A Degron Blocking Strategy Towards Improved CRL4CRBN Recruiting PROTAC Selectivity.

Small molecules inducing protein degradation are important pharmacological tools to interrogate complex biology and are rapidly translating into clinical agents. However, to fully realise the potential of these molecules, selectivity remains a limiting challenge. Herein, we addressed the issue of selectivity in the design of CRL4CRBN recruiting PROteolysis TArgeting Chimeras (PROTACs). Thalidomide derivatives used to generate CRL4CRBN recruiting PROTACs have well described intrinsic monovalent degradation profiles by inducing the recruitment of neo-substrates, such as GSPT1, Ikaros and Aiolos. We leveraged structural insights from known CRL4CRBN neo-substrates to attenuate and indeed remove this monovalent degradation function in well-known CRL4CRBN molecular glues degraders, namely CC-885 and Pomalidomide. We then applied these design principles on a previously published BRD9 PROTAC (dBRD9-A) and generated an analogue with improved selectivity profile. Finally, we implemented a computational modelling pipeline to show that our degron blocking design does not impact PROTAC-induced ternary complex formation. We believe that the tools and principles presented in this work will be valuable to support the development of targeted protein degradation.

SimPLIT: Simplified Sample Preparation for Large-Scale Isobaric Tagging Proteomics.

Large scale proteomic profiling of cell lines can reveal molecular signatures attributed to variable genotypes or induced perturbations, enabling proteogenomic associations and elucidation of pharmacological mechanisms of action. Although isobaric labeling has increased the throughput of proteomic analysis, the commonly used sample preparation workflows often require time-consuming steps and costly consumables, limiting their suitability for large scale studies. Here, we present a simplified and cost-effective one-pot reaction workflow in a 96-well plate format (SimPLIT) that minimizes processing steps and demonstrates improved reproducibility compared to alternative approaches. The workflow is based on a sodium deoxycholate lysis buffer and a single detergent cleanup step after peptide labeling, followed by quick off-line fractionation and MS2 analysis. We showcase the applicability of the workflow in a panel of colorectal cancer cell lines and by performing target discovery for a set of molecular glue degraders in different cell lines, in a 96-sample assay. Using this workflow, we report frequently dysregulated proteins in colorectal cancer cells and uncover cell-dependent protein degradation profiles of seven cereblon E3 ligase modulators (CRL4CRBN). Overall, SimPLIT is a robust method that can be easily implemented in any proteomics laboratory for medium-to-large scale TMT-based studies for deep profiling of cell lines.

Investigating the phosphinic acid tripeptide mimetic DG013A as a tool compound inhibitor of the M1-aminopeptidase ERAP1.

ERAP1 is a zinc-dependent M1-aminopeptidase that trims lipophilic amino acids from the N-terminus of peptides. Owing to its importance in the processing of antigens and regulation of the adaptive immune response, dysregulation of the highly polymorphic ERAP1 has been implicated in autoimmune disease and cancer. To test this hypothesis and establish the role of ERAP1 in these disease areas, high affinity, cell permeable and selective chemical probes are essential. DG013A 1, is a phosphinic acid tripeptide mimetic inhibitor with reported low nanomolar affinity for ERAP1. However, this chemotype is a privileged structure for binding to various metal-dependent peptidases and contains a highly charged phosphinic acid moiety, so it was unclear whether it would display the high selectivity and passive permeability required for a chemical probe. Therefore, we designed a new stereoselective route to synthesize a library of DG013A 1 analogues to determine the suitability of this compound as a cellular chemical probe to validate ERAP1 as a drug discovery target.

Lenalidomide induces ubiquitination and degradation of CK1α in del(5q) MDS.

Lenalidomide is a highly effective treatment for myelodysplastic syndrome (MDS) with deletion of chromosome 5q (del(5q)). Here, we demonstrate that lenalidomide induces the ubiquitination of casein kinase 1A1 (CK1α) by the E3 ubiquitin ligase CUL4-RBX1-DDB1-CRBN (known as CRL4(CRBN)), resulting in CK1α degradation. CK1α is encoded by a gene within the common deleted region for del(5q) MDS and haploinsufficient expression sensitizes cells to lenalidomide therapy, providing a mechanistic basis for the therapeutic window of lenalidomide in del(5q) MDS. We found that mouse cells are resistant to lenalidomide but that changing a single amino acid in mouse Crbn to the corresponding human residue enables lenalidomide-dependent degradation of CK1α. We further demonstrate that minor side chain modifications in thalidomide and a novel analogue, CC-122, can modulate the spectrum of substrates targeted by CRL4(CRBN). These findings have implications for the clinical activity of lenalidomide and related compounds, and demonstrate the therapeutic potential of novel modulators of E3 ubiquitin ligases.

Experience of valgus osteotomy for neglected and failed osteosynthesis in fractures neck of femur.

PURPOSE: There is vast literature supporting valgus osteotomy in fracture neck of femur. However, little or no distinction has ever been made to evaluate the success of the procedure in these two different scenarios-non-unions due to failed osteosynthesis and neglected fractures neck of femur. The aim of our study was to compare the results of valgus osteotomy in neglected neck femur fractures and non-union fractures of neck of femur. METHODS: This is a single tertiary centre-based retrospective study. The records of all patients aged less than 45 years who underwent valgus osteotomy for neck of femur fractures from 2012 to 2017 were evaluated. Patients with fracture neck of femur of over one month's duration, where no previous surgical intervention was undertaken were placed in neglected fracture group. Patients with failed primary osteosynthesis surgery, either cannulated cancellous screw or dynamic hip screw, were placed in fixation failure group. There were 23 patients in neglected group and 17 patients in fixation failure group. Demographical details, fracture patterns, and preoperative radiograph, surgery time, blood loss, post-operative complications, union time, and non-unions were studied in both groups. RESULTS: Osteotomy site united in mean time of 11 weeks in fixation failure group and 11.3 weeks in neglected group (p = .434). Time to radiological union of fracture was 16 weeks (12-23 weeks) for neglected fracture group compared to 25 weeks (20-32 weeks) for fixation failure group which was statistically significant (p = .02). Seven out of 17 fractures did not unite in fixation failure group compared to one non-union out of 23 patients in neglected group. (p = .004) There were two loss of fixation with implant failure in fixation failure group compared to none in neglected group (p = .174). Neither of the groups had any surgical site infection. CONCLUSION: Valgus osteotomy results in excellent union rates for neglected fractures of neck of femur. However, the union rates of valgus osteotomy are lower in neck femur fractures with failed implants compared to neglected fractures and the procedure should be cautiously used in such circumstances.

Sternoclavicular tuberculosis: an atypical imitator of refractory shoulder pain.

INTRODUCTION: Sternoclavicular joint tuberculosis is rare and has been presented in literature with few sporadic case reports or small case series. Rarity of the condition, nonspecific symptoms, difficulty to visualise the area on X-rays, and minimal clinical signs make diagnosis of sternoclavicular tuberculosis extremely difficult. Delay in diagnosis is therefore the common feature of all presented reports in literature. We here present our experience of treating 19 cases of sternoclavicular tuberculosis at our centre. MATERIALS AND METHOD: This is an observational study from 2010 to 2017 in a tertiary care referral hospital. All patients with clinical tenderness of sternoclavicular joint and shoulder joint pain of over three week duration were subjected to MRI. Patients who showed radiological lesions (radiography/MRI) were subjected to core biopsy under image guidance. A total of 26 patients had biopsy confirmed sternoclavicular tuberculosis (TB) during this period. RESULTS: All patients had improvement in shoulder function after treatment completion. Mean CSS pre-treatment was 29 which improved to mean of 8 after 18 months of ATT. Eight patients had excellent results, seven good, three fair, and one patient poor result. High initial ESR, late commencement of ATT from initial symptoms, and surgery of the involved joint were considered poor prognostic factors. DISCUSSION: Sternoclavicular tuberculosis is a rare disease with controversial etiology. Both haematogenous spread through suprascapular artery and contiguous spread through latent disease in apical lungs has been postulated. Delay in diagnosis is common to most reports in literature. Early MRI is useful in diagnosis of the lesion. The treatment for sternoclavicular joint in literature is controversial with proponents of both surgery and conservative management. CONCLUSION: Primary sternoclavicular tuberculosis is rare condition and requires a high index of suspicion for an early diagnosis. A focused sternoclavicular MRI and early biopsy may help in timely diagnosis. Early commencement of ATT has overall good clinical and functional results.

A critical evaluation of the approaches to targeted protein degradation for drug discovery.

There is a great deal of excitement around the concept of targeting proteins for degradation as an alternative to conventional inhibitory small molecules and antibodies. Protein degradation can be undertaken by bifunctional molecules that bind the target for ubiquitin mediated degradation by complexing them with Cereblon (CRBN), von Hippel-Lindau or other E-3 ligases. Alternatively, E-3 ligase receptors such as CRBN or DCAF15 can also be used as a 'template' to bind IMiD or sulphonamide like compounds to degrade multiple context specific proteins by the selected E-3 ligases. The 'template approach' results in the degradation of neo-substrates, some of which would be difficult to drug using conventional approaches. The chemical properties necessary for drug discovery, the rules by which neo-substrates are selected by E-3 ligase receptors and defining the optimal components of the ubiquitin proteasome for protein degradation are still to be fully elucidate. Theis review will aim to critically evaluate the different approaches and principles emerging for targted protein degradation.

Cereblon modulator iberdomide induces degradation of the transcription factors Ikaros and Aiolos: immunomodulation in healthy volunteers and relevance to systemic lupus erythematosus.

OBJECTIVES: IKZF1 and IKZF3 (encoding transcription factors Ikaros and Aiolos) are susceptibility loci for systemic lupus erythematosus (SLE). The pharmacology of iberdomide (CC-220), a cereblon (CRBN) modulator targeting Ikaros and Aiolos, was studied in SLE patient cells and in a phase 1 healthy volunteer study. METHODS: CRBN, IKZF1 and IKZF3 gene expression was measured in peripheral blood mononuclear cells (PBMC) from patients with SLE and healthy volunteers. Ikaros and Aiolos protein levels were measured by Western blot and flow cytometry. Anti-dsDNA and anti-phospholipid autoantibodies were measured in SLE PBMC cultures treated for 7 days with iberdomide. Fifty-six healthy volunteers were randomised to a single dose of iberdomide (0.03-6 mg, n=6 across seven cohorts) or placebo (n=2/cohort). CD19+ B cells, CD3+ T cells and intracellular Aiolos were measured by flow cytometry. Interleukin (IL)-2 and IL-1ß production was stimulated with anti-CD3 and lipopolysaccharide, respectively, in an ex vivo whole blood assay. RESULTS: SLE patient PBMCs expressed significantly higher CRBN (1.5-fold), IKZF1 (2.1-fold) and IKZF3 (4.1-fold) mRNA levels compared with healthy volunteers. Iberdomide significantly reduced Ikaros and Aiolos protein levels in B cells, T cells and monocytes. In SLE PBMC cultures, iberdomide inhibited anti-dsDNA and anti-phospholipid autoantibody production (IC50 ≈10 nM). Single doses of iberdomide (0.3-6 mg) in healthy volunteers decreased intracellular Aiolos (minimum mean per cent of baseline: ≈12%-28% (B cells); ≈0%-33% (T cells)), decreased absolute CD19+ B cells, increased IL-2 and decreased IL-1ß production ex vivo. CONCLUSIONS: These findings demonstrate pharmacodynamic activity of iberdomide and support its further clinical development for the treatment of SLE. TRIAL REGISTRATION NUMBER: NCT01733875; Results.

In Vivo Degradation of Crosslinked Hyaluronic Acid Fillers by Exogenous Hyaluronidases.

BACKGROUND: An advantage of hyaluronic acid (HA)-based fillers is reversibility. OBJECTIVE: To evaluate the ability of 2 hyaluronidases to degrade 3 HA-based fillers using a novel in vivo model. MATERIALS AND METHODS: Rats were injected with 3 HA fillers (HYC-24L+, VYC-20L, and RES-L) to create a projecting bolus. After 4 days, recombinant human hyaluronidase (HX) or ovine hyaluronidase (VIT) was administered at (1) varying doses (5 U, 10 U, or 30 U per 0.1 mL filler) or (2) different dilutions (10 U diluted 3-fold). The impact of tissue integration was assessed by administering 10 U/0.1 mL filler 4 weeks after filler injection. Three-dimensional images quantified projection loss over 72 hours. RESULTS: Complete loss of projection was achieved for all fillers with the highest HX and VIT doses; lower doses achieved less degradation. No difference in degradation was observed between HYC-24L+ and VYC-20L using HX or VIT. RES-L was slightly more degraded with 10 U VIT but not with 10 U HX. Enzyme dilution resulted in less degradation. Tissue integration did not impact the degree of degradation. CONCLUSION: This model incorporates the biological system while controlling variables including filler depth and volume and location of hyaluronidase delivery. Hyaluronic acid filler degradation by exogenous hyaluronidase was not hindered by differences among fillers.

Activity of lenalidomide in mantle cell lymphoma can be explained by NK cell-mediated cytotoxicity.

Lenalidomide is an immunomodulatory agent that has demonstrated clinical benefit for patients with relapsed or refractory mantle cell lymphoma (MCL); however, despite this observed clinical activity, the mechanism of action (MOA) of lenalidomide has not been characterized in this setting. We investigated the MOA of lenalidomide in clinical samples from patients enrolled in the CC-5013-MCL-002 trial (NCT00875667) comparing single-agent lenalidomide versus investigator's choice single-agent therapy and validated our findings in pre-clinical models of MCL. Our results revealed a significant increase in natural killer (NK) cells relative to total lymphocytes in lenalidomide responders compared to non-responders that was associated with a trend towards prolonged progression-free survival and overall survival. Clinical response to lenalidomide was independent of baseline tumour microenvironment expression of its molecular target, cereblon, as well as genetic mutations reported to impact clinical response to the Bruton tyrosine kinase inhibitor ibrutinib. Preclinical experiments revealed lenalidomide enhanced NK cell-mediated cytotoxicity against MCL cells via increased lytic immunological synapse formation and secretion of granzyme B. In contrast, lenalidomide exhibited minimal direct cytotoxic effects against MCL cells. Taken together, these data provide the first insight into the clinical activity of lenalidomide against MCL, revealing a predominately immune-mediated MOA.

Lenalidomide augments actin remodeling and lowers NK-cell activation thresholds.

As multiple myeloma (MM) progresses, natural killer (NK)-cell responses decline against malignant plasma cells. The immunomodulatory drug lenalidomide is widely used for treatment of MM but its influence on NK-cell biology is unclear. Here, we report that lenalidomide lowers the threshold for NK-cell activation, causing a 66% decrease in the 50% effective concentration (EC50) for activation through CD16, and a 38% decrease in EC50 for NK group 2 member D (NKG2D)-mediated activation, allowing NK cells to respond to lower doses of ligand. In addition, lenalidomide augments NK-cell responses, causing a twofold increase in the proportion of primary NK cells producing interferon-γ (IFN-γ), and a 20-fold increase in the amount of IFN-γ produced per cell. Importantly, lenalidomide did not trigger IFN-γ production in unstimulated NK cells. Thus, lenalidomide enhances the NK-cell arm of the immune response, without activating NK cells inappropriately. Of particular clinical importance, lenalidomide also allowed NK cells to be activated by lower doses of rituximab, an anti-CD20 monoclonal antibody (mAb) widely used to treat B-cell malignancies. This supports combined use of lenalidomide and rituximab in a clinical setting. Finally, superresolution microscopy revealed that lenalidomide increased the periodicity of cortical actin at immune synapses, resulting in an increase in the area of the actin mesh predicted to be penetrable to vesicles containing IFN-γ. NK cells from MM patients also responded to lenalidomide in this way. This indicates that nanometer-scale rearrangements in cortical actin, a recently discovered step in immune synapse assembly, are a potential new target for therapeutic compounds.

RAP-011 improves erythropoiesis in zebrafish model of Diamond-Blackfan anemia through antagonizing lefty1.

Diamond-Blackfan Anemia (DBA) is a bone marrow failure disorder characterized by low red blood cell count. Mutations in ribosomal protein genes have been identified in approximately half of all DBA cases. Corticosteriod therapy and bone marrow transplantation are common treatment options for patients; however, significant risks and complications are associated with these treatment options. Therefore, novel therapeutic approaches are needed for treating DBA. Sotatercept (ACE-011, and its murine ortholog RAP-011) acts as an activin receptor type IIA ligand trap, increasing hemoglobin and hematocrit in pharmacologic models, in healthy volunteers, and in patients with ß-thalassemia, by expanding late-stage erythroblasts through a mechanism distinct from erythropoietin. Here, we evaluated the effects of RAP-011 in zebrafish models of RPL11 ribosome deficiency. Treatment with RAP-011 dramatically restored hemoglobin levels caused by ribosome stress. In zebrafish embryos, RAP-011 likely stimulates erythropoietic activity by sequestering lefty1 from erythroid cells. These findings identify lefty1 as a signaling component in the development of erythroid cells and rationalize the use of sotatercept in DBA patients.

CC-122, a pleiotropic pathway modifier, mimics an interferon response and has antitumor activity in DLBCL.

Cereblon (CRBN), a substrate receptor of the Cullin 4 RING E3 ubiquitin ligase complex, is the target of the immunomodulatory drugs lenalidomide and pomalidomide. Recently, it was demonstrated that binding of these drugs to CRBN promotes the ubiquitination and subsequent degradation of 2 common substrates, transcription factors Aiolos and Ikaros. Here we report that CC-122, a new chemical entity termed pleiotropic pathway modifier, binds CRBN and promotes degradation of Aiolos and Ikaros in diffuse large B-cell lymphoma (DLBCL) and T cells in vitro, in vivo, and in patients, resulting in both cell autonomous as well as immunostimulatory effects. In DLBCL cell lines, CC-122-induced degradation or short hairpin RNA-mediated knockdown of Aiolos and Ikaros correlates with increased transcription of interferon (IFN)-stimulated genes independent of IFN-α, -ß, and -γ production and/or secretion and results in apoptosis in both activated B-cell (ABC) and germinal center B-cell DLBCL cell lines. Our results provide mechanistic insight into the cell-of-origin independent antilymphoma activity of CC-122, in contrast to the ABC subtype selective activity of lenalidomide.

Significance of Chilaiditi sign and Chilaiditi syndrome.

Chilaiditi sign and syndrome are uncommon conditions and often misdiagnosed. They are clinically significant, however, because they can result in a range of complications, including bowel volvulus, perforation and obstruction. When patients are symptomatic, treatment is usually conservative and surgery is rarely indicated unless there is a suspicion of ischaemia, or if conservative management does not resolve other signs and symptoms. This article describes Chilaiditi sign and syndrome, and presents four case studies to illustrate the relevant signs and symptoms.

Pomalidomide in combination with dexamethasone results in synergistic anti-tumour responses in pre-clinical models of lenalidomide-resistant multiple myeloma.

Pomalidomide is an IMiD(®) immunomodulatory agent, which has shown clinically significant benefits in relapsed and/or refractory multiple myeloma (rrMM) patients when combined with dexamethasone, regardless of refractory status to lenalidomide or bortezomib. (Schey et al, ; San Miguel et al, 2013; Richardson et al, 2014; Scott, ) In this work, we present preclinical data showing that the combination of pomalidomide with dexamethasone (PomDex) demonstrates potent anti-proliferative and pro-apoptotic activity in both lenalidomide-sensitive and lenalidomide-resistant MM cell lines. PomDex also synergistically inhibited tumour growth compared with single-agent treatment in xenografts of lenalidomide-resistant H929 R10-1 cells. Typical hallmarks of IMiD compound activity, including IKZF3 (Aiolos) degradation, and the downregulation of interferon regulatory factor (IRF) 4 and MYC, seen in lenalidomide-sensitive H929 MM cell lines, were also observed in PomDex-treated lenalidomide-resistant H929 MM cells. Remarkably, this resulted in strong, synergistic effects on the induction of apoptosis in both lenalidomide-sensitive and resistant MM cells. Furthermore, gene expression profiling revealed a unique differential gene expression pattern in PomDex-treated samples, highlighted by the modulation of pro-apoptotic pathways in lenalidomide-resistant cells. These results provide key insights into molecular mechanisms of PomDex in the lenalidomide-resistant setting.

A phase I dose-escalation study to assess safety, tolerability, pharmacokinetics, and preliminary efficacy of the dual mTORC1/mTORC2 kinase inhibitor CC-223 in patients with advanced solid tumors or multiple myeloma.

BACKGROUND: The mammalian target of rapamycin (mTOR) pathway is essential for tumor development, yet mTOR inhibitors have yielded modest results. This phase 1 study investigated the mTORC1/mTORC2 inhibitor CC-223 in patients with advanced cancer. METHODS: Patients with advanced solid tumors or multiple myeloma received an initial dose of 7.5-60 mg of CC-223, followed by oral daily dosing in 28-day cycles until disease progression. The primary objective was to determine the safety, tolerability, nontolerated dosage, maximum tolerated dosage (MTD), and preliminary pharmacokinetic profile. Secondary objectives were to evaluate pharmacodynamic effects and to describe preliminary efficacy. RESULTS: Twenty-eight patients were enrolled and received ≥1 dose of CC-223. The most common treatment-related grade 3 adverse events were hyperglycemia, fatigue, and rash. Four patients had dose-limiting toxicities, including hyperglycemia, rash, fatigue, and mucositis. Therefore, 45 mg/d was determined to be the MTD. The pharmacokinetics of CC-223 demonstrated a mean terminal half-life ranging from 4.86 to 5.64 hours and maximum observed plasma concentration ranging from 269 to 480 ng/mL in patients who received CC-223 ≥45 mg/d. Phosphorylation of mTORC1/mTORC2 pathway biomarkers in blood cells was inhibited by CC-223 ≥30 mg/d with an exposure-response relationship. Best responses included 1 partial response (breast cancer; response duration 220 days; 30-mg/d cohort), stable disease (8 patients across ≥15 mg/d cohorts; response duration range, 36-168 days), and progressive disease (12 patients). The disease control rate was 32%. CONCLUSIONS: CC-223 was tolerable, with manageable toxicities. Preliminary antitumor activity, including tumor regression, and evidence of mTORC1/mTORC2 pathway inhibition were observed.

Congenital Epulis: A Rare Benign Jaw Tumour in a 2-Day-Old Male Baby.

BACKGROUND: Congenital epulis is a rare benign jaw tumor. It is a reactive or degenerative lesion having a mesenchymal origin; presenting as an obvious mass arising from the gingival mucosa of the maxilla or mandible, presenting in neonates. Its etiology, histopathogenesis and natural history are still not well established. It is seen usually in the female gender and mostly involves the maxillary alveolar ridge. MATERIAL/MEHODS: We report a case of a 2.7 kg male baby born with growth on his mandibular ridge which was excised and was proved to be epulis on histopathology. RESULTS: Congenital epulis is often misdiagnosed before surgery because of its rarity and a lack of awareness of the condition by clinicians. It is important for the attending pediatricians, pediatric surgeon to be aware of this rare but benign congenital tumor. CONCLUSIONS: Congenital epulis is often misdiagnosed before surgery because of its rarity and a lack of awareness of the condition by clinicians. As the clinical presentation of this congenital tumor can be distressing due to its size and aggressive appearance, it is important for the attending pediatricians, pediatric surgeon to be aware of this rare but benign congenital tumor.

An activin receptor IIA ligand trap promotes erythropoiesis resulting in a rapid induction of red blood cells and haemoglobin.

Sotatercept (ACE-011), a recombinant human fusion protein containing the extracellular domain of the human Activin receptor IIA, binds to and inhibits activin and other members of the transforming growth factor -ß (TGF-ß) superfamily. Administration of sotatercept led to a rapid and sustained increase in red blood cell (RBC) count and haemoglobin (Hb) in healthy volunteers (phase I clinical trials), but the mechanism is not fully understood. Mice treated with RAP-011 (murine ortholog of ACE-011) respond with a rapid (within 24 h) increase in haematocrit, Hb, and RBC count. These effects are accompanied by an equally rapid stimulation of late-stage erythroid precursors in the bone marrow (BM). RAP-011 also induces a significant increase in erythroid burst-forming units and erythropoietin, which could contribute to additional, sustained effects on RBC production. Further in vitro co-culture studies demonstrate that BM accessory cells are required for RAP-011 effects. To better understand which TGF-ß family ligand(s) mediate RAP-011 effects, we evaluated the impact of several of these ligands on erythroid differentiation. Our data suggest that RAP-011 may act to rescue growth differentiation factor 11/Activin A-induced inhibition of late-stage erythropoiesis. These data define the mechanism of action of a novel agent that regulates RBC differentiation and provide the rationale to develop sotatercept for the treatment of anaemia and ineffective erythropoiesis.

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.).

Cereblon (CRBN), the molecular target of lenalidomide and pomalidomide, is a substrate receptor of the cullin ring E3 ubiquitin ligase complex, CRL4(CRBN) . T cell co-stimulation by lenalidomide or pomalidomide is cereblon dependent: however, the CRL4(CRBN) substrates responsible for T cell co-stimulation have yet to be identified. Here we demonstrate that interaction of the transcription factors Ikaros (IKZF1, encoded by the IKZF1 gene) and Aiolos (IKZF3, encoded by the IKZF3 gene) with CRL4(CRBN) is induced by lenalidomide or pomalidomide. Each agent promotes Aiolos and Ikaros binding to CRL4(CRBN) with enhanced ubiquitination leading to cereblon-dependent proteosomal degradation in T lymphocytes. We confirm that Aiolos and Ikaros are transcriptional repressors of interleukin-2 expression. The findings link lenalidomide- or pomalidomide-induced degradation of these transcriptional suppressors to well documented T cell activation. Importantly, Aiolos could serve as a proximal pharmacodynamic marker for lenalidomide and pomalidomide, as healthy human subjects administered lenalidomide demonstrated Aiolos degradation in their peripheral T cells. In conclusion, we present a molecular model in which drug binding to cereblon results in the interaction of Ikaros and Aiolos to CRL4(CRBN) , leading to their ubiquitination, subsequent proteasomal degradation and T cell activation.

Measuring cereblon as a biomarker of response or resistance to lenalidomide and pomalidomide requires use of standardized reagents and understanding of gene complexity.

Cereblon, a member of the cullin 4 ring ligase complex (CRL4), is the molecular target of the immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide and is required for the antiproliferative activity of these agents in multiple myeloma (MM) and immunomodulatory activity in T cells. Cereblon's central role as a target of lenalidomide and pomalidomide suggests potential utility as a predictive biomarker of response or resistance to IMiD therapy. Our studies characterized a cereblon monoclonal antibody CRBN65, with high sensitivity and specificity in Western analysis and immunohistochemistry that is superior to commercially available antibodies. We identified multiple cereblon splice variants in both MM cell lines and primary cells, highlighting challenges with conventional gene expression assays given this gene complexity. Using CRBN65 antibody and TaqMan quantitative reverse transcription polymerase chain reaction assays, we showed lack of correlation between cereblon protein and mRNA levels. Furthermore, lack of correlation between cereblon expression in MM cell lines and sensitivity to lenalidomide was shown. In cell lines made resistant to lenalidomide and pomalidomide, cereblon protein is greatly reduced. These studies show limitations to the current approaches of cereblon measurement that rely on commercial reagents and assays. Standardized reagents and validated assays are needed to accurately assess the role of cereblon as a predictive biomarker.