Acute Myeloid Leukemia Stem Cells Research Articles

A novel LGALS1-depended and immune-associated fatty acid metabolism risk model in acute myeloid leukemia stem cells

Leukemia stem cells (LSCs) are recognized as the root cause of leukemia initiation, relapse, and drug resistance. Lipid species are highly abundant and essential component of human cells, which often changed in tumor microenvironment. LSCs remodel lipid metabolism to sustain the stemness. However, there is no useful lipid related biomarker has been approved for clinical practice in AML prediction and treatment. Here, we constructed and verified fatty acid metabolism-related risk score (LFMRS) model based on TCGA database via a series of bioinformatics analysis, univariate COX regression analysis, and multivariate COX regression analysis, and found that the LFMRS model could be an independent risk factor and predict the survival time of AML patients combined with age. Moreover, we revealed that Galectin-1 (LGALS1, the key gene of LFMRS) was highly expressed in LSCs and associated with poor prognosis of AML patients, and LGALS1 repression inhibited AML cell and LSC proliferation, enhanced cell apoptosis, and decreased lipid accumulation in vitro. LGALS1 repression curbed AML progression, lipid accumulation, and CD8+ T and NK cell counts in vivo. Our study sheds light on the roles of LFMRS (especially LGALS1) model in AML, and provides information that may help clinicians improve patient prognosis and develop personalized treatment regimens for AML.

The Potential of Nanotechnology to Replace Cancer Stem Cells.

Stem cells, which were initially identified in the 1900s, are distinct cells with the potential to replenish themselves as well as differentiate into specialised cells with certain forms and functions. Cancer stem cells play a significant role in the growth and recurrence of the tumours and, similar to normal stem cells, are capable of proliferating and differentiating. Traditional cancer treatments are ineffective against cancer stem cells, which leads to tumour regrowth. Cancer stem cells are thought to emerge as a result of epithelial-to-mesenchymal transition pathways. Brain, prostate, pancreatic, blood, ovarian, lung, liver, melanomas, AML, and breast cancer stem cells are among the most prevalent cancer forms. This review aims to comprehend the possibility of using specific forms of nanotechnology to replace cancer stem cells. In terms of nanotechnology, magnetic nanoparticles can deliver medications, especially to the target region without harming healthy cells, and they are biocompatible. In order to kill glioma cancer stem cells, the gold nanoparticles bond with DNA and function as radio sensitizers. In contrast, liposomes can circulate and traverse biological membranes and exhibit high therapeutic efficacy, precise targeting, and better drug release. Similar to carbon nanotubes, grapheme, and grapheme oxide, these substances can be delivered specifically when utilized in photothermal therapy. Recent treatments including signaling pathways and indicators targeted by nanoparticles are being researched. Future research in nanotechnology aims to develop more effective and targeted medicinal approaches. The results of the current investigation also showed that this technology's utilization will improve medical therapy and treatment.

Acute myeloid leukemia stem cell signature gene EMP1 is not an eligible therapeutic target.

Relapse in pediatric acute myeloid leukemia (pedAML) patients is known to be associated with residual leukemic stem cells (LSC). We have previously shown that epithelial membrane protein 1 (EMP1) is significantly overexpressed in LSC compared to hematological stem cell fractions. EMP1 was also documented as part of the 17-gene stemness score and a 6-membrane protein gene score, both correlating high EMP1 expression with worse overall survival. However, its potential as a therapeutic target in pedAML is still unexplored. Association analyses of EMP1 expression with clinical and molecular AML characteristics were performed. Expression of EMP1 was evaluated in pedAML and cord blood samples. Expression in normal blood cells and tissues was evaluated by flow cytometry and immunohistochemistry, respectively. In silico analyses showed variable mRNA expression of EMP1 in multiple pedAML datasets, and a significant correlation between high EMP1 transcript levels and the presence of inv(16). Flow cytometry showed overexpression of EMP1 in pedAML samples, as well as expression in normal blood subsets. Importantly, immunohistochemistry revealed EMP1 expression in multiple normal tissues. Although EMP1 presents as an interesting membrane-associated target in pedAML, its abundant expression in normal blood cells and tissues will impede it from further exploration as a therapeutic target. EMP1 is highly expressed in multiple cancer types, but expression in acute myeloid leukemia (AML) and normal tissues is unexplored. As EMP1 is investigated in other cancer types, expression in normal tissues and blood cells is relevant in predicting the success of EMP1-targeted therapies. In this study, we showed expression of EMP1 in multiple tissues, predicting high on-target off-tumor toxicity, which will warn other researchers of possible toxicities when generating EMP1-targeted therapy. Finally, we showed that high EMP1 expression is associated with better overall survival of pediatric AML patients, reducing the need for EMP1-targeted therapy.

BRD4 degraders may effectively counteract therapeutic resistance of leukemic stem cells in AML and ALL.

Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) are life-threatening hematopoietic malignancies characterized by clonal expansion of leukemic blasts in the bone marrow and peripheral blood. The epigenetic reader BRD4 and its downstream effector MYC have recently been identified as potential drug targets in human AML and ALL. We compared anti-leukemic efficacies of the small-molecule BET inhibitor JQ1 and the recently developed BRD4 degraders dBET1 and dBET6 in AML and ALL cells. JQ1, dBET1, and dBET6 were found to suppress growth and viability in all AML and ALL cell lines examined as well as in primary patient-derived AML and ALL cells, including CD34+/CD38- and CD34+/CD38+ leukemic stem and progenitor cells, independent of the type (variant) of leukemia or molecular driver expressed in leukemic cells. Moreover, we found that dBET6 overcomes osteoblast-induced drug resistance in AML and ALL cells, regardless of the type of leukemia or the drug applied. Most promising cooperative or even synergistic drug combination effects were seen with dBET6 and the FLT3 ITD blocker gilteritinib in FLT3 ITD-mutated AML cells, and with dBET6 and the multi-kinase blocker ponatinib in BCR::ABL1+ ALL cells. Finally, all BRD4-targeting drugs suppressed interferon-gamma- and tumor necrosis factor-alpha-induced expression of the resistance-related checkpoint antigen PD-L1 in AML and ALL cells, including LSC. In all assays examined, the BRD4 degrader dBET6 was a superior anti-leukemic drug compared with dBET1 and JQ1. Together, BRD4 degraders may provide enhanced inhibition of multiple mechanisms of therapy resistance in AML and ALL.

Targeting Acute Myeloid Leukemia Stem Cells Through Perturbation of Mitochondrial Calcium.

Acute myeloid leukemia stem cells (LSCs) are uniquely reliant on oxidative phosphorylation (OXPHOS) for survival. Moreover, maintenance of OXPHOS is dependent on BCL-2, creating a therapeutic opportunity to target LSCs using the BCL-2 inhibitor venetoclax. While venetoclax-based regimens have shown promising clinical activity, the emergence of drug resistance is prevalent. Thus, in the present study, we investigated how mitochondrial properties may influence venetoclax responsiveness. Our data show that utilization of mitochondrial calcium is fundamentally different between drug-responsive and non-responsive LSCs. By comparison, venetoclax-resistant LSCs demonstrate a more active metabolic (i.e. OXPHOS) status with relatively high levels of calcium. Consequently, we tested genetic and pharmacological approaches to target the mitochondrial calcium uniporter, MCU. We demonstrate that inhibition of calcium uptake reduces OXPHOS and leads to eradication of venetoclax-resistant LSCs. These findings demonstrate a central role for calcium signaling in LSCs and provide an avenue for clinical management of venetoclax resistance.

Single cell and bulk RNA expression analyses identify enhanced hexosamine biosynthetic pathway and O-GlcNAcylation in acute myeloid leukemia blasts and stem cells.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults with an overall poor prognosis and high relapse rate. Multiple factors including genetic abnormalities, differentiation defects and altered cellular metabolism contribute to AML development and progression. Though the roles of oxidative phosphorylation and glycolysis are defined in AML, the role of the hexosamine biosynthetic pathway (HBP), which regulates the O-GlcNAcylation of cytoplasmic and nuclear proteins, remains poorly defined. We studied the expression of the key enzymes involved in the HBP in AML blasts and stem cells by RNA sequencing at the single-cell and bulk level. We performed flow cytometry to study OGT protein expression and global O-GlcNAcylation. We studied the functional effects of inhibiting O-GlcNAcylation on transcriptional activation in AML cells by Western blotting and real time PCR and on cell cycle by flow cytometry. We found higher expression levels of the key enzymes in the HBP in AML as compared to healthy donors in whole blood. We observed elevated O-GlcNAc Transferase (OGT) and O-GlcNAcase (OGA) expression in AML stem and bulk cells as compared to normal hematopoietic stem and progenitor cells (HSPCs). We also found that both AML bulk cells and stem cells show significantly enhanced OGT protein expression and global O-GlcNAcylation as compared to normal HSPCs, validating our in silico findings. Gene set analysis showed substantial enrichment of the NF-κB pathway in AML cells expressing high OGT levels. Inhibition of O-GlcNAcylation decreased NF-κB nuclear translocation and the expression of selected NF-κB-dependent genes controlling cell cycle. It also blocked cell cycle progression suggesting a link between enhanced O-GlcNAcylation and NF-κB activation in AML cell survival and proliferation. Our study suggests the HBP may prove a potential target, alone or in combination with other therapeutic approaches, to impact both AML blasts and stem cells. Moreover, as insufficient targeting of AML stem cells by traditional chemotherapy is thought to lead to relapse, blocking HBP and O-GlcNAcylation in AML stem cells may represent a novel promising target to control relapse.

Abstract 1646: IL-23R is a regulator of the mitotic spindle and is critical for cell viability in AML

Abstract To identify novel biological vulnerabilities in AML, we searched for gene ontologies that were upregulated in AML compared to normal hematopoietic cells. Of the upregulated processes, the mitotic spindle ontology was a top hit. Unexpectedly, expression of the IL-23 receptor (IL-23R) showed a strong positive correlation with the mitotic spindle ontology. IL-23R is a cell surface cytokine receptor that is canonically expressed on T cells, but not known to have a role in AML. In 7 of 7 tested AML cell lines and 15 of 20 primary AML patient samples, IL-23R protein was expressed >2 fold higher compared to mean IL-23R in normal mononuclear hematopoietic cells (n=5) and CD34+ cells (n=3). IL-23R is classically a T cell surface receptor and we confirmed localization to the cell surface in T cells. However, only small amounts of IL-23R were present on the cell surface of AML cell lines and primary AML samples. Rather, in AML cells, primary AML samples, and AML stem cells, IL-23R was located intracellularly as determined by 4 different modalities and 4 different antibodies directed against 4 different epitopes of the receptor. Of note, only the full-length IL-23R protein was detected by immunoblotting. We also detected IL-23R’s heterodimeric partner, IL12Rβ1, in AML cells. To understand the function of intracellular IL-23R, we used BioID mass spectrometry to identify proteins that interacted with IL-23R. Pathway analysis of interacting proteins identified mitotic spindle formation as the top pathway. Using Proximity Ligation Assays, we validated interactions with mitotic spindle proteins, and BioID hits, NUMA, TMEM201, TACC1, and BAG6 in AML cells. By confocal microscopy, we demonstrated that IL-23R colocalized with the mitotic spindle and centrosomes in AML cells and primary AML samples. IL-23R interacted with the mitotic spindle via its (S/T)x(I/L)P motif (amino acids 588-591). IL-12Rβ1 was also found in complex with IL-23R at the mitotic spindle in AML cells. Additionally, it was discovered that the cytokine, IL-23, promoted translocation of IL-23R to the mitotic spindle through clathrin-mediated endocytosis. Knockdown and knockout of IL-23R in AML cells led to dysregulation of the mitotic spindle with multipolarity, lagging chromosomes, and other spindle defects. Knockdown of IL-23R also reduced proliferation, clonogenic growth, and marrow engraftment of AML cells and primary AML samples. In contrast, knockdown of IL-23R in normal CD34+ cells did not impair engraftment. Likewise, constitutive homozygote IL-23R knockout mice had no difference in complete blood counts and stem cell number/function compared to wild type. In summary, IL-23R interacts with the mitotic spindle and centrosome where it regulates mitotic spindle formation and is critical for AML cell viability. Thus, we discover a novel function for IL-23R and a potential therapeutic target for this disease. Citation Format: Nathan Duong, Dilshad H. Khan, Geethu E. Thomas, Rose Hurren, Jong Bok Lee, Jonathan St-Germain, Lily Drimmer, Yongran Yan, Neil Maclean, Marcela Gronda, Vincent Rondeau, Brandon D. Brown, Courtney L. Jones, Hong Chang, Andrea Arruda, Mark D. Minden, Li Zhang, Steven M. Kornblau, Brian Raught, Vito Spadavecchio, Aaron D. Schimmer. IL-23R is a regulator of the mitotic spindle and is critical for cell viability in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1646.

Abstract 2372: ATG-102, a novel LILRB4 x CD3 T cell engager, targeting two nonoverlapping epitopes of LILRB4, for the treatment of monocytic AML

Abstract Background: Acute myeloid leukemia (AML) is the most common acute leukemia in adults, and the treatment of AML, especially monocytic AML subtypes still has a poor outcome. T cell engagers (TCE) have been well used for the treatment of hematological malignancies, such as B cell leukemia and multiple myeloma. However, lack of ideal target antigens that only express on AML and leukemic stem cells (LSCs) but not on normal hematopoietic stem cells (HSCs) hampers the development of TCE therapy for AML. Leukocyte immunoglobulin-like receptor B4 (LILRB4), an inhibitory receptor belonging to the LILR family, is highly and restrictedly expressed on monocytic M4/M5 AML subtypes and LSCs, but not on normal HSCs which makes it an ideal target for TCE. Here we report a novel LILRB4 x CD3 bispecific T cell engager based on AnTenGagerTM platform, ATG-102. It targets two distinct epitopes of LILRB4, inducing potent T cell-dependent cellular cytotoxicity (TDCC) with low CRS, resulting in a potent anti-tumor efficacy in vitro and in vivo. Method: ATG-102 was constructed by introducing a high affinity anti-CD3 single chain fragment variable (scFv) to the hinge region of one of the heavy chains of a LILRB4 monoclonal antibody. A scFv targeting another epitope of LILRB4 was connected to the C terminal of the same heavy chain by GSSS linker, enabling a trivalent, dual epitope recognition of LILRB4. ATG-102 was evaluated in a series of preclinical studies for binding epitope and affinity, T cell activation, T cell dependent cytotoxicity (TDCC), and cytokine release. The in vivo antitumor efficacy of ATG-102 was evaluated in a humanized PBMCs and THP-1-luciferase cells engrafted NCG mice model. Results: ATG-102 binds to LILRB4 positive cells with a single-digit nM affinity. It binds to two distinct and nonoverlapping epitopes of LILRB4. ATG-102 showed limited binding capability to CD3+ cells before LILRB4 crosslinking. However, with the presence of LILRB4 positive THP-1 cell, ATG-102 strongly activated primary T cells, upregulating early and later markers of T cell activation, CD69 and CD25, respectively. ATG-102 induced strong TDCC against LILRB4-high expressing THP-1 cells, and it induced more potent TDCC against LILRB4-low expressing MOLM-13 cells than a benchmark antibody. While compared with benchmark, ATG-102 induced significantly lower release of IL-6, which is one of the major cytokines involved in cytokine release syndrome (CRS). In addition, ATG-102 demonstrated more potent in vivo anti-tumor efficacy compared with the benchmark antibody at 1 mg/kg and 0.1 mg/kg dose level in PBMC-humanized NCG mice bearing THP-1-luciferase cells. Conclusion: ATG-102 demonstrates trivalent, dual epitope recognition of LILRB4, conditionally redirects and activates T cells to recognize and kill monocytic AML cells. It demonstrates potent in vitro and in vivo anti-tumor efficacy with low risk of CRS. Citation Format: Ao Sun, Mengshi Gao, Rong Guo, Huiling Liu, Zaoshun Hu, Enlin Zheng, Hui Yuwen, Peng Chen, Jay Mei, Bo Shan, Bing Hou. ATG-102, a novel LILRB4 x CD3 T cell engager, targeting two nonoverlapping epitopes of LILRB4, for the treatment of monocytic AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2372.

Abstract 5944: Discovery of BH-30236: A novel macrocyclic CLK inhibitor targeting alternative splicing in cancers

Abstract Aberrant alternative splicing is a newly recognized hallmark of cancer, that has been shown to play a critical role in tumorigenesis, cancer progression, and therapeutic resistance via multiple mechanisms, including increased proliferation, decreased apoptosis, promoted migration, enhanced metastatic potential, and induced evasion of immune surveillance. Serine and arginine-rich splicing factors (SRSFs) are RNA-binding proteins (RBPs) that regulate constitutive and alternative splicing. SRSFs are often mutated or overexpressed in cancers, resulting in widespread alterations in splicing patterns. The Cdc2-like kinase (CLK) family and dual-specificity tyrosine-regulated kinase (DYRK) phosphorylate SRSFs, influencing the assembly of spliceosome machinery, exon recognition, and splicing. Therefore, targeting CLK/DYRK kinases can modulate cancer specific splicing isoforms, opening avenues for new therapeutic interventions. BH-30236 was designed as a novel orally bioavailable, ATP-competitive, macrocyclic inhibitor of CLK with IC50s of 0.134, 0.165, and 0.446 nM against CLK1, CLK2, and CLK4, respectively in enzymatic kinase assays. At clinically relevant concentrations, BH-30236 also inhibited DYRK1A/1B/2, proviral insertion site of moloney murine leukemia virus kinase 3 (PIM3), and FMS-like tyrosine kinase 3 (FLT3) with IC50s of 0.111, 0.148, 0.562, 0.115 and 0.248 nM, respectively. In cancer cells, BH-30236 impaired the phosphorylation of SRSFs, Tau and 4EBP1, the direct downstream substrates of CLK, DYRK, and PIM kinases with IC50s of 40-60 nM, ~50 nM, and ~80 nM, respectively. Furthermore, BH-30236 potently inhibited the FLT3 phosphorylation with an IC50 of 0.16 nM. Overall, BH-30236 regulated alternative splicing by primarily inducing skipped exons in favor of anti-tumor isoforms, leading to cancer cell death and growth suppression in a broad panel of cancer cell lines and in vivo efficacy studies. For example, BH-30236 potently inhibited cell proliferation with an IC50 of 0.62 nM in FLT3-ITD positive MV-4-11 cells and achieved complete tumor regression in the MV-4-11 cell-derived xenograft tumor model, even after dose cessation for 4 weeks. In MV-4-11 cells, BH-30236 increased pro-apoptosis splicing variant BCLxS, downregulated RNA expression of BCL2, MCL1, and AML stem cell markers CD33 and CD123. In addition, BH-30236 has demonstrated good human ADME and preclinical safety profiles. Collectively, the preclinical study results strongly support the clinical applications of the novel multikinase CLK inhibitor BH-30236 in hematological malignancies and solid tumors, as a single agent or in combination with other therapies. Citation Format: J. Jean Cui, Ping Jiang, Wei Deng, Dayong Zhai, Nancy Ling, Danan Li, Zhenping Wang, Yue Hu, Levan Darjania, Jeff Whitten, Evan Rogers, Eugene Rui. Discovery of BH-30236: A novel macrocyclic CLK inhibitor targeting alternative splicing in cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5944.

An immunophenotype-coupled transcriptomic atlas of human hematopoietic progenitors.

Analysis of the human hematopoietic progenitor compartment is being transformed by single-cell multimodal approaches. Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) enables coupled surface protein and transcriptome profiling, thereby revealing genomic programs underlying progenitor states. To perform CITE-seq systematically on primary human bone marrow cells, we used titrations with 266 CITE-seq antibodies (antibody-derived tags) and machine learning to optimize a panel of 132 antibodies. Multimodal analysis resolved >80 stem, progenitor, immune, stromal and transitional cells defined by distinctive surface markers and transcriptomes. This dataset enables flow cytometry solutions for in silico-predicted cell states and identifies dozens of cell surface markers consistently detected across donors spanning race and sex. Finally, aligning annotations from this atlas, we nominate normal marrow equivalents for acute myeloid leukemia stem cell populations that differ in clinical response. This atlas serves as an advanced digital resource for hematopoietic progenitor analyses in human health and disease.

An arms-race against resistance: leukemic stem cells and lineage plasticity.

Acute myeloid leukemia (AML) therapy is undergoing rapid development, but primary and acquired resistance to therapy complicates the prospect of a durable cure. Recent functional and single-cell multi-omics approaches have greatly expanded our knowledge of the diversity of lineage trajectories in AML settings. AML cells range from undifferentiated stem-like cells to more differentiated myeloid or megakaryocyte/erythroid cells. Current clinically relevant drugs predominantly target the myeloid progenitor lineage, while monocyte- or stem cell-like states can evade current AML treatment and may be targeted in the future with lineage-specific inhibitors. The extent of aberrant lineage plasticity upon therapeutic pressure in AML cells in conjunction with hijacking of normal differentiation pathways is still a poorly understood topic. Insights into the mechanisms of lineage plasticity of AML stem cells could identify both therapy-specific and cross-drug resistance pathways and reveal novel strategies to overcome them.

BRG1/BRM inhibitor targets AML stem cells and exerts superior preclinical efficacy combined with BET or menin inhibitor

AbstractBRG1 (SMARCA4) and BRM (SMARCA2) are the mutually exclusive core ATPases of the chromatin remodeling BAF (BRG1/BRM-associated factor) complexes. They enable transcription factors/cofactors to access enhancers/promoter and modulate gene expressions responsible for cell growth and differentiation of acute myeloid leukemia (AML) stem/progenitor cells. In AML with MLL1 rearrangement (MLL1r) or mutant NPM1 (mtNPM1), although menin inhibitor (MI) treatment induces clinical remissions, most patients either fail to respond or relapse, some harboring menin mutations. FHD-286 is an orally bioavailable, selective inhibitor of BRG1/BRM under clinical development in AML. Present studies show that FHD-286 induces differentiation and lethality in AML cells with MLL1r or mtNPM1, concomitantly causing perturbed chromatin accessibility and repression of c-Myc, PU.1, and CDK4/6. Cotreatment with FHD-286 and decitabine, BET inhibitor (BETi) or MI, or venetoclax synergistically induced in vitro lethality in AML cells with MLL1r or mtNPM1. In models of xenografts derived from patients with AML with MLL1r or mtNPM1, FHD-286 treatment reduced AML burden, improved survival, and attenuated AML-initiating potential of stem-progenitor cells. Compared with each drug, cotreatment with FHD-286 and BETi, MI, decitabine, or venetoclax significantly reduced AML burden and improved survival, without inducing significant toxicity. These findings highlight the FHD-286–based combinations as a promising therapy for AML with MLL1r or mtNPM1.

Efficacy of novel agents against cellular models of familial platelet disorder with myeloid malignancy (FPD-MM)

Germline, mono-allelic mutations in RUNX1 cause familial platelet disorder (RUNX1-FPD) that evolves into myeloid malignancy (FPD-MM): MDS or AML. FPD-MM commonly harbors co-mutations in the second RUNX1 allele and/or other epigenetic regulators. Here we utilized patient-derived (PD) FPD-MM cells and established the first FPD-MM AML cell line (GMR-AML1). GMR-AML1 cells exhibited active super-enhancers of MYB, MYC, BCL2 and CDK6, augmented expressions of c-Myc, c-Myb, EVI1 and PLK1 and surface markers of AML stem cells. In longitudinally studied bone marrow cells from a patient at FPD-MM vs RUNX1-FPD state, we confirmed increased chromatin accessibility and mRNA expressions of MYB, MECOM and BCL2 in FPD-MM cells. GMR-AML1 and PD FPD-MM cells were sensitive to homoharringtonine (HHT or omacetaxine) or mebendazole-induced lethality, associated with repression of c-Myc, EVI1, PLK1, CDK6 and MCL1. Co-treatment with MB and the PLK1 inhibitor volasertib exerted synergistic in vitro lethality in GMR-AML1 cells. In luciferase-expressing GMR-AML1 xenograft model, MB, omacetaxine or volasertib monotherapy, or co-treatment with MB and volasertib, significantly reduced AML burden and improved survival in the immune-depleted mice. These findings highlight the molecular features of FPD-MM progression and demonstrate HHT, MB and/or volasertib as effective agents against cellular models of FPD-MM.

PI3Kγ maintains the self-renewal of acute myeloid leukemia stem cells by regulating the pentose phosphate pathway

AbstractAcute myeloid leukemia (AML) is an aggressive hematological malignancy originating from transformed hematopoietic stem or progenitor cells. AML prognosis remains poor owing to resistance and relapse driven by leukemia stem cells (LSCs). Targeting molecules essential for LSC function is a promising therapeutic approach. The phosphatidylinositol 3-kinase (PI3K)/AKT pathway is often dysregulated in AML. We found that although PI3Kγ is highly enriched in LSCs and critical for self-renewal, it was dispensable for normal hematopoietic stem cells. Mechanistically, PI3Kγ-AKT signaling promotes nuclear factor erythroid 2-related factor 2 (NRF2) nuclear accumulation, which induces 6-phosphogluconate dehydrogenase (PGD) and the pentose phosphate pathway, thereby maintaining LSC stemness. Importantly, genetic or pharmacological inhibition of PI3Kγ impaired expansion and stemness of murine and human AML cells in vitro and in vivo. Together, our findings reveal a key role for PI3Kγ in selectively maintaining LSC function by regulating AKT-NRF2-PGD metabolic pathway. Targeting the PI3Kγ pathway may, therefore, eliminate LSCs without damaging normal hematopoiesis, providing a promising therapeutic strategy for AML.

TMIGD2 is an orchestrator and therapeutic target on human acute myeloid leukemia stem cells

Acute myeloid leukemia (AML) is initiated and sustained by a hierarchy of leukemia stem cells (LSCs), and elimination of this cell population is required for curative therapies. Here we show that transmembrane and immunoglobulin domain containing 2 (TMIGD2), a recently discovered co-stimulatory immune receptor, is aberrantly expressed by human AML cells, and can be used to identify and enrich functional LSCs. We demonstrate that TMIGD2 is required for the development and maintenance of AML and self-renewal of LSCs but is not essential for normal hematopoiesis. Mechanistically, TMIGD2 promotes proliferation, blocks myeloid differentiation and increases cell-cycle of AML cells via an ERK1/2-p90RSK-CREB signaling axis. Targeting TMIGD2 signaling with anti-TMIGD2 monoclonal antibodies attenuates LSC self-renewal and reduces leukemia burden in AML patient-derived xenograft models but has negligible effect on normal hematopoietic stem/progenitor cells. Thus, our studies reveal the function of TMIGD2 in LSCs and provide a promising therapeutic strategy for AML.

Cryptanshinone extract of Salvia miltiorrhiza stimulates pediatric acute myeloid leukemia stem cell apoptosis and the anti-inflammatory mechanism via accelerating microRNA-211-5p to supress Janus kinase 2/signal transducer and activator of transcription 3 signaling pathway activation.

This study was designed to explore cryptanshinone (CPT) extract of Salvia miltiorrhiza stimulating pediatric acute myeloid leukemia (AML) stem cell (LSC) apoptosis and anti-inflammatory mechanism via accelerating microRNA (miR)-211-5p to restrain Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway activation. Obtaining blood samples from pediatric acute myeloid leukemia patients and healthy volunteers and detecting miR-211-5p and JAK2 were performed. Purchase of the human AML cell line KG1a was conducted, and sorting of KG1a cells was to gain LSC. Test of miR-211-5p and JAK2, the phosphorylation of JAK2/STAT3 was implemented. Pretreatment of LSCs was with CPT. Variation of miR-211-5p and JAK2 in LSCs was via plasmid transfection to explore their actions in cell advancement with apoptosis and inflammation. Identification of the targeting of miR-211-5p with JAK2 was implemented. In results: MiR-211-5p was declined in endometrial cancer, while JAK2 was elevated; CPT was available to boost LSC apoptosis and restrain the inflammation; elevated miR-211-5p or repressive JAK2 was available to strengthen the acceleration of CPT on LSCs apoptosis and the repression of inflammation; MiR-211-5p targeted JAK2; augmented JAK2 was available to turn around the action of elevated miR-211-5p. We conclude that CPT extract of Salvia miltiorrhiza stimulated pediatric LSC apoptosis and restrained the inflammation via accelerating microRNA (miR)-211-5p to suppress JAK2/STAT3 pathway activation.

Minimal Residual Disease in Acute Myeloid Leukemia Following Induction Chemotherapy Can be Effectively Eradicated By Targeting Mitochondrial Metabolism

Acute myeloid leukemia (AML) stem cells (AMLSCs) AMLSCs and residual cytarabine (AraC)-resistant AML cells (constituting minimal residual disease, MRD) thought to be responsible for chemoresistance and treatment failure, were shown to be highly dependent on mitochondrial function for survival and thus are vulnerable to pharmacological blockade of the oxidative phosphorylation (OXPHOS) (Farge et al. Cancer Discov, 2017). Efficacy of OXPHOS inhibitor IACS-010759 (OXPHOS-i) was previously reported, demonstrating potent inhibition of mitochondrial complex I, OXPHOS suppression and growth inhibition of AML cells (Molina, et al. Nat Med, 2018). Here we evaluated OXPHOS dependency of AML MRD cells and determined impact of OXPHOS blockade on residual AML cells surviving standard chemotherapy (Doxorubicin/AraC, DA). Our results demonstrated that AML cell lines treated with AraC or DA induced accumulation of reactive oxygen species, mitochondrial superoxides, increased mitochondrial mass and mitochondrial membrane potential. AraC- and DA therapies in vitro were significantly enhanced by OXPHOS-i. OXPHOS dependency shown as a significantly increased basal and maximal oxygen consumption rate after AraC and DA treatment, was fully inhibited by OXPHOS-i, leading to complete mitochondrial collapse. OXPHOS inhibition in combination with DA translated into reduction of viable cell numbers, induction of apoptosis and differentiation in AraC-sensitive and AraC-resistant cell lines models. In vitro efficacy was also observed in engineered p53-mutated MOLM13 model, indicating that combination of OXPHOS-i and DA might successfully overcome p53 mutation-driven chemoresistance. These effects were further validated in a subset of AraC-resistant primary patient samples. Mechanistically, induction of ROS caused by OXPHOS-i addition upon DA contributed to differentiation and cell death, and was partially reversed by ROS scavengers. Next, the efficacy of IACS-010759 together with DA chemotherapy was evaluated in several chemotherapy-sensitive and -resistant animal models in vivo. DA/IACS-010759 combination significantly reduced leukemia burden and extended survival in OCI-AML3/Luc/GFP model and in FLT3-ITD + AML PDX model. In the latter, IACS-010759 led to the reduction of leukemia burden and delayed leukemia recurrence when administered post completion of DA. At the single-cell level, CyTOF analysis demonstrated that this combination reduced frequency of CD34 +CD38 lowCD123 +AML LSCs and facilitated differentiation of immature subpopulations (Fig.1A). Furthermore, addition of OXPHOS-i to DA extended survival of mice inoculated with chemoresistant PDX AML models. Finally, OXPHOS-i administered during consolidation phase significantly extended mice survival compared to standard of care arm, supporting clinical utility of OXPHOS inhibitors in AML (Fig.1B). In conclusion, our findings indicate that chemotherapy fosters mitochondrial respiration in AML, which could be abrogated by OXPHOS inhibitor at the LSCs and MRD level , in vitro and in vivo. While IACS-010759 (Yap et al. Nat Med 2023) showed toxicities impeding its clinical utility, our data advocate for combining mitochondrial targeting strategies with chemotherapy as a part of induction and consolidation treatment for improved control of MRD, eradication of AMLSC and extended response duration. Thus, further studies to identify compounds with improved safety profile are warranted.

A STAT3 Degrader Demonstrates Pre-Clinical Efficacy in Venetoclax Resistant Acute Myeloid Leukemia

Introduction: Acute Myeloid leukemia (AML) is an aggressive hematological malignancy resulting from the transformation of immature myeloid progenitor cells followed by an uncontrolled clonal proliferation and accumulation of the transformed cells. AML is the most common myeloid malignancy in the elderly [1]. Venetoclax (Ven) is a selective inhibitor of the anti-apoptotic BCL2 protein. Ven is FDA approved in combination with hypomethylating agents (HMA's) or low dose cytarabine for the treatment of de-novo AML in patients > 75 years or those ineligible for standard induction therapies. Despite the recent FDA approval of HMA/Ven and other novel therapies, 5-year survival in newly diagnosed AML remain less than 30% and disease relapse is inevitable without a bridge to allogeneic stem cell transplantation [2]. Signal transducer and activator of transcription 3 (STAT3) belonging to the STAT family of transcription factors is well known to be inappropriately activated in several malignancies [3]. On stimulation by cytokines such as IL-6, STAT3 undergoes dimerization and phosphorylation. Phosphorylation at Tyr 705 leads to its translocation to the nucleus whereas phosphorylation at Ser 727 translocates it to the mitochondria [4]. Previous data from our lab demonstrated de-methylation and overexpression of STAT3 in MDS & AML stem cells, that is associated with an adverse prognosis [5]. We have also reported that STAT3 controls several important leukemic drivers such as the anti-apoptotic protein myeloid cell leukemia-1 (MCL1). MCL1 overexpression is the central mechanism of resistance to BCL2 inhibition (Ven) in AML [5]. While MCL1 is a well-known direct transcriptional target of STAT3, the role of STAT3 in venetoclax resistance (Ven-res) is unknown. Methods & Results: To understand the role of STAT3 in Venetoclax resistance (Ven-res), Ven-res AML cell lines (MOLM-13, MV-4-11) as well as a Ven-res large cell lymphoma cell line (SU-DHL-1) were generated, which exhibited increased levels of both total STAT3, phospho-STAT3 and its downstream effector, MCL1, when compared to their parental cell lines (Fig. 1A). Data from > 90 AML patients treated with prior Ven also showed high expression of total STAT3 along with enhanced phosphorylation for p Ser727 and p Tyr-705 STAT3, that strongly correlated with worse overall survival (OS) and reduced remission duration (RemDur) (p<0.05). A highly specific potent heterobifunctional degrader of STAT3 (degrader) resulted in a dose dependent and selective degradation of STAT3 in both parental and Ven-res hematological malignancy cell lines. Treatment with degrader showed a significant (>60%) decrease in p Tyr-705 as wellas p Ser-727 STAT3 levels in MOLM-13 parental as well as MOLM-13 Ven-res cells. STAT3 degradation also led to induction of apoptosis in both parental and Ven-res AML cell lines (p < 0.001). Importantly, colony assay of Ven-res AML patient samples showed effective degradation of STAT3 (>90%) together with the increased erythroid and myeloid differentiation (~ 2 fold) on treatment with degrader. Interestingly, no differentiation was observed in healthy samples, suggesting the specificity of the degrader to AML stem and progenitor cells. In patients with Ven-res AML, erythroid colony counts were seen to increase (~1.5 fold) with a concomitant decrease in myeloid colony counts (>2.5 fold), on treatment with degrader. This observation supports the clinical significance of our studies in AML patients with anemia. Although BH3 profiling of Ven-res MOLM-13 supports an increased dependency on MCL1, we have observed that treatment with degrader led to ~20% reduction in the MCL1 dependent mitochondrial depolarization. Additionally, cell derived xenograft (CDX) models of Ven-res showed significant reduction of p Tyr-705 STAT3(~60%), total STAT3 (>90%) and MCL1 (~70%), on treatment with STAT3 degrader - KT-333 (currently in an early phase clinical trial:NCT05225584), as early as week 2 (Fig 1B). Significant decrease in intracellular STAT3 in the spleen cells was also observed (p < 0.05), with ongoing survival analysis. Conclusion: Our study suggests that targeting STAT3 and the downstream MCL1 represents a novel and effective strategy for Ven-resistant AML patients in clinic, with strong mechanistic rationales that can spur further clinical development of STAT3 degraders especially given the significant side effects of direct MCL1 inhibitors.

Inhibition of mitochondria induces apoptosis and reduces telomere length and activity in acute myeloid leukemia stem cells.

Acute myeloid leukemia (AML) is a highly lethal hematological malignancy in adults and children. Abnormal proliferation of leukemia stem cells (LSC) with CD34+ and CD38- phenotypes are the main clinical features of AML. Patients with AML face drug resistance and treatment failure due to a default in stem and progenitor cells. Therefore, defining LSC properties is necessary for targeting leukemia-initiating cells. Mitochondrial mass and activity increase in AML initiating cells compared with normal stem cells. This idea has offered the inhibition of the mitochondrial translation machinery to reduce the number of leukemia-initiating cells in patients with AML Tigecycline is an FDA-approved microbial antibiotic that inhibits oxidative phosphorylation in mitochondria, resulting in the suppression of leukemia cell proliferation with little toxicity to normal cells. Thus, the present study was conducted to evaluate whether LSC is influenced by mitochondrial inhibition. We measured the IC50 of tigecycline in KG-1a AML cell lines. KG-1a AML cell lines were separated into CD34+ and CD34- cells by MACS. In the following, these cells were treated with 20 µM (IC50) tigecycline. The expression of Annexin/PI, Caspase 3, apoptotic genes (BCL2, BCLX, BAX, BAD, and P53) and proteins (P53, BAX, BCL2 and Caspase 9) was evaluated in CD34+ , CD34- and KG-1a AML cells. In addition, the telomere length and expression of hTERT were evaluated in this study. The results indicated that BCl2 (gene and protein) and BCLX gene dramatically decreased. In addition, BAD, BAX, and P53 gene and protein expression significantly increased in CD34+ AML cells compared to CD34- AML cells. The results also suggested that tigecycline induced intrinsic (Cleaved-caspase 9/Pro-Caspase 9 ratio) and p53-mediated apoptosis. Furthermore, hTERT gene expression and telomere length decreased in the tigecycline-treated groups. Taken together, our findings indicate that inhibition of mitochondrial activity with tigecycline can induce apoptosis in cancer stem cells and can be used as a novel method for cancer therapy.

Monoclonal Antibodies Targeting LIGHT Block Ltbr Signalling and Eliminate Acute Myeloid Leukemia Stem Cells

Introduction Acute myeloid leukemia (AML) is a molecular heterogeneous disease that originates from the malignant transformation of a hematogenic stem- or progenitor cells resulting in a leukemia stem cell (LSC). LSCs are a main cause of resistance to therapy and relapse. Despite recent advances in targeted therapy and immunotherapy, prognosis for elderly patients not fit do undergo intensive therapy remains poor. We recently documented that targeting CD70, the ligand of the tumor necrosis factor receptor (TNFR) CD27, by the antibody-dependent cell-mediated cytotoxicity (ADCC)-enhanced monoclonal antibody (mAb) cusatuzumab eliminates LSC. We now studied the role of the related TNFR lymphotoxin b receptor (LTbR) with its ligand LIGHT in the pathogenesis of AML in mice and humans and developed a LIGHT targeting mAb to specifically eliminate LSCs. Experimental design We investigated the impact of LIGHT/LTbR signalling in AML development by using the retrovirally-induced MLL-AF9 syngeneic AML mouse model. Light-, Ltbr -deficient and control MLL-AF9 transduced LSCs were injected intravenously into non-irradiated BL/6 recipient mice. AML development was evaluated by analysing MLL-AF9-GFP-positive leukemic cells in blood, spleen, and bone marrow (BM). BM LSC were phenotypically characterized by FACS and functionally by performing colony forming unit (CFU) assays in vitro and by secondary transplantations of LSCs including limited dilution assays (LDA) in vivo. Correlative survival analysis for LIGHT and LTbR expression was performed on a publicly available gene dataset (GSE6891). LIGHT and LTBR protein expression was analysed on primary AML CD34 + leukemic stem and progenitor cells (LSPCs) and control human CD34 + hematopoietic stem cell and progenitor cells (HSCPs). LIGHT/LTbR signalling was blocked by either siRNA treatment or by treatment with novel LIGHT-targeting mAb. The effect of blocking LIGHT/LTbR-signalling was analysed in vitro by CFU assays, RNA-sequencing and xenotransplant experiments in vivo. LIGHT-targeting mAbs were generated by immunization of llamas and phage display selections followed by cloning of FABs into a human backbone (hIgG1). Clones were selected based on their blocking potency of receptor/ligand interaction using competition ELISA and FACS-based cell binding affinity assays. ADCC-enhanced anti-LIGHT mAbs were generated via POTELLIGENT® technology. Anti-LIGHT mAbs that specifically block the interaction of LIGHT/LTBR with deficiency in effector functions (Fc-death) were produced by amino acid substitutions in the CH2 domain of the parental clone. Results LTbR was expressed by HSCs and AML LSCs. In contrast, LIGHT was only expressed on murine and human LSCs but not normal HSCs. The absence of LIGHT or its receptor LTbR on LSCs resulted in significantly prolonged survival and decreased numbers of LSC in BM of AML mice. Light -/- and Ltbr -/- LSCs had a reduced CFU capacity compared to control LSCs. Similarly, inhibition of LIGHT-signalling on LSCs via LTbR-Ig significantly reduced colony formation. Moreover, secondary transplantation of Light -/-LSCs resulted in prolonged survival compared to Light-proficient LSCs. An LDA analysis revealed a 11-fold reduction of the LSC frequency in BM in the absence of LIGHT or LTBR. Similarly, inhibiting LIGHT signalling by siLIGHT RNA in human AML LSPCs resulted in reduced CFU capacity compared to control siRNA treatment. In addition, a GEO analysis revealed that low LIGHT or LTbR mRNA levels correlated with improved overall survival in AML patients. After immunizations of llamas and phage-display selection three humanized anti-LIGHT mAbs with high blocking and high binding efficiency were selected (mAb10F11 and 12C2 specific for human LIGHT and the cross-reactive murine/human 9F6 mAB). Incubation of LSCs with the blocking mAbs significantly reduced CFU capacity. HSPCs from healthy donors remained unaffected by the treatment. A gene expression analysis revealed that blocking LIGHT/LTbR-signalling in LSCs reduced stemness and promoted differentiation. In addition, ADCC-enhanced mAbs activated NK cells to eliminate LIGHT-expressing LSCs. Our findings reveal that LIGHT/LTbR-signalling is crucial for the pathogenesis of AML and especially for the maintenance and expansion of LSCs. We developed and validated novel LIGHT targeting mAbs in vitro and in vivo for further development in clinical trials.