Mapping the Degradable Kinome Provides a Resource for Expedited Degrader Development.

Targeted protein degradation (TPD) refers to the use of small molecules to induce ubiquitin-dependent degradation of proteins. TPD is of interest in drug development, as it can address previously inaccessible targets. However, degrader discovery and optimization remains an inefficient process due to a lack of understanding of the relative importance of the key molecular events required to induce target degradation. Here, we use chemo-proteomics to annotate the degradable kinome. Our expansive dataset provides chemical leads for ∼200 kinases and demonstrates that the current practice of starting from the highest potency binder is an ineffective method for discovering active compounds. We develop multitargeted degraders to answer fundamental questions about the ubiquitin proteasome system, uncovering that kinase degradation is p97 dependent. This work will not only fuel kinase degrader discovery, but also provides a blueprint for evaluating targeted degradation across entire gene families to accelerate understanding of TPD beyond the kinome.

Differential Analysis of Cereblon Neosubstrates in Rabbit Embryos Using Targeted Proteomics.

Targeted protein degradation is the selective removal of a protein of interest through hijacking intracellular protein cleanup machinery. This rapidly growing field currently relies heavily on the use of the E3 ligase cereblon (CRBN) to target proteins for degradation, including the immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide which work through a molecular glue mechanism of action with CRBN. While CRBN recruitment can result in degradation of a specific protein of interest (e.g., efficacy), degradation of other proteins (called CRBN neosubstrates) also occurs. Degradation of one or more of these CRBN neosubstrates is believed to play an important role in thalidomide-related developmental toxicity observed in rabbits and primates. We identified a set of 25 proteins of interest associated with CRBN-related protein homeostasis and/or embryo/fetal development. We developed a targeted assay for these proteins combining peptide immunoaffinity enrichment and high-resolution mass spectrometry and successfully applied this assay to rabbit embryo samples from pregnant rabbits dosed with three IMiDs. We confirmed previously reported in vivo decreases in neosubstrates like SALL4, as well as provided evidence of neosubstrate changes for proteins only examined in vitro previously. While there were many proteins that were similarly decreased by all three IMiDs, no compound had the exact same neosubstrate degradation profile as another. We compared our data to previous literature reports of IMiD-induced degradation and known developmental biology associations. Based on our observations, we recommend monitoring at least a major subset of these neosubstrates in a developmental test system to improve CRBN-binding compound-specific risk assessment. A strength of our assay is that it is configurable, and the target list can be readily adapted to focus on only a subset of proteins of interest or expanded to incorporate new findings as additional information about CRBN biology is discovered.

Multiple myeloma and its treatment contribute to increased platelet reactivity.

Multiple myeloma (MM) and its precursor states, smoldering myeloma (SM) and monoclonal gammopathy of undetermined significance (MGUS) are associated with increased incidence of thrombosis, however the cause of this is unknown. Lenalidomide treatment of MM substantially improves patient survival, although significantly increases thrombotic risk by an unknown mechanism. This pilot study aimed to establish the impact of MM and its treatment with Lenalidomide on platelet function. We analyzed platelet function in MGUS, SM and MM compared to healthy controls. We report an increase in platelet reactivity in MGUS, SM, and MM where increases in fibrinogen binding, P-selectin exposure, altered receptor expression, elevated levels of aggregation and enhanced sensitivity to agonist stimulation were observed. We also demonstrate an increase in patient platelet reactivity post Lenalidomide treatment compared to pre-treatment. We show Lenalidomide treatment of platelets ex vivo increased reactivity that was associated with formation of larger thrombi at arterial shear rates but not venous shear rates. This study demonstrates a clear increase in platelet reactivity and prothrombotic potential in patients with MGUS, SM and MM which is elevated further upon treatment with Lenalidomide. Our observations suggest that more detailed studies are warranted to determine mechanisms of thrombotic complications to enable the development of new preventative strategies that specifically target platelets.

What is the context?Multiple myeloma is associated with increased risk of thrombosis, although the potential role of platelets in this has not been evaluated.What is new?We show in this pilot study that multiple myeloma and its precursor states of smoldering myeloma and monoclonal gammopathy of undetermined significance are associated with increased levels of platelet responses. This is further exacerbated by treatment with the immunomodulatory drug lenalidomide.What is the impact?This study suggests that more detailed studies are warranted to explore the mechanisms that cause these effects in a larger population of patients, since this may reveal new approaches to prevent myeloma-associated thrombotic complications.

Assessment of metabolic stability and pharmacokinetics by LC-MS/MS and establishment of the safe dose of IMID-2, a novel anticancer molecule under drug discovery.

Pyruvate kinase (PK) M2 activators ramp up glycolysis in cancer cells, leading to a reversal of the Warburg effect in cancer cells. A promising PKM2 activator molecule, IMID-2, developed by the National Institute of Pharmaceutical Education and Research-Ahmedabad showed promising anticancer activity against MCF-7 and COLO-205 cell lines, which represent breast and colon cancer. Its physicochemical properties, like solubility, ionization constant, partition coefficient and distribution constant, have already been established. Its metabolic pathway is also well established through in vitro and in vivo metabolite profiling and reported previously. In this study, we have evaluated the metabolic stability of IMID-2 using LC-MS/MS and investigated the safety aspect of the molecule through an acute oral toxicity study. In vivo studies in rats confirmed that the molecule is safe even at a dose level of 175 mg/kg. Furthermore, a pharmacokinetic study of IMID-2 was also carried out using LC-MS/MS to understand its absorption, distribution, metabolism, and excretion profile. The molecule was found to have promising bioavailability through the oral route. This research work is thus another step in the drug testing of this promising anticancer molecule. The molecule can be considered to be a potential anticancer lead based on the earlier report substantiated by current findings.

The role of E3 ubiquitin ligase in multiple myeloma: potential for cereblon E3 ligase modulators in the treatment of relapsed/refractory disease.

INTRODUCTION: Insights into the mechanisms of protein homeostasis and proteasomal degradation have led to new strategies of redirecting the ubiquitin-proteasome system (UPS) to reduce or eliminate proteins or survival factors key to malignant pathobiology, multiple myeloma (MM) in particular. These strategies have enabled researchers to target proteins that were previously considered difficult to modulate by pharmacological means. AREAS COVERED: This review provides a brief overview of UPS biology, particularly the role of the CRL4CRBN E3 ubiquitin ligase complex, and summarizes current strategies for co-opting the UPS, including CELMoD compounds, SNIPERs, PROTACs, and degronimids. A detailed discussion is provided on lead CELMoD compounds iberdomide and mezigdomide, which are currently being evaluated in clinical trials in patients with MM. EXPERT OPINION: Since a high proportion of patients develop drug resistance, it is vital to have novel therapeutic agents for treating relapsed patients with MM more effectively. It is encouraging that the expanding pathophysiological insight into cellular signaling pathways in MM increasingly translates into the development of novel therapeutic agents such as targeted protein degraders. This holds promise for improving outcomes in MM and beyond.

Evaluation of humoral and cellular response to third dose of BNT162b2 mRNA COVID-19 vaccine in patients treated with B-cell depleting therapy.

OBJECTIVE: to investigate the responses to mRNA COVID-19 vaccines in a cohort of immunosuppressed patients affected by immune-mediated inflammatory diseases (IMID). METHODS: we have measured humoral and cellular immunity using quantitative IgG anti-SARS-CoV-2 Spike antibody (anti-S-IgG), neutralization assays and specific interferon-gamma (IFN-g) release assay (IGRA) before and after the third dose of BNT162b2. The response of those on anti-CD20 (n = 18) was then compared with healthy controls (HC, n = 18) and IMID naïve to anti-CD20 drugs (n = 13). RESULTS: a third BNT162b2 dose is highly immunogenic in IMID patients naïve to anti-CD20, as 100% of the subjects seroconverted compared to the 55% in anti-CD20. The rate of IGRA response was of 79% in anti-CD20, 50% in IMID naïve to anti-CD20, 100% in HC. Among those who have seroconverted, IMID patients had significantly reduced anti-S-IgG and neutralization titers compared to HC, whereas no significant difference was observed when comparing anti-CD20 and HC. Furthermore, 13% of anti-CD20 and 7.7% of IMID were simultaneously negative for both neutralizing antibodies and IGRA after three doses. CONCLUSION: these data draw attention to the immunogenicity of COVID-19 vaccination in treated IMID, taking specific groups into consideration for vaccination program.

Chilblains in immune-mediated inflammatory diseases: a review.

Chilblains were first described over a hundred years ago as cutaneous inflammatory lesions, typically on the digits, occurring on cold exposure. Chilblains can be primary, or secondary to a number of conditions such as infections, including COVID-19, and immune-mediated inflammatory disorders (IMIDs) with SLE being the commonest. Chilblain lupus erythematosus (CHLE) was first described in 1888 as cold-induced erythematous lesions before the terms 'chilblains' or 'perniosis' were coined. Diagnostic criteria exist for both chilblains and CHLE. Histopathologically, CHLE lesions show interface dermatitis with perivascular lymphocytic infiltrate. Immunofluorescence demonstrates linear deposits of immunoglobulins and complement in the dermo-epidermal junction. This narrative review focuses on chilblains secondary to immune-mediated inflammatory disorders, primarily the epidemiology, pathogenesis and treatment of CHLE.

Information and Communication Technologies in Patients With Immune-Mediated Inflammatory Diseases: Cross-sectional Survey.

BACKGROUND: Information and communication technologies (ICTs) are changing the traditional health care model and redefining personalized health. ICTs offer effective communication and real-time monitoring of patients and provide additional data to support clinical decision-making, improve the quality of care, and contribute to the empowerment of patients. However, evidence on the use of ICTs and digital preferences of immune-mediated inflammatory disease (IMID) patients is scarce. OBJECTIVE: The aim of this study is to describe the degree of use of ICTs in patients with IMIDs (including rheumatic diseases, inflammatory bowel diseases, and psoriasis), identify their needs, and analyze their interest in the use of apps as tools for better management of their disease. METHODS: A questionnaire was created by a multidisciplinary team including pharmacists, rheumatologists, gastroenterologists, dermatologists, and nurses with experience in ICTs applied to the field of IMID. The survey included 27 questions organized into 3 blocks: (1) sociodemographic characteristics, (2) ICT use for health-related information, and (3) patient expectations about mobile health. RESULTS: A total of 472 questionnaires were analyzed. Overall, 52.9% (250/472) of patients were diagnosed with a rheumatologic disease, 39.4% (186/472) with inflammatory bowel disease, and 12.3% (58/472) with psoriasis. The state of health was considered good by 45.6% (215/472) of patients. Patients were interested in staying informed about health issues in 86.9% (410/427) of cases and sought health-related information mainly from the internet (334/472, 70.8%) and health care professionals (318/472, 67.4%). Overall, 13.6% (64/472) did not trust the health information they found in internet. Of the patients, 42.8% (202/472) had a health app, and 42.2% (199/472) had found it on their own. Patients would like a health app to help mainly to manage appointments (281/472, 59.5%), obtain information about their diseases and treatments (274/472, 58.1%), and get in contact with health professionals (250/472, 53.0%). Overall, 90.0% (425/472) of patients reported they would use an app to manage their IMID if their health professional recommended it, and 58.0% (274/472) would pay or probably be willing to pay for it. CONCLUSIONS: IMID patients were very interested in finding health-related information via ICTs, especially using smartphones and apps recommended by health professionals. Appointment management, advice on disease and treatment management, and personalized communication with health professionals were the most desired app features identified. Health professionals should play an essential role in recommending and validating these tools to ensure they are of high quality.

Phase 1 study of pomalidomide in children with recurrent, refractory, and progressive central nervous system tumors: A Pediatric Brain Tumor Consortium trial.

BACKGROUND: Central nervous system (CNS) malignancies are the most common solid tumors among children, and novel therapies are needed to help improve survival. Pomalidomide is an immunomodulatory agent that displays antiangiogenic and cytotoxic activity, making it an appropriate candidate to explore in pediatric CNS tumors. METHODS: A phase 1 first in pediatric trial of pomalidomide was conducted in children with recurrent, progressive, and refractory CNS tumors. The primary objective was to determine the maximum tolerated dose (MTD) and/or recommended phase 2 dose (RP2D) when given orally once daily for 21 consecutive days of a 28-day cycle. Once the MTD was established, 12 additional patients were enrolled on expansion cohorts based on age and steroid use. RESULTS: Twenty-nine children were enrolled and 25 were evaluable for dose-limiting toxicity (DLT). The MTD was 2.6 mg/m2 (dose level 2). Four DLTs were observed in three patients at dose level 3 (3.4 mg/m2 ) includeding grade 3 diarrhea, grade 3 thrombocytopenia, grade 3 lung infection, and grade 4 neutropenia. The most common adverse events were grade 1 and 2 myelosuppression. One patient with an oligodendroglioma had stable disease for nine cycles, and a second patient with an anaplastic pleomorphic xanthoastrocytoma achieved a sustained partial response. Immunologic analyses suggested that pomalidomide triggers immunomodulation. CONCLUSIONS: The MTD of pomalidomide is 2.6 mg/m2 . It was well tolerated, and immune correlates showed a serum immune response. These data led to an industry-sponsored phase 2 trial of pomalidomide monotherapy in children with recurrent brain tumors (NCT03257631).

COVID-19 vaccine safety and efficacy in patients with immune-mediated inflammatory disease: Review of available evidence.

Dermatologists diagnose and treat many immune-mediated inflammatory diseases (IMID). Understanding the inherent immune dysregulation of these diseases as well as the additional disruption that comes as a result of IMID treatments has been important during the COVID-19 pandemic. With vaccines becoming widely available, dermatologists need to be familiar with the risks and benefits of vaccination in these patients, particularly those taking biologics, in order to have informed discussions with their patients. In this review, we present the current evidence related to COVID-19 vaccine safety and efficacy in patients with IMID and review existing recommendations for vaccination against SARS-CoV-2. Given the current evidence, there is minimal concern that these patients are at any greater risk of harm from COVID-19 vaccination compared to healthy controls. For most, the benefit of avoiding severe COVID-19 through vaccination will outweigh the theoretical risk of these vaccines. A question that is still outstanding is whether patients on biologics will generate a sufficient immune response to the vaccine, which may be dependent on the specific biologic therapy and indication being treated. This underscores the importance of following patients with IMID after vaccination to determine the safety, efficacy, and duration of the vaccine in this population.

Metabolite profiling of IMID-2, a novel anticancer molecule of piperazine derivative: In silico prediction, in vitro and in vivo metabolite characterization using UPLC-QTOF-MS/MS.

IMID-2, a newly identified piperazine-based anticancer molecule, has been shown to be cytotoxic against various cancer cell lines. The primary aim of this research was to identify and characterize possible metabolites of the molecule formed during biotransformation. A metabolite identification study was first executed using an in silico tool to predict the possible metabolism sites of IMID-2. Thereafter, metabolites generated in vitro (rat liver microsomes, rat S9 fractions and human liver microsomes) and in vivo (rat plasma, urine and feces) were identified and characterized employing UPLC-QTOF-MS/MS. A total of eight metabolites, among which were six in phase I and two in phase II reactions, were recognized. The plausible structure of the metabolites and probable metabolic pathway have been established based on the mass fragmentation pattern, mass ppm error, ring double bond calculation and nitrogen rule. The majority of phase I metabolites were generated by N-oxidation, hydroxylation, oxidative deamination followed by reduction, oxidative dechlorination, N-dearylation, and N-dealkylation. Glucuronidation played a significant role in the formation of phase II metabolites of the molecule.

Phenyl-Glutarimides: Alternative Cereblon Binders for the Design of PROTACs.

Targeting cereblon (CRBN) is currently one of the most frequently reported proteolysis-targeting chimera (PROTAC) approaches, owing to favorable drug-like properties of CRBN ligands, immunomodulatory imide drugs (IMiDs). However, IMiDs are known to be inherently unstable, readily undergoing hydrolysis in body fluids. Here we show that IMiDs and IMiD-based PROTACs rapidly hydrolyze in commonly utilized cell media, which significantly affects their cell efficacy. We designed novel CRBN binders, phenyl glutarimide (PG) analogues, and showed that they retained affinity for CRBN with high ligand efficiency (LE >0.48) and displayed improved chemical stability. Our efforts led to the discovery of PG PROTAC 4 c (SJ995973), a uniquely potent degrader of bromodomain and extra-terminal (BET) proteins that inhibited the viability of human acute myeloid leukemia MV4-11 cells at low picomolar concentrations (IC50 =3 pM; BRD4 DC50 =0.87 nM). These findings strongly support the utility of PG derivatives in the design of CRBN-directed PROTACs.

Discovery of Novel Imidazopyridine GSK-3ß Inhibitors Supported by Computational Approaches.

The interest of research groups and pharmaceutical companies to discover novel GSK-3ß inhibitors has increased over the years considering the involvement of this enzyme in many pathophysiological processes and diseases. Along this line, we recently reported on 1H-indazole-3-carboxamide (INDZ) derivatives 1-6, showing good GSK-3ß inhibition activity. However, they suffered from generally poor central nervous system (CNS) permeability. Here, we describe the design, synthesis, and in vitro characterization of novel imidazo[1,5-a]pyridine-1-carboxamide (IMID 1) and imidazo[1,5-a]pyridine-3-carboxamide (IMID 2) compounds (7-18) to overcome such liability. In detail, structure-based approaches and fine-tuning of physicochemical properties guided the design of derivatives 7-18 resulting in ameliorated absorption, distribution, metabolism, and excretion (ADME) properties. A crystal structure of 16 in complex with GSK-3ß enzyme (PDB entry 6Y9S) confirmed the in silico models. Despite the nanomolar inhibition activity, the new core compounds showed a reduction in potency with respect to INDZ derivatives 1-6. In this context, Molecular Dynamics (MD) and Quantum Mechanics (QM) based approaches along with NMR investigation helped to rationalize the observed structure activity relationship (SAR). With these findings, the key role of the acidic hydrogen of the central core for a tight interaction within the ATP pocket of the enzyme reflecting in good GSK-3ß affinity was demonstrated.

Peripheral Immunotype Correlates with Minimal Residual Disease Status and Is Modulated by Immunomodulatory Drugs in Multiple Myeloma.

Data indicate reversal of immune dysfunction with active treatment; however, the precise contribution of specific immune effector and immune suppressor components to achieve a minimal residual disease (MRD) state and immunomodulatory drug-mediated immunomodulatory effects in multiple myeloma (MM) patients remains poorly understood. In this prospective proof-of-principle study we sought to determine the dynamic alterations in natural killer (NK), NK-T, and T cells, including maturation and activating/inhibitory repertoire associated with MRDpos versus MRDneg status after autologous stem cell transplantation (ASCT) and during lenalidomide-based maintenance therapy. Of the 46MM patients enrolled, 36 had bone marrow MRD assessment 60+ days post-ASCT, 30 had longitudinal blood immunotyping during maintenance (pretherapy and after cycles 1, 3, and 6), and 20 had both MRD assessment and longitudinal immunotyping. Multicolor flow cytometry was used for MRD and immunotyping. Although the absolute number of NK cells was significantly lower in patients with MRDpos response, phenotypically NK cells in these patients displayed higher expression of activating receptors KIRDS4 and decreased expression of inhibitory molecules NKG2A compared with the MRDneg group. Furthermore, we observed significantly lower frequencies of T cells displaying KIR3DL1 in MRDpos versus MRDneg patients. Longitudinal immunotyping during lenalidomide maintenance showed loss of mature NK effector function, augmentation of NK-T effector function, and acquisition of PD1 independent anergic state. Our findings also suggest skewing of T cells toward an exhausted state during the maintenance phase in MRDpos patients. Put together, these observations provide a distinctive signature for MRDneg and MRDpos groups. These data support exploration of immune profiling in prospective clinical trials according to MRD-defined responses to identify patients that may benefit from maintenance intensification/modification or maintenance withdrawal.

Doxorubicin, vinblastine, dacarbazine and lenalidomide for older Hodgkin lymphoma patients: final results of a German Hodgkin Study Group (GHSG) phase-I trial.

About 30% of all Hodgkin lymphoma (HL) patients are ≥60 years old. As lenalidomide has promising single agent activity in multiple relapsed HL, we replaced bleomycin in ABVD with lenalidomide in this phase-I trial. Patients aged ≥60 years with early-unfavourable- or advanced-stage HL (Eastern Cooperative Oncology Group performance status ≤2, Cumulative Illness Rating Scale for Geriatrics score 0-7) received 4-8 cycles of AVD (doxorubicin, vinblastine, dacarbazine) and lenalidomide in escalation with overdose control. Dose-limiting toxicities (DLTs) included thromboembolism ≥grade 2, severe haematological toxicity, neutropenic fever and prolonged therapy delay. Twenty-five patients with a median age of 68 years were included, 68% had advanced-stage HL. A pre-defined stopping criterion for dose escalation after DLT evaluation of 20/24 patients suggested a recommended phase II dose (RPTD) of 20 mg. DLTs occurred in 10/24 evaluable patients, all treated with ≥20 mg, however, median relative dose intensity was 97% (interquartile range 49-104%). Grade 3 or higher toxicities occurred in all 22 patients at ≥20 mg lenalidomide but no treatment-related deaths occurred. Overall response rate was 80% for all patients (20/25) and 86% (19/22) at ≥20 mg lenalidomide. Three-year estimates for progression-free survival and OS were 69·7% (95% CI: 50·3-89·1%) and 83·8% (95%-CI: 69·3-98·4%), respectively. In conclusion, AVD with lenalidomide 20 mg is feasible and highly effective in older HL patients.

Prolonged follow-up on lenalidomide-based treatment for mucosa-associated lymphoid tissue lymphoma (MALT lymphoma)-Real-world data from the Medical University of Vienna.

Based on results of two pilot trials, lenalidomide (LEN) was found to be active and safe as monotherapy and showed an increased response rate of 80% in combination with rituximab (R) for patients with mucosa-associated lymphoid tissue (MALT) lymphoma. While initial results were promising, there are currently no data on long-term outcome, and larger international phase II/III trials on LEN for indolent lymphoma lack specific subgroup analyses. Thus, we have systematically analyzed 50 patients treated with LEN-based therapy (LEN-monotherapy n = 16, R-LEN n = 34) at the Medical University of Vienna 2009 to 2019 and investigated long-term outcome and relapse patterns. At a follow-up of more than 5 years (median 68 months), 54% of patients are free of relapse, and estimated median progression-free survival (PFS) was 72 months (95%CI 49-96). There was no difference in PFS according to stage of disease, i.e. localized versus disseminated disease (P = .67) and previous systemic treatment (P = .16). Interestingly, but with the caveat of the limited number of patients included in this series, primary extragastric disease had a superior PFS compared with gastric lymphoma (P = .04) and also depth of response, i.e. complete or partial response versus stable disease was associated with significantly prolonged PFS (P = .01). We documented four patients (8%) with pronounced improvement of response during follow-up including three patients initially rated as partial remission and finally achieving complete remission at 12 to 32 months. This highlights the potential of delayed responses to LEN treatment. Estimated overall survival at 5 years was excellent at 92%. These "real-world" data confirm long-term activity of LEN in MALT lymphoma.

Rabex-5 is a lenalidomide target molecule that negatively regulates TLR-induced type 1 IFN production.

Immunomodulatory drugs (IMiDs) are a family of compounds derived from thalidomide. Binding of the IMiD molecule to the Lon protease Cereblon initiates the degradation of substrates via the ubiquitin proteasome pathway. Here, we show that Cereblon forms a complex with Rabex-5, a regulator of immune homeostasis. Treatment with lenalidomide prevented the association of Cereblon with Rabex-5. Conversely, mutation of the IMiD binding site increased Cereblon-Rabex-5 coimmunoprecipitation. The thalidomide binding region of Cereblon therefore regulates the formation of this complex. Knockdown of Rabex-5 in the THP-1 macrophage cell line up-regulated Toll-like receptor (TLR)-induced cytokine and type 1 IFN production via a STAT1/IRF activating pathway. Thus, we identify Rabex-5 as a IMiD target molecule that functions to restrain TLR activated auto-immune promoting pathways. We propose that release of Rabex-5 from complex with Cereblon enables the suppression of immune responses, contributing to the antiinflammatory properties of IMiDs.

Immunomodulatory drugs inhibit TLR4-induced type-1 interferon production independently of Cereblon via suppression of the TRIF/IRF3 pathway.

Thalidomide and its derivatives, collectively referred to as immunomodulatory drugs (IMiDs), are effective inhibitors of inflammation and are known to inhibit TLR-induced TNFα production. The identification of Cereblon as the receptor for these compounds has led to a rapid advancement in our understanding of IMiD properties; however, there remain no studies addressing the role of Cereblon in mediating the suppressive effect of IMiDs on TLR responses. Here, we developed Cereblon-deficient mice using the CRISPR-Cas9 system. TLR-induced cytokine responses were unaffected by Cereblon deficiency in vivo Moreover, IMiD treatment inhibited cytokine production even in the absence of Cereblon. The IMiD-induced suppression of cytokine production therefore occurs independently of Cereblon in mice. Further investigation revealed that IMiDs are potent inhibitors of TLR-induced type-1 interferon production via suppression of the TRIF/IRF3 pathway. These data suggest that IMiDs may prove effective in the treatment of disorders characterized by the ectopic production of type-1 interferon. Significantly, these properties are mediated separately from thalidomide's teratogenic receptor, Cereblon. Thus, certain therapeutic properties of Thalidomide can be separated from its harmful side effects.

Expression of the cereblon binding protein argonaute 2 plays an important role for multiple myeloma cell growth and survival.

BACKGROUND: Immunomodulatory drugs (IMiDs), such as lenalidomide, are therapeutically active compounds that bind and modulate the E3 ubiquitin ligase substrate recruiter cereblon, thereby affect steady-state levels of cereblon and cereblon binding partners, such as ikaros and aiolos, and induce many cellular responses, including cytotoxicity to multiple myeloma (MM) cells. Nevertheless, it takes many days for MM cells to die after IMiD induced depletion of ikaros and aiolos and thus we searched for other cereblon binding partners that participate in IMiD cytotoxicity. METHODS: Cereblon binding partners were identified from a MM cell line expressing histidine-tagged cereblon by pulling down cereblon and its binding partners and verified by co-immunoprecipitation. IMiD effects were determined by western blot analysis, cell viability assay, microRNA array and apoptosis analysis. RESULTS: We identified argonaute 2 (AGO2) as a cereblon binding partner and found that the steady-state levels of AGO2 were regulated by cereblon. Upon treatment of IMiD-sensitive MM cells with lenalidomide, the steady-state levels of cereblon were significantly increased, whereas levels of AGO2 were significantly decreased. It has been reported that AGO2 plays a pivotal role in microRNA maturation and function. Interestingly, upon treatment of MM cells with lenalidomide, the steady-state levels of microRNAs were significantly altered. In addition, silencing of AGO2 in MM cells, regardless of sensitivity to IMiDs, significantly decreased the levels of AGO2 and microRNAs and massively induced cell death. CONCLUSION: These results support the notion that the cereblon binding partner AGO2 plays an important role in regulating MM cell growth and survival and AGO2 could be considered as a novel drug target for overcoming IMiD resistance in MM cells.