Sibo Zhao

Preservation of KIT genotype in a novel pair of patient-derived orthotopic xenograft mouse models of metastatic pediatric CNS germinoma

Metastatic intracranial germinoma is difficult to treat. Although the proto-oncogene KIT is recognized as one of the most frequent genetic abnormalities in CNS germinoma, the development of new target therapeutic agents for CNS germinoma is hampered by the lack of clinically-relevant animal models that replicate the mutated or over-expressed KIT. CNS germinoma tumor cells from five pediatric patients were directly implanted into the brains of Rag2/severe combined immune deficiency mice. Once established, the xenograft tumors were sub-transplanted in vivo in mouse brains. Characterization of xenograft tumors were performed through histologic and immunohistochemical staining, and KIT mutation analysed with quantitative pyro-sequencing. Expression of putative cancer stem cell markers (CD133, CD15, CD24, CD44, CD49f) was analyzed through flow cytometry. Two patient-derived orthotopic xenograft (PDOX) models (IC-6999GCT and IC-9302GCT) were established from metastatic germinoma and serially sub-transplanted five times in mouse brains. Similar to the original patient tumors, they both exhibited faint expression (+) of PLAP, no expression (−) of β-HCG and strong (+++) expression of KIT. KIT mutation (D816H), however, was only found in IC-9320GCT. This mutation was maintained during the five in vivo tumor passages with an increased mutant allele frequency compared to the patient tumor. Expression of putative cancer stem cell markers CD49f and CD15 was also detected in a small population of tumor cells in both models. This new pair of PDOX models replicated the key biological features of pediatric intracranial germinoma and should facilitate the biological and pre-clinical studies for metastatic intracranial germinomas.

Concurrent Inhibition of Neurosphere and Monolayer Cells of Pediatric Glioblastoma by Aurora A Inhibitor MLN8237 Predicted Survival Extension in PDOX Models

Purpose: Pediatric glioblastoma multiforme (pGBM) is a highly aggressive tumor in need of novel therapies. Our objective was to demonstrate the therapeutic efficacy of MLN8237 (alisertib), an orally available selective inhibitor of Aurora A kinase (AURKA), and to evaluate which in vitro model system (monolayer or neurosphere) can predict therapeutic efficacy in vivo. Experimental Design: AURKA mRNA expressions were screened with qRT-PCR. In vitro antitumor effects were examined in three matching pairs of monolayer and neurosphere lines established from patient-derived orthotopic xenograft (PDOX) models of the untreated (IC-4687GBM), recurrent (IC-3752GBM), and terminal (IC-R0315GBM) tumors, and in vivo therapeutic efficacy through log rank analysis of survival times in two models (IC-4687GBM and IC-R0315GBM) following MLN8237 treatment (30 mg/kg/day, orally, 12 days). Drug concentrations in vivo and mechanism of action and resistance were also investigated. Results: AURKA mRNA overexpression was detected in 14 pGBM tumors, 10 PDOX models, and 6 cultured pGBM lines as compared with 11 low-grade gliomas and normal brains. MLN8237 penetrated into pGBM xenografts in mouse brains. Significant extension of survival times were achieved in IC-4687GBM of which both neurosphere and monolayer were inhibited in vitro, but not in IC-R0315GBM of which only neurosphere cells responded (similar to IC-3752GBM). Apoptosis-mediated MLN8237 induced cell death, and the presence of AURKA-negative and CD133+ cells appears to have contributed to in vivo therapy resistance. Conclusions: MLN8237 successfully targeted AURKA in a subset of pGBMs. Our data suggest that combination therapy should aim at AURKA-negative and/or CD133+ pGBM cells to prevent tumor recurrence. Clin Cancer Res; 24(9); 2159–70. ©2018 AACR.

Xenotransplantation of pediatric low grade gliomas confirms the enrichment of BRAF V600E mutation and preservation of CDKN2A deletion in a novel orthotopic xenograft mouse model of progressive pleomorphic xanthoastrocytoma

To identify cellular and molecular changes that driver pediatric low grade glioma (PLGG) progression, we analyzed putative cancer stem cells (CSCs) and evaluated key biological changes in a novel and progressive patient-derived orthotopic xenograft (PDOX) mouse model. Flow cytometric analysis of 22 PLGGs detected CD133+ (<1.5%) and CD15+ (20.7 ± 28.9%) cells, and direct intra-cranial implantation of 25 PLGGs led to the development of 1 PDOX model from a grade II pleomorphic xanthoastrocytoma (PXA). While CSC levels did not correlate with patient tumor progression, neurosphere formation and in vivo tumorigenicity, the PDOX model, IC-3635PXA, reproduced key histological features of the original tumor. Similar to the patient tumor that progressed and recurred, IC-3635PXA also progressed during serial in vivo subtransplantations (4 passages), exhibiting increased tumor take rate, elevated proliferation, loss of mature glial marker (GFAP), accumulation of GFAP−/Vimentin+ cells, enhanced local invasion, distant perivascular migration, and prominent reactive gliosis in normal mouse brains. Molecularly, xenograft cells with homozygous deletion of CDKN2A shifted from disomy chromosome 9 to trisomy chromosome 9; and BRAF V600E mutation allele frequency increased (from 28% in patient tumor to 67% in passage III xenografts). In vitro drug screening identified 2/7 BRAF V600E inhibitors and 2/9 BRAF inhibitors that suppressed cell proliferation. In summary, we showed that PLGG tumorigenicity was low despite the presence of putative CSCs, and our data supported GFAP−/Vimentin+ cells, CDKN2A homozygous deletion in trisomy chromosome 9 cells, and BRAF V600E mutation as candidate drivers of tumor progression in the PXA xenografts.

Novel histone deacetylase inhibitor N25 exerts anti-tumor effects and induces autophagy in human glioma cells by inhibiting HDAC3

N25, a novel histone deacetylase inhibitor, was created through structural modification of suberoylanilide hydroxamic acid. To evaluate the anti-tumor activity of N25 and clarify its molecular mechanism of inducing autophagy in glioma cells, we investigated its in vitro anti-proliferative effect and in vivo anticancer effect. Moreover, we detected whether N25 induces autophagy in glioma cells by transmission electron microscope and analyzed the protein expression level of HDAC3, Tip60, LC3 in glioma samples by western blot. We additionally analyzed the protein expression level of HDAC3, Tip60, ULK1 (Atg1), and Beclin-1 (Atg6) after treatment with N25 in glioma cells. Our results showed that the anti-tumor activity of N25 in glioma cells is slightly stronger than SAHA both in vitro and in vivo. We found that N25 induced autophagy, and HDAC3 was significantly elevated and Tip60 and LC3 significantly decreased in glioma samples compared with normal brain tissues. Nevertheless, N25 inhibited HDAC3 and up-regulated the protein expression of Tip60, ULK1 (Atg1), and Beclin-1 (Atg6) after treatment of glioma cells with N25. In conclusion, these data suggest that N25 has striking anti-tumor activity in part due to inhibition of HDAC3. Additionally, N25 may induce autophagy through inhibiting HDAC3.

Systems biology–based drug repositioning identifies digoxin as a potential therapy for groups 3 and 4 medulloblastoma

Systematic drug repositioning identifies digoxin as a potential treatment for groups 3 and 4 medulloblastoma. Digoxin on the brain Groups 3 and 4 medulloblastoma (MB) are highly heterogeneous in nature and have therefore proven difficult to target, resulting in corresponding meagre survival rates. Using a sophisticated systematic drug repositioning approach, Huang et al. identified the already-approved drug digoxin as a possible treatment for these MB subtypes. Application of digoxin to orthotopic patient-derived xenograft models produced an increase in survival; this increase in survival was further extended upon combining digoxin treatment with radiation, and, importantly, occurred at blood concentrations of digoxin that might be feasible in patients. These findings could mean a possible inroads in improving outcome for patients with these hard-to-treat cancers. Medulloblastoma (MB) is the most common malignant brain tumor of childhood. Although outcomes have improved in recent decades, new treatments are still needed to improve survival and reduce treatment-related complications. The MB subtypes groups 3 and 4 represent a particular challenge due to their intragroup heterogeneity, which limits the options for “rational” targeted therapies. Here, we report a systems biology approach to drug repositioning that integrates a nonparametric, bootstrapping-based simulated annealing algorithm and a 3D drug functional network to characterize dysregulated driver signaling networks, thereby identifying potential drug candidates. From more than 1300 drug candidates studied, we identified five members of the cardiac glycoside family as potentially inhibiting the growth of groups 3 and 4 MB and subsequently confirmed this in vitro. Systemic in vivo treatment of orthotopic patient-derived xenograft (PDX) models of groups 3 and 4 MB with digoxin, a member of the cardiac glycoside family approved for the treatment of heart failure, prolonged animal survival at plasma concentrations known to be tolerated in humans. These results demonstrate the power of a systematic drug repositioning method in identifying a potential treatment for MB. Our strategy could potentially be used to accelerate the repositioning of treatments for other human cancers that lack clearly defined rational targets.

Abstract 5058: Lysine specific demethylase-1 (LSD-1) Inhibitor SYC-836 in combination with radiation prolongs animal survival in patient-derived posterior fossa ependymoma xenograft mouse models

Background: Ependymoma (EPN) is the third most common malignant pediatric brain tumor. Current standard therapy include maximally safe surgical resection followed by radiation and lead to a 5-year overall survival of 50-71%. Recent molecular subgrouping of EPN has identified one group, posterior fossa A (PFA), which accounts for 45% of all EPN cases, to have one of the worst prognosis and it is driven by epigenetic changes, suggesting targeting epigenetic changes in PFA EPN can potentially be effective. In this study, we examined the therapeutic efficacy of SYC-836, a novel LSD-1 inhibitor compound developed at Baylor College of Medicine, both in vitro and in vivo in PDOX models of posterior fossa EPN. Methods: To examine in vitro anti-tumor activities, paired primary cultured cells (both as attached cells and neurospheres) from an established PDOX model of posterior fossa EPN (ICb-4423EPN) were subjected to SYC-836 at various concentrations (0-25uM). Cell viability and proliferation were measured using Cell Counting Kit-8 assay at 5 different time points over 14 days. To validate the drug’s in vivo efficacy, two established posterior fossa EPN PDOX models, ICb-4423EPN and ICb-2002EPN, were utilized. 40 eight weeks old SCID mice per model were implanted with tumor cells. They were divided into 4 treatment groups (10 mice/group) each: 1) control (DPBS, 10uL/kg IP daily x 28 days), 2) radiation/standard therapy (2 Gy focal XRT daily x 5 days), 3) SYC-836 only (15mg/kg IP daily x 28 days), and 4) combination (radiation + SYC-836 per regimen above). Animal survival times were analyzed using log rank analysis. Changes of histone lysine methylation were examined through western hybridization. Results: SYC-836 demonstrated effective cell killing in vitro against both attached and neurosphere cultured cells in both time- and dose-dependent manner. IC50 was ~7.5uM. In vivo experiment was completed in 1 of the 2 EPN PDOX models (ICb-2002EPN) with the second model ongoing. Median survival times for each group is as followed: control 136 days, radiation 148 days, SYC-836 only 136 days, combination 180 days. There were no survival benefit with either XRT only (P=0.205) or SYC-836 only (P=0.186) when compared to the control group; however, when used in combination, the treatment strategy lead to significant improvement in animal survival (P=0.004). SYC-836 was well tolerated in mice. Conclusion: Our data showed that combining SYC-836 with current standard therapy of radiation synergistically prolongs animal survival significantly, although as a single agent SYC-836 was not effective against posterior fossa ependymoma. Our data suggest that SYC-836 may have a role in the clinical setting by either reducing radiation dosages, or be a potential adjuvant agent to other chemotherapy drugs in our treatment approach for ependymoma. Citation Format: Sibo Zhao, Huiyuan Zhang, Lin Qi, Holly Lindsay, Yuchen Du, Mari Kogiso, Frank Braun, Sarah Injac, Laszlo Perlaky, Donald W. Parsons, Murali Chintagumpala, Adekunle Adesina, Yongcheng Song, Xiao-Nan Li. Lysine specific demethylase-1 (LSD-1) Inhibitor SYC-836 in combination with radiation prolongs animal survival in patient-derived posterior fossa ependymoma xenograft mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5058. doi:10.1158/1538-7445.AM2017-5058

Abstract B12: Establishment of paired primary neurosphere and monolayer cells of mouse xenograft derived from pediatric brain tumors

Background: Stable and reproducible in vitro cell lines derived from clinical samples are currently considered as the best research tools to study human cancers in vitro. Currently there is very limited availability of tumor cell lines, especially paried neurosphere and nomolayer cells. Patient tumor is difficult to obtain and cannot be harvested by multiple times. Direct use of patient tumor is associated with low success rate in in vitro culture. Here we sought to establish a set of paired primary neurosphere and monolayer cells in vitro by taking advantage of patient derived orthotopic xenograft models we have already established. Materials and Methods: We have established murine xenograft models that biologically and molecularly reciprocate their human counterparts. Those human tumor xenografts are able to provide a sustained and ample supply of tumor cells in ex vivo culture. Primary tumor cells could be continuously harvested from human tumor xenografts and are maintained both in serum-free medium to enrich neurosphere cells and in fetal bovine serum (FBS)-containing DMEM medium where tumor cells grow in monolayer. Results: We have already developed in primary culture a panel of paired neurosphere and monolayer cells derived from pediatric glioblastoma (GBM), medulloblastoma (MB)and ependymoma (EPN) at different culture stage including three different GBM cells over 60 passages and four over 20 passages, one EPN cell over 40 passages and one MB cell at early stage of around 20 passages. In vitro cultures demonstrated typical cell morphology of adherent and sphere cells for all pediatric brain tumor cell lines we cultured so far. These cultured cells are tumorigenic even with small number of tumor cells and the relative abundances of putative cancer stem cells are different between neurospheres and monolayer culture. Furthermore, our preliminary data from one pair of GBM neurospheres and monolayer cells by high throughput-out drug screening employing hundred thousands of already-identified molecules showed a panel of promising target candidates. Neurospheres and monolayer cells do not respond equally toward the same library of chemotherapeutic agents. Conclusion: Patient derived orthotopic xenograft (PDOX) can be a good source of human tumor cells for the establishment of in vitro model systems of pediatric brain tumors. The neurosphere and monolayer cells are morphologically and biologically different, which suggest that in future drug screening for both type of cells should be included. Citation Format: Yuchen Du, Lin Qi, Huiyuan Zhang, Mari Kogiso, Holly B. Lindsay, Sibo Zhao, Frank Braun, Xiaonan Li. Establishment of paired primary neurosphere and monolayer cells of mouse xenograft derived from pediatric brain tumors. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B12.

Abstract 2410: Recurrent pediatric ependymomas exhibit increased tumorigenicity in mouse brains of patient-derived xenograft models

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Ependymoma is the third most common malignant pediatric brain tumor, accounting for ∼10% all intracranial tumors in children. Current standard treatment for ependymoma patients include maximally safe surgical resection followed by radiation therapy. The degree of gross total resection remains the best prognostic factor, although more molecular characterization has been completed in recent years demonstrating several subgroups of ependymoma that can more accurately predict clinical outcome. 5-year event free survival is 40-85% and varies depending on the extent of surgical resection, tumor grade, and other prognostic factors. Prognosis is even more dismal in those patients with recurrent ependymomas which occurs in nearly half of the patients, and there are no known curative options to offer these patients other than palliative re-irradiation and additional surgeries. Chemotherapies and molecular targeted therapies have not been proven to increase survival outcomes. Therefore, it is imperative to learn more about the biology of recurrent diseases and identify the cellular driver of ependymoma recurrence, as well as understanding the mechanisms of therapy resistance. We hypothesize that ependymoma recurrence is driven by a subpopulation of therapy-resistant tumors cells with enhanced tumorigenicity in SCID mice. Between the years of 2005 to present, we have collected a total of 77 pediatric ependymoma patient tumor samples from across the country. Of those, we identified 9 patients with recurrent ependymomas in which we have collected tumor samples from each recurrence. The median age of these patients is 6 years old (ranges from 2 - 10 years) and has a 2:1 male to female ratio. The median time to first recurrence is 35 months (ranges from 9 - 61 months). 5 patients had one recurrence, 2 patients had two recurrences, 1 patient had 3 recurrences, and 1 patient had more than 5 recurrences and we have tumor tissues from this particular patient over the span of 6-year period. To study the tumorigenicity of these brain tumors at the different clinical stages, whether at diagnosis, first recurrence, second recurrence, or beyond, we directly implanted tumor cells (10,000 cells/mouse) from all 9 patients into the matched locations in the brains of SCID mice (e.g., human cerebral tumors to mouse cerebrum, human cerebellar tumors to mouse cerebellum). In 3 of the 9 models, tumor formation was confirmed in mouse brains from the latest recurrent patient tumor, while samples from the same patient from earlier stages either did not form tumors in mouse brains or are currently pending. Tumor formation in the remaining models are still pending. In conclusion, our preliminary findings demonstrate progressive enhancement of tumorigenicity during ependymoma recurrence. This has become a very unique and extremely valuable platform to study brain tumor recurrence. Citation Format: Sibo Zhao, Huiyuan Zhang, Laszlo Perlaky, Adekunle Adesina, Ching Lau, Donald W. Parsons, Murali Chintagumpala, Xiao-Nan Li. Recurrent pediatric ependymomas exhibit increased tumorigenicity in mouse brains of patient-derived xenograft models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2410.

Abstract 4390: SVV-001 prolongs animal survival in PDOX adult GBM models

Background: Glioblastoma (GBM) is the most malignant and aggressive brain tumor, and survival of patients affected by GBM has remained virtually unchanged over numerous years. GBM is only minimally responsive to aggressive standard therapies including radical surgery and concurrent chemo-radiation treatment with temozolomide (TMZ). Seneca Valley virus (SVV-001) is a non-pathogenic oncolytic virus that can be systemically administered and can pass through the blood-brain barrier. In this study, we developed a new panel of patient derived orthotopic xenograft (PDOX) models to examine the therapeutic efficacy of SVV-001 combined with irradiation. In addition, we explored the mechanism of tumor cell infection with SVV-001 in malignant gliomas. Material and Methods: Surgical GBM tumor samples were obtained from 17 patients and directly implanted into the right cerebrum of NOD/SCID mice (1×105 cells suspended in 2 uL growth medium). Once tumor formation was confirmed, we performed HE 4 of these samples have since been serially sub-transplanted for a total of 5 generations, while 4 intracerebral xenografts are in generation 2. The tumorigenicity of 8 additional samples is pending. These xenograft models precisely replicated histopathologic characteristics of their parental human tumors. SVV-001 at a multiplicity of infection of 0.5 to 25 replicated in and effectively killed primary cultures, pre-formed neurospheres, and monolayer glioma cells derived from adult glioma xenograft models in vitro. A single I.V. injection of SVV-001 (1 ×1011 viral particles/kg) administered immediately after radiation or 1 week after radiation led to the infection of orthotopic xenografts without harming normal mouse brain cells and resulted in significantly prolonged survival in permissive mouse models. Additionally, SVV-001 injected immediately after radiation significantly improved the overall survival of animals compared to administration of the virus 1 week after radiation, indicating that dose timing was crucial for full efficacy of the combinatory therapy. Conclusion: Our results demonstrated that SVV-001 possesses potent anti-tumor activity against adult malignant high-grade gliomas. Because this study was performed in a panel of patient tumor-derived orthotopic xenograft models, it provides a pre-clinical rationale that supports the consideration of SVV-001 for clinical trials against adult gliomas. Citation Format: Huiyuan Zhang, Lin Qi, Yuchen Du, Mari Kogiso, Frank K. Braun, Sibo Zhao, Holly Lindsay, Nabil Ahmed, Akash J. Patel, Patricia A. Baxter, Jack M. Su, Xiao-nan Li. SVV-001 prolongs animal survival in PDOX adult GBM models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4390.