Neratinib plus dasatinib is highly synergistic in HER2-positive breast cancer in vitro and in vivo.

BACKGROUND: HER2-targeted therapies have revolutionised the treatment of HER2-positive breast cancer. However, de novo resistance or the emergence of acquired resistance is a persistent clinical problem. Here we report that neratinib, an irreversible pan-HER inhibitor, in combination with the multi-kinase inhibitor dasatinib, currently used to treat certain leukemias, has strong anti-proliferative effects against models of HER2-positive breast cancer that are innately resistant to trastuzumab or have acquired resistance to neratinib. METHODS: Neratinib plus dasatinib was examined in a panel of 20 breast cancer cell lines, including HER2-positive, estrogen-receptor-positive, triple negative, and acquired HER2-targeted therapy resistant models. Drug effects on migration and apoptosis induction was evaluated and signaling alterations were determined by reverse phase protein array (RPPA). In vivo efficacy was examined using orthotopically-implanted HCC1954 cells. RESULTS: Synergy was observed in cell lines innately resistant to trastuzumab, models with acquired resistance to neratinib, and in triple negative breast cancer cell lines. Further investigation showed that neratinib plus dasatinib induced apoptosis and inhibited cell migration to a greater degree than either drug alone. RPPA revealed that the combination caused suppression of key survival signaling through EGFR, Akt, and MAPK inhibition. In vivo, neratinib plus dasatinib was well tolerated and had a prolonged anti-tumor effect against HCC1954 xenografts. CONCLUSIONS: This study provides a strong pre-clinical rationale for the clinical investigation neratinib and dasatinib in HER2+ breast cancer.

Protein Tyrosine Phosphatase Non-Receptor 11 (PTPN11/Shp2) as a Driver Oncogene and a Novel Therapeutic Target in Non-Small Cell Lung Cancer (NSCLC).

PTPN11 encodes the SHP2 protein tyrosine phosphatase that activates the mitogen-activated protein kinase (MAPK) pathway upstream of KRAS and MEK. PTPN11/Shp2 somatic mutations occur frequently in Juvenile myelomonocytic leukaemia (JMML); however, the role of mutated PTPN11 in lung cancer tumourigenesis and its utility as a therapeutic target has not been fully addressed. We applied mass-spectrometry-based genotyping to DNA extracted from the tumour and matched the normal tissue of 356 NSCLC patients (98 adenocarcinomas (LUAD) and 258 squamous cell carcinomas (LUSC)). Further, PTPN11 mutation cases were identified in additional cohorts, including TCGA, Broad, and MD Anderson datasets and the COSMIC database. PTPN11 constructs harbouring PTPN11 E76A, A72D and C459S mutations were stably expressed in IL-3 dependent BaF3 cells and NSCLC cell lines (NCI-H1703, NCI-H157, NCI-H1299). The MAPK and PI3K pathway activation was evaluated using Western blotting. PTPN11/Shp2 phosphatase activity was measured in whole-cell protein lysates using an Shp2 assay kit. The Shp2 inhibitor (SHPi) was assessed both in vitro and in vivo in a PTPN11-mutated cell line for improved responses to MAPK and PI3K targeting therapies. Somatic PTPN11 hotspot mutations occurred in 4/98 (4.1%) adenocarcinomas and 7/258 (2.7%) squamous cells of 356 NSCLC patients. Additional 26 PTPN11 hotspot mutations occurred in 23 and 3 adenocarcinomas and squamous cell carcinoma, respectively, across the additional cohorts. Mutant PTPN11 significantly increased the IL-3 independent survival of Ba/F3 cells compared to wildtype PTPN11 (p < 0.0001). Ba/F3, NCI-H1703, and NCI-H157 cells expressing mutant PTPN11 exhibited increased PTPN11/Shp2 phosphatase activity and phospho-ERK1/2 levels compared to cells expressing wildtype PTPN11. The transduction of the PTPN11 inactivating mutation C459S into NSCLC cell lines led to decreased phospho-ERK, as well as decreased phospho-AKT in the PTPN11-mutated NCI-H661 cell line. NCI-H661 cells (PTPN11-mutated, KRAS-wild type) were significantly more sensitive to growth inhibition by the PI3K inhibitor copanlisib (IC50: 13.9 ± 4.7 nM) compared to NCI-H1703 (PTPN11/KRAS-wild type) cells (IC50: >10,000 nM). The SHP2 inhibitor, in combination with the PI3K targeting therapy copanlisib, showed no significant difference in tumour development in vivo; however, this significantly prevented MAPK pathway induction in vitro (p < 0.0001). PTPN11/Shp2 demonstrated the in vitro features of a driver oncogene and could potentially sensitize NSCLC cells to PI3K inhibition and inhibit MAPK pathway activation following PI3K pathway targeting.

Preclinical evaluation of the CDK4/6 inhibitor palbociclib in combination with a PI3K or MEK inhibitor in colorectal cancer.

BACKGROUND: Studies have demonstrated the efficacy of Palbociclib (CDK 4/6 inhibitor), Gedatolisib (PI3K/mTOR dual inhibitor) and PD0325901 (MEK1/2 inhibitor) in colorectal cancer (CRC), however single agent therapeutics are often limited by the development of resistance. METHODS: We compared the anti-proliferative effects of the combination of Gedatolisib and Palbociclib and Gedatolisib and PD0325901 in five CRC cell lines with varying mutational background and tested their combinations on total and phosphoprotein levels of signaling pathway proteins. RESULTS: The combination of Palbociclib and Gedatolisib was superior to the combination of Palbociclib and PD0325901. The combination of Palbociclib and Gedatolisib had synergistic anti-proliferative effects in all cell lines tested [CI range: 0.11-0.69] and resulted in the suppression of S6rp (S240/244), without AKT reactivation. The combination of Palbociclib and Gedatolisib increased BAX and Bcl-2 levels in PIK3CA mutated cell lines. The combination of Palbociclib and Gedatolisib caused MAPK/ERK reactivation, as seen by an increase in expression of total EGFR, regardless of the mutational status of the cells. CONCLUSION: This study shows that the combination of Palbociclib and Gedatolisib has synergistic anti-proliferative effects in both wild-type and mutated CRC cell lines. Separately, the phosphorylation of S6rp may be a promising biomarker of responsiveness to this combination.

Comparative analysis of deeply phenotyped GBM cohorts of 'short-term' and 'long-term' survivors.

BACKGROUND: Glioblastoma (GBM) is an aggressive brain cancer that typically results in death in the first 15 months after diagnosis. There have been limited advances in finding new treatments for GBM. In this study, we investigated molecular differences between patients with extremely short (≤ 9 months, Short term survivors, STS) and long survival (≥ 36 months, Long term survivors, LTS). METHODS: Patients were selected from an in-house cohort (GLIOTRAIN-cohort), using defined inclusion criteria (Karnofsky score > 70; age < 70 years old; Stupp protocol as first line treatment, IDH wild type), and a multi-omic analysis of LTS and STS GBM samples was performed. RESULTS: Transcriptomic analysis of tumour samples identified cilium gene signatures as enriched in LTS. Moreover, Immunohistochemical analysis confirmed the presence of cilia in the tumours of LTS. Notably, reverse phase protein array analysis (RPPA) demonstrated increased phosphorylated GAB1 (Y627), SRC (Y527), BCL2 (S70) and RAF (S338) protein expression in STS compared to LTS. Next, we identified 25 unique master regulators (MR) and 13 transcription factors (TFs) belonging to ontologies of integrin signalling and cell cycle to be upregulated in STS. CONCLUSION: Overall, comparison of STS and LTS GBM patients, identifies novel biomarkers and potential actionable therapeutic targets for the management of GBM.

Proteomic time course of breast cancer cells highlights enhanced sensitivity to Stat3 and Src inhibitors prior to endocrine resistance development.

To prevent the development of endocrine-resistant breast cancer, additional targeted therapies are increasingly being trialled in combination with endocrine therapy. The molecular mechanisms facilitating cancer cell survival during endocrine treatment remain unknown but could help direct selection of additional targeted therapies. We present a novel proteomic timecourse dataset, profiling potential drug targets in a population of MCF7 cells during 1 year of tamoxifen treatment. Reverse phase protein arrays profiled >70 proteins across 30 timepoints. A biphasic response to tamoxifen was evident, which coincided with changes in growth rate. Tamoxifen strongly impeded cell growth for the first 160 days, followed by gradual growth recovery and eventual resistance development. The growth-impeded phase was distinguished by the phosphorylation of Stat3 (y705) and Src (y527). Tumour tissue from patients treated with neo-adjuvant endocrine therapy (<4 months) also displayed increased Stat3 and Src signalling. Inhibitors of Stat3 (napabucasin) and Src (dasatinib), were effective at killing tamoxifen-treated MCF7 and T47D cells. Sensitivity to both drugs was significantly enhanced once tamoxifen had induced the growth-impeded phase. This novel proteomic resource identifies key mechanisms enabling cell survival during tamoxifen treatment. It provides valuable insight into potential drug combinations and timing that may prevent the development of endocrine resistance.

A Systems Biology Approach to Investigate Kinase Signal Transduction Networks That Are Involved in Triple Negative Breast Cancer Resistance to Cisplatin.

Triple negative breast cancer (TNBC) remains a therapeutic challenge due to the lack of targetable genetic alterations and the frequent development of resistance to the standard cisplatin-based chemotherapies. Here, we have taken a systems biology approach to investigate kinase signal transduction networks that are involved in TNBC resistance to cisplatin. Treating a panel of cisplatin-sensitive and cisplatin-resistant TNBC cell lines with a panel of kinase inhibitors allowed us to reconstruct two kinase signalling networks that characterise sensitive and resistant cells. The analysis of these networks suggested that the activation of the PI3K/AKT signalling pathway is critical for cisplatin resistance. Experimental validation of the computational model predictions confirmed that TNBC cell lines with activated PI3K/AKT signalling are sensitive to combinations of cisplatin and PI3K/AKT pathway inhibitors. Thus, our results reveal a new therapeutic approach that is based on identifying targeted therapies that synergise with conventional chemotherapies.

BRCA mutations lead to XIAP overexpression and sensitise ovarian cancer to inhibitor of apoptosis (IAP) family inhibitors.

BACKGROUND: We tested the hypothesis that inhibitor of apoptosis family (IAP) proteins may be altered in BRCA1-mutated ovarian cancers and that could affect the sensitivity to IAP inhibitors. METHODS: The levels of IAP proteins were evaluated in human cancers and cell lines. Cell lines were used to determine the effects of IAP inhibitors. The in vivo effects of treatments were evaluated in PDX mouse models. RESULTS: Expression of X-linked inhibitor of apoptosis (XIAP) is increased in BRCA1-mutated cancers and high levels are associated with improved patient outcomes after platinum chemotherapy. XIAP overexpression is mediated by NF-kB activation and is associated with an optimisation of PARP. BRCA1-mutated cell lines are particularly sensitive to IAP inhibitors due to an inhibitory effect on PARP. Both a BRCA1-mutated cell line with acquired resistance to PARP inhibitors and one with restored BRCA1 remain sensitive to IAP inhibitors. Treatment with IAP inhibitors restores the efficacy of PARP inhibition in these cell lines. The IAP inhibitor LCL161 alone and in combination with a PARP inhibitor, exhibited antitumour effects in PDX mouse models of resistant BRCA2 and 1-mutated ovarian cancer, respectively. CONCLUSION: A clinical trial may be justified to further investigate the utility of IAP inhibitors.

Functional Antagonism of Junctional Adhesion Molecule-A (JAM-A), Overexpressed in Breast Ductal Carcinoma In Situ (DCIS), Reduces HER2-Positive Tumor Progression.

Breast ductal carcinoma in situ (DCIS) is clinically challenging, featuring high diagnosis rates and few targeted therapies. Expression/signaling from junctional adhesion molecule-A (JAM-A) has been linked to poor prognosis in invasive breast cancers, but its role in DCIS is unknown. Since progression from DCIS to invasive cancer has been linked with overexpression of the human epidermal growth factor receptor-2 (HER2), and JAM-A regulates HER2 expression, we evaluated JAM-A as a therapeutic target in DCIS. JAM-A expression was immunohistochemically assessed in patient DCIS tissues. A novel JAM-A antagonist (JBS2) was designed and tested alone/in combination with the HER2 kinase inhibitor lapatinib, using SUM-225 cells in vitro and in vivo as validated DCIS models. Murine tumors were proteomically analyzed. JAM-A expression was moderate/high in 96% of DCIS patient tissues, versus 23% of normal adjacent tissues. JBS2 bound to recombinant JAM-A, inhibiting cell viability in SUM-225 cells and a primary DCIS culture in vitro and in a chick embryo xenograft model. JBS2 reduced tumor progression in in vivo models of SUM-225 cells engrafted into mammary fat pads or directly injected into the mammary ducts of NOD-SCID mice. Preliminary proteomic analysis revealed alterations in angiogenic and apoptotic pathways. High JAM-A expression in aggressive DCIS lesions and their sensitivity to treatment by a novel JAM-A antagonist support the viability of testing JAM-A as a novel therapeutic target in DCIS.

Targeting the PI3K and MAPK pathways to improve response to HER2-targeted therapies in HER2-positive gastric cancer.

BACKGROUND: Aberrant PI3K signalling is implicated in trastuzumab resistance in HER2-positive gastric cancer (GC). The role of PI3K or MEK inhibitors in sensitising HER2-positive GCs to trastuzumab or in overcoming trastuzumab resistance is unclear. METHODS: Using mass spectrometry-based genotyping we analysed 105 hotspot, non-synonymous somatic mutations in PIK3CA and ERBB-family (EGFR, ERBB2, ERBB3 and ERBB4) genes in gastric tumour samples from 69 patients. A panel of gastric cell lines (N87, OE19, ESO26, SNU16, KATOIII) were profiled for anti-proliferative response to the PI3K inhibitor copanlisib and the MEK1/2 inhibitor refametinib alone and in combination with anti-HER2 therapies. RESULTS: Patients with HER2-positive GC had significantly poorer overall survival compared to HER2-negative patients (15.9 months vs. 35.7 months). Mutations in PIK3CA were only identified in HER2-negative tumours, while ERBB-family mutations were identified in HER2-positive and HER2-negative tumours. Copanlisib had anti-proliferative effects in 4/5 cell lines, with IC50s ranging from 23.4 (N87) to 93.8 nM (SNU16). All HER2-positive cell lines except SNU16 were sensitive to lapatinib (IC50s 0.04 µM-1.5 µM). OE19 cells were resistant to trastuzumab. The combination of lapatinib and copanlisib was synergistic in ESO-26 and OE-19 cells (ED50: 0.83 ± 0.19 and 0.88 ± 0.13, respectively) and additive in NCI-N87 cells (ED50:1.01 ± 0.55). The combination of copanlisib and trastuzumab significantly improved growth inhibition compared to either therapy alone in NCI-N87, ESO26 and OE19 cells (p < 0.05). CONCLUSIONS: PI3K or MEK inhibition alone or in combination with anti-HER2 therapy may represent an improved treatment strategy for some patients with HER2-positive GC, and warrants further investigation in a clinical trial setting.

AMPK-regulated miRNA-210-3p is activated during ischaemic neuronal injury and modulates PI3K-p70S6K signalling.

Progressive neuronal injury following ischaemic stroke is associated with glutamate-induced depolarization, energetic stress and activation of AMP-activated protein kinase (AMPK). We here identify a molecular signature associated with neuronal AMPK activation, as a critical regulator of cellular response to energetic stress following ischaemia. We report a robust induction of microRNA miR-210-3p both in vitro in primary cortical neurons in response to acute AMPK activation and following ischaemic stroke in vivo. Bioinformatics and reverse phase protein array analysis of neuronal protein expression changes in vivo following administration of a miR-210-3p mimic revealed altered expression of phosphatase and tensin homolog (PTEN), 3-phosphoinositide-dependent protein kinase 1 (PDK1), ribosomal protein S6 kinase (p70S6K) and ribosomal protein S6 (RPS6) signalling in response to increasing miR-210-3p. In vivo, we observed a corresponding reduction in p70S6K activity following ischaemic stroke. Utilizing models of glutamate receptor over-activation in primary neurons, we demonstrated that induction of miR-210-3p was accompanied by sustained suppression of p70S6K activity and that this effect was reversed by miR-210-3p inhibition. Collectively, these results provide new molecular insight into the regulation of cell signalling during ischaemic injury, and suggest a novel mechanism whereby AMPK regulates miR-210-3p to control p70S6K activity in ischaemic stroke and excitotoxic injury.

Systems analysis of protein signatures predicting cetuximab responses in KRAS, NRAS, BRAF and PIK3CA wild-type patient-derived xenograft models of metastatic colorectal cancer.

Antibodies targeting the human epidermal growth factor receptor (EGFR) are used for the treatment of RAS wild-type metastatic colorectal cancer. A significant proportion of patients remains unresponsive to this therapy. Here, we performed a reverse-phase protein array-based (phospho)protein analysis of 63 KRAS, NRAS, BRAF and PIK3CA wild-type metastatic CRC tumours. Responses of tumours to anti-EGFR therapy with cetuximab were recorded in patient-derived xenograft (PDX) models. Unsupervised hierarchical clustering of pretreatment tumour tissue identified three clusters, of which Cluster C3 was exclusively composed of responders. Clusters C1 and C2 exhibited mixed responses. None of the three protein clusters exhibited a significant correlation with transcriptome-based subtypes. Analysis of protein signatures across all PDXs identified 14 markers that discriminated cetuximab-sensitive and cetuximab-resistant tumours: PDK1 (S241), caspase-8, Shc (Y317), Stat3 (Y705), p27, GSK-3ß (S9), HER3, PKC-α (S657), EGFR (Y1068), Akt (S473), S6 ribosomal protein (S240/244), HER3 (Y1289), NF-κB-p65 (S536) and Gab-1 (Y627). Least absolute shrinkage and selection operator and binominal logistic regression analysis delivered refined protein signatures for predicting response to cetuximab. (Phospo-)protein analysis of matched pretreated and posttreated models furthermore showed significant reduction of Gab-1 (Y627) and GSK-3ß (S9) exclusively in responding models, suggesting novel targets for treatment.

Targeting c-Met in triple negative breast cancer: preclinical studies using the c-Met inhibitor, Cpd A.

Introduction Triple negative breast cancer (TNBC) represents a heterogeneous subtype of breast cancer that carries a poorer prognosis. There remains a need to identify novel drivers of TNBC, which may represent targets to treat the disease. c-Met overexpression is linked with decreased survival and is associated with the basal subtype of breast cancer. Cpd A, a kinase inhibitor selective/specific for Met kinase has demonstrated preclinical anti-cancer efficacy in TNBC. We aimed to assess the anti-cancer efficacy of Cpd A when combined with Src kinase, ErbB-family or hepatocyte growth factor (HGF) inhibitors in TNBC cell lines. Methods We determined the anti-proliferative effects of Cpd A, rilotumumab, neratinib and saracatinib tested alone and in combination in a panel of TNBC cells by acid phosphatase assays. We performed reverse phase protein array analysis of c-Met and IGF1Rß expression and phosphorylation of c-Met (Y1234/1235) in TNBC cells and correlated their expression/phosphorylation with Cpd A sensitivity. We examined the impact of Cpd A, neratinib and saracatinib tested alone and in combination on invasive potential and colony formation.Results TNBC cells are not inherently sensitive to Cpd A, and neither c-Met expression nor phosphorylation are biomarkers of sensitivity to Cpd A. Cpd A enhanced the anti-proliferative effects of neratinib in vitro; however, this effect was limited to cell lines with innate sensitivity to Cpd A. Cpd A had limited anti-invasive effects but it reduced colony formation in the TNBC cell line panel.Conclusions Despite Cpd A having a potential role in reducing cancer cell metastasis, identification of strong predictive biomarkers of c-Met sensitivity would be essential to the development of a c-Met targeted treatment for an appropriately selected cohort of TNBC patients.

Identification and clinical impact of potentially actionable somatic oncogenic mutations in solid tumor samples.

BACKGROUND: An increasing number of anti-cancer therapeutic agents target specific mutant proteins that are expressed by many different tumor types. Successful use of these therapies is dependent on the presence or absence of somatic mutations within the patient's tumor that can confer clinical efficacy or drug resistance. METHODS: The aim of our study was to determine the type, frequency, overlap and functional proteomic effects of potentially targetable recurrent somatic hotspot mutations in 47 cancer-related genes in multiple disease sites that could be potential therapeutic targets using currently available agents or agents in clinical development. RESULTS: Using MassArray technology, of the 1300 patient tumors analysed 571 (43.9%) had at least one somatic mutation. Mutations were identified in 30 different genes. KRAS (16.5%), PIK3CA (13.6%) and BRAF (3.8%) were the most frequently mutated genes. Prostate (10.8%) had the lowest number of somatic mutations identified, while no mutations were identified in sarcoma. Ocular melanoma (90.6%), endometrial (72.4%) and colorectal (66.4%) tumors had the highest number of mutations. We noted high concordance between mutations in different parts of the tumor (94%) and matched primary and metastatic samples (90%). KRAS and BRAF mutations were mutually exclusive. Mutation co-occurrence involved mainly PIK3CA and PTPN11, and PTPN11 and APC. Reverse Phase Protein Array (RPPA) analysis demonstrated that PI3K and MAPK signalling pathways were more altered in tumors with mutations compared to wild type tumors. CONCLUSIONS: Hotspot mutational profiling is a sensitive, high-throughput approach for identifying mutations of clinical relevance to molecular based therapeutics for treatment of cancer, and could potentially be of use in identifying novel opportunities for genotype-driven clinical trials.

Combined targeting EGFR and SRC as a potential novel therapeutic approach for the treatment of triple negative breast cancer.

BACKGROUND: Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited therapeutic options. Epidermal growth factor receptor (EGFR) has been shown to be over-expressed in TNBC and represents a rational treatment target. METHODS: We examined single agent and combination effects for afatinib and dasatinib in TNBC. We then determined IC50 and combination index values using Calcusyn. Functional analysis of single and combination treatments was performed using reverse phase protein array and cell cycle analysis. Finally, we determined the anticancer effects of the combination in vivo. RESULTS: A total of 14 TNBC cell lines responded to afatinib with IC50 values ranging from 0.008 to 5.0 µM. Three cell lines, belonging to the basal-like subtype of TNBC, were sensitive to afatinib. The addition of afatinib enhanced response to the five other targeted therapies in HCC1937 and HDQP1 cells. The combination of afatinib with dasatinib caused the greatest growth inhibition in both cell lines. The afatinib/dasatinib combination was synergistic and/or additive in 13/14 TNBC cell lines. Combined afatinib/dasatinib treatment induced G1 cell cycle arrest. Reverse phase protein array results showed the afatinib/dasatinib combination resulted in efficient inhibition of both pERK(T202/T204) and pAkt(S473) signalling in BT20 cells, which was associated with the greatest antiproliferative effects. High baseline levels of pSrc(Y416) and pMAPK(p38) correlated with sensitivity to afatinib, whereas low levels of B-cell lymphoma 2 (Bcl2) and mammalian target of rapamycin (mTOR) correlated with synergistic growth inhibition by combined afatinib and dasatinib treatment. In vivo, the combination treatment inhibited tumour growth in a HCC1806 xenograft model. CONCLUSIONS: We demonstrate that afatinib combined with dasatinib has potential clinical activity in TNBC but warrants further preclinical investigation.

Systems biology analysis identifies molecular determinants of chemotherapy-induced diarrhoea.

Chemotherapy-induced diarrhoea (CID) is a common dose-limiting adverse event in patients with cancer. Here, we hypothesise that chemotherapy evokes apoptosis in normal gut epithelium, contributes to CID and that patients with increased risk of CID can be identified using a systems model of BCL-2 protein interactions (DR_MOMP) that calculates the sensitivity of cells to undergo apoptosis. Normal adjacent gut epithelium tissue was collected during resection surgery from a cohort of 35 patients with stage II-III colorectal cancer (CRC) who were subsequently treated with capecitabine, XELOX or FOLFOX. Clinical follow-up, type and grade of adverse events during adjuvant chemotherapy were recorded. The level of five BCL-2 proteins required for the calculation of the DR_MOMP score was quantified together with 62 additional signalling proteins related to apoptotic pathways. Odds ratios for the occurrence of diarrhoea were determined using multinomial logistic regression (MLR). Patients treated with capecitabine who had a DR_MOMP score equal or higher than the mean had a significantly lower frequency of diarrhoea significantly compared to patients below the mean. High DR_MOMP scores indicate high apoptosis resistance. No statistical difference was observed in patients treated with XELOX or FOLFOX. Using MLR, we found that levels of apoptosis-related proteins caspase-8, p53 and XIAP statistically interacted with the DR_MOMP stress dose. Markers of MAPK signalling were prognostic for diarrhoea independently of DR_MOMP. In conclusion, apoptosis sensitivity and MAPK signalling status of the adjacent normal gut epithelium of chemotherapy-naïve patients represent promising biomarkers to identify patients with CRC with increased risk of CID.

Implementing Reverse Phase Protein Array Profiling as a Sensitive Method for the Early Pre-Clinical Detection of Off-Target Toxicities Associated with Sunitinib Malate.

PURPOSE: The tyrosine kinase inhibitor (TKI) sunitinib is a multi-targeted agent approved across multiple cancer indications. Nevertheless, since approval, data has emerged to describe a worrisome side effect profile including hypertension, hand-foot syndrome, fatigue, diarrhea, mucositis, proteinuria, and (rarely) congestive heart failure. It has been hypothesized that the observed multi-parameter toxicity profile is related to "on-target" kinase inhibition in "off-target" tissues. EXPERIMENTAL DESIGN: To interrogate off-target effects in pre-clinical studies, a reverse phase protein array (RPPA) approach is employed. Mice are treated with sunitinib (40 mg kg-1 ) for 4 weeks, following which critical organs are removed. The Zeptosens RPPA platform is employed for protein expression analysis. RESULTS: Differentially expressed proteins associated with damage and/or stress are found in the majority of organs from treated animals. Proteins differentially expressed in the heart are associated with myocardial hypertrophy, ischaemia/reperfusion, and hypoxia. However, hypertrophy is not evidenced on histology. Mild proteinuria is observed; however, no changes in renal glomerular structure are visible via electron microscopy. In skin, proteins associated with cutaneous inflammation, keratinocyte hyper-proliferation, and increased inflammatory response are differentially expressed. CONCLUSIONS AND CLINICAL RELEVANCE: It is posited that pre-clinical implementation of a combined histopathological/RPPA approach provides a sensitive method to mechanistically elucidate the early manifestation of TKI on-target/organ off-target toxicities.

Markers of MEK inhibitor resistance in low-grade serous ovarian cancer: EGFR is a potential therapeutic target.

BACKGROUND: Although low-grade serous ovarian cancer (LGSC) is rare, case-fatality rates are high as most patients present with advanced disease and current cytotoxic therapies are not overly effective. Recognizing that these cancers may be driven by MAPK pathway activation, MEK inhibitors (MEKi) are being tested in clinical trials. LGSC respond to MEKi only in a subgroup of patients, so predictive biomarkers and better therapies will be needed. METHODS: We evaluated a number of patient-derived LGSC cell lines, previously classified according to their MEKi sensitivity. Two cell lines were genomically compared against their matching tumors samples. MEKi-sensitive and MEKi-resistant lines were compared using whole exome sequencing and reverse phase protein array. Two treatment combinations targeting MEKi resistance markers were also evaluated using cell proliferation, cell viability, cell signaling, and drug synergism assays. RESULTS: Low-grade serous ovarian cancer cell lines recapitulated the genomic aberrations from their matching tumor samples. We identified three potential predictive biomarkers that distinguish MEKi sensitive and resistant lines: KRAS mutation status, and EGFR and PKC-alpha protein expression. The biomarkers were validated in three newly developed LGSC cell lines. Sub-lethal combination of MEK and EGFR inhibition showed drug synergy and caused complete cell death in two of four MEKi-resistant cell lines tested. CONCLUSIONS: KRAS mutations and the protein expression of EGFR and PKC-alpha should be evaluated as predictive biomarkers in patients with LGSC treated with MEKi. Combination therapy using a MEKi with EGFR inhibition may represent a promising new therapy for patients with MEKi-resistant LGSC.

Germline single nucleotide polymorphisms in ERBB3 and BARD1 genes result in a worse relapse free survival response for HER2-positive breast cancer patients treated with adjuvant based docetaxel, carboplatin and trastuzumab (TCH).

Breast cancer is the leading cause of cancer related deaths in women worldwide and is classified into subtypes based on the cancer's receptor status. Of these subtypes, those expressing the human epidermal growth factor receptor 2 (HER2) receptor were traditionally associated with poor prognosis. Several advances have been made in the treatment of HER2-positive breast cancer, yet issues of resistance and poor response to therapy remains prevalent. In this study we explored the impact of HER-family and homologous recombination deficiency SNPs on response to patients who received TCH-based (docetaxel (T), carboplatin (C), and trastuzumab (H)) treatment versus those who received other treatment regimens. Using Cox regression analysis, we identified 6 SNPs that correlate with recurrence free survival in our patients and supported our findings using support vector machines. We also used reverse phase protein array analysis to examine the impact ERBB3 SNPs may have on both the PI3K/AKT and MAPK/ERK signaling pathways. Finally, using cell line models, we correlated SNP status with sensitivity to platinum based drugs and docetaxel. We found that patients on a TCH based regimen with the minor allele of the ERBB3 (rs2229046 and rs773123) and BARD1 (rs2070096) SNPs, were significantly more likely to relapse than those women who were not. Additionally, we observed that patients with these ERBB3 SNPs had shown elevated protein expression/phosphorylation of Src kinase, c-MET (Y1234/1235), GSK-3ß (S9) and p27, indicating that these SNPs are associated with non-PI3K/AKT signaling. Finally, using cell line models, we demonstrate that the BARD1 SNP (rs2229571) is associated with greater sensitivity to both carboplatin and cisplatin. The BARD1 and ERBB3 SNPs can potentially be used to determine those patients that will have a worse response to TCH based treatment, an effect that may arise from the SNPs impact on altered cellular signaling.

Frequency, impact and a preclinical study of novel ERBB gene family mutations in HER2-positive breast cancer.

BACKGROUND: Somatic mutations in the ERBB genes (epidermal growth factor receptor: EGFR, ERBB2, ERBB3, ERBB4) promote oncogenesis and lapatinib resistance in metastatic HER2+ (human epidermal growth factor-like receptor 2) breast cancer in vitro. Our study aimed to determine the frequency of mutations in four genes: EGFR, ERBB2, ERBB3 and ERBB4 and to investigate whether these mutations affect cellular behaviour and therapy response in vitro and outcomes after adjuvant trastuzumab-based therapy in clinical samples. METHODS: We performed Agena MassArray analysis of 227 HER2+ breast cancer samples to identify the type and frequency of ERBB family mutations. Of these, two mutations, the somatic mutations ERBB4-V721I and ERBB4-S303F, were stably transfected into HCC1954 (PIK3CA mutant), HCC1569 (PIK3CA wildtype) and BT474 (PIK3CA mutant, ER positive) HER2+ breast cancer cell lines for functional in vitro experiments. RESULTS: A total of 12 somatic, likely deleterious mutations in the kinase and furin-like domains of the ERBB genes (3 EGFR, 1 ERBB2, 3 ERBB3, 5 ERBB4) were identified in 7% of HER2+ breast cancers, with ERBB4 the most frequently mutated gene. The ERBB4-V721I kinase domain mutation significantly increased 3D-colony formation in 3/3 cell lines, whereas ERBB4-S303F did not increase growth rate or 3D colony formation in vitro. ERBB4-V721I sensitized HCC1569 cells (PIK3CA wildtype) to the pan class I PI3K inhibitor copanlisib but increased resistance to the pan-HER family inhibitor afatinib. The combinations of copanlisib with trastuzumab, lapatinib, or afatinib remained synergistic regardless of ERBB4-V721I or ERBB4-S303F mutation status. CONCLUSIONS: ERBB gene family mutations, which are present in 7% of our HER2+ breast cancer cohort, may have the potential to alter cellular behaviour and the efficacy of HER- and PI3K-inhibition.

Development of a personalized therapeutic strategy for ERBB-gene-mutated cancers.

Background: The application of genomic technologies to patient tumor samples identified groups of signaling pathways which acquire activating mutations. Some cancers are dependent on these mutations and the aberrant proteins resulting from these mutations can be targeted by novel drugs which can eradicate the cancer. Methods: We used www.cbioportal.org to determine the frequency of ERBB mutations in solid tumors. We then determined the sensitivity of a panel of cell lines to clinically available PI3K inhibitors. Using proliferation and apoptosis assays as well as functional interrogation with reverse phase protein arrays we demonstrated the impact of targeting ERBB-mutant cancers with the combination of a PI3K inhibitor and the pan-HER family inhibitor afatinib. Results: In over 14,000 patients we found that 12% of their tumors have an ERBB family gene mutation (EGFR, ERBB2, ERBB3 and ERBB4). In cancers not commonly associated with HER family protein overexpression, such as ovarian, endometrial, melanoma and head and neck cancers (n = 2116), we found that ERBB family mutations are enriched, occurring at rates from 14% to 34% and commonly co-occur with PIK3CA mutations. Importantly, we demonstrate that ERBB family mutant cancers are sensitive to treatment with PI3K inhibitors. Finally we show that the combination of afatinib and copanlisib represents a novel therapeutic strategy for patients whose cancers harbor both ERBB family and PIK3CA mutation. Conclusions: We demonstrate that ERBB family mutations are common in cancers not associated with overexpression or amplification of HER family proteins. These ERBB family mutant cancers are sensitive to treatment with PI3K inhibitors, and when combined with pan-HER inhibitors have synergistic antiproliferative effects.