Infant leukemias and chemotherapy related leukemias characterized by translocations of the MLL gene at chromosome band 11q23 are devastating, often fatal diseases with unique clinical and biological features. MLL encodes a complex oncoprotein with transcriptional repression and activation functions. The translocations involve many partner genes and generate 5'-MLL-Partner-3'rearrangements, 5'-Partner-MLL-3'rearrangements and MLL haploinsufficiency due to involvement of one allele in the translocation. Unique attributes of zebrafish embryos enable in vivo visualization of normal and abnormal hematopoietic developmental processes in intact animals like no other animal models. Moreover, zebrafish embryos are well suited to model MLL because MLL translocations in infant leukemogenesis originate in utero. Until now, no studies of mll had been done in zebrafish. We cloned the zebrafish mll ortholog and showed high conservation of all of the critical functional domains of human MLL. We found that mll transcripts are maternally supplied to the embryo, expressed during the entire zebrafish lifespan, and detectable in hematopoietic cells and other tissues where Mll is expresed in mice. We demonstrated that mll morpholino knockdown phenocopies the external phenotype, apoptosis and anemia of Mll-/- mice. We made the striking observations that the resulting mll depletion caused not only reduced homeobox cofactor and altered cell cycle gene expression, but also overexpression and underexpression of many hematopoietic genes in different blood cell compartments, suggesting new links of mll to previously unknown targets. Furthermore, the changes in blood cell gene expression caused profound blood cell dysmorphologies. While overexpressed genes in the precursor/myeloid compartment featured lmo2, scl, cmyb, ikaros, rag2 and gata1, reduced expression of gata1 and other red cell genes was detected in the erythroid fraction, linking the anemia from mll depletion to deregulation of an entire erythroid program. This leads to the hypothesis that MLL has profound multi-lineage roles in the developmental control of the hematopoietic system, that temporal and cell-type specific regulation of gene expression by MLL including repression and activation is required for orderly specification of hematopoietic progenitor and stem cell development, and that loss of MLL causes ectopic and asynchronous overexpression and underexpression of blood cell lineage genes and ineffective development of the hematopoietic system. This project endeavors to investigate this hypothesis by exploiting whole organism studies and cellular and molecular studies on cells from whole organisms that are only possible in zebrafish to pinpoint the cells where MLL is most important and when MLL translocations first become transforming.

Public Health Relevance

MLL is a critically important gene for blood cell development, abnormalities of which called translocations have a significant impact on morbidity and mortality in the infant population affected with leukemia and in the patient population affected with leukemia as a complication of certain anti-cancer drugs. This project will exploit the tremendous potential of zebrafish to understand the dynamics of how normal blood cell development is regulated by MLL in space and time from the earliest developmental timepoints in the living embryo, and how the same dynamics are deregulated in disease. The exploitation of zebrafish to model the functions of human MLL in the regulation of normal and malignant blood cell development will streamline future inroads to new molecularly targeted therapies for patients with MLL leukemia.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA153348-05
Application #
8606829
Study Section
Cancer Molecular Pathobiology Study Section (CAMP)
Program Officer
Howcroft, Thomas K
Project Start
2010-04-01
Project End
2015-01-31
Budget Start
2014-02-01
Budget End
2015-01-31
Support Year
5
Fiscal Year
2014
Total Cost
$356,810
Indirect Cost
$115,475
Name
Children's Hospital of Philadelphia
Department
Type
DUNS #
073757627
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Pendleton, Maryjean; Lindsey Jr, R Hunter; Felix, Carolyn A et al. (2014) Topoisomerase II and leukemia. Ann N Y Acad Sci 1310:98-110
Robinson, Blaine W; Germano, Giuseppe; Song, Yuanquan et al. (2011) mll ortholog containing functional domains of human MLL is expressed throughout the zebrafish lifespan and in haematopoietic tissues. Br J Haematol 152:307-21