Despite these encouraging findings, investigators have worried that inclusion of
HOXB4 in gene transfer vectors could carry significant risks, particularly in the context of evolving concerns regarding vector-related insertional mutagenesis (
6,
7). The strong enhancers contained in retroviral genomes can activate adjacent cellular genes following integration, but the risk posed by one or a few integrated vectors per cell was estimated to be very low. However, these assumptions were shattered in 2002, when French researchers announced that a child who had shown unequivocal clinical benefit in the HSPC gene therapy trial for X-linked SCID (SCID-X) (
8,
9) developed a vector-related T cell acute leukemia three years after infusion of retrovirally modified CD34
+ cells (
10). Since then, three other children in the French trial and, more recently, one patient in a similar SCID-X study conducted by British researchers have developed leukemias. In at least three cases, the tumor cells had a clonal vector insertion activating the LIM domain only 2 (
LMO2) transcription factor gene (
11). It was hypothesized that cooperation between
LMO2 and the growth-promoting effect of the corrective IL-2 receptor γ (
IL2RG) transgene resulted in leukemic transformation, perhaps limiting the problem to SCID-X (
12). However, 2 patients with chronic granulomatous disease treated in an HSPC gene therapy protocol have developed abnormal hematopoiesis and clonal expansion of vector-containing cells due to activation of protooncogenes (e.g.,
MDS-EVI1), despite the lack of growth-promoting activity of the corrective transgene (
13). Our group also reported the development of vector-associated myeloid sarcoma in a rhesus macaque five years following transplantation of HSPCs transduced with a vector containing only a marker gene and a drug-resistance gene (
14).
It has been demonstrated that high-level HOXB4 expression, achieved with retroviral (
5) or adenoviral (
15) vectors in human CD34
+ cells, perturbed the myeloid differentiation program both in vitro (
5,
15) an in vivo (
5) without frank leukemia. Similarly, disturbed myeloid differentiation was observed in a multipotent hematopoietic cell line in vitro (
16). Ectopic HOXB4 expression also proved to be key in the production of primitive mouse hematopoietic cells from embryonic stem cells then able to engraft irradiated recipients; however, these mice had abnormal hematopoiesis with enforced myeloid and suppressed lymphoid development (
17).
Despite these warning signs and continued concern regarding potential cooperativity between growth-altering transgenes and vector insertion sites, investigators continued to pursue the potential of the HOXB4 approach. In a study published in 2006, Kiem and his research group at the Fred Hutchinson Cancer Research Center reported preliminary results in their powerful non-human primate competitive repopulation model (
18). They transduced monkey CD34
+ cells with either a
HOXB4-expressing vector or a control vector expressing only a marker gene and analyzed the competitive repopulating ability of these cells in vivo. As hoped, there was a very significant advantage for the
HOXB4-transduced cells early following engraftment of the monkeys, but in contrast to mouse studies, a much less significant advantage for the
HOXB4-transduced cells was observed long-term after transplantation (
18).
In their study in this issue of the
JCI, the same group now reports the first instances of leukemia linked to HOXB4 expression, both in the original group of monkeys now followed longer term and in dogs that received cells transduced with a
HOXB4-expressing vector (
19). Some 2 years after transplantation, 3 of 4 animals (2 of 2 dogs and 1 of 2 non-human primates) developed acute myeloid leukemias. Several lines of evidence strongly implicated HOXB4 in leukemogenesis. High-level HOXB4 expression was confirmed in the leukemic cells. Also, 3 of 4 animals followed long-term in this study developed leukemia, compared with none of more than 40 dogs and monkeys transplanted with cells transduced with similar vectors not expressing HOXB4 by the same research group and only 1 of 80 macaques followed long-term by our group (
14). The most compelling evidence for the pivotal role of HOXB4 was provided by deriving a cell line from the leukemic cells of one animal and using short hairpin RNAs to knockdown HOXB4 expression in these cells. Profound growth inhibition and rapid cell death occurred with downregulation of the
HOXB4 transgene.
Was HOXB4 expression sufficient as a single event to cause leukemia? To gain further insights, the authors localized vector integration sites in the genome (
19). Interestingly, insertion sites in all tumors were near or within several protooncogenes, including
c-myb and
PRDM16 (already implicated in the chronic granulomatous disease trial described above), leading to aberrant expression of these genes. This suggests cooperativity between
HOXB4 and the nearby activated protooncogenes, similar to the proposed cooperativity between
LMO2 vector activation and the
IL2RG transgene in the SCID-X trials. Overall,
HOXB4 can be considered as a catalyst for leukemia development following retroviral gene transfer (Figure ).
This study underscores the importance of large animal models in further development of gene and other novel HSPC-directed therapies. Perhaps no leukemias linked to HOXB4 overexpression developed in mice because too few gene-modified HSPCs could be given to these small animals, much fewer than the number of transduced HSPCs transplanted in clinical trials or larger animal models. Or perhaps a long latency period, longer than the lifespan of a mouse, was required before evidence of leukemic transformation could be seen. HOXB4 or similar growth-promoting genes are not a sine qua non for the development of malignancies following HSPC gene transfer, but they appear to significantly increase the probability of such an event.
See the article"
