DNA REPAIR IN PROTECTING AGAINST SECONDARY LEUKEMIAS

DNA修复蛋白，O 6-烷基鸟嘌呤-DNA烷基转移酶（AGT）已被证明可以保护细胞免受烷化剂（包括氯乙基亚硝基脲，甲基化剂和环磷酰胺）的毒性作用。 大多数原发性肿瘤具有高AGT活性，这是这些药物的有效性有限的主要原因。 严重骨髓抑制和治疗诱导的髓性白血病是临床使用烷化剂时遇到的危及生命的副作用。AGT蛋白在人造血祖细胞中的活性非常低，因此有理由认为这是其对烷化剂的骨髓毒性和致突变作用高度敏感的基础。 为了提高烷基亚硝基脲的治疗效果，我们开发了O 6-苄基鸟嘌呤（BG）。 该化合物是AGT修复蛋白的有效、选择性灭活剂，目前作为烷化剂的调节剂处于I/II期临床试验中。 虽然BG与烷化剂联合使用可增加对多种肿瘤细胞的杀伤，但主要问题是BG会损害未成熟造血细胞修复烷化剂诱导的损伤的能力，进而导致继发性白血病发病率增加。 为了支持这一点，我们的BG和BCNU I期试验的初步数据表明骨髓抑制是剂量限制性毒性。 本提案的主题是确定BG是否会增加可能导致继发性白血病诱导的烷化剂的致突变性，更重要的是，利用此信息制定治疗方案，以防止这种破坏性副作用。 具体目标1将使用体外和体内模型研究环磷酰胺、BCNU和替莫唑胺抑制AGT活性是否导致Hprt基因座突变频率增加。 我们将确定表达BG耐药AGT蛋白的细胞是否会受到BG加烷化剂诱导突变的保护。具体目标2将直接解决BG与烷化剂联合使用是否会增加治疗相关白血病发生率的问题。 杂合Nf 1基因敲除小鼠提供了一种极好的环磷酰胺诱导的髓性白血病体内模型。 具体目标3和4描述了对抗这种毁灭性副作用的方法。 我们将产生过表达突变AGT的转基因小鼠品系，与Nf 1小鼠杂交，以确定是否有预防白血病发病的保护作用。 在最后一个具体目标中，我们提出将突变的烷基转移酶基因引入造血干细胞。 引入含有突变agt基因的干细胞和高剂量烷化剂化疗将使我们能够降低肿瘤负荷，改善无病生存期，降低继发性白血病的可能性。

The DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT) has been shown to protect cells from the toxic effects of alkylating agents including chloroethylnitrosoureas, methylating agents and cyclophosphamide. Most primary tumors have high AGT activity which is primarily responsible for the limited usefulness of these drugs. Severe myelosuppression and therapy-induced myeloid leukemia are life-threatening side effects encountered with the clinical use of alkylating agents. The activity of the AGT protein in human hematopoietic progenitors is very low and it is reasonable to suggest that this is the basis of their high sensitivity to the myelotoxic and mutagenic effects of alkylating agents. In efforts to increase the therapeutic efficacy of alkylnitrosoureas, we have developed O6-benzylguanine (BG). This compound is a potent, selective inactivator of the AGT repair protein that is presently in phase I/II clinical trials as a modulator of alkylating agents. While combining BG with alkylating agents increases killing of a variety of tumor cells, a major concern is that BG will impair the ability of immature hematopoietic cells to repair alkylator-induced damage and that this will, in turn, result in an increase in the incidence of secondary leukemias. In support of this, preliminary data from our phase I trial of BG and BCNU indicates myelosuppression as the dose limiting toxicity. The subject of this proposal is to determine whether BG increases the mutagenicity of alkylating agents which may contribute to the induction of secondary leukemias and more importantly, to use this information to generate therapies to protect against this devastating side effect. Specific aim 1 will address whether inhibition AGT activity results in an increase in mutation frequency at the Hprt locus by cyclophosphamide, BCNU and temozolomide using an in vitro and in vivo model. We will establish whether cells expressing BG-resistant AGT proteins will be protected from BG plus alkylating agent-induced mutations. Specific aim 2 will directly address the question of whether the use of BG with alkylating agents will increase the incidence of therapy related leukemias. Heterozygous Nf1 knockout mice provide an excellent in vivo model of cyclophosphamide-induced myeloid leukemia. Specific aim 3 and 4 describe approaches to combat this devastating side effect. We will generate lines of transgenic mice that over-express mutant AGT to cross with Nf1 mice to determine if there is protection against the incidence of leukemias. In the last specific aim we are proposing the introduction of mutant alkyltransferase genes into hematopoietic stem cells. The introduction of stem cells containing mutant agt genes with high dose alkylating agent chemotherapy will allow us to decrease tumor burden and improve upon the duration of disease free survival and decrease the likelihood of secondary leukemias.