Abstract:

Chronic myelogenous leukemia (CML) is a disease that arises as a direct consequence of a balanced reciprocal translocation between two chromosomes in hematopoietic cells. The resulting fusion between the BCR and ABL1 (c-Abl) genes gives rise to a BCR-ABL1 fusion protein, a constitutively active tyrosine kinase that controls intracellular signaling pathways. BCR-ABL1 confers survival and proliferation advantages to hematopoietic cells in vitro and induces leukemogenesis in vivo .

In this dissertation, we show that RIN1 is necessary for in vitro transformation of primary bone marrow cells by oncogenic ABL1 fusion proteins. Primary bone marrow cells from wild type mice are transformed to growth factor independence by BCR-ABL1 and TEL-ABL1, but bone marrow cells from RIN1-null mice fail to proliferate under the same conditions. BCR-ABL1T315I , a patient-derived mutant resistant to STI571 (imatinib) and other competitive inhibitors of the ABL kinase domain, was also unable to transform Rin1-/- bone marrow cells. Importantly, the requirement for Rin1 was cell autonomous, as expression of human RIN1 in the Rin1-/- hematopoietic cells restored the transforming activity of BCR-ABL1. These results suggest that RIN1 is necessary to unleash the full activity and transforming potential of ABL1. These studies also have ramifications for the broader field of kinase inhibitor therapeutics.

Our first approach to blocking the transforming properties of BCR-ABL1 was to employ the ABL binding domain (ABD) of RIN1 for the delivery of a protein tyrosine phosphatase (PTP) directly to the oncogenic tyrosine kinase. We tested the catalytic domains of several phosphatases implicated as ABL regulators in the creation of ABD-PTP type "escort-phosphatases". We then demonstrated that an escort-phosphatase could block transformation of primary mouse hematopoietic cells and reverse the transformed phenotype of a human leukemia cell line.

We have also developed a novel application of time-resolved fluorescence resonance energy transfer (TR-FRET) to create a quantitative assay for the interaction of RIN1 and ABL1. Initial experiments showed robust properties amenable to a HTS (Z'-factor > 0.5) and identified two compounds that inhibited the interaction signal by more than three standard deviations.