Résumé
The monoclonal antibody (mAb) Daratumumab (Dara) targets CD38 to trigger cytotoxicity against myeloma cells. Dara achieves good response rates in Multiple Myeloma (MM) patients and has been approved as a first line therapy. However, relapse due to downregulation of CD38 in myeloma cells is frequently observed (Saltarella et al. Cells 2020). Identification of regulators of CD38 expression such as EZH2 (Chemlal et al. Leukemia 2023), KDM6A (Liu et al. Nat Comms 2024) and RARα, product of the gene RARA (Nijhof et al. Leukemia 2015), provided rationale for combination therapies that could help overcome Dara-resistance. A clinical trial evaluated the combination of Dara with All-Transretinoic Acid (ATRA), a RARα agonist, showing limited improvement in Dara-resistant patients (Frerichs et al. 2021 Blood Adv).
Here, we performed a genome-wide CRISPR-Cas9 knock-out (CRISPR-ko) screen to identify novel CD38 regulators in myeloma cells. Counterintuitively, RARA deletion led to an increase in CD38, which can be explained by the fact that RARα is a ligand-dependent transcription factor. In absence of a ligand, RARα represses transcription, whereas RARα bound to ATRA activates transcription (Géhin et al. Chem Biol 1999), including CD38 gene. Therefore, RARα inhibition could also be used to reinduce CD38 expression in Dara-resistant myeloma cells. Currently, a new potent and selective antagonist of RARα, YCT-529, is under evaluation as male contraception as it reversibly disrupts spermatogenesis by blocking access to vitamin A in the testes (Mannowetz et al. Commun Med 2025). The results of the Phase I clinical trial indicate good tolerability to YCT-529 with no serious adverse effects (NCT06094283). Thus, our study aims to evaluate the therapeutic interest of combining YCT-529 with Dara to improve cytotoxicity and revert resistance to anti-CD38 mAb.
JJN3-Cas9 inducible human MM cell line (HMCL) expressing very low levels of CD38 was transduced with the Brunello CRISPR-ko sgRNA library. After 2 weeks, CD38+/- cells were purified by FACS sorting and sequenced to identify the genes related to CD38 upregulation. The effect of RARα antagonist YCT-529 and agonist ATRA on CD38 expression was compared in 3 HMCLs and primary samples from 6 MM patients by western blot and/or flow cytometry. Antibody-dependent cellular cytotoxicity (ADCC) in vitro assay was conducted using HMCL treated with YCT-529 and ATRA in presence or not of anti-CD38 mAb.
Genome wide CRISPR-ko screen using HMCL JJN3 identified RARA as the CD38 negative regulator with the strongest CRISPR score. In 3 HMCLs, treatment with YCT-529 (RARα antagonist) and ATRA (RARα agonist) at nM concentrations significantly increased CD38 RNA and protein levels, without significant cellular toxicity or effect on cell proliferation. Elevated CD38 levels were confirmed by flow cytometry and western blotting. In 5 of 6 primary samples from MM patients, both RARα agonist and antagonist moderately increased CD38 levels in myeloma cells. In addition, 3 HMCLs were treated with YCT-529 or ATRA for 48 h, drugs were washed away and cells were put back in culture with fresh medium for up to 8 days. Quantification by flow cytometry showed that treatment-induced increase in CD38 protein expression rapidly decreased in ATRA-treated cells, whereas it remained elevated in YCT-529-treated cells. Pretreatment of HMCLs with YCT-529 and ATRA increased CD38 levels, which correlated with enhanced cytotoxicity capacity of anti-CD38 mAb in in vitro ADCC assays with NK lymphocytes.
Our preliminary results show that both RARα activator ATRA and inhibitor YCT-529 increase CD38 expression in myeloma cells at transcriptional and protein levels. Furthermore, CD38 induction seems more stable in time in YCT-529-treated cells than in ATRA-treated ones. More analyses are ongoing to dissect the epigenetic mechanisms that control CD38 expression in myeloma cells through RARα. In addition, the elevated levels of CD38 upon RARα activation/inhibition correlate with better anti-CD38 mAb cytotoxic activity in vitro, validating previous work on ATRA and showing for the first time a potential anti-myeloma role for YCT-529. Given the encouraging results of low toxicity and side effects of the Phase I clinical trial on YCT-529, further pre-clinical tests should be performed in order to evaluate the therapeutic interest of its combination with anti-CD38 mAb, especially in Dara-resistant myeloma.