Reverse Phase Protein Analysis Research Articles

Abstract B019: Clinical and molecular correlates of circulating tumor DNA (ctDNA) dynamics in breast tumors resistant to neoadjuvant therapy (NAT)

Abstract Purpose: We examined the utility of serial ctDNA testing for refining risk stratification of NAT- resistant tumors (RTs, defined as residual cancer burden, RCB-II/III) and predicting early treatment response. We also characterized the molecular correlates of ctDNA dynamics in RTs to elucidate the mechanisms associated with increased metastatic potential. Methods: We performed serial ctDNA testing using a tumor-informed personalized assay (SignateraTM, Natera Inc.) in 723 patients (pts) with high-risk early-stage breast cancer receiving NAT (I-SPY2). Tumors were stratified according to RCB class and ctDNA dynamics (clearance patterns), and the distant recurrence-free survival (DRFS) between groups was compared. We examined the predictive value of ctDNA dynamics using the RCB index (the continuous measure of RCB) as the response endpoint. Serial whole exome sequencing (WES), gene expression profiling (GEP), and reverse-phase protein analysis (RPPA) data were analyzed to elucidate the molecular biology of RTs. Results: Of the 723 pts, 300 (41%) had hormone receptor (HR)+HER2-, 237 (33%) triple-negative (TN), and 186 (26%) HER2+ breast cancers with pretreatment ctDNA-positivity rates of 76%, 92%, and 77%, respectively. 321 (45%) pts had responding tumors (RCB-0/I), and 55% had RTs: RCB-II (n=255) and RCB-III (n=132). CtDNA dynamics revealed heterogeneity in the metastatic potential of RTs, including tumors with a reduced propensity to metastasize (persistent ctDNAneg and early clearance). Failure to clear ctDNA after NAT was associated with an increased risk of metastatic recurrence and death (3-yr DRFS rates: no clearance=35% vs. persistent ctDNAneg=98%, p<0.001). Early clearance of ctDNA, 3 weeks after treatment initiation, skewed the distributions toward lower RCB indices (favorable response), including in arms containing immune checkpoint inhibitors. Of the 387 RTs, 316 had ctDNA data post-NAT, of which only 51 (16%) were ctDNA+. We hypothesized that changes in the mutational landscape during NAT could have impacted the detection of patient-specific ctDNA targets selected from WES of pretreatment tumors. However, serial WES (n=100) of RTs showed that these targets remained detectable in solid tissue over time, even in persistent ctDNAneg. To investigate the biology of persistent ctDNA positivity, we used logistic regression to analyze serial GEP (n=174) and RPPA (n=98) data in RTs that were ctDNA+ at pretreatment, comparing those with ctDNA clearance by surgery vs. those that remained ctDNA+. We found subtype- specific associations: In HR+HER2-, failure to clear ctDNA during NAT was associated with low pretreatment ER/AR, high early on-treatment proliferation, and low HER2-signaling after NAT, while in TN, fewer associations were observed; these included high pretreatment levels of MYC and proliferation. Conclusions: ctDNA dynamics refined risk stratification of RTs and enabled early prediction of treatment response. Understanding the molecular profiles of RTs based on ctDNA dynamics could aid treatment selection to overcome resistance to NAT. Citation Format: Mark Jesus M. Magbanua, Denise M. Wolf, Samuel Rivera-Hinojosa, Ziad Ahmed, Nayelis A. Manon, Rosalyn Sayaman, Antony Tin, Ekaterina Kalashnikova, Lamorna Brown Swigart, Gillian L. Hirst, Christina Yau, Wen Li, Claudine Isaacs, Rebecca A. Shatsky, Amy L. Delson, Charuta C. Palsuledesai, Angel Rodriguez, Minetta C. Liu, Paula R. Pohlmann, Laura J. Esserman, Hope S. Rugo, Angela DeMichele, Laura van 't Veer. Clinical and molecular correlates of circulating tumor DNA (ctDNA) dynamics in breast tumors resistant to neoadjuvant therapy (NAT) [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B019.

Bone Marrow Endothelial Cells Increase Prostate Cancer Cell Apoptosis in 3D Triculture Model of Reactive Stroma.

Simple SummaryProstate cancer (PCa) metastasizes preferentially to the bone marrow where it becomes difficult to treat. PCa cells in the bone marrow may survive, dormant and undetected for many years before patients eventually relapse with metastatic disease. Bone marrow is a complex tissue that initially is hostile to the PCa cells, Understanding how cancer cells survive in the bone marrow and what changes to the bone microenvironment permit them to switch to an actively growing state could offer new therapeutic strategies to combat metastatic PCa. In this study, we describe a method to culture PCa cells with two other cell types from the bone marrow, stromal cells and endothelial cells, as a way to study the interactions among these cell types. We found that factors produced by bone marrow endothelial cells, but not endothelial cells from other tissues, trigger PCa cells to either die or enter a dormant state, similar to what has been observed in patients when PCa cells initially colonize the bone marrow. Further analysis of the cell interactions within the culture model described in this study will offer increased understanding of PCa interaction with the bone marrow environment.The bone marrow tumor microenvironment (BMTE) is a complex network of cells, extracellular matrix, and sequestered signaling factors that initially act as a hostile environment for disseminating tumor cells (DTCs) from the cancerous prostate. Three-dimensional (3D) culture systems offer an opportunity to better model these complex interactions in reactive stroma, providing contextual behaviors for cancer cells, stromal cells, and endothelial cells. Using a new system designed for the triculture of osteoblastic prostate cancer (PCa) cells, stromal cells, and microvascular endothelial cells, we uncovered a context-specific pro-apoptotic effect of endothelial cells of the bone marrow different from those derived from the lung or dermis. The paracrine nature of this effect was demonstrated by observations that conditioned medium from bone marrow endothelial cells, but not from dermal or lung endothelial cells, led to PCa cell death in microtumors grown in 3D BMTE-simulating hydrogels. Analysis of the phosphoproteome by reverse phase protein analysis (RPPA) of PCa cells treated with conditioned media from different endothelial cells identified the differential regulation of pathways involved in proliferation, cell cycle regulation, and apoptosis. The findings from the RPPA were validated by western blotting for representative signaling factors identified, including forkhead box M1 (FOXM1; proliferation factor), pRb (cell cycle regulator), and Smac/DIABLO (pro-apoptosis) among treatment conditions. The 3D model presented here thus presents an accurate model to study the influence of the reactive BMTE, including stromal and endothelial cells, on the adaptive behaviors of cancer cells modeling DTCs at sites of bone metastasis. These findings in 3D culture systems can lead to a better understanding of the real-time interactions among cells present in reactive stroma than is possible using animal models.

Acute exercise in 5‐Fluorouracil cardiotoxicity: Effects on cardiac function and reverse phase protein analysis

Background5‐Fluorouracil (5FU) is a commonly administered chemotherapeutic agent used in the treatment of numerous cancers. Though efficacious in treatment of malignancies, 5FU is associated with dose limiting cardiotoxicity that often necessitates deviation from preferred treatment regimens and increases reliance on other, less effective treatment alternatives. The mechanisms driving these toxicities are poorly understood, but leading theories include direct myocardial damage and coronary vasospasm. Data to support these theories have historically been derived from pre‐clinical animal models utilizing i.p injection of 5FU rather than protocols designed to mimic the drug delivery route utilized in clinical practice (i.v. infusion). Further, others have demonstrated the effectiveness of acute exercise in prevention of cardiotoxicity induced by other chemotherapies, however there are no studies evaluating the efficacy of exercise in prevention of 5FU cardiotoxicity.Purpose & HypothesisThe present investigation assessed the effect of 4 moderate intensity treadmill running bouts prior to administration of a clinically relevant dose of 5FU on the functional cardiovascular wellbeing of rats. We tested the hypotheses that a 2‐hr infusion of 5FU would reduce echocardiographic measures of ejection fraction (EF) and fractional shortening (%FS) compared to a saline infusion, and that completion of an acute exercise protocol prior to 5FU treatment would mitigate these cardiotoxic effects.MethodsMale Sprague‐Dawley rats were randomized to receive 5FU (n=15) or saline (CON, n=11) via a 50 mg/kg jugular bolus followed by a 2‐hr 265 mg/kg continuous infusion. A subset of each group (n=7 ex5FU, n=4 exCON) completed 4 bouts of moderate intensity treadmill running (25 min each) commencing 3 days prior to 5FU/saline treatment with the final run ending 1‐hr prior to treatment onset. The remaining animals acted as sedentary controls (n=7 s5FU, n=8 sCON). Echocardiographic variables were compared between groups at baseline and 2‐hr time points (mixed‐model design). Reverse phase protein analysis (RPPA) was conducted on cardiac tissue excised immediately following completion of the infusion for insight into molecular alterations induced by 5FU and/or exercise.ResultsEF at 2‐hr was reduced in sCON (92±2 vs 86±3%, P=.01), s5FU (91±2 vs 86±2%, P=.03), and ex5FU (87±2 vs 82±1%, P=.02), but not in exCON (91±2 vs 87±1%, P=.35) with no timepoint differences between groups (P=0.23). Similarly, %FS decreased from baseline to 2‐hr in sCON (60±3 vs 52±4%, P=.00), s5FU (59±3 vs 51±3%, P=.03), and ex5FU (53±3 vs 45±1%, P=.04), but not in exCON (58±4 vs 52±1, P=.28) with no differences between groups at either time point (P=0.97). RPAA analysis identified graded key changes in CAV‐1, SMAD, and BCL2L1.ConclusionThe present findings suggest small reductions in EF and %FS following completion of a 2‐hr infusion of 5FU or saline, with no differences between the treatments. Further, reductions in EF and %FS were mitigated by prior exercise in exCON, but not in ex5FU. Future work will place emphasis on quantifying the molecular changes indicated by RPPA to better understand alterations induced by 5FU within the heart at the cellular level.

Effects of Exercise on Aortic Stiffness Associated with 5‐Flurouracil in Rats

Background5‐fluorouracil (5‐FU) is a chemotherapy drug that is often prescribed in treatment regimens for malignancies of the gastrointestinal tract, breast, and head/neck. However, despite its clear efficacy in cancer treatment, it is also the second most common chemotherapeutic implicated in the development of short and/or long‐term cardiotoxicity that can lead to a less efficacious cancer treatment regimen. Specifically, 5‐FU is associated with increases in arterial stiffness, leading to increased risks for developing heart failure and cardiovascular disease. Exercise, both acutely and following long‐term training, has exhibited cardiovascular health benefits and protection in healthy and disease populations, including reductions in e.g., pulse wave velocity (PWV), a clinical measure of arterial stiffness. Therefore, the purpose of this study was to investigate 5‐FU induced arterial stiffness (via PWV) and evaluate the efficacy of multiple acute bouts of exercise to mitigate arterial stiffness associated with 5‐FU. Further, we utilized reverse phase protein analysis (RPPA) to investigate mechanisms associated with 5‐FU induced arterial stiffness.MethodsMale Sprague Dawley rats aged 6‐8 months (n=26) were used in this series of experiments. All animals were first acclimated to treadmill exercise, and randomized into four groups: Control (CON, n=8), 5‐FU only (5‐FU=7), exercise (EX, n=4), and 5‐FU with exercise (5‐FUEX, n=7). All animals were surgically instrumented with catheters in the external jugular vein and tail artery. Animals received a clinically relevant dose of 5‐FU with the combination of a bolus (50mg/kg), followed immediately by a 2h continuous infusion of 5‐FU (265mg/kg) through the jugular catheter. This was done to better imitate clinical drug delivery rather than i.p injections typically used in pre‐clinical models. Baseline and 2h measurements of PWV were made from the descending to lower abdominal aorta. Both exercise groups underwent four, 20–30‐minute acute bouts of moderate intensity exercise (20m/min, 5% incline) on consecutive days, 24h apart, with the last being ~10 minutes before surgical instrumentation. RPPA analysis was performed on abdominal aorta from each animal for analysis of changes in protein expression.ResultsThere were no differences in PWV between groups at either time point (p>0.05). However, a significant increase in PWV from baseline to the 2h time point was evident in the 5‐FU group alone (CON ‐1.1±4.9, 5‐FU 14.8±4.4, EX 4.6±5.7, 5‐FUEX 5.4.9±2.2%, p<0.05). Preliminary RPPA analysis indicated 1.5log2fold changes in 5‐FU animals in multiple factors, several of which have been associated with increased vascular stiffness including Steroid Receptor Co‐activator (SRC‐1) and mitogen‐activated protein kinase (MAPK1/MAPK3) that will require further investigation.ConclusionThis study suggests that 5‐FU induces aortic stiffness in rats that can be attenuated with prior acute bouts of exercise. These findings provide further efficacy of exercise therapy in cancer standards of care besides its established benefits for reducing fatigue and improving quality of life in cancer patients. Lastly, the potential to mitigate cardiovascular complications of 5‐FU therapy is clinically important as it can possibly prevent early cessation or reductions of this efficacious therapy.

Deregulation of AKT–mTOR Signaling Contributes to Chemoradiation Resistance in Lung Squamous Cell Carcinoma

Abstract Lung squamous cell carcinoma (LUSC) accounts for one of three of non–small cell lung carcinoma (NSCLC) and 30% of LUSC patients present with locally advanced, unresectable/medically inoperable disease, who are commonly treated with definitive chemoradiation. However, disease relapse in the radiation fields occurs in one of three cases. We aim to explore the underlying molecular mechanisms of chemoradiation resistance of LUSC. Patient-derived xenograft (PDX) models of LUSC were established in immunodeficient mice, followed by treatment with cisplatin in combination with clinically relevant courses of ionizing radiation (20, 30, and 40 Gy). The recurrent tumors were extracted for functional proteomics using reverse phase protein analysis (RPPA). We found that phospho-AKT-S473, phospho-AKT-T308, phospho-S6-S235/6, and phospho-GSK3β-S9 were upregulated in the chemoradiation-resistant 20 Gy + cisplatin and 40 Gy + cisplatin tumors compared with those in the control tumors. Ingenuity pathway analysis of the RPPA data revealed that AKT–mTOR signaling was the most activated signaling pathway in the chemoradiation-resistant tumors. Similarly, elevated AKT–mTOR signaling was observed in stable 40 Gy and 60 Gy resistant HARA cell lines compared with the parental cell line. Accordingly, pharmacologic inhibition of mTOR kinase by Torin2 significantly sensitized LUSC cell lines to ionizing radiation. In conclusion, using chemoradiation-resistant PDX models coupled with RPPA proteomics analysis, we revealed that deregulation of AKT–mTOR signaling may contribute to the chemoradiation resistance of LUSC. Implications: Clonal selection of subpopulations with high AKT–mTOR signaling in heterogeneous tumors may contribute to relapse of LUSC after chemoradiation. mTOR kinase inhibitors may be promising radiosensitizing agents in upfront treatment to prevent acquired resistance.

Targeted Therapy Given after Anti-PD-1 Leads to Prolonged Responses in Mouse Melanoma Models through Sustained Antitumor Immunity.

Immunotherapy (IT) and targeted therapy (TT) are both effective against melanoma, but their combination is frequently toxic. Here, we investigated whether the sequence of IT (anti-PD-1)→ TT (ceritinib-trametinib or dabrafenib-trametinib) was associated with improved antitumor responses in mouse models of BRAF- and NRAS-mutant melanoma. Mice with NRAS-mutant (SW1) or BRAF-mutant (SM1) mouse melanomas were treated with either IT, TT, or the sequence of IT→TT. Tumor volumes were measured, and samples from the NRAS-mutant melanomas were collected for immune-cell analysis, single-cell RNA sequencing (scRNA-seq), and reverse phase protein analysis (RPPA). scRNA-seq demonstrated that the IT→TT sequence modulated the immune environment, leading to increased infiltration of T cells, monocytes, dendritic cells and natural killer cells, and decreased numbers of tumor-associated macrophages, myeloid-derived suppressor cells, and regulatory T cells. Durable responses to the IT→TT sequence were dependent on T-cell activity, with depletion of CD8+, but not CD4+, T cells abrogating the therapeutic response. An analysis of transcriptional heterogeneity in the melanoma compartment showed the sequence of IT→TT enriched for a population of melanoma cells with increased expression of MHC class I and melanoma antigens. RPPA analysis demonstrated that the sustained immune response induced by IT→TT suppressed tumor-intrinsic signaling pathways required for therapeutic escape. These studies establish that upfront IT improves the responses to TT in BRAF- and NRAS-mutant melanoma models.

Topoisomerase 2B Decrease Results in Diastolic Dysfunction via p53 and Akt: A Novel Pathway.

Diastolic dysfunction is condition of a stiff ventricle and a function of aging. It causes significant cardiovascular mortality and morbidity, and in fact, three million Americans are currently suffering from this condition. To date, all the pharmacological clinical trials have been negative. The lack of success in attenuating/ameliorating diastolic dysfunction stems from lack of duplication of myriads of clinical manifestation in pre-clinical settings. Here we report, a novel genetically engineered mice which may represents a preclinical model of human diastolic dysfunction to some extent. Topoisomerase 2 beta (Top2b) is an important enzyme in transcriptional activation of some inducible genes through transient double-stranded DNA breakage events around promoter regions. We created a conditional, tissue-specific, inducible Top2b knockout mice in the heart. Serendipitously, echocardiographic parameters and more invasive analysis of left ventricular function with pressure–volume loops show features of diastolic dysfunction. This was also confirmed histologically. At the cellular level, the Top2b knockdown showed morphological changes and molecular signaling akin to human diastolic dysfunction. Reverse phase protein analysis showed activation of p53 and inhibition of, Akt, as the possible mediators of diastolic dysfunction. Finally, activation of p53 and inhibition of Akt were confirmed in myocardial biopsy samples obtained from human diastolic dysfunctional hearts. Thus, we report for the first time, a Top2b downregulated preclinical mice model for diastolic dysfunction which demonstrates that Akt and p53 are the possible mediators of the pathology, hence representing novel and viable targets for future therapeutic interventions in diastolic dysfunction.

AML-211: Active Galectin 3/CD74 Axis is Associated with Poor Survival Outcome in AML

Context Galectin 3 (LGALS3) participates in diverse survival pathways that support many pro-tumor molecules, especially RAS. However, the role of LGALS3 in acute myeloid leukemia (AML) is not clear. Objective We previously found suppression of LGALS3 in AML cell lines sensitized cells to chemotherapy drugs. Here, we examine protein expression of LGALS3 in 511 AML patients and identify an active LGALS3 protein network. We used reversephase protein analysis for the study. Patients Peripheral blood and bone marrow specimens were collected from 511 patients with newly diagnosed AML. Samples were acquired in accordance with the regulations and protocols (Lab 01-473) approved by the IRB of MDACC. Results LGALS3 was significantly elevated in AML blast cells compared to normal CD34+ counterpart cells and highest in monocytic AML patients. Pearson correlation of LGALS3 with other proteins identified a distinct set of 23 proteins positively and 13 proteins negatively correlated with LGALS3 expression levels. LGALS3 positively correlated with the anti-apoptotic BCL2 family member MCL-1. RPPA also identified novel positive association of LGALS3 with CD74. Pearson correlation with the other proteins analyzed identified 23 proteins, including LGALS3, that were positively correlated with CD74, and 8 proteins that were negatively associated with CD74. The protein associations of LGALS3 and CD74 were used to assemble protein clusters that were used for analysis of patient survival. LGALS3 and CD74 were collectively analyzed as part of a group of T-cell regulating proteins analyzed on this array. Within that group, 4 different expression patterns (clusters) were defined by hierarchical clustering with LGALS3 being active in 2 of them. The protein cluster identifying active LGALS3 was prognostic for shorter remission duration. AML patients who exhibited both LGALS3 and CD74 active clusters exhibited very poor survival characteristics. For AML patients with both LGALS3 and CD74 active protein cluster, median overall survival was only 24.3 weeks, median remission duration was 17.8 weeks, and no patient survived beyond one year. Conclusions These data suggest that LGALS3 supports AML cell survival via diverse pathways, including positive regulation of MCL-1, and support of a CD74 protein network that identifies a new at-risk AML population.

Abstract 1671: Deregulation of mTOR signaling contributes to chemoradiation resistance in lung squamous cell carcinoma

Abstract Non-small cell lung carcinoma (NSCLC) represents ~ 85% of lung cancer, and ~1/3 of these patients present with locally-advanced, unresectable/medically-inoperable disease. Patients who present with locally-advanced NSCLC are commonly treated with definitive chemoradiation. Even though an initial response to concurrent chemoradiation is commonly observed, disease relapse in the radiation fields occurs in 1/3 of cases, likely driven by therapy-resistant tumor cells. However, the underlying molecular mechanisms of chemoradiation resistance of NSCLC remains largely unknown. We herein report that upregulation of mTOR signaling plays an important role in the chemoradiation resistance of NSCLC. Patient-derived xenograft (PDX) models of lung squamous cell carcinoma (LUSC) were established in the flanks of NOD-SCID gamma (NSG) mice. Chemoradiation resistant tumor models were developed by treating the tumors with a regimen of 2-4 weeks of chemoradiation (20, 30 or 40 Gy at 2 Gy per day, concurrent with cisplatin). Recurrent tumor models were collected and subjected to functional proteomics using reverse phase protein analysis (RPPA). Several LUSC tumors recurred in the 20 Gy+cisplatin, and 40 Gy+cisplatin groups. RPPA analysis of the recurrent and parental (untreated) PDX tumors showed that there were enhanced mTOR signaling in the chemoradiation resistant 20 Gy+cisplatin and 40 Gy+cisplatin tumors compared to that in the control tumors. Of note, phospho-AKT-S473, phospho-AKT-S308, phospho-S6-S235/6 and phospho-GSK3β-S9/21 were the highest upregulated signaling and were validated by immunoblotting. Consistently, pharmacological inhibition of mTOR kinase by Torin2 significantly sensitized LUSC cell lines to ionizing radiation. Our results demonstrate that utilization of PDX models is a useful method to elucidate the molecular mechanisms of chemoradiation resistance of NSCLC and deregulation of mTOR signaling contributes to the chemoradiation resistance of NSCLC. Our findings suggest that clonal selection of subpopulations with high mTOR signaling in heterogeneous NSCLC tumors might contribute to the relapse of NSCLC after chemoradiation and that mTOR kinase inhibitors are promising agents for the upfront treatment of NSCLC patients in combination with chemoradiation. Citation Format: Changxian Shen, Duan-Liang Shyu, Min Xu, Linlin Yang, Wenrui Duan, Terence M. Williams. Deregulation of mTOR signaling contributes to chemoradiation resistance in lung squamous cell carcinoma [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 1671.

Abstract 232: Comprehensive metabolic profiling and vulnerabilities to metabolic inhibitors among small cell lung cancer subtypes

Abstract Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumor that constitutes approximately 14% of all lung cancers diagnosed in the United States. Despite recent advances in treatment options, recurrence is a major challenge and long-term survival remains poor underscoring the importance of investigating mechanisms of treatment resistance and therapeutic strategies. Interestingly, our laboratory has recently uncovered evidence for the classification of SCLC into distinct subtypes, each with unique gene expression patterns and therapeutic vulnerabilities. These subtypes are characterized by differential expression of the transcription factors ASCL1, NEUROD1, or POU2F3. A fourth subtype is negative for all three transcription factors and has prominent expression of immune genes, thus is termed the Inflamed subtype. These subtypes are associated with differences in drug sensitivity, biomarker signatures, and mechanisms of cellular growth. One facet of tumor biology that has not been explored across the subtypes, or in SCLC in general, is the metabolic mechanisms of energy generation. Although metabolic reprogramming from mitochondrial respiration to glycolysis is considered a major hallmark of cancer, increasing evidence suggests that mitochondrial or amino acid dependencies may also contribute to treatment resistance. Therefore, understanding the metabolic demand, particularly among the subtypes, is critical for the development of new therapies. To address this, gene set enrichment analysis was applied to 81 human SCLC tumors categorized by subtype, and revealed differential gene expression correlating to pathways such as fatty acid metabolism in POU2F3 and reactive oxygen species in Inflamed that was confirmed by reverse phase protein analysis (RPPA). Further, we evaluated nutrient requirements, glycolysis dependence, and mitochondrial function in human SCLC cell lines and found differences in glucose uptake and subsequent lactate generation between the subtypes suggesting that glycolysis inhibition may be effective in particular subsets. Based on this, we then tested the efficacy of PFK-158 (glycolysis inhibitor) on cellular proliferation and metabolic function in these cell lines, which revealed variations in susceptibility dependent on subtype classification. Together, this data serves to enhance the current understanding of SCLC mechanisms of growth and provides evidence for metabolic inhibition as a potential therapeutic opportunity. Citation Format: Kasey R. Cargill, Carl M. Gay, Robert J. Cardnell, You-Hong Fan, Qi Wang, Lixia Diao, Jing Wang, Lauren A. Byers. Comprehensive metabolic profiling and vulnerabilities to metabolic inhibitors among small cell lung cancer subtypes [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 232.

MicroRNA-509-3p inhibits cellular migration, invasion, and proliferation, and sensitizes osteosarcoma to cisplatin

Osteosarcoma (OS) is the most common primary pediatric malignancy of the bone having poor prognosis and long-term survival rates of less than 30% in patients with metastasis. MicroRNA-509 was reported to be downregulated in OS. We and others previously published that miR-509-3p can strongly attenuate cellular migration/invasion and sensitize ovarian cancer to cisplatin. Here, we show that overexpression of miR-509-3p inhibited migration of primary OS cell lines U2OS, HOS, and SaOS2 as well as metastatic derivatives 143B and LM7. miR-509-3p overexpression also inhibited proliferation and invasion of HOS and 143B cells and sensitized cells to cisplatin. Luciferase reporter assays using 3′-UTRs of predicted miR-509-3p targets associated with metastatic phenotypes revealed ARHGAP1 could be one of the downstream effectors of miR-509-3p in HOS. To find the global impact of miR-509-3p overexpression and cisplatin treatment we performed Reverse Phase Protein Analysis (RPPA). AXL, which has been reported to play a critical role in cisplatin resistance and confirmed as direct target of miR-509-3p was downregulated upon miR-509-3p treatment and further down-regulated upon miR-509-3p + cisplatin treatment. We propose that the miR-509-3p/AXL and miR-509-3p/ARHGAP1 axes have the potential to uncover new druggable targets for the treatment of drug resistant metastatic osteosarcoma.

168P - Results from TRIO030, a pre-surgical tissue-acquisition study to evaluate molecular alterations in human breast cancer tissue following short-term exposure to the androgen receptor antagonist darolutamide

BackgroundThe role of the androgen receptor (AR) in breast cancer (BC) is not fully elucidated. TRIO030 was conducted to identify molecular changes in BC tissue following short-term exposure to darolutamide (DAR), a new next-generation AR antagonist. MethodsTRIO030 was a multicenter, tissue-acquisition trial in women with treatment-naïve early BC, T≥1.0cm, N0-1. Primary objective: identify molecular alterations in BC tissue following preoperative exposure to DAR (600mg bid for 14-35 days). Formalin-fixed paraffin-embedded (FFPE) blocks and fresh frozen tissue (FFT) were collected before DAR and at surgery (or pre neoadjuvant therapy). AR, ER, PgR, HER2, Ki67, Cytokeratin 5-6 were assessed by IHC. Gene expression microarray (GEx) and Reverse Phase Protein Analysis (RPPA) were done on FFT. Differentially expressed genes (DEGs) were found by ANOVA from an integrated gene expression analysis software (Resolver). A gene enrichment search by Gene Ontology (GO) analysis was done using Database for Annotation, Visualization and Integrated Discovery (DAVID). Results36 patients (pt) (median age 61.5) were enrolled (20HR+, 7 TNBC, and 9 HER2+ per central lab). Median duration of DAR was 14.5 days. 32 pts were evaluable by RPPA, 31 by GEx. Based on AR level changes in both RPPA and GEx in pre vs. post-DAR samples, cases were grouped: AR upregulated (≥1.2 fold increase, n=14), AR downregulated (≥1.2 fold decrease, n=11), AR unchanged (n=6). 233 unique DEGs were detected with 84 identified as forming two GEx profiles among the 3AR groups. GO analysis revealed that DEGs profiles were enriched in an immune (IMM) signature or cell proliferation (CP) signature which were anti-correlated (e.g., if CP is upregulated, IMM is down). No correlation between AR groups and BC subtypes was found. One pt had a grade 3-4 adverse event (non-serious ALT-AST increase). ConclusionsTRIO030 suggests an association between DAR and immune regulation genes. There was no association between pre/post-DAR AR level changes and BC subtype. Future studies will likely be needed to further investigate the role for AR blockade in BC as well as the mechanism of action for DAR at molecular level. Clinical trial identification2016-004151-79. Legal entity responsible for the studyTranslational Research in Oncology. FundingBayer (provided the study drug), Translational Research in Oncology (not for profit organization, funded other aspects of the trial). DisclosureAll authors have declared no conflicts of interest.

LGALS3 is connected to CD74 in a previously unknown protein network that is associated with poor survival in patients with AML

BackgroundGalectin 3 (LGALS3) gene expression is associated with poor survival in acute myeloid leukemia (AML) but the prognostic impact of LGALS3 protein expression in AML is unknown. LGALS3 supports diverse survival pathways including RAS mediated cascades, protein expression and stability of anti-apoptotic BCL2 family members, and activation of proliferative pathways including those mediated by beta Catenin. CD74 is a positive regulator of CD44 and CXCR4 signaling and this molecule may be critical for AML stem cell function. At present, the role of LGALS3 and CD74 in AML is unclear. In this study, we examine protein expression of LGALS3 and CD74 by reverse phase protein analysis (RPPA) and identify new protein networks associated with these molecules. In addition, we determine prognostic potential of LGALS3, CD74, and their protein networks for clinical correlates in AML patients. MethodsRPPA was used to determine relative expression of LGALS3, CD74, and 229 other proteins in 231 fresh AML patient samples and 205 samples were from patients who were treated and evaluable for outcome. Pearson correlation analysis was performed to identify proteins associated with LGALS3 and CD74. Progeny clustering was performed to generate protein networks. String analysis was performed to determine protein:protein interactions in networks and to perform gene ontology analysis. Kaplan-Meir method was used to generate survival curves. FindingsLGALS3 is highest in monocytic AML patients and those with elevated LGALS3 had significantly shorter remission duration compared to patients with lower LGALS3 levels (median 21.9 vs 51.3 weeks, p = 0.016). Pearson correlation of LGALS3 with 230 other proteins identifies a distinct set of 37 proteins positively correlated with LGALS3 expression levels with a high representation of proteins involved in AKT and ERK signaling pathways. Thirty-one proteins were negatively correlated with LGALS3 including an AKT phosphatase. Pearson correlation of proteins associated with CD74 identified 12 proteins negatively correlated with CD74 and 16 proteins that are positively correlated with CD74. CD74 network revealed strong association with CD44 signaling and a high representation of apoptosis regulators. Progeny clustering was used to build protein networks based on LGALS3 and CD74 associated proteins. A strong relationship of the LGALS3 network with the CD74 network was identified. For AML patients with both the LGALS3 and CD74 protein cluster active, median overall survival was only 24.3 weeks, median remission duration was 17.8 weeks, and no patient survived beyond one year. InterpretationThe findings from this study identify for the first time protein networks associated with LGALS3 and CD74 in AML. Each network features unique pathway characteristics. The data also suggest that the LGALS3 network and the CD74 network each support AML cell survival and the two networks may cooperate in a novel high risk AML population. FundLeukemia Lymphoma Society provided funds to SMK for RPPA study of AML patient population. Leukemia Texas provided funds to PPR and SMK to study CD74 and LGALS3 expression in AML patients using RPPA. No payment was involved in the production of this manuscript.

AXL Expression in Pediatric AML Is Associated with Putative LSC and Correlates with a Distinct Set of Proteins Associated with Cell Metabolism, Cell Cycle, and Unfolded Protein Response

Background: The receptor tyrosine kinase AXL is emerging as a critical regulator of survival signaling and cell cycle regulators in AML. Our previous work using AXL inhibitor ONO-7475 in treating AML cells demonstrated that the drug reduced proteins associated with cell cycle regulation such as CDK1 and PLK1 and PI3K/AKT/mTOR signaling (Ruvolo et al Haematologica 2017). PI3K/AKT/mTOR signaling supports cell survival from stress challenge including response to unfolded protein (UPR). CDK1 and PLK1 have also been implicated as having survival roles during UPR stress. At present an understanding of AXL expression in primary pediatric AML samples is unknown. Reverse phase protein analysis (RPPA) is a powerful proteomic tool that allows for the measurement of protein expression in clinical samples.Methods: ± the proteasome inhibitor bortezomib. Bioinformatic analysis was performed by calculating the Pearson correlation coefficients. Proteins were identified as significantly positively correlated with AXL or significantly negatively correlated with AXL based on p < 0.05 & r ≥ 0.25 or r ≤ -0.25, respectively. All p-values were Bonferroni corrected (0.05 / 291 = 0.0002). String analysis to determine protein: protein interactions was performed on sets associated with positive and negative correlation.Results: Expression of AXL appears to be lower in AML cells compared to healthy donor CD34+ cells (p < 0.001). The expression of AXL was not prognostic for clinical outcome . AXL expression was higher in AML patients with favorable cytogenetics category (p < 0.001) and in low risk pediatric patients (p = 0.002). AXL expression was significantly different in cytogenetic categories with AXL expression elevated in inv(16) and monosomy 7 and lower in 11q23 (p = 0.0014) . AXL expression is higher in putative leukemia stem cell (LSC) population (CD34+ CD38 -) compared to bulk AML (CD34+,CD38+)(p < 0.001). AXL expression in the pediatric AML cells was compared to 290 proteins by RPPA. Fifty-five protein displayed positive correlation with AXL including the immune checkpoint inhibitor protein PD-L1. Other proteins positively correlated with AXL included PLK1 and FOXM1. Our previous study using the ONO-7475 AXL inhibitor demonstrated reduction of PLK1 in cells treated with the inhibitor. Positive association of FOXM1 and PLK1 with AXL would be consistent with a regulatory network where AXL regulates PLK1 via FOXM1. Thirty-three proteins were negatively correlated with AXL expression including HNRNPK, STAT3, XPO1, and phosphorylated EIF2S1. Phosphorylation of EIF2S suppresses protein translation during stress events including unfolded protein response (UPR). String analysis to determine protein: protein interactions determined high interaction (p < 10 e-16) in both positively correlated and negatively correlated sets. KEGG pathways associated with proteins correlated with AXL include mTOR signaling (Pathway ID 4150), PI3K/AKT signaling (Pathway ID 4151), HIF signaling (Pathway ID 4066), and focal adhesion (Pathway ID 4510). Biological pathways associated with AXL networks included regulation of cellular metabolism (Pathway ID GO.0031325), regulation of gene expression (Pathway ID GO.0010604), and cellular response to unfolded protein (Pathway ID G0:0034620).Conclusions: These results suggest that AXL may be prominently expressed in putative LSC in pediatric AML. Though AXL expression appears to be associated with favorable risk cytogenetics, the correlation of AXL with PD-L1 suggests it may be involved in how the AML cell responds to immune surveillance. Finally, a distinct set of proteins are clearly associated with AXL expression and represent a number of different survival pathways including regulation of cell metabolism, cell cycle, and UPR that could contribute to AML cell survival. DisclosuresAndreeff:AstraZeneca: Research Funding.

Role of MSC-derived galectin 3 in the AML microenvironment

In acute myeloid leukemia (AML), high Galectin 3 (LGALS3) expression is associated with poor prognosis. The role of LGALS3 derived from mesenchymal stromal cells (MSC) in the AML microenvironment is unclear; however, we have recently found high LGALS3 expression in MSC derived from AML patients is associated with relapse. In this study, we used reverse phase protein analysis (RPPA) to correlate LGALS3 expression in AML MSC with 119 other proteins including variants of these proteins such as phosphorylated forms or cleaved forms to identify biologically relevant pathways. RPPA revealed that LGALS3 protein was positively correlated with expression of thirteen proteins including MYC, phosphorylated beta-Catenin (p-CTNNB1), and AKT2 and negatively correlated with expression of six proteins including integrin beta 3 (ITGB3). String analysis revealed that proteins positively correlated with LGALS3 showed strong interconnectivity. Consistent with the RPPA results, LGALS3 suppression by shRNA in MSC resulted in decreased MYC and AKT expression while ITGB3 was induced. In co-culture, the ability of AML cell to adhere to MSC LGALS3 shRNA transductants was reduced compared to AML cell adhesion to MSC control shRNA transductants. Finally, use of novel specific LGALS3 inhibitor CBP.001 in co-culture of AML cells with MSC reduced viable leukemia cell populations with induced apoptosis and augmented the chemotherapeutic effect of AraC. In summary, the current study demonstrates that MSC-derived LGALS3 may be critical for important biological pathways for MSC homeostasis and for regulating AML cell localization and survival in the leukemia microenvironmental niche.

Anexelekto/MER tyrosine kinase inhibitor ONO-7475 arrests growth and kills FMS-like tyrosine kinase 3-internal tandem duplication mutant acute myeloid leukemia cells by diverse mechanisms.

Nearly one-third of patients with acute myeloid leukemia have FMS-like tyrosine kinase 3 mutations and thus have poor survival prospects. Receptor tyrosine kinase anexelekto is critical for FMS-like tyrosine kinase 3 signaling and participates in FMS-like tyrosine kinase 3 inhibitor resistance mechanisms. Thus, strategies targeting anexelekto could prove useful for acute myeloid leukemia therapy. ONO-7475 is an inhibitor with high specificity for anexelekto and MER tyrosine kinase. Herein, we report that ONO-7475 potently arrested growth and induced apoptosis in acute myeloid leukemia with internal tandem duplication mutation of FMS-like tyrosine kinase 3. MER tyrosine kinase-lacking MOLM13 cells were sensitive to ONO-7475, while MER tyrosine kinase expressing OCI-AML3 cells were resistant, suggesting that the drug acts via anexelekto in acute myeloid leukemia cells. Reverse phase protein analysis of ONO-7475 treated cells revealed that cell cycle regulators like cyclin dependent kinase 1, cyclin B1, polo-like kinase 1, and retinoblastoma were suppressed. ONO-7475 suppressed cyclin dependent kinase 1, cyclin B1, polo-like kinase 1 gene expression suggesting that anexelekto may regulate the cell cycle, at least in part, via transcriptional mechanisms. Importantly, ONO-7475 was effective in a human FMS-like tyrosine kinase 3 with internal tandem duplication mutant murine xenograft model. Mice fed a diet containing ONO-7475 exhibited significantly longer survival and, interestingly, blocked leukemia cell infiltration in the liver. In summary, ONO-7475 effectively kills acute myeloid leukemia cells in vitro and in vivo by mechanisms that involve disruption of diverse survival and proliferation pathways.

Abstract 1813: Bevacizumab potentiates the proteomic response to neoadjuvant chemotherapy in breast cancer patients: Rppa exploration of consecutive tumor samples in the NeoAva randomized phase II trial

Abstract Antiangiogenic therapy using bevacizumab has proven effective for a number of cancers; however, in breast cancer there is an unmet need to identify patients that benefit from such treatment. Sampling of tumor biopsies before and during treatment, as well as at the time of surgery enables the assessment of response at multiple molecular levels. At the proteomic level reverse phase protein analysis (RPPA) support expression of numerous cancer associated proteins simultaneously, which can further be used to unravel molecular mechanisms associated with clinical response to bevacizumab treatment. In this phase II clinical trial, patients with HER2 negative primary tumors of ≥25 mm were treated with neoadjuvant chemotherapy (4 x FEC100 + 12 weeks of taxane-based therapy) and randomized (1:1) to receive bevacizumab or not. Mammography, ultrasound and MR imaging were used for response evaluation, in addition to final pathology assessment. Tumor responses were evaluable in 132 patients; of which 66 received bevacizumab. Ratio of the tumor size at final pathology assessment, and at inclusion was calculated to obtain a continuous scale of response reflecting the percentage of tumor shrinkage in response to therapy. Tumor biopsies were removed before start of treatment, at week 12 at the start of taxane-based tharapy and at the time of surgery. Lysates from each sample was analyzed on reverse phase protein arrays (RPPA) for expression levels of 210 proteins of which 54 were phospho-specific. The addition of bevacizumab to the chemotherapy do not alter proteomic response from week 0 to 25 to such extent that this patient group cluster naturally together. While the proteomic response from week 0 to 12 in both treatment arms had an overall similar profile regarding up- and down-regulated proteins, the combination treatment (FEC100 + bevacizumab) induced substantially more effect on the regulation of each protein. This suggests that bevacizumab treatment have the capability to potentiate the effects of the anthracyclin based chemotherapy from week 0 to 12. Conversely, from week 12-25 (taxane-based therapy + bevacizumab) this effect was lost or even reversed, possibly due to a de-vascularized and less accessible tumor. An exception to this observation was a few phospho-proteins that do seem to have sustained stronger regulation over the whole treatment period. We are in the process of analyzing in more detail the impact of phosphorylation and thus protein activation states on treatment response. Deciphering molecular response and activity regulation at the proteomic level is a promising approach and may reveal novel knowledge with potential important clinical relevance. Citation Format: Mads H. Haugen, Ole Christian Lingjaerde, Marit Krohn, Wei Zhao, Evita M. Lindholm, Laxmi Silwal-Pandit, Elin Borgen, Øystein Garred, Anne Fangberget, Marit M. Holmen, Ellen Schlichting, Helle K. Skjerven, Steinar Lundgren, Erik Wist, Bjørn Naume, Gunhild M. Maelandsmo, Yiling Lu, Anne-Lise Boerresen-Dale, Gordon B. Mills, Olav Engebraaten. Bevacizumab potentiates the proteomic response to neoadjuvant chemotherapy in breast cancer patients: Rppa exploration of consecutive tumor samples in the NeoAva randomized phase II trial [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 1813. doi:10.1158/1538-7445.AM2017-1813

Pharmacological Inhibition of Lck is Able to Revert Glucocorticoid Resistance in Pediatric T-ALL

The AIEOP-BFM group has traditionally used the peripheral blood blast cells count after a 7-day glucocorticoids (GC) prephase to classify patients as Prednisone Good Responders (PGR) or Prednisone Poor Responders (PPR). As described by Schrappe M. in 2011, PPR patients tend to have a worse prognosis, despite the fact that all of them are assigned to the High Risk protocol. Little is known about the molecular mechanisms that lead to GC resistance, guiding our research to the identification of new specific molecular targets in order to develop new approaches to improve therapy efficacy in these patients.To this end, we performed a Reverse Phase Protein Analysis (RPPA) of 54 PGR and 33 PPR pediatric T-ALL patients at diagnosis, and studied the activation or expression of 87 proteins involved in key cellular signaling pathways. Interestingly, we found a higher expression of LCK phosphorylated at Y505 (inhibited form) in PGR patients (p=0.001), together with a higher phosphorylation of SRC Y416 (active form) in PPR patients (p=0.01). Total LCK and LCK RNA expression were not differentially expressed in the two subgroups of patients, suggesting an increased activation of LCK in PPR patients. Indeed, in agreement with these results, also LCK downstream target PLCγ, phosphorylated at Y783, resulted hyperactivated in PPR compared to PGR patients (p=0.05), confirmed also by a positive correlation between PLCγ Y783 and SRC Y416 (r=0.51, p=0.01). Taken together, these results indicate a hyperactivation of the LCK pathway in PPR patients compared to PGR ones. LCK is part of the TCR multiprotein complex together with the GC receptor. In normal T lymphocytes, after GC treatment the complex is disrupted, LCK activation is decreased and downstream prosurvival signaling inhibited, thus leading to cell death. In this light, in GC resistant patients hyperactivated LCK might sustain cell survival regardless of GC activity.We then tested if FDA-approved or recently developed LCK inhibitors would revert GC resistance in T-ALL cells. GC resistant cell lines ALL-SIL, T-ALL1 and CEM were treated with Dasatinib, Bosutinib, Nintedanib and WH-4-023 alone or in combination with Dexamethasone (Dex). All four inhibitors alone are able to decrease cell survival, and all of them strongly synergize with Dex, bringing to the sensitization of these cells to GC treatment. We also tested these compounds alone or in combination with Dex in 4 PPR T-ALL patients cells derived from xenograft mice. Also in these cases we observed an enhanced sensitization of cells to GC treatment. Finally, corroborating the crucial role of LCK in GC resistance, we observed a strong decrease in cell viability after specific LCK gene silencing and Dex treatment in ALL-SIL cells, together with an increased GC resistance following LCK hyperactivation in P12-ICHIKAWA GC sensitive cells.Thus, our results strongly suggest that the inhibition of LCK using clinically approved drugs could represent a promising new additional therapeutic strategy to revert drug resistance in high-risk pediatric T-ALL patients. DisclosuresIndraccolo:OncoMed Pharmaceuticals, Inc.: Research Funding.

Abstract 4391: Overcoming primary ibrutinib resistance in mantle cell lymphoma

Abstract Mantle cell lymphoma (MCL) is a rare and incurable subtype of B-cell lymphoma. In a phase II study of ibrutinib in MCL patients, most of the patients responded and had long durable remissions; however, 22.7% of patients were considered to be non-responsive to ibrutinib, and an additional 22/110 patients displayed initial positive responses to ibrutinib but also experienced disease progression within 12 months of treatment, with both responses classified as primary ibrutinib resistant. Therefore, understanding the mechanisms mediating primary ibrutinib resistance may identify new prognostic and predictive markers and potential therapeutic targets. Bruton's tyrosine kinase (BTK) plays an important role in B-cell development, activation, and differentiation. Upon B-cell receptor (BCR) activation, BTK is phosphorylated and activated by SYK. Phosphoinositide 3-kinase (PI3K) is recruited to the BCR, and the induction of these signaling activities leads to the downstream activation of multiple effector proteins, including nuclear factor-kB (NF-kB), AKT, RAS, mTOR and mitogen-activated protein kinase (MAPK). BTK and PI3K have been shown to function independently to mediate BCR signaling, suggesting that PI3K signaling activities may underlie ibrutinib resistance independently of BTK. PI3K has also been associated with primary ibrutinib resistance. To further elucidate the mechanisms underlying ibrutinib resistance, we conferred primary ibrutinib resistance using both MCL cell lines and MCL-bearing patient-derived xenograft (PDX) mouse models and used whole exome sequencing (WES) and reverse phase protein analysis (RPPA) to identify any genetic and expression changes associated with primary ibrutinib resistance. WES did not reveal any mutations in BTK or within the proximal BCR pathway, consistent with WES data on primary ibrutinib resistant MCL cases. RPPA analysis showed a significant increase in the PI3K/AKT/mTOR/MCL-1 compensatory pathway component levels in ibrutinib-resistant cell lines when compared with their parental cells as well as ibrutinib-resistant PDXs. To determine whether inhibiting these pathways would overcome primary ibrutinib resistance, we tested various therapeutic combinations targeting these pathways. Ibrutinib plus the PI3K inhibitor idelalisib, the AKT inhibitor ACP-319, the mTOR inhibitors AZD8055 or BEZ235 as well as the proteasome inhibitor carfilzomib inhibited tumor growth in vitro and in vivo in the PDX mouse models, demonstrating that targeting these alternative pathways may overcome ibrutinib resistance. This work strongly suggests that targeting the PI3K/AKT/mTOR/MCL-1 compensatory pathway successfully inhibits the viability of ibrutinib-resistant MCL tumor cells both in vitro and in vivo and identified potential therapies that can be used to treat ibrutinib-resistant patients in clinical practice. Citation Format: Leo Zhang, Lan Pham, Hui Zhang, Jingmeng Xie, Wenjing Tao, Taylor Bell, Zhihong Chen, Krystle Nomie, Bingliang Fang, Michael Wang. Overcoming primary ibrutinib resistance in mantle cell lymphoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4391.

Abstract 5155: Integrated genomic analysis of colorectal cancer progression reveals activation of EGFR through demethylation of the EREG promoter

Abstract The molecular drivers that underlie transformation of colonic epithelium into colorectal adenocarcinoma (CRC) have been well described. However, the mechanisms through which some of the clinically targeted pathways are activated during CRC progression have yet to be elucidated. Here, we employed an integrative genomics approach to examine CRC progression. Transcriptional profiling of laser capture microdissected colonic crypt cells, differentiated surface epithelium, adenomas, carcinomas, and metastases, showed distinctive patterns in the activation of developmental and oncogenic pathways, including the clinically important EGFR axis. We observed a dramatic up-regulation of the EGFR ligand EREG in primary and metastatic cancer cells as compared to normal and adenomatous tissues that was indicative of autocrine tumor production, and confirmed this pattern of gene expression by in situ hybridization. Global methylation analysis indicated that up-regulation of EREG during the adenoma-carcinoma transition was associated with de-methylation of two key sites within the EREG promoter and this was accompanied by an increase in the levels of EGFR phosphorylation, as assessed by reverse phase protein analysis. In a clinical trial setting, we observed that low levels of EREG methylation in patients who received cetuximab as part of a Phase II study were associated with high expression of the ligand and a favorable response to therapy. Conversely, high levels of promoter methylation and low levels of EREG expression were observed in tumors that progressed after treatment. We also noted an inverse correlation between EREG methylation and expression levels in several other cancers from the TCGA datasets, including those of the head and neck, lung, and bladder. We propose that up-regulation of EREG expression through promoter de-methylation might be an important means of activating the EGFR pathway during the genesis of CRC and, potentially, other types of cancer. Citation Format: Xueping Qu, Thomas Sandmann, Henry Frierson, Ling Fu, Eloisa Fuentes, Kimberly Walter, Kwame Okrah, Craig Rumpel, Christopher Moskaluk, Shan Lu, Yulei Wang, Richard Bourgon, Elicia Penuel, Andrea Pirzkall, Lukas Amler, Mark Lackner, Josep Tabenero, Garret Hampton, Omar Kabbarah. Integrated genomic analysis of colorectal cancer progression reveals activation of EGFR through demethylation of the EREG promoter. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5155.