Multiple Myeloma Cells with Increased Proteasomal and ER Stress Are Hypersensitive to ATX-101, an Experimental Peptide Drug Targeting PCNA

Camilla Olaisen; Lisa Marie Røst; Animesh Sharma; Caroline Krogh Søgaard; Tiffany Khong; Sigrid Berg; Mi Jang; Aina Nedal; Andrew Spencer; Per Bruheim; Marit Otterlei

doi:10.3390/cancers16233963

Multiple Myeloma Cells with Increased Proteasomal and ER Stress Are Hypersensitive to ATX-101, an Experimental Peptide Drug Targeting PCNA

Cancers (Basel). 2024 Nov 26;16(23):3963. doi: 10.3390/cancers16233963.

Authors

Camilla Olaisen¹, Lisa Marie Røst², Animesh Sharma³, Caroline Krogh Søgaard¹, Tiffany Khong^{4

5}, Sigrid Berg¹, Mi Jang², Aina Nedal¹, Andrew Spencer^{4

5}, Per Bruheim², Marit Otterlei^{1

6

7}

Affiliations

¹ Department of Clinical and Molecular Medicine, Faculty of Medicine and Health Sciences, NTNU Norwegian University of Science and Technology, NO-7491 Trondheim, Norway.
² Department of Biotechnology and Food Science, Faculty of Natural Sciences, NTNU Norwegian University of Science and Technology, NO-7491 Trondheim, Norway.
³ Proteomics and Modomics Experimental Core Facility (PROMEC), NTNU Norwegian University of Science and Technology, NO-7491 Trondheim, Norway.
⁴ Australian Centre for Blood Diseases, Monash University, Melbourne 3004, Australia.
⁵ Department of Malignant Haematology and Stem Cell Transplantation, Alfred Hospital, Melbourne 3004, Australia.
⁶ Clinic of Surgery, St. Olavs Hospital, Trondheim University Hospital, NO-7006 Trondheim, Norway.
⁷ APIM Therapeutics A/S, Rådhusveien 12, NO-7100 Rissa, Norway.

Abstract

Objectives: To examine the regulatory role of PCNA in MM, we have targeted PCNA with the experimental drug ATX-101 in three commercial cell lines (JJN3, RPMI 1660, AMO) and seven in-house patient-derived cell lines with a more primary cell-like phenotype (TK9, 10, 12, 13, 14, 16 and 18) and measured the systemic molecular effects. Methods: We have used a multi-omics untargeted approach, measuring the gene expression (transcriptomics), a subproteomics approach measuring mainly signalling proteins and proteins in complex with these (signallomics) and quantitative metabolomics. These results are supplemented with traditional analysis, e.g., viability, Western and ELISA analysis. Results: The sensitivity of the cell lines to ATX-101 varied, including between three cell lines derived from the same patient at different times of disease. A trend towards increased sensitivity to ATX-101 during disease progression was detected. Although with different sensitivities, ATX-101 treatment resulted in numerous changes in signalling and metabolite pools in all cell lines. Transcriptomics and signallomics analysis of the TK cell lines revealed that elevated endogenous expression of ribosomal genes, elevated proteasomal and endoplasmic reticulum (ER) stress and low endogenous levels of NAD+ and NADH were associated with ATX-101 hypersensitivity. ATX-101 treatment further enhanced the ER stress, reduced primary metabolism and reduced the levels of the redox pair GSH/GSSG in sensitive cells. Signallome analysis suggested that eleven proteins (TPD52, TNFRS17/BCMA, LILRB4/ILT3, TSG101, ZNRF2, UPF3B, FADS2, C11orf38/SMAP, CGREF1, GAA, COG4) were activated only in the sensitive MM cell lines (TK13, 14 and 16 and JJN3), and not in nine other cancer cell lines or in primary monocytes. These proteins may therefore be biomarkers of cells with activated proteasomal and ER stress even though the gene expression levels of these proteins were not elevated. Interestingly, carfilzomib-resistant cells were at least as sensitive to ATX-101 as the wild-type cells, suggesting both low cross-resistance between ATX-101 and proteasome inhibitors and elevated proteasomal stress in carfilzomib-resistant cells. Conclusions: Our multi-omics approach revealed a vital role of PCNA in regulation of proteasomal and ER stress in MM.

Keywords: PPP; glycolysis; metabolites; redox status; ribosomal gene expression.

Grants and funding

This work was supported by grants from the Medical Technology and the Enabling Biotechnology programs at NTNU, Trondheim, Norway, and the Joint Research Committee between St. Olavs and Faculty of Medicine and Health Science, NTNU, and APIM Therapeutics AS. PROMEC is a member of the National Network of Advanced Proteomics Infrastructure (NAPI), which is funded by the RCN INFRASTRUKTUR-program (295910). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.