Inhibition Of Tyrosine Kinase Activity Research Articles

Combinatorial Approaches to Overcome Plasma Protein Inhibition of FLT3 Tyrosine Kinase Inhibitors

Background: The FMS-like tyrosine kinase 3 (FLT3) is the most frequently mutated gene in acute myeloid leukemia (AML) and also results in poor prognosis for adult and pediatric patients, and thus represents an attractive target for tyrosine kinase inhibitors (TKI). The activity of FLT3-targeted TKI is inhibited to varying extents by plasma protein binding. Staurosporine (STS)-derived TKI are inhibited almost exclusively by the plasma protein alpha-1 acid glycoprotein (AGP), an acute-phase reactant. We studied the impact of AGP binding on the other STS-derivatives and report the development of a novel method to overcome this binding.Methods: We assayed the impact of human AGP upon the activity of the STS-derived TKI (midostaurin, lestaurtinib, TTT-3002) against proliferation of the FLT3-ITD dependent cell line MOLM-14 and upon the parent compound (staurosporine) against the non-FLT3-dependent cell line HL-60. These experiments were repeated, co-incubating with drugs that competitively bind AGP to identify those that may restore TKI activity.Results: The TKI are inhibited in a linear AGP-dependent manner (fold change increase of IC50 per mg/dL AGP: midostaurin 3.00-fold, lestaurtinib 11.73-fold, TTT-3002 0.33-fold) across the range of AGP concentrations observed in human plasma. These results correspond to the drug-protein binding constants for the TKI: midostaurin 12.6 µM-1, lestaurtinib 49.2 µM-1, TTT-3002 1.41 µM-1, all validated by competitive fluorescence displacement of the AGP-binding dye, 1-anilino-8-naphthalenesulfonate. These results predict that in vivo IC50 values for these FLT3 TKI will be significantly higher than those measured under typical (10% FCS) in vitro culture conditions: midostaurin 4.7 µM, lestaurtinib 4.8 µM, TTT-3002 34 nM. By comparison, activity of the parent compound, staurosporine, against HL60 is completely inhibited by AGP. Assays using bovine plasma, serum or purified AGP do not demonstrate similar inhibition of FLT3 TKI. We are developing a murine model to overcome this experimental limitation.We have developed a mathematical model describing the interactions of AGP with FLT3 TKI using classical mass action relationships that match experimental results and furthermore describe the effects of competitive plasma protein binding by unrelated agents. These models predict that disinhibition of TKI may be achievable in vivo, and define the properties of such “rescue” agents.Mifepristone binds AGP (2-10 fold greater than STS-derived TKI) and has no independent effect upon FLT3-dependent cell growth. Co-treatment with mifepristone restores the IC50 of TTT-3002 from 12 nM with AGP to < 0.1 nM. Disinhibition is seen for lestaurtinib (IC50 shift reduced from >1000-fold to 50-fold) and midostaurin (300-fold reduced to 80-fold). This results in predicted in vivo IC50 that are clinically relevant, and serve as a proof-of-principle for this method. Using this principle we have screened a library of FDA-approved compounds for the ability to rescue TKI activity despite the presence of potentially inhibitory plasma proteins. This screen has identified 40 potential agents that may displace STS-derived TKI from AGP, and an additional 90 agents that may restore TKI activity through off-target effects. Several agents have already been validated in vitro, and found to decrease the IC50 of midostaurin and other TKI to clinically achievable ranges despite the presence of inhibiting proteins.Conclusions: The failure of FLT3 TKI in previous clinical trials has been linked to a lack of plasma drug activity. This work provides biochemical confirmation of this effect. Furthermore, these results indicate that this is a property of the class as a whole, including midostaurin. Indeed, for midostaurin, the predicted in vivo IC50 is higher than steady state levels suggesting that in clinical trials it likely acts through non-FLT3 mechanisms. Disinhibition of TKIs by mifepristone suggests a novel combinatorial approach restore TKI activity using unrelated compounds. We are currently examining other agents for similar synergy. By improving TKI potency in the face of inhibitory plasma protein binding, such combinations would be expected to improve their clinical efficacy by reducing the dosages necessary to thoroughly inhibit FLT3. Finally, this report provides a method for predicting at least one factor that affects the success or failure of FLT3 TKI in clinical trials. DisclosuresNo relevant conflicts of interest to declare.

Neonatal genistein exposure disrupts ovarian and uterine development in the mouse by inhibiting cellular proliferation.

Soy-based formula contains high concentrations of the isoflavone genistein. Genistein possesses estrogenic and tyrosine kinase inhibitory activity and interferes with cellular proliferation and development. To date, the acute and chronic effects of genistein on ovarian and uterine development have not been fully elucidated. In this study, mice at postnatal day 1 were subcutaneously injected with 100 mg/kg genistein for 10 consecutive days, and then their ovaries and uteri were collected on days 10, 21, and 90. Histological evaluation was performed after hematoxylin and eosin staining. The proliferating activity was indicated by the proliferating indicator protein Ki67. Results showed that the subcutaneous injection of genistein to neonatal mice induced the formation of multi-oocyte follicles and delayed the primordial follicle assembly in the ovaries. Genistein significantly enlarged the cross-sectional area of the uterine cavity and wall and disrupted the regularity between the uterine stroma and myometrium. Genistein exposure inhibited proliferative activity because fewer Ki67-positive nuclei were detected in ovarian and uterine cell populations than in the control. Furthermore, most ovaries from adult mice given neonatal genistein were without corpora lutea, and there appeared to be cystic follicles and hypertrophy of the theca, and cortical and medullary layers. Considering the high concentration of isoflavone in soy-based infant formulas and livestock feed, we suggest that the use of isoflavone-rich diets in humans and livestock receive closer examination.

Effects and mechanisms of betulinic acid on improving EGFR TKI-resistance of lung cancer cells.

Epidermal growth factor receptor (EGFR) mutations have been identified in approximately 55% of lung cancer patients in Taiwan. Gefitinib (Iressa) and Erlotinib (Tarceva) are the first-generation targeting drugs to patients with EGFR gene mutants a work by inhibiting tyrosine kinase activity. However, resistance in EGFR-mutated patients to first-generation tyrosine kinase inhibitor (TKI) therapy after 8-11 months of treatment has occurred. Betulinic acid (BetA) is a pentacyclic triterpenoid natural product derived from widespread plants. BetA has been reported to have a cytotoxic effect in several cancers. The purpose of this study is to investigate the effects and mechanisms of BetA on dampening EGFR TKI-resistance of lung cancer cells. Our study has demonstrated by MTT assay that combining BetA and an EGFR TKI increased the cytotoxicity against EGFR TKI-resistance lung cancer cells. Based on flow cytometry, combination treatments of BetA with an EGFR TKI enhanced Sub-G1 accumulation, induced apoptosis and induced mitochondrial membrane potential loss. Using western blotting, BetA and EGFR TKI combined treatments inhibited cell cycle related protein and triggered apoptosis- and autophagy- related protein expression. Taken together, our data suggests that a target therapy combining BetA with an EGFR TKI improves drug efficacy in EGFR TKI-resistant lung cancer cells.

Antitumor activity of BJ-1207, a 6-amino-2,4,5-trimethylpyridin-3-ol derivative, in human lung cancer

Enhanced expression of NADPH oxidase (NOX) and the subsequent production of reactive oxygen species (ROS) are associated with lung cancer. In the present study, fifty 6-amino-2,4,5-trimethylpyridin-3-ol derivatives were screened for anticancer activity by targeting NOX2-derived ROS. The compounds suppressed ROS production and decreased cancer cell viability (R2 = 0.79). Among the derivatives, the compound coded BJ-1207, which contained a 4-(hydroxydiphenylmethyl)piperidine moiety, exhibited the most effective anticancer activity against A549 lung cancer cell line and eight other cancer cell lines, including H1299, MCF-7, MDA-MB-231, HT-29, SW620, Mia PaCa-2, PANC-1, and U937. BJ-1207 also showed significantly lower inhibitory effects on kinase insert domain receptor (KDR) and c-KIT tyrosine kinase but higher inhibitory activity on NOX than those of sunitinib, a multi-receptor tyrosine kinase (RTK) inhibitor. In addition, BJ-1207-induced inhibition of RTK-downstream signaling pathways, such as ROS production, and expression of target genes, such as stem cell factor and transforming growth factor-α, were similar to those induced by sunitinib. In the xenograft chick tumor model, BJ-1207 inhibited lung tumor growth to a similar or much greater extent than that of sunitinib or cisplatin, respectively. Overall, the present study showed that BJ-1207, a vitamin B6-derived 2,4,5-trimethylpyridin-3-ol compound with azacyclonol moiety at C (6)-position of the pyridine ring, inhibited NOX activity and that it is a promising lead compound for developing anticancer drugs against lung cancer.

Abstract 1675: Design and synthesis of novel tyrosine kinase enzyme inhibitors and evaluating their anti-colorectal cancer activity

Abstract Targeting tyrosine kinases is attracting considerable attention for cancer therapeutics, nonetheless colorectal cancer. Herein we rationally designed and synthesized a group of 4-(3-methoxy-5-methylphenyl)-5-(pyridine-4-yl)pyrimidine-2-amine derivatives as potential inhibitors for tyrosine kinase. Synthesized compounds share a general structure and vary in substituents at position-2 of the terminal pyridine ring. The hydroxy-analogue of each methoxy derivative was also prepared to evaluate the effect of the methoxy group, at the phenyl ring in position-4 of the pyrimidine moiety, on activity. Compounds 1 (2-chloropyridyl and methoxyphenyl), 2 (2-chloropyridyl and hydroxyphenyl), 3 (2-(pyridine-3-yl)pyridyl and methoxyphenyl), 4 (2-(pyridine-3-yl)pyridyl and hydroxyphenyl), 5 (2-phenylpyridyl and hydroxyphenyl), 6 (2-(4-cyanophenyl)pyridyl and hydroxyphenyl) and 7 (2-(4-acetylphenyl)pyridyl and hydroxyphenyl) showed 29% to 79% tyrosine kinase inhibitory activity. Yet, cytotoxicity of all compounds were assessed against colorectal cancer cells (HT29 and HCT116) using SRB assay. Any compound (at 10 μM concentration) induced considerable viability drop was further assessed using detailed dose response curve and its cytotoxic parameters were calculated. Compounds 8 (2-(4-(N,N-dimethylamino)phenyl)pyridyl and hydroxyphenyl), 5, 6 and 7 showed considerable cytotoxic activities against HCT116 cells with IC50's of 9.18, 10.37, 9.47 and 16.60 μM, respectively. In addition, compounds 9 (2-chloropyridyl, methoxyphenyl and free 2-amino), 2, 3, 4, 5 and 6 showed considerable cytotoxicity against HT29 cells with IC50's of 9.31, 7.48, 11.26, 6.37, 9.89 and 7.14 μM, respectively. The mechanisms of cell killing effect of compounds with considerable anti-proliferative/cytotoxic activity were further assessed using cell cycle analysis (DNA content flowcytometry) and Annexin-V/FITC apoptosis assessment. Compound 2 induced antiproliferative effect which might be attributed to induction of cell cycle arrest at the G0/G1 phase; cell population in G0/G1 phase after treatment with compound 2 was 59.7±0.7% compared to 53.4±1.6% for control untreated cells. Compounds 4, 5, 6 and 7 induced significant cell cycle arrest at G2/M phase with cell populations equals 25.1±0.6%, 24.8±0.4%, 24.0±0.7% and 25.9±0.1%, respectively, compared to 19.3±1.6% for control untreated cells. Cell cycle arrest induced by compounds 1, 2, 3, 4, 6 and 7 resulted in significant apoptosis in colorectal cancer cells. In conclusion, this group of tyrosine kinase inhibitors showed promising anti-proliferative/cytotoxic effects in colorectal cancer cell lines attributed to cell cycle arrest. Further studies for the different cell cycle check point proteins and kinases are undergoing to clarify the exact underlying mechanism of cell killing attributed to each one of these compounds. Citation Format: Hanan A. Assiri, Hawazen A. BinMahfouz, Fahad A. Alabbasi, Marc Devocelle, Ibrahim M. El-deeb, Ahmed M. Al-Abd. Design and synthesis of novel tyrosine kinase enzyme inhibitors and evaluating their anti-colorectal cancer activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1675.

Combinations of Bevacizumab and Erlotinib Show Activity in Colorectal Cancer Independent of RAS Status.

Purpose: There is extensive cross-talk between VEGF- and EGFR-pathway signaling in colorectal cancer. However, combinations of VEGF- and EGFR-targeted monoclonal antibodies (mAb) show disappointing activity, in particular for patients with mutant RAS Previous results show that tyrosine kinase inhibitors (TKI) can be active in colorectal cancer models resistant to mAbs. This prompted us to examine whether the activity of bevacizumab can be increased by combination with erlotinib.Experimental Design: The antitumor activity of bevacizumab, erlotinib, and their combination was determined in colorectal cancer models with different RAS status and bevacizumab sensitivity. EGFR/VEGF pathway activation was characterized by immunohistochemistry, Western blot, and ELISA assays. The influence of cetuximab and erlotinib on EGF-mediated migration and the EGFR-EGF ligand feedback loop was established in colorectal cancer cell lines with different RAS status.Results: The addition of erlotinib increased bevacizumab activity in all models independent of RAS status. Bevacizumab exposure was accompanied by marked EGFR activation in tumor cells as well as in tumor-associated endothelial cells (TECs) and resulted in strong accumulation of intracellular EGFR, which could be attenuated by erlotinib. In cellular models, erlotinib was able to attenuate EGF-mediated functions in all cell lines independent of RAS status while cetuximab only showed activity in RAS wild-type cells.Conclusions: These results should provide a molecular framework to better understand the increased activity of the bevacizumab-erlotinib combination, compared with bevacizumab alone, in the GERCOR DREAM phase III clinical trial. Differential activity of mAbs and TKIs targeting the same signaling pathway is likely applicable for other tumor types. Clin Cancer Res; 24(11); 2548-58. ©2018 AACR.

Medical Treatment Options for Patients with Epidermal Growth Factor Receptor Mutation-Positive Non-Small Cell Lung Cancer Suffering from Brain Metastases and/or Leptomeningeal Disease.

Brain metastases and/or leptomeningeal disease (LMD) with associated central nervous system (CNS) metastases are known complications of advanced epidermal growth factor receptor (EGFR) mutation-positive non-small cell lung cancer (NSCLC). It is important, therefore, to assess the activity of EGFR tyrosine kinase inhibitors (TKIs) versus such CNS complications. This review explores the literature reporting the intracranial activity of EGFR TKIs, and finds that there is evidence for varying efficacy of the approved agents, erlotinib, gefitinib, afatinib, and osimertinib in patients with CNS metastases. Other EGFR TKIs in development, such as AZD3759, may have a future role as therapeutic options in this setting. Emerging evidence indicates that the second- and third-generation EGFR TKIs, afatinib and osimertinib, effectively penetrate the blood-brain barrier, and therefore represent viable treatment options for CNS lesions, and can reduce the risk of CNS progression. These agents should therefore be considered as first-line treatment options in patients with EGFR mutation-positive NSCLC who have brain metastases and/or LMD. While there are currently no prospective data comparing the intracranial efficacy of second- and third-generation EGFR TKIs in this setting, CNS activity and protection offered by different EGFR TKIs should be an additional consideration when making decisions about the optimal sequence of treatment with EGFR TKIs in order to maximize survival benefit in individual patients.

Real-world experience with cabozantinib in metastatic clear cell renal cell carcinoma (mccRCC).

684 Background: Cabozantinib (C) inhibits tyrosine kinase activity at MET, AXL, and VEGF receptors and is approved in mccRCC after progression on prior VEGFR-TKI. Little off-protocol data exists on tolerability and response. Methods: We retrospectively gathered baseline and treatment details on mccRCC patients treated with C at MD Anderson from 2015-present. Blinded radiologist determined best response by RECIST 1.1. Descriptive statistics were utilized. Results: We report on 38 patients with mccRCC treated with C in a “real-world” setting. Median age at mccRCC diagnosis was 58 years (range 37-71). 25 patients (66%) were male. 22 (58%) had de-novo metastatic disease. 4 patients had sarcomatoid dedifferentiation. 32 (84%) underwent nephrectomy. Cohort had a median of 3 sites of metastatic disease, including: bone 22 (57%), liver 9 (23%), and brain 6 (16%). 6 patients (16%) received C as 2nd line therapy, 10 (26%) as 3rd line, and 22 (58%) as 4th line or later. By IMDC risk score, 7 (18%) were favorable, 22 (58%) were intermediate, and 9 (24%) were poor risk. All patients had received at least 1 prior VEGFR-TKI and 24 (63%) had received prior immune checkpoint inhibitor. At C initiation, 20 (53%) patients had unfavorable neutrophil to lymphocyte ratio (NLR) of ≥ 3. 12 (32%) patients had favorable shift of NLR to &lt; 3 on C. 22 patients (58%) required dose reduction and 24 (63%) required treatment break due to C-related toxicity. At time of analysis, 26 patients had discontinued treatment, with median time on treatment of 32.2 weeks (range 15.7-130.7 weeks). For the 12 patients remaining on C, median duration of therapy has been 54.3 weeks (range 26.6-68.9 weeks). 1 (3%) patient had CR, 11 patients (29%) had PR, 23 (60%) had SD, and 3 (8%) had PD as best response to C. Conclusions: C has a high disease control rate (92%) even as a late line of therapy in mccRCC. However, careful monitoring is warranted as many patients require treatment breaks and dose reductions for therapy-related toxicity. [Table: see text]

An exosomal- and interfacial-biosensing based strategy for remote monitoring of aberrantly phosphorylated proteins in lung cancer cells.

It is a well-known phenomenon that cancer cells release key biological information such as DNA, RNA or proteins into body fluids (e.g., blood, urine or saliva). The analysis of these molecules-often encapsulated within nanovesicules called exosomes-is highly attractive, because it could replace current surgical biopsies, which are painful, costly and potentially risky for patients. For example, current strategies in lung cancer diagnosis involve genetic analyses from tumour tissues to detect the presence of underlying DNA mutations, known to alter the phosphorylation status and function of proteins. This information is used to direct therapy, as aberrantly phosphorylated proteins are the main targets of current drugs such as Tyrosine Kinase Inhibitors (TKIs). An alternative and less invasive strategy would be the remote analysis of these phospho-proteins by isolating them from cancer-derived exosomes. This would allow evaluating not only their phosphorylation status at diagnosis, but also the timely restoration of protein phosphorylation levels during therapy with TKIs. Yet, this proteomic approach remains vastly unexplored. Herein, we demonstrate that key lung cancer phosphoproteins, such as EGFR and ERK, are expressed in lung cancer exosomes and we outline a new exosomal proteomic-based approach for their fast and convenient detection. This approach, which could complement current genetic analysis for lung cancer detection, easily detects the phosphorylation status of lung cancer exosomal proteins within minutes after their extraction, bringing hope of circumventing the need for tissue biopsy and costly and cumbersome DNA sequencing techniques. It exploits the fact that phosphorylation induces protein conformational changes, which in turn alter protein's ability to effectively interact with bare gold surfaces. This leads to phosphorylated and non-phosphorylated protein isoforms displaying different gold-adsorption profiles. Using single-use and inexpensive, gold (Au) screen-printed electrodes (SPEs), we demonstrate the successful detection of aberrantly phosphorylated EGFR and ERK protein isoforms derived from lung cancer cell exosomes with a sensitivity down to 15 ng μL-1 in samples with up to 90% excess of their non-phosphorylated (wild-type) forms. We further show the applicability of this strategy for monitoring the action of Tyrosine Kinase Inhibitors over time. We believe that this non-invasive technique will open up new avenues for facilitating cancer diagnosis and time-point monitoring of therapeutic responses.

Are Soy Products Effective in DMD?

INTRODUCTION:In addition to their nutritional value, processed soy bean extracts contain several activities with potential therapeutic benefits. These include anti-oxidants, and tyrosine kinase and protease inhibitory activity. There are also anecdotal reports of health benefits of soy products in alleviating DMD symptoms.METHODS:Mdx mice were fed a control soy-free diet or the same diet containing either a proprietary soy preparation (Haelan 951), purified soy isoflavones, purified Bowman-Birk protease inhibitor or a combination of isoflavones and Bowman-Birk inhibitor. Mice were tested for their wire hanging ability at the start of the diet regimen and every 4 weeks until week 12 of treatment.RESULTS AND DISCUSSION: The diet containing Bowman-Birk inhibitor was the only one to show a significant and sustained improvement over the 12 weeks of the study. All other dietary additions; Haelan 951, isoflavones and isoflavones with Bowman-Birk inhibitor, were not significantly different from each other or from control. The effectiveness of Bowman-Birk inhibitor in mdx mice clearly warrants further study.

Design, Synthesis and Preliminary Biological Evaluation of Novel Benzyl Sulfoxide 2-Indolinone Derivatives as Anticancer Agents.

In this work, a series of novel benzyl sulfoxide 2-indolinone derivatives was designed and synthesized as potent anticancer agents. Tyrosine kinase inhibitory activity assay indicated that most of the compounds showed significant activity. The in vitro antiproliferative activity of these compounds was further investigated against five human cancer cell lines (HeLa, HepG2, MCF-7, SCC-15, and A549). Several compounds exhibited evident activities. Among them, (Z)-3-(((4-bromobenzyl)sulfinyl)methylene)indolin-2-one (6j) and (Z)-3-((benzylsulfinyl)methylene)-5-bromoindolin-2-one (6o) were found to be effective tyrosine kinase inhibitors (IC50 = 1.34 and 2.69 μM, respectively) in addition to having noteworthy antitumor potential (the average IC50 value of 6j or 6o was less than 40 μM). This class of novel derivatives has promising potential for further development as anticancer agents.

Predictive value of quantitative HER2, HER3 and p95HER2 levels in HER2-positive advanced breast cancer patients treated with lapatinib following progression on trastuzumab.

Lapatinib is a HER1 and HER2 tyrosine kinase inhibitor (TKI) approved in second line treatment of advanced or metastatic breast cancer following progression on trastuzumab-containing therapy. Biomarkers for activity of lapatinib and other TKIs are lacking.Formalin-fixed, paraffin-embedded primary tumor samples were obtained from 189 HER2-positive patients treated with lapatinib plus capecitabine following progression on trastuzumab. The HERmark® Breast Cancer Assay was used to quantify HER2 protein expression. HER3 and p95HER2 protein expression was quantified using the VeraTag® technology.Overall survival (OS) was inversely correlated with HER2 (HR = 1.9/log; P = 0.009) for patients with tumors above the cut-off positivity level by the HERmark assay. OS was significantly shorter for those with above median HER2 levels (HR = 1.7; P = 0.015) and trended shorter for those below the cut-off level of positivity by the HERmark assay (HR = 1.7; P = 0.057) compared to cases with moderate HER2 overexpression. The relationship between HER2 protein expression and OS was best captured with a U-shaped parabolic function (P = 0.004), with the best prognosis at moderate levels of HER2 protein overexpression. In a multivariate model including HER2, increasing p95HER2 expression was associated with longer OS (HR = 0.35/log; P = 0.027). Continuous HER3 did not significantly correlate with OS.Patients with moderately overexpressed HER2 levels and high p95HER2 expression may have best outcomes while receiving lapatinib following progression on trastuzumab. Further study is warranted to explore the predictive utility of quantitative HER2 and p95HER2 in guiding HER2-directed therapies.

Synthesis of piperazine-based thiazolidinones as VEGFR2 tyrosine kinase inhibitors inducing apoptosis.

VEGFR2 tyrosine kinase is a main target in suppressing cancer growth and metastasis. Materials& methods: Piperazine-based thiazolidinones were synthesized and screened for their anticancer and VEGFR2 tyrosine kinase inhibitory activity.Results: Compounds 11, 13 and 16 displayed potent anticancer activity against HepG-2 with IC50 values 0.03-0.06μM. They were safe on normal human fibroblasts with selectivity indices 8.09, 11.40 and 4.37, respectively. Also, these compounds showed VEGFR2 tyrosine kinase inhibitory activities more than the reference staurosporine with IC50 values <0.3μM. Lineweaver-Burk plot revealed that these compounds behaved as uncompetitive VEGFR2 tyrosine kinase inhibitors. They also induced caspase-dependent apoptosis in HepG-2. In addition, these compounds revealed good binding within VEGFR2 tyrosine kinase enzyme in comparison with sorafenib reference. Compounds 11, 13 and 16 comprise a new promising scaffold of selective VEGFR2 tyrosine kinase inhibitors with caspase-dependent apoptotic activities.

Discovery and Pharmacological Characterization of JNJ-42756493 (Erdafitinib), a Functionally Selective Small-Molecule FGFR Family Inhibitor.

Fibroblast growth factor (FGF) signaling plays critical roles in key biological processes ranging from embryogenesis to wound healing and has strong links to several hallmarks of cancer. Genetic alterations in FGF receptor (FGFR) family members are associated with increased tumor growth, metastasis, angiogenesis, and decreased survival. JNJ-42756493, erdafitinib, is an orally active small molecule with potent tyrosine kinase inhibitory activity against all four FGFR family members and selectivity versus other highly related kinases. JNJ-42756493 shows rapid uptake into the lysosomal compartment of cells in culture, which is associated with prolonged inhibition of FGFR signaling, possibly due to sustained release of the inhibitor. In xenografts from human tumor cell lines or patient-derived tumor tissue with activating FGFR alterations, JNJ-42756493 administration results in potent and dose-dependent antitumor activity accompanied by pharmacodynamic modulation of phospho-FGFR and phospho-ERK in tumors. The results of the current study provide a strong rationale for the clinical investigation of JNJ-42756493 in patients with tumors harboring FGFR pathway alterations. Mol Cancer Ther; 16(6); 1010-20. ©2017 AACR.

Retrospective analysis of clinical outcomes of early stage ALK-positive (ALK+) non-small cell lung cancer (NSCLC).

8536 Background: ALK rearrangements are important oncogenic drivers in NSCLC. However, the prognostic implications of these rearrangements are unclear due to 1) conflicting results from small series of patients (pts) with early-stage ALK+ NSCLC, and 2) use of highly effective ALK tyrosine kinase inhibitors (TKI) in the metastatic setting. To assess the prognostic significance of ALK rearrangements in resected NSCLC, we performed a retrospective analysis of survival outcomes among pts with resected ALK+, EGFR+, or KRAS+ NSCLC treated at two institutions. Methods: We reviewed charts of pts that underwent resection for stage 1-3 NSCLC at Massachusetts General Hospital or Memorial Sloan Kettering Cancer Center between 1/2009 and 12/2012. Recurrence-free survival (RFS) was estimated for each genotype. Results: Among 764 pts (480 KRAS+, 255 EGFR+, 29 ALK+), we identified 555 (73%), 101 (13%), and 108 (14%) pts with stage I, 2, and 3 NSCLC, respectively. ALK+ pts were distributed across all stages: 10 (34%) stage I, 6 (21%) stage 2, and 13 (45%) stage 3 NSCLCs. Chemotherapy was administered to 14 ALK+ ( 0% stage 1, 67% stage 2, 77% stage 3), 45 EGFR+ (3% stage 1, 44% stage 2, 81% stage 3), and 96 KRAS+ pts (4% stage 1, 56% stage 2, 71% stage 3), respectively, for early-stage NSCLC. Thirteen (7%) stage 1 EGFR+ patients received adjuvant EGFR TKI. Although median RFS was not reached for EGFR+ pts, it was 24.3 months (95%CI 11.4 to 65.3) for ALK+ pts and 72.9 months (95%CI 59.7 to undefined) for KRAS+ pts. RFS for ALK+ NSCLC was significantly shorter than the other groups (HR 2.9, 95%CI: 1.75-4.89 vs. EGFR and HR 1.8, 95%CI: 1.12-2.93 vs. KRAS). When adjusted for stage, ALK+ NSCLC remained associated with worse RFS compared to EGFR+ NSCLC (HR 1.8, 95%CI: 1.09-3.12), but not when compared to KRAS+ NSCLC (HR 1.30, 95%CI: 0.79-2.12). Conclusions: Early stage ALK+ NSCLC is associated with shorter RFS than EGFR+ NSCLC. The propensity for relapse and the significant anti-tumor activity of ALK TKIs in pts with metastatic NSCLC suggest that enrollment of patients on trials of adjuvant ALK TKIs should be prioritized.

Strategies targeting angiogenesis in advanced non-small cell lung cancer.

Tumor angiogenesis is a frequent event in the development and progression of non-small cell lung cancer (NSCLC) and has been identified as a promising therapeutic target. The vascular endothelial growth factor (VEGF) family and other angiogenic factors, including fibroblast growth factor and platelet-derived growth factor, promote the growth of newly formed vessels from preexisting vessels and change the tumor microenvironment. To date, two antiangiogenic monoclonal antibodies, bevacizumab and ramucirumab, which target VEGF-A and its receptor VEGF receptor-2, respectively, have been approved for the treatment of locally advanced or metastatic NSCLC when added to first-line standard chemotherapy. Numerous oral multitargeting angiogenic small molecule tyrosine kinase inhibitors (TKIs) have been widely evaluated in advanced NSCLC, but only nintedanib in combination with platinum-based doublet chemotherapy has demonstrated a survival benefit in the second-line setting. Additionally, small-molecule TKIs remain the standard of care for patients with mutated EGFR, ALK or ROS1. Moreover, immune checkpoint inhibitors that target the programmed cell death protein 1 (PD-1) and programmed cell death protein ligand 1 (PD-L1) are changing the current strategy in the treatment of advanced NSCLC without driver gene mutations. The potential synergistic activity of antiangiogenic agents and TKIs or immunotherapy is an interesting topic of research. This review will summarize the novel antiangiogenic agents, antiangiogenic monotherapy, as well as potential combination therapeutic strategies for the clinical management of advanced NSCLC.

Two pairs of rare naturally occurring 4-hydroxy-4-methyl-2,5-heptanedione derivatives from the red alga Chondria crassicaulis

Two pairs of rare naturally occurring racemic lipids, (±)-4,7-dihydroxy-4-methyl-2,5-heptanedione (1), and (±)-7-butoxy-4-hydroxy-4-methyl-2,5-heptanedione (2) were isolated from the red alga Chondria crassicaulis Harv. The structures of the racemic mixtures of 1 and 2 were elucidated by detailed spectroscopic techniques, including 1D and 2D NMR (1H and 13C NMR, 1H–1H COSY, HSQC, and HMBC) as well as mass spectrometry and optical rotation experiments, and by comparison with data for related known analogs. This is the first report of naturally occurring 4-hydroxy-4-methyl-2,5-heptanedione derivatives. Antifungal, PTP1B inhibitory, and receptor tyrosine kinase inhibitory activities of these two compounds were investigated. The results showed that compounds 1 and 2 exhibited good selective inhibition against RET tyrosine kinase activity with IC50 values of 9.56 and 8.93 μM, respectively. Compound 1 also displayed moderate antifungal activity against Cryptococcus neoformans (32609), showing a MIC80 value of 32 μg/ml.

18F-Fluoromisonidazole Kinetic Modeling for Characterization of Tumor Perfusion and Hypoxia in Response to Antiangiogenic Therapy.

Multiparametric imaging of tumor perfusion and hypoxia with dynamic 18F-fluoromisonidazole (18F-FMISO) PET may allow for an improved response assessment to antiangiogenic therapies. Cediranib (AZD2171) is a potent inhibitor of tyrosine kinase activity associated with vascular endothelial growth factor receptors 1, 2, and 3, currently in phase II/III clinical trials. Serial dynamic 18F-FMISO PET was performed to investigate changes in tumor biomarkers of perfusion and hypoxia after cediranib treatment. Methods: Twenty-one rats bearing HT29 colorectal xenograft tumors were randomized into a vehicle-treated control group (0.5% methylcellulose daily for 2 d [5 rats] or 7 d [4 rats]) and a cediranib-treated test group (3 mg/kg daily for 2 or 7 d; 6 rats in both groups). All rats were imaged before and after treatment, using a 90-min dynamic PET acquisition after administration of 42.1 ± 3.9 MBq of 18F-FMISO by tail vein injection. Tumor volumes were delineated manually, and the input function was image-derived (abdominal aorta). Kinetic modeling was performed using an irreversible 1-plasma 2-tissue compartmental model to estimate the kinetic rate constants K1, K1/k2, and k3-surrogates for perfusion, 18F-FMISO distribution volume, and hypoxia-mediated entrapment, respectively. Tumor-to-blood ratios (TBRs) were calculated on the last dynamic frame (80-90 min). Tumors were assessed ex vivo by digital autoradiography and immunofluorescence for microscopic visualization of perfusion (pimonidazole) and hypoxia (Hoechst 33342). Results: Cediranib treatment resulted in significant reduction of mean voxelwise 18F-FMISO TBR, K1, and K1/k2 in both the 2-d and the 7-d groups (P < 0.05). The k3 parameter was increased in both groups but reached significance only in the 2-d group. In the vehicle-treated groups, no significant change in TBR, K1, K1/k2, or k3 was observed (P > 0.2). Ex vivo tumor analysis confirmed the presence of hypoxic tumor regions that nevertheless exhibited relatively lower 18F-FMISO uptake. Conclusion:18F-FMISO kinetic modeling reveals a more detailed response to antiangiogenic treatment than a single static image is able to reveal. The reduced mean K1 reflects a reduction in tumor vascular perfusion, whereas the increased k3 reflects a rise in hypoxia-mediated entrapment of the radiotracer. However, if only late static images are analyzed, the observed reduction in 18F-FMISO uptake after treatment with cediranib may be mistakenly interpreted as a global decrease, rather than an increase, in tumor hypoxia. These findings support the use of 18F-FMISO kinetic modeling to more accurately characterize the response to treatments that have a direct effect on tumor vascularization and perfusion.

Impact of pharmacokinetics (PK) of sunitinib (SU) and its metabolite SU12662 on clinical outcome and toxicity in metastatic renal cell cancer (mRCC) patients.

506 Background: Pharmacological activity of SU is usually attributed to SU and its active metabolite SU12662, known to exhibit similar potency in inhibiting tyrosine kinase activity and cellular proliferation in vitro. However, equivalence is not clearly demonstrated towards clinical endpoints. The objective of this phase 2 study was to determine the impact of SU and SU12662 on clinical outcomes and toxicity in mRCC patients (NCT00943839). Methods: Patients with mRCC, eligible for SU in the first line setting, were prospectively included. SU was given orally at 50 mg qd for 4 weeks on/2 weeks off until progression or unacceptable toxicity. Minimal concentration at steady-state (CSSmin) was evaluated by high-performance liquid chromatography at day 28 of each cycle for both SU and SU12662. Data for tumor response, survival, and toxicity were recorded. Kaplan-Meyer’s curves with log-rank tests and cox regression models were used to identify relationships between PK parameters and survival. Chi2 tests and student t-tests were used to identify relationships with toxicity. Results: 41 patients in 7 French centers were included between 2009 and 2012. 16 patients had favourable prognosis, 13 intermediate prognosis, and 1 poor prognosis according to IMDC risk groups (Heng, J Clin Oncol 2009). Median follow-up was 30.6 months. Median progression-free survival (PFS) was 20.2 months and median overall survival (OS) was 39.5 months. Mean CSSmin for SU and SU12662 were 57.2 and 29.8 ng/ml, respectively. CSSmin of SU12662 was independently correlated to poor PFS and OS (p = 0.01 and 0.003 respectively). Patients with a mean CSSmin of SU12662 higher than 42.5 ng/ml had a worse PFS and OS compared to patients with lower concentrations: 5.7 months and 5.7 months versus 20.6 months and 39.5 months (p = 0.01 and 0.002 respectively). High CSSmin of both SU and SU12662 were associated with increased incidence of asthenia (p&lt;0.001). Conclusions: High exposure to SU12662 was associated with poor outcomes and increased asthenia for patients with mRCC treated with SU in the first line setting. It may be related to a higher metabolism and unequal activity between SU and SU12662 in vivo. Clinical trial information: NCT00943839.

Metabolic adaptations to targeted therapy in FLT3 mutated acute myeloid leukaemia

BackgroundAcute myeloid leukaemia has a dismal outlook. FLT3 tyrosine kinase (TK) activating mutations (FLT3mut) are present in 30% of cases and are predictive of a worse outcome. Although metabolic reprogramming has been recognised as a hallmark of cancer cells, little is known about its role in acute myeloid leukaemia. Published gene expression datasets show that glycolytic, citric acid cycle, and oxidative phosphorylation genes are upregulated in FLT3mut patient samples at diagnosis. We aimed to study metabolic changes in FLT3mut disease upon targeted inhibition of its TK activity in an attempt to unveil novel therapeutic vulnerabilities. MethodsLiquid chromatography coupled to mass spectrometry, using stable isotope-based carbon flux tracing, and an extracellular flux analyser (Seahorse, Agilent Technologies, Santa Clara, CA, USA) were used to assess metabolic changes in FLT3mut human and murine cells after FLT3 TK inhibition by the specific inhibitor AC220. Gene expression changes were measured in the same conditions by RNA sequencing. Fluorescence-activated cell sorting was used to measure changes in viability and reactive oxygen species after inhibition of TK activity in various culture conditions. FindingsWe confirmed in both human and murine cells that FLT3mut cells displayed an increased glycolytic (maximal glycolytic capacity, extracellular acidification rate of 80·64 mpH/min in FLT3mut vs 59·09 in FLT3wildtype, p<0·001) and respiratory capacity (maximal respiratory capacity, oxygen consumption rate of 326·4 pmoles/min in FLT3mut vs 180·9 in FLT3wildtype, p<0·001). Upon treatment with AC220, these metabolic phenotypes were reversed. However, whereas glucose uptake was significantly reduced upon TK inhibition (p<0·001), glutamine uptake was not affected. Metabolic flux analysis with [U−13C]glutamine suggested that glutamine was diverted to glutathione production upon AC220 treatment. FLT3mut cells displayed a large increase in concentration of reactive oxygen species upon AC220 treatment when grown in the absence of glutamine (fold increase of reactive oxygen species normalised to untreated cells 4·59 without glutamine vs 2·74 with glutamine, p<0·001), and these changes correlated with a significant reduction in viability (relative viability normalised to untreated 34·6% without glutamine vs 62·5% with glutamine, p<0·001) in the same conditions. InterpretationOur data suggest that FLT3mut acute myeloid leukaemia cells have increased respiratory and glycolytic capacity that is reversed by TK inhibition. Upon AC220 treatment glutamine metabolism becomes a metabolic dependency of FLT3mut disease, as glutamine is channelled towards glutathione production and reactive oxygen species detoxification, and protects them from cell death. This process could be exploited therapeutically by a combination of glutaminolysis or glutathione biosynthesis inhibitors with FLT3 TK inhibitors. FundingWellcome Trust.