Pivotal role of the endoplasmic reticulum stress-related XBP1s/miR-22/SIRT1 axis in acute myeloid leukemia apoptosis and response to chemotherapy

Malignant growth relies on rapid protein synthesis frequently leading to endoplasmic reticulum (ER) overload and accumulation of unfolded or misfolded protein in this cellular compartment. In the ER, protein homeostasis is finely regulated by a mechanism called the unfolded protein response (UPR), involving the activation of signalization pathways mediated by three transmembrane proteins, namely PERK, IRE1 and ATF6. IRE1 endoribonuclease activation leads in particular to the splicing of the cytosolic mRNA encoding the key UPR-specific transcription factor XBP1s. Our study shows that sustained activation of XBP1s expression in acute myeloid leukemia (AML) cells induces apoptosis in vitro and in vivo, whereas a moderate XBP1s expression sensitizes cells to chemotherapeutic treatments. ChIP-seq experiments identified specific XBP1s target genes including the MIR22HG lncRNA, the precursor transcript of microRNA-22-3p. miR-22-3p upregulation by XBP1s or forced expression of miR-22 significantly decreases cell’s viability and sensitizes leukemic cells to chemotherapy. We found that miR-22-3p intracellular effects result at least partially from the targeting of the mRNA encoding the deacetylase sirtuin-1 (SIRT1), a well-established pro-survival factor. Therefore, this novel XBP1s/miR-22/SIRT1 axis identified could play a pivotal role in the proliferation and chemotherapeutic response of leukemic cells.


Generation of inducible cell lines and shRNA-Mediated Gene Knockdown
The XBP1 spliced isoform coding sequence was PCR amplified with the proofreading Taq polymerase Phusion (Thermofisher) using the primer pair Xba_XBP1_Fw (GGTCTAGA- TCTTCTAAATCTACCACTT). Stably transduced cells expressing the shRNA were selected by treatment with 1 µg/mL of puromycin.miR-22-inducible models were generated using wild-type OCI-AML2 and OCI-AML3 cells stably transduced with shMIMIC Inducible Lentiviral microRNA (Horizon; reference VSH6906-224634676; miRNA sequence: AA-GCUGCCAGUUGAAGAACUGU).All transductions were performed in the presence of retronectin TM reagent, according to the manufacturer's instructions (Clontech).

Murine xenograft models and following of tumor growth in vivo
NSG mice were treated by an intraperitoneal injection of busulfan (20 mg/kg) to induce medullar aplasia.24h after busulfan treatment, mice were intravenously injected with 2 10 6 OCI-AML3, OCI-AML2 or HL-60 XBP1s-inducible cells or Tet-On control cells.At different time points after engraftment, doxycycline was added at 1mg/mL in drinking water and refreshed every 3 days.Mice daily monitoring was performed to detect symptoms of disease (ruffled coat, hunched back, weakness, and reduced motility).For in-vivo-chemosensitivity assay, 3 days after doxycycline-treatment onset mice were intraperitoneally injected daily with aracytine at 30mg/kg for 5 days.For subcutaneous xenografts, a total of 2.5.10 6 OCI-AML3 XBP1s cells were injected into both flanks of nude mice.Mouse body weight and tumor volumes were measured every day.Doxycycline (different concentration) was added in the drinking water and refreshed every 3 days.At the end of the experiment, mice were humanely sacrificed and subcutaneous tumors were harvested and proteins extracted for further analyses.

Chromatin Immunoprecipitation (ChIP)
ChIP was performed using the ChIP-IT® Express kit from Active Motif.In order to cross-link proteins to DNA, cells were treated with 1% formaldehyde for 10min at room temperature, then with glycine according to the manufacturer's protocol.Chromatin was sheared by sonication into relatively uniform 300pb fragments ("Input" fraction).This chromatin fraction was subsequently immunoprecipitated with two different XBP1 antibodies (Santa Cruz, sc-7160 and Biolegend, 619502), using magnetic beads.IgG isotype (Biolegend, 910801) was used as immunoprecipitation control.DNA was eluted, and then de-crosslinking and purified using phenol-chloroform extraction.The immunopurified fractions obtained with this protocol were then used for ChIP-sequencing and for ChIP-qPCR experiments.For ChIP-qPCR, 1/10 (2μl) of input or immunoprecipitated DNA was analyzed by qPCR with SYBR green (Takara Bio Inc.) on the StepOnePlus real-time PCR system (Applied Biosystems).Results obtained with the immunoprecipitated chromatin fractions are expressed as the percentage of input and represent the mean value of at least three independent ChIP experiments.For each ChIP experiment specificity of the immunoprecipitation was controlled by a parallel ChIP performed with an irrelevant antibody (igG).

RNA-seq et miRnome
Total RNA from OCI-AML3 Tet-ON and XBP1s treated with 4ng or 10 ng of doxycycline for 48h were purified using the Trizol (Ambion) extraction protocol according to the manufacturer's instructions.RNA quantification and purity were determined by using a Clariostar.RNA integrity and quality were determined by using a Fragment Analyzer.Only samples with RNA Integrity Number (RIN) >9 were sent for RNA-Sequencing.Library preparation and sequencing were performed by Eurofins GATC Biotech company.Paired-end, 120 million strandspecific reads of ~125 nucleotides/ sample were generated on an Illumina HiSeqTM2000 for RNA sequencing and 10 million reads of 50 nucleotides /sample were generated for miRnome analysis.

Binding site motifs analysis
Motif analysis was performed following peak calling, using FASTA files as input for the MEME-Chip online tool (meme-suite.org).The program finds motifs centrally enriched and analyses them for similarity.The JASPAR vertebrate's motif database was used as a reference.Default settings were used for this analysis with MEME Motif width between 6 and 30, DREME Motif E-value<0.05,CentriMo motif match score ≥5, and CentriMo E-value threshold <10.

Biotin Pull Down Assay
After a 24h transfection with the biotinylated microRNA-22 mimic the cells were centrifuged and resuspended in an immunoprecipitation (IP)-Buffer (25mM Tris-HCL pH7.4,200mM NaCl, 0.2% Triton TM X-100, 5mM Mg Acetate, 1mM DTT) supplemented with RNAseOUT (Thermo Fisher) and Complete TM protease inhibitor cocktail (Roche).Samples were sonicated (2 times 10s each separated by a 30s incubation on ice using VCX 130 Ultrasonic Processor (Sonics) at 25% of its power) and lysates were used for pull down.Pierce™ Magnetic beads from ThermoFisher were washed with IP-buffer and blocked with yeast tRNA and BSA for 1h at 4°C.Beads were washed twice and incubated with the lysate for at least 1h30 at 4°C on an end-to-end rotator.Beads was washed 5 times with IP buffer and RNA extraction was performed using TRIzol® total RNA isolation reagent (Invitrogen), following the manufacturer's protocol.Then, 11µL of immunoprecipitated RNA extract was used for RT with the Superscript III reverse transcription kit (Invitrogen).RT reactions were diluted 10-fold prior to qPCR (see primer list in the Supplementary Table 2).

Supplementary Table 1: Clinical and mutational features of the AML cell lines used in this study
Supplementary Table 2: List of PCR primers used in these studies

MIR22HG
5'-CCTCGTGCAGCAACCCC-3' 5'-GTGAGGGCGTGAGAGGAAC-3' the pIRES2GFP vector from Clontech as a template.These two PCR products were digested respectively by Xba1/Sal1 and Sal1/Bgl2 and ligated into the pTRIPz-TRE-Tight plasmid (Open Biosystem) digested by Xba1/ Bgl2 leading to the fusion of the XBP1s open reading frame to the EMCV IRES GFP sequence downstream of a doxycycline responsive promoter.Constructs were validated by sequencing and used to transduce OCI-AML2-, OCI-AML3-, MV4-11-, MOLM-14-, THP1-and HL-60-TET-On cell lines, first established in the lab by transduction with pTet-On vector (Clontech) which allows the constitutive expression of the rtTA (reverse tetracycline Transactivator) protein.Selection of XBP1s-inducible cells was based on EGFP expression and performed by flow cytometry after treatment with a low dose of doxycycline during 10H.For generation of stable XBP1 knockdown, wild-type cells were transduced with the lentivirus pTRIPZ-