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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.

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.

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

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.

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.

Sustained reliance on androgen receptor (AR) after failure of AR-targeting androgen deprivation therapy (ADT) prevents effective treatment of castration-recurrent (CR) prostate cancer (CaP). Interfering with the molecular machinery by which AR drives CaP progression may be an alternative therapeutic strategy but its feasibility remains to be tested. Here, we explore targeting the mechanism by which AR, via RhoA, conveys androgen-responsiveness to serum response factor (SRF), which controls aggressive CaP behavior and is maintained in CR-CaP. Following a siRNA screen and candidate gene approach, RNA-Seq studies confirmed that the RhoA effector Protein Kinase N1 (PKN1) transduces androgen-responsiveness to SRF. Androgen treatment induced SRF-PKN1 interaction, and PKN1 knockdown or overexpression severely impaired or stimulated, respectively, androgen regulation of SRF target genes. PKN1 overexpression occurred during clinical CR-CaP progression, and hastened CaP growth and shortened CR-CaP survival in orthotopic CaP xenografts. PKN1’s effects on SRF relied on its kinase domain. The multikinase inhibitor lestaurtinib inhibited PKN1 action and preferentially affected androgen regulation of SRF over direct AR target genes. In a CR-CaP patient-derived xenograft, expression of SRF target genes was maintained while AR target gene expression declined and proliferative gene expression increased. PKN1 inhibition decreased viability of CaP cells before and after ADT. In patient-derived CaP explants, lestaurtinib increased AR target gene expression but did not significantly alter SRF target gene or proliferative gene expression. These results provide proof-of-principle for selective forms of ADT that preferentially target different fractions of AR’s transcriptional output to inhibit CaP growth.

Myelofibrosis (MF) is a myeloproliferative neoplasm (MPN) characterized by bone marrow fibrosis and clonal myeloid proliferation which can eventually evolve into acute myeloid leukemia. Dysregulate...

FLT3 mutations are one of the most common findings in acute myeloid leukemia (AML). FLT3 inhibitors have been in active clinical development. Midostaurin as the first-in-class FLT3 inhibitor has been approved for treatment of patients with FLT3-mutated AML. In this review, we summarized the preclinical and clinical studies on new FLT3 inhibitors, including sorafenib, lestaurtinib, sunitinib, tandutinib, quizartinib, midostaurin, gilteritinib, crenolanib, cabozantinib, Sel24-B489, G-749, AMG 925, TTT-3002, and FF-10101. New generation FLT3 inhibitors and combination therapies may overcome resistance to first-generation agents.

FMS-like tyrosine kinase 3 (FLT3) is one of the most commonly mutated genes in AML. FLT3 is mutated in ~30% of patients with AML, either by internal tandem duplications (FLT3-ITD) of the juxta-membrane domain or by a point mutation, usually involving the tyrosine kinase domain. Several FLT3 tyrosine kinase inhibitors are being evaluated in multiple studies aiming at improving outcomes. The most widely used is sorafenib, a potent multikinase inhibitor approved for hepatocellular carcinoma and renal cell carcinoma. Sorafenib monotherapy or in combination with conventional chemotherapy, has been evaluated in various settings in AML, including front-line, relapsed or refractory disease including post-allograft failures and, more recently, as post-transplant maintenance therapy. Encouraging data have emerged with several other agents like lestaurtinib, midostaurin, crenolanib, gilteritinib and quizartinib. Although transient responses to FLT3 inhibitors are often observed in case of disease relapse, the most promising approach is the use of FLT3 inhibitors either in combination with induction chemotherapy or as consolidation/maintenance therapy after allogeneic hematopoietic cell transplantation. In this review, we summarize the clinical data on sorafenib and other FLT3 inhibitors in AML.

Microtubule-associated serine/threonine kinase 1 (MAST1) plays a key role in cisplatin resistance of human cancers. However, the mechanism underlying MAST1 regulation remains elusive. We identified the hsp90B as a direct MAST1 binding partner essential for its stabilization via a proteomics screen. Targeting hsp90B in cancer cells sensitizes cisplatin treatment mainly through MAST1 destabilization. Mechanistically, hsp90B protects MAST1 from ubiquitination at lysine 317 and 545 by the E3 ubiquitin ligase CHIP and prevents proteasomal degradation. The role of the hsp90B-MAST1-CHIP signaling axis in cisplatin resistance was clinically validated in cancer patients. Furthermore, a combination of hsp90 inhibitor and MAST1 inhibitor lestaurtinib further inhibited MAST1 activity and consequently potentiated the cisplatin effect in a patient-derived xenograft model. Our study not only reveals the mechanism of MAST1 regulation in tumors but also demonstrates a promising combinatorial therapy to overcome cisplatin resistance in human cancers. Citation Format: Chaoyun Pan, Jie Li, Austin Boese, Sumin Kang. Hsp90B protects MAST1 from CHIP-mediated ubiquitination and degradation providing cisplatin resistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4082.

Anaplastic thyroid cancer (ATC) is a rare and lethal human malignancy with no known effective therapies in the majority of cases. Despite the use of conventional treatments such as chemotherapy, radiation and surgical resection, this disease remains almost universally fatal. In the present study, we identified the JAK2 inhibitor Lestaurtinib as a potent compound when testing against 13 ATC cell lines. Lestaurtinib demonstrated a potent antiproliferative effect in vitro at nanomolar concentrations. Furthermore, Lestaurtinib impeded cell migration and the ability to form colonies from single cells using scratch-wound and colony formation assays, respectively. Flow cytometry was used for cell cycle analysis following drug treatment and demonstrated arrest at the G2/M phase of the cell cycle, indicative of a cytostatic effect. In vivo studies using the chick chorioallantoic membrane xenograft models demonstrated that treatment with Lestaurtinib resulted in a significant decrease in endpoint tumor volume and vascularity using power Doppler ultrasound imaging. Overall, this study provides evidence that Lestaurtinib is a potent antiproliferative agent with potential antiangiogenic activity that warrants further investigation as a targeted therapy for ATC.

Advanced prostate cancer is treated with androgen deprivation, but most patients eventually progress and need new therapy. Recent genomic/exomic sequencing identified SPOP as the most frequently mutated gene in 6% - 15% of prostate cancer. Based on the function of SPOP as a ubiquitin ligase in protein degradation, it was hypothesized that loss-of-function mutations of SPOP led to accumulation of SPOP substrates that enhance androgen receptor activity and facilitate prostate cancer formation. SPOP substrates could thus be potential targets for treatment of androgen-sensitive prostate cancer. PubMed and BLAST search identified that Gli, SRC-3, and AWP1 are SPOP substrates, and that inhibition of PRK-1, a binding partner of AWP1, by lestaurtinib suppressed androgen receptor activity. LNCaP, PC3, DU145 and 22RV1 prostate cancer cells were used to evaluate the effect of lestaurtinib. LNCaP cells, an androgen-sensitive prostate cancer cell line, were the most sensitive. SRC-3 protein decreased when LNCaP cells were treated with lestaurtinib; whereas PRK-1 increased in nucleus after lestaurtinib treatment. These data suggest that lestaurtinib modulates SRC-3 and PRK-1 to induced cell death in androgen-sensitive prostate cancer, and could be a useful agent for future development for prostate cancer with SPOP mutations.

A recently discovered receptor-rich lipid raft, which is linked to numerous transmembrane signal transduction pathways, has emerged as a medically significant biomaterial for screening antitumor agents. Compounds that interact with the biomaterial might potentially inhibit cancer cell growth, and thus be valuable as antitumor agents. Two standard anticancer drugs, lestaurtinib and gefitinib, interacted with the tropomyosin-related tyrosine kinase A (TrkA) receptor-rich lipid raft. The fact that they are both well known inhibitors of TrkA receptor strengthened the observed linkage. There is now a considerable interest in developing other related biomaterials to serve a similar purpose, and more importantly, support the concept of multitarget drug discovery. It is expected that new anticancer drugs strategically act on multiple pathways to achieve optimal therapeutic efficacies and decreased stimulation of acquired resistance. However, the current conventional approaches such as chemical screening systems and in silico methods cannot fully satisfy the increasing demand for the therapeutic agents. It has therefore become imperative to explore other alternatives to increase the number of clinically important antitumor agents. Here, we report the prospect of establishing lipid raft biomaterial with well endowed multiple cancer-related receptors for screening antitumor leads that affect multiple pathways. This review also examines receptor–ligand interactions in the hunt for novel antitumor agents and the associated key receptors.

We have recently shown that staurosporine mediates the conversion of small cell lung carcinoma (SCLC) cells into a neuron-like process-bearing phenotype. Here, we have extended these studies to the staurosporine analogs K252a, lestaurtinib, PKC412, stauprimide, and UCN-01 and analyzed their influence on process extension, cell cycle distribution, and induction of polyploidy in four SCLC cell lines. In GLC-2 cells, all compounds provoked extensive process formation with the exception of PKC412 that showed no response. In H1184 cells, process formation was predominantly induced by staurosporine and, to lesser extent, in lestaurtinib-, stauprimide-, and UCN-01-treated cells. In the presence of K252a or PKC412, cells became bipolar and spindle shaped or showed pronounced cell flattening. In GLC-36 and SCLC-24H cells, only cell flattening was detectable. Process formation was reversible upon drug removal as shown for GLC-2 and H1184 cells. Fluorescence-activated cell sorting (FACS) and fluorescence in situ hybridization (FISH) analysis indicated the induction of polyploidy in all staurosporine and in two out of four stauprimide-treated SCLC cell lines. For other staurosporine analogs, polyploidy was observed only in UCN-01-treated GLC-36 cells and in K252a-treated H1184 and GLC-36 cells. The presence of staurosporine or its analogs did not alter the constitutive activation pattern of the canonical Akt/PI3K or MEK/extracellular signal-regulated kinase (ERK)1/2 signaling pathways nor could we detect an influence of stauprimide application on the expression level of the c-Myc oncogene. These data demonstrate that in SCLC cells, albeit a higher substrate specificity, staurosporine analogs can induce staurosporine-comparable effects.

Acute myeloid leukemia remains associated with poor outcomes despite advances in our understanding of the complicated molecular events driving leukemogenesis and malignant progression. Those patients harboring mutations in the FLT3 receptor tyrosine kinase have a particularly poor prognosis; however, significant excitement has been generated by the emergence of a variety of targeted inhibitors capable of suppressing FLT3 signaling in vivo. Here we will review results from preclinical studies and early clinical trials evaluating both first- and second-generation FLT3 inhibitors. Early FLT3 inhibitors (including sunitinib, midostaurin, and lestaurtinib) demonstrated significant promise in preclinical models of FLT3 mutant AML. Unfortunately, many of these compounds failed to achieve robust and sustained FLT3 inhibition in early clinical trials, at best resulting in only transient decreases in peripheral blast counts. These results have prompted the development of second-generation FLT3 inhibitors, epitomized by the novel agent quizartinib. These second-generation inhibitors have demonstrated enhanced FLT3 specificity and have been generally well tolerated in early clinical trials. Several FLT3 inhibitors have reached phase III clinical trials, and a variety of phase I/II trials exploring a role for these novel compounds in conjunction with conventional chemotherapy or hematopoietic stem cell transplantation are ongoing. Finally, molecular insights provided by FLT3 inhibitors have shed light upon the variety of mechanisms underlying the acquisition of resistance and have provided a rationale supporting the use of combinatorial regimens with other emerging targeted therapies.

// Bao Nguyen 1,* , Allen B. Williams 1,* , David J. Young 1,2 , Hayley Ma 1 , Li Li 1 , Mark Levis 1 , Patrick Brown 1,2 and Donald Small 1,2 1 Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, USA 2 Department of Pediatrics Johns Hopkins University School of Medicine, Baltimore, MD, USA * These authors have contributed equally to this work Correspondence to: Donald Small, email: // Keywords : acute myeloid leukemia, mutant FLT3, activation loop, tyrosine kinase inhibitors Received : December 16, 2016 Accepted : December 25, 2016 Published : January 06, 2017 Abstract Fms -like tyrosine kinase-3 (FLT3) is a receptor tyrosine kinase that normally functions in hematopoietic cell survival, proliferation and differentiation. Constitutively activating mutations of FLT3 map predominately to the juxtamembrane domain (internal tandem duplications; ITD) or the activation loop (AL) of the kinase domain and are detected in about 1/3 of de novo acute myeloid leukemia (AML) patients. Small molecule tyrosine kinase inhibitors (TKI) effectively target FLT3/ITD mutations, but some activating mutations, particularly those on the AL, are relatively resistant to many FLT3 TKI. We reproduced many of the AL or other non-ITD activating mutations and tested 13 FLT3 TKI for their activity against these and wild-type FLT3. All 13 TKI tested inhibited BaF3/ITD cell proliferation in a concentration-dependent manner as reported, but most TKI exhibited a wide range of differential activity against AL and other point mutants. Western blotting results examining inhibition of FLT3 autophosphorylation and signaling pathways indicate that many AL mutations reduce TKI binding. Most FLT3 TKI effectively target wild-type FLT3 signaling. As a demonstration of this differential activity, treatment of BaF3 D835Y cells transplanted in BALB/c mice with sorafenib showed no effect in vivo against this mutant whereas lestaurtinib proved effective at reducing disease burden. Thus, while FLT3 TKI have been selected based on their ability to inhibit FLT3/ITD, the selection of appropriate TKI for AML patients with FLT3 AL and other activating point mutations requires personalized consideration.

Background/Objectives Infants with ALL have poor outcomes, especially the 70-80% with KMT2A rearrangements (KMT2A-r). Intensification of chemotherapy has reached the limit of tolerability, and stem cell transplantation (SCT) has not improved outcomes. The FLT3 kinase is overexpressed and constitutively activated in KMT2A-r ALL. FLT3 inhibitors (FLT3i) selectively kill KMT2A-r ALL cells in vitro and in vivo, and synergize with chemotherapy. COG AALL0631 was a randomized trial that tested the hypothesis that the addition of the FLT3i lestaurtinib to post-induction chemotherapy would improve outcomes for KMT2A-r ALL.As previously reported, the addition of lestaurtinib did not improve outcomes [3-year disease-free/overall survival (DFS/OS) 39%/46% without lestaurtinib vs. 37%/45% with lestaurtinib, log-rank p=0.78/0.92]. We hypothesized that correlative laboratory measures of FLT3i pharmacodynamics and ex vivo FLT3i sensitivity would enhance our understanding of the clinical responses for KMT2A-r infants treated with lestaurtinib. Design/Methods FLT3i pharmacodynamics (PD) were determined using plasma inhibitory activity (PIA) measured at 5 trough time points during lestaurtinib treatment, using Western blotting and densitometry (normalized to pre-treatment levels). PIA ranged from 0% (no evidence of FLT3 inhibition) to 100% (full inhibition). Mean PIA of 5 troughs was calculated for each patient, and was categorized based on pre-defined protocol criteria as "adequate", or FLT3 inhibited (≥85%) vs "inadequate", or FLT3 uninhibited ( Results Of 210 eligible patients, 146 were KMT2A-r, and 78 were assigned to receive lestaurtinib. Of these, data were available for FLT3i PD (n=73), FLT3i EVS (n=62), MRD (n=55), and DFS/OS (n=78). FLT3i PD and FLT3i EVS significantly correlated with both survival and MRD. For FLT3i PD, 27 were "FLT3 inhibited" and 46 were "FLT3 uninhibited". The DFS/OS for inhibited was 59%/67% vs. 28%/37% for uninhibited (p=0.01/0.02). The week 10 MRD-negative rate was 90% for inhibited vs. 52% for uninhibited (p=0.003). For FLT3i EVS, of the 62 lestaurtinib-treated patients with data, 42 were "sensitive" and 20 were "resistant". The DFS/OS for sensitive was 52%/62% vs. 5%/11% for resistant (p Conclusion While the addition of lestaurtinib to chemotherapy did not improve outcomes, patients achieving potent pharmacodynamic inhibition of FLT3 and those whose leukemia cells were sensitive to ex-vivo FLT3-inhibitor induced cytotoxicity did benefit from the addition of lestaurtinib. Selection of patients with FLT3-sensitive leukemia, real-time intra-patient dose escalation of patients with inadequate pharmacodynamics, and/or using a more potent FLT3 inhibitor may enhance the efficacy of FLT3 inhibition for KMT2A-r infant ALL. Disclosures Raetz: Pfizer: Research Funding. Loh:Medisix Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees. Hunger:Bristol Myers Squibb: Consultancy; Amgen: Consultancy, Equity Ownership; Novartis: Consultancy; Jazz: Honoraria. Borowitz:Beckman Coulter: Honoraria. Small:Pharos I, B & T: Consultancy, Research Funding; InSilico Medicine: Membership on an entity's Board of Directors or advisory committees.

We demonstrate the usefulness of synthetic lethal screening of a conditionally BCL6-deficient Burkitt lymphoma cell line, DG75-AB7, with a library of small molecules to determine survival pathways suppressed by BCL6 and suggest mechanism-based treatments for lymphoma. Lestaurtinib, a JAK2 inhibitor and one of the hits from the screen, repressed survival of BCL6-deficient cells in vitro and reduced growth and proliferation of xenografts in vivo. BCL6 deficiency in DG75-AB7 induced JAK2 mRNA and protein expression and STAT3 phosphorylation. Surface IL10RA was elevated by BCL6 deficiency, and blockade of IL10RA repressed STAT3 phosphorylation. Therefore, we define an IL10RA/JAK2/STAT3 pathway each component of which is repressed by BCL6. We also show for the first time that JAK2 is a direct BCL6 target gene; BCL6 bound to the JAK2 promoter in vitro and was enriched by ChIP-seq. The place of JAK2 inhibitors in the treatment of diffuse large B-cell lymphoma has not been defined; we suggest that JAK2 inhibitors might be most effective in poor prognosis ABC-DLBCL, which shows higher levels of IL10RA, JAK2, and STAT3 but lower levels of BCL6 than GC-DLBCL and might be usefully combined with novel approaches such as inhibition of IL10RA.

FLT3 is frequently mutated in acute myeloid leukemia (AML), but resistance has limited the benefit of tyrosine kinase inhibitors (TKI). We demonstrate that statins can impair FLT3 glycosylation, thus leading to loss of surface expression and induction of cell death, as well as mitigation of TKI resistance. Immunofluorescence microscopy confirms a reduction in surface localization and an increase in intracellular FLT3/internal tandem duplication (ITD) accumulation. This aberrant localization was associated with increased STAT5 activation but inhibition of both MAPK and AKT phosphorylation. Growth inhibition studies indicate that FLT3/ITD-expressing cells were killed with an IC50 within a range of 0.2-2μM fluvastatin. Several mechanisms of resistance could be circumvented by fluvastatin treatment. An increase in the IC50 for inhibition of phosphorylated FLT3/ITD by lestaurtinib caused by exogenous FLT3 ligand, resistance to sorafenib caused by the D835Y or FLT3/ITD N676K mutations, and activation of the IL-3 compensatory pathway were all negated by fluvastatin treatment. Finally, fluvastatin treatment in vivo reduced engraftment of BaF3 FLT3/ITD cells in Balb/c mice. These results demonstrate that statins, a class of drugs already approved by the US Food and Drug Administration, might be repurposed for the management of FLT3 mutant acute myeloid leukemia cases either alone or in conjunction with FLT3 TKI.

FMS-like tyrosine kinase 3 (FLT3) normally functions in the survival/proliferation of hematopoietic stem/progenitor cells, but its constitutive activation by internal tandem duplication (ITD) mutations correlates with a poor prognosis in AML. The development of FLT3 tyrosine kinase inhibitors (TKI) is a promising strategy, but resistance that arises during the course of treatment caused by secondary mutations within the mutated gene itself poses a significant challenge. In an effort to predict FLT3 resistance mutations that might develop in patients, we used saturation mutagenesis of FLT3/ITD followed by selection of transfected cells in FLT3 TKI. We identified F621L, A627P, F691L and Y842C mutations in FLT3/ITD that confer varying levels of resistance to FLT3 TKI. Western blotting confirmed that some FLT3 TKI were ineffective at inhibiting FLT3 autophosphorylation and signaling through MAP kinase, STAT5 and AKT in some mutants. Balb/c mice transplanted with the FLT3/ITD Y842C mutation confirmed resistance to sorafenib in vivo but not to lestaurtinib. These results indicate a growing number of FLT3 mutations that are likely to be encountered in patients. Such knowledge, combined with known remaining sensitivity to other FLT3 TKI, will be important to establish as secondary drug treatments that can be substituted when these mutants are encountered.

Background Releasing drug molecules at the targeted location could increase the clinical outcome of a large number of anti-tumor treatments which require low systemic damage and low side effects. Nano-carriers of drugs show great potential for such task due to their capability of accumulating and releasing their payload specifically, at the tumor site. Results FLT3 inhibitor - gold nanoparticle conjugates were fabricated to serve as vehicles for the delivery of anti-tumor drugs. Lestaurtinib, midostaurin, sorafenib, and quizartinib were selected among the FLT3 inhibitor drugs that are currently used in clinics for the treatment of acute myeloid leukemia. The drugs were loaded onto nanoparticle surface using a conjugation strategy based on hydrophobic-hydrophobic interactions with the Pluronic co-polymer used as nanoparticle surface coating. Optical absorption characterization of the particles in solution showed that FLT3 inhibitor-incorporated gold nanoparticles were uniformly distributed and chemically stable regardless of the drug content. Drug loading study revealed a high drug content in the case of midostaurin drug which also showed increased stability. Drug release test in simulated cancer cell conditions demonstrated more than 56 % release of the entrapped drug, a result that correlates well with the superior cytotoxicity of the nano-conjugates comparatively with the free drug. Conclusions This is a pioneering study regarding the efficient loading of gold nanoparticles with selected FLT3 inhibitors. In vitro cytotoxicity assessment shows that FLT3-incorporated gold nanoparticles are promising candidates as vehicles for anti-tumor drugs and demonstrate superior therapeutic effect comparatively with the bare drugs. Electronic supplementary material The online version of this article (doi:10.1186/s11671-015-1154-2) contains supplementary material, which is available to authorized users.

In this issue of Blood, Knapper et al analyzed 2 consecutive randomized Medical Research Council (MRC) trials in newly diagnosed patients with mutant FMS-like tyrosine kinase-3 (FLT3) acute myeloid leukemia (AML) and suggested no benefit for the addition of the kinase inhibitor lestaurtinib to chemotherapy.1

Acute myeloid leukaemia (AML) is the most common type of adult leukaemia. It is an aggressive disease that involves rapid growth of abnormal leukaemic cells in the bone marrow, resulting in failure of production of normal blood cells (Dormer et al, 1980). Fms-like tyrosine kinase 3 (FLT3), a cell surface receptor belonging to the class III receptor tyrosine kinase family, has a pivotal role in the differentiation and survival of haematopoietic stem cells in the bone marrow (Scheijen and Griffin, 2002; Markovic et al, 2005). Fms-like tyrosine kinase 3 is frequently overexpressed and mutated in AML patients and is a major driver in the pathogenesis of AML cancer cells (Stirewalt and Radich, 2003; Kiyoi et al, 2005). Activating mutations of FLT3 that result in constitutive FLT3 tyrosine kinase activity lead to activation of downstream signal molecules, including signal transducer and activator of transcription 5 (STAT5), Ras, MAPK, PI3K, and Akt, to subsequently stimulate the survival and proliferation of leukaemic cells (Zhang and Broxmeyer, 1999; Hayakawa et al, 2000; Mizuki et al, 2000; Brandts et al, 2005; Sallmyr et al, 2008). Two types of activating FLT3 mutations, namely FLT3-ITD (an internal tandem duplication mutation in the juxtamembrane domain) and FLT3-KDM (a missense mutation at the Asp835 residue within the kinase domain), account for ∼30% of AML patients (Nakao et al, 1996; Kiyoi et al, 1998; Yamamoto et al, 2001; Gilliland and Griffin, 2002a, 2002b). Clinically, these mutations are associated with poor prognosis for AML patients receiving traditional chemotherapy (Thiede et al, 2002; Yanada et al, 2005). Therefore, activated FLT3 (due to FLT3-ITD and/or FLT3-KDM) is a promising molecular target for AML therapies. A number of small molecule FLT3 inhibitors have entered clinical trials. These include sorafenib (BAY 43-9006), sunitinib (SU11248), midostaurin (PKC412), lestaurtinib (CEP-701), tandutinib (MLN518), ABT-869, KW-2449, and AC220 (Wong et al, 2009; Kindler et al, 2010; Wiernik, 2010). However, approval of these agents for FLT3-associated diseases is still challenging, which was suspected to be due to the failure to fully inhibit FLT3 in tumours and undesirable drug properties (Ustun et al, 2009). The purpose of this study was to characterise the pharmacological profile of a novel FLT3 kinase inhibitor, BPR1J-097 (N1-(3-3-[(phenylsulphonyl)amino]phenyl-1H-5-pyrazolyl)-4-(4-methylpiperazino) benzamide) (Figure 1), that was discovered by a rational design strategy. BPR1J-097 with a novel sulphonamide pharmacophore exhibits potent FLT3-inhibitory activity and has potent growth-inhibitory effects on FLT3-ITD leukaemic cells. According to our SAR studies, we found that sulphonamide pharmacophore preferred at meta-position of the phenyl ring, and that replacement the sulphonamide group of BPR1J-097 with the urea group resulted in a significant loss in cellular potency (data not shown). Inhibition of FLT3 resulted in blockage of FLT3 and STAT5 phosphorylation and triggered apoptosis in cancer cells relying on FLT3 signalling for survival. In addition, BPR1J-097 showed favourable pharmacokinetic properties and significant dose-dependent tumour reduction in FLT3-ITD murine xenograft models. Figure 1 Chemical structure of BPR1J-097 (N1-(3-3-[(phenylsulphonyl)amino]phenyl-1H-5-pyrazolyl)-4-(4-methylpiperazino) benzamide). These results demonstrate the potential of BPR1J-097 as a therapeutic candidate for treatment of AML patients. Further preclinical and clinical studies in AML patients are warranted.

The transforming JAK2V617F kinase is frequently associated with myeloproliferative neoplasms and thought to be instrumental for the overproduction of myeloid lineage cells. Several small molecule drugs targeting JAK2 are currently in clinical development for treatment in these diseases. We performed a high-throughput in vitro screen to identify point mutations in JAK2V617F that would be predicted to have potential clinical relevance and associated with drug resistance to the JAK2 inhibitor ruxolitinib (INCB018424). Seven libraries of mutagenized JAK2V617F cDNA were screened to specifically identify mutations in the predicted drug-binding region that would confer resistance to ruxolitinib, using a BaF3 cell-based assay. We identified five different non-synonymous point mutations that conferred drug resistance. Cells containing mutations had a 9- to 33-fold higher EC(50) for ruxolitinib compared with native JAK2V617F. Our results further indicated that these mutations also conferred cross-resistance to all JAK2 kinase inhibitors tested, including AZD1480, TG101348, lestaurtinib (CEP-701) and CYT-387. Surprisingly, introduction of the 'gatekeeper' mutation (M929I) in JAK2V617F affected only ruxolitinib sensitivity (fourfold increase in EC(50)). These results suggest that JAK2 inhibitors currently in clinical trials may be prone to resistance as a result of point mutations and caution should be exercised when administering these drugs.

Lestaurtinib (CEP-701), a multi-kinase inhibitor with potent activity against the Trk family of receptor tyrosine kinases, has undergone early phase clinical evaluation in children with relapsed neuroblastoma. We studied the interaction of CEP-701 with isotretinoin (13cRA) and fenretinide (4HPR), two retinoids that have been studied in children with high-risk neuroblastoma.In vitro growth inhibition was assessed following a 72-hour drug exposure using the sulforhodamine B (SRB) assay in eight neuroblastoma cell lines with variable TrkB expression. When appropriate, the combination index (CI) of Chou-Talalay was used to characterize the interaction of 13cRA (non-constant ratio) or 4HPR (constant ratio) with CEP-701.The median (range) IC(50) of single-agent CEP-701 across all cell lines was 0.09 (0.08-0.3) μM. The combination of 13cRA and CEP-701 resulted in additive to synergistic interactions in four of the five cell lines studied. Addition of 1 or 5 μM of 13cRA decreased the median (range) CEP-701 IC(50) 1.5-fold (1.1-2.8-fold) and 1.7-fold (1.5-1.8-fold), respectively. With 10 μM 13cRA, less than 50% of cells survived when combined with various concentrations of CEP-701. The combination of 4HPR and CEP-701 trended toward being antagonistic, with a median (range) CI at the ED(50) of 1.3 (1.1-1.5).The combination of 13cRA and CEP-701 was additive or synergistic in a spectrum of neuroblastoma cell lines, suggesting that these agents can be potentially studied together in the setting of minimal residual disease following intensive chemoradiotherapy for children with high-risk neuroblastoma.