Chemoprotection of murine hematopoietic cells by combined gene transfer of cytidine deaminase (CDD) and multidrug resistance 1 gene (MDR1)
© Brennig et al. 2015
Received: 1 April 2015
Accepted: 16 November 2015
Published: 12 December 2015
Hematologic toxicity represents a major side effect of cytotoxic chemotherapy frequently preventing adequately dosed chemotherapy application and impeding therapeutic success. Transgenic (over)expression of chemotherapy resistance (CTX-R) genes in hematopoietic stem- and progenitor cells represents a potential strategy to overcome this problem. To apply this concept in the context of acute myeloid leukemia and myelodysplasia, we have investigated the overexpression of the multidrug resistance 1 (MDR1) and the cytidine deaminase (CDD) gene conferring resistance to anthracyclines and cytarabine (Ara-C), the two most important drugs in the treatment of these diseases.
State-of-the-art, third generation, self-inactivating (SIN) lentiviral vectors were utilized to overexpress a human CDD-cDNA and a codon-optimized human MDR1-cDNA corrected for cryptic splice sites from a spleen focus forming virus derived internal promoter. Studies were performed in myeloid 32D cells as well as primary lineage marker negative (lin−) murine bone marrow cells and flow cytometric analysis of suspension cultures and clonogenic analysis of vector transduced cells following cytotoxic drug challenge were utilized as read outs.
Efficient chemoprotection of CDD and MDR1 transduced hematopoietic 32D as well as primary lin− cells was proven in the context of Ara-C and anthracycline application. Both, CTX-R transduced 32D as well as primary hematopoietic cells displayed marked resistance at concentrations 5–20 times the LD50 of non-transduced control cells. Moreover, simultaneous CDD/MDR1 gene transfer resulted in similar protection levels even when combined Ara-C anthracycline treatment was applied. Furthermore, significant enrichment of transduced cells was observed upon cytotoxic drug administration.
Our data demonstrate efficient chemoprotection as well as enrichment of transduced cells in hematopoietic cell lines as well as primary murine hematopoietic progenitor cells following Ara-C and/or anthracycline application, arguing for the efficacy as well as feasibility of our approach and warranting further evaluation of this concept.
Cytotoxic chemotherapy represents a major component of anti-cancer treatment strategies. However, cytotoxic agents not only affect the malignant target cells, but also are associated with substantial damage to healthy cells and tissues. In particular hematologic toxicity represents a major and frequently dose-limiting side effect that may cause severe infectious complications and thrombocytopenias, prohibit further application of chemotherapy on time, and impede therapeutic success. Transgenic (over)expression of chemotherapy resistance (CTX-R) genes in hematopoietic stem- and progenitor cells, also referred to as chemo- or myeloprotective gene therapy, represents a potential strategy to overcome this problem [1, 2]. In this regard, a number of CTX-R genes have been identified and studied for their myeloprotective properties . Of note, recently effective protection from alkylating agent-induced myelotoxicity has been demonstrated following gene transfer of the CTX-R gene mutant O6-methylguanine methyltransferase (mutMGMT) in a cohort of glioblastoma patients treated with temozolomide [3, 4]. In this study, profound in vivo enrichment of genetically modified hematopoietic cells and clinical benefits in comparison to a disease matched control group was demonstrated, establishing a proof-of-concept for the clinical applicability and efficacy of myeloprotective gene therapy approaches. Other myeloprotective strategies interfere with antimetabolite type cytotoxic drugs. This includes mutant forms of dihydrofolate reductase (mutDHFR), protecting cells from antifolate drugs such as methotrexate and trimetrexate [5–7] as well as knock-down of hypoxanthine phosphoribosyl transferase (HPRT) expression protecting cells from purine analogs like 6-thioguanine .
Multi-drug resistance 1 (MDR1) and cytidine deaminase (CDD) represent two other well-studied CTX-R genes. The human MDR1 gene encodes the cellular efflux protein ABCB1 (P-glycoprotein, P-gp), a member of the ATP-binding cassette superfamily, which confers resistance to a wide variety of clinically relevant chemotherapeutic agents including anthracyclines, epipodophyllotoxins, taxoids or vinca-alkaloids . Transgenic expression of MDR1 by γ-retroviral vectors protects murine and human hematopoietic cells from the toxic effects of paclitaxel, vincristine, etoposide, doxorubicin or daunorubicin in vitro [10–15]. Moreover, for various of these agents in vivo protection of murine and human hematopoietic cells has been demonstrated following γ-retroviral gene-transfer to HSCs in murine [10, 16–18] and humanized (NOD/SCID)  transplant models, respectively, and chemoprotection of human hematopoietic progenitor cells in vitro also has been reported following lentiviral mediated gene transfer of MDR1 [20, 21]. Furthermore, MDR1 has been successfully used as a selection marker during hematopoietic stem cell gene therapy (HSC-GT) [18, 22]. Despite these pre-clinical achievements early clinical trials with MDR1 in the late 1990s showed only moderate success primarily due to low gene transfer efficacy [23–27] or aberrant splicing of the MDR1 gene . CDD codes for an enzyme of the nucleotide salvage pathway and protects cells against such clinically relevant agents as cytosine-arabinoside (Ara-C), gemcitabine, decitabine and azacytidine . Meanwhile, CDD-mediated drug resistance and enrichment of transduced cells following γ-retroviral gene transfer has been established in murine and human hematopoietic cells in vitro [30–32] as well as murine long-term reconstituting hematopoietic stem cells (HSC) [33, 34]. Though, a potential lymphotoxicity of CDD overexpression was noted in one of these studies , this problem was circumvented when doxycycline-induced transgene expression from a lentiviral vector backbone was employed .
Combinations of Ara-C and anthracyclines as in the classical “3 + 7” or TAD regimen  are highly effective in the treatment of acute myeloid leukemia or high-risk myelodysplasia and represent the backbone of chemotherapy in these disease entities. However, these regimens are associated with a profound and long-lasting myelosuppression. This may create problems particularly in relapsed disease situations and/or in the elderly, where an already compromised hematopoietic stem cell compartment even aggravates these side effects. To overcome this dilemma hematopoietic stem cell gene therapy allowing for the combined overexpression of CDD and MDR1 appears as a logical strategy to protect the lymphohematopoietic system from combined chemotherapy. Thus, we here have evaluated this concept applying state-of-the-art lentiviral gene transfer technology. Our data demonstrate highly efficient chemoprotection as well as enrichment of transduced cells in hematopoietic cell lines as well as primary murine hematopoietic progenitor cells following combined Ara-C/anthracycline application.
Lentiviral vector constructs and preparations
Lentiviral vectors were based on 3rd generation SIN lentiviral vectors modified with a woodchuck hepatitis virus-derived xposttranscriptional-regulatory element [37, 38]. RRL.PPT.SFFV.hMDR1.IRES.GFPpre* (referred to as LV.SFFV.MDR1) contained a human codon-optimized multidrug resistance gene 1 (hMDR1) cDNA additionally modified by removing (cryptic) splice donor and acceptor sites to increase tRNA usage and thereby increase expression levels. The hMDR1-cDNA was inserted with AgeI/SalI restriction enzymes into RRL.PPT.SFFV.pre* construct. Subsequently, an enhanced green fluorescent protein (eGFP) was inserted together with an IRES site via SalI. RRL.PPT.SFFV.hCDD.IRES.GFP.pre* and RRL.PPT.SFFV.hCDD.IRES.dtomato.pre* (referred to as LV.SFFV.CDD) were generated by cloning of the cDNA of human cytidine deaminase (hCDD) (former OpenBiosystems, IHS1380-OB-97652440, Epsom, UK) via AgeI/SalI followed by SalI mediated insertion of an IRES site linked to a green (GFP) or red (dtomato) fluorescent protein. RRL.PPT.SFFV.hCDD.P2A.hMDR1.IRES.GFP.pre* (referred to as LV.SFFV.CDD.2A.MDR1) was generated by insertion of hCDD and hMDR1 and a porcine teschovirus-1 (P2A) linker sequence using overlap/extension PCR. Subsequently, the hCDD fragment was inserted into LV.SFFV.MDR1 by AgeI followed by introduction of IRES.GFP via SalI. RRL.PPT.SFFV.GFP.pre* (referred to as LV.SFFV.GFP) was cloned by insertion of GFP reporter into RRL.PPT.SFFV.pre*. Production as well as titration was performed as previously described . Titers (TU/mL) ranged from 2×106 - 9×106 for LV.SFFV.MDR1, 1×106 - 1×107 for LV.SFFV.CDD.2A.MDR1, 3×107 - 2×108 for LV.SFFV.CDD, and 2×107 - 2×108 for LV.SFFV.GFP control vector.
Experiments with 32D myeloid cells
Murine 32D cells were cultured in RPMI-1640 supplemented with 10 % fetal calf serum (Biochrom, Berlin, Germany), 100 U/ml penicillin/streptomycin (Pen/Strep), 2 mmol/l glutamine (all Life Technologies) and 2 ng/ml mIL-3 (Peprotech, Hamburg, Germany).
Genetic modification of 32D cells was performed by adding viral supernatant to cells in the presence of 10 μg/ml protaminsulfate (Carl Roth, Karlsruhe, Germany) at 37 °C. Twenty-four hours after transduction, cells were washed, expanded for several days and subsequently sorted for fluorescent reporter gene expression (FACS AriaIIu, Becton Dickinson) to establish transgenic 32D cells of purity ≥ 90 %.
In vitro protection
Chemoprotection was carried out by seeding 1.5×105 cells in 2 ml complete medium and cytotoxic drugs were added in given concentrations. Following three days incubation, cell survival was analyzed by flow cytometry (using FSC/SSC exclusion). Stock solutions of cytotoxic drugs were prepared by central pharmacy of Hannover Medical School.
Transgene expression analysis
To analyze expression of hMDR1, RNA was isolated from cells previously treated with cytotoxic drugs as well as non-treated controls using TRIsure reagent (Bioline, London, UK) as recommended by the manufacturer. Following DNase I digestion, RNA was reverse transcribed using RevertAid reverse transcriptase and oligo(dT) primers (all Thermo Scientific, Schwerte, Germany). Subsequently, 25 ng cDNA was used for quantification by SYBR Green-based qRT-PCR on a StepOnePlus Real-Time PCR System (Thermo Scientific, Schwerte, Germany Biosystems). Expression of hDMR1 mRNA is given relative to non-treated untransduced control cells (normalized to endogenous murine β-actin). Expression of hCDD protein was analyzed by Western Blot as described previously  using cells previously treated with cytotoxic drugs as well as non-treated controls.
In vitro proliferation assay
Cell proliferation analysis of (transduced) 32D cells was determined using cell proliferation dye eFluor 670 (eBioscience, San Diego, CA, USA). In brief, cells were incubated with the proliferation dye as recommended by the manufacturer and analyzed for initial dye uptake using flow cytometry (FL4 channel, FACS Calibur, Becton Dickinson). Subsequently, cells were applied to an in vitro protection assay as described above and three days later analyzed by flow cytometry.
Experiments with primary murine hematopoietic cells
Bone marrow of C57BL/6 (Janvier Laboratories, Saint Berthevin Cedex, France and Central animal facility, Hannover Medical School, Hannover, Germany) mice was harvested from femora and tibiae and lineage negative (lin−) cells were purified using MACS separation (Lineage Cell depletion kit, Miltenyi, Bergisch Gladbach, Germany). Cells were cultured and prestimulated for 24 h in StemSpan medium (StemCell Technologies, Cologne, Germany) supplemented with 10 ng/ml rmSCF, 20 ng/ml rmTPO, 20 ng/ml rmIGF and 10 ng/ml rhFGF (all PeproTech) prior to transduction.
Transduction of lin− cells with lentiviral vectors was carried out using retronection (10 mg/cm2; Takara, Otsu, Japan) -coated dishes as recommended by the manufacturer. Approximately three to four days later, non-sorted cells were applied to myeloid in vitro differentiation experiments or cells were sorted for GFP reporter gene expression and subjected to a clonogenic progenitor assays.
Clonogenic progenitor assays
Clonogenic growth of hematopoietic progenitor cells were assessed by incubating 1.500 transduced lin- cells previously sorted for eGFP expression. Clonogenic cultures were performed in 1 ml IMDM/1.3 % methylcellulose supplemented with 15 % fetal calf serum, 2 % bovine serum albumin, 2 mM L-glutamine, 50 μM 2-Mercaptoethanol, 10 μg/ml rh-insulin, 200 μg/ml human transferrin, 50 ng/ml rm-SCF, 10 ng/ml rm-IL3, 10 ng/ml rm-IL6 and 5 IU/ml rh-EPO (HSC007, R&D Systems, Wiesbaden-Nordenstadt, Germany) in the presence of different concentrations of cytotoxic drugs. Colonies of more than 50 cells were counted after 6–7 days.
Myeloid in vitro differentiation
In vitro protection and selection was performed using 5×104 non-sorted transduced lin− cells. Cells were cultured in RPMI-1640 supplemented with 10 % fetal calf serum, 100 U/ml penicillin/streptomycin (Pen/Strep), 2 mmol/l glutamine and addition of 20 ng/ml rmIL-3 and 100 ng/mL rh-G-CSF (Peprotech, Hamburg, Germany). Cells were treated with cytotoxic drugs in the given concentration for three days and analyzed by flow cytometry. For in vitro selection, data are given as fold increase in % GFP+ cells with non-treated cells = 1.
All graphs were created using Prism V5 (GraphPad) and statistical analysis was performed using Prism V6 software (GraphPad, La Jolla, CA, USA). Unless otherwise noted analysis of variance (ANOVA) with recommended post hoc testing was performed.
Generation of lentiviral vectors
Efficient protection of 32D cells from cytotoxic drug treatment
Next, we evaluated the functionality of combined CDD/MDR1 expression in 32D cells. To this point, cells were either co-transduced with the two single CTX-R vectors (LV.SFFV.CDD and LV.SFFV.MDR1) or modified with the LV.SFFV.CDD.2A.MDR1 vector expressing both genes, CDD and MDR1, from a single construct. Again, transgenic cells were enriched by FACS and thereafter treated with daunorubicin or Ara-C. Both LV.SFFV.CDD/LV.SFFV.MDR1 as well as LV.SFFV.CDD.2A.MDR1 gene-modified cells were protected against daunorubicin monotherapy (≥125 nM), whereas control cells were susceptible from 25 nM onwards (Fig. 2c). Similar results were observed when cells were treated with Ara-C, except for a slightly reduced resistance to very high Ara-C concentrations (2000 nM) in LV.SFFV.CDD.2A.MDR1 transduced cells (Fig. 2d). Finally, cells expressing CDD and MDR1 were treated with a combination of daunorubicin and Ara-C. While non-transduced 32D control cells were highly susceptible to all drug combinations applied, LV.SFFV.CDD/LV.SFFV.MDR1 as well as LV.SFFV.CDD.2A.MDR1 gene-modified cells demonstrated marked resistance to the daunorubicin/Ara-C combination with > 80 % of cells surviving even at the highest doses tested. Likewise percentages of surviving cells were significantly increased ranging from 17-100-fold for LV.SFFV.CDD/ LV.SFFV.MDR1 and 19-110-fold for LV.SFFV.CDD.2A.MDR1, respectively (Fig. 2e).
Results of these cytotoxicity studies were confirmed by cell proliferation analysis of e670 labelled 32D cells. In these studies susceptibility of 32D cells to daunorubicin as well as Ara-C was clearly associated with cell cycle arrest, whereas MDR1 or CDD transgene expression reconstituted the proliferative potential of 32D cells also in the presence of the respective drugs (Additional file 3: Figure S3).
Therefore, our data clearly show that expression of CDD and MDR1 alone or in combination results in increased myeloprotection in the context of anthracycline and/or nucleoside-analogue therapy.
Chemoprotection of primary hematopoietic progenitor cells
Next, we employed our lentiviral vectors in primary murine hematopoietic progenitor cells. To this point, lin− murine bone marrow cells were transduced and following FACS-based enrichment for GFP expression, seeded into clonogenic assays in the presence of cytotoxic drugs. Median transduction rates of primary cells used for clonogenic as well as suspension experiments (s. below) as determined by GFP reporter expression were lower for vectors containing MDR1 (4.6 % and 4.9 % for LV.SFFV.MDR1 and LV.CDD.2A.MDR1, respectively) than for LV.SFFV.CDD (49 %) and LV.SFFV.GFP control (50 %).
Marked selection of CTX-R gene-modified cells following chemotherapy application
Marked and significant in vitro selection also was observed for the LV.SFFV.CDD.2A.MDR1 construct. While 3-20 % of gene marking were observed following transduction this was increased to 36-84 % respective 23-66 % following exposure to daunorubicin or Ara-C alone (Fig. 5d, e and Additional file 5: Table S1) and to 59-85 % upon combined drug application. This correlated to a 10.7 ± 4.4 (30/300) and 7.7 ± 2.8 -fold (45/300) enrichment upon treatment with the two daunorubicin/Ara-C combinations (Fig. 5f). No enrichment has been observed in LV.SFFV.GFP transduced control cells. Taken together, these data clearly show an enhanced survival and substantial in vitro selection of CTX-R gene-modified cells upon cytotoxic drug treatment applied as single agent as well as combination therapy.
Transplantation of ex vivo CTX-R gene modified hematopoietic stem-and progenitor cells may reduce cytotoxic drug induced myelotoxicity and allow for dose-intensified or prolonged chemotherapy application. Towards this objective, we have generated lentiviral vectors expressing MDR1 as well as CDD to genetically engineer murine hematopoietic cells for increased inherent resistance to anthracycline- and nucleoside-analogue-type cytotoxic drugs. Utilizing these vectors, we demonstrate highly efficient chemoprotection as well as in vitro enrichment of transduced hematopoietic cells in the context of Ara-C as well as anthracycline application. Moreover, and of particular importance, dual expression of the CDD as well as the MDR1 transgene conferred significant cellular resistance to combined nucleoside analogue/anthracycline application.
Transgenic expression of CDD successfully conferred resistance to Ara-C in myeloid 32D as well as primary murine bone marrow cells. The degree of Ara-C resistance achieved was comparable to that described with LTR-driven γ-retroviral vectors in the past [30, 32, 35, 41]. Similarly, resistance to MDR1 associated drugs conferred to 32D and primary hematopoietic cells by transgenic MDR1 (over)expression was in the same range as reported in most studies before [10–12]. On this ground we also would expect similar in vivo efficacy of our constructs as previously demonstrated for –retroviral vectors in murine or humanized transplant models [18, 19, 33, 34]. Most importantly, however, effective protection from combined Ara-C/anthracycline application by the CDD/MDR1 combination was shown in our experiments demonstrating the feasibility to functionally co-express both genes in hematopoietic cells. Given that anti-cancer chemotherapy usually is applied as a combination of multiple agents, simultaneous transfer of CTX-R genes, represents a logical strategy for myeloprotection, and particularly combinations including MDR1 have been studied. Combination partners have been, glutathione S-transferase (GST), MGMT or mutDHFR to allow for additional protection against alkylators such as nitrosoureas, temozolomide (MGMT), chlorambucil, melphalan (GST), as well as folate antagonists [13, 14, 20, 42, 43]. Similarly, CDD has been combined with GST  and mutDHFR [45–47] to add nitrogen mustards and folate antagonists to the resistance spectrum. In our attempt to transfer the protection concept to AML and MDS treatment settings, we now for the first time demonstrate the successful combination of CDD and MDR1.
In our study, safety-optimized 3rd generation SIN lentiviral vectors were utilized. While effective myeloprotection was observed for all constructs, transduction efficiency and titers were reduced with vectors containing MDR1, most likely due to the size of the transgene cassette. As this may be problematic for in vivo applications, gene transfer efficacy needs to be improved. Here, addition of transduction enhancers, such as rapamycin or cylosporin A, engineering of the vector surface phenotype, or suitable ex vivo selection strategies prior to transplant may increase efficacy [48–51]. Co-expression of MDR1 and CDD was accomplished with co-transduction of cells with single gene vectors (in case of 32D cells) or by using the LV.SFFV.CDD.2A.MDR1 combination vector. In this vector, CDD and MDR1 are linked via a porcine teschovirus-1 (P2A) linker sequence that, in contrast to internal ribosomal entry (IRES) sites, should result in equimolar expression of both transgenes [52, 53]. In our experiments however, LV.SFFV.CDD.2A.MDR1 transduction, though highly effective, resulted in moderately reduced Ara-C resistance compared to double-transduced or LV.SFFV.CDD -transduced cells indicating reduced CDD expression from the LV.SFFV.CDD.2A.MDR1 vector. In this context, position dependency of transgene expression resulting in altered protection levels recently was shown for vectors combining MDR1 and MGMT via an F2A site. In addition, the residual “2A” tag on the upstream CDD protein may negatively influence transgene function .
In our study, efficient selection of CTX-R expressing cells was demonstrated following cytotoxic drug administration. This may be of considerable relevance for the clinical translation of our strategy, given the relative low transduction levels achieved with our MDR1 vector constructs so far. Though a myeloproliferative syndrome has been described in mice transplanted with MDR1 gene-modified hematopoietic cells following extensive ex vivo expansion, this almost certain was related to the LTR-driven design of the γ-retroviral vector as well as the applied culture conditions utilized in this study and has not been reproduced with modern safety-improved vector constructs . Efficient selection of MDR1 gene-modified cells convincingly has been demonstrated for murine as well as human hematopoietic cells [18, 48, 55] and selection also has been demonstrated for the CDD/Ara-C system in vitro [32, 56] and in a murine transplant model .
Given the therapeutic efficacy of Ara-C/anthracycline combinations in AML as well as high-risk MDS, combined CDD/MDR1 gene-transfer as a clinical scenario clearly is directed towards these disease entities. Here, consolidation treatment of high-risk disease states (e.g. second remission or even refractory disease) following secondary transplantation can be envisioned as the primary scenario. In these highly unfavorable disease situations, the potential benefits of additional chemotherapy given with few hematologic effects on disease outcome as well as live quality would clearly offset the potential genotoxic risks associated with the procedure. Moreover, an inadvertent transduction of leukemic cells by the CTX-R transgenes would be prevented by the allogeneic setting. If proven effective and safe, myeloprotection by the CDD/ MDR1 combination may have potential to improve disease outcome in the elderly, especially following alloHSCT employing reduced intensity conditioning regimen. Here the application of additional chemotherapy is associated with increased hematotoxicity frequently resulting in profound and long-lasting myelosuppression and severe infectious complication. Due to its primarily hematological side effects, at least at conventional doses, cytarabine in the context of CDD/MDR1 induced myeloprotection may be applied even at enhanced doses [57, 58], while dose-intensification of anthracyclines will be limited by its cardiotoxicity .
Certainly safety constitutes a critical aspect also of myeloprotective gene therapy approaches, and insertional mutagenesis clearly represents a concern with the integrating vectors. While the SFFV promoter used in this study provides high level of transgene expression in human HSCs when incorporated into lentiviral vectors , it is also associated with a higher genotoxic risk compared to weaker physiological promoters . In this context it is reassuring that a markedly reduced genotoxic potential has been demonstrated for novel generation SIN lentiviral vectors such as the ones used in our studies . Moreover, no genotoxic events such as profound clonal dominance or leukemias have been encountered in the current generation of hematopoietic gene therapy trial employing safety-optimized y-retroviral or lentiviral SIN vectors [63, 64]. This is of particular importance in the setting of myeloprotective gene therapy, since insertional mutagenesis may give rise to drug-resistant leukemic cells. Incorporation of suicide genes such as herpes simplex virus thymidine kinase (HSV-TK) or inducible caspase 9 should be considered as a fail-safe mechanism [65, 66].
In conclusion, we have demonstrated significant in vitro protection and selection of CDD and MDR1 gene-modified hematopoietic cells utilizing a safety-improved 3rd generation lentiviral gene transfer system as a first step towards the concept of myeloprotective gene therapy in the context of AML and MDS treatment. Now, the next step to further provide evidence for the efficacy as well as safety of this approach will require in vivo studies in relevant animal system including treatment studies in murine leukemia models.
The authors thank Christopher Jürgens and his team of the central pharmacy of Hannover Medical School for preparing cytotoxic drug stock solutions as well as Matthias Ballmaier and his team from the Core Facility Cell- Sorting of Hannover Medical School for cell sorting. Furthermore, the authors thank Doreen Lüttge for excellent technical assistance and Adele Mucci for help with gene expression analysis. This work was supported by grants from the Deutsche Forschungsgemeinschaft: Cluster of Excellence REBIRTH (Exc 62/1), SPP1230 grant MO 886/3-1 (T.M.), grant MO 886/4-1 (T.M.), SFB 738 (A.S.) and Hannover Biomedical Research School (HBRS; DFG, GSC 108).
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