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MCL-1 in human hematopoiesis
culture conditions and without being reflected by their MCL-1 addiction. This indicates that although there is a conserved role of MCL-1 for survival of murine and human HSPC, its function in human cells might not be restricted to inhibition of PUMA.
We wondered whether also the most immature stem cells, those able to self-renew, depend on MCL-1 expres- sion. While it is one limitation of in vitro model systems that dormant stem cells cannot be analyzed reliably, we were able to show by serial colony-forming assays that cells able to self-renew were dependent on BCL-XL rather than on MCL-1 expression. This is in contrast to findings published by Campbell et al., who attributed the loss of immature stem and progenitor cells upon MCL-1 inhibi- tion to their reduced propensity to self-renew.42
While human cord blood-derived HSPC were severely affected by MCL-1 inhibition in our hands, other authors claimed good tolerability of S63845 in non-malignant hematopoietic cells.33-35 We hypothesized that this dis- crepancy was caused by cell-intrinsic differences between cord blood HSPC used in our study and bone marrow HSPC derived from aged persons used in other studies.33 We therefore compared CD34+ cells isolated from cord blood and bone marrow with regards to protein expres- sion of anti-apoptotic BCL-2 proteins and their suscepti- bility to different BH3-mimetics. Adult HSPC had higher levels of BCL-2. Nevertheless, BCL-2 inhibition had no negative effects on colony-forming potential of aged HSPC. MCL-1 and BCL-XL levels were similar between the two types of HSPC while BFL1/A1 was not expressed. Importantly, inhibition of either MCL-1 or BCL-XL significantly impeded colony formation of both neonatal and adult HSPC and no cell type-specific differ- ence could be noted.
What do our results imply? First, the resistance of mature blood cells to S63845 suggests that immediate hematologic side effects might be mild in patients treated with MCL-1 inhibitors. The depletion of immature pro- genitor cells can, however, be associated with a relevant risk of severe cytopenias, although these might not occur immediately. To avoid excessive or even permanent bone marrow damage, repeated bone marrow analyses might be useful to detect hypocellularity as early as possible. Second, MCL-1 and BCL-XL inhibitors have synergistic rather than additive effects on human CD34+ HSPC. Synthetic lethality of two BH3-mimetics was shown for multiple tumors: The combination of BCL-2 and MCL-1 inhibitors showed synergistic effects in a vast variety of malignancies, including mantle cell lymphoma, T-cell pro- lymphocytic leukemia, multiple myeloma, high-risk B- cell acute lymphoblastic leukemia, AML and melanoma. BCL-XL and MCL-1 inhibitors were successfully com-
bined in multiple myeloma, melanoma, prostate cancer and multiple pediatric tumors.35,43-50 These observations make the combination of different BH3-mimetics very attractive. However, our results point strongly towards synthetic lethality also in healthy tissues. Specifically, combined BCL-XL and MCL-1 inhibition might be detri- mental to healthy hematopoietic tissue. This fact should be kept in mind when new clinical trials are designed.
Most data available so far, including our own, were acquired either in vitro or in artificial mouse models.33-35,51 Clinical data gathered from already initiated phase I trials (e.g., NCT03218683, NCT02675452, NCT03465540, NCT02979366) will provide better insight into both the anticancer efficacy of MCL-1 inhibitors and their frequent side effects.52 Importantly, some trials using small molecule MCL-1 inhibitors were placed on hold by the FDA in sep- tember 2019 because of cardiac toxicity.53 No data on hematologic toxicities have been published yet. Based on the very promising preclinical data28,34,54 one could speculate that the overall benefit-to-risk profile of MCL-1 inhibitors will be favorable, especially for tumors otherwise refracto- ry to chemotherapy. In case of severe irreversible hematopoietic damage created by MCL-1 inhibitors, these compounds could still be used within high-dose chemotherapy regimens given prior to autologous or allo- geneic hematopoietic stem cell transplantation.
Disclosures
No conflicts of interest to disclose.
Contributions
SB, SA, JFO, EMD, CM, YW, JMW and VRM designed and performed experiments; SB, SA, JFO, EMD, YW and ME analyzed and interpreted data; SB, SA and ME wrote the man- uscript; LK, HS and MK provided human samples.
Acknowledgments
We thank the members of the DFG-FOR2036 consortium for insightful discussions, Nora Kaltenbach and Caroline Ambs for excellent technical assistance, the animal care-takers of the Center for Experimental Models and Transgenic Services (CEMT) for animal care and the Lighthouse Fluorescence Technologies Core Facility, Freiburg for cell sorting and mainte- nance of flow cytometers. We are grateful to Heike Pahl for pro- viding us lentiviral constructs and Albert Gründer for support during initial cloning.
Funding
This work was supported by grants from the German Academic Exchange Service (DAAD) (funding program 57048249 to SA) and the German Research Foundation (DFG- FOR2036, ER599/3-1 and ER599/3-2 to ME).
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