Epigenetic reprogramming by organic arsenic CZ2 elicits potent antitumor responses in DLBCL through TMS1 restoration.

[OBJECTIVE] Diffuse large B-cell lymphoma (DLBCL) presents a clinical challenge due to its high recurrence risk and therapeutic resistance driven by epigenetic factors. To address this gap, we developed CZ2 (N-(4-(1,3,2-dithiarsinan-2-yl)phenyl)-3-(benzo[d][1,3]dioxol-5-yl)acrylamide), a novel organic arsenical compound, and explored its therapeutic efficacy through a multimodal mechanistic approach in DLBCL.

[MATERIALS AND METHODS] The anti-DLBCL activity of CZ2 was systematically evaluated using multiple approaches. In vitro cytotoxicity was assessed using CCK-8 assays on DLBCL cell lines (SUDHL-4, SUDHL-6) and the crucial control of normal human peripheral blood lymphocytes (PBMCs). Apoptosis was quantified via Annexin V-FITC/PI staining and flow cytometry. Mitochondrial function was assessed using the JC-1 probe for membrane potential and Caspase-Glo® 3/7/9 assays. Epigenetic modulation was studied through RT-PCR and Western blot analysis of TMS1 and DNMTs, along with methylation-specific PCR (MSP) and ChIP-qPCR validation. Finally, in vivo validation was conducted using the SUDHL-4 xenograft model.

[RESULTS] CZ2 significantly reduces the survival of DLBCL cell lines in a dose- and time-dependent manner, exhibiting a superior potency with an IC₅₀ value of 0.67 ± 0.08 μM-approximately 3.3-fold lower than that of arsenic trioxide (ATO, 2.21 ± 0.15 μM). Crucially, CZ2 showed a favorable safety profile. Mechanistically, CZ2 effectively induces apoptosis in DLBCL cells by disrupting mitochondrial integrity and activating the intrinsic caspase cascade (3/7/9). Further investigation demonstrated that CZ2 inhibits the enzymatic activity of DNMT1, DNMT3A, and DNMT3B, leading directly to the demethylation and subsequent restoration of the TMS1 tumor suppressor gene expression. In vivo experiments confirmed CZ2's potent anti-tumor efficacy, significantly inhibiting tumor growth, reducing tumor weight, and improving survival in xenograft models.

[CONCLUSION] Our study establishes that CZ2, a novel organic arsenical, exhibits superior therapeutic efficacy and high selectivity in DLBCL by targeting DNMT. Our findings suggest that CZ2 represents a promising strategy to overcome epigenetic-mediated resistance, offering a potential therapeutic intervention for relapsed/refractory DLBCL and supporting its further development in rational combination regimens.