Regulation of mir-196b by MLL and its overexpression by MLL fusions contributes to immortalization

Abstract Chromosomal translocations involving the Mixed Lineage Leukemia (MLL) gene produce chimeric proteins that cause abnormal expression of a subset of HOX genes and leukemia development. Here, we show that MLL normally regulates expression of mir-196b, a hematopoietic microRNA located within the HoxA cluster, in a pattern similar to that of the surrounding 5′ Hox genes, Hoxa9 and Hoxa10, during embryonic stem (ES) cell differentiation. Within the hematopoietic lineage, mir-196b is most abundant in short-term hematopoietic stem cells and is down-regulated in more differentiated hematopoietic cells. Leukemogenic MLL fusion proteins cause overexpression of mir-196b, while treatment of MLL-AF9 transformed bone marrow cells with mir-196–specific antagomir abrogates their replating potential in methylcellulose. This demonstrates that mir-196b function is necessary for MLL fusion-mediated immortalization. Furthermore, overexpression of mir-196b was found specifically in patients with MLL associated leukemias as determined from analysis of 55 primary leukemia samples. Overexpression of mir-196b in bone marrow progenitor cells leads to increased proliferative capacity and survival, as well as a partial block in differentiation. Our results suggest a mechanism whereby increased expression of mir-196b by MLL fusion proteins significantly contributes to leukemia development.

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Functional Hematopoietic Cells Derived From Mouse Embryonic Stem Cells.

Blood ◽

10.1182/blood.v120.21.2304.2304 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2304-2304

Author(s):

Cheng Li ◽

Daniel R. George ◽

Nichole M. Havey ◽

Jeffery M. Klco ◽

Timothy J. Ley

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Peripheral Blood ◽

Bone Marrow Cells ◽

Embryonic Stem ◽

Post Injection ◽

Spleen Cells ◽

Short Term ◽

Marrow Cells

Abstract Abstract 2304 Despite two decades of effort, deriving long-term repopulating hematopoietic stem/progenitor cells (HSPCs) from embryonic stem cells (ESCs) has proven to be extremely difficult. Both embryoid body (EB)-based and stroma-based methods have been extensively explored. However, robust production of HSPCs from C57BL/6J-derived mouse ESCs (mESCs) has not yet been reported. Furthermore, in vivo engraftment of mES-derived HSCs (from any strain) has been achieved only with forced expression of HoxB4 or related oncogenes, which creates significant limitations for most studies. Here, we describe a stroma-based co-culture method to differentiate HSCs and progenitor populations from C57BL/6J-derived mESCs. After simple co-culture on OP9 stroma cells for one week, C57BL/6J-derived mESC lines differentiate into cells that mark as HSCs, CMPs, GMPs, and MEPs (by immunophenotyping); these cells are capable of giving rise to erythrocytes, monocytes, and mast cells (by morphology and immunophenotyping) after another week of culture in methylcellulose with hematopoietic cytokines (SCF, IL-3, IL-6, and Epo). Similar in vitro hematopoietic differentiation has been achieved in several different C57BL/6J-derived mESCs (B6/Blu, B6-GFP, LK1, and B6 albino), B6/SVJ129 mESCs (R1), various SVJ129-derived mESCs (SWT16, EDJ22, and SCC10), and five independent C57BL/6J mouse embryonic fibroblast (MEF)-derived induced pluripotent stem cell (iPSC) lines. C57BL/6J ESCs derived from CAGGS-GFP transgenic mice (B6-GFP ESCs, which express high levels of GFP in all hematopoietic lineages) were used to determine whether we could obtain long-term engraftment of the OP9 differentiated ESCs. B6-GFP ESCs cultured for 7 days on OP9 cells were sorted by Kit+ surface staining. Sorted cells (1×105, 2×105, 4×105) were transferred into immunocompromised NSG mice via retro orbital injection (n=1 mouse per dose). Peripheral blood from the recipients injected with 2×105 and 4×105 cells showed 5% GFP positivity in the peripheral blood at weeks 12 and 16 post-transplant, while recipients injected with 1×105 cells had no detectable GFP+ cells in the periphery. Bone marrow cells and spleens were harvested at week 22. The recipient injected with 4×105 cells showed 5% GFP positivity in the bone marrow and 20% in the spleen. Engraftment was multi-lineage. Myeloid compartments (CD34+, CD11b+, Kit+, and Gr-1+) showed similar or less GFP positivity than overall bone marrow and spleen cells. Lymphoid (CD3+ and B220+) and erythroid (Ter119+) compartments also showed similar GFP positivity compared to overall bone marrow cells. However, lymphoid and erythroid compartments contained significantly higher GFP positivity (30–60%) than overall spleen cells. We have now modified the procedure to transfer 1×106 unfractionated B6-GFP ESCs grown for 7 days on OP9 stroma directly into NSG recipients. We have detected short-term engraftment 4 weeks post-injection in the peripheral blood of one recipient and multilineage splenic engraftment 8 weeks post-injection in two recipients, confirming that short-term repopulating cells are indeed generated by this method. Secondary transplants using the GFP+ bone marrow cells from the long-term engrafted mouse have been performed. This approach could be a valuable tool for studying the hematopoietic development of a variety of mESC lines, and potentially, iPSC lines as well. Disclosures: No relevant conflicts of interest to declare.

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Human embryonic stem cell–derived hematopoietic cells are capable of engrafting primary as well as secondary fetal sheep recipients

Blood ◽

10.1182/blood-2005-05-1922 ◽

2006 ◽

Vol 107 (5) ◽

pp. 2180-2183 ◽

Cited By ~ 93

Author(s):

A. Daisy Narayan ◽

Jessica L. Chase ◽

Rachel L. Lewis ◽

Xinghui Tian ◽

Dan S. Kaufman ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Flow Cytometry ◽

Bone Marrow Cells ◽

Hematopoietic Cells ◽

Embryonic Stem ◽

Xenograft Model ◽

Fetal Sheep ◽

Hematopoietic Stem ◽

Hematopoietic Activity

The human/sheep xenograft model has proven valuable in assessing the in vivo hematopoietic activity of stem cells from a variety of fetal and postnatal human sources. CD34+/lineage- or CD34+/CD38- cells isolated from human embryonic stem cells (hESCs) differentiated on S17 feeder layer were transplanted by intraperitoneal injections into fetal sheep. Chimerism in primary transplants was established with polymerase chain reaction (PCR) and flow cytometry of bone marrow and peripheral blood samples. Whole bone marrow cells harvested from a primary recipient were transplanted into a secondary recipient. Chimerism was established as described before. This animal was stimulated with human GM-CSF, and an increase in human hematopoietic activity was noted by flow cytometry. Bone marrow aspirations cultured in methylcellulose generated colonies identified by PCR to be of human origin. We therefore conclude that hESCs are capable of generating hematopoietic cells that engraft primary recipients. These cells also fulfill the criteria for long-term engrafting hematopoietic stem cells as demonstrated by engraftment and differentiation in the secondary recipient.

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In vivo prostaglandin E2 treatment alters the bone marrow microenvironment and preferentially expands short-term hematopoietic stem cells

Blood ◽

10.1182/blood-2009-03-205823 ◽

2009 ◽

Vol 114 (19) ◽

pp. 4054-4063 ◽

Cited By ~ 58

Author(s):

Benjamin J. Frisch ◽

Rebecca L. Porter ◽

Benjamin J. Gigliotti ◽

Adam J. Olm-Shipman ◽

Jonathan M. Weber ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Prostaglandin E2 ◽

Bone Marrow Cells ◽

Negative Impact ◽

Hematopoietic Progenitors ◽

Short Term ◽

Hematopoietic Stem ◽

Marrow Cells

Abstract Microenvironmental signals can determine hematopoietic stem cell (HSC) fate choices both directly and through stimulation of niche cells. In the bone marrow, prostaglandin E2 (PGE2) is known to affect both osteoblasts and osteoclasts, whereas in vitro it expands HSCs and affects differentiation of hematopoietic progenitors. We hypothesized that in vivo PGE2 treatment could expand HSCs through effects on both HSCs and their microenvironment. PGE2-treated mice had significantly decreased number of bone trabeculae, suggesting disruption of their microarchitecture. In addition, in vivo PGE2 increased lineage− Sca-1+ c-kit+ bone marrow cells without inhibiting their differentiation. However, detailed immunophenotyping demonstrated a PGE2-dependent increase in short-term HSCs/multipotent progenitors (ST-HSCs/MPPs) only. Bone marrow cells transplanted from PGE2 versus vehicle-treated donors had superior lymphomyeloid reconstitution, which ceased by 16 weeks, also suggesting that ST-HSCs were preferentially expanded. This was confirmed by serial transplantation studies. Thus in vivo PGE2 treatment, probably through a combination of direct and microenvironmental actions, preferentially expands ST-HSCs in the absence of marrow injury, with no negative impact on hematopoietic progenitors or long-term HSCs. These novel effects of PGE2 could be exploited clinically to increase donor ST-HSCs, which are highly proliferative and could accelerate hematopoietic recovery after stem cell transplantation.

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CCL3 Regulates Normal Hematopoiesis but Is Not Essential for the Maintenance of a Long-Term Engrafting Hematopoietic Stem Cell

Blood ◽

10.1182/blood.v128.22.1482.1482 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1482-1482

Author(s):

Rhonda J. Staversky ◽

Lila Yang ◽

Alexandra N. Goodman ◽

Mary A Georger ◽

Michael W. Becker ◽

...

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Bone Marrow Cells ◽

Flow Cytometric Analysis ◽

Hematologic Malignancies ◽

Myelogenous Leukemia ◽

Short Term ◽

Hematopoietic Stem ◽

Flow Cytometric

Abstract Background/Rationale: Hematologic malignancies are known to remodel the bone marrow microenvironment, reducing support for normal hematopoiesis while increasing support for the malignant clone. The chemokine CCL3 has been demonstrated to play a role in microenvironmental dysfunction in multiple malignancies including myeloma, acute myelogenous leukemia, chronic myelogenous leukemia, and myelodysplastic syndrome. In addition, CCL3 has been shown to be critical for the progression of chronic mylogenous leukemia in murine models. However, to consider anti-CCL3 therapy as an option for hematologic malignancies we must first understand its role in the regulation of normal hematopoiesis. To date the role of CCL3 in this process is poorly understood. Methods/Results: In these experiments we utilized genetically altered mice with a global loss of CCL3 (CCL3KO) on a C57bl/6 background. Peripheral blood counts revealed that monocytes, granulocytes, and red blood cells were all significantly decreased in the peripheral blood of CCL3KO mice as compared to WT controls at 12 weeks of age (9.78 ± 0.3 vs. 8.06 ± 0.2 RBCs*106/μl, WT vs. CCL3KO p≤0.001 n=8 mice/group). CCL3KO mice also demonstrate a 2-fold increase in the frequency and number of phenotypic long-term hematopoietic stem cells (LT-HSCs: Lin-sca1+ckit+flt3-CD150+CD48-) at 12 weeks of age in the bone marrow by flow cytometric analysis (0.0053 ± 0.0005 vs. 0.0106 ± 0.0007 % of cells, WT vs. CCL3KO p≤0.0001 n=8 mice/group). A significant increase was also seen in short-term HSCs (ST-HSCs), but not in multipotent progenitor (MPP) populations (data not shown), suggesting that CCL3 regulates the most immature hematopoietic cells. To quantify functional hematopoietic stem cells in the marrow of CCL3KO mice competitive transplants were performed using whole bone marrow cells. In primary transplants CCL3KO mice demonstrated a small but significant decrease in engraftment over 22 weeks when compared to WT littermate controls (2-way ANOVA, p≤0.0001 over 22 weeks, n=8 mice/group). Decreased engraftment was seen in B cells, T cells, and myeloid cells in the peripheral blood. Upon secondary transplantation the decrease in engraftment of HSCs from CCL3KO donor mice was much more profound. At 16 weeks post-transplant HSCs from CCL3KO donors contributed to hematopoiesis at a rate 5 times lower than WT littermate controls (64.67 ± 1.967 vs. 11.97 ± 5.322 % of cells, WT vs. CCL3KO p≤0.0001 n=10 mice/group). These results were seen in both male and female mice and suggest that, although phenotypic HSCs were increased in the bone marrow of CCL3KO mice, those HSCs were defective. To test this hypothesis we sorted Lineage-Sca1+Ckit+Flt3- (Flt3-LSK) bone marrow cells enriched for LT-HSCs in order to establish stem cell activity on a per cell basis through competitive transplantation. As with the whole bone marrow transplants, primary transplant of sorted Flt3-LSK cells resulted in reduced engraftment of CCL3KO cells as compared to WT littermate controls (2-way ANOVA, p≤0.0001 over 22 weeks, n=8 mice/group). Surprisingly, upon secondary transplantation, CCL3KO Flt3-LSK donor cells performed better than the WT littermate controls (2-way ANOVA, p<0.05 over 16 weeks, n=8 mice/group). This result suggests that a transplantable population of cells excluded by the Flt3-LSK sorting parameters is responsible for repression of long-term engraftment capacity of marrow from CCL3KO mice. In establishing the mechanism by which CCL3 regulates hematopoiesis we investigated the rate of apoptosis by quantification of caspase 3 activation, as well as cell cycle status by quantification of Ki67 positivity and DNA content by flow cytometric analysis. We found no difference in the rate of apoptosis, however there was a significant decrease in the fraction of short term HSCs (ST-HSCs) (Flt3-CD48-CD150-LSK) that were actively cycling (2.06 ± 0.43 vs. 1.23±0.44 % of ST-HSCs WT vs. CCL3KO p<0.05 n=3 mice/group). This suggests that CCL3 regulates the proliferation of hematopoietic progenitor cells downstream of the LT-HSC. Conclusions: These results highlight a role for the chemokine CCL3 in the maintenance of the hematopoietic system under benign, physiologic conditions. However, a long-term engrafting HSC population is clearly maintained even in the complete absence of CCL3 suggesting that anti-CCL3 therapy would be well tolerated by the hematopoietic system. Disclosures No relevant conflicts of interest to declare.

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Short-Term Fasting Induces Cell Cycle Arrest of Immature Hematopoietic Cells and Increases the Number of Naive T Cells in the Bone Marrow of Mice

Blood ◽

10.1182/blood.v128.22.5047.5047 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5047-5047

Author(s):

Teruhito Takakuwa ◽

Yasuhiro Nakashima ◽

Hideo Koh ◽

Takahiko Nakane ◽

Hirohisa Nakamae ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

T Cells ◽

Progenitor Cells ◽

Research Funding ◽

Bone Marrow Cells ◽

Short Term ◽

Hematopoietic Stem ◽

And Control

Abstract Calorie restriction has long been studied not only as a way to prolong longevity but also as an approach to improve cancer prevention and treatment. Dietary restriction may prevent senescence of the immune and hematopoietic systems. In addition, short-term fasting before chemotherapy can reduce hematological toxicity in cancer patients. We studied the influence of fasting on immune cells, hematopoietic stem cells, and progenitor cells in the bone marrow and spleen of mice. Six-to-twelve-week old C57BL/6 mice were starved for 48 hours before analysis. We collected bone marrow and splenic cells from starved and control mice. After 48 hours of fasting, the body weight significantly decreased by an average of 24.1% compared to that of normal control mice. Calorie restriction caused a significant decrease in peripheral white blood cell count by an average of 48.3%, but hemoglobin level and platelet count were less affected. The averaged total number of bone marrow cells in the fasting group was significantly lower than that in the normal control group (2.45×107 versus 3.14×107, P < 0.01). In fasted mice there was a significant reduction in the hematopoietic stem cell count, using detection based on the lineage- c-kit+ Sca-1+, compared to control mice (0.83×105 versus 1.24×105, P < 0.05). In contrast, there was no significant difference for progenitor cells detected based on the lineage- c-kit+ Sca-1- (6.81×105 versus 7.75×105, P = 0.11). We performed colony assays with bone marrow cells from fasted and control mice. There was no difference between the two groups for not only the primary colony assay but also for the secondary and tertiary assays. Annexin V and 7-AAD analysis by flow cytometry showed that the rates of early and late apoptosis were almost identical in hematopoietic stem cells and progenitor cells, on comparing fasted and control mice. Furthermore, DNA cell cycle analysis of progenitor cells showed that short-term fasting caused a significant increase in the percentage in G0/G1 phase (83.1% versus 70.7%, P < 0.05) and decreases in the S and G2/M phases. These results imply that immature bone marrow cells retained their proliferative capacity by maintaining cell cycle arrest after short-term fasting, a finding that may account for the protective effect of starvation against chemotherapy in cancer patients. Calorie restriction caused a significant decrease in B cells in bone marrow (5.38×106 versus 8.1×106, P < 0.05) and especially in the spleen (6.65×106 versus 33.0×106, P < 0.001), and also significantly decreased T cells in the spleen (3.91×106 versus 14.5×106, P < 0.01). To our surprise, we detected a remarkable increase in the number of T cells in the bone marrow of fasted mice (1.25×106 versus 0.91×106, P < 0.01). Of greatest significance CD44- naive CD8+ T cells were dramatically increased in fasted bone marrow (1.74×106 versus 0.47×106, P < 0.01), and CD44- naive CD4+ T cells were also increased (0.23×106 versus 0.07×106, P < 0.05). In contrast, the numbers of CD62L- CD44+ effector memory and CD62L+ CD44+central memory T cells were not substantially changed after starvation. The increased naive T cells had no activated markers and appear to have migrated into bone marrow in a resting state without proliferating there. Short-term fasting decreased the number of hematopoietic stem cells but progenitor cells remained in a relatively quiescent state, with a prolonged DNA cell cycle and retention of colony-forming capabilities. The number of T cells in the bone marrow of fasted mice also increased dramatically, especially naive CD8+ T cells which probably migrated in from other lymphoid tissues. These findings imply that immature hematopoietic cells and some lymphoid cells can survive starvation while maintaining their function. The mechanisms by which T lymphoid cells promptly accumulate in bone marrow during starvation are under investigation. Disclosures Koh: Pfizer: Consultancy, Honoraria. Nakane:Mundipharma KK: Research Funding. Nakamae:Mochida Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Novartis Pharma KK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel/accommodation/meeting expenses, Research Funding. Hino:Nippon Shinyaku: Honoraria, Speakers Bureau; Pfizer: Honoraria, Research Funding; Alexion: Honoraria, Research Funding.

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Histone Alterations Are Associated with Hematopoietic Stem Cell (HSC) Differentiation and Aging

Blood ◽

10.1182/blood.v120.21.1188.1188 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1188-1188

Author(s):

Min Luo ◽

Mira Jeong ◽

Deqiang Sun ◽

Hui Wang ◽

Rui Chen ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Transcription Factors ◽

Cancer Progression ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Embryonic Stem ◽

Hematopoietic Stem ◽

Chromatin Regulators ◽

Repressive Mark

Abstract Abstract 1188 Chromatin regulators, including both Trithorax-group (trxG) and Polycomb-group (PcG) families, have been reported to maintain hematopoietic stem cells (HSC) self-renewal and differentiation. However, their primary targets in HSC remain to be fully identified. Moreover, those histone modifiers have been recently found to control longevity in C.elegans, which leads us to further investigate their roles in HSC aging. HSCs increase in number, decrease in regeneration capacity, and exhibit a myeloid biased differentiation with age. In this study, we profiled global mRNA (RNA-seq) and chromatin changes (ChIP-seq) in highly purified young (4 month) and old (24 month) murine bone marrow-derived HSCs (SP-KSL-CD150+). One key challenge in this study is that mouse HSCs represent less than 0.01% of all bone marrow cells. Thus, we first developed and optimized a method for successful application of ChIP-seq to a limited number of cells (<20,000 cells). This method allowed us to generate the binding profiles for H3K4me3, H3K27me3 and H3K36me3 in young and old HSCs, differentiated granulocytes (Gr1+) and B (B220+) cells. In young HSCs, we determined ∼18,000 peaks for the active gene mark H3K4me3. For the repressive mark H3K27me3, we identified ∼6000 peaks across the genome, 2591 of which were present in the promoter region defined as the transcriptional start site (TSS) ± 100 bp. Strikingly, 70.2% (1820 out of 2591) of H3K27me3-enriched genes were also bound by H3K4me3. These so-called “Bivalent genes”, with both H3K4me3 and H3K27me3, are also found in embryonic stem cell (ESC) where they represent master regulators for lineage development. Here we found that these 1820 genes in HSC are prevalent in development, transcriptional regulation and RNA metabolism. They include many lineage-specific transcription factors, such as Cebpa, Ebf-1, Pax5, Gata3, Tbx21, Runx3 and Eomes. During HSC differentiation to granulocytes or B cells, HSC pluripotency regulators acquired the H3K27me3 repressive mark, while lineage regulators lost it in differentiated lineages. In addition, we observed with HSC differentiation alternative promoter usage on many epigenetic modifiers or transcription factors, such as Dnmt3a, Dnmt3b and Kdm6b (Jmjd3) and Runx3. The different isoforms may have different functions in hematopoiesis. Compared to HSC differentiation, HSC aging showed less extensive chromatin changes. Increased H3K4me3 binding at the TSS was identified for ∼300 genes, including Selp, Nupr1, Sdpr, Plscr2, Slamf1 (CD150) and Mt1. Interestingly, several genes in the Hoxb cluster showed increased H3K4me3 binding and higher expression with age. For H3K27me3, although PcG family member EZh2 expression decreases with HSC aging, we did not detect a global H3K27me3 decrease. On the contrary, H3K27me3 binding increased at ∼500 genes and decreased at ∼100 genes. Among them, the lymphoid transcription factors Pax5 and Ebf1 exhibited increased H3K27me3 binding. The enhanced repression of these lymphoid regulators may explain the myeloid-skewed differentiation that occurs with age. We also observed increased H3K27me3 on several Wnt ligands, including Wnt2a, Wnt8a and Wnt8b. As Wnt signaling is required for active HSC cycling, the increased H3K27me3 binding on these ligands may reinforce HSC quiescence. One well known target of the PcG family in aging is Cdkn2a (p16), which showed progressive loss of H3K27me3 repression with aging in neural stem cell (NSC). In contrast, our results revealed that p16 is repressed by H3K27me3 in both 4M and 24M HSC, and we did not observe its expression increase with aging. However, another cell cycle regulator p21 expression increases with aging, accompanied by a decrease of H3K27me3. In summary, we mapped chromatin state alterations with HSC differentiation and aging in this study. These findings will advance our fundamental understanding of HSC biology. Furthermore, chromatin regulators, such as Bmi1 and EZh2, have been implicated in leukemia transformation. This study provides a mechanistic link into how deregulation of these factors correlates with cancer progression. Disclosures: No relevant conflicts of interest to declare.

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The MLL PHD Fingers of MLL Block MLL Fusion Protein Mediated Transformation.

Blood ◽

10.1182/blood.v110.11.976.976 ◽

2007 ◽

Vol 110 (11) ◽

pp. 976-976

Author(s):

Andrew G. Muntean ◽

Jay L. Hess

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Fusion Protein ◽

Fusion Proteins ◽

Hox Genes ◽

Bone Marrow Cells ◽

Nurd Complex ◽

Inhibition Of Proliferation ◽

Phd Finger ◽

Histone H3k4

Abstract Mixed Lineage Leukemia (MLL) is a histone H3K4 methyltransferase that is rearranged in both acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). MLL is required for the maintenance of Hox gene expression. Deregulation of Hox genes by MLL fusion proteins, which fuse MLL in frame to one of over 50 different translocation partners, is critical for transformation. In these translocations, the DNMT homology (CXXC) domain is always included, but the set of adjacent plant homeodomains (PHD), which includes four PHD fingers and a bromodomain, is invariably excluded. PHD fingers have recently been described to bind tri-methylated histone H3K4 and others report PHD domains binding transcriptional co-repressors, such as Mi-2a of the NuRD complex. However, the role of the PHD fingers in MLL is not well understood. To determine the function of the PHD fingers in MLL, we performed bone marrow transduction and colony assays with the MLL fusion protein MLL-AF9, engineered to contain the PHD domain region (MLL-PHD-AF9). These experiments showed that inclusion of the PHD fingers inhibited immortalization as shown by the absence of compact colonies in methylcellulose replating assays and inhibition of proliferation in liquid cultures. Initial experiments with PHD finger deletions to map the inhibiting activity suggest inclusion of any PHD fingers beyond the first PHD finger, results in inhibition of transformation. To monitor the transcriptional activity of the retrovirally infected bone marrow cells, total RNA was isolated from cells harvested after the second replating, when significant differences were seen in colony morphology and size. Consistent with the transformation inhibition, Hoxa9 gene expression was found to be significantly repressed with respect to expression detected in transformed MLL-AF9 cells as determined by qPCR. To confirm this effect is directly due to the MLL fusion proteins, we performed luciferase assays with an MLL responsive myc E-box luciferase construct in MLL −/− MEFs. We found a specific and robust activation of the reporter in the presence of MLL-AF9, which was severely compromised by the inclusion of the PHD fingers. Together, these results suggest the PHD fingers act as transcriptional repressors that inhibit transformation. Our results provide an explanation for the finding that translocations including the coding region for C terminal PHD fingers do not occur in human leukemias and suggest that this region is also involved in the regulation of wild type MLL. We are currently studying the mechanisms of transcriptional repression mediated by the PHD fingers by isolating interacting proteins and assessing their effect on transcription and transformation.

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The PAF Complex Synergizes with MLL Fusion Proteins at .Hox Loci to Promote Leukemogenesis.

Blood ◽

10.1182/blood.v114.22.1277.1277 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1277-1277 ◽

Cited By ~ 1

Author(s):

Andrew G. Muntean ◽

Jiaying Tan ◽

Venkatesha Basrur ◽

Kojo S.J. Elenitoba-Johnson ◽

Jay Hess

Keyword(s):

Stem Cell ◽

Fusion Protein ◽

Fusion Proteins ◽

Hox Genes ◽

Conflicts Of Interest ◽

Embryonic Stem ◽

Cell Phenotype ◽

Mouse Bone Marrow ◽

Hematopoietic Stem ◽

Paf Complex

Abstract Abstract 1277 Poster Board I-299 Mixed lineage leukemia (MLL) is a histone H3 lysine 4 methyltransferase that is required to maintain a normal hematopoietic stem cell compartment. MLL functions to maintain expression of HOX genes as well as the HOX co-factor MEIS1, which play significant roles in regulating hematopoiesis. MLL is involved in chromosomal translocations with up to sixty different partners in both acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). HOXA9 and MEIS1, are directly regulated by MLL fusion proteins and are crucial for MLL fusion protein mediated transformation. The deregulated expression of target genes in AML is dependent on specific protein-protein interactions and functional domains of MLL. For example, the tumor suppressor Menin bridges LEDGF to the extreme N-terminus of MLL and both of these interactions are necessary for transformation. Furthermore, a DNA methyltransferase homology region (CxxC domain) of MLL is essential for binding to non-methylated CpG islands and MLL-fusion protein oncogenesis. We have found that sequences downstream of the CxxC domain, termed the RD2 region, that interact with the Polymerase Associated Factor (PAF) complex are also required for MLL fusion protein mediated transformation. The PAF complex interacts with RNA polymerase II and is required for H2B mono-ubiquitination and subsequent histone H3K4 and H3K79 methylation. Together the PAF complex has been shown to be involved in transcriptional initiation, elongation and termination. Interaction of MLL with the PAF complex is mediated through direct contacts with two subunits: Ctr9 and PAF1. The PAF complex synergizes with MLL-AF9 to augment transcriptional activation of the Hoxa9 promoter. Furthermore, MLL fusion proteins recruit high levels of the PAF complex to the Hoxa9 promoter. Importantly, deletions of the MLL RD2 region that abolish interactions with the PAF complex eliminate MLL-AF9 mediated transformation of mouse bone marrow cells. Transcription of PAF components is dramatically downregulated during differentiation of hematopoietic cells, consistent with recent data showing a requirement for the PAF complex to maintain an embryonic stem cell phenotype. Knock down and transplantation experiments are underway to further define how the PAF complex regulates normal MLL function and cooperates with MLL fusion proteins to promote leukemogenesis. Disclosures No relevant conflicts of interest to declare.

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Hoxa9 Suppresses Cellular Senescence and Sustains the Development of Leukemic Stem Cells Induced by Fusion Proteins In the Absence of Bmi1.

Blood ◽

10.1182/blood.v116.21.1578.1578 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1578-1578

Author(s):

Lan-Lan Smith ◽

Jenny Yeung ◽

Bernd B Zeisig ◽

Ivo Huijbers ◽

Nik Popov ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Cellular Senescence ◽

Fusion Proteins ◽

Hox Genes ◽

Leukemic Stem Cells ◽

Self Renewal ◽

Hematopoietic Stem ◽

Fold Reduction

Abstract Abstract 1578 While self renewal is an essential feature for the maintenance of both normal hematopoietic stem cells (HSCs) and leukemic stem cells (LSCs), very little is known about the underlying molecular pathways. Here we report a critical functional interplay between Bmi1 and Hox in establishment of HSCs and LSCs. Using Bmi1-/- bone marrow cells, we observe that leukemia-associated fusion proteins have distinctive Bmi1 requirements. AML1-ETO (AE) and PLZF-RARα (PR) fail to transform Bmi1-/- primary hematopoietic cells, and induce expression of p16/Arf leading to oncogene-induced senescence (OIS). In contrast, MLL-AF9 driving expression of multiple Hox genes can bypass oncogene-induced senescence and exhibits modest Bmi1-dependence for establishment of LSCs, which can induce leukemia upon serial transplants. Since members of Hox genes with proclaimed self-renewal property are specifically up-regulated by MLL fusions in patient samples and our murine models, we asked the question if these Hox genes may partly compensate the functions associated with the loss of Bmi1. To this end, we generated compound Bmi1-/-Hoxa9-/- mice, which have even more compromised hematopoietic stem cell/progenitor compartments than those of Bmi1-/- or Hoxa9-/- mice. Bmi1-/-Hoxa9-/- mice have a greater than eight-fold reduction in the absolute number of Lin-Sca+kit+ (LSK) in the bone marrow as compared to Bmi1-/- mice and a very significant forty-fold reduction for long term hematopoietic stem cells (LT-HSC). More importantly, while MAF9 is able to transform wild type, Bmi1-/- and Hoxa9-/-, it fails to transform Bmi1-/-Hoxa9-/- cells for establishment of LSCs, which can however be resurrected by re-expression of either Bmi1 or Hoxa9, indicating a critical functional interplay between these protein in development of MLL LSCs. Consistent with the known function of Bmi1 in suppressing cellular senescence and the expression of p16/Arf loci, we showed that Hoxa9 alone can also inhibit replicative senescence and Ras-induced senescence in primary human fibroblast. Forced expression of Hoxa9 can suppress p16/Arf expression, as well as cellular senescence induced by AE and PR in Bmi1-/- cells. Together, these results reveal a previously unrecognized functional interplay between Hox and Bmi1 in regulating cell senescence and development of LSCs induced by fusion proteins, which also suggests that synergistic targeting of both molecules may be required for certain LSCs. Disclosures: No relevant conflicts of interest to declare.

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Synergism Between HOXA9 and Mutant JAK3 (M511I) Leads to Rapid Leukemia Development within an in Vivo Murine Bone Marrow Transplant Model

Blood ◽

10.1182/blood.v124.21.1078.1078 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1078-1078

Author(s):

Charles E de Bock ◽

Sandrine Degryse ◽

Sofie Demeyer ◽

Bram Sweron ◽

Olga Gielen ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Transplant ◽

Marrow Transplant ◽

Double Positive ◽

Hoxa Cluster ◽

Hematopoietic Stem ◽

Single Positive ◽

Leukemia Development ◽

Transplant Model

Abstract Activation mutations in JAK3 occur in 16% of T-cell acute lymphoblastic leukemia (T-ALL) cases, and co-occur frequently with HOXA cluster rearrangement. Genomic rearrangement of the HOXA cluster results in increased expression of HOXA9 and HOXA10. However it remains unclear if either HOXA9 or HOXA10 can cooperate with activating JAK3 mutations during oncogenic transformation and leukemogenesis. We have previously shown that JAK3 mutations lead to cell transformation and cause a long latency T-ALL in vivo using a mouse bone marrow transplant model. In this study we demonstrate that co-expression of the activating JAK3(M511I) protein with HOXA9 cooperate to develop leukemia within 30 days of transplant using an in vivo bone marrow transplant model. In our cooperative model, murine hematopoietic stem / progenitor cells were co-transduced with either both retroviral vectors encoding JAK3(M511I)/GFP and HOXA9/mCherry or each individually and then injected into sub-lethally irradiated recipient mice. Mice transplanted with bone marrow cells expressing JAK3(M511I) mutant alone developed T-ALL in 120 to 150 days. In sharp contrast, mice transplanted with cells expressing both JAK3(M511I) and HOXA9 showed rapid leukemia development within 30 days after transplant. Leukemia development was characterized by the rapid and specific increase in GFP-mCherry double positive cells. These animals showed high WBC, and splenomegaly and accumulation of immature CD8 single positive cells in the thymus. Similar experiments with HOXA10 did not show cooperation suggesting that HOXA9 is the more important oncogene in HOXA rearranged leukemias when a JAK3 activating mutation is present. To determine the underlying genetic mechanism for cooperation between HOXA9 and JAK3(M511I) the single positive JAK3 and double positive JAK3/HOXA9 expressing cells were isolated from thymi of leukemic mice for both epigenomic profiling using ATAC-seq and gene expression profiling. These analyses identified genetic pathways activated by the co-expression of HOXA9 and JAK3(M511I) mutation and provide mechanistic insight into the synergistic interaction between these two factors in driving leukemia development. Treatment of the animals with a JAK kinase inhibitor resulted in delayed leukemia development, confirming that the leukemia cells remain sensitive to the JAK inhibitor. This mouse model provides insight in the cooperation between oncogenes in leukemia development and provides a model for the study of targeted agents in this setting. Disclosures No relevant conflicts of interest to declare.

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