Suppression of TRIP13 Induces Metabolic Changes and Potentiates Ferroptosis in Multiple Myeloma

Introduction

Despite the remarkable success of combination treatments, multiple myeloma (MM) still poses significant challenges, with a high percentage of patients experiencing relapse over time. Additional metabolic alterations are found in relapsed and refractory MM; however, their impacts have not been fully elucidated. Expression of thyroid hormone receptor interactor 13 (TRIP13) has been identified by Gene Expression Profile (GEP)-70 to correlate with poor prognosis in MM. TRIP13 is an AAA ATPase that regulates the spindle assembly checkpoint in mitosis. Based on GEP data from 34 paired baseline and relapse samples, TRIP13 continuously increased from baseline to first and to second relapse. Overexpression of TRIP13 induces bortezomib resistance. In this study, we aimed to investigate how TRIP13 might regulate MM cell metabolism and to use this information to develop a novel combination strategy to overcome MM resistance.

Methods

Knockout (KO) of TRIP13 in OPM2 and MM1.S cells was generated by dual gRNAs CRISPR-Cas9 editing deleting a fragment between exon 4 and exon 5. Cell growth curves and response to bortezomib were recorded in the control and TRIP13-KO cells. Metabolomics was performed to reveal cellular metabolic alterations. Seahorse analysis was used to test the mitochondrial stress and glycolysis stress. To investigate whether TRIP13 KO promotes ferroptosis, an intracellular iron-dependent cell death characterized by the accumulation of lipid peroxides, we applied C11-BODIY staining after treatment with Erastin, a ferroptosis-inducer. Expression levels of cyclooxygenase 2 (encoded by PTGS2, a hallmark of ferroptosis), cystine transporter (xCT), and intracellular glutathione peroxidase 4 (GPX4) were analyzed. In addition, the protein levels of iron metabolic molecules, including iron importer transferrin receptor protein 1 (TFRC), iron exporter ferroportin 1 (FPN1), ferritin heavy chain 1 (FTH1), and nuclear receptor coactivator 4 (NCOA4) were assessed. To study whether TRIP13 KO delays MM progression in an immune-competent MM mouse model, Vκ12653 murine MM cells were genetically modified by CRISPR-mediated TRIP13-KO and then injected into C57BL/6 mice. Mouse survival was monitored.

Results

The cell-doubling rate of TRIP13-KO cells was decreased by 50% compared to control cells. After 24 hours of treatment with 5 nM bortezomib, cell viability of TRIP13-KO cells decreased by 31% compared with control cells. The metabolomic studies revealed that TRIP13-KO OPM2 and MM1.S cells both had significantly decreased intracellular L-serine (FC = 0.57) but increased pyruvate (FC = 1.79), citric acid (FC = 1.76), and isocitric acid (FC = 1.64), indicating TRIP13 may regulate serine metabolism, mitochondrial function, and cellular redox balance. Seahorse analysis indicated that TRIP13-KO MM cells showed decreased glycolysis (decreased by 30%, p < 0.001) and glycolytic capacity (decreased by 33%, p < 0.001) while oxidative phosphorylation showed little change. Moreover, lipid peroxidation and PTGS2 were increased in TRIP13-KO cells after Erastin treatment, indicating that these cells were more vulnerable to ferroptosis. Mechanistically, inhibition of TRIP13 decreased GPX4, FPN1, FTH1, and increased NCOA4, while overexpression of TRIP13 showed the opposite changes, suggesting that targeting TRIP13 sensitizes MM cells to ferroptosis by impairing cellular redox balance and increasing the labile-iron pool. Lastly, TRIP13-KO significantly prolonged mouse survival in the Vκ12653 mouse model (median survival: TRIP13-KO 93 days vs. Control 60.5 days, p < 0.001).

Conclusion

Our study revealed that TRIP13 plays a critical role in regulating MM cell metabolism. By suppressing TRIP13, we observed increased sensitivity of MM cells to bortezomib, leading to enhanced ferroptosis-mediated cell death and prolonged mouse survival. These findings strongly suggest that targeting TRIP13 could be a promising approach in MM therapy. Moving forward, our future investigations will concentrate on unraveling the precise molecular mechanisms underlying TRIP13-mediated alterations in cell metabolism. Additionally, we plan to explore the potential synergistic effects of TRIP13 inhibition in combination with metabolic targeted treatments, such as venetoclax, to further enhance therapeutic outcomes in MM.