Overall Survival In AML Patients Research Articles

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<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.

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 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.

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.

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.

Overexpression of BRD4 Is an Adverse Prognostic Factor in Acute Myeloid Leukemia

Background:Bromodomain containing protein 4 (BRD4), a member of the bromodomain and extra terminal domain (BET) protein family, has been implicated in the pathogenesis of a number of hematological and solid tumors. BRD4 is an epigenetic reader that plays a central role in transcriptional regulation, cellular growth control, and cell cycle progression. As a consequence, BET inhibitors elicit anticancer activity in numerous malignant contexts. While deregulation of BRD4 activity has been shown to be essential for the maintenance of AML, its expression profile and prognostic value in AML have not been investigated. Additionally, how BRD4 is regulated in cancer cells remains largely unknown.Methodology: We therefore evaluated the level of BRD4 expression using Reverse Phase Protein Array (RPPA) technology in marrow and blood samples from 511 newly diagnosed AML patients, and determined a correlation between BRD4 expression and clinical characteristics and overall survival in AML patients. We also correlated expression of BRD4 with 232 other proteins (encompassing signal transduction, metabolism, apoptosis and cellular proliferation pathways) that were included in RPPA. Potential role of molecular chaperone heat shock protein 90 (Hsp90) in regulating BRD4 protein stability was also investigated.Results: BRD4 expression was similar in peripheral blood and bone marrow and expression was found to be higher at diagnosis than at relapse (P = .035). Compared with normal bone marrow derived CD34+ cells, BRD4 expression in AML was higher in 28% of patients and that higher BRD4 levels significantly predicted shorter overall survival (P = .002). There was no significant difference in BRD4 expression on the basis of age, sex, or performance status, antecedent malignancies, and previous chemotherapy and/or radiotherapy. BRD4 expression levels were not strongly correlated with French-American-British classification (FAB), although levels were slightly higher in M5 subtype. BRD4 levels were found to be significantly higher in patients with NPM1 mutation (P = .003) or FLT3-ITD (P = .057) or between those with both FLT3-D835F and NPM1 mutation (P = .01). BRD4 levels were not associated with the attainment of complete remission (CR), although rates were somewhat lower for those with high. However, patients with high levels of BRD4 had significantly inferior remission duration (25.9 vs 50 weeks; P = 0.03) compared with those with normal levels of BRD4. The overall survival (OS) of patients with higher levels of BRD4 was significantly inferior compared with those with normal levels of BRD4 (28.3 vs 55.3 weeks; P = .002). Patients with unfavorable cytogenetics and above-normal levels of BRD4 had inferior survival compared with those with normal levels of BRD4 (22.4 vs 38.9 weeks; P = .0043; Figure 3C). BRD4 expression was found to be strongly correlated (R > 0.2 and P < 0.0001 or 0.00001) either positively (n = 48) or negatively (n = 14) with proteins known to be important in AML. BRD4 expression was positively correlated with various cell proliferation-related proteins (Hsp90, pSTAT5, nucleolin and mTOR), anti-apoptotic protein (Bcl-2), oncoproteins (FLI1, CREB and ELK1), proteins important for stem cell functions (Ash2L and LSD1), hematopoietic transcription factors (ERG and PU.1/Spi1), and Smad proteins, signal transducers for the members of the transforming growth factor-β (TGF-β) superfamily. The stability, activity and function of several mutated, chimeric, and overexpressed signaling proteins crucial for oncogenesis are often maintained by Hsp90. Our study also demonstrates that BRD4 stability is dependent upon Hsp90 activity and pharmacologic inhibition of Hsp90 promoted ubiquitination followed by proteasome-dependent degradation of BRD4 protein.Conclusions: These findings suggest BRD4 as a prognostic biomarker for unfavorable outcomes and supports the clinical rational for using BRD4 inhibitors in AML. Future studies could identify mechanistic insights into BRD4-associated proteins as biomarkers for response to BET inhibition. DisclosuresKarnad:ADC Therapeutics: Research Funding. Weitman:Tolero Pharmaceuticals, Inc.: Employment. Iyer:Takeda: Research Funding; Genentech: Research Funding.

Predictors of Response and Survival in 206 AML Patients Treated with Guadecitabine in a Phase 2 Study

Background: Guadecitabine is a next generation hypomethylating agent (HMA) resistant to degradation by cytidine deaminase which results in prolonged in vivo exposure to the active metabolite decitabine. We conducted a prospective phase 2 study testing different schedules of guadecitabine in 206 AML patients. We present here the results of multiple logistic regression, and Cox regression analyses of predictors of composite Complete Response or CRc (CR+CRp+CRi) and overall survival (OS).Methods: Multiple logistic regression analysis of response (CRc), and Cox regression analysis of OS were conducted with inclusion of the following baseline variables: disease state (relapsed/refractory (r/r) vs. treatment naïve (TN) AML); guadecitabine schedule (10-day vs. 5-day); age (<75 vs. ≥75); ECOG PS (0-1 vs. ≥2); Cytogenetics (others vs. poor risk); baseline BM blasts (≤40% vs. >40%); baseline Peripheral blood (PB) blasts (≤30% vs. >30%); baseline WBCs count (<20,000/µL vs. ≥20,000/µL); Flt-3 ITD, NPM, and TP53 mutations (each present vs. not detected). Cutoff values for blasts % for BM and PB were chosen based on the median or mean values respectively, and cutoff value for WBCs count was chosen as a common cutoff used for proliferative AML. Backward elimination method with alpha =0.05 was used to reach the final models.Results: We treated 206 AML patients (103 patients each for TN or r/r AML); 101 with 5-day schedule and 105 with 10-day schedule. There were 91 patients (44%) ≥75 y; 53 (26%) with ECOG PS≥2; 85 (41%) with poor risk cytogenetics; 99 (48%) with baseline BM blasts >40%; 65 (32%) with baseline PB blasts >30%; 20 (9.7%) with WBCs ≥20,000/µL. Flt-3, NPM, and TP53 mutations were present in 8%, 9%, and 4% of patients respectively. The final logistic regression model indicated that patients with ECOG PS 0-1 and those with baseline PB blasts ≤ 30% have two-fold higher odds of response than those with ECOG PS ≥2 and PB blasts >30% (Odds ratio 2.18; and 2.03 respectively; p<0.05 for both). Patients with TN AML had five-fold higher odds of response to guadecitabine than r/r AML (Odds ratio of response for r/r AML 0.22; p <0.0001). The final Cox regression model showed that both ECOG PS 0-1 and PB blasts ≤30% retained their significance for OS (HR 0.69 with p=0.03; and 0.61 with p= 0.004 respectively). However disease state (r/r AML vs TN AML) lost significance for OS while cytogenetics risk level (others vs poor risk) became significant with a HR for OS of 0.68, p=0.016.Summary/Conclusions: In a prospective series of AML patients treated with guadecitabine in a phase 2 study, better ECOG PS 0-1 and lower baseline PB blasts ≤30% were associated with a significantly higher likelihood of response and a longer OS. Patients with TN AML had significantly higher likelihood of response than those with r/r AML but this was not a significant factor for OS when other factors are present in the model such as ECOG PS, cytogenetics risk and PB blasts. On the other hand, the presence of poor risk cytogenetics did not alter the likelihood of response to guadecitabine but still had shorter survival compared to patients with other cytogenetics risk levels. Other variables such as age, baseline BM blasts %, and baseline WBCs count did not significantly impact response or OS in AML patients treated with guadecitabine when all other factors are present in the models. The analysis of genetic mutations was limited by the small number of patients where these mutations were present. The analysis of the treatment schedule is limited by the different effect of the schedule on r/r AML compared to TN AML where the 10-day schedule did better than the 5-day schedule in r/r AML but not in TN AML patients. DisclosuresKantarjian:Novartis: Research Funding; ARIAD: Research Funding; Delta-Fly Pharma: Research Funding; Amgen: Research Funding; Bristol-Meyers Squibb: Research Funding; Pfizer: Research Funding. Roboz:Cellectis: Research Funding; AbbVie, Agios, Amgen, Amphivena, Array Biopharma Inc., Astex, AstraZeneca, Celator, Celgene, Clovis Oncology, CTI BioPharma, Genoptix, Immune Pharmaceuticals, Janssen Pharmaceuticals, Juno, MedImmune, MEI Pharma, Novartis, Onconova, Pfizer, Roche Pharmace: Consultancy. Kropf:Takeda: Honoraria; Celgene: Honoraria; Astex: Consultancy. Jabbour:Bristol-Myers Squibb: Consultancy. Yee:Oncoethix: Research Funding; Celgene Canada: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Canada: Honoraria; Astex: Research Funding; Karyopharm: Research Funding. O'Connell:Astex Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees; Otsuka: Membership on an entity's Board of Directors or advisory committees. Stock:Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees. Podoltsev:Ariad: Consultancy; Incyte: Consultancy; CTI biopharma/Baxalta: Consultancy; Alexion: Consultancy. Hao:Astex Pharmaceuticals, Inc.: Employment. Ahanonu:Astex Pharmaceuticals, Inc: Employment. Azab:Astex Pharmaceuticals, Inc.: Employment. Issa:Astex Pharmaceuticals, Inc.: Consultancy, Research Funding; Teva: Consultancy, Research Funding; GSK: Consultancy.

Efficacy and Relapse Prediction Model of Allogeneic Peripheral Blood Stem Cell Transplantation in Adult Acute Leukemia

To observe the clinical efficacy of allogeneic peripheral blood stem cell transplantation（allo-HSCT） on the treatment of adult acute leukemia patients, moreover, to establish and evaluate a Logistic model to predict the risk of relapse in adult acute leukemia patients after allo-HSCT. The clinical data of 145 adult acute leukemia patients treated by peripheral blood stem cell transplantation in the First Affiliated Hospital of Xi'an Jiaotong University from January 2010 to December 2019 was enrolled and analyzed retrospectively. Complications and survival of patients were observed. The relationship between patients' age, diagnosis, leukocyte count at onset, risk stratification, time of diagnosis to transplantation, HCT-CI, minimal residual disease pre-transplantation, donor-recipient sex relationship, HLA match degree, prophylaxis of graft versus host disease(GVHD), donor age, number of transfused mononuclear cells, CD34 positive cells, engraftment time, acute and chronic GVHD, CMV, EBV infection, and hemorrhagic cystitis and recurrence after transplantation were analyzed by logistic regression. Relapse prediction model was established and evaluated according to the results. Among 145 acute leukemia patients, 81 with acute myeloid leukemia, 64 with acute lymphocytic leukemia, 18 with EBV infection, 2 with post-transplant lymphoproliferative disorder(PTLD), 85 with CMV, 26 with hemorrhagic cystitis, 65 patients developed acute GVHD, 51 patients developed chronic GVHD and 45 patients relapsed. The overall survival （OS） rates in one and three years were 86.4% and 61.8%, and the progress-free survival （PFS） rates in one and three years were 67.5% and 62.4%, respectively. There were significant differences in OS and PFS between relapsed and non-relapsed patients, as well as AML and ALL patients. Univariate analysis revealed that patient's age, risk stratification, time to transplantation, HCT-CI index, ATG based GVHD prophylaxis, minimal residual disease pre-transplantation, GVHD prophylaxis, and acute and chronic GVHD were associated with the relapse of disease, multivariate logistic regression analysis showed that pre-transplantation minimal residual disease showed positively correlation with relapse of the disease, while chronic GVHD showed negatively correlation. The relapse rate of adult acute leukemia patients treated with allo-HSCT in our hospital is 31.0%, and OS of AML patients is better than ALL patients'. OS of relapsed patients is significantly lower than non-relapsed patients'. Pre-transplantation minimal residual disease is a risk factor of relapse. The risk of relapse is reduced in patients with chronic GVHD.

Integrative study of EZH2 mutational status, copy number, protein expression and H3K27 trimethylation in AML/MDS patients

BackgroundMutations in the EZH2 gene are recurrently found in patients with myeloid neoplasms and are associated with a poor prognosis. We aimed to characterize genetic and epigenetic alterations of EZH2 in 58 patients (51 with acute myeloid leukemia and 7 with myelodysplastic or myeloproliferative neoplasms) by integrating data on EZH2 mutational status, co-occurring mutations, and EZH2 copy number status with EZH2 protein expression, histone H3K27 trimethylation, and EZH2 promoter methylation.ResultsEZH2 was mutated in 6/51 acute myeloid leukemia patients (12%) and 7/7 patients with other myeloid neoplasms. EZH2 mutations were not overrepresented in patients with chromosome 7q deletions or losses. In acute myeloid leukemia patients, EZH2 mutations frequently co-occurred with CEBPA (67%), ASXL1 (50%), TET2 and RAD21 mutations (33% each). In EZH2-mutated patients with myelodysplastic or myeloproliferative neoplasms, the most common co-mutations were in ASXL1 (100%), NRAS, RUNX1, and STAG2 (29% each). EZH2 mutations were associated with a significant decrease in EZH2 expression (p = 0.0002), which was similar in patients with chromosome 7 aberrations and patients with intact chromosome 7. An association between EZH2 protein expression and H3K27 trimethylation was observed in EZH2-unmutated patients (R2 = 0.2, p = 0.01). The monoallelic state of EZH2 was not associated with EZH2 promoter hypermethylation. In multivariable analyses, EZH2 mutations were associated with a trend towards an increased risk of death (hazard ratio 2.51 [95% confidence interval 0.87–7.25], p = 0.09); similarly, low EZH2 expression was associated with elevated risk (hazard ratio 2.54 [95% confidence interval 1.07–6.04], p = 0.04).ConclusionsPerturbations of EZH2 activity in AML/MDS occur on different, genetic and non-genetic levels. Both low EZH2 protein expression and, by trend, EZH2 gene mutations predicted inferior overall survival of AML patients receiving standard chemotherapy.

TERT rs2853669 as a predictor for overall survival in patients with acute myeloid leukaemia.

Introductionthe aim of the study was to investigate the contribution of TERT rs2736100 and rs2853669 gene polymorphisms in defining the genetic predisposition to acute myeloid leukaemia (AML), their association with different prognostic markers, and their impact on survival, outcome, and the prognosis of affected patients. Also, we investigated the association of TERT SNPs in AML in the presence or absence of DNMT3A (R882), NPM1, and FLT3 mutations.Material and methodsA total of 509 participants were enrolled in our study, consisting of 146 AML patients and 363 healthy participants, with no history of malignancy. TERT rs2736100 and rs2853669 polymorphisms were genotyped by using TaqMan SNP genotyping assay FLT3 (ITD, D835), DNMT3A (R882), and NPM1 c.863_864insTCTG (type A) mutations were analised in each AML case.ResultsTERT rs2736100 and rs2853669 were not associated with AML risk in the codominant, dominant, recessive, or allelic models. Multivariate Cox regression showed that TERT rs2853669 was a significant predictor for overall survival in AML patients. After adjusting for age, gender, cytogenetic risk group, ECOG status, FLT3, DNMT3A, NPM1 mutation, AML subtype, and treatment, the estimated adjusted hazard ratio (HR adjusted = 1.54, 95% CI: 1.01–2.35) showed that the TERT rs2853669 variant genotype had a negative influence on survival time.ConclusionsTERT rs2853669 and rs2736100 polymorphisms were not risk factors for developing AML in the Romanian population, but the TERT rs2853669 variant genotype had a negative effect on AML patients’ overall survival in the presence of other known prognostic factors.

Novel prognostic genes and subclasses of acute myeloid leukemia revealed by survival analysis of gene expression data

BackgroundAcute myeloid leukemia (AML) is biologically heterogeneous diseases with adverse prognosis. This study was conducted to find prognostic biomarkers that could effectively classify AML patients and provide guidance for treatment decision making.MethodsWeighted gene co-expression network analysis was applied to detect co-expression modules and analyze their relationship with clinicopathologic characteristics using RNA sequencing data from The Cancer Genome Atlas database. The associations of gene expression with patients’ mortality were investigated by a variety of statistical methods and validated in an independent dataset of 405 AML patients. A risk score formula was created based on a linear combination of five gene expression levels.ResultsThe weighted gene co-expression network analysis detected 63 co-expression modules. The pink and darkred modules were negatively significantly correlated with overall survival of AML patients. High expression of FNDC3B, VSTM1 and CALR was associated with favourable overall survival, while high expression of PLA2G4A was associated with adverse overall survival. Hierarchical clustering analysis of FNDC3B, VSTM1, PLA2G4A, GOLGA3 and CALR uncovered four subgroups of AML patients. The cluster1 AML patients showed younger age, lower cytogenetics risk, higher frequency of NPM1 mutations and more favourable overall survival than cluster3 patients. The risk score was demonstrated to be an indicator of adverse prognosis in AML patientsConclusionsThe FNDC3B, VSTM1, PLA2G4A, GOLGA3, CALR and risk score may serve as key prognostic biomarkers for the stratification and ultimately guide rational treatment of AML patients.

Prospective Randomized Comparative Study of Myeloablative Fludarabine/Busulfan (Flu/Bu4) and Fludarabine/Busulfan/Total Body Irradiation (Flu/Bu4/TBI) Conditioning Regimen for Patients with Myeloid Diseases

Introduction A retrospective study (Russell JA et al. BBMT 2010) showed additional 400 cGy of total body irradiation (TBI) to Flu/Bu regimen improved both overall survival and disease-free survival. To investigate the role of additional TBI 400 cGy in Flu/Bu4 conditioning regimen, we conducted a prospective randomized comparative study of fludarabine/busulfan (Flu/Bu4) and fludarabine/busulfan/total body irradiation (Flu/Bu4/TBI) conditioning regimen in allogeneic hematopoietic stem cell transplantation for patients with myeloid diseases. Methods We prospectively enrolled patients with myeloid diseases and randomized to Flu/Bu4 or Flu/Bu4/TBI between July 2010 and August 2019. Patients were stratified by diagnosis and disease risk. Patients received fludarabine 40 mg/m2 on days -6 to -3 followed by IV busulfan 130 mg/m2 on days -6 to -3 (Flu/Bu4). Flu/Bu4/TBI group had additional 400 cGy TBI in two fractions on day -1. Recipients had rATG 4 mg total dose over 3 days on days -3 to -1. All patients received post-transplant immunosuppression with tacrolimus and methotrexate 5 mg/m2 on days +1, 3, 6, and 11. We analyzed overall survival (OS), leukemia free survival (LFS), non-relapse mortality (NRM), relapse, acute- and chronic graft versus-host disease (GvHD). Results Forty-six (27 male and 19 female) patients with myeloid diseases were randomized into Flu/Bu4 (24 patients) or Flu/Bu4/TBI (22 patients). Patients had AML (34 patients), CML (1 patient), MDS (5 patients), MF (3 patients), and CMML (3 patients) with mean age 49.2 (22-63 years) (Table 1). There were no graft failures. One patient died within 100 days of transplantation due to organ failure. Among 46 patients, eleven patients died within a year of transplantation (5 patients in Flu/Bu4, 6 patients in Flu/Bu4/TBI), 5 from disease progression, 3 from acute GVHD, 1 from infection, 1 from fall, and 1 from organ failure. Day 100 grade II-IV and III-IV acute GVHD rates were 45.7% (95% CI [31.3, 60.1]) and 10.9% (95% CI [1.9, 30.8]), respectively; 1-year chronic GVHD and extensive chronic GVHD rates were 39.1% (95% CI [25.0, 53.2]) and 13.0% (95% CI [3.3, 22.8]), respectively. With a median follow up of 22.6 months (range, 1.8-114.7), NRM at day 100 was 2.2% (n=1) and 10.9% at 1 year. Overall survival (OS), leukemia-free survival (LFS) and relapse at 1-year were 74.4%, 41.9%, and 30.2%, respectively. Among 34 AML patients, LFS for all AML patients was 52.9% at 1 years. LFS was higher in patients undergoing Flu/Bu4/TBI with 56.3% versus Flu/Bu4 with 50.0% at 1 year (p=0.4). AML relapse with Flu/Bu4 and Flu/Bu4/TBI regimen was 38.9% versus 18.8% in the first year, 44.4% versus 25.0% in three years (RR=0.61 95% CI [0.20, 1.91]) (Fig 1). However, Flu/Bu4/TBI regimen increased the risk of NRM in AML patients (60.0% versus 14.3%, p=0.22). Overall survival (OS) for all AML patients was 67.6% at 1 year. OS of AML patients was similar in patients undergoing Flu/Bu4 with 72.2% versus Flu/Bu4/TBI with 62.5% at 1 year (p=0.4). The median survival was 26.9 months and 21.4 months, respectively. Conclusions Addition of TBI 400 cGy to Flu/Bu4 conditioning regimen resulted in reduction of relapse, contributing to a better LFS. However, Flu/Bu4/TBI group did not have a better OS due to the regimen related mortality. These findings suggest the additional TBI 400 cGy to Flu/Bu4 should be cautiously selected for patients who is fit but has a high risk of relapsed disease. Disclosures Rowley: FATE Therapeutics: Consultancy; AbbVie: Current equity holder in publicly-traded company. Skarbnik:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kite Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CLL Society: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Speakers Bureau; Verastem: Speakers Bureau; Beigene: Speakers Bureau; Alexion: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Vesole:Janssen: Speakers Bureau; Sanofi: Speakers Bureau; BMS: Speakers Bureau; Amgen: Speakers Bureau; Takeda: Speakers Bureau. Donato:Genomics Cooperative: Current equity holder in publicly-traded company.

Genome-wide CRISPR screen reveals cancer cell resistance to NK cells induced by NKderived IFN-γ

Abstract The anti-leukemia activity of NK cells is important for preventing relapse during HSCT in leukemia patients. However, the factors that determine susceptibility of leukemia cells in the context of NK-mediated killing are not well established. Here we performed a genome wide CRIPSR screen in the human CML cell line K562 to identify genes that regulate vulnerability of leukemia cells to killing by primary human NK cells. Distribution of gRNAs in K562 cells that survived co-incubation with NK cells showed that loss of NCR3LG1 (encodes B7H6), the ligand of the activating receptor NKp30, predominantly protected K562 cells from killing. It indicates that B7H6 is a major ligand on K562 cells. In contrast, loss of genes that regulate pathways for antigen-presentation (B2M, HLA-I, PSMB5, TAP1, TAPBP) and IFN-γ signaling (IFNGRs, JAKs, STATs) increased the vulnerability of K562 cells. MHC-I on K562 cells was upregulated after co-culture with NK. Addition of IFN-γ neutralizing antibody during the co-culture increased the susceptibility of K562 cells to NK-mediated lysis. The results suggest that cancer cells respond to NK-derived IFN-γ and lead to NK-resistance and immune evasion. Moreover, Analysis of RNA-seq data from The Cancer Genome Atlas (TCGA) showed that low IFNGR2 expression in cancer tissues associated with improved overall survival in AML patients, indicating that the IFNGR-JAK-STAT pathway can be a contributor to worse prognosis of cancer patients, potentially related to IFN-γ–induced NK-resistance, despite the fact that IFN-γ signaling promotes T cell mediated immune surveillance. Our results suggest that targeting IFN-γ responses might be a promising approach to enhance NK cell anti-cancer efficacy.

Comparison of Post-Transplant Cyclophosphamide and Tacrolimus and Methotrexate Graft Versus Host Disease Prophylaxis in AML Patients Receiving Myeloablative Fractionated Busulfan Conditioning Regimen

Background Myeloablative conditioning can be given safely to older patients by simply administering busulfan over a longer period (fractionated busulfan regimen) than the standard four day regimen. (Popat et al Lancet Haematology 2018). Subsequently, we added post-transplant cyclophosphamide (PTCy) GVHD prophylaxis to our fractionated regimen with a hypothesis that it will reduce GVHD and improve outcomes. Herein, we report the results of our retrospective analysis comparing outcomes of patients with AML who received fractionated busulfan myeloablative conditioning and PTCy versus standard tacrolimus (Tac) and methotrexate (Mtx) GVHD prophylaxis. Methods Patients with AML between 18-70 years of age with adequate organ function and 8/8-HLA matched related or unrelated donor, who were treated on 4 consecutive protocols using fractionated busulfan were included in the analysis. All patients received two fixed doses of busulfan 80mg/m2 as outpatient either on days -13 and -12, or on days -20 and-13 followed by busulfan on day -6 to -3 dosed to achieve target area under the curve (AUC) of 20,000 mol/min for the whole course based on pharmacokinetic studies. Fludarabine ± cladribine were given on day -6 to -3. GVHD prophylaxis was PTCy 50mg/kg on days 3 and 4 and Tac ± mycophenolate mofetil in PTCy cohort (n=53), or standard Tac+ Mtx (n=53). Patients in both the cohorts were matched 1:1 by propensity score. Results Baseline characteristics were similar between the PTCy and Tac/Mtx cohorts. The median age was 60 (range, 18-70) and 58 (range, 24-70) years, respectively, (P=0.3). Forty-nine percent were in CR, and 30% had comorbidity index >3 in each arm. Similar proportion of patients had ELN adverse risk in both arms (43% vs 40%, respectively P=0.7). The median follow up was 19 months (range, 3-36) in the PTCy and 46 months (range, 14-73) in Tac/Mtx cohort. Overall survival at 2-year was 78% vs 58% (P=0.03), non-relapse mortality was 6% vs 13% (P=0.2), incidence of grade 3-4 acute GVHD at 1-year was 6% vs 19% (P=0.07) and chronic GVHD at 2-year was 10% vs 29% (P=0.03) [Table 1 and figure 1]. The median time to neutrophil engraftment was prolonged by 3 days (15 vs 12 days; P Conclusion As compared to Tac/Mtx, PTCy-based GVHD prophylaxis not only reduced severe acute GVHD and chronic GVHD, but also improved overall survival in AML patients up to the age of 70 years who received myeloablative fractionated busulfan conditioning. Prospective randomized trial is warranted to validate these findings.