Pulsed reduced-dose rate re-irradiation for patients with recurrent grade 2 gliomas.

Background: Patients with grade 2 glioma exhibit highly variable survival. Re-irradiation for recurrent disease has limited mature clinical data. We report treatment results of pulsed reduced-dose rate (PRDR) radiation for patients with recurrent grade 2 glioma. Methods: A retrospective analysis of 58 patients treated with PRDR from 2000 to 2021 was performed. Radiation was delivered in 0.2 Gy pulses every 3 minutes encompassing tumor plus margin. Survival outcomes and prognostic factors on outcome were Kaplan-Meier and Cox regression analyses. Results: The median survival from the date of initial surgery was 8.6 years (95% CI: 5.5-11.8 years). 69% of patients showed malignant transformation to grade 3 (38%) or grade 4 (31%) glioma. Overall survival following PRDR was 12.6 months (95% CI: 8.3-17.0 months) and progression-free survival was 6.2 months (95% CI: 3.8-8.6 months). Overall response rate based on post-PRDR MRI was 36%. In patients who maintained grade 2 histology at recurrence, overall survival from PRDR was 22.0 months with 5 patients remaining disease-free, the longest at 8.2 and 11.4 years. PRDR was generally well tolerated. Conclusions: To the best of our knowledge, this is the largest reported series of patients with recurrent grade 2 gliomas treated with PRDR radiation for disease recurrence. We demonstrate promising survival and acceptable toxicity profiles following re-irradiation. In the cohort of patients who maintain grade 2 disease, prolonged survival (>5 years) is observed in selected patients. For the entire cohort, 1p19q codeletion, KPS, and longer time from initial diagnosis to PRDR were associated with improved survival.

Case report: Radiographic complete response of radiation-induced glioblastoma to front-line radiotherapy: A report and molecular characterization of two unique cases.

Radiation-induced gliomas (RIGs) are an uncommon disease type and a known long-term complication of prior central nervous system radiation exposure, often during childhood. Given the rarity of this malignancy subtype, no clinical trials have explored optimal therapy for these patients, and the literature is primarily limited to reports of patient cases and series. Indeed, the genomic profiles of RIGs have only recently been explored in limited numbers, categorizing these gliomas into a unique subset. Here, we describe two cases of RIG diagnosed as glioblastoma (GB), IDH-wildtype, in adults who had previously received central nervous system radiation for childhood cancers. Both patients demonstrated a surprising complete radiographic response of the postoperative residual disease to front-line therapy, a phenomenon rarely observed in the management of any GB and never previously reported for the radiation-induced subgroup. Both tumors were characterized by next-generation sequencing and chromosomal microarray to identify potential etiologies for this response as well as to further add to the limited literature about the unique molecular profile of RIGs, showing signatures more consistent with diffuse pediatric-type high-grade glioma, H3-wildtype, and IDH-wildtype, WHO grade 4. Ultimately, we demonstrate that treatment utilizing a radiation-based regimen for GB in a previously radiated tissue can be highly successful despite historical limitations in the management of this disease.

Human Colon Organoids and Other Laboratory Strategies to Enhance Patient Treatment Selection.

OPINION STATEMENT: Though many advancements in personalized medicine have been made, better methods are still needed to predict treatment benefit for patients with colorectal cancer. Patient-derived cancer organoids (PDCOs) are a major advance towards true personalization of treatment strategies. A growing body of literature is demonstrating the feasibility of PDCOs as an accurate and high-throughput preclinical tool for patient treatment selection. Many studies demonstrate that these cultures are readily generated and represent the tumors they were derived from phenotypically and based on their mutation profile. This includes maintenance of the driver muatations giving the cancer cells a selective growth advantage, and also heterogeneity, including molecular and metabolic heterogeneity. Additionally, PDCOs are now being utilized to develop patient biospecimen repositories, perform high to moderate-throughput drug screening, and to potentially predict treatment response for individual patients that are undergoing anti-cancer treatments. In order to develop PDCOs as a true clinical tool, further studies are required to determine the reproducibility and accuracy of these models to predict patient response.

Novel C-terminal heat shock protein 90 inhibitors target breast cancer stem cells and block migration, self-renewal, and epithelial-mesenchymal transition.

In patients with triple-negative breast cancer (TNBC), evidence suggests that tumor-initiating cells (TIC) have stem cell-like properties, leading to invasion and metastasis. HSP90 plays a critical role in the conformational maintenance of many client proteins in TIC development. Therefore, we hypothesize that the novel C-terminal HSP90 inhibitors KU711 and KU758 can target TIC and represent a promising strategy for overcoming metastasis. Human breast cancer cells (MDA-MB-468LN, MDA-MB-231) treated with the HSP90 inhibitors KU711, KU758, and 17-AAG showed a 50-80% decrease in TIC markers CD44 and aldehyde dehydrogenase (P < 0.01) as assessed by flow cytometry. A decrease in sphere formation, which was used to assess self-renewal, was observed after the treatment of TNBC cells starting at 2.5 µm KU711 and 0.31 µm KU758. KU compounds also blocked the invasion and migration of TNBC cells in a dose-dependent manner. The knockdown of HSP90 clients was observed without any change in prosurvival HSP70 levels. In vivo, in a murine orthotopic breast cancer model, treatment with KU758 and KU711 yielded an approximately twofold and a fourfold reduction in tumor volumes versus control, respectively, without demonstrated toxicity. In conclusion, C-terminal HSP90 inhibitors are potent novel therapeutics against TNBC in vitro and in vivo as they target TICs and block invasion, EMT transition, and self-renewal.

Novel natural withanolides induce apoptosis and inhibit migration of neuroblastoma cells through down regulation of N-myc and suppression of Akt/mTOR/NF-κB activation.

Despite recent advances in intensive chemotherapy treatments, long-term success is achieved in less than 30% of children with high-risk neuroblastoma (NB). Key regulatory pathways including the PI3K/Akt, mTOR and NF-κB are implicated in the pathogenesis of NB. Although drugs targeting these individual pathways are in clinical trials, they are not effective due to the activation of compensatory mechanisms. We have previously reported that natural novel withanolides from Physalis longifolia can potently inhibit these key regulatory pathways simultaneously. In the present study, we examined the efficacy and mechanisms through which novel withanolides and their acetate derivatives (WGA-TA and WGB-DA) from P.longifolia kill NB cells. The results from the study demonstrated that our novel acetate derivatives are highly effective in inhibiting the proliferation, shifting the cell cycle and inducing apoptosis in a dose dependent manner. Analysis of oncogenic pathway proteins targeted by withanolides indicated induction of heat shock response due to oxidative stress. Dose dependent decrease in clients of HSP90 chaperone function due to suppression of Akt, mTOR, and NF-κB pathways led to decrease in the expressions of target genes such as cyclin D1, N-myc and Survivin. Additionally, there was a dose dependent attenuation of the migration and invasion of NB cells. Furthermore, the lead compound WGA-TA showed significant reduction in tumor growth of NB xenografts. Taken together, these results suggest that withanolides are an effective therapeutic option against NBs.

Effects of peritumoral nanoconjugated cisplatin on laryngeal cancer stem cells.

OBJECTIVES/HYPOTHESIS: To evaluate the efficacy of peritumoral hyaluronic acid (HA)-cisplatin therapy in a murine model of laryngeal squamous cell carcinoma and to evaluate its effect on cancer stem cells (CSCs). STUDY DESIGN: An orthotopic murine study utilizing University of Michigan squamous cell carcinoma-12 (UMSCC-12) laryngeal cancer cells was conducted in randomized controlled fashion with three treatment arms: saline, systemic cisplatin, and peritumoral HA-cisplatin. METHODS: UMSCC-12 laryngeal cancer cells were inoculated into the buccal mucosa of athymic nude mice followed by weekly treatment with saline, systemic cisplatin, or peritumoral HA-cisplatin for 3 weeks. Tumor response and animal weight was monitored and change in CD44 proportion was analyzed ex vivo. RESULTS: HA-cisplatin demonstrated superior antitumor efficacy and greater reduction in CD44 positivity on ex vivo analysis. CONCLUSIONS: Peritumoral nanoconjugated HA-cisplatin provides superior antitumor efficacy compared to standard cisplatin therapy in an in vivo laryngeal cancer model. There was also selective targeting of CD44+ cancer cells with HA-cisplatin. This therapeutic strategy could represent the first selective laryngeal CSC-targeted therapy. Further preclinical investigation is warranted to evaluate its role for locally advanced head and neck cancer treatment. LEVEL OF EVIDENCE: NA Laryngoscope, 126:E184-E190, 2016.

Triazole Containing Novobiocin and Biphenyl Amides as Hsp90 C-Terminal Inhibitors.

Hsp90 C-terminal inhibitors are advantageous for the development of new cancer chemotherapeutics due to their ability to segregate client protein degradation from induction of the prosurvival heat shock response, which is a major detriment associated with Hsp90 N-terminal inhibitors under clinical investigation. Based upon prior SAR trends, a 1,2,3-triazole side chain was placed in lieu of the aryl side chain and attached to both the coumarin and biphenyl scaffold. Antiproliferative studies against SKBr3 and MCF-7 breast cancer cell lines demonstrated these triazole-containing compounds to exhibit improved activity. These compounds were shown to manifest Hsp90 inhibitory activity through Western blot analysis and represent a new scaffold upon which more potent inhibitors can be pursued.

Oxidative cytotoxic agent withaferin A resensitizes temozolomide-resistant glioblastomas via MGMT depletion and induces apoptosis through Akt/mTOR pathway inhibitory modulation.

Temozolomide (TMZ) has remained the chemotherapy of choice in patients with glioblastoma multiforme (GBM) primarily due to the lack of more effective drugs. Tumors, however, quickly develop resistance to this line of treatment creating a critical need for alternative approaches and strategies to resensitize the cells. Withaferin A (WA), a steroidal lactone derived from several genera of the Solanaceae plant family has previously demonstrated potent anti-cancer activity in multiple tumor models. Here, we examine the effects of WA against TMZ-resistant GBM cells as a monotherapy and in combination with TMZ. WA prevented GBM cell proliferation by dose-dependent G2/M cell cycle arrest and cell death through both intrinsic and extrinsic apoptotic pathways. This effect correlated with depletion of principle proteins of the Akt/mTOR and MAPK survival and proliferation pathways with diminished phosphorylation of Akt, mTOR, and p70 S6K but compensatory activation of ERK1/2. Depletion of tyrosine kinase cell surface receptors c-Met, EGFR, and Her2 was also observed. WA demonstrated induction of N-acetyl-L-cysteine-repressible oxidative stress as measured directly and through a subsequent heat shock response with HSP32 and HSP70 upregulation and decreased HSF1. Finally, pretreatment of TMZ-resistant GBM cells with WA was associated with O6-methylguanine-DNA methyltransferase (MGMT) depletion which potentiated TMZ-mediated MGMT degradation. Combination treatment with both WA and TMZ resulted in resensitization of MGMT-mediated TMZ-resistance but not resistance through mismatch repair mutations. These studies suggest great clinical potential for the utilization of WA in TMZ-resistant GBM as both a monotherapy and a resensitizer in combination with the standard chemotherapeutic agent TMZ.

Efficacy of protracted temozolomide dosing is limited in MGMT unmethylated GBM xenograft models.

BACKGROUND: Temozolomide (TMZ) is important chemotherapy for glioblastoma multiforme (GBM), but the optimal dosing schedule is unclear. METHODS: The efficacies of different clinically relevant dosing regimens were compared in a panel of 7 primary GBM xenografts in an intracranial therapy evaluation model. RESULTS: Protracted TMZ therapy (TMZ daily M-F, 3 wk every 4) provided superior survival to a placebo-treated group in 1 of 4 O(6)-DNA methylguanine-methyltransferase (MGMT) promoter hypermethylated lines (GBM12) and none of the 3 MGMT unmethylated lines, while standard therapy (TMZ daily M-F, 1 wk every 4) provided superior survival to the placebo-treated group in 2 of 3 MGMT unmethylated lines (GBM14 and GBM43) and none of the methylated lines. In comparing GBM12, GBM14, and GBM43 intracranial specimens, both GBM14 and GBM43 mice treated with protracted TMZ had a significant elevation in MGMT levels compared with placebo. Similarly, high MGMT was found in a second model of acquired TMZ resistance in GBM14 flank xenografts, and resistance was reversed in vitro by treatment with the MGMT inhibitor O(6)-benzylguanine, demonstrating a mechanistic link between MGMT overexpression and TMZ resistance in this line. Additionally, in an analysis of gene expression data, comparison of parental and TMZ-resistant GBM14 demonstrated enrichment of functional ontologies for cell cycle control within the S, G2, and M phases of the cell cycle and DNA damage checkpoints. CONCLUSIONS: Across the 7 tumor models studied, there was no consistent difference between protracted and standard TMZ regimens. The efficacy of protracted TMZ regimens may be limited in a subset of MGMT unmethylated tumors by induction of MGMT expression.

Cytotoxicity of withaferin A in glioblastomas involves induction of an oxidative stress-mediated heat shock response while altering Akt/mTOR and MAPK signaling pathways.

Withaferin A (WA), a steroidal lactone derived from the plant Vassobia breviflora, has been reported to have anti-proliferative, pro-apoptotic, and anti-angiogenic properties against cancer growth. In this study, we identified several key underlying mechanisms of anticancer action of WA in glioblastoma cells. WA was found to inhibit proliferation by inducing a dose-dependent G2/M cell cycle arrest and promoting cell death through both intrinsic and extrinsic apoptotic pathways. This was accompanied by an inhibitory shift in the Akt/mTOR signaling pathway which included diminished expression and/or phosphorylation of Akt, mTOR, p70 S6K, and p85 S6K with increased activation of AMPKα and the tumor suppressor tuberin/TSC2. Alterations in proteins of the MAPK pathway and cell surface receptors like EGFR, Her2/ErbB2, and c-Met were also observed. WA induced an N-acetyl-L-cysteine-repressible enhancement in cellular oxidative potential/stress with subsequent induction of a heat shock stress response primarily through HSP70, HSP32, and HSP27 upregulation and HSF1 downregulation. Taken together, we suggest that WA may represent a promising chemotherapeutic candidate in glioblastoma therapy warranting further translational evaluation.

Antiproliferative withanolides from Datura wrightii.

A new withanolide, named withawrightolide (1), and four known withanolides (2-5) were isolated from the aerial parts of Datura wrightii. The structure of compound 1 was elucidated through 2D NMR and other spectroscopic techniques. In addition, the structure of withametelin L (2) was confirmed by X-ray crystallographic analysis. Using MTS viability assays, withanolides 1-5 showed antiproliferative activities against human glioblastoma (U251 and U87), head and neck squamous cell carcinoma (MDA-1986), and normal fetal lung fibroblast (MRC-5) cells with IC50 values in the range between 0.56 and 5.6 µM.

ATM inhibitor KU-55933 increases the TMZ responsiveness of only inherently TMZ sensitive GBM cells.

Ataxia telangiectasia mutated (ATM) kinase is critical in sensing and repairing DNA double-stranded breaks (DSBs) such as those induced by temozolomide (TMZ). ATM deficiency increases TMZ sensitivity, which suggests that ATM inhibitors may be effective TMZ sensitizing agents. In this study, the TMZ sensitizing effects of 2 ATM specific inhibitors were studied in established and xenograft-derived glioblastoma (GBM) lines that are inherently sensitive to TMZ and derivative TMZ-resistant lines. In parental U251 and U87 glioma lines, the addition of KU-55933 to TMZ significantly increased cell killing compared to TMZ alone [U251 survival: 0.004 ± 0.0015 vs. 0.08 ± 0.01 (p < 0.001), respectively, and U87 survival: 0.02 ± 0.005 vs. 0.04 ± 0.002 (p < 0.001), respectively] and also elevated the fraction of cells arrested in G2/M [U251 G2/M fraction: 61.8 ± 1.1 % vs. 35 ± 0.8 % (p < 0.001), respectively, and U87 G2/M fraction 25 ± 0.2 % vs.18.6 ± 0.4 % (p < 0.001), respectively]. In contrast, KU-55933 did not sensitize the resistant lines to TMZ, and neither TMZ alone or combined with KU-55933 induced a G2/M arrest. While KU-55933 did not enhance TMZ induced Chk1/Chk2 activation, it increased TMZ-induced residual γ-H2AX foci in the parental cells but not in the TMZ resistant cells. Similar sensitization was observed with either KU-55933 or CP-466722 combined with TMZ in GBM12 xenograft line but not in GBM12TMZ, which is resistant to TMZ due to MGMT overexpression. These findings are consistent with a model where ATM inhibition suppresses the repair of TMZ-induced DSBs in inherently TMZ-sensitive tumor lines, which suggests an ATM inhibitor potentially could be deployed with an improvement in the therapeutic window when combined with TMZ.

Oxygen is a master regulator of the immunogenicity of primary human glioma cells.

With recent approval of the first dendritic cell (DC) vaccine for patient use, many other DC vaccine approaches are now being tested in clinical trials. Many of these DC vaccines employ tumor cell lysates (TL) generated from cells cultured in atmospheric oxygen (∼20% O2) that greatly exceeds levels found in tumors in situ. In this study, we tested the hypothesis that TLs generated from tumor cells cultured under physiologic oxygen (∼5% O2) would be more effective as a source for DC antigens. Gene expression patterns in primary glioma cultures established at 5% O2 more closely paralleled patient tumors in situ and known immunogenic antigens were more highly expressed. DCs treated with TLs generated from primary tumor cells maintained in 5% O2 took up and presented antigens to CD8 T cells more efficiently. Moreover, CD8 T cells primed in this manner exhibited superior tumoricidal activity against target cells cultured in either atmospheric 20% O2 or physiologic 5% O2. Together, these results establish a simple method to greatly improve the effectiveness of DC vaccines in stimulating the production of tumoricidal T cells, with broad implications for many of the DC-based cancer vaccines being developed for clinical application.

Superior efficacy of tumor cell vaccines grown in physiologic oxygen.

PURPOSE: Atmospheric oxygen (∼20% O(2)) has been the universal condition employed to culture tumor cells used as vaccine antigen. We tested the hypothesis that reducing oxygen tension would increase the efficacy of tumor cell lysate vaccines. EXPERIMENTAL DESIGN: GL261 glioma cells and EMT6 breast carcinoma cells were grown in 5% or 20% O(2). Syngeneic tumor-bearing mice were vaccinated with these tumor cell lysates mixed with CpG oligodeoxynucleotides as an adjuvant. Tumor infiltrating T cells and apoptotic GL261 cells were quantified by immunohistochemistry. Tumor-reactive immunoglobulin was detected by Western blot. Ovalbumin and gp100-derived peptides were mixed with GL261 lysates as marker antigens to detect changes in presentation of exogenous antigen on MHC class I in vitro, and in vivo following adoptive transfer of gp100-specific CD8(+) T cells. RESULTS: Mice bearing orthotopic glioma and breast carcinoma survived significantly longer when vaccinated with 5% O(2) lysates. Antigen-specific CTL activation was significantly enhanced following stimulation with lysates derived from GL261 cells grown in 5% O(2) versus 20% O(2) through a mechanism that involved enhanced cross-presentation of exogenous antigen on MHC I. Vaccination with 5% O(2) GL261 cell lysates caused a significant increase in CTL proliferation, tumoricidal function, and trafficking into brain tumor sites, whereas 20% O(2) lysate vaccines predominantly evoked an antibody response. CONCLUSIONS: Tissue culture oxygen functions as an "immunologic switch" by dictating the cellular and humoral immune responses elicited by tumor cell lysates. These results have profound implications for cancer vaccines that utilize tumor cells as the source of antigen.

Anti-tumor immune response correlates with neurological symptoms in a dog with spontaneous astrocytoma treated by gene and vaccine therapy.

Gene therapy and vaccination have been tested in malignant glioma patients with modest, albeit encouraging results. The combination of these therapies has demonstrated synergistic efficacy in murine models but has not been reported in large animals. Gemistocytic astrocytoma (GemA) is a low-grade glioma that typically progresses to lethal malignancy despite conventional therapies. Until now there has been no useful animal model of GemA. Here we report the treatment of a dog with spontaneous GemA using the combination of surgery, intracavitary adenoviral interferon gamma (IFNgamma) gene transfer, and vaccination with glioma cell lysates mixed with CpG oligodeoxynucleotides. Surgical tumor debulking and delivery of Ad-IFNgamma into the resection cavity were performed. Autologous tumor cells grew slowly in culture, necessitating vaccination with allogeneic tumor lysate in four of the five vaccinations. Transient left-sided blindness and hemiparesis occurred following the fourth and fifth vaccinations. These neurological symptoms correlated with a peak in the levels of tumor-reactive IgG and CD8(+) T cells measured in the blood. All symptoms resolved and this dog remains tumor-free over 450 days following surgery. This case report preliminarily demonstrates the feasibility of treating dogs with spontaneous glioma using immune-based therapy and warrants further study using this therapeutic approach.

Radiosensitizing effects of temozolomide observed in vivo only in a subset of O6-methylguanine-DNA methyltransferase methylated glioblastoma multiforme xenografts.

PURPOSE: Concurrent temozolomide (TMZ) and radiation therapy (RT) followed by adjuvant TMZ is standard treatment for patients with glioblastoma multiforme (GBM), although the relative contribution of concurrent versus adjuvant TMZ is unknown. In this study, the efficacy of TMZ/RT was tested with a panel of 20 primary GBM xenografts. METHODS AND MATERIALS: Mice with intracranial xenografts were treated with TMZ, RT, TMZ/RT, or placebo. Survival ratio for a given treatment/line was defined as the ratio of median survival for treatment vs. placebo. RESULTS: The median survival ratio was significantly higher for O6-methylguanine-DNA methyltransferase (MGMT) methylated tumors versus unmethylated tumors following treatment with TMZ (median survival ratio, 3.6 vs. 1.5, respectively; p = 0.008) or TMZ/RT (5.7 vs. 2.3, respectively; p = 0.001) but not RT alone (1.7 vs. 1.6; p = 0.47). In an analysis of variance, MGMT methylation status and p53 mutation status were significantly associated with treatment response. When we analyzed the additional survival benefit conferred specifically by combined therapy, only a subset (5 of 11) of MGMT methylated tumors derived substantial additional benefit from combined therapy, while none of the MGMT unmethylated tumors did. Consistent with a true radiosensitizing effect of TMZ, sequential treatment in which RT (week 1) was followed by TMZ (week 2) proved significantly less effective than TMZ followed by RT or concurrent TMZ/RT (survival ratios of 4.0, 9.6 and 12.9, respectively; p < 0.0001). CONCLUSIONS: Concurrent treatment with TMZ and RT provides significant survival benefit only in a subset of MGMT methylated tumors and provides superior antitumor activity relative to sequential administration of RT and TMZ.

Effective sensitization of temozolomide by ABT-888 is lost with development of temozolomide resistance in glioblastoma xenograft lines.

Resistance to temozolomide and radiotherapy is a major problem for patients with glioblastoma but may be overcome using the poly(ADP-ribose) polymerase inhibitor ABT-888. Using two primary glioblastoma xenografts, the efficacy of ABT-888 combined with radiotherapy and/or temozolomide was evaluated. Treatment with ABT-888 combined with temozolomide resulted in significant survival prolongation (GBM12: 55.1%, P = 0.005; GBM22: 54.4%, P = 0.043). ABT-888 had no effect with radiotherapy alone but significantly enhanced survival in GBM12 when combined with concurrent radiotherapy/temozolomide. With multicycle therapy, ABT-888 further extended the survival benefit of temozolomide in the inherently sensitive GBM12 and GBM22 xenograft lines. However, after in vivo selection for temozolomide resistance, the derivative GBM12TMZ and GBM22TMZ lines were no longer sensitized by ABT-888 in combination with temozolomide, and a similar lack of efficacy was observed in two other temozolomide-resistant tumor lines. Thus, the sensitizing effects of ABT-888 were limited to tumor lines that have not been previously exposed to temozolomide, and these results suggest that patients with newly diagnosed glioblastoma may be more likely to respond to combined temozolomide/poly(ADP-ribose) polymerase inhibitor therapy than patients with recurrent disease.

Induction of MGMT expression is associated with temozolomide resistance in glioblastoma xenografts.

Temozolomide (TMZ)-based therapy is the standard of care for patients with glioblastoma multiforme (GBM), and resistance to this drug in GBM is modulated by the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT). Expression of MGMT is silenced by promoter methylation in approximately half of GBM tumors, and clinical studies have shown that elevated MGMT protein levels or lack of MGMT promoter methylation is associated with TMZ resistance in some, but not all, GBM tumors. In this study, the relationship between MGMT protein expression and tumor response to TMZ was evaluated in four GBM xenograft lines that had been established from patient specimens and maintained by serial subcutaneous passaging in nude mice. Three MGMT unmethylated tumors displayed elevated basal MGMT protein expression, but only two of these were resistant to TMZ therapy (tumors GBM43 and GBM44), while the other (GBM14) displayed a level of TMZ sensitivity that was similar in extent to that seen in a single MGMT hypermethylated line (GBM12). In tissue culture and animal studies, TMZ treatment resulted in robust and prolonged induction of MGMT expression in the resistant GBM43 and GBM44 xenograft lines, while MGMT induction was blunted and abbreviated in GBM14. Consistent with a functional significance of MGMT induction, treatment of GBM43 with a protracted low-dose TMZ regimen was significantly less effective than a shorter high-dose regimen, while survival for GBM14 was improved with the protracted dosing regimen. In conclusion, MGMT expression is dynamically regulated in some MGMT nonmethylated tumors, and in these tumors, protracted dosing regimens may not be effective.