The healthspan of mice is enhanced by killing senescent cells using a transgenic suicide gene. Achieving the same using small molecules would have a tremendous impact on quality of life and the burden of age-related chronic diseases. Here, we describe the rationale for identification and validation of a new class of drugs termed senolytics, which selectively kill senescent cells. By transcript analysis, we discovered increased expression of pro-survival networks in senescent cells, consistent with their established resistance to apoptosis. Using siRNA to silence expression of key nodes of this network, including ephrins (EFNB1 or 3), PI3Kδ, p21, BCL-xL, or plasminogen-activated inhibitor-2, killed senescent cells, but not proliferating or quiescent, differentiated cells. Drugs targeting these same factors selectively killed senescent cells. Dasatinib eliminated senescent human fat cell progenitors, while quercetin was more effective against senescent human endothelial cells and mouse BM-MSCs. The combination of dasatinib and quercetin was effective in eliminating senescent MEFs. In vivo, this combination reduced senescent cell burden in chronologically aged, radiation-exposed, and progeroid Ercc1−/Δ mice. In old mice, cardiac function and carotid vascular reactivity were improved 5 days after a single dose. Following irradiation of one limb in mice, a single dose led to improved exercise capacity for at least 7 months following drug treatment. Periodic drug administration extended healthspan in Ercc1−/Δ mice, delaying age-related symptoms and pathology, osteoporosis, and loss of intervertebral disk proteoglycans. These results demonstrate the feasibility of selectively ablating senescent cells and the efficacy of senolytics for alleviating symptoms of frailty and extending healthspan.
Chronic, low grade, sterile inflammation frequently accompanies aging and age-related diseases. Cellular senescence is associated with the production of proinflammatory chemokines, cytokines, and extracellular matrix (ECM) remodeling proteases, which comprise the senescence-associated secretory phenotype (SASP). We found a higher burden of senescent cells in adipose tissue with aging. Senescent human primary preadipocytes as well as human umbilical vein endothelial cells (HUVECs) developed a SASP that could be suppressed by targeting the JAK pathway using RNAi or JAK inhibitors. Conditioned medium (CM) from senescent human preadipocytes induced macrophage migration in vitro and inflammation in healthy adipose tissue and preadipocytes. When the senescent cells from which CM was derived had been treated with JAK inhibitors, the resulting CM was much less proinflammatory. The administration of JAK inhibitor to aged mice for 10 wk alleviated both adipose tissue and systemic inflammation and enhanced physical function. Our findings are consistent with a possible contribution of senescent cells and the SASP to age-related inflammation and frailty. We speculate that SASP inhibition by JAK inhibitors may contribute to alleviating frailty. Targeting the JAK pathway holds promise for treating age-related dysfunction.JAK/STAT pathway | cellular senescence | ruxolitinib | interleukin-6 | frailty
Senescent cells accumulate in fat with aging. We previously found genetic clearance of senescent cells from progeroid INK-ATTAC mice prevents lipodystrophy. Here we show that primary human senescent fat progenitors secrete activin A and directly inhibit adipogenesis in non-senescent progenitors. Blocking activin A partially restored lipid accumulation and expression of key adipogenic markers in differentiating progenitors exposed to senescent cells. Mouse fat tissue activin A increased with aging. Clearing senescent cells from 18-month-old naturally-aged INK-ATTAC mice reduced circulating activin A, blunted fat loss, and enhanced adipogenic transcription factor expression within 3 weeks. JAK inhibitor suppressed senescent cell activin A production and blunted senescent cell-mediated inhibition of adipogenesis. Eight weeks-treatment with ruxolitinib, an FDA-approved JAK1/2 inhibitor, reduced circulating activin A, preserved fat mass, reduced lipotoxicity, and increased insulin sensitivity in 22-month-old mice. Our study indicates targeting senescent cells or their products may alleviate age-related dysfunction of progenitors, adipose tissue, and metabolism.DOI: http://dx.doi.org/10.7554/eLife.12997.001
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) ...
Bcl-2 is phosphorylated on Ser70 after treatment of cells with spindle poisons. Based on the cellular effects of overexpressing Bcl-2 S70E or S70A mutants, various studies have concluded that Ser70 phosphorylation either enhances or diminishes Bcl-2 function. In the present study, the ability of phosphorylated Bcl-2, as well as the S70E and S70A mutants, to bind and neutralize proapoptotic Bcl-2 family members under cell-free conditions and in intact cells was examined in an attempt to resolve this controversy. Surface plasmon resonance indicated that phosphorylated Bcl-2, Bcl-2 S70E and Bcl-2 S70A exhibit enhanced binding to Bim and Bak compared to unmodified Bcl-2. This enhanced binding reflected a readily detectable conformation change in the loop domain of Bcl-2. Further, Bcl-2 S70E and S70A bound more Bak and Bim than wildtype Bcl-2 in pulldowns and afforded greater protection against several chemotherapeutic agents. Importantly, binding of endogenous Bcl-2 to Bim also increased during mitosis, when Bcl-2 is endogenously phosphorylated; and disruption of this mitotic Bcl-2/ Bim binding with navitoclax or ABT-199, like Bcl-2 downregulation, enhanced the cytotoxicity of paclitaxel. Collectively these results provide not only a mechanistic basis for the enhanced anti-apoptotic activity of phosphorylated Bcl-2, but also an explanation for the ability of BH3 mimetics to enhance taxane sensitivity.
Mitochondrial outer membrane permeabilization (MOMP), a key step in the intrinsic apoptotic pathway, is incompletely understood. Current models emphasize the role of BH3-only BCL2 family members in BAX and BAK activation. Here we demonstrate concentration-dependent BAK autoactivation under cell-free conditions and provide evidence that this autoactivation plays a key role in regulating the intrinsic apoptotic pathway in intact cells. In particular, we show that up to 80% of BAK (but not BAX) in lymphohematopoietic cell lines is oligomerized and bound to anti-apoptotic BCL2 family members in the absence of exogenous death stimuli. The extent of this constitutive BAK oligomerization is diminished by BAK knockdown and unaffected by BIM or PUMA down-regulation. Further analysis indicates that sensitivity of cells to BH3 mimetics reflects the identity of the anti-apoptotic proteins to which BAK is constitutively bound, with extensive BCLX L •BAK complexes predicting navitoclax sensitivity, and extensive MCL1•BAK complexes predicting A1210477 sensitivity. Moreover, high BAK expression correlates with sensitivity of clinical acute myelogenous leukemia to chemotherapy, whereas low BAK levels correlate with resistance and relapse. Collectively, these results inform current understanding of MOMP and provide new insight into the ability of BH3 mimetics to induce apoptosis without directly activating BAX or BAK.
The mechanism of cytotoxicity of farnesyltransferase inhibitors is incompletely understood and seems to vary depending on the cell type. To identify potential determinants of sensitivity or resistance for study in the accompanying clinical trial ( IntroductionFarnesyltransferase inhibitors (FTIs) are currently undergoing extensive clinical testing in various hematologic malignancies. [1][2][3] These agents inhibit farnesyltransferase, an enzyme that transfers the 15-carbon farnesyl group from farnesyl pyrophosphate to a variety of polypeptide acceptors, including the chaperone heat shock protein 40/HDJ-2; the nuclear intermediate filament proteins prelamin A and lamin B; the centromere protein CENP E; and small GTP-binding proteins of the Ras, Rho, and Rheb families. 4,5 Collectively, inhibition of farnesylation of these polypeptides leads to diminished cell proliferation. In addition, FTIs induce cell death in some model systems under certain conditions. These cytotoxic effects have been attributed to FTI-induced inhibition of prosurvival signaling by Akt,6,7 signal transducers and activators of transcription, [8][9][10] mitogen-activated protein kinases (MAPKs), 9,[11][12][13] or the Rheb target mammalian target of rapamycin. 14 Recent work has especially emphasized the role of Rheb inhibition as a mechanism of FTI-induced antilymphoma effects in murine lymphomas and leukemia. 15 Alternatively, it has been suggested that FTIs induce apoptosis by causing up-regulation of the proapoptotic Bcl-2 family members Bax, 16 Bak, 17 or Puma. 18 Although FTIs were initially developed based on the premise that inhibition of farnesylation would abrogate signaling by mutant Ras proteins, 19 these agents have demonstrated little efficacy in solid tumors. [20][21][22] In contrast, tantalizing activity was observed in several hematologic malignancies. [1][2][3] In particular, the orally bioavailable nonpeptidimimetic FTI tipifarnib 23 demonstrated activity in adults with acute leukemia. The initial phase 1 trial not only established a maximum tolerated dose in patients with relapsed and refractory acute leukemias but also determined that tipifarnib levels in bone marrow were 1.6-8 nmol/mg of tissue at this dose, demonstrated FT inhibition in leukemia cells in situ, and provided evidence of activity in relapsed AML. 24 Subsequent phase 2 and phase 3 studies have demonstrated response rates of 11%-23% in elderly patients with previously untreated poor risk acute myeloid leukemia (AML). 25,26 In an effort to select the subset of AML patients most likely to respond, Raponi et al empirically identified a 2-transcript signature, characterized by a high ratio of mRNA encoding the Ras guanine nucleotide exchange factor RasGRP1 27 relative to mRNA encoding the repair protein aprataxin, that had a 92% negative predictive value and a 28% positive predictive value in 2 single-agent phase 2 tipifarnib AML trials. 28 Based on these results, gene signature-guided trials of tipifarnib in acute leukemia are being initiated.Tipifarnib also has...
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