IGF-1R Tyrosine Kinase Inhibitors Research Articles

IGF1R-IRS1/2 Pharmacological Inhibitors Act By Distinct Cellular and Molecular Mechanisms and Reveals Vulnerabilities for Treatment of Acute Myeloid Leukemia

Background: Preclinical rationale for targeting the insulin-like growth factor 1 (IGF1R)-Insulin Receptor Substrates 1 and 2 (IRS1/2) signaling in acute myeloid leukemia (AML), particularly in cells harboring the FLT3-ITD mutation, has been recently provided [Blood (2018) 132 Supp: 1512 and [Blood (2019) 134 Supp: 2689]. However, little is known about the non-canonical molecular mechanisms regulated by IGF1R-IRS1/2 signaling and pharmacological inhibition of this pathway in AML. Aims: To depict distinctive non-explicit molecular effects of linsitinib (IGF1R tyrosine kinase inhibitor) and NT157 (IGF1R-IRS1/2 allosteric inhibitor) treatment in FLT3-ITD-mutated AML cells. Material and methods: The MOLM-13 (homozygous) and MV4-11 (heterozygous) FLT3-ITD-mutated AML cell lines were treated with linsitinib (10 µM) or NT157 (1 µM) for 24 hours and used for label-free proteomic quantification analysis (n=3). Raw MS/MS data were processed using the SORCERER system and proteins were identified with built-in Andromeda search engine based on the human Uniprot proteome database. False discovery rate cutoffs were set to 1% on peptide, protein, and site decoy level, only allowing high quality identification to pass. Expression values were normalized across experimental conditions by quantile normalization based on the Limma-Voom pipeline, and then systematically compared similarities and differences in protein expression across experimental conditions by applying the Benjamin-Hochberg correction for multiple comparisons. To depict pathways associated to IGF1R, IRS1 and IRS2 gene expression related to processes identified by the proteomic data, we performed a gene-set enrichment analysis (GSEA) using the curated genesets for oncogenic events and molecular functions (MSigDB, Broad Institute) from RNA-seq data of the Cancer Genome Atlas AML cohort (n=173). Results: Considering a ≥ 2-fold change difference in both directions, linsitinib treatment downregulated 6 and 18 and upregulated 13 and 116 proteins in MOLM-13 and MV4-11 cells, respectively. Likewise, NT157 downregulated 12 and 126 and upregulated 204 and 297 proteins. When compared directly, linsitinib reduced expression of 11 and 35 and increased expression of 110 and 70 proteins in MOLM-13 and MV4-11 cells, respectively. Gene ontology identified that linsitinib resulted in upregulation of 7 molecular functions, while the NT157 ensued the upregulation of 18 and downregulation of 17 molecular functions pathways in a consistently manner between all comparisons. Of note, linsitinib activates post-transcriptional regulatory mechanisms, RNA metabolism (RNA binding P=1.15E-12; RNA processing P=8.64E-7) and reduced the protein and macromolecule metabolism (cellular protein metabolism P=3.86E-6). NT157 affected several of mitochondrial functions (increasing proton transmembrane transport activity P=1.55E-12, reducing oxidoreductase activity P=9.11E-10, and oxidative phosphorylation P=5.19E-8). Altogether, these data highlighted that NT157 profounder cytotoxic effect is a result of reprogramming of cellular energetics metabolism, and that linsitinib altered transcription and translation processes, probably as a result of autophagy, a mechanism originally described by our group [Blood (2017) 130 Supp: 3966]. GSEA analysis revealed that high IGF1R expression is positively enriched with RPS14 signature (Normalized Enriched Score [NES]=2.23; FDR-q<0.001), a ribosomal protein related to pathophysiology of myeloid neoplasms related to chromosome 5q deletion. Both IRS1 and IRS2 transcriptional signatures were associated with cellular growth signaling, such as AKT (NES=1.86; FDR-q= 0.006) and MYC (NES=1.67; FDR-q= 0.005), mitochondrial function [mitochondrial gene expression (NES=1.71; FDR-q= 0.001)]. Conclusion: Our proteomic data shed light on new and non-explicit mechanisms related to IGF1R-IRS1/2 inhibitors. Linsitinib modulates molecular processes related to RNA transcription and translation, while NT157 profoundly affect the cellular energetics, and, at least in part, explain the differential pre-clinical efficiency. Moreover, allosteric pharmacological inhibition of IGF1R-IRS1/2 pathway seems a more promising strategy than the tyrosine kinase inhibition, especially for AML subgroup more dependent of mitochondrial metabolism, such as AML with FLT3 mutation. DisclosuresNo relevant conflicts of interest to declare.

IGF1R as a Potential Pharmacological Target in Allergic Asthma.

Background: Asthma is a chronic lung disease characterized by reversible airflow obstruction, airway hyperresponsiveness (AHR), mucus overproduction and inflammation. Although Insulin-like growth factor 1 receptor (IGF1R) was found to be involved in asthma, its pharmacological inhibition has not previously been investigated in this pathology. We aimed to determine if therapeutic targeting of IGF1R ameliorates allergic airway inflammation in a murine model of asthma. Methods: C57BL/6J mice were challenged by house dust mite (HDM) extract or PBS for four weeks and therapeutically treated with the IGF1R tyrosine kinase inhibitor (TKI) NVP-ADW742 (NVP) once allergic phenotype was established. Results: Lungs of HDM-challenged mice exhibited a significant increase in phospho-IGF1R levels, incremented AHR, airway remodeling, eosinophilia and allergic inflammation, as well as altered pulmonary surfactant expression, all of being these parameters counteracted by NVP treatment. HDM-challenged lungs also displayed augmented expression of the IGF1R signaling mediator p-ERK1/2, which was greatly reduced upon treatment with NVP. Conclusions: Our results demonstrate that IGF1R could be considered a potential pharmacological target in murine HDM-induced asthma and a candidate biomarker in allergic asthma.

Differential Effects of IGF-1R Small Molecule Tyrosine Kinase Inhibitors BMS-754807 and OSI-906 on Human Cancer Cell Lines.

Simple SummaryWe have tested the effects of IGF-1R tyrosine kinase inhibitors BMS-754807 (BMS) and OSI-906 (OSI) on human colon, pancreatic carcinoma cell, and glioblastoma cell lines and primary cultures. Although OSI and BMS are able to inhibit IGF-1R activity at low doses, the differential effect on cell proliferation and cell-cycle phase distribution shown by both compounds probes that many effects observed are mediated by BMS off-target interactions. Using MAPKs ELISAs and phospho-RTK array analysis, we have identified several BMS regulated putative kinases able to mediate BMS off-target effects. Interestingly, molecular docking assays suggest that BMS could affect these kinases not only by blocking their ATP-binding domain, but also by means of allosteric interactions. Since BMS has an important antineoplastic effect on these poor prognosis types of cancer, these compounds could be taken in consideration for treatment independently of IGF-1R status.We have determined the effects of the IGF-1R tyrosine kinase inhibitors BMS-754807 (BMS) and OSI-906 (OSI) on cell proliferation and cell-cycle phase distribution in human colon, pancreatic carcinoma, and glioblastoma cell lines and primary cultures. IGF-1R signaling was blocked by BMS and OSI at equivalent doses, although both inhibitors exhibited differential antiproliferative effects. In all pancreatic carcinoma cell lines tested, BMS exerted a strong antiproliferative effect, whereas OSI had a minimal effect. Similar results were obtained on glioblastoma primary cultures, where HGUE-GB-15, -16 and -17 displayed resistance to OSI effects, whereas they were inhibited in their proliferation by BMS. Differential effects of BMS and OSI were also observed in colon carcinoma cell lines. Both inhibitors also showed different effects on cell cycle phase distribution, BMS induced G2/M arrest followed by cell death, while OSI induced G1 arrest with no cell death. Both inhibitors also showed different effects on other protein kinases activities. Taken together, our results are indicative that BMS mainly acts through off-target effects exerted on other protein kinases. Given that BMS exhibits a potent antiproliferative effect, we believe that this compound could be useful for the treatment of different types of tumors independently of their IGF-1R activation status.

Reduced brain fractalkine-CX3CR1 signaling is involved in the impaired cognition of streptozotocin-treated mice

Patients with diabetes mellitus are predisposed to cognitive impairment. Fractalkine-CX3CR1 in the brain signaling represents a primary neuron-microglia inter-regulatory system for several brain functions including learning and memory processes. The present study addressed whether fractalkine-CX3CR1 signaling in the hippocampus contributes to the cognitive deficits observed in streptozotocin (STZ)-treated mice. Our results showed that STZ-treated mice exhibited significant cognitive deficits in the Y-maze test, and a decrease in fractalkine and CX3CR1 levels in the hippocampus. Moreover, intracerebroventricular injection of the CX3CR1 antagonist 18a in normal mice induced significant cognitive deficits in the Y-maze test. STZ-treated mice showed a significant increase in plasma corticosterone levels and a decrease in plasma and hippocampal levels of insulin-like growth factor-1 (IGF-1). Therefore, we examined the effects of corticosterone and IGF-1 on regulation of fractalkine and CX3CR1 expression. Dexamethasone (DEX) application significantly decreased the mRNA expression of fractalkine in primary neuron and astrocyte cultures, and of CX3CR1 in primary microglia cultures. On the other hand, IGF-1 application significantly increased the mRNA expression of fractalkine in primary neuron cultures and CX3CR1 in primary microglia cultures. In addition, administration of DEX and the IGF-1 receptor tyrosine kinase inhibitor picropodophyllin significantly reduced the mRNA expression of fractalkine and CX3CR1 in the hippocampus. These findings indicate that impaired cognition in STZ-treated mice is associated with reduced fractalkine-CX3CR1 signaling in the hippocampus which may be induced by an increase in corticosterone and a decrease in IGF-1.

Discovery of 3-(thiophen/thiazole-2-ylthio)pyridine derivatives as multitarget anticancer agents

A series of novel 3-(thiophen/thiazole-2-ylthio)pyridine derivatives were designed and synthesized as IGF-1R tyrosine kinase inhibitors. All the target compounds were tested for their IGF-1R kinase inhibitory activities and cytotoxicities against five cancer cell lines (K562, Hep-G2, HCT-116, WSU-DLCL2, and A549). Although all these compounds exhibited moderate to potent cancer cell proliferation inhibitory activities (the most potent compound 43 showed IC50 value of 1.3 ± 0.9 μM against WSU-DLCL2 cell line), IGF-1R inhibition were not observed. In order to identify the exact target of these analogues, selected compounds were further screened for various kinases. The results indicated that this series of compounds may exert their anticancer activities through inhibiting various kinases including FGFR 3, EGFR, JAK, and RON. In addition, cell cycle analysis of compound 43 on Hep-G2 cells showed cell cycle arrest at G1/G0 phase. All the experiments validated the potential of 3-(thiophen/thiazole-2-ylthio)pyridine analogues as multi-target anticancer agents.

Abstract 2718: Insulin-like growth factor-1 signaling promotes mitochondrial turnover and protection in cancer cells

Abstract Mitochondrial activity and cellular metabolic reprogramming may influence cancer phenotype and contribute to therapy resistance. Our previous work showed that Insulin-like Growth Factor 1 (IGF-1) induces expression of a mitochondrial carrier protein for nucleotides (PNC1/SLC25A33). PNC1 has an essential function in maintenance of mitochondrial RNA, DNA and in cell growth 1, 2. This suggested an underlying role for IGF-1 signalling in mitochondrial homeostasis. To explore this further we investigated (1) the signalling pathways by which IGF-1 promotes mitochondrial maintenance, and (2) investigated whether PNC1 expression levels influence the phenotype of cancer cells. We found that activation of the IGF-1 pathway promotes mitochondrial biogenesis through induction of the transcriptional co-activators PGC-1β and PRC in a range of cell lines3. MCF-7 breast cancer cells with acquired resistance to an IGF-1R tyrosine kinase inhibitor and consequently reduced IGF-1R activity (MCF-7R cells) exhibit lower expression of PGC-1β and PRC and impaired mitochondrial biogenesis compared to controls3. Interestingly, MCF-7R cells also exhibit mitochondrial dysfunction and a strong dependence on glycolysis for survival. IGF-1 also induces expression of the Nrf2/NFE2L2 gene, which is implicated in mitochondrial biogenesis, anti-oxidant responses, and regulating expression of the mitophagy mediator BNIP3. Interestingly, suppression of Nrf2 impaired the induction of BNIP3 in response to IGF-1. This suggests that Nrf2 integrates IGF-1-stimulated mitochondrial biogenesis with induction of mitophagy to regulate mitochondrial turnover and promote the survival of cancer cells. To investigate the role of IGF-1-induced PNC1 in cancer cell metabolism we manipulated its expression in different cell lines. PNC1 suppression was associated with loss of cell growth, acquisition of an invasive phenotype, and impaired mitochondrial function indicated by reduced respiration. Ectopic expression of PNC1 was sufficient to restore mitochondrial function, and to suppress clonogenic growth and invasive characteristics. Overall we conclude that IGF-1 signalling protects mitochondrial function in cancer cells by stimulating biogenesis and turnover. This mitochondria protective signal is likely to strongly influence responses to therapy and the phenotypic evolution of cancer.

Abstract 1794: The FES-related tyrosine kinase associates with and activates the insulin-like growth factor 1 receptor at sites of cell adhesion

Abstract Insulin-like Growth Factor-1 signaling has a well-described function in facilitating tumourigenesis and promoting tumour growth. Attempts to suppress IGF signals at the level of the IGF-1 Receptor have been disappointing, with kinase inhibitors and blocking antibodies generally showing poor efficacy in clinical trials. To address the mechanisms for this lack of efficacy we sought to identify proteins that modulate the cytotoxic response to IGF-1R tyrosine kinase inhibition using a functional siRNA screen. We identified the non-receptor tyrosine FES-related (FER) kinase as a mediator of sensitivity to the IGF-1R tyrosine kinase inhibitor in MCF-7 cells. We found that FER and the IGF-1R co-locate and can be co-immunoprecipitated from different cell types. Ectopic expression of FER strongly enhanced IGF-1R expression and phosphorylation on the atuophosphorylation sites at tyrosines 950 and 1131. FER phosphorylated these sites in an IGF-1R kinase-independent manner and also enhanced IGF-1-mediated phosphorylation of SHC, and activation of either the AKT or MAPK signaling pathways in breast cancer cell lines. The IGF-1R, β1 Integrin, FER and its substrate cortactin were all observed to be co-located in cell adhesion complexes, the disruption of which reduced IGF-1R expression and activity. High FER expression correlates with phosphorylation of SHC in breast cancer cell lines and with a poor prognosis in patient cohorts. FER and SHC phosphorylation and IGF-1R expression could be suppressed with a known ALK inhibitor that shows high specificity for FER kinase. Overall, we conclude that FER-enhances IGF-1R expression, phosphorylation and signaling to promote cooperative growth and adhesion signaling that may facilitate cancer progression and resistance to IGF-1R inhibition. Citation Format: Joanna Stanicka, Leonie Rieger, Orla T. Cox, Sandra O'Shea, Michael Coleman, Ciara O'Flanagan, Barbara Addario, Nuala McCabe, Richard Kennedy, Rosemary O'Connor. The FES-related tyrosine kinase associates with and activates the insulin-like growth factor 1 receptor at sites of cell adhesion [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 1794.

β-catenin/TCF activity regulates IGF-1R tyrosine kinase inhibitor sensitivity in colon cancer.

The availability of large-scale drug screening data on cell line panels provides a unique opportunity to identify predictive biomarkers for targeted drug efficacy. Analysis of diverse drug data on ~990 cancer cell lines revealed enhanced sensitivity of insulin-like growth factor 1 receptor/ Insulin Receptor (IGF-1R/IR) tyrosine kinase inhibitors (TKIs) in colon cancer cells. Interestingly, β-catenin/TCF(T cell factor)-responsive promoter activity exhibited a significant positive association with IGF-1R/IR TKI response, while the mutational status of direct upstream genes, such as CTNNB1 and APC, was not significantly associated with the response. The β-catenin/TCF activity high cell lines express components of IGF-1R/IR signaling more than the low cell lines explaining their enhanced sensitivity against IGF-1R/IR TKI. Reinforcing β-catenin/TCF responsive promoter activity by introducing CTNNB1 gain-of-function mutations into IGF-1R/IR TKI-resistant cells increased the expression and activity of IGF-1R/IR signaling components and also sensitized the cells to IGF-1R/IR TKIs in vitro and in vivo. Analysis of TCGA data revealed that the stronger β-catenin/TCF responsive promoter activity was associated with higher IGF-1R and IGF2 transcription in human colon cancer specimens as well. Collectively, compared to the mutational status of upstream genes, β-catenin/TCF responsive promoter activity has potential to be a stronger predictive positive biomarker for IGF-1R/IR TKI responses in colon cancer cells. The present study highlights the potential of transcriptional activity as therapeutic biomarkers for targeted therapies, overcoming the limited ability of upstream genetic mutations to predict responses.

Inhibiting Insulin-Like Growth Factor-1 Receptor Attenuates Cell Proliferation and Vascular Endothelial Growth Factor Production in Insulin-Like Growth Factor-1 Receptor Over-Expressing Epidermal Growth Factor Receptor Mutant Non-Small Cell Lung Cancer Cells

SESSION TITLE: Lung Cancer SESSION TYPE: Original Investigation Slide PRESENTED ON: Wednesday, November 1, 2017 at 07:30 AM - 08:30 AM PURPOSE: The aim of the present study was to demonstrate the role of insulin-like growth factor-1 receptor (IGF-1R) tyrosine kinase inhibitors (TKIs) in IGF-1R expressed epidermal growth factor receptor (EGFR) mutant cells. METHODS: Human lung adenocarcinoma PC9, HCC827 and H1975 cells were exposed to a combination of IGF-1, gefitinib, or linsitinib. Cell viability was assessed by the MTT assay. The expression of EGFR, IGF-1R, AKT, extracellular regulated kinases 1 and 2 (ERK1/2), cleaved poly ADP ribose polymerase (PARP), cleaved caspase 3, and hypoxia-inducible factor (HIF)-1α were measured by Western blot. The concentrations of vascular endothelial growth factor (VEGF) were measured using an enzyme-linked immunosorbent assay kit. RESULTS: Cell growth in PC9 and HCC827 cells was synergistically suppressed by co-treatment with gefitinib and linsitinib. Gefitinib did not affect H1975 cell growth; however, linsitinib suppressed cell proliferation. Co-treatment with gefitinib and linsitinib inhibited pAKT and pERK, and linsitinib treatment profoundly reduced IGF-1-induced pIGF-1R expression in PC9 and HCC827 cells. Dual treatment increased the number of Annexin-V positive HCC827 and H1975 cells, and expression of cleaved caspase 3, and cleaved PARP increased in H1975 cells following linsitinib treatment. Gefitinib inhibited HIF-1α and VEGF expression in HCC827 cells, and linsitinib inhibited VEGF production in H1975 cells. CONCLUSIONS: IGF-1R TKIs had modest anti-tumor efficacy and their effects were explained by blocking the EGFR and IGF-1R pathway in IGF-1R expressing EGFR-sensitive cells. IGF-1R TKI had pro-apoptotic activity and inhibited cellular growth in EGFR-resistant cells. CLINICAL IMPLICATIONS: Addition of IGF-1R TKI to standard treatment could be a better therapeutic option to overcome EGFR resistance, which remains to be seen in clinical trials. DISCLOSURE: The following authors have nothing to disclose: Yeo Chang Dong No Product/Research Disclosure Information

IGF-1R tyrosine kinase inhibitors and Vitamin K1 enhance the antitumor effects of Regorafenib in HCC cell lines.

The recent RESORCE trial showed that treatment with Regorafenib after Sorafenib failure provided a significant improvement in overall survival in HCC patients. Preclinical and clinical trial data showed that Regorafenib is a more potent drug than Sorafenib. In this study we aimed at improving Regorafenib actions and at reducing its toxicity, by targeting parallel pathways or by combination with Vitamins K (VKs). We investigated the effects of Regorafenib administrated at low concentrations and in combination with either VK1 and/or with GSK1838705A or OSI-906, two IGF1-R inhibitors, on HCC cell growth and motility. Our results showed that both IGF1-R inhibitors potentiated the antiproliferative and pro-apoptotic effects of Regorafenib and/or VK1 in HCC cell lines. Moreover we provide evidence that the combined treatment with IG1-R antagonists and Regorafenib (and/or VK1) also caused a significant reduction and depolymerization of actin resulting in synergistic inhibition exerted on cell migration. Thus, simultaneous blocking of MAPK and PI3K/Akt cascades with IGF1-R inhibitors plus Regorafenib could represent a more potent approach for HCC treatment.

The role of insulin growth factor-1 on the vascular regenerative effect of MAA coated disks and macrophage-endothelial cell crosstalk.

The IGF-1 signaling pathway and IGF-1-dependent macrophage/endothelial cell crosstalk was found to be critical features of the vascular regenerative effect displayed by implanted methacrylic acid -co-isodecyl acrylate (MAA-co-IDA; 40% MAA) coated disks in CD1 mice. Inhibition of IGF-1 signaling using AG1024 an IGF1-R tyrosine kinase inhibitor abrogated vessel formation 14 days after disk implantation in a subcutaneous pocket. Explanted tissue had increased arginase 1 expression and reduced iNOS expression consistent with the greater shift from "M1" ("pro-inflammatory") macrophages to "M2" ("pro-angiogenic") macrophages for MAA coated disks relative to control MM (methyl methacrylate-co-IDA) disks; the latter did not generate a vascular response and the polarization shift was muted with AG1024. Invitro, medium conditioned by macrophages (both human dTHP1 cells and mouse bone marrow derived macrophages) had elevated IGF-1 mRNA and protein levels, while the cells had reduced IGF1-R but elevated IGFBP-3 mRNA levels. These cells also had reduced iNOS and elevated Arg1 expression, consistent with the invivo polarization results, including the inhibitory effects of AG1024. On the other hand, HUVEC exposed to dTHP1 conditioned medium migrated and proliferated faster suggesting that the primary target of the macrophage released IGF-1 was endothelial cells. Although further investigation is warranted, IGF-1 appears to be a key feature underpinning the observed vascularization. Why MAA based materials have this effect remains to be defined, however.

Inhibiting IGF-1R attenuates cell proliferation and VEGF production in IGF-1R over-expressing EGFR mutant non-small cell lung cancer cells

ABSTRACTPurpose: The aim of the present study was to demonstrate the role of insulin-like growth factor-1 receptor (IGF-1R) tyrosine kinase inhibitors (TKIs) in IGF-1R expressed epidermal growth factor receptor (EGFR) mutant cells. Materials and Methods: Human lung adenocarcinoma PC9, HCC827, and H1975 cells were exposed to a combination of IGF-1, gefitinib, or linsitinib. Cell viability was assessed by the MTT assay. The expression of EGFR, IGF-1R, AKT, extracellular regulated kinases 1 and 2 (ERK1/2), cleaved poly ADP ribose polymerase (PARP), cleaved caspase 3, and hypoxia-inducible factor (HIF)-1α were measured by Western blot. The concentrations of vascular endothelial growth factor (VEGF) were measured using an enzyme-linked immunosorbent assay kit. Results: Cell growth in PC9 and HCC827 cells was synergistically suppressed by co-treatment with gefitinib and linsitinib. Gefitinib did not affect H1975 cell growth; however, linsitinib suppressed cell proliferation. Co-treatment with gefitinib and linsitinib inhibited pAKT and pERK, and linsitinib treatment profoundly reduced IGF-1-induced pIGF-1R expression in PC9 and HCC827 cells. Dual treatment increased the number of Annexin-V-positive HCC827 and H1975 cells, and expression of cleaved caspase 3 and cleaved PARP increased in H1975 cells following linsitinib treatment. Gefitinib inhibited HIF-1α and VEGF expression in HCC827 cells, and linsitinib inhibited VEGF production in H1975 cells. Conclusion: IGF-1R TKIs had modest anti-tumor efficacy and their effects were explained by blocking the EGFR and IGF-1R pathway in IGF-1R expressing EGFR-sensitive cells. IGF-1R TKI had pro-apoptotic activity and inhibited cellular growth in EGFR-resistant cells.

Thyrotropin and Insulin-Like Growth Factor 1 Receptor Crosstalk Upregulates Sodium-Iodide Symporter Expression in Primary Cultures of Human Thyrocytes.

Major regulation of thyroid gland function is mediated by thyrotropin (TSH) activating the TSH receptor (TSHR) and inducing upregulation of genes involved in thyroid hormone synthesis. Evidence suggests that the insulin-like growth factor 1 (IGF-1) receptor (IGF-1R) may play a role in regulating TSHR functional effects. This study examined the potential role of TSHR/IGF-1R crosstalk in primary cultures of human thyrocytes. TSH/IGF-1 co-treatment elicited additive effects on thyroglobulin (TG), thyroperoxidase (TPO), and deiodinase type 2 (DIO2) mRNA levels but synergistic effects on sodium-iodide symporter (NIS) mRNA. Similar cooperativity was seen on the level of TG protein secretion (additive) and NIS protein expression (synergistic). The IGF-1R tyrosine kinase inhibitor linsitinib inhibited TSH-stimulated upregulation of NIS but not TG, indicating that NIS regulation is in part IGF-1R dependent and occurs via receptor crosstalk. Cooperativity was not seen at the level of cAMP/protein kinase A (PKA) signaling, IGF-1R phosphorylation, or Akt activation. However, TSH and IGF-1 synergistically activated ERK1/2. Pharmacological inhibition of ERK1/2 by the MEK1/2 inhibitor U0126 and of Akt by MK-2206 virtually abolished NIS stimulation by TSH and the synergistic effect of IGF-1. As linsitinib inhibited upregulation of NIS stimulated by TSH alone, it is concluded that crosstalk between TSHR and IGF-1R, without agonist activation of IGF-1R, plays a role in NIS regulation in human thyrocytes via a mechanism involving ERK1/2 and/or Akt. Fully understanding the nature of this crosstalk has clinical implications for the treatment of thyroid diseases, including thyroid cancer.

Phase 2 Study of Erlotinib in Combination With Linsitinib (OSI-906) or Placebo in Chemotherapy-Naive Patients With Non–Small-Cell Lung Cancer and Activating Epidermal Growth Factor Receptor Mutations

IntroductionFirst-line epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor treatment of advanced non–small-cell lung cancer with EGFR-activating mutations improves outcomes compared with chemotherapy, but resistance develops in most patients. Compensatory signaling through type 1 insulin-like growth factor 1 receptor (IGF-1R) may contribute to resistance; dual blockade of IGF-1R and EGFR may improve outcomes. Patients and MethodsWe performed a randomized, double-blind, placebo-controlled phase II study of linsitinib, a dual IGF-1R and insulin receptor tyrosine kinase inhibitor, plus erlotinib versus placebo plus erlotinib in chemotherapy-naive patients with EGFR-mutation positive, advanced non–small-cell lung cancer. Patients received linsitinib 150 mg twice daily or placebo plus erlotinib 150 mg once daily on continuous 21-day cycles. The primary end point was progression-free survival. ResultsAfter randomization of 88 patients (44 each arm), the trial was unblinded early owing to inferiority in the linsitinib arm. The median progression-free survival for the linsitinib versus the placebo group was 8.4 months versus 12.4 months (hazard ratio, 1.37; P = .29). Overall response rate (47.7% vs. 75.0%; P = .02) and disease control rate (77.3% vs. 95.5%; P = .03) were also inferior. Whereas most adverse events were ≤ grade 2, linsitinib plus erlotinib was associated with increased adverse events that led to decreased erlotinib exposure (median days, 228 vs. 305). No drug-drug interaction was suggested by pharmacokinetic and pharmacodynamic results. ConclusionAdding linsitinib to erlotinib resulted in inferior outcomes compared with erlotinib alone. Further understanding of the signaling pathways and a biomarker that can predict efficacy is needed prior to further clinical development of IGF-1R inhibitors in lung cancer.

IGF-1R inhibition sensitizes breast cancer cells to ATM-related kinase (ATR) inhibitor and cisplatin.

The complexity of the IGF-1 signalling axis is clearly a roadblock in targeting this receptor in cancer therapy. Here, we sought to identify mediators of resistance, and potential co-targets for IGF-1R inhibition. By using an siRNA functional screen with the IGF-1R tyrosine kinase inhibitor (TKI) BMS-754807 in MCF-7 cells we identified several genes encoding components of the DNA damage response (DDR) pathways as mediators of resistance to IGF-1R kinase inhibition. These included ATM and Ataxia Telangiectasia and RAD3-related kinase (ATR). We also observed a clear induction of DDR in cells that were exposed to IGF-1R TKIs (BMS-754807 and OSI-906) as indicated by accumulation of γ-H2AX, and phosphorylated Chk1. Combination of the IGF-1R/IR TKIs with an ATR kinase inhibitor VE-821 resulted in additive to synergistic cytotoxicity compared to either drug alone. In MCF-7 cells with stably acquired resistance to the IGF-1R TKI (MCF-7-R), DNA damage was also observed, and again, dual inhibition of the ATR kinase and IGF-1R/IR kinase resulted in synergistic cytotoxicity. Interestingly, dual inhibition of ATR and IGF-1R was more effective in MCF-7-R cells than parental cells. IGF-1R TKIs also potentiated the effects of cisplatin in a panel of breast cancer cell lines. Overall, our findings identify induction of DDR by IGF-1R kinase inhibition as a rationale for co-targeting the IGF-1R with ATR kinase inhibitors or cisplatin, particularly in cells with acquired resistance to TKIs.

Role of G protein-coupled receptors (GPCR), matrix metalloproteinases 2 and 9 (MMP2 and MMP9), heparin-binding epidermal growth factor-like growth factor (hbEGF), epidermal growth factor receptor (EGFR), erbB2, and insulin-like growth factor 1 receptor (IGF-1R) in trenbolone acetate-stimulated bovine satellite cell proliferation.

Implanting cattle with steroids significantly enhances feed efficiency, rate of gain, and muscle growth. However, the mechanisms responsible for these improvements in muscle growth have not been fully elucidated. Trenbolone acetate (TBA), a testosterone analog, has been shown to increase proliferation rate in bovine satellite cell (BSC) cultures. The classical genomic actions of testosterone have been well characterized; however, our results indicate that TBA may also initiate a quicker, nongenomic response that involves activation of G protein-coupled receptors (GPCR) resulting in activation of matrix metalloproteinases 2 and 9 (MMP2 and MMP9) that release membrane-bound heparin-binding epidermal growth factor-like growth factor (hbEGF), which then binds to and activates the epidermal growth factor receptor (EGFR) and/or erbB2. Furthermore, the EGFR has been shown to regulate expression of the IGF-1 receptor (IGF-1R), which is well known for its role in modulating muscle growth. To determine whether this nongenomic pathway is potentially involved in TBA-stimulated BSC proliferation, we analyzed the effects of treating BSC with guanosine 5'-O-2-thiodiphosphate (GDPβS), an inhibitor of all GPCR; a MMP2 and MMP9 inhibitor (MMPI); CRM19, a specific inhibitor of hbEGF; AG1478, a specific EGFR tyrosine kinase inhibitor; AG879, a specific erbB2 kinase inhibitor; and AG1024, an IGF-1R tyrosine kinase inhibitor on TBA-stimulated proliferation rate (H-thymidine incorporation). Assays were replicated at least 9 times for each inhibitor experiment using BSC cultures obtained from at least 3 different animals. Bovine satellite cell cultures were obtained from yearling steers that had no previous exposure to androgenic or estrogenic compounds. As expected, BSC cultures treated with 10 n TBA showed ( < 0.05) increased proliferation rate when compared with control cultures. Additionally, treatment with 5 ng hbEGF/mL stimulated proliferation in BSC cultures ( < 0.05). Treatment with GDPβS, MMPI, CRM197, AG1024, AG1478, and/or AG879 all suppressed ( < 0.05) TBA-induced increases in proliferation. These data indicate that TBA likely initiates a nongenomic response involving GPCR, MMP2 and MMP9, hbEGF, EGFR, erbB2, and IGF-1R, which may play a role in TBA-mediated increases in BSC proliferation.

Targeting the insulin-like growth factor receptor and Src signaling network for the treatment of non-small cell lung cancer.

BackgroundTherapeutic interventions in the insulin-like growth factor receptor (IGF-1R) pathway were expected to provide clinical benefits; however, IGF-1R tyrosine kinase inhibitors (TKIs) have shown limited antitumor efficacy, and the mechanisms conveying resistance to these agents remain elusive.MethodsThe expression and activation of the IGF-1R and Src were assessed via the analysis of a publicly available dataset, as well as immunohistochemistry, Western blotting, RT-PCR, and in vitro kinase assays. The efficacy of IGF-1R TKIs alone or in combination with Src inhibitors was analyzed using MTT assays, colony formation assays, flow cytometric analysis, and xenograft tumor models.ResultsThe co-activation of IGF-1R and Src was observed in multiple human NSCLC cell lines as well as in a tissue microarray (n = 353). The IGF-1R and Src proteins mutually phosphorylate on their autophosphorylation sites. In high-pSrc-expressing NSCLC cells, linsitinib treatment initially inactivated the IGF-1R pathway but led a Src-dependent reactivation of downstream effectors. In low-pSrc-expressing NSCLC cells, linsitinib treatment decreased the turnover of the IGF-1R and Src proteins, ultimately amplifying the reciprocal co-activation of IGF-1R and Src. Co-targeting IGF-1R and Src significantly suppressed the proliferation and tumor growth of both high-pSrc-expressing and low-pSrc-expressing NSCLC cells in vitro and in vivo and the growth of patient-derived tissues in vivo.ConclusionsReciprocal activation between Src and IGF-1R occurs in NSCLC. Src causes IGF-1R TKI resistance by acting as a key downstream modulator of the cross-talk between multiple membrane receptors. Targeting Src is a clinically applicable strategy to overcome resistance to IGF-1R TKIs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0392-3) contains supplementary material, which is available to authorized users.

A tale of two receptors: insulin and insulin-like growth factor signaling in cancer.

Inhibition of the type I IGF receptor (IGF1R) has been the focus of numerous clinical trials. Two reports in this issue describe the results of phase I trials of an IGF1R tyrosine kinase inhibitor OSI-906. This commentary will describe the complex endocrine changes induced by these types of agents.

Abstract 1741: Inhibition of type 1 insulin-like growth factor receptor (IGF-1R) influences processing of replication-associated DNA double-strand breaks (DSBs) and induces schedule-dependent sensitization of human melanoma to temozolomide (TMZ)

Abstract IGF-1R is frequently up-regulated in cancers including melanoma, and is implicated in mediating resistance to chemotherapy and targeted agents via regulation of apoptosis. Here, we investigated responses to temozolomide (TMZ), a methylating agent that generates toxic O6-methylguanine (O6meG) adducts, which are removed by O6-methylguanine-DNA methyltransferase (MGMT). Persistent O6meG adducts trigger futile cycles of mismatch repair, ultimately leading to formation of replication-associated DSBs. In a first approach to test links between IGF-1R and response to TMZ, we used a panel of 10 human melanoma cell lines in which we characterized MGMT expression, activation and expression of IGF axis components, and growth inhibitory effects of TMZ. We found that TMZ GI50 values correlated with MGMT levels (r=0.79, p=0.009), and in 7 MGMT-proficient cell lines, with phospho-IGF-1R (r=0.81, p=0.038) suggesting a link between TMZ resistance and IGF-1R activation. In a second approach we used IGF-1R tyrosine kinase inhibitors (TKIs) AZ12253801 and linsitinib (OSI-906), which is currently undergoing clinical evaluation, to assess effects of blocking IGF signaling. These IGF-1R TKIs were capable of sensitizing wild-type and mutant BRAF melanoma cells to TMZ; lack of correlation with apoptosis induction suggested that other factors contributed to this effect. IGF-1R inhibition did not influence MGMT expression or activity, was not epistatic with MGMT inhibition, and TMZ-sensitized MGMT-null cells, suggesting that chemo-sensitization was MGMT-independent. We found that IGF-1R TKI sensitized melanoma cells to inhibition of poly(ADP ribose) polymerase (PARP), and influenced processing of TMZ-induced DSBs, with increased RPA focus formation within 24hr of TMZ application, and persistence of RAD51 foci at 72hr. These data suggest functional impairment of homologous recombination (HR)-mediated DSB repair. Compared with simultaneous or prior IGF-1R TKI, TMZ sensitization was greater when IGF-1R TKI followed TMZ, consistent with a model in which IGF-1R inhibitor pre-treatment reduces DSB yield. In mice bearing melanoma xenografts with a clinically relevant (V600E BRAF, wild-type p53) genotype, this sequential (TMZ→linsitinib) combination treatment was tolerable, and linsitinib achieved peak plasma levels comparable to clinical Cmax values. TMZ and linsitinib caused minor inhibition of tumor growth (gradient reduction of 13% and 25% respectively), while the combination caused supra-additive growth delay (72%) that was significantly different from control (p&amp;lt;0.01), TMZ (p&amp;lt;0.01) and linsitinib (p&amp;lt;0.05) groups. In summary, IGF-1R inhibition influences HR repair of TMZ-induced DSBs and sensitizes melanomas to TMZ using treatment schedules relevant to clinical practice. Citation Format: Roger N. Ramcharan, Tamara Aleksic, Shan Gao, Jordan Tanner, Nicholas Darvill, Esther Bridges, Ruth Asher, Amanda J. Watson, Geoffrey P. Margison, Emmanouela Repapi, Ji-Liang Li, Mark R. Middleton, Valentine M. Macaulay. Inhibition of type 1 insulin-like growth factor receptor (IGF-1R) influences processing of replication-associated DNA double-strand breaks (DSBs) and induces schedule-dependent sensitization of human melanoma to temozolomide (TMZ). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1741. doi:10.1158/1538-7445.AM2014-1741

Abstract 1716: Targeting the insulin-like growth factor receptor/Insulin receptor and Src signaling network for the treatment of non-small cell lung cancer

Abstract Insulin-like growth factor I receptor (IGF-1R)-mediated signaling plays an important role in the proliferation, survival, and metastasis of cancer cells. The IGF-1R-targeting anticancer agents including monoclonal antibodies and small molecule tyrosine kinase inhibitors (TKIs) have been developed, but their antitumor effects have been marginal and limited in clinical trials. Therefore, the mechanism underlying resistance to the IGF-1R-targeting therapies and the rational combination strategies to overcome potential drug resistance need to be investigated. In this study, we demonstrated the association of Src with the resistance to the IGF-1R TKI in non-small cell lung cancer (NSCLC). We found the co-activation of IGF-1R/IR and Src in various human NSCLC cell lines, The mRNA expression and phosphorylation of IGF-1R and Src were also significantly correlated with each other in NSCLC databases from a public dataset and a tissue microarray (n=353). Next, we found Src can be activated through multiple pathways including EGFR and integrin β3 signaling and function as an alternative kinase for phosphorylation of IGF-1R, especially at Tyr1135/36, but not Tyr1131. Src is activated in lung cancer cells possessing both primary and acquired resistance to an IGF-1R TKI. Consistent with the results, inhibition of Src significantly attenuated activation of IGF-1R/IR and co-targeting of IGF-1R and Src effectively suppressed cell proliferation, colony formation, and tumor growth in vitro and in vivo. Taken together, these results suggest that Src causes resistance to IGF-1R TKIs by functioning as a key downstream modulator of multiple signaling pathways for IGF-1R phosphorylation and thus co-targeting IGF-1R and Src could be an effective therapeutic strategy for NSCLC. Citation Format: Hye-Young Min, Hye Jeong Yun, Hyo-Jong Lee, Jaebeom Cho, Hyun-Ji Jang, Kyung Min Kim, Woo-Young Kim, Seung-Hyun Oh, Diane Liu, J. Jack Lee, Waun Ki Hong, Ignacio I. Wistuba, Ho-Young Lee. Targeting the insulin-like growth factor receptor/Insulin receptor and Src signaling network for the treatment of non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1716. doi:10.1158/1538-7445.AM2014-1716