Approximately half of glioblastoma and more than two-thirds of grade II and III glioma tumors lack the DNA repair protein O 6 -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.
The chemoselective functionalization of polyfunctional aryl linchpins is crucial for rapid diversification. Although well-explored for Csp2 and Csp nucleophiles, the chemoselective introduction of Csp3 groups remains notoriously difficult and is virtually undocumented using Ni catalysts. To fill this methodological gap, a “haloselective” cross-coupling process of arenes bearing two halogens, I and Br, using ammonium alkylbis(catecholato)silicates, has been developed. Utilizing Ni/photoredox dual catalysis, Csp3–Csp2 bonds can be forged selectively at the iodine-bearing carbon of bromo(iodo)arenes. The described high-yielding, base-free strategy accommodates various protic functional groups. Selective electrophile activation enables installation of a second Csp3 center and can be done without the need for purification of the intermediate monoalkylated product.
Graphical Abstract Procedure A. Cyclohexyltrimethoxysilane (2)A 250 mL, oven-dried, two-necked, round-bottomed flask is charged with a 3.2 cm, Tefloncoated magnetic oval stir bar and coupled with a 50 mL dropping funnel. Both the dropping funnel and the round-bottomed flask are sealed with a rubber septum. The system is evacuated for 10 min and back-filled with nitrogen. This process is repeated twice more, and then, the flask is charged by syringe with pentane (180 mL), anhydrous pyridine (21.0 mL, 20.5 g, 260 mmol, 4 equiv), and anhydrous methanol (10.5 mL, 8.3 g, 260 mmol, 4 equiv) (Notes 1-3).Additionally, a solution of cyclohexyltrichlorosilane 1 (14.14 g, 65.0 mmol, 1.0 equiv) in pentane (37 mL) is prepared in an addition funnel. (Figure 1, left) The stirred, homogeneous solution (Note 4) is cooled to 0 °C (external temperature) via an ice-water bath while under a nitrogen atmosphere (Figure 1). After cooling for 10 min the solution in the dropping funnel is added dropwise to the flask over 35 min (Notes 5,6,and 7). A voluminous white 1 Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, 231 S. 34th Street, Philadelphia, PA 19104-6323. 2 Pyridine was purchased from Fisher scientific (Certified ACS grade, ≥99%) and stored over KOH pellets.1 The following reagents in this section were purchased from commercial sources and used without further purification: Pentane (≥99%, Acros Organics) and methanol (extra dry over 4 Å molecular sieves, 99.8%, Acros Organics). 3 Although pentane is used in this protocol, a number of other solvents can be used (e.g., n-hexane, n-heptane, diethyl ether, tetrahydrofuran) with similar results.. For simple aliphatic alkyltrichlorosilanes hydrocarbon solvents are favored because of ease of product isolation. 4 The solution is stirred at 500 rpm throughout the reaction. 5 The solution in the funnel has a milky color. Figure 1, right) forms upon addition of 1. Following complete addition of 1, the reaction mixture is stirred at 0 °C for 5 min. The ice bath is removed, and the heterogeneous solution is stirred for 3 h at room temperature. HHS Public AccessUpon confirmation that the reaction is complete by crude 1 H NMR, the stirring is stopped, and the solids are allowed to settle (Figure 2) (Note 8). The reaction mixture is decanted from the solid pyridinium hydrochloride and transferred to a 1000 mL separatory funnel (Note 9). The white pyridinium salt is washed with pentane (100 mL) to assist in transferring all of the product to the funnel. The separatory funnel is charged with deionized H 2 O (250 mL). The layers are separated, and the aqueous layer is extracted with pentane (2 × 125 mL). The combined organic layers are washed with 2 M aqueous HCl (2 × 100 mL), saturated aqueous NaHCO 3 (150 mL), deionized water (150 mL), and then saturated aqueous NaCl (150 mL). The organic layer is then dried over sodium sulfate (25 g), and after filtration, the solvent is removed in vacuo by rotary evaporation (Note 10) to furnish pure 2 (12.49 g, 94%)...
Glioblastoma (GBM) is a lethal brain cancer with a five-year survival rate of <5%. Approximately half of GBM tumors lack the DNA repair protein O6-methylguanine DNA methyltransferase (MGMT), which reverses O6-alkylguanine (O6G) lesions. Patients presenting MGMT– GBM are treated with surgery followed by radiation therapy and temozolomide (TMZ), an imidazotetrazine prodrug that produces O6-methylguanine (O6MeG) lesions. However, ~50% of these patients will develop TMZ resistance by silencing of the DNA mismatch repair (MMR) pathway. We recently reported that the novel N3-(2-fluoroethyl)imidazotetrazine “KL-50” is efficacious and well-tolerated in murine models of TMZ-resistant GBM (Lin et al. Science 2022, 377, 502). Herein, we rigorously establish that KL-50 generates DNA interstrand crosslinks (ICLs) by DNA alkylation to generate O6-(2-fluoroethyl)guanine (O6FEtG), displacement of fluoride to form an N1,O6-ethanoguanine (N1,O6EtG) intermediate, and ring-opening by the adjacent cytidine. 2-Chloroethylating agents, such as lomustine and mitozolomide (MTZ), generate the same ICL by an analogous mechanism. However, DNA ICLs form >10-fold more slowly from O6FEtG than O6ClEtG, and this slower rate of cross-linking allows MGMT to reverse the initial O6FEtG in healthy tissue while also reducing MGMT–DNA cross-links arising from addition of MGMT to the N1,O6EtG intermediate. KL- 50 is efficacious in an intracranial patient-derived murine xenograft of TMZ-resistant, MGMT–/MMR– GBM (mOS = 205, 28, and 26 d for KL-50, TMZ, and vehicle-treated control, respectively) and in murine models of newly-diagnosed MGMT–/MMR+ GBM, suggesting its use in recurrent and up-front settings, respectively. These studies underscore the significance of considering the rates of chemical DNA modification and biochemical DNA repair in the design of systemic DNA alkylation agents.
DNA damage response (DDR) defects are common in cancer, and it is well established that these tumor-associated DNA repair vulnerabilities can be exploited for a therapeutic gain. Small molecule inhibitors of DDR proteins have been developed to target these cancers, which are now FDA-approved and/or currently being tested in clinical trials. Our team recently reported on an entirely new approach to exploit DDR defects in cancer, via DNA modification, eliminating the need to target proteins (Lin et al., Science 2022). In this recent study, we demonstrated that our new class of “DNA modifiers (DMs)” can be engineered to exploit loss of O6-methylguanine methyltransferase (MGMT), which leads to robust and selective killing of MGMT-deficient glioma tumors, both in vivo and in vitro. We also found that our approach could overcome acquired and intrinsic defects in another DDR pathway, mismatch repair (MMR), which is a known resistance mechanism to standard-of-care cytotoxic therapies in glioma, including TMZ. Here, we sought to determine the extent to which our approach can be expanded to selectively target a wide range of cancers beyond glioma in which loss of MGMT, as well as acquired and intrinsic MMR defect, are commonly seen at various frequencies. To this end, we profiled over 900 cell line models, as well as several collections of patient-derived cultures derived from several tumor types for sensitivity to our DMs based on MGMT and MMR status in vitro. We then validated our findings in cell line xenograft and patient-derived xenograft models, in vivo. In parallel, we compared the activity of DMs to commonly used, standard-of-care cytotoxic drugs, in vitro and in vivo. Our data indicate that DMs can effectively target MGMT loss, as well as intrinsic and acquired MMR defects, in multiple non-CNS tumor types. Taken together, the findings reported here support an entirely new approach to selectively exploit DDR defects via direct DNA modification. This new approach has the potential to overcome known resistance mechanisms to currently used small molecule inhibitors of DDR proteins, across a broad range of cancers. Citation Format: Bruce Ruggeri, Kyle Tarantino, Ranjini Sundaram, Kingson Lin, Seth Herzon, Joseph Park, Spenser Johnson, Susan Gueble, Ranjit S. Bindra. Exploiting MGMT loss using a new class of DNA modifiers which selectively target tumor DNA and overcome therapy resistance mechanisms across multiple tumor types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1132.
The DNA damage repair protein O6-methylguanine methyl transferase (MGMT) reverses O6-alkylguanine lesions via SN2 transfer of the alkyl lesion to an active site cysteine which restores DNA to its native state. MGMT is ubiquitously expressed in healthy tissue but is silenced by promoter hypermethylation in approximately half of glioblastomas and up to 75% of lower grade gliomas. As such, patients with MGMT deficient (MGMT-) tumors benefit from DNA alkylation agents such as temozolomide (TMZ), an imidazotetrazine that deposits methyl lesions at O6-guanine. However, the cytotoxicity of TMZ requires an intact DNA mismatch repair (MMR) pathway and acquired resistance to TMZ via MMR silencing negates the efficacy of the drug. We recently reported the synthesis, discovery, and in vivo evaluation of KL-50, an imidazotetrazine derivative that overcomes this resistance mechanism while maintaining high selectivity for MGMT- cells in vitro and in vivo. Our preliminary mechanistic data suggest that KL-50 induces cell death independent of MMR status via the deposition of 2-fluoroethyl lesions at O6-guanine to give an O6-(2-fluoroethyl)guanine (O6FEtG) lesion that evolves to a deadly DNA interstrand cross-link (ICL). We hypothesize that this occurs via a two-step pathway involving slow cyclization of the initial O6FEtG lesion to an electrophilic N1,O6-ethanoguanine intermediate and subsequent nucleophilic ring-opening by the base-paired cytidine residue to yield a guanine(N1)-cytidine(N3) ethyl ICL. Our data suggest the high therapeutic index of KL-50 derives from the relative rates of MGMT reversal of O6FEtG and its conversion to an ICL, specifically that MGMT proficient healthy cells can repair the O6FEtG before it evolves to the ICL. However, direct MGMT reversal of O6FEtG has not been reported, and the rates of ICL formation are not known. Additionally, the formation of the putative ICL has not been demonstrated. Herein, we demonstrate that MGMT can repair the O6FEtG lesion. Kinetics studies using well-defined, photocaged oligonucleotides containing a single O6FEtG lesion show that ICL formation occurs on the order of hours. Furthermore, addition of MGMT following photo-deprotection prevents the formation of ICLs via rapid reversal of O6FEtG. Finally, we have detected a mass corresponding to the putative ICL in pUC19 DNA treated with KL-50 via LCMS . These findings bolster our mechanistic model and suggest strategies that exploit the relative rates of DNA damage and repair may be extensible to other tumor types harboring specific DNA repair defects. Citation Format: Eric D. Huseman, Anna Lo, Olga Fedorova, Kingson Lin, Susan Gueble, Ranjini Sundaram, Anna M. Pyle, Ranjit S. Bindra, Seth Herzon. Mechanistic studies of KL-50, a novel imidazotetrazine for the treatment of MGMT-/MMR- gliomas and glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1568.
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