Laboratory of Tumor Cell Genetics

The project is relevant for basic science and for biomedical applications. The most effective approaches in the treatment of oncological diseases are based on simultaneous exposure to several targets or signaling pathways of the cell at once. This approach to multi-target therapy is explained by the large number of molecular damage to the tumor cell, as well as the heterogeneity of the tumor. Multi-target therapy can be implemented through exposure to the main target of the cell, DNA. Therefore, there is currently interest in developing drugs aimed at damaging DNA functions in tumor cells. Studies of non-covalent interactions of small molecules with a narrow groove of DNA remain an urgent area of search for new potential therapeutic compounds. However, DNA-damaging agents have a serious limitation in clinical use due to their genotoxicity, due to the risk of secondary malignant tumors. Therefore, low molecular weight compounds are considered the most promising, capable of non-covalently and site-specifically binding to the narrow groove of double-stranded DNA (DNA) - narrow-stranded ligands, without causing damage to it and, as a result, inhibit transcription, modulate signaling pathways, and thus lead to the death of a tumor cell. The novelty of the project is to study the biological, including antitumor activity of unique fluorescent monomeric and dimeric bisbenzimidazole pyrroles of the new DB2Py(n) series. The 15-stage synthesis of the DB2Py(n) series will be finalized and published. It is proposed to expand the number of new compounds within the series to select the most active compound with the optimal linker size (n), and to synthesize new bisbenzimidazoles DB2Py(n). This will make it possible to more accurately select a biologically active molecule. The DB2Py(n) leader compounds will be synthesized in preparative amounts. The general methodology of this study of the biological activity of compounds is to study in vivo and in vitro: in cell-free and cellular models, as well as in laboratory animals. We believe that this approach will make it possible to more fully characterize new compounds and assess their prospects. It is proposed to investigate the antiproliferative properties of new compounds both on a panel of various human tumor lines and on primary ones obtained from surgical material of patients diagnosed with glioblastoma, to study the interactions of new compounds with DNA, DNA and RNA-dependent enzymes (topoisomerases I and II, DNA polymerase, revertase). Small molecules bind to the chromatin of the cell, altering the conformation of DNA and destabilizing the nucleosomes. It is proposed to study the effect of new substances on the histone chaperone FACT in the cell and, in particular, on the SSRP1 and SPT16 subunits and answer the question “Is there a direct correlation between cytotoxicity and inhibition of FACT?". A separate block of experiments will allow us to study mutagenicity, genotoxicity, and antitumor activity in vivo. The results obtained will allow us to evaluate the potential prospects of using new monomeric and dimeric bisbenzimidazole-pyrroles as antitumor drugs.

Multidrug resistance (MDR) continues to be a serious barrier to successful chemotherapy of tumors, including multiple myeloma (MM). P-glycoprotein (P-gp, ABCB1) is one of the key proteins responsible for the formation of MDR tumor cells. P-gp is expressed on the cell surface, releasing various compounds, including anticancer drugs. It was previously demonstrated, both by foreign authors and in our laboratory, that the second-generation proteasome inhibitor carfilzomib (cfz), used in the treatment of refractory MM, leads to an increase in P-gp expression. Our results showed that cfz interacts with P-gp, and also that treatment of resistant MM cells with a P-gp inhibitor sensitizes them to the action of cfz. However, there is a known problem that none of the developed P-gp inhibitors has found its place in clinical practice either due to inefficiency or due to serious toxicity. Then one of the options for reducing P-gp expression in cells may be the inhibition of signaling pathways leading to its activation. Here it is possible to repurpose low molecular weight kinase inhibitors already used in the clinic. However, the molecular mechanisms and signaling pathways that lead to an increase in P-gp expression in carfilzomib-resistant MM cells have not been sufficiently investigated. We plan to dedicate our project to solving this scientific problem. The project involves separating the P-gp-negative and P-gp-positive subpopulation of 3 cell lines with different resistance to carfilzomib using a cell sorter: AMO-1, AMO-1/CFZ 1 (10-fold resistance), AMO-1/CFZ 2 (100-fold resistance). Next, these 6 subpopulations, as well as bortezomib-resistant AMO-1/BTZ and ixazobime AMO-1/IXZ, will be sent for RNA sequencing. Based on the results of sequencing, followed by verification of the results using real-time PCR and Western blot, 1-2 signaling pathways will be identified that are most likely to lead to overexpression of P-glycoprotein. At the last stage, experiments will be conducted to suppress the activity of these pathways and evaluate the expression of P-glycoprotein and cell sensitivity to carfilzomib. A search will be conducted for drugs that inhibit these pathways and are already used in clinical practice in order to be able to repurpose their use in the future.