OncoImmunology ISSN: (Print) 2162-402X (Online) Journal homepage: https://www.tandfonline.com/loi/koni20 IMiDs mobilize acute myeloid leukemia blasts to peripheral blood through downregulation of CXCR4 but fail to potentiate AraC/Idarubicin activity in preclinical models of non del5q/5q- AML Belen Lopez-Millan, Rafael Diaz de la Guardia, Heleia Roca-Ho, Eduardo Anguita, Abul B. M. M. K. Islam, Damia Romero-Moya, Cristina Prieto, Francisco Gutierrez-Agüera, Jose Antonio Bejarano-Garcia, Jose Antonio Perez-Simon, Paula Costales, Montse Rovira, Pedro Marín, Silvia Menendez, Mar Iglesias, Jose Luis Fuster, Alvaro Urbano-Ispizua, Fernando Anjos- Afonso, Clara Bueno & Pablo Menendez To cite this article: Belen Lopez-Millan, Rafael Diaz de la Guardia, Heleia Roca-Ho, Eduardo Anguita, Abul B. M. M. K. Islam, Damia Romero-Moya, Cristina Prieto, Francisco Gutierrez- Agüera, Jose Antonio Bejarano-Garcia, Jose Antonio Perez-Simon, Paula Costales, Montse Rovira, Pedro Marín, Silvia Menendez, Mar Iglesias, Jose Luis Fuster, Alvaro Urbano-Ispizua, Fernando Anjos-Afonso, Clara Bueno & Pablo Menendez (2018) IMiDs mobilize acute myeloid leukemia blasts to peripheral blood through downregulation of CXCR4 but fail to potentiate AraC/Idarubicin activity in preclinical models of non del5q/5q- AML, OncoImmunology, 7:9, e1477460, DOI: 10.1080/2162402X.2018.1477460 To link to this article: https://doi.org/10.1080/2162402X.2018.1477460 © 2018 Taylor & Francis Group, LLC View supplementary material Published online: 26 Jul 2018. 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M. M. K. Islam c, Damia Romero-Moyaa, Cristina Prieto a, Francisco Gutierrez-Agüeraa, Jose Antonio Bejarano-Garciad,e, Jose Antonio Perez-Simond, Paula Costalesf, Montse Rovira g, Pedro Maríng, Silvia Menendezh, Mar Iglesiash, Jose Luis Fusteri, Alvaro Urbano-Ispizuaa,g,j, Fernando Anjos-Afonsok, Clara Buenoa, and Pablo Menendeza,j,l aDepartment of Biomedicine, Josep Carreras Leukemia Research Institute-Campus Clinic, School of Medicine, University of Barcelona, Barcelona, Spain; bHematology Department, Hospital Clínico San Carlos, IdISSC, Universidad Complutense de Madrid, Madrid, Spain; cDepartment of Genetic Engineering and Biotechnology, University of Dhaka, Dhaka, Bangladesh; dHematology department, Universidad de Sevilla, Instituto de Biomedicina de Sevilla (IBiS) Hospital Universitario Virgen del Rocío, CSIC, Seville, Spain; eHematology Department, Hospital Universitario Virgen del Rocío, Seville, Spain; fEntrechem SL, Oviedo, Spain; gHematology Department, Hospital Clínico de Barcelona, Barcelona, Spain; hPathology Service, Hospital del Mar, Barcelona, Spain; iOncohematology department, Sección de Oncohematología Pediátrica, Hospital Clínico Virgen de Arrixaca, Murcia, Spain; jISCIII, Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Barcelona, Spain; kCardiff School of Biosciences, European Cancer Stem Cell Research Institute, Cardiff, UK; lInstituciò Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain ABSTRACT Treatment for acute myeloid leukemia (AML) remains suboptimal and many patients remain refractory or relapse upon standard chemotherapy based on nucleoside analogs plus anthracyclines. The crosstalk between AML cells and the BM stroma is a major mechanism underlying therapy resistance in AML. Lenalidomide and pomalidomide, a new generation immunomodulatory drugs (IMiDs), possess pleio- tropic anti-leukemic properties including potent immune-modulating effects and are commonly used in hematological malignances associated with intrinsic dysfunctional BM such as myelodysplastic syn- dromes and multiple myeloma. Whether IMiDs may improve the efficacy of current standard treatment in AML remains understudied. Here, we have exploited in vitro and in vivo preclinical AML models to analyze whether IMiDs potentiate the efficacy of AraC/Idarubicin-based standard AML chemotherapy by interfering with the BM stroma-mediated chemoresistance. We report that IMiDs do not exert cytotoxic effects on either non-del5q/5q- AML cells nor BM-MSCs, but they enhance the immunomodulatory properties of BM-MSCs. When combined with AraC/Idarubicin, IMiDs fail to circumvent BM stroma- mediated resistance of non-del5q/5q- AML cells in vitro and in vivo but induce robust extramedullary mobilization of AML cells. When administered as a single agent, lenalidomide specifically mobilizes non- del5q/5q- AML cells, but not healthy CD34+ cells, to peripheral blood (PB) through specific down- regulation of CXCR4 in AML blasts. Global gene expression profiling supports a migratory/mobilization gene signature in lenalidomide-treated non-del5q/5q- AML blasts but not in CD34+ cells. Collectively, IMiDs mobilize non-del5q/5q- AML blasts to PB through CXCR4 downregulation, but fail to potentiate AraC/Idarubicin activity in preclinical models of non-del5q/5q- AML. ARTICLE HISTORY Received 8 January 2018 Revised 10 May 2018 Accepted 12 May 2018 KEYWORDS AML; BM-MSC; IMiDs; lenalidomide; pomalidomide; AraC; Idarubicin; xenografts Introduction Acute myeloid leukemia (AML) comprises a biologically and genetically heterogeneous group of disorders characterized by the rapid expansion of immature myeloid blasts in bone marrow (BM).1,2 In contrast to acute lymphoblastic leukemia (ALL), treatment of AML has not improved substantially over the last two decades and many patients still fail to respond to standard intensive chemotherapy based on nucleoside analogs such as cytarabine (AraC) or fludarabine and anthracyclines such as idarubicin and daunorubicin.3,4 Failure of current therapies to eradicate leukemia-initiating/propagating cells (LICs) and chemotherapy refractoriness are the major mechanisms under- lying AML progression/relapse. In addition, the BM stroma has been involved in the pathogenesis of a variety of hematologic malignances including AML.5 The interaction of AML cells with the BM microenvironment in functional niches is a major mechanism underlying leukemia maintenance and therapy resistance.5,6 Thus, the high rate of mortality/morbidity and chemo-resistance in AML guides the search for new compounds with higher efficiency and lower toxicity. New generation of immunomodulatory drugs (IMiDs) such as lenalidomide and pomalidomide are thalidomide ana- logs, which possess pleiotropic anti-leukemic properties CONTACT Pablo Menéndez pmenendez@carrerasresearch.org; Belén López-Millán blopez@carrerasresearch.org Josep Carreras Leukemia Research Institute School of Medicine, University of Barcelona, Casanova 143, 08036 Barcelona, Spain Belen Lopez-Millan and Rafael Diaz de la Guardia contributed equally to this work. Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/koni. Supplemental data for this article can be accessed here. ONCOIMMUNOLOGY 2018, VOL. 7, NO. 9, e1477460 (14 pages) https://doi.org/10.1080/2162402X.2018.1477460 © 2018 Taylor & Francis Group, LLC This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way. http://orcid.org/0000-0002-7274-0855 http://orcid.org/0000-0002-0812-8100 http://orcid.org/0000-0002-6403-3317 http://www.tandfonline.com/koni https://doi.org/10.1080/2162402X.2018.1477460 https://crossmark.crossref.org/dialog/?doi=10.1080/2162402X.2018.1477460&domain=pdf&date_stamp=2018-08-24 including anti-proliferative, anti-angiogenic but also immune- modulating effects.7-9 They are approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA). Lenalidomide is approved for both 5q- mye- lodysplastic syndrome (MDS) and multiple myeloma (MM) whereas pomalidomide is approved for MM, which are hema- tological malignances associated with intrinsic dysfunctional BM.10,11 Some studies reported that lenalidomide has modest (pre)-clinical activity in lymphoma and AML with manage- able toxicity.9,12,13 Ongoing Phase I clinical trials assaying low- and high-dose lenalidomide for both newly diagnosed and relapsed/refractory AML also support that lenalidomide displays modest activity when given either alone or combined with cytarabine and anthracyclines.14 Whether and how IMiDs may benefit current standard treat- ment in AML needs further investigation. To bring IMiDs to the forefront of AML therapeutic arsenal, a better understanding of its mechanism of action in AML is required. In fact, it has recently been shown that BM mesenchymal stromal cells (BM-MSCs) derived from diagnostic AML patients are potent immunomodu- lators and provide similar chemoprotection of AML cells to cytarabine/idarubicin than normal BM-MSCs.5 Although IMiDs may have an anti-proliferative effect on leukemic cells, they may likely exert pleiotropic and immunomodulatory effects at the stroma-leukemic cell crosstalk, thus improving standard AML intensive chemotherapy by either potentiating AraC/anthracy- cline efficacy or reducing BM-MSC-derived chemoresistance. Here, we have exploited in vitro and in vivo AML preclinical models to analyze whether IMiDs potentiate the efficacy of AraC/Idarubicin-based standard chemotherapy by lowering the BM stroma-mediated chemoresistance. Lenalidomide and pomalidomide are not cytotoxic for either non-del5q/5q- AML cells or BM-MSCs, but they enhance the immuno-modulatory capacity of BM-MSCs.When administered alone or in combina- tion with AraC and Idarubicin, IMiDs fail to circumvent BM stroma-mediated resistance of non-del5q/5q- AML cells in vitro and in vivo but induce robust mobilization of non-del5q/5q- AML cells, but not healthy CD34+ cells, to peripheral blood (PB) and spleen likely through specific downregulation of CXCR4 in AML blasts. Global gene expression profiling supports a migra- tory/mobilization gene signature in lenalidomide-treated AML primary blasts. We conclude that IMiDs mobilize non-del5q/5q- AML blasts to PB through downregulation of CXCR4 but do not improve AraC/Idarubicin activity in a preclinical model of AML. Material and methods AML cell lines and primary leukemic and healthy cells HL60 and MOLM-13 cells (kindly provided by Prof. Luciano Di Croce, CRG, Barcelona and Prof Michael Andreeff, MD Anderson, Houston, TX, respectively) were cultured as in RPMI medium supplemented with 10% fetal calf serum (FCS) and antibiotics (Gibco). Patient AML samples (>90% of blasts) were obtained from fresh BM from the Hospital Clínico San Carlos (Madrid, Spain) and Hospital Virgen del Rocío (Seville, Spain). AML diagnosis was based on French-American-British (FAB)15 and World Health Organization (WHO) classifications,16,17 Table 1, summarizes patient’s main clinical/cytogenetic/molecular characteristics. Fresh peripheral blood (PB) and cord blood (CB) units were obtained from healthy donors from the Catalonia Blood Tissue Bank following the institutional guidelines approved by our local Institutional Review Board. G-SCF-mobilized PB and BM were obtained from clinical leftovers from the Hospital Clínic of Barcelona (HCB). PBMCs were isolated using Ficoll-Paque Plus (GE Healthcare) density gradient centrifugation, and CD34+ cells were MACS-purified using the human CD34 MicroBead kit and the AutoMACS device (Miltenyi Biotec, Madrid, Spain) as reported.18,19 The purity of the CD34+ fraction was assessed and was consistently >95%. Primary AML and CD34+ cells were cultured in Stemspam medium (Stem Cell Technologies, Vancouver, Canada) supplemented with the hematopoietic cytokines Stem Cell Factor (100 ng/ mL), FLT3 ligand (100 ng/mL), IL3 (10 ng/mL, all from PeproTech) and antibiotics (Gibco). BM-MSCs were obtained, grown and characterized as extensively described by our group.5,20 Cultures were maintained in a humidified atmosphere with 5% CO2 at 37⁰C. The study was IRB- approved by the HCB (HCB/2014/0687) and samples received upon signed informed consent. Table 1. Biological and cytogenetic-molecular characteristics of blasts from diagnostic AML patients. Patient ID Diagnostic Cytogenetics Molecular Age (years) Gender Blasts (%) AML#1 AML-M4 46, XX FLT3-ITD, NPM1MUT 52 F 81 AML#2 AML-M7 46, XX, 20% 47, XX, +8 t(8;21) AML1-ETO 1 F 52 AML#3 AML-M0/ M1 46, XY - 9 M 86 AML#4 AML 46, XY, del(7)(q22) - 61 M 87 AML#5 AML-M4 46, XY FLT3-ITD, NPM1MUT 48 M 80 AML#6 AML 46–47, XY, t(12;13) BCR-ABL (Ph+) 60 M 37 AML#7 AML 46, XY - 43 M 60 AML#8 AML-M5 46, XY - 47 M 54 AML#9 AML-M5a 46, XX, 92,3% 47, XX, +8, +i(8)(q10) x2, t(8;21), t(9;11)(q22;q23) AML1-ETO, MLL1-AF9, ABL+,WT-1+ 6 F 95 AML#10 AML-M2 44, XX, del(5)(q13q33), −7, del(13)(q12q14), −15[20] - 87 F 40 AML#11 AML 45, X, -Y, del(5)(q13q33), 3 ~ 8dmin[7]/44, idem, −12, der(17)t(12;17)(q11;p11), 3 ~ 34dmin[11]/46,XY[2] - 73 M 71 AML#12 AML 44, XX,del(5)(q13q33), −7, del(13)(q12q14), −15[20] - 79 F 39 AML#13 AML-M4 46, XY, del(3)(q21)[26]/46, idem, del(5)(q22)[3]/46, XY[1] FLT3-ITD, WT-1+ 16 M 75 M, male; F, female; dmin, double minute chromosome; -, no mutations found for FLT3, NMP1, cEBPα, WT and IDH1. e1477460-2 B. LOPEZ-MILLAN ET AL. Drugs AraC (Cytarabine®, Pfizer), idarubicin (Zavedos®, Pfizer) and the CXCR4 antagonist AMD3100 (Sigma) were reconstituted with PBS. Lenalidomide and pomalidomide were kindly pro- vided by Celgene Corp (San Diego, CA), reconstituted in DMSO as per supplier´s guidelines and stored in aliquots at −20⁰C. Cytotoxicity and apoptosis assays The cytotoxic effect of the different drugs on HL-60 and MOLM-13 cell was assessed using Cell Counting Kit-8 based on monosodium salt WST-8 according to the manu- facturer’s protocol (Sigma-Aldrich). Briefly, 5.000 cells were plated in 96-well plate and incubated with increasing con- centrations of the corresponding drug for 48h. After incu- bation time, absorbance was measured at 450 nm using a microplate reader. The cytotoxic effect of IMiDs on BM- MSCs was assessed using MTT assays as previously described.21,22 Briefly, 2 × 104 cells were cultured with increasing drug concentrations in 96-well plates for 48 h. To determine the chemoprotective effect of BM-MSCs a total of 2 × 104 BM-MSCs were plated in 96-well plates 18 h before addition of AML cells (2 × 104 for cell lines and 2 × 105 for primary cells). BM-MSC:AML co-cultures were treated with IC25 concentrations of the AraC (77nM) and Idarubicin (7nM) and 10 µM of lenalidomide/pomalido- mide for 48–72 h. Apoptosis of CD33+ AML cells was measured using the annexin-V/7-AAD apoptosis detection kit (BD Biosciences) on a FACSCanto-II cytometer using FACSDiva software (BD Biosciences) as previously described.23 Immunosuppressive assays and cytokine detection The effect of IMiDs on the BM-MSC-mediated immunosup- pressive properties and cytokine release (TNFα, IL1β, IL10, IL6 and SDF1) was determined by CSFE and Luminex Multiplex assays as previously described.5,24,25 FACS analysis of CXCR4 and SDF1 CXCR4 expression was determined in AML and CD34+ cells treated with IMiDs. A total of 0.2 × 106 (for cell lines) or 1 × 106 (for primary cells) cells were incubated with 10 µM of IMiDs (the CXCR4 antagonist AMD3100 was used as a posi- tive control). Seven days later (48 h–72 h for primary AML cells) treated cells were stained with anti-human CD45-APC. Cy7, CD33-APC, CD34-PECy7 and CD184-BV421 (BD Biosciences) and the mean fluorescence intensity (MFI) of CD184 within the blast population (CD45+CD33+) was FACS-quantified using a FACS Canto II cytometer. The expression of the CXCR4 ligand SDF1 was analyzed in BM- MSC (0.5 x 106) 48 h after treatment with 10 µM IMiDs. BM- MSCs were trypsinized and stained with anti-human CXCL12/SDF-1 (R&D Systems) using the Cell Permeabilization Kit (FIX&PERM®).26 NSGmice xenotransplantation and analysis of engraftment All experimental procedures were approved by the Animal Care Committee of The Barcelona Biomedical Research Park (HRH-16–0037). Eight-to-14-week-old NOD.Cg- PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice housed under patho- gen free conditions were used. In order to recapitulate a BM stroma milieu, 1 × 105 HL60 (n = 31 mice) or MOLM-13 (n = 33 mice) cells were intra-BM transplanted into non- irradiated NSG mice together with 3 × 105 irradiated BM- MSCs.25,27 HL60 AML grafts were monitored by BM aspira- tion and mice were randomized into treatment groups when graft was >1% in the contralateral BM (~2–3 weeks post- transplant). For MOLM-13 AML grafts, mice were rando- mized for treatment 3 days after intra-BM transplant due to its aggressiveness. At the end of the treatment period, mice were sacrificed and cells from the BM, spleen and PB were stained with anti-human HLA-ABC-FITC, CD33-PE and CD45-APC (BD Biosciences) to analyze human leukemic engraftment by flow cytometry. Similarly, healthy CD34+ HSPCs (0.1 × 106 cells) were intra-BM-transplanted into sub- lethally irradiated (2.25 Gy) NSG mice (n = 10) together with 3 × 105 irradiated BM-MSCs. Six weeks later, when human healthy chimerism was evident, mice were treated with lena- lidomide. At the end of lenalidomide treatment, mice were sacrificed and cells from the BM, spleen and PB were stained as above to analyze human normal chimerism by flow cyto- metry. Multilineage engraftment was assessed using CD33- APC, CD19-PE and CD34-PE.Cy7 (BD Biosciences). Spleen infiltration of AML cells was analyzed by immunohistochem- istry using an anti-human-CD45. In vivo chemotherapy and imids treatment Ara-C and idarubicin were administrated following a well- established 5 + 3 treatment schedule, consisting on intrave- nous (i.v.) co-delivery of 30 mg/Kg of Ara-C and 0.1 mg/Kg of idarubicin for 3 days followed by intraperitoneal injection (i.p.) of Ara-C for two further days (A + I treatment).28 Lenalidomide (25 mg/kg) given as single agent was admini- strated i.p. for 10 days. When combined with A + I treat- ment, lenalidomide (25 mg/kg) was administrated alone during days 1–3 and 9–10 and with A + I on days 4–8. 1% DMSO/PBS solution was always used as vehicle treatment. Mice were weighted throughout treatment and drug doses were recalculated as necessary. Microarray gene expression profiling (GEP) Non-del5q/5q- AML primary samples (n = 3) and BM-CD34+ HSPCs (n = 2) were FACS purified (CD45+CD33+ purity >98%) and cultured with 10 µM lenalidomide for 48h before global gene expression profiling (GEP) as described.29 Total RNA was then extracted using a Maxwell® RSC simplyRNA Cells Kit in a Maxwell® RSC Instrument (Promega) and its quality checked in the Agilent 2100 Bioanalyzer. Total RNA samples were labeled with Cy3 using the Quick-Amp Labeling Kit and hybridized with the Gene Expression Hybridization Kit to the GeneChip Human Gene 2.0 ST (Affymetrix) following Manufacturer’s instructions. ONCOIMMUNOLOGY e1477460-3 Hierarchical clustering of genes was performed with the one minus correlation metric and the unweighted average distance. Only genes showing >1.5-fold change expression and p-value<0.05 were considered differentially expressed and were subjected to gene ontology (GO) term analysis using Gorilla30,31 publicly available at http://cbl-gorilla.cs.technion.ac.il. Microarray data were deposited in the public Gene Expression Omnibus database, accession number GSE106748. Results BM stroma/milieu confers resistance of AML cells to AraC/idarubicin Chemotherapy based on the combination of Ara-C with anthracyclines, either idarubicin or daunorubicin, is the first-line induction treatment in diagnostic AML.3,4 Here, viability in vitro assays revealed that both cytotoxic drugs, Ara-C and idarubicin, are equally toxic in different AML cell lines with an IC50 of ~10 −7M (Fig 1S). Exposure to Ara- C plus idarubicin (Ara-C/Ida) resulted in 80%–95% cell death of AML cell lines, measured by Annexin V staining (Figure 1A). However, when co-cultured with BM-MSCs, AML cell lines were chemo-resistant with only 40–50% cell death (Figure 1A). To further test whether the BM stroma/ milieu protects AML cells from Ara-C/Ida treatment, AML cell lines were co-transplanted via intra BM transplant (IBMT) into NSG mice with human BM-MSCs which largely increase AML engraftment (Fig 2S), as reported in several hematologic malignances.32-35 When engraftment was >1% in non-injected tibia, mice were treated with Ara-C/Ida admi- nistrated following the well-established 5 + 3 treatment schedule.28 Similar to the in vitro results, Ara-C/Ida signifi- cantly (2-3-fold) reduced AML engraftment/survival in Figure 1. BM microenvironment protects HL60 and MOLM-13 cells from AraC+Idarubicin-based chemotherapy in vitro (A) and in vivo (B). (n = 3). *p < 0.05. **p < 0.01. e1477460-4 B. LOPEZ-MILLAN ET AL. http://cbl-gorilla.cs.technion.ac.il extramedular BM hematopoietic tissues and in contralateral BM (CL) whereas the co-transplanted human BM-MSCs con- ferred chemoresistant to AML cells in injected tibia (IT) (Figure 1B). Imids exert no toxicity on non-del5q/5q- AML cells but increase immunosuppressive properties of BM-MSCs The immune-modulating properties of BM stroma/BM- MSCs are a major mechanism underlying BM stroma- mediated chemoresistance in hematological malignances.5 Lenalidomide and pomalidomide possess pleiotropic anti- tumor properties including also tumor-controlling immune- modulating effects in other hematological BM malignances such as MDS and MM.9-11 We therefore set out to assess the anti-leukemia effects of lenalidomide and pomalidomide in AML. Strikingly, as single agents, lenalidomide and pomalidomide display no cytotoxic effects (assayed at 48h and 72h after drug exposure) on neither AML cell lines (Figure 2A) nor diagnostic primary AMLs (n = 9) covering different cytogenetic/molecular AML subtypes other than del5q/5q- AMLs (Figure 2A, Table 1). Importantly, despite lenalidomide/pomalidomide had neither cytotoxic effects on BM-MSCs (Figure 2A) they did enhance the immunosup- pressive capacity of BM-MSCs (Figure 2B). In fact, IMiDs boosted the BM-MSC-mediated inhibition of activated T cells (Figure 2B) through blocking T-cell release of pro- inflammatory cytokines such as TNFα and IL1β while enhancing the production of the master anti-inflammatory cytokine IL10 (Figure 2B). Lenalidomide/pomalidomide also inhibited the release of the pro-inflammatory cytokine IL6 by BM-MSC (Figure 2C). The potent immune-modulatory properties of IMiDs on BM-MSCs24 led us to combine IMiDs with Ara-C/Ida-based standard AML treatment as a potential therapeutic approach to bypass the BM-MSC- mediated chemoresistance to Ara-C/Ida. Imids fail to overcome BM-MSC/stroma-mediated chemoresistance of non-del5q/5q- AML cells but induce specific mobilization of AML cells to PB Neither lenalidomide nor pomalidomide were unable to over- come the BM-MSC-mediated resistance of non-del5q/5q- AML cells to Ara-C/Ida chemotherapy either in vitro (Figure 3A) or in vivo (Figure 3B). Surprisingly, when lenali- domide was combined with chemotherapy we observed a robust (3–4-fold) mobilization of non-del5q/5q- AML cells into extramedular BM hematopoietic sites including PB and spleen (Figure 3B). Next, we performed similar in vivo experi- ments by treating AML-engrafted NSG mice with lenalido- mide as single agent and confirmed that it induces a robust mobilization of non-del5q/5q- AML cells into PB (Figure 4A) and spleen (Figure 4B). Specific mobilization of leukemic cells from BM to PB is commonly desirable in the clinical setting because circulat- ing blasts are more sensitive to treatment in part due to their detachment from the chemoprotective BM niche.4,36,37 However, mobilization of normal hematopoietic stem/pro- genitor cells (HSPCs) to PB is not desired because it would accelerate BM aplasia and subsequent hematopoietic defects. Thus, we next analyzed whether lenalidomide also mobilizes normal HSPCs to PB. CB CD34+ cells were IBM- transplanted in NSG mice and total (CD45+HLA-ABC+) and immature (CD45+HLA-ABC+CD34+) engraftment was analyzed 6–7 weeks later. Interestingly, lenalidomide failed to mobilize normal HSPC indicating that lenalidomide-dri- ven mobilization into PB is specific for AML cells (Figure 4C). Lenalidomide-mediated mobilization of AML cells to PB is associated to downregulation of CXCR4 and a migratory transcriptomic signature in AML cells We next sought to gain insights into the mechanisms under- lying lenalidomide-mediated mobilization of AML cells. Clearly, the CXCR4/SDF1 axis plays a major role in the interaction of HSPCs and leukemic cells within BM niches. CXCR4 is highly expressed in HSPCs/leukemic cells whereas SDF1 is a cytokine commonly released by BM-MSCs,37,38 (Figure 5A). Consequently, we first treated both AML cell lines and primary non-del5q/5q- AML blasts with lenalido- mide and pomalidomide and found a significant (25%–50%) reduction in the levels of CXCR4 in AML cells similar to treatment with the specific CXCR4 inhibitor AMD3100 (Figure 5B). In line with the IMiDs-mediated specific mobi- lization of AML blasts (Figure 4), we confirmed that lenali- domide/pomalidomide do not down-regulate CXCR4 expression in healthy PB- or BM-derived HSPCs (Figure 5B). In contrast, lenalidomide and pomalidomide treatment did not impact the either expression levels or cytokine release of SDF1 by human BM-MSCs (Figure 5C). Together, these results suggest that IMiDs specifically med- iate the mobilization of AML cells to PB through down- regulation of CXCR4. To identify patterns of gene expression that might provide a molecular explanation for the IMiDs-induced mobilization of AML cells, we performed whole-genome GEP in lenalidomide- treated versus DMSO-treated AML blasts (blast purity>95%, n = 3 de novo non-del5q/5q- AMLs). A heatmap representation of hierarchical clustering of genes differentially expressed (1.5- fold regulation, p-value<0.05) between lenalidomide- versus DMSO-treated AML cells is represented in Figure 6A. A total of 323 genes were differentially expressed between lenalido- mide- and DMSO-treated AML cells. Of these, 196 (61%) were upregulated and 127 (39%) downregulated in lenalido- mide-treated AML cells, establishing a lenaledomide-specific transcriptomic signature. To get insights into the biological functions affected by differentially expressed genes, we used the Gorilla software,30,31 and found that many significant bio- logical processes predicted to be activated in the lenalidomide- treated AML cells were associated with “migration/motility/ mobilization/chemotaxis” (Figure 6A). In contrast, exposure of CB-CD34+ HSPCs to lenalidomide did not affect such bio- logical functions (Figure 6B). This lenalidomide-induced tran- scriptomic signature supports a CXCR4-mediated mechanism underlying the mobilization of non-del5q/5q- AML cells to PB in response to IMiDs. ONCOIMMUNOLOGY e1477460-5 Figure 2. Effects of IMiDs on primary AML blasts and BM-MSC. (A) Lenalidomide and pomalidomide do not have a cytotoxic effect on either AML cell lines (n = 3) (top panel), primary AML blasts (n = 9, bottom left panels) or BM-MSC (n = 3) (right bottom panels). (B) Left panel: IMiDs potentiate the immunosuppressive effect of BM-MSCs, measured as percentage of CFSE+ non-proliferating cells. CSFE-labeled PBMCs were stimulated with PHA and then co-cultured with BM-MSCs in the presence of lenalidomide or pomalidomide for 5 days (n = 3). Middle panels: representative flow-cytometry histograms of cycling (CSFElow) PBMCs. Right panels: Concentration of the indicated cytokines in cell-culture supernatants determined by Luminex Multiplex assays. PBMCs were co-cultured with BM-MSCs in the presence/absence of IMiDs. Error bars indicate the SEM values of the n = 3 biological replicates. (C) IMiDs diminish IL6 production by BM-MSC (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. e1477460-6 B. LOPEZ-MILLAN ET AL. Figure 3. IMiDs combined with AraC-Idarubicin do not circumvent BM- microenvironment-mediated resistance of AML cells but induce robust extramedullary mobilization of AML cells. (A) Lenalidomide and pomalidomide do not overcome BM-MSC-mediated resistance of AML cell lines to intensive chemotherapy (n = 3). (B) IMiDs combinedwith AraC +Idarubicin highly mobilize AML blasts to PB and spleen in AML xenograft models (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. ONCOIMMUNOLOGY e1477460-7 Lenalidomide overcomes BM-MSC/stroma-mediated chemoresistance of AML cells from del5q/5q- patients A phase II clinical trial has recently reported promising results for lenalidomide combined with AraC/daunorubicine in both MDS and AML patients with 5q alterations (del5q/5q-).39 Thus, we next focused our efforts on this cytogenetic sub- group of AML patients as potential candidate patients benefiting from the combination of IMiDs with AraC/idaru- bicine. Primary cells from del5q/5q- AML patients were trea- ted with lenalidomide, AraC/Ida or their combination for 48-72h, in the presence/absence of BM-MSCs (Figure 7A). Opposite to non-del5q/5q- AML cells (Figure 2A), lenaledo- mide used as single agent displayed a slight, but consistent, 20% toxicity in del5q/5q- AML cells (Figure 7B). More Figure 4. When administered as a single agent, lenalidomide highly mobilizes AML cells, but not healthy CD34+ cells, to PB. (A,B) Lenalidomide administered alone mobilizes AML cells to PB (A) and spleen (B). AML infiltration in PB and spleen was detected by FACS and immunohistochemistry, respectively. (C) Lenalidomide fails to mobilize healthy CD34+ cells (n = 3). *p < 0.05. **p < 0.01. e1477460-8 B. LOPEZ-MILLAN ET AL. importantly, lenaledomide significantly boosted the toxicity of del5q/5q- AML cells when combined with AraC/Ida, over- coming the BM-MSC/stroma-mediated chemoresistance of AML cells from del5q/5q- patients (Figure 7C). Interestingly, lenalidomide did not down-regulate CXCR4 expression on del5q/5q- AML cells (Figure 7D), indicating that Figure 5. IMiDs mobilizes AML cells to PB through CXCR4 downregulation. (A) Cartoon of the CXCR4-SDF1 crosstalk between leukemic blasts and BM stromal cells. (B) Effect of IMiDs on the expression levels of CXCR4 in AML cell lines, primary AML cells (top panels), PB-derived CD34+ cells and BM-derived CD34+ cells (bottom panels). (C) Effect of IMiDs on the expression (Left panel) and production levels (right panel) of SDF1 by BM-MSCs. The specific CXCR4 inhibitor AMD3100 was used as positive control. ONCOIMMUNOLOGY e1477460-9 Figure 6. Global GEP reveals a migratory signature in lenalidomide-treated AML blasts. (A, B) Left panels: Heatmap representation of hierarchical clustering of genes differentially expressed between lenalidomide-treated and untreated AML blasts (n = 3 leukemias) (top panel) and BM-CD34+ cells (n = 2 healthy donors) (bottom panel). Right panel: Statistically significant biological functions identified using IPA on genes differentially expressed in lenalidomide-treated versus untreated AML blasts (top panel) and CD34+ cells (bottom panel). They are ranked by z-score. A z-score >2 indicates a predicted activation of that biological function. Biological functions associated with ‘cell migration/movement/motility’ are shown in black. e1477460-10 B. LOPEZ-MILLAN ET AL. lenalidomide partially overcomes BM-MSC/stroma-mediated chemoresistance of AML cells from del5q/5q- patients likely at the expense of blast mobilization. Discussion AML represents a clinically and genetically very heteroge- neous group of diseases characterized by an accumulation of proliferative, abnormally differentiated hematopoietic cells in the BM and other tissues, leading to interference of normal hematopoiesis and BM failure. AML is one of the most com- mon hematopoietic malignances with ~25.000 new cases per year in United States and Europe.40 Overall, AML has an unfavorable clinical outcome, largely determined by age, cyto- genetic/genetic lesions as well as patient´s initial response to standard treatment.2 The large genetic/cytogenetic and phe- notypic heterogeneity together with a median age at diagnosis of ~65–67 years old have long represented a limitation towards the development of better treatments.4 Despite many clinical trials have been conducted over the last two decades, the standard current treatment in AML remains unchanged, and relies on intensive induction chemotherapy based on nucleoside analogs such as Ara-C or fludarabine and anthracyclines such as idarubicin and daunorubicin followed by high doses Ara-C consolidation.41 Although complete remission rates (CRR) of ~80% are commonly achieved with conventional chemotherapy,4,42 the median survival time is short and the outcome of relapsed patients is dismal.43-45 Novel agents or better treatment combinations/schedules with higher efficacy and less toxicity are therefore in high demand for de novo and relapsed AML. Failure of current therapies to eradicate LICs and che- motherapy refractoriness are the major mechanisms under- lying AML progression/relapse, being in part because the BM provides a protective microenvironment to support the survi- val of LICs/bulk leukemic cells, hence promoting drug resis- tance and influencing clinical outcome. In AML, the high rate of relapse may in part be a result of the inability of current treatments to effectively overcome the protective influence of the BM niche.5,46 In fact, it has been recently shown that BM mesenchymal stromal cells (BM-MSCs) derived from diag- nostic AML patients are strong immunomodulators and pro- vide similar or even higher chemoprotection of AML cells to cytarabine/idarubicin than normal BM-MSCs.5 Therefore, new compounds or improved combinatory regimens targeting the interaction between the BM microenvironment and AML leukemic cells are very attractive candidates. The new generation of immunomodulatory drugs (IMiDs) such as lenalidomide and pomalidomide are thalidomide ana- logs, which possess pleiotropic anti-leukemic properties including anti-proliferative, anti-angiogenic but also immune-modulating effects.7-9 They are approved by the FDA and EMA for 5q- MDS and/or MM which are hemato- logical malignances associated with intrinsic dysfunctional BM with remarkable clinical responses.10,11 However, whether IMiDs may potentiate current standard treatment in AML remains an issue of debate. To bring IMiDs to the forefront of AML therapeutic arsenal, a better understanding of its mechanism of action in AML cells and in the interphase Figure 7. Effects of Lenalidomide on primary blasts from patients with del5q/5q- AML. (A) Scheme depicting the experimental design. (B) Lenalidomide toxicity on primary del5q/5q- AML cells (n = 4). (C) When combined with standard chemotherapy lenalidomide largely overcomes BM-MSC-mediated resistance of del5q/5q- AMLs to AraC+Idarubicin (n = 4). (D) Effect of lenalidomide on the expression levels of CXCR4 in del5q/5q- AML cells (n = 4). AMD3100 was used as positive control. *p < 0.05; n.s, no statistical differences. ONCOIMMUNOLOGY e1477460-11 tumor-stroma is required. In this study, we hypothesized that the testing of IMiDs may be facilitated by in vitro and in vivo assays that model the tumor-BM stroma interactions.47-49 Hence, we used human primary BM-MSCs and intra BM co-transplantation with AML cells into NSG mice to mimic the interaction of AML cells with the BM stroma as a platform to assess whether IMiDs add efficiency to current standard AML chemotherapy. We report that lenalidomide and poma- lidomide, when administered as single agents, have cytotoxic effects on neither non-del5q/5q AML cells nor BM-MSCs, even at doses as high as 100 µM. This contrasts the anti- proliferative effects reported for IMiDs in other hematopoietic malignances, evidencing the immature nature of AML cells (progenitor-like cells) and the complex biology of AML.9-11 However, IMiDs enhanced the immunomodulatory properties of BM-MSCs as demonstrated by the suppression activated T cells proliferation, inhibition of the production of pro-inflam- matory cytokines including TNFα, IL1β and IL6 and increased production of the anti-inflammatory cytokine IL10, suggesting that IMiDs may represent safe candidate drugs interfering with the protective BM stroma for combina- tion with current AML chemotherapy. Unfortunately, when combined with AraC and Idarubicin, both lenalidomide and pomalidomide fail to circumvent BM stroma-mediated resistance of non-del5q/5q AML cells in in vitro and in vivo AML models. The clinical efficacy of lenali- domide has been explored as either low-dose or high-dose lenalidomide as monotherapy50-54 and in combination with azacitidine55,56 for both newly diagnosed and relapsed/refrac- tory AML. Overall, these clinical trials were somewhat dis- appointing with CRR as low as 11%–20% and remission duration below 5–7 months.14 Our preclinical studies mechanistically support previous modest clinical activity reported for IMiDs in AML, and discourages future clinical trials using either lenalidomide or pomalidomide for non- del5q/5q AML either as monotherapy or polytherapy. Of note, a recent Phase II clinical study of lenalidomide com- bined with intensive chemotherapy in AML has reported that only del5q/5q- AML patients show promising CRR.39 Here, our pre-clinical studies confirm that lenaledomide used as single agent displays ~20% toxicity only in del5q/5q- AML cells and it overcomes BM-MSC/stroma-mediated chemore- sistance of AML cells exclusively from del5q/5q- patients. A potential mechanism of action of lenalidomide involves the degradation of casein kinase 1A1 (CK1α).57 In fact, del5q MDS patients possess a heterozygous deletion CK1α.58 Collectively, our data is in line with Ades and colleagues39 and suggest that the mechanisms of action of lenalidomide on AML may depend on cell intrinsic/autonomous genetic makeup, further pinpointing genetic and biological heteroge- neity in AML. Despite the overall limited activity of lenalido- mide during induction/consolidation therapy its efficiency during AML maintenance therapy has yet to be tested. Intriguingly, we found that lenalidomide, administered either as monotherapy or combined with conventional che- motherapy, induces robust mobilization of non-del5q/5q AML cells to extramedullary hematopoietic tissues including PB and spleen, suggesting that IMiDs might function as plerix- afor, a small molecule inhibitor of the CXCR4/SDF1 axis, extensively used for chemosensitazion of AML blasts by dis- rupting the interaction of leukemic blasts with the environment.4,36,37,59 In fact, lenalidomide mediated the mobi- lization of AML cells to PB through downregulation of CXCR4 in non-del5q/5q AML cells but does not affect either the expression or the production of its ligand SDF1 by BM-MSCs. In contrast to plerixafor, lenalidomide does not mobilize nor- mal CD34+ HSPCs to PB. Whole-genome GEP revealed a transcriptomic migratory signature of lenalidomide-treated non-del5q/5q AML primary blasts but not lenalidomide-treated CD34+ cells, confirming that the IMiD-mediated mobilization is specific of AML blasts. We cannot rule out that other signal- ling pathways/mechanisms contribute to the IMiD-induced mobilization phenotype. Importantly, in contrast to plerixafor, IMiDs do not chemosensitize AML blasts to chemotherapy, further supporting that the addition of IMiDs to cytotoxic chemotherapy seems not feasible in AML.36,59 Lenalidomide- attenuated pro-inflammatory cytokines including TNFα, γIFN, IL1β and macrophage inhibitory factor (MIF) have been pre- viously described to interact with master pathways/molecules such as CXCR4-SDF1 axis, collagenases, integrins and eicosa- noids in controlling cell migration and mobilization.60-63 Thus, the immunomodulatory effects exerted by IMiDs seem to influ- ence the BM niche in a way non-del5q/5q AML cells physically detach from BM stroma. Future in vivo imaging studies will precisely define the mechanisms underlying the lenalidomide- mediated AML chemotaxis. Acknowledgments This work was supported by the European Research Council (CoG-2014- 646903 to P.M), the Spanish Ministry of Economy and Competitiveness (SAF-SAF2013-43065, RTC-2016-4603-1 to P.M), the Asociación Española Contra el Cáncer (AECC-CI-2015), FERO Foundation, and the ISCIII/FEDER (PI14-01191) to C.B and the ‘‘Fundación Hay Esperanza’’ to E.A. P.M also acknowledges financial support from The Obra Social La Caixa-Fundaciò Josep Carreras, The Inocente Inocente Foundation and The Generalitat de Catalunya (SGR330). P.M an inves- tigator of the Spanish Cell Therapy cooperative network (TERCEL). We thank Celgene Corporation (San Diego, CA) for providing IMiDs. We thank Judit Sopena and Mariano Graupera (IDIBELL, Barcelona) for their technical assistance with immunohistochamical analysis Disclosure statement The authors have nothing to disclose. Funding This work was supported by the EC | European Research Council (ERC) [CoG-2014-646903]; Spanish Ministry of Economy and Competitiveness [SAF-SAF2013-43065, RTC-2016-4603-1]; The Generalitat de Catalunya [SGR330]; ISCIII/FEDER [PI14-01191]; Asociación Española Contra el Cancer [AECC-CI-2015]. Author contributions BLM: designed and performed experiments, analyzed data and wrote the manuscript. HRH, RDG, DRM, CP, FG, PC, AI, CB: designed and performed experiments, and analyzed data. EA, JAPS, JABG, FAA, MR, PM, AUI: provided leukemic samples. PM: conceived the study, designed experiments, analyzed data and wrote the manuscript. e1477460-12 B. LOPEZ-MILLAN ET AL. ORCID Abul B. M. M. K. Islam http://orcid.org/0000-0002-7274-0855 Cristina Prieto http://orcid.org/0000-0002-0812-8100 Montse Rovira http://orcid.org/0000-0002-6403-3317 References 1. Porwit A, BeneMC. 2015. Acute leukemias of ambiguous origin. Am J Clin Pathol 144:361–376. doi:10.1309/AJCPSTU55DRQEGTE. 2. 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LOPEZ-MILLAN ET AL. https://doi.org/10.1016/j.stem.2014.02.014 https://doi.org/10.1182/blood-2008-06-162123 https://doi.org/10.1182/blood-2008-06-162123 https://doi.org/10.1007/s12185-016-2123-y https://doi.org/10.1007/s12185-016-2123-y https://doi.org/10.1084/jem.185.1.111 https://doi.org/10.3324/haematol.2016.151894 https://doi.org/10.3324/haematol.2016.151894 https://doi.org/10.1200/JCO.2010.30.1820 https://doi.org/10.1200/JCO.2010.30.1820 https://doi.org/10.1182/blood-2015-08-604520 https://doi.org/10.1182/blood-2015-08-604520 https://doi.org/10.1182/blood.V98.5.1302 https://doi.org/10.1182/blood-2004-01-0326 https://doi.org/10.1182/blood-2004-01-0326 https://doi.org/10.1056/NEJMoa1010222 https://doi.org/10.1056/NEJMoa1010222 https://doi.org/10.1182/blood-2016-02-699363 https://doi.org/10.1182/blood-2016-02-699363 https://doi.org/10.1038/nchembio.1367 https://doi.org/10.1038/nm.2112 https://doi.org/10.1038/nm.2112 https://doi.org/10.1038/nrd3870 https://doi.org/10.1038/nrd3870 https://doi.org/10.1200/JCO.2010.30.3339 https://doi.org/10.1016/j.clml.2012.04.001 https://doi.org/10.1182/blood-2010-07-297143 https://doi.org/10.1182/blood-2010-07-297143 https://doi.org/10.3324/haematol.2010.039669 https://doi.org/10.3324/haematol.2010.039669 https://doi.org/10.1182/blood-2011-02-337303 https://doi.org/10.1038/leu.2011.294 https://doi.org/10.1038/leu.2012.214 https://doi.org/10.1038/leu.2012.214 https://doi.org/10.1038/nature14610 https://doi.org/10.1016/S2352-3026(15)00050-2 https://doi.org/10.1182/blood-2011-10-383406 https://doi.org/10.1002/stem.569 https://doi.org/10.1016/j.stemcr.2017.05.018 https://doi.org/10.1016/j.stemcr.2017.05.018 https://doi.org/10.1038/leu.2011.197 https://doi.org/10.1038/leu.2010.216 Abstract Introduction Material and methods AML cell lines and primary leukemic and healthy cells Drugs Cytotoxicity and apoptosis assays Immunosuppressive assays and cytokine detection FACS analysis of CXCR4 and SDF1 NSG mice xenotransplantation and analysis of engraftment In vivo chemotherapy and imids treatment Microarray gene expression profiling (GEP) Results BM stroma/milieu confers resistance of AML cells to AraC/idarubicin Imids exert no toxicity on non-del5q/5q- AML cells but increase immunosuppressive properties of BM-MSCs Imids fail to overcome BM-MSC/stroma-mediated chemoresistance of non-del5q/5q- AML cells but induce specific mobilization of AML cells to PB Lenalidomide-mediated mobilization of AML cells to PB is associated to downregulation of CXCR4 and a migratory transcriptomic signature in AML cells Lenalidomide overcomes BM-MSC/stroma-mediated chemoresistance of AML cells from del5q/5q- patients Discussion Acknowledgments Disclosure statement Funding Author contributions References