Bone Marrow Of AML Patients Research Articles

Uncovering the cellular and omics characteristics of natural killer cells in the bone marrow microenvironment of patients with acute myeloid leukemia

BackgroundAcute myeloid leukemia (AML) is a highly heterogeneous hematologic malignancy and the most frequently acute leukemia of stem cell precursors and the myeloid derivatives in adult. Longitudinal studies have indicated the therapeutic landscape and drug resistance for patients with AML are still intractable, which largely attribute to the deficiency of detailed information upon the pathogenesis.MethodsIn this study, we compared the cellular phenotype of resident NK cells (rAML-NKs, rHD-NKs) and expanded NK cells (eAML-NKs, eHD-NKs) from bone marrow of AML patients (AML) and healthy donors (HD). Then, we took advantage of the co-culture strategy for the evaluation of the in vitro cytotoxicity of NK cells upon diverse tumor cell lines (e.g., K562, Nalm6, U937). With the aid of RNA-sequencing (RNA-SEQ) and bioinformatics analyses (e.g., GOBP analysis, KEGG analysis, GSEA, volcano plot), we verified the similarities and differences of the omics features between eAML-NKs and eHD-NKs.ResultsHerein, we verified the sharp decline in the content of total resident NK cells (CD3−CD56+) in rAML-NKs compared to rHD-NKs. Differ from the expanded eHD-NKs, eAML-NKs revealed decline in diverse NK cell subsets (NKG2D+, CD25+, NKp44+, NKp46+) and alterations in cellular vitality but conservations in cytotoxicity. According to transcriptomic analysis, AML-NKs and HD-NKs showed multifaceted distinctions in gene expression profiling and genetic variations.ConclusionsCollectively, our data revealed the variations in the cytobiological and transcriptomic features between AML-NKs and HD-NKs in bone marrow environment. Our findings would benefit the further development of novel biomarkers for AML diagnosis and NK cell-based cytotherapy in future.

High-Throughput Identification of Graft-Vs-Leukemia TCRs in AML Patient Samples after Allogeneic Stem Cell Transplantation

For patients with intermediate and high-risk AML/MDS, allogeneic stem cell transplantation (aSCT) is a potentially curative therapy. Efficacy is driven by the graft-versus-leukemia (GvL) response, in which donor-derived T cells recognize peptide:MHC antigens presented by leukemia cells, and subsequently kill them. The antigens that drive GvL responses are attractive therapeutic targets for engineered cell therapies and/or biologics. However, only a small number of GvL antigens have been identified, limiting the therapeutic potential of targeted immunotherapies. Deep interrogation of the T cell repertoires of patients who have responded favorably to aSCT presents an opportunity to identify bona fide GvL antigens, but high-throughput methods need to be developed to do this effectively. A popular method to identify antigen-specific T cells is fluorescent peptide:MHC tetramer staining, whereby T cells whose T cell receptors (TCR) bind a particular peptide:MHC antigen become fluorescent and identifiable by flow cytometry. However, a major challenge to identifying GvL T cells using tetramers is that the prediction of immunogenic antigens - that is, those that induce a T cell response in vivo - is poor; studies of solid tumors suggest that only ~1% of predicted MHC-binding neoantigens are immunogenic in patients. In AML/MDS aSCT patients, there are thousands of peptides that are predicted to bind MHCI and represent potential GvL antigens, but due to practical limitations of patient blood and bone marrow sample quantities, only a small subset can be screened by flow cytometry. Other higher throughput methods using heavy-metal tagged peptide:MHC tetramers followed by mass cytometry, or DNA-barcoded tetramers followed by bulk sequencing have been employed to increase antigen screening scale, but these methods are limited in sensitivity and destroy the T cell without providing TCR sequences. To overcome these limitations and rapidly identify GvL T cells in patient samples, we developed a single cell sequencing based method that enables the screening of thousands of peptide:MHC antigens simultaneously, using a single patient sample. We generated peptide: MHCI monomers using UV-mediated peptide exchange and generated large panels of combinatorial DNA-barcoded peptide:MHCI tetramers at microscale. We integrated our methodology into the 10X Genomics Single Cell Immune Profiling workflow to enable simultaneous recovery of TCR antigen-specificity, paired TCR alpha beta sequences, and single cell RNA-Seq data. These methods were used to screen post-transplant AML patient blood and bone marrow, and we identified expanded GvL T cell clonotypes that target novel minor histocompatibility antigens, overexpressed “aberrant self” proteins, and other leukemia associated antigens. We generated primary CD8 T cells that retrovirally express these TCRs and demonstrate that they are potent and have the expected antigen specificities. The methods described are highly sensitive, specific, efficient, and scalable, and enable high confidence identification of antigen specific clonotypes in patient samples. They markedly expand the throughput compared to existing methods, and simultaneously yield paired alpha beta TCR sequences, facilitating a full characterization of the antigen-TCR drivers of GvL. Furthermore, these methods have broad utility beyond SCT, as they are applicable to studying T cell responses to other immune settings, including anti-cancer, autoimmune, and anti-microbial responses.

Multimodal Atlas of Paired Diagnosis and Relapse AML Samples Enables Novel Therapeutic Targeting of Surface Antigens

Note: A.H. and M.U. share co-first authorship. AML is an aggressive clonal malignancy characterized by combinations of chromosomal abnormalities, gene mutations, and cell surface antigen (Ag) expression profiles. This heterogeneity contributes to refractory or relapsed disease that presents a major challenge when treating AML. Previous single cell sequencing studies of primary AML samples have provided remarkable insight into the clonal architecture of AML cell populations and how their mutational, transcriptomic, and surface Ag profiles vary between patients. However, information addressing clonal shifts during progression from diagnosis to relapse in large cohorts is lacking. Here, we adapted single cell RNA sequencing with surface Ag feature barcoding to analyze more than 450,000 cells from 28 paired AML patient bone marrow mononuclear cell samples collected at diagnosis and relapse (56 samples). To our knowledge, it is the largest and most comprehensive single cell AML atlas to date. This atlas contains rich clinical metadata including cytogenetics, mutation status of canonical AML genes, treatment history, and survival information. We leveraged these data to identify potential correlations with clonal heterogeneity during progression to relapse and to propose novel strategies for targeting the surface of AML cells. The feature barcoding panel consists of a comprehensive list of 81 surface Ags reported in clinicaltrials.gov or mined from literature, including large proteomics mass spectrometry datasets of AML patient bone marrow samples (deBoer et. al. Cancer Cell 2018; Jayavelu et al, Cancer Cell 2022). Feature barcoding produces antibody derived tag (ADT) counts which are interpreted as relative values but alone does not indicate absolute number of Ags per cell. We addressed this limitation by measuring absolute Ag density of AML Ags, CD33, CLL1, CD123, and EMR2 in patient samples using the flow cytometric QuantiBRITE assay. Focusing on these four Ags, we modeled the relationship between ADT expression and antibodies per cell (ABC) Ag density from QuantiBRITE. The model was applied to impute absolute Ag density for the remaining 77 Ags in all samples. UMAP visualization after sample transcriptome integration revealed distinct clustering of CD45-dim myeloblasts, T cells, B cells, and erythroid cells. Longitudinal analysis identified 28 surface Ags that were differentially expressed (absolute log 2FC > 1, p < 0.01) between relapse and diagnosis myeloblasts across multiple patients (Fig. 1A). An additional 19 Ags were differentially expressed in no more than one patient while the other 19 had no significant change in any patient, highlighting the inter-and intra- heterogeneity of AML at diagnosis and relapse in these patients. Targeting multiple antigens simultaneously may help address the issue of antigen heterogeneity of tumor cells and help avoid potential antigen escape. Because multi-targeting therapies are a potential solution to this, weleveraged the single-cell resolution of our atlas to systematically identify Ags that in combination provide maximum coverage and are significantly co-expressed in myeloblasts. From this analysis, we found 48 and 52 co-expressed Ag pairs in diagnosis and relapse blasts, respectively (r > 0.4, p < 0.01; Fig. 1B). Of note, CD44 and EMR2 were co-expressed at both diagnosis (r = 0.49 p < 0.01) and relapse (r = 0.3, p < 0.01) with >90% of their blasts expressing CD44, EMR2, or both at targetable antigen levels of >1000 Ags/cell. Furthermore, 10 samples had >40% of their blasts co-expressing both Ags simultaneously at >1000 Ags/cell at diagnosis or relapse. 6 of these patients had a >10% increase in co-expression at relapse, 3 had >10% decrease at relapse, and one patient had no co-expression change. Our comprehensive profiling of AML has provided high resolution insight on cell surface at diagnosis and during disease progression and produced novel hypotheses about targetable combinations of surface Ags based on their expression changes between timepoints, co-expression, and surface densities. Furthermore, our reference atlas can be further dissected to characterize the transcriptome and mutational profiles of myeloblasts at diagnosis and relapse thereby prodding an important resource to create surface antigen protein - transcript correlations within AML blasts and to trace molecular signatures during disease progression.

Systematic Evaluation of AML-Associated Antigens Identifies Novel Anti-U5 snRNP200 Therapeutic Antibodies for the Treatment of AML

Numerous antibody-based immunotherapies are in development for AML including antibodies targeting CD47, CD70, CD123, CD33, and TIM3. However, systematic evaluation of the distribution of AML-associated antigens in combination with Fc receptors and the immune microenvironment required for antibody-mediated therapeutic responses have been limited. Additionally, efforts to maximize antibody Fc receptor engagement on immune effector cells in AML patients via Fc region engineering have not been explored. Here we applied a custom 36-color flow cytometry panel tailored for AML patient bone marrow along with proteogenomic assessment via CITE-seq to simultaneously assess known and novel antibody target expression on AML cells, immune microenvironment, and Fc receptor expression across immune subsets. Application to samples from 7 age-matched healthy donors and 51 newly diagnosed AML patients allowed for unbiased identification of the malignant blast population via UMAP projection. Quantification of differential antigen expression on blasts and leukemic stem cells versus normal hematopoietic stem and progenitor cell (HSPC) subsets, identified that out of 9 AML putative antibody targets (CD123, TIM3, CD33, CD47, CD90, CD38, CD25, CD70, U5 snRNP200), only U5 snRNP200 is expressed on blasts but not normal HSPCs (Fig. A). Characterization of the distribution of Fc receptors revealed that AML patients exhibit reduced cell-surface activating Fc receptors (CD16, CD32A) on NK and non-classical monocytes with concomitant increases in inhibitory (CD32B) Fc receptors on monocytes and B-cells. Moreover, AML patients had fewer classical monocytes and type 2 conventional dendritic (cDC2) cells than normal age-match control marrow. Given the unique cell surface expression of U5 snRNP200 on AML blasts compared with normal HSPCs, we deeply interrogated the distribution of this marker throughout normal and malignant hematopoiesis. While U5 snRNP200 expression was not seen on normal HSPCs, U5 snRNP200 was present on mature B-cells and a subset of NK cells in normal adult human and mouse hematopoietic cells. U5 snRNP200 is an RNA helicase which is an essential conserved component of the spliceosome. Its known function is limited to the nucleus and it is therefore unexpected that it would be expressed on the cell surface. To rigorously confirm expression of U5 snRNP200 on the cell membrane, we knocked in a HaloTag at the N-terminus of the locus encoding U5 snRNP200 within K562 cells and traced expression of tagged endogenous U5 snRNP200 protein across cellular compartments. This clearly revealed cell surface localization of the full-length protein on the surface of AML cells. We next conducted genome-wide CRISPR screens in two human AML cell lines to identify regulators of surface U5 snRNP200 expression. In both cell lines, the top gene required for cell surface U5 snRNP200 expression was CD32A. CRISPR knockout of CD32A in U937 cells reduced U5 snRNP200 surface expression, while restoration of CD32A rescued U5 snRNP200 surface expression. AML-associated expression of U5 snRNP200 was present on a number of syngeneic immunocompetent mouse models of AML including MLL-AF9, RN2 (MLL-AF9 + NRAS G12D), transgenic EVI1 rearranged/SF3B1K700E mutant, and Tet2/Zrsr2 double knockout mice. To test the therapeutic potential of targeting cell surface U5 snRNP200, we engineered several anti-murine U5 snRNP200 antibody variants featuring modified Fc regions to assess the possible roles of engagement of Fc receptors on immune cell subsets to enhance AML cell killing. In both the RN2 and EVI1/SF3B1 mutant models, anti-U5 snRNP200 antibodies featuring the Fc region modified to engage activating Fc receptors and induce antibody-dependent cellular cytotoxicity (IgG2a) provided superior survival benefit compared to IgG1 or IgG2b U5 snRNP200 antibodies or a mutant IgG1 antibody (IgG1 D265A) which does not bind Fc receptors (Fig. B). These data provide a high-density roadmap of the distribution of known and novel AML-associated antigens together with the Fc receptor distribution and immune microenvironment in AML. The results motivated discovery of a novel antibody-therapeutic targeting aberrant cell-surface U5 snRNP200 and research into mechanisms for cell surface trafficking of U5 snRNP200. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

STMN1 promotes the proliferation and inhibits the apoptosis of acute myeloid leukemiacells by activating the PI3K/Akt pathway

Recent studies have shown that the microtubule disrupting protein Stathmin 1 (STMN1) is differentially expressed in AML patients and healthy control. The aim of this study was to explore the effects and molecular mechanism of STMN1 in AML. Here, the expression of STMN1 in peripheral blood cells (PBMCs) and bone marrow of AML patients and healthy volunteers was detected by RT-PCR and Western blot. STMN1 expression was regulated by transfected with STMN1 overexpressed plasmid or shRNA in two human leukemia cell lines K562 and HL60. Cell proliferation was examined by CCK8 and Edu staining. Annexin V and TUNEL assays were applied to test cell apoptosis. Flow cytometry was used to test the cell cycle distribution. The activation of the PI3K signaling pathway and the expression levels of cell cycle and cell apoptosis-related protein were determined by Western blot. In this study, we found that STMN1 was overexpressed in PBMCs and bone marrow of AML patients. STMN1 expression was closely related to FAB subtypes, risk stratification, disease-free survival, and overall survival of AML. Functional assays showed that overexpression of STMN1 in HL60 and K562 cells enhanced cell proliferation, decreased cell apoptosis, and caused G1 phase arrest. In contrast, suppression of STMN1reduced cell proliferation and enhanced cell apoptosis in both HL60 and K562 cells. Moreover, the PI3K/Akt pathway was activated by STMN1, while suppression of STMN1 dysregulated the PI3K/Akt pathway and upregulating the levels of caspases3 and Bax expression. In conclusion, STMN1 was confirmed to promote the proliferation and inhibit the apoptosis of HL60 and K562 cells by modulating the PI3K/Akt pathway. STMN1 might be a novel molecular target for treating AML.

STMN1 promotes the proliferation and inhibits the apoptosis of acute myeloid leukemiacells by activating the PI3K/Akt pathway

Recent studies have shown that the microtubule disrupting protein Stathmin 1 (STMN1) is differentially expressed in AML patients and healthy control. The aim of this study was to explore the effects and molecular mechanism of STMN1 in AML. Here, the expression of STMN1 in peripheral blood cells (PBMCs) and bone marrow of AML patients and healthy volunteers was detected by RT-PCR and Western blot. STMN1 expression was regulated by transfected with STMN1 overexpressed plasmid or shRNA in two human leukemia cell lines K562 and HL60. Cell proliferation was examined by CCK8 and Edu staining. Annexin V and TUNEL assays were applied to test cell apoptosis. Flow cytometry was used to test the cell cycle distribution. The activation of the PI3K signaling pathway and the expression levels of cell cycle and cell apoptosis-related protein were determined by Western blot. In this study, we found that STMN1 was overexpressed in PBMCs and bone marrow of AML patients. STMN1 expression was closely related to FAB subtypes, risk stratification, disease-free survival, and overall survival of AML. Functional assays showed that overexpression of STMN1 in HL60 and K562 cells enhanced cell proliferation, decreased cell apoptosis, and caused G1 phase arrest. In contrast, suppression of STMN1reduced cell proliferation and enhanced cell apoptosis in both HL60 and K562 cells. Moreover, the PI3K/Akt pathway was activated by STMN1, while suppression of STMN1 dysregulated the PI3K/Akt pathway and upregulating the levels of caspases3 and Bax expression. In conclusion, STMN1 was confirmed to promote the proliferation and inhibit the apoptosis of HL60 and K562 cells by modulating the PI3K/Akt pathway. STMN1 might be a novel molecular target for treating AML.

Combinatorial Inhibition of Focal Adhesion Kinase and BCL-2 in AML

Focal adhesion kinase (FAK), activated by integrins and cytokines, promotes cell growth, migration and metastasis. We previously reported that a selective FAK inhibitor, VS-4718, inhibited cell growth and induced cell death in leukemia cell lines in vitro, effectively reduced leukemia burden in peripheral blood and tissues including bone marrow, spleen, liver and lung, and significantly prolonged survival time in a human xenograft murine model (Carter BZ et al., MCT, 2017), 16(6):1133).Although BCL-2 inhibition by the BCL-2 inhibitor ABT-199 (Venetoclax) has demonstrated antileukemia activities, an acquired drug resistance frequently ensues. When combined with ABT-199, VS-4718 significantly potentiated ABT-199-induced apoptosis in AML cell lines and primary AML cells. VS-4718 treatment of Molm14 AML cells decreased levels of p-STAT5, MCL-1, and BCL-XL (Wang et al., ASH 2016) that play important roles in the acquired resistance to ABT-199.To further examine the effect of VS-4718 as a single agent and in combination with ABT-199 on primary AML cells in vivo, we generated patient-derived xenografts (PDX) from AML patient bone marrow cells expanded in NSGS (NOD-SCID IL2Rgnull-3/GM/SF, NSG-SGM3) mice. The xenograft used AML cells from a patient who had failed multiple chemotherapies and had NPM1, FLT3-ITD, TET2, DNMT3A, and WT1 gene mutations and a complex karyotype. The combination of VS-4718 and ABT-199 synergistically killed the PDX cells in vitro even under MSC co-culture conditions. Once PDX cells were engrafted into NSGS mice, animals were randomized into 4 groups (N=10/group) and treated with vehicle, VS-4718 (75 mg/kg, twice daily), ABT-199 (100 mg/kg, once per day), or the combination via oral gavage for 29 days. We observed that ABT-199 had a stronger anti-leukemic effect on peripheral blood than on tissue-resident cells, while VS-4718 was more effective in tissues. At the end of the treatments, VS-4718 significantly reduced circulating human CD45+ cells (P=0.0003), and ABT-199 alone (P=5.90E-07) and the ABT-199/VS-4718 combination (P=5.38E-07) were ever more effective. VS-4718 alone or in combination with ABT-199 reduced leukemia burden in spleen and liver and effectively reduced splenomegaly, while ABT-199 alone did not. VS-4718-treated mice survived significantly longer than the untreated controls (median survival 72 vs. 36 days, P=0.0002), while ABT-199 did not prolong survival (median survival 35 days). Also, the combination of VS-4718 with ABT-199 significantly prolonged survival (median survival 65.5 days, P=0.011 vs. control and P=0.0072 vs. ABT-199). No statistical difference in survival was found between VS-4718 and the combination treatment groups (P=0.33) (Fig. 1). All the mice had large spleen at death except the combination group: the 1st dead mouse had small and the 2nd to 4th ones had slightly enlarged spleen and only the last two had large spleen as others suggesting that additional reasons such as toxicity at the dose used may have contributed to the death of the 1st 4 mice in the combination group, hence shortened the median survival in this group.We determined expression of FAK and BCL-2 family proteins in cells from mouse spleens at the end of treatment and found that the combination of VS-4718 and ABT-199 decreased protein levels of FAK. Consistent with our in vitro results, the increased level of MCL-1 by ABT-199 was antagonized by the combination with VS-4718 in vivo suggesting the combination potentially be more effective.Conclusions: These results suggest a potential clinical benefit of combining a FAK inhibitor with Venetoclax for patients with AML. [Display omitted] DisclosuresPachter:Verastem, Inc.: Employment. Weaver:Verastem, Inc.: Employment. Carter:Daiichi Sankyo: Research Funding; novartis: Research Funding.

Genetic Features of AML with MLL-Rearrangement and NPM1 Mutation: An Interim-Analysis of HM-Screen-Japan 01

Genetic Features of AML with MLL-Rearrangement and NPM1 Mutation: An Interim-Analysis of HM-Screen-Japan 01

Abstract 469: Selective targeting of splicing deregulation in pediatric acute myeloid leukemia stem cells

Abstract While 5 year survival rates for acute lymphoblastic leukemia (ALL) in childhood are 90% (https://www.cancer.org/cancer/leukemia-in-children/detection-diagnosis-staging/survival-rates.html), acute myeloid leukemia survival rates have lagged behind at 65 to 70%. Thus, AML is the leading cause of childhood leukemic mortality and is a heterogeneous disease characterized by diverse mutations many occurring at low frequencies. Whole exome sequencing analyses have not been sufficient to predict relapse in many pediatric patients at least in part because of transcriptomic and epitranscriptomic alterations that drive therapy resistant leukemia stem cell (LSC) propagation. Recent results reported by ourselves and others suggest that LSC in AML harbor unique mRNA splicing profiles characterized by intron retention and exon skipping. In adult AML, whole transcriptome RNA sequencing data revealed global spliceosome disruption that uniquely distinguished LSCs from normal age-matched hematopoietic stem and progenitor cells (Crews et al Cell Stem Cell 2016). However, the role of splicing deregulation in LSC propagation in human pediatric AML had not been clearly elucidated. Recently, we have developed 17S-FD-895, a small molecule compound targeting SF3B1, which modulates mRNA splicing. To date, we have evaluated the effects of this splicing modulator on both self-renewal as well as pro-survival splice variants in CD34+ cells derived from both peripheral blood as well as bone marrow of pediatric AML patients. Splice isoform specific qRT-PCR demonstrated a dose-dependent increase in SF3B1 intron retention following treatment. Furthermore, splicing modulation induced MCL1 exon 2 skipping, producing pro-apoptotic MCL1-S transcripts. Hematopoietic progenitor assays demonstrated a dose-dependent reduction in LSC clonogenicity and self-renewal. In these assays, LSC were found to be sensitive to nanomolar concentrations that spared normal hematopoietic stem and progenitor cells. To further dissect the role of pre-mRNA splicing in pediatric AML, we developed a lentiviral fluorescent splicing reporter that switches from GFP to RFP expression following an exon skipping or intron retention event. In addition, whole transcriptome RNA sequencing (RNA-seq) was performed on FACS purified stem cells (CD34+/CD38-/Lin-) as well as progenitor cells (CD34+/CD38+/Lin-) from both pediatric AML as well as aged-matched normal bone marrow samples and samples were transplanted into immunocompromised mice followed by 17S-FD-895 treatment to assess sensitivity to splicing modulation in vivo. As a result of these studies, we have demonstrated LSC splicing patterns in pediatric AML that may inform novel biomarker identification as well as development of 17S-FD-895 for pediatric AML. Citation Format: Inge Van der Werf, Phoebe Mondala, Raymond Diep, Larisa Balaian, Cayla Mason, Gertjan Kaspers, Jacqueline Cloos, Jim La Clair, Peggy Wentworth, Tom Whisenant, Katie Fisch, Michael Burkart, Catriona Jamieson. Selective targeting of splicing deregulation in pediatric acute myeloid leukemia stem cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 469.

STAG2-mutated AML patients: ASXL1 cohesin binding motif status and mutation landscape.

7538 Background: ASXL1 and the cohesin complex ( STAG2, RAD21, SMC1A, and SMC3) are commonly mutated chromatin regulators with significant clinical implications in AML. The ASXL1-cohesin interactome regulates gene expression through chromatin accessibility via ASXL1’s cohesin binding motif (CBM). ASXL1 variants are most commonly located in the ASXM1 domain and onwards, and characteristically lead to loss of the PHD domain. Gain-of-functions in truncated ASXL1 are suggested to increase catalytic activity of BAP1, which binds the ASXH domain at AA 351, and to gain an interaction with BRD4, which binds somewhere between the ASXN and ASXH domains, to drive H3K4Me3 and H2AK119Ub. Methods: 2463 suspected AML patient bone marrow, peripheral blood, or FFPE tissue samples were evaluated using an all exon amplicon-based 27 gene NGS panel. Patients with a VAF <10% in ASXL1 were excluded to avoid reporting artifacts, particularly in variant c. 1934dup. Statistics were performed using Fisher’s exact test. Results: Mutations in STAG2-mutated patients were enriched for sAML, as evidenced by the higher number of mutations in ASXL1, SRSF2, and BCOR (associated with sAML) compared to NPM1, DNMT3A, and PTPN11 (pAML). STAG2 mutations were found in 173 samples representing 93.5% of cohesin mutations. Of all ASXL1 mutations (VAF 10.1– 54.5%; median 32.2%) 4.0% occurred in the CBM. While 23.5% of samples with mutations outside ASXL1 CBM had concomitant mutations in STAG2, none of the 18 samples with CBM mutations (VAF 11.3 – 51.7%; median 42.5%) had any cohesin gene mutation (P = 0.0174). The proportion of BCOR (27.8% vs 9.2%; P = 0.024) and CEBPA (27.8% vs 8.2%; p = 0.016) mutated patients in the CBM+ group was significantly higher than the CBM- group. JAK2 (16.7% vs 5.4%), KRAS (22.2% vs 13.6%), EZH2 (22.2% vs 13.6%), and RUNX1 (38.9% vs 27.7%) mutations were also higher though not significantly in this group. Mutations throughout all of ASXL1, the 13 amino acids after the CBM, and hotspot variants all had STAG2 mutations at a frequency of 20.9-44.4%, further suggesting mutual exclusivity. Conclusions: STAG2 mutations and mutations in the CBM were mutually exclusive events and harbored different co-mutation frequencies. In compromised ASXL1 CBM cases, BCOR and CEBPA transcriptional regulators are significantly more mutated, but in cases of ASXL1 mutation outside the CBM, cohesin mutations are preferred, suggesting alternative chromatin accessibility mechanisms driving leukemogenesis. This observation has not been previously reported in the literature to our knowledge.

A Signature of T Cell Exhaustion Is Enriched in the Bone Marrow (BM) of AML Patients and Shared with Immune Exhaustion Signatures of Solid Tumors

Background: T cells are key players in immunotherapy but our understanding of their role and function in AML is limited. We have previously shown that following induction chemotherapy, the phenotypic and transcriptional profiles of peripheral blood (PB) CD8+ T cells in AML diverge between complete responders (CR) and non-responders (NR) to treatment (Knaus et al, JCI insight 2018). Further defining AML-induced T cell dysfunction is critical for immune monitoring and developing novel immunotherapeutic strategies. Aims: 1. To characterize transcriptional differences between PB vs BM T cells of AML patients, similar to concept of circulating vs tumor infiltrating lymphocytes (TILs), and compare them to established immunologic signatures of solid tumors. 2. To examine the cumulative expression of multiple inhibitory receptors (IRs) on AML CD8+ T cells and their dynamics in PB vs BM at diagnosis (PRE-treatment) and POST-induction chemotherapy. Methods: To study transcriptional signatures, we FACS-purified CD8+ T cells from BM of 6 AML patients (3 CR and 3 NR) PRE- and POST-induction chemotherapy whose PB T cells we analyzed and published previously (Knaus et al, JCI insight 2018). Samples were hybridized to the Human Prime View Gene Expression Array (Affymetrix). Expression fold change (FC), p-values and FDR were calculated. We compared our data set to those of dysfunctional T-cell signatures from non-small cell lung cancer (NSCLC: Guo et al, Nature 2018), colorectal cancer (CRC; Zhang et al, Nature 2018) and hepatocellular carcinoma (HCC; Zheng et al, Cell 2017) using gene set enrichment analyses (GSEA) and considered a normalized enrichment score NES>2 and FDR<0.1 as enriched. Flow cytometry data on paired PB and BM samples from AML patients (n=32) PRE- and POST-treatment and healthy controls (HC; n=21) were used to calculate the IR-score (Thommen et al, Cancer Immunol Res 2015). The IR-score summarizes the cumulative relative amount of expression of 7 different IRs on CD8+ T cells. To determine the predictive ability of the IR score to differentiate CR from NR, Receiver Operating Characteristic (ROC) analysis was conducted using logistic regression, and the area under the ROC curve (AUC) was calculated. Results: Immune signatures of AML CD8+ T cells at diagnosis, particularly in the BM, overlapped with transcriptomic exhaustion signatures of TILs from other malignancies (Figure 1-Overlap CRC, HCC, NSCLC), in particular that of CRC. Further, AML BM but not PB T cells showed a high enrichment for a common immune exhaustion signature shared by all 3 solid tumor entities and included IRs like HAVCR2, PDCD1, CTLA4, LAG3 and TIGIT, and other genes (e.g. TNFRSF9, CD27, IFNG, FASLG, CD39/ENTPD1) (Figure 1). Interestingly, this signature seemed to discriminate CR from NR patients only in the BM compartment, and at both PRE- and POST-treatment, thus providing a rationale for future exploration as a predictive biomarker of response. We found that the flow-cytometry-based IR-score strongly discriminated between CR and NR in both the PB and the BM compartment POST-treatment with an AUC of 0.70 and 0.84, respectively. While IR-score including all 7 markers provided the best discriminatory value in the PB, the combination of only two senescence markers, CD57 and KLRG1, discriminated CR from NR patients in both PB and BM with an AUC of 0.69 and 0.79, respectively. The combination of 5 exhaustion markers (2B4, BTLA, Tim3, PD-1, and CD160) discriminated CR from NR patients only in the BM (AUC 0.79). Conclusion: We found that in AML, T cells display a signature of immune exhaustion which is more prominent in the tumor milieu (e.g BM), similar to exhaustion signatures of solid cancers, can discriminate CR from NR, and persists in CR. Multiple genes, such as TNFRSF9, CD27, IFNG, FASLG, and CD39/ENTPD1, were higher expressed in CR patients both PRE and POST-treatment, suggesting that this signature also includes genes that could be useful for discriminating less dysfunctional T cell states. The phenotypic IR score was increased only in NR POST-treatment, indicating that assessment of a limited number of phenotypic markers may not be sufficient to address overall changes within T cells. Further exploration of AML-imposed T cell dysfunction is ongoing to inform better integration of immune-based therapies to augment anti-leukemia immunity. Disclosures Zeidner: Celgene: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Honoraria; Tolero: Honoraria, Research Funding; Pfizer: Honoraria; AsystBio Laboratories: Consultancy; Merck: Research Funding; Takeda: Research Funding; AbbVie: Honoraria; Agios: Honoraria. Gojo:Jazz: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Merck: Research Funding; Juno: Research Funding; Amgen Inc: Consultancy, Honoraria, Research Funding; Amphivena: Research Funding. Luznik:Merck: Research Funding, Speakers Bureau; Genentech: Research Funding; AbbVie: Consultancy; WindMiL Therapeutics: Patents & Royalties: Patent holder.

Newly Diagnosed AML Patient Samples Demonstrate High Degree of Concordance in Identification of Pathogenic Mutations By Next Generation Sequencing (NGS) Performed at Enrolling Institutions Compared to Central Laboratory Results in the Beat AML Master Trial

Newly Diagnosed AML Patient Samples Demonstrate High Degree of Concordance in Identification of Pathogenic Mutations By Next Generation Sequencing (NGS) Performed at Enrolling Institutions Compared to Central Laboratory Results in the Beat AML Master Trial

Role of Arsenic Trioxide in EVI-1 Apoptosis in Patients with Acute Myeloid Leukaemia

Background:High expression of the ecotropic viral integration site-1 (EVI-1) is an independent negative prognostic indicator of survival in leukaemia patients. Zebrafish (Danio rerio) is a vertebrate animal model commonly used to examine haematopoiesis and myeloid malignancies. To clarify the molecular mechanisms of EVI-1, we previously introduced the human EVI-1 gene into embryonic zebrafish through a heat-shock promoter and established the stable germ-line Tg(EVI-1: HSE: EGFP) zebrafish (Shen et al, 2013). Arsenic trioxide (As2O3, ATO) is one of the effective anticancer drugs, especially for patients with leukaemia (Udupa et al, 2017). We thus aimed to explore the anticancer effects of ATO and the underlying functions associated with EVI-1 in an in vivo zebrafish model and in AML cells in vitro.Results:We determined EVI-1 expression in mononuclear cells isolated from the bone marrow and peripheral blood of AML patients and healthy donors by RT-qPCR and Western blot analysis. EVI-1 was highly expressed in primary AML (Fig 1A). Then, EVI-1 expression was detected in five leukaemia cell lines (K562, HL-60, U937, THP-1 and MV4-11) and normal PBMCs. Among these five leukaemia cell lines, THP-1 has the highest EVI-1 expression (Fig 1B and Fig1C).Primary acute monocytic leukaemia cells from one patient with high expression of EVI-1 were treated with ATO. We found ATO could significantly decrease EVI-1 mRNA (Fig 2A). Between the ATO-treated groups and the control group, the expression of EVI-1 were significantly reduced in the THP-1 cell line (Fig 2B). Next, we evaluated the EVI-1 expression in Tg(EVI-1: HSE; EGFP) transgenic zebrafish embryos over dose courses of ATO exposure (Fig 2C). Consistent with the results of our in vitro study, ATO decreased EVI-1 expression in a dose-dependent manner after 72 h (Fig 2C). Taken together, these results indicate that ATO is an inhibitor of EVI-1 expression both in vivo and in vitro.We investigate whether the reduction of THP-1 cells viability is due to apoptosis, THP-1 cells were incubated with 3 µM of ATO for 24 h, 48 h or 72 h. In the light microscopy images, THP-1 cells exhibited typical apoptotic characteristics (Fig 3A). The proportion of apoptotic cells was represented as early apoptotic cells (annexin V+/PI- staining, the lower right quadrant) plus late apoptotic cells (annexin V+/PI+ staining, the upper right quadrant) (Fig 3B). In cytometric analysis, ATO increased the percentage of apoptotic THP-1 cells in a dose- and time-dependent manner. We found that ATO increased the expressions of JNK, p-JNK, p-P53, PUMA, Bax, caspase-9 and caspase-3 (including cleaved caspase-9 and -3) but decreased the expressions of Bcl-2 and Bcl-xl (Fig 3C).To further verify the role of the JNK pathway in ATO-mediated THP-1 cell apoptosis, we examined if the inhibitor of JNK (SP600125) could reverse ATO-induced apoptosis in THP-1 cells. We found SP600125 not only decreased the pro-apoptotic effect of ATO in the THP-1 cell line (Fig 4A and Fig 4B) but also decreased the activation of the JNK-mediated apoptotic signalling pathway (Fig 4C). SP600125 silenced the activation of JNK by completely inhibiting the phosphorylation of JNK but had little effect on EVI-1 expression (Fig 4C).To test whether EVI-1 modulates apoptosis via the JNK signalling pathway, we transiently transfected THP-1 cells with EVI-1 siRNA which significantly reduced EVI-1 expression (Fig 5A). Silencing EVI-1 had a significant effect on the activation of the JNK pathway and the induction of THP-1 cell apoptosis (Fig 5B and Fig 5C).Conclusion:Our study demonstrated that the apoptotic pathway in THP-1 cells induced by ATO is closely associated with the oncogene EVI-1, the pro-apoptotic protein JNK, p-JNK, p-P53, PUMA, Bax, caspase-9 and caspase-3 (including cleaved caspase-9 and cleaved caspase-3), and the anti-apoptotic proteins Bcl-2 and Bcl-xL. ATO can downregulate EVI-1 mRNA and oncoprotein and block the repression of EVI-1 in the JNK pathway. Furthermore, the activated JNK signalling pathway regulated the expression level of apoptosis-associated proteins, including p-P53, PUMA, Bax, Bcl‐xL, Bcl‐2, Bax, caspase-9 and caspase-3(Fig 6). These findings may provide a novel theoretical basis for the development of personalized medical strategies for the treatment of EVI-1 positive AML patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Targeting Antagonists of Retinoic Acid Signaling in Acute Myeloid Leukemia

Introduction: Retinoic acid (RA), the active metabolite of vitamin A, influences biological processes by activating the retinoic acid receptor (RAR). RARs are ligand-controlled transcription factors that function as heterodimers with retinoid X receptors (RXRs) to regulate homeostasis of cellular growth. The success of RAR modulation in the treatment of acute promyelocytic leukemia (APL) particularly by the use of all-trans retinoic acid (ATRA) has stimulated considerable interest in the development of small molecules that can modulate RAR and RXR. Recent studies have demonstrated that RA can also activate the peroxisome proliferator-activated receptor β/δ (PPARβ/δ). In the aqueous intracellular milieu, RA is transported by the cellular retinoid-binding protein CRABP-II, or by the fatty-acid-binding protein FABP5, depending on the ratio of FABP5 to CRABP-II. In cells expressing high CRABP-II and low FABP5, RA activates the RAR, whereas in the presence of the reverse ratio, RA activates PPARβ/δ (Fig 1). These two different modes of RA delivery due to the different ratio of these two cargos leads to opposite cellular outcomes. Cells harboring high level of CRABP-II, RA is delivered to RAR leading to apoptosis, growth arrest, and anticancer activity. However, when FABP5 expression is high, RA is delivered to PPARβ/δ resulting in survival, proliferation, and tumor growth. In both cases, retinoid X receptor (RXR) is the indispensable partner of the nuclear receptor involved. Therefore, preventing FABP5 from such antagonism may be a novel therapeutic strategy for AML. Here we report the development of a therapeutic strategy based on a highly specific FABP5 inhibitor (iFABP5) (Fig 2) that will allow the delivery of RA by CRABP-II to activate tumor suppressor function of RAR and RXR. Methods: An iterative approach of design synthesis and activity were employed to select the most potent hit, iFABP5, for further experiments. Expression levels were analyzed by western blot analysis and qRT-PCR. Colony forming assays were used to analyze iFABP5 activity against AML cell lines. Flow cytometry based cell differentiation assay were performed to assess the efficacy of iFABP5 and ATRA combination treatment. Results: The analysis of TCGA data set revealed that a certain class of AML patients (pts) (Trisomy 8 AML) have low levels of CRABP-II and high levels of FABP5, presumably due to gene duplication, that in part explains the inability of ATRA to induce terminal differentiation in AML cells. To test our hypothesis, we screened AML and APL patient (pt) bone marrow cells and found that a number of AML pts bone marrow have high FABP5 and low CRABP-II protein levels while the ATRA responding APL pts has opposite ratio determined by western blotting. Therefore, low CRABP-II and high FABP5 levels in a subset of AML pts lead to the activation of pro-survival PPARβ/δ pathway that promotes proliferation and opposes the differentiation. We also analyzed AML and APL pt samples along with different AML cell lines for mRNA expression using qRT-PCR. High FABP5 levels were observed in the majority of the AML cell lines. Efficacy of novel small molecule FABP5 inhibitor as a single agent and in combination with ATRA was evaluated in HL-60 cells. The FABP5 inhibitor iFABP5 was found to increase differentiation at 72 hours as assessed by both CD11b and CD14 levels in HL-60 cells. To confirm that iFABP5 is targeting FABP5 and indirectly targeting the PPARβ/δ pathway, levels of RAR and PPARβ/δ target genes were evaluated in the absence and presence of iFABP5. Changes in the gene expression of RAR and PPARβ/δ target genes in the presence and absence of iFABP5 were also examined in shFABP5, shPPARβ/δ, and shCRABP2 versus wild type cells. Conclusion: We demonstrated that a small molecule inhibitor of FABP5 synergizes with ATRA and induces the differentiation in AML cells. High FABP5 levels (mRNA and protein) were observed in the majority of the AML cell lines. Hence, FABP5 can be a therapeutic target in AML. Utilizing virtual screening and structurally guided design, we developed a small molecule FABP5 inhibitor that induces monocytic differentiation as observed by increased CD14 surface expression as a single agent and in combination with ATRA. FABP5 is not only a strong target to treat AML pts but also an excellent approach for developing a novel therapeutic for pts where FABP5 expression and activity is high. [Display omitted] DisclosuresMaciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy. Carraway:Agios: Consultancy, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Balaxa: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; FibroGen: Consultancy; Jazz: Speakers Bureau; Novartis: Speakers Bureau.

Radio-pharmaceuticals for cancer treatment: are they ready for prime time yet?

Radio-pharmaceuticals for cancer treatment: are they ready for prime time yet?

Abstract 1501: Developing novel strategy for the treatment of acute myeloid leukemia by targeting retinoic acid signaling pathways

Abstract Retinoic acid (RA), the active metabolite of vitamin A, influences biological processes by activating the retinoic acid receptor (RAR). RARs are ligand-controlled transcription factors that function as heterodimers with retinoid X receptors (RXRs) to regulate cell growth and survival. The success of RAR modulation in the treatment of acute promyelocytic leukaemia (APL) particularly by the use of all-trans retinoic acid (atRA) has stimulated considerable interest in the development of small molecules that can modulate RAR and RXR. Recent studies have demonstrated that RA can also activate the peroxisome proliferator-activated receptor β/δ (PPARβ/δ). In the aqueous intracellular milieu, RA is transported by the cellular retinoid-binding protein CRABP-II, or by the fatty-acid-binding protein FABP5, depending on the ratio of FABP5 to CRABP-II. In cells expressing high CRABP-II and low FABP5, RA activates the RAR, whereas in the presence of the reverse ratio, RA activates PPAR β/δ. These two different mode of RA delivery due to different ratio of these two cargos leads to opposite cellular outcomes. Cells harboring high level of CRABP-II, RA is delivered to RAR leading to apoptosis, growth arrest, and anticancer activity. However, when FABP5 expression is high RA is delivered to PPAR β/δ resulting in survival, proliferation, and tumor growth. In both cases, retinoid X receptor (RXR) is the indispensable partner of the nuclear receptor involved. The analysis of TCGA data set revealed that a certain class of AML patients have low level of CRABP-II and high level of FABP5, that in part explain the inability of at-RA to induce terminal differentiation in AML cells. To test our hypothesis, we screened AML and APL patient bone marrow cells and found that a number of AML patients bone marrow have high FABP5 and low CRABP-II protein levels while the atRA responding APL patients has opposite ratio determined by western blotting. Therefore, low CRABP-II and high FABP5 levels in a subset of AML patients lead to the activation of pro-survival PPAR β/δ pathway that promotes proliferation and opposes the differentiation. We also analyzed different AML cell lines for mRNA expression using qRT-PCR and protein by western blotting in using highly specific antibodies against FABP5 and CRABP-II. High FABP5 levels were observed in the majority of the AML cell lines. Efficacy of novel small molecule FABP5 inhibitor as a single agent and in combination with atRA was evaluated in HL-60 cells. Here, we demonstrate that a small molecule inhibitor of FABP5 synergizes with atRA and induces the differentiation in AML cells. Citation Format: Metis Hasipek, Dale Grabowski, James G. Phillips, Yihong Guan, Hetty Carraway, Jaroslaw Maciejewski, Babal K. Jha. Developing novel strategy for the treatment of acute myeloid leukemia by targeting retinoic acid signaling pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1501.

Abstract 4522: An optimized NGS workflow for detection of FLT3 internal tandem duplication (ITD) in AML samples

Abstract FLT3 ITD, a prognostic marker and drug target, is present in ~19% of AML patients. FLT3 ITD is a tandem repeat of the entire or partial exon 13-15 region. FLT3 ITDs range in size from 3bp to above 300bp and impact RTK signaling. Recent publications have shown that FLT3 ITD can be detected at single nucleotide resolution by NGS panels. However, detection of large FLT3 ITD and accurate reporting of ITD frequency remain challenging for NGS methods. False negative ITD results or inaccurate mutant/wild type allele ratio measurements could negatively alter treatment decisions for AML patients. The Illumina TruSight Myeloid panel (TMP) is a commercial NGS assay that covers 54 genes involved in myeloid malignancies. Although the TMP assay can detect SBS and small indel, medium to large size FLT3 ITD cannot be detected. Here we present an enhanced workflow to overcome these challenges. Positive controls, dynamic range, accuracy, and inter-run reproducibility were validated for the detection of SBS, indel, and FLT3 ITD using reference standards and DNA extracted from cell lines and AML patient EDTA blood and bone marrow aspirate samples. NGS libraries were prepared following Illumina's standard protocol. 275bp X2 sequencing instead of the standard 150bp X2 was run on MiSeq flowcell v3. Data analysis was performed using a MolecularMD custom pipeline. The analysis of FLT3 ITD was conducted using three methods including two off-the-shelf programs, Pindel and ITDseek, as well as Cigar, a MolecularMD developed ITD detection algorithm. FLT3 ITDs at 126 bp and 21 bp in PL-21 and MOLM-13 cell lines, respectively, were detected by TMP assay using the three algorithms. In contrast to ITDseek which only reports ITD sequence without frequency information, Pindel and Cigar report both ITD sequence and frequency. Compared to Pindel, ITD frequency reported by the custom algorithm is closer to that determined by the fragment analysis. Serial dilution of the 126bp ITD into wild type DNA showed the 0.55% ITD (from fragment analysis) can be reproducibly detected at ~0.6% by the Cigar algorithm. FLT3 ITDs ranging in size from 6bp to 300bp were detected in clinical AML samples using the enhanced TMP workflow and confirmed by a fragment analysis method. Limit of detection, reproducibility and accuracy of SBS and indel detection in other key genes by the TMP assay have also been validated. Low-level mutations, i.e. NRAS G12D at 1.7%, NRAS G13D at 2%, and IDH2 R140Q at 1.4%, were detected by TMP panel from AML samples and further confirmed with an independent assay. A long deletion (52bp) in exon 9 of CALR was also detected by the custom pipeline. In summary, the enhanced TMP workflow allows for accurate detection and reporting of SBS, indel and FLT3 ITDs including large duplications. Optimized detection of FLT3 ITDs will improve AML patient selection for targeted therapy. Citation Format: Xiaodong Wang, Zhenyu Yan, Peng Fang, Weihua Liu, Scott Glynn, Jennifer Biroschak, Chad Galderisi, Cindy Spittle, Jin Li. An optimized NGS workflow for detection of FLT3 internal tandem duplication (ITD) in AML samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4522.

Enforced miR-144-3p Expression as a Non-Invasive Biomarker for the Acute Myeloid Leukemia Patients Mainly by Targeting NRF2.

MicroRNAs (miRNAs) are endogenous non-coding RNAs of 19-25 nucleotides in length. Abnormal miRNA expression has been identified in various types of cancer. However, the aberrant expression of miR-1244 has not been reported in acute myeloid leukemia. First, the expression of miR-144-3p was explored in the bone marrow and peripheral blood of AML patients and healthy control. Then, we also evaluated the level of miR-144-3p in HL-60 cells. The possible target gene of miR-144-3p was predicted using TargetScan. Dual luciferase reporter assay was applied to validate the target gene of miR-144-3p. Cell viability and apoptosis on miR-144-3p was explored. Western blot analysis was applied to identify the downstream signaling of miR-144-3p. Our data showed that miR-144-3p was markedly increased in both the peripheral blood and bone marrow of AML patients compared with healthy controls. Moreover, we also found an increased expression of miR144-3p in HL-60 cells. In comparison, NRF2 protein expression was significantly decreased in HL-60 cells. Dual luciferase reporter assays demonstrated that miR-144-3p significantly suppressed the relative luciferase reporter activity of a pmirGLO-NRF2-3'UTR. In accordance with the downstream effects of NRF2 overexpression, inhibition of miR-144-3p reduced cell viability and prompted apoptosis. More importantly, we found that the inhibition of miR-144-3p in HL-60 cells could not enhance Caspase-3 activation when NRF2 protein expression was silenced. These findings suggest a potential oncogenic function of miR-144-3p in HL-60 cells, which is mainly achieved by targeting NRF2.

Role of Rheb in Human Acute Myeloid Leukemia

To investigate the role of Rheb (mTOR activator) in AML development by measuring Rheb expression in bone marrow of adult AML patients and in AML cell line HL-60. Real-time PCR assay was used to measure the Rheb mRNA expression in 27 AML patients and 29 ITP patients as control. The relationship between Rheb mRNA expression and age, AML subtype, fusion gene, splenomegaly, hepatomegaly and survival of AML patients was analyzed and compared. In addition, HL-60 cell line over-expressing Rheb was established, and the HL-60 cells and HL-60 cells with overexpression of Rheb were treated with Ara-C of different concentrations, the proliferation level was detected by CCK-8 method, and the IC50 was calculated. The mRNA level of Rheb in AML patients was similar to that in ITP patients (control). Interestingly, higher expression of Rheb was associated with better survival and was sensitive to Ara-C treatment. However, the expression level of Rheb was not associated with age, AML subtype, fusion gene, and hepatomegaly of patients. Lower expression level of Rheb was associated with splenomegaly. In vitro analysis of HL-60 line indicated that overexpression of Rheb could increased the cell sensitivity to Ara-C treatment (IC50=0.54 µmol/L) and caused HL-60 cell apoptosis. The lower Rheb expression is a poor prognostic indicator for AML patients, which is associated with AML splenomegaly, the patients and HL-60 cells with low expression of Rheb are insensitive to Ara-C treatment.

Neuropathy correlated with imbalanced Foxp3/IL-17 in bone marrow microenvironment of patients with acute myeloid leukemia.

Bone marrow (BM) neural tissues are important components of bone marrow microenvironment and play important roles in normal hematopoiesis. Neuropathy of BM can cause immunological alteration in hematopoietic microenvironment. It also can induce the impairment of normal hematopoiesis and promote the development of hematologic diseases. In the present study, we determined the expression levels and clinical significances of nerve-related molecules [nestin, tyrosine hydroxylase (TH), Glial Fibrillary Acidic protein (GFAP) and S100B] and T helper-related molecules (IL-17, Foxp3) in BM of AML patients and controls by immunohistochemical analysis and RT-PCR. Our results showed that the positive rates and expression levels of nestin, TH, GFAP and IL-17 were significantly decreased while Foxp3 and the ratio of Foxp3/IL-17 were statistically elevated in BM of AML patients. We found that there were significantly positive correlations between nestin with TH and IL-17 in BM of AML patients. We also observed significantly negative correlations between nestin with TH and Foxp3/IL-17 ratio. Moreover, the expression of nestin was positively correlated with the overall survival of AML patients. Our study suggests that neuropathy together with imbalanced T helper immunology in bone marrow might play important roles in AML.