Targeting Leukemia Stem Cells Research Articles

Casticin inhibits the hedgehog signaling and leads to apoptosis in AML stem-like KG1a and mature KG1 cells

Abstract Objectives Acute myeloid leukemia (AML) is a severe blood cancer with less than 50 % long-term survival. Despite advancements in treatment options, relapse is still the major obstacle. The main reason of this problem is ineffective targeting of leukemic stem cells (LSCs), which play an important role in tumor development and relapse. In our previous studies, we found that casticin, the major polyphenolic component of Vitex trifolia’s fruit, targets both leukemic cells and LSCs without affecting healthy tissues. Therefore, in this study, we aimed to investigate the effect of casticin-mediated cell death in relation to the LSCs-favored survival pathways at gene and protein expression levels using in vitro LSC-like and parental leukemic cell models. Methods We validated the LSC character of KG1a and KG1 cells (84.55 % CD34+, CD38- and 93.55 % CD34+, CD38+, respectively) by flow cytometry. For the investigation of casticin’s mechanism of action, we employed real time-PCR, western blotting and bioinformatics analyses. Results Our results showed an increase in cleaved PARP/β-actin ratio but no change in LC3BI/II and SQSTM/β-actin ratios. Our gene expression, bioinformatics and immunoblotting analyses represented significant decrease in Shh, Gli and Wnt levels. We also elucidated a possible crosstalk between Hedgehog and other oncogenic cascades via the Gli, Notch, YAP, p38, Mcl-1, and Myc proteins in casticin mediated anti-leukemic effect. Conclusions In conclusion, we found that casticin induces apoptosis in both LSC-like and parental leukemia cells mainly by suppressing Shh signaling, which is crucial for LSC survival and AML relapse.

Translating molecular insights into clinical success: alkaloid-based therapies for leukemia.

Alkaloids, a diverse class of naturally occurring compounds, have shown significant potential in the treatment of leukemia by targeting key molecular pathways and cellular mechanisms. This review discusses several potent alkaloids, such as homoharringtonine, chaetominine, matrine, and jerantinine B, which induce apoptosis, cell cycle arrest, and autophagy and inhibit signaling pathways including PI3K/Akt/mTOR, MAPK, and NF-κB. For instance, homoharringtonine induces apoptosis in acute myeloid leukemia (AML) cells via the SP1/TET1/5hmC/FLT3/MYC axis, while chaetominine enhances chemosensitivity by inhibiting the PI3K/Akt/Nrf2 pathway. In addition, targeting leukemia stem cells (LSCs) with alkaloids such as zalypsis offers promise due to its ability to induce apoptosis without significantly affecting normal hematopoietic stem cells. The modulation of the immune response, such as the inhibition of NF-κB activation by noscapine, further underscores the potential of alkaloids in overcoming treatment resistance. Various studies have demonstrated the efficacy of alkaloids across different leukemia types. For example, jerantinine B targets AML cells, while vincristine has shown success in lymphocytic leukemia. Clinical trials have also highlighted the benefits of alkaloids, including homoharringtonine, which achieved a 79.9% complete remission rate in AML patients. However, adverse effects such as neutropenia and hepatotoxicity necessitate careful management. Collectively, these findings emphasize the need for further research into alkaloid-based combination therapies to enhance efficacy and minimize toxicity, providing a promising avenue for innovative leukemia treatments.

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. Although 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 nonresponsive LSCs. By comparison, venetoclax-resistant LSCs demonstrate an 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. 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. Significance: We identify increased utilization of mitochondrial calcium as a distinct metabolic requirement of venetoclax-resistant LSCs and demonstrate the potential of targeting mitochondrial calcium uptake as a therapeutic strategy.

Abstract 4664: Therapeutic targeting of TP53-mutated acute myeloid leukemia by inhibiting cell cycle checkpoint

Abstract Acute myeloid leukemia (AML) is a hematological malignancy characterized by various somatic mutations leading to abnormal proliferation and differentiation of hematopoietic cells. Mutations on the TP53 gene occur in 5% to 10% of patients with de novo AML, with higher frequency in patients with relapsed and refractory (R/R) AML. Patients with TP53-mutated AML typically exhibit a poor response to conventional induction therapy and have a short overall survival (OS) rate, with a median duration of 5-9 months. Although various target agents have been developed for the treatment of AML, treatment options for TP53-mutated AML remain limited. Here, we have discovered a novel drug combination that can enhance the efficacy of the conventional therapeutic regimen for TP53-mutated AML patients. For in vitro analysis, we used the human AML cell line Kasumi-1(AML-M2), which harbors a homozygous TP53 R248Q mutation. We treated kasumi-1 cells with a combination of cytarabine and Idarubicin to produce drug tolerant persisters (DTPs) which is a minor population that survived the drug treatment. The Kasumi-1 DTPs displayed temporal G2/M phase cell cycle arrest and significant resistance to further repeated drug treatment. To discover drug candidates targeting TP53-mutated DTPs, we first examined the differentially expressed genes (DEGs) of TP53-mutated AML patients using patients’ RNA expression data deposited in the cancer genome atlas (TCGA) database. Using selected DEGs, drug candidates potentially affecting TP53-mutated AML cells were extracted from the connectivity map (CMap build 02) database. In addition, through a literature search, drugs with the ability to reactivate mutant p53, drugs that directly target mutant p53, synthetic lethal partners with mutant p53, and drugs that target leukemia stem cells were selected as candidate groups. As a result, we selected 46 drug candidates that would be effective for TP53-mutated AML through in silico analysis and therapy strategies targeting p53. Using in vitro cytotoxicity assay, we screened 46 candidate compounds at multiple doses and confirmed several drugs that effectively eliminate DTPs. The drug screening showed that cell cycle checkpoint inhibitors not only resulted in a significant reduction of G2/M phase arrested cells but also induction of apoptotic cells at the subG1 phase. Furthermore, the zero interaction potency (ZIP) model of SynergyFinder (https://synergyfinder.fimm.fi) analysis showed that cell cycle checkpoint inhibitors synergistically enhanced the cytotoxic effect of cytarabine on Kasumi-1 DTPs. Collectively, this study suggests that novel combination treatment of cell cycle checkpoint inhibitors with conventional drugs selectively inhibits TP53-mutated AML DTPs. Citation Format: Daehyeon Gwak, Dongchan Kim, Heejun Jang, Jun Liu, Suji Min, Ja Min Byun, Youngil Koh, Junshik Hong, Sung-soo Yoon, Dong-Yeop Shin, Department of Internal Medicine, Seoul National University Hospital, Seoul, Korea, Republic of. Therapeutic targeting of TP53-mutated acute myeloid leukemia by inhibiting cell cycle checkpoint [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 4664.

Abstract 629: Activity of VIP943 on AML patient-derived leukemic blasts and healthy donor-derived bone marrow hematopoietic stem cells

Abstract Introduction: In patients with acute myeloid leukemia (AML), CD123 has been shown to have high expression on blast cells and leukemic stem cells (LSC) compared with normal hematopoietic stem cells (HSCs) and other more mature CD34+ subsets (Zhou J, World J Stem Cells 2014). LSCs are inherently resistant to standard of care chemotherapeutics and LSC persistence after chemotherapy is associated with disease relapse. VIP943 is an ADC consisting of an anti-CD123 antibody, a unique linker cleaved intracellularly by legumain which is required for release, and activation of a novel kinesin spindle protein inhibitor (KSPi) payload that accumulates inside the cell. Herein, we evaluated the potential of VIP943 to target LSC and progenitor cells. Methods: Cytotoxicity assay: Bone marrow aspirates (BMA) and peripheral blood from previously untreated patients with primary or transformed from myelodysplastic syndrome AML were used in this experiment. Cells were treated with VIP943 (66 nM or 330 nM). After the end of the 48 h or 72 h incubation, the CD123+ cells and the CD34+CD38- LSC were detected by flow cytometry. Characterization of progenitor cell population: Fresh BM samples from healthy volunteers (HVBM) were stained with specific monoclonal antibodies to identify progenitor cell populations. Depletion assay: The effect of VIP943 on HVBM was evaluated by measuring the depletion of CD34+ progenitor cell populations in comparison to the anti-CD33-ADC, gemtuzumab ozogamicin (gem-oz). At the end of the incubation time, red blood cells were lysed; the remaining cells were stained with a cocktail of antibodies to discriminate between progenitor cells and analyzed by flow cytometry. Results: Treatment of patient-derived CD123+ leukemic blasts with VIP943 resulted in a 50% reduction after 48 h and up to 80% at 72 h. A reduction of >70% of CD123+CD34+CD38- LSCs derived from BM aspirates of untreated patients with AML was achieved after incubation with VIP943 up to 72h. In addition, HVBM samples derived from five healthy volunteers were treated with VIP943 or gem-oz in a depletion assay, where gem-oz was toxic to CD34+ cells with an EC50 of 0.16 µM in contrast to VIP943 with an EC50 value of 8.83 µM. Conclusions: In this in vitro analysis of primary samples from patients with AML, activity of VIP943 is observed in AML patient-derived CD123+ blasts as well as chemoresistant LSCs. At anticipated pharmacologic levels, VIP943 shows no adverse effects on HSCs unlike gem-oz suggesting an improved therapeutic index. In the ongoing first-in-human dose-escalation study in subjects with advanced CD123+ hematologic malignancies, VIP943 demonstrates a promising safety profile (NCT06034275). Citation Format: Beatrix Stelte-Ludwig, Tibor Schomber, Melanie M. Frigault, Joseph Birkett, Amy J. Johnson, Anne-Sophie Rebstock, Sebastian Ludwig, Raquel Izumi, Ahmed Hamdy. Activity of VIP943 on AML patient-derived leukemic blasts and healthy donor-derived bone marrow hematopoietic stem cells [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 629.

A CD38-directed, single-chain T-cell engager targets leukemia stem cells through IFN-γ–induced CD38 expression

AbstractTreatment resistance of leukemia stem cells (LSCs) and suppression of the autologous immune system represent major challenges to achieve a cure in acute myeloid leukemia (AML). Although AML blasts generally retain high levels of surface CD38 (CD38pos), LSCs are frequently enriched in the CD34posCD38neg blast fraction. Here, we report that interferon gamma (IFN-γ) reduces LSCs clonogenic activity and induces CD38 upregulation in both CD38pos and CD38neg LSC-enriched blasts. IFN-γ–induced CD38 upregulation depends on interferon regulatory factor 1 transcriptional activation of the CD38 promoter. To leverage this observation, we created a novel compact, single-chain CD38-CD3 T-cell engager (BN-CD38) designed to promote an effective immunological synapse between CD38pos AML cells and both CD8pos and CD4pos T cells. We demonstrate that BN-CD38 engages autologous CD4pos and CD8pos T cells and CD38pos AML blasts, leading to T-cell activation and expansion and to the elimination of leukemia cells in an autologous setting. Importantly, BN-CD38 engagement induces the release of high levels of IFN-γ, driving the expression of CD38 on CD34posCD38neg LSC-enriched blasts and their subsequent elimination. Critically, although BN-CD38 showed significant in vivo efficacy across multiple disseminated AML cell lines and patient-derived xenograft models, it did not affect normal hematopoietic stem cell clonogenicity and the development of multilineage human immune cells in CD34pos humanized mice. Taken together, this study provides important insights to target and eliminate AML LSCs.

The potential use of recombinant immunotoxin and BCL-2 inhibitors for leukemic stem cell elimination

Leukemia is a type of hematological malignancy caused by the dysfunctional proliferation of leukocyte cells. According to the Global Burden of Disease 2019 report, there were over 643,579 new cases of leukemia cancer worldwide in 2020, killing 334,592 people. Chemotherapy, an important part of the treatment of leukemia, not only harm cancer cells but also harm healthy cells, which can lead to negative side effects. This article examines the possibility for cutting-edge therapy using targeted recombinant immunotoxins and BCL-2 inhibitors as targeted therapy against leukemic stem cells. Recombinant immunotoxin is a protein-based therapeutic agent composed of toxins combined with specific antibodies or other molecules such as growth factors, cytokines, or toxins. Diphtheria Toxin (DT) is the most commonly used toxin because it is easily expressed, making the toxin easier to take. The toxin kills cancer cells by inhibiting enzymatic cell protein synthesis. While immunotoxins enhance therapeutic outcomes by targeting leukemia stem cells, their efficacy can be compromised in certain patients due to apoptosis resistance mediated by proteins like BCL-2. The presence of anti-apoptotic protein BCL-2 enables cell survival even after exposure to immunotoxins. Consequently, combining recombinant immunotoxins and BCL-2 inhibitors holds promise for eradicating LSC and preventing relapse.

CD38-Directed, Single-Chain T Cell-Engager Targets Leukemia Stem Cells through IFNγ-Induced CD38 Expression

Leukemia stem cells (LSCs), enriched in the CD34+CD38- AML population, are considered the main drivers of relapse in acute myeloid leukemia (AML). While more differentiated progenitors and bulk AML blasts reside in the CD34+CD38+ fraction, targeting CD38 in AML is less pursued, as LSCs are mainly CD38-. We show IFNγ (10 ng/ml) induced an increase in CD38 mRNA (mean fold change IFNγ over vehicle = 7.7, p=0.03, n=3) and surface CD38 (mean CD38 High% population IFNγ 56%±17 vs vehicle 16%±10, p<0.0001) in both CD34+ and CD34- AML primary blasts, and decreased the clonogenic activities of primary AML cells (colony forming unit IFNγ 50±36 vs vehicle 216±117, p=0.002, n=4) regardless of cytogenetics. scRNA and total RNA sequencing data in CD34+ AML primary blasts showed that upon IFNγ treatment, CD38 and Interferon Regulatory Factor 1 (IRF-1) were consistently the most upregulated genes compared to vehicle (>2.5 Log 2 Fold Change; p=0; FDR=0). Furthermore, gene set enrichment analysis (FDR=0) indicated IFNγ induced an upregulation of genes involved in apoptosis and DNA repair, and a suppression of genes in the pro-oncogenic Wnt beta catenin pathways. CD38 luciferase-based reporter assays, chromatin immunoprecipitation, and IRF-1 knockdown experiments in AML cells showed that, upon IFNγ treatment, IRF1 is pivotal in CD38 transcriptional regulation. Because CD4+T helper type 1 and CD8+ cytotoxic T cells release IFNγ once engaged on their target, we hypothesized a CD38-directed T cell engager will convert CD34+CD38- LSC into CD34+CD38+ blasts via the secreted IFNγ to kill LSCs and blasts. We created a single chain construct (BN-CD38), inserting a CD38 nanobody between the light and heavy chains of an anti-CD3 Fab using two short peptide linkers. Based on modeling, BN-CD38 reproduces the distance between T cell receptor and the major histocompatibility complex, mimicking the natural immunological synapse. Surface plasmon resonance and cytometry studies showed BN-CD38 had strong binding affinity to both CD38 (K D 4.77xE -10 M) and CD3 (K D 4.27xE -8 M). The IC 50 for BN-CD38 at 1:1 E:T at 24hrs in different CD38+ AML cell lines (n=6) was within the 0.01 to 0.1 ng/ml range, whereas the IC 50 of a non-binding CD38 construct (BN-CD38Mut) was 100-fold higher. By 24hrs of treatment, we noted >60% CD4+ and CD8+ T cell activation (CD69+, CD25+, IFNγ+) with BN-CD38 (e.g., CD69: 60%±27) in contrast to controls (e.g., CD69: IgG 7%±0.8 and BN-CD38Mut 11%±10, p<0.0001). In primary AML samples (n=10) BN-CD38 activated >70% T cells against autologous leukemia cells (CD69: BN-CD38 73%±16, BN-CD38Mut 10%±9, IgG 11%±10, p<0.0001). CyTOF analysis showed BN-CD38 but not controls significantly reduced CD34+CD38+ AML blasts (%change to IgG range: BN-CD38 -27 to -82% vs BN-CD38Mut 1% to 7%, p = 0.01) and CD34+CD38- LSCs (%change to IgG range: BN-CD38 -78 to -99% vs BN-CD38Mut -7 to 11%, p= 0.0003) and expanded CD8+ effector memory (p<0.01), CD8+ terminally differentiated effector memory CD45RA+ (p<0.05), NKT cells (p<0.01), and central memory Tregs (p<0.0001). Anti-human-IFNγ neutralizing antibody reverted BN-CD38 induced expression of CD38 and BN-CD38 killing activity on CD34+CD38- AML cells (p<0.05). BN-CD38 (2.5 mg/kg, i.v, n=6/group) co-injected weekly with healthy 3x10 6 T cells induced near-complete disease eradication in mice xenografted with luciferase-CD38+ THP1 (p=0.003) and U937 (p=0.004) cells. Long-term survival analysis showed 38% of BN-CD38-treated THP-1 engrafted mice achieved complete cure compared to IgG (OS median: BN-CD38 61.5d, control IgG 29d, p=0.0005). Consistently, BN-CD38 suppressed AML cell engraftment, splenomegaly, and prolonged survival in three patient-derived xenografts (PDX) with complex karyotypes including TP53 mutation (PDX1: OS median BN-CD38 undefined, BN-CD38Mut 28d (p=0.002), and IgG 27d (p=0.002); PDX2: OS median BN-CD38 39d, BN-CD38Mut and control IgG 32d (p<0.01); PDX3: OS median BN-CD38 undefined, BN-CD38Mut 92d (p=0.001), and IgG 81d (p=0.0007)). BN-CD38 did not affect normal hematopoietic stem cell clonogenicity and the development of multi-lineage human immune cells in CD34+ humanized mice. In sum, BN-CD38 induces an effective T cell synapse and IFNγ release, while concomitantly blocking the clonogenicity and inducing CD38 expression on LSCs. These studies suggest a new mechanism to unmask and target LSCs, providing the rationale for using BN-CD38 to effectively treat AML.

Targeting CDK8/CDK19 to Disrupt Leukemic Stem Cell-like Population in Acute Myeloid Leukemia: Exploring RVU120 As a Promising Frontline Therapy

Leukemic stem cells (LSCs) are implicated in the initiation, maintenance, and relapse of leukemia. The failure of current standard induction chemotherapy, which primarily targets dividing cells, has been linked to the quiescence of LSCs, leading to resistance to treatment. Consequently, there is an urgent need for novel therapies specifically aimed at eradicating LSCs to achieve more effective and durable treatment responses. CDK8 and its paralog CDK19 associate with the mediator complex and regulate gene expression programs linked to cancer and stem cell pluripotency. RVU120, a highly selective and potent CDK8/19 inhibitor, is currently undergoing clinical trials for AML and high-risk myelodysplastic syndrome (HR-MDS) (NCT04021368). Previous studies have demonstrated its potential mechanism of action on LSCs, wherein inhibition of CDK8/CDK19 led to the activation of lineage commitment genes in sensitive AML cells. Through recent gene expression analysis, it was discovered that inhibition of CDK8/CDK19 in transformed CD34+ cord blood cells resulted in the downregulation of stemness and quiescence-associated genes, while simultaneously upregulating genes associated with hematopoietic lineage-committed progenitors. Building on these findings, this study further investigates the potential of CDK8/CDK19 inhibition as a means to target LSCs, providing a rationale for RVU120 as a frontline treatment option in AML. The impact of RVU120 on LSCs has been investigated using an in vitro hierarchical AML-8227 model. This primary model maintains distinct populations in culture conditions, including CD34+CD38- cells with quiescent LSC potential, CD34+CD38+ cells enriched in progenitors, and CD34- more mature blasts (Lechman et al., 2016, Cancer Cell). The CD34+CD38- LSC population was further characterized by flow cytometry showing the expression of LSC surface markers, namely CD45RA, CD123, CD44, and negative expression for CD90, rendering it a suitable model for mechanistic studies. Next, AML-8227 cells were subjected to RVU120 treatment, resulting in a noticeable reduction in cell proliferation and viability. Flow cytometric analysis demonstrated a significant decrease in the percentage and total cell number of LSC-enriched/progenitor populations within the AML hierarchical model upon inhibition of CDK8/CDK19. Furthermore, RVU120 treatment induced the differentiation of cells, as evidenced by a sequential increase in the percentage of early myeloid differentiation marker, CD64, and late myeloid differentiation marker, CD14 (Fig. 1). In order to deconvolute the activity and mechanism of action of RVU120 in different cell populations, single-cell RNAseq analysis on AML-8227 cells has been performed at 24h and 3 days of treatment using technology from 10xGenomics. Additionally, analyzed AML-8227 cells have been enriched in the CD34+ population to evaluate the effect of RVU120 on stem and progenitor fraction at the single-cell level. The bioinformatic analysis of the performed experiment is ongoing and its results will be integrated into the data described above. CDK8 activity can be conveniently tracked by measuring specific phosphorylation of its target S726 on STAT5 protein. Dose and exposure-dependent inhibition of this marker has been established in cells exposed to RVU120. Levels of STAT5 S726 have been measured in different cell populations in patients from the first-in-human study of RVU120 in AML/HR-MDS. Consistent with established roles of CDK8 in LSCs, significantly elevated levels of pSTAT5 were observed in CD34+ stem/progenitor-enriched population when compared to CD34- blast cells measured at baseline before the start of the treatment (p<0.0001; ratio paired t-test; updated data from ASH 2022; Fig. 2). In summary, RVU120 displays cytotoxic and differentiating effects on well-characterized LSC populations in various models. Additionally, the CDK8 activity marker is directly associated with LSC-enriched bone marrow cells in AML patients, highlighting RVU120's potential as a promising therapeutic for targeting AML LSCs. Moreover, single-cell studies offer insights into its inhibitory effects on LSC-enriched populations and capacity for inducing differentiation. Overall, these results support RVU120 as a frontline candidate in AML therapy, countering therapeutic failure caused by persistent LSCs.

Human ILC1s Target Leukemia Stem Cells and Control Development of AML

Acute myeloid leukemia (AML) is a devastating disease with a median 5-year survival of only 40-45% for patients younger than age 65 who are treated with standard chemotherapy. Although in some cases allogeneic stem cell transplantation has proven to be curative, the treatment-related mortality and the risk for disease relapse due to persistence of leukemia stem cells (LSCs) remains relatively high. Therefore, safer and more effective novel therapeutic approaches are needed to improve the clinical outcomes of patients with AML. Innate lymphoid cell (ILC) is critical in mediating immune responses and regulating tissue homeostasis and inflammation. We recently reported that mouse ILC1s contribute to the control of AML by eliminating LSCs and inhibiting their differentiation into myeloid blasts, and functional impairment of mouse ILC1s in AML leads to the outgrowth of LSCs and disease relapse (Li et al., Nature Immunology. 2022). However, the full role and mechanistic characterization of human ILC1s in anti-tumor responses to AML remains to be fully explored. Upon analysis of ILC1s in the blood of patients with AML at the onset of disease, we observed a highly significant reduction in the total ILC1s count among lineage-negative cells (Lin −, defined asdepletion of CD3, CD4, CD8, CD14, CD15, CD16, CD19, CD20, CD33, CD34, CD203c, FceRI, and CD56 positive cells) relative to healthy donors ( p = 0.031, n = 6 healthy donors; n = 4 patients with AML). Further, functional ILC1s positive for IFNγ and DNAM-1 were significantly reduced in the patients with AML compared to healthy donors. Analysis of 106 AML cases from the Cancer Genome Atlas (TCGA) showed that AML patients with a high ILC1 gene signature had a significantly prolonged overall survival compared to AML patients with a low ILC1 gene signature. By directly interacting with LSCs, human ILC1s can eliminate LSCs via the production of IFNγ. Through Wright-Giemsa staining, we observed that ILC1s blocked the differentiation of CD34 +CD38 − cells into macrophage-like leukemia-supporting cells, which were previously reported to support the growth of leukemic cells rather than inhibit them. Flow cytometry of these differentiated cells showed that some exhibited the tumor-promoting phenotype with expression of CD11b and CD206. These macrophage-like leukemia-supporting cells significantly decreased when co-cultured with ILC1s. This differentiation is at least partially dependent on TNF secreted by ILC1s. We also performed an in vivo transplantation experiment, in which human CD34 +CD38 − cells and human ILC1s were co-injected intravenously (i.v.) into NOD.Cg- Prkdc scid Il2rg tm1WjlTg (NSG-SGM3) mice that express human IL3, GM-CSF, and SCF to support the stable engraftment of myeloid lineages. Injection of human ILC1s from healthy individuals reduced the LSC engraftment into these mice and suppressed the progression of AML. This was evidenced by a significant decrease in the number of CD45 +CD33 + blast cells, CD34 +CD38 − LSCs, and significantly prolonged survival of the mice ( p = 0.0118, n = 5/group). These results were all in comparison to mice that did not receive an injection of ILC1s. Although ILC1s target LSCs, they have no toxicity on normal HSCs. For a therapeutic purpose, we were able to derive ILC1s from umbilical cord blood (UCB) CD34 + hematopoietic stem cells (HSCs) with over 700-fold expansion and the expanded ILC1s demonstrate authentic ILC1 phenotypes and functions. Moreover, arming the expanded ILC1s with a chimeric antigen receptor (CAR) targeting FLT3, which we previously constructed (Chen et al., Leukemia. 2017), enhanced their effector function. In conclusion, ILC1s in patients with AML are impaired, while a high expression of the ILC1 gene signature is associated with better overall survival. Human ILC1s can both eliminate LSCs via production of IFNγ and block LSC differentiation into tumor-promoting macrophage-like cells through TNF. These effects converge to limit leukemogenesis in vivo. The use of UCB CD34 + HSCs to generate ILC1s, especially after being engineered with a CAR, could allow for a readily available supply of ILC1s to be produced for human adoptive transfer studies. Our findings provide evidence that targeting human ILC1s may be a promising therapeutic approach for extending disease-free survival in patients with AML.

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

Engineered mesenchymal stem cell exosomes loaded with miR-34c-5p selectively promote eradication of acute myeloid leukemia stem cells

Most patients with acute myeloid leukemia (AML) relapse eventually because of the inability to effectively eliminate leukemia stem cells (LSCs), prompting the search of new therapies to eradicate LSCs. Our previous study demonstrated that miR-34c-5p promotes the clearance of LSCs in an AML mouse model, highlighting its potential as a therapeutic target for eradicating LSCs, but the effective delivery of miR-34c-5p to LSCs remains a great challenge. Here, we employed simultaneous two-step modifications to engineer mesenchymal stem cells (MSCs) and MSC-derived exosomes to create exosomes overexpressing the fused protein lysosome-associated membrane protein 2-interleukin 3 (Lamp2b-IL3) and hematopoietic cell E-selectin/L-selectin ligand (HCELL), and demonstrated that the engineered exosomes exhibited an enhanced ability for bone marrow homing and selective targeting of LSCs. Additionally, using a humanized AML mouse model, we confirmed that the engineered exosomes, loaded with miR-34c-5p, could selectively promote eradication of LSCs and impede the AML development in vivo. In summary, we successfully designed an effective delivery system and provided new insights into the development of novel therapies for delivering miRNA or other molecules to LSCs with greater cellular targeting specificity.

MiR-30e-5p regulates leukemia stem cell self-renewal through the Cyb561/ROS signaling pathway.

Leukemia stem cells (LSC) represent a crucial and rare subset of cells present in acute myeloid leukemia (AML); they play a pivotal role in the initiation, maintenance, and relapse of this disease. Targeting LSC holds great promise for preventing AML relapse and improving long-term outcomes. However the precise molecular mechanisms governing LSC self-renewal are still poorly understood. Here, we present compelling evidence that the expression of miR-30e-5p, a potential tumor-suppressive microRNA, is significantly lower in AML samples than in healthy bone marrow samples. Forced expression of miR- 30e effectively inhibits leukemogenesis, impairs LSC self-renewal, and delays leukemia progression. Mechanistically, Cyb561 acts as a direct target of miR-30e-5p in LSC, and its deficiency restricts the self-renewal of LSC by activating reactive oxygen series signaling and markedly prolongs recipients' survival. Moreover, genetic or pharmacological overexpression of miR-30e-5p or knockdown of Cyb561 suppresses the growth of human AML cells. In conclusion, our findings establish the crucial role of the miR-30e-5p/Cyb561/ROS axis in finely regulating LSC self-renewal, highlighting Cyb561 as a potential therapeutic target for LSC-directed therapies.

Therapeutic targeting of leukemia stem cells in acute myeloid leukemia.

One of the distinguishing properties of hematopoietic stem cells is their ability to self-renew. Since self-renewal is important for the continuous replenishment of the hematopoietic stem cell pool, this property is often hijacked in blood cancers. Acute myeloid leukemia (AML) is believed to be arranged in a hierarchy, with self-renewing leukemia stem cells (LSCs) giving rise to the bulk tumor. Some of the earliest characterizations of LSCs were made in seminal studies that assessed the ability of prospectively isolated candidate AML stem cells to repopulate the entire heterogeneity of the tumor in mice. Further studies indicated that LSCs may be responsible for chemotherapy resistance and therefore act as a reservoir for secondary disease and leukemia relapse. In recent years, a number of studies have helped illuminate the complexity of clonality in bone marrow pathologies, including leukemias. Many features distinguishing LSCs from normal hematopoietic stem cells have been identified, and these studies have opened up diverse avenues for targeting LSCs, with an impact on the clinical management of AML patients. This review will discuss the role of self-renewal in AML and its implications, distinguishing characteristics between normal and leukemia stem cells, and opportunities for therapeutic targeting of AML LSCs.

Treatment-free remission after discontinuation of imatinib, dasatinib, and nilotinib in patients with chronic myeloid leukemia

Patients with chronic myeloid leukemia (CML) in the chronic phase receiving tyrosine kinase inhibitor (TKI) therapy are expected to have long-term survival outcomes comparable to those of the general population. Many clinical trials have confirmed that some patients sustain molecular responses without continuing TKI therapy. Treatment-free remission (TFR) is a new goal in treating chronic CML. The safety and outcome of TFR were studied in clinical trials after discontinuing imatinib or the second-generation TKIs dasatinib or nilotinib. TFR was safe in approximately 50% of patients who achieved a deep molecular response to TKI therapy. Patients who relapsed after discontinuing TKI responded immediately to the reintroduction of TKI. The mechanism by which TFR increases the success rate still needs to be understood. The hypothesis that the modulation of immune function and targeting of leukemic stem cells could improve the TFR is under investigation. Despite the remaining questions, the TFR has become a routine consideration for clinicians in the practice of molecular remission in patients with CML.

FBXO22 promotes leukemogenesis by targeting BACH1 in MLL-rearranged acute myeloid leukemia

BackgroundSelectively targeting leukemia stem cells (LSCs) is a promising approach in treating acute myeloid leukemia (AML), for which identification of such therapeutic targets is critical. Increasing lines of evidence indicate that FBXO22 plays a critical role in solid tumor development and therapy response. However, its potential roles in leukemogenesis remain largely unknown.MethodsWe established a mixed lineage leukemia (MLL)-AF9-induced AML model with hematopoietic cell-specific FBXO22 knockout mice to elucidate the role of FBXO22 in AML progression and LSCs regulation, including self-renewal, cell cycle, apoptosis and survival analysis. Immunoprecipitation combined with liquid chromatography-tandem mass spectrometry analysis, Western blotting and rescue experiments were performed to study the mechanisms underlying the oncogenic role of FBXO22.ResultsFBXO22 was highly expressed in AML, especially in MLL-rearranged (MLLr) AML. Upon FBXO22 knockdown, human MLLr leukemia cells presented markedly increased apoptosis. Although conditional deletion of Fbxo22 in hematopoietic cells did not significantly affect the function of hematopoietic stem cells, MLL-AF9-induced leukemogenesis was dramatically abrogated upon Fbxo22 deletion, together with remarkably reduced LSCs after serial transplantations. Mechanistically, FBXO22 promoted degradation of BACH1 in MLLr AML cells, and overexpression of BACH1 suppressed MLLr AML progression. In line with this, heterozygous deletion of BACH1 significantly reversed delayed leukemogenesis in Fbxo22-deficient mice.ConclusionsFBXO22 promotes MLLr AML progression by targeting BACH1 and targeting FBXO22 might be an ideal strategy to eradicate LSCs without influencing normal hematopoiesis.

Abstract PR004: High-density CRISPR screens reveal mechanisms of chromatin regulation of stemness networks in acute myeloid leukemia

Abstract Several leukemia-associated oncoproteins activate transcriptional circuits resembling a stem-like state in acute myeloid leukemia (AML). This activation of “stemness” genes is achieved by enlisting the activity of specialized components of the epigenetic machinery. We sought to comprehensively map epigenetic regulators critical for perpetuating these stemness networks in AML, as they may represent new therapeutic targets. To this end, we used a GFP-reporter knocked into the endogenous locus of the key leukemia oncogene and self-renewal-associated gene MEIS1 and conducted a pooled domain-focused CRISPR screen targeting >600 epigenetic modifiers. Through this screen, we identified and validated multiple members of eight distinct chromatin-modifying complexes that were required for sustaining MEIS1 expression in diverse AML subtypes. These included several novel MEIS1 regulators such as TAF6, LDB1, KAT2A, AFF2, JADE3, casein kinase 2 (CK2), ENY2 and SGF29, in addition to previously characterized regulators such as DOT1L, AF10, ENL and HBO1. A secondary pooled CRISPR screen, coupled with a single-cell transcriptome readout (CROP-seq) revealed that the deletion of several of these MEIS1 activators not only reversed MEIS1 activation but also reduced expression of stem-cell associated genes, including genes of the HOXA cluster, BMI1, SATB1 and Musashi 2, and concomitantly activated expression of differentiation-associated genes. Of particular interest to us was the TUDOR domain chromatin reader SGF29, a key component of the SAGA (Spt-Ada-Gcn5 acetyltransferase) complex. CRISPR-ko of SGF29 significantly reduced the proliferation of cells with distinct HOX-activating mutations including KMT2A rearranged and AF10-rearranged AML cells. ChIP-seq studies showed that SGF29 occupied the promoters and enhancers of key leukemia-oncogenes in a TUDOR-domain-dependent manner and SGF29 deletion selectively attenuated their transcription. Importantly, chromatin proteomics showed that SGF29 deletion led to the eviction of the SAGA complex subunit KAT2A from chromatin and into the cytoplasm. Further, SGF29 knockout impaired blast colony formation and induced differentiation in the KMT2A-MLLLT3, KMT2A-AF10, and CALM-AF10 mouse AML models but did not affect normal hematopoietic colony formation. Lastly, SGF29 deletion delayed disease latency in two distinct human AML cell line models as well as a patient-derived xenograft model in vivo and resulted in striking antiproliferative effects. Our study revealed several new attractive nodes for therapeutic targeting of leukemia stem cells in AML, including the chromatin reader SGF29, and provides a framework to identify vulnerabilities against recalcitrant oncogenic networks. Citation Format: Karina O. Barbosa Guerra, Anagha Deshpande, Ping Xiang, Anna Minkina, Fiorella Schischlik, Adam Brown, Neil A. Robertson, John Doench, Peter D. Adams, Keith Humphries, Eytan Ruppin, Jay Shendure, Prashant Mali, Aniruddha Deshpande. High-density CRISPR screens reveal mechanisms of chromatin regulation of stemness networks in acute myeloid leukemia. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr PR004.

VIP943 Is a Novel CD123 Antibody Drug Conjugate with in Vitro and In Vivo Efficacy in Acute Myeloid Leukemia (AML) Models

Background: Current treatment options (cytarabine with anthracyclines) for patients with acute myeloid leukemia (AML) are often associated with severe and barely tolerable toxicities. In patients who tolerate induction therapy, the complete remission rate is 60%-80%; however, 50% of patients relapse due to the presence of leukemic stem cells (LSC). Antibody-drug conjugates (ADCs) are putatively designed to deliver potent drugs directly to cancer cells while sparing normal tissue; however, first-generation ADCs are associated with severe bone marrow and liver toxicity. VIP943 is a CD123-targeting ADC which uses a kinesin spindle protein inhibitor (KSPi) as a new payload class and a unique legumain linker designed to overcome liabilities of currently approved ADC therapies. The payload is delivered with a drug-antibody ratio of 6. It consists of the KSPi warhead which is modified with a CellTrapperTM moiety so it accumulates within the CD123-targeted tumor cell and cannot enter normal cells. Non-human primate toxicology studies demonstrated a highly favorable and differentiated safety profile for VIP943 (Kirchhoff et al, Cancers 2020). Here we characterize VIP943 in various patient-derived AML models. Methods: For co-localization experiments, MOLM-13 AML cell line was stained with labelled anti-CD123 antibody or isotype control and CytoPainter LysoGreen indicator reagent and analyzed by imaging flow cytometry. AML primary patient mononuclear cells from blood and bone marrow were treated with VIP943 for 24, 48, and 72 hours. Flow cytometry immunophenotyping for AML blasts and CD34+/CD38- LSCs was used to analyze these populations. An in vivo patient-derived AML PDX mouse model was treated with VIP943 (5 mg/kg IV every 7 days) or in combination with 5-azacytidine (2.5 mg/kg SC days 1-5 x 3) and venetoclax (50 mg/kg PO days 1-5 x 3). Cytokine release syndrome was analyzed comparing VIP943 to isotype control ADC, anti-CD123 antibody, and positive comparator antibodies by in vitro cytokine release assays (CRA) in two different assay formats using whole blood and isolated mononuclear cells from 10 healthy donors. Flow cytometric lymphocyte and leukemic immunophenotyping was performed from blood of non-human primates. A leukemia cell line panel drug screen with VIP943 was evaluated and baseline genomic features were correlated with response. Results: Co-localization studies confirm specific internalization and efficient intracellular trafficking to the lysosome of VIP943. In vitro treatment with VIP943 (66 nM, continuous exposure) delivers blast reductions of 37% after a 24h incubation with VIP943, 45% after 48h incubation and 83% after an 72h incubation. In the CD34+/CD38- cell population, a reduction of 71% was achieved. In a patient-derived AML model, VIP943 with 5-azacytidine and venetoclax led to complete tumor growth inhibition after 21 days showing superiority of this triplet therapy (complete regression: 8/9 mice) with high tolerability (maximum body weight loss -4.8 %). The immunotoxicological evaluation in both CRA formats and with the tested antibody concentrations (100µg, 10µg, 1µg) exerts no induction of cytokines by VIP943 compared to controls. Immunophenotyping demonstrates no impact of VIP943 treatment on lymphocytic subpopulations, whereas the CD123-positive basophils are reduced after 24h followed by an almost complete recovery. VIP943 IC50 ranges from 0.1nM-300nM in a leukemia cell line panel screen and features associated with sensitivity are investigated. Conclusions: VIP943 is a next-generation ADC with a differentiated safety profile from currently approved ADCs, including lack of in vitro cytokine release. In vitro and in vivo studies using patient-derived AML cells show VIP943 has favorable monotherapy and combination efficacy including targeting of leukemic stem cells, which drive relapse. These findings warrant evaluating VIP943 in clinical trials.

Targeting Leukemic Stem Cells in Acute Myeloid Leukemia Using a CpG-Linked Anti-Mir-126 Oligonucleotide

Introduction: Acute myeloid leukemia (AML) is a lethal hematopoietic malignancy, characterized by the accumulation of clonal myeloid progenitor cells arrested in development. AML patients have dismal survival odds with a 5-year overall survival (2011 - 2017) of 29.5% (SEER). The main cause of AML treatment failure is the persistence of leukemia stem cells (LSCs). Despite recent approval of new drugs, LSCs are not specifically targeted by AML therapies. MicroRNA (miR)-126 is a non-coding regulatory RNA that is necessary for the maintenance of LSCs in the bone marrow (BM) niche, where vascular endothelial cells (ECs) supply LSCs with miR-126 and promote LSC quiescence. Several lines of evidence validate miR-126 as a target in AML. Preclinical development of CpG-anti-miR126 oligonucleotide, termed miRisten, an inhibitor of miR-126, as a drug to treat AML is described. Hypothesis: Systemic administration of miRisten will result in pharmacological depletion of miR-126 levels in the ECs, AML blast cells and LSCs, leading to the eradication of LSCs. Objective: To investigate the pharmacological properties of miRisten to establish a dosing strategy that would result in prolonged knockdown of miR-126. Experimental Plan: Pharmacokinetic/pharmacodynamic (PK/PD) studies were carried out in healthy wild type mice. miRisten was administered as a bolus or 30 min continuous intravenous infusion (CIVI) at 50 mg/kg (mpk) b.i.d. and the resulting PK parameters compared in plasma. miR126 expression was evaluated in BM mononuclear cells (MNC), lung, liver, kidney, heart and stomach/intestines prior to the fourth dose of miRisten (-30 min; start of infusion = 0 min) and at 30, 60, 90, 120, and 160 min after initiation of infusion. A 21-day preliminary toxicology study was carried out in mice using the same dosing regimen. Results: Plasma miRisten levels revealed a Cmax = 4098 nM (bolus) and 556.1 nM (CIVI), t½ = 0.16h (bolus) and 0.4h (CIVI) and area under the curve (AUC) of 465.0 nMxhr (bolus) and 508.4 nMxhr (CIVI). Lung, which contains a high concentration of ECs, had the highest relative levels of miR-126 normalized by Sno234 "housekeeping gene", followed by the heart and kidney. BMMNC had the lowest relative levels. In all cases, the nadir of miR-126 levels occurred approximately 1 hour after start of infusion. In BMMNC, the nadir of miR-126 was lower than 0.1% of basal level when miRisten was given CIVI while as a bolus, the nadir was ~33% of basal levels. In all tissues except lung and stomach, the level of miR-126 prior to the 4th dose was lower than at basal levels prior to miRisten exposure. The recovery of miR-126 levels varied by organ but was more pronounced in the lung and stomach, possibly reflecting a more dynamic regulation of miR-126 in these tissues and shorter intracellular t½ of the miR-126. No significant toxicities were observed by cage-side observation of behavior, monitoring of food/water intake and weight or by histopathological examination of tissues following 21 days of treatment. Conclusion: These data are consistent with widespread tissue distribution of miRisten and supports administering as CIVI at 50 mpk b.i.d.as miRisten effectively sustains knockdown of miR-126 with this regimen. The AUC was comparable for bolus vs CIVI but the Cmax was ~7.5 fold lower with the CIVI which therefore might be safer. When given for 21 day,s miRisten showed no overt or histologically evident toxicity.

Therapeutic inhibition of PPARα-HIF1α-PGK1 signaling targets leukemia stem and progenitor cells in acute myeloid leukemia

Treatment of acute myeloid leukemia (AML) with chemotherapeutic agents fails to eliminate leukemia stem cells (LSC)，and thus patients remain at high risk for relapse. Therefore, the identification of agents that target LSC is an important consideration for the development of new therapies. Enhanced glycolysis in LSC contributes to the aggressiveness of AML, which is difficult to be targeted. In this study, we showed that targeting peroxisome-proliferator-activated receptor α (PPARα), a ligand-activated transcription factor by chiglitazar provided a promising therapeutic approach. We first identified that chiglitazar reduced cell viability and proliferation of the leukemia stem-like cells population in AML. Treatment with chiglitazar blocked the ubiquitination of PPARα and increased its expression, resulting in the inhibition of glucose metabolism and apoptosis of AML cells. Consistent with its anti-leukemia stem-like cells activity in vitro, chiglitazar treatment in vivo resulted in the significant killing of leukemia stem-like cells as demonstrated in AML patient-derived xenograft (PDX) models. Mechanistically, PPARα overexpression inhibited the expression and promoter activity of PGK1 through blocking HIF1-α interaction on the PGK1 promoter. Thus, we concluded that targeting PPARα may serve as a novel approach for enhancing stem and progenitor cells elimination in AML.