scholarly journals Expert Curation of Somatic FLT3 Variants By the Clingen Somatic Hematologic Cancer Taskforce (ClinGen HCT)

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4387-4387
Author(s):  
Xiangqiang Shao ◽  
Shruti Rao ◽  
Coumarane Mani ◽  
Jason Saliba ◽  
Rong He ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Atp Binding ◽  
Evidence Based ◽  
Type I ◽  
Type Ii ◽  
Prediction Of Response ◽  
Clinical Interpretation ◽  
Hematologic Cancer ◽  
Flt3 Inhibitor ◽  
Flt3 Inhibitors

Abstract Clinical significance of somatic gene variants needs to be comprehensively characterized for their diagnostic, prognostic and/or therapeutic actionability in patient management. However, challenges remain due to discrepancies in interpretation and reporting of these somatic variants among different testing labs. Therefore, standardized curation, clinical interpretation and reporting of somatic variants in hematologic cancers is critical. To address this issue, the Hematologic Cancer Taskforce (HCT), composed of 52 multi-disciplinary experts including oncologists, molecular pathologists, lab directors, genomic scientists and biocurators, was formed in January 2020 within the ClinGen Somatic Cancer Clinical Domain Working Group (CDWG) with a goal to undertake systematic curation and evidence-based clinical interpretation of genes/somatic variants associated with hematologic malignancies. In collaboration with the Clinical Interpretation of Variants in Cancer (CIViC) (civicdb.org) knowledgebase, HCT members curate, edit, and verify Evidence Items for each variant extracted from peer-reviewed publications. Monthly discussions based on these Evidence Items lead to the preparation of variant Assertions, which summarize the state of the field consensus variant interpretation and include tiering based on the AMP/ASCO/CAP guidelines (PMID: 27993330). FMS-like tyrosine kinase 3 (FLT3) encodes a class III receptor tyrosine kinase expressed in hematopoietic cells. FLT3 mutations, including both internal tandem duplication (ITD) and mutations in the tyrosine kinase domain (TKD), are the most common mutations in acute myeloid leukemia (AML), occurring in approximately 30% of all AML cases. Implementing FLT3 tyrosine kinase inhibitors (TKIs) in different treatment regimens for FLT3 mutated AML patients has led to significantly improved overall survival. Type I FLT3 inhibitors, including midostaurin, gilteritinib, sunitinib, lestaurtinib, and crenolanib, bind to the ATP-binding site when the receptor is in active conformation. Type II FLT3 inhibitors, including sorafenib, ponatinib, and quizartinib, interact with a hydrophobic region directly adjacent to the ATP-binding domain that is only accessible when the receptor is inactive, which prevents receptor activation. Generally in AML cells, type I FLT3 inhibitors prevent activity for both ITD and TKD mutations, while Type II inhibitors target ITD but lack efficiency against TKD mutations. The development of TKD mutations in AML cells with ITD have proved to be a mechanism of acquired, or secondary resistance to Type II FLT3 inhibitors. The HCT is piloting curation assessments of FLT3 alterations, including ITD, TKD and non-TKD variants, in AML. So far, the HCT has curated 75 evidence items for FLT3 somatic variants. FLT3-ITD, as well as D835 and I836 were asserted as tier 1 level A variants based on the prediction of response to gilteritinib in relapsed/refractory AML (PMIDs: 27993330, 31665578, 28645776, 28516360, 27908881). Recent curation activities are focused on FLT3 D839G and N676K, as clinical trials using large AML patient cohorts are lacking in their ability to validate drug response/resistance associations of these two TKD variants due to their low frequency. Functional studies showed both variants result in increased proliferation and protection from apoptosis, supporting the oncogenic potential of these two variants (PMIDs: 26891877, 2468088). FLT3 D839G combined with ITD confers resistance to pexidartinib and ponatinib, both Type II FLT3 inhibitors (PMIDs: 25847190, 23430109). FLT3 N676K predicts response to the Type I FLT3 inhibitor, gilteritinib, when N676K is present alone or in combination with ITD. Interestingly, FLT3 N676K in the absence of ITD predicts response to sorafenib, a Type II FLT3 inhibitor (PMIDs: 32040554, 32984009). However, these results are mostly derived from in vitro studies. Two separate Tier II, Level D Assertions have been submitted for FLT3-ITD&D839G for its response to pexidartinib and ponatinib, and more evidence is being collected to form an Assertion for FLT3 N676K. The complexity of the prediction of response/resistance associated with FLT3 D839G and N676K supports the importance of evidence-based curation and collection for these variants in the context of the overall mutation profile, disease context and specific FLT3 TKIs to clearly define their clinical impact. Disclosures Pullarkat: Stemline Therapeutics: Honoraria.

scholarly journals SAMHD1 Phosphorylation Coordinates the Anti-HIV-1 Response by Diverse Interferons and Tyrosine Kinase Inhibition

mBio ◽  
2018 ◽  
Vol 9 (3) ◽  
Author(s):  
Matthew A. Szaniawski ◽  
Adam M. Spivak ◽  
James E. Cox ◽  
Jonathan L. Catrow ◽  
Timothy Hanley ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Kinase Inhibitors ◽  
Restriction Factor ◽  
Type I ◽  
Dependent Manner ◽  
Type Ii ◽  
Type Iii ◽  
Type Iii Ifn ◽  
Hiv 1

ABSTRACTMacrophages are susceptible to human immunodeficiency virus type 1 (HIV-1) infection despite abundant expression of antiviral proteins. Perhaps the most important antiviral protein is the restriction factor sterile alpha motif domain and histidine/aspartic acid domain-containing protein 1 (SAMHD1). We investigated the role of SAMHD1 and its phospho-dependent regulation in the context of HIV-1 infection in primary human monocyte-derived macrophages and the ability of various interferons (IFNs) and pharmacologic agents to modulate SAMHD1. Here we show that stimulation by type I, type II, and to a lesser degree, type III interferons share activation of SAMHD1 via dephosphorylation at threonine-592 as a consequence of signaling. Cyclin-dependent kinase 1 (CDK1), a known effector kinase for SAMHD1, was downregulated at the protein level by all IFN types tested. Pharmacologic inhibition or small interfering RNA (siRNA)-mediated knockdown of CDK1 phenocopied the effects of IFN on SAMHD1. A panel of FDA-approved tyrosine kinase inhibitors potently induced activation of SAMHD1 and subsequent HIV-1 inhibition. The viral restriction imposed via IFNs or dasatinib could be overcome through depletion of SAMHD1, indicating that their effects are exerted primarily through this pathway. Our results demonstrate that SAMHD1 activation, but not transcriptional upregulation or protein induction, is the predominant mechanism of HIV-1 restriction induced by type I, type II, and type III IFN signaling in macrophages. Furthermore, SAMHD1 activation presents a pharmacologically actionable target through which HIV-1 infection can be subverted.IMPORTANCEOur experimental results demonstrate that SAMHD1 dephosphorylation at threonine-592 represents a central mechanism of HIV-1 restriction that is common to the three known families of IFNs. While IFN types I and II were potent inhibitors of HIV-1, type III IFN showed modest to undetectable activity. Regulation of SAMHD1 by IFNs involved changes in phosphorylation status but not in protein levels. Phosphorylation of SAMHD1 in macrophages occurred at least in part via CDK1. Tyrosine kinase inhibitors similarly induced SAMHD1 dephosphorylation, which protects macrophages from HIV-1 in a SAMHD1-dependent manner. SAMHD1 is a critical restriction factor regulating HIV-1 infection of macrophages.

Monocytic Maturation Induced by FLT3 Inhibitor Therapy of Acute Myeloid Leukemia: Morphologic and Immunophenotypic Characteristics

2019 ◽  
Vol 51 (5) ◽  
pp. 478-483
Author(s):  
Cade D Arries ◽  
Sophia L Yohe
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Cell Population ◽  
Myeloid Leukemia ◽  
Inhibitor Therapy ◽  
Molecular Abnormalities ◽  
Flt3 Inhibitor ◽  
Flt3 Inhibitors ◽  
Acute Myeloid

Abstract Background FMS-like tyrosine kinase-3 (FLT3-ITD) mutations are some of the most common mutations in acute myeloid leukemia (AML), and patient outcomes have improved since the advent of tyrosine kinase inhibitors. First, granulocytic differentiation was described in FLT3-positive AML treated with FLT3 inhibitors, and more recently, monocytic differentiation was reported. Methods Two patients with myelomonocytic cells in their bone marrow were identified during routine follow-up after AML treatment that included FLT3 inhibitors. The bone marrow study was done as standard of care. Results Both patients had FLT3-ITD+ AML and showed an atypical maturing monocytic cell population and a decrease in the leukemic blast cell population after FLT3 inhibitor therapy. Concurrent genetic testing revealed persistent genetic abnormalities. Conclusions These cases illustrate monocytic maturation in FLT3+ AML after FLT3 inhibitor treatment. It is critical for pathologists and clinicians to be aware of the differentiation phenomenon, as these patients have persistent molecular abnormalities despite response to treatment and normalization of blast counts.

Acquired resistance to MET inhibition in MET driven NSCLC.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. 9030-9030
Author(s):  
Richard Riedel ◽  
Carina Heydt ◽  
Andreas H. Scheel ◽  
Hannah Lea Tumbrink ◽  
Johannes Brägelmann ◽  
...  
Keyword(s):  
Partial Response ◽  
Tyrosine Kinase ◽  
Single Agent ◽  
Acquired Resistance ◽  
Kinase Domain ◽  
Type I ◽  
Type Ii ◽  
Low Level ◽  
Met Amplification ◽  
High Level

9030 Background: MET mutations ( MET∆ex14), amplifications or translocations can activate oncogenic signaling in lung cancer and are sensitive to MET inhibition. Acquired resistance to therapy with MET tyrosine kinase inhibitors (TKI) occurs inevitably. Methods: Between 2015 and 2018, eighteen patients with MET-driven NSCLC were treated with capmatinib or crizotinib as single agent at our site. Rebiopsy samples from five patients were analyzed by NGS and fluoreszenz-in-situ hybridization (FISH) at time of progression. Results: Of the five patients with rebiopsy samples at time of progression, two had initially a MET amplification (one patient with low-level and one patient with high-level amplification), two patients had a MET∆ex14 and one patient had a KIF5B-MET fusion. Patient 1 (low-level MET amplification) showed a partial response to crizotinib. The rebiopsy revealed an acquired KRAS mutation as a potential mechanism of resistance. Patient 2 (high-level MET amplification) showed stable disease as best response to capmatinib and patient 3 (MET∆ex14) showed a partial response to capmatinib. Both patients developed acquired HER2 amplifications. Patient 4 ( MET∆ex14) showed initially a partial response to crizotinib. The rebiopsy sample revealed an acquired MET kinase domain mutation (p.D1246N). As preclinical findings suggested that D1246N confers resistance to type I MET inhibitors but remains sensitive to type II inhibitors, cabozantinib was started. A CT six weeks after therapy initiation showed progressive disease. Patient 5 ( KIF5B-MET) had a partial response to crizotinib. An acquired MET p.Y1248H mutation was found at time of progression. Therapy was changed to cabozantinib. A new CT scan is pending. Conclusions: Resistance to MET inhibition is heterogeneous with on- and off-target-mechanisms occurring. We found HER2 amplification as a potential new bypass mechanism. The MET mutation D1246N conferred resistance to type I and type II inhibitors. We describe the first case of an acquired mutation of the MET tyrosine kinase domain in a patient with an oncogenic MET fusion. Further investigations are needed to collect comprehensive data to understand resistance mechanisms in MET inhibition and to develop novel therapeutic strategies.

scholarly journals Patterns of Resistance Differ in Patients with Acute Myeloid Leukemia Treated with Type I versus Type II FLT3-inhibitors

2020 ◽  
pp. bloodcandisc.0143.2020
Author(s):  
Ahmad S. Alotaibi ◽  
Musa Yilmaz ◽  
Rashmi Kanagal-Shamanna ◽  
Sanam Loghavi ◽  
Tapan M. Kadia ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Type I ◽  
Type Ii ◽  
Flt3 Inhibitors ◽  
Acute Myeloid

scholarly journals Evidence-based analysis of odontoid fracture management

2000 ◽  
Vol 8 (6) ◽  
pp. 1-6 ◽  
Author(s):  
Terrence D. Julien ◽  
Bruce Frankel ◽  
Vincent C. Traynelis ◽  
Timothy C. Ryken
Keyword(s):  
Selection Criteria ◽  
Odontoid Fracture ◽  
Evidence Based ◽  
Type I ◽  
Type Ii ◽  
Fracture Type ◽  
Class Iii ◽  
Odontoid Fractures ◽  
Type Iii ◽  
Fracture Management

Object The management of odontoid fractures remains controversial. Evidence-based methodology was used to review the published data on odontoid fracture management to determine the state of the current practices reported in the literature. Methods The Medline literature (1966–1999) was searched using the keywords “odontoid,” “odontoid fracture,” and “cervical fracture” and graded using a four-tiered system. Those articles meeting selection criteria were divided in an attempt to formulate practice guidelines and standards or options for each fracture type. Evidentiary tables were constructed by treatment type. Ninety-five articles were reviewed. Five articles for Type I, 16 for Type II, and 14 for Type III odontoid fractures met selection criteria. All studies reviewed contained Class III data (American Medical Association data classification). Conclusions There is insufficient evidence to establish a standard or guideline for odontoid fracture management. Given the extent of Class III evidence and outcomes reported on Type I and Type III fractures, a well-designed case-controlled study would appear to provide sufficient evidence to establish a practice guideline, suggesting that cervical immobilization for 6 to 8 weeks is appropriate management. In cases of Type II fracture, analysis of the Class III evidence suggests that both operative and nonoperative management remain treatment options. A randomized trial or serial case-controlled studies will be required to establish either a guideline or treatment standard for this fracture type.

scholarly journals Pseudoscience in medicine: cautionary recommendations

1970 ◽  
Vol 19 (4) ◽  
pp. 3118-3126
Author(s):  
Chris Callaghan
Keyword(s):  
Social Science ◽  
Real Life ◽  
Evidence Based ◽  
Type I ◽  
Type Ii ◽  
Scientific Publications ◽  
Moral Failure ◽  
Medical Theory ◽  
Uncritical Acceptance ◽  
Based Medicine

Introduction: Certain real life applications of scientific and social science ideas that knowingly reject accumulated empirical biomedical evidence have been termed ‘pseudoscience,’ or empirical rejectionism. An uncritical acceptance of empiricism, or even of evidence-based medicine, however, can also be problematic.Objectives: With reference to a specific type of medical denialism associated with moral failure, justified by dissident AIDS and anti-vaccine scientific publications, this paper seeks to make the argument that this type of denialism meets certain longstanding definitions for classification as pseudoscience.Methods: This paper uses a conceptual framework to make certain arguments and to juxtapose arguments for evidence-based approaches to medicine against literature that highlights certain limitations of an unquestioning approach to empiricism. Results: Discussions of certain real life examples are used to derive the important insight that, under certain conditions, moral failure can result in the violation both Type I and Type II scientific error types, with catastrophic consequences.Conclusion: It is argued that the validity of all theory should not be assumed before sufficient empirical evidence has accumulated to support its validity across contexts. However, caution is required, to avoid the consequences of an unquestioning approach to empiricism.Keywords: Pseudoscience; denialism; medical practice; medical theory; empiricism.

Understanding the Practice, Application, and Limitations of Meta-Analysis

2019 ◽  
Vol 64 (1) ◽  
pp. 74-96
Author(s):  
Mike Allen
Keyword(s):  
Type I Error ◽  
Meta Analysis ◽  
Future Research ◽  
Evidence Based ◽  
Type I ◽  
Type Ii ◽  
Type Ii Error ◽  
Statistical Process ◽  
Basic Assumptions ◽  
To Receive

This article discusses the basic assumptions and practices for meta-analysis as well as describing options and innovations for implementing this tool. Meta-analysis represents a family of techniques with different assumptions and procedures. I discuss some of the ongoing debates and limitations of the methods that continue to receive attention. In an era of “evidence-based” applications and educational material, meta-analysis (in all the forms) represents the best way to reduce Type II error and identify Type I error. Use of the technique provides facilitates the formulation and evaluation theoretical arguments as well as identifying the means to optimally generate future research efforts. The process of meta-analysis takes the scientist back to the future by reminding the community of the original premises that guided the formation of the statistical process.

scholarly journals Cap Analysis of Gene Expression (CAGE) Sequencing Reveals Alterations of the Transcriptional Signatures of FLT3-ITD with Secondary D835 TKD Mutations in Acute Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1530-1530
Author(s):  
Yoko Tabe ◽  
Kazumasa Sekihara ◽  
Kaori Saitoh ◽  
Norikazu Monma ◽  
Kazuho Ikeo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Tyrosine Kinase ◽  
Research Funding ◽  
Kinase Inhibitor ◽  
Mapk Signaling ◽  
Mtor Signaling ◽  
Type I ◽  
Type Ii ◽  
Ras Pathway ◽  
Cap Analysis

Abstract Mutations in the gene encoding Fms-like tyrosine kinase-3 (FLT3) are found in approximately 30% of acute myeloid leukemia (AML) patients, with two distinct groups of mutations; the internal tandem duplication mutations in juxtamembrane domain (FLT3-ITD) and the point mutations within the tyrosine kinase domains (FLT3-TKD). The second-generation tyrosine kinase inhibitor (TKI) quizartinib which primarily targets an inactive (type II) kinase conformation, has demonstrated responses in relapsed/refractory FLT3-ITD AML patients. However, duration of response to quizartinib is limited largely by secondary mutations in FLT3-TKD, most commonly at D835 activation loop, emerging in at least 20% of patients. To elucidate the alterations of transcript signatures of quizartinib-resistant FLT3-ITD/D835 mutants in AML, we performed cap analysis of gene expression (CAGE) sequencing utilizing 11 FLT3-ITD and 6 FLT3-ITD/D835 primary AML cells with wild type NPM1. CAGE identifies and quantifies the 5' ends of capped mRNA transcripts, which enables the identification of transcription start sites (TSS) and allows investigating promoter structures necessary for gene expression. The TSS of genes altered in AML cells with FLT3-ITD/D835 compared with FLT3-ITD were mapped, and CAGE detected upregulation of 124 genes and downregulation of 25 genes in FLT3-ITD/D835 cells (FDR < 0.05). Specifically, several genes closely linked with activation of MAPK signaling (MAPK6, TCF4), AKT/mTOR signaling (NBN) and both (TNFAIP6) were upregulated in the D835 acquired cells. Expression of inflammatory response cytokine genes IL1B and IL8 were also increased in FLT3-ITD/D835 cells. The FLT related functional pathway analysis by KEGG ontology indicated that Ras pathway with its downstream Raf/MAPK and PI3K/AKT/mTOR signaling including transcription factor NFKB are the most significantly activated pathways in FLT3-ITD/D835 cells compared to FLT3-ITD cells. No additional activation of STAT5 pathway, a direct downstream signaling of mutated FLT3, was found in FLT3-ITD/D835 cells. To assess the underlying molecular basis of transcription signature, we performed an unbiased search for enriched sequence motifs using HOMER software. HOMER revealed upregulation of 80 motifs and downregulation of 51 motifs in AML cells with FLT3-ITD/D835 compared with FLT3-ITD (q < 0.05). Motifs of AP-1, JUN, CEBP, ATF and NFKB were substantially enriched in FLT3-ITD/D835 compared to FLT3-ITD cells. ETS, SPI-1 and RUNX1 were the most common binding motifs altered (both increased and decreased) by D835 mutation acquisition. Of note, AP-1 modulates inflammatory response and is tightly regulated by MAPK. To validate the transcriptional changes and the highlighted gene ontology results of AML primary samples, we utilized paired isogenic FLT3-ITD or FLT3-ITD/D835Y transfected Ba/F3 cell lines. As predicted, type II kinase inhibitor quizartinib was active against FLT3-ITD Ba/F3 cells (IC50; 0.6nM) but not FLT3-ITD/D835Y Ba/F3 cells (IC50; > 5000nM). Type I kinase inhibitor crenolanib was highly active against both FLT3-ITD (IC50; 2.1nM) and FLT3-ITD/D835Y cells (IC50; 16.4nM). Focusing on FLT related Ras pathway, we first investigated MAPK activation in these cells. Immunoblot analysis demonstrated substantial reduction of phospho- (p-) ERK after quizartinib (5nM) or crenolanib (50nM) treatment both in FLT3-ITD and FLT3-ITD/D835Y cells, irrespective of their susceptibility. We next examined p-S6, the target of PI3K/AKT/mTOR and MAPK signaling, and observed that crenolanib but not quizartinib successfully diminished p-S6 expression in FLT3-ITD/D835Y cells. FLT3-ITD/D835 cells showed higher baseline expression of NFkB regulator p-IkB compared to FLT3-ITD cells, which was not repressed by crenolanib. In conclusion, our study showed that the acquired D835 mutation in FLT3-ITD AML cells facilitates transcriptional network activation to induce proinflammatory genes through Ras-MAPK-NF-κB/AP-1 pathway and Ras-PI3K-AKT-mTOR signaling. This transcriptional modulation promotes oncogenic pro-survival and proliferative signaling in AML cells and defines differential sensitivity against Type I and Type II kinase inhibitors. Disclosures Shah: ARIAD: Research Funding; BMS: Research Funding; Daiichi-Sankyo: Research Funding; Pfizer: Research Funding; Plexxikon: Research Funding. Konopleva:Reata Pharmaceuticals: Equity Ownership; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Stemline: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Calithera: Research Funding.

FLT3 Inhibitor Treatment in FLT3-Mutated AML Is Associated with Development of Secondary FLT3-TKD Mutations

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1493-1493 ◽  
Author(s):  
Yesid Alvarado ◽  
Hagop M. Kantarjian ◽  
Farhad Ravandi ◽  
Rajyalakshmi Luthra ◽  
Gautam Borthakur ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tyrosine Kinase ◽  
Median Survival ◽  
Research Funding ◽  
Complete Response ◽  
Mutation Status ◽  
Flt3 Mutation ◽  
Flt3 Inhibitor ◽  
Flt3 Inhibitors ◽  
Bone Marrow Blasts

Abstract Abstract 1493 Background: The two most frequent mutations in the FMS-like tyrosine kinase 3 (FLT3) receptor are internal tandem duplication (ITD) of the juxtamembrane region and mutations involving the D835/I836 residues of the tyrosine kinase domain (TKD). They are present in approximately 30% and 7% of AML cases respectively, and have been associated with higher relapse rate and worse overall survival. ITD and TKD mutations produce constitutive activation of the FLT3 receptor; however they appear to have different downstream effectors and different clinical significance. Currently, several FLT3 kinase inhibitors are in development and although some have shown promising clinical activity, responses tend to be transient and insufficient to induce a durable response. Sensitivity of different mutants to diverse FLT3 inhibitors is variable (Zhang et al. JNCI 2008; 100: 184). Aim: To determine the change in mutation status of patients with FLT3 ITD treated with FLT3 inhibitors. Methods: We analyzed 67 patients with FLT3 mutation positive AML, treated in our institution in clinical trials with different FLT3 inhibitors from October 2002 to July 2011 and in whom we obtained mutational assessment before and after treatment. Results: At baseline 58 (87%) patients had an ITD mutation, 5 (7%) had a D835/I836 mutation and 4 (6%) had combined ITD and codon D835/I836 mutations. Thirty three patients were male, median age was 55 years (range 18–87), and the median number of prior leukemia treatments was 2 (range 1–6). Karyotype was diploid in 36%, miscellaneous in 17%, complex in 14%, not done in 23%, and insufficient in 9% of patients. Concomitant NPM1, RAS, and CEBPA mutations were observed in 21%, 13%, and 4% of the patients, respectively. Patients remained on therapy for a median time of 50 days (range 18–561) before progression, 45 (67%) did not achieve response, 13 (19%) cleared their bone marrow blasts, 6 (9%) achieved a partial response, 4 (6%) a complete response and 1 (1%) patient died during treatment. None of the patients with D835/I836 or ITD+D835/I836 achieved a response. Treatment was always discontinued due to progressive disease, except in 9 (13%) patients, 6 who cleared bone marrow blasts and proceeded to hematopoietic stem cell transplantation and 3 who discontinued due to toxicity. One of them was in complete response but progressed within 4 months, and 2 who cleared their bone marrow blasts and were also bridged with other treatments to transplant. All patients in this cohort had assessment of their FLT3 mutation status at the time FLT3 inhibitor was discontinued, 14 (21%) patients progressed from a single ITD mutation to have combined ITD+D835/I836 mutations, 7 (10%) patients (6 ITD and 1 D835/I836) were negative for FLT3 mutation, and 46 (69%) patients had unchanged mutation status. At the time of this analysis, 10 patients were alive, 1 was lost to follow and 56 had died. The median survival in the entire group from the time of FLT3 inhibitor start was 5.3 months, and from time of treatment discontinuation 3.2 months. In those patients in whom the FLT3 mutation became negative the median survival was 7 months, in those with unchanged mutation 4.5 months, and in those with ITD-D835/I836 mutations the median survival was 6.1 months. Further therapy with another FLT3 inhibitor was attempted in 6 out of 14 patients with ITD-D835/I836 mutations, 5 failed to respond and 1 had a transient decrease in bone marrow blasts and peripheral white blood cell count (2 months) with a combination of a FLT3 inhibitor and a hypomethylating agent. Conclusion: There is evidence that a secondary TKD mutations arise after the use of FLT3 inhibitors in patients with single FLT3-ITD mutated AML, a phenomenon that is associated with a poor prognosis. Disclosures: Ravandi: Bayer: Research Funding; Onyx: Research Funding. Cortes:Ambit: Research Funding; Novartis: Research Funding.

Combination of Crenolanib with Sorafenib Produces Synergistic Pro-Apoptotic Effects in FLT3-ITD-Inhibitor-Resistant Acute Myelogenous Leukemias with FLT3 Mutations

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3591-3591 ◽  
Author(s):  
Chen Gao ◽  
Weiguo Zhang ◽  
Rodrigo Jacamo ◽  
Abhijit Ramachandran ◽  
Donald Small ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Single Agent ◽  
Point Mutations ◽  
Type I ◽  
Type Ii ◽  
Apoptotic Effects ◽  
Resistant Cells

Abstract Abstract 3591 Activating mutations in the FLT3 gene, including internal tandem duplications (ITDs) and missense point mutations of the tyrosine kinase domain (TKD), are frequently observed in AML patients and confer poor prognosis (1). Targeting FLT3 ITD mutations using the multi-kinase inhibitor Sorafenib (a type II kinase inhibitor, which binds to inactive conformation of a kinase ATP pocket)(2) showed impressive anti-leukemia effects in FLT3-ITD mutated AML in Phase I/II clinical trials (3) However, resistance/relapse develops regularly during prolonged Sorafenib therapy (4), in part through acquired point mutations of TKD domains. We postulated that the conformational change of FLT3 protein resulting from acquired point mutations limits the accessibility of sorafenib and leads to resistance (5, 6). Recently, Crenolanib, a novel PDGFRβ tyrosine kinase inhibitor, showed impressive anti-tumor effects by targeting the active conformation of a kinase ATP pocket of FLT3 protein (a type I kinase inhibitor). Therefore, we hypothesize that targeting different sites of FLT3 protein simultaneously using different types of kinase inhbitors may be effective in overcoming sorafenib resistance. We here report that Crenolanib has anti-leukemic activity in Sorafenib-resistant cells which harbor both ITD and acquired TKD point mutations i, and that its combination with Sorafenib in Sorafenib-resistant cells exerts synergistic pro-apoptotic effects. The anti-leukemic activity of Crenolanib was assessed by measuring cell viability (trypan Blue exclusion) and apoptosis induction (annexin V/propidium iodide staining) in isogenic murine Ba/F3 AML cell lines with stable transfection of human FLT3-ITD mutations, in Sorafenib resistant Ba/F3-ITD-Res cells derived from long-term, low-dose exposure of Ba/F3-ITD to Sorafenib in vitro, which harbor N676D and Y842C mutations, and Sorafenib-resistant cell lines Ba/F3-ITD+676, Ba/F3-ITD+842 and Ba/F3-ITD+676/842 which carry ITD and TKD point mutations (N676D, Y842C and N676D/Y842C mutations, respectively). Effects of combinatorial regimen employing Crenolanib and Sorafenib were analyzed using CalcuSyn software (combination index (CI) : CI<1 = synergistic, CI>1 = antagonistic effects). Results show that single agent Crenolanib induced cell growth arrest in leukemia cells Ba/F3-ITD, Ba/F3-ITD+676, Ba/F3-ITD+842 and Ba/f3-ITD+842/676, at IC50s of 0.012, 0.012 0.037 and 0.038uM, respectively, and induced apoptosis (EC50s) at 0.17, 0.23, 0.19, and 0.22uM, respectively, after 72 hours of treatment. Western Blot showed that Crenolanib profoundly suppressed phosphorylation levels of FLT3 protein and its downstream targets ERK and AKT and induced cleavage of caspase 3. Sorafenib-resistant cells Baf3-ITD+Res and Baf3-ITD+842/676 (EC50s for Sorafenib were 4.2 ± 1.50 and 6.6 ± 0.53 μM, respectively) were exposed to submicromolar concentrations of Crenolanib and Sorafenib concomitantly for 48 h, resulting in impressive synergistic pro-apoptotic effects (CIs were 0.56 ± 0.12 and 0.36 ± 0.04, respectively), implying high synergistic potency of Type I and Type II FLT3 kinase inhibitors, when given concomitantly. In vivo experiments are in progress. Our findings provide therapeutic rationale for a combinatorial treatment strategy with Crenolanib and Sorafenib of FLT-ITD inhibitor-refractory AML. Disclosures: No relevant conflicts of interest to declare.

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