Protease-Activated Receptor 1 (PAR-1) Inhibits Proliferation but Enhances Leukemia Stem Cell Activity in Acute Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2730-2730 ◽  
Author(s):  
Susumu Goyama ◽  
Mahesh Shrestha ◽  
Janet Schibler ◽  
Leah Rosenfeldt ◽  
Whitney Miller ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Clinical Investigation ◽  
Cell Activity ◽  
Stem Cell Activity ◽  
Acute Myeloid Leukemia Cells ◽  
Transplantation Assay ◽  
Acute Myeloid

Abstract Leukemic stem cells (LSCs) are capable of limitless self-renewal and indefinitely propagating leukemia. Eradication of LSCs is the ultimate goal of treating acute myeloid leukemia (AML). Using a mouse model of AML induced by the MLL-fusion protein MLL-AF9, we recently showed that the combined loss of Runx1/Cbfb inhibited the development of leukemia in vivo (Goyama S…Mulloy JC. Transcription factor RUNX1 promotes survival of acute myeloid leukemia cells. Journal of Clinical Investigation 123(9): 3876-3888, 2013). However, LSC-enriched cells with immature surface phenotype (cKit+Gr1-) remained viable in Runx1/Cbfb-deleted MLL-AF9 cells, indicating that RUNX targeting may not eradicate the most immature LSCs. Gene expression analyses of Runx1/Cbfb-deleted MLL-AF9 cells revealed the upregulation of thrombin pathway genes including a thrombin-activatable receptor PAR-1. Interestingly, both overexpression and knockout of PAR-1 inhibit leukemogenesis but do so through distinct mechanisms. Similar to the effect of Runx1/Cbfb-depletion, PAR-1 overexpression induced p21 expression and attenuated proliferation in MLL-AF9 cells. To our surprise, PAR-1-deficiency also prevented leukemia development induced by a small number of MLL-AF9 LSCs in vivo. Re-expression of PAR-1 in PAR-1-deficient cells combined with a limiting-dilution transplantation assay demonstrated the cell-dose dependent role of PAR-1 in MLL-AF9 leukemia: PAR-1 inhibited rapid leukemic proliferation when there are a large number of LSCs, while a small numbers of LSCs required PAR-1 for their growth. Mechanistically, PAR-1 increased adhering properties of MLL-AF9 cells and promoted their engraftment to bone marrow. PAR-1-deficiency also reduced leukemogenicity of AML1-ETO-induced leukemia. Together, these data reveal a multifaceted role for PAR-1 in leukemogenesis, and highlight this receptor as a potential target to eradicate primitive LSCs in AML. Disclosures No relevant conflicts of interest to declare.

CXCR4 Inhibitors Selectively Eliminate CXCR4 Expressing Human Acute Myeloid Leukemia Cells in NOG Mouse Model

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1881-1881
Author(s):  
Yanyan Zhang ◽  
Satyananda Patel ◽  
Monika Wittner ◽  
Stephane De Botton ◽  
Eric Solary ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Leukemic Cell ◽  
Cxcr4 Expression ◽  
Leukemic Cells ◽  
Acute Myeloid Leukemia Cells ◽  
Acute Myeloid

Abstract Abstract 1881 The chemokine receptor CXCR4 favors the interaction of acute myeloid leukemia (AML) cells with their niche but the extent to which it participates to pathogenesis is unclear. Here we show that CXCR4 expression at the surface of leukemic cells allowed distinguishing CXCR4high (25/47; 53%) from CXCR4neg/low (22/47, 47%) AML patients. Leukemic engraftment in NOD/Shi-scid/IL-2Rnull (NOG) mice was observed for both the CXCR4high and CXCR4neg/low groups. When high levels of CXCR4 are expressed at the surface of AML cells, blocking the receptor function with small molecule inhibitors could promote leukemic cell death and reduce NOG leukemia-initiating cells (LICs). Conversely, these drugs had no efficacy when AML cells do not express CXCR4 or when they do not respond to CXCL12. Mechanisms of this anti-leukemic effect included interference with the retention of LICs with their supportive bone marrow microenvironment niches, as indicated by a mobilization of LICs in response to drugs, and increased apoptosis of leukemic cells in vitro and in vivo. CXCR4 expression level on AML blast cells and their migratory response to CXCL12 are therefore predictive of the response to the inhibitors and could be used as biomarkers to select patients that could potentially benefit from the drugs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A KDM4A-PAF1-mediated epigenomic network is essential for acute myeloid leukemia cell self-renewal and survival

2021 ◽  
Vol 12 (6) ◽  
Author(s):  
Matthew E. Massett ◽  
Laura Monaghan ◽  
Shaun Patterson ◽  
Niamh Mannion ◽  
Roderick P. Bunschoten ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Cell Activity ◽  
Leukemia Cell ◽  
Gene Signature ◽  
Pathological Feature ◽  
Self Renewal ◽  
Stem Cell Activity ◽  
Molecular Mechanism Of Action ◽  
Acute Myeloid

AbstractEpigenomic dysregulation is a common pathological feature in human hematological malignancies. H3K9me3 emerges as an important epigenomic marker in acute myeloid leukemia (AML). Its associated methyltransferases, such as SETDB1, suppress AML leukemogenesis, whilst H3K9me3 demethylases KDM4C is required for mixed-lineage leukemia rearranged AML. However, the specific role and molecular mechanism of action of another member of the KDM4 family, KDM4A has not previously been clearly defined. In this study, we delineated and functionally validated the epigenomic network regulated by KDM4A. We show that selective loss of KDM4A is sufficient to induce apoptosis in a broad spectrum of human AML cells. This detrimental phenotype results from a global accumulation of H3K9me3 and H3K27me3 at KDM4A targeted genomic loci thereby causing downregulation of a KDM4A-PAF1 controlled transcriptional program essential for leukemogenesis, distinct from that of KDM4C. From this regulatory network, we further extracted a KDM4A-9 gene signature enriched with leukemia stem cell activity; the KDM4A-9 score alone or in combination with the known LSC17 score, effectively stratifies high-risk AML patients. Together, these results establish the essential and unique role of KDM4A for AML self-renewal and survival, supporting further investigation of KDM4A and its targets as a potential therapeutic vulnerability in AML.

scholarly journals The Salt-Inducible Kinase inhibitor YKL-05-099 suppresses MEF2C function and acute myeloid leukemia progressionin vivo

2019 ◽  
Author(s):  
Yusuke Tarumoto ◽  
Shan Lin ◽  
Jinhua Wang ◽  
Joseph P. Milazzo ◽  
Yali Xu ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Disease Progression ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Clinical Investigation ◽  
Genetic Targeting ◽  
Acute Myeloid

AbstractLineage-defining transcription factors (TFs) are compelling targets for leukemia therapy, yet they are among the most challenging proteins to modulate directly with small molecules. We previously used CRISPR screening to identify a Salt-Inducible Kinase 3 (SIK3) requirement for the growth of acute myeloid leukemia (AML) cell lines that overexpress the lineage TF MEF2C. In this context, SIK3 maintains MEF2C function by directly phosphorylating histone deacetylase 4 (HDAC4), a repressive cofactor of MEF2C. Here, we evaluated whether inhibition of SIK3 with the tool compound YKL-05-099 can suppress MEF2C function and attenuate disease progression in animal models of AML. Genetic targeting of SIK3 or MEF2C selectively suppressed the growth of transformed hematopoietic cells underin vitroandin vivoconditions. Similar phenotypes were obtained when exposing cells to YKL-05-099, which caused cell cycle arrest and apoptosis in MEF2C-expressing AML cell lines. An epigenomic analysis revealed that YKL-05-099 rapidly suppressed MEF2C function by altering the phosphorylation state and nuclear localization of HDAC4. Using a gatekeeper allele ofSIK3, we found that the anti-proliferative effects of YKL-05-099 occurred through on-target inhibition of SIK3 kinase activity. Based on these findings, we treated two different mouse models of MLL-AF9 AML with YKL-05-099, which attenuated disease progressionin vivoand extended animal survival at well-tolerated doses. These findings validate SIK3 as a therapeutic target in MEF2C-positive AML and provide a rationale for developing drug-like inhibitors of SIK3 for definitive pre-clinical investigation and for studies in human patients with leukemia.Key PointsAML cells are uniquely sensitive to genetic or chemical inhibition of Salt-Inducible Kinase 3in vitroandin vivo.A SIK inhibitor YKL-05-099 suppresses MEF2C function and AMLin vivo.

Rig-I Activates Expression of Interferon-Stimulated Genes (ISGs) and Inhibits the Proliferation of Acute Myeloid Leukemia Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2846-2846 ◽  
Author(s):  
Nan-Nan Zhang ◽  
Lei Chen ◽  
Wu Zhang ◽  
Xian-Yang Li ◽  
Lin-Jia Jiang ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Leukemia Cells ◽  
Mrna Levels ◽  
Terminal Part ◽  
Granulocytic Differentiation ◽  
Acute Myeloid Leukemia Cells ◽  
Acute Myeloid

Abstract Acute promyelocytic leukemia (APL) is initiated by the formation of PML/RARα oncogenic fusion protein, a potent transcriptional repressor. Retinoid acid (RA) at pharmacological dosage can physically bind to the PML/RARα protein, ushering in the unfolding of downstream programs normally regulated by the wild type RARα. However, through what particular regulatory pathways RA inhibits APL malignant hematopoiesis has remained largely obscured. Rig-I is one of the genes whose mRNA levels were highly up-regulated, along with all-trans-RA (ATRA)-induced terminal granulocytic differentiation of APL cell line NB4 cells in vitro. Based on the analysis of a Rig-I−/− mouse model, recently we have reported a critical regulatory role of Rig-I in normal granulopoiesis. To understand the functional contribution of Rig-I induction in RA-mediated leukemia cell differentiation, we converted a pair of previously reported Rig-I RNAi-duplex sequences into a miR30a-based small hairpin-encoding sequence, which was expressed under the CMV enhancer/promoter within a lentiviral vector. As expected, Rig-I shRNAmir30 infection induced a significant knockdown of Rig-I protein level, and accordingly its delivery into HL-60 cells partially inhibited ATRA-induced granulocytic differentiation, growth inhibition/cell cycle arrest and apoptosis induction, suggesting that Rig-I upregulation participates in RA-induced granulocytic differentiation of acute myeloid leukemia cells. In order to investigate the effect of Rig-I induction on the proliferation of APL cells in vivo, we transduced PML/RARα-harboring leukemic cells with vector or Rig-I-expressing retrovirus, and then transplanted these cells into the syngeneic mice. The vector-transduced APL cells readily expanded in vivo, but the proliferation of Rig-I-transduced cells was apparently prohibited. Moreover, we found that the forced expression of Rig-I induced the expression of numerous ISGs in APL cells, which was recapitulated by the transduction of the C terminal part of Rig-I, but not by the N terminal part. In line with this, during the in vitro short-term culture post-IFNγ or IFNα stimulation, Stat1 phosphorylation at p701 in Rig-I−/− granulocytes was significantly inhibited. In parallel, the induction of multiple ISGs by IFNs was also significantly impaired. In conclusion, our findings indicate that the Rig-I induction inhibited APL reconstitution potentially through up-regulating a number of ISGs via regulating Stat1Tyr701 phosphorylation.

RANKL Expressed by Acute Myeloid Leukemia Cells Impairs NK Cell-Mediated Immune Surveillance

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2164-2164
Author(s):  
Benjamin J Schmiedel ◽  
Constantin M Wende ◽  
Tina Baessler ◽  
Carolin Scheible ◽  
Stefan Wirths ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Nk Cells ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Immune Surveillance ◽  
Surface Protein ◽  
Leukemia Cells ◽  
Acute Myeloid Leukemia Cells ◽  
Acute Myeloid

Abstract Abstract 2164 NK cells play an important role in tumor immunosurveillance, especially of leukemia. Their reactivity is governed by various activating and inhibitory molecules expressed by their targets including multiple members of the TNF family. The TNF family member Receptor Activator of NF-κB ligand (RANKL) and its receptors RANK and osteoprotegerin (OPG) are key regulators of bone remodelling, but recently have also been shown to influence progression of hematopoetic malignancies. Here we studied the yet unkown role of the RANK/RANKL molecule system in NK cells and their reactivity against acute myeloid leukemia (AML). Primary leukemia cells from AML patients were found to substantially express RANKL mRNA and surface protein in 75% of the investigated cases (n=40). Reverse signaling via surface-expressed RANKL into AML blasts induced the release of soluble factors including the immunoregulatory cytokines TNF and IL-10, which impaired NK cell anti-tumor reactivity. Moreover, we observed upregulation of RANK on NK cells among PBMC of healthy donors upon exposure to IL-10. This was not caused by direct effects on NK cells, but was rather due to yet unidentified factors released by monocytes among the PBMC upon IL-10 exposure and could be prevented by the activating cytokine IL-2. Furthermore, functional experiments with NK cells and RANKL transfectants or RANKL-negative controls revealed that forward signaling into RANK-expressing NK cells by tumor-expressed RANKL also directly impaired NK cytotoxicity and IFN-γ production. In line, blocking RANK-RANKL interaction using anti-RANKL antibodies or RANK-Fc fusion protein increased cytotoxicity and cytokine production of allogenic NK cells in cultures with RANKL-positive primary AML cells. Our data indicate that RANKL expression enables immune evasion of leukemia cells both by directly inhibiting reactivity of RANK-expressing NK cells and by orchestrating a reciprocal interplay between AML cells, monocytes and NK cells resulting in an immunosuppressive cytokine milieu. Thus, therapeutic modulation of the RANK/RANKL system, e.g. with Denosumab/AMG162, which is presently being evaluated for treatment of both non-malignant and malignant osteolysis, holds promise to reinforce NK reactivity against hematopoietic malignancies. Disclosures: No relevant conflicts of interest to declare.

Activity of the Hypoxia-Activated Prodrug, TH-302, in Human Acute Myeloid Leukemia Models

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3611-3611
Author(s):  
Scott Portwood ◽  
Deepika Lal ◽  
Yung-Chun Hsu ◽  
Rodrigo Vargas ◽  
Meir Wetzler ◽  
...  
Keyword(s):  
Overall Survival ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Flow Cytometric Analysis ◽  
Chemotherapeutic Agents ◽  
Scid Mice ◽  
Hypoxic Conditions ◽  
Acute Myeloid

Abstract Abstract 3611 Recent evidence has demonstrated the bone marrow (BM) microenvironment, the principal site of acute myeloid leukemia (AML) initiation and expansion, is characterized by intrinsically low oxygen tension. Theoretically, such microenvironmental changes may lead to the selective outgrowth of AML clones which are “better adapted” to survive within a severely hypoxic microenvironment and/or may confer resistance to chemotherapeutic agents, similar to solid tumor cells. We report here that human AML cells (HL60, ML-2) cultured under chronic hypoxic conditions mimicking the marrow microenvironment (1% O2, 72 hours) exhibited reduced sensitivity to cytarabine-induced apoptosis as compared with normoxic cells, as determined by flow cytometric analysis, western blot analysis, and cell viability assays. Similar results were noted in primary AML samples treated with cytarabine under normoxic and hypoxic conditions in colony formation assays (n=3 samples, p=0.01). In order to improve upon chemotherapy outcomes, we investigated the effects of TH-302, a hypoxia-activated bromo-isophosphoramidate mustard prodrug, which is currently undergoing clinical trial evaluation in multiple tumor types. Treatment of AML cell lines (HL60, HEL) and primary AML samples with TH-302 (at doses ranging from 0.1– 5 mM, p values ranging from <0.05–0.0001) resulted in dose- and hypoxic-dependent inhibition of AML proliferation and apoptosis. In vivo TH-302 treatment significantly decreased disease burden, as measured by total animal bioluminescence, and prolonged overall survival in two systemic human AML xenograft models (HEL-luciferase, HL60-luciferase) (Figure 1). Immunohistochemical studies demonstrated that TH-302 treatment reduced numbers of hypoxic (pimonidazole-positive) cells within the leukemic marrow microenvironment. Because prior data in animal models has shown that AML progression within the marrow is associated with expansion of hypoxic BM areas, we examined the effects of TH-302 treatment on systemic AML growth when initiated early (prior to AML inoculation) or late (several days following AML engraftment) in the disease process. TH-302 was equally effective at both time points. Although anti-vascular therapy has been shown to enhance tumor hypoxia in other cancer types, we noted no synergistic or additive in vivo effects when TH-302 therapy was combined with sorafenib, an inhibitor of vascular endothelial growth factor receptors (VEGFR), in our models. TH-302 therapy administered for two weeks in non-leukemic and leukemia-engrafted mice was not associated with hematologic toxicities. In summary, our results demonstrate the anti-leukemic activity of TH-302 in preclinical AML models and suggest that the efficacy of this and other drugs for AML therapy may be uniquely affected by the BM microenvironment. Further clinical development of TH-302 and other hypoxia-targeted drugs for AML therapy are warranted. Based on our data, higher TH-302 doses and/or chronic drug administration may be needed for optimal in vivo anti-leukemic activity. Figure 1. Effects of TH-302 treatment on systemic AML growth and overall survival in HL60-luciferase engrafted SCID mice. Figure 1. Effects of TH-302 treatment on systemic AML growth and overall survival in HL60-luciferase engrafted SCID mice. Disclosures: No relevant conflicts of interest to declare.

CXCR4 Invalidation Limits the MLL-ENL Induced Leucemogenesis in Vivo

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4089-4089
Author(s):  
Yanyan Zhang ◽  
Hadjer Abdelouahab ◽  
Aline Betems ◽  
Monika Wittner ◽  
William Vainchenker ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Survival Time ◽  
Median Survival ◽  
Median Survival Time ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Hematopoietic Stem ◽  
Acute Myeloid

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

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