34A combination of the BCL-2 inhibitors ABT-263 and A-1210477 inhibited cell proliferation 35 in the HeLa, C33A, SiHa and CaSki human cervical cancer cell lines. Drug sensitivity was 36 initially tested using 2-dimensional (2D) cell culture models. As ABT-263 binds to both BCL-37 2 and BCL-XL at high affinity, it was unclear whether the synergism of the drug combination 38 was driven either by singly inhibiting BCL-2 or BCL-XL, or inhibition of both. Therefore, we 39 used the BCL-2 selective inhibitor ABT-199 and the BCL-XL selective inhibitor A1331852 to 40 resolved the individual antitumor activities of ABT-263 into BCL-2 and BCL-XL dependent 41 mechanisms. A-1210477 was substituted with the orally bioavailable S63845. The SiHa, C33A 42 and CaSki cell lines were resistant to single agent treatment of all three drugs, suggesting that 43 none of these anti-apoptotic proteins singly mediate survival of the cells. HeLa cells were 44 resistant to single agent treatment of ABT-199 and A1331852 but were sensitive to S63845 45 indicating that they depend on MCL-1 for survival. Co-inhibition of BCL-XL and MCL-1 with 46 A1331852 and S63845 significantly inhibited cell proliferation of all four cell lines. Similar 47 data were obtained with 3-dimensional spheroid cell culture models generated from two 48 cervical cancer cell lines in vitro. Treatment with a combination of A1331852 and S63845 49 resulted in inhibition of growth and invasion of the 3D spheroids. Co-inhibition of BCL-2 and 50 MCL-1 with ABT-199 and S63845, also inhibited cell proliferation of all cancer cell lines, 51 except SiHa. However, the effect of the combination was not as pronounced as combination of 52 A1331852 and S63845. Collectively, our data demonstrate that the combination of MCL-1-53 selective inhibitors with either selective inhibitors of either BCL-XL or BCL-2 may be 54 potentially useful as treatment strategies for the management of cervical cancer. 55 56