Protection of Hematopoietic Stem Cells from Pemetrexed Toxicity by Retroviral Gene Transfer with a Mutant Dihydrofolate Reductas

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Protection of hematopoietic stem cells from pemetrexed toxicity by retroviral gene transfer with a mutant dihydrofolate reductase-mutant thymidylate synthase fusion gene Gina M Capiaux,1,2 Tulin Budak-Alpdogan,2 Onder Alpdogan,2 William Bornmann,2 Naoko Takebe,2,a Debabrata Banerjee,2,b Frank Maley,3 and Joseph R Bertino1,2,b 1Graduate School of Medical Sciences, Cornell University, New York, NY 10021, USA; 2Program of Molecular Pharmacology and Therapeutics, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA; and 3New York State Department of Health, Wadsworth Center, Albany, NY 12201, USA.

Myelosuppression is one of the major side effects of most anticancer drugs. To confer myeloprotection, our laboratory generated drug-resistant mutants of select target human enzymes for gene transfer to the bone marrow. Mutants of two of these enzymes, dihydrofolate reductase (DHFR F/S) and thymidylate synthase (TS G52S), were previously shown to confer resistance to methotrexate and 5-FU, respectively, and recently a fusion cDNA of both mutant enzymes (DHFR F/S-TS G52S) was shown to confer dual resistance to both antimetabolites. In this study, we examined the sensitivity of the DHFR F/S-TS G52S fusion protein to the multitargeted antifolate, pemetrexed (LY231514, Alimta), which targets both DHFR and TS and is currently in phase III trials for the treatment of solid tumors and in combination with cisplatin has been shown to be an advance in the treatment of mesothelioma.

The Ki for the DHFR F/S portion of the purified fusion protein to pemetrexed was increased by greater than 9000-fold when compared to wtDHFR (8000 versus 0.86 nM), while the Ki for the TS G52S portion of the fusion protein to pemetrexed was similar to that of wtTS (2.8 versus 3.1 nM). When the fusion gene was retrovirally transduced into NIH 3T3 fibroblasts, the IC50 to pemetrexed was three- to four-fold higher than cells transduced with DHFR F/S or TS G52S alone (163 versus 53 and 45 nM, respectively). Similarly, expression of the DHFR F/S–TS G52S fusion gene in retrovirally transduced mouse marrow cells resulted in an increased survival of CFU-GM colonies when compared to cells transduced with either of the mutants alone. Co-expression of mutant DHFR and TS enzymes has additive effects in conferring resistance to pemetrexed-induced toxicity. This construct may be useful for conferring myeloprotection to patients receiving this drug. Cancer Gene Therapy (2004) 11, 767–773. doi:10.1038/sj.cgt.7700683 Published online 10 September 2004

Keywords: gene transfer; dihydrofolate reductase; thymidylate synthase; pemetrexed

ince the introduction of aminopterin and subsequently tions and may prevent or slow the development of drug Smethotrexate (MTX) into the clinic, many promising resistance. analogs have been developed and their use in combination Pemetrexed is a novel folate antagonist that exerts its chemotherapy has become a standard mode of treatment activity through the inhibition of multiple folate coen- for a wide range of malignancies. The basis of this zyme-dependent enzyme targets involved in the de novo approach is that drugs with different mechanisms of purine and pyrimidine pathways. These enzymes include action or targets when combined may allow additive or dihydrofolate reductase (DHFR),1 thymidylate synthase synergistic effects on the tumor. This offers a broader (TS), and glycinamide ribonucleotide formyltransferase range of efficacy against heterogeneous tumor popula- (GARFT).2 Phase II and III studies have shown that pemetrexed has promising antitumor activity against a variety of solid tumor types including mesothelioma, non- Received July 15, 2003. small-cell lung carcinoma, and colon, pancreatic, and Address correspondence and reprint requests to: Dr Joseph R breast cancers.2,3 Additionally, xenograft studies with Bertino, Cancer Institute of New Jersey, 195 Little Albany Street, human breast and non-small-cell lung carcinomas showed New Brunswick, NJ 08903, USA. E-mail: [email protected]. that combinations of pemetrexed with platinum com- aCurrent address: Department of Medicine and Greenebaum Cancer pounds, MTX and 5-fluorouracil (5FU), produced Center, University of Maryland School of Medicine, Baltimore, MD 4 21201, USA. additive or synergistic effects. bCurrent address: Cancer Institute of New Jersey, Robert Wood As with many chemotherapy regimens, a significant Johnson Medical School, 195 Little Albany Street, New Brunswick, limitation to combination chemotherapy is myelosuppres- NJ 08903, USA. sion. Furthermore, with combination chemotherapy the Marrow protection by a mDHFR/mTS fusion gene GM Capiaux et al 768 myelosuppressive effects may be additive. Standard incubation at 251C to initiate the reaction. TS activity was treatments for reducing the duration and intensity of determined spectrophotometrically at 301C, where con- myelosuppression involve the use of recombinant growth version of substrate (dUMP) and cofactor (CH2FH4)to 13 factors and cytokine therapy, that is, G-CSF or GM-CSF. dTMP and FH2 was monitored at 340 nm. The assay With the use of drug-resistant genes and retroviral gene mixture contained the enzyme, 50 mM Tris-HCl, pH 7.4, transfer into hematopoietic stem cells (HSC), expression 25 mM MgCl2, 6.5 mM formaldehyde, 1 mM EDTA, of resistant target enzymes in bone marrow is another 75 mM b-mercaptoethanol, and 100 mM dUMP substrate; strategy that has been considered for overcoming 6-R-CH2FH4 cofactor (100 mM, Eprova AG, Schaffhau- chemotherapy-induced myelosuppression.5 The advan- sen, Switzerland) was subsequently added after a 5 minute tage to this approach is that it could provide resistance pre-incubation at 301C to initiate the 1 mL reaction. against specific chemotherapeutic agents, potentially allowing for higher dose intensity and/or shorter intervals between treatments. Furthermore, co-expression of multi- Determination of Km values via steady-state kinetics ple drug-resistant genes could provide myeloprotection Michaelis constants (Km) for the DHFR substrate H2 against combination chemotherapies with two or more 14 drugs. folate were determined as described previously. Briefly, Previously, we reported on the construction and using the above assay for DHFR activity, the initial characterization of a novel fusion gene consisting of a velocity measurements of wtDHFR (5–10 nM) or DHFR- mutant dihyrofolate reductase (DHFR F/S) and mutant F/S-TS G52S (50 nM) were measured at a saturating thymidylate synthase (TS G52S) cDNA, which when concentration of cofactor NADPH (50 mM), while vary- transduced into murine confers resistance to both MTX ing the concentration of H2 folate over the range of 0.25– 1 10 mM. Km values for the TS substrate dUMP were and 5FU. As many malignancies, including breast 15 cancer,6 colon cancer,7,8 and head and neck cancer9 are determined as described previously. Using the above effectively treated with either or both of these agents, assay for TS activity, the initial velocity measurements of protection of hematopoietic precursor cells using this wtTS (200 nM), TS G52S (100 nM) or DHFR-F/S-TS construct would allow safer use of these drugs and G52S (80 nM) were measured at a saturating concentra- perhaps increased doses. We now report that this fusion tion of cofactor, 6-R-CH2H4 folate (400 mM), while gene also confers resistance to the multitargeted anti- varying the concentration of dUMP over the range of folate, pemetrexed, with additive resistance in murine 5–500 mM. Similarly, Km values for TS cofactor were bone marrow compared to cells expressing the individual determined at a saturating concentration of dUMP mutant genes alone. (500 mM), while varying the concentration of 6-R- CH2H4 folate over the range of 2–500 mM.

Methods Determination of Ki values Materials Pemetrexed Ki values using both the DHFR and TS Pemetrexed and pemetrexed triglutamate were supplied assays were determined from steady-state inhibition by Eli Lilly (Indianapolis), and were also synthesized by reaction rates. For the determination of Ki values, enzyme Dr William Bornmann (Memorial Sloan-Kettering Can- preparations were pre-incubated as indicated for DHFR cer Center). or TS activities and the reactions initiated by adding a pre-mixture of pemetrexed (varying concentrations), with Cell lines and culture conditions saturating levels of either FH2 (100 mM) for DHFR 10 NIH 3T3 murine fibroblasts and GP-envAM12 cells evaluation or CH2FH4 (400 mM) for TS evaluation. A were cultured in Dulbecco’s modified Eagle’s medium competitive tight-binding mode of inhibition was as- 16,17 (DMEM) containing high glucose (4.5 g/L) supplemented sumed and an iterative fitting of the data to the with 10% fetal bovine serum, 2 mM L-glutamine, 100 mg/ Morrison equation was performed using the DA- 18 mL streptomycin, and 100 U/mL penicillin, and main- TADNRP V2.31 program to derive values for Ki. tained at 371C/5% CO2.

Enzyme activities Generation of retroviral producer cell lines DHFR activity was determined spectrophotometrically at SFG retroviral plasmid DNA constructs were transfected þ 251C. Conversion of NADPH and FH2 to NADP and into GP-envAM12 cells using SuperFectt (QIAGEN 11 FH4 was monitored at 340 nm. The assay mixture (final Inc.). After 24 hours, transfected cells were exposed to volume 1 mL) contained the enzyme, MATS assay 150 nM MTX or 750 mg/mL G418 for 10 days and buffer12 (25 mM MES, 25 mM acetic acid, 50 mM Tris individual colonies were isolated and expanded into base, 100 mM sodium chloride) (pH 7.4), supplemented producer cell lines. Titers of these producer cell lines with 50 mg/mL BSA, 1 mM DTT, 50 mM b-mercaptoetha- were determined with NIH3T3 cells as target cells, and 5 6 nol, and 100 mM NADPH cofactor; FH2 substrate cell lines with high titers (10 –10 CFU/mL) were used for (50 mM) was subsequently added after a 5 minute pre- further experiments.

Cancer Gene Therapy Marrow protection by a mDHFR/mTS fusion gene GM Capiaux et al 769 Transduction of NIH3T3 cells were expressed as a percentage of the control without 5 drug. NIH3T3 cells (1 Â 10 ) were plated in 100 mm dishes. After 24 h, the media was removed and replaced with filtered (0.45 mm) AM12 retroviral supernatant containing CFU-C colony PCR. Individual colonies were har- polybrene (8 mg/mL) at a multiplicity of infection (MOI) vested, resuspended in 500 mL of PBS, and centrifuged of 1 for an additional 24 h. Transduced 3T3 cells were at 3000 r.p.m. for 10 minutes at room temperature. Cells selected for 3–4 days in either a medium containing were then resuspended in 30 mL of PCR lysis buffer (5 mM 150 nM MTX (DHFR F/S) and 10% dialyzed FBS Tris, pH 8.1, 0.45% Tween 20, 100 mg/mL proteinase K) (dFBS) or in a medium containing 750 mg/mL G418 and incubated at 501C for 90 minutes, followed by a (Neor) and 10% FBS. After selection, the medium was 5 minute proteinase K inactivation at 951C. A volume of changed to a non-drug-containing media containing 10% 2 mL of each sample was used to amplify the transgene, as FBS, and 4–5 days later polyclonal populations of drug- well as mouse b-actin as a control for DNA, in a 50 mL resistant colonies were used to perform clonogenic assays. PCR reaction containing 200 mM dNTP, 300 ng each primer, 5 U Taq polymerase, and 1 Â Taq polymerase buffer containing 15 mM MgCl2. As described pre- Clonogenic assay viously,1 three sets of gene-specific primers were used to Parental and retrovirally transduced NIH3T3 cells were amplify a 628 bp segment of the DHFR F/S-TS G52S seeded in duplicate in six-well plates at a concentration of fusion gene, the entire 940 bp TS cDNA within the 1000 cells/well. The following day, the medium was bicistronic TS G52S-IRES-DHFR F/S cDNA, and a replaced with a medium containing pemetrexed at various 633 bp fragment within the neomycin phosphotransferase concentrations in the presence of 10% dialyzed fetal gene of all constructs containing the individual mutant or bovine serum. After 7–10 days, colonies were stained with wild-type DHFR and TS genes. b-actin primers were used Crystal Violet and individual colonies of 50 or more cells to amplify a 752 bp fragment within the genomic mouse b- were counted. IC50 values were calculated from sigmoidal- actin gene. dose response curve fits of the data using the GraphPad Prisms software V3.0. Results Mouse bone marrow transduction The fusion protein has reduced affinity for pemetrexed Bone marrow cells from the long bones of 8–11-week-old (glu)3 male C57Bl/6J mice were harvested in IMDM medium The purified DHFR F/S-TS G52S fusion protein, supplemented with 20% FBS and filtered through a 70 mm previously shown to retain the enzymatic properties of nylon cell strainer to remove contaminating bone frag- the individual mutant enzymes DHFR F/S and TS G52S, ments, as previously described. The cells were then was evaluated for its affinity for pemetrexed.1 As the centrifuged at 1000 r.p.m. for 10 minutes followed by polyglutamate form of this drug is the predominant resuspension in IMDM media containing 10% WEHI-3B intracellular form and has a much higher affinity for TS conditioned medium, 20% FBS, 50 ng/mL rSCF (Am- than the monoglutamate,2 the triglutamate form of this gen), 50 ng/mL hIL-6 (Gibco), and 4 mg/mL polybrene. compound, pemetrexed (glu)3, was used for the determi- Cells were then added to T75-flasks containing 80% nation of inhibition constants (Ki). While there was no confluent AM12 producer cells previously irradiated at significant difference in affinity of the TS G52S portion of 1500 cGy. After 48 hours of coculture with AM12 the fusion protein for pemetrexed (glu)3 when compared producer cells, nonadherent bone marrow cells were to that of TS G52S and TS wt (Ki; 2.8, 2.5, 3.1 nM, harvested from each flask and counted to determine BM respectively) (Table 1), the affinity of the DHFR F/S recovery and viability. Experiments with all cell lines were component of the fusion protein for pemetrexed (glu)3 performed at the same time with the same source of bone was reduced dramatically when compared to wtDHFR, marrow. with greater than 9000-fold reduction in Ki (Ki; 8000 versus 0.86 nM), as was DHFR F/S with greater than CFU-C assay 7000-fold reduction in Ki (Ki; 6382 nM versus 0.86 nM) (Table 2). Bone marrow cells were collected from coculture and were plated in duplicate at 55,000 cells per 35 mm plate. Cells were plated in thymidine-phosphorylase-treated methyl- Retroviral gene transfer of the DHFR F/S–TS G52S fusion cellulose19 culture medium (MethoCult GF M3434; Stem into 3T3 cells confers additive resistance to pemetrexed Cell Technologies Vancouver, BC, Canada) without and The NIH 3T3 cell lines were generated by retroviral with pemetrexed at or above the concentration (100 nM) transduction using supernatant from AM12 retroviral previously determined to be 100% lethal for mock- producer cell lines containing the retroviral constructs transduced mouse bone marrow-derived colonies shown in Figure 1. Following a 3–4-day selection and (100 nM). The plates were stored at 371C in a 5% CO2 PCR analyses to verify the presence of the transduced incubator, and colonies of more than 50 cells were gene (Fig 2), clonogenic assays were performed and IC50 counted after 12 days. The surviving colonies in drug values for pemetrexed were determined.

Cancer Gene Therapy Marrow protection by a mDHFR/mTS fusion gene GM Capiaux et al 770 Table 1 TS kinetics and affinity of DHFR F/S-TS G52S versus TS G52S and wt TS enzymes for pemetrexed

Km (mM) Ki (nM) CH2H4 folate dUMP Kcat (second-1) Pemetrexed

DHFR 1671.5 4.971.6 2.170.5 2.871.3

FS-TS G52S X φ 3T3 Par 3T3 wtDHFR.I.Neo 3T3 TS G52S.I.Neo 3T3 DHFR F/S.I.Neo 3T3 wtTS.I.Neo TS G52S 8.671.8 3.870.9 2.570.8 2.570.9 3T3 TS G52S.I.DHFR F/S 3T3 DHFR F/S-TS G52S wtTS 9.170.1 4.370.3 1.770.8 3.171.6 Data are presented as the mean7standard deviations of at least three independent experiments.

Table 2 DHFR kinetics and affinity of DHFR F/S-TS G52S for pemetrexed Figure 2 PCR analyses demonstrating transgene presence in Km (mM) Ki (nM) retrovirally transduced NIH 3T3 cells. See Methods for details. FH2 Kcat (second-1) Pemetrexed

DHFR FS-TS G52S 0.4470.2 2.270.3 80007500 7 7 7 DHFR F/S 0.44 0.1 1.6 0.5 6400 180 IC wtDHFRa 0.08 12.7 0.04 50 100 29 3T3 Parental aFrom Ercikan-Abali.13 Data are presented as the mean7 53 DHF standard deviations of at least three independent experiments. 45 TS G52S-i-Neo 40 TS G52S-i-F/S 163 DHFR F/S-TS G52S MPSV 25 wtDHFR-i-Neo 50 23 wtTS-i-Neo 5’-LTR DHFR F/S LINKER TS G52S 3’-LTR

MPSV % survival

5’-LTR TS G52S IRES DHFR F/S 3’-LTR MPSV 0 1 10 100 1000 10000 5’-LTR DHFR F/S IRES NeoR 3’-LTR Pemetrexed (nM) MPSV Figure 3 NIH 3T3 cells retrovirally transduced with the fusion gene confer additive resistance to pemetrexed; K cells expressing DHFR 5’-LTR TS G52S IRES NeoR 3’-LTR F/S-TS G52S (IC50 ¼ 163 nM), E cells expressing TS G52S.i.DHFR n MPSV F/S (IC50 ¼ 40 nM), cells expressing wtTS.i.Neo (IC50 ¼ 23 nM), & cells expressing wtDHFR.i.Neo (IC50 ¼ 25 nM), J cells expressing TS G52S.i.Neo (IC ¼ 45 nM), m cells expressing DHFR F/S.i.Neo 5’-LTR wt DHFR IRES NeoR 3’-LTR 50 (IC50 ¼ 53 nM), and ’ NIH 3T3 parental cells (IC50 ¼ 29 nM). See MPSV Methods for details.

5’-LTR wt TS IRES NeoR 3’-LTR wtTS (23 nM) or wtDHFR (25 nM) (Fig 3). These cells Figure 1 Retroviral constructs used to generate AM12 producer cell were also two-to three-foldmore resistant to pemetrexed lines. Black arrows indicate the chimeric 30 LTR containing the MPSV than cells expressing each of the individual mutant enhancer. enzymes either alone, DHFR F/S (53 nM) or TS G52S (45 nM), or together from the bicistronic gene (40 nM). Neither wtDHFR (25 nM) or wtTS (23 nM) alone, when DHFR and TS activity levels in each of the cell lines expressed in 3T3 cells, provided any protection against were determined as a control for differences in enzyme pemetrexed as compared to parental 3T3 cells (29 nM). activity levels between the cell lines. These results1 indicated that the DHFR and TS activity levels in cells expressing the DHFR F/S-TS G52S fusion protein were Retroviral gene transfer of DHFR F/S and TS G52S into similar to the DHFR and TS activity levels in cells mouse bone marrow protects from pemetrexed toxicity expressing DHFR F/S and TS G52S alone, respectively. The LD100 for pemetrexed in hematopoietic progenitor The IC50 value for pemetrexed in cells expressing the cells was determined by performing a titration of DHFR F/S-TS G52S fusion protein was 163 nM. This nontransduced mouse bone marrow cells in methylcellu- value was five-to six-foldhigher than cells expressing lose with increasing doses of pemetrexed (Fig 4). CFU-C

Cancer Gene Therapy Marrow protection by a mDHFR/mTS fusion gene GM Capiaux et al 771 assays were then performed using pemetrexed at and effects of these drugs, with the most common side effect above the LD100. being myelosuppression. Gene transfer of drug resistance In CFU-C assays, dual expression of DHFR F/S and genes or resistant gene variants into hematopoietic stem TS G52S resulted in significant colony formation over cells is a strategy that is being investigated to increase cells expressing the individual wt or mutant enzymes bone marrow tolerance to chemotherapy.5 Our laboratory alone. At 300 nM pemetrexed, cells expressing either has previously generated a variety of drug resistance DHFR F/S or TS G52S alone had 44 and 79% resistant genes, and combinations thereof, conferring resistance to colonies, respectively (Table 3), while cells expressing both antimetabolites such as MTX, trimetrexate, 5FU, ralti- enzymes together as a fusion or separately from the IRES trexed, thymitaq, and cytosine arabinoside.14,20–24 In this element of the bicistronic gene had 104 and 84% resistant study, we show that the transfer of a DHFR F/S-TS G52S colonies, respectively. At 1000 nM pemetrexed, the fusion gene into hematopoietic progenitor cells results in number of colonies formed in bone marrow transduced resistance to a promising new antifolate, pemetrexed. with DHFR F/S-TS G52S and TS G52S-ires-DHFR F/S Resistance to pemetrexed was evaluated in NIH 3T3 was 44 and 18%, respectively, and at 3000 nM peme- cells and mouse bone marrow cells expressing the DHFR trexed, colony formation in these cells was 31 and 8% F/S-TS G52S fusion protein and compared to cells respectively. In contrast, there was little to no colony expressing the individual mutant or wild-type enzymes formation above 300 nM in bone marrow cells transduced alone. Comparison was also performed against cells to express the individual mutant and wt enzymes alone. In expressing both enzyme variants as separate proteins addition, PCR performed on cells cultured in the presence from a bicistronic gene. In clonogenic assays, NIH3T3 of drug indicated that 100% of drug-resistant colonies cells expressing each of the individual mutant enzymes contained the gene of interest. alone, DHFR F/S or TS G52S, provided only a two-fold resistance to pemetrexed compared to parental 3T3 cells. Although the affinity (Ki) of purified DHFR F/S for pemetrexed (glu) was dramatically reduced (47000- Discussion 3 fold), this drug is known to mainly target TS; thus, only a two-fold decreased sensitivity in cells expressing DHFR The need for effective new drugs to treat cancers parallels F/S was not surprising. However, the two-fold decrease in the need for effective new strategies to manage the side sensitivity to pemetrexed in cells expressing TS G52S was 125 not expected, since the affinity of purified TS G52S for pemetrexed (glu)3 was the same as wtTS. Of importance, cells expressing the DHFR F/S-TS G52S fusion protein 100 provided an additional three-to four-foldincrease in resistance to pemetrexed compared to cells expressing 75 either of the mutant enzymes alone or cells expressing both enzymes from the IRES element of the bicistronic 50 gene.

% Survival In CFU-C assays, expression of both mutant enzymes 25 together, DHFR F/S and TS G52S, from either the fusion gene or from the bicistronic gene provided significant 0 colony survival at pemetrexed concentrations up to 3 mM 0 1 3 1 3 0 0 0 0 03 . . 0 0 0 0 compared to the expression of either of these mutant . 0 0 1 3 1 3 00 00 0 1 3 enzymes alone. These results are consistent with the Pemetrexed nM additive effects of the fusion protein in conferring Figure 4 Dose response data for pemetrexed toxicity in naı¨ve resistance to pemetrexed in clonogenic assays. Although mouse hematopoietic progenitor cells. expression of both mutant enzymes together from either

Table 3 Resistance of retrovirally transduced mouse bone marrow to pemetrexed in CFU-C assays % Pemetrexed-resistant colonies

Vector Transduction efficiency % 300 nM 1000 mM 3000 mM wtTS-IRES-NeoR 3273.4 57728 070 1.371.3 TS G52S-IRES-NeoR 2977.7 79753 1.771.4 2.071.6 wt DHFR-IRES-NeoR 3477.3 15712 070070 DHFR F/S-IRES-NeoR 2371.5 44736 070070 TS G52S-IRES-DHFR F/S 3674.4 84744 1877.0 8.075.5 TS G52S- DHFR F/S 3776.6 104731 44712 31711 eGFP-IRES-NeoR 7477.5 2.572.5 070070 The number of drug-resistant colonies are expressed as the percentage of gene-containing (PCR+) control colonies. Data are presented as the mean7SEM of at least three independent experiments.

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