A 72-year-old woman with relapsed FLT3-ITD-positive acute myeloid leukemia was treated with gilteritinib and achieved complete remission with incomplete hematological recovery. However, two months later, she developed optic nerve infiltration and lost vision in her right eye while maintaining hematological remission on gilteritinib. Intrathecal injection of cytotoxic drugs reduced the number of blasts in the cerebrospinal fluid (CSF), but her vision did not recover. At the onset of optic nerve infiltration, at a dose of 80 mg/day gilteritinib, the plasma trough and CSF levels of gilteritinib were 151.9 ng/ml and 1.9 ng/ml, respectively, with a central nervous system (CNS) penetration rate of 1.3%. Hematologic progressive disease (PD) was detected after 40 days, and the patient died one month later. Target sequencing at the time of hematologic PD revealed the FLT3 F691L mutation, which is known to confer resistance to gilteritinib. In this patient, pharmacokinetic (low CNS penetration of gilteritinib) and pharmacodynamic (acquisition of a drug resistance mutation) mechanisms were thought to be responsible for the CNS relapse and hematologic PD, respectively. We believe this is a valuable case to report considering the scarcity of data on CNS penetration of FLT3 inhibitors and their effects on CNS disease in the literature.

BACKGROUND: FLT3-ITD AML is associated with an increased risk of relapse, leading many patients to receive an allogeneic hematopoietic stem cell transplantation (alloHCT) after induction. Unfortunately, relapse rate after alloHCT remains high and strategies are needed to improve outcomes.
METHODS: We performed a retrospective analysis of adult patients with FLT3-ITD AML who received alloHCT from 6/1/2016 to 12/31/2020 and received gilteritinib (GILT) or sorafenib (SORA)as post-transplant maintenance, outside of a clinical trial.
RESULTS: A total of 55 patients were treated with either GILT (n = 27) or SORA (n = 29) for post-HCT maintenance. One patient was treated with SORA after first alloHCT and GILT after second alloHCT. Patient characteristics were comparable between groups. FLT3 inhibitors were utilized in pre-alloHCT therapy in all but 1 patient. The median duration of time that patients remained on GILT was 385 days (range, 10-804) and on SORA 315 days (range, 3-1777). 1-year PFS and relapse incidence were similar between GILT and SORA; PFS was 66% versus 76% (P = .4) and relapse incidence was 19% versus 24% (P = .6), respectively.Both groups had high incidence of Grade 3-4 hematological toxicity, including neutropenia (45% GILT and 34% SORA) and thrombocytopenia (30% GILT and 52% SORA). Only 44% and 14% patients who received GILT and SORA did not discontinue maintenance, respectively.
CONCLUSIONS: Our results revealed comparable PFS and a similar toxicity profile when SORA and GILT are used as post- HCT maintenance therapy.

ETV6::ABL1 is a rare fusion gene that found in MPN, ALL, and AML. It has a complex and diverse formation mechanism due to the reciprocal orientations of the ETV6 and ABL1 genes relative to the centromeres. NPM1 is frequently mutated in adult AML, often accompanied by FLT3-ITD, which suggests molecular synergisms in AML pathogenesis. Previous reports on ETV6::ABL1 mostly focus on FLT3-ITD. In this study, we present a case of AML with ETV6::ABL1, along with NPM1 and FLT3-ITD. The patient showed a rapid increase in primitive cells at the initial stage, along with the presence of immature granulocytes and erythrocytes. Through cytogenetic analysis, fluorescence in situ hybridization (FISH), and RNA-seq, we elucidated the mechanism behind the formation of the ETV6::ABL1 fusion gene. Despite conventional chemotherapy failure and rapid tumor proliferation, we attempted to add FLT3 inhibitor sorafenib to the treatment, along with chemotherapy bridging to haploidentical transplantation. After haplo-HSCT, a combination of sorafenib and dasatinib was administered as maintenance therapy. The patient achieved complete remission (CR) and maintained it for 11 months. The intricate genetic landscape observed in this case presents diagnostic dilemmas and therapeutic challenges, emphasizing the importance of a comprehensive understanding of its implications for disease classification, risk stratification, and treatment selection.

BACKGROUND: FLT3 gene mutations are genetic abnormality that caused leukemogenesis. Furthermore, presence of FLT3 mutations is associated with poor prognosis in AML. This study aimed to identify FLT3 gene mutations so that it can be used as a genetic reference for the AML patients in Indonesian population.
METHODS: This cross-sectional study recruited 63 AML de novo patients between August 2021 and July 2023 at Cipto Mangukusumo General Hospital and Dharmais Cancer Hospital. We collected peripheral blood from the patients for DNA isolation. FLT3 gene mutation was detected using PCR method, then followed by the Sanger sequencing. Novel mutation in exon-14 continued to in silico study using SWISS MODEL server for modelling protein and PyMOL2 software for visualizing the protein model.
RESULTS: Frequency FLT3-ITD mutation was 22% and 6 (10%) patients had a novel mutation on juxtamembrane domain. The number of FLT3-ITD insertions was 24 bp to 111 bp, with a median of 72 bp. Novel mutation indicated a change in the protein sequence at amino acid number 572 from Tyrosine to Valine and formed a stop codon (UGA) at amino acid position ins572G573. In-silico study from novel mutation showed the receptor FLT3 protein was a loss of most of the juxtamembrane domain and the entire kinase domain.
CONCLUSIONS: A novel FLT3 gene mutation was found in this study in the juxtamembrane domain. Based on the sequencing analysis and in silico studies, this mutation is likely to affect the activity of the FLT3 receptor. Therefore, further studies on this novel mutation are needed.

Fms-like tyrosine kinase 3 (FLT3) mutations, present in over 30% of acute myeloid leukemia (AML) cases and dominated by FLT3-internal tandem duplication (FLT3-ITD), are associated with poor outcomes in patients with AML. While tyrosine kinase inhibitors (TKIs; e.g., gilteritinib) are effective, they face challenges such as drug resistance, relapse, and high costs. Here, we report that metformin, a cheap, safe, and widely used anti-diabetic agent, exhibits a striking synergistic effect with gilteritinib in treating FLT3-ITD AML. Metformin significantly sensitizes FLT3-ITD AML cells (including TKI-resistant ones) to gilteritinib. Metformin plus gilteritinib (low dose) dramatically suppresses leukemia progression and prolongs survival in FLT3-ITD AML mouse models. Mechanistically, the combinational treatment cooperatively suppresses polo-like kinase 1 (PLK1) expression and phosphorylation of FLT3/STAT5/ERK/mTOR. Clinical analysis also shows improved survival rates in patients with FLT3-ITD AML taking metformin. Thus, the metformin/gilteritinib combination represents a promising and cost-effective treatment for patients with FLT3-mutated AML, particularly for those with low income/affordability.

FLT3-ITD is a type of poor prognostic factors in acute myeloid leukemia (AML) disease. Gilteritinib, the second-generation FLT3 tyrosine kinase inhibitor, improved the overall survival of patients with relapsed/refractory FLT3-mutated AML in the ADMIRAL phase III trial. However, few data are available on the efficacy and safety of gilteritinib-based therapy for FLT3-mutated AML with central nervous system (CNS) involvement. We performed gilteritinib to treat a patient with CNS relapsed AML after allogeneic hematopoietic stem cell transplantation. The positive antileukemic effect of gilteritinib may bring new hope for the treatment of FLT3-mutated AML with CNS relapse.

BACKGROUND: FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is a common mutation type in acute myeloid leukemia (AML) and is usually associated with poor patient prognosis. With advancements in molecular diagnostics and the development of tyrosine kinase inhibitors (TKI), the overall survival (OS) of AML patients with FLT3-ITD mutations has been prolonged to some extent, but relapse and drug resistance are still substantial challenges. Ningetinib is a novel TKI against various kinases in relation to tumour pathogenesis and is undergoing clinical trials of lung cancer. In this study, we explored the antitumor activity of ningetinib against AML with FLT3 mutations both in vivo and in vitro.
METHODS: Cell proliferation assays were performed in AML cell lines and Ba/F3 cells expressing various FLT3 mutations to validate the antileukemic activity of ningetinib in vitro. Immunoblot assays were used to verify the effect of ningetinib on the FLT3 protein and downstream pathways. Molecular docking and CETSA were used to validate the interaction of ningetinib with target proteins. The survival benefit of ningetinib in vivo was assessed in Ba/F3-FLT3-ITD-, MOLM13, Ba/F3-FLT3-ITD-F691L-, MOLM13-FLT3-ITD-F691L-induced leukemia mouse models. We also used patient-derived primary cells to determine the efficacy of ningetinib.
RESULTS: Ningetinib inhibited cell proliferation, blocked the cell cycle, induced apoptosis and bound FLT3 to inhibit its downstream signaling pathways, including the STAT5, AKT and ERK pathways, in FLT3-ITD AML cell lines. In the mouse models with FLT3-ITD and FLT3-ITD-F691L mutation, ningetinib showed superior anti-leukemia activity to existing clinical drugs gilteritinib and quizartinib, significantly prolongating the survival of mice. In addition, ningetinib exhibited activity against patient-derived primary cells harboring FLT3-ITD mutations.
CONCLUSIONS: Overall, our study confirmed the therapeutic role of ningetinib in AML with FLT3-ITD mutations, providing a potential new option for clinically resistant patients.

Hyperactive FMS-like receptor tyrosine kinase-3 mutants with internal tandem duplications (FLT3-ITD) are frequent driver mutations of aggressive acute myeloid leukemia (AML). Inhibitors of FLT3 produce promising results in rationally designed cotreatment schemes. Since FLT3-ITD modulates DNA replication and DNA repair, valid anti-leukemia strategies could rely on a combined inhibition of FLT3-ITD and regulators of cell cycle progression and DNA integrity. These include the WEE1 kinase which controls cell cycle progression, nucleotide synthesis, and DNA replication origin firing. We investigated how pharmacological inhibition of FLT3 and WEE1 affected the survival and genomic integrity of AML cell lines and primary AML cells. We reveal that promising clinical grade and preclinical inhibitors of FLT3 and WEE1 synergistically trigger apoptosis in leukemic cells that express FLT3-ITD. An accumulation of single and double strand DNA damage precedes this process. Mass spectrometry-based proteomic analyses show that FLT3-ITD and WEE1 sustain the expression of the ribonucleotide reductase subunit RRM2, which provides dNTPs for DNA replication. Unlike their strong pro-apoptotic effects on leukemia cells with FLT3-ITD, inhibitors of FLT3 and WEE1 do not damage healthy human blood cells and murine hematopoietic stem cells. Thus, pharmacological inhibition of FLT3-ITD and WEE1 might become an improved, rationally designed therapeutic option.

FMS-like tyrosine kinase 3 (FLT3) mutations occur in approximately one third of acute myeloid leukemia (AML) patients. FLT3-Internal tandem duplication (FLT3-ITD) mutations are the most common FLT3 mutations and are associated with a poor prognosis. Gilteritinib is a FLT3 inhibitor that is US FDA approved for treating adult patients with relapsed/refractory AML and a FLT3 mutation. While gilteritinib monotherapy has improved patient outcome, few patients achieve durable responses. Combining gilteritinib with venetoclax (VEN) appears to make further improvements, though early results suggest that patients with prior exposure to VEN fair much worse than those without prior exposure. MRX-2843 is a promising inhibitor of FLT3 and MERTK. We recently demonstrated that MRX-2843 is equally potent as gilteritinib in FLT3-ITD AML cell lines in vitro and primary patient samples ex vivo. In this study, we investigated the combination of VEN and MRX-2843 against FLT3-ITD AML cells. We found that VEN synergistically enhances cell death induced by MRX-2843 in FLT3-mutated AML cell lines and primary patient samples. Importantly, we found that VEN synergistically enhances cell death induced by MRX-2843 in FLT3-ITD AML cells with acquired resistance to cytarabine (AraC) or VEN+AraC. VEN and MRX-2843 significantly reduce colony-forming capacity of FLT3-ITD primary AML cells. Mechanistic studies show that MRX-2843 decreases Mcl-1 and c-Myc protein levels via transcriptional regulation and combined MRX-2843 and VEN significantly decreases oxidative phosphorylation in FLT3-ITD AML cells. Our findings highlight a promising combination therapy against FLT3-ITD AML, supporting further in vitro and in vivo testing.

Fms-like tyrosine receptor kinase 3 (FLT3) proteolysis targeting chimeras (PROTACs) emerge as a promising approach to overcome the limitations of FLT3 inhibitors, while the development of orally bioavailable FLT3-PROTACs faces great challenges. Here, we report the rational design and evaluation of a series of Gilteritinib-based FLT3-PROTACs. Among them, B3-2 exhibited the strongest antiproliferative activity against FLT3-ITD mutant AML cells, and significantly induced FLT3-ITD protein degradation. Mechanistic investigations demonstrated that B3-2 induced FLT3-ITD degradation in a ubiquitin-proteasome-dependent manner. More importantly, B3-2 exhibited an oral bioavailability of 5.65%, and oral administration of B3-2 showed good antitumor activity in MV-4-11 xenograft models. Furthermore, B3-2 showed strong antiproliferative activity against FLT3 resistant mutations, highlighting its potential in overcoming drug resistance.