BH3-only protein BIM: An emerging target in chemotherapy

Accepted Manuscript Title: BH3-only protein BIM: An emerging target in chemotherapy Authors: Shatrunajay Shukla, Sugandh Saxena, Brijesh Kumar Singh, Poonam Kakkar PII: DOI: Reference:

S0171-9335(17)30160-7 https://doi.org/10.1016/j.ejcb.2017.09.002 EJCB 50976

To appear in: Received date: Revised date: Accepted date:

21-6-2017 1-9-2017 19-9-2017

Please cite this article as: Shukla, Shatrunajay, Singh, Brijesh Kumar, Kakkar, Poonam, BH3-only emerging target in chemotherapy.European Journal https://doi.org/10.1016/j.ejcb.2017.09.002

Saxena, Sugandh, protein BIM: An of Cell Biology

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

  BH3-only protein BIM: An emerging target in chemotherapy Shatrunajay Shuklaa, Sugandh Saxenaa, Brijesh Kumar Singhb, Poonam Kakkara,* a

Herbal Research Laboratory, Food Drug & Chemical Toxicology Group, CSIR-Indian Institute of Toxicology

Research (CSIR-IITR), Vishvigyan Bhawan 31, Post Box No. 80,Mahatma Gandhi Marg, Lucknow-226001, India. b

Laboratory of Hormonal Regulation, Duke-NUS Graduate Medical School, No 8 College Road, Singapore-

169857

*

Correspondence to:

Prof. (Dr) Poonam Kakkar Chief Scientist Herbal Research Laboratory Food Drug & Chemical Toxicology Group CSIR- Indian Institute of Toxicology Research P.O. Box- 80, M.G. Marg, Lucknow-226001, (U.P.), India Tel: (+91)-0522-2616762, 2627586*269 Fax: (+91)-0522-2628227 Email: [email protected]; [email protected]

Abstract BH3-only proteins constitute major proportion of pro-apoptotic members of B-cell lymphoma 2 (Bcl-2) family of apoptotic regulatory proteins and participate in embryonic development, tissue homeostasis and immunity. Absence of BH3-only proteins contributes to autoimmune disorders and tumorigenesis. Bim (Bcl-2 Interacting Mediator of cell death), most important member of BH3-only proteins, shares a BH3-only domain (9-16 aa)

1   

  among 4 domains (BH1-BH4) of Bcl-2 family proteins and highly pro-apoptotic in nature. Bim initiates the intrinsic apoptotic pathway under both physiological and patho-physiological conditions. Reduction in Bim expression was found to be associated with tumor promotion and autoimmunity, while overexpression inhibited tumor growth and drug resistance as cancer cells suppress Bim expression and stability. Apart from its role in normal homeostasis, Bim has emerged as a central player in regulation of tumorigenesis, therefore gaining attention as a plausible target for chemotherapy. Regulation of Bim expression and stability is complicated and regulated at multiple levels viz. transcriptional, post-transcriptional, post-translational (preferably by phosphorylation and ubiquitination), epigenetic (by promoter acetylation or methylation) including miRNAs. Furthermore, control over Bim expression and stability may be exploited to enhance chemotherapeutic efficacy, overcome drug resistance and select anticancer drug regimen as various chemotherapeutic agents exploit Bim as an executioner of cell death. Owing to its potent anti-tumorigenic activity many BH3 mimetics e.g. ABT-737, ABT-263, obatoclax, AT-101and A-1210477 have been developed and entered in clinical trials. It is more likely that in near future strategies commanding Bim expression and stability ultimately lead to Bim based therapeutic regimen for cancer treatment. Keywords: Bcl-2 family; Bim; Mitochondria; Apoptosis; Cell signalling; Cancer

Abbreviations: A1:

Bcl-2-related protein A1

Apaf-1:

Apoptotic protease-activating factor 1

ASK1:

Apoptosis Signal Regulated Kinase 1

Bad:

Bcl-2 associated agonist of cell death

Bak:

Bcl-2 homologous antagonist/killer

Bax:

Bcl-2-associated X protein

Bcl-2:

B-cell lymphoma 2

Bcl-w:

Bcl-2-like protein 2 (BCL2L2)

Bcl-XL:

B-cell lymphoma-extra large

BET:

Bromodomain and extra-terminal

BH domain: Bcl-2 homology domain Bid:

BH3 interacting-domain death agonist

Bik:

Bcl-2-interacting killer 2 

 

 

Bim:

Bcl-2 interacting mediator of cell death

Bmf:

Bcl-2 modifying factor

Bok:

Bcl-2 related ovarian killer

C/EBPα:

CCAAT/enhancer-binding protein alpha

CBP:

CREB-binding protein

CHOP:

C/EBP homologous protein

DP family: Dimerization partner protein family DR3/4/5:

Death receptor 3/4/5

E2F:

E2 factor family protein

EBV: ER:

Epstein-Barr virus Endoplasmic reticulum

ERK:

Extracellular signal-regulated kinase

FasR:

Fas receptor of tumor necrosis factor (TNF) family

FoxO:

Forkhead box family protein belonging to ‘O’ subclass

HDAC:

Histone deacetylase

HIF-1α:

Hypoxia-inducible factor 1-alpha

HoxB8:

Homeobox B8

Hrk:

Activator of apoptosis harakiri

Hsc:

Heat shock cognate protein

HSP:

Heat shock protein

IFN:

Interferon

JAK:

Janus Kinase

JNK: LRF:

c-Jun N-terminal Kinase Leukemia/lymphoma-related factor or Pokemon

Mcl-1:

Myeloid cell leukemia 1

miR:

Micro-RNA

MOM:

Mitochondrial Outer Membrane

MOMP:

Mitochondrial outer membrane permeabilization 3 

 

 

NF-Y:

Nuclear transcription factor-Y

NF-κB: NGF:

Nuclear factor kappa-light-chain-enhancer of activated B cells Nerve Growth factor

Noxa:

Phorbol-12-myristate-13-acetate-induced protein 1

PKA:

Protein Kinase A

PP2A:

Protein Phosphatase 2A

PRC:

Polycomb repressive complex

Puma:

p53 upregulated modulator of apoptosis

Rb:

Retinoblastoma

Runx3:

Runt-related transcription factor 3

SCF:

Skp Cullin F-box

SNAI2:

Snail family zinc finger 2

Sp:

Specificity protein

STAT-1:

Signal Transducer and activator of Transcription 1

TGF-β:

Transforming growth factor beta

TNFR1: TNFα:

Tumor necrosis factor receptor 1 (TNFR1) Tumor necrosis factor alpha

Trim33: UPR:

Tripartite motif containing 33 Unfolded Protein Response

YY1:

Yin Yang 1

Zbtb7a:

Zinc finger and BTB domain-containing protein 7A

β-TrCP:

Beta-transducin repeats-containing protein

Introduction Mammals, in general, have two distinct and disciplined signaling pathways of apoptosis (A morphologically distinct and programmed form of cell death); the intrinsic (Kapoor et al., 2013) and the extrinsic pathway (Elmore, 2007;). In the intrinsic mode of apoptosis, signals include a variety of extracellular and developmental stimulus which evokes mitochondrial outer membrane permeabilization followed by the

4   

  release of various apoptogenic proteins and caspases activation; while in the extrinsic pathway, apoptosis is initiated by the stimulation of death receptors including FasR, TNFR1, DR3, and DR4/DR5. The Bcl-2 family proteins constitute critical intracellular checkpoints and key regulators of cell death that can either suppress or promote apoptosis, a process often dysregulated in cancer and other diseases (Akiyama and Tanaka, 2011). The Bcl-2 family of proteins is classified into one anti-apoptotic group and two pro-apoptotic groups based upon family members participated in inhibition and activation of apoptosis respectively (fig.1 (A)). The anti-apoptotic or pro-survival members include multidomain [Bcl-2 Homology domains (BH domain), numbers of which ranging from BH1 to BH4] Bcl-2, Bcl-XL, Bcl-w, Mcl-1 and A1 proteins, whereas the pro-apoptotic members are further classified into the multidomain pro-apoptotic group (Bax, Bak, and Bok) and the BH3-only group (Bim, Puma, Noxa, Hrk, Bmf, Bad, Bik and Bid) (Um, 2015). The anti-apoptotic group of proteins prevents the pro-apoptotic Bcl-2 family of proteins, including Bcl-2-associated X protein (Bax) and Bcl-2 antagonist/killer (Bak), from inducing the mitochondrial outer membrane permeabilization, release of apoptotic proteins from mitochondria and subsequent caspases activation. Bim (Bcl-2 Interacting Mediator of cell death), classified under single BH3 domain i.e. the Bcl-2-homology domain 3-only (BH3-only) proteins, is a potent inducer of apoptosis. BH3-only proteins provoke apoptosis either by direct activation of pro-apoptotic Bax/Bak or by neutralizing anti-apoptotic Bcl-2 proteins (Bcl-2, Bcl-XL, Bcl-w, Mcl-1 and A-1) via their BH3 domain (Brunelle and Letai., 2009). Once activated either directly or indirectly by BH3-only proteins, Bax gets inserted into the mitochondrial outer membrane (MOM), oligomerizes into pores with Bax/Bak, permeabilizes the outer membrane and facilitates the release of intermembrane space proteins such as cytochrome c (cyt-c) into the cytoplasm. In cytoplasm, cyt-c then binds to apoptotic protease-activating factor 1 (Apaf-1) and allows a (d)ATP dependent conformational change in Apaf-1, which results in its oligomerization into a heptameric caspase-activating complex, known as the Apaf-1 apoptosome (Bratton and Salvesen, 2010). The apoptosome assembly sequentially recruits and activates the initiator caspase-9, which then recruit executioner caspase-3, that targets >800 cellular substrates for proteolytic cleavage (fig.2). In this way BH3-only proteins either directly activate pro-apoptotic proteins or indirectly by neutralizing anti-apoptotic proteins resulting into release of pro-apoptotic proteins, induce mitochondrial outer membrane permeabilization (MOMP), apoptosome assembly formation, caspases activation culminating into cell death (Kim et al., 2009; Mahajan et al., 2014). The multiplicity and complex regulation of mammalian BH3-only proteins allow exquisite control over apoptosis and made it possible to manipulate apoptosis at various nodal points. Studies reported that binding and

5   

  affinity of BH3-only proteins to various anti-apoptotic proteins differ significantly among each other and Bim/Puma/tBid are the only members capable to bind and antagonize the action of all anti-apoptotic Bcl-2 proteins with equal affinity, therefore considered as the most potent effectors of apoptotic pathway (Doerflinger et al., 2015). In general, Bim is sequestered in its inactive form to the dynein complex associated with microtubular cytoskeleton or present as an inactive heterodimer with different anti-apoptotic Bcl-2 family proteins but discharged by various death signals (Mariana et al., 2002). Bim is an impressive pro-apoptotic member of the BH3-only proteins and alternative mRNA splicing generates three major isoforms (total 19 different isoforms) of Bim; BimEL (extra-large, 198 aa and 22kDa), BimL (large, 138 aa and 15.8kDa) and BimS (small, 112 aa and 12.3kDa) (Liu et al., 2002; Liu et al., 2007) (fig.1(B)). BimEL is the predominant isoform in body tissues while BimS is the most potent inducer of apoptosis among all isoforms. More than 12 minor isoforms of Bim have been cloned from humans and recently another isoform BimAD has been identified that lacks the dynein binding domain and has poor pro-apoptotic efficacy compared to its longer isoforms (Marani et al., 2002). The physiological significance of these minor isoforms is still unexplored (Marani et al., 2002). Bim alongwith tBid and Puma are known to directly activate Bax/Bak mediated cytochrome c release, followed by initiation of apoptotic cascade, hence a tight control over Bim expression and stability are required in order to prevent cells to undergo apoptosis. The present review focuses on diverse modes of Bim regulation at first. Subsequently, it reviews potential role of Bim modulation in tumor promotion/resistance and chemotherapy. The diversity of Bim regulatory network builds upon particular cell type, type of stimulus and pathological conditions. Bim expression and pro-apoptotic activity can be regulated at multiple levels i.e. epigenetic, transcriptional, post-transcriptional and post-translational level. Epigenetic and transcriptional regulation of Bim Bim expression is controlled at transcriptional level by epigenetic modifications and transcription factors (Ridinger-Saison et al., 2013). Latency-associated virus gene products hyper-methylated the large CpG island located at the 5’ end of Bim promoter, inhibited Bim expression and found to be associated with enhanced cell survival in human B cells infected with Epstein-Barr virus (EBV) (Harada and Grant, 2012; Paschos et al., 2009)), while Bim promoter acetylation was linked to increased Bim expression in multiple myeloma and aggravated apoptosis (De Bruyne et al., 2010). EBV mediated Bim repression was also linked with reduced acetylation of histones H3 and H4 (Paschos et al., 2009). Forkhead-box class O (FoxO) family proteins centrally govern transcriptional upregulation of Bim in growth factor deficiency, cytokine withdrawal and chemotherapy (Harada and Grant, 2012; Plötz and Eberle,

6   

  2014), while transcriptional repression of Bim is controlled by a number of transcription factors including Yin Yang 1 (YY-1), Spi-1/PU.1, Tripartite motif containing 33 (Trim33), Pokemon, Hypoxia-inducible factor 1alpha (HIF-1α), Specificity Protein 1 (Sp1) and Bromodomain-containing protein 4 (Brd4) (Lee et al., 2014; Li et al., 2016; Ridinger-Saison et al., 2013; Wang et al., 2015; Whelan et al., 2010; Xu et al., 2016). Factors commanding transcriptional regulation of Bim are outlined below. FoxO transcription factors have been reported to play role in diverse cellular processes viz. glucose metabolism, reactive oxygen species detoxification, DNA repair, cell cycle arrest and different modes of cell death i.e. apoptosis and autophagy. FoxO proteins are important downstream targets of PI3K/Akt-signaling axis, which is frequently over-expressed in a variety of cancers and Akt (serine/threonine kinase) mediated phosphorylation of FoxOs at different residues results in FoxOs exclusion from nucleus, association with 14-3-3 protein and cytoplasmic degradation. Growth factor deficiency, cytokines deprivation and chemotherapeutic agent such as cisplatin inhibit Akt-mediated phosphorylation of FoxOs, resulting into FoxOs nuclear retention and FoxO-dependent transcriptional activation of BH3-only protein Bim, culminating into induction of apoptosis (Liu et al., 2014; Shukla et al., 2014). FoxO3a binds within Bim promoter region on FHRE (Forkhead responsive element) and usually acts in concert with Activator protein-1 (AP-1) (Essafi et al., 2005; Sunters et al., 2003). Nuclear transcription factor Y (NF-Y) is a hetero-trimeric transcription factor, composed of NF-YA, NF-YB and NF-YC subunits necessary to form active NF-Y complex, which binds to CCAAT box in promoter regions of various genes including Bim in eukaryotes (Ly et al., 2013). CCAAT box is one of the most commonly occurring cis-acting elements in promoter and enhancer region of genes in mammals. It is reported that NF-Y collaborates with FoxO3a along with transcriptional co-activators p300 and CBP to bind to the CCAAT box of Bim promoter that is essential for transcriptional activation of Bim following NGF (Nerve growth factor) withdrawal induced cell death in sympathetic neurons (Hughes et al., 2011). RUNX3, a downstream target of transforming growth factor-β (TGF-β), is an important tumor suppressor which is inactivated in many cancer types including gastric cancer. RUNX3 is responsible for transforming growth factor-β (TGF-β)-induced gastric epithelial cells apoptosis. RUNX3 upregulates Bim transcription in association with FoxO3a (Physical interaction of RUNX3 and FoxO3a on the Bim promoter activate transcription of Bim) resulting into TGF-β−induced apoptosis in gastric cancer cells (Yamamura et al., 2006). RUNX2 may also regulate Bim expression through zinc finger transcription factor SNAI2 (Snail family

7   

  zinc finger 2), which binds to Bim promoter and induces Bim expression in breast tumor cells (Merino et al., 2014). Nerve growth factor (NGF) is an essential requirement for the survival of sympathetic neurons and its deprivation induce apoptosis which is mediated by activation of c-Jun N-terminal kinases (JNK) and c-Jun, induction of the Bcl-2 homology 3-only protein BimEL, Bax-dependent loss of mitochondrial membrane potential, release of cytochrome c, and caspase activation (Whitfield et al., 2001). Studies documented that Smad3, a transcription factor activated by TGF-β, induces Bim expression in B lymphocytes. Co-exposure of TGF-β and TNF-α also induced Smad3 dependent Bim expression in human gastric carcinoma cells (Wildey et al., 2003). Stress stimuli upholds unfolded protein response (UPR) and endoplasmic reticulum stress culminating into cell death in a variety of cell types both in vitro and in vivo, where Bim plays a critical role in apoptosis induction. It was found that BH3-only protein Bim is essential for ER stress mediated apoptosis in macrophages and thymocytes etc. while it may also be induced by other members of BH3-only proteins including PUMA and Noxa in other cell types e.g. neuroblastoma, colon carcinoma and mouse embryonal fibroblasts (Li et al., 2006; Puthalakath et al., 2007; Reimertz et al., 2003). Studies reported that during ER stress signaling transcriptional activation of Bim was directly governed by CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) and its heterodimeric partner C/EBPα. In addition, during ER stress Bim expression level increases post-translationally by protein phosphatase 2A (PP2A)-mediated dephosphorylation, which obstructs ubiquitindependent proteasomal degradation of Bim (Puthalakath et al., 2007). The E2F family of transcription factors is mainly involved in transcriptional activation of genes required for cell cycle progression and includes six structurally related E2Fs (E2F1-6) which function as heterodimers with members of the DP family (DP-1 and DP-2). Studies reported that ectopic expression of E2F1 leads to induction of Bim expression and apoptosis in tissue culture cells and transgenic mice (Hershko and Ginsberg, 2004; Holmberg et al., 1998). E2F transcriptional activity is negatively controlled by its interaction with the product of the retinoblastoma tumor suppressor gene, pRB, and related proteins p107and p130, collectively referred to as pocket proteins. E2F1 also induces expression of Apoptosis Signal Regulated Kinase 1 (ASK1) protein, which further induces E2F1 dependent Bim expression via inhibition of Rb. ASK1 knockdown resulted in reduced E2F1-induced Bim transcription and apoptosis in HDAC inhibitor treated cancer cells (Tan et al., 2006). In addition, Angiotensin II, a key pro-apoptotic factor in fibrosis induced apoptosis,

8   

  promotes cell death through E2F1 mediated Bim induction as Angiotensin II regulates hyperphosphorylation of Rb protein and concomitant release of E2F1 for transcriptional activation (Kim and Day, 2012). Signal transducer and activator of transcription 1 (STAT-1) protein plays a crucial role in Janus kinase (JAK)/STAT signalling cascade and has been known for its essential role in mediating responses to type I, type II, or type III interferons (IFN). STAT-1 has been reported to induce Bim transcription during TNF-α and IFNϒ activated pancreatic β- cell death. IFN-ϒ is reported to induce Bim transcription and TNF-α stimulated JNK activation, which positively regulate Bim protein stability, followed by apoptotic cell death (Barthson et al., 2011). Bim promoter region from -686 to -385 is reported to have two direct STAT-1 binding sites, TTCtacGAA and TTCttgGAA (Barthson et al., 2011). High glucose induced apoptosis in retinal pericytes followed by diabetic retinopathy involved STAT-1 mediated increase in Bim expression (Shin et al., 2014). STAT-1 induced Bim transcription has also been reported in chronic lymphoblastic leukemia after IL-21 exposure, which induced B cell apoptosis (Gowda et al., 2008). YY1, a downstream target gene of NF-κB which form a complex with Rel-A (p65) subunit of NF-κB, has been reported to repress Bim transcription. Deletion of either YY1 or RelA subunit resulted in enhanced Bim expression with subsequent apoptosis induction in multiple myeloma cells (Potluri et al., 2013). YY1-RelA complex binds between nucleotides -300 to -156 on Bim promoter and needs to be co-expressed for transcriptional repression of Bim (Potluri et al., 2013). Spi-1/PU.1, a transcription factor, represses Bim transcription by binding to Bim promoter region and inducing trimethylation of histone H3 at Lys27 resulting in inhibition of apoptosis in erythroleukemia (Ridinger-Saison et al., 2013). The same study reported that Polycomb repressive complex 2 (PRC2) is responsible for histone tri-methylation (Ridinger-Saison et al., 2013). Trim 33, a transcription cofactor, is recruited on Bim promoter by PU.1 transcription factor is reported to involve in Bim repression and B lymphoblastic leukemia cells evasion from apoptosis (Wang et al., 2015). Homeobox B8 (HoxB8), a member of the Antp homeobox family, overexpression is linked with Bim repression due to c-myc dependent upregulation of miR-17-92 cluster, which binds to 3’ UTR of Bim mRNA in hematopoietic stem cells (HSCs) (Salmanidis et al., 2013). POZ-Krüppel family transcription factor, LRF (also known as Zbtb7a/Pokemon), is a proto-oncogene, which plays an essential role in the B versus T lymphoid cellfate decision. LRF (Pokemon) is a master regulator of erythroid development and maturation. LRF was reported to directly repress Bim transcription by binding on a tandem-binding site on Bim promoter (Maeda et al., 2009; Maeda et al., 2007). LRF/Pokemon is a downstream target of GATA-1 transcription factor which governs erythroid differentiation and maturation via activation of LRF expression in T/B cells (Maeda et al., 2009). Sp1

9   

  is a member of Specificity Protein (Sp) family of transcription factors, others include Sp3 and Sp4, which controls the expression of a number of genes associated with cancer cell proliferation, differentiation, and metastasis (T Sankpal et al., 2012). Earlier studies reported that Sp-1 is highly overexpressed in various cancers including pancreatic cancer (Luo et al., 2015; T Sankpal et al., 2012). Six GGGCGG motifs on Bim promoter serve as a binding site for Sp1 transcription factor (Bouillet et al., 2001). A curcumin analogue dibenzylideneacetone inhibited Sp1 followed by increased Bim expression and apoptosis in mucoepidermoid carcinoma, revealed that Sp1 acts as a suppressor for Bim expression (Lee et al., 2014). Mithramycin A, an anticancer drug, also inhibits Sp1 transcription factor and induced Bim expression and apoptosis (Lee et al., 2014). Brd4, a chromatin regulator, is a family member of bromodomain and extra-terminal (BET) protein family and contains two bromodomains in tandem which allow recognition and binding to acetylated histones and recruitment of various cofactors (including pTEFb) for RNA polymerase II dependent transcription elongation. A study reported that Bim expression is repressed by Brd4 in Malignant Peripheral Nerve Sheath Tumors (MPNSTs), a highly aggressive form of sarcoma. In addition, Brd4 also upregulate c-myc and antiapoptotic Bcl-2 expression in MPNSTs. Brd4 inhibition by JQ1, I-BET 151 and CPI203 (Selective small molecule inhibitors) led to induction of apoptosis by Bim induction and Bcl-2 downregulation in MPNSTs (Patel et al., 2014). JQ1 co-treatment with HDAC inhibitor panobinostat synergistically reduced c-myc and Bcl2 expression and potentiated Bim dependent apoptosis in acute myeloid leukemia blast progenitor cells (Fiskus et al., 2014). Another Brd4 inhibitor, I-BET 151 also induced Bim dependent apoptosis in melanoma cells (Gallagher et al., 2014). Hypoxia-inducible factor-1α (HIF-1α) inhibited apoptosis induction in sympathetic neurons followed by NGF withdrawal due to suppression of Bim expression (Xie et al., 2005). Human Tlymphotropic virus type 1 (HTLV-1) encoded protein Tax inhibited Bim transcription by HIF-1α upregulation in T cells (Mühleisen et al., 2014). In addition, HIF-1α might also be able to reduce Bim expression via ERK dependent phosphorylation and subsequent degradation (Whelan et al., 2013). EBNA3A and EBNA3C, oncoproteins from Epstein-Barr virus (EBV), repress Bim transcription and contribute to the development of a variety of cancers including Burkitt’s lymphoma (Anderton et al., 2008). EBNA3C binds near the transcriptional site of Bim promoter and recruits PRC2 subunits, which is essential to repress Bim transcription (Paschos et al., 2012). Post-transcriptional control of Bim expression Expression of Bim is negatively regulated by a plethora of microRNAs (group of approximately 20-24 nucleotides single strand RNA molecules which negatively regulate gene expression) silencing pathways.

10   

  miRNAs silencing is a very well regulated system of protein expression with mRNA degradation or translational repression in a sequence-specific manner. miRNA-mediated gene silencing is a highly conserved system of gene regulation in almost all eukaryotes including mammals (Ji et al., 2017). MicroRNAs (miRs), miR-17-92 and miR106b-25 cluster have been found to suppress Bim expression in multiple myeloma cells, human ovarian cancer cells and in esophageal adenocarcinoma (Kan et al., 2009; Xiao et al., 2008; Zhang et al., 2012). Nine potential binding sites for miR-17-92 family members have been reported to occur in 3’–UTR of Bim mRNA (Koralov et al., 2008). Over-expression of this cluster in mice resulted in lymphoproliferative disorder mimicking Bim knockout mice and animals died prematurely, while absence of miR-17-92 in mice aggravated Bim expression (Ventura et al., 2008; Xiao et al., 2008). Dexamethasone treatment resulted in decreased expression of miR-17-92 cluster and concomitant increase in Bim expression resulting into aggravated apoptosis (Harada et al., 2012; Molitoris et al., 2011). Similarly, overexpression of miR-106b-25 cluster inhibited TGF-β induced Bim expression and apoptosis in gastric cancer cells. The same study also revealed that miR-25, not miR-106b or miR-93, was essential for Bim repression (Petrocca et al., 2008). miR-20, miR-92, 92a and miR302 have also been found to repress Bim expression and important for epiblast stem cells and glioma cell survival (Niu et al., 2012; Pernaute et al., 2014). miR-192 has recently been found to target 3’-UTR of Bim gene and inhibited protein expression of Bim in esophageal squamous cell carcinoma (Li et al., 2015). miR-148a downregulates Bim mRNA expression in activated T helper type 1 (Th1) cells, while inhibition of miR-148a resulted in enhanced Bim mRNA expression and apoptosis of Th1 cells and glioblastoma cells (Haftmann et al., 2015; Kim et al., 2014). In addition, miR-9 and miR-181a negatively regulate Cbl E3 ubiquitin ligase, an important E3 ubiquitin ligase for bone formation and homeostasis regulation. In tibial plateau fractures, elevated levels of Cbl induced Bim ubiquitination and degradation, resulting into osteoclast survival (Wang et al., 2014). β-adrenergic mediated activation of PKA (protein kinase A) leads to activation of CBP and binding to Bim promoter through its interaction with c-Myc which results into histone acetylation and demethylation, ultimately enhancing Bim gene expression (Lee et al., 2013). Stability of Bim mRNA is governed by heat shock cognate protein 70 (Hsc70), which selectively binds to AU-rich region in the 3′-untranslated region and enhances Bim mRNA stability upon cytokines deprivation as the binding efficiency of Hsc70 is negatively regulated by cytokines activated Ras signalling. Thus, activation of cytokines signalling leads to destabilization of Bim mRNA and ultimately enhanced cell survival (Harada and Grant, 2012). Post-translational regulation of Bim

11   

  Post-translational modifications on proteins (viz. phosphorylation, ubiquitination) are very common that result in conformational changes to cause release from an inactive complex and increased affinity or accessibility to other BCL-2 family proteins (Huang and Strasser, 2000). Pro-apoptotic activity of Bim is regulated by different post-translational modifications as discussed below. Phosphorylation Recent studies have suggested that Bim has sites for phosphorylation, ubiquitination and other post-translational modifications. ERK1/2 mediated phosphorylation of BimEL at Serine residues, Ser59/Ser69/Ser77 in human and Ser55/Ser65/Ser73 in mouse triggers Bim ubiquitylation, reduced interaction with pro-survival proteins or multi-domain pro-apoptotic proteins and allowed its 26S proteasome-mediated degradation (fig.4) (Akiyama et al., 2009; Wiggins et al., 2011). Moreover, ERK1/2 mediated phosphorylation of Bim at Ser65 in mouse resulted in loss of binding to pro-apoptotic Bax and dissociation from Mcl-1 and Bcl-XL (Ewings et al., 2007a; Ewings et al., 2007b; Hinds et al., 2007; Ley et al., 2004). Recent mutation studies reported that mutation of phosphorylation sites of Bim at Ser55/Ser65/Ser73 in mouse leads to reduced proteasomal degradation and increased apoptosis, while mutation of Bim at Thr112 in mouse resulted in decreased binding to anti-apoptotic protein Bcl-2 and reduced apoptosis (Hübner et al., 2008). Interleukin-3 (IL-3), a hematopoietic survival factor, induces ERK-mediated phosphorylation of Bim in mouse at three serine sites (S55, S65, and S100) which hindered its binding to pro-apoptotic Bax, showed that BH3-only protein Bim is crucial for the cytokinemediated homeostasis of hematopoietic cells (Harada et al., 2004). ERK1/2 mediated phosphorylation of human BimEL at Ser69 induced cytokine mediated phosphorylation of Bim on Ser93/Ser94/ Ser98 resulting in its binding to βTrCP (F box protein) and degradation via SCF-βTrCP complex (Skp, Cullin, F-box or SCF complex is

a

multi-protein

E3 ubiquitin

ligase complex

which

catalyses ubiquitination of

proteins

and

ultimately proteasomal degradation). The same study also found that ERK1/2 co-operates with ribosomal S6 kinases (RSK1/2) to phosphorylate BimEL and its binding to F box protein βTrCP resulting in polyubiquitination of BimEL, while silencing of either βTrCP or RSK1/2 leads to Bim induced apoptosis in NSCLC (non-small cell lung cancer) cells (fig.4) (Dehan et al., 2009). Quite the contrary, c-Jun N-terminal kinase mediated phosphorylation of Bim at Threonine 112 (Thr112) escalates the pro-apoptotic activity of Bim by causing its release from the dynein motor complex (phosphorylation site occupy the position within DLC1binding domain and its phosphorylation compromise protein-protein interaction) as BimEL and BimL remain sequestered to the microtubule-associated dynein motor complex through its interaction with the dynein light chain LC8 (Harada and Grant, 2012). UV exposure to human embryonic kidney (HEK) 293T cells resulted into

12   

  Bax-dependent apoptosis due to JNK mediated phosphorylation of BimL at Thr56, which does not bind DLC1 and released from dynein motor complex (Lei and Davis, 2003). In addition, it is also reported that JNK mediated phosphorylation of BimEL at Ser100, Thr112 and Ser114 enhanced its stability, redistribution from cytoskeletal to membrane fraction, interaction with Bcl-2 and Bcl-xL and more effective activation of Bak/Bax in response to apoptotic stimuli (Geissler et al., 2013). Another member of Mitogen-Activated Protein Kinases, p38 is also reported to enhance the apoptotic activity of Bim by phosphorylation at Ser65 in PC12 cells (Cai et al., 2006). p38 may potentiate Bim transcription through positive regulation of transcription factors including FoxO3a, Runx2 and c-jun (Cai and Xia, 2008; Heidari et al., 2012). Similarly, another up-stream modulator for Bim phosphorylation, protein kinase A which phosphorylates BimEL at Ser87 in human and Ser83 in mouse [cyclic AMP (cAMP)-regulated protein kinase A (PKA) regulatory subunit-a (PRKAR1A) serves as a binding partner] stabilizes BimEL, increase its half-life and apoptotic potential (Moujalled et al., 2011; Zhang and Insel, 2004). Growth factor induced Akt (protein kinase-B) activation resulted into FoxOs phosphorylation, exclusion from nucleus, cytoplasmic degradation and reduced Bim transcription (Yamagata et al., 2008; ., ). In another study, phosphorylation of BimEL, was correlated with Akt, and not ERK activation. The PI3K inhibitor, LY294002, blocked IL-3-stimulated Akt activity and partially blocked BimEL phosphorylation. Study revealed that Akt directly phosphorylates BimEL at Ser87 and a mutation at this site dramatically increased its apoptotic potency. Therefore, an additional mechanism exists to phosphorylate ERK wherein Bim EL is targeted by Akt to attenuate its pro-apoptotic action (Qi et al., 2006). A study on HT-1080 fibrosarcoma cells demonstrated the role of PINCH-1 (Particularly interesting cystein-histidine-rich protein-1), an integrin-proximal scaffolding protein, which suppresses Bim both transcriptionally and post-transcriptionally by promoting the activation of Src family kinase and ERK1/2 mediated phosphorylation. ERK1/2-mediated Ser69 phosphorylation of Bim, a crucial event for Bim turnover is suppressed by the removal of PINCH-1 (Chen et al., 2008). Knockdown of PINCH-1 resulted in 10-fold increase in Bim EL mRNA and Bim-mediated cell death in multiple types of cancer cells (Chen et al., 2008). Ubiquitination Bim up-regulation and enhanced stability have been reported to show correlation with hindered tumor growth in vitro and size in vivo, while ERK dependent phosphorylation, ubiquitination and degradation has been linked to tumorigenesis, confirming its tumor suppressor character (Zantl et al., 2007). Anaphase promoting complex (APC) regulates timely cell cycle progression in G1 and M phase and forms complexes APCCdc20 (E3 ubiquitin ligase) and APCCdh1. Cdc20 is found to be frequently over expressed in many cancers and inactivating APCCdc20

13   

  promoted cell death in cancer cells. BH3-only protein, Bim was reported a ubiquitin substrate of APCCdc20 as APCCdc20 interacts with BimEL and induces its proteasomal degradation and rescues from ubiquitin dependent degradation upon APCCdc20 inhibition imparts to apoptosis induction and chemo-radiation sensitization in cancers of different origin (Wan et al., 2014). It was also found that Bim stability and abundance was decreased during mitosis when APCCdc20 activity was at peak (Wan et al., 2014). Bim activity and stability are kept under control during mitosis by Aurora A kinase and protein phosphatase 2A (PP2A) as Bim is a pro-apoptotic protein implicated in numerous apoptotic stimuli. When mitosis occurs, BimEL is phosphorylated on Ser69 and Ser93/Ser94/Ser98 by Aurora A kinase and creates a binding site for the F-box protein βTrCP1 leading to ubiquitin dependent Bim degradation (Moustafa-Kamal et al., 2013). Bim level is again maintained to normal value in the cells by PP2A, which dephosphorylates BimELon Ser93/Ser94/Ser98 and prevents its ubiquitin dependent degradation after mitotic exit (Moustafa-Kamal et al., 2013). Role of Bim as a chemotherapeutic target: Bim is required to induce apoptosis by various chemotherapeutic agents i.e. paclitaxel, imatinib, dasatinib, nilotinib, gefitinib, erlotinib and bortezomib (Akiyama et al., 2009). Bcr-Abl is a fusion oncoprotein, formed as a result of reciprocal t(9;22)(q34;q11) chromosomal translocation (Philadelphia chromosome; Ph1) in chronic myeloid leukemia (CML) (Song et al., 2015). The mutant Bcr-Abl oncoprotein activates a number of signaling pathways i.e. ERK1/2, PI3k/Akt and the Janus kinase/signal transducer and activator of transcription (JAK-STAT) pathway, essential for CML and Ph1-positive acute lymphoblastic leukemia (ALL) progression. Thus, Bcr-Abl represents a novel target for CML and ALL targeted chemotherapy. Imatinib has shown significant improvement in CML and ALL patients especially for early chronic phase CML patients. Bim is reported to play a pivotal role in imatinib induced apoptosis of Bcr-Abl expressing cells via a mitochondrial mediated pathway (Kuribara et al., 2004; Kuroda et al., 2006). Imatinib has been shown to increase Bim and Bcl-2-modifying factor (Bmf) protein transcriptionally as well as post-transcriptionally. Knockdown of Bim in Bcr-Abl positive human leukemia cell lines reverses imatinib induced cell death (Kuroda et al., 2006). Dasatinib and nilotinib, newer generation chemotherapeutic agents, are developed to treat imatinib resistant cases of CML/ALL and induce cell death in a Bim dependent manner (Bhamidipati et al., 2013). ERK signaling, a major regulator of Bim stability, has also been implicated in dasatinib induced cell death of resistant leukemic cells and ERK inhibitor (PD184352) sensitizes the myelogenous leukemic cells K562 to dasatinib activity (Nguyen et al., 2007b). These findings reveal that number of first-generation and second-generation Bcr-Abl kinase

14   

  inhibitor target CML/ALL cells in a Bim dependent manner, where Bim act in conjugation with other BH3-only proteins such as BOD/BAK. Mutation in Epidermal Growth Factor Receptor (EGFR) has been associated with variety of carcinomas of lung, breast and colon. Gefitinib, an EGFR targeting chemotherapeutic agent, has shown significant clinical efficacy in non-small cell lung cancer (NSCLC) (Kim et al., 2016a; Zhu et al., 2016). Gefitinib and erlotinib are reported to induce cell death in NSCLC via a mitochondria-mediated intrinsic pathway of apoptosis. Gefitinib treatment causes massive increase in Bim expression level in NSCLC cell lines NCI-H358, NCI-441, NCI-H1650 through both transcriptional and post-translational mechanisms including inhibition of MEK/ERK kinase downstream of EGFR (Cragg et al., 2007). Erlotinib, another EGFR targeting drug, inhibits cell proliferation of lung cancer cell lines and tumor formation in transgenic mice through increased expression of Bim (Costa et al., 2007; Gong et al., 2007). Bim knockdown in NSCLC cells harbouring primary EGFR mutations partially restored cell viability and reversed cell death induced by erlotinib or gefitinib. Mutations in EGFR are likely to be associated with gefitinib resistant advanced lung adenocarcinoma. Thr790Met is a gatekeeper mutation of ATP binding site of EGFR kinase domain and abolishes Bim activation and mitochondria mediated apoptosis induced by gefitinib (Costa et al., 2007). These findings outline the importance of Bim as an important key effector of gefitinib- or erlotinib-induced cell death. Bortezomib (Velcade, PS-341) is a first therapeutically approved 20S proteasome inhibitor for the treatment of multiple myeloma and mantle cell lymphoma (Chen et al., 2014). As discussed earlier, ERK mediated Bim phosphorylation potentiates Bim degradation via proteasome-ubiquitination channel. Bortezomib is reported to enhance Bim half-life by abrogating its proteasomal degradation as well as through decreased binding of Bim with Mcl-1, a member of the multidomain anti-apoptotic Bcl2 family (Gomez-Bougie et al., 2007). Displacement of Bim from Mcl-1 promotes Bax/Bak dependent mitochondrial permeabilization and intrinsic mode of apoptosis. It has also been shown that mouse embryonic fibroblasts (MEFs) deficient in Bim and Bik were resistant to bortezomib-induced cell death (Nikrad et al., 2005). Knockdown of Bim and Bik in LNCaP prostate cancer cells impaired TRAIL induced apoptosis in the presence of bortezomib (Nikrad et al., 2005). Thus, Bim seems indispensable for bortezomib induced cell death of number of cancer cell types. The v-raf-1 murine leukemia viral oncogene homolog B1 (BRAF) and rat sarcoma virus oncogene (RAS) mutations have been identified as a typical signature of certain human cancers including malignant melanoma (Kim et al., 2016b). Tumors having BRAF/RAS mutations are strongly addicted to ERK1/2 signaling for cancer progression and ERK1/2 (MEK1/2) inhibitor PD184352 has showed significant clinical efficacy in pre-clinical

15   

  models (LoRusso et al., 2005). Now more selective inhibitors of MEK1⁄2–ERK1⁄2 pathway; PD0325901and AZD6244 (ARRY-142886) are developed (Davies et al., 2007; Solit et al., 2006). It is now evident that Bim is the prime executioner of cell death program in response to RAS-RAF-MEK-ERK1/2 inhibitors (Wickenden et al., 2008). Bim silencing in colorectal cancer cells partially reversed AZD6244 induced cell death and disclosed the prime importance of Bim as a key effector protein (Wickenden et al., 2008). Treatment of PD184352 or the BRAF600E-selective inhibitor PLX4720 in melanoma cells harbouring BRAF600E mutations enhanced cancer cell death and efficacy of the treatment is due to elevated Bim expression (Cartlidge et al., 2008). Similar results were also reported in colorectal and melanoma cancers (Cragg et al., 2008) and once again, Bim was the central culprit for observed anti-cancer property. Bim alone or in conjugation with other members of BH3-only proteins were found to be responsible for MEK1/2 inhibitors induced melanoma cell death in number of different studies (Boisvert-Adamo and Aplin, 2008; Sheridan et al., 2008; Wang et al., 2007). Later it was also reported that MEK1/2 inhibitors derived Bim induction alone was not capable for efficient killing of cancer cells. Bim activation alongwith growth factors/serum deprivation were associated with dramatic increase of apoptosis in melanoma and colorectal cancer cells (Cartlidge et al., 2008; Wickenden et al., 2008). These findings suggested that inhibition of growth factors/serum derived survival kinases i.e. PI3K/Akt/mTOR might also be required to work in cooperation with MEK1/2 inhibitors to attain optimal cell death of tumor cells outlined a rationale for the combined use of MEK1⁄2 inhibitors and PI3K/Akt pathway inhibitors. Inhibitor of mTOR, a downstream key protein to PI3K/Akt axis, showed synergistic effects with MEK1/2 inhibitor PD0325901 to promote cell death in melanoma model (Dankort et al., 2009). Synergistic interaction between other kinase signaling inhibitors and MEK1/2 inhibitors have also been documented (Pei et al., 2007). UCN-01, a competitive inhibitor of ATP binding site on various kinases i.e. cyclin dependent kinases (CDKs), checkpoint protein 1 (CHK1), 3’phosphoinositide-dependent kinase-1 (PDK1) and protein kinase C (PKC) potentiated tumor cell death when coadministered with MEK1⁄2 inhibitor PD184352 (Pei et al., 2007).

Future perspective in developing Bim-targeted chemotherapy: As we have discussed above, a number of potential signals and pathways have been reported to regulate Bim expression and stability. The aim of modern chemotherapeutic agents that target pathways affecting Bim expression and half-life is the selective induction of apoptosis in cancer cells. As a potent initiator of cell death, Bim has been implicated in chemotherapy with a number of such chemotherapeutic agents as the main pro-apoptotic initiator on one hand, and as part of relevant resistance mechanisms, when lost, on the other hand. Bim functions as a tumor suppressor and is a crucial regulator of tumorigenesis, frequently induced upon

16   

  treatment with anticancer therapeutics. Most of the treatments involved with metastatic malignancies remain unsatisfactory because cancerous cells pose resistance to apoptosis. Resistance to apoptosis in cancer chemotherapy is the most often due to faulty Bim function that might be due to disruption of any upstream regulatory pathway of Bim, which include diminished transcription of Bim due to PI3K/Akt hyperactivation, enhanced expression of transcriptional repressors, decreased Bim translation due to elevated expression of microRNAs, epigenetic silencing including Bim promoter hyper-methylation, reduced Bim stability because of hyperactivation of survival kinases (Ras-MEK-ERK1/2) and sequestration of Bim with other anti-apoptotic proteins, making it unavailable to execute intrinsic mode of apoptosis. Fortunately many chemotherapeutic interventions have been evolved to restore Bim stability and activity, which act upon various points either upstream or downstream to Bim regulatory pathways. Considerable number of reports now exist implying that interventions which lead to Bim upregulation, increased stability via post-translational modulation may aggravate lethality of a chemotherapeutic agent or contribute to synergistic interactions, targeting cancers of different origin. Recently, attention has turned to the development of a new class of molecule which can mimic the action of BH3-only proteins and disrupt Bcl-2 complexes. BH3 mimetics mimic the BH3 domain of Bim and disturb the interaction of BH3-only proteins with other anti-apoptotic Bcl-2 proteins resulting into release of BH3-only proteins to initiate intrinsic mode of apoptosis. In addition, BH3-only proteins get displaced as a result of BH3-mimetic treatment may bind and inhibit Mcl-1, providing further activation of BAX⁄BAK. Prosurvival members of Bcl-2 family are thus captured with BH3-mimetics and failed to neutralize Bax/Bak activation. Owing to its potent anti-tumorigenic activity, many BH3 mimetics e.g. Gossypol, obatoclax (GX15070), AT-101, ABT-737, ABT-263, ABT-199 and A-1210477 have been developed and entered in clinical trials (Cleary et al., 2014; Faber et al., 2015; Kipps et al., 2015; Leverson et al., 2015a; Moore et al., 2007; Nguyen et al., 2007a; Oltersdorf et al., 2005; Trudel et al., 2007; Wilson et al., 2010). Obatoclax (GX15-070), cotton-seed extract gossypol and its synthetic derivative AT-101 neutralized anti-apoptotic Bcl-2 proteins Bcl-2, Bcl-XL and Mcl-1 with modest affinity and exerted limited potential into clinical studies (Baggstrom et al., 2011; Langer et al., 2014; Ready et al., 2011; Sonpavde et al., 2012). ABT-737, ABT-263 (Navitoclax) and ABT-199 (Venetoclax) are new agents to BH3-mimetics class and showed remarkable specificity and antitumoral activity in a variety of cancer type (Hata et al., 2015). ABT-737 and ABT-263 are designed to bind Bcl-2, Bcl-XL and Bcl-W with high affinity, but are unable to neutralize Mcl-1 and A1 (Oltersdorf et al., 2005; Tse et al., 2008). On the other hand, ABT-199 binds to Bcl-2 protein with high affinity as ABT-737, but lack the affinity to bind with Bcl-XL, Bcl-W, Mcl-1 or A1 in nano-molar range (Souers et al., 2013). In preclinical studies, ABT-737 was found to be very effective as a single agent for treatment of haematopoietic and lymphoid malignancies.

17   

  However, overexpression of both Mcl-1 and A1 anti-apoptotic proteins cause ABT-737 resistance as the binding affinity of ABT-737 to Mcl-1 is weak (Akiyama et al., 2009). Mcl-1 is found to be overexpressed in various tumors, so more work has to be needed to increase coverage. ABT-263 and ABT-199 are currently being evaluated for their clinical efficacy in chronic lymphocytic leukemia (Davids et al., 2013; Roberts et al., 2012). ABT-199 is reported to possess an advantage of avoiding dose-dependent thrombocytopenia, a serious drawback of ABT-263 due to the dependence of mature platelets on Bcl-XL for survival (Cang et al., 2015;). S44563, a new generation Bcl-2/Bcl-XL inhibitor has been reported to elicit promising result in-vivo in preclinical models of uveal melanoma (Némati et al., 2014). S55746, another Bcl-2 specific inhibitor is presently under evaluation in a Phase 1 dose-escalation study in B-cell non-Hodgkin Lymphoma (ISRCTN04804337). Till date, dual Bcl-XL/Mcl-1 inhibitors and Specific Bcl- XL inhibitors WEHI-539, A1155463 and A-1331852 have also been added to BH3-mimetic class but their clinical utility is not defined yet (Lessene et al., 2013; Leverson et al., 2015b; Sleebs et al., 2013; Tao et al., 2014). Finally selective Mcl-1 inhibitor A-1210477 has also been synthesized and reports outline its utility in breast cancer cells having a TNBC phenotype (Bruncko et al., 2015; Fang et al., 2014; Leverson et al., 2015a). Recently a BH3 α-helical mimetic agent, BH3-M6, has been developed by Kazi et al (Kazi et al., 2011) that has ability to bind with Bcl-XL, Bcl-2 and Mcl-1 and release Bax, Bak, Bad and Bim leading to cytochrome C release, PARP cleavage and activation of executioner caspases. Another group developed a hydrocarbon-stapled Bim peptide, which targets anti-apoptotic proteins of Bcl-2 family members with high affinity and induced cell death in resistant hematologic cancers (LaBelle et al., 2012). BH3-mimetics can also synergise with available anti-cancer drugs and chemotherapeutic agents. Down-modulation of Bim expression and stability is central to chemotherapy resistance and tumor metastasis. Some of available BH3-mimetics or Bim targeting agents can be used to restore chemotherapy sensitivity or anchorage-dependence. Hence, we can reduce chemotherapy side effects and advocate use of some new cocktail regimens of chemotherapeutic agents. It is possible that in near future strategies enhancing Bim stability or Bim mimetics will be able to be approved for clinical studies. Conflict of interest statement Nothing declared.

18   

  Acknowledgements Research of our group is supported by Council of Scientific & Industrial Research network project BSC-0106 and BSC-0111. Shatrunajay Shukla and Sugandh Saxena gratefully acknowledge Council of Scientific and Industrial Research and Department of Biotechnology, Government of India, respectively for award of research fellowship.

References:

1. Akiyama,  T.,  Dass, C.R., Choong,  P.F.,  2009.  Bim‐targeted  cancer therapy:  a  link  between  drug  action  and  underlying  molecular  changes.  Molecular  cancer  therapeutics  8,  3173‐ 3180.    2. Akiyama, T., Tanaka, S., 2011. Bim: guardian of tissue homeostasis and critical regulator of  the  immune  system,  tumorigenesis  and  bone  biology.  Archivum  immunologiae  et  therapiae experimentalis 59, 277‐287.    3. Anderton,  E.,  Yee,  J.,  Smith,  P.,  Crook,  T.,  White,  R.,  Allday,  M.,  2008.  Two  Epstein–Barr  virus  (EBV)  oncoproteins  cooperate  to  repress  expression  of  the  proapoptotic  tumour‐ suppressor Bim: clues to the pathogenesis of Burkitt's lymphoma. Oncogene 27, 421‐433.    4. Baggstrom, M.Q., Qi, Y., Koczywas, M., Argiris, A., Johnson, E.A., Millward, M.J., Murphy,  S.C., Erlichman, C., Rudin, C.M., Govindan, R., 2011. A phase II study of AT‐101 (Gossypol)  in  chemotherapy‐sensitive  recurrent  extensive‐stage  small  cell  lung  cancer.  Journal  of  Thoracic Oncology 6, 1757‐1760.    5. Barthson, J., Germano, C.M., Moore, F., Maida, A., Drucker, D.J., Marchetti, P., Gysemans,  C.,  Mathieu,  C.,  Nuñez,  G.,  Jurisicova,  A.,  2011.  Cytokines  tumor  necrosis  factor‐α  and  interferon‐γ  induce  pancreatic  β‐cell  apoptosis  through  STAT1‐mediated  Bim  protein  activation. Journal of Biological Chemistry 286, 39632‐39643.      6. Bhamidipati,  P.K.,  Kantarjian,  H.,  Cortes,  J.,  Cornelison,  A.M.,  Jabbour,  E.,  2013.  Management  of  imatinib‐resistant  patients  with  chronic  myeloid  leukemia.  Therapeutic  advances in hematology 4, 103‐117.      7. Boisvert‐Adamo, K., Aplin, A., 2008. Mutant B‐RAF mediates resistance to anoikis via Bad  and Bim. Oncogene 27, 3301‐3312.    8. Bouillet,  P.,  Zhang,  L.C.,  Huang,  D.C.,  Webb,  G.C.,  Bottema,  C.D.,  Shore,  P.,  Eyre,  H.J.,  Sutherland,  G.R.,  Adams,  J.M.,  2001.  Gene  structure,  alternative  splicing,  and  19   

  chromosomal  localization  of  pro‐apoptotic  Bcl‐2  relative  Bim.  Mammalian  Genome  12,  163‐168.    9. Bratton,  S.B.,  Salvesen,  G.S.,  2010.  Regulation  of  the  Apaf‐1–caspase‐9  apoptosome.  Journal of cell science 123, 3209‐3214.    10. Bruncko,  M.,  Wang,  L.,  Sheppard,  G.S.,  Phillips,  D.C.,  Tahir,  S.K.,  Xue,  J.,  Erickson,  S.,  Fidanze,  S.,  Fry,  E.,  Hasvold,  L.,  2015.  Structure‐guided  design  of  a  series  of  MCL‐1  inhibitors with high affinity and selectivity. Journal of medicinal chemistry 58, 2180‐2194.    11. Brunelle,  J.K.,  Letai,  A.,  2009.  Control  of  mitochondrial  apoptosis  by  the  Bcl‐2  family.  Journal of cell science 122, 437‐441.      12. Cai,  B.,  Chang,  S.H.,  Becker,  E.B.,  Bonni,  A.,  Xia,  Z.,  2006.  p38  MAP  kinase  mediates  apoptosis through phosphorylation of BimEL at Ser‐65. Journal of Biological Chemistry 281,  25215‐25222.    13. Cai,  B.,  Xia,  Z.,  2008.  p38  MAP  kinase  mediates  arsenite‐induced  apoptosis  through  FOXO3a activation and induction of Bim transcription. Apoptosis 13, 803‐810.    14. Cang, S., Iragavarapu, C., Savooji, J., Song, Y., Liu, D., 2015. ABT‐199 (venetoclax) and BCL‐2  inhibitors in clinical development. Journal of hematology & oncology 8, 129.      15. Cartlidge, R.A., Thomas, G., Cagnol, S., Jong, K.A., Molton, S.A., Finch, A.J., McMahon, M.,  2008. Oncogenic BRAFV600E inhibits BIM expression to promote melanoma cell survival.  Pigment cell & melanoma research 21, 534‐544.    16. Chen, K., Tu, Y., Zhang, Y., Blair, H.C., Zhang, L., Wu, C., 2008. PINCH‐1 regulates the ERK‐ Bim pathway and contributes to apoptosis resistance in cancer cells. Journal of Biological  Chemistry 283, 2508‐2517.    17. Chen, S., Zhang, Y., Zhou, L., Leng, Y., Lin, H., Kmieciak, M., Pei, X.‐Y., Jones, R., Orlowski,  R.Z., Dai, Y., 2014. A Bim‐targeting strategy overcomes adaptive bortezomib resistance in  myeloma through a novel link between autophagy and apoptosis. Blood 124, 2687‐2697.    18. Cleary, J.M., Lima, C.M.S.R., Hurwitz, H.I., Montero, A.J., Franklin, C., Yang, J., Graham, A.,  Busman,  T.,  Mabry,  M.,  Holen,  K.,  2014.  A  phase  I  clinical  trial  of  navitoclax,  a  targeted  high‐affinity Bcl‐2 family inhibitor, in combination with gemcitabine in patients with solid  tumors. Investigational new drugs 32, 937‐945.    19. Costa,  D.B.,  Halmos,  B.,  Kumar,  A.,  Schumer,  S.T.,  Huberman,  M.S.,  Boggon,  T.J.,  Tenen,  D.G., Kobayashi, S., 2007. BIM mediates EGFR tyrosine kinase inhibitor‐induced apoptosis  in lung cancers with oncogenic EGFR mutations. PLoS Med 4, e315.    20. Cragg, M.S., Jansen, E.S., Cook, M., Harris, C., Strasser, A., Scott, C.L., 2008. Treatment of B‐ RAF  mutant  human  tumor  cells  with  a  MEK  inhibitor  requires  Bim  and  is  enhanced  by  a  BH3 mimetic. The Journal of clinical investigation 118, 3651‐3659.    21. Cragg, M.S., Kuroda, J., Puthalakath, H., Huang, D.C., Strasser, A., 2007. Gefitinib‐induced  killing of NSCLC cell  lines expressing  mutant  EGFR requires BIM  and  can be  enhanced  by  BH3 mimetics. PLoS Med 4, e316.  20   

    22. Dankort, D., Curley, D.P., Cartlidge, R.A., Nelson, B., Karnezis, A.N., Damsky Jr, W.E., You,  M.J., DePinho, R.A., McMahon, M., Bosenberg, M., 2009. BrafV600E cooperates with Pten  loss to induce metastatic melanoma. Nature genetics 41, 544‐552.    23. Davids, M.S., Pagel, J.M., Kahl, B.S., Wierda, W.G., Miller, T.P., Gerecitano, J.F., Kipps, T.J.,  Anderson, M.‐A., Huang, D.C., Rudersdorf, N.K., 2013. Bcl‐2 inhibitor ABT‐199 (GDC‐0199)  monotherapy  shows  anti‐tumor  activity  including  complete  remissions  in  high‐risk  relapsed/refractory  (R/R)  chronic  lymphocytic  leukemia  (CLL)  and  small  lymphocytic  lymphoma (SLL). Blood 122, 872‐872.    24. Davies,  B.R.,  Logie,  A.,  McKay,  J.S.,  Martin,  P.,  Steele,  S.,  Jenkins,  R.,  Cockerill,  M.,  Cartlidge,  S.,  Smith,  P.D.,  2007.  AZD6244  (ARRY‐142886),  a  potent  inhibitor  of  mitogen‐ activated  protein  kinase/extracellular  signal‐regulated  kinase  kinase  1/2  kinases:  mechanism  of  action  in  vivo,  pharmacokinetic/pharmacodynamic  relationship,  and  potential  for  combination  in  preclinical  models.  Molecular  cancer  therapeutics  6,  2209‐ 2219.    25. De Bruyne, E., Bos, T.J., Schuit, F., Van Valckenborgh, E., Menu, E., Thorrez, L., Atadja, P.,  Jernberg‐Wiklund, H., Vanderkerken, K., 2010. IGF‐1 suppresses Bim expression in multiple  myeloma via epigenetic and posttranslational mechanisms. Blood 115, 2430‐2440.    26. Dehan,  E.,  Bassermann,  F.,  Guardavaccaro,  D.,  Vasiliver‐Shamis,  G.,  Cohen,  M.,  Lowes,  K.N., Dustin, M., Huang, D.C., Taunton, J., Pagano, M., 2009. βTrCP‐and Rsk1/2‐mediated  degradation of BimEL inhibits apoptosis. Molecular cell 33, 109‐116.    27. Doerflinger, M., Glab, J.A., Puthalakath, H., 2015. BH3‐only proteins: a 20‐year stock‐take.  FEBS Journal 282, 1006‐1016.    28. Elmore, S., 2007. Apoptosis: a review of programmed cell death. Toxicologic pathology 35,  495‐516.    29. Essafi,  A.,  de  Mattos,  S.F.,  Hassen,  Y.A.,  Soeiro,  I.,  Mufti,  G.J.,  Thomas,  N.S.B.,  Medema,  R.H., Lam, E.W., 2005. Direct transcriptional regulation of Bim by FoxO3a mediates STI571‐ induced apoptosis in Bcr‐Abl‐expressing cells. Oncogene 24, 2317‐2329.    30. Ewings, K.E., Hadfield‐Moorhouse, K., Wiggins, C.M., Wickenden, J.A., Balmanno, K., Gilley,  R.,  Degenhardt,  K.,  White,  E.,  Cook,  S.J.,  2007a.  ERK1/2‐dependent  phosphorylation  of  BimEL promotes its rapid dissociation from Mcl‐1 and Bcl‐xL. The EMBO journal 26, 2856‐ 2867.    31. Ewings,  K.E.,  Wiggins,  C.M.,  Cook,  S.J.,  2007b.  Bim  and  the  pro‐survival  Bcl‐2  proteins:  opposites attract, ERK repels. Cell Cycle 6, 2236‐2240.    32. Faber, A.C., Farago, A.F., Costa, C., Dastur, A., Gomez‐Caraballo, M., Robbins, R., Wagner,  B.L., Rideout, W.M., Jakubik, C.T., Ham, J., 2015. Assessment of ABT‐263 activity across a  cancer cell line collection leads to a potent combination therapy for small‐cell lung cancer.  Proceedings of the National Academy of Sciences 112, E1288‐E1296.    33. Fang,  C.,  D’Souza,  B.,  Thompson,  C.F.,  Clifton,  M.C.,  Fairman,  J.W.,  Fulroth,  B.,  Leed,  A.,  McCarren, P., Wang, L., Wang, Y., 2014. Single diastereomer of a macrolactam core binds  specifically  to  myeloid  cell  leukemia  1  (MCL1).  ACS  medicinal  chemistry  letters  5,  1308‐ 1312.  21   

    34. Fiskus,  W.,  Sharma,  S.,  Qi,  J.,  Valenta,  J.A.,  Schaub,  L.J.,  Shah,  B.,  Peth,  K.,  Portier,  B.P.,  Rodriguez,  M.,  Devaraj,  S.G.,  2014.  Highly  active  combination  of  BRD4  antagonist  and  histone deacetylase inhibitor against human acute myelogenous leukemia cells. Molecular  cancer therapeutics 13, 1142‐1154.    35. Gallagher,  S.J.,  Mijatov,  B.,  Gunatilake,  D.,  Tiffen,  J.C.,  Gowrishankar,  K.,  Jin,  L.,  Pupo,  G.M.,  Cullinane,  C.,  Prinjha,  R.K.,  Smithers,  N.,  2014.  The  epigenetic  regulator  I‐BET151  induces BIM‐dependent apoptosis and cell cycle arrest of human melanoma cells. Journal  of Investigative Dermatology 134, 2795‐2805.    36. Geissler, A.,  Haun, F.,  Frank, D., Wieland, K., Simon, M., Idzko, M.,  Davis, R., Maurer,  U.,  Borner,  C.,  2013.  Apoptosis  induced  by  the  fungal  pathogen  gliotoxin  requires  a  triple  phosphorylation of Bim by JNK. Cell Death & Differentiation 20, 1317‐1329.    37. Gomez‐Bougie, P., Wuillème‐Toumi, S., Ménoret, E., Trichet, V., Robillard, N., Philippe, M.,  Bataille,  R.,  Amiot,  M.,  2007.  Noxa  up‐regulation  and  Mcl‐1  cleavage  are  associated  to  apoptosis induction by bortezomib in multiple myeloma. Cancer research 67, 5418‐5424.    38. Gong, Y., Somwar, R., Politi, K., Balak, M., Chmielecki, J., Jiang, X., Pao, W., 2007. Induction  of  BIM  is  essential  for  apoptosis  triggered  by  EGFR  kinase  inhibitors  in  mutant  EGFR‐ dependent lung adenocarcinomas. PLoS Med 4, e294.    39. Gowda,  A.,  Roda,  J.,  Hussain,  S.‐R.A.,  Ramanunni,  A.,  Joshi,  T.,  Schmidt,  S.,  Zhang,  X.,  Lehman,  A.,  Jarjoura,  D.,  Carson,  W.E.,  2008.  IL‐21  mediates  apoptosis  through  up‐ regulation  of  the  BH3  family  member  BIM  and  enhances  both  direct  and  antibody‐ dependent  cellular  cytotoxicity  in  primary  chronic  lymphocytic  leukemia  cells  in  vitro.  Blood 111, 4723‐4730.    40. Haftmann,  C.,  Stittrich,  A.B.,  Zimmermann,  J.,  Fang,  Z.,  Hradilkova,  K.,  Bardua,  M.,  Westendorf, K., Heinz, G.A., Riedel, R., Siede, J., 2015. miR‐148a is upregulated by Twist1  and  T‐bet  and  promotes  Th1‐cell  survival  by  regulating  the  proapoptotic  gene  Bim.  European journal of immunology 45, 1192‐1205.    41. Harada, H., Grant, S., 2012. Targeting the regulatory machinery of BIM for cancer therapy.  Critical Reviews™ in Eukaryotic Gene Expression 22.    42. Harada,  H.,  Quearry,  B.,  Ruiz‐Vela,  A.,  Korsmeyer,  S.J.,  2004.  Survival  factor‐induced  extracellular  signal‐regulated  kinase  phosphorylates  BIM,  inhibiting  its  association  with  BAX  and  proapoptotic  activity.  Proceedings  of  the  National  Academy  of  Sciences  of  the  United States of America 101, 15313‐15317.    43. Harada, M., Pokrovskaja‐Tamm, K., Söderhäll, S., Heyman, M., Grander, D., Corcoran, M.,  2012.  Involvement  of  miR17  pathway  in  glucocorticoid‐induced  cell  death  in  pediatric  acute lymphoblastic leukemia. Leukemia & lymphoma 53, 2041‐2050.    44. Hata,  A.N.,  Engelman,  J.A.,  Faber,  A.C.,  2015.  The  BCL2  family:  key  mediators  of  the  apoptotic response to targeted anticancer therapeutics. Cancer discovery 5, 475‐487.    45. Heidari,  N.,  Miller,  A.,  Hicks,  M.,  Marking,  C.,  Harada,  H.,  2012.  Glucocorticoid‐mediated  BIM induction and apoptosis are regulated by Runx2 and c‐Jun in leukemia cells. Cell death  & disease 3, e349.    22   

  46. Hershko, T., Ginsberg, D., 2004. Up‐regulation of Bcl‐2 homology 3 (BH3)‐only proteins by  E2F1 mediates apoptosis. Journal of Biological Chemistry 279, 8627‐8634.    47. Hinds, M., Smits, C., Fredericks‐Short, R., Risk, J., Bailey, M., Huang, D., Day, C., 2007. Bim,  Bad  and  Bmf:  intrinsically  unstructured  BH3‐only  proteins  that  undergo  a  localized  conformational  change  upon  binding  to  prosurvival  Bcl‐2  targets.  Cell  Death  &  Differentiation 14, 128‐136.    48. Holmberg, C., Helin, K., Sehested, M., Karlström, O., 1998. E2F‐1‐induced p53‐independent  apoptosis in transgenic mice. Oncogene 17, 143‐155.    49. Huang,  D.C.,  Strasser,  A.,  2000.  BH3‐only  proteins—essential  initiators  of  apoptotic  cell  death. Cell 103, 839‐842.    50. Hübner, A., Barrett, T., Flavell, R.A., Davis, R.J., 2008. Multisite phosphorylation regulates  Bim stability and apoptotic activity. Molecular cell 30, 415‐425.    51. Hughes, R., Kristiansen, M., Lassot, I., Desagher, S., Mantovani, R., Ham, J., 2011. NF‐Y is  essential for expression of the proapoptotic bim gene in sympathetic neurons. Cell Death  & Differentiation 18, 937‐947.    52. Ji, W., Sun, B., Su, C., 2017. Targeting MicroRNAs in Cancer Gene Therapy. Genes 8, 21.    53. Kan, T., Sato, F., Ito, T., Matsumura, N., David, S., Cheng, Y., Agarwal, R., Paun, B.C., Jin, Z.,  Olaru,  A.V.,  2009.  The  miR‐106b‐25  polycistron,  activated  by  genomic  amplification,  functions as an oncogene by suppressing p21 and Bim. Gastroenterology 136, 1689‐1700.    54. Kapoor, R., Rizvi, F., Kakkar, P., 2013. Naringenin prevents high glucose‐induced  mitochondria‐mediated apoptosis involving AIF, Endo‐G and caspases. Apoptosis 18, 9‐27.    55. Kazi,  A.,  Sun,  J.,  Doi,  K.,  Sung,  S.‐S.,  Takahashi,  Y.,  Yin,  H.,  Rodriguez,  J.M.,  Becerril,  J.,  Berndt, N., Hamilton, A.D.,  2011. The  BH3 α‐helical mimic  BH3‐M6 disrupts Bcl‐XL,  Bcl‐2,  and MCL‐1 protein‐protein interactions with Bax, Bak, Bad, or Bim and induces apoptosis  in a Bax‐and Bim‐dependent manner. Journal of Biological Chemistry 286, 9382‐9392.    56. Kim, H., Jang, J., Sun, J., Ahn, M., Kim, D., Jung, I., Lee, K., Kim, J., Lee, D., Kim, S., 2016a. A  randomized,  phase  II  study  of  gefitinib  alone  versus  nimotuzumab  plus  gefitinib  after  platinum‐based  chemotherapy  in  advanced  non‐small  cell  lung  cancer  (KCSG  LU12‐01).  Oncotarget 8, 15943.    57. Kim, H., Tu, H.‐C., Ren, D., Takeuchi, O., Jeffers, J.R., Zambetti, G.P., Hsieh, J.J.‐D., Cheng,  E.H.‐Y.,  2009.  Stepwise  activation  of  BAX  and  BAK  by  tBID,  BIM,  and  PUMA  initiates  mitochondrial apoptosis. Molecular cell 36, 487‐499.      58. Kim,  H.K.,  Lee,  S.,  Kim,  K.,  Heo,  M.H.,  Lee,  H.,  Cho,  J.,  Kim,  N.K.,  Park,  W.,  Lee,  S.J.,  Kim,  J.H., 2016b. Efficacy of BRAF Inhibitors in Asian Metastatic Melanoma Patients: Potential  Implications of Genomic Sequencing in BRAF‐Mutated Melanoma. Translational Oncology  9, 557‐564.    23   

  59. Kim, J., Zhang, Y., Skalski, M., Hayes, J., Kefas, B., Schiff, D., Purow, B., Parsons, S., Lawler,  S.,  Abounader,  R.,  2014.  microRNA‐148a  is  a  prognostic  oncomiR  that  targets  MIG6  and  BIM to regulate EGFR and apoptosis in glioblastoma. Cancer research 74, 1541‐1553.    60. Kim, Y.‐C., Day, R.M., 2012. Angiotensin II regulates activation of Bim via Rb/E2F1 during  apoptosis: involvement of interaction between AMPKβ1/2 and Cdk4. American Journal of  Physiology‐Lung Cellular and Molecular Physiology 303, L228‐L238.    61. Kipps, T.J., Eradat, H., Grosicki, S., Catalano, J., Cosolo, W., Dyagil, I., Yalamanchili, S., Chai,  A.,  Saharanaman,  S.,  Punnoose,  E.,  2015.  A  phase  2  study  of  the  BH3  mimetic  BCL2  inhibitor  navitoclax  (ABT‐263)  with  or  without  rituximab,  in  previously  untreated  B‐cell  chronic lymphocytic leukemia. Leukemia & lymphoma 56, 2826‐2833.    62. Koralov, S.B., Muljo, S.A., Galler, G.R., Krek, A., Chakraborty, T., Kanellopoulou, C., Jensen,  K.,  Cobb,  B.S.,  Merkenschlager,  M.,  Rajewsky,  N.,  2008.  Dicer  ablation  affects  antibody  diversity and cell survival in the B lymphocyte lineage. Cell 132, 860‐874.    63. Kuribara, R., Honda, H., Matsui, H., Shinjyo, T., Inukai, T., Sugita, K., Nakazawa, S., Hirai, H.,  Ozawa,  K.,  Inaba,  T.,  2004.  Roles  of  Bim  in  apoptosis  of  normal  and  Bcr‐Abl‐expressing  hematopoietic progenitors. Molecular and cellular biology 24, 6172‐6183.    64. Kuroda,  J.,  Puthalakath,  H.,  Cragg,  M.S.,  Kelly,  P.N.,  Bouillet,  P.,  Huang,  D.C.,  Kimura,  S.,  Ottmann,  O.G.,  Druker,  B.J.,  Villunger,  A.,  2006.  Bim  and  Bad  mediate  imatinib‐induced  killing  of  Bcr/Abl+  leukemic  cells,  and  resistance  due  to  their  loss  is  overcome  by  a  BH3  mimetic. Proceedings of the National Academy of Sciences 103, 14907‐14912.    65. LaBelle, J.L., Katz, S.G., Bird, G.H., Gavathiotis, E., Stewart, M.L., Lawrence, C., Fisher, J.K.,  Godes,  M.,  Pitter,  K.,  Kung,  A.L.,  2012.  A  stapled  BIM  peptide  overcomes  apoptotic  resistance in hematologic cancers. The Journal of clinical investigation 122, 2018‐2031.    66. Langer, C.J., Albert, I., Ross, H.J., Kovacs, P., Blakely, L.J., Pajkos, G., Somfay, A., Zatloukal,  P.,  Kazarnowicz,  A.,  Moezi,  M.M.,  2014.  Randomized  phase  II  study  of  carboplatin  and  etoposide  with  or  without  obatoclax  mesylate  in  extensive‐stage  small  cell  lung  cancer.  Lung Cancer 85, 420‐428.    67. Lee,  H.‐E.,  Choi,  E.‐S.,  Jung,  J.‐Y.,  You,  M.‐J.,  Kim,  L.‐H.,  Cho,  S.‐D.,  2014.  Inhibition  of  specificity protein 1 by dibenzylideneacetone, a curcumin analogue, induces apoptosis in  mucoepidermoid  carcinomas  and  tumor  xenografts  through  Bim  and  truncated  Bid.  Oral  oncology 50, 189‐195.    68. Lee, Y., Moujalled, D., Doerflinger, M., Gangoda, L., Weston, R., Rahimi, A., de Alboran, I.,  Herold, M., Bouillet, P., Xu, Q., 2013. CREB‐binding protein (CBP) regulates β‐adrenoceptor  (β‐AR)− mediated apoptosis. Cell Death & Differen a on 20, 941‐952.    69. Lei, K., Davis, R.J., 2003. JNK phosphorylation of Bim‐related members of the Bcl2 family  induces Bax‐dependent apoptosis. Proceedings of the National Academy of Sciences 100,  2432‐2437.    70. Lessene,  G.,  Czabotar,  P.E.,  Sleebs,  B.E.,  Zobel,  K.,  Lowes,  K.N.,  Adams,  J.M.,  Baell,  J.B.,  Colman, P.M., Deshayes, K., Fairbrother, W.J., 2013. Structure‐guided design of a selective  BCL‐XL inhibitor. Nature chemical biology 9, 390‐397.    24   

  71. Leverson, J., Zhang, H., Chen, J., Tahir, S., Phillips, D., Xue, J., Nimmer, P., Jin, S., Smith, M.,  Xiao,  Y.,  2015a.  Potent  and  selective  small‐molecule  MCL‐1  inhibitors  demonstrate  on‐ target  cancer  cell  killing  activity  as  single  agents  and  in  combination  with  ABT‐263  (navitoclax). Cell death & disease 6, e1590.    72. Leverson,  J.D.,  Phillips,  D.C.,  Mitten,  M.J.,  Boghaert,  E.R.,  Diaz,  D.,  Tahir,  S.K.,  Belmont,  L.D., Nimmer, P., Xiao, Y., Ma, X.M., 2015b. Exploiting selective BCL‐2 family inhibitors to  dissect  cell  survival  dependencies  and  define  improved  strategies  for  cancer  therapy.  Science translational medicine 7, 279ra40‐279ra40.    73. Ley, R., Ewings, K.E., Hadfield, K., Howes, E., Balmanno, K., Cook, S.J., 2004. Extracellular  signal‐regulated kinases  1/2 are serum‐stimulated “BimEL kinases” that bind to the BH3‐ only  protein  BimEL  causing  its  phosphorylation  and  turnover.  Journal  of  Biological  Chemistry 279, 8837‐8847.    74. Li, G.‐Q., Guo, W.‐Z., Zhang, Y., Seng, J.‐J., Zhang, H.‐P., Ma, X.‐X., Zhang, G., Li, J., Yan, B.,  Tang,  H.‐W.,  2016.  Suppression  of  BRD4  inhibits  human  hepatocellular  carcinoma  by  repressing MYC and enhancing BIM expression. Oncotarget 7, 2462.    75. Li,  J.,  Lee,  B.,  Lee,  A.S.,  2006.  Endoplasmic  reticulum  stress‐induced  apoptosis  multiple  pathways and activation of p53‐up‐regulated modulator of apoptosis (puma) and noxa by  p53. Journal of Biological Chemistry 281, 7260‐7270.    76. Li,  S.,  Li,  F.,  Niu,  R.,  Zhang,  H.,  Cui,  A.,  An,  W.,  Wang,  X.,  2015.  Mir‐192  suppresses  apoptosis and promotes proliferation in esophageal aquamous cell caicinoma by targeting  Bim. International journal of clinical and experimental pathology 8, 8048.    77. Liu,  H.,  Yin,  J.,  Wang,  C.,  Gu,  Y.,  Deng,  M.,  He,  Z.,  2014.  FOXO3a  mediates  the  cytotoxic  effects of cisplatin in lung cancer cells. Anti‐cancer drugs 25, 898‐907.    78. Liu,  J.‐W.,  Chandra,  D.,  Tang,  S.‐H.,  Chopra,  D.,  Tang,  D.G.,  2002.  Identification  and  characterization  of  Bimγ,  a  novel  proapoptotic  BH3‐only  splice  variant  of  Bim.  Cancer  research 62, 2976‐2981.  79. Liu, L., Chen, J., Zhang, J., Ji, C., Zhang, X., Gu, S., Xie, Y., Mao, Y., 2007. Overexpression of  BimSs3,  the  novel  isoform  of  Bim,  can  trigger  cell  apoptosis  by  inducing  cytochrome  c  release from mitochondria. ACTA BIOCHIMICA POLONICA‐ENGLISH EDITION‐ 54, 603.    80. LoRusso, P.M., Adjei, A.A., Varterasian, M., Gadgeel, S., Reid, J., Mitchell, D.Y., Hanson, L.,  DeLuca, P., Bruzek, L., Piens, J., 2005. Phase I and pharmacodynamic study of the oral MEK  inhibitor CI‐1040 in patients with advanced malignancies. Journal of clinical oncology  23,  5281‐5293.    81. Luo, J., Wang, X., Xia, Z., Yang, L., Ding, Z., Chen, S., Lai, B., Zhang, N., 2015. Transcriptional  factor  specificity  protein  1  (SP1)  promotes  the  proliferation  of  glioma  cells  by  up‐ regulating midkine (MDK). Molecular biology of the cell 26, 430‐439.    82. Ly,  L.L.,  Yoshida,  H.,  Yamaguchi,  M.,  2013.  Nuclear  transcription  factor  Y  and  its  roles  in  cellular processes related to human disease. American journal of cancer research 3, 339.    83. Maeda, T., Ito, K., Merghoub, T., Poliseno, L., Hobbs, R.M., Wang, G., Dong, L., Maeda, M.,  Dore, L.C., Zelent, A., 2009. LRF is an essential downstream target of GATA1 in erythroid  development and regulates BIM‐dependent apoptosis. Developmental cell 17, 527‐540.    25   

  84. Maeda, T., Merghoub, T., Hobbs, R.M., Dong, L., Maeda, M., Zakrzewski, J., van den Brink,  M.R.,  Zelent,  A.,  Shigematsu,  H.,  Akashi,  K.,  2007.  Regulation  of  B  versus  T  lymphoid  lineage fate decision by the proto‐oncogene LRF. Science 316, 860‐866.    85. Mahajan,  I.M.,  Chen,  M.‐D.,  Muro,  I.,  Robertson,  J.D.,  Wright,  C.W.,  Bratton,  S.B.,  2014.  BH3‐only protein BIM mediates heat shock‐induced apoptosis. PloS one 9, e84388.    86. Marani,  M.,  Tenev,  T.,  Hancock,  D.,  Downward,  J.,  Lemoine,  N.R.,  2002.  Identification  of  novel  isoforms  of  the  BH3  domain  protein  Bim  which  directly  activate  Bax  to  trigger  apoptosis. Molecular and cellular biology 22, 3577‐3589.    87. Merino,  D.,  Best,  S.,  Asselin‐Labat,  M.,  Vaillant,  F.,  Pal,  B.,  Dickins,  R.,  Anderson,  R.,  Strasser, A., Bouillet, P., Lindeman, G., 2014. Pro‐apoptotic Bim suppresses breast tumor  cell metastasis and is a target gene of SNAI2. Oncogene 34, 3926‐3934.      88. Molitoris, J.K., McColl, K.S., Distelhorst, C.W., 2011. Glucocorticoid‐mediated repression of  the  oncogenic  microRNA  cluster  miR‐17∼  92  contributes  to  the  induction  of  Bim  and  initiation of apoptosis. Molecular Endocrinology 25, 409‐420.    89. Moore,  V.D.G.,  Brown,  J.R.,  Certo,  M.,  Love,  T.M.,  Novina,  C.D.,  Letai,  A.,  2007.  Chronic  lymphocytic leukemia requires  BCL2 to  sequester  prodeath  BIM,  explaining  sensitivity  to  BCL2 antagonist ABT‐737. Journal of Clinical Investigation 117, 112‐121.    90. Moujalled, D., Weston, R., Anderton, H., Ninnis, R., Goel, P., Coley, A., Huang, D.C., Wu, L.,  Strasser,  A.,  Puthalakath,  H.,  2011.  Cyclic‐AMP‐dependent  protein  kinase  A  regulates  apoptosis by stabilizing the BH3‐only protein Bim. EMBO reports 12, 77‐83.    91. Moustafa‐Kamal, M., Gamache, I., Lu, Y., Li, S., Teodoro, J., 2013. BimEL is phosphorylated  at  mitosis  by  Aurora  A  and  targeted  for  degradation  by  βTrCP1.  Cell  Death  &  Differentiation 20, 1393‐1403.    92. Mühleisen,  A.,  Giaisi,  M.,  Köhler,  R.,  Krammer,  P.,  Li‐Weber,  M.,  2014.  Tax  contributes  apoptosis resistance to HTLV‐1‐infected T cells via suppression of Bid and Bim expression.  Cell death & disease 5, e1575.    93. Némati,  F.,  de  Montrion,  C.,  Lang,  G.,  Kraus‐Berthier,  L.,  Carita,  G.,  Sastre‐Garau,  X.,  Berniard, A., Vallerand, D., Geneste, O., de Plater, L., 2014. Targeting Bcl‐2/Bcl‐X L Induces  Antitumor Activity in Uveal Melanoma Patient‐Derived Xenografts. PloS one 9, e80836.    94. Nguyen, M., Marcellus, R.C., Roulston, A., Watson, M., Serfass, L., Madiraju, S.M., Goulet,  D., Viallet, J., Bélec, L., Billot, X., 2007a. Small molecule obatoclax (GX15‐070) antagonizes  MCL‐1  and  overcomes  MCL‐1‐mediated  resistance  to  apoptosis.  Proceedings  of  the  National Academy of Sciences 104, 19512‐19517.    95. Nguyen,  T.K.,  Rahmani,  M.,  Harada,  H.,  Dent,  P.,  Grant,  S.,  2007b.  MEK1/2  inhibitors  sensitize Bcr/Abl+ human leukemia cells to the dual Abl/Src inhibitor BMS‐354/825. Blood  109, 4006‐4015.    96. Nikrad,  M.,  Johnson,  T.,  Puthalalath,  H.,  Coultas,  L.,  Adams,  J.,  Kraft,  A.S.,  2005.  The  proteasome inhibitor bortezomib sensitizes cells to killing by death receptor ligand TRAIL  via BH3‐only proteins Bik and Bim. Molecular Cancer Therapeutics 4, 443‐449.    26   

  97. Niu, H., Wang, K., Zhang, A., Yang, S., Song, Z., Wang, W., Qian, C., Li, X., Zhu, Y., Wang, Y.,  2012. miR‐92a is a critical regulator of the apoptosis pathway in glioblastoma with inverse  expression of BCL2L11. Oncology reports 28, 1771‐1777.    98. Oltersdorf,  T.,  Elmore,  S.W.,  Shoemaker,  A.R.,  Armstrong,  R.C.,  Augeri,  D.J.,  Belli,  B.A.,  Bruncko,  M.,  Deckwerth,  T.L.,  Dinges,  J.,  Hajduk,  P.J.,  2005.  An  inhibitor  of  Bcl‐2  family  proteins induces regression of solid tumours. Nature 435, 677‐681.    99. Paschos, K., Parker, G.A., Watanatanasup, E., White, R.E., Allday, M.J., 2012. BIM promoter  directly  targeted  by  EBNA3C  in  polycomb‐mediated  repression  by  EBV.  Nucleic  acids  research 40, 7233‐7246.    100. Paschos,  K.,  Smith,  P.,  Anderton,  E.,  Middeldorp,  J.M.,  White,  R.E.,  Allday,  M.J.,  2009.  Epstein‐barr virus latency in B cells leads to epigenetic repression and CpG methylation  of the tumour suppressor gene Bim. PLoS Pathog 5, e1000492.    101. Patel,  A.J.,  Liao,  C.‐P.,  Chen,  Z.,  Liu,  C.,  Wang,  Y.,  Le,  L.Q.,  2014.  BET  bromodomain  inhibition triggers apoptosis of NF1‐associated malignant peripheral nerve sheath tumors  through Bim induction. Cell reports 6, 81‐92.    102. Pei,  X.‐Y.,  Dai,  Y.,  Tenorio,  S.,  Lu,  J.,  Harada,  H.,  Dent,  P.,  Grant,  S.,  2007.  MEK1/2  inhibitors potentiate UCN‐01 lethality  in human multiple myeloma cells through  a Bim‐ dependent mechanism. Blood 110, 2092‐2101.    103. Pernaute,  B.,  Spruce,  T.,  Smith,  K.M.,  Sánchez‐Nieto,  J.M.,  Manzanares,  M.,  Cobb,  B.,  Rodríguez, T.A., 2014. MicroRNAs control the apoptotic threshold in primed pluripotent  stem cells through regulation of BIM. Genes & development 28, 1873‐1878.    104. Petrocca,  F.,  Visone,  R.,  Onelli,  M.R.,  Shah,  M.H.,  Nicoloso,  M.S.,  de  Martino,  I.,  Iliopoulos, D., Pilozzi, E., Liu, C.‐G., Negrini, M., 2008. E2F1‐regulated microRNAs impair  TGFβ‐dependent cell‐cycle arrest and apoptosis in gastric cancer. Cancer cell 13, 272‐286.    105. Plötz,  M.,  Eberle,  J.,  2014.  BH3‐only  proteins–possible  proapoptotic  triggers  for  melanoma therapy. Experimental dermatology 23, 375‐378.    106. Potluri,  V.,  Noothi,  S.K.,  Vallabhapurapu,  S.D.,  Yoon,  S.‐O.,  Driscoll,  J.J.,  Lawrie,  C.H.,  Vallabhapurapu, S., 2013. Transcriptional repression of Bim by a novel YY1‐relA complex  is essential for the survival and growth of multiple myeloma. PloS one 8, e66121.    107. Puthalakath,  H.,  O'Reilly,  L.A.,  Gunn,  P.,  Lee,  L.,  Kelly,  P.N.,  Huntington,  N.D.,  Hughes,  P.D.,  Michalak,  E.M.,  McKimm‐Breschkin,  J.,  Motoyama,  N.,  2007.  ER  stress  triggers  apoptosis by activating BH3‐only protein Bim. Cell 129, 1337‐1349.    108. Qi, X.‐J., Wildey, G.M., Howe, P.H., 2006. Evidence that Ser87 of BimEL is phosphorylated  by Akt and regulates BimEL apoptotic function. Journal of Biological Chemistry 281, 813‐ 823.    109. Ready,  N.,  Karaseva,  N.A.,  Orlov,  S.V.,  Luft,  A.V.,  Popovych,  O.,  Holmlund,  J.T.,  Wood,  B.A.,  Leopold,  L.,  2011.  Double‐blind,  placebo‐controlled,  randomized  phase  2  study  of  the proapoptotic agent AT‐101 plus docetaxel, in second‐line non‐small cell lung cancer.  Journal of Thoracic Oncology 6, 781‐785.    27   

  110. Reimertz,  C.,  Kögel,  D.,  Rami,  A.,  Chittenden,  T.,  Prehn,  J.H.,  2003.  Gene  expression  during  ER  stress–induced  apoptosis  in  neurons  induction  of  the  BH3‐only  protein  Bbc3/PUMA and activation of the mitochondrial apoptosis pathway. The Journal of cell  biology 162, 587‐597.    111. Ridinger‐Saison,  M.,  Evanno,  E.,  Gallais,  I.,  Rimmelé,  P.,  Selimoglu‐Buet,  D.,  Sapharikas,  E.,  Moreau‐Gachelin,  F.,  Guillouf,  C.,  2013.  Epigenetic  silencing  of  Bim  transcription  by  Spi‐1/PU. 1 promotes apoptosis resistance in leukaemia. Cell Death & Differentiation 20,  1268‐1278.    112. Roberts,  A.W., Seymour,  J.F.,  Brown,  J.R., Wierda,  W.G.,  Kipps,  T.J.,  Khaw,  S.L., Carney,  D.A.,  He,  S.Z.,  Huang,  D.C.,  Xiong,  H.,  2012.  Substantial  susceptibility  of  chronic  lymphocytic  leukemia  to  BCL2  inhibition:  results  of  a  phase  I  study  of  navitoclax  in  patients with relapsed or refractory disease. Journal of Clinical Oncology 30, 488‐496.    113. Salmanidis, M., Brumatti, G., Narayan, N., Green, B., van den Bergen, J., Sandow, J., Bert,  A., Silke, N., Sladic, R., Puthalakath, H., 2013. Hoxb8 regulates expression of microRNAs  to control cell death and differentiation. Cell Death & Differentiation 20, 1370‐1380.    114. Sheridan,  C.,  Brumatti,  G.,  Martin,  S.J.,  2008.  Oncogenic  B‐RafV600E  inhibits  apoptosis  and  promotes  ERK‐dependent  inactivation  of  Bad  and  Bim.  Journal  of  Biological  Chemistry 283, 22128‐22135.    115. Shin,  E.,  Huang,  Q.,  Gurel,  Z.,  Palenski,  T.,  Zaitoun,  I.,  Sorenson,  C.,  Sheibani,  N.,  2014.  STAT1‐mediated Bim expression promotes the apoptosis of retinal pericytes under high  glucose conditions. Cell death & disease 5, e986.    116. Shukla, S., Rizvi, F., Raisuddin, S., Kakkar, P., 2014. FoxO proteins’ nuclear retention and  BH3‐only protein Bim induction evoke mitochondrial dysfunction‐mediated apoptosis in  berberine‐treated HepG2 cells. Free Radical Biology and Medicine 76, 185‐199.    117. Sleebs,  B.E.,  Kersten,  W.J.,  Kulasegaram,  S.,  Nikolakopoulos,  G.,  Hatzis,  E.,  Moss,  R.M.,  Parisot,  J.P.,  Yang,  H.,  Czabotar,  P.E.,  Fairlie,  W.D.,  2013.  Discovery  of  potent  and  selective benzothiazole hydrazone inhibitors of Bcl‐XL. Journal of medicinal chemistry 56,  5514‐5540.    118. Solit, D.B., Garraway, L.A., Pratilas, C.A., Sawai, A., Getz, G., Basso, A., Ye, Q., Lobo, J.M.,  She,  Y.,  Osman,  I.,  2006.  BRAF  mutation  predicts  sensitivity  to  MEK  inhibition.  Nature  439, 358‐362.    119. Song, T., Chai, G., Liu, Y., Xie, M., Chen, Q., Yu, X., Sheng, H., Zhang, Z., 2015. Mechanism  of  synergy  of  BH3  mimetics  and  paclitaxel  in  chronic  myeloid  leukemia  cells:  Mcl‐1  inhibition. European Journal of Pharmaceutical Sciences 70, 64‐71.    120. Sonpavde, G., Matveev, V., Burke, J., Caton, J., Fleming, M., Hutson, T., Galsky, M., Berry,  W., Karlov, P., Holmlund, J., 2012. Randomized phase II trial of docetaxel plus prednisone  in combination with placebo or AT‐101, an oral small molecule Bcl‐2 family antagonist, as  first‐line therapy for metastatic castration‐resistant prostate cancer. Annals of Oncology  23, 1803‐1808.    121. Souers,  A.J.,  Leverson,  J.D.,  Boghaert,  E.R.,  Ackler,  S.L.,  Catron,  N.D.,  Chen,  J.,  Dayton,  B.D.,  Ding,  H.,  Enschede,  S.H.,  Fairbrother,  W.J.,  2013.  ABT‐199,  a  potent  and  selective  28   

  BCL‐2 inhibitor, achieves antitumor activity while sparing platelets. Nature medicine 19,  202‐208.    122. Sunters,  A.,  de  Mattos,  S.F.,  Stahl,  M.,  Brosens,  J.J.,  Zoumpoulidou,  G.,  Saunders,  C.A.,  Coffer,  P.J.,  Medema,  R.H.,  Coombes,  R.C.,  Lam,  E.W.‐F.,  2003.  FoxO3a  transcriptional  regulation of Bim controls apoptosis in paclitaxel‐treated breast cancer cell lines. Journal  of Biological Chemistry 278, 49795‐49805.    123.

  124.

  125.

  126.

  127.

  128.

  129.

  130.

  131.

  132.

  133.

T  Sankpal,  U.,  Maliakal,  P.,  Bose,  D.,  Kayaleh,  O.,  Buchholz,  D.,  Basha,  R.,  2012.  Expression  of  specificity  protein  transcription  factors  in  pancreatic  cancer  and  their  association in prognosis and therapy. Current medicinal chemistry 19, 3779‐3786.  Tan, J., Zhuang, L., Jiang, X., Yang, K.K., Karuturi, K.M., Yu, Q., 2006. Apoptosis signal‐ regulating  kinase  1  is  a  direct  target  of  E2F1  and  contributes  to  histone  deacetylase  inhibitorinduced  apoptosis  through  positive  feedback  regulation  of  E2F1  apoptotic  activity. Journal of Biological Chemistry 281, 10508‐10515.  Tao,  Z.‐F.,  Hasvold,  L.,  Wang,  L.,  Wang,  X.,  Petros,  A.M.,  Park,  C.H.,  Boghaert,  E.R.,  Catron, N.D., Chen, J., Colman, P.M., 2014. Discovery of a potent and selective BCL‐XL  inhibitor with in vivo activity. ACS medicinal chemistry letters 5, 1088‐1093.  Trudel, S., Li, Z.H., Rauw, J., Tiedemann, R.E., Wen, X.Y., Stewart, A.K., 2007. Preclinical  studies of the pan‐Bcl inhibitor obatoclax (GX015‐070) in multiple myeloma. Blood 109,  5430‐5438.  Tse,  C.,  Shoemaker,  A.R.,  Adickes,  J.,  Anderson,  M.G.,  Chen,  J.,  Jin,  S.,  Johnson,  E.F.,  Marsh, K.C., Mitten, M.J., Nimmer, P., 2008. ABT‐263: a potent and orally bioavailable  Bcl‐2 family inhibitor. Cancer research 68, 3421‐3428.  Um, H., 2015. Bcl‐2 family proteins as regulators of cancer cell invasion and metastasis:  a review focusing on mitochondrial respiration and reactive oxygen species. Oncotarget  7, 5193‐5203.  Ventura,  A.,  Young,  A.G.,  Winslow,  M.M.,  Lintault,  L.,  Meissner,  A.,  Erkeland,  S.J.,  Newman,  J.,  Bronson,  R.T.,  Crowley,  D.,  Stone,  J.R.,  2008.  Targeted  deletion  reveals  essential  and  overlapping  functions  of  the  miR‐17∼  92  family  of  miRNA  clusters.  Cell  132, 875‐886.  Wan, L., Tan, M., Yang, J., Inuzuka, H., Dai, X., Wu, T., Liu, J., Shaik, S., Chen, G., Deng, J.,  2014.  APC  Cdc20  Suppresses  Apoptosis  through  Targeting  Bim  for  Ubiquitination  and  Destruction. Developmental cell 29, 377‐391.  Wang, E., Kawaoka, S., Roe, J.‐S., Shi, J., Hohmann, A.F., Xu, Y., Bhagwat, A.S., Suzuki,  Y.,  Kinney,  J.B.,  Vakoc,  C.R.,  2015.  The  transcriptional  cofactor  TRIM33  prevents  apoptosis  in  B  lymphoblastic  leukemia  by  deactivating  a  single  enhancer.  eLife  4,  e06377.  Wang, S., Tang, C., Zhang, Q., Chen, W., 2014. Reduced miR‐9 and miR‐181a expression  down‐regulates  Bim  concentration  and  promote  osteoclasts  survival.  International  journal of clinical and experimental pathology 7, 2209.  Wang,  Y.F.,  Jiang,  C.C.,  Kiejda,  K.A.,  Gillespie,  S.,  Zhang,  X.D.,  Hersey,  P.,  2007.  Apoptosis  induction  in  human  melanoma  cells  by  inhibition  of  MEK  is  caspase‐ 29 

 

  independent and mediated by the Bcl‐2 family members PUMA, Bim, and Mcl‐1. Clinical  cancer research 13, 4934‐4942.    134.

  135.

  136.

  137.

  138.

  139.

  140.

  141.

  142.

  143.

  144.

 

Whelan,  K.A.,  Caldwell,  S.A.,  Shahriari,  K.S.,  Jackson,  S.R.,  Franchetti,  L.D.,  Johannes,  G.J.,  Reginato,  M.J.,  2010.  Hypoxia  suppression  of  Bim  and  Bmf  blocks  anoikis  and  luminal  clearing  during  mammary  morphogenesis.  Molecular  biology  of  the  cell  21,  3829‐3837.  Whelan, K.A., Schwab, L.P., Karakashev, S.V., Franchetti, L., Johannes, G.J., Seagroves,  T.N.,  Reginato,  M.J.,  2013.  The  oncogene  HER2/neu  (ERBB2)  requires  the  hypoxia‐ inducible  factor  HIF‐1  for  mammary  tumor  growth  and  anoikis  resistance.  Journal  of  Biological Chemistry 288, 15865‐15877.  Whitfield, J., Neame, S.J., Paquet, L., Bernard, O., Ham, J., 2001. Dominant‐negative c‐ Jun  promotes  neuronal  survival  by  reducing  BIM  expression  and  inhibiting  mitochondrial cytochrome c release. Neuron 29, 629‐643.  Wickenden,  J.,  Jin,  H.,  Johnson,  M.,  Gillings,  A.,  Newson,  C.,  Austin,  M.,  Chell,  S.,  Balmanno, K., Pritchard, C., Cook, S., 2008. Colorectal cancer cells with the BRAFV600E  mutation  are addicted to  the ERK1/2  pathway  for growth factor‐independent survival  and repression of BIM. Oncogene 27, 7150‐7161.  Wiggins,  C.M.,  Tsvetkov,  P.,  Johnson,  M.,  Joyce,  C.L.,  Lamb,  C.A.,  Bryant,  N.J.,  Komander, D., Shaul, Y., Cook, S.J., 2011. BIMEL, an intrinsically disordered protein, is  degraded  by  20S  proteasomes  in  the  absence  of  poly‐ubiquitylation.  Journal  of  cell  science 124, 969‐977.  Wildey, G.M., Patil, S., Howe, P.H., 2003. Smad3 potentiates transforming growth factor  β (TGFβ)‐induced apoptosis and expression of the BH3‐only protein Bim in WEHI 231 B  lymphocytes. Journal of Biological Chemistry 278, 18069‐18077.  Wilson, W.H., O'Connor, O.A., Czuczman, M.S., LaCasce, A.S., Gerecitano, J.F., Leonard,  J.P., Tulpule, A., Dunleavy, K., Xiong,  H., Chiu, Y.‐L., 2010. Navitoclax, a targeted high‐ affinity inhibitor of BCL‐2, in lymphoid malignancies: a phase 1 dose‐escalation study of  safety,  pharmacokinetics,  pharmacodynamics,  and  antitumour  activity.  The  lancet  oncology 11, 1149‐1159.  Xiao,  C.,  Srinivasan,  L.,  Calado,  D.P.,  Patterson,  H.C.,  Zhang,  B.,  Wang,  J.,  Henderson,  J.M., Kutok, J.L., Rajewsky, K., 2008. Lymphoproliferative disease and autoimmunity in  mice with increased miR‐17‐92 expression in lymphocytes. Nature immunology 9, 405‐ 414.  Xie, L., Johnson, R.S., Freeman, R.S., 2005. Inhibition of NGF deprivation–induced death  by low oxygen involves suppression of BIMEL and activation of HIF‐1. The Journal of cell  biology 168, 911‐920.  Xu,  Z.,  Sharp,  P.,  Yao,  Y.,  Segal,  D.,  Ang,  C.,  Khaw,  S.,  Aubrey,  B.,  Gong,  J.,  Kelly,  G.,  Herold, M., 2016. BET inhibition represses miR17‐92 to drive BIM‐initiated apoptosis of  normal and transformed hematopoietic cells. Leukemia 30, 1531‐1541.  Yamagata,  K.,  Daitoku, H.,  Takahashi,  Y.,  Namiki, K.,  Hisatake,  K., Kako,  K.,  Mukai,  H.,         Kasuya,  Y.,  Fukamizu,  A.,  2008.  Arginine  methylation  of  FOXO  transcription  factors  inhibits their phosphorylation by Akt. Molecular cell 32, 221‐231.  30 

        145. Yamamura,  Y.,  Lee,  W.L.,  Inoue,  K.‐i.,  Ida,  H.,  Ito,  Y.,  2006.  RUNX3  cooperates  with  FoxO3a to induce apoptosis in gastric cancer cells. Journal of Biological Chemistry 281,  5267‐5276.    146. Zantl,  N.,  Weirich,  G.,  Zall,  H.,  Seiffert,  B.,  Fischer,  S.,  Kirschnek,  S.,  Hartmann,  C.,  Fritsch,  R.,  Gillissen,  B.,  Daniel,  P.,  2007.  Frequent  loss  of  expression  of  the  pro‐ apoptotic  protein  Bim  in  renal  cell  carcinoma:  evidence  for  contribution  to  apoptosis  resistance. Oncogene 26, 7038‐7048.    147. Zhang, H., Zuo, Z., Lu, X., Wang, L., Wang, H., Zhu, Z., 2012. MiR‐25 regulates apoptosis  by targeting Bim in human ovarian cancer. Oncology reports 27, 594‐598.    148. Zhang,  L.,  Insel,  P.A.,  2004.  The  pro‐apoptotic  protein  Bim  is  a  convergence  point  for  cAMP/protein  kinase  A‐and  glucocorticoid‐promoted  apoptosis  of  lymphoid  cells.  Journal of Biological Chemistry 279, 20858‐20865.    149. Zhu,  Y.,  Du,  Y.,  Liu,  H.,  Ma,  T.,  Shen,  Y.,  Pan,  Y.,  2016.  Study  of  efficacy  and  safety  of  pulsatile  administration  of high‐dose gefitinib or  erlotinib  for  advanced  non‐small cell  lung cancer patients with secondary drug resistance: A single center, single arm, phase  II clinical trial. Thoracic Cancer 7, 663‐669. 

Figure legends Fig.1(A) The Bcl-2 protein family; Members of Bcl-2 family can be divided into three broad groups depending upon presence of different BH domains and nature of function performed. The BH3-only proteins, potent inducers of apoptosis, contain only BH3 domain and comprise family members BIM, PUMA, NOXA, BMF, tBID, HRK, BAD, BIK. The pro-survival (Anti-apoptotic) BCL2-like proteins have all four Bcl-2 homology domains (BH1-4) and comprise family members BCL2, BCL-XL, BCL-W, MCL1 and A1. Finally, the multidomain pro-apoptotic proteins BAX, BAK, and possibly BOK, all of which contain four BH domains. Most of the BCL2 family members also have a transmembrane domain (TM), which enables them to anchor to the mitochondria.

Fig.1(B) Pre-dominant Bim isoforms that are subject to post-translational modifications. The domain structure of some of main Bim isoforms (BimEL, BimL and BimS) arising from alternate splicing of Bim is shown. The numbers in brackets represent the number of constituent amino acids according to the human sequence. BimELpossesses ERK1/2 phosphorylation sites whereas BimEL and BimLboth splice variants have JNK

31   

  phosphorylation sites. BimS possesses only BH3 domain and devoid of ERK1/2 and JNK phosphorylation domain. Fig.2 Induction of apoptosis by BH3-only protein Bim. Following exposure to a pro-apoptotic stimulus (chemotherapeutic agent, growth factor withdrawal etc.) intracellular level of free Bim is increased, which might be due to increased transcription and/or translation, increased alternative splicing, and/or release of Bim from sequestered intracellular complexes) initiates the intrinsic pathway of mitochondria induced apoptosis. Bim induces apoptosis either by direct activation of pro-apoptotic Bax/Bak or by neutralizing different anti-apoptotic Bcl-2 proteins via their BH3 domain. Activated Bax gets inserted into mitochondrial outer membrane (MOM), oligomerizes into pores with Bax/Bak, permeabilizes the outer membrane and facilitate the release of inter membrane space proteins i.e. cytochrome c (cyt c) into the cytoplasm. In cytoplasm, cyt c complexes with apoptotic protease-activating factor 1 (Apaf-1) and forms apoptosome, which sequentially recruits and activates the initiator caspase-9 and executioner caspase 3 culminating into cell death. Fig.3 Regulation of Bim expression by a range of transcription factors. Bim expression is tightly regulated by plethora of transcription factors, whose activities itself controlled precisely. Transcription factors, which upregulate Bim expression (lower green region in figure 3) include Runx2/3, FoxO3a, SNAI2, NF-Y/CBP/P300 complex, E2F1, c-JUN, Smad3, CHOP-C/EBPα and STAT-1, are cell/tissue type specific and their potential to upregulate Bim, depend upon type of stimulus. Proteins which negatively regulate Bim expression (upper region in figure 3) include Spi-1/PU.1, Trim33, Pokemon, HIF-1α, Sp1, Brd4, RelA/YY-1 complex, PINCH-1 and EBNA3A/3C, thus providing a growth advantage and evasion from apoptosis to the tumor cells. Fig.4 Regulation of Bim stability by mitogen-activated protein kinase ERK1/2. The pro-apoptotic Bim is overexpressed following a number of toxic insults and binds to anti-apoptotic proteins of Bcl-2 family thereby discharging Bax or Bak to induce intrinsic mode of apoptosis. Bim is directly phosphorylated by ERK1/2, a survival kinase of MAPK family, on three different phosphorylation sites that inhibits binding of Bim with antiapoptotic proteins of Bcl-2 family. ERK1/2-mediated phosphorylation also potentiates Bim for phosphorylation by RSK1/2 providing a binding site for the F box protein βTrCP. βTrCP1/2 promotes poly-ubiquitination of Bim and targeting 26S proteasome mediated Bim degradation via multi-protein E3 ubiquitin ligase complex.

32   

 

33   

 

34   

 

35   

 

36