AURKA Regulates the Mechanism of ATR Mediated DNA Damage Response about Drug Resistance in Multiple Myeloma

Objective: Multiple myeloma (MM) is a cancer formed by malignant plasma cells. So far, drug resistance and relapse are major clinical problems in MM. Our previous studies showed that some chromosomal instability genes are involved in drug resistance, which AURKA expression is closely related survival. But the correlation between AURKA expression and clinical and how to participate in the mechanism of proliferation and drug resistance are unknown.

Methods: Analyzing the Gene expression profiling (GEP) data and the relationship between AURKA expression and prognosis in MM. Bone marrow plasma cells from MM patients and health donor were collected by CD138. Detecting oncogene AURKA in presence of deletion and amplification by FISH. Detecting expression of mRNA by real-time qPCR. Immunofluorescence (IF) was used to detect the expression of protein. Comet assay was used to evaluate DNA repair. Then using the AURKA inhibitors and targeting AURKA, CCK-8 is used to detect the proliferation, WB detection of apoptosis, DNA Damage protein, IF detection of spindle formation and analyzing whether the AURKA is correlated with chromosome instability and drug resistance. In vivo and in vitro, combined with clinical medication bortezomib, Melphalan, lenalidomide. We detect apoptosis and DNA damage related protein and tumor volume in NOD/SCID mice. Changes in the culture supernatant of DNA damage response factor is used by real-time qPCR technique and ELASA detection of cell mass.

Results: 1: Analyzing 350 patients with GEP data, the prognosis of patients with the AURKA high expression are better than the lowers (P=0). Patients with high expression of AURKA are mainly distributed in the high proliferation group. 2: Detecting the expression of mRNA by Real time qPCR technique, AURKA expression is significantly higher in newly diagnosed patients with 27.5% (n =11/n=40). The expression of AURKA protein is detected by IF assay. The results show that AURKA expression was higher in newly diagnosed (36.84%, n=14/n=38) and relapsed (50%, n=20/n=40) than that in normal donors. Patients with high expression of AURKA are closely related to the clinical relevance of anemia, bone destruction, high CR and high LDH. To further detect the relationship between AURKA expression and chromosome DNA copy number, the results show that AURKA copy number is normal in newly diagnosed patients (n=20). But there are 10% patients (n=2/n=20) with AURKA amplified in relapsed patients. 3: In vivo and in vitro, AURKA inhibitor combined with Bomtzomib, lenalidomide and Melphalan could enhance the sensitivity of BTZ and xenograft tumor biopsy showed reduced group AURKA, Ki67 expression. After over expression of AURKA, the proportion of cells with a single nucleus was decreased and the formation of multiple nuclei increased. Knocking down the AURKA, spindle formation is disorder. The expression of gamma-H2AX molecules and 53BP1 related DNA damage is abnormal. WB suggests that DNA damage is inhibited, phosphorylation of ATR, ATM, CHK2 reduction, phosphorylation of gamma-H2AX and BRCA increased. Further, real time qPCR technique and ELASA detection of DNA damage response factor in cell culture supernatant is mainly changed, such as IL-1 and IL6.

Conclusion: High expression of AURKA is associated with poor prognosis in MM; Inhibition of AURKA can promote apoptosis, DNA damage and reduce the secretion of IL-6 that could enhance the sensitivity to bortezomib, lenalidomide and Melphalan. The abnormal expression of AURKA related spindle formation disorder and cell nuclear phenomena that cause chromosomal instability. AURKA is mainly involved in the drug resistance through the DNA damage response ATR-CHK1-gammaH2AX pathway.