Chloroethylnitrosourea analogue HJ03 overcomes temozolomide resistance of glioblastoma in mice through induction of ferroptosis and apoptosis

Surgery followed by adjuvant chemoradiation is the standard treatment for glioblastoma multiforme (GBM). Temozolomide (TMZ) is the only first-line chemotherapeutic drug approved by the US Food and Drug Administration for the treatment of GBM. Acquired chemoresistance to TMZ is the primary cause of treatment failure, resulting in recurrence and a poor prognosis. O6-methylguanine DNA methyltransferase (MGMT) overexpression and loss of function mutations targeting mismatch repair (MMR) are the major mechanisms TMZ-resistance found in GBM cells. In this study we developed a new alkylating agent that could overcome TMZ resistance. We designed a new chloroethylnitrosourea analog HJ03, and demonstrated that HJ03 was more potent than TMZ in inhibiting GBM cell lines U251 (MGMT⁻, MMR⁺), U87 (MGMT⁻, MMR⁻) and T98G (MGMT⁺, MMR⁺) as well as colorectal cancer cell line HCT116 (MGMT⁺, MMR⁻) with MGMT overexpression or MMR dysfunction. Furthermore, HJ03 exerted a dual synergistic attack effect by inducing DNA damage and ferroptosis in U251, U87 and T98G cells. We showed that HJ03 caused U251 and T98G cells to be arrested in G2/M phase and undergo apoptosis by inducing the formation of DNA adducts and interstrand crosslinks. In U251, U87 and T98G cells, HJ03 promoted extensive ferroptosis by upregulating p53 and ATF3 expression while downregulating SLC7A11, leading to intracellular accumulation of ROS and Fe²⁺ along with increased MDA levels. Pharmacokinetic study showed that HJ03 crossed the blood-brain barrier more efficiently than TMZ and exhibited lower levels of bone marrow toxicity. In model mice bearing orthotopic CT2A GBM tumors, administration of HJ03 (20 mg/kg, i.g. 5 consecutive days per week for 3 weeks) significantly prolonged the lifespan. Furthermore, HJ03 synergized with radiotherapy and an anti-PD-1 monoclonal antibody to dramatically prolong mouse survival. Thus, HJ03 emerges as a promising novel candidate for GBM treatment, particularly for patients with TMZ-resistant tumors.