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Interdisciplinary Biomedical Sciences Program
B.S. 2006, Hebei Medical University, China
Thesis Advisor: Ian Whitehead, Ph.D.
Department of Microbiology and Molecular Genetics
Thursday, January 23, 2014
2:00 P.M., Cancer Center-G1196
Aims: Previous studies have demonstrated that chronic myelogenous leukemia is associated with the fusion oncoprotein p210 BCR/ABL. Our preliminary data indicate that p210 BCR/ABL interacts directly with the XPB protein, and that loss of XPB binding attenuates the ability of p210 BCR/ABL to induce myeloproliferation in a bone marrow transplantation (BMT) model. In the present study we investigate the molecular and cellular mechanisms through which loss of XPB binding regulates p210 BCR/ABL induced leukemogenesis.
Methods and results: With ex vivo colony formation assays and progenitor flow cytometry analysis, we demonstrated that loss of XPB binding reduced the ability of p210 BCR/ABL to drive the proliferation of granulocyte macrophage progenitors (GMPs), which are considered to be the leukemic stem cells in the blast crisis of CML. We determined with comet assays that XPB binding does not affect the ability of p210 BCR/ABL to affect nucleotide excision repair in both myeloid and lymphoid cell lines. Using an RNA synthesis study, an up-regulated general transcription was observed in cells expressing the XPB binding mutant. We further determined by western blot analysis that the expression level of c-Myc, which is up-regulated by p210 BCR/ABL, is significantly down-regulated in cells expressing the XPB binding mutant. Using an affinity binding assay, loss of XPB binding was found to increase RhoGEF activity, and resulted in increased activity of RhoA, which is further confirmed by the western blot analysis of downstream effectors of the RhoA signaling pathway. We then constructed a double mutant with impaired XPB binding, and abolished RhoGEF activity, and tested its ability to drive myeloproliferation in the murine BMT model for CML. Recipient mice transplanted with the double mutant developed myeloproliferation which is mainly caused by an expansion of neutrophils. The double mutant transplanted mice displayed a prolonged lifespan compared with p210 BCR/ABL transplanted mice (average 54 days vs. 28 days), but not as long as mice transplanted with the XPB binding mutant alone (average 78 days).
Conclusion: P210 BCR/ABL regulates c-MYC transcription and RhoGEF activity through an interaction with XPB, which in turn regulates myeloid progenitor populations and disease progression in CML.
Keywords: chronic myelogenous leukemia, p210 BCR/ABL, XPB, NER, c-MYC, RhoA