Abstract Relapse and chemoresistance remain major challenges in paediatric acute myeloid leukaemia (PAML), particularly in KMT2A-rearranged (KMT2A-r) subtypes where conventional markers such as CD34 are often absent, complicating measurable residual disease (MRD) detection. Leukaemia stem/regenerating cells (LSC/LRC) drive disease initiation, progression, and relapse, sharing stemness and chemoresistance properties that make them critical therapeutic targets. Using high-dimensional spectral flow cytometry, we identified CD180, a Toll-like receptor-like surface protein, as highly expressed on blasts and stem-like populations in KMT2A-r AML, while near absent on normal haematopoietic stem cells (HSCs). PAML KMT2A-r exhibits an unconventional immunophenotype dominated by CD34 ⁻ CD180 ⁺ populations. Integrated single-cell transcriptomics and functional profiling revealed CD180 high clusters enriched for quiescence, oxidative phosphorylation, and KMT2A/LSC stemness signatures. CD180 ⁺ cells demonstrated robust leukaemia-initiating capacity in xenograft models and persisted through therapy, re-emerging at relapse with phenotypic plasticity. Epigenomic analysis showed CD180 is a direct transcriptional target of the KMT2A::MLLT3 fusion complex, regulated by intragenic enhancers and downregulated by menin and BET inhibitors. Longitudinal single-cell analysis confirmed persistence and clonal evolution of CD180 ⁺ populations during treatment and relapse, underscoring their mechanistic role in chemoresistance and disease progression. In summary, CD180 marks dynamic, relapse-driving populations in KMT2A-r PAML, persists through therapy, and importantly is near absent on normal HSCs, offering a selective therapeutic window. These findings position CD180 as a clinically actionable biomarker for MRD detection and a compelling therapeutic target for eradicating chemoresistant, stem-like cells in paediatric AML. Main Points CD180 marks chemoresistant, relapse-driving stem-like blasts in KMT2A-r paediatric AML, overcoming CD34-based MRD limitations. Absent on normal HSCs, CD180 is a KMT2A::MLLT3 target and actionable for MRD, relapse prediction, and CD180-directed therapies. Novelty This study introduces CD180 as a novel biomarker and therapeutic target in AML, particularly KMT2A-rearranged subtypes where conventional markers are often absent. Unlike MRD strategies focused on bulk blasts, CD180 marks chemoresistant, stem-like populations driving relapse, critical reservoirs poorly defined in paediatric AML. This work fills a major gap in prognostic assessment and therapy by enabling precise detection of relapse-driving cells and offering a selective therapeutic window.

Whilst it is recognised that targeting self-renewal is an effective way to functionally impair the quiescent leukaemic stem cells (LSC) that persist as residual disease in chronic myeloid leukaemia (CML), developing therapeutic strategies to achieve this have proved challenging. We demonstrate that the regulatory programmes of quiescent LSC in chronic phase CML are similar to that of embryonic stem cells, pointing to a role for wild type p53 in LSC self-renewal. In support of this, increasing p53 activity in primitive CML cells using an MDM2 inhibitor in combination with a tyrosine kinase inhibitor resulted in reduced CFC outputs and engraftment potential, followed by loss of multilineage priming potential and LSC exhaustion when combination treatment was discontinued. Our work provides evidence that targeting LSC self-renewal is exploitable in the clinic to irreversibly impair quiescent LSC function in CML residual disease - with the potential to enable more CML patients to discontinue therapy and remain in therapy-free remission.

Macrophages are fundamental cells of the innate immune system that support normal haematopoiesis and play roles in both anti-cancer immunity and tumour progression. Here we use a chimeric mouse model of chronic myeloid leukaemia (CML) and human bone marrow (BM) derived macrophages to study the impact of the dysregulated BM microenvironment on bystander macrophages. Utilising single-cell RNA sequencing (scRNA-seq) of Philadelphia chromosome (Ph) negative macrophages we reveal unique subpopulations of immature macrophages residing in the CML BM microenvironment. CML exposed macrophages separate from their normal counterparts by reduced expression of the surface marker CD36, which significantly reduces clearance of apoptotic cells. We uncover aberrant production of CML-secreted factors, including the immune modulatory protein lactotransferrin (LTF), that suppresses efferocytosis, phagocytosis, and CD36 surface expression in BM macrophages, indicating that the elevated secretion of LTF is, at least partially responsible for the supressed clearance function of Ph- macrophages.

High expression of the long non-coding RNA (lncRNA) FAM30A has been previously associated with leukemic stem cell (LSC) activity and poor prognosis in both adult and paediatric acute myeloid leukaemia (AML) patients, yet it has not been functionally studied. This study provides the first cellular characterization of FAM30A focussing on an internal tandemly organised region, referred to as FAM30A repeats. FAM30A levels correlated with canonical AML LSC signatures and FAM30A depletion decreased cell viability as well as increased sensitivity to chemotherapeutics. It also inhibited colony formation, promoted granulocytic differentiation and abrogated leukemic engraftment in murine bone marrow in vivo . Overexpression of FAM30A repeats in this setting enhanced stemness, proliferation, chemoresistance, and engraftment thus highlighting the biological relevance of this region for LSC biology. On the molecular level, FAM30A repeats interact with the pro-LSC regulator Musashi-2 (MSI2), positively influencing expression of its targets including RUNX1 isoforms. We herein uncover that this FAM30A -MSI2-RUNX1 regulatory loop is of potential relevance for LSC maintenance in AML. These findings provide valuable insights into FAM30A 's cellular role and highlight its targeting potential for eliminating LSCs and improving treatment outcomes in AML patients. ### Competing Interest Statement The authors have declared no competing interest.

Supplementary Table S6. ChIP-sequencing datasets used in the present study.

Supplementary Table S1. Genome-wide analysis of mRNA expression levels in HPCs, HSCs, LSCs and LPCs (E-MTAB-2581, Affymetrix HuGe 1.0 ST).

Supplementary Table S2. mRNA expression changes in CML and normal CD34+ cells resulting from treatment of EZHi (GSK343) (EMTAB-2893, MTAB-3552; Affymetrix HuGe 1.0 ST).

Supplementary Table S3. Pathway analysis of mRNA expression changes resulting from treatment of CML CD34+ cells with EZH2i (GSK343).

Supplementary Table S4. Comparison of the efficacy of EZH2i with inhibitors of other novel CML drug targets (4-15) in like-for-like assays.

Supplementary Figure S1. mRNA levels of PRC2 components in CML and normal samples used in this study. Supplementary Figure S2. Effect of TKI treatment on mRNA levels of PRC2 components on CML cells in vitro. Supplementary Figure S3. Analysis of promoters reprogrammed for H3K27me3 levels at promoters. Supplementary Figure S4. Relationship between changes in levels of H3K27me3 and changes in mRNA expression at H3K27me3 targets. Supplementary Figure S5. EZH2i treatment of CML and normal CD34+ cells. Supplementary Figure S6. EZH2i treatment of CML CD34+CD38- cells (LSCs). Supplementary Figure S7. Validation of EZH2 shRNAs in a CML cell line. Supplementary Figure S8. The effect of treatment of CML or normal CD34+ cells with EZH2i (GSK343; 1000 nM) on the global mRNA levels of H3K27me3 and H3K27me1 targets. Supplementary Figure S9. Both imatinib and EZH2i selectively target upregulation of H3K27me3 target genes. Supplementary Figure S10. Nilotinib, dasatinib and EZH2i selectively target upregulation of H3K27me3 target genes. Supplementary Figure S11. mRNA expression levels for H3K27me3 target genes in HSC and LSCs and their behaviour after TKI treatment. Supplementary Figure S12. mRNA expression levels for H3K27me3 target genes in HPC and LPCs (â‰^ CML CD34+) and their behaviour after EZH2i treatment. Supplementary Figure S13. Primary CML cells engrafted in NSG mice treated in vivo with EZH2i and TKI, alone and in combination. Supplementary Figure S14. Validation of mRNA expression changes determined by Affymetrix GeneChip analysis (E-MTAB-2581, Affymetrix HuGe 1.0 ST). Supplementary Figure S15. PRC2 mis-regulation in the three CML patient samples used for Affymetrix GeneChIP and ChIP-sequencing in this study. Supplementary Figure S16. Determining thresholds for identifying H3K27me3 target genes. Supplementary Figure S17. Validation of seqMiner(3) promoter classifications.

Supplementary Table S5. Primary CML and normal samples that were used in this study.

Tyrosine kinase inhibitors (TKI) have revolutionised the treatment of CML. However, TKI do not eliminate the leukaemia stem cells (LSC), which can re-initiate the disease. Thus, finding new therapeutic targets in CML LSC is key to finding a curative treatment. Using microarray datasets, we defined a list of 227 genes that were differentially expressed in CML LSC compared to the healthy controls but were not affected by TKI in vitro. Two of them, CD33 and PPIF, are targeted by gemtuzumab–ozogamicin and cyclosporin A, respectively. We treated CML and the control CD34+ cells with either drug with or without imatinib to investigate the therapeutic potential of the TKI-independent gene expression programme. Cyclosporine A, in combination with imatinib, reduced the number of CML CFC compared with non-CML controls, but only at supra-therapeutic concentrations. Gemtuzumab–ozogamicin showed an EC50 of 146 ng/mL, below the plasma peak concentration of 630 ng/mL observed in the AML patients and below the EC50 of 3247 ng/mL observed in the non-CML cells. Interestingly, gemtuzumab–ozogamicin seems to promote cell cycle progression in CML CD34+ cells and demonstrated activation of the RUNX1 pathway in an RNAseq experiment. This suggests that targeting the TKI-independent genes in CML LSC could be exploited for the development of new therapies in CML.

Inhibition of autophagy has been proposed as a potential therapy for individuals with cancer. However, current lysosomotropic autophagy inhibitors have demonstrated limited efficacy in clinical trials. Therefore, validation of novel specific autophagy inhibitors using robust preclinical models is critical. In chronic myeloid leukemia (CML), minimal residual disease is maintained by persistent leukemic stem cells (LSCs), which drive tyrosine kinase inhibitor (TKI) resistance and patient relapse. Here, we show that deletion of autophagy-inducing kinase ULK1 (unc-51–like autophagy activating kinase 1) reduces growth of cell line and patient-derived xenografted CML cells in mouse models. Using primitive cells, isolated from individuals with CML, we demonstrate that pharmacological inhibition of ULK1 selectively targets CML LSCs ex vivo and in vivo, when combined with TKI treatment. The enhanced TKI sensitivity after ULK1-mediated autophagy inhibition is driven by increased mitochondrial respiration and loss of quiescence and points to oxidative stress–induced differentiation of CML LSCs, proposing an alternative strategy for treating patients with CML.

Although it has been recognized for many years that cancer stem cells and embryonic stem cells (ESC) share molecular features, identifying ways to exploit this therapeutically has proved challenging. To date, these shared features have not been examined in the leukemic stem cells (LSC) found in patients with chronic myeloid leukemia (CML).

Epigenomic dysregulation is a common pathological feature in human hematological malignancies. H3K9me3 emerges as an important epigenomic marker in acute myeloid leukemia (AML). Its associated methyltransferases, such as SETDB1, suppress AML leukemogenesis, whilst H3K9me3 demethylases KDM4C is required for mixed-lineage leukemia rearranged AML. However, the specific role and molecular mechanism of action of another member of the KDM4 family, KDM4A has not previously been clearly defined. In this study, we delineated and functionally validated the epigenomic network regulated by KDM4A. We show that selective loss of KDM4A is sufficient to induce apoptosis in a broad spectrum of human AML cells. This detrimental phenotype results from a global accumulation of H3K9me3 and H3K27me3 at KDM4A targeted genomic loci thereby causing downregulation of a KDM4A-PAF1 controlled transcriptional program essential for leukemogenesis, distinct from that of KDM4C. From this regulatory network, we further extracted a KDM4A-9 gene signature enriched with leukemia stem cell activity; the KDM4A-9 score alone or in combination with the known LSC17 score, effectively stratifies high-risk AML patients. Together, these results establish the essential and unique role of KDM4A for AML self-renewal and survival, supporting further investigation of KDM4A and its targets as a potential therapeutic vulnerability in AML.

For two decades, leukaemia stem cells (LSCs) in chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML) have been advanced paradigms for the cancer stem cell field. In CML, the acquisition of the fusion tyrosine kinase BCR–ABL1 in a haematopoietic stem cell drives its transformation to become a LSC. In AML, LSCs can arise from multiple cell types through the activity of a number of oncogenic drivers and pre-leukaemic events, adding further layers of context and genetic and cellular heterogeneity to AML LSCs not observed in most cases of CML. Furthermore, LSCs from both AML and CML can be refractory to standard-of-care therapies and persist in patients, diversify clonally and serve as reservoirs to drive relapse, recurrence or progression to more aggressive forms. Despite these complexities, LSCs in both diseases share biological features, making them distinct from other CML or AML progenitor cells and from normal haematopoietic stem cells. These features may represent Achilles’ heels against which novel therapies can be developed. Here, we review many of the similarities and differences that exist between LSCs in CML and AML and examine the therapeutic strategies that could be used to eradicate them. This Review discusses many of the similarities and differences between leukaemia stem cells (LSCs) in chronic myeloid leukaemia and acute myeloid leukaemia and examines the therapeutic strategies that could be used to eradicate these LSCs.

Background:The BCR‐ABL1 chimeric oncoprotein drives chronic myeloid leukaemia (CML) pathogenesis. The introduction of tyrosine kinase inhibitors (TKIs) has transformed clinical outcomes for patients with CML, with over 80% of those treated with imatinib (IM) surviving for more than 10 years. Second and third‐generation TKIs are more potent and can impede the emergence of resistance, inducing deep molecular responses in both untreated and IM‐resistant CML patients. Despite the success of TKIs, up to 35% of patients discontinue their TKI due to intolerance or develop resistance and this remains a key area of unmet clinical need. Asciminib (formerly ABL001) is a potent and selective allosteric inhibitor of ABL1 that binds to the myristoyl binding pocket of ABL1 to hold BCR‐ABL1 in an inactive conformation. Preclinical studies have shown that asciminib selectively inhibits the growth of BCR‐ABL1 positive (+) cells regardless of the presence of BCR‐ABL1 point mutations1. Clinical trials for patients with CML or Ph+ acute lymphoblastic leukaemia, testing asciminib alone and in combination with TKIs are currently underway and preliminary results are promising.Aims:To assess if dual inhibition of BCR‐ABL1 leads to improved treatment outcomes in preclinical studies.Methods:Here, we assess the effects of asciminib, alone and in combination with ATP‐competitive TKIs (IM, nilotinib (NIL) and ponatinib (PON)) in CML cell lines and primary CD34+ chronic phase (CP)‐CML stem/progenitor cells (HSPC). We assessed synergy using resazurin readouts using CompuSyn Software. We performed cell counts, apoptosis, cell‐cycle and proliferation assays to determine the effect of drug combinations in CML cell lines and primary samples; and confirmed effects on primitive cells using colony‐forming cell (CFC) and long‐term culture‐initiating cell (LTC‐IC) assays in vitro.Results:Dose‐response studies using the resazurin assays in CML cell lines (Bv173, K562, KCL22) indicate that asciminib is potent at low nanomolar concentration, even in cells that express the BCR‐ABL1 T315I point mutation (KCL22). Apoptosis and cell cycle assays’ assessed by FACS showed that the inhibitory effects of asciminib were maintained in KCL22T315I‐expressing cells when asciminib was used in combination with PON. Washout studies with asciminib in KCL22WT/T315I‐expressing cells demonstrated a prolonged phenotypic response using low‐nanomolar doses of asciminib as the cells failed to regrow and had irreversible cell‐cycle damage.Primary CD34+ CML HSPCs demonstrated proliferation arrest and increased apoptosis (70–100% increase relative to control; p < 0.001) when treated for up to 72 hours with asciminib, alone and in combination with IM or NIL. LTC‐IC and CFC assays, determining the functional activity of primitive CML HSPCs in vitro, demonstrated that the combination of asciminib with IM or NIL reduced colony outputs (60–90% decrease relative to controll, p < 0.001), beyond that achieved with each drug alone (40–80% relative to control, p < 0.001), and in separate experiments, with minimal effect on normal HSPC.Summary/Conclusion:These results suggest that asciminib represents a novel therapeutic approach with effects on primitive CP‐CML HSPCs both as a single agent and in combination with TKI and has efficacy in cells expressing the multi‐TKI resistant T315I mutation. We are now investigating the mechanism of action for asciminib, alone and in combination with NIL, by RNA‐sequencing.

Background: In chronic myeloid leukaemia (CML), BCR-ABL tyrosine kinase inhibitors (TKI) fail to eliminate leukaemia stem cells (LSC) which can serve as a reservoir to drive relapse, TKI resistance and promote disease progression. Therefore, identification of pathways/targets that promote LSC survival is essential for the development of curative therapies. Our previous transcriptomics analysis pointed to neurotransmitter pathways as being aberrantly expressed in LSC compared to normal haemopoietic stem cells. Aims: This led us to hypothesise that LSCs require neurotransmitter pathways to maintain their stem cell potential. The aim of our study was therefore to understand whether modulating these pathways would affect LSC survival. Methods: To identify drug-able targets within these pathways, we performed compound screens in BCR-ABL-/+ cell lines and primary CML CD34+ cells using 658 compounds known to modulate neurotransmitter signalling, many of which are used clinically to treat neuro-psychiatric disorders. Hits selective for BCR-ABL-expressing cells were further evaluated in phenotypic assays using primary CML CD34+ versus non-CML CD34+ cells. Western blotting and FACs analysis was used to determine the effects that these compounds had on cellular pathways known to affect LSC survival. Results: 3-CPMT, a selective dopamine reuptake inhibitor (SDRI), and paroxetine, a clinical grade selective serotonin reuptake inhibitor (SSRI), that target SLC6A3 and SLC6A4 respectively, emerged as compounds that selectively reduced CML CD34+ cell counts (P = 0.0007 and 0.04 respectively) and inhibited CFC outputs (P = 0.001 and 0.04, respectively). FACs analysis has shown that LSC and CML cell lines express 3–4-fold higher levels of SLC6A3 and SLC6A4 than their non-CML counterparts, with expression differences for SLC6A4 reaching significance (CML CD34+CD38- versus non-CML CD34+CD38- cells, P = 0.04; BaF3 BCR-ABL+ versus wild type BaF3, P = 0.04). Targeting of SLC6A3 or SLC6A4 with 3-CPMT or paroxetine +/- nilotinib inhibits a number of known LSC survival factors including PRKCH, FOXO3a, p38 MAPK and c-Myc. Furthermore, we have demonstrated that paroxetine in combination with nilotinib is highly effective at eradicating CD34+CD38-CD90+ LSC compared to nilotinib alone in patient-derived xenograft mouse models (P = 0.01). Summary/Conclusion: In conclusion, our pre-clinical data suggest that repurposing drugs that target neurotransmitter transporters may represent a novel way to target LSC in CML patients.