Constitutive Autophosphorylation Research Articles

PRDM16 Induces Methylation of FLT3 to Promote FLT3-ITD Signaling and Leukemia Progression.

Internal tandem duplication (ITD) in the FMS-like receptor tyrosine kinase-3 (FLT3) is one of the most frequent mutations in acute myeloid leukemia (AML) and is associated with poor prognosis. FLT3-ITD mutations result in endoplasmic reticulum (ER) retention and constitutive autophosphorylation of FLT3. The PR/SET domain 16 (PRDM16) is highly expressed in FLT3-ITD+ AML patients, suggesting it might play a role in leukemogenesis. Here, we revealed that genetic and pharmacological suppression of PRDM16 greatly slowed the progression of FLT3-ITD-driven leukemia, sensitized leukemic cells to tyrosine kinase inhibitors (TKIs), and extended the survival of leukemic mice. PRDM16 enhanced activation of oncogenic FLT3-ITD and ligand-dependent activation of wild-type FLT3 in leukemic cells. Mechanistically, PRDM16 mediated monomethylation of FLT3-ITD at lysine 614 and promoted its ER localization, resulting in enhanced FLT3 signaling in leukemia cells. Moreover, pharmacological suppression of FLT3-ITD methylation in combination with TKIs increased the elimination of FLT3-ITD+ AML cells. Altogether, these results suggest that PRDM16 boosts oncogenic FLT3 signaling in leukemic cells by prompting FLT3-ITD methylation. Therefore, PRDM16 may serve as a therapeutic target for AML.

Tyrosine kinase inhibitors for the treatment of indolent systemic mastocytosis: Are we there yet?

Indolent systemic mastocytosis (ISM) is the most prevalent form of systemic mastocytosis. Many patients with ISM suffer from mast cell (MC) mediator-related symptoms. In a small number of patients, hematologic progression is seen in the follow-up. In some patients with ISM, symptoms arising from MC-derived mediators including gastrointestinal symptoms, anaphylaxis, and neuropsychiatric symptoms are kept under control with conventional mediator-targeting drugs or MC-stabilizing agents. However, in a substantial number of patients, the symptoms are refractory to such conventional therapy. For these patients, novel drugs and targeted approaches are considered. One reasonable approach may be to apply tyrosine kinase inhibitors directed against KIT and other key kinase targets expressed in neoplastic MCs in ISM. Because MCs in more than 90% of all patients with typical ISM display the KIT D816V mutant receptor, clinically effective KIT-targeting drugs have to be active against this mutant form of KIT. The 2 such most effective and well-studied agents currently available are midostaurin and avapritinib. Other KIT-targeting drugs, such as imatinib or masitinib, are less effective or even noneffective against KIT D816V and are thus recommended for use only in patients with other KIT mutant forms (noncodon 816 mutations) or with wild-type KIT. In the present article, we review the current state in the treatment of ISM with tyrosine kinase inhibitors, with special emphasis on treatment responses and potential adverse effects. In fact, all of these agents also have unique and common adverse effects, and their use to treat patients with ISM should be balanced against their toxicity and short- and long-term safety.

Potential of Withaferin-A, Withanone and Caffeic Acid Phenethyl ester as ATP-competitive inhibitors of BRAF: A bioinformatics study.

Serine/threonine-protein kinase B-raf (BRAF) plays a significant role in regulating cell division and proliferation through MAPK/ERK pathway. The constitutive expression of wild-type BRAF (BRAFWT) and its mutant forms, especially V600E (BRAFV600E), has been linked to multiple cancers. Various synthetic drugs have been approved and are in clinical trials, but most of them are reported to become ineffective within a short duration. Therefore, combinational therapy involving multiple drugs are often recruited for cancer treatment. However, they lead to toxicity and adverse side effects. In this computational study, we have investigated three natural compounds, namely Withaferin-A (Wi-A), Withanone (Wi-N) and Caffeic Acid Phenethyl ester (CAPE) for anti-BRAFWT and anti-BRAFV600E activity. We found that these compounds could bind stably at ATP-binding site in both BRAFWT and BRAFV600E proteins. In-depth analysis revealed that these compounds maintained the active conformation of wild-type BRAF protein by inducing αC-helix-In, DFG-In, extended activation segment and well-aligned R-spine residues similar to already known drugs Vemurafenib (VEM), BGB283 and Ponatinib. In terms of binding energy, among the natural compounds, CAPE showed better affinity towards both wild-type and V600E mutant proteins than the other two compounds. These data suggested that CAPE, Wi-A and Wi-N have potential to block constitutive autophosphorylation of BRAF and hence warrant in vitro and in vivo experimental validation.

Therapeutic Potential of PI3K/AKT/mTOR Pathway in Gastrointestinal Stromal Tumors: Rationale and Progress.

Simple SummaryMost gastrointestinal stromal tumors (GISTs) arise due to gain-of-function mutations of KIT and PDGFRA, encoding the receptor tyrosine kinase (RTK). The introduction of the RTK inhibitor imatinib has significantly improved the management of GISTs; however, drug resistance remains a challenge. Constitutive autophosphorylation of RTKs is associated with the activation of the PI3K/AKT/mTOR pathway. Especially, this pathway plays a pivotal role in mRNA translation initiation, directly regulated by eukaryotic initiation factors (eIFs). This review highlights the progress for targeting PI3K/AKT/mTOR-dependent mechanisms in GISTs and explores the relationship between mTOR downstream eIFs and the development of GISTs, which may be a promising future therapeutic target for this tumor entity.Gastrointestinal stromal tumor (GIST) originates from interstitial cells of Cajal (ICCs) in the myenteric plexus of the gastrointestinal tract. Most GISTs arise due to mutations of KIT and PDGFRA gene activation, encoding the receptor tyrosine kinase (RTK). The clinical use of the RTK inhibitor imatinib has significantly improved the management of GIST patients; however, imatinib resistance remains a challenge. The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway is a critical survival pathway for cell proliferation, apoptosis, autophagy and translation in neoplasms. Constitutive autophosphorylation of RTKs has an impact on the activation of the PI3K/AKT/mTOR pathway. In several preclinical and early-stage clinical trials PI3K/AKT/mTOR signaling inhibition has been considered as a promising targeted therapy strategy for GISTs. Various inhibitory drugs targeting different parts of the PI3K/AKT/mTOR pathway are currently being investigated in phase Ι and phase ΙΙ clinical trials. This review highlights the progress for PI3K/AKT/mTOR-dependent mechanisms in GISTs, and explores the relationship between mTOR downstream signals, in particular, eukaryotic initiation factors (eIFs) and the development of GISTs, which may be instrumental for identifying novel therapeutic targets.

<p>Quizartinib (AC220): a promising option for acute myeloid leukemia</p>

Quizartinib is an effective therapy for patients with FLT3-ITD acute myeloid leukemia (AML) by continuing to inhibit the activity of FLT3 gene, leading to apoptosis of tumor cells. Multiple clinical trials have proved that it is effective in relapsed or refractory AML with an FLT3-ITD mutation. In this review, we focus on the characteristics of FLT3/ITD mutations, the mechanism and pharmacokinetics of quizartinib, and the mechanisms of resistance to quizartinib. We also summarize clinical experiences and adverse effects with quizartinib and recommend crucial approaches of quizartinib in the therapy of patients with newly diagnosed AML and patients with relapsed/refractory AML, particularly those with FLT3-ITD mutation. Quizartinib presents its advantages as a very promising agent in the treatment of AML, especially in patients with FLT3-ITD mutations. FLT3/ITD mutation can lead to constitutive autophosphorylation of FLT3 and activation of its downstream effectors including RAS/RAF/MEK, MAPK/ERK, PI3K/AKT/mTOR and JAK/STAT5 signal pathways, while Quizartinib can inhibit these downstream pathways through specific FLT3 inhibition. Quizartinib has received US Food and Drug Administration breakthrough therapy designation in patients with relapsed/refractory FLT3-ITD AML based on clinical trials. A larger sample of clinical trials are needed to verify its safety and efficacy, and the efficacy of quizartinib combined with chemotherapy or allogeneic hematopoietic cell transplantation should also be estimated in clinical trials. Meanwhile, for the side effects of quizartinib, further studies are needed to find a way to reduce its toxicity.

Anti-tumor effect of neratinib against lung cancer cells harboring HER2 oncogene alterations.

Human epidermal growth factor receptor 2 (HER2) is a member of the ErbB family of receptor tyrosine kinases. Numerous studies have reported the amplification and overexpression of HER2 in several types of cancer, including non-small cell lung cancer (NSCLC). However, the benefits of HER2-targeted therapy have not been fully established. In the present study, the anti-tumor effect of neratinib, an irreversible pan-HER tyrosine kinase inhibitor (TKI), against NSCLC cells harboring HER2 alterations was investigated. The sensitivity of normal bronchial epithelial cells (BEAS-2B) ectopically overexpressing wild-type or mutant HER2 to neratinib was assessed. Furthermore, the anti-tumor activity of neratinib in several NSCLC cell lines harboring HER2 alterations was determined in vitro and in vivo, and the association between their genetic alterations and sensitivity to neratinib treatment was investigated. BEAS-2B cells ectopically overexpressing wild-type HER2 or mutants (A775insYVMA, G776VC, G776LC, P780insGSP, V659E, G660D and S310F) exhibited constitutive autophosphorylation of HER2, as determined by western blotting. While these BEAS-2B cells were sensitive to neratinib, they were insensitive to erlotinib, a first-generation epidermal growth factor receptor-TKI. Neratinib also exerted anti-proliferative effects on HER2-altered (H2170, Calu-3 and H1781) NSCLC cell lines. Neratinib was also demonstrated to exert strong tumor growth inhibitory activity in mouse xenograft models using HER2-altered lung cancer cells. The results of the present study strongly suggest that neratinib has potential as a promising therapeutic option for the treatment of HER2-altered NSCLC.

Abstract 4777: Antitumor effect of neratinib in lung cancers harboring HER2 oncogene alterations

Abstract Background The HER2-targeted therapy for non-small cell lung cancer (NSCLC) harboring HER2 oncogene alterations is one of promising strategy to improve the clinical outcome of treatment for NSCLC. In this study, we investigated the antitumor effect of neratinib, an irreversible pan-HER tyrosine kinase inhibitor (TKI) in NSCLC cells harboring HER2 alterations including mutation and amplification. Materials and Methods We examined the sensitivity of neratinib against normal bronchial epithelial cells BEAS-2B which ectopically overexpressing wild-type or mutant HER2. Furthermore, we examined the antitumor activity of neratinib in addition to afatinib in several NSCLC cell lines harboring HER2 or EGFR alterations in in vitro and in vivo experiments, and investigated the association between their genetic alterations and sensitivity to neratinib treatment. Results BEAS-2B cells ectopically overexpressing wild-type HER2 or mutants (A775insYVMA, G776VC, G776LC, P780insGSP, V659E, G660D, and S310F) showed constitutive autophosphorylation of HER2 and activation of downstream signaling by Western blotting. These BEAS-2B cells were sensitive to neratinib, but insensitive to erlotinib, a first generation EGFR-TKI. Neratinib also showed antiproliferative effects on both HER2-altered (H2170, Calu-3, and H1781) and EGFR-mutant (HCC827, PC9, and HCC4006) NSCLC cell lines. Neratinib administration showed strong antitumor effect on tumor growth in mouse xenograft model using HER2-altered lung cancer cell lines. Conclusions Our study strongly suggests that neratinib is a promising therapeutic option for the treatment of HER2-altered NSCLC. Citation Format: Yusuke Ogoshi, shinichi Toyooka, Jyunichi Soh, hiromasa Yamamoto, Kazuhiko Shien, Hidejiro torigoe, Hiroki sato, Takahiro Yoshioka, Kei Namba, Yuta Takahashi, Eisuke Kurihara. Antitumor effect of neratinib in lung cancers harboring HER2 oncogene alterations [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 4777.

Whole exome sequencing in families at high risk for Hodgkin lymphoma: identification of a predisposing mutation in the KDR gene.

Hodgkin lymphoma shows strong familial aggregation but no major susceptibility genes have been identified to date. The goal of this study was to identify high-penetrance variants using whole exome sequencing in 17 Hodgkin lymphoma prone families with three or more affected cases or obligate carriers (69 individuals), followed by targeted sequencing in an additional 48 smaller HL families (80 individuals). Alignment and variant calling were performed using standard methods. Dominantly segregating, rare, coding or potentially functional variants were further prioritized based on predicted deleteriousness, conservation, and potential importance in lymphoid malignancy pathways. We selected 23 genes for targeted sequencing. Only the p.A1065T variant in KDR (kinase insert domain receptor) also known as VEGFR2 (vascular endothelial growth factor receptor 2) was replicated in two independent Hodgkin lymphoma families. KDR is a type III receptor tyrosine kinase, the main mediator of vascular endothelial growth factor induced proliferation, survival, and migration. Its activity is associated with several diseases including lymphoma. Functional experiments have shown that p.A1065T, located in the activation loop, can promote constitutive autophosphorylation on tyrosine in the absence of vascular endothelial growth factor and that the kinase activity was abrogated after exposure to kinase inhibitors. A few other promising mutations were identified but appear to be "private". In conclusion, in the largest sequenced cohort of Hodgkin lymphoma families to date, we identified a causal mutation in the KDR gene. While independent validation is needed, this mutation may increase downstream tumor cell proliferation activity and might be a candidate for targeted therapy.

Antitumor effect of afatinib, as a human epidermal growth factor receptor 2-targeted therapy, in lung cancers harboring HER2 oncogene alterations.

Human epidermal growth factor receptor 2 (HER2) is a member of the HER family of proteins containing four receptor tyrosine kinases. It plays an important role in the pathogenesis of certain human cancers. In non-small-cell lung cancer (NSCLC), HER2 amplification or mutations have been reported. However, little is known about the benefit of HER2-targeted therapy for NSCLCs harboring HER2 alterations. In this study, we investigated the antitumor effect of afatinib, an irreversible epidermal growth factor receptor (EGFR)-HER2 dual inhibitor, in lung cancers harboring HER2 oncogene alterations, including novel HER2 mutations in the transmembrane domain, which we recently identified. Normal bronchial epithelial cells, BEAS-2B, ectopically overexpressing wild-type HER2 or mutants (A775insYVMA, G776VC, G776LC, P780insGSP, V659E, and G660D) showed constitutive autophosphorylation of HER2 and activation of downstream signaling. They were sensitive to afatinib, but insensitive to gefitinib. Furthermore, we examined the antitumor activity of afatinib and gefitinib in several NSCLC cell lines, and investigated the association between their genetic alterations and sensitivity to afatinib treatment. In HER2-altered NSCLC cells (H2170, Calu-3, and H1781), afatinib downregulated the phosphorylation of HER2 and EGFR as well as their downstream signaling, and induced an antiproliferative effect through G1 arrest and apoptotic cell death. In contrast, HER2- or EGFR-non-dependent NSCLC cells were insensitive to afatinib. In addition, these effects were confirmed in vivo by using a xenograft mouse model of HER2-altered lung cancer cells. Our results suggest that afatinib is a therapeutic option as a HER2-targeted therapy for NSCLC harboring HER2 amplification or mutations.

Aptamer targeting EGFRvIII mutant hampers its constitutive autophosphorylation and affects migration, invasion and proliferation of glioblastoma cells.

Glioblastoma Multiforme (GBM) is the most common and aggressive human brain tumor, associated with very poor survival despite surgery, radiotherapy and chemotherapy.The epidermal growth factor receptor (EGFR) and the platelet-derived growth factor receptor β (PDGFRβ) are hallmarks in GBM with driving roles in tumor progression. In approximately half of the tumors with amplified EGFR, the EGFRvIII truncated extracellular mutant is detected. EGFRvIII does not bind ligands, is highly oncogenic and its expression confers resistance to EGFR tyrosine kinase inhibitors (TKIs). It has been demonstrated that EGFRvIII-dependent cancers may escape targeted therapy by developing dependence on PDGFRβ signaling, thus providing a strong rationale for combination therapy aimed at blocking both EGFRvIII and PDGFRβsignaling.We have recently generated two nuclease resistant RNA aptamers, CL4 and Gint4.T, as high affinity ligands and inhibitors of the human wild-type EGFR (EGFRwt) and PDGFRβ, respectively.Herein, by different approaches, we demonstrate that CL4 aptamer binds to the EGFRvIII mutant even though it lacks most of the extracellular domain. As a consequence of binding, the aptamer inhibits EGFRvIII autophosphorylation and downstream signaling pathways, thus affecting migration, invasion and proliferation of EGFRvIII-expressing GBM cell lines.Further, we show that targeting EGFRvIII by CL4, as well as by EGFR-TKIs, erlotinib and gefitinib, causes upregulation of PDGFRβ. Importantly, CL4 and gefitinib cooperate with the anti-PDGFRβ Gint4.T aptamer in inhibiting cell proliferation.The proposed aptamer-based strategy could have impact on targeted molecular cancer therapies and may result in progresses against GBMs.

Abstract 2588: Anti-tumor effect of afatinib, an irreversible EGFR/HER2 dual inhibitor, in lung cancers harboring HER2 oncogene

Abstract HER2 is a member of the EGFR family of tyrosine kinase receptors and plays important roles in the pathogenesis of certain human cancers. In non-small cell lung cancer (NSCLC), HER2 amplification or mutations are reported. However, little is known about the benefit of HER2-targeted therapy for NSCLC harboring HER2 alteration. In this study, we investigated the anti-tumor effect of afatinib, an irreversible EGFR/HER2 dual inhibitor, in lung cancers harboring HER2 oncogene alteration, including the novel HER2 mutations in transmembrane domain which we recently identified. Ectopically overexpressing HER2 wild-type or mutant (G776V,Cins, A775insYVMA, G660D, and V659E) in normal bronchial epithelial cells, BEAS2B, showed constitutive autophosphorylation of HER2 and activation of downstream signaling. They were sensitive to afatinib, but insensitive to EGFR-TKI (gefitinib). Furthermore, we examined anti-tumor activity of afatinib and gefitinib in several NSCLC cell lines, and investigated the association between their genetic alteration and sensitivity to afatinib treatment. Afatinib down-regulated the phosphorylation of HER2, EGFR, and their downstream signalings, and induced anti-proliferative effect through a G1 arrest and apoptotic cell death in HER2 altered cells (H2170, Calu-3, and H1781). Whereas, HER2 non-dependent NSCLC cells (A549, H1299, and H1993) were insensitive to afatinib. Our results strongly suggest that afatinib is a therapeutic option for NSCLC patients with HER2 alteration. Citation Format: Ken Suzawa, Shinichi Toyooka, Masakiyo Sakaguchi, Tomoaki Ohtsuka, Mototsugu Watanabe, Shinsuke Hashida, Yuho Maki, Hiromasa Yamamoto, Junichi Soh, Hiroaki Asano, Kazunori Tsukuda, Shinichiro Miyoshi. Anti-tumor effect of afatinib, an irreversible EGFR/HER2 dual inhibitor, in lung cancers harboring HER2 oncogene. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2588. doi:10.1158/1538-7445.AM2015-2588

The involvement of reactive oxygen species derived from NADPH oxidase-1 activation on the constitutive tyrosine auto-phosphorylation of RET proteins

Reactive oxygen species (ROS) play a key role in neoplastic growth and tumor invasion is supported by various experimental data. In this study, we analyzed the participation of ROS in the RET tyrosine auto-phosphorylation. The NIH3T3 cell lines transfected with cRET, MEN2A, and MEN2B individually (designated NIH3T3cRET, NIH3T3 RET-MEN2A, and NIH3T3RET-MEN2B) showed the elevated levels of intracellular ROS, and concomitantly increased Rac1 expression, as well as down-regulation of Mn SOD and Cu/Zn SOD in comparison with the parental cell line expressing RET. H2O2 enhanced the constitutive tyrosine auto-phosphorylation of RET-MEN2A and RET-MEN2B proteins, and this increase was attenuated by treatment with the NOX inhibitor diphenyliodonium (DPI) or catalase. We also showed that DPI inhibited dimerization of RET-MEN2A. Elevated ROS derived from NOX1 activation and downregulation of SOD in NIH3T3RET-MEN2A and NIH3T3RET-MEN 2B cells may be involved in RET constitutive tyrosine auto-phosphorylation, and scavengers of ROS such as catalase and blocking NOX1 are useful for targeting RET tyrosine kinase activation in cancer.

Bone marrow stroma‐mediated resistance to FLT3 inhibitors in FLT3‐ITD AML is mediated by persistent activation of extracellular regulated kinase

A consistent pattern of response has been observed when FMS-like tyrosine kinase 3 (FLT3) tyrosine kinase inhibitors (TKIs) have been used as monotherapy to treat patients with relapsed or refractory FLT3- internal tandem duplication (ITD) acute myeloid leukaemia (AML). Circulating blasts are cleared from the peripheral blood, while bone marrow blasts are either unaffected or are cleared from the marrow at a much slower rate. We used an in vitro model of FLT3-ITD AML blasts co-cultured with normal human bone marrow stromal cells to investigate the basis for this dichotomous response pattern to FLT3 inhibitors. We have found that in blasts on stroma, potent FLT3 inhibition predominantly results in cell cycle arrest rather than apoptosis. The anti-apoptotic effect is mediated through a combination of direct cell-cell contact and soluble factors. The addition of exogenous FLT3 ligand (FL) augments the protection, primarily by shifting the 50% inhibitory concentration for FLT3 inhibition upwards. Cytokine-activated extracellular regulated kinase (ERK), rather than STAT5, appears to be the most important downstream signalling protein mediating the protective effect, and inhibition of MEK significantly abrogates stromal-mediated resistance. These findings explain the phenomenon of peripheral blood versus bone marrow blast responses and suggest that the combination of potent FLT3 inhibition and MEK inhibition is a promising strategy for the treatment of FLT3-ITD AML.

Sa1745 Critical Residues in the Transmembrane Helical Bundle Domains of the Human Motilin Receptor for Motilin and Erythromycin Binding and Activity

Background: Activated pancreatic stellate cells (PSC) play an important role in chronic pancreatitis to exacerbate fibrosis and increase the risk of pancreatic cancer. Systemic dysregulation of glucose metabolism, increased rates of obesity and type II diabetes are rising at an alarming pace in the Western world. Hyperinsulinemia and glucose intolerance associated with these disorders may have a variety of deleterious effects on the pancreas, an organ with a vital role to regulate glucose in the whole body. However, the extent to which these effects involve PSC has not been determined. Methods: we analyzed the expression of insulin/ IGF-1 signaling pathway components and their operation as profibrotic and/or proliferative pathways within a novel immortalized human pancreatic stellate cell line (ihPSC) and assessed the potential contributions of these cells to a procarcinogenic microenvironment. Results: ihPSC express receptors for both insulin (IR) and insulin-like growth factor 1 (IGF-1R), and respond to insulin/IGF-1 stimulation with increased tyrosine phosphorylation at specific autophosphorylation sites. Whereas routine culture conditions (DMEM/F12 supplemented with 10% FBS) maintained constitutive tyrosine autophosphorylation of IR/IGF-1R, and activity of ERK1/2 and Akt pathways, serum starvation (24 h in fresh medium without FBS) diminished the Akt Ser473 and Thr308 phosphorylation to ,20% of basal. In serum-starved PSC, insulin at 100 nM induced peak stimulation of Akt phosphorylation that remained elevated at levels of 5-8 fold above basal from 10 min to at least 1 h. IGF-1 at 10 nM also produced peak stimulation, maximally at 10 min. Whereas the basal level of ERK1/2 phosphorylation was high even in serum-starved cells, stimulation with either 100 nM insulin or 10 nM IGF-1 produced transient increases which peaked at approximately 3 min. Interestingly, we also found effects of different culture conditions on mRNA and protein expression of IR/IGF-1R β subunits. In particular, in cells grown to 50% confluence and then shifted to physiologic glucose level (5 mM), marked enhancement of IGF-1R expression levels was exhibited within 24 h, as assessed by Western blot analysis. Also under these conditions, ihPSC express galectin-1, galectin-3 and IGF-1 ligand, and insulin/IGF-1 stimulation enhanced fibronectin production. Interestingly, whereas low glucose was associated with a sustained enhancement in IR/IGF-1R β subunit as well as IGF-1 mRNA expression, exposure to a hyperglycemic condition (25 mM glucose) with or without insulin appeared to destabilize these mRNA and protein expression levels. Conclusions: our data support a role of insulin/IGF-1 in PSC proliferative and profibrotic signaling and suggest that hyperglycemia/ hyperinsulinemia negatively modulate responsiveness to, as well as production of, IGF-1 in PSC.

Exploring Novel Therapeutic Targets in GIST: Focus on the PI3K/Akt/mTOR Pathway

Gastrointestinal stromal tumors (GISTs) are the most common soft tissue sarcoma, and most feature abnormalities in two genes encoding the receptor tyrosine kinases (RTKs), KIT, and PDGFRA. The RTK inhibitor imatinib revolutionized treatment in GIST; however, drug resistance remains a challenge. Constitutive autophosphorylation of RTKs is linked to phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway hyperactivation, which is central to oncogenic signaling, and known to be dysregulated in GIST. Preclinical experiments have confirmed that inhibiting the PI3K/Akt/mTOR pathway is a rational target for therapy. Early studies using mTOR inhibitors have shown limited success, which may be due to the activation of Akt that occurs following mTORC1 inhibition. Therefore, targeting PI3K or Akt, which lie upstream of mTORC1, may translate into more complete pathway inhibition. Several treatment strategies are currently being developed in phase 1 and 2 clinical trials. Compounds currently in development include pan-Class I PI3K inhibitors, dual PI3K/mTOR inhibitors, and Akt inhibitors. The aim of this review is to highlight the evidence for targeting PI3K/Akt/mTOR-dependent mechanisms in GIST and to evaluate the existing preclinical and clinical data supporting this strategy.

Abstract 856: Biomarkers of sensitivity and resistance to the BTK inhibitor PCI-32765 in mantle cell lymphoma

Abstract Bruton's tyrosine kinase (Btk) is a central regulator of BCR signaling in B-cell lymphomas. PCI-32765 is a potent and selective inhibitor of Btk which has shown good clinical activity, particularly in mantle cell lymphoma. But its mechanism of action in this disease is poorly understood. In this study, we explored the role of Btk and the activity of PCI-32765 on BCR signaling in PBMC samples from patients, which contain a high percentage of tumor cells, as well as in several MCL lines, including Granta-519, Jeko-1, JVM-2, JVM-13, Maver-1, Mino, NCEB-1, Rec-1 and Z-138. In this work, we present a detailed characterization of signalling pathways inhibited by PCI-32765 downstream of the B-cell receptor in cell lines and tumor cells, studied by gene expression profiling, flow cytometry, and proteomic analyses of signaling pathways and effectors. Correlation with PCI-32765 activity in tumor cells and patients leads to the identification of biomarkers of biomarkers of sensitivity and resistance. Results: Btk and surface IgM protein expression was detected in all MCL lines at variable levels. In a 3-day proliferation assay, JVM-2 & MINO emerged as the most sensitive lines to PCI-32765 (GI50: 1.75-4.4uM). Rec-1 was resistant to PCI-32765 alone (11.3uM) but became much more sensitive (1.45uM) upon BCR stimulation using anti-IgM (10ug/mL). Other lines such as Maver-1, Granta-519 & Jeko-1 all required >10uM of PCI-32765 for inhibition and BCR stimulation did not make much difference. When signaling pathways downstream of BCR activation were studied, intracellular calcium flux following stimulation with IgM was observed in all lines (except JVM-2) and was inhibited at <100nM PCI-32765 in most of them, but no correlation between this and growth inhibition was observed. Constitutive BTK autophosphorylation was observed in all lines and was completely abolished by PCI-32765. BCR stimulation increased p-BTK which was also blocked by PCI-32765 in all lines. Mino and JVM-2 showed constitutive p-ERK activity, which was slightly increased upon BCR stimulation and could be blocked with PCI-32765, whereas the more resistant lines such as Maver-1 and Rec-1 had low endogeneous levels of p-ERK, but which was increased by BCR stimulation and only partially or not reversed by PCI-32765 at 5uM. Little change was observed in levels of p-PLCg1 or p-NF-kB p65. Additionally, in all cell lines stimulation with anti-IgM led to an increased secretion of the chemokines CCL3 and CCL4, which was inhibited by PCI-32765. Blockade of Btk function using PCI-32765 inhibits MCL cell proliferation, BCR signaling and chemokine secretion. These findings highlight the importance of BCR signaling and Btk in MCL, help explain the activity of the Btk inhibitor PCI-32765 in MCL patients, and provide biomarkers that may be of value in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 856. doi:1538-7445.AM2012-856

Abstract A141: Analysis of cellular kinase activity of resistance-related MET mutants using transmembrane-domain (TMD) activated kinase variants.

Abstract Kinases are important targets in oncology. However, the frequent emergence of resistance (gatekeeper) mutants in target kinases seriously limits the patient's response time to a given drug. Secondary drugs need to be developed that specifically target mutant enzymes. The discovery of such kinase inhibitors requires relevant assay systems. In order to efficiently analyze whether novel inhibitors target gatekeeper mutants of a given kinase in a cellular context we set out to establish a suitable cellular test system for the receptor tyrosine kinase MET. Our aim was to minimize variations such as cellular background and ligand dependent activation while putting the focus on the mutation of interest. In an approach to uncouple kinase activity from ligand activation, we screened a set of artificial transmembrane domains (TMD) to identify a TMD that induces constitutive MET autophosphorylation in transient transfections. Subsequently, Rat1 cells were transduced to express the tag-labelled intracellular wild-type (wt) MET domain harbouring the activating TMD while lacking the extracellular domain. Characterization of TMD-MET-wt in Rat1 cells showed constitutive MET expression and phosphorylation. ELISA studies to analyze the inhibition of MET phosphorylation by cognate MET inhibitors revealed comparable IC50 values to those obtained with wild-type MET naturally over expressed in MKN45 gastric carcinoma cells. Moreover, expression of TMD-MET-wt in Rat1 cells led to cell transformation as assessed by growth in soft agar as well as in vivo as a subcutaneous xenograft. Interestingly, the same artificial TMD induced constitutive activity of various gatekeeper mutants of MET when transduced into Rat1 cells. This observation allowed for the comparative analysis of various small molecule MET inhibitors on multiple MET mutants in an identical cellular background and without the need of ligand-induced kinase activation. Using a straight forward ELISA-based approach, we generated profiles of various MET inhibitors on cellular MET mutant phosphorylation and compared these with inhibition profiles obtained in biochemical MET activity assays. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A141.

Abstract 736: Mechanisms of EGFR and c-Met inhibitor resistance in NSCLC

Abstract While tyrosine kinase inhibitors (TKI's) such as gefitinib and erlotinib against EGFR have positive therapeutic effects in a subset of Non-Small Cell Lung Cancer (NSCLC) patients, their clinical efficacies are limited due to drug resistance, which may be due to secondary T790M mutations in EGFR and/or c-MET gene amplification. Current clinical trials using both c-Met/EGFR-TKIs have found longer progression free states. However, since resistance has been observed in a subset of patients, it is vital to determine the mechanism of combined TKI acquired resistance. To study this mechanism further, we established two NSCLC cell lines (H2170 and H358) which are resistant to EGFR-TKI, erlotinib and c-Met-TKI SU11274 and had IC50 concentrations which were of 10-20 fold higher. To determine the key pathways involved in TKI resistance, immunoblots were performed with the IC50 of resistant cell lines (erlotinib 8µM and SU11274 9µM) in the presence and absence of EGF (15ng/ml), HGF (40ng/ml) or both for 2.5 minutes. We found a 2 and 5 fold decrease in expression of p-c-Met in H2170 ESR and H358 ESR cells respectively in the presence of HGF compared to parental control cells. Downregulation of p-c-Met in both resistant cell lines would imply that these cells were not selected to harbor amplified c-MET expression. These results indicate that EGFR-TKI resistant NSCLC could be due to other factors besides selection of c-Met amplified cells as has been currently suggested and is thus a novel concept which needs further studies. c-Met independent pathways such as VEGF or IGF-1 could also mediate tumor proliferation in the presence of low levels of c-Met. In H2170 ESR cells, EGFR was constitutively autophosphorylated at Tyr1068 (19 fold increase) which resulted in the upregulation of downstream signaling protein p-ERK (Thr202/Tyr204) (2 Fold) in the absence of EGF. We also found upregulation of p-mTOR (Ser2448) (2.5 fold) and phospho p70S6 kinase (Thr389) (2 fold) in the presence and absence of erlotinib which could be modulated by p-ERK or p-mTOR. Modulation of these proteins could result in increased proliferation and cause resistance in NSCLC. Conversely, in H358 ESR, an increase in p-ERK (Thr202/Tyr204) (1.3 fold) was seen in the absence of constitutive autophosphorylation of EGFR. Similar to H2170, an upregulation of p-mTOR (Ser2448) (2 fold) and phospho p70S6 kinase (Thr389) (4 fold) was observed in H358 ESR in the presence of erlotinib in comparison to the parental cell line. These results indicate that other pathways described above may have an important role in mediating resistance. No changes in total protein levels of EGFR, ERK or p70S6 kinase were observed. Thus, in these resistant lines, the p-mTOR pathway may mediate downstream EGFR and c-Met signaling, and our current preliminary studies with rapamycin indicate that mTOR may play a major role in resistance to EGFR/c-Met TKI's. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 736. doi:10.1158/1538-7445.AM2011-736

Combining the FLT3 Inhibitor PKC412 and the Triterpenoid CDDO-Me Synergistically Induces Apoptosis in Acute Myeloid Leukemia with the Internal Tandem Duplication Mutation

Mutations of the FLT3 receptor tyrosine kinase consisting of internal tandem duplications (ITD) have been detected in blasts from 20% to 30% of patients with acute myeloid leukemia (AML) and are associated with a poor prognosis. FLT3/ITD results in constitutive autophosphorylation of the receptor and factor-independent survival in leukemia cell lines. The C-28 methyl ester of the oleane triterpenoid (CDDO-Me) is a multifunctional molecule that induces apoptosis of human myeloid leukemia cells. Here, we report that CDDO-Me blocks targeting of NFkappaB to the nucleus by inhibiting IkappaB kinase beta-mediated phosphorylation of IkappaBalpha. Moreover, CDDO-Me blocked constitutive activation of the signal transducer and activator of transcription 3. We report the potent and selective antiproliferative effects of CDDO-Me on FLT3/ITD-positive myeloid leukemia cell lines and primary AML cells. The present studies show that CDDO-Me treatment results in caspase-3-mediated induction of apoptosis of FLT3/ITD-expressing cells and its antiproliferative effects are synergistic with PKC412, a FLT3-tyrosine kinase inhibitor currently in clinical trials. Taken together, our studies indicate that CDDO-Me greatly enhanced the efficacy of the FLT3 inhibitor PKC412, suggesting that combining two separate pathway inhibitors might be a viable therapeutic strategy for AML associated with a FLT3/ITD mutation.

Abstract LB-309: EML4-ALK signaling is required for the maintenance of neoplastic phenotype of non-small cell lung cancer cells: Novel strategy for lung cancer-tailored therapies

Abstract Lung cancer is the most common cancer in the world, and is lethal in 90% of the cases. In non-small cell lung cancer (NSCLC), deregulated receptor tyrosine kinases (RTKs) stand out among the causal dominant oncogenes. Consequently, the genomic and/or pharmacological ablation of RTK signaling has emerged as a novel tailored therapeutic strategy. Nevertheless, this approach has serious constrains, probably due to the frequent co-activation of multiple RTKs in a considerable subsets of solid tumors. It has become also evident that during TK inhibitor-based regimens, RTK switching and/or the selection of resistant clones can frequently occur representing a serious and problematic drawback. In a subset of NSCLC, the Anaplastic Lymphoma Kinase (ALK) gene has been described to be translocated and fused to EML4. This determines the ALK constitutive dimerization and autophosphorylation leading to cellular transformation. Here, we investigated the ALK oncogenic addiction of human NSCLC and studied the putative co-operative role of other kinases. We first demonstrated that the ectopic expression of EML4-ALK in ALK positive NSCLC cell lines (H2228 and H3122) resulted in the activation of multiple signaling pathways, in manner similar to that described for other known ALK fusions. Although EML4-ALK can induce transformation in lung in vitro and in vivo, ALK inhibition via shRNA or small molecule inhibitors (CEPs, Cephalon) induced only the apoptosis of H3122 cells, whereas in H2228 it caused cell growth arrest. Moreover, the treatment with ALK inhibitors led to tumor regression, but not tumor eradication, in vivo. Based on Phosphoproteomic analyses we demonstrated that the phosphorylation status of several tyrosine kinases (such as, EGFR, Met, FGFR, Jak1 or IGFR) was affected by the ALK inhibition only in H2228 cell line, but not in H3122. Notably, the combined treatment with anti-ALK (CEP14083, CEP2550, CEP28122) and -EGFR inhibitors resulted in an increased cell death of H2228 cells to values similar to those observed for ALK treated H3122 cells. Finally, gene expression analyses showed that known EGFR substrates were specifically down regulated upon the combined treatment in ALK positive H2228 cells. Our findings suggest that ALK signaling is required for the maintenance of the neoplastic phenotype of some ALK positive NSCLC cells and that its abrogation could represent a novel strategy for the treatment of a well-defined subset of human lung cancer (complete ALK addiction). More importantly, we showed that the tumor survival and maintenance of ALK positive neoplastic cells might relay on the concomitant activation of multiple RTKs. These findings further support the notion that molecular and functional signatures are required for designing molecular-based “patient specific” therapeutic protocols in lung cancer patients. 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 LB-309.