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523 M2 Macrophages Drive Resistance to Phagocytosis and Improve Mitochondrial Metabolism in Acute Myeloid Leukemia Facilitating Leukemic Transformation and In Vivo Engraftment

Program: Oral and Poster Abstracts
Type: Oral
Session: 506. Bone Marrow Microenvironment: Contribution of Immune Signaling to the Microenvironment
Hematology Disease Topics & Pathways:
AML, Acute Myeloid Malignancies, Fundamental Science, Research, Translational Research, Non-Biological therapies, APL, Combination therapy, genomics, Clinical Research, immune mechanism, Diseases, patient-reported outcomes, immunology, Therapies, metabolism, Pharmacology, Biological Processes, Myeloid Malignancies, molecular biology, Technology and Procedures, profiling, omics technologies
Sunday, December 11, 2022: 12:00 PM

Isabel Weinhaeuser, PhD1*, Diego Pereira-Martins, PhD2*, Luciana Yamamoto de Almeida, PhD3*, Jacobien R Hilberink, BSc4*, Douglas RA Silveira, MD5,6*, Lynn Quek, MBBCh, DPhil, MRCP (UK), FRCPath5,7,8*, César Alexander Ortiz, BSc9*, Antonio R. Lucena-Araujo, Prof. PhD10*, Nienke Visser11*, Emanuele Ammatuna, MD. PhD2*, Gerwin A. Huls, MD, PhD12, Eduardo M Rego, MD, PhD13,14,15,16,17 and Jan Jacob Schuringa, PhD2

1Experimental Hematology, University Medical Center Groningen, Groningen, AL, Netherlands
2Department of Hematology, Cancer Research Centre Groningen, University Medical Centre Groningen, University of Groningen, Groningen, Netherlands
3University of Sao Paulo, Ribeirao Preto, Brazil
4Department of Experimental Hematology, University Medical Center Groningen, Groningen, Netherlands
5School of Cancer and Pharmaceutical Sciences, King's College London, London, United Kingdom
6School of Cancer and Pharmaceutical Sciences, King's College London, London, ENG, United Kingdom
7MRC Molecular Haematology Unit and Oxford Biomedical Research Centre, University of Oxford and Oxford University Hospitals, Oxford, United Kingdom
8Department of Haematological Medicine, King's College Hospital NHS Foundation Trust, London, United Kingdom
9Center of Cell-based Therapy, University of Sao Paulo, Medical School of Ribeirao Preto, RIBEIRAO PRETO, BRA
10Department of Genetic, Federal University of Pernambuco, Recife - PE, AC, BRA
11Experimental Hematology, University Medical Center Groningen, Groningen, NLD
12University Medical Center, University Groningen, Groningen, GZ, Netherlands
13Division of Hematology, Transfusion Medicine and Cell Therapy, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil
14Medical Research Laboratory on Molecular Hematology (LIM-31), University of São Paulo, São Paulo, São Paulo, Brazil
15Center for Cell-based Therapy and Regional Hemotherapy Center, University of Sao Paulo at Ribeirao Preto Medical School, Ribeirao Preto, Sao Paulo, Brazil
16University of Sao Paulo, Sao Paulo, Brazil
17University of São Paulo Medical School, Instituto do Câncer do Estado de São Paulo (ICESP), São Paulo, Brazil

Acute myeloid leukemia (AML) is a heterogeneous disease, which also extends to its tumor microenvironment (TME). Using single cell RNA-seq data (GSE116256), we performed differential gene expression analysis on macrophages from AML patients (AML-derived macrophages, AdMs) compared to healthy bone marrow macrophages. Thus, 306 upregulated genes in AdMs were identified as involved in different biological pathways such as proliferation, inflammation and cellular metabolism. Down-regulated genes (232) were mainly associated with the regulation of apoptosis. Single cell gene set enrichment analysis associated AdMs with an M2-like phenotype and an immunosuppression transcriptional program. We then investigated the macrophage landscape in a large cohort of AML patients by flow cytometry (n = 61) and immunohistochemistry, which revealed a large heterogeneity whereby AML patients with a predominant M2-like TME showed poor prognosis.

Next, we evaluated the immune function of AdMs compared to healthy donor-derived macrophages. Thus, we showed that AdMs displayed decreased phagocytic capacity towards AML cells compared to healthy macrophages. Since AdMs might directly descend from the mutated leukemic clones we evaluated whether these mutations could affect the immune function of macrophages. Cord blood-derived CD34+ cells were lentivirally transduced with FLT3-ITD, differentiated towards macrophages, and these FLT3-ITD+ macrophages displayed decreased phagocytic activity.

We questioned whether endogenous murine macrophages that are still present in immunodeficient mouse strains that are often used for human studies might form a barrier that needs to be taken to achieve efficient engraftment of human cells. Macrophages were isolated from NSG mice and were used in phagocytosis assays. The percentage of phagocytosed primary AML blasts by murine macrophages ranged between 3.2-51.4%, similar to phagocytosis levels observed when using human macrophages for the same AML samples. While performing these phagocytosis assays, we noted that the cells that remained and were not phagocytosed had an altered appearance and were very viable, as if these cells had gained fitness. We tested this further by exposing leukemic blasts to M2 macrophages for 48 h, after which the remaining cells were analyzed functionally. We observed that the 48 h “training” of primary leukemic blasts significantly enhanced their mitochondrial metabolism (in part mediated via mitochondrial transfer from macrophages to leukemic blasts), enhanced their in vitro and in vivo homing capacity and that they became resistant to phagocytosis. As a most striking example we used primary APL cells that are notoriously difficult to engraft in NSG mice, which we hypothesized could be in line with their high intrinsic susceptibility to macrophage-mediated phagocytosis. APL cells were exposed to M2 macrophages for 48h resulting in two cellular fates: 91.7% of cells were phagocytosed, but the remaining 8.3% APL blasts were able to induce fatal leukemia when transplanted into NSGS mice in all evaluated cases (n = 6), while prior to training these cells did not engraft long-term and were not able to confer disease. We also tested the tumor supportive capacity on M2 macrophages by performing intrafemoral injection in NSGS mice after which non-trained APL blasts were injected intravenously. Again, efficient engraftment and fatal leukemia development were observed, but only in mice co-injected with M2 macrophages.

Strategies to target the supportive AdM subpopulation would be of interest. In our scRNA-seq analysis, we identified the metabolic enzyme Nicotinamide phosphoribosyltransferase (NAMPT) to be upregulated in AdMs. Ex vivo studies revealed that while conventional chemotherapeutics, AraC/venetoclax, efficiently target the AML blasts (CD34+/CD117+) with minimum effects on the AdMs, NAMPT inhibition with KPT-9274 was able to efficiently target both the AML blasts and the AdM fractions. Functionally, treatment of M2 macrophages with KPT-9274 shifted their polarization towards an M1 phenotype, with reduced support to AML cells in co-culture assays.

In conclusion, we uncover the heterogeneity in the macrophage landscape in AML patients, show the functional relevance of M2-polarized macrophages for leukemic transformation, and provide alternative approaches for targeting aimed at the tumor-supportive microenvironment.

Disclosures: No relevant conflicts of interest to declare.

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