RESEARCH ARTICLE Avadomide Induces Degradation of ZMYM2 Fusion Oncoproteins in Hematologic Malignancies Aline Renneville1,2,3, Jessica A. Gasser1,2, Daniel E. Grinshpun1,2, Pierre M. Jean Beltran1, Namrata D. Udeshi1, Mary E. Matyskiela4, Thomas Clayton4, Marie McConkey2, Kaushik Viswanathan2, Alexander Tepper1,2, Andrew A. Guirguis1,2,5, Rob S. Sellar2,6, Sophie Cotteret7, Christophe Marzac7, Véronique Saada7, Stéphane De Botton8, Jean-Jacques Kiladjian9, Jean-Michel Cayuela10, Mark Rolfe4, Philip P. Chamberlain4, Steven A. Carr1, and Benjamin L. Ebert1,2,11 Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 26, 2021. Copyright 2021 American Copyright 2021 by AssociationAmerican for Association Cancer Research. for Cancer Research. ABSTRACT Thalidomide analogues exert their therapeutic effects by binding to the CRL4CRBN E3 ubiquitin ligase, promoting ubiquitination and subsequent proteasomal degrada- tion of specific protein substrates. Drug-induced degradation of IKZF1 and IKZF3 in B-cell malignancies demonstrates the clinical utility of targeting disease-relevant transcription factors for degradation. Here, we found that avadomide (CC-122) induces CRBN-dependent ubiquitination and proteasomal degradation of ZMYM2 (ZNF198), a transcription factor involved in balanced chromosomal rear- rangements with FGFR1 and FLT3 in aggressive forms of hematologic malignancies. The minimal drug- responsive element of ZMYM2 is a zinc-chelating MYM domain and is contained in the N-terminal portion of ZMYM2 that is universally included in the derived fusion proteins. We demonstrate that avadomide has the ability to induce proteasomal degradation of ZMYM2–FGFR1 and ZMYM2–FLT3 chimeric oncoproteins, both in vitro and in vivo. Our findings suggest that patients with hematologic malignancies harboring these ZMYM2 fusion proteins may benefit from avadomide treatment. SIGNIFICANCE: We extend the potential clinical scope of thalidomide analogues by the identification of a novel avadomide-dependent CRL4CRBN substrate, ZMYM2. Avadomide induces ubiquitination and degradation of ZMYM2–FGFR1 and ZMYM2–FLT3, two chimeric oncoproteins involved in hematologic malignancies, providing a proof of concept for drug-induced degradation of transcription factor fusion proteins by thalidomide analogues. INTRODUCTION IKZF3, accounting for their therapeutic efficacy in multiple myeloma due to addiction of certain B-cell malignancies to Thalidomide and its analogues, including lenalidomide these transcription factors (2, 4, 9). Lenalidomide-induced and pomalidomide, have demonstrated extraordinary clinical degradation of casein kinase 1 alpha (Ck1α) explains the utility mediated by drug-induced targeted protein degrada- clinical efficacy of lenalidomide in myelodysplastic syn- tion. Thalidomide analogues bind the Cullin-RING E3 ubiq- drome with deletion of chromosome 5q [del(5q); ref. 3]. uitin ligase CUL4–RBX1–DDB1–CRBN (CRL4CRBN) complex, Avadomide (CC-122) is a novel thalidomide analogue with promoting ubiquitination and subsequent proteasomal deg- potent antitumor and immunomodulatory activities, par- radation of nonnative target proteins, called neosubstrates ticularly in diffuse large B-cell lymphoma, through degrada- (1–5). Thalidomide analogues bind a shallow hydrophobic tion of IKZF1/3 (10). Phase I trials revealed that avadomide pocket on the surface of CRBN to create a compound– monotherapy has acceptable safety and favorable pharma- protein interface for substrate recruitment to the CRL4CRBN cokinetics (10–12). complex, thereby acting as a “molecular glue” (6–8). Using quantitative mass spectrometry–based proteomic Thalidomide, lenalidomide, pomalidomide, and other profiling, we found decreased ZMYM2 abundance following thalidomide analogues induce degradation of IKZF1 and avadomide treatment. ZMYM2, formerly known as ZNF198, is a zinc finger protein that may function as part of a transcriptional corepressor complex (13). ZMYM2 has long 1Broad Institute of MIT and Harvard, Cambridge, Massachusetts. 2Depart- been known for its implication in a chimeric fusion gene ment of Medical Oncology, Dana-Farber Cancer Institute, Boston, with fibroblast growth factor receptor-1 FGFR1( ), generated Massachusetts. 3INSERM U1287, Gustave Roussy Cancer Campus, Villejuif, by a t(8;13)(p11;q12) chromosomal translocation (14–19). France. 4Celgene/Bristol-Myers Squibb Corporation, San Diego, California. ZMYM2 is the most frequent FGFR1 partner gene, account- 5 6 Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. Depart- ing for 40% to 50% of cases of FGFR1-rearranged myeloid/ ment of Haematology, UCL Cancer Institute, London, United Kingdom. 7Département de Biologie et Pathologie, Gustave Roussy Cancer Campus, lymphoid neoplasms (18, 20), a distinct entity in the 2016 Villejuif, France. 8Département d’Hématologie, Gustave Roussy Cancer revision of the WHO classification of myeloid neoplasms Campus, Villejuif, France. 9Université de Paris, AP-HP, Hôpital Saint-Louis, and acute leukemia (21). More recently, another fusion tran- Centre d’Investigations Cliniques CIC 1427, INSERM, Paris, France. script involving ZMYM2, ZMYM2–FLT3, resulting from a 10Hematology Laboratory and EA3518, University Hospital Saint-Louis, Université de Paris, Paris, France. 11Howard Hughes Medical Institute, cytogenetically cryptic inversion on chromosome 13, has Dana-Farber Cancer Institute, Boston, Massachusetts. been identified by RNA sequencing in patients with myelo- Note: Supplementary data for this article are available at Blood Cancer proliferative neoplasms with eosinophilia (22) and Philadel- Discovery Online (https://bloodcancerdiscov.aacrjournals.org/). phia chromosome–like acute lymphoblastic leukemia (23). In Corresponding Author: Benjamin L. Ebert, Dana-Farber Cancer Institute, this study, we report for the first time that ZMYM2–FGFR1 450 Brookline Avenue, D1610A, Boston, MA 02215. Phone: 617–632– and ZMYM2–FLT3 oncoproteins are avadomide-dependent 1902; E-mail: [email protected] CRBN neosubstrates, suggesting patients with hematologic Blood Cancer Discov 2021;2:1–16 malignancies harboring these ZMYM2 rearrangements may doi: 10.1158/2643-3230.BCD-20-0105 benefit from treatment with avadomide or novel thalidomide ©2021 American Association for Cancer Research. analogues that selectively degrade ZMYM2. MAY 2021 BLOOD CANCER DISCOVERY | OF2 Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 26, 2021. Copyright 2021 American Association for Cancer Research. RESEARCH ARTICLE Renneville et al. RESULTS Fig. S2I), consistent with a nontranscriptional mechanism of regulation. Avadomide Decreases ZMYM2 Protein Level To identify novel targets of avadomide, we profiled Avadomide-Induced Ubiquitination of ZMYM2 changes in protein abundance in Hep3B cells, a hepato- Depends on CRBN cellular carcinoma cell line, treated with varying concen- trations of avadomide, pomalidomide, or vehicle control To investigate the mechanism of avadomide-mediated (Supplementary Fig. S1). Samples were analyzed by liquid decrease in ZMYM2 protein level, we treated Hep3B cells chromatography/tandem mass spectrometry (LC/MS-MS) with avadomide or the vehicle control for 12 hours in the employing isobaric chemical labeling and multiplexing presence or absence of small-molecule inhibitors of the using tandem mass tag (TMT) reagents for precise rela- ubiquitin-proteasome system. Cotreatment with avadomide tive quantification and offline fractionation for deep-scale and the E1 (UBA1) inhibitor MLN7243, which blocks all cellular ubiquitination by inhibiting the initial step of the protein analysis. Of the ∼10,000 proteins quantified in Hep3B cells, 8 had decreased abundance following avado- ubiquitination cascade, abrogated ZMYM2 depletion. Like- mide treatment relative to vehicle control, with a log fold wise, addition of MLN4924, a NEDD8-activating enzyme 2 inhibitor that interferes with the activity of Cullin-RING change < –0.8 and a Padj value < 0.01 (Fig. 1A; Supplemen- tary Fig. S2A–S2C; Supplementary Table S1). Among those E3 ubiquitin ligases, or the proteasome inhibitor MG-132, significant hits were ZFP91, RAB28, and WIZ, previously prevented ZMYM2 from being degraded in the presence of identified as targets of thalidomide analogues (2, 24, 25), avadomide (Fig. 1G). and two other proteins not previously published as drug- We next sought to determine whether ubiquitination and induced CRL4CRBN substrates, PDE6D and ZMYM2. Given degradation of ZMYM2 was dependent on CRBN, the molec- the implication of ZMYM2 in two fusion oncoproteins ular target of thalidomide analogues. We utilized CRISPR/ involved in hematologic malignancies, we decided to prior- Cas9 gene editing in Hep3B cells to generate biallelic inacti- itize this target for validation and follow-up studies. Poma- vation of CRBN. In CRBN-knockout Hep3B cells, avadomide lidomide, whose chemical structure is closely related to that did not induce degradation of ZMYM2 (Fig. 1H). We next examined whether ZMYM2 binds CRBN and is ubiquit- of avadomide, had a less pronounced effect on ZMYM2 CRBN protein abundance (Fig. 1B). inated by CRL4 E3 ubiquitin ligase. We observed coim- To validate the proteomic findings, we performed immu- munoprecipitation of endogenous CRBN with V5-tagged noblot analysis in Hep3B cells and tested a series of tha- ZMYM2 only in the presence of avadomide (Fig. 1I). Analysis lidomide analogues. Avadomide was the only drug tested of V5-tagged ZMYM2 in Hep3B cells