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The eIF2-alpha kinase HRI is a novel therapeutic target in multiple myeloma
Abstract
Dexamethasone (dex) induces apoptosis in multiple myeloma (MM) cells and is a frontline treatment for this disease. However resistance to dex remains a major challenge and novel treatment approaches are needed. We hypothesized that dex utilizes translational pathways to promote apoptosis in MM and that specific targeting of these pathways could overcome dex-resistance. Global unbiased profiling of mRNA translational profiles in MM cells treated with or without dex revealed that dex significantly repressed eIF2 signaling, an important pathway for regulating ternary complex formation and protein synthesis. We demonstrate that dex induces the phosphorylation of eIF2α resulting in the translational upregulation of ATF4, a known eIF2 regulated mRNA. Pharmacologic induction of eIF2α phosphorylation via activation of the heme-regulated eIF2α kinase (HRI) induced apoptosis in MM cell lines and in primary MM cells from patients with dex-resistant disease. In addition, co-culture with marrow stroma failed to protect MM cells from apoptosis induced by targeting the eIF2 pathway. Combination therapy with rapamycin, an mTOR inhibitor, and BTdCPU, an activator of HRI, demonstrated additive effects on apoptosis in dex-resistant cells. Thus, specific activation of the eIF2α kinase HRI is a novel therapeutic target in MM that can augment current treatment strategies.
... Experimentally, dominant negative PKR mutants, or eIF2α mutants that cannot be phosphorylated on serine 51, have been found to induce malignant transformation in NIH 3T3 cells [35]. In many cancers, eIF2α expression is increased when compared to normal tissue [32,36,37] and likely reflects the heightened translational demand in tumor cells. In some early-stage cancers, an increase in eIF2α kinase activity is observed and is associated with improved survival [38]. ...
... In a separate study, BTdCPU also demonstrated activity in multiple myeloma (MM) cells in vitro. In this study, BTdCPU treatment lead to the rapid phosphorylation of eIF2α, expression of CHOP, and induction of apoptosis in both dexamethasone-sensitive andresistant cells [32]. Proteasome inhibitors, which have potent clinical activity in MM, also induce eIF2α phosphorylation and expression of CHOP in MM cells [53,58]. ...
... Thus, while the induction of eIF2α phosphorylation via HRI is a viable anticancer strategy, the inhibition of eIF2α phosphorylation to augment effectiveness Burwick of PI3K-Akt blockade has also been proposed as a potential therapeutic combination [65]. Whether HRI activation results in similar cross talk is less clear, though in one study inhibition of mTOR augmented anticancer efficacy in combination with HRI activation in vitro [32]. The upregulation of ATF4 via eIF2α phosphorylation may itself serve to attenuate mTOR activity through the induction of mTOR repressors including Sestrin2 and REDD1 [66][67][68]. ...
Introduction: The eIF2α kinase heme-regulated inhibitor (HRI) is one of four well-described kinases that phosphorylate eIF2α in response to various cell stressors, resulting in reduced ternary complex formation and attenuation of mRNA translation. Although HRI is well known for its role as a heme sensor in erythroid progenitors, pharmacologic activation of HRI has been demonstrated to have anti-cancer activity across a wide range of tumor sub-types. Here, the potential of HRI activators as novel cancer therapeutics is explored.
Areas covered: We provide an introduction to eIF2 signaling pathways in general, and specifically review data on the eIF2α kinase HRI in erythroid and non-erythroid cells. We review aspects of targeting eIF2 signaling in cancer and highlight promising data using HRI activators against tumor cells.
Expert opinion: Pharmacologic activation of HRI inhibits tumor growth as a single agent without appreciable toxicity in vivo. The ability of HRI activators to provide direct and sustained eIF2-alpha phosphorylation without inducing oxidative stress or broad eIF2-alpha kinase activation may be especially advantageous for tolerability. Combination therapy with established therapeutics may further augment anti-cancer activity to overcome disease resistance.
... Expression of HRI was previously thought to be restricted to erythroid cells [36], however, recent findings suggest that HRI may be expressed more broadly, including in some hematologic malignancies such as MM and Ph + B-cell ALL (B-ALL), where HRI activity has been implicated in the regulation of apoptosis [39,40]. Indeed, in MM, pharmacological activation of HRI led to increased phospho-eIF2α and caused cell death in both cell lines and patient samples [39]. ...
... Expression of HRI was previously thought to be restricted to erythroid cells [36], however, recent findings suggest that HRI may be expressed more broadly, including in some hematologic malignancies such as MM and Ph + B-cell ALL (B-ALL), where HRI activity has been implicated in the regulation of apoptosis [39,40]. Indeed, in MM, pharmacological activation of HRI led to increased phospho-eIF2α and caused cell death in both cell lines and patient samples [39]. Similarly, pharmacological activation of HRI in B-ALL cell lines resulted in a suppression of the pro-survival protein MCL1 (a critical effector of the mitochondrial apoptotic pathway) to sensitize B-ALL cells to the activity of BH3-mimetics (potent inducers of apoptosis) [40]. ...
While numerous targeted therapies have been recently adopted to improve the treatment of hematologic malignancies, acquired or intrinsic resistance poses a significant obstacle to their efficacy. Thus, there is increasing need to identify novel, targetable pathways to further improve therapy for these diseases. The integrated stress response is a signaling pathway activated in cancer cells in response to both dysregulated growth and metabolism, and also following exposure to many therapies that appears one such targetable pathway for improved treatment of these diseases. In this review, we discuss the role of the integrated stress response in the biology of hematologic malignancies, its critical involvement in the mechanism of action of targeted therapies, and as a target for pharmacologic modulation as a novel strategy for the treatment of hematologic malignancies.
... The authors also showed that BTdCPU potently inhibited the growth of xenografted human breast carcinoma without any major toxicity [50]. Burwick et al. also reported that diarylurea compound BTdCPU treatment induced phosphorylation of eIF2α in multiple myeloma (MM) cells and upregulated mRNA and protein levels of CHOP [55], which is known to play a role in ER stress-induced apoptosis [56,57]. In addition, it was demonstrated that BTdCPU induced cell death in both dex-sensitive (MM1.S, H929) and dexresistant (MM1.R, RPMI8266, U266) MM cells [55]. ...
... Burwick et al. also reported that diarylurea compound BTdCPU treatment induced phosphorylation of eIF2α in multiple myeloma (MM) cells and upregulated mRNA and protein levels of CHOP [55], which is known to play a role in ER stress-induced apoptosis [56,57]. In addition, it was demonstrated that BTdCPU induced cell death in both dex-sensitive (MM1.S, H929) and dexresistant (MM1.R, RPMI8266, U266) MM cells [55]. Zhang and coworkers recently reported 1-phenyl-3-((1,4-trans)-4 phenoxycyclohexyl)urea series as promising new activators of HRI (Fig. 3). ...
Heme-regulated inhibitor (HRI) kinase is a serine–threonine kinase, controlling the initiation of protein synthesis via phosphorylating α subunit of eIF2 on serine 51 residue, mainly in response to heme deprivation in erythroid cells. However, recent studies showed that HRI is also activated by several diverse signals, causing dysregulations in intracellular homeostatic mechanisms in non-erythroid cells. For instance, it was reported that the decrease in protein synthesis upon the 26S proteasomal inhibition by MG132 or bortezomib is mediated by increased eIF2α phosphorylation in an HRI-dependent manner in mouse embryonic fibroblast cells. The increase in eIF2α phosphorylation level through the activation of HRI upon 26S proteasomal inhibition is believed to protect cells against the buildup of misfolded and ubiquitinated proteins, having the potential to trigger the apoptotic response. In contrast, prolonged and sustained HRI-mediated eIF2α phosphorylation can induce cell death, which may involve ATF4 and CHOP expression. Altogether, these studies suggest that HRI-mediated eIF2α phosphorylation may be cytoprotective or cytotoxic depending on the cells, type, and duration of pharmacological agents used. It is thus hypothesized that both HRI activators, inducing eIF2α phosphorylation or HRI inhibitors causing disturbances in eIF2α phosphorylation, may be effective as novel strategies in cancer treatment if the balance in eIF2α phosphorylation is shifted in favor of autophagic or apoptotic response in cancer cells. It is here aimed to review the role of HRI in various biological mechanisms as well as the therapeutic potentials of recently developed HRI activators and inhibitors, targeting eIF2α phosphorylation in cancer cells.
... salubrinal, an inhibitor of GaDD34, prevented eiF2α-P dephosphorylation, increasing the sensitivity of resistant MCF-7 cells to doxorubicin 281 . treatment with 1-(benzo[d] [1,2,3]thiadiazol-6-yl)-3-(3,4-dichlorophenyl)urea (BtdCPu) activated haem-regulated inhibitor (Hri) in dexamethasone-resistant multiple myeloma cell lines and patient-derived primary cells, inducing apoptosis via eiF2α-P 282 . an additive effect was observed for treatment of dexamethasone-resistant multiple myeloma with a combination of BtdCPu and rapamycin 282 . ...
... treatment with 1-(benzo[d] [1,2,3]thiadiazol-6-yl)-3-(3,4-dichlorophenyl)urea (BtdCPu) activated haem-regulated inhibitor (Hri) in dexamethasone-resistant multiple myeloma cell lines and patient-derived primary cells, inducing apoptosis via eiF2α-P 282 . an additive effect was observed for treatment of dexamethasone-resistant multiple myeloma with a combination of BtdCPu and rapamycin 282 . Finally, svC112, an inhibitor of translation elongation, showed strong antiproliferative effects on head and neck squamous cell carcinoma cells by targeting cancer stem cells and synergizing with radiation therapy 283 . in conclusion, targeting several circuits of translational control in combination with current standard-of-care cancer therapies opens new windows of opportunity to overcome drug resistance and increase options for precision-based medical care. ...
Translational control of mRNAs during gene expression allows cells to promptly and dynamically adapt to a variety of stimuli, including in neoplasia in response to aberrant oncogenic signalling (for example, PI3K–AKT–mTOR, RAS–MAPK and MYC) and microenvironmental stress such as low oxygen and nutrient supply. Such translational rewiring allows rapid, specific changes in the cell proteome that shape specific cancer phenotypes to promote cancer onset, progression and resistance to anticancer therapies. In this Review, we illustrate the plasticity of mRNA translation. We first highlight the diverse mechanisms by which it is regulated, including by translation factors (for example, eukaryotic initiation factor 4F (eIF4F) and eIF2), RNA-binding proteins, tRNAs and ribosomal RNAs that are modulated in response to aberrant intracellular pathways or microenvironmental stress. We then describe how translational control can influence tumour behaviour by impacting on the phenotypic plasticity of cancer cells as well as on components of the tumour microenvironment. Finally, we highlight the role of mRNA translation in the cellular response to anticancer therapies and its promise as a key therapeutic target.
... Burwick et al., for instance, identified that Dex treatment increased the phosphorylation of eIF2α in MM1.S cells, which inhibited translation initiation [88]. Pharmacological induction of eIF2α phosphorylation using BTdCPU further induced cell death in Dex-responsive and Dex-resistant MM cell lines and primary MM cells. ...
... Pharmacological induction of eIF2α phosphorylation using BTdCPU further induced cell death in Dex-responsive and Dex-resistant MM cell lines and primary MM cells. Even co-cultures of Dex-sensitive or Dex-resistant MM cell lines with BM stromal or endothelial cells were not able to protect the MM cells from a killing by BTdCPU [88]. ...
Glucocorticoids are essential drugs in the treatment protocols of lymphoid malignancies. These steroidal hormones trigger apoptosis of the malignant cells by binding to the glucocorticoid receptor (GR), which is a member of the nuclear receptor superfamily. Long term glucocorticoid treatment is limited by two major problems. The development of glucocorticoid-related side effects, which hampers patient quality of life, and the emergence of glucocorticoid resistance, which is a gradual process that is inevitable in many patients. This emphasizes the need to reevaluate and optimize the widespread use of glucocorticoids in lymphoid malignancies. To achieve this goal, a deep understanding of the mechanisms governing glucocorticoid responsiveness is required, yet, a recent comprehensive overview is currently lacking. In this review, we examine how glucocorticoids mediate apoptosis by detailing GR's genomic and non-genomic action mechanisms in lymphoid malignancies. We continue with a discussion of the glucocorticoid-related problems and how these are intertwined with one another. We further zoom in on glucocorticoid resistance by critically analyzing the plethora of proposed mechanisms and highlighting therapeutic opportunities that emerge from these studies. In conclusion, early detection of glucocorticoid resistance in patients remains an important challenge as this would result in a timelier treatment reorientation and reduced glucocorticoid-instigated side effects.
... After H 2 S interven- tion, the expression of P-eif2α (p = 0.0084) was down-regulated, the PI3K (p = 0.0092)/AKT1 (p = 0.0092) autophagy pathway was activated, and the pyroptosis-related proteins Caspase1 (p = 0.0374) and Caspase3 (p = 0.0360) were also significantly downregulated; the expression levels of the above proteins in the cardiomyocytes of the H 2 S and control groups did not change significantly, the difference was not statistically significant (p > 0.05). However, in the H 2 S intervention group, eif2α phosphorylation (p = 0.0029) activation (BTdCPU: 10 µm [18]) was added, and PI3K ...
This study aimed to assess the effects of exogenous hydrogen sulfide (H2S) on abdominal aorta coarctation (AAC) induced myocardial fibrosis (MF) and autophagy in rats. Forty-four Sprague-Dawley rats were randomly divided into control group, AAC group, AAC + H2S group, and H2S control group. After a model of rats with AAC was built surgically, AAC + H2S group and H2S group were injected intraperitoneally with H2S (100 μmol/kg) daily. The rats in the control group and the AAC group were injected with the same amount of PBS. We observed that H2S can improve left ventricular function and the deposition of myocardial collagen fibers, inhibit pyroptosis, down-regulate the expression of P-eif2α in myocardial tissue, and inhibit cell autophagy by activating the phosphatidylinositol 3-kinase (PI3K)/AKT1 signaling pathway (p < 0.05). In addition, angiotensin II (1 μM) H9c2 cardiomyocytes were injured in vitro experiments, and it was also observed that pyroptosis was inhibited after H2S (400 μmol/kg) intervention, the expression of P-eif2α in cardiomyocytes was significantly down-regulated, and the PI3K/AKT1 signaling pathway was activated at the same time. Therefore, increasing the expression of P-eif2α reverses the activation of the PI3K/AKT1 signaling pathway by H2S. In conclusion, these findings suggest that exogenous H2S can ameliorate MF in rats with AAC by inhibiting pyroptosis, and the mechanism may be associated with inhibiting the phosphorylation of eif2α and activating the PI3K/AKT1 signaling pathway to inhibit excessive cell autophagy.
... BtdCPU and related N,N' diarylureas have largely been developed to increase apoptotic signaling downstream of HRI for the treatment of hematologic cancers including multiple myeloma and acute leukemia (Burwick et al, 2017;Smith et al, 2021). However, the mechanism of BtdCPU-dependent HRI activation was previously undefined. ...
The integrated stress response (ISR) is a network of eIF2alpha kinases, comprising PERK, GCN2, HRI, and PKR, that induce translational and transcriptional signaling in response to diverse insults. The PERK ISR kinase regulates mitochondria in response to endoplasmic reticulum (ER) stress. Deficiencies in PERK signaling lead to mitochondrial dysfunction and contribute to the pathogenesis of numerous diseases. We define the potential for pharmacologic activators of other ISR kinases to rescue ISR signaling and promote mitochondrial adaptation in cells lacking PERK. We show that the HRI activator BtdCPU and the GCN2 activator halofuginone activate ISR signaling and restore ER stress sensitivity in Perk-deficient cells. However, these compounds differentially impact mitochondria. BtdCPU induces mitochondrial depolarization, leading to mitochondrial fragmentation and ISR activation through the OMA1-DELE1-HRI signaling axis. In contrast, halofuginone promotes mitochondrial elongation and altered mitochondrial respiration, mimicking the regulation induced by PERK. This shows halofuginone can compensate for deficiencies in PERK activity and promote adaptive mitochondrial remodeling, highlighting the potential for pharmacologic ISR activation to mitigate mitochondrial dysfunction and motivating the pursuit of highly-selective ISR activators.
... In the Eukaryotic Initiation Factor 2 (eIF2) pathway, phosphorylation of eIF2a has been shown to play a significant role in maintaining normal cellular homeostasis and regulating cell growth [91], with dysregulation of eIF2 signaling pathway stimulating the cancerous tumors transformation [92]. The overexpression of eIF2a has been observed in several cancers, such as gastrointestinal cancer [93] and non-Hodgkin's lymphomas [94] and has been proposed as a potential therapeutic target [95]. ...
Background
Prioritization of breast cancer patients based on the risk of resistance to tamoxifen plays a significant role in personalized therapeutic planning and improving disease course and outcomes.
Methods
In this work, we demonstrate that a genome-wide pathway-centric computational framework elucidates molecular pathways as markers of tamoxifen resistance in ER+ breast cancer patients. In particular, we associated activity levels of molecular pathways with a wide spectrum of response to tamoxifen, which defined markers of tamoxifen resistance in patients with ER+ breast cancer.
Findings
We identified five biological pathways as markers of tamoxifen failure and demonstrated their ability to predict the risk of tamoxifen resistance in two independent patient cohorts (Test cohort1: log-rank p-value = 0.02, adjusted HR = 3.11; Test cohort2: log-rank p-value = 0.01, adjusted HR = 4.24). We have shown that these pathways are not markers of aggressiveness and outperform known markers of tamoxifen response. Furthermore, for adoption into clinic, we derived a list of pathway read-out genes and their associated scoring system, which assigns a risk of tamoxifen resistance for new incoming patients.
Interpretation
We propose that the identified pathways and their read-out genes can be utilized to prioritize patients who would benefit from tamoxifen treatment and patients at risk of tamoxifen resistance that should be offered alternative regimens.
Funding
This work was supported by the Rutgers SHP Dean's research grant, Rutgers start-up funds, Libyan Ministry of Higher Education and Scientific Research, and Katrina Kehlet Graduate Award from The NJ Chapter of the Healthcare Information Management Systems Society.
... One of the most notable findings of our study was that eIF2 signaling was the most significantly affected canonical pathway unique to the Ix-resistant cells. eIF2 signaling is involved in regulating ternary complex formation and protein synthesis 19 . Earlier studies have shown that inhibition of the 26 S proteasome results in a rapid decrease in the rate of protein synthesis owing to phosphorylating alpha subunit of the eukaryotic translation initiation factor 2 (eIF2α) by the heme-regulated inhibitor kinase (HRI) 20 . ...
Extensive inter-individual variation in response to chemotherapy (sensitive vs resistant tumors) is a serious cause of concern in the treatment of multiple myeloma (MM). In this study, we used human myeloma cell lines (HMCLs), and patient-derived CD138+ cells to compare kinetic changes in gene expression patterns between innate proteasome inhibitor (PI)-sensitive and PI-resistant HMCLs following test dosing with the second-generation PI Ixazomib. We found 1553 genes that changed significantly post treatment in PI-sensitive HMCLs compared with only seven in PI-resistant HMCLs (p < 0.05). Genes that were uniquely regulated in PI-resistant lines were RICTOR (activated), HNF4A, miR-16-5p (activated), MYCN (inhibited), and MYC (inhibited). Ingenuity pathway analysis (IPA) using top kinetic response genes identified the proteasome ubiquitination pathway (PUP), and nuclear factor erythroid 2-related factor 2 (NRF2)-mediated oxidative stress response as top canonical pathways in Ix-sensitive cell lines and patient-derived cells, whereas EIF2 signaling and mTOR signaling pathways were unique to PI resistance. Further, 10 genes were common between our in vitro and ex vivo post-treatment kinetic PI response profiles and Shaughnessy’s GEP80-postBz gene expression signature, including the high-risk PUP gene PSMD4. Notably, we found that heat shock proteins and PUP pathway genes showed significant higher upregulation in Ix-sensitive lines compared with the fold-change in Ix-resistant myelomas.
... Our results suggest that the majority of the ATF4 increase occurs at the posttranscriptional level. 21 The phospho-eIF2a/ATF4 pathway may be activated in isolation or as a component of a coordinated process known as the UPR in the setting of endoplasmic reticulum stress. In addition to the phospho-eIF2a/ATF4 pathway, the other branches of the UPR consist of ATF6 cleavage and IRE1-mediated XBP1 splicing. ...
Key Points
Atovaquone induces AML blast apoptosis and prolongs survival in AML xenografts. Atovaquone induces proapoptotic signaling and inhibits the mTOR pathway through upregulation of ATF4 and also suppresses OXPHOS.
... Recent discovery of small molecule activators of HRI [9e12] provided scientific community with tools to better understand the role of this kinase as well as eIF2ɑ phosphorylation in normal-and patho-biology. For example, these agents allowed us and others to study HRI's regulation of fibroblast growth factor 21 (FGF21) activity and its role in diabetes and non-alcoholic fatty liver disease and interaction of HRI/eIF2ɑ-P/ATF-4 pathway with the PPAR-b/d pathway [13,14], regulation of host/intracellular pathogen interactions [15,16], and treatment of therapy resistant multiple myeloma cancers [8,17]. Importantly, when employed in combination with other eIF2a kinase activators, HRI activators are invaluable tools for dissecting contribution of eIF2a vs other substrates of eIF2a kinases to normal-and patho-biology. ...
Heme-regulated inhibitor (HRI), a eukaryotic translation initiation factor 2 alpha (eIF2α) kinase, is critically important for coupling protein synthesis to heme availability in reticulocytes and adaptation to various environmental stressors in all cells. HRI modifies the severity of several hemoglobin misfolding disorders including β-thalassemia. Small molecule activators of HRI are essential for studying normal- and patho-biology of this kinase as well as for the treatment of various human disorders for which activation of HRI or phosphorylation of eIF2α may be beneficial. We previously reported development of 1-((1,4-trans)-4-aryloxycyclohexyl)-3-arylureas (cHAUs) as specific HRI activators and demonstrated their potential as molecular probes for studying HRI biology and as lead compounds for treatment of various human disorders. To develop more druglike cHAUs for in vivo studies and drug development and to expand the chemical space, we undertook bioassay guided structure-activity relationship studies replacing cyclohexyl ring with various 4-6-membered rings and explored further substitutions on the N-phenyl ring. We tested all analogs in the surrogate eIF2α phosphorylation and cell proliferation assays, and a subset of analogs in secondary mechanistic assays that included endogenous eIF2α phosphorylation and expression of C/EBP homologous protein (CHOP), a downstream effector. Finally, we determined specificity of these compounds for HRI by testing their anti-proliferative activity in cells transfected with siRNA targeting HRI or mock. These compounds have significantly improved cLogPs with no loss of potencies, making them excellent candidates for lead optimization for development of investigational new drugs that potently and specifically activate HRI.
... Dexamethasone treatment also leads to suppression of protein synthesis via the phosphorylation of eIF2-alpha and induction of REDD1, an inhibitor of mTOR [26]. Moreover, inhibitors of mTOR sensitize multiple myeloma cells to dexamethasone-induced apoptosis, an effect mediated by inhibition of cap-dependent translation [27]. ...
Glucocorticoids are a backbone of treatment for multiple myeloma in both the upfront and relapsed/refractory setting. While glucocorticoids have single agent activity in multiple myeloma, in the modern era, they are paired with novel agents to induce high clinical response rates. On the other hand, toxicities of steroid therapy limit high dose delivery and impact patient quality of life. We provide a history of steroid use in multiple myeloma with the aim to understand how steroids have emerged and persisted in the treatment of multiple myeloma. We review mechanisms of glucocorticoid sensitivity and resistance and highlight potential future directions to evaluate steroid responsiveness. Further research in this area will aid in optimizing steroid utilization and help determine when glucocorticoid therapy may no longer benefit patients.
... Ours is the first study to identify HRI as the BZ-activated eIF2α kinase in pancreatic cancer models, and our data are consistent with the results of Fournier et al. [55] who implicated HRI in the BZ-induced formation of stress granules. Whereas other groups have begun activating HRI to sensitize MM cells to therapy [56], our data provide a strong rationale for inhibiting HRI as a means of sensitizing pancreatic cancer cells to proteasome inhibitors. Notably, disruption of HRI signaling appears to be well-tolerated in vivo, as HRI −/− mice were found to be completely normal and fertile, with only mild hematologic abnormalities [10]. ...
Human cancer cells display extensive heterogeneity in their sensitivities to the proteasome inhibitor bortezomib (Velcade). The molecular mechanisms underlying this heterogeneity remain unclear, and strategies to overcome resistance are limited. Here, we discover that inherent differences in eIF2α phosphorylation among a panel of ten human pancreatic cancer cell lines significantly impacts bortezomib sensitivity, and implicate the HRI (heme-regulated inhibitor) eIF2α kinase as a novel therapeutic target. Within our panel, we identified a subset of cell lines with defective induction of eIF2α phosphorylation, conferring a high degree of sensitivity to bortezomib. These bortezomib-sensitive cells exhibited impaired translation attenuation followed by toxic accumulation of protein aggregates and reactive oxygen species (ROS), whereas the bortezomib-resistant cell lines displayed increased phosphorylation of eIF2α, decreased translation, few protein aggregates, and minimal ROS production. Importantly, we identified HRI as the primary bortezomib-activated eIF2α kinase, and demonstrated that HRI knockdown promoted cell death in the bortezomib-resistant cells. Overall, our data implicate inducible HRI-mediated phosphorylation of eIF2α as a central cytoprotective mechanism following exposure to bortezomib and provide proof-of-concept for the development of HRI inhibitors to overcome proteasome inhibitor resistance.
... HRI (a heme regulated kinase that phosphorylates the translation initiation factor elF2-α) is a key factor in protein synthesis regulation. HRI activators such as BTdCPU reduce the abundance of the elF2-GTP-tRNAi Met ternary complex, and thus inhibit cancer cell proliferation [46]; multiple myeloma seems to be a particularly attractive application [47]. However, eIF-2α kinases are key mediators of general stress response in cells, and modulators should be able to act much more broadly. ...
A snapshot of noteworthy recent developments in the patent literature of relevance to pharmaceutical and medical research and development.
Heme-Regulated Inhibitor (HRI) is one of the four mammalian kinases which phosphorylates eIF2α to facilitate a cellular response to stress through the regulation of mRNA translation. Originally identified for its role as a heme sensor in erythroid progenitor cells, it has since materialised as a potential therapeutic target in both cancer and neurodegeneration. Here we characterise two modes of HRI inhibition of using structural mass spectrometry, biochemical and biophysical techniques. We demonstrate that several ATP-mimetic compounds, including BRAF inhibitors and a compound, GCN2iB, thought to be specific to GCN2, are capable of potently inhibiting HRI. We demonstrate that hemin, a haem-like molecule, inactivates HRI structurally using hydrogen-deuterium exchange mass spectrometry (HDX-MS), and this results in wide-spread structural rearrangement of the protein and how that impacts on the kinase domain through a series of allosteric interactions. This inhibition mainly impacts autophosphorylation, which includes tyrosine phosphorylation, not observed before in the eIF2α kinases.
Multiple myeloma (MM) is an incurable cancer of plasma cells with a 5-year survival rate of 59%. Dysregulation of fatty acid (FA) metabolism is associated with MM development and progression; however, the underlying mechanisms remain unclear. Acyl-CoA synthetase long-chain family members (ACSLs) convert free long-chain fatty acids into fatty acyl-CoA esters and play key roles in catabolic and anabolic fatty acid metabolism. The Cancer Dependency Map data suggested that ACSL3 and ACSL4 were among the top 25% Hallmark Fatty Acid Metabolism genes that support MM fitness. Here, we show that inhibition of ACSLs in human myeloma cell lines using the pharmacological inhibitor Triascin C (TriC) causes apoptosis and decreases proliferation in a dose- and time-dependent manner. RNA-seq of MM.1S cells treated with TriC for 24 h showed a significant enrichment in apoptosis, ferroptosis, and ER stress. Proteomics of MM.1S cells treated with TriC for 48 h revealed that mitochondrial dysfunction and oxidative phosphorylation were significantly enriched pathways of interest, consistent with our observations of decreased mitochondrial membrane potential and increased mitochondrial superoxide levels. Interestingly, MM.1S cells treated with TriC for 24 h also showed decreased mitochondrial ATP production rates and overall lower cellular respiration.
Implications:
Overall, our data support the hypothesis that suppression of ACSL in human MM cells inhibit their growth and viability, indicating that ACSL proteins may be promising therapeutic targets in treating myeloma progression.
Cellular senescence is a complex process characterized by irreversible cell cycle arrest. Senescent cells accumulate with age, promoting disease development, yet the absence of specific markers hampers the development of selective anti-senescence drugs. The integrated stress response (ISR), an evolutionarily highly conserved signaling network activated in response to stress, globally downregulates protein translation while initiating the translation of specific protein sets including transcription factors. We propose that ISR signaling plays a central role in controlling senescence, given that senescence is considered a form of cellular stress. Exploring the intricate relationship between the ISR pathway and cellular senescence, we emphasize its potential as a regulatory mechanism in senescence and cellular metabolism. The ISR emerges as a master regulator of cellular metabolism during stress, activating autophagy and the mitochondrial unfolded protein response, crucial for maintaining mitochondrial quality and efficiency. Our review comprehensively examines ISR molecular mechanisms, focusing on ATF4-interacting partners, ISR modulators, and their impact on senescence-related conditions. By shedding light on the intricate relationship between ISR and cellular senescence, we aim to inspire future research directions and advance the development of targeted anti-senescence therapies based on ISR modulation.
Translational control of mRNAs during gene expression allows cells to promptly and dynamically adapt to a variety of stimuli, including in neoplasia in response to aberrant oncogenic signalling (for example, PI3K–AKT–mTOR, RAS–MAPK and MYC) and microenvironmental stress such as low oxygen and nutrient supply. Such translational rewiring allows rapid, specific changes in the cell proteome that shape specific cancer phenotypes to promote cancer onset, progression and resistance to anticancer therapies. In this Review, we illustrate the plasticity of mRNA translation. We first highlight the diverse mechanisms by which it is regulated, including by translation factors (for example, eukaryotic initiation factor 4F (eIF4F) and eIF2), RNA-binding proteins, tRNAs and ribosomal RNAs that are modulated in response to aberrant intracellular pathways or microenvironmental stress. We then describe how translational control can influence tumour behaviour by impacting on the phenotypic plasticity of cancer cells as well as on components of the tumour microenvironment. Finally, we highlight the role of mRNA translation in the cellular response to anticancer therapies and its promise as a key therapeutic target. The translational control of mRNAs during gene expression allows rapid, specific changes in the cell proteome. This Review describes the mechanisms underlying changes in mRNA translation in response to oncogenic signalling and microenvironmental stress, and how these changes can promote cancer onset, progression and resistance to anticancer therapies.
Under physiological and pathological conditions, cells activate the unfolded protein response (UPR) to deal with the accumulation of unfolded or misfolded proteins in the endoplasmic reticulum. Multiple myeloma (MM) is a hematological malignancy arising from immunoglobulin-secreting plasma cells. MM cells are subject to continual ER stress and highly dependent on the UPR signaling activation due to overproduction of paraproteins. Mounting evidence suggests the close linkage between ER stress and oxidative stress, demonstrated by overlapping signaling pathways and inter-organelle communication pivotal to cell fate decision. Imbalance of intracellular homeostasis can lead to deranged control of cellular functions and engage apoptosis due to mutual activation between ER stress and reactive oxygen species generation through a self-perpetuating cycle. Here, we present accumulating evidence showing the interactive roles of redox homeostasis and proteostasis in MM pathogenesis and drug resistance, which would be helpful in elucidating the still underdefined molecular pathways linking ER stress and oxidative stress in MM. Lastly, we highlight future research directions in the development of anti-myeloma therapy, focusing particularly on targeting redox signaling and ER stress responses.
The respiratory tract and its resident immune cells face daily exposure to stress, both from without and from within. Inhaled pathogens, including severe acute respiratory syndrome coronavirus 2, and toxins from pollution trigger a cellular defence system that reduces protein synthesis to minimise viral replication or the accumulation of misfolded proteins. Simultaneously, a gene expression programme enhances antioxidant and protein folding machineries in the lung. Four kinases (PERK, PKR, GCN2 and HRI) sense a diverse range of stresses to trigger this “integrated stress response”. Here we review recent advances identifying the integrated stress response as a critical pathway in the pathogenesis of pulmonary diseases, including pneumonias, thoracic malignancy, pulmonary fibrosis and pulmonary hypertension. Understanding the integrated stress response provides novel targets for the development of therapies.
La caractérisation des tumeurs malignes et la compréhension des mécanismes de résistance aux traitements anticancéreux sont essentielles pour la découverte de nouvelles cibles thérapeutiques. Les criblages génétiques, devenus encore plus puissants avec la technologie d’édition du génome CRISPR-Cas9, le séquençage nouvelle génération et la bioinformatique, sont des outils formidables pour décrypter de nouveaux mécanismes cellulaires, dont la résistance au traitement. La leucémie myéloïde chronique (LMC) est un syndrome myéloprolifératif qui est caractérisé par l’anomalie génétique t(9;22). Cette aberration chromosomique est à l’origine du gène de fusion BCR-ABL1 qui code l’oncogène du même nom responsable de la prolifération anarchique des cellules. L’imatinib mesylate, un inhibiteur de tyrosine kinase, élimine de manière spécifique les cellules leucémiques en ciblant et en bloquant l’activité kinase de cette protéine. Malheureusement, comme pour tout type de thérapie ciblée, une résistance au traitement survient chez certains patients. Afin de repérer des nouvelles voies de résistance à cet inhibiteur de tyrosine kinase, nous avons effectué un criblage génétique avec la librairie « genome-scale CRISPR knock-out » (GeCKO v2) in vitro dans la lignée cellulaire K562. Nous avons découvert plusieurs gènes qui semblent être essentiels pour la réponse au traitement par imatinib, tels que les facteurs pro-apoptotiques BIM et BAX, ou le répresseur de la voie des MAPK, SPRED2. Le rétablissement spécifique de l’apoptose dans les cellules BIM knock-out (KO) par des BH3-mimétiques, ou l’inhibition ciblée de la voie MAPK dans la lignée SPRED2 KO sensibilise de nouveau les lignées résistantes. Dans ce travail, nous avons découvert des mécanismes de résistance déjà connus (l’apoptose, la voie MAPK…) mais nous avons également démontré l’implication de voies peu connues telles que le complexe Mediator, la maturation de ARNm et l’ubiquitinylation de protéines. Spécifiquement cibler ces lésions génétiques avec des thérapies ciblées combinées peut permettre de surmonter les phénotypes de résistance et ouvre la porte à l’utilisation de l’oncologie de précision.
As essential components of hemoglobin, iron and heme play central roles in terminal erythropoiesis. The impairment of this process in iron/heme deficiency results in microcytic hypochromic anemia, the most prevalent anemia globally. Heme-regulated eIF2a kinase, also known as heme-regulated inhibitor (HRI), is a key heme-binding protein that senses intracellular heme concentrations to balance globin protein synthesis with the amount of heme available for hemoglobin production. HRI is activated during heme deficiency to phosphorylate eIF2a (eIF2aP), which simultaneously inhibits the translation of globin mRNAs and selectively enhances the translation of activating transcription factor 4 (ATF4) mRNA to induce stress response genes. This coordinated translational regulation is a universal hallmark across the eIF2a kinase family under various stress and is termed the integrated stress response (ISR). Inhibition of general protein synthesis by HRI-eIF2aP in erythroblasts is necessary to prevent proteotoxicity and maintain protein homeostasis in both the cytoplasm and mitochondria. Additionally, the HRI-eIF2aP-ATF4 pathway represses mTORC1 signaling specifically in the erythroid lineage as a feedback mechanism of erythropoietin-stimulated erythropoiesis during iron/heme deficiency. Furthermore, ATF4 target genes are most highly activated during iron deficiency to maintain mitochondrial function, redox homeostasis, and to enable erythroid differentiation. Thus, heme and translation regulate erythropoiesis through two key signaling pathways, ISR and mTORC1, coordinated by HRI to circumvent ineffective erythropoiesis. HRI-ISR is also activated to reduce the severity of b-thalassemia intermedia in the Hbbth1/th1 murine model. Recently, HRI has been implicated in the regulation of human fetal hemoglobin production. HRI-ISR has therefore emerged as a potential therapeutic target for hemoglobinopathies.
Introduction: Despite rapid advances in myeloma treatment with the development of new drugs, curative therapies remain elusive. Relapsed/refractory disease related to progressive dysregulation of immune system and acquired genetic abnormalities continues to be a major obstacle in achieving cure. Immune-based therapy harnessing the host defense mechanism of natural killer (NK) cells is a promising avenue in the treatment of myeloma.
Areas covered: Here, we discuss the biology and cytotoxic activity of NK cells and the potential role of these innate immune cells in defense against cancer and specifically multiple myeloma. We also discuss the role of NK cells in the anti-myeloma effects of autologous and allogenic stem cell transplantation, various novel drugs, and treatment modalities such as chimeric antigen receptor therapy. Immune evasion, either directly or indirectly involving NK cell dysfunction, may be a key and under-recognized mechanism in myeloma progression. We reviewed extensive literature identified using the key words immunotherapy, natural killer cells, and multiple myeloma.
Expert Opinion: Novel treatment approaches in myeloma utilizing the immunomodulatory and cytotoxic properties of NK cells to eradicate resistant and quiescent clones could pave the way for potentially curative interventions.
The unfolded protein response (UPR) of the endoplasmic reticulum (ER) is a highly conserved system by which cells regulate multiple pathways during misfolded protein accumulation. Acute UPR signaling inhibits translation, induces chaperone expression, and activates proteolysis, whereas chronic UPR signaling can lead to apoptosis. Each of the canonical functions of UPR serves as a mechanism that can limit or facilitate tumorigenesis. Tumor cells are able to coopt UPR signaling to facilitate proliferation, transformation, and epithelial-to-mesenchymal transition (EMT) under hypoxia and glucose starvation, potentially causing metastasis. UPR signaling is typically initiated by Glucose-Regulated Protein 78 (GRP78/BiP) binding to unfolded proteins, causing GRP-78 to dissociate from each of the three primary UPR sensors on the ER membrane: protein kinase R-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring protein 1α (IRE1α). Recent studies highlight the complexity of the signaling interactions involved, but also potential clinical opportunities to target unique molecular interfaces. This review discusses the current understanding of UPR pathways, ongoing clinical approaches to manipulate UPR signaling, and future avenues by which cancer therapy may be advanced by utilizing approaches that target the molecules involved in UPR signaling.
Multiple myeloma is an incurable malignancy of plasma cells, and its pathogenesis is poorly understood. Here we report the massively parallel sequencing of 38 tumour genomes and their comparison to matched normal DNAs. Several new and unexpected oncogenic mechanisms were suggested by the pattern of somatic mutation across the data set. These include the mutation of genes involved in protein translation (seen in nearly half of the patients), genes involved in histone methylation, and genes involved in blood coagulation. In addition, a broader than anticipated role of NF-κB signalling was indicated by mutations in 11 members of the NF-κB pathway. Of potential immediate clinical relevance, activating mutations of the kinase BRAF were observed in 4% of patients, suggesting the evaluation of BRAF inhibitors in multiple myeloma clinical trials. These results indicate that cancer genome sequencing of large collections of samples will yield new insights into cancer not anticipated by existing knowledge.
REDD1 (Regulated in development and DNA damage response 1) has been proposed to inhibit the mechanistic target of rapamycin complex 1 (MTORC1) during in vitro hypoxia. REDD1 expression is low under basal conditions but is highly increased in response to several catabolic stresses like hypoxia and glucocorticoids. However, REDD1 function seems to be tissue and stress-dependent and its role in skeletal muscle in vivo has been poorly characterized. We investigated here the effect of REDD1 deletion on skeletal muscle mass, protein synthesis, proteolysis and MTORC1 signalling pathway under basal conditions and after glucocorticoids administration. While skeletal muscle mass and typology were unchanged between wild-type (WT) and REDD1 null mice, oral gavage with dexamethasone (DEX) during 7 days reduced tibialis anterior and gastrocnemius muscle weights as well as tibialis anterior fiber size only in WT. Similarly, REDD1 deletion prevented the inhibition of protein synthesis and MTORC1 activity (assessed by S6, 4EBP1 and ULK1 phosphorylation) observed in gastrocnemius muscle of WT mice following single DEX administration for 5 hours. However, our results suggest that REDD1-mediated inhibition of MTORC1 in skeletal muscle is not related to the modulation of the binding between TSC2 and 14-3-3. In contrast our data highlight a new mechanism involved in MTORC1 inhibition linking REDD1, Akt and PRAS40. Altogether, these results demonstrated in vivo that REDD1 is required for glucocorticoids-induced inhibition of protein synthesis via MTORC1 down-regulation. Inhibition of REDD1 may thus be a strategy to limit muscle loss in glucocorticoids-mediated atrophy.
Despite promising preclinical results with mTOR kinase inhibitors in multiple myeloma (MM), resistance to these drugs may arise via feedback activation loops. This concern is especially true for IGF-1 receptor (IGF-1R), because IGF-1R signaling is downregulated by multiple AKT and mTOR feedback mechanisms. We have tested this hypothesis in MM using the novel selective mTOR kinase inhibitor AZD8055. We evaluated p-mTOR S2481 as the readout for mTORC2/Akt activity in MM cells in the context of mTOR inhibition via AZD8055, or rapamycin. We next validated AZD8055 inhibition of mTORC1 and mTORC2 functions in MM cells alone or in culture with BMSCs and growth factors. Unlike rapamycin, AZD8055 resulted in apoptosis of MM cells. AZD8055 treatment however induced upregulation of IGF-1R phosphorylation in p-Akt S473 expressing MM cell lines. Furthermore, exposure of AZD8055-treated cells to IGF-1 induced p-Akt S473 and rescued MM cells from apoptosis despite mTOR kinase inhibition and TORC2/Akt blockage. The addition of blocking IGF-1R antibody resulted in reversing this effect and increased AZD8055-induced apoptosis. Our study suggests that combination treatment with AZD8055 and IGF-1R blocking agents is a promising strategy in MM with potential IGF-1R/Akt signaling mediated survival.
Cell signaling in response to an array of diverse stress stimuli converges on the phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF2). Phosphorylation of eIF2α on serine 51 results in a severe decline in de novo protein synthesis and is an important strategy in the cell's armory against stressful insults including viral infection, the accumulation of misfolded proteins, and starvation. The phosphorylation of eIF2α is carried out by a family of four kinases, PERK (PKR-like ER kinase), PKR (protein kinase double-stranded RNA-dependent), GCN2 (general control non-derepressible-2), and HRI (heme-regulated inhibitor). Each primarily responds to a distinct type of stress or stresses. Thus, while significant sequence similarity exists between the eIF2α kinases in their kinase domains, underlying their common role in phosphorylating eIF2α, additional unique features determine the regulation of these four proteins, that is, what signals activate them. This review will describe the structure of each eIF2α kinase and discuss how this is linked to their activation and function. In parallel to the general translational attenuation elicited by eIF2α kinase activation the translation of stress-induced mRNAs, most notably activating transcription factor 4 (ATF4) is enhanced and these set in motion cascades of gene expression constituting the integrated stress response (ISR), which seek to remediate stress and restore homeostasis. Depending on the cellular context and concurrent signaling pathways active, however, translational attenuation can also facilitate apoptosis. Accordingly, the role of the kinases in determining cell fate will also be discussed.
The mammalian target of rapamycin (mTOR) kinase is a master regulator of protein synthesis that couples nutrient sensing to cell growth and cancer. However, the downstream translationally regulated nodes of gene expression that may direct cancer development are poorly characterized. Using ribosome profiling, we uncover specialized translation of the prostate cancer genome by oncogenic mTOR signalling, revealing a remarkably specific repertoire of genes involved in cell proliferation, metabolism and invasion. We extend these findings by functionally characterizing a class of translationally controlled pro-invasion messenger RNAs that we show direct prostate cancer invasion and metastasis downstream of oncogenic mTOR signalling. Furthermore, we develop a clinically relevant ATP site inhibitor of mTOR, INK128, which reprograms this gene expression signature with therapeutic benefit for prostate cancer metastasis, for which there is presently no cure. Together, these findings extend our understanding of how the 'cancerous' translation machinery steers specific cancer cell behaviours, including metastasis, and may be therapeutically targeted.
Diamond-Blackfan anemia (DBA) is associated with developmental defects and profound anemia. Mutations in genes encoding a ribosomal protein of the small (e.g., RPS19) or large (e.g., RPL11) ribosomal subunit are found in more than half of these patients. The mutations cause ribosomal haploinsufficiency, which reduces overall translation efficiency of cellular mRNAs. We reduced the expression of Rps19 or Rpl11 in mouse erythroblasts and investigated mRNA polyribosome association, which revealed deregulated translation initiation of specific transcripts. Among these were Bag1, encoding a Hsp70 cochaperone, and Csde1, encoding an RNA-binding protein, and both were expressed at increased levels in erythroblasts. Their translation initiation is cap independent and starts from an internal ribosomal entry site, which appeared sensitive to knockdown of Rps19 or Rpl11. Mouse embryos lacking Bag1 die at embryonic day 13.5, with reduced erythroid colony forming cells in the fetal liver, and low Bag1 expression impairs erythroid differentiation in vitro. Reduced expression of Csde1 impairs the proliferation and differentiation of erythroid blasts. Protein but not mRNA expression of BAG1 and CSDE1 was reduced in erythroblasts cultured from DBA patients. Our data suggest that impaired internal ribosomal entry site-mediated translation of mRNAs expressed at increased levels in erythroblasts contributes to the erythroid phenotype of DBA.
Measuring the properties of endogenous cell proteins, such as expression level, subcellular localization, and turnover rates, on a whole proteome level remains a major challenge in the postgenome era. Quantitative methods for measuring mRNA expression do not reliably predict corresponding protein levels and provide little or no information on other protein properties. Here we describe a combined pulse-labeling, spatial proteomics and data analysis strategy to characterize the expression, localization, synthesis, degradation, and turnover rates of endogenously expressed, untagged human proteins in different subcellular compartments. Using quantitative mass spectrometry and stable isotope labeling with amino acids in cell culture, a total of 80,098 peptides from 8,041 HeLa proteins were quantified, and their spatial distribution between the cytoplasm, nucleus and nucleolus determined and visualized using specialized software tools developed in PepTracker. Using information from ion intensities and rates of change in isotope ratios, protein abundance levels and protein synthesis, degradation and turnover rates were calculated for the whole cell and for the respective cytoplasmic, nuclear, and nucleolar compartments. Expression levels of endogenous HeLa proteins varied by up to seven orders of magnitude. The average turnover rate for HeLa proteins was ~20 h. Turnover rate did not correlate with either molecular weight or net charge, but did correlate with abundance, with highly abundant proteins showing longer than average half-lives. Fast turnover proteins had overall a higher frequency of PEST motifs than slow turnover proteins but no general correlation was observed between amino or carboxyl terminal amino acid identities and turnover rates. A subset of proteins was identified that exist in pools with different turnover rates depending on their subcellular localization. This strongly correlated with subunits of large, multiprotein complexes, suggesting a general mechanism whereby their assembly is controlled in a different subcellular location to their main site of function.
Translation initiation plays a critical role in cellular homeostasis, proliferation, differentiation and malignant transformation. Consistently, increasing the abundance of the eIF2-GTP-tRNA(i)(Met) translation initiation complex transforms normal cells and contributes to cancer initiation and the severity of some anemias. The chemical modifiers of the eIF2-GTP-tRNA(i)(Met) ternary complex are therefore invaluable tools for studying its role in the pathobiology of human disorders and for determining whether this complex can be pharmacologically targeted for therapeutic purposes. Using a cell-based assay, we identified N,N'-diarylureas as unique inhibitors of ternary complex accumulation. Direct functional-genetic and biochemical evidence demonstrated that the N,N'-diarylureas activate heme-regulated inhibitor kinase, thereby phosphorylating eIF2α and reducing the abundance of the ternary complex. Using tumor cell proliferation in vitro and tumor growth in vivo as paradigms, we demonstrate that N,N'-diarylureas are potent and specific tools for studying the role of eIF2-GTP-tRNA(i)(Met) ternary complex in the pathobiology of human disorders.
In response to different environmental stresses, phosphorylation of eukaryotic initiation factor-2 (eIF2) rapidly reduces
protein synthesis, which lowers energy expenditure and facilitates reprogramming of gene expression to remediate stress damage.
Central to the changes in gene expression, eIF2 phosphorylation also enhances translation of ATF4, a transcriptional activator
of genes subject to the integrated stress response (ISR). The ISR increases the expression of genes important for alleviating
stress or alternatively triggering apoptosis. One ISR target gene encodes the transcriptional regulator CHOP whose accumulation
is critical for stress-induced apoptosis. In this study, we show that eIF2 phosphorylation induces preferential translation
of CHOP by a mechanism involving a single upstream ORF (uORF) located in the 5′-leader of the CHOP mRNA. In the absence of stress and low eIF2 phosphorylation, translation of the uORF serves as a barrier that prevents translation
of the downstream CHOP coding region. Enhanced eIF2 phosphorylation during stress facilitates ribosome bypass of the uORF due to its poor start
site context, and instead it allows scanning ribosomes to translate CHOP. This new mechanism of translational control explains how expression of CHOP and the fate of cells are tightly linked to the levels of phosphorylated eIF2 and stress damage.
The effects of dexamethasone on the growth of four human multiple myeloma cell lines were studied. In addition, the effects on the expression of interleukin-6 (IL-6) and IL-6 receptor (IL-6R) genes were investigated by the use of reverse-transcriptase polymerase chain reaction. Dexamethasone (Dex) concentrations of 10(-7) to 10(-6) mol/L inhibited IL-6 gene expression in three of four cell lines studied, whereas the higher concentration of the hormone inhibited also IL-6R gene expression. Dex effects were modulated through the glucocorticoid receptor (GR). Dex treatment resulted in killing of sensitive cells associated with DNA fragmentation, which could be reversed by concomitant treatment with IL-6. The reversal of Dex-mediated effects by IL-6 did not result from an inhibition of GR function as measured by receptor nuclear translocation or Dex-regulated reporter gene function. These results indicate that blockage of the IL-6 signaling pathway is essential for effective myeloma cell kill by Dex.
The immunosuppressant rapamycin blocks p70s6k/p85s6k activation and phosphorylation of 40S ribosomal protein S6 in Swiss 3T3 cells. The same net result is obtained when the macrolide is added 3 hr after serum stimulation. In stimulated cells p70s6k/p85s6k inactivation is achieved within minutes, whereas S6 dephosphorylation requires 1-2 hr, supporting the concept that S6 dephosphorylation results from kinase inactivation. In parallel, rapamycin treatment causes a small, but significant, reduction in the initiation rate of protein synthesis, as measured both by [35S]methionine incorporation into protein and by recruitment of 80S ribosomes into polysomes. More striking, analysis of individual mRNA transcripts revealed that rapamycin selectively suppresses the translation of a family of mRNAs that is characterized by a polypyrimidine tract immediately after their N7-methylguanosine cap, a motif that can act as a translational modulator. This family includes transcripts for ribosomal proteins, elongation factors of protein synthesis, and proteins of as-yet-unknown function. The results imply that (i) 40S ribosomes containing phosphorylated S6 may selectively recognize this motif or proteins which bind to it and (ii) rapamycin may inhibit cell growth by blocking S6 phosphorylation and, thus, translation of these mRNAs.
Translation initiation is a complex process in which initiator tRNA, 40S, and 60S ribosomal subunits are assembled by eukaryotic initiation factors (eIFs) into an 80S ribosome at the initiation codon of mRNA. The cap-binding complex eIF4F and the factors eIF4A and eIF4B are required for binding of 43S complexes (comprising a 40S subunit, eIF2/GTP/Met-tRNAi and eIF3) to the 5' end of capped mRNA but are not sufficient to promote ribosomal scanning to the initiation codon. eIF1A enhances the ability of eIF1 to dissociate aberrantly assembled complexes from mRNA, and these factors synergistically mediate 48S complex assembly at the initiation codon. Joining of 48S complexes to 60S subunits to form 80S ribosomes requires eIF5B, which has an essential ribosome-dependent GTPase activity and hydrolysis of eIF2-bound GTP induced by eIF5. Initiation on a few mRNAs is cap-independent and occurs instead by internal ribosomal entry. Encephalomyocarditis virus (EMCV) and hepatitis C virus epitomize distinct mechanisms of internal ribosomal entry site (IRES)-mediated initiation. The eIF4A and eIF4G subunits of eIF4F bind immediately upstream of the EMCV initiation codon and promote binding of 43S complexes. EMCV initiation does not involve scanning and does not require eIF1, eIF1A, and the eIF4E subunit of eIF4F. Initiation on some EMCV-like IRESs requires additional noncanonical initiation factors, which alter IRES conformation and promote binding of eIF4A/4G. Initiation on the hepatitis C virus IRES is even simpler: 43S complexes containing only eIF2 and eIF3 bind directly to the initiation codon as a result of specific interaction of the IRES and the 40S subunit.
Our previous studies have characterized Dexamethasone (Dex)-induced apoptotic signaling pathways in multiple myeloma (MM) cells; however, related transcriptional events are not fully defined. In the present study, gene expression profiles of Dex-treated MM cells were determined using oligonucleotide arrays. Dex triggers early transient induction of many genes involved in cell defense/repair-machinery. This is followed by induction of genes known to mediate cell death and repression of growth/survival-related genes. The molecular and genetic alterations associated with Dex resistance in MM cells are also unknown. We compared the gene expression profiles of Dex-sensitive and Dex-resistant MM cells and identified a number of genes which may confer Dex-resistance. Finally, gene profiling of freshly isolated MM patient cells validates our in vitro MM cell line data, confirming an in vivo relevance of these studies. Collectively, these findings provide insights into the basic mechanisms of Dex activity against MM, as well as mechanisms of Dex-resistance in MM cells. These studies may therefore allow improved therapeutic uses of Dex, based upon targeting genes that regulate MM cell growth and survival.
Smac, second mitochondria-derived activator of caspases, promotes apoptosis via activation of caspases. Heat shock protein 27 (Hsp27) negatively regulates another mitochondrial protein, cytochrome c, during apoptosis; however, the role of Hsp27 in modulating Smac release is unknown. Here we show that Hsp27 is overexpressed in both dexamethasone (Dex)-resistant multiple myeloma (MM) cell lines (MM.1R, U266, RPMI-8226) and primary patient cells. Blocking Hsp27 by an antisense (AS) strategy restores the apoptotic response to Dex in Dex-resistant MM cells by triggering the release of mitochondrial protein Smac, followed by activation of caspase-9 and caspase-3. Moreover, AS-Hsp27 overcomes interleukin-6 (IL-6)-mediated protection against Dex-induced apoptosis. These data demonstrate that Hsp27 inhibits the release of Smac, and thereby confers Dex resistance in MM cells.
Various mitogenic or growth inhibitory stimuli induce a rapid change in the association of terminal oligopyrimidine (TOP) mRNAs with polysomes. It is generally believed that such translational control hinges on the mammalian target of rapamycin (mTOR)-S6 kinase pathway. Amino acid availability affects the translation of TOP mRNAs, although the signaling pathway involved in this regulation is less well characterized. To investigate both serum- and amino acid-dependent control of TOP mRNA translation and the signaling pathways involved, HeLa cells were subjected to serum and/or amino acid deprivation and stimulation. Our results indicate the following. 1). Serum and amino acid deprivation had additive effects on TOP mRNA translation. 2). The serum content of the medium specifically affected TOP mRNA translation, whereas amino acid availability affected both TOP and non-TOP mRNAs. 3). Serum signaling to TOP mRNAs involved only a rapamycin-sensitive pathway, whereas amino acid signaling depended on both rapamycin-sensitive and rapamycin-insensitive but wortmannin-sensitive events. 4). Eukaryotic initiation factor-2alpha phosphorylation increased during amino acid deprivation, but not following serum deprivation. Interestingly, rapamycin treatment suggests a novel connection between the mTOR pathway and eukaryotic initiation factor-2alpha phosphorylation in mammalian cells, which may not, however, be involved in TOP mRNA translational regulation.
This study compared bortezomib with high-dose dexamethasone in patients with relapsed multiple myeloma who had received one to three previous therapies.
We randomly assigned 669 patients with relapsed myeloma to receive either an intravenous bolus of bortezomib (1.3 mg per square meter of body-surface area) on days 1, 4, 8, and 11 for eight three-week cycles, followed by treatment on days 1, 8, 15, and 22 for three five-week cycles, or high-dose dexamethasone (40 mg orally) on days 1 through 4, 9 through 12, and 17 through 20 for four five-week cycles, followed by treatment on days 1 through 4 for five four-week cycles. Patients who were assigned to receive dexamethasone were permitted to cross over to receive bortezomib in a companion study after disease progression.
Patients treated with bortezomib had higher response rates, a longer time to progression (the primary end point), and a longer survival than patients treated with dexamethasone. The combined complete and partial response rates were 38 percent for bortezomib and 18 percent for dexamethasone (P<0.001), and the complete response rates were 6 percent and less than 1 percent, respectively (P<0.001). Median times to progression in the bortezomib and dexamethasone groups were 6.22 months (189 days) and 3.49 months (106 days), respectively (hazard ratio, 0.55; P<0.001). The one-year survival rate was 80 percent among patients taking bortezomib and 66 percent among patients taking dexamethasone (P=0.003), and the hazard ratio for overall survival with bortezomib was 0.57 (P=0.001). Grade 3 or 4 adverse events were reported in 75 percent of patients treated with bortezomib and in 60 percent of those treated with dexamethasone.
Bortezomib is superior to high-dose dexamethasone for the treatment of patients with multiple myeloma who have had a relapse after one to three previous therapies.
To determine if thalidomide plus dexamethasone yields superior response rates compared with dexamethasone alone as induction therapy for newly diagnosed multiple myeloma.
Patients were randomly assigned to receive thalidomide plus dexamethasone or dexamethasone alone. Patients in arm A received thalidomide 200 mg orally for 4 weeks; dexamethasone was administered at a dose of 40 mg orally on days 1 to 4, 9 to 12, and 17 to 20. Cycles were repeated every 4 weeks. Patients in arm B received dexamethasone alone at the same schedule as in arm A.
Two hundred seven patients were enrolled: 103 were randomly assigned to thalidomide plus dexamethasone and 104 were randomly assigned to dexamethasone alone; eight patients were ineligible. The response rate with thalidomide plus dexamethasone was significantly higher than with dexamethasone alone (63% v 41%, respectively; P = .0017). The response rate allowing for use of serum monoclonal protein levels when a measurable urine monoclonal protein was unavailable at follow-up was 72% v 50%, respectively. The incidence rates of grade 3 or higher deep vein thrombosis (DVT), rash, bradycardia, neuropathy, and any grade 4 to 5 toxicity in the first 4 months were significantly higher with thalidomide plus dexamethasone compared with dexamethasone alone (45% v 21%, respectively; P < .001). DVT was more frequent in arm A than in arm B (17% v 3%); grade 3 or higher peripheral neuropathy was also more frequent (7% v 4%, respectively).
Thalidomide plus dexamethasone demonstrates significantly superior response rates in newly diagnosed myeloma compared with dexamethasone alone. However, this must be balanced against the greater toxicity seen with the combination.
The mammalian target of rapamycin (mTOR), a critical modulator of cell growth, acts to integrate signals from hormones, nutrients, and growth-promoting stimuli to downstream effector mechanisms involved in the regulation of protein synthesis. Dexamethasone, a synthetic glucocorticoid that represses protein synthesis, acts to inhibit mTOR signaling as assessed by reduced phosphorylation of the downstream targets S6K1 and 4E-BP1. Dexamethasone has also been shown in one study to up-regulate the expression of REDD1 (also referred to RTP801, a novel stress-induced gene linked to repression of mTOR signaling) in lymphoid, but not nonlymphoid, cells. In contrast to the findings of that study, here we demonstrate that REDD1, but not REDD2, mRNA expression is dramatically induced following acute dexamethasone treatment both in rat skeletal muscle in vivo and in L6 myoblasts in culture. In L6 myoblasts, the effect of the drug on mTOR signaling is efficiently blunted in the presence of REDD1 RNA interference oligonucleotides. Moreover, the dexamethasone-induced assembly of the mTOR regulatory complex Tuberin. Hamartin is disrupted in L6 myoblasts following small interfering RNA-mediated repression of REDD1 expression. Finally, overexpression of Rheb, a downstream target of Tuberin function and a positive upstream effector of mTOR, reverses the effect of dexamethasone on phosphorylation of mTOR substrates. Overall, the data support the conclusion that REDD1 functions upstream of Tuberin and Rheb to down-regulate mTOR signaling in response to dexamethasone.
Motivation: The processing of the Affymetrix GeneChip data has been a recent focus for data analysts. Alternatives to the original procedure have been proposed and some of these
new methods are widely used.
Results: The affy package is an R package of functions and classes for the analysis of oligonucleotide arrays manufactured by Affymetrix. The package is currently in its second release, affy provides the user with extreme flexibility when carrying out an analysis and make it possible to access and manipulate probe
intensity data. In this paper, we present the main classes and functions in the package and demonstrate how they can be used
to process probe-level data. We also demonstrate the importance of probe-level analysis when using the Affymetrix GeneChip platform.
mTORC1 is a complex of proteins that includes the mammalian target of rapamycin (mTOR) and several regulatory proteins. It
is activated by a variety of hormones (e.g. insulin) and nutrients (e.g. amino acids) that act to stimulate cell growth and proliferation and repressed by hormones (e.g. glucocorticoids) that act to reduce cell growth. Curiously, mTORC1 signaling is reported to be rapidly (e.g. within 1-2 h) activated by inhibitors of protein synthesis that act on either mRNA translation elongation or gene transcription.
However, the basis for the mTORC1 activation has not been satisfactorily delineated. In the present study, mTORC1 signaling
was found to be activated in response to inhibition of either the initiation or elongation phases of mRNA translation. Changes
in mTORC1 signaling were inversely proportional to alterations in the expression of the mTORC1 repressor, REDD1, but not the
expression of TRB3 or TSC2. Moreover the cycloheximide-induced increase in mTORC1 signaling was significantly attenuated in
cells lacking REDD1, showing that REDD1 plays an integral role in the response. Finally, the half-life of REDD1 was estimated
to be 5 min or less. Overall, the results are consistent with a model in which inhibition of protein synthesis leads to a
loss of REDD1 protein because of its rapid degradation, and in part reduced REDD1 expression subsequently leads to de-repression
of mTORC1 activity.
When resting (G
0
) mouse 3T6 fibroblasts are serum stimulated to reenter the cell cycle, the rates of synthesis of rRNA and ribosomal proteins increase, resulting in an increase in ribosome content beginning about 6 h after stimulation. In this study, we monitored the content, metabolism, and translation of ribosomal protein mRNA (rp mRNA) in resting, exponentially growing, and serum-stimulated 3T6 cells. Cloned cDNAs for seven rp mRNAs were used in DNA-excess filter hybridization studies to assay rp mRNA. We found that about 85% of rp mRNA is polyadenylated under all growth conditions. The rate of labeling of rp mRNA relative to total polyadenylated mRNA changed very little after stimulation. The half-life of rp mRNA was about 11 h in resting cells and about 8 h in exponentially growing cells, values which are similar to the half-lives of total mRNA in resting and growing cells (about 9 h). The content of rp mRNA relative to total mRNA was about the same in resting and growing 3T6 cells. Furthermore, the total amount of rp mRNA did not begin to increase until about 6 h after stimulation. Since an increase in rp mRNA content did not appear to be responsible for the increase in ribosomal protein synthesis, we determined the efficiency of translation of rp mRNA under different conditions. We found that about 85% of pulse-labeled rp mRNA was associated with polysomes in exponentially growing cells. In resting cells, however, only about half was associated with polysomes, and about 30% was found in the monosomal fraction. The distribution shifted to that found in growing cells within 3 h after serum stimulation. Similar results were obtained when cells were labeled for 10.5 h. About 70% of total polyadenylated mRNA was in the polysome fraction in all growth states regardless of labeling time, indicating that the shift in mRNA distribution was species specific. These results indicate that the content and metabolism of rp mRNA do not change significantly after growth stimulation. The rate of ribosomal protein synthesis appears to be controlled during the resting-growing transition by an alteration of the efficiency of translation of rp mRNA, possibly at the level of protein synthesis initiation.
Oxidative stress is pathogenic in neurological diseases, including stroke. The identity of oxidative stress-inducible transcription factors and their role in propagating the death cascade are not well known. In an in vitro model of oxidative stress, the expression of the bZip transcription factor activating transcription factor 4 (ATF4) was induced by glutathione depletion and localized to the promoter of a putative death gene in neurons. Germline deletion of ATF4 resulted in a profound reduction in oxidative stress-induced gene expression and resistance to oxidative death. In neurons, ATF4 modulates an early, upstream event in the death pathway, as resistance to oxidative death by ATF4 deletion was associated with decreased consumption of the antioxidant glutathione. Forced expression of ATF4 was sufficient to promote cell death and loss of glutathione. In ATF4(-/-) neurons, restoration of ATF4 protein expression reinstated sensitivity to oxidative death. In addition, ATF4(-/-) mice experienced significantly smaller infarcts and improved behavioral recovery as compared with wild-type mice subjected to the same reductions in blood flow in a rodent model of ischemic stroke. Collectively, these findings establish ATF4 as a redox-regulated, prodeath transcriptional activator in the nervous system that propagates death responses to oxidative stress in vitro and to stroke in vivo.
The effects of dexamethasone on the growth of four human multiple myeloma cell lines were studied. In addition, the effects on the expression of interleukin-6 (IL-6) and IL-6 receptor (IL-6R) genes were investigated by the use of reverse-transcriptase polymerase chain reaction. Dexamethasone (Dex) concentrations of 10(-7) to 10(-6) mol/L inhibited IL-6 gene expression in three of four cell lines studied, whereas the higher concentration of the hormone inhibited also IL-6R gene expression. Dex effects were modulated through the glucocorticoid receptor (GR). Dex treatment resulted in killing of sensitive cells associated with DNA fragmentation, which could be reversed by concomitant treatment with IL-6. The reversal of Dex-mediated effects by IL-6 did not result from an inhibition of GR function as measured by receptor nuclear translocation or Dex-regulated reporter gene function. These results indicate that blockage of the IL-6 signaling pathway is essential for effective myeloma cell kill by Dex.
The mammalian target of rapamycin plays an important role in multiple myeloma. The allosteric mammalian target of rapamycin inhibitor everolimus has long been approved for immunosuppression and has shown activity in certain cancers. This investigator-initiated phase I trial explores everolimus in relapsed and/or refractory multiple myeloma patients with ≥ 2 lines of prior treatment. Following a dose-escalation design, it called for a fixed dose of oral everolimus. Blood drug levels were monitored and the biological activity of everolimus was evaluated in bone marrow. Seventeen patients were enrolled (age range: 52 to 76 years). All were previously treated with stem cell transplantation, proteasome inhibitors and almost all with immunomodulatory drugs. No dose limiting toxicity was observed reaching the intended final daily dose of 10 mg. Only one severe adverse event was assessed as possibly related to the study drug, namely atypical pneumonia. Remarkably few infections were observed. Although the trial was mainly designed to evaluate feasibility, anti-myeloma activity defined as clinical benefit was documented in 10 of 15 evaluable patients at every dose level including 8 patients with stable disease, 1 minor and one partial remission. However, the median time to progression was 90 days (range: 13 to 278 days). The biomarker study documented on-target activity of everolimus in malignant plasma cells as well as the microenvironment. The observed responses are promising and allow considering further studies including combination strategies addressing escape pathways. Trial is registered with EudraCT number 2006-002675-41.
Copyright © 2015, Ferrata Storti Foundation.
Cells have evolved exquisite mechanisms to fine-tune the rate of protein synthesis in response to stress. Systemic mapping of start-codon positions and precise measurement of the corresponding initiation rate would transform our understanding of translational control. Here we present quantitative translation initiation sequencing (QTI-seq), with which the initiating ribosomes can be profiled in real time at single-nucleotide resolution. Resultant initiation maps not only delineated variations of start-codon selection but also highlighted a dynamic range of initiation rates in response to nutrient starvation. The integrated data set provided unique insights into principles of alternative translation and mechanisms controlling different aspects of translation initiation. With RiboTag mice, QTI-seq permitted tissue-specific profiling of initiating ribosomes in vivo. Liver cell-specific ribosome profiling uncovered a robust translational reprogramming of the proteasome system in fasted mice. Our findings illuminated the prevalence and dynamic nature of translational regulation pivotal to physiological adaptation in vivo.
Everolimus, an oral mammalian target of rapamycin (mTOR) inhibitor, has been studied in multiple myeloma (MM) but lacks significant single agent activity. Based on preclinical studies showing synergistic activity of mTOR inhibitors with lenalidomide, we studied the combination of lenalidomide and everolimus in relapsed or refractory MM in a phase I clinical trial. We assessed patient samples using gene expression, Western blotting and immunohistochemistry to probe the mTOR pathway. Twenty-six patients were evaluable for toxicity. Dose-limiting toxicities included grade 4 neutropenia and thrombocytopenia. The maximum tolerated dose was lenalidomide 15 mg and everolimus 5 mg for 21 d with a 7 d rest period. Grade 3/4 adverse events included thrombocytopenia (35%) and neutropenia (42%). The overall response rate was 65% (1 complete response + 4 partial response + 10 minimal response). The median progression-free survival was 5·5 months and median overall survival was 29·5 months. Biomarker data demonstrated downregulation of phosphorylated p70S6K. Gene expression profiling suggested activation of mTOR in responders versus non-responders. The combination of lenalidomide and everolimus was well tolerated with predictable toxicities and showed responses in a heavily pretreated population. When confirmed with larger patient numbers, this analysis may guide patient selection for future clinical trials of mTOR inhibition in MM.
Heme-regulated inhibitor kinase (HRI), an eukaryotic translation initiation factor 2 alpha (eIF2α kinase, plays critical roles in cell proliferation, differentiation, and adaptation to cytoplasmic stress. HRI is also a critical modifier of hemoglobin disorders such as β-thalassemia. We previously identified N,N'-diarylureas as potent activators of HRI suitable for studying biology of this important kinase. To expand the repertoire of chemotypes that activate HRI we screened a ~1,900 member N,N'-disubstituted urea library in the surrogate eIF2α phosphorylation assay identifying N-aryl,N'-cyclohexylphenoxyurea as a promising scaffold. We validated hit compounds as a bona-fide HRI activators in secondary assays and explored contributions of substitutions on the N-aryl and N'-cyclohexylphenoxy groups to their activity by studying focused libraries of complementing analogs. We tested these N-aryl,N'-cyclohexylphenoxyureas in the surrogate eIF2α phosphorylation and cell proliferation assays, demonstrating significantly improved bioactivities and specificities. We consider these compounds to represent lead candidates for the development of potent and specific HRI activators.
The role of stromal cells and the tumour microenvironment in general in modulating tumour sensitivity is increasingly becoming a key consideration for the development of active anticancer therapeutics. Here, we discuss how these tumour-stromal interactions affect tumour cell signalling, survival, proliferation and drug sensitivity. Particular emphasis is placed on the ability of stromal cells to confer - to tumour cells - resistance or sensitization to different classes of therapeutics, depending on the specific microenvironmental context. The mechanistic understanding of these microenvironmental interactions can influence the evaluation and selection of candidate agents for various cancers, in both the primary site as well as the metastatic setting. Progress in in vitro screening platforms as well as orthotopic and 'orthometastatic' xenograft mouse models has enabled comprehensive characterization of the impact of the tumour microenvironment on therapeutic efficacy. These recent advances can hopefully bridge the gap between preclinical studies and clinical trials of anticancer agents.
Multiple myeloma (MM) is a clonal disorder of plasma cells that remains, for the most part, incurable despite the advent of several novel therapeutic agents. Tumor cells in this disease are cradled within the bone marrow (BM) microenvironment by an array of adhesive interactions between the BM cellular residents, the surrounding extracellular matrix (ECM) components such as fibronectin (FN), laminin, vascular cell adhesion molecule-1 (VCAM-1), proteoglycans, collagens and hyaluronan, and a variety of adhesion molecules on the surface of MM cells including integrins, hyaluronan receptors (CD44 and RHAMM) and heparan sulfate proteoglycans. Several signaling responses are activated by these interactions, affecting the survival, proliferation and migration of MM cells. An important consequence of these direct adhesive interactions between the BM/ECM and MM cells is the development of drug resistance. This phenomenon is termed "cell adhesion-mediated drug resistance" (CAM-DR) and it is thought to be one of the major mechanisms by which MM cells escape the cytotoxic effects of therapeutic agents. This review will focus on the adhesion molecules involved in the cross-talk between MM cells and components of the BM microenvironment. The complex signaling networks downstream of these adhesive molecules mediated by direct ligand binding or inside-out soluble factors signaling will also be reviewed. Finally, novel therapeutic strategies targeting these molecules will be discussed. Identification of the mediators of MM-BM interaction is essential to understand MM biology and to elucidate novel therapeutic targets for this disease.
Multiple myeloma is still uncurable. Myeloma cells become resistant to common drugs and patients eventually die of tumour progression. Therefore, new targets and drugs are urgently needed. NVP-BGT226 is a novel, orally bioavailable small-molecule inhibitor of phosphoinositol-3-kinase and mammalian target of rapamycin. Here, we show that NVP-BGT226 inhibits growth in common myeloma cell lines and primary myeloma cells at nanomolar concentrations in a time-dependent and dose-dependent manner. Western blots for the detection of caspase 3 cleavage and annexin-V-fluorescein isothiocyanate/propidium iodide assays revealed induction of apoptosis in common myeloma cells lines. Induction of apoptosis was accompanied by upregulation of proapoptotic Bim and a moderate upregulation of Mcl-1 and Bad and a downregulation of Bcl-2, Bax and Bcl-Xl. Inhibition of cell growth was mainly due to inhibition of myeloma cell proliferation, as shown by the 5-bromo-2'-deoxyuridine assay. Cell cycle analysis revealed induction of cell cycle arrest in the G1 phase, which was due to downregulation of cyclin D1, cyclin D2, pRb and cdc25a. NVP-BGT226 inhibited phosphorylation of protein kinase B (Akt), P70S6k and 4E-BP-1 in a time-dependent and dose-dependent manner. Furthermore, we show that the stimulatory effect of insulin-like growth factor 1, interleukin-6 and conditioned medium of HS-5 stromal cells on myeloma cell growth is completely abrogated by NVP-BGT226. Overall, inhibition of phosphoinositol-3-kinase/mammalian target of rapamycin by NVP-BGT226 is highly effective, and NVP-BGT226 represents a potential new candidate for targeted therapy in multiple myeloma.
Historically, the ribosome has been viewed as a complex ribozyme with constitutive rather than regulatory capacity in mRNA translation. Here we identify mutations of the Ribosomal Protein L38 (Rpl38) gene in mice exhibiting surprising tissue-specific patterning defects, including pronounced homeotic transformations of the axial skeleton. In Rpl38 mutant embryos, global protein synthesis is unchanged; however the translation of a select subset of Homeobox mRNAs is perturbed. Our data reveal that RPL38 facilitates 80S complex formation on these mRNAs as a regulatory component of the ribosome to confer transcript-specific translational control. We further show that Rpl38 expression is markedly enriched in regions of the embryo where loss-of-function phenotypes occur. Unexpectedly, a ribosomal protein (RP) expression screen reveals dynamic regulation of individual RPs within the vertebrate embryo. Collectively, these findings suggest that RP activity may be highly regulated to impart a new layer of specificity in the control of gene expression and mammalian development.
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Intermittent courses of dexamethasone (DEX) were administered to 112 consecutive, previously untreated patients with multiple myeloma (MM). Using criteria based on a 75% or greater reduction of calculated tumor mass, the overall response rate was 43%. Among comparable patients, response rate were approximately 15% less than those observed previously with vincristine-doxorubicin by continuous infusion with intermittent DEX (VAD) and similar to those with melphalan-prednisone. The projected survival times with VAD or DEX were similar. Results indicated that DEX accounted for most of the plasma cell reduction achieved with VAD. Serious complications occurred in 27% of patients treated with VAD, but in only 4% of those who received DEX. In view of the similar outcome with fewer serious complications, DEX provided a simple, effective, and safe primary treatment for a large fraction of patients with MM. Patients who appear most likely to benefit include those with hypercalcemia or pancytopenia, or who require simultaneous radiotherapy for a pathologic fracture.
In multiple myeloma cells resistant to glucocorticoids, we have previously identified a variant glucocorticoid receptor (GR) transcript (P. A. Moalli et al., Cancer Res., 53: 3877-3879, 1993). Here, we report a reverse transcription-PCR assay to assess whether this aberrant GR transcript is present in myeloma patients. We detected both the wild-type and variant GR transcripts in the patient isolate that was the source of our myeloma cell lines, in patients refractory to steroid treatment, and in healthy control subjects. Simultaneous amplification of wild-type and variant GR mRNAs indicates that the variant GR is more highly expressed in cells that are resistant to glucocorticoids. We hypothesize that the variant GR is a normal mRNA transcript that acts to modulate glucocorticoid responsiveness, and increased expression contributes to a resistant phenotype.
Dexamethasone (Dex), which is often used for the treatment of multiple myeloma, produces rapid reductions in tumor mass and improvement in disease symptoms; however, it is not curative, and drug-resistant cells eventually emerge. To elucidate this apparent paradox, we tested the effect of the bone marrow environment on myeloma cell response to this drug. To determine whether bone marrow stroma provides sufficient amounts of interleukin (IL)-6 to protect myeloma cells against the effects of Dex, we compared the production of IL-6 by marrow stromal cells from four myeloma patients before, during, and after exposure to 10(-7) M Dex, and found that even in the presence of this drug, stromal cells continued to produce IL-6, albeit in reduced concentrations. We tested the ability of stromal cells to protect myeloma cells, purified from the bone marrow of seven patients by cell sorting on the basis of CD38 and CD45 expression, and two light-scatter parameters, from Dex-induced apoptosis. In contrast to mature CD38+CD45- cells, which were not protected, coculture with stroma very effectively protected immature CD38+CD45+ myeloma cells from Dex. These data may explain the palliative efficacy of Dex treatment and provide a rationale for combining IL-6 antagonists with Dex to overcome the IL-6-mediated resistance of immature tumor cells.
The synthesis of many mammalian proteins associated with the translational apparatus is selectively regulated by mitogenic and nutritional stimuli, at the translational level. The apparent advantages of the regulation of gene expression at the translational level are the speed and the readily reversible nature of the response to altering physiological conditions. These two features enable cells to rapidly repress the biosynthesis of the translational machinery upon shortage of amino acids or growth arrest, thus rapidly blocking unnecessary energy wastage. Likewise, when amino acids are replenished or mitogenic stimulation is applied, then cells can rapidly respond in resuming the costly biosynthesis of the translational apparatus. A structural hallmark, common to mRNAs encoding many components of the translational machinery, is the presence of a 5' terminal oligopyrimidine tract (5'TOP), referred to as TOP mRNAs. This structural motif comprises the core of the translational cis-regulatory element of these mRNAs. The present review focuses on the mechanism underlying the translational control of TOP mRNAs upon growth and nutritional stimuli. A special emphasis is put on the pivotal role played by ribosomal protein S6 kinase (S6K) in this mode of regulation, and the upstream regulatory pathways, which might be engaged in transducing external signals into activation of S6K. Finally, the possible involvement of pyrimidine-binding proteins in the translational control of TOP mRNAs is discussed.
Protein kinases that phosphorylate the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) are activated in stressed cells and negatively regulate protein synthesis. Phenotypic analysis of targeted mutations in murine cells reveals a novel role for eIF2alpha kinases in regulating gene expression in the unfolded protein response (UPR) and in amino acid starved cells. When activated by their cognate upstream stress signals, the mammalian eIF2 kinases PERK and GCN2 repress translation of most mRNAs but selectively increase translation of Activating Transcription Factor 4 (ATF4), resulting in the induction of the downstream gene CHOP (GADD153). This is the first example of a mammalian signaling pathway homologous to the well studied yeast general control response in which eIF2alpha phosphorylation activates genes involved in amino acid biosynthesis. Mammalian cells thus utilize an ancient pathway to regulate gene expression in response to diverse stress signals.
Eukaryotic cells respond to unfolded proteins in their endoplasmic reticulum (ER stress), amino acid starvation, or oxidants by phosphorylating the alpha subunit of translation initiation factor 2 (eIF2alpha). This adaptation inhibits general protein synthesis while promoting translation and expression of the transcription factor ATF4. Atf4(-/-) cells are impaired in expressing genes involved in amino acid import, glutathione biosynthesis, and resistance to oxidative stress. Perk(-/-) cells, lacking an upstream ER stress-activated eIF2alpha kinase that activates Atf4, accumulate endogenous peroxides during ER stress, whereas interference with the ER oxidase ERO1 abrogates such accumulation. A signaling pathway initiated by eIF2alpha phosphorylation protects cells against metabolic consequences of ER oxidation by promoting the linked processes of amino acid sufficiency and resistance to oxidative stress.
Plasma cells are the terminally differentiated, non-dividing effector cells of the B-cell lineage. They are cellular factories devoted to the task of synthesizing and secreting thousands of molecules of clonospecific antibody each second. To respond to microbial pathogens with the necessary specificity and rapidity, B cells are exquisitely regulated with respect to both development in the bone marrow and activation in the periphery. This review focuses on the terminal differentiation of B cells into plasma cells, including the different subsets of B cells that become plasma cells, the mechanism of regulation of this transition, the transcription factors that control each developmental stage and the characteristics of long-lived plasma cells.
Cells reprogram gene expression in response to environmental changes by mobilizing transcriptional activators. The activator protein Gcn4 of the yeast Saccharomyces cerevisiae is regulated by an intricate translational control mechanism, which is the primary focus of this review, and also by the modulation of its stability in response to nutrient availability. Translation of GCN4 mRNA is derepressed in amino acid-deprived cells, leading to transcriptional induction of nearly all genes encoding amino acid biosynthetic enzymes. The trans-acting proteins that control GCN4 translation have general functions in the initiation of protein synthesis, or regulate the activities of initiation factors, so that the molecular events that induce GCN4 translation also reduce the rate of general protein synthesis. This dual regulatory response enables cells to limit their consumption of amino acids while diverting resources into amino acid biosynthesis in nutrient-poor environments. Remarkably, mammalian cells use the same strategy to downregulate protein synthesis while inducing transcriptional activators of stress-response genes under various stressful conditions, including amino acid starvation.
Mammalian target of rapamycin (mTOR) inhibitors, such as rapamycin and CCI-779, have shown preclinical potential as therapy for multiple myeloma. By inhibiting expression of cell cycle proteins, these agents induce G1 arrest. However, by also inhibiting an mTOR-dependent serine phosphorylation of insulin receptor substrate-1 (IRS-1), they may enhance insulin-like growth factor-I (IGF-I) signaling and downstream phosphatidylinositol 3-kinase (PI3K)/AKT activation. This may be a particular problem in multiple myeloma where IGF-I-induced activation of AKT is an important antiapoptotic cascade. We, therefore, studied AKT activation in multiple myeloma cells treated with mTOR inhibitors. Rapamycin enhanced basal AKT activity, AKT phosphorylation, and PI3K activity in multiple myeloma cells and prolonged activation of AKT induced by exogenous IGF-I. CCI-779, used in a xenograft model, also resulted in multiple myeloma cell AKT activation in vivo. Blockade of IGF-I receptor function prevented rapamycin's activation of AKT. Furthermore, rapamycin prevented serine phosphorylation of IRS-1, enhanced IRS-1 association with IGF-I receptors, and prevented IRS-1 degradation. Although similarly blocking IRS-1 degradation, proteasome inhibitors did not activate AKT. Thus, mTOR inhibitors activate PI3-K/AKT in multiple myeloma cells; activation depends on basal IGF-R signaling; and enhanced IRS-1/IGF-I receptor interactions secondary to inhibited IRS-1 serine phosphorylation may play a role in activation of the cascade. In cotreatment experiments, rapamycin inhibited myeloma cell apoptosis induced by PS-341. These results provide a caveat for future use of mTOR inhibitors in myeloma patients if they are to be combined with apoptosis-inducing agents.
Multiple myeloma (MM) is a clonal B-lymphocyte malignancy, which is characterized by the accumulation of terminally differentiated antibody-producing cells in the bone marrow. Because current treatments offer only a median survival of 3 years, investigators continue to search for novel therapeutic strategies to combat the disease. Rational drug design is enhanced by understanding MM cell proliferation and key signaling pathways employed. In addition, a model system for preclinical evaluation of novel therapeutics is critical. Our laboratory has developed MM cell lines to study drug action and resistance, cell proliferation, and apoptosis. These cell lines are widely used in MM research. From a single MM patient, three separate cell lines were established that parallel the progression of the disease. These three cell lines, designated MM1.S, MM1.R(E), and MM1.R(L), can be distinguished on the basis of their sensitivity to steroid hormones such as glucocorticoids (GCs). Utilization of these cell lines to study the etiology of MM, effects of chemotherapeutic agents, and development of clinical resistance, will provide us with vital information for the evolution of new and more efficacious therapeutics. The aim of this review is to summarize the morphological, biochemical, and growth characteristics of these cells, and to review the results from investigations of the MM.1 signaling pathways. This information will enhance the study, treatment, and eventual eradication of MM.
Stem cell differentiation involves changes in transcription, but little is known about translational control during differentiation. We comprehensively profiled gene expression during differentiation of murine embryonic stem cells (ESCs) into embryoid bodies by integrating transcriptome analysis with global assessment of ribosome loading. While protein synthesis was parsimonious during self-renewal, differentiation induced an anabolic switch, with global increases in transcript abundance, polysome content, protein synthesis, and protein content. Furthermore, 78% of transcripts showed increased ribosome loading, thereby enhancing translational efficiency. Transcripts under exclusive translational control included the transcription factor ATF5, the tumor suppressor DCC, and the beta-catenin agonist Wnt1. We show that a hierarchy of translational regulators, including mTOR, 4EBP1, and the RNA-binding proteins DAZL and GRSF1, control global and selective protein synthesis during ESC differentiation. Parsimonious translation in pluripotent state and hierarchical translational regulation during differentiation may be important quality controls for self-renewal and choice of fate in ESCs.
The role of tumour-stromal interactions in modifying drug response: challenges and opportunities
- D W Mcmillin
- J M Negri
- C S Mitsiades
D.W. McMillin, J.M. Negri, C.S. Mitsiades, The role of tumour-stromal
interactions in modifying drug response: challenges and opportunities, Nat.
Rev. Drug Discov. 12 (3) (2013) 217-228, http://dx.doi.org/10.1038/nrd3870.
Regulation of plasma-cell development
- M Shapiro-Shelef
- K Calame
M. Shapiro-Shelef, K. Calame, Regulation of plasma-cell development, Nat.
Rev. Immunol. 5 (3) (2005) 230-242, http://dx.doi.org/10.1038/nri1572.
Identification of genes regulated by dexamethasone in multiple myeloma cells using oligonucleotide arrays
- D Chauhan
- D Auclair
- E K Robinson
D. Chauhan, D. Auclair, E.K. Robinson, et al., Identification of genes regulated
by dexamethasone in multiple myeloma cells using oligonucleotide arrays,
Oncogene 21 (9) (2002) 1346-1358, http://dx.doi.org/10.1038/sj.onc.
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Affy-analysis of affymetrix GeneChip data at the probe level
- L Gautier
- L Cope
- B M Bolstad
- R A Irizarry
L. Gautier, L. Cope, B.M. Bolstad, R.A. Irizarry, Affy-analysis of affymetrix
GeneChip data at the probe level, Bioinformatics 20 (3) (2004) 307-315,
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Limma: Linear Models for Microarray Data
- V C R Gentleman
- S Dudoit
- R Irizarry
- W Huber
V.C.R. Gentleman, S. Dudoit, R. Irizarry, W. Huber, Limma: Linear Models for
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ATF4 is an oxidative stress-inducible, prodeath transcription factor in neurons in vitro and in vivo
- P S Lange
- J C Chavez
- J T Pinto
P.S. Lange, J.C. Chavez, J.T. Pinto, et al., ATF4 is an oxidative stress-inducible,
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