Variable expression of features of normal and neoplastic stem cells in patients with thrombocytosis

Abstract Recent studies indicate that osteoblasts play an important role in maintaining hematopoietic stem cells (HSCs) niche in the bone marrow microenvironment. The aim of study was to test the effect of osteoclasts on the fate of HSCs in a long term co-culture assay. To generate osteoclasts, peripheral blood mononuclear cells from mobilized donors were cultured for 6–10 days in αMEM media supplemented with 10% FCS, M-CSF and RANKL. After removal of non-adherent cells, the cultures contained 95% multinucleated osteoclasts and their precursors. These osteoclasts expressed TRAP and formed resorption pits on bone slices (Yaccoby et al., Cancer Res., 2004). CD34+ cells were purified from donor PBSCs and cord blood using immunomagnetic beads separation (>95% purity). Adult and cord blood HSCs were co-cultured with osteoclasts for up to 3 and 10 months, respectively, in media lacking any cytokines. Because osteoclasts do not survive long without M-CSF and RANKL, the HSCs were transferred to fresh osteoclast cultures every 6–10 days. Unlike their tight adherence to stromal cells, HSCs did not adhere to the osteoclasts and were easily recovered from co-cultures by gentle pipetting. Following 1 to 3 weeks of co-culture, committed HSCs rapidly differentiated into various hematopoietic cell lineage, followed by phagocytosis of terminal differentiated hematopoietic cells by the osteoclasts. The remaining HSCs were highly viable (>90% by trypan blue exclusion) and gradually lost their CD34 expression, so that the cultures contained subpopulations of HSCs expressing CD34−/lowCD38+ and CD34−/lowCD38−. Quantitive real time RT-PCR (qRT-PCR) revealed loss of expression of CD34 and reduced expression of CD45 by HSCs co-cultured with osteoclasts longer than 6 weeks. Variable expression of CD34 on HSCs was previously reported in murine but not human HSCs (Tajima et al., Blood, 2001). The co-cultured HSCs showed reduced capacity of generating in vitro hematopoietic colonies, and did not differentiate into osteoclasts upon stimulation with M-CSF and RANKL. We next tested the long term engraftment of these co-cultured HSCs in 2 animal models. In the first model, cord blood and adult HSCs from 2 donors recovered after >6 weeks in co-culture were injected I.V. into irradiated NOD/SCID mice. In the second novel model, co-cultured cord blood and adult HSCs from 2 donors were injected directly into rabbit bones implanted subcutaneously in SCID mice (SCID-rab model), 6–8 weeks after rabbit bone implantation. After 2–4 months, 10%±3% human CD45-expressing cells were identified in the NOD/SCID mice femora and 8%±4% in the SCID-rab mice rabbit bone. Our study suggests that osteoclasts promote rapid differentiation of committed HSCs and induce conversion of CD34+ cells to CD34− stem cells with self renewal potential. Intriguingly, long term co-culture of primary CD138-selected myeloma plasma cells (n=16) with osteoclasts resulted in dedifferentiation of tumor cells from a mature CD45− phenotype to an immature, CD45-expressing cells, suggesting a common mechanism of osteoclast-induced HSC and myeloma cell plasticity. This indicates that osteoclasts are important bone marrow component regulating human HSC niche, plasticity and fate.

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Normal and Neoplastic Stem Cells

Cold Spring Harbor Symposia on Quantitative Biology ◽

10.1101/sqb.2016.81.030965 ◽

2016 ◽

Vol 81 ◽

pp. 1-9 ◽

Cited By ~ 4

Author(s):

Melissa N. McCracken ◽

Benson M. George ◽

Kevin S. Kao ◽

Kristopher D. Marjon ◽

Tal Raveh ◽

...

Keyword(s):

Stem Cells ◽

Neoplastic Stem Cells

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EZH2 Mutations Are Drivers of Clonal Hematopoiesis and Leukemic Transformation in a Mouse Model of Primary Myelofibrosis

Blood ◽

10.1182/blood.v124.21.3211.3211 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3211-3211

Author(s):

Ioanna Triviai ◽

Thomas Stuebig ◽

Anita Badbaran ◽

Silke Zeschke ◽

Victoria Panagiota ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mouse Model ◽

Conflicts Of Interest ◽

Primary Myelofibrosis ◽

Bone Marrow Niche ◽

Leukemic Transformation ◽

Hematopoietic Stem ◽

Neoplastic Stem Cells ◽

Calr Mutations

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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