FMS-related Tyrosine Kinase 3 Inhibitor Research Articles

STI-8591, a Novel Tyrosine Kinase Inhibitor, Shows Excellent Antileukemic Activity in FLT3-Mutated (mut) Acute Myeloid Leukemia (AML)

FMS-related tyrosine kinase 3 ( FLT3) mutations, including internal tandem duplications ( FLT3-ITD) and tyrosine kinase domain ( FLT3-TKD), are found in approximately 30% of AML patients. FLT3 has emerged as a promising molecular target in therapy of AML. Herein, we report on the activity of STI-8591, a novel kinase inhibitor developed by Hangzhou ACEA Pharmaceutical Research Co, in AML models carrying FLT3 mut, including those associated with TKI resistance [e.g., FLT3-ITD(F691L)]. We first determined the in vitro antileukemic effects of STI-8591 in FLT3mut human AML cell lines (MV4-11, MOLM-13), a murine cell line Ba/F3 transduced with FLT3-TKD(D835Y)[Ba/F3 FLT3-TKD(D835Y)] or FLT3-ITD-TKD(D835Y) [Ba/F3 FLT3-ITD-TKD(D835Y)] or FLT3-ITD/F691L [Ba/F3 FLT3-ITD(F691L), which has been associated with drug-resistant, and primary blasts from FLT3mut AML patients. STI-8591 effectively inhibited cell proliferation and induced apoptosis in FLT3-ITD/TKDmut cell lines (IC 50: 0.33 to 9.19nM at 24 hours). Of note, STI-8591 demonstrates 4-7-fold higher antileukemic activity on otherwise resistant Ba/F3 FLT3-ITD(F691L) cells than the currently available FLT3 inhibitors, Gilteritinib and Quizartinib. Similar antileukemic activity of STI-8591 was observed on primary FLT3mut AML blasts (median IC 50: 13.67nM, n=5). The antileukemic mechanism of STI-8591 is associated with inhibition of the phosphorylation of the mutated FLT3 protein and its downstream targets (i.e., STAT5, AKT, and ERK) as shown by western blot analysis. To test the in vivo antileukemic activity of STI-8591, we utilized 4 FLT3mut AML murine and human models, obtained by transplanting: 1. BM mononuclear cells (MNCs) from CD45.2 MLLPTD/WTFLT3ITDITD AML mice congenic CD45.1 B6 mice; 2. murine cell lines [i.e. Ba/F3 FLT3-TKD(D835Y) , Ba/F3 FLT3-ITD-TKD(D835Y), or Ba/F3 FLT3-ITD(F691L), 3. Luciferase-expressing FLT3mut MOLM13 cells (MOLM13 Luci) into NCG mice; 4. primary blasts from FLT3 mut AML patients into NSGS mice. We treated these mice for 3-4 weeks [3 to 30mg/kg/day(d), oral gavage]. For the MLLPTD/WTFLT3ITDITD model (n=10 mice/group), compared with Vehicle(V)-treated mice [median survival: 44 d], we observed significantly lower engraftment and longer overall survival (OS) in mice treated with STI-8591 10mg/kg (52 d, p<0.0001) or 30mg/kg (57 d, p<0.0001) or Gilteritinib 10mg/kg (49 d, p=0.0029). At 10mg/kg dosage, STI-8591-treated mice lived longer than Gilteritinib-treated mice (p=0.0195). Treatment of STI-8591 also demonstrated significant benefits to mice transplanted with FLT3mut Ba/F3 cells. For the TKI-resistant Ba/F3 FLT3-ITD(F691L) model (n=10/group), STI-8591 (median:19 and 37 d for 10 and 30 mg/kg) and Gilteritinib (17 and 24 d for 10 and 30 mg/kg) -treated mice had longer OS than V-treated (12 d) mice. At equivalent dosage (10 and 30mg/kg), STI-8591-treated mice lived longer than Gilteritinib-treated mice (p=0.0013 and p<0.0001). For the MOLM13 Luci+ model (n=5/group), mice receiving STI-8591 or Gilteritinib treatment demonstrated a significant reduction in disease burden as observed by bioluminescence imaging (BLI), and prolonged OS. Compared with V-treated mice (median: 23 d), we observed longer OS in Gilteritinib (10mg/kg, 33 d, p=0.0023) and STI-8591 (3mg/kg, 27 d, p=0.0179; 10mg/kg, 34 d, p=0.0023; and 30mg/kg, 35 d, p=0.0023) -treated mice. At 10 mg/kg dosage, STI-8591- and Gilteritinib- treated mice showed no significant difference in OS (p=0.2411). Similar experiments are ongoing in FLT3 mut primary AML patient-derived xenografts (PDXs) and will be reported at the ASH meeting. To evaluate antileukemic activity of STI-8591 in mice bearing disease in central nervous system (CNS), we implanted 5×10 5 /3 µL MOLM13 cells intracranially into SCID mice to construct the CNS leukemia model (n=9/group). Compared with V-treated mice (median: 11 d), we observed a longer OS in mice treated with Gilteritinib (10 mg/kg, 18 d, p=0.0004) or STI-8591 (10 mg/kg, 23 d, p<0.0001; and 30 mg/kg, 26 d, p<0.0001). At 10 mg/kg dosage, STI-8591-treated mice lived longer than Gilteritinib-treated mice (p=0.005). In conclusion, STI-8591, a novel TKI exhibited a potent antileukemic activity in various FLT3mut AML models, including those carrying FLT3 mut associated with drug resistance. These results supported further clinical development of STI-8591 for treatment of AML.

Characteristics of FLT3-ITD and Efficacy of Sorafenib Treatment in Pediatric AML: A Report from AML-BFM Study Group

Introduction Internal tandem duplication (ITD) of FMS-related tyrosine kinase 3 ( FLT3) gene is one of the most common somatic mutations in acute myeloid leukemia (AML). We and others have previously shown that the prognostic significance of FLT3-ITD in children and young adults relies on its characteristics e.g., co-occurrence with WT1 mutationsand NUP98::NSD1 fusions (Niktoreh et al. 2019) or higher allelic ratio (AR) (Tarlock et al. 2018) lead to worse survival outcomes. Also, recent studies suggested that treatment with FLT3 inhibitors, including sorafenib, might be effective in children with FLT3-ITD positive AML. In this report, we summarize the characteristics of FLT3-ITD and outcomes of treatment with sorafenib in patients recruited in AML-BFM consecutive trials. Methods Patients aged 0-21 years old, diagnosed with FLT3-ITD positive de novo AML between Jan. 2004 and Dec. 2021 in Germany and recruited in AML-BFM 2004 and 2012 studies or the 2012 and 2017 registries were retrospectively analyzed. All patients with FLT3-ITD with high- or intermediate risk stratification (HR or IR) were recommended to receive 100 mg/m² sorafenib taken orally twice daily on days 7 to 21 of their induction and consolidation chemotherapy blocks, the final decision however was made by the treating physicians. Patient samples were examined in AML-BFM reference laboratory in Essen, Germany using standard methods. Patients with Down syndrome and AML-M3 were excluded from the current analysis and survival rates were calculated using Kaplan-Meier method and Log-Rank test (significance threshold p<0.05). Results In total, 137 patients (51% male) with median age of 7.78 (1.3-19, IQR=7.27) years at diagnosis were included. Most of the patients were initially stratified in IR group (70%, N=95) followed by HR (26%, N=35). FABs M2, M4 and M1 consisted 74% of all morphologic subtypes with 26%, 25% and 23% of patients, respectively and with a median of 64,000 (500 -700,400, IQR=53,178) x10 9/L, the majority of patients showed a leukocytosis at diagnosis. The median ITD length in the 107 patients with available data was 51 (9-111, IQR=39) bps and 54% of the 91 patients with available data had an AR above 0.5. WT1 mutations and NUP98::NSD1 were detected in 25% (N=34) and 7% (N=10) of patients, respectively and 4% (N=6), harbored both mutations simultaneously. The three-year probability of overall and event-free survival (3y-OS and 3y-EFS, respectively) in all FLT3-ITD patients were 68.8% (±4.3) and 47.6% (±4.5), respectively and although harboring ITDs longer than the median length had a tendency to worsen the 3y-OS (54.4% in ITD>51 bps vs. 73.3% in ITDs<51 bps, p=0.071), it did not influence the 3-y EFS (41.7% vs. 54.2%, p=0.204). Similarly, an AR higher than 0.5 did not affect the survival outcomes (3y-OS: 67.4% vs. 60.4%, p=0.744, 3y-EFS: 46.8% vs. 47%, p=0.618). In total, 36 patients (27%) were treated with sorafenib of which, 23 (63%) received a consecutive HSCT. We observed no survival advantage between sorafenib-treated and sorafenib-naive groups of patients (3y-OS: 73.7% vs. 67.3%, p=0.408, 3y-EFS: 48.3% vs. 47.5%, p=0.929). Patients with longer ITDs did not benefit from sorafenib treatment (3y-OS: 68% vs. 54.3%, p=0.279, 3y-EFS: 48.9% vs. 38.4 %, p=0.649) but higher AR, showed a tendency of improved survival outcomes when sorafenib treatment was implicated (3y-OS: 80.8 % vs. 61.9 %, p=0.159, 3y-EFS: 67.3 % vs. 39.9 %, p=0.131). Although the number of patients were limited, we observed a possible survival advantage in double or triple FLT3-ITD, NUP98::NSD1 and WT1 mutated patients who received Sorafenib: from 28 patients with WT1 mutations only, four patients received sorafenib three of which, are currently alive and in CR but contrarily, from the 24 untreated patients, 14 (58%) are deceased (78% due to relapse). Additionally, from 10 patients with NUP98::NSD1 (w/o WT1 mutations), two who were treated with sorafenib are alive and in CR, whereas from the remaining 8 patients, 6 (75%) experienced relapse and unfortunately did not survive. Conclusions Our preliminary analysis implicates that although addition of sorafenib to the intensive chemotherapy of children and adolescence with FLT3-ITD positive AML, did not improve the survival outcomes in allpatients; specific genetic subgroups might benefit from this treatment. These results must be validated in bigger cohorts of patients with longer follow-ups.

Clinical Outcomes in Patients with FLT3-ITD-Mutated Relapsed/Refractory Acute Myelogenous Leukemia Undergoing Hematopoietic Stem Cell Transplantation after Quizartinib or Salvage Chemotherapy in the QuANTUM-R Trial

Despite the substantial clinical activity of fms-related tyrosine kinase 3 (FLT3) inhibitors in relapsed or refractory (R/R) FLT3-ITD‒positive acute myelogenous leukemia (AML), durable remissions and prolonged survival in this population require allogeneic hematopoietic stem cell transplantation (allo-HSCT). Quizartinib, a once-daily oral, highly potent, and selective FLT3 inhibitor, significantly prolonged overall survival (OS) and improved clinical benefit compared with salvage chemotherapy (median OS, 6.2 months versus 4.7 months; hazard ratio [HR], .76; 95% confidence interval [CI], .58 to .98; P=.018; composite complete remission [CRc] rate, 48% versus 27%; median duration of CRc, 2.8 months versus 1.2 months; mortality rate, .8% versus 14% by day 30, 7% versus 24% by day 60) in patients with R/R FLT3-ITD AML in the phase 3 QuANTUM-R trial. In this post hoc analysis, we described the characteristics of and clinical outcomes in patients who underwent on-study HSCT in QuANTUM-R at the investigator's discretion and institutional practices. Of 367 randomized patients, 78 (32%) in the quizartinib arm and 14 (11%) in the salvage chemotherapy arm underwent on-study allo-HSCT without any intervening therapy for AML after quizartinib or study-specified salvage chemotherapy. Pooled data of patients from both treatment arms showed a longer median overall survival (OS) in transplant recipients versus those treated without allo-HSCT (12.2 months versus 4.4 months; HR, .315; 95% CI, .233 to .427). Pooled data also showed a longer median OS in patients with a last recorded response of CRc before allo-HSCT versus patients without a CRc (20.1 months versus 8.8 months; HR, .506; 95% CI, .296 to .864). By treatment arm, the median OS was 25.1 months with quizartinib and 20.1 months with salvage chemotherapy in patients with a last recorded response of CRc before allo-HSCT. Forty-eight patients in the quizartinib arm continued quizartinib treatment after allo-HSCT. In the 31 patients with a last recorded response of CRc before allo-HSCT who continued quizartinib after allo-HSCT, the median OS was 27.1 months. Continuation of quizartinib after allo-HSCT was tolerable, and no new safety signals were identified. These results suggest that post-transplantation survival following salvage chemotherapy and quizartinib treatment are similar. However, quizartinib response occurs more frequently than with salvage chemotherapy, potentially allowing more patients to undergo transplantation and achieve durable clinical benefit. In addition, post-transplant quizartinib was found to be tolerable and may be associated with prolonged survival in some patients, highlighting its potential value in the management of patients with FLT3-ITD R/R AML.

Multiparametric Flow-Cytometry Is a Reliable Tool for Measurable Residual Disease Assessment and Risk-Stratification of FLT3-Mutated AML Patients

Background: Mutations of the gene encoding Fms Related Tyrosine Kinase 3 (FLT3), at the juxta-membrane level (ITD), represent the most common lesions found in Acute Myeloid Leukemia (AML), identifying a subgroup of patients (pts) with unfavorable prognosis. FLT3-ITD mutations are considered an unreliable tool for measurable residual disease (MRD) monitoring, due to their intraclonal heterogeneity and instability during the course of disease. Instead, multiparametric flow cytometry (MFC) may represent an alternative to monitor MRD in this molecular subset. In fact, through the recognition and monitoring of leukemia associated immunophenotypes, MFC is applicable to > 90% of AML patients with a sensitivity of 10-4. Aims: The aim of our study was to investigate the reliability of MFC in MRD assessment of 72 FLT3-ITD positive pts whose treatment allocation was prospectively decided according to the genetic/cytogenetic profile at diagnosis and post consolidation MRD. FLT3-ITD pts were to receive, after induction and consolidation, allogeneic stem cell transplant (ASCT), whatever the source of stem cells. In this subgroup analysis, we investigated if FLT3-ITD mutated pts have a different propensity to achieve high quality (e.g. MRD negative) complete remission as compared to FLT3 wildtype ones. Furthermore, we seek for a correlation between different levels of MRD and overall (OS) and disease-free survival (DFS). Methods: We included in the analysis 72 pts with de novo AML carrying FLT3-ITD mutations whose MRD assessment at the post-consolidation timepoint was available. Pts were defined as MRD-negative, when obtaining a residual leukemic cells count below the threshold of 3.5x10-4 (0.035%). MRD positive pts (with MRD ≥ 3.5x10-4 RLC) were stratified into 3 classes according to the levels of MRD (0.035%-0.1%; >0.1%-1%; >1%). We compared the MRD status and clinical outcome with a matched group of FLT3 wildtype AML (n = 203) treated in the same protocol. Results: Overall median age was 49 (range 18-60.9). The 2 cohorts were balanced in terms of age and sex distribution. In the FLT3-ITD group, 80/126 (64%) cases carried a concomitant NPM1 mutation vs 107/374 (28.6%) of FLT3 wildtype ones (p <0.001). Furthermore, FLT3 mutated pts had a median WBC count of 35x109/L vs 9.5x109/L of those FLT3 wildtype (p < 0.001). MRD determination after consolidation cycle was available in 72/126 FLT3-ITD pts (57%) and in 203/374 FLT3 wildtypeones (54.3%), respectively. After having received induction and consolidation course, 47/72 FLT3-ITD pts (65,2%) were submitted to allogenic stem cells transplantation (ASCT). At the post-consolidation time-point, MRD negativity rate was significantly lower in FTL3-ITD pts (27/72, 37.5%) as compared to those FLT3 wildtype (94/203, 46.3%). Furthermore, 38/72 (52.8%) and 10/72 (13.9%) FLT3-ITD pts had a level of MRD > 0.1% and > 1%, respectively as compared to 65/203 (33.0%) and 15/203 (7.4%) of FLT3 wildtypeones, respectively (p=0.017). When considering the different MRD stratification levels of FLT3-ITD pts, OS probability at 24 months was 57.2% (27 pts), 71.4% (7 pts), 53.6% (28 pts) and 20% (10 pts), for the MRD categories <0.035%, 0.035%-0.1%, >0.1%-1%, >1%, respectively (p=0.028). DFS probability at 24 months was 53.8% (27 pts), 71.4% (7 pts), 34.9% (27 pts) and 20% (10 pts), for the MRD categories <0.035%, 0.035%-0.1%, >0.1%-1%, >1%, respectively (p=0.038). Summary/Conclusion: We demonstrated that MRD determination by MFC is a reliable tool to assess remission quality and prognosis in FLT-ITD positive patients. This subpopulation shows a lower propensity to obtain a MRD negative CR, with the majority of pts maintaining an amount of MRD > 0.1% after standard treatment. Even though most of these pts were addressed to ASCT, post-consolidation MRD maintained its negative impact on OS and DFS, particularly for those pts with MRD >1%. In the attempt to improve the quality of response, prevent leukemia recurrence and pursue a durable remission, delivery of FLT3 inhibitors as a maintenance after transplant may represent a promising option. Disclosures Venditti: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy; Astellas: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees. Buccisano:Janssen: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Midostaurin In Acute Myeloid Leukemia: An Evidence-Based Review And Patient Selection.

Fms-related-tyrosine kinase 3 (FLT3) mutations occur in approximately a third of acute myeloid leukemia (AML) patients and confer an adverse prognosis. Numerous studies have evaluated FLT3 targeting as single agent and in combination approaches in frontline and relapsed AML. At this time, midostaurin, a multikinase inhibitor, is the only FLT3-inhibitor that is US FDA approved to be used in combination with induction therapy in the frontline FLT3-mutated AML setting based on improved overall survival noted in the RATIFY Phase III trial. The utility of midostaurin in maintenance post stem cell transplantation has shown promising results and further studies are still ongoing. In this review, we discuss the studies that led to the inception of midostaurin as a targeted kinase inhibitor, its evaluation in AML, the early clinical trials and the large Phase III clinical trial that led to its eventual US FDA-approval in FLT3-mutated AML. Our review also discusses data on midostaurin adverse effects, mechanisms of resistance and limitations of its utility. We further discuss emerging second-generation FLT3 inhibitors, with a focus on quizartinib and gilteritinib and future directions to enhance FLT3-inhibitor efficacy and overcome mechanisms of resistance.

PS1011 MEASURABLE RESIDUAL DISEASE BY MULTIPARAMETRIC FLOW‐CYTOMETRY IS A RELIABLE TOOL FOR RISK‐STRATIFICATION OF FLT3‐MUTATED AML PATIENTS

Background:Mutations of the gene encoding Fms Related Tyrosine Kinase 3 (FLT3), both at the tyrosine kinase domain (TKD) and the juxta‐membrane level (ITD), represent the most common lesions found in Acute Myeloid Leukemia (AML),identifying a subgroup of patients with mainly unfavorable prognosis.FLT3 mutations are considered an unreliable tool for measurable (previously minimal) residual disease (MRD) monitoring, due to their intraclonal heterogeneity and instability during the course of disease. Instead, multiparametric flow cytometry (MFC) may represent a reliable tool to monitor MRD in this molecular subset. In fact,through the recognition and monitoring of leukemia associated immunophenotypes,MFC is applicable in up to 90% of AML patients with a sensitivity up to 10–4.Aims:The aim of our study was to investigate the feasibility of MRD assessment by MFC in FLT3 mutated AML. Furthermore, we investigated if MRD persistence in FLT3 mutated AML affects prognosis in terms of overall (OS) and disease‐free survival (DFS), as compared with FLT3 wildtype ones.Methods:We retrospectively analyzed a series of 39 patients with de novo AML carrying FLT3 mutations (34/39,87% ITD, 2/39, 5% TKD, 3/39, 8% ITD/TKD). We compared MRD status and clinical outcome with a matched group of FLT3 wildtype AML (n = 128). Both groups were treated according to intensive EORTC/GIMEMA protocols. MRD was measured by MFC at the post‐consolidation timepoint. Patients were defined as MRD‐negative, when obtaining a residual leukemic cells (RLCs) count below the threshold of 3.5x10–4 (0.035%).Results:Overall median age was 47 (range 17–72). The 2 cohorts were balanced in terms of age and sex distribution. In the FLT3 mutated group, 22 out of 37 (59%) cases carried a concomitant NPM1 mutation vs 32 out of 124 (26%) of FLT3 wildtype ones (p <0.001). Furthermore, FLT3 mutation was significantly associated with a WBC count >50x109/L (20/39, 51%) vs 30/128 (23%) of those FLT3 wildtype (p < 0.001). Post remission treatment allocation was different between FLT3 mutated patients as compared to. FLT3 wildtype ones; 16 out of 39 (41%) FLT3 mutated patients were submitted to allogenic stem cells transplantation (ASCT) as compared to 34 out of 128 (27%) FLT3 wildtype ones (p = 001). At the post‐consolidation time‐point, MRD negativity rate was significantly lower in FTL3 mutated patients (4/39, 10%) as compared to those FLT3 wildtype (40/128, 31%), (p = 0.009). In particular, 32 out of 35 (91%) FLT3 mutated patients were above the ELN recommended threshold of positivity (0.1%) and 11/35 (31%) were above 1% of RLC.Among FLT3 mutated patients, OS was significantly longer for patients obtaining MRD‐negativity as compared to those who did not (67% vs. 31%, p = 0.9). As for as DFS, we observed the same difference for MRD negative patients vs. MRD positive ones (67% vs. 29%, p = 0.8). The negative impact of MRD was proportional to the amount of RLC at post‐consolidation time‐point (Figure 1) (p = 0.045). However, among FLT3 mutated patients, ASCT was the only post remission treatment allowing a prolonged OS to be achieved as compared to autologous SCT or standard chemotherapy (4‐years OS 63% vs. 0% vs. 15%, p = 0.019, respectively).Summary/Conclusion:In conclusion, MRD as measured by MFC is a biomarker of response also in FLT3 mutated patients. In these patients, ASCT delivery is associated with a satisfactory long term outcome, however a longer duration of response can be pursued by administering FLT3 inhibitors before or after transplant, in order to improve the quality of remission and prevent leukemic recurrence.image

Abstract 2721: Prospective whole exome and whole transcriptome sequencing of hematologic cancers for biomarker-driven treatment : Preliminary results of a randomized pragmatic clinical trial

Abstract Introduction: Biomarker-driven early clinical trials of molecularly targeted agents have reported improvement of the response rate in various malignancies. In this pragmatic clinical trial, we aimed to assess whether biomarker-driven treatment strategy could improve outcomes of incurable hematologic cancer patients. Methods: This is an open-label, randomized, pragmatic trial in adult patients with hematologic cancer. Patients with any kind of hematologic cancer refractory to standard-of-care are eligible. We randomly assigned these patients to receive a matched molecularly targeted agent (experimental group) or treatment at physician’s choice (control group). Randomizations are determined by central block randomization in 2:1 ratio. Clinicians and patients are not masked to allocation. Whole exome and whole transcriptome sequencing (WES/WTS) are applied for detecting all classes of genomic alterations: single nucleotide variation (SNV), insertions and deletions (INDEL), copy number alterations, Fms-Related Tyrosine Kinase 3 (FLT3) internal tandem duplication (ITD), Mixed-lineage leukemia partial tandem duplication and gene rearrangements. The primary endpoint is response rate in the intention-to-treat population. Results: Twenty-three patients with median age of 53 years and male to female ratio of 11:12 were enrolled between December 2015 and October 2016; 15 patients in experimental group, 8 patients in control group. Actionable mutations were identified in four patients in experimental arm. A 78-year-old female with acute myelogenous leukemia (AML) had FLT3 ITD at exon 14, which was identified as a long insertion mutation in SNV/INDEL calling. She received ASP2215 (FLT3 inhibitor), and showed a dramatic reduction in blast counts of peripheral blood. A 75-year-old female with myelodysplastic syndrome had TSC1P39A mutation. Because mTOR exists in downstream signaling pathway of TSC1, everolimus was administered and regression of disease activity, reflected by reduction of C-reactive protein level, was observed. NOTCH2 c.17′ and KITD815V mutation were identified in two patient; 69-year-old female with multiple myeloma and 42-year-old male with AML, respectively. Among 8 patients in control arm, seven patients received cytotoxic chemotherapy. One undergone another comprehensive genomic profiling called FoundationOne Heme, however no actionable mutations was identified. Conclusions: Among patients with incurable hematologic malignancy, actionable mutations could be identified in one fourth of patients for whom WES/WTS were performed. They showed favorable initial response to molecularly targeted agents. This randomized pragmatic clinical trial is currently ongoing(NCT02758080). Citation Format: Sanga Kim, Ryul Kim, Youngil Koh, Choong Hyun Sun, Hogune Im, Hong Seok Yun, Dong-Yeop Shin, Inho Kim, Sung-soo Yoon. Prospective whole exome and whole transcriptome sequencing of hematologic cancers for biomarker-driven treatment : Preliminary results of a randomized pragmatic clinical trial [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 2721. doi:10.1158/1538-7445.AM2017-2721

Internal Tandem Duplication in FLT3 Attenuates Proliferation and Regulates Resistance to the FLT3 Inhibitor AC220 by Modulating p21Cdkn1a and Pbx1 in Hematopoietic Cells.

Internal tandem duplication (ITD) mutations in the Fms-related tyrosine kinase 3 (FLT3) gene (FLT3-ITD) are associated with poor prognosis in patients with acute myeloid leukemia (AML). Due to the development of drug resistance, few FLT3-ITD inhibitors are effective against FLT3-ITD+ AML. In this study, we show that FLT3-ITD activates a novel pathway involving p21Cdkn1a (p21) and pre-B cell leukemia transcription factor 1 (Pbx1) that attenuates FLT3-ITD cell proliferation and is involved in the development of drug resistance. FLT3-ITD up-regulated p21 expression in both mouse bone marrow c-kit+-Sca-1+-Lin- (KSL) cells and Ba/F3 cells. The loss of p21 expression enhanced growth factor-independent proliferation and sensitivity to cytarabine as a consequence of concomitantly enriching the S+G2/M phase population and significantly increasing the expression of Pbx1, but not Evi-1, in FLT3-ITD+ cells. This enhanced cell proliferation following the loss of p21 was partially abrogated when Pbx1 expression was silenced in FLT3-ITD+ primary bone marrow colony-forming cells and Ba/F3 cells. When FLT3-ITD was antagonized with AC220, a selective inhibitor of FLT3-ITD, p21 expression was decreased coincident with Pbx1 mRNA up-regulation and a rapid decline in the number of viable FLT3-ITD+ Ba/F3 cells; however, the cells eventually became refractory to AC220. Overexpressing p21 in FLT3-ITD+ Ba/F3 cells delayed the emergence of cells that were refractory to AC220, whereas p21 silencing accelerated their development. These data indicate that FLT3-ITD is capable of inhibiting FLT3-ITD+ cell proliferation through the p21/Pbx1 axis and that treatments that antagonize FLT3-ITD contribute to the subsequent development of cells that are refractory to a FLT3-ITD inhibitor by disrupting p21 expression.

Constitutive Activation of FLT3 Is a Positive Prognostic Factor in Infants with MLL-Rearranged Acute Lymphoblastic Leukemia

Constitutive activation of the FMS-related tyrosine kinase-3 (FLT3) is a common feature of acute leukemias which can be caused by activating mutations or by high expression levels. Aberrant FLT3 downstream signaling is mediated via the RAS/MAPK, PI-3-Kinase/AKT, and STAT5 pathways leading to proliferation, survival, and therapy resistance. In MLL-rearranged acute lymphoblastic leukemia (ALL) activating FLT3 tyrosine kinase domain mutations (TKDs) affecting codons D835 and I836 are frequently identified (Armstrong et al., 2003; Taketani et al., 2004). In addition, MLL-rearranged ALLs are consistently characterized by an exceptionally high FLT3 expression (Armstrong et al., 2002) which has been shown to be associated with ligand independent signaling (Stam et al., 2005). However, the prognostic impact of a constitutive activation of FLT3 in this ALL subset remains controversial.Here, we report on the FLT3 mutational status and gene transcription levels in a large cohort of 95 infants and 72 children with MLL-rearranged ALL. Results obtained were further complemented by a high resolution melting (HRM) screen for common activating NRAS and KRAS mutations at codons 12, 13, and 61. All patients were enrolled in the multicenter trials ALL-BFM 86, 90, 95, 2000, and AIEOP-BFM ALL 2009 as well as Interfant-99 and Interfant-06.In infants, FLT3-TKD mutations were identified in 12/95 patients (12.6%) including one novel insertion/deletion involving codons D835 to S838. In only 2/95 patients (2.1%) an alteration in the juxtamembrane domain of FLT3 was detected. Of the 12 infants with mutation only 2 suffered from a relapse, 2-years cumulative incidence of relapse (CIR) 18%± 12%. In children, only one FLT3 aberration (FLT3-TKD D835 mutation) was detected (1/72, 1.4%). FLT3 transcript levels were analyzed by quantitative real-time PCR in 124 patients (69 infants and 55 children) with available RNA. When we separated the infant cohort into two groups according to the median RQ value, FLT3high and FLT3low, the CIR was significantly different (CIR 19%±7% vs. 66%± 9%, Gray p=0.0001). Of the 6 patients with low FLT3 transcription level, but with presence of a mutation, only one had a relapse. These results indicate that activating FLT3 mutations may compensate for the high relapse risk of patients with a low FLT3 expression. Accordingly, we could show that the CIR was significantly reduced for infants with a low FLT3activation (low transcription, no mutation 19%±7%) compared to those with a high FLT3 activation (high transcription or mutation 75%±9%, Gray p<0.0001, Figure 1). In multivariate analysis, the high prognostic impact of the FLT3 transcription level in infants was independent of age (< vs. ≥6 month), white blood cell count at diagnosis (WBC ≥ vs. <300000), or prednisone response (poor vs. good). This influence of the FLT3 expression could not be seen in children: CIR 13%±7% for low transcription vs. 12%±7% for high transcription.The only other known recurrent mutations in pediatric MLL-rearranged ALL are activating N- and KRAS mutations at codons 12, 13, and 61 (Liang et al., 2006; Andersson et al., 2015). As RAS signaling is considered as a putative downstream target of FLT3, we investigated the frequency of these mutations and their impact in the context of the prognostic value of FLT3 activation. We identified non-synonymous N/KRAS mutations in 21/95 (22.1%) infants and in 10/72 (13.9%) children. The presence of activating RAS mutations correlated with a higher rate of relapse and a lower probability of event-free survival (pEFS). For infants alone, constitutive activation of N/KRAS resulted in a lower pEFS (43%±6% wt vs. 11%±8%, p=0.01), but there were no significant differences in the CIR (40%±6% wt vs. 51±12%, p=0.40).In summary, we confirm that MLL-rearranged infant ALL represents a biologically distinctive entity with unique molecular genetic features. However, in contrast to published studies, we report that hyperactivation of FLT3 signaling is associated with a good prognosis in MLL-rearranged infant ALL. Our data has important implications for the design of rational therapies in MLL-rearranged ALL as the use of small molecule FLT3 inhibitors may be disadvantageous in some infants depending on FLT3 expression levels and FLT3 and RAS mutational status. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Exome Sequencing Identifies Recurring FLT3 N676K Mutations in Core Binding Factor Leukemia

AbstractAbstract 404Inversion inv(16) or translocation t(16;16) and translocation t(8;21) are recurring rearrangements in acute myeloid leukemia (AML), which result in the fusion genes CBFB/MYH11 or RUNX1/RUNX1T1, respectively. These rearrangements are found in 15–20% of adult de novo AML cases and are associated with favourable prognosis. CBFB and RUNX1 form the core binding factor (CBF), which is a transcription factor essential for normal hematopoiesis and myeloid development. By disrupting the physiological transcription factor activity of CBF the fusion proteins causes repression of the CBF-target genes resulting in a block of differentiation. Since expression of CBFB/MYH11 or RUNX1/RUNX1T1 on their own is not sufficient to cause leukemia it is likely that additional mutations are required for malignant transformation.To systematically identify mutations which may collaborate with CBFB/MYH11 during leukemogenesis we performed exome sequencing of an AML sample with an inv(16). The sample was selected based on availability and absence of known additional genetic alterations. By comparing the AML exome sequence with the exome sequence of a remission sample from the same patient we were able to identify leukemia-specific sequence variants as described previously (Greif et al., 2012, Blood). Using this approach we found an N676K mutation in the ATP-binding domain (TKD1) of the fms-related tyrosine kinase 3 (FLT3) gene. Mutations affecting N676 resulting in variable amino acid changes (N676D or N676S) were initially discovered in a screen for resistance to tyrosine kinase inhibitors (TKI) in FLT3 internal tandem duplication (ITD) expressing Ba/F3 cells (Cools et al., 2004, Cancer Res). An N676K point mutation has been reported in a cytogenetically normal (CN) AML patient with FLT3-ITD and TKI-resistance (Heidel et al., 2006, Blood). In contrast, our patient with inv(16) and the FLT3 N676K did not carry an additional FLT3-ITD. In a cohort of 69 patients with inv(16) we found a total of 4 patients with FLT3 N676K mutations (4/69, 6%). In 14 patients with t(16;16) and in 36 patients with t(8;21) we identified one patient each with FLT3 N676K (1/14, 7% and 1/36, 3%). Thus, the overall mutation frequency for patients with CBFB/MYH11 rearrangement was 6% (5/83). None of the CBF AML patients with FLT3 N676K mutation had an additional FLT3-ITD. In 90 CN-AML patients we detected only a single FLT3 N676K mutation and the affected patient had a concurrent FLT3-ITD. We are currently testing the frequency of FLT3 N676K mutations in independent CBF-AML cohorts.To test the transforming potential we expressed the FLT3 N676K mutant in Ba/F3 cells. As controls we expressed FLT3 wild type (WT), FLT3 mutants D835Y or ITD in parallel. Cell surface expression of N676K was similar to WT, but increased compared to D835Y and ITD. Cell proliferation assays were done in presence and absence of IL-3 or FLT3 ligand (FL). FLT3 N676K leads to IL-3 and FL independent cell growth reaching 25% of IL-3 mediated growth. FLT3 inhibition by AC220 or PKC412 abrogates this proliferation, but N676K is slightly more resistant to inhibition than ITD. In contrast to ITD expression that results in STAT5 phosphorylation, N676K expression leads to phosphorylation of MAPK and AKT.Gene expression profiling of patients with inv(16) revealed that patients with additional FLT3 N676K mutation show a significant enrichment of gene sets including ubiquitin mediated proteolysis, adherens junction and JAK/STAT signaling pathway.According to a structural model, the N676K mutation stabilizes the fold of the kinase domain between the juxtamembrane domain (JMD) and a hydrophobic pocket that is the target of FLT3 inhibitors. N676K mutations could therefore reduce the autoinhibition of FLT3 by the JMD and also negatively affect the binding affinity of FLT3 inhibitors.Ours is the first report of recurring FLT3 N676 mutations in the absence of FLT3-ITD. Our findings point towards a specific association of FLT3 N676K mutations with CBF leukemia. Based on our functional assays N676K acts as a gain-of-function mutation. Although FLT3 has been known for more than a decade to be mutated in one third of AML patients, it appears that the spectrum of FLT3 mutations is still not fully understood, in particular, in defined cytogenetic subgroups of AML. Unbiased mutation screening by exome sequencing allows the detection of novel sequence variations even in extensively studied genes. Disclosures:Krebs:Illumina: Honoraria. Greif:Illumina: Honoraria.

Sorafenib treatment of FLT3-ITD+ acute myeloid leukemia: favorable initial outcome and mechanisms of subsequent nonresponsiveness associated with the emergence of a D835 mutation

Internal tandem duplication (ITD) of the fms-related tyrosine kinase-3 (FLT3) gene occurs in 30% of acute myeloid leukemias (AMLs) and confers a poor prognosis. Thirteen relapsed or chemo-refractory FLT3-ITD(+) AML patients were treated with sorafenib (200-400 mg twice daily). Twelve patients showed clearance or near clearance of bone marrow myeloblasts after 27 (range 21-84) days with evidence of differentiation of leukemia cells. The sorafenib response was lost in most patients after 72 (range 54-287) days but the FLT3 and downstream effectors remained suppressed. Gene expression profiling showed that leukemia cells that have become sorafenib resistant expressed several genes including ALDH1A1, JAK3, and MMP15, whose functions were unknown in AML. Nonobese diabetic/severe combined immunodeficiency mice transplanted with leukemia cells from patients before and during sorafenib resistance recapitulated the clinical results. Both ITD and tyrosine kinase domain mutations at D835 were identified in leukemia initiating cells (LICs) from samples before sorafenib treatment. LICs bearing the D835 mutant have expanded during sorafenib treatment and dominated during the subsequent clinical resistance. These results suggest that sorafenib have selected more aggressive sorafenib-resistant subclones carrying both FLT3-ITD and D835 mutations, and might provide important leads to further improvement of treatment outcome with FLT3 inhibitors.

Novel targeted therapy for acute myeloid leukemia with a dual FLT3 and JAK2 inhibitor

Acute myeloid leukemia (AML) is a highly malignant hematopoietic tumor. The use of all-trans retinoic acid (ATRA) and arsenic trioxide, which began from China, has resulted in revolution of the acute promyelocytic leukemia (APL) that appears curable in more than 70% of patients1. However, the treatment regimen for non-APL AML particularly in older patients has progressed little in the past two decades. Intensive efforts have been made toward the development of novel target agents, which are based on newfound pathophysiological events crucial for cancers. FMS-related tyrosine kinase 3 (FLT3), which is mutated in about 30% patients of AML, is the most ideal target for molecular therapy. Over the past decade, a number of FLT3 inhibitors have been studied in clinical trials. However, the results were often transient because of secondary resistance developed2. In addition, genetic lesions and aberrations involving Janus-associated kinase 2 (JAK2) have been found to be associated with a wide spectrum of hematological malignancies3. Recently, S Hart et al reported that pacritinib (SB1518), a dual JAK2/FLT3 inhibitor, emerged as an ideal new therapeutic agent for acute myelogenous leukemia in a preclinical study4. Pacritinib is a potent and selective inhibitor of FLT3 and JAK2 with IC50 of 23 and 22 nmol/L, respectively5. In the study, the authors demonstrated pacritinib led to a dose-dependent decrease of FLT3 auto-phosphorylation and downstream effectors of STAT5, ERK1/2, AKT phosphorylation in FLT3-internal-tandem duplication (ITD) cell lines (MV4-11, MOLM-13) and in FLT3-wt-bearing cell line (RS4;11). The agent inhibited the proliferation of MV4-11, MOLM-13 and RS4; 11 cells with IC50 of 47, 67, and 930 nmol/L, respectively. Furthermore, the JAK2V617F-harboring cell line, SET-2, was also very sensitive to pacritinib (IC50=220 nmol/L). Flow cytometry analysis showed that the agent could induce G1 arrest and caspase-dependent apoptotsis. Pacritinib inhibited the proliferation of 14 primary AML samples with the IC50 ranging from 190 nmol/L to 1300 nmol/L, with concomitant inhibition of phosphorylation of FLT3, STAT3, and STAT5. The two samples harboring the FLT3-ITD mutation were among the most sensitive. Moreover, pacritinib was also highly active in in vivo models of FLT3-ITD driving cell lines. In MV4-11 tumor-bearing mice, pacritinib (once daily for 21 consecutive days) induced dose-dependent inhibition of tumor growth. Complete regression was observed in 3/10 and 8/8 mice in the groups receiving 50 and 100 mg·kg−1·day−1, respectively. In the MOLM-13 model, pacritinib treatment (150 mg/kg bid for 7 consecutive days) resulted in tumor growth inhibition of 83%. Finally, higher activity of JAK/STAT signaling was confirmed in FLT3-linifanib/ABT-869 resistant cells (MV4-11-R). Pacritinib was highly effective in the resistant cell lines. A combination of FLT3 inhibitor linifanib with JAK family inhibitor ruxolitinib showed the synergistic effect on MV4-11 cells. Interestingly, pacritinib entered the clinic in 2008 has completed Phase 2 trials for myelofibrosis. It has shown promising clinical activity and a favorable safety profile. The agent has received orphan drug designation from the US and the EU regulatory authorities. In summary, the preliminary results of the dual inhibitor of FLT3 and JAK2 were promising. Hope remains that pacritinib will be become an effective therapeutic adjunct to our current treatment approach to AML. Results from near future clinical trials will answer the question whether the dual inhibitor of FLT3 and JAK2 will be a real successful targeted agent. As the next generation sequence technology advanced, more genetic and molecular changes in cancers including AML were discovered6. Multiple different genetic changes may cooperate in cancers. New mutations in signaling pathway or alternate pathway will emerge when treated by only one specific target agent7. Thus, from a clinical point of view there are at least two important aspects in this study. First, the resistant of target therapy could be possible reversed by simultaneously blocking two or more signaling pathways. It indicates a new strategy to screen for agents for an effective targeted cancer therapy. Second, since there are a handful of FLT3, JAK2 inhibitors or other kinase inhibitors have already been discovered and are currently being developed for clinical trials. It is rationale to designing trials in a more effective manner by combining one targeted drugs along with other agents that target alternate mechanisms of disease pathogenesis.

Isolation of human mAbs that directly modulate FMS-related tyrosine kinase 3 signaling

FMS-related tyrosine kinase 3 (FLT3) is a class III receptor tyrosine kinase that plays important roles in hematopoiesis, including early progenitors and dendritic cell development. FLT3 is expressed at high levels in 70-100% of cases of AML and in virtually all cases of B-lineage acute lymphoblastic leukemia. FLT3 is regarded as a molecular target in the development of novel therapies for acute leukemia patients. Currently, many small-molecule FLT3 inhibitors have been developed, but clinical trials have resulted in limited antileukemia effects because of off-target toxicities and drug resistance. The development of anti-FLT3 Abs might overcome these difficulties and enhance the antileukemia efficacy of FLT3 inhibitors. In the present study, we demonstrate the isolation of novel human mAbs against FLT3 with antagonistic or agonistic activities. An antagonistic Ab, designated A2, continuously inhibits FLT3 ligand (FL)-induced phosphorylation of FLT3 and MAPK. A2 cooperatively induces apoptosis with daunorubicin, even in the presence of FL. An agonistic Ab, designated 3E6, surprisingly induces the phosphorylation of FLT3 and MAPK, and supports the growth of a factor-dependent cell line independently of FL addition. In addition, A2 showed complement-dependent cytotoxicity activity, but was devoid of Ab-dependent cell mediated cytotoxicity. Finally, we evaluated Ab internalization in a cell line. Immunofluorescence and flow cytometry analyses revealed that A2 is efficiently internalized. Collectively, these data demonstrate that A2 is a potent human Ab that might be capable of delivering cytotoxic reagents and that has antagonistic effects on FLT3 signaling. In addition, 3E6 might be a potential scaffold for novel dendritic cell-based immunotherapies.