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Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders, representing high risk of progression to acute myeloid leukaemia, and frequently associated to somatic mutations, notably in the epigenetic regulator TET2. Natural Killer (NK) cells play a role in the anti-leukemic immune response via their cytolytic activity. Here we show that patients with MDS clones harbouring mutations in the TET2 gene are characterised by phenotypic defects in their circulating NK cells. Remarkably, NK cells and MDS clones from the same patient share the TET2 genotype, and the NK cells are characterised by increased methylation of genomic DNA and reduced expression of Killer Immunoglobulin-like receptors (KIR), perforin, and TNF-α. In vitro inhibition of TET2 in NK cells of healthy donors reduces their cytotoxicity, supporting its critical role in NK cell function. Conversely, NK cells from patients treated with azacytidine (#NCT02985190; https://clinicaltrials.gov/) show increased KIR and cytolytic protein expression, and IFN-γ production. Altogether, our findings show that, in addition to their oncogenic consequences in the myeloid cell subsets, TET2 mutations contribute to repressing NK-cell function in MDS patients.

The extrafollicular immune response is essential to generate a rapid but transient wave of protective antibodies during infection. Despite its importance, the molecular mechanisms controlling this first response are poorly understood. Here, we demonstrate that enhanced Cxcr4 signaling caused by defective receptor desensitization leads to exacerbated extrafollicular B-cell response. Using a mouse model bearing a gain-of-function mutation of Cxcr4 described in 2 human hematologic disorders, warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome and Waldenström macroglobulinemia, we demonstrated that mutant B cells exhibited enhanced mechanistic target of rapamycin signaling, cycled more, and differentiated more potently into plasma cells than wild-type B cells after Toll-like receptor (TLR) stimulation. Moreover, Cxcr4 gain of function promoted enhanced homing and persistence of immature plasma cells in the bone marrow, a phenomenon recapitulated in WHIM syndrome patient samples. This translated in increased and more sustained production of antibodies after T-independent immunization in Cxcr4 mutant mice. Thus, our results establish that fine-tuning of Cxcr4 signaling is essential to limit the strength and length of the extrafollicular immune response.

Innate lymphoid cells (ILCs) - which include cytotoxic Natural Killer (NK) cells and helper-type ILC - are important regulators of tissue immune homeostasis, with possible roles in tumor surveillance. We analyzed ILC and their functionality in human lymph nodes (LN). In LN, NK cells and ILC3 were the prominent subpopulations. Among the ILC3s, we identified a CD56

WHIM Syndrome (WS) is a rare immunodeficiency caused by gain-of-function CXCR4 mutations. Here we report for the first time a substantial decrease in bone mineral density in 25% of WS patients and bone defects leading to osteoporosis in a WS mouse model. Reduction in bone content involved impaired CXCR4 desensitization that disrupts cell cycle progression and osteogenic specification of mouse bone marrow (BM)-residing skeletal stromal/stem cells (SSCs). This was also evidenced in BM stromal cells from WS patients. Consistent with this, chronic treatment with the CXCR4 antagonist AMD3100 normalized in vitro osteogenic fate of mutant SSCs and reversed in vivo loss in skeletal cells, thus demonstrating that proper CXCR4 desensitization is required for the osteogenic specification of BM SSCs. Our study provides novel mechanistic insights into how CXCR4 signaling regulates the osteogenic fate of BM SSCs.

Mesenchymal stromal cells (MSC) are a rare, heterogeneous and multipotent population that can be isolated from several tissues. MSC were originally discovered in the bone marrow and studied for their capacity to maintain hematopoietic cells. We will describe here methods to isolate, culture, and bank MSC from human bone marrow. Then, characterization protocols by flow cytometry, clonogenic assays and doubling time evaluation will be developed. Finally, in vitro MSC culture and differentiation into osteoblasts, adipocytes, and chondrocytes will be explained. Thus, this chapter will detail all bases to work on MSC with consensus and clear methods and protocols.

Bone marrow (BM) microenvironment contributes to the regulation of normal hematopoiesis through a finely tuned balance of self-renewal and differentiation processes, cell-cell interaction and secretion of cytokines that during leukemogenesis are altered and favor tumor cell growth. In pediatric acute myeloid leukemia (AML), chemotherapy is the standard of care, but still >30% of patients relapse. The need to accelerate the evaluation of innovative medicines prompted us to investigate the mesenchymal stromal cells (MSCs) role in the leukemic niche to define its contribution to the mechanisms of leukemia escape. We generated humanized three-dimensional (3D) niche with AML cells and MSCs derived from either patients (AML-MSCs) or healthy donors. We observed that AML cells establish physical connections with MSCs, mediating a reprogrammed transcriptome inducing aberrant cell proliferation and differentiation, and severely compromising their immunomodulatory capability. We confirmed that AML cells modulate h-MSCs transcriptional profile promoting functions similar to the AML-MSCs when co-cultured in vitro, thus facilitating leukemia progression. Conversely, MSCs derived from BM of patients at time of disease remission showed recovered healthy features, at transcriptional and functional levels, including the secretome. We proved that AML blasts alter MSCs activities in the BM niche, favoring disease development and progression. We discovered that a novel AML-MSCs selective CaV1.2 channel blocker drug, Lercanidipine, is able to impair leukemia progression in 3D niche both in vitro and when implanted in vivo, if used in combination with chemotherapy, supporting the hypothesis that synergistic effects can be obtained by dual targeting approaches.

Context: Systemic inflammatory and auto-immune diseases (SIAD) are observed in 25% of patients with MDS or CMML, who harbor a high frequency of mutations in TET2, IDH1/2 and SRSF2 as we previously reported (Zhao et al, Leukemia 2021). Recently, the identification of mutations in UBA1 in patients with autoinflammatory disorders, some of whom have dysplastic bone marrow, defined novel disease called VEXAS syndrome (Beck et al, NEJM 2020). Objective: To explore the prevalence of UBA1 mutations in our initial cohort of MDS/CMML patients with SIAD (n=85) (Zhao et al, Leukemia 2021). Design and Patients: All male patients for whom material was available (n=33) were analyzed for the presence of UBA1 mutations. Primary Outcome: UBA1 mutations screening with Sanger sequencing. Results: Four out of 33 (12%) patients had UBA1 mutations. All of them had MDS without excess of blasts. Cytomorphologic review of bone marrow smears showed numerous vacuoles in precursors in all 4 cases. Karyotype was normal in 3/4 patients, and one patient had deletion 9q. One patient also had somatic mutations in DNMT3A (VAF 11%) and TP53 (VAF 2%), and another patient had a mutation of TET2 (VAF 18%). Three patients in our MDS/CMML SIAD cohort had relapsing polychondritis, and 2 of them had UBA1 mutations, while 6 patients from our initial SIAD cohort had a diagnosis of Sweet syndrome, including 2 with UBA1 mutations. Three out of 4mut patients had steroid refractory SIAD and were heavily treated with other immunosuppressive therapies including methotrexate, azathioprine or cytokine targeting agents. At last follow-up, none of the 4 patients with UBA1 mutations had MDS progression, and one of them had died from a stroke, 2.4 years after MDS diagnosis. Conclusions: Our retrospective study represents the first assessment of VEXAS syndrome among patients with myeloid malignancies associated with SIAD. The low prevalence of UBA1 mutations in our cohort suggests that other pathological mechanisms may drive inflammation in the large majority of MDS/CMML patients with associated SIAD, likely in relation with mutations in epigenetic regulators TET2/IDH and SRSF2 as we previously proposed.

Mesenchymal stromal cells (MSC) are a rare, heterogeneous and multipotent population that can be isolated from several tissues. MSC were originally discovered in the bone marrow and studied for their capacity to maintain hematopoietic cells. We will describe here methods to isolate, culture, and bank MSC from human bone marrow. Then, characterization protocols by flow cytometry, clonogenic assays and doubling time evaluation will be developed. Finally, in vitro MSC culture and differentiation into osteoblasts, adipocytes, and chondrocytes will be explained. Thus, this chapter will detail all bases to work on MSC with consensus and clear methods and protocols.

Bone marrow (BM) microenvironment contributes to the regulation of normal hematopoiesis through a finely tuned balance of self-renewal and differentiation processes, cell-cell interaction, and secretion of cytokines that during leukemogenesis are altered and favor tumor cell growth. In pediatric acute myeloid leukemia (AML), chemotherapy is the standard of care, but30% of patients still relapse. The need to accelerate the evaluation of innovative medicines prompted us to investigate the role of mesenchymal stromal cells (MSCs) in the leukemic niche to define its contribution to the mechanism of leukemia drug escape. We generated a humanized 3-dimensional (3D) niche with AML cells and MSCs derived from either patients (AML-MSCs) or healthy donors. We observed that AML cells establish physical connections with MSCs, mediating a reprogrammed transcriptome inducing aberrant cell proliferation and differentiation and severely compromising their immunomodulatory capability. We confirmed that AML cells modulate h-MSCs transcriptional profile promoting functions similar to the AML-MSCs when cocultured in vitro, thus facilitating leukemia progression. Conversely, MSCs derived from BM of patients at time of disease remission showed recovered healthy features at transcriptional and functional levels, including the secretome. We proved that AML blasts alter MSCs activities in the BM niche, favoring disease development and progression. We discovered that a novel AML-MSC selective CaV1.2 channel blocker drug, lercanidipine, is able to impair leukemia progression in 3D both in vitro and when implanted in vivo if used in combination with chemotherapy, supporting the hypothesis that synergistic effects can be obtained by dual targeting approaches.

cAMP response element binding protein (CREB) is frequently overexpressed in acute myeloid leukemia (AML) and acts as a proto-oncogene; however, it is still debated whether such overactivation alone is able to induce leukemia as its pathogenetic downstream signaling is still unclear. We generated a zebrafish model overexpressing CREB in the myeloid lineage, which showed an aberrant regulation of primitive hematopoiesis, and in 79% of adult CREB-zebrafish a block of myeloid differentiation, triggering to a monocytic leukemia akin the human counterpart. Gene expression analysis of CREB-zebrafish revealed a signature of 20 differentially expressed human homologous CREB targets in common with pediatric AML. Among them, we demonstrated that CREB overexpression increased CCAAT-enhancer-binding protein-δ (C/EBPδ) levels to cause myeloid differentiation arrest, and the silencing of CREB-C/EBPδ axis restored myeloid terminal differentiation. Then, C/EBPδ overexpression was found to identify a subset of pediatric AML affected by a block of myeloid differentiation at monocytic stage who presented a significant higher relapse risk and the enrichment of aggressive signatures. Finally, this study unveils the aberrant activation of CREB-C/EBPδ axis concurring to AML onset by disrupting the myeloid cell differentiation process. We provide a novel in vivo model to perform high-throughput drug screening for AML cure improvement.

Chemotherapy still remains the pillar of treatment of children with AML, a disease in which refinements in diagnostic approaches, minimal residual disease monitoring, and patient stratification have resulted into remarkable progresses during the past decade. However, most of the recently tested, novel anti-leukemia agents failed during pre-clinical and clinical validation phases, and one main limit in AML field is the inappropriateness of current preclinical models used to study drug efficacy, this jeopardizing the advance of phase II and III clinical trials, especially for children. In light of this consideration, we aimed at creating novel robust in vitro and in vivo approaches to discover or to re-assess alternative treatments to improve the portfolio of agents active in childhood AML. For this purpose, we developed new protocols for long-term 3D-AML cultures to perform more predictable high throughput drug screening in vitro, and, once identified the best compounds, to create new pre-clinical in vivo models. We set up the bone marrow (BM) endosteal niche by using a biomimetic 3D structure, made up of engineered hydroxyapatite and collagen I, where we seeded mesenchymal stromal cells derived either from AML patients (AML-MSCs) or from healthy BM donors (h-MSCs), together with osteoblasts, endothelial cells and finally AML blasts. We studied AML cell proliferation and clonogenicity cultured in 3D. We obtained results from twenty 3D long-term cultures of different primary AML, confirming blast proliferation up to 21 days. Clonogenic potential and immunophenotype preservation of the original AML blasts was also documented. At the same time, we compared AML-MSCs with h-MSCs, finding that AML-MSCs exhibited a higher proliferation rate (40% increase proliferation at 72 and 96 hours, p Disclosures Locatelli: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria; bluebird bio: Consultancy.

In acute myeloid leukemia (AML), the assessment of post-induction minimal residual disease (MRD) is largely utilized for choosing post-remission therapies aimed at maintaining complete remission (CR) and preventing relapse. This latter is still the major cause of treatment failure in pediatric AML, and even if several efforts have been spent to validate MRD as a prognostic marker, numerous studies demonstrated that MRD negativity cannot be considered a completely reliable surrogate biomarker predicting outcome, since it does not exclude a relapse. The current interpretation is that disease relapse is due to mechanisms leading to therapy resistance mainly depending on driver chimeric or oncogenic protein-coding genes, which are monitored during treatment, and does not consider that chemotherapy resistance may arise from other genetic markers, phenomenon linked to methylation and non-coding RNAs genomic pressure. We, thus, hypothesized that other markers need to be explored to re-interpret leukemia progression. We showed an overall hyper-expression of the lncRNA BALR2 in 132 de novo AML bone marrow samples collected at diagnosis and analyzed the gene expression profile (GEP) of 58 cases. By unsupervised clustering analysis, we produced important advances in identifying BALR2 as a robust novel molecular marker of a new subgroup of AML characterized by a high rate of resistance to induction therapy, independently from the genetic lesions detected at diagnosis and any other prognostic clinical and genetic features. We demonstrated in vitro that BALR2 has a direct role in controlling bi-directionally its own and of its neighbor gene CDK6 promoter activity. This latter finding of high CDK6 expression was shown to sustain its complex with RUNX1 in order to inhibit RUNX1 binding to its target promoters, thus preventing the process of hematopoietic differentiation progression. To support BALR2 as a new proto-oncogene involved in the control of the myeloid differentiation program, we ranked the genes across the expression profile obtaining a signature of 337 transcripts able to cluster CD34+ human stem cell precursors (HSCPs) separately from more mature CD14+ cells. These in silico findings were validated in vitro by showing that, after BALR2 depletion, CD34+ cells had a skewed myeloid differentiation. Furthermore, we found that AML differentiation toward mature myeloid cells with increased phagocytic capacity was obtained through BALR2 level reduction, and enhanced by combinatorial differentiation stimuli. Our findings attribute a distinct role to BALR2 in the block of myeloid stem cell differentiation occurring during leukemogenesis. At the same time, we interrogated GEP ontology, finding that enrichments of genes involved in mitochondrial synthesis pathways were significantly correlated to patients with highest BALR2 levels, and confirmed the same mitochondriogenesis profile in the immature CD34+ HSCPs. We moved to deconvolute this feature and demonstrated that BALR2, by controlling mitochondria gene balance, was directly controlling the mitochondrial mass, which dramatically decreased after BALR2 silencing, this supporting the hypothesis that BALR2 would maintain mitochondrial functions to confer AML resistance to cytotoxicity. Consistently with this line of reasoning, we inhibited mitochondria by tigecycline, demonstrating that its activity was dramatically strengthened in BALR2 depleted cells, when used either alone or in combination with cytosine-arabinoside (Ara-C). Concomitantly, tigecycline treatment in BALR2 silenced AML cells reduced mitochondria depolarization, and increased the number of differentiated M-CFU colonies formation, confirming that BALR2, together with CDK6, forms novel transcriptional networks to create a circuit able to impair myeloid differentiation and to lower chemo-sensitivity in AML. We speculate that a novel therapeutic window of mitochondrial targeting in defined AML subgroups, identified through assessment of BALR2 levels at diagnosis or persistent MRD levels, could be envisaged to optimize the outcome of childhood AML. Disclosures Locatelli: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria.

The proto-oncogene c-KIT, which encodes a receptor for stem cell factor (SCF), belongs to the type-III receptor of the tyrosine kinase subfamily and is characterized by five extracellular immunoglobulin-like domains, a single transmembrane helix (TM), a cytoplasmic juxtamembrane domain(JMD), and a kinase domain. Abnormal activation of c-KIT/SCF growth signal has been frequently documented to occur in cancers, including hematological malignancies, and has been frequently associated with poor prognosis in adults with acute myeloid leukemia (AML) harboring aberrancies at core-binding factor genes (CBF). c-KIT mutations have been reported in pediatric CBF-rearranged AML at frequencies ranging from 15 to 54.5%; however, their prognostic significance is still debated. Mutations of c-KIT occur in the extracellular portion of the receptor implicated in dimerization within exon 8, in the TM-JMD domain within exon 11 and in the activation loop of the tyrosine kinase domain within exon 17, this mediating the constitutive activation of the receptor.

Purpose. Among pediatric acute myeloid leukemia (AML), the t(6;11)(q27;q23) MLL-AF6 translocation accounts for 26% of MLL-rearranged AML, and is associated with a worse prognosis (event-free survival of 23.3% at 3-years) compared to other forms of MLL-rearranged AML1. Gene expression profile analysis revealed a specific transcriptional signature, and this peculiarity has been explained by the mislocalization of AF6 protein into the nucleus with a consequent hyperactivation of the RAS pathway in these patients. The uncovered involvement of the RAS pathway in this AML subgroup provides the rationale for searching new therapeutical strategies to selectively target MLL-AF6-rearranged cells. Methods. We established a cell-based drug screening assay, by testing a library of 1,280 pharmacologically active compounds (Lopac library, Sigma-Aldrich) on t(6;11)-rearranged ML2 and SHI-1 cell lines. Compounds (used at 10μM) which decreased cell viability by at least 50% (by ATP measurement) were further tested in different AML cell lines (HL60, as well as NOMO1 and THP1, both t(9;11)MLL-AF9 rearranged), to exclude those with broad anti-leukemic activity and to focus specifically over MLL-AF6 action. Finally, functional studies were performed for the compounds resulted selective for the MLL-AF6 rearrangement in cell lines and patient's primary blast cultures and the most promising drug was tested in vivo using NSG mice. Results. Of 1,280 compounds, 104 and 93 impaired cell proliferation of ML2 and SHI-1, respectively. 73 were found efficacious over HL60 and, thus, excluded. Then, the remaining 20 compounds were evaluated in other MLL-cell lines (NOMO-1 and THP-1), and finally 10/20 resulted active selectively on t(6;11)-rearranged cell lines. The selected compounds were Arvanil, CP-100356 monohydrochloride, Fluspirilene, CID2858522, Eupatorin, ANA-12, BAY 61-3606 hydrochloride hydrate, Ara-G hydrate, Tyrphostin 47, Thioridazine hydrochloride and were also confirmed to impair viability over t(6;11) primary blast cultures from patients. Among them, we were particularly interested in Fluspirilene and Thioridazine, these compounds being both antipsychotics working as dopamine receptor (DR) antagonists and FDA approved. By flow cytometry we showed the DRs (DR-1 to DR-5) expression in ML2, SHI-1, NOMO-1, THP1 and SKNO-1 whereas HL60 resulted devoid of DRs. Blasts from t(6;11)-rearranged patients (n=3) expressed DRs as well. Treatment of the cell lines with Fluspirilene and Thioridazine triggered apoptosis induction in SHI-1, and to a lesser extent in ML2, due to autophagy activation, whereas no effects were observed on HL60, NOMO-1, THP1 and SKNO-1. Clonogenic assay showed that after 24 hours of treatment self-renewal ability of SHI-1 and ML-2 significantly decreased, with no effects observed in other cell lines. Same results were obtained in primary cultures from patients t(6;11), without toxic effects on healthy bone marrow cells, confirming the drug specific activity over leukemia proliferation, and with Thioridazine being more active. NSG mice were then flank injected with t(6;11) cells and treated with Thioridazine 12 mg/kg; treatment significantly inhibited tumor growth in vivo (compared to mice treated with DMSO, p Conclusions. This study led to the identification of DRs expression in myeloid blasts, and revealed their role in leukemia maintenance exclusively of the t(6;11)-rearranged AML. We identified a series of new compounds to be prioritized for further analysis in MLL-AML; in particular Thioridazine deserves further investigation as a novel therapeutic strategy to improve outcome of t(6;11)-rearranged patient's. 1 Pigazzi M, et al Leukemia. 2011 Mar;25(3):560-3. Disclosures Indraccolo: OncoMed Pharmaceuticals, Inc.: Research Funding.

t(8;21)(q22;q22)RUNX1-RUNX1T1 is a recurrent somatic lesion detected at diagnosis in approximately 12-15% of children with acute myeloid leukemia (AML). Children with this isolated translocation are usually considered at standard risk, but our last multicenter trial revealed a higher than expected cumulative incidence of relapse for these patients1. Genetic and epigenetic heterogeneity is emerging as a fundamental property of AML in the context of the clonal architecture dynamic evolution. In view of this observation, we hypothesized that within t(8;21) patients there may coexist a complex mosaic of cells containing combinations of the same genetic t(8,21) lesion together with different epigenetic variants, and that epigenetic complexity may play a crucial role in predisposing patients to relapse. The importance of the identification of molecular markers distinctive of t(8,21)-rearranged patients prone to develop relapse could be instrumental to improve their cure rate. We performed high throughput DNA methylation profiling (RRBS-seq) and integrated results with gene expression profiling (Affymetrix HTA 2.0) of 16 isolated t(8;21) AML samples collected at diagnosis, and analyzed data by comparing patients who did or did not experience relapse. We applied a logistic regression algorithm to identify differentially methylated regions (DMRs) considering a minimum change in methylation level of 25%. We validated results in a proteome context by reverse phase protein array (RPPA) in an independent cohort of 35 t(8;21) AML patients. DNA methylation profiling analysis identified 337 DMRs able to correctly predict t(8;21) patients who did relapse from those who did not. In particular, 23 DMRs (7%) were located at promoters, while most of them were equally distributed between intronic (48%) and exonic (45%) regions. Globally, we found hypomethylated DMRs being significantly enriched in relapsed patients, in particular in repetitive elements regions of the genome (LINE, SINE, DNA transposon: 38.9% vs 52.4%; p We then considered the role of methylation over gene expression and found a weak correlation between DMRs (mostly at promoters) and their associated gene levels (14.5% of DMRs with an inverse correlation r These data show that the methylation signature may be considered a novel, emerging diagnostic tool making possible to better stratifying t(8,21)-rearranged patients through the identification, already at diagnosis, of those who are prone to relapse . Preliminary data of functional analysis suggest that epigenome of t(8;21) blasts may control cell adhesion properties at bone marrow niche and treatment response, contributing to patients relapse. 1 Pession A, Blood. 2013;122(2):170-8. Disclosures No relevant conflicts of interest to declare.

Purpose. While most of the recurrent molecular marker of AML have been already discovered and used for risk stratification in clinical protocols, post-treatment monitoring of these abnormalities can be useful in the clinical management of patients. In particular, monitoring of minimal residual disease (MRD), largely used in acute lymphoblastic leukemia, is gaining popularity also in AML, in the perspective of optimizing risk stratification of patients in terms of subsequent clinical relapse. While MRD monitoring in AML is mainly based on flow-cytometry approaches, molecular MRD measurements are not currently routinely used for taking clinical decision. Here we examine if the molecular MRD and Allelic Ratio (AR) levels monitoring may influence the survival of FLT3 internal tandem duplication (ITD)-mutated AML patients. Patients and methods. We retrospectively analyzed 507 children with de novo AML for FLT3ITD mutation by RT-PCR. Mutation was sequenced and the AR was calculated by Genescan. Bone marrow samples after induction treatment were analyzed for MRD levels by Real-Time PCR. We correlated these parameters with both patient event-free survival (EFS) and gene expression profile (GEP) findings. Results. 54/507 patients (10.6%) harboured FLT3ITD mutation. AR was calculated both at cDNA and DNA levels showing a reliable correlation (R=0.68), even if only AR measured on cDNA was found to be a significant poor prognostic feature. This latter observation supports the concept that expression of the mutation is more important than genetic bulk architecture at diagnosis. Patients with high AR showed a significant worse EFS as compared to those with low AR (19.2% for AR>0.51, vs 63.5% for AR Conclusion. Our results show that ITD-AR and MRD status are important independent prognostic factors for the management of of FLT3ITD patients, to be taken into consideration in planning the post-induction treatment. The high expression of the FLT3ITD mutation and the persistence of the mutated clone confer an aberrant hyperactivation of the downstream oncogenic FLT3 pathway influencing the outcome within these AML. Transcriptional profiles opens for further consideration of epigenetic targeting for FLT3ITD patients. Disclosures No relevant conflicts of interest to declare.

Introduction and aim: Childhood cytogenetically normal acute myeloid leukemia (CN-AML) is a subgroup of pediatric AML lacking any known cytogenetic and abnormality. CN-AML accounts for 20% of pediatric AML and exhibits a very heterogeneous response to treatment and, consequently, variable outcome. With the aim of providing new insights into the molecular lesions of this subset of AML, we analyzed, through RNA-seq, 13 cases of pediatric CN-AML, corroborating our findings in an independent cohort of 152 AML patients enrolled in the AIEOP AML 2002/01 clinical trial. Methods: Paired-end RNA-seq (75×2) was performed on PoliA(+) RNA extracted from blasts at diagnosis. ChimeraScan, deFuse and FusionMap algorithms were used for chimeric transcript detection. Results: An alteration that was present in 2 out 13 cases was a chimeric transcript involving the genes nucleoporin 98kDa (NUP98) and PHD finger protein 23 (PHF23), resulting from a cryptic translocation t(11;17)(p15;p13). Both patients showed an in-frame fusion between exon 13 of NUP98 and exon 4 of PHF23 that was confirmed by RT-PCR analysis and Sanger sequencing. To date, the cryptic translocation t(11;17)(p15;p13) was never described before in pediatric AML and has been reported only once in an adult AML patient (Reader, et al. Leukemia 2007). In both our patients the same breakpoint in PHF23 gene at the beginning of exon 4 was present, which was different from that present in the adult patient. To assess the incidence of NUP98-PHF23 fusion in pediatric CN-AML, then we studied by RT-PCR and Sanger sequencing a validation cohort of 152 CN-AML children negative for the most common recurrent cytogenetic and molecular lesions, i.e. those involving MLL, CBFB, NPM1 and FLT3. Overall, 2 out of the 152 CN-AML cases were found positive for NUP98-PHF23, demonstrating that this genomic aberrancy is not rare in pediatric CN-AML (tentative frequency 2.4%). Fluorescence in situ hybridization analysis confirmed the cryptic chromosomal translocation t(7;11)(p15;p13) leading to the fusion between NUP98 and PHF23 in all cases. Conclusions: Lately, genome-wide approaches have been widely used to investigate the mutational landscape of CN-AML in adults. However, the genetic profile of childhood CN-AML still needs to be defined. Here, for the first time, we report the identification of a NUP98-PHF23 chimeric transcript in pediatric CN-AML. Together with recently published data demonstrating that NUP98-PHF23 promoted leukemogenesis and the observation that treatment with inhibitors of PHD-domain-mediated H3K4me3 binding, such as disulfiram (an FDA-approved drug), could selectively killed cells expressing NUP98-PHF23 (Gough, et al Cancer Discovery 2014), our findings enforce the role of epigenetic regulators in pediatric AML and suggest screening for this fusion gene at diagnosis in CN-AML pediatric patients. Citation Format: Marco Togni, Riccardo Masetti, Martina Pigazzi, Annalisa Astolfi, Daniele Zama, Valentina Indio, Salvatore Serravalle, Elena Manara, Valeria Bisio, Sergio Rutella, Franca Fagioli, Giuseppe Basso, Andrea Pession, Franco Locatelli. NUP98-PHF23 is a novel fusion gene in pediatric cytogenetically normal acute myeloid leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-211. doi:10.1158/1538-7445.AM2015-LB-211

Background The proto-oncogene c-KIT encodes a receptor tyrosine kinase characterized by five extracellular immunoglobin-like domains, such as a single transmembrane helix, a cytoplasmic juxtamembrane domain (JMD), and a kinase domain. Mutations of c-KIT have been shown to occur in the extracellular portion of the receptor within exon 8, in the JMD domain within exon 11, and in the activation loop of the kinase domain within exon 17. All mutations led to the constitutive activation of the protein and promote development of human cancer, including hematological malignancies. Moreover, they have been frequently described to be associated with poor prognosis in adults with acute myeloid leukemia (AML) harboring aberrancies of the core binding factors (CBF). c-KIT mutations are also found in pediatric CBF-rearranged AML, but their incidence and prognostic impact are still debated. The AIEOP AML2001/02 protocol assigned patients with CBF rearrangements who reach complete remission (CR) at the end of the first course of induction therapy to the standard-risk (SR) group. These patients showed an incidence of relapse higher than expected (24%). New independent prognostic factors are thus desirable for improving the prognosis of this group of AML. In this study, we investigated the prognostic impact of c-KIT mutations. Materials and Methods We retrospectively analyzed the bone marrow of 49 and 30 patients carrying either the t(8;21) or inv(16)(p13;q22). Screening for mutations of c-KIT was assessed by PCR amplification followed by Sanger sequencing of the exons 8, 11 and 17. The prognostic impact was assessed through the calculation of the probability of event-free survival (EFS). Results c-KIT mutations analyzed in the study are: the point mutation at D816 residue known to affect the activation loop of the kinase domain (exon 17); the internal tandem duplication located at exon 11, and small deletions or insertions, or combinations of deletions and insertions, of variable size at exon 8. The screening showed that 2/49 (4%) t(8;21) patients were positive for the point mutation affecting the codon D816V/Y; 2/49 (4%) for internal tandem duplication of exon 11, and 7/49 (14.3%) for small deletions and/or insertions of variable size in the extracellular portion of the receptor (exon 8). We found that the t(8;21) positive patients mutated for cKIT (18.3%) had a significant lower EFS (45.7%) than wild type patients (78.4%; p = 0.025). On the contrary, c-KIT mutations were rarely found in the cohort of inv(16) positive patients. In particular, 1/30 harbored the D816V, and 2/30 (6.6 %) had insertions at exon 8. No mutations at exon 11 were found. Conclusions c-KIT mutations are frequently found in t(8;21) postitive patients and they confer a worse prognosis. Targeted therapy with tyrosine kinase inhibitors may be considered as a new and promising therapeutic strategy for cKIT mutated patients in order to improve their outcome. Disclosures: No relevant conflicts of interest to declare.

Abstract 2494 Introduction. Acute myeloid leukemia (AML) is an heterogeneous disease with known specific recurrent genetic aberrations. The continuous and increasing identification of new genetic mutations has permitted to identify new subgroups with different prognosis. In the present work we evaluated the incidence of rare genetic abnormalities in pediatric AML such as del(4)(q12)FIP1L1-PDGFRA, t(16;21)(p11;q22)FUS-ERG, t(8;16)(p11;p13)MOZ-CBP, t(11;17)(q23;q12–21)MLL-AF17, t(4;11)(q35;q23)MLL-ArgB2, t(5;11)(q35;p15.5)NUP98-NSD1, t(3;5)(q25;q34)NPM1-MLF1, and MLLPTD. Methods. We selected 306 patients with AML other than acute promyelocytic leukemia, negative for known recurrent genetic abnormalities involving MLL, CBF-beta and FLT3 genes. RNA was extracted from fresh bone marrow at diagnosis, and multiplex RT-PCR was employed. Sequencing by Sanger method was applied to all positive cases to characterize breakpoints of fusion. The Kaplan-Meier method was used for estimating the probability of event-free survival (EFS). Results. We identified one patient each positive for t(16;21)(p11;q22)FUS-ERG, t(11;17)(q23;q12–21)MLL-AF17, and t(4;11)(q35;q23)MLL-ArgB2, respectively, this suggesting that these rearrangements are rare in pediatric AML. 2/306 patients had del(4)(q12)FIP1L1/PDGFRA, and 4/306 the t(8;16)(p11;p13)MOZ-CBP; both these anomalies should be investigated in larger cohorts for definining their prognostic value. Interestingly 6/306 (2%) patients had the t(3;5)(q25;q34)NPM1-MLF1, 6/306 (2%) the MLLPTD, and 8/306 (2.6%) were found to carry the t(5;11)(q35;p15.5)NUP98-NSD1. Since the t(5;11) fusion was recently associated to FLT3ITD, we enlarged the screening to 42 de novo AML harbouring FLT3ITD mutation enrolled in the AIEOP-LAM 2002 protocol finding that 6 of them (14%) had the NUP98-NSD1 fusion gene. We documented a poor EFS for patients with t(5;11)NUP98-NDS1 (n=12) as compared to patients negative for molecular lesions and enrolled in the LAM 2002-AIEOP protocol (25% vs 53.1% at 3 years, p Conclusions. We provide evidence that NUP98-NSD1 may be considered a recurrent translocation in pediatric AML with poor prognosis. Being cryptic to conventional karyotyping, we confirmed the need of using molecular approaches for a proper identification of this anomaly. We also suggest that the NUP98-NSD1 fusion gene be considered for a better evaluation of the FLT3ITD+ patients. Disclosures: No relevant conflicts of interest to declare.

Nucleoporin 98 (NUP98) is part of a family of proteins that are involved in the nuclear pore complex known to control trafficking of many molecules between nucleus and cytoplasm. However, NUP98 has been discovered to play a critical role in gene regulation, being involved in several chromosomal translocations in hematopoietic disorders. Up to thirty different NUP98 partner genes have been identified among patients with myelodysplastic syndrome and acute myeloid leukemia (AML). The chimeric NUP98 protein has the N-terminal of NUP98 and the C-terminal of its partner gene. Partners, if belonging to the homeobox genes conserved the DNA-binding domain, if not, they maintained chromatin interaction domains, mediating in any case a transcriptional regulatory function of the chimera, as recently described for NUP98-NSD1 and NUP98-JARID1A fusions. Here, we report the results of a study aimed at identifying the more frequent NUP98 fusion proteins present at diagnosis in children with AML treated in Italy with the AIEOP protocols. We performed a molecular screening of 10 partner genes of NUP98, namely, NSD1, HOXC11, PHF23, HOXA9, JARID1A, HOXD13, LEDGF, DDX10, ADD3 and HHEX in 191 patients affected by de novo AML enrolled in the AIEOP LAM 2002/01 protocol, and previously found to be negative for known recurrent genetic abnormalities involving MLL, CBFB, and FLT3 genes. We found 32 positive patients (17%) harboring one of the NUP98 chimeric fusion studied, this abnormality allowing to identify a new AML subgroup. By considering the whole number of children treated with the Italian pediatric AML 2002/01 protocol, 32 out of 482 enrolled patients carried a NUP98 rearrangement, the final frequency of NUP98 aberrancies at diagnosis being 6.6 %. In detail, 12 patients were found positive for t(5;11)(q35;p15.5)NUP98-NSD1 (6 out of 12 were also FLT3ITD mutated), 5 for t(11;12)(p15;q13)NUP98-HOXC11, 3 for t(10;11) NUP98-HHEX, 3 for t(11;17)(p15.5;p13)NUP98-PHF23, 3 for t(5;11)(q35;p15.5)NUP98-JARID1A, 2 for t(10;11)NUP98-ADD3, 2 for t(7;11)(p15;p15)NUP98-HOXA9, one patient harbored t(2;11)(q31;p15)NUP98-HOXD13, one t(9;11)(p22;p15.5)NUP98-LEDGF. We then considered the NUP98 rearrangements for their clinical impact and found that the 8-year event-free survival (EFS) was significantly worse in patients with a NUP98 rearrangement (34.3%, SE 8.3) than the remaining patients negative for known recurrent genetic abnormalities (n=159, EFS = 52.3%, SE 4.3; p = 0.007). This observation was confirmed when we considered patients with NUP98 rearrangements excluding those with the FLT3ITD mutation (n=26, EFS = 34.6%). In conclusion, we found that NUP98 is recurrently found to be involved in somatic translocations leading to childhood AML . NUP98 rearrangements identify a new subgroup of pediatric AML with a similar, dismal prognosis, deserving further consideration in novel biological studies aimed at targeting NUP98 to improve outcome. Disclosures No relevant conflicts of interest to declare.