DNA REPAIR IN PROTECTING AGAINST SECONDARY LEUKEMIAS

DNA修复蛋白O6-烷基鸟嘌呤-DNA烷基转移酶(AGT)被证明可以保护细胞免受烷化剂包括氯乙基亚硝脲、甲基化试剂和环磷酰胺的毒性作用。大多数原发肿瘤具有较高的AGT活性，这是这些药物用处有限的主要原因。严重的骨髓抑制和治疗诱导的髓系白血病是临床使用烷基化药物时遇到的危及生命的副作用。人造血祖细胞的AGT蛋白活性很低，这是其对烷基化药物的骨髓毒性和致突变作用高度敏感的基础。为了提高烷基亚硝脲的治疗效果，我们开发了O6-苄基鸟嘌呤(BG)。这种化合物是一种有效的、选择性的AGT修复蛋白失活剂，目前正处于I/II期临床试验中，作为烷化剂的调节剂。虽然BG与烷化剂联合使用会增加对多种肿瘤细胞的杀伤力，但一个主要的担忧是，BG会削弱未成熟的造血细胞修复烷化剂诱导的损伤的能力，这反过来会导致继发性白血病的发生率增加。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。这项建议的主题是确定BG是否增加了可能导致继发性白血病的烷化剂的诱变性，更重要的是，利用这些信息来产生治疗方法，以防止这种毁灭性的副作用。具体目标1将使用体外和体内模型研究抑制AGT活性是否会导致环磷酰胺、BCNU和替莫唑胺增加HPRT基因突变频率。我们将确定表达BG耐药AGT蛋白的细胞是否会受到BG加烷化剂诱导的突变的保护。具体目标2将直接解决将BG与烷化剂一起使用是否会增加与治疗有关的白血病的发生率的问题。杂合子NF1基因敲除小鼠为环磷酰胺诱导的髓系白血病提供了一个很好的体内模型。具体目标3和4描述了对抗这种毁灭性副作用的方法。我们将产生过量表达突变AGT的转基因小鼠品系，与NF1小鼠杂交，以确定是否对白血病的发生有保护作用。在最后一个具体目标中，我们建议将突变的烷基转移酶基因引入造血干细胞。将含有突变AGT基因的干细胞与大剂量烷化剂化疗相结合，将使我们能够减轻肿瘤负担，提高无病生存时间，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.