Differences in heat sensitivity between normal and acute myeloid leukemic stem cells: feasibility of hyperthermic purging of leukemic cells from autologous stem cell grafts

OBJECTIVES: In autologous stem cell transplantation contamination of the graft with malignant cells is frequently noticed and necessitates the use of in vivo or in vitro purging modalities. The hematopoietic recovery after transplantation depends on the number of stem and progenitor cells in the transplant. Therefore, in the present study the effects of hyperthermic treatment on the human normal and acute myeloid leukemic (AML) stem cell compartment were investigated. METHODS: Normal bone marrow and AML blasts were heat treated up to 120 minutes at 43 degrees C. The surviving fractions of the different stem cell subsets were determined using in vitro methylcellulose and cobblestone area-forming cell (CAFC) clonogenic assays, as well as the in vivo NOD/SCID repopulating assay. The leukemic nature of the colonies from AML cells was confirmed by RT-PCR analysis. In order to increase the therapeutic index of the hyperthermic purging modality, the heat treatment was preceded by a 3-hour incubation at 37 degrees C with the ether lipid ET-18-OCH(3) (25 microg/mL). RESULTS: It could be demonstrated that normal progenitor cells are far more resistant to hyperthermia than leukemic progenitor cells (56%+/-7% vs 9.9%+/-2.6% survival after 60 minutes at 43 degrees C, respectively). Furthermore, normal hematopoietic stem cells appear to be extremely resistant to the heat treatment (94%+/-9% survival after 60 minutes at 43 degrees C). In contrast, in the leukemic stem cell compartment no significant differences in heat sensitivity between the stem cells and progenitor subsets could be observed (12.3%+/-2.9% vs 9.9%+/-2.6% survival after 60 minutes at 43 degrees C, respectively). The combined treatment resulted in a survival for normal progenitor and stem cells of 32%+/-6% and 85%+/-15% after 60 minutes at 43 degrees C, respectively. Under these conditions the number of leukemic stem cells was reduced to 1%+/-0.3%. After 120 minutes at 43 degrees C, no AML-colonies could be detected anymore. CONCLUSIONS: Our data demonstrate that leukemic stem cells have an increased hyperthermic sensitivity compared to their normal counterparts and that this difference can be further increased in combination with ET-18-OCH(3). These striking differences in heat sensitivity warrant the use of hyperthermia as a clinically applicable purging modality in autologous stem cell transplantation.     

The antitumor ether lipid ET-18-OCH(3) induces apoptosis through translocation and capping of Fas/CD95 into membrane rafts in human leukemic cells.

The antitumor ether lipid ET-18-OCH(3) promotes apoptosis in tumor cells through intracellular activation of Fas/CD95. Results of this study showed that ET-18-OCH(3) induces cocapping of Fas and membrane rafts, specialized plasma membrane regions involved in signaling, before the onset of apoptosis in human leukemic cells. Patches of membrane rafts accumulated Fas clusters in leukemic cells treated with ET-18-OCH(3). Sucrose gradient centrifugation of Triton X-100 cell lysates showed that Fas translocated into membrane rafts following ET-18-OCH(3) treatment of T-leukemic Jurkat cells. Disruption of membrane raft integrity by methyl-beta-cyclodextrin or filipin inhibited ET-18-OCH(3)-induced apoptosis in leukemic primary cells and cell lines. Fas clustering was also inhibited by methyl-beta-cyclodextrin. These data indicate that ET-18-OCH(3) reorganizes membrane rafts to trigger apoptosis in human leukemic cells, and that Fas coaggregation with membrane rafts is required for ET-18-OCH(3)-induced apoptosis. This translocation of Fas into membrane rafts may provide a mechanism for amplifying Fas signaling by reorganization of membrane microdomains.     

Alpha-interferon broadens the difference between surviving fractions of normal and leukemic progenitor cells in vitro by heat: its application to marrow purging.

Alpha-interferon (IFN) may inhibit the proliferation of human leukemic progenitor cells (L-CFU) in vitro and enhance the anti-tumor effects by heat. In this study, the combined effects of IFN and hyperthermia on the growth of L-CFU and human granulocyte-macrophage progenitors (CFU-GM) were examined to determine if this combination resulted in a greater selective killing of L-CFU than that obtained by heat treatment alone. The survival of normal CFU-GM without IFN decreased at elevated temperatures (42-44 degrees C). However, IFN added during heating (42 and 43 degrees C) appeared significantly to protect against the hyperthermic killing of CFU-GM in vitro leaving over 50% of CFU-GM surviving. The optimal dose to protect CFU-GM in vitro dropped to a rather low dose (100 U/ml). On the other hand, the addition of IFN to leukemic cell suspensions enhanced the hyperthermic killing of myeloid leukemic cell lines (HEL and KG-1) as well as a T lymphoblastic cell line (CEM) in a dose-related manner. In addition, similar results were observed in the study of L-CFU from patients with acute myelogenous leukemia. These results suggest that IFN can be used to broaden the difference between surviving fractions of CFU-GM and L-CFU by heat. Thus, this combination could be applied effectively and safely for the elimination of residual clonogenic leukemic cells in autologous remission marrow graft before autologous bone marrow transplantation.     

Effects of human FLT3 ligand on myeloid leukemia cell growth: heterogeneity in response and synergy with other hematopoietic growth factors

A novel hematopoietic growth factor for primitive hematopoietic progenitor cells, the ligand for the flt3/flk2 receptor, (FL), has been recently purified and its gene has been cloned. In the present study, we investigated the effects of FL on the proliferation and differentiation of normal and leukemic myeloid progenitor cells. We demonstrate that FL is a potent stimulator of the in vitro growth of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin- 3 (IL-3), or G-CSF-dependent granulocyte-macrophage committed precursors from Lin- CD34+ bone marrow cells of normal donors. By contrast, FL does not affect the growth of erythroid-committed progenitors even in the presence of erythropoietin. The effect of FL on the proliferation and on the in vitro growth of clonogenic leukemic precursor cells was studied in 54 acute myeloid leukemia (AML) cases. Fresh leukemia blasts from 36 of 45 patients with AML significantly responded to FL without any relation to the French-American-British (FAB) subtype. FL stimulated the proliferation of leukemic blasts in a dose-dependent fashion. Synergistic activities were seen when FL was combined with G-CSF, GM-CSF, IL-3, or stem cell factor (SCF). FL as a single factor induced or increased significantly colony formation by clonogenic precursor cells from 21 of 24 patients with AML. In the presence of suboptimal and optimal concentrations of G-CSF, GM-CSF, IL3, SCF, or a combination of all factors, FL strongly enhanced the number of leukemic colonies (up to 18-fold). We also evaluated the induction of tyrosine phosphorylated protein on FL stimulation in fresh AML cells. We demonstrate that, on FL stimulation, a band of phosphorylated protein(s) of about 90 kD can be detected in FL- responsive, but not in FL-unresponsive cases. This study suggests that FL may be an important factor for the growth of myeloid leukemia cells, either as a direct stimulus or as a synergistic factor with other cytokines.     

Differential cytotoxicity of an ether lipid on lymphoma and bone marrow cells and its role in purging malignant lymphoid cells from remission bone marrow contaminated with tumor cells.

Four human clonogenic malignant lymphoid cell lines (CEM, Su-DHL-4, Li-A, and Raji) as well as normal human bone marrow stem cell progenitor cells were investigated for clonal in vitro growth before and after incubation with the ether lipid ET-18-OCH3 for various times (1, 4, and 18 h) and at increasing concentrations of the drug (25, 50, 75, and 100 micrograms/ml). The clonal growth of the malignant lymphoid cell lines was inversely correlated with concentrations and times of drug incubation. The antineoplastic effect of ET-18-OCH3 was further amplified by subsequent cryopreservation. In a situation of 4-h exposure to less than or equal to 50 micrograms/ml ET-18-OCH3 and subsequent cryopreservation, in which greater than 50% of the normal human bone marrow progenitor cells survived, 1-3 logs of the malignant lymphoblastoid cells were killed, indicating a potential value of this drug for bone marrow purging in lymphoid malignancy. In order to simulate the situation of autologous bone marrow transplantation (ABMT) in complete remission of the disease, we contaminated normal human bone marrow cells with malignant CEM or Su-DHL-4 lymphoid cells at a ratio of 100:1. Results show that 4 h of incubation with 75 micrograms/ml ET-18-OCH3 and subsequent cryopreservation can eliminate 2-3 logs of clonogenic cells of the malignant lymphoblastoid cell lines under conditions that allow recovery of greater than 50% of the normal human hematopoietic progenitors.     

Synergistic effects of heat and ET-18-OCH3 on membrane expression of hsp70 and lysis of leukemic K562 cells

We previously reported that cell surface expression of hsp70, the major stress inducible member of the 70-kDa heat shock protein family, is inducible by nonlethal heat as well as by treatment with the membrane-interactive compound alkyl-lysophospholipid 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) selectively on human tumor cell lines. Plasma membrane expression of hsp70 increases selectively the sensitivity of tumor cells to lysis and, therefore, might play an important role in the antitumor immune response. Here, we demonstrate that a combined treatment consisting of sublethal heat (41.8 degrees C) and a noncytotoxic concentration of ET-18-OCH3 (25 micrograms/mL) results in a synergistic increase in the amount of cell membrane-bound hsp70 on leukemic K562 cells and on freshly isolated bone marrow of a chronic myelogeneous leukemia (CML) patient, but not on peripheral blood lymphocytes or CD34+ hematopoietic progenitor cells of healthy human individuals. Under these conditions the repopulating capacity of progenitor cells was not influenced. The increased hsp70 membrane expression on leukemic K562 cells results in a significantly increased sensitivity to lysis mediated by natural killer cells. In contrast to leukemic cells, the lysis of peripheral blood lymphocytes and CD34+ progenitor cells that lack expression of hsp70 on their plasma membrane was not negatively influenced by this treatment. A nonspecific disruption of the plasma membrane could be excluded, because treatment with a nontoxic concentration of the detergent Tween20 did not have an influence on hsp70 cell surface expression or on the sensitivity to lysis. Our findings might have further clinical implications with respect to purging of bone marrow from patients suffering from leukemia at sublethal conditions to induce a tumor-selective immune response.     

In vitro sensitivity of normal and leukemic myeloid clonogenic cells to hyperthermia: absence of selective effect

The in vitro heat sensitivity of myeloid clonogenic cells was tested in 22 normal marrows (granulo-monocytic progenitors) and in 40 marrows of patients with acute myelogenous leukemia (leukemic progenitors). Cells were treated for 1 h at 42 degrees, 43 degrees, 44 degrees, or 45 degrees C prior to plating. A temperature-dependent inhibition of growth was seen without a selective effect on the two kinds of progenitors. Because these two kinds of progenitors have the same heat sensitivity, hyperthermia should not be used alone as a technique for in vitro depletion of residual myeloid leukemic cells.     

Leukemic cells from murine myeloid leukemia display an intrinsic ability for autonomous proliferation

OBJECTIVE: Human acute myeloid leukemia (AML) cells can proliferate in vitro in the absence of added growth factors when cultured at high cell density. Autocrine growth factor production is a postulated mechanism of autonomous growth. We sought to examine this using murine AML cells. MATERIALS AND METHODS: We have utilized a Moloney murine leukemia virus (M-MuLV) model of AML to investigate the nature of autonomous in vitro growth of myeloid leukemic cells. RESULTS: Like human AML, M-MuLV-induced myeloid leukemic cells displayed autonomous growth in unstimulated high cell density cultures. However, replating of individual, primary, growth factor autonomous colonies of leukemic cells demonstrated the presence of clonogenic cells capable of autonomous growth when cultured at low cell density. In addition, there was heterogeneity in the progeny of these cells: both factor-dependent leukemic cells and cells autonomous of exogenous factor were observed. CONCLUSION: We propose that clonogenic cells capable of autonomous growth at low cell density represent leukemic progenitors while the majority of leukemic cells derived from these "autonomous" leukemic cells are factor-dependent.     

Purging of peripheral blood stem cell transplants in AML: a predictive model based on minimal residual disease burden

OBJECTIVE: Minimal residual disease (MRD) present in peripheral blood stem cell (PBSC) products of AML patients may contribute to relapse. Our goal was to 1) predict leukemia recurrence based on the frequency of MRD present in PBSC products, 2) establish the efficacy of different purging procedures, and 3) integrate this into a model that enables to predict whether or not to purge. METHODS: Minimal residual disease was measured with flow cytometry using leukemia-associated phenotypes as established at diagnosis. Toxicity of purging procedures was established using clonogenic assays. Purging procedures used were cryopreservation, hyperthermia, ether lipid ET-18-OCH3, and combinations. RESULTS: Minimal residual disease in PBSC products correlated significantly with relapse-free survival (n=24, p=0.003). At a cut-off value of 0.05% MRD the relative risk of relapse was 4.6 times lower in the group with less than 0.05% MRD. As measured in 54 PBSC products, the MRD level was less than 0.05% in 17 of 54 cases, between 0.05% and 0.5% in 19 of 54 cases, and higher than 0.5% in 18 of 54 cases. Based on the MRD cut-off of 0.05%, the log tumor reduction needed to achieve this threshold is zero for the 17 of 54 cases in which MRD was below 0.05%, less than or equal to 1 log in 19 of 54 cases, and greater than 1-2 log in 18 of 54 cases. When applying purging with 25 mug/mL ET-18-OCH3 combined with cryopreservation at 10% DMSO and hyperthermia at 42 degrees C combined with cryopreservation at 10% or 4% DMSO, there was greater than or equal to 1 log depletion of AML blasts. CONCLUSION: This study establishes (1) a threshold level for MRD above which prognosis is worse, (2) that stem cell products from 69% of patients have higher than this "safe" MRD level, and (3) that ET-18-OCH3 and hyperthermia may be used to purge products in part of these patients.     

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

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

Purging murine leukemic marrow with alkyl-lysophospholipids

Autologous bone marrow transplantation is potentially curative in the treatment of acute leukemia if residual leukemic cells in the marrow can be eliminated prior to transplantation. We studied the purging effects of a synthetic alkyl-lysophospholipid (ALP) on marrow containing leukemic cells from a transplantable myelomonocytic leukemia (WEHI-3B) in BALB/c mice. Simulated remission bone marrow containing 2% leukemic cells treated in vitro with 20 and 100 micrograms/mL of ET-18- OCH3 (1-octadecyl-2-methyl-sn-glycerol-3-phosphocholine) significantly prolonged survival of lethally irradiated transplanted recipients. At a dose of 100 micrograms/mL, 88% of the mice survived for the duration of the experiment (approximately five months). Autopsies showed that 25% of these survivors had microscopic evidence of leukemia. Thus, in vitro treatment of marrow eliminated leukemic blasts and spared sufficient normal stem cells to allow hematologic reconstitution. The effect of ET- 18-OCH3 is not entirely selective for leukemic cells. A spleen colony assay showed that ALP has some cytotoxic effect on normal hematopoietic stem cells.     

Antileukemic activity of rapamycin in acute myeloid leukemia

The mammalian target of rapamycin (mTOR) is a key regulator of growth and survival in many cell types. Its constitutive activation has been involved in the pathogenesis of various cancers. In this study, we show that mTOR inhibition by rapamycin strongly inhibits the growth of the most immature acute myeloid leukemia (AML) cell lines through blockade in G0/G1 phase of the cell cycle. Accordingly, 2 downstream effectors of mTOR, 4E-BP1 and p70S6K, are phosphorylated in a rapamycin-sensitive manner in a series of 23 AML cases. Interestingly, the mTOR inhibitor markedly impairs the clonogenic properties of fresh AML cells while sparing normal hematopoietic progenitors. Moreover, rapamycin induces significant clinical responses in 4 of 9 patients with either refractory/relapsed de novo AML or secondary AML. Overall, our data strongly suggest that mTOR is aberrantly regulated in most AML cells and that rapamycin and analogs, by targeting the clonogenic compartment of the leukemic clone, may be used as new compounds in AML therapy.     

Enhanced effect of mutant granulocyte-colony-stimulating factor (KW-2228) on the growth of normal and leukemic hemopoietic progenitor cells in comparison with recombinant human granulocyte-colony-stimulating factor (G-CSF).

We tested the in vitro effect of a novel granulocyte colony-stimulating factor (G-CSF) derivative (KW-2228) on the growth of G-CSF-dependent hemopoietic progenitor cells: granulocyte precursor cells (CFU-G), leukemic blast progenitors freshly obtained from 9 patients with acute myeloblastic leukemia (AML) and cells of a G-CSF-dependent human AML cell line (OCI/AML 1a). KW-2228 showed a higher stimulating effect than recombinant human G-CSF (rhCSF) on CFU-G; 3 out of 9 leukemic blast progenitors and OCI/AML 1a cells. The difference in biochemical stability between rhG-CSF and KW-2228 was considered to explain the superior colony-stimulating activity of KW-2228. The results show that KW-2228 will be a new granulopoietic factor.     

Autocrine growth mechanisms of the progenitors of blast cells in acute myeloblastic leukemia

Autocrine growth mechanisms of leukemic blast progenitors in acute myeloblastic leukemia (AML) were investigated. Colony formation of leukemic blast progenitors was observed in 14 of 14 patients tested when purified blast cell fraction depleted of both T cells and monocytes was plated in methylcellulose without any colony-stimulating factor (CSF). However, there existed a minimal cell density required to initiate blast progenitor growth with marked patient-to-patient variation. To clarify the role of cell density on the spontaneous growth of blast progenitors, we tested whether leukemic cells produced and secreted some stimulatory humoral factor(s). Production of colony- stimulating activity (CSA) by blast cells was observed in 17 of 18 patients tested. Following further depletion of monocytes, the CSA levels decreased markedly in 14 patients, indicating that blast cells with monocytoid differentiation were responsible for CSA production. We also confirmed granulocyte colony-stimulating factor (G-CSF) and/or granulocyte macrophage-colony-stimulating factor (GM-CSF) production by leukemic blasts using specific immunologic assays. When leukemic cells were divided into nonadherent nonphagocytic cell fraction and adherent cell fraction, only nonadherent nonphagocytic cells showed clonogenecity and adherent blast cells lacked the colony-forming capacity. The results indicate that there are at least two blast cell subpopulations in AML: one is proliferating subpopulation with self- renewal capacity and the other is supporting subpopulation with functions such as CSF production. The quite intimate relationship between these two blast cell subpopulations in AML may play an important role on the growth of leukemic blast progenitors in vitro.     

Normal and malignant human myeloid progenitors differ in their sensitivity to hyperthermia

The effect of in vitro hyperthermia on normal human bone marrow granulocyte-macrophage progenitor cells (granulocyte-macrophage colony-forming units, CFU-GM) was compared to its effect on clonogenic acute nonlymphocytic leukemic (ANLL) cells. Mononuclear normal bone marrow cells, blasts from patients with ANLL, and HL-60 cells were incubated at room temperature (control) and at 42 degrees-44 degrees C for 0-120 min prior to assay in methylcellulose. The heat sensitivity of the leukemic cells was significantly greater than that of normal bone marrow progenitors. Two-h exposure to 43 degrees C, for example, resulted in survival of 52% of normal marrow CFU-GM, whereas only 3% of leukemic CFU-GM survived (p less than 0.001 for HL-60 cells and p less than 0.005 for patient blast cells). To determine the effect of hyperthermia on more primitive progenitors and on marrow stromal cells, long-term cultures of normal bone marrow were established using control and heat-treated cells. Generation of CFU-GM was detected in the nonadherent fraction of hyperthermia-treated samples throughout the 5-week culture period. Although stromal development was slightly delayed, hyperthermia-treated cells were able to establish stromal layers similar to control cells. These results indicate that normal bone marrow committed progenitor cells are more resistant to hyperthermia than are myeloid leukemic cells. Normal stromal cells and primitive cells assayed in long-term culture are also resistant to hyperthermia that is toxic for leukemic cells. Because of this differential sensitivity to heat, ex vivo hyperthermia may be applicable for removing residual leukemic cells from bone marrow harvested for autologous transplantation.     

Purging of acute myeloid leukaemia cells from stem cell grafts by hyperthermia: enhancement of the therapeutic index by the tetrapeptide AcSDKP and the alkyl-lysophospholipid ET-18-OCH(3)

Hyperthermia has been shown to be a potential purging modality in autologous stem cell transplantation settings owing to its selective toxicity towards leukaemic cells. We describe two approaches to further increase the therapeutic index of the hyperthermic purging modality by using normal murine bone marrow cells and a murine model for acute myeloid leukaemia. First, the tetrapeptide AcSDKP was used to protect the normal haematopoietic progenitor cells against hyperthermic damage. Pretreatment for 8 h at 37 degrees C with 1 x 10(-9) mol/l AcSDKP resulted in a decrease in hyperthermic sensitivity of only normal haematopoietic progenitor cells. This combined treatment protocol revealed a therapeutic index (ratio of surviving fractions of normal vs. leukaemic cells) of > 500, which was considered to be sufficient for purging. This was confirmed in vivo by the survival of lethally irradiated recipients transplanted with purged simulated remission bone marrow (1 x 10(6) normal bone marrow cells and 5 x 10(4) leukaemic cells). A further increase of the therapeutic index cells was achieved by the alkyl-lysophospholipid ET-18-OCH(3). An incubation for 4 h at 37 degrees C with 25 microg/ml in the presence of 5% fetal calf serum preferentially enhanced the cytotoxic effect towards the leukaemic stem cell. The combination of AcSDKP and ET-18-OCH(3) with hyperthermia resulted in a therapeutic index of > 5000. This enabled a reduction of the hyperthermic treatment and will further minimize the toxicity to normal haematopoietic stem cell subsets, while a therapeutic index far above the required value is achieved. This tripartite purging treatment therefore offers a safe and fast purging protocol for the elimination of residual leukaemic cells in autografts.     

A novel CD34-specific T-cell engager efficiently depletes acute myeloid leukemia and leukemic stem cells in vitro and in vivo

Less than a third of patients with acute myeloid leukemia (AML) are cured by chemotherapy and/or hematopoietic stem cell transplantation, highlighting the need to develop more efficient drugs. The low efficacy of standard treatments is associated with inadequate depletion of CD34⁺ blasts and leukemic stem cells, the latter a drug-resistant subpopulation of leukemia cells characterized by the CD34⁺CD38^- phenotype. To target these drug-resistant primitive leukemic cells better, we have designed a CD34/CD3 bi-specific T-cell engager (BTE) and characterized its anti-leukemia potential in vitro, ex vivo and in vivo. Our results show that this CD34-specific BTE induces CD34-dependent T-cell activation and subsequent leukemia cell killing in a dose-dependent manner, further corroborated by enhanced T-cell-mediated killing at the singlecell level. Additionally, the BTE triggered efficient T-cell-mediated depletion of CD34⁺ hematopoietic stem cells from peripheral blood stem cell grafts and CD34⁺ blasts from AML patients. Using a humanized AML xenograft model, we confirmed that the CD34-specific BTE had in vivo efficacy by depleting CD34⁺ blasts and leukemic stem cells without side effects. Taken together, these data demonstrate that the CD34-specific BTE has robust antitumor effects, supporting development of a novel treatment modality with the aim of improving outcomes of patients with AML and myelodysplastic syndromes.