Targeting PGC-1α Exerts An Anti-myeloma Effect Via Regulation Of Oxidative Phosphorylation

PART I Expression level of oxidative phosphorylation gene set in multiple myeloma and its prognostic significanceObjective: To analyze the expression level of oxidative phosphorylation(OXPHOS)gene set in patients with multiple myeloma(MM)and its effect on prognosis.Methods: The integrated transcriptome data of bone marrow CD138+ cells in a total of 247 newly diagnosed patients with MM and 25 healthy donors were obtained,following the gene expression profiles of GSE6477,GSE13591,and GSE47552 merged and the batch effect eliminated.To compare the expression of OXPHOS gene set between both groups,we performed gene set variation analysis(GSVA)and gene set enrichment analysis(GSEA).Gene expression profiles and survival information of 559 newly diagnosed patients with MM were obtained from GSE2658,and Kaplan-Meier analysis was used to analyze the correlation between the expression levels of OXPHOS genes and the survival of MM patients.Real-time quantitative polymerase chain reaction(q RT-PCR)and Western blot were conducted to verify the expression levels of several representative subunits of OXPHOS complexes in human MM cell lines(U266 and MM.1S).Results: The OXPHOS gene set was significantly enriched in MM patients compared to healthy controls.The overexpression of several representative subunits of OXPHOS complexes were associated with shorter survival time in MM.Consistently,the m RNA and protein levels of several OXPHOS genes were increased in both U266 and MM.1S cells.Conclusion: The OXPHOS gene set is generally highly expressed in human MM cell lines and MM samples,and associated with poorer survival in MM,providing potential molecular markers for the prognostic evaluation of MM.Part II The effect of transcriptional coactivator PGC-1α on the energy status via regulation of oxidative phosphorylation in multiple myelomaObjective: To clarify the expression of PGC-1α in MM patients,and explore its effect on the expression of OXPHOS gene set,OXPHOS metabolism and energy status in MM cells.Methods: The expression values of PGC-1α in bone marrow CD138+ cells of the healthy controls and MM patients were extracted from the abovementioned integrated transcriptome data,and the difference of PGC-1α expression was analyzed.The expression of PGC-1α was verified in MM cell lines(U266 and MM.1S)by q RT-PCR and Western blot.MM patients in the abovementioned integrated transcriptome data were divided into high-PGC-1α expression and low-PGC-1α expression groups,and the gene sets associated with high PGC-1α expression were obtained by GSEA.The 559 newly diagnosed MM patients from GSE2658 were analyzed in the same way.To screen out the biology processes involved by PGC-1α in MM,the gene sets associated with higher expression of PGC-1α in both analyses were overlapped.Following treatment U266 and MM.1S cells with the selective inhibitor of PGC-1α(SR18292),we measured the expression of OXPHOS genes by q RT-PCR and Western blot,observed the changes of mitochondrial structure by transmission electron microscopy,detected the oxygen consumption rate(OCR)of MM cells using the Seahorse XF24 extracellular flux analyzer,assessed the mitochondrial membrane potential(MMP)by JC-1 staining,detected the total intracellular ATP levels by the ATP detection kits,and evaluated the status of AMPK activation by Western blot.Results: The expression of PGC-1α was significantly increased in 247 newly diagnosed patients with MM and both MM cell lines compared to healthy controls.The OXPHOS gene set was markedly enriched in MM patients with higher PGC-1α expression from different MM database.SR18292,a selective inhibitor of PGC-1α,reduced the m RNA and protein expression levels of OXPHOS genes in both MM cells,and induced mitochondria to a round,shriveled shape with disorganized or lost cristae.The baseline OCR,ATP-linked OCR,maximal OCR were all significantly suppressed after SR18292 treatment.In addition,SR18292 decreased the levels of MMP and intracellular ATP,as well as increased the phosphorylation of AMPK in a concentration-dependent manner.Conclusion: The expression of PGC-1α is aberrantly upregulated in human MM cell lines and MM patients,and drives the overall high expression of OXPHOS gene set,increasing OXPHOS metabolism and energy supply.PGC-1α may serve as a new target for reversing the enhanced OXPHOS activity in MM.Part III Targeting PGC-1α by selective inhibitor SR18292 exerts antimyeloma effect in vitro and in vivoObjective: To investigate the effect of SR18292(a selective PGC-1α inhibitor)on the survival of MM cells and the underlying mechanism,as well as evaluate its efficacy and safety for the treatment of MM in vivo.Methods: MM cell lines(U266 and MM.1S)were treated with gradient concentration of SR18292(0 μM、10 μM、20 μM、30 μM、40 μM、50 μM、60 μM、70 μM)for 24 h,48 h and 72 h,and the cell viability was assessed by CCK-8.Following exposure of MM cells to SR18292 for indicated time,we measured the cell cycle distribution and the apoptotic rates by flow cytometry,detected the expression levels of cell cycle arrest associated protein (p-cdc2 and p-cdc25C)and the pro-apoptosis proteins(PARP and Cleaved-caspase3)by Western blot,and measured the level of intracellular reactive oxygen species(ROS)by DCFH-DA staining using flow cytometry.After pretreatment with antioxidant N-Acetyl-Lcysteine(NAC)for 1 h,U266 and MM.1S cells were treated with SR18292 for 48 h,and the effect of ROS on apoptosis were evaluated by flow cytometry and Western blot.A murine model of MM was established,via subcutaneous injection of U266 cells into NOD/SCID mice.SR18292(45 mg/kg)was injected intraperitoneally and the tumor volume as well as the viability of the mice were monitored daily.The pretein expression of Cleaved-caspase3,p-cdc2,several representative OXPHOS genes and p-AMPK were measured by immunohistochemical staining(IHC),following SR18292 injection for 18 days.The biochemical indicators and HE staining of heart,liver and kidney in mice were performed to evaluate the in vivo toxicity of SR18292.Results: SR18292 inhibited the proliferation of both MM cells in a time-dependent and concentration-dependent manner.Compared with the control group,SR18292 increased the proportion of cells in the G2 phase and the expression of proteins associated with G2/M phase arrest(p-cdc2 and p-cdc25C),as well as elevated the apoptosis rates and the expression of pro-apoptotic proteins(PARP and Cleaved-caspase3).SR18292 also increased intracellular ROS levels,and the inhibition of ROS levels significantly reversed SR18292-mediated apoptosis.Moreover,compared to the control group,the weight,volume,and growth rate of xenograft tumors were significantly reduced in the SR18292-treated mice.The protein expression levels of Cleaved-caspase3,p-cdc2,and p-AMPK were increased,as well as the expression of OXPHOS genes were decreased in SR18292-treated group.In addition,biochemical indicators and histopathological analysis showed no evidence of tissue(heart,liver,or kidney)damage in mice treated with SR18292.Conclusion: SR18292(a selective PGC-1α inhibitor)inhibits the proliferation of MM cells,arrests cell cycle progression due to energy depletion,and induces ROS-dependent apoptosis. SR18292 can decrease the expression of OXPHOS genes and increase the expression of energy stress biomarker in vivo,which also exerts significant anti-myeloma effect in MM model mice,without significant systemic toxicity,offering a potential tangible avenue for MM therapy.