Reciprocal Relationship between O6-Methylguanine-DNA Methyltransferase P140K Expression Level and Chemoprotection of Hematopoietic Stem Cells

Abstract

Retroviral-mediated delivery of the P140K mutant O6-methylguanine-DNA methyltransferase (MGMTP140K) into hematopoietic stem cells (HSC) has been proposed as a means to protect against dose-limiting myelosuppressive toxicity ensuing from chemotherapy combining O6-alkylating agents (e.g., temozolomide) with pseudosubstrate inhibitors (such as O6-benzylguanine) of endogenous MGMT. Because detoxification of O6-alkylguanine adducts by MGMT is stoichiometric, it has been suggested that higher levels of MGMT will afford better protection to gene-modified HSC. However, accomplishing this goal would potentially be in conflict with current efforts in the gene therapy field, which aim to incorporate weaker enhancer elements to avoid insertional mutagenesis. Using a panel of self-inactivating gamma-retroviral vectors that express a range of MGMTP140K activity, we show that MGMTP140K expression by weaker cellular promoter/enhancers is sufficient for in vivo protection/selection following treatment with O6-benzylguanine/temozolomide. Conversely, the highest level of MGMTP140K activity did not promote efficient in vivo protection despite mediating detoxification of O6-alkylguanine adducts. Moreover, very high expression of MGMTP140K was associated with a competitive repopulation defect in HSC. Mechanistically, we show a defect in cellular proliferation associated with elevated expression of MGMTP140K, but not wild-type MGMT. This proliferation defect correlated with increased localization of MGMTP140K to the nucleus/chromatin. These data show that very high expression of MGMTP140K has a deleterious effect on cellular proliferation, engraftment, and chemoprotection. These studies have direct translational relevance to ongoing clinical gene therapy studies using MGMTP140K, whereas the novel mechanistic findings are relevant to the basic understanding of DNA repair by MGMT.

F2A sequence linking MGMTP140K and MDR1 in a bicistronic lentiviral vector enables efficient chemoprotection of haematopoietic stem cells

“…This implies that increased amounts of MGMT P140K protein are not required for efficient protection against DNA damage induced by alkylating drugs. This result is in line with the findings by Milsom et al 26 demonstrating that in contrast to a strong viral promoter a weaker cellular promoter was sufficient for an in vivo protection after treatment with BCNU or TMZ.…”

“…66 Comparison of a strong viral and a weaker cellular promoter revealed that use of the cellular promoter resulted in sufficient expression and detoxification efficiency of MGMT P140K protein and in better engraftment of transduced HSCs in mice. 26 Promoter methylation and silencing has been described as a disadvantage of the spleen focus-forming virus promoter, 67,68 which we used in our vector. To circumvent this problem, replacement of the spleen focus-forming virus promoter by the methylation-resistant ubiquitous chromatin opening element should enable stable and long-term expression of transgenes in HSCs.…”

“…23,24 As for MDR1 no chemoprotection could be achieved in nonhuman primates and in humans. 25,26 Simultaneous gammaretroviral transfer of both resistance genes was performed only in vitro into cell lines or primary hematopoietic cells to assay for chemoprotection against various combinations of certain MDR1 substrate drugs with alkylating agents. [27][28][29][30] However, we showed for the first time efficient chemoprotection of HSCs using a third-generation lentiviral SIN vector for transfer of MDR1 and the MGMT P140K mutant.…”

Mechanisms controlling titer and expression of bidirectional lentiviral and gammaretroviral vectors

“…In contrast, mCherry expression from GV and LV bidirectional vectors did not differ but was 44 GV and LV bidirectional vectors were constructed harboring MGMT-P140K and eGFP in either orientation. We chose the modest human PGK promoter as high MGMT-P140K expression was shown to inhibit proliferation of hematopoietic stem/progenitor cells.…”

“…We chose the modest human PGK promoter as high MGMT-P140K expression was shown to inhibit proliferation of hematopoietic stem/progenitor cells. 44 Pilot experiments performed with GV and LV vectors showed that only the expression of MGMT-P140K in sense orientation was strong enough to yield a large amount of double-positive cells (data not shown). When a murine myeloid progenitor cell line, 32D, was transduced with these vectors (Figure 5a According to the coexpressed eGFP (Figure 5c), GV and LV transduced cells could be stepwise enriched from 20 to 93% or 43 to 93% by increasing BCNU concentrations from 0 to 35 mM, respectively.…”