EGFR amplification and outcome in a randomised phase III trial of chemotherapy alone or chemotherapy plus panitumumab for advanced gastro-oesophageal cancers.

OBJECTIVE: Epidermal growth factor receptor (EGFR) inhibition may be effective in biomarker-selected populations of advanced gastro-oesophageal adenocarcinoma (aGEA) patients. Here, we tested the association between outcome and EGFR copy number (CN) in pretreatment tissue and plasma cell-free DNA (cfDNA) of patients enrolled in a randomised first-line phase III clinical trial of chemotherapy or chemotherapy plus the anti-EGFR monoclonal antibody panitumumab in aGEA (NCT00824785). DESIGN: EGFR CN by either fluorescence in situ hybridisation (n=114) or digital-droplet PCR in tissues (n=250) and plasma cfDNAs (n=354) was available for 474 (86%) patients in the intention-to-treat (ITT) population. Tissue and plasma low-pass whole-genome sequencing was used to screen for coamplifications in receptor tyrosine kinases. Interaction between chemotherapy and EGFR inhibitors was modelled in patient-derived organoids (PDOs) from aGEA patients. RESULTS: EGFR amplification in cfDNA correlated with poor survival in the ITT population and similar trends were observed when the analysis was conducted in tissue and plasma by treatment arm. EGFR inhibition in combination with chemotherapy did not correlate with improved survival, even in patients with significant EGFR CN gains. Addition of anti-EGFR inhibitors to the chemotherapy agent epirubicin in PDOs, resulted in a paradoxical increase in viability and accelerated progression through the cell cycle, associated with p21 and cyclin B1 downregulation and cyclin E1 upregulation, selectively in organoids from EGFR-amplified aGEA. CONCLUSION: EGFR CN can be accurately measured in tissue and liquid biopsies and may be used for the selection of aGEA patients. EGFR inhibitors may antagonise the antitumour effect of anthracyclines with important implications for the design of future combinatorial trials.

A systematic molecular and pharmacologic evaluation of AKT inhibitors reveals new insight into their biological activity.

BACKGROUND: AKT, a critical effector of the phosphoinositide 3-kinase (PI3K) signalling cascade, is an intensely pursued therapeutic target in oncology. Two distinct classes of AKT inhibitors have been in clinical development, ATP-competitive and allosteric. Class-specific differences in drug activity are likely the result of differential structural and conformational requirements governing efficient target binding, which ultimately determine isoform-specific potency, selectivity profiles and activity against clinically relevant AKT mutant variants. METHODS: We have carried out a systematic evaluation of clinical AKT inhibitors using in vitro pharmacology, molecular profiling and biochemical assays together with structural modelling to better understand the context of drug-specific and drug-class-specific cell-killing activity. RESULTS: Our data demonstrate clear differences between ATP-competitive and allosteric AKT inhibitors, including differential effects on non-catalytic activity as measured by a novel functional readout. Surprisingly, we found that some mutations can cause drug resistance in an isoform-selective manner despite high structural conservation across AKT isoforms. Finally, we have derived drug-class-specific phosphoproteomic signatures and used them to identify effective drug combinations. CONCLUSIONS: These findings illustrate the utility of individual AKT inhibitors, both as drugs and as chemical probes, and the benefit of AKT inhibitor pharmacological diversity in providing a repertoire of context-specific therapeutic options.

Inhibitors in AKTion: ATP-competitive vs allosteric.

Aberrant activation of the PI3K pathway is one of the commonest oncogenic events in human cancer. AKT is a key mediator of PI3K oncogenic function, and thus has been intensely pursued as a therapeutic target. Multiple AKT inhibitors, broadly classified as either ATP-competitive or allosteric, are currently in various stages of clinical development. Herein, we review the evidence for AKT dependence in human tumours and focus on its therapeutic targeting by the two drug classes. We highlight the future prospects for the development and implementation of more effective context-specific AKT inhibitors aided by our increasing knowledge of both its regulation and some previously unrecognised non-canonical functions.

Mathematical modeling identifies optimum lapatinib dosing schedules for the treatment of glioblastoma patients.

Human primary glioblastomas (GBM) often harbor mutations within the epidermal growth factor receptor (EGFR). Treatment of EGFR-mutant GBM cell lines with the EGFR/HER2 tyrosine kinase inhibitor lapatinib can effectively induce cell death in these models. However, EGFR inhibitors have shown little efficacy in the clinic, partly because of inappropriate dosing. Here, we developed a computational approach to model the in vitro cellular dynamics of the EGFR-mutant cell line SF268 in response to different lapatinib concentrations and dosing schedules. We then used this approach to identify an effective treatment strategy within the clinical toxicity limits of lapatinib, and developed a partial differential equation modeling approach to study the in vivo GBM treatment response by taking into account the heterogeneous and diffusive nature of the disease. Despite the inability of lapatinib to induce tumor regressions with a continuous daily schedule, our modeling approach consistently predicts that continuous dosing remains the best clinically feasible strategy for slowing down tumor growth and lowering overall tumor burden, compared to pulsatile schedules currently known to be tolerated, even when considering drug resistance, reduced lapatinib tumor concentrations due to the blood brain barrier, and the phenotypic switch from proliferative to migratory cell phenotypes that occurs in hypoxic microenvironments. Our mathematical modeling and statistical analysis platform provides a rational method for comparing treatment schedules in search for optimal dosing strategies for glioblastoma and other cancer types.

IQGAP1 controls tight junction formation through differential regulation of claudin recruitment.

IQGAP1 is a scaffolding protein previously implicated in adherens junction formation. However, its role in the establishment or maintenance of tight junctions (TJs) has not been explored. We hypothesized that IQGAP1 could regulate TJ formation by modulating the expression and/or localization of junctional proteins, and we systematically tested this hypothesis in the model Madin-Darby canine kidney (MDCK) cell line. We find that IQGAP1 silencing enhances a transient increase in transepithelial electrical resistance (TER) observed during the early stages of TJ formation (Cereijido et al., 1978). Quantitative microscopy and biochemical experiments suggest that this effect of IQGAP1 on TJ assembly is accounted for by reduced expression and TJ recruitment of claudin 2, and increased TJ recruitment of claudin 4. Furthermore, we show that IQGAP1 also regulates TJ formation through its interactor CDC42, because IQGAP1 knockdown increases the activity of the CDC42 effector JNK and dominant-negative CDC42 prevents the increase in TER caused by IQGAP1 silencing. Hence, we provide evidence that IQGAP1 modulates TJ formation by a twofold mechanism: (1) controlling the expression and recruitment of claudin 2 and recruitment of claudin 4 to the TJ, and (2) transient inhibition of the CDC42-JNK pathway.

Identification of the JNK signaling pathway as a functional target of the tumor suppressor PTEN.

Although most oncogenic phenotypes of PTEN loss are attributed to AKT activation, AKT alone is not sufficient to induce all of the biological activities associated with PTEN inactivation. We searched for additional PTEN-regulated pathways through gene set enrichment analysis (GSEA) and identified genes associated with JNK activation. PTEN null cells exhibit higher JNK activity, and genetic studies demonstrate that JNK functions parallel to and independently of AKT. Furthermore, PTEN deficiency sensitizes cells to JNK inhibition and negative feedback regulation of PI3K was impaired in PTEN null cells. Akt and JNK activation are highly correlated in human prostate cancer. These findings implicate JNK in PI3K-driven cancers and demonstrate the utility of GSEA to identify functional pathways using genetically defined systems.

Glucose deprivation activates a metabolic and signaling amplification loop leading to cell death.

The altered metabolism of cancer can render cells dependent on the availability of metabolic substrates for viability. Investigating the signaling mechanisms underlying cell death in cells dependent upon glucose for survival, we demonstrate that glucose withdrawal rapidly induces supra-physiological levels of phospho-tyrosine signaling, even in cells expressing constitutively active tyrosine kinases. Using unbiased mass spectrometry-based phospho-proteomics, we show that glucose withdrawal initiates a unique signature of phospho-tyrosine activation that is associated with focal adhesions. Building upon this observation, we demonstrate that glucose withdrawal activates a positive feedback loop involving generation of reactive oxygen species (ROS) by NADPH oxidase and mitochondria, inhibition of protein tyrosine phosphatases by oxidation, and increased tyrosine kinase signaling. In cells dependent on glucose for survival, glucose withdrawal-induced ROS generation and tyrosine kinase signaling synergize to amplify ROS levels, ultimately resulting in ROS-mediated cell death. Taken together, these findings illustrate the systems-level cross-talk between metabolism and signaling in the maintenance of cancer cell homeostasis.

The phosphatase and tensin homolog regulates epidermal growth factor receptor (EGFR) inhibitor response by targeting EGFR for degradation.

The phosphatase and tensin homolog (PTEN) is a tumor suppressor that is inactivated in many human cancers. PTEN loss has been associated with resistance to inhibitors of the epidermal growth factor receptor (EGFR), but the molecular basis of this resistance is unclear. It is believed that unopposed phosphatidylinositol-3-kinase (PI3K) activation through multiple receptor tyrosine kinases (RTKs) can relieve PTEN-deficient cancers from their "dependence" on EGFR or any other single RTK for survival. Here we report a distinct resistance mechanism whereby PTEN inactivation specifically raises EGFR activity by impairing the ligand-induced ubiquitylation and degradation of the activated receptor through destabilization of newly formed ubiquitin ligase Cbl complexes. PTEN-associated resistance to EGFR kinase inhibitors is phenocopied by expression of dominant negative Cbl and can be overcome by more complete EGFR kinase inhibition. PTEN inactivation does not confer resistance to inhibitors of the MET or PDGFRA kinase. Our study identifies a critical role for PTEN in EGFR signal termination and suggests that more potent EGFR inhibition should overcome resistance caused by PI3K pathway activation.

HER2/neu kinase-dependent modulation of androgen receptor function through effects on DNA binding and stability.

Given the role of the EGFR/HER2 family of tyrosine kinases in breast cancer, we dissected the molecular basis of EGFR/HER2 kinase signaling in prostate cancer. Using the small molecule dual EGFR/HER2 inhibitor PKI-166, we show that the biologic effects of EGFR/HER-2 pathway inhibition are caused by reduced AR transcriptional activity. Additional genetic and pharmacologic experiments show that this modulation of AR function is mediated by the HER2/ERBB3 pathway, not by EGFR. This HER2/ERBB3 signal stabilizes AR protein levels and optimizes binding of AR to promoter/enhancer regions of androgen-regulated genes. Surprisingly, the downstream signaling pathway responsible for these effects appears to involve kinases other than Akt. These data suggest that the HER2/ERBB3 pathway is a critical target in hormone-refractory prostate cancer.

The tyrosine phosphatase PTPRD is a tumor suppressor that is frequently inactivated and mutated in glioblastoma and other human cancers.

Tyrosine phosphorylation plays a critical role in regulating cellular function and is a central feature in signaling cascades involved in oncogenesis. The regulation of tyrosine phosphorylation is coordinately controlled by kinases and phosphatases (PTPs). Whereas activation of tyrosine kinases has been shown to play vital roles in tumor development, the role of PTPs is much less well defined. Here, we show that the receptor protein tyrosine phosphatase delta (PTPRD) is frequently inactivated in glioblastoma multiforme (GBM), a deadly primary neoplasm of the brain. PTPRD is a target of deletion in GBM, often via focal intragenic loss. In GBM tumors that do not possess deletions in PTPRD, the gene is frequently subject to cancer-specific epigenetic silencing via promoter CpG island hypermethylation (37%). Sequencing of the PTPRD gene in GBM and other primary human tumors revealed that the gene is mutated in 6% of GBMs, 13% of head and neck squamous cell carcinomas, and in 9% of lung cancers. These mutations were deleterious. In total, PTPRD inactivation occurs in >50% of GBM tumors, and loss of expression predicts for poor prognosis in glioma patients. Wild-type PTPRD inhibits the growth of GBM and other tumor cells, an effect not observed with PTPRD alleles harboring cancer-specific mutations. Human astrocytes lacking PTPRD exhibited increased growth. PTPRD was found to dephosphorylate the oncoprotein STAT3. These results implicate PTPRD as a tumor suppressor on chromosome 9p that is involved in the development of GBMs and multiple human cancers.

Targeting un-MET needs in advanced non-small cell lung cancer.

Lung cancer classification has been radically transformed in recent years as genomic profiling has identified multiple novel therapeutic targets including MET exon 14 (METex14) alterations and MET amplification. Utilizing targeted therapies in patients with molecularly-defined NSCLC leads to remarkable objective response rates and improved progression-free survival. However, acquired resistance is inevitable. Several recent phase II trials have confirmed that METex14 NSCLC can be treated effectively with MET kinase inhibitors, such as crizotinib, capmatinib, tepotinib, and savolitinib. However, response rates for many MET TKIs are modest relative to the activity of targeted therapy in other oncogene-driven lung cancers, where ORRs are more consistently greater than 60%. In spite of significant gains in the field of MET inhibition in NSCLC, challenges remain: the landscape of resistance mechanisms to MET TKIs is not yet well characterized, and there may be intrinsic and acquired resistance mechanisms that require further characterization to enable increased MET TKI activity. In this review, we overview MET pathway dysregulation in lung cancer, methods of detection in the clinic, recent clinical trial data, and discuss current mechanisms of TKI resistance, exploring emerging strategies to overcome resistance.

DUSP4 protects BRAF- and NRAS-mutant melanoma from oncogene overdose through modulation of MITF.

MAPK inhibitors (MAPKi) remain an important component of the standard of care for metastatic melanoma. However, acquired resistance to these drugs limits their therapeutic benefit. Tumor cells can become refractory to MAPKi by reactivation of ERK. When this happens, tumors often become sensitive to drug withdrawal. This drug addiction phenotype results from the hyperactivation of the oncogenic pathway, a phenomenon commonly referred to as oncogene overdose. Several feedback mechanisms are involved in regulating ERK signaling. However, the genes that serve as gatekeepers of oncogene overdose in mutant melanoma remain unknown. Here, we demonstrate that depletion of the ERK phosphatase, DUSP4, leads to toxic levels of MAPK activation in both drug-naive and drug-resistant mutant melanoma cells. Importantly, ERK hyperactivation is associated with down-regulation of lineage-defining genes including MITF Our results offer an alternative therapeutic strategy to treat mutant melanoma patients with acquired MAPKi resistance and those unable to tolerate MAPKi.

Clinical Development of AKT Inhibitors and Associated Predictive Biomarkers to Guide Patient Treatment in Cancer Medicine.

The serine/threonine kinase AKT is a critical effector of the phosphoinositide 3-kinase (PI3K) signaling cascade and has a pivotal role in cell growth, proliferation, survival, and metabolism. AKT is one of the most commonly activated pathways in human cancer and dysregulation of AKT-dependent pathways is associated with the development and maintenance of a range of solid tumors. There are multiple small-molecule inhibitors targeting different components of the PI3K/AKT pathway currently at various stages of clinical development, in addition to new combination strategies aiming to boost the therapeutic efficacy of these drugs. Correlative and translational studies have been undertaken in the context of clinical trials investigating AKT inhibitors, however the identification of predictive biomarkers of response and resistance to AKT inhibition remains an unmet need. In this review, we discuss the biological function and activation of AKT, discuss its contribution to tumor development and progression, and review the efficacy and toxicity data from clinical trials, including both AKT inhibitor monotherapy and combination strategies with other agents. We also discuss the promise and challenges associated with the development of AKT inhibitors and associated predictive biomarkers of response and resistance.

Inhibition of the phosphatidylinositol 3-kinase-Akt pathway enhances gamma-2 herpesvirus lytic replication and facilitates reactivation from latency.

Cellular signalling pathways are critical in regulating the balance between latency and lytic replication of herpesviruses. Here, we investigated the effect of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway on replication of two gamma-2 herpesviruses, murine gammaherpesvirus-68 (MHV-68) and human herpesvirus-8/Kaposi's sarcoma-associated herpesvirus (HHV-8/KSHV). We found that de novo infection of MHV-68 induced PI3K-dependent Akt activation and the lytic replication of MHV-68 was enhanced by inhibiting the PI3K-Akt pathway with both chemical inhibitors and RNA interference technology. Inhibiting the activity of Akt using Akt inhibitor VIII also facilitated the reactivation of KSHV from latency. Both lytic replication and latency depend on the activity of viral transactivator RTA and we further show that the activity of RTA is increased by reducing Akt1 expression. The data suggest that the PI3K-Akt pathway suppresses the activity of RTA and thereby contributes to the maintenance of viral latency and promotes tumorigenesis.

Epidermal growth factor receptor inhibitors in oncology.

PURPOSE OF REVIEW: Inhibition of the epidermal growth factor receptor (EGFR) by small molecules or antibodies has been pursued as a paradigm to treat human cancers for over two decades. It is now clear that these agents can induce tumor regressions in a variety of human cancers, proving the critical role of EGFR signals for tumor maintenance in subsets of patients with these cancers. Clinically meaningful responses, however, are only transient and further refinement of EGFR-targeted therapies is urgently needed. RECENT FINDINGS: Several studies provide new insights into the molecular basis of EGFR kinase inhibitor resistance, including co-activation of the MET growth factor receptor, loss of the phosphatase and tensin homolog (PTEN) tumor suppressor, and KRAS mutation. Potential strategies are emerging to overcome acquired EGFR kinase inhibitor resistance associated with the T790 M EGFR mutation, including a new compound identified in a chemical library screen and a combination regimen of an anti-EGFR antibody plus a small molecule EGFR kinase inhibitor. Lastly, pulsatile dosing schedules are being pursued to accomplish more complete target inhibition. SUMMARY: Current data point toward a strong association between EGFR genotype and EGFR kinase inhibitor response, similar to the findings with other oncogenic kinases (BCR-ABL, HER2, KIT, PDGFRA, BRAF). This relationship is less obvious for antibodies targeting EGFR. More complete inhibition of EGFR in tumor cells and more focused clinical drug development remain important goals toward further success with this class of anticancer agents.

Assessing the significance of chromosomal aberrations in cancer: methodology and application to glioma.

Comprehensive knowledge of the genomic alterations that underlie cancer is a critical foundation for diagnostics, prognostics, and targeted therapeutics. Systematic efforts to analyze cancer genomes are underway, but the analysis is hampered by the lack of a statistical framework to distinguish meaningful events from random background aberrations. Here we describe a systematic method, called Genomic Identification of Significant Targets in Cancer (GISTIC), designed for analyzing chromosomal aberrations in cancer. We use it to study chromosomal aberrations in 141 gliomas and compare the results with two prior studies. Traditional methods highlight hundreds of altered regions with little concordance between studies. The new approach reveals a highly concordant picture involving approximately 35 significant events, including 16-18 broad events near chromosome-arm size and 16-21 focal events. Approximately half of these events correspond to known cancer-related genes, only some of which have been previously tied to glioma. We also show that superimposed broad and focal events may have different biological consequences. Specifically, gliomas with broad amplification of chromosome 7 have properties different from those with overlapping focalEGFR amplification: the broad events act in part through effects on MET and its ligand HGF and correlate with MET dependence in vitro. Our results support the feasibility and utility of systematic characterization of the cancer genome.

Orally bioavailable CDK9/2 inhibitor shows mechanism-based therapeutic potential in MYCN-driven neuroblastoma.

The undruggable nature of oncogenic Myc transcription factors poses a therapeutic challenge in neuroblastoma, a pediatric cancer in which MYCN amplification is strongly associated with unfavorable outcome. Here, we show that CYC065 (fadraciclib), a clinical inhibitor of CDK9 and CDK2, selectively targeted MYCN-amplified neuroblastoma via multiple mechanisms. CDK9 - a component of the transcription elongation complex P-TEFb - bound to the MYCN-amplicon superenhancer, and its inhibition resulted in selective loss of nascent MYCN transcription. MYCN loss led to growth arrest, sensitizing cells for apoptosis following CDK2 inhibition. In MYCN-amplified neuroblastoma, MYCN invaded active enhancers, driving a transcriptionally encoded adrenergic gene expression program that was selectively reversed by CYC065. MYCN overexpression in mesenchymal neuroblastoma was sufficient to induce adrenergic identity and sensitize cells to CYC065. CYC065, used together with temozolomide, a reference therapy for relapsed neuroblastoma, caused long-term suppression of neuroblastoma growth in vivo, highlighting the clinical potential of CDK9/2 inhibition in the treatment of MYCN-amplified neuroblastoma.

Murine cell lines derived from Pten null prostate cancer show the critical role of PTEN in hormone refractory prostate cancer development.

PTEN mutations are among the most frequent genetic alterations found in human prostate cancers. Our previous works suggest that although precancerous lesions were found in Pten heterozygous mice, cancer progression and metastasis only happened when both alleles of Pten were deleted. To understand the molecular mechanisms underlying the role of PTEN in prostate cancer control, we generated two pairs of isogenic, androgen receptor (AR)-positive prostate epithelial lines from intact conditional Pten knock-out mice that are either heterozygous (PTEN-P2 and -P8) or homozygous (PTEN-CaP2 and PTEN-CaP8) for Pten deletion. Further characterization of these cells showed that loss of the second allele of Pten leads to increased anchorage-independent growth in vitro and tumorigenesis in vivo without obvious structural or numerical chromosome changes based on SKY karyotyping analysis. Despite no prior exposure to hormone ablation therapy, Pten null cells are tumorigenic in both male and female severe combined immunodeficiency mice. Furthermore, knocking down PTEN can convert the androgen-dependent Myc-CaP cell into androgen independence, suggesting that PTEN intrinsically controls androgen responsiveness, a critical step in the development of hormone refractory prostate cancer. Importantly, knocking down AR by shRNA in Pten null cells reverses androgen-independent growth in vitro and partially inhibited tumorigenesis in vivo, indicating that PTEN-controlled prostate tumorigenesis is AR dependent. These cell lines will serve as useful tools for understanding signaling pathways controlled by PTEN and elucidating the molecular mechanisms involved in hormone refractory prostate cancer formation.

Molecular determinants of the response of glioblastomas to EGFR kinase inhibitors.

BACKGROUND: The epidermal growth factor receptor (EGFR) is frequently amplified, overexpressed, or mutated in glioblastomas, but only 10 to 20 percent of patients have a response to EGFR kinase inhibitors. The mechanism of responsiveness of glioblastomas to these inhibitors is unknown. METHODS: We sequenced kinase domains in the EGFR and human EGFR type 2 (Her2/neu) genes and analyzed the expression of EGFR, EGFR deletion mutant variant III (EGFRvIII), and the tumor-suppressor protein PTEN in recurrent malignant gliomas from patients who had received EGFR kinase inhibitors. We determined the molecular correlates of clinical response, validated them in an independent data set, and identified effects of the molecular abnormalities in vitro. RESULTS: Of 49 patients with recurrent malignant glioma who were treated with EGFR kinase inhibitors, 9 had tumor shrinkage of at least 25 percent. Pretreatment tissue was available for molecular analysis from 26 patients, 7 of whom had had a response and 19 of whom had rapid progression during therapy. No mutations in EGFR or Her2/neu kinase domains were detected in the tumors. Coexpression of EGFRvIII and PTEN was significantly associated with a clinical response (P<0.001; odds ratio, 51; 95 percent confidence interval, 4 to 669). These findings were validated in 33 patients who received similar treatment for glioblastoma at a different institution (P=0.001; odds ratio, 40; 95 percent confidence interval, 3 to 468). In vitro, coexpression of EGFRvIII and PTEN sensitized glioblastoma cells to erlotinib. CONCLUSIONS: Coexpression of EGFRvIII and PTEN by glioblastoma cells is associated with responsiveness to EGFR kinase inhibitors.