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Cisplatin, 5FU and docetaxel (TPF) are the most common chemotherapy regimen used for advanced OSCC. However, many cancer patients experience relapse, continued tumor growth, and spread due to drug resistance, which leads to treatment failure and metastatic disease. Here, using a CRISPR/Cas9 based kinome knockout screening, Misshapen-like kinase 1 (MINK1) is identified as an important mediator of 5FU resistance in OSCC. Analysis of clinical samples demonstrated significantly higher MINK1 expression in the tumor tissues of chemotherapy non-responder as compared to chemotherapy responders. The in-vitro and xenograft experiments indicate that knocking out MINK1 restores 5FU mediated cell death in chemoresistant OSCC. An antibody based phosphorylation array screen revealed MINK1 as a negative regulator of p53. Mechanistically, MINK1 modulates AKT phosphorylation at Ser473, which enables p-MDM2 (Ser 166) mediated degradation of p53. We also identified lestaurtinib as a potent inhibitor of MINK1 kinase activity. Lestaurtinib significantly induces 5FU mediated cell death in chemoresistant OSCC lines. The patient derived chemoresistant cell based xenograft data suggest that lestaurtinib restores 5FU sensitivity and facilitates a significant reduction of tumor burden. Overall, our study suggests that MINK1 is a major driver of 5FU resistance in OSCC. The novel combination of MINK1 inhibitor lestaurtinib and 5FU needs further clinical investigation in advanced OSCC. One sentence summary Lestaurtinib reverses 5FU sensitivity in drug resistant OSCC.

Lestaurtinib, also called CEP‐701, is an inhibitor of tyrosine kinase, causes haematological remission in patients with AML possessing FLT3‐ITD (FLT3 gene) internal tandem duplication and strongly inhibits tyrosine kinase FLT3. Treatment with lestaurtinib modulates various signalling pathways and leads to cell growth arrest and programmed cell death in several tumour types. However, the effect of lestaurtinib on glioma remains unclear. In this study, we examined lestaurtinib and TRAIL interactions in glioma cells and observed their synergistic activity on glioma cell apoptosis. While U87 and U251 cells showed resistance to TRAIL single treatment, they were sensitized to apoptosis induced by TRAIL in the presence of lestaurtinib because of increased death receptor 5 (DR5) levels through CHOP‐dependent manner. We also demonstrated using a xenograft model of mouse that the tumour growth was absolutely suppressed because of the combined treatment compared to TRAIL or lestaurtinib treatment carried out singly. Our findings reveal a potential new strategy to improve antitumour activity induced by TRAIL in glioma cells using lestaurtinib through a mechanism dependent on CHOP.

The FMS-like tyrosine kinase 3 (FLT3) gene is mutated in approximately one third of patients with acute myeloid leukemia (AML), either by internal tandem duplications (FLT3-ITD), or by a point mutation mainly involving the tyrosine kinase domain (FLT3-TKD). Patients with FLT3-ITD have a high risk of relapse and low cure rates. Several FLT3 tyrosine kinase inhibitors have been developed in the last few years with variable kinase inhibitory properties, pharmacokinetics, and toxicity profiles. FLT3 inhibitors are divided into first generation multi-kinase inhibitors (such as sorafenib, lestaurtinib, midostaurin) and next generation inhibitors (such as quizartinib, crenolanib, gilteritinib) based on their potency and specificity of FLT3 inhibition. These diverse FLT3 inhibitors have been evaluated in myriad clinical trials as monotherapy or in combination with conventional chemotherapy or hypomethylating agents and in various settings, including front-line, relapsed or refractory disease, and maintenance therapy after consolidation chemotherapy or allogeneic stem cell transplantation. In this practical question-and-answer-based review, the main issues faced by the leukemia specialists on the use of FLT3 inhibitors in AML are addressed.

Post-therapeutic relapse, poor survival rates and increasing incidence justify the search for novel therapeutic targets and strategies in cutaneous malignant melanoma (CMM). Within this context, a potential oncogenic role for TrkA in CMM is suggested by reports of NTRK1 amplification, enhanced TrkA expression and intracellular TrkA activation associated with poor prognosis. TrkA, however, exhibits tumour-suppressing properties in melanoma cell lines and has recently been reported not to be associated with CMM progression. To better understand these contradictions, we present the first analysis of potential oncogenic alternative TrkA mRNA splicing, associated with TrkA immunoreactivity, in CMMs, and compare the behaviour of fully spliced TrkA and the alternative TrkAIII splice variant in BRAF(V600E)-mutated A375 melanoma cells. Alternative TrkA splicing in CMMs was associated with unfolded protein response (UPR) activation. Of the several alternative TrkA mRNA splice variants detected, TrkAIII was the only variant with an open reading frame and, therefore, oncogenic potential. TrkAIII expression was more frequent in metastatic CMMs, predominated over fully spliced TrkA mRNA expression in ≈50% and was invariably linked to intracellular phosphorylated TrkA immunoreactivity. Phosphorylated TrkA species resembling TrkAIII were also detected in metastatic CMM extracts. In A375 cells, reductive stress induced UPR activation and promoted TrkAIII expression and, in transient transfectants, promoted TrkAIII and Akt phosphorylation, enhancing resistance to reductive stress-induced death, which was prevented by lestaurtinib and entrectinib. In contrast, fully spliced TrkA was dysfunctional in A375 cells. The data identify fully spliced TrkA dysfunction as a novel mechanism for reducing melanoma suppression, support a causal relationship between reductive stress, UPR activation, alternative TrkAIII splicing and TrkAIII activation and characterise a targetable oncogenic pro-survival role for TrkAIII in CMM.

The free-living amebae Naegleria, Acanthamoeba, and Balamuthia cause rare but life-threatening infections. All three parasites can cause meningoencephalitis. Acanthamoeba can also cause chronic keratitis and both Balamuthia and Acanthamoeba can cause skin and systemic infections. There are minimal drug development pipelines for these pathogens despite a lack of available treatment regimens and high fatality rates. To identify anti-amebic drugs, we screened 159 compounds from a high-value repurposed library against trophozoites of the three amebae. Our efforts identified 38 compounds with activity against at least one ameba. Multiple drugs that bind the ATP-binding pocket of mTOR and PI3K are active, highlighting these compounds as important inhibitors of these parasites. Importantly, 24 active compounds have progressed at least to phase II clinical studies and overall 15 compounds were active against all three amebae. Based on central nervous system (CNS) penetration or exceptional potency against one amebic species, we identified sixteen priority compounds for the treatment of meningoencephalitis caused by these pathogens. The top five compounds are (i) plicamycin, active against all three free-living amebae and previously U.S. Food and Drug Administration (FDA) approved, (ii) TG02, active against all three amebae, (iii and iv) FDA-approved panobinostat and FDA orphan drug lestaurtinib, both highly potent against Naegleria, and (v) GDC-0084, a CNS penetrant mTOR inhibitor, active against at least two of the three amebae. These results set the stage for further investigation of these clinically advanced compounds for treatment of infections caused by the free-living amebae, including treatment of the highly fatal meningoencephalitis., Graphical abstract Image 1, Highlights • Focused screen of ReFRAME library (159 compounds) on three free-living amebae. • 16 priority compounds selected based on potency, advanced clinical studies and CNS penetration. • Identified compounds with fast killing kinetics against Naegleria and Balamuthia. • Identified compounds capable of delaying Balamuthia trophozoite recrudescence. • 5 compounds of high interest for treatment of CNS infections with free-living amebae.

Agonists of glucocorticoid receptor (GR) are frequently given to cancer patients with platinum-containing chemotherapy to reduce inflammation, but how GR influences tumor growth in response to platinum-based chemotherapy such as cisplatin through inflammation-independent signaling remains largely unclear. Combined genomics and transcription factor profiling reveal that MAST1, a critical platinum resistance factor that reprograms the MAPK pathway, is upregulated upon cisplatin exposure through activated transcription factor GR. Mechanistically, cisplatin binds to C622 in GR and recruits GR to the nucleus for its activation, which induces MAST1 expression and consequently reactivates MEK signaling. GR nuclear translocation and MAST1 upregulation coordinately occur in patient tumors collected after platinum treatment, and align with patient treatment resistance. Co-treatment with dexamethasone and cisplatin restores cisplatin-resistant tumor growth, whereas addition of the MAST1 inhibitor lestaurtinib abrogates tumor growth while preserving the inhibitory effect of dexamethasone on inflammation in vivo. These findings not only provide insights into the underlying mechanism of GR in cisplatin resistance but also offer an effective alternative therapeutic strategy to improve the clinical outcome of patients receiving platinum-based chemotherapy with GR agonists., Glucocorticoid receptor (GR) agonists - used in the treatment of solid malignant tumors to reduce inflammation - could potentially affect the anti-tumor activity of chemotherapy. Here, the authors identify a mechanism of cisplatin resistance observed with GR agonist treatment, and show the binding and activation of GR by cisplatin, which leads to MAST1 activation and subsequent MAPK re-activation.

FMS-like tyrosine kinase 3- internal tandem duplication (FLT3-ITD) remains as one of the most frequently mutated genes in acute myeloid leukemia (AML), especially in those with normal cytogenetics. The FLT3-ITD and FLT3-TKD (tyrosine kinase domain) mutations are biomarkers for high risk AML and are associated with drug resistance and high risk of relapse. Multiple FLT3 inhibitors are in clinical development, including lestaurtinib, tandutinib, quizartinib, midostaurin, gilteritinib, and crenolanib. Midostaurin and gilteritinib have been approved by FDA for Flt3 mutated AML. Gilteritinib (ASP2215, Xospata) is a small molecule dual inhibitor of FLT3/AXL. The ADMIRAL study showed that longer overall survival and higher response rate are associated with gilteritinib in comparison with salvage chemotherapy for relapse /refractory (R/R) AML. These data from the ADMIRAL study may lead to the therapy paradigm shift and establish gilteritinib as the new standard therapy for R/R FLT3-mutated AML. Currently, multiple clinical trials are ongoing to evaluate the combination of gilteritinib with other agents and regimens. This study summarized clinical trials of gilteritinib for AML.

Microtubule-associated serine/threonine kinase 1 (MAST1) is a central driver of cisplatin resistance in human cancers. However, the molecular mechanism regulating MAST1 levels in cisplatin-resistant tumors is unknown. Through a proteomics screen, we identified the heat shock protein 90 B (hsp90B) chaperone as a direct MAST1 binding partner essential for its stabilization. Targeting hsp90B sensitized cancer cells to cisplatin predominantly through MAST1 destabilization. Mechanistically, interaction of hsp90B with MAST1 blocked ubiquitination of MAST1 at lysines 317 and 545 by the E3 ubiquitin ligase CHIP and prevented proteasomal degradation. The hsp90B-MAST1-CHIP signaling axis and its relationship with cisplatin response were clinically validated in cancer patients. Furthermore, combined treatment with a hsp90 inhibitor and the MAST1 inhibitor lestaurtinib further abrogated MAST1 activity and consequently enhanced cisplatin-induced tumor growth arrest in a patient-derived xenograft model. Our study not only uncovers the regulatory mechanism of MAST1 in tumors but also suggests a promising combinatorial therapy to overcome cisplatin resistance in human cancers.

INTRODUCTION Mutations of the FLT3 gene are among most common genetic abnormalities occurring in acute myeloid leukemia (AML) and are associated with dismal prognosis. Tremendous effort has been devoted to developing clinically effective FLT3 inhibitors. First generation inhibitors consisted of multi-kinase inhibitors (sorafenib, lestaurtinib, midostaurin), which blocked FLT3 as well as multiple other kinase receptors. The failure of these agents to induce durable responses led to the development of second generation FLT3 tyrosine kinase inhibitors (quizartinib, crenolinib, gilteritinib) exhibiting high potency and specificity for mutant FLT3 kinases and sustained in vivo FLT3 inhibition. These myriad FLT3 inhibitors possess diverse kinase inhibitory properties, toxicity profiles, and pharmacokinetics, which impact on their incorporation into therapeutic regimens. Areas covered: This article reviews the medical literature on current and future FLT3 inhibitors for AML therapy. We provide algorithms for which kinase inhibitor should be utilized for different FLT3 mutations (ITD±TKD) and clinical scenarios (de novo, relapsed/refractory, fit vs. unfit) and discuss novel FLT3 targeted therapeutic approaches. Expert commentary: Integration of clinically active FLT3 inhibitors into all stages of therapy for all individuals with FLT3 mutant AML promises to significantly improve outcomes for this poor prognosis disease.

Determining which FLT3 inhibitor holds the greatest promise is a difficult task, as the drugs vary according to potency, specificity, protein-binding, drug interactions, and side effect profile. The best choice depends on when in the course of the disease the inhibitor will be used. Moreover, as the results of ongoing trials become available, newer agents could supplant former 'best' drugs. This paper reviews FLT3 inhibitors in combination with chemotherapy early in the disease in FLT3 mutant patients, as single agents or in combination in advanced disease, or in the post-transplant setting to provide separate answers to the main question.

Infants with KMT2A‐rearranged acute lymphoblastic leukemia (KMT2A-r ALL) have a poor prognosis. KMT2A-r ALL overexpresses FLT3, and the FLT3 inhibitor (FLT3i) lestaurtinib potentiates chemotherapy‐induced cytotoxicity in preclinical models. Children’s Oncology Group (COG) AALL0631 tested whether adding lestaurtinib to post‐induction chemotherapy improved event-free survival (EFS). After chemotherapy induction, KMT2A-r infants received either chemotherapy only or chemotherapy plus lestaurtinib. Correlative assays included FLT3i plasma pharmacodynamics (PD), which categorized patients as inhibited or uninhibited, and FLT3i ex vivo sensitivity (EVS), which categorized leukemic blasts as sensitive or resistant. There was no difference in 3-year EFS between patients treated with chemotherapy plus lestaurtinib (n = 67, 36 ± 6%) vs. chemotherapy only (n = 54, 39 ± 7%, p = 0.67). However, for the lestaurtinib-treated patients, FLT3i PD and FLT3i EVS significantly correlated with EFS. For FLT3i PD, EFS for inhibited/uninhibited was 59 ± 10%/28 ± 7% (p = 0.009) and for FLTi EVS, EFS for sensitive/resistant was 52 ± 8%/5 ± 5% (p

Background FMS-like tyrosine kinase 3 (FLT3) is the most frequent mutation in AML. With two FLT3 inhibitors recently approved by the FDA (midostaurin and gilteritinib), there is a need to evaluate these targeted agents. Purpose To assess the clinical effectiveness of FLT3 inhibitors in AML patients. Methods Standard systematic review methods were utilised. Searches were conducted to July 2020 for completed and in-progress randomised controlled trials of FLT3 inhibitors in AML. A fixed-effect meta-analysis was undertaken. Results Eight completed trials involving 2656 patients and assessing five different FLT3 inhibitors (sorafenib, lestaurtinib, midostaurin, gilteritinib and quizartinib) were included. The pooled results were as follows (FLT3 inhibitor/control): overall survival hazard ratio (HR) = 0.83 (95% confidence interval [CI] 0.75 to 0.92, p = 0.0005), event-free survival HR = 0.85 (95% CI 0.77 to 0.94, p = 0.002), relapse-free survival HR = 0.76 (95% CI 0.64 to 0.90, p = 0.001), complete remission relative risk (RR) = 1.11 (95% CI 1.00 to 1.22. p = 0.05) and 60-day mortality RR = 1.04 (95% CI 0.77 to 1.40, p = 0.79). Relative risk of grade 3 and above vascular, dermatological, respiratory and hepatobiliary adverse events were found to be statistically significantly higher in the FLT3 inhibitor group compared to control, but the actual numbers of events were relatively small. Nineteen ongoing trials are still in progress, only one of which specifically targets older patients with AML. Conclusions There is evidence to support the use of FLT3 inhibitors in patients with AML, but more data is needed to verify the optimum use of the drugs regarding type of inhibitor, disease stage and patient characteristics, not only in relation to disease control, but adverse events and quality of life. There are a large number of ongoing trials; therefore, the results of this review are not a fait accompli; thus, is it recommended that the review be updated in a couple of years’ time. Given the challenges in extracting the complete data set required to assess clinical effectiveness, it is highly recommended that ongoing and future trials improve transparency and consistency of reporting of all trial outcomes, particularly disease control and adverse events, to enable a global clinical effectiveness assessment. Systematic review registration PROSPERO CRD42017055581

Mutations in the FMS-like tyrosine kinase 3 (FLT3) gene arise in 25-30% of all acute myeloid leukemia (AML) patients. These mutations lead to constitutive activation of the protein product and are divided in two broad types: internal tandem duplication (ITD) of the juxtamembrane domain (25% of cases) and point mutations in the tyrosine kinase domain (TKD). Patients with FLT3 ITD mutations have a high relapse risk and inferior cure rates, whereas the role of FLT3 TKD mutations still remains to be clarified. Additionally, growing research indicates that FLT3 status evolves through a disease continuum (clonal evolution), where AML cases can acquire FLT3 mutations at relapse - not present in the moment of diagnosis. Several FLT3 inhibitors have been tested in patients with FLT3-mutated AML. These drugs exhibit different kinase inhibitory profiles, pharmacokinetics and adverse events. First-generation multi-kinase inhibitors (sorafenib, midostaurin, lestaurtinib) are characterized by a broad-spectrum of drug targets, whereas second-generation inhibitors (quizartinib, crenolanib, gilteritinib) show more potent and specific FLT3 inhibition, and are thereby accompanied by less toxic effects. Notwithstanding, all FLT3 inhibitors face primary and acquired mechanisms of resistance, and therefore the combinations with other drugs (standard chemotherapy, hypomethylating agents, checkpoint inhibitors) and its application in different clinical settings (upfront therapy, maintenance, relapsed or refractory disease) are under study in a myriad of clinical trials. This review focuses on the role of FLT3 mutations in AML, pharmacological features of FLT3 inhibitors, known mechanisms of drug resistance and accumulated evidence for the use of FLT3 inhibitors in different clinical settings.

Repurposing drugs to enhance skeletal muscle repair mechanisms may unveil novel treatments for certain muscular pathophysiological states while maintaining high safety and efficacy standards at lower costs. The anti-cancer drug lestaurtinib appears to induce fusion and differentiation of myoblasts in vitro, inviting an attempt in the present study to determine if these results are replicable in vivo. To initiate skeletal muscle regeneration, tibialis anterior muscles were injured in both legs of C57BL/6J mice (n = 30) using the myotoxin, cardiotoxin (CTX). At 3 days post-CTX, the affected area on the right leg was treated with lestaurtinib at a concentration of either 40 nM or 200 nM; the left leg was used as a vehicle-treated control. Muscles were excised at either 4, 7, or 14 days post-CTX and then histologically examined to determine the effect of lestaurtinib on muscle regeneration. Fibre cross-sectional area, collagen-positive area (%), embryonic myosin heavy chain (emb-MyHC) positive area (%), and number of emb-MyHC-positive cells were assessed. The regeneration process appeared to be mostly unaffected following lestaurtinib treatment as indicated by no difference in fibre cross-sectional area, number of newly regenerated myofibres, and emb-MyHC-positive area. However, significantly reduced collagen-positive area was observed in lestaurtinib-treated muscle at 7 and 14 days post-CTX. Divergent effects on collagen content were observed between lestaurtinib-treated muscles, suggesting that each drug level exhibited opposing effects on collagen synthesis and/or degradation. These data suggest that single dose treatment of 40 or 200 nM of lestaurtinib at 3 days post-injury does not augment muscle repair, although the 200 nM dose may reduce collagen accumulation. Further investigation of lestaurtinib treatment on muscle regeneration in myopathic states may reveal utility in suppressing muscle fibrosis; however, further study is needed to understand the mechanisms by which lestaurtinib exerts its effects on muscle in vivo.

Human adenovirus (HAdV) can cause severe disease in certain at-risk populations such as newborns, young children, the elderly and individuals with a compromised immune system. Unfortunately, no FDA-approved antiviraldrug is currently available for the treatment of HAdV infections. Within the nucleus of infected cells, the HAdV genome associates with histones and forms a chromatin-like structure during early infection, and viral gene expression appears to be regulated by cellular epigenetic processes. Thus, one potential therapeutic strategy to combat HAdV disease may be to target the cellular proteins involved in modifying the viral nucleoprotein structure and facilitating HAdV gene expression and replication. We have screened a panel of small molecules that modulate the activity of epigenetic regulatory proteins for compounds affecting HAdV gene expression. Several of the compounds, specifically chaetocin, gemcitabine and lestaurtinib, reduced HAdV recovery by 100- to 1000-fold, while showing limited effects on cell health, suggesting that these compounds may indeed be promising as anti-HAdV therapeutics.

Flt3 is expressed by early myeloid and lymphoid progenitor cells and it regulates the proliferation and differentiation of hematopoietic cells. Flt3 is activated by the Flt3 ligand, the monomeric form of which is a polypeptide of about 200 amino acid residues. Both membrane-associated and soluble Flt3 ligands, which are a product of the same gene, function as noncovalent dimers. FLT3 mutations occur in about one-third of newly diagnosed acute myelogenous leukemia (AML) patients. This disease is a malignancy of hematopoietic progenitor cells with a variable clinical course; the incidence of this disorder is more than twice that of patients with chronic myelogenous leukemias (20,000 vs. 8500 new patients per year, respectively, in the United States). FLT3 internal tandem duplication (ITD) results from the head-to-tail duplication of from one to more than 100 amino acids within the juxtamembrane domain and such duplication occurs in about 20-25 % of patients with acute myelogenous leukemias. FLT3 tyrosine kinase (FLT3 TK) mutations, usually within the activation segment, occur in 5-10 % of these patients. The mainstay for the care of acute myelogenous leukemias include daunorubicin or idarubicin and cytarabine. Older patients who are not candidates for such traditional therapy are usually given 5-azacitidine, decitabine, or clofarabine. The addition of orally effective small molecule Flt3 inhibitors to these therapies may prolong event-free and overall survival, a subject of ongoing clinical studies. Midostaurin is US FDA-approved in combination with standard cytarabine and daunorubicin for first-line induction chemotherapy and in combination with cytarabine for second-line consolidation chemotherapy in the treatment of acute myelogenous leukemias with FLT3-postive mutations. Moreover, gilteritinib is a Flt3 multikinase inhibitor that is also FDA approved for the care of adult patients with relapsed or refractory acute myelogenous leukemias with FLT3 mutations. Quizartinib is a Flt3 multikinase inhibitor that was approved by the Ministry of Health, Labor and Welfare (MHLW) of Japan for the treatment of adult patients with relapsed/refractory Flt3-positive acute myelogenous leukemias. Gilteritinib and quizartinib bind to Flt3 with the inactive DFG-Dout structure and are classified as type II inhibitors. Furthermore, ponatinib is a multikinase inhibitor that is approved as therapy for Philadelphia chromosome-positive acute lymphoblastic and chronic myelogenous leukemias; it is used off label for the treatment of patients with acute myelogenous leukemias. Moreover, sorafenib is FDA-approved for the treatment of hepatocellular, renal cell, and differentiated thyroid cancers and it is used off label as maintenance therapy following allogeneic hematopoietic stem cell transplantation in the treatment of acute myelogenous leukemias. Other drugs that are in clinical trials for the treatment of this disorder include sunitinib, crenolanib, FF10101, and lestaurtinib. Unlike chronic myelogenous leukemias, which result solely from the formation of the BCR-Abl chimeric protein kinase, acute myelogenous leukemias result from multi-factorial causes and are prone to be resistant to both cytotoxic and targeted therapies. Consequently, there is a pressing need for better understanding the etiologies of acute myelogenous leukemias and for the development of more effective therapies.

The oncogenic mutation V600E in B-Raf activation loop (A-loop) has been frequently observed to cause drug resistance in colorectal cancer chemotherapy. Here, the molecular mechanism of V600E-induced conformational flipping of B-Raf activation loop (A-loop) is investigated systematically via continuum electrostatic analysis. It is found that substitution of the electroneutral Val600 residue with negatively charged glutamic acid Glu600 electrostatically destabilizes the inactive DFG-out conformation of B-Raf kinase and promotes its shifting to active DFG-in conformation. This is analogous with natural phosphorylation of Thr598 and/or Ser601 residues in A-loop to activate the kinase, that is, both the mutation and phosphorylation can introduce negative charge to B-Raf A-loop and then trigger the loop flipping. Energetic analysis reveals that the V600E mutation can affect inhibitor binding indirectly via regulation of kinase conformation. Type-I and type-II inhibitors respond distinctly to V600E mutation; the former is sensitized by the mutation, while the latter generally shows a low sensitivity to the mutation. Based on this guideline, the sophisticated type-I pan-kinase inhibitor Staurosporine as well as its analogs Midostaurin and Lestaurtinib are identified as potent mutant-selective inhibitors by modeling analysis and kinase assay, which exhibit a moderate or high selectivity for B-RafV600E over B-RafWT (3.7-fold, 6.1-fold and > 3.1-fold, respectively).

Controversy exists whether internal tandem duplication of FMS-like tyrosine kinase 3 (FLT3-internal tandem duplication [ITD]) allelic ratio (AR) and/or length of the ITD should be taken into account for risk stratification of pediatric acute myeloid leukemia (AML) and whether it should be measured on RNA or DNA. Moreover, the ITD status may be of relevance for selecting patients eligible for FLT3 inhibitors. Here, we included 172 pediatric AML patients, of whom 36 (21%) harbored FLT3-ITD as determined on both RNA and DNA. Although there was a good correlation between both parameters AR(spearman) = 0.62 (95% confidence interval, 0.22-0.87) and ITDlength(spearman) = 0.98 (95% confidence interval, 0.90-1.00), only AR >= 0.5 and length >= 48 base pairs (bps) based on RNA measurements were significantly associated with overall survival (AR: P-logrank = .008; ITDlength: P-logrank = .011). In large ITDs (>156 bp on DNA) a remarkable 90-bp difference exists between DNA and RNA, including intron 14, which is spliced out in RNA. Ex vivo exposure (n = 30) to FLT3 inhibitors, in particular to the FLT3-specific inhibitor gilteritinib, showed that colony-forming capacity was significantly more reduced in FLT3-ITD-AR >= 0.5 compared with ITD-AR-low and ITD2 patient samples (P

Here we assembled a novel metabolic drug library covering 243 compounds allowing to systematically identify metabolic dependencies in high-throughput phenotypic screens. The CeMM Library of Metabolic Drugs (CLIMET) was compiled in a stepwise fashion, starting from 8000 candidate compounds, after a survey of public drug-target databases, and ending with 243 highly-curated compounds including extensive crosschecking for approval status, structural information, compound's potency and selectivity for the intended target, pathway/target redundancy, and commercial availability. To assess the potency of the compounds in CLIMET, we screened the full collection against a panel of 15 diverse myeloid leukemia cell lines. Each compound was tested for its effect on cell growth and survival in a 10,000-fold concentration range (1nM to 10uM) enabling the generation of dose response curves and calculation of area under the curve values (AUC) for each drug. We, further, functionally grouped the cell lines and drugs based on metabolic drug efficacy patterns and associated them with distinct genomic and metabolic attributes. Analysis of the metabolic drug response profiles revealed that 77 compounds (32%) affected cell viability with the top effective compounds targeting nucleotide metabolism, oxidative stress, and the PI3K/mTOR pathway. Unsupervised hierarchical clustering of the drug sensitivity profiles stratified the cell lines in 5 functional taxonomic groups, with the activity of 19 compounds significantly contributing to the cell line grouping (e.g. PF-02545920, GW 4064, mTOR inhibitors, daporinad). Comparison of the oxygen consumption rate and extracellular acidification rate showed that the examined cell lines have analogous baseline metabolic phenotypes, suggesting that the mitochondrial function of the cells as assessed by Seahorse analysis did not significantly influence the clustering. Genotype to phenotype associations were identified between FLT3mutations and sensitivity to 5-FU, lestaurtinib, and PF-02545920. Moreover, RAS mutations negatively correlated to mTOR and mitochondrial respiration inhibitor sensitivity, whereas TP53 mutations conferred a resistance phenotype to PI3K pathway inhibitors and antineoplastic agents. Selective sensitivities were detected to the lactate transporter (SLC16A1) inhibitor AZD3965, the PI3K inhibitor pictilisib, and the fatty acid synthase inhibitor GSK2194069, which could be explained by varied gene expression in sensitive cell lines and target/process dependency. CLIMET allows for identification of metabolic susceptibilities, grouping of cancer cells based on metabolic dependencies, as well as understanding of context-dependent mechanism of action of drugs. Functional drug testing may provide a rapid and robust approach to identify metabolic vulnerabilities, responding patients, and prioritize compounds for clinical evaluation as illustrated with our study. Disclosures Staber: Janssen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Astra Zeneca: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Research Funding; Celgene/ BMS: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; msd: Consultancy, Honoraria.

9 ISSN 2045-1393 10.2217/IJH.12.32 © 2013 Future Medicine Ltd Int. J. Hematol. Oncol. (2013) 2(1), 9–11 Approximately one-quarter of acute myeloid leukemia (AML) cases are characterized by an internal tandem duplication (ITD) mutation in the FLT3 gene. Patients with FLT3-ITD AML carry an especially poor prognosis, with few patients surviving 5 years or longer [1,2]. Point mutations at the D835 locus on the FLT3 gene occur in approximately 8% of adult AML cases, although the prognostic value of de novo FLT3 D835 mutations remains a topic of debate [3–5]. A growing number of smallmolecule tyrosine kinase inhibitors (TKIs) are being developed as FLT3 inhibitors and used in clinical trials, primarily for the treatment of FLT3-ITD AML. Early failures of FLT3 inhibitors in clinical trials prompted speculation that the ITD may be a passenger mutation. However, in a Phase II study of the FLT3 inhibitor quizartinib, there was a population of FLT3-ITD AML patients who initially exhibited blast reduction to

Multiple voltage-gated Na+ (Nav) channelopathies can be ascribed to subtle changes in the Nav macromolecular complex. Fibroblast growth factor 14 (FGF14) is a functionally relevant component of the Nav1.6 channel complex, a causative link to spinocerebellar ataxia 27 (SCA27) and an emerging risk factor for neuropsychiatric disorders. Yet, how this protein:channel complex is regulated in the cell is still poorly understood. To search for key cellular pathways upstream of the FGF14:Nav1.6 complex, we have developed, miniaturized and optimized an in-cell assay in 384-well plates by stably reconstituting the FGF14:Nav1.6 complex using the split-luciferase complementation assay. We then conducted a high-throughput screening (HTS) of 267 FDA-approved compounds targeting known mediators of cellular signaling. Of the 65 hits initially detected, 24 were excluded based on counter-screening and cellular toxicity. Based on target analysis, potency and dose-response relationships, 5 compounds were subsequently repurchased for validation and confirmed as hits. Among those, the tyrosine kinase inhibitor lestaurtinib was highest ranked, exhibiting submicromolar inhibition of FGF14:Nav1.6 assembly. While providing evidence for a robust in-cell HTS platform that can be adapted to search for any channelopathy-associated regulatory proteins, these results lay the potential groundwork for repurposing cancer drugs for neuropsychopharmacology.

The clinical benefit of adding FMS-like tyrosine kinase-3 (FLT3)-directed small molecule therapy to standard first-line treatment of acute myeloid leukemia (AML) has not yet been established. As part of the UK AML15 and AML17 trials, patients with previously untreated AML and confirmed FLT3-activating mutations, mostly younger than 60 years, were randomly assigned either to receive oral lestaurtinib (CEP701) or not after each of 4 cycles of induction and consolidation chemotherapy. Lestaurtinib was commenced 2 days after completing chemotherapy and administered in cycles of up to 28 days. The trials ran consecutively. Primary endpoints were overall survival in AML15 and relapse-free survival in AML17; outcome data were meta-analyzed. Five hundred patients were randomly assigned between lestaurtinib and control: 74% had FLT3-internal tandem duplication mutations, 23% FLT3-tyrosine kinase domain point mutations, and 2% both types. No significant differences were seen in either 5-year overall survival (lestaurtinib 46% vs control 45%; hazard ratio, 0.90; 95% CI 0.70-1.15; P = .3) or 5-year relapse-free survival (40% vs 36%; hazard ratio, 0.88; 95% CI 0.69-1.12; P = .3). Exploratory subgroup analysis suggested survival benefit with lestaurtinib in patients receiving concomitant azole antifungal prophylaxis and gemtuzumab ozogamicin with the first course of chemotherapy. Correlative studies included analysis of in vivo FLT3 inhibition by plasma inhibitory activity assay and indicated improved overall survival and significantly reduced rates of relapse in lestaurtinib-treated patients who achieved sustained greater than 85% FLT3 inhibition. In conclusion, combining lestaurtinib with intensive chemotherapy proved feasible in younger patients with newly diagnosed FLT3-mutated AML, but yielded no overall clinical benefit. The improved clinical outcomes seen in patients achieving sustained FLT3 inhibition encourage continued evaluation of FLT3-directed therapy alongside front-line AML treatment. The UK AML15 and AML17 trials are registered at www.isrctn.com/ISRCTN17161961 and www.isrctn.com/ISRCTN55675535 respectively.

Fms-like tyrosine kinase 3 (Flt3) is a hematopoietic growth factor receptor expressed on lymphomyeloid progenitors and frequently, by AML blasts. Its ligand, Flt3L, has non-hematopoietic and lymphoid origins, is detectable during homeostasis and increases to high levels in states of hypoplasia due to genetic defects or treatment with cytoreductive agents. Measurement of Flt3L by ELISA reveals that Flt3+AML, is associated with depletion of Flt3L to undetectable levels. After induction chemotherapy, Flt3L is restored in patients entering CR, but remains depressed in those with refractory disease. Weekly sampling reveals marked differences in the kinetics of Flt3L response during the first 6 weeks of treatment, proportionate to the clearance of blasts and cellularity of the BM. In the UK NCRI AML17 trial, Flt3L was measured at day 26 in a subgroup of 135 patients with Flt3 mutation randomized to the tyrosine kinase inhibitor lestaurtinib. In these patients, attainment of CR was associated with higher Flt3L at day 26 (Mann-Whitney p < 0.0001). Day 26 Flt3L was also associated with survival: Flt3L ≤ 291pg/ml was associated with inferior event-free survival; and, Flt3L >1185pg/ml was associated with higher overall survival (p = 0.0119). Serial measurement of Flt3L in patients who had received a hematopoietic stem cell transplant for AML further illustrated the potential value of declining Flt3L to identify relapse. Together these observations suggest that measurement of Flt3L provides a non-invasive estimate of progenitor cell mass in most patients with AML, with the potential to inform clinical decisions.Graphical abstract

Fms-like tyrosine kinase 3 (Flt3) is expressed on progenitor cells and acute myeloid leukemia (AML) blasts. Fms-like tyrosine kinase 3 ligand (Flt3L) is detectable during homeostasis and increases in hypoplasia due to genetic defects or treatment with cytoreductive agents. Conversely, Flt31 AML is associated with depletion of Flt3L to undetectable levels. After induction chemotherapy, Flt3L is restored in patients entering complete remission (CR) but remains depressed in those with refractory disease. Weekly sampling reveals marked differences in the kinetics of Flt3L response during the first 6 weeks of treatment, proportionate to the clearance of blasts and cellularity of the bone marrow. In the UK NCRI AML17 trial, Flt3L was measured at day 26 in a subgroup of 140 patients with Flt3 mutation randomized to the tyrosine kinase inhibitor lestaurtinib or placebo. In these patients, attainment of CR was associated with higher Flt3L at day 26 (Mann-Whitney U P, .0001). Day 26 Flt3L was also associated with survival; Flt3L #291 pg/mL was associated with inferior event-free survival (EFS), and Flt3L .1185 pg/mL was associated with higher overall survival (OS; P 5 .0119). The separation of EFS and OS curves increased when minimal residual disease (MRD) status was combined with Flt3L measurement, and Flt3L retained a near-significant association with survival after adjusting for MRD in a proportional hazards model. Serial measurement of Flt3L in patients who had received a hematopoietic stem cell transplant for AML illustrates the potential value of monitoring Flt3L to identify relapse. Measurement of Flt3L is a noninvasive test with the potential to inform clinical decisions in patients with AML.