IGF-1R Tyrosine Kinase Inhibitors Research Articles

How to Train Your Biomarker

Type I insulin-like growth factor (IGF) receptor (IGF1R) inhibitors are new cancer therapies. Pitts and colleagues used in vitro data to "train" a predictive biomarker for an IGF1R tyrosine kinase inhibitor. Given the complexity of IGF signaling, additional layers of biomarker analysis will likely be needed to develop predictive factors.

Abstract 4127: EGFR and K-Ras mutations and resistance of lung cancer to the IGF-1R tyrosine kinase inhibitors

Abstract Background: The majority of patients with non-small cell lung cancer (NSCLC) has responded poorly to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). We investigated (1) the involvement of insulin-like growth factor 1 receptor (IGF-1R) signaling in primary resistance to EGFR TKIs and (2) the molecular determinants of resistance to IGF-1R TKIs. Methods: Phosphorylated IGF-1R/insulin receptor (pIGF-1R/IR) was immunohistochemically evaluated in NSCLC tissue microarrays. The antitumor effects of IGF-1R TKIs (PQIP, OSI906), either alone or in combination with small-molecular inhibitors or siRNA targeting K-Ras or MAPK/extracellular signal-regulated kinase kinase (MEK) were analyzed in vitro and in vivo in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-transformed human bronchial epithelial (HBE) cells and in NSCLC cells with variable histologic features and mutations in EGFR or K-Ras. Results: pIGF-1R/IR expression in NSCLC specimens was positively correlated with presence of a history of tobacco smoking, squamous cell carcinoma, mutant (mut) K-Ras, and wild-type (wt) EGFR, all of which have been strongly associated with poor response to EGFR TKIs. IGF-1R TKIs exhibited significant antitumor activity in NNK-transformed HBE cells and in NSCLC cells harboring wt EGFR and wt K-Ras, but not those with mutations in these genes. Introduction of mut K-Ras attenuated the effects of IGF-1R TKIs on wt K-Ras-expressing NSCLC cells. Conversely, inactivation of MEK restored sensitivity to IGFR-TKI in cells carrying mut K-Ras. Conclusions: The mutation status of both EGFR and K-Ras could be a predictive marker for response to IGF-1R TKIs. Also, MEK antagonism can abrogate primary resistance of NSCLC to IGF-1R TKIs. This work was supported by NIH grants R01 CA-109520-01 and CA-100816 (all to H-YL.) and in part by DOD grant W81XWH-04-1-0142 VITAL and W8XWH-06-1-0303 BATTLE (W-K H.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4127.

The potential role of insulin-like growth factor receptor inhibitors in the treatment of advanced non-small cell lung cancer

Importance of the field: Lung cancer is the leading cause of cancer-related mortality worldwide. NSCLC accounts for > 80% of all lung cancers. The treatment of advanced fit NSCLC patients seems to have reached a plateau. Considerable efforts have been initiated to identify new biological agents.Areas covered in this review: Diagnosis of NSCLC histotype is becoming extremely important to address treatment. While non-squamous histology could start to benefit from the administration of several new drugs only recently, non-adenocarcinoma subtype seems to benefit from the administration of figitumumab (CP-751,871) a fully human anti-IGF 1 receptor (IGF-1R) mAb. In this paper, we reviewed the IGF-1R pathway and its inhibitors.What the reader will gain: Approaches targeting IGF-1R include small-molecule IGF-1R tyrosine kinase inhibitors (TKIs), which are in preclinical and early clinical phases of development, and the mAbs, among which figitumumab is being investigated in Phase III trials of advanced NSCLC.Take home message: Figitumumab reported interesting results in the treatment of advanced non-adenocarcinoma NSCLC patients. Overall, in order to administer the optimal treatment to patients, a more definite histological diagnosis is mandatory.

Complete IGF Signaling Blockade by the Dual-Kinase Inhibitor, BMS-754807, Is Sufficient To Overcome Tamoxifen and Letrozole Resistance In Vitro and In Vivo.

Abstract Resistance to hormonal therapy is a clinically unmet need in breast cancer. IGF signaling has been identified as a major mechanism of resistance to hormonal therapy in breast cancer. As components of the IGF signaling pathway are expressed in most breast cancers, the development of IGF-1R monoclonal antibody (mAb) and tyrosine kinase inhibitors (TKI) are active areas of clinical investigations. A key distinction between the mAb and TKIs are their differences in their ability to inhibit the Insulin Receptor (InsR). While targeting the InsR with TKIs may have a theoretical liability of hyperglycemia, targeting only the IGF-1R may have the theoretical liability of incompletely blocking IGF signaling. As InsR isoform A expression, which can transduce IGF-II-mediated proliferation, is higher in breast cancers compared to normal breast tissue, we investigated whether IGF-1R or IGF-1R/InsR inhibition was sufficient for overcoming resistance to hormonal therapy. To determine the optimal combination strategies for clinical investigations, we tested the hypothesis that IGF signaling inhibition could overcome primary (or de novo/intrinsic) and secondary (or acquired/selected) resistance to hormonal therapy. For these studies, we used either hormone therapy-naïve or hormone therapy-resistant variants of the breast cancer model, MCF-7/AC-1, which has been engineered to stably express full-length human aromatase. We employed and compared a novel, potent dual kinase inhibitor of the IGF-1R and InsR, BMS-754807, which is currently in early clinical investigations, with the IGF-1R antibody mAb391. BMS-754807 has been shown to induce apoptosis more potently than mAb391 in Rh41 human rhabdomyosarcoma cells. In vitro, BMS-754807 demonstrated profound synergy in combination with tamoxifen and letrozole (median effect combination index <0.1). In vivo, BMS-754807 enhanced the anti-tumor activity of tamoxifen and letrozole in hormone-naïve tumors and induced regression of tumors resistant to tamoxifen or letrozole when combined with letrozole. This activity was not observed with mAb therapy, which resulted in greater up-regulation of InsR-A and erbB receptor expression and activation. This suggested a greater susceptibility to resistance pathways with mAb therapy. Dual IGF-1R/InsR blockade alone or in combination was tolerated by the animals and has no significant change in glucose homeostasis. Gene expression profiling experiments to compare the difference between the effects of tamoxifen in combination with BMS-754807 and with mAb revealed alternative pathway signaling is one of the potential mechanisms of resistance.In summary, combined hormonal therapy with BMS-754807 overcomes primary and secondary resistance to tamoxifen and letrozole and was well tolerated. IGF-1R blockade with a mAb alone is insufficient to overcome resistance and induces InsR over-expression. Thus, IGF signaling through either InsR or IGF-1R may be a major mechanism of resistance to hormonal therapy. These data suggest that blockade of IGF-1 and IGF-II from activation of IGF-1R and InsR, with agents such as BMS-754807 have promise in extending the benefits of hormonal therapy in breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 402.

Potential Benefit of an Agonistic Anti IGFIR Antibody (scFv-Fc) as an Effective Breast Cancer Therapy.

Abstract Multiple monoclonal antibodies targeting the type-1 insulin-like growth factor receptor (IGFIR) are currently in different phases of clinical trials. All the reported antibodies inhibit biochemical activation of IGFIR and block cell proliferation. In contrast, we have reported an agonistic anti-IGFIR antibody scFv-Fc that stimulates IGFIR and its downstream signaling of PI3K and MAPK pathways to promote cell proliferation in vitro. Like the purely antagonistic antibodies, our previous work has shown that scFv-Fc can down regulate IGFIR levels, partially inhibit xenograft growth of MCF-7 cells in athymic mice, and render MCF-7 cells refractory to the mitogenic effects of IGF-I after the initial wave of agonistic activity. MCF-7 cells maintained long term in scFv-Fc have low levels of IGFIR expression, yet the remaining receptor is activated and inhibited by an IGFIR tyrosine kinase inhibitor (TKI) AEW541. In this study, we have further explored the future potential of using this antibody as breast cancer therapy. The common characteristic effect of anti-IGFIR antibodies is to down regulate IGFIR. Since scFv-Fc can activate IGFIR signaling in a manner different than the natural ligand IGF-I, we examined if this antibody could render cells more sensitive to TKI. In MCF-7 cells, IC50 of the IGF1R tyrosine kinase inhibitor AEW541 was decreased 5 to 10 fold when cultured in scFv-Fc. ER negative MDA-MB-231 cells are insensitive to both inhibitory antibody (AVE1642) and TKI (AEW541) in the presence or absence of IGF-I, which may due to the very low levels of IGF1R that are weakly activated by IGF-I. MDA-MB-231 cells were not affected by AEW541 in monolayer growth assays. After chronic treatment of MDA-MB-231 with scFv-Fc for 2 months, AEW541 significantly inhibited monolayer cell growth by 30% in the presence or absence of IGF-I. We also examined the effect of scFv-Fc's effect on AEW541 inhibition of cell motility in MDA-MB-231 cells. Both acute and chronic treatment of scFv-Fc dramatically inhibited cell migration by 90%.Inhibitory antibodies AVE 1642 and MK 0646 had no effect on cell migration. Both IGF-I and excess amounts of AVE 1642/MK 0646 did not reverse scFv-Fc inhibition on cell migration, suggesting other cell signaling system may be involved. In MDA231-BO, a metastatic variant of MDA-MB-231, scFv-Fc had less inhibitory effect on basal cell migration, but in combination of AEW541 treatment, cell migration was significantly decreased by 70%. In conclusion, our data suggests an antibody with agonistic activity may have different properties than purely inhibitory antibodies. Specifically, initiating biochemical signaling followed by inhibition of tyrosine kinase activity and receptor downregulation may be a potent way to disrupt IGF mediated breast cancer cell biology. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3125.

Abstract C22: Identification of candidate microRNAs that correlate with sensitivity to the IGF-1R/IR inhibitor, OSI-906, in colorectal cancer (CRC) cell lines

Abstract Background: MicroRNAs (miRNAs) are small ∼20–22 nucleotide noncoding RNAs, thatwhich function as antisense regulators of gene expression by targeting specific mRNAs, and thereby modulate a variety of cellular signaling pathways. Deregulation of miRNA expression contributes to specific oncogenic events in human cancers and therefore may contribute to have a role in resistance to targeted cancer therapies. This study was based upon the hypothesis Here, we hypothesized that miRNA expression profiles wouldcan predict sensitivity to the novel IGF-1R tyrosine kinase inhibitor, OSI-906, in colorectal cancer (CRC) cell lines. Materials and Methods: To evaluate the sensitivity of CRC cells to OSI-906, a panel of 27 lines was exposed to increasing concentrations of the drug and assessed for proliferation using the sulforhodamine B assay. Six cell lines were sensitive (S, IC50 < 1.5 mol/L) and 21 were resistant (R, IC50 > 1.5 mol/L) to OSI-906. Baseline total RNA was isolated from 4 S and 4 R cell lines for miRNA and mRNA profiling on Dharmacon miRNA microarray and Affymetrix HG-U133 Plus 2.0 microarray platforms, respectively. For miRNA microarray data pre-processing, 342 miRNA probes with relative intensity of p ≤ 0.01 in at least 2 of the 8 experiments were kept for further data analysis. For mRNA microarray analysis, probe sets representing the same gene were collapsed based on maximum values and the expression levels were converted to a rank-based matrix. Anti-sense and mimic-miRNAs were used to study the effects of loss- and gain-of-function for the identified candidate miRNAs. Results: Significance Analysis of Microarray was used to determine the differentially expressed miRNAs in cell lines S or R to OSI-906 S vs. R cell lines profiles. Thirteen13 miRNAs were identified which showed a positive correlation with OSI-906 sensitivity. Three and 10 miRNAs were under- orand over-expressed in OSI-906 S cell lines, while respectively. gGene expression analysis revealed 78 and 42 genes under- orand over-expressed in OSI-906 S cell lines (p < 0.005), respectively. By comparing the predicted miRNA targets and differentially expressed genes, we identified two candidate miRNAs (hsa-miR-181a and hsa-miR-224) where the expression of their potential target genes, metallothioneins 1E and 2A (MT1E and MT2A) showed an inverse correlation with OSI-906 sensitivity. This observation was confirmed by qRT-PCR on both miRNAs and their target genes. To assess the loss- and gain-of-function effects of the candidate miRNAs, we delivered mimic- and anti-sense miRNAs to the OSI-906 R and S cell lines, respectively. Interestingly, over-expression of the miR-181a and miR-224 mimics in the OSI-906 R cell lines resulted in increased sensitivity to the drug, whereas. However, silencing of these miRNAs in the S cell lines did not demonstrateshow the reverse effect. Conclusion: These results suggest that loss of the candidate miRNA expression can confer OSI-906 resistance. Restoring the expression of these candidate miRNAs may induce sensitivity to OSI-906 in resistant CRC cell lines and may have clinical significant application in the targeted therapy of patients with advanced CRC. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C22.

Abstract CN07-02: Disrupting insulin-like growth factor signaling with monoclonal antibodies

Abstract The insulin-like growth factor (IGF) system is composed of a network of ligands, receptors, and binding proteins. The type I IGF receptor (IGF1R) is a transmembrane tyrosine kinase receptor and shares a high degree of homology with the insulin receptor (InsR). Data from many sources implicate the IGF system in cancer biology. Because of the role for InsR in glucose homeostasis, specific targeting of IGF1R with monoclonal antibodies has been deemed desirable. Numerous monoclonal antibodies have been developed against this receptor and share similar properties in preclinical model systems. To date, all of the antibodies have been shown to bind IGF1R and cause its internalization and downregulation. Early clinical studies have shown that IGF1R antibodies have activity as single agents in some diseases (sarcoma) and have activity in combination with cytotoxic chemotherapy. These early promising results suggest that inhibition of this receptor will emerge as a new cancer therapy. In addition, these early clinical results raise additional questions regarding optimizing their use in cancer treatment. In a xenograft model, monoclonal antibodies to IGF1R do not suppress tumor growth of the MDA-435/LCC6 cell line yet inhibit tumor metastasis. These data suggest that a commonly measured clinical phenotype, tumor growth, may not be detected in early phase II clinical trials. Clinical trials have also shown that IGF1R monoclonal antibody therapy results in elevation of growth hormone, IGF-I, and insulin levels. Since InsR may also mediate signaling in the IGF system, it is possible that InsR is also a target for cancer therapy. To address this issue, we have used shRNA to downregulate InsR expression while leaving IGF1R levels intact. In the MDA-435/LCC6 cells, disruption of InsR decreases tumor growth and inhibits pulmonary metastases. We have also selected cells in increasing concentrations of IGF1R antibodies. Finally, long term incubation of cells with IGF1R monoclonal antibodies results in cells with low levels of receptor expression, yet the receptor remains biochemically functional and can be inhibited by IGF1R tyrosine kinase inhibitors. Thus, disruption of IGF1R signaling has promise as a new cancer therapy. Future consideration of the role of the ligands, the InsR, and combination therapies with other signaling disruptors will allow us to optimize this therapeutic strategy. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):CN07-02.

MCF-7 cells selected for acquired resistance to an anti IGF-IR antibody remain sensitive to fulvestrant and to an IGFIR tyrosine kinase inhibitor (TKI).

Abstract Abstract #72 Monoclonal antibodies and tyrosine kinase inhibitors (TKI) targeting the type-1 Insulin-like growth factor receptor (IGFIR) are currently in clinical trials. Resistance to several targeted therapies has been observed and is not well understood. Estrogen receptor (ER) has been shown to interact with IGFIR signaling to promote cell growth. To examine acquired resistance to IGF1R monoclonal antibodies, we selected ER positive MCF7L cells in increasing concentrations of an IGFIR inhibitory antibody (scFvFc, treated over 2 years). These cells (MCF7L-scFvFc) had downregulated IGFIR levels, increased basal phosphorylation of IRS-1 with constitutive activation of Akt. MCF7L-scFvFc cells had enhanced basal growth and were no longer responsive to IGF-I and estradiol (E2). To examine the relationship between IGF1R and ER function, we used the pure steroidal anti-estrogen fulvestrant to examine effects on growth of the parent and antibody-resistance cells. In parent MCF7L cells, fulvestrant inhibited growth responses to IGF-I and E2 treatment when each ligand was given individually, but when both ligands were given together, the growth inhibition was reversed. In MCF7L-scFvFc, fulvestrant inhibited growth response to IGF-I, but cells still responded to E2 even in the presence of fulvestrant. Fulvestrant inhibition of MCF7L-scFvFc basal growth was associated with downregulation of ER and IGFIR system components. Fulvestrant treatment induced PARP cleavage in MCF7L, but not in MCF7L-scFvFc cells. IGF-I partially protected cells against fulvestrant induced PARP cleavage, and E2 treatment has full protective effects on these cells. These data suggest that MCF7L-scFvFc cells were more resistant to cell apoptosis induced by fulvestrant. Other monoclonal antibodies directed against IGF1R were ineffective in reversing the resistance in MCF7L-scFvFc cells. To determine if activated IGF1R was responsible for the behavior of MCF7L-scFvFc cells, we used the IGF1R TKI AEW541. AEW541 inhibited growth of these antibody resistant cells and a combination of fulvestrant and AEW541 completely inhibited cell growth and IGF signaling in both MCF7L and MCF7L-scFvFc cells. Thus, activated IGFIR signaling was still required for the growth of MCF7L-scFvFc cells. In conclusion, MCF7L-scFvFc had low levels of IGF1R but retained activated IGF1R signaling that was inhibited by TKI (AEW541). In addition, the ER antagonist fulvestrant also inhibited growth of these cells. In these ER-positive cells, combined blockade of ER and IGF1R could provide a more prolonged inhibition of cell growth. Moreover, cells selected for resistance to an IGF1R antibody remain sensitive to IGF1R TKIs. Thus, concurrent or sequential targeting of IGF1R and ER function should be examined in breast cancer clinical trials of ER positive breast cancer. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 72.

Insulin-Like Growth Factor 1 Receptor Targeted Therapeutics: Novel Compounds and Novel Treatment Strategies for Cancer Medicine

The insulin-like growth factor 1 receptor (IGF-1R) and its associated signalling system has provoked considerable interest over recent years as a novel therapeutic target in cancer. A brief outline of the IGF-1R signalling system and the rationale for its use in cancer medicine is given. This is followed by a discussion of the different possible targets within the IGF-1R system, and drugs developed to interact at each target. A systems-based approach is then used to review the in vitro and in vivo data in the published literature of the following compounds targeting IGF-1R components using specific examples: growth hormone releasing hormone antagonists (e.g. JV-1-38), growth hormone receptor antagonists (e.g. pegvisomant), IGF-1R antibodies (e.g. CP-751,871, AVE1642/EM164, IMC-A12, SCH-717454, BIIB022, AMG 479, MK-0646/h7C10), and IGF-1R tyrosine kinase inhibitors (e.g. BMS-536942, BMS-554417, NVP-AEW541, NVP-ADW742, AG1024, potent quinolinyl-derived imidazo (1,5-a)pyrazine PQIP, picropodophyllin PPP, Nordihydroguaiaretic acid Insm-18/NDGA). The following tumour types are specifically discussed: lung, breast, colorectal, pancreatic, NETs, sarcoma, prostate, leukaemia, multiple myeloma. Other tumour types are mentioned briefly: squamous cell carcinoma of the head and neck, melanoma, glioblastoma, ovary, gastric and mesothelioma. Results of early stage clinical trials, involving recently patented drugs. are included where appropriate. We then outline the current understanding of toxicity related to IGF-1R targeted therapy, and finally outline areas for further research.

Optimization of 4,6-bis-anilino-1 H-pyrrolo[2,3- d]pyrimidine IGF-1R tyrosine kinase inhibitors towards JNK selectivity

The SAR of C5′ functional groups with terminal basic amines at the C6 aniline of 4,6-bis-anilino-1 H-pyrrolo[2,3- d]pyrimidines is reported. Examples demonstrate potent inhibition of IGF-1R with 1000-fold selectivity over JNK1 and 3 in enzymatic assays.

Introduction: Uveal melanoma

Introduction: Uveal melanoma

HoxA9 induces insulin-like growth factor-1 receptor expression in B-lineage acute lymphoblastic leukemia

The homeobox (Hox) gene family encodes a group of transcription factors preferentially expressed during embryonic development and hematopoiesis. Deregulation of Hox gene expression is frequently associated with acute leukemia. HoxA9 is the most commonly overexpressed Hox gene in acute leukemia. However, little is known regarding specific pathways regulated by HoxA9 that promote the growth and survival of leukemic cells. We have generated a conditional model of HoxA9 activity in the stromal cell dependent, HoxA9 negative, pre-B-cell line B-lineage-2 (BLIN-2). Conditional HoxA9 activation in BLIN-2 resulted in increased proliferation in the presence and absence of stromal cell support. Stimulation of HoxA9 activity resulted in increased expression of the c-Myb transcription factor and induction of insulin-like growth factor-1 receptor (IGF-1R) surface expression. HoxA9-mediated proliferative effects in BLIN-2 cells were abrogated when the cells were treated with specific IGF-1R tyrosine kinase inhibitors or with an IGF-1R mAb (A12). IGF-1R expression correlated with endogenous HoxA9 expression in a small panel of mixed lineage leukemia (MLL)/AF4 cell lines. siRNA knockdown of endogenous HoxA9 expression in the MLL/AF4-positive cell line RS4;11 resulted in loss of IGF-1R expression. These data indicate that HoxA9 overexpression induces IGF-1R expression and subsequently promotes leukemic cell growth.

HoxA9 Induces Insulin-Like Growth Factor-1 Receptor (IGF-1R) Expression in Acute Leukemia.

The homeobox (Hox) gene family encodes a group of transcription factors preferentially expressed during embryonic development and hematopoiesis. Overexpression of Hox genes is frequently associated with acute leukemia. HoxA9 is the most commonly overexpressed Hox gene in acute leukemia and forms part of a genetic signature that characterizes AML and ALL bearing MLL translocations. Despite frequent involvement in leukemia, pathways downstream of HoxA9 overexpression are not well characterized. In previous studies, we found that a B-lineage cell line bearing the MLL/AF4 oncogene, but lacking HoxA9 expression, retained B-lineage developmental capacity and normal apoptotic sensitivity to growth factor/stromal cell withdrawal. This led to the hypothesis that HoxA9 regulates pathways that promote leukemic cell proliferation and apoptotic resistance. This hypothesis was tested herein, by generating a conditional model of HoxA9 activity in the stromal cell dependent, HoxA9 negative pre-B ALL cell line, BLIN-2. HoxA9 was fused to the hormone binding domain of the estrogen receptor and cloned into a GFP/IRES retroviral construct. This construct was used to generate stably transduced BLIN-2/HoxA9:ER cells in which HoxA9 activity could be induced through the addition of the estrogen analog 4-hydroxytamoxifen (4HT) to the media. Cell fractionation experiments revealed that the presence of 4HT resulted in stabilization of HoxA9:ER protein and its nuclear localization in BLIN-2/HoxA9:ER cells. Electrophoretic mobility shifts performed with BLIN-2/HoxA9:ER cell nuclear extracts demonstrated that the HoxA9:ER fusion protein was capable of binding to the HoxA9 consensus DNA binding sequence. Conditional HoxA9 activation in BLIN-2/HoxA9:ER cells resulted in increased proliferation in the presence and absence of stromal cell support as compared with parental BLIN-2 cells, or BLIN-2 cells transduced with an empty vector (BLIN-2/MigR1). BLIN-2/HoxA9:ER cells survived in the absence of stromal support for an additional 3 to 5 days as compared to control cells. Stimulation of HoxA9 activity resulted in induction of IGF-1R protein expression on the surface of BLIN-2/HoxA9:ER cells. Induction of IGF-1R expression was further verified by western blot and RT-PCR analysis. HoxA9-mediated proliferative and survival effects were abrogated when BLIN-2/HoxA9:ER cells were treated with an IGF-1R tyrosine kinase inhibitor (AG1024) or with an IGF-1R monoclonal antibody (A12). IGF-1R expression correlated with endogenous HoxA9 expression in a small panel of MLL/AF4 positive ALL cell lines. Treatment of the MLL/AF4 positive cell line RS4;11 with the anti-IGF-1R antibody (A12) resulted in reduced proliferation. This effect was abrogated with supraphysiologic amounts of IGF-1. siRNA knock-down of endogenous HoxA9 expression in RS4;11 cells resulted in loss of IGF-1R expression. These data indicate that HoxA9 overexpression induces IGF-1R expression and subsequently promotes leukemic cell growth. These data provide novel mechanistic insight into how HoxA9 overexpression in acute leukemia promotes proliferation and apoptotic resistance through regulation of the IGF-1R pathway.

The insulin-like growth factor 1 receptor in cancer: Old focus, new future

The importance of insulin-like growth factor 1 receptor (IGF-1R) signalling in malignant behaviour of tumour cells is well established. Currently, development of drugs targeting the IGF-1R as anticancer treatment is emerging. Several IGF-1R targeting strategies are being investigated in phases I and II clinical trials. Interactions of IGF-1R with insulin receptor, however, might complicate efficiency and tolerability of such drugs. This review describes mechanisms, recent developments and potential limitations of IGF-1R antibodies and tyrosine kinase inhibitors.

The PI3-K/AKT/mTOR pathway as a target for breast cancer therapy

3511 Background: The PI3K/Akt/mTOR pathway is found dysregulated in multiple tumours, including breast cancer (BC). RAD001 is a recently developed drug which blocks mTOR. To study this compound’s anti-tumour effect and interactions with the aforementioned pathway, we set up a translational research program which integrates basic and clinical research data. Methods: The preclinal studies performed in BC cell lines and xenografts included the use of proliferation assays, western blot (WB), small interfering RNA and immunohistochemistry (IHC) analysis. The clinical studies consisted in a phase I study with escalating doses and collection of tumour biospies at different time points Results: Our first achievement was validating the antibodies used in WB and IHC and directed towards different components of the PI3K/Akt/mTOR pathway. We first reported that sensitivity to RAD001 correlated to basal levels of p-Akt. We then observed that RAD001 inhibits mTOR downstream, pS6 and eIF4G, but increases the levels of p-Akt, both in vitro and in patients’tumour biopsies. We demonstrated that knock down of the pS6 kinase mimics the effect of RAD001. Indeed, by combining the PI3K inhibitors and the genomic approch of p85 dominant negative vector, we demonstrated that RAD001 induced p-Akt is through PI3K. Besides the p-Akt induction we also found a RAD001 induction of IRS-1, the pivot mediator of the Insulin like Growth factor-1 Receptor (IGF-1R). We reported that RAD001 resulted in a prolonged activation of IRS-1 and in a sustained association with the p85 subunit of PI3K. Thus RAD001 disrupts an inhibitory mechanism impinging on the PI3K signalling, resulting in sustained activation of the IGF-1R pathway. Accordingly we demonstrated that combination of RAD001 with coumpounds directed against the IGF-1R, i.e. both IGF-1R tyrosine kinase inhibitors and monoclonal antibodies, prevented RAD001 induced p-Akt and resulted in a supradditive growth inhibitory effects both in vitro and in MCF-7 human BC derived xenografts.We are now conducting a phase I study with a monoclonal antibody directed at the IGF-1R that blocks IGF-1R signalling. Conclusions: Dual inhibition of mTOR and IGF-1R completely blocks the PI3K/Akt/mTOR pathway and provide a mechanistic-based combined approach. Clinical studies in BC patients are being planned. No significant financial relationships to disclose.

Insulin-like growth factor-I receptor kinase inhibitor NVP-AEW541 is active in breast cancer cells and enhances growth inhibition by herceptin through an increase in cell cycle arrest

21077 Background: Targeting ErbB2 with the monoclonal antibody trastuzumab (Herceptin) has shown to be active in breast cancer. However, a proportion of patients do not benefit from this treatment due to primary resistance. Mechanisms proposed for this resistance include activation of other receptors involved in proliferation as is the case of the insulin-like growth factor receptor (IGF-1R).Different strategies have been proposed for the inhibition of IGF-1R such as antibodies or small tyrosine-kinase inhibitors. In this study we analyzed the mechanism of action and the antiproliferative activity of an specific IGF-1R tyrosine-kinase inhibitor termed NVP-AEW541 alone and in combination with Herceptin. Methods: MCF7, SKBR3, BT474, and MDA-MB-231 breast cancer cells were used for this study. Proliferation and apoptosis were analyzed by MTT uptake assays or Annexin-V-FITC, respectively. The levels of different signalling proteins were analyzed by Western blotting. Microarray studies were performed using the HU-133A oligonucleotide arrays (Affymetrix, Santa Clara, CA). Real time quantitative PCR was used to confirm the differentially regulated genes. Results: The IGF-1R was expressed in all the cell lines studied. Exogenous addition of IGF-1 increased the tyrosine phosphorylation of IGF-1R, and this was prevented by preincubation with NVP- AEW541. The latter also decreased MTT uptake, and provoked a slight increase in apoptosis of BT474 cells. Combination of NVP-AEW541 and Herceptin had a synergistic effect on the inhibition of BT474 proliferation. Cell cycle analyses indicated that the combined addition of NVP- AEW541 and Herceptin increased the number of cells in the G0/G1 phases. These data were complemented with an increase in p27 levels upon treatment with both compounds. Microarray analyses identified several genes whose expression was modified by each agent alone and by the combination. Conclusions: Our results indicate that the combined targeting of two receptor tyrosine kinases known to play important roles in tumor cell proliferation may be more efficient than individual targeting of these receptors. No significant financial relationships to disclose.

Enhancement of Sensitivity of Human Lung Cancer Cell Line to TRAIL and Gefitinib by IGF-1R Blockade

Background: TRAIL is a cytokine that selectively induces apoptosis in various cancer cell lines. Gefitinib is new targeted drug applied in lung cancer that selectively inhibits EGFR tyrosine kinase. However, lung cancers have shown an initial or acquired resistance to these drugs. This study examined the effect of IGF-1R and its blockade on enhancing the sensitivity of lung cancer cell lines to TRAIL and gefitinib. Methods: Two lung cancer cell lines were used in this study. NCI H460 is very sensitive to TRAIL and gefitinib. On the other hand, A549 shows moderate resistance to TRAIL and gefitinib. The IGF-1R blockade was performed using adenoviruses expressing the dominant negative IGF-1R and shRNA to IGF-1R and AG1024 (IGF-1R tyrosine kinase inhibitor). Results: The adenovirus expressing dominant negative IGF-1R(950st) induced the increased expression of defective IGF-1R on the lung cancer cell surface, and the adenovirus-shIGF-1R effectively decreased the level of IGF-1R expression on cell surface. The genetic blockade of IGF-1R by the adenovirus-dnIGF-1R and AG1024 increased the sensitivity of A549 cells to TRAIL. The reduction of IGF-1R by transduction with ad-shIGF-1R also increased the sensitivity of the A549 cells to gefitinib. Conclusion: The blockade of IGF-1R through various mechanisms increased the sensitivity of the lung cancer cell line that was resistant to TRAIL and gefitinib. However, further studies using other cell lines showing acquired resistance as well as in vivo animal experiments will be needed.

Paradoxical effects of the phage display-derived peptide antagonist IGF-F1-1 on insulin-like growth factor-1 receptor signaling

The insulin-like growth factor binding proteins (IGFBPs) represent a unique class of IGF antagonists regulating the bioavailability of the IGFs extracellularly. Accordingly, they represent an important class of proteins for cancer therapeutics and chemoprevention. IGF-F1-1 is a cyclic hexadecapeptide identified by high throughput phage display that binds to the IGFBP-binding domain on IGF-1. It acts as an IGFBP-mimetic, capable of inhibiting IGF-1 binding to the IGFBPs. To further examine the utility of IGF-F1-1 as an IGF-1 antagonist we tested its ability to inhibit IGFBP-2 and IGFBP-3 binding to IGF-1, 125I-IGF-1 binding to IGF-1Rs and to block IGF-1 induced Akt activation, cell cycle changes and [ 3H]thymidine incorporation in MCF-7 cells. These biological activities were inhibited by treatment with IGFBP-2, wortmannin or the IGF-1R tyrosine kinase inhibitor, NVP-AEW541, but not by IGF-F1-1. Our findings confirm previous studies indicating that IGF-F1-1 is a weak antagonist of IGF-1 binding to the IGFBPs and the IGF-1R and suggest that it does not effectively inhibit downstream events stimulated by IGF-1. We further demonstrated that IGF-F1-1 treatment of MCF-7 cells results in the paradoxical activation of Akt, S-phase transition and [ 3H]thymidine incorporation. These results suggest that IGF-F1-1 is a weak agonist, exhibiting mitogenic actions. IGF-F1-1 may act in conjunction with IGF-1 at the IGF-1R or independently of IGF-1 at the IGF-1R or another receptor.

Blockade of IGF-1 receptor tyrosine kinase has antineoplastic effects in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is one of the most common cancer-related causes of death worldwide. Due to very poor 5-year-survival new therapeutic approaches are mandatory. Most HCCs express insulin-like growth factors and their receptors (IGF-R). As IGF-1R-mediated signaling promotes survival, oncogenic transformation and tumor growth and spread, it represents a potential target for innovative treatment strategies of HCC. Here we studied the antineoplastic effects of inhibiting IGF-1R signaling in HCC cells by the novel IGF-1R tyrosine kinase inhibitor NVP-AEW541. Methods and results NVP-AEW541 induced a time- and dose-dependent growth inhibition in the human hepatoblastoma and hepatocellular carcinoma cell lines SK-Hep-1, Hep-3B, Hep-G2 and Huh-7. Measurement of LDH-release showed that the antineoplastic effect of NVP-AEW541 was not due to cytotoxicity. Instead NVP-AEW541 induced apoptosis as evidenced by both caspase-3 and -8 activation as well as by apoptosis-specific morphological and mitochondrial changes. In addition, nuclear degradation was monitored by DNA-laddering. NVP-AEW541-treatment suppressed the expression of the antiapoptotic proteins Bcl-2 and survivin, while the expression of the proapoptotic protein BAX was stimulated in a dose-dependent manner. Moreover, NVP-AEW541 arrested the cell cycle at the G1/S checkpoint. When NVP-AEW541 was combined with cytotoxic chemotherapy or with a specific epidermal growth factor receptor antibody additive antiproliferative effects were observed. Interpretation Inhibition of IGF-1R tyrosine kinase (IGF-1R-TK) by NVP-AEW541 induces growth inhibition, apoptosis and cell cycle arrest in human HCC cell lines without accompanying cytotoxicity. Thus, IGF-1R-TK inhibition may be a promising novel treatment approach in HCC.

The insulin-like growth factor receptor 1 is a promising target for novel treatment approaches in neuroendocrine gastrointestinal tumours

Gastrointestinal neuroendocrine tumours (NET) represent a heterogeneous tumour entity. The anti-neoplastic therapy of advanced NET disease is still unsatisfactory and innovative therapeutic approaches are needed. As NET frequently express insulin-like growth factors (IGFs) and their receptors (IGFR), known to promote survival, oncogenic transformation, tumour growth and spreading, the inhibition of the IGF/IGF-receptor system may offer possibilities for novel targeted treatment strategies of NET. Here, we studied the anti-neoplastic effects of an inhibition of the IGF-I receptor (IGF-1R) signalling in NET cells by the novel IGF-1R tyrosine kinase (TK) inhibitor NVP-AEW541, whose anti-neoplastic potency has not yet been tested in NET disease. Using two human NET cell lines with different growth characteristics, we demonstrated that NVP-AEW541 dose-dependently inhibited the proliferation of NET cells by inducing apoptosis and cell cycle arrest. Anti-neoplastic effects of NVP-AEW541 were also detected in primary cultures of human neuroendocrine gastrointestinal tumours. Apoptosis was characterized by activation of the apoptotic key enzyme, caspase-3, as well as by detection of changes in the expression of the pro- and anti-apoptotic proteins, BAX and Bcl-2, after NVP-AEW541 treatment. Cell cycle was arrested at the G1/S checkpoint. The anti-neoplastic effects of NVP-AEW541 involved the inactivation of ERK1/2. Induction of immediate cytotoxicity did not account for the anti-neoplastic effects of NVP-AEW541, as shown by measurement of lactate dehydrogenase release. Moreover, additive anti-neoplastic effects were observed when NVP-AEW541 was combined with cytostatics such as doxorubicin or the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, fluvastatin. This is the first report on the induction of apoptosis and cell cycle arrest by the IGF-1R-TK inhibitor, NVP-AEW541, in NET cells. The inhibition of the IGF/IGFR system appears to be a promising novel approach for future treatment strategies of NET disease.