Chemoprotection in Brain Tumor Patients: Another Success for Stem Cell Gene Therapy
Authors: Brian P Sorrentino
Journal: Molecular Therapy (2012)

Chemotherapy with alkylating agents such as 1,3-bis (2 chloroethyl)-1-nitrosourea (BCNU) and temozolomide is a mainstay for treating malignant brain tumors; however, as a result of tumor cell resistance and progressive toxicity in normal tissues, these drugs invariably fail to eradicate high-grade glioblastomas. Drug resistance in tumor cells can be overcome with sensitization agents, but this strategy is limited by concomitant sensitization of the hematopoietic system, resulting in delays in therapy, drug dose reductions, and the occurrence of significant side effects. One approach to addressing this problem is to transfer appropriate resistance genes into transplanted hematopoietic stem cells (HSCs) to preserve blood formation during chemotherapy.1Sorrentino BP Gene therapy to protect haematopoietic cells from cytotoxic cancer drugs.Nat Rev Cancer. 2002; 2: 431-441Crossref PubMed Scopus (64) Google Scholar This approach has been validated in preclinical animal models but until recently had not been successfully implemented in human patients. In this regard, it is notable that Adair and colleagues recently conducted a pioneering human gene therapy trial in adults with high-grade gliomas2Adair JE Beard BC Trobridge GD Neff T Rockhill JK Silbergeld DL et al.Extended survival of glioblastoma patients after chemoprotective HSC gene therapy.Sci Transl Med. 2012; 4: 133ra57Crossref PubMed Scopus (75) Google Scholar and show that this approach prevents hematopoietic suppression and may improve outcomes associated with this highly lethal and treatment-resistant cancer.2Adair JE Beard BC Trobridge GD Neff T Rockhill JK Silbergeld DL et al.Extended survival of glioblastoma patients after chemoprotective HSC gene therapy.Sci Transl Med. 2012; 4: 133ra57Crossref PubMed Scopus (75) Google Scholar Drug resistance in glioblastoma is commonly due to high-level expression of O6-methylguanine methyltransferase (MGMT) in the tumor cells. MGMT is a DNA-repair protein that protects cells by reversing the DNA damage caused by BCNU, temozolomide, and certain other alkylating agents. Although this resistance can be overcome by administration of the substrate analog O6-benzylguanine (6BG), which depletes MGMT in the tumor cells, the combined use of 6BG with alkylating agents is limited by severe hematopoietic toxicity that results in drug dose attenuation and significant treatment-related side effects.3Quinn JA Jiang SX Reardon DA Desjardins A Vredenburgh JJ Rich JN et al.Phase II trial of temozolomide plus O6-benzylguanine in adults with recurrent, temozolomide-resistant malignant glioma.J Clin Oncol. 2009; 27: 1262-1267Crossref PubMed Scopus (235) Google Scholar This hematopoietic toxicity has been overcome in animal models through retroviral-mediated transfer of a 6BG-resistant MGMT mutant gene into HSCs (Figure 1), which also allows potent in vivo selection of MGMT-transduced HSC clones.4Sawai N Zhou S Vanin EF Houghton P Brent TP Sorrentino BP Protection and in vivo selection of hematopoietic stem cells using temozolomide, O6-benzylguanine, and an alkyltransferase-expressing retroviral vector.Mol Ther. 2001; 3: 78-87Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar,5Neff T Beard BC Peterson LJ Anandakumar P Thompson J Kiem HP Polyclonal chemoprotection against temozolomide in a large-animal model of drug resistance gene therapy.Blood. 2005; 105: 997-1002Crossref PubMed Scopus (85) Google Scholar Toxicity in tissues other than the hematopoietic system is minimal with increased doses of temozolomide and 6BG, allowing significant dose escalation before secondary toxicities ensue. In the new study, three patients were treated with standard surgical resection and cranial irradiation before receiving a nonmyeloablative dose of BCNU to condition the subjects for engraftment with their own genetically modified hematopoietic cells and to treat residual brain tumor cells. Only patients with unmethylated MGMT promoter patterns in tumor tissue were enrolled. This epigenetic activation marker is known to identify patients with relatively high levels of MGMT expression, tumor cell drug resistance, and a relatively poor prognosis.6Hegi ME Liu L Herman JG Stupp R Wick W Weller M et al.Correlation of O6-methylguanine methyltransferase (MGMT) promoter methylation with clinical outcomes in glioblastoma and clinical strategies to modulate MGMT activity.J Clin Oncol. 2008; 26: 4189-4199Crossref PubMed Scopus (635) Google Scholar Autologous CD34+ cells, mobilized into the peripheral blood with granulocyte colony-stimulating factor, were transduced with a gammaretrovirus vector expressing a 6BG-resistant MGMT mutant gene and reinfused after BCNU therapy. Relatively high levels of engraftment with marked cells were obtained, ranging from 18% to 85% in peripheral blood leukocytes and 27% to 94% in myeloid progenitors from the bone marrow. This degree of engraftment and marking is notable, particularly considering the absence of fully myeloablative conditioning. After hematopoietic reconstitution, all patients received multiple cycles of chemotherapy with 6BG and temozolomide administered approximately every four to five weeks. Most treatment cycles were well tolerated, with rapid recovery of granulocytes and platelet counts in the peripheral blood, thereby reducing the risk of infection or bleeding that otherwise can limit the delivery of chemotherapy. There was no evidence of progressive hematopoietic exhaustion with repeated treatment cycles, which is commonly seen when this treatment regimen is used in the absence of chemoprotection. One patient tolerated nine courses of chemotherapy, which would be very unusual without functional protection of the hematopoietic system. Stable disease was noted in two subjects one year after diagnosis, and one of these subjects was recently reported to be a long-term survivor. Although the number of cases in this study is too small to enable statistical comparisons with historical controls, these results are very encouraging with regard to the overall clinical benefit of this approach. Despite clear evidence of chemoprotection, proof of in vivo selection of transduced HSCs was less clear. In two cases, transient increases in marked peripheral blood cells were observed immediately following drug therapy but decayed over time to lower baseline levels. Because it is impossible to ascertain what the long-term marking level would have been if no chemotherapy had been administered, evidence for stable in vivo selection remains ambiguous. One application of MGMT gene transfer is to select and amplify genetically transduced HSC clones that express a linked therapeutic gene for monogenetic disorders.7Zhao H Pestina TI Nasimuzzaman M Mehta P Hargrove PW Persons DA Amelioration of murine beta-thalassemia through drug selection of hematopoietic stem cells transduced with a lentiviral vector encoding both gamma-globin and the MGMT drug resistance gene.Blood. 2009; 113: 5747-5756Crossref PubMed Scopus (39) Google Scholar,8Beard BC Trobridge GD Ironside C McCune JS Adair JE Kiem HP Efficient and stable MGMT-mediated selection of long-term repopulating stem cells in nonhuman primates.J Clin Invest. 2010; 120: 2345-2354Crossref PubMed Scopus (88) Google Scholar Transient, rather than stable, selection has also been noted in nonhuman primate models using temozolomide and 6BG (ref. 9Larochelle A Choi U Shou Y Naumann N Loktionova NA Clevenger JR et al.In vivo selection of hematopoietic progenitor cells and temozolomide dose intensification in rhesus macaques through lentiviral transduction with a drug resistance gene.J Clin Invest. 2009; 119: 1952-1963PubMed Google Scholar) and may reflect the lack of clearance of unmodified HSCs with this particular drug combination. Stem cell selection in primates is more efficient using BCNU plus 6BG; however, BCNU is associated with more toxicity and may therefore be problematic for HSC selection outside of oncology applications. The demonstration of chemoprotection in these patients does not necessarily contradict a lack of in vivo HSC selection. If expression of the resistance gene occurs in a significant proportion of hematopoietic progenitors, increased regeneration and recovery could occur without selection at the HSC level. An important finding of this study is that when a sufficient mass of hematopoietic progenitors are rendered drug-resistant, hematopoietic toxicity can be abrogated and increased drug dose intensity becomes possible. Vector insertion-site analysis done using whole-genome sequencing revealed that, in one case, different retrovirus insertion sites were detected at serial time points. This observation suggests that either fluctuating repopulating clones composed the majority of marked cells, or—perhaps more likely—the whole-genome sequencing assay does not capture all insertion events because of sampling errors.10Gabriel R Eckenberg R Paruzynski A Bartholomae CC Nowrouzi A Arens A et al.Comprehensive genomic access to vector integration in clinical gene therapy.Nat Med. 2009; 15: 1431-1436Crossref PubMed Scopus (146) Google Scholar In the two other cases, clonal insertion events were detected at serial time points, and the proportion of certain clones increased over time. Two independent integrations into the PRDM16 locus were detected in one case, and these clones increased proportionally to compose the majority of marked cells about one year after transplant. These insertions led to activation of PRDM16 expression, presumably because the vector-encoded enhancers led to activation of the PRDM16 promoter. This apparent “clonal dominance” must be qualified by the observation that the absolute number of marked cells did not increase over time and that this clone represented only about 1% of all peripheral blood granulocytes at the latest time point. By contrast, the clonal outgrowth due to LMO2 activation that was noted in the lymphoblastic leukemia cases seen in the X-linked severe combined immunodeficiency trials11Fischer A Hacein-Bey Abina S Thrasher A von Kalle C Cavazzana-Calvo M LMO2 and gene therapy for severe combined immunodeficiency [letter].N Engl J Med. 2004; 350: 2526-2527Crossref PubMed Scopus (1) Google Scholar,12Hacein-Bey-Abina S von Kalle C Schmidt M McCormack MP Wulffraat N Leboulch P et al.LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1.Science. 2003; 302: 415-419Crossref PubMed Scopus (2995) Google Scholar progressively replaced the entire hematopoietic system. Nonetheless, these findings reinforce the dangers of gammaretroviral integration events, especially considering that PRDM16, and other integration sites detected—such as SETBP1 and HMGA2—are proto-oncogenes and well-defined hotspots for retroviral vector integration.13Ott MG Schmidt M Schwarzwaelder K Stein S Siler U Koehl U et al.Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1.Nat Med. 2006; 12: 401-409Crossref PubMed Scopus (981) Google Scholar,14Cavazzana-Calvo M Payen E Negre O Wang G Hehir K Fusil F et al.Transfusion independence and HMGA2 activation after gene therapy of human β-thalassaemia.Nature. 2010; 467: 318-322Crossref PubMed Scopus (1030) Google Scholar Although the use of gammaretroviral vectors in glioblastoma patients offers an acceptable risk-to-benefit ratio, it will be important to determine whether the use of enhancer-free lentiviral vectors will be less prone to causing clonal dominance and/or hematopoietic malignancies. The use of appropriately designed lentiviral vectors will be an important next step in chemoprotection trials for brain tumors. Another important question will be whether abrogation of MGMT-mediated resistance in brain tumor cells will lead to long-term cures. Although it is clear that MGMT is a clinically relevant resistance mechanism in glioblastoma,6Hegi ME Liu L Herman JG Stupp R Wick W Weller M et al.Correlation of O6-methylguanine methyltransferase (MGMT) promoter methylation with clinical outcomes in glioblastoma and clinical strategies to modulate MGMT activity.J Clin Oncol. 2008; 26: 4189-4199Crossref PubMed Scopus (635) Google Scholar it is possible that other mechanisms of resistance, such as lesions in DNA mismatch repair and downstream apoptosis pathways, may also contribute to temozolomide resistance.15Happold C Roth P Wick W Schmidt N Florea AM Silginer M et al.Distinct molecular mechanisms of acquired resistance to temozolomide in glioblastoma cells.J Neurochem. 2012; (e-pub ahead of print 7 May 2012)PubMed Google Scholar The degree to which these more distal resistance mechanisms are clinically significant should be evident after MGMT-mediated resistance in the tumors has been effectively overcome without dose-limiting toxicity. The study by Adair and colleagues2Adair JE Beard BC Trobridge GD Neff T Rockhill JK Silbergeld DL et al.Extended survival of glioblastoma patients after chemoprotective HSC gene therapy.Sci Transl Med. 2012; 4: 133ra57Crossref PubMed Scopus (75) Google Scholar using MGMT gene transfer to increase tolerance to 6BG plus temozolomide represents a major milestone toward this goal.