CRIP1 INDUCES PIS RESISTANCE BY FORMING A COMPLEX WITH USP7 /PA200 AND ENHANCING PROTEASOME ACTIVITY AND AUTOPHAGY IN MULTIPLE MYELOMA

Abstract

Introduction: Multiple myeloma (MM) is a plasma malignancy characterized by an accumulation of misfolded immunoglobulins in the cell. Although proteasome inhibitor (PI) can treat MM by inhibiting ubiquitin-proteasome system (UPS), autophagy as a compensatory protein clearance mechanism, it still leads to drug resistance. The discovery of new targets that can simultaneously inhibit UPS and autophagy will provide great potential for the treatment of MM. Methods: The proliferation, invasion, migration, proteasome activity and autophagy were detected in CRIP1- knockdown /overexpression and control MM cells. Co-IP (Co-Immunoprecipitation) with TAP/MS (Tandem affinity purification/Mass spectrum) was performed to detect the binding of USP7, CRIP1 and PA200. Myeloma xenograft model was used to determine the role of CRIP1 to promote proliferation of MM cells and induce BTZ resistance in vivo. Results: The levels of CRIP1 were significantly increased in plasma cells from NDMM (newly diagnosed multiple myeloma) compared to healthy donors, and further increased in patients with RRMM (relapsed/refractory multiple myeloma). Moreover, the high expression of CRIP1 is significantly related to the poor prognosis of MM patients, especially those treated with BTZ. Our results in vitro and in vivo showed that CRIP1 knockdown noteworthy inhibited the cell growth, invasion, migration. MM cells were more sensitive to PIs-induced cell growth arrest when CRIP1 knockdown. The proteasome activity and the protein level of LC3B-Ⅱ of the MM cells were significantly reduced with CRIP1 knockdown. Co-IP analysis showed that CRIP1 formed a complex with USP7/PA200. The protein level of CRIP1 decreased and the ubiquitination CRIP1 increased with USP7 inhibition. These data suggested that CRIP1 is the substrate of USP7.CRIP1 promotes the de-ubiquitination and stabilization of PA200 mediated by USP7 by interacting with USP7 and PA200, which in turn promotes MM cell survival and drug resistant. Functional inhibition of CRIP1 by knocking down of USP7 or PSME4 in CRIP1 OE MM cell line led to a decrease of the activity of proteasome and autophagy, thus enhancing the sensitivity to PIs in intro and in vivo. Conclusion: CRIP1 plays a critical role in MM progression and PIs resistance by formation of CRIP1/USP7/PA200 complex. High level of CRIP1 is a biomarker for high-risk MM patients. CRIP1 is a potential target for MM therapy, can simultaneously inhibit proteasome activity and autophagy. Encore Abstract - previously submitted to EHA 2023 The research was funded by the following foundations:(a) CAMS Innovation Fund for Medical Sciences (CIFMS) (2021-I2M-1-040 and 2022-I2M-1-022), (b) National NaturalScience Foundation of China (82170194, 81920108006, 82270175) and Natural Science Foundation of Fujian Province of China (2021J02040). Keywords: Diagnostic and Prognostic Biomarkers, Multiple Myeloma, Tumor Biology and Heterogeneity No conflicts of interests pertinent to the abstract.