Leukemic Stem Cell Assessment in Remission Bone Marrow of Acute Myeloid Leukemia Patients Is a New Prognostic Parameter.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 399-399 ◽  
Author(s):  
Monique Terwijn ◽  
Angèle Kelder ◽  
Arjo P Rutten ◽  
Alexander N Snel ◽  
Willemijn Scholten ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Stem Cell ◽  
Myeloid Leukemia ◽  
Prognostic Information ◽  
Cell Compartment ◽  
Hematopoietic Stem ◽  
Stem Cell Compartment ◽  
Acute Myeloid

Abstract Abstract 399 In acute myeloid leukemia (AML), relapses originate from the outgrowth of therapy surviving leukemic blasts know as minimal residual disease (MRD). Accumulating evidence shows that leukemia initiating cells or leukemic stem cells (LSCs) are responsible for persistence and outgrowth of AML. Monitoring LSCs during and after therapy might thus offer accurate prognostic information. However, as LSCs and hematopoietic stem cells (HSCs) both reside within the immunophenotypically defined CD34+CD38- compartment, accurate discrimination between LSCs and HSCs is required. We previously showed that within the CD34+CD38- stem cell compartment, LSCs can be discriminated from HSC by aberrant expression of markers (leukemia associated phenotype, LAP), including lineage markers like CD7, CD19 and CD56 and the novel LSC marker CLL-1 (van Rhenen, Leukemia 2007, Blood 2007). In addition, we reported that flowcytometer light scatter properties add to even better detection of LSCs, allowing LSCs detection in AML cases lacking LAP (ASH abstract 1353, 2008). Using this gating strategy, we determined LSC frequency in 64 remission bone marrow samples of CD34+ AML patients. A stem cell compartment was defined as a minimum of 5 clustered CD34+CD38- events with a minimal analyzed number of 500,000 white blood cells. After first cycle of chemotherapy, high LSC frequency (>1 × 10-3) clearly predicted adverse relapse free survival (RFS, figure 1a). LSC frequency above cut-off led to a median RFS of 5 months (n=9), while patients with LSC frequency below cut-off (n=22) showed a significantly longer median RFS of >56 months (p=0.00003). In spite of the relatively low number of patients, again a high LSC frequency (>2 × 10-4) after the second cycle and after consolidation therapy predicted worse RFS: after second cycle, median RFS was 6 months (n=9) vs. >43 months for patients with LSC frequency below cut-off (p=0.004). After consolidation, these figures were 6 months (n=7) vs. >32 months (n=6, p=0.03). Although total blast MRD (leukemic blasts as % of WBC) is known to predict survival (N.Feller et al. Leukemia 2004), monitoring LSCs as compared to total blast MRD has two major advantages: the specificity is higher (van Rhenen et al. Leukemia 2007) and well-known LSC makers like CLL-1, CD96 and CD123 can in principle be used for LSC monitoring, but not for total blast MRD detection since these markers are also expressed on normal progenitor cells. On the other hand, LSCs constitute only a small fraction of all leukemic blasts and therefore monitoring total blast MRD may have the advantage of a higher sensitivity. We thus tested the hypothesis that even more accurate prognostic information could be obtained by combining LSC frequency with total blast MRD. Total blast MRD after first cycle was predictive for survival with borderline significance (p=0.08): a cut-off of 0.3% resulted in two patient groups with median RFS of 9 months vs. >56 months. Figure 1b shows the result of the combined data of LSC and MRD frequency after first cycle therapy. We used the terms LSC+ and MRD+ for cell frequencies above cut-off and LSC- and MRD- for those below cut-off. We could clearly identify that apart from LSC+/MRD+ patients, LSC+/MRD- patients too have very poor prognosis, while MRD+/LSC- patients show an adverse prognosis as compared to LSC-/MRD- patients. These results from the first study on the in vivo fate of LSCs during and after therapy, strongly support the hypothesis that in CD34+ AML the leukemia initiating capacity originates from the CD34+CD38- population and is important for tumor survival and outgrowth. These results show that LSC frequency might be superior in predicting prognosis of AML patients in CR as compared to MRD total blast frequency, while the combination of both may offer the most optimal parameter to guide future intervention therapies. This work was supported by Netherlands Cancer Foundation KWF. Disclosures: No relevant conflicts of interest to declare.

scholarly journals CXCR4 Antagonists as Stem Cell Mobilizers and Therapy Sensitizers for Acute Myeloid Leukemia and Glioblastoma?

Biology ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 31 ◽  
Author(s):  
Vashendriya V.V. Hira ◽  
Cornelis J.F. Van Noorden ◽  
Remco J. Molenaar
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Stem Cell ◽  
Myeloid Leukemia ◽  
Patient Survival ◽  
Hematopoietic Stem ◽  
Poor Patient ◽  
Acute Myeloid ◽  
Poor Patient Survival

Glioblastoma is the most aggressive and malignant primary brain tumor in adults and has a poor patient survival of only 20 months after diagnosis. This poor patient survival is at least partly caused by glioblastoma stem cells (GSCs), which are slowly-dividing and therefore therapy-resistant. GSCs are localized in protective hypoxic peri-arteriolar niches where these aforementioned stemness properties are maintained. We previously showed that hypoxic peri-arteriolar GSC niches in human glioblastoma are functionally similar to hypoxic peri-arteriolar hematopoietic stem cell (HSC) niches in human bone marrow. GSCs and HSCs express the receptor C-X-C receptor type 4 (CXCR4), which binds to the chemoattractant stromal-derived factor-1α (SDF-1α), which is highly expressed in GSC niches in glioblastoma and HSC niches in bone marrow. This receptor–ligand interaction retains the GSCs/HSCs in their niches and thereby maintains their slowly-dividing state. In acute myeloid leukemia (AML), leukemic cells use the SDF-1α–CXCR4 interaction to migrate to HSC niches and become slowly-dividing and therapy-resistant leukemic stem cells (LSCs). In this communication, we aim to elucidate how disruption of the SDF-1α–CXCR4 interaction using the FDA-approved CXCR4 inhibitor plerixafor (AMD3100) may be used to force slowly-dividing cancer stem cells out of their niches in glioblastoma and AML. Ultimately, this strategy aims to induce GSC and LSC differentiation and their sensitization to therapy.

Results of Hematopoietic Stem Cell Transplantation for Acute Myeloid Leukemia of the FAB M0 Subtype in First Complete Remission: A Survey of the Acute Leukemia Working Party of the European Bone Marrow Transplant Group (EBMT).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 5131-5131
Author(s):  
Andree-Laure Herr ◽  
Myriam Labopin ◽  
Rosy Reiffers ◽  
Donald Bunjes ◽  
Didier Blaise ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Complete Remission ◽  
Myeloid Leukemia ◽  
Hematopoietic Stem ◽  
First Complete Remission ◽  
Acute Myeloid

Abstract Hematopoietic stem cell transplantation (HSCT) is a potentially curative treatment for patients with acute myeloid leukemia (AML). AML of the FAB M0 subtype is rare, often associated with a complex karyotype and a poor prognosis. Results of HSCT for this AML subtype have never been reported separately from other subtypes. We did a survey of the results of 274 HSCT in adults with M0 AML in first complete remission (CR1), performed in EBMT centres since January 1990 until 2002. One hundred fifty patients were transplanted with an HLA identical donor (HLA-id), 30 with an HLA-matched unrelated donor (MUD) and 94 received an autologous transplant (auto). The median age was 45 years (16–71), the median interval from diagnosis to HSCT was 4 months for HLA-id, 6 months for MUD and 5 months for auto HSCT. The median follow-up time (range) was 20 months (1–109), 12 (2–53) and 10 months (1–96) for HLA-id, MUD and auto-HSCT respectively. The source of stem cells was peripheral blood stem cells for 67% of cases, and bone marrow for the remaining. The majority of grafts were non-T-cell depleted. Acute GVHD (grade I–IV) occurred in 56% of HLA-id and in 64% of MUD cases. The table shows the outcomes at two years according to the type of transplant. In conclusion, outcomes after HLA identical HSCT and MUD in adult patients with AML FAB subtype M0 in CR1 are encouraging. In comparison to allogeneic transplant cases, LFS is decreased in patients receiving an autologous transplant due to a high relapse incidence, reflecting the probable role of a graft-versus-leukemia effect in this FAB subtype. Results of HSCT in AML M0 CR1 patients Outcomes HLA-id n=150 MUD n= 30 Auto n=94 LFS: leukemia free survival; OS: overall survival; RI: relapse incidence; TRM: treatment-related mortality 2y LFS 50% 45% 33% 2y OS 58% 50% 49% 2y RI 25% 40% 57% 2y TRM 24% 14% 9%

Specific Detection of Aberrant and Normal Stem Cells in Acute Myeloid Leukemia Patients Opens the Way for Defining Highly Specific Targets for Stem Cell Therapy.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1353-1353
Author(s):  
J Monique Terwi ◽  
Angèle Kelder ◽  
Arjo P Rutten ◽  
Sonja Zweegman ◽  
Gert J Ossenkoppele ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Stem Cell ◽  
Myeloid Leukemia ◽  
The Other ◽  
Specific Detection ◽  
Cell Compartment ◽  
Stem Cell Compartment ◽  
Marker Expression ◽  
Acute Myeloid

Abstract In acute myeloid leukemia (AML), a small fraction of blast cells contains the tumor initiating cells, further referred to as leukemic stem cells (LSCs). LSC resemble hematopoietic stem cells (HSCs) with respect to self renewal capacity and quiescence. Therefore, LSCs are proposed to be therapy resistant. In order to optimally target LSCs and sparing HSC and to monitor therapy, discrimination between LSC is HSC is required. We showed that within the CD34+CD38− stem cell compartment, LSCs can be discriminated from HSC by aberrant expression of markers, including lineage markers like CD7, CD19 and CD56 and the novel LSC marker CLL-1 (van Rhenen et al., Leukemia 2007 and Blood 2007). Too low aberrant marker expression, however, hampers discrimination in part of the cases. Therefore, we investigated additional parameters that would allow to distinguish LSCs from HSCs in CD34 positive AML patients. In 14 out of 48 cases studied, flow cytometry revealed a double population within the CD34+CD38− compartment, characterized by a small but clear difference in forward scatter (FSC, reflecting cell size) and sideward scatter (SSC, reflecting granularity). In 7/14 cases with high marker expression, FSChighSSChigh population coincided completely with the population with aberrant marker expression. In the other cases, marker expression was too moderate to show a complete overlap. The FSClowSSClow population within the CD34+CD38− stem cell compartment is the minor population at diagnosis (median 16%, range 0.2%–92%; n=14), had no expression of aberrant markers and, moreover, closely resembled the FSC/SSC characteristics of normal BM HSCs. In addition, in these patients, the normality of the FSClowSSClow population was also supported by the fact that the CD34 and CD45 antigen density was similar to that of normal BM HSCs. Altogether, this enabled to use FSC/SSC characteristics together with aberrant CD34 and CD45 expression to discriminate between LSC and HSC in cases with low or absent aberrant marker expression (8/48). In addition, the malignant character of the FSChighSSChigh population and the normal character of the FSClowSSClow population could be proven in three AML patients with cytogenetic aberrancies. Patient 1 had a t(8;21) translocation and presented with a CD34+CD38−- population that was CD19 positive (81% of the stem cell compartment) and had FSChighSSChigh properties. FACSsorted cells contained the translocation in 90% of the cells. The CD19 negative population (19% of the stem cell compartment) had FSClowSSClow characteristics and contained 0% t(8;21) cells. In two other AML cases with a cytogenetic aberrancy (t(8;21) and t(15;17), respectively), FSC/SSC characteristics, CD34/CD45 antigen density and aberrant marker expression (CD56 in one case and CLL-1 in the other) were partly overlapping (estimated LSC contribution to the CD34+CD38− compartment was 85% in both cases). Cell sorting on the highest FSC/SSC and marker expression nevertheless resulted in enrichment of cytogenetically aberrant cells (63% and 73%, respectively), while the corresponding FSClowSSClow cells, which missed CD56 and CLL-1 expression, were enriched for cytogenetically normal HSCs (87% and 67%, respectively). The marker and scatter parameters discussed above have generated the possibility to discriminate between LSCs and HSCs and now allows specific detection of LSC in >75 % of the patients. Discrimination between LSCs and HSCs in AML might not only facilitate to establish the therapeutic window of current therapies in terms of LSC specificity, but also allow the identification of new highly AML stem cells specific therapeutic targets. This should ultimately result in more selective therapies, which would be highly effective for AML stem cells, while leaving the normal HSC intact. This work was supported by Netherlands Cancer Foundation KWF.

scholarly journals Runx2 induces acute myeloid leukemia in cooperation with Cbfβ-SMMHC in mice

Blood ◽  
2009 ◽  
Vol 113 (14) ◽  
pp. 3323-3332 ◽  
Author(s):  
Ya-Huei Kuo ◽  
Sayyed K. Zaidi ◽  
Svetlana Gornostaeva ◽  
Toshihisa Komori ◽  
Gary S. Stein ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Stem Cell ◽  
Myeloid Leukemia ◽  
Cell Compartment ◽  
Hematopoietic Stem ◽  
Myeloid Progenitor ◽  
Stem Cell Compartment ◽  
Targeting Signal ◽  
Leukemia Development ◽  
Acute Myeloid

Abstract The core-binding factor (CBF) is a master regulator of developmental and differentiation programs, and CBF alterations are frequently associated with acute leukemia. The role of the CBF member RUNX2 in hematopoiesis is poorly understood. Genetic evidence suggests that deregulation of Runx2 may cause myeloid leukemia in mice expressing the fusion oncogene Cbfb-MYH11. In this study, we show that sustained expression of Runx2 modulates Cbfβ-smooth muscle myosin heavy chain (SMMHC)–mediated myeloid leukemia development. Expression of Runx2 is high in the hematopoietic stem cell compartment and decreases during myeloid differentiation. Sustained Runx2 expression hinders myeloid progenitor differentiation capacity and represses expression of CBF targets Csf1R, Mpo, Cebpd, the cell cycle inhibitor Cdkn1a, and myeloid markers Cebpa and Gfi1. In addition, full-length Runx2 cooperates with Cbfβ-SMMHC in leukemia development in transplantation assays. Furthermore, we show that the nuclear matrix–targeting signal and DNA-binding runt-homology domain of Runx2 are essential for its leukemogenic activity. Conversely, Runx2 haplo-insufficiency delays the onset and reduces the incidence of acute myeloid leukemia. Together, these results indicate that Runx2 is expressed in the stem cell compartment, interferes with differentiation and represses CBF targets in the myeloid compartment, and modulates the leukemogenic function of Cbfβ-SMMHC in mouse leukemia.

A leukemic stem cell with intrinsic drug efflux capacity in acute myeloid leukemia

Blood ◽  
2001 ◽  
Vol 98 (4) ◽  
pp. 1166-1173 ◽  
Author(s):  
Gerald G. Wulf ◽  
Rui-Yu Wang ◽  
Ingrid Kuehnle ◽  
Douglas Weidner ◽  
Frank Marini ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Stem Cell ◽  
Myeloid Leukemia ◽  
Side Population ◽  
Fluorescence Emission ◽  
Drug Efflux ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Acute Myeloid

The hematopoietic stem cell underlying acute myeloid leukemia (AML) is controversial. Flow cytometry and the DNA-binding dye Hoechst 33342 were previously used to identify a distinct subset of murine hematopoietic stem cells, termed the side population (SP), which rapidly expels Hoechst dye and can reconstitute the bone marrow of lethally irradiated mice. Here, the prevalence and pathogenic role of SP cells in human AML were investigated. Such cells were found in the bone marrow of more than 80% of 61 patients and had a predominant CD34low/− immunophenotype. Importantly, they carried cytogenetic markers of AML in all 11 cases of active disease examined and in 2 out of 5 cases in complete hematological remission. Comparison of daunorubicin and mitoxantrone fluorescence emission profiles revealed significantly higher drug efflux from leukemic SP cells than from non-SP cells. Three of 28 SP cell transplants generated overt AML-like disease in nonobese diabetic–severe combined immunodeficient mice. Low but persistent numbers of leukemic SP cells were detected by molecular and immunological assays in half of the remaining mice. Taken together, these findings indicate that SP cells are frequently involved in human AML and may be a target for leukemic transformation. They also suggest a mechanism by which SP cells could escape the effects of cytostatic drugs and might eventually contribute to leukemia relapse.

scholarly journals 7-Ketocholesterol Promotes Oxiapoptophagy in Bone Marrow Mesenchymal Stem Cell from Patients with Acute Myeloid Leukemia

Cells ◽  
2019 ◽  
Vol 8 (5) ◽  
pp. 482 ◽  
Author(s):  
Jessica Liliane Paz ◽  
Debora Levy ◽  
Beatriz Araujo Oliveira ◽  
Thatiana Correia de Melo ◽  
Fabio Alessandro de Freitas ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Cholesterol Oxidation ◽  
Specific Cell ◽  
Hematopoietic Stem ◽  
Shh Pathway ◽  
Number Of Cells ◽  
Acute Myeloid

7-Ketocholesterol (7-KC) is a cholesterol oxidation product with several biological functions. 7-KC has the capacity to cause cell death depending on the concentration and specific cell type. Mesenchymal stem cells (MSCs) are multipotent cells with the ability to differentiate into various types of cells, such as osteoblasts and adipocytes, among others. MSCs contribute to the development of a suitable niche for hematopoietic stem cells, and are involved in the development of diseases, such as leukemia, to a yet unknown extent. Here, we describe the effect of 7-KC on the death of bone marrow MSCs from patients with acute myeloid leukemia (LMSCs). LMSCs were less susceptible to the death-promoting effect of 7-KC than other cell types. 7-KC exposure triggered the extrinsic pathway of apoptosis with an increase in activated caspase-8 and caspase-3 activity. Mechanisms other than caspase-dependent pathways were involved. 7-KC increased ROS generation by LMSCs, which was related to decreased cell viability. 7-KC also led to disruption of the cytoskeleton of LMSCs, increased the number of cells in S phase, and decreased the number of cells in the G1/S transition. Autophagosome accumulation was also observed. 7-KC downregulated the SHh protein in LMSCs but did not change the expression of SMO. In conclusion, oxiapoptophagy (OXIdative stress + APOPTOsis + autophagy) seems to be activated by 7-KC in LMSCs. More studies are needed to better understand the role of 7-KC in the death of LMSCs and the possible effects on the SHh pathway.

scholarly journals Gpr44 Mediates the Selenium-Dependent Anti-Leukemic Effect in Acute Myeloid Leukemia

2020 ◽  
Vol 4 (Supplement_2) ◽  
pp. 1835-1835
Author(s):  
Fenghua Qian ◽  
Fenghua Qian ◽  
Diwakar Tukaramrao ◽  
Jiayan Zhou ◽  
Nicole Palmiero ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Murine Model ◽  
Myeloid Leukemia ◽  
Hematopoietic Stem ◽  
Cell Counts ◽  
Pparγ Agonist ◽  
Acute Myeloid

Abstract Objectives The relapse of acute myeloid leukemia (AML) remains a significant concern due to persistent leukemia stem cells (LSCs) that are not targeted by existing therapies. LSCs show sensitivity to endogenous cyclopentenone prostaglandin J (CyPG) metabolites that are increased by dietary trace element selenium (Se), which is significantly decreased in AML patients. We investigated the anti-leukemic effect of Se supplementation in AML via mechanisms involving the activation of the membrane-bound G-protein coupled receptor 44 (Gpr44) and the intracellular receptor, peroxisome proliferator-activated receptor gamma (PPARγ), by endogenous CyPGs. Methods A murine model of AML generated by transplantation of hematopoietic stem cells (HSCs- WT or Gpr44−/−) expressing human MLL-AF9 fusion oncoprotein, in the following experiments: To investigate the effect of Se supplementation on the outcome of AML, donor mice were maintained on either Se-adequate (Se-A; 0.08–0.1 ppm Se) or Se-supplemented (Se-S; 0.4 ppm Se) diets. Complete cell counts in peripheral blood were analyzed by hemavet. LSCs in bone marrow and spleen were analyzed by flow cytometry. To determine the role of Gpr44 activation in AML, mice were treated with Gpr44 agonists, CyPGs. LSCs in bone marrow and spleen were analyzed. Mice transplanted with Gpr44−/- AML cells were compared with mice transplanted with wild type AML cells and the progression of the disease was followed as above. To determine the role of PPARγ activation in AML, PPARγ agonist (Rosiglitazone, 6 mg/kg, i.p, 14 d) and antagonist (GW9662, 1 mg/kg, i.p. once every other day, 7 injections) were applied to Se-S mice transplanted with Gpr44−/- AML cells and disease progression was followed. Results Se supplementation at supraphysiological levels alleviated the disease via the elimination of LSCs in a murine model of AML. CyPGs induced by Se supplementation mediate the apoptosis in LSCs via the activation of Gpr44 and PPARγ. Conclusions Endogenous CyPGs produced upon supplementation with Se at supraphysiological levels improved the outcome of AML by targeting LSCs to apoptosis via the activation of two receptors, Gpr44 and PPARg. Funding Sources NIH DK 07,7152; CA 175,576; CA 162,665. Office of Dietary Supplements, USDA Hatch funds PEN04605, Accession # 1,010,021 (KSP, RFP).

TIM-3 Is a Promising Target to Eradicate Acute Myeloid Leukemia Stem Cells Sparing Normal Hematopoietic Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 559-559
Author(s):  
Toshihiro Miyamoto ◽  
Yoshikane Kikushige ◽  
Takahiro Shima ◽  
Koichi Akashi
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Hematopoietic Stem Cells ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Cytotoxic Activities ◽  
Antibody Treatment ◽  
Hematopoietic Stem ◽  
Acute Myeloid

Abstract Abstract 559 Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), an ultimate therapeutic target for permanent cure. To selectively kill AML LSCs sparing normal hematopoietic stem cells (HSCs), one of the most practical approaches is to target the AML LSCs-specific surface or functionally indispensable molecules. Based on differential transcriptome analysis of prospectively-purified CD34+CD38− LSCs from AML patient samples and normal HSCs, we found that T-cell immunoglobulin mucin-3 (TIM-3) was highly expressed in AML LSCs but not in normal HSCs (Kikushige et al., Cell Stem Cell, 2010). In normal hematopoiesis, TIM-3 is mainly expressed in mature monocytes and a fraction of NK cells, but not in granulocytes, T cells or B cells. In the bone marrow, TIM-3 is expressed only in a fraction of granulocyte/macrophage progenitors (GMPs) at a low level, but not in HSCs, common myeloid progenitors, or megakaryocyte/erythrocyte progenitors. In contrast, in human AML, TIM-3 was expressed on cell surface of the vast majority of CD34+CD38− LSCs and CD34+CD38+ leukemic progenitors in AML of most FAB types, except for acute promyelocytic leukemia (M3). FACS-sorted TIM-3+ but not TIM-3− AML cells reconstituted human AML in the immunodeficient mice, indicating that the TIM-3+ population contains most of functional LSCs. To selectively eradicate TIM-3-expressing AML LSCs, we established an anti-human TIM-3 mouse IgG2a antibody, ATIK2a, possessing antibody-dependent cellular cytotoxic and complement-dependent cytotoxic activities in leukemia cell lines transfected with TIM-3. We first tested the effect of ATIK2a treatment on reconstitution of normal HSCs in a xenograft model. ATIK2a was intraperitoneally injected to the mice once a week after 12 hours of transplantation of human CD34+ cells. Injection of ATIK2a did not affect reconstitution of normal human hematopoiesis except removing TIM-3-expressing mature monocytes. In contrast, injection of TIM-3 to the mice transplanted with human AML samples markedly reduced leukemic repopulation. In some mice transplanted with AML bone marrow, only normal hematopoiesis was reconstituted after anti-TIM-3 antibody treatment, suggesting that the antibody selectively killed AML cells, sparing residual normal HSCs. To further test the inhibitory effect of ATIK2a on established human AML, eight weeks after transplantation of human AML cells, engraftment of human AML cells was confirmed by blood sampling and thereafter ATIK2a was injected to these mice. In all cases tested, ATIK2a treatment significantly reduced human TIM-3+ AML fraction as well as the CD34+CD38− LSCs fraction. In addition, to verify the anti-AML LSCs effect of ATIK2a treatment, human CD45+AML cells from the primary recipients were re-transplanted into secondary recipients. All mice transplanted from primary recipients treated with control IgG developed AML, whereas none of mice transplanted with cells from ATIK2a-treated primary recipients developed AML, suggesting that functional LSCs were effectively eliminated by ATIK2a treatment in primary recipients. Thus, TIM-3 is a promising surface molecule to target AML LSCs. Our experiments strongly suggest that targeting this molecule by monoclonal antibody treatment is a practical approach to eradicate human AML. Disclosures: No relevant conflicts of interest to declare.

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