Dual agent chemoprotection by retroviral co‐expression of either MDR1 or MRP1 with the P₁₄₀K mutant of O⁶‐methylguanine‐DNA‐methyl transferase

Abstract

Tumour resistance to chemotherapeutic agents results in most chemotherapy being administered in a multi-agent fashion that is often associated with a high level of toxicity in highly proliferative tissues such as the haematopoietic compartment. Thus, whilst many genetic manipulation strategies aim to protect normal tissue against a single component of a multi-agent regime, it is clearly preferable to protect normal cells against all toxicities. In this study we have used retroviral gene transfer to achieve co-expression of either p-glycoprotein (MDR1) or multi-drug resistance-related protein 1 (MRP1) with the P140K mutant form of O6-methylguanine-DNA-methyl transferase (MGMT) which, unlike the wild-type protein, is insensitive to inactivation by tumour sensitisers such as O6-benzylguanine (O6-BeG) or PaTrin2. The combination of certain MDR1/MRP1 substrate drugs with O6-alkylating agents (against which MGMT confers resistance) is particularly myelotoxic. We show here that haematopoietic progenitors co-expressing mutant MGMT with an ABC-transporter exhibit resistance to combination chemotherapy in vitro. This combination of drug transporter and DNA repair function may provide an effective in vivo protection of the haematopoietic compartment during tumour ablation using combination chemotherapy.

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

“…33,55 Especially for MGMT, it was shown that the IRES-dependent expression in a retroviral vector resulted only in a tenth of active protein compared with CAP-dependent expression. 27 A high expression rate of the MGMT protein is important for efficient dealkylation of O 6 -guanines as each MGMT molecule is irreversibly inactivated after binding of one alkyl group. 8 The transgene itself might influence the IRES-dependent translation owing to its structure or sequence.…”

“…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. 31 This special form of MGMT is resistant against O 6 -benzylguanine (O 6 -BG), 32 an inhibitor of wild-type MGMT, which can be applied to block MGMT activity in tumor cells rendering them sensitive for an alkylating agent while HSCs expressing the mutant form are efficiently protected.…”

A New O6-Alkylguanine-DNA Alkyltransferase Inhibitor Associated with a Nitrosourea (Cystemustine) Validates a Strategy of Melanoma-Targeted Therapy in Murine B16 and Human-Resistant M4Beu Melanoma Xenograft Models

“…Genetic chemoprotection of hematopoietic and progenitor stem cells with mutant AGT has been explored (Southgate et al, 2006). However, this approach does not consider extra-hematopoietic toxicities, and it needs to be optimized before any clinical application can be envisioned.…”

Clinical Trials Using LV-P140K-MGMT for Gliomas

“…MDR1 and mutant MGMT have been integrated into one bicistronic vector to provide modified cells with protection against BG/ACNU or BG/TMZ and vincristine or paclitaxel treatments [27,74,75]. MDR1 and mutant MGMT have been integrated into one bicistronic vector to provide modified cells with protection against BG/ACNU or BG/TMZ and vincristine or paclitaxel treatments [27,74,75].…”