Protein Pathway Activation Mapping Research Articles

Protein signaling and drug target activation signatures to guide therapy prioritization: Therapeutic resistance and sensitivity in the I-SPY 2 Trial

Molecular subtyping of breast cancer is based mostly on HR/HER2 and gene expression-based immune, DNA repair deficiency, and luminal signatures. We extend this description via functional protein pathway activation mapping using pre-treatment, quantitative expression data from 139 proteins/phosphoproteins from 736 patients across 8 treatment arms of the I-SPY 2 Trial (ClinicalTrials.gov: NCT01042379). We identify predictive fit-for-purpose, mechanism-of-action-based signatures and individual predictive protein biomarker candidates by evaluating associations with pathologic complete response. Elevated levels of cyclin D1, estrogen receptor alpha, and androgen receptor S650 associate with non-response and are biomarkers for global resistance. We uncover protein/phosphoprotein-based signatures that can be utilized both for molecularly rationalized therapeutic selection and for response prediction. We introduce a dichotomous HER2 activation response predictive signature for stratifying triple-negative breast cancer patients to either HER2 or immune checkpoint therapy response as a model for how protein activation signatures provide a different lens to view the molecular landscape of breast cancer and synergize with transcriptomic-defined signatures.

Abstract PD5-08: PD5-08 Drug target activation mapping of matched triple negative primary and axillary lymph node metastases identifies and links the ER-AR-GR steroid hormone receptor axis with downstream AKT activation

Abstract Title: Drug target activation mapping of matched triple negative primary and axillary LN metastases identifies and links the ER-AR-GR steroid hormone receptor axis with downstream AKT activation Background: Triple negative breast cancer (TNBC) is comprised of heterogeneous subtypes that arise from various molecular alterations that result in TNBC having the poorest overall prognosis. There is growing interest in more sensitive ways to measure estrogen receptor (ER) and HER2 levels in the ER “negative” and HER2 “low/negative” settings, as there are patients with TNBC whose tumors are actually ER and/or HER2 “expressing/active”, and who may benefit from ER- and/or HER2-targeted therapies. Given the limitations of IHC in reliably quantitating ER and HER2 in low (0-1+) expressing tumors, we utilized highly sensitive reverse phase protein array (RPPA) to quantitate expression of these therapeutic targets along with downstream signaling activation mapping in a unique pilot set of patient-matched primary (P) and axillary lymph node (LN) metastases obtained synchronously. The quantitative expression of steroid hormone receptor expression/activation (ER, glucocorticoid receptor (GR), androgen receptor (AR)) along with their signaling architecture and dynamics were analyzed. Methods: A 12 P-LN paired tumor set was chosen for analysis (N=24). Manual macrodissection was used to enrich for tumor epithelium prior to RPPA analysis that quantitatively measured 155 proteins/phosphoprotein drug targets in key signaling pathways known to be involved in steroid hormone receptor biology and tumorigenesis. Statistical analysis using paired sample t-testing or Wilcoxon rank testing was performed (p< 0.05 uncorrected). Results: Protein pathway activation mapping and unsupervised clustering analysis revealed a subset of TNBCs from 3 of the 12 (25%) patients (N=4: 1 LN and 1 P, and one patient-matched LN and P) that was characterized by high relative levels of total ER. Interestingly, these same tumors also had amongst the highest relative levels of both total AR and activated (phosphorylated) GR compared to the rest of the 20 tumor samples. Downstream signaling analysis found significant (p< 0.05) activation/phosphorylation of AKT (S473 and/or T308) associated with the ER-AR-GR/steroid hormone activated/”high” TNBC signature compared to the 20 tumors that were steroid hormone “low”. Conclusions: Functional protein pathway activation mapping of a unique study set of synchronously obtained TNBC primary and LN metastasis revealed a steroid hormone receptor expression/activation signature of co-incidentally activated/expressed ER, GR and AR, that was also characterized by increased AKT activation (p< 0.05). This steroid hormone receptor “activated” signature was not observed to be significantly differentially expressed in LN compared to P tissues in cohort or matched pair analysis, suggesting that this signature may be present as an intrinsic event in the P, and not acquired in LN metastases. Recent data from stage I-III TNBC patients treated in the neoadjuvant setting with an AKT inhibitor1 revealed that responding TNBC patients had pre-treatment tumor biopsies that were enriched for both high AR expression and high phosphoAKT (S473 and/or T308) levels. If these observations that a subset of TNBCs are steroid hormone receptor “activated” despite being ER “negative” are confirmed in larger study sets, we hypothesize that this subgroup, identified by RPPA analysis of both P and LN metastases, could be potentially sensitive to AKT inhibition in combination with agents that target steroid hormone receptors. 1Shi Z, et al. Functional Mapping of AKT Signaling and Biomarkers of Response from the FAIRLANE Trial of Neoadjuvant Ipatasertib plus Paclitaxel for Triple-Negative Breast Cancer. Clin Cancer Res. 2022 Mar 1;28(5):993-1003 Citation Format: Julia Wulfkuhle, Page Blas, Heather Williams, Claudius Mueller, Rosa I. Gallagher, Mariaelena Pierobon, Joyce O’Shaughnessy, Emanuel F. Petricoin. PD5-08 Drug target activation mapping of matched triple negative primary and axillary lymph node metastases identifies and links the ER-AR-GR steroid hormone receptor axis with downstream AKT activation [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD5-08.

Multi‐omic molecular profiling guide’s efficacious treatment selection in refractory metastatic breast cancer: a prospective phase II clinical trial

This prospective phase II clinical trial (Side Out 2) explored the clinical benefits of treatment selection informed by multi‐omic molecular profiling (MoMP) in refractory metastatic breast cancers (MBCs). Core needle biopsies were collected from 32 patients with MBC at trial enrollment. Patients had received an average of 3.94 previous lines of treatment in the metastatic setting before enrollment in this study. Samples underwent MoMP, including exome sequencing, RNA sequencing (RNA‐Seq), immunohistochemistry, and quantitative protein pathway activation mapping by Reverse Phase Protein Microarray (RPPA). Clinical benefit was assessed using the previously published growth modulation index (GMI) under the hypothesis that MoMP‐selected therapy would warrant further investigation for GMI ≥ 1.3 in ≥ 35% of the patients. Of the 32 patients enrolled, 29 received treatment based on their MoMP and 25 met the follow‐up criteria established by the trial protocol. Molecular information was delivered to the tumor board in a median time frame of 14 days (11–22 days), and targetable alterations for commercially available agents were found in 23/25 patients (92%). Of the 25 patients, 14 (56%) reached GMI ≥ 1.3. A high level of DNA topoisomerase I (TOPO1) led to the selection of irinotecan‐based treatments in 48% (12/25) of the patients. A pooled analysis suggested clinical benefit in patients with high TOPO1 expression receiving irinotecan‐based regimens (GMI ≥ 1.3 in 66.7% of cases). These results confirmed previous observations that MoMP increases the frequency of identifiable actionable alterations (92% of patients). The MoMP proposed allows the identification of biomarkers that are frequently expressed in MBCs and the evaluation of their role as predictors of response to commercially available agents. Lastly, this study confirmed the role of MoMP for informing treatment selection in refractory MBC patients: more than half of the enrolled patients reached a GMI ≥ 1.3 even after multiple lines of previous therapies for metastatic disease.

Selinexor in Combination with Induction and Consolidation Therapy in Older Adults with AML Is Highly Active

Background: Acute myeloid leukemia (AML) is an aggressive myeloid cancer of the hematopoietic system that primarily affects older adults and is characterized by therapy resistance and dismal outcomes. Novel approaches to treat AML are desperately needed. Selinexor is a small-molecule inhibitor of the nuclear export protein XPO1. Treatment of AML cells with selinexor was shown to sensitize them to chemotherapy in part by blocking the nuclear export of topoisomerase II, resulting in increased DNA strand breaks when an anthracycline is present. Furthermore, selinexor has shown encouraging results when combined with chemotherapy in the relapsed setting. This abstract reports on the initial results of a randomized phase II study of induction and consolidation with or without selinexor in newly diagnosed patients with AML 60 years of age or older. Methods: Patients 60 years of age or older with newly diagnosed de novo AML were randomized between 7+3 or 7+3+selinexor induction. Responding patients could then go on to high dose cytarabine consolidation with or without selinexor as per their initial treatment assignment. Patients in the selinexor arm who completed all planned consolidation could then move to maintenance therapy with selinexor alone. During induction cytarabine was dosed at 100 mg/m2 by continuous infusion for 7 days and daunorubicin was dosed at 60 mg/m2 on days 1-3. In consolidation cytarabine was dosed at 1.5 gm/m2 given Q12 hours on days 1, 3 and 5. Selinexor was dosed at 60 mg PO on days 1, 3, 8, 10, 15 and 17 during induction and consolidation and on days 1 and 8 every 21 days in maintenance. Optional blood samples were collected just prior to and at several time points after selinexor during induction. Mononuclear cells were isolated from these samples, lysed and lysates evaluated for protein pathway drug target activation mapping by reverse phase phosphoprotein array (RPPA). Results: Thirteen patients have been enrolled to date with 12 available for evaluation. Of the 12 evaluable patients 6 were randomized to each arm. Baseline demographics are listed in Table 1. Overall the combination was well tolerated with no patients discontinuing selinexor secondary to treatment related adverse events. In the standard arm 3 of 6 patients achieved a complete remission. Of the 3 responders 1 has relapsed, 1 has gone on to transplant and one has completed consolidation chemotherapy. In the selinexor arm 5 of 6 patients achieved a complete remission and 1 patient achieved a morphologic leukemia free state. All patients given selinexor achieved a response. Of the 5 responders 3 have gone on to transplant, 1 is on maintenance therapy (completed 14 cycles) and one is completing consolidation therapy. In the standard arm no patients died during induction and 3 of 6 patients have died from progressive disease. In the selinexor arm 1 patient died during induction and none of the remaining patients have progressed (Figure 1 and Table 2). No difference in the AE profile was noted between arms and no unexpected side effects were observed. The patient on long term maintenance selinexor is tolerating it without significant AEs to date. Blood samples were available from 3 patients from the selinexor arm. RPPA based protein pathway activation mapping analysis of PBMCs revealed a significant increase in phosphorylation of AMPK, BAD and LC3B at one-hour post treatment that returned to baseline by 2 hours suggesting a transient increase in these pathways that was subsequently suppressed (Figure 2). Conclusions: Selinexor in combination with standard induction and consolidation therapy appears highly active in older de novo AML patients. Selinexor may increase response by modulation of AMPK, autophagy and BAD phosphorylation. Enrollment is ongoing. Disclosures Pardee: Spherix Intellectual Property: Research Funding; CBM Bipharma: Membership on an entity's Board of Directors or advisory committees; Amgen: Speakers Bureau; Celgene: Speakers Bureau; Pharmacyclics/Janssen: Speakers Bureau; Karyopharm: Research Funding; Rafael Pharmaceuticals: Consultancy, Research Funding. Manuel:Novartis: Speakers Bureau; Jazz Pharmaceuticals: Speakers Bureau. Powell:Pfizer: Consultancy, Research Funding; Rafael Pharmaceuticals: Consultancy, Research Funding; Novartis: Consultancy, Speakers Bureau; Jazz Pharmaceuticals: Consultancy, Research Funding, Speakers Bureau; Janssen: Research Funding. OffLabel Disclosure: Selinexor is not approved for the treatment of AML

Antigen-Presenting Signaling Events in the Tumor Ecology Associate with Response to Anti-PD-1 Treatment in Lung Cancer

Immune-checkpoint blockade has emerged as a powerful therapeutic strategy in lung cancer. However, the molecular mechanisms associated with response are still poorly understood. Using lung cancer as a model system, we employed protein pathway activation mapping and multiplex immunofluorescence to explore pathway-centered signaling events and tumor-immune spatial interactions associated with response to Nivolumab. Signaling data were collected from laser dissected tumor epithelia and the surrounding tumor-stroma interface (TSI) of 28 lung cancer patients treated with Nivolumab using the Reverse Phase Protein Microarray. As expected, quantitative PD-L1 measurements associated with response to treatment. Pathway-centered analysis revealed increased TLR4-based signaling events, pro-inflammatory molecules, and tumor-associated antigens in patients that benefitted from treatment. CD68+/PD-L1+ antigen-presenting cells spatial distribution was also associated with response to treatment. Taken together, these results suggest that a tumor ecology that favors immune activation may play a primary role in modulating response to compounds targeting the tumor-immune axis.

Abstract 3290: The Side-Out Foundation Metastatic Breast Cancer Database, an open-access portal for multi-omic molecular data and more

Abstract Introduction: With the high volume of molecular data being generated daily from human malignancies, there is an increasing need to create novel internet-based portals where this information is readily and easily accessible to physicians, scientists, and the general public. Although numerous databases have been created to capture the molecular characteristics of primary tumors, limited resources are available when it comes to the metastatic lesions. We have developed a novel open-access database to capture demographic, clinical, and pathological information, outcome data, and multi-omic based molecular profiles from metastatic breast cancer (MBC) patients. Materials and Methods: The portal was created using the open-source relational database management system MySQL and the custom-codes were written using the PHP server-side scripting language. User interface, management, and authentication were created in WordPress. The database is primarily used to record information collected through the Side-Out clinical trials, a series of prospective Phase II trials targeting refractory MBC (NCT01074814, NCT01919749, NCT03195192). De-identified patients' information includes patients' demographics, treatment history, pathological characteristics of the primary tumor, outcome data, and multi-omic molecular profiles. Molecular information collected from each lesion include genomic (NGS-based whole/targeted exome sequencing), transcriptomic (RNA microarray/RNA Seq), proteomic (protein expression by IHC), and phosphoproteomic (protein pathway activation mapping by Reverse Phase Protein Microarray) data. Over 700 data fields are collected for each patient. A higher level of security for the recorded information is achieved by using a secondary database along with custom-codes during the data entry process. Investigators are exploring how Block-chain database design can be used to make portion of the data public while encrypting patient identifiers and key variables. Results: We present an overview of the portal, its usability, how access can be requested by interested third party, the user-friendly interface for downloading clinical, pathological and molecular data, and a few examples of how these data can be used to explore different aspects of metastatic breast cancers. Conclusions: To our knowledge, this is the first web-based publicly accessible database portal where broad multi-omic profiles are captured from the MBC patients along with demographic, clinical and pathological data. This open-access portal represents a unique and highly valuable tool as it integrates different aspects of the disease and can be used for correlative analyses and hypothesis-generating studies. Finally, this web-based portal allows free-of-charge dissemination of data from existing or upcoming clinical and translational studies targeting breast cancer patients. Citation Format: Elisa Baldelli, Shiva Ghaemi, Sanja Avramovic, Janusz Wojtusiak, Lance A. Liotta, Farrokh Alemi, Emanuel F. Petricoin, Bryant Dunetz, Mariaelena Pierobon. The Side-Out Foundation Metastatic Breast Cancer Database, an open-access portal for multi-omic molecular data and more [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 3290.

Protein drug target activation homogeneity in the face of intra-tumor heterogeneity: implications for precision medicine.

Introduction: Recent studies indicated tumors may be comprised of heterogeneous molecular subtypes and incongruent molecular portraits may emerge if different areas of the tumor are sampled. This study explored the impact of intra-tumoral heterogeneity in terms of activation/phosphorylation of FDA approved drug targets and downstream kinase substrates.Material and methods: Two independent sets of liver metastases from colorectal cancer were used to evaluate protein kinase-driven signaling networks within different areas using laser capture microdissection and reverse phase protein array.Results: Unsupervised hierarchical clustering analysis indicated that the signaling architecture and activation of the MAPK and AKT-mTOR pathways were consistently maintained within different regions of the same biopsy. Intra-patient variability of the MAPK and AKT-mTOR pathway were <1.06 fold change, while inter-patients variability reached fold change values of 5.01.Conclusions: Protein pathway activation mapping of enriched tumor cells obtained from different regions of the same tumor indicated consistency and robustness independent of the region sampled. This suggests a dominant protein pathway network may be activated in a high percentage of the tumor cell population. Given the genomic intra-tumoral variability, our data suggest that protein/phosphoprotein signaling measurements should be integrated with genomic analysis for precision medicine based analysis.

Abstract PP01-01: A funny thing happened to me while trying to cure ovarian cancer; I became a cancer prevention research specialist

Abstract PP01-01: A funny thing happened to me while trying to cure ovarian cancer; I became a cancer prevention research specialist

Phosphorylation, Signaling, and Cancer: Targets and Targeting

Phosphorylation, Signaling, and Cancer: Targets and Targeting

Abstract 3465: Impact of intratumoral protein signaling network activation heterogeneity: Implication for precision medicine

Abstract Background: The evaluation of intratumor heterogeneity has become a major topic in the oncological field and it has major implications in the delivery of individualized therapy. While a number of genomic studies have reported significant intratumoral heterogeneity the analysis of the activation of protein kinase-driven signaling networks, which constitute the targets of most of the targeted compounds, has been sparse, or did not focus specifically on the tumor cells. This study evaluated the activation/phosphorylation of the protein signaling networks and a number of major precision medicine-oriented drug target to assess whether the intratumoral heterogeneity could impact patient stratification for targeted treatment in the context of metastatic disease. Methods: 3 independent study sets were used for this analysis: 1) metastatic colorectal cancer (mCRC) liver biopsies were collected from 6 patients; for each biopsy, 2 different regions were microdissected, 2) 18 needle biopsies were collected from 2 different regions of the same lesion of 9 mCRC liver metastases, 3) 7 serial liver biopsies were collected from 3 mCRC before and after treatment. Specimens were frozen upon collection, stored at -80°C and subjected to laser capture microdissection to isolate pure tumor epithelium. Protein pathway activation mapping was performed using reverse phase protein microarray. To assess changes in the activation level of the members of the MAPK and the AKT-mTOR pathway, unsupervised hierarchical clustering analysis and fold change between the different areas of the tumors were evaluated. Results: Hierarchical clustering analysis of the activation of 47 key signaling protein drug targets revealed that the signaling activation of any given protein or pathway was largely the same within a patient's tumor, no matter where the biopsy was obtained, but large interpatient variation was observed. When we evaluated activation of the MAPK and AKT-mTOR pathways, we found that the relative levels of pathway activation were found to be similar in different regions of the tumor, while each patient looked distinct. Conclusions: This first of its kind protein drug target activation analysis of microdissected tumor cells from human metastatic tumors revealed that the activated signaling architecture is largely maintained within the same tumor while each patient's tumor has a distinct patient-specific signaling portrait. The data suggest that interpatient protein drug target activation heterogeneity is much larger than intratumoral heterogeneity and point to the need to determine precision medicine based profiling on a patient-by-patient basis. Citation Format: Erika M. Parasido, Alessandra Silvestri, Claudio Belluco, Vincenzo Canzonieri, Maria Grazia Diodoro, Massimo Milione, Lance A. Liotta, Emanuel F. Petricoin, Mariaelena Pierobon. Impact of intratumoral protein signaling network activation heterogeneity: Implication for precision medicine. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3465. doi:10.1158/1538-7445.AM2014-3465

Abstract 3246: Mapping of functional protein pathway modulations in non-sun exposed skin of healthy volunteers using solar simulated light: A new model for pharmacodynamic testing of skin cancer chemopreventive drugs

Abstract Efficient secondary prevention methods for non-melanoma skin cancers are needed to supplement primary prevention in high-risk individuals. Pharmacologic effects in UV-induced keratinocyte signaling may predict efficacy of new drugs. In order to understand effects of solar light on signal transduction networks in human skin, we exposed buttocks skin of healthy volunteers to solar-simulated light (SSL) at doses 2x-3x of minimum erythemal dose (MED) using a Multiport UV Solar Simulator (Solar Light Co.). Punch biopsies (6 mm) were collected at baseline, 5 min, 1 hr, 5 hr, and 24 hr post-exposure. Biopsies were split with half fixed in formalin and half snap-frozen for Reverse Phase Protein Microarray (RPMA) analysis to identify relevant signaling networks activated by SSL exposure. 24 subjects ≥ 18 y.o. with Fitzpatrick skin type II or III, with no concurrent illness, cancer, or use of photosensitizing drugs were recruited. 12 were exposed to SSL at 2x MED, 6 at 2.5x MED, and 6 at 3x MED. The activation/phosphorylation or total levels of 128 key signaling proteins and drug targets were measured for each sample. Coordinate network-based analysis was performed on specific signaling pathways that included PI3k/Akt/mTOR, Ras/Raf/MEK/Erk, and Fyn/RSK2. Analyte levels were compared at baseline to those at 5 min, 1 hr, 5 hr, and 24 hr after SSL exposure. Unsupervised and supervised statistical analysis was used with Bonferroni multiple comparison adjustment (p &amp;lt; 0.01). Pathway activation maps were constructed using p values to indicate time-dependence of pathway activation. Differences in MED did not significantly affect expression, so all 24 subjects were analyzed independent of SSL dose. Most pathway modulation occurred within the first 5 hr, with cell death and apoptosis-related endpoints maximal at 24 hr. Many kinases were activated within 5 min and activity increased at 1-5 hr before reversing to baseline or lower at 24 hr. Early and sustained activation of p38/SAPK/ ERK pathways started at 5 min, continued through 5 hr and was sustained at 24 hr. Systemic AKT-mTOR pathway activation was observed from 5 min-1 hr, sustained through 5 hr, and decreased at 24 hr. EGFR-HER3 activation followed a similar pattern. COX2 expression increased at 1 hr and was sustained through 24 hr. AMPK was activated early and sustained through 24 hr since LKB1, the dominant AMPK kinase was activated within 5 min-1hr and sustained. Correlation-based network maps were generated and revealed time-dependent SSL induced pathway activation linkages. This work shows that protein pathway activation mapping of phosphorylated proteins in relevant signaling pathways can be used in future studies to determine pharmacodynamic activity of selective topical agents administered in a test area exposed to SSL to determine drug-induced modification of skin carcinogenesis pathways. Citation Format: Steven P. Stratton, Clara Curiel-Lewandrowski, Janine G. Einspahr, Valerie Calvert, Chengcheng Hu, Yira Bermudez, David S. Alberts, George T. Bowden, Emanuel F. Petricoin. Mapping of functional protein pathway modulations in non-sun exposed skin of healthy volunteers using solar simulated light: A new model for pharmacodynamic testing of skin cancer chemopreventive drugs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3246. doi:10.1158/1538-7445.AM2014-3246

Pilot phase I/II personalized therapy trial for metastatic colorectal cancer: evaluating the feasibility of protein pathway activation mapping for stratifying patients to therapy with imatinib and panitumumab.

This nonrandomized phase I/II trial assessed the efficacy/tolerability of imatinib plus panitumumab in patients affected by metastatic colorectal cancer (mCRC) after stratification to treatment by selection of activated imatinib drug targets using reverse-phase protein array (RPPA). mCRC patients presenting with a biopsiable liver metastasis were enrolled. Allocation to the experimental and control arms was established using functional pathway activation mapping of c-Kit, PDGFR, and c-Abl phosphorylation by RPPA. The experimental arm received run-in escalation therapy with imatinib followed by panitumumab. The control arm received panitumumab alone. Seven patients were enrolled in the study. For three of the seven patients, sequential pre- and post-treatment biopsies were used to evaluate the effect of the therapeutic compounds on the drug targets and substrates. A decrease in the activation level of the drug targets and downstream substrates was observed in two of three patients. Combination therapy increased the activation of the AKT-mTOR pathway and several receptor tyrosine kinases. This study proposes a novel methodology for stratifying patients to personalized treatment based on the activation level of the drug targets. This workflow provides the ability to monitor changes in the signaling pathways after the administration of targeted therapies and to identify compensatory mechanisms.

Abstract P6-04-01: Protein pathway activation mapping of LCM tumor epithelium from I-SPY 1 TRIAL surgical specimens reveals activation of receptor tyrosine kinase drug target signaling networks in the non-responding patient cohort

Abstract Background: Identification of alternative therapeutic strategies is critically needed for patients who do not achieve complete clinical response in the neoadjuvant setting, and in particular, from that cohort with the most aggressive disease (shortest relapse-free survival (RFS)). The I-SPY 1 TRIAL (CALGB 150007/150012, ACRIN 6657) is a trial of neoadjuvant anthracycline- and taxane-based chemotherapy that provides longitudinal biopsy specimens and extensive molecular and clinical/pathological characterization of non-responding patients. Methods: Tumor epithelium was procured by Laser Capture Microdissection (LCM) from frozen surgical specimens of 27 patients who did not achieve complete pathological response in the I-SPY1 TRIAL. Reverse Phase Protein Microarray (RPPA) technology was used to measure the levels of 120 key signaling proteins including drug targets for Phase I-III and FDA-approved therapeutics. Cox proportional hazard univariate and multivariate analyses were used to evaluate RFS correlates. Significance threshold was p = &amp;lt;0.05. Results: Univariate analysis of pathway activation revealed a number of actionable/druggable targets, particularly receptor tyrosine kinase (RTK) activation, where increased phosphorylation of ALK (Y1586), EGFR (Y1173), ERBB3 (Y1289), cABL (T735), and FAK (Y576/Y577) was significantly associated with aggressive disease (shorter RFS). Higher levels of SRC (Y527) inhibitory phosphorylation were also significantly associated with early recurrence. Coordinate activation of downstream targets of these RTKs, such as RAS-GRF S916, SHC (Y317), MEK (S217/221) and the Aurora kinases were also associated with early relapse in this study set. In multivariate analyses controlling for receptor subtype (HR+/HER2-, triple-negative (TN), HER2+), the associations between shorter RFS and RTK pathway activation including EGFR, ERBB3, cABL and FAK activation and SRC inhibition, as well as RAS-GRF and SHC phosphorylation remained significant. Conclusions: This unique functional protein pathway/proteomic analysis of LCM-isolated tumor epithelium by RPPA in surgical specimens taken from patients who did not achieve complete pathological response provides evidence for a significant number of druggable RTK pathway targets in patients with residual disease who are most at risk to die from breast cancer. Activation of ALK, EGFR, ERBB3, ABL, FAK, along with downstream RAS-ERK and Aurora kinase signaling activation in this cohort provide new insights into tumor progression along with distinct, actionable information on new therapeutic modalities to test in patients with progressing disease. Identification of new therapeutic strategies for patients who progress or recur early after neoadjuvant therapy is critical and, if validated in larger independent cohorts, could lead to rationalized personalized therapy-based trials. This strategy is being utilized in the I SPY 2 TRIAL as we introduce pathway-directed therapeutics in the neoadjuvant setting and are employing the RPPA platform to determine whether these agents are impacting their intended pathway. This work was partially supported by NIH grants CA31946 and CA33601. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-01.

An heregulin-EGFR-HER3 autocrine signaling axis can mediate acquired lapatinib resistance in HER2+ breast cancer models.

IntroductionThe human epidermal growth factor receptor 2 (HER2) receptor tyrosine kinase (RTK) oncogene is an attractive therapeutic target for the treatment of HER2-addicted tumors. Although lapatinib, an FDA-approved small-molecule HER2 and epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), represents a significant therapeutic advancement in the treatment of HER2+ breast cancers, responses to lapatinib have not been durable. Consequently, elucidation of mechanisms of acquired therapeutic resistance to HER-directed therapies is of critical importance.MethodsUsing a functional protein-pathway activation mapping strategy, along with targeted genomic knockdowns applied to a series of isogenic-matched pairs of lapatinib-sensitive and resistant cell lines, we now report an unexpected mechanism of acquired resistance to lapatinib and similar TKIs.ResultsThe signaling analysis revealed that whereas HER2 was appropriately inhibited in lapatinib-resistant cells, EGFR tyrosine phosphorylation was incompletely inhibited. Using a targeted molecular knockdown approach to interrogate the causal molecular underpinnings of EGFR-persistent activation, we found that lapatinib-resistant cells were no longer oncogene addicted to HER2-HER3-PI3K signaling, as seen in the parental lapatinib-sensitive cell lines, but instead were dependent on a heregulin (HRG)-driven HER3-EGFR-PI3K-PDK1 signaling axis. Two FDA-approved EGFR TKIs could not overcome HRG-HER3-mediated activation of EGFR, or reverse lapatinib resistance. The ability to overcome EGFR-mediated acquired therapeutic resistance to lapatinib was demonstrated through molecular knockdown of EGFR and treatment with the irreversible pan-HER TKI neratinib, which blocked HRG-dependent phosphorylation of HER3 and EGFR, resulting in apoptosis of resistant cells. In addition, whereas HRG reversed lapatinib-mediated antitumor effects in parental HER2+ breast cancer cells, neratinib was comparatively resistant to the effects of HRG in parental cells. Finally, we showed that HRG expression is an independent negative predictor of clinical outcome in HER2+ breast cancers, providing potential clinical relevance to our findings.ConclusionsMolecular analysis of acquired therapeutic resistance to lapatinib identified a new resistance mechanism based on incomplete and "leaky" inhibition of EGFR by lapatinib. The selective pressure applied by incomplete inhibition of the EGFR drug target resulted in selection of ligand-driven feedback that sustained EGFR activation in the face of constant exposure to the drug. Inadequate target inhibition driven by a ligand-mediated autocrine feedback loop may represent a broader mechanism of therapeutic resistance to HER TKIs and suggests adopting a different strategy for selecting more effective TKIs to advance into the clinic.

Dynamic Protein Pathway Activation Mapping of Adipose-Derived Stem Cell Differentiation Implicates Novel Regulators of Adipocyte Differentiation

Next to embryonic stem cell research, adult stem cell research is providing a promising alternative for enhanced tissue regeneration and transplantation. The key biochemical networks controlling the differentiation processes regulating stem cell biology remain largely disputed and or undefined, contributing to a lack of knowledge of the principle phosphoregulatory events propagating signal transduction. To effectively monitor these events relative to adipocyte differentiation, this study utilized a high throughput reverse phase protein microarray platform and characterized adult adipose-derived stem cell (ASC) differentiation through the monitoring of ∼100 phosphospecific endpoints with 33 distinct time points examined across 14 days. This kinetic-based analysis showed time ordered signal transduction ultimately implicating pathways correlated with adipogenic differentiation. To further validate the causal significance of these network activations, pharmacological targeting was implemented to include the chemical inhibitors MAPK inhibitor PD169316, rapamycin, and HNMPA-(AM)3 yielding partial or complete disruption of adipocytic differentiation, as noted by a decrease or lack of lipid formation within the mature adipocytes. Based on this analysis, v-crk sarcoma virus CT10 oncogene homolog (CRKII) and c-abl oncogene 1, non-receptor tyrosine kinase (c-ABL) were implicated as novel key regulators of adipocyte differentiation, with v-akt murine thymoma viral oncogene (AKT), mammalian target of rapamycin (mTOR), and SMAD family member (SMAD) pathways being implicated as secondary regulators. This dynamic molecular profiling provides a novel insight into the signaling architecture of mesenchymal stem cell differentiation and may be useful in the development of therapeutic modulators for clinical applications; in addition to advancing the collective understanding of key cellular processes, ultimately contributing to more confident stem cell manipulation.

Reverse-phase protein microarray highlights HER2 signaling activation in immunohistochemistry/FISH/HER2-negative breast cancers

Evaluation of: Wulfkuhle JD, Berg D, Wolff C et al. Molecular analysis of HER2 signaling in human breast cancer by functional protein pathway activation mapping. Clin. Cancer Res. 18(23), 6426–6435 (2012).Exhaustive characterization and mapping of pivotal molecules and downstream effectors deregulated in breast cancer is of fundamental clinical value to define the most effective therapy. Wulfkuhle et al. applied reverse-phase protein microarray, a highly sensitive immunoassay able to perform quantitative and multiplexed analysis of total and/or modified cellular proteins, to assess protein levels and activation/phosphorylation status of the HER family (EGFR, HER2, HER3) and downstream signaling molecules in HER2+ and HER2- breast cancers. The research was performed using laser capture microdissected tumor epithelial cells from frozen samples and formalin-fixed paraffin embedded specimens, which were also analyzed by immunohistochemistry (IHC) and FISH. This study identified a subgroup of IHC/FISH/HER2- patients with HER2 activation/phosphorylation levels comparable with those obtained from IHC/FISH/HER2+ tumors. HER2 signaling activation was independent from total HER2 expression and involved HER3 and EGFR activation. These findings indicate that molecular characterization by reverse-phase protein microarray of HER2 and its partners/effectors in the signaling cascade enables the identification of a subgroup of IHC/FISH/HER2- patients showing HER2 signaling activation. These patients, currently excluded from targeted therapy administration, could potentially benefit from this and it could improve prognosis and survival.

A pilot study utilizing molecular profiling to find potential targets and select individualized treatments for patients with metastatic breast cancer.

TPS11123 Background: The objective of this prospective pilot study was to determine if treatment selected by molecular profiling (MP) of metastatic breast cancer (MBC) tumors at time of disease progression provides greater clinical benefit than empiric treatment selection. Methods: Eligible pts had MBC, ≥ 3 prior lines of therapy, and &gt; 6 wks - &lt; 6 months time to progression on last treatment prior to study entry. Fresh core biopsy samples were taken from the metastatic site for MP. Analysis by IHC, FISH, DNA microarray and reverse phase protein microarray (RPMA) a quantitative protein pathway activation mapping technique using laser capture micro dissected tumor cells was done, with results in ≤14 days reviewed by a Treatment Selection Committee. The primary objective was to compare the progression-free survival (PFS) using a MP selected treatment regimen to the preceding PFS. Clinical benefit for the pt is defined by PFS ratio (PFS on MP selected therapy/PFS on prior therapy) is ≥ 1.3, (JCO,28:4877-83:2010). N= 25 was determined (exact single-stage design for phase II studies, type I error rate of 5% [one-sided], power of 90%). MP selected therapy would warrant further investigation if ≥ 35% of the pts demonstrate a PFS ratio of ≥ 1.3. Results: Three U.S. sites enrolled 25 evaluable pts who were treated based on MP results. Pts were heavily pretreated with 4 -11 prior therapies (median 7.24). Ten pts (40%) met or exceeded the PFS ratio of 1.3. The most common targets used in drug treatment selection were TOPO1, TS, ER/PR, TOP2A, HER2. Sixty percent of pts’ samples had activation of drug targets determined by RPMA in 3 major clusters: pan-HER-AKT; EGFR/SRC/ERK/mTOR; IGFR/RAF/MEK/PLK1. Days on MP selected treatments range from 9 - 816+. 3 pts continue on treatment for 199, 254 and 816 days. Conclusions: This study demonstrates the feasibility of a highly multiplexed genomic and proteomic MP study for treatment selection in a timely fashion. Patient-specific target driven treatment selection based on MP of a metastatic lesion provided clinical benefit for 10 of 25 heavily pretreated MBC pts. Thus, this approach merits further investigation in future studies. Funded by The Side-Out Foundation. Clinical trial information: NCT01074814.

Protein network mapping of platinum-resistant and poor-survival ovarian cancer.

5560 Background: Epithelial ovarian carcinoma (EOC) is the fifth leading cause of tumor related death in the female population, with only 30% of patients alive at 5 years after diagnosis. Platinum resistance is a major cause of treatment failure. The aim of the study was to perform broad-scale drug target activation mapping of EOC to identify new druggable targets for personalized therapy. Methods: 72 ovarian primary lesions collected from chemo-naïve EOC patients were analyzed. Highly enriched tumor epithelial cells were isolated by laser capture microdissection, lysed and subjected to reverse phase protein microarray analysis for multiplexed protein pathway activation mapping. The activation/phosphorylation level of 156 key signaling proteins was analysed. Based on the disease-free interval to platinum therapy, 61 stage II-IV patients were segregated into platinum-resistant (&lt;6 months), platinum-sensitive (6-12 months), and platinum-supersensitive disease (&gt;12 months). One-way analysis of variance was used to detect significant differences among the three groups in the drug target activation profile. Results: Expression of the drug target PDGF Receptor β and activation of ErbB2/HER2 (Y1248) were significantly higher in patients with resistant disease compared to sensitive groups (respectively, p 0.0033 and p 0.0134), while the expression of Estrogen receptor α was greater in the supersensitive group (p 0.0295). Moreover, overall survival analysis including all stages revealed that the expression level of Cox2 is significantly higher in patients with shorter survival (HR: 2.48, p 0.0179). Conclusions: Functional drug target activation mapping revealed the unique signaling architecture of platinum-resistant EOC. If confirmed in independent study sets, these results suggest that the utilization of drugs targeting PDGF Receptor β and ErbB2/HER2 could be evaluated in platinum resistant EOC and/or in combination with platinum therapy in order to overcome acquired resistance. Finally, this study indicates that Cox2 may play an important role in aggressive EOC, and that the addition of Cox-inhibitors to standard of care could be rationally evaluated as a novel therapeutic regimen for ovarian cancer.

Abstract 2043: Protein pathway activation mapping analysis of metastatic breast cancer reveals potential new targets for personalized therapy.

Abstract Introduction: It is well known that the development of distant metastasis is the strongest prognostic factor associated with cancer mortality. Recent genomic and proteomic studies have demonstrated that the molecular profile of the metastatic lesions significantly differs from the primary tumor. For this reason, to improve response to therapy the identification of new drug targets needs to be based on the molecular profile of the metastatic lesions rather than the primary tumor. Since most targeted therapies work by modulating hyperactivated protein kinase signaling, the aim of this study was to utilize reverse phase protein arrays (RPPA) to quantitatively measure the drug target signaling architecture to identify new therapeutic options for Stage IV breast cancer patients based on the molecular profile of the metastatic lesion. Methods: Snap frozen material collected from 25 metastatic breast cancer patients enrolled in a prospective phase II trial (“Side Out” 1) were used for this study. Sites of metastasis were: liver (n=12), skin (n=5), lymph nodes (n=5), breast (n=2), and pelvis (n=1). All samples were subjected to Laser Capture Microdissection (LCM) and RPPA whereby the activation/phosphorylation level of 55 drug targets and linked substrates for FDA cleared/experimental therapies were measured. Results: Unsupervised hieratical clustering analysis revealed distinct patient subgroups driven largely by pathway activation more than by site of metastasis. Based on the activation level of the drug targets and the downstream effectors, three major clusters were identified. Each cluster appeared to be driven by a specific subset of drug targets: a) pan-HER family members, b) EGFR/SRC/ERK/mTOR, and c) IGFR/RAF/MEK/PLK1. Conclusion: This is the first study evaluating the activation status of FDA approved/experimental drug targets and downstream effectors in a relatively large number of breast cancer metastatic lesions. This type of approach provides a unique opportunity for identifying new drug targets for this group of patients and provides initial insights on which pathways should be considered in the delivery of targeted therapy to this group of patients. 1. A Pilot Study Utilizing Molecular Profiling by IHC, FISH, DNA Microarray, and Reverse Phase Protein Microarray (RPMA) of Patients’ Tumors to Find Potential Targets and Select Treatments for Patients with Metastatic Breast Cancer, sponsored by the Side Out Foundation Citation Format: Mariaelena Pierobon, Kimberly A. Reeder, Gayle Jameson, Nicholas Robert, Stephen P. Anthony, Jasgit Sachdev, Linda Vocila, Julia Wulfkuhle, Elisa Baldelli, Lance Liotta, Emanuel F. Petricoin. Protein pathway activation mapping analysis of metastatic breast cancer reveals potential new targets for personalized therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2043. doi:10.1158/1538-7445.AM2013-2043

Abstract P3-04-01: Protein pathway activation mapping of the I-SPY 1 biopsy specimens identifies new network focused drug targets for patients with triple negative tumors

Abstract Background: Identification of new therapies and predictive biomarkers is needed for patients with triple negative (TN) breast cancer. Elucidating pathway activation in cancer is critical as these proteins represent targets for many of the new molecular therapeutics and companion diagnostic markers. The I-SPY TRIAL (CALGB 150007/150012, ACRIN 6657) is a trial of neoadjuvant anthracycline- and taxane-based chemotherapy that provides longitudinal biopsy specimens and molecular and clinical/pathological characterization. Methods: Tumor epithelium was procured by Laser Capture Microdissection from 149 pretreatment frozen biopsy specimens (T1) and 102 frozen biopsy specimens collected 1–4 days after first chemotherapy treatment (T2). Reverse Phase Protein Microarray was used to measure the activation of 39 and 100 signaling proteins in the T1 and T2 biopsies, respectively, including drug targets for Phase I-III and FDA cleared therapeutics. Associations between pathway activation and response to chemotherapy (RCB 0/1 vs. 2/3) and relapse-free survival (RFS) were evaluated for the triple-negative (TN) patient population at each time point and for changes between T1 and T2. Significance thresholds for response and RFS were as follows: Wilcoxon rank sum test p &amp;lt; 0.05; Cox proportional hazard model, likelihood ratio p &amp;lt; 0.05. Results: We focused our analysis on protein changes in patients who had poor clinical outcomes (Table 1) We observed systemic activation of receptor tyrosine kinases such as MET and ALK in the T2 biopsies from patients that recurred, and VEGFR activation was seen in the T1 relapsed tumors. Activation in the AURORA-PLK1 cell cycle signaling network was seen with increased activation in the T2 biopsy from patients who did not respond to therapy and had poor RFS. Dynamic changes in ERK activation were revealed with decreased ERK activation between T1 and T2 time points associated with recurrence. Conclusions: Our analysis has revealed activation and increased expression of proteins involved in proliferation (Ki67), DNA repair (phosphorylated p53) and protein kinases in patients with TN disease who had poor clinical response. These events, if validated, point to potential treatment options that could be considered for non-responding TN breast cancer patients. VEGFR, ALK and MET, have FDA cleared therapeutics that could be rationally proposed for rapid clinical investigation, and there are numerous investigational agents that target AURORA and PLK1 in the clinic. Further validation is required to explore the significance of these ongoing findings with the hope that the analysis could lead to molecularly rationalized therapies for patients with TN tumors. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-04-01.