Embryonic ectoderm development (EED) inhibitor APG-5918 overcomes immunomodulatory drug (IMiD) resistance as monotherapy and synergizes with IMiDs/cereblon E3 ligase modulators (CELMoDs) in preclinical models of multiple myeloma (MM) Free

Introduction IMiDs (eg, lenalidomide, pomalidomide) are approved for the treatment of patients with MM and other hematologic malignancies. IMiDs bind cereblon (CRBN), the CRL4^CRBNE3 ubiquitin ligase complex substrate receptor, and promote ubiquitination and degradation of neo-substrates such as B-cell transcription factors Ikaros (IKZF1) and Aiolos (IKZF3), which suppresses key MM cell survival factor IRF4. CELMoDs, next-generation CRBN-targeting agents, are promising for IMiD-refractory MM but prone to acquired resistance, warranting rational combination regimens. Aberrant activation of the EZH2 PRC2 catalytic subunit, which mediates transcriptional repression by catalyzing H3K27 trimethylation (H3K27me3), contributes to cancer progression and resistance. APG-5918, a potent investigational inhibitor of EED (the core scaffolding subunit of PRC2 essential for maintaining its methyltransferase activity), disrupts PRC2 function, reduces H3K27me3, reactivates tumor suppressor genes, and reverses epigenetic drug resistance. We assessed antitumor activity and mechanisms of APG-5918 (± IMiDs or CELMoDs) in preclinical MM models.

Methods A genetically diverse panel of MM cell lines, including IMiD-resistant (KMS11, MOLP-8, U266, RPMI-8226) and -sensitive (AMO-1, OPM-2, MM.1S) lines, were used to assess in vitro antiproliferative activity of APG-5918 (± pomalidomide or CELMoD CC-92480). Cell viability was measured by CellTiter-Glo®assay, apoptosis by annexin V-FITC assay and flow cytometry, and western blotting to analyze protein expression. A subcutaneous KMS-11 CDX model was used to assess in vivo antitumor efficacy.

Results MM.1S, OPM-2, and AMO-1 MM cell lines were IMiD sensitive, with low IC₅₀ values for pomalidomide (51.11, 63.59, and 251.7 nM, respectively), while KMS-11, MOLP-8, RPMI-8226, and U266 cells were IMiD resistant (IC₅₀ > 10,000 nM for pomalidomide or lenalidomide). APG-5918 showed potent antiproliferative activity across most IMiD-sensitive and -resistant cell lines, with low nanomolar to submicromolar IC₅₀ values, indicating efficacy regardless of IMiD sensitivity and superior antiproliferative effects vs. EZH2 inhibitor tazemetostat. Combined with pomalidomide or CC-92480, APG-5918 synergistically inhibited proliferation in all 7 (IMiD-sensitive and -resistant) lines, and concomitant treatment with pomalidomide further synergistically induced apoptosis in IMiD-resistant KMS-11 and RPMI-8226 lines.

In the KMS-11 CDX model, pomalidomide (10/20 mg/kg) or CC-92480 (1 mg/kg) was administered for 28 days and exerted no or minimal antitumor activity (T/C: 132.95% and 73.65%, respectively); and APG-5918 (100 mg/kg) showed modest activity (T/C: 68.18%). However, APG-5918 plus pomalidomide (T/C: 37.28%; synergy index:2.43) or, more strikingly, plus CC-92480 (T/C: 0.67%; synergy index: 75.31) produced marked tumor regression, indicating strong in vivo synergy. No significant body weight changes were observed between treatment and vehicle groups, suggesting a favorable safety profile.

Mechanistically, APG-5918 downregulated core PRC2 components (EED, EZH2, SUZ12) and H3K27me3 in IMiD-resistant lines (KMS-11, RPMI-8226), confirming target engagement; and upregulated BIM, supporting its role in activating the intrinsic apoptotic pathway. Despite functional CRBN (pomalidomide-induced degradation of IKZF1/IKZF3), no significant suppression of IRF4 expression was observed, suggesting a decoupling of IKZF1/IKZF3-mediated regulation of IRF4 in the IMiD resistant setting. APG-5918 plus pomalidomide restored IRF4 downregulation, suggesting reversal of IMiD resistance. The combination also reduced pRb and CDK4 expression and upregulated cleaved PARP-1 and caspase-3, indicating synergistic induction of apoptosis and cell cycle arrest.

Conclusions APG-5918 overcomes IMiD resistance in preclinical MM models, demonstrating in vitro efficacy as a single agent, and both in vitro and in vivo synergy when combined with IMiDs or CELMoDs. Mechanistically, APG-5918 disrupts the PRC2 complex and activates BIM-mediated apoptosis. Combined with pomalidomide, it restores IMiD signaling via IRF4 suppression and reinforces cell cycle arrest by downregulating CDK4/pRb, resulting in synergistic antitumor activity. These findings support clinical development of APG-5918-based regimens for IMiD-resistant MM.