Fms-like Tyrosine Kinase 3 ITD Research Articles

An Ex-Vivo Combination Drug Sensitivity Platform for Predicting Sensitivity to FLT3 Inhibitor-Based Combination in Acute Myeloid Leukemia

Introduction Acute myeloid leukemia (AML) is a clonally heterogenous group of disorders with many treatment options. Despite the increase in available options including targeted therapy, challenges remain in identifying patients who may respond to a given regimen. The ability to accurately predict responses to treatment is crucial given the varying response rates to standard treatments, adverse effects and increasing drug cost. In this study, we explore the ability of an ex vivo combination drug sensitivity platform, quadratic phenotypic optimization platform (QPOP), in identifying potential responders and non-responders to possible combination therapies in FMS-like tyrosine kinase-3 (FLT3) mutant AML. We further investigate the mechanism behind selective sensitivity and resistance to FLT3 inhibitor-based combinations. Methods FLT3-ITD mutant or WT AML cell lines were treated with a set panel of drugs recommended by physicians including FLT3 inhibitors (FLT3i), midostaurin and gilteritinib. QPOP drug combination scores and ranking were derived from analysis of cell viability response in cancer cells following treatment with a series of test combinations derived from an orthogonal array composite design. Molecular analysis was conducted to determine the accuracy of QPOP predicted response and FLT3 mutant status. Subsequently, we performed a prospective cohort pilot study of QPOP analysis in AML. AML patients from the National University Hospital,Singapore, with disease amenable to blood or marrow aspiration, were recruited between 26th November 2019 and 15th July 2022 with a median follow up of 4 months. CD33 or CD34 selection was performed to enrich for malignant blasts cells, depending on the sample. Patients were treated with current standard of care treatment options as guided by clinician opinion. The primary outcomes were to determine the ability of QPOP to predict sensitivity to FLT3i-based therapies. Results QPOP analysis on 8 AML cell lines was accurately able to stratify FLT3i responders and non-responders including differential responses between an FLT3-ITD isogenic parental and FLT3i resistant line. We recruited 25 patients (2 patients having repeat QPOP evaluations post treatment) with a median age of 59 years and 2 prior lines of treatment. QPOP responses were evaluable for 18 independent treatments in 15 patients (n=3 FLT3 ITD or TKD (20%), n=12 FLT3 WT (80%)). The median turn-around time for QPOP analysis was 8 (±2.1) days. Patients with either FLT3i among the top 5 ranking 2 or 3 drug combinations were suggested to be sensitive to the use of FLT3i. QPOP analysis of FLT3 mutant patients had predicted 2 patients with the FLT3-ITD mutation to be responsive to a 2-drug combination of midostaurin-venetoclax. The third FLT3-mutant patient with a TKD point mutation was predicted to be respond to a combination of gilteritinib-dasatinib. While FLT3 WT patients did not predict sensitivity to either FLT3 inhibitor as single agents or 2-drug combinations, 4/12 (33.3%) of the WT patients also showed sensitivity to the use of gilteritinib in3-drug combination rankings. This indicates that gilteritinib may be useful for FLT3 WT patients when used in the appropriate combination therapy. Furthermore, QPOP was also able to accurately identify increased sensitivity to FLT3i-based combinations in accordance with a gain of FLT3 ITD mutation. Serial QPOP analysis in subsequent samples following midostaurin-based therapy indicated a decrease in FLT3i sensitivity that was concordant with increasing FLT3 allelic burden and development of resistance. The development of resistance to FLT3i-based combinations was correlated to an increase in phosphorylated AKT expression in both AML cell lines and the patient who developed FLT3 resistance. Conclusion QPOP analysis predicts sensitivity to drug-combinations for AML in a clinically applicable timeframe. Here we show that QPOP can be used as a tool to identify sensitivity to FLT3i-based combination therapy without prior knowledge of FLT3 mutation status. AML cell lines and patient samples can be stratified according to FLT3 inhibitor response sensitivity which parallels their mutational background and identify potential drug resistance. Further clinical study of the safety and efficacy of QPOP-derived FLT3i-based combinations as well as inquiry into the potential mechanisms of acquired resistance to these combinations will be necessary. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Venetoclax Plus Hypomethylating Agent (HMA) Based Induction for Treatment-Naive Older/Unfit Patients with FLT3-Mutated Acute Myeloid Leukemia (AML)

Introduction: FMS-like tyrosine kinase 3 (FLT3) mutation (m) is one of the most prevalent mutations in acute myeloid leukemia (AML), and it is associated with an increased risk of relapse and decreased overall survival (OS). The standard of care therapy for physically fit treatment naïve patients is intensive chemotherapy with FLT3 inhibitor (i) (midostaurin). For older/unfit patients, venetoclax-based induction is preferred as per NCCN guidelines. We explored the clinical outcome of FLT3m AML patients who were ineligible for intensive chemotherapy and treated with a hypomethylating agent (HMA) plus venetoclax (Ven) based induction at Mayo Clinic. Methods: Thirty FLT3m AML patients who were ineligible for intensive chemotherapy and received HMA+Ven induction at Mayo Clinic between June 2019 and May 2022 were analyzed for clinical characteristics and treatment outcomes. Patients who received HMA+Ven for the relapsed or refractory disease were excluded. All patients received at least one cycle of HMA, either azacitidine (75 mg/m2 from day 1 to 7) or decitabine (20 mg/m2 from day 1 to 5). Responses were assessed according to the 2022 European Leukemia Net (ELN) criteria. Results: The baseline characteristics are summarized in Table 1. Most of the patients (n=27, 90%) were ≥ 65 years old with a median age of 74 years (range, 59 to 88). Majority of the patients (n=16, 53%) had de novo AML, 8 (27%) patients had secondary AML, 4 patients (13%) had primary AML with MDS-related changes, and 2 patients (7%) were diagnosed with therapy-related AML. Two-thirds of the patients (n=20, 67%) had diploid cytogenetics (CG) and 10 (33%) patients had additional cytogenetic (CG) abnormality at diagnosis. Twenty-six (87%) and 4 (13%) patients had FLT3 ITD and TKD mutation subtypes, respectively. The median baseline FLT3m allelic ratio was 0.39 (range, 0.1-0.7). Most frequent reported mutations (≥ 5%) were DNMT3A (40%), NPM1 (37%), spliceosome mutations (SF3B1, U2AF1, and SRSF2; 30%), RUNX1 (23%), and TET2 (17%). Seventy percent of the patients achieved complete remission with or without count recovery (CR/CRi) (n=21), 13% (n=4) of the patients had partial remission, and 17% (n=5) of the patients had refractory disease to HMA+Ven induction. The median time to achieve CR/CRi was 35 days (range, 22-230). Most CR/CRi patients (86%, n=18) continued consolidation therapy with HMA+Ven. Until changing the HMA+Ven regimen for any reason, the median number of HMA+Ven cycles received was 6 (range, 1-13). Only one patient received allogeneic stem cell transplant allo-HSCT after achieving remission and received gilteritinib maintenance therapy post-transplant. Six (29%) of the CR/CRi patients had relapse with a median time to relapse of 6 months (range, 1-10). The median duration of response was 13 months (95% CI: 6.71-19.28) (Figure 1A), and the median OS was 15 months (95% CI: 8.89-21.10) (Figure-1B). FLT3i (gilteritinib) was received in 6 patients (20%) post HMA+Ven; 2/6 received as maintenance therapy and 4/6 for relapse disease. In the univariate analysis for OS, diploid cytogenetics (p<0.0001) and non-high risk cytogenetics (p=0.04) were associated with significantly better OS (Figure-1D/E). AML sub-type (de novo vs sAML) (p=0.97), NPM1 mutation at diagnosis (p=0.39), FLT3m subtype (ITD vs TKD) (p=0.79) or allelic ratio at diagnosis (<0.5 vs >0.5) (p=0.89), and HMA sub-type (azacitidine vs decitabine) (p=0.43) were not significantly associated with difference in OS (Figure-1C/F/G/H, respectively). Conclusions: In summary, HMA+ Ven showed significant activity in treatment naïve FLT3m AML patients who were ineligible for intensive chemotherapy. HMA+Ven can be used as a backbone in combination with FLT3 inhibitor to improve outcome further and for successful transition to curative intent therapy such as allo-HSCT. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Intensive Therapy for NPM1 Mutant AML Patients: Negative Impact of FLT3-ITDhighbut Not FLT3-ITDlow and FLT3-TKD and Role of Transplant in Patients over 60 Years of Age

Introduction: Nucleophosmin 1 (NPM1) mutations without an FMS-like tyrosine kinase-3 ITD (ITD) mutation are associated with a favorable prognosis in younger (age <60 years) patients (pts) with acute myeloid leukemia (AML). However, the prognostic impact of NPM1 mutations with concurrent low ITD allelic ratio (ITDlow) is unclear. Furthermore, the benefits of hematopoietic stem cell transplant (SCT) in NPM1+ AML in pts >60 is not known. In this retrospective study, we investigated the impact of FLT3 mutational status in pts with NPM1+ AML and the role of SCT in patients >60 years with ITD negative (ITDneg). Methods: We included patients from January 2013 to August 19, 2021 from 7 centers with NPM1+ AML who received induction chemotherapy with an anthracycline and cytarabine. Data was collected by chart review and mutational status was determined by next generation sequencing at each institution according to local guidelines. ITDhigh was defined as an allelic ratio (AR) of ≥0.5; AR <0.5 was considered ITDlow. The log-rank test was used to compare overall survival between groups. Results: A total of 345 pts met the inclusion criteria. The mean age was 58 years (range 18-79) and 56% were female. Normal karyotype was seen in 80%. ITD was detected in 142 of the 345 (41%); a FLT3-TKD mutation was detected in 48 (14%) pts. Common co-mutations included DNMT3A (42%), TET2 (23%), IDH2 (19%), IDH1 (19%), and RAS (19%). The complete response (CR) rates for NPM1+/FLT3neg pts was 79.8%; the CR rates for the NPM1/ITDpos pts treated with IC+TKI, IC alone, and NPM1+/ITDneg/TKD+ were 73.3%, 74.1%, and 80%, respectively. In the entire ITD+ population, AR was known in 42.3% (N=60) of pts. The 2-year overall survival (OS) for the NPM1+/ITDhigh (N=29) was 48.6% (90% CI: 35.0%, 67.5%) and the 2-year duration of response (DOR) was 56.1% (90% CI: 38.7%, 81.5%), while the NPM1+/ITDlow group (N=31) had a 2-year OS of 66.4% (90% CI: 53.5%, 82.4%) and 2-year DOR of 62.0% (90% CI: 46.6%, 82.3%). The comparison of OS in these two groups by log-rank was not significant (p=0.31). In the ITDlow group, pts who had SCT (N=15) had a 2-year OS of 71.8% (90% CI: 52.6%, 94.5%) and a 2-year DOR of 76.2% (90% CI: 58.7%, 98.8%); those treated with chemotherapy alone (N=14) had a 2-year OS of 62.9% (90% CI: 44.4%, 88.9%) and 2-year DOR of 50.8% (90% CI: 28.7%, 90.0%). OS was not significantly different (p=0.35). Comparing survival between FLT3 groups with a Cox PH model, FLT3-ITDhigh was significantly associated with inferior OS vs. FLT3neg(HR = 2.30, p=0.005). However, FLT3-ITDlow and FLT3-TKD+ were not associated with inferior OS vs. FLT3neg (HR=1.56, p=0.17 and HR=1.65, p=0.08, respectively). In pts >60 and NPM1+/ITDneg (N=104), the mean age was 67 years (range 60-78), median WBC count at baseline 7.7 (range 0-127), and 48.1% of pts were female. The CR rate was 75.0% (N=78). A total of 32 pts underwent SCT and 40 pts received a median of 3 cycles of consolidation. The 2-year OS and DOR in the SCT group was 71.1% (90% CI: 58.8%, 85.8%) and 53.2% (90% CI: 40.1%, 70.7%), respectively as compared to 77.3% (90% CI: 66.4%, 89.9%) and 51.4% (90% CI: 39.1%, 67.6%), respectively in the non-SCT group. Albeit with small numbers SCT did not improve OS in this age group (p=0.2) In the entire cohort, the five most common co-mutations other than FLT3 were evaluated for influence on survival. Using the log-rank test, only DNMT3A and TET2 were associated with inferior OS (P=0.026 and 0.039, respectively), while IDH1, IDH2, and RAS were not significantly associated with survival (p-values of 0.43, 0.6, and 0.69, respectively). Conclusion: In our multicenter cohort of pts with NPM1+ AML, OS was significantly inferior in pts with ITDhigh v. FLT3neg, but not in ITDlow and TKD. Co-mutations in DNMT3A and TET2 adversely affected overall survival. Our data did not find a significant difference in OS in pts >60 with NPM1+/FLT3-ITDneg who were consolidated with chemotherapy versus SCT, though larger follow up studies are needed. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Difference in FLT3 Mutational Status between Diagnosis and Relapsed/Refractory Acute Myeloid Leukemia: A Meta-Analysis

Introduction Research in recent years has shown that the class III receptor tyrosine kinase Fms-like tyrosine kinase 3 (FLT3) gene mutation was identified in 30% of Acute Myeloid Leukemia (AML) patients. The FLT3 internal tandem duplication (FLT3-ITD) is positive for 25% of AML cases, while FLT3 tyrosine kinase domain (FLT3-TKD) is present in 5% of cases. Some studies have showed that patients with AML with FLT3 mutation may maintain the alteration in the relapse/refractory (RR) disease, although this is not always the rule. This report is a review about the genetic change of FLT3 mutation in the RR AML scenario. Methods It is a retrospective meta-analysis research. A search in June/2020 was performed in PubMed to identify studies with the terms FLT3 (title/abstract) AND Acute Myeloid Leukemia without a restricted date. The papers were select based in title and abstract. Inclusion criteria were adult AML, language in English, Spanish or Portuguese. Exclusion criteria were studies in children and acute promyelocytic anemia. Data were collected from retrospective or prospective studies in which patients with RR AML had the data about FLT3-ITD or FLT-TKD in the diagnosis and relapse. Results and Discussion 1201 papers were found, in which 688 were excluded by reading the title, and 506 eliminated after the title and abstract revision. From these, 17 were eligible for full text reading. From eligible papers, 6 were rejected. All the studies used PCR as the method of evaluation of FLT3 mutational status. Six studies assessed FLT3 ITD and TKD mutations, four assessed only FLT3-ITD and one checked only FLT3-TKD mutation. Six articles described mutation status in both FLT3 alterations. The total of 272 patients with RR AML were analyzed in these studies. 74 (27%) were positive for FLT3-ITD at diagnosis and 15 (5%) for FLT3-TKD. In the relapse, 81 (29%) were FLT3-ITD positive and 11 (4%) FLT3-TKD positive. The change in mutation status occurred in 47 (17%) of patients, 3 patients of these change from FLT3-TKD to FLT3-ITD mutation. Three works had patients with acute promyelocytic leukemia, a total of 10 patients. In one work 42 children were included in the study, representing 15% of the total population. Mutational status change from negative to FLT3-ITD in 20 (7%) patients, FLT3-ITD to negative in 14 (5%). From those that were FLT3-ITD positive, 60 (22%) remained positive in the relapse. In patients with FLT3-TKD, change in mutation status from positive to negative occurred in 6 (2%) of the patients, 4 (1%) were negative and gained the mutation, 6 (2%) maintained positive in diagnosis and relapse. 159 (58%) were negative in all the disease course. When we analyzed only FLT3-ITD mutation, a total of 607 patients were studied at diagnosis and relapsed, 30% were FLT3-ITD positive in relapse. Change in mutational status occurred in 12%. Meanwhile, 392 patients had their mutational status available when considering only FLT3-TKD. 89% were negative in diagnosis and relapsed. 31 (8%) patients had their status changed. We described FLT3 mutations status in paired samples from diagnosis to relapsed/refractory AML. Our data demonstrate that 41% of patients were positive for FLT3 mutation in some point of the disease. One in six patients had their mutational status altered. Some patients may have had small clones with mutated cells that were below the limit of the detection by assay and proliferated after treatment. Another hypothesis is that ade novomutation was acquired during the disease course. From the population in the study, 7% were FLT3 positive/negative, which could represent the loss of the mutation post chemotherapy and a selection of a new clone originating from the relapsed disease. The information about FLT3 status is important due to a possible prognostic value in patients with relapse/refractory disease and because the patient can benefit from a novel therapeutic treatment with FLT3 inhibitors. Conclusion FLT3 mutation is an important mutation in AML at diagnosis and relapse/refractory scenario and the mutational status is variable during the disease. Since one in six patients had their mutational status altered in the study, reassessment should be mandatory in any case of relapse and refractoriness. More studies are needed to evaluate the prognostic value of those changes and the development of more sensitive methods could help to find small clones already present in AML at diagnosis. Disclosures No relevant conflicts of interest to declare.

FLT3-ITD and CD135 Over-Expression are Frequent Findings of Poor Survival in Adult Patients with Acute Leukemias

Fms-like tyrosine kinase 3 (FLT3) expression and mutation have been considered a poor prognostic factor in acute myeloid leukemia (AML). FLT3-ITD mutation is present in 30% of adult patients with AML and 2-5% in childhood acute lymphoblastic leukemia (ALL). The impact of these mutations on the prognosis of ALL patients, has not yet been established. Moreover, a limited number of publications regarding the level of expression of the FLT3 receptor (CD135) in both leukemias exist. This study aimed to analyze the clinical outcomes associated to the presence of FLT3-ITD mutation and the expression of CD135. 82 adult patients with newly diagnosed acute leukemia (39 with AML and 43 with ALL) were included. Flow cytometry and RT-PCR were done to analyze the expression of CD135 and the presence of FLT3 ITD mutation, respectively. FLT3-ITD was present in 14 (36%) of AML and 15 (35%) of ALL patients. Disease free survival (DFS) and overall survival (OS) were lower in ALL patients having a CD135 expression >3000cells/μL. There was a trend for poor OS in AML patients expressing FLT3 ITD. OS was worse in AML patients with high expression of CD135. A higher (35%) frequency of FLT3-ITD was found in adult ALL patients. The presence of FLT3-ITD was associated with a trend of poor OS in AML cases, and overexpression of CD135 was correlated with poor DFS in ALL cases and poor OS in both acute leukemias.

Abstract 537: Preclinical development and activity of Anti-FLT3 nanoworms in acute myeloid leukemia

Abstract Introduction: Mutations in FMS-like tyrosine kinase 3 (FLT3) occur in ~30% of acute myeloid leukemia (AML) cases causing high relapse and short survival. Acquired resistance limits the efficacy of current tyrosine kinase inhibitors against FLT3. Developing FLT3 target-specific antibodies has gained interest as a therapeutic approach for AML. Single-chain antibody fragments (scFv) are as specific as full-length monoclonal antibodies (mAbs), yet they outperform mAbs due to lower immunogenicity and a faster development process via recombinant protein engineering. However, scFvs have short circulating half-lives in humans and are difficult to purify. To overcome this, we developed anti-FLT3 scFv fused with an elastin-like polypeptide (ELP), A192, which aids in protein purifyication and extending its half life. Methods: The α-FLT3 scFv gene was fused to the amino terminus of (VPGAG)192 (A192), in the pET-25b(+) vector, purified and refolded for maximum biological activity. Binding specificity of α-FLT3-A192 was analyzed using FLT3 ITD+ AML cell lines MOLM-13 and MV4-11 and FLT3−U937 cells via flow cytometry. Viability and apoptosis assays were performed via trypan blue and Annexin-V at 72 hours, respectively. Immunoblot analyses were performed to assess the effect of α-FLT3-A192 on FLT3/STAT5/ERK signaling pathway. In vivo activity was analyzed by engrafting 106 MOLM-13 cells/ NOD scid mouse, treating with 220 uM of A192 or α-FLT3-A192 (N=8) on day 7, 10, 13 and 16, and sacrificing on day 17. Engraftment was analyzed in peripheral blood and bone marrow by HuCD45 stain and measured by flow cytometry. Kaplan Meier survival analysis was performed in MOLM-13 murine model. Results: Binding of α-FLT3-A192 (10 μM) vs. A192 (25 μM) was observed in MOLM-13 (MFI 1.0 vs 2.0; p&amp;lt;0.0001) and MV4-11 cells (MFI 1.0 vs 1.36; p=0.03). Contrarily, U937 cells had no binding. Cell viability decreased in MOLM-13 cells treated with α-FLT3-A192 vs. A192 at 10µM (p=0.0002, 23%), and 25µM (p&amp;lt;0.0001, 34%) and in MV4-11 cells at 10 µM (p&amp;lt;0.0001, 71%), and 25 µM (p&amp;lt;0.0001, 80%) vs. A192. Consistently, apoptosis increased in cells treated with 25 µM α-FLT3-A192 vs A192 (MOLM-13: p=0.007, 42.76% vs 5.27%; (MV4-11: p=0.003, 62.77% vs 25.36%). Mechanistically, α-FLT3-A192 inhibited both phospho-STAT5 and phospho-ERK in MOLM-13 and MV4-11 cells. In vivo analyses showed that spleens of mice treated with α-FLT3-A192 weighed less compared with A192 (0.48 vs 126 mg, p=0.03). Additionally, α-FLT3-A192 reduced engraftment in the peripheral blood (%huCD45- 17.5 vs 43.44, p=0.007) and bone marrow (%huCD45- 10.2 vs 26.1%, p&amp;lt;0.0001). Mice treated with α-FLT3-A192 had significantly longer OS than A192 group (median survival: 36 vs 30 Days, p=0.0015). Pharmacokinetic (PK) analysis showed that the terminal half-life of α-FLT3-A192 in NSG mice was ~ 14.9 hours. Conclusion: α-FLT3-A192 nanoworms present an effective FLT3 targeting strategy with high pharmacological activity and PK properties in cellular and xenograft murine models of AML. V.P.V. and M.P. contributed equally to the manuscript Citation Format: Vijaya Pooja Vaikari, Mincheol Park, John Andrew Mackay, Houda Alachkar. Preclinical development and activity of Anti-FLT3 nanoworms in acute myeloid leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 537.

HM43239, a Novel Potent Small Molecule FLT3 Inhibitor, in Acute Myeloid Leukemia (AML) with FMS-like Tyrosine Kinase 3 (FLT3) Mutations: Phase 1 /2 Study

Background: HM43239 is an orally active small molecule inhibitor of FLT3 that selectively inhibits not only FLT3 wild type, ITD mutants or TKD mutations, but also FLT3 ITD/TKD double mutations. HM43239 directly inhibits the kinase activity of FLT3 as a reversible Type I inhibitor and effectively modulates downstream p-STAT5 and p-ERK. HM43239 also demonstrated inhibition of SYK, JAK1/2 and TAK1, known to be involved in tumor cell proliferation and/or differentiation HM43239 monotherapy induced dose-dependent regression of tumor growth in FLT3 wild-type and FLT3 mutated leukemia cancer cell lines (Poster presented at AACR 2018; Abst.804 and also prolonged survival in a FLT3 ITD/TKD double mutated xenograft mouse model (Poster presented at AACR 2019; Abst.1293). The anti-leukemic activity of HM43239 monotherapy was evaluated in in vivo studies that suggested the ability of this agent to potentially target LSCs (Poster presented at AACR 2019; Abst.1293). Preclinical studies suggest that HM43239 may be an effective agent in patients with hematologic malignancies including AML, to target various mutant types of FLT3. Herein, we present a clinical trial design to assess the potential clinical activity and safety of HM43239 in patients with AML. This phase I/II clinical trial (NCT03850574) consists of dose escalation (Part A) and dose expansion (Part B) stages to access the overall safety and efficacy findings. Trial Design: This is an ongoing, open-label, multicenter, first in human, phase I/II trial enrolling adult patient with AML who relapsed or have refractory disease after at least one prior line of therapy. Patients were treated with HM43239 once daily (QD) in 28-day cycle except for the first 30-day cycle including a single PK sampling period. This trial comprises two parts: dose escalation and dose expansion. During dose escalation, the study follows an accelerated titration design, with around 50% dose increments and 1 patient per dose level. Accelerated titration will continue until 1 patient experiences a dose-limiting toxicity (DLT) or moderate toxicity (MT), drug-related grade 2 adverse event (except for hematologic toxicities), at any dose level, after which, a 3 + 3 dose escalation design will be used. If a patient achieves a clinical response [complete remission (CR), complete remission with low platelets (CRp), complete remission with incomplete hematologic recovery (CRi) or partial remission (PR)] at any dose level in escalation cohort, then an expansion cohort will be open at that dose level. Based on the evaluation of DLTs and composite complete remissions (CRc) from the dose escalation cohorts, additional subjects may be recruited in expansion cohorts at each dose level selected for expansion. At least 10 subjects with FLT3 mutations (ITD or activating point mutations such as D835Y, D835V, I836) will be enrolled to each dose level selected for expansion (including the subjects from the dose escalation cohort). Blood samples are collected for pharmacokinetics (PK) &amp; pharmacodynamics (PD) evaluation and for exploratory biomarker analysis in both cohorts. A 2-parameter Bayesian logistic regression will be used to model the dose-toxicity relationship on DLT in dose escalation and expansion parts and the estimated DLT rate will be provided as supportive reference for dose escalation procedure and safety monitoring. The primary endpoint is the assessment of pharmacokinetics, safety and tolerability in order to determine the recommended phase 2 dose (RP2D). Secondary endpoints include best response rate [CRc (CRc=CR+CRp=CRi)+ PR], duration of response, event free survival, overall survival, cumulative incidence of relapse of HM43239. PK-PD relationships and PD evaluated by plasma inhibitory assay (PIA) are exploratory endpoints. At first, this trial was initiated in Jan 2019 and an enrolled patient has been evaluated since May 2019. Dose level 1 (20 mg) was completed without any DLTs; enrollment to next dose level (40 mg) was initiated in June 2019 . This new cohort is currently recruiting patients in the Republic of Korea and the USA. Clinical trial information: NCT03850574. 3,800 characters allowed 3,580 characters in abstract Disclosures Daver: Pfizer: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Agios: Consultancy; Otsuka: Consultancy; Forty-Seven: Consultancy; Immunogen: Consultancy, Research Funding; Sunesis: Consultancy, Research Funding; NOHLA: Research Funding; Glycomimetics: Research Funding; Karyopharm: Consultancy, Research Funding; Jazz: Consultancy; Hanmi Pharm Co., Ltd.: Research Funding; Celgene: Consultancy; BMS: Consultancy, Research Funding; Astellas: Consultancy; Incyte: Consultancy, Research Funding; Servier: Research Funding; Novartis: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding.

Quizartinib (AC220) reverses ABCG2-mediated multidrug resistance: In vitro and in vivo studies.

Previous reports have shown that some tyrosine kinase inhibitors (TKIs) could inhibit the ATP-binding cassette (ABC) transporters involved in multidrug resistance (MDR). Quizartinib (AC220), a potent class III receptor tyrosine kinase inhibitor (TKI), was synthesized to selectively inhibit FMS-like tyrosine kinase-3 (FLT3), a target in the treatment of acute myeloid leukemia (AML). Quizartinib is currently under clinical trials for FLT3 ITD and wild-type AML and is tested in combination with chemotherapy. While non-toxic to cell lines, quizartinib at 3 μM showed significant reversal effect on wild-type and mutant ABCG2 (R482T)-mediated MDR, and only a moderate reversal effect on mutant ABCG2 (R482G)-mediated MDR. Results also showed that quizartinib reversed MDR not by reducing the expression of ABCG2 protein, but by antagonizing the drug efflux function and increasing the intracellular accumulation of substrate anticancer drugs in ABCG2-overexpressing cells. Importantly, quizartinib at 30 mg/kg strongly enhanced the effect of topotecan (3 mg/kg) in ABCG2-overexpressing (H460/MX20) xenografts in athymic nude mice. These results demonstrated that quizartinib potentiates the antineoplastic activity of wild-type and R482T mutant ABCG2 substrates. These findings may be useful in clinical practice for cancer combination therapy with quizartinib.

Feasibility of Allogeneic Hematopoietic Stem Cell Transplant for High Risk FLT3-ITD Mutant Patients with Acute Myeloid Leukemia in CR1- a Real Word Analysis

Background: Hematopoietic stem cell transplantation (HCT) remains the most effective postremission therapy for high-risk acute myeloid leukemia (AML). FMS-like tyrosine kinase3 (FLT3) mutations occur in about 30% of patients with cytogenetically normal AML. FLT3-ITD (internal tandem duplications) mutations are associated with short-lived remissions and poor outcomes. These adverse effects can be offset by allogeneic HCT in first complete remission (CR1). We carried out this study to get a real world perspective on feasibility of allogeneic HCT in CR1 for FLT3-ITD mutant AML patients. Methods: The Mayo Clinic AML database was queried for all patients that had tested positive for the FLT3 ITD, from January 2003 to December 2014. 75 patients were identified, of which 28 (37%) underwent allogeneic HCT in CR1. 12 were excluded as they were transplant ineligible from an age standpoint, 6 patients actually had MDS and 2 were excluded for primary induction failure. Clinical and outcomes data were abstracted retrospectively. Results: 29 patients were included in the final analysis, with a median age of 57 years (range 21-68), 38% males. Median time from diagnosis to last follow up was 339 days. 24(83%) had normal cytogenetics. 26(90%) received treatment with anthracycline + cytarabine based therapy, 3(10%) were enrolled on a clinical trial. 25(87%) patients went on to receive consolidation therapy. 22 (75%) of the patients relapsed after achieving a CR1, with a median time to relapse of 134 days (range 27-1710 days). Only 8 (26%) of the 22 patients were able to achieve a CR2 with salvage chemotherapy, of which 6 (75 %) successfully underwent allogeneic HCT.2 (25%) out of 8 patients received FLT3 inhibitor in 1st relapse. We then assessed the reasons why relapsed patients did not undergo HCT in CR1. The most common being; relapse during evaluation/work up for HCT in 8(27%) patients, 5 (17%) had comorbidities that precluded HCT, 3 (10%) were never referred for HCT due to personal preferences. At last follow up 19(65%) patients were dead. Conclusion: While allogeneic HCT is an effective consolidative strategy for FLT3 ITD mutant AML patients, in the real world, a large number are not transplanted in CR1 due to delays in donor search and transplant scheduling strategies resulting in disease relapse. Effective strategies to avoid delays in scheduling, rapid donor searches and the use of alternative donor sources are much needed for these patients. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Dual E-Selectin/CXCR4 Antagonist GMI-1359 Exerts Efficient Anti-Leukemia Effects in a FLT3 ITD Mutated Acute Myeloid Leukemia Patient-Derived Xenograft Murine Model

Background: Acute myeloid leukemia (AML) is a clinically and molecularly heterogeneous disease with poor outcome. Targeted therapy of FMS-like tyrosine kinase-3 (FLT3)-mutated AML patients using small molecular inhibitors including sorafenib showed clinical success in reducing leukemia blasts in peripheral blood, but has limited effect on leukemic stem cells in the bone marrow (BM) microenvironment (1, 2). The BM is presumed to be the reservoir for leukemia stem cells (LSCs) which persist under targeted therapy and mediate disease relapse. The interaction of leukemic blasts with the BM microenvironment mediated by receptor-ligand axes such as CXCR4/SDF-1, E-selectin/HECA-452, and cell-cell contact have also been associated with drug resistance in FLT3 mutated AML (3-5). We recently reported that targeting CXCR4/E-selectin with the dual inhibitor GMI-1359 (GlycoMimetics, Inc., Rockville, MD) showed efficient mobilization of leukemia cells into the circulation and significant prolongation of survival of mice in FLT3-ITD mutant AML cells-xenografted murine model (Zhang et al., ASH Abstract #3790, 2015; Zhang et al., AACR Abstract #3284, 2016).Methods: In the present study, we further evaluated anti-leukemia effects of the dual CXCR4/E-selectin inhibitor GMI-1359 in a patient-derived xenograft (PDX) murine model. Primary FLT3 ITD mutated AML cells were isolated from BM aspirates and injected into irradiated NSG mice. Human CD45+/CD33+ cells were isolated from spleens of secondary engrafted mice after 3-6 months. The PDX leukemic cells were injected into irradiated NSG mice via tail vein (2x106 cells/mouse) and mice received either GMI-1359 (40mg/kg) or sorafenib (10mg/kg), or combination treatment starting at Day 18 after injection of leukemia cells. Vehicle treated mice served as controls. Leukemic cell burden was evaluated by flow cytometry (hCD45+ cells), histology and immunohistochemistry.Results: GMI-1359 treatment alone efficiently mobilized leukemic cells into blood circulation (absolute hCD45+ cells: 23.24k/UL vs. 9.06k/UL, respectively, in GMI-1359 vs. vehicle treatment groups after 71 days of treatment). Sorafenib alone or combined with GMI-1359 enhanced apoptosis induction and markedly reduced hCD45+ cells in circulation, BM and liver. The combination treatment profoundly reduced spleens leukemia cell infiltration (immunohistochemistry of CD45). A novel and unexpected finding of this study was that the combination of GMI-1359 with sorafenib significantly increased the regeneration of normal bone marrow cell populations (erythropoiesis, myelopoiesis and megakaryopoiesis) in the PDX model analyzed by histomorphology. Morphology and frequency of the hematopoiesis were identified in normal bone marrow of non-tumor bearing mice, suggesting a partial treatment response to the combination of GMI-1359 and sorafenib. In addition, GMI-1359 alone, sorafenib alone, or the combination treatment profoundly extended survival of PDX mice compared to vehicle treatment (98, 98 and 98 days vs 60.5 days, respectively (p = 0.04)).Conclusions: The dual CXCR4/E-selectin inhibitor GMI-1359 alone or in combination with the FLT3 ITD inhibitor sorafenib demonstrated anti-leukemia effects in a PDX model of FLT3 ITD mutated AML. Interestingly, combining GMI-1359 with sorafenib strikingly increased normal hematopoiesis in the bone marrow. The underlying mechanisms are under investigation. DisclosuresFogler:GlycoMimetics, Inc.: Employment. Magnani:GlycoMimetics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Responsiveness of FLT3-ITD+ AML to Kinase Inhibition Correlates with High Allele Ratio and Relapsed Disease but Not to Inhibition of Canonical Targets

Fms-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase and is the most commonly mutated gene in acute myeloid leukemia (AML). Second generation FLT3 inhibitors such as quizartinib (AC220) are clinically active in relapsed FLT3-ITD+ patients. However, not all patients respond and to date primary resistance has not been characterized. A previous study proposed that AML cells from patients with relapsed FLT3 mutant AML and samples with high allelic burden for FLT3 ITD are more sensitive to FLT3 inhibitor cytotoxicity (Pratz KW, et al Blood 2010). We performed studies to test this hypothesis and determine if cells with homodimeric FLT3 ITD are more heavily dependent on FLT3 ITD for growth and survival than cells expressing heterodimeric FLT3 WT:FLT3 ITD. 16 primary AML samples that contain FLT3 ITD mutations were incubated in increasing concentrations of the second generation FLT3 inhibitor crenolanib and assayed for survival in short term liquid culture assays. Only 6 samples demonstrated greater than 30% inhibition of survival in this culture system whereas 10 samples showed little or no cytotoxic response. Consistent with previous results (Pratz, et. al, Blood 2010), responding samples tended to be from relapsed patients and to have higher FLT3 ITD allelic ratios. We then analyzed FLT3 expression and phosphorylation levels as well as inhibition of and crenolanib inhibition of FLT3 phosphorylation, as well as the canonical downstream signal transduction pathways STAT5, ras/MAPK, and PI3K/AKT/mTOR in 13 FLT3-ITD+ primary AML samples. For 11/13 samples, crenolanib strongly inhibited phosphorylation of FLT3 kinase. However, neither FLT3 protein expression nor baseline phosphorylation level correlated with cytotoxic response in liquid culture assays. Crenolanib inhibited phosphorylation of STAT5, ribosomal S6 and ERK to varying degrees and inhibition of none of these pathways consistently correlated with cytotoxicity. Overall, these results are consistent with the hypothesis that FLT3 ITD mutant AML with high allele burden or relapsed samples are more addicted to FLT3 ITD. To further examine this topic, we studied AML cell lines that express only wild type FLT3 (THP1), both FLT3-ITD and WT FLT3 (MOLM14) or FLT3-ITD but not WT-FLT3 (Mv4;11, TF1-ITD). Growth of all three cell lines expressing FLT3-ITD but not THP1 cells was inhibited by crenolanib. Crenolanib inhibited tyrosine phosphorylation and activation of downstream signaling pathways in all three FLT3-ITD+ cell lines. Importantly, crenolanib was active at 10 nM in TF1-ITD cells but higher concentrations were required to inhibit signaling in Mv4;11 cells. This suggests that allele ratio alone does not determine sensitivity to FLT3 inhibitors. Interestingly, FLT3 ligand (FL) impairs inhibition of FLT3 by kinase inhibitor and, again, mutant cell lines are similarly responsive to FL in the presence of kinase inhibitor. In summary, these data demonstrate that AML cells have variable dependence on mutant FLT3 for survival. Samples with high allele ratio and relapsed samples behave in a manner consistent with oncogene addiction and are likely to show cytotoxicity to FLT3 inhibition. However, the basis for oncogene addiction is unclear and does not depend on activation of the canonical signal transduction pathways known to be downstream of FLT3. Interestingly, a recent unbiased screen of phosphorylated proteins in FLT3-ITD+ AML to predict clinical response to AC220 did not identify tyrosine phosphorylation of canonical FLT3 targets, but correlated clinical response with serine phosphorylation on EEPD1-S160, BCL11A-S630, and RANBP3-S333 (Schaab C, et al. Leukemia 2014). Analysis of the effects of FLT3 inhibitors upon these proteins in FLT3 mutant primary samples and cell lines is ongoing. DisclosuresPerl:Arog pharmaceuticals: Consultancy; Ambit Biosciences: Consultancy.

Results of a Phase I Open-Label Study of Decitabine in Combination with Midostaurin (PKC412) for Elderly (Age ≥ 60) Newly Diagnosed or Relapsed/Refractory Adult Patients with Acute Myeloid Leukemia,

Abstract 3610The activated FMS-like tyrosine kinase 3 (FLT3) receptor promotes proliferation, survival and differentiation of primitive hematopoietic progenitor cells, with downstream targets including the Raf/MEK/ERK pathway. Methylation-associated silencing inactivates certain tumor suppressor genes as effectively as some mutations potentially acting as one of the cancer predisposing hits of the two-hit = leukemogenesis hypothesis. Methods:We present data from a phase I, single-center study designed to determine the MTD and recommended Phase II dose of midostaurin (PKC412), a FLT3 inhibitor, combined either sequentially (days 8–21) or concurrently (days 1–28) with decitabine 20 mg/m2 days 1–5 in AML patients. Additional endpoints: tolerability; exploratory activity assessments. Adult AML patients with relapsed or refractory (R/R) disease or elderly (≥ 60 years) patients unable to receive standard induction chemotherapy, with ECOG PS ≤2, were eligible and received treatment for 3 cycles or until disease progression. Previous hypomethylating agents were allowed. Results:Enrollment is complete with 16 patients (Table 1) with 15 evaluable for safety (1 pt withdrew prior to receiving any midostaurin). One patient in cohort 1 remains on study in cycle 14. Median number of cycles completed was 2 with 5 patients receiving 3 or more cycles. Median age was 68 years (range 48–81) with 9 males. The number of newly diagnosed and R/R patients was evenly split at 8 each. Of note, 3 of 8 patients (38%) with R/R disease had relapsed subsequent to allogeneic hematopoietic stem cell transplant (HSCT). Only 2 of 16 patients (13%) had FLT3 ITD mutations and no patient had KIT mutations. Additionally, 44% of patients had received prior therapy with either azacitidine or decitabine.Table 1Age and GenderCohortAML StatusMutation StatusMidostaurin DoseDecitabine DoseNumber of Cycles completedPrior hypomethylating therapyBest Response (CR, PR, SD, PD)Reason for Study Withdrawal81/M1NewFLT3 WT; KIT-4NoCRVoluntary74/F1R/RFLT3 WT; KIT-25 mg bid (d 8–21)2YesSDPoor QOL71/M1R/RFLT3 WT; KIT-14NoPRStill on study66/F2R/RFLT3 WT; KIT-—YesPDProgression63/F2NewFLT3 MUT; KIT-—NoNot evaluableDLT (QTc > 500)70/F2NewFLT3 WT; KIT-—NoNot evaluableVoluntary48/M2R/RFLT3 MUT; KIT-4NoCRHSCT64/M2NewFLT3 WT; KIT-50 mg bid (d 8–21)20mg/m2 IV (d 1–5)2YesCRiHSCT74/M2R/RFLT3 WT; KIT-7YesSDProgression68/M2NewFLT3 WT; KIT-1NoPRFungal Pneumonia65/F2R/R (HSCT)FLT3 WT; KIT-1NoSDViral Pneumonia54/F2R/R (HSCT)FLT3 WT; KIT-1NoSDFungal Pneumonia66/M2NewFLT3 WT; KIT-3NoSDProgression68/F3R/R (HSCT)FLT3 WT; KIT-1YesPDProgression76/M3NewFLT3 WT; KIT-50 mg bid (d 1–28)—YesNot evaluableDLT (Pulmonary)80/M3NewFLT3 WT; KIT-—YesNot evaluableDLT (Pulmonary) Safety profile:All patients whom received treatment (15) experienced adverse events (AE), the majority of which were Grade 1/2 in severity and included fatigue, nausea, mouth sores, and insomnia. Most AEs were hematologic, including anemia, thrombocytopenia, and neutropenia as expected in this population. Three patients developed dose limiting toxicities: 2 patients in cohort 3 developed pulmonary edema requiring mechanical ventilation and 1 patient in cohort 2 developed a prolonged QTc > 500 msec. All but two of 15 patients required admission for neutropenic fever. Three patients developed fungal (2) or viral pneumonia (1) and expired. However, most study treatments were given in the clinic allowing patients to spend a majority of the time as an outpatient. Response:Of twelve patients evaluable for response, 10 (83%) achieved stable disease (SD) or better while on trial. Three of the 12 patients (25%) had a complete response (CR or CRi) after 1 cycle (28 days) and 2 of these patients were able to undergo allogeneic HSCT. Of the two patients with FLT3 ITD mutations, one patient was evaluable for response and achieved a CR (cohort 2). Pharmacokinetic analysis is ongoing. Conclusion:These data indicate that the combination of decitabine with sequential midostaurin is possible without significant unexpected toxicity. The MTD for this combination was determined to be decitabine 20mg/m2 IV daily × 5 days (days 1–5) followed by midostaurin 50mg po bid days 8–21 given every 4 weeks. In addition, the combination shows promising clinical activity in R/R and untreated AML in elderly patients. Disclosures:No relevant conflicts of interest to declare.

Anchoring of FLT3 in the endoplasmic reticulum alters signaling quality

AbstractThe mechanism of cell transformation by Fms-like tyrosine kinase 3 (FLT3) in acute myeloid leukemia (AML) is incompletely understood. The most prevalent activated mutant FLT3 ITD exhibits an altered signaling quality, including strong activation of the STAT5 transcription factor. FLT3 ITD has also been found partially retained as a high-mannose precursor in an intracellular compartment. To analyze the role of intracellular retention of FLT3 for transformation, we have generated FLT3 versions that are anchored in the perinuclear endoplasmic reticulum (ER) by appending an ER retention sequence containing a RRR (R3) motif. ER retention of R3, but not of corresponding A3 FLT3 versions, is shown by biochemical, fluorescence-activated cell sorting, and immunocytochemical analyses. ER anchoring reduced global autophosphorylation and diminished constitutive activation of ERK1/2 and AKT of the constitutively active FLT3 versions. ER anchoring was, however, associated with elevated signaling to STAT3. Transforming activity of the FLT3 D835Y mutant was suppressed by ER anchoring. In contrast, ER-anchored FLT3 ITD retained STAT5-activating capacity and was transforming in vitro and in vivo. The findings highlight another aspect of the different signaling quality of FLT3 ITD: It can transform cells from an intracellular location.

Tyrosine Phosphorylation Regulates Maturation of Receptor Tyrosine Kinases

Constitutive activation of receptor tyrosine kinases (RTKs) is a frequent event in human cancer cells. Activating mutations in Fms-like tyrosine kinase 3 (FLT-3), notably, internal tandem duplications in the juxtamembrane domain (FLT-3 ITD), have been causally linked to acute myeloid leukemia. As we describe here, FLT-3 ITD exists predominantly in an immature, underglycosylated 130-kDa form, whereas wild-type FLT-3 is expressed predominantly as a mature, complex glycosylated 150-kDa molecule. Endogenous FLT-3 ITD, but little wild-type FLT-3, is detectable in the endoplasmic reticulum (ER) compartment. Conversely, cell surface expression of FLT-3 ITD is less efficient than that of wild-type FLT-3. Inhibition of FLT-3 ITD kinase by small molecules, inactivating point mutations, or coexpression with the protein-tyrosine phosphatases (PTPs) SHP-1, PTP1B, and PTP-PEST but not RPTPalpha promotes complex glycosylation and surface localization. However, PTP coexpression has no effect on the maturation of a surface glycoprotein of vesicular stomatitis virus. The maturation of wild-type FLT-3 is impaired by general PTP inhibition or by suppression of endogenous PTP1B. Enhanced complex formation of FLT-3 ITD with the ER-resident chaperone calnexin indicates that its retention in the ER is related to inefficient folding. The regulation of RTK maturation by tyrosine phosphorylation was observed with other RTKs as well, defines a possible role for ER-resident PTPs, and may be related to the altered signaling quality of constitutively active, transforming RTK mutants.

Phase 1 Study of PKC412, an Oral FLT3 Kinase Inhibitor, in Sequential and Concomitant Combinations with Daunorubicin and Cytarabine (DA) Induction and High-Dose Cytarabine (HDAra-C) Consolidation in Newly Diagnosed Patients with AML.

The Fms-like tyrosine kinase 3 (FLT3), a receptor expressed in 80% of AML patients, is constitutively activated in 25–35% of adult AML patients due to mutations, in the form of either ITDs or activating loop point mutations, most commonly at D835Y. PKC412 is an oral multi-targeted tyrosine kinase inhibitor known to potently inhibit both activated wild type (WT) and mutated (mut) FLT3 kinase. PKC412 (100 mg po bid) was tested in a phase IB study in newly diagnosed AML patients between 18 and 60 years of age (both WT and mut FLT3) in combinations with DA for induction therapy (up to 2 cycles) and HDAra-C for consolidation (up to 3 cycles).Study design: PKC412 was given daily beginning on Day 8 (Arm 1) or Day 1 (Arm 2) of induction chemoRX in cycle 1. Primary endpoints were PK, tolerability, and safety as defined by fewer than 2 drug-related deaths in the initial cohort of 6 pts per arm (stage 1) and fewer than 4 drug-related deaths among the total 20 pts per arm (stage 2) during induction.Results: 15 pts were enrolled (median age 50; 1 FLT3 ITD+, 14 WT; ): 7 in Arm 1 and 8 in Arm 2. Cytogenetic prognostic categories of the 15 patients were: 0 (good), 9 (intermediate) and 6 (bad). Of the 15 pts, 14 were evaluable for toxicity; 1 pt did not receive PKC412; no drug-related deaths were observed during induction therapy. The most common G3 drug-related toxicities included: nausea (46%), vomiting (32%), transaminitis (18%), and hyperbilirubinemia (18%), all were transient and/or reversible. 13/15 patients were evaluable for response: 2 pts were not evaluated: one pt never received PKC412 and one pt did not complete the full-course of induction chemoRx; 3/7 (43%) achieved a CR in Arm 1 and 3/6 (50%) achieved a CR in Arm 2. 3 pts have completed the study (Days 143, 141, 132) and one pt is ongoing (Day 127). Because of the high rate of G3 nausea and vomiting, the study was amended, (5 patients are ongoing) such that PKC412 is given with each cycle of chemoRx on either Days 1–7 and 15–21 (Arm 1), or on Days 8–21 (Arm 2).Conclusions: PKC412 can be given safely in newly diagnosed AML patients in combinations with DA and HDAra-C chemoRx. An update of this study, along with PK data of PKC412, daunorubicin, and Ara-C levels will be presented.

CHIR258: A Multitargeted Growth Factor Receptor Tyrosine Kinase (RTK) Inhibitor Induces Significant Apoptosis of Human FLT3 ITD Acute Myelogenous Leukemia Cells In Vitro and in Vivo.

Fms-like tyrosine kinase 3 (FLT3) encodes a receptor tyrosine kinase (RTK) for which activating mutations have been identified in a proportion of acute myelogenous leukemia (AML) patients and associated with poor clinical prognosis. CHIR258, a novel, orally active, small molecule exhibits potent activity against FLT3 kinase and other RTKs involved in myeloid leukemias as well as endothelial and solid tumor cell proliferation (biochemical IC50 for FLT3 = 1 nM; cKIT = 2 nM; VEGFR1/2/3 ~ 8 nM, FGFR1/3 ~ 10nM, PDGFRβ = 27 nM, CSF-R1 = 36 nM). CHIR258 was tested in two human leukemic cell lines with differing FLT3 mutational status (MV4;11, FLT3 ITD vs. RS4;11, FLT3 WT). Cell cycle analysis of leukemic cells incubated with CHIR258 (0.1 μM, 48 h) showed a significant increase in the sub-Go/G1 cell population in MV4;11cells compared to the RS4;11 cells. The pro-apoptotic activity of CHIR258 against MV4;11 cells in culture was confirmed by AnnexinV staining, and appearance of cleaved poly ADP-ribose polymerase (PARP) after 24 h. Interestingly, cleaved PARP fragments were detected with either pulsed (1 h) or continuous incubations of MV4;11 cells with CHIR258. In addition, cell cycle arrest and apoptosis was consistent with degradation of survivin and loss of phosphorylation of Retinoblastoma (Rb) protein observed at CHIR258 concentrations ≥ 0.1 μM. The mechanism of action of CHIR258 was examined in a subcutaneous (s.c.) xenograft model of leukemia in SCID-NOD mice. In s.c. xenografts, daily oral doses of CHIR258 resulted in dose-dependent increased efficacy against FLT3-ITD MV4;11 tumors compared to RS4;11 WT tumors. Modulation of FLT3 downstream signaling molecules (p-FLT3, p-ERK) was achieved in MV4;11 tumors at biologically active doses in vivo, confirming molecular mechanism of action. Target modulation (p-FLT3) was maintained for up to 24 h post-dose. MV4;11 tumor responses to CHIR258 were evidenced within 1–2 days of initiating drug treatment. Immunohistochemical analyses of MV4;11 tumors revealed that early responses to CHIR258 correlated well with decreased tumor cellularity, and proliferation. Tumors stained positive for active caspase-3 and cleaved PARP suggesting cell death was mainly mediated via apoptosis. Efficacy was also demonstrated in an intravenous MV4;11 bone marrow (BM) engraftment model, wherein daily CHIR258 treatments substantially increased the life span of mice, with 33% long term survivors or “cures”. Strikingly, CHIR258-treated mice were devoid of MV4;11 tumor cells in BM as confirmed by flow cytometry and immunohistochemical analyses. In conclusion, our data indicates that CHIR258 might become a highly effective therapy in AML associated with FLT3 expression and warrants clinical trials with AML patients.

Effects of MLN518, a dual FLT3 and KIT inhibitor, on normal and malignant hematopoiesis

AbstractInternal tandem duplications (ITDs) of the FMS-like tyrosine kinase 3 (FLT3) receptor tyrosine kinase are found in approximately 30% of patients with acute myelogenous leukemia (AML) and are associated with a poor prognosis. FLT3 ITD mutations result in constitutive kinase activation and are thought to be pathogenetically relevant, implicating FLT3 as a plausible therapeutic target. MLN518 (formerly CT53518) is a small molecule inhibitor of the FLT3, KIT, and platelet-derived growth-factor receptor (PDGFR) tyrosine kinases with significant activity in murine models of FLT3 ITD-positive leukemia. Given the importance of FLT3 and KIT for normal hematopoietic progenitor cells, we analyzed the effect of MLN518 on murine hematopoiesis under steady-state conditions, after chemotherapy-induced myelosuppression, and during bone marrow transplantation. In these assays, we show that MLN518 has mild toxicity toward normal hematopoiesis at concentrations that are effective in treating FLT3 ITD-positive leukemia in mice. We also demonstrate that MLN518 preferentially inhibits the growth of blast colonies from FLT3 ITD-positive compared with ITD-negative patients with AML, at concentrations that do not significantly affect colony formation by normal human progenitor cells. In analogy to imatinib mesylate in BCR-ABL–positive acute leukemia, MLN518-induced remissions may not be durable. Our studies provide the basis for integrating this compound into chemotherapy and transplantation protocols.