Abstract
Deregulated cell death and survival pathways contribute to leukemogenesis and treatment failure of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients. The intrinsic apoptosis pathway is regulated at the mitochondrial level by different pro- and anti-apoptotic molecules. Members of the BCL-2 family are key regulators of mitochondrial apoptosis signaling. Pro-apoptotic BH3-only proteins like BIM and BID activate pro-death proteins such as BAX and BAK leading to cell death. Anti-apoptotic BCL-2 family members including BCL-2, BCL-XL and MCL-1 bind to and sequester pro-apoptotic molecules, prevent activation of pro-death proteins and counter-regulate apoptosis induction. Small molecule inhibitors have been developed that block binding to anti-apoptotic molecules like BCL-2, leading to release of pro-apoptotic proteins and cell death induction. In particular, the BCL-2-specific inhibitor venetoclax (VEN) has demonstrated substantial anti-cancer activity and became an approved drug for the treatment of CLL patients. Investigating different, individual BCP-ALL samples, we and others recently identified heterogeneous sensitivities for VEN, suggesting that BCP-ALL cells might also depend on other pro-survival BCL-2 family proteins including MCL-1, leading to VEN insensitivity and resistance. A novel BH3-mimetic, S63845, that selectively targets MCL-1 has been reported.
Here, we assessed the activity of S63845 and addressed a potential synergism of simultaneous blockage of BCL-2 and MCL-1 by VEN and S63845 (S) in BCP-ALL.
The activity of the MCL-1 inhibitor was analyzed in a panel of BCP-ALL cell lines (N=6) and a series of primary, patient-derived BCP-ALL primograft samples (N=27) determining half-maximal effective concentrations (EC50) upon exposure to increasing concentrations of S and analysis of cell death induction. We observed heterogeneous sensitivities to S with EC50 values ranging from 16 nM to almost 10 µM. Protein expression of MCL-1 and other BCL-2 family members BCL-2, BCL-XL and BCL-W was assessed by western blot analysis and quantified, however neither association of MCL-1 levels nor expression of the other regulators and S sensitivity was found in cell lines and primograft leukemias. Moreover, we also compared sensitivities for both inhibitors but found independent activities of S and VEN in individual ALL samples.
Next, we addressed the role of MCL-1 for VEN sensitivity and generated two MCL-1 knock out BCP-ALL cell lines by CRISPR/Cas9 gene editing. In both lines, clearly increased VEN sensitivities were observed upon depletion of MCL-1, indicating that MCL-1 is contributing to activity of the BCL-2 inhibitor VEN.
Based on these findings, we investigated the effects of pharmacological MCL-1 inhibition for VEN sensitivity and incubated all 6 cell lines with VEN and S at increasing concentrations and observed clear synergistic effects upon combined BCL-2 and MCL-1 inhibition indicated by combination indices (CI) below 0.1. Moreover, we investigated 7 primograft BCP-ALL samples and found that MCL-1 inhibition by S clearly synergized with VEN activity (CI < 0.3).
To investigate the anti-leukemia activity of co-targeting BCL-2 and MCL-1 in vivo in a pre-clinical setting, a high-risk leukemia derived from an infant, MLL/ENL rearranged pro-B ALL case was transplanted onto NOD/SCID mice. Upon ALL manifestation (presence of >5% human blasts in blood), recipients were treated with either VEN, S, the combination of both, or vehicle for 10 days. After treatment, leukemia loads were analyzed showing significantly reduced loads in the co-treated group as compared to vehicle, VEN or S alone in spleen, bone marrow, and central nervous system (p-values < 0.05), indicating synergistic activity of co-inhibition of BCL-2 and MCL-1 in vivo.
Taken together, our data show heterogeneous sensitivity of individual BCP-ALL samples to MCL-1 inhibition by S, which is not associated with MCL-1 protein expression levels or VEN sensitivity. Both, genetic depletion and inhibition of MCL-1 by S synergizes with VEN leading to increased anti-leukemia activity in vitro and ex vivo. Importantly, co-targeting BCL-2 and MCL-1 significantly reduced leukemia infiltration in spleen, BM and CNS in a pre-clinical model of high-risk BCP-ALL, warranting further evaluation and possible clinical application of targeting MCL-1 alone and in combination with BCL-2 inhibition.
Disclosures
No relevant conflicts of interest to declare.
Overcoming apoptosis resistance in high risk acute lymphoblastic leukemia by SMAC mimetics in a preclinical ALL xenograft model in vivo