AML-derived Cells Research Articles

Cardiac glycoside ouabain efficiently targets leukemic stem cell apoptotic machinery independent of cell differentiation status

BackgroundAcute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by an accumulation of immature leukemic myeloblasts initiating from leukemic stem cells (LSCs)—the subpopulation that is also considered the root cause of chemotherapy resistance. Repurposing cardiac glycosides to treat cancers has gained increasing attention and supporting evidence, but how cardiac glycosides effectively target LSCs, e.g., whether it involves cell differentiation, remains largely unexplored.MethodsDigoxin, a user-designed digitoxigenin-α-L-rhamnoside (D6-MA), and ouabain were tested against various human AML-derived cells with different maturation phenotypes. Herein, we established two study models to specifically determine the effects of cardiac glycosides on LSC death and differentiation—one allowed change in dynamics of LSCs and leukemic progenitor cells (LPCs), while another maintained their undifferentiated status. Regulatory mechanisms underlying cardiac glycoside-induced cytotoxicity were investigated and linked to cell cycle distribution and apoptotic machinery.ResultsPrimitive AML cells containing CD34+ LSCs/LPCs were very responsive to nanomolar concentrations of cardiac glycosides, with ouabain showing the greatest efficiency. Ouabain preferentially induces caspase-dependent apoptosis in LSCs, independent of its cell differentiation status, as evidenced by (i) the tremendous induction of apoptosis by ouabain in AML cells that acquired less than 15% differentiation and (ii) the higher rate of apoptosis in enriched LSCs than in LPCs. We sorted LSCs and LPCs according to their cell cycle distribution into G0/G1, S, and G2/M cells and revealed that G0/G1 cells in LSCs, which was its major subpopulation, were the top ouabain responders, indicating that the difference in ouabain sensitivity between LSCs and LPCs involved both distinct cell cycle distribution and intrinsic apoptosis regulatory mechanisms. Further, Mcl-1 and c-Myc, which were differentially expressed in LSCs and LPCs, were found to be the key apoptosis mediators that determined ouabain sensitivity in AML cells. Ouabain induces a more rapid loss of Mcl-1 and c-Myc in LSCs than in LPCs via the mechanisms that in part involve an inhibition of Mcl-1 protein synthesis and an induction of c-Myc degradation.ConclusionsOur data provide new insight for repurposing cardiac glycosides for the treatment of relapsed/refractory AML through targeting LSCs via distinct cell cycle and apoptosis machinery.53GxHmRcFt-KhVdMmSCJrsVideo

Synthesis and anti-tumor activities in human leukemia-derived cells of polyenylpyrroles with a methyl group at the conjugated polyene terminus

To develop novel drugs for treating T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML) which are highly malignant hematological tumors, a series of analogs having a polyenylpyrrole structure of natural compounds (rumbrin and auxarconjugatin B) were synthesized and investigated their structure–activity relationships (SAR) of in vitro anti-T-ALL and anti-AML activities. We obtained three findings: (1) introduction of a methyl group at the conjugated polyene terminus enhanced anti-T-ALL activity, (2) analogs with a 3-chloropyrrole moiety had even higher selectivity for T-ALL cells, and (3) some analogs were effective against AML-derived cells. Among the studied compounds, 3-chloro-2-(8-ethoxycarbonylnona-1,3,5,7-tetraenyl) pyrrole 4e was the most promising candidate of T-ALL- and AML-treating drug. This study provides useful structural information for designing novel drugs treating T-ALL and AML.

Anticancer Effects of ZnO/CNT@Fe3O4 in AML-Derived KG1 Cells: Shedding Light on Promising Potential of Metal Nanoparticles in Acute Leukemia.

Background: Therapeutic approaches for acute myeloid leukemia (AML) have remained largely unchanged for over 40 years and cytarabine and an anthracycline (e.g., daunorubicin) backbone is the main induction therapy for these patients. Resistance to chemotherapy is the major clinical challenge and contributes to short-term survival with a high rate of disease recurrence. Given the established efficacy of nanoparticles in cancer treatment, this study was designed to evaluate the anticancer property of our novel nanocomposite in the AML-derived KG1 cells. Materials and Methods: To assess the anti-leukemic effects of our nanocomposite on AML cells, we used MTT and trypan blue assays. Flow cytometric analysis and q-RT-PCR were also applied to evaluate the impact of nanocomposite on cell cycle and apoptosis. Results: Our results outlined that ZnO/CNT@Fe3O4 decreased viability and metabolic activity of KG1 cells through induction of G1 arrest by increasing the expression of p21 and p27 cyclin-dependent kinase inhibitors and decreasing c-Myc transcription. Moreover, ZnO/CNT@Fe3O4 markedly elevated the percentage of apoptotic cells which was coupled with a significant alteration of Bax and Bcl-2 expressions. Synergistic experiments showed that ZnO/CNT@Fe3O4 enhances the cytotoxic effects of Vincristine on KG1 cells. Conclusion: In conclusion, this study sheds light on the potent anti-leukemic effects of ZnO/CNT@Fe3O4 and provides evidence for the application of this agent in the treatment of acute myeloid leukemia.

Cytotoxic Effects of Britannin on Acute and Chronic Myeloid Leukemia Cells Through Inducing p21-Mediated Apoptotic Cell Death.

Following the success of natural compounds for treating solid tumors, interest in applying such agents for treating hematologic malignancies has been fired up more strikingly. Thus far, anti-leukemic effects of several compounds have been examined in different leukemia cell lines, especially in acute lymphoblastic leukemia. The agent that has recently attracted tremendous attention is Britannin, which is derived from Inula aucheriana DC., a plant that grows in Iran (Azerbaijan) and Türkiye. In this study, we evaluated the effects of this compound in myeloid leukemia for the first time. We treated chronic myeloid leukemia (CML)-derived K562 and acute myeloid leukemia (AML)-derived U937 cells with different concentrations of britannin. We used several assays, including trypan blue, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, bromodeoxyuridine/5-bromo-2'-deoxyuridine, flow cytometry, and quantitative real-time polymerase chain reaction analysis, to study anti-leukemic effects of the compound. Our results show that while britannin remarkably reduced the survival of both cell lines in a concentrations-dependent manner, it had cytotoxic effects neither on mouse fibroblast-derived L929 cells nor on normal peripheral mononuclear cells. Moreover, among the tested cell lines, the viability of CML-derived K562 cells was inhibited at higher concentrations of the compound compared with AML-derived U937 cells. We found that britannin induced apoptotic cell death in both cell lines by altering the expression of anti- and pro-apoptotic genes. Britannin also hampered proliferative capacity of the cells in a p21/p27-dependent manner. Overall, we suggest that based on the lack of toxicity on the normal cells and valuable anti-leukemic activities, britannin could be a promising agent in the treatment strategies of both CML and AML. However, further investigations must more precisely study this compound's mechanism of action and evaluate its safety profile.

Scalable Enrichment of Immunomodulatory Human Acute Myeloid Leukemia Cell Line-Derived Extracellular Vesicles.

Acute myeloid leukemia (AML) cells can secrete trophic factors, including extracellular vesicles (EVs), instructing the stromal leukemic niche. Here, we introduce a scalable workflow for purification of immunomodulatory AML-EVs to compare their phenotype and function to the parental AML cells and their secreted soluble factors. AML cell lines HL-60, KG-1, OCI-AML3, and MOLM-14 released EVs with a peak diameter of approximately 80 nm in serum-free particle-reduced medium. We enriched EVs >100x using tangential flow filtration (TFF) and separated AML-derived soluble factors and cells in parallel. EVs were characterized by electron microscopy, immunoblotting, and flow cytometry, confirming the double-membrane morphology, purity and identity. AML-EVs showed significant enrichment of immune response and leukemia-related pathways in tandem mass-tag proteomics and a significant dose-dependent inhibition of T cell proliferation, which was not observed with AML cells or their soluble factors. Furthermore, AML-EVs dose-dependently reduced NK cell lysis of third-party K-562 leukemia targets. This emphasizes the peculiar role of AML-EVs in leukemia immune escape and indicates novel EV-based targets for therapeutic interventions.

Acute myeloid leukemia maturation lineage influences residual disease and relapse following differentiation therapy

Acute myeloid leukemia (AML) is a malignancy of immature progenitor cells. AML differentiation therapies trigger leukemia maturation and can induce remission, but relapse is prevalent and its cellular origin is unclear. Here we describe high resolution analysis of differentiation therapy response and relapse in a mouse AML model. Triggering leukemia differentiation in this model invariably produces two phenotypically distinct mature myeloid lineages in vivo. Leukemia-derived neutrophils dominate the initial wave of leukemia differentiation but clear rapidly and do not contribute to residual disease. In contrast, a therapy-induced population of mature AML-derived eosinophil-like cells persists during remission, often in extramedullary organs. Using genetic approaches we show that restricting therapy-induced leukemia maturation to the short-lived neutrophil lineage markedly reduces relapse rates and can yield cure. These results indicate that relapse can originate from therapy-resistant mature AML cells, and suggest differentiation therapy combined with targeted eradication of mature leukemia-derived lineages may improve disease outcome.

Cell-Autonomous Feedback Loop of RUNX1-p53-CBFB in Acute Myeloid Leukemia Cells

Although runt-related transcription factor 1 (RUNX1) and its associating core binding factor-β (CBFB) play pivotal roles in leukemogenesis and inhibition of RUNX1 has now been widely recognized as a novel strategy in anti-leukemic therapies, it remains elusive how leukemic cells acquire resistance to RUNX1 inhibition therapies. Here, we show evidence that p53 (TP53) and CBFB are sequentially up-regulated in response to RUNX1 depletion in acute myeloid leukemia (AML) cells. The up-regulated CBFB expression in turn contributes to the stabilization of RUNX1, thereby constituting the RUNX1-p53-CBFB auto-regulatory feedback loop. To address RUNX1 depletion-mediated cellular responses in AML cells, we first performed shRNA-mediated knockdown experiments in human AML-derived MV4-11 and MOLM-13 cells, and investigated the expression of other CBF family proteins such as RUNX2, RUNX3 and CBFB. As we have previously reported, the expression of RUNX2 and RUNX3 were consistently elevated upon RUNX1 depletion. It has been known that CBFB stabilizes the RUNX transcription complex and enhances its DNA-binding capability. Based on this knowledge, we had expected that depletion of RUNX1 possibly disrupts the stable RUNX1/CBFB complex and thus down-regulates CBFB protein expression by destabilizing it. Contrary to our expectations, however, the expression of CBFB was also significantly up-regulated in response to RUNX1 -silencing in AML cells. Knockdown of RUNX2 and/or RUNX3 in addition to RUNX1 further induced CBFB expressions. Considering that the anti-tumor potency of RUNX gene silencing is highly dependent on functional p53 with the sequence-specific transactivation capability, we have sought to examine a possible involvement of p53 in the transcriptional regulation of CBFB. Indeed, series of RUNX family knockdown experiments elucidated that the expression levels of CBFB induced are proportional to the extent of p53 induction. In addition, we found that the expression levels of p53 and CBFB are positively-correlated in de novo AML patients from two independent studies (GSE22845; n = 154, GSE21261; n = 96). In a good agreement with these observations, knockdown of p53 in AML cells caused obvious reduction in CBFB relative to the control. Furthermore, ChIP-qPCR assay validated the actual binding of p53 at the promoter regions of CBFB. Given these information, RUNX1 depletion-mediated up-regulation of CBFB is under the control of the accumulated p53, and thus creating an autonomous RUNX1-p53-CBFB feedback regulatory system in AML cells. Moreover, AML cells derived from relapsed cases exhibited higher CBFB expression levels compared to those from primary AML cells at diagnosis (GSE17855 and GSE52891, n = 23). Consistent with these observations, RUNX1 inhibition treatment-resistant MV4-11 cells established from 4-month in vitro selection with the presence of specific RUNX inhibitors expressed higher CBFB relative to RUNX1 inhibition-sensitive ones. Besides, the ectopic expression of CBFB conferred resistance to RUNX1 inhibition therapy. Although the majority of primary de novo AML cases harbor wild-type p53, a small number of AML patients carry p53 mutations. According to our RUNX1-p53-CBFB loop working model, the stabilized mutant p53 possibly augments this feedback regulatory loop through the direct transactivation of CBFB and potentially contributes to the acquired resistance to RUNX1-inhibition therapy. We thus asked whether p53 mutants could up-regulate CBFB expression. To this end, we have prepared 3 representative p53 mutants (R175H, R248W, R273C) and transfected them into HEK293T cells. Intriguingly, these mutants significantly induced CBFB expressions. In addition, deep sequencing of genomic DNA extracted from the above-mentioned RUNX1 inhibition-resistant AML cells revealed that the majority of them acquire p53-stabilizing point mutations during treatment, possibly contributing to the enhanced transactivation of CBFB . These observations strongly suggest the RUNX inhibition-mediated treatment permits the selective proliferation of p53 -mutated AML cells and these cells acquire the enhanced tumorigenicity through the RUNX1-p53-CBFB axis. Collectively, our present results underscore the importance of RUNX1-p53-CBFB regulatory loop in the development and/or maintenance of AML cells, which could be targeted in strategizing anti-leukemia therapies. DisclosuresNo relevant conflicts of interest to declare.

RUNX1 Enhances Leukemia Cell Engraftment in the Vascular Niche through up-Regulating E-Selectin

Although the function of runt-related transcription factor 1 (RUNX1) has been well-characterized in leukemogenesis and regarded as an ideal target in anti-leukemia strategies, neither the effect of RUNX1 inhibition therapy on the bone marrow niche cells nor its impact on the engraftment of acute myeloid leukemia (AML) cells has largely been unknown. Here we show evidence that RUNX1 inhibition attenuates the expression of E-selectin in the vascular endothelial cells of bone marrow niche, thereby disrupting the homing of AML cells to the bone marrow niche, ultimately extending the overall survival period in AML mice models. In order to investigate the effect of RUNX1 inhibition on endothelial vascular niche cells, firstly we identified and focused on E-selectin from significant affected factors, such as E-selectin, P-selectin, VCAM 1 and TIE 2 through the screening the expressions of representative surface molecules in human umbilical vein endothelial cells (HUVEC) treated with our selective RUNX inhibitor : Chb-M'. E-selectin has recently been recognized as a vital component of vascular niche and fundamentally involved in the housing of hematopoietic stem cells (HSCs). We also genetically attenuated the expression of RUNX1 in HUVEC with doxycycline-inducible short hairpin RNA (shRNA). As we have expected, the expression of E-selectin was tightly and most profoundly down-regulated both at the transcript and surface expression levels upon RUNX1 -silencing. Of note, neither Chb-M'-treatment nor shRNA-mediated RUNX1 knockdown resulted in significant change in the expressions of niche-associated surface molecules in AML-derived MV4-11 cells. In addition, neither Chb-M'-treatment nor RUNX1 knockdown resulted in significant change in the expression of osteoblastic niche components in osteosarcoma-derived HOS cells. These results suggest that RUNX1 potentially and primordially be involved in the regulation of E-selectin in the endothelial vascular niche cells. Besides, the activity of E-selectin proximal promoter was increased upon additional RUNX1 expression, while the modified E-selectin promoter which lacks RUNX consensus binding site (5'-TGTGGT-3') dramatically decreased its luciferase reporter activity, confirming that RUNX1 directly transactivates E-selectin expression. We next examined the effect of RUNX1 attenuation on E-selectin expression in murine endothelial vascular niche cells through several assays. Treatment of C56BL/6 mice for 2 weeks with either E-selectin inhibitor: andrographolide, A205804 or RUNX-inhibitor: Chb-M' showed effective decreasing of E-selectin expression on endothelial vascular niche cells. We next retrovirally-transduced MLL-ENL to c-kit+ mice bone marrow cells and obtained mice leukemic cells. Using these mice leukemic cells, we investigated the effect of Runx1 inhibition therapy on the homing ability of leukemia cells to their bone marrow niche. We studied and confirmed the effect of RUNX1 inhibition on the homing ability of leukemic cells by both in vitro static assays and in vivo mouse models. Mise treated with andrographolide, A 205804 or Chb-M' for 2 weeks, mouse leukemic cells of MLL-ENL+ mice were transplanted. All these treatments significantly decreased the number of homed leukemic cells in the bone marrow compared to the vehicle-treated mice, underscoring the importance of E-selectin in the engraftment of AML cells in the vascular niche. Consistent with these observations, conditioning bone marrow vascular niche with Chb-M' before transplantation of leukemic cells significantly prolonged the overall survival period of AML mice with Runx inhibition therapy by Chb-M' or vehicle control treatment. Our work collectively indicates that RUNX -silencing drugs such as Chb-M' not only directly control the growth of AML cells themselves, but also indirectly enhance its anti-leukemic potential through attenuating E-selectin expressions in the bone marrow vascular niche. These drugs potentially bring the leukemic stem-like cells out from deep in the bone marrow niche to the peripheral blood stream and expose these primordial leukemia cells to chemotherapeutic drugs at much higher concentrations. DisclosuresNo relevant conflicts of interest to declare.

Controlling Hematopoietic Stem Cell Quiescence By Ablating the TGFβ-Responsive, Hematopoietic-Hpecific Enhancer of CDKN1C (p57)

Introduction: The cyclin-dependent kinase inhibitor (CDKI) CDKN1C (p57) is a putative tumor suppressor gene with strong differential expression in both human and murine hematopoietic stem cells (HSCs). The most immature, quiescent HSCs are marked by p57 expression and p57 is rapidly downregulated as HSCs enter cell cycle and differentiate. Genetic deletion of p57 leads to abnormal HSC cell cycle entry and, in murine models, a propensity for HSC exhaustion indicating that p57 is an essential gatekeeper of HSC function. TGFβ signaling is an important mediator of HSC quiescence and mandates the withdrawal of HSCs from cell cycle following hematopoietic stress. We have found that p57 is a direct transcriptional target of TGFβ in HSCs where it serves as an essential downstream mediator of TGFβ signaling. In HSCs, genetic deletion of p57 phenocopies the effects of TGFβ blockade in vivo. The regulation of p57 is complex and interesting. In almost all epithelial cell types, p57 is poorly expressed and is not a transcriptional target of TGFβ. This is also true for most differentiated hematopoietic lineages. The mechanism for the tissue-type and differentiation stage-specific expression of p57 and its control by TGFβ in HSCs is not known. We hypothesized that a hematopoietic enhancer for p57 must be responsible for TGFβ induction in HSCs.Methods: We performed chromatin immunoprecipitation (ChIP/ChIP-chip/ChIP-seq) in human, AML-derived M091 cells that faithfully upregulate p57 in response to TGFβ signaling in a manner indistinguishable from human cord blood derived CD34+ cells. Cultured cells were studied while proliferating and following treatment with TGFβ for various times. ChIP was performed for histone modifications that mark enhancer elements (histone H3 lysine 4 mono-methylation, H3K4me1, and H3K27-acetlylation, H3K27ac) and promoters (histone H3 lysine 4 tri-methylation, H3K4me2, and H3K27-acetlylation, H3K27ac). Genomic localization of receptor activated phospho-Smad2 was also identified before and after TGFβ stimulation. Putative enhancers were cloned into reporter constructs that were then studied after stable incorporation into the genome of M091 and K562 cells.Results: We identified putative enhancer elements more than 100 KB from the p57 gene body (Fig. A). A cluster of three small elements fulfilled our biochemical criterion for a hematopoietic-specific enhancer: enhancer histone marks (H3K4me1/H3K27ac); hyper-acetylation and phospho-Smad2 binding in response to TGFβ (Fig. B, D). When subcloned and tested in reporter assays, the elements had enhancer function and two of the three were induced by TGFβ (Fig. C). Interestingly, the ENCODE consortium identified the genomic region of our putative enhancers as overlapping with a DNAseI hypersensitive site and harboring the chromatin signature of a “Strong Enhancer” only in hematopoietic cells (K562) but not in other cell type tested. Nonetheless, unequivocal classification of this element as a p57 enhancer required in vivo analysis of HSCs with targeted deletion of the enhancer element.We designed and cloned, ten guide-RNAs (gRNAs) targeting regions upstream and downstream of this putative enhancer cluster. These gRNAs were then tested in HeLa cells by transient transfection with wild-type Cas9 nuclease. Using this approach, we selected a pair of gRNAs with the highest efficiency in deleting the entire enhancer cluster as assessed by ddPCR (~30% alleles). These gRNAs or non-targeting control gRNAs and Cas9 were then transfected into M091 cells along with a GFP reporter. Transfected GFP+ cells were then sorted 36 hrs later. A second lot of unsorted cells were grown in bulk cultures. Allelic ratios of this enhancer element were tracked by ddPCR to assess the relative outgrowth of genomically unmodified cells compared to those that had this putative p57 enhancer deleted. The targeted MO91 population has exhibited resistance to TGFβ as assessed using a growth curve assay.Conclusions: A hematopoietic-specific enhancer lying ~100 kb from the p57 gene body is important for TGFβ-mediated upregulation of p57 and consequent cell cycle withdrawal in immature hematopoietic cells. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

PI3K Abrogation Using Pan-PI3K Inhibitor BKM120 Gives Rise to a Significant Anticancer Effect on AML-Derived KG-1 Cells by Inducing Apoptosis and G2/M Arrest

Objective:The association between PI3K overexpression and the acquisition of chemoresistance has attracted tremendous attention to this axis as an appealing target to revolutionize the conventional treatment strategies of human cancers. In the present study, we aimed to survey the inhibitory impact of the pan-PI3K inhibitor BKM120 on both cellular and molecular aspects of acute myeloid leukemia (AML)-derived KG-1 and U937 cells.Materials and Methods:We designed various assays to survey the antitumor impacts and molecular mechanisms underlying the action of BKM120 for the treatment of AML, and we performed experiments to check the effect of BKM120 in combination with idarubicin.Results:We found that PI3K inhibition diminished cell viability and metabolic activity and exerted a concentration-dependent growth-suppressive effect on the cells. Moreover, we suggested that the ability of BKM120 to induce its antiproliferative properties was mediated through the induction of p21-mediated G2/M cell-cycle arrest. Investigating the effect of inhibitor on the molecular features revealed not only that BKM120 reduced the expression of NF-κB antiapoptotic targets, but also that NF-κB suppression using bortezomib profoundly enhanced the cytotoxicity of the inhibitor, highlighting that the antileukemic effects of BKM120 are mediated, at least partly, through the modulation of the NF-κB pathway. Interestingly, we found that the single agent of BKM120 was unable to significantly alter the expression level of c-Myc; however, the capability of BKM120 to reduce the survival rate of AML cells was potentiated upon c-Myc inhibition using 10058-F4, suggestive of the plausible contribution of c-Myc in leukemic cell response to the PI3K inhibitor.Conclusion:Taken together, the results of this study reveal the efficacy of BKM120 as a therapeutic approach for AML; however, further investigations should be undertaken to determine the expediency of this inhibitor.

Evaluation of a Bifunctional Sirpα-CD123 Fusion Antibody for the Elimination of Acute Myeloid Leukemia Stem Cells

Background: Despite considerable advances in the development of novel strategies for the treatment of acute myeloid leukemia (AML) the relapse rate is still high with only limited treatment options. Relapse occurs due to the persistence of chemotherapy-resistant leukemic stem cells (LSCs), which re-initiate outgrowth of the disease, highlighting the need of targeting LSCs to improve overall survival. Immunotherapies represent a promising strategy to target chemotherapy-resistant LSCs in AML. LSCs are characterized by the expression of the interleukin-3 receptor α, also known as CD123. CD123 is expressed on AML blasts and LSCs, and shows only a moderate expression on normal hematopoietic stem cells, claiming CD123 as a suitable target antigen (Haubner et al, Leukemia 2019). CD47, known as a marker of self, is also highly expressed on LSCs as immune escape mechanism. CD47 transmits a "don't eat me" signal upon its interaction with the myeloid-specific signal regulatory protein alpha (SIRPα) receptor on macrophages, thus inhibiting phagocytosis. In order to efficiently eliminate LSCs and provide AML patients a possibility for prolonged relapse-free survival, we have designed a bifunctional antibody that specifically targets CD123 and simultaneously blocks CD47. Importantly, our strategy restricts the benefits of the CD47 blockade to CD123 positive AML cells. Thus, we hypothesize a lower risk for on-target off-leukemia toxicity. Methods: The bifunctional SIRPα-CD123 antibody was generated by fusing the endogenous extracellular domain of SIRPα, which functions as the CD47 blocking domain, to an CD123 antibody CD123. We assessed the selective binding of the bifunctional antibody to CD123+CD47+ AML-derived cells and the ability to block CD47 on CD123+ cells in vitro. Furthermore, the biological activity of the SIRPα-CD123 antibody was examined using the AML-derived cell line MOLM-13, patient-derived xenografted (PDX) AML cells as well as primary cells from patients with newly diagnosed or relapsed AML. Results: We engrafted the endogenous SIRPα V-like domain to an antibody targeting CD123, which improved the binding of the bifunctional SIRPα-CD123 antibody to AML cells compared to a conventional CD123 antibody (MFI ratioCD123 = 2.46 0.25 vs MFI ratioSIRPα-CD123 = 4.44 0.60). The SIRPα-CD123 antibody enhanced the elimination of the AML-derived MOLM-13 cells by antibody-dependent cellular cytotoxicity (EC50CD123 = 38.5 pM vs EC50SIRPα-CD123 = 10.1 pM, n = 9). Additionally, the cytotoxicity was confirmed using primary patient-derived AML cells ex vivo. Further, an improved ex vivo cytotoxicity towards AML PDX cells was observed with the SIRPα-CD123 antibody (% lysis at 100 nM: 14.27 5.40 vs 42.94 10.21 for CD123 and SIRPα-CD123 antibodies respectively, n = 3). With regards to the inhibition of CD47 signaling, we were able to show a blockade of CD47 on CD123+CD47+ positive cells by the SIRPα-CD123 antibody. Correspondingly, a significant increase in phagocytosis of primary patient-derived AML cells mediated by monocyte-derived macrophages was observed in allogenic as well as autologous settings (% phagocytosis, normalized to isotype control and maximum phagocytosis in an autologous setting: 20.11 4.59 vs 90.37 6.22, n = 5 for CD123 and SIRPα-CD123 antibodies, respectively). We were further able to show a preferential binding to MOLM-13 in the presence of a 20-fold excess of red blood cells indicating a potential low on-target off-leukemia toxicity. Taken together, our in vitro data supports the elimination of the CD123+CD47+ positive AML LSC compartment by a synergistic effect of avidity-dependent binding to CD123 and CD47 and the simultaneous inhibition of the innate immune CD47-SIRPα signaling pathway. Conclusions: The SIRPα-CD123 is a bifunctional antibody with the potential to deplete CD123+CD47+ AML LSCs by a dual mode of action mechanism resulting in NK cell dependent cytotoxicity and macrophage-mediated phagocytosis. By combining a high affinity binding to CD123+ cells and a low affinity CD47 blockade that is restricted to CD123+ cancer cells we effectively minimize the risk for CD47-related on-target off-leukemia toxicity. The results of our in vitro assays using AML cell lines are consistent with the data from PDX and primary AML samples and support further preclinical testing of the SIRPα-CD123 antibody in vivo. Disclosures Subklewe: Miltenyi: Research Funding; Pfizer: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Research Funding; AMGEN: Consultancy, Honoraria, Research Funding; Oxford Biotherapeutics: Research Funding; Roche: Consultancy, Research Funding; Celgene: Consultancy, Honoraria; Morphosys: Research Funding; Janssen: Consultancy.

Effect of Fms-like tyrosine kinase 3 (FLT3) ligand (FL) on antitumor activity of gilteritinib, a FLT3 inhibitor, in mice xenografted with FL-overexpressing cells.

Therapeutic effects of FLT3 inhibitors have been reported in acute myeloid leukemia (AML) with constitutively activating FLT3 mutations, including internal tandem duplication (ITD) and point mutation, which are found in approximately one-third of AML patients. One of the critical issues of treatment with FLT3 inhibitors in FLT3-mutated AML is drug resistance. FLT3 ligand (FL) represents a mechanism of resistance to FLT3 inhibitors, including quizartinib, midostaurin, and sorafenib, in AML cells harboring both wild-type and mutant FLT3 (FLT3wt/FLT3mut). Here, we investigated the effect of FL on the efficacy of gilteritinib, a FLT3 inhibitor, in AML-derived cells in vitro and in mice. In contrast to other FLT3 inhibitors, FL stimulation had little effect on growth inhibition or apoptosis induction by gilteritinib. The antitumor activity of gilteritinib was also comparable between xenograft mouse models injected with FL-expressing and mock MOLM-13 cells. In the FLT3 signaling analyses, gilteritinib inhibited FLT3wt and FLT3-ITD to a similar degree in HEK293 and Ba/F3 cells, and similarly suppressed FLT3 downstream signaling molecules (including ERK1/2 and STAT5) in both the presence and absence of FL in MOLM-13 cells. Co-crystal structure analysis showed that gilteritinib bound to the ATP-binding pocket of FLT3. These results suggest that gilteritinib has therapeutic potential in FLT3-mutated AML patients with FL overexpression.

AML Differentiation Plasticity May Contribute to Relapse.

Differentiated AML-derived cells are able to revert into leukemogenic states.

2000 - MONOLINEAGE ORIGIN OF RELAPSE FOLLOWING MULTILINEAGE DIFFERENTIATION THERAPY OF ACUTE MYELOID LEUKEMIA

Acute myeloid leukaemia (AML) is characterized by the accumulation of transformed immature myeloid blasts. While most AML patients treated with standard therapy have poor outcomes, in the APL disease subtype retinoic acid induces leukaemia maturation and can be curative in combination with arsenic trioxide. Recently approved mutant IDH1/2 inhibitors also induce AML maturation, renewing interest in AML differentiation therapy. To examine differentiation therapy dynamics in vivo we have generated a novel mouse AML model driven by reversible RNAi-mediated knockdown of the myeloid transcription factor PU.1. Restoration of endogenous PU.1 in established AML in vivo triggers synchronous differentiation of leukemic blasts and disease clearance. However, despite near-complete remission, mice reproducibly relapse with immature AML. Notably, in vivo time course studies reveal that one week after PU.1 restoration leukemic blasts differentiate into two mature myeloid lineages with distinct immunophenotype and morphology. AML-derived SSClowLy6G+ cells resembling neutrophils initially predominate but are rapidly eradicated in vivo. In contrast, high resolution flow and imaging indicates that mature AML-derived SSChighF4/80+SigF+ eosinophil-like cells persist at low numbers in specific organs during disease remission and appear to seed relapse. In mice transplanted with AML blasts lacking the essential eosinophil lineage transcription factor GATA1, in vivo PU.1 restoration triggers neutrophil but not eosinophil lineage differentiation and thereby eliminates residual disease. These results demonstrate that AML differentiation therapy can produce long-lived sublineages of mature AML-derived cells from which relapse can originate. Understanding the multilineage potential of AML blasts in individual patients may inform new strategies to improve differentiation therapy outcomes.

SAMHD1 inhibits epithelial cell transformation in vitro and affects leukemia development in xenograft mice

ABSTRACTSterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a mammalian dNTP hydrolase (dNTPase) and functions as a negative regulator in the efficacy of cytarabine treatment of acute myeloid leukemia (AML). We have reported that SAMHD1 knockout (KO) increased the activity of phosphoinositide 3-kinase (PI3K) in AML-derived THP-1 cells and attenuated their ability to form subcutaneous tumors in xenografted immunodeficient mice. However, the functional significance of SAMHD1 in controlling AML leukemogenesis remains unclear. Previous studies show that in vitro transformation of Madin-Darby canine kidney (MDCK) epithelial cells by the Jaagsiekte sheep retrovirus (JSRV) envelope protein requires activation of the PI3K/Akt oncogenic signaling pathway. Using this cell transformation model, we demonstrated that ectopic expression of wild-type human SAMHD1 or a dNTPase-defective SAMHD1 mutant (HD/AA) significantly inhibited MDCK cell transformation, but did not affect cell proliferation. To visualize and quantify THP-1 cell growth and metastasis in xenografted immunodeficient mice, we generated luciferase-expressing stable SAMHD1 KO THP-1 cells and control THP-1 cells, which were injected intravenously into immunodeficient mice. Bioluminescence imaging and quantification analysis of xenografted mice revealed that SAMHD1 KO cell-derived tumors had similar growth and metastatic potential compared with control cells at 35 days post-injection. However, mice xenografted with SAMHD1 KO cells showed greater survival compared with mice injected with control cells. Our data suggest that exogenous SAMHD1 expression suppresses in vitro cell transformation independently of its dNTPase activity, and that endogenous SAMHD1 affects AML tumorigenicity and disease progression in vivo.

SAMHD1 modulates in vitro proliferation of acute myeloid leukemia-derived THP-1 cells through the PI3K-Akt-p27 axis

ABSTRACTSterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a mammalian dNTP hydrolase that acts as a negative regulator in the efficacy of cytarabine treatment against acute myeloid leukemia (AML). However, the role of SAMHD1 in AML development and progression remains unknown. We have reported that SAMHD1 knockout (KO) in the AML-derived THP-1 cells results in enhanced proliferation and reduced apoptosis, but the underlying mechanisms are unclear. Here we show that SAMHD1 KO in THP-1 cells increased PI3K activity and reduced expression of the tumor suppressor PTEN. Pharmacological inhibition of PI3K activity reduced cell proliferation specifically in SAMHD1 KO cells, suggesting that SAMHD1 KO-induced cell proliferation is mediated via enhanced PI3K signaling. However, PI3K inhibition did not significantly affect SAMHD1 KO-reduced apoptosis, implicating the involvement of additional mechanisms. SAMHD1 KO also led to enhanced phosphorylation of p27 at residue T157 and its mis-localization to the cytoplasm. Inhibition of PI3K activity reversed these effects, indicating that SAMHD1 KO-induced changes in p27 phosphorylation and localization is mediated via PI3K-Akt signaling. While SAMHD1 KO significantly enhanced THP-1 cell migration in vitro, SAMHD1 KO attenuated the ability of THP-1 cells to form subcutaneous tumors in xenografted immunodeficient mice. This effect correlated with significantly increased expression of tumor necrosis factor α (TNF-α) in tumors, which may suggest that TNF-α-mediated inflammation could account for the decreased tumorigenicity in vivo. Our findings implicate that SAMHD1 can regulate AML cell proliferation via modulation of the PI3K-Akt-p27 signaling axis, and that SAMHD1 may affect tumorigenicity by downregulating inflammation.

Micro-RNA Profiling of Exosomes from Marrow-Derived Mesenchymal Stromal Cells in Patients with Acute Myeloid Leukemia: Implications in Leukemogenesis

Gene regulatory networks in AML may be influenced by microRNAs (miRs) contained in exosomes derived from bone marrow mesenchymal stromal cells (MSCs). We sequenced miRs from exosomes isolated from marrow-derived MSCs from patients with AML (n = 3) and from healthy controls (n = 3; not age-matched). Known targets of mIRs that were significantly different in AML-derived MSC exosomes compared to controls were identified. Of the five candidate miRs identified by differential packaging in exosomes, only miR-26a-5p and miR-101-3p were significantly increased in AML-derived samples while miR-23b-5p, miR-339-3p and miR-425-5p were significantly decreased. Validation of the predicted change in gene expression of the potential targets was investigated by interrogating gene expression levels from public datasets of marrow-derived CD34-selected cells from patients with AML (n = 69) and healthy donors (n = 40). Two molecules with decreased gene expression in AML (EZH2 and GSK3β) were predicted by the miR profiling and have been previously implicated in AML while three molecules were increased in AML-derived cells and have not been previously associated with leukemogenesis (KRBA2, RRBP1 and HIST2H 2BE). In summary, profiling miRs in exosomes from AML-derived MSCs allowed us to identify candidate miRs with potential relevance in AML that could yield new insights regarding leukemogenesis or new treatment strategies.

The Transcription Factor PU.1 Controls a Reversible Differentiation Program in Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is an aggressive malignancy characterized by clonal expansion of transformed myeloid precursors that fail to differentiate into mature cells. Since myeloid lineage maturation curbs self-renewal and is considered irreversible, engaging this process in AML is an attractive therapeutic strategy.Results: Normal myeloid differentiation requires the transcription factor PU.1 (SPI1), which is functionally compromised in several AML subtypes and is directly inhibited by the recurrent fusion oncoproteins AML1-ETO and PML-RARA. To examine the importance of PU.1 suppression in AML maintenance in vivo, we have combined RNAi-mediated PU.1 inhibition with p53 deficiency to drive highly aggressive AML in mice. Using these models we find that restoring endogenous PU.1 activity in established AML in vivo is sufficient to trigger robust transcriptional, immunophenotypic, and morphological differentiation of leukemic blasts, yielding polymorphonuclear, neutrophil-like cells. Maturation of AML is associated with significant loss of cell viability and yields sustained disease clearance in vivo. Although PU.1 restoration is potently anti-leukemic, remarkably we find that subsequent suppression of PU.1 in mature neutrophil-like cells reverts them to a transformed state within several days. While mature AML-derived cells are slower to form blast colonies in methylcellulose cultures, their clonogenic frequency is only reduced four-fold relative to AML blasts suggesting highly efficient de-differentiation.Conclusions: These results demonstrate that triggering myeloid differentiation can effectively resolve a p53-deficient model of treatment resistant AML, but also identify a previously unrecognised ability of AML cells to bidirectionally transition between transformed and differentiated states based on the activity of a single transcription factor. Our findings challenge the concept of 'terminal differentiation' in AML and highlight the importance of therapeutically eradicating leukemia cells at all stages of myeloid lineage maturation. DisclosuresNo relevant conflicts of interest to declare.

Phenotypic, genotypic, and functional characterization of normal and acute myeloid leukemia-derived marrow endothelial cells.

In addition to participation in homing, egress, and transmigration of hematopoietic cells, marrow endothelium also contributes to cell proliferation and survival. Endothelial cells from multiple vascular beds are able to prevent spontaneous or therapy-induced apoptosis in acute myelogenous leukemia (AML) blasts. Marrow-derived endothelial cells from leukemia patients have not been well-characterized, and in this work, endothelial cells were purified from marrow aspirates from normal subjects or from newly diagnosed AML patients to compare these cells phenotypically and functionally. By reverse transcription polymerase chain reaction, these cells express CD31, Tie-2, vascular endothelial growth factor (VEGF), and endothelial nitric oxide synthase (eNOS), supporting endothelial origin. They take up acetyl low-density lipoprotein and are able to form tubular structures. Culture of AML cells with endothelial cells from both normal and AML subjects supported adhesion, transmigration, and leukemia colony-forming unit outgrowth. RNA-sequencing analysis revealed 130 genes significantly up- or downregulated in AML-derived endothelial cells as compared with those derived from normal marrow. The genes differentially expressed (p < 0.001) were included in biological function categories involving cancer, cell development, cell growth and proliferation, cell signaling, inflammatory response, and cell death and survival. Further pathway analysis revealed upregulation of c-Fos and genes involved in chemotaxis such as CXCL16. AML-derived endothelial cells are similar in phenotype and function to their normal marrow-derived counterparts, but genomic analysis suggests a differential signature with altered expression of genes, which could play a role in leukemogenesis or leukemia cell maintenance in the marrow microenvironment.

Expression analysis of BECN1 in acute myeloid leukemia: association with distinct cytogenetic and molecular abnormalities.

In acute myeloid leukemia (AML), it has been shown that AML-derived cells often remain sensitive to autophagy-inducing stimuli, leading to the idea that harnessing the autophagy can be pertinent to AML cytotoxic therapy. Despite this promising notion, to date, there is no comprehensive study addressing autophagy-related genes expression status in AML. As a critical mediator, BECN1 influences the onset and advance of autophagy and several studies have pointed to the BECN1 recurrent allelic deletion and expression variation in a broad range of tumors. To explore this caveat, we chose this alteration-prone gene to investigate in our study. We have analyzed the expression status of BECN1 in a series of 128 de novo AML patients using real-time quantitative polymerase chain reaction (qRT-PCR). In our favorable subgroup, BECN1 expression did not alter (P = 0.301), but in intermediate and unfavorable patients, we have had BECN1 low expression compared to the normal controls (P = 0.008 and P < 0.001, respectively). We found evidence for the association of reduced expression of BECN1 with FLT3-ITD mutation (19 of 27 patients), monosomal karyotype (all of 11 patients), higher age, and WBC count. Overall, remarkable association of reduced expression of BECN1 with FLT3-ITD mutation and monosomal karyotype and their functional relationship is interesting which should be addressed and verified in future studies.