Disrupting Mitochondrial Copper Distribution Inhibits Leukemic Stem Cell Self-Renewal

Abstract Knockdown of the expression of the myeloid master regulator PU.1 leads to the development of an immature acute myeloid leukemia (AML) in mice. Recent reports suggest that functional inactivation of PU.1 might also play a role in human AML. However, the molecular mechanisms underlying PU.1-mediated malignant transformation are unknown. We examined leukemic PU.1 knockdown mice and found a 3-fold expansion of lin-, c-kit+, Sca1+ (KLS) hematopoietic stem cells (HSC) as compared to wildtype controls, which was not observed during the preleukemic phase. When we transplanted double-sorted leukemic KLS-HSC into NOD-SCID mice the recipients developed AML after 9–12 weeks indicating that the leukemic stem cells derive from the HSC compartment. This finding prompted us to examine the transcriptome of PU.1 knockdown preleukemic HSC to identify early transcriptional changes underlying their malignant transformation. After lineage-depletion and FACS sorting of preleukemic KLS-HSC we performed linear amplification of RNA by 2 cycles of RT-IVT and hybridized the cRNA with Affymetrix Mouse Genome 430 2.0 arrays. Principal component analysis as well as hierarchical cluster analysis clearly distinguished PU.1 knockdown and wildtype HSC. Several in-vitro targets of PU.1 such as c-Fes, BTK, TFEC, CSF2R, and Ebi3 were downregulated demonstrating that those are also affected in HSC in vivo. Differential expression of 16 genes was corroborated by qRT-PCR. Strikingly, several Jun family transcription factors including c-Jun and JunB were downregulated. Retroviral restoration of c-Jun expression in bone marrow cells of preleukemic mice rescued the PU.1-initiated myelomonocytic differentiation block in this early phase. To target cells in the leukemic stage we applied lentiviral vectors expressing c-Jun or JunB. While c-Jun did not affect leukemic proliferation, lentiviral restoration of JunB led to an 80% reduction of clonogenic growth and a loss of leukemic self-renewal capacity in serial replating assays. Expression analysis of 285 patients with AML confirmed the correlation between PU.1 and JunB downregulation and suggests its relevance in human disease. These results delineate a transcriptional pattern that precedes leukemic transformation in PU.1 knockdown HSC and demonstrate that downregulation of c-Jun and JunB contribute to the development of PU.1-induced AML by blocking differentiation (c-Jun) and increasing self-renewal (JunB). Therefore, examination of disturbed gene expression in preleukemic HSC can identify genes whose dysregulation is essential for leukemic stem cell function and are potential targets for therapeutic interventions.

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Co-Existence of LMPP-Like and GMP-Like Leukemia Stem Cells In Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v116.21.91.91 ◽

2010 ◽

Vol 116 (21) ◽

pp. 91-91

Author(s):

Nicolas Goardon ◽

Emmanuele Marchi ◽

Lynn Quek ◽

Anna Schuh ◽

Petter Woll ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Acute Myeloid Leukemia ◽

Stem Cell ◽

Myeloid Leukemia ◽

Expression Profiles ◽

Leukemic Stem Cell ◽

Self Renewal ◽

Two Populations ◽

Acute Myeloid

Abstract Abstract 91 In normal and leukemic hemopoiesis, stem cells differentiate through intermediate progenitors into terminal cells. In human Acute Myeloid Leukemia (AML), there is uncertainty about: (i) whether there is more than one leukemic stem cell (LSC) population in any one individual patient; (ii) how homogeneous AML LSCs populations are at a molecular and cellular level and (iii) the relationship between AML LSCs and normal stem/progenitor populations. Answers to these questions will clarify the molecular pathways important in the stepwise transformation of normal HSCs/progenitors. We have studied 82 primary human CD34+ AML samples (spanning a range of FAB subtypes, cytogenetic categories and FLT3 and NPM1 mutation states) and 8 age-matched control marrow samples. In ∼80% of AML cases, two expanded populations with hemopoietic progenitor immunophenotype coexist in most patients. One population is CD34+CD38-CD90-CD45RA+ (CD38-CD45RA+) and the other CD34+CD38+CD110-CD45RA+ (GMP-like). Both populations from 7/8 patients have leukemic stem cell (LSC) activity in primary and secondary xenograft assays with no LSC activity in CD34- compartment. The two CD34+ LSC populations are hierarchically ordered, with CD38-CD45RA+ LSC giving rise to CD38+CD45RA+ LSC in vivo and in vitro. Limit dilution analysis shows that CD38-CD45RA+LSCs are more potent by 8–10 fold. From 18 patients, we isolated both CD38-CD45RA+ and GMP-like LSC populations. Global mRNA expression profiles of FACS-sorted CD38-CD45RA+ and GMP-like populations from the same patient allowed comparison of the two populations within each patient (negating the effect of genetic/epigenetic changes between patients). Using a paired t-test, 748 genes were differentially expressed between CD38-CD45RA+ and GMP-like LSCs and separated the two populations in most patients in 3D PCA. This was confirmed by independent quantitative measures of difference in gene expression using a non-parametric rank product analysis with a false discovery rate of 0.01. Thus, the two AML LSC populations are molecularly distinct. We then compared LSC profiles with those from 4 different adult marrow normal stem/progenitor cells to identify the normal stem/progenitor cell populations which the two AML LSC populations are most similar to at a molecular level. We first obtained a 2626 gene set by ANOVA, that maximally distinguished normal stem and progenitor populations. Next, the expression profiles of 22 CD38-CD45RA+ and 21 GMP-like AML LSC populations were distributed by 3D PCA using this ANOVA gene set. This showed that AML LSCs were most closely related to their normal counterpart progenitor population and not normal HSC. This data was confirmed quantitatively by a classifier analysis and hierarchical clustering. Taken together, the two LSC populations are hierarchically ordered, molecularly distinct and their gene expression profiles do not map most closely to normal HSCs but rather to their counterpart normal progenitor populations. Finally, as global expression profiles of CD38-CD45RA+ AML LSC resemble normal CD38-CD45RA+ cells, we defined the functional potential of these normal cells. This had not been previously determined. Using colony and limiting dilution liquid culture assays, we showed that single normal CD38-CD45RA+ cells have granulocyte and macrophage (GM), lymphoid (T and B cell) but not megakaryocyte-erythroid (MK-E) potential. Furthermore, gene expression studies on 10 cells showed that CD38-CD45RA+ cells express lymphoid and GM but not Mk-E genes. Taken together, normal CD38-CD45RA+ cells are most similar to mouse lymphoid primed multi-potential progenitor cells (LMPP) cells and distinct from the recently identified human Macrophage Lymphoid progenitor (MLP) population. In summary, for the first time, we show the co-existence of LMPP-like and GMP-like LSCs in CD34+ AML. Thus, CD34+ AML is a progenitor disease where LSCs have acquired abnormal self-renewal potential (Figure 1). Going forward, this work provides a platform for determining pathological LSCs self-renewal and tracking LSCs post treatment, both of which will impact on leukemia biology and therapy. Disclosures: No relevant conflicts of interest to declare.

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PRMT1 Activates Leukemic Stem Cell Program in MLL-Rearranged Leukemia

Blood ◽

10.1182/blood.v124.21.3493.3493 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3493-3493

Author(s):

Wing Chi Lui ◽

Yuen Fan Chan ◽

Ray Ng

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Fusion Protein ◽

Fusion Proteins ◽

Leukemic Cells ◽

Leukemic Stem Cell ◽

Self Renewal ◽

Mll Gene ◽

Acute Myeloid

Abstract In MLL-rearranged leukemia, the Mixed Lineage Leukemia (MLL) gene undergoes chromosomal translocation that results in the loss of C-terminal histone methyltransferase SET domain, whereas the N-terminal of MLL gene fuses in-frame with one of the 60 identified partner genes. The resultant MLL fusion proteins lead to a characteristic aberrant gene expression pattern in human acute myeloid and lymphoblastic leukemia. Epigenetic dysregulation mediated by MLL fusion proteins has been suggested to be a key event in MLL-rearranged leukemia. It has been demonstrated that MLL-EEN/PRMT1 oncogenic complex induces transformation of primary myeloid progenitors via introduction of aberrant H4R3me2 at target Hoxloci. PRMT1 is the predominant protein arginine methyltransferase in mammals and is responsible for over 85% of arginine methylation activity in mammalian cells. Dysregulation of PRMT1 has been implicated in different cancers such as leukemia, suggesting the expression of PRMT1 is positively correlated with cancer progression and clinical parameters. Nevertheless, the leukemogenic role of PRMT1 in the establishment of leukemic stem cell (LSC) remains unclear. Previously we have demonstrated that a MLL fusion protein, MLL-EEN, can strongly enhance the self-renewal ability of murine primary hematopoietic cells through multiple rounds of replating assays. We have created a conditional Mll-Een invertor mouse model (MllEen/+) in which the expression of fusion protein is restricted to hematopoietic progenitors. Immunophenotypic analysis demonstrated a significant increase in the immature myeloid cell population (c-kit+Mac-1+) in bone marrow of MllEen/+ mice, suggesting that the expression of Mll-Een induces the development of acute myeloid leukemia. We have also established an Mll-Een expressing cell line from the bone marrow of MllEen/+ mouse. These leukemic cells can persistently form colonies and they also demonstrated deregulation of Hox genes, which is frequently observed in human leukemia cases. The leukemogenicity of Mll-Een is closely associated with Prmt1, which was demonstrated through knockdown of Prmt1. Strikingly, we discovered a subpopulation of CD41+Mll-Een expressing cells, which showed enhanced self-renewal ability in the serial colony forming assays. The percentage of CD41+ leukemic cells is reduced once Prmt1 was knocked down, suggesting that Prmt1 is crucial in the maintenance of this subpopulation of cells. In addition, the CD41+ cells showed enhanced expression of genes associated with hematopoietic stem cell (HSC) activities (Bmi-1, Runx1, Tal-1 and Lmo2), implying that part of the HSC transcriptional program has been re-activated in these cells. We therefore speculate that the CD41+ cells may represent a group of MLL leukemic cells that harbors strong stem cell features, and presumably functions as LSCs. The CD41+ leukemic cells will be further characterized by their LSC functions and CD41 can potentially serve as a novel LSC marker in MLL-rearranged leukemia. Taken together, studies on the role of PRMT1 can provide novel insights on the establishment of LSC and the development of effective clinical treatment for MLL-rearranged leukemia. Disclosures No relevant conflicts of interest to declare.

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Faculty Opinions recommendation of Acute myeloid leukemia originates from a hierarchy of leukemic stem cell classes that differ in self-renewal capacity.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1019877.231455 ◽

2004 ◽

Author(s):

Alan Houghton

Keyword(s):

Acute Myeloid Leukemia ◽

Stem Cell ◽

Myeloid Leukemia ◽

Leukemic Stem Cell ◽

Self Renewal ◽

Acute Myeloid

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Drug Induced MLL Fusion Degradation Via Hsp90 Inhibition

Blood ◽

10.1182/blood.v126.23.4850.4850 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4850-4850

Author(s):

Sandra Cantilena ◽

Nicholas Goulden ◽

Owen Williams ◽

Jasper de Boer

Keyword(s):

Stem Cell ◽

Fusion Protein ◽

Target Genes ◽

Fusion Gene ◽

Leukemic Stem Cell ◽

Down Regulation ◽

Drug Induced ◽

Acute Leukemias ◽

Self Renewal

Abstract The survival rate for infants is less than 50%. The fast majority of infant acute leukemias are characterized cytogenetically by balanced chromosomal translocations involving the mixed lineage leukemia (MLL) gene. Leukemic therapies that degrade the driver oncogene are associated with loss of cancer cell self-renewal and excellent cure rates. Therefore, therapy that degrades the MLL fusion gene would offer new hope to these patients. Recently it was shown that the Drosophila Trithorax gene, an analogue of the human MLL, is degraded by Radicicol. Radicicol is a natural compound and a well-known Hsp90 inhibitor. Here, we show that Radicicol is able to induce a dose dependent degradation of the MLL-fusion protein in a panel of human MLL rearranged cell lines and in human cord blood-derived MLL-AF9 immortalised myeloid cells. This drug induced degradation of the MLL-fusion gene results in down-regulation of the expression of MLL target genes, including HOXA9, MEIS1 and c-MYB. Functionally, this results in a loss of self-renewal of the leukemic stem cells, as shown by methylcellulose colony forming assays. Radicicol proved ineffective and too toxic for in vivo use. One of the best tolerated Hsp90 inhibitors is Ganetespib. It is currently in phase II/III clinical trials. We extended our Radicicol data to Ganetespib. Like Radicicol, Ganetespib induces MLL-fusion protein degradation and downregulation of MLL target genes. Treatment of MLL rearranged leukemia with Ganetespib results in a loss of leukemic stem cell activity. In conclusion, we show how the inactivation of the MLL-fusion and down-regulation of MLL target genes results in a block of leukemic stem cell self- renewal. We will validate these findings in a pre-clinical in vivo model in the near future. Disclosures No relevant conflicts of interest to declare.

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Acute myeloid leukemia originates from a hierarchy of leukemic stem cell classes that differ in self-renewal capacity

Nature Immunology ◽

10.1038/ni1080 ◽

2004 ◽

Vol 5 (7) ◽

pp. 738-743 ◽

Cited By ~ 624

Author(s):

Kristin J Hope ◽

Liqing Jin ◽

John E Dick

Keyword(s):

Acute Myeloid Leukemia ◽

Stem Cell ◽

Myeloid Leukemia ◽

Leukemic Stem Cell ◽

Self Renewal ◽

Acute Myeloid

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Metabolism and the leukemic stem cell

Journal of Experimental Medicine ◽

10.1084/jem.20100523 ◽

2010 ◽

Vol 207 (4) ◽

pp. 677-680 ◽

Cited By ~ 57

Author(s):

Omar Abdel-Wahab ◽

Ross L. Levine

Keyword(s):

Stem Cell ◽

Metabolic Pathways ◽

Myeloid Leukemia ◽

Leukemic Cells ◽

Oxygen Species ◽

Leukemic Stem Cell ◽

Hematopoietic Differentiation ◽

Acute Leukemias ◽

Self Renewal ◽

Leukemic Clone

Acute leukemias are clonal disorders of hematopoiesis wherein a leukemic stem cell (LSC) acquires mutations that confer the capacity for unlimited self-renewal, impaired hematopoietic differentiation, and enhanced proliferation to the leukemic clone. Many recent advances in understanding the biology of leukemia have come from studies defining specific genetic and epigenetic abnormalities in leukemic cells. Three recent articles, however, further our understanding of leukemia biology by elucidating specific abnormalities in metabolic pathways in leukemic hematopoiesis. These studies potentially converge on the concept that modulation of reactive oxygen species (ROS) abundance may influence the pathogenesis and treatment of acute myeloid leukemia (AML).

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