Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Jun 11;6(1):vdae095.
doi: 10.1093/noajnl/vdae095. eCollection 2024 Jan-Dec.

NEO212, temozolomide conjugated to NEO100, exerts superior therapeutic activity over temozolomide in preclinical chemoradiation models of glioblastoma

Radu O Minea  1   2 , Thu Zan Thein  1 , Zhuoyue Yang  3 , Mihaela Campan  4 , Pamela M Ward  5 , Axel H Schönthal  3 , Thomas C Chen  6   1   5   2
Affiliations

NEO212, temozolomide conjugated to NEO100, exerts superior therapeutic activity over temozolomide in preclinical chemoradiation models of glioblastoma

Radu O Minea et al. Neurooncol Adv. .

Abstract

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.

Keywords: O6-guanine-DNA methyltransferase; chemoradiation; mismatch repair deficiency; radiosensitization; temozolomide.

PubMed Disclaimer

Conflict of interest statement

T.C.C. is founder and stakeholder of NeOnc Technologies, Los Angeles, California, USA. All other authors declare no conflict of interest.

Figures

Figure 1.
Figure 1.
The GB cell models used in this study display different mechanisms of resistance to TMZ. (A) Cell lysates were analyzed by Western blot to determine MGMT protein expression in the various cell models used in this study. T98G cells were included as a positive control and LN229 cells as a negative control; actin was used as the loading control. (B) MSH2 and MSH6 are proteins necessary for MMR function. Western blot comparison of parental LN229 and derived LN229TR2 cells reveals very low MSH2 levels, and near absence of MSH6 protein, indicating MMR deficiency. (C) Analysis of microsatellite instability (MSI) shows evidence of instability in LN229TR2 cells, with novel alleles displayed in 2/8 markers (NR-21 and BAT60), which is yet another indication of MMR deficiency.
Figure 2.
Figure 2.
NEO212 synergizes with radiation therapy and prolongs survival of multiple GB models. Kaplan–Meier survival plots were generated to display animal survival in response to various treatments. Groups of 5 mice were treated with NEO212 (25 mg/kg/day) or TMZ (25 mg/kg/day) alone or in combination with radiation therapy (2 Gy/day). Control groups of mice received vehicle only. (A) The USC04 primary GB stem cell model represents the “baseline” model, as it lacks obvious treatment resistance mechanisms. (B) The U251M model expresses high levels of exogenous MGMT and therefore represents a TMZ-resistant model. (C) The LN229TR model represents a model where TMZ resistance is based on MMR deficiency. (D) The USC02 primary GB stem cell model represents a strongly treatment-resistant model, based on expression of endogenous MGMT, along with relative radioresistance. The log-rank (Mantel-Cox) test was used for statistical comparisons. ns: not significant. Some graphical elements used for this figure were imported from BioRender.com.
Figure 3.
Figure 3.
Chemoradiation with NEO212 causes profound DNA damage and apoptosis in tumor tissue but spares normal brain tissue. Mice harboring USC02 tumors were subjected to the same treatment regimen as described in the legend in Figure 2. At the end of the 10-day treatment period, brains were collected and separated into the ipsilateral hemisphere (containing the tumor) and contralateral (tumor-free) hemisphere. Brains were sectioned and analyzed. (A) Sections were stained with AF647-labeled γH2AX antibody to detect DNA damage, and counterstained with DAPI to visualize cell nuclei. Green fluorescence identifies tumor cells, which express a green fluorescent protein (GFP). Scale bars are 100 μm. (B) The graph presents the quantification of DNA damage staining in tumor tissue and tumor-free normal brain tissue. This was done in 30 noncontiguous fields per treatment group with each data point representing the average for one field. (C) Sections were subjected to TUNEL staining to reveal apoptotic cells, and counterstained with DAPI. Scale bars are 200 µm. (D) Quantitative representation of apoptosis in tumor tissue. ns: not significant; 3 asterisks (***): P < .001.
Figure 4.
Figure 4.
Chemoradiation with NEO212 causes anti-angiogenic effects. Mice harboring USC02 tumors were subjected to the same treatment regimen as described in the legend in Figure 2. At the end of the 10-day treatment period, brain tumors were collected, and sections were analyzed for microvessel density (MVD). (A) Sections were stained with AF647-conjugated CD31 antibody to visualize endothelial cells and z-stacks are shown to illustrate the 3-D microvessel structures. DAPI was used as the counterstain, and the presence of green fluorescent protein (GFP) was used to identify tumor cells. Scale bars are 200 µM. (B) Chart showing the quantitative analysis of CD31 staining. This was done in 30 noncontiguous z-stacks per treatment group with each data point representing the average for one z-stack. ns: not significant; one asterisk (*): P < .05.
Figure 5.
Figure 5.
Chemoradiation with NEO212 does not cause bone marrow toxicity. Mice harboring USC02 tumors were subjected to the same treatment regimen as described in the legend in Figure 2. At the end of the 10-day treatment period, bone marrow and blood were collected and analyzed for signs of toxicity. (A) Bone marrow was subjected to H&E staining (to show general cellularity), along with immunohistological analysis with AF647-conjugated γH2AX antibody (to reveal DNA damage) and AF555-conjugated CD45 antibody (to identify hematopoietic cells). DAPI was used as the counterstain. Scale bars are 50 µm for the immunostains and 100 µm for the H&E stains. (B) Quantitative analysis of γH2AX staining. (C) Quantitative analysis of CD45+ cell counts. (D) Number of white blood cells (WBC) in the peripheral blood. (E) Number of red blood cells (RBC). ns: not significant.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Stupp R, Mason WP, van den Bent MJ, et al.; European Organisation for Research and Treatment of Cancer Brain Tumor and Radiotherapy Groups. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med. 2005;352(10):987–996. - PubMed
    1. Adegboyega G, Kanmounye US, Petrinic T, et al.; InciSion UK Collaborative. Global landscape of glioblastoma Multiforme management in the Stupp protocol era: Systematic review protocol. Int J Surg Protoc. 2021;25(1):108–113. - PMC - PubMed
    1. McMahon DJ, Gleeson JP, O’Reilly S, Bambury RM.. Management of newly diagnosed glioblastoma multiforme: Current state of the art and emerging therapeutic approaches. Med Oncol. 2022;39(9):129. - PubMed
    1. Hegi ME, Diserens AC, Gorlia T, et al.. MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med. 2005;352(10):997–1003. - PubMed
    1. Kaina B. Temozolomide, Procarbazine and Nitrosoureas in the therapy of malignant gliomas: Update of mechanisms, drug resistance and therapeutic implications. J Clin Med. 2023;12(23):7442. - PMC - PubMed

LinkOut - more resources