Alkynyl nicotinamides show antileukemic activity in drug-resistant acute myeloid leukemia.

Activating mutations of FLT3 contribute to deregulated hematopoietic stem and progenitor cell (HSC/Ps) growth and survival in patients with acute myeloid leukemia (AML), leading to poor overall survival. AML patients treated with investigational drugs targeting mutant FLT3, including Quizartinib and Crenolanib, develop resistance to these drugs. Development of resistance is largely due to acquisition of cooccurring mutations and activation of additional survival pathways, as well as emergence of additional FLT3 mutations. Despite the high prevalence of FLT3 mutations and their clinical significance in AML, there are few targeted therapeutic options available. We have identified 2 novel nicotinamide-based FLT3 inhibitors (HSN608 and HSN748) that target FLT3 mutations at subnanomolar concentrations and are potently effective against drug-resistant secondary mutations of FLT3. These compounds show antileukemic activity against FLT3ITD in drug-resistant AML, relapsed/refractory AML, and in AML bearing a combination of epigenetic mutations of TET2 along with FLT3ITD. We demonstrate that HSN748 outperformed the FDA-approved FLT3 inhibitor Gilteritinib in terms of inhibitory activity against FLT3ITD in vivo.

Repression of SMAD3 by STAT3 and c-Ski induces conventional dendritic cell differentiation.

A pleiotropic immunoregulatory cytokine, TGF-ß, signals via the receptor-regulated SMADs: SMAD2 and SMAD3, which are constitutively expressed in normal cells. Here, we show that selective repression of SMAD3 induces cDC differentiation from the CD115+ common DC progenitor (CDP). SMAD3 was expressed in haematopoietic cells including the macrophage DC progenitor. However, SMAD3 was specifically down-regulated in CD115+ CDPs, SiglecH- pre-DCs, and cDCs, whereas SMAD2 remained constitutive. SMAD3-deficient mice showed a significant increase in cDCs, SiglecH- pre-DCs, and CD115+ CDPs compared with the littermate control. SMAD3 repressed the mRNA expression of FLT3 and the cDC-related genes: IRF4 and ID2. We found that one of the SMAD transcriptional corepressors, c-SKI, cooperated with phosphorylated STAT3 at Y705 and S727 to repress the transcription of SMAD3 to induce cDC differentiation. These data indicate that STAT3 and c-Ski induce cDC differentiation by repressing SMAD3: the repressor of the cDC-related genes during the developmental stage between the macrophage DC progenitor and CD115+ CDP.

CXCR4-Mediated Codelivery of FLT3 and BCL-2 Inhibitors for Enhanced Targeted Combination Therapy of FLT3-ITD Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is often associated with poor prognosis and survival. Small molecule inhibitors, though widening the treatment landscape, have limited monotherapy efficacy. The combination therapy, however, shows suboptimal clinical outcomes due to low bioavailability, overlapping systemic toxicity and drug resistance. Here, we report that CXCR4-mediated codelivery of the BCL-2 inhibitor venetoclax (VEN) and the FLT3 inhibitor sorafenib (SOR) via T22 peptide-tagged disulfide cross-linked polymeric micelles (TM) achieves synergistic treatment of FLT3-ITD AML. TM-VS with a VEN/SOR weight ratio of 1/4 and T22 peptide density of 20% exhibited an extraordinary inhibitory effect on CXCR4-overexpressing MV4-11 AML cells. TM-VS at a VEN/SOR dosage of 2.5/10 mg/kg remarkably reduced leukemia burden, prolonged mouse survival, and impeded bone loss in orthotopic MV4-11-bearing mice, outperforming the nontargeted M-VS and oral administration of free VEN/SOR. CXCR4-mediated codelivery of BCL-2 and FLT3 inhibitors has emerged as a prospective clinical treatment for FLT3-ITD AML.

Discovery of a potent Gilteritinib-based FLT3-PROTAC degrader for the treatment of Acute myeloid leukemia.

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.

FLT3-ITD regulation of the endoplasmic reticulum functions in acute myeloid leukemia.

The FLT3-ITD mutation represents the most frequent genetic alteration in newly diagnosed acute myeloid leukemia (AML) patient and is associated with poor prognosis. Mutation result in the retention of a constitutively active form of this receptor in the endoplasmic reticulum (ER) and the subsequent modification of its downstream effectors. Here, we assessed the impact of such retention on ER homeostasis and found that mutant cells present lower levels of ER stress due to the overexpression of ERO1α, one of the main proteins of the protein folding machinery at the ER. Overexpression of ERO1α resulted essential for ITD mutant cells survival and chemoresistance and also played a crucial role in shaping the type of glucose metabolism in AML cells, being the mitochondrial pathway the predominant one in those with a higher ER stress (non-mutated cells) and the glycolytic pathway the predominant one in those with lower ER stress (mutated cells). Our data indicate that FLT3 mutational status dictates the route for glucose metabolism in an ERO1α depending on manner and this provides a survival advantage to tumors carrying these ITD mutations.

FLT3-selective PROTAC: Enhanced safety and increased synergy with Venetoclax in FLT3-ITD mutated acute myeloid leukemia.

Acute myeloid leukemia (AML) patients carrying Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutations often face a poor prognosis. While some FLT3 inhibitors have been used clinically, challenges such as short efficacy and poor specificity persist. Proteolytic targeting chimera (PROTAC), with its lower ligand affinity requirement for target proteins, offers higher and rapid targeting capability. Gilteritinib, used as the ligand for the target protein, was connected with different E3 ligase ligands to synthesize several series of PROTAC targeting FLT3-ITD. Through screening and structural optimization, the optimal lead compound PROTAC Z29 showed better specificity than Gilteritinib. Z29 induced FLT3 degradation through the proteasome pathway and inhibited tumor growth in subcutaneous xenograft mice. We verified Z29's minimal impact on platelets in a patient-derived xenografts (PDX) model compared to Gilteritinib. The combination of Z29 and Venetoclax showed better anti-tumor effects, lower platelet toxicity, and lower hepatic toxicity in FLT3-ITD+ models. The FLT3-selective PROTAC can mitigate the platelet toxicity of small molecule inhibitors, ensuring safety and efficacy in monotherapy and combination therapy with Venetoclax. It is a promising strategy for FLT3-ITD+ patients, especially those with platelet deficiency or liver damage.

Discovery of a Novel Orally Bioavailable FLT3-PROTAC Degrader for Efficient Treatment of Acute Myeloid Leukemia and Overcoming Resistance of FLT3 Inhibitors.

Fms-like tyrosine receptor kinase 3 (FLT3) proteolysis-targeting chimeras (PROTACs) represent a promising approach to eliminate the resistance of FLT3 inhibitors. However, due to the poor druggability of PROTACs, the development of orally bioavailable FLT3-PROTACs faces great challenges. Herein, a novel orally bioavailable FLT3-ITD degrader A20 with excellent pharmacokinetic properties was discovered through reasonable design. A20 selectively inhibited the proliferation of FLT3-ITD mutant acute myeloid leukemia (AML) cells and potently induced FLT3-ITD degradation through the ubiquitin-proteasome system. Notably, oral administration of A20 resulted in complete tumor regression on subcutaneous AML xenograft models. Furthermore, on systemic AML xenograft models, A20 could completely eliminate the CD45+CD33+ human leukemic cells in murine and significantly prolonged the survival time of mice. Most importantly, A20 exerted significantly improved antiproliferative activity against drug-resistant AML cells compared to existing FLT3 inhibitors. These findings suggested that A20 could serve as a promising drug candidate for relapsed or refractory AML.

Narazaciclib, a novel multi-kinase inhibitor with potent activity against CSF1R, FLT3 and CDK6, shows strong anti-AML activity in defined preclinical models.

CSF1R is a receptor tyrosine kinase responsible for the growth/survival/polarization of macrophages and overexpressed in some AML patients. We hypothesized that a novel multi-kinase inhibitor (TKi), narazaciclib (HX301/ON123300), with high potency against CSF1R (IC50 ~ 0.285 nM), would have anti-AML effects. We tested this by confirming HX301's high potency against CSF1R (IC50 ~ 0.285 nM), as well as other kinases, e.g. FLT3 (IC50 of ~ 19.77 nM) and CDK6 (0.53 nM). An in vitro proliferation assay showed that narazaciclib has a high growth inhibitory effect in cell cultures where CSF1R or mutant FLT3-ITD variants that may be proliferation drivers, including primary macrophages (IC50 of 72.5 nM) and a subset of AML lines (IC50 < 1.5 µM). In vivo pharmacology modeling of narazaciclib using five AML xenografts resulted in: inhibition of MV4-11 (FLT3-ITD) subcutaneous tumor growth and complete suppression of AM7577-PDX (FLT3-ITD/CSF1Rmed) systemic growth, likely due to the suppression of FLT3-ITD activity; complete suppression of AM8096-PDX (CSF1Rhi/wild-type FLT3) growth, likely due to the inhibition of CSF1R ("a putative driver"); and nonresponse of both AM5512-PDX and AM7407-PDX (wild-type FLT3/CSF1Rlo). Significant leukemia load reductions in bone marrow, where disease originated, were also achieved in both responders (AM7577/AM8096), implicating that HX301 might be a potentially more effective therapy than those only affecting peripheral leukemic cells. Altogether, narazaciclib can potentially be a candidate treatment for a subset of AML with CSF1Rhi and/or mutant FLT3-ITD variants, particularly second generation FLT3 inhibitor resistant variants.

Discovery of FLT3-targeting PROTACs with potent antiproliferative activity against acute myeloid leukemia cells harboring FLT3 mutations.

Acute myeloid leukemia (AML) patients harboring Fms-like tyrosine kinase 3 (FLT3) mutations often suffer from poor prognosis and relapse. Targeted protein degradation utilizing proteolysis targeting chimeras (PROTACs) is considered as a novel therapeutic strategy in drug discovery and may be a promising modality to target FLT3 mutations for the development of potent anti-AML drugs. Herein, a kind of FLT3-targeting PROTACs was rationally developed based on a FLT3 inhibitor previously reported by us. The representative compound 35 showed potent and selective antiproliferative activities against AML cells harboring FLT3 mutations. Western blot assay demonstrated that compound 35 effectively induced the degradation of FLT3-ITD and decreased the phosphorylation levels of FLT3-ITD, AKT, STAT5 and ERK in MV4-11 cells in a dose-dependent manner. Flow cytometry analysis illustrated that compound 35 strongly induced apoptosis and cell cycle arrest in MV4-11 cells in a dose-dependent manner. Moreover, compound 35 displayed favorable metabolic stability in in-vitro liver microsomes studies. Comparative molecular dynamic (MD) simulation studies further elucidated the underlying mechanism of compound 35 to stabilize the dynamic ensemble of the FLT3-compound 35-cereblon (CRBN) ternary complex. Taken together, compound 35 could serve as a lead molecule for developing FLT3 degraders against AML.

High FLT3 expression increases immune-cell infiltration in the tumor microenvironment and correlates with prolonged disease-free survival in patients with non-small cell lung cancer.

Most of the currently used cancer immunotherapies inhibit the programmed cell death protein 1 (PD1)-programmed cell death 1 ligand 1 (PDL1) axis of T-cells. However, dendritic cells (DCs) controlled by natural killer (NK) cells via the FMS-related tyrosine kinase 3 (FLT3) axis are necessary for activation of T-cells. The aim of the study was to evaluate FLT3 as a prognostic factor and determine its role in immune infiltration (with emphasis on NK cells and DCs). Using The Cancer Genome Atlas (TCGA) database, we performed bioinformatic analysis of the gene expression datasets of 501 lung squamous cell carcinoma (LUSC) and 515 lung adenocarcinoma (LUAD) patient who had corresponding clinical data [analysis was performed in R (version 4.2.0)]. Disease-free survival (DFS) differed between the FLT3-low and FLT3-high expression groups, respectively, in LUSC (61.0 vs 71.3 months P = 0.075) and LUAD (32.7 vs 47.5 months P = 0.045). A tumor microenvironment (TME) with high immune infiltration and rich in T-cell exhaustion markers was observed in the FLT3-high group. We showed overexpression of NK cell and DC gene signatures in the FLT3-high expression group as well as overexpression of key effector genes of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes protein (STING) pathway, which is crucial in response to radiotherapy. High expression of FLT3 in the TME was associated with immune cell infiltration (especially of NK cells and DCs), increased expression of T-cell exhaustion markers and expression of effector genes of the cGAS-STING pathway, which may consequently increase susceptibility to immunotherapy and radiotherapy. High FLT3 expression correlated with prolonged DFS in the LUSC and LUAD cohorts.

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.

ATP1A1/BCL2L1 predicts the response of myelomonocytic and monocytic acute myeloid leukemia to cardiac glycosides.

Myelomonocytic and monocytic acute myeloid leukemia (AML) subtypes are intrinsically resistant to venetoclax-based regimens. Identifying targetable vulnerabilities would limit resistance and relapse. We previously documented the synergism of venetoclax and cardiac glycoside (CG) combination in AML. Despite preclinical evidence, the repurposing of cardiac glycosides (CGs) in cancer therapy remained unsuccessful due to a lack of predictive biomarkers. We report that the ex vivo response of AML patient blasts and the in vitro sensitivity of established cell lines to the hemi-synthetic CG UNBS1450 correlates with the ATPase Na+/K+ transporting subunit alpha 1 (ATP1A1)/BCL2 like 1 (BCL2L1) expression ratio. Publicly available AML datasets identify myelomonocytic/monocytic differentiation as the most robust prognostic feature, along with core-binding factor subunit beta (CBFB), lysine methyltransferase 2A (KMT2A) rearrangements, and missense Fms-related receptor tyrosine kinase 3 (FLT3) mutations. Mechanistically, BCL2L1 protects from cell death commitment induced by the CG-mediated stepwise triggering of ionic perturbation, protein synthesis inhibition, and MCL1 downregulation. In vivo, CGs showed an overall tolerable profile while impacting tumor growth with an effect ranging from tumor growth inhibition to regression. These findings suggest a predictive marker for CG repurposing in specific AML subtypes.

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.

Targeting FLT3-TAZ signaling to suppress drug resistance in blast phase chronic myeloid leukemia.

BACKGROUND: Although the development of BCR::ABL1 tyrosine kinase inhibitors (TKIs) rendered chronic myeloid leukemia (CML) a manageable condition, acquisition of drug resistance during blast phase (BP) progression remains a critical challenge. Here, we reposition FLT3, one of the most frequently mutated drivers of acute myeloid leukemia (AML), as a prognostic marker and therapeutic target of BP-CML. METHODS: We generated FLT3 expressing BCR::ABL1 TKI-resistant CML cells and enrolled phase-specific CML patient cohort to obtain unpaired and paired serial specimens and verify the role of FLT3 signaling in BP-CML patients. We performed multi-omics approaches in animal and patient studies to demonstrate the clinical feasibility of FLT3 as a viable target of BP-CML by establishing the (1) molecular mechanisms of FLT3-driven drug resistance, (2) diagnostic methods of FLT3 protein expression and localization, (3) association between FLT3 signaling and CML prognosis, and (4) therapeutic strategies to tackle FLT3+ CML patients. RESULTS: We reposition the significance of FLT3 in the acquisition of drug resistance in BP-CML, thereby, newly classify a FLT3+ BP-CML subgroup. Mechanistically, FLT3 expression in CML cells activated the FLT3-JAK-STAT3-TAZ-TEAD-CD36 signaling pathway, which conferred resistance to a wide range of BCR::ABL1 TKIs that was independent of recurrent BCR::ABL1 mutations. Notably, FLT3+ BP-CML patients had significantly less favorable prognosis than FLT3- patients. Remarkably, we demonstrate that repurposing FLT3 inhibitors combined with BCR::ABL1 targeted therapies or the single treatment with ponatinib alone can overcome drug resistance and promote BP-CML cell death in patient-derived FLT3+ BCR::ABL1 cells and mouse xenograft models. CONCLUSION: Here, we reposition FLT3 as a critical determinant of CML progression via FLT3-JAK-STAT3-TAZ-TEAD-CD36 signaling pathway that promotes TKI resistance and predicts worse prognosis in BP-CML patients. Our findings open novel therapeutic opportunities that exploit the undescribed link between distinct types of malignancies.

Simvastatin Preferentially Targets FLT3/ITD Acute Myeloid Leukemia by Inhibiting MEK/ERK and p38-MAPK Signaling Pathways.

BACKGROUND: The FLT3/ITD mutation exists in many acute myeloid leukemia (AML) patients and is related to the poor prognosis of patients. In this study, we attempted to evaluate the antitumor activity of simvastatin, a member of the statin class of drugs, in vitro and in vivo models of FLT3/ITD AML and to identify the potential mechanisms. METHODS: Cell Counting Kit-8 (CCK-8) and Annexin V/propidium iodide (PI) staining kits were used to detect cell viability and apoptosis, respectively. Subsequently, Western blot and rescue experiment were applied to explore the potential molecular mechanism. In vivo anti-leukemia activity of simvastatin was evaluated in xenograft mouse models. RESULTS: In vitro experiments revealed that simvastatin inhibited AML progression in a dose- and time-dependent manner, while in vivo experiments showed that simvastatin significantly reduced tumor burden in FLT3/ITD xenograft mouse models. After simvastatin treatment of FLT3/ITD AML cells, intracellular Rap1 was downregulated and the phosphorylation levels of its downstream targets MEK, ERK and p38 were significantly inhibited. The rescue experiment showed that mevalonate, an intermediate product of the metabolic pathway of mevalonate, and its downstream geranylgeranyl pyrophosphate (GGPP) played a key role in this process. Finally, we demonstrate that simvastatin can induce apoptosis of primary AML cells, while having no effect on peripheral blood mononuclear cells from normal donors. CONCLUSIONS: Simvastatin can selectively and effectively eradicate FLT3/ITD AML cells in vitro and in vivo, and its mechanism may be related to the disruption of the HMG-CoA reductase pathway and the downregulation of the MEK/ERK and p38-MAPK signaling pathways.

FLT3-directed UniCAR T-cell therapy of acute myeloid leukaemia.

Adaptor chimeric antigen receptor (CAR) T-cell therapy offers solutions for improved safety and antigen escape, which represent main obstacles for the clinical translation of CAR T-cell therapy in myeloid malignancies. The adaptor CAR T-cell platform 'UniCAR' is currently under early clinical investigation. Recently, the first proof of concept of a well-tolerated, rapidly switchable, CD123-directed UniCAR T-cell product treating patients with acute myeloid leukaemia (AML) was reported. Relapsed and refractory AML is prone to high plasticity under therapy pressure targeting one single tumour antigen. Thus, targeting of multiple tumour antigens seems to be required to achieve durable anti-tumour responses, underlining the need to further design alternative AML-specific target modules (TM) for the UniCAR platform. We here present the preclinical development of a novel FMS-like tyrosine kinase 3 (FLT3)-directed UniCAR T-cell therapy, which is highly effective for in vitro killing of both AML cell lines and primary AML samples. Furthermore, we show in vivo functionality in a murine xenograft model. PET analyses further demonstrate a short serum half-life of FLT3 TMs, which will enable a rapid on/off switch of UniCAR T cells. Overall, the presented preclinical data encourage the further development and clinical translation of FLT3-specific UniCAR T cells for the therapy of AML.

RUNX1/ETO regulates reactive oxygen species (ROS) levels in t(8,21) acute myeloid leukaemia via FLT3 and RAC1.

Reactive oxygen species (ROS) homeostasis is crucial for leukaemogenesisand deregulation would hamper leukaemic progression. Although the regulatory effects of RUNX1/ETO has been extensively studied, its underlying molecular mechanims in ROS production in t(8,21) AML is yet to be fully elucidated. Here, we report that RUNX1/ETO could directly control FLT3 by occupying several DNA elements on FLT3 locus. The possible hijacking mechanism by RUNX1/ETO over FLT3 mediated ROS modulation in AML t(8;21) was made apparent when suppression of RUNX1/ETO led to decrement in ROS levels and the direct oxidative marker FOXO3 but not in FLT3 and RAC1 suppressed t(8,21) AML cell line Furthermore, nuclear import of RUNX1/ETO was aberrated following RUNX1/ETO and RAC1 suppression suggesting association in ROS control. A different picture was depicted in non t(8;21) cells where suppression of RAC1 and FLT3 led to decreased levels of FOXO3a and ROS. Results alltogether indicate a possible dysregulation of ROS levels by RUNX1/ETO in t(8,21) AML.

Design, synthesis, and biological evaluation of a series of indolone derivatives as novel FLT3 inhibitors for the treatment of acute myeloid leukemia.

FLT3-ITD mutant has been extensively studied as a drug discovery target for acute myeloid leukemia. Based on our previous discovered FLT3 inhibitor (2), a series of urea group based indolone derivatives were designed, synthesized, and biological evaluated as novel FLT3 inhibitors for the treatment of FLT3-ITD positive AML. Among them, compound LC-3 exhibited potent inhibitory effects against FLT3 (IC50 = 8.4 nM) and significantly inhibited the proliferation of FLT3-ITD positive AML cells MV-4-11 (IC50 = 5.3 nM). In the cellular context, LC-3 strongly inhibited FLT3-mediated signaling pathways and induced cellular apoptosis by arresting cell cycle in G1 phase. In the in vivo studies, LC-3 significantly suppressed the tumor growth on MV-4-11 xenograft models (10 mg/kg/day, TGI = 92.16%) without exhibiting obvious toxicity. These results suggested that compound LC-3 might be a potential drug candidate for FLT3-ITD positive AML.

Novel spiroindoline quinazolinedione derivatives as anticancer agents and potential FLT3 kinase inhibitors.

Despite considerable recent progress in therapeutic strategies, cancer still remains one of the leading causes of death. Molecularly targeted therapies, in particular those focused on blocking receptor tyrosine kinases have produced promising outcomes in recent years. In this study, a new series of spiro[indoline-3,2'-quinazoline]-2,4'(3'H)-dione derivatives (5a-5l) were synthesized and evaluated as potential kinase inhibitors with anticancereffects. The anti-proliferative activity was measured by MTT assay, while the cell cycle was studied using flow cytometry. Moreover, kinase inhibition profiles of the most promising compounds were assessed against a panel of 25 oncogenic kinases. Compounds 5f,5g,5i, and 5jshowed anti-proliferative effect against EBC-1, A549, and HT-29 solid tumor models in addition to leukemia cell line K562. In particular, compound 5f, bearing 4-methylphenyl pendant on the isatin ring displayed considerable potency with IC50 values of 2.4 to 13.4 µM against cancer cells. The most potent derivatives also altered the distribution of cells in different phases of cell cycle and increased the sub-G1 phase cells in K562 cells. Moreover, kinase inhibition assays identified FLT3 kinase was as the primary targetof these derivatives. Compound 5f at 25 µM concentration showed inhibitory activities of 55% and 62% against wild-type FLT3 and its mutant, D835Y, respectively. Finally, the docking and simulation studies revealed the important interactions of compound 5f with wild type and mutant FLT3. The results of this study showed that some novel spiroindoline quinazolinedione compounds could be potential candidates for further development as novel targeted anticancer agents.