The Role of Dominant-Negative IKAROS Mutations In the Pathogenesis and Treatment Responsiveness of BCR-ABL1 positive Acute Lymphoblastic Leukemia

Abstract 540

Expression of the constitutively active tyrosine kinase BCR-ABL1 is the hallmark of two diseases with distinct pathologic and clinical features: chronic myeloid leukemia (CML), an expansion of relatively mature granulocytes that typically responds well to kinase inhibition, and pre-B cell acute lymphoblastic leukemia (ALL), an aggressive malignancy of lymphoid progenitors that has a dismal prognosis. The basis for this dichotomy has been poorly understood. Recent studies profiling genome-wide DNA copy number alterations in CML and ALL have identified common deletions of IKZF1 (encoding the lymphoid transcription factor IKAROS) in de novo BCR-ABL1 positive ALL, and at the progression of CML to lymphoid blast crisis, suggesting that perturbation of IKAROS activity is a key event in the pathogenesis of BCR-ABL1 lymphoid leukemia. The IKAROS alterations commonly involve coding exons 3–6, resulting in expression of a dominant negative IKAROS isoform, IK6. Moreover, the presence of IKZF1 alterations is associated with poor outcome in BCR-ABL1 ALL. We have previously shown in a retroviral bone marrow transplant model of BCR-ABL1 ALL that Ikzf1 loss results in increased penetrance of leukemia, but the role of IK6 in the pathogenesis of ALL has not been studied. Here, we have examined the effect of the expression of Ik6 in a retroviral bone marrow transplant model of murine BCR-ABL1 B-progenitor ALL. Unmanipulated marrow from C57BL/6 Arf null mice was transduced with MSCV retrovirus coexpressing p185 BCR-ABL1 and luciferase, plated for 8 days to derive pre-B cells, then transduced with MSCV retrovirus expressing either wildtype Ikaros (Ik1-RFP), Ik6-RFP, or empty vector. Expression of Ik1 was not tolerated and resulted in cell death and apoptosis. IK6 expression led to increased proliferation of p185+Arf null cells with reduced sensitivity to the BCR-ABL1 kinase inhibitor dasatinib compared to cells transduced with empty vector. Intracellular phosphosignaling analysis of Crkl phosphorylation demonstrated that this reduced sensitivity to dasatinib was independent of ABL1 inhibition. Gene expression profiling of p185+Arf null-Ik6 cells revealed a gene expression signature similar to that of human BCR-ABL1+ ALL with enrichment of hematopoietic stem cells genes as well as genes involved in B-cell receptor, Notch, and Jak-Stat signaling pathways. To test the role of Ik6 in leukemogenesis and treatment responsiveness in vivo, p185 BCR-ABL1-luciferase Arf null cells were transduced with MSCV retrovirus expressing GFP alone, Ik1-GFP, or Ik6-GFP then transplanted into lethally irradiated C57BL/6 recipients. Expression of Ik6 in vivo led to accelerated tumorigenesis and decreased survival with tumors uniformly of pre-B immunophenotype. Moreover, mice transplanted with Ik6-expressing marrow were less sensitive to dasatinib therapy (10mg/kg QD initiated 7 days post-BMT) compared to control mice (19d vs. 31.5d, p<0.001), suggesting that expression of the dominant-negative Ikaros isoform Ik6 may play a key role in resistance to therapy and poor outcome in human BCR-ABL1 positive ALL. These results indicate that perturbation of IKAROS activity is a key event in the pathogenesis of BCR-ABL1 positive ALL, and that expression of dominant negative IKZF1 isoforms influences tumor responsiveness.