Holly Lindsay

Co-inheritance of mild hemophilia A and heterozygosity for type 2N von Willebrand disease: A diagnostic and therapeutic challenge

Hemophilia A and von Willebrand disease are the two most common inherited bleeding disorders. Despite their frequency, however, there are very few reports of co‐inheritance of the two disorders. We present the first report of a patient with mild hemophilia A and heterozygosity for type 2N von Willebrand disease (VWD). We discuss the patients phenotype and highlight the diagnostic and therapeutic challenges caused by this co‐inheritance. Pediatr Blood Cancer 2014; 61:1888–1890.

c-Fos over-expression promotes radioresistance and predicts poor prognosis in malignant glioma

c-Fos is a major component of activator protein (AP)-1 complex. It has been implicated in cell differentiation, proliferation, angiogenesis, invasion, and metastasis. To investigate the role of c-Fos in glioma radiosensitivity and to understand the underlying molecular mechanisms, we downregulated c-Fos gene expression by lentivirus-mediated shRNA in glioma cell lines and subsequently analyzed the radiosensitivity, DNA damage repair capacity, and cell cycle distribution. Finally, we explored its prognostic value in 41 malignant glioma patients by immunohistochemistry. Our results showed that silencing c-Fos sensitized glioma cells to radiation by increasing radiation-induced DNA double strand breaks (DSBs), disturbing the DNA damage repair process, promoting G2/M cell cycle arrest, and enhancing apoptosis. c-Fos protein overexpression correlated with poor prognosis in malignant glioma patients treated with standard therapy. Our findings provide new insights into the mechanism of radioresistance in malignant glioma and identify c-Fos as a potentially novel therapeutic target for malignant glioma patients.

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.

Cefepime-Induced Neurotoxicity Despite Dose Adjustment for Renal Disease: A Brief Report and Review of the Literature.

Cefepime is increasingly used as empiric treatment for fever in the setting of neutropenia. We present a patient with acute-on-chronic renal disease who received cefepime at the appropriate dose for his glomerular filtration rate but developed cefepime-associated encephalopathy. Here, we review neurologic toxicities of cefepime and present suggestions for work-up and management.

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.

Work efficiency improvement of >90% after implementation of an annual inpatient blood products administration consent form

Paediatric haematology, oncology and bone marrow transplant (BMT) patients frequently require transfusion of blood products. Our institution required a new transfusion consent be obtained every admission. The objectives of this project were to: revise inpatient blood products consent form to be valid for 1 year, decrease provider time spent consenting from 15 to <5 min per admission, and improve provider frustration with the consent process. Over 6 months, we determined the average number of hospitalisations requiring transfusions in a random sampling of haematology/oncology/BMT inpatients. We surveyed nurses and providers regarding frustration levels and contact required regarding consents. Four and 12 months after implementation of the annual consent, providers and nurses were resurveyed, and new inpatient cohorts were assessed. Comparison of preintervention and postintervention time data allowed calculation of provider time reduction, a surrogate measure of improved work efficiency. Prior to the annual consent, >33 hours were spent over 6 months obtaining consent on 40 patients, with >19 hours spent obtaining consent when no transfusions were administered during admission. Twelve months after annual consent implementation, 97.5% (39/40) of analysed patients had a completed annual blood products transfusion consent and provider work efficiency had improved by 94.6% (>30 hours). Although several surveyed variables improved following annual consent implementation, provider frustration with consent process remained 6 out of a max score of 10, the same level as prior to the intervention. Development of an annual inpatient blood products consent form decreased provider time from 15 to <1 min per admission, decreased consenting numbers and increased work efficiency by >90%.

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