Kmt2c Limits the Self-Renewal Capacity of Multiply Divided HSCs By Promoting Sensitivity to Interleukin-1

Under normal homeostatic conditions, adult hematopoietic stem cells (HSCs) are usually quiescent. Hematopoietic stress, such as blood loss, infection, inflammation, or chemotherapy, can drive HSCs into cycle. When adult HSCs divide multiple times, they lose self-renewal capacity. Inflammatory cytokines, such as interleukin-1 (IL-1), can accelerate the loss of HSC self-renewal capacity by activating PU.1 and promoting myeloid commitment. This raises the question of whether intrinsic tumor suppressor genes can modulate sensitivity to inflammatory cytokines, and whether loss of these tumor suppressors can allow HSCs to evade commitment programs that would otherwise limit self-renewal capacity. The KMT2C tumor suppressor is located on chromosome 7q within a region that is frequently deleted in myelodsplastic syndrome (MDS) and therapy-related acute myeloid leukemia (AML). It encodes MLL3, a histone methyltransferase that activates enhancer elements and promotes transcription. Haploid KMT2C deletion has previously been shown to activate self-renewal programs and accelerate AML formation. This raised the question of whether KMT2C/MLL3 regulates normal HSC self-renewal and whether KMT2C deletion conveys a selective advantage to HSCs in contexts that would otherwise deplete the HSC pool. By protecting HSCs from exhaustion, KMT2C deletions may indirectly facilitate 7q-deficient MDS/AML. To understand whether and how Kmt2c regulates HSC self-renewal, we developed novel germline and conditional Kmt2c knockout mouse alleles. Mono- and bi-allelic Kmt2c deletions led to a modest increase in adult HSC numbers and a significant reduction in committed hematopoietic progenitors (HPCs). Kmt2c deletions markedly enhanced HSC self-renewal capacity, but HSC proliferation rates were not altered. To mimic conditions that lead to therapy-related AML, we deleted a single Kmt2c allele in a minority of HSCs. We then tested whether the Kmt2c-deleted HSC population expanded, relative to wild type HSCs, under native and stressed conditions. Under native conditions, the percentage of Kmt2c-heterozygous HSCs remained stable. However, after several cycles of chemotherapy, the Kmt2c mutant HSCs predominated within the marrow. In mechanistic studies, RNA-sequencing showed that Kmt2c-deficient HSCs expressed genes associated with innate immune signaling, including the receptor for interleukin-1 (IL1R), at lower levels than wild type HSCs. This suggested that Kmt2c mutations might sustain self-renewal capacity in multiply divided HSCs by dampening IL-1 driven myeloid commitment. In support of this hypothesis, Kmt2c-deficient HSCs retained multilineage potential when they were cultured with IL-1, and they failed to activate JNK and p38 upon exposure to IL-1. Altogether, our data suggest a mechanism to explain how KMT2C deletions, in the context of larger 7q deletions, may promote therapy related MDS/AML. When HSCs acquire a KMT2C deletion, they can then escape IL-1-mediated exhaustion when they are driven into cycle by chemotherapy or other stressors. In lieu of chemotherapy-induced stress, the same clones may remain relatively indolent. Disclosures No relevant conflicts of interest to declare.

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Non-Canonical NF-Kb Signaling Regulates Hematopoietic Stem Cell Self-Renewal and Microenvironment Interactions

Blood ◽

10.1182/blood.v118.21.859.859 ◽

2011 ◽

Vol 118 (21) ◽

pp. 859-859 ◽

Cited By ~ 1

Author(s):

Chen Zhao ◽

Yan Xiu ◽

John M Ashton ◽

Lianping Xing ◽

Yoshikazu Morita ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Wild Type ◽

Double Knockout ◽

Self Renewal ◽

Hematopoietic Stem ◽

Physiological Processes ◽

Marrow Cells

Abstract Abstract 859 RelB and NF-kB2 are the main effectors of NF-kB non-canonical signaling and play critical roles in many physiological processes. However, their role in hematopoietic stem/progenitor cell (HSPC) maintenance has not been characterized. To investigate this, we generated RelB/NF-kB2 double-knockout (dKO) mice and found that dKO HSPCs have profoundly impaired engraftment and self-renewal activity after transplantation into wild-type recipients. Transplantation of wild-type bone marrow cells into dKO mice to assess the role of the dKO microenvironment showed that wild-type HSPCs cycled more rapidly, were more abundant, and had developmental aberrancies: increased myeloid and decreased lymphoid lineages, similar to dKO HSPCs. Notably, when these wild-type cells were returned to normal hosts, these phenotypic changes were reversed, indicating a potent but transient phenotype conferred by the dKO microenvironment. However, dKO bone marrow stromal cell numbers were reduced, and bone-lining niche cells supported less HSPC expansion than controls. Further, increased dKO HSPC proliferation was associated with impaired expression of niche adhesion molecules by bone-lining cells and increased inflammatory cytokine expression by bone marrow cells. Thus, RelB/NF-kB2 signaling positively and intrinsically regulates HSPC self-renewal and maintains stromal/osteoblastic niches and negatively and extrinsically regulates HSPC expansion and lineage commitment through the marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

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hnRNP K: A Tumor Suppressor or Oncogene?

Blood ◽

10.1182/blood.v124.21.259.259 ◽

2014 ◽

Vol 124 (21) ◽

pp. 259-259

Author(s):

Miguel Gallardo ◽

Hun Ju Lee ◽

Carlos E. Bueso-Ramos ◽

Xiaorui Zhang ◽

Laura R. Pageon ◽

...

Keyword(s):

Bone Marrow ◽

Tumor Suppressor ◽

Conflicts Of Interest ◽

Chromosome 9 ◽

Wild Type ◽

Differentiation Potential ◽

Hematopoietic Stem ◽

Hnrnp K ◽

Differentiation And Proliferation

Abstract Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is an RNA and DNA binding protein that regulates critical pathways controlling differentiation and proliferation programs. While alterations in hnRNP K expression are associated with neoplastic malignancies, we currently do not understand how changes in hnRNP K expression contribute to tumor phenotypes in vivo. Previous biochemical and cell line studies demonstrate that hnRNP K transcriptionally regulates p53-dependent activities, suggesting it functions as a potential tumor suppressor. However, hnRNP K has also been shown to positively regulate c-Myc expression, indicating it may behave as an oncogene. The HNRNP K gene maps to a region of chromosome 9 (9q21.32), which is lost in a subset of patients with acute myeloid leukemia (AML). RNA expression analyses of patient samples with AML that harbor 9q21.32 deletions revealed a significant reduction in HNRNP K expression compared to wild type control samples, supporting the notion that hnRNP K acts as a tumor suppressor (Figure 1A). However, patients with AML who do not harbor a 9q21.32 deletion displayed a significant increase in hnRNP K expression (Figure 1A). Thus, to examine the association between altered hnRNP K expression and disease status in patients with AML, we performed reverse phase protein array (RPPA) analysis on CD34+ bone marrow cells from 415 de novo AML patient as well as healthy donor controls. Interestingly, we observed a significant correlation between elevated hnRNP K levels and poor outcomes, which supports the idea that hnRNP K has oncogenic potential (Figure 1A). Together, these observations indicate that any change in hnRNP K expression may contribute to the etiology of AML and supports the idea that hnRNP K may potentially act as either a haploinsufficient tumor suppressor or oncogene in AML. To directly interrogate these possibilities in vivo, we generated mouse models that either harbor a deletion of one hnRNP K allele (hnRNP K+/-) or overexpressed hnRNP K (hnRNP KTg) in the hematological compartment. Western blot analyses demonstrated that hnRNP K haploinsufficiency results in a significant reduction in hnRNP K expression while tissue-specific activation of hnRNP K resulted in overexpression of hnRNP K. Similar to our observation in AML patients, either hnRNP K haploinsufficiency or overexpression resulted in similar phenotypes in vitro and in vivo. Lin-CD117+ hematopoietic stem cells (HSCs) from hnRNP K+/- and hnRNP KTg mice had significant increases in differentiation and proliferation potential as determined by colony formation assays. In these experiments, we observed a significant increase in the number of total colonies and number of cells per colony in both hnRNP K+/- and hnRNP KTg HSCs as compared to wild type HSCs (Figure 1B). In vivo analyses of the hnRNP K+/- and hnRNP KTg mice revealed a significant increase in myeloid hyperplasia in the peripheral blood and bone marrow, increased tumor formation, genomic instability, and decreased survival compared to wild type mice (Figure 1C). Interestingly, both increased and decreased hnRNP K expression resulted in alterations in similar pathways that regulate differentiation and proliferations potential (e.g.; p53 and c-Myc pathways and alterations in C/EBP expression). Together, these clinical and animal model studies illustrate that either over-expression or under-expression of hnRNP K lead to strikingly similar phenotypes that directly impact the etiology of AML. Furthermore, these data not only implicate that hnRNP K behaves as both a tumor suppressor and oncogene, but also suggest that it functions as a master toggle that dictates the proliferation and differentiation potential of HSCs. We are currently using Whole Transcriptome Shotgun Sequencing (RNA-Seq) and ChIP-Seq to evaluate the mechanisms by which increased and decreased hnRNP K expression impact hematologic malignancies. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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A Mouse Model of Hematopoietic Stem Cell Failure Associated with p53-Loss and Defective Fbw7-Dependent Cyclin E Regulation.

Blood ◽

10.1182/blood.v120.21.2297.2297 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2297-2297

Author(s):

Ka Tat Siu ◽

Yanfei Xu ◽

Mitra Bhattacharyya ◽

Alexander C. Minella

Keyword(s):

Cell Cycle ◽

Mouse Model ◽

Conflicts Of Interest ◽

Cyclin E ◽

Cell Cycle Control ◽

Control Mechanisms ◽

Wild Type ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Abstract 2297 Recent findings have challenged the notion that increased proliferation of hematopoietic stem cells (HSCs) necessarily restricts their self-renewal capacity. We have studied the physiologic consequences to HSCs of ablating a key cell cycle regulatory mechanism, Fbw7-dependent cyclin E ubiquitination, using germline knock-in of a cyclin ET74A T393A allele. Fbw7 is a tumor suppressor that regulates the abundance of several oncoprotein substrates by ubiquitin-mediated proteolysis, including cyclin E, Notch, and c-Myc. Cyclin E overexpression in vivo is associated with increased proliferation in some cellular contexts as well as a variety of deleterious consequences, including genomic instability, senescence, or apoptosis. In HSCs, Fbw7-loss has been shown to induce self-renewal and multi-lineage reconstitution defects, and the effect of Fbw7-loss in HSCs has been ascribed to dysregulated Myc and Notch expression. Using the cyclin ET74A T393A mouse model, we tested the hypothesis that impaired Fbw7-mediated regulation of cyclin E, specifically, promotes HSC exhaustion due to loss of self-renewal capacity. We first examined bone marrow HSC counts and their cell cycle kinetics in cyclin E knock-in and wild-type control mice at steady state and following hematologic injury induced by 5-fluorouracil treatment. We found that cyclin E dysregulation reduces numbers of quiescent HSCs and increases cells in S/G2/M-phases, while decreasing total numbers of HSCs, phenotypes made more severe after recovery from hematologic stress. Using bromodeoxyuridine labeling studies, we found that excess cyclin E activity causes DNA hyper-replication in cyclin ET74A T393A HSCs in a cell autonomous manner. By enumerating multi-potent progenitors (MPPs), we ruled out increased rate of transit from HSC-to-MPP as a cause of the apparent exhaustion of cyclin E knock-in HSCs. Thus, dysregulated cyclin E in HSCs promotes both increased proliferation and depletion of the HSC pool. Serial transplantation further revealed peripheral blood reconstitution defects associated with cyclin ET74A T393A HSCs. Recently, we have found that p53 is activated by dysregulated cyclin E in hematopoietic cells in vivo, in association with phosphorylation of both p53 and Chk1 proteins, resembling a DNA damage-type response. Interestingly, p53-loss has been found to be associated with a gain of HSC self-renewal activity. We therefore hypothesized that p53-loss would rescue the self-renewal defect of cyclin E knock-in HSCs. Surprisingly, we discovered that cyclin ET74A T393A; p53-null HSCs showed evidence of significantly worse self-renewal and peripheral reconstitution, compared to p53-null HSCs, defects that are more severe than those associated with impaired Fbw7-mediated cyclin E control in the setting of wild-type p53 (Chi-squared test, p<0.0001). Thus, our data are consistent with the concept that intact p53 function, in the setting of oncogenic insult, can preserve partial HSC self-renewal capacity, and its loss in vivo is detrimental to HSC viability when accompanied by defects in cell cycle control mechanisms. Disclosures: No relevant conflicts of interest to declare.

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Modeling MLL-PTD Related Myelodysplastic Syndromes in Mouse.

Blood ◽

10.1182/blood.v120.21.2811.2811 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2811-2811

Author(s):

Xiaomei Yan ◽

Yue Zhang ◽

Goro Sashida ◽

Aili Chen ◽

Xinghui Zhao ◽

...

Keyword(s):

De Novo ◽

Conflicts Of Interest ◽

Gene Dosage ◽

Fetal Liver ◽

Loss Of Function ◽

Wild Type ◽

Self Renewal ◽

Hematopoietic Stem ◽

De Novo Aml

Abstract Abstract 2811 MLL partial tandem duplication (MLL-PTD) is found in 5–8% of human MDS, secondary acute myeloid leukemia (s-AML) and de novo AML. The molecular and clinical features of MLL-PTD+ AML are different from MLL-fusion+ AML, although they share similar worse outcomes. Mouse knock-in model of Mll-PTD has been generated to understand its underlining mechanism (Dorrance et al. JCI. 2006). Using this model, we've recently reported hematopoietic stem/progenitor cell (HSPC) phenotypes of MllPTD/WT mice. Their HSPCs showed increased apoptosis and reduced cell number, but they have a proliferative advantage over wild-type HSPCs. Furthermore, the MllPTD/WT–derived phenotypic ST-HSCs/MPPs and even GMPs have self-renewal capabilities. However, MllPTD/WT HSPCs never develop MDS or s-AML in primary or transplanted recipient mice, suggesting that additional genetic and/or epigenetic defects are necessary for transformation (Zhang et al. Blood. 2012). Recently, high frequent co-existences of both MLL-PTD and RUNX1 mutations have been reported in several MDS, s-AML and de novo AML clinical cohorts, which strongly suggest a potential cooperation for transformation between these two mutations. Our previous study has shown that MLL interacts with and stabilizes RUNX1 (Huang et al. Blood. 2011). Thus, we hypothesize that reducing RUNX1 dosage may facilitate the MLL-PTD mediated transformation toward MDS and/or s-AML. We first generated the mice containing one allele of Mll-PTD in a Runx1+/− background and assessed HSPCs of MllPTD/wt/Runx1+/− double heterozygous (DH) mice. The DH newborns are runty; they frequently die in early postnatal stage and barely survive to adulthood, compared to the normal life span of wild type (WT) or single heterozygous (Mllwt/wt/Runx1+/− and MllPTD/wt/Runx1+/+) mice. We studied DH embryos fetal liver hematopoiesis and found reduced LSK and LSK/SLAM+ cells, partly because of increased apoptosis. Enhanced proliferation was found in DH fetal liver cells (FLCs) in vitro CFU replating assays over WT and MllPTD/wt/Runx1+/+ controls. DH FLCs also showed dominant expansion in both serial competitive and serial non-competitive BMT assays compared to WT controls. The DH derived phenotypic ST-HSCs/MPPs and GMPs also have enhanced self-renewal capabilities, rescuing hematopoiesis by giving rise to long-term repopulating cells in recipient mice better than cells derived from MllPTD/wt/Runx1+/+ mice. However, DH HSPCs didn't develop MDS or s-AML in primary or in serial BMT recipient mice. We further generated MllPTD/wt/Runx1Δ/Δ mice using Mx1-Cre mediated deletion. These mice showed thrombocytopenia 1 month after pI-pC injection, and developed pancytopenia 2–4 months later. All these MllPTD/wt/Runx1Δ/Δ mice died of MDS induced complications within 7–8 months, and tri-lineages dysplasias (TLD) were found in bone marrow aspirate. However, there are no spontaneous s-AML found in MllPTD/wt/Runx1Δ/Δ mice, which suggests that RUNX1 mutants found in MLL-PTD+ patients may not be simply loss-of-function mutations and present gain-of-function activities which cooperate with MLL-PTD in human diseases onsets. In conclusion, our study demonstrates that: 1) RUNX1 gene dosage reverse-correlates with HSPCs self-renewal activity; 2) Runx1 complete deletion causes MDS in Mll-PTD background. Future studies are needed to fully understand the collaboration between MLL-PTD and RUNX1 mutations for MDS development and leukemic transformation, which should facilitate improved therapies and patient outcomes. Disclosures: No relevant conflicts of interest to declare.

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Haploid Kmt2c Deletions Enhance Hematopoietic Stem Cell Mobilization in Response to Granulocyte-Colony Stimulating Factor

Blood ◽

10.1182/blood-2021-147914 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2762-2762

Author(s):

Ran Chen ◽

Riddhi M Patel ◽

Emily B Casey ◽

Jeffrey A. Magee

Keyword(s):

Bone Marrow ◽

Autologous Transplantation ◽

Selective Advantage ◽

The Body ◽

Granulocyte Colony Stimulating Factor ◽

Colony Stimulating Factor ◽

Wild Type ◽

Self Renewal ◽

Hematopoietic Stem ◽

Stimulating Factor

Abstract KMT2C is one of several tumor suppressor genes deleted in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) as part of larger chromosome 7q deletions. These deletions are particularly common in therapy-related MDS/AML, raising the question of whether loss of one or more 7q genes conveys a selective advantage to hematopoietic stem cells (HSCs) in the setting of chemotherapy-induced stress. We recently showed that haploid Kmt2c deletions do indeed enhance HSC self-renewal capacity. However, the deletions do not drive HSCs into cycle; instead, a proliferative stress such as chemotherapy is required to create a context in which Kmt2c deletion convey a selective advantage. We have also identified a mechanism, to explain this phenotype. Kmt2c encodes MLL3, a SET domain protein that binds enhancers and facilitates transcription. We have shown that Kmt2c/MLL3 deletions impairs enhancer recruitment during HSC differentiation, therefore blunting HSC commitment. Our findings suggest that acquired or pre-existing 7q (KMT2C) deletions may select for HSCs that could give rise to MDS/AML in the setting of autologous-transplantation. Granulocyte-colony stimulating factor (G-CSF) is a cytokine that is often used to expedite neutrophil recovery after chemotherapy and to mobilize HSCs for collection and transplantation. We considered the possibility that 7q deletions, and KMT2C deletions in particular, may promote disproportionate mobilization of the mutant HSCs in response to G-CSF. To test this, we treated Kmt2c f/f; Vav1-Cre and Kmt2c f/+; Vav1-Cre mice with G-CSF, and we assessed HSC mobilization to the spleen and bone marrow. A far greater proportion of HSCs with heterozygous and homozygous Kmt2c deletions mobilized in response to G-CSF, relative to wild type HSCs. Kmt2c deletion also enhanced colony forming unit frequency in the peripheral blood after G-CSF treatment. Total body HSC numbers did not change in the body after G-CSF treatment on any genetic background, indicating that Kmt2c deletions enhanced HSC mobilization in response to G-CSF rather than self-renewal. To more faithfully recapitulate clinical conditions, we used Fgd5-CreER to delete a single Kmt2c allele in only a minority of HSCs. We then tested whether the mutant HSCs mobilized more efficiently than wild type HSCs. Surprisingly, Kmt2c deletions did not enhance HSC mobilization in this context. This raised the question of whether Kmt2c deletion in a non-HSC population could promote HSC mobilization in the Kmt2c f/+; Vav1-Cre mice. Indeed, analysis of mice chimeric for wild type and Kmt2c f/+; Vav1-Cre bone marrow suggested that Kmt2c non-cell autonomously regulates HSC mobilization. Finally, Kmt2c deletions did not enhance mobilization following exposure to plerixafor, a CXCR4 antagonist that acts directly on HSCs to promote mobilization rather than indirectly via monocyte populations, as occurs with G-CSF. Additional studies are needed to elucidate the mechanism by which Kmt2c non-cell autonomously regulates HSC mobilization. Our findings provide reassurance that, in a clinical setting, rare KMT2C-mutant HSCs will not disproportionately mobilize prior to apheresis. Furthermore, the data suggest that transient inhibition of MLL3, or its targets, may enhance HSC mobilization and negate selective advantages that 7q-deleted HSCs may acquire after chemotherapy treatment. Disclosures No relevant conflicts of interest to declare.

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Mutation in DNA Methyltransferase DNMT3A Confers Enhanced Self-Renewal Capacity Onto Multipotent Progenitor Cells and Predisposes to Acute Myeloid Leukemia (AML)

Blood ◽

10.1182/blood-2018-99-111560 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2569-2569

Author(s):

Matthew Loberg ◽

Rebecca Bell ◽

Tim Stearns ◽

Leslie Goodwin ◽

Kira Young ◽

...

Keyword(s):

Myeloid Leukemia ◽

Dna Methyltransferase ◽

Hematologic Malignancy ◽

Selective Advantage ◽

Wild Type ◽

Self Renewal ◽

Hematopoietic Stem ◽

Npm1 Mutation ◽

Clonal Hematopoiesis ◽

Acute Myeloid

Abstract The clinical significance of clonal hematopoiesis (CHIP or ARCH) remains a barrier to predicting the risk of hematologic malignancy. DNMT3A is a de novo DNA methyltransferase frequently mutated in clonal hematopoiesis, myelodysplastic syndrome and acute myeloid leukemia. Loss of or mutation in DNMT3A has been demonstrated to enhance self-renewal of hematopoietic stem cells (HSCs), suggesting that this is the predominant cell population driving clonal hematopoiesis. How DNMT3A-mutant cells become at risk for transformation is unclear, in part due to our limited understanding of how DNMT3A mutation confers a selective advantage and the cooperating mechanisms required for progression to MDS or AML. To address this gap in knowledge, we generated a cre-inducible Dnmt3a-LSL-R878H mouse model (representing the DNMT3A-R882H mutation commonly found in human AML), in which wild-type Dnmt3a expression is preserved prior to recombination. Heterozygous Dnmt3aR878H mice exhibit an expansion in both HSCs and multipotent progenitor (MPP) cell subsets with distinct kinetics. Transcriptional profiling of sorted HSC and MPP populations by RNA-seq revealed distinct transcriptional signatures indicating that different mechanisms underlie expansion of Dnmt3aR878H/+ HSCs and MPPs. Dnmt3a-mutant HSCs exhibit downregulation of genes important for differentiation, while Dnmt3a-mutant MPPs exhibit upregulation of genes associated with stem cell self-renewal, including Jam2 and Ryk. Functionally, we observe that Dnmt3a-mutant MPPs have enhanced serial replating capacity in in vitro colony assays. These data suggest that mutation in DNMT3A may cause clonal hematopoietic expansion through distinct mechanisms dependent on the cell-of-origin which incurs this mutation. To determine whether clonal hematopoiesis driven by Dnmt3aR878H/+ was sufficient to predispose to a hematologic malignancy, we generated an independent, Flp-inducible Npm1-FSF-cA mouse model (representing the NPM1cA mutation commonly found in human AML), in which wild-type Npm1 expression is preserved prior to recombination. Inducing Npm1cA mutation in hematopoietic stem and progenitor cells carrying Dnmt3aR878H caused development of a fully penetrant myeloproliferative disorder upon transplant into recipient mice. Transplantation of these cells into secondary recipient mice led to a fully penetrant AML with accelerated disease kinetics compared to primary transplant recipients. These data suggest that the combination of DNMT3A mutation followed by NPM1 mutation is sufficient to cause AML. In summary, this study reveals a novel cell context-specificity of how DNMT3A mutation confers a selective advantage and demonstrates that NPM1 mutation can cooperate with DNMT3A mutation to cause AML. This work has implications for predicting individuals at risk of progression from clonal hematopoiesis to AML. Disclosures No relevant conflicts of interest to declare.

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The TLE1 Tumor Suppressor Regulates Myc Induced Leukemogenesis

Blood ◽

10.1182/blood.v118.21.2473.2473 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2473-2473

Author(s):

David Sweetser ◽

Selvi Ramasamy ◽

Jessica S Blackburn ◽

David M. Langenau

Keyword(s):

Cell Proliferation ◽

Tumor Suppressor ◽

Gene Family ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Hematopoietic Cells ◽

Wild Type ◽

Hematopoietic Stem ◽

Over Expression ◽

Cell Fate Decisions

Abstract Abstract 2473 The Groucho/TLE family of corepressors has been described as master regulatory genes during development, affecting multiple cell fate decisions. These proteins bind to a variety of transcription factors and recruit inhibitory proteins to repress transcription. We previously identified TLE1 as a novel tumor suppressor gene that is deleted or methylated in subgroups of acute myeloid leukemia (AML) and other hematological malignancies. We find the loss of Tle1 alone is insufficient to induce leukemia in mice and apparently requires cooperation with additional oncogenes. Our studies, and those from other groups, have shown that over-expression of TLE1 in leukemia cells slows cell cycle progression, colony formation and tumor growth in xenografts, while silencing results in increased cell proliferation. The pathways by which TLE1 affects oncogenesis is unclear, but this gene family is capable of interacting with effectors of Myc, Wnt, Notch, TGFB signaling–prominent pathways dysregulated in malignancies. Myc is important for hematopoietic stem cell proliferation, survival and differentiation and is over-expressed in most AML samples. The TLE homologue Groucho binds and represses Drosophila Myc expression of target genes, thus we postulated that TLE1 could be an important regulator of Myc activity in leukemia. Using hematopoietic progenitor cells from Tle1 knockout and wild-type fetal livers we found that the loss of Tle1 dramatically increased proliferation and serial replating efficiency. Expression of N-Myc by itself in wild type fetal liver cells triggered significant cell death and apoptosis. However, when N-Myc expression was combined with the additional loss of Tle1, not only was N-Myc induced apoptosis inhibited, but a dramatic cell proliferation, well in excess of that seen with Tle1 loss by itself, was seen. Furthermore, mice transplanted with N-Myc transduced hematopoietic cells from Tle1 knockout mice fetal liver developed a more aggressive leukemia, compared to N-Myc transfected wild type mice fetal liver hematopoietic cells, with increased proliferation of leukemic cells as demonstrated by in vitro colony assays and higher secondary transplantability. We extended these studies to a zebrafish model of Rag2-Myc mediated T-ALL. Using these zebrafish we demonstrated over-expression of the TLE homologue, Groucho, completely blocked the initiation and progression of Myc induced leukemia development. Expression of a truncated version of Groucho reduced the initiation of T-ALL and prolonged the survival of fish developing leukemia. These studies demonstrate TLE1 can inhibit the oncogenicity of Myc, and suggests modulation of expression of this gene family may be of importance for a variety of malignancies. Disclosures: No relevant conflicts of interest to declare.

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Enhanced NF-Kb Non-Canonical Signaling Impairs Hematopoietic Stem Cell Self-Renewal

Blood ◽

10.1182/blood.v126.23.2367.2367 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2367-2367

Author(s):

Yan Xiu ◽

Chen Zhao

Keyword(s):

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Critical Role ◽

Wild Type ◽

Bone Marrow Failure Syndrome ◽

Self Renewal ◽

Hematopoietic Stem ◽

Marrow Cellularity ◽

Mutant Cells

Abstract We previously demonstrated that the NF-B non-canonical signaling way positively and intrinsically regulates hematopoietic stem/progenitor cell (HSPC) self-renewal and maintains stromal/osteoblastic niches (Stem Cells 2012 30:709-18). These results lead us to think that persistent activation of NF-B non-canonical signaling would have favorable effects on the HSPC pool size and self-renewal capacity. NF-B-inducing kinase (NIK) plays a critical role in non-canonical NF-B signaling by directly phosphorylating IKK. It is constitutively degraded by TRAF3 in unstimulated cells to prevent unwanted NF-B activation. To investigate the enhanced NF-kB non-canonical signaling specifically in hematopoietic cells, we crossed Vav-Cre mice with a mouse strain in which a mutated form of NIK lacking the TRAF3-binding domain is expressed under the control of the ROSA26 promoter after Cre-mediated deletion of the LoxP-flanked STOP cassette (NIKΔT3Cre mice). In contrast to what we expected in these preliminary studies, the NIKΔT3Cre mice rapidly developed anemia, pancytopenia, with a reduced HSPC pool and marrow cellularity and postnatal lethality, mimicking many of the findings in humans with bone marrow failure syndrome, and different from recently published mice with deficiency in A20, which also activates NF-B signaling. Furthermore, the NIK activated HSPCs have profoundly impaired engraftment and self-renewal activity after transplantation into wild-type recipients. Further analysis showed that the mutant cells are proliferate faster and predispose to apoptosis than wild type cells. These observations suggest that finely controlled NF-B activity is crucial for HSC maintenance. Currently, we are focusing on the analysis of the underlying molecular mechanisms. Disclosures No relevant conflicts of interest to declare.

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Deletion of PTPN1 Promotes the Development of Myelofibrosis

Blood ◽

10.1182/blood.v128.22.795.795 ◽

2016 ◽

Vol 128 (22) ◽

pp. 795-795

Author(s):

Fatoumata Jobe ◽

Bhumika Patel ◽

Hideki Makishima ◽

Bartlomiej P Przychodzen ◽

Robert E Hutchison ◽

...

Keyword(s):

Stem Cells ◽

Tumor Suppressor ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Tyrosine Phosphatase ◽

Wild Type ◽

Hematopoietic Stem ◽

Flow Cytometric ◽

Obesity And Diabetes

Abstract Deletion of chromosome 20q [del(20q)] is a common chromosomal abnormality associated with myeloid neoplasms including myeloproliferative neoplasms (MPN), myelodysplastic syndrome (MDS), MDS/MPN overlap disorders and acute myeloid leukemia (AML). The del(20q) lesion is often associated with myeloproliferative features; it is present in patients with myelofibrosis (MF) at a high frequency (24%) and thus considered to be one of the most frequent cytogenetic abnormalities in MF (Wassie et al., Br J Haematol. 2015). The del(20q) lesion can also coexist with JAK2V617F mutation in MPN/MF. However, the target tumor-suppressor gene(s) within chromosome 20q involved in the pathogenesis of MF remains unknown. The PTPN1 locus maps to human chromosome 20q13.1-q13.2. PTPN1 (also known as PTP1B) is a ubiquitously expressed non-receptor tyrosine phosphatase that has been linked to metabolism and cancer. Mice deficient in Ptpn1 exhibit resistance to diet-induced obesity and diabetes. Both oncogenic and tumor suppressor functions for PTPN1 have been suggested. PTPN1 can negatively regulate the JAK/STAT signaling, which is frequently found activated in MPN. Here, we report the identification and functional consequences of PTPN1 deletion in the pathogenesis of MF. Deletion of PTPN1 was identified in 14% cases of MF. Conditional deletion of Ptpn1 in the mouse hematopoietic compartment resulted in significant increases in white blood cell and neutrophil counts in the peripheral blood and enlargement of spleen size. Flow cytometric analyses showed significant expansion of myeloid (Gr-1+/Mac-1+) precursors in the bone marrow (BM) and spleens of Ptpn1-deleted mice compared with control animals. Megakaryocytic (CD41+/CD61+) precursors were also significantly increased in the spleens of Ptpn1-deleted mice. Flow cytometric analyses also revealed significant increases in absolute numbers of LSK cells (Lin-Sca1+c-kit+) and its subsets including long-term hematopoietic stem cells (LT-HSC), short-term HSC (ST-HSC) and multi-potent progenitors (MPP) in the spleens of Ptpn1-deleted mice. Hematopoietic progenitor colony assays showed significant increases in myeloid (CFU-GM) and megakaryocytic (CFU-Mk) colonies in the BM of Ptpn1-deleted mice compared with control mice BM. Histopathologic analysis demonstrated fibrosis (grade 2) in the BM and spleens of Ptpn1-deleted mice at 52 weeks after induction, whereas control animals did not exhibit fibrosis at that age. Together, these results suggest that deletion of Ptpn1 induces an MPN-like phenotype, which progresses to MF over time. Moreover, transplantation of Ptpn1-deficient BM into lethally irradiated wild-type animals resulted in fibrosis at 18 weeks after transplantation, demonstrating that the effect of Ptpn1 loss in the development of myelofibrosis is cell-intrinsic. Competitive repopulation assays using BM from control or Ptpn1-deficient (CD45.2+) mice with wild type congenic (CD45.1+) mice showed that deletion of Ptpn1 enhances the repopulation capacity of hematopoietic stem cells. Biochemical analyses revealed that depletion of Ptpn1 enhanced JAK2/STAT5, AKT and ERK signaling in the BM of Ptpn1-deleted mice. Furthermore, we observed that deletion of Ptpn1 in Jak2V617F knock-in mice accelerates the development of myelofibrosis. In conclusion, our results establish a tumor-suppressor function for PTPN1 in MF. Disclosures No relevant conflicts of interest to declare.

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Ing4 Deficiency Reprograms Multipotent Progenitor Cells to Confer Self-Renewal Capabilities

Blood ◽

10.1182/blood-2020-142902 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 30-30

Author(s):

Katie L Kathrein ◽

Zanshe Thompson ◽

Seth Gabriel ◽

Melanie Rodriguez ◽

Georgina Anderson

Keyword(s):

Stem Cell ◽

Tumor Suppressor ◽

Progenitor Cells ◽

Conflicts Of Interest ◽

Post Transplantation ◽

Self Renewal ◽

Hematopoietic Stem ◽

Multipotent Progenitor Cells ◽

Deficient Mice

A network of transcription factors and associated complexes regulate the process of hematopoiesis and are required for maintenance and development of the hematopoietic program. Ing4, a tumor suppressor protein, was identified in a screen for epigenetic regulators of hematopoiesis in zebrafish as required for specification of hematopoietic stem and progenitor cells (HSPCs). Recent work has shown that Ing4 is inactivated in various cancer cells. This inactivation promotes stem cell-like qualities in malignant cells. Ing4 plays an inhibitory role in the NF-κB pathway, conferring, in part, Ing4's tumor-suppressor capability. Loss of Ing4 is correlated to diminished hematopoietic stem cell (HSC) specification in zebrafish and increased NF-κB target gene expression. NF-κB knockdown assays in zebrafish embryos suggest inhibition of NF-κB remediates loss of Ing4 expression, with HSC rescue efficacy varying directly with concentration of inhibitor. Similarly, the necessity of Ing4 in murine hematopoiesis has been observed. Here, Ing4 deficiency impairs HSC function, while simultaneously enhancing the regenerative capacity of multipotent progenitor cells (MPPs). Characterization of bone marrow from Ing4-deficient mice shows abnormal hematopoiesis, with a striking decrease in MPPs as compared to wildtype mice (47.9% vs 19.3%). Hematopoiesis under stress conditions is also altered in Ing4-deficient mice, as observed following competitive HSC transplantation. In a surprising finding, MPPs from Ing4-deficient mice showed a dramatic increase in peripheral blood multilineage chimerism compared to wildtype mice up to 9 months post-transplantation in a competitive transplant assay (19% vs. 59%). This supports the hypothesis that MPPs from Ing4-deficient mice have enhanced self-renewal capacity. Additionally, we have observed a subpopulation of Ing4-deficient MPPs that express lower levels of CD34, CD34+/mid. This population of CD34+/mid cells was also shown to express CD229 (85% positive), while very few WT MPPs express both CD34+/mid and CD229 (5.0%). Reduced levels of CD34 expression combined with CD299 are known to be markers of HSCs, and so we hypothesize that a subset of CD34+/midCD229+ MPPs in Ing4-deficient mice retain their self-renewal capacity. Taken together, our data suggest Ing4 typically functions as a suppressor of genes necessary for self-renewal and developmental potency of MPPs. Additionally, cell cycle analysis combined with Ki-67 expression showed Ing4-deficient MPPs have enhanced ability to maintain quiescence, with 15.2% of cells found to be in G0 phase as compared to 6.5% of wildtype MPPs in G0. Finally, after 5-FU treatment, levels of MPPs in WT mice were similar pre- and post-treatment. Future experiments will seek to elucidate this observation in consideration of the pro-inflammatory environment. These findings suggest Ing4 is a critical regulator of hematopoiesis, and these data provide important clues for further characterization of the pathways and functions of Ing4. Our data show that Ing4 deficiency promotes stem cell-like properties in MPPs, suggesting it has crucial regulatory functions in both stem cell self-renewal and maintenance. Disclosures No relevant conflicts of interest to declare.

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