The reactive thiol in cysteine is used for coupling maleimide linkers in the generation of antibody conjugates. To assess the impact of the conjugation site, we engineered cysteines into a therapeutic HER2/neu antibody at three sites differing in solvent accessibility and local charge. The highly solvent-accessible site rapidly lost conjugated thiol-reactive linkers in plasma owing to maleimide exchange with reactive thiols in albumin, free cysteine or glutathione. In contrast, a partially accessible site with a positively charged environment promoted hydrolysis of the succinimide ring in the linker, thereby preventing this exchange reaction. The site with partial solvent-accessibility and neutral charge displayed both properties. In a mouse mammary tumor model, the stability and therapeutic activity of the antibody conjugate were affected positively by succinimide ring hydrolysis and negatively by maleimide exchange with thiol-reactive constituents in plasma. Thus, the chemical and structural dynamics of the conjugation site can influence antibody conjugate performance by modulating the stability of the antibody-linker interface.
Glioblastoma (GBM), a WHO grade IV malignant glioma, is the most common and lethal adult primary brain tumor. Median survival rates range from 12-15 months. The current standard of care for GBM has evolved from resection followed by adjuvant radiotherapy to resection, concurrent adjuvant chemotherapy (temozolomide) and radiation, and additional adjuvant chemotherapy. The expression of specific molecular biomarkers, especially O-6-methylguanine methyltransferase (MGMT) status, may determine the response of the tumor to treatment, and helps in identifying the magnitude of benefit from this regimen. By identifying further biological subtypes of GBM at the molecular level, specific targeted therapies could be developed and used in the future for more individualized therapeutic regimens. This article will review the current therapies for GBM and the investigation of new molecular and targeted therapies, such as EGFR inhibitors, mTOR/PI3Kinase inhibitors, and anti-angiogenesis agents.
Purpose To identify potential molecular hubs that regulate oncogenic kinases and target them to improve treatment outcomes for glioblastoma (GBM) patients. Experimental Design Data mining of The Cancer Genome Atlas (TCGA) datasets identified Nicotinamide-N-methyl transferase (NNMT) as a prognostic marker for GBM, an enzyme linked to the reorganization of the methylome. We tested our hypothesis that NNMT plays a crucial role by modulating protein methylation leading to inactivation of tumor suppressors and activation of oncogenes. Further experiments were performed to understand the underlying biochemical mechanisms using GBM patient samples, established, primary, and isogenic cells. Results We demonstrate that NNMT outcompetes leucine carboxyl methyl transferase 1 (LCMT1) for methyl transfer from principal methyl donor SAM in biological systems. Inhibiting NNMT increased the availability of methyl groups for LCMT1 to methylate PP2A, resulting in the inhibition of oncogenic serine/threonine kinases (STKs). Further, NNMT inhibition retained the radiosensitizer nicotinamide and enhanced radiation sensitivity. We have provided the biochemical rationale of how NNMT plays a vital role in inhibiting tumor suppressor PP2A while concomitantly activating STKs. Conclusion We report the intricate novel mechanism in which NNMT inhibits tumor suppressor PP2A by reorganizing the methylome both at epigenome and proteome levels and concomitantly activating pro-survival STKs. In GBM tumors with NNMT expression, activation of PP2A can be accomplished by FDA approved perphenazine (PPZ) which is currently used to treat mood disorders such as schizophrenia, bipolar disorder, etc. This study forms a foundation for further GBM clinical trials using PPZ with standard of care treatment.
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