SciCombinator

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Concept: Proteasome

187

Cervical cancer is one of the leading causes of cancer death in women worldwide. The causative agents of cervical cancers, high-risk human papillomaviruses (HPVs), cause cancer through the action of two oncoproteins, E6 and E7. The E6 oncoprotein cooperates with an E3 ubiquitin ligase (UBE3A) to target the p53 tumour suppressor and important polarity and junctional PDZ proteins for proteasomal degradation, activities that are believed to contribute towards malignancy. However, the causative link between degradation of PDZ proteins and E6-mediated malignancy is largely unknown. We have developed an in vivo model of HPV E6-mediated cellular transformation using the genetic model organism, Drosophila melanogaster. Co-expression of E6 and human UBE3A in wing and eye epithelia results in severe morphological abnormalities. Furthermore, E6, via its PDZ-binding motif and in cooperation with UBE3A, targets a suite of PDZ proteins that are conserved in human and Drosophila, including Magi, Dlg and Scribble. Similar to human epithelia, Drosophila Magi is a major degradation target. Magi overexpression rescues the cellular abnormalities caused by E6+UBE3A coexpression and this activity of Magi is PDZ domain-dependent. Drosophila p53 was not targeted by E6+UBE3A, and E6+UBE3A activity alone is not sufficient to induce tumorigenesis, which only occurs when E6+UBE3A are expressed in conjunction with activated/oncogenic forms of Ras or Notch. Finally, through a genetic screen we have identified the insulin receptor signaling pathway as being required for E6+UBE3A induced hyperplasia. Our results suggest a highly conserved mechanism of HPV E6 mediated cellular transformation, and establish a powerful genetic model to identify and understand the cellular mechanisms that underlie HPV E6-induced malignancy.

Concepts: Oncology, Drosophila, Proteasome, Cervical cancer, Model organism, Drosophila melanogaster, Human papillomavirus, Cancer

183

Background Daratumumab, a human IgGκ monoclonal antibody that targets CD38, induces direct and indirect antimyeloma activity and has shown substantial efficacy as monotherapy in heavily pretreated patients with multiple myeloma, as well as in combination with bortezomib in patients with newly diagnosed multiple myeloma. Methods In this phase 3 trial, we randomly assigned 498 patients with relapsed or relapsed and refractory multiple myeloma to receive bortezomib (1.3 mg per square meter of body-surface area) and dexamethasone (20 mg) alone (control group) or in combination with daratumumab (16 mg per kilogram of body weight) (daratumumab group). The primary end point was progression-free survival. Results A prespecified interim analysis showed that the rate of progression-free survival was significantly higher in the daratumumab group than in the control group; the 12-month rate of progression-free survival was 60.7% in the daratumumab group versus 26.9% in the control group. After a median follow-up period of 7.4 months, the median progression-free survival was not reached in the daratumumab group and was 7.2 months in the control group (hazard ratio for progression or death with daratumumab vs. control, 0.39; 95% confidence interval, 0.28 to 0.53; P<0.001). The rate of overall response was higher in the daratumumab group than in the control group (82.9% vs. 63.2%, P<0.001), as were the rates of very good partial response or better (59.2% vs. 29.1%, P<0.001) and complete response or better (19.2% vs. 9.0%, P=0.001). Three of the most common grade 3 or 4 adverse events reported in the daratumumab group and the control group were thrombocytopenia (45.3% and 32.9%, respectively), anemia (14.4% and 16.0%, respectively), and neutropenia (12.8% and 4.2%, respectively). Infusion-related reactions that were associated with daratumumab treatment were reported in 45.3% of the patients in the daratumumab group; these reactions were mostly grade 1 or 2 (grade 3 in 8.6% of the patients), and in 98.2% of these patients, they occurred during the first infusion. Conclusions Among patients with relapsed or relapsed and refractory multiple myeloma, daratumumab in combination with bortezomib and dexamethasone resulted in significantly longer progression-free survival than bortezomib and dexamethasone alone and was associated with infusion-related reactions and higher rates of thrombocytopenia and neutropenia than bortezomib and dexamethasone alone. (Funded by Janssen Research and Development; ClinicalTrials.gov number, NCT02136134 .).

Concepts: Spinal cord compression, Hematology, Proteasome, Dexamethasone, Bortezomib, Lenalidomide, Thalidomide, Multiple myeloma

170

Carfilzomib, an irreversible proteasome inhibitor, has a favorable safety profile and significant antitumor activity in patients with relapsed and refractory multiple myeloma (MM). Here we summarize the clinical pharmacokinetics (PK), metabolism, and drug-drug interaction (DDI) profile of carfilzomib. The PK of carfilzomib, infused over 2-10 minutes, was evaluated in patients with solid tumors or MM. Metabolites of carfilzomib were characterized in patient plasma and urine samples. In vitro drug metabolism and DDI studies were conducted in human liver microsomes and hepatocytes. A clinical DDI study was conducted in patients with solid tumors to evaluate the effect of carfilzomib on CYP3A activity. Plasma concentrations of carfilzomib declined rapidly and in a biphasic manner after intravenous administration. The systemic half-life was short and the systemic clearance rate was higher than hepatic blood flow. Carfilzomib was cleared largely extrahepatically via peptidase cleavage and epoxide hydrolysis. Cytochrome P450-mediated metabolism played a minor role, suggesting that coadministration of P450 inhibitors or inducers is unlikely to change its PK profile. Carfilzomib showed direct and time-dependent inhibition of CYP3A in human liver microsome preparations and exposure to carfilzomib resulted in reductions in CYP3A and 1A2 gene expression in cultured human hepatocytes. However, administration of carfilzomib did not affect the PK of midazolam in patients with solid tumors, and there were no safety signals indicative of potential drug interactions. We conclude that the rapid systemic clearance and short half-life of carfilzomib limit clinically significant DDI.

Concepts: Proteasome, Glycogen, Cytochrome P450, Hepatocyte, Cancer, Drug metabolism, Pharmacology, Liver

168

The 11 members of the Membrane-associated RING-CH (MARCH) ubiquitin ligase family are relatively unexplored. Upon exogenous (over)expression, a number of these ligases can affect the trafficking of membrane molecules, but only in case of MARCH-1 endogenous functions have been demonstrated. Of the other endogenous MARCH proteins, no functions or substrates are known. We report here that TRAIL-receptor ®1 is a physiological substrate of the endogenous MARCH-8 ligase. The two human TRAIL death receptors play a role in immunosurveillance and are targets for cancer therapy, since they selectively induce apoptosis in tumor cells. We demonstrate that TRAIL-R1 is downregulated from the cell surface, with great preference over TRAIL-R2, by exogenous expression of MARCH ligases that are implicated in endosomal trafficking, such as MARCH-1 and -8. MARCH-8 attenuated TRAIL-R1 cell surface expression and apoptosis signaling by virtue of its ligase activity. This suggested that ubiquitination of TRAIL-R1 was instrumental in its downregulation by MARCH-8. Indeed, in cells with endogenous MARCH expression, TRAIL-R1 was ubiquitinated at steady-state, with the conserved membrane-proximal lysine 273 as interaction and potential acceptor site. This residue was also essential for the interaction of TRAIL-R1 with MARCH-1 and MARCH-8 and its downregulation by these ligases. Gene silencing identified MARCH-8 as the endogenous ligase that ubiquitinates TRAIL-R1 and attenuates its cell surface expression. These findings reveal that endogenous MARCH-8 regulates the steady-state cell surface expression of TRAIL-R1.

Concepts: Cancer, Posttranslational modification, Ubiquitin ligase, DNA, Proteasome, Ubiquitin, Enzyme, Protein

165

JNJ-26854165 (serdemetan) has previously been reported to inhibit the function of the E3 ligase human double minute-2, and we initially sought to characterize its activity in models of mantle cell lymphoma (MCL) and multiple myeloma (MM). Serdemetan induced a dose dependent inhibition of proliferation in both wild-type (wt) and mutant (mut) p53 cell lines, with IC50’s from 0.25 μ/L to 3 μ/L, in association with an S phase cell cycle arrest. Caspase-3 activation was primarily seen in wtp53 bearing cells, but also occurred in mutp53 bearing cells, albeit to a lesser extent. 293T cells treated with JNJ-26854165, and serdemetan-resistant fibroblasts displayed accumulation of cholesterol within endosomes, a phenotype reminiscent of that seen in the ATP-binding cassette sub-family A member-1 (ABCA1) cholesterol transport disorder, Tangiers disease. MM and MCL cells had decreased cholesterol efflux and electron microscopy demonstrated the accumulation of lipid whorls, confirming the lysosomal storage disease phenotype. JNJ-26854165 induced induction of cholesterol regulatory genes sterol regulatory element-binding transcription factor-1 and -2, liver X receptors α and β, along with increased expression of Niemann-Pick disease type-C1 and -C2. However, JNJ-26854165 induced enhanced ABCA1 turnover despite enhancing transcription. Finally, ABCA1 depletion resulted in enhanced sensitivity to JNJ-26854165. Overall, these findings support the hypothesis that serdemetan functions in part by inhibiting cholesterol transport, and that this pathway is a potential new target for the treatment of MCL and MM.

Concepts: Golgi apparatus, Proteasome, DNA, Chromosome, Protein, Mantle cell lymphoma, Cell cycle, Gene

158

A20 has been suggested to limit NF-κB activation by removing regulatory ubiquitin chains from ubiquitinated substrates. A20 is a ubiquitin-editing enzyme that removes K63-linked ubiquitin chains from adaptor proteins, such as RIP1, and then conjugates them to K48-linked polyubiquitin chains to trigger proteasomal degradation. To determine the role of the deubiquitinase function of A20 in downregulating NF-κB signaling, we have generated a knock-in mouse that lacks the deubiquitinase function of A20 (A20-OTU mice). These mice are normal and have no signs of inflammation, have normal proportions of B, T, dendritic, and myeloid cells, respond normally to LPS and TNF, and undergo normal NF-κB activation. Our results thus indicate that the deubiquitinase activity of A20 is dispensable for normal NF-κB signaling.

Concepts: Normal distribution, Ubiquitin, Mouse, Normality, Protein, Gene, Proteasome, Enzyme

151

Heat-shock protein 70 (HSP70) is known to function as a protective molecular chaperone that is massively induced in response to misfolded proteins following cerebral ischemia. The objective of this study was to characterize HSP70 induction by Z-ligustilide and explore its potential role in protection against cerebral ischemia-reperfusion injury. Our results demonstrated that the intranasal administration of Z-ligustilide reduced infarct volume and improved neurological function in a rat stroke model. Meanwhile, Z-ligustilide enhanced the cell viability of PC12 cells insulted by oxygen glucose deprivation-reoxygenation (OGD-Reoxy) and decreased apoptotic and necrotic cell death. Importantly, Z-ligustilide induced HSP70 expression both in vitro and in vivo. Although heat-shock factor 1 (HSF1) nuclear translocation was promoted by Z-ligustilide, HSP70-based heat-shock element (HSE)-binding luciferase activity was not activated, and HSP70 expression responsive to Z-ligustilide was not attenuated by HSE decoy oligonucleotides. However, Z-ligustilide significantly activated the phosphorylation of mitogen-activated protein kinases (MAPKs). Further inhibition of MAPK activity by specific inhibitors attenuated HSP70 induction by Z-ligustilide. Meanwhile, downregulation of HSP70 using KNK437, an HSP70 synthesis inhibitor, or small hairpin RNA (shRNA) significantly attenuated the protection of Z-ligustilide against OGD-Reoxy-induced injury. Moreover, the application of specific inhibitors of MAPKs also achieved similar results. Finally, Z-ligustilide alleviated the accumulation of ubiquitinated proteins induced by OGD-Reoxy, which was inhibited by HSP70-shRNA. Taken together, our results demonstrated that Z-ligustilide may induce protective HSP70 expression via the activation of the MAPK pathway, but not canonical HSF1 transcription. HSP70 plays a key role in the protection of Z-ligustilide against OGD-Reoxy-induced injury.

Concepts: Heat shock protein, Proteasome, Mitogen-activated protein kinase, Enzyme, Chaperone, Adenosine triphosphate, Signal transduction, Protein

139

Kidney fibrosis and fibrogenesis significantly exacerbate chronic kidney disease (CKD) progression and are essential therapeutic targets. Bortezomib (BZM) is a proteasome inhibitor used for the treatment of multiple myeloma (MM). Several studies have demonstrated that BZM attenuates renal impairment in patients with MM, although this effect is generally considered to be the result of MM remission. Recently, several studies on BZM reported anti-fibrotic effects on liver and skin in experimental animal models. However, its effect on renal fibrosis has yet to be examined. Here, we investigated the anti-fibrotic effects of BZM in an experimental mouse model of fibrosis that uses aristolochic acid I (AA). Ten weeks of AA administration with BZM treatment twice a week significantly attenuated AA-induced renal dysfunction and albuminuria, reduced the expression of renal fibrosis-related proteins and kidney injury markers, such as αSMA, Kim1, and Ngal, and prevented renal fibrosis at the level of histopathology. Furthermore, pathological activation of TGFβ1-Smad3 signaling and apoptosis, essential pathophysiological causes of AA-induced nephropathy (AAN), were ameliorated by BZM, suggesting this mechanism may be involved in improving fibrosis in AAN. In conclusion, BZM directly inhibits renal fibrosis in CKD via suppression of TGFβ1-Smad3 signaling and is promising in terms of drug repositioning.

Concepts: Animal model, Renal failure, Multiple myeloma, Kidney, Nephrology, Proteasome, Bortezomib, Chronic kidney disease

55

Background Multiple myeloma cells uniformly overexpress CD38. We studied daratumumab, a CD38-targeting, human IgG1κ monoclonal antibody, in a phase 1-2 trial involving patients with relapsed myeloma or relapsed myeloma that was refractory to two or more prior lines of therapy. Methods In part 1, the dose-escalation phase, we administered daratumumab at doses of 0.005 to 24 mg per kilogram of body weight. In part 2, the dose-expansion phase, 30 patients received 8 mg per kilogram of daratumumab and 42 received 16 mg per kilogram, administered once weekly (8 doses), twice monthly (8 doses), and monthly for up to 24 months. End points included safety, efficacy, and pharmacokinetics. Results No maximum tolerated dose was identified in part 1. In part 2, the median time since diagnosis was 5.7 years. Patients had received a median of four prior treatments; 79% of the patients had disease that was refractory to the last therapy received (64% had disease refractory to proteasome inhibitors and immunomodulatory drugs and 64% had disease refractory to bortezomib and lenalidomide), and 76% had received autologous stem-cell transplants. Infusion-related reactions in part 2 were mild (71% of patients had an event of any grade, and 1% had an event of grade 3), with no dose-dependent adverse events. The most common adverse events of grade 3 or 4 (in ≥5% of patients) were pneumonia and thrombocytopenia. The overall response rate was 36% in the cohort that received 16 mg per kilogram (15 patients had a partial response or better, including 2 with a complete response and 2 with a very good partial response) and 10% in the cohort that received 8 mg per kilogram (3 had a partial response). In the cohort that received 16 mg per kilogram, the median progression-free survival was 5.6 months (95% confidence interval [CI], 4.2 to 8.1), and 65% (95% CI, 28 to 86) of the patients who had a response did not have progression at 12 months. Conclusions Daratumumab monotherapy had a favorable safety profile and encouraging efficacy in patients with heavily pretreated and refractory myeloma. (Funded by Janssen Research and Development and Genmab; ClinicalTrials.gov number, NCT00574288 .).

Concepts: Plasma cell, Proteasome inhibitor, Clinical trial, Lenalidomide, Thalidomide, Bortezomib, Proteasome, Multiple myeloma

50

Highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) has developed strategies to inhibit host immune recognition. We identify cellular E3 ubiquitin ligase ring-finger and CHY zinc-finger domain-containing 1 (RCHY1) as an interacting partner of the viral SARS-unique domain (SUD) and papain-like protease (PL(pro)), and, as a consequence, the involvement of cellular p53 as antagonist of coronaviral replication. Residues 95-144 of RCHY1 and 389-652 of SUD (SUD-NM) subdomains are crucial for interaction. Association with SUD increases the stability of RCHY1 and augments RCHY1-mediated ubiquitination as well as degradation of p53. The calcium/calmodulin-dependent protein kinase II delta (CAMK2D), which normally influences RCHY1 stability by phosphorylation, also binds to SUD. In vivo phosphorylation shows that SUD does not regulate phosphorylation of RCHY1 via CAMK2D. Similarly to SUD, the PL(pro)s from SARS-CoV, MERS-CoV, and HCoV-NL63 physically interact with and stabilize RCHY1, and thus trigger degradation of endogenous p53. The SARS-CoV papain-like protease is encoded next to SUD within nonstructural protein 3. A SUD-PL(pro) fusion interacts with RCHY1 more intensively and causes stronger p53 degradation than SARS-CoV PL(pro) alone. We show that p53 inhibits replication of infectious SARS-CoV as well as of replicons and human coronavirus NL63. Hence, human coronaviruses antagonize the viral inhibitor p53 via stabilizing RCHY1 and promoting RCHY1-mediated p53 degradation. SUD functions as an enhancer to strengthen interaction between RCHY1 and nonstructural protein 3, leading to a further increase in in p53 degradation. The significance of these findings is that down-regulation of p53 as a major player in antiviral innate immunity provides a long-sought explanation for delayed activities of respective genes.

Concepts: Ubiquitin ligase, SARS coronavirus, Ubiquitin, Enzyme inhibitor, Posttranslational modification, Proteasome, Coronavirus, Severe acute respiratory syndrome