Xeroderma pigmentosum (XP) is an orphan autosomal recessive disorder of DNA repair. When exposed to genotoxic stress, XP patients have reduced capacity to remove bulky adducts by nucleotide excision repair and are thus greatly predisposed to cancer. Unfortunately, given the nature of their underlying genetic defect, tumor-bearing XP patients cannot be treated with conventional DNA damaging therapies. Engineered strains of the poxvirus Vaccinia have been shown to cure cancer in numerous preclinical models, and based on promising Phase I/II clinical trials have recently been approved for late phase evaluation in humans. As poxviruses are nongenotoxic, we investigated whether clinical-candidate strains of Vaccinia can safely and effectively treat cancers arising from XP. In vitro, Vaccinia virus was highly cytotoxic against tumor-derived cells from XP patients, on average 10- to 100-fold more so than on nontumor derived control cells from similar patients. In vivo, local or systemic administration of Vaccinia virus led to durable tumor resolution in both xenograft and genetic models of XP. Importantly, Vaccinia virus was well tolerated in the genetic models, which are each null for a critical component of the DNA repair process. Taken together, our data suggest that oncolytic Vaccinia virus may be a safe and effective therapy for cancers arising from XP, and raise the possibility of similar therapeutic potential against tumors that arise in patients with other DNA repair disorders.
Xeroderma pigmentosum (XP) is an orphan autosomal recessive disorder that results in a >1,000-fold increased predisposition towards cancer.1–3 The molecular basis of XP is deficient nucleotide excision repair,1 a process that is critical for fixing damaged DNA.4 As a consequence, individuals with XP are at extreme risk for developing cancer when exposed to DNA mutagens. Unfortunately, by the same token these individuals cannot be treated with conventional genotoxic therapies for their tumors. This “double whammy” is best exemplified with XP-induced skin cancer. XP decreases the latency at which skin neoplasms develop from an average age of 60 years to an average age of 8 years, but these patients cannot be treated with standard therapies.5 Although some preventative strategies exist, such as retinoids, UV protective clothing, and sunscreen, they are marginally effective and ∼60% of individuals with XP nonetheless develop metastatic skin cancer in childhood and die before reaching adulthood. Clearly, new nongenotoxic treatment approaches are desperately needed for this underserved patient population.
Natural and genetically modified replicating “oncolytic” viruses have shown great promise for the treatment of cancer.6, 7 We previously noted that Vaccinia virus is highly cytolytic in skin cancer cells from the NCI60 panel,8 and others have demonstrated that it can replicate within and induce tumor regression in a small group of skin cancer patients.9 Moreover, the life cycle of Vaccinia virus occurs entirely in the cytoplasm of infected cells,10 and thus there is no opportunity for genotoxicity. Given these two characteristics, as well as its advanced position in clinical development, we evaluated whether the clinical-candidate strains of Vaccinia virus can safely and effectively treat cancers arising from the genetic DNA repair disorder xeroderma pigmentosum.
Material and Methods
The clinical candidate attenuated Vaccinia viruses have been described elsewhere.11, 12 Briefly, vvΔTK (vaccinia virus missing thymidine kinase) is derived from the Wyeth strain and contains an engineered deletion of thymidine kinase, which improves tumor selectivity by attenuating virus replication in nondividing cells. The m-vvΔTK (mouse-vvΔTK) expresses the mouse cytokine granulocyte–monocyte colony-stimulating factor (GM-CSF) and is the “mouse homologue” of JX-594 (or h-vvΔTK, vvΔTK expressing human GM-CSF). The closely related vvDD (vaccinia virus doubly deleted) is derived from the Western Reserve strain and contains, in addition to the TK deletion, an additional deletion in vaccinia growth factor that further enhances its safety and therapeutic index. The m-vvDD strain is the mouse homologue of JX-963 (or h-vvDD, vvDD expressing human GM-CSF). Viruses were grown, purified and titered using standard procedures.
XP knock out models and chemical tumor induction
XP-A, XP-C and XP-V null animals have been described elsewhere.13–15 For safety studies, mice were treated intravenously (tail vein) with a single dose of Vaccinia virus (m-vvΔTK, half log increments from 3e6 to 3e8). Mice were monitored daily for weight loss, respiratory distress and other signs of morbidity, including piloerection as a surrogate for fever. For therapeutic studies, we used the two-step DMBA/TPA chemical carcinogen model16 on the XP-A mouse background. Eight- to twelve-week-old XP-A mice were given a single topical application of the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA; 100 nmol in 200 μL of acetone) to the dorsal skin. One week after DMBA treatment, the mice received thrice weekly treatments with the tumor promoter 12-O-tetradecanolyphorbol-13-acetate (TPA; 17 nmol in 200 μL of acetone). Tumors began to develop within 5 weeks of the initial DMBA treatment, most of which resembled papillomas (soft, sometimes pedunculated, sometimes having multiple projections but often just a single rounded or “raspberry-like” mass) with a small-percentage resembling squamous cell carcinomas (scaly, crusty or ulcerated lesions and/or projections) (Supporting Information Fig. S1). At approximately the midpoint of tumor burden, mice were treated with three doses of Vaccinia virus (m-vvΔTK, 1e8 pfu/dose IV, each dose separated by 48 hr). Approximately 10 weeks later, some tumors reappeared in the treated animals, and the mice were treated again with three doses of Vaccinia virus (m-vvΔTK, 1e8 pfu/dose IV + 5e7 pfu/dose IT into all visible tumors, each dose separated by 48 hr). Animals were monitored daily, as described above, and were euthanized by animal care technicians (who were blinded to the treatments) when tumors became overly ulcerated and/or their overall body condition deteriorated significantly (e.g., weight loss >20%, severe dehydration, very poor grooming, etc.). Tumor volume was measured with calipers and calculated using (L × W2)/2. For more materials and methods, please see Supporting Information.
ddVV: double-deleted (TK and Vaccinia growth factor) Vaccinia virus; DMBA: 7,12-dimethylbenz[a]anthracene; EGFR: epidermal growth factor receptor; FLUC: firefly luciferase; GFP: green fluorescent protein; GM-CSF: granulocyte–monocyte colony stimulating factor; GM-CSF, m-vvDD: ddVV expressing mouse; GM-CSF, m-vvΔTK: vvΔTK expressing mouse; moi: multiplicity of infection; PBS: phosphate buffered saline; pfu: plaque forming units; TPA: 12-O-tetradecanolyphorbol-13-acetate; vvΔTK: vaccinia virus missing thymidine kinase; XP: xeroderma pigmentosum; XP-A, XP-C, XP-V: XP mouse rendered null for complementation group A, C, V
Oncolytic Vaccinia virus is a potent killer of XP-derived melanoma cells
To begin, we first investigated the safety profile of several clinically relevant attenuated Vaccinia variants in a series of normal diploid cells derived from XP patients. Similar survival was observed in primary skin fibroblasts derived from patients with XP-V, XP-A and XP-C inactivating gene mutations infected with Vaccinia virus (EC50 = 0.01–0.5 moi, Fig. 1a) as compared to those derived from a non-affected individual (GM38, EC50 = 0.1 moi, Fig. 1a). These data demonstrate that Vaccinia virus-induced lysis is not affected by the DNA repair deficits present in cells derived from patients with XP. Next, we interrogated the capacity of Vaccinia virus to kill the only established melanoma cell line derived from a patient with XP (GM13030) as well as an SV40 transduced XP cell line (GM4429). As compared to the primary diploid cells, Vaccinia virus was 10–100 fold more lytic in the tumor-derived GM13030 cells and the transformed GM4429 cells (EC50 = 0.01–0.0005, Fig. 1a and representative image in Fig. 1b). Taken together, these cell-based data show that Vaccinia virus preferentially kills tumor versus normal cells derived from XP patients and, unlike standard genotoxic agents, cytotoxicity in normal cells is not altered by DNA repair defects.
Systemic and local-regional delivery of oncolytic Vaccinia virus effectively treats tumors in a xenograft model of XP
Next, we sought to determine the utility of oncolytic Vaccinia virus for treating XP-derived tumors in vivo. We therefore developed a subcutaneous xenograft model with GM13030 cells (Figs. 2a and 2b). CD-1 nude mice were injected subcutaneously with GM13030 cells stably expressing Firefly luciferase (FLUC), which grew into palpable tumors (50–125 mm3) in ∼80% of mice by 14–21 days (Fig. 2a), and were easily measured with bioluminescent IVIS imaging (Fig. 2b). We next determined whether various clinical-candidate Vaccinia virus variants could selectively infect and replicate within the tumor bed of GM13030-bearing mice, when delivered systemically. Attenuated clinical-candidate Vaccinia virus variants vvDD, m-vvDD and m-vvΔTK expressing either a FLUC or GFP marker were easily detected in tumors derived from parental (i.e., not expressing FLUC) GM13030 cells 24 hr postintravenous (IV) inoculation, and were not observed in nontumor tissues (Fig. 2c). We thus tested the potency of these oncolytic Vaccinia variants in the GM13030 xenograft model. Beginning 14 days following subcutaneous implantation of GM13030 cells into one (unilateral model) or both (bilateral model) flanks of CD-1 nude mice, Vaccinia virus variants were delivered intratumorally (IT) or IV thrice weekly for 2 weeks. Vaccinia virus was well tolerated in CD-1 animals with minimal weight loss (<5%) and no mortality resulting from viral dosing. We also made the following qualitative observations of the mice treated with Vaccinia virus: some pox lesions (resembling raised pustular skin lesions) on the hindlimb foot pads of several of the treated animals; a slight change in respiration rate (mild hyperventilation); however, these symptoms quickly resolved after the treatments stopped. While tumor growth progressed unabated in PBS treated animals, all Vaccinia virus variants led to a significant resolution of tumors by Day 28 and durable cures in most animals (Figs. 2d–2f). In a small number of animals (see Material and Methods), tumor relapse occurred. We redosed these animals at Days 44, 46, 50 and 52, after which time the tumors regressed permanently. Upon postmortem examination, we noticed that mock treated animals had a large primary tumor burden as well as metastasis to the liver, lungs and rib cage (representative image in Fig. 2f). In contrast, the tumors in animals treated with Vaccinia variants were nonvascularized and accumulated inflammatory exudate prior to complete resolution, and the animals were devoid of detectable metastases. Collectively, these data demonstrate that clinical-candidate oncolytic Vaccinia virus variants are highly effective at treating XP-derived melanomas and preventing metastatic spread in our GM13030 xenograft model.
Oncolytic Vaccinia virus induces melanoma regression in a genetic model of XP
Finally, we evaluated whether Vaccinia virus could treat chemically induced tumors in a genetic model of XP. To begin, we examined the safety profile of Vaccinia virus in animals rendered null for XP-A, XP-C and XP-V. Intravenous dosing in half log increments beginning at 3e6 and ending at 3e8 demonstrated that oncolytic Vaccinia virus (m-vvΔTK) is safe in XP-A-, XP-C- and XP-V-null mice, at least at doses up to three times higher than our standard therapeutic dose of 1e8 for immunocompetent mice.17 All animals survived these safety studies, regardless of genetic background (Supporting Information Fig. S2). At the highest doses tested (1e8 and 3e8), all animals displayed mild weight loss (<5%) 24–72 hr post-treatment, which returned to baseline within a week (Supporting Information Fig. S2), but had no other evidence of morbidity. These data corroborate the cell-based safety results and demonstrate that a therapeutically relevant dose of Vaccinia virus is well tolerated in XP knockout mice.
To induce tumor growth, the carcinogen DMBA and the tumor promoter TPA were applied topically to the dorsal skin of XP-A mice.16 In a small percentage of animals, tumor growth became apparent ∼5 weeks later, after which time the mice were treated with three IV doses of Vaccinia virus (m-vvΔTK, or PBS) separated by 48-hr each. Tumor growth was relatively steady in the PBS control mice, which reached endpoint ∼10 weeks later due to weight loss (>20%), poor body condition and/or ulcerated tumors (Figs. 3a–3c). In contrast, the tumors in mice treated with Vaccinia virus regressed substantially. Importantly, in response to regrowth of several of the treated tumors ∼10 weeks later, a second round of Vaccinia therapy led to complete tumor resolution and long-term survival (Figs. 3a–3c). Collectively, our data indicate that oncolytic Vaccinia virus is a safe and effective therapeutic agent against tumors induced in an immunocompetent genetic model of XP. Moreover, following tumor recurrence, repeated treatment with Vaccinia virus is equally if not more potent than the original doses.
Herein, we have shown that several clinical-candidate oncolytic Vaccinia virus variants can safely and effectively treat tumors derived from both an immunocompromised xenograft as well as an immunocompetent chemical-genetic model of XP. The potency with which Vaccinia virus destroys XP-derived tumors is likely due to several factors. First, oncolytic vaccinia variants are tumor selective in large part due to their dependence on activated EGFR and ras pathways,18 and the GM13030 tumor-derived cells have a strongly activating ras mutation.19 Thus, it appears that oncolytic Vaccinia variants may have inherently high tropism for XP-derived tumor cells. Furthermore, we noted upon postmortem examination that the XP-derived tumors were highly vascularized. As part of its multimodal mechanism of tumor destruction, Vaccinia virus induces a focal inflammatory response to the tumor vasculature that leads to vessel occlusion and impaired tumor blood flow (i.e., “vascular shutdown”),20 which can effectively starve a tumor. Finally, several of the Vaccinia virus variants used in this study were ‘armed’ with the cytokine GM-CSF, which has been shown to stimulate antitumor immunity.21 Thus, immune awakening to tumor-associated antigens, released upon Vaccinia virus-mediated tumor cell lysis, may also have contributed to tumor resolution and durable disease-free status.
A key finding in this study was that recurrent cancers in the XP mice models were effectively treated with a subsequent regimen of Vaccinia virus therapy. As treating a primary cancer in patients with XP is purely symptomatic management that does not address the underlying genetic defect, cancer relapse is inevitable. A theoretical disadvantage of oncolytic virus-based therapies for XP patients is the strong adaptive antiviral immune response that would be elicited upon initial virus exposure, which could greatly impair oncolytic efficacy against recurrent tumors. However, being somewhat resistant to complement and antibodies,18, 22, 23 Vaccinia virus is in fact a compelling oncolytic agent for treating relapse in the same patient. As an example, in a completed Phase I study, a patient who had been successfully treated for hepatocarcinoma with the clinical-candidate Vaccinia virus JX-594 developed a secondary head and neck tumor. Direct intratumoral injection of JX-594 led to robust reduction in tumor burden despite the presence of high anti-Vaccinia antibody titers.23 Moreover, Hwang et al. recently demonstrated that Vaccinia virus can efficiently replicate in solid tumors in patients with metastatic melanoma well after the induction of neutralizing antibodies.24 Our data in mice suggests that Vaccinia virus could be used in the same manner to treat recurrent tumors in patients with XP.
One limitation of our work is that, for the genetic studies, we performed our therapeutic analyses in but one mouse model (XP-A null) using a single skin carcinogen (DMBA/TPA). It is well known that disparities in sensitivity to carcinogens exist between the mouse models missing one of the various XP complementation groups,25 and that different carcinogens (e.g., UV versus DMBA) induce unique, albeit somewhat overlapping, tumor profiles in the XP mouse models.13, 15 We opted for the XP-A null mouse treated with DMBA/TPA because others have shown that this is the quickest method to induce skin tumors on an XP mouse model.13 This protocol led to the development of tumors that visually resembled both papillomas and SCCs, all of which were eradicated by virus treatment. However, future work using different carcinogens (e.g., UV) on the other XP mouse models is clearly warranted, to gain a clearer picture of the potential clinical utility of oncolytic Vaccinia virus for human XP.
Because Phase I/II clinical evaluation is complete and late-stage multicenter trials have begun, it is a conceivable that the Vaccinia virus variant JX-594 will be FDA approved within the next 2–4 years. Given the small market for orphan diseases such as XP, there is little focus on therapeutic development for the cancers that arise in these patients. We have now demonstrated that several clinical-candidate Vaccinia virus variants are safe in mouse models of XP, and can potently treat primary and recurrent tumors that develop therein. Although further evaluation is clearly warranted, our data point towards Vaccinia virus as a therapeutic option for cancers arising in XP, and possibly other autosomal recessive DNA repair disorders such as Fanconi's anemia, Werner's syndrome, Bloom's syndrome and ataxia talengiectasia.
The authors acknowledge Dr. Errol Friedberg (University of Texas Southwestern) for kindly providing XPA and XPC animals and Dr. Raju Kucherlapatie (Harvard Medical School) for kindly providing XPV animals.