LMO2 Oncoprotein Stability in T-Cell Leukemia Requires Direct LDB1 Binding

Abstract

LMO2 is a component of multisubunit DNA-binding transcription factor complexes that regulate gene expression in hematopoietic stem and progenitor cell development. Enforced expression of LMO2 causes leukemia by inducing hematopoietic stem cell-like features in T-cell progenitor cells, but the biochemical mechanisms of LMO2 function have not been fully elucidated. In this study, we systematically dissected the LMO2/LDB1-binding interface to investigate the role of this interaction in T-cell leukemia. Alanine scanning mutagenesis of the LIM interaction domain of LDB1 revealed a discrete motif, R³²⁰LITR, required for LMO2 binding. Most strikingly, coexpression of full-length, wild-type LDB1 increased LMO2 steady-state abundance, whereas coexpression of mutant proteins deficient in LMO2 binding compromised LMO2 stability. These mutant LDB1 proteins also exerted dominant negative effects on growth and transcription in diverse leukemic cell lines. Mass spectrometric analysis of LDB1 binding partners in leukemic lines supports the notion that LMO2/LDB1 function in leukemia occurs in the context of multisubunit complexes, which also protect the LMO2 oncoprotein from degradation. Collectively, these data suggest that the assembly of LMO2 into complexes, via direct LDB1 interaction, is a potential molecular target that could be exploited in LMO2-driven leukemias resistant to existing chemotherapy regimens.

ACKNOWLEDGMENTS

We thank our colleagues Stephen Brandt and Ying Cai for discussions and for LDB1 and LMO2 cDNAs, Mark Koury for advice on cell lines and for supplies and reagents, David Cortez and Nancy Zhao for cDNAs, Scott Hiebert for discussions and pGIPZ shRNA vectors, Yan Guo for assistance with next-generation sequencing data, Rati Tripathi for technical assistance, and Sabine Wenzel for positive encouragement. We thank Derya Unutmaz for pH163 vectors, John Strouboulis for pBirA-Zeo, Mark Chiang for KOPT-K1 cells, Charles Mullighan for CCRF-CEM cells, Michelle Churchman for other cell lines, and Ronald Levy for the anti-LMO2 hybridoma. J.H.L. thanks H. P. Erickson for a generous gift helpful to past, present, and future works. J.H.L. acknowledges P. A. Weil for sharing his passion about macromolecular transcription factors and for his steadfast advocacy of attentive conception, design, and execution of experiments, especially advocacy of effective control(s).

This work was supported by the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development (BX001799-01A1); the American Society of Hematology; and the Vanderbilt Ingram Cancer Center (P30 CA68485) (U.P.D.). J.H.L. was supported by NIH training grant T32CA093240 to Michael Freeman.

We declare that we have no conflicts of interest with the contents of this article.

J.H.L. conceived of and performed experiments, interpreted data, and wrote the paper; U.P.D. conceived of experiments, interpreted data, and wrote the paper; C.E.A. performed all flow cytometry and sorting; and W.H.M. performed mass spectrometry and matching spectra to proteomic databases.