Tumor-targeted inhibition of the DNA repair protein MGMT for enhanced chemotherapy overcoming tumor resistance and reducing systemic toxicity

DNA修复蛋白O6-烷基鸟嘌呤-DNA烷基转移酶(MGMT)是肿瘤细胞对O6烷基化抗癌药物替莫唑胺(TMZ)、前卡巴津(Procarbazin)、达卡巴津(DTIC)、尼莫司汀(Nimustine)、司马汀(Semustine)、卡莫司汀和洛莫司汀(Lomustine，CCNU)耐药的主要因素。烷化剂对表达MGMT的细胞的细胞毒作用被MGMT抑制剂预处理后显著增强，MGMT抑制剂具有很高的选择性，本身没有毒性。然而，在临床应用中，MGMT抑制剂迄今未能产生预期的治疗效果，原因是它们抑制正常组织和肿瘤组织中的MGMT，从而导致全身增敏，这反过来又需要减少抗癌药物的剂量。到目前为止，用于临床试验的MGMT抑制剂是O6-苄基鸟嘌呤(O6-BG)和O6-(4-溴乙烯基)鸟嘌呤(O6-BTG，lomgutrib)。由于这些抑制剂增加了烷化剂的骨髓毒性，我们需要设计一种MGMT的肿瘤靶向策略。一种成功的肿瘤靶向MGMT抑制剂的策略对其他抑制修复的物质也具有重要意义，并可以在不增加全身毒性的情况下促进选择性地降低化疗耐药性。葡萄糖衍生物正被用于肿瘤成像，这是因为它们在高葡萄糖消耗的细胞(大多数肿瘤细胞)中积累。申请人打算遵循类似的策略，将MGMT抑制剂O6-BG和O6-BTG偶联到？-葡萄糖(偶联物是O6-BG-Glu和O6-BTG-Glu)，以便用这些抑制剂丰富肿瘤细胞，并主要在肿瘤中抑制MGMT。申请人之前所做的工作表明，葡萄糖结合物有效地抑制MGMT，无毒，并使肿瘤细胞对烷基化剂敏感；同时，研究表明，肿瘤细胞系可以在很大程度上增敏。然而，目前尚不清楚转运蛋白的身份，它在生物体内储存和分配葡萄糖结合物，我们也不知道与正常组织相比肿瘤的反应。本后续研究旨在明确地回答以下问题：1)确定O6-BG-Glu和O6-BTG-Glu的转运体(进出)；2)是否有可能增强TMZ和CCNU在小鼠移植瘤模型中的抗肿瘤作用；3)提供TMZ和CCNU通过应用葡萄糖抑制剂降低全身毒性的证据，并与非结合抑制剂进行比较；4)测定O6-BG-Glu和O6-BTG-Glu在移植小鼠体内的分布；5)一旦确定转运体，确定其在肿瘤细胞系和组织/肿瘤中的表达，以确定那些对Glu-MGMT靶向抑制MGMT特别敏感的肿瘤组。

The DNA repair protein O6-alkylguanine-DNA alkyltransferase (MGMT) is the main factor in the resistance of tumor cells to the cytotoxic effects of O6-alkylating anticancer drugs such as temozolomide (TMZ), procarbazin, dacarbazin (DTIC), nimustine, semustine, carmustine and lomustine (CCNU). The cytotoxic effect alkylating agents on cells which express MGMT is drastically enhanced by pretreatment with MGMT inhibitors, which are quite selective without being toxic themselves. Clinically applied, however, MGMT inhibitors have thus far failed to produce the expected therapeutic results, the reason being that they inhibit MGMT in normal tissue as well as tumor tissue and thus lead to a general sensitization, which in turn requires a dose-reduction of anticancer drugs. MGMT inhibitors, which have thus far been used in clinical trials, are O6-benzylguanine (O6-BG) and O6-(4-bromothenyl)guanine (O6-BTG, lomeguatrib). Since these inhibitors increase the bone marrow toxicity of alkylating agents, we need to devise a tumor-targeting strategy of MGMT. A successful strategy for tumor-targeting of an MGMT inhibitor would also be significant for other substances that inhibit repair and could facilitate a selective reduction of chemo-resistance without adding to the systemic toxicity. Glucose derivatives are being used for tumor-imaging, facilitated by the fact that they accumulate in cells with high glucose consumption (most tumor cells). The applicants intend to follow a similar strategy by conjugating the MGMT inhibitors O6-BG and O6-BTG to ß-glucose (the conjugates are O6-BG-Glu and O6-BTG-Glu), in order to enrich tumor cells with these inhibitors and inhibit MGMT primarily in tumors. Previous work done by the applicant shows that glucose conjugates efficiently inhibit MGMT, are not toxic, and sensitise tumor cells to alkylating agents; at the same time it was shown that tumor cell lines can be sensitised to a large degree. Still unknown, however, is the identity of the transporter, which deposits and distributes the glucose conjugates within the organism, nor do we know the tumor response compared to normal tissue. The present follow-on project aims to decisively answer the following questions: 1) identify the transporters (in- and efflux) for O6-BG-Glu and O6-BTG-Glu; 2) is it possible to increase the anti-tumor-effect of TMZ and CCNU in a mouse xenograft model; 3) provide proof of the reduced systemic toxicity of TMZ and CCNU by applying glucose inhibitors and compare it with the non-conjugated inhibitors; 4) determine the distribution of O6-BG-Glu and O6-BTG-Glu within the organs of xeno-transplanted mouse; 5) once the transporter has been identified, determine the expression in tumor cell lines and tissue/tumors in order to identify those tumor groups, which are particularly susceptible to MGMT inhibition by Glu-MGMT-targeting.