Regulation of Bim in Health and Disease

The BH3-only Bim protein is a major determinant for initiating the intrinsic apoptotic pathway under both physiological and pathophysiological conditions. Tight regulation of its expression and activity at the transcriptional, translational and post-translational levels together with the induction of alternatively spliced isoforms with different pro-apoptotic potential, ensure timely activation of Bim. Under physiological conditions, Bim is essential for shaping immune responses where its absence promotes autoimmunity, while too early Bim induction eliminates cytotoxic T cells prematurely, resulting in chronic inflammation and tumor progression. Enhanced Bim induction in neurons causes neurodegenerative disorders including Alzheimer's, Parkinson's and Huntington's diseases. Moreover, type I diabetes is promoted by genetically predisposed elevation of Bim in β-cells. On the contrary, cancer cells have developed mechanisms that suppress Bim expression necessary for tumor progression and metastasis. This review focuses on the intricate network regulating Bim activity and its involvement in physiological and pathophysiological processes.

1.1. General Aspects of Bim The BH3-only proteins participate in vital biological processes, and their absence contributes to autoimmunity and neoplasia [ 1 , 2 ]. Bim is a Bcl-2 homology 3 (BH3)-only protein that was discovered by O'Connor et al. [ 3 ] in 1998, while screening for proteins binding the anti-apoptotic Bcl-2 protein, giving raise to its name B cl-2 i nteracting m ediator of cell death. In the same year, Hsu et al. [ 4 ] discovered the same gene using Mcl-1 as a bait and termed the gene Bcl-2 related ovarian death agonist (BOD). Its official gene name is now Bcl-2-like 11 (Bcl-2L11/apoptosis facilitator). The Bim gene is conserved in diverse mammalian species [ 4 ]. Bim function seems also to be conserved in non-mammalian vertebrates. A zebrafish ortholog of mammalian Bim was found to be the most toxic product of the zebrafish BH3-only genes examined, sharing this characteristic with the mammalian Bim gene [ 5 ]. Bim proteins are expressed in a wide variety of tissues including brain, heart, kidney, liver, lung, ovary, testis, spleen, thymus and trachea, but are most prominently expressed by cells of hematopoietic origin [ 6 ]. In the yeast two-hybrid protein-protein interaction assay, Bim interacts with the anti-apoptotic Bcl-2 proteins Mcl-1, Bcl-2, Bcl-xL, Bcl-w, Bfl-1 and Epstein-Barr virus (EBV) BHRF-1, but not with the pro-apoptotic Bcl-2 proteins Bad, Bak, Bok and Bax [ 4 ]. In mammalian cells, Bim can engage all anti-apoptotic proteins of the Bcl-2 superfamily, making it an efficient killer [ 1 ]. The BH3 domain becomes inserted into a hydrophobic groove of the pro-survival relatives [ 1 ]. The hydrophobic C-terminal part of Bim (MVILRLLRYIVRLVW) targets the protein to intracellular membranes [ 3 ]. In addition, Bim can directly interact with Bax and Bak, leading to mitochondrial outer membrane permeabilization [ 7 - 11 ]. Genetic analyses by Merino et al. [ 11 ] showed that only the Bim BH3 domain, but not other BH3 sequences inserted into the Bim protein, could rescue the leukocyte accumulation and autoimmune phenotype of Bim knockout (KO) mice, emphasizing the unique killing potency of Bim BH3. Thus, Bim may promote apoptosis by both acting as a death agonist and survival antagonist [ 8 ]. Other pro-apoptotic members of the BH3-only family include Bad, Bik, Bmf, Bid, Hrk, Noxa and Puma [ 12 ]. Bim often acts in concert with these pro-apoptotic proteins, which may explain why apoptosis is only partly compromised in Bim KO mice, as seen for glucocorticoid-induced apoptosis of thymocytes [ 2 ]. In contrast to normal cells, apoptosis of cancer cells often shows a complete dependence on Bim [ 13 , 14 ]. The BH3-only pro-apoptotic proteins act upstream of the multi-domain pro-apoptotic proteins Bak, Bax and Bok, which have three BH domains (BH1, BH2 and BH3). Double knockout of Bax and Bak often leads to complete resistance to apoptosis mediated by the intrinsic apoptotic pathway [ 15 ]. The multiple defects in Bcl-2 −/− mice can be prevented by loss of Bim, including the severe lymphopenia [ 16 ]. Even the loss of only one Bim allele was sufficient for the correction of the disorders, suggesting that the Bim levels set the threshold for initiation of apoptosis in several tissues [ 16 ].

Ng et al. [ 31 ] observed that a common intronic 2903bp-deletion polymorphism within intron 2 in the Bim gene switched Bim splicing from exon 4 to exon 3 leading to the preferential expression of Bimγ. This deletion polymorphism occurs naturally in 12.9% of East-Asian individuals and is observed in some individuals with chronic myeloid leukemia (CML) or epidermal growth factor receptor (EGFR)-mutated non-small-cell lung cancer (NSCLC) that conferred resistance to tyrosine kinase inhibitors such as gefitinib [ 31 , 32 ]. Of note, the resistance could be overcome by the BH3-mimetic drug ABT-737 [ 31 ] or by combining gefitinib with the histone deacetylase inhibitor vorinostat that increased the splicing to exon 4, leading to augmented expression of the pro-apoptotic BH3-containing Bim [ 32 ]. The intronic deletion polymorphism may explain the heterogeneic response of cancer patients to tyrosine kinase inhibitors [ 31 ]. The progression free survival following EGFR tyrosine kinase inhibitor treatment of NSCLC was significantly shorter in patients with Bim polymorphism (6.6 months) than those with wild-type Bim (11.9 months) [ 31 ]. However, others couldn't find an association of Bim deletion polymorphism and intrinsic resistance to tyrosine kinase inhibitors [ 33 ]. Another study showed that acute lymphoblastic leukemia patients harboring the single nucleotide BimC29201T (rs724710) polymorphism in exon 4 had shorter overall survival [ 34 ]. Overall survival was even shorter in patients with both Bim polymorphism and Mcl-1 gene polymorphism (G486T) [ 34 ]. The single nucleotide polymorphism C>T in Bim affected the inclusion of exon 3 and seems to contribute to drug resistance [ 34 ].

2.1. Indirect and Direct Apoptosis Induction by Bim Bim has been implicated in the regulation of intrinsic cell death induced by a large number of stimuli, including growth factor or cytokine deprivation, calcium flux, ligation of antigen receptors on T and B cells, loss of adhesion (anoikis), glucocorticoids, microtubule perturbation and tyrosine kinase inhibitors (Tables 1 , 2 , 3 , 4 , 5 , 6 , 7 ). It has been shown to be critical for apoptosis in B and T lymphocytes, macrophages and granulocytes [ 53 ]. The pioneer studies by O'Connor et al. [ 3 ] and Hsu et al. [ 4 ], showed that overexpression of Bim in Chinese hamster ovary (CHO) cells or 293T human embryonic kidney cells led to apoptosis that could be prevented by the baculoviral caspase inhibitor P35, and the anti-apoptotic Bcl-2, Bcl-xL and Bcl-w proteins. However, the more distant viral homologues adenovirus E1B19K and Epstein-Barr virus BHRF-1 were unable to prevent the pro-apoptotic effect of Bim [ 3 ]. The BH3 domain is essential for its pro-apoptotic function [ 3 , 4 ]. A mutant Bim protein lacking the BH3 domain did not interact with Bcl-2, Bcl-xL or Bcl-w [ 3 ]. Although all three Bim isoforms in mouse bind Bcl-2, Bim S antagonizes Bcl-2 more effectively than Bim L , while Bim EL was the least potent [ 3 ]. Besides neutralizing the anti-apoptotic proteins, Bim promotes apoptosis by binding to Bax leading to a conformation change in Bax that leads to its activation [ 8 , 9 ] (Figure 2 ). Table 1 Bim Function in The Immune System Apoptotic Stimulus Remarks References Negative selection of self-reactive thymocytes • Thymocytes lacking Bim are refractory to apoptosis induced by TCR-CD3 stimulation. • Bim is required for apoptosis of CD4 + CD8 + thymocytes induced by high-affinity antigens. • Bim is involved in clonal deletion of thymocytes recognizing tissue-restricted antigens (TRAs), but not superantigen-mediated apoptosis. • Autoreactive NODBim −/− thymocytes receiving strong TCR signals that would normally delete them, escape apoptosis and are diverted into T reg cells. • Bim is essential for deletion of CD4 + CD8 − CD24 − thymocytes in response to TCR ligation. [ 40 , 46 , 401 , 402 , 404 - 406 , 416 ] Activated T cell death • Bim is a key regulator of T cell apoptosis during the contraction phase of CD8 + T cell response. • Vβ8 + T cells from Bim-deficient mice are resistant to staphylococcus enterotoxin B-induced T cell death. • While the Bim levels did not change after exposure to staphylococcal enterotoxin B, the Bcl-2 levels decreased. • Shutdown of an acute T cell response to herpes simplex virus involved Bim. • Bim deficiency increases antigen-specific CD8 + T cell responses during viral infection. [ 47 , 395 , 398 , 399 ] B7-H1 (PD-L1)-induced apoptosis of effector T cells • B7-H1 (PD-L1) engagement with its receptor PD-1 promotes apoptosis of effector T cells through upregulation of Bim. • More memory CD8 + T cells were generated in B7-H1-deficient mice following immunization. [ 409 ] Elimination of poorly functional Th1 responder cells • Rescued Bim −/− CD4 + memory T cells showed deficient effector functions, poor sensitivity to antigen and an inability to respond to secondary challenge. • Bim mediates T cell death in the absence of appropriate TCR-driven activation and differentiation. • Bim shapes the CD4 + memory T cell repertoire by eliminating Th1 effector cells with suboptimal function, thereby ensuring the emergence of highly functional CD4 + memory cells. [ 408 ] Regulation of T memory cells • The absence of Bim increased the effector CD8 + T cell population with more memory potential. • Survival of memory T cells depends on TRAF1-mediated Bim downregulation. • The absence of Bim-mediated death of lymphocytic choriomeningitis virus-specific CD4 + and CD8 + T cells in vivo leads to increased differentiation, even of CD127 lo T cells, into memory T cells. [ 411 , 422 , 471 ] Regulation of T regulatory cells • In the absence of Bim, T regulatory cells accumulate rapidly, accounting for >25% of the CD4 + T cells in aged mice. • Rapid peripheral T regulatory cell turnover depends on Bim. • Induced regulatory T cells show decreased Bcl-2 expression and increased Bim expression and were more prone to apoptosis. • Rag −/− hosts repopulated with Bim −/− conventional CD4 + T cells resulted in a larger induction of regulatory T cells than mice given wild-type conventional CD4 + cells. • Bim deficient mice showed increased numbers of CD25 low Foxp3 + cells in the thymus and peripheral lymph tissue. The CD25 low Foxp3 + CD4 + cells were anergic and had weaker regulatory function than CD25 high Foxp3 + CD4 + T cells from the same mice. [ 37 - 39 , 394 , 413 ] B cell antigen receptor (BCR) stimulation-induced apoptosis • B lymphocytes lacking Bim are refractory to apoptosis induced by BCR ligation. • The loss of Bim also inhibited deletion of autoreactive B cells in vivo in a B cell tolerance model. • Siglecs induce tolerance to cel

Han et al. [ 95 ] described an intriguing interplay between Mcl-1 and Bim in Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. In the absence of pro-apoptotic stimuli, Bim is sequestrated to Mcl-1 in tumor cells. Upon stimulation with TRAIL, caspase 8 is activated and promotes degradation of Mcl-1, resulting in the release of Bim that triggers Bax-dependent apoptosis. The Mcl-1 expression level at the mitochondrial outer membrane determines the release efficiency for the apoptogenic proteins cytochrome C, Second mitochondria-derived activator of caspase (Smac), and High temperature requirement serine protease A2 (HtrA2) in response to Bim [ 95 ]. Earlier studies by Herrant et al. [ 96 ] showed that Mcl-1 is cleaved by caspases during the induction of apoptosis in various cancer cells. The Mcl-1 cleavage results in the loss of the BH4 homolgy domain required for its anti-apoptotic activity [ 96 ]. Similarly, Mcl-1 could be degraded by the T cell granule serine protease granzyme B, again releasing Bim that promotes apoptosis [ 97 ]. Mcl-1 is subjected to tight regulation at the level of protein stability. The degradation of Mcl-1 is often, but not always, required for initiation of apoptosis [ 64 , 98 - 100 ]. The mRNA level of Mcl-1 decreases in response to various apoptotic stimuli such as UV irradiation and staurosporine [ 98 ]. Mcl-1 is a short-lived protein, positively regulated by the mTOR signaling pathway, while negatively by the E3 ligases Mcl-1 ubiquitin ligase E3 (Mule), F-Box and WD repeat domain containing 7 (Fbw7), Tripartite motif containing 17 (Trim17) and β-transducin repeat containing E3 ubiquitin protein ligase 1 (β-TrCP1), that promote its degradation [ 101 - 106 ]. Phosphorylation of Mcl-1 by Glycogen synthase kinase 3 (GSK3) triggers the interaction of Mcl-1 with Fbw7 [ 106 ]. The microtubule-targeting agents Paclitaxel and Vinblastine induce phosphorylation-dependent, Fbw7-mediated degradation of Mcl-1 [ 107 ]. The mTOR complex 2 (mTORC2) stabilizes Mcl-1 by suppressing the GSK3-dependent and Fbw7-mediated degradation [ 108 ]. It is noteworthy that GSK3 is activated upon glucocorticoid-induced apoptosis, leading to interaction of GSK3 with Bim and induction of Bim-dependent apoptosis of lymphoma cells [ 63 ]. Thus, GSK3 has a dual function, the first is to reduce Mcl-1 levels, and the other to enhance Bim activity, thereby fortifying apoptosis. The VTLISFG in the BH1 domain of Mcl-1 seems to be important for regulating its degradation [ 109 ]. The deubiquitinase Ubiquitin-Specific Protease 9X (USP9X) stabilizes Mcl-1, thereby promotes cell survival [ 110 ]. Increased USP9X expression correlates with increased Mcl-1 protein in human follicular lymphomas and diffuse large B cell lymphomas [ 110 ]. Moreover, patients with multiple myeloma overexpressing USP9X have a poor prognosis [ 110 ]. Importantly, targeting USP9X attenuates B cell acute lymphoblastic leukemia cell survival and overcomes glucocorticoid resistance [ 111 ], a death process dependent on Bim [ 14 ]. A competing BH3-ligand derived from Bim interacted with Mcl-1 and prevented its interaction with Mule, leading to increased Mcl-1 expression [ 112 ]. This suggests that Bim needs to be released from Mcl-1 prior to its degradation by Mule [ 112 ]. However, binding of the p53-regulated pro-apoptotic Noxa protein to Mcl-1 leads to its degradation [ 113 ]. The Noxa-mediated degradation of Mcl-1 required Mule, but is also mediated through interruption of the deubiquitinase USP9X with Mcl-1 [ 113 ]. Also, Beclin-1 leads to destabilization of Mcl-1 through competitive binding to USP9X [ 114 ]. USP9X inhibition using WP1130 led to Mcl-1 inhibition and sensitization of solid tumors to various chemotherapeutic agents [ 115 ].

3.1.1.1. FKHR-L1/FoxO3a The forkhead transcription factor FKHR-L1/FoxO3a was found to be essential for the transcriptional activation of Bim, especially after cytokine withdrawal and various chemotherapeutic agents [ 118 , 121 , 122 ]. Dijkers et al. [ 122 ] demonstrated that cytokine withdrawal led to FoxO3a activation and Bim induction in T lymphocytes accompanied by apoptosis. Cytokines promote lymphocyte survival by inhibiting the activity of FoxO3a, thus preventing Bim expression [ 122 ]. Overexpression of FoxO transcription factors induces Bim expression and promotes death of sympathetic neurons in a Bim-dependent manner [ 117 ]. In neuroblastoma cells, the activation of FoxO3 triggers the intrinsic death pathway via induction of Bim and Noxa [ 123 ]. Inhibition of Breakpoint cluster region-Abelson murine leukaemia (Bcr-Abl) kinase by STI571 in Bcr-Abl expressing cells results in FoxO3a activation, induction of Bim expression and apoptosis [ 121 ]. The microtubule interfering drug paclitaxel induced Bim expression in breast cancer cells that expressed high basal levels of FoxO3a, but not in those with low basal FoxO3a levels [ 118 ]. Knockdown of FoxO3a prevented Bim induction and apoptosis induced by paclitaxel [ 118 ]. In neuroblastoma, brain-derived neurotrophic factor (BDNF) activates the PI3K/Akt pathway resulting in suppression of FoxO3a activity and Bim induction, thereby preventing paclitaxel-induced apoptosis [ 124 ]. Simultaneous activation of the MEK/ERK pathway further reduced Bim expression at the protein level [ 124 ]. FoxO3 binds to a FoxO-binding site (FHRE) within the bim promoter [ 118 , 121 ]. FoxO3a acts in concert with the Activator protein-1 (AP-1) transcription factor [ 116 , 117 , 125 ]. High expression levels of FoxO3a correlated with long-term survival in breast cancer patients [ 126 ], and nuclear localization of FoxO3a is associated with longer luminal-like breast cancer survival and longer distant metastasis free interval [ 127 ]. FoxO3a expression was lower in nasopharyngeal carcinoma than in the normal nasopharyngeal tissues [ 128 ]. Nasopharyngeal carcinoma patients with low FoxO3a and high Hypoxia-inducible factor 1α (HIF-1α) expression had poorer prognosis than patients with high FoxO3a and low HIF-1α levels [ 128 ]. Low levels of FoxO factors are associated with poor prognosis in liver cancer [ 129 ] and gastric adenocarcinoma [ 130 ]. Prostate cancer with increasing Gleason grade showed marked cytoplasmic accumulation of FoxO3a, in contrast to exclusive nuclear accumulation in benign prostate cells [ 131 ]. Thus, FoxO factors act as tumor suppressors that are frequently downregulated in advanced cancers. Since FoxO3a is an important regulator of Bim, factors affecting FoxO3a expression have direct effect on tumorigenesis. For instance, ERK phosphorylates FoxO3a at Ser294, Ser344 and Ser425, which leads to Mdm2-mediated degradation of FoxO3a via the ubiquitin-proteasome system [ 132 ]. Sirtuin 1 (SIRT1) deacetylates FoxO3a leading to its ubiquitination and degradation, thus protecting against oxidative stress-induced apoptosis of endothelial cells [ 133 ]. Similarly, IκB kinase (IKK) and Akt (PKB) phosphorylate and cause proteolysis of FoxO3a [ 134 , 135 ]. Akt phosphorylation of FoxO3a leads to cytoplasmic sequestration of FoxO3a to 14-3-3 proteins, thereby preventing the transcription of genes required for apoptosis [ 136 ] Akt phosphorylates FoxO3a at Thr32 and Ser253, while the Serum- and glucocorticoid-inducible kinase (SGK) phosphorylates it at Thr32 and Ser315. Cytoplasmic FoxO3a correlated with expression of IKKβ or phosphorylated Akt in many tumors and was associated with poor survival in breast cancer [ 134 ]. Inhibition of both the mTOR and MEK/ERK pathways led to increased nuclear accumulation and activation of FoxO3a that promoted differentiation and reduced tumorigenicity of glioblastoma cancer stem-like cells [ 137 ]. p38 MAPK phosphorylates FoxO3a at Ser7 leading to its nuclear accumulation [ 138 ], and induction of Bim expression [ 139 ]. In a recent publication, Chatterjee et al. [ 140 ] described an additional level of FoxO3a/Bim regulation (Figure 4 ). Namely, the Casein kinase II (CK2), which is activated under various stress conditions and is abnormally deregulated in many human malignancies, indirectly suppresses FoxO3a activity by promoting phosphorylation of the Promyelocytic leukemia (PML) protein at Ser517, resulting in its ubiquitin-mediated proteasomal degradation [ 141 ]. PML is required for the functional interaction of activated phosphorylated Akt and its phosphatases (e.g., PHLPP2) inside the nucleus, that leads to inactivation of Akt [ 142 ] and proper functioning of FoxO3a [ 140 ]. In the absence of PML, Akt is aberrantly activated, leading to nuclear exclusion of FoxO3a. As a consequence, the FoxO3 target genes p27 Kip1 , p21 Cip1 and Bim are downregulated, while VEGF gene expression, which is suppressed by FoxO3a [ 143 ], becomes

Overexpression of Smad3, a transcription factor activated by TGFβ, increased the expression of Bim in B lymphocytes [ 152 ]. Activation of the pro-survival transmembrane glycoprotein CD40 abrogated TGFβ-mediated Bim induction and apoptosis [ 152 ]. Smad3 was also found to be involved in the transcriptional activation of Bim in human gastric carcinoma cells when simultaneously exposed to both TGFβ and TNFα [ 153 ]. Under these conditions the Bim protein was stabilized by a JNK-dependent mechanism [ 153 ]. TGFβ-induced apoptosis of human hepatoma cells was also mediated by a Smad3/Smad4-mediated upregulation of Bim [ 154 ]. TGFβ also activates FoxO3 dephosphorylation of Thr32, leading to its nuclear translocation [ 155 ]. These authors further showed that the TGFβ-mediated upregulation of Bim and apoptosis was dependent on Casein kinase Iε (CKIε) [ 155 ]. Moreover, the TGFβ-activated FoxO3 cooperated with Smad2/3 to mediate Bim upregulation and apoptosis [ 155 ].

E2F transcription factors are best known for their involvement in the timely activation of genes required for cell cycle progression. E2F activity is negatively regulated through its interaction with the retinoblastoma (Rb) tumor suppressor. Human tumors with inactive Rb pathway, often show deregulated E2F1 activity. Ectopic expression of E2F1 resulted in induction of Bim expression and apoptosis [ 168 ]. Also the BH3-only proteins PUMA, Noxa and Hrk/DP5 were upregulated by E2F1 [ 168 ]. Inhibitors of histone deacetylases (HDACi) were shown to target the Rb/E2F1 pathway for apoptosis induction through activation of Bim [ 169 ]. Cancer cells with elevated E2F1 activity were highly sensitive to HDACi-induced cell death [ 169 ]. E2F1 also transactivates apoptosis signal-regulated kinase 1 (ASK1) that further enhances E2F1-mediated Bim transcription through inhibition of Rb [ 170 ]. ASK1 knockdown led to reduced E2F1-induced Bim transcription and reduced apoptosis in response to the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) [ 170 ]. Spinal cord injury led to strong induction of E2F1 and Bim followed by neuronal apoptosis that could be prevented by inhibition of E2F1 [ 171 ]. Angiotensin II, a key pro-apoptotic factor in fibrosis, mediates apoptosis in primary pulmonary artery endothelial cells through E2F1-mediated upregulation of Bim [ 172 ]. Angiotensin II regulates the association of AMP-regulated protein kinase β1/2 (AMPKβ1/2) with cyclin-dependent kinase 4 (Cdk4) leading to hyperphosphorylation of Rb and the release of E2F1 for transcriptional activation [ 172 ]. E2F1 also induces the expression of the oncogenic polycomb histone methyltransferase enhancer of zeste homolog 2 (EZH2), that antagonizes the induction of Bim by E2F1 [ 173 ] as well as the transcription of Runx3 [ 174 ] that also upregulates Bim [ 164 ]. Thus, the apoptotic activity of E2F1 is restrained in human cancer by the concomitant induction of EZH2 and Bim [ 173 ]. Moreover, deregulated E2F1 activity in gastric cancer may confer resistance to TGFβ-induced apoptosis through upregulation of the miR-106∼25 cluster that targets Bim [ 175 ]. Thus, E2F1 has opposing effects on Bim expression. Upregulated Bim expression in prostate and breast cancer cells was dependent on E2F1, where E2F1 silencing led to loss of Bim [ 176 ]. These authors identified eight endogenous E2F1-binding sites in the Bim promoter. However, FoxO3a didn't bind to the Bim promoter in these cancer cells [ 176 ]. Interestingly, Gogada et al. [ 176 ] observed that Bim silencing or microinjection of anti-Bim antibodies into the cell cytoplasm of breast cancer cells resulted in cell rounding, detachment, and subsequent apoptosis, suggesting that Bim might have a pro-survival role in addition to being pro-apoptotic. The low pro-apoptotic activity of Bim in the epithelial cancer cells was explained by sequestration of Bim to microtubules, Bcl-xL and Mcl-1 together with low expression of Bax and Bax and elevated expression of X-linked inhibitor of apoptosis protein (XIAP) that inhibits caspase 9 and caspase 3 [ 176 ]. Since Bim associates with the microtubule, it could be that Bim is important for stabilizing the cytoskeleton required for survival. Alteration in cytoskeleton integrity might affect mitochondrial respiration and apoptosis [ 177 ].

The transcription factor Signal transducer and activator of transcription 1 (STAT-1) has been shown to be involved in the induction of Bim during TNFα- and IFNγ-induced pancreatic β-cell apoptosis [ 183 ]. IFNγ induces Bim transcription [ 183 ], while TNFα activates JNK that phosphorylates, and thereby upregulates Bim protein expression by protein stabilization [ 184 ]. IFNγ activates signal transduction pathways that involve the tyrosine Janus kinases JAK1 and JAK2, which phosphorylate and induce the dimerization of STAT-1 [ 185 ]. Silencing of Bim or STAT-1 protected β-cells from cytokine-induced apoptosis [ 183 , 186 ]. STAT-1 directly binds to the bim promoter in positions −686 to −385 [ 183 ]. This region contains two STAT-1 binding sites, TTCtacGAA and TTCttgGAA [ 183 ]. Knockdown of the PTPN2 phosphatase, a candidate gene for type 1 diabetes [ 187 ], led to increased phosphorylation and activation of STAT-1 in β-cells, and increased cytokine-induced Bim expression [ 183 , 188 ]. Diabetic retinopathy caused by high glucose-induced apoptosis of retinal pericytes involved STAT-1-mediated Bim expression [ 189 ]. TNFα was responsible for the high glucose-mediated activation of STAT-1, while Bim was responsible for the high glucose-induced ROS production [ 189 ]. STAT-1-dependent Bim transactivation was also observed in chronic lymphoblastic leukemia cells after exposure to IL-21, a gamma-chain receptor cytokine family member that promotes B cell apoptosis [ 190 ].

The nuclear factor erythroid-derived 2, like 2 (Nrf2) is a key regulator of the antioxidant defense system that is activated under hyperprolifeation conditions in the liver. Activated Nrf2 delays proliferation and induces apoptosis of hepatocytes in the regenerating liver through direct transcriptional upregulation of Bim [ 204 ].

In hematopoietic progenitor cells, Homeobox B8 (HoxB8) overexpression repressed Bim through a c-Myc-dependent upregulation of miR-17∼92 that binds to the 3′UTR of Bim mRNA [ 207 ]. HoxB8 overexpression immortalizes hematopoietic progenitor cells in a growth factor-dependent manner and co-operates with IL-3 to induce acute myeloid leukemia. Downregulation of HoxB8, in the presence of IL-3, caused cell-cycle arrest and apoptosis that was dependent on Bax and Bak, and in part, on Bim [ 207 ]. The requirement of miR-17∼92 to suppress Bim expression by HoxB8 is evident in the strong selection against deletion of miR-17∼92 in HoxB8-immortalized myeloid cells [ 207 ].

Particularly interesting cysteine-histidine-rich protein-1 (PINCH-1) is a cytoplasmic component of cell-extracellular matrix adhesions required for protection of multiple types of cancer cells from apoptosis. Although PINCH-1 is not a transcription factor, its knockdown leads to a 10-fold increase in Bim EL mRNA levels, suggesting for a role of PINCH-1 in suppressing Bim transcription [ 210 ]. Depletion of Bim blocked apoptosis induced by the loss of PINCH-1 [ 210 ]. Besides affecting Bim transcription, PINCH-1 promotes activating phosphorylation of Src family kinases and ERK1/2 that leads to Ser69 phosphorylation of Bim resulting in enhanced degradation [ 210 ].

SP-1 (Specificity Protein 1) is often overexpressed in cancer and may be a negative prognostic factor for the overall survival [ 213 , 214 ]. The bim promoter contains six GGGCGG motifs that are recognized by the transcription factor SP-1 [ 17 ]. Inhibition of SP-1 by the curcumin analogue dibenzylideneacetone, led to increased expression of Bim and apoptosis in mucoepidermoid carcinomas [ 215 ], suggesting that SP-1 acts as a suppressor of Bim expression. Also mithramycin A, another SP-1 inhibitor induced Bim expression and apoptosis [ 215 ]. SP-1 may positively regulate the anti-apoptotic Mcl-1 expression [ 216 ], such that inhibition of SP-1 has dual pro-apoptotic effects.

Sympathetic neurons exposed to low O 2 tension upregulate hypoxia-inducible factor-1α (HIF-1α) that protects against NGF-deprivation-induced apoptosis through suppression of Bim expression [ 222 ]. The Tax protein of human T-lymphotropic virus type 1 (HTLV-1) downregulates Bim expression at the transcriptional level through induction of HIF-1α [ 223 ]. Knockdown of HIF-1α or chemical inhibition of its transactivation activity resulted in increased Bim expression and sensitization of HTLV-1-infected leukemic T cells to CD95/TRAIL- and anticancer drug-induced apoptosis [ 223 ]. HIF-1α may also reduce Bim protein expression through ERK-dependent phosphorylation and degradation [ 224 ].

As described in Section 1.3, the Bim transcripts undergo extensive alternative splicing, giving rise to a variety of Bim isoforms with different intrinsic toxicities and modes of regulation [ 3 , 25 , 28 ]. Splicing of exon 3 and splicing of exon 4 usually occur in a mutual exclusive manner, and due to the presence of a stop codon and a polyadenylated signal within exon 3, its inclusion leads to a pre-mature Bim protein that lacks the BH3 domain required for apoptosis [ 30 , 31 , 228 ]. Intron 2 deletion polymorphism led to preferential splicing of exon 3 over exon 4, thus excluding the BH3 encoded by exon 4 [ 31 ]. Bimγ formed by the splicing of exon 3 had a very short half-life of less than an hour [ 31 ]. The splicing factor oncoprotein SRSF1 (also known as SFSR1 or SF2/ASF) promoted alternative splicing of Bim to produce isoforms that lack pro-apoptotic functions [ 229 ]. Two new isoforms termed Bimγ1 and Bimγ2 were generated due to the inclusion of an alternative 3′ exon and exclusion of exons E4 and E5, thus lacking the BH3 domain [ 229 ]. The expression of the isoforms Bim EL , Bim L and Bim S was concomitantly reduced [ 229 ]. SRSF1 is frequently upregulated in breast cancers and its overexpression leads to the formation of larger acinar cells, due to increased proliferation and delayed apoptosis during acinar morphogenesis [ 229 ]. The alternative splicing of Bim is important for the reduced apoptosis of acinar cells [ 229 ]. High SRSF1 expression occurs frequently in tumors overexpressing c-Myc, and these tumors are of higher histological grade that those with low c-Myc and SRSF1 expression [ 229 ]. Further studies showed that SRSF1 is a target gene of c-Myc [ 230 ]. Loss of SRSF1 induces G2 cell cycle arrest and apoptosis [ 231 ]. Knockout of the serine/arginine-rich (SR)-related protein Pinin (Pnn) leads to early embryonic lethality, and its knockdown in breast cancer cells leads to apoptosis due to reduced expression of SRSF1 and increased expression of Bim [ 232 ]. The human Krüppel-like zinc finger protein Gli-similar (GLIS) 3 whose mutations lead to neonatal diabetes, seem to indirectly affect Bim splicing [ 233 ]. Knockdown of GLIS3 leads to preferential expression of Bim S and β-cell apoptosis that was further aggravated by cytokines [ 233 ]. Simultaneous knockdown of Bim abrogated the pro-apoptotic effect of GLIS3 [ 233 ]. One mechanism by which GLIS3 prevents Bim S isoform formation is through induction of the splicing factor SRp55 (encoded by the gene splicing factor arginine/serine (RS)-rich 6 ( SFRS6 )) [ 233 ]. Overexpression of GLIS3 in β-cells antagonized cytokine-induced apoptosis [ 233 ] and SRp55 expression was modified in human islets following cytokine treatment [ 234 ]. The observation by Nogueira et al. [ 233 ] showing that downregulation of SRp55 in β-cells leads to increased expression of Bim S and apoptosis, opposes the report of Jiang et al. [ 235 ] showing that SRp55 appears necessary for the preferential increase in Bim S splicing observed after treating B-Raf V600E mutant human melanoma cells with the B-Raf V600E inhibitor PLX4720. The apparent discrepancy could be due to cell-specific effects. Also, Hara et al. [ 236 ] observed that SRp55/SFRS6 leads to preferential upregulation of the Bim S isoform following treatment of human neuroblastoma cells with Zn 2+ , that induces apoptosis of these cells. They identified a SFRS6 binding site in the intronic region adjacent to exon 4 [ 236 ]. Juan et al. [ 237 ] studied the 2,903-bp deletion polymorphism within Bim intron 2 that biased towards exon 3, leading to impaired Bim-dependent apoptosis. They observed that this region has many cis-acting elements that repress exon 3 inclusion [ 237 ]. A 23-nt intronic splicing silencer at the 3′-end of the deletion was found to be important for the exon 3 exclusion [ 237 ]. They demonstrated that Polypyrimidine tract binding protein 1 (PTBP1) and Heterogeneous nuclear ribonucleoprotein C (hnRNP C) repress exon 3 inclusion, and downregulation of PTBP1 inhibits Bim-mediated imatinib-induced apoptosis [ 237 ]. The catalytic subunit Brm (Brahma) of the SWI/SNF related, matrix associated, subfamily a, member 2 (also known as mating-type switch/sucrose nonfermenting) complex involved in chromatin remodeling on promoters was shown to associate with several components of the spliceosome and with Sam68, an ERK-activated enhancer of variant exon inclusion [ 238 ]. Brm favors the inclusion of variant exons in the mRNA of Bim, in addition to increasing the Bim expression level [ 238 ]. The BH3-containing exon 4 (that they coined exon 5) was included between exon 2 and 5 (that they coined exon 7), giving rise to the Bim L and Bim EL isoforms [ 238 ]. The Brm gene codes for an ATPase catalytic subunit that shifts histones and opens the chromatin and is considered to be a tumor suppressor gene [ 239 ]. As Bim S can directly bind and activate Bax [ 9 , 23 , 24 ], and is not sequestrated to the cytos

3.2.1. Regulation of Bim mRNA through the 3′-Untranslated Region (3′-UTR) Translation of Bim mRNA can be regulated through the 3′-untranslated region (3′-UTR) [ 253 , 254 ]. This region binds microRNAs and RNA-binding proteins (RBPs) that regulate mRNA stability and/or translation [ 255 ]. Cytokines negatively regulate the steady-state levels of Bim through heat-shock cognate protein 70 (Hsc70), which binds to AU-rich elements (AREs) in the 3′-UTR [ 253 ]. The RNA binding potential of Hsc70 is regulated by the co-chaperones Bag-4 (SODD), CHIP, Hip, and Hsp40. Cytokines regulate the expression or function of these co-chaperones by activating Ras pathways. Thus, exposure of cells to cytokines ultimately leads to destabilization of Bim mRNA and promotion of cell survival [ 253 ]. Heat shock protein 27 (Hsp27) prevents oxidative stress-induced cell death in cerebellar granule neurons by binding to the 3′-UTR of the Bim mRNA, thereby preventing translation of Bim protein [ 256 ]. Oxidative stress induced by hydrogen peroxide led to Hsp27 depletion and increased Bim expression, resulting in subsequent neuronal death [ 256 ]. Of note, HMG-CoA reductase inhibitors (statins) increased Hsp27 expression [ 257 ] and prevented oxidative stress-induced apoptosis of endothelial progenitor cells by inhibiting FoxO4-mediated upregulation of Bim [ 258 ].

The 3′-UTR of Bim mRNA contains nine potential binding sites for miR-17∼92 family members with at least one binding site for each of the three distinct microRNA seeds related to the miR-17∼92 cluster [ 260 ]. miR-92, and to a lesser extent miR-19, targets the Bim 3′-UTR in reporter assays in HeLa cells [ 263 ]. Genetically engineered mice with higher expression of miR-17∼92 in lymphocytes developed lymphoproliferative disease and autoimmunity and died prematurely [ 263 ]. Lymphocytes from these mice showed more proliferation and less activation-induced cell death [ 263 ]. In contrast, mice deficient for miR-17∼92 die shortly after birth with lung hypoplasia and a ventricular septal defect as a result of increased Bim expression [ 264 ]. miR-17 and the miR-17∼92 cluster have also been shown to cause resistance of pediatric acute lymphoblastic leukemia to glucocorticoid-induced apoptosis through prevention of Bim expression [ 265 , 266 ]. Dexamethasone treatment led to reduced expression of the miR-17∼92 cluster, with concomitant upregulation of Bim and apoptotic sensitization [ 265 , 266 ]. miR-92a promotes glioma cell survival through repression of Bim [ 267 ]. miR-20, miR-92 and miR-302 are important for epiblast stem cell survival through repression of Bim [ 268 ]. Bim knockout rescued the cell death phenotype in epiblasts of Dicer −/− embryos [ 268 ]. miR-25 of the miR-106b∼25 cluster prevents Bim expression in human ovarian cancer cells, promoting their survival [ 269 ]. miR-25 also suppresses Bim expression in gastric cancer [ 175 ]. The miR-106b∼25 polycistron is activated by genomic amplification and is potentially involved in esophageal neoplastic progression [ 270 ]. Repression of the miR-106b∼25 cluster by PKR-like endoplasmic reticulum kinase (Perk) was required for ER stress-induced apoptosis that is mediated by Bim [ 271 ]. The HDAC inhibitor trichostatin suppressed miR-106b∼93∼25 expression through downregulation of c-Myc, thereby inducing apoptosis in human endometrial cancer cells [ 272 ]. Besides c-Myc, E2F1 positively regulates the expression of the intronic microRNAs 106b∼93∼25 [ 175 ]. Of note, myc is a direct target gene of E2F1 [ 273 ]. Overexpression of the miR-106b∼25 cluster in gastric cancer prevented TGFβ-induced Bim expression and apoptosis [ 175 ]. Further studies showed that miR-25, but not miR-106b or miR-93, was responsible for the downregulation of Bim [ 175 ]. microRNAs 106b and 93, however, prevent E2F1 expression, forming a negative feedback loop [ 175 ].

Upregulation of miR-32 by 1,25-dihydroxyvitamin D3 in human myeloid leukemia cells leads to targeting of Bim and prevention of arabinocytosine (AraC)-induced apoptosis [ 276 ]. miR-32 was also found to target Bim in prostate cancer [ 277 ]. miR-301a promotes pancreatic cancer cell proliferation by inhibiting Bim expression [ 278 ], while miR-363 supports human glioblastoma stem cell survival for the same reason [ 279 ]. NGF increases the expression of miR-221/222 in PC12 cells through the ERK pathway. These microRNAs bind to the 3′-UTR of Bim mRNA, thereby preventing Bim translation [ 254 ]. miR-124 that is abundantly expressed in midbrain dopaminergic neurons, was downregulated in a mouse model of Parkinson's disease, accompanied by an increase in Bim expression and death of the neurons [ 280 ]. miR-24 was found to suppress cardiomyocyte apoptosis in part through repressing Bim [ 281 ]. Forced overexpression of miR-24 in a mouse myocardial infarction model inhibited cardiomyocyte apoptosis, attenuated infarct size and reduced cardiac dysfunction [ 281 ].

3.3.1. Regulation of Bim Activity by Protein Kinases The phosphorylation status of Bim controls its pro-apoptotic activity and stability [ 7 , 25 , 295 ]. Especially, the MAP kinases are involved in the regulation of the pro-apoptotic activity of Bim [ 25 ]. In general, phosphorylation of Bim by ERK1/2 leads to the degradation of Bim through the ubiquitin-proteasome pathway, while its phosphorylation by JNK or p38 increases its activity. The Akt/mTOR pathway indirectly affects Bim through phosphorylation and inactivation of the FoxO transcription factors and upregulation of the anti-apoptotic Mcl-1 protein. Thus, receptor signaling or oncogenic addictions activating ERK, Akt and/or mTOR signaling pathways confer apoptotic resistance through reducing Bim expression or antagonizing its activity. Mutation studies showed that mutation of the phosphorylation site Thr112 in mouse Bim caused decreased binding of Bim to the anti-apoptotic protein Bcl-2 and increased cell survival [ 295 ]. However, mutation of the phosphorylation sites Ser55, Ser65, and Ser73 in mouse Bim caused increased apoptosis because of reduced proteasomal degradation of Bim [ 295 ]. The amino acid positions differ somewhat between human and mouse Bim (Figure 7 ), so we have adapted in the text the numbers described in the cited references. 3.3.1.1. ERK1/2 Phosphorylation of BIM EL by ERK1/2 at Ser69 in human or at Ser65 in mouse leads to K48-linked ubiquitin-dependent 26S proteasome-mediated degradation of Bim EL [ 295 - 298 ]. Bim EL can also be degraded by the 20S proteasome in the absence of poly-ubiquitination, as shown by mutating the only two lysine residues Lys3 and Lys108 in rat Bim [ 298 ]. This might be related to the fact that Bim EL is an intrinsically unstructured protein [ 299 ]. The degradation in absence of poly-ubiquitination is prevented when Bim EL is bound to Mcl-1 [ 298 ]. Since exon E2B encodes the ERK1/2-docking domain and ERK1/2 phosphorylation sites, only Bim EL , but not Bim L or Bim S , is subject to phosphorylation by the MEK/ERK pathway [ 296 , 300 ]. In addition, ERK phosphorylation of mouse Bim at Ser65 prevents its binding to Bax [ 296 , 300 ], and leads to its dissociation from Mcl-1 and Bcl-xL [ 301 , 302 ]. Binding of Bim to Mcl-1 stabilizes the latter [ 303 ]. This is in contrast to the binding of Noxa to Mcl-1 that promotes Mcl-1 degradation [ 303 ]. Thus, Bim dissociation from Mcl-1 leads to destabilization of both the pro-apoptotic Bim and the anti-apoptotic Mcl-1, the net effect determining the cell fate. The hematopoietic survival factor Interleukin-3 (IL-3) induces ERK-mediated phosphorylation of mouse Bim on three serine residues (Ser55, Ser65 and Ser100) that prevents Bim from interacting with Bax [ 7 ]. Following IL-3 withdrawal, only the non-phosphorylated form of Bim interacts with Bax [ 7 ]. Survival is often promoted in tyrosine kinase-driven cancers such as CML and EGFR NSLCL, through repression of Bim transcription and through targeting the Bim protein for proteasomal degradation following Mitogen-activated protein kinase 1 (MAPK1/ERK2)-dependent phosphorylation [ 304 - 308 ]. Targeting EGFR using kinase inhibitors (e.g., erlotinib and gefitinib) led to suppression of PI3K/mTORC and MEK/ERK signaling, followed by an increase in Bim expression [ 307 , 309 , 310 ] and a decrease in Mcl-1 [ 310 ]. NGF induces MEK/MAPK-mediated phosphorylation of rat Bim EL at Ser109 and Thr110 in sympathetic neurons [ 311 ]. Dehan et al. [ 312 ] observed that ERK1/2 co-operates with ribosomal S6 kinase (RSK) to phosphorylate Bim EL , allowing binding of the F-box proteins β-TrCP1/2, which promotes BIM EL poly-ubiquitination. Silencing of either β-TrCP or RSK1/2 resulted in Bim EL -mediated apoptosis of both gefitinib-sensitive and gefitinib-insensitive NSCLC cells [ 312 ]. Phosphorylation of Ser69 of human Bim EL by ERK1/2 promoted cytokine-induced phosphorylation on Ser93, Ser94 and Ser98, that was required for the binding of Bim EL with β-TrCP1 [ 312 ]. RSK1 and RSK2 were responsible for the phosphorylation on Ser93/94/98 [ 312 ]. Elevated expression of urokinase plasminogen activator (uPA) in EGFR-positive glioblastoma cells leads to apoptosis resistance to EGFR tyrosine kinase inhibitors through ERK1/2-dependent repression of Bim expression [ 313 ]. Tyrosine kinase inhibitor-resistant glioblastomas can be resensitized to EGFR tyrosine kinase inhibitors by pharmacologic inhibition of MEK or a BH3 mimetic drug to replace Bim function [ 313 ]. ERK1/2 inhibition leads to Bim EL stabilization and increased tumor cell death [ 314 , 315 ]. Similarly, MEK inhibitors could potentiate dexamethasone lethality of acute lymphoblastic leukemia cells through upregulating Bim expression [ 316 ]. Bim accumulated by this treatment interacted with Bcl-xL and Mcl-1, resulting in the release of Bak [ 316 ]. Combined treatment of inhibitors of the PI3K/Akt and MEK/ERK1/2 pathways led to Bim-dependent leukemia cell death [ 317 ].

Phosphorylation of Bim EL at Ser65 by p38 increased its pro-apoptotic activity [ 327 ]. Sodium arsenite-induced apoptosis in PC12 cells may be due to the direct phosphorylation of Bim EL at Ser65 by p38 [ 327 ]. p38 may indirectly increase Bim transcription through positive regulation of FoxO3a [ 139 ], Runx2 and c-Jun [ 159 ].

Treatment of T lymphoma cells with the Protein kinase A (PKA) agonist 8-CPT-cAMP led to the phosphorylation of cAMP response element-binding protein (CREB) and induction of Bim, followed by apoptosis [ 341 ]. The cytotoxic effect of 8-CPT-cAMP was dependent on PKA and Bim [ 342 ]. The PRKAR1A regulatory subunit of cyclin-dependent PKA was found to interact with Bim EL [ 343 ]. Phosphorylation of mouse Bim EL at Ser83 (corresponding to Ser87 in human) by PKA leads to stabilization of the Bim protein and induction of apoptosis [ 343 ]. Of note, Ser83 resides within the D-box consensus sequence recognized by the Anaphase promoting complex (APC cdc20 ) E3 ligase [ 344 ] (Figure 7 , and see Section 3.3.2).

Bim is important for determining the lifespan of myeloid and lymphoid cells, as these cells are increased in numbers in mice lacking Bim [ 2 ]. Bim is also required for further homeostatic regulation in situations such as the proper termination of the innate and adaptive immune responses [ 47 ]. Alterations in Bim expression are associated with several diseases (Figure 8 ). Too rapid or too slow elimination of activated T and B cells as a result of too high or too low Bim expression, respectively, leads to chronic infection due to incomplete immune responses or autoimmune diseases. Bim downregulation is involved in cell transformation and reduces the sensitivity of cancerous cells to various chemotherapeutic drugs [ 121 , 246 , 304 - 307 ]. Increased Bim expression contributes to increased cardiomyocyte and neuronal cell death following ischemia [ 281 , 351 ], increased β-cell death resulting in diabetes [ 40 ] and enhanced neuronal cell death in Alzheimer's disease [ 352 ]. Decreased Bim expression confers protection from viral-induced hepatitis and sepsis-related mortality [ 353 , 354 ]. Below we will discuss these aspects in more detail. Figure 8 Abberant Bim expression in diseases Elevated Bim expression is associated with neuronal degenerative diseases, diabetes, fibrosis and liver damage, while too low or absent Bim expression is associated with cancer (Section 4). Absent Bim expression in the immune system may lead to autoimmune diseases due to lack of elimination of autoreactive immune cells, but is, in part, compensated by an increase in regulatory T cells. Treatment of neurodegenerative diseases and diabetes should aim in preventing Bim expression, while treatment of cancer should aim in increasing Bim expression. Too dramatic increase in Bim during cancer treatment might thus have adverse effects on the nerve system, liver and endocrine system, that needs to be taken into account. Targeting cancer-specific pro-survival pathways would reduce the adverse effects. 4.1. Regulation of Bim by Growth Factor Withdrawal Bim expression is induced de novo following withdrawal of survival factors (cytokines, growth factors, trophic factors) or serum from primary sympathetic neurons [ 125 , 355 ], lymphocytes [ 2 , 122 , 356 ], hematopoietic progenitor cells [ 26 ], granulocytes [ 357 ], mast cells [ 358 , 359 ], osteoclasts [ 360 ], fibroblasts [ 361 ] and multiple myeloma [ 247 ], while its expression is repressed by the PI3K/Akt or MEK/ERK signaling pathways [ 117 , 122 , 359 , 361 ] (Figure 9 ). In neurons the JNK/c-Jun pathway is required for Bim mRNA expression following withdrawal of NGF [ 125 , 355 , 362 ], but this pathway does not seem to be important for fibroblasts [ 361 ]. Bim induction after NGF withdrawal is reduced in sympathetic neurons in mice carrying a mutant c-Jun gene that lacks activating Ser63/Ser73 phosphorylation sites [ 363 ]. Further studies showed that induction of Bim in NGF-deprived cells requires expression and activity of Cdk4 and consequent de-repression of E2 promoter binding factor (E2F)-regulated genes including members of the Myb transcription factor family [ 364 ]. Mutation in the two Myb binding sites in the bim promoter abolished Bim induction following NGF deprivation [ 364 ]. In lymphocytes, Bim mRNA expression is promoted by the FoxO3a transcription factor, which is normally repressed by Akt/PKB-mediated phosphorylation. Consequently, inhibition of PI3K or withdrawal of survival factors is sufficient to induce Bim expression in these cells [ 122 ]. Figure 9 Growth factor-mediated cell survival versus apoptosis induction by growth factor deprivation Growth factor receptor activation leads to activation of the Ras-Raf-MEK-ERK1/2 and PI3K-Akt pathways that co-operate in inhibiting Bim activity. ERK1/2-mediated phosphorylation of Bim EL leads to its subsequent phosphorylation by RSK1/2 and ubiquitin-mediated proteasomal degradation. Akt-mediated phosphorylation of Bim leads to its sequestration to 14-3-3 proteins. Akt also inactivates Mst1 and FoxO3a. Following growth factor withdrawal, the lack of activation of the Ras-Raf-MEK-ERK1/2 and PI3K-Akt pathways leads to Mst1 and FoxO3a activation, resulting in the transcriptional upregulation of Bim. In addition, JNK phosphorylates Bim resulting in its activation and apoptosis induction. Inhibition of growth factor receptor signaling (e.g., by EGFR, ALK, c-Met and B-Raf V600E inhibitors) is a major cancer-specific targeting strategy that aims to promote Bim-dependent apoptosis. Addition of growth factors usually leads to downregulation of Bim [ 26 , 311 , 360 ]. IL-3 promotes survival of hematopoietic progenitors through downregulation of Bim [ 26 ]. The IL-3-dependent downregulation of Bim is mediated through activation of Raf/MAPK and PI3K/mTOR pathways [ 26 ]. IL-3 led to Akt activation and Bim EL phosphorylation at Ser87 in Ba/F3 cells, leading to attenuation of its pro-apoptotic function [ 336 ]. IL-7, that is important

Examination of human brains of post-mortem Alzheimer's disease patients showed that Bim is upregulated in vulnerable entorhinal cortical neurons, but not cerebellum, a region usually unaffected by the disease [ 352 ]. They further demonstrated that Bim is required for β-amyloid-induced neuronal apoptosis [ 352 ]. Cdk4 and its downstream effector B-Myb were required for β-amyloid-dependent Bim induction and death in cultured neurons [ 352 ]. Cdk4 inhibitors were recently shown to be neuroprotective [ 384 ]. Also, FoxO3a and AP-1 (c-Jun/c-Fos) are involved in the β-amyloid-induced upregulation of Bim [ 388 , 389 ]. The activation of FoxO3a by β-amyloid is in part due to reduced Akt-mediated FoxO3a phosphorylation and increased Mst1-mediated FoxO3a phosphorylation [ 388 ]. Estrogen protects against β-amyloid peptide-induced apoptosis by upregulating Bcl-w and downregulating Bim [ 390 ]. In addition to parenchymal accumulation and neuronal degeneration in the brain of Alzheimer's disease patients, β-amyloid accumulates in the cerebrovascular wall leading to cerebral amyloid angiopathy. Amyloid-β peptide-induced death in cerebral endothelial cells is preceded by mitochondrial dysfunction and signaling events characteristic of apoptosis. The expression of Bim , but not other BH3-only members, was selectively increased in cerebral microvessels isolated from 18-month-old APPsw (Tg2576) mice, a model of cerebral amyloid angiopathy, suggesting a pivotal role for Bim in β-amyloid-induced cerebrovascular degeneration in vivo [ 391 ]. β-Amyloid increases FoxO3a activity through activation of PP2A that negatively regulates Akt [ 391 ]. Suppression of PP2A attenuated Bim expression and cell death in cerebral endothelial cells [ 391 ].

Within the thymus, Bim L/EL expression was observed in the cortex where the double positive thymocytes are located, while Bim was absent from the thymic medulla [ 6 ]. Bim is required for the apoptosis of double positive thymocytes induced by high-affinity antigens [ 401 ]. In immature murine thymocytes, Bim is associated with mitochondria before stimulation and its level is not increased following CD3ε cross-linking, a treatment stimulating signals through the TCR [ 402 ]. However, CD3ε cross-linking led to rapid phosphorylation of Bim EL in CD4 + CD8 + thymocytes [ 402 ]. In contrast to TCR signaling, dexamethasone did not lead to Bim phosphorylation in CD4 + CD8 + thymocytes [ 402 ]. Bim is overexpressed in pro-apoptotic, pre-TCR-deficient thymocytes [ 403 ]. Pre-TCR signaling suppresses Bim expression through PI3K/Akt-mediated inhibition of FoxO3a [ 403 ]. Bim is also essential for the deletion of CD4 + CD8 − CD24 + thymocytes in response to TCR ligation [ 404 ]. TCR ligation upregulates Bim expression in thymocytes and promotes the interaction of Bim with Bcl-xL, leading to thymocyte killing [ 46 ]. Bim is also required for the activation of autoreactive T cells [ 41 ]. Deletion of Bim in hematopoietic cells rendered mice resistant to autoimmune encephalomyelitis and diabetes, and Bim-deficient T cells showed diminished cytokine production. The Bim-deficient T cells showed defective calcium signaling upon T cell receptor activation, that was associated with an increase in the formation of an inhibitory complex containing Bcl-2 and the inositol triphosphate receptor (IP3R). Thus, in addition to mediating the death of auto-reactive T cells, Bim controls T cell activation through the inositol triphosphate receptor (IP3R)/calcium/nuclear factor of activated T cells (NFAT) pathway [ 41 ]. This function may explain why Bim-deficient mice do not reject their own organs despite lacking thymic negative selection [ 41 ]. Bim is required for negative selection of thymocytes recognizing tissue-restricted (TRA), but not ubiquitous (UbA), self-antigens [ 401 , 405 , 406 ]. In polyclonal Bim KO mice, there is an increase in anergic phenotype CD4 + T cells, which may indicate an increase in self-specificities due to a block of clonal deletion [ 405 ]. Also, Bim deficiency in Vβ5 transgenic mice led to impaired peripheral deletion of CD4 + T cells, resulting in more Recombination activating gene (RAG)-expressing, revising CD4 + T cells [ 407 ]. Selective clonal deletion of the CD4 + T cell compartment during the transition from effector to memory is accompanied by enhanced expression of Bim. Bim deficiency enables the survival of poorly functional Th1 responders that are normally eliminated during contraction [ 408 ]. Bim −/− CD4 + memory T cells showed deficient effector functions, poor sensitivity to antigen and an inability to respond to secondary challenge [ 408 ]. Thus, Bim plays a key role in shaping the CD4 + memory T cell repertoire, ensuring the emergence of highly functional CD4 + memory T cells and the elimination of Th1 effector cells with sub-optimal function [ 408 ]. Bim was also found to be involved in the apoptosis of effector T cells following interaction of B7-H1 (PD-L1) with its receptor PD-1 [ 409 ]. B7-H1 is expressed on primary and metastatic tumor cells as well as antigen-presenting cells and exerts immunosuppressive functions on CD8 + T cells. Interruption of the PD-L1/PD-1 interaction using blocking antibodies to either of the two molecules has proven effective in increasing the anti-tumor immune responses [ 410 ]. More memory CD8 + T cells were generated in B7-H1-deficient mice following immunization as compared to wild-type mice [ 409 ]. At the peak of the expansion phase, CD8 + T cells expressed lower levels of Bim in the B7-H1-deficient mice than wild-type mice [ 409 ]. Bim is a key regulator of T cell apoptosis during the contraction phase of CD8 + T cell response [ 399 ]. Thus, B7-H1 negatively regulates CD8 + T cell memory by enhancing the depletion of effector CD8 + T cells through upregulation of Bim [ 409 ]. Also, Kurtulus et al. [ 411 ] observed that Bim controls T cell memory development by limiting the survival of pre-memory effector cells. The absence of Bim increased the effector CD8 + T cell population with more memory potential, which was due to increased IL-15-dependent survival of memory precursors [ 411 ]. Although Bim was critical to limit survival of killer-cell lectin like receptor G1 (KLRG1) hi CD127 lo T effector cells, the absence of Bim enriched for KLRG1 lo CD127 hi pre-memory T cells as the response progressed [ 411 ]. Also the production of regulatory T cells (T reg ) was affected by Bim [ 37 - 39 ]. Regulatory T cells accumulate dramatically in aged mice, which is due to reduced Bim expression [ 38 ]. Bim expression naturally declines in peripheral wild-type CD4 + T cells during aging [ 412 ], which is co-incident with the increasing numbers of T reg cells [ 37 ].

Bim is phosphorylated and upregulated during Toll-like receptor (TLR) stimulation of macrophages [ 430 ]. Inhibition of the MAPK p38 reduced both upregulation of Bim and apoptosis [ 430 ]. Bim upregulation was induced by LPS that stimulates TLR4, the bacterial lipopeptide Pam 3 Cys that stimulates TLR2, and CpG oligodeoxynucleotide that stimulates TLR9 [ 430 ]. It should be noted that upregulation of Bim per se was insufficient in inducing apoptosis, that required an additional signal [ 430 ]. The adaptor protein MyD88 was both necessary and sufficient for the induction of Bim [ 430 ]. FoxO3a seems to be involved in Bim induction, as it translocates into the nucleus upon TLR stimulation [ 430 ]. Of note, Bim −/− macrophages were resistant to E. coli phagocytosis-induced apoptosis [ 430 ]. TLR stimulation might also promote Bim degradation through ERK-mediated phosphorylation of Bim at Ser55, Ser65 and Ser100 [ 431 ]. Bim KO mice had an increased number of dendritic cells [ 432 ]. Bim is expressed at low levels in dendritic cells, but is significantly upregulated by signaling from CD40 or TLRs [ 432 ]. Simultaneously with the upregulation of Bim, the anti-apoptotic Mcl-1 is also upregulated by TLR signaling, that antagonizes the pro-apoptotic function of Bim during the early stages of dendritic cell activation [ 432 ]. GM-CSF treatment of dendritic cells led to reduced Bim expression and increased dendritic cell survival [ 432 ]. Bim-deficient dendritic cells showed decreased spontaneous cell death and induced more robust T cell activation, thus being more immunogenic [ 432 ]. Adoptive transfer of Bim-deficient dendritic cells led to the induction of autoantibody production that may contribute to the spontaneous systemic autoimmunity observed in Bim KO mice [ 432 ].

Natural killer (NK) cells undergo antigen-driven expansion to become memory cells after mouse cytomegalovirus (MCMV) infection. Bim −/− Ly49H + NK cells expanded normally during the early infection phase, but showed reduced contraction after the expansion phase leading to higher NK cell numbers than wild-type cells [ 436 ]. The inability to reduce the effector pool leads to larger Bim −/− NK memory subsets, which displays a less mature phenotype (CD11b lo , CD27 + ) and lower levels of NK cell memory-associated markers KLGR1 and Ly6C [ 436 ]. Bim −/− memory NK cells showed a reduced response to the MCMV-encoded ligand m157-mediated stimulation and were less effective than wild-type NK cells in protecting against MCMV infection [ 436 ].

Bim-deficient CD34 − /c-kit + /Sca-1 + /Lin − hematopoietic stem cells and megakaryocytes are resistant to apoptosis induced by cytokine depletion [ 440 ]. Platelet recovery after 5-fluorouracil-induced thrombocytopenia is delayed in Bim KO mice, which is reflected in reduced number of megakaryocytes [ 440 ]. Bim-deficient c-Kit + /Lin − progenitor cells poorly proliferate and differentiate into CD41 + cells in response to thrombopoietin, but once differentiated into megakaryocytes, these cells mature normally. The transition from G 1 to S phase is delayed in Bim-deficient hematopoietic stem cells, suggesting a role for Bim in regulating cell cycle progression in hematopoietic progenitors during megakaryopoiesis [ 440 ].

Bim contributes to cytokine- [ 183 , 188 ], virus- [ 449 ] and high glucose-induced [ 450 , 451 ] pancreatic β-cell apoptosis. Bim can be regulated by pro-inflammatory cytokines at the transcriptional level through STAT1 [ 183 ] and by phosphorylation [ 188 ]. Puma and Bax are also involved in high glucose-induced apoptosis of islet cells [ 450 , 451 ]. Loss of Bid, Noxa or Bak had no impact on glucose-induced apoptosis [ 451 ]. Islets deficient in both Bim and Puma, but not Bim or Puma alone, were protected from killing induced by the mitochondrial reactive oxygen species donor rotenone [ 450 ]. Non-obese diabetic (NOD) mice that develop spontaneous autoimmune diabetes, show decreased thymic clonal deletion associated with diminished induction of Bim and Nur77 [ 452 ]. These mice show defective thymic enrichment of CD4 + CD25 + T reg cells [ 452 ]. Bim deficient NOD mice developed less insulitis and were protected from diabetes despite substantial defects in the deletion of autoreactive thymocytes [ 40 ]. Bim deficiency didn't impair effector T cell function, but NOD Bim −/− mice had increased numbers of antigen-specific T reg cells both in the thymus and peripheral lymphoid tissues [ 40 ]. It is likely that the absence of Bim in the β-cells prevents their cell death, thereby avoiding the trigger of the immune system, which often happens due to cell death-released factors such as High-mobility group box 1 (HMGB1) that activates the receptor for advanced glycation end products (RAGE) and spliceosome-associated protein 130 (SAP130) activating C-type lectin 4e (Clec4e) [ 453 ]. The Bim gene Bcl2l11 lies within the Idd13 diabetes susceptibility locus [ 40 , 452 ]. As described in Sections 3.1.3 and 3.1.1.7, diabetes-related GLIS deficiency leads to preferential upregulation of the Bim S isoform [ 233 ], and diabetes-related PTPN2 deficiency leads to increased activation of STAT-1 resulting in increased Bim transcription [ 183 ]. Also, other candidate risk genes for type 1 diabetes have been described that protects β-cells from apoptosis through prevention of Bim upregulation. For instance, deficiency in the basic leucine zipper transcription factor 2 (BACH2) leads to increased phosporylation of JNK1 by mitogen-activated protein kinase kinase 7 (MKK7) and downregulation of PTPN2. These changes, in turn, led to increased Bim expression [ 454 ]. The candidate gene cathepsin H prevents β-cell apoptosis by suppressing JNK and p38 signaling and reducing Bim expression [ 455 ]. Pro-inflammatory cytokines decreased the expression of cathepsin H in human islets, and overexpression of cathepsin H protected β-cells against cytokine-induced apoptosis [ 455 ]. Islets of human donors with type 2 diabetes had higher mRNA levels of Bim and Puma, suggesting that these pro-apoptotic proteins contribute to β-cell death [ 450 ]. High concentrations of glucose led to increased expression of the transcription factor CHOP (C/ERB homologous protein) [ 450 ], that co-operates with FoxO3a to regulate Bim and Puma expression [ 181 ]. The increased apoptosis of β-cells in pancreatic duodenal homeobox-1 (Pdx1)-haploinsufficient mice could be prevented by simultaneous knockout of either Bim or Puma [ 456 ]. Both Bim and Puma expression is upregulated in β-cells following Pdx1 suppression [ 456 ]. Similarly, Bim mediates β-cell death upon Insulin receptor substrate 2 (IRS2) deficiency [ 457 ]. Dexamethasone induced Pdx1 downregulation and Bim activation in β-cells by a mechanism dependent on glucocorticoid receptor activation, but independent of FoxO1 [ 458 ]. Diabetogenic conditions lead to the upregulation of Mst1 in β-cells of both human and mouse islets, resulting in the induction of Bim expression and apoptosis [ 459 ]. Mst1 phosphorylates Pdx1 at Thr11, resulting in ubiquitination and degradation of Pdx-1 [ 459 ].

The expression of Bim was reduced in macrophages from synovial tissue of patients suffering from rheumatoid arthritis [ 461 ]. Macrophages from Bim KO mice displayed elevated expression of inflammation markers and secreted more IL-1β in response to LPS or thioglycollate, suggesting for a role of Bim in limiting the activation of macrophages [ 461 ]. A Bim-BH3 mimetic (TAT-BH3) peptide induced apoptosis of myeloid cells and reduced the symptoms of arthritis in mice treated with K/BxN serum [ 461 ].

Epithelial cells organize into cyst-like structures that contain a spherical monolayer of cells that enclose a central lumen. Reginato et al. [ 325 ] showed a requirement for Bim in selectively triggering apoptosis of the centrally localized acinar cells, leading to controlled lumen formation. In a three-dimensional basement membrane culture model in which mammary epithelial cells form hollow, acinus-like structures, Bim was not detectable during early stages of mammary acinar morphogenesis, but was later highly upregulated in acinar cells [ 325 ]. Inhibition of Bim expression by RNA interference transiently blocked luminal apoptosis and delayed lumen formation [ 325 ]. Oncogenes that induce acinar luminal filling, such as ErbB2 and v-Src, suppressed Bim expression through ERK-dependent pathway [ 325 ]. Bim is also involved in the death of alveolar cells during involution of the mammary gland after cessation of lactation [ 478 ].

Reduced Bim expression is a hallmark for carcinogenesis. Tumor cells have evolved different mechanisms to suppress Bim expression and/or activity thereby overcoming the apoptotic barrier that else would have led to their eradication. The efficacy of many anti-cancer drugs depend on Bim, and insufficient Bim induction or Bim function is often an underlying cause of therapy failure. Many cancer cells have developed one or more mechanisms for preventing Bim from acting, intervention of which may result in the reactivation of the apoptotic process. Determination of the specific survival dependency pathway in each cancer case is important for choosing the right targeting drug therapy. 4.11.1. A Tumor Suppressive Function of Bim Several studies suggest that Bim functions as a tumor suppressor. In mice, inactivation of one allele of Bim accelerates Myc-induced B cell leukemia [ 480 ]. In this experimental system, Bim is induced by Myc, and inactivation of one Bim allele was sufficient for Myc-induced tumor development. Whereas the p19 Arf /p53 pathway is frequently mutated in tumors arising in Bim +/+ Eμ-Myc mice, it was unaffected in most Bim-deficient tumors, indicating that Bim reduction is an effective alternative to loss of p53 function [ 480 ]. Similarly, Bim deficiency in mice overexpressing the Eμ-vAbl oncogene accelerated the development of plasmacytomas [ 481 ]. The v-Abl-expressing plasmacytomas frequently harbor a rearranged c-Myc gene [ 481 ]. Another example is the formation and growth of tumors derived from baby mouse kidney epithelial (BMK) cells transformed by E1A and dominant negative p53, that is facilitated by simultaneous Bim deficiency [ 482 ], suggesting a role for Bim in preventing epithelial cancer cell formation. These authors [ 482 ] also showed that Bim-deficiency led to paclitaxel-resistant tumor cells. An interesting study by Merino et al. [ 163 ] showed that Bim deficiency in PyMT (MMTV-Polyoma middle-T) female mice didn't affect primary breast tumor growth, but rather increased the survival of metastatic cells within the lung. They further showed a correlation between Bim expression and the EMT transcription factor SNAI2 at the proliferative edge of the tumor. Chromatin immunoprecipitation analysis suggests that Bim is a target of SNAI2 [ 163 ]. These data suggest a role for Bim in the suppression of breast cancer metastasis. Bim is frequently eliminated in human cancer, providing a growth advantage to the tumor cells. For instance, homozygous deletions of the Bim locus have been observed in mantel cell lymphomas and methylation of the bim promoter has been found in certain Burkitt's lymphoma and diffuse large B cell lymphoma [ 246 , 483 ]. Bim is downregulated in a subset of colorectal cancers through Cox2/PGE 2 signaling that activates the c-Raf/MEK/ERK1/2 pathway [ 484 ]. Treatment of Cox2-expressing colorectal carcinoma with selective Cox2 inhibitors induced Bim expression [ 484 ]. Downregulation of Bim expression was associated with tumor progression towards an anchorage-independent phenotype [ 484 ]. When the ERK pathway is constitutively activated in tumor cells, Bim is degraded, and confers chemoresistance. This was demonstrated by Tan et al. [ 482 ] where apoptotic sensitivity to paclitaxel could be restored in H-Ras/ERK-dependent tumor cells, by treatment with the proteasome inhibitor bortezomib that promoted Bim-dependent tumor regression [ 482 ]. Bortezomib also sensitized prostate and colon cancer cells to TRAIL-mediated cell death through a mechanism dependent on Bik and Bim [ 485 ]. Similarly, Bim levels are low in NSCLC cells harboring activating EGFR mutations [ 306 - 309 ]. Inhibition of EGFR tyrosine kinase activity by drugs such as gefitinib, results in Bim EL accumulation and induction of apoptosis. Metastatic melanomas had lower levels of Bim than dysplastic nevi [ 486 ]. Reduced Bim expression was significantly correlated with poor 5-year survival of melanoma patients [ 486 ]. The single point mutation V600E in B-Raf, frequently observed in melanoma, inhibited Bim expression through ERK-dependent phosphorylation and degradation [ 314 , 487 ]. This might be one mechanism for the anti-apoptotic actions of B-Raf V600E [ 487 ]. Petrocca et al. [ 175 ] observed that Bim expression is higher in gastric primary tumors than normal tissues. This might be due to upregulation of Bim by oncogenic stress stimuli [ 480 ]. Bim EL and Bim L were also found to be expressed at higher levels in prostate cancer cells than normal prostate tissue [ 30 ].

The Bcr-Abl fusion protein overexpressed in CML suppresses Bim expression [ 305 ]. The Bcl-Abl kinase inhibitor imatinib (Gleevec/STI-571) induces killing of Bcr-Abl + CML through a mechanism that depends on Bim and Bad, but not Bmf or Puma [ 304 , 305 , 501 ]. Cytokines antagonized imatinib- and nilotinib-induced apoptosis that was related to reduced Bim accumulation [ 501 ]. Drug resistance due to loss of Bim could be overcome by the BH3 mimetic ABT-737 [ 304 ]. Similarly, insulin-like growth factor 1 (IGF-1), which acts as a growth and survival factor in multiple myeloma (MM), downregulates Bim expression in these malignant cells [ 247 ]. IGF-1 prevented Bim transcription through Akt-mediated inactivation of FoxO3a, and increased proteasomal degradation of Bim EL by activating MAPK [ 247 ]. In addition, IGF-1 reduced histone H3 tail Lys9 (H3K9) acetylation and increased H3K9 dimethylation, both contributing to Bim silencing [ 247 ]. The deacetylase inhibitor LBH589 induced Bim expression [ 247 ]. High levels of Baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5/Survivin) expression in multiple myeloma cells correlates with reduced Bim transcription in response to IL-6 deprivation and shorter overall survival of patients [ 502 ]. Bim was shown to be epigenetically silenced in NPM/ALK + anaplastic large cell lymphoma (ALCL) [ 503 ]. The Bim silencing involved the recruitment of the methyl-CpG-binding protein MeCP2 and the SIN3a/histone deacetylase 1/2 (HDAC1/2) co-repressor complex [ 503 ]. The deacetylase inhibitor (HDACi) Trichostatin A restored histone acetylation, with concomitant upregulation of Bim [ 503 ]. While NPM/ALK induces de novo Bim 5′UTR methylation, its later silencing or inhibition by using crizotinib (PF02341066) did not lead to reacetylation of the Bim locus [ 503 ]. Treatment resistance in T cell acute lymphoblastic leukemia (T-ALL) is associated with PTEN deletions which result in the activation of the PI3K-Akt pathway, and Notch1-mediated c-Myc overexpression [ 504 , 505 ]. Treatment with c-Myc or PI3K/Akt pathway inhibitors induced Bim upregulation and apoptosis, indicating that Bim is repressed downstream of c-Myc and PI3K/Akt in high-risk T-ALL [ 506 ]. Restoring Bim function in human T-ALL cells using a stapled peptide mimetic of the Bim BH3 domain had beneficial therapeutic effect [ 506 ]. Multiple myeloma cells often express high basal Bim levels that are important for bortezomib-induced apoptosis [ 507 ]. Repeated exposure to bortezomib led to marked Bim downregulation, Mcl-1 upregulation and drug resistance [ 507 , 508 ]. The drug resistance could be overcome by combining HDAC inhibitors that upregulate Bim, with ABT-737 that releases Bim from the anti-apoptotic proteins [ 507 ]. Chloroquine, that interrupts the autophagic process, could further enhance the cytotoxic effect [ 507 ]. 3-Phosphoinositide protein kinase 1 (PDPK1) that is frequently activated in multiple myeloma cells, suppresses Bim expression and is associated with reduced overall survival [ 509 ]. IL-6 and IGF-1 in the microenvironment further promote drug resistance through activation of the PI3K/Akt pathway that leads to Bim downregulation and Mcl-1 upregulation [ 86 ]. Similarily, the CD28 receptor on multiple myeloma cells promote survival following interaction with its ligand CD80/CD86 on dendritic cells that is related to PI3K activation and Bim downregulation [ 510 ]. Interruption of the CD28-CD80/CD86 interaction sensitized multiple myeloma cells to chemotherapy [ 510 ]. Multiple myeloma cells also show enhanced NFκB activity, and the NFκB target gene YY-1 is often hyperexpressed resulting in transcriptional repression of Bim transcription [ 205 ]. B cell receptor (BCR)- and microenvironmental-derived signals promote survival of chronic lymphoblastic leukemia (CLL) cells through increased proliferation and decreased apoptosis. Surface IgM stimulation increased phosphorylation of Bim EL and/or Bim L by an MEK1/2-dependent mechanism [ 511 ]. Other studies have shown that BCR engagement in CLL leads to Akt activation in addition to ERK1/2 activation, both pathways being pro-survival [ 512 ]. CLL cells are also characterized by high expression of Mcl-1 that antagonizes the pro-apoptotic effects of Bim [ 511 , 512 ], and high Mcl-1 expression is associated with poor clinical outcome [ 513 ]. This is in contrast to normal B cells where Bim mediates BCR-induced apoptosis [ 36 ], unless the MEK/ERK signaling pathway is activated by mitogenic stimuli [ 514 ]. Bim is also highly sequestered to Bcl-2 in CLL cells that make Bim rapidly available upon treatment with the BH3 mimetics ABT-737 [ 515 ]. Also, ALL cells with Bcl-2 dependency respond well to ABT-737 [ 516 ].

Bim expression was found to be lost in a large part of renal cell carcinoma (RCC) [ 539 ]. Apoptosis sensitivity correlated with Bim protein levels [ 539 ]. Inhibition of histone deacetylation restored Bim expression in RCC cell lines [ 539 ]. Bim expression was lower in AJCC II-IV stages of melanoma than in AJCC I-II stages [ 540 ]. Loss of PTEN in B-Raf V600E -mutated melanoma cells confers resistance to the B-Raf inhibitor PLX4720 through the suppression of Bim expression [ 541 ]. PLX4720 stimulated Akt activity in PTEN − , but not in PTEN + cells [ 541 ]. The treatment resistance of PTEN − cells could be overcome by co-treatment of PLX4720 with a PI3K inhibitor that leads to enhanced Bim expression [ 541 ]. G1P3, a survival protein induced by interferons and a contributor to poor outcomes in estrogen receptor (ER)-positive breast cancer patients, attenuated the induction of Bim [ 542 ]. Elevated expression of G1P3 was associated with decreased relapse-free and overall survival in ER-positive breast cancer patients [ 542 ]. Abnormal overexpression of the chaperone-associated E3-ligase C terminus of Hsc70-interacting protein (CHIP) induced apoptosis resistance in breast cancer cells by activating the Akt pathway with subsequent prevention of FoxO-dependent Bim and PTEN transcription [ 543 ]. CHIP activates the Akt pathway through targeting PTEN for proteasomal degradation [ 543 , 544 ]. Pyruvate kinase M2 (PKM2) overexpressed in hepatocellular carcinoma promotes Bim degradation and is associated with poor outcome [ 545 ]. Depletion of PKM2 induced apoptosis that could be prevented by simultaneous silencing of Bim [ 545 ]. Bim expression was low or intermediate in 64% of EGFR mutation-positive NSCLC and high in 36% of the patients. Those with higher Bim expression showed longer progression-free survival when treated with the EGFR tyrosine kinase inhibitor erlotinib and longer overall survival [ 546 - 548 ]. EGFR signaling prevents Bim expression through activation of the MAPK-ERK and PI3K-Akt signaling pathways. Imatinib that inhibits c-Kit activity in gastrointestinal stromal tumors, leads to an increase in the dephosphorylated and deubiquitinated form of Bim in addition to increased FoxO3a-induced Bim transcription [ 549 ]. Overexpression of the differentiation-related gene-1 (Drg1) promotes metastasis of colorectal cancer and confers resistance to the topoisomerase inhibitor irinotecan (CPT-11) [ 550 ]. Drg1 interacts with Bim and negatively regulates its stability by promoting its interaction with the ElonginB-Cullin2-CIS ubiquitin-protein ligase complex resulting in proteasomal degradation [ 550 ]. In the absence of Drg1, Bim was stabilized and bound more abundantly to Hsp70, thereby sensitizing the tumor cells to irinotecan [ 550 ]. Similarily, the Receptor for activated C kinase 1 (RACK1) has been shown to confer paclitaxel resistance to breast cancer cells by binding to both the dynein light chain 1 and Bim EL upon exposure to paclitaxel [ 551 ]. RACK1 then promotes the degradation of Bim EL through the ElonginB/C-Cullin2-CIS complex [ 551 ]. RACK1 is frequently overexpressed in solid tumors and the CIS protein level was negatively correlated with Bim EL in cancer specimens [ 551 ]. 4.11.5.1. Role of Bim in TGFβ-induced apoptosis TGFβ is a pleiotropic cytokine that can induce various signal transduction pathways, ultimately leading to cell growth, apoptosis or tumor progression, dependent on the cellular context [ 552 ] (Figure 10 ). TGFβ induces apoptosis of normal epithelial cells, osteoclasts and lymphoid cells by a mechanism that depends on Bim and Bmf [ 42 , 152 , 153 , 166 , 553 - 555 ]. Acquisition of resistance to TGFβ-induced apoptosis is a critical step for carcinogenesis in many organs. Figure 10 The decision between TGFβ-induced apoptosis and cell transformation TGFβ may induce either apoptosis or cell transformation dependent on the cellular context (Section 4.11.5.1). TGFβ induces Bim expression by activating the Runx1/2/3, Smad3/4 and FoxO3a transcription factors. Also, BDH was found to be important for Bim induction. The simultaneous induction of DUSP4 that antagonizes ERK signaling, leads to stabilization of the Bim protein. TGFβ may also promote cell survival through Akt and MSK1-dependent mechanisms. Upon oncogenic switch, Bim expression is downregulated by reduced transcription, increased degradation and inhibition of Bim translation by the oncomiRs miR-106∼25 and miR-181. miR-106∼25 is upregulated by E2F1, c-Myc and Six1, while TGFβ itself may upregulate miR-181. The transcription factors Smad3/4 and Runx1/3, the p38 MAPK signaling pathway, and the generation of ROS are all involved in the TGFβ-mediated upregulation of Bim transcription [ 152 , 164 , 553 ]. Runx1 cooperates with FoxO3 to transcriptionally induce Bim [ 165 ]. TGFβ also induces the expression of the MAPK phosphatase MKP2/DUSP4 to rapidly increase Bim EL by inactivation of ERK1/2 [ 166 ]. The Birt-Hogg-Dubé (BHD)

The use of BH3 mimetics is hampered by high hepatotoxicity, which limits their applications. Also, the dependency on sufficient Bim expression for apoptosis induction makes BH3 mimetics unsuitable for overcoming tumor resistance caused by low or absent Bim expression [ 577 ]. In many cases, Bim expression is repressed through activation of the PI3K-Akt-mTOR and/or Ras-MEK-ERK1/2 signaling pathways, and inhibition of these pathways might be sufficient for Bim upregulation and induction of apoptosis. Usually combined treatment of drugs targeting different pathways is advantageous. The mTOR inhibitors rapamycin (Sirolimus) and RAD001 (Everolimus) have long been known to increase the sensitivity of malignant hematopoietic cells to chemotherapeutic drugs such as glucocorticoids [ 519 , 533 , 534 , 597 - 599 ], rituximab [ 600 ] and IFNα [ 601 ], and solid tumors to cisplatin [ 602 ], mitoxantrone [ 603 ], carboplatin [ 604 ], vinorelbine [ 604 ] and taxoids [ 603 , 604 ]. One of the mechanisms by which rapamycin sensitizes tumor cells is through downregulation of Mcl-1 with simultaneous upregulation of Bim [ 533 , 534 ]. Inhibition of glucose metabolism or mTORC1 leads to decreased Mcl-1 expression and upregulation of Bim, thereby sensitizing diffuse large B cell leukemic cells to ABT-737 [ 605 ]. As rapamycin inhibits TORC1, but not TORC2, what leads to a feedback loop activating the Akt pathway [ 606 ] and further Mcl-1 stabilization [ 108 ], dual TORC1/TORC2 inhibitors have been developed with enhanced synergistic effect on other chemotherapeutics. In the case of rapamycin sensitization of oral squamous cell carcinoma cells to cisplatin, rapamycin increased FoxO3a protein stability and cisplatin inhibited the feedback activation of Akt by rapamycin [ 602 ]. This resulted in FoxO3a activation and Bim induction [ 602 ]. Similarily, the Akt inhibitor MK-2206 enhanced the anti-tumor effect of rapamycin on neuroblastoma cells [ 607 ]. The dual TORC1/2 inhibitor AZD8055 sensitized colorectal cancers with K-Ras or B-Raf mutations to ABT-263 by suppressing Mcl-1 [ 608 ]. AZD8055 induced apoptosis in laryngeal carcinoma by upregulating Bid, Bad and Bim [ 609 ]. The dual mTORC1/TORC2 inhibitor OSI-027 induced apoptosis in specimens from B cell acute lymphoblastic leukemia, mantle cell lymphoma and marginal zone lymphoma by a mechanism that was dependent on Puma and Bim [ 610 ]. The PI3K/mTOR dual inhibitor NVP-BEZ235 reduced Mcl-1 expression and sensitized ovarian carcinoma cells to ABT-737 through a Bim-dependent mechanism [ 575 ]. Inhibition of the PI3K/Akt pathway also sensitized lymphoid malignancies to glucocorticoid-induced apoptosis, which can be explained by the requirement for both GSK3 and Bim [ 63 ]. Sorafenib, a potent multikinase inhibitor, induces apoptosis of human acute myeloid leukemia (AML) cells through downregulating Mcl-1 and enhancing binding of Bim to Bcl-2 and Bcl-xL [ 611 ]. Sorafenib may also upregulate Bim expression [ 612 ]. Combined treatment of sorafenib with obatoclax or ABT-737 had a synergistic effect in reducing tumor growth [ 611 , 612 ]. Similarly, combining obatoclax with the pan-CDK inhibitor flavopiridol increased apoptosis of both drug-naïve and drug-resistant multiple myeloma cells in a Bim- and Noxa-dependent mechanism [ 613 ]. Flavopiridol inhibited Mcl-1 transcription, but increased transcription of Bim and its binding to Bcl-2 and Bcl-xL [ 613 ]. Obatoclax prevented Mcl-1 recovery and caused release of Bim from Bcl-2, Bcl-xL and Mcl-1, accompanied by activation of Bak and Bax [ 613 ]. Simultaneous targeting of PI3K and mTOR using NVP-BGT226 induced apoptosis in multiple myeloma cells by upregulating Bim [ 614 ]. The growth stimulatory effect of IGF1 and IL-6 on multiple myeloma cells was completely abrogated by NPV-BGT226 [ 614 ]. NVP-BGT226 has also been shown to exert cytotoxic effects against other cancer cell types such as ALK-positive anaplastic large cell lymphoma [ 615 ] and hepatocellular carcinoma [ 616 ] and has entered Phase I/II clinical trials for breast cancer [ 617 ]. Inhibition of EGFR using the tyrosine kinase inhibitors (TKIs) erlotinib or gefitinib could induce apoptosis of NSCLC with mutant constitutively active EGFR (ΔL747-S752 or L858R) through a process dependent on Bim [ 306 , 307 , 309 ]. The three Bim isoforms Bim EL , Bim L and Bim S were induced by erlotinib in single mutated EGFR-sensitive cells, but not in PTEN-deficient or double mutated EGFR (additional T790M mutation)-insensitive cells [ 307 , 308 , 546 ]. Pretreatment mRNA levels of Bim predicted the capacity of EGFR inhibitors to induce apoptosis in EGFR-mutant cancer cells [ 618 ]. TKI-resistance usually develops upon repeated treatment which was shown in some cases to be due to overexpression of paxillin [ 619 ] or neutrophil gelatinase-associated lipocalin (NGAL) [ 620 ]. Both paxillin and NGAL activate ERK, resulting in enhanced Bim degradation and increased Mcl-1 expression [ 619 , 620

In this review we have attempted to survey the various signal transduction pathways regulating the expression and activity of Bim. The multiple regulatory mechanisms affecting Bim, make it critical for determining the cell fate in response to any changes in the cell's microenvironment or in the intrinsic cell signal pathways. Many of these pathways can also affect Mcl-1 expression, often in an inverse manner than Bim, thereby tipping the Bim/Mcl-1 balance towards either apoptosis or survival. As an essential component of the intrinsic apoptotic pathway, alterations in Bim expression affect almost every process in the body from regulating the immune system, the nerve system, β-cell and liver physiology to affecting mammary lumen formation and cancer progression. Therefore there is no wonder why abnormal Bim expression causes a range of pathological conditions (Figure 8 ). As Bim expression is regulated differentially in different cell types, each pathological condition needs to be considered separately. For instance, an increase in intracellular cAMP levels makes malignant lymphoid cells more susceptible to an apoptotic stimulus, while in insulin-producing β-cells and neurons it is protective. Another example of a cell-specific effect is JNK activation. In neuronal cells and β-cells excessive JNK activation is detrimental, activating Bim-dependent cell death, while in lymphoid cells, JNK activation actually antagonizes apoptosis. This cell-specific effect may be explained by Bim sequestration to microtubules in the former cell types where JNK phosphorylates and releases Bim, while in lymphoid cells pre-made Bim is tonically sequestered to anti-apoptotic proteins rather than microtubules. Death of various immune cells can be prevented by respective cytokines. For instance, IL-7 can rescue T cell death, whereas IL-4 and BAFF prevent B cell death. In the context of cancer, increased survival of tumor-specific cytotoxic T cells can be achieved by interrupting the PD-1L/PD-1 or enhancing the 4-1BB signaling using specific antibodies, thereby increasing the anti-tumor effect [ 410 ]. On the contrary, in autoimmune diseases, elimination of autoreactive T and B cells is desirable. Several approaches have been examined that can increase apoptosis of autoreactive immune cells. These include the use of ABT-737 mimetics [ 640 ], monoclonal antibody against IL-6R (e.g., tocilizumab or siltiximab) [ 641 , 642 ], mTOR inhibition [ 643 ] or activation of the PD-1 pathway [ 644 ], all tipping the Bim/Mcl-1 balance in favor of Bim. Another approach is to prevent the death of target cells affected by the autoimmune cells. This is best exemplified by the intrinsic higher β-cell death in diabetes-predisposed patients that triggers insulitis which, in turn, promotes further β-cell death, thereby forming a bad negative feedback loop. As we have learned, some of the diabetes susceptibility genes e.g., GLIS3, PTPN2, BACH2 and Cathepsin H, regulate Bim expression. Thus, preventing Bim upregulation in β-cells should reduce the propensity to develop diabetes. Similarly, preventing β-amyloid aggregation should restrain the development of Alzheimer's disease. On the other side of the pathological spectrum lies the cancer issue. Each tumor cell has often developed its own characteristic dependency on specific protein kinases that antagonizes Bim expression, while fortifying Mcl-1 expression. Enormous efforts have been made to develop drugs that specifically target the specific upstream kinase responsible for tumor cell survival. Classical examples are the Bcr-Abl/c-Kit inhibitor imatinib, the EGFR inhibitors erlotinib and gefitinib, the Flt3 inhibitor ponatinib and the B-Raf V600E inhibitor vemurafenib, all inducing Bim expression. Although initial clinical response is often seen when using these drugs, adaptive resistance mechanisms frequently develop, leading to cancer recurrence. The main resistance mechanisms involve the single or combined reactivation of the PI3K-Akt-mTOR and Ras-Raf-MEK-ERK1/2 survival pathways that prevent Bim expression. The current trend is to combine the specific targeting therapies with inhibitors targeting the downstream survival protein kinases. This combined treatment ensures proper reactivation of Bim. Other approaches include the combined treatment of a tyrosine kinase inhibitor with an HDAC inhibitor that aims to synergistically increase Bim expression. In summary, drug-induced apoptosis of tumor cells that depends on the intrinsic apoptotic pathway, needs a sufficient amount of Bim. Any mechanism that prevents Bim expression in cancer cells should therefore be targeted.