Developing a clinically relevant radiosensitizer for temozolomide-resistant gliomas

Abstract

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.

NEO212, a Perillyl Alcohol-Temozolomide Conjugate, Triggers Macrophage Differentiation of Acute Myeloid Leukemia Cells and Blocks Their Tumorigenicity

“…In our AML mouse models, 25 mg/kg NEO212 achieved curative outcomes ( Figure 8 ). At the same time, this dose did not cause any detectable side effects, as we have extensively documented in the past with the use of complete blood counts (CBC) with differential, blood chemistry and liver/kidney panels, histopathological organ analysis, or behavioral observations [ 6 , 7 , 38 , 44 , 49 ].…”

“…In our earlier studies, we had established that NEO212 retains the alkylating potency of its TMZ subunit [ 38 , 48 ], and therefore myelosuppression might be of concern. However, our previous studies with NEO212 in mice were unable to detect significant alterations in white blood cell (WBC) counts or other markers of bone marrow toxicity; similarly, no changes in body weight or behavior, or any signs of pathological organ damage could be documented [ 6 , 7 , 38 , 44 , 49 ]. In an effort to approach the limits of NEO212 tolerability, we switched to rats, because this rodent model allowed us to administer substantially higher oral dosages of drug, far above what is needed for therapeutic purposes.…”

“…Actin was used as the loading control. The following controls were included to ascertain specificity of the MGMT antibody: (i) HL60 and THP1 cells were treated for 18 h with 15 µM O6BG, an MGMT inhibitor known to down-regulate MGMT protein levels; (ii) T98G are glioblastoma cells known to be positive for MGMT expression; (iii) LN229 are glioblastoma cells known to be negative for MGMT, and LN229-M are a subline infected with a construct expressing MGMT cDNA [ 44 ]. Original blots see File S1 .…”

The use of radiosensitizing agents in the therapy of glioblastoma multiforme—a comprehensive review

“…Unfortunately, tumors expressing O6-methylguanine DNA methyltransferase (MGMT) show increased potential of repairing these lesions, which leads to a decreased effectiveness of TMZ therapy in patients with nonmethylated MGMT promotor [ 115 ], resulting in poorer prognosis [ 116 ]. Scientific studies currently try to overcome these limitations for example by using a compound-drug to increase the alkylating effects in MGMT-expressing tumor tissue [ 117 ].…”

Potentially Curative Therapeutic Activity of NEO212, a Perillyl Alcohol-Temozolomide Conjugate, in Preclinical Cytarabine-Resistant Models of Acute Myeloid Leukemia

“…The rationale for conjugating POH to TMZ was based on our in silico studies indicating superior blood–brain barrier penetration of the NEO212 molecule as compared to either of its individual components [ 16 ]. While this prediction turned out to be correct [ 17 ], we further established that NEO212 exerted promising anticancer activity in a number of preclinical mouse tumor models, including glioblastoma, brain-localized breast cancer, melanoma, and others [ 3 , 4 , 5 ]. NEO212’s robust therapeutic impact prompted us to investigate additional tumor types with significant need for better therapies, which led us to AML.…”