Repetitive Busulfan Administration Uncovers a Competitive Advantage for Hematopoietic Clones with a Retrovirus Insertion Near the HDAC7A Locus

Abstract

The recent report of the development of leukemia in four children receiving CD34+ cells transduced with a retroviral vector expressing a corrective IL2 receptor transgene has focused interest on the mechanisms and risks of insertional mutagenesis. In our primate model, we have found evidence for highly non-random integration of retroviral vectors into areas of the genome that could activate genes implicated in leukemogenesis. In a steady-state, any proliferative “pre-malignant” advantage of cells harboring these insertions takes years to manifest. Under hematopoietic stress, abnormalities of behavior of these clones may become evident. The alkylating agent busulfan is known to have profound effects on stem cell behavior and specifically stem cell numbers. We hypothesized that repeated doses of busulfan will result in significant hematopoietic stress at a stem cell level, allowing us to analyze the impact on clonal patterns of retrovirally-transduced cells in vivo and investigate whether clones containing a single insertion proximal to an oncogene gain a competitive advantage, a potentially pre-leukemic situation tendency. Seven years ago, rhesus macaque RQ2297 underwent autologous transplantation of CD34+ cells transduced with standard MLV-derived retrovirus vectors carrying the NeoR gene marker. The animal received a single dose of busulfan 4mg/Kg approximately 2.5 years after transplant, and four additional monthly busulfan injections (4mg/kg, 4 mg/kg, 6mg/kg and 6mg/kg) were administered 4 years post-transplant. LAM-PCR was used to follow integration sites in granulocytes before and after busulfan administration. Repetitive busulfan administration resulted in prolonged decrease in three-lineage blood counts, with a complete recovery to baseline four months after the last infusion of busulfan. The animal was initially highly polyclonal with over 50 independent integrations prior to busulfan, including two independent insertions in the Mds1/Evi1 locus, previously implicated in clonal expansion and preferential engraftment. Following repetitive busulfan, the clonal diversity decreased markedly. Surprisingly, the Mds1-Evi1 clones disappeared, but instead a highly dominant clone emerged, characterized by a vector insertion 5.7 kb upstream of the HDAC7A gene. This clone was detected prior to busulfan, but was not dominant, accounting for 3.2% of shotgun cloned LAM-PCR insertions. 2.5–2.8 years later, this insertion constituted 71% and 94% of the cloned sequences, respectively, and the overall number of independent clones had decreased to only 3–8 at the same time points. HDAC7A belongs to class IIa histone deacetylases. While no direct alterations of HDAC7 genes have been demonstrated in human cancers, its association with various oncogenes and tumor suppressor genes is well established. No evidence of malignancy has been detected in this animal to date. Under conditions of stem cell stress, stem cell clones containing retroviral vectors integrated in the proximity of the HDAC7A locus gained a selective advantage, suggesting immortalization or preferential survival and engraftment of cells with activation of the HDAC7A locus. This model could both accelerate analysis of genotoxicity related to vector insertions, as well as help assess the safety of administering myelotoxic chemotherapy in patients previously engrafted with vector-containing cells.