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Hematopoietic homeostasis is maintained by hematopoietic stem cells (HSCs), and it is tightly controlled at multiple levels to sustain the self-renewal capacity and differentiation potential of HSCs. Dysregulation of self-renewal and differentiation of HSCs leads to the development of hematologic diseases, including acute myeloid leukemia (AML). Thus, understanding the underlying mechanisms of HSC maintenance and the development of hematologic malignancies is one of the fundamental scientific endeavors in stem cell biology. N  6-methyladenosine (m6A) is a common modification in mammalian messenger RNAs (mRNAs) and plays important roles in various biological processes. In this study, we performed a comparative analysis of the dynamics of the RNA m6A methylome of hematopoietic stem and progenitor cells (HSPCs) and leukemia-initiating cells (LICs) in AML. We found that RNA m6A modification regulates the transformation of long-term HSCs into short-term HSCs and determines the lineage commitment of HSCs. Interestingly, m6A modification leads to reprogramming that promotes cellular transformation during AML development, and LIC-specific m6A targets are recognized by different m6A readers. Moreover, the very long chain fatty acid transporter ATP-binding cassette subfamily D member 2 (ABCD2) is a key factor that promotes AML development, and deletion of ABCD2 damages clonogenic ability, inhibits proliferation, and promotes apoptosis of human leukemia cells. This study provides a comprehensive understanding of the role of m6A in regulating cell state transition in normal hematopoiesis and leukemogenesis, and identifies ABCD2 as a key factor in AML development.

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Leukemia-initiating cells (LICs) are regarded as the origin of leukemia relapse and therapeutic resistance. Identifying direct stemness determinants that fuel LIC self-renewal is critical for developing targeted approaches. Here, we show that the RNA-editing enzyme ADAR1 is a crucial stemness factor that promotes LIC self-renewal by attenuating aberrant double-stranded RNA (dsRNA) sensing. Elevated adenosine-to-inosine editing is a common attribute of relapsed T cell acute lymphoblastic leukemia (T-ALL) regardless of molecular subtype. Consequently, knockdown of ADAR1 severely inhibits LIC self-renewal capacity and prolongs survival in T-ALL patient-derived xenograft models. Mechanistically, ADAR1 directs hyper-editing of immunogenic dsRNA to avoid detection by the innate immune sensor melanoma differentiation-associated protein 5 (MDA5). Moreover, we uncover that the cell-intrinsic level of MDA5 dictates the dependency on the ADAR1-MDA5 axis in T-ALL. Collectively, our results show that ADAR1 functions as a self-renewal factor that limits the sensing of endogenous dsRNA. Thus, targeting ADAR1 presents an effective therapeutic strategy for eliminating T-ALL LICs.

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The Wnt/ß-catenin pathway's significance in cancer initiation, progression, and stem cell biology underscores its therapeutic potential. However, the clinical application of Wnt inhibitors remains limited due to challenges posed by off-target effects and complex cross-talk of Wnt signaling with other pathways. In this study, we leveraged a zebrafish model to perform a robust and rapid drug screening of 773 FDA-approved compounds to identify Wnt/ß-catenin inhibitors with minimal toxicity. Utilizing zebrafish expressing a Wnt reporter, we identified several drugs that suppressed Wnt signaling without compromising zebrafish development. The efficacy of the top hit, Erlotinib, extended to human cells, where it blocked Wnt/ß-catenin signaling downstream of the destruction complex. Notably, Erlotinib treatment reduced self-renewal in human T-cell Acute Lymphoblastic Leukemia cells, which rely on active ß-catenin signaling for maintenance of leukemia-initiating cells. Erlotinib also reduced leukemia-initiating cell frequency and delayed disease formation in zebrafish models. This study underscores zebrafish's translational potential in drug discovery and repurposing and highlights a new use for Erlotinib as a Wnt inhibitor for cancers driven by aberrant Wnt/ß-catenin signaling.

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Introduction: Refractory/relapsed pediatric acute leukemia are still clinically challenging and new therapeutic strategies are needed. Interactions between Natural Killer Group 2D (NKG2D) receptor, expressed in cytotoxic immune cells, and its ligands (NKG2DL), which are upregulated in leukemic blasts, are important for anti-leukemia immunosurveillance. Nevertheless, leukemia cells may develop immunoescape strategies as NKG2DL shedding and/or downregulation. Methods: In this report, we analyzed the anti-leukemia activity of NKG2D chimeric antigen receptor (CAR) redirected memory (CD45RA-) T cells in vitro and in a murine model of T-cell acute lymphoblastic leukemia (T-ALL). We also explored in vitro how soluble NKG2DL (sNKG2DL) affected NKG2D-CAR T cells' cytotoxicity and the impact of NKG2D-CAR T cells on Jurkat cells gene expression and in vivo functionality. Results: In vitro, we found NKG2D-CAR T cells targeted leukemia cells and showed resistance to the immunosuppressive effects exerted by sNKG2DL. In vivo, NKG2D-CAR T cells controlled T cell leukemia burden and increased survival of the treated mice but failed to cure the animals. After CAR T cell treatment, Jurkat cells upregulated genes related to proliferation, survival and stemness, and in vivo, they exhibited functional properties of leukemia initiating cells. Discussion: The data here presented suggest, that, in combination with other therapeutic approaches, NKG2D-CAR T cells could be a novel treatment for pediatric T-ALL.

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T-cell lymphoblastic acute leukemia (T-ALL) is an aggressive blood cancer, characterized by restricted cellular subsets with enriched leukemia initiating cells (LICs). Recently, Ephrin receptors (Eph) were described to be highly expressed in cancer stem cells. Here, using public RNA-Seq datasets of human T-ALL, we reported that EphB6 was the only member within the Eph family overexpressed in over 260 samples. We also found the highest level of EphB6 in a minor cell subpopulation within bulk tumors of patient-derived xenografts, obtained through the injection of primary patient biopsy material into immunocompromised NOD-Scid/IL2Rγc-/- (NSG) mice. Interestingly, this EphB6 positive (EphB6+) subset showed an enriched LIC activity after in vivo transplantation into NSG mice. Additionally, gene expression data at the single-cell level of primary patients' leukemic cells revealed that EphB6 + cells were significantly selected in minimal residual disease up to 30 days from the standard treatments and characterized by high levels of markers related to cell proliferation and poor clinical outcome, such as CCNB1 and KIF20A. Taken together, our data suggest that EphB6 supports LICs' maintenance and progression in T-ALL and, thus, targeting EphB6 + cells could be therapeutically relevant for the treatment of T-ALL patients.

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Rewiring of redox metabolism has a profound impact on tumor development, but how the cellular heterogeneity of redox balance affects leukemogenesis remains unknown. To precisely characterize the dynamic change in redox metabolism in vivo, we developed a bright genetically encoded biosensor for H2O2 (named HyPerion) and tracked the redox state of leukemic cells in situ in a transgenic sensor mouse. A H2O2-low (HyPerion-low) subset of acute myeloid leukemia (AML) cells was enriched with leukemia-initiating cells, which were endowed with high colony-forming ability, potent drug resistance, endosteal rather than vascular localization, and short survival. Significantly high expression of malic enzymes, including ME1/3, accounted for nicotinamide adenine dinucleotide phosphate (NADPH) production and the subsequent low abundance of H2O2. Deletion of malic enzymes decreased the population size of leukemia-initiating cells and impaired their leukemogenic capacity and drug resistance. In summary, by establishing an in vivo redox monitoring tool at single-cell resolution, this work reveals a critical role of redox metabolism in leukemogenesis and a potential therapeutic target.

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BACKGROUND: Mixed lineage leukemia 1-rearranged (MLL1-r) acute leukemia patients respond poorly to currently available treatments and there is a need to develop more effective therapies directly disrupting the Menin‒MLL1 complex. Small-molecule-mediated inhibition of the protein‒protein interaction between Menin and MLL1 fusion proteins is a potential therapeutic strategy for patients with MLL1-r or mutated-nucleophosmin 1 (NPM1c) acute leukemia. In this study, we preclinically evaluated the new compound DS-1594a and its salts. METHODS: We evaluated the preclinical efficacy of DS-1594a as well as DS-1594a·HCl (the HCl salt of DS-1594a) and DS-1594a·succinate (the succinic acid salt of DS-1594a, DS-1594b) in vitro and in vivo using acute myeloid leukemia (AML)/acute lymphoblastic leukemia (ALL) models. RESULTS: Our results showed that MLL1-r or NPM1c human leukemic cell lines were selectively and highly sensitive to DS-1594a·HCl, with 50% growth inhibition values < 30 nM. Compared with cytrabine, the standard chemotherapy drug as AML therapy, both DS-1594a·HCl and DS-1594a·succinate mediated the eradication of potential leukemia-initiating cells by enhancing differentiation and reducing serial colony-forming potential in MLL1-r AML cells in vitro. The results were confirmed by flow cytometry, RNA sequencing, RT‒qPCR and chromatin immunoprecipitation sequencing analyses. DS-1594a·HCl and DS-1594a·succinate exhibited significant antitumor efficacy and survival benefit in MOLM-13 cell and patient-derived xenograft models of MLL1-r or NPM1c acute leukemia in vivo. CONCLUSION: We have generated a novel, potent, orally available small-molecule inhibitor of the Menin-MLL1 interaction, DS-1594a. Our results suggest that DS-1594a has medicinal properties distinct from those of cytarabine and that DS-1594a has the potential to be a new anticancer therapy and support oral dosing regimen for clinical studies (NCT04752163).

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N6-methyladenosine (m6A), the most prevalent internal modification in mammalian mRNAs, is involved in many pathological processes. METTL16 is a recently identified m6A methyltransferase. However, its role in leukemia has yet to be investigated. Here, we show that METTL16 is a highly essential gene for the survival of acute myeloid leukemia (AML) cells via CRISPR-Cas9 screening and experimental validation. METTL16 is aberrantly overexpressed in human AML cells, especially in leukemia stem cells (LSCs) and leukemia-initiating cells (LICs). Genetic depletion of METTL16 dramatically suppresses AML initiation/development and maintenance and significantly attenuates LSC/LIC self-renewal, while moderately influencing normal hematopoiesis in mice. Mechanistically, METTL16 exerts its oncogenic role by promoting expression of branched-chain amino acid (BCAA) transaminase 1 (BCAT1) and BCAT2 in an m6A-dependent manner and reprogramming BCAA metabolism in AML. Collectively, our results characterize the METTL16/m6A/BCAT1-2/BCAA axis in leukemogenesis and highlight the essential role of METTL16-mediated m6A epitranscriptome and BCAA metabolism reprograming in leukemogenesis and LSC/LIC maintenance.

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In leukemia, a distinct subpopulation of cancer-initiating cells called leukemia stem cells (LSCs) is believed to drive population expansion and tumor growth. Failing to eliminate LSCs may result in disease relapse regardless of the amount of non-LSCs destroyed. The first step in targeting and eliminating LSCs is to identify and characterize them. Acute precursor B lymphoblastic leukemia (B-ALL) cells derived from patients were incubated with fluorescent glucose analog 2-(N-(7-Nitrobenz-2-oxa-1, 3-diazol-4-yl) Amino)-2-Deoxyglucose (NBDG) and sorted based on NBDG uptake. Cell subpopulations defined by glucose uptake were then serially transplanted into mice and evaluated for leukemia initiating capacity. Gene expression profiles of these cells were characterized using RNA-Sequencing (RNA-Seq). A distinct population of NBDG-low cells was identified in patient B-ALL samples. These cells are a small population (1.92% of the entire leukemia population), have lower HLA expression, and are smaller in size (4.0 to 7.0 µm) than the rest of the leukemia population. All mice transplanted with NBDG-low cells developed leukemia between 5 and 14 weeks, while those transplanted with NBDG-high cells did not develop leukemia (p ≤ 0.0001-0.002). Serial transplantation of the NBDG-low mouse model resulted in successful leukemia development. NBDG-medium (NBDG-med) populations also developed leukemia. Interestingly, comprehensive molecular characterization of NBDG-low and NBDG-med cells from patient-derived xenograft (PDX) models using RNA-Seq revealed a distinct profile of 2,162 differentially-expressed transcripts (DETs) (p<0.05) with 70.6% down-regulated in NBDG-low cells. Hierarchical clustering of DETs showed distinct segregation of NBDG-low from NBDG-med and NBDG-high groups with marked transcription expression alterations in the NBDG-low group consistent with cancer survival. In conclusion, A unique subpopulation of cells with low glucose uptake (NBDG-low) in B-ALL was discovered. These cells, despite their quiescence characteristics, once transplanted in mice, showed potent leukemia initiating capacity. Although NBDG-med cells also initiated leukemia, gene expression profiling revealed a distinct signature that clearly distinguishes NBDG-low cells from NBDG-med and the rest of the leukemia populations. These results suggest that NBDG-low cells may represent quiescent LSCs. These cells can be activated in the appropriate environment in vivo, showing leukemia initiating capacity. Our study provides insight into the biologic mechanisms of B-ALL initiation and survival.

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The existence of "leukemia-initiating cells" (LICs) in chronic lymphocytic leukemia (CLL) remains controversial due to the difficulty in isolating and identifying the tumor-initiating cells. Here, we demonstrate a microchannel electroporation (MEP) microarray that injects RNA-detecting probes into single live cells, allowing the imaging and characterization of heterogeneous LICs by intracellular RNA expression. Using limited-cell FACS sequencing (LC-FACSeq), we can detect and monitor rare live LICs during leukemogenesis and characterize their differential drug sensitivity. Disease-associated mutation accumulation in developing B lymphoid but not myeloid lineage in CLL patient hematopoietic stem cells (CLL-HSCs), and development of independent clonal CLL-like cells in murine patient-derived xenograft models, suggests the existence of CLL LICs. Furthermore, we identify differential protein ubiquitination and unfolding response signatures in GATA2high CLL-HSCs that exhibit increased sensitivity to lenalidomide and resistance to fludarabine compared to GATA2lowCLL-HSCs. These results highlight the existence of therapeutically targetable disease precursors in CLL.

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The mechanism by which redox metabolism regulates the fates of acute myeloid leukemia (AML) cells remains largely unknown. Using a highly sensitive, genetically encoded fluorescent sensor of nicotinamide adenine dinucleotide phosphate (NADPH), iNap1, we find three heterogeneous subpopulations of AML cells with different cytosolic NADPH levels in an MLL-AF9-induced murine AML model. The iNap1-high AML cells have enhanced proliferation capacities both in vitro and in vivo and are enriched for more functional leukemia-initiating cells than iNap1-low counterparts. The iNap1-high AML cells prefer localizing in the bone marrow endosteal niche and are resistant to methotrexate treatment. Furthermore, iNap1-high human primary AML cells have enhanced proliferation abilities both in vitro and in vivo. Mechanistically, the MTHFD1-mediated folate cycle regulates NADPH homeostasis to promote leukemogenesis and methotrexate resistance. These results provide important clues for understanding mechanisms by which redox metabolism regulates cancer cell fates and a potential metabolic target for AML treatments.

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PURPOSE OF REVIEW: Acute leukemias represent a tremendous threat to public health around the globe and the main cause of death due to disease in scholar age children from developing nations. Here, we review their current status in Mexico, as a paradigm of study, and the major challenges to control systemic diseases like childhood cancer. RECENT FINDINGS: A unique molecular epidemiology, late/low precision diagnosis, limited access to treatment, toxicity associated with therapy, continuous exposure to environmental risk factors, and the high frequency of early relapses are some of the factors cooperating to low rates of survival in low-to-medium-income countries. Deliberative dialogues and exhaustive programs have emerged as promising means of advancing evidence-informed policy, by providing a structured forum for key stakeholders to integrate scientific and pragmatic knowledge about complex health concerns. A system-wide strategy based on the comprehensive leukemia identity is essential for a meaningful decline in early childhood mortality.

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Leukemia-initiating cells, also known as leukemic stem cells (LSCs), are experimentally defined by their ability to engraft immunocompromised mice and are believed to be a major cause of relapse in acute myeloid leukemia (AML). Despite the aggressive characteristics of acute leukemia, AML blasts are difficult to culture once removed from the patient, and LSCs, which are a minor fraction of the blast population, are especially difficult to transplant after culture. This impedes development of new therapies for AML that target LSCs. Here, we present a simple strategy to culture LSCs in cytokine-free medium and to perform flow cytometric analysis of the resulting cell population for the characterization of LSCs maintenance and differentiation.

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T-cell acute lymphoblastic leukemia (T-ALL), a T-cell malignant disease that mainly affects children, is still a medical challenge, especially for refractory patients for whom therapeutic options are scarce. Recent advances in immunotherapy for B-cell malignancies based on increasingly efficacious monoclonal antibodies (mAbs) and chimeric antigen receptors (CARs) have been encouraging for non-responding or relapsing patients suffering from other aggressive cancers like T-ALL. However, secondary life-threatening T-cell immunodeficiency due to shared expression of targeted antigens by healthy and malignant T cells is a main drawback of mAb-or CAR-based immunotherapies for T-ALL and other T-cell malignancies. This review provides a comprehensive update on the different immunotherapeutic strategies that are being currently applied to T-ALL. We highlight recent progress on the identification of new potential targets showing promising preclinical results and discuss current challenges and opportunities for developing novel safe and efficacious immunotherapies for T-ALL.

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BACKGROUND: Tyrosine kinase inhibitors (TKIs) are the standard treatment for chronic myeloid leukemia (CML). Despite their clinical success, TKIs are faced with challenges such as treatment resistance, which may be driven by kinase domain mutations, and frequent disease relapse upon the cessation of treatment. The combination of arsenic trioxide (ATO) and interferon-α (IFN) was previously demonstrated to inhibit proliferation and induce apoptosis in CML cell lines, prolong the survival of primary wild-type CML mice, and dramatically decrease the activity of leukemia-initiating cells (LICs). METHODS: The ATO/IFN combination was tested in vitro on imatinib (IMN)-resistant K562-R and Ar230-R cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays were used to evaluate proliferation and apoptosis, respectively. The acridine orange assay was used to assess autophagy, and quantitative reverse transcription-polymerase chain reaction was used to assess the involvement of the hedgehog (Hh) pathway. In vivo, a retroviral transduction/transplantation T315I BCR-ABL CML mouse model was used to assay the effect of the treatment on survival, tumor burden (histopathology and blood counts), and LIC activity (secondary transplantation). RESULTS: In vitro, ATO/IFN synergized to inhibit proliferation and induce apoptosis of IMN-resistant cells with variant modes of resistance. Furthermore, the preclinical effects of ATO/IFN were associated with induction of autophagy along with inhibition of the Hh pathway. Most remarkably, ATO/IFN significantly prolonged the survival of primary T315I-CML mice and displayed a dramatic impairment of disease engraftment in secondary mice, which reflected decreased LIC activity. CONCLUSIONS: Collectively, the ATO/IFN strategy has been demonstrated to have the potential to lead to durable remissions in TKI-resistant CML preclinical models and to overcome various TKI-specific mechanisms of resistance.

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The metabolic properties of leukemia-initiating cells (LICs) in distinct bone marrow niches and their relationships to cell-fate determinations remain largely unknown. Using a metabolic imaging system with a highly responsive genetically encoded metabolic sensor, SoNar, we reveal that SoNar-high cells are more glycolytic, enriched for higher LIC frequency, and develop leukemia much faster than SoNar-low counterparts in an MLL-AF9-induced murine acute myeloid leukemia model. SoNar-high cells mainly home to and locate in the hypoxic endosteal niche and maintain their activities through efficient symmetric division. SoNar can indicate the dynamics of metabolic changes of LICs in the endosteal niche. SoNar-high human leukemia cells or primary samples have enhanced clonogenic capacities in vitro or leukemogenesis in vivo. PDK2 fine-tunes glycolysis, homing, and symmetric division of LICs. These findings provide a unique angle for the study of metabolisms in stem cells, and may lead to development of novel strategies for cancer treatment.

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In addition to acting as building blocks for biosynthesis, amino acids might serve as signaling regulators in various physiological and pathological processes. However, it remains unknown whether amino acid levels affect the activities of hematopoietic stem cells (HSCs). By using a genetically encoded fluorescent sensor of the intracellular levels of branched-chain amino acids (BCAAs), we could monitor the dynamics of BCAA metabolism in HSCs. A mitochondrial-targeted 2C-type Ser/Thr protein phosphatase (PPM1K) promotes the catabolism of BCAAs to maintain MEIS1 and p21 levels by decreasing the ubiquitination-mediated degradation controlled by the E3 ubiquitin ligase CDC20. PPM1K deficiency led to a notable decrease in MEIS1/p21 signaling to reduce the glycolysis and quiescence of HSCs, followed by a severe impairment in repopulation activities. Moreover, the deletion of Ppm1k dramatically extended survival in a murine leukemia model. These findings will enhance the current understanding of nutrient signaling in metabolism and function of stem cells.

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Acute myeloid leukemia (AML) is a devastating illness which carries a very poor prognosis, with most patients living less than 18 months. Leukemia relapse may occur because current therapies eliminate proliferating leukemia cells but fail to eradicate quiescent leukemia-initiating cells (LICs) that can reinitiate the disease after a period of latency. While we demonstrated that p53 target gene Necdin maintains hematopoietic stem cell (HSC) quiescence, its roles in LIC quiescence and response to chemotherapy are unclear. In this study, we utilized two well-established murine models of human AML induced by MLL-AF9 or AML1-ETO9a to determine the role of Necdin in leukemogenesis. We found that loss of Necdin decreased the number of functional LICs and enhanced myeloid differentiation in vivo, leading to delayed development of leukemia induced by MLL-AF9. Importantly, Necdin null LICs expressing MLL-AF9 were less quiescent than wild-type LICs. Further, loss of Necdin enhanced the response of MLL-AF9+ leukemia cells to chemotherapy treatment, manifested by decreased viability and enhanced apoptosis. We observed decreased expression of Bcl2 and increased expression of p53 and its target gene Bax in Necdin null leukemia cells following chemotherapy treatment, indicating that p53-dependent apoptotic pathways may be activated in the absence of Necdin. In addition, we found that loss of Necdin decreased the engraftment of AML1-ETO9a+ hematopoietic stem and progenitor cells in transplantation assays. However, Necdin-deficiency did not affect the response of AML1-ETO9a+ hematopoietic cells to chemotherapy treatment. Thus, Necdin regulates leukemia-initiating cell quiescence and chemotherapy response in a context-dependent manner. Our findings suggest that pharmacological inhibition of Necdin may hold potential as a novel therapy for leukemia patients with MLL translocations.

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T-cell acute lymphoblastic leukemia (T-ALL) is a hematological malignancy characterized by the clonal proliferation of immature T-cell precursors. T-ALL has many similar pathophysiological features to acute myeloid leukemia, which has been extensively studied in the establishment of the cancer stem cell (CSC) theory, but the CSC concept in T-ALL is still debatable. Although leukemia-initiating cells (LICs), which can generate leukemia in a xenograft setting, have been found in both human T-ALL patients and animal models, the nature and origin of LICs are largely unknown. In this review, we discuss recent studies on LICs in T-ALL and the potential mechanisms of LIC emergence in this disease. We focus on the oncogenic transcription factors TAL1, LMO2, and NOTCH1 and highlight the significance of the transcriptional regulatory programs in normal hematopoietic stem cells and T-ALL.