Glioblastoma (GBM) is the most aggressive primary brain tumor, exhibiting a high rate of recurrence and poor prognosis. Surgery and chemoradiation with temozolomide (TMZ) represent the standard of care, but, in most cases, the tumor develops resistance to further treatment and the patients succumb to disease. Therefore, there is a great need for the development of well-tolerated, effective drugs that specifically target chemoresistant gliomas. NEO214 was generated by covalently conjugating rolipram, a PDE4 (phosphodiesterase 4) inhibitor, to perillyl alcohol, a naturally occurring monoterpene related to limonene. Our previous studies in preclinical models showed that NEO214 harbors anticancer activity, is able to cross the blood-brain barrier (BBB), and is remarkably well tolerated. In the present study, we investigated its mechanism of action and discovered inhibition of macroautophagy/autophagy as a key component of its anticancer effect in glioblastoma cells. We show that NEO214 prevents autophagy-lysosome fusion, thereby blocking autophagic flux and triggering glioma cell death. This process involves activation of MTOR (mechanistic target of rapamycin kinase) activity, which leads to cytoplasmic accumulation of TFEB (transcription factor EB), a critical regulator of genes involved in the autophagy-lysosomal pathway, and consequently reduced expression of autophagy-lysosome genes. When combined with chloroquine and TMZ, the anticancer impact of NEO214 is further potentiated and unfolds against TMZ-resistant cells as well. Taken together, our findings characterize NEO214 as a novel autophagy inhibitor that could become useful for overcoming chemoresistance in glioblastoma.Abbreviations: ATG: autophagy related; BAFA1: bafilomycin A₁; BBB: blood brain barrier; CQ: chloroquine; GBM: glioblastoma; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MGMT: O-6-methylguanine-DNA methyltransferase; MTOR: mechanistic target of rapamycin kinase; MTORC: MTOR complex; POH: perillyl alcohol; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; TMZ: temozolomide.

We developed a bacterial expression system to produce a recombinant disintegrin, vicrostatin (VCN), whose structure is based on a natural disintegrin isolated from southern copperhead snake venom. Our goal is to develop VCN for potential clinical translation as an anti-cancer agent. VCN is a peptide of 69 amino acids with a single tyrosine residue. We have employed VCN as integrin-targeted radionuclide therapy (brachytherapy) for treatment of glioblastoma (GBM, glioma). GBM is a deadly brain cancer that doesn’t discriminate between sexes and knows no age limit. We established that the tyrosine residue in VCN can be radioiodinated with full retention of bioactivity. ¹³¹I-VCN was utilized for integrin-targeted radionuclide therapy using mouse models of glioma. The combination of radioiodinated VCN plus temozolomide (a DNA alkylating agent) significantly prolonged survival of glioma-bearing mice. We also obtained similar results using an immunocompetent mouse model and a murine glioma cell line. In summary, as demonstrated in studies reported here we have shown that VCN as targeted radionuclide therapy for GBM has significant translational potential for therapy of this deadly disease.

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 O⁶-methylguanine DNA methyltransferase (MGMT), which specifically reverses O⁶-methylguanine lesions. Theoretically, non-O⁶-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.

Many patients with acute myeloid leukemia (AML) are still dying from this disease. In the past, the alkylating agent temozolomide (TMZ) has been investigated for AML and found to be partially effective; however, the presence of O6-methylguanine DNA methyltransferase (MGMT; a DNA repair enzyme) in tumor cells confers profound treatment resistance against TMZ. We are developing a novel anticancer compound, called NEO212, where TMZ was covalently conjugated to perillyl alcohol (a naturally occurring monoterpene). NEO212 has revealed robust therapeutic activity in a variety of preclinical cancer models, including AML. In the current study, we investigated its impact on a panel of human AML cell lines and found that it exerted cytotoxic potency even against MGMT-positive cells that were highly resistant to TMZ. Furthermore, NEO212 strongly stimulated the expression of a large number of macrophage-associated marker genes, including CD11b/ITGAM. This latter effect could not be mimicked when cells were treated with TMZ or an equimolar mix of individual agents, TMZ plus perillyl alcohol. The superior cytotoxic impact of NEO212 appeared to involve down-regulation of MGMT protein levels. In a mouse model implanted with TMZ-resistant, MGMT-positive AML cells, two 5-day cycles of 25 mg/kg NEO212 achieved an apparent cure, as mice survived >300 days without any signs of disease. In parallel toxicity studies with rats, a 5-day cycle of 200 mg/kg NEO212 was well tolerated by these animals, whereas animals that were given 200 mg/kg TMZ all died due to severe leukopenia. Together, our results show that NEO212 exerts pleiotropic effects on AML cells that include differentiation, proliferation arrest, and eventual cell death. In vivo, NEO212 was well tolerated even at dosages that far exceed the therapeutic need, indicating a large therapeutic window. These results present NEO212 as an agent that should be considered for development as a therapeutic agent for AML.

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.

Background: The chemotherapeutic standard of care for patients with glioblastoma (GB) is radiation therapy (RT) combined with temozolomide (TMZ). However, during the twenty years since its introduction, this so-called Stupp protocol has revealed major drawbacks, because nearly half of all GBs harbor intrinsic treatment resistance mechanisms. Prime among these are the increased expression of the DNA repair protein O6-guanine-DNA methyltransferase (MGMT) and cellular deficiency in DNA mismatch repair (MMR). Patients with such tumors receive very little, if any, benefit from TMZ. We are developing a novel molecule, NEO212 (TMZ conjugated to NEO100), that harbors the potential to overcome these limitations. Methods: We used mouse models that were orthotopically implanted with GB cell lines or primary, radioresistant human GB stem cells, representing different treatment resistance mechanisms. Animals received NEO212 (or TMZ for comparison) without or with RT. Overall survival was recorded, and histology studies quantified DNA damage, apoptosis, microvessel density, and impact on bone marrow. Results: In all tumor models, replacing TMZ with NEO212 in a schedule designed to mimic the Stupp protocol achieved a strikingly superior extension of survival, especially in TMZ-resistant and RT-resistant models. While NEO212 displayed pronounced radiation-sensitizing, DNA-damaging, pro-apoptotic, and anti-angiogenic effects in tumor tissue, it did not cause bone marrow toxicity. Conclusions: NEO212 is a candidate drug to potentially replace TMZ within the standard Stupp protocol. It has the potential to become the first chemotherapeutic agent to significantly extend overall survival in TMZ-resistant patients when combined with radiation.