Abstract 988: Targeting immunological and apoptotic cell death to improve therapeutic efficacy in melanoma
Authors: Savaraj Niramol , You Min , Li Ying-Ying , Wu Chunjing , Wangpaichitr Medhi , Miguel Saurez , Chicco Alfred , Kuo Macus T. , Feun Lynn G. 
Published: July 2021

Apoptotic and immunological cell death are the two major pathways that can lead to demise of cancer cells. Targeting both pathways by combining chemotherapy and checkpoint inhibitors has proven to be more effective in treating lung cancer but not in melanoma, due to the fact that melanoma cells often express high levels of antiapoptotic proteins Bcl-2 ( 60%) which contribute to their resistance to chemotherapy. We have previously shown that melanoma cell lines with high Bcl-2 expression , regardless of their BRAF mutation, do not possess high PD-L1 ( AACR abstract #4747 2019). This is also seen in melanoma tissue obtained from patients. Here, we attempt to explore whether there is any relationship between Bcl-2 and PD-L1 in melanoma cells using knock in and knock out methods. Two melanoma cells derived from patients ( mel1220 which possess high levels of Bcl-2 and Mel-F which possess high levels of PD-L1 ) and their BRAF/MEK counterparts, Mel-1220 BMR and MelF BMR, respectively, were used for the study. Interestingly, cell lines with high Bcl-2 , their BMR counterparts showed no changes in Bcl-2 levels, However, in cell lines with high PD-L1 expression, the levels of PD-L1 is even higher when they become BMR. This could explain why after failure to BRAF/MEK inhibitor , the clinical response to anti PD-1 check point inhibitors varies. Next, we transfected PD-L1 using the SP-GFP-CD274-NU Vector ( purchased from Addgene )into the BcL-2 over expressing cell lines. Three clones were selected. Two clones ( 1220.PD-L1/1 and 1220 PD-L1/3) showed only slight decreased Bcl-2 expression ( less than 1 fold) and in one clone ( 1220.PD-L1/2), there is no effect seen. Furthermore, there is no discernable change in other antiapoptotic proteins ( Bcl-xl, BAX, Mcl-2 survivin, NOXA and PUMA ) by westernblot noted. Similar results were obtained with their BMR counterparts. Total knock down of Bcl-2 using CRISPR knockout ( kit purchased from Origene) results in cell death. In contrast, transfection of Bcl2 into PD-L1 overexpression results in decreased PD-L1 expression by 2-4 fold both by PCR and by westernblot in both clones. however , there is no discernable changes on surface PD-L1 by flow cytometery. Transfection of BCl-2 also does not affect other BCL-2 family proteins. Similar results were obtained with their BMR counterparts. From these data, it appears that the presence of Bcl-2 ( RNA/protein) could affect intrinsic PD-L1 expression, however, the surface PD-L1 which is the key to bind PD-1 did not change. On the other hand, the presence of PD-L1 RNA/protein has no discernable effect on BcL-2 expression. Whether partial knock down BCl-2 or treatment with Bcl-2 inhibitor can lead to increased PD-L1 expression is not known and is under investigation. Overall, it appears that combination of Bcl-2 inhibitor and check point inhibitor may be more effective in melanoma patients with high Bcl-2 expression. (Supported by VA merit review award; 1BX003328)

Citation Format: Niramol Savaraj, Min You, Ying-Ying Li, Chunjing Wu, Medhi Wangpaichitr, Saurez Miguel, Alfred Chicco, Macus T. Kuo, Lynn G. Feun. Targeting immunological and apoptotic cell death to improve therapeutic efficacy in melanoma [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 988.