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

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.

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A New Flowcytometry-Based Method to Discriminate Malignant From Normal Stem Cells in CML.

Blood ◽

10.1182/blood.v114.22.3245.3245 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3245-3245

Author(s):

Jeroen J.W.M. Janssen ◽

Wendy Deenik ◽

Karlijn G.M. Smolders ◽

Monique Terwijn ◽

Angele Kelder ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Risk Scores ◽

Fish Analysis ◽

Leukemic Stem Cells ◽

Aberrant Expression ◽

Hematopoietic Stem ◽

Culture Techniques

Abstract Abstract 3245 Poster Board III-182 Tyrosine kinase inhibitor (TKI) insensitivity of CML hematopoietic stem cells prevents eradication of the disease by these drugs and is presumably implicated in development of TKI resistance. Probably, improvement of treatment results will involve leukemic stem cell directed therapy. Therefore, more knowledge of stem cell specific targets would be instrumental. Previously, leukemic stem cells could only be identified indirectly by using culture techniques. We developed a new flowcytometric approach that enables to directly distinguish CML stem cells from their normal counterparts within single patient samples. In 24 newly diagnosed CML patients CML CD34+CD38- stem cells could be discriminated from normal stem cells by higher CD34 and CD45 expression and different forward/sideward light scatter properties, reflecting differences in size and granularity. In addition, aberrant expression of CD7, CD11b and CD56 was demonstrated on malignant stem cells, allowing clear discrimination from benign stem cells, that were always negative for these markers. Above all, in all tested CML patients we were able to demonstrate that high CD90 expression is a specific feature of CML stem cells, while CD90 expression is low on their normal counterparts. FISH analysis on FACS sorted cells proved that populations were BCR-ABL positive (in case of high CD34 and CD45 expression and high CD90 expression) or negative (in case of low CD34 and CD45 expression and low CD90 expression), while long term liquid culture assays with subsequent CFU assays and FISH analysis proved their malignant/normal stem cell character. Patients with a large proportion of non-leukemic stem cells had significantly lower clinical risk scores (Sokal, Euro) than patients with few remaining normal stem cells. This new technique will expand our possibilities to identify new CML stem cell specific targets and may improve efficacy assessment of CML treatment as well. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v116.21.4836.4836 ◽

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.

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Early and Late Leukemic Stem Cells in Murine Leukemia Models

Blood ◽

10.1182/blood.v128.22.1544.1544 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1544-1544

Author(s):

Fang Dong ◽

Haitao Bai ◽

Shanshan Zhang ◽

Xiaofang Wang ◽

Jinhong Wang ◽

...

Keyword(s):

Stem Cells ◽

Progenitor Cells ◽

Conflicts Of Interest ◽

Gene Mutations ◽

Leukemic Cells ◽

Mouse Bone Marrow ◽

Leukemic Stem Cells ◽

Hematopoietic Stem ◽

Murine Leukemia

Abstract Acute leukemia is uncontrolled proliferation of leukemic stem cells (LSCs). Murine models of leukemia suggest that LSCs arise from lineage-committed progenitor cells. However, whether LSCs also directly arise from long-term (LT) and short-term (ST) hematopoietic stem cells (HSCs) and other hematopoietic progenitor cells (HPCs) and whether differentiation potentials influence the leukemia types are poorly understood. In this study, we used two murine leukemia models (AML with MLL-AF9 fusion protein and T-ALL with Notch-1 intracellular domain, ICN-1). Two HSC and three HPC populations were sorted from B6 (CD45.1) mouse bone marrow (BM) by flow cytometry: HSC1, CD150+CD41-CD34-Lin-Sca-1+c-Kit+ (LSK) cells; HSC2, CD150-CD41-CD34-LSK cells; HPC1, CD150+CD41+CD34-LSK cells; HPC2, CD150+CD41+CD34+LSK cells; HPC3, CD150-CD41-CD34+LSK cells. HSC1, HSC2, HPC1, and HPC3 are enriched in LT-HSCs, ST-HSCs, repopulating common myeloid progenitors, and lymphoid-primed multipotent progenitors, respectively. 400-600 cells were sorted from each population, pre-stimulated for 24 hrs, and transduced with MLL-AF9 or ICN-1 retrovirus for another 24 hrs. Cells were mixed with 3-5 x 105 BM cells (CD45.2), and injected into the lethally irradiated mice (CD45.2). The recipient mice were monitored by detection of GFP+ cells in the peripheral blood (PB). When the recipient mice showed > 50% GFP+ cells in the PB or became ill, mice were killed and analyzed for leukemia. In the MLL-AF9 AML model, leukemia developed in all recipient mice injected with HSC1, 2, HPC1, 2, or 3 around 6 weeks after transplantation. Leukemic cells in PB and BM appeared positive for Mac-1/Gr-1, but negative for CD3 and B220. We detected a new LSC population in the BM: Lin-Mac-1+c-Kit+Sca-1+CD150-CD16/32+ cells. Both CD34-negative and CD34-positive cells were detected in this population. We named this population as "early LSCs" because it was phenotypically similar to ST-HSCs and/or LMPPs in normal mouse BM cells except Mac-1 expression. Early LSCs differed from previously identified LSCs (L-GMP) because they are Sca-1-positive. We now consider L-GMP as one of "late LSC" types. Early LSCs formed leukemic colonies in vitro and initiated the AML in serial transplantation. These results show the similar AML develops regardless of HSC and HPC types transduced. Both early and late LSCs likely play a role in establishment of AML. In the ICN-1 T-ALL model, leukemia developed in all recipient mice injected with HSC1, 2, HPC1, 2, or 3 around 4 weeks after transplantation. Most leukemic cells in PB and BM appeared positive for CD4/CD8, but negative for Mac-1/Gr-1 and B220. We did not detect early LSCs in BM. To identify late LSCs in BM, we injected the CD3+CD8+CD4- and CD3+CD8+CD4+ cells into the sub-lethally irradiated mice. T-ALL developed from both populations (median latency, 23 vs 35 days). These results show the similar T-ALL develops regardless of HSC and HPC types transduced. In this case, late LSCs can be directly generated without early LSCs from HSC or HPC populations. Taken together, we found that the similar types of leukemia develop regardless of different types of initiating cells in both models. We also found new LSC populations, early LSCs in the AML model and late LSCs in the T-ALL model. Both early and late LSCs were able to re-initiate leukemia after secondary transplantation. In conclusion, highly purified murine HSCs and HPCs were used for the first time to initiate leukemia. Both MLL-AF9 AML and ICN-1 T-ALL models suggest that gene mutations at all differentiation stages from HSCs to HPCs potentially induce the similar types of leukemia. Of note is that distinct lineage differentiation potentials of HSCs and HPCs do not affect leukemia types. More importantly, early LSCs may serve as the earliest event in leukemogenesis in AML. In the case of ALL, gene mutations seem carried over until reaching the developmental stage of late LSCs. Our results suggest that both early and late LSCs should be eradicated to achieve complete remission and prevent relapse. Disclosures No relevant conflicts of interest to declare.

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JMJD1c, a Histone Demethylase, Is Required for the Maintenance of MLL-AF9 AML Leukemic Stem Cells

Blood ◽

10.1182/blood.v124.21.3530.3530 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3530-3530 ◽

Cited By ~ 1

Author(s):

Halina Leung ◽

Jianlong Wang ◽

Jenny Yingzi Wang

Keyword(s):

Stem Cells ◽

Hox Genes ◽

Conflicts Of Interest ◽

Therapeutic Potential ◽

Mrna Level ◽

Histone Demethylase ◽

Leukemic Cells ◽

Leukemic Stem Cells ◽

Hematopoietic Stem

Abstract While most leukemic cells are initially sensitive to chemo- and radiotherapy, leukemic stem cells (LSC) persist and are therefore considered to be the basis for disease relapse in acute myeloid leukemia (AML) (Nat Biotechnol, 25: 1315-1321, 2007). The discovery of epigenetic mutations being the cause of a number of cancers has increased interest in their therapeutic potential (Nat Rev Genet, 7:21-33, 2006; Cell, 128: 683-692, 2007). Although the clinical importance of epigenetic dysregulation has been recognised in various cancers, including MLL-rearranged AML, this topic is currently a novel area of study. Our microarray analysis comparing genes differentially expressed between LSC and normal hematopoietic stem cells (HSC) identified JMJD1c, a histone demethylase, as a potential LSC-specific target. An analysis of a comprehensive patient outcome database showed that high levels of JMJD1c were associated with significantly poorer survival in patients with AML (P = 0.0029), making it clinically relevant and emphasising the therapeutic potential of JMJD1c. Furthermore, the JMJD1c mRNA level was significantly higher in MLL-rearranged than in non-MLL AML patient xenograft samples. Western blot and quantitative PCR confirmed the microarray results, where JMJD1c was significantly upregulated in MLL-AF9 LSC compared to Hoxa9/Meis1a derived LSC and HSC. Overexpression of JMJD1c in Hoxa9/Meis1a pre-LSC significantly increased the clonogenic activity and proliferation compared to control cells. Subsequent microarray experiment identified Hox genes as downstream targets of this epigenetic regulator (fold change > 2, P < 0.05), suggesting the regulatory role of JMJD1c in the self-renewal pathway. Consistent with in vitro observations, there was a significant increase in disease progression as shown by leukemia onset in mice with Hoxa9/Meisa1 pre-LSC transduced with JMJD1c cDNA compared to control (P < 0.0001; at least 12 mice per cohort). Stable knockdown of JMJD1c in MLL-AF9 cells using shRNA significantly impaired the proliferation, clonogenic activity and induced cell differentiation in vitro. In vivo survival studies in mice showed that JMJD1c knockdown severely impaired the maintenance of MLL-AF9 induced leukemia (P < 0.0001; 12 mice per cohort). In conclusion, our studies have identified JMJD1c as an important driver in leukemia maintenance and strongly suggest that JMJD1c possesses an oncogenic role in MLL-AF9 leukemogenesis. Targeting this oncogene may enable us to directly target LSC as a strategy for overcoming drug resistance in AML patients. Disclosures No relevant conflicts of interest to declare.

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Targeting BCR-ABL Kinase Activity-Independent Signaling Pathways and Leukemia Stem Cells Is Essential for Curative Therapy of Philadelphia Chromosome Positive (Ph+) Leukemia.

Blood ◽

10.1182/blood.v106.11.1990.1990 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1990-1990 ◽

Cited By ~ 2

Author(s):

Shaoguang Li ◽

Yiguo Hu ◽

Sarah Swerdlow ◽

Theodore M. Duffy ◽

Roberto Weinmann ◽

...

Keyword(s):

Stem Cells ◽

Signaling Pathways ◽

Kinase Activity ◽

Blast Crisis ◽

Philadelphia Chromosome ◽

Leukemic Cells ◽

Src Kinases ◽

Leukemic Stem Cells ◽

Abl Kinase ◽

Philadelphia Chromosome Positive

Abstract Therapeutic efforts for Philadelphia chromosome positive (Ph+) leukemia have focused on targeting mainly BCR-ABL kinase activity with kinase inhibitors, since it has generally been believed that shutting down BCR-ABL kinase activity will completely inhibit its functions, leading to inactivation of downstream signaling pathways. Inhibition of BCR-ABL kinase activity by imatinib mesylate (Gleevec) is highly effective in treating human Ph+ chronic myeloid leukemia (CML) in chronic phase, but not Ph+ B-cell acute lymphoblastic leukemia (B-ALL) and CML blast crisis. The reasons for this are not well understood, but the fact that imatinib is a strong inhibitor of BCR-ABL kinase activity suggests that BCR-ABL kinase activity-independent pathways also play a critical role in the development of both forms of Ph+ leukemia. We have previously shown that the SRC family kinases LYN, HCK, and FGR are activated by BCR-ABL in pre-B leukemic cells and are required for the development of B-ALL (Hu et al, Nat Genet36:453, 2004). Others have shown that cells from imatinib-resistant patients imatinib expressed an activated form of LYN (Donato et al, Blood101:690, 2003), and that a BCR-ABL mutant with no kinase activity was still able to activate HCK (Warmuth et al, J Biol Chem272:33260, 1997). Based on these observations, we hypothesized that inhibition of BCR-ABL kinase by imatinib might not inactivate SRC kinases activated by BCR-ABL in pre-B leukemic cells, which may explain the relatively poor activity of imatinib against Ph+ B-ALL and lymphoid blast crisis CML. We find that SRC kinases activated by BCR-ABL remain fully active in imatinib-treated mouse leukemic cells and this BCR-ABL kinase activity-independent pathway is essential for leukemic cell survival and proliferation. Blockade of this pathway also prevents CML transition to lymphoid blast crisis. In mice with B-ALL, inhibition solely of BCR-ABL kinase activity by imatinib is not curative, but inhibition of both SRC and BCR-ABL kinase activities by the novel, oral, multi-targeted kinase inhibitor dasatinib (BMS-354825), while not killing leukemic stem cells, affords complete remission, maintained as long as treatment is continued. In these mice, we identified the B-ALL leukemic stem cells as B220+CD43+ pro-B cells. CML mice treated with dasatinib lived significantly longer than those treated with imatinib, which correlated with significantly lower numbers of BCR-ABL-expressing leukemic cells in bone marrow, peripheral blood, and spleens of dasatinib-treated CML mice versus placebo- or imatinib-treated mice. However, neither dasatinib nor imatinib were curative in these mice, which was attributed to an inability of both drugs to completely kill Lin-c-kit+CD34-Hoe- CML stem cells. Our studies indicate that complete eradication of leukemic cells in B-ALL and CML mice requires not only targeting BCR-ABL kinase activity-dependent and SRC-dependent pathways, but also killing BCR-ABL-expressing stem cells insensitive to both imatinib and dasatinib. However, the rapid and striking hematologic response of B-ALL mice to dasatinib suggests that the pro-B progenitors with acquired self-renewal capacity are the major source of highly proliferating B-lymphoid leukemic cells in B-ALL mice, and that complete inhibition of growth of this leukemic population with dasatinib could achieve long-term survival in B-ALL.

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Disruption of the Peripheral Lymphoid Niche Contributes to Lymphopenia in CML Patients Undergoing Imatinib Treatments

Blood ◽

10.1182/blood.v126.23.5165.5165 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5165-5165

Author(s):

Stephanie Thiant ◽

Moutuaata M.Moutuou ◽

Philippe Laflamme ◽

Radia Sidi Boumedine ◽

Fanny Larochelle ◽

...

Keyword(s):

Stem Cells ◽

Imatinib Mesylate ◽

Median Time ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Philadelphia Chromosome ◽

Chronic Phase ◽

Myelogenous Leukemia ◽

Hematopoietic Stem ◽

Gm Csf

Abstract PURPOSE: Chronic myelogenous leukemia (CML) is a disorder affecting early hematopoietic stem cells (HSC) and is characterized by excessive proliferation and accumulation of myeloid progenitors and progeny in the periphery. During the chronic phase of the disease, CML patients are normally at low risk of developing infections but such complications tend to rise during the progression of the disease. Gleevec (imatinib mesylate) is currently administered as first line therapy for patients with Philadelphia chromosome-positive CML. Despite the relative high specificity of tyrosine kinase inhibitor (TKI) treatment towards the BCR-ABL fusion protein, off-target multikinase inhibitory effects occur and can interfere with normal hematopoiesis. This study was conducted to evaluate how myeloid and lymphoid immune homeostasis are affected by Imatinib mesylate. METHODS: Healthy volunteer donors (n=25) and CML patients were recruited during their first visit at our CML clinic. Seven CML patients were treated with Imatinib (400mg). The median time of Gleevec treatment was 2.9 years (range: 0.5-10.9). The median time of remission post TKI was 1.1 years (range: 0.3-3). Phenotypic analysis of dendritic cell (DCs) subsets: plasmacytoid (pDCs) and myeloid type 1, 2 and 3 (mDC1, mDC2, mDC3) were evaluated by flow cytometry. Percentage and absolute numbers of naive and memory CD4+ and CD8+ T cells, NK cells and B cells were also evaluated. DCs were differentiated from purified CD34+ cells culturedwith GM-CSF (800 U/ml) or Flt3-L (50ng/ml), IL-4 (10 U/ml) and TNFa (50 U/ml), in the presence of varying concentrations of Imatinib mesylate (0 to 5µM/mL). TCR and IL-7 signaling were evaluated based on ERK-phosphorylation (-p) and STAT5-p after incubation with 3µM of Imatinib. RESULTS AND CONCLUSION: At diagnosis, several CML patients have a deficit in DCs resulting from a severe skewing affecting BM progenitor cells. After initiating Gleevec therapy, normalization of stem cell progenitors occurs but DC counts remain well below normal levels in all CML patients. We demonstrated a direct and dose dependent interference of Imatinib on GM-CSF and Flt3-L pathways for DC differentiation from CD34+ stem cells. For T lymphocytes, Imatinib interfered with TCR and IL-7 signaling through the inhibition of ERK and STAT5 phosphorylation respectively. The failure to maintain adequate numbers of DCs combined to diminished homeostatic response to cytokines and TCR stimuli explains T cell lymphopenia in these patients. Such immune dysfunction is at least in part responsible for infectious complications that are often increased in patients treated with Imatinib. Disclosures No relevant conflicts of interest to declare.

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The quiescent fraction of chronic myeloid leukemic stem cells depends on BMPR1B, Stat3 and BMP4-niche signals to persist in patients in remission

Haematologica ◽

10.3324/haematol.2019.232793 ◽

2020 ◽

Vol 106 (1) ◽

pp. 111-122 ◽

Cited By ~ 4

Author(s):

Sandrine Jeanpierre ◽

Kawtar Arizkane ◽

Supat Thongjuea ◽

Elodie Grockowiak ◽

Kevin Geistlich ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Tyrosine Kinase ◽

Stromal Cells ◽

Kinase Inhibitor ◽

Bmp Signaling ◽

Myelogenous Leukemia ◽

Leukemic Stem Cells ◽

Hematopoietic Stem

Chronic myelogenous leukemia arises from the transformation of hematopoietic stem cells by the BCR-ABL oncogene. Though transformed cells are predominantly BCR-ABL-dependent and sensitive to tyrosine kinase inhibitor treatment, some BMPR1B+ leukemic stem cells are treatment-insensitive and rely, among others, on the bone morphogenetic protein (BMP) pathway for their survival via a BMP4 autocrine loop. Here, we further studied the involvement of BMP signaling in favoring residual leukemic stem cell persistence in the bone marrow of patients having achieved remission under treatment. We demonstrate by single-cell RNA-Seq analysis that a sub-fraction of surviving BMPR1B+ leukemic stem cells are co-enriched in BMP signaling, quiescence and stem cell signatures, without modulation of the canonical BMP target genes, but enrichment in actors of the Jak2/Stat3 signaling pathway. Indeed, based on a new model of persisting CD34+CD38- leukemic stem cells, we show that BMPR1B+ cells display co-activated Smad1/5/8 and Stat3 pathways. Interestingly, we reveal that only the BMPR1B+ cells adhering to stromal cells display a quiescent status. Surprisingly, this quiescence is induced by treatment, while non-adherent BMPR1B+ cells treated with tyrosine kinase inhibitors continued to proliferate. The subsequent targeting of BMPR1B and Jak2 pathways decreased quiescent leukemic stem cells by promoting their cell cycle re-entry and differentiation. Moreover, while Jak2-inhibitors alone increased BMP4 production by mesenchymal cells, the addition of the newly described BMPR1B inhibitor (E6201) impaired BMP4-mediated production by stromal cells. Altogether, our data demonstrate that targeting both BMPR1B and Jak2/Stat3 efficiently impacts persisting and dormant leukemic stem cells hidden in their bone marrow microenvironment.

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MiR-150 Suppresses MLL-AF9 Associated Leukemia Through Simultaneously Targeting Multiple Oncogenes,

Blood ◽

10.1182/blood.v118.21.3461.3461 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3461-3461

Author(s):

Beiyan Zhou

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Target Genes ◽

Conflicts Of Interest ◽

Expression Patterns ◽

Gene Profiling ◽

Fusion Genes ◽

Leukemic Stem Cells ◽

Hematopoietic Stem ◽

Mll Gene

Abstract Abstract 3461 The mixed lineage leukemia (MLL) gene codes for an evolutionarily conserved histone methyltransferase that is crucial for early hematopoiesis. As a result of a chromosomal translocation involving locus 11q23 results in formation of chimeras composed of the 5' part of the MLL gene fused with more than 60 partner genes lead to disruption of normal function of MLL as a histone methytransferase and acquisition of transcriptional properties conferred by the partner genes. MLL fusion genes (MLL-FG) are often the causal mutations for aggressive acute myeloid and lymphoid leukemias (AML and ALL) that correlated with poor prognosis. In order to treat or even eliminate MLL-associated leukemias, extensive studies on the regulatory mechanism underlying MLL associated transformation and progression have been carried out. Leukemic stem cells (LSC) can derive from either hematopoietic stem or progenitor cells with the recruitment of MLL-fusion genes (MLL-FG) and wild type MLL protein. We report that miR-150, a key hematopoietic regulatory microRNA (miRNA) and one of the most downregulated miRNAs in MLL-associated leukemias, acts as a tumor suppressor to block the leukemogenic potency of leukemic stem cells. When expression of miR-150 was restored, a significantly suppressed leukemic stem cell potency of MLL-AF9 cells was observed both in vivo and in vitro. Gene profiling analysis demonstrated that elevated miR-150 altered various aspects of gene expression patterns in MLL-AF9 cells, including stem cell signatures, cancer pathways, and cell survival. By screening more than 30 predicted target genes, we identified multiple leukemia-associated oncogenes as bona fide miR-150 targets, and knockdown of these genes by shRNAs recapitulated the tumor suppressive effects observed after ectopically expression of miR-150 in MLL-AF9 cells. Disclosures: No relevant conflicts of interest to declare.

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Hypoxia-Adapted Myeloma Cells Possess Stem Cell Character

Blood ◽

10.1182/blood.v122.21.3088.3088 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3088-3088

Author(s):

Eishi Ashihara ◽

Yoko Nakagawa ◽

Hisayuki Yao ◽

Asumi Yokota ◽

Yasuo Miura ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Conflicts Of Interest ◽

Flow Cytometric Analysis ◽

Scid Mice ◽

Myelogenous Leukemia ◽

Leukemic Cells ◽

Hematopoietic Stem ◽

Hypoxic Conditions ◽

Arterial Blood

Abstract The prognosis of patients with multiple myeloma (MM) has been improved by the emergence of new molecular targeting agents including proteasome inhibitors and immunomodurating agents. Nevertheless, MM remains incurable at present because it is likely that MM stem cells are resistant to these targeting agents. Thus, it is important to further investigate the biology of MM stem cells to cure the MM patients. We have demonstrated that b-catenin is a novel and attractive target against MM (Ashihara et al. Clin Cancer Res, 2009; Yao et al. Blood Cancer J, 2011). We next investigate novel targets focused on the hypoxic bone marrow (BM) environment. BM is known to have low levels of oxygen, particularly at the epiphysis, which is distant form the BM arterial blood supply. Normal hematopoietic stem cells (HSCs) reside in this hypoxic epiphyseal region “niche”, and HSCs are protected from DNA damage induced by reactive oxygen species. We have previously found that chronic myelogenous leukemia (CML) cells engrafted in the BM survived and proliferated in the severely hypoxic environment and that these hypoxia-adapted (HA) leukemic cells acquired stem cell-like characters (Takeuchi et al. Cell Death Differ, 2010). In this study, we investigated the characteristics of hypoxia-adapted MM (HA-MM) cells. We first confirmed oxygen status in the BM of the MM cell-engrafted mice. Irradiated NOD/SCID mice were inoculated with 2 x 106 AMO-1 cells. After 2 or 4 weeks transplantation, we sacrificed mice and confirmed engraftment. The inoculated MM cells engrafted in the epiphysis in recipient mice after 2 weeks transplantation, and populated endosteum of epiphysis after 4 weeks. These MM cells were positive for pimonidazole, which specifically accumulated in hypoxic cells (< 1.3% O2 concentration). These observations suggested that MM cells resided in the BM are hypoxic. We then established AMO-1, OPM-2, and IM-9 HA-MM cells cultured under hypoxic conditions (O2 1%). These HA cells can continue to proliferate in hypoxic conditions for more than six months. In flow cytometric analysis, the G0 fraction cells as well as side population fraction cells significantly increased in HA-MM cells compared with those in the parental MM cells. We next transplanted parental or HA-MM AMO-1 cells with same cell numbers into irradiated NOD/SCID mice. The survival durations of mice transplanted with HA-AMO-1 cells were significantly shorter than that of mice transplanted with parental cells. Moreover, in serial transplantation experiments, all 5 HA-MM cell-transplanted mice died of MM whereas 1 out of 5 parental MM cell-transplanted mice (Figure 1). Quantitative RT-PCR analysis demonstrated that Sox2, Oct3, and Nanog mRNA transcripts increased in the HA-MM AMO-1 cells (Figure 2). We next investigated the signaling pathway activated in HA-MM cells. Interestingly, phosphorylated Smad2 expression was increased in HA-AMO-1 cells. These findings suggest that HA-MM cells possess stem cell-like character, and these cells may provide a useful model to investigate the mechanism of MM stem cells (myeloma-initiating cells) resistant to molecular target agents. Disclosures: No relevant conflicts of interest to declare.

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MiR-150 Inhibits MLL-AF9 Associated Leukemia By Suppressing Leukemic Stem Cells

Blood ◽

10.1182/blood.v122.21.3764.3764 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3764-3764

Author(s):

Patali S Cheruku ◽

Marina Bousquet ◽

Guoqing Zhang ◽

Guangtao Ge ◽

Wei Ying ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Progenitor Cells ◽

Colony Formation ◽

Conflicts Of Interest ◽

Expression Patterns ◽

Gene Profiling ◽

Leukemic Stem Cells ◽

Hematopoietic Stem ◽

Signature Genes

Abstract Leukemic stem cells (LSCs) are derived from hematopoietic stem or progenitor cells and often share gene expression patterns and specific pathways. Characterization and mechanistic studies of LSCs are critical as they are responsible for the initiation and potential relapse of leukemias, however the overall framework, including epigenetic regulation, is not yet clear. We previously identified microRNA-150 (miR-150) as a critical regulator of mixed lineage leukemia (MLL) -associated leukemias by targeting oncogenes. Our additional results suggest that miR-150 can inhibit LSC survival and disease initiating capacity by suppressing more than 30% of “stem cell signature genes,” hence altering multiple cancer pathways and/or stem cell identities. MLL-AF9 cells derived from miR-150 deficient hematopoietic stem/progenitor cells displayed significant proliferating advantage and enhanced leukemic colony formation. Whereas, with ectopic miR-150 expression, the MLL-AF9 associated LSC population (defined as Lin-ckit+sca1- cells) was significantly decreased in culture. This is further confirmed by decreased blast leukemic colony formation in vitro. Furthermore, restoration of miR-150 levels in transformed MLL-AF9 cells, which often display loss of miR-150 expression in AML patients with MLL-fusion protein expressing, completely blocked the myeloid leukemia development in a transplantation mouse model. Gene profiling analysis demonstrated that an increased level of miR-150 expression down regulates 30 of 114 stem cell signature genes by more than 1.5 fold, partially mediated by the suppressive effects of miR-150 on CBL, c-Myb and Egr2 oncogenes. In conclusion, our results suggest that miR-150 is a potent MLL-AF9 leukemic inhibitor that may act by suppressing the survival and leukemic initiating potency of MLL-AF9 LSCs. Disclosures: No relevant conflicts of interest to declare.

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