A Review of the Role of Puma, Noxa and Bim in the Tumorigenesis, Therapy and Drug Resistance of Chronic Lymphocytic Leukemia

REVIEW A review of the role of Puma, Noxa and Bim in the tumorigenesis, therapy and drug resistance of chronic lymphocytic leukemia

L-N Zhang, J-Y Li and W Xu

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults in the Western countries. The entire pathogenesis of CLL is not clear now, but defective regulation of apoptosis seems to be more important than uncontrolled cell proliferation in CLL. There are two main pathways of apoptosis: the extrinsic pathway and the intrinsic pathway. It is worth noting that the intrinsic pathway, rather than the extrinsic pathway, appears to be the key mediator of impaired apoptosis in CLL as a result of B-cell lymphoma 2 (Bcl-2) upregulation. One subclass of pro-apoptotic members within the Bcl-2 family are Bcl-2 homology 3 (BH3)-only proteins. They can regulate directly and/or indirectly the remaining Bcl-2 proteins to endanger mitochondria and induce apoptosis. We chose three molecules from the BH3-only family, Puma, Noxa and Bim, respectively, which had shown an exciting antitumor potential in previous reports, to explore their characters and functions in tumorigenesis, therapy and drug resistance of CLL.

Cancer Gene Therapy (2013) 20, 1–7; doi:10.1038/cgt.2012.84; published online 23 November 2012 Keywords: BH3-only; Puma; Noxa; Bim; chronic lymphocytic leukemia

INTRODUCTION proteins, but recently they have also been suggested containing a 8 Chronic lymphocytic leukemia (CLL) is the most common leukemia BH4 domain. Many Bcl-2 proteins also contain a hydrophobic in adults in Western countries. It is a heterogeneous disease transmembrane domain, which is not necessarily present in each characterized by the accumulation of mature CD5 þ CD19 þ member (Figure 1). CD23 þ B lymphocytes in peripheral blood, bone marrow, lymph nodes and spleen.1 Although the entire pathogenesis of CLL has not been clariﬁed, it was previously assumed that CLL was THE MECHANISM OF BH3-ONLY FAMILY REGULATING THE associated with a defective regulation of apoptosis, rather than INTRINSIC PATHWAY uncontrolled cell proliferation, as the vast majority of tumor cells 2 When cells receive intrinsic death signals, there will be a did not display a high proliferative capacity. Therefore, we paid transcriptional upregulation and/or activation of pro-apoptotic more attention to the restoration of the apoptotic pathway as a BH3-only proteins such as Bim, Bid, Bad, Bmf, Noxa and Puma. novel approach for treating CLL. It is well known that there are These proteins are able to bind anti-apoptotic members of the two main pathways of apoptosis: the extrinsic pathway triggered family (for example, Bcl-2, Bcl-XL, Bcl-W and/or Mcl-1) and inhibit by death signals from cell surface and the intrinsic or B-cell their activity. In addition to inhibiting anti-apoptotic proteins, lymphoma 2 (Bcl-2) family-regulated pathway started with the some direct activators (for example, Bid and Bim) also bind and perturbation of mitochondria. Bcl-2 homology 3 (BH3)-only activate the effectors Bak and Bax. Once activated, Bak and Bax proteins are pro-apoptotic members within the Bcl-2 family that can form an oligomer and subsequently punch pores in the outer can regulate directly and/or indirectly the remaining anti- and pro- mitochondrial membrane, leading to mitochondrial outer mem- apoptotic Bcl-2 proteins to endanger mitochondria and induce 3 brane permeabilization. Afterwards, pro-apoptotic proteins such apoptosis. Here we chose Puma, Noxa and Bim from BH3-only as cytochrome c and second mitochondria-derived activator family to explore their characters and functions in CLL. caspase (SMAC) are released from the intermembrane space into the cytosol where they initiate a cascade of caspase activation events that result in apoptosis (Figure 2).3–7,9 MEMBERS OF THE BCL-2 FAMILY The BH3-only proteins are often divided into two subgroups Members of the Bcl-2 family are able to regulate cell death in according to their ability of interacting with the anti-apoptotic and response to a wide range of intracellular stress signals, including effectors of Bcl-2 family. Members of the ﬁrst subgroup are termed hypoxia, growth factor or cytokine deprivation, DNA damage, the direct activators, including Bid and Bim, that can bind and certain anticancer drugs and so on.4,5 This protein family contains activate the effectors Bak and Bax directly.6,10 However, it is three structurally and functionally distinct subgroups important to note that the combined genetic deletion of Bid and characterized by structural motives called BH domains (Table 1). Bim only results in a slight decline of the intrinsic apoptosis, Anti-apoptotic members share four such homology domains suggesting the presence of either additional direct activators or (BH1–4), whereas pro-apoptotic members possess either three BH alternate means of Bax/Bak activation.11 The second subgroup of domains (BH1–3) or only the BH3 domain.4–7 Those that carry BH3-only proteins is known to be the sensitizers/de-repressors.10 BH1–3 are considered to be the effectors of pro-apoptotic Bcl-2 They inhibit the anti-apoptotic Bcl-2 network rather than directly

Department of Hematology, The First Afﬁliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Nanjing, China. Correspondence: Dr W Xu, Department of Hematology, The First Afﬁliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Nanjing, 210029, China. E-mail: [email protected] Received 17 May 2012; revised 2 October 2012; accepted 4 October 2012; published online 23 November 2012 The role of Puma, Noxa and Bim in CLL L-N ZHANG et al 2 Table 1. Members of the Bcl-2 family4–9

Structure Function Members

Four BH domains (BH1–4) Anti-apoptosis Bcl-2, Bcl-XL, Bcl-W, A1 and Mcl-1 Three BH domains (BH1–3) Pro-apoptosis Bax, Bak BH3-only Pro-apoptosis Bik, Bid, Bad, Noxa, Bmf, Puma, Bim and Hrk Abbreviations: Bcl-2, B-cell lymphoma 2; BH, Bcl-2 homology.

interact within the Bcl-2 family network remains controversial. It is unlikely that one pro-apoptotic Bcl-2 protein is sufﬁcient to start apoptosis, but the breakage of balance between anti- and pro- apoptotic factors will decide the cell’s fate.

PUMA, NOXA AND BIM ARE THREE IMPORTANT MEMBERS OF THE BH3-ONLY FAMILY Puma (p53 upregulated modulator of apoptosis) Once transcriptionally activated, the Puma gene produces two transcripts: Puma-a and Puma-b, both of which contain the BH3 domain that can interact with other members of the Bcl-2 family.13 Among all the transcriptional regulators of Puma, p53 is the most famous one. Puma is a critical mediator of p53-dependent and p53-independent apoptosis induced by a wide variety of stimuli, including deregulated oncogene expression, toxins, growth factor/ cytokine withdrawal, infection and so on.13 For example, in the event of DNA damage, p53 is activated by the ataxia Figure 1. The structure of B-cell lymphoma 2 (Bcl-2) family. BH3, Bcl- telangiectasia-mutated (ATM) and ataxia telangiectasia and 2 homology 3; TM, transmembrane domain. Rad3-related (ATR) pathways and then induces the production of Puma as a transcription factor.17 Puma can also be activated independently of p53 and thus plays an important role in p53- independent apoptosis. The p53 homolog p73 can also regulate Puma expression by binding to the same p53-responsive elements in the Puma promoter. The forkhead family member forkhead box O3A (FoxO3a), C/EBP homologous protein (CHOP) and E2F1 are also involved in Puma upregulation by a p53-independent way.18 Moreover, general transcription factors, including C/EBPb, CREB, c-Jun and Sp1, have been implicated in Puma induction, some of which may do so by cooperating with p53 or p73.13 On the other hand, Puma transcription can also be negatively regulated through the p53 pathway, as p53 activates the transcriptional repressor Slug, which in turn inhibits the transcription of Puma.19 Some alternative splice products of p73 (DNp73 and p73a) or p63 (DNp63) and microRNA can cut down the expression of Puma as well.20 Recently, posttranscriptional regulation of Puma has been reported to be the phosphorylation at multiple sites, which promotes its proteasomal degradation.21 It is worth noting that Puma seems to be more powerful than p53 as the deletion of Puma can protect against apoptosis, rivaling the degree of protection when p53 is deleted.22 Puma is believed to bind and/or activate Bax and Bak through Figure 2. There are two main pathways of apoptosis: the extrinsic 6,10,13 pathway and the intrinsic pathway. The extrinsic pathway is Bid and Bim. However, it has been suggested that Puma can also sponsor apoptosis by directly activating Bax, or through triggered by many kinds of death signals from cell surface and we 23 take FAS/FAS ligand (FASL) for example. The intrinsic pathway is cytoplasmic p53 in some cells. Although we cannot identify regulated by B-cell lymphoma 2 (Bcl-2) homology 3 (BH3)-only whether Puma is able to active Bax and Bak directly, there are no family members and it is the main focus of this article. FADD, FAS- signs that the follow-up would change. In addition, Puma also associated protein with death domain; SMAC, second mitochondria- functions through Bax to induce mitochondrial autophagy in derived activator caspase. response to mitochondrial perturbations, and under some circumstances, the autophagy can enhance apoptosis.24 activate Bak or Bax. Sensitizer/de-repressor proteins contain Bad, Bmf, Hrk Harakiri, Noxa and Puma.11–16 Furthermore, the Noxa interaction between certain BH3-only proteins and anti- A decade after its initial identification, Noxa was defined as a apoptotic Bcl-2 family members is selective. Bid, Bim and Puma p53-inducible gene that took part in DNA damage and hypoxia- are able to bind all the anti-apoptotic Bcl-2 members, whereas induced apoptosis.14,22 Similar to Puma, more recent work has Bad, Noxa and Bmf can only bind some specific ones.12–16 At demonstrated a p53-independent induction of Noxa involving present, the exact mechanism(s) by which the BH3-only proteins either E2F1 or p73.25,26

Cancer Gene Therapy (2013), 1 – 7 & 2013 Nature America, Inc. The role of Puma, Noxa and Bim in CLL L-N ZHANG et al 3 It is not surprising that Noxa is less effective than Puma in Bcl-2 family members is disturbed and apoptosis can be p53-mediated apoptosis, for Puma (like Bim) can bind to all the prevented. anti-apoptotic Bcl-2 family members, whereas Noxa antagonizes only Mcl-1 and A1. Nevertheless, the functional overlap of Noxa and Puma in apoptosis caused by DNA damage indicates that, to TREATMENT TARGETING THE BH3-ONLY REGULATED some extent, they may cooperate in the progress of apoptosis. APOPTOSIS PATHWAY IS A BREAKTHROUGH IN THE When the function of Puma is compromised, the level and/or ANTI-TUMOR THERAPY OF CLL 27 activation state of Noxa is heightened. Noxa has been proven as The role of BH3-only proteins in the treatment for CLL can be a particularly relevant therapeutic target for CLL because it divided into two parts. First, Puma, Noxa and Bim are the interacts speciﬁcally with Mcl-1, thus neutralizing its anti-apoptotic executioners of some current drugs for CLL. On the other activity.28 It is interesting that the ratio of Noxa/Mcl-1 in peripheral 29 hand, several BH3-only mimetic molecules that inhibit anti- versus lymph node CLL cell is correlated with survival capacity. apoptotic Bcl-2 protein activity are now attractive approaches This fact clearly indicates that the Noxa/Mcl-1 axis is an attractive for treating CLL. target for apoptosis-based therapeutic strategies in CLL.

Puma, Noxa and Bim are the executioners of some current drugs Bim (Bcl-2-interacting mediator of cell death) for CLL Three isoforms of Bim exist because of alternative mRNA splicing, The clinical course of CLL is highly variable, and chemotherapy is which are usually described as short, long and extra-long (BimS, usually not applied in early and stable stage. So far, many effective BimL and BimEL, respectively). The pro-apoptotic activity of Bim is drugs have been applied to clinical practice, including chloram- regulated by transcriptional as well as posttranscriptional mechan- bucil, purine nucleoside analogs (fludarabine, cladribine and isms. The forkhead-like transcription factor FOXO3a and pentostatin), corticosteroids, cyclophosphamide, monoclonal antitumor suppressor gene runt-related protein 3 (Runx3) are key bodies and some other novel drugs. transcriptional regulators of Bim.30 Bim can be regulated Cyclophosphamide. Cyclophosphamide can kill tumor cells by posttranscriptionally through a series of phosphorylation events. causing DNA damaging, which is thought to occur largely through Recently, Bim has been suggested to interact with all the the activation of the intrinsic apoptosis pathway by the tumor prosurvival members of the Bcl-2 family. Of all BH3-only proteins suppressor p53. Puma and Noxa are direct transcriptional targets examined, only Bim and Bid have the capacity to act as direct of p53, and hence they were naturally thought to take part in this activators of Bax and Bak. Bim and Puma also have clearly mechanism. Unexpectedly, not only Puma and Noxa but also 38 overlapping functions in p53-dependent and p53-independent Bim, which was not known to be a direct p53 target, contributed apoptosis. In vitro, Bim is essential for apoptosis of various cell to the progress. These three BH3-only proteins do cooperate in types, including lymphocytes (especially germinal center-derived cyclophosphamide-induced lymphoma cell death in vitro and memory B cells and antibody-forming cells), mast cells, osteo- in vivo, and the level of drug resistance observed in these triple- clasts, epithelial cells, endothelial cells and neurons.31,32 knockout lymphomas was as severe as that seen with the loss of p53.38 Surprisingly, with the loss of both Puma and Noxa, lymphomas became more sensitive to cyclophosphamide in vitro, which is perhaps because of a rebalance between pro- THE DELETION OF PUMA, NOXA OR BIM MAY INCREASE THE survival Bcl-2 family members and BH3-only proteins.39 TUMOR BURDEN BUT DO NOT CONTRIBUTE TO Fludarabine. Fludarabine is a kind of the purine analog currently TUMORIGENESIS applied in CLL patients, and it removes CLL cells by activating p53 Several experiments confirmed that Puma deficiency could both and then downstream BH3-only family members, consequent on increase B-lineage cells and accelerate the development of B the induction of DNA damage. However, which one of the BH3- lymphoma. Meanwhile, decreased Noxa or Bim expression was only family members is the key factor in this apoptotic pathway is also reported in several human malignancies, including B-cell still unknown. We incubated CLL cells with an active metabolite of 33,34 lymphoma, colon cancer and small-cell lung cancer. Naturally, fludarabine, 9-b-D-arabinosyl-2-fluoroadenine (F-ara-A), in vitro and animals lacking these three genes can be logically expected to be then analyzed the role of Puma, Noxa and Bim in p53-mediated tumor prone. However, this is exactly not the case as Puma À / À , apoptosis of CLL cells.37 We found that only Puma was the Noxa À / À , Bim À / À or even Puma À / À Noxa À / À mice do essential factor, and Noxa and Bim seemed to play a more not spontaneously form tumors.33,35,36 Only in the Em-myc mouse restricted role. Moreover, functional p53 was necessary in model of B-cell lymphoma, the loss of single Puma or Bim fludarabine-induced apoptosis. Puma expression was strongly sensitizes the mice to tumorigenesis.33,35 In order to clarify augmented in a p53-dependent manner, and this was more whether the three molecules were able to initiate tumorigenesis, prominent in cells with mutated immunoglobulin heavy-chain we detected the mRNA levels of Puma, Noxa, Bim and Bcl-2 genes variable region (IGHV) genes, which indicated a significantly better in 100 CLL patients and 10 normal controls, and then found that overall survival rate than patients with unmutated IGHV genes. the expression level of Puma was not significantly distinct with Histone deacetylase inhibitors (HDACis). HDACis are a new class of normal controls, but the levels of Noxa, Bim and Bcl-2 were targeted anticancer agents that exert antitumor activity by their overexpressed in CLL cells.37 Furthermore, we noticed that low ability to induce growth arrest, differentiation and apoptosis.40,41 Puma expression level was significantly correlated with variant The precise mechanism whereby HDACis induce apoptosis has not clinical features and biocharacteristics that could intimate a poor been clarified, but most studies suggest that they rely on the prognosis. Together, we hypothesize that the lower expression of intrinsic pathway.42 Recent studies show that there is a rapid and Puma, Noxa and/or Bim is related to the high tumor burden extensive upregulation of Noxa and Bim caused by HDACis in CLL (proliferation of lymphocytes) but does not contribute to and other leukemic cells.43 Moreover, HDACi-mediated tumorigenesis. There appeared to be some redundancy in Noxa upregulation of Noxa is associated with the amount of Mcl-1 and Bim regulation of apoptosis, which was overcome by Bcl-2 immunoprecipitated.43 It is valuable for us to ponder this overexpression. It implies that tumorigenesis is a complex process, phenomenon. Increased Noxa may neutralize Mcl-1 and and the balance between pro- and anti-apoptotic regulators inactivate the major member of anti-apoptotic Bcl-2 family determines the fate of a cell. When anti-apoptotic Bcl-2 family members. This is significant in tumors such as CLL, where Mcl-1 members are overexpressed, the ratio of pro- and anti-apoptotic is proposed to be an important anti-apoptotic protein.44

& 2013 Nature America, Inc. Cancer Gene Therapy (2013), 1 – 7 The role of Puma, Noxa and Bim in CLL L-N ZHANG et al 4 The Noxa/Mcl-1 ratio may be a key prognosis factor in CLL the antimetabolite drug arabinoside, the microtubule-targeting patients treated with HDACis. agents paclitaxel and vincristine, the glucocorticoid dexametha- Glucocorticoids. Glucocorticoids are valid inducers of apoptosis in sone and the anti-inflammatory agent thalidomide, especially the CLL cells, through a caspase-dependent mechanism. However, proteasome inhibitors bortezomib or carfilzomib.53,56,71 ABT-737 their mechanism of apoptosis remains unknown. Current studies plus bortezomib induced significant apoptosis in primary samples using microarray approaches demonstrate that Bim mRNA and of mantle cell lymphoma, diffuse large B-cell lymphoma, and CLL protein are both upregulated by glucocorticoids in CLL cells.45–47 but no significant cytotoxic effect was observed in peripheral Other reports also offer evidence that the induction of Bim by blood mononuclear cells from healthy donors.71 Thus, combina- glucocorticoids is essential for the complete apoptotic response in tion therapy with ABT-737 may lower the doses of conventional acute lymphoblastic leukemia cell lines and xenografts.48,49 In an anticancer agents required for clinical responses to reduce colla- opposite manner, Bim À / À T lymphocytes are partially resistant teral damage to normal cells, or ensure more stable remissions to glucocorticoid-induced apoptosis.50 The specific mechanism for without lowering doses. the induction of Bim by glucocorticoids remains unclear. However, Gossypol is the first compound that shows inhibition of Bcl-2, it could be an indirect mechanism, as the human Bim promoter Bcl-XL and Mcl-1. Gossypol exhibits a type of enantiomerism that does not contain a clear glucocorticoid response element.46 arises from restricted rotation: the ( À )gossypol isomer shows Besides, the downregulation of Mcl-1 has also existed when greater cytotoxicity than the ( þ )isomer in several human cancer glucocorticoids upregulate the expression of Bim. We suppose cell lines.72 Gossypol and its analogs can mainly bind and interact that it is because of Bim’s combination with Mcl-1, which inhibits with the anti-apoptotic proteins Bcl-2 and Bcl- XL, and afterwards the anti-apoptotic activity of Mcl-1. Analogously, the Bim/Mcl-1 induce cytochrome c release and Bak activation in whole cells as ratio may be a prognosis factor in CLL patients treated with well as in isolated mitochondria that express high levels of Bcl-2.73 glucocorticoids. Further studies show that gossypol also induces Bax/Bak- independent activation of apoptosis and cytochrome c release 74 BH3-only mimetic molecules are gene-targeted therapies for CLL via a conformational change in Bcl-2. Although this compound Several BH3-only mimetic drugs that inhibit anti-apoptotic has been primarily studied as an inhibitor of Bcl-2, its other proteins activity have been developed. These molecules are mechanisms of action have also been proposed. An extrinsic cell designed to bind the hydrophobic groove of anti-apoptotic Bcl-2 death pathway through upregulation of Fas/Fas ligand could proteins and oligomerize Bax or Bak, which can subsequently mediate by gossypol as well. It has been reported earlier that depolarize mitochondrial membrane potential to release cyto- gossypol also induced apoptosis in chronic lymphocytic leukemia through the generation of reactive oxygen species that in turn chrome c. This new approach carries conspicuous advantages, as 75 restraining the anti-apoptotic proteins using BH3 mimetics should mediate the release of cytochrome c, thus causing apoptosis. be more valid than targeting the upstream signaling steps, which It is worth noting that the apoptotic effect of gossypol in CLL cells are often defective because of oncogenic alterations. In recent is not associated with the Rai stage of the disease, prior treatment, ZAP-70 or immunoglobulin status, IGHV mutation, p53 status, years, these BH3-only mimetic drugs have displayed the greatest 75 success in clinical trials of many kinds of cancers, especially CLL, as chromosomal trisomy or ATM mutation. Hence, patients with Bcl-2 family proteins were confirmed highly expressed in CLL poor prognosis will benefit a lot from gossypol. As a single agent lymphocytes (Table 2). or in combination with chemotherapy, gossypol is effective in Among all these mimetics, ABT-737 and Gossypol are likely to lymphoma cells. be the most promising ones for CLL therapy. ABT-737 and its oral form, ABT-263, have been reported to have the strongest binding affinities to the anti-apoptotic members Bcl-2, Bcl-XL and Bcl-W. PUMA, NOXA AND BIM ARE ABLE TO RESTORE THE Mechanistic studies reveal that ABT-737 is similar to the BH3 SENSITIVITY OF CLL CELLS TO CHEMOTHERAPEUTIC AGENTS domain of Bad.70 Importantly, unlike most of the other BH3 Drug resistance in CLL contributes to poor clinical outcomes and mimetics, ABT-737-induced apoptosis is Bak/Bax dependent, as remains a significant obstacle to successful chemotherapy. At least evidenced by the finding that Bak À / À Bax À / À mouse two mechanisms contribute to the development of resistance to embryonic fibroblasts are resistant to the treatment.53 ABT-737 drugs: acquired mutations resulting in a dysfunctional p53 exhibits cytotoxicity against a broad panel of lymphoma cell lines, response76,77 and shifts in the balance between apoptosis- including mantle cell lymphoma, diffuse large B-cell lymphoma regulating proteins.78 Strategies to overcome resistance and CLL.51–56 For most tumor cell lines, especially those derived mechanisms are therefore highly needed. from solid tumors, the cell-killing activity of ABT-737 is not Inactivation of p53 reportedly occurs in 15–25% of CLL patients, satisfying as a single agent.70 However, it exhibits striking synergy which is caused either by p53 mutation79 or inactivation of the when combined with a variety of anticancer agents, including gene encoding ATM,80 a kinase that regulates p53. Restoration of genotoxic agents etoposide, doxorubicin, cisplatin and melphalan, the p53 pathway by activating p53 itself or p53 downstream

Table 2. Bcl-2 family inhibitors

Bcl-2 inhibitors Binding to Bcl-2 members Cancers Clinical trials

51–56 ABT-737 Bcl-2, Bcl-XL, Bcl-W CLL, small-cell lung cancer, lymphoma, AML, multiple myeloma Phase I/II 57–59 Gossypol (AT-101) Bcl-2, Bcl-XL, Mcl-1 CLL, non-Hodgkin’s lymphoma, glioblastoma, pancreatic cancer, Phase II/III breast cancer, melanoma 60–64 GX-15-070 Bcl-2, Bcl-XL, Bcl-W, Mcl-1 Leukemia, lymphoma, multiple myeloma, non-small-cell Phase I/II lung cancer, CLL, myeloﬁbrosis 65,66 072RB Bcl-XL, Mcl-1 AML, CLL Preclinical 67–69 TW-37 Bcl-XL, Mcl-1, Bcl-2 CLL, non-Hodgkin’s lymphoma, head and neck cancer, pancreatic Preclinical cancer, small-cell lung cancer Abbreviations: AML, acute myeloid leukemia; Bcl-2, B-cell lymphoma 2; CLL, chronic lymphocytic leukemia.

Cancer Gene Therapy (2013), 1 – 7 & 2013 Nature America, Inc. The role of Puma, Noxa and Bim in CLL L-N ZHANG et al 5 targets has been explored to improve the efficacy of anticancer ACKNOWLEDGEMENTS therapies. Puma is an essential mediator of p53-dependent and This study was supported by National Natural Science Foundation of China 22 p53-independent apoptosis, and hence it turns into the most (30871104, 30971296, 81170488), Natural Science Foundation of Jiangsu Province promising candidate. Low dose of Ad-Puma was confirmed to (BK2010584), Key Projects of Health Department of Jiangsu Province (K201108), restore significantly the sensitivity of cancer cells to Jiangsu Province’s Medical Elite Program (RC2011169), University Doctoral Founda- chemotherapeutic agents in vitro and in vivo through restoration tion of the Ministry of Education of China (20093234110010), the Program for of the p53 pathway in human melanoma81 and choriocarcinoma82 Development of Innovative Research Team in the First Affiliated Hospital of NJMU, cell lines. Conversely, targeted deletion of Puma in human and the project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. colorectal cancer cells resulted in resistance to apoptosis induced by p53, the DNA-damaging agent adriamycin and hypoxia.83 The reason why Puma is able to ameliorate the drug resistance of CLL can be divided into three aspects. 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& 2013 Nature America, Inc. Cancer Gene Therapy (2013), 1 – 7