BAY 43-9006, a multikinase inhibitor that targets Raf, prevents tumor cell proliferation in vitro and inhibits diverse human tumor xenografts in vivo. The mechanism of action of BAY 43-9006 remains incompletely defined. In the present study, the effects of BAY 43-9006 on the antiapoptotic Bcl-2 family member Mcl-1 were examined. Treatment of A549 lung cancer cells with BAY 43-9006 diminished Mcl-1 levels in a time- and dose-dependent manner without affecting other Bcl-2 family members. Similar BAY 43-9006-induced Mcl-1 downregulation was observed in ACHN (renal cell), HT-29 (colon), MDA-MB-231 (breast), KMCH (cholangiocarcinoma), Jurkat (acute T-cell leukemia), K562 (chronic myelogenous leukemia) and MEC-2 (chronic lymphocytic leukemia) cells. Mcl-1 mRNA levels did not change in BAY 43-9006-treated cells. Instead, BAY 43-9006 enhanced proteasome-mediated Mcl-1 degradation. This Mcl-1 downregulation was followed by mitochondrial cytochrome c release and caspase activation as well as enhanced sensitivity to other proapoptotic agents. The caspase inhibitor Boc-D-fmk inhibited BAY 43-9006-induced caspase activation but not cytochrome c release. In contrast, Mcl-1 overexpression inhibited cytochrome c release and other features of BAY 43-9006-induced apoptosis. Conversely, Mcl-1 downregulation by short hairpin RNA enhanced BAY 43-9006-induced apoptosis. Collectively, these findings demonstrate that drug-induced Mcl-1 downregulation contributes to the proapoptotic effects of BAY 43-9006.

Interaction between the hydrophobic Bak BH3-binding groove and the BH3 domain of activator proteins is a key step in initiating Bak oligomerization and activation.

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Nonalcoholic steatohepatitis is characterized by hepatic steatosis, elevated levels of circulating free fatty acids (FFA), endoplasmic reticulum (ER) stress, and hepatocyte lipoapoptosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor 5 (DR5) is significantly elevated in patients with nonalcoholic steatohepatitis, and steatotic hepatocytes demonstrate increased sensitivity to TRAIL-mediated cell death. Nonetheless, a role for TRAIL and/or DR5 in mediating lipoapoptotic pathways is unexplored. Here, we examined the contribution of DR5 death signaling to lipoapoptosis by free fatty acids. The toxic saturated free fatty acid palmitate induces an increase in DR5 mRNA and protein expression in Huh-7 human hepatoma cells leading to DR5 localization into lipid rafts, cell surface receptor clustering with subsequent recruitment of the initiator caspase-8, and ultimately cellular demise. Obesity and insulin resistance, cardinal features of the metabolic syndrome, are associated with enhanced lipolysis in adipose tissue (1, 2). This excessive lipolysis results in increased serum concentrations of free fatty acids (FFA), 2 augmenting their delivery to non-adipose tissues such as liver, heart, and pancreatic ␤-cells (2). Inundation of these tissues with FFA overwhelms fatty acid oxidative pathways with toxic cellular consequences. An advanced response to toxic FFA is cellular demise by apoptosis, termed lipoapoptosis (3). Hepatocyte lipotoxicity is particularly germane to injury in the liver, where it contributes to the syndrome of nonalcoholic fatty liver disease (NAFLD) (4). For example, the magnitude of hepatocyte lipoapoptosis correlates with hepatic disease severity (4). Thus, the mechanisms initiating lipoapoptosis are of biomedical interest and human health relevance. Lipotoxicity is likely multifactorial. Sustained endoplasmic reticulum (ER) stress (5-7), c-Jun N-terminal kinase (JNK) activation (8, 9), and oxidative stress (10) have all been implicated in lipotoxicity. Despite the fact that death receptors are potent mediators of cytotoxicity (11), especially in hepatic diseases, their contribution to lipotoxicity is incompletely defined. Fas, the prototype death receptor, has been implicated in adipocyte toxicity and inflammation (12), and its hepatic expression is increased in NAFLD (4). Tumor necrosis factor-␣ (TNF-␣) and TNF receptor-1 (TNFR-1) have also been implicated in hepatic steatosis (13)(14)(15). In contrast, the role of TNF-related apoptosis-inducing ligand (TRAIL) and its cognate death receptors (DR)-4 and -5 in lipotoxicity is understudied. Yet, increasing data implicate a critical role for DR5 in lipotoxicity. For example, DR5 expression is increased in steatotic hepatocytes and sensitizes hepatocytes to exogenous TRAIL cytotoxicity (16). Thus, the role of DR5 in lipoapoptosis merits further investigation.In this study, we explored the potential contribution of DR5 death signaling during lipoapoptosis by saturated FFA. The results implicate TRAIL death signaling by DR...

Mcl-1 is an antiapoptoticThe Bcl-2 family member Mcl-1 (myeloid cell leukemia 1) promotes cell viability at a critical upstream point in the apoptotic cascade (1). Mcl-1 and other antiapoptotic members of this family (Bcl-2, Bcl-x L , A1, and Bcl-w) interrupt cell death signals by binding to pro-apoptotic family members (e.g. Bim, Noxa, and others in the case of Mcl-1) (2-5). A distinctive characteristic of Mcl-1 is its short half-life (ϳ2-4 h in various cell types) (6 -8), which probably relates to the presence of a long proline-, glutamic acid-, serine-, and threonine-rich (PEST) 2 region upstream of the Bcl-2 homology domains (9 -11).Alterations that affect Mcl-1 expression can have severe, potentially detrimental consequences. Conditional deletion of Mcl-1 results in bone marrow failure in mice caused by apoptosis of precursor cells (12)(13)(14). Conversely, forced Mcl-1 overexpression predisposes hematopoietic cells to malignant transformation (15). In addition, enhanced Mcl-1 expression has been observed in multiple human cancers, often in association with poor prognosis, disease recurrence, or drug resistance (7, 16 -21). Thus, the mechanisms that regulate Mcl-1 play an important role both in controlling cell survival in normal tissues and in preventing the emergence of tumors.Mcl-1 expression is regulated at the transcriptional and posttranslational levels. The transcription factors SRF/ETS, STAT3, cAMP-responsive element-binding protein, and PU.1 have been found to regulate Mcl-1 gene expression in various cell types (22)(23)(24)(25)(26). At the post-translational level, Mcl-1 is targeted for proteasomal degradation after ubiquitylation by an E3 ligase termed MULE (11). This susceptibility of Mcl-1 to degradation is phosphorylation-dependent. In particular, ERKmediated phosphorylation of Mcl-1 at Thr 163 within the PEST region prolongs the Mcl-1 half-life (6). In contrast, following Thr 163 phosphorylation, Ser 159 phosphorylation by GSK-3␤ reportedly enhances Mcl-1 ubiquitylation and degradation (8). Consistent with the latter results, Akt (cellular homolog of v-Akt/protein kinase B) inhibition, which activates GSK-3␤, results in Mcl-1 down-regulation (7). Mcl-1 is also downregulated by inhibitors of CDK9 that diminish its synthesis (20,27,28).Mcl-1 phosphorylation is also known to be stimulated at other, as-yet unidentified sites during the G 2 /M phase of the * This work was supported by National Institutes of Health Grants DK 59427 (to G. J. G.), CA 69008 (to S. H. K.), and CA 57359 (to R. W. C.) and funds from the Mayo and Palumbo Foundations (to G. J. G.) and the Japanese Society for Science Promotion (to S. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. 2 The abbreviations used are: PEST, proline-, glutamic acid-, serine-, and threonine-rich; CDK, cyclin-dependent kinase; DAPI, 4Ј,6-diamidino-2-phenylindole dihydrochloride; ERK, extracellular signal-regul...

Bcl-2, the founding member of a family of apoptotic regulators, was initially identified as the protein product of a gene that is translocated and overexpressed in greater than 85% of follicular lymphomas (FLs). Thirty years later we now understand that Bcl-2 modulates the intrinsic apoptotic pathway by binding and neutralizing the mitochondrial permeabilizers Bax and Bak as well as a variety of pro-apoptotic proteins, including the cellular stress sensors Bim, Bid, Puma, Bad, Bmf and, under some conditions, Noxa. Despite extensive investigation of all of these proteins, important questions remain. For example, how Bax and Bak breach the outer mitochondrial membrane remains poorly understood. Likewise, how the functions of anti-apoptotic Bcl-2 family members such as eponymous Bcl-2 are affected by phosphorylation or cancer-associated mutations has been incompletely defined. Finally, whether Bcl-2 family members can be successfully targeted for therapeutic advantage is only now being investigated in the clinic. Here we review recent advances in understanding Bcl-2 family biology and biochemistry that begin to address these questions.

Key Points• BCL2 mutations in FL correlate with activationinduced cytidine deaminase expression and frequently alter the amino acid sequence of the protein.• Mutations in the BCL2 coding sequence at diagnosis are associated with shortened time to transformation and earlier death due to lymphoma.Follicular lymphoma (FL), an indolent neoplasm caused by a t(14;18) chromosomal translocation that juxtaposes the BCL2 gene and immunoglobulin locus, has a variable clinical course and frequently undergoes transformation to an aggressive lymphoma. Although BCL2 mutations have been previously described, their relationship to FL progression remains unclear. In this study, we evaluated the frequency and nature of BCL2 mutations in 2 independent cohorts of grade 1 and 2 FLs, along with the correlation between BCL2 mutations, transformation risk, and survival. The prevalence of BCL2 coding sequence mutations was 12% in FL at diagnosis and 53% at transformation (P < .0001). The presence of these BCL2 mutations at diagnosis correlated with an increased risk of transformation (hazard ratio 3.6; 95% CI, 2.0-6.2; P < .0001) and increased risk of death due to lymphoma (median survival of 9.5 years with BCL2 mutations vs 20.4 years without; P 5 .012).In a multivariate analysis, BCL2 mutations and high FL international prognostic index were independent risk factors for transformation and death due to lymphoma. Some mutant Bcl-2 proteins exhibited enhanced antiapoptotic capacity in vitro. Accordingly, BCL2 mutations can affect antiapoptotic Bcl-2 function, are associated with increased activationinduced cytidine deaminase expression, and correlate with increased risk of transformation and death due to lymphoma. (Blood. 2015;125(4):658-667) Introduction Follicular lymphoma (FL) has a highly variable clinical course. [1][2][3] Although some patients do well for decades, often with limited therapy, at some point 30% to 50% of patients experience histologic transformation to a more aggressive lymphoma, usually diffuse large B-cell lymphoma (DLBCL). [4][5][6][7][8][9][10][11] This transformation, which is thought to reflect the acquisition of new genetic abnormalities leading to further genomic instability, [12][13][14][15][16] has generally been associated with a poor clinical outcome. 17 Retrospective analyses from the prerituximab era have reported a median survival of only 1 to 2 years after transformation, 18,19 although a recent prospective observational study suggests somewhat better survival after transformation in the rituximab era. 20 At the present time, the FL international prognostic index (FLIPI), which integrates patient characteristics at diagnosis, is the gold standard for predicting FL clinical outcome. 21,22 There is, however, considerable interest in identifying characteristics of the FL cells themselves that might also impact prognosis. 22,23 The BCL2 gene is critical for FL pathogenesis. 24,25 Originally identified because of its translocation to the immunoglobulin heavy chain (IGH) locus as a part of the t(14;18) ...

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