The PRC2 component JARID2 is dispensable for hematopoietic stem cells, but critical for maintenance of leukemic stem cells

2016 ◽  
Vol 44 (9) ◽  
pp. S47
Author(s):  
Anne-Katrine Frank ◽  
Anton Willer ◽  
Tanja Lyholm Jensen ◽  
Teresa D'Altri ◽  
Nicolas Rapin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem

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 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.

Selective Expression of a Number of ABC Transporter Genes in Normal CD34+CD38− Versus CD34+CD38− Cells, with a Reverse Pattern in a Subgroup of AML Patients.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4291-4291
Author(s):  
Dorina M. van der Kolk ◽  
Susan D.P.W.M. Peeters ◽  
Gerald de Haan ◽  
Leonid Bystrykh ◽  
Elisabeth G.E. de Vries ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Mrna Expression ◽  
Abc Transporter ◽  
Abc Transporters ◽  
Cholesterol Synthesis ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Reverse Pattern ◽  
Abc Transporter Genes

Abstract Several ABC transporters involved in drug transport have been identified in hematopoietic stem cells, including ABCB1, ABCC1 and ABCG2. The ABC transporters play a role in chemotherapy resistant AML, although the relevant information is mostly obtained from the total AML cell population instead of the leukemic stem cells characterized by the CD34+CD38− phenotype. In this study we investigated which ABC transporters are selectively expressed in normal CD34+CD38− hematopoietic stem cells versus CD34+CD38+ cells, and to what extent lineage-restricted modulation is aberrantly regulated in AML stem cells. We first investigated murine microarray expression data of 29 ABC transporter genes in lin−sca-1+c-kit+ cells (available on www.webqtl.org). Based on these data 7 of the 29 ABC transporters were selected with a high expression profile (abcg1, abcb2, abca2, abcd1, abcc3, abcc5, and abcg2). Based on data published at www.sciencemag.org/cgi/content/full/1073823/DC1, concerning the lineage restricted expression of genes in lin−AA4.1+ + c-kit+sca-1+ murine stem cells, 6 additional stem cell-related ABC transporters (abcb1, abcb11, abcc1b, abcd4, abce1 and abcf2) were selected. The mRNA expression of the 13 ABC transporters was analyzed in the CD34+CD38− versus CD34+CD38+ fraction of human normal bone marrow cells (n=10) by quantitative RT-PCR. Five ABC transporter genes were not detectable in the human CD34+CD38− and CD34+CD38+cells (ABCA2, ABCB11, ABCC3, ABCD1 and ABCF2). Three ABC transporters were expressed equally in both fractions (ABCC5, ABCE1 and ABCG2). However, five ABC transporters were differentially expressed, with a higher expression in the CD34+CD38− cells, (ABCB1, ratio of CD34+CD38+/CD34+CD38− expression of 0.22, p<0.001; ABCG1, 0.27, p<0.001; ABCC1, 0.52, p<0.001; ABCD4, 0.60, p<0.001; and ABCB2, 0.71, p<0.02). Additionally these five ABC transporters were studied in sorted AML subpopulations (n=7). In the sorted AML cells (CD34+CD38− versus CD34+CD38+) a more heterogeneous expression pattern was observed as compared to normal CD34+CD38− cells. In general, the expression levels of ABCB1 and ABCC1 in the AML subpopulations were lower than in normal CD34+CD38− cells, ABCB2 expression was higher in the AML fractions and ABCG1 and ABCD4 were expressed similar in AML and normal CD34+CD38− cells. Downregulation of the ABC transporters in the leukemic CD34+CD38+ cells was observed in 50%–60% of the samples, the reverse pattern was observed for the remaining cases, independent of FAB classification. Since ABCG1 plays a prominent role in cholesterol transport and was strongly downregulated in normal CD34+CD38+ cells (ratio 0.27, p<0.001), the mRNA expression of a number of additional cholesterol synthesis genes was investigated. PPARβ, LXRα and HMCGCoA reductase appeared to be downregulated in the CD34+CD38+ cells (ratios of 0.59, p=0.002, 0.32, p<0.001 and 0.59, p= 0.002 respectively). In conclusion, these results indicate that cholesterol synthesis and transport might play an important role in hematopoietic stem cells. Furthermore, a number of ABC transporter genes appeared to be predominantly expressed in hematopoietic stem cells, and are downregulated upon maturation, whereas the reverse pattern is observed in about 40% of the AML patients suggesting that these more committed leukemic cells might have gained some properties of the leukemic stem cells.

High Aldehyde Dehydrogenase Activity (ALDH) Is a General Marker for Normal Hematopoietic Stem Cells but Not Leukemic Stem Cells in Acute Myeloid Leukemia (AML). .

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4035-4035
Author(s):  
Linda Smit ◽  
Lisa A Min ◽  
Monique Terwijn ◽  
Angele Kelder ◽  
Alexander N Snel ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Alkylating Agents ◽  
Therapeutic Window ◽  
Leukemic Stem Cells ◽  
Aldh Activity ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Blast Cells

Abstract Abstract 4035 Poster Board III-971 Only a minority of cells, the leukemic stem cells (LSCs), within AML are responsible for tumor growth and maintenance. Many patients experience a relapse after therapy which is thought to originate from the outgrowth of therapy resistant LSC. Therefore, eradication of the LSCs is likely necessary to cure AML. Both the normal hematopoietic stem cells (HSCs) and LSCs co-exist in the bone marrow (BM) of leukemia patients and therefore success of an anti-stem-cell strategy relies on specific induction of LSC death while sparing the normal HSC. In AML, apart from the CD34+CD38- and the side population (SP) compartment, the high ALDH activity compartment contains the LSCs. The SP and ALDH defined compartments may include both CD34+ and CD34- HSCs and LSCs. ALDH is a detoxifying enzyme responsible for the oxidation of intracellular aldehydes and high ALDH activity results in resistance to alkylating agents such as the active derivatives of cyclophosphamide. Recent data has shown that ALDH is highly expressed in both normal progenitor and stem cells and in AML blast cells. In view of the applicability of LSC specific therapies the detoxification by ALDH might be of importance. Therefore, a difference in ALDH activity between the normal HSC and the malignant LSC might be used to preferentially kill the LSC and spare the HSC. We have previously shown that CD34+CD38- and SP LSCs can be identified and discriminated from HSCs using stem cell-associated cell surface markers, including C-type lectin-like molecules (CLL-1), lineage markers, such as CD7, CD19, and CD56 and recently cell size characteristics as measured by flow cytometry (Terwijn, Blood 111: 487,2008). Here we have analyzed ALDH activity in 23 AML cases. In 7 AML cases, a high SSCloALDHbr cell population was identified (median: 10,9%, range 5,24-15,29%). In 16 cases there were rare (<5%) SSCloALDHbr cells. We have analyzed ALDH activity in aberrant marker defined HSCs and LSCs, both present within the same BM samples in 18 AML patients (summarized in Figure 1). In 9 BM AML samples, defined as CD34-, the CD34+ compartment contained only normal CD34+CD38- HSCs. The ALDH activity in the CD34- cells, which includes by definition in this AML subgroup the LSC, is a factor 4,4 (range 1,7-18,9) lower than in the HSC (Figure 1, panel 1). The ALDHbrSSClo cells present in these CD34- AML cases contained both normal CD34+ and CD34- cells. The activity of the normal HSC within this AML BM is similar to that of the normal HSC in NBM of healthy donors (Figure 1, panel 3). In addition, 9 BM AML patients, defined as CD34+ AML and with both marker negative, normal HSCs and marker positive LSCs present, were analyzed for ALDH activity. We show that the marker positive CD34+CD38- LSCs have 7,7 fold (range 1,73-29,2 fold) lower ALDH activity than the marker negative CD34+CD38- HSCs (Figure 1, panel 2). Altogether, we show that, although malignant AML blast cells have varying ALDH activity, a common feature of all AML cases is that the normal HSCs that co-exist with leukemic (stem) cells in the BM of AML patients have a higher ALDH activity as compared to their malignant counterparts (summarized in figure 1). In conclusion, high ALDH activity is an unique marker of normal HSC within the AML BM (irrespective of AML phenotype, CD34+ or CD34-) at diagnosis. Consequently, AML patients with high ALDH activity in the normal HSC might benefit from treatment with alkylating agents such as cyclophosphamide, whereby the difference between the ALDH activity in LSC and HSC defines the therapeutic window. At present, drugs, known to be dependent on low ALDH for proper activity, are tested for their LSC specific killing while sparing the normal HSC. Additionally, transcriptional profiles are obtained from purified ALDH+ HSC and ALDH- LSC. This will enable us to use this general discriminating property to identify molecules that differ between the LSC and HSC and can function as LSC specific therapeutic targets. Disclosures: No relevant conflicts of interest to declare.

Competition In Engraftment of Normal Hematopoietic Stem Cells and Leukemic Stem Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4836-4836
Author(s):  
Gyeongsin Park ◽  
Michael Heuser ◽  
Tobias Berg ◽  
R. Keith Humphries
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Leukemic Cell ◽  
Fixed Number ◽  
Leukemic Cells ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem

Abstract Abstract 4836 Engraftment is a process including homing to bone marrow, implantation and proliferation. Implantation implies interactions with specialized microenvironments, niches, in which hematopoietic stem cells (HSCs) live and are regulated. Studies have demonstrated the possibility that leukemic stem cells (LSCs) interact with niches in a similar manner to HSCs. We investigated whether HSCs and LSCs compete with each other in their engraftment. We employed a mouse transplantation assay with unmanipulatated bone marrow cells (BMCs) as a source of normal HSCs and LSCs generated by transduction of BMCs with Meningioma 1 (MN1), a potent oncogene causing myeloid leukemia in mice. In irradiated recipients (750 cGy), cotransplantation of leukemic cells (1×105) with various numbers of BMCs (1×105, 1×106 and 1×107) demonstrated that the engraftment level of leukemic cells is influenced by BMCs in a dose dependant manner (5.2%, 41.3% and 82.2% at 2-weeks; 52.3%, 69.5% and 86.9% at 4weeks; mice died before the 5 weeks bleeding, 94.9% and 97.5% at 5weeks, respectively). Cotransplantation of various numbers of leukemic cells (1×104, 1×105 and 1×106) with a fixed number of BMCs (1×106) demonstrated a similar pattern of leukemic engraftment (7.0%, 59.5% and 87.1% at 2weeks; 62.0%, 85.7% at 4 weeks, and mice died before the four week bleeding, respectively). To further elucidate the competition between HSCs and LSCs, we transplanted the cells at different time intervals. Transplantation of normal BMCs (1×106) 2 days prior to transplantation of LSCs (1×105) resulted in much reduced levels of leukemic engraftment compared to that seen in mice simultaneously transplanted (3.5% vs 59.5% at 2 weeks; 73.1% vs 85.76% at 4weeks). This competitive suppression of leukemic engraftment was further enhanced by transplanting larger numbers of normal BMCs (2×107) as little as 12 hours prior LSC transplantation (5×105) compared to simultaneous injection (0% vs 7.26% at 2weeks, 0.9% vs 35.3% at 3 weeks, and 6.0% vs 60.6% at 4 weeks). When BMCs (1×105) or leukemic cells (1×105) were transplanted at equal doses of 1×105 together with normal helper cells (1×106) the leukemic cells expanded 280-fold compared to only 7.3 fold for normal BMCs at 2 weeks (total cell count from two femurs and two tibias per 1×105 transplanted cells). Thus the competitive suppression of leukemic cell growth seen upon sequential transplantation of normal BMCs is not readily explained by enhanced kinetics of normal BMC growth but rather by competition at the level of initial engraftment. In conclusion, our data demonstrate that there is a competition between normal and leukemic cells during the engraftment process, suggesting niche competition of HSCs and LSCs. Disclosures: No relevant conflicts of interest to declare.

Oncogenic Nras Grants Apoptosis Resistance to Hematopoietic Stem Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2451-2451
Author(s):  
Kevin B Yang ◽  
Victor Ng ◽  
Gina Ney ◽  
Lu Liu ◽  
Xi Jin ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Reactive Oxygen ◽  
Clonal Expansion ◽  
Oxygen Species ◽  
Leukemic Stem Cells ◽  
Apoptosis Resistance ◽  
Hematopoietic Stem ◽  
Pkc Signaling

Abstract Genetic lesions hijack the regulatory programs of hematopoietic stem cells and progenitors (HSC/P) to transform them into leukemic stem cells (LSC). These initiating mutations confer a competitive advantage to promote clonal expansion of pre-leukemic stem cells (pre-LSC). Pre-LSCs are believed to serve as a reservoir for leukemia relapses and a cure for leukemia likely depends on the eliminating of pre-LSCs. Better understanding of how mutations dysregulate HSCs to transform them into pre-LSCs will identify new therapeutic targets to eliminate pre-LSCs. Activating RAS mutations are highly prevalent in hematopoietic malignancies. We have previously shown that a single allele of oncogenic NrasG12D promotes clonal expansion in hematopoietic stem cells (HSCs) through increased proliferation and self-renewal, but the role apoptosis plays in the clonal dominance of NrasG12D HSCs remains unclear. Here we report that oncogenic NRasG12D protects HSCs from cellular stress. Upon cytokine starvation and γ-irradiation, HSCs fromMx1-cre; LSL-NrasG12D/+ mice display reduced apoptosis as measured by Annexin V staining and Caspase 3/7 activation. This NRasG12D-mediated HSC survival is not dependent on autophagy since Bafilomycin A, an inhibitor previously shown to inhibit autophagy in HSCs, did not rescue the phenotype. Moreover, NrasG12D HSCs exhibits decreased levels of cellular reactive oxygen species (ROS), and restoration of ROS levels with buthionine sulfoximine significantly blocked the survival of NrasG12D HSCs. We next sought to identify the signaling activated by NRasG12D to promote HSC survival. Although our previous studies show that STAT5 is required for NRasG12D-mediated HSC proliferation, STAT5 is dispensable for NRasG12D-mediated HSC survival. We then determined whether inhibition of the canonical Ras effector pathways blocks NRasG12D-mediated HSC survival. To our surprise, inhibition of PI3K/AKT, MEK/ERK, or mTOR signaling did not abrogate the pro-survival effect of NRasG12D in HSCs. However, inhibition of protein kinase C (PKC) with two structurally independent inhibitors rescued the pro-survival phenotype of NrasG12D/+ HSCs. PKC inhibition also led to increased ROS levels in NrasG12D mutant HSCs, suggesting that NRasG12D reduces ROS and protects HSCs from stress through activation of PKC signaling. Taken together, we discover that in addition to increasing HSC proliferation, oncogenic NRasG12D promotes HSC survival under stress conditions. Furthermore, we reveal a pathway NRasG12D relies on to evade apoptosis and manage reactive oxygen species in HSCs independent of the STAT5 signaling that governs cell proliferation. Targeting PKC signaling, alone or in combination with STAT5 signaling, may have therapeutic benefit in eliminating pre-LSCs. Disclosures No relevant conflicts of interest to declare.

scholarly journals Constitutively active AKT depletes hematopoietic stem cells and induces leukemia in mice

Blood ◽  
2010 ◽  
Vol 115 (7) ◽  
pp. 1406-1415 ◽  
Author(s):  
Michael G. Kharas ◽  
Rachel Okabe ◽  
Jared J. Ganis ◽  
Maricel Gozo ◽  
Tulasi Khandan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Leukemic Stem Cells ◽  
Myeloproliferative Disease ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Akt Activation ◽  
Marrow Cells

Abstract Human cancers, including acute myeloid leukemia (AML), commonly display constitutive phosphoinositide 3-kinase (PI3K) AKT signaling. However, the exact role of AKT activation in leukemia and its effects on hematopoietic stem cells (HSCs) are poorly understood. Several members of the PI3K pathway, phosphatase and tensin homolog (Pten), the forkhead box, subgroup O (FOXO) transcription factors, and TSC1, have demonstrated functions in normal and leukemic stem cells but are rarely mutated in leukemia. We developed an activated allele of AKT1 that models increased signaling in normal and leukemic stem cells. In our murine bone marrow transplantation model using a myristoylated AKT1 (myr-AKT), recipients develop myeloproliferative disease, T-cell lymphoma, or AML. Analysis of the HSCs in myr-AKT mice reveals transient expansion and increased cycling, associated with impaired engraftment. myr-AKT–expressing bone marrow cells are unable to form cobblestones in long-term cocultures. Rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR) rescues cobblestone formation in myr-AKT–expressing bone marrow cells and increases the survival of myr-AKT mice. This study demonstrates that enhanced AKT activation is an important mechanism of transformation in AML and that HSCs are highly sensitive to excess AKT/mTOR signaling.

scholarly journals Selective activation of STAT5 unveils its role in stem cell self-renewal in normal and leukemic hematopoiesis

2005 ◽  
Vol 202 (1) ◽  
pp. 169-179 ◽  
Author(s):  
Yuko Kato ◽  
Atsushi Iwama ◽  
Yuko Tadokoro ◽  
Kazuya Shimoda ◽  
Mayu Minoguchi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Leukemic Stem Cells ◽  
Myeloproliferative Disease ◽  
Self Renewal ◽  
Hematopoietic Stem

Although the concept of a leukemic stem cell system has recently been well accepted, its nature and the underlying molecular mechanisms remain obscure. Constitutive activation of signal transducers and activators of transcription 3 (STAT3) and STAT5 is frequently detected in various hematopoietic tumors. To evaluate their role in normal and leukemic stem cells, we took advantage of constitutively active STAT mutants to activate STAT signaling selectively in hematopoietic stem cells (HSCs). Activation of STAT5 in CD34–c-Kit+Sca-1+ lineage marker– (CD34–KSL) HSCs led to a drastic expansion of multipotential progenitors and promoted HSC self-renewal ex vivo. In sharp contrast, STAT3 was demonstrated to be dispensable for the HSC maintenance in vivo, and its activation facilitated lineage commitment of HSCs in vitro. In a mouse model of myeloproliferative disease (MPD), sustained STAT5 activation in CD34–KSL HSCs but not in CD34+KSL multipotential progenitors induced fatal MPD, indicating that the capacity of STAT5 to promote self-renewal of hematopoietic stem cells is crucial to MPD development. Our findings collectively establish a specific role for STAT5 in self-renewal of normal as well as leukemic stem cells.

scholarly journals Targeting miRNA-551b, a "Stemness"-like microRNA, to Eradicate AML (Stem) Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1494-1494
Author(s):  
Tània Martiáñez ◽  
Noortje Van Gils ◽  
David Christian De Leeuw ◽  
Eline Vermue ◽  
Arjo Rutten ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Research Funding ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Inhibition Of Proliferation ◽  
Novel Target

Abstract Despite high complete remission (CR) rates achieved after chemotherapy, only 30-40% of patients with Acute Myeloid Leukemia (AML) survive five years after diagnosis. The main cause of this treatment failure is insufficient eradication of a subpopulation of chemotherapy-resistant leukemia cells with stem cell properties, named "leukemic stem cells" (LSCs). LSCs use a variety of mechanisms to resist chemotherapy and targeting them is one of the major challenges in AML treatment. Since miRNAs can target multiple genes/pathways simultaneously, their modulation (downregulation or upregulation) may have great potential for the successful elimination of therapy-resistant leukemic (stem) cells (Martiañez Canales et al. Cancers 2017). Here, we show that miRNA-551b, previously identified by us as a stem cell-like miRNA, can be a potential novel target to specifically eradicate AML stem-like cells. Aiming at identification of miRNA-based therapy to specifically eradicate LSCs, while sparing normal Hematopoietic Stem Cells (HSCs), we determined expression of miRNAs in normal HSCs, Leukemic Stem Cells (LSCs) and leukemic progenitors (LP) all derived from the same AML patient's bone marrow. Using this approach, we identified miRNA-551b as being highly expressed in normal HSCs residing both in healthy and AML bone marrows. In AML, high expression of miR551b demonstrated to be associated with an adverse prognosis. Moreover, miRNA-551b was highly expressed in immature AML cases and its expression in a cohort of patients coincided with the expression of stem cell genes (De Leeuw et al. Leukemia 2016). To further elucidate the link between miRNA-551b and AML "stemness" and to test whether downregulation of miRNA-551b affects the survival of AML (stem/progenitor) cells, proliferation and the balance between differentiation and "stemness", we reduced miRNA-551b expression, either by lentiviral transduction of antagomirs or by adding locked nucleotide acid (LNA)-oligonucleotides to AML cell lines and primary AML cells. Downregulation of miRNA-551b in the stem cell-like AML cell line KG1a led to inhibition of cell growth in vitro, which was due to inhibition of proliferation rather than induction of apoptosis. KG1a tumor growth in an in vivo mouse model was also reduced when miRNA-551b was downregulated. In primary AML, miRNA-551b knockdown resulted in a significant decrease in the survival of leukemic progenitors and LSCs, while hematopoietic stem cells (HSCs) and normal progenitors from healthy bone marrows were not affected. These results suggest that a therapeutic approach inhibiting miRNA-551b expression might specifically eradicate leukemic progenitors and LSCs from primary AML, while sparing HSCs. We are currently studying miRNA-551b targets which can be responsible for this specific LSCs elimination. In conclusion, our results suggest that inhibition of miRNA-551b could be a promising approach to eliminate stem cell-like AML cells, thereby decreasing relapse rates and improving AML treatment outcome. Disclosures Ossenkoppele: Pfizer: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Genentech: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Research Funding; Roche: Consultancy, Honoraria; Celgene: Honoraria, Research Funding; Johnson & Johnson: Consultancy, Honoraria, Research Funding; Genmab: Research Funding.

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