Overcoming IMiD resistance in T-cell lymphomas through potent degradation of ZFP91 and IKZF1

T-cell lymphomas (TCLs) comprise ∼10% of all non-Hodgkin lymphomas (NHLs) in Western countries.¹ The World Health Organization currently divides TCLs into 29 different subtypes based on histologic and immunophenotypic features that include both peripheral (PTCL) and cutaneous (CTCL) entities.² Essentially, every T-cell subset (eg, follicular helper T, regulatory T [Treg], TH1, TH2) has a malignant counterpart with overlapping transcriptional and immunophenotypic features.

Treatment of TCLs to date has been largely derivative or empiric. Cyclophosphamide, doxorubicin, vincristine, and prednisolone–based regimens remain the standard for first-line therapy in most patients.³ Except for anaplastic large cell lymphomas (ALCL), which benefit from the addition of brentuximab vedotin,⁴ over 75% of PTCLs fail to respond or relapse after first-line therapy. PTCL not otherwise specified, angioimmunoblastic T-cell lymphoma (AITL), ALCL that lack ALK rearrangements (ALK⁻ ALCL), and natural killer/TCL, the most common subtypes of PTCL, as well as late-stage CTCL are each associated with <30% 5-year overall survival.⁵ Long-term survivors of rare subtypes of TCL like hepatosplenic TCL and monomorphic epitheliotropic intestinal TCL are extremely uncommon. Currently, available second line agents like histone deacetylase inhibitors and pralatrexate induce responses in <30% of patients and have progression-free survival <4 months.⁶ The imperative is clearly to define new and important vulnerabilities that can be targeted in subsets of TCL.

The immunomodulatory agents (IMiD) thalidomide, lenalidomide, and pomalidomide were the first drugs found to promote the ubiquitination and degradation of specific substrates by an E3 ubiquitin ligase complex.^7,8 These compounds function by increasing the affinity of the CRL4^CRBN E3 ubiquitin ligase for a small set of protein substrates. In myelodysplastic syndrome with loss of chromosome 5q, the critical target for lenalidomide activity is casein kinase 1α (CK1α).^9,10 In multiple myeloma and B-cell lymphomas, the key drug targets are the master lymphocyte transcription factors IKZF1 (Ikaros) and IKZF3 (Aiolos).^7,8,11

Through both cell-intrinsic programs and feed-forward cytokine loops, the IKZF family transcription factors play important roles in the differentiation and function of many T-cell subsets, including TH1, TH2, TH17, follicular helper T, and Treg cells.¹² Yet, IMiDs have very limited activity in TCL. In a trial of single-agent lenalidomide in 54 patients with relapsed or refractory PTCL, the overall response rate was 22% and median progression-free survival was only 2.5 months.¹³ Lenalidomide also failed to improve efficacy when added to cyclophosphamide, doxorubicin, vincristine, and prednisolone chemotherapy in the first line.¹⁴ Using a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screen, we previously showed that IKZF1 is a vulnerability within a subset of TCL cell lines that are resistant to IMiDs.¹⁵ That resistance could result from inadequate degradation of IKZF1 by available IMiDs (ie, pharmacologic failure). However, TCLs are typically resistant to both lenalidomide and pomalidomide at doses >30 µM, suggesting a fundamental difference in the role of IKZF1 and potentially other known substrates within malignant TCL cells.

Among the other known substrates of lenalidomide and pomalidomide is zinc finger protein 91 (ZFP91), which is targeted for degradation through its 4th C2H2 zinc finger (ZnF4) domain.¹⁶ ZFP91 is broadly expressed across tissues, with highest expression in lymph nodes and testis.¹⁷ ZFP91 shows structural characteristics highly suggestive of transcription factor (TF) activity: 5 zinc-finger domains, a leucine zipper, and several nuclear localization signals.¹⁸ Multiple studies have demonstrated that ZFP91 is dispensable for survival in myeloid and B-cell malignancies.^19,20 Similarly, our previous CRISPR screen failed to identify ZFP91 as a selective vulnerability in TCL lines. Here, we further explore the roles of IKZF1 and ZFP91 in TCL survival, proliferation, and resistance to IMiDs.

Further details are available in supplemental Methods.

Cell lines were cultured in RPMI-1640 media supplemented with 10% to 20% fetal bovine serum and 1% penicillin/streptomycin. Cell lines were routinely tested for mycoplasma (ATCC Universal Mycoplasma Detection Kit), and authenticity was validated by short tandem repeat profiling.

Proliferation assays were performed twice in triplicate. Cells were seeded at 1:5 ratio, and tissue culture medium containing drugs or dimethyl sulfoxide (DMSO) were replaced every 48 hours. Cells were stained with 1 µg/mL propidium iodide (PI) solution (Sigma-Aldrich # P4864). Cell numbers and PI⁺ population were detected by flow cytometry every 48 hours.

Animal work was performed in NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ mice (JAX; stock #005557) with approval of the Dana-Farber Cancer Institute Institutional Animal Care and Use Committee.

Data are presented as mean plus or minus standard deviation (SD) unless otherwise indicated in the figure legends. Data were tested using a 2-tailed Student t test (when comparing 2 groups) or 2-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc comparison (to test multiple groups), as indicated in the figure legends. Kaplan-Meier survival curves were analyzed using a log-rank test. Overall survival for patient-derived xenograft (PDX) experiments was calculated from the time of treatment initiation to the time of death and compared using a log-rank test. Violin plots show full range of data points with thick line at median and thin lines at 25th and 75th percentiles. Unless indicated otherwise, P values were derived from paired, 2-tailed Student t tests. For all tests, significance was determined with a 95% confidence interval (*P < .05; **P < .01; ***P < .001) on GraphPad Prism, version 8.

First, we tested the activity of IMiDs across 20 available TCL cell lines.¹⁵ Consistent with the low response rate in patients with TCL,^13,21 lenalidomide was active in only the ALK-rearranged (ALK⁺) ALCL cell lines SU-DHL-1 and KI-JK. The more potent agent pomalidomide was active only in SU-DHL-1, KI-JK, and the ALK⁻ ALCL cell line DL40 (Figure 1A; supplemental Figure 1A).

To identify the substrates of IMiDs in TCL cells, we treated SU-DHL-1 cells with 2.5 μM lenalidomide, 1 μM pomalidomide, or DMSO control for 5 hours, then assessed protein abundance by quantitative mass spectrometry (MS)-based proteomics. Lenalidomide induced twofold downregulation of only IKZF1, RAB28, and IL10. Pomalidomide-treated cells had greater than twofold reductions in these proteins as well as ZFP91 (Figure 1B). Degradation of IKZF1 and ZFP91 but not RAB28 was confirmed by immunoblotting (Figure 1C-D; supplemental Figure 1B). Even 10 μM lenalidomide only partially degraded IKZF1 and had limited effect on ZFP91. Lenalidomide decreased IKZF1 protein levels without altering IKZF1 messenger RNA expression (Figure 1E), consistent with a posttranslational mechanism of regulation. However, lenalidomide induced a decrease in IL10 transcript within 2 hours (Figure 1E), consistent with previous reports that IKZF1 directly regulates IL10 expression.²² Knockout of CRBN reversed IMiD-mediated toxicity, confirming CRBN dependence of IMiDs in TCL cells (supplemental Figure 1C-D).

To evaluate whether decreased IKZF1 expression drives the sensitivity of SU-DHL-1 and KI-JK cells to lenalidomide, we transduced with lentiviral vectors expressing CRISPR-associated protein 9 and a doxycycline-inducible sgRNA²³ cassette. Doxycycline treatment resulted in near complete loss of IKZF1 protein and phenocopied the effects of IMiDs on survival in both lines (Figure 1F-G; supplemental Figure 1E-F).

To better understand how TCL cells acquire resistance to IMiDs, we generated lenalidomide-resistant TCL cells by long-term culture with increasing concentrations of lenalidomide. These cells maintained stable resistance (len-regrown cells) after lenalidomide withdrawal for >2 months (Figure 2A-B; supplemental Figure 2A; supplemental Table 1). Exome sequencing demonstrated no mutations or copy number alterations in CRBN or additional factors previously described to modulate CRL4^CRBN E3 ubiquitin ligase activity^24,25 (supplemental Figure 2B; supplemental Table 1). Lenalidomide treatment decreased IKZF1 protein levels to a similar extent in len-regrown and parental cells (Figure 2C; supplemental Figure 2C). However, knockout of IKZF1 had markedly reduced effects on proliferation and survival in len-regrown cells compared with parental cells (Figure 2D-E), indicating that len-regrown cells are tolerant to IKZF1 loss.

Interestingly, the len-regrown cells remained sensitive to pomalidomide treatment (Figure 2A-B; supplemental Figure 2A). Based on the MS data (Figure 1B), we hypothesized that ZFP91 degradation contributes to pomalidomide sensitivity. Similar to parental cells, pomalidomide but not lenalidomide induced ZFP91 loss in len-regrown cells (supplemental Figure 2C). ZFP91 knockout did not affect the growth or survival of len-regrown cells (supplemental Figure 2D-E) but significantly increased the effects of both lenalidomide and IKZF1 knockout in len-regrown cells (Figure 2F-I).

To directly address the role of ZFP91 in resistance, we mutated glycine 406 to alanine (G406A), which disrupts the pomalidomide-mediated interaction of the ZFP91-ZnF4 domain with CRBN.¹⁶ As expected, ZFP91 G406A was not degraded by pomalidomide (supplemental Figure 2F). Expression of ZFP91 G406A reduced the effect of pomalidomide in lenalidomide-resistant TCLs (supplemental Figure 2G-I). These results demonstrate that selective resistance to lenalidomide can involve codependence on IKZF1 and ZFP91.

As noted above, pomalidomide induces near complete degradation of target proteins in both sensitive and acquired len-resistant TCLs, which is associated with cell killing. However, pomalidomide led to significantly less degradation of the same targets in multiple pomalidomide-resistant TCLs (Figure 3A). Importantly, double knockout of IKZF1 and ZFP91 markedly reduced cell growth and survival in these pomalidomide-resistant TCLs compared with knockout of either target alone (Figure 3B-D; supplemental Figure 3A-C). This suggests that the failure of pomalidomide within these lymphomas may be pharmacologic rather than due to a lack of dependence on IKZF1 and ZFP91.

To address possible causes of pharmacologic failure, we compared gene expression of 33 known regulators of IMiD activity²⁴ with the extent of lenalidomide-induced IKZF1 degradation across TCL lines (supplemental Figure 3D). Strikingly, expression of CRBN was the most correlated with IKZF1 degradation (r = 0.8166; Figure 3E). To test the direct contribution of CRBN levels to IMiD sensitivity, we transduced pomalidomide-resistant cells with a viral vector overexpressing CRBN, which led to a greater than fivefold increase in CRBN protein levels. CRBN overexpression increased the degree of degradation for both IKZF1 and ZFP91, causing a markedly greater sensitivity to pomalidomide in TCLs with primary resistance (supplemental Figure 3E-J). We therefore hypothesized that a more potent degrader of IKZF1 and ZFP91 could overcome pomalidomide resistance resulting from low CRBN expression.

CC-92480, a novel CEreblon E3 Ligase MoDulator (CELMoD) agent,²⁶ shares the same glutarimide ring with IMiDs but contains an additional arylpiperazine moiety. We found that CC-92480 (1 μM) treatment led to almost complete degradation of IKZF1 and ZFP91 across pomalidomide-resistant TCL lines (Figure 3A). The newer-generation agent CC-220/iberdomide²⁷ had intermediate activity (Figure 3A). Strikingly, the improved degradation by CC-92480 was associated with induction of apoptosis and reduced cell proliferation across all 20 TCL cell lines, even at a dose of 0.1 μM (Figure 3F; supplemental Figure 4A-B).

To directly compare substrate selectivity in TCL cells, we treated SUP-M2 cells with 1 μM CC-92480 or IMiDs for 5 hours, then assessed protein abundance by quantitative MS. CC-92480 induced 18.3-fold and 6.7-fold downregulation of IKZF1 and ZFP91, respectively (Figure 3G). Pomalidomide had similar selectivity but less effect (6.1-fold of IKZF1, 2.6-fold of ZFP91). Lenalidomide only induced 2.6-fold reduction of IKZF1, with no effect on ZFP91 (Figure 3G). As expected, both CC-92480 and IMiDs decreased their target protein levels without altering messenger RNA expression, whereas IL10 downregulation was a secondary effect induced by IKZF1 degradation (supplemental Figure 4C).

To define mechanisms of killing by IMiDs and CC-92480, we performed RNA sequencing (RNA-seq) and gene set variation analysis in pomalidomide-resistant TCLs treated with vehicle, lenalidomide, pomalidomide, or CC-92480. Across 3 lines, CC-92480 treatment resulted in an enrichment of signatures for interferon response, p53 pathway, TNFα signaling, and apoptosis with impaired cell cycle compared with DMSO or the other 2 agents (supplemental Figure 4D-E). Together, these data suggest that more potent degradation of both IKZF1 and ZFP91 can overcome pomalidomide resistance in TCL cells through upregulation of pathways previously implicated for IMiDs in B- and myeloid diseases.^23,28,29

To assess the in vivo efficacy of CC-92480, we first used an ALK⁺ ALCL subcutaneous model with SUP-M2 cells, which are resistant to pomalidomide in vitro (Figure 3F). After engraftment, mice were randomized to receive pomalidomide (3 mg/kg), CC-92480 (1 mg/kg), or vehicle by mouth. All the mice were euthanized when any tumor reached 2 cm in the longest dimension (Figure 4A). Treatment with CC-92480 markedly reduced tumor growth compared with either vehicle- or pomalidomide-treated mice (Figure 4B).

Next, we engrafted mice with a systemic AITL (DFTL-78024) PDX that primarily involves the spleen and treated for 8 days with vehicle, pomalidomide, or CC-92480 (Figure 4C). CC-92480 potently induced degradation of both IKZF1 and ZFP91 in the PDX cells in vitro (Figure 4D). Splenic AITL involvement was reduced >95% in CC-92480–treated mice after only 8 days of treatment, and ∼60% of the residual AITL cells were apoptotic (Figure 4E). In contrast, pomalidomide had very limited effects (Figure 4D-E).

To test the effects of these agents on survival, we engrafted ATLL (DFTL-69579) or T-cell prolymphocytic leukemia (DFTL-28776) PDXs and treated with pomalidomide, CC-92480, or vehicle (Figure 4F-K). Spleen size and lymphoma involvement were markedly reduced in CC-92480–treated mice in both models based on splenic ultrasound or luciferase imaging (Figure 4G,J). In contrast, pomalidomide had very limited effects (Figure 4G,J). Treatment with CC-92480 for 21 days significantly improved survival in both models compared with pomalidomide treatment or vehicle (Figure 4H,K). Both pomalidomide and CC-92480 treatment were very well tolerated (supplemental Figure 4F). Together, these results demonstrate that CC-92480 is highly active against a range of TCL subtypes and has superior activity compared with pomalidomide both in vitro and in vivo.

Next, we took advantage of the paired parental and len-regrown SU-DHL1 cells to examine the role of ZFP91 in TCL cells. Upregulated genes in len-regrown compared with parental cells were enriched in pathways for chromatin modifications, hormone signaling, cell-cell interactions, and Wnt signaling (supplemental Figure 5A-B; P < .01). Consistent with these upregulated pathways, len-regrown cells but not parental cells had constitutive phosphorylation of extracellular signal-regulated kinase (ERK) and could form single-cell colonies (Figure 5A-B). Strikingly, ZFP91 knockout had minimal effects in parental cells (n = 38 genes with fold change >1.5 and adjusted P < .05) but markedly affected transcription in len-regrown cells (n = 1139 genes) (Figure 5C; supplemental Table 2). The same enriched, upregulated pathways in len-regrown cells were downregulated upon ZFP91 knockout (supplemental Figure 5C-D), indicating that loss of ZFP91 reverses the transcriptional differences between len-regrown and parental cells.

To gain further insight into ZFP91 function, we mapped ZFP91 binding genome-wide using chromatin immunoprecipitation sequencing (ChIP-seq) (supplemental Table 3). Analysis of ZFP91 ChIP-seq signal (n = 1419) demonstrated broadly increased binding of ZFP91 on chromatin in len-regrown cells compared with parental cells (Figure 5D). We observed peaks of active histone marks H3K27ac and H3K4me3 at these loci (supplemental Figure 6A-B). Notably, the levels of these active marks increased significantly in len-regrown cells at sites of elevated ZFP91 binding (supplemental Figure 6C).

To precisely map the effects of ZFP91 at both enhancers and promoters, we directly quantified nascent RNA synthesis using precision nuclear run-on sequencing (PRO-seq). This assay revealed that the increased ZFP91 binding at distal peaks corresponded to increased transcription of eRNAs near bound sites (Figures 5E; supplemental Figure 6C). Moreover, higher-level binding of ZFP91 in len-regrown cells was associated with increased transcription of the closest genes as evidenced by both RNA-seq and PRO-seq signals (Figure 5F). To directly confirm the activity of ZFP91 as a transcription factor, we cloned its predicted binding sequence into a luciferase reporter. Both pomalidomide treatment and ZFP91 knockout reduced luciferase activity (supplemental Figure 6D-E), demonstrating that ZFP91 can function as a transcriptional activator in lenalidomide-resistant TCL cells.

Notably, ZFP91 binding sites by ChIP-seq were highly overlapping (35.4% ∼ 53.1%) between acquired len-regrown SU-DHL1 cells and TCLs with primary resistance to lenalidomide (Figure 5G). Kyoto Encyclopedia of Genes and Genomes and gene ontology analysis revealed that the potential target genes of ZFP91 were mainly enriched in pathways for MAPK, NF-κB, Wnt signaling, and histone modification (supplemental Figure 7A-B). We then focused on target genes directly activated by ZFP91 in TCL cells. We analyzed high-confidence target genes, in which ZFP91 binds within or surrounding (<10 kb) the gene locus, and the expression was significantly downregulated by ZFP91 knockout (fold change <0.67 and adjusted P < .05). Among these genes were MAP3K12 (MAPK pathway), FZD2 (Wnt signaling pathway), HDAC5 (histone modification), GHDC, and IKBKE (NF-κB pathway) (Figure 5H). A very similar set of targets from the same pathways were identified in len-regrown cells (supplemental Figure 7C). We confirmed ZFP91 binding at multiple loci by ChIP–quantitative polymerase chain reaction across multiple TCL lines (supplemental Figure 7D-E). PRO-seq analysis demonstrated transcription of eRNAs near peaks of ZFP91 and active histone modifications at these loci (Figure 5H). Thus, ZFP91 functions as a transcriptional activator that drives a program in multiple TCLs associated with activation of MAPK, NF-κB, and WNT signaling.

Finally, we addressed how acquired len-resistant TCLs become dependent on the combination of ZFP91 and IKZF1. Motif analysis at sites of ZFP91 binding demonstrated a joint ZFP91/IKZF1 motif in both len-regrown cells and in multiple primary resistant TCL lines (Figure 6A). Unlike in resistant cells, a joint ZFP91/AP-1 motif was identified in lenalidomide-sensitive cells (Figure 6A). In line with this motif change, JUN (encoding the AP-1 transcription factor c-Jun) was one of the top downregulated genes in len-regrown cells compared with parental cells (supplemental Figure 8A; supplemental Table 4). c-Jun protein was also decreased in len-regrown cells. The residual c-Jun had increased phosphorylation at serine 249 (Figure 6B-C), which is known to reduce DNA binding and leads to autodownregulation of JUN transcription.³⁰ To test whether the redistribution of ZFP91 binding is regulated by c-Jun, we generated JUN-knockout cells. Compared with parental (wild-type) cells, JUN-knockout recapitulated the profile of ZFP91 rewiring observed in len-regrown cells by significantly increasing ZFP91 binding (P < .0001, Figure 6D-E), particularly at the target genes identified in len-regrown cells (Figure 6F; supplemental Figure 8B).

To address the mechanism of c-Jun downregulation in len-regrown cells, we analyzed exome sequencing data and found an increased copy number for a region of chromosome 6p21.33 compared with parental cells (Figure 6G). In fact, 11 of the top 20 upregulated genes from RNA-seq of len-regrown cells were within this region (Figure 6G; supplemental Table 5). We confirmed 6p21.33 amplification by fluorescence in situ hybridization (supplemental Figure 8C; supplemental Table 6).

CSNK2B, which encodes the casein kinase 2 (CK2) regulatory subunit CK2b, was one of the genes included within the 6p21.33 amplicon (Figure 6G). CSNK2B was a vulnerability in our previous CRISPR screen of TCL cell lines (Figure 6G) and is highly expressed in patient samples and PDXs across multiple PTCL entities (supplemental Figure 8D-E). Previous studies have established that CK2 phosphorylates c-Jun at serine 249,³¹ so we hypothesized that overexpression of CSNK2B would promote c-Jun S249 phosphorylation. We overexpressed CSNK2B in lenalidomide-sensitive cells at levels comparable to the increased expression in len-regrown cells (supplemental Figure 8F-G). CSNK2B overexpression increased c-Jun phosphorylation at serine 249, decreased c-Jun protein levels, and significantly reduced lenalidomide sensitivity (Figure 6H-K). The opposite effects were observed with CSNK2B knockout in len-regrown cells (supplemental Figure 8H-J). At the same time, CSNK2B knockout in TCL cells with primary resistance to IMiDs decreased c-Jun S249 phosphorylation and increased its total protein levels (supplemental Figure 8K-L), suggesting that CK2 also regulates c-Jun in these cells.

Despite the role of IKZF1 in T-cell differentiation and cytokine signaling, currently available IMiDs have disappointing activity.^13,14 It has remained an open question whether the “primary resistance” to lenalidomide and pomalidomide represents a lack of dependence on target proteins or is simply a pharmacologic failure. We found a close correlation between levels of CRBN expression and the extent of IKZF1 degradation induced by lenalidomide (supplemental Figure 3E). More definitively, overexpression of CRBN in IMiD-resistant TCLs markedly extended the degree of target protein degradation and resensitized cells to pomalidomide (supplemental Figure 3E-J), suggesting that a more potent degrader could overcome IMiD-resistance in TCLs.

CC-92480, a novel CELMoD agent, shares the same glutarimide rings with IMiDs for binding to cereblon and substrates. However, CC-92480 contains additional arylpiperazine moiety and thus creates a hotspot on the surface of CRBN for direct ligase-target protein interactions.²⁶ Consistent with its subtle structure, we found that CC-92480 exhibited a unique profile in IMiD-resistant TCLs: (1) enhanced efficacy to drive the degradation of target proteins, which requires only ∼100-fold lower concentrations and shorter times, and (2) resulted in much higher levels of immune response, apoptosis, and cell cycle arrest. This improved potency may explain the superior cell autonomous activity of CC-92480 and its ability to function in primary IMiD-resistant backgrounds. In fact, CC-92480 was broadly effective against multiple TCL subtypes, and this was solely through degradation of IKZF1 and ZFP91 (Figure 3E-F).

Currently, CC-92480 are in clinical development for evaluating the safety and efficacy in newly diagnosed multiple myeloma and relapsed or refractory multiple myeloma. In the present study, we tested CC-92480 in our preclinical PDX models that recapitulate features of human TCL. We noted that CC-92480 given daily (1 mg/kg) was well tolerated by xenografted mice and was able to maintain lower levers of IKZF1 and ZFP91 expression in vivo compared with either vehicle or pomalidomide regimen. In systemic PDX models, only 8 to 10 days treatment with CC-92480 could decrease the spleen size to near normal range. CC-92480 as a single-agent delayed tumor progression and significantly prolong survival in PDX models, strongly suggesting that novel CELMoD agents, like CC-92480, could be an attractive therapeutic strategy for the treatment of TCL patients.

Previous findings that lenalidomide lead to degradation of IKZF1 and CK1α were important steps in exploiting the central role of IKZF1 and CK1α and hence provided a mechanistic basis for the therapeutic window of lenalidomide in B-cell malignance and myelodysplastic syndrome.⁹ Previous CRISPR screen revealed IKZF1 as one of the key vulnerability in TCLs.¹⁵ A subset of TCL lines, including SU-DHL-1 and KI-JK, are dependent on IKZF1 alone. It is also worth noting that IKZF1 is dispensable for the other half of the lines from the same screen. Moreover, in our present study, codependence on IKZF1 and ZFP91 was found to be existed in both acquired and primary IMiD-resistant scenarios. This finding highlighted that a greater and broader toxicity could be achieved by cotargeting IKZF1 and ZFP91 in TCLs. Consistently, double knockout of IKZF1 and ZFP91 fully recapitulated the effect of CC-92480, suggesting that both IKZF1 and ZFP91 play critical roles and contribute to IMiD-resistance in TCL cells.

Using a combination of ChIP-seq, PRO-seq, and RNA-seq, we showed that ZFP91 binds at promoters and enhancers and promotes transcription of both genes and enhancer RNAs. Based on its structure, ZFP91 was initially thought to act as a TF.³⁴ Supporting this, ZFP91 acted as a cofactor with NF-κB/p65 to drive transcriptional activation of HIF1³⁵ and as an insulin-responsive TF in hepatocytes that required an intact Pck1p insulin-response sequence for binding at target genes.³⁶ These studies suggest that ZFP91 promotes transcription as a coactivator with another TF. In that regard, we noted that ZFP91 binding frequently occurred in sequences with motifs for either AP-1 (in IMiD-sensitive cells) or IKZF1 (in IMiD-resistant cells). Loss of c-Jun resulted in redistribution of ZFP91, suggesting that ZFP91 is not a pioneer factor but instead modulates transcription in concert with other TFs.

Of interest, multiple studies have reported that ZFP91 has E3 ubiquitin ligase activity. Jin et al reported that ZFP91 promoted K63-linked ubiquitination of NF-κB-inducing kinase in HEK293 cells.³⁷ In Treg cells, ZFP91 loss did not affect K63-linked ubiquitination of NF-κB-inducing kinase.³⁸ Instead, ZFP91 rapidly translocated from the nucleus to the cytoplasm in response to T-cell receptor stimulation and then mediated K63-linked ubiquitination of BECN1 and formation of the autophagosome initiation complex.³⁸ Mice with Treg-specific deletion of ZFP91 developed severe autoimmunity affecting multiple tissues as well as inflammation-induced colon carcinogenesis. In effector T cells, deletion of ZFP91 increased mTORC1-driven cell glycolysis and boosted antitumor T-cell responses,³⁹ suggesting that targeting ZFP91 may also improve the efficacy of cancer immunotherapy. In fact, the effects on nonmalignant T cells from concurrent loss of IKZF1 and ZFP91, whether genetic or through potent small molecule degraders, remains an important and unaddressed area for investigation. It also remains unclear whether ZFP91 could promote the ubiquitination of TFs or other factors bound to chromatin and thereby modulate transcription of target genes. Overall, we discovered a critical role of ZFP91 as a transcription factor that coregulates cell survival of IMiD-resistant TCLs with IKZF1. Newer-generation degrader, by potently targeting IKZF1 and ZFP91, could be an attractive therapeutic strategy for the treatment of TCL patients.

The authors thank Katia Georgopoulos, Cedric Menard, and members of the Weinstock laboratory for thoughtful comments. They thank Wenjing Wu for help with preparation of graphics. They thank the Nascent Transcriptomics Core at Harvard Medical School, Boston, MA for assistance with PRO-seq library construction and data analysis. They thank the Flow Cytometry Core facility at Dana-Farber Cancer Institute for assistance with cell sorting and flow cytometry analysis.

This work was supported in part by the National Cancer Institute (NCI), National Institutes of Health R35 CA231958 and P01 CA248384 (both to D.M.W.), R01 CA214608 (to E.S.F.), and the Ludwig Center at Harvard (to K.A.).

Contribution: W.W., E.S.F., K.A., and D.M.W. were responsible for conception and design of the study; W.W., R.K., K.A.D., R.P.N., T.B.H.-F., H.L., J.D., M.K.J., L.Y., S.A., N.L.D., and F.P. were responsible for acquisition of data; W.W., G.M.N., K.A.D., K.E.S., T.B.H.-F., M.K.J., A.J.N., S.Y.N., G.W., S.J., R.X., P.R.H., L.d.L., P.G., J.I., A.T., E.S.F., K.A., and D.M.W. interpreted and analyzed data; and W.W and D.M.W. wrote the manuscript. All authors critically revised the article and approved the final version.

Conflict-of-interest disclosure: E.S.F. is a founder, scientific advisory board (SAB), and equity holder in Civetta Therapeutics, Jengu Therapeutics (board member), and Neomorph, Inc. E.S.F. is an equity holder in C4 Therapeutics and reports personal fees from Novartis, Sanofi, EcoR1 capital, Deerfield and Astellas during the conduct of the study and outside the submitted work. The Fischer lab receives or has received research funding from Novartis, Ajax and Astellas. K.A. is a consultant for Syros Pharmaceuticals, is on the SAB of CAMP4 Therapeutics, and receives research funding from Novartis not related to this work. S.J. is supported by the National Cancer Institute K08 Career Development Award (K08CA230498). K.A.D. is a consultant to Kronos Bio and Neomorph Inc. G.W. is a Fellow of The Leukemia & Lymphoma Society. D.M.W. is a founder and SAB member for Ajax Pharmaceuticals and a consultant or Scientific Advisory Board member for Bantam Pharmaceuticals, Secura Biopharma, Daiichi Sankyo, Travera, Trillium, ASELL and Astra Zeneca. The Weinstock lab receives research funding from Daiichi Sankyo, Verastem and Abcuro. No other disclosures were reported. The remaining authors declare no competing financial interests.

Correspondence: David M. Weinstock, Dana-Farber Cancer Institute, 450 Brookline Avenue, Dana 510B, Boston, MA 02215; e-mail: davidm_weinstock@dfci.harvard.edu