scholarly journals A pharmacodynamic study of the FLT3 inhibitor KW-2449 yields insight into the basis for clinical response

Blood ◽  
2009 ◽  
Vol 113 (17) ◽  
pp. 3938-3946 ◽  
Author(s):  
Keith W. Pratz ◽  
Jorge Cortes ◽  
Gail J. Roboz ◽  
Niranjan Rao ◽  
Omotayo Arowojolu ◽  
...  
Keyword(s):  
Quantitative Measurement ◽  
Tandem Duplication ◽  
Kinase Inhibitor ◽  
Phase 1 ◽  
Internal Tandem Duplication ◽  
Flt3 Inhibitor ◽  
Flt3 Inhibitors ◽  
Targeted Agent ◽  
Insight Into

AbstractInternal tandem duplication mutations of FLT3 (FLT3/ITD mutations) are common in acute myeloid leukemia (AML) and confer a poor prognosis. This would suggest that FLT3 is an ideal therapeutic target, but FLT3 targeted therapy has produced only modest benefits in clinical trials. Due to technical obstacles, the assessment of target inhibition in patients treated with FLT3 inhibitors has been limited and generally only qualitative. KW-2449 is a novel multitargeted kinase inhibitor that induces cytotoxicity in Molm14 cells (which harbor an FLT3/ITD mutation). The cytotoxic effect occurs primarily at concentrations sufficient to inhibit FLT3 autophosphorylation to less than 20% of its baseline. We report here correlative data from a phase 1 trial of KW-2449, a trial in which typical transient reductions in the peripheral blast counts were observed. Using quantitative measurement of FLT3 inhibition over time in these patients, we confirmed that FLT3 was inhibited, but only transiently to less than 20% of baseline. Our results suggest that the failure to fully inhibit FLT3 in sustained fashion may be an underlying reason for the minimal success of FLT3 inhibitors to date, and stress the importance of confirming in vivo target inhibition when taking a targeted agent into the clinical setting. The clinical studies are registered on www.clinicaltrials.gov as NCT00346632.

scholarly journals Concurrent FLT3 Inhibitor and PP2A Activating Drug Treatment Induces Synergistic Cytotoxicity in Acute Myeloid Leukemia Cells with FLT3 Internal Tandem Duplication through Proteasomal Degradation of Pim-1 and c-Myc

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3951-3951
Author(s):  
Mario Scarpa ◽  
Shivani Kapoor ◽  
Danilo Perrotti ◽  
Maria R. Baer
Keyword(s):  
Combination Treatment ◽  
Tandem Duplication ◽  
Retroviral Vectors ◽  
Concurrent Treatment ◽  
Internal Tandem Duplication ◽  
Flt3 Inhibitor ◽  
Synergistic Cytotoxicity ◽  
Flt3 Inhibitors ◽  
Drug Treatments ◽  
Flt3 Internal Tandem Duplication

Abstract Introduction: In 30% of acute myeloid leukemia (AML) patients, internal tandem duplication of fms-like tyrosine kinase 3 (FLT3-ITD) causes constitutive and aberrant FLT3 signaling, and these patients have short relapse-free and overall survival. FLT3 inhibitors have limited and transient efficacy, but their efficacy may be enhanced by combination with other drugs targeting FLT3 signaling. FLT3 activation also inhibits the tumor suppressor protein phosphatase 2A (PP2A). FLT3 inhibitors and PP2A-activating drugs have been shown to induce synergistic cytotoxicity in cells with FLT3-ITD. To address mechanisms underlying this effect, we studied effects of combination therapy on the oncogenic serine/threonine kinase Pim-1 and the transcription factor c-Myc, both of which are upregulated in cells with FLT3-ITD and are also PP2A substrates. Methods: Ba/F3-ITD and MV4-11 cells and AML patient blasts with FLT3-ITD were cultured with a FLT3 inhibitor, gilteritinib (ASP2215) or quizartinib (AC220), and/or the PP2A-activating drug fingolimod (FTY720) at pharmacologically relevant concentrations, or DMSO control. Drug combination effects were measured by combination index determined by the Chou-Talalay method using CompuSyn software. Apoptosis was measured by Annexin V/propidium iodide staining detected by flow cytometry. c-Myc and GAPDH control mRNA was measured by real-time polymerase chain reaction. Pim-1 kinase, c-Myc, phospho-c-MycSer62, phospho-c-MycThr58, phospho-STAT5Tyr694, STAT5, phospho-PP2ATyr307, PP2A, phospho-BADSer112 and BAD levels were measured by immunoblotting. Cycloheximide treatment was used to assess protein stability. Protein expression and stability were measured with and without the proteasome inhibitor MG-132. Pim-1 kinase was inhibited with the pan-Pim inhibitor AZD1208. Ba/F3-ITD cells were infected with pMX-Flag-K67M kinase-dead (KD) Pim-1 and empty pMX retroviral vectors and with pBABE-ER-cMYC and with empty pBABE-ER retroviral vectors. Results: Concurrent treatment with 15 nM gilteritinib or 1 nM quizartinib and FTY720 2 µM in cell lines and 4 µM in patient samples decreased growth and increased apoptosis of cells with FLT3-ITD, relative to single drug treatments, and produced synergistic cytotoxicity. FLT3 inhibition was confirmed by decrease in phospho-STAT5 and PP2A activation by decreased phospho-PP2A. Concurrent treatment decreased expression of both Pim-1 and c-Myc protein, but not c-Myc mRNA, in Ba/F3-ITD and MV4-11 cells and AML patient blasts with FLT3-ITD, relative to single drug treatments. Additionally, selective decrease in phospho-MycSer62, a stable c-Myc phosphoprotein that is dephosphorylated by PP2A, was seen, with persistence of phospho-c-MycThr58. FLT3 inhibitor and PP2A activator combination treatment was found to decrease stability of c-Myc and Pim-1 protein, in relation to single drugs. Moreover, pretreatment with the proteasome inhibitor MG-132 abrogated downregulation of Pim-1 and c-Myc protein expression and decrease in Pim-1 and c-Myc protein stability in Ba/F3-ITD cells treated with FLT3 inhibitor and PP2A activator. Pretreatment with the pan-Pim kinase inhibitor AZD1208, with Pim-1 inhibition confirmed by decreased phospho-BADS112 had no effect on c-Myc downregulation, and c-Myc was similarly downregulated in Pim-1 kinase-dead cells as in parental and empty-vector cells, demonstrating that combination treatment effects on c-Myc are not Pim-1 kinase-dependent. Additionally, FLT3 inhibitor and PP2A-activating drug combination induced apoptosis in 30% of cells with c-Myc overexpression, compared to 60% of parental and empty vector-infected cells. Finally, c-Myc overexpression did not abrogate Pim-1 downregulation by combination treatment. Conclusions: Concurrent FLT3 inhibitor and PP2A activating drug treatment induces synergistic cytotoxicity in AML cells with FLT3 internal tandem duplication through proteasomal degradation of Pim-1 and c-Myc, and effects on Pim-1 and c-Myc are independent. The data support in vivo testing of FLT3 inhibitor and PP2A-activating drug combinations and development of a clinical trial. Disclosures No relevant conflicts of interest to declare.

The Multi-Kinase Inhibitor TG02 targets CD34+CD38-CD123+ AML Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1823-1823
Author(s):  
Monica Pallis ◽  
Francis Burrows ◽  
Nigel H. Russell
Keyword(s):  
Kinase Inhibitor ◽  
In Vitro Toxicity ◽  
The Novel ◽  
In Vivo Models ◽  
Erk Activation ◽  
Akt Phosphorylation ◽  
Flt3 Inhibitor ◽  
Flt3 Inhibitors

Abstract Abstract 1823 In clinical trials, FLT3 inhibitors are reported to kill circulating AML blasts, but the bone marrow is protected. We have previously reported that niche-like conditions (fibronectin and a cytokine cocktail) significantly reduced the in vitro toxicity of the FLT3 inhibitor AG1296 to AML cells. Moreover, the toxicity of AG1296 to the chemoresistant leukaemic CD34+CD38-CD123+ subset was completely abolished under niche-like conditions. The novel multi-kinase inhibitor TG02 has selectivity against cell cycle and transcriptional CDKs and JAK2 as well as FLT3. TG02 has efficacy in in vivo models and induces apoptosis in primary AML cells. We have now evaluated its in vitro toxicity under niche-like conditions in bulk AML cells and in the CD34+CD38-CD123+ subset. In a cohort of six FLT3-ITD and five FLT3-wildtype samples, 100nM TG02 induced decreases of 30% in bulk cells and 32% in CD34+CD38-CD123+ cells, whereas AG1296 (5μM) induced a median 21% decrease in bulk cells under niche-like conditions but a 0% decrease in CD34+CD38-CD123+ cells. Lestaurtinib, sorafenib and sunitinib were used as comparators (all at 100 nM) and induced, respectively, 13%, 4% and 13% decrease in bulk cells and 10%, 0% and 8% decrease in CD34+CD38-CD123+ cells. FLT3 wildtype as well as ITD samples were targeted. In order to establish the molecular pathways involved in niche-mediated chemoresistance and its reversal, we treated primary AML samples with TG02 or AG1296 for 3 hours in the presence and absence of niche proteins; we measured activating phosphorylations of STAT3 (tyr705), STAT5 (tyr694), ERK1/2 (thr202/tyr404) and AKT(ser473). Basal levels of activating phosphorylations were generally higher in the bulk cells than the CD34+CD38-CD123+ cells, possibly reflecting the increased quiescence of the latter subset. STAT3, STAT5 and ERK1/2 phosphorylation were reduced by TG02 to a slightly greater degree than by AG1296 in bulk cells. However, in CD34+CD38-CD123+ cells this contrast was enhanced, such that AG1296 was ineffective, whereas TG02 was at least as effective as in bulk cells. Niche-like conditions induced an increase in phosphorylation of STAT5, but not of the other proteins tested. TG02 reduced this to basal levels in both bulk cells and CD34+CD38-CD123+ cells. AG1296 partially blocked niche-induced STAT5 phosphorylation in bulk cells, but not in CD34+CD38-CD123+ cells. It had no effect on ERK signalling. AKT phosphorylation was not informative. In conclusion, TGO2 is more cytotoxic than comparatively selective FLT3 inhibitors towards CD34+CD38-CD123+ AML cells as well as bulk cells under niche conditions and the toxicity is associated with downregulation of STAT3, STAT5 and ERK activation. Disclosures: Pallis: Tragara Pharmaceuticals: Research Funding. Burrows:Tragara Pharmaceuticals: Employment.

A FLT3-targeted tyrosine kinase inhibitor is cytotoxic to leukemia cells in vitro and in vivo

Blood ◽  
2002 ◽  
Vol 99 (11) ◽  
pp. 3885-3891 ◽  
Author(s):  
Mark Levis ◽  
Jeffrey Allebach ◽  
Kam-Fai Tse ◽  
Rui Zheng ◽  
Brenda R. Baldwin ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Point Mutations ◽  
Internal Tandem Duplication ◽  
Activating Mutations ◽  
Flt3 Inhibitor

Constitutively activating internal tandem duplication (ITD) and point mutations of the receptor tyrosine kinase FLT3 are present in up to 41% of patients with acute myeloid leukemia (AML). These FLT3/ITD mutations are likely to be important because their presence is associated with a poor prognosis. Both types of mutations appear to activate the tyrosine kinase activity of FLT3. We describe here the identification and characterization of the indolocarbazole derivative CEP-701 as a FLT3 inhibitor. This drug potently and selectively inhibits autophosphorylation of wild-type and constitutively activated mutant FLT3 in vitro in FLT3/ITD-transfected cells and in human FLT3-expressing myeloid leukemia–derived cell lines. We demonstrate that CEP-701 induces a cytotoxic effect on cells in a dose-responsive fashion that parallels the inhibition of FLT3. STAT5 and ERK1/2, downstream targets of FLT3 in the signaling pathway, are inhibited in response to FLT3 inhibition. In primary leukemia blasts from AML patients harboring FLT3/ITD mutations, FLT3 is also inhibited, with an associated cytotoxic response. Finally, using a mouse model of FLT3/ITD leukemia, we demonstrate that the drug inhibits FLT3 phosphorylation in vivo and prolongs survival. These findings form the basis for a planned clinical trial of CEP-701 in patients with AML harboring FLT3- activating mutations.

scholarly journals Terminal myeloid differentiation in vivo is induced by FLT3 inhibition in FLT3/ITD AML

Blood ◽  
2012 ◽  
Vol 120 (20) ◽  
pp. 4205-4214 ◽  
Author(s):  
Amy Sexauer ◽  
Alexander Perl ◽  
Xiaochuan Yang ◽  
Michael Borowitz ◽  
Christopher Gocke ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Normal Karyotype ◽  
Myeloid Differentiation ◽  
Internal Tandem Duplication ◽  
Cycle Arrest ◽  
Flt3 Inhibitor ◽  
Differentiation Block

Abstract A hallmark of cancer is the disruption of differentiation within tumor cells. Internal tandem duplication mutations of the FLT3 kinase (FLT3/ITD) occur commonly in acute myeloid leukemia (AML) and are associated with poor survival, leading to efforts to develop FLT3 kinase inhibitors. However, FLT3 inhibitors have thus far met with limited success, inducing only a clearance of peripheral blasts with minimal BM responses. Quizartinib is a novel potent and selective FLT3 inhibitor currently being studied in clinical trials. In 13 of 14 FLT3/ITD AML patients with normal karyotype treated with quizartinib, we observed terminal myeloid differentiation of BM blasts in association with a clinical differentiation syndrome. The single patient whose blasts failed to differentiate had a preexisting C/EBPα mutation and another developed a C/EBPα mutation at disease progression, suggesting a mechanism of resistance to FLT3 inhibition. In vitro, in primary blasts cocultured with human BM stroma, FLT3 inhibition with quizartinib induced cell-cycle arrest and differentiation rather than apoptosis. The present study is the first description of terminal differentiation of cancer cells in patients treated with a tyrosine kinase inhibitor. These data highlight the importance of the differentiation block in the patho-genesis of AML.

scholarly journals A Mixed Type 1 and Type 2 Kinase Inhibitor That Overrides FLT3 F691 and D835 Resistance Mutations

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1074-1074 ◽  
Author(s):  
Jack Lin ◽  
Ying Zhang ◽  
Bernice Matusow ◽  
Adam Mumy ◽  
Garson Tsang ◽  
...  
Keyword(s):  
Mixed Type ◽  
Kinase Inhibitor ◽  
Binding Mode ◽  
Resistance Mutations ◽  
Flt3 Inhibitor ◽  
Flt3 Inhibitors

Abstract Introduction: Mutations in FLT3, a class III receptor tyrosine kinase predominantly expressed on hematopoietic progenitor cells, represent the most common genetic alteration in patients with acute myeloid leukemia (AML). Approximately 25% of patients with AML harbor internal tandem duplications (ITD) within the juxtamembrane domain of FLT3. Quizartinib, the first selective FLT3 tyrosine kinase inhibitor (TKI) to show clinical activity, frequently achieves a composite complete remissions (CRc) rate of approximately 50% in relapsed and refractory FLT3-ITD+AML patients. However, patient samples obtained at the time of relapse with quizartinib revealed resistance-conferring FLT3 mutations at two kinase domain residues (F691L and D835V/Y/F). The crystal structure of the FLT3-quizartinib complex indicates that quizartinib binding relies on essential edge-to-face aromatic interactions with the gatekeeper F691 residue, and with F830 within the DFG motif in the activation loop (A-loop). This reliance makes quizartinib responses critically vulnerable to gatekeeper (F691) and A-loop (D835) substitutions. This has led to the identification of TKIs that have activity against quizartinib-resistant FLT3 mutants, including type 1 inhibitors (e.g. crenolanib, ASP2215) targeting specifically D835 mutants, and type 2 inhibitors (e.g. pexidartinib, ponatinib) active against F691L. Until now, no FLT3 inhibitor has demonstrated potent inhibition of both classes of resistance mutations. Methods: To identify FLT3 inhibitors with activity against both gatekeeper and A-loop mutants, we used a modular approach to systematically alter the constituents of quizartinib to optimize interactions with the mutant proteins guided by X-ray structures and modeling. The compounds were profiled through a panel of in vitro growth assays and in vivo studies using Ba/F3 cells engineered to express FLT3-ITD/F691L and FLT3-ITD/D835Y, and MOLM14 quizartinib-resistant clones (MOLM14/F691L and MOLM14/D835Y) that have acquired the same mutations after chronic exposure to quizartinib. When injected into the tail vein of nude mice, the engineered Ba/F3 cells home to the spleen and proliferate, causing marked splenomegaly, whereas MOLM14 parental cells and MOLM14 resistant clones significantly reduce the overall survival of mice. Ba/F3-induced splenomegaly is directly dependent on the activity of the expressed mutant FLT3 and inhibition of splenomegaly measures target engagement in vivo. Analysis of efficacy in the MOLM14 survival models was done by Kaplan-Meier estimates and the logrank statistic. Results: Structure-guided lead optimization generated a series of FLT3 inhibitors with a mixed type 1 and type 2 binding mode and equal potency against the two classes of resistant mutants. Further optimization to improve pharmaceutical properties and maximize in vivo efficacy led to development candidate PLX3623. PLX3623 potently inhibited the growth of two Ba/F3 cell lines expressing F691L and D835Y mutants, with IC50 values of 0.18 (±0.04) and 0.15 (±0.03) nM, respectively. PLX3623 had a comparable antiproliferative effect on the MOLM14 resistant clones, with IC50values of 0.52 (±0.14) and 0.26 (±0.08) nM for the F691L and D835Y clones, respectively. In vivo PLX3623 treatment resulted in dose-dependent reduction in Ba/F3-induced splenomegaly, achieving >90% inhibition at 3 mg/kg dose. PLX3623 at 3mg/kg increased the length of survival of MOLM14 parental cell recipients by 120%, compared to the 80% increase afforded by 3 mg/kg quizartinib. At 3mg/kg dose, PLX3623 extended the survival of mice that received the two MOLM14 resistant clones by 80%; quizartinib at the same dose provided no survival benefit. Conclusion: Clonal evolution and tumor heterogeneity present a major challenge to targeted therapy and personalized cancer medicine. While acquired mutations individually may be actionable targets, often multiple inhibitors are required to combat the clonal diversity in a refractory setting. PLX3623, a FLT3 inhibitor with a mixed type 1 and type 2 binding mode, is the first TKI that is active against mutations conferring resistance through independent structural mechanisms. The in vitro and in vivo potency and favorable nonclinical safety profile of PLX3623 support its clinical development as a potentially best-in-class therapy to overcome clinical resistance to first generation FLT3 inhibitors. Disclosures Lin: Plexxikon Inc.: Employment. Zhang:Plexxikon Inc.: Employment. Matusow:Plexxikon Inc.: Employment. Mumy:Plexxikon Inc.: Employment. Tsang:Plexxikon Inc.: Employment. Zhang:Plexxikon Inc.: Employment. Powers:Plexxikon Inc.: Employment. Spevak:Plexxikon Inc.: Employment. Severson:Plexxikon Inc.: Employment. Tsai:Plexxikon Inc.: Employment. Bollag:Plexxikon Inc.: Employment. Zhang:Plexxikon Inc.: Employment.

scholarly journals ATM/G6PD-driven redox metabolism promotes FLT3 inhibitor resistance in acute myeloid leukemia

2016 ◽  
Vol 113 (43) ◽  
pp. E6669-E6678 ◽  
Author(s):  
Mark A. Gregory ◽  
Angelo D’Alessandro ◽  
Francesca Alvarez-Calderon ◽  
Jihye Kim ◽  
Travis Nemkov ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Mitochondrial Oxidative Stress ◽  
Flt3 Inhibitor ◽  
A Genome ◽  
Flt3 Inhibitors ◽  
Acute Myeloid

Activating mutations in FMS-like tyrosine kinase 3 (FLT3) are common in acute myeloid leukemia (AML) and drive leukemic cell growth and survival. Although FLT3 inhibitors have shown considerable promise for the treatment of AML, they ultimately fail to achieve long-term remissions as monotherapy. To identify genetic targets that can sensitize AML cells to killing by FLT3 inhibitors, we performed a genome-wide RNA interference (RNAi)-based screen that identified ATM (ataxia telangiectasia mutated) as being synthetic lethal with FLT3 inhibitor therapy. We found that inactivating ATM or its downstream effector glucose 6-phosphate dehydrogenase (G6PD) sensitizes AML cells to FLT3 inhibitor induced apoptosis. Examination of the cellular metabolome showed that FLT3 inhibition by itself causes profound alterations in central carbon metabolism, resulting in impaired production of the antioxidant factor glutathione, which was further impaired by ATM or G6PD inactivation. Moreover, FLT3 inhibition elicited severe mitochondrial oxidative stress that is causative in apoptosis and is exacerbated by ATM/G6PD inhibition. The use of an agent that intensifies mitochondrial oxidative stress in combination with a FLT3 inhibitor augmented elimination of AML cells in vitro and in vivo, revealing a therapeutic strategy for the improved treatment of FLT3 mutated AML.

scholarly journals AZD1208, a potent and selective pan-Pim kinase inhibitor, demonstrates efficacy in preclinical models of acute myeloid leukemia

Blood ◽  
2014 ◽  
Vol 123 (6) ◽  
pp. 905-913 ◽  
Author(s):  
Erika K. Keeton ◽  
Kristen McEachern ◽  
Keith S. Dillman ◽  
Sangeetha Palakurthi ◽  
Yichen Cao ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Myeloid Leukemia ◽  
Tandem Duplication ◽  
Kinase Inhibitor ◽  
Wild Type ◽  
Internal Tandem Duplication ◽  
Rna Translation ◽  
Key Points ◽  
Messenger Rna Translation ◽  
Flt3 Internal Tandem Duplication

Key Points AZD1208 is a selective pan-Pim kinase inhibitor with efficacy in AML cells, xenografts, and Flt3-internal tandem duplication or Flt3 wild-type patient samples. AML cell growth inhibition is associated with suppression of p70S6K, 4EBP1 phosphorylation, and messenger RNA translation.

scholarly journals Blocking DNA Damage Repair May Be Involved in Stattic (STAT3 Inhibitor)-Induced FLT3-ITD AML Cell Apoptosis

2021 ◽  
Vol 9 ◽  
Author(s):  
Yuxuan Luo ◽  
Ying Lu ◽  
Bing Long ◽  
Yansi Lin ◽  
Yanling Yang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Myeloid Leukemia ◽  
Tandem Duplication ◽  
Dna Damage Repair ◽  
Underlying Mechanism ◽  
Internal Tandem Duplication ◽  
Mrna Sequencing ◽  
Damage Repair ◽  
Flt3 Inhibitors ◽  
Flt3 Internal Tandem Duplication

The FMS-like tyrosine kinase 3 (FLT3)- internal tandem duplication (ITD) mutation can be found in approximately 25% of all acute myeloid leukemia (AML) cases and is associated with a poor prognosis. The main treatment for FLT3-ITD-positive AML patients includes genotoxic therapy and FLT3 inhibitors, which are rarely curative. Inhibiting STAT3 activity can improve the sensitivity of solid tumor cells to radiotherapy and chemotherapy. This study aimed to explore whether Stattic (a STAT3 inhibitor) affects FLT3-ITD AML cells and the underlying mechanism. Stattic can inhibit the proliferation, promote apoptosis, arrest cell cycle at G0/G1, and suppress DNA damage repair in MV4-11cells. During the process, through mRNA sequencing, we found that DNA damage repair-related mRNA are also altered during the process. In summary, the mechanism by which Stattic induces apoptosis in MV4-11cells may involve blocking DNA damage repair machineries.

Internal tandem duplication mutation of FLT3 blocks myeloid differentiation through suppression of C/EBPα expression

Blood ◽  
2004 ◽  
Vol 103 (5) ◽  
pp. 1883-1890 ◽  
Author(s):  
Rui Zheng ◽  
Alan D. Friedman ◽  
Mark Levis ◽  
Li Li ◽  
Edward G. Weir ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Poor Prognosis ◽  
Tandem Duplication ◽  
Myeloid Differentiation ◽  
Internal Tandem Duplication ◽  
Activating Mutations ◽  
Flt3 Inhibitor ◽  
Enhancer Binding Protein ◽  
Differentiation Block ◽  
Proliferative Signaling

AbstractConstitutively activating mutations of FMS-like tyrosine kinase 3 (FLT3) occur in approximately one third of patients with acute myeloid leukemia (AML) and are associated with poor prognosis. Altered FLT3 signaling leads to antiapoptotic and proliferative signaling pathways. We recently showed that these mutations can also contribute to the differentiation arrest that characterizes leukemia. In this report we investigated the mechanism by which internal tandem duplication (ITD) mutation of FLT3 signaling blocks differentiation. Normally, myeloid differentiation requires the induction of CCAAT/enhancer-binding protein α (C/EBPα) and PU.1 expression. Expression of both genes was repressed by FLT3/ITD signaling in 32Dcl3 (32D) cells and this repression was overcome by treatment with a FLT3 inhibitor, allowing differentiation to proceed. We also observed increased expression of C/EBPα and PU.1 accompanied by signs of differentiation in 2 of 3 primary AML samples from patients with FLT3/ITD mutations receiving a FLT3 inhibitor, CEP-701, as part of a clinical trial. Forced expression of C/EBPα was also able to overcome FLT3/ITD-mediated differentiation block, further proving the importance of C/EBPα in this process.

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