Search

mp3 file (11 MB). In the May edition of the Cancer Discovery podcast, Science Writer Elizabeth McKenna talks with Ingo K. Mellinghoff about his paper, which suggests that the disappointing clinical activity of first-generation EGFR inhibitors in glioblastoma versus lung cancer may be attributed to the different conformational requirements of mutant EGFR in these two cancer types.

PDF file - 689K

PDF file - 104K

PDF file - 133K

Supplementary Table 1, Figures 1-4 from Murine Cell Lines Derived from Pten Null Prostate Cancer Show the Critical Role of PTEN in Hormone Refractory Prostate Cancer Development

The epidermal growth factor receptor (EGFR) is commonly amplified, overexpressed, and mutated in glioblastoma, making it a compelling molecular target for therapy. We have recently shown that coexpression of EGFRvIII and PTEN protein by glioblastoma cells is strongly associated with clinical response to EGFR kinase inhibitor therapy. PTEN loss, by dissociating inhibition of the EGFR from downstream phosphatidylinositol 3-kinase (PI3K) pathway inhibition, seems to act as a resistance factor. Because 40% to 50% of glioblastomas are PTEN deficient, a critical challenge is to identify strategies that promote responsiveness to EGFR kinase inhibitors in patients whose tumors lack PTEN. Here, we show that the mammalian target of rapamycin (mTOR) inhibitor rapamycin enhances the sensitivity of PTEN-deficient tumor cells to the EGFR kinase inhibitor erlotinib. In two isogenic model systems (U87MG glioblastoma cells expressing EGFR, EGFRvIII, and PTEN in relevant combinations, and SF295 glioblastoma cells in which PTEN protein expression has been stably restored), we show that combined EGFR/mTOR kinase inhibition inhibits tumor cell growth and has an additive effect on inhibiting downstream PI3K pathway signaling. We also show that combination therapy provides added benefit in promoting cell death in PTEN-deficient tumor cells. These studies provide strong rationale for combined mTOR/EGFR kinase inhibitor therapy in glioblastoma patients, particularly those with PTEN-deficient tumors. (Cancer Res 2006; 66(16): 7864-9)

Supplementary Table 1 from Mammalian Target of Rapamycin Inhibition Promotes Response to Epidermal Growth Factor Receptor Kinase Inhibitors in PTEN-Deficient and PTEN-Intact Glioblastoma Cells

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. [Cancer Res 2007;67(13):6083–91]

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.

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.

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.

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.

Aims Despite improved understanding of effector T-cell trafficking into the central nervous system, initial trials with anti-PD1/PD-L1 immune checkpoint inhibitors (ICIs) have failed to meet their primary endpoints. PTEN loss of function is frequent in GBM and has been correlated with not only poor overall prognosis, but also impaired antitumour responses, including reduced T cell infiltration into tumour and reduced efficacy of ICIs. Ipatasertib is a novel, potent, selective, small-molecule inhibitor of Akt. We have shown that Ipatasertib efficiently depletes FOXP3+ regulatory T cells from the tumour microenvironment (TME) resulting in increased infiltration of effector T cells in solid tumours (Lopez 2020, AACR). We hypothesize that the use of AKT inhibition in PTEN glioblastomas may deplete the TME of suppressive immune cells, and render malignant brain tumours more responsive to ICIs. We present updated data for the combination of Ipat+ATZ in patients with glioblastoma. Method Patients with relapsed WHO grade IV GBM with stable neurological symptoms ≥5 days prior to enrolment, requiring The Ice-CAP A2 cohort assessed safety, pharmacodynamic, and preliminary clinical activity of Ipat (200mg or 400mg OD) + ATZ (1200mg Q3W) in pts with potentially resectable relapsed WHO Grade IV GBM. Pts had a 14-21-day run-in phase of Ipat then surgical tumour resection. Combination Ipat+ATZ commenced post surgery. Patients who declined surgery or who were deemed high risk for surgery proceeded directly to combination. Patients in the expansion cohort B3 commenced directly on Ipat+ATZ at the RP2D of 400mg Ipat with ATZ. Results 16 evaluable recurrent GBM pts were enrolled across two cohorts. Median age 56 yrs (25-71 yrs). Median ECOG PS 1. Median lines of prior therapy 1 (range 1-4). 10 pts had PTEN loss by IHC (H No DLTs, treatment-related (TR) serious adverse events (AEs), or immune-related AEs were observed. Most common TR AEs were G1 diarrhoea (44%), mucositis (17%), rash (28%). Clinical benefit rate (CR, PR and SD> 6 cycles) at clinical cutoff date (23/02/21) in patients with PTEN aberration was 30% (3/10). A 58-year-old man with PTEN loss had MRI at Cycle 5 showing worsening enhancement suggestive of disease progression. Resection of the lesion showed intense lymphocyte infiltration and pathological CR. He is currently on Cycle 22 with no evidence of disease. Two other patients with PTEN loss with radiological stable disease per RANO criteria remain well on study for >6 cycles. Conclusion Combination Ipat+ATZ appears safe and tolerable in GBM pts, with 400mg Ipatasertib OD + 1200mg ATZ Q3W declared as RP2D. Early efficacy signals were detected with PTEN loss being a promising predictive biomarker for response to combination. An expansion cohort enriched with pts with PTEN loss is ongoing.

Background: Hyperactivation of the PI3K/AKT pathway correlates with impaired antitumour response, including reduced T cell infiltration into tumour and reduced efficacy of immune checkpoint inhibitors (ICIs). PTEN loss of function, often observed in GBM, may contribute to refractoriness of ICIs in this disease. Methods: The Ice-CAP A2 cohort assessed safety, pharmacodynamic, and preliminary clinical activity of Ipa (200mg or 400mg OD) + A (1200mg Q3W) in pts with potentially resectable relapsed WHO Grade IV GBM. Key inclusion criteria were stable neurological symptoms ≥5 days prior to enrolment, steroid requirement 12wks, both on 400mg Ipa. A 58-year-old man with PTEN loss had MRI at Cycle 5 showing worsening enhancement suggestive of disease progression. Resection of the lesion showed intense lymphocyte infiltration and pathological CR. He is currently on Cycle 18 with no evidence of disease. Conclusion: Combination Ipa+A appears safe and tolerable in GBM pts, with 400mg Ipa OD + 1200mg A Q3W declared as RP2D. PTEN loss may be a promising predictive biomarker for response to combination. An expansion cohort enriched with pts with PTEN loss is ongoing. Cytokine and FACS data will be presented at AACR Table 1.Clinical Benefit Rate of glioblastoma patients stratified according to PTEN aberrationsPTEN statusnBest responseClinical Benefit RatePTEN loss on IHC (H12 wks, ongoingb3 PDPTEN aberration on NGS but PTEN protein expression pending11 PDcPTEN loss of heterozygozity on PCR11 PDWild type PTEN on NGS or IHC (H≥30)33 PDLegend: IHC = immunohistochemistry; NGS = next generation sequencing; PCR = polymerase chain reaction;pCR = pathologic complete response; SD = stable disease; PD = progressive diseaseaExceptional responder with PTEN H=5 on IHC and splice site 75_79+2delGACCTGT on NGSb PTENY68*; c PTENQ298* Citation Format: Crescens Tiu, Andrea Biondo, Liam C. Welsh, Timothy L. Jones, Anna Zachariou, Toby Prout, Alison J. Turner, Robert Daly, Igor Vivanco, Christina Yap, Ben Jenkins, Mateus Crespo, Ruth Riisnaes, Suzanne Carreira, Bora Gurel, Nina Tunariu, Anna Minchom, Udai Banerji, Johann S. de Bono, Juanita S. Lopez. Results of the glioblastoma multiforme (GBM) cohort of phase 1 trial Ice-CAP (NCT03673787): Preliminary evidence of antitumour activity of Ipatasertib (Ipa) and Atezolizumab (A) in patients (pts) with PTEN loss [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT120.

In 2016 there were 5250 brain cancer deaths in the UK. Standard treatment is surgical resection followed by chemo-radiotherapy. In most cases of diffuse glioma, complete tumour resection is not feasible. Many chemotherapy drugs have untested penetration through the blood brain barrier, potentially leading to sub-therapeutic concentrations in the tumour. There is need to refine current treatment strategies in relation to the understanding of tumour biology, and rapidly introduce and evaluate novel therapeutic approaches and agents through delivering rigorous clinical trials. The TESSA JOWELL BRAIN MATRIX Study will evaluate the feasibility of delivering precision medicine for brain cancer patients within the NHS. A multicentre, platform feasibility study of 1200 patients with diffuse glioma will build on the 100,000 genome project to develop and evaluate an infrastructure to collect and integrate: 1) real time comprehensive integrated molecular analysis, including whole genome sequencing and epigenetic classification; 2) serial sampling and annotation of tumours; 3) collection of matched clinical data; 4) assessment of patient quality of life; 5) centralised radiological review and response assessment as per RANO criteria. Once developed this will allow rapid introduction of therapeutic trials to specific patient groups. Secondary objectives include: understanding the association between extent of resection and molecular stratification to refine the role of surgery; optimisation and harmonisation of protocols to best collect, manage and store tissue, clinical data, and radiological images in order to provide a resource for researchers, both within and outside of the study. Improve patient recruitment by identifying and removing recruitment barriers and improve the information and consent processes for patients. Promote the development of a national network with expertise in brain cancer. Enrolment of the first patient is expected in late 2019. For further information, please contact the Brain Matrix Trial Office BrainMatrix@trials.bham.ac.uk.

INTRODUCTION The brain tumour community has seen significant progress in the discovery of new therapeutic targets and anticancer drugs. Unfortunately, advances in how to deliver drugs to the brain lag behind. The blood-brain barrier restricts the entry of many small-molecule drugs and nearly all large molecule drugs that have been developed to treat brain disorders. METHODS Following an international CNS drug delivery workshop in 2016, we were awarded funding from Children with Cancer UK to launch the Children’s Brain Tumour Drug Delivery Consortium (CBTDDC; www.cbtddc.org; @cbtddc). RESULTS The CBTDDC launched in 2017 (in Europe and the US) to raise awareness of the challenge of drug delivery in childhood brain tumours, and to initiate and strengthen research collaborations to accelerate the development of drug delivery systems. We ran a Workshop on Drug Delivery to the Brain, attracting 52 delegates from the UK, Belgium, Spain and Portugal. We liaised with UK-based funders over the drug delivery agenda, and with UK policy makers. In the US, we jointly organised the SIGN2019 meeting and we are currently liaising with the leads of Project ‘All In’ DIPG about how we can lend our support to this project. As of June 2019, 150 individuals have registered with the consortium, representing researchers, clinicians, charities, patient groups and industry. These stakeholders represent 70 research institutions, covering 15 countries (France, UK, Italy, Sweden, The Netherlands, USA, Greece, Germany, Belgium, Cuba, Denmark, Spain, Portugal, Israel and Egypt). We host a freely accessible online collaborative research database, containing the details of over 70 researchers. CONCLUSION We believe that collaboration between clinicians and multi-disciplinary researchers is vital to solving the brain tumour drug delivery challenge. We hope to raise awareness of the CBTDDC, and to extend our invitation for collaborators to join the consortium, through SCIDOT’s unrivalled drug delivery platform.

3118 Background: EB1, a protein located on the plus-ends of microtubules is involved in microtubule function and has been associated with glioblastoma (GBM) stem-cell-ness and more aggressive disease. Lisavanbulin (BAL101553) is a prodrug of the lipophilic small molecule BAL27862, that promotes tumor cell death by modulating the spindle assembly checkpoint and has been shown in rodents to efficiently penetrate the brain. Data from GBM mouse models and recent phase 1 clinical data (Lopez et al. ESMO 2020) suggest that EB1 is a response-predictive marker for lisavanbulin in GBM. A phase 2 study is ongoing to confirm this hypothesis (NCT02490800). A proof-of-concept in GBM would support an expansion of EB1-directed lisavanbulin clinical development in non-GBM tumors, which requires prevalence estimates of EB1-positivity in non-GBM tumor types. Methods: Tissue samples from GBM and other tumor types were stained for EB1 using a CE-marked immunohistochemistry Clinical Trial Assay (Targos Molecular Pathology GmbH, Kassel Germany). EB1-positivity was assessed by a board-certified pathologist based on the percentage of tumor cells showing moderate or strong staining for EB1, using thresholds of ≥50%, ≥60% and ≥70% of tumor cells with EB1 positivity. Whole transcriptome sequencing (WTS) using RNAseq was performed in a subset of tissue samples to develop a potential RNA-based predictive response signature for lisavanbulin. Results: 73 GBM tissue samples and 333 tissue samples from 13 other cancer types were stained for EB1. The strongest overall signal for EB1-positivity was obtained for medulloblastoma, neuroblastoma and GBM. In addition, moderate or strong EB1-staining in ≥50% of tumor cells was observed in samples from colorectal cancer (CRC), non small-cell lung cancer (NSCLC), metastatic melanoma, small-cell lung cancer (SCLC) and triple-negative breast cancer (TNBC). An expanded staining campaign is ongoing in these cancer types. Initial results from the ongoing WTS analyses show marked differences in gene expression profiles between EB1-positive and -negative cases. Conclusions: Strong EB1-positivity is infrequent but occurs in a variety of tumor types, with the strongest signals in medulloblastoma, neuroblastoma and GBM. A phase 2 study is ongoing to assess prospectively whether EB1 is a response-predictive biomarker for lisavanbulin in GBM.[Table: see text]

Background: Hyperactivation of the PI3K/AKT pathway correlates with impaired anti-tumor responses, including reduced T cell infiltration into tumor, and reduced efficacy of immune checkpoint inhibitors. Blockade of this pathway synergizes with PD-L1/PD-1 axis blockade preclinically. Methods: This Phase I clinical trial (NCT03673787) assessed the safety, pharmacodynamic, and preliminary clinical activity of Ipa (200mg or 400mg OD) given in combination with A 1200mg q3 wk in refractory pts. Serial paired blood and tumor samples were analysed to interrogate the effect of Ipa on the tumor micro-environment and host immune system prior to the addition of the immune check point inhibitor, A. Results: 18 adult pts were treated in dose escalation. Median age 49 yrs. All pts had ECOG PS 0-1 and median 7 prior therapies. Most common TRAEs (>15%) were mild Gr1-2 diarrhea (56%), rash (50%), fatigue (33%), nausea (33%), raised ALT/AST (33%), headache (28%) and arthralgia (22%). 1 pt had G2 systemic immune activation; 2 pts had G3 rash, both rapidly reversible. 1 DLT of G3 raised ALT seen at 200mg (1 DLT/9 evaluable pts) but none at 400mg (0 DLT/6). Of 14 RECIST evaluable patients, there were 2 confirmed PRs, and 5 SD (clinical benefit rate 50%). Reductions of CD4+FOXP3+ Tregs in tumor microenvironment were seen after 2wks of single agent Ipa, regardless of PIK3/AKT somatic mutation status (Table 1). Responding pts had a >400% median increase in intra-tumoral CD8+ Teff cell infiltration, effectively switching from a desert phenotype to an inflamed phenotype. Paired changes in FACS, transcriptome and cytokine will also be presented.Conclusions: The RP2D of Ipa 400mg OD combination with A was well tolerated with early efficacy signals. Further biomarker work is ongoing and will be evaluated in expansion cohorts. Table 1:Changes in immune cell populations as assessed by multicolour Immunofluorescence in paired biopsies of breast/gynae patients, % change in cell number/mm2 from baseline (median [min,max$])&Post 2 weeks single agent Ipatasertib(n=9)Post 1 cycle of combination Ipatasertib and Atezolizumab(n=7)CD4+FOXP3+Tregs cellsCD 8+ Teff cellsCD4+FOXP3+Tregs cellsCD 8+ Teff cellsIntra-tumourstromaIntra-tumourstromaIntra-tumourstromaIntra-tumourstromaAll patients-23.9*[-89.7, BL0]-30.0*[-91.6, BL0]-37.7*[-84.4, -24.5]-28.4[-92.4, 259.8]335.9[-44.0,BL0]45.4[-51.0, BL0]59.6[-60.6,493.3]64.7[-51.7,293.3]Stratified by somatic PI3K/AKT/PTEN mutational statusPathogenic mutations (mt)11.1[-82.2, BL0]#-10.7[-91.6, BL0]Φnsnsnsns-30.5[-60.6,-0.5]11.3[-51.7,50.0]Wildtype (wt)-63.1[-89.7,19.0]#-47.5[-77.0,11.1]Φnsnsnsns426.5[59.6,493.3]126.7[79.4,293.3]Stratified by responseResponders (PR + SD>4 cycles). 1 ER+ HER2+ breast cancer (wt), 1 ER+ HER2- breast cancer (wt)459.9[426.5,493.3]@103.1[79.4,126.7]Non-responders (PD at 4 cycles) 1 cervical cancer, 4 ER+ breast cancer-0.5[-60.6, 59.6]@30.6[-51.7,293.3]*significant change (p≤0.05; Wilcoxon sign-rank test) from baseline, $maximum values denoted by BL0indicate that the baseline value was zero, and so percentage change from baseline is not defined. For the analysis, the baseline value has been replaced by a nominal value of 0.1 so that a large percentage increase is associated with these cases. Note that these large percentage increases do not affect the non-parametric statistical tests used.#no significant difference in distribution of reduction in intra-tumoural CD4+ FOXP3+Tregsbetween pts with pathogenic mutations in PI3K/AKT and those without (p=0.30; Wilcoxon rank-sum test)Φno significant difference in distribution of reduction in stromal CD4+FOXP3+Tregsbetween pts with pathogenic mutations in PI3K/AKT and those without (p=0.44; Wilcoxon rank-sum test) @ difference between responders and non-responders p=0.083; Wilcoxon rank-sum test)mt pathogenic mutations in PI3K/AKT and PTEN as per COSMIC database present in tumour or PTEN loss by IHC. wt no pathogenic mutations in PI3K/AKT and PTEN as per COSMIC database detected in tumour and intact PTEN expression by IHC. &exploratory analyses with no adjustment for multiple testing Citation Format: Juanita S. Lopez, Andrea Biondo, Crescens Tiu, Mariana Scaranti, Malaka Ameratunga, Anna Zachariou, Alison Turner, Nina Tunariu, Toby Prout, Mona Parmar, Hannah Badham, Karen Swales, Wei Yuan, Ricardo Morilla, Mateus Crespo, Rob Daly, Ines Figueiredo, Bora Gurel, Rita Pereira, Ruth Riisnaes, Igor Vivanco, Anna Minchom, Ben Jenkins, Christina Yap, Udai Banerji, Johann De Bono. Proof-of-concept evidence of immune modulation by blockade of the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway in the phase I dose escalation study of Ipatasertib (Ipa) in combination with atezolizumab (A) in patients (pts) with advanced solid tumors (Ice-CAP) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT140.

Transport of macromolecules through the nuclear pore by importins and exportins plays a critical role in the spatial regulation of protein activity. How cancer cells co-opt this process to promote tumorigenesis remains unclear. The epidermal growth factor receptor (EGFR) plays a critical role in normal development and in human cancer. Here we describe a mechanism of EGFR regulation through the importin β family member RAN-binding protein 6 (RanBP6), a protein of hitherto unknown functions. We show that RanBP6 silencing impairs nuclear translocation of signal transducer and activator of transcription 3 (STAT3), reduces STAT3 binding to the EGFR promoter, results in transcriptional derepression of EGFR, and increased EGFR pathway output. Focal deletions of the RanBP6 locus on chromosome 9p were found in a subset of glioblastoma (GBM) and silencing of RanBP6 promoted glioma growth in vivo. Our results provide an example of EGFR deregulation in cancer through silencing of components of the nuclear import pathway., The epidermal growth factor receptor (EGFR) signalling is regulated at multiple levels. Here the authors show that the importin RanBP6 acts as a tumor suppressor in Glioblastoma and regulates EGFR signalling through promoting translocation of STAT3 to the nuclei and repressing EGFR transcription.

Cancer cells depend on a finite number of critical signals for their survival. Oncogene addiction, that is, the acquired dependence of a cancer cell on the activity of a single oncogenic gene product, has been the basis for the targeted therapy paradigm, and operationally defines such signals. Additionally, cancer cells have altered metabolic requirements that create addictions to specific nutrients such as glucose and glutamine. In this review, I will discuss the therapeutic opportunities that these two types of molecular addictions offer, focusing on lessons learned from targeting members of the epidermal growth factor receptor family of kinases, and components of MAPK pathway. I will also discuss the challenges in simultaneously harnessing two types of molecular addictions for therapeutic benefit, and the importance of understanding not only the effects of oncogenic signal transduction on metabolism, but also the impact of metabolic states on signal transduction.

Activation of the epidermal growth factor receptor (EGFR) in glioblastoma (GBM) occurs through mutations or deletions in the extracellular (EC) domain. Unlike lung cancers with EGFR kinase domain (KD) mutations, GBMs respond poorly to the EGFR inhibitor erlotinib. Using RNAi, we show that GBM cells carrying EGFR EC mutations display EGFR addiction. In contrast to KD mutants found in lung cancer, glioma-specific EGFR EC mutants are poorly inhibited by EGFR inhibitors that target the active kinase conformation (e.g., erlotinib). Inhibitors that bind to the inactive EGFR conformation, however, potently inhibit EGFR EC mutants and induce cell death in EGFR-mutant GBM cells. Our results provide first evidence for single kinase addiction in GBM and suggest that the disappointing clinical activity of first-generation EGFR inhibitors in GBM versus lung cancer may be attributed to the different conformational requirements of mutant EGFR in these 2 cancer types. Significance: Approximately 40% of human glioblastomas harbor oncogenic EGFR alterations, but attempts to therapeutically target EGFR with first-generation EGFR kinase inhibitors have failed. Here, we demonstrate selective sensitivity of glioma-specific EGFR mutants to ATP-site competitive EGFR kinase inhibitors that target the inactive conformation of the catalytic domain. Cancer Discov; 2(5); 458–71. ©2012 AACR. Read the Commentary on this article by Park and Lemmon, p. 398. This article is highlighted in the In This Issue feature, p. 377.

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.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.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.

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., Author summary In vivo inhibition of tumor expansion requires a sufficient amount of therapeutic agent to be present in the tumor tissue. A number of factors affect drug concentrations including the maximum tolerated dose, pharmacokinetics and pharmacodynamics profiles. We present a computational modeling platform incorporating both in vitro data and published clinical trial data to investigate the efficacy of lapatinib as a function of different dosing schedules for inhibiting glioblastoma tumor cell growth. The goal of our method is to find the best dosing schedule balancing both toxicity and efficacy. Our modeling approach identifies continuous dosing as the best clinically feasible strategy for slowing down tumor growth even when taking into consideration intratumor heterogeneity, drug resistance and reduced lapatinib concentrations in the tumor due to the blood brain barrier.

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.

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 ≈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 focal EGFR 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.

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.

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.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.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 (P0.001; 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.Coexpression of EGFRvIII and PTEN by glioblastoma cells is associated with responsiveness to EGFR kinase inhibitors.

One signal that is overactivated in a wide range of tumour types is the production of a phospholipid, phosphatidylinositol (3,4,5) trisphosphate, by phosphatidylinositol 3-kinase (PI3K). This lipid and the protein kinase that is activated by it — AKT — trigger a cascade of responses, from cell growth and proliferation to survival and motility, that drive tumour progression. Small-molecule therapeutics that block PI3K signalling might deal a severe blow to cancer cells by blocking many aspects of the tumour-cell phenotype.

Amplification and overexpression of the epidermal growth factor receptor (EGFR) are common in glioblastoma (GBM) and frequently associated with silencing of the phosphatase and tensin homologue (PTEN) tumor suppressor. PTEN silencing has been associated with clinical resistance to EGFR tyrosine kinase inhibitors, in part by raising EGFR levels. Here, we investigated the effect of PTEN on the EGFR signaling complex by EGFR affinity immunopurification and mass spectrometry with and without PTEN knockdown. We identified Ran binding protein 6 (RanBP6), a 125-kDa protein of previously unknown functions, as EGFR interacting protein in PTEN expressing, but not PTEN knockdown cells. Further studies of the effect of RanBP6 on EGFR revealed that RanBP6 depletion by shRNA or CRISPR/Cas9-mediated gene silencing resulted in increased EGFR mRNA levels and upregulation of EGFR promoter activity. Consistent with a model of a negative EGFR regulation by RanBP6, we observed an inverse correlation between RanBP6 and EGFR mRNA levels in PTEN wildtype but not PTEN altered cancer cells in a large panel of human cancer cell lines (Cancer Cell Line Encyclopedia). To further understand the mechanism of how RanBP6 negatively regulates EGFR mRNA level, we found that RanBP6 interacted with nuclear Ran-GTPase and repressed EGFR transcription by promoting nuclear import of Signal transducer and activator of transcription 3 (STAT3). Lastly, RanBP6 appeared to be frequently deleted on chromosome 9p in GBM. We showed that RanBP6 silencing raised EGFR levels and signal output and accelerated in-vivo glioma growth. Our results establish a novel function of RanBP6 as a link between EGFR signaling and the Ran-mediated nuclear import pathway, and identify RanBP6 as candidate tumor suppressor on chromosome 9p. Citation Format: Wan-Ying Hsieh, Barbara Oldrini, Hediye Erdjument-Bromage, Paolo Codega, Maria S. Carro, Igor Vivanco, Dan Rohle, Carl Campos, Craig Bielski, Barry Taylor, Paul Tempst, Massimo Squatrito, Ingo K. Mellinghoff. Identification of Ran binding protein 6 as a novel negative regulator of EGFR and candidate tumor suppressor in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1032. doi:10.1158/1538-7445.AM2017-1032

The serine–threonine kinase AKT regulates proliferation and survival by phosphorylating a network of protein substrates. In this study, we describe a kinase-independent function of AKT. In cancer cells harboring gain-of-function alterations in MET, HER2, or Phosphatidyl-Inositol-3-Kinase (PI3K), catalytically inactive AKT (K179M) protected from drug induced cell death in a PH-domain dependent manner. An AKT kinase domain mutant found in human melanoma (G161V) lacked enzymatic activity in vitro and in AKT1/AKT2 double knockout cells, but promoted growth factor independent survival of primary human melanocytes. ATP-competitive AKT inhibitors failed to block the kinase-independent function of AKT, a liability that limits their effectiveness compared to allosteric AKT inhibitors. Our results broaden the current view of AKT function and have important implications for the development of AKT inhibitors for cancer. National Institutes of Health (1R01NS080944–01, U54CA143798, F32 GM096558), the James S McDonnell Foundation, and National Brain Tumor Society Defeat GBM initiative

In search of novel genes expressed in metastatic prostate cancer, we subtracted cDNA isolated from benign prostatic hypertrophic tissue from cDNA isolated from a prostate cancer xenograft model that mimics advanced disease. One novel gene that is highly expressed in advanced prostate cancer encodes a 339-amino acid protein with six potential membrane-spanning regions flanked by hydrophilic amino- and carboxyl-terminal domains. This structure suggests a potential function as a channel or transporter protein. This gene, named STEAP for s ix- t ransmembrane e pithelial a ntigen of the p rostate, is expressed predominantly in human prostate tissue and is up-regulated in multiple cancer cell lines, including prostate, bladder, colon, ovarian, and Ewing sarcoma. Immunohistochemical analysis of clinical specimens demonstrates significant STEAP expression at the cell–cell junctions of the secretory epithelium of prostate and prostate cancer cells. Little to no staining was detected at the plasma membranes of normal, nonprostate human tissues, except for bladder tissue, which expressed low levels of STEAP at the cell membrane. Protein analysis located STEAP at the cell surface of prostate-cancer cell lines. Our results support STEAP as a cell-surface tumor-antigen target for prostate cancer therapy and diagnostic imaging.

In cancer cells dependent upon glucose for survival, glucose withdrawal activates a positive feedback loop involving reactive oxygen species (ROS), ROS-mediated inhibition of tyrosine phosphatases, and tyrosine kinase signaling. This loop amplifies ROS to toxic levels, resulting in cell death., In cancer cell lines dependent on glucose for survival, glucose withdrawal induces supra-physiological levels of phospho-tyrosine signaling, even in cells expressing constitutively active tyrosine kinases. Unbiased, mass spectrometry-based phospho-tyrosine profiling demonstrates that glucose withdrawal induces a unique signature of phospho-tyrosine signaling associated with focal adhesions. The glucose withdrawal-induced phospho-tyrosine signature results from a positive feedback loop in which reactive oxygen species (ROS) oxidize and inhibit protein tyrosine phosphatases, causing increased tyrosine kinase signaling, thereby inducing further ROS generation until cells undergo ROS-mediated cell death. The glucose withdrawal-initiated positive feedback loop illustrates the complex, systems-level integration of metabolism and tyrosine kinase signaling in cancer cell homeostasis., 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.

Mutation of the gene PARK2, which encodes an E3 ubiquitin ligase, is the most common cause of early-onset Parkinson's disease1, 2, 3. In a search for multisite tumor suppressors, we identified PARK2 as a frequently targeted gene on chromosome 6q25.2–q27 in cancer. Here we describe inactivating somatic mutations and frequent intragenic deletions of PARK2 in human malignancies. The PARK2 mutations in cancer occur in the same domains, and sometimes at the same residues, as the germline mutations causing familial Parkinson's disease. Cancer-specific mutations abrogate the growth-suppressive effects of the PARK2 protein. PARK2 mutations in cancer decrease PARK2's E3 ligase activity, compromising its ability to ubiquitinate cyclin E and resulting in mitotic instability. These data strongly point to PARK2 as a tumor suppressor on 6q25.2–q27. Thus, PARK2, a gene that causes neuronal dysfunction when mutated in the germline, may instead contribute to oncogenesis when altered in non-neuronal somatic cells.

EGFR is a major anticancer drug target in human epithelial tumors. One effective class of agents is the tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib. These drugs induce dramatic responses in individuals with lung adenocarcinomas characterized by mutations in exons encoding the EGFR tyrosine kinase domain, but disease progression invariably occurs. A major reason for such acquired resistance is the outgrowth of tumor cells with additional TKI-resistant EGFR mutations. Here we used relevant transgenic mouse lung tumor models to evaluate strategies to overcome the most common EGFR TKI resistance mutation, T790M. We treated mice bearing tumors harboring EGFR mutations with a variety of anticancer agents, including a new irreversible EGFR TKI that is under development (BIBW-2992) and the EGFR-specific antibody cetuximab. Surprisingly, we found that only the combination of both agents together induced dramatic shrinkage of erlotinib-resistant tumors harboring the T790M mutation, because together they efficiently depleted both phosphorylated and total EGFR. We suggest that these studies have immediate therapeutic implications for lung cancer patients, as dual targeting with cetuximab and a second-generation EGFR TKI may be an effective strategy to overcome T790M-mediated drug resistance. Moreover, this approach could serve as an important model for targeting other receptor tyrosine kinases activated in human cancers.

DNA damage checkpoints are critical for preventing tumorigenesis and regulating the response of cells to genotoxic agents. It is believed that the coordinated actions of a number of effectors underlie proper checkpoint function. The kinase Chk2, p21 and 14-3-3sigma have each been shown to be independent effectors of the G(2) DNA damage checkpoint. However, the relative roles of these proteins remain unclear. To help elucidate this question, we have perturbed each of these 3 genes in combination in human cells. We show that Chk2 depletion causes markedly increased sensitivity to DNA damage in p21(-/-), 14-3-3sigma(-/-) cells but not in cells lacking only one or none of these genes. This greater sensitivity was due to an increase in apoptosis following DNA damage and not due to exacerbation of G(2) checkpoint defects. Pharmacologic inhibition of Chk2 in p21(-/-), 14-3-3sigma(-/-) cells also resulted in greater sensitivity to DNA damage. Our data indicates that p21 and 14-3-3sigma synergize as molecular determinants of sensitivity to DNA damage following Chk2 inhibition, and Chk2 modulates the biological rheostat that determines whether a cancer cell undergoes arrest versus death after treatment with a chemotherapeutic agent. These findings have implications for the targeting of Chk2 in 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.

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. [Cancer Res 2007;67(13):6083–91]

Background Protein tyrosine kinases are important regulators of cellular homeostasis with tightly controlled catalytic activity. Mutations in kinase-encoding genes can relieve the autoinhibitory constraints on kinase activity, can promote malignant transformation, and appear to be a major determinant of response to kinase inhibitor therapy. Missense mutations in the EGFR kinase domain, for example, have recently been identified in patients who showed clinical responses to EGFR kinase inhibitor therapy. Methods and Findings Encouraged by the promising clinical activity of epidermal growth factor receptor (EGFR) kinase inhibitors in treating glioblastoma in humans, we have sequenced the complete EGFR coding sequence in glioma tumor samples and cell lines. We identified novel missense mutations in the extracellular domain of EGFR in 13.6% (18/132) of glioblastomas and 12.5% (1/8) of glioblastoma cell lines. These EGFR mutations were associated with increased EGFR gene dosage and conferred anchorage-independent growth and tumorigenicity to NIH-3T3 cells. Cells transformed by expression of these EGFR mutants were sensitive to small-molecule EGFR kinase inhibitors. Conclusions Our results suggest extracellular missense mutations as a novel mechanism for oncogenic EGFR activation and may help identify patients who can benefit from EGFR kinase inhibitors for treatment of glioblastoma., Ingo Mellinghoff and colleagues sequenced theEGFR gene in glioblastoma samples and cell lines and identified missense mutations in the extracellular domain that suggest a new mechanism for EGFR activation., Editors' Summary Background. Normally, cell division (which produces new cells) and cell death are finely balanced to keep the tissues and organs of the human body in working order. But sometimes, cells acquire changes (mutations) in their genetic material that allow them to divide uncontrollably to form cancers—life-threatening, disorganized masses of cells. Cancer treatments often involve drugs that kill rapidly dividing cells but, although these hit cancer cells hardest, they also damage some normal tissues. Now, though, some of the specific changes that allow cancer cells to divide uncontrollably have been identified and drugs that attack only these abnormal cells are being developed. One of these—erlotinib—inhibits the activity of epidermal growth factor receptor (EGFR), a “receptor tyrosine kinase” that sits in the cell membrane. The interaction of epidermal growth factor (EGF)—a messenger protein—with the extracellular portion (or domain) of EGFR activates its intracellular part (a kinase enzyme). This adds phosphate groups to tyrosine (an amino acid) in proteins that form part of a signaling cascade that tells cells to divide. Cancer cells often have alterations in EGFR signaling. Some have extra copies of the EGFR gene (EGFR amplification); others make a short version of EGFR that is always active because it lacks the extracellular domain that binds EGF; yet others contain EGFR that is permanently active because of mutations in its kinase domain. Why Was This Study Done? Erlotinib can help only patients whose tumor growth is dependent on EGFR signaling. To identify these patients it is necessary to have a detailed catalog of the mutations that occur in EGFR in tumors and to know which mutations drive uncontrolled cell growth. In this study, the researchers have catalogued and characterized the mutations in EGFR that occur in glioblastoma, a deadly type of brain tumor. The researchers chose this tumor type for their study because EGFR amplification and loss of the extracellular domain of EGFR are both common in glioblastomas and because about one in five patients with glioblastoma responds well to EGFR kinase inhibitors. What Did the Researchers Do and Find? The researchers sequenced the whole coding sequence of the EGFR gene in more than 100 glioblastomas. Nearly 15% of the tumors contained missense mutations—changes that alter the amino acid sequence of EGFR. Only one tumor had a mutation in the EGFR kinase domain; the rest had mutations in its extracellular domain. To test whether these newly identified mutations might contribute to cancer development (oncogenesis), the researchers introduced mutated or normal EGFR genes into nontumorigenic mouse cells. Only the cells that contained the mutated EGFR genes formed tumors when injected into mice, indicating that the nontumorigenic cells had been “transformed” into cancer cells by the mutated EGFR genes. Finally, the researchers showed that EGFR containing the extracellular missense mutations had kinase activity in the absence of EGF when expressed in human and mouse cells, and that the growth of cells transformed by expression of the mutated genes was sensitive to erlotinib. What Do These Findings Mean? These findings identify missense mutations in the extracellular domain of EGFR as a new way to oncogenically activate this protein. Until now researchers have concentrated on the kinase domain of this and other receptor tyrosine kinases in their search for oncogenic mutations, but the results of this study suggest that future searches should be much broader. The distribution of EGFR missense mutations in glioblastoma contrasts with that in lung cancer, in which alterations in EGFR signaling are also implicated in cancer development but all the oncogenic mutations are in the kinase domain. Fortunately, EGFR kinase inhibitors like erlotinib have broad activity: They inhibit the growth of cells transformed by the expression of EGFR containing extracellular domain mutations or kinase mutations, or by the expression of the short EGFR variant. This bodes well for the use of these drugs in patients with glioblastoma. However, before these inhibitors become a standard part of cancer treatments, sensitive techniques need to be developed to analyze tumors for these mutations so that the patients who will benefit from these targeted therapies can be identified. Additional Information. Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030485. MedlinePlus encyclopedia entries on cancer and on brain tumors US National Cancer Institute information for patients and professionals on brain tumors Wikipedia pages on protein kinases, epidermal growth factor receptor, and erlotinib (note that Wikipedia is a free online encyclopedia that anyone can edit)

The epidermal growth factor receptor (EGFR) is commonly amplified, overexpressed, and mutated in glioblastoma, making it a compelling molecular target for therapy. We have recently shown that coexpression of EGFRvIII and PTEN protein by glioblastoma cells is strongly associated with clinical response to EGFR kinase inhibitor therapy. PTEN loss, by dissociating inhibition of the EGFR from downstream phosphatidylinositol 3-kinase (PI3K) pathway inhibition, seems to act as a resistance factor. Because 40% to 50% of glioblastomas are PTEN deficient, a critical challenge is to identify strategies that promote responsiveness to EGFR kinase inhibitors in patients whose tumors lack PTEN. Here, we show that the mammalian target of rapamycin (mTOR) inhibitor rapamycin enhances the sensitivity of PTEN-deficient tumor cells to the EGFR kinase inhibitor erlotinib. In two isogenic model systems (U87MG glioblastoma cells expressing EGFR, EGFRvIII, and PTEN in relevant combinations, and SF295 glioblastoma cells in which PTEN protein expression has been stably restored), we show that combined EGFR/mTOR kinase inhibition inhibits tumor cell growth and has an additive effect on inhibiting downstream PI3K pathway signaling. We also show that combination therapy provides added benefit in promoting cell death in PTEN-deficient tumor cells. These studies provide strong rationale for combined mTOR/EGFR kinase inhibitor therapy in glioblastoma patients, particularly those with PTEN-deficient tumors. (Cancer Res 2006; 66(16): 7864-9)

SummaryAlthough 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.

Prostate stem cell antigen (PSCA) is emerging as an important diagnostic marker and therapeutic target in prostate cancer. Previous studies indicated that PSCA was directly regulated by androgens, but the mechanism has not been elucidated. Here we describe the identification of a compact cell-specific and androgen-responsive enhancer between 2.7 and 3 kb upstream of the transcription start site. The enhancer functions autonomously when positioned immediately adjacent to a minimal promoter. Deoxyribonuclease I footprinting analysis with recombinant androgen receptor (AR) reveals that the enhancer contains two AR binding sites at one end. Mutational analysis of the AR binding sites revealed the importance of the higher affinity one. The dissociation constant of the high affinity binding site (androgen response element I) was determined to be approximately 87 nm. The remainder of the enhancer contains elements that function synergistically with the AR. We discuss the structural organization of the PSCA enhancer and compare it with that found in other AR-regulated genes.

We identified TMPRSS2 as a gene that is down-regulated in androgen-independent prostate cancer xenograft tissue derived from a bone metastasis. Using specific monoclonal antibodies, we show that the TMPRSS2-encoded serine protease is expressed as a Mr 70,000 full-length form and a cleaved Mr 32,000 protease domain. Mutation of Ser-441 in the catalytic triad shows that the proteolytic cleavage is dependent on catalytic activity, suggesting that it occurs as a result of autocleavage. Mutational analysis reveals the cleavage site to be at Arg-255. A consequence of autocatalytic cleavage is the secretion of the protease domain into the media by TMPRSS2-expressing prostate cancer cells and into the sera of prostate tumor-bearing mice. Immunohistochemical analysis of clinical specimens demonstrates the highest expression of TMPRSS2 at the apical side of prostate and prostate cancer secretory epithelia and within the lumen of the glands. Similar luminal staining was detected in colon cancer samples. Expression was also seen in colon and pancreas, with little to no expression detected in seven additional normal tissues. These data demonstrate that TMPRSS2 is a secreted protease that is highly expressed in prostate and prostate cancer, making it a potential target for cancer therapy and diagnosis.

Funder: FP7 People: Marie-Curie Actions; FundRef: http://dx.doi.org/10.13039/100011264; Grant(s): CIG 334261, 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.