IntroductionThe modification of cellular proteins by ubiquitin (Ub) and ubiquitin-like (UbL) molecules and the degradation of polyubiquitinated substrates by the proteasome control a broad spectrum of cellular processes that regulate cell proliferation, functional differentiation and apoptosis (reviewed in [1] Abstract The ubiquitin C-terminal hydrolase-L1 (UCH-L1) is a deubiquitinating enzyme that catalyses the hydrolysis of polyubiquitin precursors and small ubiquitin adducts. UCH-L1 has been detected in a variety of malignant and metastatic tumours but its biological function in these cells is unknown. We have previously shown that UCH-L1 is highly expressed in Burkitt's lymphoma (BL) and is up-regulated upon infection of B lymphocytes with Epstein-Barr virus (EBV). Here we show that knockdown of UCH-L1 by RNAi inhibits the proliferation of BL cells in suspension and semisolid agar and activates strong LFA-1-dependent homotypic adhesion. Induction of cell adhesion correlated with cation-induced binding to ICAM-1, clustering of LFA-1 into lipid rafts and constitutive activation of the Rap1 and Rac1GTPases. Expression of a catalytically active UCH-L1 promoted the proliferation of a UCH-L1-negative EBV transformed lymphoblastoid cell line (LCL) and inhibited cell adhesion, whereas a catalytic mutant had no effect, confirming the requirement of UCH-L1 enzymatic activity for the regulation of these phenotypes. Our results identify UCH-L1 as a new player in the signalling pathways that promote the proliferation and invasive capacity of malignant B cells.

The oligopeptidase tripeptidyl-peptidase II (TPP II) is upregulated Burkitt's lymphoma (BL) cells that overexpress the c-myc proto-oncogene and is required for their growth and survival. Here we show that overexpression of TPP II induces accelerated growth and resistance to apoptosis in human embryonic kidney 293 cells. This correlates with the appearance of multiple chromosomal aberrations, numerical and structural centrosome abnormalities, and multipolar cell divisions. Similar mitotic aberrations were also observed in a panel of BL lines and were suppressed, in parallel with TPP II down-regulation, upon reversion of BL-like characteristics in EBV-immortalized B lymphocytes carrying a tetracyclineregulated c-myc. Functional TPP II knockdown by small interfering RNA expression in BL cells caused the appearance of giant polynucleated cells that failed to complete cell division. Collectively, these data point to a role of TPP II in the regulation of centrosome homeostasis and mitotic fidelity suggesting that this enzyme may be a critical player in the induction and/or maintenance of genetic instability in malignant cells. (Cancer Res 2005; 65(4): 1361-8)

Retroviral vectors are an efficient and widely employed means of introducing an exogenous expression cassette into target cells. These vectors have been shown to integrate semi-randomly into the cellular genome, and can be associated with genotoxicity due to impact on expression of proximate genes. Therefore, efficient and accurate integration site analysis, while quantifying contributions of individual vector-containing clones, is desirable. Linear amplification-mediated polymerase chain reaction (LAM-PCR) is a widely used technique for identifying integrated proviral and host genomic DNA junctions. However, LAM-PCR is subject to selection bias inherent in the reliance of the assay on the presence of a restriction enzyme-cutting site adjacent to a retrievable integration site, and it is further limited by an inability to discriminate prior to sequencing between the flanking genomic DNA of interest and uninformative internal vector DNA. We report a modified restriction enzyme-free LAM-PCR (Re-free LAM-PCR) approach that is less time and labor intensive compared to conventional LAM-PCR, but in contrast to some other nonrestrictive methods, compares in efficiency and sensitivity, excludes retrieval of uninformative internal vector sequences, and allows retrieval of integration sites unbiased by the presence of nearby restriction sites. However, we report that Re-free LAM-PCR remains inaccurate for quantitation of the relative contributions of individual integration site-containing clones in a polyclonal setting, suggesting that bias in LAM-PCR retrieval of integration sites is not wholly explained by restriction enzyme-related factors.

The risk of genotoxicity of retroviral vector-delivered gene therapy targeting hematopoietic stem cells (HSCs) has been highlighted by the development of clonal dominance and malignancies in human and animal gene therapy trials. Large-animal models have proven invaluable to test the safety of retroviral vectors, but the detection of clonal dominance may require years of follow-up. We hypothesized that hematopoietic stress may accelerate the proliferation and therefore the detection of abnormal clones in these models. We administered four monthly busulfan (Bu) infusions to induce hematopoietic stress in a healthy rhesus macaque previously transplanted with CD34 þ cells transduced with retroviral vectors carrying a simple marker gene. Busulfan administration resulted in significant cytopenias with each cycle, and prolonged pancytopenia after the final cycle with eventual recovery. Before busulfan treatment there was highly polyclonal marking in all lineages. After Bu administration clonal diversity was markedly decreased in all lineages. Unexpectedly, we found no evidence of selection of the MDS1=EVI1 clones present before Bu administration, but a clone with a vector integration in intron 1 of the histone deacetylase-7 (HDAC7) gene became dominant in granulocytes over time after Bu administration. The overall marking level in the animal was increased significantly after Bu treatment and coincident with expansion of the HDAC7 clone, suggesting an in vivo advantage for this clone under stress. HDAC7 expression was upregulated in marrow progenitors containing the vector. Almost 5 years after Bu administration, the animal developed progressive cytopenias, and at autopsy the marrow showed complete lack of neutrophil or platelet maturation, with a new population of approximately 20% undifferentiated blasts. These data suggest that chemotherapeutic stress may accelerate vector-related clonal dominance, even in the absence of drug resistance genes expressed by the vector. This model may both accelerate the detection of abnormal clones to facilitate analysis of genotoxicity for human gene therapy, and help assess the safety of administering myelotoxic chemotherapeutic agents in patients previously engrafted with vector-containing cells.

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Retroviral vector insertion induced mutagenesis has been demonstrated to be associated with the development of leukemias and other tumors in human and animals. In a steady-state, any proliferative advantage of “pre-malignant” cells harboring retroviral insertions takes years to manifest. We hypothesized that, under hematopoietic stress, behavior of some potentially abnormal clones may rapidly become evident. Busulfan (Bu) is known to have profound effects on stem cell behavior, specifically stem cell numbers, thus may function as hematopoietic stressor. In this study, we analyzed the impact of repetitive doses of Bu on hematopoietic clones with retroviral vector insertion in vivo in two rhesus macaques, RQ2297 and RQ2314. Eight years ago, RQ2297 and RQ2314 underwent autologous transplantation of CD34+ cells transduced with standard MLV-derived retrovirus vectors (G1Na and LNL6) carrying bacterial NeoR gene marker. The two animals received a single dose of Bu 4mg/Kg approximately 2.5 years after transplant, and three to four additional monthly Bu injections (4mg/kg, 4 mg/kg, 6mg/kg and 6mg/kg for RQ2297, and 4mg/kg, 4 mg/kg and 6mg/kg for RQ2314) were administered 4 and 7 years post transplant for RQ2297 and RQ2314, respectively. LAM-PCR and Gene Scanning were employed to profile retroviral integration sites (RIS) in peripheral white blood cells. The results showed that repetitive Bu administration resulted in prolonged (15–20 weeks) decrease in three-lineage blood counts followed by complete recovery to baseline approximately four months after the last infusion of Bu in both animals except for persistent thrombocytopenia in RQ2297. RQ2297 harbored high polyclonality prior to Bu with 68, 75 and 63 independent integrations in granulocytes, T and B cells, respectively, including two independent clones with retroviral insertions in the Mds1/Evi1 locus, previously implicated in clonal expansion and preferential engraftment in granulocytes. Following repetitive Bu, the clonal diversity decreased markedly, the Mds1- Evi1 clones disappeared, but instead a highly dominant clone emerged, characterized by a vector insertion within intron 1 of the HDAC7 gene, which was not dominant prior to repetitive Bu, accounting for 3% of shotgun cloned LAM-PCR insertions, and constituted 71% and 94% of the cloned sequences, 2.5–2.8 years post Bu, respectively. The overall number of independent clones had decreased to only 3–9 in granulocytes at the same time points. Surprisingly, the NeoR level increased up to 11%–28.2% 2.5 and 2.8 years post Bu compared to 0.1% prior to Bu (p =0.006 and 0.003) only in granulocytes whereas the NeoR level was not significantly changed in T and B cells (1%–2%), indicating that the clone with retroviral insertion within HDAC7 gene became highly dominant and dramatically expanded following Bu-induced stress. Compared with non-transplanted animals, HDAC7 expression measured by quantitative RT-PCR was 4.3-fold increased in CFU enriched for the HDAC7 clone by G418 selection, but a similar increase was also detected in CFU not enriched for the HDAC7 clone, indicating no upregulation or downregulation of HDAC7 expression. While there has been no report about direct relevance of HDAC7 to cancers, its association with various oncogenes and tumor suppressor genes has been well established. No evidence of malignancy and other hematopoietic disorders has been detected in RQ2297 to date. Those data indicated immortalization or preferential survival and engraftment of cells with retroviral insertion within HDAC7A locus. For RQ2314, the number of clones with RIS decreased to 12 six months post Bu administration from 80 prior to Bu in granulocytes, however no highly dominant clones have emerged yet. The NeoR level in granulocyes 6 months post Bu increased to 7.5% from 2.7% prior to Bu (p=. 0.038), indicating that some clones expanded. The data suggest that repetitive busulfan administration induced emergence of dominant and consequently expanding hematopoietic clones with retroviral insertion in rhesus macaques. This model could both accelerate analysis of genotoxicity related to vector insertions, as well as help assess the safety of administering myelotoxic chemotherapy in patients previously engrafted with vector-containing cells.

The recent report of the development of leukemia in four children receiving CD34+ cells transduced with a retroviral vector expressing a corrective IL2 receptor transgene has focused interest on the mechanisms and risks of insertional mutagenesis. In our primate model, we have found evidence for highly non-random integration of retroviral vectors into areas of the genome that could activate genes implicated in leukemogenesis. In a steady-state, any proliferative “pre-malignant” advantage of cells harboring these insertions takes years to manifest. Under hematopoietic stress, abnormalities of behavior of these clones may become evident. The alkylating agent busulfan is known to have profound effects on stem cell behavior and specifically stem cell numbers. We hypothesized that repeated doses of busulfan will result in significant hematopoietic stress at a stem cell level, allowing us to analyze the impact on clonal patterns of retrovirally-transduced cells in vivo and investigate whether clones containing a single insertion proximal to an oncogene gain a competitive advantage, a potentially pre-leukemic situation tendency. Seven years ago, rhesus macaque RQ2297 underwent autologous transplantation of CD34+ cells transduced with standard MLV-derived retrovirus vectors carrying the NeoR gene marker. The animal received a single dose of busulfan 4mg/Kg approximately 2.5 years after transplant, and four additional monthly busulfan injections (4mg/kg, 4 mg/kg, 6mg/kg and 6mg/kg) were administered 4 years post-transplant. LAM-PCR was used to follow integration sites in granulocytes before and after busulfan administration. Repetitive busulfan administration resulted in prolonged decrease in three-lineage blood counts, with a complete recovery to baseline four months after the last infusion of busulfan. The animal was initially highly polyclonal with over 50 independent integrations prior to busulfan, including two independent insertions in the Mds1/Evi1 locus, previously implicated in clonal expansion and preferential engraftment. Following repetitive busulfan, the clonal diversity decreased markedly. Surprisingly, the Mds1-Evi1 clones disappeared, but instead a highly dominant clone emerged, characterized by a vector insertion 5.7 kb upstream of the HDAC7A gene. This clone was detected prior to busulfan, but was not dominant, accounting for 3.2% of shotgun cloned LAM-PCR insertions. 2.5–2.8 years later, this insertion constituted 71% and 94% of the cloned sequences, respectively, and the overall number of independent clones had decreased to only 3–8 at the same time points. HDAC7A belongs to class IIa histone deacetylases. While no direct alterations of HDAC7 genes have been demonstrated in human cancers, its association with various oncogenes and tumor suppressor genes is well established. No evidence of malignancy has been detected in this animal to date. Under conditions of stem cell stress, stem cell clones containing retroviral vectors integrated in the proximity of the HDAC7A locus gained a selective advantage, suggesting immortalization or preferential survival and engraftment of cells with activation of the HDAC7A locus. This model could both accelerate analysis of genotoxicity related to vector insertions, as well as help assess the safety of administering myelotoxic chemotherapy in patients previously engrafted with vector-containing cells.