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Mutational loss of CDKN2A (encoding p16INK4A) tumor-suppressor function is a key genetic step that complements activation of KRAS in promoting the development and malignant growth of pancreatic ductal adenocarcinoma (PDAC). However, pharmacologic restoration of p16INK4A function with inhibitors of CDK4 and CDK6 (CDK4/6) has shown limited clinical efficacy in PDAC. Here, we found that concurrent treatment with both a CDK4/6 inhibitor (CDK4/6i) and an ERK–MAPK inhibitor (ERKi) synergistically suppresses the growth of PDAC cell lines and organoids by cooperatively blocking CDK4/6i-induced compensatory upregulation of ERK, PI3K, antiapoptotic signaling, and MYC expression. On the basis of these findings, a Phase I clinical trial was initiated to evaluate the ERKi ulixertinib in combination with the CDK4/6i palbociclib in patients with advanced PDAC (NCT03454035). As inhibition of other proteins might also counter CDK4/6i-mediated signaling changes to increase cellular CDK4/6i sensitivity, a CRISPR-Cas9 loss-of-function screen was conducted that revealed a spectrum of functionally diverse genes whose loss enhanced CDK4/6i growth inhibitory activity. These genes were enriched around diverse signaling nodes, including cell-cycle regulatory proteins centered on CDK2 activation, PI3K–AKT–mTOR signaling, SRC family kinases, HDAC proteins, autophagy-activating pathways, chromosome regulation and maintenance, and DNA damage and repair pathways. Novel therapeutic combinations were validated using siRNA and small-molecule inhibitor–based approaches. In addition, genes whose loss imparts a survival advantage were identified (e.g., RB1, PTEN, FBXW7), suggesting possible resistance mechanisms to CDK4/6 inhibition. In summary, this study has identified novel combinations with CDK4/6i that may have clinical benefit to patients with PDAC. Significance: CRISPR-Cas9 screening and protein activity mapping reveal combinations that increase potency of CDK4/6 inhibitors and overcome drug-induced compensations in pancreatic cancer.

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Reverse phase protein microarray analysis was used to identify cell signaling derangements in squamous cell carcinoma (SCC) compared with actinic keratosis (AK) and upper inner arm (UIA). We analyzed two independent tissue sets with isolation and enrichment of epithelial cells by laser capture microdissection. Set 1 served as a pilot and a means to identify protein pathway activation alterations that could be further validated in a second independent set. Set 1 was comprised of 4 AK, 13 SCC, and 20 UIA. Set 2 included 15 AK, 9 SCCs, and 20 UIAs. Activation of 51 signaling proteins, known to be involved in tumorigenesis, were assessed for set 1 and showed that the MEK–ERK [mitogen-activated protein (MAP)/extracellular signal-regulated (ERK; MEK)] pathway was activated in SCC compared with AK and UIA, and that epidermal growth factor receptor (EGFR) and mTOR pathways were aberrantly activated in SCC. Unsupervised two-way hierarchical clustering revealed that AK and UIA shared a common signaling network activation architecture while SCC was dramatically different. Statistical analysis found that prosurvival signaling through phosphorylation of ASK and 4EBP1 as well as increased Bax and Bak expression was higher in AK compared with UIA. We expanded pathway network activation mapping in set 2 to 101 key signaling proteins, which corroborated activation of MEK–ERK, EGFR, and mTOR pathways through discovery of a number of upstream and downstream signaling molecules within these pathways to conclude that SCC is indeed a pathway activation–driven disease. Pathway activation mapping of SCC compared with AK revealed several interconnected networks that could be targeted with drug therapy for potential chemoprevention and therapeutic applications. Cancer Prev Res; 5(3); 403–13. ©2012 AACR.

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Supplementary Data and Materials

Mutational loss of CDKN2A (encoding p16INK4A) tumor-suppressor function is a key genetic step that complements activation of KRAS in promoting the development and malignant growth of pancreatic ductal adenocarcinoma (PDAC). However, pharmacologic restoration of p16INK4A function with inhibitors of CDK4 and CDK6 (CDK4/6) has shown limited clinical efficacy in PDAC. Here, we found that concurrent treatment with both a CDK4/6 inhibitor (CDK4/6i) and an ERK–MAPK inhibitor (ERKi) synergistically suppresses the growth of PDAC cell lines and organoids by cooperatively blocking CDK4/6i-induced compensatory upregulation of ERK, PI3K, antiapoptotic signaling, and MYC expression. On the basis of these findings, a Phase I clinical trial was initiated to evaluate the ERKi ulixertinib in combination with the CDK4/6i palbociclib in patients with advanced PDAC (NCT03454035). As inhibition of other proteins might also counter CDK4/6i-mediated signaling changes to increase cellular CDK4/6i sensitivity, a CRISPR-Cas9 loss-of-function screen was conducted that revealed a spectrum of functionally diverse genes whose loss enhanced CDK4/6i growth inhibitory activity. These genes were enriched around diverse signaling nodes, including cell-cycle regulatory proteins centered on CDK2 activation, PI3K–AKT–mTOR signaling, SRC family kinases, HDAC proteins, autophagy-activating pathways, chromosome regulation and maintenance, and DNA damage and repair pathways. Novel therapeutic combinations were validated using siRNA and small-molecule inhibitor–based approaches. In addition, genes whose loss imparts a survival advantage were identified (e.g., RB1, PTEN, FBXW7), suggesting possible resistance mechanisms to CDK4/6 inhibition. In summary, this study has identified novel combinations with CDK4/6i that may have clinical benefit to patients with PDAC.Significance:CRISPR-Cas9 screening and protein activity mapping reveal combinations that increase potency of CDK4/6 inhibitors and overcome drug-induced compensations in pancreatic cancer.

Supplementary Tables

XLSX file - 12K, Tumor Sample Information.

XLSX file - 17K, Antibody Information.

PDF file - 197K, Protein/Phosphoprotein comparison between matched LCM and non-LCM samples by unsupervised hierarchical clustering.

PDF file - 129K, Schematic of potential protein sources and contribution to overall signal from tissue analysis.

The efficacy of molecular targeted therapy depends on expression and enzymatic activity of the target molecules. As radiotherapy modulates gene expression and protein phosphorylation dependent on dose and fractionation, we analyzed the long-term effects of irradiation on the post-radiation efficacy of molecular targeted drugs. We irradiated prostate cancer cells either with a single dose (SD) of 10 Gy x-ray or a multifractionated (MF) regimen with 10 fractions of 1 Gy. Whole genome arrays and reverse phase protein microarrays were used to determine gene expression and protein phosphorylation. Additionally, we evaluated radiation-induced pathway activation with the Ingenuity Pathway Analysis software. To measure cell survival and sensitivity to clinically used molecular targeted drugs, we performed colony formation assays. We found increased activation of several pathways regulating important cell functions such as cell migration and cell survival at 24 h after MF irradiation or at 2 months after SD irradiation. Further, cells which survived a SD of 10 Gy showed a long-term upregulation and increased activity of multiple molecular targets including AKT, IGF-1R, VEGFR2, or MET, while HDAC expression was decreased. In line with this, 10 Gy SD cells were more sensitive to target inhibition with Capivasertib or Ipatasertib (AKTi), BMS-754807 (IGF-1Ri), or Foretinib (VEGFR2/METi), but less sensitive to Panobinostat or Vorinostat (HDACi). In summary, understanding the molecular short- and long-term changes after irradiation can aid in optimizing the efficacy of multimodal radiation oncology in combination with post-irradiation molecularly-targeted drug treatment and improving the outcome of prostate cancer patients.

While genes and RNA encode information about cellular status, proteins are considered the engine of the cellular machine, as they are the effective elements that drive all cellular functions including proliferation, migration, differentiation, and apoptosis. Consequently, investigations of the cellular protein network are considered a fundamental tool for understanding cellular functions.Alteration of the cellular homeostasis driven by elaborate intra- and extracellular interactions has become one of the most studied fields in the era of personalized medicine and targeted therapy. Increasing interest has been focused on developing and improving proteomic technologies that are suitable for analysis of clinical samples. In this context, reverse-phase protein microarrays (RPPA) is a sensitive, quantitative, high-throughput immunoassay for protein analyses of tissue samples, cells, and body fluids.RPPA is well suited for broad proteomic profiling and is capable of capturing protein activation as well as biochemical reactions such as phosphorylation, glycosylation, ubiquitination, protein cleavage, and conformational alterations across hundreds of samples using a limited amount of biological material. For these reasons, RPPA represents a valid tool for protein analyses and generates data that help elucidate the functional signaling architecture through protein-protein interaction and protein activation mapping for the identification of critical nodes for individualized or combinatorial targeted therapy.

While genes and RNA encode information about cellular status, proteins are considered the engine of the cellular machine, as they are the effective elements that drive all cellular functions including proliferation, migration, differentiation, and apoptosis. Consequently, investigations of the cellular protein network are considered a fundamental tool for understanding cellular functions.Alteration of the cellular homeostasis driven by elaborate intra- and extracellular interactions has become one of the most studied fields in the era of personalized medicine and targeted therapy. Increasing interest has been focused on developing and improving proteomic technologies that are suitable for analysis of clinical samples. In this context, reverse-phase protein microarrays (RPPA) is a sensitive, quantitative, high-throughput immunoassay for protein analyses of tissue samples, cells, and body fluids.RPPA is well suited for broad proteomic profiling and is capable of capturing protein activation as well as biochemical reactions such as phosphorylation, glycosylation, ubiquitination, protein cleavage, and conformational alterations across hundreds of samples using a limited amount of biological material. For these reasons, RPPA represents a valid tool for protein analyses and generates data that help elucidate the functional signaling architecture through protein-protein interaction and protein activation mapping for the identification of critical nodes for individualized or combinatorial targeted therapy.

Plasticity of the ErbB family of receptors and ligands is a hallmark of many human tumors and drives resistance to EGFR and HER2 targeting agents such as cetuximab or trastuzumab. Pan-HER is a unique antibody mixture designed to counter ErbB family plasticity by simultaneously targeting EGFR, HER2 and HER3. Pan-HER contains pairs of antibodies against each of the receptors, six antibodies in total, and induces efficient internalization and degradation of all three receptors. Here, the anti-tumor activity of Pan-HER was investigated in a panel of 57 patient-derived epithelial tumor models (PDXs) of various origin including pancreatic (18), colorectal (11), triple negative breast (11), lung (6), squamous head and neck (6), ovarian (3), and gastric (2) cancer, and the level of activity was correlated to baseline protein and phosphoprotein status using laser capture microdissection (LCM) and Reverse Phase Protein Arrays (RPPA). This panel has been preselected to represent hard-to-treat cancers and that are not overtly HER family positive. Pan-HER demonstrated anti-tumor activity across all indications except the gastric models and induced tumor regression in 26 of the 57 PDX models tested. The LCM-RPPA analysis of over 150 proteins and phosphoproteins that are known to be central for receptor tyrosine kinase signaling, survival, apoptosis, growth, inflammation, motility and autophagy identified both broad and indication specific signatures of response to Pan-HER and points to a key role for the KRAS/BRAF/ERK and PIK3CA/AKT pathway activation that associate with therapeutic sensitivity. In conclusion, our results demonstrate that Pan-HER has broad anti-tumor activity across indications and identify phosphoprotein pathway activation signatures predicting for sensitivity or resistance that should be explored clinically. Citation Format: Valerie S. Calvert, Camilla Fröhlich, Thomas T. Poulsen, Mikkel W. Pedersen, Michael Kragh, Ivan D. Horak, Emanuel F. Petricoin. Protein pathway activation analysis of a panel of patient-derived epithelial tumor models identifies signatures of response to ErbB1-3 co-targeting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-216.

Scientists employing microarray profiling technology to compare sample sets generate data for a large number of endpoints. Assuming the experimental design minimized sources of bias, and the analytical technology was reliable, precise, and accurate, how does the experimentalist determine which endpoints are meaningfully different between the groups? Comparison of two population means for individual analysis measurements is the most common statistical problem associated with microarray data analysis. This chapter focuses on the hands-on procedures using SAS software to describe how to choose statistical methods to find the statistically significantly different endpoints between two groups of data generated from reverse phase protein microarrays. The four methods outlined are: (a) two-sample t-test, (b) Wilcoxon rank sum test, (c) one-sample t-test, and (d) Wilcoxon signed rank test. Two sample t-test is used for two independently normally distributed groups. One-sample t-test is used for a normally distributed difference of paired observations. Wilcoxon rank sum test is considered a nonparametric version of the two-sample t-test, and Wilcoxon signed rank test is considered a nonparametric version of the one-sample t-test.

MicroRNAs (miRs) are small molecules that post-transcriptionally regulate expression of target mRNAs and are thought to play a major role in prostate cancer (CaP) tumorigenesis. We compared miR expression in a progressive prostate cancer cell model consisting of the immortalized p69 human prostate cell line compared to M12, a highly tumorigenic, metastatic subline, and F6, a weakly tumorigenic variant using Exiqon's miRCURY LNA™ miR human panels. Most notable was a decrease in expression of miR-125b and miR-17-3p, which was confirmed by single miR analysis via RT-qPCR. Previously, miR array screens have been done on RNA extracted from a variety of whole tumors. However, due to the heterogeneous nature of prostate tumors, it is impossible to determine which cell-type is contributing to differential miR expression. To address this concern, we have developed laser capture microdissection (LCM) to validate miR expression in RNA extracted from the individual cell-types (normal stroma, normal epithelial, PIN, and tumor cell of increasing Gleason score) that comprise human prostate tumors. This analysis validated that the expression of miR-17-3p and miR-125b is reduced in the prostate tumor cell compared to normal epithelium. Previously, we have shown that miR-17-3p targets vimentin and IGF1R. Restoration of miR-17-3p expression in the M12 cell line resulted in decreased cell motility and invasion in transwell (Boyden) chamber assays as well as decreased growth of subcutaneous and orthotopic tumors in male, athymic nude mice confirming miR-17-3p is behaving as a tumor suppressor. Previously, data from miRNA array screens coupled to gene arrays have been used to predict mRNA targets. This approach assumes that all miRNAs regulate gene expression by degrading their respective targets, which is not always the case. Since the end result of miRNA targeting is reduced protein synthesis, we propose that proteomics using RPMA, Reverse Phase Protein MicroArrays, is a better approach for deducing targets of miR regulation. Proteomic analysis of these same cell lines confirmed a correlative increase in pErB2/ErB3 and vimentin protein levels in cell lines where miR-125b and miR-17-3p expression are low. MiR-125b is proposed to target ErbB2/ErbB3. Interestingly, a network analysis of oncogenic miRNA targets in the prostate revealed that the EGFR family of which ErB2/ErB3 is thought to be a major driver of CaP was one of the most highly connected proteins. Currently, we are using a variety of in vitro and in vivo experiments to prove that miR-125b can function as a tumor suppressor as we have shown for miR-17-3p. Altogether this multi-factorial approach of miRNA array analysis coupled to proteomics, validated by LCM analysis of human tumor samples and additional experiments has identified two new tumor suppressors, miR-17-3p and miR-125b, which could be relevant biomarkers for CaP. Restoration of miR-17-3p and/or miR-125 expression could provide new targets/treatments to block prostate cancer progression. Citation Format: William T. Budd, Danielle E. Weaver, Valerie Calvert, Emanuel F. Petricoin, III, Joy L. Ware, Zendra E. Zehner. miR-125b and miR-17-3p: Newly identified tumor suppressors for prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B54.