Survival In AML Patients Research Articles

The Role of FLT3-ITD Mutation, PI3K/AKT Pathway, and Leukemia Stem Cells in D3A7 Induction therapy – the Outcomes of Adult Indonesian Patients with Acute Myeloid Leukemia

Objective. This cohort study aimed to examine the impact of the FLT3-ITD mutation on the downstream signaling pathway of PI3K/AKT pathway, the percentage of leukemia stem cells, and the survival of patients receiving D3A7 induction therapy. Method. Bone marrow mononuclear cells were collected from 20 adult AML patients who had completed D3A7 induction therapy at Cipto Mangunkusumo National General Hospital and Dharmais Cancer Hospital. FLT3-ITD gene mutation was examined by the PCR-sequencing method. Expression of phosphorylated PI3K and AKT was detected using the sandwich ELISA method. Flow cytometry was used for detecting the number of apoptosis and proliferation cells, and biomarkers of leukemia stem cells. Result. The expression levels of PI3K and AKT proteins were higher in FLT3-ITD, both in the mutant group compared to the non-mutation group, and in the patient group with treatment failure outcomes compared to the patient group with treatment response. The percentage of the leukemia stem cell population did not differ significantly between the FLT3-ITD mutation group and the wild type group, and between the treatment failure outcome group and the response outcome group. Conclusion. This study presents the important role of FLT3-ITD mutation via its downstream signaling (PI3K/AKT) in the outcome of D3A7 induction therapy. The FLT3-ITD mutation plays an important role in the 12-month survival of AML patients after D3A7 therapy. However, the outcome of D3A7 therapy and FLT3-ITD mutation were not associated with leukemia stem cells.

6-month survival of patients with acute myeloid leukemia who received modified chemotherapy at Wahidin Sudirohusodo Hospital

Background and aim. Standard chemotherapy for AML is daunorubicin combined with cytarabine. However, the combination chemotherapy also has side effects that are quite dangerous for AML patients. The purpose of this study is to assess the survival rates of AML patients treated at Wahidin Sudirohusodo Hospital in Makassar using modified chemotherapy regimens. Methods and material. This study was conducted at Wahidin Sudirohusodo Hospital in Makassar using a prospective cohort design. The study population included AML patients aged 18 years or older who had undergone modified chemotherapy for six months. Data on life expectancy, chemotherapy modifications, age, gender, and AML subtype were collected. Data were analyzed using SPSS version 22.0. Survival analysis was performed using the Kaplan-Meier curve, log-rank, and statistic test. Results. The cohort consisted of 25 males (51%) and 24 females (49%) with a mean age of 42 years. The 6-month survival rates were 68% for females and 50% for males. Age-based survival rates were 61.3% for patients <45 years, and 55.6 for those >45 years. Survival rates by AML subtype were 40% for M1, 63.3% for M2, 0% for M3, 60% for M4, and 66.7% for M5. The survival curve for the therapy regimen showed a significant relationship (p <0.05), with 86.7% survival for daunorubicin and 47.1% for cytarabine. Conclusions. Administration of daunorubicin and cytarabine chemotherapy is a variable that significantly determines the 6-month survival of AML patients.

Targeting NLRP3 inhibits AML progression by inducing PERK/eIF2-mediated apoptosis

BackgroundAcute myeloid leukemia (AML) is characterized by the abnormal proliferation of myeloid precursor cells and presents significant challenges in treatment due to its heterogeneity. Recently, the NLRP3 inflammasome has emerged as a potential contributor to AML pathogenesis, although its precise mechanisms remain poorly understood.MethodsPublic genome datasets were utilized to evaluate the expression of NLRP3 inflammasome-related genes (IL-1β, IL-18, ASC, and NLRP3) in AML patients compared to healthy individuals. CRISPR/Cas9 technology was employed to generate NLRP3-deficient MOLM-13 AML cells, followed by comprehensive characterization using real-time PCR, western blotting, FACS analysis, and transmission electron and immunofluorescence microscopy. Proteomic analyses were conducted to identify NLRP3-dependent alterations in protein levels, with a focus on the eIF2 kinase PERK-mediated signaling pathways. Additionally, in vivo studies were performed using a leukemic mouse model to elucidate the pathogenic role of NLRP3 in AML.ResultsElevated expression of NLRP3 was significantly associated with diminished overall survival in AML patients. Genetic deletion, pharmacological inhibition and silencing by RNA interference of NLRP3 led to decreased AML cell survival through the induction of apoptosis. Proteomic analyses uncovered NLRP3-dependent alterations in protein translation, characterized by enhanced eIF2α phosphorylation in NLRP3-deficient AML cells. Moreover, inhibition of PERK-mediated eIF2α phosphorylation reduced apoptosis by downregulating pro-apoptotic Bcl-2 family members. In vivo studies demonstrated reduced leukemic burden in mice engrafted with NLRP3 knockout AML cells, as evidenced by alleviated leukemic symptoms.ConclusionOur findings elucidate the involvement of the NLRP3/PERK/eIF2 axis as a novel driver of AML cell survival. Targeting NLRP3-induced signaling pathways, particularly through the PERK/eIF2 axis, presents a promising therapeutic strategy for AML intervention. These insights into the role of the NLRP3 inflammasome offer potential avenues for improving the prognosis and treatment outcomes of AML patients.

A novel prognostic scoring system for AML patients undergoing allogeneic hematopoietic stem cell transplantation with real world validation

ObjectivesThis study aims to develop a robust predictive model for survival in AML patients undergoing allo-HSCT. MethodsIt was performed a retrospective analysis of 336 AML patients who underwent allo-HSCT at Peking University First Hospital between September 2003 and March 2023. Univariable and multivariable Cox regression analyses were conducted to determine hazard ratios (HR) for overall survival. A predictive model was developed based on multivariable analysis results. Internal validation was carried out through bootstrap resampling, and the model’s performance was assessed using the Concordance Index (C-index), Receiver Operating Characteristics (ROC) curve, calibration plots, and Decision Curve Analysis (DCA). ResultsOur prognostic model, which includes age, disease stage, donor/recipient gender, mononuclear cell counts, and the Hematopoietic Cell Transplantation Comorbidity Index (HCT-CI), effectively stratified patients into low-risk and high-risk groups. The two groups showed significant differences in overall survival (P<0.0001), disease-free survival (P<0.0001), non-relapse mortality (NRM) (P<0.0001), and relapse rates (P=0.08). The model achieved a C-index of 0.71. Calibration plots and DCA confirmed strong alignment between predicted and observed outcomes. Subgroup analysis revealed that overall survival was significantly lower in the high-risk group compared to the low-risk group in both measurable residual disease (MRD) negative and MRD positive subgroups (P=0.015 for both). ConclusionThe developed prognostic model, which integrates comprehensive disease and patient characteristics, enhances risk stratification for AML patients undergoing allo-HSCT. This model effectively stratifies risk in both MRD-negative and MRD-positive subgroups and may facilitate more informed MRD-based treatment decisions.

FLT3-MUTATED ACUTE MYELOID LEUKEMIA OUTCOMES IN A NORTHEAST BRAZILIAN UNIVERSITY HOSPITAL

Mutations in the FMS-like tyrosine kinase 3 (FLT3) gene occur in approximately 25-45% of new diagnoses of Acute myeloid leukemia. The addition of FLT3 inhibitors to conventional protocols improves overall survival in this condition. Objectives: To assess the incidence of FLT3 gene mutation among patients diagnosed with AML at Walter Cantídio University Hospital; Describe access to FLT3 inhibitors and bone marrow transplantation (BMT), and the overall survival of this group. Methodology: Retrospective evaluation of medical records of patients treated for AML between 2020 and 2022. Statistical analysis was performed using the Kaplan-Meier method to estimate survival probability. Results: 47 patients were diagnosed with AML during this period, of whom 17% had FLT3+ mutation. 3/8 patients accessed FLT3 inhibitors. The median survival with FLT3+ mutation was 9.1 months vs. 12.9 months in FLT3- (p = 0.196). The overall survival of AML patients was 30.9% at 2 years. 11/47 patients underwent allogeneic BMT. Conclusion: The addition of targeted therapies and BMT may contribute to reduce mortality in AML. Elderly patients and those not undergoing HSCT have worse outcomes.

Monoclonal Antibodies Targeting LIGHT Block Ltbr Signalling and Eliminate Acute Myeloid Leukemia Stem Cells

Introduction Acute myeloid leukemia (AML) is a molecular heterogeneous disease that originates from the malignant transformation of a hematogenic stem- or progenitor cells resulting in a leukemia stem cell (LSC). LSCs are a main cause of resistance to therapy and relapse. Despite recent advances in targeted therapy and immunotherapy, prognosis for elderly patients not fit do undergo intensive therapy remains poor. We recently documented that targeting CD70, the ligand of the tumor necrosis factor receptor (TNFR) CD27, by the antibody-dependent cell-mediated cytotoxicity (ADCC)-enhanced monoclonal antibody (mAb) cusatuzumab eliminates LSC. We now studied the role of the related TNFR lymphotoxin b receptor (LTbR) with its ligand LIGHT in the pathogenesis of AML in mice and humans and developed a LIGHT targeting mAb to specifically eliminate LSCs. Experimental design We investigated the impact of LIGHT/LTbR signalling in AML development by using the retrovirally-induced MLL-AF9 syngeneic AML mouse model. Light-, Ltbr -deficient and control MLL-AF9 transduced LSCs were injected intravenously into non-irradiated BL/6 recipient mice. AML development was evaluated by analysing MLL-AF9-GFP-positive leukemic cells in blood, spleen, and bone marrow (BM). BM LSC were phenotypically characterized by FACS and functionally by performing colony forming unit (CFU) assays in vitro and by secondary transplantations of LSCs including limited dilution assays (LDA) in vivo. Correlative survival analysis for LIGHT and LTbR expression was performed on a publicly available gene dataset (GSE6891). LIGHT and LTBR protein expression was analysed on primary AML CD34 + leukemic stem and progenitor cells (LSPCs) and control human CD34 + hematopoietic stem cell and progenitor cells (HSCPs). LIGHT/LTbR signalling was blocked by either siRNA treatment or by treatment with novel LIGHT-targeting mAb. The effect of blocking LIGHT/LTbR-signalling was analysed in vitro by CFU assays, RNA-sequencing and xenotransplant experiments in vivo. LIGHT-targeting mAbs were generated by immunization of llamas and phage display selections followed by cloning of FABs into a human backbone (hIgG1). Clones were selected based on their blocking potency of receptor/ligand interaction using competition ELISA and FACS-based cell binding affinity assays. ADCC-enhanced anti-LIGHT mAbs were generated via POTELLIGENT® technology. Anti-LIGHT mAbs that specifically block the interaction of LIGHT/LTBR with deficiency in effector functions (Fc-death) were produced by amino acid substitutions in the CH2 domain of the parental clone. Results LTbR was expressed by HSCs and AML LSCs. In contrast, LIGHT was only expressed on murine and human LSCs but not normal HSCs. The absence of LIGHT or its receptor LTbR on LSCs resulted in significantly prolonged survival and decreased numbers of LSC in BM of AML mice. Light -/- and Ltbr -/- LSCs had a reduced CFU capacity compared to control LSCs. Similarly, inhibition of LIGHT-signalling on LSCs via LTbR-Ig significantly reduced colony formation. Moreover, secondary transplantation of Light -/-LSCs resulted in prolonged survival compared to Light-proficient LSCs. An LDA analysis revealed a 11-fold reduction of the LSC frequency in BM in the absence of LIGHT or LTBR. Similarly, inhibiting LIGHT signalling by siLIGHT RNA in human AML LSPCs resulted in reduced CFU capacity compared to control siRNA treatment. In addition, a GEO analysis revealed that low LIGHT or LTbR mRNA levels correlated with improved overall survival in AML patients. After immunizations of llamas and phage-display selection three humanized anti-LIGHT mAbs with high blocking and high binding efficiency were selected (mAb10F11 and 12C2 specific for human LIGHT and the cross-reactive murine/human 9F6 mAB). Incubation of LSCs with the blocking mAbs significantly reduced CFU capacity. HSPCs from healthy donors remained unaffected by the treatment. A gene expression analysis revealed that blocking LIGHT/LTbR-signalling in LSCs reduced stemness and promoted differentiation. In addition, ADCC-enhanced mAbs activated NK cells to eliminate LIGHT-expressing LSCs. Our findings reveal that LIGHT/LTbR-signalling is crucial for the pathogenesis of AML and especially for the maintenance and expansion of LSCs. We developed and validated novel LIGHT targeting mAbs in vitro and in vivo for further development in clinical trials.

Vaccination Using an Allogeneic Leukemia-Derived Dendritic Cell Vaccine, Maintains and Improves Frequencies of Circulating Antigen Presenting Dendritic Cells Correlating with Relapse Free and Overall Survival in AML Patients

Background. Active immunotherapy relies on the patient's immune system for induction of an effective immune response. Not only the frequency, number and exhaustion or activation status of CD8 T cells is important for an efficacious tumor cell lysis, but the quality, number and distribution of different antigen presenting cells is of equal importance. Vididencel is an allogeneic leukemia-derived dendritic cell vaccine used in AML patients in first complete remission (CR1/CRi), but with MRD positivity, to mount an immune response through intradermal vaccination (ADVANCE-II, Clintrials.gov: NCT03697707). Intradermal vaccination is known to induce a specific immune response, by recruitment of skin and blood derived dendritic cells, which process antigen and present them to the immune system (Zuo et al., 2021). In this study circulating dendritic cells and monocytes have been investigated before and during vaccination to investigate the effect of vididencel treatment on the composition of antigen presenting cells in relation to clinical responses. Methods. A total of 20 evaluable AML patients, in CR1/CRi, with measurable residual disease (MRD +) and ineligible for HSCT at inclusion, received 4 biweekly doses of vididencel, followed by 2 booster doses at week 14 and 18. Peripheral blood mononuclear cells were taken at baseline, week 6, 11, 14, 18, 20 and 32, ficoll isolated and cryopreserved in liquid nitrogen. Analysis of immune cell composition was performed by spectral flow cytometry, using a single 40-marker panel. Immune subset analysis of dendritic (live, single, CD45+, Lin-(CD3, 56, 19), CD16-, CD15-) cells was performed and correlated to clinical response, relapse free and overall survival, by high dimensional analysis using FlowSOM. Results. Dendritic cell subsets increased upon vaccination, with levels before and during vaccination being the highest in patients remaining in CR. Gating of the dendritic cell subsets by spectral flow cytometry analyses could be hampered by presence of leukemic blasts in the peripheral blood, expressing for example CD123, a marker required for gating of pDCs. Using gating on the lineage negative, CD15-, CD16- and HLA-DR+ cells, a deep immunophenotyping was performed on dendritic cells in peripheral blood. Baseline analysis showed a significantly higher frequency of CD45RA+, HLA-DR+ cells in patients who remain in CR. Using tSNE and UMAP plots generated to analyse changes in the Lin-, CD14-, CD15-, CD16- and CD11b-, HLA-DR+ population; changes and increases in several subsets were observed, most notably in subsets with cDC1, cDC2 and pDC characteristics, like expression of CD141, CLEC9A, CD1c and CD123. Higher frequencies at baseline of dendritic cells (cDC1 and cDC2) correlated positively with both relapse-free and overall survival. In patients remaining in CR, vaccination further increased or maintained dendritic cell subset frequencies, of which classical cDC1 and cDC2 correlated with longer overall survival (p&amp;lt;0.05). Conclusion: Patients who remain in CR after vididencel treatment had highest baseline levels of HLA-DR+ CD45RA+ cells, which could be myeloid cells able to differentiate into dendritic cell subsets, as in general CD45RA+ is lost during maturation from precursor to matured dendritic cells. Vaccination might improve and induce maturation of dendritic cell subsets, such as cDC1, cDC2 and pDC, which enhance antigen capturing, processing and presentation to tumor-reactive T cells, ultimately leading to improved survival.

AML Treatment By the AXL Inhibitor Bemcentinib in Combination with Cytarabine Shows Clinical Efficacy Related to TNFα and Cytotoxic Immune Cells: A Single-Cell Translational Study from the BGBC003 Trial

Acute myeloid leukemia can be treated by intensive chemotherapy. However, this treatment is unsuitable for a subset of patients because of age and/or co-morbidities. Such patients benefit from hypomethylating agents and venetoclax but frequently develop resistance to this treatment regimen. This calls for the development of novel drugs that can prolong survival in AML patients unfit for intensive chemotherapy. The receptor tyrosine kinase AXL is a negative prognostic factor in AML. Its activation promotes survival, chemoresistance and proliferation of AML blasts. In addition, it is also present on innate immune cells and contributes to the formation of immunosuppressive environments. Together, this makes AXL a promising target for the treatment of AML. Bemcentinib is a first-in-class, orally available, selective inhibitor of AXL. Here, we report a single-cell translational sub-study in a sub-cohort of patients unfit for intensive chemotherapy (B2+B5) from the phase Ib/II clinical trial BGBC003 (NCT02488408). This sub-cohort was treated by a combination of bemcentinib and low-dose cytarabine (LDAC). To gain first insights into the mechanisms underlying treatment response, we conducted a translational study evaluating 13 participating patients. Among 32 patients evaluated for efficacy, 8 patients responded to their treatment (objective response rate: 25%). Among patients who received previous therapy ( i.e. relapsed/refractory patients), objective response rate was 18.5% (5/27). The median overall survival was 8.0 months, with a notable survival benefit (median overall survival of 24.8 months) in responders. Relapsed/refractory patients exhibited a median overall survival of 7.8 months. These data highlight that bemcentinib-LDAC benefits a considerable subset of AML patients unfit for intensive chemotherapy. In order to gain mechanistic insights into the efficacy of bemcentinib-LDAC, we profiled cells from bone marrows of 13 participating patients (6 responders, 7 non-responders, according to best response) using single-cell transcriptomics and multi-omics (CITE-seq). Cell type annotation highlighted various immune cell populations next to AML blasts. Successful treatment was associated with stronger TNFα signaling in blasts before treatment. A tight link between TNFα and AXL could subsequently be established in vitro as several AML cell lines up-regulated expression of AXL upon exposure to TNFα. Inhibiting AXL in these cell lines using bemcentinib increased expression of TNFα. This indicates a potential negative feedback loop between these two players. Furthermore, cytotoxic immune cells (CD8 + effector T cells, γδ T cells and natural killer cells) from responders displayed evidence for increased pro-inflammatory signaling upon bemcentinib-LDAC treatment. This included TNFα signaling programs next to IFNα and IFNγ signaling programs. Thus, our data indicate that bemcentinib-LDAC promotes the activity of such cytotoxic cells. In line with this, we observed increasing crosstalk between immune cells upon bemcentinib-LDAC treatment in responders. Importantly, cells from non-responders generally exhibited the opposite trend, highlighting the aforementioned cell types and pathways as factors that distinguish responders from non-responders. Together, our results indicate a potential role of TNFα and cytotoxic immune cells in the successful application of bemcentinib-LDAC. In conclusion, our findings warrant further clinical development of bemcentinib-LDAC for AML patients unfit for intensive chemotherapy. Additionally, extended research on TNFα and cytotoxic immune cells may lead to a better understanding of what mechanisms cause a treatment response. This may ultimately enable an accurate selection of patients who will benefit from bemcentinib-LDAC.

Abstract 6248: Inhibiting β-catenin in AML by targeting DDX5

Abstract Despite the use of high-dose chemotherapy and HSCT, less than 70% of adult AML patients enter complete remission and most patients relapse in less than two years. These poor outcomes have motivated intensive searches for effective and safe targeted agents. Asp-Glu-Ala-Asp (DEAD)-box protein DDX5 was found to be highly expressed in several solid cancers. DDX5, especially its phosphorylated form at Y593, has been reported to promote β-catenin cytoplasm-to-nucleus shuttling and subsequently facilitate the transcriptional activities of the β-catenin/T-cell factor (TCF) complex leading to enhanced expression of genes that are critical for growth, including MYC and CCND1. Knockdown of DDX5 has also been reported to reduce the growth of AML cells, indicating that DDX5 is a potential therapeutic target in AML. However, it remains unknown whether the growth dependence of AML on DDX5 is mainly mediated by β-catenin signaling. Importantly, drugs that selectively target DDX5 have not been tested in AML. In this study, we first analyzed the TCGA AML dataset and found that DDX5 gene is significantly overexpressed in the AML cohort compared to normal bone marrow samples. High DDX5 expression is associated with worse overall survival in AML patients. Moreover, DDX5 correlates with CTNNB1 which encodes β-catenin, at transcript levels. We further found that DDX5 is constitutively phosphorylated at Y593 and predominantly present in the nucleus of 10 AML cell lines tested. These results indicate the clinical relevance of DDX5 whose functional impact may link to β-catenin signaling in AML. We then determined the therapeutic efficacy of targeting DDX5 in human AML using RX-5902, a quinoxalinyl-piperazine compound. RX-5902 was previously shown to interfere with the interaction between phosphorylated DDX5 and β-catenin, inhibiting β-catenin signaling in some solid tumors. We found that RX-5902 attenuated phosphorylation of DDX5 at Y593, led to accumulation of β-catenin in the cytoplasm and consequently downregulated c-MYC in human AML cells within 24 hours. Treatment with RX-5902 dramatically increased cellular ROS level, induced cleaved-caspase3 mediated apoptosis and eventually inhibited cell growth of most human AML cell lines and primary AML cells in culture. These results mirrored what we observed following DDX5 depletion by shRNAs in AML cell lines. Also, dosing of RX-5902 in vivo significantly suppressed the growth of AML xenotransplants in immunocompromised mice and prolonged survival. Finally, we performed dynamic BH3 profiling and showed that RX-5902 increased apoptotic priming and BCL-2 dependence in AML cells. Therefore, we reasoned that simultaneous targeting DDX5 and BCL-2 may improve the therapeutic efficacy in AML. Indeed, concurrent treatment using RX-5902 and Venetoclax, a BCL-2 inhibitor, synergistically induced apoptosis in AML cells. Collectively, therapeutic targeting of DDX5 may be a novel and effective approach in AML and warrants further study. Citation Format: WEI NI, Swati Garg, Fen Zhu, Ellen L. Weisberg, Basudev Chowdhury, Martin Sattler, Amulya Jakkani, Suiyang Liu, Dana M. Sanchez, Lizi Wu, Richard M. Stone, Matthew S. Davids, James D. Griffin. Inhibiting β-catenin in AML by targeting DDX5 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6248.

The trace that is valuable: serum copper and copper to zinc ratio for survival prediction in younger patients with newly diagnosed acute myeloid leukaemia

PurposeNo data on predicting the survival of AML patients based on the level of trace elements in the serum have been presented to date. The aims of this prospective cohort study were as follows: (i) to evaluate the serum Cu and Zn levels in people from Northeast China, (ii) to assess the association between the serum Cu level (SCL) and Cu to Zn ratio (SCZR) and clinical and nutrition data, and (iii) to investigate the predictive values of the SCL and SCZR in newly diagnosed de novo AML patients.MethodsA total of 105 newly diagnosed AML patients and 82 healthy controls were recruited. The serum Cu and Zn levels were determined by inductively coupled plasma spectrometry. The associations of SCL and SCZR with the survival of these AML patients were assessed by Cox proportional hazards models.ResultsBoth SCL and SCZR were positively related to the blast percentage of bone marrow and C-reactive protein, negatively related to albumin level and CEBPA double mutation and were significantly associated with worse overall survival and disease-free survival. Meanwhile, patients with higher SCL had worse CTCAE levels, and patients with higher SCZR showed less complete remission during the first course of induction chemotherapy. Moreover, higher SCZR was positively associated with ELN risk stratification, and was negatively associated with haemoglobin level and prognostic nutritional index (PNI).ConclusionThe SCL and SCZR are associated with long-term survival in patients with newly diagnosed AML undergoing intensive induction and may serve as important predictive biomarkers.

The Exportin, XPO2, Is a Novel Therapeutic Target in Pediatric, Adolescent and Young Adult AML

The active transport of proteins across the nuclear membrane is regulated by nuclear transport receptors termed importins and exportins, depending on their direction of transport. Nuclear transport receptors contribute to cancer development, through abnormal localization of protein cargos. Increased expression of the exportin, exportin-1/chromosome region maintenance 1 (XPO1/CRM1), has been associated with high risk AML and shorter survival times and XPO1 inhibitors show signs of efficacy in AML. While previous data demonstrate a functional role for XPO1 in AML, less is known about the exportin, XPO2, (alias CSE1L). Despite XPO2 RNAi depletion screens demonstrating a higher genetic dependence in leukemia versus solid tumors/non-hematologic malignancies (p = 0.0394), the functional impact of inhibiting XPO2 has never been studied in leukemia. We analyzed a public database of CRISPR/Cas9 depletion screens in 26 AML cell lines. XPO2 ranked in the top 1.4% of essential genes in AML, along with KPNB1 and XPO1 (DepMap 22Q2 Public+Score, Chronos, www.depmap.org). An RNAi depletion screen confirmed these findings, with XPO2 identified within the top 0.2% of essential genes in AML (Achilles+DRIVE+Marcotte, DEMETER2, www.depmap.org). Gene dependency scores indicated a greater reliance on XPO2, compared to XPO1 (p <0.0001). Increased expression of XPO2 mRNA was associated with decreased event free survival (p = 0.0014) and overall survival (p = 0.00091) in pediatric and adolescent/young adult (AYA) AML patients from the TARGET dataset. Analyzing the BeatAML dataset, increased XPO2 mRNA expression was also associated with decreased overall survival in AML patients <40 years, but not in patients >40 years (p <0.0001). We analyzed protein expression databases and identified XPO2 as the highest expressed member of the exportin family in AML cells lines and AML primary patient samples. By immunoblot analysis, increased expression of XPO2 was detected in 7/7 primary AML samples and 8 AML cell lines (HL60, MV4-11, MOML13, NB4, OCI-AML2, OCI-M2, TEX and U937), compared to normal hematopoietic cells. XPO2 mRNA was also increased in 5/5 primary AML samples and 8/8 AML cell lines, compared to normal hematopoietic cells, as measured by qRT-PCR. To confirm the functional importance of XPO2 in AML, we knocked down XPO2 with shRNA in AML cell lines. Genetic silencing of XPO2 decreased growth and viability of OCI-AML2, TEX and NB4 cells. Gambogic acid (GA), a natural product from Garcinia hanburyi, has been identified as a covalent inhibitor of XPO2-mediated nuclear transportation. In OCI-AML2, TEX and NB4 cell lines, gambogic acid decreased growth and viability of AML cell lines at nanomolar concentrations (TEX cells IC50 = 369nM, OCI-AML2 cells IC50 = 433nM, NB4 cells IC50 = 161nM). To uncover the mechanism of how XPO2 mediates cell death in AML we interrogated a publicly available BioID database (PXD007976) and performed gene set enrichment analysis on XPO2 targets using gprofiler. Processes related to ribosome biogenesis were significantly enriched in XPO2 cargo, whereas XPO1 cargo were enriched for processes related to snRNA transcription and transport (p <0.05). In summary, we identified XPO2 as an essential gene for AML. XPO2 is preferentially increased in AML cell lines and primary patient samples compared to normal hematopoietic cells. Increased XPO2 expression is associated with worse clinical outcomes in pediatric and AYA patients with AML. XPO2 may function to enhance ribosomal biogenesis, demonstrating a critical role in leukemia initiation and progression. Thus, this work demonstrates a new dependency of AML on XPO2, as well as highlights a new therapeutic target for this disease.

Loss of FBXO11 Function Establishes a Stem Cell Program in Acute Myeloid Leukemia through Dysregulation of the Mitochondrial Protease LONP1

Despite advances in targeted therapies, survival for AML patients remains poor. Molecular classification systems attempt to stratify patients into risk groups, but 20-40% of patients do not fall under established genomic subgroups (Papaemmanuil et al., N Engl J Med 2016) We analyzed a total of 1,627 unique patient samples from the AML-PMP, TCGA, Beat AML, and TARGET datasets and identified loss-of-function mutations in ubiquitin ligase family genes in 10% of patient samples (n = 163). Among recurrently mutated genes, expression of FBXO11 was the most significantly reduced component of the SKP1-CUL1-Fbox (SCF) E3 ligase complex in AML samples compared to normal CD34+ hematopoietic stem/progenitor cells (HSPC). RNA sequencing (RNA-seq) following knockdown (KD) of FBXO11 by shRNA in cord blood derived CD34+ HSPCs resulted in enrichment of hematopoietic stem (HSC) and leukemia stem cell (LSC) signatures accompanied by a diminishment in hematopoietic progenitor cell signatures. Consistent with enrichment of a quiescent stem cell phenotype, cell cycle analysis showed that FBXO11 KD increased the G0 fraction of the CD34+ population. In liquid culture, FBXO11 KD HSPCs displayed myeloid-biased output. Fbxo11 KD in mouse HSPCs co-operated with AML1-ETO (RUNX1-RUNX1T1) fusion to generate serially transplantable AML in mice. The combination of FBXO11 KD with AML1-ETO and a KRASG12D mutant in human cord blood HSPC also promoted stem cell maintenance and myeloid malignancy in a human xenotransplant model, but AML1-ETO with KRASG12D without FBXO11 KD did not. Mass spectrometry analysis of proteins co-immunoprecipitating with FLAG-FBXO11 in myeloid cells identified the the mitochondria protease and chaperone protein LONP1. LONP1 protein expression remained unchanged with FBXO11 overexpression or CRISPR/Cas9-mediated targeting, suggesting that LONP1 is not targeted for degradation by the SCFFBXO11 complex. FBXO11 has been demonstrated to facilitate ubiquitination (Xu et al., Blood 2021) and neddylation (Abida et al., J Biol Chem 2007) of protein targets. FBXO11 expression resulted in activating K63 ubiquitination as well as neddylation of LONP1, but only ubiquitination inhibition resulted in reversal of an FBXO11-overexpression phenotype. Further, proteasome inhibitors also did not reverse the FBXO11-overexpression phenotype, consistent with FBXO11 mediating an activating ubiqutination of LONP1. FBXO11 or LONP1 KD in cord blood HSPCs promoted myeloid biased output and reduced mitochondria membrane potential, a characteristic of long-term HSCs (Mansell et al., Cell Stem Cell 2021). To validate these findings and direction of the FBXO11-LONP1 relationship, we performed RNA-seq on cord blood HSPCs following lentiviral transductions to combinatorially deplete or overexpress FBXO11 and LONP1. Unsupervised clustering of the samples demonstrated overexpression of LONP1 clustered with controls while knockdown of LONP1 reversed the effect of FBXO11 overexpression. We observed upregulation of a mitochondria respiratory defect gene set with depletion of FBXO11 or LONP1, indicating improper assembly of the electron transport chain, a reported consequence of impaired LONP1 function (Ghosh et al., Oncogene 2019). We also determined enrichment in several HSC and LSC genesets with KD of either FBXO11 or LONP1, while overexpression of FBXO11 downregulated these genesets. In contrast to normal HSCs which utilize glycolysis for energy production (Simsek et al., Cell Stem Cell 2010), LSCs from AML patients, are dependent on oxidative phosphorylation for energy metabolism but remain in a quiescent state (Jones et al., Cancer Cell 2018). Interestingly, FBXO11 depletion resulted in downregulated glycolysis genesets and upregulated quiescent stem cell signatures. Taken together, these findings suggest that FBXO11 depletion dysregulates LONP1 function to establish a mitochondrial state reminiscent of quiescent HSC. In summary, we demonstrate that the ubiquitin ligase family is frequently mutated in AML. We show that gene expression of a component of the SCF E3 ubiquitin ligase, FBXO11, is reduced in AML, and that FBXO11 depletion cooperates with other driver mutations to initate AML in mouse and human xenotransplant models. Finally, we establish a novel link between FBXO11 and LONP1, and provide evidence that loss of FBXO11 regulation of LONP1 promotes a myeloid-biased HSC/LSC-like state.

Dermatologic adverse events associated with IDH inhibitors ivosidenib and enasidenib for the treatment of acute myeloid leukemia

Two novel inhibitors of isocitrate dehydrogenase (IDHi), ivosidenib and enasidenib, significantly improve survival for AML patients with an IDH1 or IDH2 mutation, respectively; however, rash has been reported as a toxicity of IDHi. The objective of our study is to determine the incidence, grade, clinical, and histopathologic features of dermatologic adverse events (DAEs) secondary to IDHi. This study is a retrospective analysis of 169 patients who were treated with either ivosidenib or enasidenib as single agent or in combination with induction chemotherapy at Memorial Sloan Kettering Cancer Center from January 1, 2013 to April 1, 2021. DAEs thought to be possibly, probably, or definitely related to IDHi occurred in 55 of 169 patients [0.32, 95 % CI: 0.25 – 0.40]. Of a total 81 DAEs observed, the most common DAE types were inflammatory dermatoses (27 %); cutaneous vascular manifestations (8%); cutaneous infections (7%); and pruritus (2%). Notably, 50% of infections and 15.5% of rashes were high grade. Knowledge of these findings is critical to optimize the treatment and quality of life of patients with AML on IDHi.

Development and Validation of an Individualized Metabolism-Related Prognostic Model for Adult Acute Myeloid Leukemia Patients

ObjectivesAcute myeloid leukemia (AML) is a highly heterogeneous hematologic malignancy with widely variable prognosis. For this reason, a more tailored-stratified approach for prognosis is urgently needed to improve the treatment success rates of AML patients.MethodsIn the investigation of metabolic pattern in AML patients, we developed a metabolism-related prognostic model, which was consisted of metabolism-related gene pairs (MRGPs) identified by pairwise comparison. Furthermore, we analyzed the predictive ability and clinical significance of the prognostic model.ResultsGiven the significant differences in metabolic pathways between AML patients and healthy donors, we proposed a metabolism-related prognostic signature index (MRPSI) consisting of three MRGPs, which were remarkedly related with the overall survival of AML patients in the training set. The association of MRPSI with prognosis was also validated in two other independent cohorts, suggesting that high MRPSI score can identify patients with poor prognosis. The MRPSI and age were confirmed to be independent prognostic factors via multivariate Cox regression analysis. Furthermore, we combined MRPSI with age and constructed a composite metabolism-clinical prognostic model index (MCPMI), which demonstrated better prognostic accuracy in all cohorts. Stratification analysis and multivariate Cox regression analysis revealed that the MCPMI was an independent prognostic factor. By estimating the sensitivity of anti-cancer drugs in different AML patients, we selected five drugs that were more sensitive to patients in MCPMI-high group than those in MCPMI-low group.ConclusionOur study provided an individualized metabolism-related prognostic model that identified high-risk patients and revealed new potential therapeutic drugs for AML patients with poor prognosis.

Revealing key lncRNAs in cytogenetically normal acute myeloid leukemia by reconstruction of the lncRNA\u2013miRNA\u2013mRNA network

Cytogenetically normal acute myeloid leukemia (CN-AML) is a heterogeneous disease with different prognoses. Researches on prognostic biomarkers and therapy targets of CN-AML are still ongoing. Instead of protein-coding genes, more and more researches were focused on the non-coding RNAs especially long non-coding RNAs (lncRNAs) which may play an important role in the development of AML. Although a large number of lncRNAs have been found, our knowledge of their functions and pathological process is still in its infancy. The purpose of this research is to identify the key lncRNAs and explore their functions in CN-AML by reconstructing the lncRNA–miRNA–mRNA network based on the competitive endogenous RNA (ceRNA) theory. We reconstructed a global triple network based on the ceRNA theory using the data from National Center for Biotechnology Information Gene Expression Omnibus and published literature. According to the topological algorithm, we identified the key lncRNAs which had both the higher node degrees and the higher numbers of lncRNA–miRNA pairs and total pairs in the ceRNA network. Meanwhile, Gene Ontology (GO) and pathway analysis were performed using databases such as DAVID, KOBAS and Cytoscape plug-in ClueGO respectively. The lncRNA–miRNA–mRNA network was composed of 90 lncRNAs,33mRNAs,26 miRNAs and 259 edges in the lncRNA upregulated group, and 18 lncRNAs,11 mRNAs,6 miRNAs and 45 edges in the lncRNA downregulated group. The functional assay showed that 53 pathways and 108 GO terms were enriched. Three lncRNAs (XIST, TUG1, GABPB1-AS1) could possibly be selected as key lncRNAs which may play an important role in the development of CN-AML. Particularly, GABPB1-AS1 was highly expressed in CN-AML by both bioinformatic analysis and experimental verification in AML cell line (THP-1) with quantitative real‐time polymerase chain reaction. In addition, GABPB1-AS1 was also negatively correlated with overall survival of AML patients. The lncRNA–miRNA–mRNA network revealed key lncRNAs and their functions in CN-AML. Particularly, lncRNA GABPB1-AS1 was firstly proposed in AML. We believe that GABPB1-AS1 is expected to become a candidate prognostic biomarker or a potential therapeutic target.

Survival prediction in acute myeloid leukemia using gene expression profiling

BackgroundAcute myeloid leukemia (AML) is a genetically heterogeneous blood disorder. AML patients are associated with a relatively poor overall survival. The objective of this study was to establish a machine learning model to accurately perform the prognosis prediction in AML patients.MethodsWe first screened for prognosis-related genes using Kaplan–Meier survival analysis in The Cancer Genome Atlas dataset and validated the results in the Oregon Health & Science University dataset. With a random forest model, we built a prognostic risk score using patient’s age, TP53 mutation, ELN classification and normalized 197 gene expression as predictor variable. Gene set enrichment analysis was implemented to determine the dysregulated gene sets between the high-risk and low-risk groups. Similarity Network Fusion (SNF)-based integrative clustering was performed to identify subgroups of AML patients with different clinical features.ResultsThe random forest model was deemed the best model (area under curve value, 0.75). The random forest-derived risk score exhibited significant association with shorter overall survival in AML patients. The gene sets of pantothenate and coa biosynthesis, glycerolipid metabolism, biosynthesis of unsaturated fatty acids were significantly enriched in phenotype high risk score. SNF-based integrative clustering indicated three distinct subsets of AML patients in the TCGA cohort. The cluster3 AML patients were characterized by older age, higher risk score, more frequent TP53 mutations, higher cytogenetics risk, shorter overall survival.ConclusionsThe random forest-based risk score offers an effective method to perform prognosis prediction for AML patients.

Construction of a Pyroptosis-Related Signature for Prognostic Prediction and Characterization of Immune Microenvironment in Acute Myelogenous Leukemia.

BackgroundAcute myelogenous leukemia (AML) is a common and fatal disease in hematology with frequent relapses and a poor prognosis. Pyroptosis, a programmed cell death mediated by inflammasomes, has been shown to be associated with leukemia recently. However, the role of pyroptosis for diagnosis and prognosis in AML remained less understood.MethodsWe downloaded three public datasets and constructed a signature of TCGA cohort using the least absolute shrinkage and selection operator (LASSO) Cox regression model to predict the overall survival of AML patients. Samples from the GEO database were treated as a validation cohort. Gone through LASSO-Cox regression analysis, an 8-PRG-related signature was developed. Used the median score from the signature, we classified patients in two subgroups. Subsequently, we employed univariate COX, multivariate Cox regression, ROC analysis and constructed a nomogram, Finally, differential analysis, GO and KEGG functional analysis, ESTIMATE algorithm and CIBERSORT algorithm were used to explore the difference between two groups.ResultsThe expression levels of 90.9% pyroptosis-related genes (PRGs) had significant difference compared AML with normal tissues. The results of univariate COX regression analysis demonstrated 10 differentially expressed genes (DEGs) were associated with patients’ OS (p < 0.05). Then, we found OS of patients in the low-risk group was more likely to be lengthened compared with their high-risk counterparts (P < 0.05 both in the TCGA and GEO cohort). After controlling clinical factors, the risk score could still remain an independent predictive element (HR > 1, P < 0.001) of OS in multivariate Cox regression analysis. Furthermore, a nomogram with prognostic value for AML was thus established. Time-dependent ROC analysis proved the predictive power of the signature. Functional analysis suggested that DEGs were mainly concentrated in immune-related pathways, such as humoral immune response and T cell proliferation. TME scores and risk scores were strongly correlated and immune status differed between the risk subgroups.ConclusionA novel PRG-related signature was established to forecast the prognosis in AML, and pyroptosis may be a potential therapeutic target for AML.

BSG (CD147) Serum Level and Genetic Variants Are Associated with Overall Survival in Acute Myeloid Leukaemia

Basigin (BSG, CD147) is a multifunctional protein involved in cancer cell survival, mostly by controlling lactate transport through its interaction with monocarboxylate transporters (MCTs) such as MCT1. Previous studies have found that single nucleotide polymorphisms (SNPs) in the gene coding for BSG and MCT1, as well as levels of the soluble form of BSG (sBSG), are potential biomarkers in various diseases. The goal of this study was to confirm BSG and MCT1 RNA overexpression in AML cell lines, as well as to analyse soluble BSG levels and selected BSG/MCT1 genetic variants as potential biomarkers in AML patients. We found that BSG and MCT1 were overexpressed in most AML cell lines. Soluble BSG was increased in AML patients compared to healthy controls, and correlated with various clinical parameters. High soluble BSG was associated with worse overall survival, higher bone marrow blast percentage, and higher white blood cell count. BSG SNPs rs4919859 and rs4682, as well as MCT1 SNP rs1049434, were also associated with overall survival of AML patients. In conclusion, this study confirms the importance of BSG/MCT1 in AML, and suggests that soluble BSG and BSG/MCT1 genetic variants may act as potential AML biomarkers.

TIFAB accelerates MLL-AF9−Induced acute myeloid leukemia through upregulation of HOXA9

SummaryWe previously showed stabilization of NIK−induced activation of NF-κB non-canonical signaling suppresses MLL-AF9−induced AML. In the current study, we demonstrate that deletion of NF-κB non-canonical RelB prevents the inhibitory effect of NIK stabilization in MLL-AF9 AML. Mechanistically, RelB suppresses its direct target, TIFAB, which is upregulated in human AML and correlates negatively with the survival of AML patients. Forced expression of TIFAB reverses NIK−induced impaired AML development through downregulation of RelB and upregulation of HOXA9. Consistent with upregulation of HOXA9, gene set enrichment analysis shows that forced expression of TIFAB blocks myeloid cell development, upregulates leukemia stem cell signature and induces similar gene expression patterns to those of HOXA9-MEIS1 and HOXA9-NUP98, and upregulates oxidative phosphorylation. Accordingly, forced expression of HOXA9 also largely releases the inhibitory impact of NIK stabilization via downregulation of RelB and upregulation of RelA. Our data suggest that NIK/RelB suppresses MLL-AF9−induced AML mainly through downregulation of TIFAB/HOXA9.

MiR-106b-25 Promotes Chemoresistance in Acute Myeloid Leukemia Via Abolishing Multiple Apoptotic Pathways

Introduction: Chemoresistance and disease relapse remain the main obstacles responsible for treatment failure in leukemia. MicroRNAs (miRNAs) play essential roles in various physiological and pathological processes, including cell proliferation, differentiation, metabolism, and cancer development. The miR-106b-25 cluster consists of three miRNAs: miR-106b, miR-93 and miR-25. We have previously reported that miR-106b-25 was associated with chemoresistance by negatively regulated EP300 in breast cancer, but its role in hematological malignancies has not yet been elucidated. Here, we aim to clarify the biological role and underlying mechanisms of miR-106b-25 on drug resistance in leukemia.Methods: To see whether the miR-106b-25 was associated with the poor prognosis of AML patients, enriched LSCs (CD34 + cells) were isolated from the bone marrow of 18 newly diagnosed AML patients, the expression of miR-106b, miR-93, and miR-25 were examined, respectively. The expression levels of miR-106b, miR-93 and miR-25 were further determined in the doxorubicin-resistant leukemia cell line K562/A02 and HL60/ADR, compared with their parental cell lines. In addition, K562 cells were transduced with lentiviral vectors carrying miR-106b-25, and cell proliferation, drug resistance, colony-forming assay, apoptosis assays were performed to explore the function of miR-106b-25 overexpression in leukemia cells in vitro. To investigate the role of miR-106b-25 on tumor growth and overall survival after drug treatment, we performed xenotransplantation in nude mice using miR-106b-25 overexpressed K562 cells. To further clarify the function of each microRNA function in this cluster, K562 cells were also transduced with lentiviral vectors carrying individual miR-25, miR-93, or miR-106b separately. Cell proliferation, colony forming assay and cell apoptosis assay were also carried out subsequently. Simultaneously, RNA-sequencing was performed to reveal the underlying mechanisms of miR-106b-25 in the chemoresistance of myeloid leukemia. To experimentally confirm the direct target of the miR-106b-25 cluster in AMLs, we further performed a dual-luciferase reporter assay.Results: Upregulated miR-106b, miR-93 and miR-25 expression in enriched LSCs were significantly associated with shortened overall survival of AML patients. We also found miR-106b, miR-93 and miR-25 were significantly upregulated in drug-resistant leukemia cell lines compared with its parental cell lines. Overexpression of miR-106b-25 cluster promoted cell proliferation, led to resistance of K562 cells to doxorubicin, imatinib and ABT-737 (BCL-2 inhibitor) in liquid culture and drug-resistant colony-forming assays. Overexpression of miR-93 or miR-106b accelerated cell growth, and all the three miRNAs can promote drug-resistant colony-forming and inhibit cell apoptosis. RNA-sequencing (RNA-Seq) data revealed that multiple critical genes related to apoptotic pathways were downregulated after overexpressing miR-25, miR-93, miR-106b as well as the whole cluster, such as TP73, BAX, BAK1, Caspase-7, CDKN1A and BTG2. RT-qPCR confirmed that these genes are reduced with or without ABT-737 treatment. Luciferase assay further identified TP73 was a direct target of miR-93 and miR106b, BAK1 was a direct target of miR-25, and CASPASE-7 was a direct target of all these three miRNAs.Conclusions: In summary, we made the novel observation that miR-106-25 is associated with AML drug-resisitance and disease prognosis and identified TP73, BAK1 caspase-7 as a novel direct target of this cluster. Further studies revealed that the biological effects of miR-106b-25 cluster on leukemic cell proliferation, chemoresistance and apoptosis were mediated through regulation of apoptotic pathway. These findings indicate a promising diagnostic biomarker and a potential target therapeutic strategy for AML patients. DisclosuresNo relevant conflicts of interest to declare.