Title: Avadomide Induces Degradation of ZMYM2 Fusion Oncoproteins in Hematologic Malignancies

Title: Avadomide induces degradation of ZMYM2 fusion oncoproteins in hematologic malignancies

Authors:

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.

Affiliations:

1 Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA;

2 Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA;

3 INSERM U1287, Gustave Roussy Cancer Campus, Villejuif, France;

4 Celgene/Bristol-Myers Squibb corporation, San Diego, California, USA;

5 Peter MacCallum Cancer Centre, Melbourne, VIC, Australia;

6 Department of Haematology, UCL Cancer Institute, London, United Kingdom;

7 Département de Biologie et Pathologie, Gustave Roussy Cancer Campus, Villejuif, France;

8 Département d’Hématologie, Gustave Roussy Cancer Campus, Villejuif, France;

9 Université de Paris, AP-HP, Hôpital Saint-Louis, Centre d’Investigations Cliniques CIC 1427,

INSERM, Paris, France;

10 Hematology Laboratory and EA3518, University Hospital Saint-Louis, Université de Paris,

Paris, France;

11 Howard Hughes Medical Institute, Dana-Farber Cancer Institute, Boston, Massachusetts,

USA.

Downloaded from https://bloodcancerdiscov.aacrjournals.orgCopyright 2021 by American Association by guest on Septemberfor Cancer 28, Research.2021. Copyright 2021 American Association for Cancer Research. Running title: Avadomide induces ZMYM2 fusion oncoprotein degradation

Keywords: hematologic malignancies, thalidomide analogs, zinc finger protein, fusion oncoproteins, ubiquitination

Corresponding author:

Benjamin L. Ebert, Dana-Farber Cancer Institute, 450 Brookline Ave, D1610A, Boston, MA

02215; USA.

E-mail: [email protected]

Work phone number: 1-617-632-1902

Conflict of interest disclosure

B.L. Ebert has received research funding from Celgene and Deerfield. He has received consulting fees from GRAIL, and he serves on the scientific advisory boards for Skyhawk

Therapeutics, Exo Therapeutics, and Neomorph Therapeutics. S.A. Carr is a member of the scientific advisory boards of Kymera, PTM BioLabs and Seer and a scientific advisor to Pfizer and Biogen.

Abstract word count: 147

Statement of significance word count: 49

Main text word count: 5808

Main text Figures: 7

Supplementary files: 2

References: 49

Thalidomide analogs exert their therapeutic effects by binding to the CRL4CRBN E3 ubiquitin ligase, promoting ubiquitination and subsequent proteasomal degradation 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 rearrangements 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 analogs 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 analogs.

Thalidomide and its analogs, including lenalidomide and pomalidomide, have demonstrated extraordinary clinical utility mediated by drug-induced targeted protein degradation. Thalidomide analogs bind the Cullin-RING E3 ubiquitin ligase CUL4-RBX1-DDB1-CRBN (CRL4CRBN) complex, promoting ubiquitination and subsequent proteasomal degradation of non-native target proteins, called neosubstrates (1–5). Thalidomide analogs bind a shallow hydrophobic pocket on the surface of CRBN to create a compound-protein interface for substrate recruitment to the

CRL4CRBN complex, thereby acting as a “molecular glue” (6–8).

Thalidomide, lenalidomide, pomalidomide, and other thalidomide analogs induce degradation of

IKZF1 and IKZF3, accounting for their therapeutic efficacy in multiple myeloma due to addiction of certain B-cell malignancies to these transcription factors (2,4,9). Lenalidomide-induced degradation of casein kinase 1 alpha (Ck1α) explains the clinical efficacy of lenalidomide in myelodysplastic syndrome with deletion of chromosome 5q (del(5q)) (3). Avadomide (CC-122) is a novel thalidomide analog with potent antitumor and immunomodulatory activities, particularly in diffuse large B-cell lymphoma, through degradation of IKZF1/3 (10). Phase 1 trials revealed that avadomide monotherapy has acceptable safety and favorable pharmacokinetics

(10–12).

Using quantitative mass spectrometry-based proteomic profiling, we found decreased ZMYM2 abundance following avadomide treatment. ZMYM2, formerly known as ZNF198, is a zinc finger protein that may function as part of a transcriptional co-repressor complex (13). ZMYM2 has long been known for its implication in a chimeric fusion gene with fibroblast growth factor receptor-1 (FGFR1), generated by a t(8;13)(p11;q12) chromosomal translocation (14–19).

ZMYM2 is the most frequent FGFR1 partner gene, accounting for 40-50% of cases of FGFR1- rearranged myeloid/lymphoid neoplasms (18,20), a distinct entity in the 2016 revision of the

Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 28, 2021. Copyright 2021 American Association for Cancer Research. WHO classification (21). More recently, another fusion transcript involving ZMYM2, ZMYM2-

FLT3, resulting from a cytogenetically cryptic inversion on chromosome 13, has been identified by RNA-sequencing in patients with myeloproliferative neoplasms with eosinophilia (22) and

Philadelphia chromosome-like acute lymphoblastic leukemia (23). In this study, we report for the first time that ZMYM2-FGFR1 and ZMYM2-FLT3 oncoproteins are avadomide-dependent

CRBN neosubstrates, suggesting patients with hematologic malignancies harboring these

ZMYM2 rearrangements may benefit from treatment with avadomide or novel thalidomide analogs that selectively degrade ZMYM2.

Avadomide decreases ZMYM2 protein level

To identify novel targets of avadomide, we profiled changes in protein abundance in Hep3B cells, a hepatocellular carcinoma cell line, treated with varying concentrations of avadomide, pomalidomide, or vehicle control (Fig. S1). Samples were analyzed by liquid chromatography- tandem mass spectrometry (LC-MS/MS) employing isobaric chemical labeling and multiplexing using tandem mass tag (TMT) reagents for precise relative quantification and off-line fractionation for deep-scale protein analysis. Of the ~10,000 proteins quantified in Hep3B cells,

8 had decreased abundance following avadomide treatment relative to vehicle control with a log2 fold-change < - 0.8 and an adjusted P-value < 0.01 (Fig. 1A; Fig. S2A-C; Table S1). Among those significant hits were ZFP91, RAB28, and WIZ, previously identified as targets of thalidomide analogs (2,24,25), and 2 other proteins not previously published as drug-induced

CRL4CRBN substrates, PDE6D and ZMYM2. Given the implication of ZMYM2 in two fusion oncoproteins involved in hematologic malignancies, we decided to prioritize this target for validation and follow-up studies. Pomalidomide, whose chemical structure is closely related to that of avadomide, had a less pronounced effect on ZMYM2 protein abundance (Fig. 1B).

To validate the proteomic findings, we performed immunoblot analysis in Hep3B cells and tested a series of thalidomide analogs. Avadomide was the only drug tested that robustly decreased ZMYM2 endogenous protein level. Pomalidomide had a relatively minor effect; and lenalidomide, iberdomide (CC-220), and CC-885 had no detectable effect on ZMYM2 protein levels (Fig. 1C). Given the relevance of ZMYM2 in hematologic malignancies, we confirmed these findings in the acute myeloid leukemia cell line TF-1 (Fig. S2D). We next showed that avadomide decreased ZMYM2 protein abundance in a dose-dependent manner in multiple cellular contexts (Fig. 1D; Fig. S2E-F). Furthermore, a time course experiment revealed that the

Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 28, 2021. Copyright 2021 American Association for Cancer Research. degradation kinetics of ZMYM2 was similar to ZFP91 in Hep3B cells (Fig. 1E), but relatively slower in TF-1 and JURKAT cells (Fig. S2G-H). Avadomide decreased ZMYM2 protein levels without decreasing ZMYM2 mRNA levels (Fig. 1F; Fig. S2I), consistent with a non- transcriptional mechanism of regulation.

Avadomide-induced ubiquitination of ZMYM2 depends on CRBN

To investigate the mechanism of avadomide-mediated decrease in ZMYM2 protein level, we treated Hep3B cells with avadomide or the vehicle control for 12 h, in the presence or absence of small-molecule inhibitors of the ubiquitin-proteasome system. Co-treatment with avadomide and the E1 (UBA1) inhibitor MLN7243, which blocks all cellular ubiquitination by inhibiting the initial step of the ubiquitination cascade, abrogated ZMYM2 depletion. Likewise, addition of

MLN4924, a NEDD8-activating enzyme inhibitor that interferes with the activity of Cullin-RING

E3 ubiquitin ligases, or the proteasome inhibitor MG-132, prevented ZMYM2 from being degraded in the presence of avadomide (Fig. 1G).

We next sought to determine whether ubiquitination and degradation of ZMYM2 was dependent on CRBN, the molecular target of thalidomide analogs. We utilized CRISPR/Cas9 gene editing in Hep3B cells to generate biallelic inactivation of CRBN. In CRBN-knockout Hep3B cells, avadomide did not induce degradation of ZMYM2 (Fig. 1H). We next examined whether ZMYM2 binds CRBN and is ubiquitinated by CRL4CRBN E3 ubiquitin ligase. We observed co- immunoprecipitation of endogenous CRBN with V5-tagged ZMYM2 only in the presence of avadomide (Fig. 1I). Analysis of V5-tagged ZMYM2 in Hep3B cells with an antibody specific to

K48-linked ubiquitin chains showed that avadomide treatment was associated with an increase in ZMYM2 carrying K48-linked polyubiquitin chains (Fig. 1J), the canonical signal for proteasomal degradation. Collectively, these results indicate that avadomide treatment causes CRBN-dependent ubiquitination and subsequent proteasomal degradation of ZMYM2.

Thalidomide analogs in complex with CRL4CRBN recognize substrates through degrons, short stretches of primary sequence that are necessary and sufficient to mediate degradation (26).

Many CRBN neosubstrates reported to date belong to the family of C2H2 zinc finger proteins, for which the drug-inducible degrons are zinc finger domains. A conserved feature of this C2H2 zinc finger degrome is the canonical CXXCG motif (27), including a key glycine residue that engages the phthalimide moiety of thalidomide analogs (8,27,28). MYM-type zinc finger proteins, such as ZMYM2, are characterized by a conserved MYM domain with the consensus motif CX CX CX CX CX CX FCX CX [F/Y] (15). By analogy to the C2H2 zinc finger 2 19-22 3 13-19 2 19-25 3 3 proteins, we examined whether the CXXCG motif was also present in ZMYM2. This motif was found twice in the entire ZMYM2 protein, both located in the second MYM domain (MYM2) (Fig.

2A). To test whether the MYM2 domain contained the degron, we employed a lentiviral degradation reporter vector that enables comparison of the fluorescence of enhanced green fluorescent protein (EGFP)-tagged ZMYM2 construct to mCherry using flow cytometry (Fig. 2B)

(27). This assay revealed that the MYM2 domain (amino acids 425 to 502) was indeed sufficient to allow degradation in cells treated with avadomide. The MYM2 N-terminal portion (amino acids

425 to 466) similarly mediated degradation, whereas the C-terminal portion (amino acids 467 to

502) did not contribute. Further deletion mapping led to the identification of a ZMYM2 degron that was more sensitive to avadomide (amino acids 423 to 466), with a dose-response almost overlapping with that of the IKZF1/3 degron (Fig. 2C). We next analyzed the effect of different thalidomide analogs on the degradation of the ZMYM2 423-466 degron in the reporter vector.

Only avadomide and pomalidomide induced efficient degradation, while the other drugs had little or no effect, even at high concentrations (Fig. 2D; Fig. S3).

To determine which specific amino acid residues are critical for ZMYM2 degradation, we performed an alanine scan, systematically mutating each amino acid within the 423-466 degron

Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 28, 2021. Copyright 2021 American Association for Cancer Research. to alanine. Hep3B and TF-1 cells stably expressing the respective mutant degron in the degradation reporter vector were treated with avadomide or the vehicle control. The alanine scan highlighted 7 residues critical for degradation in Hep3B and TF-1 cells, including 4 zinc- coordinating cysteine residues and 3 non-structural amino acid residues, R424, I427, and the key glycine G429 (Fig. 2E). Of note, the ZMYM2 RCXICG motif is not found in any other MYM- type zinc finger proteins (https://prosite.expasy.org/scanprosite/), in accordance with the finding that other proteins in this family are not degraded upon avadomide treatment in our LC-MS/MS data (Table S2).

To confirm that this minimal degron is sufficient for drug-induced ubiquitination by the CRL4CRBN, we performed in vitro ubiquitination assays. Indeed, we observed that the ZMYM2 423-466 degron can be ubiquitinated by CRL4CRBN in the presence of avadomide or pomalidomide, demonstrating that ZMYM2 is a primary target of CRL4CRBN. As expected, the G429A mutant completely prevented ubiquitination (Fig. 2F). Immunoblot validation in Hep3B cells confirmed that deletion of the MYM2 domain or introduction of the G429A point mutation in full-length

ZMYM2 protein conferred resistance to avadomide-induced degradation (Fig. 2G). Collectively, these data demonstrate that the MYM2 domain is necessary and sufficient for drug-induced degradation of ZMYM2.

Structural modeling of CRBN bound to avadomide and ZMYM2 degron

To validate our findings and gain insight into the role of specific amino acid residues within

ZMYM2 degron, we built a model of ZMYM2 recruitment to the DDB1-CRBN complex using in silico structural modeling. The model revealed the predicted ZMYM2 domain (amino acids 421-

453) is highly compatible with the surface of CRBN and binds in a cleft between the N-terminal

Lon-like domain and the C-terminal thalidomide-binding domain of CRBN (6,7). The ZMYM2 β- hairpin was predicted to bind CRBN through the structural degron reported for other

Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 28, 2021. Copyright 2021 American Association for Cancer Research. neosubstrates, consistent with our deletion mapping and mutagenesis studies (8,27,28). This binding mode places the critical glycine residue G429 in the conserved position against the bound avadomide molecule. The G429A mutant should not be tolerated in this binding mode, consistent with our degradation reporter and in vitro ubiquitination results. Of the other amino acid residues found to be critical for ZMYM2 degradation in our functional studies, R424 is proximal to avadomide in the ternary complex, and I427 makes direct contacts with the CRBN surface (Fig. 3). Therefore, both of these residues likely contribute to stabilization of the ternary complex needed for efficient ubiquitin transfer. Overall, this model confirms the interaction between the MYM2 domain of ZMYM2 and avadomide-bound CRBN. The model rationalizes the importance of the 3 functionally-identified non-structural amino acid residues in ZMYM2 degron for CRBN binding and degradation.

Avadomide promotes ZMYM2-FGFR1 and ZMYM2-FLT3 fusion protein degradation

ZMYM2 is implicated in two chimeric fusion oncoproteins involved in hematologic malignancies.

In both fusion proteins, the N-terminal portion of ZMYM2, including the drug-responsive degron, is fused to the C-terminal portion of either FGFR1 or FLT3, including their respective tyrosine kinase domains (Fig. 4A) (14,15,22,23). No cancer cell lines harboring ZMYM2-FGFR1 or

ZMYM2-FLT3 rearrangements are documented. To address whether the fusion proteins could be degraded in cells in a manner similar to the full-length ZMYM2 protein, we stably overexpressed the V5-tagged fusion proteins in the leukemia line TF-1 and treated these cells with avadomide for 24 h. Immunoblot analysis using antibodies against the ZMYM2 N-terminus,

FGFR1 or FLT3 C-terminus, and C-terminal V5 tag consistently showed that the ZMYM2-

FGFR1 and ZMYM2-FLT3 proteins were both degraded upon avadomide treatment in a dose- dependent manner. The G429A point mutation within ZMYM2 degron rendered both fusion proteins resistant to degradation, indicating that ZMYM2-FGFR1 and ZMYM2-FLT3 are degraded through the same mechanism as the full-length ZMYM2 protein (Fig. 4B-C).

Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 28, 2021. Copyright 2021 American Association for Cancer Research. Ba/F3 is a murine lymphoid leukemia cell line dependent on interleukin-3 (IL-3), and its removal induces cell apoptosis. Like other tyrosine kinase oncogenes (29), ZMYM2-FGFR1 transforms

Ba/F3 cells to IL-3 independence (30,31), thereby rendering Ba/F3 cells dependent on the fusion oncoprotein for their survival and proliferation. We transformed Ba/F3 cells with ZMYM2-

FGFR1 bearing the wild-type degron (ZMYM2WT-FGFR1) or its G429A mutant counterpart

(ZMYM2G429A-FGFR1) (Fig. 5A-B). Since mouse cells are intrinsically resistant to thalidomide and its analogs, transformed Ba/F3 cells were subsequently transduced to express either human CRBN or the humanized mouse CrbnI391V mutant, which are both capable of mediating drug-induced degradation of Ikzf1/3, Ck1α, and Zfp91 in murine cells (3,32). Expression of human CRBN allowed robust degradation of ZMYM2WT-FGFR1 upon avadomide treatment.

However, expression of CrbnI391V was not sufficient to promote degradation of human

ZMYM2WT-FGFR1 (Fig. 5C), consistent with the lack of cytotoxic effects of avadomide in these cells (Fig. S4).

Ba/F3 transformation by ZMYM2-FGFR1 is mediated, at least in part, by activation or subversion of signaling pathways employed by the Jak/Stat pathway downstream of activated receptor tyrosine kinase signaling (30). Immunoblot analysis showed that Stat1, and to a lesser extent Stat5, were phosphorylated in ZMYM2-FGFR1 transformed Ba/F3 cells. In Ba/F3 cells transformed by ZMYM2WT-FGFR1 and expressing human CRBN, avadomide treatment led to

ZMYM2WT-FGFR1 degradation and a concomitant decrease in phospho-Stat1 and Stat5 levels

(Fig. 5D). In Ba/F3 cells transformed with the drug-resistant ZMYM2G429A-FGFR1 fusion, the effects of avadomide on phospho-Stat1 and Stat5 were absent or decreased (Fig. 5E). In Ba/F3 cells transformed by ZMYM2WT-FGFR1 and expressing human CRBN, avadomide reduced cell viability and proliferation in a dose-dependent manner. This effect was completely rescued in

Ba/F3 cells transformed by the ZMYM2G429A-FGFR1 mutant, confirming the cytotoxic effect of avadomide was due to ZMYM2WT-FGFR1 depletion. Ba/F3 cells transformed by either

Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 28, 2021. Copyright 2021 American Association for Cancer Research. ZMYM2WT-FGFR1 or ZMYM2G429A-FGFR1 were all sensitive to ponatinib, a multi-tyrosine kinase inhibitor with activity against FGFR1 (33), regardless of CRBN status (Fig. 5F).

In vivo validation of ZMYM2-FGFR1 degradation by avadomide

To address whether avadomide could inhibit the proliferation of ZMYM2-FGFR1 dependent leukemia cells in vivo, we performed a syngeneic transplant experiment in BALB/c mouse recipients (34,35), the mouse strain from which Ba/F3 cells were originally derived. As previously described with BCR-ABL transformed BaF3 cells (34,35), we found that injection of

ZMYM2-FGFR1 transformed Ba/F3 cells into BALB/c mice caused a highly aggressive disease, with all mice dying secondary to a diffuse leukemic process within 2 weeks after transplant. To assess the effect of avadomide in this model, sublethally irradiated BALB/c mice were injected with ZMYM2WT-FGFR1 transformed Ba/F3 cells expressing human CRBN (with co-expression of GFP), and dosed twice per day with avadomide or the vehicle control for 7 consecutive days

(Fig. 6A) Spleen weight, which reflects tumor burden, was significantly lower in mice who received avadomide compared to those who received the vehicle control (Fig. 6B). Compared to the vehicle control, avadomide treatment was associated with a lower white blood cell count and a higher hemoglobin level, even though blood count values at baseline did not differ between the two groups (Fig. 6C-E). Additionally, the percentages of GFP positive cells in peripheral blood, bone marrow, and spleen were all strikingly lower in the avadomide group compared to the control group (Fig. 6F-H). Altogether, these results demonstrate the efficacy of avadomide to inhibit the growth of ZMYM2-FGFR1 dependent Ba/F3 leukemia cells in vivo.

Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 28, 2021. Copyright 2021 American Association for Cancer Research. Validation in primary human hematopoietic stem/progenitor cells

To verify our findings from experiments with ectopically expressed fusion proteins in primary samples with endogenous expression of ZMYM2 fusions, we collected diagnostic bone marrow samples from patients with a hematologic malignancy harboring a t(8;13)(p11;q12) translocation, which generates the ZMYM2-FGFR1 fusion. The clinical characteristics of the 4 patients are summarized in Table S3. As normal controls, we used bone marrow from healthy donors. CD34+ stem and progenitor cells from healthy donors and leukemia patients were expanded in culture and treated with 1 µM avadomide or the vehicle control. In normal CD34+ cells, avadomide treatment did not significantly affect cell viability or proliferation (Fig. 7A). In contrast, in bone marrow CD34+ cells isolated from patients with ZMYM2-FGFR1-positive hematologic malignancy, avadomide treatment significantly reduced the number of viable cells compared to the vehicle control (Fig. 7B). Altogether, these data indicate that avadomide has anti-proliferative effects in primary cells from patients with hematologic malignancy harboring

ZMYM2-FGFR1 rearrangement, with a broad therapeutic window.

In this study, we established ZMYM2 as a direct avadomide-dependent substrate of the

CRL4CRBN E3 ubiquitin ligase. ZMYM2 is implicated in two chimeric fusion oncoproteins involved in hematologic malignancies, ZMYM2-FGFR1 and ZMYM2-FLT3. We demonstrated that avadomide has the ability to induce proteasomal degradation of both fusion oncoproteins utilizing the same mechanism as the ZMYM2 full-length protein. We validated these findings in primary cells from patients with hematologic malignancy harboring ZMYM2-FGFR1 rearrangements. This is the first description of fusion oncoproteins that can be targeted for degradation by a thalidomide analog.

We characterized a novel type of drug-inducible degron for thalidomide analogs containing a C4 motif, in which the 4 cysteines represent the zinc-coordinating residues that belong to the consensus motif of MYM-type zinc finger domains. The ZMYM2 degron shares similarities with

C2H2 zinc finger degrons, including the critical glycine residue, but is differentiated in terms of drug sensitivity. Unlike IKZF1/3 degron, ZMYM2 degron is not uniformly degraded by active thalidomide analogs. Novel thalidomide analogs have the potential to induce ZMYM2 degradation with greater potency and selectivity, thereby reducing the competition with other neosubstrates for CRBN occupancy, which is a limiting factor for drug-induced protein degradation (36). Moreover, our findings raise the possibility that other zinc finger proteins containing a C4 motif, such as the LIM, PHD, or RING-type zinc finger proteins, may also be degraded in the presence of new thalidomide analogs, providing new therapeutic opportunities.

Avadomide is a novel therapeutic agent that is currently in phase 1/2 clinical trials for patients with lymphomas, given its IKZF1/3 degradation activity. Our data from cell lines and primary patient samples suggest that avadomide could also be repurposed for the treatment of hematologic malignancies with ZMYM2-FGFR1 or ZMYM2-FLT3 rearrangements. Chimeric

Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 28, 2021. Copyright 2021 American Association for Cancer Research. fusion genes are recognized as strong genetic drivers in hematologic malignancies, sometimes even pathognomonic for specific clinical subtypes of disease, and often represent the primary event in the leukemic process, which makes them attractive targets for diagnosis and treatment

(21,37). In immunodeficient mice, ZMYM2-FGFR1 alone is capable of initiating a myeloproliferative-like disorder, with increased cellular proliferation, blast accumulation, bone marrow fibrosis, and increased extramedullary hematopoiesis, consistent with features observed in patients with ZMYM2-FGFR1 neoplasms (17,38). Given the recent discovery of

ZMYM2-FLT3, no data are available regarding its role in the pathogenesis of hematologic malignancies or its prognostic significance. However, it is likely that this chimeric fusion gene similarly drives the malignant phenotype (22,23,39).

The prognosis of FGFR1-rearranged myeloid/lymphoid neoplasms is poor despite intensive chemotherapy. Tyrosine kinase inhibitors targeting FGFR1, such as midostaurine (40), ponatinib (41), or pemigatinib (42), have been tested in monotherapy in patients with FGFR1- rearranged myeloid/lymphoid neoplasms, but hematologic response, if any, is typically partial and transient. Allogeneic stem cell transplantation currently remains the only option to achieve long-term remission and possibly cure these diseases (20,41). As opposed to reversible small molecule inhibitors that lose efficacy as drug concentration drops, protein destruction is able to drive long-lasting target depletion and strong biological effects (9). Moreover, small molecule degraders and inhibitors will have distinct mechanisms of resistance, providing additional rationale for combining these drugs. Therefore, we speculate that, in patients with hematologic malignancies with ZMYM2-FGFR1 or ZMYM2-FLT3, a combination therapy associating a degrader such as avadomide or a novel thalidomide analog that selectively degrades ZMYM2 and a tyrosine kinase inhibitor targeting FGFR1 or FLT3 could help to achieve deep and durable responses, whilst preventing the emergence of drug resistance.

Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 28, 2021. Copyright 2021 American Association for Cancer Research. ZMYM2-FGFR1 and ZMYM2-FLT3 are thought to be rare genetic abnormalities, but their prevalence might be underestimated, especially as the latter is not detectable by conventional cytogenetics. Retrospective and prospective screening of patient samples using FISH analysis,

RNA-sequencing, or other assays, may reveal additional cases that would not have been identified by cytogenetic and molecular diagnostic tests used routinely at diagnosis, but could benefit from targeted therapies.

Hundreds of recurrent fusion genes have been reported in hematologic malignancies and solid tumors (43). These fusion genes are often powerfully oncogenic and strong genetic dependencies in the resulting malignancies. Few of the corresponding fusion proteins can be specifically targeted by small molecule therapies. The targeting of BCR-ABL and PML-RARA in chronic myeloid leukemia and acute promyelocytic leukemia, respectively, have yielded extraordinarily successful therapies (37,43,44). However, the vast majority of fusion oncoproteins are still considered undruggable. Our studies of avadomide-induced degradation of ZMYM2 fusion proteins highlights the broader potential of targeted protein degradation as a therapeutic strategy for fusion oncoproteins in cancer.

Quantitative proteomic profiling by liquid chromatography tandem mass spectrometry

(LC-MS/MS)

Early passage parental Hep3B cells were treated with DMSO vehicle control, 1 µM pomalidomide for 24 h, or 1 µM or 10 µM avadomide for 12 h or 24 h, with 2 biological duplicates for all conditions. Cells were harvested by trypsinization, washed with 1X PBS, and cell pellets were snap frozen and stored at - 80°C until use. Proteins were extracted from cell pellets through lysis in 8M urea buffer, followed by reduction, alkylation, and digestion with endoproteinase LysC and trypsin (Fig. S1). Tryptic peptides were labeled using the TMT 11plex reagent (ThermoFisher Scientific), and combined as previously described (45). TMT-labeled peptides were fractionated by offline basic reverse-phase chromatography into 24 fractions as described (46). Multiplexed peptide fractions were analyzed by LC-MS/MS using an Orbitrap

Fusion Lumos Mass Spectrometer. MS/MS data was processed using Spectrum Mill v7 prerelease (Agilent Technologies), and protein quantitative values were further analyzed using

R. Details on proteomic sample processing, LC-MS/MS conditions, and data analysis are provided in the Supplemental Data. The original mass spectra and the protein sequence database used for searches have been deposited in the public proteomics repository MassIVE

(http://massive.ucsd.edu) under the identifier MSV000086902 and are accessible at ftp://massive.ucsd.edu/MSV000086902/.

Western immunoblotting

Cells were washed with 1X-PBS and lysed for 30 min on ice in the following buffer: 150 mM

NaCl, 25 mM Tris-HCl pH 8.0, 1% NP-40, 5% glycerol, 0.02 mM ZnCl, 1X Halt Protease and

Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). The lysates were centrifuged at

Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 28, 2021. Copyright 2021 American Association for Cancer Research. 13,000 g at 4°C for 15 min, and protein concentrations were quantified with the Pierce BCA

Protein Assay Kit (Thermo Fisher Scientific). Samples were boiled and reduced in a mixture containing Laemmli (6X, SDS-Sample Buffer, Reducing) (Boston BioProducts) and lysis buffer.

Protein lysates were run on Bolt 8% Bis-Tris Plus Gels (Thermo Fisher Scientific) by SDS-

PAGE at a constant voltage. Proteins were transferred onto nitrocellulose membranes (Thermo

Fisher Scientific) utilizing a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad) in 1X Tris-

Glycine Buffer (Bio-Rad) at 40 V overnight. Membranes were blocked in 5% non-fat dry milk in

TBST for 1 h and probed with the indicated primary antibody at 4°C overnight. The primary antibodies used are listed in Table S4. The membranes were washed in TBST, 4 times for 5 min, before 1 h incubation with the secondary anti-rabbit or anti-mouse IgG horseradish peroxidase-conjugated antibody (Cell Signaling Technology) at room temperature. After 4 final washes in TBST for 5 min, the blots were subjected to enhanced chemiluminescence (ECL) detection using the SuperSignal West Dura Extended Duration Substrate (Thermo Fisher

Scientific). Blots were exposed to CL-XPosure films (Thermo Fisher Scientific), which were developed using the Medical Film Processor SRX-101A (Konica Minolta). For re-probing, blots were stripped in Restore PLUS Western Blot stripping buffer (Thermo Fisher Scientific) and re- blocked. Uncropped immunoblots can be found in Fig. S5.

RNA extraction and real-time quantitative PCR

The RNeasy Plus Mini Kit (Qiagen) was used to isolate mRNA. Reverse transcription was performed using the SuperScript IV VILO Master Mix (Thermo Fisher Scientific), according to the manufacturer’s instructions. The qRT-PCR analysis was performed using the TaqMan Gene

Expression Master Mix and the GAPDH (Hs99999905_m1) and ZMYM2 (Hs01547045_g1) primer-probe sets from Thermo Fisher Scientific. Each RT-qPCR was performed in triplicate in a

384-well plate on a QuantStudio 6 Flex System instrument (Thermo Fisher Scientific). The

Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 28, 2021. Copyright 2021 American Association for Cancer Research. mean threshold cycle (Ct) for each assay was used for further calculations. Relative ZMYM2 gene expression levels were calculated using the ΔΔCt method.

Cloning

Empty backbones pDONR223, pLX302, pLX304, pLX306, as well as pLX311-Cas9, and human

CRBN isoform 2 (ccsbBroadEn_08244) in pDONR223, were acquired from the Broad Genetic

Perturbation Platform. CRBN gRNA1 in pXPR003 and IKZF1/3 amino acids (AA) 146-168 in

Cilantro 2 (Addgene #74450) were generated as described (27,47). ZMYM2 double-stranded

DNA sequences encoding for AA 425-502, 425-466, 467-502, 425-452, or 423-466 and appended with BsmBI restriction sites were in vitro synthesized by IDT, digested with BsmbI

(NEB), purified using the QIAquick PCR Purification Kit (Qiagen), and ligated into Artichoke

(Addgene #73320) reporter vector using the T4 DNA ligase (Thermo Fisher Scientific). For the alanine scan of the ZMYM2 423-466 degron, double-stranded DNA sequences were in vitro synthesized by Twist Bioscience and cloned into Artichoke, as described above. ZMYM2 coding sequence (#BC036372) was PCR amplified from a human cDNA clone purchased from

Transomic Technologies, cloned into pDONR223 by BP recombination reaction using the

Gateway BP Clonase II Enzyme mix (Thermo Fisher Scientific), and then shuttled into pLX302 and pLX304 by LR recombination reactions using the Gateway LR Clonase II Enzyme mix

(Thermo Fisher Scientific). ZMYM2, with deletion of the MYM2 domain (AA 425-502), was in vitro synthesized by IDT and cloned in pDONR223 and pLX302. The FGFR1 3’ sequence encoding AA 429 to 822 was in vitro synthesized by IDT and fused by Gibson assembly (NEB) to the ZMYM2 5’ sequence encoding AA 1 to 913 to create the ZMYM2-FGFR1 chimeric fusion, using the breakpoints described by Reiter et al.(15). Likewise, the FLT3 3’ sequence encoding

AA 594 to 993 was in vitro synthesized by IDT and fused by Gibson assembly to the ZMYM2 5’ sequence encoding AA 1 to 1059 to create the ZMYM2-FLT3 chimeric fusion, as reported by

Jawhar et al. (22). Both fusion sequences were then cloned into pDONR223, and into the

Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 28, 2021. Copyright 2021 American Association for Cancer Research. destination vectors pLX302, pLX304 or pLX306. Wild-type mouse Crbn isoform 2 was PCR amplified from a retroviral backbone previously described (3) and cloned into pDONR223. To generate the G429A point mutation in ZMYM2, ZMYM2-FGFR1, or ZMYM2-FLT3, and the mouse CrbnI391V mutant, we performed mutagenesis in pDONR223 vectors using the Q5 site- directed mutagenesis kit (NEB) with primers designed using the NEBaseChanger™

(https://nebasechanger.neb.com/). Wild-type human CRBN and CrbnI391V were then cloned into the Gateway destination vector « GW » plenti.SFFV.gateway.IRES.EGFP that was generated in- house.

Lentiviral production and infection

Lentiviral expression plasmids were transfected together with the packaging plasmids pVSV-G

(Addgene #12259) and psPAX2 (Addgene #12260) and using TRANS-LTI (Mirrus) into

HEK293T cells. The viral supernatant was collected 48 h after transfection, cleared by centrifugation at 500 g for 5 min, and then filtered through a Millex-HV Syringe Filter Unit, 0.45

µm (EMD Millipore). Cells were transduced by spin-infection at 30°C for 2 h at 1000 g. To select for infected cells, puromycin (2 µg/ml for at least 4 days) or blasticidin (10 µg/ml for at least 7-10 days) was added to the culture media 24 h after transduction. Cell sorting (Sony cell sorter) for

GFP was performed to select Ba/F3 cells stably integrated with the GW lentiviral vector.

Co-immunoprecipitation of CRBN

3 x 106 wild-type Hep3B cells were plated onto 15 cm dishes and transfected with 35 µg empty vector or pLX304-ZMYM2, which encodes ZMYM2-V5. Cells were treated with DMSO or 20 µM avadomide in the presence of 10 µM MG-132 for the last 3 h before harvest. Cells were harvested 48 h post-transfection and lysed in lysis buffer (as described above) containing 20 µM avadomide or DMSO. Whole cell extracts were cleared by centrifugation at 13,000 g at 4°C for

15 min, normalized, and incubated with anti-V5-tag magnetic beads (MBL International) at 4°C

Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 28, 2021. Copyright 2021 American Association for Cancer Research. for 16 h. After washing the beads 3 times in wash buffer (150 mM NaCl, 50 mM Tris-HCl pH 8.0,

1% glycerol, 1X Halt Protease and Phosphatase Inhibitor Cocktail) containing 20 µM avadomide or DMSO, anti-V5 immunoprecipitates were eluted by boiling in Laemmli SDS sample buffer at

95°C for 5 min, and then subjected to immunoblot analysis for CRBN.

In vivo ubiquitination

For assessment of endogenous ubiquitination of ZMYM2, 3 x 106 wild-type Hep3B cells were plated onto 15 cm dishes and transfected with 35 µg empty vector or pLX304-ZMYM2, which encodes ZMYM2-V5. Cells were treated with DMSO, 1 or 10 µM avadomide and in the presence of 10 µM MG-132 for the last 5 h before harvest. Cells were harvested 48 h post- transfection. Cell lysates were cleared by centrifugation, normalized, and incubated with anti-

V5-tag magnetic beads (MBL International) at 4°C overnight. After washing the beads 3 times in wash buffer, proteins were eluted by boiling in Laemmli SDS sample buffer at 95°C for 5 min, then subjected to immunoblot analysis using a K48-linkage specific polyubiquitin antibody.

In vitro ubiquitination assays

Purified E1, E2, ubiquitin, CUL4A–RBX1, cereblon–DDB1, and ZMYM2 proteins were used to reconstitute the ubiquitination of ZMYM2 in vitro. ZMYM2 degron (amino acids 423-466) was expressed in BL21 star DE3 E. coli as a MBP fusion. Cultures were grown to an OD of 0.6 in

2XYT media, induced with 0.4 mM IPTG, and harvested following a 16-hour incubation at 16°C.

Cells were lysed via B-PER Complete (Thermo Fisher), diluted with lysis buffer (50 mM Tris pH7.5 200 mM NaCl 3mM TCEP 10%Gly, 50uM Zinc Acetate, 2X protease inhibitors), batch bound to maltose affinity resin (NEB), washed extensively with lysis buffer, and eluted with lysis buffer supplemented with 10 mM maltose. Human CRBN (amino acids 40–442) and full-length

DDB1 were co-expressed in SF9 insect cells in the presence of 50 μM zinc acetate, and purified by nickel affinity resin (Qiagen), HiTrap ANX column ion exchange (GE Healthcare), and

Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 28, 2021. Copyright 2021 American Association for Cancer Research. Sephacryl 400 16/60 size-exclusion chromatography (GE healthcare), as described above.

Human full-length CUL4A and RBX1 were co-expressed in SF9 insect cells and purified by nickel affinity resin and Superdex 200 16/60 size-exclusion chromatography. Purified recombinant human Ube1 E1 (E-305), UbcH5a E2 (E2-616), and ubiquitin (U-100H) were purchased from R&D systems. Components were mixed to final concentrations of 1 μM Ube1,

25 μM UbcH5a, 200 μM Ub, 1 μM CUL4–RBX1, 42 μM ZMYM2, and 1 μM cereblon–DDB1, with or without 10 μM avadomide, or 10 μM pomalidomide in ubiquitination assay buffer (20 mM

HEPES (pH 7.5), 150 mM NaCl, 10 mM MgCl2). After preincubation for 10 min at room temperature, ubiquitination reactions were started by addition of ATP to a final concentration of

10 mM. Reactions were incubated at 30 °C for 2.5 h before separation by SDS-PAGE followed by Coomassie staining or immunoblot analysis for MBP-ZMYM2.

Protein degradation reporter analysis

Cells stably expressing the respective construct in the protein degradation reporter vector

Artichoke or Cilantro 2 were seeded at a density of 10,000 to 20,000 cells per well in 96-well culture plates with 200 µl of media per well. Different concentrations of thalidomide analogs were printed in 3-4 biological replicates using the Tecan D300e Digital Dispenser. After 20 h of drug treatment, flow cytometry using the CytoFLEX instrument (Beckman Coulter) was performed to quantify EGFP and mCherry fluorescence. In FlowJo software, a parameter was derived which calculated the EGFP/mCherry fluorescence ratio on a single-cell basis. The geometric mean of the ratio for a given drug-treated sample was normalized to the average of

DMSO-treated controls.

In silico structural modeling

Our findings from the reporter assay suggested ZMYM2 recruitment to CRBN occurs through the MYM-type zinc finger residues 423-466. In the absence of a ZMYM2 structure, a homology

Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 28, 2021. Copyright 2021 American Association for Cancer Research. model for the ZMYM2 degron residues 421-453 was built in Maestro software (Schrödinger)

(48) using the structure of the N-terminal MYM-type zinc finger of human ZMYM5 (Protein Data

Bank [PBD] 2DAS) as a template. ZMYM2 residues 454-466 could not be modeled due to a lack of structural coverage in PDB 2DAS. A model of avadomide bound to DDB1-CRBN (PDB

4TZ4) was generated in Maestro. The ZMYM2 ternary complex model was assembled in

PyMOL (Schrödinger) (49), superimposing the CRBN-avadomide model with CRBN from PDB

5HXB and then superimposing ZMYM2 homology model residues 424-431 onto GSPT1 residues 570-577 from PDB 5HXB.

Ba/F3 cell proliferation and viability assays

Cell number and viability were determined using a hemocytometer by trypan blue staining. As an assessment of cytotoxicity, Ba/F3 cells were seeded at a density of 1,000 cells per well in

96-well culture plates with 200 µl of media per well. Different concentrations of avadomide or ponatinib were printed in 6 biological replicates using the Tecan D300e Digital Dispenser. After

48 h of drug treatment, total cellular ATP content was measured using CellTiter-Glo®

Luminescent Cell Viability Assay (Promega) according to the manufacturer protocol.

Luminescence was assessed by an Envision Microplate Reader (PerkinElmer). Results were normalized to the vehicle control.

Mouse experiments

6-8 week old female BALB/cByJ mice were obtained from The Jackson laboratory. All mice were housed in a pathogen-free animal facility and experiments were conducted with the ethical approval of the Harvard Medical Area Standing Committee on animals and according to an

IACUC approved protocol at Dana Farber Cancer Institute in Boston. Ba/F3 cells transformed by wild-type ZMYM2-FGFR1 in pLX304 and expressing human CRBN (with co-expression of

GFP) were used for in vivo experiments. Mice were sublethally irradiated using a Gamma-cell

Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 28, 2021. Copyright 2021 American Association for Cancer Research. irradiator at a dose of 550 Rads, retro-orbitally injected with 100,000 Ba/F3 cells/mouse, and dosed with avadomide (50 mg/kg) (n = 15) or the DMSO vehicle control (n = 15) suspended in

0.5% carboxymethyl cellulose 0.25% Tween-80 in deionized water(10) by oral gavage bi-daily from day 3 to day 9 post-transplantation. Complete blood counts were determined using the

Element HT5 Hematology Analyzer (Heska) and flow cytometry was performed to monitor the percentage of GFP positive cells in peripheral blood samples collected from the retro-orbital cavity on day 3 and day 10 post-transplantation. Mice were euthanized at day 10 post- transplantation. At the time of sacrifice, spleen weight was recorded, and bone marrow cells harvested from femurs and spleen were analyzed by flow cytometry.

Primary human hematopoietic cell experiments

Cryopreserved primary CD34+ hematopoietic stem and progenitor cells from healthy donors were obtained from Lonza. Cryopreserved bone marrow samples collected at the time of diagnosis from patients with ZMYM2-FGFR1-positive hematologic malignancy were obtained from Gustave Roussy or Paris Saint-Louis Hospital biobanks. The studies were approved by institutional review boards and performed in accordance with the Declaration of Helsinki. All patients gave their informed written consent. Cytogenetic and molecular analyses performed in bone marrow patient samples are described in the Supplemental Data (Supplementary

Methods; Table S5). Patient CD34+ cells were purified from bone marrow total or mononuclear cells using the AutoMACS and MACS CD34 microbeads (Miltenyi Biotec). Following enrichment, CD34+ cells were cultured in StemSpan Serum-Free Expansion Medium II (SFEM

II) (StemCell Technologies) supplemented with 1% penicillin-streptomycin, 1% L-glutamine (Life

Technologies), and human cytokines (25 ng/mL SCF, 50 ng/mL thrombopoietin, 40 ng/mL FLT3 ligand, 10 ng/mL IL-3) (Miltenyi Biotec) and incubated at 37°C with 5% CO2. After expansion for

3 to 5 days, cells were plated in 96-well plates (200-500 cells / well) and dosed with 1 µM avadomide or 0.01% DMSO for 2, 4, or 6 days, with 4-6 biological replicates for each condition.

Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 28, 2021. Copyright 2021 American Association for Cancer Research. Complete media change was performed every 48 h during drug treatment. Viable cell counts were determined using Trypan blue staining.

Statistical analysis

Graphpad Prism 8.3.0 software was used to represent and analyze data from cell viability assays, reporter assays, and mouse experiments. Comparison of quantitative variables was performed using the 2-tailed Student t-test. P < 0.05 was considered as statistically significant.

B.L. Ebert has received research funding from Celgene and Deerfield. He has received consulting fees from GRAIL, and he serves on the scientific advisory boards for Skyhawk

Therapeutics, Exo Therapeutics, and Neomorph Therapeutics. S.A. Carr is a member of the scientific advisory boards of Kymera, PTM BioLabs and Seer and a scientific advisor to Pfizer and Biogen.

Authors’ contributions

A. Renneville: Conceptualization, formal analysis, supervision, validation, investigation, visualization, methodology, writing-original draft, writing-review and editing. J.A. Gasser:

Resources and investigation. D. E. Grinshpun: Formal analysis and investigation. P.M. Jean

Beltran: Formal analysis, investigation, visualization, methodology. N.D. Udeshi: Resources and methodology. M.E. Matyskiela: Investigation, visualization, methodology, writing-review and editing. T. Clayton: Investigation. M. McConkey: Investigation. K. Viswanathan:

Investigation. A. Tepper: Investigation. A.A. Guirguis: Resources, writing-review and editing.

R.S. Sellar: Resources. S. Cotteret: Resources. V. Saada: Resources. C. Marzac: Resources.

S. De Botton: Resources. J.J. Kiladjian: Resources. J.M. Cayuela: Resources. M. Rolfe:

Resources and funding acquisition. P.P. Chamberlain: Resources, supervision, writing-review and editing. S.A. Carr: Resources, supervision, funding acquisition, methodology, writing-review and editing. B.L. Ebert: Conceptualization, resources, supervision, funding acquisition, methodology, writing-review and editing, project administration.

Acknowledgements

We would like to thank Anna Molineaux and J. Dora Levin for administrative support and assistance. The authors are grateful to Ebert lab and Carr lab (D.R. Mani and Karsten Krug) members for advice and helpful discussions. This work was supported by the NIH

Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 28, 2021. Copyright 2021 American Association for Cancer Research. (R01HL082945, P01CA108631, and P50CA206963), the Howard Hughes Medical Institute, and

Celgene to B.L. Ebert. S.A. Carr was supported by grants from the National Cancer Institute

(NCI) Clinical Proteomic Tumor Analysis Consortium grants NIH/NCI U24-CA210986 and

NIH/NCI U01 CA214125. R.S. Sellar was supported by an Intermediate Fellowship grant from the Kay Kendall Leukaemia Fund. We thank Gustave Roussy’s and Saint-Louis Hospital’s CRB

Biobanks for managing patient biological samples.

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Figure 1. Avadomide promotes ZMYM2 degradation in a CRBN- and proteasome- dependent manner. A and B, Volcano plot illustrating changes in protein abundance assessed by proteomic analysis in Hep3B cells after 12 h of treatment with 10 µM avadomide (A) or 24 h of treatment with 1 µM pomalidomide (B). The plots represent statistical significance versus the fold change in protein abundance relative to the DMSO vehicle control. Results from 2 biological replicates are shown. Average log2 fold-change in ZMYM2 protein abundance: -0.858, adjusted

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ZFP91, and GSPT1 protein levels after 24 h treatment with DMSO, 1 µM lenalidomide (LEN), 1

µM pomalidomide (POM), 1 µM avadomide (AVA), 1 µM iberdomide (IBER), or 0.005 µM CC-

885 in Hep3B cells. ZFP91 and GSPT1 are shown as positive controls for degradation. D,

Immunoblot analysis of ZMYM2 protein level in Hep3B cells treated with the indicated concentrations of avadomide for 24 h. E, Time course of avadomide treatment in Hep3B cells for ZMYM2 protein level. F, Time course of avadomide treatment in Hep3B cells for ZMYM2 mRNA levels assessed by real-time q-PCR. Data are mean ± s.d., n = 3 technical replicates. G,

Immunoblot analysis of ZMYM2 protein levels in Hep3B cells treated for 12 h with DMSO or 20

µM avadomide alone or in the presence of 0.5 µM MLN7243, or 5 µM MLN4924, or 2 µM MG-

132. H, Immunoblot analysis of ZMYM2 protein level in wild-type or CRBN knockout Hep3B cells treated with avadomide for 24 h. I, Immunoprecipitation of ZMYM2-V5 in Hep3B cells treated for 3h with DMSO or 10 µM avadomide in the presence of 10 µM MG-132. J,

Ubiquitination analysis of ZMYM2-V5 in Hep3B cells treated with DMSO or the indicated concentrations of avadomide for 6 h in the presence of 10 µM MG-132. EV, empty vector.

Results in panels C to J are representative of 3 independent experiments.

Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 28, 2021. Copyright 2021 American Association for Cancer Research. Figure 2. Identification of ZMYM2 degron and amino acids critical for drug-induced degradation. A, Sequence alignment of ZMYM2 MYM2 domain and the different peptides tested for degradation using the reporter assay. The cysteine residues that coordinate the zinc ions are highlighted in gray. The glycine residues following a CXXC motif are highlighted in yellow. B, Schematic of the protein degradation reporter vector used to identify ZMYM2 degron.

IRES, internal ribosome entry site. C, Hep3B cells stably expressing the ZMYM2 constructs in the degradation reporter were treated for 20 h with DMSO or avadomide and analyzed by flow cytometry to quantify the DMSO-normalized ratio of EGFP/mCherry fluorescence. The dose response curve for IKZF1/3 zinc finger 2 (ZF2) domain is represented as a reference (mean ± s.d, n = 4 biological replicates); AA, amino acid. D, Effect of different thalidomide analogs on the degradation of the ZMYM2 423-466 wild-type degron in the reporter vector in Hep3B cells

(mean ± s.d., 4 = 4 biological replicates). IC50, inhibitory concentration 50; n.r., not reached. E,

Alanine scan of the ZMYM2 423-466 degron using the reporter assay in Hep3B and TF1 cells treated with 1 µM avadomide for 20 h. Amino acids that reduced degradation above 0.4 when mutated into an alanine are figured in red characters (mean ± s.d., n = 3 biological replicates).

F, MBP-tagged ZMYM2 amino acids 423-466 wild-type or G429A were tested for in vitro ubiquitination by CRBN-CRL4 in the absence or presence of 10 µM pomalidomide or avadomide. Ubiquitination reactions were separated by SDS-PAGE followed by anti-MBP. G,

Immunoblot validation of the ZMYM2 degron in Hep3B cells transfected with the empty vector,

ZMYM2 wild-type, ZMYM2 with deletion of the MYM2 domain (del 425-502), or ZMYM2 binding mutant G429A and treated with DMSO or 10 µM avadomide for 24 h. Results in C to G are representative of 3 independent experiments.

Figure 3. Structural model of ZMYM2 recruitment to DDB1-CRBN bound to avadomide. A model of ZMYM2 amino acids 421-453 (blue), with the positions of G429 highlighted in red and

R424 and I427 in orange, shown bound to the complex of DDB1 (purple), CRBN (green), and

Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 28, 2021. Copyright 2021 American Association for Cancer Research. avadomide (yellow). Zinc ions are shown as gray spheres. The glycine-containing β-hairpin degron of ZMYM2 was docked based on the structures of other glycine-containing degron substrates recruited to CRBN (8,27,28).

Figure 4. Avadomide promotes ZMYM2-FGFR1 and ZMYM2-FLT3 fusion protein degradation. A, Schematic representation of ZMYM2, FGFR1, FLT3, and the chimeric fusion proteins ZMYM2-FGFR1 and ZMYM2-FLT3. The indicated breakpoints reported in previous studies (15,22) have been used to clone both fusion sequences in expression vectors for immunoblot validation. In the context of ZMYM2-FGFR1 fusion, the ZMYM2 proline/valine (P/V) rich domain has been shown to mediate oligomerization and tyrosine kinase constitutive activation (31). NLS, nuclear localization site; CL, Cre-like domain; Ig, Immunoglobulin-like domain; TM, transmembrane domain; TK, tyrosine kinase domain; JM, juxtamembrane domain.

B and C, Immunoblot validation of the chimeric fusion protein degradation in TF1 cells stably expressing the V5-tagged ZMYM2-FGFR1 (B) or ZMYM2-FLT3 (C) fusion protein, either with the WT or the G429A mutant degron, and treated with avadomide for 24 h. Results are representative of 3 independent experiments. EV, empty vector; WT, wild-type.

Figure 5. Avadomide affects viability of ZMYM2-FGFR1 transformed Ba/F3 cells expressing human CRBN. A, Growth curves assessed by Trypan blue staining after mIL3 withdrawal in Ba/F3 cells transduced with lentiviruses expressing the EV, ZMYM2WT-FGFR1 or

ZMYM2G429A-FGFR1 (mean ± s.d., n =3-4 biological replicates). B, Growth curves assessed by

Trypan blue staining in ZMYM2-FGFR1 transformed Ba/F3 cells in the absence of mIL3, in comparison with Ba/F3 parental and the EV control in the presence of mIL3 (mean ± s.d., n = 3-

4 biological replicates). C, Immunoblot analysis in Ba/F3 cells transformed by V5-tagged

ZMYM2WT-FGFR1 in pLX304 and subsequently transduced with lentiviruses expressing either

Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 28, 2021. Copyright 2021 American Association for Cancer Research. human CRBN or the humanized mouse CrbnI391V mutant in GW vector. Ba/F3 cells transduced with both EV pLX304 and EV GW are shown as a negative control. Ba/F3 cells were treated with DMSO or 10 µM avadomide for 24 h. D and E, Immunoblot analysis in Ba/F3 cells transduced with EV pLX304 and EV GW or human CRBN in GW, and Ba/F3 cells transformed by V5-tagged ZMYM2WT-FGFR1 (D) or ZMYM2G429A-FGFR1 (E) in pLX304 and subsequently transduced with EV GW or human CRBN in GW. Ba/F3 cells were treated with DMSO, 1 µM or

10 µM avadomide for 24 h. F, Cell viability assessed by Cell Titer Glo in Ba/F3 cells transformed by ZMYM2WT-FGFR1 or ZMYM2G429A-FGFR1 and transduced with EV GW or human CRBN in

GW. Ba/F3 cells were treated with different concentrations of avadomide or ponatinib for 48 h

(mean ± s.d., n = 6 biological replicates). Results in panels A to F are representative of 3 independent experiments. EV, empty vector; GW, Gateway; WT, wild-type; mIL-3, murine interleukin-3; Z-F1, ZMYM2-FGFR1.

Figure 6. Effects of avadomide treatment on ZMYM2-FGFR1 transformed Ba/F3 cells in vivo. A, Experimental design of the mouse experiment. ZMYM2WT-FGFR1 transformed Ba/F3 cells expressing human CRBN were retro-orbitally injected into sublethally irradiated BALB/c mice. n =15 mice for both treatment group. PB, peripheral blood. B, Spleen weight at day 10 post-transplantation. C, White blood cell count, (D) hemoglobin level, and (E) platelet count measured in peripheral blood samples collected before and after treatment. F to H, Percentage of GFP positive cells assessed by flow cytometry in (F) peripheral blood, (G) bone marrow, and

(H) spleen. P-values are from an unpaired two-sided t-test. ns, non-statistically significant. ****,

P < 0.0001; ***, P < 0.001; *, P < 0.05.

Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 28, 2021. Copyright 2021 American Association for Cancer Research. Figure 7. Effects of avadomide treatment on primary human CD34+ hematopoietic stem and progenitor cells ex vivo. Viable cell counts assessed by Trypan blue staining in bone marrow CD34+ cells collected from (A) healthy donors and (B) patients with ZMYM2-FGFR1- positive hematologic malignancy are represented after 2, 4 or 6 days of treatment by 1 µM avadomide or the vehicle control (mean ± s.d., n = 4-6 biological replicates). P-values are from an unpaired two-sided t-test. ns, non-statistically significant. ****, P < 0.0001; **, P < 0.01; *, P <

0.05. T-ALL, T acute lymphoblastic leukemia; AML-5, acute myeloid leukemia 5 (monoblastic);

MPN-Eo, myeloproliferative neoplasm with eosinophilia.

Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 28, 2021. Copyright 2021 American Association for Cancer Research. Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 28, 2021. Copyright 2021 American Association for Cancer Research. Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 28, 2021. Copyright 2021 American Association for Cancer Research. Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 28, 2021. Copyright 2021 American Association for Cancer Research. Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 28, 2021. Copyright 2021 American Association for Cancer Research. Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 28, 2021. Copyright 2021 American Association for Cancer Research. Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 28, 2021. Copyright 2021 American Association for Cancer Research. Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 28, 2021. Copyright 2021 American Association for Cancer Research. Avadomide induces degradation of ZMYM2 fusion oncoproteins in hematologic malignancies

Aline RENNEVILLE, Jessica A Gasser, Daniel E Grinshpun, et al.

Blood Cancer Discov Published OnlineFirst March 10, 2021.

Updated version Access the most recent version of this article at: doi: 10.1158/2643-3230.BCD-20-0105

Supplementary Access the most recent supplemental material at: Material http://bloodcancerdiscov.aacrjournals.org/content/suppl/2021/03/09/2643-3230.BCD-20-0105.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

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