The novel retinoid WYC-209 sensitizes multiple myeloma to carfilzomib via epigenetically upregulating ZMYND8

Abstract

Carfilzomib (CFZ) is a cornerstone therapy for patients with relapsed multiple myeloma (MM); however, poor prolonged treatment response remains a major clinical limitation. ZMYND8 overexpression increases the sensitivity of MM cells to CFZ. Therefore, pharmacological activation of ZMYND8 may offer an emerging strategy to improve CFZ efficacy. In this study, we identified a novel retinoic acid analog, WYC-209, as an epigenetic activator of ZMYND8 expression in MM cells. The additive cytotoxic effects of WYC-209 and CFZ were analyzed using flow cytometry, transmission electron microscopy, and drug synergy assays. The in vivo therapeutic synergy was assessed in a mouse xenograft model using tumor burden and survival analysis. We found that both all-trans retinoic acid (ATRA) and WYC-209 significantly upregulated the transcriptional expression of ZMYND8 by remodeling the epigenetic landscape, particularly via H3K27ac. Combined treatment with CFZ and either ATRA or WYC-209 exhibited pronounced synergistic effects in killing primary MM cells. Moreover, WYC-209 synergized with CFZ to inhibit cell viability, induce apoptosis, and exacerbate endoplasmic reticulum dilation in MM cells, whereas the depletion of ZMYND8 markedly attenuated these effects. In vivo experiments confirmed that WYC-209 potentiated the antitumor efficacy of CFZ by upregulating ZMYND8, thereby ameliorating tumor burden in NSG mice. These findings establish that targeting ZMYND8 with the novel retinoid WYC-209 potently enhances the efficacy of CFZ and holds translational promise for improving clinical outcomes in patients with MM.

Supplementary Information

The online version contains supplementary material available at 10.1186/s40164-026-00770-8.

To the Editor,

Multiple myeloma (MM) is a malignant proliferative disorder involving terminally differentiated plasma cells [1]. Carfilzomib (CFZ) is the cornerstone of the treatment of newly diagnosed high-risk or relapsed/refractory MM. Nonetheless, poor prognosis due to an inadequate response to CFZ poses a significant challenge, creating an urgent need to investigate novel strategies to enhance its efficacy. Previously, we demonstrated that the H3K36me2 reader ZMYND8 activates CEBPE to inhibit the adaptive unfolded protein response and significantly improves the sensitivity of MM cells to CFZ [2]. Consequently, ZMYND8 agonists may constitute a novel therapeutic approach to potentiate the efficacy of CFZ in MM treatment.

ZMYND8 is reportedly a responsive target gene of all-trans retinoic acid (ATRA) in other disease models [3, 4]. Therefore, we first treated MM cells with ATRA alone or in combination with CFZ for transcriptomic sequencing to validate its downstream effects (Fig. 1A). Differentially expressed gene (DEG) analysis confirmed that ZMYND8 is a core gene exhibiting consistent upregulation across different treatment conditions, especially in the ATRA + CFZ versus CFZ groups (Figs. 1B and S1A). The endoplasmic reticulum (ER) stress response pathway was significantly enriched in the combination treatment group (Fig. 1C). Subsequently, we conducted a dual-luciferase reporter gene assay to determine the most potent retinoid compound that activated ZMYND8 transcription. In addition to ATRA, WYC-209 exhibited superior transcriptional activation of ZMYND8 compared to the other compounds (Fig. 1D). ATRA promoted concentration-dependent upregulation of ZMYND8 and its downstream target gene CEBPE in MM cells (Fig. 1E and F, S1B), and this effect was further potentiated upon combination with CFZ (Figures S1C, S1D). WYC-209 is a newly developed retinoid analog that targets retinoic acid receptors (RARs) (Figure S2A) [5, 6]. Similarly, WYC-209 combined with CFZ synergistically increased ZMYND8 expression (Figure S2B).

Fig. 1.

Retinoic acid treatment significantly upregulates ZMYND8 transcription in MM cells. A Hierarchical clustering heatmap illustrating differentially expressed genes (DEGs) in H929 cells treated with DMSO, ATRA, CFZ, or ATRA plus CFZ (Comb). RNA sequencing was performed on three biologically independent replicates per treatment group. B Volcano plot comparing DEGs between Comb and ATRA-treated groups, highlighting ZMYND8 as significantly upregulated in the Comb group. C Gene Ontology (GO) enrichment analysis of DEGs identified in the Comb vs. CFZ comparison. Bubble size corresponds to gene count, while color intensity reflects the adjusted P-value. D Dual-luciferase reporter assay assessing ZMYND8 transcriptional activity in H929 cells exposed to standard MM therapeutics or various retinoic acid derivatives. E, F Quantitative PCR (E) and Western blot (F) analyses of ZMYND8 and CEBPE expression in H929 and ARP-1 cells following 24-hour ATRA treatment at indicated concentrations. G Schematic representation of the ZMYND8 promoter region depicting the positions of predicted retinoic acid response elements (RAREs, P1-P3) as identified by the JASPAR database (https://jaspar.genereg.net). TSS denotes the transcriptional start site. H CUT&Tag sequencing profiles of H3K27ac enrichment at the ZMYND8 locus in H929 cells treated with DMSO, CFZ (10 nM), ATRA (1 µM), ATRA + CFZ, WYC-209 (1 µM), or WYC-209 + CFZ for 24 h. I ChIP-qPCR analysis of H3K27ac occupancy at the ZMYND8 promoter in H929 cells under the indicated treatments for 24 h. J ChIP-qPCR quantification of H3K27me3, H3K9ac, and H3K4me3 enrichment at the P3 region of the ZMYND8 promoter in treated H929 cells. Fold enrichment was calculated compared to the IgG negative controls in ChIP-qPCR analysis. Data are presented as mean ± SD

To further investigate the mechanism by which ATRA and WYC-209 regulate ZMYND8 transcription, we used the JASPAR database to predict the top-scoring retinoic acid response element (RARE) regions within the ZMYND8 promoter. (Fig. 1G). CUT&Tag analysis revealed that both ATRA and WYC-209 significantly increased H3K27ac deposition at the ZMYND8 promoter in MM cells, with greater accumulation observed upon co-treatment with CFZ (Fig. 1H). This is consistent with the role of ATRA in promoting active promoter conformation [7, 8]. However, ERN1, a crucial ER stress gene downstream of ZMYND8, was not epigenetically regulated by either ATRA or WYC-209 (Figure S3A). ChIP assays confirmed a significant increase in H3K27ac enrichment within the P1-3 regions of the ZMYND8 promoter after treatment with ATRA or WYC-209 (Fig. 1I, S3B). Moreover, we observed that transcriptional activation markers, such as H3K9ac and H3K4me3, were significantly enriched after combination treatment, whereas the enrichment of the transcriptional repressive marker H3K27me3 was diminished (Figs. 1J, S3C). Furthermore, we found that the retinoid-induced switch from H3K27me3 to H3K27ac at the ZMYND8 promoter was primarily mediated by the activation of KDM6A (Figure S4). Taken together, these results demonstrate that ATRA and WYC-209 significantly activate ZMYND8 transcription by remodeling the epigenetic landscape.

We investigated the possible synergistic effect of ATRA and WYC-209 on CFZ, as well as their potential dependence on ZMYND8. The synergistic pro-apoptotic effects of ATRA or WYC-209 combined with CFZ were confirmed in primary cells from patients newly diagnosed with MM (Fig. 2A). Using the CRISPR-Cas9 system, we generated ZMYND8-knockout (ZMYND8-KO) MM cell lines (Figure S5). Combined treatment with ATRA/WYC-209 and CFZ synergistically reduced the viability of MM cells, whereas depletion of ZMYND8 significantly attenuated this synergistic effect (Fig. 2B). WYC-209/ATRA and CFZ exhibited potent synergistic inhibitory effects on MM cells. This synergy was particularly striking at CFZ concentrations ranging from 1 to 10 nM for WYC-209, surpassing that achieved with ATRA (10–30 nM) (Figs. 2C and S6). We observed that WYC-209 enhanced MM cell sensitivity to CFZ more effectively than did ATRA; therefore, we chose WYC-209 for further investigation. Moreover, WYC-209 and CFZ acted synergistically in a ZMYND8-dependent manner to promote cell apoptosis, with increased cleaved Caspase-3 and BAX expression and decreased BCL-2 expression (Figs. 2D and S7). Transmission electron microscopy revealed that co-treatment with WYC-209 and CFZ markedly exacerbated ER dilation and vesiculation relative to treatment with CFZ alone. Conversely, ZMYND8-KO alleviated the ER morphological alterations induced by WYC209 + CFZ proteotoxicity (Fig. 2E).

Fig. 2.

The combination of WYC-209 and CFZ demonstrates potent synergistic effects on MM in a ZMYND8-dependent manner. A Apoptosis rates of primary MM cells derived from patients treated with DMSO, CFZ (10 nM), ATRA (1 µM), ATRA + CFZ, WYC-209 (1 µM), or WYC-209 + CFZ (n = 8) for 48 h. B Cell viability assays following 48-hour treatment with DMSO, CFZ (10 nM), ATRA (1 µM), ATRA + CFZ, WYC-209 (1 µM), or WYC-209 + CFZ in ZMYND8-knockout (ZMYND8-KO) and non-targeting control (sgNC) H929 (left) and ARP-1 (right) cells. C Synergistic interactions between WYC-209 and CFZ in H929 cells were assessed using SynergyFinder. Cells were exposed to varying concentrations of WYC-209 (0–3000 nM) and CFZ (0–300 nM), and viability was determined via CCK-8 assay. The dose-response matrix (left panel) illustrates inhibition ratios across different concentration combinations. Synergistic effects were visualized using heatmaps (middle and right panels), with a ZIP synergy score > 10 indicating strong synergy. The white dashed rectangle highlights the concentration range exhibiting maximal synergy. D Apoptosis rates in ZMYND8-KO and sgNC H929 cells after 48-hour treatment with DMSO, CFZ (10 nM), ATRA (1 µM), ATRA + CFZ, WYC-209 (1 µM), or WYC-209 + CFZ. E Transmission electron microscopy analysis of endoplasmic reticulum (ER) ultrastructure in ZMYND8-KO and sgNC H929 cells following 48-hour treatment with DMSO, CFZ, or WYC-209 + CFZ. Red arrows denote representative ER structures. Scale bars: 0.5 μm. F Schematic representation of the experimental timeline for tumor cell inoculation and drug administration in NSG mice. G Relative ZMYND8 mRNA expression in CD138 + MM cells isolated from murine bone marrow. H Kaplan-Meier curves of overall survival for tumor-bearing mice across treatment groups (n = 6 per group). P value is for the comparison of the Comb group with the Comb+sgZMYND8 group or with the CFZ group. I Serum levels of human immunoglobulin light chain measured by ELISA on day 30 after tumor injection. J Proportion of MM cell infiltration in murine bone marrow across experimental groups. Data are presented as mean ± SD

To evaluate the synergistic therapeutic effect of WYC-209 and CFZ on MM progression in vivo, we established a xenograft model in NSG mice by intravenously injecting H929 cells stably expressing sgNC and sgZMYND8 (Fig. 2F). WYC-209 and CFZ combination therapy had no obvious effects on body weight, blood cell counts, or hepatotoxicity, and no severe adverse effects were observed (Figure S8 and Table S1). However, WYC-209 treatment significantly upregulated ZMYND8 expression in isolated MM cells, and the effect was enhanced by CFZ co-administration (Fig. 2G). The combination of WYC-209 and CFZ robustly improved mouse survival (Fig. 2H), reduced tumor burden (Fig. 2I), and diminished MM cell infiltration in the bone marrow (Fig. 2J). These synergistic therapeutic effects were inhibited upon ZMYND8 depletion. Collectively, these findings indicated that WYC-209 works synergistically with CFZ by activating ZMYND8 to suppress MM tumor growth.

ATRA is a well-known first-line drug for the treatment of acute promyelocytic leukemia that induces cell differentiation [9]. Despite being well tolerated, ATRA monotherapy has demonstrated limited clinical activity against MM [10]. Current research on MM has largely focused on the combined use of ATRA and other therapeutic agents. Gu et al. previously showed that the induction of differentiation by ATRA can increase the efficacy of bortezomib in MM cells, but the underlying synergistic effect remains unclear [11]. A recent study indicated that ATRA sensitizes myeloma cells to CFZ treatment through activation of the RARα-IRF1-OAS1 pathway [12]. Our data demonstrated that retinoic acid efficiently upregulates the expression of ZMYND8, which is essential for augmenting CFZ-induced MM killing. This synergy could be attributed to CFZ exacerbating the reliance of MM cells on ER stress due to proteasome inhibition, while retinoids exploit this dependency by transcriptionally activating ZMYND8 to suppress the adaptive unfolded protein response, leading to additive cell death. Our findings provide a new basis for the combined use of CFZ with retinoic acids in clinical practice. Furthermore, we identified WYC-209, a novel synthetic retinoid analog, as a more effective partner than ATRA for CFZ, not only yielding greater synergistic antimyeloma activity but also exhibiting stronger dependence on ZMYND8.

In conclusion, ZMYND8 plays a pivotal role in mediating the sensitization of MM cells to CFZ with retinoic acid. We further propose a combination strategy using a novel ZMYND8 agonist, WYC-209, to augment CFZ efficacy, which offers therapeutic potential for high-risk or relapsed MM patients.

Abbreviations

MM

Multiple myeloma

ZMYND8

Zinc-finger MYND-type-containing 8

ATRA

All-trans retinoic acid

CFZ

Carfilzomib

ChIP

Chromatin immunoprecipitation

ZIP

Zero interaction potency

DEG

Differentially expressed gene

ER

Endoplasmic reticulum

RAR

Retinoic acid receptor

RARE

Retinoic acid response element

Author contributions

JX performed the research and wrote the manuscript. JY, HZ, JD, YW, and JQ assisted with experiments and data analysis. QZ, XD, and BC supervised the project. All authors have read and approved the final version of this manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (82504899, 82304987, and 82273954) and Basic Research Program of Jiangsu Province (BK20250773).

Data availability

Original data used and/or analyzed during the current study are available from the corresponding authors on reasonable request.

Declarations

Ethics approval and consent to participate

All animal experiments were approved by the Animal Ethical and Welfare Committee of Nanjing University (IACUC-2302005).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.