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S. Bohler et al.
Colony-forming assays and differentiating culture
One thousand CD34+ cells were seeded in MethoCult SF- H4436 medium. After 10-11 days, colony types (identified by light microscopy) and total cell counts were determined. The percentages of HSPC, erythroid and myeloid cells were deter- mined by flow cytometry (Online Supplementary Table S2).
Proliferating culture
CD34+ cells were cultured for 5-11 days in StemPro-34 medi- um supplemented with 10% ES-FBS, SCF, FLT3L, TPO and IL-3.. The medium was refreshed every 3 days. Cells were analyzed for GFP and immature populations (Online Supplementary Table S2).
Quantitative reverse transcription polymerase chain reaction
RNA was isolated by a Quick RNA Micro Prep kit (Zymo Research) and reversely transcribed to cDNA (Quantitec- Reverse transcription kit, QIAgen). Quantitative reverse tran- scription polymerase chain reaction (qRT-PCR) was performed by using BIO-RAD (CFX96 Touch) a RT-PCR detection system and SYBR Green master mix (Thermofisher). Expression of the gene of interest was normalized to the expression of either 18S or 36B4.
Reverse transcriptase multiplex ligation dependent probe amplification
RNA samples from CD34+ cells cultured under different con- ditions were obtained by a Quick RNA Micro Prep kit (Zymo Research). Reverse transcriptase multiplex ligation dependent probe amplification (RT-MLPA) was performed according to the manufacturer’s instructions (MRC Holland, R011-C1). The resultant amplicons were separated by capillary electrophoresis (ABI-3130xl Genetic Analyzer) and Sequence Pilot (JSI Medical Systems) was used for the analysis. The sum of all peaks was taken as 100%, and the values of the single peaks were normal- ized accordingly.
Xenotransplantation
All experiments were performed after approval from the local ethics committee and in compliance with German law. Rag2−/−gc−/− mice were kept under specific pathogen-free condi- tions. Newborn mice were sub-lethally irradiated with 2.5 Gy.30 After 6 h, the progeny of 1x105 transduced or untransduced human CD34+ cells were injected intrahepatically. Mice were sacrificed for analysis after 8 weeks.
Flow cytometry
Single cell suspensions obtained from colony-forming assays or hematopoietic organs from mice were surface stained with monoclonal antibodies: CD34 PE-Cy7(581), CD38 APC (HIT2), CD10 PE/APC (HI10a), CD45RA PerCP-Cy5.5 (HI100), CD90 APC-Cy7 (5E10), CD117 PE-Cy7 (104D2), CD71 APC (CY1G4), CD33 PE (WM53), CD14 APC (M5E2), CD115 BV421 (4D2- 1E4), CD15 PeCy5 (W6D3), CD66b PerCP-Cy5.5 (G10F5), CD19 PE-Cy7 (HIB19), IgM APC-Cy7 (MHM-88), CD45 Biotin (HI30), CD45 PE-Cy7/V500 (30-F11) (Biolegend), and CD235a BV421 (HIR2) (BD Biosciences). Streptavidin PerCP-Cy5.5/V450 (Biolegend) was used as a secondary antibody. BD LSRFortessa and FlowJo were used for flow cytometry and analyses, respec- tively. The gating strategy was published earlier.31
Western blot
Purified proteins were size fractioned by 12% sodium dode- cylsulfate polyacrylamide gel electrophoresis under reducing
conditions and transferred onto polyvinylidene difluoride mem- branes. The antibodies used were: MCL-1 (D2W9E) rabbit monoclonal antibody, BCL-XL (54H6), BCL-2 (D17C4), BFL1/A1 (D1A1C), α/b-tubulin or b-actin (13E5) (all rabbit, Cell Signaling). A peroxidase-coupled goat anti-rabbit IgG secondary antibody was used (sc-2004, Santa Cruz).
Statistics
Statistical analyses were performed using the unpaired Mann- Whitney test in GraphPad Prism 7 software. P values less than 0.05 were considered statistically significant. Synergy of BCL-XL and MCL-1 inhibitors was calculated using the Bliss synergy score and the program SynergyFinder (https://synergyfinder.fimm.fi).32
Results
MCL-1 knockdown sensitizes human CD34+ cells selectively to endoplasmic reticulum stress
Putative shRNA sequences specific for MCL-1 were tested in transfected HEK293T cells. For shRNA delivery into human CD34+ cells, a lentiviral system that allowed stable expression of the shRNA and GFP was used. Two different shRNA sequences, both binding to exon 3 of the human MCL-1 gene, were identified to reduce MCL- 1 mRNA expression to 25-55% in HEK293T and by 41- 55% in human cord blood-derived CD34+ HSPC (Figure 1A). Knockdown of the Luciferase (Luci) gene was used as a negative control. Efficient MCL-1 knockdown was confirmed on a protein level in HEK293T and CD34+ cells and showed, at least in CD34+ cells, no relevant dif- ferences between the two shRNA sequences used (Figure 1B, C). Transduction efficiency was similar for the differ- ent viruses (Online Supplementary Figure S1B) but CD34+ cells transduced with shRNA specific for MCL-1 showed increased apoptosis rates 24 h after transduction (Figure 1D). The surviving cells were cultured and treated for 24 h and 48 h with different cytotoxic drugs including the DNA damaging agent etoposide, the mitotic spindle inhibitor taxol and different compounds inducing endo- plasmic reticulum (ER) stress (i.e., tunicamycin, thapsi- gargin and brefeldin A) (Figure 1E, F). Interestingly, MCL- 1 inhibition selectively increased the sensitivity of CD34+ cells to ER stress (Figure 1E, F). While transduced GFP+ cells expressing MCL-1 shRNA were killed in a dose- dependent manner (Online Supplementary Figure S2A), GFP+ cells expressing Luci shRNA or non-transduced GFP- cells did not undergo increased cell death upon ER stress (Online Supplementary Figure S2A, B). These find- ings indicate that MCL-1 inhibition increased cellular sensitivity in a stress-dependent and cell-intrinsic man- ner. Since knockdown of MCL-1 was not complete in CD34+ cells, we hypothesized that ER stress itself reduced MCL-1 levels, thereby leading to critical deple- tion of this protein once combined with the gene knock- down. Indeed, we found significant and dose-dependent downregulation of MCL-1 mRNA when CD34+ cells were treated with tunicamycin (Figure 1G). At the same time, tunicamycin led to downregulation of BCL-XL mRNA and upregulation of the BH3-only proteins PUMA and BMF (Online Supplementary Figure S2C). Upregulation of CHOP and PERK mRNA confirmed the presence of ER stress (Online Supplementary Figure S2D). In summary, ER stress shifts the BCL-2 equilibrium towards apoptosis, which is initiated once levels of
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