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3097 Phenotyping of Disease-Initiating CD34+/CD38 Stem Cells in BCR-ABL1 MPN Reveals Expression of Multiple Cytokine Receptors and Resistance-Related Antigens

Program: Oral and Poster Abstracts
Session: 635. Myeloproliferative Syndromes: Basic Science: Poster III
Hematology Disease Topics & Pathways:
Diseases, MPN, Polycythemia vera, thrombocythemia, Myeloid Malignancies
Monday, December 7, 2020, 7:00 AM-3:30 PM

Daniel Ivanov, MSc1,2*, Jelena D. Milosevic Feenstra1*, Gregor Eisenwort, MD, PhD1,2*, Robert Spörk, MSc1*, Alexandra Keller, PhD2*, Barbara Peter1,2*, Michael Willmann1,3*, Georg Greiner1,4*, Gabriele Stefanzl1,2*, Katharina Slavnitsch1,5*, Gregor Hoermann1,4,6, Sybille Hofer7*, Wolfgang R. Sperr, MD1,2*, Heinz Sill, MD7*, Peter Bettelheim, MD8*, Klaus Geissler, MD9*, Elisabeth Koller10*, Michael Fillitz, MD11*, Michael Pfeilstocker, MD1,10, Thomas Rülicke1,5*, Thamer Sliwa, MD10*, Felix Keil, MD1,10*, Robert Kralovics, PhD4,12 and Peter Valent, MD1,2

1Ludwig Boltzmann Institute for Hematology and Oncology, Medical University of Vienna, Vienna, Austria
2Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Vienna, Austria
3Department for Companion Animals and Horses, Clinical Unit of Internal Medicine, University of Veterinary Medicine Vienna, Vienna, Austria
4Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria
5Institute of Laboratory Animal Science, University of Veterinary Medicine Vienna, Vienna, Austria
6MLL Munich Leukemia Laboratory, Munich, Germany
7Department of Internal Medicine, Division of Hematology, Medical University of Graz, Graz, Austria
8Elisabethinen Hospital, Linz, Upper Austria, Austria
95th Medical Department with Hematology, Oncology and Palliative Medicine, Hospital Hietzing, Vienna, Austria
10Third Medical Department for Hematology and Oncology, Hanusch Hospital, Vienna, Austria
11Third Department of Internal Medicine, Hanusch Hospital, Vienna, Austria
12CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria

The classical BCR-ABL1-negative myeloproliferative neoplasms (MPN) are characterized by over-production of myeloid cells, disease-related mutations in certain driver-genes (JAK2, CALR, MPL) and an increased risk to transform to secondary acute myeloid leukemia (sAML). Although considered stem cell-derived neoplasms, little is known about the phenotype and functional properties of disease-initiating neoplastic stem cells (NSC) in MPN and sAML. Recent data suggest that MPN NSC reside in a CD34+ fraction of the malignant clone. Therefore, these cells are considered most critical target populations to be examined for expression of molecular and immunological targets with the aim to develop improved or even curative NSC-eliminating therapies, such as antibody-based or CAR-T cell approaches. Using a panel of monoclonal antibodies (n=40) and multicolor flow cytometry, we established the immunological phenotype and target expression profiles of putative CD34+/CD38 NSC and CD34+/CD38+ progenitor cells in patients with polycythemia vera (PV, n=18), essential thrombocythemia (ET, n=29), primary myelofibrosis (PMF, n=38) and post-MPN sAML (n=11). In almost all patients, the putative MPN stem cells expressed the stem cell invasion receptors Hermes (CD44) and ADGRE5 (CD97), C1qR1 (CD93), the migration/adhesion receptor MIC2 (CD99), and the stem cell antigen AC133 (CD133). Contrasting normal stem cells, MPN NCS and sAML stem cells failed to express Thy-1 (CD90). Among the cytokine receptors tested, MPN NSC invariably displayed the TGFßR-related antigen endoglin (CD105), TPOR (CD110), SCFR KIT (CD117), IL-3RA (CD123), CXCR4 (CD184) and IGF-1R (CD221). NSC expressed particularly high levels of KIT and low levels of TPOR and IGF-1R. The IL-2RA (CD25) was identified on NSC in most patients with PMF and sAML, and in a few with ET, but not in patients with PV. Similarly, the GM-CSFR (CD116) was found to be expressed on NSC in most patients with PMF, a few with ET and no with PV. MPN NSC did not exhibit substantial amounts of M-CSFR (CD115), IL-3RB (CD131), FLT3 (CD135), NGFR (CD271) VEGFR-2 KDR (CD309), EPOR, MET or OSMRB. The CD34+/CD38+ MPN progenitor cells displayed a similar profile of cytokine receptors. In addition, MPN and sAML progenitor cells expressed IL-1RAP and CLL-1 in most donors examined. We next examined the expression of various immunological targets and resistance-mediating immune checkpoint antigens on NSC and MPN progenitor cells. In all MPN patients and all sAML patients tested, NSC were found to express substantial amounts of Siglec-3 (CD33) and low levels of Campath-1 (CD52) and MDR-1 (CD243). In addition, MPN NSC and sAML stem cells invariably displayed the "don’t eat" me checkpoint IAP (CD47) and the classical checkpoint PD-L1 (CD274). Exposure to interferon-gamma (200 U/ml, 24 hours) resulted in an upregulation of PD-L1 on NSC. In a subset of patients, MPN NSC expressed low levels of HB15 (CD83). In contrast, MPN NSC and sAML stem cells failed to express B7-1 (CD80), B7-2 (CD86), PD-L2 (CD273) and PD1 (CD279). MPN progenitor cells and sAML progenitors expressed an identical profile of cell surface targets and checkpoint antigens. Finally, we confirmed the disease-initiating capacity of MPN stem- and progenitor cells (CD34+ cells) using primary PMF cells in xenotransplantation experiments employing NSGS mice expressing human interleukin-3 (IL-3), granulocyte/macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF). After 28 weeks post injection, engraftment of human CD45+ cells in the bone marrow of NSGS mice was found in 15/15 mice injected with bulk mononuclear cells (MNC) containing CD34+ cells and in 0/15 NSGS mice injected with MNC depleted of CD34+ cells. Together, MPN NSC reside in a CD34+ fraction of the malignant clone and display a unique phenotype, including cytokine receptors, immune checkpoint molecules and other target antigens. The phenotypic characterization of neoplastic stem cells should facilitate their enrichment and the development of NSC-eradicating treatment concepts in MPN.

Disclosures: Valent: Allcyte GmbH: Research Funding; Pfizer: Honoraria; Cellgene: Honoraria, Research Funding.

*signifies non-member of ASH