Pyrroline-5-Carboxylate Reductase 1: A Novel Target for Sensitizing Myeloma to Cytotoxic Agents By Inhibition of PRAS40-Mediated Protein Synthesis

Introduction

Multiple myeloma (MM) remains an incurable cancer despite advances in therapy. Therefore, the search for new targets is still essential to uncover potential treatment strategies. Metabolic changes, induced by the hypoxic bone marrow, contribute to both cancer cell survival and drug resistance. In this study, we aimed to identify which metabolic changes and downstream pathways are involved in myeloma cell growth and persistence.

Methods

Correlation of pyrroline-5-carboxylate reductase 1 and 2 (PYCR1 and PYCR2) with overall survival was investigated in the gene-expression data of MM patients (MMRF CoMMpass trial). To perform a tracer study, RPMI-8226 cells were supplemented with ¹³C-glutamine for 48h in both normoxia and hypoxia (<1% O ₂, by chamber). For further in vitro investigation, 2 human MM cell lines (OPM-2 and RPMI-8226) were used. Proline concentrations in cell lysates were measured by ELISA-based proline assay kit. We used siRNA to establish a knockdown of PYCR1 and/or PYCR2. Levels of apoptosis were measured using AnnexinV and 7-AAD positivity on flow cytometry. Differential protein expression was evaluated with western blot. Proliferation was measured by assessing BrdU incorporation through flow cytometry. Pargyline was used as a PYCR1 inhibitor. All in vitro experiments were performed in hypoxic conditions. For the in vivo murine experiment, C57BL/KalwRij mice were inoculated with 1 million of eGFP+ 5TGM1 cells, and treated with vehicle, bortezomib (0.6 mpk, 2x/week, starting day 14), pargyline (100 mpk, 5x/week, starting day 1) or combination of both. Tumor burden was measured by flow cytometry when vehicle mice reached end-stage.

Results

Pyrroline-5-carboxylate reductase 1 and 2 (PYCR1 and PYCR2) are 2 mitochondrial enzymes that facilitate the last step in the enzymatic conversion of glutamine to proline. High expression of both enzymes correlated with a lower overall survival in the CoMMpass trial. Moreover, MM cells from relapse/refractory patients expressed significant higher levels of PYCR1.

We performed a tracer study with RPMI-8226 cells, revealing an increased conversion of ¹³C-glutamine to proline in hypoxia compared to normoxia. We confirmed these results by increased proline production after 48h of hypoxic culture.

SiRNA-mediated knockdown of PYCR1 or both PYCR1/2 combined with bortezomib increased apoptotic cell death in OPM-2 and RPMI-8226, which we confirmed by detecting upregulation of cleaved PARP and cleaved CASPASE 3 levels. In contrast, PYCR2 knockdown combined with bortezomib did not significantly alter apoptosis. Further investigation revealed that PYCR1 knockdown reduced proliferation, and led to a decrease in p-AKT, p-p42/44 MAPK and c-MYC levels. Mechanistically, we found that PYCR1 silencing affected protein synthesis, as shown by a downregulation of p-PRAS40, p-MTOR, p-p70, p-S6, p-4EBP1 and p-EIF4e levels.

Next, we evaluated whether the clinically relevant anti-hypertensive agent and PYCR1 inhibitor, pargyline, was capable of inducing myeloma cell death. In vitro, pargyline reduced proline production, MM viability and increased apoptotic cell death. Pargyline was also capable of reducing viability in CD138+ cells of primary patient samples . Finally, in vivo combination of pargyline with bortezomib significantly reduced tumor burden in the 5TGM1 model. On protein level, we also observed a significant decrease in p-4EBP1 and p-EIF4e in the freshly isolated 5TGM1 cells for the combination therapy.

Conclusion

Hypoxia increased glutamine-to-proline conversion in myeloma cells by stimulating PYCR activity. Knockdown of PYCR1 and PYCR1/2 increased bortezomib efficacy and inhibited proliferation. Mechanistically, PYCR1 interference reduced PRAS40-mediated protein synthesis. Pargyline, a PYCR1 inhibitor, also reduced MM viability and increased apoptosis. In vivo, pargyline combined with bortezomib significantly reduced tumor burden in the 5TGM1 model compared to both single agents. In conclusion, this study identifies PYCR1 as a novel target in MM therapy.