Suppression of Cdc37 Induces Bortezomib Resistance through Autophagy Activation and Plasma Cell Immaturation in Multiple Myeloma

Introduction: Cell division cycle protein 37 (Cdc37) is considered as a key co-chaperone for heat shock protein 90 (HSP90) to guide the stabilization and activation of protein kinases, which is critical for cell signaling transduction, proliferation and survival. Our previous study suggested that the population of low expression of cdc37 was correlated with bortezomib 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.

Material and method: 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 purified CD138⁺ tumor cells by qRT-PCR. Cdc37 was down-regulated by lentivirus in MM cell line NCI-H929. Sensitivity of bortezomib was evaluated by CCK-8 and apoptosis analysis. Autophagosome was detected by electron microscope. Bone marrow MM cells from MM clinical samples were enriched by negative selection for CD2 (T and NK cells) and CD14 (monocytes) using FACS. Tumor cells were gated into subpopulations by CD38 and CD138 status. CD38⁺ CD138⁺ plasma cells, CD38⁺ CD138^- plasmablasts and CD38^- CD138^- B cells were examined for xbp1s and cdc37 protein expression by IF.

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 (Data base GEP). Cdc37 was suppressed in bortezomib 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 compared with their counterparts. Primary tumor cells from five relapsed MM patients after bortezomib treatment also showed low expression of cdc37 compared with five MM samples that were sensitive to bortezomib treatment. Indeed, suppression cdc37 in MM cell line NC-H929 by shRNA lentivirus (cdc37-shRNA) resulted in a higher IC50 of bortezomib, and lower apoptosis rate after bortezomib treatment compared with scramble. These results indicated that cdc37 may participate in bortezomib resistance in MM samples and in vitro.

Autophagy has recently emerged as a new mechanism involved in bortezomib resistance. Our results also showed that autophagy associated proteins Beclin-1 and LC-3b were increased in ANBL-6.BR compared with ANBL-6.WT. Besides, Beclin-1 and LC-3b were up-regulated in NCI-H929- cdc37 shRNA cells. What's more, there were more autophagosomes in NCI-H929- cdc37 shRNA cells detected by electron microscope. Furthermore, combinatorial application of autophagy inhibitor chloroquine and bortezomib would partially rescue bortezomib resistance after cdc37 down-regulation.

Moreover, cdc37 expression was also found to be positively correlated with xbp1s in 62 newly diagnosed, 25 relapsed and 18 remission MM patients. Xbp1s, CHOP and BLIMP1, which were essential for the plasma cell differentiation and maturation, were also lower expression in 5 bortezomib resistant primary MM patients compared with the sensitive ones detected by qRT-PCR. Then CD38⁺ CD138⁺ plasma cells, CD38⁺ CD138^- plasmablasts and CD38^- CD138^- B cells were examined for xbp1s and cdc37 protein expression by IF 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, together with xbp1s, may induce bortezomib resistance through plasma cell immaturation.

Conclusion: Our results demonstrated that cdc37 downregulation may result in bortezomib resistance through compensatory increasing of autophagy level and the immaturation of plasma cells. Further study will be focused on how cdc37 interacts with xbp1s to participate in autophagy and promotion plasma cell maturation in MM.