Mechanism-based design of agents that selectively target drug-resistant glioma

Abstract

Approximately half of glioblastoma and more than two-thirds of grade II and III glioma tumors lack the DNA repair protein O⁶-methylguanine methyl transferase (MGMT). MGMT-deficient tumors respond initially to the DNA methylation agent temozolomide (TMZ) but frequently acquire resistance through loss of the mismatch repair (MMR) pathway. We report the development of agents that overcome this resistance mechanism by inducing MMR-independent cell killing selectively in MGMT-silenced tumors. These agents deposit a dynamic DNA lesion that can be reversed by MGMT but slowly evolves into an interstrand cross-link in MGMT-deficient settings, resulting in MMR-independent cell death with low toxicity in vitro and in vivo. This discovery may lead to new treatments for gliomas and may represent a new paradigm for designing chemotherapeutics that exploit specific DNA repair defects.

Fig. 1.. Overview of mechanistic strategy and structures of agents used in this study.

(A) Underlying mechanistic design. Systemic administration of a bifunctional agent is envisioned to form a primary lesion that is rapidly resolved by healthy (DDR+) but not DDR-deficient (DDR−) cells. The persistence of the primary lesion allows it to evolve slowly to a more toxic secondary lesion. (B) Under physiological conditions, TMZ (1a) converts to MTIC (1b), which in turn decomposes to methyl diazonium (1c).(C) O⁶-Methylguanine (3) is the most clinical significant alkyl nucleobase generated by methyl diazonium (1c) and is rapidly reverted to dG (2) by MGMT. (D) Conversion of KL-50 (4a) to KL-85 (4b) in aqueous solution followed by decomposition to 2-fluoroethyl diazonium ion (4c).(E) 2-fluoroethyl diazonium ion (4c), derived from KL-50 (4a), is proposed to lead to the formation of G(N1)–C(N3)Et ICL 8 specifically in MGMT− cells. This mechanism is independent of MMR status. (F) Structures of the triazenes 9 to 13, mitozolomide 12a, and lomustine (CCNU; 14). Syntheses are available in the supplementary materials.

Fig. 2.. KL-50 (4a) displays previously unseen MGMT-dependent, MMR-independent cytotoxicity in multiple isogenic cell models.

(A) IC₅₀ values derived from short-term viability assays in LN229 MGMT+/−, MMR+/− cells treated with TMZ (1a) derivatives. ^aMGMT TI = IC₅₀ (MGMT+/MMR+) divided by IC₅₀ (MGMT−/MMR+). ^bMMR resistance index (RI) = IC50 (MGMT−/MMR−) divided by IC50 (MGMT−/MMR+). (B) Short-term viability assay curves for TMZ (1a), CCNU (14), KL-85 (4b), and KL-50 (4a) in LN229 MGMT+/−, MMR+/− cells. (C) Clonogenic survival curves for TMZ (1a) in LN229 MGMT+/−, MMR+/− cells, with representative images of wells containing 1000 plated cells treated with 30 μM TMZ (1a).(D) Clonogenic survival curves for KL-50 (4a) in LN229 MGMT+/−, MMR+/− cells, with representative images of wells containing 1000 plated cells treated with 30 μM KL-50 (4a). (E) Short-term viability assay curves for TMZ (1a) in DLD1 MSH6-deficient cells pretreated with 0.01% dimethyl sulfoxide (DMSO) control (CTR) or 10 μM O⁶BG (+O⁶BG) for 1 hour before TMZ (1a) addition to deplete MGMT. (F) Short-term viability assay curves for KL-50 (4a) in DLD1 MSH6-deficient cells pretreated with 0.01% DMSO control (CTR) or 10 μM O⁶BG (+O⁶BG) for 1 hour before KL-50 (4a) addition. (G) Short-term viability assay curves for TMZ (1a) in HCT116 MLH1−/− cells or HCT116 cells complemented with chromosome 3 carrying wild-type MLH1 (+Chr3) pretreated with 0.01% DMSO control or 10 μM O⁶BG (+O⁶BG) for 1 hour before TMZ (1a) addition. (H) Short-term viability assay curves for KL-50 (4a) in HCT116 MLH1−/− cells or HCT116 cells complemented with chromosome 3 carrying wild-type MLH1 (+Chr3) pretreated with 0.01% DMSO control or 10 μM O⁶BG (+O⁶BG) for 1 hour before KL-50 (4a) addition. For (B) to (H), points indicate the mean, and error bars indicate SD; n ≥ 3 technical replicates.

Fig. 3.. Unrepaired primary KL-50 (4a) lesions convert to DNA ICLs in the absence of MGMT.

(A) Scatter dot plots of the percent DNA in tail upon single-cell alkaline gel electrophoresis performed on LN229 MGMT−/MMR+ and MGMT−/MMR− cells treated with 0.2% DMSO control, 200 μM TMZ (1a), 200 μM KL-50 (4a), or 0.1 μM MMC (MMC*) for 24 hours or with 50 μM MMC (MMC**) for 2 hours. After cell lysis, comet slides were irradiated with 0 or 10 grays (Gy) before alkaline electrophoresis. Lines indicate median; error bars indicate 95% confidence interval (CI); n ≥ 160 comets per condition. (B) Representative comet images from (A). (C) Scatter dot plots of the percent DNA in tail upon single-cell alkaline gel electrophoresis performed on LN229 MGMT−/MMR− cells treated with 0.2% DMSO control, 200 μM MTZ (12a), 200 μM TMZ (1a), or 200 μM KL-50 (4a) for 2, 8, or 24 hours. After cell lysis, comet slides were irradiated with 10 Gy before alkaline electrophoresis. Lines indicate median; error bars indicate 95% CI; n ≥ 230 comets per condition. Data from samples treated with 0 Gy are shown in fig. S4, C and D. (D) Representative comet images from (C). (E) Denaturing gel electrophoresis of genomic DNA isolated from LN229 MGMT−/MMR+ cells treated with 0.2% DMSO control, 200 μM KL-50 (4a), 200 μM TMZ (1a), 200 μM KL-85 (4b), or 200 μM MTIC (1b) for 24 hours or with 50 μM MMC or 200 μM CCNU (14) for 2 hours. (F) Denaturing gel electrophoresis of linearized 100 ng pUC19 plasmid DNA treated in vitro with 100 μM cisplatin (36 hours), 100 μM MMS (36 hours), 100 μM of KL-50 (4a), or 12b for 6 to 36 hours. For (E) and (F), bands indicating cross-linked DNA are indicated with arrows. Quantification of bands in (F) is provided in fig. S4E.

Fig. 4.. KL-50 (4a) activates DDR pathways and cycle arrest in MGMT− cells, independent of MMR, and cells deficient in ICL or HR repair are sensitized to KL-50 (4a).

(A) γH2AX, (B) 53BP1, and (C) pRPA foci formation quantified by percent of cells with ≥10 foci in LN229 MGMT+/−, MMR+/− cells treated with 0.1% DMSO control, 20 μM KL-50 (4a), or 20 μM TMZ (1a) for 48 hours. Columns indicate the mean; error bars indicate SD; n > 5 technical replicates. Additional time course data are presented in fig. S6, B to D. (D) Representative foci images of data in (A) to (C). (E) Percentage of cells in G₁, S, and G₂ cell cycle phases after treatment as in (A) to (C), measured by using integrated nuclear (Hoechst) staining intensity. Columns indicate the mean; error bars indicate SD; n = 3 independent analyses. Additional time course data, cell cycle controls, and representative histograms are presented in fig. S7. (F) Change in percent cells with ≥1 micronuclei from baseline (DMSO control) after treatment as in (A) to (C). Columns indicate the mean; error bars indicate SD; n ≥ 15 technical replicates; ****P < 0.0001; ns, not significant. Additional validation is presented in fig. S9, A and B. (G) Short-term viability assay curves for KL-50 (4a) in PD20 cells, deficient in FANCD2 (FANCD2−/−) or complemented with FANCD2 (+FANCD2). (H) Short-term viability assay curves for KL-50 (4a) in PEO4 (BRCA2+) and PEO1 (BRCA2−/−) cells pretreated with 0.01% DMSO control or 10 mM O⁶BG (+O⁶BG) for 1 hour before KL-50 (4a) addition. (I) Short-term viability assay curves for KL-50 (4a) in DLD1 BRCA2+/− and BRCA2−/− cells pretreated with 0.01% DMSO control or 10 mM O⁶BG (+O⁶BG) for 1 hour before KL-50 (4a) addition. For (G) to (I), points indicate the mean, and error bars indicate SD; n = 3 technical replicates.

Fig. 5.. KL-50 (4a) appears to be safe and efficacious in both MGMT−/MMR+ and MGMT−/MMR− flank tumors over a wide range of treatment regimens and conditions.

(A) Xenograft LN229 MGMT−/MMR+ flank tumors treated with three weekly cycles of oral administration of 10% cyclodextrin control (n = 7 mice), TMZ (1a) (n = 7 mice, 5 mg/kg) or KL-50 (4a) (n = 6 mice, 5 mg/kg) on Monday, Wednesday, and Friday (individual spider plots in fig. S10A). (B) Xenograft LN229 MGMT−/MMR− flank tumors treated with three weekly cycles of oral administration of 10% cyclodextrin control (n = 6 mice), TMZ (1a) (n = 5 mice, 5 mg/kg), or KL-50 (4a) (n = 5 mice, 5 mg/kg) on Monday, Wednesday, and Friday (individual spider plots in fig. S10B). (C) Mean body weight of mice during LN229 flank tumor experiments in (A) and (B). (D to F) Xenograft (D) LN229 MGMT−/MMR+ and (E) LN229 MGMT−/MMR− flank tumors treated with oral administration of 10% cyclodextrin control (n = 7 mice), KL-50 (4a) (n = 6 mice, three cycles of 15 mg/kg on Monday, Wednesday, and Friday), KL-50 (4a) (n = 6 mice, 1 cycle of 25 mg/kg Monday through Friday), or intraperitoneal administration of KL-50 (4a) (n = 7 mice, three cycles of 5 mg/kg on Monday, Wednesday, and Friday) revealed equal efficacy with no observable increases in toxicity, (F) as measured with mice systemic weights (individual spider plots are provided in fig. S10C). (G) Xenograft LN229 MGMT−/MMR+ and LN229 MGMT−/MSH6− flank tumors with a larger average starting tumor size of ~400 mm³ and ~350 mm³, respectively, treated with three weekly cycles of oral administration of 10% cyclodextrin (n = 4 mice) or KL-50 (4a) (n = 4 mice, 25 mg/kg on Monday, Wednesday, and Friday). The study period was limited by control groups that had to be euthanized for exceeding the ethical maximum allowed tumor size, thus ending the study. In (A) to (G), points indicate the mean, and error bars indicate SEM; *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.

Fig. 6.. KL-50 (4a) is efficacious in an LN229 MGMT−/MMR− intracranial model and is well tolerated with limited myelosuppression at supratherapeutic doses.

(A) Mean tumor size as measured with bioluminescent imaging as relative light units (RLU; photons/s) with SEM of xenograft LN229 MGMT−/MMR− intracranial tumors treated with 5 consecutive days of oral administration of 10% cyclodextrin control (n = 10 mice), TMZ (1a) (n = 11 mice, 25 mg/kg), or KL-50 (4a) (n = 11 mice, 25 mg/kg) (individual spider plots are provided in fig. S10D). (B) Kaplan-Meier analysis of intracranial xenograft tumor-bearing mice in (A), treated with 5 consecutive days of oral administration of 10% cyclodextrin control (n = 10 mice), TMZ (1a) (n = 11 mice, 25 mg/kg), or KL-50 (4a) (n = 11 mice, 25 mg/kg) demonstrating a significant survival benefit for KL-50 (4a) compared with both control and TMZ (1a) groups. (C) Mean body weight change with SEM of mice during maximum tolerated dose experiment in non-tumor-bearing mice. (D) Complete blood counts for mice before treatment and 7 days after treatment with escalations of single-dose KL-50 (4a) delivered orally. White blood cells (WBC) lower limit of normal (LLN), 2.2 k/μL; neutrophil LLN, 0.42 k/μl; lymphocyte LLN, 1.7 k/μl; red blood cells (RBC) LLN, 3.47 M/μl; platelet LLN, 155 k/μl. *P < 0.05; ****P < 0.0001; n.s.; not significant.