ATP-binding cassette (ABC) transporters, such as breast cancer resistance protein (BCRP), are key players in resistance to multiple anti-cancer drugs, leading to cancer treatment failure and cancer-related death. Currently, there are no clinically approved drugs for reversal of cancer drug resistance caused by ABC transporters. This study investigated if a novel drug candidate, SCO-201, could inhibit BCRP and reverse BCRP-mediated drug resistance. We applied in vitro cell viability assays in SN-38 (7-Ethyl-10-hydroxycamptothecin)-resistant colon cancer cells and in non-cancer cells with ectopic expression of BCRP. SCO-201 reversed resistance to SN-38 (active metabolite of irinotecan) in both model systems. Dye efflux assays, bidirectional transport assays, and ATPase assays demonstrated that SCO-201 inhibits BCRP. In silico interaction analyses supported the ATPase assay data and suggest that SCO-201 competes with SN-38 for the BCRP drug-binding site. To analyze for inhibition of other transporters or cytochrome P450 (CYP) enzymes, we performed enzyme and transporter assays by in vitro drug metabolism and pharmacokinetics studies, which demonstrated that SCO-201 selectively inhibited BCRP and neither inhibited nor induced CYPs. We conclude that SCO-201 is a specific, potent, and potentially non-toxic drug candidate for the reversal of BCRP-mediated resistance in cancer cells.

The calcium binding protein ALG-2 is upregulated in several types of cancerous tissues and cancer cell death may be a consequence of ALG-2 downregulation. Novel research suggests that ALG-2 is involved in membrane repair mechanisms, in line with several published studies linking ALG-2 to processes of membrane remodeling and transport, which may contribute to the fitness of cells or protect them from damage. To investigate the involvement of ALG-2 in cell recovery after membrane damage we disrupted the PDCD6 gene encoding the ALG-2 protein in DT-40 cells and exposed them to electroporation. ALG-2 knock-out cells were more sensitive to electroporation as compared to wild type cells. This phenotype could be reversed by reestablishing ALG-2 expression confirming that ALG-2 plays an important role in cell recovery after plasma membrane damage. We found that overexpression of wild type ALG-2 but not a mutated form unable to bind Ca2+ partially protected HeLa cells from digitonin-induced cell death. Further, we were able to inhibit the cell protective function of ALG-2 after digitonin treatment by adding a peptide with the ALG-2 binding sequence of ALIX, which has been proposed to serve as the ALG-2 downstream target in a number of processes including cell membrane repair. Our results suggest that ALG-2 may serve as a novel therapeutic target in combination with membrane damaging interventions.

Introduction: Annually, more than 1 million people are diagnosed with Colorectal Cancer (CRC) and more than 500.000 die from CRC. Only three types of chemotherapy are currently being used worldwide to treat CRC patients: 5FU, oxaliplatin and irinotecan. However, chemotherapy resistance is present or develops in the majority of metastatic CRC patients. Resistance to anti-cancer drugs represents the main cause of cancer-related deaths. Thus, re-sensitization of chemotherapy resistant cancer cells constitutes a highly unmet medical need. Materials and Methods: The drug, SCO-101, is an oral drug and has previously passed 4 Phase I clinical trials where it demonstrated excellent PK (T1/2=15 hours) and favourable safety profile. We used two pairs of parental (sensitive) and SN38-resistant (SN38 is the active metabilote of irinotecan) human colon cancer cell lines (HT29 and LoVo). Cell viability in vitro during 72h exposure to SCO-101 and/or SN38 was adressed by MTT assays. The level of the SN38 efflux transpoter, ABCG2/BCRP, was investigated in the presence or absence of SCO-101. Additionally, various flux assays were applied to investigate the effect of SCO-101 on ABCG2/BCRP activity. A kinase screen revealed that the kinase SRPK1 was inhibited by SCO-101 and the importance of SRPK1 activity in re-sensitization of SN38 resistant colon cancer cells was investigated by two different synthetic SRPK1 inhbitors. Results: In parental HT29 and LoVo cells, SCO-101 and SN38 each had a dose-dependent effect on cell viability and no combinational effects were observed between the drugs. When exposing SN38-resistant cells to the combination of SCO-101 and SN38, a significant inhibitory effect on cell viability compared to either drug alone was observed, suggesting an additive or synergistic effect between these two drugs with SCO-101 re-sensitizing the resistant cells to SN38. Flux assays with ABCG2 /BCRP substrates clearly demonstrated that SCO-101 inhibited the activity of ABCG2 and protein analysis further demonstrated that SCO-101 causes degradation of ABCG2/BCRP. Cell viability assays with two different SRPK1 inhibitors demonstrated that this kinase is involved in re-sensitization to SN38. Conclusion and future perspectives: These preclinical studies demonstrate that SCO-101 sensitizes SN38-resistant colon cancer cells to SN38 through two different mechanisms of actions e.g. inhibition of the SRPK1 kinase and degradation of the ABCG2/BCRP drug efflux pump. We are now preparing a clinical phase II study enrolling metastatic CRC patients, who have developed acquired resistance to irinotecan containing treatment. These patients will be re-exposed to the combination of irinotecan containing treatment and SCO-101. Primary end-points will be safety and objective response rate according to RECIST 1.1. Secondary end-points will be clinical benefit rate. The study has been approved by the Danish Medicines Agency. In conclusion, SCO-101 represents a unique drug with an innovative dual mechanism of action. Citation Format: Jan Stenvang, Sophie Ambjørner, Khwajanezrabodin Sedighi, Peter Michael Vestlev, Lasse Saaby, Birger Brodin, Nils Brunner. Re-sensitization of irinotecan/SN38 resistant colorectal cancer cells by SCO-101 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6340.