Persistence Of Leukemic Stem Cells Research Articles

Nutrient-sensitizing drug repurposing screen identifies lomerizine as a mitochondrial metabolism inhibitor of chronic myeloid leukemia.

In chronic myeloid leukemia (CML), the persistence of leukemic stem cells (LSCs) after treatment with tyrosine kinase inhibitors (TKIs), such as imatinib, can lead to disease relapse. It is known that therapy-resistant LSCs rely on oxidative phosphorylation (OXPHOS) for their survival and that targeting mitochondrial respiration sensitizes CML LSCs to imatinib treatment. However, current OXPHOS inhibitors have demonstrated limited efficacy or have shown adverse effects in clinical trials, highlighting that identification of clinically safe oxidative pathway inhibitors is warranted. We performed a high-throughput drug repurposing screen designed to identify mitochondrial metabolism inhibitors in myeloid leukemia cells. This identified lomerizine, a US Food and Drug Administration (FDA)-approved voltage-gated Ca2+ channel blocker now used for the treatment of migraines, as one of the top hits. Transcriptome analysis revealed increased expression of voltage-gated CACNA1D and receptor-activated TRPC6 Ca2+ channels in CML LSCs (CD34+CD38-) compared with normal counterparts. This correlated with increased endoplasmic reticulum (ER) mass and increased ER and mitochondrial Ca2+ content in CML stem/progenitor cells. We demonstrate that lomerizine-mediated inhibition of Ca2+ uptake leads to ER and mitochondrial Ca2+ depletion, with similar effects seen after CACNA1D and TRPC6 knockdown. Through stable isotope-assisted metabolomics and functional assays, we observe that lomerizine treatment inhibits mitochondrial isocitrate dehydrogenase activity and mitochondrial oxidative metabolism and selectively sensitizes CML LSCs to imatinib treatment. In addition, combination treatment with imatinib and lomerizine reduced CML tumor burden, targeted CML LSCs, and extended survival in xenotransplantation model of human CML, suggesting this as a potential therapeutic strategy to prevent disease relapse in patients.

Integrated drug profiling and CRISPR screening identify BCR::ABL1-independent vulnerabilities in chronic myeloid leukemia

BCR::ABL1-independent pathways contribute to primary resistance to tyrosine kinase inhibitor (TKI) treatment in chronic myeloid leukemia (CML) and play a role in leukemic stem cell persistence. Here, we perform exvivodrug screening of CML CD34+ leukemic stem/progenitor cells using 100 single drugs and TKI-drugcombinations and identify sensitivities to Wee1, MDM2, and BCL2 inhibitors. These agents effectively inhibit primitive CD34+CD38- CML cells and demonstrate potent synergies when combined with TKIs. Flow-cytometry-baseddrug screening identifies mepacrine to induce differentiation of CD34+CD38- cells. We employ genome-wide CRISPR-Cas9 screening for six drugs, and mediator complex, apoptosis, and erythroid-lineage-related genes are identified as key resistance hits for TKIs, whereas the Wee1 inhibitor AZD1775 and mepacrine exhibit distinct resistance profiles. KCTD5, a consistent TKI-resistance-conferring gene, is found to mediate TKI-induced BCR::ABL1 ubiquitination. In summary, we delineate potential mechanismsfor primary TKI resistance and non-BCR::ABL1-targeting drugs, offering insights for optimizing CML treatment.

Unveiling IL6R and MYC as Targeting Biomarkers in Imatinib-Resistant Chronic Myeloid Leukemia through Advanced Non-Invasive Apoptosis Detection Sensor Version 2 Detection.

The management of chronic myelogenous leukemia (CML) has seen significant progress with the introduction of tyrosine kinase inhibitors (TKIs), particularly Imatinib. However, a notable proportion of CML patients develop resistance to Imatinib, often due to the persistence of leukemia stem cells and resistance mechanisms independent of BCR::ABL1 This study investigates the roles of IL6R, IL7R, and MYC in Imatinib resistance by employing CRISPR/Cas9 for gene editing and the Non-Invasive Apoptosis Detection Sensor version 2 (NIADS v2) for apoptosis assessment. The results indicate that Imatinib-resistant K562 cells (K562-IR) predominantly express IL6R, IL7R, and MYC, with IL6R and MYC playing crucial roles in cell survival and sensitivity to Imatinib. Conversely, IL7R does not significantly impact cytotoxicity, either alone or in combination with Imatinib. Further genetic editing experiments confirm the protective functions of IL6R and MYC in K562-IR cells, suggesting their potential as therapeutic targets for overcoming Imatinib resistance in CML. This study contributes to understanding the mechanisms of Imatinib resistance in CML, proposing IL6R and MYC as pivotal targets for therapeutic strategies. Moreover, the utilization of NIADS v2 enhances our capability to analyze apoptosis and drug responses, contributing to a deeper understanding of CML pathogenesis and treatment options.

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.

CircFAM193B interaction with PRMT6 regulates AML leukemia stem cells chemoresistance through altering the oxidative metabolism and lipid peroxidation.

Most forms of chemotherapy for acute myeloid leukemia (AML) are often ineffective in eliminating leukemic stem cells (LSCs), as their underlying mechanisms remain unclear. Here, we have identified circFAM193B, which regulates the redox biology of LSCs and is associated with unfavorable outcomes in AML patients. In vitro and in vivo assays suggested that circFAM193B significantly inhibits LSCs chemotherapy resistance and AML progression. Knockdown circFAM193B enhances mitochondrial OXPHOS function and inhibits the accumulation of reactive oxygen species and lipid peroxidation mediated by chemotherapy, which protects AML cells from oxidative stress-induced cell death. Mechanistically, circFAM193B physically interacts with arginine methyltransferase PRMT6 catalytic domain and enhances the transcription efficiency of key lipid peroxidation factor ALOX15 by decreasing H3R2me2a modification. In summary, we have identified circFAM193B was downregulated in LSCs to promote the survival of LSC by modulating energy metabolism and the redox balance in the postchemotherapy persistence of LSC. Our studies provide a conceptual advance and biological insights regarding the drug resistance of LSCs via circRNA mediated PRMT6-deposited methylarginine signaling.

Pharmacology of a CpG-Linked Anti-Mir-126 Oligonucleotide Targeting Acute Myeloid Leukemia (AML) in Rats and Non-Human Primates (NHP)

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 outcomes with a 5-year overall survival (2011 - 2017) of 29.5% (SEER). The persistence of leukemia stem cells (LSCs), a treatment-resistant primitive cell population able to inititiate and maintain AML, is thought to underlie relapse. Even newly approved drugs do not target LSCs efficiently. MicroRNA (miR)-126 is a non-coding regulatory RNA that contributes to LSC homeostasis in the bone marrow niche. Several lines of evidence validate miR-126 as a target in AML. Thus, we designed and made a novel anti-miR-126 oligonucleotide (oligo) linked to a CpG moiety, termed miRisten. miRisten incorporates modifications such as 2'-fluoro and phosphorothioate substituting phosphodiester in the sugar moiety of the nucleotide to enhance in vivo stability. miRisten eradicates LSCs in mouse models of AML and synergizes with FDA-approved AML drugs (PMID: 29505034, 34372909). Here, the results of IND-enabling toxicokinetic (TK) studies of miRisten in rats and NHP are presented. Objective: To investigate the pharmacological properties of miRisten to establish a safe dosing strategy to achieve prolonged knockdown of miR-126 for a first-in-human trial. Experimental Plan and Results: Good laboratory practice (GLP) TK studies were carried out in Sprague-Dawley rats. miRisten was given IV b.i.d. for 19 days at human equivalent doses (HED) of 1.6 and 4 mg/kg. miRisten was measured in plasma after the first dose. Plasma miRisten C max average was 3622 ± 335 and 6010 ± 175 nM post first doses of 1.6 and 4mg/kg, respectively. Plasma t 1/2 averaged 20.0 ± 3.8 minutes across both doses. The area under the curve (AUC 0 - ¥) were 638.9 ± 32 nMxhr (1.6 mg/kg) and 1313.5 ± 439 (4 mg/kg). A parallel toxicology study was undertaken documenting cage-side observations and body weight; complement activation, blood chemistries, urine and hematology were also evaluated. There were no unscheduled deaths in the toxicology segment. Four single NHP exploratory studies were also carried out (Fig. 1: Treatment schema). In each study, miRisten was given IV as a slow bolus (~5') b.i.d. and PK parameters evaluated in plasma. Study #1 & #2: 1 wk at 4mg/kg; #3: 1 wk at 5 mg/kg; #4: 18 days at 4 mg/kg. In all studies, miRisten dosage was ramped up (1, 2, 4 mg/kg for all and 5 mg/kg at dose 4 for Study #3). Solumedrol was given as premedication for the first 3 doses in all studies to prevent known phosphorothioate-mediated complement activation and cytokine release syndrome. Blood draws for PK were carried out immediately following doses 3 and 12 (Studies 1 - 3) and after the last dose on Day 18 (Study 4). The average area under the curve AUC was 3860 ± 1189 (4 mg/kg) and 3954 ± 1569 nMxhr (5 mg/kg) which was greater than observed in mice given the equivalent HED in bolus doses (~6x, 465.0). The t 1/2 of plasma miRisten was 11.7 min on Day 3 and 12.0 min on Day 7, comparable to mouse (9.6 min) and rat (20 min). Cage-side observations, body weight, complement activation, blood chemistries, urine and hematology were also evaluated. There was no terminal sacrifice of NHP however, all animals tolerated the treatment without observable adverse events. Conclusion: The dose ramp-up in NHP combined with pre-medication with solumedrol were undertaken to prevent possible acute inflammatory reactivity leading potentially to death. There were no unscheduled deaths in any species studied. These data are consistent with the safety of miRisten given IV with a dose ramping regimen combined with solumedrol. The greater AUC in NHP may predict better potency in humans. In mice, miRisten at 1.6 mg/kg HED q.d. effectively eradicated LSCs [PMID 29505034].

Deciphering the Role of RAS Pathway Mutations in the Biology of Human Acute Myeloid Leukemia Using In Vivo Models

Acute myeloid leukemia (AML) is a genetically heterogeneous hematologic malignancy for which the mutational profile of the leukemic cells is the most important prognostic factor for disease-free survival and overall survival. The failure of chemotherapy to completely eradicate the leukemic clones is the most common cause of death in AML. The genetic events that can contribute to chemoresistance are therefore important factors for the clinical outcome, but the relative importance of these events on chemoresistance and persistence of leukemia stem cells (LSC) remains poorly defined. MLL rearranged AMLs account for 5-10% of all AMLs and the RAS pathway is the most frequently mutated pathway in this sub-group of AMLs. The goal of this project is to study the role and biology of RAS pathway mutations in clonal evolution in human MLL-rearranged AML. Methods We have previously shown that human cord blood CD34+ cells transduced with a retrovirus encoding the MLL-AF9 fusion gene with an EGFP maker generate human AML when injected into immunodeficient NSG-SGM3 mice. Using a similar experimental design, we added a second hit to the CD34 transduced cells before their injection into NSG-SGM3 mice. MLL-AF9 cells were electroporated with Cas9 enzyme for the control and with Cas9 enzyme plus a single guide RNA (sgRNA) designed to induce the KRAS G13D or the NRAS G12D mutation. Mice were sacrificed when they showed sign of disease and the bone marrow and spleen were collected and analyzed. Kaplan-Meier curves, flow cytometry analyses, RNA and exome sequencing and secondary transplantation were performed on the samples. Results Mice injected with MLL-AF9 + KRAS G13D got sick around day 80 after injection and around day 60 for MLL-AF9 NRAS G12D, whereas it took ~150 days for control mice (MLL-AF9 KRAS WT) to get sick (fig 1). In multiple experiment, the latency was roughly cut by half by the KRAS G13D and NRAS G12D mutations. The cell surface phenotype and morphology were not significantly different between MLL-AF9 WT and MLL-AF9 KRAS/NRAS mutated. The variant allele frequency (VAF) for the KRAS mutation invariably increased from the day of injection to the day of sacrifice, where the percentages varied between 20-40%, which is typically observed in AML patients. In contrast, the VAF for NRAS mutated cells was lower (3-9%) but seems to be very aggressive into mice. We performed limiting dilution analyses in secondary mice to quantify the LSC frequencies in MLL-AF9 WT and KRAS G13D / NRAS G12D leukemia. The average frequencies of control MLL-AF9 AMLs were between 1/510 000 to 1/1 450 000, while MLL-AF9+KRAS G13D AMLs frequencies were >1/45 000 and 1/7 800 for MLL-AF9+NRAS G12D AMLs, demonstrating that these 2 mutations increase the LSC frequency by >20-100 fold in our modelled MLL-AF9 AML. Using transcriptome analyses, we looked at the gene expression changes induced by the KRAS G13D mutation in comparison to MLL-AF9 leukemias. These experiments are in progress for the NRAS G13D mutation. Many more genes were overexpressed in MLL-AF9 KRAS G13D vs MLL-AF9 KRAS WT than down regulated. Genes from the hedgehog (Hh) pathway such as Patched1 (PTCH1) and Smoothened (SMO) were amongst the genes the most differentially overexpressed by the KRAS mutation. It is particularly interesting because they have been shown important in stem cell biology and could explain the increase in LSC. Some SMO inhibitors have been tested in clinical trial and Glasdegib is FDA approved in combination with low dose cytarabine in elderly patients. Its effect on MLL leukemias or on KRAS mutated AMLs has not been tested/reported in a clinical study. Experiments are currently in progress to test the effect of Glasgebib on engraftment of MLL-AF9 KRAS mutated cells in our experimental setting. Single cell RNA sequencing data on the three conditions will be presented at the meeting. Given the success of generating these two point mutations, we are currently generating more RAS pathway mutations, including other KRAS mutations, PNTP11 and NF1 mutations. Conclusion These experiments showed: 1) It is possible to induce 2 oncogenic hits in human primary cells and get leukemia in vivo; 2) the KRAS G13D and NRAS G12D mutations shorten the latency of the disease and 3) increase the LSC frequency in secondary mice; 4) a possible involvement of the Hh pathway on stemness/LSC in RAS mutated cells; 5) our experimental approach is robust and very promising to decipher the RAS pathway in human MLL leukemias.

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.

Cytokine profile in patients with chronic myeloid leukemia

Resistance to tyrosine kinase inhibitors (TKIs) is currently an important clinical problem in the management of patients with chronic myeloid leukemia (CML). Recent studies suggested that aberrant cytokine secretion may be among the BCR/ABL-independent mechanisms of resistance, thus contributing to the persistence of leukemic stem cells in spite of continuous targeted therapy. The aim of the study was to evaluate concentration of cytokines in the serum of patients with CML depending on the efficiency of therapy.Quantitative determination of the cytokines (TNFα, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-17, IL-18, IFNα and VEGF) in blood serum of patients with chronic-phase CML (n = 84) and healthy subjects (n = 30) was performed using enzyme immunoassay (ELISA). The patients with CML were divided into 3 groups depending on the duration of therapy: group I, newly diagnosed patients (n = 10); group II, patients receiving therapy for < 12 months (n = 10); group III included patients receiving therapy for more than 12 months (n = 64).The results of our study showed that cytokine concentration among CML patients significantly differed, depending on the duration of therapy. Significantly higher concentration of IL-17, IL-6, IL-1β, IL-10, IL-18, IL-2 and TNFα was found in group I compared with control group. Group II patients also demonstrated significantly higher concentrations of TNFα, IL-6, IL-10, IL-18 and IFNα by comparison with control group, as well as higher concentration of IFNα compared with in groups I and III. In group III, concentrations of IL-17, IL-1β, TNFα, IL-6, IL-10, IL-18 were significantly higher than in control group. When compared with group I, it was found that concentrations of IL-1β, IL-2 and IL-18 were significantly lower. A direct correlation was found between expression levels of chimeric BCR/ABL gene, (a marker of CML malignancy), and concentrations of IL-1β and IL-17. ROC-analysis demonstrated high-quality models which showed an association between achievement of major molecular response (MMR) and low serum concentrations of IL-1β, IL-6 and IL-17.Hence, the results of our study have shown that determination of IL-1β, IL-6 and IL-17 concentrations may be a prognostic marker for assessing the efficiency of therapy and probability of achieving MMR in CML.

Bcl-2 inhibition combined with PPARα activation synergistically targets leukemic stem cell-like cells in acute myeloid leukemia

Persistence of leukemic stem cells (LSCs) is one of the determining factors to acute myeloid leukemia (AML) treatment failure and responsible for the poor prognosis of the disease. Hence, novel therapeutic strategies that target LSCs are crucial for treatment success. We investigated if targeting Bcl-2 and peroxisome proliferator activated receptor α (PPARα), two distinct cell survival regulating mechanisms could eliminate LSCs. This study demonstrate that the Bcl-2 inhibitor venetoclax combined with the PPARα agonist chiglitazar resulted in synergistic killing of LSC-like cell lines and CD34+ primary AML cells while sparing their normal counterparts. Furthermore, the combination regimen significantly suppressed AML progression in patient-derived xenograft (PDX) mouse models. Mechanistically, chiglitazar-mediated PPARα activation inhibited the transcriptional activity of the PIK3AP1 gene promoter and down-regulated the PI3K/Akt signaling pathway and anti-apoptotic Bcl-2 proteins, leading to cell proliferation inhibition and apoptosis induction, which was synergized with venetoclax. These findings suggest that combinatorial Bcl-2 inhibition and PPARα activation selectively eliminates AML cells in vivo and vitro, representing an effective therapy for patients with relapsed and refractory AML.

Myelofibrosis at diagnosis is associated with the failure of treatment-free remission in CML patients.

The management of patients with chronic myeloid leukemia (CML) has been revolutionized by the introduction of tyrosine kinase inhibitors (TKIs), which induce deep molecular responses so that treatment can eventually be discontinued, leading to treatment-free remission (TFR) in a subset of patients. Unfortunately, leukemic stem cells (LSCs) often persist and a fraction of these can again expand in about half of patients that attempt TKI discontinuation. In this study, we show that presence of myelofibrosis (MF) at the time of diagnosis is a factor associating with TFR failure. Fibrotic transformation is governed by the action of several cytokines, and interestingly, some of them have also been described to support LSC persistence. At the cellular level, these could be produced by both malignant cells and by components of the bone marrow (BM) niche, including megakaryocytes (MKs) and mesenchymal stromal cells (MSCs). In our cohort of 57 patients, around 40% presented with MF at diagnosis and the number of blasts in the peripheral blood and BM was significantly elevated in patients with higher grade of MF. Employing a CML transgenic mouse model, we could observe higher levels of alpha-smooth muscle actin (α-SMA) in the BM when compared to control mice. Short-term treatment with the TKI nilotinib, efficiently reduced spleen weight and BCR::ABL1 mRNA levels, while α-SMA expression was only partially reduced. Interestingly, the number of MKs was increased in the spleen of CML mice and elevated in both BM and spleen upon nilotinib treatment. Analysis of human CML-vs healthy donor (HD)-derived MSCs showed an altered expression of gene signatures reflecting fibrosis as well as hematopoietic support, thus suggesting MSCs as a potential player in these two processes. Finally, in our cohort, 12 patients qualified for TKI discontinuation, and here we observed that all patients who failed TFR had BM fibrosis at diagnosis, whereas this was only the case in 25% of patients with achieved TFR, further supporting the link between fibrosis and LSC persistence.

Molecular genetic and cytofluorimetric prognostic factors in the development of acute myeloid leukemia relapse in children after allogeneic hematopoietic stem cell transplantation

Relapse of acute myeloid leukemia (AML) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains one of the main causes of reduced long-term survival. Modern methods for predicting the risk of AML relapse after allo-HSCT take into account the data on the pre-transplant level of minimal residual disease (MRD) determined by flow cytometry and molecular biological studies of recurrent genetic abnormalities, which are currently widespread in clinical practice. Recent studies of the expression of genes characteristic of leukemic stem cells (LSCs) have shown prognostic significance for children with AML in relation to treatment response and the risk of relapse. The study of LSC persistence in order to predict the risk of recurrence after allo-HSCT in children with AML in addition to standard MRD detection methods may be of great importance. The aim of the work was to evaluate the impact of MRD status, both using classic methods and taking into account the genes characteristic of LSC, on the results of allo-HSCT in children with AML. The study was approved by the Independent Ethics Committee and the Scientific Council of the I.P. Pavlov First Saint Petersburg State Medical University of Ministry of Healthcare of the Russian Federation. To assess MRD using standard diagnostic methods, we analyzed the data of 95 children with AML in their 1st–2nd remission (cohort 1). MRD status was negative in 67 (70.6 %) patients; in 28 (29.4 %) children, MRD status was positive according to molecular genetic studies and / or immunophenotyping results. For pre-transplant evaluation of the expression of genes characteristic of LSC, we investigated bone marrow samples of 50 patients (cohort 2) using real-time polymerase chain reaction. The DNMT3B, GPR56, CD34, SOCS2, SPINK2, FAM30A, and ABL genes were studied by real-time polymerase chain reaction, followed by calculation of the pLSC6 value using the formula: DNMT3b × 0.189 + GPR56 × 0.054 + CD34 × 0.0171 + SOCS2 × 0.141 + SPINK2 × 0.109 + FAM30A × 0.0516. At the time of allo-HSCT, 37 (74 %) children with AML were in their 1st or 2nd remission of the disease, 13 (26 %) were out of the 1st–2nd remission. With a median follow-up of 5 years in the group of patients with a positive MRD status, determined by standard methods (cohort 1), overall survival (OS) was 67.9 % vs 73.1 % for patients with a negative MRD status (p = 0.83). The cumulative incidence of relapse was 50 % and 22 %, respectively; p = 0.012. When assessing the level of expression of genes characteristic of LSC (cohort 2), a pLSC6 level was above the median in 18/37 (49 %) patients. The results of linear regression showed that the pre-transplant level of expression of genes characteristic of LSC was not associated with the number of blasts/MRD (odds ratio 1.002; 95 % confidence interval 0.979–1.025). One-year OS rates did not differ significantly in children in the 1st–2nd remission of AML, depending on pLSC6 level: 84.2% in patients with low pLSC6 and 72.2 % – with high pLSC6 (p = 0.4), event-free survival in the corresponding groups – 68.4 % and 61.1 %, respectively (p = 0.34). The cumulative incidence of early relapse after allo-HSCT in the group of AML patients with a high pLSC6 score was significantly higher than in children with a low pLSC6 score before allo-HSCT (22 % and 0 %, respectively; p = 0.03). MRD does not have a statistically significant effect on OS. However, MRD positivity before allo-HSCT increases cumulative incidence of relapse. The level of expression of genes characteristic of LSC, determined before allo-HSCT, showed a prognostic significance in relation to the development of early AML relapse after allo-HSCT.

Mast cell deficiency prevents BCR::ABL1 induced splenomegaly and cytokine elevation in a CML mouse model

The persistence of leukemic stem cells (LSCs) represents a problem in the therapy of chronic myeloid leukemia (CML). Hence, it is of utmost importance to explore the underlying mechanisms to develop new therapeutic approaches to cure CML. Using the genetically engineered ScltTA/TRE-BCR::ABL1 mouse model for chronic phase CML, we previously demonstrated that the loss of the docking protein GAB2 counteracts the infiltration of mast cells (MCs) in the bone marrow (BM) of BCR::ABL1 positive mice. Here, we show for the first time that BCR::ABL1 drives the cytokine independent expansion of BM derived MCs and sensitizes them for FcεRI triggered degranulation. Importantly, we demonstrate that genetic mast cell deficiency conferred by the Cpa3Cre allele prevents BCR::ABL1 induced splenomegaly and impairs the production of pro-inflammatory cytokines. Furthermore, we show in CML patients that splenomegaly is associated with high BM MC counts and that upregulation of pro-inflammatory cytokines in patient serum samples correlates with tryptase levels. Finally, MC-associated transcripts were elevated in human CML BM samples. Thus, our study identifies MCs as essential contributors to disease progression and suggests considering them as an additional target in CML therapy.Mast cells play a key role in the pro-inflammatory tumor microenvironment of the bone marrow. Shown is a cartoon summarizing our results from the mouse model. BCR::ABL1 transformed MCs, as part of the malignant clone, are essential for the elevation of pro-inflammatory cytokines, known to be important in disease initiation and progression.

Microenvironmental CXCL12 deletion enhances Flt3-ITD acute myeloid leukemia stem cell response to therapy by reducing p38 MAPK signaling.

Fms-like tyrosine kinase 3 (Flt3) tyrosine kinase inhibitors (Flt3-TKI) have improved outcomes for patients with Flt3-mutated acute myeloid leukemia (AML) but are limited by resistance and relapse, indicating persistence of leukemia stem cells (LSC). Here utilizing a Flt3-internal tandem duplication (Flt3-ITD) and Tet2-deleted AML genetic mouse model we determined that FLT3-ITD AML LSC were enriched within the primitive ST-HSC population. FLT3-ITD LSC showed increased expression of the CXCL12 receptor CXCR4. CXCL12-abundant reticular (CAR) cells were increased in Flt3-ITD AML marrow. CXCL12 deletion from the microenvironment enhanced targeting of AML cells by Flt3-TKI plus chemotherapy treatment, including enhanced LSC targeting. Both treatment and CXCL12 deletion partially reduced p38 mitogen-activated protein kinase (p38) signaling in AML cells and further reduction was seen after treatment in CXCL12 deleted mice. p38 inhibition reduced CXCL12-dependent and -independent maintenance of both murine and human Flt3-ITD AML LSC by MSC and enhanced their sensitivity to treatment. p38 inhibition in combination with chemotherapy plus TKI treatment leads to greater depletion of Flt3-ITD AML LSC compared with CXCL12 deletion. Our studies support roles for CXCL12 and p38 signaling in microenvironmental protection of AML LSC and provide a rationale for inhibiting p38 signaling to enhance Flt3-ITD AML targeting.

Iadademstat Combination with Azacitidine Is a Safe and Effective Treatment in First Line Acute Myeloid Leukemia. Final Results of the Alice Trial

Background: Acute myeloid leukemia (AML) is a heterogenous disease with a broad spectrum of cytogenetic and molecular aberrations. Emerging targeted therapies have improved overall survival (OS) for some specific subgroups. Outcomes, however, remain poor, particularly for patients (pts) with high-risk characteristics and not eligible for intensive treatments. Combinations with hypomethylating agents are the standard of care (SoC) for unfit patients. Azacitidine (aza) plus venetoclax has shown an OS of 14.7 months (mo) and a complete remission or complete remission with incomplete hematological recovery (CR/CRi) rate of 66% (VIALE-A trial). The presence of mutations in TP53 or FLT3-ITD predicts resistance to this combination. Overall, 50% of AML pts relapse after first line treatments, generally attributed to the persistence of leukemic stem cells (LSC) and/or their clonal evolution, emphasizing the need to develop new strategies targeting LSC, to improve outcomes. One such investigational strategy is iadademstat (iada), an oral, potent and selective inhibitor of the Lysine-Specific Demethylase 1 (LSD1/KDMA1) enzyme. This enzyme works as a master regulator of transcription by a double mechanism of action: 1) removing methyl groups from histone 3 lysines (epigenetic eraser); and 2) disrupting multiprotein transcriptional complexes on genes governing cell differentiation and stemness. In myeloid cells, LSD1 provides a scaffold for assembly of the GFI1/CoREST transcriptional repressor complex, which regulates hematopoietic differentiation. Iada-induced gene expression shifts from a proliferation to differentiation signature has been preclinically characterized in AML cells (Maes, et al., Cancer Cell 2018) and in the clinic in a Ph1 study in Relapsed/Refractory AML (Salamero, et al., JCO 2020). To date, iada has been administered to more than 100 oncology pts in Ph1 and Ph2 trials, showing manageable toxicity and encouraging preliminary activity. Herein, we present the results of the Phase 2 ALICE study (EudraCT 2018-000482-36) testing the combination of iada plus aza for the frontline treatment of unfit AML patients, with a median follow-up of 6.9 mo (range 0.7-41.9) (cut-off June 30, 2022). The final data (cut-off September 30, 2022) will be presented at the ASH meeting. Methods: Unfit adult patients with AML per WHO 2017 classification who had not received previous treatment were enrolled. Two cohort doses of iada at 60 or 90μg/m2/d (PO, 5d ON, 2d OFF weekly) were explored in combination with aza (75mg/m2 SC for 7d, either d1-5, 8-9, or d1-7 every cycle) in a 28d cycle. Primary endpoints are safety, tolerability, and establishment of the recommended phase 2 dose (RP2D); secondary endpoints include remission rates, time to response (TTR), duration of responses (DoR) and OS. Additional assessments include measurable residual disease (MRD), PK/PD determinations and molecular correlations with response. Results: Baseline characteristics for 34 per protocol pts out of 36 accrued are shown in Table 1. Safety and efficacy results are detailed in Table 2. Main safety events remain consistent with previous updates, with the most frequently reported adverse reactions (AR) being platelet reduction in 53% of pts (58% of pts entered the study with grade ≥3 thrombocytopenia). Only 2 drug-related SAEs have occurred to date. Of 27 pts evaluable for efficacy, 81% responded (64% of those with CR/CRi). Responses were rapid (91% by end of C2). 71% of CR/CRi pts became transfusion independent and 82% of CR/CRi pts evaluable for MRD were negative by flow cytometry. Mean/median DoR (CR/CRi) are 9.3 mo (SE: 1.0 mo) / NE (95% CI 10.1mo, NE). Mean/median OS are 8.2 mo (SE: 0.80 mo)/9.3 mo (95% CI 11.0 mo, NE). Median OS for pts achieving CR/CRi is ~14.3 mo. Based on PK/PD, safety and efficacy, iada at 90 μg/m2/d is the RP2D for the combination. Response assessment per specific mutations shows that 3 out of 3 (3/3) with FLT3 mutations, 7/7 with RAS pathway mutations, and 6/8 pts with TP53 mutations responded. Gene expression and cell differentiation analyses are ongoing and correlations with clinical responses will be presented. Conclusion: The combination of iada with aza produces robust, rapid and durable responses in previously untreated unfit AML patients, including those with high-risk features, with a manageable toxicity profile. Further research with iada in combination with SoC treatments for AML is warranted. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The AP-1/ETS Transcription Factor Network Regulates Stem Cell Quiescence and Chemotherapy Resistance in Acute Myeloid Leukemia

Despite of the continuous improvements in the treatment for acute myeloid leukemia (AML), therapy resistance and disease relapse have been persistent and attributed to leukemia stem cells (LSCs). Recent single cell RNA sequencing (Galen et al, Bernstein 2019) and deconvoluted bulk RNA sequencing analysis (Zeng et al, Dick 2022) show hierarchical cellular organization in individual AML patient specimens, with LSCs, and quiescent LSCs in particular, at its apex. However, the mechanisms controlling LSCs and their quiescence remain elusive, hindering the development of effective targeted therapies. Here, using human patient AML specimens, we show that activator protein 1 (AP-1) and E26 transformation-specific (ETS) family transcription factors (TFs) coordinately regulate LSC quiescence and disease persistence after chemotherapy. A chemical label tracing technique using carboxyfluorescein succinimidyl ester (CFSE) has emerged as a powerful tool to identify quiescent LSCs, and using this technique, we and others have demonstrated that CFSE-label retaining quiescent LSCs isolated from patient leukemia cells exhibit potent leukemia initiating property, resistance to conventional chemotherapy, and epigenetic plasticity (Takao et al, Kentsis 2018 ASH, Ebinger et al, Jeremias 2016 and 2020). Here, we combined this method with functional genomic approaches to elucidate the mechanisms controlling quiescent LSCs and identify novel therapeutic targets to overcome chemotherapy resistance. First, to identify the candidate genes for LSC quiescence regulators, we performed gene expression and chromatin accessibility profiling of label-retaining quiescent LSCs isolated from human patient leukemia cell-engrafted NSG mice in diverse subtypes of AMLs using mRNA sequencing (RNA-seq) and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). We identified that several transcription factors important for normal hematopoiesis, which included AP-1 TFs, such as JUN and JUNB, and ETS TFs, such as ETS1 and FLI1, were differentially expressed in quiescent LSCs. Chromatin accessibility profiling further revealed the predominant occupancy of AP-1 and ETS TF family binding motifs in differentially accessible chromatin loci in quiescent LSCs. Importantly, gene expression signatures detected in quiescent LSCs showed significant similarity to gene expression profiles of residual leukemia cells observed in chemotherapy-treated AML patients (GSE116256, Galen et al. 2019), supporting the clinical relevance of our models. Next, to develop a functional screen system to identify the regulators of quiescent LSCs in human patient AML-bearing mice, we designed a lentiviral vector encoding mCherry fluorescent protein and doxycycline-inducible cDNAs marked by specific barcode sequences. cDNA library screening using this lentiviral vector identified several TFs as regulators for AML LSC quiescence, which included JUN as a positive regulator, and ETS1 and FLI1 as negative regulators (Figure 1). Finally, given the evidence of transcriptional activity of JUN to regulate LSC quiescence, we hypothesized that JUN activity may be also associated with chemotherapy resistance and disease persistence in patients. To investigate this, we analyzed JUN expression in individual AML cells in diverse patients before and after chemotherapy treatment (GSE116256, Galen et al. 2019). We found JUN to be highly expressed in persistent AML cells upon induction chemotherapy, especially in leukemia cells with features of hematopoietic stem cells (HSC-like), while ETS1 and FLI1 expression was not (Figure 2). These results argue that JUN activity may be required for chemotherapy resistance, LSC persistence, and ultimate AML relapse. In all, human AML LSC quiescence and therapy response can be dynamically regulated by a distinct transcription factor network. This work defines molecular mechanisms controlling AML LSC quiescence. We anticipate that these findings will lead to the elucidation of essential properties of LSC quiescence and the design of therapeutic strategies for their clinical identification and control. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

In Search of Drivers of CD34+/CD38-/CD26+ Leukemia Stem Cells Persistence in CML Patients

Introduction: In Chronic Myeloid Leukemia (CML) patients, we confirmed that the expression of CD26 discriminates CML Leukemic Stem Cells (LSCs) from normal Hematopoietic Stem Cells (HSCs) or from LSCs of other myeloid neoplasms. CD34+CD38-CD26+ cells are easily measurable in Peripheral Blood (PB) at diagnosis, and circulating residual CD26+LSCs persist in most patients during treatment with Tyrosine Kinase Inhibitors (TKIs) and after successful TKI discontinuation. The reason for LCSs persistence may be correlated to mechanisms helping their survival, such as resistance to apoptosis (via BCL-2 or CD47 expression) or immune checkpoint escaping (via PD-L1). Moreover, few is still known about the "activity state" or "quiescence" of CML LSCs. Aim of the study: to unravel phenotypic characteristics that could help CML LSCs persistence, evaluating by flow-cytometry the expression level of BCL-2 protein, PD-L1, CD47 and Ki67 on CD26+ LSCs population. Methods: Flow-cytometry assays were performed on PB from newly diagnosed CML patients by using lyophilized reagents (CD34-FITC, CD26-PE, CD38-APC, CD45-V500) and liquid reagents (CD47-V450, PD-L1-Cy7, BCL-2-FITC, Ki67-PerCP-Cy 5.5). Analysis have been done using a BD FACSLyric cytometer. Results: 47 newly diagnosed CML patients entered this study. With regards to PD-L1 expression on CD26+LSCs, 22/47 (47%) scored negative while 25/47 (53%) resulted positive, albeit with a quite variable percentage of expression (median 28.5 % of CD26+ cells, range 12-82.8 %). BCL-2 protein was expressed in 47/47 (100%) CD26+ LSCs, and the level of expression evaluated comparing the Mean Fluorescent Index (MFI) was found much lower on CD26+ LSCs than lymphocytes (median 5.98, range 1.90-32.14; median 17.20, range 7-76, respectively, p value <0.05). Regarding CD47 expression, 47/47 (100%) patients scored positive, with an MFI that is double on lymphocytes compared to CD26+ LSCs (median 26.58, range 5.70-87.09; median 13.22, range 5.64-33.22, respectively). Lastly, all 47/47 (100%) CD26+ LSCs were found Ki-67 positive. Conclusions: These preliminary results show that in newly diagnosed CML patients, Ki67 is highly expressed on circulating CD26+ LSCs, suggesting an unexpected "active proliferative state" of CD26+ LSCs, rather than a "quiescent" status. The latter may explain the rapid reduction of the bulk of CD26+LSCs we observe already at 3 months of TKI treatment. Both BCL-2 protein and CD47 are expressed at a higher level on lymphocytes than on CD26+ CML LSCs, indicating that the "don't eat me signal" that blocks apoptosis may not be relevant for CML LSCs persistence. On the other hand, CD26+/PD-L1+ LSCs (found in about 53% of CML patients) could theoretically escape the immune checkpoint controls, an aspect that could be taken in consideration for a future Treatment Free Survival (TFR) approach in CML patients as it may lead to a loss of molecular response compared to patients with CD26+ LSCs scoring negative for PD-L1. Additional studies need to be performed on a larger cohort of patients with a longer follow-up to support these hypotheses; however, these preliminary data may start to pave the way to understand the relationship between CD26+ LSC and immunity of CML patients.

Chimeric antigen receptor T-cells targeting IL-1RAP: a promising new cellular immunotherapy to treat acute myeloid leukemia

BackgroundAcute myeloid leukemia (AML) remains a very difficult disease to cure due to the persistence of leukemic stem cells (LSCs), which are resistant to different lines of chemotherapy and are...

Abstract 3153: Imatinib mesylate treatment improves reprogramming efficiency, and morphology of chronic myeloid leukemia derived induced pluripotent stem cells

Abstract Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder characterized by the BCR-ABL1 fusion gene with constitutively active tyrosine kinase activity. Although the tyrosine kinase inhibitors (TKI) have revolutionized the treatment for CML, withdrawal of TKI therapy in patients on deep molecular response causes disease relapse, primarily due to the persistence of leukemic stem cells (LSCs) that are insensitive to TKI. As LSCs are a rare population of cells, it is challenging to understand the molecular basis of the disease relapse to tailor strategies to eliminate them selectively. Induced pluripotent stem cells (iPSCs) derived from primary CML are increasingly used for disease modeling and high through drug screening. Cryopreserved CD34+ from three chronic phase CML patients (n=3) and CD34+ cells from a normal donor after mobilization were expanded in SFEM II supplemented with CD34+ Expansion Supplement (10X) including UM729 for 3-days and were nucleofected with episomal reprogramming plasmids as described previously (Manian et al., 2018). The nucleofection efficiency was 15% in the expanded CD34+ CML and normal cells. While the normal CD34+ cells formed iPSCs with the characteristic flat morphology between 20-24 days, the CML CD34+ cells formed several dome-shaped colonies after 30 days. Two normal CD34+ iPSC colonies continued to maintain their morphology for 12 passages. All the CML iPSC colonies (n=11) expressed the same type of BCR-ABL1 transcript as the CD34+ cells and did not carry any mutations in the BCR-ABL1 kinase domain. However, only three of the 11 CML-iPSC colonies could be maintained without significant differentiation after five passages. They lacked typical iPSC morphology and appeared as small cell aggregates or dome-shaped colonies. To understand whether this atypical morphology of the CML iPSC colonies was due to the expression of the BCR-ABL fusion protein within the cells, the medium was supplemented with 10μM imatinib. There was an increase in cell proliferation rate and gain of typical iPSCs morphology similar to normal iPSCs within two days after adding IM. Withdrawal of IM from the IM-treated CML iPSC resulted in a dome-shaped morphology, reduced proliferation rate, and reduction in TRA1-60 expression after 96 hrs. The CML iPSC cultured in the presence of IM showed significant downregulation of phospho-CRKL, a BCR-ABL1 downstream signaling protein, compared to the colonies cultured in the absence of IM. These results suggest that suppression of BCR-ABL1 by treatment with IM produced stable CML iPSC-like phenotype while IM withdrawal resulted in dome-shaped colonies and pushed the cells towards differentiation. This proof-of-concept study identified a unique CML LSCs mimetic model. It would serve as an excellent platform for screening small molecules to eliminate LSCs as it thrives on TKI therapy. Citation Format: Esther Benjamin, Gaurav Joshi, Bharathi Rajamani, Krittika Nandy, Shaji Velayudhan, Poonkuzhali Balasubramanian. Imatinib mesylate treatment improves reprogramming efficiency, and morphology of chronic myeloid leukemia derived induced pluripotent stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3153.

Synergy of Venetoclax and 8-Chloro-Adenosine in AML: The Interplay of rRNA Inhibition and Fatty Acid Metabolism.

Simple SummaryTreatment failures of acute myeloid leukemia (AML) have been attributed to the persistence of leukemia stem cells (LSCs), which are refractory to conventional treatments. Venetoclax (VEN), currently FDA-approved in combination with low-dose cytarabine or hypomethylating agents, is highly effective in inducing disease remission in patients with de novo AML; however, most of these patients eventually relapse; thus, novel VEN combinations are urgently needed. In this regard, 8-chloro-adenosine (8-Cl-Ado) is a novel RNA-directed nucleoside analog that targets AML cells, including LSCs. We demonstrate that VEN and 8-Cl-Ado cooperate in targeting ribosomal RNA synthesis and mitochondrial metabolism in LSCs, thereby decreasing LSC survival. Given the emerging concept that LSC behavior is strongly associated with protein synthesis regulation and mitochondrial metabolism, our results suggest that the VEN/8-Cl-Ado combination is a promising regimen for the treatment of patients with relapsed AML.It is known that 8-chloro-adenosine (8-Cl-Ado) is a novel RNA-directed nucleoside analog that targets leukemic stem cells (LSCs). In a phase I clinical trial with 8-Cl-Ado in patients with refractory or relapsed (R/R) AML, we observed encouraging but short-lived clinical responses, likely due to intrinsic mechanisms of LSC resistance. LSC homeostasis depends on amino acid-driven and/or fatty acid oxidation (FAO)-driven oxidative phosphorylation (OXPHOS) for survival. We recently reported that 8-Cl-Ado and the BCL-2-selective inhibitor venetoclax (VEN) synergistically inhibit FAO and OXPHOS in LSCs, thereby suppressing acute myeloid leukemia (AML) growth in vitro and in vivo. Herein, we report that 8-Cl-Ado inhibits ribosomal RNA (rRNA) synthesis through the downregulation of transcription initiation factor TIF-IA that is associated with increasing levels of p53. Paradoxically, 8-Cl-Ado-induced p53 increased FAO and OXPHOS, thereby self-limiting the activity of 8-Cl-Ado on LSCs. Since VEN inhibits amino acid-driven OXPHOS, the addition of VEN significantly enhanced the activity of 8-Cl-Ado by counteracting the self-limiting effect of p53 on FAO and OXPHOS. Overall, our results indicate that VEN and 8-Cl-Ado can cooperate in targeting rRNA synthesis and OXPHOS and in decreasing the survival of the LSC-enriched cell population, suggesting the VEN/8-Cl-Ado regimen as a promising therapeutic approach for patients with R/R AML.