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Regulatory T cells (Tregs) reside in nonlymphoid tissues where they carry out unique functions. The molecular mechanisms responsible for Treg accumulation and maintenance in these tissues are relatively unknown. Using an unbiased discovery approach, we identified LAYN (layilin), a C-type lectin-like receptor, to be preferentially and highly expressed on a subset of activated Tregs in healthy and diseased human skin. Expression of layilin on Tregs was induced by TCR-mediated activation in the presence of IL-2 or TGF-β. Mice with a conditional deletion of layilin in Tregs had reduced accumulation of these cells in tumors. However, these animals somewhat paradoxically had enhanced immune regulation in the tumor microenvironment, resulting in increased tumor growth. Mechanistically, layilin expression on Tregs had a minimal effect on their activation and suppressive capacity in vitro. However, expression of this molecule resulted in a cumulative anchoring effect on Treg dynamic motility in vivo. Taken together, our results suggest a model whereby layilin facilitates Treg adhesion in skin and, in doing so, limits their suppressive capacity. These findings uncover a unique mechanism whereby reduced Treg motility acts to limit immune regulation in nonlymphoid organs and may help guide strategies to exploit this phenomenon for therapeutic benefit.

Background: Multi-omic single cell studies are revolutionizing knowledge of the tumor immune microenvironment and positioned to detect rare cell subsets that correlate with response to immunotherapy. DNA-barcoded antibody panels enable the unambiguous classification of cell subsets via simultaneous measurement of surface proteins and transcriptome. However, single-cell sequencing performs best on fresh samples while multi-center clinical trials routinely obtain frozen specimens. Peripheral blood mononuclear cells (PBMCs) are commonly used tissues for charactering immune cell types, dynamics, and signaling in human studies. Our group has pioneered analyses of the tumor micro-environment in several cancer types, and also developed genomic capabilities with the resolution of flow cytometry over thousands of markers. We hypothesized that the development of advanced bioinformatics tools and large antibody panels (137 antibodies) can augment the analysis of frozen samples. Methods: Single cell genomics on matched fresh and frozen human PBMCs was performed using the TotalSeq-C human universal cocktail (BioLegend) and the 10x Genomics Chromium single-cell platform. We created bioinformatics pipelines to compare the quality control metrics, isotype matched control performance, and cell subtype analyses for fresh and frozen samples. Advanced visualization and analysis tools were developed to allow interrogation of the data in a flow cytometry paradigm, including complex gating strategies to identify rarer cell subtypes. Results: In terms of library quality, fresh and frozen cells had similar quality control parameters, although mean reads per cell were moderately lower for frozen vs. fresh tissue in both the RNA and antibody arms of the study. RNA and antibody data were integrated, clustered, and annotated for cell type and subtype. Cell type proportions were comparable across fresh and frozen samples. A panel of 7 antibody isotype controls demonstrated limited non-specific binding. Comparison of individual surface proteins across fresh and frozen samples revealed similar distributions in most cases, although signal was attenuated for a small number of epitopes in frozen samples. Our bioinformatics approaches allowed rare cell subtype identification with sensitivity and selectivity rivaling flow cytometry. Conclusions: Multi-modal single cell genomics, including a 137 antibody panel, and novel bioinformatics analytics reveal that fresh and frozen PBMC samples are largely concordant at both the transcriptome and cell-surface protein levels. Our pilot study is being expanded to larger scale clinical settings and may help characterize important cell populations that are associated with disease status, pharmacodynamics, and therapeutic response. Citation Format: Nathan O. Siemers, Jasmine Chen, Parminder Mankoo, Shazia Ilyas. Evaluation of large antibody panels in single-cell genomic immunophenotyping of fresh and preserved human leukocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2244.

TGFβ is a potent immunosuppressive cytokine, acting on multiple cell types of both the innate and adaptive immune systems. Emerging preclinical and clinical data implicate TGFβ in tumor immune evasion, poor prognosis and resistance to PD1/PDL1 directed checkpoint therapy. Thus, neutralization of TGFβ is suggested to relieve immunosuppression via mechanisms that are distinct yet complementary to checkpoint inhibitors such as PD1. SAR439459 is a potent human pan-neutralizing anti-TGFβ antibody that has entered first-in-human studies in advanced solid tumors (NCT03192345*). I. Biomarkers for indication selection: A gene expression signature of TGFβ pathway activation was derived from analysis of TGFβ-stimulated versus naïve cancer cell lines. Queries of the signature against The Cancer Genome Atlas (TCGA) revealed that primary head and neck, ovarian, and colorectal cancers are enriched for activation. II. Biomarkers for patient selection: An analysis of the patients enriched for activation revealed that mesenchymal tumors predominate. Machine learning methods were applied to panels of gene expression data for colorectal (CRC), ovarian serous and head and neck squamous cell carcinoma to derive a two-gene biomarker for selection of patients with mesenchymal tumors. Implementation in a logistic regression model achieves good for prediction in CRC, and similar performance in other indications. Patient cohorts are enriched over three-fold for the mesenchymal subtype and false-negative rates are acceptable. Implementation of the two-gene biomarker on a low complexity platform and its validation on an independent panel of samples is described. III. Biomarkers for assessment of the tumor microenvironment: In partnership with NeoGenomics, the immune contexture of patient tumors was evaluated using MultiOmyx, a multiplex IHC assay, on CRC and melanoma. Multiplexing was conducted with 12 biomarkers (jointly describing 22 immune cell types) on single FFPE section from each tumor sample. Pilot studies included a range of inflammation to evaluate how well the analytics assess each tumor type and correlate to possible treatment effects. Statistical methods were developed to assess differences at the cell population level, including replicate concordance, volcano plots for analyses of variance, and correlation matrices. The MultiOmyx assay demonstrated excellent technical reproducibility and precision, a favorable dynamic range, inflammation status differences in select immune cells and regions of interest, and included both positive and negative correlations between cell populations. * https://clinicaltrials.gov/ct2/show/NCT03192345 Citation Format: Joachim Theilhaber, Jean Cavallo, Stephen L. Madden, Charlene Manning, Sherry Cao, Parminder Mankoo, Robert Pomponio, Hongjing Qu, Natalia Malkova, Gary Shapiro, Christopher Winter, Dmitri Wiederschain, Michele Sanicola-Nadel, Fangxian Sun, Tun Tun Lin, Richard C. Gregory, Jack Pollard. Translational biomarkers for SAR439459, an anti-TGFβ antibody for cancer immunotherapy [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 5550.

There are currently few therapeutic options for patients with pancreatic cancer, and new insights into the pathogenesis of this lethal disease are urgently needed. Toward this end, we performed a comprehensive genetic analysis of 24 pancreatic cancers. We first determined the sequences of 23,219 transcripts, representing 20,661 protein-coding genes, in these samples. Then, we searched for homozygous deletions and amplifications in the tumor DNA by using microarrays containing probes for ∼10 6 single-nucleotide polymorphisms. We found that pancreatic cancers contain an average of 63 genetic alterations, the majority of which are point mutations. These alterations defined a core set of 12 cellular signaling pathways and processes that were each genetically altered in 67 to 100% of the tumors. Analysis of these tumors' transcriptomes with next-generation sequencing-by-synthesis technologies provided independent evidence for the importance of these pathways and processes. Our data indicate that genetically altered core pathways and regulatory processes only become evident once the coding regions of the genome are analyzed in depth. Dysregulation of these core pathways and processes through mutation can explain the major features of pancreatic tumorigenesis.

The recombinant fusion protein aflibercept (ziv-aflibercept in the United States) binds VEGF-A, VEGF-B, and placental growth factor (PlGF). The monoclonal antibody bevacizumab binds VEGF-A. Recent studies hypothesized that dual targeting of VEGF/PlGF is more beneficial than targeting either ligand. We compared activity of aflibercept versus bevacizumab in 48 patient-derived xenograft (PDX) colorectal cancer models. Nude mice engrafted subcutaneously with PDX colorectal cancer tumors received biweekly aflibercept, bevacizumab, or vehicle injections. Differential activity between aflibercept and bevacizumab, determined by mouse (m), human (h), VEGF-A, and PlGF levels in untreated tumors, was measured. Aflibercept induced complete tumor stasis in 31 of 48 models and bevacizumab in 2 of 48. Based on statistical analysis, aflibercept was more active than bevacizumab in 39 of 48 models; in 9 of 39 of these models, bevacizumab was considered inactive. In 9 of 48 remaining models, aflibercept and bevacizumab had similar activity. Tumor levels of hVEGF-A (range 776–56,039 pg/mg total protein) were ∼16- to 1,777-fold greater than mVEGF-A (range 8–159 pg/mg total protein). Tumor levels of mPlGF (range 104–1,837 pg/mg total protein) were higher than hPlGF (range 0–543 pg/mg total protein) in 47 of 48 models. Tumor cells were the major source of VEGF; PlGF was primarily produced by tumor stroma. Because tumor levels of hVEGF-A were far greater than mVEGF-A, bevacizumab's inability to bind mVEGF-A is unlikely to explain higher and more consistent aflibercept activity. Neutralizing PlGF and VEGFR-1 activation may be a factor and should be investigated in future studies. In these colorectal cancer PDX models, aflibercept demonstrated greater antitumor activity than bevacizumab. Mol Cancer Ther; 13(6); 1636–44. ©2014 AACR.

International audience; ModBase (http://salilab.org/modbase) is a database of annotated comparative protein structure models. The models are calculated by ModPipe, an auto- mated modeling pipeline that relies primarily on Modeller for fold assignment, sequence-structure alignment, model building and model assessment (http://salilab.org/modeller/). ModBase currently contains almost 30 million reliable models for domains in 4.7 million unique protein sequences. ModBase allows users to compute or update com- parative models on demand, through an interface to the ModWeb modeling server (http://salilab.org/ modweb). ModBase models are also available through the Protein Model Portal (http://www.prote inmodelportal.org/). Recently developed associated resources include the AllosMod server for modeling ligand-induced protein dynamics (http://salilab.org/ allosmod), the AllosMod-FoXS server for predicting a structural ensemble that fits an SAXS profile (http://salilab.org/allosmod-foxs), the FoXSDock server for protein–protein docking filtered by an SAXS profile (http://salilab.org/foxsdock), the SAXS Merge server for automatic merging of SAXS profiles (http://salilab.org/saxsmerge) and the Pose& Rank server for scoring protein–ligand complexes (http://salilab.org/poseandrank). In this update, we also highlight two applications of ModBase: a PSI:Biology initiative to maximize the structural coverage of the human alpha-helical transmem- brane proteome and a determination of structural determinants of human immunodeficiency virus-1 protease specificity.

Somatic mutations in PIK3CA (phosphati-dylinositol-3 kinase, catalytic subunit, alpha isoform) are reported in breast and other human cancers to concentrate at hotspots within its kinase and helical domains. Most of these mutations cause kinase gain of function in vitro and are associated with oncogenicity in vivo. However, little is known about the mechanisms driving tumor development. We have performed computational structural studies on a homology model of wildtype PIK3CA plus recurrent H1047R, H1047L, and P539R mutations, located in the kinase and helical domains, respectively. The time evolution of the structures show that H1047R/L mutants exhibit a larger area of the catalytic cleft between the kinase N- and C-lobes compared with the wildtype that could facilitate the entrance of substrates. This larger area might yield enhanced substrate-to-product turnover associated with oncogenicity. In addition, the H1047R/L mutants display increased kinase activation loop mobility, compared with the wildtype. The P539R mutant forms more hydrogen bonds and salt-bridge interactions than the wildtype, properties that are associated with enhanced thermostability. Mutant-specific differences in the catalytic cleft and activation loop behavior suggest that structure-based mutant-specific inhibitors can be designed for PIK3CA-positive breast cancers.

Glioblastoma multiforme (GBM) is the most common and lethal type of brain cancer. To identify the genetic alterations in GBMs, we sequenced 20,661 protein coding genes, determined the presence of amplifications and deletions using high-density oligonucleotide arrays, and performed gene expression analyses using next-generation sequencing technologies in 22 human tumor samples. This comprehensive analysis led to the discovery of a variety of genes that were not known to be altered in GBMs. Most notably, we found recurrent mutations in the active site of isocitrate dehydrogenase 1 ( IDH1 ) in 12% of GBM patients. Mutations in IDH1 occurred in a large fraction of young patients and in most patients with secondary GBMs and were associated with an increase in overall survival. These studies demonstrate the value of unbiased genomic analyses in the characterization of human brain cancer and identify a potentially useful genetic alteration for the classification and targeted therapy of GBMs.

Gastric cancer is the second leading cause of cancer deaths in the world. The genomics of gastric cancers is unique in that they harbor significantly more copy number alterations compared to point mutations, yet the functional importance of these genetic alterations in tumor maintenance is not known. To better understand oncogenic drivers of gastric cancer and identify potential therapeutic targets we performed negative selection RNAi screens in ten well annotated gastric cancer cell lines. Screens were performed using two different but overlapping shRNA libraries. The first library was the Decipher Human Module I pool from Cellecta composed of 27500 shRNAs targeting 5043 genes. The second library was a custom designed focused pool with 6500 shRNAs targeting 608 genes. In addition to screening the two shRNA libraries in vitro, the focused pool was also screened in subcutaneous xenograft tumor models in eight of the gastric cancer cell lines. The screens revealed distinct genetic vulnerabilities that correlated with the corresponding genomic alteration in the specific cell lines. In particular we found that KRAS amplifications confer dependency to the same degree as activating KRAS mutations. This KRAS dependency was further validated with additional shRNAs in KRAS amplified and mutated cell lines. Furthermore, we identified AMPK which is focally amplified in 9% of gastric cancer as a critical oncogenic driver. Multiple subunits of the AMPK holoenzyme scored in the screen and dependency on AMPK alpha and beta subunits was demonstrated with independent shRNAs in two cell lines from the primary screen. Consistent with the screen results we find that LMSU, a gastric cancer cell line not part of the primary screen but annotated as amplified for the AMPK alpha subunit shows elevated expression levels and is sensitive to knockdown of AMPK. These observations have identified AMPK as a novel oncogenic driver in gastric cancer with therapeutic potential. Citation Format: Meghana M. Kulkarni, Sushma Gurumurthy, Oleg Schmidt-Kittler, Jason Berglund, Christopher H. Hulton, David J. Wilson, David Jakubosky, Daniel Michaud, Robert E. Jones, Nicole M. Sjoblom, Russell McSweeney, Hongwei Zhou, Annapurna Venkatakrishnan, Karin J. Jensen, Jingxin Zhang, Parminder K. Mankoo, Jack Pollard, Christopher Winter, Pasi A. Jänne, Kwok-Kin Wong, Victoria M. Richon, Jessie M. English, Mark A. Bittinger. Functional genomics reveals genetic dependencies in gastric 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 1110. doi:10.1158/1538-7445.AM2015-1110

Background: The recombinant fusion protein aflibercept (afl) binds VEGF-A, -B, and placental growth factor (PlGF). Based on an OS benefit observed in the phase 3 VELOUR trial, aflibercept in combination with FOLFIRI was approved for patients with metastatic colorectal cancer (mCRC) following a prior oxaliplatin-containing regimen. In vitro, afl inhibited VEGFR signaling more potently and blocked cell migration more effectively vs. bevacizumab (bev) which only binds VEGF-A. Recent in vivo studies comparing the anti-tumor activity of afl vs. bev were conducted in patient-derived xenograft (PDX) CRC models. The PDX models are clinically relevant as they represent common genetic backgrounds in CRC and were derived from primary tumors or from metastases. A sequential treatment regimen of aflibercept post-bev vs. continuation of bev treatment was investigated. Methods: Forty eight different CRC adenocarcinoma tumors were engrafted subcutaneously (SC) into NMRI nude mice to generate 48 distinct PDX CRC models. ELISA and Luminex measured the expression of mouse and human VEGF-A and mouse and human PlGF in untreated tumors. Tumor-bearing mice (8 mice/group) were dosed with afl, bev, or placebo 2x/wk for 3 wks and SC tumor measurements were recorded 2x or 3x/wk. In 3 PDX models, mice that were treated with bev were randomized and either switched to afl treatment or continued to receive bev. Activity was expressed as relative change in tumor volume between treatment (ΔT) and control (ΔC) at 3 wks. A ΔT/ΔC ≤0% indicated complete tumor stasis. Student's t tests evaluated statistical differences in final tumor volumes. Results: Aflbercept induced complete tumor stasis in 31/48 PDX CRC models and bev in 2/48 PDX CRC models. The PDX models were characterized into 2 phenotypes based on statistical analysis: phenotype A, where afl activity was greater than bev activity (39 /48 PDX), and phenotype B, where there was no significant difference between the 2 drugs (9/48 PDX). Three models in which bev treatment resulted in eventual tumor escape vs. continuous inhibition by afl were selected for sequential treatment of bev followed by afl. Afl treatment began after bev treatment resulted in a greater than 2-fold increase in tumor volume. Greater anti-tumor activity occurred when afl was administered after bev vs. continuous bev treatment. At the end of the study, the tumor volumes were statistically significantly different between the afl and bev groups (T test p values < 0.0001). Analysis of angiogenic factors revealed that the tumor cells were the major source of VEGF whereas PlGF was primarily produced by the tumor stroma. Conclusions: Aflibercept induced complete tumor stasis more frequently in CRC PDX models than bevacizumab. As tumor levels of hVEGF-A were far greater than mVEGF-A, the inability of bevacizumab to bind mVEGF-A is unlikely to explain the higher and more consistent activity of aflibercept. In all 3 PDX models tested, switching to aflibercept treatment after bev treatment resulted in a greater tumor response than continuous bev treatment. The greater response to aflibercept than bev after an initial treatment with bev suggests that individual patients may derive more benefit from aflibercept than bev in second line treatment after an initial bev-based regimen. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B2. Citation Format: Marielle Chiron, Rebecca G. Bagley, Jack Pollard, Christophe Henry, Parminder Mankoo, Loic Vincent, Catherine Geslin, Tatjana Kloss, Donald A. Bergstrom. Switching to aflibercept treatment resulted in greater tumor responses than continuous bevacizumab treatment in patient-derived xenograft models of colorectal cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B2.