Using Genome Engineering To Prospectively Investigate The Pathogenesis Of MLL Translocations In Infant Acute Lymphoblastic Leukemia

Introduction Chromosomal rearrangements involving the MLL gene occur in both primary and treatment-related leukemias and convey a poor prognosis. Infants with acute lymphoblastic leukemia (ALL) containing an MLL translocation have a 5-year event free survival of 35% as compared to 69% for infants with germline MLL (Kang et al., Blood 2012). While animal models of MLL-AF9 translocations typically found in adult AML have improved our understanding of the role of MLL translocations in leukemia pathogenesis, modeling the MLL-AF4 translocation commonly found in infant ALL has proven difficult. We hypothesized that recent advances in genome engineering would facilitate induction of specific MLL translocations in primary hematopoietic stem cells to recapitulate the initiating event that leads to infant leukemia. A better understanding of the pathogenesis of the MLL-AF4 translocation will allow for the development of targeted therapies to improve outcome in MLL -rearranged infant leukemia. Methods Transcription activator-like effector nucleases (TALENs) were designed to recognize a patient-specific translocation site within the break point cluster region of the MLL gene and the AF4 gene, respectively. These TALEN pairs were then co-expressed in either K562 cells or primary human CD34+ cells isolated from umbilical cord blood to induce patient-specific double strand breaks. Traditional PCR, nested PCR, and high throughput next generation sequencing were used to detect MLL-AF4 translocations, reciprocal AF4-MLL translocations, as well as other MLL translocations. Clonal analysis was performed to determine the translocation efficiency resulting from two site-specific double strand breaks. Primary cells were maintained in long-term culture to assess for potential survival advantage conferred by the translocations. Finally, primary human CD34+ cells with an induced MLL-AF4 translocation were transplanted into NSG mice to assess for leukemic potential. Results We successfully generated TALENs that induce a patient-specific double strand break within the breakpoint cluster regions of the endogenous MLL and AF4 genes. Co-expression of the MLL and AF4 TALENs in K562 cells as well as in primary human hematopoietic stem cells isolated from umbilical cord blood resulted in de novo MLL-AF4 translocations as well as the reciprocal AF4-MLL translocation. The MLL-AF4 translocation efficiency was appreciably higher in K562 cells (10-4) compared to primary hematopoietic stem cells (10-5). Long-term culture of the primary human hematopoietic cells showed an increase in the frequency of the MLL-AF4 translocation-positive cells, suggesting a survival advantage for the cells containing the translocation. These cells were subsequently transplanted into NSG mice to assess their ability to induce leukemia. These studies are currently ongoing. Conclusions Advances in genome engineering have provided the tools necessary to study leukemia pathogenesis in a prospective manner. We have designed an experimental system to recapitulate the process of leukemogenesis. TALENs are effective to induce a specific MLL translocation in primary human hematopoietic stem cells with minimal off target effects, under the control of the endogenous promoter, and in the presence of the reciprocal translocation. This system will allow us to prospectively investigate the downstream effects of this initiating event on gene expression, epigenetic regulation, and genome stability in order to understand the key steps critical for the pathogenesis of MLL -rearranged leukemias. Disclosures: No relevant conflicts of interest to declare.