Leukemia Progression Research Articles

P92 Cutaneous manifestations of myelodysplastic syndrome: a systematic review

Abstract A variety of associations between cutaneous manifestations and myelodysplastic syndrome (MDS) have been described, but the frequency is unknown. The presence of cutaneous manifestations may have therapeutic and prognostic significance, as the presence thereof has been reported to be associated with poor prognosis or acute myeloid leukaemia (AML) progression, or may precede the diagnosis of MDS [Farah C, Bulai Livideanu C, Jegu J et al. Prevalence and prognostic value of cutaneous manifestations in patients with myelodysplastic syndrome. J Eur Acad Dermatol Venereol 2010; 24:1171–5; Ghoufi L, Ortonne N, Ingen-Housz-Oro S et al. Histiocytoid Sweet syndrome is more frequently associated with myelodysplastic syndromes than the classical neutrophilic variant: a comparative series of 62 patients. Medicine (Baltimore) 2016; 95:e3033]. We aimed to determine the types and prevalence of cutaneous manifestations in MDS, treatment options and prognosis associated with specific cutaneous manifestations and MDS subtypes. PubMed, MEDLINE and Embase were searched from inception up to 26 January 2023 for studies reporting cutaneous manifestations of MDS in adults aged ≥ 18 years, as per the World Health Organization classification. Case reports and case series were included. The review was registered with PROSPERO (CRD42021293140). In total, 92 studies were included (case reports n = 71, case series n = 21), consisting of 140 patients [98 male, mean age 61.0 (SD 15.4)]. We identified six common cutaneous manifestations: neutrophilic, e.g. Sweet syndrome and pyoderma gangrenosum (n = 66); vasculitis, e.g. leucocytoclastic vasculitis and Behçet disease (n = 21); granulomatous, e.g. granuloma annulare and interstitial granulomatous dermatitis (n = 8); connective tissue disease (CTD), e.g. dermatomyositis (n = 7); panniculitis (n = 4); immunobullous (n = 2); and other (n = 30). Sweet syndrome (81.8%) and pyoderma gangrenosum (18.2%) were the commonest presentations of neutrophilic dermatosis. Cutaneous features either occurred at time of MDS diagnosis in 24.7%, preceded the diagnosis in 36.1% (mean 17 months; range 0.5–216), or after diagnosis in 39.2% (mean 20.6 msonths; range 1–132). Prognosis was poor (40.2% death) with 34.1% progressing to acute myeloid leukaemia (AML), 50% of which had neutrophilic dermatosis. Death occurred in 38.5% of neutrophilic dermatoses (median time from onset of cutaneous manifestation: 12 months), 50% of vasculitis (7.5 months), 62.5% of granulomatous (15.5 months), 14.3% of CTD (7 months), and none reported in immunobullous and panniculitis. Granulomatous skin lesions had the poorest prognosis, 60% of which had progressed to AML. This systematic review highlights the range of cutaneous manifestations associated with myelodysplastic syndrome. Of note, we found that the majority of cases progressing to AML were associated with neutrophilic dermatoses overall; however, granulomatous skin lesions had the poorest prognosis with the highest proportion progressing to AML. We speculate that these manifestations may confer an increase in risk of transformation, compared with other cutaneous changes.

Circ_0104700 contributes to acute myeloid leukemia progression by enhancing MCM2 expression through targeting miR-665

ABSTRACTObjectiveAcute myeloid leukemia (AML) is a common blood cancer associated with poor prognosis and high mortality. In this study, we investigated the role and underlying mechanism of action of circ_0104700 in the pathogenesis of AML.MethodsCirc_0104700 was screened from the GEO database and detected in AML samples and cell lines. The effect of circ_0104700 on AML was analyzed using a methylcellulose colony assay, CCK-8 assay, and cell cycle and apoptosis analyses. The mechanism was explored using bioinformatic analysis, quantitative reverse transcription-PCR, dual-luciferase reporter assays, northern blotting and western blot analysis in AML cells.ResultsCirc_0104700 expression was higher in AML patients and AML cell lines. Functionally, circ_0104700 depletion attenuated cell viability and induced apoptosis in MV-4-11 and Kasumi-1 cells. Circ_0104700 depletion enhanced the G0/G1-phase proportion but reduced the proportion of S-phase cells in MV-4-11 and Kasumi-1 cells. circ_0104700 served as a competing endogenous RNA of miR-665 and enhanced MCM2 expression by sponging miR-665 in MV-4-11 and Kasumi-1 cells. Silencing circ_0104700 repressed the proliferation and cell cycle and induced apoptosis of MV-4-11 and Kasumi-1 cells by inhibiting miR-665. MCM2 depletion alleviated the proliferation and cell cycle and enhanced the apoptosis of MV-4-11 and Kasumi-1 cells by inactivating JAK/STAT signaling. JAK/STAT signaling was involved in circ_0104700-mediated malignant phenotypes of MV-4-11 and Kasumi-1 cells.Conclusioncirc_0104700 contributed to AML progression by enhancing MCM2 expression by targeting miR-665. Our findings provide novel potential therapeutic targets for AML, including circ_0104700, miR-665, and MCM2.

RUNX1/ETO regulates reactive oxygen species (ROS) levels in t(8,21) acute myeloid leukaemia via FLT3 and RAC1.

Reactive oxygen species (ROS) homeostasis is crucial for leukaemogenesisand deregulation would hamper leukaemic progression. Although the regulatory effects of RUNX1/ETO has been extensively studied, its underlying molecular mechanims in ROS production in t(8,21) AML is yet to be fully elucidated. Here, we report that RUNX1/ETO could directly control FLT3 by occupying several DNA elements on FLT3 locus. The possible hijacking mechanism by RUNX1/ETO over FLT3 mediated ROS modulation in AML t(8;21) was made apparent when suppression of RUNX1/ETO led to decrement in ROS levels and the direct oxidative marker FOXO3 but not in FLT3 and RAC1 suppressed t(8,21) AML cell line Furthermore, nuclear import of RUNX1/ETO was aberrated following RUNX1/ETO and RAC1 suppression suggesting association in ROS control. A different picture was depicted in non t(8;21) cells where suppression of RAC1 and FLT3 led to decreased levels of FOXO3a and ROS. Results alltogether indicate a possible dysregulation of ROS levels by RUNX1/ETO in t(8,21) AML.

Gene SH3BGRL3 regulates acute myeloid leukemia progression through circRNA_0010984 based on competitive endogenous RNA mechanism.

Introduction: Acute myeloid leukemia (AML) is a malignant proliferative disease affecting the bone marrow hematopoietic system and has a poor long-term outcome. Exploring genes that affect the malignant proliferation of AML cells can facilitate the accurate diagnosis and treatment of AML. Studies have confirmed that circular RNA (circRNA) is positively correlated with its linear gene expression. Therefore, by exploring the effect of SH3BGRL3 on the malignant proliferation of leukemia, we further studied the role of circRNA produced by its exon cyclization in the occurrence and development of tumors. Methods: Genes with protein-coding function obtained from the TCGA database. we detected the expression of SH3BGRL3 and circRNA_0010984 by real-time quantitative polymerase chain reaction (qRT-PCR). We synthesized plasmid vectors and carried out cell experiments, including cell proliferation, cell cycle and cell differentiation by cell transfection. We also studied the transfection plasmid vector (PLVX-SHRNA2-PURO) combined with a drug (daunorubicin) to observe the therapeutic effect. The miR-375 binding site of circRNA_0010984 was queried using the circinteractome databases, and the relationship was validated by RNA immunoprecipitation and Dual-luciferase reporter assay. Finally, a protein-protein interaction network was constructed with a STRING database. GO and KEGG functional enrichment identified mRNA-related functions and signaling pathways regulated by miR-375. Results: We identified the related gene SH3BGRL3 in AML and explored the circRNA_0010984 produced by its cyclization. It has a certain effect on the disease progression. In addition, we verified the function of circRNA_0010984. We found that circSH3BGRL3 knockdown specifically inhibited the proliferation of AML cell lines and blocked the cell cycle. We then discussed the related molecular biological mechanisms. CircSH3BGRL3 acts as an endogenous sponge for miR-375 to isolate miR-375 and inhibits its activity, increases the expression of its target YAP1, and ultimately activates the Hippo signaling pathway involved in malignant tumor proliferation. Discussion: We found that SH3BGRL3 and circRNA_0010984 are important to AML. circRNA_0010984 was significantly up-regulated in AML and promoted cell proliferation by regulating miR-375 through molecular sponge action.

Aberrant expression of T cell activation markers and upregulation of Tregs in bone marrow and peripheral blood in acute myeloid leukemia patients

ABSTRACTBackground:T cells’ function and activation and the immunosuppressive effect of regulatory T cells (Tregs) play a pivotal role in the occurrence and progression of acute myeloid leukemia (AML). In this study, we investigate the expression of T cell activation markers and quantity of Tregs in bone marrow (BM) and peripheral blood (PB) from AML patients and further characterized their correlation with BM leukemic blasts.Methods:Expression of CD25, CD38, CD69, and HLA-DR on the surfaces of CD4+ and CD8+ T cells and the quantity of Tregs in BM and PB from new diagnosed (ND), relapsed-refractory (RR), complete remission (CR) AML patients were measured via flow cytometry.Results:Compared to normal controls (NC), we found higher proportion of CD4+ CD69+ T cells, CD8+ CD69+ T cells and Tregs in PB. CD8+ CD38+ T cells and CD8+ HLA-DR+ T cells in RR were significantly higher than ND, CR and NC). Tregs were normalized when AML patients achieved CR. Moreover, there was a minor positive correlation between AML blasts and CD8+ CD25+ T cells or Tregs, while AML blasts had a minor negative correlation with CD4+ CD69+ T cells.Conclusion:Abnormal activation markers of T cells and Tregs may be involved in the pathological mechanism of ND and RR AML. Our results indicated that CD8+ CD38+ T cells and CD8+ HLA-DR+ T cells might be RR markers of AML patients. Furthermore, Tregs could be used as clinical indicators to evaluate prognosis for AML patients.

PARP-1 inhibits DNMT1-mediated promoter methylation and promotes linc01132 expression in benzene-exposed workers and hydroquinone-induced malignant transformed cells

Hydroquinone (HQ), one of the main active metabolites of benzene, can induce the abnormal expression of long non-coding RNA (lncRNA). Studies have shown that lncRNA plays an important role in the occurrence of hematologic tumors induced by benzene or HQ. However, the molecular mechanism remains to be elucidated. Here, we investigated the molecular mechanism by which poly(ADP-ribose)polymerase 1 (PARP-1) interacts with DNA methyltransferase 1 (DNMT1) to regulate promoter methylation mediated linc01132 expression in HQ-induced TK6 malignant transformed cells (HQ-MT). The results revealed that the expression of linc01132 was increased in benzene-exposed workers and HQ-MT cells. The methylation of linc01132 promoter region was inhibited. Furthermore, in HQ-MT cells treated with 5-Aza-2′-deoxycytidine (5-AzaC) (DNA methyltransferase inhibitor) or trichostatin A (TSA) (histone deacetylation inhibitor), the expression of linc01132 was increased due to the regulation of DNA promoter methylation level by inhibiting DNMT1 expression. The methylation level of linc01132 promoter was correlated negatively with the expression of linc01132 in benzene-exposed workers, indicating that DNA methylation may contribute the expression of linc01132. Knockout of DNMT1, not DNMT3b, increased the expression of linc01132 as well as the demethylation of linc01132 promoter in HQ-MT cells. It was found that by knockdown PARP-1, the expression of DNMT1 in the nucleus was increased by immunofluorescence confocal microscopy, leading to the inhibition of hypermethylation in the promoter region of linc01132. Therefore, PARP-1 inhibits DNA methyltransferase (DNMT)-mediated promoter methylation and plays a role in linc01132 expression in benzene-exposed workers or HQ-MT cells, and is associated with benzene or HQ induced leukemia progression.

EIF4A3-induced Circ_0001187 facilitates AML suppression through promoting ubiquitin-proteasomal degradation of METTL3 and decreasing m6A modification level mediated by miR-499a-5p/RNF113A pathway

Aberrant expression of circRNAs has been proven to play a crucial role in the progression of acute myeloid leukemia (AML); however, its regulatory mechanism remains unclear. Herein, we identified a novel circRNA, Circ_0001187, which is downregulated in AML patients, and its low level contributes to a poor prognosis. We further validated their expression in large-scale samples and found that only the expression of Circ_0001187 was significantly decreased in newly diagnosed (ND) AML patients and increased in patients with hematological complete remission (HCR) compared with controls. Knockdown of Circ_0001187 significantly promoted proliferation and inhibited apoptosis of AML cells in vitro and in vivo, whereas overexpression of Circ _0001187 exerted the opposite effects. Interestingly, we found that Circ_0001187 decreases mRNA m6A modification in AML cells by enhancing METTL3 protein degradation. Mechanistically, Circ_0001187 sponges miR-499a-5p to enhance the expression of E3 ubiquitin ligase RNF113A, which mediates METTL3 ubiquitin/proteasome-dependent degradation via K48-linked polyubiquitin chains. Moreover, we found that the low expression of Circ _0001187 is regulated by promoter DNA methylation and histone acetylation. Collectively, our findings highlight the potential clinical implications of Circ _0001187 as a key tumor suppressor in AML via the miR-499a-5p/RNF113A/METTL3 pathway.

SYT7 regulates the progression of chronic lymphocytic leukemia through interacting and regulating KNTC1

BackgroundChronic lymphocytic leukemia (CLL) is one of the most frequent occurring types of leukemia. It typically occurs in elderly patients and has a highly variable clinical course. At present, the molecular mechanism driving the pathogenesis and progression of CLL is not fully understood. The protein Synaptotagmin 7 (SYT7) encoded by the SYT7 gene has been found to be closely related to the development of various solid tumors, but its role in CLL is unclear. In this study, we investigated the function and molecular mechanism of SYT7 in CLL.MethodsThe expression level of SYT7 in CLL was determined by immunohistochemical staining and qPCR. The role of SYT7 in promoting CLL development was verified by in vivo and in vitro experiments. The molecular mechanism of SYT7 in CLL was elucidated by methods such as GeneChip analysis and Co-immunoprecipitation assay.ResultsMalignant behaviors such as proliferation, migration, and anti-apoptosis of CLL cells were significantly inhibited after SYT7 gene knockdown. In contrast, SYT7 overexpression promoted CLL development in vitro. Consistently, the knockdown of SYT7 also inhibited xenograft tumor growth of CLL cells. Mechanistically, SYT7 promoted CLL development by inhibiting SYVN1-mediated KNTC1 ubiquitination. The KNTC1 knockdown also attenuated the effects of SYT7 overexpression on development of CLL.ConclusionsSYT7 regulates the progression of CLL through SYVN1-mediated KNTC1 ubiquitination, which has potential value for molecular targeted therapy of CLL.

CircPLXNB2 regulates acute myeloid leukemia progression through miR-654-3p/CCND1 axis

ABSTRACTBackgroundAcute myeloid leukemia (AML) is a common hematological malignancy. Circular RNAs (circRNAs) are newly discovered endogenous non-coding RNAs that play important roles in regulating gene expression in cancer biology. The aim of this study was to identify novel prognostic and therapeutic targets associated with AML.MethodsThe expression levels of circRNA Plexin B2 (circPLXNB2), microRNA-654-3p (miR-654-3p) and cyclin D1 protein (CCND1) mRNA were detected by real-time quantitative polymerase chain reaction (qRT-PCR). The stability of circPLXNB2 was determined by RNase R and Actinomycin D assays. Cell counting kit-8 (CCK-8) and 5′-ethynyl-2′-deoxyuridine (EDU) assays were used to detect cell proliferation. Western blot was used to detect protein content. Cell apoptosis and cell cycle distribution were detected by flow cytometry. The relationship between miR-654-3p and circPLXNB2 or CCND1 was confirmed by dual-luciferase reporter assay.ResultsCircPLXNB2 was highly expressed in AML patients and cells, and circPLXNB2 was more stable than linear PLXNB2. Knockdown of circPLXNB2 affected the proliferation, apoptosis and cell cycle distribution of AML cells. CircPLXNB2 acted as a sponge for miR-654-3p and affected the progression of AML cells by targeting miR-654-3p. CCND1 was targeted by miR-654-3p, and miR-654-3p suppressed AML progression by targeting CCND1. CircPLXNB2 regulated CCND1 expression through sponging miR-654-3p.ConclusionIn conclusion, circPLXNB2 was highly expressed in AML patients and cells and modulated tumor progression by regulating the circPLXNB2/miR-654-3p/CCND1 axis, suggested that circPLXNB2 might be a new therapeutic target for AML treatment.

EE282 Financial Burden of Progression of Chronic Myeloid Leukemia (CML) Patients Who Failed Two or More Tyrosine Kinase Inhibitors Treatment in the Brazilian Private Market Perspective

To analyze the financial burden associated with the management of chronic myeloid leukemia (CML) patients who failed treatment with two or more tyrosine kinase inhibitors (TKI) through the Brazilian private market’s perspective.

Preclinical evaluation of targeting PRAME with TCR mimic CAR T cells in AML.

2553 Background: Chimeric antigen receptor (CAR) T cell therapy has revolutionized cancer treatment, but has had limited success against AML in part due to overlap of cell surface antigens expressed in AML and normal hematopoietic cells. To identify AML-restricted targets, we interrogated the transcriptome from over 3000 AML patients and found PRAME (Preferentially Expressed Antigen in Melanoma), an intracellular protein, to be a highly expressed AML-restricted target. Using a novel approach to target intracellular antigens, we developed a PRAME CAR T cell using a TCR mimic (mTCR) antibody, which recognizes the PRAME peptide/HLA-A2 complex on the tumor cell surface. To conduct final IND-enabling studies, we generated an in vivo patient derived xenograft (PDX) model to treat with PRAME mTCRCAR T cells. Here we demonstrate the in vivo activity of PRAME mTCRCAR T cells against a PDX AML model. Methods: We used the VL and VH sequences from the PRAME TCR mimic antibody (Pr20) to construct the single chain fragment variable domain into the 41-BB/CD3ζ CAR vector. The PDX model was derived from a PRAME+/HLA-A2+ pediatric patient with AML. PDX cells were transduced with ffluciferase for noninvasive bioluminescent IVIS imaging to monitor leukemic progression. PDX leukemia-bearing mice were treated with unmodified T-cells or PRAME mTCRCAR T cells at 5x106 cells (1:1 CD4:CD8) per mouse 1 week after leukemia injection. Leukemia burden was measured by IVIS imaging and peripheral blood analysis. Results: Treatment with PRAME mTCRCAR T cells led to eradication of leukemia with all mice alive >100 days post-treatment, while control mice (unmodified T cells) had disease progression at Day 50 (p=0.001). Average leukemia burden in the control cohort was 3.5% at week 6 and 41.75% at week 11, while no leukemia was detected following CAR T cell treatment. In addition, the control arm had a marked expansion of leukemia with a 7.75 and 429.6 fold increase in radiance by IVIS at 6 and 11 weeks post-treatment. No significant increase in radiance was seen in the PRAME mTCRCAR T cell treated group. Human T cells (CD45+/CD3+) were detectable in the peripheral blood at Day 7 and 14 post-treatment in both the unmodified and CAR T cell treated groups. Conclusions: We demonstrate the therapeutic potential of targeting PRAME with mTCRCAR T cells in AML. We show potent efficacy with eradication of leukemia in PDX-bearing mice following treatment with PRAME mTCRCAR T cells resulting in prolonged survival. These results provide a novel approach to target PRAME with CAR T cells and provide compelling data to evaluate PRAME mTCRCAR T cells for use in clinical trials against AML. [Table: see text]

Glutamate dehydrogenase 1: A novel metabolic target in inhibiting acute myeloid leukaemia progression.

Glutamine metabolic reprogramming in acute myeloid leukaemia (AML) cells contributes to the decreased sensitivity to antileukemic drugs. Leukaemic cells, but not their myeloid counterparts, largely depend on glutamine. Glutamate dehydrogenase 1 (GDH1) is a regulation enzyme in glutaminolysis. However, its role in AML remains unknown. Here, we reported that GDH1 was highly expressed in AML: high GDH1 was one of the independent negative prognostic factors in AML cohort. The dependence of leukaemic cells on GDH1 was proved both in vitro and in vivo. High GDH1 promoted cell proliferation and reduced survival time of leukaemic mice. Targeting GDH1 eliminated the blast cells and delayed AML progression. Mechanistically, GDH1 knockdown inhibited glutamine uptake by downregulating SLC1A5. Moreover, GDH1 invalidation also inhibited SLC3A2 and abrogated the cystine-glutamate antiporter system Xc- . The reduced cystine and glutamine disrupted the synthesis of glutathione (GSH) and led to the dysfunction of glutathione peroxidase-4 (GPX4), which maintains the lipid peroxidation homeostasis by using GSH as a co-factor. Collectively, triggering ferroptosis in AML cells in a GSH depletion manner, GDH1 inhibition was synthetically lethal with the chemotherapy drug cytarabine. Ferroptosis induced by inhibiting GDH1 provides an actionable therapeutic opportunity and a unique target for synthetic lethality to facilitate the elimination of malignant AML cells.

PAX5 fusion genes in acute lymphoblastic leukemia: A literature review.

Acute lymphoblastic leukemia (ALL) is a common cancer affecting children worldwide. The development of ALL is driven by several genes, some of which can be targeted for treatment by inhibiting gene fusions. PAX5 is frequently mutated in ALL and is involved in chromosomal rearrangements and translocations. Mutations in PAX5 interact with other genes, such as ETV6 and FOXP1, which influence B-cell development. PAX5/ETV6 has been observed in both B-ALL patients and a mouse model. The interaction between PAX5 and FOXP1 negatively suppresses the Pax5 gene in B-ALL patients. Additionally, ELN and PML genes have been found to fuse with PAX5, leading to adverse effects on B-cell differentiation. ELN-PAX5 interaction results in the decreased expression of LEF1, MB1, and BLNK, while PML-PAX5 is critical in the early stages of leukemia. PAX5 fusion genes prevent the transcription of the PAX5 gene, making it an essential target gene for the study of leukemia progression and the diagnosis of B-ALL.

New Flow Cytometric Methods for Monitoring STAT5 Signaling Reveal Responses to SARS-CoV-2 Antigen-Specific Stimulation in FOXP3+ Regulatory T Cells also in Patients with Advanced Chronic Lymphocytic Leukemia

Increased frequency of CD4+CD25+ regulatory T-cells (Treg) has been associated with disease progression in chronic lymphocytic leukemia (CLL). Flow cytometric methods, which allow for the simultaneous analysis of their specific transcription factor Foxp3 and activated STAT proteins, together with proliferation can help to elucidate the signaling mechanisms driving Treg expansion and suppression of FOXP3- conventional CD4+T-cells (Tcon). Herein, we first report a novel approach in which STAT5 phosphorylation (pSTAT5) and proliferation (BrdU-FITC incorporation) could be analyzed specifically in FOXP3+ and FOXP3- responding cells after CD3/CD28 stimulation. The addition of magnetically purified CD4+CD25+ T-cells from healthy donors to cocultured autologous CD4+CD25- T-cells resulted in suppression of Tcon cell cycle progression accompanied by a decrease in pSTAT5. Next, a method using imaging flow cytometry is presented for the detection of cytokine-dependent pSTAT5 nuclear translocation in FOXP3-expressing cells. Finally, we discuss our experimental data obtained by combining Treg pSTAT5 analysis and antigen-specific stimulation with SARS-CoV-2 antigens. Applying these methods on samples from patients revealed Treg responses to antigen-specific stimulation and significantly higher basal pSTAT5 in CLL patients treated with immunochemotherapy. Thus, we speculate that through the use of this pharmacodynamic tool, the efficacy of immunosuppressive drugs and their possible off-target effects can be assessed.

Combined inhibition of XIAP and autophagy induces apoptosis and differentiation in acute myeloid leukaemia.

Perturbations in autophagy, apoptosis and differentiation have greatly affected the progression and therapy of acute myeloid leukaemia (AML). The role of X-linked inhibitor of apoptosis (XIAP)-related autophagy remains unclear in AML therapeutics. Here, we found that XIAP was highly expressed and associated with poor overall survival in patients with AML. Furthermore, pharmacologic inhibition of XIAP using birinapant or XIAP knockdown via siRNA impaired the proliferation and clonogenic capacity by inducing autophagy and apoptosis in AML cells. Intriguingly, birinapant-induced cell death was aggravated in combination with ATG5 siRNA or an autophagy inhibitor spautin-1, suggesting that autophagy may be a pro-survival signalling. Spautin-1 further enhanced the ROS level and myeloid differentiation in THP-1 cells treated with birinapant. The mechanism analysis showed that XIAP interacted with MDM2 and p53, and XIAP inhibition notably downregulated p53, substantially increased the AMPKα1 phosphorylation and downregulated the mTOR phosphorylation. Combined treatment using birinapant and chloroquine significantly retarded AML progression in both a subcutaneous xenograft model injected with HEL cells and an orthotopic xenograft model injected intravenously with C1498 cells. Collectively, our data suggested that XIAP inhibition can induce autophagy, apoptosis and differentiation, and combined inhibition of XIAP and autophagy may be a promising therapeutic strategy for AML.

Sox13 and M2-like leukemia-associated macrophages contribute to endogenous IL-34 caused accelerated progression of acute myeloid leukemia

Interleukin 34 (IL-34) mainly plays physiologic and pathologic roles through the sophisticated multi-ligand signaling system, macrophage colony-stimulating factor (M-CSF, CSF-1)/IL-34-CSF-1R axis, which exhibits functional redundancy, tissue-restriction and diversity. This axis is vital for the survival, differentiation and function of monocytic lineage cells and plays pathologic roles in a broad range of diseases. However, the role of IL-34 in leukemia has not been established. Here MLL-AF9 induced mouse acute myeloid leukemia (AML) model overexpressing IL-34 (MA9-IL-34) was used to explore its role in AML. MA9-IL-34 mice exhibited accelerated disease progression and short survival time with significant subcutaneous infiltration of AML cells. MA9-IL-34 cells showed increased proliferation. In vitro colony forming assays and limiting dilution transplantation experiments demonstrated that MA9-IL-34 cells had elevated leukemia stem cell (LSC) levels. Gene expression microarray analysis revealed a panel of differential expressed genes including Sex-determining region Y (SRY)-box 13 (Sox13). Furthermore, a positive correlation between the expressions of IL-34 and Sox13 was detected human datasets. Knockdown of Sox13 rescued the enhanced proliferation, high LSC level and subcutaneous infiltration in MA9-IL-34 cells. Moreover, more leukemia-associated macrophages (LAMs) were detected in MA9-IL-34 microenvironment. Additionally, those LAMs showed M2-like phenotype since they expressed high level of M2-associated genes and had attenuated phagocytic potential, suggesting that LAMs should also contribute to IL-34 caused adverse phenotypes. Therefore, our findings uncover the intrinsic and microenvironmental mechanisms of IL-34 in AML and broadens the knowledge of M-CSF/IL-34-CSF-1R axis in malignancies.

Germline Neurofibromin 1 mutation enhances the anti-tumour immune response and decreases juvenile myelomonocytic leukaemia tumourigenicity.

Juvenile myelomonocytic leukaemia (JMML) is an aggressive paediatric leukaemia characterized by mutations in five canonical RAS pathway genes, including the NF1 gene. JMML is driven by germline NF1 gene mutations, with additional somatic aberrations resulting in the NF1 biallelic inactivation, leading to disease progression. Germline mutations in the NF1 gene alone primarily cause benign neurofibromatosis type 1 (NF1) tumours rather than malignant JMML, yet the underlying mechanism remains unclear. Here, we demonstrate that with reduced NF1 gene dose, immune cells are promoted in anti-tumour immune response. Comparing the biological properties of JMML and NF1 patients, we found that not only JMML but also NF1 patients driven by NF1 mutations could increase monocytes generation. But monocytes cannot further malignant development in NF1 patients. Utilizing haematopoietic and macrophage differentiation from iPSCs, we revealed that NF1 mutations or knockout (KO) recapitulated the classical haematopoietic pathological features of JMML with reduced NF1 gene dose. NF1 mutations or KO promoted the proliferation and immune function of NK cells and iMacs derived from iPSCs. Moreover, NF1-mutated iNKs had a high capacity to kill NF1-KO iMacs. NF1-mutated or KO iNKs administration delayed leukaemia progression in a xenograft animal model. Our findings demonstrate that germline NF1 mutations alone cannot directly drive JMML development and suggest a potential cell immunotherapy for JMML patients.

Telomere loss is accompanied by decreased pool of shelterin proteins TRF2 and RAP1, elevated levels of TERRA and enhanced glycolysis in imatinib-resistant CML cells

Telomere length may be maintained by telomerase nucleoprotein complex and shelterin complex, namely TRF1, TRF2, TIN2, TPP1, POT1 and RAP1 proteins and modulated by TERRA expression. Telomere loss is observed during progression of chronic myeloid leukemia (CML) from the chronic phase (CML-CP) to the blastic phase (CML-BP). The introduction of tyrosine kinase inhibitors (TKIs), such as imatinib (IM), has changed outcome for majority of patients, however, a number of patients treated with TKIs may develop drug resistance. The molecular mechanisms underlying this phenomenon are not fully understood and require further investigation. In the present study, we demonstrate that IM-resistant BCR::ABL1 gene-positive CML K-562 and MEG-A2 cells are characterized by decreased telomere length, lowered protein levels of TRF2 and RAP1 and increased expression of TERRA in comparison to corresponding IM-sensitive CML cells and BCR::ABL1 gene-negative HL-60 cells. Furthermore, enhanced activity of glycolytic pathway was observed in IM-resistant CML cells. A negative correlation between a telomere length and advanced glycation end products (AGE) was also revealed in CD34+ cells isolated from CML patients. In conclusion, we suggest that affected expression of shelterin complex proteins, namely TRF2 and RAP1, TERRA levels, and glucose consumption rate may promote telomere dysfunction in IM-resistant CML cells.

Single-cell transcriptome analyses reveal critical roles of RNA splicing during leukemia progression.

Leukemogenesis is proposed to be a multistep process by which normal hematopoietic stem and progenitor cells are transformed into full-blown leukemic cells, the details of which are not fully understood. Here, we performed serial single-cell transcriptome analyses of preleukemic and leukemic cells (PLCs) and constructed the cellular and molecular transformation trajectory in a Myc-driven acute myeloid leukemia (AML) model in mice, which represented the transformation course in patients. We found that the Myc targets were gradually up-regulated along the trajectory. Among them were splicing factors, which showed stage-specific prognosis for AML patients. Furthermore, we dissected the detailed gene network of a tipping point for hematopoietic stem and progenitor cells (HSPCs) to generate initiating PLCs, which was characterized by dramatically increased splicing factors and unusual RNA velocity. In the late stage, PLCs acquired explosive heterogeneity through RNA alternative splicing. Among them, the Hsp90aa1hi subpopulation was conserved in both human and mouse AML and associated with poor prognosis. Exon 4 skipping of Tmem134 was identified in these cells. While the exon skipping product Tmem134β promoted the cell cycle, full-length Tmem134α delayed tumorigenesis. Our study emphasized the critical roles of RNA splicing in the full process of leukemogenesis.

FLI1 Regulates Histamine Decarboxylase Expression to Control Inflammation Signaling and Leukemia Progression.

Histamine decarboxylase (HDC) catalyzes decarboxylation of histidine to generate histamine. This enzyme affects several biological processes including inflammation, allergy, asthma, and cancer, although the underlying mechanism is not fully understood. The present study provides a novel insight into the relationship between the transcription factor FLI1 and its downstream target HDC, and their effects on inflammation and leukemia progression. Promoter analysis combined with chromatin immunoprecipitation (ChIp) was used to demonstrate binding of FLI1 to the promoter of HDC in leukemic cells. Western blotting and RT-qPCR were used to determine expression of HDC and allergy response genes, and lentivirus shRNA was used to knock-down target genes. Proliferation, cell cycle, apoptosis assays and molecular docking were used to determine the effect of HDC inhibitors in culture. An animal model of leukemia was employed to test the effect of HDC inhibitory compounds in vivo. Results presented herein demonstrate that FLI1 transcriptionally regulates HDC by direct binding to its promoter. Using genetic and pharmacological inhibition of HDC, or the addition of histamine, the enzymatic product of HDC, we show neither have a discernable effect on leukemic cell proliferation in culture. However, HDC controls several inflammatory genes including IL1B and CXCR2 that may influence leukemia progression in vivo through the tumor microenvironment. Indeed, diacerein, an IL1B inhibitor, strongly blocked Fli-1-induced leukemia in mice. In addition to allergy, FLI1 is shown to regulate genes associated with asthma such as IL1B, CPA3 and CXCR2. Toward treatment of these inflammatory conditions, epigallocatechin (EGC), a tea polyphenolic compound, is found strongly inhibit HDC independently of FLI1 and its downstream effector GATA2. Moreover, the HDC inhibitor, tetrandrine, suppressed HDC transcription by directly binding to and inhibiting the FLI1 DNA binding domain, and like other FLI1 inhibitors, tetrandrine strongly suppressed cell proliferation in culture and leukemia progression in vivo. These results suggest a role for the transcription factor FLI1 in inflammation signaling and leukemia progression through HDC and point to the HDC pathway as potential therapeutics for FLI1-driven leukemia.