The Molecular Basis of Human Leukemia: Evaluation by the Application of Forward and Reverse Genetics

The development of leukemia involves a sequential series of mutational events involving primitive cells within the bone marrow that normally serve as stem cells capable of both self-renewal (i.e. sustaining a small reserve population of undifferentiated cells) and differentiating into all the different blood cell lineages. The initiating events frequently arise by specific chromosome breakages and fusions that cause inappropriate association of two different genes. Additional mutations involve progression from a preleukemic disorder such as myelodysplastic syndrome (MDS) progressing to acute leukemia or chronic leukemia progressing to blastic crisis. Most commonly these secondary mutations occur in genes involved in important signaling pathways (e.g Ras) or in stem cell growth factor receptors (e.g Flt3 or cKit activating mutants). With two sets of mutations the marrow stem cells fail to produce mature blood cells, generating instead large numbers of primitive “blast” cells. A third change is also required, namely the acquisition of immortality associated with stabilization of telomeres and up regulation of telomerase (see section of telomere biology).

Leukemogenic gene transfer is being used to “transform” normal human and murine hematopoietic stem cells. Hematopoietic stem cells isolated from human umbilical cord blood or murine bone marrow are transduced with retroviral or lentiviral vectors expressing the leukemogenic gene of interest, together with a marker gene (green fluorescence protein) that allows transduced cells to be isolated and monitored. Shortly after transduction, RNA is isolated and the profile of gene expression (genes upregulated or downregulated) is determined by Affymetrix gene chip analysis. We have generated data on the cellular and genetic changes associated with introduction of three types of “fusion” gene, AML1/ETO (found in ~20% of AML), Nup98/HOXA9 (found in 2-5% of AML and MDS), and Bcr/Abl (found in >95% of CML). Introduction of these leukemogenic genes into normal blood stem cells provided them with significant growth advantage over control stem cells, and in long-term cultures over many weeks or months the “transformed” stem cells exhibited a number of preleukemic features, including altered or delayed differentiation and modified growth factor responsiveness and enhanced proliferation. Homeobox genes are important for regulating the processes of stem cell self-renewal and differentiation. Nup98 is a nucleoporin gene whose protein regulates passage of molecules, including certain RNA species, between the nucleus and the cytoplasm. When fused with the homeobox gene HOXA9, the two genes transform stem cells and generate a preleukemic phenotype. HOXA9 alone when over-expressed in normal stem cells can provide an initiating signal for the process of leukemia development and we have shown that levels of the HOXA9 protein are normally closely regulated by ubiquitination. Specific pathways exist for ubiquitination of the homeobox proteins and we have demonstrated that by inactivating the CUL4A ligase (using siRNA) we could stabilize the levels of HOXA9 protein in stem cells resulting in blockade of their normal differentiation and increased stem cell self-renewal. We have found that the HOXA9 fusion with NUP98 is protected from the ubiquitination-degradation system (possibly by some physical interference), resulting in more prolonged and elevated stimulation of the pathways favoring proliferation at the expense of differentiation.

Of additional interest, the Nup98/HoxA9 fusion protein acts as a dominant negative, inhibiting the function of normal Nup98 and disrupting movement of molecules across the nuclear membrane. We are currently determining if stable integration of siRNA targeting normal NUP98, and of HOXA9 with mutations removing the site responsible for binding the ubiquitination machinery (i.e. stabilizing the HOXA9 protein), will produce the same preleukemic changes as the fusion gene.

Our work with the “second hit” mutants have progressed furthest with the Flt3 constitutively active mutants (FLT3ITD), found in ~35% of AML cases. Mutations cause the receptor to provide a continuous downstream signal to the stem cell in the absence of the growth factor that normally binds to the receptor and regulates stem cell proliferation. Genomic analysis revealed that an important signaling pathway, STAT5, was strongly activated by the mutant receptor. We then made a constitutively activated form of STAT5 and introduced that into normal stem cells and showed a similar pattern of changes in stem cell behavior and altered differentiation as found with the mutated receptor alone. Taking this genetic analysis still further we compared the pattern of gene up- and down-regulation following introduction of either Flt3ITD or STAT5 into normal stem cells versus control stem cells. We have identified a number of genes and gene pathways that we will be investigating for their roles in leukemic dysregulation. One of these genes (RAC3) is a member of the RhoGTPase family that plays a role in migration of stem cells and their interaction with critical regulatory niches within the bone marrow environment. We have developed lentiviral vectors expressing active forms of RAC3 or siRNA inhibitors of RAC3 and we will evaluate the ability of these to either generate or inhibit preleukemic changes in stem cells.

Our objectives at this stage are to combine within the same stem cell, the fusion gene (Nup98/HOXA9, or AML1/ETO or Bcr/Abl), a second hit gene (Flt3ITD or mutated Ras), and telomerase (hTERT) and evaluate both in vitro or following transplantation into immunodeficient mice that support human leukemic cell proliferation. We predict that these three genetic changes will be sufficient to transform the normal stem cell into an acute myeloid leukemic cell. We shall confirm this by close comparison of the phenotype, the self-renewal and differentiation potential, and the gene profile of the genetically modified stem cells, with leukemic cells obtained from patients.

Reverse genetics:

This approach is based upon availability of marrow and blood samples from patients undergoing treatment for leukemia at Memorial Hospital. Under an approved Hospital protocol and with patient informed consent, samples of leukemic cells at diagnosis and relapse, as well as marrow from patients with preleukemia/MDS are obtained. We have shown that only a small fraction (<1%)of the leukemic cell population is responsible for the continuous growth of the leukemia population.

These rare cells are the leukemic equivalent of marrow stem cells. We have developed techniques for purifying these rare leukemic stem cells and have developed in vitro assays for measuring their numbers and function. In addition we have undertaken gene chip analysis of the purified leukemic stem cells compared to the rest of the leukemic population, and also compared to purified populations of normal human marrow stem/progenitor cells. The data we are generating has identified some genes that we have already implicated in the leukemic process (e.g. homeobox genes) as well as novel genes with as yet no known function. We are using gene “knock down” and “knock in” strategies to evaluate the role of these genes in the process of leukemia development.

The science of proteomics complements that of genomics, allowing investigation of the pattern of proteins displayed by particular cells. We are particularly interested in cell surface proteins since these include many receptors and adhesion molecules that are involved in cell regulation. To this end, we have initiated a study using phage antibody display using leukemic stem cell targets. By generating leukemia-specific and leukemia-associated antibodies and by using nuclear magnetic resonance strategies to further identify the surface “epitopes” being recognized by the antibodies, we hope to gain insight into differences between leukemic and normal stem cells. In addition, specific antibody binding to leukemic cell surface structures may serve to block their function and inhibit leukemic cell proliferation, possibly in a leukemia-specific manner. We have considerable experience in the use of monoclonal antibodies targeting human leukemic cells and we have demonstrated their therapeutic efficacy in human leukemia growing in immunodeficient mice (antibodies targeting the vascular endothelial growth factor receptors, antibodies against the Insulin-like growth factor receptor 1, antibodies against the Flt3 receptor).

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Zhang Y, Morrone G, Zhang J, Chen X, Lu X, Ma L, Moore M, Zhou P. CUL-4A stimulates ubiquitylation and degradation of the HOXA9 homeodomain protein. EMBO J. 2003, 22:6057-6067.

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Nimer SD, Moore MAS. Effects of the leukemia-associated AML1/ETO protein on hematopoietic stem and progenitor cells. Oncogene. 2004, 23:4249-54.

Moldenhauer A, Frank RC, Pinilla-Ibarz J, Holland G, Boccuni P, Scheinberg D, Salama A, Seeger K, Moore MA, Nimer SD. Histone deacetylase inhibition improves dendritic cell differentiation of leukemic blasts with AML1-containing fusion proteins. J Leukoc Biol. 2004, 76:623-633.

Schuringa JJ, Chung KY, Morrone G, Moore MAS. Constitutive activation of STAT5 promotes human hematopoietic stem cell self-renewal and erythroid differentiation. J Exp Med. 2004, 2000:623-635.

Schuringa JJ, Wu K, Morrone G, Moore MA. Enforced activation of STAT5A facilitates the generation of embryonic stem-derived hematopoietic stem cells that contribute to hematopoiesis in vivo. Stem Cells. 2004, 22:1191-1204.

Chung KY, Morrone G, Schuringa JJ, Wong B, Dorn DC, Moore MA. Enforced expression of an Flt3 internal tandem duplication in human CD34+ cells confers properties of self-renewal and enhanced erythropoiesis. Blood. 2005, 105:77-84.

Mulloy JC, Cammenga J, Berguido FJ, Wu K, Zhou P, Comenzo RL, Jahnwar S, Moore MAS, Nimer SD. Maintaining the self-renewal and differentiation potential of human CD34+ hematopoietic stem cells using a single genetic element. Blood. 2003, 102:4369-76.

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