Homeobox B4 Research Articles

Transmission and expansion of HOXB4-induced leukemia in two immunosuppressed dogs: Implications for a new canine leukemia model

There are currently no large animal models to study the biology of leukemia and development of novel antileukemia therapies. We have previously shown that dogs transplanted with homeobox B4 (HOXB4)-transduced autologous CD34(+) cells developed myeloid leukemia associated with HOXB4 overexpression. Here we describe the transmission, engraftment, and expansion of these canine leukemia cells into two genetically unrelated, immunosuppressed dogs. Two dogs immunosuppressed after major histocompatibility complex-haploidentical hematopoietic cell transplantation and exhibiting mixed donor-host chimerism were accidentally infused trace amounts of HOXB4-overexpressing leukemia cells from a third-party dog. Six weeks after infusion of HOXB4-overexpressing leukemia cells, these two dogs rapidly developed myeloid leukemia consisting of marrow and organ infiltration, circulating blasts, and, in one dog, chloromatous masses. Despite neither of these dogs sharing any dog leukocyte antigen haplotypes with the sentinel case, the HOXB4-transduced clones engrafted and proliferated without difficulty in the presence of immunosuppression. Chimerism studies in both dogs confirmed that donor and, in one case, host hematopoietic cell engraftment was lost and replaced by third-party HOXB4 cells. The engraftment and expansion of these leukemia cells in dogs will allow studies into the biology of leukemia and development and evaluation of novel antileukemia therapies in a clinically relevant large animal model.

In vivo selection of hematopoietic progenitor cells and temozolomide dose intensification in rhesus macaques through lentiviral transduction with a drug resistance gene

Major limitations to gene therapy using HSCs are low gene transfer efficiency and the inability of most therapeutic genes to confer a selective advantage on the gene-corrected cells. One approach to enrich for gene-modified cells in vivo is to include in the retroviral vector a drug resistance gene, such as the P140K mutant of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT*). We transplanted 5 rhesus macaques with CD34+ cells transduced with lentiviral vectors encoding MGMT* and a fluorescent marker, with or without homeobox B4 (HOXB4), a potent stem cell self-renewal gene. Transgene expression and common integration sites in lymphoid and myeloid lineages several months after transplantation confirmed transduction of long-term repopulating HSCs. However, all animals showed only a transient increase in gene-marked lymphoid and myeloid cells after O6-benzylguanine (BG) and temozolomide (TMZ) administration. In 1 animal, cells transduced with MGMT* lentiviral vectors were protected and expanded after multiple courses of BG/TMZ, providing a substantial increase in the maximum tolerated dose of TMZ. Additional cycles of chemotherapy using 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) resulted in similar increases in gene marking levels, but caused high levels of nonhematopoietic toxicity. Inclusion of HOXB4 in the MGMT* vectors resulted in no substantial increase in gene marking or HSC amplification after chemotherapy treatment. Our data therefore suggest that lentivirally mediated gene transfer in transplanted HSCs can provide in vivo chemoprotection of progenitor cells, although selection of long-term repopulating HSCs was not seen.

Integrative Genomic Analysis Reveals Aberrant Epigenetic Marks in MDS That Can Be Seen in Peripheral Blood Leucocytes

Myelodysplasia (MDS) is a clonal hematopoietic disorder that leads to ineffective hematopoiesis and peripheral cytopenias. DNMT inhibitors such as azacytidine have led to clinical responses in patients, though global epigenetic alterations in MDS have not been well described. The transmission of these epigenetic marks during hematopoietic differentiation and their role in disease pathophysiology is also unknown. We first compared global methylation profiles of 8 bone marrow samples with peripheral leucocytes by using a recently described novel method, the HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR; Khulan et al, Genome Res. 2006 Aug;16(8)) that uses differential methylation-specific digestion by HpaII and MspI followed by amplification, two color labeling and hybridization to quantitatively determine individual promoter CpG island methylation. A whole genome human promoter array (Nimblegen) was used to determine the level of methylation of 25626 gene promoters by calculating HpaII/MspI cut fragment intensity ratio. We observed a high correlation (r=0.89–0.96) of epigenetic marks between bone marrow and peripheral blood samples suggesting that a majority of epigenetic marks can be also be seen in differentiated cells. We subsequently compared peripheral blood leucocytes from 20 patients with MDS with 10 age-matched normal and anemic controls. Parallel gene expression analysis was performed using 37K oligo maskless arrays on cDNA from the same samples. Analysis showed that whole genome methylation profiling has greater discriminatory power in separating clusters of MDS samples from normal and anemic controls when compared to gene expression analysis. Epigenetic profiling demonstrated two clusters of MDS based on similarity of aberrant epigenetic changes. Overall, there was a trend towards hypermethylation in MDS, albeit not statistically significant given the large number of relatively unchanged genes. Detailed analysis revealed several novel differentially methylated genes that had corresponding changes in gene expression, when MDS samples were compared to the controls with a low false discovery rate of analysis. Interesting genes getting hypomethylated and overexpressed included TNF superfamily member 9, granulocyte pep A, microsomal glutathione S-transferase, homeo box B4, mitochondrial RPL11, and others. Similarly, the set of genes that were getting hypermethylated with associated decrease in gene expression included Evi-1, DAPK, HOXB3, Protein Phosphatase 1, CEBPB, mutated in colorectal cancer (MCC), myeloid-lymphoid or mixed-lineage leukemia 5 (MLL5), plasminogen-related protein B, ovarian cancer related protein 1 (ORP1), and others. In addition, we did array-based comparative genome hybridization (aCGH) to look at exact genome copy number changes in these samples. We found changes that were not detectable by conventional karyotyping in all samples. Commonly seen alterations were del(14q11), del(20q11), del(5q13), del(8p23), amp(1q42), amp(5q11), amp(17q12), amp(19q13) and amp(7q22). Integrative analysis revealed sets of genes that were either silenced by methylation or deletion in different patients.Thus, our data demonstrates that promoter DNA methylation changes are an important phenomenon in MDS evolution, and are associated with changes in expression of genes playing important roles in cancer development and/or progression. We also show that previously unrecognizable changes in copy number exist in most patients with MDS. In addition, our work shows that whole genome methylation assays, even when done on peripheral blood leukocytes, can be used for potential biomarker studies in the diagnosis of MDS.

High incidence of leukemia in large animals after stem cell gene therapy with a HOXB4-expressing retroviral vector

Retroviral vector-mediated HSC gene therapy has been used to treat individuals with a number of life-threatening diseases. However, some patients with SCID-X1 developed retroviral vector-mediated leukemia after treatment. The selective growth advantage of gene-modified cells in patients with SCID-X1 suggests that the transgene may have played a role in leukemogenesis. Here we report that 2 of 2 dogs and 1 of 2 macaques developed myeloid leukemia approximately 2 years after being transplanted with cells that overexpressed homeobox B4 (HOXB4) and cells transduced with a control gammaretroviral vector that did not express HOXB4. The leukemic cells had dysregulated expression of oncogenes, a block in myeloid differentiation, and overexpression of HOXB4. HOXB4 knockdown restored differentiation in leukemic cells, suggesting involvement of HOXB4. In contrast, leukemia did not arise from the cells carrying the control gammaretroviral vector. In addition, leukemia did not arise in 5 animals with high-level marking and polyclonal long-term repopulation following transplantation with cells transduced with an identical gammaretrovirus vector backbone expressing methylguanine methyltransferase. These findings, combined with the absence of leukemia in many other large animals transplanted with cells transduced with gammaretroviral vectors expressing genes other than HOXB4, show that HOXB4 overexpression poses a significant risk of leukemogenesis. Our data thus suggest the continued need for caution in genetic manipulation of repopulating cells, particularly when the transgene might impart an intrinsic growth advantage.

HOXB4 and retroviral vectors: adding fuel to the fire

The transcription factor homeobox B4 (HOXB4) is a promising agent capable of providing a growth advantage to genetically modified hematopoietic stem and progenitor cells (HSPCs). In this issue of the JCI, Zhang and colleagues overexpressed HOXB4 in HSPCs from large animals using retroviral vectors (see the related article beginning on page 1502). Two years after transplantation, most animals developed leukemia, a consequence of combined HOXB4 and deregulated protooncogene expression. These results highlight the risks of combining integrating vectors and growth-promoting genes for clinical applications.

Development of Novel Multigene Lentiviral Vectors.

Lentiviral vectors are efficient in transducing dividing and non-dividing cells. Many gene transfer applications require vectors that express more than one protein which include but not limited to therapeutic gene plus a selectable marker gene, multiple genes encoding different subunits of a complex protein or multiple independent genes that cooperate functionally. Previous strategies in constructing multigene lentiviral vectors by other groups included splicing, multiple internal promoters, internal ribosome entry site (IRES), and fusion proteins. In this study we explored the feasibility of expressing homeobox B4 (HOXB4), O6-methylguanine-DNA-methyltransferase (MGMT), and enhanced green fluorescent protein (EGFP) under the control of a single promoter with the use of IRES sequence from encephalomyocarditis (EMCV) virus and/or foot and mouth disease virus (FMDV) 2A cleavage factor in a HIV-1 based self-inactivating lentiviral vector. In bicistronic vectors the small FMDV 2A sequence can be used efficiently as an alternative to IRES. In case of tricistronic vectors the 2A sequence can be used in combination with IRES to obtain simultaneous expression of three cDNAs under the control of a single internal promoter. Monocistronic, bicistronic and tricistronic vectors were constructed and a three-plasmid expression system was used to generate vesicular stomatitis virus glycoprotein pseudotyped lentiviral vector particles. Viral titers were measured by quantification of HIV p24 gag by ELISA. The relative efficiency of transgene expression in transduced cells by various vectors was compared by appropriate methods including fluorescence microscopy, flow cytometry, immunocytochemistry, biochemical assay, and western blotting. All the multigene vectors produced high titer viral particles and were able to simultaneously express two or three transgenes in transduced cells. However, the expression of EGFP from monocistronic vector as measured by mean fluorescence intensity was 3–8 times and 10–20 times higher than that of bicistronic and tricistronic vectors respectively. Notably expression of second gene encoded by the bicistronic vector containing FMDV 2A was 2–3 fold higher than that of IRES-based vector. Expression of MGMT in monocistronic and bicistronic constructs was also 4 to 20 times higher than tricistronic vectors. The FMDV 2A cleavage factor efficiently mediated the generation of the expected cleavage products from the artificial fusion protein. These vectors can be used in a wide range of applications including the expression of: multiple subunits of a functional protein, tumor antigen and co-stimulatory molecule(s), multiple tumor antigens for immunotherapy applications, and co-expression of selectable marker gene/s with therapeutic gene/s.

In vitro expansion of hematopoietic stem cells by recombinant TAT-HOXB4 protein.

Hematopoietic stem cells (HSCs) can self-renew extensively after transplantation. The conditions supporting their in vitro expansion are still being defined. Retroviral overexpression of the human homeobox B4 (HOXB4) gene in mouse bone marrow cells enables over 40-fold expansion of HSCs in vitro. To circumvent the requirement for retroviral infection, we used recombinant human TAT-HOXB4 protein carrying the protein transduction domain of the HIV transactivating protein (TAT) as a potential growth factor for stem cells. HSCs exposed to TAT-HOXB4 for 4 d expanded by about four- to sixfold and were 8-20 times more numerous than HSCs in control cultures, indicating that HSC expansion induced by TAT-HOXB4 was comparable to that induced by the human HOXB4 retrovirus during a similar period of observation. Our results also show that TAT-HOXB4-expanded HSC populations retain their normal in vivo potential for differentiation and long-term repopulation. It is thus feasible to exploit recombinant HOXB4 protein for rapid and significant ex vivo expansion of normal HSCs.

NF-Y cooperates with USF1/2 to induce the hematopoietic expression of HOXB4

AbstractThe transcription factor homeobox B4 (HOXB4) is preferentially expressed in immature hematopoietic cells and implicated in the transition from primitive hematopoiesis to definitive hematopoiesis as well as in immature hematopoietic cell proliferation and differentiation. We previously identified Hox response element 1 (HxRE-1) and HxRE-2/E-box as 2 critical DNA-binding sites of the HOXB4 promoter active in hematopoietic cells and demonstrated that upstream stimulating factor 1 and 2 (USF1/2) activate HOXB4 transcription through their binding to the E-box site. Here we report that the trimeric regulatory complex nuclear factor Y (NF-Y) is the factor that recognizes HxRE-1 and activates the HOXB4 promoter in hematopoietic cells. We further show that NF-Y interacts biochemically with USF1/2 on the HOXB4 promoter, and that the formation of this NF-Y/USF1/2 complex is required for the full activity of the HOXB4 promoter. Most important, NF-Ya subunit protein levels are found to be lower in c-Kit-Gr-1+ granulocytic bone marrow (BM) cells than in c-Kit+ immature BM cells, in parallel with a reduction of NF-Y occupancy on the HOXB4 promoter as shown by chromatin immunoprecipitation (ChIP) assay. These results suggest that NF-Y is a developmentally regulated inducer of the HOXB4 gene in hematopoietic cells. (Blood. 2003;102:2420-2427)

High-level ectopic HOXB4 expression confers a profound in vivo competitive growth advantage on human cord blood CD34+ cells, but impairs lymphomyeloid differentiation

Ectopic retroviral expression of homeobox B4 (HOXB4) causes an accelerated and enhanced regeneration of murine hematopoietic stem cells (HSCs) and is not known to compromise any program of lineage differentiation. However, HOXB4 expression levels for expansion of human stem cells have still to be established. To test the proposed hypothesis that HOXB4 could become a prime tool for in vivo expansion of genetically modified human HSCs, we retrovirally overexpressed HOXB4 in purified cord blood (CB) CD34+ cells together with green fluorescent protein (GFP) as a reporter protein, and evaluated the impact of ectopic HOXB4 expression on proliferation and differentiation in vitro and in vivo. When injected separately into nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice or in competition with control vector-transduced cells, HOXB4-overexpressing cord blood CD34+ cells had a selective growth advantage in vivo, which resulted in a marked enhancement of the primitive CD34+ subpopulation (P =.01). However, high HOXB4 expression substantially impaired the myeloerythroid differentiation program, and this was reflected in a severe reduction of erythroid and myeloid progenitors in vitro (P <.03) and in vivo (P =.01). Furthermore, HOXB4 overexpression also significantly reduced B-cell output (P <.01). These results show for the first time unwanted side effects of ectopic HOXB4 expression and therefore underscore the need to carefully determine the therapeutic window of HOXB4 expression levels before initializing clinical trials.