The molecular mechanism underlying the granulocytic maturation arrest that characterizes Acute Promyelocytic Leukemia (APL) remains obscure. Most patients with APL manifest the t(15;17) chromosomal translocation, which results in expression of the PML-RAR fusion protein. Competing models suggest that PML-RAR acts as a dominant negative for PML, a dominant negative for wild type retinoic acid receptor alpha (RARa), or a rogue transcriptional activator. In an effort to gain greater insight into the mechanism underlying APL we have studies an APL patient with a t(5;17) chromosomal translocation. We show that this translocation links 5' elements of the nucleolar protein nucleophosmin (NPM) to 3' elements of RARa. In our preliminary data we identify and sequence the chimeric cDNA, and demonstrate that it encodes a 52 kd protein. We hypothesize that this fusion affects the function of the retained RAR domains through a novel mechanism of changing the nuclear compartment in which the RAR domains segregate. We hypothesize that NPM motifs redirect RAR domains into an aberrant nuclear compartment, where the fusion binds to and sequesters RAR heterodimerization partners. By this novel mechanism of redirecting its fusion partner into an unaccustomed architectural region of the nucleus, NPM converts the linked RAR domain into a dominant negative for RARa, and thereby blocks myeloid maturation. To test this hypothesis we will complete the sequencing of the full open reading frame for NPM-RAR, and establish that the fusion protein is indeed expressed in the t(5;17) leukemic cells. We will determine whether NPM-RAR reproduces the APL phenotype by investigating the effects of forced expression of NPM-RAR on the differentiation potential of HL-60 cells. We will determine whether NPM-RAR expression modulates wild type RARa function, and whether NPM-RAR complexes with RARa heterodimerization partners. We will determine by immunofluorescence the distribution of NPM-RAR in the cell, and whether its expression affects the architectural compartment in which RARa dimerization partners localize. Finally, we will determine whether an artificial construct that directs RAR to an unaccustomed nuclear architectural domain similarly blocks myeloid differentiation. These investigations will test the hypothesis that disrupted retinoic acid signaling underlies the maturational block in APL. In addition, they will serve as seminal studies for future work mapping nuclear functional compartments.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29CA067346-03
Application #
2443148
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1995-07-01
Project End
2000-06-30
Budget Start
1997-07-01
Budget End
1998-06-30
Support Year
3
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Pittsburgh
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
053785812
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Pollock, Sheri L; Rush, Elizabeth A; Redner, Robert L (2014) NPM-RAR, not the RAR-NPM reciprocal t(5;17)(q35;q21) acute promyelocytic leukemia fusion protein, inhibits myeloid differentiation. Leuk Lymphoma 55:1383-7
Dorritie, Kathleen A; Redner, Robert L; Johnson, Daniel E (2014) STAT transcription factors in normal and cancer stem cells. Adv Biol Regul 56:30-44
Rush, Elizabeth A; Schlesinger, Kimberly W; Watkins, Simon C et al. (2006) The NPM-RAR fusion protein associated with the t(5;17) variant of APL does not interact with PML. Leuk Res 30:979-86
Redner, R L; Chen, J D; Rush, E A et al. (2000) The t(5;17) acute promyelocytic leukemia fusion protein NPM-RAR interacts with co-repressor and co-activator proteins and exhibits both positive and negative transcriptional properties. Blood 95:2683-90
Redner, R L; Corey, S J; Rush, E A (1997) Differentiation of t(5;17) variant acute promyelocytic leukemic blasts by all-trans retinoic acid. Leukemia 11:1014-6
Redner, R L; Rush, E A; Faas, S et al. (1996) The t(5;17) variant of acute promyelocytic leukemia expresses a nucleophosmin-retinoic acid receptor fusion. Blood 87:882-6