scholarly journals Leukemic Stem Cells: From Leukemic Niche Biology to Treatment Opportunities

2021 ◽  
Vol 12 ◽  
Author(s):  
Tony Marchand ◽  
Sandra Pinho
Keyword(s):  
Stem Cells ◽  
Myeloid Leukemia ◽  
Metabolic Profile ◽  
Leukemic Cells ◽  
Intensive Chemotherapy ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Bidirectional Interactions ◽  
Intense Research ◽  
The Way

Acute myeloid leukemia (AML) is one of the most common types of leukemia in adults. While complete remission can be obtained with intensive chemotherapy in young and fit patients, relapse is frequent and prognosis remains poor. Leukemic cells are thought to arise from a pool of leukemic stem cells (LSCs) which sit at the top of the hierarchy. Since their discovery, more than 30 years ago, LSCs have been a topic of intense research and their identification paved the way for cancer stem cell research. LSCs are defined by their ability to self-renew, to engraft into recipient mice and to give rise to leukemia. Compared to healthy hematopoietic stem cells (HSCs), LSCs display specific mutations, epigenetic modifications, and a specific metabolic profile. LSCs are usually considered resistant to chemotherapy and are therefore the drivers of relapse. Similar to their HSC counterpart, LSCs reside in a highly specialized microenvironment referred to as the “niche”. Bidirectional interactions between leukemic cells and the microenvironment favor leukemic progression at the expense of healthy hematopoiesis. Within the niche, LSCs are thought to be protected from genotoxic insults. Improvement in our understanding of LSC gene expression profile and phenotype has led to the development of prognosis signatures and the identification of potential therapeutic targets. In this review, we will discuss LSC biology in the context of their specific microenvironment and how a better understanding of LSC niche biology could pave the way for new therapies that target AML.

scholarly journals The Role of NADPH Oxidase 2 in Normal and Malignant Hematopoiesis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1079-1079
Author(s):  
Biniam Adane ◽  
Haobin Ye ◽  
Shanshan Pei ◽  
Nabilah Khan ◽  
Mohammad Minhajuddin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Myeloid Leukemia ◽  
Advisory Board ◽  
Leukemic Cells ◽  
Leukemic Stem Cells ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Knock Out

Abstract NADPH dependent oxidase 2 (NOX2) is the founding member of a family of multimeric, oxido-reductase enzymes that catalyze the production of superoxides by transferring a single electron from the cofactor NADPH to molecular oxygen. It is primarily utilized in neutrophils and macrophages to generate copious amount of reactive oxygen species (ROS) to facilitate the neutralization of engulfed particulates during phagocytosis. In sharp contrast to this specialized function however, recent evidence implies a non-phagocytic role for NADPH oxidases in which physiologic levels of ROS generated by these enzymes modulate key signaling proteins and transcription factors to exert profound biological effects. Based on this information we decided to investigate the potential role of NOX2 in normal and leukemic stem cells. Using transgenic NOX2 knock out mice, genetically defined murine models of myeloid leukemia and primary human acute myeloid leukemia (AML) specimens, we show that NOX2 is critical for the proper function of normal and malignant hematopoietic stem cells. In silico analysis using published transcriptional profiles of hematopoietic populations revealed that multiple subunits of the NOX2 complex are expressed at low levels in hematopoietic stem cells (HSCs) and at relatively higher levels in multipotent progenitors (MPPs). Next, we characterized the different hematopoietic compartments from age and sex matched wild type (WT) and transgenic NOX2 knock out (KO) mice. Our studies revealed that in the bone marrow of KO mice, a subset of multipotent progenitor populations (MPP2 & MPP3), which often have biased myelo-erythroid output are markedly expanded relative to their wild type counterparts. Consistently, we found increased levels of granulocytes and monocytes in the peripheral circulation of NOX2 KO mice. To test whether NOX2 has a functional, biological role in the self-renewal of HSCs, we performed competitive transplantation assays using equal numbers of whole BM cells from WT and KO mice to co-repopulate lethally irradiated hosts. Analysis of engrafted mice showed that the contribution from NOX2 KO HSCs was severely compromised in all lineages and developmental stages of hematopoiesis examined. Collectively, these results suggest a critical biological role for NOX2 in maintaining the quiescence and long term self-renewal of HSCs. Similar to normal hematopoiesis, we found out that NOX2 is also widely expressed by functionally defined leukemic stem cells in a murine model of myeloid leukemia generated by expressing the oncogenic translocations BCR-ABL and NUP98-HOXA9. To evaluate the role of NOX2 in leukemogenesis, we established the BCR-ABL/NUP98-HOXA9 model using primitive cells derived from either WT or KO. Intriguingly, NOX2 KO leukemic cells generated a much less aggressive disease upon transplantation into primary and subsequently into secondary recipients. Furthermore, leukemic cells in which NOX2 is suppressed displayed aberrant mitotic activity and altered developmental potential marked by loss of quiescence, enhanced entry into cycle and terminal differentiation. To gain mechanistic insight into the observed phenotype, we isolated leukemic stem cells and performed whole genome expression analysis. The data showed that deficiency of NOX2 leads to downregulation of the cell cycle inhibitor CDKN2C (p18) and robust activation of the granulocyte fate determining transcription factor CEBPε. Thus we conclude that loss of NOX2 impacts leukemogenesis through rewiring of the cell cycle machinery and developmental programs in leukemic stem cells. Finally, we found that in CD34+ primary human AML cells, NOX2 and the other subunits of the complex are abundantly expressed. Furthermore, pharmacologic inhibition of NOX2 with VAS2870, a selective NADPH oxidase inhibitor, reduced the level of ROS and limited the in vitro proliferation and survival of leukemic cells. Next we genetically suppressed the expression of NOX2 in primary human AML cells using sh-RNAs and transplanted these cells into immune compromised mice. Consistent with the murine leukemia, NOX2 knocked down AML cells failed to engraft and expand in vivo. Taken together, our results firmly establish a hitherto unrecognized, prominent regulatory role for NOX2 in the biology of normal and malignant hematopoietic stem cells and imply a potential therapeutic opportunity that can get exploited to treat AML. Disclosures Pollyea: Celgene: Other: advisory board, Research Funding; Ariad: Other: advisory board; Pfizer: Other: advisory board, Research Funding; Glycomimetics: Other: DSMB member; Alexion: Other: advisory board.

scholarly journals KLF4 Controls Leukemic Stem Cell Self-Renewal in MLL-AF9-Induced Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1231-1231
Author(s):  
Andrew Lewis ◽  
Chun Shik Park ◽  
Monica Puppi ◽  
H. Daniel Lacorazza
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Myeloid Leukemia ◽  
Leukemic Cells ◽  
Epigenetic Mechanisms ◽  
Leukemic Stem Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Limiting Dilution

Acute myeloid leukemia (AML) develops from sequential mutations which transform hematopoietic stem and progenitor cells (HSPCs) in the bone marrow into leukemic stem cells (LSCs) which drive the progression of frank leukemia. Especially poor outcomes in elderly patients coupled with frequent relapse have led to a dismal 28.3% 5-year survival, warranting the need for innovative therapeutic approaches. Successful targeted therapy will selectively eliminate LSCs, which possess distinct characteristics enabling self-renewal and chemotherapeutic resistance, while sparing normal HSPCs. We theorized that KLF4, a zinc finger transcription factor, maintains key self-renewal pathways in LSCs due to its known importance in preserving stemness in embryonic and cancer stem cells. KLF4 alters gene transcription through its activating and repressing domains as well as remodeling chromatin through various epigenetic mechanisms, and work from our lab has demonstrated that loss of KLF4 in leukemia driven by the BCR-ABL fusion oncogene results in depletion of LSCs (Park et. al in revision) while enhancing self-renewal of hematopoietic stem cells. To address this hypothesis, mice featuring floxed Klf4 gene (Klf4fl/fl) were crossed with transgenic Vav-iCre mice to produce mice with hematopoietic-specific deletion of Klf4 (Klf4Δ/Δ). The murine t(9;11)(p21;q23) translocation (MLL-AF9 or MA9) transduction model has previously been shown to reflect clinical disease attributes, and represents the MLL-rearranged human patient subset with particularly poor prognosis and relatively higher levels of KLF4. Lin−Sca-1+c-Kit+ (LSK) cells from Klf4fl/fl and Klf4Δ/Δ mice were transduced with retrovirus containing MA9 and GFP reporter and transplanted into lethally-irradiated wild-type (WT) mice to generate trackable Klf4fl/fl and Klf4Δ/ΔAMLs. Recipients of both MA9Klf4fl/fl and Klf4Δ/Δ cells developed a rapid expansion of leukemic cells with myeloid immunophenotype by flow cytometric analysis (CD11b+Gr-1+; 68-91%), characterized as AML with latency of approximately 44.5 days. To quantify the defect induced by loss of KLF4 in the leukemic stem cell population, we performed secondary transplant of multiple limiting-dilution cell doses of primary transformed leukemic bone marrow from moribund mice. Klf4Δ/Δ AML mice exhibited significantly improved survival in all dose-cohorts, in some cases presenting no detectable leukemic cells at completion of monitoring (225 days). Limiting dilution analysis using the ELDA online software tool demonstrated a 7-fold reduction from 1 in 513 in Klf4fl/fl to 1 in 3836 in Klf4Δ/Δ AML bone marrow cells capable of leukemic initiation function (p<0.001), a hallmark of LSCs. Using the ERCre-tamoxifen inducible deletion system, Klf4 deletion 15 days post-transplant of AML significantly improved survival of Klf4Δ/Δ mice compared to controls, demonstrating KLF4 promotes maintenance of disease. Plating of leukemic bone marrow from Klf4Δ/Δ mice in methylcellulose medium revealed a reduction in serial colony-forming ability, further supporting a defect in self-renewal. To further determine the mechanisms connected to this reduction in functional LSCs, we isolated leukemic granulocyte-macrophage progenitors (L-GMPs), a population previously reported to be highly enriched for functional LSCs and representing a comparable cellular subset in human clinical samples, from Klf4fl/fl and Klf4Δ/Δ AMLs and conducted RNA-Seq to identify potential transcriptional targets of KLF4 with therapeutic promise. Taken together, these data suggest a novel function of the stemness transcription factor KLF4 in the preservation of leukemic stem cells in AML. Whereas prior models based on KLF4 expression in human cell lines and bulk AML samples have proposed a tumor suppressive role, our work suggests KLF4 supports expansion of leukemic cells with a stem cell phenotype and serial assays suggest an effect on LSC self-renewal. Further studies are being conducted to define the transcriptional and epigenetic mechanisms governing these findings. Understanding the molecular changes induced by loss of KLF4 presents promise for development of new therapies selectively targeting LSCs. Disclosures No relevant conflicts of interest to declare.

scholarly journals Evidence for malignant transformation in acute myeloid leukemia at the level of early hematopoietic stem cells by cytogenetic analysis of CD34+ subpopulations

Blood ◽  
1995 ◽  
Vol 86 (8) ◽  
pp. 2906-2912 ◽  
Author(s):  
D Haase ◽  
M Feuring-Buske ◽  
S Konemann ◽  
C Fonatsch ◽  
C Troff ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Hematopoietic Stem Cells ◽  
Malignant Transformation ◽  
Cell Sorting ◽  
Myeloid Leukemia ◽  
De Novo ◽  
Leukemic Cells ◽  
Hematopoietic Stem ◽  
Acute Myeloid

Acute myeloid leukemia (AML) is a heterogenous disease according to morphology, immunophenotype, and genetics. The retained capacity of differentiation is the basis for the phenotypic classification of the bulk population of leukemic blasts and the identification of distinct subpopulations. Within the hierarchy of hematopoietic development and differentiation it is still unknown at which stage the malignant transformation occurs. It was our aim to analyze the potential involvement of cells with the immunophenotype of pluripotent stem cells in the leukemic process by the use of cytogenetic and cell sorting techniques. Cytogenetic analyses of bone marrow aspirates were performed in 13 patients with AML (11 de novo and 2 secondary) and showed karyotype abnormalities in 10 cases [2q+, +4, 6p, t(6:9), 7, +8 in 1 patient each and inv(16) in 4 patients each]. Aliquots of the samples were fractionated by fluorescence-activated cell sorting of CD34+ cells. Two subpopulations, CD34+/CD38-(early hematopoietic stem cells) and CD34+/CD38+ (more mature progenitor cells), were screened for karyotype aberations as a marker for leukemic cells. Clonal abnormalities and evaluable metaphases were found in 8 highly purified CD34+/CD38-populations and in 9 of the CD34+/CD38-specimens, respectively. In the majority of cases (CD34+/CD38-, 6 of 8 informative samples; CD34+/CD38+, 5 of 9 informative samples), the highly purified CD34+ specimens also contained cytogenetically normal cells. Secondary, progression-associated chromosomal changes (+8, 12) were identified in the CD34+/CD38-cells of 2 patients. We conclude that clonal karyotypic abnormalities are frequently found in the stem cell-like (CD34+/CD38-) and more mature (CD34+/CD38+) populations of patients with AML, irrespective of the phenotype of the bulk population of leukemic blasts and of the primary or secondary character of the leukemia. Our data suggest that, in AML, malignant transformation as well as disease progression may occur at the level of CD34+/CD38-cells with multilineage potential.

The Target Receptor Siglec-3 (CD33) Is Expressed on AML Stem Cells in a Majority of All Patients with AML.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4324-4324
Author(s):  
Alexander W. Hauswirth ◽  
Stefan FLorian ◽  
Maria-Theresa Krauth ◽  
Gerit-Holger Schernthaner ◽  
Edgar Selzer ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Staining Technique ◽  
Scid Mice ◽  
Leukemic Cells ◽  
Cytostatic Drug ◽  
Leukemic Stem Cells ◽  
Targeting Therapy ◽  
Acute Myeloid

Abstract The cell surface antigen Siglec-3 = CD33 is becoming increasingly important as target of therapy in acute myeloid leukemia (AML). In particular, a conjugate consisting of the humanized CD33 antibody P67.6 (gemtuzumab) and the cytostatic drug calicheamicin has been developed for clinical use and was found to work as an effective antileukemic agent (Mylotarg®) in patients with CD33+ AML. In normal myelopoiesis, expression of CD33 is restricted to advanced stages of differentiation, whereas primitive stem cells do not express CD33 (Siglec-3). In line with this notion, CD33-targeting therapy is a non-myeloablative approach. In the present study, we asked whether leukemic stem cells in patients with AML express CD33. For this purpose, a multicolor-staining technique was applied in eleven patients with AML. Leukemic stem cells were defined as CD34+/CD38−/CD123+ cells. In all patients in whom the majority of myeloblasts expressed CD33 (=CD33+ AML, n=8), the AML progenitor cells reacted with the CD33 antibody P67.6. Repopulation experiments utilizing NOD/SCID mice confirmed that the AML stem cells in these patients reside within the CD33+ subpopulation of leukemic cells. Moreover, AML stem cells (CD34+/CD38−/CD123+ cells) highly purified (&gt;98% purity) from patients with (CD33+) AML by cell sorting, were found to express CD33 mRNA in RT-PCR analyses. To demonstrate that AML stem cells can also reside within the CD33-negative fraction of the AML clone, we purified CD33-negative cells in a patient with AML in whom a majority of leukemic stem cells were found to lack CD33. In this particular patient, the CD33-negative cells were found to repopulate NOD/SCID mice with leukemias in the same way as the entire leukemic clone did. The CD33 antigen was neither detectable on CD34+/CD38− cells in the normal bone marrow nor on leukemic stem cells in patients with CD33-negative AML. In summary, our data show that leukemic stem cells in patients with CD33+ AML frequently express the target receptor CD33. This observation is in favor of novel treatment concepts employing CD33-targeting antibodies (Mylotarg®) in acute myeloid leukemia.

The Transcription Factor ELF4 Promotes Survival of Myeloid Leukemic Stem Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1209-1209
Author(s):  
Chun Shik Park ◽  
Koramit Suppipat ◽  
H. Daniel Lacorazza
Keyword(s):  
Stem Cells ◽  
Transcription Factor ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Blast Crisis ◽  
Philadelphia Chromosome ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem

Abstract Abstract 1209 Chronic myeloid leukemia (CML) is a myeloproliferative disease that originate in hematopoietic stem cells (HSCs) as a result of the t(9;22) translocation, giving rise to the Ph (Philadelphia chromosome) and BCR-ABL oncoprotein. Although treatment of CML patients with tyrosine kinase inhibitor can efficiently eliminate most leukemic cells, chemoresistant leukemic stem cells (LSCs) can survive and drive recurrence of CML in these patients. A number of genes have been described to promote or inhibit proliferation of LSCs. Some of them have similar roles in normal HSCs. The transcription factor ELF4 promotes cell cycle entry of quiescent HSCs during homeostasis (Lacorazza et al., 2006). Thus, to investigate the function of ELF4 in CML initiation and maintenance, we developed a BCR-ABL-induced CML-like disease using retroviral transfer of BCR-ABL in Elf4-null bone marrow (BM) cells. We first investigated whether ELF4 is required for the induction of CML. Recipient mice of BCR-ABL-transduced WT BM cells developed CML and died with a latency 16–23 days, whereas recipient mice of BCR-ABL-transduced Elf4-/- BM cells showed longer latency of 45–47 days (n=20; p<0.0005). Progression of leukemia was monitored in peripheral blood, BM and spleen by flow cytometry. In mice transplanted with BCR-ABL-transduced Elf4-null BM cells, Gr-1+ leukemic cells expanded the first two weeks after BM transplantation followed by a decline at expense of a secondary expansion of B220+ cells. In contrast, Gr-1+ leukemic cells continuously expanded in mice receiving BCR-ABL-transduced WT BM cells. These results suggest that loss of ELF4 causes a profound abrogation in BCR-ABL-induced CML, while allowing progression of B-cell acute lymphocytic leukemia. Since loss of Elf4 led to impaired maintenance of myeloid leukemic cells, we postulated that ELF4 may affect survival of LSCs. Thus, we analyzed the frequency of Lin-c-Kit+Sca-1+ (LSK) cells that are BCR-ABL positive in BM and spleen. We found that BCR-ABL+ LSK cells were significantly reduced in recipients of BCR-ABL-transduced Elf4-/- BM cells. These studies indicate that ELF4 is essential to maintain the LSC pool in CML acting as a molecular switch between myeloid and lymphoid blast crisis. Disclosures: No relevant conflicts of interest to declare.

Gfi1b-A Novel Tumor Suppressor In Acute Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3795-3795
Author(s):  
Lacramioara Botezatu ◽  
Judith Hönes ◽  
Amos Zeller ◽  
Lars C. Michel ◽  
Andre Görgens ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Transcription Factors ◽  
Tumor Suppressor ◽  
Myeloid Leukemia ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Blast Cells ◽  
Leukemia Development ◽  
Acute Myeloid

Abstract The proper differentiation of hematopoietic stem cells is regulated by a concert of different so called transcription factors. Growth Factor Independence 1b (Gfi1b) is a repressing transcription factor, which is pivotal for the proper emergence and maturation of erythrocytes and platelets. Furthermore, Gfi1b controls quiescence as well as cell cycle progression of hematopoietic stem cells and early progenitor cells. It has been shown for other transcription factors that a disturbed function of these transcription factors can be the basis of malignant diseases such acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). MDS is characterized by disturbed differentiation of one or several hematopoietic lineages. The accumulation of malignant blast cells, which are arrested in their development, is a key feature of AML. Since transcription factors play a role in AML development, we sought to investigate whether Gfi1b might also play a role in the development and progression of AML. Based on published gene expression arrays and own patient samples, we observed that Gfi1b is expressed at a lower level in leukemic blasts and leukemic stem cells compared to the non-malignant control cells. We correlated Gfi1b expression level in blast cells of patients from Essen and we found out that patients with high Gfi1b levels had a poor prognosis and an increased risk of relapse. In contrast low levels of Gfi1b expression were associated with a good prognosis. To test how different levels of Gfi1b might influence initiation of AML we have mouse strains available expressing Gfi1b at different expression levels. We have Gfi1b wt mice with one allele of Gfi1b deleted (Gfi1b het) and Gfi1b conditional mice, in which the expression of Gf1b (Mx Cre tg Gfi1b fl/fl) can be abrogated after injecting these mice with pIpC. To explore the role of Gfi1b in leukemia development, we used different murine AML models, resembling human leukemia. First these mice were crossed with Nup98HoxD13 transgenic mice, a mouse strain that develops a disease similar to the human MDS. We observed that Gfi1b heterozygosity (n=15) accelerated AML development (p=0,03) compared to wt mice (n=16). More importantly, complete absence of Gfi1b (n=8) results in a substantially earlier onset of AML, with a median survival time of about 50 days (p=0.002). To confirm our findings, we used a different murine AML model. Recurrent so called oncofusion proteins such as AML1-Eto9a, CBFbeta-Myh11 or MLL-AF9 are characteristic for certain subtypes of AML. We transduced Lineage negative (Lin-) bone marrow cells from wt, Gfi1b heterozygous (Gfi1b het) and Gfi1b deficient (MxCre Gfi1b fl/fl) mice with retroviruses encoding either AML1-Eto9a or MLL-AF9 oncofusion -proteins. Transduced Gfi1b heterozygous or Gfi1b deficient cells generated more colonies and higher cell number than wt transduced cells. We also used mice transgenically expressing CBFbeta-Myh11. Deletion of Gfi1b accelerated leukemia formation in these mice compared to mice in which Gfi1b was still expressed. On a molecular level, we found that loss of Gfi1b leads to increased levels of ROS level. It has been shown by other groups, that increased levels of Gfi1b contribute to leukemia development. In addition, Gfi1b represses the expression of Integrin beta 3 (ITGB3). Absence of Gfi1b leads to higher expression level of ITGB3. ITGB3 has been shown to promote growth and expansion of leukemic stem cells, which play an important role in AML development. Thus we report here that Gfi1b acts as a novel tumor suppressor in AML development. Disclosures: No relevant conflicts of interest to declare.

scholarly journals PRMT5 Inhibition Selectively Targets Acute Myeloid Leukemia Stem Cells Though a p53-Dependent Mechanism

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4061-4061
Author(s):  
Emma Toulmin ◽  
Stefan Sonderegger ◽  
Loretta Cerruti ◽  
Andrej Terzic ◽  
Feng Yan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Advisory Committee ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Cancer Therapeutics ◽  
Leukemic Stem Cells ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Dependent Mechanism

Abstract Background: Targeting leukemic stem cells without detrimental effects on hematopoietic stem cells is a major goal for improving cure rates for acute myeloid leukemia (AML). Strategies include targeting leukemia specific mutations or pathways where leukemic cells are more dependent, leading to so-called synthetic lethality. One potential target for the latter is PRMT5, an arginine methyltransferase that methylates arginine on histones and a large number of non-histone proteins including components of the spliceosome. PRMT5 is essential for the maintenance of normal hematopoietic stem cells, through p53-dependent and independent mechanisms. Aim: To determine the relative importance of PRMT5 in normal and leukemic stem cells using genetic and pharmacologic approaches. Hypothesis: Survival of leukemic stem cells will be more dependent on PRMT5 than normal hematopoietic stem cells. Results: Using a conditional-null allele, we deleted Prmt5 in two mouse models of AML; AML1-ETO and MLL-ENL. Deletion of Prmt5 in AML1-ETO leukemia dramatically improved survival, with relapse only occurring in the setting of Prmt5-expressing cells. In contrast, deletion of Prmt5 in MLL-ENL leukemia had little effect. After screening a 350,000-compound library, we developed a potent and selective SAM-dependent inhibitor (CTx293) of PRMT5 similar to that reported by Chan-Penebre E. at al. Nat. Chem. Biol. 2015. We tested the in vivo activity of CTx293 in the AML1-ETO model generated on either a p53 wild-type or p53-/- background. Twice daily administration of CTx293 for 14 days demonstrated absolute p53 dependent activity, with prolongation of mean survival from 35 to 130 days (Figure 1A). To directly compare the effects of PRMT5 inhibition on leukemic and normal progenitors, we examined the numbers of cells within the same animal during treatment with CTx293. After 3 days, there was a two-fold reduction in both leukemic and normal progenitors. However, after 7 days treatment, leukemic progenitors had reduced more than 1000-fold whilst normal progenitors (in the same mouse) had recovered (Figure 1B). To understand this differential effect on normal and leukemic progenitors, we FACS-isolated cells after 3 days therapy. While, there was evidence of p53 activation in both normal and leukemic progenitors, the downstream effects were quite distinct, with leukemic progenitors showing activation of apoptosis. We tested the potency of CTx293 on primary human AML cells using a 14-day semi-solid agar growth assay. This demonstrated greater sensitivity of most AML samples (LD<30 nM) compared with healthy CD34+ cells (LD>100 nM). Of note, TP53-mutant samples were more resistant. Finally, we demonstrate activity of single agent CTx293 in 4 patient-derived xenografts. Conclusion: We have used both genetic and pharmacologic approaches to show that PRMT5 is an attractive target for eliminating leukemic stem cells through a p53-dependent mechanism without toxicity to healthy stem cells. Disclosures Toulmin: CRC Cancer Therapeutics: Research Funding. Sonderegger:CRC Cancer Therapeutics: Research Funding. Cerruti:CRC Cancer Therapeutics: Research Funding. Street:CRC Cancer Therapeutics: Employment, Patents & Royalties; MERCK: Membership on an entity's Board of Directors or advisory committees. Stupple:CRC Cancer Therapeutics: Employment. Jane:CRC Cancer Therapeutics: Patents & Royalties. Wei:Novartis: Honoraria, Other: Advisory committee, Research Funding, Speakers Bureau; Celgene: Honoraria, Other: Advisory committee, Research Funding; Abbvie: Honoraria, Other: Advisory board, Research Funding, Speakers Bureau; Amgen: Honoraria, Other: Advisory committee, Research Funding; Pfizer: Honoraria, Other: Advisory committee; Servier: Consultancy, Honoraria, Other: Advisory committee, Research Funding. Altura:MERCK: Employment. Nicholson:MERCK: Employment. Curtis:MERCK: Membership on an entity's Board of Directors or advisory committees; CRC Cancer Therapeutics: Patents & Royalties, Research Funding.

scholarly journals Novel Therapeutic Targeting of UHRF1 Restores 5'-Azacytidine Response in Resistant Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1356-1356
Author(s):  
Anup Kumar Singh ◽  
Xiaochun Yu
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Antiproliferative Effect ◽  
Leukemic Cells ◽  
Hematopoietic Stem ◽  
Dna Strand ◽  
Acute Myeloid

Abstract DNA hypermethylation plays a pivotal role in the pathogenesis of acute myeloid leukemia (AML). Most of the recurrent driver mutations and chromosomal translocations in AML involve genes encoding chromatin modifiers and DNA methylation relevant enzymes. Hypo-methylating drugs such as 5-Azacytidine (AZA) that target DNMTs prolong overall survival in AML patients. However, their long term treatments lead to emergence of acquired therapy resistance mostly through unknown mechanisms and hence there is an urgent need for alternate therapeutics to address AZA resistance in AML patients. Recently, it has been shown that AZA resistant leukemic cells are relatively quiescent with higher expression of many components of DNA methylation machinery that also includes UHRF1 (ubiquitin-like with PHD and ring finger domains 1). UHRF1 is a key epigenetic modulator that regulates DNA methylation and gene expression. It is a multi-domain nuclear protein with an SRA (SET-and-RING-associated) domain to recognize hemi-methylated DNA immediately after replication. It plays a crucial role in the maintenance of DNA methylation by recruiting DNMT1 to replication sites and facilitates methylation on newly synthesized DNA strand. UHRF1 is frequently overexpressed in multiple human neoplasms including AML and in the absence of UHRF1, hematopoietic stem cells undergo erythroid-biased differentiation at the expense of self-renewal capacity. Despite UHRF1 being key a therapeutic target against AML, specific, and cell-permeable inhibitors of UHRF1 have not been identified yet. In this study, we hypothesized that targeting UHRF1 using novel small molecule inhibitor will interfere with DNMT1-dependent DNA methylation at newly synthesized DNA strand, which may further synergize with antiproliferative effect of classical DNMT inhibitors in AML cells. In this study, we used in silico strategy to discover novel putative UHRF1 inhibitors by screening NCI compound database. For in vitro validation, we have first purified the SRA domain of UHRF1 followed by analysis of total DNA methylation levels using 5'-methyl cytosine (5mC) dot blot in the presence of each inhibitor. After a series of stringent in vitro and cell based assays we have identified lead compound 20 (C20) as a potent UHRF1 inhibitor which suppresses DNA methylation without affecting DNMTs in leukemic cells. Specificity of C20 against SRA domain was further established by isothermal titration calorimetry (ITC). We next found that C20 treatment significantly decreased UHRF1 and DNMT1 foci formation in the nucleus of mouse embryonic fibroblast and stem cells. Based on the its critical role in DNA methylation and enhanced expression in resistant cells, we assumed that AZA resistance in AML may be mediated by UHRF1 and C20 might restore AZA sensitivity by attenuating enhanced UHRF1 activity. To validate this, we pretreated AZA resistant leukemic cells (HL60R) with suboptimal dose of C20 followed by AZA treatment. Interestingly, we found a synergistic increase in antiproliferative effect by flow cytometry and colony formation assay. By analyzing the surface expression of myeloid differentiation markers, we found that C20 treatment promotes differentiation and decreases quiescent leukemic cell population. In conclusion, we report a novel UHRF1 inhibitor as a sensitizer of resistant AML cells towards AZA treatment potentially by promoting differentiation, suggesting a novel combination approach for future clinical evaluations. Disclosures No relevant conflicts of interest to declare.

scholarly journals Fluorouracil selectively spares acute myeloid leukemia cells with long- term growth abilities in immunodeficient mice and in culture

Blood ◽  
1996 ◽  
Vol 88 (6) ◽  
pp. 1944-1950 ◽  
Author(s):  
W Terpstra ◽  
RE Ploemacher ◽  
A Prins ◽  
K van Lom ◽  
K Pouwels ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Severe Combined Immunodeficiency ◽  
Scid Mice ◽  
Leukemic Cells ◽  
Human Leukemia ◽  
Hematopoietic Stem ◽  
Acute Myeloid

A subset of leukemic cells is assumed to maintain long-term growth of acute myeloid leukemia (AML) in vivo. Characterization of these AML progenitor cells may further define growth properties of human leukemia. In vitro incubations with 5-fluorouracil (5-FU) have been used for enrichment of normal primitive hematopoietic stem cells. By analogy to normal hematopoiesis, it was hypothesized that primitive leukemic stem cells might be kinetically more inactive than colony- forming cells (colony-forming units-AML [CFU-AML]). To examine this hypothesis, conditions were established for incubation with 5-FU that eliminated all CFU-AML. These conditions selected a 5-FU-resistant AML fraction that was evaluated for its capacity for long-term growth by transplantation into mice with severe combined immunodeficiency (SCID) and long-term culture in the quantitative cobblestone area-forming cell (CAFC) assay. Transplantation of the 5-FU-resistant fraction of four cases of AML into SCID mice resulted in growth of AML. Whereas no CFU- AML survived, 31% to 82% of primitive (week-6) CAFC were recovered from the 5-FU-treated cells. Hematopoietic cells proliferating in the CAFC assay were shown to be leukemic by cytologic, cytogenetic, or molecular analysis. The reduction of AML growth as determined by outgrowth of AML in SCID mice was in the same order of magnitude as the primitive (week- 6) CAFC reduction. This indicates that both assays measure closely related cell populations and that the CAFC assay can be used to study long-term growth of AML. These results show a hierarchy of AML cells that includes 5-FU-resistant progenitors. These cells are characterized as primitive (week-6) CAFC and as leukemia-initiating cells in SCID mice.

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