Internal Tandem Duplication Of FLT3-ITD Research Articles

Identification Of Tet2 Gene Level Of Expression In Acute Leukemias

Objective: TET2 gene (4q24) is one of the most important regulatory factors of the methylation process in human genome. The homologous protein is directly involved in convertion of 5-methylcytosine into 5-hydroximethylcytosine (1). Loss of function mutations in TET2 gene are considered to be driver mutations (initiating mutations) for leukemogenesis. In some cases they can be found in early clonal cells of Acute Myeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL) or myelodisplastic syndromes (MDS) (2). The aim of this study is to determinate the frequency of TET2 mutations in Acute Leukemia (AL) patients. Materials and methods: This retrospective study enrolled 24 patients newly diagnosed with AML (19 patients) and ALL (5 pacients). By using Real-Time PCR we obtained a relative cuantification of TET2 gene level of expression taking ABL gene as reference. Outcomes: Low levels of TET2 gene expression were found in 6 patients (25%), 3 of them being diagnosed with AML and 3 with ALL. No cytogenetic anomalies were noticed. However, all these patients had blastocytosis and higher white blood cells count than pacients with normal levels of expression. There was one case with mutations in both TET2 gene and NPM1 gene and one case that associated a defective TET2 gene and an internal tandem duplication of FLT3-ITD. Conclusions: In our study, TET2 gene loss of function mutations occured on a specific background which involved some characteristic mutations for AL and some particular features of cell morphology. We aim to continue this study by investigating the long term effects of this epigenetic mutation in AL patients.

Assessment of 2022 European LeukemiaNet risk classification system in real-world cohort from China.

The European LeukemiaNet (ELN) risk classification system for acute myeloid leukemia (AML) patients has been used worldwide. In 2022, the ELN risk classification system modified risk genes including CEBPA mutation status, myelodysplasia-related (MR) gene mutations and internal tandem duplications of FLT3 (FLT3-ITD). We include newly diagnosed de novo AML patients at our center from January 2017 to December 2021, regardless of the further treatment received. Clinical data and date of survival were included. Survival analysis were performed using the Kaplan-Meier method, and the log-rank test was used to compare survival between different risk groups. We include 363 newly diagnosed de novo AML patients from 2017 to 2021 to assess the accuracy of the ELN risk classification system. Their survival results show that the ELN-2022 risk classification system is not superior to the ELN-2017 version; for patients with FLT3-ITD mutations but without FLT3 inhibitor treatment, their survival is similar to the ELN-2022 adverse risk group. The ELN-2022 risk classification system cannot accurately clarify ECOG performance status (PS) 2-4 patients, especially in the ELN-2022 favorable risk group. The ELN-2022 risk stratification system may not be appropriate for patients unable to receive intensive therapy or FLT3 inhibitor; more real-world data is needed to straify patients with worse ECOG PS and inferior intensive therapy.

Ubtf tandem Duplications Define a Novel Subtype of Acute Myeloid Leukemia Associated with Younger Age, WT1 Mutations and HOXA9 Marked Overexpression

Background Upstream Binding Transcription Factor ( UBTF) gene plays an important role in ribosomal RNA transcription. UBTF tandem duplications (TD) have been recently described as a recurrent genetic lesion in acute myeloid leukemia (AML). UBTF-TDs appear to be mutually exclusive with other class-defining lesions in AML and display a unique comutational signature. UBTF-mutated AML affects predominantly younger patients and is associated with trisomy 8, internal tandem duplication of FLT3 ( FLT3-ITD), mutations in WT1 and poor prognosis. Moreover, HOX and MEIS genes seem to be overexpressed in AML with UBTF-TD similarly to NPM1-mutated ( NPM1m) or KMT2A-rearragend ( KMT2Ar) AML, suggesting potential sensitivity to menin inhibitors. In this study we sought to characterize UBTF-TDs in our AML cohort. Methods We studied 456 adult patients (median age 54 years; range 18-70) diagnosed with de novo AML and included in AML-12 protocol from CETLAM group. All patients from the 2012-2017 period (n=403) were consecutively studied and we also selected 53 cases of WT1-mutated AML from the 2017-2020 period, given the strong association with UBTF-TD. Screening for UBTF-TD was performed on bone marrow (BM) or peripheral blood (PB) genomic DNA samples. Exon 13 of UBTF gene was amplified and analysed by Polymerase chain reaction (PCR) and subsequent fragment length analysis by capillary electrophoresis. Mutated samples were confirmed through Sanger sequencing. Further, RNA from 9 UBTF-TD cases was available and HOXA9 and MEIS1 gene expression analysis was performed by Real-Time Polymerase Chain Reaction (RT-qPCR). Clinical and biological data were obtained from CETLAM registry. Results UBTF-TD was found in 13 patients from our cohort whose characteristics are detailed in Table 1. UBTF-TDs range from 45 base pairs (bp) to 365 bp, being the 48 bp the most recurring size. As described, although small UBTF-TDs are in frame insertions, the largest ones are not multiple of 3. Notably, most UBTF-TDs presented small insertions and deletions among the duplication. Presence of UBTF-TD was associated with younger age (36 vs 55 years; p<0.001) and BM dysplastic changes (80% of U BTFMUT). UBTF-TD was particularly frequent (8.9% of all cases) in patients below 40 years, compared to other lesions as CEBPA mutations (1.1%). Eleven (85%) U BTFMUT patients presented intermediate cytogenetics, being trisomy 8 (5), normal karyotype (3) and 9q deletion (2) the most recurrent findings. Two cases of adverse karyotype with MECOM-rearrangements were found. As previously described, there was a strong association between UBTF-TD and of FLT3-ITD (38.5%). Other recurrent genetic lesions in AML like NPM1 and CEBPA mutations or core-binding factor (CBF) rearrangements were exclusive of UBTF wild type patients. Further, we found that UBTFMUT leukemias showed marked overexpression of HOXA9 compared with other AML subtypes (22.1-fold vs 5.0-fold; p<0.001; Fig. 1) and similar to NPM1m AML (22.3-fold). On the other hand, MEIS expression did not differ significantly among groups. Conclusions In our cohort of adult AML patients, UBTF-TDs are found in 1.8% of cases and are especially frequent in younger individuals in absence of other class-defining genetic lesions. The presence of UBTF-TD is strongly associated with cytomorphological dysplasia and co-occurrence of FLT3-ITD and WT1 mutations, while NPM1 and CEBPA mutations and CBF rearrangements appear to be mutually exclusive. HOXA9 upregulation could be a leukemogenic event in UBTF-TD AML, as described in NPM1-m and KMT2A-r AML, suggesting potential utility of menin inhibitors in this subset of patients. Screening for UBTF-TD is going to be incorporated in our molecular diagnostic panel for new AML cases.

Genome-wide DNA methylation analysis in pediatric acute myeloid leukemia

AbstractWe investigated genome-wide DNA methylation patterns in 64 pediatric patients with acute myeloid leukemia (AML). Based on unsupervised clustering with the 567 most variably methylated cytosine guanine dinucleotide (CpG) sites, patients were categorized into 4 clusters associated with genetic alterations. Clusters 1 and 3 were characterized by the presence of known favorable prognostic factors, such as RUNX1-RUNX1T1 fusion and KMT2A rearrangement with low MECOM expression, and biallelic CEBPA mutations (all 8 patients), respectively. Clusters 2 and 4 comprised patients exhibiting molecular features associated with adverse outcomes, namely internal tandem duplication of FLT3 (FLT3-ITD), partial tandem duplication of KMT2A, and high PRDM16 expression. Depending on the methylation values of the 1243 CpG sites that were significantly different between FLT3-ITD+ and FLT3-ITD− AML, patients were categorized into 3 clusters: A, B, and C. The STAT5-binding motif was most frequently found close to the 1243 CpG sites. All 8 patients with FLT3-ITD in cluster A harbored high PRDM16 expression and experienced adverse events, whereas only 1 of 7 patients with FLT3-ITD in the other clusters experienced adverse events. PRDM16 expression levels were also related to DNA methylation patterns, which were drastically changed at the cutoff value of PRDM16/ABL1 = 0.10. The assay for transposase-accessible chromatin sequencing of AMLs supported enhanced chromatin accessibility around genomic regions, such as HOXB cluster genes, SCHIP1, and PRDM16, which were associated with DNA methylation changes in AMLs with FLT3-ITD and high PRDM16 expression. Our results suggest that DNA methylation levels at specific CpG sites are useful to support genetic alterations and gene expression patterns of patients with pediatric AML.

Impact of FLT3 internal tandem duplication and NPM1 mutations in acute myeloid leukemia treated with allogeneic hematopoietic cell transplantation

Background aimsThe internal tandem duplication of FLT3 (FLT3ITD) and NPM1 mutations (NPM1mut) are well-established prognostic factors in cytogenetically intermediate-risk acute myeloid leukemia (AML) when treated with chemotherapy alone. However, their prognostic value in the setting of allogeneic hematopoietic cell transplantation (HCT) is controversial. MethodsFLT3 and NPM1 mutational status was determined at diagnosis using single-gene polymerase chain reaction or next-generation sequencing in 247 adult patients with cytogenetically intermediate-risk AML who underwent myeloablative HCT. Multivariate Fine–Gray and Cox regression was used to analyze the cumulative incidence of relapse (CIR), relapse-free survival (RFS) and overall survival (OS). ResultsFLT3ITD and NPM1mut were present in 74 of 247 (30%) and 79 of 247 (32%) patients, respectively. There was no significant difference between patients without a FLT3ITD or NPM1mut (FLT3NONITD/NPM1WT) and patients with a FLT3ITD mutation alone (FLT3ITD/NPM1WT) with regard to CIR (P = 0.60), RFS (P = 0.91) or OS (P = 0.66). Similarly, there was no significant difference between FLT3NONITD/NPM1WT and FLT3NONITD/NPM1mut patients with regard to CIR (P = 0.70), RFS (P = 0.75) or OS (P = 0.95). The presence of a concurrent mutation in NPM1 did not appear to modify the impact of having a FLT3ITD mutation. ConclusionsIn contrast to chemotherapy-only treatment, FLT3 and NPM1 mutational status does not appear to predict outcomes in patients with cytogenetically intermediate-risk AML following HCT. These results suggest that HCT may ameliorate the poor prognostic effect of FLT3ITD mutation and that HCT should be considered over chemotherapy-only treatment in FLT3ITD-mutated AML.

Synergistic cytotoxicity of dual PI3K/mTOR and FLT3 inhibition in FLT3-ITD AML cells.

Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy, characterized by a heterogeneous genetic landscape and complex clonal evolution, with poor outcomes. Mutation at the internal tandem duplication of FLT3 (FLT3-ITD) is one of the most common somatic alterations in AML, associated with high relapse rates and poor survival due to the constitutive activation of the FLT3 receptor tyrosine kinase and its downstream effectors, such as PI3K signaling. Thus, aberrantly activated FLT3-kinase is regarded as an attractive target for therapy for this AML subtype, and a number of small molecule inhibitors of this kinase have been identified, some of which are approved for clinical practice. Nevertheless, acquired resistance to these molecules is often observed, leading to severe clinical outcomes. Therapeutic strategies to tackle resistance include combining FLT3 inhibitors with other antileukemic agents. Here, we report on the preclinical activity of the combination of the FLT3 inhibitor quizartinib with the dual PI3K/mTOR inhibitor PF-04691502 in FLT3-ITD cells. Briefly, we show that the association of these two molecules displays synergistic cytotoxicity in vitro in FLT3-ITD AML cells, triggering 90% cell death at nanomolar concentrations after 48h.

Discovery and structure − activity relationship exploration of pyrazolo[1,5-a]pyrimidine derivatives as potent FLT3-ITD inhibitors

Internal tandem duplications of FLT3 (FLT3-ITD) occur in approximately 25% of all acute myeloid leukemia (AML) cases and confer a poor prognosis. Optimization of the screening hit 1 from our in-house compound library led to the discovery of a series of pyrazolo[1,5-a]pyrimidine derivatives as potent and selective FLT3-ITD inhibitors. Compounds 17 and 19 displayed potent FLT3-ITD activities both with IC50 values of 0.4 nM and excellent antiproliferative activities against AML cell lines. Especially, compounds 17 and 19 inhibited the quizartinib resistance-conferring mutations, FLT3D835Y, both with IC50 values of 0.3 nM. Moreover, western blot analysis indicated that compounds 17 and 19 potently inhibited the phosphorylation of FLT3 and attenuated downstream signaling in AML cells. These results indicated that pyrazolo[1,5-a]pyrimidine derivatives could be promising FLT3-ITD inhibitors for the treatment AML.

Prognostic impact of DNMT3A mutation in acute myeloid leukemia with mutated NPM1

AbstractThe negative prognostic impact of internal tandem duplication of FLT3 (FLT3-ITD) in patients with acute myeloid leukemia with mutated NPM1 (AML-NPM1) is restricted to those with a higher FLT3-ITD allelic ratio (FLT3high; ≥0.5) and considered negligible in those with a wild-type (FLT3WT)/low ITD ratio (FLT3low). Because the comutation of DNMT3A (DNMT3Amut) has been suggested to negatively influence prognosis in AML-NPM1, we analyzed the impact of DNMT3Amut in FLT3-ITD subsets (absent, low, and high ratios). A total of 164 patients diagnosed with AML-NPM1 included in 2 consecutive CETLAM protocols and with DNMT3A and FLT3 status available were studied. Overall, DNMT3Amut status did not have a prognostic impact, with comparable overall survival (P = .2). Prognostic stratification established by FLT3-ITD (FLT3WT = FLT3low >FLT3high) was independent of DNMT3Amut status. Measurable residual disease (MRD) based on NPM1 quantitative polymerase chain reaction was available for 94 patients. DNMT3Amut was associated with a higher number of mutated NPM1 transcripts after induction (P = .012) and first consolidation (C1; P < .001). All DNMT3Amut patients were MRD+ after C1 (P < .001) and exhibited significant MRD persistence after C2 and C3 (MRD+ vs MRD−; P = .027 and P = .001, respectively). Finally, DNMT3Amut patients exhibited a trend toward greater risk of molecular relapse (P = .054). In conclusion, DNMT3Amut did not modify the overall prognosis exerted by FLT3-ITD in AML-NPM1 despite delayed MRD clearance, possibly because of MRD-driven preemptive intervention.

Multiplexed single\u2010cell mass cytometry reveals distinct inhibitory effects on intracellular phosphoproteins by midostaurin in combination with chemotherapy in AML cells

BackgroundFms-related tyrosine kinase 3 (FLT3) receptor serves as a prognostic marker and therapeutic target in acute myeloid leukemia (AML). Approximately one-third of AML patients carry mutation in FLT3, associated with unfavourable prognosis and high relapse rate. The multitargeted kinase inhibitor midostaurin (PKC412) in combination with standard chemotherapy (daunorubicin and cytarabine) was recently shown to increase overall survival of AML patients. For that reason, PKC412 has been approved for treatment of AML patients with FLT3-mutation. PKC412 synergizes with standard chemotherapy, but the mechanism involved is not fully understood and the risk of relapse is still highly problematic.MethodsBy utilizing the unique nature of mass cytometry for single cell multiparameter analysis, we have explored the proteomic effect and intracellular signaling response in individual leukemic cells with internal tandem duplication of FLT3 (FLT3-ITD) after midostaurin treatment in combination with daunorubicin or cytarabine.ResultsWe have identified a synergistic inhibition of intracellular signaling proteins after PKC412 treatment in combination with daunorubicin. In contrast, cytarabine antagonized phosphorylation inhibition of PKC412. Moreover, we found elevated levels of FLT3 surface expression after cytarabine treatment. Interestingly, the surface localization of FLT3 receptor increased in vivo on the blast cell population of two AML patients during day 3 of induction therapy (daunorubicin; once/day from day 1–3 and cytarabine; twice/day from day 1–7). We found FLT3 receptor expression to correlate with intracellular cytarabine (AraC) response. AML cell line cultured with AraC with or without PKC412 had an antagonizing phosphorylation inhibition of pAKT (p = 0.042 and 0.0261, respectively) and pERK1/2 (0.0134 and 0.0096, respectively) in FLT3high compared to FLT3low expressing cell populations.ConclusionsOur study provides insights into how conventional chemotherapy affects protein phosphorylation of vital signaling proteins in human leukemia cells. The results presented here support further investigation of novel strategies to treat FLT3-mutated AML patients with PKC412 in combination with chemotherapy agents and the potential development of novel treatment strategies.

Quizartinib-resistant FLT3-ITD acute myeloid leukemia cells are sensitive to the FLT3-Aurora kinase inhibitor CCT241736

Internal tandem duplication of FLT3 (FLT3-ITD) is one of the most common somatic mutations in acute myeloid leukemia (AML); it causes constitutive activation of FLT3 kinase and is associated with high relapse rates and poor survival. Small-molecule inhibition of FLT3 represents an attractive therapeutic strategy for this subtype of AML, although resistance from secondary FLT3 tyrosine kinase domain (FLT3-TKD) mutations is an emerging clinical problem. CCT241736 is an orally bioavailable, selective, and potent dual inhibitor of FLT3 and Aurora kinases. FLT3-ITD+ cells with secondary FLT3-TKD mutations have high in vitro relative resistance to the FLT3 inhibitors quizartinib and sorafenib, but not to CCT241736. The mechanism of action of CCT241736 results in significant in vivo efficacy, with inhibition of tumor growth observed in efficacy studies in FLT3-ITD and FLT3-ITD-TKD human tumor xenograft models. The efficacy of CCT241736 was also confirmed in primary samples from AML patients, including those with quizartinib-resistant disease, which induces apoptosis through inhibition of both FLT3 and Aurora kinases. The unique combination of CCT241736 properties based on robust potency, dual selectivity, and significant in vivo activity indicate that CCT241736 is a bona fide clinical drug candidate for FLT3-ITD and TKD AML patients with resistance to current drugs.

Allogeneic stem cell transplantation benefits for patients ≥ 60 years with acute myeloid leukemia and FLT3 internal tandem duplication: a study from the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation.

Intermediate-risk cytogenetic acute myeloid leukemia with an internal tandem duplication of FLT3 (FLT3-ITD) is associated with a high risk of relapse, and is now a standard indication for allogeneic stem cell transplantation. Nevertheless, most studies supporting this strategy have been performed in young patients. To address the benefit of allogeneic transplantation in the elderly, we made a selection from the European Society for Blood and Marrow Transplantation registry of de novo intermediate-risk cytogenetic acute myeloid leukemia harboring FLT3-ITD in patients aged 60 or over and transplanted from a related or unrelated donor between January 2000 and December 2015. Two hundred and ninety-one patients were identified. Most patients received a reduced-intensity conditioning (82%), while donors consisted of an unrelated donor in 161 (55%) patients. Two hundred and twelve patients received their transplantation in first remission, 37 in second remission and 42 in a more advanced stage of the disease. The 2-year leukemia-free survival rate was 56% in patients in first remission, 22% in those in second remission and 10% in patients with active disease, respectively (P<0.005). Non-relapse mortality for the entire cohort was 20%. In multivariate analysis, disease status at transplantation was the most powerful predictor of worse leukemia-free survival, graft-versus-host disease and relapse-free survival, and overall survival. In this elderly population, age was not associated with outcome. Based on the current results, allogeneic transplantation translates into a favorable outcome in fit patients ≥ 60 with FLT3-ITD acute myeloid leukemia in first remission, similarly to current treatment recommendations for younger patients.

Inhibition of the phosphatase STS1 in acute myeloid leukemia

The class III receptor tyrosine kinases (RTKs) FLT3 and c-KIT play a central role in normal and malignant hematopoiesis. After binding to their natural ligands, these RTKs dimerize, become autophosphorylated and activate an intracellular signaling cascade. Negative RTK regulation by dephosphorylation, ubiquitination and degradation are equally important to prevent uncontrolled kinase activity and downstream signaling in normal and malignant hematopoiesis. Recently, we identified STS1/STS2 as the phosphatases of FLT3 and c-KIT (Zhang J. et al, Stem Cell Reports, 2015). Loss of STS1/STS2 causes hyperphosphorylation of FLT3, enhanced AKT signaling and a strong proliferative advantage of hematopoietic stem and progenitors cells in STS1/ STS2 knockout mice. This indicates that STS1 and STS2 serve as negative regulators for wild-type FLT3 kinase activity and normal hematopoiesis. Internal tandem duplications of FLT3 (FLT3-ITD) are found in up to 30% of patients with acute myeloid leukemia (AML). Here, we investigated the function of STS1/ STS2 in FLT3-ITD induced malignant transformation. Surprisingly we found that loss of STS1 decreased the transforming capacity of the FLT3-ITD mutant, demonstrated by both reduced cell proliferation and colony forming ability. Importantly, the overexpression of STS1 enhanced FLT3-ITD induced cytokine independent cell growth in vitro in a phosphatase-dependent manner. Bone marrow transplantation results recapitulated the collaboration between FLT3-ITD and STS1/STS2, manifested by the increased engraftment of FLT3-ITD and STS1/STS2 co-expressing cells in vivo. Biological analyses showed prolonged half-life of FLT3-ITD, as well as increased downstream signaling in STS1 over-expressing cells. Mechanistically, the E3 ligase activity of CBL was negatively regulated by STS1. In summary, STS1/STS2 demonstrated two distinct roles in the context of FLT3wt and FLT3-ITD. Our data suggests that the phosphatase inhibition of STS1/STS2 may both improve hematopoietic recovery of normal hematopoiesis and decrease the leukemic potential of FLT3-ITD expressing blasts. Inhibiting STS1 may provide a novel therapeutic in FLT3-ITD positive AML.

Allogeneic Stem Cell Transplantation for Elderly Patients with Intermediate-Risk Cytogenetic Acute Myeloid Leukemia and Internal Tandem Duplication of FLT3 (FLT3-ITD); A Study from the Acute Leukemia Working Party (ALWP) of the European Society of Blood and Marrow Transplantation (EBMT)

Introduction: The presence of an internal tandem duplication of FLT3 (FLT3-ITD) confers a higher risk of relapse and is now a current indication of allogeneic stem cell transplantation (SCT) in patients with intermediate-risk cytogenetic acute myeloid leukemia (IRC-AML) in first complete remission (CR1). Most studies encouraging this strategy have been performed in patients below 60 year-old, after a myeloablative conditioning (MAC) and using a sibling donor. Because age remains associated with a worse outcome after SCT, we decided to analyse outcomes of SCT in patients aged 60 year-old or older with intermediate-risk AML and FLT3-ITD.Methods: Using the EBMT registry, we selected de novo acute myeloid leukemia (AML) harboring IRC-AML and FLT3-ITD in patients transplanted from a related or matched unrelated donor (9/10 or 10/10) between January 2000 and July 2014.Results: Two hundred and five patients have been allocated. Median age at the time of SCT was 64 (range, 60-75) year-old and median follow-up was 20 (range, 2-139) months. Ninety-four percent of the patients had a good performance status (Karnofsky at SCT ≥ 80%). Most patients had a normal karyotype at diagnosis (90% versus 10% with other intermediate-risk karyotype) and NPM1 status was reported in 131 patients out of which 100 (76%) were mutated. Thirty-four patients received a MAC, 142 had a reduced-intensity conditioning (RIC) and 29 a non-myeloablative conditioning (NMA). One hundred forty-six patients received their SCT in first remission (CR1), 24 in second remission (CR2) and 35 in more advanced stage of the disease, respectively. The 2-year leukemia-free survival (LFS) was 52% in patients in CR1, 17% in those in CR2 and 11% in patients with advanced disease, respectively (p<0.005). Similarly, the 2-year overall survival (OS) was 54% in CR1, 24% in CR2 and 11% in advanced disease, respectively (p<0.005). The 2-year non-relapse mortality (NRM) for the all cohort was 20%. The cumulative incidence of grade II to IV acute graft-versus-host disease (GvHD) was 24% and that of chronic GvHD was 30%.In multivariate analysis, disease status at SCT was the most powerful predictor of worse LFS and OS. Age, as a continuous variable, was not significantly associated with outcomes. Donor type (unrelated versus sibling donor) and donor CMV positivity correlated with worse OS and higher NRM. Next, we performed a second analysis focusing on patients transplanted in CR1. A multivariate analysis performed in this subgroup showed that age, as a continuous variable, did not translate into worse LFS, OS or NRM. Interval from diagnosis to CR1 was significantly associated with LFS, being 63% in patients achieving CR1 in less than 43 days and 45% in patients achieving CR1 in more than 43 days, respectively (p<0.005). NMA conditioning was associated with a trend to a worse OS.Conclusion: Allogeneic SCT in elderly (≥60 up to 75 year-old) patients with IRC-AML harboring the FLT3-ITD mutation, similar to current strategy in younger patients, appears as a good treatment strategy if performed in CR1, independently of age, with somewhat inferior outcome in transplants from unrelated donors. Nevertheless, indication for transplantation should still be evaluated taking into account donor type, co-morbidities and performance status. DisclosuresCraddock:Celgene: Consultancy, Honoraria, Research Funding; Pfizer: Speakers Bureau; Sunesis: Honoraria; Johnson and Johnson: Consultancy. Russell:Therakos: Other: shares.

Low frequency of mutations in Chinese with acute myeloid leukemia: Different disease or different aetiology?

Mutations in FLT3, DNMT3A, NRAS, NF1 and TP53 occur in persons of predominately European descent with acute myeloid leukemia (AML). Some, such as internal tandem duplication of FLT3 (FLT3-ITD) and point mutations in DNMT3A and NRAS, are especially frequent whereas others such as NF1 and TP53 are less so. Frequencies of these mutations in persons with seemingly similar AML from other genetic groups are largely unknown. We studied 269 Chinese (mostly Han) with de novo AML. FLT3-ITD was detected in 51 subjects (23%; 95% CI, 17–28%), R882 mutation of DNMT3A in 17 (6%; 95% CI, 3–9%) and NRAS mutation in 17 (7%; 95% CI, 3–9%). No mutations in NF1 and only 1 mutation in TP53 (1%, 95% CI, <2.5%) were detected. Except for FLT3-ITD, frequencies of these mutations are significantly less than those in persons of predominately European descent with AML. The reason(s) for this disparity is unknown but may offer clues to the aetiology of AML in different populations or may indicate some mutations associated with AML in persons of predominately European descent are not fundamental to the aetiology of the disease.

H2.0-Like Homeobox (HLX) Causes Pre-Leukemic Myeloid Expansion and Initiates AML In Cooperation With FLT3-ITD

Acute myeloid leukemia (AML) is an aggressive disease associated with poor clinical outcome. Less than one third of patients achieve durable remission with current treatment regimens, and prognostication and risk stratification are challenging. We have recently reported that the non-clustered homeobox gene, H2.0-like homeobox (HLX), is 2 to 16 fold overexpressed in more than 80% of patients with AML, across all major disease subtypes, and higher levels of HLX are associated with poor overall survival in AML. Inhibition of HLX in both murine and human AML cells has a significant anti-leukemic and differentiation-inducing effect suggesting HLX and its downstream targets as novel therapeutic targets in AML.In order to better understand the role of Hlx at the stem cell level and in myeloid differentiation in vivo, we generated knock-in mice conditionally overexpressing Hlx from the Rosa26 locus and bred them to mice that bear Cre recombinase under the control of the pIpC-inducible, hematopoietic specific promoter, Mx1. Animals overexpressing HLX exhibit elevated WBC counts and abnormal myeloid cells in the peripheral blood. Analysis of the bone marrow reveals expansion of the granulocyte-macrophage progenitor population (lin- ckit+ cd34+ CD16/32high) and expansion of immature myelocytes (ckit+ cd34+ CD16/32high Gr1int). Hlx knock-in bone marrow cells, and specifically immature granulocyte precursors, exhibit enhanced serial clonogenicity in methylcellulose colony assays, and a differentiation block and maintenance of immaturity in response to GM-CSF.Internal tandem duplications of FLT3 (FLT3-ITD) are seen in approximately 30% of all AML patients, and frequently co-occur with elevated HLX levels. Correlative analyses showed that AML patients with mutant FLT3 and low HLX have overall survival similar to WT FLT3 patients, and survive significantly longer than patients with mutant FLT3 and high HLX (p=0.005), demonstrating that FLT3 mutations confer poor prognosis only if HLX is highly expressed, and suggesting that HLX and mutant FLT3 functionally cooperate. We retrovirally co-expressed HLX and FLT3-ITD, or FLT3-ITD alone (plus an empty control), in primary Lin-Kit+cells and transplanted them into congenic recipient animals. Four weeks after transplantation, donor chimerism was 4-fold increased on average in the peripheral blood (PB) and bone marrow (BM), and by 12 weeks post-transplantation mice expressing FLT3-ITD and HLX developed AML with large numbers of leukemic blasts in the peripheral blood and bone marrow.We then crossed our new Hlx knock-in mouse model with previously generated FLT3-ITD knock-in mice. Strikingly, heterozygous double-transgenic mice expressing both the knock-in FLT3-ITD mutation and HLX develop acute myeloid leukemia after a latency of 2 months. Morphological and flow cytometric analysis revealed large numbers of blasts circulating in the peripheral blood and replacing the marrow, as well as substantial leukemic infiltrates in the spleen and liver.Our studies reveal a critical role for HLX in conferring a differentiation block and increased clonogenicity at the pre-leukemic stem and progenitor cell level in a genetic in vivo model. Furthermore, a novel compound knock-in mouse model of Hlx overexpression and FLT3-ITD demonstrates that Hlx can initiate AML in cooperation with FLT3-ITD in vivo. Disclosures:No relevant conflicts of interest to declare.

Activities and Mechanism Based Combinations Of Omacetaxine In Acute Myeloid Leukemia

Omacetaxine, an inhibitor of translation, was recently granted accelerated approval for the treatment of chronic myeloid leukemia (CML). Omacetaxine blocks translation elongation by competing with the incoming aminoacyl-tRNAs for binding to the A-site cleft in the peptidyl-transferase center. Our previous studies showed that by transiently inhibiting translation, omacetaxine reduced the expression of the key, short-lived oncoproteins Bcr/Abl and Mcl-1, leading to cell death in the CML cells. This action sensitized the cells to the Abl kinase inhibitor and killed the CML cells synergistically. Further, as omacetaxine acts in a different mechanism than the Abl kinase inhibitors, it overcame resistance to TKI that was associated with kinase domain mutations. These studies paved the foundation for the clinical development of omacetaxine in CML. We also demonstrated that omacetaxine was active in chronic lymphocytic leukemia by translational inhibition of Mcl-1 expression. In contrast to normal tissues, the fact that the leukemia cells are critically dependent on the oncogene activity for survival provided a biologic context for a positive therapeutic index. As the biological features of acute myeloid leukemia (AML) rely largely on the overexpressed oncoproteins or constitutively activated kinases, we hypothesized that omacetaxine would have therapeutic benefit in AML either alone or in mechanism based combinations.To test this hypothesis, first, we compared omacetaxine to AC220, a potent FLT3 inhibitor, in AML cell lines OCI-AML3 and MV4-11. OCI-AML3 cells harbor the signature mutation of NPM1, whereas MV4-11 is a widely used model for the internal tandem duplications of FLT3 (FLT3-ITD), a common FLT3 mutation that constitutively activates the receptor tyrosine kinase. AC220 was selectively toxic to the MV4-11 cells, but had no effect on the viability of OCI-AML3. This is consistent with the biological context of MV4-11, but not OCI-AML3, that is addicted to the sustained activity of FLT3 for survival. In contrast, omacetaxine induced apoptosis in both cell lines with IC50s less than 100 nM. Protein synthesis was inhibited in both lines, measured by the incorporation of tritiated leucine. Apoptosis was induced rapidly within 24 h by omacetaxine, whereas AC220 required 72 h to kill the leukemia cells. These results indicated a common dependence on the continued protein synthesis in the AML lines, suggesting a potentially broad application of omacetaxine in AML patients with diverse genetic backgrounds. Over-expression of the anti-apoptotic protein Mcl-1 is associated with AML disease maintenance and resistant to therapy. Both Mcl-1 and FLT3 turn-over rapidly and are vulnerable targets of transient translation inhibition. Immunoblots showed that omacetaxine reduced the levels of both FLT3 and Mcl-1 in the MV4-11 cells. This activity augmented the effect of AC220 on FLT3 kinase, and induced synergistic apoptosis. Same synergistic combination was observed with omacetaxine and sunitinib, an inhibitor of FLT3, KIT and PDGF-R. Dose reduction index derived from these analyses showed that omacetaxine greatly potentiated the activity of both AC220 and sunitinib, resulting in profound apoptosis.Both Bcl-2 and Mcl-1 are pro-survival proteins that regulate apoptosis by interacting with the BH3 motifs of their pro-apoptotic partners. BH3 mimetics, such as ABT-199, bind with high affinity to Bcl-2 and block this interaction, but not to Mcl-1. Resistance to BH3 mimetics in AML cells is associated with upregulation of Mcl-1. Since ABT-199 inhibits Bcl-2 but spares Mcl-1, and omacetaxine reduces Mcl-1 without affecting Bcl-2 expression, we hypothesized that their combination would target the two parallel arms of apoptosis control and kill the AML cells synergistically. Indeed, omacetaxine reduced Mcl-1 in the OCI-AML3 cells, leading to loss of mitochondrial membrane potential and apoptosis. ABT-199 blocked Bcl-2 function and also induced the intrinsic pathway of apoptosis. Their combination induced greater mitochondrial damage and apoptosis than either drug alone. The median effect analysis showed that they potentiate each other and exhibited strong synergy. Taken together, these results demonstrated that omacetaxine is active in AML cells alone and in mechanism based combinations. These actions provide rationale that warrants investigation in the clinic. Disclosures:No relevant conflicts of interest to declare.

Translational Research From the Tropical Forest: Silvestrol, a Natural Product From the Plant Aglaia Foveolata inhibits the Expression of Tyrosine Kinases and Shows a Significant In Vivo Activity in Acute Myeloid Leukemia (AML)

Abstract 2616Despite the progress made in the diagnosis, classification, prognostication and treatment of AML, the majority of patients still fail to achieve long-term survival and die of their disease. Several cytogenetic and molecular markers including gene mutations and aberrant expression have been shown to impact the outcome of AML patients and some of these are also potential therapeutic targets. Receptor tyrosine kinases (RTKs) regulate cell survival, proliferation and differentiation in normal hematopoiesis. Gain-of-function mutations of genes encoding RTKs, such as internal tandem duplication of FLT3 (FLT3-ITD) and mutations of KIT, result in constitutive tyrosine kinase activity and have been associated with poor outcome in distinct cytogenetic (i.e., normal karyotype and core binding factor, respectively) subsets of AML. In addition, overexpression of FLT3 and wild type (wt) KIT was also found to negatively impact outcome. Tyrosine kinase (TK) inhibitors (Is) (e.g., imatinib, dasatinib or PKC412) have been shown to suppress the aberrant activity of these mutants, but their activities as single agents have been limited by their relatively elevated IC50 values for myeloid blasts and early onset of resistance. Indeed, even in combination with chemotherapy, TKIs have not yet been shown to impact favorably on AML patient outcome. This supports the need for development of novel strategies that effectively interfere with aberrant TK activity in leukemia blasts. Silvestrol, a cyclopenta[b]benzofuran derivative isolated from Aglaia foveolata, a plant that grows in the tropical rain forests of Southeast Asia, has been shown to exhibit potent inhibitory activity against a panel of human cancer cell lines and shows in vitro and in vivo efficacy in B-cell malignancies. The mechanism of action has been thought to involve inhibition of translation initiation, which in turn results in selective depletion of actively translated, short half-lives proteins. We hypothesized that silvestrol-mediated inhibition of the translation of mutated and/or overexpressed RTKs results in a significant antileukemic activity in AML cells that rely on the aberrant TK activity of these proteins. Silvestrol showed antiproliferative and proapoptotic activities in representative AML cell lines harboring activated TK proteins: FLT3-ITD-positive MV4-11, FLT3-overexpressing THP-1 KIT-mutated Kasumi-1 and also BCR-ABL-positive K562, an erythroblastic cell line notoriously resistant to chemotherapy. Importantly, silvestrol treatment reduced the expression levels of FLT3 in MV4-11 (92 %) and THP-1 (84 %), KIT in Kasumi-1 (46 %), and BCR-ABL in K562 (64 %), compared to the respective vehicle-treated controls. The silvestrol IC50 at 48 hr was less than 10nM for all cell lines (MTS assay). Silvestrol also showed antiproliferative activity in FLT3-ITD-positive AML patient blasts (IC50 24 nM at 48 hr). To evaluate in vivo activity, NOD-SCID Gamma (NSG) mice were engrafted with FLT3-ITD-positive MV4-11 cells. Twenty-one days post-engraftment, mice were treated intraperitoneally with vehicle or silvestrol (1.5 mg/kg every 48 hr for 3 weeks). After 3 doses (day 6 of treatment), spleens were obtained from three mice from each group. Spleens from silvestrol treated mice were 60% decreased in size (P=0.016) and showed 80% FLT3 protein downregulation (P=0.002) compared to vehicle-treated controls. Silvestrol treated leukemic mice (n=10) survived significantly longer than vehicle treated control (n=6) (median survival: 55.5 days vs. 29 days from engraftment day respectively; P<0.0001). All vehicle-treated mice died by day 31 from engraftment (day 11 from treatment start), while 50% of silvestrol-treated mice are still alive on day 60 from engraftment (day 40 from treatment start; updated survival curves will be provided). We conclude that silvestrol has a potent in vitro and in vivo antileukemic activity in AML through downregulation of the expression and activity of FLT3 and other TKs driving leukemia. Given the excellent antileukemic activity of silvestrol via a novel mechanism of TK inhibition that is potentially complementary to that of TKIs (i.e., silvestrol-induced suppression of TK protein expression vs. TKI-induced protein enzymatic inhibition), a rapid translation of this natural product to the clinic as a single agent or in combination with other TKIs ± chemotherapy seems warranted. Disclosures:No relevant conflicts of interest to declare.

Methylated Retroviral Integration Mutagenesis (MRIM) in Mice and Comparative Analysis in Human AML Identify Reduced PTP4A3 Expression As a Prognostic Indicator

Abstract 746Acute myeloid leukemia (AML) is a complex disease driven by multiple cytogenetic abnormalities, aberrant expression levels and mutations of different genes. Frequent occurrence of chromosomal deletions suggests that haplo-insufficiencies contribute to the pathogenesis of AML. However, because deleted regions often harbor numerous genes, it remains difficult to pin point critical haplo-insufficient genes (HIGs) involved in the pathogenesis of AML. Retroviral insertion mutagenesis in mouse models has been used to discover novel genes involved in the development of different types of cancer. Only a small minority of identified genes in these screens has been classified as tumor suppressor genes or HIGs, based on disruption of coding sequences by proviral integrations. Gene therapy studies using murine leukemia virus based vectors have shown that epigenetic changes of long terminal repeats (LTRs) of integrated proviruses often result in silencing of therapeutic genes, and that preventing methylation of the CpG islands within LTRs overcomes this problem. Based on these observations, we hypothesized that methylation of viral sequences not only results in silencing of retroviral genes themselves but may also affect host genes located proximal to proviral integrations. Methylated LTRs located in proximity of promoter regions may thus identify genes that are deregulated leading to haplo-insufficiency.To identify novel HIGs, genomic DNA of 6 murine leukemias induced by the Graffi 1.4 murine leukemia virus, was isolated and digested. Subsequently, methylated fragments were immunoprecipitated using methylated DNA immunoprecipitation (MeDIP), followed by inverse PCR to amplify fragments flanking methylated viral integrations (mVIS). Amplified fragments were labeled, fragmented and hybridized on murine promoter DNA arrays. DNA methylated viral integration sites were detected using a novel algorithm, hypergeometric analysis of tiling arrays (HAT) [1]. All integrations were confirmed by directed PCR and Sanger sequencing. Next, their methylation status was confirmed with methylation sensitive restriction analysis. We refer to this strategy as Methylated Virus Integration Mutagenesis (MRIM). In the murine leukemias, expression levels of genes identified by MRIM, relative to Tbp (TATA box binding protein) expression, were determined by quantitative PCR (qPCR). Survival analysis was performed in a cohort of 454 de novo AML patients under the age of 60 using a Cox-regression model. For this analysis, MAS5 normalised, log2 transformed PTP4A3 gene expression levels were used, determined by gene expression profiling.Using MRIM, 6 genes that were flanked by a methylated LTR were identified. Of these genes, Ptp4a3 was transcriptionally down regulated relative to normal bone marrow. Ptp4a3 encodes a tyrosine phosphatase that is implicated in Flt3 signaling. Studies in a human AML patient cohort under the age of 60 (n=454) revealed that PTP4A3 expression is negatively correlated with prognostic outcome, both for overall survival (p-value <0.0001, HR = 1.269) and event-free survival (p-value <0.0001, HR = 1.261). Additional analysis showed that this negative correlation with survival was independent of other prognostic parameters as age, white blood cell count, cytogenetic risk and mutation status of NPM1, CEBPA and internal tandem duplication of FLT3 (FLT3ITD).In conclusion, by mapping DNA methylated viral integration sites in murine leukemias induced by retroviral integration mutagenesis (MRIM), followed by expression level analysis, we identified Ptp4a3 as a candidate HIG. PTP4A3 has recently been proposed to have a role in drug-resistance in AMLs with FLT3ITD. This finding, together with the observation that high PTP4A3 expression negatively correlates with prognostic outcome, marks PTP4A3 as a potential therapeutic target in AML.1. E. Taskesen et al., HAT: hypergeometric analysis of tiling-arrays with application to promoter-GeneChip data. BMC Bioinformatics, 2010. 11: p. 275. Disclosures:No relevant conflicts of interest to declare.

Sensitive measurement of quantity dynamics of FLT3 internal tandem duplication at early time points provides prognostic information

BackgroundThe level of minimal residual disease (MRD) in acute myeloid leukemia (AML) at early time points (TPs) may be an important prognostic factor. Although internal tandem duplication of FLT3 (FLT3-ITD) as an MRD marker has been questioned for its instability based on semi-quantitative methods, we hypothesized that FLT3-ITD dynamics measured by sensitive quantitative real-time PCR at early TPs before appearance of instability may provide prognostic information. Patients and methodsWe measured mutant quantity in 493 serial samples from 55 patients with a median follow-up time of 64.8 months. The FLT3-ITD quantities after induction (TP1) and after the first post-induction chemotherapy (TP2) were analyzed. ResultsWe found that lower FLT3-ITD levels at TP2 predicted longer overall survival (OS) and disease-free survival (DFS) regardless of cytogenetic risk. Multivariate analysis showed that ≥3 log reduction of FLT3-ITD at TP2 independently predicted better DFS and a trend toward better OS. FLT3-ITD disappeared at relapse in 16.7% of patients and none in those harboring mutant NPM1 compared with 29.4% in those with wild-type NPM1 (P = 0.032). ConclusionsThough the mutation may disappear at relapse in a few patients, FLT3-ITD levels at early TPs after chemotherapy provide prognostic information. FLT3-ITD is significantly more stable in those with mutant NPM1.

Impact of Allogeneic Transplantation From Matched Related and Unrelated Donors on Clinical Outcome In Younger Adult AML Patients with FLT3 Internal Tandem Duplications

AbstractAbstract 909 Background:Internal tandem duplications of FLT3 (FLT3-ITD) occur in approximately one third of acute myeloid leukemia (AML) patients and are associated with poor outcome. It has been suggested that allogeneic hematopoietic stem cell transplantation (allo-HSCT) from a matched related donor (MRD) can overcome the negative impact of FLT3-ITD. However, for those patients without MRD it is unclear, whether the adverse impact can be abrogated by allo-HSCT from a matched unrelated donor (MUD). Aims:To assess clinical outcome in AML with FLT3-ITD after allo-HSCT from MRD and MUD. Methods:The study included 437 adult patients (median age, 47 years; range, 16–60 years) with newly diagnosed, FLT3-ITD positive AML enrolled on five prospective treatment trials of the German-Austrian AML Study Group (AMLSG) between 1993 and 2008. Patients with acute promyelocytic and core binding factor leukemia were excluded. FLT3-ITD was diagnosed by genomic DNA PCR and Genescan analysis. Mutant to wild-type FLT3-ITD ratio (allelic ratio) was available in 78%, and FLT3-ITD insertion site was determined by direct sequencing in 59% of the patients.The strategy of allo-HSCT changed in November 2006. Before the amendment, only patients with MRD were intended to receive allo-HSCT in first complete remission (CR), whereas thereafter, the spectrum of donors was extended to MUD. To account for the time dependency of allo-HSCT, landmark analyses for univariable analyses and Andersen Gill models for multivariable analyses were used. Results:CR rates before and after the amendment were 72% and 68%, respectively (p=0.63). Before amendment, 71 of 234 (30%) patients received allo-HSCT (48 MRD, 23 MUD), after amendment, 58 of 77 (74%) patients (16 MRD, 42 MUD). After amendment, the cohort was characterized by a significantly higher white blood count (WBC) (p=0.002), higher allelic ratio (p=0.04) and a higher frequency of FLT3-ITD insertion site in the beta-1-sheet (p=0.05). Comparing the two cohorts before and after amendment, there was no significant difference in relapse-free survival (RFS) (p=0.51) and overall survival (OS) (p=0.45). To account for the differences in the frequencies of unfavorable prognostic markers, we performed multivariable analyses in the whole population including the following variables: age, log(WBC), allo-HSCT from MRD or MUD, NPM1 mutation and median dichotomized allelic ratio. This model revealed allo-HSCT from MRD (HR 0.64, p=0.03) and age (difference of ten years) (HR 1.19, p=0.02) as significant prognostic parameters, whereas allo-HSCT from MUD was not significant (HR 0.82, p=0.31). In subset analysis, based on the availability of FLT3-ITD insertion site, the beneficial impact of allo-HSCT from MRD (HR 0.48, p=0.02) was even more pronounced, and FLT3-ITD insertion site in the beta-1-sheet appeared as a significant variable (HR 1.67, p=0.01). Landmark analyses for RFS at five months revealed a beneficial impact of allo-HSCT from both MRD and MUD, whereas at later time points the beneficial impact of allo-HSCT from MUD disappeared. There was no significant difference in cumulative incidence of relapse (p=0.25) and death (p=0.50) between allo-HSCT from MRD and MUD. Of 167 patients who relapsed, four received allo-HSCT from a haplo-identical family donor (HID), 54 from MUD and 12 from MRD. Overall survival for relapsed patients without transplantation and those with allo-HSCT from MRD was below 10% after one year and was significantly inferior compared to patients receiving allo-HSCT from MUD or HID with 49% after one year (p<0.0001). Conclusions:Allo-HSCT from MRD and also from MUD, if performed early during first CR, improves survival of younger adult patients with FLT3-ITD positive AML. Outcome after relapse was generally very poor, with allo-HSCT from MUD or HID having a beneficial effect. Disclosures:No relevant conflicts of interest to declare.