Epigenetic Signaling Is Required for HoxA9-Based Leukemic Transformation.

Abstract Abstract 3966 Poster Board III-902 Since oncogenic activation of HoxA9 is induced by multiple chromosomal translocations affecting MLL1 (11q23)(e.g. MLL-Af9) or Nup98 (11p15)(e.g. Nup98-HoxA9 or Nup98-Nsd1), the function of the HoxA9 transcription factor is of critical interest in human acute myeloid leukemia (AML). HoxA9 forms heterodimeric DNA binding complexes with members of the Pbx and/or Meis family of homeodomain proteins. Importantly, the direct transcriptional targets of endogenous HoxA9 that mediate transformation remain largely unknown. The Growth factor independent-1 (Gfi1) transcriptional repressor is known to induce granulopoiesis and inhibit myeloid progenitor proliferation. GFI1 is mutated in patients with severe congenital neutropenia (SCN). SCN patients are at increased risk for AML. In a transcriptional circuit conserved to Drosophila, we have recently shown that Gfi1 represses HoxA9, Meis1 and Pbx1 expression, that Gfi1 and HoxA9 demonstrate dramatic epistatic relationships, and that Gfi1 loss of function is potently preleukemic. Our new bioinformatic, biochemical and expression data reveal microRNA genes to be targets of endogenous HoxA9 versus Gfi1 antagonism. Moreover, these miR are activated by Hox-signaling leukemia oncoproteins. Next, in both murine leukemia models and primary human AML samples, antagomir-mediated inhibition of microRNA function specifically disrupts oncogenic signaling by HoxA9, Nup98-HoxA9 and MLL-Af9 (but not AML-ETO which does not signal through HoxA9). In vivo, antagomir treatment blocked MLL-Af9-initiated leukemia lethality. These data establish microRNA genes as functional downstream targets of endogenous HoxA9, and implicate epigenetic signaling as critical client/mediators of Hox-based leukemia oncoproteins. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Intrinsic Requirement of MicroRNA In Hox-Based Leukemia Initiating Cell Maintenance

Blood ◽

10.1182/blood.v116.21.4192.4192 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4192-4192 ◽

Cited By ~ 1

Author(s):

Aditya Chaubey ◽

Chinavenmeni Subramani Velu ◽

Shane Horman ◽

Anil Jegga ◽

Monica L. Guzman ◽

...

Keyword(s):

Research Funding ◽

Bone Marrow Cells ◽

Human Leukemia ◽

Increased Risk ◽

Elevated Expression ◽

Bona Fide ◽

Lysine Demethylase ◽

Microrna Genes ◽

Microrna Function

Abstract Abstract 4192 Mouse models of MLL-Af9 leukemia have been exploited to roughly determine leukemia initiating cell (LIC) characteristics and biology. Each colony formed by MLL-Af9 leukemic splenocytes is capable of initiating leukemia upon transplantation. MLL-fusion oncoproteins signal through elevated expression of HoxA9, Meis and Pbx proteins. The HoxA9 transcription factor is of critical interest in human AML because it is; 1) directly affected by chromosomal translocations, 2) upregulated by leukemia oncoproteins, and 3) the level of HoxA9 in cytogenetically normal human AML predicts outcome. However, the direct transcriptional targets of endogenous HoxA9 that mediate transformation remain largely unknown. The Growth factor independent-1 (Gfi1) transcriptional repressor is known to induce granulopoiesis and inhibit myeloid progenitor proliferation. GFI1 is mutated in patients with severe congenital neutropenia (SCN). SCN patients are at increased risk for AML. We have shown that Hox and Gfi1 orthologs antagonize each other during Drosophila anterior posterior patterning, and that in mammalian myeloid progenitors Gfi1 directly represses the expression of Hoxa9, Pbx1 and Meis1. Moreover, Gfi1 regulates the expression of miR-21 and miR-196b, and forced expression of these miR blocks G-CSF instructed granulopoiesis. Here we demonstrate that microRNA genes are the targets of endogenous HoxA9 versus Gfi1 antagonism and that this antagonism is relevant in the context of human leukemia. Moreover, we also show that miR-196b and miR-21 are activated by Hox-signaling leukemia oncoproteins. Next, in both murine leukemia models and primary human AML samples, antagomir-mediated inhibition of microRNA function specifically disrupts colony replating potential of Lin- bone marrow cells transformed by HoxA9, Nup98-HoxA9 and MLL-Af9, as well as bona fide MLL-Af9 leukemias. In contrast, cells transformed by AML-ETO (which does not signal through HoxA9) are not affected. In vivo, antagomir treatment blocked MLL-Af9-initiated leukemia (but not AML-ETO leukemia) lethality. Finally, limiting dilution analyses demonstrate that antagomirs inhibit Hox-based transformation by targeting the LIC. Antagomir treatment induces the re-expression of a histone lysine demethylase, downregulation of gene expression associated with maintenance of MLL-Af9 leukemia and associated H3K4me3 marks on these genes. Our data establish microRNA genes as functional downstream targets of endogenous HoxA9, and implicate epigenetic signaling as critical client/mediators of Hox-based leukemia oncoproteins in LIC maintenance. Disclosures: Carroll: Sanofi Aventis Corporation: Research Funding; Kyowa Hakko Kirin Pharmaceuticals: Research Funding; Agios Pharmaceuticals: Research Funding.

Download Full-text

Tet2 Deficiency Rejuvenates Hematopoietic Stem and Progenitor Cells during Ageing

Blood ◽

10.1182/blood-2020-141705 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 37-37

Author(s):

Tingting Hong ◽

Shengli Li ◽

Shaohai Fang ◽

Anna Guzman ◽

Wei Han ◽

...

Keyword(s):

Stem Cells ◽

Conflicts Of Interest ◽

Pool Size ◽

Loss Of Function ◽

Self Renewal ◽

Hematopoietic Stem ◽

Increased Risk ◽

Age Dependent

Ageing is accompanied by a significant reduction of hematopoietic competence driven by various causes including epigenetic alternations [1-4]. Ten-eleven translocation 2 (TET2) has been well delineated as a critical epigenetic regulator that affects hematopoietic progenitor and stem cells (HSPCs) function. Tet2 deficiency confers advantages in clonal expansion of HSPCs and skews myeloid lineage differentiation, giving rise to increased risk of hematological malignancy transformation [5-8]. TET2 loss-of-function mutations are frequently detected in aged HSPCs [10-11], thereby raising the question of how Tet2 deficiency affects HSPCs self-renewal and lineage specification during ageing. To address this question, we harvested HSPCs from wild-type (WT) or Tet2KO young and aged donor mice, followed by competitive bone marrow transplantations to monitor age-dependent functional alterations. Despite the enlargement of the HSC pool size (the number of cells with regenerative potential) in aged mice, the aged WT HSPCs exhibited lower self-renewal capability and displayed impaired hematopoietic differentiation when competed against young stem cells. However, we found that both aged and young Tet2-deficient HSPCs shared comparable peripheral blood reconstitution, indicating no engraftment defects were caused by age for Tet2-deficient HSPCs. In parallel, scRNA-seq analysis revealed that Tet2 deficiency and age promoted the expansion of HSC compartment in a synergistic manner, leading to the largely augmented pool size of Tet2-deficient aged HSCs. But unlike aged WT stem cells, these expanded aged Tet2-null stem cells retained high self-renewal potential and possessed a competitive advantage of lineage outputs both in vitro and in vivo. Overall, through conducting repopulation assays and single-cell transcriptomes analysis, we have demonstrated that Tet2 ablation alters age-dependent HSC functional decline, revealing a disparate ageing process in the Tet2-deficient haemopoietic system. Disclosures No relevant conflicts of interest to declare.

Download Full-text

GFI1 Is a Tumor Suppressor in Myeloid Progenitors

Blood ◽

10.1182/blood.v112.11.2942.2942 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2942-2942

Author(s):

Aditya Chaubey ◽

Shane Hormon ◽

Chinavenmeni S. Velu ◽

Tristan Bourdeau ◽

Jinfang Zhu ◽

...

Keyword(s):

Dna Sequences ◽

Myeloid Leukemia ◽

Dependent Manner ◽

Loss Of Function ◽

Increased Risk ◽

Myeloid Leukemias ◽

Dose Dependent ◽

Anterior Posterior

Abstract In severe congenital neutropenia (SCN) patients and mice with Growth factor independent-1 (Gfi1) loss of function, arrested progenitors are suspended in a hyperproliferative state while terminal granulpoiesis is blocked. SCN patients are at increased risk for the development of acute myeloid leukemia. We demonstrate that Gfi1 directly targets HoxA9, Pbx1 and Meis1 during normal myelopoiesis. Gfi1−/− progenitors exhibit elevated levels of HoxA9, Pbx1 and Meis1, exaggerated HoxA9-Pbx1-Meis1 activity, and increased persistence in vivo and in vitro. Limiting HoxA9 alleles corrects, in a dose dependent manner, in vivo and in vitro phenotypes observed with loss of Gfi1. Moreover, in a manner conserved in Drosophila anterior/posterior patterning, we demonstrate that these factors can compete for occupancy of DNA sequences encoding composite Gfi1-HoxA9-Pbx1-Meis1 binding sites. Finally, the expression of Gfi1 and HoxA9 are inverse and stratify human myeloid leukemias, suggesting a role for HoxA9- Gfi1 antagonism in human AML. In agreement with this, a myeloproliferative disorder progresses into a rapid, lethal and transplantable myeloid leukemia in a Gfi1−/− setting. We conclude that the lifespan and oncogenic transformation of hematopoietic progenitor cells is regulated through a conserved competition between Gfi1 and HoxA9-Pbx1-Meis1.

Download Full-text

Role of Notch Signaling in Hematopoietic Stem Cell Function

Blood ◽

10.1182/blood.v118.21.sci-15.sci-15 ◽

2011 ◽

Vol 118 (21) ◽

pp. SCI-15-SCI-15

Author(s):

Lluis Espinosa ◽

Anna Bigas

Keyword(s):

Stem Cell ◽

Cell Function ◽

Conflicts Of Interest ◽

Notch Pathway ◽

Cell Types ◽

Loss Of Function ◽

Hematopoietic Stem ◽

On Chip ◽

Stem Cell Populations

Abstract Abstract SCI-15 The Notch pathway controls the generation of different cell types in most tissues including blood, and dysregulation of this pathway is strongly associated with oncogenic processes. In many systems, Notch is also required for the maintenance of the stem cell populations. However, in the adult hematopoietic system this link between Notch and stemness has not been established. Instead, work of several groups, including ours, has clearly demonstrated that Notch has a prominent role in the generation of hematopoietic stem cells (HSC) during embryonic development. Although the first wave of blood cells appears in the mouse embryo around day 7.5 of development and is independent of Notch function, embryonic HSC are formed around day 10 of development from endothelial-like progenitors that reside in the embryonic aorta surrounded by the gonad and mesonephros, also called AGM region. By analyzing different Notch pathway mutant mouse embryos, we have demonstrated the involvement of the Jagged1-Notch1-GATA2 axis in this event. However, the formal demonstration that Notch regulates the GATA2 gene during HSC generation is still lacking. We have now found that GATA2 is a direct Notch target in vivo during embryonic HSC generation. However, whereas Notch positively activates GATA2 transcription in the HSC precursors, it simultaneously activates hes1 transcription, which acts a repressor of the same GATA2 gene. This finding directly implicates hes1 in the regulation of HSC development although further studies using loss-of-function mutant embryos are still needed. Altogether, our results indicate that both Notch and hes1 are required to finely regulate the levels, distribution, and likely the timing of GATA2 expression through an incoherent feed-forward loop. In parallel, we have identified other downstream targets of Notch in the AGM region by ChIP-on-chip and expression microarray analysis that we are currently characterizing. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Oncogenic KRASG12D in the Hematopoietic System Causes NLRP3 Inflammasome Activation Leading to Myeloproliferative Syndrome

Blood ◽

10.1182/blood-2018-99-114161 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2618-2618

Author(s):

Shaima'A Hamarsheh ◽

Miriam Erlacher ◽

Lena Osswald ◽

Oliver Gorka ◽

Justus Duyster ◽

...

Keyword(s):

Bone Marrow ◽

Nlrp3 Inflammasome ◽

Immune Modulation ◽

Conflicts Of Interest ◽

Tamoxifen Treatment ◽

Juvenile Myelomonocytic Leukemia ◽

Inflammasome Activation ◽

Oncogenic Signaling ◽

Nlrp3 Inflammasome Activation

Abstract Oncogenic Ras mutations occur frequently in myelodysplastic (MDS) and myeloproliferative syndromes (MPN). It was unclear if besides the direct transforming effect via constant RAS/MEK/ERK signaling, an inflammation related effect of KRAS contributes to the development of the disease. To address this question we performed a microarray based analysis of bone marrow derived from Rosa26CreERT2; LSL-KrasG12D mice after induction of KrasG12D with tamoxifen. We found that the NLRP3 inflammasome and related genes were upregulated. In agreement with increased NLRP3 sensitivity, KrasG12D bone marrow derived dendritic cells (BMDCs) showed increased inflammasome activation upon stimulation with LPS and ATP as quantified by intracellular levels of IL-1β and caspase-1 p20 subunit. To validate the functional role of the NLRP3 inflammasome for the MDS phenotype in vivo we generated Rosa26CreERT2; LSL-KrasG12D; NLRP3-/- mice. We transferred BM (CD45.2) from WT mice, Rosa26CreERT2; LSL-KrasG12D or Rosa26CreERT2; LSL-KrasG12D; NLRP3-/- mice into C57BL/6 WT recipients (CD45.1) after myeloablative conditioning and induced KrasG12D by tamoxifen treatment. Rosa26CreERT2; LSL-KrasG12D BM recipient mice developed MPN stigmata including anemia, leukopenia and thrombocytopenia. The mice also presented weight loss, splenomegaly, and an increase in myeloid cells in peripheral blood, spleen and BM, as well as increased CD11b+ckit+ immature myeloid progenitors. In contrast, Rosa26CreERT2; LSL-KrasG12D; NLRP3-/- recipient mice developed no disease phenotype. BMDCs isolated from Rosa26CreERT2; LSL-KrasG12D recipient mice exhibited strong NLRP3 inflammasome activation and high IL-1β levels which was not found in BMDCs of Rosa26CreERT2; LSL-KrasG12D; NLRP3-/- mice. CD11b+ cells of a Juvenile myelomonocytic leukemia (JMML) patient with an activating KRAS mutation showed increased inflammasome activation as compared to a non-mutant patient, which is in agreement with our observations in mouse models. Our findings support the concept that oncogenic KrasG12D does not only act via its oncogenic driver function, but also enhances activation of the NLRP3/IL-1β axis. This could lead to novel therapeutic approaches combining inhibition of oncogenic signaling and immune modulation via IL-1β blockade. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Pten Inactivating Mutations Promote Loss of MYC “Oncogene Addiction” in a Conditional Zebrafish Model of T-ALL.

Blood ◽

10.1182/blood.v114.22.3977.3977 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3977-3977

Author(s):

Alejandro Gutierrez ◽

Hui Feng ◽

Adele Faucherre ◽

Jeroen Den Hertog ◽

Edward V. Prochownik ◽

...

Keyword(s):

Conflicts Of Interest ◽

Tumor Regression ◽

Lymphoblastic Leukemia ◽

Loss Of Function ◽

Oncogene Addiction ◽

Zebrafish Model ◽

Pten Loss ◽

Myc Oncogene ◽

Tumor Onset

Abstract Abstract 3977 Poster Board III-913 The MYC oncogene is overexpressed in most human cases of T-cell acute lymphoblastic leukemia (T-ALL), and our laboratory previously developed a zebrafish model of MYC-induced T-ALL that closely recapitulates the most common subtype of human T-ALL, which demonstrates overexpression of TAL1 and LMO2. We have now developed a conditional zebrafish model of MYC-induced T-ALL, based on the rag2-driven expression of a MYC-estrogen receptor (MYC-ER) fusion transgene, in which T-ALL is induced by 4-hydroxytamoxifen treatment. Most cases of T-ALL in this model exhibit 'oncogene addiction' to MYC, as evidenced by the regression of established tumors when 4HT treatment is discontinued. However, the genetic determinants of 'addiction' to the MYC oncogene are unknown. Deletion of the PTEN tumor suppressor has been found to be a recurrent abnormality in human T-ALL, and we have now identified abnormalities in PTEN, PI3K or AKT in 48% of human T-ALL. Due to a partial genome duplication that occurred during evolution, zebrafish have two pten genes, pten-A and pten-B, which are both ubiquitously expressed and are largely redundant. In order to examine the consequences of pten inactivation in MYC-induced T-ALL, we mated zebrafish loss-of-function mutations in pten-A and pten-B into our rag2-MYC-ER line that also carries the rag2-GFP transgene, allowing the in vivo monitoring of tumor onset and regression. Zebrafish were raised in 4-hydroxytamoxifen beginning at 5 days post-fertilization, and tumor onset was monitored via fluorescence microscopy. At the time of tumor onset, zebrafish were removed from 4-hydroxytamoxifen, and tumor regression was monitored weekly. Although pten loss-of-function mutations did not accelerate the onset of MYC-induced T-ALL, these did affect tumor regression. We found that T-ALL underwent regression in 68% (21/31) of zebrafish whose germline pten status was wild-type, while regression occurred in only 21% (7/32) of zebrafish harboring loss of any of their germline pten alleles (p=0.0003). The effect of pten inactivation on tumor regression was phenocopied by the expression of a constitutively active murine Akt2 transgene in the MYC-ER line, indicating that the effect of pten inactivation is mediated through the activation of AKT. Our studies provide new evidence that the PTEN-PI3K-AKT pathway is an important modulator of MYC ‘oncogene addiction’ in zebrafish T-ALL. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

NR2F1 is a barrier to dissemination of early-evolved mammary cancer cells

10.1101/2021.01.29.428822 ◽

2021 ◽

Author(s):

Carolina Rodriguez-Tirado ◽

Nupura Kale ◽

Maria Jose Carlini ◽

Nitisha Shrivastava ◽

Bassem Khalil ◽

...

Keyword(s):

Cancer Cells ◽

Early Cancer ◽

Mapk Pathway ◽

Tumor Formation ◽

Orphan Nuclear Receptor ◽

Loss Of Function ◽

Oncogenic Signaling ◽

Laminin 5 ◽

Her2 Signaling

SummaryCancer cells disseminate during very early and sometimes asymptomatic stages of tumor progression. Granted that biological barriers to tumorigenesis exist, there must also be limiting steps to early dissemination, all of which remain largely unknown. We report that the orphan nuclear receptor NR2F1/COUP-TF1 serves as a barrier to early dissemination. High-resolution intravital imaging revealed that loss of function of NR2F1 in HER2+ early cancer cells increased in vivo dissemination without accelerating mammary tumor formation. NR2F1 expression was positively regulated by the tumor suppressive MMK3/6-p38-MAPK pathway and downregulated by HER2 and Wnt4 oncogenic signaling. NR2F1 downregulation by HER2 in early cancer cells led to decreased E-cadherin expression and β-catenin membrane localization, disorganized laminin 5 deposition, and increased expression of CK14, TWIST1, ZEB1 and PRRX1. Our findings reveal the existence of an inhibitory mechanism of dissemination regulated by NR2F1 downstream of HER2 signaling.

Download Full-text

The Association Between SERPINC1 C.883G>a and VT in the Chinese Population

Blood ◽

10.1182/blood.v126.23.3505.3505 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3505-3505

Author(s):

Jingdi Liu ◽

Liang Tang ◽

Wei Zeng ◽

Yu Hu ◽

Han Liu

Keyword(s):

Venous Thrombosis ◽

Chinese Population ◽

Case Control Study ◽

Conflicts Of Interest ◽

Hot Spot ◽

Case Control ◽

Missense Mutations ◽

Increased Risk ◽

Control Study

Abstract Background: Antithrombin(AT) is a major anticoagulation molecule in vivo and is encoded by the gene SERPINC1. AT plays a key role as an inhibitor of physiological haemostasis by inhibiting the procoagulation factors, especially the factor Xa and thrombin. Objectives: To explore the variations of SERPINC1 gene associated with venous thrombosis in the Chinese population. Methods: SERPINC1 gene sequencing was carried out. A case-control study involving 1335 patients diagnosed with VT and 1315 Age- and sex-matched control individuals without a history of thrombosis were further carried out. Furthermore, plasma AT activity, AT antigen, and thrombin generation tests (TGT) were performed to evaluate the influences of the mutations. Results: Four different missense mutations were identified in an unreported hot spot region of SERPINC1. They were c.880C>T(p.Arg294Cys), c.881G>T(p.Arg294Leu), c.881G>A(p.Arg294His) and c.883G>A(p.Val295Met). All of the affected individuals were heterozygotes. In addition, c.883G>A was found to be a predominant mutation. In the case-control study, the mutation was proved to be a strong risk factor for venous thrombosis with an OR of 10.92(p<0.01, 95%CI 1.41-84.68). Functional assays showed that both the activities and antigens of plasma AT decreased mildly. Conclusion: A hot spot mutation region of SERPINC1 gene was discovered. The predominant mutation of SERPINC1 c.883G>A is the most frequent cause of AT deficiency and is associated with an increased risk of venous thrombosis in the Chinese population. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Gfi1 integrates progenitor versus granulocytic transcriptional programming

Blood ◽

10.1182/blood-2008-09-179747 ◽

2009 ◽

Vol 113 (22) ◽

pp. 5466-5475 ◽

Cited By ~ 48

Author(s):

Shane R. Horman ◽

Chinavenmeni S. Velu ◽

Aditya Chaubey ◽

Tristan Bourdeau ◽

Jinfang Zhu ◽

...

Keyword(s):

Dependent Manner ◽

Loss Of Function ◽

Lineage Specification ◽

Myeloid Progenitor ◽

Myeloid Progenitor Cells ◽

Dose Dependent ◽

Transcription Program ◽

Myeloid Progenitors

AbstractIn patients with severe congenital neutropenia (SCN) and mice with growth factor independent-1 (Gfi1) loss of function, arrested myeloid progenitors accumulate, whereas terminal granulopoiesis is blocked. One might assume that Gfi-null progenitors accumulate because they lack the ability to differentiate. Instead, our data indicate that Gfi1 loss of function deregulates 2 separable transcriptional programs, one of which controls the accumulation and lineage specification of myeloid progenitors, but not terminal granulopoiesis. We demonstrate that Gfi1 directly represses HoxA9, Pbx1, and Meis1 during normal myelopoiesis. Gfi1−/− progenitors exhibit elevated levels of HoxA9, Pbx1 and Meis1, exaggerated HoxA9-Pbx1-Meis1 activity, and progenitor transformation in collaboration with oncogenic K-Ras. Limiting HoxA9 alleles corrects, in a dose-dependent manner, in vivo and in vitro phenotypes observed with loss of Gfi1 in myeloid progenitor cells but did not rescue Gfi1−/− blocked granulopoiesis. Thus, Gfi1 integrates 2 events during normal myeloid differentiation; the suppression of a HoxA9-Pbx1-Meis1 progenitor program and the induction of a granulopoietic transcription program.

Download Full-text

Characterizing the Disease-Initiating Cell Population In a Murine Model of Jak2V617F+ Myeloproliferative Neoplasm

Blood ◽

10.1182/blood.v116.21.317.317 ◽

2010 ◽

Vol 116 (21) ◽

pp. 317-317

Author(s):

Ann Mullally ◽

Steven W. Lane ◽

Luke Poveromo ◽

Fatima Al-Shahrour ◽

Jeffery Kutok ◽

...

Keyword(s):

Cell Population ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Functional Characterization ◽

Myeloproliferative Neoplasm ◽

Hematopoietic Stem ◽

Myeloid Progenitor

Abstract Abstract 317 The JAK2V617F mutation is the most common somatic mutation in BCR-ABL negative myeloproliferative neoplasms (MPN) and small molecule JAK2 kinase inhibitors are currently being evaluated in clinical trials in MPN patients. We recently reported a Jak2V617F conditional knockin mouse MPN model in which expression of Jak2V617F was under the control of the endogenous Jak2 promoter and the phenotype closely recapitulated the features of human polycythemia vera (PV) (Mullally and Lane et al, Cancer Cell 2010). In this model the MPN phenotype developed as a result of expansion and erythroid skewing of committed myeloid progenitor populations, while the hematopoietic stem cell (HSC) compartment (enriched within LineagelowSca1+cKithigh, LSK cells) was not expanded. In contrast to the expanded progenitor population, only LSK cells had the unique capacity to initiate the MPN. Notably, treatment with a JAK2 kinase inhibitor ameliorated the MPN phenotype, but did not eliminate the disease-initiating population. In light of these findings, we wished to further characterize MPN-initiating LSK cells in this model, with a view to gaining understanding of the differential molecular circuitry of JAK2V617F mutant and normal HSC. The LSK compartment, although enriched for HSC, may be further defined by immunophenotype into long-term HSC (LT-HSC, LSKCD150+CD48-), short-term HSC (ST–HSC, LSKCD150-CD48-), or multipotent progenitor cells (MPP, LSKCD150-CD48+). Gene expression analysis of total LSK cells revealed enrichment for markers of myeloid progenitor differentiation in Jak2V617F LSK cells compared to Jak2 wild-type (WT) controls, although there were no quantitative differences in the LSK sub-populations between the two groups. We therefore sought to evaluate whether the MPN-initiating cell population was contained within the LT-HSC compartment. Employing single cell sorting multi-parameter flow cytometry and limiting dilution bone marrow transplantation assays, we provide further functional characterization of the MPN-initiating population in this model. We demonstrate that Jak2V617F LT-HSCs are highly enriched for MPN-initiating activity in vivo and are able to maintain the MPN through multiple rounds of serial transplantation for periods greater than one year. The MPN phenotype remains stable during this time without evolution to myelofibrosis or leukemia. These findings provide important biological insights into MPN initiation and maintenance and have implications for the development of curative therapeutic strategies for MPN patients. Disclosures: No relevant conflicts of interest to declare.

Download Full-text