Citations

Oncolytic viruses (OVs) are a class of immunotherapeutic agents with promising preclinical results for the treatment of glioblastoma (GBM) but have shown limited success in recent clinical trials. Advanced bioengineering principles from disciplines such as synthetic and systems biology are needed to overcome the current challenges faced in developing effective OV-based immunotherapies for GBMs, including off-target effects and poor clinical responses. Synthetic biology is an emerging field that focuses on the development of synthetic DNA constructs that encode networks of genes and proteins (synthetic genetic circuits) to perform novel functions, whereas systems biology is an analytical framework that enables the study of complex interactions between host pathways and these synthetic genetic circuits. In this review, the authors summarize synthetic and systems biology concepts for developing programmable, logic-based OVs to treat GBMs. Programmable OVs can increase selectivity for tumor cells and enhance the local immunological response using synthetic genetic circuits. The authors discuss key principles for developing programmable OV-based immunotherapies, including how to 1) select an appropriate chassis, a vector that carries a synthetic genetic circuit, and 2) design a synthetic genetic circuit that can be programmed to sense key signals in the GBM microenvironment and trigger release of a therapeutic payload. To illustrate these principles, some original laboratory data are included, highlighting the need for systems biology studies, as well as some preliminary network analyses in preparation for synthetic biology applications. Examples from the literature of state-of-the-art synthetic genetic circuits that can be packaged into leading candidate OV chassis are also surveyed and discussed.

Effective treatments for brain tumors remain one of the most urgent and unmet needs in modern oncology. This is due not only to the presence of the neurovascular unit/blood–brain barrier (NVU/BBB) but also to the heterogeneity of barrier alteration in the case of brain tumors, which results in what is referred to as the blood–tumor barrier (BTB). Herein, we discuss this heterogeneity, how it contributes to the failure of novel pharmaceutical treatment strategies, and why a “whole brain” approach to the treatment of brain tumors might be beneficial. We discuss various methods by which these obstacles might be overcome and assess how these strategies are progressing in the clinic. We believe that by approaching brain tumor treatment from this perspective, a new paradigm for drug delivery to brain tumors might be established.

Abstract PURPOSE Post-therapy recurrent glioblastoma (GBM) patient-derived xenografts (PDX), developed from corresponding treatment-naïve PDX, could serve as useful resources for identifying therapeutics with activity against recurrent GBM. The goal of this study was to determine whether treatment-naïve intracranial GBM PDX, in mice receiving radiotherapy (RT) and/or temozolomide (TMZ), acquire the same mutations that occur in post-RT+TMZ GBMs from patients. METHODS Luciferase-modified, treatment-naïve GBM PDX were engrafted in the brains of athymic nude mice, followed by treatment with RT only (2 Gy/day x 5), TMZ only (10 mg/kg/day x 5), or RT+TMZ. Bioluminescence imaging was used to monitor intracranial tumor growth, response to treatment, and recurrence from treatment. Some mice with recurrent tumors received additional TMZ treatment. When mice became symptomatic, intracranial tumors were resected and engrafted subcutaneously in a new mouse host, then sequentially propagated subcutaneously into additional host mice. After the third passage, whole-exome sequencing (WES) was done, comparing post-therapy with treatment-naïve PDX sequence variants. RESULTS Analysis of PDX WES showed the following: 1) TMZ consistently caused more genes to incur coding sequence mutations than RT, as much as 13x more; 2) TMZ-treated tumor mutations were mostly G-C to A-T transitions (71-92%), consistent with the known mutagenic effect of TMZ; and 3) post-therapy PDX acquire similar mutations as do recurrent GBMs in patients, for example involving DNA mismatch repair gene MSH6. One of the derivative PDX with MSH6 mutation has been retested for response to RT and TMZ, with results showing its having become TMZ, but not RT resistant. CONCLUSIONS The mutation profiles of RT+TMZ-treated PDX are similar to those reported for GBMs that recur after RT+TMZ in patients. The new PDX resources described here may prove useful for identifying effective treatments against recurrent GBM.

Abstract Pediatric glioblastoma and diffuse intrinsic pontine glioma are high-grade gliomas of children (pHGG) with a median overall survival of under 15 months and among the most lethal cancers. Mutations in histone H3.3 and H3.1 occur as an early event in pHGG. H3.3G34R/V-mutations occur in pHGG of cerebral hemispheres, and H3.3K27M mutations occur in midline pHGGs. Post-translational histone modifications (PTMs) serve to regulate gene expression by relaxing or compacting chromatin and by recruiting proteins, with subsequent silencing or activating effects. H3.3 Serine 31 (S31) shows reduced phosphorylation during mitosis in H3.3G34R/V and H3.3K27M mutant cell. Phosphorylation at S31 is restored in wildtype H3.3K27 CRISPR revertants. Serine to alanine (A) mutant H3.3 S31A are nonphosphorylatable in vitro. To study the influence of histone mutations and the role of altered PTM and including the loss of methylation and phosphorylation on tumorigenesis, we have developed an innovative model based on the RCAS/N-TVA mouse model. In this system, the expression of an oncogenic driver is linked to mutant histone expression using a self-cleaving peptide, and tumors develop following viral delivery to neural stem cells in newborn mice. This approach is necessary, as otherwise, clonal selection could prevent tumors from forming with mutations detrimental to growth. To establish the model, N-TVA mice were injected with RCAS H3.3K27M-P2A-PDGFB, RCAS H3.3G34R-P2A-PDGFB, or H3.3WT-P2A-PDGFB. The mean survival of mice injected with H3.3K27M and H3.3S31A was 81 and 68 days, respectively, and 100% of S31A mice developed HGG. In contrast, H3.3WT caused only low-grade tumors in 46% of the mice, and all mice survived until 100 days. In ongoing experiments with H3.3G34R, 23% of mice succumb to tumors by 80 days. These results provide mechanistic insights into the early establishment of pHGGs and established a new mouse model to study the role of histone mutation and PTMs in tumor development.

Abstract Non-genotoxic reactivation of p53 by MDM2 inhibitors represents a promising therapeutic strategy for tumors with wild-type TP53, particularly tumors harboring MDM2 amplification. MDM2 controls p53 levels by targeting it for degradation, while disruption of the MDM2-p53 interaction causes rapid accumulation of p53 and activation of the p53 pathway. We examined the efficacy of the small molecule MDM2 inhibitor KRT-232, alone and in combination with radiation therapy (RT), in MDM2-amplified and/or p53 wildtype patient-derived xenograft (PDX) models of glioblastoma in vitro and in vivo. In vitro, glioblastoma PDX explant cultures showed sensitivity to KRT-232, both tumors with MDM2 amplification (GBM108 and G148) and non-amplified but TP53-wildtype lines (GBM10, GBM14, and GBM39), with IC50s ranging from 300-800 nM in FBS culture conditions. A TP53 p.F270C mutant PDX (GBM43) was inherently resistant, with IC50 >3000 nM. In the MDM2-amplified GBM108 line, KRT-232 led to a robust (5-6 fold) induction of p53-target genes p21, PUMA, and NOXA, with initiation of both apoptosis and senescence. Expression of p21 and PUMA was greater with KRT-232 in combination with RT (25-35 fold induction), while stable knock-down of p53 in GBM108 led to complete resistance to KRT-232. In contrast, GBM10 showed lower induction of p21 and PUMA (2-3 fold) and was more resistant to KRT-232. In an orthotopic GBM108 xenograft model, treatment with KRT-232 +/- RT for one week extended survival from 22 days (placebo) to 46 days (KRT-232 alone); combination KRT-232 + RT further extended survival (77 days) over RT alone (31 days). KRT-232 is an effective treatment in a subset of glioblastoma pre-clinical models alone and in combination with RT. Further studies are underway to understand the mechanisms conferring innate sensitivity or resistance to KRT-232.

Abstract In Glioblastoma (GBM), tumor spreading is driven by tumor cells’ ability to infiltrate healthy brain parenchyma, which prevents complete surgical resection and contributes to tumor recurrence. GBM molecular subtypes, classical, proneural and mesenchymal, were shown to strongly correlate with specific genetic alterations (Mesenchymal: NF1; Classical: EGFRVIII; Proneural: PDGFRA). Here we tested the hypothesis that a key mechanistic difference between GBM molecular subtypes is that proneural cells are slow migrating and mesenchymal cells are fast migrating. Using Sleeping Beauty transposon system, immune-competent murine brain tumors were induced by SV40-LgT antigen in combination with either NRASG12V (NRAS) or PDGFB (PDGF) overexpression. Cross-species transcriptomic analysis revealed NRAS and PDGF-driven tumors correlate with human mesenchymal and proneural GBM, respectively. Similar to human GBM, CD44 expression was higher in NRAS tumors and, consistent with migration simulations of varying CD44 levels, ex vivo brain slice live imaging showed NRAS tumors cells migrate faster than PDGF tumors cells (random motility coefficient = 30µm2/hr vs. 2.5µm2/hr, p < 0.001). Consistent with CD44 function as an adhesion molecule, migration phenotype was independent of the tumor microenvironment. NRAS and human PDX/MES tumor cells were found to migrate faster and have larger cell spread area than PDGF and human PDX/PN tumors cells, respectively, in healthy mouse brain slices. Furthermore, traction force microscopy revealed NRAS tumor cells generate larger traction forces than PDGF tumors cells which further supports our theoretical mechanism driving glioma migration. Despite increased migration, NRAS cohort had better survival than PDGF which was attributed to enhanced antitumoral immune response in NRAS tumors, consistent with increased immune cell infiltration found in human mesenchymal GBM. Overall our work identified a potentially actionable difference in migration mechanics between GBM subtypes and establishes an integrated biophysical modeling and experimental approach to mechanically parameterize and simulate distinct molecular subtypes in preclinical models of cancer.

Abstract BACKGROUND Radiotherapy, combined with surgical resection and chemotherapy, remains a first-line treatment for infiltrative gliomas. However, these tumors are not surgically curable, and often recur, even within the prior radiation field, and may demonstrate a more aggressive phenotype. Importantly, high grade gliomas display diverse molecular phenotypes, and whether this genetic variability leads to divergent behaviour in the radiated tumor microenvironment is unknown. Herein, we characterize the effects of the irradiated brain microenvinroment on nine additional unique GBM cell lines to better understand the nuances of how tumor molecular phenotypes influence cellular dynamics. METHODS Female athymic nude mice were randomly divided into cranial radiation (15 Gy) and non-radiated groups. Mice then underwent intracranial implantation with one of the selected patient-derived xenograft (PDX) GBM cell lines (GBM 6, 10, 12, 39, 46, 76, 123, 164, 196; total n=8-15, per group, per line). Kaplan-Meyer (K-M) and log-rank tests were performed to compare the survival between irradiated and non-irradiated groups. RESULT Of nine previously untested human GBM lines, we found that five demonstrated shorter survival in the pre-radiated brain (GBM 6, 46, 76, 164, 196). However, two lines yielded prolonged survival in the pre-radiated brain (GBM 10, 12); GBM 39, 123 whose rate of growth was not impacted by the radiated brain. CONCLUSION These results highlight the likely critical impact of the irradiated microenvironment on tumor behaviour, yet illustrate that different tumors may exhibit opposing responses. Although further evaluation will be needed to understand mechanisms of divergent behavior, our data suggest the increased rate of growth in the radiated microenvironment may not apply to the fastest-growing tumor lines, which could instead demonstrate a paradoxical response.

Abstract Radio-resistance mechanisms limit the benefit of radiation therapy (RT) for melanoma brain metastases. A key pathway for radiation-induced DNA double-strand break repair is non-homologous end joining, which is critically mediated by DNA-dependent protein kinase (DNA-PKcs). Here we evaluated radio-sensitizing effects of M3814, a selective oral inhibitor of DNA-PKcs, in patient-derived xenografts (PDXs) of melanoma brain metastases. In a clonogenic survival assay, M3841 augmented RT-induced killing of M12 cells at concentrations of ≥300 nM, and a minimum of 16 h exposure with ~300 nM M3814 was required for effective sensitization. M3814 inhibited RT-induced (5 Gy) auto-phosphorylation of serine-2056 of DNA-PKcs in primary cultures of M12, M15 and M27 PDX lines. Interestingly, inhibition of RT-induced DNA-PKcs by M3814 coincided with increased KAP1 phosphorylation, a DNA damage signaling regulated via ATM. Persistent γH2AX foci were observed in 28% M12 cells at 24 hours after co-treatment with M3814 and RT as compared to 12% cells following RT alone. In vivo pharmacokinetic analysis after single oral dose of 20 mg/kg M3814, showed reasonably short half-life (~2.44 hours) and poor brain distribution in wild-type FBV mice (Kpuu, 0.027). Consistent with an efflux liability, brain distribution of M3814 in triple knockout mice for BCRP/MDR1A/B was ~11 fold higher (Kpuu, 0.215). Compared to normal brain, much higher M3814 concentrations were detected in intracranially implanted M12 tumors (~23 fold and ~20 fold) 2 and 6 hours after a single oral dose of 50mg/kg respectively. The relative exclusion of M3814 from normal brain as compared to brain metastases suggests that this drug may have a favorable toxicity profile when combined with radiation for treatment of melanoma brain metastases, and this hypothesis is being tested in ongoing efficacy studies.

Abstract There is an unmet need to identify novel targets that can sensitize temozolomide (TMZ) or prevent resistance in GBM. We have demonstrated that retinoblastoma binding protein 4 (RBBP4) interacts with p300 to modulate expression of genes involved in homologous recombination (HR), including RAD51. In vitro, RBBP4- or p300-shRNA significantly sensitized TMZ in patient derived xenograft (PDX) GBM43 cells (relative fluorescence for 100µM TMZ treated control shNT cells was 0.89 ± 0.1 vs 0.47± 0.09 and 0.39 ± 0.01 for shRBBP4 and shp300, respectively (p< 0.01)). TMZ sensitization increased DNA damage signaling through phosphorylation of KAP1 (p-KAP1) and p-CHK1. Moreover, RBBP4- or p300-shRNA delayed the repair of TMZ-induced DSBs evidenced by persistent gH2AX. Silencing RBBP4 or p300 reduced acetylation of lysine 27 of histone H3 (H3K27Ac) within promoters of HR genes regulated by RBBP4/p300 complex. Thus, RBBP4/p300 complex controls gene expression through p300-mediated histone acetyltransferase (HAT) activity, suggesting that p300 inhibitors could sensitize GBM to TMZ. Accordingly, CBP/p300 inhibitor CPI1612 significantly suppressed H3K27Ac and HR repair genes, including RAD51. Moreover, CPI1612 sensitized TMZ in GBM43 (synergy score = 258), and TMZ/CPI1612 significantly suppressed growth of GBM39 PDX cells compared with either drug alone (confluence (%) was 92 ± 1.0 (DMSO), 76.5 ± 4.6 (10 µM TMZ), 62 ± 3.4 (10 nM CPI1612) and 21.9 ± 3.2 (TMZ 10 µM/CPI-CPI1612 10 nM). CPI1612 enhanced TMZ-induced DSBs with increased damage signaling through p- KAP1 and persistent gH2AX. Pharmacodynamics studies in GBM39 orthotopic mice models revealed that oral CPI1612 penetrates the brain and accumulate in tumor regions and suppresses H3K27Ac without significant weight loss in mice that received placebo, TMZ, CPI1612 alone or combined TMZ/CPI-1612, demonstrating good animal tolerability. Collectively, these findings are encouraging that CBP/p300 inhibition by the brain penetrant CPI-1612 is a potential strategy for enhancing the efficacy of TMZ in GBM.

Abstract Glioblastoma (GBM) is invariably fatal due to failure of current chemoradiation (Stupp) regimes. Biomarkers such as MGMT have proven to predict response to Temozolomide (TMZ). An equivalent biomarker for radiation (RT) has not yet been identified. Transglutaminase-2 (TGM2) has been implicated in driving radiation resistance; but the mechanism is poorly understood. We have investigated how exposure to neoadjuvant TMZ in glioma stem cells (GSCs) with different levels of TGM2 would affect the response to RT. MATERIALS/METHODS: Primary GSCs lines with different TGM2 levels (high: 1123, 83; low: 528, OPK49) were used to explore the role of TGM2 in RT response and modulation of expression by TMZ in vitro and in-vivo. RESULTS: We showed that TGM2 drives radioresistance in GSCs through restriction of p53 mediated repression of RAD51 expression. We demonstrate that exposure of GSCs to TMZ drives rapid downregulation of TGM2 in vitro and this phenomenon is recapitulated in vivo. Interestingly, we confirm that RT is able to drive reciprocal changes in TGM2 and promotes reactivation of TGM2 in TGM2-high tumours but not TGM2-low tumours. Given these observations, we hypothesized that exposure to neoadjuvant TMZ in TGM2-low tumours would increase the efficacy of subsequent RT in these tumours. Comparison of the effect of standard treatment consisting of 3 weeks of concurrent TMZ and RT (Stupp) to a novel regime (neo-Stupp) consisting of 1 week of neoadjuvant TMZ followed by two weeks of TMZ and hypofractionated RT revealed a superior survival benefit of this novel regime in TGM2-low tumours but not in TGM2-high tumours. Utilization of the TGM2 inhibitor GK921 in combination with neo-Stupp prevented rapid relapse previously observed in TGM2-high tumours. CONCLUSION: We provide evidence that TGM2 is a biomarker of RT response and can be used to tailor chemoradiation protocols to the unique biology of each individual GBM patient.