Abrogation of a Histone Chaperone Pathway Mitigates Inflammation-Driven AML Progression

Abstract Background: Genetic heterogeneity makes clinical interventions challenging for acute myeloid leukemia (AML) patients. Identifying and targeting microenvironment-driven pathways that are active across AML genetic subtypes should allow the development of more broadly effective therapies. Previously, we have shown that AML microenvironment is rich in proinflammatory cytokine interleukin-1β (IL-1β) and significantly promotes the growth of AML progenitors while suppressing healthy progenitors. To elucidate this paradoxical effect, we performed transcriptome (RNA-seq) analysis from IL-1β-stimulated CD34+ AML and normal progenitors and found that ASF1B (anti-silencing function-1B) is one of the most differentially expressed genes. ASF1B is a histone chaperone, which recruits H3-H4 histones onto the replication fork during S-phase. This process is regulated by tousled-like kinase 1 and 2 (TLKs). TLKs and ASF1B are overexpressed in multiple solid tumors and associated with poor prognosis. However, their functional roles in hematopoiesis and inflammation-driven leukemia are unexplored. Here, we reveal a novel molecular mechanism that IL-1β promotes leukemia progression by activating the TLK-ASF1B pathway. Methods and Results: We first confirmed that IL-1β stimulation upregulates ASF1B expression at both mRNA and protein levels in FLT3-ITD, MLL-ENL, and NPM1 positive primary AML samples. ASF1B upregulation is abolished upon treatment with a p38MAPK inhibitor, further suggesting that ASF1B is downstream of IL-1β/p38 signaling. Next, we stably knocked down ASF1B in an AML cell line MOLM-14 using doxycycline-inducible shRNA. ASF1B-depleted AML cells exhibited reduced cell viability, inhibited growth, blocked cell cycle progression, and impaired colony formation ability. We xenografted shASF1B expressing AML cells into NSG mice and induced knockdown in vivo. Flow cytometry analysis of bone marrow cells 3 weeks post-engraftment showed 80 percent reduction in leukemia burden following ASF1B silencing compared to controls. To determine whether upregulation of ASF1B contributes to IL-1β-driven leukemic growth, we overexpressed ASF1B with MLL-ENL oncogene in a murine bone marrow transplantation model. We found that daily IL-1β exposure accelerated leukemia progression compared to vehicle-treated group (median survival = 64 vs. 85 days, p<0.05), and this effect was phenocopied by overexpression of ASF1B (median survival = 62 vs. 85 days, p<0.05). Conversely, heterozygous and complete Asf1b deletion in the MLL-ENL AML model delayed the leukemia progression compared to wildtype mice. Furthermore, Asf1b deletion attenuated IL-1β-mediated AML progression compared to wildtype controls (median survival = 63 vs. 47 days, p <0.01). Immunophenotyping of Asf1b-deficient mice using flow cytometry suggested that ASF1B is dispensable for normal hematopoiesis. Together, these data suggested that targeting of the ASF1B pathway may spare healthy cells. Additionally, we found that TLK2 which regulates ASF1B activation, is also upregulated in AML progenitors compared to healthy cells at the baseline levels and upon IL-1β stimulation. We next knocked down TLKs in human AML cells and observed a similar pattern with growth arrest, higher replication stress and DNA damage response. Finally, we generated Vav-Cre+ Tlk2 mice allowing Tlk2 deletion only in hematopoietic cells and found that Tlk2 deletion prolongs survival of leukemic mice in a dose dependent manner with and without IL-1β stimulation. Conclusions: We demonstrate that increased TLK-ASF1B expression promotes survival of AML cells. We also provide the first in vitro and in vivo evidence that the TLK-ASF1B pathway plays a critical role in potentiating IL-1β-dependent AML growth. Therefore, we establish TLK-ASF1B pathway as a novel route for therapeutic strategy to suppress inflammation-driven growth in various AML genetic subtypes. Disclosures No relevant conflicts of interest to declare.

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Epigenetic Targeting of the Myeloma-Bone Microenvironment in 3D

Blood ◽

10.1182/blood-2018-99-119649 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 246-246 ◽

Cited By ~ 1

Author(s):

Juraj Adamik ◽

Yerneni Saigopalakrishna ◽

Sree Pulugulla ◽

Quanhong Sun ◽

Philip E Auron ◽

...

Keyword(s):

Bone Marrow ◽

Inflammatory Responses ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Marrow Cell ◽

Critical Role ◽

Physical Interaction ◽

Bone Microenvironment ◽

Selective Protection

Abstract EZH2, the methyltransferase subunit of Polycomb Repressive Complex 2 (PRC2), catalyzes H3K27me3 histone modifications and epigenetically regulates genes involved in cellular pluripotency and differentiation. EZH2 plays a role in myeloma (MM) cell proliferation, survival, stemness and its elevated expression correlates with poor prognosis in MM patients. We have previously shown that EZH2 plays a critical role in preventing osteoblast differentiation of myeloma-exposed bone marrow stromal cells (BMSCs). Here we show that GSK126 blocks MM-induced hyperactivation of osteoclast precursors (OCLp). RNA-seq profiling revealed that inhibition of EZH2 prevented RANKL-induced repression of genes associated with bivalent and/or H3K27me3 promoter signatures including OCL inhibitory factors MafB, Irf8, Bcl6b and Arg1. In contrast, we found that OCLp expansion in MM1.S-conditioned media induced significant gene expression changes, which correlated with TNF and IKK signaling, inflammatory responses and CXC-chemokine receptor pathways. Several classes of small molecule EZH2 inhibitors exhibited anti-MM effects, but their efficacy has been primarily studied in 2-dimensional (2D) cell culture systems or subcutaneous MM-tumor models in vivo. Therefore, we evaluated the effectiveness of GSK126 in the context of the bone microenvironment in a novel 3D model of MM co-cultures (3D-MM). We combined basement membrane extract (BME) hydrogels with devitalized bone slices to mimic the 3D setting of MM with the OCL-resorbing endosteal surface. This enabled us to test GSK126 alone or in combination with bortezomib simultaneously on MM survival and OCLp differentiation and resorption. Differentiating OCLp did not protect MM cells from GSK126 anti-MM effects, nor did the MM cells prevent GSK126 from blocking OCL differentiation. However, mature OCL added to 3D-MM co-cultures increased the IC50 MM inhibition dose of both bortezomib and GSK126 by 40% and 50%, respectively. Further their synergy on MM cells was reduced by 70%. 3D-MM co-cultures with total bone marrows harvested from mice of different ages (1-12 months old) showed selective protection from GSK126, but not bortezomib, on MM viability. Furthermore, the resistance to GSK126 was age-dependent. Cultures of bone marrows from older mice exhibited enhanced protection of MM cells from GSK126 as compared to younger marrows. Using confocal microscopy, we found that in addition to soluble factors, physical interaction between MM and bone marrow cells reduced the effectiveness of the epigenetic drug targeting against MM. Depletion of the BMSC population from the total bone marrows using CD45+ selection before establishing the 3D MM co-cultures resulted in diminished protection of MM cell survival from GSK126. Consistent with this, addition of both primary murine and MM-patient derived BMSCs to MM cultures significantly protected MM cells from EZH2 inhibition. In addition to cell-cell contacts, the pro-survival factor IL6 released by mature OCL and BMSCs, has been implicated in mediating chemo-resistance of MM cells. In agreement with this, addition of soluble IL6 to MM-3D cultures significantly protected MM cells from GSK126 inhibition. Here we show that various cell compartments of the bone microenvironment exhibit differential and drug-specific protection for MM cells from EZH2 inhibition. In addition to direct bone marrow-MM cell interactions, soluble IL6 also exhibits resistance to GSK126. Our novel 3D-MM system enables us to rapidly screen drug combinations, and simultaneously evaluate the influence of bone-microenvironmental interactions on MM drug resistance and bone marrow cell responses to the drugs. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

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Bone marrow-derived mesenchymal stem cells inhibit T follicular helper cell in lupus-prone mice

Lupus ◽

10.1177/0961203317711013 ◽

2017 ◽

Vol 27 (1) ◽

pp. 49-59 ◽

Cited By ~ 10

Author(s):

X Yang ◽

J Yang ◽

X Li ◽

W Ma ◽

H Zou

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

Lupus Nephritis ◽

Bone Marrow Cells ◽

Tfh Cells ◽

Follicular Helper

Background The objective of this paper is to analyze the role of bone marrow-derived mesenchymal stem cells (BM-MSCs) on the differentiation of T follicular helper (Tfh) cells in lupus-prone mice. Methods Bone marrow cells were isolated from C57BL/6 (B6) mice and cultured in vitro, and surface markers were identified by flow cytometry. Naïve CD4+ T cells, splenocytes and Tfh cells were isolated from B6 mice spleens and co-cultured with BM-MSCs. The proliferation and the differentiation of CD4+ T cells and Tfh cells were analyzed by flow cytometry. Lupus-prone MRL/Mp-lpr/lpr (MRL/lpr) mice were treated via intravenous injection with expanded BM-MSCs, the differentiation of Tfh cells was detected, and the relief of lupus nephritis was analyzed. Results MSCs could be successfully induced from bone marrow cells, and cultured BM-MSCs could inhibit T cell proliferation dose-dependently. BM-MSCs could prevent Tfh cell development from naïve CD4+ T cells and splenocytes. BM-MSCs could inhibit IL-21 gene expression and cytokine production and inhibit isolated Tfh cells and STAT3 phosphorylation. In vivo study proved that BM-MSCs intravenous injection could effectively inhibit Tfh cell expansion and IL-21 production, alleviate lupus nephritis, and prolong the survival rate of lupus-prone mice. Conclusions BM-MSCs could effectively inhibit the differentiation of Tfh cells both in vitro and in vivo. BM-MSC treatment could relieve lupus nephritis, which indicates that BM-MSCs might be a promising therapeutic method for the treatment of SLE.

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Dynamic Patterns of Migration and Expansion of Hematopoiesis during MGMT Mediated Drug Selection.

Blood ◽

10.1182/blood.v104.11.156.156 ◽

2004 ◽

Vol 104 (11) ◽

pp. 156-156 ◽

Cited By ~ 3

Author(s):

Yuan Lin ◽

Perrin Cheung ◽

David L. Wilson ◽

Stanton L. Gerson

Keyword(s):

Bone Marrow ◽

In Vivo Imaging ◽

Bone Marrow Cells ◽

Clonal Expansion ◽

Dependent Manner ◽

Drug Selection ◽

Hematopoietic Stem ◽

Mouth Disease Virus ◽

Marrow Cells

Abstract While hematopoietic engraftment kinetics are well appreciated after lethal irradiation in the mouse, most observations have been limited to blood samples or terminal examination of marrow or spleen. The development of non-invasive bioluminescence in vivo imaging technology allows a dynamic picture of engraftment and clonal expansion to be defined. We have extended this technology to the process of drug resistance gene therapy. We hypothesized that drug selection would profoundly affect the extent and dynamics of hematopoietic stem cells (HSC) engraftment and clonal expansion after lentiviral mediated gene transfer of the P140KMGMT gene into murine HSC. In previous studies, we have shown that P140KMGMT gene containing retroviral and lentiviral transduced bone marrow cells provided significant protection against chemotherapeutic drugs BCNU and TMZ given with BG (O6-Benzylguanine), in vitro and in vivo. We generated a bicistronic lentiviral vector containing P140KMGMT gene and firefly luciferase gene linked by 2A sequence of FMDV(Foot-and-Mouth Disease Virus), which will cleave itself during ribosomal translation. Whole bone marrow cells was collected from BALB/c mice 4 days after 5-FU treatment and transduced with P140KMGMT-luc lentiviruses at MOI of 1.4. Transduced bone marrow cells were transplanted into lethally irradiated or non-myeloablated syngeneic recipient mice at different cell numbers. Initial bioluminescent signal emerged 6–8 days after transplantation in both lethally irradiated and non-myeloablated recipients. The onset of bioluminescent foci after transplantation occurred in a cell dose dependent manner. The initial signal emitted predominantly from bone marrow, especially femurs, humeri and vertebrae during the early stage of clonal expansion. Intense signal appeared in spleen at days 12–14 and became weaker or even disappeared by days 20–28. Clonal expansion and engraftment greatly increased after a single course of BG+TMZ treatment and initiated strong hematopoiesis in non-myeloablated recipients. Total body bioluminescence intensity of drug treated mice increased 24 fold and 7 fold compared to non-treated mice in both non-myeloablated and lethally irradiated recipients, respectively. A transient phase suggesting migration through the lymphatic system and in the spleen occurred in most mice and was exacerbated by drug selection, but this was less clear in lethally irradiated mice, where engraftment was more confined to the marrow spaces. Bioluminescence in vivo imaging reveals active migration between the bone marrow and the spleen during hematopoiesis. Drug selection has a significant impact on the patterns of engraftment and clonal expansion of HSC and progenitor cells after transplantation.

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The Role of gas6 in Venous Thrombosis In Vivo.

Blood ◽

10.1182/blood.v114.22.3057.3057 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3057-3057

Author(s):

Richard Robins ◽

Peter Carmeliet ◽

Mark Blostein

Keyword(s):

Bone Marrow ◽

Venous Thrombosis ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Recovery Period ◽

Vena Cava ◽

Specific Gene ◽

Combined Genotypes

Abstract Abstract 3057 Poster Board II-1033 Gas6 is the vitamin-K dependent protein product of growth arrest specific gene 6. A genetic deficiency of this protein protects mice against experimentally induced thrombosis without causing a bleeding diathesis. Protection from thrombosis results from a deficiency in platelet aggregation and secretion. In addition to being expressed by platelets, Gas6 and its receptors are also expressed by vascular cells including the endothelium, an organ known to play a role in the hemostatic balance. While endothelial Gas6 has been shown to promote inflammation and cell survival, it remains unknown if it contributes to the pathophysiology of venous thrombosis. To answer this question, we employed a bone marrow transplantation (BMT) strategy using wild type and Gas6 null mice to create chimeric mice with combined genotypes in the vascular and platelet compartments. Mice were exposed to a dose of radiation optimized to maximize both survival and ablation of recipient marrow. Irradiated mice were then infused with bone marrow cells isolated from the femurs and tibias of donor mice and were allowed a one month recovery period for hematologic reconstitution. Success of marrow uptake was confirmed by PCR. They were then subjected to the Ferric Chloride model of venous thrombosis in the Inferior Vena Cava (IVC). Four groups of transplanted mice were studied. Results from these BMT experiment show a contributing effect by both endothelial as well as platelet Gas6 to thrombus formation (n=8, p<0.01). Mice with combined genotypes (Gas6-/- into WT and WT into Gas6 -/-) show an intermediate thrombus weight suggesting that both vascular and platelet derived Gas6 are both responsible for thrombosis pathology. Therefore, Gas6 at both sites could be potential targets in treating venous thrombosis. Disclosures No relevant conflicts of interest to declare.

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Quantitative Phosphoproteomics Identified a New Syk-Lyn-Axl Signalling Pathway Involved In Resistance to Nilotinib In Chronic Myeloid Leukemia Cells.

Blood ◽

10.1182/blood.v116.21.3376.3376 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3376-3376

Author(s):

Romain Gioia ◽

Cedric Leroy ◽

Claire Drullion ◽

Valérie Lagarde ◽

Serge Roche ◽

...

Keyword(s):

Chronic Myeloid Leukemia ◽

Tyrosine Kinase ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Critical Role ◽

Dependent Manner ◽

Stable Isotope Labelling ◽

Resistant Cells

Abstract Abstract 3376 Nilotinib has been developed to overcome resistance to imatinib, the first line treatment of chronic myeloid leukemia (CML). To anticipate resistance to nilotinib, we generate nilotinib resistant CML cell lines in vitro to characterize mechanisms and signaling pathways that may contribute to resistance. Among the different mechanisms of resistance identified, the overexpression of the Src-kinase Lyn was involved in resistance both in vitro, in a K562 cell line (K562-rn), and in vivo, in nilotinib-resistant CML patients. To characterize how Lyn mediates resistance, we performed a phosphoproteomic study using SILAC (Stable Isotope Labelling with Amino acid in Cell culture). Quantification and identification of phosphotyrosine proteins in the nilotinib resistant cells point out two tyrosine kinases, the spleen tyrosine kinase Syk and the UFO receptor Axl. The two tyrosine kinase Syk and Axl interact with Lyn as seen by coimmunopreciptation. Syk is phosphorylated on tyrosine 323 and 525/526 in Lyn dependent manner in nilotinib resistant cells. The inhibition of Syk tyrosine kinase by R406 or BAY31-6606 restores sensitivity to nilotinib in K562-rn cells. In parallel, the inhibition of Syk expression by ShRNA in K562-rn cells abolishes Lyn and Axl phosphorylation and then interaction between Lyn and Axl leading to a full restoration of nilotinib efficacy. In the opposite, the coexpression of Lyn and Syk in nilotinib sensitive K562 cells induced resistance to nilotinib whereas a Syk kinase dead mutant did not. These results highlight for the first time the critical role of Syk in resistance to tyrosine kinase inhibitors in CML disease emphasizing the therapeutic targeting of this tyrosine kinase. Moreover, Axl, which is already a target in solid tumor, will be also an interesting pathway to target in CML. Disclosures: No relevant conflicts of interest to declare.

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Treatment with 8F4, An Anti-PR1/HLA-A2 T Cell Receptor-Like Antibody, Reduces Established Acute Myeloid Leukemia and Eliminates Leukemia Stem Cells in Mice

Blood ◽

10.1182/blood.v118.21.766.766 ◽

2011 ◽

Vol 118 (21) ◽

pp. 766-766

Author(s):

Anna Sergeeva ◽

Hong He ◽

Kathryn Ruisaard ◽

Karen Clise-Dwyer ◽

Lisa S St. John ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Flow Cytometry ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Cell Receptor ◽

Leukemia Stem Cells ◽

Nsg Mice ◽

Secondary Transfer

Abstract Abstract 766 PR1 (VLQELNVTV) is an HLA-A2-restricted leukemia-associated peptide from proteinase 3 and neutrophil elastase that is recognized by PR1-specific cytotoxic T lymphocytes that contribute to cytogenetic remission of myeloid leukemia. We developed a high affinity T cell receptor (TCR)-like mouse monoclonal antibody (8F4) that binds to a conformational epitope of the PR1/HLA-A2 complex. Flow cytometry and confocal microscopy of 8F4-labeled cells showed significantly higher PR1/HLA-A2 expression on AML blasts compared with normal leukocytes. Moreover, 8F4 mediated complement dependent cytolysis of AML blasts and Lin−CD34+CD38− leukemia stem cells (LSC), but not normal leukocytes. To investigate in vivo biological effects 8F4 on established leukemia, we established xenografts of primary human HLA-A2-positive AML in sublethally irradiated NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Leukemia engraftment was monitored in peripheral blood by flow cytometry. Mice with established PR1/HLA-A2-expressing leukemia were treated with twice-weekly intravenous injections of 200 μg 8F4 or isotype control antibody. Flow cytometry and histology analysis of tissues was used to assess leukemia burden and level of engraftment. After 5 weeks of treatment AML was reduced 300-fold in bone marrow of 8F4-treated mice compared to isotype-treated control animals (0.07 ± 0.06% hCD45+cells versus 20.4 ± 4.1%, n=5 mice per group). Moreover, leukemia stem cells (LSC, CD34+CD38−Lin-) were no longer detected in bone marrow of 8F4-treated mice, compared to 0.88 ± 0.24% in isotype-treated mice. Equally, AML was evident in the liver and spleen of isotype-treated mice (1.1 ± 0.16% and 0.32 ± 0.17%, respectively), but was undetectable in 8F4-treated mice (p<0.001). Similar results were obtained with AML from two additional patients, one with secondary AML (CMML) and one with AML-M7. Bone marrow contained 6.2 ± 3.0% (n=3) AML versus 41 ± 15% (n=2 mice; p=0.06) in the first case and 0.16 (n=1) versus 7.0 ± 4.1 (n=2) in the second case after 2–3 weeks of twice-weekly injection. To confirm 8F4-mediated elimination of LSC, we performed secondary transfer experiment with 1×106 bone marrow cells from 8F4- and isotype-treated mice, transplanted into recipient NSG mice, irradiated with 250 cGy. AML was undetectable in mice that received bone marrow from 8F4-treated animals versus 4.1 ± 2.4% (n=4) in bone marrow of mice that received cells from isotype- treated mice, determined at 16 weeks after secondary transfer. Because PR1/HLA-A2 expression on normal hematopoietic cells (HSC) is similar to LSC in AML patients, we sought to determine whether 8F4 treatment of NSG mice xenografted with CD34-selected umbilical cord blood resulted in elimination of xenograft. Fourteen weeks after transplant stable chimerism (4.1 - 7.7% hCD45+ cells) was established, mice were treated with 50 μg 8F4 intravenously and peripheral blood was monitored weekly for chimerism. Human CD45+ cells decreased to 0.35 – 0.95% by week 1, but increased to 1.9 – 2.1 % hCD45+ cells at week 3. Bone marrow at week three contained myeloid (CD13+CD33+) and lymphoid (CD19+) cells showing that while 8F4 has off- target effects against normal hematopoietic cells, HSC are preserved. This is consistent with our previous studies that showed no 8F4-mediated effect on colony formation of normal bone marrow cells. In conclusion, these results show that anti-PR1/HLA-A2 monoclonal antibody 8F4 is biologically active in vivo and selectively eliminates LSC, but not normal HSC. This justifies continued study of 8F4 as a novel therapy for AML. Disclosures: No relevant conflicts of interest to declare.

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Egr1 and Apc Haploinsufficiency Cooperate in the Pathogenesis of Myeloid Neoplasia: A Mouse Model for Therapy-Related Myeloid Neoplasms with a Del(5q)

Blood ◽

10.1182/blood.v120.21.117.117 ◽

2012 ◽

Vol 120 (21) ◽

pp. 117-117 ◽

Cited By ~ 1

Author(s):

Angela Stoddart ◽

Jianghong Wang ◽

Anthony Fernald ◽

John Anastasi ◽

Michelle Lebeau

Keyword(s):

Bone Marrow ◽

Mouse Models ◽

Median Survival ◽

De Novo ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Macrocytic Anemia ◽

Complete Inactivation ◽

Myeloid Neoplasms ◽

Single Allele

Abstract Abstract 117 Heterozygous deletions of the long arm of chromosome 5 are among the most common abnormalities in de novo (∼15% of patients) and therapy-related myeloid neoplasms (t-MN) (∼40% of patients). Two minimally deleted segments have been identified - the minimally deleted segment within 5q31.2 is associated with de novo AML and t-MNs, whereas the other spans 5q33.1 and is associated with MDS with an isolated del(5q). Current studies support a haploinsufficiency model, in which loss of a single allele of more than one gene on 5q contributes to the development of myeloid neoplasms. Using mouse models, we previously showed that haploinsufficiency of Egr1 (5q31.2) or Apc (5q22-frequently deleted in t-MN) independently recapitulates some features of human myelodysplastic syndromes (MDS). To test the hypothesis that reduced levels of EGR1 and APC cooperate in the pathogenesis of MDS/AML, we generated mice expressing a single allele of Egr1 and Apc: Mx1-Cre+Apcfl/+Egr1+/−(Apcdel/+Egr1+/−). At 2 mos of age, we induced deletion of a single allele of Apc by injection of 3 doses of pI-pC. Survival curves clearly show that Egr1 and Apc haploinsufficiency cooperate in the development of disease with a median survival of 129 days for Apcdel/+Egr1+/− mice and 296 days for Apcdel/+mice (P<0.0001). Although disease latency was significantly shorter for Apcdel/+Egr1+/− mice, their phenotype was similar to Apcdel/+ mice, with only two exceptions. For both cohorts, mice typically developed splenomegaly and a lethal macrocytic anemia with monocytosis. Anemic mice had an increased proportion of CD71+Ter119+ erythroblasts, indicating a block in erythroid development between the early and late basophilic erythroblast stage. Two mice displayed anemia and leukocytosis (WBC 51–72 k/mL) with an increased proportion of Mac1+ cells in the spleen and Kit+ cells in the bone marrow (1 mouse). As anticipated, mice with wild type levels of Apc (Mx1-Cre-Apcfl/+) or with loss of one allele of Egr1 showed no signs of anemia. Mutations in TP53 are commonly found in t-MNs with a del(5q) and loss of Tp53 in mouse models has been shown to promote AML by enabling aberrant self renewal. To test the hypothesis that loss of TP53 may adversely advance disease development, we crossed Tp53+/− to Egr1+/− and Apcdel/+ mice. Similar to Apcdel/+Egr1+/− mice, Apcdel/+Tp53+/− mice rapidly developed macrocytic anemia with a median survival of 144 days, suggesting that partial loss of TP53 function accelerates the Apcdel/+ -induced macrocytic anemia. Triple heterozygous mice (Apcdel/+Tp53+/−Egr1+/−) had a median survival of 178 days, but survival was not statistically different than Apcdel/+Egr1+/− mice (P=0.35) suggesting that Egr1 and Tp53 loss play redundant roles in the development of disease in Apcdel/+ mice. Thus, in the context of Apc haploinsufficiency, loss of Egr1 or Tp53 function promotes erythroid failure. These results are in contrast to the setting of ribosomal protein haploinsufficiency, as is the case in MDS with an isolated del(5q), where induction of TP53 is essential for erythroid failure. It has been proposed that inactivation of TP53 (through additional TP53 mutations) would be required for progression to AML, in the setting of a 5q deletion. To this end we transduced Egr1+/−Apcdel/+ bone marrow cells with a Tp53-specific shRNA, known to reduce Tp53 transcripts by ∼90%, and transplanted them into lethally irradiated C57BL/6 mice. Although penetrance of disease was low, 2 out of 13 mice (15%) developed an aggressive AML, as compared to 0 of 12 mice transplanted with Egr1+/−Apcdel/+ cells transduced with control shRNA. These data suggest that EGR1 and APC haploinsufficiency cooperate in the development of myeloid disorders, characterized by ineffective erythropoiesis, and that further mutations, such as that achieved by complete inactivation of TP53, are required for progression to AML. Disclosures: No relevant conflicts of interest to declare.

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CXCR4 Invalidation Limits the MLL-ENL Induced Leucemogenesis in Vivo

Blood ◽

10.1182/blood.v120.21.4089.4089 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4089-4089

Author(s):

Yanyan Zhang ◽

Hadjer Abdelouahab ◽

Aline Betems ◽

Monika Wittner ◽

William Vainchenker ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Survival Time ◽

Median Survival ◽

Median Survival Time ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Hematopoietic Stem ◽

Acute Myeloid

Abstract Abstract 4089 The receptor CXCR4 and its ligand SDF-1 play major physiological roles especially on hematopoietic stem cells homing and retention. Many studies have implicated CXCR4 in the invasion by tumor cells of organs that produce SDF-1. In acute myeloid leukemia, the physiological role of CXCR4 is not fully understood. We used retrovirus to express MLL-ENL oncogene in CXCR4+/+ and CXCR4−/− hematopoietic primitive cells (Lin- isolated from fetal liver) and showed that CXCR4 is dispensable for generation of immortalized colonies in vitro. To determine CXCR4 function in vivo, CXCR4+/+ and CXCR4−/− transformed cells were transplanted into lethally irradiated mice. Whatever their phenotype, the recipient developed a myelo-monocytique leukemia characterized by their expression of Gr-1 and Mac-1. As expected, all recipients of MLL-ENL transduced CXCR4+/+ cells were moribund within 35 to 80 days post transplant (median survival time: 50 days). Strikingly, recipients of MLL-ENL transduced CXCR4−/− cells showed significantly increased lifespan, with a median survival time of 90 days. The cellularity of the peripheral blood of recipients of MLL-ENL transduced cells displayed considerable increases over time although this increase was much lower in CXCR4−/− than in CXCR4+/+ chimera. Bone marrow of MLL-ENL transduced CXCR4−/− chimera had moderately decreased numbers of mononuclear cells. There were important numbers of leukemic CD45.2+/Gr1+/Mac1+/c-kit+ cells in spleen from MLL-ENL CXCR4+/+ chimera which suggested that CXCR4 is important for leukemic progenitors cells retention in the bone marrow and especially in the spleen. The homing capacity of transduced CXCR4+/+ cells is comparable to the CXCR4−/− cells. Finally, more DNA damages were found in the BM cells of MLL-ENL CXCR4−/− chimera. All these results were confirmed by treating of MLL-ENL CXCR4+/+ chimera with CXCR4 inhibitor (TN140). These results demonstrated that in absence of CXCR4, the cells transduced by oncogene MLL-ENL are capable of generating leukemia in the recipients. However, mice transplanted with MLL-ENL transduced CXCR4−/− FL cells developed acute myeloid leukemia with reduced aggressiveness and organ infiltration, which is associated with induced differentiation and DNA instability. These results indicated that the MLL-ENL progenitors are dependent on CXCR4 for their maintenance in the BM and spleen suggesting that CXCR4 inhibitors might have potential therapeutic applications. Disclosures: No relevant conflicts of interest to declare.

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Recipient CD8+ DC Delete Alloreactive Donor CTL and Promote Leukemic Relapse after Allogeneic BMT

Blood ◽

10.1182/blood.v126.23.4279.4279 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4279-4279

Author(s):

Kate A Markey ◽

Rachel D Kuns ◽

Renee J Robb ◽

Motoko Koyama ◽

Kate Helen Gartlan ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Antigen Presentation ◽

Median Survival ◽

Conflicts Of Interest ◽

Critical Role ◽

Allogeneic Bone Marrow Transplantation ◽

Allogeneic Bone ◽

Leukemic Relapse

Abstract Allogeneic bone marrow transplantation (BMT) remains the therapy of choice for many haematological malignancies, but despite the curative benefit of the immunological graft-versus-leukemia (GVL) effect, relapse remains a key cause of death. We have investigated the role of recipient dendritic cells (DC) in antigen presentation to donor CD8 cytotoxic T cells (CTL) in a model of BMT where GVHD and GVL are directed to multiple minor histocompatibility antigens (mHA) and survival reflects GVL activity. C3H.Sw bone marrow and purified CD8 T cell grafts were transplanted with B6-derived MLL-AF9 induced primary acute myeloid leukemia (AML) into lethally irradiated B6.CD11c.DOG recipients (diphtheria toxin receptor (DTR), ovalbumin and GFP expression driven off the CD11c promoter) such that recipient DC can be deleted by DT administration. Surprisingly, depletion of recipient DC resulted in improved leukemic control (median survival 43 vs 31 days, P <0.001). The use of IRF8-/- BMT recipients (in which the CD8+ DC subset is absent) confirmed that recipient CD8+ DC were critical for regulating these GVL effects (median survival 43 vs 34 days, P = 0.0005). Conversely, when recipient CD8+ DC were expanded in a B6 to B6D2F1 model with bcr-abl/Nup98-HoxA9 induced primary AML, by using Flt3-L treatment for 10 days prior to BMT, GVL effects were completely eliminated, rendering relapse rate equivalent to that seen in the recipients of T cell depleted (TCD) grafts (median survival 11 days in BM+T and TCD groups where recipients were pre-treated with Flt3-L, vs. >45 days in the saline treated BM+T group). The use of B6.CD11c-Rac1 transgenic BMT recipients (who cannot process and present exogenously acquired antigen) confirmed that this effect was the result of endogenous alloantigen presentation by recipient DC and independent of cross-presentation.Using the same depletion strategies in an antigen-specific model (with donor OT-I T cells and B6.CD11c.DOG x DBA/2 F1 recipients) we confirmed that recipient DC invoked effector donor CTL activation, differentiation (CD25+ CD69+ CD62L-) and subsequent apoptosis (as measured by Annexin V; 52.4% vs. 23.9% in DC replete vs. depleted recipients, P = 0.01). There was a consequent profound contraction of the donor CTL compartment by day 10 in DC replete recipients. This contraction of the CTL compartment was associated with reduced expression of the cytolytic molecule granzyme B (MFI 1922 vs 1097, P = 0.02). Antigen presentation has a critical role in the initiation of donor T cell alloreactivity and GVL after BMT. Here we demonstrate that endogenous alloantigen presentation by recipient CD8+ DC to donor T cells leads to activation induced death of donor CTL early after BMT, which in turn facilitates leukemic relapse. This concept has critical implications for the design of therapies that target DC in the peri-transplant period and confirms that recipient DC regulate GVL effects. Disclosures No relevant conflicts of interest to declare.

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Divisional Memory Drives Hematopoietic Stem Cell Functional Diversity

Blood ◽

10.1182/blood-2021-153296 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 20-20

Author(s):

James Bartram ◽

Baobao (Annie) Song ◽

Juying Xu ◽

Nathan Salomonis ◽

H. Leighton Grimes ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Functional Decline ◽

Functional Heterogeneity ◽

Hematopoietic Stem ◽

Donor Cells ◽

Marrow Cells

Abstract Hematopoietic stem cells are endowed with high regenerative potential but their actual self-renewal capacity is limited. Studies using the H2B-retention labeling system show HSC functional decline at each round of division (Qiu, Stem Cell Reports 2014). We have shown that mitochondria drive HSC functional decline with division history after transplantation (Cell Stem Cell 2020). Here we examined the link between mitochondrial metabolism, in vivo division at steady state, and HSC functions using the GFP label-Histone 2B (GFP-H2B) mouse model driven by a doxycycline-inducible promoter. Five months after doxycycline removal, mitochondrial membrane potential (MMP) was examined using TMRE in HSC with varying GFP intensity. HSC were separated into an H2B-labeled retention population and an H2B-labeled population. Interestingly, within the H2B-labeled retention population, HSC could be further subdivided into GFP high, medium, and low. MMP increased in a stepwise fashion with GFP dilution in HSC. We noted the presence of 2 TMRE peaks within each GFP high and medium populations leading to 5 populations: GFP-high;MMP-low (G1), GFP-high;MMP-high (G2), GFP-medium;MMP-low (G3), GFP-medium;MMP-high (G4), GFP-low;MMP-high (G5). We examined the repopulation activity of each population in a serial competitive transplant assay. G1 and G2 maintained higher peripheral blood chimerism up to 24 weeks post-transplant than G3 and G4. G5 did not engraft at all. However, only G1 reconstituted high frequency of HSC in primary recipients. In secondary recipients, G1, G2, G3 but not G4 gave rise to positive engraftment. Interestingly, G1 and G2 grafts showed myeloid/lymphoid balanced engraftment whereas the G3 graft was myeloid-bias, suggesting that myeloid skewing can be acquired upon HSC division. We further examined lineage fate maps of bone marrow cells derived from G1 or G3 population in vivo, using single cell RNA sequencing, 10X genomics. Surprisingly, G3-derived bone marrow cells displayed a distinct myeloid cell trajectory from G1-derived bone marrow cells, in which G3 gave rise to increased immature neutrophils but fewer myeloid precursors. Remarkably, each lineage population derived from G3 donor cells had different gene expression signatures than those derived from G1 donor cells. Therefore, HSC that have divided in vivo in the same bone marrow microenvironment are intrinsically and molecularly different such that not only do they exhibit lineage potential differences but they also produce progeny that are transcriptionally different. These findings imply that cellular division rewires HSC and that this rewiring is passed down to their fully differentiated progeny. When G1 and G3 single HSC were cultured in-vitro, G1 had a slower entry into cell-cycle which has been associated with increased stemness. Additionally, when single HSC from G1 and G3 were assessed for their multipotency in a lineage differentiation assay, G1 HSC had a higher propensity to produce all four myeloid lineages (megakaryocytes, neutrophils, macrophages, and erythroid), further supporting increased stemness in G1 compared to G3 HSC. Finally, HSC from G1, G2, G3 and G4 populations carried mitochondria that were morphologically different, and express distinct levels of Sca-1, CD34 and EPCR, with Sca-1 high, CD34-, EPCR+ cells more enriched in G1. In summary, this study suggests that HSC transition into distinct metabolic and functional states with division history that may contribute to HSC diversity and functional heterogeneity. It also suggests the existence of a cell-autonomous mechanism that confers HSC divisional memory to actively drive HSC functional heterogeneity and aging. Disclosures No relevant conflicts of interest to declare.

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