FLT3 tyrosine kinase inhibition modulates PRC2 and promotes differentiation in acute myeloid leukemia.

Internal tandem duplication mutations in fms-like tyrosine kinase 3 (FLT3-ITD) are recurrent in acute myeloid leukemia (AML) and increase the risk of relapse. Clinical responses to FLT3 inhibitors (FLT3i) include myeloid differentiation of the FLT3-ITD clone in nearly half of patients through an unknown mechanism. We identified enhancer of zeste homolog 2 (EZH2), a component of polycomb repressive complex 2 (PRC2), as a mediator of this effect using a proteomic-based screen. FLT3i downregulated EZH2 protein expression and PRC2 activity on H3K27me3. FLT3-ITD and loss-of-function mutations in EZH2 are mutually exclusive in human AML. We demonstrated that FLT3i increase myeloid maturation with reduced stem/progenitor cell populations in murine Flt3-ITD AML. Combining EZH1/2 inhibitors with FLT3i increased terminal maturation of leukemic cells and reduced leukemic burden. Our data suggest that reduced EZH2 activity following FLT3 inhibition promotes myeloid differentiation of FLT3-ITD leukemic cells, providing a mechanistic explanation for the clinical observations. These results demonstrate that in addition to its known cell survival and proliferation signaling, FLT3-ITD has a second, previously undefined function to maintain a myeloid stem/progenitor cell state through modulation of PRC2 activity. Our findings support exploring EZH1/2 inhibitors as therapy for FLT3-ITD AML.

Synergy and antagonism between azacitidine and FLT3 inhibitors.

Synergetic interactions between drugs can make a drug combination more effective. Alternatively, they may allow to use lower concentrations and thus avoid toxicities or side effects that not only cause discomfort but might also reduce the overall survival. Here, we studied whether synergy exists between agents that are used for treatment of acute myeloid leukaemia (AML). Azacitidine is a demethylation agent that is used in the treatment of AML patients that are unfit for aggressive chemotherapy. An activating mutation in the FLT3 gene is common in AML patients and in the absence of specific treatment makes prognosis worse. FLT3 inhibitors may be used in such cases. We sought to determine whether combination of azacitidine with a FLT3 inhibitor (gilteritinib, quizartinib, LT-850-166, FN-1501 or FF-10101) displayed synergy or antagonism. To this end, we calculated dose-response matrices of these drug combinations from experiments in human AML cells and subsequently analysed the data using a novel consensus scoring algorithm. The results show that combinations that involved non-covalent FLT3 inhibitors, including the two clinically approved drugs gilteritinib and quizartinib were antagonistic. On the other hand combinations with the covalent inhibitor FF-10101 had some range of concentrations where synergy was observed.

Identifying novel therapeutic inhibitors to target FMS-like tyrosine kinase-3 (FLT3) against acute myeloid leukemia: a molecular docking, molecular dynamics, and DFT study.

Around 30% of acute myeloid leukemia (AML) patients have triggering mutations in Feline McDonough Sarcoma (FMS)-like tyrosine kinase 3 (FLT3), which has been suggested as a possible therapeutic candidate for AML therapy. Many tyrosine kinase inhibitors are available and have a wide variety of applications in the treatment of cancer by inhibiting subsequent steps of cell proliferation. Therefore, our study aims to identify effective antileukemic agents against FLT3 gene. Initially, well-known antileukemic drug candidates have been chosen to generate a structure-based pharmacophore model to assist the virtual screening of 217,77,093 compounds from the Zinc database. The final hits compounds were retrieved and evaluated by docking against the target protein, where the top four compounds have been selected for the analysis of ADMET. Based on the density functional theory (DFT), the geometry optimization, frontier molecular orbital (FMO), HOMO-LUMO, and global reactivity descriptor values have been evaluated that confirming a satisfactory profile and reactivity order for the selected candidates. In comparison to control compounds, the docking results revealed that the four compounds had substantial binding energies (-11.1 to -11.5 kcal/mol) with FLT3. The physicochemical and ADMET (adsorption, distribution, metabolism, excretion, toxicity) prediction results corresponded to the bioactive and safe candidates. Molecular dynamics (MD) confirmed the better binding affinity and stability compared to gilteritinib as a potential FLT3 inhibitor. In this study, a computational approach has been performed that found a better docking and dynamics score against target proteins, indicating potent and safe antileukemic agents, furthermore in-vivo and in-vitro investigations are recommended.Communicated by Ramaswamy H. Sarma.

TP-0184 inhibits FLT3/ACVR1 to overcome FLT3 inhibitor resistance and hinder AML growth synergistically with venetoclax.

We identified activin A receptor type I (ACVR1), a member of the TGF-ß superfamily, as a factor favoring acute myeloid leukemia (AML) growth and a new potential therapeutic target. ACVR1 is overexpressed in FLT3-mutated AML and inhibition of ACVR1 expression sensitized AML cells to FLT3 inhibitors. We developed a novel ACVR1 inhibitor, TP-0184, which selectively caused growth arrest in FLT3-mutated AML cell lines. Molecular docking and in vitro kinase assays revealed that TP-0184 binds to both ACVR1 and FLT3 with high affinity and inhibits FLT3/ACVR1 downstream signaling. Treatment with TP-0184 or in combination with BCL2 inhibitor, venetoclax dramatically inhibited leukemia growth in FLT3-mutated AML cell lines and patient-derived xenograft models in a dose-dependent manner. These findings suggest that ACVR1 is a novel biomarker and plays a role in AML resistance to FLT3 inhibitors and that FLT3/ACVR1 dual inhibitor TP-0184 is a novel potential therapeutic tool for AML with FLT3 mutations.

Identification of a Selective FLT3 Inhibitor with Low Activity against VEGFR, FGFR, PDGFR, c-KIT, and RET Anti-Targets.

FLT3 is mainly expressed in immune and various cancer cells and is a drug target for acute myeloid leukemia (AML). Recently, FLT3 has also been identified as a potential target for treating chronic pain. Most FLT3 inhibitors (FLT3i) identified to date, including approved drugs such as gilteritinib, midostaurin, ponatinib, quizartinib, and FLT3i in clinical trials, such as quizartinib and crenolanib, also inhibit closely-related kinases that are important for immune (c-KIT), cardiovascular (KDR/VEGFR2, FGFR, PDGFR) or kidney (RET) functions. While the aforementioned FLT3i may increase survival rates in AML, they are neither ideal for AML maintenance therapy nor for non-oncology applications, such as for the treatment of chronic pain, due to their promiscuous inhibition of many kinase anti-targets. Here, we report the identification of new FLT3i compounds that have low activities against kinases that have traditionally been difficult to differentiate from FLT3 inhibition, such as KDR/VEGFR, FGFR, PGFR, c-KIT, and RET. These selective compounds could be valuable chemical probes for studying FLT3 biology in the context of chronic pain and/or may represent good starting points to develop well-tolerated FLT3 therapeutics for non-oncology indications or for maintenance therapy for AML.

Sorafenib plus triplet therapy with venetoclax, azacitidine and homoharringtonine for refractory/relapsed acute myeloid leukemia with FLT3-ITD: A multicenter phase 2 study.

BACKGROUND: Patients with relapsed or refractory acute myeloid leukemia (R/R AML) and FLT3-internal tandem duplication (FLT3-ITD) respond infrequently to salvage chemotherapy. OBJECTIVE: To investigate the efficacy of sorafenib plus triplet therapy with venetoclax, azacitidine, and homoharringtonine (VAH) as a salvage therapy in this population. METHODS: This multicenter, single-arm, phase 2 study was conducted at 12 hospitals across China. Eligible patients had R/R AML with FLT3-ITD (aged 18-65 years) who were treated with VAH. The primary endpoint was composite complete remission (CRc) after two cycles. Secondary outcomes included the overall response rate (ORR), safety, and survival. RESULTS: Between July 9, 2020, and March 19, 2022, 58 patients were assessed for eligibility, 51 of whom were enrolled. The median patient age was 47 years (interquartile range [IQR] 31-57). CRc was 76.5% with ORR of 82.4%. At a median follow-up of 17.7 months (IQR, 8.7-24.7), the median duration of CRc was not reached (NR), overall survival was 18.1 months (95% confidence interval [CI], 11.8-NR) and event-free survival was 11.4 months (95% CI, 5.6-NR). Grade 3 or 4 adverse events occurring in ≥10% of patients included neutropenia in 47 (92.2%), thrombocytopenia in 41 (80.4%), anemia in 35 (68.6%), febrile neutropenia in 29 (56.9%), pneumonia in 13 (25.5%), and sepsis in 6 (11.8%) patients. Treatment-related death occurred in two (3.9%) patients. CONCLUSIONS: The sorafenib plus VAH regimen was well tolerated and highly active against R/R AML with FLT3-ITD. This regimen may be a suitable therapeutic option for this population, but larger population trials are needed to be explored. TRIAL REGISTRATION: Clinical Trials Registry: NCT04424147.

A combinatorial therapeutic approach to enhance FLT3-ITD AML treatment.

Internal tandem duplication mutations of the FMS-like tyrosine kinase-3 (FLT3-ITDs) occur in 25%-30% of patients with acute myeloid leukemia (AML) and are associated with dismal prognosis. Although FLT3 inhibitors have demonstrated initial clinical efficacy, the overall outcome of patients with FLT3-ITD AML remains poor, highlighting the urgency to develop more effective treatment strategies. In this study, we reveal that FLT3 inhibitors reduced protein stability of the anti-cancer protein p53, resulting in drug resistance. Blocking p53 degradation with proteasome inhibitors restores intracellular p53 protein levels and, in combination with FLT3-ITD inhibitors, shows superior therapeutic effects against FLT3-ITD AML in cells, mouse models, and patients. These data suggest that this combinatorial therapeutic approach may represent a promising strategy to target FLT3-ITD AML.

PDP1 is a key metabolic gatekeeper and modulator of drug resistance in FLT3-ITD-positive acute myeloid leukemia.

High metabolic flexibility is pivotal for the persistence and therapy resistance of acute myeloid leukemia (AML). In 20-30% of AML patients, activating mutations of FLT3, specifically FLT3-ITD, are key therapeutic targets. Here, we investigated the influence of FLT3-ITD on AML metabolism. Nuclear Magnetic Resonance (NMR) profiling showed enhanced reshuffling of pyruvate towards the tricarboxylic acid (TCA) cycle, suggesting an increased activity of the pyruvate dehydrogenase complex (PDC). Consistently, FLT3-ITD-positive cells expressed high levels of PDP1, an activator of the PDC. Combining endogenous tagging of PDP1 with genome-wide CRISPR screens revealed that FLT3-ITD induces PDP1 expression through the RAS signaling axis. PDP1 knockdown resulted in reduced cellular respiration thereby impairing the proliferation of only FLT3-ITD cells. These cells continued to depend on PDP1, even in hypoxic conditions, and unlike FLT3-ITD-negative cells, they exhibited a rapid, PDP1-dependent revival of their respiratory capacity during reoxygenation. Moreover, we show that PDP1 modifies the response to FLT3 inhibition. Upon incubation with the FLT3 tyrosine kinase inhibitor quizartinib (AC220), PDP1 persisted or was upregulated, resulting in a further shift of glucose/pyruvate metabolism towards the TCA cycle. Overexpression of PDP1 enhanced, while PDP1 depletion diminished AC220 resistance in cell lines and peripheral blasts from an AC220-resistant AML patient in vivo. In conclusion, FLT3-ITD assures the expression of PDP1, a pivotal metabolic regulator that enhances oxidative glucose metabolism and drug resistance. Hence, PDP1 emerges as a potentially targetable vulnerability in the management of AML.

Constitutively Synergistic Multiagent Drug Formulations Targeting MERTK, FLT3, and BCL-2 for Treatment of AML.

PURPOSE: Although high-dose, multiagent chemotherapy has improved leukemia survival rates, treatment outcomes remain poor in high-risk subsets, including acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) in infants. The development of new, more effective therapies for these patients is therefore an urgent, unmet clinical need. METHODS: The dual MERTK/FLT3 inhibitor MRX-2843 and BCL-2 family protein inhibitors were screened in high-throughput against a panel of AML and MLL-rearranged precursor B-cell ALL (infant ALL) cell lines. A neural network model was built to correlate ratiometric drug synergy and target gene expression. Drugs were loaded into liposomal nanocarriers to assess primary AML cell responses. RESULTS: MRX-2843 synergized with venetoclax to reduce AML cell density in vitro. A neural network classifier based on drug exposure and target gene expression predicted drug synergy and growth inhibition in AML with high accuracy. Combination monovalent liposomal drug formulations delivered defined drug ratios intracellularly and recapitulated synergistic drug activity. The magnitude and frequency of synergistic responses were both maintained and improved following drug formulation in a genotypically diverse set of primary AML bone marrow specimens. CONCLUSIONS: We developed a nanoscale combination drug formulation that exploits ectopic expression of MERTK tyrosine kinase and dependency on BCL-2 family proteins for leukemia cell survival in pediatric AML and infant ALL cells. We demonstrate ratiometric drug delivery and synergistic cell killing in AML, a result achieved by a systematic, generalizable approach of combination drug screening and nanoscale formulation that may be extended to other drug pairs or diseases in the future.

Clinical impact of the genomic landscape and leukemogenic trajectories in non-intensively treated elderly acute myeloid leukemia patients.

To characterize the genomic landscape and leukemogenic pathways of older, newly diagnosed, non-intensively treated patients with AML and to study the clinical implications, comprehensive genetics analyses were performed including targeted DNA sequencing of 263 genes in 604 patients treated in a prospective Phase III clinical trial. Leukemic trajectories were delineated using oncogenetic tree modeling and hierarchical clustering, and prognostic groups were derived from multivariable Cox regression models. Clonal hematopoiesis-related genes (ASXL1, TET2, SRSF2, DNMT3A) were most frequently mutated. The oncogenetic modeling algorithm produced a tree with five branches with ASXL1, DDX41, DNMT3A, TET2, and TP53 emanating from the root suggesting leukemia-initiating events which gave rise to further subbranches with distinct subclones. Unsupervised clustering mirrored the genetic groups identified by the tree model. Multivariable analysis identified FLT3 internal tandem duplications (ITD), SRSF2, and TP53 mutations as poor prognostic factors, while DDX41 mutations exerted an exceptionally favorable effect. Subsequent backwards elimination based on the Akaike information criterion delineated three genetic risk groups: DDX41 mutations (favorable-risk), DDX41wildtype/FLT3-ITDneg/TP53wildtype (intermediate-risk), and FLT3-ITD or TP53 mutations (high-risk). Our data identified distinct trajectories of leukemia development in older AML patients and provide a basis for a clinically meaningful genetic outcome stratification for patients receiving less intensive therapies.

Midostaurin plus daunorubicin or idarubicin for young and older adults with FLT3-mutated AML: a phase 3b trial.

The pivotal RATIFY study demonstrated midostaurin (50 mg twice daily) with standard chemotherapy significantly reduced mortality in adult patients (<60 years) with newly diagnosed (ND) FLT3mut acute myeloid leukemia (AML). Considering that AML often present in older patients who show poor response to chemotherapy, this open-label, multicenter phase 3b trial was designed to further assess safety and efficacy of midostaurin plus chemotherapy in induction, consolidation, and maintenance monotherapy in young (≤60 years) and older (>60 years) patients with FLT3mut ND-AML. Compared with RATIFY, this study extended midostaurin treatment from 14 days to 21 days, substituted anthracyclines (idarubicin or daunorubicin), and introduced variation in standard combination chemotherapy dosing ("7+3" or "5+2" in more fragile patients). Total 301 patients (47.2% >60 years and 82.7% with FLT3-ITDmut) of median age 59 years entered induction phase. Overall, 295 patients (98.0%) had at least 1 adverse event (AE), including 254 patients (84.4%) with grade ≥3 AE. The grade ≥3 serious AEs occurred in 134 patients. No difference was seen in AE frequency between age groups, but grade ≥3AE frequency was higher in older patients. Overall, complete remission (CR) rate including incomplete hematologic recovery (CR + CRi) (80.7% [95% confidence interval, 75.74-84.98]) was comparable between age groups (≤60 years [83.5%]; >60 to ≤70 years [82.5%]; in patients >70 years [64.1%]) and the type of anthracycline used in induction. CR + CRi rate was lower in males (76.4%) than females (84.4%). Overall, the safety and efficacy of midostaurin remains consistent with previous findings, regardless of age, sex, or induction regimen. The trial is registered at www.clinicaltrials.gov as #NCT03379727.

Targeting conditioned media dependencies and FLT-3 in chronic lymphocytic leukemia.

The importance of the stromal microenvironment in chronic lymphocytic leukemia (CLL) pathogenesis and drug resistance is well established. Despite recent advances in CLL therapy, identifying novel ways to disrupt interactions between CLL and its microenvironment may identify new combination partners for the drugs currently in use. To understand the role of microenvironmental factors on primary CLL cells, we took advantage of an observation that conditioned media (CM) collected from stroma was protective of CLL cells from spontaneous cell death ex vivo. The cytokine in the CM-dependent cells that most supports CLL survival in short-term ex vivo culture was CCL2. Pretreatment of CLL cells with anti-CCL2 antibody enhanced venetoclax-mediated killing. Surprisingly, we found a group of CLL samples (9/23 cases) that are less likely to undergo cell death in the absence of CM support. Functional studies revealed that CM-independent (CMI) CLL cells are less sensitive to apoptosis than conventional stroma-dependent CLL. In addition, a majority of the CMI CLL samples (80%) harbored unmutated immunoglobulin heavy-chain variable (IGHV) region. Bulk-RNA sequence analysis revealed upregulation of the focal adhesion and RAS signaling pathways in this group, along with expression of fms-like tyrosine kinase 3 (FLT3) and CD135. Treatment with FLT3 inhibitors caused a significant reduction in cell viability among CMI samples. In summary, we were able to discriminate and target 2 biologically distinct subgroups of CLL based on CM dependence with distinct microenvironmental vulnerabilities.

Ferroptosis vulnerability in FLT3-mutant leukemia.

Resistance to FLT3 inhibition is a prevalent challenge in managing FLT3-mutant acute myeloid leukemia (AML). A recent study by Sabatier et al. discovered ferroptosis vulnerability in FLT3-mutant AML and they propose a promising therapeutic approach of combining FLT3 inhibitors with ferroptosis inducers for treating this type of cancer.

Improved efficacy of quizartinib in combination therapy with PI3K inhibition in primary FLT3-ITD AML cells.

Acute myeloid leukemia is a heterogeneous hematopoietic malignancy, characterized by uncontrolled clonal proliferation of abnormal myeloid progenitor cells, with poor outcomes. The internal tandem duplication (ITD) mutation of the Fms-like receptor tyrosine kinase 3 (FLT3) (FLT3-ITD) represents the most common genetic alteration in AML, detected in approximately 30% of AML patients, and is associated with high leukemic burden and poor prognosis. Therefore, this kinase has been regarded as an attractive druggable target for the treatment of FLT3-ITD AML, and selective small molecule inhibitors, such as quizartinib, have been identified and trialled. However, clinical outcomes have been disappointing so far due to poor remission rates, also because of acquired resistance. A strategy to overcome resistance is to combine FLT3 inhibitors with other targeted therapies. In this study, we investigated the preclinical efficacy of the combination of quizartinib with the pan PI3K inhibitor BAY-806946 in FLT3-ITD cell lines and primary cells from AML patients. We show here that BAY-806946 enhanced quizartinib cytotoxicity and, most importantly, that this combination increases the ability of quizartinib to kill CD34+ CD38-leukemia stem cells, whilst sparing normal hematopoietic stem cells. Because constitutively active FLT3 receptor tyrosine kinase is known to boost aberrant PI3K signaling, the increased sensitivity of primary cells to the above combination can be the mechanistic results of the disruption of signaling by vertical inhibition.

Beta Elemene induces cytotoxic effects in FLT3 ITD-mutated acute myeloid leukemia by modulating apoptosis.

OBJECTIVE: ß-Elemene, a sesquiterpene with a broad anti-cancer spectrum, is particularly effective against drug-resistant and complex tumors. It can also be efficient against FLT3-expressed acute myeloid leukemia. This research aims to determine whether ß-Elemene has cytotoxic effects on FLT3 ITD-mutated AML cells. MATERIALS AND METHODS: Cytotoxicity, cell morphology, mRNA analysis with apoptotic markers, and analysis of 43 distinct protein markers related to cell death, survival, and resistance were all performed to elucidate its mechanism. Additionally, in order to understand how ß-Elemene and FLT3 interact, molecular docking, molecular dynamics simulations, and computational ADME investigations were performed. RESULTS: ß-Elemene exhibited cytotoxic activity against FLT3-mutated MV4-11 and FLT3 wild-type THP-1 cells, with an IC50 of around 25 µg/ml. The molecular studies revealed that ß-Elemene inhibited cell proliferation by inducing p53, and the involvement of p21, p27, HTRA, and HSPs were also demonstrated. The interactive inhibition in proliferation was confirmed via molecular docking and dynamics analyses. ß-Elemene occupied the FLT3 enzymatic pocket with good stability at the FLT3 active site. CONCLUSIONS: We concluded from our observations that ß-Elemene causes cell death in ITD mutant AML cells, together with the effects of stress factors and inhibiting cell division.

IGF2BP2 promotes lncRNA DANCR stability mediated glycolysis and affects the progression of FLT3-ITD + acute myeloid leukemia.

Internal tandem duplication (ITD) is the most common type of FLT3 mutation (FLT3-ITD), accounting for about 25% of AML patients. The expression of DANCR in FLT3-ITD AML had not been paid attention to, and whether its regulatory relationship with IGF2BP2 can affect the progression of FLT3-ITD AML was unclear. Our study sought to verify the biological role of IGF2BP2 as an m6A reading protein in FLT3-ITD AML. To further explore the role and mechanism of DANCR in AML, and provide a basis for the screening of biomarkers and the development of targeted drugs. The results show that IGF2BP2 was upregulated in FLT3-ITD+ AML patients and cells. Si-IGF2BP2 could inhibit the proliferation, glycolytic and promote the apoptosis in MV4-11 cells. IGF2BP2 could promote the DANCR RNA stability. This discovery will provide new horizons for early screening and targeted therapy of FLT3-ITD+ AML.

Causal linkage of presence of mutant NPM1 to efficacy of novel therapeutic agents against AML cells with mutant NPM1.

In AML with NPM1 mutation causing cytoplasmic dislocation of NPM1, treatments with Menin inhibitor (MI) and standard AML chemotherapy yield complete remissions. However, the causal and mechanistic linkage of mtNPM1 to the efficacy of these agents has not been definitively established. Utilizing CRISPR-Cas9 editing to knockout (KO) or knock-in a copy of mtNPM1 in AML cells, present studies demonstrate that KO of mtNPM1 from AML cells abrogates sensitivity to MI, selinexor (exportin-1 inhibitor), and cytarabine. Conversely, the knock-in of a copy of mtNPM1 markedly sensitized AML cells to treatment with MI or cytarabine. Following AML therapy, most elderly patients with AML with mtNPM1 and co-mutations in FLT3 suffer AML relapse with poor outcomes, creating a need for novel effective therapies. Utilizing the RNA-Seq signature of CRISPR-edited AML cells with mtNPM1 KO, we interrogated the LINCS1000-CMap data set and found several pan-HDAC inhibitors and a WEE1 tyrosine kinase inhibitor among the top expression mimickers (EMs). Additionally, treatment with adavosertib (WEE1 inhibitor) or panobinostat (pan-HDAC inhibitor) exhibited synergistic in vitro lethal activity with MI against AML cells with mtNPM1. Treatment with adavosertib or panobinostat also reduced AML burden and improved survival in AML xenograft models sensitive or resistant to MI.

Discovery of 4-(4-aminophenyl)-6-phenylisoxazolo[3,4-b]pyridine-3-amine derivatives as novel FLT3 covalent inhibitors for the intervention of acute myeloid leukemia.

Small molecule covalent drugs have proved to be desirable therapies especially on drug resistance related to point mutations. Secondary mutations of FLT3 have become the main mechanism of FLT3 inhibitors resistance which further causes the failure of treatment. Herein, a series of 4-(4-aminophenyl)-6-phenylisoxazolo[3,4-b]pyridine-3-amine covalent derivatives were synthesized and optimized to overcome the common secondary resistance mutations of FLT3. Among these derivatives, compound F15 displayed potent inhibition activities against FLT3 (IC50 = 123 nM) and FLT3-internal tandem duplication (ITD) by 80% and 26.06%, respectively, at the concentration of 1 µM. Besides, F15 exhibited potent activity against FLT3-dependent human acute myeloid leukemia (AML) cell lines MOLM-13 (IC50 = 253 nM) and MV4-11 (IC50 = 91 nM), as well as BaF3 cells with variety of secondary mutations. Furthermore, cellular mechanism assays indicated that F15 inhibited phosphorylation of FLT3 and its downstream signaling factors. Notably, F15 could be considered for further development as potential drug candidate to treat AML.

Outcomes in Patients with FLT3-Mutated Relapsed/ Refractory Acute Myelogenous Leukemia Who Underwent Transplantation in the Phase 3 ADMIRAL Trial of Gilteritinib versus Salvage Chemotherapy.

The fms-like tyrosine kinase 3 (FLT3) inhibitor gilteritinib improved the survival of patients with relapsed or refractory (R/R) FLT3-mutated acute myelogenous leukemia (AML) in the phase 3 ADMIRAL trial. In this study, we assessed survival and relapse rates of patients in the ADMIRAL trial who underwent hematopoietic stem cell transplantation (HSCT), as well as safety outcomes in patients who received post-transplantation gilteritinib maintenance therapy. ADMIRAL was a global phase 3 randomized controlled trial that enrolled adult patients with FLT3-mutated R/R AML. Patients with R/R AML who harbored FLT3 internal tandem duplication mutations in the juxtamembrane domain or D835/I836 point mutations in the tyrosine kinase domain were randomized (2:1) to gilteritinib (120 mg/day) or to preselected high- or low-intensity salvage chemotherapy (1 or 2 cycles). Patients in the gilteritinib arm who proceeded to HSCT could receive post-transplantation gilteritinib maintenance therapy if they were within 30 to 90 days post-transplantation and had achieved composite complete remission (CRc) with successful engraftment and no post-transplantation complications. Adverse events (AEs) during HSCT were recorded in the gilteritinib arm only. Survival outcomes and the cumulative incidence of relapse were assessed in patients who underwent HSCT during the trial. Treatment-emergent AEs were evaluated in patients who restarted gilteritinib as post-transplantation maintenance therapy. Patients in the gilteritinib arm underwent HSCT more frequently than those in the chemotherapy arm (26% [n = 64] versus 15% [n = 19]). For all transplantation recipients, 12- and 24-month overall survival (OS) rates were 68% and 47%, respectively. Despite a trend toward longer OS after pretransplantation CRc, post-transplantation survival was comparable in the 2 arms. Patients who resumed gilteritinib after HSCT had a low relapse rate after pretransplantation CRc (20%) or CR (0%). The most common AEs observed with post-transplantation gilteritinib therapy were increased alanine aminotransferase level (45%), pyrexia (43%), and diarrhea (40%); grade ≥3 AEs were related primarily to myelosuppression. The incidences of grade ≥III acute graft-versus-host disease and related mortality were low. Post-transplantation survival was similar across the 2 study arms in the ADMIRAL trial, but higher remission rates with gilteritinib facilitated receipt of HSCT. Gilteritinib as post-transplantation maintenance therapy had a stable safety and tolerability profile and was associated with low relapse rates. Taken together, these data support a preference for bridging therapy with gilteritinib over chemotherapy in transplantation-eligible patients.

Dual inhibition of CHK1/FLT3 enhances cytotoxicity and overcomes adaptive and acquired resistance in FLT3-ITD acute myeloid leukemia.

FLT3 inhibitors (FLT3i) are widely used for the treatment of acute myeloid leukemia (AML), but adaptive and acquired resistance remains a primary challenge. Inhibitors simultaneously blocking adaptive and acquired resistance are highly demanded. Here, we observed the potential of CHK1 inhibitors to synergistically improve the therapeutic effect of FLT3i in FLT3-mutated AML cells. Notably, the combination overcame adaptive resistance. The simultaneous targeting of FLT3 and CHK1 kinases may overcome acquired and adaptive resistance. A dual FLT3/CHK1 inhibitor 30 with a good oral PK profile was identified. Mechanistic studies indicated that 30 inhibited FLT3 and CHK1, downregulated the c-Myc pathway and further activated the p53 pathway. Functional studies showed that 30 was more selective against cells with various FLT3 mutants, overcame adaptive resistance in vitro, and effectively inhibited resistant FLT3-ITD AML in vivo. Moreover, 30 showed favorable druggability without significant blood toxicity or myelosuppression and exhibited a good oral PK profile with a T1/2 over 12 h in beagles. These findings support the targeting of FLT3 and CHK1 as a novel strategy for overcoming adaptive and acquired resistance to FLT3i therapy in AML and suggest 30 as a potential clinical candidate.