Lineage commitment pathways epigenetically oppose oncogenic Gαq/11-YAP signaling in dormant disseminated uveal melanoma.

The mechanisms driving late relapse in uveal melanoma (UM) patients remains a medical mystery and major challenge. Clinically it is inferred that UM disseminated cancer cells (DCCs) persist asymptomatic for years-to-decades mainly in the liver before they manifest as symptomatic metastasis. Here we reveal using Gαq/11 mut /BAP wt human uveal melanoma models and human UM metastatic samples, that the neural crest lineage commitment nuclear receptor NR2F1 is a key regulator of spontaneous UM DCC dormancy in the liver. Using a quiescence reporter, RNA-seq and multiplex imaging we revealed that rare dormant UM DCCs upregulate NR2F1 expression and genes related to neural crest programs while repressing gene related to cell cycle progression. Gain and loss of function assays showed that NR2F1 silences YAP1/TEAD1 transcription downstream of Gαq/11 signaling and that NR2F1 expression can also be repressed by YAP1. YAP1 expression is repressed by NR2F1 binding to its promoter and changing the histone H3 tail activation marks to repress YAP1 transcription. In vivo CRISPR KO of NR2F1 led dormant UM DCCs to awaken and initiate relentless liver metastatic growth. Cut&Run and bulk RNA sequencing further confirmed that NR2F1 epigenetically stimulates neuron axon guidance and neural lineage programs, and it globally represses gene expression linked to G-protein signaling to drive dormancy. Pharmacological inhibition of Gαq/11 mut signaling resulted in NR2F1 upregulation and robust UM growth arrest, which was also achieved using a novel NR2F1 agonist. Our work sheds light on the molecular underpinnings of UM dormancy revealing that transcriptional programs driven by NR2F1 epigenetically short-circuit Gαq/11 signaling to its downstream target YAP1. Highlights: Quiescent solitary uveal melanoma (UM) DCCs in the liver up- and down-regulate neural crest and cell cycle progression programs, respectively.NR2F1 drives solitary UM DCC dormancy by antagonizing the Gαq/11-YAP1 pathway; small molecule Gαq/11 inhibition restores NR2F1 expression and quiescence. NR2F1 short-circuits oncogenic YAP1 and G-protein signaling via a chromatin remodeling program. Loss of function of NR2F1 in dormant UM DCCs leads to aggressive liver metastasis.

Randomized phase II study of preoperative afatinib in untreated head and neck cancers: predictive and pharmacodynamic biomarkers of activity.

There is no strong and reliable predictive biomarker in head and neck squamous cell carcinoma (HNSCC) for EGFR inhibitors. We aimed to identify predictive and pharmacodynamic biomarkers of efficacy of afatinib, a pan-HER tyrosine kinase inhibitor, in a window-of-opportunity trial (NCT01415674). Multi-omics analyses were carried out on pre-treatment biopsy and surgical specimen for biological assessment of afatinib activity. Sixty-one treatment-naïve and operable HNSCC patients were randomised to afatinib 40 mg/day for 21-28 days versus no treatment. Afatinib produced a high rate of metabolic response. Responders had a higher expression of pERK1/2 (P = 0.02) and lower expressions of pHER4 (P = 0.03) and pRB1 (P = 0.002) in pre-treatment biopsy compared to non-responders. At the cellular level, responders displayed an enrichment of tumor-infiltrating B cells under afatinib (P = 0.02). At the molecular level, NF-kappa B signaling was over-represented among upregulated genes in non-responders (P < 0.001; FDR = 0.01). Although exploratory, phosphoproteomics-based biomarkers deserve further investigations as predictors of afatinib efficacy.

Patient Derived Xenografts (PDX) Models as an Avatar to Assess Personalized Therapy Options in Uveal Melanoma: A Feasibility Study.

Uveal melanoma is the most common primary intraocular malignancy in adults. Up to 50% of UM patients develop metastatic disease, usually in the liver. When metastatic, the prognosis is poor, and few treatment options exist. Here, we investigated the feasibility of establishing patient-derived xenografts (PDXs) from a patient's tumor in order to screen for therapies that the patient could benefit from. Samples obtained from 29 primary tumors and liver metastases of uveal melanoma were grafted into SCID mice. PDX models were successfully established for 35% of primary patient tumors and 67% of liver metastases. The tumor take rate was proportional to the risk of metastases. PDXs showed the same morphology, the same GNAQ/11, BAP1, and SF3B1 mutations, and the same chromosome 3 and 8q status as the corresponding patient samples. Six PDX models were challenged with two compounds for 4 weeks. We show that, for 31% of patients with high or intermediate risk of metastasis, the timing to obtain efficacy results on PDX models derived from their primary tumors was compatible with the selection of the therapy to treat the patient after relapse. PDXs could thus be a valid tool ("avatar") to select the best personalized therapy for one third of patients that are most at risk of relapse.

Immunohistochemical characterisation of the immune landscape in primary uveal melanoma and liver metastases.

BACKGROUND: The immune landscape of uveal melanoma liver metastases (UMLM) has not been sufficiently studied. METHODS: Immune cell infiltrates (ICIs), PD-1 and PD-L1 were characterised in 62 UMLM and 28 primary uveal melanomas (PUM). ICI, PD-1 and PD-L1 were scored as: (1) % tumoral area occupied by tumour-infiltrating lymphocytes or macrophages (TILs, TIMs) and (2) % perTumoral (perT) area. ICIs and other variables including histopathologic growth patterns (HGPs), replacement and desmoplastic, of UMLM were analysed for their prognostic value. RESULTS: ICIs recognised by haematoxylin-eosin-saffron (HES) and IHC (e.g., T cells (CD3), B cells (CD20). Macrophages (CD68), (CD163), were primarily localised to the perT region in PUM and UMLM and were more conspicuous in UMLM. HES, CD3, CD4, FoxP3, CD8, CD20, PD-1 TILs were scant (<5%). TIMs were more frequent, particularly in UMLM than in PUM. Both CD68+ TIMs and HGPs remained significant on multivariate analysis, influencing overall (OS) and metastasis-specific overall survival (MSOS). CD68 + , CD163+ and CD20+ perT infiltrates in UMLM predicted increased OS and MSOS on univariate analysis. CONCLUSIONS: TILs and PD-L1 have no predictive value in PUM or UMLM. CD68+ and CD163+TIMs, CD20+ perT lymphocytes, and HGPs are important prognostic factors in UMLMs.

Nivolumab plus ipilimumab in metastatic uveal melanoma: a real-life, retrospective cohort of 47 patients.

Although combined PD-1/CTLA-4 inhibition showed limited efficacy in single-arm, phase II trials in metastatic uveal melanoma (mUM), such combination appears frequently used in mUM patients. We here report our experience with nivolumab/ipilimumab in mUM. A retrospective cohort of 47 mUM patients, 24 men and 23 women, received nivolumab/ipilimumab between October 2019 and December 2021, mostly first line (94%). Two regimens were used: nivolumab 1 mg/kg + ipilimumab 3 mg/kg (nivo1ipi3, 49% of patients) and nivolumab 3 mg/kg + ipilimumab 1 mg/kg (nivo3ipi1, 51% of patients). Median follow-up was 37 and 88 weeks in nivo3ipi1 and nivo1ipi3 cohorts, respectively. We observed partial response in two patients (4%) and stable disease in 14 patients (30%), with no significant difference between the two regimens. Median progression-free survival was 13.6 weeks and 11.9 weeks in the nivo1ipi3 and nivo3ipi1 cohorts, respectively (p = 0.49). Severe adverse events (grade 3 or 4) were observed in seven patients (15%) among which five treated with nivo1ipi3 (22%) and two treated with nivo3ipi1 (8%). These data suggest that nivolumab/ipilimumab combination does not improve clinical outcomes compared to other therapies but is more toxic. In the absence of controlled clinical trials, we would not recommend this combination as a standard treatment in all mUM patients but rather as an option. Patients for whom the benefit-risk ratio could justify the combination need to be defined.

Helping Patients Communicate With Oncologists When Cancer Treatment Resistance Occurs to Develop, Test, and Implement a Patient Communication Aid: Sequential Collaborative Mixed Methods Study.

BACKGROUND: Most cancer-related deaths result from disseminated diseases that develop resistance to anticancer treatments. Inappropriate communication in this challenging situation may result in unmet patient information and support needs. Patient communication aids such as question prompt lists (QPLs) may help. OBJECTIVE: This study aims to develop and pilot-test a specific QPL in the following two contrasting clinical contexts in France after cancer resistance has developed: triple-negative and luminal B metastatic breast cancer (MBC) and metastatic uveal melanoma (MUM). METHODS: A sequential study design with a mixed methods collaborative approach will be applied. The first step aims to build a specific QPL. Step 1a will explore oncologist-patient communication issues from oncology professionals' interviews (n=20 approximately). Step 1b will appraise information and support needs experienced by patients with MBC or MUM both quantitatively (n=80) and qualitatively (n=40 approximately). These data will be used to develop and pilot-test a QPL specific to patients with cancer experiencing initial or acquired resistance to treatment. We expect to obtain a core QPL that comprises questions and concerns commonly expressed by patients with resistant cancer and is complemented by specific issues for either MBC or MUM cancer sites. In step 1c, 2 focus groups of patients with any type of metastatic cancer (n=4) and health care professionals (n=4) will be conducted to revise the content of a preliminary QPL and elaborate an acceptable and feasible clinical implementation. In step 1d, the content of the QPL version 1 and implementation guidance will be validated using a Delphi process. Step 2 will pilot-test the QPL version 1 in real practice with patients with MBC or MUM (n=80). Clinical utility will be assessed by comparing responses to questionnaires administered in step 1b (QPL-naive historical control group) and step 2 (QPL intervention group). RESULTS: This study received grants in March and December 2019 and was approved by the French national ethics committee in July 2019. As of October 2021, interviews with oncology professionals have been conducted and analyzed (N=26 to reach saturation), and 39 and 27 patients with MBC and MUM, respectively, have been recruited. CONCLUSIONS: A clinically and culturally tailored QPL is expected to facilitate patients' participation in consultations, improve oncologists' responses to patients' information and support needs, and thus foster patients' psychological adjustment to the diagnosis and follow-up of cancer resistance to treatment. TRIAL REGISTRATION: ClinicalTrials.gov NCT04118062; http://clinicaltrials.gov/ct2/show/NCT04118062. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/26414.

CENP-A Subnuclear Localization Pattern as Marker Predicting Curability by Chemoradiation Therapy for Locally Advanced Head and Neck Cancer Patients.

Effective biomarkers predictive of the response to treatments are key for precision medicine. This study identifies the staining pattern of the centromeric histone 3 variant, CENP-A, as a predictive biomarker of locoregional disease curability by chemoradiation therapy. We compared by imaging the subnuclear distribution of CENP-A in normal and tumoral tissues, and in a retrospective study in biopsies of 62 locally advanced head and neck squamous cell carcinoma (HNSCC) patients treated by chemoradiation therapy. We looked for predictive factors of locoregional disease control and patient's survival, including CENP-A patterns, Ki67, HPV status and anisokaryosis. In different normal tissues, we reproducibly found a CENP-A subnuclear pattern characterized by CENP-A clusters both localized at the nuclear periphery and regularly spaced. In corresponding tumors, both features are lost. In locally advanced HNSCC, a specific CENP-A pattern identified in pretreatment biopsies predicts definitive locoregional disease control after chemoradiation treatment in 96% (24/25) of patients (OR = 17.6 CI 95% [2.6; 362.8], p = 0.002), independently of anisokaryosis, Ki67 labeling or HPV status. The characteristics of the subnuclear pattern of CENP-A in cell nuclei revealed by immunohistochemistry could provide an easy to use a reliable marker of disease curability by chemoradiation therapy in locally advanced HNSCC patients.

Prognostic Implications of MRI Melanin Quantification and Cytogenetic Abnormalities in Liver Metastases of Uveal Melanoma.

To evaluate the prognostic implications of melanin quantification assessed by magnetic resonance imaging (MRI) with respect to the clinical, pathological, and genetic features of liver metastases of uveal melanoma (LMUM). This single-center retrospective cohort study included 63 patients eligible for margin-free resection of LMUM between 2007 and 2018. Comparative genomic hybridization of resected liver metastases on microarrays was performed for genetic risk classification. Metastases exhibiting monosomy 3 with any type of gain of chromosome 8 (M3/8g) were considered high-genetic-risk. MRI melanin quantification using the mean T1 signal (mT1s) in liver metastases was assessed quantitatively on preoperative imaging examination and compared to that of gross pathological evaluation. The association between MRI melanin quantification and overall survival (OS) was assessed by multivariate analysis using the Cox proportional hazards model. Gross pathological assessment of melanin content and MRI melanin quantification were strongly correlated (r = 0.8, p < 0.001). Independent prognostic factors associated with OS were disease-free interval ≤ 24 months (HR = 3.1; 95% CI, 1.6-6.0; p < 0.001), high-genetic-risk (HR = 2.2; 95% CI, 1.1-4.8; p = 0.04), mT1s > 1.1 (HR = 2.3; 95% CI, 1.2-4.7; p = 0.019), and complete hepatic resection (HR = 0.3; 95% CI, 0.2-0.7; p = 0.004). In patients with high-genetic-risk, mT1s showed a significant association with OS (HR = 3.7; 95% CI, 1.5-9.3; p = 0.006). The median OS was 17.5 months vs. 27 months for >1.1 and ≤1.1 mT1s tumors, respectively (p = 0.003). We showed that the level of pigmentation in M3/8g LMUM identified two subsets that were correlated with distinct clinical outcomes.

Characterization of Signalling Pathways That Link Apoptosis and Autophagy to Cell Death Induced by Estrone Analogues Which Reversibly Depolymerize Microtubules.

The search for novel anti-cancer compounds which can circumvent chemotherapeutic drug resistance and limit systemic toxicity remains a priority. 2-Ethyl-3-O-sulphamoyl-estra-1,3,5(10)15-tetraene-3-ol-17one (ESE-15-one) and 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16) are sulphamoylated 2-methoxyestradiol (2-ME) analogues designed by our research team. Although their cytotoxicity has been demonstrated in vitro, the temporal and mechanistic responses of the initiated intracellular events are yet to be determined. In order to do so, assays investigating the compounds' effects on microtubules, cell cycle progression, signalling cascades, autophagy and apoptosis were conducted using HeLa cervical- and MDA-MB-231 metastatic breast cancer cells. Both compounds reversibly disrupted microtubule dynamics as an early event by binding to the microtubule colchicine site, which blocked progression through the cell cycle at the G1/S- and G2/M transitions. This was supported by increased pRB and p27Kip1 phosphorylation. Induction of apoptosis with time-dependent signalling involving the p-JNK, Erk1/2 and Akt/mTOR pathways and loss of mitochondrial membrane potential was demonstrated. Inhibition of autophagy attenuated the apoptotic response. In conclusion, the 2-ME analogues induced a time-dependent cross-talk between cell cycle checkpoints, apoptotic signalling and autophagic processes, with an increased reactive oxygen species formation and perturbated microtubule functioning appearing to connect the processes. Subtle differences in the responses were observed between the two compounds and the different cell lines.

Proliferation Genes Repressed by TGF-ß Are Downstream of Slug/Snail2 in Normal Bronchial Epithelial Progenitors and Are Deregulated in COPD.

Slug/Snail2 belongs to the Epithelial-Mesenchymal Transition (EMT)-inducing transcription factors involved in development and diseases. Slug is expressed in adult stem/progenitor cells of several epithelia, making it unique among these transcription factors. To investigate Slug role in human bronchial epithelium progenitors, we studied primary bronchial basal/progenitor cells in an air-liquid interface culture system that allows regenerating a bronchial epithelium. To identify Slug downstream genes we knocked down Slug in basal/progenitor cells from normal subjects and subjects with COPD, a respiratory disease presenting anomalies in the bronchial epithelium and high levels of TGF-ß in the lungs. We show that normal and COPD bronchial basal/progenitors, even when treated with TGF-ß, express both epithelial and mesenchymal markers, and that the epithelial marker E-cadherin is not a target of Slug and, moreover, positively correlates with Slug. We reveal that Slug downstream genes responding to both differentiation and TGF-ß are different in normal and COPD progenitors, with in particular a set of proliferation-related genes that are among the genes repressed downstream of Slug in normal but not COPD. In COPD progenitors at the onset of differentiation in presence of TGF-ß,we show that there is positive correlations between the effect of differentiation and TGF-ß on proliferation-related genes and on Slug protein, and that their expression levels are higher than in normal cells. As well, the expression of Smad3 and ß-Catenin, two molecules from TGF-ßsignaling pathways, are higher in COPD progenitors, and our results indicate that proliferation-related genes and Slug protein are increased by different TGF-ß-induced mechanisms.

PARP inhibitors and radiation potentiate liver cell death in vitro. Do hepatocellular carcinomas have an achilles' heel?

BACKGROUND: A promising avenue for cancer treatment is exacerbating the deregulation of the DNA repair machinery that would normally protect the genome. To address the applicability of poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) combined with radiotherapy for the treatment of hepatocellular carcinoma (HCC) two approaches were used: firstly, the in vitro sensitivity to the PARPi Veliparib and Talazoparib +/- radiation exposure was determined in liver cell lines and the impact of the HBV X protein (HBx) that deregulates cellular DNA damage repair via SMC5/6 degradation was investigated. Secondly, PARP expression profiles and DNA damage levels using the surrogate marker gammaH2AX were assessed in a panel of control liver vs HCC tissues. METHODS: Cell cytotoxicity was measured by clonogenic survival or relative cell growth and the DNA damage response using immunological-based techniques in Hep3B, PLC/PRF/5, HepG2- and HepaRG-derived models. Transcriptome changes due to HBx expression vs SMC6 loss were assessed by RNA sequencing in HepaRG-derived models. PARP and PARG transcripts (qPCR) and PARP1, H2AX and gammaH2AX protein levels (RPPA) were compared in control liver vs HBV-, HCV-, alcohol- and non-alcoholic steatohepatitis-associated HCC (tumor/peritumor) tissues. RESULTS: PARPi cytotoxicity was significantly enhanced when combined with X-rays (2Gy) with Talazoparib having a greater impact than Veliparib in most in vitro models. HBx expression significantly lowered survival, probably driven by SMC5/6 loss based on the transcriptome analysis and higher DNA damage levels. PARP1 and PARP2 transcript levels were significantly higher in tumor than peritumor and control tissues. The HBV/HCV/alcohol-associated tumor tissues studied had reduced H2AX but higher gammaH2AX protein levels compared to peritumor and control tissues providing evidence of increased DNA damage during liver disease progression. CONCLUSIONS: These proof-of-concept experiments support PARPi alone or combined with radiotherapy for HCC treatment, particularly for HBV-associated tumors, that warrant further investigation.

Integrative analysis of multi-platform reverse-phase protein array data for the pharmacodynamic assessment of response to targeted therapies.

Reverse-phase protein array (RPPA) technology uses panels of high-specificity antibodies to measure proteins and protein post-translational modifications in cells and tissues. The approach offers sensitive and precise quantification of large numbers of samples and has thus found applications in the analysis of clinical and pre-clinical samples. For effective integration into drug development and clinical practice, robust assays with consistent results are essential. Leveraging a collaborative RPPA model, we set out to assess the variability between three different RPPA platforms using distinct instrument set-ups and workflows. Employing multiple RPPA-based approaches operated across distinct laboratories, we characterised a range of human breast cancer cells and their protein-level responses to two clinically relevant cancer drugs. We integrated multi-platform RPPA data and used unsupervised learning to identify protein expression and phosphorylation signatures that were not dependent on RPPA platform and analysis workflow. Our findings indicate that proteomic analyses of cancer cell lines using different RPPA platforms can identify concordant profiles of response to pharmacological inhibition, including when using different antibodies to measure the same target antigens. These results highlight the robustness and the reproducibility of RPPA technology and its capacity to identify protein markers of disease or response to therapy.

Genetic markers and phosphoprotein forms of beta-catenin pß-Cat552 and pß-Cat675 are prognostic biomarkers of cervical cancer.

BACKGROUND: Cervical cancer (CC) remains a leading cause of gynaecological cancer-related mortality world wide and constitutes the third most common malignancy in women. The RAIDs consortium (http://www.raids-fp7.eu/) conducted a prospective European study [BioRAIDs (NCT02428842)] with the objective to stratify CC patients for innovative treatments. A "metagene" of genomic markers in the PI3K pathway and epigenetic regulators had been previously associated with poor outcome [2]. METHODS: To detect new, more specific, targets for treatment of patients who resist standard chemo-radiation, a high-dimensional Cox model was applied to define dominant molecular variants, copy number variations, and reverse phase protein arrays (RPPA). FINDINGS: Survival analysis on 89 patients with all omics data available, suggested loss-of-function (LOF) or activating molecular alterations in nine genes to be candidate biomarkers for worse prognosis in patients treated by chemo-radiation while LOF of ATRX, MED13 as well as CASP8 were associated with better prognosis. When protein expression data by RPPA were factored in, the supposedly low molecular weight and nuclear form, of beta-catenin, phosphorylated in Ser552 (pß-Cat552), ranked highest for good prognosis, while pß-Cat675 was associated with worse prognosis. INTERPRETATION: These findings call for molecularly targeted treatments involving p53, Wnt pathway, PI3K pathway, and epigenetic regulator genes. Pß-Cat552 and pß-Cat675 may be useful biomarkers to predict outcome to chemo-radiation, which targets the DNA repair axis. FUNDING: European Union's Seventh Program for research, technological development and demonstration (agreement N°304,810), the Fondation ARC pour la recherche contre le cancer.

The high protein expression of FOXO3, but not that of FOXO1, is associated with markers of good prognosis.

To better define the role of FOXO1 and FOXO3 transcriptional factors in breast carcinogenesis, we performed a comparative study of their expression at both the RNA and protein levels in a series of human breast tumors. We used qRT-PCR assay to quantify mRNA expression and Reverse Phase Protein Arrays (RPPA) to quantify protein expression in 218 breast tumors from patients with known clinical/pathological status and outcome. Weak correlations were observed between mRNA and protein expressions for both FOXO1 and FOXO3 genes. High expression of FOXO3 protein, but not FOXO1 protein, was a good prognostic marker, negatively correlated with KI67 and markers of activity of the PI3K/AKT/mTOR oncogenic pathway, and positively correlated with p53, a marker of apoptosis. Moreover, FOXO3 protein expression, but not FOXO1 protein expression, was also negatively correlated with various proteins involved in different DNA repair mechanisms. FOXO3 protein, but not FOXO1 protein, appears to be a tumor suppressor that inhibits breast cancer by altering DNA damage response (DDR), thereby inducing p53-dependent apoptosis. This antitumor effect appears to be suppressed by excessive activity of the PI3K/AKT/mTOR pathway. High FOXO3 protein expression could be a biomarker of deficient DDR in breast tumors.

Quantification of biomarkers for beef meat qualities using a combination of Parallel Reaction Monitoring- and antibody-based proteomics.

We and others have identified biomarker candidates of tenderness or marbling, two major attributes of bovine meat-eating qualities for consumers' satisfaction. In this study, Reverse Phase Protein Arrays (RPPA) and targeted mass spectrometry assays using Parallel Reaction Monitoring (PRM) were developed to test whether 10 proteins pass the sequential qualification and verification steps of the challenging biomarker discovery pipeline. At least MYH1, TPI1, ALDH1A1 and CRYAB were qualified by RPPA or PRM as being differentially abundant according to marbling values of longissimus thoracis and semimembranosus muscles. Significant mathematical relationships between the individual abundance of each of the four proteins and marbling values were verified by linear or logistic regressions. Four proteins, TNNT1, MDH1, PRDX6 and ENO3 were qualified and verified for tenderness, and the abundance of MDH1 explained 49% of the tenderness variability. The present PRM and RPPA results pave the way for development of useful meat industrial multiplex-proteins assays.

Response to mTOR and PI3K inhibitors in enzalutamide-resistant luminal androgen receptor triple-negative breast cancer patient-derived xenografts.

Luminal androgen receptor (LAR) breast cancer accounts for 10% of all triple-negative breast cancers (TNBC). Anti-androgen therapy for this subtype is in development, but yields only partial clinical benefits. In this study, we aimed to characterize the genomic alterations of LAR TNBC, to analyze activation of the PI3K signaling pathway and to compare the response to PI3K pathway inhibitors with that to anti-androgen therapy in patient-derived xenografts (PDX) of LAR TNBC. Methods: Four LAR PDX models were identified, on the basis of their transcriptomic profiles, in a cohort of 57 PDX models of TNBC. The expression of AR-related genes, basal and luminal cytokeratins and EMT genes was analyzed by RT-PCR and IHC. AKT1 and PIK3CA mutations were identified by targeted NGS, and activation of the PI3K pathway was analyzed with a reverse-phase protein array. Three LAR PDXs with a PIK3CA or AKT1 mutation were treated with the AR inhibitor enzalutamide, a PI3K inhibitor, a dual PI3K-mTOR inhibitor and a mTORC1-mTORC2 inhibitor. Finally, we screened a clinical cohort of 329 TNBC for PIK3CA and AKT1 hotspot mutations. Results: LAR TNBC PDXs were significantly enriched in PIK3CA and AKT1 mutations, and had higher levels of luminal-androgen-like gene expression and a higher PI3K pathway protein activation score than other TNBC subtypes. Immunohistochemistry analysis revealed strong expression of the luminal cytokeratin CK18 and AR in three LAR PDX models. We found that mTOR and PI3K inhibitors had marked antitumor activity in vivo in PDX harboring genomic alterations of PIK3CA and AKT1 genes that did not respond to the AR antagonist enzalutamide. PIK3CA mutations were detected in more than one third of AR+ TNBC from patients (38%), and only 10% of AR-negative TNBC. Conclusion: Our results for PDX models of LAR TNBC resistant to enzalutamide indicate that PIK3CA and AKT1 are potential therapeutic targets.

Genomic Profiling of Metastatic Uveal Melanoma and Clinical Results of a Phase I Study of the Protein Kinase C Inhibitor AEB071.

Up to 50% of patients with uveal melanoma (UM) develop metastatic disease, for which there is no effective systemic treatment. This study aimed to evaluate the safety and efficacy of the orally available protein kinase C inhibitor, AEB071, in patients with metastatic UM, and to perform genomic profiling of metastatic tumor samples, with the aim to propose combination therapies. Patients with metastatic UM (n = 153) were treated with AEB071 in a phase I, single-arm study. Patients received total daily doses of AEB071 ranging from 450 to 1,400 mg. First-cycle dose-limiting toxicities were observed in 13 patients (13%). These were most commonly gastrointestinal system toxicities and were dose related, occurring at doses ≥700 mg/day. Preliminary clinical activity was observed, with 3% of patients achieving a partial response and 50% with stable disease (median duration 15 weeks). High-depth, targeted next-generation DNA sequencing was performed on 89 metastatic tumor biopsy samples. Mutations previously identified in UM were observed, including mutations in GNAQ, GNA11, BAP1, SF3B1, PLCB4, and amplification of chromosome arm 8q. GNAQ/GNA11 mutations were observed at a similar frequency (93%) as previously reported, confirming a therapeutic window for inhibition of the downstream effector PKC in metastatic UM.In conclusion, the protein kinase C inhibitor AEB071 was well tolerated, and modest clinical activity was observed in metastatic UM. The genomic findings were consistent with previous reports in primary UM. Together, our data allow envisaging combination therapies of protein kinase C inhibitors with other compounds in metastatic UM.

Analytical Platforms 1: Use of Cultured Cells and Fluorescent Read-Out Coupled to NormaCurve Normalization in RPPA.

The analytic platform described in this chapter uses proteins extracted from cultured cells as an infinite source of material to set up, validate, and quality control an RPPA platform. Readout of the arrays uses near-infrared fluorescence labeling and data normalization is performed using the bioinformatics package NormaCurve.In the first part, we will describe the advantages, drawbacks, and different applications of cell line material for RPPA. In the second part, we will describe how the staining protocol, the method of readout, and the normalization method applied afterward are interconnected and should be considered together. Finally, we will describe the NormaCurve package, which is freely available, and its requirements for implementation.Four protocols are provided in this chapter: (1) Protein lysis of cell lines using a homemade Laemmli buffer, (2) RPPA staining for fluorescent readout including a signal amplification step, (3) total protein staining in the visible spectrum for normalization purposes, and (4) total protein staining in the near-infrared spectrum for normalization purposes.

Induction of Acquired Resistance towards EGFR Inhibitor Gefitinib in a Patient-Derived Xenograft Model of Non-Small Cell Lung Cancer and Subsequent Molecular Characterization.

In up to 30% of non-small cell lung cancer (NSCLC) patients, the oncogenic driver of tumor growth is a constitutively activated epidermal growth factor receptor (EGFR). Although these patients gain great benefit from treatment with EGFR tyrosine kinase inhibitors, the development of resistance is inevitable. To model the emergence of drug resistance, an EGFR-driven, patient-derived xenograft (PDX) NSCLC model was treated continuously with Gefitinib in vivo. Over a period of more than three months, three separate clones developed and were subsequently analyzed: Whole exome sequencing and reverse phase protein arrays (RPPAs) were performed to identify the mechanism of resistance. In total, 13 genes were identified, which were mutated in all three resistant lines. Amongst them the mutations in NOMO2, ARHGEF5 and SMTNL2 were predicted as deleterious. The 53 mutated genes specific for at least two of the resistant lines were mainly involved in cell cycle activities or the Fanconi anemia pathway. On a protein level, total EGFR, total Axl, phospho-NFκB, and phospho-Stat1 were upregulated. Stat1, Stat3, MEK1/2, and NFκB displayed enhanced activation in the resistant clones determined by the phosphorylated vs. total protein ratio. In summary, we developed an NSCLC PDX line modelling possible escape mechanism under EGFR treatment. We identified three genes that have not been described before to be involved in an acquired EGFR resistance. Further functional studies are needed to decipher the underlying pathway regulation.