Science Spotlight

Insertional mutagenesis reveals role of new gene in blood cell development

The integration of retroviral genes into host chromosomal DNA can occur at promoter or enhancer sites that drive the increase in proto-oncogene expression, leading to malignant transformation of the cell. Novel proto-oncogenes are identified through retroviral integration analysis. The function of these proto-oncogenes is determined by isolating and expressing the gene in an animal model to monitor gene expression changes and changes to cellular development.
Image provided by John Kowalski
A major safety concern of human gene therapy is the inadvertent development of cancer brought about by mutagenesis of chromosomal DNA, which halted a pivotal gene therapy clinical trial in the early 2000s. Retroviral-based vectors randomly insert genes in the chromosomes of target cells, which can lead to upregulation of proto-oncogenes or disruption of tumor suppressors. These side effects hinder the success of gene therapy with retroviruses; however, retroviral integration studies have proven to be a successful discovery tool in cancer research. Newer sequencing techniques allow researchers to elucidate where provirus integration takes place in the genome, providing a way to identify genes that influence cell survival, proliferation, and differentiation. In a study published in Experimental Hematology, researchers in the laboratory of Dr. Hans-Peter Kiem (Clinical Research Division) used retroviral integration analysis from stem cell gene therapy studies to identify and study a novel gene, HOXC6, in abnormal blood cell development.


The Kiem laboratory uses preclinical animal models to study hematopoietic stem cell (HSC) gene therapy. As an extension of this research, the investigators analyze the retroviral integration sites in HSC clones that repopulate the animals in the long-term after transplant. If the provirus inserts into a promoter or enhancer region, genes nearby are also upregulated and can influence HSC self-renewal. Through these studies, the researchers identified HOXC6, a member of the homeobox (HOX) gene family of transcription factors. While 22 of 39 individual HOX genes are expressed in mouse HSC, the role of many of these genes in hematopoietic differentiation is unknown. Wurm et al. studied the function of HOXC6 by isolating and expressing the gene in mouse HSC with a retroviral-based vector. HOXC6 overexpression enhanced the comparative repopulation of the bone marrow in a murine hematopoietic stem cell transplantation model threefold to fivefold. HOXC6 overexpression also increased the formation of hematopoietic colonies in an in vitro assay. This effect was stronger than observed by overexpressing HOXB4, a HOX gene known to be involved in HSC self-renewal.


The researchers then examined how HOXC6 overexpression specifically altered blood cell development. The percentage of T cells, B cells, and myeloid cells in the peripheral blood were analyzed by flow cytometry 6, 12, and 16 weeks after transplant. HOXC6 expression significantly biased differentiation towards the myeloid lineage (p = 0.0013) and decreased the number of B cells (p = 0.0037) compared to HOXB4 expression. The researchers further confirmed that HOXC6 overexpression inhibits B cell differentiation using an in vitro assay. In addition, HOXC6-overexpressing cells were retained in the bone marrow and significantly increased the numbers of early blood cell lineages known as common myeloid progenitors (CMP) and granulocyte macrophage progenitors (GMP). This suggests that HOXC6 regulates self-renewal or induces long-term proliferation of hematopoietic progenitor cells. Importantly, the authors demonstrated that HOXC6 is endogenously expressed in murine hematopoietic progenitor cells, strongly supporting a role for HOXC6 in early lineage development.


In mouse models with HOXC6 overexpression, 20% of the mice developed leukemia or a myeloproliferative disorder with increased immature myeloid cells. To determine the mechanisms of altered myeloid differentiation in HOXC6-expressing versus control cells, the researchers used gene expression analysis on sorted myeloid progenitor cells. HOXC6 induced gene expression signatures consistent with stem/progenitor cells and similar to those found in acute myeloid leukemia (AML) patient cells. Furthermore, the researchers found the HOXC6-expressing retroviral vector integrated near a gene involved in myeloid malignancies, Meis1, which could contribute to leukemia formation. Notably, HOXC6 expression is increased in human AML and acute lymphoid leukemia bone marrow.


According to Dr. Kiem "Through retroviral integration analysis from previous stem cell gene therapy studies, we identified HOXC6 as a gene that could potentially expand blood stem cells. The study determined that HOXC6 acts as a regulator in hematopoiesis and was implicated in malignant transformation. When overexpressed in a murine model it caused leukemia with a profile akin to AML." While normal levels of HOXC6 maintain the self-renewal of stem cells, overexpression may promote leukemia formation by keeping cells in an immature stage. Taken together, this study shows the utility of using insertional mutagenesis to find novel genes involved in cancer development. As for gene therapy studies, improved viral vectors are being developed to more safely integrate genes without driving the expression of neighboring oncogenes.


Wurm, M, Kowalski, J, Heckl, D, Zhang, XB, Nelson, V, Beard, BC, Kiem, H-P. (2014) Ectopic expression of HOXC6 blocks myeloid differentiation and predisposes to malignant transformation. Exp Hematol 42: 114-125 e4.


See also: Baron BW1, Anastasi J, Hyjek EM, Bies J, Reddy PL, Dong J, Joseph L, Thirman MJ, Wroblewski K, Wolff L, Baron JM. 2012. PIM1 gene cooperates with human BCL6 gene to promote the development of lymphomas. Proc Natl Acad Sci U S A 10:5735-5739.


Kustikova OS1, Geiger H, Li Z, Brugman MH, Chambers SM, Shaw CA, Pike-Overzet K, de Ridder D, Staal FJ, von Keudell G, Cornils K, Nattamai KJ, Modlich U, Wagemaker G, Goodell MA, Fehse B, Baum C. 2007. Retroviral vector insertion sites associated with dominant hematopoietic clones mark "stemness" pathways. Blood 109:1897-1907.


Deneault E1, Cellot S, Faubert A, Laverdure JP, Fréchette M, Chagraoui J, Mayotte N, Sauvageau M, Ting SB, Sauvageau G. 2009. A functional screen to identify novel effectors of hematopoietic stem cell activity. Cell 137:369-379.