Breakthrough Study on KL-50 in Treating Recurrent Glioblastoma

Background

Glioblastoma (GBM) is a highly malignant brain tumor, particularly in patients with IDH wild-type (IDHwt), who have a very poor prognosis. Although the current standard treatment includes surgical resection, radiotherapy, and Temozolomide (TMZ) chemotherapy, most patients eventually experience recurrence. A key mechanism of recurrence is the acquisition of mutations in DNA mismatch repair (MMR) enzyme genes, leading to resistance to TMZ. TMZ’s efficacy depends on the function of MMR enzymes, so tumor cells with MMR defects become insensitive to TMZ. Addressing this clinical challenge, the development of new treatment strategies is urgently needed.

KL-50 is a novel DNA cross-linking agent specifically designed to induce DNA interstrand cross-links (ICLs) in cells lacking O-6-methylguanine-DNA methyltransferase (MGMT), leading to double-strand breaks (DSBs) independent of the MMR pathway. Previous studies have shown that KL-50 has significant anti-tumor effects in MMR-deficient glioma cells both in vitro and in vivo. However, its activity in patient-derived recurrent GBM models remains unclear. This study aims to evaluate the potential of KL-50 in treating newly diagnosed and recurrent MMR-deficient GBM.

Source of the Study

This study was conducted by Matthew McCord, Thomas Sears, Wenxia Wang, and other co-authors from institutions such as Northwestern University and the Mayo Clinic. The research was published on December 10, 2024, in Neuro-Oncology, a leading journal in the field of neuro-oncology.

Research Process and Results

1. Generation of MMR-Deficient GBM Patient-Derived Xenograft (PDX) Models

Researchers first constructed MMR-deficient GBM PDX models by repeatedly exposing them to TMZ. The specific process is as follows: - Tumor Cell Culture: IDHwt, EGFRvIII-amplified GBM6 and EGFR-amplified GBM12 tumor tissues were obtained from the Mayo Clinic PDX repository and cultured in serum-free media. - Intracranial Transplantation in Mice: 100,000 tumor cells were injected into the right frontal lobe of 8-10-week-old NOD-SCID-Gamma (NSG) mice. - TMZ Treatment: 27 days after tumor transplantation, mice received a week-long TMZ treatment (2.5 mg/kg, intraperitoneal injection, 5 days a week), followed by a week of rest, and then two additional cycles of the same TMZ treatment. - Tumor Recurrence and Passage: After treatment, mice were monitored for tumor recurrence and euthanized upon symptom onset. Fresh brain tissue was harvested, homogenized, mixed with Matrigel, and injected into the flanks of a new cohort of NSG mice for propagation and expansion.

Through this process, researchers successfully constructed the MMR-deficient GBM6R-M185 model and validated the absence of MMR proteins (MSH6, MSH2, MLH1, PMS2) via Western blot.

2. Evaluation of KL-50 Activity in TMZ-Naïve and Recurrent MMR-Deficient GBM PDX Models

Researchers evaluated the efficacy of KL-50 in TMZ-naïve and recurrent MMR-deficient GBM PDX models: - TMZ-Naïve GBM6 and GBM12 Models: KL-50 significantly extended the median survival of mice, from 32.5 days to 57 days in the GBM6 model and from 33 days to 71 days in the GBM12 model. Additionally, low-dose radiotherapy (4 Gy) further enhanced the efficacy of KL-50, extending the median survival in the GBM12 model to 80 days. - Recurrent MMR-Deficient GBM6R-M185 Model: KL-50 significantly extended the median survival of mice (140 days vs. 37 days) and outperformed TMZ (108 days).

3. In Vitro Activity of KL-50 in MMR-Deficient GBM Cells

Researchers constructed MSH6 knockout (KO) GBM6 and GBM12 cell lines using CRISPR technology and evaluated the in vitro activity of KL-50: - TMZ Resistance: MSH6 KO significantly increased TMZ resistance in GBM6 and GBM12 cells, with IC50 values increasing by 4.9-fold and 11.9-fold, respectively. - KL-50 Activity: Unlike TMZ, KL-50 remained highly effective in MSH6 KO cells, with IC50 values decreasing by 10-80%. Additionally, KL-50 induced more DNA double-strand break markers (p-H2A.X and p-P95) in MSH6 KO cells.

4. Synergistic Effect of KL-50 and Radiotherapy

In the GBM12 model, low-dose radiotherapy (4 Gy) significantly enhanced the efficacy of KL-50, further extending the median survival of mice. This result suggests that KL-50 and radiotherapy have a synergistic effect, potentially offering a new combination strategy for clinical treatment.

Conclusion and Significance

This study demonstrates that KL-50 has significant anti-tumor activity in treating newly diagnosed and recurrent MMR-deficient GBM, particularly in MGMT-deficient tumors. KL-50 not only overcomes TMZ resistance but also synergizes with radiotherapy to further extend survival. These findings provide important experimental evidence for developing new GBM treatment strategies and support the advancement of KL-50 into clinical trials for recurrent GBM patients.

Research Highlights

1. Breakthrough Treatment Strategy: KL-50 overcomes TMZ resistance by inducing DNA interstrand cross-links, offering a new treatment option for recurrent GBM patients.
2. Enhanced Efficacy in MMR Deficiency: Unlike TMZ, KL-50 remains highly effective in MMR-deficient tumor cells and may even enhance its anti-tumor activity.
3. Synergistic Effect with Radiotherapy: The synergistic effect of KL-50 and low-dose radiotherapy provides a new combination strategy for clinical treatment, potentially improving efficacy further.

Additional Valuable Information

The success of this study was supported by ModifiBio, which provided the KL-50 compound and funded the experiments. Additionally, the animal care team at Northwestern University played a crucial role in ensuring the smooth progress of the experiments.