Magdalena Strachowska

The adhesion G protein-coupled receptor F5 (ADGRF5) has been implicated in modulating immune responses in cancer; however, its role in inflammatory bowel diseases (IBDs), particularly colitis, remains largely unexplored. In this study, we aimed to design and characterize novel peptide agonists derived from the ADGRF5 Stachel sequence, as well as to evaluate their therapeutic potential in preclinical colitis models. In silico analysis and single amino acid substitutions within the ADGRF5 tethered agonist sequence, combined with functional assays in ADGRF5-overexpressing cells, including calcium mobilization and inositol phosphate production, were employed to assess the activity of novel ADGRF5 agonists. Western blot technique and murine model of colitis were used to evaluate downstream signaling pathways and immunomodulatory effects of ADGRF5 ligands. We identified a series of peptides exhibiting significantly enhanced ADGRF5 agonist activity, achieving up to a 6-fold increase in potency over the wild-type version. We identified critical substitutions within the Stachel sequence, namely S11N and D13S, as essential for improving agonistic activity. Finally, using these novel ADGRF5 agonists, we demonstrated their potent anti-inflammatory effects in vivo, showing that ADGRF5 activation ameliorates experimental colitis, as evidenced by reduced macroscopic damage scores and improved colon architecture. These findings establish ADGRF5 as a potential therapeutic target for colitis and highlight the promise of Stachel-derived peptide agonists for the development of novel anti-inflammatory therapies.

Multidrug resistance of cancer cells is attributed to drug-induced alteration of numerous intracellular processes. Using clinically relevant models of triple-negative breast and non-small lung cancer cells we previously showed that these cells respond to repeated paclitaxel exposure by inter alia lysosome enrichment in ABCC3, ABCC5 and ABCC10, which contribute to drug sequestration in these organelles and reduced drug cytotoxicity. In this study we provide experimental evidence that transcription of above mentioned ABCC genes is enabled by BRG1-based SWI/SNF chromatin remodeling complex. Pharmacological inhibition of SWI/SNF with PFI3 or ACBI1, the PROTAC degrader of SMARCA2/4, substantially decline transcription of ABCC3, ABCC5 and ABCC10. Similar effect is caused by transient silencing of SMARCA4 (BRG1), but not SMARCA2 (BRM). The deficiency of BRG1 led to extralysosomal distribution of anticancer drugs, their deeper penetration of spheroids and substantial increase in drug cytotoxicity. Interestingly, in BRG1 deficient cell line paclitaxel triggered mutations, which reverted BRG1 truncating deletion in SMARCA4, thereby restoring SWI/SNF ATPase expression in paclitaxel-resistant cells and increasing transcription of ABCC. Acquisition of drug resistance was associated with BRG1 redistribution in the genome, de novo occurrence at the promoters of genes functionally linked to endo-lysosomal system and stronger co-occurrence with EP300. Our study indicates possible target - SWI/SNF complex for anticancer combinatorial interventions in paclitaxel-induced multidrug resistant phenotypes.

Although cisplatin-based therapies are common among anticancer approaches, they are often associated with the development of cancer drug resistance. This phenomenon is, among others, caused by the overexpression of ATP-binding cassette, membrane-anchored transporters (ABC proteins), which utilize ATP to remove, e.g., chemotherapeutics from intracellular compartments. To test the possible molecular basis of increased expression of ABCC subfamily members in a cisplatin therapy mimicking model, we generated two cisplatin-resistant cell lines derived from non-small cell lung cancer cells (A549) and triple-negative breast cancer cells (MDA-MB-231). Analysis of data for A549 cells deposited in UCSC Genome Browser provided evidence on the negative interdependence between the occurrence of the CoREST complex at the gene promoters and the overexpression of ABCC genes in cisplatin-resistant lung cancer cells. Pharmacological inhibition of CoREST enzymatic subunits—LSD1 and HDACs—restored gene responsiveness to cisplatin. Overexpression of CoREST-free ABCC10 in cisplatin-resistant phenotypes was caused by the activity of EP300 that was enriched at the ABCC10 promoter in drug-treated cells. Cisplatin-induced and EP300-dependent transcriptional activation of ABCC10 was only possible in the presence of p53. In summary, the CoREST complex prevents the overexpression of some multidrug resistance proteins from the ABCC subfamily in cancer cells exposed to cisplatin. p53-mediated activation of some ABCC genes by EP300 occurs once their promoters are devoid of the CoREST complex.

The increased level of hydrogen peroxide accompanies some modes of macrophage specification and is linked to ROS-based antimicrobial activity of these phagocytes. In this study, we show that activation of toll-like receptors with bacterial components such as LPS is accompanied by the decline in transcription of hydrogen peroxide decomposing enzyme-catalase, suppression of which facilitates the polarization of human macrophages towards the pro-inflammatory phenotype. The chromatin remodeling at the CAT promoter involves LSD1 and HDAC1, but activity of the first enzyme defines abundance of the two proteins on chromatin, histone acetylation status and the CAT transcription. LSD1 inhibition prior to macrophage activation with LPS prevents CAT repression by enhancing the LSD1 and interfering with the HDAC1 recruitment to the gene promoter. The maintenance of catalase level with LSD1 inhibitors during M1 polarization considerably limits LPS-triggered expression of some pro-inflammatory cytokines and markers such as IL1β, TNFα, COX2, CD14, TLR2, and IFNAR, but the effect of LSD1 inhibitors is lost upon catalase deficiency. Summarizing, activity of LSD1 allows for the CAT repression in LPS stimulated macrophages, which negatively controls expression of some key pro-inflammatory markers. LSD1 inhibitors can be considered as possible immunosuppressive drugs capable of limiting macrophage M1 specialization.

The high expression of some ATP-binding cassette (ABC) transporters is linked to multidrug resistance in cancer cells. We aimed to determine if I-CBP112, which is a CBP/p300 bromodomain inhibitor, altered the vulnerability of the MDA-MB-231 cell line to chemotherapy drugs, which are used in neoadjuvant therapy in patients with triple negative breast cancer (TNBC). MDA-MB-231 cells represent TNBC, which is negative for the expression of estrogen and progesterone receptors and HER2 protein. An I-CBP112-induced decrease in the expression of all the studied ABCs in the breast, but also in the lung (A549), and hepatic (HepG2) cancer cell lines was associated with increased accumulation of doxorubicin, daunorubicin, and methotrexate inside the cells as well as with considerable cell sensitization to a wide range of chemotherapeutics. Gene promoters repressed by I-CBP112 in MDA-MB-231 cells, such as ABCC1 and ABCC10, were characterized by enhanced nucleosome acetylation and, simultaneously, by considerably lower trimethylation in the transcription-promoting form of H3K4me3. The CBP/p300 bromodomain inhibitor induced the recruitment of LSD1 to the gene promoters. The inhibition of this demethylase in the presence of I-CBP112 prevented the repression of ABCC1 and ABCC10 and, to a considerable extent, cancer cells’ sensitization to drugs. In conclusion, the CBP/p300 bromodomain inhibitor I-CBP112 can be considered as a potent anti-multidrug-resistance agent, capable of repressing key ABC transporters responsible for drug efflux in various cancer types.