MGMT-inhibitor in combination with TGF-ßRI inhibitor or CDK 4/6 inhibitor increases temozolomide sensitivity in temozolomide-resistant glioblastoma cells.

BACKGROUND: Glioblastoma (GB) remains an incurable and deadly brain malignancy that often proves resistant to upfront treatment with temozolomide. Nevertheless, temozolomide remains the most commonly prescribed FDA-approved chemotherapy for GB. The DNA repair protein methylguanine-DNA methyl transferase (MGMT) confers resistance to temozolomide. Unsurprisingly temozolomide-resistant tumors tend to possess elevated MGMT protein levels or lack inhibitory MGMT promotor methylation. In this study, cultured human temozolomide resistance GB (43RG) cells were introduced to the MGMT inhibitor O6-benzylguanine combined with temozolomide and either LY2835219 (CDK 4/6 inhibitor) or LY2157299 (TGF-ßRI inhibitor) seeking to overcome GB treatment resistance. METHODS: Treatment effects were assessed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, western blot, cell viability, and cell cycle progression. RESULTS: Our in vitro study demonstrated that sequential treatment of O6-Benzylguanine with either LY2385219 or LY2157299-enhanced temozolomide enhanced sensitivity in MGMT+ 43RG cells. Importantly, normal human neurons and astrocytes remained impervious to the drug therapies under these conditions. Furthermore, LY2835219 has additional anti-proliferative effects on cell cycling, including induction of an RB-associated G (1) arrest via suppression of cyclin D-CDK4/6-Rb pathway. LY2157299 enhances anti-tumor effect by disrupting TGF-ß-dependent HIF-1α signaling and by activating both Smad and PI3K-AKT pathways towards transcription of S/G2 checkpoints. CONCLUSION: This study establishes the groundwork for the development of a combinatorial pharmacologic approach by using either LY2385219 or LY2157299 inhibitor plus O6-Benzylguanine to augment temozolomide response in temozolomide-resistant GB cells.

Evaluating anti-tumor activity of palbociclib plus radiation in anaplastic and radiation-induced meningiomas: pre-clinical investigations.

PURPOSE: Meningiomas are common brain tumors, the majority of which are considered benign. Despite surgery and/or radiation therapy, recurrence rates are approximately 8-10%. One likely cause is the dysregulation of cyclin D-cyclin-dependent kinases 4 and 6 (CDK4/6)-retinoblastoma (Rb) pathway, which controls the cell cycle restriction point. This pathway is commonly dysregulated in anaplastic meningioma cell lines (AM) and radiation-induced meningioma cells (RIM), making it a rational target for anti-meningioma therapy. In this study, we investigate the effect of a CDK4/6 inhibitor, palbociclib, with radiation in relevant pre-clinical models. METHODS: In vitro cell culture, ex vivo slice culture and in vivo cell line-derived orthotopic xenograft animal models of AM/RIM were utilized to assess treatment efficacy with palbociclib plus radiation. Treatment effects were examined by immunoblot, cell viability, apoptosis, and cell cycle progression. RESULTS: The in vitro and ex vivo studies demonstrate that palbociclib plus radiation treatment reduced proliferation and has additional effects on cell cycling, including induction of an RB-associated G (1) arrest in Rb+ AM and RIM cells, but not in Rb- cells. Our results also demonstrated reduced CDK4 and CDK6 expression as well as reduced E2F target gene expression (CCNA2 and CCNE2) with the combination therapy. MRI results in vivo demonstrated reduced tumor size at 5 weeks when treated with 14 days palbociclib (10 mg/kg) plus 6 Gy radiation compared to saline-treated tumors. Finally, no hepatic toxicity was found after treatments. CONCLUSION: A pre-clinical murine model provides preclinical evidence for use of palbociclib plus radiation as a therapeutic agent for Rb+ meningiomas.

Ganoderic acid A/DM-induced NDRG2 over-expression suppresses high-grade meningioma growth.

PURPOSE: N-myc downstream-regulated gene 2 (NDRG2) is down-regulated in grade-III meningioma [anaplastic meningioma (AM)] and associated with clinically aggressive behavior. Current therapies in the treatment of high-grade meningioma are lacking with limited success. This study aims to validate the effect of NDRG2-targeted therapy using structurally related bioactive triterpene compounds derived from the edible mushroom Ganoderma lucidum (ganoderic acid A:GA-A/ganoderic acid DM:GA-DM) in human AM in relevant pre-clinical models. METHODS: Tissue samples from the AM tumor regions of three human patients and control non-tumor samples were used to analyze the expression pattern of NDRG2. In vitro cell culture and in vivo cell-line-derived orthotopic xenograft animal models of AM were utilized to assess efficacy of treatment with GA-A/DM. RESULTS: Downregulation of NDRG2 expression was observed in surgically resected high-grade meningiomas compared to normal brain. These results prompt us to use NDRG2-targeting agents GA-A/DM. In vitro results showed that 72-h treatments of 25 µM GA-A/DM induced AM cell death, upregulate NDRG2 protein expression, downregulate NDRG2 promoter methylation in meningioma cells as compared to azacitidine and decitabine, the most commonly used demethylating agents. Our results also demonstrated that GA-A/DM does not have any detrimental effect on normal human neurons and arachnoid cells. GA-A/DM promoted apoptotic factors (Bax) while suppressing MMP-9, p-P13K, p-AKT, p-mTOR, and Wnt-2 protein expression. RNAi-mediated knockdown of NDRG2 protein expression increased tumor proliferation, while forced expression of wt-NDRG2 decreased proliferation in an in vitro model. Magnetic resonance (MR) imaging and Hematoxylin (H&E) staining demonstrated gross reduction of tumor volume in GA-A/DM treated mice at 5 weeks when compared with saline-treated orthotopic AM xenografted controls. There was an overall decrease in tumor cell proliferation with increased survival in GA-A/DM-treated animals. Enzyme assays showed that GA-A/DM did not negatively impact hepatic function. CONCLUSION: GA-A/DM may be a promising natural therapeutic reagent in the treatment of AM by suppressing growth via NDRG2 modulation and altering of intracellular signal pathways. We have shown it could potentially be an effective treatment for AM with decreased cellular proliferation in vitro, decreased tumor volume and increased survival in vivo.