Abstract SY08-02: Normal and neoplastic stem cells

Abstract Primary Myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by aberrant myeloid differentiation, associated with disruption of the bone marrow niche with subsequent fibrosis development and a high risk of leukemic transformation. The phenotypical complexity observed in PMF likely reflects the heterogeneous mutation profile of the neoplastic stem cells driving the disease. In our former work, we identified a CD133+ hematopoietic stem / progenitor cell (HSPC) population from patient peripheral blood that can drive major PMF morbidity parameters in a xenotransplantation mouse model. Mutational analysis of the JAK2 locus at the single cell level within the CD133+ population showed highly variable levels of cells with a JAK2+/+, JAK2V617F/+, or JAK2V617F/V617F genotype, indicating that clonality is unlikely driven by JAK2 mutations. In two of these patient samples, and in a third patient sample with CALR-fs* mutations, we identified a high load of missense mutations in EZH2 (45 to 95%), suggesting they may be critical for the clonal expansion of the neoplastic stem cell compartment. EZH2 mutations are found in circa 7% of PMF patients and are correlated with poor prognosis. EZH2 is a critical enzymatic subunit of the Polycomb Repressor Complex 2, which initiates gene repression of select genes through its intrinsic activity for methylating lysine-27 of histone H3 (H3K27). To date, the exact contribution of EZH2 mutations to PMF evolution or AML transition has not been clarified. CD133+ HSPC carrying EZH2 mutations either with JAK2 or CALR mutations were transplanted into immunodeficient NOD-scid-gamma (NSG) mice. Mice engrafted with patient samples carrying either EZH2-Y633C and JAK2-V617F or EZH2-Y733* and CALR-fs* mutations showed a strikingly similar phenotype, including high human cell engraftment (10-20%), skewed myelopoiesis, dysplastic human megakaryocytes, splenomegaly, anemia, and fibrosis in either the BM or spleen. In the case of xenotransplanted mice receiving CD133+ cells with a low JAK2 burden and EZH2-D265H mutations, we observed the highest engraftment in our mouse model (62-95%) and in one case AML transition with >50% CD133+ human blasts in murine bone marrow. Notably, AML arose from a CD133+ EZH2D265H/+ cell that lacked JAK2V617Fmutation. We thus conclude that EZH2 mutations confer to CD133+ neoplastic stem cells a predisposition to clonal aberrant hematopoiesis; whereas acquisition of JAK2V617F or CALR mutations likely leads to the observed myeloproliferation and disruption of megakaryocytic and erythroid regulation . Moreover, our results demonstrate that epigenetic mutations (like EZH2D265) and not JAK2V617F are critical for AML transition. Our data underscore the importance of post-transcriptional modifiers of histones in altering the epigenetic landscape of neoplastic stem cells, whose clonal growth sustains aberrant myelopoiesis and expansion of pre-leukemic clones. Disclosures No relevant conflicts of interest to declare.

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Evaluation of in vitro effects of various targeted drugs on plasma cells and putative neoplastic stem cells in patients with multiple myeloma

Oncotarget ◽

10.18632/oncotarget.11593 ◽

2016 ◽

Vol 7 (40) ◽

pp. 65627-65642 ◽

Cited By ~ 6

Author(s):

Katharina Blatt ◽

Harald Herrmann ◽

Gabriele Stefanzl ◽

Wolfgang R. Sperr ◽

Peter Valent

Keyword(s):

Stem Cells ◽

Multiple Myeloma ◽

Plasma Cells ◽

Targeted Drugs ◽

Neoplastic Stem Cells ◽

In Vitro Effects

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

Blood ◽

10.1182/blood-2020-140477 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 53-53

Author(s):

Daniel Ivanov ◽

Jelena D. Milosevic Feenstra ◽

Gregor Eisenwort ◽

Robert Spörk ◽

Alexandra Keller ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Progenitor Cells ◽

Immune Checkpoint ◽

Research Funding ◽

Cytokine Receptors ◽

Increased Risk ◽

Cd34 Cells ◽

Neoplastic Stem Cells ◽

Low Levels

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.

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