Nucleophosmin Research Articles

Characterization and dynamics of specific T cells against nucleophosmin-1 (NPM1)-mutated peptides in patients with NPM1-mutated acute myeloid leukemia

Nucleophosmin(NPM1)-mutated protein, a leukemia-specific antigen, represents an ideal target for AML immunotherapy. We investigated the dynamics of NPM1-mutated-specific T cells on PB and BM samples, collected from 31 adult NPM1-mutated AML patients throughout the disease course, and stimulated with mixtures of 18 short and long peptides (9-18mers), deriving from the complete C-terminal of the NPM1-mutated protein. Two 9-mer peptides, namely LAVEEVSLR and AVEEVSLRK (13.9–14.9), were identified as the most immunogenic epitopes. IFNγ-producing NPM1-mutated-specific T cells were observed by ELISPOT assay after stimulation with peptides 13.9–14.9 in 43/85 (50.6%) PB and 34/80 (42.5%) BM samples. An inverse correlation between MRD kinetics and anti-leukemic specific T cells was observed. Cytokine Secretion Assays allowed to predominantly and respectively identify Effector Memory and Central Memory T cells among IFNγ–producing and IL2–producing T cells. Moreover, NPM1-mutated-specific CTLs against primary leukemic blasts or PHA-blasts pulsed with different peptide pools could be expanded ex vivo from NPM1-mutated AML patients or primed in healthy donors. We describe the spontaneous appearance and persistence of NPM1-mutated-specific T cells, which may contribute to the maintenance of long-lasting remissions. Future studies are warranted to investigate the potential role of both autologous and allogeneic adoptive immunotherapy in NPM1-mutated AML patients.

Mutated nucleophosmin 1 as immunotherapy target in acute myeloid leukemia.

The most frequent subtype of acute myeloid leukemia (AML) is defined by mutations in the nucleophosmin 1 (NPM1) gene. Mutated NPM1 (ΔNPM1) is an attractive target for immunotherapy, since it is an essential driver gene and 4 bp frameshift insertions occur in the same hotspot in 30%-35% of AMLs, resulting in a C-terminal alternative reading frame of 11 aa. By searching the HLA class I ligandome of primary AMLs, we identified multiple ΔNPM1-derived peptides. For one of these peptides, HLA-A*02:01-binding CLAVEEVSL, we searched for specific T cells in healthy individuals using peptide-HLA tetramers. Tetramer-positive CD8+ T cells were isolated and analyzed for reactivity against primary AMLs. From one clone with superior antitumor reactivity, we isolated the T cell receptor (TCR) and demonstrated specific recognition and lysis of HLA-A*02:01-positive ΔNPM1 AML after retroviral transfer to CD8+ and CD4+ T cells. Antitumor efficacy of TCR-transduced T cells was confirmed in immunodeficient mice engrafted with a human AML cell line expressing ΔNPM1. In conclusion, the data show that ΔNPM1-derived peptides are presented on AML and that CLAVEEVSL is a neoantigen that can be efficiently targeted on AML by ΔNPM1 TCR gene transfer. Immunotherapy targeting ΔNPM1 may therefore contribute to treatment of AML.

Cellular therapy against public neoantigens.

Neoantigen-targeted therapies have typically been based upon personalized neoantigen-specific vaccines; however, in this issue of JCI, van der Lee et al. describe the development of a potential cellular immunotherapy targeting a "public" neoantigen derived from nucleophosmin 1 (NPM1), which is mutated in approximately 30% of acute myeloid leukemias (AMLs). The authors use reverse immunology to predict, and biochemically confirm, NPM1-derived neoepitopes (ΔNPM1) and then generate high-avidity T cell clones and retrovirally transduced T cell populations that kill NPM1-mutated AML. This study provides a general approach to adoptive cellular therapy that can be applied to targeting other tumors with public neoantigens.

Computational modeling of early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) to identify personalized therapy using genomics

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) is an aggressive hematological malignancy for which optimal therapeutic approaches are poorly characterized. Using computational biology modeling (CBM) in conjunction with genomic data from cell lines and individual patients, we generated disease-specific protein network maps that were used to identify unique characteristics associated with the mutational profiles of ETP-ALL compared to non-ETP-ALL (T-ALL) cases and simulated cellular responses to a digital library of FDA-approved and investigational agents. Genomics-based classification of ETP-ALL patients using CBM had a prediction sensitivity and specificity of 93% and 87%, respectively. This analysis identified key genomic and pathway characteristics that are distinct in ETP-ALL including deletion of nucleophosmin-1 (NPM1), mutations of which are used to direct therapeutic decisions in acute myeloid leukemia. Computational simulations based on mutational profiles of 62 ETP-ALL patient models identified 87 unique targeted combination therapies in 56 of the 62 patients despite actionable mutations being present in only 37% of ETP-ALL patients. Shortlisted two-drug combinations were predicted to be synergistic in 11 profiles and were validated by in vitro chemosensitivity assays. In conclusion, computational modeling was able to identify unique biomarkers and pathways for ETP-ALL, and identify new drug combinations for potential clinical testing.

Allogeneic hematopoietic stem cells transplantation improves the survival of intermediate-risk acute myeloid leukemia patients aged less than 60years.

The prognosis of acute myeloid leukemia (AML) with normal karyotype is further determined by specific genetic alterations. The optimal post-remission therapy (PRT) in younger patients within this group after first complete remission (CR1) remains to be determined. We report a retrospective evaluation of PRT approaches in 223 patients under the age of 60years old with intermediate-risk AML in CR1. Patients receiving allogenic hematopoietic stem cell transplantation (alloHSCT) obtained improved overall survival (OS) than patients who treated with chemotherapy (5-year 61.6 ± 5.2% versus 41.1 ± 5.3%, p = 0.004). AlloHSCT led to fewer cases of relapse (hazard ratio [HR] 0.14, p < 0.001) and increased the relapse-free survival (RFS, HR 0.45, p < 0.001). With alloHSCT, the outcome of patients who reached negative minimal residual disease after 2cycles of consolidation could be further improved with an increased OS of 66% and RFS of 61%. Nucleophosmin-1 (NPM1) mutation negative, CCAAT/enhancer binding protein alpha (CEBPA) double mutation negative, and FLT-3 internal tandem duplication negative (NPM1mut-negCEBPAdm-negFLT3-ITDneg) patients had a significantly longer RFS with alloHSCT. In conclusion, our results provide additional evidence that alloHSCT is preferential PRT in patients with intermediate-risk AML that are under the age of 60years old in CR1.

Clinicopathological significance of common genetic alterations in patients with acute myeloid leukaemia 1-eight twenty-one of acute myeloid leukaemia: a retrospective cohort study

Objectives Acute myeloid leukaemia 1-eight twenty-one (AML1-ETO) is the most frequent abnormality seen in ∼40% of patients with acute myeloid leukaemia with French-American-British-M2 morphology and has received much attention over the past decade. We performed this study to investigate the prevalence and clinical significance of AML1-ETO in a cohort in Assam, India. Coprevalence and clinical significance of FMS-like tyrosine kinase 3, nucleophosmin 1 (NPM1), and p53 mutations, expression of epidermal growth factor receptor (EGFR), and flow markers were also documented and co-related with disease progress. Materials and methods Peripheral blood/bone marrow aspirates were collected from 165 de novo acute myeloid leukaemia patients. Reverse transcriptase PCR and real-time PCR assays were used for detection. Statistical analyses were carried out using statistical packages SPSS 22.0 and Epi Info 2000. Results AML1-ETO t(8;21)(q22;q22) were detected in 24 of 165 (14.60%, 95% confidence interval: 9.77–20.56) samples. The morphologic finding of bone marrow in AML1-ETO-positive patients revealed a higher occurrence of Auer rods. Prominent golgi and abnormal granules were abundantly found with an increasing number of large blasts. The prevalence of NPM1, p53 and EGFR were detected in 7/24 (29.50%), 2/24 (10.50%), and 4/24 (17%) patients, respectively. There was no significant difference in the overall survival (P=0.68) between AML1-ETO-positive and AML1-ETO-negative patients. White blood cell (WBC) count (P=0.00), platelet count (P=0.00), haemoglobin level (P=0.03) and blast count (P=0.01) showed a significant difference between AML1-ETO alive and dead patients. NPM1 mutation was associated with a high WBC count. Association of EGFR and NPM1 in AML-ETO-positive patients was not significant (P=0.75 and 0.88, respectively) compared with AML1-ETO-negative patients. Conclusion In this cohort study, the remission rate of AML1-ETO t(8;21) was not as high as compared with western countries. The presence of NPM1 mutants in AML1-ETO patents is a striking observation, but NPM1 and EGFR had no significant impact on AML1-ETO patients.

Clinico-Haematologic association and prognostic relevance of NPM1 and FLT3-ITD mutations in acute Myeloid Leukaemia.

Background & Objectives:Molecular genetic abnormalities have a significant role not only in diagnosis but also in determining the clinical course and prognosis. Nucleophosmin-1 (NPM-1) is associated with good prognosis while internal tandem duplication of the fms-like tyrosine kinase-3 gene (FLT3-ITD) confers a poor prognosis. Knowledge of the status of these mutations in AML patients not only guides treatment decisions but also helps in predicting response to frontline induction and consolidation chemotherapy as well as the risk of relapse and overall survival. Our objectives were to determine the prevalence, clinico-haematological features and immunophenotypic characteristics of AML patients with FLT3-ITD and NPM1 mutation and to evaluate the response to induction therapy (CR) and disease free survival (DFS) in this cohort of patients.Methods:Patients diagnosed as AML from March 2015 to March 2017 at Armed Forces Institute of Pathology Rawalpindi were included in the study. Clinico-haematologic and immunophenotypic parameters were noted and molecular analysis for FLT3-ITD and NPM1 mutation was performed. Any correlation with cytogenetics or other molecular markers was also studied. Response to standard induction chemotherapy and disease-free survival were assessed.Results:A total of 108 cases of AML were analyzed. Median age was 35 years and 64.8% were males. The median age of the study group was 35 years. Of these, 70 (64.8%) were males while 38 (35.2%) were females. Twenty-nine (26.9%) patients were NPM1 positive, twelve (11.1%) were FLT3-ITD positive while eight (7.4%) were positive for both mutations. Patients with NPM1 mutations were associated with female gender, higher haemoglobin level and platelet counts while those with FLT3-ITD mutations were predominantly seen in male patients and had significantly higher WBC counts, bone marrow blasts, biopsy cellularity and LDH levels. CR rates of NPM1 positive, FLT3-ITD positive and both mutation positive groups were 72%, 60% and 71%, respectively. The median disease-free survival was significantly lower in the FLT3-ITD positive group (7.1 months) as compared to the NPM1 positive group (16.1 months). The median disease-free survival was 12 months and 11.9 months in the NPM1 positive/FLT3-ITD positive and the NPM1 negative/FLT3-ITD negative groups, respectively.Conclusion:AML patients harbouring NPM1 and FLT3-ITD mutations have distinct clinical and haematological characteristics. NPM1 mutations have a better CR and DFS as compared to FLT3-ITD group.

RNA-Binding Protein HuR Regulates Rac1 Nucleocytoplasmic Shuttling Through Nucleophosmin in the Intestinal Epithelium.

Background & AimsThe mammalian intestinal epithelium is a rapidly self-renewing tissue in the body, and its homeostasis is tightly regulated via well-controlled mechanisms. The RNA-binding protein HuR is essential for maintaining gut epithelial integrity, and targeted deletion of HuR in intestinal epithelial cells (IECs) disrupts mucosal regeneration and delays repair after injury. Here, we defined the role of HuR in regulating subcellular distribution of small guanosine triphosphatase Rac1 and investigated the implication of nucleophosmin (NPM) as a molecular chaperone in this process.MethodsStudies were conducted in intestinal epithelial tissue-specific HuR knockout (IE-HuR-/-) mice and cultured IEC-6 cells, derived from rat small intestinal crypts. Functions of HuR and NPM in vitro were investigated via their gene silencing and overexpression.ResultsThe abundance of cytoplasmic Rac1 in the small intestinal mucosa increased significantly in IE-HuR-/- mice, although HuR deletion did not alter total Rac1 levels. HuR silencing in cultured IECs also increased the cytoplasmic Rac1 levels, without an effect on whole-cell Rac1 content. In addition, HuR deficiency in the intestinal epithelium decreased the levels of NPM in IE-HuR-/- mice and cultured IECs. NPM physically interacted with Rac1 and formed the NPM/Rac1 complex. NPM silencing decreased the NPM/Rac1 association and inhibited nuclear accumulation of Rac1, along with an increase in cytoplasmic abundances of Rac1. In contrast, ectopically expressed NPM enhanced Rac1 nuclear translocation and restored Rac1 subcellular localization to near normal in HuR-deficient cells.ConclusionsThese results indicate that HuR regulates Rac1 nucleocytoplasmic shuttling in the intestinal epithelium by altering NPM expression.

Quantitative Assay of Mutated Nucleophosmin in Acute Myeloid Leukemia.

BackgroundIn our previous work, we provided strong evidence that nucleophosmin (NPM) gene mutation has an important role in leukemogenesis of primary acute myeloid leukemia (AML). Furthermore, we speculated a new targeted therapy in patients with primary AML and bearing mutated NPM (mNPM). Based on these results together with findings of other researchers, it was essential to develop a method for accurate detection of mNPM.MethodsOur method based on utilizing the most recent flow cytometeric techniques and instruments in measuring mNPM. Attributed to their availability and technical feasibility, we used human leukemia cell lines to validate our method.ResultsThe main findings were differential expression of wild-type NPM (wtNPM) within the same sample. Furthermore flow cytometry (FCM) was a simple straightforward tool for quantitative assay of mNPM.ConclusionsIn this work we developed an innovative technique that could enable quantitative assay of mNPM, and ease its use as a biomarker in cytogenetic and molecular prognostication of primary AML. In addition the study suggested that FCM could differentiate mNPM expression within cells of the same patient thus could be used for monitoring of minimal residual disease.

Allogeneic transplant for FLT3-ITD mutated AML: a focus on FLT3 inhibitors before, during, and after transplant.

FMS-like tyrosine kinase 3 (FLT3) mutations are one of the most frequently encountered genetic alterations in acute myeloid leukemia (AML), and are generally associated with unfavorable outcomes. Several tools are currently available to provide an accurate prognosis for patients with these mutations, including FLT3 mutation type (internal tandem duplication versus tyrosine kinase domain), mutation allelic ratio (high versus low), and concurrent nucleophosmin-1 (NPM1) mutation, to help decide on optimal treatment. Recent advances in targeted therapies have paved the way for modern treatment strategies, such as the development of FLT3 kinase inhibitors. These novel drugs can be incorporated into any treatment component, including induction and consolidation, the relapse/refractory setting, bridging for transplant, salvage post-transplant, and as prophylactic long-term post-transplant maintenance. Many challenges remain though, such as their intolerability with high-dose chemotherapy in frail patients; whether their optimal use involves watchful waiting for molecular or hematologic relapse compared with prophylactic use as maintenance; and the exact role and indication for allogeneic stem cell transplantation, which arguably remains the only curative option for these high-risk patients.

Nucleophosmin Phosphorylation as a Diagnostic and Therapeutic Target for Ischemic AKI.

Background Ischemic AKI lacks a urinary marker for early diagnosis and an effective therapy. Differential nucleophosmin (NPM) phosphorylation is a potential early marker of ischemic renal cell injury and a therapeutic target.Methods Differential NPM phosphorylation was assessed by mass spectrometry in NPM harvested from murine and human primary renal epithelial cells, fresh kidney tissue, and urine before and after ischemic injury. The biologic behavior and toxicity of NPM was assessed using phospho-NPM mutant proteins that either mimic stress-induced or normal NPM phosphorylation. Peptides designed to interfere with NPM function were used to explore NPM as a therapeutic target.Results Within hours of stress, virtually identical phosphorylation changes were detected at distinct serine/threonine sites in NPM harvested from primary renal cells, tissue, and urine. A phosphomimic NPM protein that replicated phosphorylation under stress localized to the cytosol, formed monomers that interacted with Bax, a cell death protein, coaccumulated with Bax in isolated mitochondria, and significantly increased cell death after stress; wild-type NPM or a phosphomimic NPM with a normal phosphorylation configuration did not. Three renal targeted peptides designed to interfere with NPM at distinct functional sites significantly protected against cell death, and a single dose of one peptide administered several hours after ischemia that would be lethal in untreated mice significantly reduced AKI severity and improved survival.Conclusions These findings establish phosphorylated NPM as a potential early marker of ischemic AKI that links early diagnosis with effective therapeutic interventions.

Anaplastic lymphoma kinase fusions: Roles in cancer and therapeutic perspectives.

Receptor tyrosine kinase (RTK) anaplastic lymphoma kinase (ALK) serves a crucial role in brain development. ALK is located on the short arm of chromosome 2 (2p23) and exchange of chromosomal segments with other genes, including nucleophosmin (NPM), echinoderm microtubule-associated protein-like 4 (EML4) and Trk-fused gene (TFG), readily occurs. Such chromosomal translocation results in the formation of chimeric X-ALK fusion oncoproteins, which possess potential oncogenic functions due to constitutive activation of ALK kinase. These proteins contribute to the pathogenesis of various hematological malignancies and solid tumors, including lymphoma, lung cancer, inflammatory myofibroblastic tumors (IMTs), Spitz tumors, renal carcinoma, thyroid cancer, digestive tract cancer, breast cancer, leukemia and ovarian carcinoma. Targeting of ALK fusion oncoproteins exclusively, or in combination with ALK kinase inhibitors including crizotinib, is the most common therapeutic strategy. As is often the case for small-molecule tyrosine kinase inhibitors (TKIs), drug resistance eventually develops via an adaptive secondary mutation in the ALK fusion oncogene, or through engagement of alternative signaling mechanisms. The updated mechanisms of a variety of ALK fusions in tumorigenesis, proliferation and metastasis, in addition to targeted therapies are discussed below.

B23/Nucleophosmin promotes reconstitution of synaptic path in hippocampus after injury

B23, also known as nucleophosmin (NPM), is multifunctional protein directly implicated in cell proliferation, cell cycle progression, and cell survival. In the current study, in addition to confirming its anti-apoptotic function in neuronal survival, we demonstrated that the spatial-temporal expression profile of B23 during development of hippocampal neurons is high in the embryonic stage, down-regulated after birth, and preferentially localized at the tips of growing neuritis and branching points. Overexpression of B23 promotes axon growth with abundant branching points in growing hippocampal neurons, but depletion of B23 impairs axon growth, leading to neuronal death. Following injury to the trisynaptic path in hippocampal slice, overexpression of B23 remarkably increased the number and length of regenerative fibers in the mossy fiber path. Our study suggests that B23 expression in developing neurons is essential for neuritogenesis and axon growth and that up-regulation of B23 may be a strategy for enhancing the reconstitution of synaptic paths after injury to hippocampal synapses.

Validation of the 2017 European LeukemiaNet classification for acute myeloid leukemia with NPM1 and FLT3-internal tandem duplication genotypes.

The revised 2017 European LeukemiaNet (ELN) classification (ELN-2017) of acute myeloid leukemia (AML) divides patients into 3 prognostic risk categories, with additional factors such as the fms-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD) allele ratio (AR) considered for risk stratification. To the best of the authors' knowledge, the prognostic usefulness of ELN-2017 in comparison with ELN-2010 in younger patients with AML has not been validated to date. The authors performed a retrospective study on patients aged <60 years who received idarubicin plus cytarabine (IA)-based induction chemotherapy for newly diagnosed AML. According to ELN-2017 criteria, the number of patients in the favorable (Fav), intermediate (Int), and adverse (Adv) risk categories was 192 patients (27%), 331 patients (46%), and 192 patients (27%), respectively. Overall survival probabilities at 5 years in the Fav, Int, and Adv groups were 57%, 37%, and 18%, respectively. In comparison, the 5-year overall survival probabilities in the Fav (169 patients), intermediate (IR)-1 (80 patients), IR-2 (306 patients), and Adv (160 patients) ELN-2010 categories were 59%, 32%, 40%, and 14%, respectively. Although ELN-2010 historically distinguishes prognosis into IR-1 and IR-2 categories in younger patients, this difference was nullified in the current study cohort. When comparing patients with a low FLT3-ITD AR with those with a high FLT3-ITD AR, no significant differences in survival were noted among patients with nucleophosmin 1 (NPM1)-mutated AML (P=.28) or wild-type NPM1 (P=.35), and in those treated with IA alone (P=.79) or those treated with IA and a FLT3 inhibitor (P=.10). The ELN-2017 more accurately distinguishes prognosis in patients with newly diagnosed AML. The lack of prognostic significance for the FLT3-ITD AR needs further evaluation in different treatment settings.

Comprehensive Single-Cell RNA-Sequencing Mapping of Primary Acute Myeloid Leukemias and Profiling of NPM1-Mutated Cells

Introduction: Acute myeloid leukemia (AML) evolution is a multistep process in which cells evolve from hematopoietic stem and progenitor cells (HSPCs) that acquire genetic anomalies, such as chromosomal rearrangements and mutations, which define distinct subgroups. Mutations in Nucleophosmin 1 (NPM1), which occur in ~30% patients, are the most frequent subgroup-defining mutations in AML and appear to be a late driver event in this disease. Bulk RNA-sequencing studies have identified differentially expressed genes between AML subgroups, but they are uninformative of the composition of cell types populating each sample. Large scale Single-cell RNA sequencing (scRNA-seq) technologies now enable a detailed characterization of intra tumoral heterogeneity, and could help to better understand the stepwise evolution from normal to malignant cells.Methods: Twelve primary human AML specimens from MSKCC and Quebec Leukemia Cell Bank, including 8 with NPM1 mutations, were included in this cohort. Cells were subjected to scRNA-seq using 10X Genomics Chromium Single Cell 3' protocols and libraries were sequenced on Illumina HiSeq or NovaSeq platforms. FASTQ files were processed using SEQC pipeline (Azizi E et al, Cell 2018), resulting in a carefully filtered count matrix of > 100,000 single cells (4877 to 11532 cells per sample).Results: Using euclidean distance metrics and t-Distributed Stochastic Neighbor Embedding (t-SNE) visualization, we explored the phenotypic overlap between samples and showed that leukemia cells from different patients were mostly dissimilar, suggesting inter-sample heterogeneity. However, samples with similar morphology and similar NPM1 mutational status were phenotypically closer (Fig A), as anticipated from bulk RNA-sequencing data (TCGA, NEJM 2013). We partitioned cells into distinct clusters using Phenograph (Levine J et al, Cell 2015) (Fig B) and measured the diversity of samples per cluster using Shannon's entropy metric, revealing that mature cell types (B/plasma cells, T/NK and erythroid cells, Fig C), presumably excluded from the tumor bulk, are transcriptionally similar across samples. Most notably, the next most diverse cluster (C36), comprising 438 cells from 11/12 samples, contains cells with a HSPC-like phenotype, as suggested by i) highest correlation of the centroid of this cluster with HSC1 (lin-/CD133+/CD34dim) population from sorted bulk RNA-sequencing data (Novershtern N et al, Cell 2011), and ii) marked GSEA enrichment for stem cell signatures (top enrichment: Jaatinen_hematopoeitic_stem_cell_up, NES = 9.04, FDR q-val = 0).To study the extent to which NPM1 or other mutations drive heterogeneity in leukemia populations, we interrogated 3‘-derived single-cell sequences for all recurrent mutations in AML and found that NPM1 gene has unique features (e.g. relatively high single-cell expression and 3‘ localization) that allow specific identification of mutations in 5 to 34% of cells per mutated sample. To control for the high frequency of false negatives caused by dropouts in scRNA-seq data, we normalized the abundance of mutated vs wild-type cells to provide an estimation of mutation frequency in different cell types (Fig D). As expected, NPM1 mutations were rare in B and T/NK lymphoid cells (also observed using RT-qPCR in sorted populations by Dvorakova D et al, Leuk Lymphoma 2013) and were found in the majority of leukemia and myeloid cells. Interestingly, these mutations were detected at various frequencies in erythroid cells, suggesting that NPM1 mutations are acquired in cells with different lineage commitment in different patients. Most notably, the HSPC-like cluster C36 also contained a subpopulation of cells that have acquired NPM1 mutations and are transcriptionally different from wild-type cells.Conclusion: This study presents a first comprehensive single-cell map of primary AML, and the first 3'-based interrogation of mutations in single cells. It led to the identification phenotypically distinct cells presenting a HSPC-like expression profile which were sub-clonally harboring NPM1 mutations, providing the means to identify deregulated genes in these important leukemia subpopulations. DisclosuresLevine:Epizyme: Patents & Royalties; Celgene: Consultancy, Research Funding; Janssen: Consultancy, Honoraria; Isoplexis: Equity Ownership; C4 Therapeutics: Equity Ownership; Prelude: Research Funding; Gilead: Honoraria; Imago: Equity Ownership; Novartis: Consultancy; Roche: Consultancy, Research Funding; Loxo: Consultancy, Equity Ownership; Qiagen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Predicting Response to BET Inhibitor in Combination with Palbociclib / Sorafenib Using a Computational Model and Its Validation: A Beat AML Project Study

Predicting Response to BET Inhibitor in Combination with Palbociclib / Sorafenib Using a Computational Model and Its Validation: A Beat AML Project Study

Identification of Novel Pathways and Small Molecules Able to Down Regulate Oncogenes Expression By Compounds Screening Approaches in Acute Leukemia and Neuroblastoma Cells

Targeting anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase initially identified as a potent oncogenic driver in anaplastic large-cell lymphoma (ALCL) in the form of nucleophosmin (NPM)-ALK fusion protein, using tyrosine kinase inhibitors has shown to be a promising therapeutic approach for ALK-expressing tumors. However, resistance to ALK inhibitors is a ubiquitous problem in ALK-expressing cell lines as well as treated patients. Amplified ALK or mutated ALK was identified in ~14% of neuroblastomas (NB), the most common and aggressive childhood malignancy, and phase I trial of ALK inhibitor such as crizotinib showed a lack of response in patients harboring certain ALK mutations. Previous reports have suggested that mechanism of resistance is mediated by mutations in the ALK kinase domain impairing binding of an inhibitor to an ALK protein. Thus, new treatment modalities are urgently needed to sensitize patients to crizotinib thereby improving the management of hematological or solid malignancies harboring ALK mutations. To identify compounds with the potential of inhibiting oncogenic activity of ALK in NB, we implemented a high throughput chemical screen in 4 NB-derived cell lines, using a curated library of ~450 compounds. In the compounds screening, JAK-STAT kinase inhibitor (cucurbitacin I) was the most discriminatory with regard to sensitivity for ALK-mutated cell lines. Since Gamma cytokine JAK/STAT system as a target in the treatment of T-cell or myeloid malignancies, we analyzed the cytotoxicity of cucurbitacin I antitumor effect using an MTT assay revealed cucurbitacin I to possess potent cytotoxic activity across a broad spectrum of hematopoietic malignancies, with T-acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) being especially responsive. For example, T-ALL cell lines, as well as AML lines, were potently inhibited by cucurbitacin I (IC50values:T-ALL lines, ICH-TALL-UK, 103.2 nM; MOLT-14, 35.6 nM; KCMC-T, 134.2 nM; Jurkat, 77 nM; ICH-TALL-SM, 47.7 nM; MOLT-4, 22.2 nM. AML lines, HEL, 66.7 nM; Kasumi-3, 4.5 nM; KG-1, 53.6 nM; THP-1, 337.2 nM). In an expanded panel of 20 NB cell lines, those with or without MYCN-amplification or 11q loss of heterozygousity which have been identified as two major oncogenic events in NB pathogenesis, especially in the high-risk group were the most sensitive to low nanomolar concentrations of cucurbitacin I. In NB cell lines harboring F1174L or R1275Q-mutated ALK, crizotinib combined with cucurbitacin I enhanced tumor responses and showed synergistic cytotoxicity.Although crizotinib and cucurbitacin I alone or combination therapy (cucurbitacin I + crizotinib) did not result in decreased viability over control compared with vehicle, the combination therapy in all of 6 cell lines with ALK aberrations and 10 of 13 ALK wild-type cell lines with MYCN amplification or 11q LOH was more effective than vehicle, crizotinib alone, and cucurbitacin I alone. Analysis of downstream signalling through MAPK, AKT and STAT3 pathways showed that NIH3T3 cells stably expressed F1174L mutated ALK or TGW cells harboring R1275Q-mutated ALK, expressed lower levels of pERK, pAKT and pSTAT3 in combination therapy compared with cells treated with cucurbitacin I or crizotinib alone. These findings may provide a indication that the combination of low-dose ALK and STAT3 inhibitors may be benefitical for the treatment of NB, by enhancing efficacy while reducing toxicity. In conclusion, our studies suggested that NB, T-ALL or AML cell lines also exhibited potent cytotoxic responses to the cucurbitacin I and the combination of ALK and JAK-STAT inhibitors could be a valuable therapeutic option for ALK mutated malignancies such as high-risk NB with potential clinical application. DisclosuresNo relevant conflicts of interest to declare.

CRISPR/Cas9 Generation of Npm1-Alk in Transplantable Murine Hematopoietic Stem Cells Accurately Models ALK-Positive Lymphoma in Recipients

Chromosomal rearrangements resulting in generation of novel fusion oncogenes are common in hematologic malignancies. These disease drivers are key therapeutic targets and form the basis of animal model development for preclinical studies. For example, retroviral introduction of the chronic myeloid leukemia (CML) fusion BCR-ABL to hematopoietic stem cells (HSCs) results in a myeloproliferative disease similar to accelerated-phase human CML when transplanted to recipient mice. This model and many others are established traditionally through retroviral or germline introduction of human fusion oncogenes to the murine genome. Recent advances in CRISPR/Cas9 technology now permit direct editing of the murine genome to create endogenous genotypes that more accurately reflect configurations found in human diseases. To date, these techniques have not been successfully applied to the modeling of fusion oncogene-driven hematologic malignancies.Anaplastic Large Cell lymphoma (ALCL) is a T-cell non-Hodgkin lymphoma common in adolescents and young adults and driven in ~70% of cases by chromosomal rearrangements involving Anaplastic Lymphoma Kinase (ALK). Most commonly, t(2:5; p23:q35) fuses the 3‘ ALK kinase domain to the 5‘ oligomerization domain of the constitutively expressed Nucleophosmin1 (NPM1) gene. An existing immunocompetent model of ALK+ lymphoma employs a CD4 promoter-driven NPM1-ALK transgene but results in immature T-lymphomas in about two-thirds and B-lineage plasma-cell lymphomas in the rest of animals. We employed CRISPR/Cas9 vectors containing guide strands designed to generate double-stranded DNA cleavage in mouse chromosomes 11 and 17 at breakpoints predicted to generate an in-frame Npm1-Alk fusion oncogene. Wild-type HSCs derived from fetal livers were divided and subjected to either transient CRISPR or mock transfection during a brief ex vivo passage followed by immediate transplantation to sub-lethally irradiated wild-type recipients.Mice initially transplanted with CRISPR-modified HSCs developed an ALK+ large cell lymphoma with a latency of ~9 months, while mock transfected controls sacrificed in parallel were phenotypically normal. qPCR analysis of lymphoid organs from mice that developed disease showed extremely high expression of Alk and Tnfrsf8, which encodes CD30. Pathologically, tumors contained large malignancy T cells with anaplastic morphology. Immunohistochemistry confirmed ALK protein expression, including nuclear localization classic for NPM1-ALK, and intense CD30 cell-surface staining. One recipient instead developed a CD30-negative ALK+ diffuse large B-cell lymphoma. Transplantation of primary T-lymphoma cells to secondary recipients resulted also in ALK+ T-cell lymphomas in recipients with more rapid onset infiltrating lymph nodes, spleen, liver, and other organs. T-cell receptor clonality analyses through β-chain deep sequencing show an oligoclonal T-cell disease in both primary and secondary recipients.We therefore demonstrate successful genomic editing of transplantable murine hematopoietic stem cells to generate a novel model of a fusion oncogene-driven hematologic malignancy. These methods are widely applicable to additional lymphomas and leukemias and could fuel development of improved in vivo preclinical model systems. DisclosuresNo relevant conflicts of interest to declare.

FLT3 and NPM1 Are Powerful Determinants of Outcome in Acute Myeloid Leukemia Patients Treated with Autologous Stem Cell Transplantation: An Analysis By the Acute Leukemia Working Party of the EBMT

Background: While intensive consolidation therapy with autologous stem cell transplantation (ASCT) can secure a remission in selected Acute Myeloid Leukemia (AML) patients with intermediate-risk cytogenetics, a substantial proportion will ultimately relapse. Knowledge of the mutational status of FMS like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) and nucleophosmin (NPM) 1 and their possible combinations could further refine a subset of intermediate cytogenetic risk patients who may benefit from ASCT. However, data are limited. We, therefore, set out to evaluate the impact of FLT3-ITD and NPM1 in a large cohort of patients undergoing ASCT.Methods: This was a retrospective analysis of the Acute Leukemia Working Parity of the European Society for Blood and Marrow Transplantation (EBMT) registry. We included 405 de-novo AML patients, from 51 European centers, with intermediate-risk cytogenetics (Grimwade et al., Blood 2010) and complete data on FLT3-ITD and NPM1 status, receiving an ASCT at first complete remission (CR1), between 2000-2014. Leukemia free survival (LFS) was the primary outcome. Secondary outcomes were overall survival (OS), transplantation-related mortality (TRM), and relapse incidence (RI). The latter two were considered as competing events. Univariate and multivariable Cox regression models, adjusted for recipient sex, age, Karnofsky performance status, FLT3/NPM1 combinations, days from diagnosis to transplantation, stem cell source -bone marrow or peripheral blood (PB), and use of total-body irradiation-based conditioning.Results: Patients included had a median age of 52 years and received an autograft at median of 5 months from diagnosis. PB-based autograft and non-TBI conditioning were used in the majority of patients (93% and 90%, respectively). FLT3-/NPM1- was the leading molecular combination (50%), followed by FLT3-/NPM1+ (30%), FLT3+/NPM1+ (11%), and FLT3+/NPM1- (9%). Age, time from diagnosis to transplantation, graft source, and use of TBI were similar between the molecular subgroups. The median year of transplantation was earlier in NPM1- patients (FLT3+/NPM1- 2008, FLT3-/NPM1- 2009, FLT3-/NPM1+ 2010, FLT3+/NPM1+ 2011, p<0.001). In the univariate analysis, age, molecular makers, time from diagnosis to transplantation, and use of TBI were associated with the risk for experiencing an LFS event. Patients in the FLT3-/NPM1+ subgroup had the most favorable outcome (5-years LFS [62%] and OS [74%]). Nonetheless, 5 years following ASCT the RI was 35% (Figure). FLT3+/NPM1- were the least likely to survive due to a tendency to relapse: 5-years LFS (21%), OS (33%),and RI (79%). In a Cox multivariable model, the combination of molecular markers were the strongest predictors of LFS: FLT3-/NPM1+ (reference), FLT3-/NPM1- HR 2.39 (1.65-3.46), FLT3+/NPM1+ HR 2.24 (1.33-3.75), FLT3+/NPM1- HR 5.09 (2.94-8.82). We did not find interactions between molecular markers and other covariates in the model.Conclusions: In this pioneering analysis of CR1-AML patients, with intermediate risk cytogenetics, receiving an ASCT as post-remission therapy, we show that the molecular subgroups of FLT3-ITD and NPM1 are highly prognostic. In line with previous publications in the allogeneic setting, patients with FLT3-/NPM1+ have promising outcomes, while those with FLT3+/NPM1- do poorly. Testing for the role of additional markers, such as isocitrate dehydrogenase (IDH), could further identify populations who may derive the most benefit from ASCT. DisclosuresBaerlocher:Novartis: Research Funding. Foà:NOVARTIS: Speakers Bureau; ABBVIE: Other: ADVISORY BOARD, Speakers Bureau; CELGENE: Other: ADVISORY BOARD, Speakers Bureau; JANSSEN: Other: ADVISORY BOARD, Speakers Bureau; ROCHE: Other: ADVISORY BOARD, Speakers Bureau; GILEAD: Speakers Bureau; CELTRION: Other: ADVISORY BOARD; AMGEN: Other: ADVISORY BOARD; INCYTE: Other: ADVISORY BOARD. Haenel:Novartis: Honoraria; Amgen: Honoraria; Takeda: Honoraria; Roche: Honoraria. Zuckerman:Cellect Biotherapeutics Ltd: Consultancy. Mohty:MaaT Pharma: Consultancy, Honoraria.

Elucidation of the IMiDs-Resistant Mechanism and Development of the Overcoming Drugs Inducing CRBN Independent G2/M Cell Cycle Arrest in Myeloma Cells

[Introduction]Recent development of novel drugs significantly improved prognosis of Multiple Myeloma (MM). Immunomodulatory drugs (IMiDs) and proteasome inhibitors play central roles in MM therapy. However expanded usage of lenalidomide (Len) has increased the number of Len-resistant patients. And limited information is obtainable with regand to Len-resistant mechanism, such as overexpression and genetic mutation of IMiDs-binding protein, celeblon (CRBN); thus, elucidating the Len-resistant mechanism and development of drugs overcoming the Len-resistance are very important for improving the outcome of MM. The purposes of this study are [1] to clarify the molecular mechanism of Len-resistance using Len-resistant MM cell lines [2] to confirm the mechanism of CRBN independent myeloma cell death by novel phthalimide-derivatives, TC11 and PEG(E)-TC11.[Method][1] In our laboratory, Len-resistant cell lines, KMS21R, KMS27R and MUM24R have been established by long-term co-culture with low-dose Len. Using these cell lines, we examined expression of CRBN and the downstream molecules, IKZF1/3, IRF4 and c-MYC by western blotting. We also examined the mutation of CRBN in KMS27R. [2] We have originally developed a novel phthalimide-derivative, TC11 and PEG(E)-TC11 synthesized for improving water solubility. We examined whether TC11 and PEG(E)-TC11 induced cell death to Len-resistance MM or not.[Result][1] First, we validated expression of CRBN and the down-stream molecules, which mediate pharmacological action of Len. Decreased expression of CRBN and subsequent up-regulation of down-stream IKZF1 were confirmed in KMS21R cell. In KMS27R cell, IKZF 1/3 expressions are increased without alteration of CRBN expression level. Thus, genetic mutation in CRBN or IKZF1 is suspected in KMS27R cells. In MUM24R cell, no significant change in the expression levels of the CRBN pathway molecules was confirmed, suggesting other molecular alternation than CRBN pathway.[2] TC11 significantly induced apoptosis of Len-resistant cells. We have previously reported that TC11 didn't bind to CRBN and TC11 directly bound to nucleophosmin1 (NPM1) and α-tubulin. It was found that TC11 induced G2/M arrest and subsequent apoptosis by inhibition of tubulin polymerization and NPM1 oligomerization. Fluorescence microscopy observation showed that TC11 treatment induced hyper duplication of centrosomes in MM cells. Water solubility and blood absorption of PEG(E)-TC11 were significantly improved compared with those of TC11. As a consequence, PEG-(E)TC11 significantly delayed tumor growth in xenograft model mice.[Discussion & Conclusion][1] Our present data suggested diversity of Len-resistant mechanism in MM patients. For example, in KMS21R, decreased expression of CRBN was likely the cause of Len-resistance. In KMS27R, genetic mutation in CRBN-IKZF1 pathway caused inhibition of IKZF1 degradation. In MUM24R, the Len-resistant mechanism didn't relate to the CRBN pathway but to unknown molecular mechanism. Len-resistant cell lines are useful tools for studying Len-resistant mechanisms and developing drugs overcoming Len-resistance.[2]TC11 abrogated tubulin polymerization and NPM1 oligomerization, induced centrosome disruption and G2/M arrest. Since G2/M check point doesn't closely rely on p53. TC11 was able to induce apoptosis of MM cells with high-risk cytogenetic mutations such as deletion of TP53 gene. TC11 and PEG-(E)TC11 are expected as a candidate compound overcoming Len-resistance and high-risk MM. DisclosuresMatsushita:Amgen: Research Funding. Hattori:Takeda: Research Funding; IDAC inc.: Research Funding.