Search

INTRODUCTION The median survival of Glioblastoma (GBM) patients is less than two years with the standard of care of maximal surgical resection, radiation, and temozolomide (TMZ) chemotherapy. Protein Arginine Methyltransferase 5 (PRMT5), which regulates cellular functions by symmetrically di-methylating arginine residues, is overexpressed in GBM. Inhibiting PRMT5 induces apoptosis in differentiated and senescence in stem-like GBM tumor cells. We inhibited PRMT5 in GBM neurospheres to determine if PRMT5 inhibition would enhance TMZ’s antitumor effect. METHODS We depleted PRMT5 activity, in vitro, using target-specific siRNA or LLY-283 and combined these with TMZ treatment. We evaluated the antitumor effect of this combination using cell viability assay, cell cycle analysis, apoptosis assay, and western blot. RESULTS TMZ reduced the viability of GBMNS with PRMT5 knockdown significantly more than the viability of PRMT5 intact GBMNS. The combination of TMZ and PRMT5 knockdown elevated the expression of cleaved caspase 3 and caspase3/7 indicating that PRMT5 knockdown enhanced the apoptotic effects of TMZ. Cell cycle analysis showed that depleting PRMT5 abrogated TMZ-induced G2/M cell cycle arrest. Further, treatment of PRMT5-depleted GBMNS with TMZ increased ɣ-H2AX expression compared PRMT5 intact GBMNS treated with TMZ, suggesting that PRMT5 depletion enhanced TMZ-induced DNA damage. PRMT5 knockdown also inhibited the symmetric di-methylation of RUVBL1 that is required for homologous recombination repair of TMZ treatment-related DNA damage. CONCLUSION Overall, PRMT5 inhibition sensitized GBMNS to TMZ and enhanced TMZ-related DNA damage and cytotoxicity. These findings support further development of this potential therapeutic combination.

Background Despite multi-model therapy of maximal surgical resection, radiation, chemotherapy, and tumor-treating fields, the median survival of glioblastoma (GBM) patients is less than 15 months. Protein arginine methyltransferase 5 (PRMT5) catalyzes the symmetric dimethylation of arginine residues and is overexpressed in GBM. Inhibition of PRMT5 causes senescence in stem-like GBM tumor cells. LB100, a first-in-class small molecular inhibitor of protein phosphatase 2A (PP2A), can sensitize therapy-resistant tumor cells. Here, we tested the anti-GBM effect of concurrent PRMT5 and PP2A inhibition. Methods Patient-derived primary GBM neurospheres (GBMNS), transfected with PRMT5 target-specific siRNA, were treated with LB100 and subjected to in vitro assays including PP2A activity and western blot. The intracranial mouse xenograft model was used to test the in vivo antitumor efficacy of combination treatment. Results We found that PRMT5 depletion increased PP2A activity in GBMNS. LB100 treatment significantly reduced the viability of PRMT5-depleted GBMNS compared to PRMT5-intact GBMNS. LB100 enhanced G1 cell cycle arrest induced by PRMT5 depletion. Combination therapy also increased the expression of phospho-MLKL. Necrostatin-1 rescued PRMT5-depleted cells from the cytotoxic effects of LB100, indicating that necroptosis caused the enhanced cytotoxicity of combination therapy. In the in vivo mouse tumor xenograft model, LB100 treatment combined with transient depletion of PRMT5 significantly decreased tumor size and prolonged survival, while LB100 treatment alone had no survival benefit. Conclusion Overall, combined PRMT5 and PP2A inhibition had significantly greater antitumor effects than PRMT5 inhibition alone.

The host immune system has developed many mechanisms to defend against and clear virus infections, including PKR, cGAS-STING and TLR-MyD88 signaling pathways. Compromised anti-viral mechanisms in tumor cells allows for infection, replication and lysis by oncolytic viruses. However, compromised antiviral mechanisms, together with immune cells and the tumor microenvironment (TME), limit the sensitivity of oncolytic virus in tumor cells. We generated a novel oncolytic Herpes simplex virus, oHSV-shPKR which disables tumor-intrinsic PKR signaling. oHSV-shPKR significantly increases oHSV infection and lysis in both oncolytic virus-resistant and sensitive glioblastomas (GBMs). Infection of oHSV-shPKR in GBMs induces G2/M cell cycle arrest and inhibits tumor cell growth. oHSV-shPKR increases activation of antigen presentation cells through type I interferon signaling activation and its immune-stimulatory function to increase tumor-antigen specific CD8 T-cell expansion, including cytotoxic T lymphocytes. Preclinical studies showed that oHSV-shPKR intra-tumoral injection significantly inhibits human GBM patient derived xenograft (PDX) tumor growth in immune-deficient NSG mice and murine orthotopic GBM tumor growth in immunocompetent mice. The results demonstrate that the novel oHSV-shPKR has the potential to be used in clinical applications for both oHSV-resistant and sensitive GBM treatment.

BACKGROUND This study aims to investigate the prognostic role of complete metabolic response (CMR) on interim 18F-fluorodeoxyglucose positron emission tomography/computed tomography (PET/CT) in patients with breast cancer (BC) receiving neoadjuvant chemotherapy (NAC) according to tumor subtypes and PET timing. PATIENTS AND METHODS Eighty-six consecutive patients with stage II/III BC who received PET/CT during or following NAC were included. Time-dependent receiver operating characteristic analysis and Kaplan-Meier analysis were used to determine correlation between metabolic parameters and survival outcomes. RESULTS The median follow-up duration was 71 months. Maximum standardized uptake value (SUVmax) on an interim PET/CT independently correlated with survival by multivariate analysis (overall survival [OS]: hazard ratio: 1.139, 95% confidence interval: 1.058-1.226, p = .001). By taking PET timing into account, best association of SUVmax with survival was obtained on PET after two to three cycles of NAC (area under the curve [AUC]: 0.941 at 1 year after initiation of NAC) and PET after four to five (AUC: 0.871 at 4 years), while PET after six to eight cycles of NAC had less prognostic value. CMR was obtained in 62% of patients (23/37) with estrogen receptor-positive (ER+)/human epidermal growth factor receptor 2-negative (HER2-) BC, in 48% (12/25) triple-negative BC (TNBC), and in 75% (18/24) HER2-positive (HER2+) tumors. Patients with CMR on an early-mid PET had 5-year OS rates of 92% for ER+/HER2- tumors and 80% for TNBC, respectively. Among HER2+ subtype, 89% patients (16/18) with CMR had no relapse. CONCLUSION CMR indicated a significantly better outcome in BC and may serve as a favorable imaging prognosticator. The Oncologist 2017;22:526-534 IMPLICATIONS FOR PRACTICE: This study shows a significantly better outcome for breast cancer (BC) patients who achieved complete metabolic response (CMR) on 18F-fluorodeoxyglucose emission tomography/computed tomography (PET/CT) during neoadjuvant chemotherapy, especially for hormone receptor-positive tumors and triple negative BC. Moreover, PET/CT performed during an early- or mid-course neoadjuvant therapy is more predictive for long-term survival outcome than a late PET/CT. These findings support that CMR may serve as a favorable imaging prognosticator for BC and has potential for application to daily clinical practice.

NOTCH signaling is a method of cell-cell communication where membrane bound NOTCH ligands on signal-sending cells can bind to and initiate cleavage of the NOTCH receptor, releasing NICD which can initiate signal transduction in adjacent “signal-receiving” cells. We have recently shown that oHSV treatment of GBM cells induces NICD cleavage and NOTCH activation in adjacent uninfected glioma cells. RNA sequencing of GBM cells post-infection also uncovered Gene Ontology NOTCH signaling pathway to be significantly upregulated. This activation was induced by viral miRNA-H16, which represses FIH-1 expression. FIH-1 was found to be a negative regulator of Mib1, a ubiquitin ligase, which activates NOTCH ligand-mediated activation of adjacent signal-receiving cells bearing the NOTCH receptor (Otani et al Clin. Can. Res. 2020). Here we have investigated the impact of oHSV-induced NOTCH signaling on the tumor microenvironment. Treatment of brain tumors in immune competent mice with oHSV and NOTCH blocking gamma secretase inhibitor (GSI) induced an anti-tumor memory immune response. Long term survivors in mice treated with the combination also completely rejected subsequent tumor re-challenge in the other hemisphere. UMAP of flow cytometry of tumor-bearing hemispheres and functional analysis of isolated cellular fractions from treated mice showed a significant influx of MDSC cells after oHSV treatment that was rescued in mice treated with oHSV and GSI. Ongoing mechanistic studies are uncovering a significant induction of NOTCH in tumor associated macrophages that aids in recruitment of MDSC cells. Overall these studies have uncovered a significant impact of oHSV therapy on GBM tumor microenvironment and presents opportunities for combination therapies that can help improve therapeutic benefit and anti-tumor immunity.

INTRODUCTION Glioblastomas (GBMs) are resistant to traditional therapies, and new treatment is urgently needed. NOTCH signaling is highly activated in glioma and activated NOTCH signaling is associated with increased tumor growth and therapeutic resistance. Effect of oncolytic virus therapy on NOTCH signaling activation has not been reported to our knowledge. METHODS Western blotting, real time Q-PCR, reporter assay, and bioluminescence mice imaging were used to evaluate oHSV-mediated activation of NOTCH signaling. High throughput analysis of HSV-1-encoded microRNAs and genes were performed to identify the activator of NOTCH signaling, and in silico analysis, 3’ untranslated region (3’UTR) assay, and immunoprecipitation were used to uncover the down-stream signaling. Anti-tumor efficacy of NOTCH inhibitor, gamma-secretase inhibitor (GSI) combined with oHSV was evaluated using intracranial glioma-bearing mouse xenografts. RESULTS oHSV infection induced NOTCH activity in glioma PDX cells. High throughput analysis uncovered this to be due to HSV-1-encoded miR-H16 (miR-H16). 3’UTR analysis of Factor Inhibiting HIF-1 (FIH-1) indicated miR-H16 directly target FIH-1 and repress its protein expression. Immunoprecipitation analysis also uncovered that FIH-1 reduction liberated Mild bomb 1 (Mib1) from FIH-1 mediated sequestration, permitting it to activate NOTCH signaling by ubiquitination of NOTCH ligands. Forced expression of either antago-miR-H16 or FIH-1 attenuated the oHSV-induced NOTCH activation. Furthermore, oHSV therapy-induced activation of NOTCH signaling sensitized tumors to GSI, resulting in significantly improved therapeutic response to oHSV therapy and prolonged the survival in several intracranial GBM xenografts. CONCLUSION In this study, we identified the mechanism of oHSV therapy-mediated activation of NOTCH signaling, and showed the therapeutic benefit of combining oHSV with GSI.

Chemoradiation following surgical resection of glioblastoma has improved overall survival (OS). Unfortunately, all patients develop progression of the disease, typically characterized as worsening radiographic appearance. Radiological imaging is limited in differentiating tumor progression from radiation-induced necrosis; therefore, re-resection is often needed to remove the abnormal tissue. There is little and contradictory data assessing the relationship between pathological evaluation (active tumor vs. radiation necrosis) and clinical outcomes. We performed a large retrospective study to analyze the prognostic value and clinical significance of the pathology evaluation following re-resection in patients with presumed tumor progression. Clinical, radiographic and pathological information was compiled from 675 patients with GBM from the Memorial Hermann Health Care System between 2010 and 2017. Second surgery was performed in 121 (18%) patients (mean age=56, median KPS=80) with presumed progression following chemoradiation. According to the pathology report following second surgery patients were classified into 2 groups (1: recurrent tumor- # of patients = 91 and 2: radiation necrosis # of patients = 30). A Kaplan-Meier survival analysis was performed comparing the OS of each group. No statistically significant differences in OS from initial diagnosis (median OS: active tumor 22.6 mo, radiation necrosis 21.3 mo, p=0.4) and suspected recurrence (median OS: active tumor 12.7 mo, radiation necrosis 13.7 mo, p=0.3) were found between the groups. The current study suggests that histopathologic findings following chemoradiation cannot predict outcomes. These findings are important and should be considered to guide treatment strategies and clinical trial endpoints.