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Activity and rational combinations of a novel, engineered chimeric, TRAIL-based ligand in diffuse large B-cell lymphoma

ABSTRACT: Background: TRAIL (TNF-related apoptosis inducing ligand) exhibits selective proapoptotic activity in multiple tumor types, while sparing normal cells. This selectivity makes TRAIL an attractive therapeutic candidate for cancer patients. However, despite encouraging activity in preclinical models, clinical trials with TRAIL mimetics/death receptor agonists demonstrated insufficient activity, largely due to emerging resistance to these agents. Herein, we investigated the cytotoxic activity of a novel, TRAIL-based chimeric protein AD-051.4 combining TRAIL and VEGFA-derived peptide sequences, in hematological malignancies. We characterize key molecular mechanisms leading to resistance and propose rational pharmacological combinations sensitizing cells to AD-051.4. Methods: Sensitivity of DLBCL, classical Hodgkin lymphoma, (cHL), Burkitt lymphoma (BL) and acute myeloid leukemia (AML) to AD-051.4 was assessed in vitro with MTS assay and apoptosis tests (Annexin V/PI staining). Markers of apoptosis were assessed using immunoblotting, flow cytometry, or fluorogenic caspase cleavage assays. Resistant cell lines were obtained by incubation with increasing doses of AD-O51. Transcriptomic analyses were performed by RNA sequencing. Sensitizing effects of selected pathway modulators (BCL2, dynamin and HDAC inhibitors) were assessed using MTS/apoptosis assays. Results: AD-051.4 exhibited low-nanomolar cytotoxic activity in DLBCL cells, but not in other lymphoid or AML cell lines. AD-051.4 induced death-receptor (DR) mediated, caspase-dependent apoptosis in sensitive DLBCL cells, but not in primary resistant cells. The presence of DRs and caspase 8 in cancer cells was crucial for AD-O51.4-driven apoptosis. To understand the potential mechanisms of resistance in an unbiased way, we engineered AD-051.4-resistant cells and evaluated resistance-associated transcriptomic changes. Resistant cells exhibited changes in the expression of multiple genes and pathways associated with apoptosis, endocytosis and HDAC-dependent epigenetic reprogramming, suggesting potential therapeutic strategies of sensitization to AD-051.4. In subsequent analyses, we demonstrated that HDAC inhibitors, BCL2 inhibitors and endocytosis/dynamin inhibitors sensitized primary resistant DLBCL cells to AD-051.4 Conclusions: Taken together, we identified rational pharmacologic strategies sensitizing cells to AD-051.4, including BCL2, histone deacetylase inhibitors and dynamin modulators. Since AD-051.4 exhibits favorable pharmacokinetics and an acceptable safety profile, further clinical development is expected. Identification of resistance mechanisms in a clinical setting might indicate a personalized pharmacological approach to override the resistance.

ORGANISM(S): Homo sapiens

PROVIDER: GSE208543 | GEO | 2022/10/18

REPOSITORIES: GEO

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Project description:Senescent cells secrete a plethora of factors with potent paracrine signaling capacity. Strikingly, senescence, which acts as a defense against cell transformation, exerts pro-tumorigenic activities through its secretome by promoting numerous tumor-specific features, such as cellular proliferation, epithelial-mesenchymal transition and invasiveness. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has the unique activity of activating cell death exclusively in tumor cells. Given that the senescence-associated secretome supports cell transformation, we asked whether factor(s) of this secretome would establish a program required for the acquisition of TRAIL sensitivity. We found that conditioned media from several types of senescent cells (CMS) efficiently sensitized pre-transformed cells to TRAIL, while the same was not observed with normal or immortalized cells. Dynamic transcription profiling analysis of CMS-exposed pre-transformed cells revealed paracrine autoregulatory loop of senescence-associated secretome factors and a dominant role of CMS-induced MYC. Sensitization to TRAIL coincided with MYC upregulation and massive changes in gene regulation. CMS-induced MYC silenced its target gene CFLAR, encoding the apoptosis inhibitor FLIPL, thus leading to the acquisition of TRAIL sensitivity. Altogether, our results reveal that senescent cell-secreted factors exert a TRAIL sensitizing effect on pre-transformed cells by modulating the expression of MYC and CFLAR. Notably, CMS dose-dependent sensitization to TRAIL was observed with TRAIL-insensitive cancer cells and confirmed in co-culture experiments. Dissection and characterization of TRAIL-sensitizing CMS factors and the associated signaling pathway(s) may provide a mechanistic insight in the acquisition of TRAIL sensitivity and lead to novel concepts for the apoptogenic therapy of pre-malignant and TRAIL-resistant tumors. Pre-transformed BJEL cells were incubated with CMS for 0, 1, 3, 6, 8, 16 or 24 h respectively. Total RNA has been extracted from each time point and used for gene expression analysis (Affymetrix Human Gene 1.0 ST Arrays).

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Activity and rational combinations of a novel, engineered chimeric, TRAIL-based ligand in diffuse large B-cell lymphoma

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