REGULAR ARTICLE Engraftment characterization of risk-stratified AML in NSGS mice Rafael Dıaz de la Guardia,1,2 Talıa Velasco-Hernandez,1 Francisco Guti uera,1 Heleia Roca-Ho,1 Oscar Molina,1 errez-Ag€ Cesar Nombela-Arrieta, Alex Bataller, Jose Luis Fuster, Eduardo Anguita, Susana Vives,1,7 Lurdes Zamora,1,7 Josep Nomdedeu,1,8 3 1,4 5 6 Marıa Teresa Go mez-Casares,9 Manuel Ramırez-Orellana,10 Helene Lapillonne,11 Vero nica Ramos-Mejia,2 Juan Carlos Rodrıguez-Manzaneque, Clara Bueno, 2 1,12 1,2 Belen Lopez-Millan, and Pablo Mene ndez1,11,13 1 Josep Carreras Leukemia Research Institute, Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, Spain; 2GENYO, Centre for Genomics and Downloaded from http://ashpublications.org/bloodadvances/article-pdf/5/23/4842/1846467/advancesadv2020003958.pdf by guest on 27 January 2023 Oncological Research, Pfizer/Universidad de Granada/Junta de Andalucıa, Granada, Spain; 3Department of Medical Oncology and Hematology, University and University Hospital Zurich, Zurich, Switzerland; 4Sevicio de Hematologıa Clınica, Hospital Clınic de Barcelona, Barcelona, Spain; 5Secci on de Oncohematologıa Pediatrica, Hospital Clınico Universitario Virgen de Arrixaca, Instituto Murciano de Investigaci on Biosanitaria, Murcia, Spain; 6Servicio de Hematologıa, Hospital Clınico San Carlos, IdISSC, Medicina UCM, Madrid, Spain; 7Hematology Department, ICO-Hospital Germans Trias i Pujol, Barcelona, Spain; 8Servicio de Hematologıa, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; 9 Servicio de Hematologıa, Hospital Universitario de Gran Canaria Dr. Negrin, Las Palmas de Gran Canaria, Spain; 10Department of Pediatric Hematology and Oncology, Hospital Infantil Universitario Ni~ no Jes on Sanitaria La Princesa, Madrid, Spain; 11Haematology Laboratory, AP-HP, H^ us, Instituto de Investigaci opital Armand Trousseau, Centre de Recherche Saint-Antoine CRSA, INSERM, Sorbonne Universit e, Paris, France; 12Centro de Investigacion Biomedica en Red-Oncologıa (CIBERONC); and 13Instituci o Catalana de Recerca i Estudis Avançats, Barcelona, Spain Key Points Acute myeloid leukemia (AML) is the most common acute leukemia in adults. Disease heterogeneity is well documented, and patient stratification determines treatment  PDXs from risk- decisions. Patient-derived xenografts (PDXs) from risk-stratified AML are crucial for stratified AML samples studying AML biology and testing novel therapeutics. Despite recent advances in PDX are crucial for studying modeling of AML, reproducible engraftment of human AML is primarily limited to high-risk AML biology and (HR) cases, with inconsistent or very protracted engraftment observed for favorable-risk testing novel therapeutics. (FR) and intermediate-risk (IR) patients. We used NSGS mice to characterize the engraft- ment robustness/kinetics of 28 AML patient samples grouped according to molecular/  We characterize cytogenetic classification and assessed whether the orthotopic coadministration of patient- human AML matched bone marrow mesenchymal stromal cells (BM MSCs) improves AML engraftment. engraftment in NSGS PDX event-free survival correlated well with the predictable prognosis of risk-stratified mice, offering a valuable platform for in AML patients. The majority (85-94%) of the mice were engrafted in bone marrow (BM) vivo testing of targeted independently of the risk group, although HR AML patients showed engraftment levels that therapies. were significantly superior to those of FR or IR AML patients. Importantly, the engraftment levels observed in NSGS mice by week 6 remained stable over time. Serial transplantation and long-term culture-initiating cell (LTC-IC) assays revealed long-term engraftment lim- ited to HR AML patients, fitter leukemia-initiating cells (LICs) in HR AML samples, and the presence of AML LICs in the CD342 leukemic fraction, regardless of the risk group. Finally, orthotopic coadministration of patient-matched BM MSCs and AML cells was dispensable for BM engraftment levels but favored peripheralization of engrafted AML cells. This comprehensive characterization of human AML engraftment in NSGS mice offers a valuable platform for in vivo testing of targeted therapies in risk-stratified AML patient samples. Submitted 7 December 2020; accepted 19 May 2021; prepublished online on Blood The full-text version of this article contains a data supplement. Advances First Edition 1 September 2021; final version published online 24 November ß 2021 by The American Society of Hematology. Licensed under Creative Commons 2021. DOI 10.1182/bloodadvances.2020003958. Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), per- Data sharing requests should be sent to Pablo Menendez (pmenendez@carrerasre- mitting only noncommercial, nonderivative use with attribution. All other rights reserved. search.org), Belen Lopez-Millan (blopez@carrerasresearch.org) or Rafael Dıaz de la Guardia (rafael.diaz@genyo.es). 4842 14 DECEMBER 2021 • VOLUME 5, NUMBER 23 Introduction term engraftment was limited to HR patients and fitter LICs in HR AML samples vs FR or IR AML samples. Orthotopic coadministration of patient-matched BM MSCs and AML cells was dispensable for BM Acute myeloid leukemia (AML), the most common acute leukemia in engraftment levels but favored peripheralization of engrafted AML adults, is a biologically and genetically heterogeneous group of disor- cells. This comprehensive characterization of human AML cell engraft- ders that is characterized by the rapid expansion and accumulation of ment in NSGS mice represents a robust platform for in vivo testing of poorly differentiated myeloid cells in the bone marrow (BM) and infil- targeted therapies for risk-stratified AML patient samples. trating tissues.1-3 Disease heterogeneity is well documented, and patients are stratified at disease presentation based on cytogenetic, molecular, and immunophenotypic data. Patient stratification is crucial Methods for prognostication AQ1and informs treatment decisions.1 AML treat- Patients ment has not improved substantially in the last 2 decades, and many Fresh BM aspirates were obtained at the time of disease presentation patients fail to respond to standard-of-care chemotherapy.2,3 Robust Downloaded from http://ashpublications.org/bloodadvances/article-pdf/5/23/4842/1846467/advancesadv2020003958.pdf by guest on 27 January 2023 from 28 patients (17 males and 11 females) with a mean age of 43 AML patient-derived xenograft (PDX) models represent a unique in years (standard error of the mean [SEM], 5 years) and cytogenetically vivo tool for studying the pathogenesis of AML and for testing novel different AMLs. Patients were diagnosed in the following hospitals: therapeutics. AML PDXs provide information about the mechanisms Hospital Clinico San Carlos, Hospital Clınico Universitario Virgen de underlying disease initiation and progression, (epi)genetic clonal evo- Arrixaca, Hospital Universitario de Gran Canaria, ICO-Hospital Ger- lution, the biology of AML leukemia-initiating cells (LICs), BM niche- mans Trias i Pujol, Hospital Infantil Universitario Ni~no Jes us, H^ opital leukemia interactions, and chemoresistance.4-9 However, in immuno- Armand Trousseau, and Hospital de la Santa Creu i Sant Pau. deficient mice, it is challenging to anticipate the engraftment of primary Molecular-cytogenetic stratification was based on European Leuke- samples derived from patients with favorable-risk (FR) and miaNet3 and World Health Organization23 classifications. Accord- intermediate-risk (IR) AML, which hinders the establishment of accu- ingly, the disease was classified as FR AML (displaying favorable rate PDX models encompassing AML heterogeneity. cytogenetics/molecular features), IR AML (normal karyotype and Advanced strains of immunodeficient mice have improved PDX absence of mutations in TP53, NPM1, FLT3, and WT), or HR AML modeling of AML.10,11 In NSG mice, the most widely used immunode- (displaying unfavorable cytogenetics/molecular features).3,23 The ficient mouse strain, reproducible xenoengraftment of human AML is main cytogenetic/molecular diagnostics and other clinical-biological primarily limited to high-risk (HR) cases; inconsistent or very pro- features of each patient are listed in Table 1. Blasts were immunophe- tracted engraftment is observed for FR and IR patients.12 It was not notyped by flow cytometry using the monoclonal antibodies (mAbs) until recently that a more consistent engraftment of FR/IR AML sam- CD45-APC-H7 (clone 2D1), CD19-BV421 (clone HIB19), and ples was achieved using NSGS mice, a more advanced immunodefi- CD34-PE-Cy7 (clone 8G12) (all from BD Biosciences), and CD33- cient mouse strain that results from crossing NSG and NSS mice; it PE (clone AC104.3E3; Miltenyi Biotec), following the manufacturer’s provides an increased myeloid potential as a result of the transgenic recommendations. The study was approved by the Institutional expression of human stem cell factor, granulocyte-macrophage col- Review Board of the Clinic Hospital of Barcelona (HCB/2014/ ony-stimulating factor, and interleukin-3.6,13,14 0687), and samples were collected upon signed informed consent. A reciprocal remodeling between the BM niche and leukemic cells is Isolation and expansion of BM MSCs well established in several hematological malignancies.15-17 In fact, AML MSCs were isolated from the BM of the 28 patients, as AML progression and chemoresistance are highly dependent on described previously in detail.24,25 AML MSC cultures were main- leukemia-BM stroma interactions.15,18 Several nonorthotopic human- tained in advanced Dulbecco’s modified Eagle medium (Gibco) sup- ized BM niche models have been explored for hematopoietic xeno- plemented with 10% fetal calf serum (Invitrogen), L-glutamine, and transplantation.19 Furthermore, the impact of orthotopic penicillin-streptomycin-amphotericin B (Gibco). Established AML cotransplantation (intra-BM transplantation) of BM mesenchymal stro- MSC cultures were characterized extensively and used at passages mal cells (MSCs) on the engraftment of primary human samples has 1 through 3.24-29 been evaluated in part in NSG mice in the context of healthy hemato- poietic stem and progenitor cells (HSPCs), multiple myeloma, and PDX models and follow-up myelodysplastic syndromes (MDSs), with contradictory Seven- to 12-week-old NSGS mice (n 5 275 with an equal distribu- results.14,17,20-22 However, it remains unknown whether intra-BM tion of males and females), housed under pathogen-free conditions, coinjection of patient-matched bone marrow mesenchymal stromal were used in this study. All experimental procedures were approved cells (BM MSCs) impacts the engraftment of cells from humans by the Animal Care Committee of The Barcelona Biomedical with primary AML. Research Park (HRH-16-0037-P2). BM mononuclear cells (MNCs) In this study, we characterized the engraftment robustness/kinetics of from AML patients were isolated by density gradient centrifugation cells from 28 AML patients, grouped according to molecular/cytoge- using Ficoll-Hypaque (Amersham Biosciences). Subsequently, sam- netic classification, in NSGS mice and assessed the contribution of ples were T cell-depleted with an anti–CD3-OKT3 antibody (LAB- patient-matched BM MSCs in AML engraftment. We report that CLINICS). To do this, cells were incubated with the antibody (5.83 PDX event-free survival (EFS) correlated well with the predictable mg per 106 MNCs) on ice for 30 minutes just prior to intra-BM prognosis of risk-stratified AML patients and that NSGS mice repre- (IBM) transplantation. A total of 106 T-cell–depleted MNCs was sent a robust model for a rapid read-out of the engraftment potential IBM transplanted into sublethally irradiated (2 Gy) mice, as previously of cells from risk-stratified AML patients. Serial transplantation and described,30-32 with or without 105 patient-matched established BM long-term culture-initiating cell (LTC-IC) assays revealed that long- MSCs.24,25 At weeks 6 and 12, BM and peripheral blood (PB) 14 DECEMBER 2021 • VOLUME 5, NUMBER 23 RISK-STRATIFIED AML ENGRAFTMENT IN NSGS MICE 4843 Table 1. Biological and cytogenetic-molecular characteristics of AML patients in this study Patient ID Diagnosis Cytogenetics Molecular Age, y Sex Blasts, % Risk group 14011 AML-M5 46, XY, inv(16) Cbfb-MYH11 1 M 50 FR 14022 AML 46, XX NPM1MUT, IDH1MUT 20 F 92 FR 14030 AML 46, XY NPM1MUT 18 M 32 FR 14046 AML-M3 46, XX t(15;17) PML-RARa 59 F 90 FR 14032 AML 46, XY NPM1MUT 80 M 49 FR 14096 AML-M3 46, XY, t(15;17) PML-RARa 25 M 90 FR 14175 AML-M4 46, XX, t(8;21) AML1-ETO 9 F 75 FR 14218 AML 46, XY NPM1MUT 62 M 25 FR Downloaded from http://ashpublications.org/bloodadvances/article-pdf/5/23/4842/1846467/advancesadv2020003958.pdf by guest on 27 January 2023 14182 AML-M5 46, XY, inv(16) Cbfb-MYH11 5 M 32 FR 14185 AML-M5 46, XY, inv(16) Cbfb-MYH11 8 M 77 FR 14008 AML 46, XY — 10 M 92 IR 14089 AML 46, XY — 77 M 45 IR 14088 AML-M4 46, XX — 61 F 71 IR 14050 AML-M4 46, XY — 86 M 50 IR 14113 AML 46, XY — 56 M 40 IR 14204 AML 46, XX — 82 F 71 IR 14137 AML-M5 46, XY — 76 M 50 IR 14202 AML-M5 46, XY — 47 M 54 IR 14119 AML-M0/M1 46, XY — 9 M 86 IR 14048 AML-M4 46, XX, t(9;11) MLL-AF9 63 F 90 HR 14057 AML-M5 46, XX NPM1MUT, FLT3-ITDhi 65 F 90 HR 14058 AML-M1 46, XX NPM1MUT, FLT3-ITDhi 73 F 90 HR 14070 AML-M1 46, XX, t(9;11) MLL-AF9 77 F 80 HR 14085 AML-M4 46, XY NPM1MUT, FLT3-ITDhi, WT1MUT 42 M 99 HR 14093 AML-M4 46, XX NPM1MUT, FLT3-ITDhi 52 F 81 HR 14160 AML-M5a 47,XX,8,1i(8)(q10)x2,t(8;21),t(9;11) MLL-AF9, ABL1, AML1-ETO, WT1MUT 6 F 66 HR 14186 AML-M5b 46,XY,t(11;17) MLL (11q23)1 MLL-RARa 1 M 87 HR 14163 AML 46, XY NPM1MUT, FLT3-ITDhi 48 M 88 HR F, female; FLT3-ITD , high FLT3-ITD allele burden; M, male; —, no mutation in TP53, FLT3, or NPM1. hi were collected by aspiration from noninjected tibiae and facial vein Biosciences). The cutoff to define engraftment was established at bleeding, respectively, and leukemic engraftment was analyzed by .0.1% in the BM. AML samples were categorized as high engrafters flow cytometry. Mice were euthanized when disease symptoms (.20%), medium engrafters (5-20%), low engrafters (0.1-5%), or were evident, leukemia engraftment was incompatible with animal wel- nonengrafters (,0.1%) based on the percentage of leukemic cells fare (.70%), or at week 18 (end point of the experiment).33 For EFS in the BM. If a mouse was euthanized before the end point (week curves, an event was called when disease symptoms with detectable 18), the engraftment levels observed at the sacrifice point were human graft in BM were observed or when the human graft was included in the following time points of the curve, not altering the .70% in BM in the absence of disease symptoms. Spleens and livers mean values for each risk group. Common leukemic genomic abnor- were collected and analyzed when euthanized. For secondary trans- malities, such as the inv(16), t(8;21), 18, and mixed lineage leukemia plantation, 106 leukemic cells from primary mice with leukemic graft (MLL) rearrangements as MLL-AF9, were confirmed by fluorescence . 60% in BM were IBM transplanted into irradiated secondary recip- in situ hybridization (FISH) in engrafted PDX samples, as described ients. For limiting dilution experiments, irradiated mice were IBM trans- previously.34-37 In mice engrafted with IR AML samples, the identity planted with as few as 105 cells from HR AML patients. of the AML graft was confirmed using targeted next-generation sequencing (NGS), as previously described.38,39 Analysis of leukemic grafts Cells retrieved from BM, PB, spleen, and liver were stained with HLA- Three-dimensional confocal microscopy of BM ABC-FITC and CD45-APC-H7 mAbs and analyzed by flow cytometry Three-dimensional imaging of mouse femoral BM cavities was per- to detect the human graft (HLA-ABC1CD451 population). Within the formed as described.40 Briefly, bones were collected, fixed, dehy- human graft, the leukemia was immunophenotyped using CD33-PE, drated, embedded in OCT, and snap frozen in liquid nitrogen. Thick CD19-BV421, and CD34-PE-Cy7 mAbs, as described above, on a slices (400-700 mm) of BM were obtained by iteratively sectioning BD FACSCanto II cytometer running FACSDiva software (BD both sides of the femur using a cryostat until the BM cavity was 4844 DIAZ de la GUARDIA et al 14 DECEMBER 2021 • VOLUME 5, NUMBER 23 Downloaded from http://ashpublications.org/bloodadvances/article-pdf/5/23/4842/1846467/advancesadv2020003958.pdf by guest on 27 January 2023 4845 RISK-STRATIFIED AML ENGRAFTMENT IN NSGS MICE Figure 1. 14 DECEMBER 2021 • VOLUME 5, NUMBER 23 bilaterally exposed. Slices were blocked and incubated with primary LTC-IC assays antibodies against endomucin (clone V.7C7; sc-65495; Santa Cruz CD451CD331CD192 AML blasts from 6 patients (2 patients from Biotechnology; 1:100) and human CD45 (clone MEM-28; Abcam; each risk group; supplemental Table 1) were stained with CD33-PE 1:200). Slices were then incubated with a secondary antibody cock- (clone AC104.3E3; Miltenyi Biotec), CD45-FITC (clone HI30; BD tail solution (Jackson ImmunoResearch) including 2 mM 4',6-diami- Biosciences), and CD19-APC (clone HIB19; BD Biosciences) anti- dino-2-phenylindole (DAPI). Finally, the slices were mounted on bodies and sorted using a FACSAria Fusion Cell Sorter (BD Bioscien- glass slides for imaging on a SP8 Leica confocal microscope ces). Sorted blasts were plated using a limiting dilution analysis of 4 equipped with hybrid detectors using 103 (HCX-PL FLUOTAR) doses (250, 500, 1000 and 2000 cells) in 15 replicates in 96-well and 203 (HC PL APO CS2) objectives. Imaging data were analyzed microplates containing a confluent irradiated (50 Gy) MS-5 mono- and videos were assembled using Imaris v8.2 software (Bitplane AG). layer. The AML blast-MS5 coculture was maintained in MyeloCult A supplemental Video is available online. H5100 culture medium (STEMCELL Technologies) supplemented with recombinant human interleukin-3, granulocyte colony- Downloaded from http://ashpublications.org/bloodadvances/article-pdf/5/23/4842/1846467/advancesadv2020003958.pdf by guest on 27 January 2023 FISH studies stimulating factor, and thrombopoietin (MS-51 3GT) (20 ng/mL PDX samples were processed as described.34-37 In brief, cells were each; PeproTech), as previously described41 at 37 C in 5% CO2. resuspended in warm hypotonic solution (0.075 mM KCl) for 20 After 5 weeks, the number of wells plated with 250 cells displaying minutes at 37 C and fixed in cold methanol/acetic acid (3:1). Samples cobblestone areas (CAs) were scored, and the long-term culture were spread onto methanol-cleaned slides and kept at 220 C until (LTC) medium was replaced by methylcellulose H4435 (STEMCELL processing. Two-color FISH experiments were performed using XL Technologies). After an additional 2 weeks, each well was scored as CBFB or XL t(8;21) (both from Metasystems Inc.) or LSI MLL positive/negative if colony-forming units were present/absent. The fre- Break-Apart (Abbott Molecular) probes to detect inv(16), t(8;21), or quency of LICs was determined using ELDA software, as previously MLL rearrangements, respectively. FISH was performed following described (http://bioinf.wehi.edu.au/software/elda/).33,42,43 standard procedures.34-37 Briefly, cells were denatured at 73 C in 70% formamide in 23 saline sodium citrate (SSC) for 2 minutes. Statistical analysis Hybridization was carried out by adding 5 mL of the corresponding Data are expressed as mean 6 SEM of different independent experi- probe mixture to preparations and incubating the slides in a humid chamber at 37 C for 16 hours. Posthybridization washes were per- ments unless otherwise specified. Statistical comparisons were per- formed using 0.43 SSC with 0.3% NP-40 at 73 C, followed by formed using unpaired a Student t test. EFS curves were estimated 23 SSC with 0.1% NP-40 at room temperature, for 1 minute each. according to the Kaplan-Meier method and were compared using a Slides were mounted with DAPI II solution (Abbott Molecular). Analy- log-rank test. Statistical analyses, normality test (Shapiro-Wilk), and ses were performed using a Nikon Ci-S/Ci-L epifluorescence micro- linear regression were performed using GraphPad Prism v6.0 (Graph- scope equipped with specific filters for DAPI, FITC, and Cy3 and a Pad Software). Statistical significance was defined as a P value , .05. dual-band pass filter for FITC and Cy3. The primary chromosomal abnormality was confirmed in $20 informative nuclei per PDX sample. Results Targeted NGS Characterization of the engraftment of risk-stratified AML cells in NSGS mice A targeted-NGS study was performed on 2 independent IR AML xen- ografts. The NGS panel included 32 genes related to myeloid disor- AML patient samples were classified as FR, IR, or HR, according to ders, including those genes known to be mutated at disease European LeukemiaNet/World Health Organization cytogenetics/ presentation.38,39 Briefly, DNA was extracted from PDX IR AML cells, molecular features (Table 1). To establish a suitable PDX model that and a library was prepared using a QIAseq FX DNA Library Kit (QIA- would allow us to characterize the AML risk groups, 106 GEN). Target regions were then captured and amplified by hybridizing T-cell–depleted mice BM MNCs were IBM transplanted into irradiated the DNA library with a Myeloid Solution Capture Kit (SOPHiA NSGS mice (n 5 112 mice), and the leukemic graft in BM was mon- GENETICS). The captured and amplified DNA was sequenced using itored by fluorescence-activated cell sorting (FACS) for 18 weeks a MiSeq platform (Illumina). The results were analyzed using a Sophia (Figure 1A). NSGS mice transplanted with HR AML cells began to DDM platform, as described elsewhere.38,39 show symptoms of disease or to exhibit engraftment levels in BM Figure 1. Characterization of risk-stratified AML engraftment in NSGS mice. (A) Experimental design (left panel) and representative FACS analysis of the engraftment (right panels). (B) Kaplan-Meier curves representing the 18-week EFS from each risk group (n 5 28 AML primary samples and 112 mice). Mice were considered “dead” when they showed disease symptoms with detectable human graft in BM or when human graft reached 70% in BM. (C) Percentage of AML engraftment in BM at euthanization for all patient samples grouped by risk (upper panel). The horizontal dotted lines indicate the cutoff for engraftment levels .0.1% (red line), between 0.1% to 5% (blue line), and .20% (green line). Each circle represents a single mouse. Gray circles depict mice with ,0.01% engraftment. The mean engraftment for the 3 or 4 mice used per patient sample is represented as a horizontal bar. Percentage of engrafted mice (leukemic cells . 0.1%) and percentage of engrafted patient samples (lower panels). Based on engraftment levels, patient samples were categorized as high (leukemic graft . 20%), medium (5-20%), or low (0.1-5%). (D) In situ 3-dimensional microscopy imaging of murine BM from a rep- resentative very low engrafted sample (0.8% by FACS; supplemental Video). (E) FISH for the indicated chromosomal abnormalities in PDX cells retrieved from mice at week 18. Magnification 1003. Yellow arrowheads depict the indicated chromosome abnormalities. (F) Engraftment kinetics in BM for each AML risk group at 6, 12, and 18 weeks (or at the end point). Data are mean (6 SEM) engraftment levels for all patients within each risk group. (G) As in (F) but showing each patient and risk group independently. Each point indicates the mean engraftment for each patient sample (3-4 mice). Individual mice euthanized before week 18 are indicated by “†”; the engraftment value observed at eutha- nization was maintained at the end point. ****P , .0001, log-rank test. NK, normal karyotype; CK, complex karyotype; D, day; IBMT, intra-BM transplantation; W, week. 4846 DIAZ de la GUARDIA et al 14 DECEMBER 2021 • VOLUME 5, NUMBER 23 Table 2. Targeted NGS for 32 genes related to myeloid disorders in 2 matched diagnostic PDX IR AML pairs Patient 14088 Patient 14204 Diagnostic PDX Gene Diagnostic (VAF range) PDX (VAF range) (VAF range) (VAF range) NRAS 1 (13.37-24.49)* 1 (1.85-42.82)* 2 2 IDH1 1 (46.18)* 1 (51.67)* 2 2 DNMT3A 1 (93.74)* 1 (99.44)* 2 1 (5.06-7.31)† ASXL1 2 1 (5.5-15.73)† 2 1 (5.71-64.27)† CEBPA 2 1 (5.24-23.77)† 2 1 (32.99-82.6)† EZH2 2 1 (19.9)† 2 1 (10.04-43.6)† JAK2 2 1 (9.3-11.16)† 2 1 (54.59-60.28)† Downloaded from http://ashpublications.org/bloodadvances/article-pdf/5/23/4842/1846467/advancesadv2020003958.pdf by guest on 27 January 2023 KMT2A 2 1 (5.2-28.71)† 2 1 (5.02-83.99)† MPL 2 1 (8.83-10.89)† 2 1 (12.56-55.8)† SETBP1 2 1 (5.2-7.4)† 2 1 (22.49-46.37)† WT1 2 1 (9.32)† 2 1 (54.93)† RUNX1 2 1 (13.19-14.64)† 1 (46.76)* 1 (12.71-65.57)* SRSF2 2 2 1 (45.23)* 2 ETV6 2 2 2 1 (6.91-13.93)† SF3B1 2 2 2 1 (16.91)† ZRSR2 2 2 2 1 (7.97-9.07)† ABL1 2 2 2 2 BRAF 2 2 2 2 CALR 2 2 2 2 CBL 2 2 2 2 CSF3 2 2 2 2 CSNK1A1 2 2 2 2 FLT3 2 2 2 2 HRAS 2 2 2 2 IDH2 2 2 2 2 KIT 2 2 2 2 KRAS 2 2 2 2 NPM1 2 2 2 2 PTPN11 2 2 2 2 TET2 2 2 2 2 TP53 2 2 2 2 U2AF1 2 2 2 2 VAF, variant allele frequency; 2 no pathogenic variants found; 1, pathogenic variants found. *Mutations in myeloid genes found in the diagnostic patient sample and PDX. †Mutations in myeloid genes found only in PDX (absent at diagnosis). that were incompatible with animal welfare (.70%) after week 5. 1C). However, although 80% of the HR AML patients showed high Follow-up of the animals confirmed a significantly lower 18-week engraftment levels (.20%) only 40% and 25% of the FR AML and EFS in mice transplanted with HR AML samples than in mice trans- IR AML patients, respectively, showed such a robust engraftment planted with FR or IR AML samples (29% vs 75% and 83%, respec- (Figure 1C). No correlation was observed between the number of tively; P , .0001; Figure 1B). This correlation between PDX EFS and AML primary cells transplanted and the engraftment level (supplemen- a predictable prognosis in risk-stratified AML patients faithfully mimics tal Figure 1). For mice that exhibited levels of engraftment , 1%, the current risk-stratification of AML patients. We then quantified the FACS analysis was confirmed by in situ 3-dimensional microscopy proportion of engrafted mice and engrafted patient samples, catego- imaging (Figure 1D; supplemental Video). rizing AML samples as high (.20%), medium (5-20%), or low (0.1-5%) engrafters or nonengrafters (,0.1%). Of note, the majority The presence of chromosomal abnormalities associated with FR and of the mice showed engraftment in BM independently of the risk group HR AML, such as inv(16), t(8;21), 18, and MLL-AF9, was detected by (87%, 85%, and 94% for FR, IR, and HR AML, respectively; Figure FISH in BM blasts from engrafted mice at euthanization (Figure 1E). 14 DECEMBER 2021 • VOLUME 5, NUMBER 23 RISK-STRATIFIED AML ENGRAFTMENT IN NSGS MICE 4847 Downloaded from http://ashpublications.org/bloodadvances/article-pdf/5/23/4842/1846467/advancesadv2020003958.pdf by guest on 27 January 2023 14 DECEMBER 2021 • VOLUME 5, NUMBER 23 Figure 2. DIAZ de la GUARDIA et al 4848 Similarly, when the leukemic identity of the human graft from mice for those 2 groups (Figure 2B). Secondary engraftment phenocopied reconstituted with IR AML cells was analyzed by targeted NGS, the primary myeloid engraftment (HLA-ABC1CD451CD331) in the pathogenic mutations found in myeloid genes at diagnosis were reca- absence of lymphoid engraftment (Figure 2C). pitulated in the matched PDX samples (Table 2). This, together with CD34 is a surrogate marker for normal HSPCs and LICs in some sub- the myeloid-skewed graft in the absence of a lymphoid graft, confirms types of AML.4,48 Because the transgenic human myeloid cytokines that it stemmed from the original leukemic clone and not from residual expressed by NSGS mice compromise the long-term engraftment healthy myeloid progenitor cells. Of interest, beyond the pathogenic of healthy CD341 HSPCs,45-47 we next wanted to trace the CD34- mutations found in the diagnostic sample, we also observed that IR expressing AML blasts before and after xenotransplantation as an indi- AML PDXs displayed multiple pathogenic mutations in myeloid genes rect approach to gain insights into the differential long-term engraft- that were absent in the diagnostic sample (Table 2), likely reflecting ment of AML cells according to the risk group (Figure 2D). Despite minor leukemic clones undetectable at diagnosis or the acquisition the heterogeneity of CD34 expression across AML patients, we of de novo environmental/niche pressure–mediated myeloid found, for HR AML, that the proportion of CD341 blasts observed Downloaded from http://ashpublications.org/bloodadvances/article-pdf/5/23/4842/1846467/advancesadv2020003958.pdf by guest on 27 January 2023 mutations. in the primary samples is retained or increased after 18 weeks of Previous studies have indicated that, by extending the time-to-engraft- expansion in NSGS mice (4.9% 6 2.6% vs 9.2% 6 2.9%; n 5 9; ment read-out of AML primary cells in NSG mice, all types of AML P . .05; Figure 2D). In contrast, the CD341 AML blasts were pro- samples, including FR AML, could be studied.5,44 Interestingly, foundly lost in PDXs transplanted with FR AML cells (18.5% 6 when the engraftment kinetics were analyzed sequentially in BM at 8.8% vs 4.4% 6 0.8%; P , .001) or IR AML cells (54.8% 6 6, 12, and 18 weeks, we observed that the engraftment levels 9.3% vs 1.9% 6 0.3%; Figure 2D). We next performed LTC-IC increased only slightly from week 6 to week 18 for HR AML samples assays using HR, IR, and FR primary AML samples (Figure 2E), and (from 34.6% 6 6% to 45.5% 6 6%; P . .05), whereas the engraft- observed that HR AML patients showed the highest number of wells ment levels observed at week 6 remained stable until week 18 for FR with CAs (Figure 2F) and the highest LIC estimation in a limiting dilu- and IR AML samples, suggesting that the stable engraftment levels tion analysis using ELDA software (Figure 2G). Together, these data observed in NSGS mice by week 6 may shorten/facilitate5 down- confirm the existence of AML-LICs in the CD342 leukemic fraction, stream studies requiring in vivo primary AML-PDXs (Figure 1F-G). regardless of the risk group, and reveal the presence of more (or fitter) Overall, these results confirm that NSGS mice allow for a rapid and LICs in HR AML samples than in FR or IR AML samples. stable read-out of the engraftment of cells from risk-stratified AML patients. Orthotopic coadministration of patient-matched BM MSCs and primary blasts is dispensable for AML Long-term engraftment of AML cells in NSGS mice is engraftment levels but favors peripheralization of limited to those from HR AML patients who have engrafted AML cells more or fitter LICs MSCs are an important cellular component of healthy and leukemic NSGS mice have been shown not to support a robust engraftment of BM microenvironments. In fact, AML progression and chemoresist- healthy CD341 HSPCs in long-term serial transplantation assays as a ance are highly dependent on leukemia–BM stroma interactions.15,18 result of the potential exhaustion of the engraftment-initiating The impact of the coadministration of BM MSCs on the engraftment of cells attributed to the long-term exposure to human myeloid cyto- primary human samples has been evaluated in the context of healthy kines.45-47 In contrast, the engraftment of MDS primary cells is sus- CD341 HSPCs, multiple myeloma, and MDS, with contradictory tained upon secondary transplantation in NSGS mice.14 To evaluate results.14,17,20-22 We next evaluated whether intra-BM coinjection of whether long-term engraftment of AML cells can be achieved in patient-matched BM MSCs affects the engraftment of risk-stratified NSGS mice, 106 cells were isolated from the BM of primary recipients AML samples. AML patient-matched MSCs were generated from pas- robustly engrafted (.60% in BM) with FR, IR, or HR AML samples sages 1 through 3, expanded, and functionally characterized in vitro and IBM transplanted into secondary NSGS recipients (n 5 24 and in vivo in previous studies from our group.24,25 A total of 105 mice; Figure 2A). We observed, 18 weeks later, that all secondary bona fide AML-BM MSCs was IBM transplanted into NSGS mice mice transplanted with HR AML samples showed a robust BM together with 106 T-cell–depleted AML cells; the engraftment was engraftment similar to that observed in primary recipients (Figure monitored in BM and PB every 6 weeks until euthanization (week 2B-C). However, for FR and IR AML samples, only 22% and 50%, 18) (Figure 3A). The presence of patient-matched BM MSCs in the respectively, of the secondary recipients had engraftment levels that NSGS BM niche did not result in enhanced AML engraftment, regard- were profoundly lower than those observed in the primary recipients less of the risk group (Figure 3B). Accordingly, follow-up of the Figure 2. Secondary transplantation of risk-stratified AML cells in NSGS mice. (A) Experimental design. 1ry, primary; 2ry, secondary. (B) Percentage of BM engraftment in secondary (2ry) recipients (n 5 24) at euthanization (18 weeks or sacrifice point) for each patient sample grouped by risk (left panel). Only the highly engrafted primary samples/mice were selected for serial transplantation (1-2 mice). The dotted line indicates the cutoff for engraftment levels . 0.1%. Each circle represents a single mouse. Gray circles depict mice with ,0.01% engraftment. Percentage of engrafted 2ry recipients (leukemic cells . 0.1%) transplanted with BM cells from primary recipients of FR, IR and HR samples (right panel). (C) Representative FACS analysis of CD45, CD33, CD19, and CD34 expression in AML patient samples and PDXs. CD34 expression within the leukemic blasts is heterogeneous. (D) Proportion of CD341 AML blasts before and after xenotransplantation in NSGS mice for all risk-stratified AML primary samples (n 5 28). For primary AML samples, each point represents a single patient. (E) Schematic diagram of the LTC-IC assays. Magnification 403. Red arrowheads depict the colonies. (F) Number of wells plated with 250 cells showing CAs after 5 weeks of blast:MS5 LTC. (G) Estimated LICs in FR, IR, and HR AML primary samples for cell doses of 250 to 2000 AML blasts. For PDX samples, each dot represents the mean 6 SEM from all mice transplanted with that sample (n 5 112 mice). Data are mean 6 SEM. **P , .01, ****P , .0001, unpaired Student t test. D, day; IBMT, intra-BM transplantation; ND, not detected; NK, normal karyotype; ns, not significant; W, week. 14 DECEMBER 2021 • VOLUME 5, NUMBER 23 RISK-STRATIFIED AML ENGRAFTMENT IN NSGS MICE 4849 Downloaded from http://ashpublications.org/bloodadvances/article-pdf/5/23/4842/1846467/advancesadv2020003958.pdf by guest on 27 January 2023 Figure 3. Orthotopic coadministration of patient-matched BM MSCs together with AML blasts does not enhance BM engraftment levels but favors peripheralization of engrafted AML cells. (A) Experimental design. (B) Kinetics of risk-stratified AML engraftment in BM at 6, 12, and 18 weeks in the presence or absence of patient-matched BM MSCs. Each point represents the mean (6SEM) of 36 to 40 mice. (C) Kaplan-Meier curves depicting 18-week EFS of mice transplanted with risk-stratified AML samples with or without BM MSCs (n 5 28 AML samples; n 5 224 mice). (D) Kinetics of risk-stratified AML engraftment in PB at 6, 12, and 18 weeks in the presence or absence of patient-matched BM MSCs. Each point represents the mean (6SEM) of 36 to 40 mice. (E) Blast infiltration in spleen and liver at euthanization for risk-stratified AML in the presence or absence of patient-matched BM MSCs. Data are mean 6 SEM. *P , .05, unpaired Student t test. D, day; IBMT, intra-BM transplantation; ns, not significant; W, week. 4850 DIAZ de la GUARDIA et al 14 DECEMBER 2021 • VOLUME 5, NUMBER 23 animals confirmed an almost identical 18-week EFS for mice trans- residual healthy myeloid progenitor cells. Our results confirm recent planted with risk-stratified AML samples in the presence or absence studies suggesting the use of the NSGS mouse strain for robust of patient-matched BM MSCs (P . .05; Figure 3C). We further engraftment of primary myeloid neoplasms6,14,45 and to reliably mimic assessed, in a limited number of NSGS mice, whether MSCs from human disease. Interestingly, IR AML PDXs also displayed multiple healthy BM donors contribute to AML engraftment, but we did not pathogenic mutations in myeloid genes that were absent in the diag- find any enhanced engraftment associated with the coadministration nostic sample, likely reflecting minor leukemic clones that were unde- of healthy donor BM MSCs (supplemental Figure 2). tectable at diagnosis or the acquisition of de novo environmental/ niche pressure–mediated myeloid mutations. Further prospective Of note, intra-BM coadministration of patient-matched BM MSCs experimental work should unravel the potential contribution of these favored the BM exit of FR and IR AML engrafted cells to the PB (Fig- de novo pathogenic myeloid mutations to leukemia initiation and main- ure 3D) and spleen and liver (Figure 3E). However, such BM tenance in NSGS mice. MSC–mediated peripheralization was not observed in NSGS mice transplanted with HR AML cells (Figure 3D-E). We hypothesized Importantly, the number of engrafted mice and the engraftment levels Downloaded from http://ashpublications.org/bloodadvances/article-pdf/5/23/4842/1846467/advancesadv2020003958.pdf by guest on 27 January 2023 that the aggressiveness and high engraftment levels of HR AML cells observed in NSGS mice by week 6 remained stable over time (up to may mask such a MSC-mediated effect. To address this, NSGS mice week 18) regardless of the risk group, confirming that NSGS mice (n 5 8) were IBM transplanted with a 10-fold lower dose of HR-AML allow for a rapid and stable read-out of the engraftment of risk- cells (105 T-cell–depleted AML cells) together with 105 patient- stratified AML samples. This contrasts with previous studies reporting matched BM MSCs. These dose-limiting in vivo assays revealed that the engraftment read-out of AML primary cells in NSG mice must that, similar to FR and IR AML samples, the coadministration of BM be extended for several months. Of note, FR AML samples engraft MSCs also favors liver and spleen infiltration of HR AML cells (supple- poorly in NSG mice, likely as the result of a defective environmental mental Figure 3). Together, the administration of patient-matched BM support. Similar to the study by Ellegast et al,6 our data revealed MSCs together with AML blasts is dispensable for BM engraftment that all (10/10) FR AML samples, including inv(16)-, PML-RARa–, levels but favors peripheralization of engrafted AML cells. AML1-ETO–, or NPM1-mutated samples, engrafted in 85% of NSGS mice. Of interest, cells from 1 NPM1/IDH comutated patient Discussion exhibited significantly lower engraftment levels than did cells from NPM1-mutated patients with germline IDH1. Although these data AML is 1 of the most common hematopoietic cancers in adults; its should be treated with caution because of the limited number of incidence increases significantly in elderly people, and it is often asso- NPM1/IDH comutated patients assayed in this study, they provide bio- ciated with a dismal prognosis.1 AML is initiated by genetic and epige- logical clues about the impact of IDH mutations on the initiation and netic lesions in HSPCs or myeloid progenitors, resulting in the maintenance of AML. accumulation of poorly differentiated myeloid cells in BM and infiltrat- ing extramedullary tissues.49 The large cytogenetic and molecular het- MDS primary cells, but not healthy CD341 HSPCs, have been shown erogeneity observed in AML contributes to patient risk stratification, to be serially transplanted in NSGS mice.45-47 In our cohort of risk- which has a major role in prognostication and contributes significantly stratified AML patients, we found that HR AML samples, but not FR to treatment decisions.2-4 AML is frequently associated with chemo- or IR AML samples, could be serially transplanted in NSGS mice. therapy refractoriness and relapse, suggesting that a failure of current One exception were cells from an inv(16)1 AML patient that could therapies to eradicate LICs is a major mechanism underlying AML pro- be successfully retransplanted, in line with a previous study showing gression/relapse.4,50,51 Moreover, BM MSCs, a major cellular compo- that inv(16)1 AML diagnostic samples may be serially transplanted nent of the BM niche, have been shown to support the growth, in NSGS mice upon intrahepatic injection.6 Previous studies have survival, and chemoresistance of AML cells.15,18 Unfortunately, treat- shown that long-term exposure to the high levels of human cytokines ment of AML has not improved substantially in the last 2 or 3 decades; occurring in NSGS mice could lead to the exhaustion of CD341 this is due, in part, to the lack of reliable in vivo models recapitulating HSPCs, affecting the long-term self-renewal activity of and impacting the disease.2,3 In this sense, robust AML-PDX models represent a the establishment of secondary grafts.43-47 Therefore, we analyzed the unique in vivo platform for studying AML pathogenesis and for testing dynamics of CD341 AML blasts before and after transplantation into novel targeted therapies.4-9 However, it has been challenging to antic- primary NSGS mice as an indirect approach to gain insights into the ipate the engraftment of primary samples derived from FR and IR AML differential serial transplantation observed for AML cells according to patients in NSG mice, hindering the establishment of reliable PDX the risk group. Despite the heterogeneity of CD34 expression across models covering AML heterogeneity.12 Here, we characterized the AML patients, the proportion of CD341 blasts observed in HR AML engraftment robustness/kinetics of cells from 28 risk-stratified AML samples was retained or slightly increased in primary NSGS mice, patients in NSGS mice6,13,14 and assessed whether the orthotopic whereas CD341 AML primary blasts were lost in PDXs transplanted coadministration of patient-matched BM MSCs improves AML with FR or IR AML samples. LTC-IC in vitro assays further confirmed engraftment. the presence of more (or fitter) LICs in HR AML samples compared with FR or IR AML samples. Differences in sensitivity to specific niche Our results show that the vast majority of NSGS mice were engrafted cues involved in maintaining leukemic growth cannot be ruled out. Our with primary AML samples independently of the risk group, with data also suggest the presence of AML-LICs in the CD342 leukemic engraftment levels significantly superior for HR AML cells compared fraction, regardless of the risk group, which is in line with previous with FR and IR AML cells. Importantly, EFS of PDXs correlated well reports.48,52,53 with the predictable prognosis of risk-stratified AML patients. Of note, AML PDXs consistently phenocopied the immunophenotype The impact of the coadministration of BM MSCs in the engraftment of of the primary AMLs, and FISH and NGS studies confirmed that the primary human samples has been evaluated in the context of normal engraftment stemmed from the original leukemic clone and not from hematopoiesis, multiple myeloma, and MDS, with inconsistent 14 DECEMBER 2021 • VOLUME 5, NUMBER 23 RISK-STRATIFIED AML ENGRAFTMENT IN NSGS MICE 4851 results.14,17,20-22 For example, Medyouf et al17 demonstrated that PI17/01028) and Asociacio n Espan~ola Contra el Ca ncer (C.B.), coinjection of MSCs from MDS patients enhanced engraftment in Health Institute Carlos III/FEDER (CPII17/00032) (V.R.-M.), and NSG mice. In contrast, Rouault-Pierre et al20 failed to find any Fundacio n Hay Esperanza (E.A.). CERCA/Generalitat de Catalu- improvement in the engraftment levels of MDS BM cells in mice coin- nya and Fundaci o Josep Carreras-Obra Social la Caixa provided jected with MSCs. Likewise, Krevvata et al14 reported that the coinjec- institutional support. B.L.-M. was supported by a Lady Tata Memo- tion of MSCs did not enhance human MDS engraftment. However, it rial Trust International Award and Asociacion Espan~ola Contra el remains elusive whether intra-BM coinjection of patient-matched BM Cancer (INVES20011LOPE).  O.M. and T.V.-H. were supported MSCs impacts the engraftment of risk-stratified AML samples. In by Asociacio n Espan~ola Contra el Cancer (INVES211226MOLI) this study, we orthotopically IBM transplanted primary blasts with and a Marie Sklodowska Curie Fellowship (792923), respectively. autologous BM MSCs from 28 risk-stratified AML patients and found P.M. is an investigator in the Spanish Cell Therapy Network. that the coinfusion of patient-matched BM MSCs in the NSGS BM niche did not result in enhanced AML engraftment, regardless of Authorship Downloaded from http://ashpublications.org/bloodadvances/article-pdf/5/23/4842/1846467/advancesadv2020003958.pdf by guest on 27 January 2023 the risk group. However, orthotopic cotransplantation of patient- matched BM MSCs favored peripheralization of engrafted AML cells, Contribution: R.D.d.l.G. and B.L.-M. conceived the study, designed as revealed by the higher levels of leukemic cells in PB, spleen, and and performed experiments, analyzed data. and wrote the manuscript; liver, especially in the FR and IR AML groups. Although the aggres- T.V.-H., F.G.-A., H.R.-H., O.M., C.N.-A., L.Z., V.R.-M., J.C.R.-M., and siveness and high-engraftment levels of HR AML cells somehow C.B. performed experiments and analyzed data; A.B., J.L.F., E.A., masked such MSC-mediated effects, a potential greater resistance S.V., J.N., M.T.G.-C., M.R.-O., and H.L. provided leukemia samples; to external cues from stroma cannot be ruled out. The capacity of and P.M conceived the study, designed experiments, wrote the man- stroma to favor mobilization has been described in several studies uscript, and financially supported the study. focusing on different chemokine axes, such as CXCR4-SDF1.7,54 In Conflict-of-interest disclosure: The authors declare no competing summary, our study provides a comprehensive in vivo characterization financial interests. of human AML in NSGS mice and reveals different inherent properties for each risk group with regard to engraftment ability, LICs, and inter- ORCID profiles: T.V.-H., 0000-0003-2183-7443; H.R.-H., 0000- actions with the stroma. The contribution of these properties to the 0001-7324-5234; O.M., 0000-0001-7585-4519; A.B., 0000- pathogenesis of AML warrants further study, which may help to 0002-6085-2745; J.L.F., 0000-0002-4881-9440; E.A., 0000-0003- improve the treatment and prognosis of AML patients. 1386-4943; J.N., 0000-0003-3399-346X; M.T.G.-C., 0000-0003- 0505-5126; H.L., 0000-0002-6900-8313; V.R.-M., 0000-0002- Acknowledgments 8013-4273; J.C.R.-M, 0000-0001-5951-7029. The authors thank Paola Romecin and Virginia Rodriguez-Cortez Correspondence: Pablo Mene ndez, Josep Carreras Leukemia for technical assistance. Research Institute, School of Medicine, University of Barcelona, Casa- This work was supported by the Spanish Ministry of Economy nova 143, 08036 Barcelona, Spain; e-mail: pmenendez@ and Competitiveness (SAF2016-80481R, PID2019-108160RB- carrerasresearch.org. Belen Lopez-Millan, Josep Carreras Leukemia I00), the Obra Social La Caixa (LCF/PR/HR19/52160011), Inter- Research Institute, School of Medicine, University of Barcelona, reg V-A programme (POCTEFA) 2014-2020 (grant PROTEO- Casanova 143, 08036 Barcelona, Spain; e-mail: blopez@ blood EFA360/19), Health Canada (H4080-144541), and carrerasresearch.org; and Rafael Dıaz de la Guardia, Josep Deutsche Josep Carreras Leuk€amie Stiftung (P.M.). Additional Carreras Leukemia Research Institute, School of Medicine, funding was provided by Consejerıa de Salud y Familia (PI- University of Barcelona, Casanova 143, 08036 Barcelona, Spain; 0119-2019) (R.D.d.l.G.), Health Institute Carlos III (ISCIII/FEDER, e-mail: rafael.diaz@genyo.es. References 1. Cancer Genome Atlas Research Network; Ley TJ, Miller C, Ding L, et al. Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. N Engl J Med. 2013;368(22):2059-2074. 2. Burnett AK. 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