Constitutive Activation Of FLT3 Research Articles

1. Microdeletion 13q12.2 in B cell acute lymphoblastic leukemia: Little but important!

Chromosomal microarray analysis (CMA) is instrumental for identification of recurrent focal deletions in patients with B cell acute lymphoblastic leukemia (B-ALL). These microdeletions include genes involved in B cell differentiation, cell cycle control and transcriptional regulation often with prognostic or therapeutic significance. Recently, a 13q12.2 microdeletion with breakpoints upstream of FLT3 and within intronic regions of the PAN3 gene was reported. Functionally, this microdeletion resulted in enhancer hijacking and constitutive activation of FLT3 in pediatric B-ALL. Seven institutions performed internal database searches to identify similar deletions. In total, 10 patients were identified, all with B-ALL, confirming the specificity for this diagnosis. This cohort included seven pediatric cases (3 male, 4 female; ages 2-14 yo, mean 5) and 3 adult cases (1 male, 2 female; ages 22, 39, 42). Subtypes included two high hyperdiploid, one hypodiploid, one iAMP21, one P2RY8::CLRF, one unbalanced rearrangement involving TFE3, one PAX5::PML, three B-ALL NOS.? Deletions ranged in size from?6.7 kb ? 136 kb (mean 76 kb); each with proximal breakpoints between FLT3 and PAN3 and 9/10 with distal breakpoints within intron 5 of the PAN3 gene. Our study is the first to report the microdeletion in adults, and unlike the discovery cohorts, our patients showed no sex bias. Our data does not confirm a strong association of the deletion with high hyperdiploidy, since 8/10 had other B-ALL subtypes. Constitutive activation of FLT3 may have significant clinical implications for prognosis and treatment; thus, detection of 13q12.2 microdeletion at diagnosis or disease relapse is paramount.

Efficacy and Synergy of Small Molecule Inhibitors Targeting FLT3-ITD+ Acute Myeloid Leukemia.

Simple SummaryFLT3-ITD mutations belong to the most frequent yet most detrimental genetic alterations in AML. Next-generation FLT3 inhibitors are potent therapeutics and often effective in AML patients carrying the FLT3-ITD driver kinase. However, AML cells are particularly quick in acquiring resistance to FLT3 kinase blockers. We aimed to identify novel therapeutic options for FLT3-ITD+ AML, to investigate possible emerging resistance mechanisms to FLT3 inhibitors and to explore alternative targeting strategies. We applied a kinase-focused drug screen to find alternative therapeutics. We identified ispinesib, a kinesin spindle blocker, and kinase blockers WS6, ponatinib and cabozantinib, as very efficacious agents against FLT3-ITD+ AML cells. Importantly, we identify the combination of cabozantinib and ispinesib as particularly potent against FLT3-ITD+ AML. We suggest that a combinatorial treatment with these drugs could overcome resistance mechanisms and kill FLT3-ITD+ AML blasts.Constitutive activation of FLT3 by ITD mutations is one of the most common genetic aberrations in AML, present in ~1/3 of cases. Patients harboring FLT3-ITD display worse clinical outcomes. The integration and advancement of FLT3 TKI in AML treatment provided significant therapeutic improvement. However, due to the emergence of resistance mechanisms, FLT3-ITD+ AML remains a clinical challenge. We performed an unbiased drug screen to identify 18 compounds as particularly efficacious against FLT3-ITD+ AML. Among these, we characterized two investigational compounds, WS6 and ispinesib, and two approved drugs, ponatinib and cabozantinib, in depth. We found that WS6, although not yet investigated in oncology, shows a similar mechanism and potency as ponatinib and cabozantinib. Interestingly, ispinesib and cabozantinib prevent activation of AXL, a key driver and mechanism of drug resistance in FLT3-ITD+ AML patients. We further investigated synergies between the selected compounds and found that combination treatment with ispinesib and cabozantinib or ponatinib shows high synergy in FLT3-ITD+ AML cell lines and patient samples. Together, we suggest WS6, ispinesib, ponatinib and cabozantinib as novel options for targeting FLT3-ITD+ AML. Whether combinatorial tyrosine kinase and kinesin spindle blockade is effective in eradicating neoplastic (stem) cells in FLT3-ITD+ AML remains to be determined in clinical trials.

Driver Mutations in Leukemia Promote Disease Pathogenesis through a Combination of Cell-Autonomous and Niche Modulation.

SummaryStudies of patients with acute myeloid leukemia (AML) have led to the identification of mutations that affect different cellular pathways. Some of these have been classified as preleukemic, and a stepwise evolution program whereby cells acquire additional mutations has been proposed in the development of AML. How the timing of acquisition of these mutations and their impact on transformation and the bone marrow (BM) microenvironment occurs has only recently begun to be investigated. We show that constitutive and early loss of the epigenetic regulator, TET2, when combined with constitutive activation of FLT3, results in transformation of chronic myelomonocytic leukemia-like or myeloproliferative neoplasm-like phenotype to AML, which is more pronounced in double-mutant mice relative to mice carrying mutations in single genes. Furthermore, we show that in preleukemic and leukemic mice there are alterations in the BM niche and secreted cytokines, which creates a permissive environment for the growth of mutation-bearing cells relative to normal cells.

Wild-type FLT3 and FLT3 ITD exhibit similar ligand-induced internalization characteristics.

Class III receptor tyrosine kinases control the development of hematopoietic stem cells. Constitutive activation of FLT3 by internal tandem duplications (ITD) in the juxtamembrane domain has been causally linked to acute myeloid leukaemia. Oncogenic FLT3 ITD is partially retained in compartments of the biosynthetic route and aberrantly activates STAT5, thereby promoting cellular transformation. The pool of FLT3 ITD molecules in the plasma membrane efficiently activates RAS and AKT, which is likewise essential for cell transformation. Little is known about features and mechanisms of FLT3 ligand (FL)‐dependent internalization of surface‐bound FLT3 or FLT3 ITD. We have addressed this issue by internalization experiments using human RS4‐11 and MV4‐11 cells with endogenous wild‐type FLT3 or FLT3 ITD expression, respectively, and surface biotinylation. Further, FLT3 wild‐type, or FLT3 ITD‐GFP hybrid proteins were stably expressed and characterized in 32D cells, and internalization and stability were assessed by flow cytometry, imaging flow cytometry, and immunoblotting. FL‐stimulated surface‐exposed FLT3 WT or FLT3 ITD protein showed similar endocytosis and degradation characteristics. Kinase inactivation by mutation or FLT3 inhibitor treatment strongly promoted FLT3 ITD surface localization, and attenuated but did not abrogate FL‐induced internalization. Experiments with the dynamin inhibitor dynasore suggest that active FLT3 as well as FLT3 ITD is largely endocytosed via clathrin‐dependent endocytosis. Internalization of kinase‐inactivated molecules occurred through a different yet unidentified mechanism. Our data demonstrate that FLT3 WT and constitutively active FLT3 ITD receptor follow, despite very different biogenesis kinetics, similar internalization and degradation routes.

Loss of Heterozygosity of FLT3-ITD Is Common in Acute Myeloid Leukemia and May be a More Consistent Prognostic Marker Than FLT3-ITD Allele Frequency

Loss of Heterozygosity of FLT3-ITD Is Common in Acute Myeloid Leukemia and May be a More Consistent Prognostic Marker Than FLT3-ITD Allele Frequency

Pediatric Acute Myeloid Leukemia (AML): From Genes to Models Toward Targeted Therapeutic Intervention.

This review aims to provide an overview of the current knowledge of the genetic lesions driving pediatric acute myeloid leukemia (AML), emerging biological concepts, and strategies for therapeutic intervention. Hereby, we focus on lesions that preferentially or exclusively occur in pediatric patients and molecular markers of aggressive disease with often poor outcome including fusion oncogenes that involve epigenetic regulators like KMT2A, NUP98, or CBFA2T3, respectively. Functional studies were able to demonstrate cooperation with signaling mutations leading to constitutive activation of FLT3 or the RAS signal transduction pathways. We discuss the issues faced to faithfully model pediatric acute leukemia in mice. Emerging experimental evidence suggests that the disease phenotype is dependent on the appropriate expression and activity of the driver fusion oncogenes during a particular window of opportunity during fetal development. We also highlight biochemical studies that deciphered some molecular mechanisms of malignant transformation by KMT2A, NUP98, and CBFA2T3 fusions, which, in some instances, allowed the development of small molecules with potent anti-leukemic activities in preclinical models (e.g., inhibitors of the KMT2A–MENIN interaction). Finally, we discuss other potential therapeutic strategies that not only target driver fusion-controlled signals but also interfere with the transformed cell state either by exploiting the primed apoptosis or vulnerable metabolic states or by increasing tumor cell recognition and elimination by the immune system.

Comprehensive Genomic Profiling of Chinese Acute Myeloid Leukemia (AML) with FLT3 ITD Using Ultra Deep Sequencing

Introduction: The FMS-like tyrosine (FLT3) gene encodes a class III receptor tyrosine kinase, belonging family members, including c-kit, c-FMS, FLT1, and PDGF-ΒR. FLT3 plays a key role in the hematopoiesis. It was discovered that in acute myeloid leukemia (AML) FLT3 is frequently mutated. Furthermore, constitutive activation of FLT3 by internal tandem duplication (ITD) mutation is one of the most common molecular variants in AML, occurring in approximately 20% to 30% of AML patients who have a comparatively poor clinical outcome and increased relapse rate. In the last years, next generation sequencing technology has allowed a high- throughput, comprehensive profiling of cancer genomes that can aid in making clinical decisions in the process of diagnosis and treatment. A few reports included somatic genomic profiling in AML with FLT3-ITD. However, none comprehensive genomic profiling data shows in chinese population. In this study, we sequenced 26 FLT3-ITD AML samples to show genomic profiling with multiple mutations types and discover the mutations associated with FLT3-ITD based on in-house bioinformatics mutation calling pipelines.Methods: DNA was extracted from blood or bone marrow of AML FLT3-ITD positive samples. Those DNA samples were subjected to comprehensive genomic profiling (CGP) assay consisting of whole exon coding region in 450 tumor actionable or cancer driver genes as well as selected introns (N=244) from 39 genes frequently involved in gene rearrangement using hybridization target capture and next generation sequencing (NGS) technologies and in house established bioinformatics pipelines. Somatic genomic alterations including SNV, short/long Indel, CNV and gene rearrangement were analyzed. Libraries were constructed with KAPA Hyper Prep kit( KAPA Biosystems), and hybridized to customized capture probes( Integrated DNA Technologies)and then sequenced on NovaSeq 6500 (Illumina). The mutation is detected with VAF of no less than 1% of point mutation, insertion and deletion, including long insertion and deletion( long indel). Long indel variants from 10bp-2kb were called with OriLindel algorithm, which is in silicon developed for structure variation calling, especially for FLT3-ITD variants calling.Results: In this study, we sequenced 26 FLT3-ITD AML samples to show genomic profiling with multiple mutations types. The average sequencing depth of 26 samples is above 700X(720-2300X). The mutation is detected with VAF of no less than 1% of point mutation, insertion and deletion, including long insertion and deletion( long indel). Long indel variants from 10bp-2kb were called with OriLindel algorithm, which is in silicon developed for structure variation calling, especially for FLT3-ITD variants calling.We discovered the previous reported high rate of mutations of NPM1(34.6%,9/26), DNMT3A(38.5%,10/26), IDH2(11.5%,3/26), NRAS(19.2%,5/26),RUNX1 (15.4%,4/26), WT1 (31%,8/26) and MLL3(KMT2C, 11.5%,3/26) which have consistent percentage with previous genomic profiling report of FLT3-ITD AML.A total of 42 mutations were detected on the FLT3 gene of 26 samples (162%, 42/26). All 36 ITD sequences of 26 samples were distributed in the protein kinase domain with lengths ranging from 18 to 207bp. 5 samples have more than two ITD variants (19.2%,5/26)). 6 point mutation of FLT3 were detected, 5 of which were located in the 835th amino acid residue of tyrosine kinase domain and 1 in the protein kinase catalytic domain. One of the samples contained two mutations, D835H and V592G. Using NGS technology and in silico bioinformatics pipeline could call multiple type mutations including point mutation, insertion and deletion of FLT3 simultaneously.We also found recurrent mutations found not often in AML, such as AXIN1 (7.7%,2/26) ,RIT1 (7.7%,2/26). AXIN1 belongs to Wnt signaling pathway. Wnt extracellular signaling pathway is one of the evolutionarily conserved pathways that regulate proliferation, polarity and cell migration. RIT1 belongs to Ras gene family. Somatic mutations of the Ras gene family are present in 20-30% of all human cancers,including15-20% of acute myeloid leukemia (AML). Ras-like-without-CAAX-1 (RIT1) gene is a new member of the family and has been found to be critical in noonan syndrome. One report shows the discovery of novel somatic mutations in the RIT1 gene in patients with myeloproliferative. DisclosuresNo relevant conflicts of interest to declare.

FLT3 receptor/CD135 expression by flow cytometry in acute myeloid leukemia: Relation to FLT3 gene mutations and mRNA transcripts

BackgroundAlterations of the FLT3 gene are the most frequent molecular aberrations seen at diagnosis of acute myeloid leukemia (AML). Two main types of FLT3 mutations have been the most commonly detected; internal tandem duplication (ITD) in the juxtamembrane domain and point mutation D835Y in the tyrosine kinase domain (TKD). Both classes of mutations result in constitutive activation of FLT3 receptor/CD135. AimTo assess the frequency of FLT3 gene mutations (ITD and TKD D835Y) and the flow cytometric expression of FLT3 receptor/CD135 among AML patients to define the role for FLT3 receptor expression in predicting FLT3 gene mutational status and mRNA transcript level. Subjects and methodsEighty AML patients at diagnosis and 20 control subjects were enrolled. FLT3 receptor/CD135 expression, FLT3 gene mutations, and FLT3 transcript level were evaluated by flow cytometry, conventional polymerase chain reaction (PCR), and quantitative real-time reverse-transcription PCR, respectively. Fluorescence in situ hybridization was done to stratify patients into favorable, intermediate, and poor cytogenetic risk groups. ResultsFLT3-ITD was detected in 22.5% AML patients, while none had FLT3-TKD D835Y mutation. A cut-off value of >17% was assigned to define FLT3 receptor/CD135+ cases. FLT3 receptor/CD135 and FLT3 transcripts were overexpressed in 100% AML patients; higher levels were found among AML-M5 subtype and poor cytogenetic group. AML patients harboring FLT3-ITD showed a trend for higher FLT3 receptor/CD135 expression and FLT3 transcript level than those with wild-type FLT3. FLT3 receptor/CD135 >49% was predictive for FLT3-ITD. A positive correlation was found between FLT3 receptor/CD135 expression and FLT3 transcript level. ConclusionEvaluation of FLT3 receptor/CD135 expression by flow cytometry at diagnosis of AML could constitute a predictor for the FLT3-ITD mutational status and FLT3 transcript level.

Abstract 574: AGS62P1, a novel site-specific antibody drug conjugate targeting FLT3 exhibits potent anti-tumor activity regardless of FLT3 kinase activation status

Abstract FLT3 is a member of the class III receptor tyrosine kinase family that includes C-KIT, C-FMS and platelet derived growth factor receptor (PDGFR). FLT3 is primarily expressed in early myeloid and lymphoid progenitors and plays an important role in their proliferation and differentiation. In human leukemia, FLT3 is expressed on 70-90% acute myeloid leukemia (AML) and most B-acute lymphoblastic leukemia (B-ALL). FLT3 genetic aberrations are commonly detected in patients with AML. The most common aberration is internal tandem duplication (ITD), which occurs in 25-30% of AML patients and causes constitutive activation of FLT3. Point mutation in codon D835 of the FLT3 tyrosine kinase domain is reported in 7-10% of AML patients and also causes constitutive activation of the receptor. FLT3 small molecule inhibitors targeting the kinase domain are predominantly active against FLT3 activated AML. The restricted normal tissue expression profile and higher differential in leukemic specimens makes FLT3 amenable to antibody-based therapeutics, requiring only target expression independent of kinase activation status. Therefore, development of an antibody-drug conjugate (ADC) may provide a therapeutic alternative for AML patients. Here, we report the development of the first FLT3specific ADC, AGS62P1, employing site-specific conjugation using the non-natural amino acid, p-acetyl phenylalanine (pAF). AGS62P1 comprises a human gamma one antibody including an inserted pAF residue in each of the heavy chains. The antibody was conjugated to a potent cytotoxic payload via an oxime bond at the pAF sites, thus creating a nearly homogeneous drug distribution, with approximately 2 drug molecules per antibody. Strong binding affinity (0.1-0.9 nM) and potent in vitro cytotoxic activity (IC50 = 0.2-12 nM) was achieved in AML cell lines. The anti-FLT3 ADC was highly efficacious in AML tumor xenografts, leading to statistically significant tumor growth inhibition of both FLT3 ITD and non-ITD models. Additional characterization of both the antibody and ADC was performed, including ligand receptor interaction, degradation, internalization, and apoptosis. In summary, we have developed a site-specific ADC targeting FLT3 that exhibits potent anti-tumor activity in xenograft models regardless of FLT3 activation status. This drug can potentially offer a new and more versatile approach in targeting FLT3-expressing leukemia through a mechanism independent of FLT3 genetic aberration. Citation Format: Nandini Rudra-Ganguly, Pia M. Challita-Eid, Christine Lowe, Mike Mattie, Sung-Ju Moon, Brian A. Mendelsohn, Monica Leavitt, Cyrus Virata, Alla Verlinsky, Linnette Capo, Mi Sook Chang, Deanna L. Russell, Baljinder Randhawa, Gao Liu, René Hubert, Mary Brodey, Hector Aviña, Chunying Zhang, Joseph D. Abad, Banmeet Anand, Sher Karki, Zili An, Roland Luethy, Fernando Doñate, Daniel S. Pereira, Kendall Morrison, Ingrid B.J. Joseph, David R. Stover. AGS62P1, a novel site-specific antibody drug conjugate targeting FLT3 exhibits potent anti-tumor activity regardless of FLT3 kinase activation status. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 574.

Discovery and Functional Validation of Novel Pediatric Specific FLT3 Activating Mutations in Acute Myeloid Leukemia: Results from the COG/NCI Target Initiative

Mutations in the FLT3 gene are among the most common somatic events in AML, with a higher prevalence in adults than in children. The most common activating mutations of FLT3 include internal tandem duplications (FLT3/ITD) in the juxtamembrane domain (JMD) or missense mutations in the tyrosine kinase domain (TKD) at the D835/I836 positions (FLT3/ALM). To date, much of the data on FLT3 mutations has been derived from adult studies and comprehensive sequencing of the FLT3 gene from recent TCGA analysis demonstrated that FLT3 activating mutations were limited to the FLT3/ITD in the JMD and D835/I836 hotspots. As part of the Children's Oncology Group (COG)/NCI TARGET AML initiative, we interrogated the genomic landscape of pediatric AML and identified and verified novel FLT3 activating events that appear to be unique to childhood AML and could provide a target for therapeutic intervention.Whole genome sequencing was performed in a discovery cohort (N=200) followed by validation with targeted exome capture for a total of 799 diagnostic specimens from children treated on contemporary COG trials. In addition to the known FLT3 mutations (FLT3/ITD, N=128 and D835/I836, N=37), we identified novel point mutations (PM; N=49) and novel insertion-deletions (in-dels; N=12). We observed a prevalence of 7.6% of novel PMs and in-dels, in addition to the FLT3/ITD and D835/I836 mutations. The total prevalence of all FLT3 mutations was 28%. In contrast to adult AML, where virtually all non-ITD activating mutations are limited to the D835/I836 region, FLT3PMs in the pediatric cohort occurred in TKD domain (N=44), but commonly occurred in the transmembrane domain (TMD) and JMD. Twelve somatic mutations were identified at distinct positions within the JMD, the region involved in regulation of activity of the kinase. Among the JMD mutations, 9 occur at novel pediatric specific sites with significant activating potential (E573D/G, L576R, T582N, D586Y, Y589H, E596K/G, E598D, Y599C, D600G).Crystal structure analysis of FLT3 variants was used to assess the potential functional significance of the newly discovered variants. This structural modeling indicated that many of the mutations within the TMD (e.g. A680V) and TKD1 (L616R, M664I, M665L) were predicted to cause JMD destabilization, resulting in dysregulated activation of FLT3. Additionally, almost all JMD mutations have the potential to significantly disrupt the auto-inhibitory conformation, resulting in constitutive FLT3 activation. Mutations causing excessive activation of the kinase may have significant oncogenic capacity. In order to assess functional implications of the mutations, we cloned and expressed 6 of the most common novel variants (E573D, L576R, Y599C, D600G, F612L, and A680V) for functional assessment. Of the 6 mutations tested, 5 (E573D, L576R, Y599C, D600G, and A680V) resulted in auto-phosphorylation of FLT3, demonstrating dysregulated and enhanced kinase activation.Acquired mutations following tyrosine kinase inhibitor (TKI) exposure are heavily implicated in resistance and are almost exclusively confined to the two TKD regions, and the D835 position is a hotspot for resistance conferring mutations. It is important to identify patients at diagnosis who harbor dual mutations as this could indicate de novo resistance to TKIs, and exposure to these agents would have no efficacy, but may result in unnecessary toxicity. The presence of dual FLT3/ITD and FLT3/ALM mutations at diagnosis has been reported to occur at a very low prevalence. We analyzed the presence of co-occurring FLT3/ITDand FLT3 PMs in our pediatric cohort. Of the 128 patients with FLT3/ITD, 18 (14%) harbored a secondary FLT3 PM. Mutations in the JMD (N=8) and TMD (N=2) accounted for 56% of co-occurring mutations. Only 22% (N=4) mutations occurred at the D835/I836 sites, with an additional 22% (N=4) located at other TKD sites. Identified TKD diagnostic mutations included F612L, F616R, N676K/S, and N841K, a few which have only been reported in the setting of post TKI relapse.We identified novel pediatric specific FLT3 mutations with significant functional capacity. Importantly, these activating mutations may be uniquely susceptible to FLT3 inhibition and provide a therapeutic target in pediatric AML. Further work is ongoing to completely understand the oncogenic potential of each unique mutation and their prognostic and therapeutic implications. DisclosuresNo relevant conflicts of interest to declare.

Development of FF-10101, a Novel Irreversible FLT3 Inhibitor, Which Overcomes Drug Resistance Mutations

Development of FF-10101, a Novel Irreversible FLT3 Inhibitor, Which Overcomes Drug Resistance Mutations

FLT3 Inhibitors for the Treatment of Acute Myeloid Leukemia: An Evaluation of Efficacy of Target Inhibition and Relationship to Disease Progression

IntroductionFLT3 is a receptor tyrosine kinase that plays a role in hematopoietic stem/progenitor cell proliferation and survival and is frequently found to be mutated in patients with acute myeloid leukemia (AML). Mutations that lead to constitutive activation of FLT3 (such as internal tandem duplications of the juxtamembrane domain or point mutations involving the kinase domain) are associated with a poor prognosis. This poor prognosis is in part due to an increased relapse rate after allogeneic hematopoietic cell transplant (HCT). Several compounds that inhibit the activity of FLT3 in vitro, including ASP2215, midostaurin and quizartinib, are now being studied in clinical trials for the treatment of AML. Inhibition of the formation of phospho-FLT3 has been correlated with clinical anti-leukemia effects and remissions.MethodsIn this pilot study we use immunohistochemistry to measure the levels of activated FLT3 in bone marrow biopsies of patients prior to and during treatment inclinical trials at our institution to determine the efficacy of these FLT3 inhibitors and correlate it with patient outcomes.ResultsThe different FLT3 compounds tested had a heterogenous effect on activated FLT3 levels. In some patients, ASP2215 caused a decrease in levels of activated FLT3 (indicated by brown nuclear staining), which corresponded to a decrease in leukemic blasts (Fig 1). Other patients, however, showed similar amounts of activated FLT3 both before and after treatment, which corresponded with no significant change in leukemic blasts. (Fig 2).Other FLT3 inhibitors also showed differences in their effects. Midostaurin reduced activated FLT3 levels, which correlated with a positive clinical response. In contrast, one patient receiving quizartinibshowed little to no decrease in activated FLT3 levels, despite remaining in clinical remission. This suggests that this FLT3 inhibitor may have alternative targets, such as the tyrosine kinases AXL or LTK.ConclusionsThe heterogeneity in the responses to ASP2215, midostaurin, and quizartinib suggests that there may be other targets for these compounds that are not currently accounted for in the clinical studies. Immunohistochemicalmeasurements of activated FLT3 in bone marrow sections before and after treatment with FLT3 inhibitors was not predictive for clinical response. [Display omitted] [Display omitted] DisclosuresLarson:Pfizer: Consultancy; Ariad: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy.

Abstract LB-325: Global analysis of the phosphoproteome of human blasts reveals predictive phosphorylation markers for the treatment of acute myeloid leukemia with quizartinib

Abstract Acute Myeloid Leukemia (AML) results from a combination of oncogenic events that can involve multiple signal transduction pathways including mutation-induced activation of tyrosine kinases. Kinase inhibitors are increasingly studied as promising targeted approaches either alone or in combination with other agents. However, only subsets of patients respond to respective targeted therapies. Internal tandem duplication (ITD) of FLT3 is one of the most common mutations in AML. It causes constitutive activation of FLT3. Quizartinib (AC220) is an example of a potent FLT3 inhibitor that was studied in a recent phase II open-label study in patients with relapsed/refractory AML. However, the presence of activating mutations within FLT3 can predict response to a certain extent only. Here, we investigated whether large-scale analyses of phosphorylation-based signalling events allows identification of more accurate markers based on the hypothesis that the read-out is closer to the mode of action of FLT3 inhibitors. Therefore, we applied quantitative mass-spectrometry to globally profile the phosphoproteome of 12 pre-treatment bone marrow aspirates obtained from AML patients treated with the quizartinib. A signature derived from this analysis consists of five phospho-sites within the proteins EEPD1, BCL11A, RANBP3, RP3, and LMN1 and it accurately predicted response to treatment with AC220 as revealed by validation in additional independent nine AML patients. Although the combined signature of five phospho-sites showed the highest prediction accuracy, we could demonstrate that in particular phosphorylation of S640 on BCL11A and S333 on RANBP3 lead to almost equally good predictions if used as individual markers. Furthermore, we could show that in case of BCL11A, EEPD1, and LMN1 the expression of the total protein correlates with its phosphorylation and thus with response. The phosphorylation markers were identified and validated in bone marrow aspirates. Although, it is clinical standard procedure to use bone marrow aspirates for diagnosis of AML patients, a predictive test that can be applied to peripheral blood samples would have many advantages. Indeed, we could show that the phosphorylation of the marker proteins strongly correlate between bone marrow and peripheral blood samples from the same patients, suggesting that the phosphorylation or protein markers can be measured and are predictive in both, bone marrow and peripheral blood samples. Citation Format: Christoph Schaab, Felix Oppermann, Martin Klammer, Heike Pfeifer, Andreas Tebbe, Thomas Oellerich, Juergen Krauter, Mark Levis, Alexander Perl, Henrik Daub, Bjoern Steffen, Klaus Godl, Hubert Serve. Global analysis of the phosphoproteome of human blasts reveals predictive phosphorylation markers for the treatment of acute myeloid leukemia with quizartinib. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-325. doi:10.1158/1538-7445.AM2014-LB-325

Inhibition of the receptor tyrosine kinase Axl impedes activation of the FLT3 internal tandem duplication in human acute myeloid leukemia: implications for Axl as a potential therapeutic target

AbstractApproximately 20% to 25% of patients with acute myeloid leukemia (AML) have a constitutively activated FLT3-internal tandem duplication (FLT3-ITD), and these patients exhibit a poor prognosis. Here, we report that Axl, a receptor tyrosine kinase (RTK) overexpressed and constitutively active in human AML, targets the RTK FLT3 in FLT3-ITD+ AML. Abrogation of Axl activation by soluble Axl chimeric protein (Axl-Fc) or small interfering RNA (siRNA) diminishes constitutive FLT3 phosphorylation in FLT3-ITD+ AML. In addition, inhibition of Axl activation by Axl-Fc interferes with the physical interaction between Axl and FLT3. We found that Axl-Fc, a pharmacologic Axl inhibitor, or siRNA targeting Axl inhibits cell growth, induces cell-cycle arrest and apoptosis, and relieves a block in myeloid differentiation of FLT3-ITD+ AML in vitro. Axl-Fc also suppresses the growth of human FLT3-ITD+ AML in vivo. Collectively, our data suggest that Axl contributes to the pathogenesis of FLT3-ITD+ AML through, at least in part, positive regulation of constitutive FLT3 activation. This also suggests that Axl should be pursued as a potential target for the treatment of FLT3-ITD+ AML.

Global Phosphoproteome Analysis of AML Bone Marrow Reveals Predictive Markers for the Treatment with AC220

AbstractAbstract ▪786▪This icon denotes a clinically relevant abstractAcute Myeloid Leukemia (AML) results from a combination of oncogenic events that can involve multiple signal transduction pathways including mutation-induced activation of tyrosine kinases. Kinase inhibitors are increasingly studied as promising targeted approaches either alone or in combination with other agents. However, only subsets of patients respond to respective targeted therapies. We hypothesize that the phosphorylation status of certain sets of proteins (phosphosignatures) can predict the clinical response. In recent years, advances in sample processing, mass spectrometry, and computer algorithms for the analyses of proteomics data have enabled the application of mass spectrometry-based proteomics to monitor phosphorylation events in a global and unbiased manner. These methods have become sufficiently sensitive and robust to identify and quantify thousands of phosphorylation sites in a single experiment. Furthermore, spiking-in a SILAC- (stable-isotope labelling by amino acids in cell culture) reference sample (Super-SILAC) allows for the precise quantification of phosphorylation events also in in-vivo samples. Internal tandem duplication (ITD) of FLT3 is one of the most common mutations in AML. It causes constitutive activation of FLT3. AC220 (Quizartinib®, Ambit) is a selective inhibitor of the receptor-type tyrosine-protein kinase FLT3 that is currently under development for the treatment of AML. In a recent phase II open-label study, patients with relapsed AML were treated with AC220. The results of this trial will be reported elsewhere. Here, we sought to identify phosphorylation events that predict clinical response with high accuracy, especially in the group of FLT3-ITD-positive patients. To this end, we established a global, quantitative phosphoproteome analysis of AML cells derived from patient bone marrow. We analyzed samples from 15 patients before treatment with AC220 and employed the obtained phosphoproteomics data to discover a predictive signature of phosphorylation markers. In total, we confidently identified and quantified roughly 10,000 phosphorylation sites. Unsupervised clustering of the data revealed two large clusters that are both characterized by stronger phosphorylation of associated protein kinases. Next, we correlated these clusters with the reported clinical response of the respective patients. Although the clustering was unsupervised regarding the response information, the two clusters almost perfectly separated responders (complete or partial remission) from non-responders (stable or progressive disease). Furthermore, we identified a signature comprising four phosphorylation sites that accurately predict the outcome of therapy based on the corresponding phosphorylations obtained before treatment. We evaluated the performance of this signature in a cross-validation set-up and in additional test samples that have not been used for training. Moreover, we validated the robustness of the selected predictive features. Our study supports that the phosphorylation of four proteins are candidate biomarkers for predicting response to AC220 in AML. Notably, the phosphorylation markers are more predictive than the FTL3-ITD status alone and will thus allow a more precise stratification of AML patients for treatment with AC220. In addition, our results demonstrate for the first time that identifying predictive phosphorylation signatures directly from clinical patient samples is possible. Disclosures:Schaab:Kinaxo, Evotec: Employment, Research Funding. Oppermann:Kinaxo, Evotec: Employment, Research Funding. Pfeifer:Ambit: Joined performance of clinical trial, Joined performance of clinical trial Other; Kinaxo, Evotec: Research Funding. Klammer:Kinaxo, Evotec: Employment, Research Funding. Tebbe:Kinaxo, Evotec: Employment, Research Funding. Oellerich:Kinaxo, Evotec: Research Funding; Ambit: Joined performance of clinical trial, Joined performance of clinical trial Other. Krauter:Ambit: Joined performance of clinical trial Other. Levis:Astellas Pharma: Consultancy; Plexxikon: Consultancy; Symphogen: Consultancy; Ambit: Joined performance of clinical trial, Joined performance of clinical trial Other. Perl:Ambit: Joined performance of clinical trial Other. Daub:Kinaxo, Evotec: Employment, Research Funding. Steffen:Kinaxo, Evotec: Research Funding; Ambit: Joined performance of clinical trial, Joined performance of clinical trial Other. Godl:Kinaxo, Evotec: Employment, Research Funding. Serve:Ambit: Joined performance of clinical trial, Joined performance of clinical trial Other; Kinaxo, Evotec: Research Funding.

Phosphoproteome analyses reveal specific implications of Hcls1, p21-activated kinase 1 and Ezrin in proliferation of a myeloid progenitor cell line downstream of wild-type and ITD mutant Fms-like tyrosine kinase 3 receptors

The tyrosine kinase receptor Flt3 (Fms-like tyrosine kinase 3) is almost always expressed in AML (acute myeloid leukemia) cells, and constitutive activation of Flt3 by ITD (internal tandem duplication) mutations is one of the most common molecular alterations known in AML, especially monocytic AML. Furthermore, Flt3-ligand (FL) was shown as an in vitro growth factor for monocytic precursors, pointing to the important role of Flt3 in the regulation of monocyte/macrophage production. To get a relevant model for studying the molecular mechanisms underlying the physiopathological role of Flt3 on monocytic lineage development, we used the IL-3 dependent murine myeloid progenitors FDC-P1 cell line to generate cells stably co-expressing murine Fms (M-CSF receptor) and human Flt3. Wild type (WT)-Flt3 expressing cells could proliferate in an FL-dependent manner, whereas those expressing Flt3-ITD all survived IL-3 deprivation and showed autonomous proliferation, whereas both types of cells could differentiate to monocytic cells in response to M-CSF. Next, by combining phosphoprotein detection or purification, comparative 2D-PAGE and mass spectrometry sequencing, we sought for downstream mediators of Flt3-WT or Flt3-ITD in FD/Fms cell proliferation. Amongst the differentially expressed and/or phosphorylated proteins, 3 showed a specific implication in FD/Fms cell proliferation: Hcls1 and the Pak1/2 in FL-dependent proliferation of Flt3-WT expressing cells and Ezrin in autonomous proliferation of Flt3-ITD expressing cells.

FLT3-ITD+ AML Blast, Progenitor and Stem Cell Populations Demonstrate Higher Sensitivity to the Hsp90 Inhibitor PU-H71,

Abstract 3500Hsp90, one of the best-characterized molecular chaperones, plays indispensable roles in folding and assembly, intracellular transport, stabilization, and degradation of proteins, and therefore, facilitating cell signaling. Hsp90 is also involved in tumorigenesis by stabilizing oncogenic client proteins. Thus, Hsp90 inhibition has been considered a promising therapeutic strategy for different types of cancer including leukemia. PU-H71 is a novel HSP90 inhibitor with high specificity for oncogenic Hsp90. We investigated the effect of PU-H71 in acute myelogenous leukemia (AML), in particularly, AML stem cells (AML-SCs) that are known to give rise to AML blasts, are refractory to conventional therapies, and thus likely to account for AML relapses.The effect of PU-H71 was evaluated using a panel of 12 leukemia cell lines. Among the 12 leukemia cell lines tested, MOLM-13 and MV4-11 cells were the most sensitive (LD50s 253 nM and 120 nM respectively). Both MV4-11 and MOLM-13 carry FLT3-ITD mutation (occurring in ∼40% AML cases) and MLL translocations (occurring in ∼20% AML cases). Both MLL and FLT3 have been reported as client proteins of Hsp90. However, other leukemia cell lines with MLL rearrangements such as THP-1 and a MLL-ENL cell line derived from MLL-ENL transformed human CD34+ cord blood cells exhibited resistance to PU-H71 treatment (LD50 > 2 μM). The data suggested that FLT3-ITD+ AML samples may display higher sensitivity to Hsp90 inhibition.To confirm the higher sensitivity of FLT3-ITD+ AML cells to Hsp90 targeted therapy, 15 primary AML patient samples (8 FLT3-ITD mutants and 7 wild type FLT3) were treated with increasing concentrations of PU-H71. Cell viability on different cell populations was evaluated using multiparameter flow cytometry at 48 hours after treatment with PU-H71. The average LD50 of PU-H71 in FLT3-ITD+ AML cells was 492 nM (95% CI, 127.636 – 856.364). In contrast, the average LD50 in AML samples with wild type FLT3 was 2.795 μM (95% CI, 1.058 – 4.532). The near 6-fold difference between LD50s for PU-H71 was significant (p=0.0068). Importantly PU-H71 also killed FLT3-ITD+ AML stem and progenitor cells more effectively. Furthermore, PU-H71 treatment decreased the ability to form colonies in FLT3-ITD+ AML specimens more effectively than FLT3 WT AMLs (97.6% and 79.3% decrease relative to control respectively; N=3; P=0.0236). Importantly, PU-H71 had minor toxicity to normal blood mononuclear cells and normal cord blood hematopoietic stem cells.FLT3-ITD+ cell lines and primary AML cells treated with 0.5 μM PU-H71 showed a substantial decrease of phosphorylated forms of Erk1/2, JNK, AKT, p70RSK, NF-κB(p65) and Stat5, which was observed within 4 hours post PU-H71 treatment, whereas the phosphorylation levels of MAPK p38 remained unaffected. Immunoblotting and phosphoflow assays corroborated the inhibition of AKT and Stat5 signaling by PU-H71 in stem and progenitor populations.In summary, FLT3-ITD+ AML cells display a stronger response to PU-H71, suggesting that the FLT3-ITD mutation results in a higher dependency on Hsp90 to stabilize the aberrant signaling elicited by constitutive activation of FLT3. Our data suggests that PU-H71 represents a novel therapy for FLT3-ITD+ AML patients with the potential to ablate AML-SCs. Disclosures:Roboz:EpiCept: Consultancy; ChemGenex: Consultancy; Celgene: Consultancy; Boehringer Ingelheim: Consultancy.

CALGB 10603 (RATIFY): A randomized phase III study of induction (daunorubicin/cytarabine) and consolidation (high-dose cytarabine) chemotherapy combined with midostaurin or placebo in treatment-naive patients with FLT3 mutated AML.

TPS199 Background: The prognosis for patients (pts) with AML is variable and dependent on the presence of mutations in the FLT3 tyrosine kinase. Internal tandem duplications (ITD) in the juxtamembrane region and mutations in the tyrosine kinase domain (TKD) cause constitutive activation of FLT3 and lead to blast proliferation. Preclinical data suggested that chemotherapy combined with FLT3 inhibitors, including midostaurin, could synergistically kill leukemic cells. In a phase Ib study of midostaurin 50 mg BID with chemotherapy, 92% of FLT3mut pts and 74% of FLT3WT pts achieved a CR. OS of FLT3mut pts was similar to FLT3WT pts (Stone, ASH 2009), supporting the rationale for the ongoing study (NCT00651261). Methods: This is a CALGB-led intergroup, international, randomized, double-blind, PBO controlled trial of adult (age 18-59), untreated FLT3mut AML pts treated with induction chemotherapy and 4 post-remission cycles of high dose ara-C with PBO or midostaurin 50 mg BID on d8-21 of each cycle, followed by ...

FLT3/ ITD regulates leukaemia cell adhesion through α4β1 integrin and Pyk2 signalling

Internal tandem duplication of FMS-like receptor tyrosine kinase 3 (FLT3/ITD) within its juxtamembrane domain is a frequent mutation in adult acute myeloid leukaemia (AML). This mutation causes constitutive activation of FLT3 and is associated with poor prognosis. The high relapse rate of FLT3/ITD-positive AML might be partly because of insufficient eradication of slow-cycling leukaemic stem cells in the bone marrow microenvironment. β1 integrin mediates haematopoietic stem and progenitor cell homing along with their retention in the bone marrow and also inhibits haematopoietic proliferation and differentiation. Here, we demonstrate that inhibition of FLT3/ITD kinase activity by a FLT3 selective inhibitor named FI-700 decreases affinity of α4β1 integrin to soluble VCAM-1. α4β1 integrin deactivation by FI-700 is independent of Rap1, which is the critical regulator of integrin inside-out signalling. In addition, selective inhibition of FLT3/ITD induces Pyk2 dephosphorylation together with the inhibition of phosphatidylinositol-3-kinase (PI3K)/Akt pathway. Both wild-type and ITD-FLT3 proteins co-immunoprecipitated with β1 integrin and Pyk2 indicating the signal crosstalk between FLT3, β1 integrin and Pyk2. These results collectively indicated that the inhibition of FLT3 kinase might contribute not only to the induction of apoptosis, but also to the leukaemia cell detachment from the bone marrow microenvironment in the treatment of AML.

Functional Characterization of FLT3 Receptor Signaling Deregulation in Acute Myeloid Leukemia by Single Cell Network Profiling (SCNP)

BackgroundMolecular characterization of the FMS-like tyrosine kinase 3 receptor (FLT3) in cytogenetically normal acute myeloid leukemia (AML) has recently been incorporated into clinical guidelines based on correlations between FLT3 internal tandem duplications (FLT3-ITD) and decreased disease-free and overall survival. These mutations result in constitutive activation of FLT3, and FLT3 inhibitors are currently undergoing trials in AML patients selected on FLT3 molecular status. However, the transient and partial responses observed suggest that FLT3 mutational status alone does not provide complete information on FLT3 biological activity at the individual patient level. Examination of variation in cellular responsiveness to signaling modulation may be more informative.Methodology/Principal FindingsUsing single cell network profiling (SCNP), cells were treated with extracellular modulators and their functional responses were quantified by multiparametric flow cytometry. Intracellular signaling responses were compared between healthy bone marrow myeloblasts (BMMb) and AML leukemic blasts characterized as FLT3 wild type (FLT3-WT) or FLT3-ITD. Compared to healthy BMMb, FLT3-WT leukemic blasts demonstrated a wide range of signaling responses to FLT3 ligand (FLT3L), including elevated and sustained PI3K and Ras/Raf/Erk signaling. Distinct signaling and apoptosis profiles were observed in FLT3-WT and FLT3-ITD AML samples, with more uniform signaling observed in FLT3-ITD AML samples. Specifically, increased basal p-Stat5 levels, decreased FLT3L induced activation of the PI3K and Ras/Raf/Erk pathways, decreased IL-27 induced activation of the Jak/Stat pathway, and heightened apoptotic responses to agents inducing DNA damage were observed in FLT3-ITD AML samples. Preliminary analysis correlating these findings with clinical outcomes suggests that classification of patient samples based on signaling profiles may more accurately reflect FLT3 signaling deregulation and provide additional information for disease characterization and management.Conclusions/SignificanceThese studies show the feasibility of SCNP to assess modulated intracellular signaling pathways and characterize the biology of individual AML samples in the context of genetic alterations.