Dynamin-2 Deficiency Causes Neutropenia and Dysplastic Bone Marrow Changes in an Age and Sex Dependent Manner in Mice

Abstract The dynamins are a family of ubiquitously expressed proteins with GTPase activity and are known for their role in membrane remodeling and intracellular trafficking. However, their exact role within various hematopoietic lineages is incompletely understood. In humans, most of the clinical cases with cytopenia in Charcot-Marie-Tooth (CMT) disease due to dynamin-2 mutations are associated with neutropenia, and CMT patients may suffer from impaired wound healing. Of interest, pregnancy notably worsens CMT disease, possibly due to hormonal changes. Antiprogesterone treatment was successfully given in a CMT rat model, and similar treatment is being considered for human CMT. We previously reported that inhibiting dynamin (DNM) activity impairs migration capability in mature megakaryocytes. We obtained the conditional deletion of Dnm2 and targeted its deletion in hematopoietic tissues with the vav-cre murine strain. Homozygous deletion of Dnm2 in blood tissues appears embryonic lethal. None of the pups born showed a Vav-cre/Dnm-2 fl/fl genotype, whereas a third of the pups born had a Vav-cre/Dnm-2 fl/wt (Dnm2 het) genotype. Bone marrow cells from the heterozygous female mice (Dnm2 het) had 35% to 50% decreased Dnm2 expression in comparison with age-matched controls (CTRL). Evaluation of the complete blood counts demonstrated that Dnm2 het female mice developed leukopenia which was detected from 40 weeks of age (average granulocyte-monocyte counts: CTRL 532/mm3 vs. Dnm2 het 300/mm3; p=0.0164). Neutropenia was unequivocal at 65 weeks of age (average neutrophil counts, CTRL 700/mm3 vs. Dnm2 het 343/mm3, p=0.016). Dnm2 het showed a trend for higher platelet counts than controls, but this was non-statistically significant. Further analysis of hematopoietic lineage maturation by flow cytometry indicated that lineage-negative cells and granulocyte-monocyte progenitors were decreased in Dnm2 het mice (average bone marrow lineage-negative cells: CTRL 2.8x10E6 vs. Dnm2 het 1.97x10E6, p =0.0056; average granulocyte-monocyte progenitors: CTRL 1.35x10E6 vs. Dnm2 het 0.85x10e6, p=0.01), along with a relative increase of common lymphoid progenitors and of megakaryocyte/erythrocyte progenitors in the bone marrow. Immunohistochemical staining for mature neutrophils with Ly6G showed an overall decreased number of mature granulocytes in the bone marrow of Dnm2 het mice (average Ly6G-positive cells: CTRL 20% vs. Dnm2 het 29%, p=0.0026). A linear pattern of distribution of Ly6G positive bone marrow cells along blood vessels was observed in fewer mice in the Dnm2 het group than in the CTRL group (25% vs. 59%, p=0.02), indicating that the migration pattern within the bone marrow is altered in the Dnm2 het group (see Figure). In addition, Dnm2 het mice developed splenomegaly (average spleen weight: Dnm2 het 146 mg vs. CTRL 99 mg, p=0.006), which was secondary to a marked florid reactive germinal center hyperplasia. Some of the Dnm2 het mice, including 5 mice whose pregnancy occurred in middle-age (p=0.005 when comparing with CTRL or young Dnm2 het mice) and 2 non-pregnant older mice, showed physical signs of distress with markedly reduced activity, poor grooming, ruffled furs, and hunched posture. Both non-pregnant sick mice showed a marked decrease in Ly6G positive mature neutrophils at 0.3% of total marrow cells (Figure), and the bone marrow from one mouse was completely effaced by immature myeloid precursors, consistent with the development of acute myeloid leukemia. In addition, a third of Dnm2 hetmice showed no distress but displayed morphological bone marrow abnormalities including megakaryocytic dysmorphology. In summary, female mice with loss of Dnm2 in the hematopoietic compartment develop persistent neutropenia as they age, with decreased granulocyte progenitor production and with migration defects. These abnormalities are associated with a risk for developing megakaryocytic dysplasia, and acute myeloid leukemia. These findings might also suggest a mechanism for chronic idiopathic neutropenia, which has a predominance in middle-aged women. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Patient-derived acute myeloid leukemia (AML) bone marrow cells display distinct intracellular kinase phosphorylation patterns

Cancer Management and Research ◽

10.2147/cmar.s5611 ◽

2009 ◽

Vol Volume 1 ◽

pp. 49-59 ◽

Cited By ~ 3

Author(s):

Michael Lahn

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Kinase Phosphorylation ◽

Acute Myeloid ◽

Marrow Cells

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GM-CSF stimulates proliferation of clonal leukemic bone marrow cells in acute myeloid leukemia (AML) in vitro

Annals of Hematology ◽

10.1007/s002770050597 ◽

1999 ◽

Vol 78 (10) ◽

pp. 449-455 ◽

Cited By ~ 4

Author(s):

H. M. Schmetzer ◽

H. H. Gerhartz ◽

W. Wilmanns

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Gm Csf ◽

Acute Myeloid ◽

Marrow Cells

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CD64-targeted HO-1 RNA interference enhances chemosensitivity in orthotopic model of acute myeloid leukemia and patient-derived bone marrow cells

Biomaterials ◽

10.1016/j.biomaterials.2019.119651 ◽

2020 ◽

Vol 230 ◽

pp. 119651 ◽

Cited By ~ 1

Author(s):

Seok-Beom Yong ◽

Jee Young Chung ◽

Seong Su Kim ◽

Hyung Seok Choi ◽

Yong-Hee Kim

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Rna Interference ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Orthotopic Model ◽

Acute Myeloid ◽

Marrow Cells

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The Growth Inhibition and Apoptosis Induction of Acute Myeloid Leukemia Blast Population by the Blocking IGF-IR Pathway.

Blood ◽

10.1182/blood.v108.11.4515.4515 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4515-4515

Author(s):

Si Sun ◽

Yanli He ◽

Xingbing Wang ◽

Wei Liu ◽

Jun Liu ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Pi3k Pathway ◽

Leukemia Cell Line ◽

Free System ◽

Acute Myeloid ◽

Marrow Cells

Abstract The insulin-like growth factor-1receptor (IGF-1R) is overexpressed in a variety of tumors and has been associated with cancer development. Here, we analysis the IGF-IR expression on the bone marrow cells from 45 newly diagnosed patients with acute myeloid leukemia (AML) by flow cytometry. IGF-1R universally expressed on AML blasts and the leukemia cell line HL-60, did not show significant correlation with FAB subtypes. However, the bone marrow cells from AML patients with high myeloblast counts (>80%) generally showed brighter IGF-IR expressions, which indicated the IGF-IR pathway might play an important role for AML blast proliferation and survival. Indeed, blocking the IGF-1R pathway by neutralizing monoclonal antibodies could reduce the proliferation of HL-60 cells by 38.28% at 48 hr. This inhibitory effect on blast growth was observed in 4 of 5 AML samples. In the same IGF-1R blocking treatment, the apoptosis of HL-60 cells was significantly induced, resulting in apoptosis of 57% of the cell population with the measurement of Annexin V vs PI staining by flow cytometry. The control contained only 20% apoptotic cells. We also demonstrated that the blockade of the IGF-1R pathway inhibited the phophorylation of the PI3K pathway component Akt in HL-60 cells when cultured in a serum free system with a supplement of 50ng/ml exogenous IGF. Since PI3K pathway activation greatly contributes to the proliferation, survival and drug resistance of AML, it is of interest to study whether blockading IGF-IR could also inhibit the PI3K pathway in primary AML blasts and synergize other anti-leukemia agents to improve the therapeutic effectiveness. Conclusions: IGF-IR may play an important role in the proliferation and survival of the AML blast population; Blocking the IGF-IR pathway could significantly inhibit the growth of AML blasts and considerably induce the apoptosis of AML blasts; IGF-IR could become a critical molecular target in anti-leukemia drug discovery.

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Acute Myeloid Leukemia Cells Generate Leukemic Endothelial Cells with Leukemogenic Potential: Blood Vessels As Sanctuaries for Leukemia Relapse

Blood ◽

10.1182/blood.v118.21.241.241 ◽

2011 ◽

Vol 118 (21) ◽

pp. 241-241

Author(s):

Christopher R Cogle ◽

Gerard J Madlambayan ◽

Devorah C Goldman ◽

Azzah Al Masri ◽

Ronald P. Leon ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Endothelial Cell ◽

Blood Vessels ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Surface Proteins ◽

Nsg Mice ◽

Acute Myeloid ◽

Marrow Cells

Abstract Abstract 241 Human hematopoietic stem cells (HSCs) possess hemangioblast activity, which is defined as the ability to generate both blood and endothelium. Whether malignant HSC counterparts such as acute myeloid leukemia (AML) also display this bipotentiality remains to be defined. To test the hemangioblast potential of AML cells we first cultured primary human AML bone marrow in conditions established by Yoder and colleagues that support the growth of functional endothelial cell (EC) progenitors. AML cultured in endothelial colony forming cell (ECFC) media generated endothelial progenitor cell colonies that showed uptake of acetylated LDL and expressed several EC surface proteins, including CD105, CD146, UEA-1 and CD144. Importantly, ECFCs derived from AML bone marrow no longer expressed CD45 or myeloid surface proteins such as CD14. When placed in Matrigel, these AML derived ECFC generated capillary-like, tubular structures. Moreover, these ECFCs contained cytogenetic mutations associated with their parental leukemias. Thus, under the appropriate conditions, AML bone marrow cells can generate cells with an endothelial-like phenotype and harboring leukemia specific mutations that will be referred to as ‘L-ECFC.' To functionally define leukemia hemangioblast activity, a xenograft model of AML was employed. Sublethally irradiated NOD/scid/IL2Rγ−/− (NSG) mice were transplanted with primary human AML cells and then sacrificed at 8–36 weeks after transplant. Significant accumulations of human AML cells were found in perivascular regions of the liver. Both tight coupling and bona fide cell fusion between AML and ECs was observed. AML derived EC that were integrated into portal vein endothelium showed induction of CD105 expression Follow-up AML xenotransplant experiments with BrdU labeling revealed almost four-fold fewer (6%) of the AML cells incorporated within blood vessels were BrdU+, as compared to AML cells not integrated in blood vessels (22%) (P=0.01). These results suggest that AML cell incorporation within the endovascular lining induces cell quiescence. Thus, leukemia-integrated ECs may be less susceptible to cell cycle active agents like cytarabine. Results from these experiments also raised the possibility that AML cells adopting an endothelial-like phenotype may serve as a reservoir for leukemic relapse. To test this hypothesis, we injected CD105+CD45- L-ECFC derived from AML patients into NSG mice. These L-ECFC generated colonies of human CD105+CD45- within spleens and bone marrow of recipient mice. We also found a distinct population of human CD45+CD19- cells comprising 5–10% of bone marrow cells. Leukemia-derived cells were confirmed by detection of cytogenetically mutant cells consistent with the parent leukemia (e.g., MLL duplications). In conclusion, this study demonstrates that AML cells can functionally generate leukemic ECs that become quiescent after incorporation in blood vessel networks and can re-emerge with a leukemogenic phenotype. Together, our results raise the strong possibility that AML cells exhibit functional hemangioblast activity and that vascular endothelium may serve as a clinically important sanctuary for occult leukemia. Our data also support endothelial cell targeting strategies as a means to eradicate AML. Disclosures: No relevant conflicts of interest to declare.

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t(6;9) in bone marrow cells in two patients with sarcoidosis and acute myeloid leukemia

Cancer Genetics and Cytogenetics ◽

10.1016/0165-4608(89)90671-7 ◽

1989 ◽

Vol 38 (2) ◽

pp. 297-300 ◽

Cited By ~ 10

Author(s):

I. Nordenson ◽

L. Bjermer ◽

G. Holmgren ◽

P. Hörnsten ◽

A. Wahlin

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Acute Myeloid ◽

Marrow Cells

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Faculty Opinions recommendation of Effect of the nonpeptide thrombopoietin receptor agonist Eltrombopag on bone marrow cells from patients with acute myeloid leukemia and myelodysplastic syndrome.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1168446.630630 ◽

2009 ◽

Author(s):

Antonio Risitano ◽

Fabiana Perna

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myelodysplastic Syndrome ◽

Myeloid Leukemia ◽

Receptor Agonist ◽

Bone Marrow Cells ◽

Thrombopoietin Receptor ◽

Thrombopoietin Receptor Agonist ◽

Acute Myeloid ◽

Marrow Cells

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Continued Expression of the NRAS(G12V) Oncogene Is Required for the Maintenance of Acute Myeloid Leukemia Induced in Cooperation with MLL-AF9.

Blood ◽

10.1182/blood.v110.11.1615.1615 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1615-1615

Author(s):

Won-Il Kim ◽

Ilze Matise ◽

Miechaleen Diers ◽

David Largaespada

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Transgenic Mice ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Scid Mice ◽

Myeloid Lineage ◽

Blast Cells ◽

Acute Myeloid ◽

Marrow Cells

Abstract To study the role of the NRAS(G12V) oncogene in the context of acute myeloid leukemia (AML) cells developing with in cooperation with MLL fusion oncogene (MLL-AF9), we used a Vav promoter-Tet transactivator (Vav-tTA)-driven repressible system of NRAS(G12V) expression in Mll-AF9 mice. Vav-tTA; TRE-NRAS(G12V); Mll-AF9 triply-transgenic mice were generated by crossing the Vav-tTA; TRE-NRAS(G12V) doubly-transgenic FVB/n and Mll-AF9 knock-in BL6 mice. The triply-transgenic FVB/n × BL6 F1 mice expressing both the NRAS(G12V) and Mll-AF9 transgenes developed AML, which showed a trend of decreased latency compared with those carrying only the Mll-AF9 knock-in transgene. Mast cell disease also occurred accordingly in the Vav-tTA; TRE-NRAS(G12V) co-transgenic mice. Since the mastocytosis disease is not transplantable, we transplanted bone marrow cells from four independent AML mice into recipient SCID mice to determine whether NRAS(G12V) expression is necessary to maintain AML in the recipient mice without mastocytosis. Continuously treating the transplanted SCID mice with doxycycline (Dox) in drinking water, we found the expression of NRAS(G12V) oncogene was required for AML persistence in three out of the four independent primary AML cells. Furthermore, we transplanted the AML bone marrow cells previously xenografted in the recipient SCID mice into other SCID mice to conditionally repress NRAS(G12V) expression only after the transplanted AML was fully established. We found the number of WBC cells was greatly decreased 4–6 days after the Dox treatment and this was correlated with the significant increase of apoptotic cells in bone marrow and peripheral bloods. The transplanted AML blast cells underwent apoptosis and were mostly removed from the circulating blood, bone marrow, and spleen after 8 days post Dox treatment. In 2–3 weeks after beginning Dox treatment and observing AML remission, Dox-resistant leukemia relapse was observed in recipient SCID mice. The relapsed leukemia failed to express NRAS(G12V) and showed significantly reduced aggressiveness along with less myelosuppression and more differentiated myeloid lineage cells than AML prior to repression of NRAS(G12V) expression. The NRAS(G12V)-independent relapsed disease histopathologically resembles an aggressive myeloproliferative disease (MPD) rather than AML, because the proportion of AML blast cells was less than 20% of myeloid lineage cells. The NRAS(G12V)-independent MPD could be transplanted into recipient SCID mice, but the subsequent anemia was greatly attenuated compared to transplant of the same AML clone expressing NRAS(G12V). We conclude that NRAS(G12V) can be a good molecular target to treat AML, because NRAS(G12V) expression is required for persistence and specific malignant features in AML induced in cooperation with MLL-AF9. Targeting NRAS(G12V) can strongly disturb the maintenance of AML blast cells and myelosuppression, although leukemia cells can relapse without NRAS(G12V) expression.

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