RTK-targeted Therapies Research Articles

Abstract 537: Validating USP27X as a drug target in lung cancer

Abstract Over the past two decades, tremendous efforts have been dedicated to developing and improving receptor tyrosine kinase (RTK) targeted therapies to combat the development and progression of different cancers. Despite the significant progress made, these therapies are not durable: not all patients respond to treatment, and primary or acquired resistances are common. Interestingly, however, while RTK-targeted therapies have no effect on resistant lung cancer cell lines, si/shRNA-mediated RTK depletion or combined treatments that affect RTK protein levels profoundly impact the growth and survival of these cells, indicating that RTKs are still critical for their proliferation. Thus, strategies to target RTK protein levels hold promise as an option for treating RTK-driven cancers. In our recent studies, we discovered that ubiquitin-specific protease 27X (USP27X) functions are crucial for properly recycling and maintaining EGFR steady-state levels in lung cancer cells. Ablation of USP27X leads to a severe decrease of EGFR steady levels, inhibition of xenograft tumor growth, and sensitization of lung cancer cell lines to targeted therapy. To fully understand the in vivo functions of USP27X in EGFR regulation and lung carcinogenesis, we created a new mouse line carrying a conditional Usp27X allele and introduced this allele into the well-established and clinically relevant EGFRL858R mouse lung cancer model. Ongoing studies will determine whether USP27X can be a critical therapeutic in EGFR-driven lung cancer. Citation Format: Shamshad Alam, Amanda Zunic, Swati Venkat, Michael E Feigin, Boyko Atanassov. Validating USP27X as a drug target in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 537.

Evaluation of NTRK Gene Fusion by Five Different Platforms in Triple-Negative Breast Carcinoma.

Triple-negative breast carcinoma (TNBC) is an aggressive disease that has a poor prognosis since it lacks effective treatment methods. Neurotrophic tyrosine receptor kinase (NTRK) fusion genes are excellent candidates for targeted RTK inhibitor therapies and there are available targeted therapy drugs for the treatment of TRK fusion-positive tumors in a tumor agnostic pattern. Our study was designed to investigate the NTRK gene fusion status in TNBC patients and to determine whether RTK-targeted therapies are suitable for TNBC patients. A total of 305 TNBC patients were enrolled in our study. IHC was employed as a prescreening method, and IHC positive cases were further submitted for evaluation by FISH, RT-PCR, and NGS methods. NTRK IHC was evaluated successfully in 287 of the 305 cases, and there were 32 (11.15%) positive cases. FISH was carried out in the 32 IHC positive cases. There were 13 FISH-positive cases if the threshold was set as >15% of the 100 counted tumor cells having a split orange and green signal with more than one signal diameter. There were only 2 FISH-positive cases if the cutoff value was defined as >15% of the counted tumor cells having a split signal with more than two signal diameter widths. One of the FISH-positive cases had a separate NTRK3 FISH signal in 88% of the tumor cells, and its IHC result was strong nuclear staining in all the tumor cells. After evaluation of the morphology, it was re-diagnosed as secretory breast carcinoma, and the NGS result confirmed that it had a NTRK3-ETV6 fusion gene. The other FISH-positive cases were all negative for NTRK gene fusion in the NGS or RT-PCR examination. The NTRK gene fusion rate was low in our TNBC cohort. NTRK gene fusion may be a rare event in TNBC. The high false-positive rate of NTRK gene fusion detected by IHC questions its role as a prescreening method in TNBC. More data may be needed to determine a suitable threshold for NTRK FISH in TNBC in the future. More studies are needed to confirm whether RTK-targeted therapies are appropriate treatments for TNBC patients.

Immuno-PET Detects Changes in Multi-RTK Tumor Cell Expression Levels in Response to Targeted Kinase Inhibition.

Receptor tyrosine kinase (RTK) coexpression facilitates tumor resistance due to redundancies in the phosphatidylinositol-3'-kinase/protein kinase B and KRAS/extracellular-signal-regulated kinase signaling pathways, among others. Crosstalk between the oncogenic RTK hepatocyte growth factor receptor (MET), epidermal growth factor receptor (EGFR), and human epidermal growth factor receptor 2 (HER2) are involved in tumor resistance to RTK-targeted therapies. Methods: In a relevant renal cell carcinoma patient-derived xenograft model, we use the 89Zr-labeled anti-RTK antibodies (immuno-PET) onartuzumab, panitumumab, and trastuzumab to monitor MET, EGFR, and HER2 protein levels, respectively, during treatment with agents to which the model was resistant (cetuximab) or sensitive (INC280 and trametinib). Results: Cetuximab treatment resulted in continued tumor growth, as well as an increase in all RTK protein levels at the tumor in vivo on immuno-PET and ex vivo at the cellular level. Conversely, after dual MET/mitogen-activated protein kinase inhibition, tumor growth was significantly blunted and corresponded to a decrease in RTK levels. Conclusion: These data show the utility of RTK-targeted immuno-PET to annotate RTK changes in protein expression and inform tumor response to targeted therapies.

Preparation of a phosphotyrosine-protein standard for use in semiquantitative western blotting with enhanced chemiluminescence.

Protein tyrosine phosphorylation is key to activation of receptor tyrosine kinases (RTK) that drive development of some cancers. One challenge of RTK-targeted therapy is identification of those tumors that express non-mutated but activated RTKs. Phosphotyrosine (pTyr) RTK levels should be more predictive of the latter than expressed total protein. Western blotting (WB) with a pTyr antibody and enhanced chemiluminescence (ECL) detection is sufficiently sensitive to detect pTyr-RTKs in human tumor homogenates. Presentation of results by comparing WB images, however, is wanting. Here we describe the preparation of a new pTyr-protein standard, pTyr-ALK48-SB (pA), derived from a commercial anaplastic lymphoma kinase (ALK) recombinant fragment, and its use to quantify pTyr-epidermal growth factor receptor (pTyr-EGFR) in commercial A431 cell lysates. Linearity of one-dimensional (1D) WB plots of pA band density versus load as well as its lower level of detection (0.1 ng, 2 fmole) were determined for standardized conditions. Adding pA to two lots of A431 cell lysates with high and low pTyr-EGFR allowed normalization and quantification of the latter by expressing results as density ratios for both 1D and 2D WB. This approach is semi-quantitative because unknown RTKs may be outside the linear range of detection. Semiquantitative ratios are an improvement over comparisons of images without a reference standard and facilitate comparisons between samples.

Roles for receptor tyrosine kinases in tumor progression and implications for cancer treatment.

Growth factors and their receptor tyrosine kinases (RTKs), a group of transmembrane molecules harboring cytoplasm-facing tyrosine-specific kinase functions, play essential roles in migration of multipotent cell populations and rapid proliferation of stem cells' descendants, transit amplifying cells, during embryogenesis and tissue repair. These intrinsic functions are aberrantly harnessed when cancer cells undergo intertwined phases of cell migration and proliferation during cancer progression. For example, by means of clonal expansion growth factors fixate the rarely occurring driver mutations, which initiate tumors. Likewise, autocrine and stromal growth factors propel angiogenesis and penetration into the newly sprouted vessels, which enable seeding micro-metastases at distant organs. We review genetic and other mechanisms that preempt ligand-mediated activation of RTKs, thereby supporting sustained cancer progression. The widespread occurrence of aberrant RTKs and downstream signaling pathways in cancer, identifies molecular targets suitable for pharmacological intervention. We list all clinically approved cancer drugs that specifically intercept oncogenic RTKs. These are mainly tyrosine kinase inhibitors and monoclonal antibodies, which can inhibit cancer but inevitably become progressively less effective due to adaptive rewiring processes or emergence of new mutations, processes we overview. Similarly important are patient treatments making use of radiation, chemotherapeutic agents and immune checkpoint inhibitors. The many interfaces linking RTK-targeted therapies and these systemic or local regimens are described in details because of the great promise offered by combining pharmacological modalities.

Abstract 2655: Oxygen tension regulates lysosomal activation and receptor tyrosine kinase degradation

Abstract Oxygen sensing is crucial for adaptation to variable habitats and physiological conditions. Low oxygen tension, or hypoxia, is a common feature of solid tumors and hypoxic tumors are often more aggressive and resistant to therapy. Here we show that, in mammalian tissue culture cells, hypoxia suppressed lysosomal acidification/activation and receptor tyrosine kinase (RTK) degradation. Hypoxia down-regulated mTORc1, reducing its ability to activate transcription factor EB (TFEB), a master regulator of v-ATPase, the lysosomal proton pump. Hypoxia prevented epidermal growth factor receptor (EGFR) degradation in tumor tissues, whereas activation of lysosomes enhanced tumor cell response to anti-EGFR treatment. Our results linkoxygen tension and lysosomal activity, provide a molecular explanation of the malignant phenotype associated with hypoxic tumors, and suggest activation of lysosomes may provide therapeutic benefit in RTK-targeted cancer therapy. Citation Format: Jaewoo Hong, P. Charles Lin, Todd Wuest, Yongfen Min. Oxygen tension regulates lysosomal activation and receptor tyrosine kinase degradation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2655.

PHLDA1 Mediates Drug Resistance in Receptor Tyrosine Kinase-Driven Cancer

SummaryDevelopment of resistance causes failure of drugs targeting receptor tyrosine kinase (RTK) networks and represents a critical challenge for precision medicine. Here, we show that PHLDA1 downregulation is critical to acquisition and maintenance of drug resistance in RTK-driven cancer. Using fibroblast growth factor receptor (FGFR) inhibition in endometrial cancer cells, we identify an Akt-driven compensatory mechanism underpinned by downregulation of PHLDA1. We demonstrate broad clinical relevance of our findings, showing that PHLDA1 downregulation also occurs in response to RTK-targeted therapy in breast and renal cancer patients, as well as following trastuzumab treatment in HER2+ breast cancer cells. Crucially, knockdown of PHLDA1 alone was sufficient to confer de novo resistance to RTK inhibitors and induction of PHLDA1 expression re-sensitized drug-resistant cancer cells to targeted therapies, identifying PHLDA1 as a biomarker for drug response and highlighting the potential of PHLDA1 reactivation as a means of circumventing drug resistance.

Splice Variants of the RTK Family: Their Role in Tumour Progression and Response to Targeted Therapy.

Receptor tyrosine kinases (RTKs) belong to a family of transmembrane receptors that display tyrosine kinase activity and trigger the activation of downstream signalling pathways mainly involved in cell proliferation and survival. RTK amplification or somatic mutations leading to their constitutive activation and oncogenic properties have been reported in various tumour types. Numerous RTK-targeted therapies have been developed to counteract this hyperactivation. Alternative splicing of pre-mRNA has recently emerged as an important contributor to cancer development and tumour maintenance. Interestingly, RTKs are alternatively spliced. However, the biological functions of RTK splice variants, as well as the upstream signals that control their expression in tumours, remain to be understood. More importantly, it remains to be determined whether, and how, these splicing events may affect the response of tumour cells to RTK-targeted therapies, and inversely, whether these therapies may impact these splicing events. In this review, we will discuss the role of alternative splicing of RTKs in tumour progression and response to therapies, with a special focus on two major RTKs that control proliferation, survival, and angiogenesis, namely, epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor-1 (VEGFR1).

Broad RTK-targeted therapy overcomes molecular heterogeneity-driven resistance to cetuximab via vectored immunoprophylaxis in colorectal cancer

The human epidermal growth factor receptor (EGFR) targeting chimeric monoclonal antibody, cetuximab (Erbitux®), is a widely used drug in the treatment of metastatic colorectal cancer. However, the activation of the extensive crosstalk among the EGFR family receptors as well as other tyrosine kinase receptors (RTKs) impairs the efficacy of the drug by fueling acquired resistance. To identify the responsible potential activation pathway underlying cetuximab resistance and generate novel treatment strategies, cetuximab-resistant colorectal cancer cell lines were generated and validated and a functional RNAi screen targeting human RTKs was used to identify extensive receptor tyrosine kinase signaling networks established in resistant cancer cells. MET, Axl, and IGF-1R were identified as contributors to the acquired resistance to cetuximab. Targeting vectored immunoprophylaxis (VIPs) to different RTKs were generated and characterized. Different VIP approaches were evaluated in vivo with parental and cetuximab-resistance xenografts and the RTKs in resistant cancer xenografts were inhibited with VIPs via re-sensitization to cetuximab treatment. Combination of VIPs was more broadly efficacious, mechanistically, due to co-blocking the EGFR/Axl/MET signaling pathway, which was cross-activated in the resistant cell lines. Moreover, a VIP-based procedural treatment strategy not only eliminated the tumor but also afforded long-lasting protection from tumor recurrence and resistance. Overall, EGFR-related RTK pathway-network activation represents a novel mechanism underlying cetuximab resistance. A broad VIP combination strategy and VIP-based procedural treatment strategy may be a recommended addition to cetuximab-based targeted therapy. Our results establish a new principle to achieve combined RTK inhibition and reverse drug resistance using a VIP approach.

Abstract 205: Analysis of resistance to RTK inhibitors using a novel RTK multiplex assay

Abstract A novel multiplex immunoassay was developed for examining acquired resistance to Receptor Tyrosine Kinase (RTK) inhibitors. RTKs are transmembrane proteins which act as receptors for growth factors, neurotrophic factors and other extracellular signaling molecules. These cell-surface kinases are activated by extracellular ligands leading to receptor dimerization and tyrosine phosphorylation at specific residues in the cytoplasmic tails to initiate RTK-mediated signal transduction. Of the >500 known protein kinases in the human genome there are approximately 60 RTKs. They are central components of cell signaling networks and play crucial roles in normal physiological processes and disease processes ranging from diabetes to cancer. Many RTKs, such as EGFR, HER2, c-Kit, PDGFR and VEGFR, have been used as targets for drug development and RTK-targeted therapies have illustrated the utility of these treatments for selected cancers. However, in many cases, compensatory RTK signaling enables cancer cells to acquire resistance to RTK inhibitors that selectively target a single RTK. For example, some EGFR and HER2 inhibitors have led to resistance and are associated with increased expression of IGF1R. In order to understand the mechanisms of resistance to RTK inhibitors, we have developed a bead-based multiplex immunoassay capable of simultaneously detecting phosphorylation of 18 different RTKs. First we demonstrate detection of RTK phosphorylation in response to growth factor stimulation using various cell lines. Next we examined the specificity of two inhibitors targeting EGFR and HER2. These inhibitors specifically reduced growth factor-stimulated phosphorylation of EGFR and HER2, without inhibiting the activation of other RTKs. Although we did not observe compensatory activation of other RTKs in response to the two EGFR and HER2 inhibitors we tested, this study demonstrates the feasibility of using this novel 18-plex RTK panel for examining the mechanism of resistance to single RTK inhibitors. In summary, the RTK multiplex panel allowed for simultaneous detection of multiple tyrosine phosphorylated RTKs in a specific, sensitive, and reproducible manner. Citation Format: Lu Chen, Anthony J. Saporita, Wen-Rong Lie, Reeti Maheshwari, Melissa Schluter, Jehangir Mistry, Joseph Hwang. Analysis of resistance to RTK inhibitors using a novel RTK multiplex assay. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 205.

Truncated RAF kinases drive resistance to MET inhibition in MET-addicted cancer cells.

Constitutively active receptor tyrosine kinases (RTKs) are known oncogenic drivers and provide valuable therapeutic targets in many cancer types. However, clinical efficacy of RTK inhibitors is limited by intrinsic and acquired resistance. To identify genes conferring resistance to inhibition of the MET RTK, we conducted a forward genetics screen in the GTL-16 gastric cancer cell line, carrying MET amplification and exquisitely sensitive to MET inhibition. Cells were transduced with three different retroviral cDNA expression libraries and selected for growth in the presence of the MET inhibitor PHA-665752. Selected cells displayed robust and reproducible enrichment of library-derived cDNAs encoding truncated forms of RAF1 and BRAF proteins, whose silencing reversed the resistant phenotype. Transduction of naïve GTL-16 cells with truncated, but not full length, RAF1 and BRAF conferred in vitro and in vivo resistance to MET inhibitors, which could be reversed by MEK inhibition. Induction of resistance by truncated RAFs was confirmed in other MET-addicted cell lines, and further extended to EGFR-addicted cells. These data show that truncated RAF1 and BRAF proteins, recently described as products of genomic rearrangements in gastric cancer and other malignancies, have the ability to render neoplastic cells resistant to RTK-targeted therapy.

Coamplification of receptor tyrosine kinases and downstream targets in Japanese gastric cancers.

41 Background: Amplifications of receptor tyrosine kinases genes (RTKs), EGFR, ERBB2, FGFR2, MET, have been associated with the pathogenesis and progression of gastric cancer (GC). Recent studies have found that coamplifications of RTKs are rare in GC. Two phase III trials using RTK targeted therapy have failed to achieve survival benefit in GC patients. Co-amplification of RTKs and downstream targets genes (DSTs), PIK3CA, KRAS, MYC, CCNE1 may be related to resistance to RTK targeted therapy. We tested the hypothesis whether RTKs and DSTs genes are coamplified in GC. Methods: DNA and RNA were extracted from 221 GC from the Kanagawa Cancer Center Hospital (Yokohama, Japan). Copy number of EGFR, ERBB2, FGFR2, PIK3CA, KRAS, MYC, CCNE1 was investigated by a newly developed multiplex ligation dependent probe amplification (MLPA) assay. RNA expression of the same genes was measured using the NanoString platform and compared to DNA copy number status. The frequency of RTK and DST co-amplifications was established and related to KRAS mutation status. Results: RTK amplification was associated with high levels of RNA expression except MYC (all p<0.05). 68% of GC had no RTK amplification. 9% of GC had RTK amplification and no DST amplification. 10% of GC had RTKs co-amplified. 23% of GC had RTK and DST co-amplified. There was no overlap between KRAS mutations and amplifications of KRAS, PIK3CA, FGFR2 or MET. Conclusions: This is the first study reporting that RTK and DST co-amplification are more frequent than RTK co-amplification in GC. These results indicate that GC patients considered for RTK targeting therapy might require DNA copy number assessment of RTKs as well as key DSTs and KRAS mutation testing in order to identify those patients who benefit most from treatment.

Abstract 967: Establishing mechanisms of acquired resistance to Met inhibitor in Met-addicted gastric cancer cell lines.

Abstract Signaling by the Met, hepatocyte growth factor (HGF) receptor tyrosine kinase (RTK) activates multiple downstream signaling pathways that promote cell migration and invasive growth. Cells that overexpress and are “addicted” to Met also require Met signaling to sustain cell survival. Thus, a number of specific small-molecule inhibitors have been developed to target Met in the clinic. Although several successes of RTK-targeted therapies are acknowledged, most have had limited long-term success clinically due to development of drug resistance. As MET amplification occurs in 10-20% of gastric cancers, and MET-amplified cell lines are often exquisitely dependent on Met signaling for proliferation and survival, we established Met inhibitor-resistant clones derived from two different gastric cancer cell lines (MKN45 and KATO II) that are sensitive to Met inhibition. Treatment with a small-molecule inhibitor, PHA-665752 (PHA), specific to Met abrogates cell proliferation and anchorage-independent growth in these cells. In vitro selection of the most tumorigenic and resistant cells was performed by isolating colonies in anchorage-independent conditions with increasing PHA concentrations. Colonies were picked and expanded in 2D cell culture conditions and PHA concentration gradually increased until the cells were able to proliferate in a PHA concentration of >1μM (10x greater than the dose required for abrogation of proliferation in the parental cell lines). DNA, RNA, and protein have been isolated from each of the clones and sequencing, microarray analyses, phospho-RTK arrays, reverse-phase protein arrays (RPPA) have been utilized to identify the changes in signalling, protein and transcript expression that allow these cells to escape PHA inhibition. The analyses from these high-throughput assays will be presented here. As several Met inhibitors are presently in all three phases of clinical trials, and Met signalling has emerged as a means of escaping treatment by other targeted small-molecule kinase inhibitors, there exists a great need to determine the mechanisms through which cells may circumvent Met inhibition. The identification of resistance mechanisms will be essential for developing multi-pronged therapeutic strategies that may prove more efficacious at combatting tumorigenesis. Citation Format: Andrea Z. Lai, Crista Thompson, Sean Cory, Hong Zhao, Naila Chughtai, Michael Hallett, Morag Park. Establishing mechanisms of acquired resistance to Met inhibitor in Met-addicted gastric cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 967. doi:10.1158/1538-7445.AM2013-967

Abstract B45: Met-Dependent Positive and Negative Signaling Cascades in Gastric Cancer Cells

Abstract Signaling by the Met, hepatocyte growth factor (HGF) receptor tyrosine kinase (RTK) activates multiple downstream signaling pathways that promote cell migration and invasive growth. Cells that overexpress and are “addicted” to Met also require Met signaling to sustain cell survival. Thus, a number of specific small-molecule inhibitors have been developed to target Met in the clinic. Although several successes of RTK-targeted therapies are acknowledged, several have limited long-term success clinically, due to development of drug resistance. Initiation of RTK signaling cascades leads to the activation of a number of downstream signaling molecules, but prolonged activation also triggers negative feedback loops that function in part to abrogate this signaling. Hence, targeted inhibition of Met, in addition to suppressing Met-dependent signaling, may also release cells from negative feedback mechanisms and allow the reactivation of signaling pathways that may promote resistance to Met inhibition. As MET amplification occurs in 20–40% of gastric cancers, to identify core Met dependent signaling pathways activated in Met dependent cancers we have used four different gastric cancer cell lines that exhibit amplification, overexpression, and constitutive activation of Met. Upon inhibition of Met with a small-molecule inhibitor, we observe abrogation of several downstream signaling pathways at both the protein phosphorylation and transcript level. Interestingly, as Met has been demonstrated to cross-talk with the EGF receptor family, we also observe a decrease in the phosphorylation of EGFR and HER3 upon treatment with Met inhibitor, and a decrease in expression of EGFR ligands. Conversely, Met inhibition results in an elevation in expression of HER3 transcript and protein in all 4 cell lines, implicating Met signaling in HER3 repression, and inhibition of Met may release HER3 from this negative regulation. As increases in HER3 phosphorylation and expression occur in other models (such as breast or lung cancer cell lines) upon treatment with EGFR, HER2, or AKT inhibitors, and high HER3 expression is strongly associated with tumor progression and poor prognosis in gastric cancer; hence, the loss of negative regulation of HER3, downstream from Met, may ultimately contribute to clinical efficacy, or lack thereof, of Met inhibitors.

Abstract 1216: c-Met protein expression is regulated by palmitoylation in prostate cancer cells

Abstract Prostate cancer (PCa) is the most common noncutaneous neoplasia diagnosed in men and represents roughly 10% of cancer-associated mortalities. The lethal phenotype of PCa is primarily characterized by progression of tumor cells to androgen-independence and metastasis. Organ-confined PCa is often curable with surgery and/or radiation therapy, however, as the disease becomes metastatic, the likelihood of survival becomes minimal. Key influences in the progression to metastasis are growth factor receptors including the receptor tyrosine kinase c-Met. Numerous bodies of evidence strongly link aberrant c-Met signaling with causation and/or progression of prostate cancer. Overexpression of c-Met can lead to activation in a ligand-independent manner and has been identified as a common mechanism of resistance to RTK-targeted therapy. In addition, multiple reports have identified the enzyme fatty acid synthase (FASN) as being commonly overexpressed in prostate cancer, and that this aberrant expression is an early event that becomes more pronounced with aggressive androgen-independent and metastatic disease. FASN is the sole enzyme responsible for de novo synthesis of the 16-carbon saturated fatty acid palmitate. In cancer, de novo lipids are more selectively partitioned into lipid rafts as phospholipids as well as utilized for post-translational acyl-modifications of signaling proteins. Previous findings have led us to identify a novel mechanism by which FASN activity regulates c-Met expression. Our work has determined that inhibition or shRNA knockdown of FASN results in a post-translational downregulation of already synthesized c-Met protein. This downregulation is prevented by the addition of exogenous palmitate. Here we provide evidence that inhibition of palmitoylation, using the palmitate analog 2-bromopalmitate, lowers total c-Met levels. This protein loss occurs post-transcriptionally and is associated with accumulation of c-Met in Golgi compartments. Using inhibitors to a number of internalization pathways, as well as surface biotinylation studies, our findings suggest the inhibition of palmitoylation reduces the stability of newly synthesized c-Met as opposed to inducing internalization and degradation. Moreover, both an acyl-biotin exchange technique and a click-chemistry based palmitate-labeling protocol have provided evidence suggesting c-Met itself is palmitoylated. From these data, we hypothesize that c-Met requires palmitoylation along a biosynthetic route to promote its stability and trafficking to the cell surface. In the absence of palmitoylation, c-Met accumulates within the Golgi and is disposed of through a yet to be determined degradation pathway. Our findings potentially reveal a novel mechanism for restricting the expression of c-Met as a means of preventing prostate cancer invasion and metastasis. 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 1216. doi:1538-7445.AM2012-1216

Abstract 3: HER2 gene-amplified human breast cancer cells harboring a gatekeeper T768M mutation in HER2 overexpress EGFR ligands and are sensitive to dual therapeutic blockade of EGFR and HER2

Abstract Intrinsic or acquired mutations in receptor tyrosine kinases (RTKs) can confer resistance to RTK-targeted therapies. A previous study reported that a ‘gatekeeper’ T768M mutation in HER2 confers resistance to the tyrosine kinase inhibitor (TKI) lapatinib. We generated BT474 cells stably expressing either T768M HER2 or GFP. Treatment with lapatinib disrupted HER3-p85 complex and blocked phosphorylation of HER2, HER3, AKT and Erk1/2 in BT474-GFP but not in BT474-T768M cells. BT474-T768M cells were also resistant to other anti-HER2 therapies such as trastuzumab. BT474-T768M cells were sensitive to the PI3K inhibitor BKM120 but not the MEK inhibitor CI1040, suggesting they still depend on the PI3K pathway for survival. Treatment with the irreversible HER2/EGFR inhibitor BIBW2992 (afatinib) eliminated P-HER2 and inhibited growth of cells expressing the mutant, suggesting they still depend on the ErbB receptor network to activate PI3K. Indeed, like in BT474-GFP (control) cells, BIBW2992 disrupted the association of p85 (PI3K) with HER3 and blocked phosphorylation of AKT. A co-culture experiment with varying proportions of BT474-GFP and BT474-T768M cells revealed that lapatinib-resistant acini emerge when as low as 5% of the population has T768M HER2. Moreover, we found that only 3.2% of the HER2 alleles had the T768M mutation in the BT474-T768M cells. This result suggests that allelic dilution will render it difficult to identify this drug resistant mutation with conventional sequencing techniques (i.e., Sanger) in patients with HER2 gene-amplified breast cancer. Next, we stably expressed either wild-type or T768M HER2 in MCF10A cells. Both WT and T768M HER2 resulted in increased phosphorylation of HER3 and AKT. MCF10A-HER2 but not MCF10A-T768M cells were sensitive to growth inhibition by lapatinib. Lapatinib blocked HER2, HER3 and tyrosine phoshorylation in MCF10A-HER2 but not in MCF10A-T768M cells. Quantitative PCR indicated that the BT474-T768M cells produce more EGFR ligands (EGF, TGFα, amphiregulin and HB-EGF) compared to BT474-GFP cells. Treatment with cetuximab, an EGFR antibody that blocks ligand binding, restored sensitivity of BT474-T768M cells to trastuzumab. Further, the BT474-T768M cells were sensitive to a combination of trastuzumab and lapatinib both in vitro and in vivo. Hence, dual blockade of HER2 and EGFR might be effective at trumping the development of HER2-overexpressing cancers bearing a gatekeeper mutation in T768M. 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 3. doi:1538-7445.AM2012-3

Abstract 1222: Met-dependent positive and negative signaling cascades in gastric cancer cells

Abstract Signaling by the Met, hepatocyte growth factor (HGF) receptor tyrosine kinase (RTK) activates multiple downstream signaling pathways that promote cell migration and invasive growth. Cells that overexpress and are “addicted” to Met also require Met signaling to sustain cell survival. Thus, a number of specific small-molecule inhibitors have been developed to target Met in the clinic. Although several successes of RTK-targeted therapies are acknowledged, several have limited long-term success clinically, due to development of drug resistance. Initiation of RTK signaling cascades leads to the activation of a number of downstream signaling molecules, but prolonged activation also triggers negative feedback loops that function in part to abrogate this signaling. Hence, targeted inhibition of Met, in addition to suppressing Met-dependent signaling, may also release cells from negative feedback mechanisms and allow the reactivation of signaling pathways, that may promote resistance to Met inhibition. As MET amplification occurs in 10-20% of gastric cancers, to identify core Met dependent signaling pathways activated in Met dependent cancers we have used four different gastric cancer cell lines that exhibit amplification, overexpression, and constitutive activation of Met. Upon inhibition of Met with a small-molecule inhibitor, we observe abrogation of several downstream signaling pathways at both the protein phosphorylation and transcript level. Interestingly, as Met has been demonstrated to cross-talk with the EGF receptor family, we also observe a decrease in the phosphorylation of EGFR and HER3 upon treatment with Met inhibitor, and a decrease in expression of EGFR ligands. Conversely, Met inhibition results in an elevation in expression of HER3 transcript and protein in all 4 cell lines, implicating Met signaling in HER3 repression, and inhibition of Met may release HER3 from this negative regulation. As increases in HER3 phosphorylation and expression occur in other models (such as breast or lung cancer cell lines) upon treatment with EGFR, HER2, or AKT inhibitors, and high HER3 expression is strongly associated with tumor progression and poor prognosis in gastric cancer; hence, the loss of negative regulation of HER3, downstream from Met, may ultimately contribute to clinical efficacy, or lack thereof, of Met inhibitors. 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 1222. doi:1538-7445.AM2012-1222

Checks and balances: Interplay of RTKs and PTPs in cancer progression

In recent years, targeted therapies for receptor tyrosine kinases (RTKs) have shown initial promise in the clinical setting for the treatment of several tumors driven by these oncogenic signaling pathways. Unfortunately, clinical relapse due to acquired resistance to these molecular therapeutics is common. An improved understanding of how tumors bypass the inhibitory effects of RTK-targeted therapies has revealed a rich myriad of possible mechanisms for acquired resistance. Protein tyrosine phosphatases (PTPs) can function as oncogenes or tumor suppressors to either enhance or suppress RTK signaling. Recent studies suggest that the loss or gain of function of PTP's can significantly impinge on RTK signaling during tumor progression. Here we review the interplay between RTKs and PTPs as an emerging mechanism for acquired resistance to RTK-targeted therapies, that may aid in the design of improved therapies to prevent and overcome resistance in treatments for cancer patients.

Subgroup classification of receptor tyrosine kinase (RTK) pathway activation in KRAS-mutated metastatic carcinoma.

e18019 Background: Molecular stratification of metastatic carcinoma in body fluids has become critical to therapy selection, as these are often the only accessible disease sites. We performed multimodality RTK pathway analysis of metastatic carcinoma in pleural fluid to assess how functional RTK dysregulation correlates with initiating mutation. Methods: Tumor samples from 45 patients with metastatic carcinoma diagnosed in pleural fluid were assessed (22 lung adenocarcinomas, 7 breast, 3 gastric, 4 ovarian, 9 others); with sequential analysis in 9 patients. Site of origin was determined by a combination of clinical history, cytomorphology, and CK7/20, CK5/6, napsin A, and TTF-1 immunostains. Mutation analysis of KRAS (codons 12, 13, and 61), BRAF, and PIK3CA (exons 1, 9, and 20) was performed with PCR-based pyrosequencing, with EGFR mutations in exons 18-21 assessed by Sanger sequencing. FISH analysis to assess copy number of the HER2, MET, ALK, and EGFR loci was performed using commercially available probes. Functional RTK pathway activation was assessed using semi-quantitative analysis using a panel of phospho(p)-antibodies including MET, EGFR, HER2 and AKT activation sites. Results: KRAS mutations were common in all tumors, including lung (11/22, 50%), breast (6/7, 86%), gastric (2/3, 67%), and ovarian (4/4, 100%) carcinomas. Cases without KRAS mutation showed EGFR mutations or BRAF mutations (lung), with PIK3CA mutations in 2 breast, 1 lung, and 1 endometrial tumor. The KRAS-mutated cases showed distinct subgroups characterized by other TK abnormalities, including genomic amplification of HER2 (17.2%), EGFR (3.4%), and MET (13.8%) and strong p-activation of MET (3.4%), EGFR (24.1%), and AKT (32.1%). Conclusions: Metastatic carcinoma in pleural fluid has high rates of KRAS mutation, regardless of site of origin Among KRAS-mutated tumors, there is a stable subgrouping/clustering of RTK activation patterns, indicative of distinct progression events. Given that KRAS mutation predicts lack of response to current RTK-targeted therapies, functional correlates of these observed genetic subgroups will be critical to designing novel treatments for this high-risk subgroup of carcinomas.

Therapeutic Implications of Detection of Amplification and Activation of HER2 and Other Receptor Tyrosine Kinases (RTKs) in Circulating Tumor Cells (CTCs) in Recurrent Breast Cancer.

Abstract Background: HER2 is one of four trans-membrane RTKs in epidermal growth factor receptor family, and HER2-positive phenotype has been associated with aggressive subtype of breast cancer with HER2 gene amplification. Approximately 15-20% of breast cancers are considered HER2-positive by IHC or FISH analysis. Recently, changes in HER2 expression status between primary tumor and CTCs found in recurrent metastatic disease have been reported to occur at a significant frequency. Methods for detecting HER2 expression and phosphorylation in serially collected CTCs may provide valuable insight into the overall disease profile shift, and therefore lead to better selection of therapy for each patient.Methods: A triple-antibody-enzyme-channeling multiplexed protein microarray platform has been developed to detect the phosphorylation on target molecules. Extremely high assay specificity was achieved by immuno-complex formation via co-localization of two detector enzyme-conjugated-antibodies once target proteins are captured on the microarray-surface. The channeling events between two detector enzymes in proximity enabled profiling of the RTKs with a single-cell level sensitivity. In order to validate the method on clinical samples, CTCs from 77 breast cancer patients on different therapy regimens were analyzed at various time points along their course of therapy.Results: Whole blood of 77 metastatic cancer patients and 60 healthy volunteers were analyzed for CTC-HER2 expression and activation. We observed significant HER2 status conversion with recurrent disease. 29% of patients with negative HER2 expression in the primary tumor showed HER2-amplification in isolated CTCs. Phosphorylated HER2 receptors were found in 52% of patients with primary HER2 negative disease. The enhancement of assay sensitivity and specificity using proximity mediated immuno-assay made the detection of HER2 activation (even without amplification) possible when isolated CTCs were stimulated with ligands to other RTKs with transactivation potential.Discussion: The multiplexed-proximity mediated immunoassay successfully detected the expression of HER2 RTKs and their degree of activation in CTCs isolated from recurrent breast cancer patients. As we hypothesize that CTCs found in metastatic stage represent the most aggressive and invasive cell population, serial CTC-profiling can lead to better therapy selection/adjustment and disease/treatment monitoring as there are available options to choose appropriate kinase inhibitors for RTK-targeted therapies. While significant number of patients acquired HER2-amplification in their CTCs, substantially higher rate of CTC-HER2 activation was found in relapsed metastatic disease. The unique triple-antibody mediated immuno-microarray analysis allowed a single cell level profiling of the CTC-HER2. The ability to profile serially collected CTCs will provide valuable information on changes occurring in tumor cells as a function of time and treatment. This method can provide guidance, not only for initial selection of targeted therapeutics, but also in subsequent monitoring for rapidly 'evolving' disease in individual patient. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3010.