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MCL-1 in human hematopoiesis
signals, BH3-mimetics are able to bypass this mode of activation.
Similar to BH3-only proteins, every BH3-mimetic avail- able so far has specific binding affinities to one or more anti-apoptotic BCL-2 proteins (Online Supplementary Figure S1A). Navitoclax (ABT-263)5 and its intravenously used precursor drug, ABT-737,6 bind to BCL-2, BCL-XL and BCL-W. The drug showed good efficacy against non-small lung carcinoma and hematologic malignancies.7,8 However, its side effects on the hematopoietic system precluded its full clinical exploration and FDA approval. This indicated that a combined inhibition of more than one pro-survival BCL-2 protein might impede survival of healthy body cells. Later, a BCL-2-specific inhibitor called venetoclax (ABT-199) found its way into clinical trials.9 Thanks to the much less severe side effects, it was approved by the FDA in 2016 as a second-line treatment for chronic lymphocytic leukemia (CLL) with 17p dele- tion, and in 2019 for the treatment of all adult CLL and small lymphocytic lymphoma patients.1 For acute myeloid leukemia (AML), venetoclax was FDA-approved only in combination with hypomethylating agents.1
Unfortunately, as for other cytotoxic drugs, tumor cell resistance poses a major problem to the efficacy of vene- toclax. Primary resistance is present when tumor cells require anti-apoptotic BCL-2 proteins other than BCL-2 for survival. Naturally, only lymphocytes10 and melanocytes11 are dependent on BCL-2 expression, as shown in BCL-2 knockout mice. This might explain why venetoclax is most effective in mature lymphoma while most other tumors show primary resistance. Such primary resistance to venetoclax can also be caused by overexpres- sion of pro-survival proteins other than BCL-2, such as BCL-XL and/or MCL-1.12,13 As shown for CLL, these BCL- 2 homologs can be induced by signals from the tumor microenvironment.14 Secondary resistance, in contrast, is acquired by tumor cells to escape previously effective BCL-2 inhibition. Several mechanisms, such as BCL-2 mutations which strongly lower venetoclax affinity,15 have been implicated in the development of secondary veneto- clax resistance.16 Alternatively, BCL-XL and MCL-1 over- expression was noted in relapsed CLL patients who had been previously treated with venetoclax.17,18 Therefore, the development and administration of MCL-1/BCL-XL inhibitors are much needed to overcome primary and sec- ondary venetoclax resistance. MCL-1 inhibitors, in partic- ular, are eagerly awaited by oncologists since this protein plays an essential role in many tumor types (e.g., AML, multiple myeloma, non-small cell lung carcinoma).19
MCL-1 was first identified during the differentiation of monocytes to macrophages in ML-1, a human myeloid leukemia cell line.20 Three isoforms of the gene have been reported; the most abundant anti-apoptotic MCL-1 long (MCL-1L)20 and two shorter pro-apoptotic isoforms (MCL- 1 short, MCL-1 extra short).21,22 In addition, a truncated isoform was shown to localize at the mitochondrial matrix where it facilitates mitochondrial fusion and ATP synthesis.23 Genetic Mcl-1 deletion in mice revealed its essential role in many tissues, both during embryogenesis and in adult mice. Specifically, constitutive MCL-1 defi- ciency resulted in peri-implantation embryonic lethality,24 while targeted deletion in the fetal hematopoietic system resulted in loss of stem cells.25 When one Mcl-1 allele was deleted in adult mice, hematopoietic stem and progenitor cells (HSPC) were depleted, leading to the death of the
animals within 2-3 weeks.26 With regard to human HSPC, there is only indirect evidence for the essential role of MCL-1, given by the BH3 profiling method: Mitochondria isolated from human CD34+ cells were highly sensitive to NOXA BH3 peptides, which typically correlated with MCL-1 dependency.27
Recently, a potent and specific MCL-1 inhibitor, S63845, was developed. This compound can efficiently kill a vari- ety of tumor cell types such as multiple myeloma, lym- phomas, leukemias and primary AML cells as well as to some extent solid cancers.28 Treatment of mice with S63845 resulted in only a few side effects in vivo,28 which was rather unexpected considering the many roles of MCL-1 during development and for tissue homeostasis. Here, we extended these studies to human cells and focused on the hematopoietic system. Understanding hematotoxicity of novel anticancer drugs is crucial since suppression of hematopoiesis accounts for most treat- ment-related morbidity and mortality. By using two differ- ent shRNA sequences and the MCL-1 inhibitor S63845, we consistently found that MCL-1 expression is essential for the survival of human stem and progenitor cells, espe- cially during early stages of differentiation. In contrast, mature blood cells are less sensitive to MCL-1 inhibition. Of note, combined inhibition of MCL-1 and BCL-XL was synergistic and already low concentrations of both drugs resulted in profound stem and progenitor cell depletion.
Methods
Lentiviruses
A pLeGO-hU6 lentiviral vector with huU6 promoter and green fluorescent protein (GFP) expression was used to generate shRNA expressing lentiviruses (Online Supplementary Table S1),29 CD34+ cells were transduced with the lentivirus (2x MOI 10, 24 h each) and knockdown efficiencies were determined 24 h later.
Isolation and culture of human CD34+ cells
Umbilical cord blood and bone marrow were obtained imme- diately after birth or from patients (age: 44-90 years) undergoing orthopedic surgery, respectively. Informed consent was obtained and the ethics committee approved the study. CD34+ cells were isolated (by magnetic activated cell sorting) from mononuclear cells to a purity >90%. Cells were used either immediately or stored in liquid nitrogen (CS10 freezing medium, Sigma) for later use. Cells were cultured in serum-free StemPro-34 medium supplemented with embryonic stem cell fetal bovine serum (ES- FBS), penicillin/streptomycin (P/S; Invitrogen), stem cell factor (SCF), FMS-like tyrosine kinase 3 ligand (FLT3L) (200 ng/mL each), thrombopoietin (TPO; 100 ng/mL) and interleukin 3 (IL-3; 20 ng/mL; Immunotools/Peprotech). Where indicated, the BH3- mimetics S63845 (SynMedChem), A-1155463 and ABT-199 (Sellekchem) were added.
Apoptosis assay
CD34+ cells were subjected to cytokine deprivation or treated with etoposide (VP16), tunicamycin, taxol, thapsigargin and brefeldin A (BFA). After 0, 24 and 48 h, cells were stained with annexin V (Biolegend) and 7-aminoactinomycin D (7-AAD; Sigma-Aldrich) to detect apoptosis. The percentage of specific apoptosis was calculated as: 100 x (% living cells under control condition - % living cells under treatment)/ % living cells under control condition. Control condition was culture with ES-FBS and cytokines.
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