p53基因及CAR受体对ONYX-015肿瘤杀伤作用的影响

目的：利用肿瘤特异性增殖型腺病毒ONYX-015分别感染具有柯萨奇病毒和腺病毒联合受体（CAR）水平正常、p53正常或突变的,以及CAR水平低下、p53突变的肿瘤细胞株,研究ONYX-015对这些肿瘤细胞的特异性增殖及杀伤能力。方法：以正常的肝细胞株L02作为对照,用细胞病变效应（CPE）实验观察ONYX-015对细胞的选择性杀伤效应;病毒增殖实验检测野生型腺病毒（Ad5）、ONYX-015在多种肿瘤细胞中的增殖能力。结果：ONYX-015对正常的肝细胞L02无杀伤性,但能够有效地杀伤p53突变的肝癌细胞Hep3B、p53正常的肝癌细胞HepG2及肺癌细胞A549,不能杀伤p53突变的人乳腺癌细胞株MDA—MB-231。在CAR受体水平正常的癌细胞株Hep3B、HepG2和A549中,Ad5和ONYX-015均可增殖。在CAR受体水平低下、p53突变的人乳腺癌细胞株MDA—MB-231中,两种病毒均不增殖。结论：CAR受体对ONYX-015的增殖力起着至关重要的作用。在CAR受体水平正常的前提下,无论肿瘤细胞的p53基因正常与否,ONYX-015均可以有效增殖并杀伤细胞;相反,如果CAR受体水平低下,即使该种肿瘤细胞p53基因突变,ONYX-015在该细胞中的增殖力也会受到限制。ONYX-015不杀伤CAR受体及p53基因均正常的正常肝细胞。     

特异性增殖型腺病毒CNHK200-mIFN-γ表达mIFN-γ及体外肿瘤杀伤实验

目的:构建增殖型腺病毒CNHK200-mIFN-γ和增殖缺陷型腺病毒AdEasy-mIFN-γ,比较两者在肿瘤细胞中表达mIFN-γ蛋白的能力以及CNHK200-mIFN-γ,ONYX-015及野生型腺病毒Ad5在正常及肿瘤细胞中的增殖力,进一步观察其抗瘤效果.方法:以病毒增殖试验检测病毒在细胞中的增殖能力;透射电镜观察CNHK200-mIFN-γ在Hep3B细胞中的增殖复制;检测病毒感染肿瘤细胞后mIFN-γ的表达;CPE实验观察增殖病毒对细胞的杀伤效应.结果:CNHK200-mIFN-γ和ONYX-015仅在肿瘤细胞中增殖,且前者增殖力较强.CNHK200-mIFN-γ在肿瘤细胞中表达mIFN-γ,且表达量与病毒增殖密切相关,而AdEasymIFN-γ在肿瘤细胞中的mIFN-γ表达几乎不能测到.ONYX-015与CNHK200-mIFN-γ对正常的细胞无杀伤性,但能有效地杀伤癌细胞.结论:外源基因mIFN-γ的插人没有改变增殖病毒在肿瘤细胞中选择性增殖的特性.增殖型腺病毒CNHK200-mIFN-γ具有良好的肿瘤选择性及增殖性,且可以有效表达mIFN-γ,并且有效杀伤癌细胞,显示其良好的临床应用前景.     

E1B55kDa缺失的增殖腺病毒抗肿瘤效果差异及其分子机制

目的:观察E1B55kDa缺失的增殖腺病毒CNHK200和CNHK200-hA对A549肺癌和MBD-231乳腺癌动物模型的抗肿瘤效果,结合研究肿瘤细胞柯萨奇病毒-腺病毒受体(CAR)的表达状态,分析腺病毒抗肿瘤效果的差别及其分子机制。方法:建立A549肺癌和MBD-231乳腺癌裸鼠模型,给予病毒总剂量1×109pfu的CNHK200和CNHK200-hA治疗。观察期结束,取肿瘤标本进行柯萨奇病毒-腺病毒受体(CAR)和腺病毒壳蛋白Hexon的免疫组化定位。结果:在CAR水平高表达的A549细胞内,CNHK200-hA和CNHK200均可以有效增殖并杀伤肿瘤细胞,产生明显的疗效。相反,在CAR水平低下的MBD-231细胞内,CNHK200-hA和CNHK200没有增殖复制能力,CNHK200几乎没有任何治疗效果,CNHK200-hA的治疗效果仅由Angiostatin基因表达所产生。结论:CAR在E1B55kDa缺失的增殖腺病毒的感染和增殖过程中起着至关重要的作用,肿瘤细胞CAR表达低下可以影响腺病毒载体的感染力和增殖力,从而降低用腺病毒进行肿瘤基因治疗的疗效。     

非增殖型腺病毒和肿瘤特异性增殖型腺病毒携带IL-12基因治疗肝癌的体外实验研究

目的 比较携带小鼠IL-12基因的增殖型腺病毒(CNHK200-mIL12)和非增殖型腺病毒(Adv-mIL12)对IL-12基因的表达以及对肝癌细胞的杀伤能力。方法 通过MTT以及病毒增殖实验．评估E1B-55000缺陷的增殖型腺病毒CNHK200-mIL12和ONYX-015(dl1520),以及非增殖型腺病毒Adv-mIL12对人正常肝细胞株LO2、人肝癌细胞株HepG2和Hep3B的杀伤能力。采用蛋白质印迹分析和ELISA法,检测CNHK200-mIL12和Adv-mill2感染HepG2和Hep3B细胞后,小鼠IL-12基因的表达情况。结果 CNHK200-mIL12感染HepG2和Hep3B细胞后大量增殖,在感染后96h时检测,分别增殖3160倍和630倍,在极低的MOI(空斑形成单位／细胞)值和极短的时间内(HepG2细胞：MOI=0．2,第4天;Hep3B细胞：MOI=0．005,第2天),可大量杀伤肿瘤细胞,而对LO2细胞无明显杀伤。CNHK200-miLl2和Adv-mIL12感染HepG2细胞后,其IL-12基因表达量,前者是后者的101倍;感染Hep3B细胞后,前者是后者的20倍。结论 增殖型腺病毒载体对肿瘤细胞的杀伤能力和目的基因的表达,明显优于传统的非增殖型腺病毒载体,应用前景广阔。     

肿瘤选择性增殖腺病毒CNHK300治疗乳腺癌的体外实验研究(英文)

目的观察肿瘤选择性增殖腺病毒 CNHK300对乳腺癌的选择性杀伤作用。方法用 RT-PCR 方法检测各种细胞株的端粒酶活性;CNHK300、ONYX-015(E1B 55 KDa 蛋白缺失的2型和5型嵌合型腺病毒)、wtAd5(野生型腺病毒)分别行病毒增殖实验和细胞生长抑制实验,验证 CNHK300选择性复制和杀伤能力;Western Blot 检测腺病毒E1A 在细胞中的表达。结果乳腺癌细胞株 MCF-7、BT-549和 SK-BR-3端粒酶 hTERT mRNA 均为阳性表达,而正常成纤维细胞株 MRC-5和 BJ 端粒酶hTERT mRNA 为阴性。CNHK300在乳腺癌细胞 MCF-7、BT-549和 SK-BR-3中48 h 复制倍数分别为40 625、1265和20000倍,与wtAd5的增殖能力相似,较 ONYX-015增殖能力强,在 MCF-7和 BT-549细胞中复制能力甚至强于野生型腺病毒。然而,在正常成纤维细胞 MRC-5和 BJ 中 CNHK300病毒增殖能力减弱,48 h 增殖倍数为63～192倍,而 wtAd5增殖仍可高达3160～4846倍CNHK300 MOI 10 PFU/cell 作用7天,可有效杀伤半数乳腺癌细胞,与 ONYX-015相比,CNHK300具有更强的肿瘤杀伤能力CNHK300对正常成纤维细胞的杀伤力较 wtAd5明显减弱,CNHK300在 MOI 100 PFU/cell 时 BJ 细胞存活率50%以上。正常成纤维细胞株中未检测到 CNHK300 E1A 基因表达,在293细胞和感染 CNHK300的乳腺癌细胞株中能够检测到 E1A 基因表达。结论 hTERT 启动子可成功地调控腺病毒 CNHK300选择性在端粒酶阳性的乳腺癌细胞中复制,并产生溶瘤作用。可望成为治疗乳癌的一种新的治疗策略。     

肿瘤细胞表面柯萨奇病毒 腺病毒受体与5型腺病毒感染效率的关系

目的〖HT5"SS〗: 探讨不同肿瘤细胞系柯萨奇病毒腺病毒受体（CAR）和整合素的表达水平与5型腺病毒感染效率的关系,为腺病毒基因治疗研究奠定基础。〖HT5W〗方法〖HT5"SS〗: 利用瞬时转染CAR的真核表达质粒提高肿瘤细胞表面CAR的表达,应用抗体封闭细胞表面的CAR和整合素后,通过流式细胞仪和荧光素酶分析法测定腺病毒Ad5CMVEGFP和Ad5CMVLuc对肿瘤细胞的感染效率和基因表达水平的变化。〖HT5W〗结果〖HT5"SS〗： 不同肿瘤细胞表面CAR和整合素的表达水平是不同的,其中SMMC7721和A549细胞CAR的表达量最高,而K562细胞CAR的表达水平最低;荧光显微镜和流式细胞仪检测Ad5CMVEGFP对肿瘤细胞的感染效率,结果显示5型腺病毒对于SMMC7721和A549细胞的感染效率最高,而对于K562细胞则很低;瞬时转染表达CAR的真核质粒可提高多种肿瘤细胞表面CAR的表达,较大幅度提高了5型腺病毒的感染效率。而抗体封闭肿瘤细胞表面的CAR或整合素后,腺病毒感染效率显著下降。〖HT5W〗结论〖HT5"SS〗:肿瘤细胞表面CAR和整合素的表达水平决定了腺病毒对肿瘤细胞的感染效率。     

一种新型增殖型腺病毒治疗肝癌的体外研究

目的评价增殖腺病毒 CNHK500对肝癌细胞的治疗效果。方法利用病毒增殖实验、细胞活力实验(MTT)、蛋白印迹分析来检测增殖病毒 CNHK500在端粒酶阳性的肝癌细胞株 HepGⅡ、Hep3B、SMMC7721及正常细胞中选择性增殖和溶解细胞的特性。结果 CNHK500感染人肝癌细胞株 HepGⅡ、Hep3B、SMMC7721细胞后大量增殖,在感染后96小时增殖倍数分别为52000、396984.9和632911.3倍,同野生型5型腺病毒(wtAd5)类似。然而在正常细胞中,CNHK500的增殖能力较 wtAd5大大减弱,感染96小时后仅增殖3.1～100倍,而 wtAd5却高达3160～17357倍。MTT 实验观察到在肝癌细胞 HepGⅡ和 Hep3B 中,感染后第7天达到半数杀伤的 MOI 值(IC50)分别为2和0.01,而在正常成纤维细胞 BJ 细胞中却高达1000。在常氧情况下,用蛋白印迹可在肿瘤细胞中检测到腺病毒 E1A 蛋白的表达,但在正常细胞却检测不到。E1B 蛋白仅在缺氧条件下(0.1%O_2)的肿瘤细胞中表达。结论实验结果表明 CNHK500能有效地选择性在肝癌细胞中增殖、复制、杀伤,而在正常细胞中增殖和溶解细胞能力却大大减弱。联合治疗基因,CNHK500可能为肝癌的治疗提供一种新的策略。     

溶瘤腺病毒SG7605-11R-P53对肝癌细胞的体外杀伤作用

目的:研究携带细胞穿膜肽11R和P53的溶瘤腺病毒SG7605-11R-P53对肝癌细胞的体外杀伤作用。方法:以本课题组前期实验构建的携细胞穿膜肽11R和P53的溶瘤腺病毒SG7605-11R-P53和不携11R的溶瘤腺病毒SG7605-P53感染肝癌细胞HepG2、SMMC-7721、Hep3B、Huh7和正常成纤维细胞株BJ,Western blotting检测感染后细胞P53和11R-P53的表达情况,TCID50法检测SG7605-11R-P53和SG7605-P53在肝癌细胞中的增殖能力,MTT法检测SG7605-11R-P53对肝癌细胞及正常细胞的杀伤作用。结果:SG7605-11R-P53和SG7605-P53能在肝癌细胞中高表达P53和11R-P53蛋白。SG7605-11R-P53可在HepG2、SMMC-7721、Hep3B和Huh7细胞中大量增殖,其增殖倍数是SG7605-P53的10~100倍,但在正常BJ细胞内几乎不增殖。SG7605-11R-P53在MOI=0.1时对Hep3B细胞的杀伤率达90%,对于正常BJ细胞只有当MOI=50时才有很弱的抑制作用;SG7605-11R-P53对4种肝癌细胞杀伤作用的大小依次为Hep3B、HepG2、Huh7和SMMC-7721细胞。结论:携带细胞穿膜肽11R和P53的SG7605-11R-P53溶瘤腺病毒体外对4种肝癌细胞株均有较好的靶向杀伤作用,尤其对Hep3B细胞的杀伤作用最强。     

装载mIFN-γ基因的溶瘤腺病毒CNHK200的抗肝癌活性

目的:研究鼠源γ-干扰素(mIFN-γ)基因插入溶瘤腺病毒CNHK200后,该病毒对不同的肝癌细胞系及其裸鼠移植瘤的增殖力及特异性杀伤作用。方法:用MTT法检测CNHK200-mIFN-γ在体外对人正常肝细胞L02、人肝癌细胞Hep3B、HepGⅡ的特异性杀伤作用;裸鼠体内试验观察CNHK200-mIFN-γ对肝癌Hep3B模型的抗肿瘤疗效。结果:CNHK200-mIFN-γ对正常人肝细胞L02无杀伤作用,但能特异性杀伤肝癌细胞,mIFN-γ的插入使病毒对肿瘤细胞的杀伤能力提高;动物体内,CNHK200-mIFN-γ具有明显的肿瘤生长抑制作用。结论:CNHK200-mIFN-γ可以高效率地杀死肝癌肿瘤细胞,而不杀伤正常细胞,可能具有良好的临床应用前景。     

5/35嵌合型溶瘤腺病毒SG635对肝癌细胞的抑制作用

目的:研究5/35嵌合型溶瘤腺病毒SG635在体外对肝癌HepG2和SMMC-7721细胞的特异性杀伤作用。方法:将SG600载体中5型腺病毒(Ad5)纤毛蛋白的knob和shaft结构域替换为35型腺病毒(Ad35)纤毛蛋白的相应结构域,构建成5/35嵌合型溶瘤腺病毒SG635。流式细胞术检测5/35嵌合型腺病毒Ad5/35-EGFP对HepG2和SMMC-7721细胞的感染效率,体外病毒增殖实验观察溶瘤腺病毒SG635的增殖能力,Western blotting检测SG635感染后肝癌细胞中E1A蛋白的表达,CCK-8实验检测SG635对肝癌HepG2和SMMC-7721细胞的杀伤作用。结果:在肝癌HepG2和SMMC-7721细胞中,Ad5/35-EGFP的感染效率明显强于5型腺病毒Ad5-EGFP;5/35嵌合型溶瘤腺病毒SG635在HepG2和SMMC-7721细胞中72 h的增殖倍数高于5型溶瘤腺病毒SG600(15 848.93vs6 309.57,6 309.57vs5 011.87,均P<0.01),而在人正常成纤维细胞BJ中几乎不增殖。SG635感染后,HepG2和SMMC-7721细胞中E1A蛋白表达高于SG600感染,在BJ中则无E1A表达。在一定MOI范围内,SG635对于HepG2细胞和SMMC-7721细胞的杀伤作用逐渐增强,且杀伤率明显强于SG600(MOI为1时,90%vs60%;MOI为10时,90%vs50%),对BJ无杀伤作用。结论:5/35嵌合型溶瘤腺病毒SG635能够高效感染并特异性杀伤肝癌细胞,具有较好的靶向性和安全性。     

Late viral RNA export, rather than p53 inactivation, determines ONYX-015 tumor selectivity

ONYX-015 is an adenovirus that lacks the E1B-55K gene product for p53 degradation. Thus, ONYX-015 was conceived as an oncolytic virus that would selectively replicate in p53-defective tumor cells. Here we show that loss of E1B-55K leads to the induction, but not the activation, of p53 in ONYX-015-infected primary cells. We use a novel adenovirus mutant, ONYX-053, to demonstrate that loss of E1B-55K-mediated late viral RNA export, rather than p53 degradation, restricts ONYX-015 replication in primary cells. In contrast, we show that tumor cells that support ONYX-015 replication provide the RNA export function of E1B-55K. These data reveal that tumor cells have altered mechanisms for RNA export and resolve the controversial role of p53 in governing ONYX-015 oncolytic selectivity.     

In vivo antitumor activity of ONYX-015 is influenced by p53 status and is augmented by radiotherapy

The E1B-deleted, replication-competent ONYX-015 (dl1520) adenovirus was originally described as being able to selectively kill p53-deficient cells due to a requirement of p53 inactivation for efficient viral replication. This hypothesis has become controversial because subsequent in vitro studies have demonstrated that the host range specificity of ONYX-015 is independent of p53 gene status. Using a pair of isogenic cell lines that differ only in their p53 status, we demonstrate here that although ONYX-015 can replicate in both p53 wild-type and mutant cells in vitro, the virus demonstrates significantly greater antitumor activity against mutant p53 tumors in vivo. Moreover, ONYX-015 viral therapy can be combined with radiation to improve tumor control beyond that of either monotherapy. The results demonstrate that ONYX-015 can discern in vivo between tumors having a different p53 status and that it may be an effective neoadjuvant to radiation therapy.     

Interactions between adenovirus proteins and the p53 pathway: the development of ONYX-015

dl1520 (ONYX-015) is an adenovirus mutant that lacks the E1b 55K gene. As a result it cannot neutralize p53. Therefore dl1520 should only grow in cells that lack p53, and should replicate selectively in cancer cells. However, there is no correlation between replication and p53 status, in cancer cells. This is for two reasons: (1) E1B 55K has additional functions, that are necessary in some tumor cells and not in others. (2) p53 function can be lost by alternative mechanisms such as loss of p14ARF. In normal cells, dl1520 induces p53, and is generally strongly attenuated for replication. ONYX-015 is currently being tested in clinical trials, and is a promising new therapeutic agent in cancer.     

Selective replication and oncolysis in p53 mutant tumors with ONYX-015, an E1B-55kD gene-deleted adenovirus, in patients with advanced head and neck cancer: a phase II trial

ONYX-015 is an E1B-55kDa gene-deleted adenovirus engineered to selectively replicate in and lyse p53-deficient cancer cells. To evaluate the selectivity of ONYX-015 replication and cytopathic effects for the first time in humans, we carried out a Phase II clinical testing of intratumoral and peritumoral ONYX-015 injection in 37 patients with recurrent head and neck carcinoma. Patients received ONYX-015 at a daily dose of 1 x 10(10) plaque-forming units (pfu) via intratumoral injection for 5 days during week 1 of each 3-week cycle (n = 30; cohort A), or 1 x 10(10) pfu twice a day for 10 days during weeks 1 and 2 of each 3-week cycle. Posttreatment biopsies documented selective ONYX-015 presence and/or replication in the tumor tissue of 7 of 11 patients biopsied on days 5-14, but not in immediately adjacent normal tissue (0 of 11 patients; P = 0.01). Tissue destruction was also highly selective; significant tumor regression (>50%) occurred in 21% of evaluable patients, whereas no toxicity to injected normal peritumoral tissues was demonstrated. p53 mutant tumors were significantly more likely to undergo ONYX-015-induced necrosis (7 of 12) than were p53 wild-type tumors (0 of 7; P = 0.017). High neutralizing antibody titers did not prevent infection and/or replication within tumors. ONYX-015 is the first genetically engineered replication-competent virus to demonstrate selective intratumoral replication and necrosis in patients. This agent demonstrates the promise of replication-selective viruses as a novel therapeutic platform against cancer.     

Oncolytic activity of the E1B-55 kDa-deleted adenovirus ONYX-015 is independent of cellular p53 status in human malignant glioma xenografts

Treatment of malignant gliomas remains a major challenge in adults and children because of high treatment failure. The E1B 55 kDa-gene deleted adenovirus, ONYX-015 (ONYX Pharmaceuticals), was demonstrated to replicate selectively in and lyse tumor cells. Currently ongoing clinical trials of ONYX-015 in head and neck tumors are promising. Here, we demonstrate ONYX-015-mediated cell lysis and antitumor activity in three of four s.c. human malignant glioma xenografts deriving from primary tumors. Intratumoral injections of ONYX-015, 1 x 10(8) plaque-forming units daily for 5 consecutive days, yielded significant tumor growth delay in the p53 mutant xenografts IGRG88 and the p53 wild-type IGRG93 and IGRG121 treated at an advanced tumor stage. The p53 wild-type tumors IGRG93 and IGRG121 experienced 45% and 82% complete tumor regressions. Four and 8 of 11 animals, respectively, survived tumor free 4 months after treatment. Widespread intratumoral adenoviral replication was observed in tumor cells of these two xenografts compared with only scattered replication in the p53-mutant tumors. In addition to a fast tumor growth rate, wild-type p53 status was associated with increased antitumor activity of the E1B-attenuated virus, and induction of functional p53 may therefore determine adenoviral cytolysis in tumor cells. In conclusion, ONYX-015 displayed a major antitumor activity in human xenografts derived from primary malignant glioma supporting its development in the treatment of these highly malignant tumors.     

ONYX-015 works synergistically with chemotherapy in lung cancer cell lines and primary cultures freshly made from lung cancer patients

p53 mutations and loss of heterozygosity (LOH) have been detected in >50% of lung cancers. Wild-type p53 can prevent replication of damaged DNA and promote apoptosis of cells with abnormal DNA. A human adenovirus, ONYX-015, which has a deletion in the E1B region, has shown tumor-specific cytolytic effect in tumor cells with nonfunctional p53 and antitumor efficacy that can be augmented by chemotherapeutic agents. A recent report from an independent group, however, indicates that wild-type p53 is necessary for the infection of this replicating virus, and it is in direct contradiction to previous observations of the ONYX group. In this study, we carried out cytopathic effect (CPE) assays using ONYX-015 on five human lung cancer cell lines with known p53 status. Two of these cell lines, NCI-H522 and NCI-H1703, have mutations and LOH in their p53 gene. Both lines were lysed in a dose-dependent manner and showed 100% cytolysis at a multiplicity of infection of 0.1. Two additional cell lines, NCI-H2347 and NCI-H838, both of which have wild-type p53 gene, showed near complete lysis at a multiplicity of infection of 1. We demonstrate here that the lung cancer cells with nonfunctional p53 are at least 10 times more sensitive to ONYX-015 cytolysis than the lung cancer cells with wild-type p53. In addition, standard chemotherapeutic agents (paclitaxol and cisplatin) showed a synergistic effect when combined with ONYX-015, and this effect was p53 mutant dependent. Furthermore, we tested the cytolytic effect of ONYX-015 on a panel (n = 7) of primary first-passage cultures made from freshly resected lung cancers. ONYX-015 lysed primary lung cancer cells in six of seven (86 %) primary cultures. Two of four primary cultures treated with chemotherapeutic agents had a synergistic effect with ONYX-015. Our data indicate that wild-type p53 is not required for the infection of this replicating virus, and also we demonstrate that ONYX-015 is effective alone and works synergistically with chemotherapeutic agents in lung cancer cell lines and primary cultures. This study suggests that ONYX-015 may be effective, especially in combination with conventional chemotherapy, in the treatment of patients with lung cancer.     

ONYX-015 selectivity and the p14ARF pathway.

ONYX-015, dl1520, is an adenovirus that lacks the E1B 55K gene and therefore lacks the capacity to neutralize p53 during infection. This virus induces high levels of p53 and fails to grow efficiently in primary epithelial cells. However, it does replicate in many tumor cells, including those expressing wild-type p53. In these cells, ONYX-015 fails to induce active p53. This is because the pathway from Ela to p53 is disrupted through loss of p14ARF. We propose that high levels of Mdm2 activity resulting from loss of p14ARF, and high levels of Mdm2 protein resulting from activated Ras prevent accumulation of functional p53 during infection of tumor cells that retain wild-type p53.     

Elucidation of the molecular mechanism underlying tumor-selective replication of the oncolytic adenovirus mutant ONYX-015

Tumor-selective replicating viruses offer appealing advantages over conventional cancer therapy. ONYX-015 (dl1520) is the prototype for oncolytic adenoviral therapy. It has undergone extensive clinical testing with proven safety and evidence of promising clinical efficacy. The strategy underlying its tumor-selective cell killing is based on deletion of the viral E1B-55K gene, which is crucial for efficient viral replication in normal cells but dispensable in tumor cells. Originally, the successful replication of ONYX-015 was thought to strictly depend on deregulated p53 signaling in tumor cells. However, recent preclinical as well as clinical evidence questions this mechanism. The study by O'Shea and colleagues is of immense importance as it sheds new light into the molecular mechanism underlying the tumor-selective replication of ONYX-015. Based on these findings, modulation of the proposed molecular mechanism by pharmacologic agents or hyperthermia may largely enhance the therapeutic index of ONYX-015 for tumor cells versus normal tissue and improve clinical efficacy. Finally, new strategies to allow successful patient stratification for future clinical trials appear to be in reach, based on the reported results.