FLT3 Activation Loop Research Articles

Understanding the Role of Activation Loop Mutants in Drug Efficacy for FLT3-ITD.

The type III receptor tyrosine kinase FLT3 is a pivotal kinase for hematopoietic progenitor cell regulation, with significant implications in acute myeloid leukemia (AML) through mutations like internal tandem duplication (ITD). This study delves into the structural intricacies of FLT3, the roles of activation loop mutants, and their interaction with tyrosine kinase inhibitors. Coupled with this, the research leverages molecular contrastive learning and protein language modeling to examine interactions between small molecule inhibitors and FLT3 activation loop mutants. Utilizing the ConPLex platform, over 5.7 million unique FLT3 activation loop mutants-small molecule pairs were analyzed. The binding free energies of three inhibitors were assessed, and cellular apoptotic responses were evaluated under drug treatments. Notably, the introduction of the Xepto50 scoring system provides a nuanced metric for drug efficacy. The findings underscore the modulation of molecular interactions and cellular responses by Y842 mutations in FLT3-KD, highlighting the need for tailored therapeutic approaches in FLT3-ITD-related malignancies.

Abstract 3969: Y842 mutations in the activation loop of FLT3 regulates drug response in FLT3-ITD positive acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is an aggressive form of blood cancer with a poor prognosis. Approximately 30% of AML patients carry mutations in the type III receptor tyrosine kinase FLT3. The most common form of FLT3 mutations includes internal tandem duplication (ITD) mutations, which are also associated with poor clinical outcomes. Several inhibitors of FLT3 have been developed and are under clinical trials. Major problems in targeting FLT3-ITD include acquired resistance to the inhibitors which is mediated by secondary point mutations in FLT3. The activity of many, but not all, tyrosine kinases is dependent on the well-conserved tyrosine residue in the activation loop for receptor activation. For instance, we have recently shown that the KIT receptor is dependent on its activation loop tyrosine for downstream signaling and receptor stability, but not for the activation of the receptor. In many AML patients, FLT3-ITD mutations in combination with mutations in the activation loop tyrosine residue (Y842) result in drug resistance. We therefore stably transduced cells with FLT3-ITD in combination with different Y842 mutants (Y842C and Y842F). Using MM-PBSA, we found that the introduction of the Y842C and Y842F point mutations changed the binding energy of the FLT3 inhibitors quizartinib, sorafenib, and midostaurin in silico. In addition, the mutants displayed a change in drug response and apoptosis levels after treatments with FLT3 inhibitors in vitro. Previous data from our lab also shows that the Y842F mutant resulted in delayed tumor formation in vivo, indicating that the phosphorylation of the activation loop tyrosine is an important event in FLT3-ITD-mediated transformation. All in all, our data suggest that the activation loop tyrosine residue in FLT3-ITD positive cells plays an important role in drug response and tumor formation. Citation Format: Lina Al Ashiri, Sausan Moharram, Rituraj Purohit, Julhash Kazi Uddin, Lars Rönnstrand. Y842 mutations in the activation loop of FLT3 regulates drug response in FLT3-ITD positive acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3969.

Abstract 2371: Oncogenic transformation of FLT3-ITD is partially dependent on tyrosine 842 in the activation loop

Abstract Acute myeloid leukemia (AML) is a heterogeneous disease of the blood. About 30 % of AML patients carry an oncogenic mutant of the type III receptor tyrosine kinase FLT3. Among the various mutations in FLT3, the internal tandem duplication (ITD) mutations in the juxtamembrane domain are the most common type of mutation, while point mutations in the kinase domain also occur. Although oncogenic FLT3 mutants are constitutively active, the wild-type receptor needs its ligand (FLT3 ligand, FL) for activation. Ligand binding to the receptor induces dimerization, auto-phosphorylation, and activation of the receptor. Tyrosine phosphorylation sites on FLT3 constitute docking sites for interacting proteins that transduce pro-survival and proliferative signals. The so-called activation loop tyrosine residue (in FLT3, Y842) is well conserved in all receptor tyrosine kinases and long been known essential for the activity of some but not all receptor tyrosine kinases. Recently we have shown that activation loop tyrosine residue in KIT is not essential for its activation but plays an important role in receptor stabilization and downstream signaling. However, so far the role of FLT3 activation loop tyrosine residue remains unknown. We generated 32D cell lines carrying a Y-to-F mutant (Y842F) of FLT3 in either the wild-type or ITD background to study the role of this tyrosine residue. We show that the Y842 residue is not necessary for FLT3 activation or stability but is required for oncogenic transformation. We observed that Y842F mutation results in reduced cell viability, proliferation and in vitro colony formation in semi-solid medium. In addition, cells expressing the Y842F mutant in FLT3-ITD background display a significant reduction in tumor volume and weight in a xenograft model in nude mice. Gene set enrichment analysis (GESA) shows that mutation of Y842 causes suppression of anti-apoptotic genes. Furthermore, we demonstrated that phosphorylated Y842 is one of the binding sites of the protein tyrosine phosphataseSHP2, which is required for activation of signaling through the RAS/ERK pathway. Taken together, our data suggest that activation loop tyrosine residue in FLT3 plays an important role in the cellular transformation which is regulated by RAS/ERK signaling. Citation Format: Julhash U. Kazi, Lars Rönnstrand. Oncogenic transformation of FLT3-ITD is partially dependent on tyrosine 842 in the activation loop [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2371. doi:10.1158/1538-7445.AM2017-2371

Tyrosine 842 Residue in the Activation Loop of FLT3-ITD Is Indespensible for Oncogenic Transformation

The receptor tyrosine kinase FLT3 is mutated in around 30% of acute myeloid leukemia patients. The so-called internal tandem duplication (ITD) mutation in the juxtamembrane domain is the most common type of mutation in FLT3. Other oncogenic mutations include point mutations in the kinase domain. One of the hallmark of receptor activation is phosphorylation on several tyrosine residues in the receptor. Oncogenic FLT3 mutants display constitutive activity leading to aberrant cell proliferation and survival. Phosphorylation on several critical tyrosine residues is known to be essential for FLT3 signaling. The tyrosine residue Y842, which is located in the activation loop of FLT3, and is well conserved in all receptor tyrosine kinases. It has been demonstrated that tyrosine phosphorylation of the activation loop is essential for the activity of some but not all receptor tyrosine kinases. In this report we show that phosphorylation on Y842 residue is dispensable for receptor activity and stability. However, Y842 plays critical roles in oncogenic FLT3-ITD mediated transformation. We have shown that a Y-to-F mutation (Y842F) results in reduced cell viability and proliferation. This mutation also led to dramatic decrease in in vitro colony formation in semi-solid medium. Furthermore, cells carrying the Y842F mutant of FLT3-ITD displayed significant delay in tumor formation in nude mice. Gene expression analysis, using microarrays, showed that mutation of Y842 causes suppression of anti-apoptotic genes. Finally, we showed that phosphorylated Y842 is a binding site for SHP2 and mutation of this site results in impaired activation of signaling through the RAS/ERK pathway. Collectively, our data suggest that Y842 in FLT3 plays an important role in FLT3-mediated RAS/ERK signaling and cellular transformation. DisclosuresNo relevant conflicts of interest to declare.

Anti-tumor activity of TAK-659, a dual inhibitor of SYK and FLT-3 kinases, in AML models.

e14091Background: TAK-659 is a reversible, dual inhibitor of SYK and FLT-3 being developed for oncology indications including Acute Myelogenous Leukemia (AML), a disease driven by SYK and/or FLT-3-mediated signaling. Loss of SYK in AML cell lines has been shown to induce myeloid differentiation. FLT-3 is a Class III receptor tyrosine kinase that is normally expressed only in hematopoietic stem and progenitor cells. However, its expression has been found in the blasts of a majority of patients with AML. Activating mutations of FLT-3, including internal tandem duplication (ITDs) within the juxtamembrane region of FLT-3 and point mutations in the FLT-3 activation loop, are observed in approximately 30% of AML patients. These FLT-3 mutations, associated with early relapse and poor survival, represent a critical prognostic factor for AML. Methods: TAK-659 is an investigational inhibitor of SYK and FLT-3 that is currently being evaluated in a Phase 1b/2 clinical trial of AML patients (NCT02323113). TAK-659 inhi...

Characterizing and Overriding the Structural Mechanism of the Quizartinib-Resistant FLT3 "Gatekeeper" F691L Mutation with PLX3397.

Tyrosine kinase domain mutations are a common cause of acquired clinical resistance to tyrosine kinase inhibitors (TKI) used to treat cancer, including the FLT3 inhibitor quizartinib. Mutation of kinase "gatekeeper" residues, which control access to an allosteric pocket adjacent to the ATP-binding site, has been frequently implicated in TKI resistance. The molecular underpinnings of gatekeeper mutation-mediated resistance are incompletely understood. We report the first cocrystal structure of FLT3 with the TKI quizartinib, which demonstrates that quizartinib binding relies on essential edge-to-face aromatic interactions with the gatekeeper F691 residue, and F830 within the highly conserved Asp-Phe-Gly motif in the activation loop. This reliance makes quizartinib critically vulnerable to gatekeeper and activation loop substitutions while minimizing the impact of mutations elsewhere. Moreover, we identify PLX3397, a novel FLT3 inhibitor that retains activity against the F691L mutant due to a binding mode that depends less vitally on specific interactions with the gatekeeper position. We report the first cocrystal structure of FLT3 with a kinase inhibitor, elucidating the structural mechanism of resistance due to the gatekeeper F691L mutation. PLX3397 is a novel FLT3 inhibitor with in vitro activity against this mutation but is vulnerable to kinase domain mutations in the FLT3 activation loop.

Preclinical and Clinical Resistance Mechanisms To The Investigational Selective FLT3 Inhibitor PLX3397 In FLT3-ITD+ Acute Myeloid Leukemia (AML)

BackgroundActivating mutations [primarily internal tandem duplication (ITD) events] in FLT3 are detected in 30% of acute myeloid leukemia (AML). The clinically active FLT3 tyrosine kinase inhibitor (TKI) AC220 (quizartinib) has achieved complete remissions in relapsed/refractory FLT3-ITD+ AML patients in a phase II study (Cortes, et al and Levis et al, ASH 2012, abstracts 48 and 673) but is vulnerable to resistance-conferring mutations in the FLT3 kinase domain (KD). The F691L “gatekeeper” substitution was the most commonly detected mutation in an in vitro mutagenesis screen for AC220 resistance (Smith et al, Nature 2012). This mutation, and substitutions at the activation loop residue D835, have been associated with acquired clinical resistance to AC220 (Smith et al, Nature 2012; Alber et al, Leukemia 2013). Mutations at gatekeeper residues such as F691 have repeatedly surfaced as mediators of clinical resistance to TKIs. Identifying TKIs that retain activity against these substitutions has consistently proven challenging. PLX3397 is a potent and selective inhibitor of FMS, KIT and FLT3-ITD with a half-life of 20 hours in humans, resulting in µM steady-state plasma concentrations at the recommended phase II dose for AML patients. PLX3397 retains activity against the AC220-resistant FLT3-ITD/F691L mutant, but not against several D835 mutants (Smith et al., ASH 2011, abstract 764). In this study, we conducted a mutagenesis screen of FLT3-ITD and FLT3-ITD/F691L to identify single and compound mutations that confer resistance to PLX3397 and may cause acquired resistance in patients. ResultsPLX3397 inhibited the proliferation of BaF3/ FLT3-ITD cells at a concentration well below that achieved in patients (IC50 0.14 µM) and retained activity against cells expressing the FLT3-ITD/F691L mutant (IC50 0.350 µM). Other AC220-resistant mutants (D835V/Y/F and Y842C/H) conferred substantial cross-resistance to PLX3397 (∼50 to 400-fold shift in IC50 of FLT3-ITD; ranging from 7.2 to >10 µM).An in vitro mutagenesis screen of FLT3-ITD identified several mutations conferring resistance to PLX3397, including novel substitutions in 3 residues which conferred ≥10X resistance relative to FLT3-ITD: D835E/G/N, D839A/G and R845G (IC50s 1.4 to 4.1 µM). Given the in vitro activity of PLX3397 against the AC220-resistant F691L mutant, it is anticipated that PLX3397 will be administered to patients who acquire resistance to AC220 or sorafenib due to this mutation; a mutagenesis screen of FLT3-ITD/F691L was therefore conducted. We identified multiple KD mutations in FLT3-ITD/F691L conferring ≥10X resistance to PLX3397 (compared to FLT3-ITD) including several mutations in the FLT3 activation loop: D835H/G/E/N, D839A/G/N, N841K, Y842S, R845G (IC50s 1.6 to >10 µM), and 2 mutations in residues located in the tyrosine kinase domain 1 (TK1) domain: N676S, a residue previously implicated in clinical resistance to the FLT3 inhibitor PKC412 (IC50 2.8 µM), and M664I, a residue not previously linked to FLT3 inhibitor resistance (IC50 2.0 µM). While all identified mutants conferred some degree of resistance to PLX3397 in the absence of an F691L mutation, most conferred a higher degree of resistance in the setting of F691L, suggesting a cooperation between the gatekeeper residue and residues in the activation loop and TK1 domain that impacts PLX3397 binding.Finally, we conducted a preliminary analysis of samples from AML patients who relapsed after an initial response to PLX3397. Using Pacific Biosciences Single Molecule Real-Time Sequencing, we identified evolution of polyclonal FLT3 KD mutations at the D835 residue at the time of relapse in 2 patients, including, in one patient, novel PLX3397-resistant D835E/H mutations identified in our mutagenesis screen. Analysis of additional patient samples for single and compound resistant mutations is ongoing and will be presented. ConclusionsPLX3397 harbors promise for the treatment of FLT3-ITD+ AML, particularly for patients who have developed resistance to FLT3 TKIs due to the gatekeeper F691L mutation. However, a mutagenesis screen reveals PLX3397 is vulnerable to mutations in the FLT3 activation loop and TK1 domain. Patients acquire secondary FLT3 KD mutations at the time of resistance to PLX3397, confirming the mechanism of action of this clinically active FLT3 inhibitor. A multi-site phase I/II study of PLX3397 in FLT3-ITD+ AML is ongoing. Disclosures:Smith:Plexxikon Inc: Research Funding. Off Label Use: Unapproved drugs for AML: AC220 and PLX3397. Le:Plexxikon Inc: Employment. Zhang:Plexxikon Inc: Employment. West:Plexxikon Inc: Employment. Shah:Ariad Pharmaceuticals: Consultancy, Research Funding; Plexxikon Inc: Research Funding; Ambit Biosciences: Research Funding.

Constitutively Activating Mutations At the FLT3 Activation Loop Residue D835 Are Associated with Clinical Resistance to AC220

Abstract 674 Background.Activating mutations in FLT3 occur in ∼30% of adult acute myeloid leukemia (AML) cases, including internal tandem duplication (ITD) mutations (∼25%) and point mutations in the tyrosine kinase domain (KD), primarily at the activation loop (AL) residue D835 (∼5%). FLT3-ITD and FLT3-AL mutations can co-occur in individual patients. AC220 (quizartinib) is a potent selective investigational FLT3/KIT inhibitor with encouraging preliminary clinical activity in FLT3-ITD+ AML, as evidenced by a composite complete remission rate of 45% in 53 chemotherapy refractory/relapsed patients in an interim analysis of a phase II study (Cortes et al, ASH 2011 abstract #614). While some patients without FLT3 mutations in the AC220 phase I dose escalation study responded, no objective response was observed in a small number of FLT3-AL mutant patients (Cortes et al, ASH 2008 abstract #767). We recently reported that secondary mutations at F691, D835 and Y842 in FLT3-ITD confer in vitro resistance to AC220, and that relapse in 8/8 AML patients who initially responded to AC220 was associated with evolution of secondary KD mutations in the FLT3-ITD+ allele (Smith et al., Nature, 2012). In 6 of 8 patients, relapse was associated with D835 substitutions, making this amino acid position the most commonly substituted residue in FLT3-ITD+ patients at the time of disease relapse. Given that D835 mutations in FLT3-ITD appear to confer a high degree of pre-clinical and clinical resistance to AC220, we hypothesized that D835 mutations in the absence of the ITD might also confer resistance to AC220 and could be associated with clinical relapse, especially in patients harboring a mixture of FLT3-WT, FLT3-ITD+, and FLT3-AL mutant blasts. Results.In vitro binding studies revealed that FLT3-D835V and FLT3-D835Y have decreased affinity for AC220 (Kd=4.8 and 7nM, respectively) compared to native FLT3 (Kd=1.8nM). In proliferation assays of BaF/3 cells, FLT3-D835V/Y mutants demonstrated increased relative AC220 resistance (IC50 24 and 6.8nM) compared to FLT3-ITD (IC50 0.13nM). To sensitively and precisely determine the frequency and phase (ITD+ vs ITD-) of mutations in clinical isolates, we utilized single molecule real-time (SMRT; Pacific Biosciences, Menlo Park, CA) sequencing, which can generate reads of sufficient length to enable focused interrogation of the KD of FLT3 ITD+ and ITD- alleles. We analyzed ITD- sequences from pretreatment and relapse samples obtained from the 8 FLT3-ITD-positive AML patients with acquired AC220 resistance reported in our previous analysis. Interestingly, all 6 patients with secondary mutations at D835 in ITD+ alleles at relapse also harbored D835 mutant ITD- alleles. All D835 mutations detected in ITD- alleles were also observed in ITD+ alleles from the same patient sample. In ITD+ alleles, we detected D835Y (n=6), D835V (n=3), and D835F (n=2) (range 2.7–50.6% of ITD+ alleles). In ITD- alleles, we found D835Y (n=6), D835V (n=2), and D835F (n=1) (range 3.8–50% of ITD- alleles). One patient had 2 D835 mutations (D835Y/F) in ITD- alleles at relapse. Although 2 of 8 patients evolved the F691L mutation in ITD+ alleles at relapse, this substitution was not detectable in ITD- sequences in any patient. In 7/8 patients, samples obtained immediately prior to AC220 administration were available for analysis. We were unable to document the presence of AC220-resistant mutations in either ITD+ or ITD- sequences in these pretreatment samples, suggesting that these mutants evolved under the selective pressure of AC220 treatment. We are currently performing single cell RT-PCR to determine if D835 mutations in ITD+ and ITD- alleles occur in distinct leukemic cells, which would suggest a polyclonal blast population at relapse. Conclusions.FLT3-AL mutations at D835 confer resistance to AC220 in vitro. The evolution of D835 substitutions in ITD- alleles in the majority of patients who respond and relapse on AC220 suggests that constitutively activating FLT3-AL mutations at residue D835 can confer acquired clinical resistance to AC220. AML patients with FLT3-D835 mutations may exhibit de novo resistance to AC220 and other FLT3 inhibitors. Our findings predict that the clinical activity of potent FLT3 inhibitors that are tolerant of D835 substitutions will mechanistically involve inhibition of D835 mutants in both the presence and absence of an ITD mutation. Disclosures:Chin:Pacific Biosciences: Employment. Levis:Astellas Pharma: Consultancy; Plexxikon: Consultancy; Symphogen: Consultancy; Ambit: Joined performance of clinical trial, Joined performance of clinical trial Other. Perl:Astellas Pharmaceuticals: Consultancy. Travers:Pacific Biosciences: Employment. Kasarskis:Pacific Biosciences: Equity Ownership. Radich:Ariad: Consultancy; Bristol-Myers Squibb: Consultancy; Pfizer: Consultancy; Novartis: Consultancy, Research Funding. Shah:Ariad: Consultancy, Research Funding.

Analysis of in Vitro Activity of the Clinically-Active ABL/FLT3 Inhibitor Ponatinib (AP24534) Against AC220-Resistant FLT3-ITD Mutants

Abstract 930 Background:Activating mutations in FLT3 are detected in approximately 30 percent of adult acute myeloid leukemia (AML) cases, most commonly in-tandem duplication (ITD) events. Ponatinib (AP24534) is a potent inhibitor of ABL and FLT3 that has shown clinical activity in CML as well as in a limited experience with FLT3-ITD-positive acute myeloid leukemia (AML) with 2/7 kinase inhibitor-naïve AML patients achieving CRi in a phase I study (Talpaz et al, ASCO 2011, abstract #6518). We recently reported that the FLT3 inhibitor AC220, which has achieved an interim composite CR rate of 43% in a phase II study in relapsed/refractory AML (Cortes, et al, EHA 2011, abstract #1019) is vulnerable to a limited number of resistance-conferring kinase domain mutations in vitro (Smith et al, AACR 2011, abstract #4737). Mutations at 2 of these residues, F691 and D835, have been identified in 9/9 FLT3-ITD+ patients who relapsed after achieving clearance of bone marrow blasts on AC220 (Smith et al, ASH 2011, submitted). Notably, AC220-resistant mutations were found to confer cross-resistance to sorafenib. Given that ponatinib retains activity against a wide range of TKI-resistant BCR-ABL mutants, we sought to test its activity against AC220-resistant mutants. Results:We assessed the activity of ponatinib against highly AC220-resistant FLT3-ITD mutations F691I/L, D835V/Y and Y842C/H. Ponatinib potently suppressed the growth of Ba/F3 cells transformed with native FLT3-ITD with an inhibitory concentration 50 (IC50) of 2 nM. Ba/F3 cells transformed with the “gatekeeper” F691I mutation retained sensitivity to ponatinib at a similar concentration (5 nM). This substitution is analogous to the BCR-ABL T315I mutation, which is clinically sensitive to ponatinib. The remaining AC220-resistant FLT3-ITD mutants, including the gatekeeper F691L substitution, conferred a substantial degree of relative cross-resistance to ponatinib. Mutations at the activation loop residue D835 confer the highest degree of resistance. In an effort to identify other substitutions in FLT3-ITD that are capable of conferring ponatinib resistance in vitro, we employed an in vitro mutagenesis strategy (Azam et al, Cell, 2003) of FLT3-ITD in varying concentrations of ponatinib. In a preliminary analysis, we have identified resistance-causing substitutions at D835, as well as other residues in the FLT3 activation loop including D839 and N841. Interestingly, one of these substitutions, D835N, confers resistance to ponatinib but not to AC220.A molecular analysis of the ponatinib/FLT3 complex based on the crystal structure of ABL/ponatinib was performed and revealed that isoleucine substitution at the FLT3 “gatekeeper” residue (F691I), as with the T315I mutation in Abl, does not creates substantial steric clash with ponatinib. In contrast, the leucine substitution, (F691L), is more bulky than isoleucine and may cause more steric hindrance. In addition, as leucine is more rigid than isoleucine, more energy would be required for the F691L mutation to adopt a conformation compatible with ponatinib binding. As ponatinib binds to the “DFG-out” inactive conformation of FLT3, activation loop mutations such as D835V/Y and Y842C/H likely destabilize the activation loop conformation of FLT3 required for ponatinib binding. Conclusions:Our studies demonstrate that gatekeeper and, in particular, activation loop substitutions in FLT3-ITD, confer a high degree of cross-resistance to the clinically-active FLT3 TKIs described to date. Ponatinib retains in vitro activity against the gatekeeper mutation FLT3-ITD/F691I, which confers a high degree of in vitro resistance to AC220, but leucine substitution at this residue notably confers a higher degree of resistance to ponatinib. Activation loop mutations confer substantial degrees of in vitro cross-resistance to ponatinib and are predicted to confer clinical resistance. Select substitutions at D835 appear to confer selective resistance to ponatinib. Categorization of the spectrum of resistance-conferring mutations may facilitate a more personalized approach toward patients with FLT3-ITD+ AML treated with clinically-active TKI therapy. Disclosures:Off Label Use: Investigational agent ponatinib will be discussed. Zhu:Ariad Pharmaceuticals: Employment. Shah:Ariad: Consultancy, Research Funding; Novartis: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding.

The Effect of ABC Transporters and FLT3 Status on the Activity of the Aurora Kinase B Inhibitor AZD1152 in AML Cells.

The aurora kinases are essential for mitotic progression. Both Aurora A and B are over-expressed in a wide variety of human haematological and epithelial malignancies. A number of Aurora kinase inhibitors have been described that show anti-tumour activity in vivo, with results suggesting that their cellular effects are largely due to the inhibition of Aurora B. Aurora B is required for histone H3 phosphorylation, chromosome segregation and cytokinesis. AZD1152 is an aurora kinase inhibitor with potent activity against Aurora B. The compound is a phosphate prodrug that is converted rapidly in vivo to the active moiety AZD1152-HQPA. Acute myeloid leukaemia (AML) is a heterogeneous malignancy that affects bone marrow haematopoietic cells of the myeloid lineage. The ABC transporter P-glycoprotein (Pgp) is a known poor prognostic factor in the treatment of AML. Breast cancer resistance protein (BCRP) has been implicated in the resistance to chemotherapy in AML. AZD1152-HQPA activity was studied in 6 myeloid cell lines: KG1a (Pgp+, BCRP−), U937 (−,−), MV4-11(−,−), MOLM-13 (−,−), OCI-AML3 (−,−) and BCRP transfected OCI-AML6.2 (−, +) at concentrations of 0–1000nM. The Pgp and BCRP status of cell lines was confirmed with R123 and BODIPY-prazocin flow cytometric accumulation assays. Flow cytometry was used to determine 24hr inhibition of phosphorylation of histone H3 (phH3) as a biomarker for AZD1152-HQPA activity as well as 72hr cell number and viability using an in-house flow cytometric method. Both the Pgp positive KG1a cells and the OCI-AML6.2 cells showed decreased sensitivity to AZD1152-HQPA compared with transporter-negative cell lines. This was noted at the level of the biomarker phH3 as well as growth inhibition. Interestingly, the most sensitive lines were the FLT3-ITD expressing MV4-11 and MOLM-13 cells, with cell viability IC50s below 10nM and complete down-regulation of phH3 at 30nM. FLT3 mutations are found in approximately 30% of AML patients, either FLT3-ITD (24%) or FLT-3 activation loop mutations (7%), making FLT3 mutation one of the most commonly mutated genes in AML. ITD mutation of FLT3 induces activating phosphorylation of the receptor in the absence of ligand. Using immunoprecipitation we were able to show a ≥40% decrease in phosphorylated FLT3 (pFLT3) expression in the ITD cell lines after 1hr treatment with 300nM AZD1152-HQPA and a similar decrease in phosphorylation of the downstream transcription factor STAT5 (measured by flow cytometry). AZD1152-HQPA activity was studied in 14 primary AML samples after 48hr pre-incubation with a cytokine cocktail to induce cycling. Pgp status of the samples was confirmed by cytometry using R123 and MRK16 antibody. BCRP and FLT3 status of the samples was determined using RT-PCR. 5 samples were positive for Pgp, 4 of which were also positive for BCRP, and 5 samples had a FLT3-ITD mutation. 20% inhibition of phH3 was achieved in all 14 samples at concentrations ≤130nM AZD1152-HQPA after 1hr. 20% cell death was achieved in 11 out of 12 samples at ≤300nM AZD1152-HQPA, including 3 out of 4 Pgp+ and 2 out of 3 BCRP+ samples. There was a statistical trend towards increased sensitivity for the ITD samples (IC20 ≤15nM; p=0.09). 40% inhibition of pSTAT5 was achieved in 2 ITD samples after 1hr treatment with 100 nM AZD1152-HQPA. In conclusion, the decreased response to AZD1152-HQPA in Pgp+ and BCRP+ cell lines is not characteristic of patient samples. AZD1152-HQPA directly targets pFLT3 in FLT3ITD cells along with its downstream target pSTAT5. AZD1152 is currently being tested in phase I clinical trials.

High incidence of FLT3 mutations in adults with Acute Promyelocytic Leukemia (APL): Correlation with diagnostic features and treatment outcome (CALGB 9710)

7002 Background: Studies have associated activating mutations of FLT3 (FLT3+) with adverse outcomes in patients (pts) with APL, but little is known about the impact of adding Arsenic Trioxide (As2O3) to frontline APL treatment on the clinical outcome of pts harboring these mutations. Methods: We examined the incidence of FLT3+ in a subset of 78 adults with newly diagnosed APL entered onto CALGB 9710, a North American Intergroup phase III randomized trial that demonstrated a significant survival benefit from adding two 25-day courses of As2O3 to post-remission consolidation therapy (Powell, et al. ASCO 2007). FLT3 internal tandem duplications (ITD) and activation loop point mutations (D835) were identified using standard methods (InVivoScribe, San Diego). FLT3+ was correlated with other diagnostic/prognostic features including PML-RARA isoform type, initial white blood cell (WBC) count, and pt age, and with treatment response. Results: FLT3+ was identified in 42 pts (54%); 28 (36%) had an ITD, 17 (22%) had a D835, and 3 (4%) had both. FLT3+ was not associated with increasing age; 55% of FLT3+ pts were male. There was a strong association of FLT3+ with high initial WBC count > 10K/μl (p= 0.0002), and with the S-isoform of PML-RARA (p < 0.0001). Overall, FLT3+ was not associated with early deaths during induction, lower CR rate, or inferior disease free survival (DFS) or survival (S). However, when analyzed by treatment arm, relapses occurred in only 2/21 (10%) FLT3+ pts who received As2O3 compared to a very high relapse rate of 13/21 (62%) among FLT3+ pts who did not receive As2O3 (p = 0.0004). Conclusions: FLT3+ occurs with high frequency (54%) in our APL cohort and is very strongly associated with high initial WBC count and S-isoform, both of which correlated with adverse outcomes for pts on CALGB 9710 (Stock et al, ASCO 07). In contrast to previous reports, we did not find an association of FLT3+ with high induction death rates, DFS, or S. Treatment with As2O3 appeared to decrease markedly the relapse rate in FLT3+ pts. These results will be examined in a larger cohort of 9710 pts. Given the high frequency of FLT3+ APL, targeted therapy with FLT3 inhibitors is an attractive strategy to further improve relapse-free survival for pts with FLT3+ APL. No significant financial relationships to disclose.

Identification of Driver and Passenger Mutations of FLT3 by High-Throughput DNA Sequence Analysis and Functional Assessment of Candidate Alleles.

Activating mutations in the juxtamembrane domain or the activation loop of the receptor tyrosine kinase FLT3 occur in many cases of acute myeloid leukemia (AML), but it is not known whether genomic alterations outside these regions contribute to leukemogenesis. High-throughput DNA sequence analysis has provided insights into the mutational profiles of various cancers and represents a promising strategy for the identification of novel therapeutic targets. However, recognizing the subset of genomic alterations that are functionally relevant has proven difficult. We used a high-throughput platform to interrogate the entire FLT3 coding sequence in a cohort of 222 adult AML patients without known FLT3 mutations and experimentally tested the functional consequences of each candidate leukemogenic allele by exogenous expression in BaF3 cells. DNA sequencing detected nine non-synonymous sequence variants in six exons that were not known single-nucleotide polymorphisms. Functional assessment of these alleles identified four novel activating mutations in the extracellular domain, the juxtamembrane domain, and the activation loop that induced constitutive kinase activity, differentially activated downstream signaling pathways, and conferred varying sensitivity to pharmacologic FLT3 inhibition. In contrast, the remaining five alleles, including mutations in highly conserved, key functional domains of FLT3, were not associated with increased kinase activity and aberrant signal transduction. These findings support the concept that acquired mutations in cancer may not contribute to malignant transformation, and underscore the importance of functional validation of candidate alleles discovered using high-throughput genomic screens, to distinguish between ‘driver' mutations underlying cancer development, and biologically neutral ‘passenger' alterations.

Gene Conversion Is a Late Event in Evolution of High Risk AML.

Evolution and progression of AML is considered a result of cooperation of multiple genetic and epigenetic alterations. FLT3 internal tandem duplication (ITD) is a common mutation in AML and is considered a secondary, proliferative event in the pathogenesis of AML. We used SNP/CGH microarrays to evaluate the mechanism of disease evolution in FLT3/ITD-positive AML. Diagnostic DNA from patients with FLT3/ITD (N=25), FLT3 activation loop mutation (FLT3/ALM, N=15) or with no FLT3 mutations (FLT3/WT, N=55) was evaluated for copy number and LOH alterations. 12/25 (48%) FLT3/ITD-positive patients had loss of heterozygosity (LOH) of a large segment of 13q arm downstream of the FLT3 gene without an associated chromosome loss, demonstrating a copy neutral LOH (CN-LOH) state. In contrast, CN-LOH was not detected in those with FLT3/ALM or FLT3/WT suggesting that FLT3/ITD is causally associated with evolution of CN-LOH. Single cell analysis of the FACS purified leukemic blasts from FLT3/ITD-positive samples that were heterozygous for 13q (no CN-LOH) by SNP/CGH arrays demonstrated the presence of 3 distinct molecular subtypes that included a subpopulation with CN-LOH, as well as heterozygous ITD and FLT3/WT, demonstrating oligoclonal nature of the leukemic blast population which prevented identification of CN-LOH. In patient samples with CN-LOH, CFU-GM and BFU-E colonies derived from CD34+/CD33- progenitors demonstrated heterozygous ITD, with no LOH or copy number alterations. In contrast, more differentiated, CD33+ leukemic blasts as well as CFU-GM colonies derived from CD34+/CD33+ progenitors had homozygous ITD and CN-LOH. This cumulative data demonstrates that CN-LOH of the 13q, as a result of gene conversion-type event mediates the pathogenesis of FLT3/ITD and is a secondary event in the evolution of high risk FLT3/ITD. Gene conversion, leading to haplo-insufficiency may be an important event in the evolution of AML.

FLT3 K663Q is a novel AML-associated oncogenic kinase: determination of biochemical properties and sensitivity to Sunitinib (SU11248)

Somatic mutations of FLT3 resulting in constitutive kinase activation are the most common acquired genomic abnormality found in acute myeloid leukemia (AML). The majority of these mutations are internal tandem duplications (ITD) of the juxtamembrane region (JM). In addition, a minority of cases of AML are associated with mutation of the FLT3 activation loop (AL), typically involving codons D835 and/or I836. We hypothesized that other novel mutations of FLT3 could also contribute to leukemogenesis. We genotyped 109 cases of AML and identified two novel gain-of-function mutations. The first mutation, N841 H, is similar to previously described mutations involving amino-acid substitutions of codon 841. The other novel mutation, FLT3 K663Q, is the first AML-associated gain-of-function mutation located outside the JM and AL domains. Of note, this mutation was potently inhibited by Sunitinib (SU11248), a previously described FLT3 kinase inhibitor. Sunitinib reduced the proliferation and induced apoptosis of transformed Ba/F3 cells expressing FLT3 K663Q. The potency of Sunitinib against FLT3 K663Q was similar to its potency against FLT3 ITD mutations. We conclude that FLT3 mutations in AML can involve novel regions of the TK1. Future studies are needed to define the incidence and prognostic significance of FLT3 mutations outside the well-established JM and AL regions.

Identification of a novel activating mutation (Y842C) within the activation loop of FLT3 in patients with acute myeloid leukemia (AML)

AbstractFms-like tyrosine kinase 3 (FLT3) receptor mutations as internal tandem duplication (ITD) or within the kinase domain are detected in up to 35% of patients with acute myeloid leukemia (AML). N-benzoyl staurosporine (PKC412), a highly effective inhibitor of mutated FLT3 receptors, has significant antileukemic efficacy in patients with FLT3-mutated AML. Mutation screening of FLT3 exon 20 in AML patients (n = 110) revealed 2 patients with a novel mutation (Y842C) within the highly conserved activation loop of FLT3. FLT3-Y842C-transfected 32D cells showed constitutive FLT3 tyrosine phosphorylation and interleukin 3 (IL-3)-independent growth. Treatment with PKC412 led to inhibition of proliferation and apoptotic cell death. Primary AML blasts bearing FLT3-Y842C mutations showed constitutive FLT3 and signal transducer and activator of transcription 5 (STAT-5) tyrosine phosphorylation. Ex vivo PKC412 treatment of primary blasts resulted in suppression of constitutive FLT3 and STAT-5 activation and apoptotic cell death. Inspection of the FLT3 structure revealed that Y842 is the key residue in regulating the switch from the closed to the open (= active) conformation of the FLT3 activation loop. Overall, our data suggest that mutations at Y842 represent a significant new activating mutation in AML blasts. Since FLT3 tyrosine kinase inhibitors (TKIs) such as PKC412 are currently being investigated in clinical trials in AML, extended sequence analysis of FLT3 may be helpful in defining the spectrum of TKI-sensitive FLT3 mutations in AML. (Blood. 2005;105:335-340)

A novel FLT3 activation loop mutation N841K in acute myeloblastic leukemia

A novel FLT3 activation loop mutation N841K in acute myeloblastic leukemia

FLT3 ITD and FLT3 Tyrosine Kinase Domain Mutants Induce Different Phenotypes in a Murine Bone Marrow Transplant Model.

Activating mutations of FLT3 are frequent in patients with acute myeloid leukemia (AML). Two distinct types of FLT3 mutations are most common: Internal tandem duplication (ITD) of the juxtamembrane domain-coding sequence in approximately 30% of patients with AML and point mutations within the second tyrosine kinase domain (TKD) in about 7% of AML patients. Patients carrying the FLT3 ITD mutation seem to have a significantly worse prognosis, whereas the impact of TKD mutations on clinical outcome has not yet been determined. Recently, point mutations within the activation loop of FLT3 were also found in a significant percentage of infant and childhood acute lymhoblastic leukemia (ALL).Previous studies demonstrate that mice receiving transplants of bone marrow retrovirally infected with FLT3 ITD develop a myeloproliferative disease. The effect of FLT3 TKD mutations in vivo has not yet been investigated.To examine the transforming properties of FLT3 TKD mutants in primary hematopoietic cells, we used a bone marrow transplant model (BMT). Therefore we transduced bone marrow with retrovirus expressing either FLT3 D835Y or FLT3 I836M+R and transplanted it to lethally irradiated syngeneic recipient mice. As control we also transplanted mice with FLT3 WT and ITD infected bone marrow, respectively. We found that mice transplanted with FLT3 ITD developed a myeloproliferative disorder in mice, as previously described. In contrast, mice transplanted with FLT3 TKD mutants developed a lymphoid disease with distinct hematologic manifestation.Most recipients of FLT3 TKD transduced bone marrow developed T lymphoma syndrome, characterized by massive enlargement of thymus and lymph nodes. Some mice developed a B lymphoid leukemia with splenomegaly and enlarged lymph nodes. Interestingly, the disease latency of 53 to 183 days (median 102 days) of FLT3 TKD mutants contrasted with FLT3 ITD mice, which succumbed myeloproliferative disease within 53 to 70 days (median 58 days).The lymphoid manifestation and longer latency of FLT3 TKD in a murine BMT model together with the absent influence of FLT3 TKD mutations on clinical outcome of AML patients suggest differences in cell signaling between FLT3 TKD mutants and FLT3 ITD. The TKD mutants seem to require lymphoid cell context for full malignant transformation, whereas FLT3 ITDs transform myeloid cells.

Flow Cytometric FLT3 Expression in Acute Leukaemias Is of Diagnostic Value but Does Not Correlate with ITD / D835Y Mutation Status.

High level expression of FLT3 in acute myeloid (AML) and lymphoid (ALL) leukaemias has been demonstrated by PCR techniques. This suggests that FLT3 and its ligand may play a significant role in the proliferation of leukaemic blast cells. Mutations of FLT3 are the most common genetic lesion in AML, occurring in 20–30% of cases, and confer an adverse prognosis. Flow cytometry was used as an alternative method to investigate FLT3 expression in acute leukaemia. Surface expression of FLT3 was measured in 111 bone marrow or peripheral blood samples of patients with AML or ALL using a phycoerythrin-labelled monoclonal IgG1 antibody (Coulter-Immunotech). Exons 14 – 15 and 20 were amplified from DNA extracted from mononuclear cells of 38 of the 111 samples. Internal tandem duplication (ITD) and mutation of the D835 position of the activation loop of FLT3 were identified by SSCP and direct sequencing. 82% (47/57) of AML and 81% (34/42) of B-lineage ALL samples showed ≥ 20% (range, 20 – 97%) positivity for FLT3 using flow cytometry. The Mean Fluorescence Intensity (MFI) was similar between the two groups and no significant difference was found between adult (82%) and paediatric (80%) samples. However, FLT3 expression in all 9 T-ALL samples (pro, intermediate and late subtypes) was negative with markedly reduced MFIs. Of the 25 AML cases tested for ITD/D835Y, 5 (20%) were positive (4 ITDs, 1 D835Y). Surface FLT3 expression in these 5 cases (% and MFI) was the same as in those with wild-type FLT3. No mutations of FLT3 were found in the ALL cases tested (9 B-ALL, 4 T-ALL). Of three biphenotypic leukaemias, one case with myeloid/B lineage positivity was FLT3+ by flow cytometry, while another with myeloid/T lineage markers, was FLT3−. A diagnostic conundrum was a patient with AML morphologically (cytogenetics failed): CD33+, MPO−, CD13− CD117−; T-lineage markers CD2+, CD5+, CD7+, as well as TdT+. The T-lineage specific marker cytoplasmic CD3 was negative. Only FLT3 positivity allowed a definitive diagnosis of AML to be made. In conclusion, surface FLT3 expression was present in > 80% of AML and B-lineage ALL, but in 0% (0/9) of T-ALL cases. Detection of this antigen should be included in the diagnostic immunophenotyping of acute leukaemias and is important where T-ALL needs to be excluded. Flow cytometric FLT3 expression cannot differentiate cases with or without ITD or point mutations of FLT3.

Clinical Course and Significance of the Novel FLT3-Y842C Mutation in a Patient with AML Treated with PKC412 Monotherapy.

We recently identified a novel mutation (Y842C) within the tyrosine kinase domain of FLT3 in a patient treated with PKC410 monotherapy (ASH 2003, # 4681). Here, we present follow up studies including the clinical course of the patient and frequency analysis in 110 patients with AML. In addition, we characterized the novel mutation using overexpression of FLT3-Y842C in 32D cells.AML M2 was diagnosed in a 63 year old, male patient in 1993. After having experienced his second relapse upon standard therapy the patient was refractory to alemtuzumab treatment. Due to reduced performance status the patient was not eligible to standard chemotherapy and was enrolled into a phase II trial investigating PKC412. On conventional FLT3 mutation analysis the patient was considered to be FLT3 wild-type. Upon 8 and 29 days of treatment complete clearance of PB blast counts and BM blast infiltration was observed, respectively. Daily substitution of G-CSF resulted in transient recovery or the patients ANC′s. Since the patient showed an excellent clinical responsiveness, we reasoned whether the patient may have a yet unidentified FLT3 mutation. Sequence analysis revealed a novel point mutation in exon 21 of FLT3 (Y842C). Protein analysis of primary AML blasts showed constitutive FLT3 tyrosine-phosphorylation, ex vivo treatment with PKC412 caused significant inhibition of FLT3 and STAT5 activation. Further, in vivo analysis of FLT3 tyrosine-phosphorylation during the course of PKC412 treatment showed complete suppression of FLT3 activation within 8 days.Overexpression of FLT3-Y842C in 32D cells resulted in constitutive activation of FLT3 and STAT5 as well as in factor independent proliferation. Treatment with PKC412 caused inhibition of FLT3 tyrosine-phosphorylation, factor independent growth and apoptotic cell death. To further investigate the clinical significance of the novel Y842C mutation, the tyrosine kinase domain of FLT3 was investigated in 110 patients with AML using sequence analysis. Altogether, the novel mutation Y842C was identified in 2 patients, FLT-ITD in 22 patients and D835 in 7 patients, respectively.It is interesting to note that the recently described crystal structure of FLT3 reveals a critical role for Y842 in regulating the switch from the closed to the open (=active) conformation of the FLT3 activation loop. Since our data is consistent with the concept that the Y842C mutation results in constitutive activation of FLT3, it is tempting to speculate that the exchange of tyrosine for cysteine at position 842 disrupts the autoinhibited state of the FLT3 activation loop. Given that the novel mutation described here could only be identified by direct sequencing, it is likely that the number of mutations in this region of FLT3 is currently underestimated. Thus, extended sequence analysis of this mutational hotspot may be helpful in further defining the spectrum of TKI-sensitive FLT3 mutations in AML.

Variable sensitivity of FLT3 activation loop mutations to the small molecule tyrosine kinase inhibitor MLN518

AbstractFLT3 is constitutively activated by internal tandem duplications (ITDs) in the juxtamembrane domain or by activation loop mutations in acute myeloid leukemia (AML). We tested the sensitivity of 8 activation loop mutations to the small molecule FLT3 inhibitor, MLN518. Each FLT3 activation loop mutant, including D835Y, D835A, D835E, D835H, D835N, D835V, D835del, and I836del, transformed Ba/F3 cells to factor-independent proliferation and had constitutive tyrosine kinase activation, as assessed by FLT3 autophosphorylation and activation of downstream effectors, including STAT5 and ERK. MLN518 inhibited FLT3 autophosphorylation and phosphorylation of STAT5 and ERK in FLT3-ITD-transformed Ba/F3 cells with an IC50 (50% inhibition of cell viability) of approximately 500 nM. However, there was a broad spectrum of sensitivity among the 8 activation loop mutants, with IC50 ranging from approximately 500 nM to more than 10 μM for the inhibition of phosphorylation of FLT3, STAT5, and ERK. The relative sensitivity of the mutants to MLN518 in biochemical assays correlated with the cellular IC50 for cytokine-independent proliferation of FLT3-transformed Ba/F3 cells in the presence of MLN518. Thus, certain activation loop mutations in FLT3 simultaneously confer resistance to small molecule inhibitors. These findings have implications for the evaluation of responses in clinical trials with FLT3 inhibitors and provide a strategy to screen for compounds that can overcome resistance.