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AKT S473 distribution based on ER expression measured by IHC for 31 lesions included of the discovery set. ER was measured in a binary scale (Panel A) and in continuous scale using the H-Score (Panel B).

Purpose: Little is known about the molecular signatures associated with specific metastatic sites in breast cancer. Using comprehensive multi-omic molecular profiling, we assessed whether alterations or activation of the PI3K–AKT–mTOR pathway is associated with specific sites of breast cancer metastasis.Experimental Design: Next-generation sequencing–based whole-exome sequencing was coupled with reverse-phase protein microarray (RPPA) functional signaling network analysis to explore the PI3K–AKT–mTOR axis in 32 pretreated breast cancer metastases. RPPA-based signaling data were further validated in an independent cohort of 154 metastatic lesions from breast cancer and 101 unmatched primary breast tumors. The proportion of cases with PI3K–AKT–mTOR genomic alterations or signaling network activation were compared between hepatic and nonhepatic lesions.Results: PIK3CA mutation and activation of AKT (S473) and p70S6K (T389) were detected more frequently among liver metastases than nonhepatic lesions (P < 0.01, P = 0.056, and P = 0.053, respectively). However, PIK3CA mutations alone were insufficient in predicting protein activation (P = 0.32 and P = 0.19 for activated AKT and p70S6K, respectively). RPPA analysis of an independent cohort of 154 tumors confirmed the relationship between pathway activation and hepatic metastasis [AKT (S473), mTOR (S2448), and 4EBP1 (S65); P < 0.01, P = 0.02, and P = 0.01, respectively]. Similar results were also seen between liver metastases and primary breast tumors [AKT (S473) P < 0.01, mTOR (S2448) P < 0.01, 4EBP1 (S65) P = 0.01]. This signature was lost when primary tumors were compared with all metastatic sites combined.Conclusions: Breast cancer patients with liver metastasis may represent a molecularly homogenized cohort with increased incidence of PIK3CA mutations and activation of the PI3K–AKT–mTOR signaling network. Clin Cancer Res; 23(16); 4919–28. ©2017 AACR.

Supplementary material legend

Concordance between PIK3CA, AKT, and PTEN mutation status and AKT (S473) and p70S6 (T389) activation.

The interest and utility of high-plex spatial profiling of RNA and protein biomarkers has increased over the last few years. The implementation of high-plex analyte spatial platforms, such as GeoMx® Digital Spatial Profiler (DSP), is increasing within discovery and development approaches for biomarkers associated with clinical outcome. The surge in spatial platforms parallels the increase of digital pathology in translational and clinical research studies. The integration of these two workflows has the potential to benefit diagnostic and therapeutic development. This study aims to facilitate the implementation of DSP in tissue analysis workflows helping those involved in drug discovery and development efforts to (1) assess platform feasibility for their research, (2) design effective DSP experiments, and (3) enable generation of high-quality, usable spatial data from large cohort studies. The BioPharma GeoMx DSP Consortium has developed consensus-based best practices incorporating the expertise of panel members via virtual meetings. Best practices guidelines for spatial profiling of tissue biopsies in drug discovery and development using GeoMx stands to advance current standard practices in tissue analysis. These best practices recommendations encompass every step of the implementation of DSP in standard tissue analysis workflows, emphasizing the importance of multidisciplinary stakeholder involvement, testing, defining experimental conditions prior to execution of large-scale studies, and considerations in assessing assay performance. This study offers a practical reference for the optimal implementation of GeoMx DSP in exploratory sample analysis for drug discovery and development studies. Citation Format: Leslie Abad, Maxine McClain, Edward Bonnevie, Benjamin Chen, Sarah Church, Gokhan Demirkan, Premi Haynes, Kelly Hunter, Anil Kersarwani, David Krull, Yan Liang, Prithwish Pal, Corinne Ramos, Deniliz Rodriguez, Jessica Runyon, Julien Tessier, Esperanza Anguiano. A BioPharma best practices framework to enable successful morphology guided spatial biology studies using NanoString GeoMx® Digital Spatial Profiler [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 986.

Background: Glioblastoma (GBM) is the most common and aggressive form of brain tumor, characterized by a poorly accessible microenvironment that render it notoriously hard to treat. The insufficient treatment success is, in large parts, due to its tremendous molecular heterogeneity, which affects the overall prognosis and response to therapies. The significant intra- and inter-tumor microenvironment (TME) composition heterogeneity plays a crucial role in GBM progression. Mostly due to this high, multifactorial immunosuppression occurring in the microenvironment, the efficacy of immunotherapy in GBM is low. Focusing on characterizing the components of different TMEs to evaluate their molecular and cellular component heterogeneity has a significant advantage to target group of cancer patients that would normally be resistant to immunotherapy. Methods: Deep spatial transcriptomics profiling was performed on non-treated glioblastoma human tissue microarrays (TMA) using NanoString's GeoMx Digital Spatial Profiler (DSP). 6 tumor tissues from 6 different patients were hybridized and analyzed with the Whole Transcriptomic Atlas (WTA) panel that includes 18,000 genes. Three regions of interest (ROIs) were selected per tissue sample. All the statistical analysis were performed with GeoMx® DSP Analysis Suite. Cell deconvolution using the SpatialDecon® algorithm (Nanostring®) was then performed to estimate mixed cell type abundance in the spatially-resolved gene expression defined segments. Results: Spatial transcriptomic analyses revealed that glioblastoma tumors varied dramatically in their global gene expression profiles and suggested the existence of differential gene expression among the tumors highlighting the inter-tumors heterogeneity of glioblastoma. Interestingly, different areas of the same tumor did not display distinct molecular profiles. Upregulation of stromal collagen genes was significantly detected in some patient specifically the fibrillar collagens type I, III and VI. Those findings indicated excessive collagen depositions in the surrounding of the tumor for some patient characteristic of tumor progression and involvement of cancer associated fibroblasts (CAFs) in the dysregulated collagen turnover leading to tumor fibrosis. Conclusion: As a cold tumor, malignant glioma has strong immunosuppression and immune escape characteristics. The TME provides the “soil” for the survival of malignant tumors, and recent studies have highlighted a major role for cancer-associated fibroblasts (CAFs) in promoting immunotherapy resistance by excluding T cells from tumors in the TME. Here the spatial transcriptomic analyses allowed a better phenotypical stratification of the cancer patient. In some, targeting CAFs would improve the TME and enhance the efficacy of immunotherapy. Citation Format: Sandra Delebecq, Adele Ponzoni, Richard Ruez, Corinne Ramos, Jonathan Stauber. Heterogeneity in glioblastoma tumor microenvironment: Opportunities for new therapies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4629.

Background: Forkhead box protein P3 (FOXP3), belonging to the forkhead/winged-helix family, is of great importance in modulating the differentiation and development of T-regulatory cells (Tregs). The numbers of FOXP3+ Tregs as well as exhaustive subsets of CD4+ and CD8+ T cells have been observed increased in solid tumor tissues of patients with colorectal and breast cancers, which helps to create an immunosuppressive environment and promotes tumor progression. However, the correlation between the presence of FOXP3+ Tregs and patients’ survival has been extensively studied in many cancer types, which remains conflicting. The approach presented here aims to absolutely quantify the level of FOXP3 in single cells and constructed effective immune risk models to predict tumor prognosis. Method: Imaging mass cytometry (IMC) was used to quantify FOXP3 inside each single cell. The indirect quantification used a free-metal titration curve in order to match the level of intensity of the metal-tagged antibodies to the level of expression of the antigen. Deep learning (StarDist QuPath extension) was used for cell segmentation and expression quantification. FOXP3 quantification in tissue or at the single cell level was validated against ELISA assay that measure FOXP3 in tissue lysate. Results: Linearity of the metal signals with an average %CV of 17% was observed emphasizing the linear correlation between free metal concentration and intensity. With the IMC technique, the minimum amount of FoxP3 detectable is 0.09 femtogram and its absolute quantification in the different cells in tumor tissues was confronted to disease prognosis in order to start building an effective immune risk model. Conclusion: With the introduction of new biologics in cancer therapy, it is imperative to follow real quantitative change in the expression of biological marker that are indicator of tumor prognosis. Indeed, although the FOXP3 distribution was already seen to be heterogenous in tumors, its absolute level of expression indicative of disease progression can be directly correlated to gain better understanding of the patient response stratification to biologics. Citation Format: Adele Ponzoni, Sandra Delebecq, Melodie Boute, Corinne Ramos, Jonathan Stauber. FoxP3 absolute quantification to build an effective risk model for cancer patient survival. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5571.

Background PD-L1 immunohistochemistry (IHC) has been traditionally used for predicting clinical responses to immune checkpoint inhibitors (ICIs). However, there are at least 4 different assays and antibodies used for PD-L1 IHC, each developed with a different ICI. Spatial transcriptomic analysis at the single cell level can be helpful for deeply characterize the complexity and heterogeneity of tumors. We set to test if in-situ hybridization-based RNA scope coupled to single cell quantification is a robust method to determine PD-L1 mRNA expression levels in each cell and furthermore efficacy of predicting response to ICIs as compared to routinely used standardized IHC procedures. Method FFPE sections from retrospectively collected surgically resected NSCLCs (adenocarcinoma and squamous cell carcinoma) tumor with progression disease or complete response from adjuvant pembrolizumab therapy were used. Target genes CD274 (PD-L1) and GZMB (Granzyme B) were assessed. For fluorescent detection, the label probe was conjugated to Opal 570 and 620 respectively. Tissues cells were counterstained with DAPI to detect the nuclei. A Digital pathology software QuPath was used for cell segmentation and dot’s detection for gene expression quantification and further classification. Results Difference in expression levels were seen between PD-L1 and GZMB across the tumor with PD-L1 staining mostly located in the tumoral area and Granzyme B in stromal area of the NSCLC tissue. This is consistent with the expected expression of PD-L1 on tumor epithelial cells and Granzyme B on T cells. This heterogeneity in the signal distribution specific to the analyzed tissue correspond to intra-tissue cells metabolism heterogeneity between cell types. This heterogeneity of expression was captured though a digital workflow capable of quantifying the number of cells expressing PD-L1 and GZMB. Among the cells expressing the markers, 96% were positive for PD-L1 for the tumor that did not respond to therapy with the threshold of positivity being set at 1 of dots, that corresponds to 1 transcript, per cell. Conclusion Our results demonstrate that PD-L1 was highly expressed in a tumor context that was not responding to PD-1 ICI inhibitor. Measurement of mRNA expression at the single cell level on tissue provides another layer of PD-L1 detection which can be exploited to predict tumor response to ICI suggesting that moving forward with this technology is a viable approach for this dynamic biomarker. Citation Format: Amandine Gerstenberg, Adele Ponzoni, Corinne Ramos, Jonathan Stauber. Absolute quantification of PD-L1 gene expression in single cell on tissue for accurately predicting response to ICIs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2183.

Purpose Non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancers. There is a strong rationale for incorporating immunotherapy into the treatment of early-stage NSCLC, given the breakthrough results with PD-1 checkpoint inhibitors in advanced-stage NSCLC. How immunotherapy should be implemented in patients who are operable is still unclear. Most of the efforts so far to identify clinically useful biomarkers do not preserve spatial information and leave us blind to the critical source of information revealed in the cell-to-cell biology of the tumor microenvironment (TME). In order to overcome these limitations, we used spatial biomarkers assays that preserve this critical information about which cells are influencing treatment response. Method Frozen sections from retrospectively collected surgically resected NSCLC (adenocarcinoma and squamous cell carcinoma) tumors treated with adjuvant pembrolizumab therapy were used. Patients were classified in two groups according to their Objective Response Rate (ORR): Complete Response (CR) and Progression Disease (PD) for spatial transcriptomic and proteomics assays. Associated to RNA In situ hybridization (ISH) technology, the NanoString GeoMx™ Digital Spatial Profiling (DSP) technology was used to determine immune markers within compartment specific defined by fluorescence localization [tumor (panCK) and leucocytes (CD45)]. Wilcoxon, Linear Mixed Model and Mann-Whitney U statistic tests were used to evaluate differences between ORR groups. Results Using the spatial analysis, we identified 4 protein markers from immune cell profiling and immune activation status modules independently associated with benefit from single-agent PD-1 checkpoint blockade in spatial context. High expression of CD4, CD40, Granzyme B and HLA-DR were significantly associated with all favorable clinical outcome, whereas upregulation of extracellular matrix (ECM) proteins related to pro-tumoral reprogramming of the stroma were associated with immunotherapy resistance. We also validated the DSP finding that high granzyme level in the stroma compartment was predictive for response to check point inhibitor in the same set of patients using ISH immunofluorescence, strengthening this marker relevance in antitumor immunity and as a predictive biomarker candidate for immunotherapy in NSCLC. Conclusion This work identifies a number of relevant spatial candidate predictors of immunotherapy outcome in spatial context for operable lung cancer that show promise for future validation in larger independent cohorts. Citation Format: Corinne Ramos, Ophelie Lemieux, Adele Ponzoni, Jonathan Stauber. Spatial distribution of granzyme B to improve the predictive power of response to immunotherapy in operable lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1116.

BACKGROUND Acquired resistance to antiepidermal growth factor receptor (anti-EGFR) therapy may be caused by EGFR–v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2 (ErbB2) heterodimerization and pathway reactivation. In preclinical studies, inhibiting ErbB2 blocked this resistance mechanism and resensitized cells to anti-EGFR therapy. Cetuximab targets EGFR, whereas lapatinib inhibits both EGFR and ErbB2. The objective of this phase 1 trial was to assess the safety, dose-limiting toxicities (DLTs), and maximum tolerated doses (MTDs) of cetuximab and lapatinib in patients with solid tumors. METHODS Patients received standard weekly cetuximab with escalating lapatinib doses of 750 mg, 1000 mg, or 1250 mg daily in 3-week cycles. DLTs were monitored through the end of cycle 2. Pretreatment and post-treatment tumor biopsies and germline DNA samples were obtained for correlative studies. RESULTS Twenty-two patients were enrolled, and 18 patients each were evaluable for toxicity and response. Fifty-nine percent of patients had received prior anti-EGFR therapy. Common toxicities included rash and diarrhea. No patient experienced a DLT at the highest dose level, and no grade 4 toxicity was observed. Response included no complete responses, 3 partial responses, 9 patients with stable disease, and 6 patients with disease progression, for an overall response rate of 17% and a clinical benefit rate of 67%. The clinical benefit rate in patients who had previously received anti-EGFR therapy was 70%. The mean treatment duration was 4.7 cycles (range, 1-14 cycles). Decreased expression of EGFR/ErbB2 pathway components after treatment was correlated with response, whereas increased expression in the PI3K, Jak/Stat, and MAPK pathways occurred in nonresponders. CONCLUSIONS The combination of cetuximab and lapatinib was well tolerated, had the expected toxicities, and exhibited notable clinical activity, including in patients who had received previous anti-EGFR therapy. Further clinical study of this combination is warranted. Cancer 2015;121:1645–1653. © 2015 American Cancer Society.

Purpose: Little is known about the molecular signatures associated with specific metastatic sites in breast cancer. Using comprehensive multi-omic molecular profiling, we assessed whether alterations or activation of the PI3K–AKT–mTOR pathway is associated with specific sites of breast cancer metastasis. Experimental Design: Next-generation sequencing–based whole-exome sequencing was coupled with reverse-phase protein microarray (RPPA) functional signaling network analysis to explore the PI3K–AKT–mTOR axis in 32 pretreated breast cancer metastases. RPPA-based signaling data were further validated in an independent cohort of 154 metastatic lesions from breast cancer and 101 unmatched primary breast tumors. The proportion of cases with PI3K–AKT–mTOR genomic alterations or signaling network activation were compared between hepatic and nonhepatic lesions. Results: PIK3CA mutation and activation of AKT (S473) and p70S6K (T389) were detected more frequently among liver metastases than nonhepatic lesions (P < 0.01, P = 0.056, and P = 0.053, respectively). However, PIK3CA mutations alone were insufficient in predicting protein activation (P = 0.32 and P = 0.19 for activated AKT and p70S6K, respectively). RPPA analysis of an independent cohort of 154 tumors confirmed the relationship between pathway activation and hepatic metastasis [AKT (S473), mTOR (S2448), and 4EBP1 (S65); P < 0.01, P = 0.02, and P = 0.01, respectively]. Similar results were also seen between liver metastases and primary breast tumors [AKT (S473) P < 0.01, mTOR (S2448) P < 0.01, 4EBP1 (S65) P = 0.01]. This signature was lost when primary tumors were compared with all metastatic sites combined. Conclusions: Breast cancer patients with liver metastasis may represent a molecularly homogenized cohort with increased incidence of PIK3CA mutations and activation of the PI3K–AKT–mTOR signaling network. Clin Cancer Res; 23(16); 4919–28. ©2017 AACR.

Nuclear pore complexes (NPCs) are large proteinaceous channels embedded in double nuclear membranes, which carry out nucleocytoplasmic exchange. The mechanism of nuclear pore assembly involves a unique challenge, as it requires creation of a long-lived membrane-lined channel connecting the inner and outer nuclear membranes. This stabilized membrane channel has little evolutionary precedent. Here we mapped inner/outer nuclear membrane fusion in NPC assembly biochemically by using novel assembly intermediates and membrane fusion inhibitors. Incubation of a Xenopus in vitro nuclear assembly system at 14°C revealed an early pore intermediate where nucleoporin subunits POM121 and the Nup107-160 complex were organized in a punctate pattern on the inner nuclear membrane. With time, this intermediate progressed to diffusion channel formation and finally to complete nuclear pore assembly. Correct channel formation was blocked by the hemifusion inhibitor lysophosphatidylcholine (LPC), but not if a complementary-shaped lipid, oleic acid (OA), was simultaneously added, as determined with a novel fluorescent dextran-quenching assay. Importantly, recruitment of the bulk of FG nucleoporins, characteristic of mature nuclear pores, was not observed before diffusion channel formation and was prevented by LPC or OA, but not by LPC+OA. These results map the crucial inner/outer nuclear membrane fusion event of NPC assembly downstream of POM121/Nup107-160 complex interaction and upstream or at the time of FG nucleoporin recruitment.

Post-mitotic reassembly of nuclear envelope (NE) and the endoplasmic reticulum (ER) has been reconstituted in a cell-free system based on interphase Xenopus egg extract. To evaluate the relative contributions of cytosolic and transmembrane proteins in NE and ER assembly, we replaced a part of native membrane vesicles with ones either functionally impaired by trypsin or N-ethylmaleimide treatments or with protein-free liposomes. Although neither impaired membrane vesicles nor liposomes formed ER and nuclear membrane, they both supported assembly reactions by fusing with native membrane vesicles. At membrane concentrations insufficient to generate full-sized functional nuclei, addition of liposomes and their fusion with membrane vesicles resulted in an extensive expansion of NE, further chromatin decondensation, restoration of the functionality, and spatial distribution of the nuclear pore complexes (NPCs), and, absent newly delivered transmembrane proteins, an increase in NPC numbers. This rescue of the nuclear assembly by liposomes was inhibited by wheat germ agglutinin and thus required active nuclear transport, similarly to the assembly of full-sized functional NE with membrane vesicles. Mechanism of fusion between liposomes and between liposomes and membrane vesicles was investigated using lipid mixing assay. This fusion required interphase cytosol and, like fusion between native membrane vesicles, was inhibited by guanosine 5′-3-O-(thio)triphosphate, soluble N-ethylmaleimide-sensitive factor attachment protein, and N-ethylmaleimide. Our findings suggest that interphase cytosol contains proteins that mediate the fusion stage of ER and NE reassembly, emphasize an unexpected tolerance of nucleus assembly to changes in concentrations of transmembrane proteins, and reveal the existence of a feedback mechanism that couples NE expansion with NPC assembly.

Assembly of the nuclear pore, gateway to the genome, from its component subunits is a complex process. In higher eukaryotes, nuclear pore assembly begins with the binding of ELYS/MEL-28 to chromatin and recruitment of the large critical Nup107-160 pore subunit. The choreography of steps that follow is largely speculative. Here, we set out to molecularly define early steps in nuclear pore assembly, beginning with chromatin binding. Point mutation analysis indicates that pore assembly is exquisitely sensitive to the change of only two amino acids in the AT-hook motif of ELYS. The dependence on AT-rich chromatin for ELYS binding is borne out by the use of two DNA-binding antibiotics. AT-binding Distamycin A largely blocks nuclear pore assembly, whereas GC-binding Chromomycin A3does not. Next, we find that recruitment of vesicles containing the key integral membrane pore proteins POM121 and NDC1 to the forming nucleus is dependent on chromatin-bound ELYS/Nup107-160 complex, whereas recruitment of gp210 vesicles is not. Indeed, we reveal an interaction between the cytoplasmic domain of POM121 and the Nup107-160 complex. Our data thus suggest an order for nuclear pore assembly of 1) AT-rich chromatin sites, 2) ELYS, 3) the Nup107-160 complex, and 4) POM121- and NDC1-containing membrane vesicles and/or sheets, followed by (5) assembly of the bulk of the remaining soluble pore subunits.

All identified membrane fusion proteins are transmembrane proteins. In the present study, we explored the post-mitotic reassembly of the NE (nuclear envelope). The proteins that drive membrane rearrangements in NE assembly remain unknown. To determine whether transmembrane proteins are prerequisite components of this fusion machinery, we have focused on nuclear reconstitution in a cell-free system. Mixing of soluble interphase cytosolic extract and MV (membrane vesicles) from amphibian eggs with chromatin results in the formation of functional nuclei. We replaced MV and cytosol with protein-free phosphatidylcholine LS (liposomes) that were pre-incubated with interphase cytosol. While later stages of NE assembly yielding functional nucleus did not proceed without integral proteins of MV, LS-associated cytosolic proteins were sufficient to reconstitute membrane targeting to the chromatin and GTP-dependent lipid mixing. Binding involved LS-associated A-type lamin, and fusion involved Ran GTPase. Thus in contrast with post-fusion stages, fusion initiation in NE assembly, like membrane remodelling in budding and fission, does not require transmembrane proteins.

The present invention discloses cell penetrating peptides and conjugates of a cell penetrating peptide and a cargo molecule.

We analyzed the genomic and phosphoproteomic profiles of breast cancer tissue obtained from six patients with estrogen receptor (ER)-positive, HER2-negative metastatic breast cancer who had highly durable (≥5 years) and, in some cases, ongoing clinical responses with capecitabine. Formalin-fixed, paraffin-embedded tissue samples from patients’ primary (n = 4) or metastatic (n = 2) breast cancers were utilized for targeted next-generation sequencing and reversed phase protein microarray. Two patients received capecitabine monotherapy. Four patients received capecitabine in combination with paclitaxel; three of these continued single-agent capecitabine after stopping paclitaxel. Capecitabine was discontinued for progressive disease after a mean of 66 months in four patients (range 54–86 months), and two patients remain on therapy, having received capecitabine for >91 months and >122 months, respectively. Three patients’ cancers (50%) had likely functional alterations in DNA repair and chromatin remodeling genes, while three other patients’ cancers had variants of unknown significance in these pathways. Mutations in PIK3CA, amplifications of FGFR1 or ZNF703, or phosphorylation of HER family receptors and their downstream proteins did not preclude exceptional responses to capecitabine. None of the patients’ tumors harbored TP53 or PTEN mutations. Four of the patients had breast cancer tissue available for PTEN immunohistochemistry, and all four patients’ cancers were positive for PTEN. These surprising findings in a group of phenotypically similar patients with ER-positive, endocrine therapy-pretreated, HER2-negative metastases, are supported by preclinical data showing that sensitivity to 5-fluorouracil is enhanced by deficiencies in chromatin remodeling and homologous recombination genes. Our findings suggest that mutations that inactivate homologous recombination and/or chromatin remodeling genes within ER-positive, HER2-negative breast cancers may predict for highly durable responses to capecitabine.

In this study, new methods are used to control cellular membrane tension to evaluate the role it plays in electrofusion. The data show that membrane tension present during the application of an electric field facilitates electro-induced membrane fusion. No enhancement was detected if the strain was applied after the pulse. Analysis of the electromechanical process of fusion revealed a synergy between the two kinds of constraints in the membrane fusion. Both mechanical and electrical constraints apparently play a key role in membrane fusion between the granule membrane and the plasma membrane, i.e. the exocytosis process.

Initial transport kinetics of isoniazid (INH) and its uptake at the plateau were studied in Mycobacterium tuberculosis H37Rv under various experimental conditions. The initial uptake velocity increased linearly with INH concentration from 2 x 10-6 M to 10-2 M. It was modified neither by addition of a protonophore that abolished proline transport, nor following ATP depletion by arsenate, which inhibited glycerol uptake, two transport processes taken as controls for secondary active transport and facilitated diffusion, respectively. Microaerobiosis or low temperature (4 °) were without effect on initial uptake. It is thus likely that INH transport in M. tuberculosis proceeds by a passive diffusion mechanism, and that catalase-peroxidase (KatG) is not involved in the actual transport. However, conditions inhibiting KatG activity (high INH concentration, microaerobiosis, low temperature) decrease cell radioactivity at the uptake plateau. It is proposed that INH transport occurs by passive diffusion. KatG is involved only in the intracellular accumulation of oxidized derivatives of INH, especially of isonicotinic acid, which is trapped inside cells in its ionized form. This model explains observed and previously known characteristics of the accumulation of radioactivity in the presence of [14C]INH for various species and strains of mycobacteria.

Background A significant percentage of patients with HER2+ breast cancer exhibit de novo resistance or develop an acquired resistance to anti-HER therapy. HER2 overexpression is currently used to guide anti-HER2 therapy by either IHC to measure the total amount of HER2 protein present, or FISH to measure the number of copies of the HER2 gene. Several potential mechanisms for this resistance have been proposed, ranging from mechanisms intrinsic to the target itself to the involvement of compensatory signaling pathways. As current therapy-guiding assays provide a measure of the level of HER2 protein, a better predictor of response may be possible through an assessment of not only HER2 activation (phosphorylation status), but also the protein levels of the other HER family members, their activation, and the activation of downstream resistance signaling pathways. To provide evidence that HER receptors are actively signaling, and to identify alternative therapies, a measure of the activation status of key signal transduction pathways downstream of these receptors was performed. The availability of molecular diagnostic assays that can evaluate phosphoproteins is an appealing approach to predicting treatment-sensitivity and to select more effective therapies. Methods De-identified breast tissue samples, including a subset of samples having their HER2 status identified by IHC and FISH were received for phosphoproteomic analysis using the TheraLink® HER Family Assay. The assay utilizes reverse-phase protein microarray (RPMA) to measure the total protein level and activation status of multiple proteins directly from a FFPE-biopsy lysate. The TheraLink® Assay measures the protein levels of EGFR, HER2, and HER3, their phosphorylation status, and the activation status of proteins in three downstream signaling pathways: AKT/mTOR; Mek/Erk; and Jak/STAT. Results The TheraLink® Assay's panel identified the cohort of tumors with known HER2 status (100% concordance between HER2 level measured by RPMA platform and IHC/FISH). Moreover, unsupervised clustering of this subset of samples identified three patterns of unique signaling. The first group showed high phosphorylation levels of HER2 and EGFR protein, suggesting the potential use of a dual kinase inhibitor therapy. The PI3 kinase and MAPK pathways were also elevated in this cohort. A second group that might not benefit from mono-therapy was observed. This group showed high levels of HER3 protein, a well-known dimerization partner for HER2. Therefore, this group might benefit from an anti-dimerization therapy, like pertuzumab. The PI3 Kinase and Jak/Stat pathways were also highly activated in this cohort. A third unique group exhibiting only activation of HER2 and HER3 was observed, with no downstream activity. Although a pan-HER kinase inhibitor has therapeutic potential, further downstream pathways, as well as other tyrosine kinase receptors, should be further examined. Conclusion The TheraLink® Assay has the potential to identify therapeutic strategies that might provide benefit to patients that are HER2 positive but failing on anti-HER2 therapy. Alternative therapeutics can be more precisely identified using the TheraLinkTM HER Family Assay's panel, based upon the molecular uniqueness of the groups described. Citation Format: Corinne Ramos, Nicholas Hoke, George J. Snipes, Pinar Yurt, Cody Thomas, Tuan Tran. A retrospective study to assess the potential for the TheraLink® HER family assay, a reverse-phase protein microarray assay, to predict treatment benefit in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-112. doi:10.1158/1538-7445.AM2015-LB-112

Cellular uptake of a family of cationic cell-penetrating peptides (examples include Tat peptides and penetratin) have been ascribed in the literature to a mechanism that does not involve endocytosis. In this work we reevaluate the mechanisms of cellular uptake of Tat 48-60 and (Arg)(9). We demonstrate here that cell fixation, even in mild conditions, leads to the artifactual uptake of these peptides. Moreover, we show that flow cytometry analysis cannot be used validly to evaluate cellular uptake unless a step of trypsin digestion of the cell membrane-adsorbed peptide is included in the protocol. Fluorescence microscopy on live unfixed cells shows characteristic endosomal distribution of peptides. Flow cytometry analysis indicates that the kinetics of uptake are similar to the kinetics of endocytosis. Peptide uptake is inhibited by incubation at low temperature and cellular ATP pool depletion. Similar data were obtained for Tat-conjugated peptide nucleic acids. These data are consistent with the involvement of endocytosis in the cellular internalization of cell-penetrating peptides and their conjugates to peptide nucleic acids.

The refolding of the prototypic fusogenic protein hemagglutinin (HA) at the pH of fusion is considered to be a concerted and irreversible discharge of a loaded spring, with no distinct intermediates between the initial and final conformations. Here, we show that HA refolding involves reversible conformations with a lifetime of minutes. After reneutralization, low pH‐activated HA returns from the conformations wherein both the fusion peptide and the kinked loop of the HA2 subunit are exposed, but the HA1 subunits have not yet dissociated, to a structure indistinguishable from the initial one in functional, biochemical and immunological characteristics. The rate of the transition from reversible conformations to irreversible refolding depends on the pH and on the presence of target membrane. Importantly, recovery of the initial conformation is blocked by the interactions between adjacent HA trimers. The existence of the identified reversible stage of refolding can be crucial for allowing multiple copies of HA to synchronize their release of conformational energy, as required for fusion.

African American women are more likely to be diagnosed with aggressive breast cancer at a younger age, have a higher risk of death, and have a higher prevalence of obesity than their Caucasian counterparts. Moreover, African American (AA) women diagnosed with triple-negative breast cancer have the lowest survival rates of all racial/ethnic groups. Although these disparities have been attributed to differences in biology, family and reproductive history, mammographic breast density, body mass index, access to healthcare, diet and environment, more etiologic studies are needed to elucidate the complex association between race, biologic factors, and socioeconomic factors. In the present study, we tested the feasibility of using high-throughput reverse phase protein microarray to identify protein(s) that are differentially expressed in the primary invasive breast cancers of premenopausal Caucasian and African American women. We examined the expression of 60 phosphorylated, total, and cleaved proteins in microdissected epithelial cells from 26 pre-treatment primary breast cancers. The impact of obesity on protein expression was also considered by stratifying samples into non-obese and obese based on a body mass index (BMI). Samples that come from women with BMI < 30 kg/m2 are classified as non-obese, while those with BMI ≥ 30 kg/m2 are considered obese. All statistical analyses were done using SAS Enterprise Guide 5.1 (SAS Institute, Cary, NC). For comparison of 2 groups, t tests were used for continuous variables and Fisher exact tests for categorical variables. ANOVA was used for comparison of more than 2 groups. Two-tailed p-value < 0.05 was considered statistically significant. Unsupervised hierarchical clustering analysis was performed on log 2-transformed values for each protein endpoint, using average linkage of the Pearson correlation coefficient. In this exploratory study, 58% of the primary breast cancers come from Caucasian women, while 42% of the samples come from AA women. The mean age and BMI for the two groups of women did not differ significantly. While the majority of the samples were of luminal subtypes, the triple-negative breast cancers were represented in majority of AA women. The expression of MEK1/2 S217/S221, PKC pan beta S660, and total PTEN were significantly higher in Caucasian women. When the impact of breast cancer subtypes (triple-negative vs. luminal A) on protein expression was examined, only total PTEN was statistically significant. Given the role of obesity in breast cancer, we determined which proteins are differentially expressed in non-obese and obese samples. Few components of the insulin and insulin-like growth factor and inflammatory signaling pathways (e.g. PTEN, acetyl CoA carboxylase, PKC pan beta S660, Stat3) were highly expressed in samples of obese women. This preliminary data demonstrates (a) the feasibility of mapping cell signaling networks in limited number of pre-treatment samples from premenopausal women and (b) the potential of future proteomic and larger cohort studies to identify which women will likely benefit from combination of targeted agents. Citation Format: Catherine Ibarra Drendall, William Barry, Corinne Ramos, Nora Tolbert, Joseph Geradts, Emanuel Petricoin, Victoria Seewaldt. Feasibility study: Protein profiling of primary breast cancers from premenopausal women. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr C16. doi:10.1158/1538-7755.DISP13-C16