TIM-3, a Leukemia Stem Cell Marker, Plays a Role In Leukemic Transformation Through Autocrine Stimulatory Signaling By Its Ligand, Galectin-9

Abstract Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), an ultimate therapeutic target for AML. We have reported that the T-cell immunoglobulin mucin-3 (TIM-3) is expressed on LSCs in most types of AML but not on normal hematopoietic stem cells (HSCs) (Kikushige et al, Cell Stem Cell, 2010). We extended the analysis of TIM-3 expression into various types of human hematological malignancies, and found that human TIM-3 is expressed in the vast majority of CD34+CD38- LSCs of human myeloid malignancies including chronic myeloid leukemia, chronic myelomonocytic leukemia and myelodysplastic syndromes (MDS). Although CD34+CD38- normal bone marrow stem cells do not express TIM-3, TIM-3 is expressed in the CD34+CD38- population in MDS, and is further up-regulated with progression into leukemia. The average percentages of TIM-3+ cells in the CD34+CD38- population was 7.8% in RCMD (n=10), 19.2% in RAEB-1 (n=10), 84.0% in RAEB-2 (n=10) and 92.2% in overt AML (n=10). The close association of TIM-3 expression with transformation into AML led us to hypothesize that TIM-3 itself has a function in AML stem cell development. TIM-3 is a type 1 cell-surface glycoprotein and has a structure that includes an N-terminal immunoglobulin variable domain followed by a mucin domain, a transmembrane domain and a cytoplasmic tail. Tyrosine residues are clustered in the cytoplasmic tail, suggesting that TIM-3 can induce signal transduction in TIM-3+ AML cells. To understand the function of TIM-3, we investigated the interaction between TIM-3 and its ligand galectin-9 in AML LSCs. We found that AML patients showed significantly higher serum galectin-9 concentration than healthy individuals (healthy controls: 18.3+4.3 pg/ml, AML patients: 139.1+33.4 pg/ml, P<0.05). Unexpectedly, we found that leukemic cells expressed a high level of galectin-9 protein, as compared to other hematopoietic cells including T cells, B cells and monocytes. Using KASUMI-3 (TIM-3+ AML cell line) and primary AML samples, we confirmed that AML cells could secrete galectin-9 after TLR stimulation in vitro. Furthermore, microarray analysis demonstrated that TIM-3 stimulation by the physiological concentration of galectin-9 induced significant gene expression changes toward pro-survival axis including up-regulation of MCL-1, the important survival factor for HSCs and LSCs. These results collectively suggest that AML cells can produce and secrete galectin-9, and galectin-9 can bind and stimulate TIM-3-expressing AML cells including LSCs in an autocrine manner to support their survival or leukemia progression. Disclosures: Miyamoto: Kyushu University Hospital: Employment.

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Leukemogenic Function of TIM-3, a Leukemia Stem Cell Marker, in Acute Myelogenous Leukemia and Myelodysplastic Syndromes.

Blood ◽

10.1182/blood.v120.21.2983.2983 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2983-2983 ◽

Cited By ~ 2

Author(s):

Yoshikane Kikushige ◽

Takahiro Shima ◽

Junichiro Yuda ◽

Toshihiro Miyamoto ◽

Koichi Akashi

Keyword(s):

Signal Transduction ◽

Stem Cells ◽

Stem Cell ◽

Myelodysplastic Syndromes ◽

Myeloid Leukemia ◽

Cytoplasmic Tail ◽

Myelogenous Leukemia ◽

Stem Cell Marker ◽

Cell Surface Glycoprotein ◽

Hematopoietic Stem

Abstract Abstract 2983 Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), an ultimate therapeutic target for AML. We have reported that the T-cell immunoglobulin mucin-3 (TIM-3) is expressed on LSCs in most types of AML but not on normal hematopoietic stem cells (HSCs). TIM-3+ AML cells reconstituted human AML in immunodeficient mice, whereas TIM3− AML cells did not, suggesting that the TIM-3+ population contains all functional LSCs. We established an anti-human TIM-3 mouse IgG2a antibody having complement-dependent and antibody-dependent cellular cytotoxic activities. This antibody did not harm reconstitution of normal human HSCs, but blocked engraftment of AML after xenotransplantation. Furthermore, when it is administered into mice grafted with human AML, this treatment dramatically diminished their leukemic burden, and eliminated LSCs capable of reconstituting human AML in secondary recipients (Kikushige et al, Cell Stem Cell, 2010).We extended the analysis of TIM-3 expression into various types of human hematological malignancies, and found that human TIM-3 is expressed in the vast majority of CD34+CD38− LSCs of human myeloid malignancies including chronic myeloid leukemia, chronic myelomonocytic leukemia and myelodysplastic syndromes (MDS). Although TIM-3 was not expressed in CD34+CD38− stem cell fraction in normal bone marrow cells, TIM-3 was progressively up-regulated in this population of MDS, along with disease progression into leukemia: The average percentages of TIM-3+ cells in the CD34+CD38− population was 7.8% in RCMD (n=10), 19.2% in RAEB-1 (n=10), 84.0% in RAEB-2 (n=10) and 92.2% in overt AML (n=10). Thus, TIM-3 might be useful to isolate malignant stem cells responsible for progression into AML in MDS patients. The close association of TIM-3 expression with transformation into AML led us to hypothesize that TIM-3 itself has a function in AML stem cell development. TIM-3 is type 1 cell-surface glycoprotein and has a structure that includes an N-terminal immunoglobulin variable domain followed by a mucin domain, a transmembrane domain and a cytoplasmic tail. Tyrosine residues are clustered in the cytoplasmic tail, suggesting that TIM-3 can induce signal transduction in TIM-3+ AML cells. Previous reports have shown that galectin-9 and HMGB-1 are the ligand of TIM-3 in lymphocytes and dendritic cells. TIM-3 is reported to signal differently in lymphocytes and myeloid cells, because TIM-3 ligation results in different patterns of tyrosine phosphorylation in these cell types, suggesting that TIM-3 has lineage- or cellular context-dependent signal transduction pathways or functions. Therefore, we considered that it should be critical to identify the function of TIM-3 in primary AML cells. We cultured TIM-3+ AML cells in the presence or absence of galectin-9 or HMGB-1, and performed cDNA microarray analysis to find genes activated in response to TIM-3 ligation. Interestingly, pro-apoptotic genes such as BAX and SIVA were significantly down-regulated in the presence of galectin-9 or HMGB-1, suggesting that TIM-3 signaling could promote survival of TIM-3-expressing LSCs. These data suggest that TIM-3 is a surface marker useful to track malignant LSCs in progression from MDS to AML, and TIM-3 may function for maintenance of LSC through inducing survival-promoting signaling. Disclosures: No relevant conflicts of interest to declare.

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CD52 Expression In Leukemic Stem/Progenitor Cells

Blood ◽

10.1182/blood.v116.21.2743.2743 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2743-2743 ◽

Cited By ~ 3

Author(s):

Vivian G. Oehler ◽

Roland B. Walter ◽

Carrie Cummings ◽

Olga Sala-Torra ◽

Derek L. Stirewalt ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Progenitor Cells ◽

Cell Lines ◽

Myeloid Leukemia ◽

Research Funding ◽

Lymphoblastic Leukemia ◽

Cell Populations ◽

Cell Surface Glycoprotein ◽

Hematopoietic Stem

Abstract Abstract 2743 CD52 is a cell surface glycoprotein of unknown function that is expressed in B and T lymphocytes, macrophages, and monocytes, but is not expressed in normal hematopoietic stem/progenitor cells. CD52 is also expressed in chronic lymphocytic leukemia (CLL), B-cell acute lymphoblastic leukemia (ALL), and some cases of T-ALL. Alemtuzumab, a recombinant humanized monoclonal antibody, targets CD52 and is used to treat CLL. In contrast to normal hematopoietic stem/progenitor cells, CD52 expression has been described in acute myeloid leukemia (AML) and in blast crisis (BC) chronic myeloid leukemia (CML). Based on these observations we were curious whether CD52 expression distinguished normal from malignant or more mature from immature stem/progenitors cells, and whether these cells were sensitive to alemtuzumab. CD52 expression was examined in three blast cell populations (CD34+/CD38-, CD34+/CD38+, and CD34-) in patients with myeloid (44) and lymphoid (18) neoplasms, and normal patients (6). In normal hematopoietic cells, stems cells are enriched in the first population; more mature cells are characterized by increasing CD38 expression and loss of CD34 expression. In AML and CML leukemia stem cells may arise within either CD34+ population and possibly in the CD34- population. Relative to normal lymphocytes average CD52 expression could be characterized as low to moderate. Using an expression cutoff of > 20%, in contrast to normal patients, CD52 was detected in at least one of three blast populations in almost all patients. Using a more stringent cutoff of > 50%, CD52 was expressed in CD34+/CD38- cells in 7/11 B-ALL and 6/7 T-ALL cases and was concordantly expressed in the other two populations. Using the same criteria in myeloid malignancies (Table 1), expression occurred more frequently in AML, AML arising from myelodysplastic syndrome (MDS), and BC CML. In AML and AML arising from MDS, CD52 was expressed in the 34+/38- population in 7/15 cases (47%) and 4/7 cases (57%), respectively; it was expressed in both BC CML patients. In AML and BC CML patients, CD52 was expressed at similar levels in the CD34+/CD38+ fraction. No clear association between CD52 expression and cytogenetic abnormalities was found. We then examined whether CD52 expression differentiated normal from malignant blasts (CD34+/CD38- and CD34+/CD38+) in two CML myeloid BC patients. FISH and quantitative PCR demonstrated that BCR-ABL was expressed in all 4 populations, which were also morphologically distinct. Colony forming unit (CFU) assays demonstrated a significantly decreased ability to form CFU (on average 5–20 fold decrease) in CD52+/CD34+/CD38- CML cells suggesting CD52 cells may be more mature. Lastly and not previously described, we found that several BC CML cell lines express CD52, and complement-mediated cell cytotoxicity was similar in the highest expressing cell lines to that seen in EHEB (B-CLL) cells known to be targeted by alemtuzumab. Thus, alemtuzumab may have clinical efficacy in BC CML. In conclusion, CD52 is expressed on blast populations enriched for leukemic stem cells. Whether the absence or presence of CD52 more precisely segregates a leukemia stem cell containing population currently remains unknown and requires functional testing in a murine model. Our preliminary experiments in CML suggest CD52 may not differentiate between normal and malignant stem/progenitor cells. However, CD52 expression may distinguish normal and malignant stem cell populations in cases where CD52 and CD38 are more highly expressed. The observation that CD52 expression is increased in acute vs. chronic leukemias raises the intriguing possibility that CD52, if not directly involved, may be a marker for genes or pathways contributing to the block in differentiation seen with progression to acute leukemia. Furthermore, given that CD52 expression is heterogeneous in chronic disorders, it is possible that CD52 expression within these populations may correlate with poor prognosis or impending leukemic conversion. Table 1. The proportion of patients (44) expressing CD52 at levels > 50% in 3 blast populations. Three populations were present in most, but not all patients. Gray shading indicates chronic myeloid diseases. MPN is myeloproliferative neoplasm; NOS is not otherwise specified; ET is essential thrombocythemia; CMML is chronic myelomonocytic leukemia; and an arrow represents progressed to. Disclosure: Oehler: Pfizer: Research Funding. Radich:Novartis: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria.

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

Blood ◽

10.1182/blood-2019-129164 ◽

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.

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The t(15;17)-Associated Fusion Protein PML/RAR Upregulates c-Kit Which Contributes to the Induction of the Leukemic Phenotype.

Blood ◽

10.1182/blood.v106.11.1608.1608 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1608-1608

Author(s):

Xiaomin Zheng ◽

Tim Beissert ◽

Brigitte Ruster ◽

Dieter Hoelzer ◽

Reinhard Henschler ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Fusion Protein ◽

Wnt Signaling ◽

Leukemic Cells ◽

Stem Cell Marker ◽

Aberrant Expression ◽

Self Renewal ◽

Hematopoietic Stem ◽

Differentiation Block

Abstract The pathogenesis of acute myeloid leukemia (AML) is strictly related to a block of terminal differentiation combined with an increased proliferation of hematopoietic progenitors (HP) in the bone marrow (BM). Furthermore an aberrant self-renewal of leukemic stem cells seems to be obligatory for the establishment of the leukemic clone in the BM. The block of differentiation is due to a deregulated function of differentiation-relevant transcription factors, whereas the proliferation is induced by aberrantly activated signaling pathways related to growth factor-dependent receptor kinases such as Flt3 or c-Kit (CD117). The aberrant self renewal can be attributed to specific pathways such as the Wnt-signaling, which play an important role also in the maintenance of the normal hematopoietic stem cell (HSC). The acute promyelocytic leukemia (APL) is characterized by the t(15;17) which leads to the expression of the PML/RAR fusion protein in the leukemic cells. PML/RAR mediates the APL phenotype given by a differentiation block at the promyelocytic level and an increased self renewal of the APL stem cells by the activation of the Wnt-signaling. The differentiation block, but not the aberrant self renewal, can be overcome by treatment with ATRA resulting ina high percentage of complete remissions. Nevertheless, if ATRA is used as a monotherapy a relapse is inevitable. APL blasts are frequently positive for constitutive activating Flt3 mutations and are constantly c-Kit-positive. Given the fact that c-Kit is a stem cell marker, the expression of c-Kit has to be considered aberrant and not related to the differentiation stage of the promyelocytes in APL. Therefore we investigated first the relationship between PML/RAR and the aberrant expression of c-Kit and then the role of aberrantly activated c-Kit for the pathogenesis of APL by studying its influence on the biology of early HSC. Here we report that i.) in contrast to the t(8;21)-associated AML-1/ETO, PML/RAR activated the c-Kit promotor in HP; ii.) the inhibition of the endogenous c-Kit kinase activity by imatinib or by AZD2171 abrogated the aberrant “replating efficiency“ of PML/RAR-positive HSC; iii.) activated c-Kit (c-Kit-D814H) accelerated cell cycle progression of Sca1+/lin- HSC; iv.) activated c-Kit blocked the differentiation of Sca1+/lin- HSC and increased their “replating efficiency“; v.) “colony forming unit-spleen“ (CFU-S) as well as “competitive repopulation“ assays“ (CRA) revealed that c-Kit-D814H strongly increased the “self renewal“-potential of Sca1+/lin- HSC; vi) c-Kit-D814H augmented the migration of Sca1+/lin- HSC into a 3D stromal spheroid model based on M2-10B4 cells, but did not have any influence on their adhesion (flow chamber on TNFalpha-stimulated HUVEC cells) as well as on their chemotaxis (SDF-1 gradient in transwell assays); vii.) c-Kit-D814H further increased the aberrant replating efficiency of PML/RAR- as well as of AML-1/ETO-positive HSC. Taken together our results strongly indicate not only that c-Kit importantly contributes to the leukemogenesis of APL, but that PML/RAR has a dual effect on c-Kit - the amplification of its expression at the promotor level as well as driving its expression in cells which normally do not have this proliferation stimulus. Thus it seems that there is a sort of positive feedback between PML/RAR and c-Kit which establishes c-Kit as a valuable therapeutic target for the treatment of APL-patients.

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The Novel Leukemia Stem Cell Marker GPR56 Discriminates Leukemic Subclones with Divergent Stem Cell Properties in Human Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v126.23.1859.1859 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1859-1859

Author(s):

Caroline Pabst ◽

Anne Bergeron ◽

Vincent-Philippe Lavallée ◽

Jonathan Yeh ◽

Patrick Gendron ◽

...

Keyword(s):

Stem Cells ◽

Acute Myeloid Leukemia ◽

Stem Cell ◽

Myeloid Leukemia ◽

Expression Profiles ◽

Stem Cell Marker ◽

Mouse Bone Marrow ◽

Rna Seq ◽

Hematopoietic Stem ◽

Acute Myeloid

Abstract Insights into the complex clonal architecture of acute myeloid leukemia (AML) unravelled by deep sequencing technologies have challenged the concept of AML as a hierarchically organised disease initiated and driven by rare self-renewing leukemic stem cells (LSCs). In contrast to normal human hematopoietic stem cells (HSCs), which are highly enriched in the CD34+ CD38- population, LSCs have also been found in the CD34- and the CD38+ fractions questioning the existence of a consistent LSC surface marker profile for AML. Besides, low LSC frequencies in primary samples, rapid onset of differentiation upon ex vivo culture, and genetic inter-specimen heterogeneity hamper the dissection of the molecular machinery that drives LSC self-renewal. We performed RNA-Sequencing of primary human AML samples and assessed LSC frequencies by limiting dilution analyses for 56 of these in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. By comparing gene expression profiles between high vs low LSC frequency leukemias, we identified the G-protein coupled receptor 56 (GPR56) has significantly more expressed in high LSC frequency leukemias. We validated the RNA-seq data with protein expression by FACS and found an excellent correlation. To determine whether GPR56 positive cells overlapped with the known LSC-associated phenotype CD34+ CD38-, we stained 45 AML samples with CD34, CD38, GPR56, and antibodies against other described LSC markers. Although CD34+ GPR56+ and CD34+ CD38- compartments identified the same population in some samples, we found in the majority of samples that GPR56 further subdivided the CD34+ CD38- compartment. Accordingly, not only the proportions of total GPR56+ and CD34+ GPR56+ cells were significantly higher in LSChigh versus LSClow samples, but also the proportion of GPR56+ cells within the CD34+ CD38- compartment was significantly different between the groups indicating that GPR56 might be of additional value to what is currently considered the best described LSC phenotype. The percentage of total CD34 positive cells did not correlate with LSC frequency clearly distinguishing GPR56 from CD34 or CD38, which are only suitable LSC markers when used in combination. We analysed other potential LSC markers (TIM3, CD96, CD44, CD123, CLL1 and CD47) in our RNA-Seq dataset and by FACS analysis in combination with CD34 as we did for GPR56 and none of them correlated with LSC frequency in our sample collection. To determine whether GPR56 discriminates engrafting LSCs from non-LSCs, we sorted GPR56+ and GPR56- cells within the CD34-positive and -negative compartments from selected specimens with known engraftment potential. We found that GPR56 identified the engrafting fraction in CD34positive AML samples, with a >50 fold enrichment in LSC in the CD34+GRP56+ fraction vs the CD34+GPR56- fraction within the same sample, demonstrating that GPR56 is a good LSC marker. Specimens with high molecular or cytogenetic risk such as chromosome 5 or 7 anomalies and EVI1- rearrangementexpressed high levels of both, GPR56 and CD34, while samples with coexistent FLT3 -ITD, DNMT3A, and NPM1 mutations displayed a unique CD34low GPR56high profile. Moreover, we found a divergent distribution of variant allele frequencies in GPR56+ versus GPR56- fractions identifying GPR56 as a discriminator of leukemic sub-clones with high and low NSG engrafting capacity. Analysis of engrafted cells re-sorted based on GPR56 after being harvested from mouse bone marrow revealed reduced complexity of the clonal composition. Most importantly, GPR56 positive cells differentiated to GPR56 negative cells in mice, which did not happen in the human niche, in which GPR56 positive and negative fractions represented two independently evolved subclones. In summary our work identifies GPR56 as a novel LSC marker in AML and also shows that GPR56 readily identifies a functionally distinct LSC-rich subclone in the majority of human AML patients and reveals hitherto unforeseen complexity in the interaction between human LSCs and the NSG mouse environment. Disclosures No relevant conflicts of interest to declare.

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CGRP Signaling via CALCRL Increases Chemotherapy Resistance and Stem Cell Properties in Acute Myeloid Leukemia

International Journal of Molecular Sciences ◽

10.3390/ijms20235826 ◽

2019 ◽

Vol 20 (23) ◽

pp. 5826 ◽

Cited By ~ 2

Author(s):

Tobias Gluexam ◽

Alexander M. Grandits ◽

Angela Schlerka ◽

Chi Huu Nguyen ◽

Julia Etzler ◽

...

Keyword(s):

Stem Cells ◽

Acute Myeloid Leukemia ◽

Stem Cell ◽

Mouse Model ◽

Cell Lines ◽

Myeloid Leukemia ◽

Leukemic Cells ◽

Hematopoietic Stem ◽

Acute Myeloid

The neuropeptide CGRP, acting through the G-protein coupled receptor CALCRL and its coreceptor RAMP1, plays a key role in migraines, which has led to the clinical development of several inhibitory compounds. Recently, high CALCRL expression has been shown to be associated with a poor prognosis in acute myeloid leukemia (AML). We investigate, therefore, the functional role of the CGRP-CALCRL axis in AML. To this end, in silico analyses, human AML cell lines, primary patient samples, and a C57BL/6-based mouse model of AML are used. We find that CALCRL is up-regulated at relapse of AML, in leukemic stem cells (LSCs) versus bulk leukemic cells, and in LSCs versus normal hematopoietic stem cells. CGRP protects receptor-positive AML cell lines and primary AML samples from apoptosis induced by cytostatic drugs used in AML therapy, and this effect is inhibited by specific antagonists. Furthermore, the CGRP antagonist olcegepant increases differentiation and reduces the leukemic burden as well as key stem cell properties in a mouse model of AML. These data provide a basis for further investigations into a possible role of CGRP-CALCRL inhibition in the therapy of AML.

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Thrombomucin, a Novel Cell Surface Protein that Defines Thrombocytes and Multipotent Hematopoietic Progenitors

The Journal of Cell Biology ◽

10.1083/jcb.138.6.1395 ◽

1997 ◽

Vol 138 (6) ◽

pp. 1395-1407 ◽

Cited By ~ 76

Author(s):

Kelly M. McNagny ◽

Inger Pettersson ◽

Fabio Rossi ◽

Ingo Flamme ◽

Andrej Shevchenko ◽

...

Keyword(s):

Stem Cell ◽

Cell Surface ◽

Single Cell Analysis ◽

Surface Glycoprotein ◽

Stem Cell Marker ◽

Cell Marker ◽

Hematopoietic Progenitors ◽

Cell Surface Glycoprotein ◽

Hematopoietic Stem ◽

Multipotent Progenitors

MEP21 is an avian antigen specifically expressed on the surface of Myb-Ets–transformed multipotent hematopoietic precursors (MEPs) and of normal thrombocytes. Using nanoelectrospray tandem mass spectrometry, we have sequenced and subsequently cloned the MEP21 cDNA and named the gene thrombomucin as it encodes a 571–amino acid protein with an extracellular domain typical of the mucin family of proteoglycans. Thrombomucin is distantly related to CD34, the best characterized and most used human hematopoietic stem cell marker. It is also highly homologous in its transmembrane/intracellular domain to podocalyxinlike protein–1, a rabbit cell surface glycoprotein of kidney podocytes. Single cell analysis of yolk sac cells from 3-d-old chick embryos revealed that thrombomucin is expressed on the surface of both lineage-restricted and multipotent progenitors. In the bone marrow, thrombomucin is also expressed on mono- and multipotent progenitors, showing an overlapping but distinct expression pattern from that of the receptor-type stem cell marker c-kit. These observations strengthen the notion that the Myb-Ets oncoprotein can induce the proliferation of thrombomucin-positive hematopoietic progenitors that have retained the capacity to differentiate along multiple lineages. They also suggest that thrombomucin and CD34 form a family of stem cell–specific proteins with possibly overlapping functions in early hematopoietic progenitors.

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CD9, a potential leukemia stem cell marker, regulates drug resistance and leukemia development in acute myeloid leukemia

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02155-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Yongliang Liu ◽

Guiqin Wang ◽

Jiasi Zhang ◽

Xue Chen ◽

Huailong Xu ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Drug Resistance ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Stem Cell Marker ◽

Hematopoietic Stem ◽

Chemotherapy Drugs ◽

And Migration ◽

Acute Myeloid

Abstract Background Leukemia stem cells (LSCs) are responsible for the initiation, progression, and relapse of acute myeloid leukemia (AML). Therefore, a therapeutic strategy targeting LSCs is a potential approach to eradicate AML. In this study, we aimed to identify LSC-specific surface markers and uncover the underlying mechanism of AML LSCs. Methods Microarray gene expression data were used to investigate candidate AML-LSC-specific markers. CD9 expression in AML cell lines, patients with AML, and normal donors was evaluated by flow cytometry (FC). The biological characteristics of CD9-positive (CD9+) cells were analyzed by in vitro proliferation, chemotherapeutic drug resistance, migration, and in vivo xenotransplantation assays. The molecular mechanism involved in CD9+ cell function was investigated by gene expression profiling. The effects of alpha-2-macroglobulin (A2M) on CD9+ cells were analyzed with regard to proliferation, drug resistance, and migration. Results CD9, a cell surface protein, was specifically expressed on AML LSCs but barely detected on normal hematopoietic stem cells (HSCs). CD9+ cells exhibit more resistance to chemotherapy drugs and higher migration potential than do CD9-negative (CD9−) cells. More importantly, CD9+ cells possess the ability to reconstitute human AML in immunocompromised mice and promote leukemia growth, suggesting that CD9+ cells define the LSC population. Furthermore, we identified that A2M plays a crucial role in maintaining CD9+ LSC stemness. Knockdown of A2M impairs drug resistance and migration of CD9+ cells. Conclusion Our findings suggest that CD9 is a new biomarker of AML LSCs and is a promising therapeutic target.

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

Blood ◽

10.1182/blood.v86.8.2906.bloodjournal8682906 ◽

1995 ◽

Vol 86 (8) ◽

pp. 2906-2912 ◽

Cited By ~ 2

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.

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