Genetic Interaction of the BCR-ABL Oncogene and the ARF Tumor Suppressor in Ph+ Lymphoid Leukemia.

BCR-ABL-associated (Ph+) B-lineage ALL confers a poor prognosis in both children and adults despite treatment with intensive chemotherapy and stem cell transplantation. Deletion of the portion of human chromosome 9p21 that contains the intimately linked p14ARF and p16INK4A tumor suppressors is the second most common cytogenetic abnormality observed in BCR-ABL-associated (Ph+) pediatric ALL. Moreover, molecular analysis of adult and pediatric Ph+ ALL cases reveals frequent mono- or bi-allelic deletions of these two tumor suppressors. Utilizing mice with a targeted deletion of the ARF tumor suppressor (p19Arf in mice) that does not affect the expression and regulation of Ink4a, we have examined the collaboration between BCR-ABL and Arf loss in models of Ph+ B-lineage ALL. Expression of BCR-ABL in normal pre-B cells induces the Arf tumor suppressor and triggers a p53 response. Culture of primary mouse pre-B cells derived from mice that contain two (wild type), one (heterozygous) or no Arf alleles (null) confirmed that Arf loss conferred a significant in vitro proliferative advantage and relative protection from apoptosis upon withdrawal of stromal support. Infection of Arf-null cells with retroviral vectors encoding green fluorescent protein (GFP) and either the p185 or p210 BCR-ABL oncoproteins conferred IL-7 independence and facilitated the rapid emergence of immortalized, factor-independent, tumorigenic pre-B cell lines. Arf−/− and, with a lesser efficiency, Arf+/− primary pre-B cells engineered to express BCR-ABL rapidly formed aggressive disseminated lympholeukemias when injected into cohorts of syngeneic immune-competent recipient mice, whereas BCR-ABL-infected wild type cells expressed p19Arf and rarely formed tumors. Loss of Arf did not nullify potent Gleevec-mediated inhibition of BCR-ABL tyrosine kinase activity in vitro, but the Gleevec sensitivity of Arf-null pre-B cells expressing either p185 or p210 BCR-ABL was significantly, but not totally, overcome in the presence of excess IL-7. Gleevec treatment of mice bearing BCR-ABL/Arf-null pre-B cells extended their median survival; yet, virtually all animals ultimately became functionally resistant to Gleevec and succumbed to disseminated disease. Utilizing retroviral vectors to transduce either p185 or p210 BCR-ABL linked to GFP in conjunction with a bone marrow transplantation protocol that favors development of B-lineage lymphoblastic leukemias in irradiated syngeneic recipients, we observed accelerated disease using Arf-null donor cells as compared to transduced wild type controls. Lympholeukemic Arf-null cells in peripheral blood expressed high levels of GFP, whereas p19Arf-expressing wild type blood cells did not. Together, these data indicate that the BCR-ABL-induced Arf tumor suppressor dampens unrestrained cell proliferation and that loss of Arf therefore collaborates with BCR-ABL in experimental models of Ph+ B lineage ALL. Although resistance to Gleevec is generally mediated by mutations in the BCR-ABL kinase, we speculate that deletion of INK4a-ARF in Ph+ ALL may well contribute to a worse clinical outcome.