Lenalidomide Stabilizes the Erythropoietin Receptor by Inhibiting the E3 Ubiquitin Ligase RNF41 Ashley A

Published OnlineFirst April 6, 2016; DOI: 10.1158/0008-5472.CAN-15-1756 Cancer Therapeutics, Targets, and Chemical Biology Research

Lenalidomide Stabilizes the Erythropoietin Receptor by Inhibiting the E3 Ubiquitin Ligase RNF41 Ashley A. Basiorka1, Kathy L. McGraw2, Leentje De Ceuninck3, Lori N. Griner4, Ling Zhang5, Justine A. Clark2, Gisela Caceres6, Lubomir Sokol2, Rami S. Komrokji2, Gary W. Reuther7, Sheng Wei8, Jan Tavernier3, and Alan F. List2

Abstract

In a subset of patients with non-del(5q) myelodysplastic syn- gene expression vectors. Steady-state EpoR expression was drome (MDS), lenalidomide promotes erythroid lineage compe- reduced in EpoR/RNF41 cells, whereas EpoR upregulation by tence and effective erythropoiesis. To determine the mechanism lenalidomide was abrogated, indicating that cellular RNF41 is by which lenalidomide promotes erythropoiesis, we investigated a critical determinant of drug-induced receptor modulation. its action on erythropoietin receptor (EpoR) cellular dynamics. Notably, shRNA suppression of CRBN gene expression failed to Lenalidomide upregulated expression and stability of JAK2-asso- alter EpoR upregulation, indicating that drug-induced receptor ciated EpoR in UT7 erythroid cells and primary CD71þ erythroid modulation is independent of cereblon. Immunohistochemical progenitors. The effects of lenalidomide on receptor turnover staining showed that RNF41 expression decreased in primary were Type I cytokine receptor specific, as evidenced by coregula- erythroid cells of lenalidomide-responding patients, suggesting tion of the IL3-Ra receptor but not c-Kit. To elucidate this that cellular RNF41 expression merits investigation as a bio- mechanism, we investigated the effects of lenalidomide on the marker for lenalidomide response. Our findings indicate that E3 ubiquitin ligase RNF41. Lenalidomide promoted EpoR/ lenalidomide has E3 ubiquitin ligase inhibitory effects that RNF41 association and inhibited RNF41 auto-ubiquitination, extend to RNF41 and that inhibition of RNF41 auto-ubiquiti- accompanied by a reduction in EpoR ubiquitination. To confirm nation promotes membrane accumulation of signaling compe- that RNF41 is the principal target responsible for EpoR stabili- tent JAK2/EpoR complexes that augment Epo responsiveness. zation, HEK293T cells were transfected with EpoR and/or RNF41 Cancer Res; 76(12); 1–10. 2016 AACR.

Introduction Lenalidomide, a second-generation immunomodulatory drug (IMiD), has greater potency and a more favorable toxicity profile Ineffective erythropoiesis manifested as refractory anemia than its parent, thalidomide (1, 5). Both lenalidomide and its remains the principal management challenge for patients with analogue, pomalidomide, promote erythroid lineage competence myelodysplastic syndromes (MDS; refs. 1, 2). Bone marrow and the expansion of primitive erythroid precursors in CD34- progenitors from patients with MDS display diminished enriched hematopoietic progenitors (6). In transfusion-depen- STAT5 activation and transcriptional response to erythropoietin dent, lower-risk patients with MDS without chromosome 5q (Epo) stimulation, despite appropriate Epo receptor (EpoR) deletion, lenalidomide restores effective erythropoiesis and red membrane display (3, 4). The precise mechanisms underlying blood cell transfusion independence in approximately 25% of the impairment in cytokine signaling remain unclear. patients who are unresponsive to treatment with recombinant erythropoietins (7). Gene expression profiling performed by Ebert and colleagues showed that lenalidomide responders display 1Department of Malignant Hematology, H. Lee Moffitt Cancer Center inherently lower expression of erythroid-specific genes and that and the Cancer Biology Ph.D. Program, University of South Florida, lenalidomide relieves repression of Epo-induced transcriptional Tampa, Florida. 2Department of Malignant Hematology, H. Lee Moffitt Cancer Center, Tampa, Florida. 3VIB Department of Medical Protein response, suggesting that lenalidomide modulates EpoR signal Research, Ghent University, Albert Baertsoenkaai, Ghent, Belgium. capacity (8). 4National Institute of Child Health and Human Development, NIH, Recent investigations revealed that the IMiDs bind to and Bethesda, Maryland. 5Department of Hematopathology, H. Lee Moffitt Cancer Center, Tampa, Florida. 6Morsani Molecular Diagnostic Labo- inhibit the function of the cereblon E3 ubiquitin ligase complex, ratory, H. Lee Moffitt Cancer Center, Tampa, Florida. 7Department of which accounts for the teratogenicity of thalidomide and the Molecular Oncology, H. Lee Moffitt Cancer Center, Tampa, Florida. antiproliferative effects of lenalidomide in multiple myeloma 8 fi Department of Immunology, H. Lee Mof tt Cancer Center, Tampa, (9–12). We previously reported that lenalidomide inhibits the Florida. function of the E3 ubiquitin ligase, murine double minute-2 Corresponding Author: Alan F. List, 12902 Magnolia Drive, SRB-CEO, Tampa, FL protein (MDM2), which stabilizes the protein and fosters its 33612. Phone: 813 745-7101; Fax: 813 745-3090; E-mail: Alan.List@Moffitt.org binding to and degradation of p53 in del(5q) MDS (13). Because doi: 10.1158/0008-5472.CAN-15-1756 EpoR turnover is regulated by ubiquitination and proteasomal 2016 American Association for Cancer Research. degradation, we evaluated the effects of lenalidomide on the E3

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ubiquitin ligase, Really Interesting New Gene (RING) finger Immunoprecipitation protein 41 [RNF41, also known as neuroregulin receptor degra- Two hundred micrograms of protein from total cell lysates was dation protein-1 (Nrdp1)]. RNF41 regulates steady-state or incubated with 2 mg of indicated antibody for 2 hours on ice. Fifty ligand-independent, JAK2-associated type I receptor surface microliters of Protein G Agarose beads (EMD Millipore) was expression, signaling, and intracellular sorting by controlling added and incubated overnight on a rotator at 4C. Bead lysate ubiquitin-specific peptidase (USP)-8 and the endosomal sorting slurries were washed three times in lysis buffer. Sample buffer was complexes required for transport (ESCRT)-0 stability (14, 15). We then added, and beads were dissociated at 95C for 5 minutes. hypothesized that lenalidomide modulates JAK2/EpoR stability Proteins were separated by SDS-PAGE and immunoblotted with through inhibition of RNF41 to enhance JAK2 competent receptor the indicated antibodies. signaling. Immunofluorescence Materials and Methods MNCs were isolated using Ficoll-Hypaque Plus gradient cen- trifugation and were treated with 1 mmol/L lenalidomide for 1 Reagents and cells hour (GE Healthcare). The cells were cytospun for 5 minutes at a UT7 cells were acquired from DSMZ and grown in MEM with 450 rpm. Slides were fixed in BD cytofix for 10 minutes at 37C, 20% FBS, 1% penicillin/streptomycin solution, and 5 ng/mL washed with PBS, then blocked in 2% BSA/PBS for 5 minutes at granulocyte macrophage colony-stimulating factor (GM-CSF). room temperature (RT). Cells were incubated with primary anti- HEK293T cells acquired from ATCC were maintained in DMEM body (1:50 for EpoR and 1:200 for CD71) for 1 hour at RT, supplemented with 10% FBS. All cell lines were authenticated washed, and incubated in secondary antibody (1:1000) for 1 hour by DSMZ and ATCC using short tandem repeat (STR) analysis at RT. Cells were washed again, DAPI and coverslip were added. fi pro ling and passaged for fewer than 6 months after receipt. Micrographs were taken using a Leica TCS SP5 AOBS Laser Normal bone marrow mononuclear cells (BM-MNC) were Scanning Confocal microscope (Leica Microsystems). Data were purchased from Lonza Walkersville. Lenalidomide was pur- analyzed on pooled cells using Image Pro Plus version 6.2 (Media fi a chased from Fisher Scienti c. Ubiquitin, IL3-R ,c-Kit,IFNAR, Cybernetics, Inc.). EpoR (sc-697), and CD71 antibodies were purchased from Santa Cruz Biotechnology. b-Actin antibody and cycloheximide Transfections were purchased from Sigma Aldrich. EpoR (ab10653) and HEK293T cells were transfected with pMET7/EpoR and RNF41 antibodies were purchased from Abcam and USP8 pMET7/RNF41 expression vectors kindly provided by Dr. Jan antibody from Bethyl Laboratories. EpoR and CRBN antibo- Tavernier. Briefly, cells were transfected with 2 mg DNA by calcium dies were provided by Amgen. Bortezomib and MG132 were phosphate method. Three hours after transfection, medium was purchased from Selleckchem. changed. Cells were either harvested for expression detection or treated after 48 hours. Scrambled and shCRBN-specific shRNA Immunoblotting (TG305228) constructs were purchased from Origene Technolo- Cells were treated as indicated, then harvested and lysed in gies, Inc. Six million UT7 cells were suspended in 100 mLof 1 RIPA buffer with 250 mmol/L NaOV4,2mg/mL aprotinin, 2 electroporation solution (Ingenio Solution, Mirus Bio LLC.) and 5 mg/mL leupeptin, 0.2 mg/mL pepstatin A, and 500 mmol/L mg of plasmid was added. Cells were electroporated using Amaxa phenylmethylsulfonylfluoride (PMSF). For detection of USP8, Nucleofector program T-20 and resuspended in 20 mL of aMEM cells were lysed in 2 SDS gel loading buffer (62.5 mmol/L with 5 ng/mL GM-CSF. Functional assays were performed 72 Tris-HCl,pH6.8,3%SDS,10%glycerol,5%b-mercaptoetha- hours after transfection. nol, and 0.01% bromophenol blue sodium salt sonicated), Luciferase reporter assays then sonicated using the Bioruptor Plus (Diagenode). Proteins HEK293T cells were transfected with 2 mg of RNF41 construct, were resolved by SDS-PAGE and transferred to polyvinylidene together with a STAT3-dependent pXP2d2-rPAPI luciferase difluoride (PVDF) membranes. Membranes were blocked in reporter plasmid (200 ng) and a b-gal reporter construct (200 5% dry milk PBST solution (PBS with 0.1% Tween-20) and ng) to correct for transfection efficiency. HEK293T cells were incubated with the indicated antibody. Membranes were washed 24 hours post-transfection, transferred to a 96-well plate washed and developed using ECL or ECLþ according to man- and left untreated or co-treated for at least 24 hours with human ufacturer's protocol (Thermo Scientific). LIF (10 ng/mL; EMD Millipore) and the indicated amount of lenalidomide. Luciferase activity from triplicate samples was Quantitative PCR measured by chemiluminescence in a TopCount luminometer RNA was isolated from UT7 cells using the RNeasy Mini Kit (PerkinElmer) and expressed as relative light units normalized for (Qiagen). cDNA was generated using High Capacity cDNA transfection efficiency. All luciferase data shown are based on at Reverse Transcription Kit per protocol (Life Technology, Applied least three independent experiments (n ¼ 3). Biosystems). ActB, 18S, and GAPDH RNA were used as endoge- nous controls. EpoR and cereblon mRNA were detected using Immunohistochemistry TaqMan Gene Expression Assays (Life Technology, Applied Bio- Paraffin-embedded bone marrow trephine biopsies were systems). Real-time quantitative PCR (qPCR) was carried out on deparaffinized using EZ Prep Solution (Ventana Medical Sys- an ABI PRISM 7900HT Sequence Detection System with triplicate tem, Inc.). Slides were stained sequentially, first with prediluted samples using TaqMan Universal PCR Master Mix with 2-minute spectrin (Cell Marque) for 16 minutes followed by secondary incubation at 50C, followed by activation of AmpliTaq Gold for for 8 minutes, detected with red chromagen. RNF41 secondary 10 minutes at 95C, then 40 cycles of 15 seconds at 95C and 1 antibody (Abcam) was added (1:400) for 1 hour, with minute at 60C. Data were analyzed using SDS software (v2.3). secondary incubation of 16 minutes, and detection by

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Lenalidomide Stabilizes EpoR via RNF41 Inhibition

3,30-diaminobenzidine (DAB) chromagen. Retrieval was done centrations of lenalidomide for 1 hour. We previously reported with cell conditioning 1 (Ventana Medical Systems, Inc.). Slides that both recombinant erythropoietin stimulation and treatment were dehydrated and cover-slipped for analysis. Slides were with lenalidomide induces lipid raft formation and aggregation in scanned using Aperio ScanScope XT with a 200/0.8NA objec- UT7 and MDS cells, accompanied by recruitment of EpoR, JAK2, tive lens via tri-liner array. Three regions from each slide were and STAT5 into raft fractions (16, 17). Immunoblot analyses manually selected by the study pathologist and extracted with- showed that lenalidomide treatment of UT7 cells increased EpoR out compression into Definiens Tissue Studio v3.0 software protein expression in a concentration-dependent manner at phys- suite for quantitative analysis. These regional images were iologically relevant concentrations ranging from 0.1 to 10 mmol/L segmented using Tissue Composer to classify colocalized (Fig. 1A and B). We next treated UT7 cells up to 24 hours and regions of interest using the red spectrin staining as the initial found that upregulation of EpoR by lenalidomide increased 2- nuclear detection marker. The cells of interest were spectrin- fold within 1 hour of treatment and increased thereafter through positive erythroid cells, which also displayed RNF41 staining. 8 hours of incubation with no significant decay until 24 hours of Therefore, each nucleus within the regions of interest was drug exposure (Fig. 1C and D). Of note, certain EpoR antibodies identified with a hematoxylin threshold of 0.16 and an IHC have been reported to detect doublets in UT7 cells, corresponding threshold of 1. Cytoplasmic border was identified outside the to suspected maturation isoforms of the receptor (18). Specificity nuclear border. The training algorithm was closely monitored of EpoR antibodies used in these studies was confirmed using by the study pathologist and applied to all images representa- additional EpoR antibodies, including one provided by Amgen. tive of the patients' slides. To determine whether EpoR upregulation was transcriptionally mediated, EpoR gene expression was assessed by qPCR after Results 1 mmol/L lenalidomide exposure. There was no change in EpoR Lenalidomide upregulates EpoR protein expression mRNA expression after drug treatment, indicating that receptor To determine the effect of lenalidomide on EpoR expression, protein upregulation is mediated by a posttranscriptional mech- UT7 erythroid progenitor cells were treated with increasing con- anism (Fig. 1E). To investigate whether lenalidomide had similar

Figure 1. Lenalidomide increases EpoR expression. A, Western blot of UT7 cells treated with increasing concentrations of lenalidomide for 1 hour showing a concentration- dependent increase in EpoR expression. B, densitometry analysis. C, Western blot of UT7 cells treated with 1 mmol/L lenalidomide over the indicated time intervals showing an increase in EpoR through 8 hours after treatment. D, densitometry analysis. E, relative expression of UT7 EpoR mRNA detectedby qPCR showing no change in transcription following treatment with 1 mmol/L lenalidomide, indicating that lenalidomide increases EpoR expression by a posttransriptional mechanism. Western blot analyses are representative of at least two independent experiments.

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receptor modulating effects in primary erythroid progenitors, BM- without lenalidomide (co-treated after 24 hours cycloheximide MNCs were isolated from three normal donors, and changes in pretreatment), and lysates were collected at the indicated time EpoR expression were assessed by fluorescence microscopy in points over a 24-hour period. Western blotting was performed erythroid precursors identified by CD71 expression (Fig. 2A). to assess levels of EpoR at each time point. Addition of lenali- Lenalidomide induced a significant increase in EpoR expression domide markedly extended the half-life of EpoR to beyond in normal, primary erythroid progenitors after 1 hour of drug 24 hours (Fig. 3A and B). These data demonstrate that lenalido- exposure (Fig. 2B). Mean fluorescence intensity (MFI) of pooled mide stabilizes the EpoR protein, which increases cellular density untreated erythroid progenitors was 1,043.5 32.5 (SE) com- of signaling competent receptors. Furthermore, to determine pared with 1,216.6 51.7 following 1 mmol/L lenalidomide 1- whether the effects of lenalidomide on receptor turnover involve hour exposure (P ¼ 0.003). only type I or both type I and II cytokine receptors, we examined the effects of lenalidomide on cellular expression of IL3 receptor Lenalidomide increases EpoR stability and type I cytokine (IL3-Ra; Type I) and c-Kit (Type II). Lenalidomide upregulated receptor expression IL3-Ra expression in a concentration-dependent manner, where- The findings that lenalidomide yielded a sustained increase in as c-Kit expression was unchanged, confirming type I receptor cellular EpoR expression suggested that lenalidomide may influ- specificity (Fig. 3C and D). ence receptor turnover. To determine whether lenalidomide increased the stability of EpoR protein, we first treated cells with Lenalidomide inhibits the E3 ubiquitin ligase activity of RNF41 cycloheximide to inhibit new protein synthesis. UT7 cells were Recent investigations have shown that steady-state turnover treated with 1 mmol/L cycloheximide for 24 hours either with or of type I receptors, such as EpoR, is regulated by the E3

Figure 2. Lenalidomide induces EpoR expression in primary erythroid progenitors. BM-MNCs were isolated from three primary normal donors, pooled, and treated with 1 mmol/L lenalidomide for 1 hour. A, representative immunoﬂuorescent micrographs (20 mm scale) of erythroid progenitors, identiﬁed as CD71þ (green). DAPI, blue; EpoR, red. The merged image illustrates colocalization. B, MFI SE showing an increase in EpoR expression in erythroid progenitors after lenalidomide treatment (P ¼ 0.003).

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Figure 3. Lenalidomide increases EpoR stability and expression of type I cytokine receptors. A, Western blot of UT7 cells treated with cycloheximide (CHX), with or without 1 mmol/L lenalidomide treatment. Treatment with lenalidomide increased EpoR stability beyond 24 hours. B, densitometry analysis. C, Western blot of UT7 cells treated with lenalidomide at increasing concentrations. D, densitometry analysis. Lenalidomide increased expression of type I receptors (IL3-Ra and EpoR) specifically, and had no effect on the type II receptor, c-Kit, confirming type I receptor specificity. Western blot analyses are representative of at least two independent experiments.

ubiquitin ligase, RNF41 (14, 19). RNF41 increases the ligand- lenalidomide on type II receptor ubiquitination were assessed independent polyubiquitination of both Epo- and IL3 recep- on an alternative receptor, that is, IFN-a/b receptor (IFNAR; tors and controls the intracellular sorting and processing of ref. 20). Ubiquitination of IFNAR was unchanged following several type I cytokine receptors by ubiquitination and sup- treatment with lenalidomide, confirming type I cytokine recep- pression of cellular USP8 expression (14, 15) We first con- tor specificity (Fig. 4G). These findings suggest that the E3 firmed that RNF41 bound to EpoR/JAK2 complexes after ubiquitin ligase inhibitory effects of lenalidomide extend to lenalidomide treatment by RNF41 immunoprecipitation (IP) RNF41. followed by EpoR and JAK2 immunoblot (IB). EpoR:RNF41 binding increased in a concentration-dependent fashion fol- RNF41 overexpression abrogates lenalidomide-induced EpoR lowing lenalidomide exposure (Fig. 4A). In addition, IP of upregulation and leukemia inhibitor factor receptor signaling EpoR followed by RNF41 IB showed similar results (data not To confirm that RNF41 is the principal target of lenalido- shown). To investigate the effects of lenalidomide on the E3 mide responsible for EpoR stabilization, we transfected ubiquitin ligase function of RNF41, we assessed ubiquitina- HEK293T cells with EpoR and/or RNF41 expression vectors tion of several RNF41 substrates after proteasomal inhibition using the calcium phosphate method. Steady-state EpoR with bortezomib or MG132, followed by lenalidomide treat- expression was lower in EpoR/RNF41 cells than in cells trans- ment. RNF41 IP followed by ubiquitin IB showed that lena- fected with EpoR alone (Fig. 5A and B). Moreover, EpoR lidomide treatment resulted in concentration-dependent sta- upregulation by lenalidomide was abrogated in EpoR/RNF41 bilization and cellular accumulation of RNF41 (Fig. 4B) due to cells, indicating that RNF41 is a critical determinant of EpoR inhibition of RNF41 auto-ubiquitination (Fig. 4C). Drug inhi- upregulation by lenalidomide. Parallel to enhanced EpoR bition of RNF41 E3 ubiquitin ligase activity was accompanied signaling (17), lenalidomide treatment increased the STAT3 by increase in cellular USP8 in HEK293T cells (Fig. 4D) and reporter activity of HEK293T cells stimulated with leukemia increased RNF41 association with EpoR and JAK2 in UT7 cells inhibitory factor (LIF; Fig. 5C). Similar to lenalidomide- (Fig. 4A). Moreover, IP of EpoR followed by ubiquitin IB after induced EpoR upregulation, this effect is lost upon ectopic lenalidomide treatment showed decreased ubiquitination of RNF41 overexpression. the receptor (Fig. 4E). Similarly, IP of IL3-Ra,whosereceptor expression is also modulated by RNF41, and ubiquitin IB EpoR upregulation by lenalidomide is independent of cereblon following lenalidomide treatment demonstrated a similar As binding of lenalidomide to the CUL4–RBX1–DDB1– concentration-dependent decrease in ubiquitination of the CRBN E3 ubiquitin ligase complex is implicated in many receptor (Fig. 4F). Given that bortezomib promotes internal- of its biologic effects in multiple myeloma and MDS with ization and the lysosomal degradation of c-Kit, the effects of deletion of chromosome 5q [del(5q)] (21, 22), we investigated

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Figure 4. Lenalidomide inhibits RNF41 ubiquitin ligase activity. A, IP of RNF41 in UT7 cells treated with lenalidomide at the indicated concentrations for 1 hour. There is a concentration-dependent increase in co-IP of EpoR with JAK2 and RNF41 after lenalidomide treatment. B, RNF41 protein expression levels increase in total cell lysates of UT7 cells treated with lenalidomide for 1 hour at the indicated concentrations, corresponding to decreased RNF41 ubiquitination. C, following treatment with 20 nmol/L bortezomib to block proteasomal degradation, RNF41 was immunoprecipitated, then immunoblotted (IB) for ubiquitin. Lenalidomide decreases the ubiquitination of RNF41 in a concentration-dependent manner. D, HEK293T cells transiently transfected with the irrelevant construct pMET7-solIL-5Ra (mock) or pMET7-RNF41 were treated with vehicle alone (DMSO) or were incubated overnight with 5 nmol/L MG132, together with increasing concentrations of lenalidomide. Total cell lysates were immunoblotted for USP8. Ubiquitination of EpoR (IP, EpoR; IB, ubiquitin; E)and IL3-Ra (IP, IL3-Ra; IB, ubiquitin; F) decreased with lenalidomide treatment via inhibition of RNF41 ligase activity. G, ubiquitination of the type II receptor IFNAR (IP, IFNAR; IB, ubiquitin) remained unchanged following lenalidomide treatment, conﬁrming type I cytokine receptor speciﬁcity. Blots are representative of at least two independent experiments.

the possible role of cereblon in EpoR modulation by lenali- RNF41 expression is decreased in lenalidomide-responsive domide. UT7 cells were transfected with shRNAs targeting MDS primary erythroid cells CRBN (Fig. 6A–C), and EpoR modulation was assessed follow- To determine the effects of lenalidomide on RNF41 expression ing treatment with 1 mmol/L lenalidomide for 1 hour. Upre- in vivo, we performed IHC staining on 26 (10 lenalidomide gulation of EpoR by lenalidomide was preserved after CRBN responders and 16 non-responders) bone marrow biopsies from knockdown (Fig. 6D and E), indicating that lenalidomide- non-del(5q) patients with MDS. The relationship between cellu- induced receptor modulation is independent of cereblon. Inter- lar RNF41 level in erythroid precursors and clinical erythroid estingly, lenalidomide treatment modestly increased CRBN response was assessed following costaining for RNF41 and the proteinexpression(Fig.6Band C), consistent with recent erythroid marker spectrin (Fig. 7A). Responding patients dem- reports that lenalidomide stabilizes CRBN (23). onstrated a reduction in cellular RNF41 expression in erythroids

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Figure 5. Overexpression of RNF41 blocks lenalidomide- induced upregulation of EpoR expression. A and B, Western blot (A) and corresponding densitometry analysis (B) of HEK293T cells transfected with EpoR (pMET7-EpoR)orEpoR and RNF41 (pMet7-EpoR/RNF41) expression vectors demonstrate a decrease in steady-state EpoR in the dual gene-transfected cells accompanied by abrogation of lenalidomide induction of EpoR that is retained only in cells transfected with EpoR alone. C, STAT3- responsive luciferase assay of HEK293T cells transiently transfected with increasing amounts of pMET7-RNF41. The pMET7–solIL–5Ra construct (15) was used to normalize the total amount of transfected DNA and load of the transcriptional and translational machinery. The next day, cells were split into 96-wells and stimulated overnight with or without LIF, simultaneously with increasing amounts of lenalidomide. Absolute luciferase counts of triplicate measurements normalized for transfection efﬁciency are representive of three separate experiments.

that approached significance (P ¼ 0.058), whereas non-respon- UT7 and primary MDS erythroid progenitors enhances phos- ders showed no discernible change (Fig. 7B). These results suggest phorylation of the Janus kinase (JAK)-2 and STAT5 in response that erythroid RNF41 expression is modulated by lenalidomide in to Epo stimulation (17). Ubiquitination and endocytosis of the patients, and the ability of lenalidomide to reduce expression in EpoR governs the amplitude and duration of cytokine signaling responding patients may be an important biologic marker of by controlling both membrane residence of signal competent therapeutic efficacy. receptors and the rapidity of receptor degradation. Our investigations show that lenalidomide stabilizes and upregulates expression of signaling competent JAK2-associated EpoR com- Discussion plexes in a concentration-dependent fashion. This is mediated Ebert and colleagues reported that bone marrow progenitors by posttranscriptional inhibition of the E3 ubiquitin ligase from lenalidomide-responsive patients with MDS display lower activity of RNF41, which regulates steady-state type I cytokine levels of expression of erythroid-specific genes that is restored receptor membrane turnover. In addition to diminishing by lenalidomide treatment. Although these findings have not RNF41 auto-ubiquitination, which results in increased RNF41 been validated in a larger cohort, the data suggest that lenali- expression, lenalidomide treatment increased USP8 expression domide positively modifies EpoR signaling. Indeed, both lena- and basal expression and signaling of EpoR and IL3 receptors, lidomide and pomalidomide promote the expansion of prim- without altering expression of type II receptors such as c-Kit and itive erythroid precursors in response to Epo stimulation (6). IFNAR. Furthermore, forced expression of RNF41 in HEK293T Moreover, we recently reported that lenalidomide treatment of cells completely abrogated the capacity of lenalidomide to

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Figure 6. EpoR upregulation by lenalidomide is independent of cereblon. A, relative expression of UT7 CRBN mRNA detected by qPCR following transfection with scrambled or CRBN-speciﬁc shRNAs. Western blot and corresponding densitometry analysis of CRBN (B and C) and EpoR (D and E) expression, respectively, in UT7 cells treated with 1 mmol/L lenalidomide for 1 hour following transfection with scrambled or CRBN-speciﬁc shRNAs. Data are representative of three independent experiments.

upregulate EpoR and LIF receptor expression, confirming that TLR-induced myeloid proliferation while enhancing erythroid RNF41 is a key determinant of receptor modulation by lena- receptor signaling (28). This notion supports our previous lidomide. Importantly, we demonstrate that drug-induced findings that lenalidomide treatment promotes proliferation EpoR upregulation occurs independent of cereblon. Further- of immature myeloid precursors at the expense of terminal more, IHC analysis of cellular RNF41 expression in bone differentiation, thereby contributing to the myelosuppressive marrow erythroid precursors of responding patients demon- effects of lenalidomide while promoting effective erythropoi- strated marked reductions in RNF41 expression following esis (29). treatment, suggesting that RNF41 expression may be a useful Our data support the recent findings that lenalidomide biomarker predictive for response to lenalidomide that merits inhibits E3 ubiquitin ligase complexes, including cereblon further investigation in a larger patient cohort. and MDM2 (9, 10, 12, 13). Collectively, these data suggest RNF41 serves as a scaffold protein coordinating ubiquitin that lenalidomide may act as a much broader RING finger E3 transfer from a ubiquitin-conjugating enzyme (E2) recruited ubiquitin ligase inhibitor than originally appreciated. For by its N-terminal RING domain to substrates that interact with instance, Rac GTPases, responsible for actin cytoskeletal reor- its C-terminal substrate–binding domain. Of particular inter- ganization and plasma membrane compartmentalization, are est, RNF41 was recently found to control Toll-like receptor activated by IMiDs by an unknown mechanism. Perhaps (TLR)-mediated responses through ubiquitination of the cen- inhibition of specific ligases that ubiquitinate these GTPases tral adaptor MyD88 and the TANK-binding kinase 1 (TBK1; underlies the drug activating effects. Furthermore, E3 ligases ref. 24). Increasing evidence implicates activation of innate are important for cellular transport, and inhibition by lenali- immune signaling in the pathobiology of MDS (25–27). These domide may have a profound effect on the spatial organization findings suggest that lenalidomide, through inhibition of of cellular machinery (30). In addition, as E3 ubiquitin ligases RNF41, may stabilize TLR signal intermediates to augment are important for chromatin remodeling, lenalidomide

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Lenalidomide Stabilizes EpoR via RNF41 Inhibition

Figure 7. RNF41 expression decreases in primary lenalidomide responders. A, representative IHC micrograph of MDS bone marrow biopsies depicting RNF41 (brown) and spectrin (red) staining, and the corresponding mean RNF41 intensity in the erythroid population. B, quantitation of the mean RNF41 intensity in the spectrin-positive cells. Lenalidomide responders (n ¼ 10) demonstrate a reduction in cellular RNF41 expression (P ¼ 0.058), whereas no signiﬁcant reduction is observed in non-responders (n ¼ 16).

inhibition of these proteins may have significant effects on of Celgene. No potential conflicts of interest were disclosed by the other gene expression (31). Recently, the E3 ubiquitin ligase authors. SMURF2 (Smad ubiquitin regulating factor 2) was shown to Authors' Contributions regulate histone 2B (H2B) ubiquitination, and consequently Conception and design: A.A. Basiorka, K.L. McGraw, A.F. List fi methylation, through inhibition of the RING nger ligase, Development of methodology: A.A. Basiorka, K.L. McGraw, A.F. List RNF20 (31). Acquisition of data (provided animals, acquired and managed patients, Notably, lenalidomide-induced degradation of casein kinase provided facilities, etc.): A.A. Basiorka, K.L. McGraw, L. De Ceuninck, L. Zhang, 1A1 (CK1a) and the lymphoid transcription factors Ikaros and G. Caceres, R.S. Komrokji, J. Tavernier, A.F. List Aiolos has been shown to occur through modulation of the Analysis and interpretation of data (e.g., statistical analysis, biostatistics, – – – computational analysis): A.A. Basiorka, K.L. McGraw, L. Sokol, R.S. Komrokji, CUL4 RBX1 DDB1 CRBN E3 ubiquitin ligase complex, con- S. Wei, J. Tavernier, A.F. List fi tributing to the therapeutic ef cacy of this agent in both MDS Writing, review, and/or revision of the manuscript: A.A. Basiorka, with deletion of chromosome 5q [del(5q)] and myeloma, K.L. McGraw, L. De Ceuninck, L. Zhang, G. Caceres, L. Sokol, R.S. Komrokji, respectively (21, 22). In these contexts, lenalidomide binds G.W. Reuther, J. Tavernier, A.F. List CRBN to induce activity of the E3 ubiquitin ligase. Therefore, it Administrative, technical, or material support (i.e., reporting or organizing is likely that the effects of lenalidomide are mediated by data, constructing databases): A.A. Basiorka, K.L. McGraw, L. De Ceuninck, L.N. Griner, J.A. Clark, G.W. Reuther, S. Wei alteration of multiple cellular regulatory networks via inhibi- Study supervision: A.A. Basiorka, K.L. McGraw, S. Wei, J. Tavernier, A.F. List tion or enhanced activation of varied ubiquitin ligases, result- ing in biologic consequences that are largely context-depen- Grant Support dent. Further investigations of ubiquitin ligases are warranted Support for this work was provided by the National Cancer Institute/NIH and will provide additional insight into the molecular actions (5 R01 CA131076-04) and by grants from the Belgian government (IUAP P6/ of this immunomodulatory agent. 36) and the Group-ID Multidisciplinary Research Partnership of Ghent University. J. Tavernier holds an ERC Advanced Grant (N 340941). The costs of publication of this article were defrayed in part by the Disclosure of Potential Conflicts of Interest payment of page charges. This article must therefore be hereby marked advertisement R.S. Komrokji has received speakers' bureau honoraria from and is a in accordance with 18 U.S.C. Section 1734 solely to indicate consultant/advisory board member of Celgene. J. Tavernier is employed with this fact. Orionis Biosciences as CTO and also reports receiving commercial research grant from Sponsored Research Collaboration. A.F. List also reports receiving Received July 2, 2015; revised November 23, 2015; accepted March 8, 2016; commercial research grant from and is a consultant/advisory board member published OnlineFirst April 6, 2016.

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OF10 Cancer Res; 76(12) June 15, 2016 Cancer Research

Lenalidomide Stabilizes the Erythropoietin Receptor by Inhibiting the E3 Ubiquitin Ligase RNF41

Ashley A. Basiorka, Kathy L. McGraw, Leentje De Ceuninck, et al.

Cancer Res Published OnlineFirst April 6, 2016.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-15-1756

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