Direct Protein Target Research Articles

Myricanol prevents aging-related sarcopenia by rescuing mitochondrial dysfunction via targeting peroxiredoxin 5.

Aging is a process that represents the accumulation of changes in organism overtime. In biological level, accumulations of molecular and cellular damage in aging lead to an increasing risk of diseases like sarcopenia. Sarcopenia reduces mobility, leads to fall-related injuries, and diminishes life quality. Thus, it is meaningful to find out novel therapeutic strategies for sarcopenia intervention that may help the elderly maintain their functional ability. Oxidative damage-induced dysfunctional mitochondria are considered as a culprit of muscle wasting during aging. Herein, we aimed to demonstrate whether myricanol (MY) protects aged mice against muscle wasting through alleviating oxidative damage in mitochondria and identify the direct protein target and its underlying mechanism. We discovered that MY protects aged mice against the loss of muscle mass and strength through scavenging reactive oxygen species accumulation to rebuild the redox homeostasis. Taking advantage of biophysical assays, peroxiredoxin 5 was discovered and validated as the direct target of MY. Through activating peroxiredoxin 5, MY reduced reactive oxygen species accumulation and damaged mitochondrial DNA in C2C12 myotubes. Our findings provide an insight for therapy against sarcopenia through alleviating oxidative damage-induced dysfunctional mitochondria by targeting peroxiredoxin 5, which may contribute an insight for healthy aging.

Heat shock cognate 70 protein is a novel target of nobiletin and its colonic metabolites in inhibiting colon carcinogenesis.

Nobiletin (NBT) is a unique flavonoid mainly found in citrus fruits and has been reported to inhibit colon carcinogenesis in multiple rodent models. However, the direct molecular targets of NBT are unknown, which greatly limits its utilization in cancer prevention and treatment. In this study, using affinity chromatography, proteomics, computer modeling and various biochemical analyses, for the first time we identified HSC70 as a direct protein target of NBT in colon cancer cells. Moreover, NBT bound to HSC70 at its ATP-binding site and inhibited its ATPase activity. Importantly, our results also demonstrated that the major colonic metabolites of NBT (generated in the colon of NBT-fed mice) produced similar inhibitory effects against HSC70-mediated pro-carcinogenic events to those of NBT. Overall, our results provide a solid basis to further investigate the implication of the interaction between NBT/NBT metabolites and HSC70 in cancer chemoprevention.

Formononetin attenuates high glucose-induced neurotoxicity by negatively regulating oxidative stress and mitochondrial dysfunction in Schwann cells via activation of SIRT3

High glucose induces Schwann cells death and neurotoxicity. Formononetin was originally found in Astragalus membranaceus and showed anti-tumor and anti-neuroinflammation properties. The aim of this study is to explore the molecular mechanism underlying the neuroprotective effects of formononetin and identify its direct protein target. The effects of formononetin on oxidative stress and mitochondrial dysfunction in Schwann cells induced by high glucose were investigated. High glucose treatment significantly induced oxidative stress, mitochondrial dysfunction and apoptosis in Schwann cells, while these effects were partially or completely prevented by co-treatment with formononetin. Mechanistically, we found that SIRT3/PGC-1α/SOD2 pathway was activated by formononetin under high glucose conditions as evidenced by western blotting. Knockdown of SIRT3 by siRNA delivery reversed the protective effects of formononetin on high glucose-induced Schwann cells injury and changes in expression profile of SIRT3 downstream target genes. Molecular docking, thermal shift assay and surface plasmon resonance assay revealed a direct binding between formononetin and SIRT3. Taken together, we identified a novel SIRT3 activator formononetin and revealed its beneficial effects on high glucose-induced neurotoxicity, suggesting that targeting SIRT3 in Schwann cells may be a new approach for treatment of peripheral nerve regeneration related diseases such as diabetic peripheral neuropathy.

The Golgi stacking protein GRASP55 is targeted by the natural compound prodigiosin

BackgroundThe bacterial secondary metabolite prodigiosin has been shown to exert anticancer, antimalarial, antibacterial and immunomodulatory properties. With regard to cancer, it has been reported to affect cancer cells but not non-malignant cells, rendering prodigiosin a promising lead compound for anticancer drug discovery. However, a direct protein target has not yet been experimentally identified.MethodsWe used mass spectrometry-based thermal proteome profiling in order to identify target proteins of prodigiosin. For target validation, we employed a genetic knockout approach and electron microscopy.ResultsWe identified the Golgi stacking protein GRASP55 as target protein of prodigiosin. We show that prodigiosin treatment severely affects Golgi morphology and functionality, and that prodigiosin-dependent cytotoxicity is partially reduced in GRASP55 knockout cells. We also found that prodigiosin treatment results in decreased cathepsin activity and overall blocks autophagic flux, whereas co-localization of the autophagosomal marker LC3 and the lysosomal marker LAMP1 is clearly promoted. Finally, we observed that autophagosomes accumulate at GRASP55-positive structures, pointing towards an involvement of an altered Golgi function in the autophagy-inhibitory effect of this natural compound.ConclusionTaken together, we propose that prodigiosin affects autophagy and Golgi apparatus integrity in an interlinked mode of action involving the regulation of organelle alkalization and the Golgi stacking protein GRASP55.8brCwiZ4E3PVMoFvaLMguTVideo

Mechanism of immunomodulatory drug resistance and novel therapeutic strategies in multiple myeloma

ABSTRACT Objective The mechanism of immunomodulatory drugs (IMiDs) resistance to multiple myeloma (MM) cells has been gradually demonstrated by recently studies, and some potential novel strategies have been confirmed to have antimyeloma activity and be associated with IMiD activity in MM. Methods This article searched the Pubmed library, reviewed some recently studies related to IMiD resistance to MM cells and summarized some potent agents to improve IMiD resistance to MM cells. Results Studies have confirmed that cereblon is a primary direct protein target of IMiDs. IRF4 not only is affected by the IKZF protein but also can directly inhibit the expression of BMF and BIM, thereby promoting the survival of MM cells. Additionally, the expression of IRF4 and MYC also plays an important role in three important signaling pathways (Wnt, STAT3 and MAPK/ERK) related to IMiD resistance. Notably, MYC, a downstream factor of IRF4, may be upregulated by BRD4, and upregulation of MYC promotes cell proliferation in MM and disease progression. Recently, some novel therapeutic agents targeting BRD4, a histone modification-related ‘reader’ of epigenetic marks, or other important factors (e.g. TAK1) in relevant signaling pathways have been developed and they may provide new options for relapse/refractory MM therapy, such as BET inhibitors, CBP/EP300 inhibitors, dual-target BET-CBP/EP300 inhibitors, TAK1 inhibitors, and they may provide new options for relapsed/refractory MM therapy. Conclusions Accumulated studies have revealed that some key factors associated with the mechanism of IMiD resistance to MM cells. Some agents represent promising new therapeutics of MM to regulate the IRF4/MYC axis by inhibiting BRD4 expression or signaling pathway activation.

Dihydromyricetin ameliorates osteogenic differentiation of human aortic valve interstitial cells by targeting c-KIT/interleukin-6 signaling pathway.

Aims: Calcific aortic valve disease (CAVD) is a chronic cardiovascular disease with high morbidity that lacks effective pharmacotherapeutics. As a natural flavonoid extracted from Ampelopsis grossedentata, dihydromyricetin (DHM) has been shown to be effective in protecting against atherosclerosis; yet, the therapeutic role of DHM in CAVD remains poorly understood. Herein, we aimed to clarify the therapeutic implications of DHM in CAVD and the underlying molecular mechanisms in human valvular interstitial cells (hVICs). Methods and Results: The protein levels of two known osteogenesis-specific genes (alkaline phosphatase, ALP; runt-related transcription factor 2, Runx2) and calcified nodule formation in hVICs were detected by Western blot and Alizarin Red staining, respectively. The results showed that DHM markedly ameliorated osteogenic induction medium (OM)–induced osteogenic differentiation of hVICs, as evidenced by downregulation of ALP and Runx2 expression and decreased calcium deposition. The SwissTargetPrediction database was used to identify the potential AVC-associated direct protein target of DHM. Protein–protein interaction (PPI) analysis revealed that c-KIT, a tyrosine-protein kinase, can act as a credible protein target of DHM, as evidenced by molecular docking. Mechanistically, DHM-mediated inhibition of c-KIT phosphorylation drove interleukin-6 (IL-6) downregulation in CAVD, thereby ameliorating OM-induced osteogenic differentiation of hVICs and aortic valve calcification progression. Conclusion: DHM ameliorates osteogenic differentiation of hVICs by blocking the phosphorylation of c-KIT, thus reducing IL-6 expression in CAVD. DHM could be a viable therapeutic supplement to impede CAVD.

Rapamycin targets STAT3 and impacts c-Myc to suppress tumor growth

Rapamycin is widely recognized as an inhibitor of mTOR, and has been approved for clinical use as an immunosuppressant. Its potencies in anti-cancer, anti-aging, and neurodegenerative diseases are emergingly established. The exploration of other targets of rapamycin will further elucidate its underlying mechanisms of action. In this study, we use a chemical proteomics strategy that has identified STAT3, a transcription factor considered to be undruggable, as a direct functional protein target of rapamycin. Together with other multi-dimensional proteomics data, we show that rapamycin treatment in cell culture significantly inhibits c-Myc-regulated gene expression. Furthermore, we show that rapamycin suppresses tumor growth along with a decreased expression of STAT3 and c-Myc in an invivo xenograft mouse model for hepatocellular carcinoma. Our data suggest that rapamycin acts directly on STAT3 to decrease its transcription activity, providing important information for the pharmacological and pharmaceutical development of STAT3 inhibitors for cancer therapy.

Exploring the Capability of Indonesia Natural Medicine Secondary Metabolite as Potential Inhibitors of SARS-CoV-2 Proteins to Prevent Virulence of COVID-19: In silico and Bioinformatic Approach

BACKGROUND: SARS-CoV-2 was causing COVID-19 disease resulting in many deaths and being a significant concern in the world today. There is an emergent need to search for possible medications for COVID-19 treatment. The key point to halt SARS-CoV-2 infection through inhibition of the virus-receptor interaction and stimulates the immune system. Utilization of the bioinformatic and in silico molecular docking a number of available medications might be proven to be effective in inhibiting SARS-CoV-2 main drug targets including the SARS-CoV2 spike glycoprotein, the 3CL protease SARS-CoV-2 active target, PD-ACE2, 2019-nCoV PLpro, and NF-kβ. AIM: This present study was conducted to identify the potential target and molecular mechanism of the major compound on Alpinia galanga extract and Citrus sinensis (L.) extract in circumventing COVID-19 using a bioinformatics approach and in silico molecular docking. RESULTS: Direct protein target of all secondary metabolite and the gene list from PubMed “Severe acute respiratory syndrome coronavirus 2” generated 2 genes (CCL2 and VEGFA) as potential therapeutics target genes (PTTG). The molecular docking was conducted by the Protein-Ligand Ant System (PLANTS) software. The results show that hesperidin, naringenin, and galangin have lower docking score for all five-protein target receptor compared with chloroquine and remdesivir. The lower docking score suggests a high affinity to bind the protein. Moreover, these compounds have a strong affinity in their inhibitory capacity for viral infection. CONCLUSION: In general, this study’s findings show that the compound of Alpinia galanga extract dan Citrus sinensis (L.) extract exhibit the best potential as an inhibitor to the development of the SARS-CoV-2 and inhibited cytokine storm through inactivation NF-kβ _pathway.

Autophagic Network Analysis of the Dual Effect of Sevoflurane on Neurons Associated with GABARAPL1 and 2.

Background Sevoflurane is commonly used as a general anesthetic in neonates to aged patients. Preconditioning or postconditioning with sevoflurane protects neurons from excitotoxic injury. Conversely, sevoflurane exposure induces neurotoxicity during early or late life. However, little is known about the underlying mechanism of the dual effect of sevoflurane on neurons. Autophagy is believed to control neuronal homeostasis. We hypothesized that autophagy determined the dual effect of sevoflurane on neurons. Methods DTome was used to identify the direct protein target (DPT) of sevoflurane. The STRING database was employed to investigate the proteins associated with the DPTs. Protein-protein interaction was assessed using Cytoscape. WebGestalt was used to analyze gene set enrichment. The linkage between candidate genes and autophagy was identified using GeneCards. Results This study found that 23 essential DPTs of sevoflurane interacted with 77 proteins from the STRING database. GABARAPL1 and 2, both of which are DPT- and autophagy-associated proteins, were significantly expressed in the brain and enriched in GABAergic synapses. Conclusions Taken together, our findings showed that the network of sevoflurane-DPT-GABARAPL1 and 2 is related to the dual effect of sevoflurane on neurons.

Cereblon and Redox in Plasma Cells

The protein Cereblon (CRBN) has recently attracted considerable interest by virtue of its ability to mediate both anti-tumor as well as teratogenic activities of immunomodulatory drugs (IMiDs) 1-3 . Certain IMiDs have been shown to re-direct CRBN-associated ubiquitylation activity towards neo-substrates such as IKZF1/3 and CK1a, thereby promoting their proteasomal degradation 4-8. This mechanism has been implicated in the anti-proliferative effects of IMiDs in multiple myeloma (MM) and del(5q)-MDS. However, the pleiotropic anti-tumor effects of IMiDs, their adverse effects such as teratogenicity and neurotoxicity, and their synergy with proteasome inhibitors suggests further mechanisms of action. Previous research on CRBN has revealed novel insights that shed light on the physiological functions of this versatile protein in plasma cells. Indeed, next to its function as a ubiquitin ligase, CRBN has been shown to exist in ubiquitin-ligase independent complexes, where it functions as a chaperone for crucial trans-membrane receptors such as CD147 (also known as Basigin) and MCT1 (monocarboxylate transporter 1) 9. This functions links CRBN to the regulation of angiogenesis, cell invasion and particularly cell metabolism by governing lactate homeostasis. Likewise, CRBN has been implicated in the regulation of TrxR (thioredoxin reductase), thereby critically determining the redox balance in plasma cells 10. These findings have important implications for our understanding of IMiD biology and for the development of novel treatment approaches and predictive biomarkers for MM. Indeed, IMiDs inhibit the chaperone function of CRBN in a competitive manner and attenuate TrxR via CRBN, thereby preventing intracellular H2O2 decomposition. In turn, CRBN-dependent degradation of IKZF1 and IKZF3 have been shown to be the result high H2O2 levels. Against this background, CRBN-regulated proteins such as CD147 and MCT1 as well as TrxR may serve as new therapeutic target structures while functional read-outs as CD147 and MCT1 destabilization or antioxidative capacity may serve as predictive biomarkers for IMiD response.1. Ito T, Ando H, Suzuki T, et al. Identification of a primary target of thalidomide teratogenicity. Science . 2010;327(5971):1345- 1350.2. Zhu YX, Braggio E, Shi CX, et al. Cereblon expression is required for the antimyeloma activity of lenalidomide and pomalidomide. Blood . 2011;118(18):4771-4779.3. Lopez-Girona A, Mendy D, Ito T, et al. Cereblon is a direct protein target for immunomodulatory and antiproliferative activities of lenalidomide and pomalidomide. Leukemia . 2012;26(11):2326-2335.4. Kronke J, Udeshi ND, Narla A, et al. Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science . 2014;343(6168):301-305.5. Lu G, Middleton RE, Sun H, et al. The myeloma drug lenalidomide promotes the cereblon-dependent destruction of Ikaros proteins. Science . 2014;343(6168):305-309.6. Gandhi AK, Kang J, Havens CG, et al. Immunomodulatory agents lenalidomide and pomalidomide co-stimulate T cells by inducing degradation of T cell repressors Ikaros and Aiolos via modulation of the E3 ubiquitin ligase complex CRL4(CRBN.). Br J Haematol . 2014;164(6):811-821.7. Zhu YX, Braggio E, Shi CX, et al. Identification of cereblon-binding proteins and relationship with response and survival after IMiDs in multiple myeloma. Blood . 2014;124(4):536-545.8. Kronke J, Fink EC, Hollenbach PW, et al. Lenalidomide induces ubiquitination and degradation of CK1alpha in del(5q) MDS. Nature . 2015;523(7559):183-188.9. Eichner R, Heider M, Fernandez-Saiz V, et al. Immunomodulatory drugs disrupt the cereblon-CD147-MCT1 axis to exert antitumor activity and teratogenicity. Nat Med . 2016;22(7):735-743.10. Sebastian S, Zhu YX, Braggio E, et al. Multiple myeloma cells' capacity to decompose H2O2 determines lenalidomide sensitivity. Blood . 2017;129(8):991-1007. DisclosuresBassermann:Celgene: Research Funding.

Berberine binds RXR\u03b1 to suppress \u03b2-catenin signaling in colon cancer cells

Berberine, an isoquinoline alkaloid, is a traditional oriental medicine used to treat diarrhea and gastroenteritis. Recently, we reported that it could inhibit the growth of intestinal polyp in animals and in patients with the familial adenomatous polyposis by downregulating β-catenin signaling. However, the intracellular target mediating the effects of berberine remains elusive. Here, we provide evidence that berberine inhibits β-catenin function via directly binding to a unique region comprising residues Gln275, Arg316 and Arg371 in nuclear receptor retinoid X receptor alpha (RXRα), where berberine concomitantly binding to and synergistically activating RXRα with 9-cis-retinoic acid (9-cis-RA), a natural ligand binding to the classical ligand-binding pocket of RXRα. Berberine binding promotes RXRα interaction with nuclear β-catenin, leading to c-Cbl mediated degradation of β-catenin, and consequently inhibits the proliferation of colon cancer cells. Furthermore, berberine suppresses the growth of human colon carcinoma xenograft in nude mice in an RXRα-dependent manner. Together, our study not only identifies RXRα as a direct protein target for berberine but also dissects their binding mode and validates that berberine indeed suppresses β-catenin signaling and cell growth in colon cancer via binding RXRα, which provide new strategies for the design of new RXRα-based antitumor agents and drug combinations.

P97/VCP promotes degradation of CRBN substrate glutamine synthetase and neosubstrates

Glutamine synthetase (GS) plays an essential role in metabolism by catalyzing the synthesis of glutamine from glutamate and ammonia. Our recent study showed that CRBN, a direct protein target for the teratogenic and antitumor activities of immunomodulatory drugs such as thalidomide, lenalidomide, and pomalidomide, recognizes an acetyl degron of GS, resulting in ubiquitylation and degradation of GS in response to glutamine. Here, we report that valosin-containing protein (VCP)/p97 promotes the degradation of ubiquitylated GS, resulting in its accumulation in cells with compromised p97 function. Notably, p97 is also required for the degradation of all four known CRBN neo-substrates [Ikaros family zinc finger proteins 1 (IKZF1) and 3 (IKZF3), casein kinase 1α (CK1α), and the translation termination factor GSPT1] whose ubiquitylation is induced by immunomodulatory drugs. Together, these data point to an unexpectedly intimate relationship between the E3 ubiquitin ligase CRL4CRBN and p97 pathways.

Glutamine Triggers Acetylation-Dependent Degradation of Glutamine Synthetase via the Thalidomide Receptor Cereblon

Cereblon (CRBN), a substrate receptor for the cullin-RING ubiquitin ligase 4 (CRL4) complex, is a direct protein target for thalidomide teratogenicity and antitumor activity of immunomodulatory drugs (IMiDs). Here we report that glutamine synthetase (GS) is an endogenous substrate of CRL4(CRBN). Upon exposing cells to high glutamine concentration, GS is acetylated at lysines 11 and 14, yielding a degron that is necessary and sufficient for binding and ubiquitylation by CRL4(CRBN) and degradation by the proteasome. Binding of acetylated degron peptides to CRBN depends on an intact thalidomide-binding pocket but is not competitive with IMiDs. These findings reveal a feedback loop involving CRL4(CRBN) that adjusts GS protein levels in response to glutamine and uncover a new function for lysine acetylation.

Chemopreventive Effects of Licorice and Its Components.

Cancer is still a major health issue worldwide and identifying novel but safe compounds for prevention and treatment is a high priority. Licorice (Glycyrrhiza) is a perennial plant that is cultivated in many countries and has been reported to exert antioxidant, anti-inflammatory and anticancer effects. However, some components of licorice exert unwanted side effects and therefore identifying safer licorice components would be ideal. The anticancer activities of many of the licorice components appear to include cycle arrest, apoptosis induction, and general antioxidant effects. Commonly reported indirect protein targets important in tumorigenesis include many cell cycle-related proteins, apoptosis-associated proteins, MMP proteins, COX-2, GSK-β, Akt, NF-κB, and MAP kinases. Importantly, several licorice components were reported to directly bind to and inhibit the activities of PI3-K, MKK4, MKK7, JNK1, mTOR, and Cdk2, resulting in decreased carcinogenesis in several cell and mouse models with no obvious toxicity. This review focuses on specific components of licorice for which a direct protein target has been identified.

Characterization Of RING Finger E3-Ubiquitin Ligase Cereblon In Hematopoietic Regulation

Characterization of RING finger E3-Ubiquitin Ligase Cereblon in Hematopoietic RegulationCereblon is a RING-domain E3 ubiquitin ligase (UbL) that is the direct protein target for thalidomide and lenalidomide. Inhibition of this molecule mediates antiproliferative activity in myeloma cells and in activated B-cell-like subtype diffuse large B-cell lymphoma. In addition to its anticancer properties, lenalidomide potentiates T-cell effector function through the engagement of the CD28-mediated co-stimulatory pathway by a CRBL-dependent mechanism that is poorly characterized. Human and animal studies show CRBL to be ubiquitously expressed in the hematopoietic compartment and in neurons, where a nonsense mutation causes a mild autosomal recessive non-syndromic intellectual disability. Ubiquitin ligase activity underlies suppression of growth factor signaling and maintains homeostasis within the hematopoietic compartment. C-Cbl, cbl-b, GRAIL, and ITCH represent four partially overlapping E3-UbL regulators of cytokine signaling and hematopoietic regulation. To understand the role of CRBL in hematopoetic development and function, we studied a CRBL-deficient mouse strain. All mice used in this study were bred and maintained under specific pathogen-free conditions according to institutional guidelines. The strategy used to create germline crbn deletion (crbl-/-) was described previously, although these mice were not investigated for aberrations in hematopoietic development. First, crbl-/- mice were viable, fertile and normal in appearance without limb malformations. Consistent with a role for CRBL in the hematopoietic development, crbl-/- mice exhibited marked changes in their hematopoietic profiles, including lymphoid hyperplasia, a markedly expanded peripheral white blood cell count and neutrophil expansion. In the T-cell compartment, splenic lymphocytes numbers were increased with demargination of the T-cells into B-cell zones by immunohistochemical staining. Double-negative thymocytes ratio were altered with an accumulation of DN1 and DN3 cells and lower DN4. In contrast, differentiated cell lineages such as mature T-cells, including CD4 and CD8 single-positive (SP) thymocytes, SP peripheral T-cells and naïve and memory T-cells were similar in number to wild-type littermates. The mature T-cells, similar to Cbl-b KO mice showed superior proliferation and IL-2 production in the absence of CD28 co-ligation. Collectively, these histological changes are indicative of a role for crbl in negatively regulating cytokine and receptor signaling events that control cell proliferation or survival. Interestingly, this phenotype overlaps partially with c-cbl and cbl-b KO mice suggesting that they could be coordinately regulating similar pathways. In summary, our findings demonstrate a novel role for crbl, the molecular target of thalidomide and lenalidomide in hematopoiesis and suggest that this molecule may act by an unknown mechanism in concert with other E3 UbLs involved in cytokine receptor signaling. This study provides the first characterization of crbl genetic deficiency in a murine model and demonstrates a unique regulatory relationship between CRBL and other known E3 UbLs. Disclosures:McDaniel:Celgene: Employment.

Long-Term Follow-up of a Phase 2 Study of Single Agent Lenalidomide in Previously Untreated, Symptomatic Chronic Lymphocytic Leukemia (CLL)

AbstractAbstract ▪718▪This icon denotes a clinically relevant abstract Introduction:In a previously reported phase 2 study of single agent lenalidomide in 25 untreated CLL patients (pts), we reported an overall response rate (ORR) of 56% (14 pts), 40% SD (10 pts) and no CR at a median follow-up of 20.7 months (Chen et al. JCO 2010;29:1175). Although an amended protocol with conservative lenalidomide dosing was used to mitigate tumor lysis and severe myelosuppression observed in the first 2 accrued pts, we continued to observe frequent toxicities of grade 3–4 neutropenia (72%) and tumor flare (TF 88%). We now report long-term efficacy and toxicity from this study at a median follow-up of 47 months (mos). Methods:Patients were eligible if previously untreated and symptomatic (cytopenias, symptomatic adenopathy/organomegaly, constitutional symptoms, lymphocyte doubling count <12 mos). The amended starting dose for lenalidomide was 2.5mg daily on days 1–21 of a 28 day cycle, with slow monthly dose escalations (2.5mg cycle 1, 5mg cycle 2, 10mg cycle 3 and if required for response, further 5mg increments to a maximum of 25mg daily were allowed).Results: Longterm toxicities:Hematologic toxicities were common: grade 3–4 neutropenia (76%), thrombocytopenia (28%), anemia (20%). With longer term use, neutropenia tended to recur (12% of all cycles) and 10 pts required GCSF support (5 routinely during each cycle). Most common non-hematologic toxicities (all grades) were TF (88%), fatigue (76%), rash (60%), muscle cramping (40%), diarrhea (40%). All non-hematologic toxicities were mild (grade 1–2), except for 1 pt each with grade 3 rash and diarrhea. Although TF was most common during cycle 1, repeat flare symptoms upon resuming lenalidomide after the 7 day rest period of each cycle were noted in 16% of all 898 cycles administered, and as late as at cycle 28. Infections were mild (most respiratory, skin) with only 2 grade 3 events (disseminated zoster, S.pneumoniae bacteremia). Other malignancies: 2 pts developed transformed large cell lymphoma 7 and 18 mos after study discontinuation, 1 pt developed squamous cell carcinoma of skin at cycle 51, and 1 pt developed recurrence of remote non-small cell lung cancer at cycle 34. Dose modifications/discontinuation:The median highest dose achieved for all 25 pts was 15 mg (range 2.5–25 mgs); 8 pts were able to escalate to the maximal 25mg dose. Ten pts (40%) required dose reductions for grade 3 cytopenias [neutropenia (2), thrombocytopenia (2), both (2)], febrile neutropenia (2), and diarrhea (2). Of all 25 pts, the median duration on therapy was 31.1 mos (range 28 days – 60.6 mos). Twelve pts (52%) currently remain on study, receiving a median of 59 cycles of therapy (range 48–66). Causes of discontinuation for 13 pts included: treatment-related toxicity (8), lack of response/progressive disease (4), and recurrence of remote lung cancer (1). Toxicities leading to discontinuation included: prolonged cytopenias (3), recurrent infections (1), atrial fibrillation (1), disseminated herpes zoster (1), persistent grade 2 diarrhea (1), and grade 3 skin rash (1). Efficacy:With extended median follow-up from 20.7 to 47 mos, the ORR improved from 56% (14 pts) to 72% (18 pts), with 3 pts in PR upgrading to CR, and 1 SD to PR. Although the median time to response was 7.7 mos, responses occurred as quickly as 1.8 mos to as late as 27.0 mos of therapy. For the 3 CR pts, prolonged therapy with an additional 14.9, 28.3 and 40.6 mos beyond the time of first response was required to achieve CR. To date, 7 pts have progressed with 3-year PFS 68.8% (95% CI:52–91%) and OS 85.3% (95% CI:71.1–100%). Correlatives:Cereblon (CRBN), recently identified as a direct protein target of lenalidomide, was evaluated by gene expression profiling and Western blot and found to be uniformly expressed in all 19 evaluable day 1 pt samples regardless of lenalidomide response. Thus, baseline CRBN expression does not appear to be a useful predictive biomarker of response in this population. The mechanism by which CRBN is linked to response is reported by Trudel et al, ASH 2012. Conclusions:Long-term followup of this study demonstrates that when using low doses of single agent lenalidomide in CLL, prolonged therapy is feasible and may be required for the achievement of durable, high quality responses. Maximal daily doses of 25mg can be reached and may also be needed for optimal response, though recurrent myelosuppression remains limiting. Disclosures:Chen:Celgene: Honoraria, Research Funding; GlaxoSmithKline: Research Funding; Lundbeck: Consultancy; Janssen: Consultancy, Research Funding. Off Label Use: Lenalidomide is not approved for use in chronic lymphocytic leukemia. Kukreti:Roche: Honoraria; Celgene: Honoraria; Janssen Ortho: Honoraria. Trudel:Celgene: Honoraria, Research Funding, Speakers Bureau.

Erratum: Cereblon is a direct protein target for immunomodulatory and antiproliferative activities of lenalidomide and pomalidomide

Erratum: Cereblon is a direct protein target for immunomodulatory and antiproliferative activities of lenalidomide and pomalidomide

Cereblon is a direct protein target for immunomodulatory and antiproliferative activities of lenalidomide and pomalidomide

Thalidomide and the immunomodulatory drug, lenalidomide, are therapeutically active in hematological malignancies. The ubiquitously expressed E3 ligase protein cereblon (CRBN) has been identified as the primary teratogenic target of thalidomide. Our studies demonstrate that thalidomide, lenalidomide and another immunomodulatory drug, pomalidomide, bound endogenous CRBN and recombinant CRBN–DNA damage binding protein-1 (DDB1) complexes. CRBN mediated antiproliferative activities of lenalidomide and pomalidomide in myeloma cells, as well as lenalidomide- and pomalidomide-induced cytokine production in T cells. Lenalidomide and pomalidomide inhibited autoubiquitination of CRBN in HEK293T cells expressing thalidomide-binding competent wild-type CRBN, but not thalidomide-binding defective CRBNYW/AA. Overexpression of CRBN wild-type protein, but not CRBNYW/AA mutant protein, in KMS12 myeloma cells, amplified pomalidomide-mediated reductions in c-myc and IRF4 expression and increases in p21WAF-1 expression. Long-term selection for lenalidomide resistance in H929 myeloma cell lines was accompanied by a reduction in CRBN, while in DF15R myeloma cells resistant to both pomalidomide and lenalidomide, CRBN protein was undetectable. Our biophysical, biochemical and gene silencing studies show that CRBN is a proximate, therapeutically important molecular target of lenalidomide and pomalidomide.

Clarifying the signal network of salvianolic acid B using proteomic assay and bioinformatic analysis

Salvianolic acid B (SB) is a natural compound with protective effect against ischemia-reperfusion heart injury. However, the signal network of SB including both direct target proteins and downstream signal-related proteins has not been clarified. In the present study, epidermal growth factor receptor (EGFR) was predicted to be the most possible direct protein target of SB by INVDOCK, a ligand-protein inverse-docking algorithm. Possible signal-related proteins of SB in H9C2 cells, including both under normal condition and under ischemia-reperfusion injury, were searched using 2-DE analysis. Totally, 14 signal-related proteins were found. Finally, signal network from EGFR to the signal-related proteins was established using bioinformatic analysis. Interestingly, 9 of the 14 signal-related proteins could be included in a network together with EGFR through direct interaction or only one intermediate partner. The signal cascade from EGFR to heat shock protein 27 (HSP27) and mitofilin (IMMT, inner membrane mitochondrial protein) might be the most important cascade. The signal network was certified by measuring the binding affinity of SB to EGFR in vitro, the effect of SB on internalization and phosphorylation of EGFR, the effect of SB on viability and proliferation of H9C2 cells, and the expression of inner membrane mitochondrial protein in the presence of EGFR inhibitor AG 1478.

Drug target pharmacogenomics: an overview.

Pharmacogenomics is a field aimed at understanding the genetic contribution to variability in drug efficacy and toxicity. The goal is to be able to select the drugs with the greatest likelihood of benefit and the least likelihood of harm in individual patients, based on their genetic make-up. Pharmacogenetics has historically been a field focused primarily on genetic polymorphisms in drug metabolizing enzymes and their impact on drug efficacy and toxicity. More recently, investigators have begun to study the relationship between drug target polymorphisms and drug efficacy and toxicity. There are now numerous examples in the literature of associations between drug target polymorphisms and drug effect. Drug targets can be broken into three main categories: the direct protein target of the drug, signal transduction cascades or downstream proteins, and disease pathogenesis proteins. While the drug target pharmacogenetics literature provides 'proof of concept' that genetic variability contributes to the variability in drug response, the data are not to the point of being clinically useful in most cases. Specific problems to date include the inability of a single polymorphism to be highly predictive of response, and inconsistencies across studies of the same polymorphism. It seems likely that an important factor in the above limitations is the approach of focusing on a single polymorphism in a single gene. Given that most drug responses involve a large number of proteins, all of whose genes could have several polymorphisms, it seems unlikely that a single polymorphism in a single gene would explain a high degree of drug response variability in a consistent fashion. Thus, it seems that a polygenic, or genomic approach will be more appropriate. Candidate gene and genome scanning approaches to pharmacogenomics have shown promise in relating drug target polymorphisms to response or toxicity, and pharmacogenomic strategies for drug discovery and drug development are now being implemented by most major pharmaceutical companies. Pharmacogenomics has the potential to significantly enhance the ability of clinicians to use medications in a safe and effective manner and, as such, represents an exciting field with tremendous clinical potential.