INHIBITORS OF ADRENOMEDULLIN FOR THE TREATMENT OF ACUTE MYELOID LEUKEMIA BY ERADICATING LEUKEMIC STEM CELLS
20230037414 · 2023-02-09
Inventors
Cpc classification
A61K31/713
HUMAN NECESSITIES
C12N15/113
CHEMISTRY; METALLURGY
International classification
C07K16/28
CHEMISTRY; METALLURGY
A61K31/713
HUMAN NECESSITIES
Abstract
The emergence of cells with drug resistant and/or stem cell features might explain frequent relapses and the poor outcome of patients with acute myeloid leukemia (AML). LSCs are heterogeneous for their phenotypes and their sensitivity to chemotherapeutic agents in vivo. Using in silico and functional approaches, the inventors uncovered that CALCRL is overexpressed in LSCs compared with normal hematopoietic cells. They further demonstrated that the CALCRL ligand adrenomedullin (ADM) is highly expressed in AML cells and that increased transcript level was markedly associated with decreased complete remission rates, 5-year overall and event7free survival. The inventors also showed that CALCRL depletion strongly affected leukemic growth in vivo and increased mice survival. Targeting ADM phenocopies the biological and anti-leukemic effects of the CALCRL depletion. These data highlight the critical role of ADM and disclose a promising therapeutic target to specifically eradicate R-LSCs and overcome relapse in AML.
Claims
1. A method of depleting leukemic stem cells in a subject suffering from acute myeloid leukemia (AML) comprising administering to the subject a therapeutically effective amount of an inhibitor of adrenomedullin activity or expression thereby depleting said leukemic stem cells.
2. A method for preventing relapse of a patient suffering from AML who was treated with chemotherapy comprising administering to the subject a therapeutically effective amount of an inhibitor of adrenomedullin activity or expression.
3. A method of treating chemoresistant acute myeloid leukemia (AML) in a patient in need thereof comprising administering to the patient a therapeutically effective amount of an inhibitor of adrenomedullin activity or expression.
4. The method of claim 3 wherein the leukemia is resistant to a combination of daunorubicin plus cytarabine (AraC), or idarubicin plus cytarabine (AraC).
5. The method according to claim 1, wherein the inhibitor of adrenomedullin activity is a polypeptide that binds to adrenomedullin and comprises all or a portion of the extracellular domains of CALCRL so as to form a soluble receptor that is capable of trapping adrenomedullin.
6. The method according to claim 1, wherein the inhibitor of adrenomedullin activity is an antibody that binds to adrenomedullin.
7. The method of claim 6 wherein the antibody binds to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 consecutive amino acids located in the sequence which ranges from the amino acid residue at position 42 to the amino acid residue at position 52 in SEQ ID NO:1.
8. The method of claim 6 wherein the antibody binds to the sequence which ranges from the amino acid residue at position 42 to the amino acid residue at position 52 in SEQ ID NO:1.
9. The method according to claim 1, wherein the inhibitor of adrenomedullin expression is an antisense oligonucleotide or a siRNA that directly blocks the translation of the mRNA encoding for the precursor of adrenomedullin by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of adrenomedullin.
Description
FIGURES
[0039]
[0040] (A) Overall survival and Event-Free Survival according to ADM H-scores. (B) Overall and Event-Free Survival according to CALCRL and ADM H-scores. *p<0.05; **p<0.01; ***p<0.001; ns, not significant.
[0041]
[0042] (A) Western blot results showing expression of ADM and 3-ACTIN proteins in MOLM-14 and OCI-AML3 four days after transduction with shADM. (B) Graph shows cell number of MOLM-14 or OCI-AML3. Three days after transduction, cells were plated at 0.3M cells per ml (DO) and cell proliferation was followed using trypan blue exclusion. (C) Graph shows the percentage of Annexin-V+ or 7-AAD+ cells 4 days after cell transduction.
[0043]
[0044] Graph shows the percentage of Annexin-V+ or 7-AAD+ cells. Three days after transduction with shADM.
[0045]
[0046] (A) Percentage of human cells in the murine bone marrow in PBS and AraC-treated mice. (B) Western-Blot and graph showing the protein expression of ADM in the bone marrow supernatant of xenografted mice treated with PBS or AraC.
EXAMPLE
[0047] Methods
[0048] Human Studies
[0049] Primary AML patient specimens are from Toulouse University Hospital (TUH), Toulouse, France]. Frozen samples were obtained from patients diagnosed with AML at TUH after signed informed consent in accordance with the Declaration of Helsinki, and stored at the HIMIP collection (BB-0033-00060). According to the French law, HIMIP biobank collection has been declared to the Ministry of Higher Education and Research (DC 2008-307, collection 1) and obtained a transfer agreement (AC 2008-129) after approbation by the Comité de Protection des Personnes Sud-Ouest et Outremer II (ethical committee). Clinical and biological annotations of the samples have been declared to the CNIL (Comité National Informatique et Libertés ie Data processing and Liberties National Committee). See Table S3 for age, sex, cytogenetics and mutation information on human specimens used in the current study.
[0050] In vivo animal studies NSG (NOD.Cg-Prkdcscid Il2rgtmlWjI/SzJ) mice (Charles River Laboratories) were used for transplantation of AML cell lines or primary AML samples. Male or Female mice ranging in age from 6 to 9 weeks were started on experiment and before cell injection or drug treatments, mice were randomly assigned to experimental groups. Mice were housed in sterile conditions using HEPA-filtered micro-isolators and fed with irradiated food and sterile water in the Animal core facility of the Cancer Research Center of Toulouse (France). All animals were used in accordance with a protocol reviewed and approved by the Institutional Animal Care and Use Committee of Region Midi-Pyrenees (France).
[0051] Cell lines and primary cultures For primary human AML cells, peripheral blood or bone marrow samples were frozen in FCS with 10% DMSO and stored in liquid nitrogen. The percentage of blasts was determined by flow cytometry and morphologic characteristics before purification. Cells were thawed in 37° C. water bath, washed in thawing media composed of IMDM, 20% FBS. Then cells were maintained in IMDM, 20% FBS and 1% Pen/Strep (GIBCO) for all experiments.
[0052] Cell Lines and Culture Conditions
[0053] Human AML cell lines were maintained in RPMI-media (Gibco) supplemented with 10% FBS (Invitrogen) in the presence of 100 U/mL of penicillin and 100 g/mL of streptomycin, and were incubated at 37° C. with 5% CO2. The cultured cells were split every 2 to 3 days and maintained in an exponential growth phase. All AML cell lines were purchased at DSMZ or ATCC, and their liquid nitrogen stock were renewed every 2 years. These cell lines have been routinely tested for Mycoplasma contamination in the laboratory. The U937 cells were obtained from the DSMZ in February 2012 and from the ATCC in January 2014. MV4-11 and HL-60 cells were obtained from the DSMZ in February 2012 and 2016. KG1 cells were obtained from the DSMZ in February 2012 and from the ATCC in March 2013. KG1a cells were obtained from the DSMZ in February 2016. MOLM14 was obtained from Pr. Martin Carroll (University of Pennsylvania, Philadelphia, Pa.) in 2011.
[0054] Mouse Xenograft Model
[0055] NSG mice were produced at the Genotoul Anexplo platform at Toulouse (France) using breeders obtained from Charles River Laboratories. Transplanted mice were treated with antibiotic (Baytril) for the duration of the experiment. For experiments assessing the response to chemotherapy in PDX models, mice (6-9 weeks old) were sublethally treated with busulfan (30 mg/kg) 24 hours before injection of leukemic cells. Leukemia samples were thawed in 37° C. water bath, washed in IMDM 20% FBS, and suspended in Hank's Balanced Salt Solution at a final concentration of 1-10×106 cells per 200 μL for tail vein injection in NSG mice. Eight to 18 weeks after AML cell transplantation and when mice were engrafted (tested by flow cytometry on peripheral blood or bone marrow aspirates), NSG mice were treated by daily intraperitoneal injection of 60 mg/kg AraC or vehicle (PBS) for 5 days. AraC was kindly provided by the pharmacy of the TUH. Mice were sacrificed at day 8 to harvest human leukemic cells from murine bone marrow. For AML cell lines, 24 hours before injection of leukemic cells mice were treated with busulfan (20 mg/kg). Then cells were thawed and washed as previously described, suspended in HBSS at a final concentration of 2×106 per 200 μL before injection into bloodstream of NSG mice. For experiments using inducible shRNAs, doxycycline (0.2 mg/ml+1% sucrose) was added to drinking water the day of cell injection or 10 days after until the end of the experiment. Mice were treated by daily intraperitoneal injection of 30 mg/kg AraC for 5 days and sacrificed at day 8. Daily monitoring of mice for symptoms of disease (ruffled coat, hunched back, weakness, and reduced mobility) determined the time of killing for injected animals with signs of distress.
[0056] Assessment of Leukemic Engraftment
[0057] At the end of experiment, NSG mice were humanely killed in accordance with European ethics protocols. Bone marrow (mixed from tibias and femurs) and spleen were dissected and flushed in HBSS with 1% FBS. MNCs from bone marrow, and spleen were labeled with anti-hCD33, anti-mCD45.1, anti-hCD45, anti-hCD3 and/or anti-hCD44 (all from BD) antibodies to determine the fraction of viable human blasts (hCD3-hCD45+mCD45.1-hCD33+/hCD44+AnnV-cells) using flow cytometry. In some experiments, we also added anti-CALCRL, anti-CD34 and anti-CD38 to characterize AML stem cells. Monoclonal antibody recognizing extracellular domain of CALCRL was generated in the lab with the help of Biotem company (France). Then antibody was labelled with R-Phycoerythrin using Lightning-Link kit (Expedeon). All antibodies were used at concentrations between 1/50 and 1/200 depending on specificity and cell density. Analyses were performed on a LSRFortessa flow cytometer with DIVA software (BD Biosciences) or CytoFLEX flow cytometer with CytoExpert software (Beckman Coulter). The number of AML cells/μL peripheral blood and number of AML cells in total cell tumor burden (in bone marrow and spleen) were determined by using CountBright beads (Invitrogen) using described protocol.
[0058] For LDA experiments, human engraftment was considered positive if at least >0.1% of cells in the murine bone marrow were hCD45+mCD45.1-hCD33+. The cut-off was increased to >0.5% for AML #31 because the engraftment was measured only based on hCD45+mCD45.1−. Limiting dilution analysis was performed using ELDA software.
[0059] Western Blot Analysis
[0060] Proteins were resolved using 4% to 12% polyacrylamide gel electrophoresis Bis-Tris gels (Life Technology, Carlsbad, Calif.) and electrotransferred to nitrocellulose membranes. After blocking in Tris-buffered saline (TBS) 0.1%, Tween 20%, 5% bovine serum albumin, membranes were immunostained overnight with appropriate primary antibodies followed by incubation with secondary antibodies conjugated to HRP. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL Supersignal West Pico; Thermo Fisher Scientific) with a Syngene camera. Quantification of chemiluminescent signals was done with the GeneTools software from Syngene.
[0061] Cell Death Assay
[0062] After treatment, 5.105 cells were washed with PBS and resuspended in 200 L of Annexin-V binding buffer (BD biosciences). Two microliters of Annexin-V-FITC (BD Biosciences) and 7-amino-actinomycin D (7-AAD; Sigma Aldrich) were added for 15 minutes at room temperature in the dark. All samples were analyzed using LSRFortessa or CytoFLEX flow cytometer.
[0063] Cell Cycle Analysis
[0064] Cells were harvested, washed with PBS and fixed in ice-cold 70% ethanol at −20° C. Cells were then permeabilized with 1×PBS containing 0.25% Triton X-100, resuspended in 1×PBS containing 10 μg/ml propidium iodide and 1 μg/ml RNase, and incubated for 30 min at 37° C. Data were collected on a CytoFLEX flow cytometer.
[0065] Clonogenic Assay
[0066] Primary cells from AML patients were thawed and resuspended in 100 μl Nucleofector Kit V (Amaxa, Cologne, Germany). Then, cells were nucleofected according to the manufacturer's instructions (program U-001 Amaxa, Cologne, Germany) with 200 nM siRNA scrambled (ON-TARGETplus Non-targeting siRNA #2, Dharmacon) or anti-CALCRL (SMARTpool ON-TARGETplus CALCRL siRNA, Dharmacon). Cells were adjusted to 1×105 cells/ml final concentration in H4230 methylcellulose medium (STEMCELL Technologies) supplemented with 10% 5637-CM as a stimulant and then plated in 35-mm petri dishes in duplicate and allowed to grow for 7 days in a humidified CO2 incubator (5% CO2, 37° C.). At day 7, the leukemic colonies (more than five cells) were scored.
[0067] shRNA, Lentiviral Production and Leukemic Cell Transduction
[0068] shRNA sequences were constructed into pLKO-TET-ON or bought cloned into pLKO vectors. Each construct (6 μg) was co-transfected using lipofectamine 2000 (20 μL) in 10 cm-dish with psPax2 (4 μg, provides packaging proteins) and pMD2.G (2 μg, provides VSV-g envelope protein) plasmids into 293T cells to produce lentiviral particles. Twenty-four hours after cell transfection, medium was removed and 10 ml opti-MEM+1% Pen/Strep was added. At about 72 hours post transfection, 293T culture supernatants containing lentiviral particles were harvested, filtered, aliquoted and stored in −80° C. freezer for future use. At the day of transduction, cells were infected by mixing 2.106 cells in 2 ml of freshly thawed lentivirus and Polybrene at a final concentration of 8 ug/ml. At 3 days post infection, transduced cells were selected using 1 μg/ml puromycin.
[0069] EC50 Experiments
[0070] The day before experiment, cells were adjusted to 3×105 cells/ml final concentration and plated in a 96-well plate (final volume: 100 l). To measure half-maximal inhibitory concentration (EC50), increased concentrations of AraC or idarubicin were added to the medium. After two days, 20 l per well of MTS solution (Promega) was added for two hours and then absorbance was recorded at 490 nm with a 96-well plate reader. The doses that decrease cell viability to 50% (EC50) were analyzed Nonlinear regression log [inhibitor] vs. normalized response-variable slope with GraphPad Prism software.
[0071] Measurement of oxygen consumption in AML cultured cells using Seahorse Assay All XF assays were performed using the XFp Extracellular Flux Analyser (Seahorse Bioscience, North Billerica, Mass.). The day before the assay, the sensor cartridge was placed into the calibration buffer medium supplied by Seahorse Biosciences to hydrate overnight. Seahorse XFp microplates wells were coated with 50 μl of Cell-Tak (Corning; Cat #354240) solution at a concentration of 22.4 μg/ml and kept at 4° C. overnight. Then, Cell-Tak coated Seahorse microplates wells were rinsed with distillated water and AML cells were plated at a density of 105 cells per well with XF base minimal DMEM media containing 11 mM glucose, 1 mM pyruvate and 2 mM glutamine. Then, 180 μl of XF base minimal DMEM medium was placed into each well and the micrcoplate was centrifuged at 80 g for 5 min. After one hour incubation at 37° C. in CO2 free-atmosphere, basal oxygen consumption rate (OCR, as a mitochondrial respiration indicator) and extracellular acidification rate (ECAR, as a glycolysis indicator) were performed using the XFp analyzer.
[0072] RNA Microarray and Bioinformatics Analyses
[0073] For primary AML samples, human CD45+CD33+ were isolated using cell sorter cytometer from engrafted BM mice (for 3 primary AML specimens) treated with PBS or treated with AraC. RNA from AML cells was extracted using Trizol (Invitrogen) or RNeasy (Qiagen). For MOLM-14 AML cell line, mRNA from 2.106 of cells was extracted using RNeasy (Qiagen). RNA purity was monitored with NanoDrop 1ND-1000 spectrophotometer and RNA quality was assessed through Agilent 2100 Bionalyzer with RNA 6000 Nano assay kit. No RNA degradation or contamination were detected (RIN>9). 100 ng of total RNA were analysed on Affymetrix GeneChip© Human Gene 2.0 ST Array using the Affymetrix GeneChip© WT Plus Reagent Kit according to the manufacturer's instructions (Manual Target Preparation for GeneChip® Whole Transcript (WT) Expression Arrays P/N 703174 Rev. 2). Arrays were washed and scanned; and the raw files generated by the scanner was transferred into R software for preprocessing (with RMA function, Oligo package), quality control (boxplot, clustering and PCA) and differential expression analysis (with eBayes function, LIMMA package). Prior to differential expression analysis, all transcript clusters without any gene association were removed. Mapping between transcript clusters and genes were done using annotation provided by Affymetrix (HuGene-2_0-st-v1.na36.hg19.transcript.csv) and the R/Bioconductor package hugene20sttranscriptcluster.db. p-values generated by the eBayes function were adjusted to control false discovery using the Benajmin and Hochberg's procedure. [RMA] Irizarry et al., Biostatistics, 2003; [Oligo package] Carvalho and Irizarry, Bioinformatics, 2010; [LIMMA reference] Ritchie et al., Nucleic Acids Research, 2015; hugene20sttranscriptcluster.db.MacDonald J W 2017, Affymetrix hugene20 annotation data (chip hugene20sttranscriptcluster); [FDR]: Benjamini et al., Journal of the Royal Statistical Society, 1995.
[0074] GSEA Analysis
[0075] GSEA analysis was performed using GSEA version 3.0 (Broad Institute). Gene signatures used in this study were from Broad Institute database, literature, or in-house built. Following parameters were used: Number of permutations=1000, permutation type=gene_set. Other parameters were left at default values.
[0076] Quantification and Statistical Analysis
[0077] We assessed the statistical analysis of the difference between two sets of data using two-tailed (non-directional) Student's t test with Welch's correction. For survival analyses, we used Log-rank (Mantel-Cox) test. For Limit Dilution Assay experiments, frequency and statistics analyses were performed using L-calc software (Stemcell technologies). A p value of less than 0.05 indicates significance. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, not significant. Detailed information of each test is in the figure legends.
[0078] All publicly accessible transcriptomic databases of AML patients used in this study: GSE30377: Eppert K, Takenaka K, Lechman E R, Waldron L, Nilsson B, van Galen P, Metzeler K H, Poeppl A, Ling V, Beyene J, Canty A J, Danska J S, Bohlander S K, Buske C, Minden M D, Golub T R, Jurisica I, Ebert B L, Dick J E. (28 Aug. 2011) Stem cell gene expression programs influence clinical outcome in human leukemia. Nat Med, 17(9), 1086-93.
[0079] GSE14468: Verhaak R G, Wouters B J, Erpelinck C A, Abbas S, Beverloo H B, Lugthart S, Lowenberg B, Delwel R, Valk P J. (January 2009) Prediction of molecular subtypes in acute myeloid leukemia based on gene expression profiling. Haematologica, 94(1), 131-4.
[0080] GSE12417: Metzeler K H, Hummel M, Bloomfield C D, Spiekermann K, Braess J, Sauerland M C, Heinecke A, Radmacher M, Marcucci G, Whitman S P, Maharry K, Paschka P, Larson R A, Berdel W E, Büchner T, Wörmann B, Mansmann U, Hiddemann W, Bohlander S K, Buske C; Cancer and Leukemia Group B; German A ML Cooperative Group. (15 Nov. 2008) An 86-probe-set geneexpression signature predicts survival in cytogenetically normal acute myeloid leukemia. Blood, 112(10), 4193-201.
[0081] GSE116256: Van Galen P, Hovestadt V, Wadsworth Ii M H, Hughes T K, Griffin G K, Battaglia S, Verga J A, Stephansky J, Pastika T J, Lombardi Story J, Pinkus G S, Pozdnyakova O, Galinsky I, Stone R M, Graubert T A, Shalek A K, Aster J C, Lane A A, Bernstein B E. Single-cell RNA-seq reveals AML hierarchies relevant to disease progression and immunity. Cell. 2019 March 7; 176(6):1265-1281.
[0082] TCGA: The Cancer Genome Atlas Research Network. (30 May 2013) Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. N Engl J Med, 368(22), 2059-74. Erratum in: N Engl J Med. 2013 Jul. 4; 369(1):98.
[0083] Results
[0084] The Receptor CALCRL and its Ligand Adrenomedullin are Expressed in AML Cells and Associated with a Poor Outcome in Patients
[0085] Using a clinically relevant chemotherapeutic model, we and others previously demonstrated that LSCs are not necessarily enriched in AraC residual AML, suggesting that these cells are also targeted by chemotherapy and LSCs are comprised of both chemosensitive and chemoresistant stem cell sub-populations (Farge et al., 2017; Boyd et al., 2018). In order to identify chemoresistant LSCs which are at the origin of AML regeneration after chemotherapy, we analyzed transcriptomic data from three different studies that (data not shown): i) identified 134 genes overexpressed in functionally defined LSC compared with a normal HSC counterpart (Eppert et al., 2011; GSE30377); ii) uncovered 114 genes of high expression associated with poor prognosis in AML (the Cancer Genome Atlas, AML cohort, 2013); and iii) selected 536 genes overexpressed at relapse compared to pairwise matched diagnosis after intensive chemotherapy (Hackl et al., 2015; GSE66525). Surprisingly, we found one unique gene common to these three independent transcriptomic databases: CALCRL encoding for a G protein-coupled seven-transmembrane domain receptor poorly documented in cancer that has been recently described as associated with bad prognosis in AML (Angenendt et al, 2019). Using three independently published cohorts of AML patients (TCGA AML cohort; GSE12417; GSE14468), we confirmed that patients with high CALCRL expression had a worse overall survival (data not shown) and are more refractory to chemotherapy (data not shown) compared to patients with low CALCRL expression. This is correlated with a greater expression in complex and normal karyotypes compared with Core Binding Factor AML (CBF) (data not shown). Furthermore, CALCRL gene expression was significantly higher at relapse compared to diagnosis in patients treated with intensive chemotherapy (data not shown). CALCRL expression was also higher in the leukemic compartment compared with normal hematopoietic cells, and more specifically in the LSC population as both functionally—(data not shown) and phenotypically—(data not shown) defined compared with the AML bulk population. Interestingly, CALCRL expression negatively correlated with increasing FAB type, suggesting that CALCRL is a marker of cell immaturity (data not shown). Then, we confirmed by flow cytometry analysis that cell surface expression of CALCRL is higher in the leukemic bulk (n=37) vs. normal bulk (n=9) population (Fold change, FC=2.3; data not shown) Next, we assessed the expression of ADM, a CALCRL ligand already described in several cancers (Berenguer-Daizé et al., 2013; Kocemba et al., 2013). TheADMgene is overexpressed in AML cells compared to normal cells (data not shown) although its expression is not altered in AML patients at relapse compared to initial diagnosis (data not shown). Using a combination of western blotting, confocal microscopy and RNA microarray, we have established that CALCRL, its three co-receptors RAMP1, RAMP2 and RAMP3, ADM (but not CGRP, another putative CALCRL ligand) are expressed in all tested AML cell lines and primary AML samples (data not shown). Then we addressed the impact of CALCRL and ADM protein level on patient outcome. Using IHC analyses, we observed that increasing protein levels of CALCRL or ADM (
[0086] The CALCRL-ADM Axis is Required for Cell Growth and Survival
[0087] Given the role of several GPCRs identified in AML such as CXCR4 or GPR56 on cell survival and proliferation (Chen et al., 2013; Daria et al., 2016; Pabst et al., 2016), we then investigated whether the CALCRL-ADM axis had an impact on these properties. CALCRL depletion was associated with a decrease of blast cell proliferation (data not shown), an increase in cell death (data not shown) in three AML (MOLM-14, OCI-AML2, OCI-AML3) cell lines. Furthermore, ADM-targeting shRNA (
[0088] CALCRL is Required for Leukemic Stem Cell Maintenance
[0089] As CALCRL expression is linked to an immature phenotype and CALCRL depletion impaired AML growth in cell lines, we next aimed to address the role of CALCRL in LSC biology. First, we analyzed previously published single cell RNA-sequencing data (van Galen et al., 2019) and observed that CALCRL is preferentially expressed in HSC-like and progenitor-like cells (Prog-like cells compared with more committed cells in 11 AML patients (data not show). Moreover, while HSC-like and Prog-like cells represent only 34.3% of the total of leukemic cells found in these patients, they accounted for more than 80% of CALCRL.sup.positive cells (data not show). Gene Set Enrichment Analysis (GSEA) confirmed that several LSC-associated gene signatures (Eppert et al., 2011; Gentles, 2010; Ng et al., 2016) (data not show) are significantly enriched in AML patients (the Cancer Genome Atlas, AML cohort, 2013) exhibiting the highest CALCRL expression compared to AML patients with the lowest CALCRL expression (data not show). To functionally investigate the role of CALCRL in LSC biology, we performed Limit Dilution Assay (LDA) from sorted CALCRL.sup.negative and CALCRL.sup.positive cell subpopulations (data not show) followed by injection of increasing cell doses in immunocompromised NSG mice and showed that the CALCRL.sup.positive cell subpopulation was enriched in LSCs compared with the CALCRL.sup.negative one (data not show). Then we performed ex vivo assays knocking down CALCRL in 4 primary AML samples followed by LDA and demonstrated that in all tested samples CALCRL invalidation significantly decreased the frequency of LSCs in vivo both in primary and secondary transplantations (data not show), demonstrating its requirement in preserving the function of LSCs.
[0090] Depletion of CALCRL Alters Cell Cycle and DNA Repair Pathways in AML
[0091] To examine regulatory pathways downstream of CALCRL, we generated and performed comparative transcriptomic and functional assays on shCTR vs shCALCRL MOLM-14 cells. CALCRL knockdown is associated with a significant decrease in the expression of 623 genes and an increase of 278 (FDR<0.05) (data not show). Data mining analyses showed significant depletion in genes involved in cell cycle and DNA integrity pathways in shCALCRL AML cells (data not show). Western blotting confirmed that CALCRL knockdown affected the expression of RAD51, CHEK1 and BCL2 protein levels in MOLM-14 and OCI-AML3 cells, in particular in the former (data not show). This was associated with an accumulation of cells in the G.sub.0/G.sub.1 phase (data not show). Interestingly, enrichment analysis showed that depletion of CALCRL affects the gene signatures of several key transcription factors such as E2F1, P53 or FOXM1 described as critical cell cycle regulators (data not show). We focused on the E2F1 transcription factor, whose importance in the biology of leukemic stem/progenitors cells has recently been shown (Pelicano et al., 2018). We first confirmed that CALCRL depletion was closely associated with a significant decrease in the activity of E2F1 (data not show). Then, we demonstrated that the knockdown of E2F1 affected protein expression of RAD51, CHK1 but not BCL2 (data not show), inhibited cell proliferation (data not show), cell cycle progression (data not show) and induced cell death in both MOLM-14 and OCI-AML3 (data not show). We further investigated whether CALCRL might regulate the proliferation of primary AML cells. Interestingly, CALCRL protein level positively correlated with clonogenic capacities in methylcellulose (data not show). As expected the depletion of CALCRL in primary samples decreased the number of colonies (data not show), and BCL2 and RAD51 protein levels (data not show). All these results suggested that CALCRL had a role in the proliferation of AML blasts and controls critical pathways involved in DNA repair processes.
[0092] CALCRL Downregulation Sensitizes Leukemic Cells to Chemotherapeutic Drugs Cytarabine and Idarubicin
[0093] Based on CALCRL-regulated target proteins such as BCL2, CHK1 or FOXM1 (David et al., 2016; Khan et al., 2017; Konopleva et al., 2016), we hypothesized that CALCRL was involved in chemoresistance. Accordingly, CALCRL depletion sensitized MOLM-14 and OCI-AML3 cells to AraC and idarubicin through the reduction of cell viability (data not show) and the induction of cell death (increased AnnV staining, data not show; and increased cleavage of apoptotic proteins Caspase-3 and PARP, data not show). Furthermore, the depletion of ADM or E2F1 also sensitized AML cells to these genotoxic agents (
[0094] To evaluate the role of CALCRL in this chemoresistance pathway in vivo, we depleted CALCRL in these cells. Knockdown of CALCRL by two different shRNAs sensitized cells to AraC and idarubicin compared to shCTR in cells treated with vehicle (data not show) or AraC alone (data not show). Remarkably, the EC50 of AraC and idarucibin in AraC-treated cells in vivo and transduced with shCALCRL was similar to that observed for MOLM-14 cells from vehicle-treated mice and then transduced with shCTR. Because mitochondrial metabolism has emerged as a critical regulator of cell proliferation and survival in basal and chemotherapy-treated conditions in AML (Li et al 2019; Molina et al., 2018; Scotland et al., 2013; Sriskanthadevan et al., 2015; Farge et al., 2017; Laganidou et al 2013; Jones et al 2018, Pollyea et al 2019), we also analyzed the impact of CALCRL depletion on mitochondrial function. GSEA showed a significant depletion in the gene signature associated with mitochondrial oxidative metabolism in the shCALCRL MOLM-14 cells (data not show). OCR measurements revealed a modest but significant reduction in basal OCR, whereas maximal respiration was conserved, indicating that mitochondria remain functional (data not show). We also consistently observed a significant decrease in mitochondrial ATP production by shCALCRL (data not show). We and other groups have previously shown that chemoresistant cells have elevated oxidative metabolism and that targeting mitochondria in combination with conventional chemotherapy may be an innovative therapeutic approach in AML (Lagadinou et al 2013; Farge et al., 2017; Kuntz et al., 2017). Since the depletion of CALCRL modestly decreased OCR and more greatly decreased mitochondrial ATP in AML cells (data not show), we assessed cellular energetic status associated with AraC. Knockdown of CALCRL significantly abrogated the AraC-induced increase of basal respiration and maximal respiration (data not show). Moreover, we observed a decrease in mitochondrial ATP production in response to AraC upon CALCRL silencing (data not show) whereas glycolysis (e.g. ECAR) was not affected (data not show).
[0095] As it has been reported that BCL2 controlled oxidative status in AML cells (Lagadinou et al., 2013), we investigated its role downstream of CALCRL. We showed that upon AraC treatment, the overexpression of BCL2 in MOLM-14 cells (data not show) is sufficient to partially rescue maximal respiration but not basal respiration (data not show). This suggests a role of the CALCRL-BCL2 axis in maintaining some aspects of mitochondrial function in AraC resistant AML cells in response to AraC. This was not related to energy production, as neither mitochondrial ATP production nor ECAR were affected (data not show). Finally, BCL2 overexpression almost entirely inhibited basal apoptosis induced by the depletion of CALCRL and by the combination with AraC or idarubicin (data not show).
[0096] Overall, these results suggest that CALCRL and its downstream signaling pathways mediate chemoresistance of AML cells in a OXPHOS and BCL2-dependent manner.
[0097] Depletion of CALCRL in Residual Disease after AraC Treatment Impedes LSC Function
[0098] To address the role of CALCRL in response to chemotherapy in primary AML samples, we used a clinically relevant PDX model of AraC treatment in AML (Farge et al., 2017). After injection of primary cells into NSG mice and after engraftment was established and confirmed, mice were treated for 5 days with 60 mg/kg/day of AraC and sacrificed at day 8 to study the minimal residual disease (MRD; data not show). We tested and analyzed 10 different PDX models and stratified them according to their response to AraC as low (FC AraC-to-Vehicle<10) or high (FC>10) responders (data not show). The percentage of cells positive for CALCRL in AML bulk was approximately doubled in the low responder group compared to the high responder group (3.6% vs 7.8%; data not show C). We also observed an inverse linear correlation between the percentage of positive cells and the tumor reduction (R.sup.2=0.418; p=0.0434; data not show). Moreover, after AraC treatment a significant increase in the percentage of positive blasts for CALCRL was observed (5.6% vs. 23%; data not show) in all CD34/CD38 subpopulations (data not show) from minimal residual disease. In addition we also observed in PDX models treated 3 days with Idarubicin (data not show) a slight enrichment in CALCRL.sup.positive cells (data not show), and an inverse correlation between the expression of CALCRL and the tumor reduction (data not shown). Although further studies will be required to confirm these results, they suggest that CALCRL expression might also predict the response to anthracyclines. We next investigated the effects of AraC on ADM secretion. For this, we evaluated the protein level of ADM in bone marrow supernatants of mice treated with PBS or AraC. Chemotherapy reduced both percentage of human cells (
DISCUSSION
[0099] Clinical efficacy of LSC-selective targeted therapies has not been proven for AML treatment due to high plasticity and heterogeneity not only for the phenotype (Taussig et al., 2010; Eppert et al., 2011; Sarry et al., 2011) but also for the drug sensitivity (Farge et al., 2017; Boyd et al., 2018) of the LSC population. However, fundamental studies focusing on intrinsic properties of this cell population such as their resistance to chemotherapy are crucially needed for the development of improved and more specific therapies in AML.
[0100] Our study provides key insights of LSC biology and drug resistance and identifies the ADM receptor CALCRL as a master regulator of R-LSCs. Our work first shows that CALCRL gene is overexpressed in the leukemic compartment compared to normal counterpart based on Eppert's study that functionally characterizes LSCs. CALCRL could be specifically upregulated by LSC-related transcription factors such as HIF1α or ATF4 (Wang et al., 2011; van Galen et al., 2018). Indeed, both ADM and CALCRL possess the consensus hypoxia-response element (HRE) in the 5′-flanking region and are HIF1α-regulated genes (Nikitenko et al., 2003). Recently, it has been demonstrated that the integrated stress response and the transcription factor ATF4 is involved in AML cell proliferation and is uniquely active in HSCs and LSCs (van Galen et al., 2018; Heydt et al., 2018). Interestingly, maintenance of murine HSCs under proliferative stress but not steady-state conditions is dependent on CALCRL signaling (Suekane et al., 2019). Accordingly, CALCRL might support leukemic hematopoiesis and overcome stress induced by the high proliferation rate of AML cells.
[0101] Our findings clearly show that targeting CALCRL expression impacts clonogenic capacities, cell cycle progression and genes related to DNA repair and genomic stability. If cancer stem cells and LSCs are predominantly quiescent thereby preserving them from chemotherapy, recent studies suggested that LSCs also display a more active cycling phenotype (Iwasaki et al., 2015; Pei et al., 2018). C-type lectin CD93 is expressed on a subset of actively cycling, non-quiescent AML cells enriched for LSC activity (Iwasaki et al., 2015). Recently, Pei et al. showed that targeting the AMPK-FIS1 axis disrupted mitophagy and induced cell cycle arrest in AML, leading to the depletion of LSC potential in primary AML. These results are consistent with the existence of different sub-populations of LSCs that differ in proliferative state. Moreover, FIS1 depletion induces the down-regulation of several genes (e.g. CCND2, CDC25A, PLK1, CENPO, AURKB) and of the E2F1 gene signature that both we also identified after CALCRL knockdown. Recently, it has been proposed that E2F1 plays a pivotal role in regulating CML stem/progenitor cells proliferation and survival status (Pellicano et al., 2018).
[0102] Several signaling pathways, for instance MAPKs, CDK/cyclin or PI3K/AKT, have been described to be stimulated by ADM/CALCRL axis and may control pRB/E2F1 complex activity (Hallstrom et al., 2008; Wang et al., 1998). Other signaling mediators activated in LSCs such as c-Myc and CEBPα regulate E2F1 transcription and allow the interaction of the E2F1 protein with the E2F gene promoters to activate genes essential for DNA replication at G1/S, cell proliferation and survival in AML (Leung et al., 2008; O'Donnell et al., 2005; Rishi et al., 2014). Therefore, this analysis of cellular signaling downstream of CALCRL uncovers new pathways crucial for the maintenance and the chemoresistance of LSCs.
[0103] The characterization of chemotherapy-spared R-LSCs, which are present at the onset of relapse, is necessary to develop new therapeutic strategies to eradicate them. Boyd and colleagues have proposed the existence of a transient state of Leukemic Regenerating Cells (LRC) during the immediate and acute response to AraC that are responsible for disease regrowth foregoing the recovery of LSC pool (Boyd et al., 2018). In this attractive model and in the dynamic of MRD post-chemotherapy, CALCRL-positive AML cells are a part of this LRC subpopulation and CALCRL is essential for the preservation of LSC potential of chemoresistant primary AML. It would be interesting to determine whether chemotherapy only spares cells that are positive for CALCRL and/or whether it induces an adaptive response to stress that increases the expression of CALCRL. Transcription factors that are activated in response to chemotherapy should be identified to improve our knowledge of the acute response to chemotherapy. Therefore, therapeutic targeting of CALCRL should be clinically investigated to specifically eradicate MRD and prevent relapse in AML. Finally, as several molecules preventing the binding of the neuropeptide CGRP to CALCRL have been developed for treatment in other diseases (Hutchings et al., 2017; Schuster and Rapoport, 2017), this facilitates future pharmacological approaches to antagonize ADM-CALCRL axis in AML.
[0104] In summary, our data clearly identify CALCRL as a new stem cell actor required to sustain AML development in vivo. This receptor regulates genes involved in chemoresistance mechanisms and its depletion sensitizes AML cells to both cytarabine and anthracyclines in vitro and in vivo. This further indicates that LSCs resistant to these drugs share common activated pathways involved in these resistance mechanisms. All of these results strongly suggest CALCRL and AMD is a new and promising candidate therapeutic target for anti-LSC therapy.
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