The prognosis for patients with glioblastoma (GB) remains grim. Concurrent temozolomide (TMZ) radiation-the cornerstone of glioma control-extends the overall median survival of GB patients by only a few months over radiotherapy alone. While these survival gains could be partly attributed to radiosensitization, this benefit is greatly minimized in tumors expressing O 6-methylguanine DNA methyltransferase (MGMT), which specifically reverses O 6methylguanine lesions. Theoretically, non-O 6-methylguanine lesions (i.e., the N-methylpurine adducts), which represent up to 90% of TMZ-generated DNA adducts, could also contribute to radiosensitization. Unfortunately, at concentrations attainable in clinical practice, the alkylation capacity of TMZ cannot overwhelm the repair of N-methylpurine adducts to efficiently exploit these lesions. The current therapeutic application of TMZ therefore faces two main obstacles: (i) the stochastic presence of MGMT and (ii) a blunted radiosensitization potential at physiologic concentrations. To circumvent these limitations, we are developing a novel molecule called NEO212-a derivatization of TMZ generated by coupling TMZ to perillyl alcohol. Based on gas chromatography/mass spectrometry and high-performance liquid chromatography analyses, we determined that NEO212 had greater tumor cell uptake than TMZ. In mouse models, NEO212 was more efficient than TMZ at crossing the blood-brain barrier, preferentially accumulating in tumoral over normal brain tissue. Moreover, in vitro analyses with GB cell lines, including TMZ-resistant isogenic variants, revealed more potent cytotoxic and radiosensitizing activities for NEO212 at physiologic concentrations. Mechanistically, these advantages of NEO212 over TMZ could be attributed to its enhanced tumor uptake presumably leading to more extensive DNA alkylation at equivalent dosages which, ultimately, allows for N-methylpurine lesions to be better exploited for radiosensitization. This effect cannot be achieved with TMZ at clinically relevant concentrations and is independent of MGMT. Our findings establish NEO212 as a superior radiosensitizer and a potentially better alternative to TMZ for newly diagnosed GB patients, irrespective of their MGMT status.

Background: Mycosis fungoides (MF) and Sézary syndrome (SS) are subtypes of primary cutaneous lymphomas and represent complex diseases regarding their physiopathology and management. Depending on the stage of the disease, different treatment regimens are applied, but there is no consensus on an optimal approach. Prognosis for patients with early stage MF is favorable, but significantly worsens in advanced disease and in SS, where patients frequently relapse and require multiple therapies. Methods: We investigated the potential anticancer effects of NEO212, a novel compound generated by covalently conjugating perillyl alcohol (a natural monoterpene) to temozolomide (an alkylating agent), on MF and SS cell lines in vitro. HUT-78, HUT-102, and MyLa cells were treated with NEO212 under different conditions, and drug effects on proliferation, viability, and apoptosis were characterized. Results: NEO212 inhibited proliferation, diminished viability, and stimulated apoptosis in all cell lines, although with varying degrees of potency in the different cell lines. It down-regulated c-myc and cyclin D1 proteins, which are required for cell proliferation, but triggered endoplasmic reticulum stress and activation of caspases. Pretreatment of cells with antioxidants ascorbic acid and beta-mercaptoethanol prevented these NEO212-induced effects. Conclusions: NEO212 exerted promising anticancer effects on SS and MF cell lines. The generation of reactive oxygen species (ROS) appears to play a key role in the NEO212-induced cell death process, because the blockage of ROS with antioxidants prevented caspase activation. We propose that NEO212 should be investigated further toward clinical testing in these tumor types.

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BackgroundCiliary defects cause heterogenous phenotypes related to mutation burden which lead to impaired development. A previously reported homozygous deletion in the Man1a2 gene causes lethal respiratory failure in newborn pups and decreased lung ciliation compared with wild type (WT) pups. The effects of heterozygous mutation, and the potential for rescue are not known.PurposeWe hypothesized that survival and lung ciliation, (a) would decrease progressively in Man1a2+/− heterozygous and Man1a2–/– null newborn pups compared with WT, and (b) could be enhanced by gestational treatment with N-Acetyl-cysteine (NAC), an antioxidant.MethodsMan1a2+/– adult mice were fed NAC or placebo from a week before breeding through gestation. Survival of newborn pups was monitored for 24 h. Lungs, liver and tails were harvested for morphology, genotyping, and transcriptional profiling.ResultsSurvival (p = 0.0001, Kaplan-Meier) and percent lung ciliation (p = 0.0001, ANOVA) measured by frequency of Arl13b+ respiratory epithelial cells decreased progressively, as hypothesized. Compared with placebo, gestational NAC treatment enhanced (a) lung ciliation in pups with each genotype, (b) survival in heterozygous pups (p = 0.017) but not in WT or null pups. Whole transcriptome of lung but not liver demonstrated patterns of up- and down-regulated genes that were identical in living heterozygous and WT pups, and completely opposite to those in dead heterozygous and null pups. Systems biology analysis enabled reconstruction of protein interaction networks that yielded functionally relevant modules and their interactions. In these networks, the mutant Man1a2 enzyme contributes to abnormal synthesis of proteins essential for lung development. The associated unfolded protein, hypoxic and oxidative stress responses can be mitigated with NAC. Comparisons with the developing human fetal lung transcriptome show that NAC likely restores normal vascular and epithelial tube morphogenesis in Man1a2 mutant mice.ConclusionSurvival and lung ciliation in the Man1a2 mutant mouse, and its improvement with N-Acetyl cysteine is genotype-dependent. NAC-mediated rescue depends on the central role for oxidative and hypoxic stress in regulating ciliary function and organogenesis during development.

The chemotherapeutic agent temozolomide (TMZ) kills tumor cells preferentially via alkylation of the O6-position of guanine. However, cells that express the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT), or harbor deficient DNA mismatch repair (MMR) function, are profoundly resistant to this drug. TMZ is in clinical use for melanoma, but objective response rates are low, even when TMZ is combined with O6-benzylguanine (O6BG), a potent MGMT inhibitor. We used in vitro and in vivo models of melanoma to characterize the early events leading to cellular TMZ resistance. Melanoma cell lines were exposed to a single treatment with TMZ, at physiologically relevant concentrations, in the absence or presence of O6BG. Surviving clones and mass cultures were analyzed by Western blot, colony formation assays, and DNA methylation studies. Mice with melanoma xenografts received TMZ treatment, and tumor tissue was analyzed by immunohistochemistry. We found that MGMT-negative melanoma cell cultures, before any drug treatment, already harbored a small fraction of MGMT-positive cells, which survived TMZ treatment and promptly became the dominant cell type within the surviving population. The MGMT-negative status in individual cells was not stable, as clonal selection of MGMT-negative cells again resulted in a mixed population harboring MGMT-positive, TMZ-resistant cells. Blocking the survival advantage of MGMT via the addition of O6BG still resulted in surviving clones, although at much lower frequency and independent of MGMT, and the resistance mechanism of these clones was based on a common lack of expression of MSH6, a key MMR enzyme. TMZ treatment of mice implanted with MGMT-negative melanoma cells resulted in effective tumor growth delay, but eventually tumor growth resumed, with tumor tissue having become MGMT positive. Altogether, these data reveal stochastic expression of MGMT as a pre-existing, key determinant of TMZ resistance in melanoma cell lines. Although MGMT activity can effectively be eliminated by pharmacologic intervention with O6BG, additional layers of TMZ resistance, although considerably rarer, are present as well and minimize the cytotoxic impact of TMZ/O6BG combination treatment. Our results provide rational explanations regarding clinical observations, where the TMZ/O6BG regimen has yielded mostly disappointing outcomes in melanoma patients.