Chemoresistance In Acute Myeloid Leukemia Research Articles

Metabolic dependencies of acute myeloid leukemia stem cells.

Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy primarily driven by an immature population of AML cells termed leukemia stem cells (LSCs) that are implicated in AML development, chemoresistance, and relapse. An emerging area of research in AML focuses on identifying and targeting the aberrant metabolism in LSCs. Dysregulated metabolism is involved in sustaining functional properties of LSCs, impeding myeloid differentiation, and evading programmed cell death, both in the process of leukemogenesis and in response to chemotherapy. This review discusses recent discoveries regarding the aberrant metabolic processes of AML LSCs that have begun to change the therapeutic landscape of AML.

Glucose Metabolism as a Potential Therapeutic Target in Cytarabine-Resistant Acute Myeloid Leukemia.

Altered glycolytic metabolism has been associated with chemoresistance in acute myeloid leukemia (AML). However, there are still aspects that need clarification, as well as how to explore these metabolic alterations in therapy. In the present study, we aimed to elucidate the role of glucose metabolism in the acquired resistance of AML cells to cytarabine (Ara-C) and to explore it as a therapeutic target. Resistance was induced by stepwise exposure of AML cells to increasing concentrations of Ara-C. Ara-C-resistant cells were characterized for their growth capacity, genetic alterations, metabolic profile, and sensitivity to different metabolic inhibitors. Ara-C-resistant AML cell lines, KG-1 Ara-R, and MOLM13 Ara-R presented different metabolic profiles. KG-1 Ara-R cells exhibited a more pronounced glycolytic phenotype than parental cells, with a weaker acute response to 3-bromopyruvate (3-BP) but higher sensitivity after 48 h. KG-1 Ara-R cells also display increased respiration rates and are more sensitive to phenformin than parental cells. On the other hand, MOLM13 Ara-R cells display a glucose metabolism profile similar to parental cells, as well as sensitivity to glycolytic inhibitors. These results indicate that acquired resistance to Ara-C in AML may involve metabolic adaptations, which can be explored therapeutically in the AML patient setting who developed resistance to therapy.

Abstract 5851: Dysregulated RNA splicing promotes AML quiescence and chemoresistance

Abstract Chemotherapy is the first-line treatment for patients with acute myeloid leukemia (AML). Although most achieve initial remission, an estimated 50% of patients will relapse with poor clinical outcome. The paucity of genetic lesions in AML patients that relapse from chemotherapy implicate non-genetic alterations that mediate chemoresistance. In this study, we conducted a longitudinal analysis of alternative splicing changes in a cohort of 19 AML patients at diagnosis and relapse post-chemotherapy treatment. We found that the long isoform of the transcription factor-encoding gene RUNX1 (also known as RUNX1C) is upregulated in AML relapse cohorts and conferred chemotherapy resistance. Mechanistically, we show that B cell translocation gene 2 (BTG2) is an RUNX1C isoform-specific transcriptional target that promote transition into a quiescent and treatment-resistant state. Direct targeting of RUNX1C mRNA augments chemotherapy response in AML patient-derived xenograft (PDX) models in vivo. These findings uncover an isoform-specific functionality that influences chemotherapy responsiveness and offers a therapeutic strategy to sensitize leukemic cells to conventional chemotherapy treatments. Citation Format: Eric Wang, Cuijuan Han, Alexander Calderon, Abimbo Lawal, Nidhi Hariramani, Edie Crosse, Robert Bradley. Dysregulated RNA splicing promotes AML quiescence and chemoresistance [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 5851.

Panobinostat sensitizes AraC-resistant AML cells to the combination of azacitidine and venetoclax

The majority of acute myeloid leukemia (AML) patients respond to intensive induction therapy, consisting of cytarabine (AraC) and an anthracycline, though more than half experience relapse. Relapsed/refractory (R/R) AML patients are difficult to treat, and their clinical outcomes remain dismal. Venetoclax (VEN) in combination with azacitidine (AZA) has provided a promising treatment option for R/R AML, though the overall survival (OS) could be improved (OS ranges from 4.3 to 9.1 months). Overexpression of c-Myc is associated with chemoresistance in AML. Histone deacetylase (HDAC) inhibitors have been shown to suppress c-Myc and enhance the antileukemic activity of VEN, as well as AZA, though combination of all three has not been fully explored. In this study, we investigated the HDAC inhibitor, panobinostat, in combination with VEN + AZA against AraC-resistant AML cells. Panobinostat treatment downregulated c-Myc and Bcl-xL and upregulated Bim, which enhanced the antileukemic activity of VEN + AZA against AraC-resistant AML cells. In addition, panobinostat alone and in combination with VEN + AZA suppressed oxidative phosphorylation and/or glycolysis in AraC-resistant AML cells. These findings support further development of panobinostat in combination with VEN + AZA for the treatment of AraC-resistant AML.

DNA-PKcs-mediated transcriptional regulation of TOP2B drives chemoresistance in acute myeloid leukemia.

Anthracyclines, topoisomerase II enzyme poisons that cause DNA damage, are the mainstay of acute myeloid leukemia (AML) treatment. However, acquired resistance to anthracyclines leads to relapse, which currently lacks effective treatment and is the cause of poor survival in individuals with AML. Therefore, the identification of the mechanisms underlying anthracycline resistance remains an unmet clinical need. Here, using patient-derived primary cultures and clinically relevant cellular models that recapitulate acquired anthracycline resistance in AML, we have found that GCN5 (also known as KAT2A) mediates transcriptional upregulation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in AML relapse, independently of the DNA-damage response. We demonstrate that anthracyclines fail to induce DNA damage in resistant cells, owing to the loss of expression of their target enzyme, TOP2B; this was caused by DNA-PKcs directly binding to its promoter upstream region as a transcriptional repressor. Importantly, DNA-PKcs kinase activity inhibition re-sensitized AML relapse primary cultures and cells resistant to mitoxantrone, and abrogated their tumorigenic potential in a xenograft mouse model. Taken together, our findings identify a GCN5-DNA-PKcs-TOP2B transcriptional regulatory axis as the mechanism underlying anthracycline resistance, and demonstrate the therapeutic potential of DNA-PKcs inhibition to re-sensitize resistant AML relapse cells to anthracycline.

The roles and mechanisms of TGFB1 in acute myeloid leukemia chemoresistance

Relapsed or Refractory (R/R) Acute Myeloid Leukemia (AML) patients usually have very poor prognoses, and drug-resistance is one of the major limiting factors. In this study, we aimed to explore the functions of Transforming Growth Factor-β1 (TGFB1) in AML drug-resistance. First, TGFB1 levels in serum and bone marrow are higher in R/R patients compared with newly diagnosed patients, this phenomenon could be due to different sources of secreted TGFB1 according to immunohistochemistry of marrow biopsies. Similarly, TGFB1 expression in AML drug-resistant cell lines is higher than that in their parental cell lines, and blocking the TGFB signaling pathway by specific inhibitors decreased resistance to chemotherapeutic agents. On the other hand, exogenous TGFB1 can also promote AML parental cells senescence and chemotherapy resistance. Next, we found SOX4 level is upregulated in drug-resistant cells, and parental cells treated with exogenous TGFB1 induced upregulation of SOX4 levels. Interference of SOX4 expression by siRNA diminished the TGFB1-induced sensitivity to chemotherapeutic agents. Finally, we conduct metabolomic analysis and find Alanine, aspartate and glutamate metabolism pathway, and Glycerophospholipid metabolism pathway are decreased after inhibiting TGFB signaling pathway or interfering SOX4 expression. This study concludes that TGFB1 level in R/R AML patients and drug-resistant strains is significantly increased. Blocking the TGFB signaling pathway can enhance the chemosensitivity of drug-resistant cells by suppressing SOX4 expression and metabolic reprogramming.

Deletion of Mettl3 in mesenchymal stem cells promotes acute myeloid leukemia resistance to chemotherapy

Acute myeloid leukemia (AML) cell survival and chemoresistance are influenced by the existence of bone marrow mesenchymal stem cells (BMMSCs); however, the pathways by which BMMSCs contribute to these processes remain unclear. We earlier revealed that methyltransferase-like 3 (METTL3) expression is significantly reduced in AML BMMSCs and that METTL3 mediates BMMSC adipogenesis to promote chemoresistance in human AML cell lines in vitro. In this investigation, we evaluated the METTL3 function in vivo. Mice exhibiting a conditional removal of Mettl3 in BMMSCs were developed by mating Prrx1-CreERT2;Mettl3fl/+ mice with Mettl3fl/fl mice using the CRISPR-Cas9 system. The Mettl3 deletion increased bone marrow adiposity, enhanced disease progression in the transplantation-induced MLL-AF9 AML mouse model, and chemoresistance to cytarabine. The removal of Mettl3 in BMMSCs resulted in a significant increase in BMMSC adipogenesis. This effect was attributed to the downregulation of AKT1 expression, an AKT serine/threonine kinase 1, in an m6A-dependent manner. The development of chemoresistance in AML is linked to the promoted adipogenesis of BMMSCs. We conclude that METTL3 expression in BMMSCs has a critical function in limiting AML progression and chemoresistance, providing a basis for the progression of therapeutic approaches for AML.

Exosome-shuttled FTO from BM-MSCs contributes to cancer malignancy and chemoresistance in acute myeloid leukemia by inducing m6A-demethylation: A nano-based investigation

Although bone marrow mesenchymal stem cells (BM-MSCs)-derived exosomes have been reported to be closely associated with acute myeloid leukemia (AML) progression and chemo-resistance, but its detailed functions and molecular mechanisms have not been fully delineated. Besides, serum RNA m6A demethylase fat mass and obesity-associated protein (FTO)-containing exosomes are deemed as important indicators for cancer progression, and this study aimed to investigate the role of BM-MSCs-derived FTO-exosomes in regulating the malignant phenotypes of AML cells. Here, we verified that BM-MSCs-derived exosomes delivered FTO to promote cancer aggressiveness, stem cell properties and Cytosine arabinoside (Ara-C)-chemoresistance in AML cells, and the underlying mechanisms were also uncovered. Our data suggested that BM-MSCs-derived FTO-exo demethylated m6A modifications in the m6A-modified LncRNA GLCC1 to facilitate its combination with the RNA-binding protein Hu antigen R (HuR), which further increased the stability and expression levels of LncRNA GLCC1. In addition, LncRNA GLCC1 was verified as an oncogene to facilitate cell proliferation and enhanced Ara-C-chemoresistance in AML cells. Further experiments confirmed that demethylated LncRNA GLCC1 served as scaffold to facilitate the formation of the IGF2 mRNA binding protein 1 (IGF2BP1)-c-Myc complex, which led to the activation of the downstream tumor-promoting c-Myc-associated signal pathways. Moreover, our rescuing experiments validated that the promoting effects of BM-MSCs-derived FTO-exo on cancer aggressiveness and drug resistance in AML cells were abrogated by silencing LncRNA GLCC1 and c-Myc. Thus, the present firstly investigated the functions and underlying mechanisms by which BM-MSCs-derived FTO-exo enhanced cancer aggressiveness and chemo-resistance in AML by modulating the LncRNA GLCC1-IGF2BP1-c-Myc signal pathway, and our work provided novel biomarkers for the diagnosis, treatment and therapy of AML in clinic.

The Splicing-Associated Network PAK1-Clk-SRRM1 Is a Critical Vulnerability to Overcome Chemoresistance in Acute Myeloid Leukemia

In Acute Myeloid Leukemia (AML), distinguishing between chemoresistance driver mechanisms and cell plasticity in response to chemotherapy is challenging due to their interconnectedness. To address this issue, we generated an MLL-AF9-driven syngeneic mouse model of AML, which we evolved to develop resistance to a front-line chemotherapy regimen by repetitive exposure to a maximally-tolerated combination of cytarabine and doxorubicin. An RNA sequencing (RNAseq)-based profiling of the chemoresistant (Chemo R) AML subpopulation and its naive (Chemo N) counterpart revealed a significant upregulation in the expression of 326 genes in Chemo R cells which were markedly enriched in gene sets related to splicing/mRNA processing. Notably, single-sample GSEA analysis of relapsed AML samples from the BEAT-AML cohort supported these findings, as they were enriched for the upregulation of genes in our chemoresistance gene signature. A combined in vivo functional screening endeavor using a pool of 2098 shRNAs targeting these upregulated genes and a motif discovery analysis performed on the set of dysregulated transcripts in Chemo R cells identified three candidate hits, Srrm1, Sf3b3, and Prpf8, which scored as chemotherapy-specific dependencies and were found to interact with central regulators of alternative splicing ( Figure 1A). Silencing these three candidate genes in our chemoresistant mouse model and the human AML cell lines, MOLM-14, MV4-11, and U937, evolved to become resistant to the combination of cytarabine and daunorubicin, revealed that the splicing regulator, SRRM1, is a critical vulnerability of chemoresistant leukemic cells. An orthogonal phospho-proteomic approach identified that PAK1 and CLK kinases are regulators of SRRM1 in chemoresistant AML cells, through the phosphorylation of multiple serine and threonine phosphosites in its arginine-rich domain. Human and murine chemoresistant cells were found to be markedly more sensitive to inhibitors of PAKs (FRAX597) and CLKs (TG003 and ML167) compared to their naive counterparts, an effect which was even further enhanced by treatment with cytarabine and daunorubicin. Notably, a whole-exome sequencing pipeline at our institution revealed a clonal variant of uncertain significance (c.1429G>T p.(Ala477Ser)) in PAK1 in an AML patient at relapse. Absence of this variant at diagnosis suggested its association with chemotherapy resistance. Biochemical and cellular assays confirmed that the A477S variant increased PAK1 activation as well as chemotherapy resistance in AML cell lines. This effect could be counteracted by suppressing SRRM1, indicating that PAK1 activation in relapsed AML confers increased vulnerability to SRRM1 suppression. Simultaneous suppression of Clk1 and Pak1 or Clk4 and Pak1 using validated shRNAs and combinations of FRAX597 and TG003, or FRAX597 and ML167 synergized to reduce the growth and the colony-forming capacity of chemoresistant AML cells and sensitized them to chemotherapy. These observations were supported by the RNAseq-based estimate of the inclusion ratios of alternative exons and introns which showed that the combination of PAK and CLK inhibitors phenocopied partially the effects of SRRM1 silencing on splicing perturbations. In preclinical studies, the combined treatment improved overall mouse survival and reduced disease burden in our chemoresistant AML mouse models compared to single-drug treatments ( Figure 1B). Moreover, AML patient cells that were primarily refractory or from a post chemotherapy relapse (n=21) exhibited a higher ex vivo sensitivity to the FRAX597 + TG003 combination in comparison with chemosensitive patients at diagnosis (n=28). Moreover, in NSG-S mice transplanted with matched samples from the same AML patient at diagnosis and relapse post-chemotherapy, combined PAK1 and CLK inhibition more effectively reduced disease progression in animals transplanted with the relapse sample than in those engrafted with the sample taken at diagnosis. In conclusion, our study advances the rationale that combined inhibition of PAK1 and CLK kinases effectively curtails malignant cell splicing adaptation to chemotherapy by affecting SRRM1 function. Combined PAK1 and CLK inhibitors either concomitant with or sequential to chemotherapy may offer greater clinical benefit and significantly improve AML patients' long-term outcomes.

MPO and Trh within Leukemia Stem Cells Increased Chemosensitivity in Acute Myeloid Leukemia: A Single-Cell RNA Sequencing Approach

Introduction The “7+3” regimen is widely recognized as the established standard induction therapy for newly diagnosed acute myeloid leukemia (AML), exhibiting a complete remission (CR) rate of 70%. However, the prognosis for patients who do not achieve CR following induction remains discouraging. Leukemia stem cells (LSCs) are recognized as major contributors to chemoresistance in AML. Hence, it is crucial to identify potential targets within LSCs that can overcome chemoresistance. Patients and methods This prospective study involved 20 consecutive de novo AML patients who underwent “7+3” induction therapy. These patients were divided into CR (n=15) and non-CR (n=5) groups. By employing single-cell RNA sequencing (scRNA-seq), we meticulously examined the cellular states of bone marrow mononuclear cells from AML patients at the time of diagnosis and identified LSCs among these cells. The genetic profiles of the LSCs were subsequently compared between the CR and non-CR groups and further validated using independent cohorts. Results The non-CR AML patients exhibited a significant increase in the proportion of immature cells during hematopoiesis within the AML cell populations. Moreover, we found that expressions of MPO and TRH within LSCs were significantly higher in the CR than non-CR groups, which were further validated by independent cohorts. Furthermore, patients with higher expression of TRH and MPO demonstrated substantially improved relapse-free survival (p = 0.009 for TRH; p = 0.002 for MPO) and overall survival (p < 0.001 for TRH; p = 0.002 for MPO). The dysregulation of the OXPHOS pathway, along with altered interferon alpha and gamma responses, disrupted cholesterol homeostasis, and aberrant MYC activity, may contribute as underlying mechanisms for the observed association between MPO or TRH and chemotherapy response. Conclusions MPO and TRH in LSCs may increase chemosensitivity in AML and serve as chemoresponse biomarkers, offering potential benefits in guiding induction strategies for newly diagnosed AML patients

ID2 Promotes Leukemogenesis and Chemoresistance of Acute Myeloid Leukemia By Modulating Cellular Oxidative Metabolism

Acute myeloid leukemia (AML) relies on quiescent leukemia stem cells (LSCs) to evade chemotherapy and drive disease relapse. Recent research indicates that a specific subset of LSCs, characterized by elevated oxidative phosphorylation and/or increased senescence, is responsible for chemotherapy resistance and relapse. While numerous genes associated with LSC maintenance and resistance have been identified, the underlying molecular mechanisms remain elusive. Inhibitor of DNA binding (ID) proteins, a group of Helix-Loop-Helix (HLH) transcription regulators, play a dominant role in regulating other HLH proteins (E proteins) required for the normal development of hematopoietic, muscle, neuronal, and other cells. Our previous investigation revealed that ID2 protects quiescent hematopoietic stem cells (HSCs) activation and exhaustion. Analysis of TCGA data sets demonstrated that high levels of ID2 expression are linked to reduced overall survival in AML patients, suggesting ID2 may be required to maintain some LSCs. Consistent with murine cell findings, we found that knocking down (KD) ID2 expression in human cord blood cells resulted in a loss of CD34+CD38- progenitors. Similarly, ID2-KD in AML cell lines (UT-7, MB-02, and M-07e) significantly reduced their leukemogenic potential in NSG mice, indicating that ID2 is essential for AML cell growth in vivo. Conversely, overexpressing ID2 in the human AML cell line M-07e enhanced their leukemic potential and burden in NSG mice, supporting the notion that ID2 may preserve LSC quiescence. Experiments with primary AML-Patient Derived Xenograft (AML-PDX) cells, which are more clinically relevant, also showed reduced leukemic potential in transplanted NSG mice upon ID2 knockdown. Additionally, ID2-KD cells displayed significantly lower levels of reactive oxygen species (ROS) and mitochondrial activity compared to control cells in both AML cell lines and PDX cells. As ROS-low cells are more susceptible to cytarabine (AraC), we investigated whether ID2-KD renders AML cells more susceptible to AraC. Indeed, MB-02-ID2-KD and UT7-ID2-KD cells showed increased sensitivity to AraC, while M-07e cells overexpressing ID2 exhibited resistance to AraC. Furthermore, AraC effectively eliminated ROS-low cells, but ROS-high cells remained resistant, supporting previous findings. To further investigate the effect of ID2-KD in AraC resistance in vivo, UT7-ID-KD and MB-02-ID2-KD transplanted NSG mice were treated with AraC and monitored for their overall survival, and it was observed that ID2-KD transplanted mice have significantly higher overall survival compared to the control cell transplanted mice. In conclusion, it can be said that ID2 plays a critical role in maintaining normal murine and human HSCs, and it likely contributes to leukemia development and chemotherapy resistance. Therefore, targeting ID2 may offer a promising therapeutic approach to selectively tackle LSCs, followed by chemotherapy and bone marrow transplantation to achieve complete remission.

The Mevalonate Pathway Is a Therapeutic Target in TP53 Mutant Acute Myeloid Leukemia

Introduction: Acute myeloid leukemia (AML) with mutations in the TP53 tumor suppressor gene is the most fatal of AMLs with a median overall survival of only six months due to chemotherapy resistance. To understand biologic differences in TP53 mutant (MT) AML, as compared to wildtype (WT) AML, we performed transcriptomic analysis on sorted patient samples. Gene set enrichment analysis demonstrated significant upregulation of the cholesterol biosynthesis or mevalonate pathway in TP53 MT AML. In TP53 MT solid tumor models, the mevalonate pathway plays a key role in tumorigenesis and metastasis. We hypothesized that the mevalonate pathway is essential for chemotherapy resistance of TP53 MT AML and represents a novel therapeutic target. Methods: We developed chemoresistant, isogenic TP53 MT AML cell lines using CRISPR/Cas9 technology in the MOLM14 AML cell line. We used primary TP53 MT AML patient samples to perform colony forming unit (CFU) assays, in vitro assays with a serum substitute and cytokines, and a patient derived xenograft (PDX) model of TP53 MT AML. Results: We first sought to determine if TP53 MT AML exhibits upregulation of mevalonate pathway activity in response to cytarabine (AraC), the backbone of AML therapy. We treated our isogenic TP53 MT AML cell lines for 24 hours (h) with AraC and assessed mevalonate pathway gene expression and metabolites using qRT-PCR and liquid chromatography high resolution mass spectrometry (LC-HRMS), respectively. Only TP53 MT cell lines respond to AraC with a significant upregulation of key mevalonate pathway genes and metabolites. We then determined if TP53 MT AML is sensitized to AraC by inhibition of the mevalonate pathway with a statin. We pretreated our isogenic cell lines with 24h rosuvastatin before 24h AraC and assessed cell viability with flow cytometry with AnnexinV/7AAD staining and XTT assays. We also performed CFU assays in TP53 MT AML patient samples treated with the two drugs alone or in combination and assessed CFUs after 14 days. These studies revealed a synergistic reduction in cell viability and CFUs in TP53 MT AML by rosuvastatin in combination with AraC. We next addressed the mechanism by which a statin sensitizes TP53 MT AML to AraC. Our group and others have demonstrated enhanced mitochondrial activity is associated with AML chemoresistance. We hypothesized that mevalonate pathway byproducts, known to be crucial for mitochondrial functions, contribute to the mitochondrial response to AraC. We treated TP53 MT AML cell lines as above with rosuvastatin and AraC and used seahorse technology to measure oxidative phosphorylation (OXPHOS). We also used electron microscopy, flow cytometry of mitochondrial protein, TOM20, and qRT-PCR for mitochondrial DNA content to quantify and characterize mitochondria. These data demonstrate that TP53 MT AML cell lines, compared to WT, exhibit a significant increase in OXPHOS after 24h that is due to an increase in total mitochondrial. These effects are abrogated by pretreatment with a statin. We also validated these findings in primary TP53 MT AML patient samples in vitro. Importantly, co-treatment of the isogenic TP53 MT AML cell lines with a soluble form of mevalonate or a downstream byproduct, geranylgeranyl pyrophosphate (GGPP), recovered OXPHOS and subsequent chemoresistance. Overall, this data supports a model where TP53 MT AML cells dynamically upregulate the mevalonate pathway to regulate OXPHOS and avoid DNA damage-induced cell death (Figure 1). Finally, we studied a PDX model of TP53 MT AML. Engrafted mice were treated with AraC for five days alone or in combination with high dose rosuvastatin. Mice were harvested on day eight with assessment of leukemic burden by flow, OXPHOS by seahorse and mevalonate byproducts by LC-HRMS. TP53 MT AML leukemic burden was only significantly reduced by the combination therapy. Consistent with in vitro findings, mice sacrificed three days after completing AraC continue to demonstrate enhanced OXPHOS and increased mevalonate byproducts. This response is blunted by rosuvastatin. Conclusions: These results demonstrate that TP53 MT AML requires the mevalonate pathway for chemotherapy resistance and targeting of this pathway in combination with chemotherapy may improve clinical responses. Analysis is ongoing to determine the role of GGPP in regulating the mitochondrial response to DNA damage in TP53 MT cells in order to elucidate novel therapeutic approaches.

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

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

Protective Function of Human Skin Mesenchymal Niches for Acute Myeloid Leukemia-Initiating Stem Cells

Leukemia cutis (LC) or leukemic cell infiltration in skin is one of the common extramedullary manifestations in acute myeloid leukemia (AML) and signifies a poorer prognosis. However, the pathogenesis and maintenance of LC have been understudied, limiting our understanding of its impact on AML progression and relapse. We recently have shown that the AML cells infiltrated in the mouse skin during steady state and after chemotherapy were capable to regenerate AML after serial transplantation (Sandhow et al., J. Exp. Med, 2023). Further characterization of the skin mesenchymal stem cells (MSCs) suggests that skin MSCs could maintain AML-initiating stem cells (LSCs) and protect them from chemotherapy, even to a greater degree than their bone marrow (BM) counterparts. These data suggest the protective role of skin mesenchymal niche in AML LSCs during chemotherapy. However, it remains unexplored whether human skin MSCs exert the same functional impact on human AML cells. We here have explored the role of human skin MSCs in AML maintenance and survival during chemotherapy by RNA sequencing and in vitro co-culture of patient-derived AML cells with primary human skin MSCs in comparison with BM MSCs. We first by prospective characterization identified human skin MSCs with a phenotype of CD45-CD235a-CD31-CD44+CD146- from healthy donors. RNA sequencing and Q-PCR revealed expression of key hematopoietic stem cell (HSC) regulatory genes including THPO, SCF, CXCL12 and LAMA4 in human skin MSCs. Interestingly, while HSC maintenance genes such as THPO and KITLG were significantly upregulated in skin MSCs (p<0.0001 and p<0.05, respectively), HSC negative regulator SPP1 was downregulated (p=0.05), pointing to a possible better hematopoiesis-supportive function of skin MSCs. In line with this, our functional studies indicated that skin MSCs displayed a superior protective effect on human AML cell line THP-1 than their BM cell counterparts during cytarabine (Ara-C) treatment in the in vitro co-culture. There were more residual chemo-resistant AML cells, reflected in the higher number of CD36+ AML cells after treatment with Ara-C in cocultures with skin MSCs, compared to that with BM MSCs (p=0.0065). This result was confirmed by functional assays showing the increased number of cobblestone area-forming cells (p=0.0298) and colony-forming units (p=0.038) within the residual AML cells co-cultured with skin MSCs. To investigate the mechanisms involved in the protective function of human skin MSCs, we have performed RNA-sequencing of the human skin and BM MSCs before and after co-culture with AML cells. This has revealed potential unique molecular pathways between AML and skin MSCs. Furthermore, there were dramatically less altered genes in the skin MSCs compared to BM MSCs after 3 day-coculture with THP1 AML cells (221 vs 1761 altered genes with adjusted p values <0.05). These data indicate that skin MSCs are likely more resistant to AML-remodeling, which could in turn maintain AML cells in quiescence status, thereby protecting AML cells from Ara-C treatment. This speculation is currently under validation. In addition, gene set enrichment analysis showed upregulated genes associated with fatty acid metabolisms and ribosome in skin MSCs compared to BM MSCs, providing molecular evidence for a potential better metabolic support of skin MSCs for AML cells. Taken together, our preliminary data indicate an important role of human skin MSCs in supporting and protecting AML cells during Ara-C treatment. We are further investigating the underlying mechanisms.

Interleukin-6 Facilitates Acute Myeloid Leukemia Chemoresistance via Mitofusin 1-Mediated Mitochondrial Fusion.

IL6 treatment induces MFN1-mediated mitochondrial fusion, promotes OXPHOS, and confers chemoresistance in AML cells. Targeting IL6 regulation in mitochondria is a promising therapeutic strategy to enhance the chemosensitivity of AML.

Distinct N7-methylguanosine profiles of circular RNAs in drug-resistant acute myeloid leukemia

Post-transcriptional methylation modifications, such as the N7-methylguanosine (m7G) modification, are increasingly acknowledged for their role in the development and resistance to chemotherapy in acute myeloid leukemia (AML). This study employed MeRIP-seq technology to investigate the m7G sites within circular RNAs (circRNAs) derived from human AML cells and drug-resistant AML cells, in order to identify these sites more comprehensively. In addition, a detailed analysis of the relationship between m7G and drug-resistant AML was conducted. The bioinformatics analysis was utilized to predict the functions of specific methylated transcripts. The findings revealed a significant difference in m7G level between AML cells and drug-resistant AML cells, suggesting a potentially critical role of m7G in circRNAs in drug-resistant AML development. The methylation of M7G could affect the circRNA-miRNA-mRNA co-expression during the development of AML resistance, which could further influence the regulation of resistance-associated target genes in AML. Furthermore, gene ontology analysis indicated that the distinct distribution pattern of circRNAs with m7G methylation in drug-resistant AML cells was correlated with metabolism-related pathways. These results suggested a potential association between drug-resistant AML and m7G methylation of circRNAs. Moreover, the results revealed a novel role of m7G RNA methylation in circRNAs in the progression of AML chemoresistance.

Single-cell transcriptomics reveals multiple chemoresistant properties in leukemic stem and progenitor cells in pediatric AML

BackgroundCancer patients can achieve dramatic responses to chemotherapy yet retain resistant tumor cells, which ultimately results in relapse. Although xenograft model studies have identified several cellular and molecular features that are associated with chemoresistance in acute myeloid leukemia (AML), to what extent AML patients exhibit these properties remains largely unknown.ResultsWe apply single-cell RNA sequencing to paired pre- and post-chemotherapy whole bone marrow samples obtained from 13 pediatric AML patients who had achieved disease remission, and distinguish AML clusters from normal cells based on their unique transcriptomic profiles. Approximately 50% of leukemic stem and progenitor populations actively express leukemia stem cell (LSC) and oxidative phosphorylation (OXPHOS) signatures, respectively. These clusters have a higher chance of tolerating therapy and exhibit an enhanced metabolic program in response to treatment. Interestingly, the transmembrane receptor CD69 is highly expressed in chemoresistant hematopoietic stem cell (HSC)-like populations (named the CD69+ HSC-like subpopulation). Furthermore, overexpression of CD69 results in suppression of the mTOR signaling pathway and promotion of cell quiescence and adhesion in vitro. Finally, the presence of CD69+ HSC-like cells is associated with unfavorable genetic mutations, the persistence of residual tumor cells in chemotherapy, and poor outcomes in independent pediatric and adult public AML cohorts.ConclusionsOur analysis reveals leukemia stem cell and OXPHOS as two major chemoresistant features in human AML patients. CD69 may serve as a potential biomarker in defining a subpopulation of chemoresistant leukemia stem cells. These findings have important implications for targeting residual chemo-surviving AML cells.

Skin mesenchymal niches maintain and protect AML-initiating stem cells.

Leukemia cutis or leukemic cell infiltration in skin is one of the common extramedullary manifestations of acute myeloid leukemia (AML) and signifies a poorer prognosis. However, its pathogenesis and maintenance remain understudied. Here, we report massive AML cell infiltration in the skin in a transplantation-induced MLL-AF9 AML mouse model. These AML cells could regenerate AML after transplantation. Prospective niche characterization revealed that skin harbored mesenchymal progenitor cells (MPCs) with a similar phenotype as BM mesenchymal stem cells. These skin MPCs protected AML-initiating stem cells (LSCs) from chemotherapy in vitro partially via mitochondrial transfer. Furthermore, Lama4 deletion in skin MPCs promoted AML LSC proliferation and chemoresistance. Importantly, more chemoresistant AML LSCs appeared to be retained in Lama4-/- mouse skin after cytarabine treatment. Our study reveals the characteristics and previously unrecognized roles of skin mesenchymal niches in maintaining and protecting AML LSCs during chemotherapy, meriting future exploration of their impact on AML relapse.

FOXP1 regulates oxidative stress, SIRT1 expression, and resistance to chemotherapies in acute myeloid leukemia cells.

Transcription factor Forkhead box P1 (FOXP1) belongs to the same protein family as the FOXOs that are well-known regulators of murine hematopoietic stem progenitor cell (HSPC) maintenance by dampening oxidative stress. FOXP1 and FOXOs can play opposite or similar roles depending on cell context; they can cross-regulate each other's expression. In a previous study, we have shown that FOXP1 contributes to normal human HSP and acute myeloid leukemia (AML) cell growth. Here we investigated the role of FOXP1 in HSPCs and AML cell oxidative stress defense in human context. FOXP1 expression level was associated with inferior survival outcome of cytogenetically normal (CN) AML patients. FOXP1 knockdown enhanced superoxide anion levels of human committed CD34+CD38+ but not stem cell-enriched CD34+CD38- HSPCs, and AML cells in vitro. It triggered enhanced NRF2 activity and increased cell oxidative stress. FOXP1 had no impact on FOXO1/3/4 expression in these cells; genetic and pharmacological inhibition of FOXOs did not change superoxide anion levels of human HSPCs and AML cells. Also, FOXP1 antioxidant activity was independent of superoxide dismutase (SOD)1-2 or catalase expression changes. Instead, FOXP1 upregulated expression of the stress sensor SIRT1 by stabilizing SIRT1 protein. FOXP1 loss sensitized AML cells to chemotherapy. Altogether, this study identified FOXP1 as a new safeguard against myeloid progenitor oxidative stress, which works independently of FOXOs but through SIRT1, and contributes to AML chemoresistance. It proposes FOXP1 expression/activity as a promising target to overcome drug-resistance of AML HSPCs.

Acute myeloid leukemia (AML)-derived mesenchymal stem cells induce chemoresistance and epithelial-mesenchymal transition-like program in AML through IL-6/JAK2/STAT3 signaling.

Acute myeloid leukemia (AML) has a high rate of treatment failure due to increased prevalence of therapy resistance. Mesenchymal stem cells (MSCs) in the leukemia microenvironment contribute to chemoresistance in AML, but the specific mechanism remains unclear. The critical role of the epithelial-mesenchymal transition (EMT)-like profile in AML chemoresistance has been gradually recognized. However, there is no research to suggest that the AML-derived bone marrow mesenchymal stem cells (AML-MSCs) induce the EMT program in AML thus far. We isolated AML-MSCs and cocultured them with AML cells. We found that AML-MSCs induced a significant mesenchymal-like morphology in drug-resistant AML cells, but it was scarce in parental AML cells. The AML-MSCs promoted growth of AML cells in the presence or absence of chemotherapeutics in vitro and in vivo. Acute myeloid leukemia MSCs also induced EMT marker expression in AML cells, especially in chemoresistant AML cells. Mechanistically, AML-MSCs secreted abundant interleukin-6 (IL-6) and upregulated IL-6 expression in AML cells. Acute myeloid leukemia cells upregulated IL-6 expression in AML-MSCs in turn. Meanwhile, AML-MSCs activated the JAK2/STAT3 pathway in AML cells. Two JAK/STAT pathway inhibitors counteracted the AML-MSCs induced morphology change and EMT marker expression in AML cells. In conclusion, AML-MSCs not only promote the emergence of chemoresistance but also enhance it once AML acquires chemoresistance. AML-MSCs induce EMT-like features in AML cells; this phenotypic change could be related to chemoresistance progression. AML-MSCs induce the EMT-like program in AML cells through IL-6/JAK2/STAT3 signaling, which provides a therapeutic target to reverse chemoresistance in AML.