Depletion Of MGMT Research Articles

Somatostatin Receptor Subtype-2 Targeting System for Specific Delivery of Temozolomide.

Temozolomide (TMZ) is a DNA alkylating agent that produces objective responses in patients with neuroendocrine tumors (NETs) when the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) is inactivated. At high doses, TMZ therapy exhausts MGMT activity but also produces dose-limiting toxicities. To reduce off-target effects, we converted the clinically approved radiotracer 68Ga-DOTA-TOC into a peptide-drug conjugate (PDC) for targeted delivery of TMZ to somatostatin receptor subtype-2 (SSTR2)-positive tumor cells. We used an integrated radiolabeling strategy for direct quantitative assessment of receptor binding, pharmacokinetics, and tissue biodistribution. In vitro studies revealed selective binding to SSTR2-positive cells with high affinity (5.98 ± 0.96 nmol/L), internalization, receptor-dependent DNA damage, cytotoxicity, and MGMT depletion. Imaging and biodistribution analysis showed preferential accumulation of the PDC in receptor-positive tumors and high renal clearance. This study identified a trackable SSTR2-targeting system for TMZ delivery and utilizes a modular design that could be broadly applied in PDC development.

Inhibition of O6‐methylguanine‐DNA‐methyltransferase (MGMT) by lomeguatrib reduces multiple myeloma cell viability and impairs DNA repair in MGMT‐proficient cells

AbstractThe function of direct DNA damage repair protein, namely MGMT in MM, and the impact of MGMT on melphalan treatment remains unclear. We showed a significantly higher MGMT mRNA expression in CD138+ myeloma cells than in matched CD138‐nontumorigenic cells derived from newly diagnosed and relapsed/refractory MM patients using qPCR. However, using gene expression databases, a similar expression of MGMT was observed during disease progression. MGMT depletion by its specific inhibitor lomeguatrib reduced myeloma cell viability, impaired S phase entry and DNA repair, and increased DNA damage and apoptosis. Apoptosis and DNA damage were further elevated by combined treatment with lomeguatrib and melphalan in RPMI 8226 cells. This is the first study demonstrating MGMT inhibition enhances DNA damage‐induced apoptosis and melphalan cytotoxicity in MGMT‐proficient MM cells and suggesting using lomeguatrib might have clinical importance in treating MM and overcoming melphalan resistance in MGMT‐proficient patients.

MGMT-Deficiency Is a Biomarker to Guide Treatment of Solid Tumors with Temozolomide and ATR Inhibitors

Temozolomide (TMZ) is an alkylating agent that methylates guanine bases at the O6 position (O6meG). O6meG-DNA methyltransferase (MGMT) is a suicide enzyme that repairs O6meG lesions by direct reversal. The promoter region of MGMT is methylated in various types of solid tumors, including glioblastoma (GBM), colorectal cancer, non-small and small-cell lung cancer, gastric cancer, and pancreatic cancer. Numerous previous studies have demonstrated that TMZ is more effective in treating tumors deficient in MGMT. However, clinical use of TMZ has been largely limited to GBM. Thus, the goal of this study was to determine what drugs are synergistic with TMZ as a novel strategy to increase the efficacy of TMZ for a wider array of tumor types. We assessed in vitro and in vivo synergistic interactions with a variety of DNA repair inhibitors and alkylating agents, using a collection MGMT-proficient and -deficient cell line models across various tumor types. We found that TMZ, unlike other alkylators tested, specifically induces growth delay, DNA damage, and G2/M-phase cell cycle arrest in MGMT-deficient cells, but not in MGMT-proficient cells. Because the protein ATR activates the G2/M checkpoint in response to DNA damage, we hypothesized and verified that TMZ activates ATR more specifically in MGMT-deficient cells. Next, we hypothesized that ATR inhibitors (ATRi's) would increase sensitivity to TMZ. In vitro, VX-970, an ATRi currently in clinical trials, increased sensitivity to TMZ by over 1000-fold in MGMT-deficient cells, but not in MGMT-proficient cells. Depletion of MGMT with a competitive inhibitor (O6-benzylguanine) conferred exquisite sensitivity to combined therapy with ATRi and TMZ in multiple MGMT-proficient cell lines, including U2OS and HeLa. In vivo, we demonstrated that combined treatment with TMZ and ATRi inhibited tumor growth in comparison to either agent alone, in both MGMT-deficient LN229 cells (GBM) and an MGMT-deficient, GBM PDX model. ATRi’s also appear to have promising activity as radiosensitizers, which prompted us to test whether we could detect their effects in combination with ionizing radiation (IR) and TMZ, specifically in MGMT-deficient cells. Indeed, we were able detect significant efficacy with this triple combination in our cell line models using a range of IR doses. Our data suggest that MGMT status can be utilized as a critical biomarker to select tumors for treatment with ATRi-based combinations. As TMZ and RT currently are standard of care for GBM, and nearly 50% of tumors harbor MGMT-deficiency, these results form the basis for clinical trials testing the integration of ATRi’s into novel chemoradiation strategies in the future. In particular, we propose that the combination of ATRi’s with RT, during both the concurrent and adjuvant TMZ phases of treatment, will have great potential. Additional studies are currently underway in our laboratory to further validate the efficacy of this “triple strategy” in GBM.

Chemotherapy sensitization of glioblastoma by focused ultrasound-mediated delivery of therapeutic liposomes

In glioblastoma, the benefit from temozolomide chemotherapy is largely limited to a subgroup of patients (30–35%) with tumors exhibiting methylation of the promoter region of the O6-methylguanine-DNA methyltransferase (MGMT) gene. In order to allow more patients to benefit from this treatment, we explored magnetic resonance image-guided microbubble-enhanced low-intensity pulsed focused ultrasound (LIFU) to transiently open the blood-brain barrier and deliver a first-in-class liposome-loaded small molecule MGMT inactivator in mice bearing temozolomide-resistant gliomas. We demonstrate that a liposomal O6-(4-bromothenyl)guanine (O6BTG) derivative can efficiently target MGMT, thereby sensitizing murine and human glioma cells to temozolomide in vitro. Furthermore, we report that image-guided LIFU mediates the delivery of the stable liposomal MGMT inactivator in the tumor region resulting in potent MGMT depletion in vivo. Treatment with this new liposomal MGMT inactivator facilitated by LIFU-mediated blood-brain barrier opening reduced tumor growth and significantly prolonged survival of glioma-bearing mice, when combined with temozolomide chemotherapy. Exploring this novel combined approach in the clinic to treat glioblastoma patients with MGMT promoter-unmethylated tumors is warranted.

Dose-dense temozolomide for recurrent high-grade gliomas: a single-center retrospective study.

There are limited treatment modalities after high-grade gliomas recurrence. MGMT depletion modulated by dose-dense temozolomide (ddTMZ) remains a debated therapy for initial TMZ responders. Patients were selected retrospectively from our practice with diagnosis of high-grade gliomas (WHO grade III or IV), and were followed since the start of ddTMZ until death or change of therapy. Twenty-one patients were reviewed, with a median age of 47 (25-61) years and a median of 5.8 (1.5-38.8) cycles of ddTMZ. The majority were males (71.4%). Sixty-six percent received 21 on/28 off ddTMZ schedule, 28.6% daily, and 1 patient received a 7days on/7days off schedule. IDH mutation status was available for 18 (85.7%) patients, with 7 (33.3%) IDH mutant and 11 (52.5%) IDH wild type. MGMT methylation was assessed in 6 (28.6%) of the patients, being MGMT methylated in 3 (14.3%) patients, and non-methylated in 3 (14.3%) patients. The majority of patients (57.1%) were receiving ddTMZ in addition to other forms of therapy, including either bevacizumab (38.1%) or tumor-treating fields (TTFields) (19.1%). Overall ddTMZ was well tolerated, with few adverse events reported. The estimated median overall survival after ddTMZ start was 11months. Median progression-free survival (PFS) was 6months. Outcomes did not vary between patients receiving ddTMZ alone or those using TTFields or bevacizumab as concomitant therapy, but there was a trend to longer survival with the use of concomitant TTFields. Our results demonstrate benefit of ddTMZ after previous treatment with standard TMZ dosing with no apparent increase in treatment-related toxicities. In summary, ddTMZ should be considered in TMZ responsive patients and warrants further investigation.

Abstract 2928: Bortezomib sensitizes glioblastoma with unmethylated MGMT promoter to temozolomide-chemotherapy through MGMT depletion and abrogated autophagy flux

Abstract Background: Glioblastoma with unmethylated O6-methyl guanine DNA methyltransferase (MGMT) promoter is highly resistant to temozolomide (TMZ) chemotherapy. Strategies that ameliorate drug resistance are sorely needed. Recent trials of the proteasome inhibitor bortezomib (BTZ) (Velcade) in combination with various drugs failed due to inappropriate schedule timing and dosing. We hypothesized that pretreatment with BTZ prior to TMZ administration may sensitize glioblastoma cell to TMZ chemotherapy. Methods: We investigated treatment efficacy through DNA damage, apoptosis and autophagy flux by flow cytometry, western blotting, long-lived protein degradation assays, electron and fluorescence microscopy in cell lines (U87, T98G, HF66) and patient biopsy-derived cells (P3, 2012-18, BG5 and BG7). Treatment efficacy and tolerability was investigated in vivo in mice implanted orthotopically with patient-derived GBM xenografts and subsequently treated with human equivalent dose (HED) of BTZ 1.3 mg/m2 on days 1, 4, 8 and 11 for two cycles during TMZ 82 mg/m2 or 164 mg/m2 by oral gavage 5 days/week for 5 weeks. MRI, mouse survival times, tandem LC-MS/MS and clinical chemistry were used to monitor tumor growth and evaluate tissue and blood samples for biomarkers of treatment efficacy and toxicity. Results: Patient-derived glioblastoma cells were universally more sensitive to BTZ than carfilzomib or MG-132 (P<0.0001), while their sensitivity to TMZ was strongly associated with MGMT promoter methylation status (P<0.0001). BTZ depleted MGMT protein (P<0.001) and mRNA (P<0.0001) in TMZ resistant tumor cells and sensitized to chemotherapy through induction of prominent DNA damage, G2-S phase cell cycle arrest and apoptosis with half the IC50 doses for both drugs. BTZ simultaneously abrogated TMZ-induced autophagic flux indicated by p62 accumulation, inhibition of utophagosome fusion and degradation of long-lived proteins. Human equivalent doses of BTZ 1.3 mg/m2 and TMZ 164 mg/m2 prolonged progression free and overall survival (P<0.0001), through diminished tumor growth (P<0.05), angiogenesis (P<0.01) and proliferation (P<0.05). Loss of structural and catalytic proteasomal subunits confirmed target inhibition at the therapeutically effective dose (P<0.01). Peroxiredoxin, thioredoxin and catalase antioxidant enzymes (P<0.01), alanine aminotransferase (P<0.05) and MGMT mRNA expression (P<0.01) were downregulated in combination treated animals, serving as accessible biomarkers of response. Conclusion: Pretreatment with BTZ chemo-sensitized resistant glioblastoma through aborted autophagic flux and MGMT depletion to augment DNA damage, apoptosis and prolonged survival. Our findings warrant a controlled investigation of the treatment schedule in selected patients. Citation Format: Mohummad A. Rahman, Andrea G. Navarro, Jorunn Brekke, Christian Bindesbøll, Agnete Engelsen, Shahin Sarowar, Marzieh Bahador, Bjørn T. Gjertsen, Dorota Goplen, Per Ø. Enger, Frode Selheim, Anne Simonsen, Martha Chekenya. Bortezomib sensitizes glioblastoma with unmethylated MGMT promoter to temozolomide-chemotherapy through MGMT depletion and abrogated autophagy flux [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2928.

Abstract 1121: Redox-mediated inactivation of MGMT DNA repair through thiolation or nitrosylation of the active site cysteine145 sensitizes human gliomas to alkylating agents in vitro and in xenograft models

Abstract O6-Methylguanine-DNA methyltransferase (MGMT) is a simple and unique antimutagenic DNA repair protein that plays a crucial role in conferring tumor resistance to various alkylating agents in brain tumor therapy. MGMT repairs O6-alkylguanines by transferring the alkyl groups to its active site cysteine (Cys145) in a stoichiometric/suicidal reaction thereby restoring the original nucleotide sequence in a single step. Although O6-benzylguanine (BG), a pseudosubstrate inhibitor, currently in clinical trials, potently inhibits MGMT and improves the efficacy of alkylating agents, unacceptable levels of bone marrow toxicity has been a great concern. For developing new MGMT inhibitors, we exploited the reactive nature of the active site Cys145 (pKa 4.5) and its susceptibility for thiolation and nitrosylation, both of which inactivate the MGMT. A new strategy involving inhibition of S-nitosoglutathione reductase (GSNOR) was also explored to increase the steady-state levels of nitrosylated MGMT. First, we designed a redox perturbing glutathione mimetic, a homoglutathione disulfide (hGTX) that has been stabilized with the addition of small amounts of cisplatin (1000:10). Spermine NONOate and S-nitroso penicillamine (SNAP) served as the protein-nitrosylating agents. N6022, a specific and reversible inhibitor of GSNOR was used with nitrosylators in some assays. Both the hGTX and spermine NONOate at 100-400 µM concentrations for 24 h inhibited the repair activity of MGMT >75% in HT29, SF188, and T98G cells. The NONOate strongly curtailed the DNA repair activity of recombinant MGMT showing a direct interaction. The inactivated MGMT protein was promptly eliminated from glioma cells. MGMT disappeared from cells much faster in spermine NONOate +N6022 treated cells, suggesting an extended steady-state of nitrosylated MGMT and its subsequent degradation. MGMT depletion was accompanied by 4-6 fold increases in cell killing by BCNU or temozolomide (TMZ). Binding of the biotinylated-O6-benzylguanine to the MGMT protein was prevented by hGTX and spermine NONOate treatments indicating the Cys145 was indeed the site of modification. In the nude mice bearing T98G or HT29-luc2 xenografts, combinations of hGTX and TMZ or spermine NONOate +TMZ produced markedly synergistic tumor regressions compared with TMZ alone. A reduction of MGMT protein as by immunoblotting and diminished MGMT activity levels in tumor tissues was verified. Apoptosis regulatory proteins were upregulated in excised tumors and H&E staining confirmed no discernible host organ toxicities. Our studies highlight the options for redox-driven therapeutic strategies for MGMT [supported by CPRIT grants RP130266 and RP170207 to KSS]. Citation Format: Debasish Basak, Kalkunte S. Srivenugopal. Redox-mediated inactivation of MGMT DNA repair through thiolation or nitrosylation of the active site cysteine145 sensitizes human gliomas to alkylating agents in vitro and in xenograft 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 1121. doi:10.1158/1538-7445.AM2017-1121

Posttranslational Regulation of O(6)-Methylguanine-DNA Methyltransferase (MGMT) and New Opportunities for Treatment of Brain Cancers.

O(6)-Methylguanine-DNA-methyltransferase (MGMT) is an antimutagenic DNA repair protein highly expressed in human brain tumors. Because MGMT repairs the mutagenic, carcinogenic and cytotoxic O(6)-alkylguanine adducts, including those generated by the clinically used anticancer alkylating agents, it has emerged as a central and rational target for overcoming tumor resistance to alkylating agents. Although the pseudosubstrates for MGMT [O(6)-benzylguanine, O(6)-(4- bromothenyl)guanine] have gained attention as powerful and clinically-relevant inhibitors, bone marrow suppression due to excessive alkylation damage has diminished this strategy. Our laboratory has been working on various posttranslational modifications of MGMT that affect its protein stability, DNA repair activity and response to oxidative stress. While these modifications greatly impact the physiological regulation of MGMT, they also highlight the opportunities for inactivating DNA repair and new drug discovery in this specific area. This review briefly describes the newer aspects of MGMT posttranslational regulation by ubiquitination, sumoylation and glutathionylation and reveals how the reactivity of the active site Cys145 can be exploited for potent inhibition and depletion of MGMT by thiol-reacting drugs such as the disulfiram and various dithiocarbamate derivatives. The possible repurposing of these nontoxic and safe drugs for improved therapy of pediatric and adult brain tumors is discussed.

Dose-Dense Temozolomide in Patients with MGMT-Silenced Chemorefractory Colorectal Cancer.

In a phase II study, we showed that temozolomide (TMZ) was tolerable and active in heavily pre-treated patients with advanced colorectal cancer (CRC) and MGMT methylation. A schedule of dose-dense TMZ may have enhanced activity due to the higher cumulative dose and induction of MGMT depletion, even in resistant tumors. Thirty-two patients with chemorefractory MGMT-methylated CRC were treated with TMZ at a daily dose of 75mg/m(2) for 21 consecutive days every 4weeks, for up to sixcycles or until the occurrence of progressive disease/unacceptable toxicity. The primary endpoint was treatment activity in terms of objective response rate (ORR). MGMT protein expression was tested by immunohistochemistry (IHC) on two pooled cohorts: patients from the previous study of standard-dose TMZ and those from the current investigation. From November 2013 to December 2014, 32 patients were treated at Fondazione IRCCS Istituto Nazionale dei Tumori. We observed only three episodes of grade 3 asthenia and no significant myelotoxicity. The ORR was 16% (all partial responses occurring in RAS-BRAF-mutated tumors). Median progression-free survival (PFS) and overall survival (OS) were 2.3 and 6.7months, respectively. Patients with MGMT-low expression by IHC had a significantly higher ORR (p < 0.0001) and PFS (p = 0.001) compared to those with MGMT-high expression, while no difference was observed in OS. Our data confirm the encouraging activity of TMZ in chemorefractory CRC patients selected for MGMT silencing, even in the RAS-BRAF-mutated population. The role of MGMT IHC as a biomarker for improving patient selection warrants further prospective confirmation.

Endoplasmic reticulum protein 29 (ERp29) confers radioresistance through the DNA repair gene, O(6)-methylguanine DNA-methyltransferase, in breast cancer cells.

Resistance of cancer cells to radiotherapy is a major clinical problem in cancer treatment. Therefore, understanding the molecular basis of cellular resistance to radiotherapy and identification of novel targets are essential for improving treatment efficacy for cancer patients. Our previous studies have demonstrated a significant role of ERp29 in breast cancer cell survival against doxorubicin-induced genotoxic stress. We here reported that ERp29 expression in the triple negative MDA-MB-231 breast cancer cells significantly increased cell survival against ionizing radiation. Methylation PCR array analysis identified that ERp29 expression increased promoter hypomethylation of the DNA repair gene, O6-methylguanine DNA-methyltransferase (MGMT), by downregulating DNA methyltransferase 1. Knockdown of MGMT in the ERp29-transfected cancer cells increased radiosensitivity, leading to a decreased post-irradiation survival. In addition, radiation treatment in the MGMT-knockdown cells elevated phosphorylation of γ-H2AX and cleavage of caspase 3, indicating that depletion of MGMT facilitates DNA double strands breaks and increases cell apoptosis. Hence, our studies prove a novel function of ERp29\\MGMT in cancer cell survival against radiation. Targeting ERp29\\MGMT axis may be useful for providing better treatment efficacy in combination with radiotherapy in breast cancer.

P-0267 - Dose-Dense Temozolomide (TMZ) in Patients with Advanced Chemorefractory Colorectal Cancer (CRC) and Mgmt Promoter Methylation

Introduction: In a previous phase II study, we showed that TMZ is tolerable and active in heavily pre-treated patients with advanced CRC and MGMT promoter methylation, particularly in the RAS-BRAF wild-type subgroup (Pietrantonio et al., Ann Oncol 2014). In fact, MAPK signaling may enhance MGMT activity and RAS-BRAF mutations may drive cellular resistance to standard dose TMZ. However, dose-dense TMZ results in prolonged depletion of MGMT in blood mononuclear cells and possibly in tumor (Tolcher et al., BJC 2003).

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.

Dose-Dense Temozolomide for Newly Diagnosed Glioblastoma: A Randomized Phase III Clinical Trial

Radiotherapy with concomitant and adjuvant temozolomide is the standard of care for newly diagnosed glioblastoma (GBM). O(6)-methylguanine-DNA methyltransferase (MGMT) methylation status may be an important determinant of treatment response. Dose-dense (DD) temozolomide results in prolonged depletion of MGMT in blood mononuclear cells and possibly in tumor. This trial tested whether DD temozolomide improves overall survival (OS) or progression-free survival (PFS) in patients with newly diagnosed GBM. This phase III trial enrolled patients older than age 18 years with a Karnofsky performance score of ≥ 60 with adequate tissue. Stratification included clinical factors and tumor MGMT methylation status. Patients were randomly assigned to standard temozolomide (arm 1) or DD temozolomide (arm 2) for 6 to 12 cycles. The primary end point was OS. Secondary analyses evaluated the impact of MGMT status. A total of 833 patients were randomly assigned to either arm 1 or arm 2 (1,173 registered). No statistically significant difference was observed between arms for median OS (16.6 v 14.9 months, respectively; hazard ratio [HR], 1.03; P = .63) or median PFS (5.5 v 6.7 months; HR, 0.87; P = .06). Efficacy did not differ by methylation status. MGMT methylation was associated with improved OS (21.2 v 14 months; HR, 1.74; P < .001), PFS (8.7 v 5.7 months; HR, 1.63; P < .001), and response (P = .012). There was increased grade ≥ 3 toxicity in arm 2 (34% v 53%; P < .001), mostly lymphopenia and fatigue. This study did not demonstrate improved efficacy for DD temozolomide for newly diagnosed GBM, regardless of methylation status. However, it did confirm the prognostic significance of MGMT methylation. Feasibility of large-scale accrual, prospective tumor collection, and molecular stratification was demonstrated.

O6-benzylguanine (BG) and temozolomide (TMZ) therapy of glioblastoma multiforme (GBM) with infusion of autologous lentiviral transduced P140KMGMT+ hematopoietic progenitors to protect hematopoiesis: A phase I study.

TPS1616 Background: The administration of radiation therapy (RT) and concomitant and adjuvant temozolomide (TMZ) for the treatment of newly-diagnosed glioblastoma (GBM) is associated with modestly prolonged survival. However, the benefit of concurrent therapy is restricted to a subgroup of patients whose tumor exhibits methylation of the promoter for methylguanine-DNA-methyltransferase (MGMT). Promoter methylation silences the expression of DNA alkyltransferase (AGT), the product of the MGMT gene which functions to repair alkylated DNA. Unfortunately, the majority of GBMs have non-methylated MGMT promoters and do not appear to benefit from adding TMZ to radiotherapy. Two potential strategies for targeting MGMT-induced chemoprotection in gliomas include depletion of tumor MGMT, and dose escalation of TMZ accomplished by minimizing TMZ-induced bone marrow toxicity. In this study, we propose to combine the irreversible MGMT inhibitor O6-benzylguanine (BG) with infusion of autologous hematopoetic stem cells (HSCs) transduced with lentiviral P140K-MGMT, an MGMT mutant which is highly resistant to inactivation by BG, with the intent to reduce or prevent the myelosuppressive effects of TMZ. The primary objective is to assess the safety and feasibility of combining these two strategies in patients with newly-diagnosed GBM. Methods: This is a three cohort, Phase I study for patients with newly resected supratentorial GBM. There will be at least four evaluable patients per cohort. Cohort 1 will receive standard RT and concomitant TMZ prior to infusion of autologous lentiviral P140K-MGMT transduced HSCs, followed by adjuvant TMZ. Cohort 2 will receive an induction dose of TMZ+BG followed by infusion of autologous LV-P140K-MGMT HSCs prior to concomitant RT +TMZ and adjuvant TMZ. Cohort 3 will include intra-patient dose escalation of TMZ in patients who exhibit detectable levels of P140K-MGMT marked cells in vivo, using the design (i.e. cohort 1 or 2) proven to be safest and most effective. The study is open and has accrued the first patient.

Abstract C119: Depletion of O6-methylguanine DNA methyltransferase (MGMT) by TriN3001 leads to remission in SW-480 xenograft mice.

Abstract Background: TriN2755 is an alkylating agent currently under investigation in a phase-I study. Pharmacokinetic evaluation showed high inter-individual variability of the plasma concentration of its metabolite TriN3001. We speculate that differences in renal and metabolic clearance of TriN2755 and different expression of MGMT had contributed to the heterogeneous responses in xenograft models. We hypothesize that administration of TriN3001 improves predictability of drug levels and broadens the therapeutic window. In this study, we investigated the anti-tumor effect of TriN3001 given intraperitoneally after treatment of TriN2755 and temozolomide (TEM) in the colon-cancer cell line SW-480. Material and Methods: The in vitro effect of TriN2755 and 3001 on the proliferation of SW-480 was evaluated by IC50 determination. To study the in vivo efficacy, 5 × 106 cells of each cell line were inoculated in the flanks or back of nude mice. SW-480 mice were treated with TriN2755 (500mg/kg, biw, ip), TEM (120mg/kg qd×5, po) and NaCl as control. Treatment stopped at day 24 for interim-analysis. Mice were then being treated once daily with TriN3001 at a dose of 67,6mg/kg (low dose, former TriN), 100mg/kg (mid dose, former TEM) and 150mg/kg (high dose, former control) for another 16 to 20 days. Antitumor activity was assessed as (i) tumor volume inhibition relative to a vehicle control group for TriN2755 and (ii) change in tumor size from start of treatment for TriN3001. Tolerability was analysed by recording mortality, body weight loss and gross necropsy. Plasma and tissues were collected for pharmacokinetic and -dynamic evaluation. Results: In vitro, TriN2755 and 3001 and TEM displayed only weak growth inhibition with IC50 values of &amp;gt;3mM, 1mM and 1.2mM respectively. Nevertheless, in vivo T/C values of 40% (TriN2755) and 29% (TEM) were achieved at day 21. TEM and TriN2755 showed acceptable tolerability with no mortality and maximum body weight losses of 7% (TriN2755) and 10% (TEM). Daily administration of TriN3001 lead to remission in all mice, ranging from 66mm3 (30,3%) in the low, 77,6mm3 (23,1%) in the mid and 224mm3 (29,4%) in the high dose. TriN3001 and its metabolites were detectable in plasma and all tissues. In tumors, depletion of the repair enzyme MGMT increased over time with 60% depletion at 90min. Body weight loss didn't appear in the mid but in the low (−6%) and high (−20%) dose. At the end of treatment, thrombocytes and erythrocytes are within the reference range, lymphocytes lower and granulocytes higher than the reference. Conclusions: Daily application of TriN3001 led to remission of SW-480 tumors in mice independent from magnitude of tumor size. The treatment caused a marked depletion of MGMT protein in tumors. A second metabolite, with an IC50 value about 10fold lower than TriN3001 may have contributed to the efficacy. Further PK/PD data will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C119.

Abstract B63: Targeting cancer cells with a glucose-conjugated DNA repair inhibitor.

Abstract Alkylating agents are important chemotherapeutic drugs used for the treatment of several types of cancers, including brain tumors, melanoma and lymphoma. These chemotherapeutic agents have a strong affinity towards oxygen atoms in DNA giving rise to the important genotoxic DNA lesions O6-methylguanine and O6-chloroethylguanine, which are responsible for the cytotoxic effects of several alkylating anticancer drugs (e.g. temozolomide and lomustine). The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) is considered as an important player of drug resistance because it removes these DNA adducts from the DNA. The MGMT protein restores guanine in the DNA by a suicide repair reaction leading to irreversible inactivation and degradation of the protein. Therefore depletion of MGMT renders cells more susceptible to treatment with alkylating anticancer drugs. There has been numerous attempts to inactivate MGMT with different inhibitors like O6-benzylguanine (O6-BG) or O6-(4-bromothenyl) guanine (O6-BTG, lomeguatrib) in glioblastoma and malignant melanoma cells. These strategies however have failed thus far, mainly due to the severe side effects associated with the systemic sensitization of the patients to chemotherapeutic agents. The application of targeting strategies for MGMT inhibitors therefore should improve chemotherapy by selective inhibition of MGMT in the tumor. In our previous work we have shown that O6-BG and O6-BTG bound to glucose is efficient in inhibiting MGMT on enzyme level and in the cell, causing sensitization to DNA alkylating N-nitrosoureas (Kaina et. al., 2004; 2010). Here we show, using a quantitative MGMT assay, that intracellular uptake of O6-benzylguanine-C8-glucose (O6-BG-Glu) occurs by a transporter-dependent mechanism and not through passive diffusion. We observed differences in the transport kinetics between several cell lines. Most cancer cell lines are able to take up the inhibitor, whereas normal cells only show marginal uptake of the glucose-conjugated inhibitor. The uptake is probably not depended on the glucose transporter GLUT since blocking of GLUT does not abolishes MGMT inhibition. Uptake of the glucose conjugated inhibitors in cancer cells leads to an increased DNA damage response, induction of apoptosis and significant decrease in surviving fraction after treatment with alkylating anticancer drugs, pointing to the usefulness of the glucose-mediated MGMT inhibitor targeting strategy.

Rechallenge with temozolomide in recurrent glioma

Despite a confirmed survival benefit associated with adjuvant radio- and chemotherapy, the majority of patients with malignant glioma relapse after initial therapy. Recurrent malignant glioma treatment has not been standardised and usually the response rate to standard chemotherapy protocols for recurrent malignant glioma is less than 30%. The growing body of evidence demonstrating the clinical importance of O6-methylguanine methyltransferase (MGMT) has generated a considerable interest in the exploration of strategies to overcome MGMT-mediated resistance to alkylating agents; for example protracted administration of Temozolomide (TMZ) may result in more extensive and sustained depletion of MGMT; for this reason a variety of dosing schedules that increase the duration of exposure and the cumulative dose of TMZ are being investigated for the treatment of patient with recurrent malignant glioma after standard treatment. The most widely studied regimens in this setting include (1) 21 of 28-day schedule at a dose of 75-100 mg/m(2)/day; (2) 7 of 14-day schedule at a dose of 150 mg/m(2)/day, also referred to as the ''one week on/one week off'' schedule; (3) Continuous daily schedule at a dose of 50 mg/m(2)/day. An alternative dosing schedule of TMZ may be a reasonable option in patients having high-grade gliomas with recurrence after standard therapy.

Inhibition of DNA repair with MGMT pseudosubstrates: phase I study of lomeguatrib in combination with dacarbazine in patients with advanced melanoma and other solid tumours

Background:The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) reverses the O6-methylguanine (O6-meG) lesion induced by dacarbazine. Depletion of MGMT can be achieved using O6-meG pseudosubstrates. Herein, we report the first phase I experience of the novel O6-meG pseudosubstrate lomeguatrib, combined with dacarbazine.Methods:This is a phase I dose-escalation study to determine the maximum tolerated dose and recommended phase II dose (RP2D) of lomeguatrib combined with a single dose of dacarbazine on a 21-day schedule.Results:The vast majority of the 41 patients enrolled had metastatic melanoma (36/41) and most had no previous chemotherapy (30/41). The most frequent non-hematological adverse events (AEs) were nausea (52%), and fatigue (42%). The most frequent AEs of grade 3–4 severity were neutropaenia (42%), leukopaenia (17%), and thrombocytopaenia (12%). Only 1 patient had a partial response and 10 patients had stable disease.Conclusion:The RP2D of lomeguatrib was 40 mg orally twice daily for 10 days combined with 400 mg m−2 of dacarbazine IV on day 2. Oral administration of lomeguatrib substantially increases the haematological toxicity of dacarbazine consistent with experience with other O6-meG pseudosubstrates.

Final results of phase I/II study of decitabine (DAC) combined with temozolomide (TMZ) in metastatic melanoma (MM).

8530 Background: TMZ is widely used in MM despite low response rates (7-13%) and no improvement in median overall survival (OS) 7.7 months (m) when compared to dacarbazine in Phase III trials. The DNA repair protein, O6-methylguanine-DNA-methyltransferase (MGMT), is implicated in melanoma resistance to TMZ and can be depleted with extended schedule TMZ. DNA mismatch repair (MMR) deficiency is another mechanism of resistance that can be reversed with DNA hypomethylators such as DAC. We sought to determine the recommended phase II Dose (RP2D), safety, and efficacy of DAC in combination with extended schedule TMZ in MM. Methods: In the Phase I portion, pts were treated with 2 dose levels (DL) of DAC: 0.075 mg/kg IV QD and 0.15 mg/kg IV QD. DAC was administered qd x 5 d/wk for 2 wks, and TMZ was administered PO at 75 mg/m2 qd for 4 wks, starting on wk 2 of each 6-wk cycle. A Simon 2-stage Phase II design with success defined as ≥5 Objective Responses (OR) in 34 evaluable patients gave µ=0.10 and 85% power. PK sampling was performed on Days 1 and 8; PBMC and tumor tissues were studied on Days 1, 8, 15 and 29 of cycle 1. Results: The RP2D is DAC 0.15 mg/kg and TMZ 75 mg/m2. 35 patients were accrued to the Phase II portion of the study, the majority with M1c disease (88%); brain metastases were present in 42%. 2/35 were not evaluable (NE) for efficacy. For the remaining 33 patients, the best RECIST responses were 1CR, 4 PR, 13 SD, and 15 PD, for an OR rate of 15% and a clinical benefit rate (CR+PR+SD) of 54%. The median PFS was 2.8 m, median OS was 15.2 m (compared to 7.7 m), and 1-year OS rate was 62% (compared to 25%). Grade 3/4 neutropenia was observed in 68% of pts but reversible, lasting >7 d in only 5 pts (DLT rate 15%). Only 2 patients discontinued therapy for hematologic toxicity. PK studies revealed no effect of DAC on the PK profile of TMZ. Conclusions: The combination of DAC and TMZ is safe and results in improved median OS from historic reference of 7.7 m to 15.2 m, and 1-year OS rate from 25% to 62%. The addition of DAC appears to improve the clinical activity of TMZ, possibly through dual modulation of DNA repair (depletion of MGMT and re-expression of MMR), and warrants further evaluation in a randomized setting.

A phase I trial of laromustine (VNP40101M) and temozolomide for patients with malignant gliomas in first relapse or progression.

2064 Background: Effective treatments for recurrent malignant gliomas are limited. MGMT is one of the mechanisms of resistance for malignant gliomas. Depletion of MGMT may improve antitumor activity by increasing activity of alkylators. We used this concept in performing a phase I trial of temozolomide (TMZ) and laromustine (chloretazine). Methods: Patients were enrolled in standard 3 x 3 phase I trial with goal of performing a phase II trial at the MTD. Patients were treated with TMZ at 75 mg/m2days 1-7 and then chloretazine at 100, 150, or 125mg/m2 on day 7 (2 hours after TMZ) of a 6 week cycle. Patients were treated until progression or intolerable side effects. Patients had MRI scans every 6 weeks with a physical exam and weekly CBC’s were performed. Results: A total of 14 pts were accrued (10 GBM, 3 AA and 1 AOA), 10 M and 4 F with a median age and KPS of 51 (25-68) and 90 (80-100). Median number of cycles was 1 (1-4). Dose level 100 mg/m2 had 7 pts with 1 grade 4 thrombocytopenia, dose level 150 mg/m2had5 pts with1 grade 4 thrombocytopenia and 1 grade 3 fatigue. Does level 125 mg/m2 had 2 pts with 1 grade 4 leukopenia and thrombocytopenia. 10 pts had PD after 1 cycles, 4 were removed for toxicity. 3 pts who did not progress had SD. PFS-6 was 7%. Conclusions: The optimal dose for phase II was felt to be TMZ 75 mg/m2 x 7days and chloretazine 100 mg/m2 on day 7. The main toxicity of this regimen attempting to take advantage of MGMT depletion strategy is myelosuppression; similar to other trials using this strategy.