Abstract 3102: Venetoclax resistance associates with high-level expression of ROR1 and cancer stemness in chronic lymphocytic leukemia
Authors: Ghia Emanuela M. , Rassenti Laura Z. , Choi Michael Y. , Quijada-Alamo Miguel , Chu Elvin , Widhopf George F. , Kipps Thomas J. 
Published: July 2021

The BH3-mimetic venetoclax is highly cytotoxic for chronic lymphocytic leukemia (CLL) cells. Nonetheless, some patients (pts) fail to clear minimal residual disease (MRD) and develop progressive disease (PD) on venetoclax-based therapy (V-Rx). Acquisition of de novo BCL2 mutations that reduce venetoclax-binding to BCL2 has been observed in CLL cells of pts who develop PD on V-Rx (Blombery et al., 2019; Blombery et al., 2020; Tausch et al., 2019). However, such BCL2 mutations generally are either subclonal or not invariably detected in CLL cells of pts who develop resistance to venetoclax (Blombery et al., 2019; Guieze et al., 2019). Moreover, CLL cells of pts with PD on V-Rx appear even more resistant to the cytotoxic effects of venetoclax when cultured under conditions mimicking stroma (Blombery et al., 2019; Thijssen et al., 2015), arguing that factors other than BCL2 mutations account for venetoclax resistance. We find that high-level expression of ROR1 and Wnt5a-induced ROR1-signaling causes enrichment in cancer-stemness gene expression in CLL. We examined whether this was associated with resistance to V-Rx. We performed flow cytometry and transcriptome analyses on CLL cells of pts prior to V-Rx (pre-Rx) and on CLL cells of the same pts after they developed PD on V-Rx after a median of 2 years of therapy. At PD, we identified de novo BCL2 mutations that were subclonal in 66% of these cases. Pre-Rx CLL cells of all 6 pts expressed high levels of ROR1, which became even higher on the CLL cells of the same pts after they developed PD on V-Rx. We found that that transcriptomes of CLL cells of pts with PD had even greater enrichment in cancer-stemness gene expression than pre-Rx CLL cells of these same pts. To examine whether expression of ROR1 could influence cancer stemness and resistance to venetoclax, we transfected the CLL-cell line MEC1 with ROR1 to generate MEC1-ROR1 cells. We found that MEC1-ROR1 had greater enrichment in cancer-stemness gene expression than MEC1 cells and had increased resistance to venetoclax in vitro. Moreover, mutant forms of BCL2 identified in CLL cells of pts with PD on V-Rx, and reducing venetoclax-binding to BCL2, were significantly more effective in protecting MEC1-ROR1 cells than MEC1 cells from the cytotoxic effects of venetoclax in vitro. We find that treatment of primary CLL cells with Wnt5a to induce ROR1-signaling could enhance their resistance to the cytotoxic effects of venetoclax in vitro; such induced-resistance could be inhibited by an anti-ROR1 mAb (cirmtuzumab), which can block Wnt5a-induced ROR1-signaling. Collectively these studies demonstrate that Wnt5a-induced ROR1-signaling in CLL promotes cancer dedifferentiation/stemness and increases resistance to Ven. Strategies that inhibit ROR1-signaling with agents such as cirmtuzumab may enhance the cytotoxic activity of venetoclax and/or mitigate risk of developing resistance to venetoclax therapy.

Citation Format: Emanuela M. Ghia, Laura Z. Rassenti, Michael Y. Choi, Miguel Quijada-Alamo, Elvin Chu, George F. Widhopf, II, Thomas J. Kipps. Venetoclax resistance associates with high-level expression of ROR1 and cancer stemness in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3102.