Candidate Gene Targets of the c-Myb Protooncogene in Human Myloid Leukemia

Reviewer: Walter F. Sall, MD
The Abramson Cancer Center of the University of Pennsylvania
Last Modified: December 8, 2001

Presenter: Susan E. Shetzline
Institution: University of Pennsylvania

Background:

The c-Myb proto-oncogene encoded transcription factor, Myb is required for normal and malignant hematopoeisis. Ectopic c-Myb expression can transform hematopoetic cells, thus Myb may play a role in leukemogenesis.
To identify potential downstream targets of Myb, which may be targets for future leukemia therapy, K562 cells were engineered to express an inducible dominant negative form of c-Myb. Downstream gene induction was assayed using micro-array technique and confirmed using PCR. 10,000 candidate genes were evaluated using this technique.

Materials and Methods

A chimeric transcription actor composed of c-Myb's DNA binding domain, the engrailed protein transcription repression domain and a mutated estrogen receptor responsive to tamoxifen was created and stably transfected into a K562 cell line.
The cells were cultured for 3 days with Tamoxifen, repressing transcription of myb regulated genes.
These cells were processed for micro-array gene expression analysis. Confirmation of gene over or under-expression was performed for several candidate genes using PCR.

Results:

Culture of this modified K562 cell line with tamoxifen led to reduced cell proliferation in-vitro. Suppression of Myb function led to:
Greater than two-fold over-expression of 34 of 10,000 tested genes. Several of these, including cdc7 and neuromedin U are associated with erythroid or megakaryocytic differentiation. Cdc7 and neuromedin U over-expression was confirmed using PCR.
Greater than two-fold under-expression of 70 genes was found.
Previously identified Myb targets including bcl-2, c-kit, CD34 and c-myb showed less than 50% change in expression.

Author's conclusions:

It is unclear why dominant negative myb expression slows cell proliferation.
A manageable number of potential myb regulated genes has been identified for further analysis.
Neuromedin U and cdc7 overexpression has been confirmed using PCR. Further investigation into the role of these genes in hematopoetic cell cycle regulation is ongoing.

Clinical/Scientific Implications:

This study has identified several new potential molecular targets which may be important in the pathogenesis of leukemia. Further study of these downstream targets of c-Myb proto-oncogene expression may yield novel genetically targeted anti-leukemia therapies in the same mold as STI-571