Retroviral Insertion and Temozolomide Selection Promote T-Cell Lymphomas in Tumor-Prone Mice.

In vivo selection is a key strategy for increasing contribution of gene-modified cells in hematopoietic stem cell gene therapy. The combination of DNA-alkylating agents such as temozolomide (TMZ) with O⁶-benzylguanine (BG) allows selection for cells expressing a drug-resistant O⁶-methylguanine-DNA-methyltransferase (MGMT) gene, resulting in hematopoietic repopulation with gene-modified cells in numerous mouse studies. TMZ is used clinically for treatment of glioma and other malignancies, with dose-limiting myelotoxicity, and has been used with BG in a phase I trial of drug-resistant MGMT gene transfer for chemoprotection in treatment of advanced solid malignancies. We report here that in transplants of lymphoma-prone LMO1 transgenic or retrovirus-transduced normal mouse marrow to study the effect of in vivo selection on insertional mutagenesis by retrovirus vectors in gene therapy, administration of BG+TMZ post-transplant increased the incidence of donor-derived T-cell lymphomas. In transplants of LMO1 transgenic mouse lineage-negative (Lin⁻) marrow progenitor cells, BG+TMZ significantly increased (p=0.0026) the incidence of lymphoma (87.5% incidence in mice surviving drug-induced hematologic toxicity; n=8) compared to mice without drug (41% incidence by day 315 post-transplant; n=17). Increased tumor incidence after BG+TMZ was observed both in transplants of untransduced LMO1 Lin⁻ cells and in LMO1 cells transduced with an MFG gamma-retroviral vector encoding a drug-resistant P140K MGMT gene and a GFP reporter gene. Additionally, in transplants of normal C57BL/6 mouse Lin⁻ cells transduced with the P140K-GFP retroviral vector, BG+TMZ significantly increased (p=0.0043) the tumor incidence (100% incidence by day 169 post-transplant in mice surviving drug toxicity; n=6) compared to mice without drug (0% incidence by day 315 post-transplant; n=9). This is a higher frequency of lymphomas than we have previous observed in numerous selection studies with MGMT transduction of normal mouse whole marrow. Administration of BG+TMZ following transplant of transduced C57BL/6 mouse cells resulted in an approximately 2-fold increase in vector-expressing cells in peripheral blood, compared to an approximately 57% decline in untreated mice, demonstrating in vivo selection of MGMT-expressing cells. However, in all cases of LMO1 or C57BL/6-derived lymphomas, the tumor cells were found to lack expression of the P140K-GFP vector, suggesting that cells expressing drug-resistant MGMT were protected from the mutagenesis caused by BG+TMZ, as well as from its hematologic toxicity. Despite the lack of transgene expression, retroviral vector DNA was detected by LAM-PCR in some of the LMO1-derived and all of the C57BL/6-derived lymphomas, suggesting that insertional mutagenesis by an integrated but non-expressing (or silenced) retroviral vector may have contributed to TMZ-associated tumorigenesis in some instances. Two cases of treatment-related secondary myelodysplastic syndrome and acute myeloid leukemia have been reported in the literature for glioma patients treated with TMZ, indicating a clinical risk of TMZ tumorigenesis in some cancer patients. Here we present evidence that TMZ can cooperate with a tumor-promoting transgene (in LMO1 marrow) or potentially with insertional mutagenesis (in gamma-retrovirus transduced C57BL/6 or LMO1 marrow) for hematopoietic transformation, and that hematopoietic cells not protected by drug-resistant MGMT expression can give rise to lymphomas in a majority of mice following TMZ treatment.