Suppression of Cdc37 Induces BTZ Resistance through XBP1s Mediated Plasma Cell Immaturation in Multiple Myeloma

Backgrounds: Cell division cycle protein 37 (Cdc37) is considered as a key co-chaperone of heat shock protein 90 (Hsp90), guiding the stabilization and activation of client kinases with time specificity and substrate selectivity. Previous studies have demonstrated that Cdc37 is over-expressed in many kinds of cancers, contributing to the tumorigenesis. However, our previous study suggested that some MM cell population with low expression of Cdc37 was correlated with BTZ resistance in clinical MM samples and in vitro study. The purpose of this study is to investigate how Cdc37 contributes to drug resistance in MM.

Materials and methods: Nineteen sequential MM samples with gene expression profiling (GSE19554) obtained from published studies were analyzed. The expression of Cdc37 was investigated in MM cell lines and primary CD138⁺ cells by qRT-PCR. Cdc37 function was then interrupted by Cdc37 shRNA or Hsp90-Cdc37 inhibitor Celastrol in MM cell line. Sensitivity of BTZ was evaluated. Bone marrow MM cells were enriched and gated into subpopulations by CD38 and CD138 status by FACS. CD38⁺CD138⁺ plasma cells, CD38⁺CD138^- plasmablasts and CD38^-CD138^- B cells were examined for Xbp1s and Cdc37 expression by qRT-PCR and IF. Then cellular morphology, supernatant light chain immunoglobulin and makers of plasma cell maturation were checked when MM cells were exposed to Celastrol or underwent Xbp1s overexpression. Finally, the in vivo verification was carried out in 5TGM1 mouse model.

Results: The GEP analysis showed that Cdc37 was down-regulated as the progression of MM (base line, pre-1^st transplantation, pre-2^nd transplantation, post-2^nd transplantation) in 13 out of 19 sequential samples. Our results also showed that some relapsed MM patients had a down-regulated Cdc37 expression compared with newly diagnosed cases (0.78±1.13 vs 0.25±1.05, p <0.05). Moreover, Cdc37 was only suppressed in bortezomib (BTZ) resistant cell line ANBL-6. BR compared with their wild type ANBL-6.WT. While there were no significant differences between doxorubicin-, dexamethasone- resistant cell line. Indeed, suppression Cdc37 in MM cell line by Cdc37-shRNA or Celastrol resulted in a lower apoptosis rate after BTZ treatment. These results indicated that Cdc37 may participate in BTZ resistance.

To explore the mechanism of Cdc37 mediating BTZ resistance, we tried to determine if Cdc37 suppression might cause immaturity of plasma cells, which is well established to cause MM cells resistant to BTZ. Cdc37 expression was first found to be positively correlated with Xbp1s in primary MM cells (r=0.61, p <0.001). Then CD38⁺CD138⁺ plasma cells, CD38⁺CD138^- plasmablasts and CD38^-CD138^- B cells were examined for Xbp1s and Cdc37 and showed that Cdc37 and Xbp1s were gradually up-regulated during the maturation of plasma cells in 3/5 patients. The results indicated that Cdc37 may induce BTZ resistancethroughXBP1s mediated plasma cell immaturation.

To certify our hypothesis, Cdc37-Hsp90 interaction were interrupted with Celastrol, NCI-H929 cells appeared smaller and rounder with larger nucleus, less cytoplasm, resembling plasmablast. ELISA analysis also demonstrated a decreased secretion of free light chain from MM cells. Partial repression of plasma cell maturation markers, such as Xbp1s, Chop, CD49e were also observed when Cdc37 was inhibited in NCI-H929 cells. However, the above changes were reversed when Xbp1s was overexpressed in MM cells. In vivo, the serum IgG2b level was (146.8±4.0) ng/ml and (166.0±13.0) ng/ml (p<0.05) in BTZ group and BTZ+Celastrol group, respectively. Although statistical significance were not reached, the BTZ+Celastrol group showed a shorter overall survival compared with BTZ group.

Conclusion: Our results demonstrated that Cdc37 downregulation may result in BTZ resistance through Xbp1s mediated plasma cell immaturation. These findings provide a certain theoretical reference for the proper combination of BTZ and other therapy, forcing BTZ resistant cells differentiation, to fight against BTZ resistance. More importantly, Cdc37 may provide a diagnostic biomarker for predicting BTZ sensitivity prior to treatment.