scholarly journals GADD45a Controls Self-Renewal in Acute Myeloid Leukemia Stem Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 31-31
Author(s):  
Nunki Hassan ◽  
Hangyu Yi ◽  
Lucie Gaspard-Boulinc ◽  
Franklin Chen ◽  
Jayvee Datuin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Cancer Cell ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Leukemia Stem Cells ◽  
Beta Catenin ◽  
Self Renewal ◽  
Acute Myeloid

Acute myeloid leukemia (AML) is a heterogenous malignancy, where the persistence of chemo-resistant leukemia stem cells (LSCs) contributes to disease relapse. We have previously demonstrated the clinical significance of WNT/β-catenin signaling in driving AML LSCs (Science, 327:1650-1653, 2010; Cancer Cell, 38:1-16, 2020). In this study, we uncover that GADD45a (growth arrest and DNA-damage inducible protein) is an essential regulator of β-catenin signaling pathway and its loss promotes LSC function and leukemia progression. Transgenic knockout of Gadd45a led to a progressive increase in aberrant self-renewal and leukemogenesis in vivo. Gadd45a-/- leukemic cells developed a more aggressive leukemia with a shorter latency than Gadd45a+/+ cells in mice, indicating the involvement of Gadd45a loss in AML initiation and progression. Subsequent serial transplantation experiments showed that Gadd45a deletion enhanced LSC self-renewal in vivo. In agreement with our findings in murine LSCs, deletion of GADD45a by CRISPR/Cas9 in AML patient-derived xenograft (PDX) cells revealed increased engraftment and tumor burden in NSG mice. Consistent with our phenotypic observations, knockout of GADD45a increased βcatenin activity and key WNT/self-renewal target genes in human AML cells. In addition, our cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) data showed that GADD45a deletion in patient-derived LSCs was associated with cell metabolism, reactive oxygen species and tumor progression, as well as poor patient outcomes in AML. Further studies are being conducted to evaluate transcriptional mechanisms discovered by our single-cell sequencing. Taken together, this study is the first to demonstrate that GADD45a loss promotes LSC potential and consequently enhances tumor growth in murine and PDX models of AML, thus showcasing GADD45a as a promising therapeutic target in AML. References: Wang Y, Krivtsov AV, Sinha AU, et al. The Wnt/beta-catenin pathway is required for the development of leukemia stem cells in AML. Science. 2010;327:1650-1653. Salik B, Yi H, Hassan N, et al. Targeting RSPO-LGR4 signaling for leukemia stem cell eradication in acute myeloid leukemia. Cancer Cell. 2020; 38:1-16. Disclosures No relevant conflicts of interest to declare.

scholarly journals Rspo-LGR4 Cooperates with HOXA9 to Sustain Self-Renewal in Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2669-2669
Author(s):  
Nunki Hassan ◽  
Basit Salik ◽  
Alastair Duly ◽  
Jenny Yingzi Wang
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Myeloid Differentiation ◽  
Beta Catenin ◽  
Leukemic Stem Cells ◽  
Self Renewal ◽  
Differentiation Block ◽  
Acute Myeloid

Acute myeloid leukemia (AML) is associated with high relapse rates and poor survival, with limited response to conventional cancer therapy and lacking effective targeting of highly self-renewing leukemic stem cells (LSCs). The mechanism underlying the high self-renewal activity of LSCs that determines the aggressiveness of disease remains poorly understood. Although we and others have previously demonstrated the clinical significance of aberrant WNT/β-catenin signaling in AML (Science, 327:1650-1653, 2010; Cancer Cell, 18:606-618, 2010), its pharmacologically tractable components essential for the regulation of LSC self-renewal have not yet been determined. Our studies discover, for the first time, a critical link between R-spondin (RSPO)-LGR4/HOXA9 and WNT/β-catenin pathways in AML LSCs. Microarray data analysis of 183 AML patient samples showed a significant positive correlation between expression of LGR4 and HOXA9 (r=0.546, P<0.0001). LGR4 exerted a cell-of-origin-specific function in promoting aberrant self-renewal and AML progression in vivo through cooperating with HOXA9, a poor prognostic predictor. We observed that LGR4 itself was not able to fully transform normal hematopoietic stem/progenitor cells (HSPCs), but instead cooperated with HOXA9 in HSPCs to accelerate disease onset producing a highly aggressive short latency AML in vivo. LGR4 and HOXA9 were epigenetically upregulated and their coexpression was an essential determinant of RSPO-LGR4 oncogenic activity. RSPO/WNT3 ligands could serve as stem cell growth factors to sustain myeloid differentiation block and to promote proliferation of CD34+ LSC-enriched subpopulations in primary AML patient specimens co-expressing LGR4 and HOXA9. Conversely, CRISPR/Cas9-mediated knockout of LGR4 not only suppressed RSPO/WNT3 signals and markedly decreased nuclear active β-catenin, but also reduced tumor burden in a patient-derived xenograft (PDX) mouse model of relapsed AML. Importantly, this study is the first to demonstrate that pharmacological inhibition of RSPO3-LGR4 signaling by a clinical-grade anti-RSPO3 monoclonal antibody induced LSC differentiation and consequently prevented tumor growth in AML PDX mice but did not affect normal human stem cell compartment in NSG mice. Together, our findings support a critical role for RSPO-LGR4 in the Wnt/β-catenin signaling pathway to promote AML leukemogenesis. Aberrant activation of RSPO-LGR4 is crucial for enhancing the self-renewal potential and myeloid differentiation block, which contribute to an aggressive leukemia phenotype through cooperating with HOXA9. Genetic and pharmacological targeting of this pathway impairs LSC self-renewal and survival and impedes AML development in murine models and patient-derived xenografts, highlighting the therapeutic value of targeting RSPO-LGR4 signaling in AML. References: Wang Y, et al. The Wnt/beta-catenin pathway is required for the development of leukemia stem cells in AML. Science. 2010;327:1650-1653. Yeung J, et al. Beta-catenin mediates the establishment and drug resistance of MLL leukemic stem cells. Cancer Cell. 2010;18:606-618. Disclosures No relevant conflicts of interest to declare.

scholarly journals Long Non-Coding RNA LINC00152 Regulates Self-Renewal of Leukemia Stem Cells and Induces Chemo-Resistance in Acute Myeloid Leukemia

2021 ◽  
Vol 11 ◽  
Author(s):  
Chunhong Cui ◽  
Yan Wang ◽  
Wenjie Gong ◽  
Haiju He ◽  
Hao Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Poor Prognosis ◽  
Underlying Mechanism ◽  
Therapy Resistance ◽  
Initial Therapy ◽  
Leukemia Stem Cells ◽  
Self Renewal ◽  
Acute Myeloid

Relapse of acute myeloid leukemia (AML) has a very poor prognosis and remains a common cause of treatment failure in patients with this disease. AML relapse is partially driven by the chemoresistant nature of leukemia stem cells (LSCs), which remains poorly understood, and our study aimed at elucidating the underlying mechanism. Accumulating evidences show that long noncoding RNAs (lncRNAs) play a crucial role in AML development. Herein, the lncRNA, LINC00152, was identified to be highly expressed in CD34+ LSCs and found to regulate the self-renewal of LSCs derived from AML patients. Importantly, LINC00152 upregulation was correlated with the expression of 16 genes within a 17-gene LSC biomarker panel, which contributed to the accurate prediction of initial therapy resistance in AML. Knockdown of LINC00152 markedly increased the drug sensitivity of leukemia cells. Furthermore, LINC00152 expression was found to be correlated with poly (ADP-ribose) polymerase 1 (PARP1) expression in AML, whereas LINC00152 knockdown significantly decreased the expression of PARP1. Upregulation of LINC00152 or PARP1 was associated with poor prognosis in AML patients. Collectively, these data highlight the importance and contribution of LINC00152 in the regulation of self-renewal and chemoresistance of LSCs in AML.

scholarly journals Self-Renewal Pathways in Acute Myeloid Leukemia Stem Cells

2020 ◽  
Author(s):  
Jonason Yang ◽  
Nunki Hassan ◽  
Sheng Xiang Franklin Chen ◽  
Jayvee Datuin ◽  
Jenny Y. Wang
Keyword(s):  
Stem Cells ◽  
Monoclonal Antibody ◽  
Acute Myeloid Leukemia ◽  
Hematopoietic Stem Cells ◽  
Myeloid Leukemia ◽  
Leukemia Stem Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Clinical Potential ◽  
Acute Myeloid

Acute myeloid leukemia (AML) is a difficult-to-treat blood cancer. A major challenge in treating patients with AML is relapse, which is caused by the persistence of leukemia stem cells (LSCs). Self-renewal is a defining property of LSCs and its deregulation is crucial for re-initiating a new leukemia after chemotherapy. Emerging therapeutic agents inhibiting aberrant self-renewal pathways, such as anti-RSPO3 monoclonal antibody discovered in our recent study, present significant clinical potential that may extend beyond the scope of leukemogenesis. In this chapter, we provide an overview of normal and malignant hematopoietic stem cells, discuss current treatments and limitations, and review key self-renewal pathways and potential therapeutic opportunities in AML.

scholarly journals Mir-125b Regulates the Self-Renewal of Acute Myeloid Leukemia Stem Cells through PTPN18 and GSK3

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 16-17
Author(s):  
Qiang Liu ◽  
Olga I. Gan ◽  
Gabriela Krivdova ◽  
Aaron Trotman-Grant ◽  
Stephanie M. Dobson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Tyrosine Phosphorylation ◽  
Myeloid Leukemia ◽  
The Self ◽  
Lower Percentage ◽  
Leukemia Stem Cells ◽  
Self Renewal ◽  
Acute Myeloid

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with poor survival, especially in older patients. Despite high remission rates after chemotherapy, relapse and death are frequent due to persistence of leukemia stem cells (LSCs), which possess properties linked to therapy resistance. Thus, there is an urgent need for a deeper understanding of the unique properties of LSCs. MicroRNAs (miRNAs) are non-coding RNAs that decrease expression of their target mRNAs by post-translational silencing. miRNA profiling of human AML samples fractionated based on LSC activity revealed that miR-125b is expressed at significantly higher levels on cell fractions enriched in LSCs. To evaluate the role of miR-125b in LSCs, expression of miR-125b was enforced in a hierarchical AML model cell line (OCI-AML-8227). miR-125b overexpression (OE) resulted in a significantly lower percentage of CD14+CD15+ differentiated myeloblasts (Figure 1A) and enhanced clonogenic potential in vitro (Figure 1B). Xenotransplantation of four AML patient samples with miR-125b OE revealed a significant increase in the proportion of CD117+ cells, a marker of hematopoietic and leukemic progenitors (Figure 1C). Secondary transplantation of cells harvested from primary engrafted mice at limiting dilution demonstrated a marked increase in LSC frequency with miR-125b OE compared to controls for the two AML samples tested (Figure 1D). Together, these data strongly suggest that miR-125b enhances the self-renewal of LSCs. To investigate the mechanisms by which miR-125b enhances self-renewal, proteomic analysis of miR-125b-OE Ba/F3 cells as well as in silico target prediction were performed and identified PTPN18 as a top putative target for miR-125b. PTPN18 is a tyrosine phosphatase that has been reported to dephosphorylate auto-phosphorylated kinases such as Her2 and Abl to prevent their activation. To evaluate whether PTPN18 OE can rescue the effects miR-125b on LSCs, we carried out transduction of an AML patient sample with control, miR-125b OE, PTPN18 OE, or both miR-125b and PTPN18 OE vectors followed by xenotransplantation. Similar to previous findings, miR-125b OE alone significantly reduced the frequency of CD11b+CD15+ differentiated myeloblasts. Co-transduction of miR-125b/PTPN18 OE vectors resulted in generation of significantly more CD11b+CD15+ cells compared to miR-125b OE alone (Figure 1E), suggesting that suppression of PTPN18 contributes to miR-125b-mediated enhancement of LSC self-renewal. To identify putative phosphotyrosines that might be altered through the miR-125b-PTPN18 signalling axis, we performed immunoprecipitation of phosphotyrosines followed by mass spectrometry in miR-125b-OE Ba/F3 cells and identified increased GSK3 tyrosine phosphorylation as a top target. Additionally, miR-125b OE was confirmed to enhance GSK3 tyrosine phosphorylation, whereas PTPN18 OE reduced it (Figure 1F), together strongly suggesting that miR-125b could enhance tyrosine phosphorylation of GSK3 by silencing PTPN18. GSK3A and GSK3B (GSK3A/B) are paralogous genes that share a high degree of sequence homology and belong to the glycogen synthase kinase 3 (GSK3) family. Tyrosine phosphorylation activates the kinase activity of GSK3, whereas serine phosphorylation inactivates it. We recently identified GSK inhibitors as top candidates targeting LSCs in a stemness-based drug screen using OCI-AML-8227 cells (data not shown). Treatment of OCI-AML-8227 cells with two selective inhibitors of GSK3 selectively reduced the proportion of CD34+ cells while concomitantly increasing expression of myeloid markers CD14 and CD15 (Figure 1G). Overall, our results support an important functional role for PTPN18 and GSK3 in LSC function, and present a potential novel therapeutic target against LSCs. This study highlights the importance of understanding the role of miRNAs and may identify a new druggable vulnerability in LSCs that could lead to the development of new treatment options for AML patients. Figure 1 Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding. Wang:Trilium Therapeutics: Patents & Royalties.

scholarly journals Targeting leukemia stem cells in vivo with antagomiR-126 nanoparticles in acute myeloid leukemia

Leukemia ◽  
2015 ◽  
Vol 29 (11) ◽  
pp. 2143-2153 ◽  
Author(s):  
A M Dorrance ◽  
P Neviani ◽  
G J Ferenchak ◽  
X Huang ◽  
D Nicolet ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Leukemia Stem Cells ◽  
Acute Myeloid

scholarly journals The Role of Gata2 in Murine Models of Acute Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1516-1516
Author(s):  
Taylor Yamauchi ◽  
Etienne Danis ◽  
Xi Zhang ◽  
Simone Riedel ◽  
Hua Huang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Murine Models ◽  
Self Renewal ◽  
Genetic Inactivation ◽  
Acute Myeloid

Abstract The importance of stem cell and self-renewal programs in Acute Myeloid Leukemia (AML) is generally accepted, but the molecular details are incompletely understood. The master transcriptional regulator GATA2 is highly expressed in hematopoietic stem cells (HSCs) and has critically important roles in the hematopoietic system. Gata2 is required for murine HSC development and maintenance, and heterozygous loss of Gata2 compromises murine HSC- and progenitor cell-function. High levels of GATA2-expression have been correlated with adverse prognosis in human AML. GATA2 is also overexpressed in human chronic myeloid leukemia. These data suggest an important role for GATA2 in normal stem cells and in leukemia. However, genetic lesions resulting in compromised GATA2 function can lead to MDS and in some cases AML. In a murine AML model driven by Flt3-ITD and inactivation of Tet2, Gata2 is strongly downregulated. Furthermore, mouse models of leukemia suggest that high-level forced expression of Gata2 can have a tumor suppressor role. To clarify the role of Gata2in AML we used homozygous genetic inactivation in established murine models of leukemia, using a a conditional allele. We initially tested the role of Gata2 in a murine leukemia mediated by forced expression of Meningioma1 (MN1). This model has a HoxA9/Meis1 transcriptional program. We recently found that MN1-driven leukemia depends on the histone methyltransferase Dot1l (J Clin Invest. 2016 Feb 29. pii: 80825). Lineage marker negative (Lin-), Sca1+, Kit-positive (LSK) bone marrow cells from mice with a floxed exon 5 in the Gata2 gene, and a ROSA26-YFP Cre-reporter allele were transduced with an MSCV-based ecotropic retroviral vector expressing MN1 and linked via an internal ribosomal entry site (IRES) the selectable marker GFP. Floxed Gata2-sequences were excised using transduction with a self-excising Cre-expressing vector (HR-Cre). Cells were sorted and plated in methylcellulose. The GFP/YFP double positive Gata2ko cells showed a replating defect compared to GFP single positive Gata2-floxed cells, both with regard to colony number and colony size. Next, we tested the role of Gata2 in disease maintenance in vivo. We established MN1 Gata2ff leukemias in primary recipients. Primary leukemias were transduced with Cre-expressing vector and Gata2ko and Gata2ff MN1 cells were transplanted. While all mice in the Gata2ffgroup developed leukemia with a median survival of 35 days, the mice in the Gata2ko cohort developed leukemia with incomplete penetrance with a latency of 249 days (p=0.0005). These data suggest an important role for Gata2in MN1 leukemia in vitro and in vivo. Genetic inactivation of Gata2 resulted in increased protein levels of p53 in vitro as detected by Western blot. Furthermore, MN1-transduced cells showed accentuated p53 stabilization and apoptosis in response to the Mdm2-antagonist and p53 stabilizer Nutlin3. We next tested the role of Gata2 in leukemia driven by the oncogenic fusion MLL-AF9. In contrast to the MN1 model, recipients of Gata2koleukemias only showed a trend towards prolonged latency in secondary recipients (median survival Gata2ff=48 days vs. Gata2ko=62 days, p=0.09). In this model, we also did not observe a substantial effect of Gata2-inactivation on p53 activation. We are currently characterizing the underlying molecular mechanisms. Our data document an important role for Gata2 in AML mediated by MN1 and to a lesser degree, MLL-AF9. The role of Gata2 in leukemia is complex and depends on expression levels and cellular context. A more detailed understanding of leukemic self-renewal, including the role of Gata2, will inform the development of more efficacious and less toxic therapies for this difficult-to-treat malignancy. Disclosures Bernt: Epizyme: Patents & Royalties: patent filed. Neff:Epizyme: Patents & Royalties: patent filed; Bristol Myers Squibb: Other: Travel; Janssen: Other: Travel.

Critical Roles Of The IDH2 Mutation In Development and Maintenance Of Acute Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 225-225
Author(s):  
Yoko Ogawara ◽  
Takuo Katsumoto ◽  
Yukiko Aikawa ◽  
Yutaka Shima ◽  
Yuki Kagiyama ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Growth And Survival ◽  
Genes Encoding ◽  
Idh2 Mutation ◽  
Acute Myeloid

Abstract Mutations in genes encoding isocitrate dehydrogenase (IDH) 1 and 2 are frequently observed in numerous cancers including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and angioimmunoblastic t-cell lymphoma (AITL). The roles of mutant IDHs in tumorigenesis remain unclear because of a lack of appropriate cancer models. Here we established a mouse AML model harboring an IDH2 mutation. IDH1/2 mutations in AML frequently occur simultaneously with mutations in other genes such as NPM, DNMT3A, and FLT3. In accordance with these observations, IDH2/R140Q, NPMc, DNMT3A/R882H and FLT3/ITD cooperatively induced AML in the mouse model. When only three out of the four mutant genes were transduced, the onset of AML was delayed in any combinations. These results clearly indicate that all four mutations are necessary for the efficient induction of AML. Gene-expression analysis indicated that IDH2/R140Q and NPMc cooperatively activate Hoxa9/Meis1 and hypoxia pathways to maintain AML cells in vivo. These two pathways are likely to be important for the IDH2/R140Q-mediated engraftment/survival of NPMc+ cells in mice. DNMT3A/R882H further upregulated the expression levels of Meis1. Furthermore, DNMT3A/R882H promoted the maintenance of cells in an undifferentiated state. Previous studies have shown that FLT3/ITD promotes cell growth and survival. Taken together, our results suggest that multiple signaling pathways are activated in this IDH-mediated AML system. The tumor-associated mutant IDHs catalyze the formation of an oncometabolite 2-hydroxyglutarate (2-HG), which dysregulates a set of α-ketoglutarate-dependent dioxygenases that includes epigenetic regulators (TETs and KDM4A), hypoxic signaling molecule (EGLN) and others (collagen prolyl 4-hydroxylases). Because mutant IDHs act via a mechanism that is completely different from those of previously described oncogenes, it has attracted increasing attention as a new therapeutic target. Small molecules that potently and selectively inhibit the mutant IDHs induced differentiation of cancer cells in vitro. However, it remains unclear whether the mutant IDHs are valid targets for cancer therapy in vivo. Here we report that AML harboring IDH2/R140Q can be blocked by conditional deletion of IDH2/R140Q, even after leukemia has developed. Deletion of IDH2/R140Q blocked 2-HG production and the maintenance of Csf-1r+ and c-Kit+ leukemia stem cells, resulting in survival of the AML mice. These results indicate that the IDH2 mutation is critical for the development and maintenance of AML stem cells, and that mutant IDHs are promising targets for anticancer therapy. Disclosures: No relevant conflicts of interest to declare.

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