Generation of Dual Resistance to 4-Hydroperoxycyclophosphamide and Methotrexate by Retroviral Transfer of the Human Aldehyde Dehydrogenase Class 1 Gene and a Mutated Dihydrofolate Reductase Gene

Abstract

The genetic transfer of drug resistance to hematopoietic cells is an attractive approach to overcoming myelosuppression caused by high-dose chemotherapy. Because cyclophosphamide (CTX) and methotrexate (MTX) are commonly used non-cross-resistant drugs, generation of dual drug resistance in hematopoietic cells that allows dose intensification may increase anti-tumor effects and circumvent the emergence of drug-resistant tumors. We constructed a retroviral vector containing both a human cytosolic ALDH-1 cDNA and a human doubly mutated DHFR cDNA (Phe22/Ser31; termed F/S in the description of constructs) to generate increased resistance to both CTX and MTX. Infection of NIH3T3 cells resulted in increased resistance to both 4-hydroperoxycyclophosphamide (4HC) (1.9 +/- 0.1-fold) and MTX (73 +/- 2.8-fold). Transduced human CD34(+) enriched hematopoietic progenitor cells were also resistant to both 4HC and MTX by CFU-GM readout. Lethally irradiated mice transplanted with SFG-ALDH-IRES-F/S or mock-transduced bone marrow cells were treated with high-dose pulse CTX or high-dose CTX/MTX. Animals receiving marrow not transduced with ALDH-1 or mutated DHFR cDNA died from CTX or CTX/MTX toxicity, whereas mice transduced with ALDH-1 and mutated DHFR cDNA-containing marrow were able to tolerate the same doses of CTX or CTX/MTX treatment posttransplant. These data taken together indicate that ALDH-1 overexpression and mutant DHFR increased both 4HC and MTX resistance in vitro and in the in vivo mouse model. This construct may be useful for protecting patients from high-dose CTX- and MTX-induced myelosuppression.

Identification of Human Aldehyde Dehydrogenase 1 Family Member A1 as a Novel CD8+ T-Cell–Defined Tumor Antigen in Squamous Cell Carcinoma of the Head and Neck

“…In addition to its role in alcohol metabolism, ALDH1A1 plays a critical role in defining resistance to a range of chemotherapeutic agents, which generate toxic aldehydes, such as cyclophosphamide and cisplatin (21,(33)(34)(35)(36). Furthermore, elevated ALDH1A1 expression endows tumors with an enhanced capability to metabolize retinal to retinoic acid, which inhibits cell growth and induces apoptosis of tumor cells (6, 7) as well as down-regulates epidermal growth factor receptor expression (37).…”

Resistance to antifolates

“…The strategy has been utilized to achieve resistance to MTX with mutated DHFR in murine progenitor cells (Banerjee et al, 1994;Flasshove et al, 1995a;Takebe et al, 2000) and human CD34 þ peripheral human cells in vitro and in vivo (Flasshove et al, 1995b;Sorrentino et al, 1999). Bicistronic retroviral vectors have been used to expand the spectrum of resistance in progenitors, as with the transduction of both mutated DHFR and Pgp into murine bone marrow progenitors (Galipeau et al, 1997), or with DHFR and cytidine deaminase conferring resistance to both MTX and cytosine arabinoside (Sauerbrey et al, 1999), or with cotransfection of mutated DHFR and wild-type aldehyde dehydrogenase to achieve resistance to both MTX and cyclophosphamide (Takebe et al, 2001). Transplantion of transduced CD34 þ cells into irradiated mice in the latter case resulted in substantial protection of the bone marrow from drug toxicity.…”

Selection based on CD133 and high aldehyde dehydrogenase activity isolates long-term reconstituting human hematopoietic stem cells

“…One promising purification strategy exploits cytosolic aldehyde dehydrogenase (ALDH), an enzyme implicated in retinoid metabolism and the resistance of HSCs to alkylating agents such as cyclophosphamide. 11,12 Murine repopulating cells 13,14 and human hematopoietic progenitors have previously been isolated based on increased activity of intracellular ALDH. 15,16 We have previously characterized a novel reconstituting HSC population from human umbilical cord blood (UCB) isolated by depletion of cells with mature lineage markers (Lin Ϫ ) and selection of cells with high ALDH activity.…”