence of drug resistance.Experimental Design:We established multiple paired BRAFV600E-mutant HGG patient-derived xenograft models based on tissues collected prior to and at relapse after molecular targeted therapy. Using these models, we dissected treatment-resistant mechanisms for molecular targeted therapy and explored therapeutic targets to overcome resistance in BRAFV600E HGG models in vitro and in vivo.Results:We found that, despite causing no major genetic and epigenetic changes, BRAF and/or MEK inhibitor treatment deregulated multiple negative feedback mechanisms, which led to the reactivation of the MAPK pathway through c-Raf and AKT signaling. This altered oncogenic signaling primarily mediated resistance to molecular targeted therapy in BRAFV600E-mutant HGG. To overcome this resistance mechanism, we performed a high-throughput drug screening to identify therapeutic agents that potently induce additive cytotoxicity with BRAF and MEK inhibitors. We discovered that HSP90 inhibition combined with BRAF/MEK inhibition coordinately deactivated the MAPK and AKT/mTOR pathways, and subsequently induced apoptosis via dephosphorylation of GSK3β (Ser9) and inhibition of Bcl-2 family proteins. This mediated potent cytotoxicity in vitro and in vivo in refractory models with acquired resistance to molecular targeted therapy.Conclusions:The combination of an HSP90 inhibitor with BRAF or MEK inhibitors can overcome the limitations of the current therapeutic strategies for BRAFV600E-mutant HGG.

Supplementary Table from HSP90 Inhibition Overcomes Resistance to Molecular Targeted Therapy in BRAFV600E-mutant High-grade Glioma

Supplementary Data from HSP90 Inhibition Overcomes Resistance to Molecular Targeted Therapy in BRAFV600E-mutant High-grade Glioma

Supplementary Figure from HSP90 Inhibition Overcomes Resistance to Molecular Targeted Therapy in BRAFV600E-mutant High-grade Glioma

Supplementary document

Supplementary Table 1 Primers used for PCR amplification and sequencing Supplementary Table 2 Results of Multiplex PCR based MGH SNAPShot assay, Pyrosequencing and Sanger sequencing in YMG6 patient and xenograft cells Supplementary Table 3 Analysis of microsatellite instability in YMG6 parent and xenograft tumors Supplementary Table 4 Quantitative analysis for MGMT methylation status in YMG6 cells Supplementary Table 5 Multiplex PCR based MGH SnaPShot assay for oligodendroglial tumors Supplementary Table 6 Correlation of xenograft lethality and phosphorylation of PI3K pathway protein Supplementary Table 7 Phosphorylation in PI3K pathway in oligodendroglioma xenograft model Supplementary Table 8 Clinical characteristics in oligodendroglial tumor patients

Supplementary Figure S1-S7

Supplementary Figure 1. A, Hematoxylin and eosin, Ki-67, and IDH1R132H staining in each specimen. B, Sanger sequencing, immunohistochemical analysis, pyrosequencing, and FISH to identify YMG6 tumors as oligodendroglial tumors. C, Multiplex Ligation-dependent Probe Amplification assay demonstrating chromosome 1p and 19q co-deletion in YMG6R3F (left) and YMG6R3T (right). Bars, 50 �m. Supplementary Figure 2. A, DNA fingerprinting showing matched DNA identification in YMG6R3F (upper), YMG6R3T (middle), and YMG6R3T xenograft (YMG6R3Tsc1, lower). Supplementary Figure 3. Sanger sequencing indicating MSH6Ala1293Ser (left, arrow) and STK11Pro281Leu (right, arrow) in YMG6I. B, Immunohistochemical analysis demonstrating MSH6 expression in YMG6I (left) and YMG6R4T (right). C, Immunohistochemical analysis demonstrating LKB1/STK11 expression in YMG6I (left) and YMG23 (right, positive control). D, Sanger sequencing indicating MSH6Gly409Glu (arrow) in YMG6R4T. E, F, Upper gastrointestinal endoscopy (E) and colonoscopy (F) demonstrating no abnormal lesion in YMG6 patient. G, FDG-PET indicating no abnormal uptake in YMG6 patient. Bars, 50 �m. Supplementary Figure 4. Microsatellite instability (MSI)-PCR indicating microsatellite stable (MSS) in YMG6R2 and YMG6R3T. MSI-H, microsatellite instability high. MSS, microsatellite stable. Supplementary Figure 5. Immunohistochemical analysis demonstrating ATRX expression and weak expression of p53 in YMG6R3T. Supplementary Figure 6. Pyrosequencing indicating PIK3CAE542K (arrows) in YMG6I and YMG6R3T. B, Immunohistochemical analysis demonstrating phospho-AKT, -4EBP1, and -S6K expression in YMG6I and YMG6R1. Supplementary Figure 7. A, Multiplex Ligation-dependent Probe Amplification assay indicating 1p and 19q co-deletion in YMG6R3Tsc1. B, Sanger sequencing showing mutation in IDH1 (arrow; c.395G>A, left) and TERT (arrow; c.-124C>T, right) of YMG6R4Tsc1. Supplementary Figure 8. A, Overview and microscopic view of hematoxylin and eosin (H&E, upper), Ki-67 (middle) and IDH1R132H (lower) in first (YMG6R3Tsc1, left), second (YMG6R3Tsc2, middle), and third (YMG6R3Tsc3, right) generation YMG6R3T xenograft. B, H&E (upper), Ki-67 (middle), and IDH1R132H (lower) in YMG6R4Tsc1 (left), YMG6R4Tsc2 (middle), and YMG6R4Tsc3 (right). C, Sanger sequencing showing mutation of IDH1 (arrows, c.395G>A, left) and TERT (arrows, c.-124C>T, right), and PIK3CA (arrows, c.1624G>A) in YMG6R3Tsc2 (upper), YMG6R4Tsc2 (middle), and YMG6R4Tsc3 (lower). D, Immunohistochemical analysis demonstrating strong expression of phospho-AKT (Ser473, left), phospho-4EBP1 (middle), and phospho-S6K (right) in YMG6R4Tsc3. Bars, 50 �m. Supplementary Figure 9. A, Upper, Sanger sequencing showing IDH1 mutation (arrow, c.395 G>A), TERT (arrow, c.-124C>T), and PIK3CA (arrow, c.3140A>G) in YMG23. Lower, MLPA demonstrating chromosome 1p/19q co-deletion and CDKN2A (chr.9p.21) loss in YMG23 patient tumor. B, Immunohistochemical analysis demonstrating negative expression of p16INK4a/CDKN2A in YMG23 (upper). YMG5 (CDKN2A intact) served as positive control. C, Sanger sequencing showing mutations of IDH1 (arrow, c.395G>A, R132H, left) and TERT (arrow, c.-124C>T) in YMG5. D, Sanger sequencing showing mutations of IDH1 (arrow, c.395G>A), TERT (arrow, c.-124C>T), and PIK3CA (arrow, c.3140A>G) in YMG23sc1. E, MLPA demonstrating chromosome 1p/19q co-deletion and CDKN2A loss (chr.9p.21) in YMG23sc1. F, immunohistochemical analysis demonstrating negative expression of p16INK4a/CDKN2A in YMG23sc1. Bars, 50 �m. Supplementary Figure 10. A, B, C, Sanger sequencing showing IDH1 mutation (arrows, c.395G>A, R132H, left) and TERT promoter mutation (c.-124C>T, C228T or c.-146C>T, C250T), and in YMG46 (A), YMG28 (B) and YMG53 (C). PIK3CA mutation (c.1406A>G, E542G, arrow, A, right) in YMG46. D, Contrast enhancing MRI showing non-enhanced tumor recurrence at right frontal lobe (left). H&E (upper) and IDH1R132H staining (lower) in YMG53 (AOD, middle). Overview of H&E staining indicating no xenograft formation in YMG53sc1 (right). Bars, 50 �m. Supplementary Figure 11. A, Overview and magnification of hematoxylin and eosin staining of second passaged YMG23 xenograft model (YMG23sc2). B, C, Immunohistochemical analysis demonstrating expression of IDHR132H (B, left), Ki-67 (B, right), phospho-AKT (C, left), phospho-4EBP1 (C, middle), and phospho-S6K (C, right) in YMG23sc2. D, Sanger sequencing showing mutations in IDH1 (arrow, c.395 G>A), TERT (arrow, c.-124C>T), and PIK3CA (arrow, c.3140A>G) in YMG23sc2. Bars, 50 �m. Supplementary Figure 12. (A-D) Contrast enhanced MRI of the indicated patients at pre-treatment (left) and post-chemotherapy with/without radiotherapy (right). Supplementary Figure 13. Kaplan Meier Curve indicating no survival difference (P = 0.9) of overall survival in PIK3CA/PIK3RI- mutant (blue) and -wild-type (red) oligodendroglial tumor patients. Supplementary Figure 14. A, Relative cell viability of YMG28 (left) and YMG46 (right) cells after 3-day treatment with FK866 combined with DMSO control (blue bars) or TMZ (200 �M, purple bars). *, P

Primary central nervous system lymphoma (PCNSL) is an isolated type of lymphoma of the central nervous system and has a dismal prognosis despite intensive chemotherapy. Recent genomic analyses have identified highly recurrent mutations of MYD88 and CD79B in immunocompetent PCNSL, whereas LMP1 activation is commonly observed in Epstein–Barr virus (EBV)-positive PCNSL. However, a lack of clinically representative preclinical models has hampered our understanding of the pathogenic mechanisms by which genetic aberrations drive PCNSL disease phenotypes. Here, we establish a panel of 12 orthotopic, patient-derived xenograft (PDX) models from both immunocompetent and EBV-positive PCNSL and secondary CNSL biopsy specimens. PDXs faithfully retained their phenotypic, metabolic, and genetic features, with 100% concordance of MYD88 and CD79B mutations present in PCNSL in immunocompetent patients. These models revealed a convergent functional dependency upon a deregulated RelA/p65-hexokinase 2 signaling axis, codriven by either mutated MYD88/CD79B or LMP1 with Pin1 overactivation in immunocompetent PCNSL and EBV-positive PCNSL, respectively. Notably, distinct molecular alterations used by immunocompetent and EBV-positive PCNSL converged to deregulate RelA/p65 expression and to drive glycolysis, which is critical for intracerebral tumor progression and FDG-PET imaging characteristics. Genetic and pharmacologic inhibition of this key signaling axis potently suppressed PCNSL growth in vitro and in vivo. These patient-derived models offer a platform for predicting clinical chemotherapeutics efficacy and provide critical insights into PCNSL pathogenic mechanisms, accelerating therapeutic discovery for this aggressive disease.Significance:A set of clinically relevant CNSL xenografts identifies a hyperactive RelA/p65-hexokinase 2 signaling axis as a driver of progression and potential therapeutic target for treatment and provides a foundational preclinical platform.

Supplementary Table S1-S12

Purpose:Oligodendroglioma has a relatively favorable prognosis, however, often undergoes malignant progression. We hypothesized that preclinical models of oligodendroglioma could facilitate identification of therapeutic targets in progressive oligodendroglioma. We established multiple oligodendroglioma xenografts to determine if the PI3K/AKT/mTOR signaling pathway drives tumor progression.Experimental Design:Two anatomically distinct tumor samples from a patient who developed progressive anaplastic oligodendroglioma (AOD) were collected for orthotopic transplantation in mice. We additionally implanted 13 tumors to investigate the relationship between PI3K/AKT/mTOR pathway alterations and oligodendroglioma xenograft formation. Pharmacologic vulnerabilities were tested in newly developed AOD models in vitro and in vivo.Results:A specimen from the tumor site that subsequently manifested rapid clinical progression contained a PIK3CA mutation E542K, and yielded propagating xenografts that retained the OD/AOD-defining genomic alterations (IDH1R132H and 1p/19q codeletion) and PIK3CAE542K, and displayed characteristic sensitivity to alkylating chemotherapeutic agents. In contrast, a xenograft did not engraft from the region that was clinically stable and had wild-type PIK3CA. In our panel of OD/AOD xenografts, the presence of activating mutations in the PI3K/AKT/mTOR pathway was consistently associated with xenograft establishment (6/6, 100%). OD/AOD that failed to generate xenografts did not have activating PI3K/AKT/mTOR alterations (0/9, P < 0.0001). Importantly, mutant PIK3CA oligodendroglioma xenografts were vulnerable to PI3K/AKT/mTOR pathway inhibitors in vitro and in vivo—evidence that mutant PIK3CA is a tumorigenic driver in oligodendroglioma.Conclusions:Activation of the PI3K/AKT/mTOR pathway is an oncogenic driver and is associated with xenograft formation in oligodendrogliomas. These findings have implications for therapeutic targeting of PI3K/AKT/mTOR pathway activation in progressive oligodendrogliomas.

Primary central nervous system lymphoma (PCNSL) is an isolated type of lymphoma of the central nervous system and has a dismal prognosis despite intensive chemotherapy. Recent genomic analyses have identified highly recurrent mutations of MYD88 and CD79B in immunocompetent PCNSL, whereas LMP1 activation is commonly observed in Epstein–Barr virus (EBV)-positive PCNSL. However, a lack of clinically representative preclinical models has hampered our understanding of the pathogenic mechanisms by which genetic aberrations drive PCNSL disease phenotypes. Here, we establish a panel of 12 orthotopic, patient-derived xenograft (PDX) models from both immunocompetent and EBV-positive PCNSL and secondary CNSL biopsy specimens. PDXs faithfully retained their phenotypic, metabolic, and genetic features, with 100% concordance of MYD88 and CD79B mutations present in PCNSL in immunocompetent patients. These models revealed a convergent functional dependency upon a deregulated RelA/p65-hexokinase 2 signaling axis, codriven by either mutated MYD88/CD79B or LMP1 with Pin1 overactivation in immunocompetent PCNSL and EBV-positive PCNSL, respectively. Notably, distinct molecular alterations used by immunocompetent and EBV-positive PCNSL converged to deregulate RelA/p65 expression and to drive glycolysis, which is critical for intracerebral tumor progression and FDG-PET imaging characteristics. Genetic and pharmacologic inhibition of this key signaling axis potently suppressed PCNSL growth in vitro and in vivo. These patient-derived models offer a platform for predicting clinical chemotherapeutics efficacy and provide critical insights into PCNSL pathogenic mechanisms, accelerating therapeutic discovery for this aggressive disease. Significance: A set of clinically relevant CNSL xenografts identifies a hyperactive RelA/p65-hexokinase 2 signaling axis as a driver of progression and potential therapeutic target for treatment and provides a foundational preclinical platform.

Purpose: Oligodendroglioma has a relatively favorable prognosis, however, often undergoes malignant progression. We hypothesized that preclinical models of oligodendroglioma could facilitate identification of therapeutic targets in progressive oligodendroglioma. We established multiple oligodendroglioma xenografts to determine if the PI3K/AKT/mTOR signaling pathway drives tumor progression. Experimental Design: Two anatomically distinct tumor samples from a patient who developed progressive anaplastic oligodendroglioma (AOD) were collected for orthotopic transplantation in mice. We additionally implanted 13 tumors to investigate the relationship between PI3K/AKT/mTOR pathway alterations and oligodendroglioma xenograft formation. Pharmacologic vulnerabilities were tested in newly developed AOD models in vitro and in vivo. Results: A specimen from the tumor site that subsequently manifested rapid clinical progression contained a PIK3CA mutation E542K, and yielded propagating xenografts that retained the OD/AOD-defining genomic alterations (IDH1R132H and 1p/19q codeletion) and PIK3CAE542K, and displayed characteristic sensitivity to alkylating chemotherapeutic agents. In contrast, a xenograft did not engraft from the region that was clinically stable and had wild-type PIK3CA. In our panel of OD/AOD xenografts, the presence of activating mutations in the PI3K/AKT/mTOR pathway was consistently associated with xenograft establishment (6/6, 100%). OD/AOD that failed to generate xenografts did not have activating PI3K/AKT/mTOR alterations (0/9, P < 0.0001). Importantly, mutant PIK3CA oligodendroglioma xenografts were vulnerable to PI3K/AKT/mTOR pathway inhibitors in vitro and in vivo—evidence that mutant PIK3CA is a tumorigenic driver in oligodendroglioma. Conclusions: Activation of the PI3K/AKT/mTOR pathway is an oncogenic driver and is associated with xenograft formation in oligodendrogliomas. These findings have implications for therapeutic targeting of PI3K/AKT/mTOR pathway activation in progressive oligodendrogliomas.

INTRODUCTION Molecular targeted therapy using BRAF and/or MEK inhibitors has been applied to BRAFV600E mutant high-grade gliomas (HGGs); however, the therapeutic effect is limited by the emergence of drug resistance. METHODS We established multiple paired BRAFV600E mutant HGG patient-derived xenograft (PDX) models based on tissues collected prior to and at relapse after molecular targeted therapy. Using these models, we dissected treatment resistant mechanisms for molecular targeted therapy and explored therapeutic targets to overcome resistance in BRAFV600E HGG models in vitro and in vivo. RESULTS We found that, despite causing no major genetic and epigenetic changes, BRAF and/or MEK inhibitor treatment deregulated multiple negative feedback mechanisms, which led to the re-activation of the MAPK pathway through c-Raf and AKT signaling. This altered oncogenic signaling primarily mediated resistance to molecular targeted therapy in BRAFV600E mutant HGG. To overcome this resistance mechanism, we performed a high-throughput drug screening to identify therapeutic agents that potently induce additive cytotoxicity with BRAF and MEK inhibitors. We discovered that HSP90 inhibition combined with BRAF/MEK inhibition coordinately deactivated the MAPK and AKT/mTOR pathways, and subsequently induced apoptosis via dephosphorylation of GSK3β (Ser9) and inhibition of Bcl-2 family proteins. This mediated potent cytotoxicity in vitro and in vivo in refractory models with acquired resistance to molecular-targeted therapy. CONCLUSIONS The combination of an HSP90 inhibitor with BRAF or MEK inhibitors can overcome the limitations of the current therapeutic strategies for BRAFV600E mutant HGG.

Introduction: The standard therapy for malignant brain tumors includes surgery and combination therapy with radiation and chemotherapy, but to provide individualized treatment based on the biological and molecular genetic background of the tumor, integrate genetic information with various functional data are required. In this study, we present an overview of our integrated approaches for translational research and clinical management. Methods: In glioma, pre-and intra-operative clinical information, including intraoperative genetic diagnosis, and intraoperative rapid immunohistochemistry is obtained, then a multidisciplinary treatment approach is started based on these integrated data. Specimens collected intraoperatively are cryopreserved for future analysis, and primary cultured cells are routinely collected. The cultured cells are transplanted into the brain of immunodeficient mice to establish patient-derived xenograft model (PDX). Genetic screening, such as IDH, TERT, BRAF, H3F3A mutation and MGMT methylation analysis are routinely assessed within a few days after surgery and used as information for integrated diagnosis. In case of PDX establishment or recurrence, we perform whole exon sequencing or comprehensive genomic assessment to identify genetic abnormalities. If genomic alterations for possible molecular targeted therapy are identified, we assess drug sensitivity test in vitro and in vivo, which are utilized for research to develop optimal molecular targeted therapy. The results, such as the therapeutic effects of molecular targeted drugs, are used for clinical applications. Results: Since the platform was established, we have treated a total of 286 patients, including 189 gliomas and 37 central nervous system lymphomas based on the integrated information. We are currently collecting clinical data to examine if this integrated approach could provide clinical benefit.Conclusion: The translational research system for malignant brain tumors plays an important role in the promotion of clinical and basic research.

The BRAFV600E mutation results in the constitutive activation of downstream mitogen activated protein kinase (MAPK) pathway that promotes tumor growth. Recently, molecular targeted therapy using BRAF/MEK inhibitor has been reported for BRAFV600E mutant high-grade glioma, but the therapeutic effect is limited by the emergence of drug resistance. Herein, we established paired BRAFV600E mutant glioblastoma (GBM) patient-derived xenograft (PDX) models, which were derived from tumors at prior to and recurrence after molecular targeted therapy. These PDX models were found to extensively recapitulate the histology, genetic abnormalities, and even the clinical course of the patients. Furthermore, BRAF/MEK inhibitor gradually caused resistance in cell lines derived from specimens that initially responded to molecular targeted therapy. In this study, genomic and epigenomic changes had little effect on the resistance mechanism. On the other hand, we found that hyperactivation of the MAPK pathway through c-Raf and the AKT/mTOR pathway primarily caused resistance to molecular targeted therapy in BRAFV600E mutant GBM. Through a high throughput drug screening, we find that HSP90 inhibitor with BRAF/MEK inhibitor coordinately deactivates MAPK pathway and AKT/mTOR pathway, and mediates potent toxicity in vitro and in vivo in refractory and acquired resistant models. These findings support that this therapeutic approach can overcome the limitation of current molecular targeted therapy in BRAFV600E mutant GBM.

Previous studies indicated that MGMT promoter methylation status with IDH and TERT promotor mutation are major prognostic factors in glioma. In addition to these molecular features, we have been assessing drug sensitivity against several chemotherapeutic agents, including temozolomide (TMZ). Here, we examined if this combined information could strongly predict drug sensitivity and the prognosis in glioma patients. One hundred and twenty-five IDH wild-type gliomas (WHO grade III and grade IV) were included in this study and retrospectively analyzed. Among them, we focused on 37 patients with partial surgical resection and biopsy to assess radiological difference on MRI. The primary cultured tumor cells were exposed with several compounds for 72 hours, then ATP based cell viability assay was performed. The favorable radiological therapeutic effect was found in 6 out of 8 (75%) with MGMT promoter methylated cases, while unfavorable in 23 of 29 (79.3%) with MGMT promoter unmethylated cases (p=0.008). The drug screening assay demonstrated that 7 of 10 cases with favorable TMZ sensitivity in vitro showed response on MRI, whereas 22 of 27 (81.5%) cases with TMZ resistance in vitro indicated tumor progression (p=0.006). Of note, all 5 cases with sensitive to TMZ and methylated MGMT promoter demonstrated favorable radiological response (p=0.002). These 5 cases tended to survive longer (median survival time, 697 days) as compared to others (median survival time, 391 days, p=0.13). These data indicate that integrated approach with genomic assessment and drug screening test may predict therapeutic response to chemotherapy and contribute selecting optimal therapy in glioma patients.

Epithelioid glioblastoma (E-GBM) predominantly arises at younger age and promotes dismal prognosis. Because of its rare etiology, pathological and genetical characterization of E-GBM remains elusive. Herein, we report 2 patient-derived E-GBM xenograft (PDX) models from young adult patients (YMG62 and YMG89) with BRAFV600E and TERT promoter mutation. The YMG62 patient received dabrafenib with trametinib, while YMG89 patient received dabrafenib monotherapy after recurrence with Stupp regimen. These molecular target therapies were initially responded, but gradually became resistant (YMG62R and YMG89R) and resulted in lethal. Treatment resistant cells were collected from CSF. These primary cells were propagated at multiple passage in vitro. Paired PDX models were established from initial and recurrent cells. All PDX tumors were preferentially disseminated and negative expression of GFAP, which were recapitulated to the patient characteristics. BRAF and MEK inhibitor moderately suppressed cell viability of YMG62 and YMG89 in vitro. However, BRAF and MEK inhibitor became resistant at recurrence in vitro. Western blotting indicated retained phospho-MEK expression after BRAF/MEK inhibitor treatment in recurrent cells, which implies crucial role of MEK activation for tumor maintenance in BRAFV600E mutant E-GBM. Together, paired E-GBM PDX models with/without molecular target therapy recapitulate patient characteristics, which may contribute to elucidate tumor biology and establish novel therapeutic target in E-GBM.

Epithelioid glioblastoma (E-GBM) arises at younger age, commonly disseminates to cerebrospinal fluid, and results in dismal prognosis. About half of E-GBM harbors BRAF V600E mutation, thus BRAF/MEK inhibitors are expected to be specifically sensitive to E-GBM like other BRAF V600E mutant carcinomas. However, therapeutic effect is limited by the emergence of drug resistance. To overcome this issue, it is crucial to elucidate the treatment resistance mechanisms by clinically representative models. Herein, we establish 2 paired E-GBM patient-derived xenograft (PDX) models from young adult patients (YMG62 and YMG89) with BRAF V600E, TERT promoter mutations and CDKN2A homozygous deletions. The YMG62 patient received dabrafenib with trametinib, while YMG89 patient received dabrafenib monotherapy after recurrence with standard treatment. The YMG62 patient was refractory to combination therapy. The YMG89 patient was initially responded to dabrafenib, but gradually became resistant and the 2 patients died due to CNS dissemination. Paired PDX models were established from tumors prior and after molecular target therapy. All PDXs were formed as CNS dissemination model, which were recapitulated to the patient characteristics. BRAF/MEK inhibitors strongly suppressed cell viability in primary tumor (YMG89P). However, BRAF/MEK inhibitors became resistant in recurrent tumor (YMG89R). YMG62 paired PDXs were resistant to molecular target therapy. Western blotting indicated retained MAPK signaling pathway and/or increased AKT phosphorylation after BRAF/MEK inhibitors treatment in refractory and recurrent cells, which indicates crucial role of re-activation in the MAPK signaling pathway and/or PI3 kinase pathway for tumor maintenance in BRAF V600E mutant E-GBM. We have done high throughput drug screening to identify compounds to overcome resistant to molecular target therapy. Our established E-GBM paired PDX models recapitulate patient characteristics, which may uncover treatment resistant mechanism and novel therapeutic target in E-GBM.

Primary central nervous system lymphoma (PCNSL) is a rare lymphoma of the central nervous system and has a dismal prognosis despite intensive chemotherapy. Recent genomic analyses have identified recurrent genetic alterations in Primary central nervous system lymphoma (PCNSL). However, lack of clinically representative PCNSL models has diminished our understanding of the pathogenic mechanisms of those genetic events. Here, we established 14 patient-derived orthotopic xenografts (PDOXs). Comprehensive analysis showed that PDOXs faithfully retained the phenotypic, metabolic, and genetic features with 100 % concordance of MYD88 and CD79B mutations present in immuno-competent PCNSL patients. Notably, orthotopic xenograft formation was consistently dependent on deregulated signaling through the RelA/p65-hexokinase 2 (HK-2) axis. MYD88/CD79B mutations and Pin1 activation, or LMP1 and Pin1 activation, converge on the RelA/p65-HK-2 signaling in immunocompetent and EBV-positive PCNSL, respectively. Genetic and pharmacological inhibition of this key signaling axis potently suppressed PCNSL tumor growth in vitro and in vivo. Additionally, our models further offer a platform for predicting clinical chemotherapeutics efficacy. Therefore, our models provide critical insights into pathogenic mechanisms and therapeutic discovery in PCNSL.

Recent genomic analyses have identified highly recurrent genetic alterations in PCNSL. However, due to the lack of clinically representative PCNSL preclinical models, the pathogenic mechanisms of these alterations remains largely unknown. Here, we established the largest panel of 12 clinically relevant PCNSL patient-derived orthotopic xenografts retained the histopathologic phenotype, lymphoma expression subtype, copy number alterations and 90% of the non-synonymous mutations of primary tumors, with 100% concordance of MYD88 and CD79B mutations, which are highly recurrent in PCNSL. Patient tumor regression with high-dose methotrexate correlated with in vitro sensitivity to methotrexate in corresponding PCNSL models. By knocking down canonical NF-kB pathway genes, we found that successful orthotopic xenograft formation was dependent on NF-kB canonical pathway activation induced by MYD88 mutation or overexpression of EBV-related LMP1. Metabolically, PCNSL xenografts phenocopied the high 18F-fluorodeoxyglucose uptake observed in patients and demonstrated glycolytic dependence, revealing new potential therapeutic strategies in PCNSL. Collectively, we found NF-kB canonical pathway activation as a crucial driver of PCNSL xenograft progression and found that NF-kB canonical pathway induced an addiction to glycolysis, revealing a novel potential therapeutic strategy. Our PCNSL xenograft panel represents a valuable and reproducible preclinical tool that has the potential to help decipher how genetic and/or epigenetic alterations contributes to lymphomagenesis and tumor maintenance and enhance the development of novel therapeutic strategies in PCNSL.

Epithelioid glioblastoma (E-GBM) predominantly arises at younger age and promotes dismal prognosis. Because of its rare etiology, pathological and genetical characterization of E-GBM remains elusive. Herein, we report unique patient-derived E-GBM xenograft (PDX) models from 3 E-GBM patients (2 BRAFV600E mutant and 1 BRAFV600E wild-type). Two BRAF mutant E-GBM cells (YMG62 and YMG89) were originated from adolescent and young adult patients and harbored TERT promoter mutation and CDKN2A homozygous deletion, while 1 BRAFV600E E-GBM cell (YMG64) was from elderly patient and had TERT wild-type. YMG62 and YMG89 could be propagated at multiple passage in vitro, while YMG64 could not be maintained. PDX models were established from YMG62, YMG89, and YMG64. All PDX tumors were preferentially disseminated and negative expression of GFAP, which were recapitulated to the patient characteristics. BRAF and MEK inhibitor mildly suppressed cell viability in vitro. Collectively, E-GBM PDX models recapitulate patient characteristics, which may be helpful to elucidate tumor biology and establish novel therapeutic target in E-GBM.

Patients with malignant brain tumors have varied degree of neurological impairments because of factors influencing activities of daily living (ADL). Those factors are related to characteristics of patients, tumor natures, and received treatment. However, factors influencing ADL of patients with malignant brain tumors are not fully elucidated. The functional independence measurement (FIM) is an ADL scoring system to determine impairment in different domains objectively. Here, we assessed ADL of 27 patients with malignant brain tumors at admission, discharge, 1 year and 2 years later using FIM scoring system, and identified factors influencing their ADL. In results, patient age and tumor pathology were identified as factors influencing ADL, while tumor site and operation type were not related to FIM scores. In conclusion, patient age and tumor pathology are identified as factors influencing ADL of patients with malignant brain tumors. Taking into account of those factors, neuro-rehabilitation program for patients with malignant brain tumors should be scheduled.