A novel irreversible FLT3 inhibitor, FF-10101, shows excellent efficacy against AML cells with FLT3 mutations

Key Points FF-10101 has selective and potent inhibitory activities against FLT3 by forming a covalent bond to the C695 residue. FF-10101 shows high efficacy against AML cells with FLT3 mutations including quizartinib-resistant activation loop mutations.

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FF-10101 Retains Potent Inhibitory Activities Against Resistant Mutations to FLT3 Inhibitors, Newly Identified in Random Mutagenesis Screens

Blood ◽

10.1182/blood-2019-124632 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2672-2672

Author(s):

Yuichi Ishikawa ◽

Koichi Saito ◽

Naomi Kawashima ◽

Michie Morimoto ◽

Hidetoshi Murao ◽

...

Keyword(s):

Inhibitory Activity ◽

Research Funding ◽

Random Mutagenesis ◽

Covalent Bond ◽

Kinase Domain ◽

Poor Prognostic Factor ◽

Flt3 Inhibitor ◽

Flt3 Inhibitors ◽

Inhibitory Activities ◽

Resistant Mutation

Background: FLT3-internal tandem duplication (FLT3-ITD) mutation is a poor prognostic factor for acute myeloid leukemia (AML). Several second-generation FLT3-targeted tyrosine kinase inhibitors with high selectivity and potency have been developed to date. Recently, gilteritinib was approved for the FLT3 mutation-positive relapsed or refractory AML patients. However, acquired mutations at the F691 residue in FLT3 kinase domain were identified in the patients who had disease progression after the treatment with gilteritinib, as with quizartinib treatment that caused resistant mutations at F691 and D835 residues. Therefore, it is very important to clarify the potencies of each FLT3 inhibitors against acquired FLT3 mutations for clinically selecting an appropriate FLT3 inhibitor according to the mutation type. In this study, we explored the resistant mutations against FLT3 inhibitors by random mutagenesis analysis and evaluated the cross-reactivity of FLT3 inhibitors against each resistant mutation. Methods: For random mutagenesis assay, 32D cells were infected with retroviruses encoding randomly mutagenized human FLT3-ITD. FLT3-ITD dependent 32D cells were established without an addition of IL-3, and then treated with FLT3 inhibitors, gilteritinib, FF-10101 and quizartinib, at concentrations of GI95 and 3 x GI95. Two weeks after treatment, full length FLT3-ITD sequences of viable clones were analyzed. The identified mutated FLT3-ITDs were introduced into 32D cells to confirm the resistance to FLT3 inhibitors. For cell growth assay, 32D transfectants were incubated with 5 FLT3 inhibitors (gilteritinib, FF-10101, quizartinib, crenolanib and midostaurin) for 3 days followed by determination of cell viability. Results: We identified the gilteritinib-resistant mutation (FLT3-ITD+D698N), quizartinib-resitant mutation (FLT3-ITD+N676T), and FF-10101-resistant mutation (FLT3-ITD+C695W) (Table). Inhibitory activity of gilteritinib against FLT3-ITD+D698N-expressing 32D cells (GI50, 27 nM) was decreased by 12-fold as compared with that against original FLT3-ITD-expressing 32D cells (GI50, 2.3nM). FF-10101 (GI50, 0.73 nM), quizartinib (GI50, 0.99 nM) and crenolanib (GI50, 19 nM) retained potency against FLT3-ITD+D698N, while midostaurin (GI50, 47 nM) did not have a potency. In FLT3-ITD+N676T-expressing 32D cells, inhibitory activities of quizartinib and midostaurin were decreased by 11 and 15-fold (GI50, 6.6 nM and 83 nM, respectively), while FF-10101 (GI50, 0.73 nM), gilteritinib (GI50, 6.6 nM) and crenolanib (GI50, 19 nM) retained potency. In FF-10101-resistant mutation (FLT3-ITD+C695W)-expressing 32D cells, the other inhibitors retained growth inhibitory activity. Conclusions: Resistant mutations to gilteritinib and quizartinib were newly identified in FLT3 kinase domain by random mutagenesis analysis. FF-10101 retained potent inhibitory activities against FLT3-ITD+N676T conferring resistance to quizartinib and midostaurin, and FLT3-ITD+D698N resistant to gilteritinib and midostaurin, although FF-10101 was vulnerable to FLT3-ITD+C695W substituted for C695 residue which forms covalent bond with FF-10101. These results indicated that FF-10101 was a promising agent for the treatment of patients with AML with FLT3 inhibitor-resistant mutations newly identified in this study. Disclosures Ishikawa: Bristol-Myers Squibb: Honoraria; Abbvie GK.: Honoraria; Celgene Co., Ltd.: Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria. Saito:FUJIFILM Corporation: Employment. Morimoto:FUJIFILM Corporation: Employment. Murao:FUJIFILM Corporation: Employment. Terada:FUJIFILM Corporation: Employment. Yamaura:FUJIFILM Corporation: Employment. Hagiwara:FUJIFILM Coporation: Employment. Kiyoi:Daiichi Sankyo Co., Ltd: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; FUJIFILM Corporation: Research Funding; Pfizer Japan Inc.: Honoraria; Bristol-Myers Squibb: Research Funding; Perseus Proteomics Inc.: Research Funding; Otsuka Pharmaceutical Co.,Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Astellas Pharma Inc.: Honoraria, Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding.

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Discovery of a Potent FLT3 Inhibitor (LT-850-166) with the Capacity of Overcoming a Variety of FLT3 Mutations

Journal of Medicinal Chemistry ◽

10.1021/acs.jmedchem.1c01196 ◽

2021 ◽

Vol 64 (19) ◽

pp. 14664-14701

Author(s):

Zhijie Wang ◽

Jiongheng Cai ◽

Jiwei Ren ◽

Yun Chen ◽

Yingli Wu ◽

...

Keyword(s):

Flt3 Inhibitor ◽

Flt3 Mutations

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No Evidence for Constitutively Activated FLT3 in Juvenile Myelo-Monocytic Leukemia.

Blood ◽

10.1182/blood.v106.11.4915.4915 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4915-4915

Author(s):

Andrica C.H. de Vries ◽

Ronald W. Stam ◽

Pauline Schneider ◽

Charlotte M. Niemeyer ◽

Elisabeth R. van Wering ◽

...

Keyword(s):

Tyrosine Kinase ◽

Kinase Inhibitors ◽

Kinase Domain ◽

Leukemic Cells ◽

Tyrosine Kinase Domain ◽

Mrna Levels ◽

Monocytic Leukemia ◽

Real Time Quantitative Pcr ◽

Flt3 Inhibitor ◽

Flt3 Mutations

Abstract Activating FLT3 mutations have been identified as prognostic factors in multiple myeloid malignancies. Recent studies have demonstrated that ligand-independent activation of FLT3 can also result from overexpression of wild-type FLT3. In addition, ligand-dependent activation has been observed in leukemic cells co-expressing FLT3 ligand (FLT3L), resulting in autocrine FLT3 signaling which is independent of FLT3 mutations. In Juvenile Myelo-Monocytic Leukemia (JMML), FLT3 internal tandem duplications (FLT3/ITDs) mutations affecting the tyrosine kinase domain (TKD) are rare. However, no data are yet available on the frequency of expression levels of FLT3 and FLT3L in JMML. If activated FLT3 occurs in JMML, one could imagine that these patients might benefit from treatment with small molecule FLT3 inhibitors, especially as to date the curative treatment of JMML is limited to allogeneous bone marrow transplantation. In 36 JMML patients FLT3 and FLT3L mRNA levels were assessed using real-time quantitative PCR (Taqman). Furthermore these samples were screened for the presence of activating FLT3/ITDs and FLT3/TKD mutations. MTT assays were performed to assess the response of JMML cells to the known FLT3 inhibitor PKC412 (Novartis). FLT3 appeared to be expressed only at basal levels and FLT3L expression was very low. In none of the 36 JMML samples FLT3/ITDs or TDK mutations were found, consistent with the observation that PKC412 was not cytotoxic in JMML samples (n=12), in contrast to leukemic cells of children with ALL which carried an activated FLT3. These data suggest that constitutively activated FLT3 does not occur in JMML. Therefore targeting FLT3 by tyrosine kinase inhibitors like PKC412 is unlikely to be effective in JMML.

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FLT3 Inhibitor Therapy for Patients with Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML): Impact On Survival According to FLT3 Status.

Blood ◽

10.1182/blood.v114.22.1026.1026 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1026-1026 ◽

Cited By ~ 4

Author(s):

Naveen Pemmaraju ◽

Hagop M. Kantarjian ◽

Farhad Ravandi ◽

Guillermo Garcia-Manero ◽

Borthakur Gautam ◽

...

Keyword(s):

Research Funding ◽

Initial Therapy ◽

Inhibitor Therapy ◽

Clinical Activity ◽

Term Outcome ◽

Long Term Outcome ◽

Flt3 Inhibitor ◽

Flt3 Mutations ◽

Flt3 Inhibitors ◽

Impact On Survival

Abstract Abstract 1026 Poster Board I-48 Background: FLT3 mutations (ITD or D835 point mutation) are frequently observed in patients (pts) with AML and they confer an adverse prognosis, particularly among pts with diploid karyotype. This has made FLT3 an important target for drug development in AML. Several FLT3 inhibitors are currently being developed (eg, sorafenib, PKC-412, AC-220, CEP-701, IMC EB10, sunitinib). Results from early trials with many of these agents suggest they have clinical activity in the treatment of MDS and AML, although most responses are represented by a marked decrease in blast counts, with few complete remissions(CR). Whether these responses ultimately improve long-term outcome of pts, and whether they may be particularly beneficial for pts with FLT3 mutations compared to those with FLT3 wild-type (WT) is being investigated. Aims: To ascertain outcomes of patients given treatment with FLT3 inhibitors, alone or in combination with other therapies, and to compare outcomes in those patients with FLT3 mutations (ITD or D835) versus those with FLT3-WT. Methods: We reviewed the records of patients with MDS and AML who were enrolled on clinical trials with FLT3 inhibitors at our institution. We compared patient outcomes in those who received a FLT3 inhibitor in both FLT3 positive and FLT3 negative patients. Pts were classified as receiving FLT3 inhibitors 1) as part of their initial therapy, 2) as first salvage, or 3) as second salvage or beyond. Results: A total of 128 pts were included: 51 (40%) with FLT3-WT, 56 (44%) with FLT3-ITD, 11 (9%) with D835, and 10 (8%) had both FLT3-ITD and D835. The overall median age was 62 yrs (range, 17-88); by FLT3 status, median age was 70 yrs (35-88) for FLT3-WT pts and 58 yrs (17-81) for FLT3 mutated. Sixty-four pts (50%) were female. Twenty-three (18%) pts received FLT3 inhibitors as part of their induction therapy (18 FLT3-WT, 5 FLT3 mutated; median age 74 yrs); 22 (17%) as first salvage (4 FLT3-WT, 18 mutated; median age 67 yrs); and 83 (65%) as second or later salvage (29 FLT3-WT, 54 mutated; median age 59 yrs). Nine pts overall, all of whom were FLT3 mutated, achieved either CR (n=6) or CRp (n=3) with FLT3 inhibitors. Eight of the nine CR/CRp have been lost with a median CR duration of 8 months (mo) (3-12+). After a median follow-up of 3.5 mo, 115 (90%) pts have died, including 47 (92%) FLT3-WT, and 68 (88%) FLT3 mutated. The median survival is 3.8 mo for the total population. Survival by mutation status and timing of FLT3 inhibitor therapy is presented in table 1. Conclusions: Despite the inferior outcome expected for pts with FLT3 mutations, and the low rate of CR/CRp with FLT3 inhibitors, these results suggest that therapy with FLT3 inhibitors has the potential to improve the outcome of pts with FLT3 mutations. Additional studies incorporating these agents in AML therapy are warranted. Disclosures: Off Label Use: Sorafenib has not been FDA approved for use in MDS and AML. Kantarjian:Novartis: Research Funding. Cortes:Ambit: Research Funding; Novartis: Research Funding; ImClone: Research Funding.

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Momelotinib is a highly potent inhibitor of FLT3-mutant AML

10.1101/2021.03.18.436047 ◽

2021 ◽

Author(s):

Mohammad Azhar ◽

Zachary Kincaid ◽

Meenu Kesarwani ◽

Tahir Latif ◽

Daniel Starczynowski ◽

...

Keyword(s):

Growth Factor ◽

Kinase Inhibitor ◽

Initial Response ◽

Leukemic Cells ◽

Activation Loop ◽

Growth Factor Signaling ◽

Durable Response ◽

Flt3 Inhibitor ◽

Flt3 Inhibitors

Kinase activating mutation in FLT3 is the most frequent genetic lesion associated with poor prognosis in acute myeloid leukemia (AML). Therapeutic response to FLT3 tyrosine kinase inhibitor (TKI) therapy is dismal, and many patients relapse even after allogeneic stem cell transplantation. Despite the introduction of more selective FLT3 inhibitors, remissions are short-lived, and patients show progressive disease after an initial response. Acquisition of resistance-conferring genetic mutations and growth factor signaling are two principal mechanisms that drive relapse. FLT3 inhibitors targeting both escape mechanisms could lead to more profound and lasting clinical responses. Here we show that the JAK2 inhibitor, momelotinib, is an equipotent type-1 FLT3 inhibitor. Momelotinib showed potent inhibitory activity on both mouse and human cells expressing FLT3-ITD, including clinically relevant resistant mutations within the activation loop at residues D835, D839, and Y842. Additionally, momelotinib efficiently suppressed the resistance mediated by FLT3 ligand (FL), and hematopoietic cytokine activated JAK2 signaling. Interestingly, unlike gilteritinib, momelotinib inhibits the expression of MYC in leukemic cells. Consequently, concomitant inhibition of FLT3 and downregulation of MYC by momelotinib treatment showed better efficacy in suppressing the leukemia in a preclinical murine model of AML. Altogether, these data provide evidence that momelotinib is an effective type-1 dual JAK2/FLT3 inhibitor and may offer an alternative to gilteritinib. Its ability to impede the resistance conferred by growth factor signaling and activation loop mutants suggests that momelotinib treatment could provide a deeper and durable response; thus, warrants its clinical evaluation.

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Selective FLT3 Inhibitor FI-700 Neutralizes Mcl-1 and Enhances p53-Mediated Apoptosis in Acute Myeloid Leukemia (AML) Cells with Activating Mutations of FLT3 Via Mcl-1/Noxa Axis.

Blood ◽

10.1182/blood.v114.22.2597.2597 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2597-2597

Author(s):

Kensuke Kojima ◽

Marina Konopleva ◽

Twee Tsao ◽

Michael Andreeff ◽

Hiroshi Ishida ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Activating Mutations ◽

Phosphatidylserine Externalization ◽

Flt3 Inhibitor ◽

Flt3 Mutations ◽

Flt3 Inhibitors ◽

Induced Apoptosis ◽

Apoptotic Effects ◽

Acute Myeloid

Abstract Abstract 2597 Poster Board II-573 Introduction: Activating mutations of the Fms-like tyrosine kinase-3 gene (FLT3) occur in approximately 30–40% of acute myeloid leukemia (AML) patients. FLT3 mutations confer numerous oncogenic properties, including dysregulated proliferation, resistance to apoptosis and a block in differentiation. FLT3 mutations result in abnormal activation of the downstream pathways, including signal transducer and activator of transcription 5 (STAT5), mitogen-activated protein kinase kinase (Mek)/extracellular signal–regulated kinase (Erk) and phosphatidylinositol-3 kinase (PI3K)/Akt. Activation of these downstream effectors has been thought to allow leukemia cells to evade apoptosis. Targeting of FLT3 mutations is a promising approach to overcome the dismal prognosis of acute myeloid leukemia (AML) with activating FLT3 mutations. Current trials are combining FLT3 inhibitors with p53-activating conventional chemotherapy. The mechanisms of cytotoxicity of FLT3 inhibitors are poorly understood. We investigated the interaction of FLT3 and p53 pathways after their simultaneous blockade using the selective FLT3 inhibitor FI-700 and the MDM2 inhibitor Nutlin-3 in AML. Results: FI-700 induced G1-phase cell cycle arrest and apoptosis as evidenced by increased sub-G1 DNA content and phosphatidylserine externalization in FLT3/ITD MOLM-13 (FLT3-ITD, wild-type (wt)-p53) and MV4-11NR (FLT3-ITD, mutated-p53) AML cells. FI-700 did not affect cell cycle distribution patterns nor did it induce apoptosis in FLT3/WT OCI-AML-3 (FLT3/WT, wt-p53) and HL-60 (FLT3/WT, del (del)-p53). Wt-p53 MOLM-13 and OCI-AML-3 cells were susceptible to Nutlin-induced apoptosis. FI-700 augmented Nutlin-induced Bax activation, mitochondrial membrane potential (MMP) loss, caspase-3 activation and phosphatidylserine externalization in MOLM-13 cells. FI-700 rapidly reduced Mcl-1 levels in FLT3/ITD cells, mainly by enhancing proteasomal Mcl-1 degradation. Levels of other Bcl-2 family proteins examined did not change significantly. Mcl-1 levels were only modestly reduced upon Nutlin treatment. The FI-700/Nutlin-3 combination profoundly reduced Mcl-1 levels. Immunoprecipitation/ immunoblotting results suggested that the drug combination results in a profound decrease in Mcl-1-bound Bim. FI-700 enhanced doxorubicin-induced apoptosis in FLT3/ITD MOLM-13 and MV4-11NR cells, suggesting that FI-700 can enhance both the p53-dependent and the p53-independent apoptotic effects of doxorubicin. Finally, cooperative apoptotic effects of FI-700/Nutlin-3 were seen in primary AML cells with FLT3/ITD. Conclusion: FLT3 inhibition by FI-700 immediately reduces anti-apoptotic Mcl-1 levels and enhances Nutlin-induced p53-mediated mitochondrial apoptosis in FLT3/ITD-expressing AML cells via the Mcl-1/Noxa axis. FLT3 inhibition, in combination with p53-inducing agents, might represent a potential therapeutic approach in AML with FLT3/ITD. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v118.21.930.930 ◽

2011 ◽

Vol 118 (21) ◽

pp. 930-930 ◽

Cited By ~ 1

Author(s):

Catherine C. Smith ◽

Lauren E. Damon ◽

Xiaotian Zhu ◽

Sara Salerno ◽

Neil Shah

Keyword(s):

Myeloid Leukemia ◽

Research Funding ◽

Cross Resistance ◽

Activation Loop ◽

In Vitro Activity ◽

Flt3 Inhibitor ◽

T315i Mutation ◽

High Degree ◽

Acute Myeloid

Abstract 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.

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

Blood ◽

10.1182/blood.v122.21.3938.3938 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3938-3938 ◽

Cited By ~ 5

Author(s):

Catherine C Smith ◽

Kimberly Lin ◽

Elisabeth Lasater ◽

Whitney Stewart ◽

Lauren E Damon ◽

...

Keyword(s):

Myeloid Leukemia ◽

Research Funding ◽

Kinase Domain ◽

Activation Loop ◽

In Vitro Mutagenesis ◽

Mutagenesis Screen ◽

Flt3 Inhibitor ◽

Clinical Resistance ◽

Acute Myeloid

Abstract Background Activating 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. Results PLX3397 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. Conclusions PLX3397 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.

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Identifying and characterizing a novel activating mutation of the FLT3 tyrosine kinase in AML

Blood ◽

10.1182/blood-2004-02-0712 ◽

2004 ◽

Vol 104 (6) ◽

pp. 1855-1858 ◽

Cited By ~ 55

Author(s):

Jingrui Jiang ◽

J. Guillermo Paez ◽

Jeffrey C. Lee ◽

Ronghai Bo ◽

Richard M. Stone ◽

...

Keyword(s):

Tyrosine Kinase ◽

Lymphoblastic Leukemia ◽

Point Mutations ◽

Sample Introduction ◽

Activation Loop ◽

Kinase Gene ◽

Flt3 Inhibitor ◽

Activating Mutation ◽

Tyrosine Kinase Gene ◽

Gene Exon

Abstract The FLT3 receptor is activated by juxtamembrane insertion mutations and by activation loop point mutations in patients with acute myeloid leukemia (AML). In a systematic tyrosine kinase gene exon resequencing study, 21 of 24 FLT3 exons were sequenced in samples from 53 patients with AML, 9 patients with acute lymphoblastic leukemia (ALL), and 3 patients with myelodysplasia samples. Three patients had novel point mutations at residue N841 that resulted in a change to isoleucine in 2 samples and to tyrosine in 1 sample. Introduction of FLT3-N841I cDNA into Ba/F3 cells led to interleukin-3 (IL-3)–independent proliferation, receptor phosphorylation, and constitutive activation of signal transducer and activator of transcription 5 (STAT5) and extracellular regulatory kinase (ERK), suggesting that the N841I mutation confers constitutive activity to the receptor. An FLT3 inhibitor (PKC412) inhibited the growth of Ba/F3-FLT3N841I cells (IC50 10 nM), but not of wild-type Ba/F3 cells cultured with IL-3. PKC412 also reduced tyrosine phosphorylation of the mutant receptor and inhibited STAT5 phosphorylation. Examination of the FLT3 autoinhibited structure showed that N841 is the key residue in a hydrogen-bonding network that likely stabilizes the activation loop. These results suggest that mutations at N841 represent a significant new activating mutation in patients with AML and that patients with such mutations may respond to small-molecule FLT3 inhibitors such as PKC412.

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FLT3-ITD and TLR9 use Bruton tyrosine kinase to activate distinct transcriptional programs mediating AML cell survival and proliferation

Blood ◽

10.1182/blood-2014-06-585216 ◽

2015 ◽

Vol 125 (12) ◽

pp. 1936-1947 ◽

Cited By ~ 33

Author(s):

Thomas Oellerich ◽

Sebastian Mohr ◽

Jasmin Corso ◽

Julia Beck ◽

Carmen Döbele ◽

...

Keyword(s):

Tyrosine Kinase ◽

Cell Survival ◽

Treatment Strategies ◽

Specific Treatment ◽

Oncogenic Signaling ◽

Flt3 Inhibitor ◽

Key Points ◽

Bruton Tyrosine Kinase ◽

Cell Survival And Proliferation

Key Points Two novel transducer modules consisting of BTK in combination with either FLT3-ITD or TLR9 induce distinct oncogenic signaling programs. This study suggests subtype-specific treatment strategies, including BTK/FLT3 inhibitor combinations, and shows how TLR9 affects AML biology.

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