FLT3-TKD Measurable Residual Disease Detection Using Droplet Digital PCR and Clinical Applications in Acute Myeloid Leukemia.

The tyrosine kinase domain of the FMS-Like tyrosine kinase 3 (FLT3-TKD) is recurrently mutated in acute myeloid leukemia (AML). Common molecular techniques used in its detection include PCR and capillary electrophoresis, Sanger sequencing and next-generation sequencing with recognized sensitivity limitations. This study aims to validate the use of droplet digital PCR (ddPCR) in the detection of measurable residual disease (MRD) involving the common FLT3-TKD mutations (D835Y, D835H, D835V, D835E). Twenty-two diagnostic samples, six donor controls, and a commercial D835Y positive control were tested using a commercial Bio-rad® ddPCR assay. All known variants were identified, and no false positives were detected in the wild-type control (100% specificity and sensitivity). The assays achieved a limit of detection suitable for MRD testing at 0.01% variant allelic fraction. Serial samples from seven intensively-treated patients with FLT3-TKD variants at diagnosis were tested. Five patients demonstrated clearance of FLT3-TKD clones, but two patients had FLT3-TKD persistence in the context of primary refractory disease. In conclusion, ddPCR is suitable for the detection and quantification of FLT3-TKD mutations in the MRD setting; however, the clinical significance and optimal management of MRD positivity require further exploration.

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 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.

The potential of triplet combination therapies for patients with FLT3-ITD -mutated acute myeloid leukemia.

INTRODUCTION: Acute myeloid leukemia (AML) encompasses a heterogeneous group of aggressive myeloid malignancies, where FMS-like tyrosine kinase 3 (FLT3) mutations are prevalent, accounting for approximately 25-30% of adult patients. The presence of this mutation is related to a dismal prognosis and high relapse rates. In the lasts years many FLT3 inhibitors have been developed. AREAS COVERED: This review provides a comprehensive overview of FLT3mut AML, summarizing the state of art of current treatment and available data about combination strategies including an FLT3 inhibitor. EXPERT OPINION: In addition, the review discusses the emergence of drug resistance and the need for a nuanced approaches in treating patients who are ineligible for or resistant to intensive chemotherapy. Specifically, it explores the historical context of FLT3 inhibitors (FLT3Is) and their impact on treatment outcomes, emphasizing the pivotal role of midostaurin, as well as gilteritinib and quizartinib, and providing detailed insights into ongoing trials exploring the safety and efficacy of novel triplet combinations involving FLT3Is in different AML settings.

Phase 1 study of azacitidine in combination with quizartinib in patients with FLT3 or CBL mutated MDS and MDS/MPN.

We conducted a phase 1 study evaluating 3 dose levels of quizartinib (30 mg, 40 mg or 60 mg) in combination with azacitidine for HMA-naïve or relapsed/refractory MDS or MDS/MPN with FLT3 or CBL mutations. Overall, 12 patients (HMA naïve: n=9, HMA failure: n=3) were enrolled; 7 (58 %) patients had FLT3 mutations and 5 (42 %) had CBL mutations. The maximum tolerated dose was not reached. Most common grade 3-4 treatment-emergent adverse events were thrombocytopenia (n=5, 42 %), anemia (n=4, 33 %), lung infection (n=2, 17 %), skin infection (n=2, 17 %), hyponatremia (n=2, 17 %) and sepsis (n=2, 17 %). The overall response rate was 83 % with median relapse-free and overall survivals of 15.1 months (95 % CI 0.0-38.4 months) and 17.5 months (95 % CI NC-NC), respectively. FLT3 mutation clearance was observed in 57 % (n=4) patients. These data suggest quizartinib is safe and shows encouraging activity in FLT3-mutated MDS and MDS/MPN. This study is registered at Clinicaltrials.gov as NCT04493138.

A systematic review of second-generation FLT3 inhibitors for treatment of patients with relapsed/refractory acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is a complex disease with diverse mutations, including prevalent mutations in the FMS-like receptor tyrosine kinase 3 (FLT3) gene that lead to poor prognosis. Recent advancements have introduced FLT3 inhibitors that have improved outcomes for FLT3-mutated AML patients, however, questions remain on their application in complex conditions such as relapsed/refractory (R/R) disease. Therefore, we aimed to evaluate the clinical effectiveness of second-generation FLT3 inhibitors in treating patients with R/R AML. METHODS: A systematic literature search of PubMed, MEDLINE, SCOPUS and Google Scholar databases was made to identify relevant studies up to January 30, 2024. This study was conducted following the guidelines of the PRISMA. RESULTS: The ADMIRAL trial revealed significantly improved overall survival and complete remission rates with gilteritinib compared to salvage chemotherapy, with manageable adverse effects. Ongoing research explores its potential in combination therapies, showing synergistic effects with venetoclax and promising outcomes in various clinical trials. The QuANTUM-R trial suggested longer overall survival with quizartinib compared to standard chemotherapy, although concerns were raised regarding trial design and cardiotoxicity. Ongoing research explores combination therapies involving quizartinib, such as doublet or triplet regimens with venetoclax, showing promising outcomes in FLT3-mutated AML patients. CONCLUSION: These targeted therapies offer promise for managing this subgroup of AML patients, but further research is needed to optimize their use. This study underscores the importance of personalized treatment based on genetic mutations in AML, paving the way for more effective and tailored approaches to combat the disease.

[Fulminant Clostridioides difficile infection during treatment with FLT3 inhibitor for acute myeloid leukemia].

An 80-year-old man with FLT3-TKD mutation-positive acute myeloid leukemia (AML) relapsed during consolidation therapy with venetoclax/azacitidine and was started on gilteritinib as salvage therapy. On the day after treatment initiation, febrile neutropenia was observed, but the fever resolved promptly after initiation of antimicrobial therapy. On the fifth day after completion of antimicrobial therapy, the patient experienced fever and watery diarrhea over 10 times a day, and a diagnosis of Clostridioides difficile infection (CDI) was made based on stool examination. The patient was treated with intravenous metronidazole, but renal dysfunction, hypotension, and hypoxemia developed, and a CT scan showed pleural and intraperitoneal effusion, significant intestinal wall thickening, and intestinal dilatation. Fidaxomicin was started under general monitoring in the intensive care unit and response was achieved. The patient was discharged from the intensive care unit on the 18th day after the onset of CDI. We report this case not only due to the rarity of fulminant CDI during AML treatment, but also because it is a valuable example of effective treatment of fulminant CDI with fidaxomicin.

Sweet syndrome induced by FLT3 inhibitors: case report and literature review.

BACKGROUND: Acute febrile neutrophilic dermatosis, also commonly referred to as Sweet syndrome, is often associated with tumors, infections, immune disorders and medications. FLT3 inhibitor-induced Sweet syndrome is a rare complication. METHODS AND RESULTS: We report a patient with relapsed and refractory acute monocytic leukemia harboring high-frequency FLT3-ITD and DNMT3a mutations. The FLT3 inhibitor gilteritinib was administered for reinduction therapy after failure of chemotherapy with a combination of venetoclax, decitabine, aclarubicin, cytarabine and granulocyte colony-stimulating factor. The leukemia patient achieved remission after 1 month of treatment. However, Sweet syndrome induced by gilteritinib, which was confirmed by skin biopsy, developed during induction therapy. Similar cases of Sweet syndrome following FLT3 inhibitor therapy for acute myeloid leukemia were reviewed. CONCLUSION: Attention should be given to this rare complication when FLT3 inhibitors are used for acute myeloid leukemia therapy, and appropriate treatments need to be administered in a timely manner.

Current knowledge about FLT3 gene mutations, exploring the isoforms, and protein importance in AML.

Acute myeloid leukaemia (AML) is a complex haematological malignancy characterised by diverse genetic alterations leading to abnormal proliferation of myeloid precursor cells. One of the most significant genetic alterations in AML involves mutations in the FLT3 gene, which plays a critical role in haematopoiesis and haematopoietic homeostasis. This review explores the current understanding of FLT3 gene mutations and isoforms and the importance of the FLT3 protein in AML. FLT3 mutations, including internal tandem duplications (FLT3-ITD) and point mutations in the tyrosine kinase domain (FLT3-TKD), occur in 25-30% in AML and are associated with poor prognosis. FLT3-ITD mutations lead to constitutive activation of the FLT3 signalling pathway, promoting cell survival and proliferation. FLT3-TKD mutations affect the tyrosine kinase domain and affect AML prognosis in various ways. Furthermore, FLT3 isoforms, including shorter variants, contribute to the complexity of FLT3 biology. Additionally, nonpathological polymorphisms in FLT3 are being explored for their potential impact on AML prognosis and treatment response. This review also discusses the development of molecular treatments targeting FLT3, including first-generation and next-generation tyrosine kinase inhibitors, highlighting the challenges of resistance that often arise during therapy. The final chapter describes FLT3 protein domain rearrangements and their relevance to AML pathogenesis.

[Acute myeloid leukemia with central nervous system involvement successfully treated with gilteritinib].

A 69-year-old woman was referred to our hospital due to hyperleukocytosis. We diagnosed acute myeloid leukemia and started induction therapy with the CAG regimen (aclarubicin, cytarabine and filgrastim). However, the patient was refractory to the initial treatment and developed quadriplegia, and a cerebrospinal fluid (CSF) test showed elevated blasts. We then performed intrathecal chemotherapy, and the number of blasts in CSF gradually decreased. But only two cycles of intrathecal therapy were possible due to severe methotrexate-induced mucositis. The leukemia cells had fms-like kinase 3-internal tandem duplication (FLT3-ITD), so we started treatment with oral gilteritinib. The patient then achieved hematological complete remission. Her paralysis was also resolving, and the CSF was clear of blasts for more than 6 months. Some reports show that gilteritinib may penetrate the CNS, and this case also supports the effectiveness of gilteritinib on CNS leukemia.

[Correlation of miR-155 Expression with Drug Sensitivity of FLT3-ITD+ Acute Myeloid Leukemia Cell Line and Its Mechanism].

OBJECTIVE: To investigate the correlation of miR-155 expression with drug sensitivity of FLT3-ITD+ acute myeloid leukemia (AML) cell line and its potential regulatory mechanism. METHODS: By knocking out miR-155 gene in FLT3-ITD+ AML cell line MV411 through CRISPR/Cas9 gene-editing technology, monoclonal cells were screened. The genotype of these monoclonal cells was validated by PCR and Sanger sequencing. The expression of mature miRNA was measured by RT-qPCR. The treatment response of doxorubicin, quizartinib and midostaurin were measured by MTT assay and IC50 of these drugs were calculated to identify the sensitivity. Transcriptome sequencing was used to analyze change of mRNA level in MV411 cells after miR-155 knockout, gene set enrichment analysis to analyze change of signaling pathway, and Western blot to verify expressions of key molecules in signaling pathway. RESULTS: Four heterozygotes with gene knockout and one heterozygote with gene insertion were obtained through PCR screening and Sanger sequencing. RT-qPCR results showed that the expression of mature miR-155 in the monoclonal cells was significantly lower than wild-type clones. MTT results showed that the sensitivity of MV411 cells to various anti FLT3-ITD+ AML drugs increased significantly after miR-155 knockout compared with wild-type clones. RNA sequencing showed that the mTOR signaling pathway and Wnt signaling pathway were inhibited after miR-155 knockout. Western blot showed that the expressions of key molecules p-mTOR, Wnt5α and ß-catenin in signaling pathway were down-regulated. CONCLUSION: Drug sensitivity of MV411 cells to doxorubicin, quizartinib and midostaurin can be enhanced significantly after miR-155 knockout, which is related to the inhibition of multiple signaling pathways including mTOR and Wnt signaling pathways.

Day-21 bone marrow findings incorrectly designate residual leukaemia in FLT3-mutated acute myeloid leukaemia treated with intensive induction plus midostaurin: a morphology-focused study.

Early induction response assessment with day-21 bone marrow (D21-BM) is commonly performed in patients with FLT3-mutated acute myeloid leukaemia (AML), where detection of residual leukaemia (RL; blasts ≥5%) typically results in the administration of a second induction course. However, whether D21-BM results predict for RL at the end of first induction has not been systematically assessed. This study evaluates the predictive role of D21-BM morphology in detecting RL following first induction. Between August 2018 and March 2022, all patients with FLT3-AML receiving 7+3 plus midostaurin, with D21-BM performed, were identified. Correlation between D21-BM morphology vs D21-BM ancillary flow/molecular results, as well as vs D28-BM end of first induction response, were retrospectively reviewed. Subsequently, D21-BMs were subjected to anonymised morphological re-assessments by independent haematopathologists (total in triplicate per patient). Of nine patients included in this study, three (33%) were designated to have RL at D21-BM, all of whom entered complete remission at D28-BM. Furthermore, only low-level measurable residual disease was detected in all three cases by flow or molecular methods at D21-BM, hence none proceeded to a second induction. Independent re-evaluations of these cases failed to correctly reassign D21-BM responses, yielding a final false positive rate of 33%. In summary, based on morphology alone, D21-BM assessment following 7+3 intensive induction plus midostaurin for FLT3-AML incorrectly designates RL in some patients; thus correlating with associated flow and molecular results is essential before concluding RL following first induction. Where remission status is unclear, repeat D28-BMs should be performed.

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 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.

Unveiling the signaling network of FLT3-ITD AML improves drug sensitivity prediction.

Currently, the identification of patient-specific therapies in cancer is mainly informed by personalized genomic analysis. In the setting of acute myeloid leukemia (AML), patient-drug treatment matching fails in a subset of patients harboring atypical internal tandem duplications (ITDs) in the tyrosine kinase domain of the FLT3 gene. To address this unmet medical need, here we develop a systems-based strategy that integrates multiparametric analysis of crucial signaling pathways, and patient-specific genomic and transcriptomic data with a prior knowledge signaling network using a Boolean-based formalism. By this approach, we derive personalized predictive models describing the signaling landscape of AML FLT3-ITD positive cell lines and patients. These models enable us to derive mechanistic insight into drug resistance mechanisms and suggest novel opportunities for combinatorial treatments. Interestingly, our analysis reveals that the JNK kinase pathway plays a crucial role in the tyrosine kinase inhibitor response of FLT3-ITD cells through cell cycle regulation. Finally, our work shows that patient-specific logic models have the potential to inform precision medicine approaches.

Inhibition of NRF2 signaling overcomes acquired resistance to arsenic trioxide in FLT3-mutated Acute Myeloid Leukemia.

De novo acute myeloid leukemia (AML) patients with FMS-like tyrosine kinase 3 internal tandem duplications (FLT3-ITD) have worse treatment outcomes. Arsenic trioxide (ATO) used in the treatment of acute promyelocytic leukemia (APL) has been reported to be effective in degrading the FLT3 protein in AML cell lines and sensitizing non-APL AML patient samples in-vitro. We have previously reported that primary cells from FLT3-ITD mutated AML patients were sensitive to ATO in-vitro compared to other non-M3 AML and molecular/pharmacological inhibition of NF-E2 related factor 2 (NRF2), a master regulator of antioxidant response improved the chemosensitivity to ATO and daunorubicin even in non FLT3-ITD mutated cell lines and primary samples. We examined the effects of molecular/pharmacological suppression of NRF2 on acquired ATO resistance in the FLT3-ITD mutant AML cell line (MV4-11-ATO-R). ATO-R cells showed increased NRF2 expression, nuclear localization, and upregulation of bonafide NRF2 targets. Molecular inhibition of NRF2 in this resistant cell line improved ATO sensitivity in vitro. Digoxin treatment lowered p-AKT expression, abrogating nuclear NRF2 localization and sensitizing cells to ATO. However, digoxin and ATO did not sensitize non-ITD AML cell line THP1 with high NRF2 expression. Digoxin decreased leukemic burden and prolonged survival in MV4-11 ATO-R xenograft mice. We establish that altering NRF2 expression may reverse acquired ATO resistance in FLT3-ITD AML.

A practical algorithm for acute myeloid leukaemia diagnosis following the updated 2022 classifications.

Disease classification of complex and heterogenous diseases, such as acute myeloid leukaemia (AML), is continuously updated to define diagnoses, appropriate treatments, and assist research and education. Recent availability of molecular profiling techniques further benefits the classification of AML. The World Health Organization (WHO) classification of haematolymphoid tumours and the International Consensus Classification of myeloid neoplasms and acute leukaemia from 2022 are two updated versions of the WHO 2016 classification. As a consequence, the European LeukemiaNet 2022 recommendations on the diagnosis and management of AML in adults have been also updated. The current review provides a practical interpretation of these guidelines to facilitate the diagnosis of AML and discusses genetic testing, disease genetic heterogeneity, and FLT3 mutations. We propose a practical algorithm for the speedy diagnosis of AML. Future classifications may need to incorporate gene mutation combinations to enable personalised treatment regimens in the management of patients with AML.