Abstract
Introduction:Although the macrolide antibiotic clarithromycin (CAM, or Biaxin) has only minimal single agent activity in MM, the regimens with addition of CAM to IMiDs and steroids, including BLT-D (Biaxin/low-dose thalidomide/dexamethasone [Dex]), BiRD (Biaxin/Revlimid [lenalidomide, Lena]/Dex), t-BiRD (thalidomide/BiRD), Car-BiRD (carfilzomib/BiRD), and ClaPD (CAM/pomalidomide/Dex), appear highly effective in treatment of newly-diagnosed and probably relapsed/refractory multiple myeloma (MM). In this context, two phase 3 trials are currently ongoing to evaluate the efficacy of BiRD vs. RD in newly-diagnosed MM in the United States and Europe, respectively. Of note, recent findings also suggests that addition of CAM to RD might overcome resistance to RD. However, despite increasing clinical evidence for its promising activity, the exact mechanism for such a combination strategy still remain largely unclear. Here, we investigated the mechanisms of action underlying the interaction between CAM and Lena and their capability to overcome drug resistance in MM cells, focusing on the cereblon (CRBN)/IKZF1,3/IRF4/Myc signaling cascade, recently identified as the novel target of IMiDs,
Materials and Methods: To test our hypothesis whether and how BiRD overcomes resistance to RD (Lena/Dex), human MM cells lines employed in this study included Dex-sensitive (MM.1S) vs -resistant (MM.1R) cells, drug-naïve RPMI8226 cells vs their Lena-resistant (R10R) or bortezomib (Btz)-resistant counterparts (DR), as well as primary CD138+MM cells isolated from bone marrow samples of newly-diagnosed and relapsed/refractory patients who had received prior IMiDs (including Lena) or Btz. Cells were exposed (72 hr) to CAM (50-100 mg/ml) ± Lena (1-10 mM), after which the CCK-8 assay and flow cytometry with annexin V/7AAD staining were performed to monitor cell viability and apoptosis, respectively. Mechanistic studies included Western blot analyses of the CRBN/IKZF1,3/IRF4/Myc signaling pathway, as well as the apoptotic caspase cascade. Cell cycle was also assessed by flow cytometry.
Results: Whereas Lena (1-10 mM) had almost no direct effects on cell viability, CAM (≥ 100 mg/ml) displayed a dose-dependent toxicity in various MM cell lines. Notably, subtoxic concentrations of CAM (e.g., 50 mg/ml) significantly potentiated lethality of Lena in MM.1S (CI value = 0.40-0.86, indicating synergism). Significantly, this effect was even more robust in Dex-resistant MM.1R cells. These events were associated with marked activation of caspase 3, 8, and 9 and PARP cleavage, accompanied by down-regulation of the anti-apoptotic proteins Bcl-2 and Bcl-xL. While add-on of CAM significantly increased lethality of Lena in RPMI8226 cells, combined treatment was strikingly more effective against Lena-resistant R10R cells. In addition, Btz-resistant RPMI8226 cells were also more sensitive to both CAM alone and in combination with Lena, compared to parental RPMI8226 cells. Consistently, markedly enhanced cell killing by the combination was also observed in primary CD138+ cells, particularly those obtained from patients relapsed after prior IMiDs. Exposure to Lena with or without CAM sharply down-regulated CRBN in MM cells, accompanied by reduced expression of IKZF1, IKZF3, IRF4, and Myc. Interestingly, Lena failed to down-regulate CRBN/IKZF1/IRF4/Myc in Lena-resistant R10R cells, while addition of CAM dramatically resensitized these cells to the action of Lena. Moreover, Lena in the presence or absence of CAM induced cell cycle arrest at G0/G1, in association with marked up-regulation of p21Cip1 and p27Kip1. Last, Lena induced LC3A-II expression (a marker of autophagy), which was clearly increased in the presence of CAM, likely in association with the capability of CAM to impair the late stage process of autophagy e.g., autophagosome clearance by lysosome.
Conclusion: Together, these findings indicate that CAM significantly increases the anti-MM activity of Lena in MM cells, especially those resistant to the first-line therapy (e.g., Dex and Btz), and notably overcomes Lena resistance. The mechanisms involves disruption of the CRBN/IKZF1/IRF4/Myc pathway, as well as activation of the apoptotic caspase cascade, induction of cell cycle arrest, and attenuation of autophagy. Collectively, these mechanistic findings support exploring the BiRD regimen in MM, particularly to overcome RD resistance.
Disclosures
No relevant conflicts of interest to declare.
Orai1 and Stim1 Mediate the Majority of Store-Operated Calcium Entry in Multiple Myeloma and Have Strong Implications for Adverse Prognosis