CancerInSilico: An R/Bioconductor package for combining mathematical and statistical modeling to simulate time course bulk and single cell gene expression data in cancer.

Bioinformatics techniques to analyze time course bulk and single cell omics data are advancing. The absence of a known ground truth of the dynamics of molecular changes challenges benchmarking their performance on real data. Realistic simulated time-course datasets are essential to assess the performance of time course bioinformatics algorithms. We develop an R/Bioconductor package, CancerInSilico, to simulate bulk and single cell transcriptional data from a known ground truth obtained from mathematical models of cellular systems. This package contains a general R infrastructure for running cell-based models and simulating gene expression data based on the model states. We show how to use this package to simulate a gene expression data set and consequently benchmark analysis methods on this data set with a known ground truth. The package is freely available via Bioconductor: http://bioconductor.org/packages/CancerInSilico/.

Cholecystokinin receptor antagonist alters pancreatic cancer microenvironment and increases efficacy of immune checkpoint antibody therapy in mice.

Advanced pancreatic ductal adenocarcinoma (PDAC) has typically been resistant to chemotherapy and immunotherapy; therefore, novel strategies are needed to enhance therapeutic response. Cholecystokinin (CCK) has been shown to stimulate growth of pancreatic cancer. CCK receptors (CCKRs) are present on pancreatic cancer cells, fibroblasts, and lymphocytes. We hypothesized that CCKR blockade would improve response to immune checkpoint antibodies by promoting influx of tumor-infiltrating lymphocytes (TILs) and reducing fibrosis. We examined the effects of CCKR antagonists or immune checkpoint blockade antibodies alone or in combination in murine models of PDAC. Monotherapy with CCKR blockade significantly decreased tumor size and metastases in SCID mice with orthotopic PDAC, and in C57BL/6 mice, it reduced fibrosis and induced the influx of TILs. Immune-competent mice bearing syngeneic pancreatic cancer (Panc02 and mT3-2D) that were treated with the combination of CCK receptor antagonists and immune checkpoint blockade antibodies survived significantly longer with smaller tumors. Tumor immunohistochemical staining and flow cytometry demonstrated that the tumors of mice treated with the combination regimen had a significant reduction in Foxp3+ T-regulatory cells and an increase in CD4+ and CD8+ lymphocytes. Masson's trichrome stain analysis revealed 50% less fibrosis in the tumors of mice treated with CCKR antagonist compared to controls and compared to checkpoint antibody therapy. CCKR antagonists given with immune checkpoint antibody therapy represent a novel approach for improving survival of PDAC. The mechanism by which this combination therapy improves the survival of PDAC may be related to the decreased fibrosis and immune cells of the tumor microenvironment.

Protein network construction using reverse phase protein array data.

In this paper, we introduce a novel computational method for constructing protein networks based on reverse phase protein array (RPPA) data to identify complex patterns in protein signaling. The method is applied to phosphoproteomic profiles of basal expression and activation/phosphorylation of 76 key signaling proteins in three breast cancer cell lines (MCF7, LCC1, and LCC9). Temporal RPPA data are acquired at 48h, 96h, and 144h after knocking down four genes in separate experiments. These genes are selected from a previous study as important determinants for breast cancer survival. Interaction networks are constructed by analyzing the expression levels of protein pairs using a multivariate analysis of variance model. A new scoring criterion is introduced to determine relevant protein pairs. Through a network topology based analysis, we search for wiring patterns to identify key proteins that are associated with significant changes in expression levels across various experimental conditions.

An unbiased in vivo functional genomics screening approach in mice identifies novel tumor cell-based regulators of immune rejection.

The clinical successes of immune checkpoint therapies for cancer make it important to identify mechanisms of resistance to anti-tumor immune responses. Numerous resistance mechanisms have been identified employing studies of single genes or pathways, thereby parsing the tumor microenvironment complexity into tractable pieces. However, this limits the potential for novel gene discovery to in vivo immune attack. To address this challenge, we developed an unbiased in vivo genome-wide RNAi screening platform that leverages host immune selection in strains of immune-competent and immunodeficient mice to select for tumor cell-based genes that regulate in vivo sensitivity to immune attack. Utilizing this approach in a syngeneic triple-negative breast cancer (TNBC) model, we identified 709 genes that selectively regulated adaptive anti-tumor immunity and focused on five genes (CD47, TGFß1, Sgpl1, Tex9 and Pex14) with the greatest impact. We validated the mechanisms that underlie the immune-related effects of expression of these genes in different TNBC lines, as well as tandem synergistic interactions. Furthermore, we demonstrate the impact of different genes with previously unknown immune functions (Tex9 and Pex14) on anti-tumor immunity. Thus, this innovative approach has utility in identifying unknown tumor-specific regulators of immune recognition in multiple settings to reveal novel targets for future immunotherapies.

Natural killer cells associate with epithelial cells in the pancreatic ductal adenocarcinoma tumor microenvironment.

Background: Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer. PDAC's poor prognosis and resistance to immunotherapy are attributed in part to its dense, fibrotic tumor microenvironment (TME), which is known to inhibit immune cell infiltration. We recently demonstrated that PDAC patients with higher natural killer (NK) cell content and activation have better survival rates. However, NK cell interactions in the PDAC TME have yet to be deeply studied. We show here that NK cells are present and active in the human PDAC TME. Methods: We used imaging mass cytometry (IMC) to assess NK cell content, function, and spatial localization in human PDAC samples. Then, we used CellChat, a tool to infer ligand-receptor interactions, on a human PDAC scRNAseq dataset to further define NK cell interactions in PDAC. Results: Spatial analyses showed for the first time that active NK cells are present in the PDAC TME, and both associate and interact with malignant epithelial cell ducts. We also found that fibroblast-rich, desmoplastic regions limit NK cell infiltration in the PDAC TME. CellChat analysis identified that the CD44 receptor on NK cells interacts with PDAC extracellular matrix (ECM) components such as collagen, fibronectin and laminin expressed by fibroblasts and malignant epithelial cells. This led us to hypothesize that these interactions play roles in regulating NK cell motility in desmoplastic PDAC TMEs. Using 2D and 3D in vitro assays, we found that CD44 neutralization significantly increased NK cell invasion through matrix. Conclusions: Targeting ECM-immune cell interactions may increase NK cell invasion into the PDAC TME.

Antibody dependent cell-mediated cytotoxicity selection pressure induces diverse mechanisms of resistance.

Targeted monoclonal antibody therapy has emerged as a powerful therapeutic strategy for cancer. However, only a minority of patients have durable responses and the development of resistance remains a major clinical obstacle. Antibody-dependent cell-mediated cytotoxicity (ADCC) represents a crucial therapeutic mechanism of action; however, few studies have explored ADCC resistance. Using multiple in vitro models of ADCC selection pressure, we have uncovered both shared and distinct resistance mechanisms. Persistent ADCC selection pressure yielded ADCC-resistant cells that are characterized by a loss of NK cell conjugation and this shared resistance phenotype is associated with cell-line dependent modulation of cell surface proteins that contribute to immune synapse formation and NK cell function. We employed single-cell RNA sequencing and proteomic screens to interrogate molecular mechanisms of resistance. We demonstrate that ADCC resistance involves upregulation of interferon/STAT1 and DNA damage response signaling as well as activation of the immunoproteasome. Here, we identify pathways that modulate ADCC sensitivity and report strategies to enhance ADCC-mediated elimination of cancer cells. ADCC resistance could not be reversed with combinatorial treatment approaches. Hence, our findings indicate that tumor cells utilize multiple strategies to inhibit NK cell mediated-ADCC. Future research and development of NK cell-based immunotherapies must incorporate plans to address or potentially prevent the induction of resistance.

Human CENP-I specifies localization of CENP-F, MAD1 and MAD2 to kinetochores and is essential for mitosis.

The kinetochore, a macromolecular complex located at the centromere of chromosomes, provides essential functions for accurate chromosome segregation. Kinetochores contain checkpoint proteins that monitor attachments between the kinetochore and microtubules to ensure that cells do not exit mitosis in the presence of unaligned chromosomes. Here we report that human CENP-I, a constitutive protein of the kinetochore that shares limited similarity with Mis6 of Schizosaccharomyces pombe, is required for the localization of CENP-F and the checkpoint proteins MAD1 and MAD2 to kinetochores. Depletion of CENP-I from kinetochores causes the cell cycle to delay in G2. Although monopolar chromosomes in CENP-I-depleted cells fail to establish bipolar connections, the cells are unable to arrest in mitosis. These cells are transiently delayed in mitosis in a MAD2-dependent manner, even though their kinetochores are depleted of MAD2. The delay is extended considerably when the number of unattached kinetochores is increased. This suggests that no single unattached kinetochore in CENP-I-depleted cells can arrest mitosis. The collective output from many unattached kinetochores is required to reach a threshold signal of 'wait for anaphase' to sustain a prolonged mitotic arrest.

Mapping the assembly pathways that specify formation of the trilaminar kinetochore plates in human cells.

We report the interactions amongst 20 proteins that specify their assembly to the centromere-kinetochore complex in human cells. Centromere protein (CENP)-A is at the top of a hierarchy that directs three major pathways, which are specified by CENP-C, -I, and Aurora B. Each pathway consists of branches that intersect to form nodes that may coordinate the assembly process. Complementary EM studies found that the formation of kinetochore trilaminar plates depends on the CENP-I/NUF2 branch, whereas CENP-C and Aurora B affect the size, shape, and structural integrity of the plates. We found that hMis12 is not constitutively localized at kinetochores, and that it is not essential for recruiting CENP-I. Our studies also revealed that kinetochores in HeLa cells contain an excess of CENP-A, of which approximately 10% is sufficient to promote the assembly of normal levels of kinetochore proteins. We elaborate on a previous model that suggested kinetochores are assembled from repetitive modules (Zinkowski, R.P., J. Meyne, and B.R. Brinkley. 1991. J. Cell Biol. 113:1091-110).

DPP inhibition alters the CXCR3 axis and enhances NK and CD8+ T cell infiltration to improve anti-PD1 efficacy in murine models of pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is projected to be the second leading cause of cancer death in the USA by 2030. Immune checkpoint inhibitors fail to control most PDAC tumors because of PDAC's extensive immunosuppressive microenvironment and poor immune infiltration, a phenotype also seen in other non-inflamed (ie, 'cold') tumors. Identifying novel ways to enhance immunotherapy efficacy in PDAC is critical. Dipeptidyl peptidase (DPP) inhibition can enhance immunotherapy efficacy in other cancer types; however, the impact of DPP inhibition on PDAC tumors remains unexplored. METHODS: We examined the effects of an oral small molecule DPP inhibitor (BXCL701) on PDAC tumor growth using mT3-2D and Pan02 subcutaneous syngeneic murine models in C57BL/6 mice. We explored the effects of DPP inhibition on the tumor immune landscape using RNAseq, immunohistochemistry, cytokine evaluation and flow cytometry. We then tested if BXCL701 enhanced anti-programmed cell death protein 1 (anti-PD1) efficacy and performed immune cell depletion and rechallenged studies to explore the relevance of cytotoxic immune cells to combination treatment efficacy. RESULTS: In both murine models of PDAC, DPP inhibition enhanced NK and T cell immune infiltration and reduced tumor growth. DPP inhibition also enhanced the efficacy of anti-PD1. The efficacy of dual anti-PD1 and BXCL701 therapy was dependent on both CD8+ T cells and NK cells. Mice treated with this combination therapy developed antitumor immune memory that cleared some tumors after re-exposure. Lastly, we used The Cancer Genome Atlas (TCGA) to demonstrate that increased NK cell content, but not T cell content, in human PDAC tumors is correlated with longer overall survival. We propose that broad DPP inhibition enhances antitumor immune response via two mechanisms: (1) DPP4 inhibition increases tumor content of CXCL9/10, which recruits CXCR3+ NK and T cells, and (2) DPP8/9 inhibition activates the inflammasome, resulting in proinflammatory cytokine release and Th1 response, further enhancing the CXCL9/10-CXCR3 axis. CONCLUSIONS: These findings show that DPP inhibition with BXCL701 represents a pharmacologic strategy to increase the tumor microenvironment immune cell content to improve anti-PD1 efficacy in PDAC, suggesting BXCL701 can enhance immunotherapy efficacy in 'cold' tumor types. These findings also highlight the potential importance of NK cells along with T cells in regulating PDAC tumor growth.

Antitumor T-cell Immunity Contributes to Pancreatic Cancer Immune Resistance.

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer death in the United States. Pancreatic tumors are minimally infiltrated by T cells and are largely refractory to immunotherapy. Accordingly, the role of T-cell immunity in pancreatic cancer has been somewhat overlooked. Here, we hypothesized that immune resistance in pancreatic cancer was induced in response to antitumor T-cell immune responses and that understanding how pancreatic tumors respond to immune attack may facilitate the development of more effective therapeutic strategies. We now provide evidence that T-cell-dependent host immune responses induce a PDAC-derived myeloid mimicry phenomenon and stimulate immune resistance. Three KPC mouse models of pancreatic cancer were used: the mT3-2D (Kras+/LSL-G12D; Trp53+/LSL-R172H; Pdx1-Cre) subcutaneous and orthotopic models, as well as the KP1 (p48-CRE/LSL-Kras/Trp53 flox/flox ) subcutaneous model. KPC cancer cells were grown in immunocompetent and immunodeficient C57BL/6 mice and analyzed to determine the impact of adaptive immunity on malignant epithelial cells, as well as on whole tumors. We found that induced T-cell antitumor immunity, via signal transducer and activator of transcription 1 (STAT1), stimulated malignant epithelial pancreatic cells to induce the expression of genes typically expressed by myeloid cells and altered intratumoral immunosuppressive myeloid cell profiles. Targeting the Janus Kinase (JAK)/STAT signaling pathway using the FDA-approved drug ruxolitinib overcame these tumor-protective responses and improved anti-PD-1 therapeutic efficacy. These findings provide future directions for treatments that specifically disable this mechanism of resistance in PDAC.

RNA interference screening methods to identify proliferation determinants and mechanisms of resistance to immune attack.

We have used RNA interference (RNAi) screening technology to reveal unknown components of biological signaling pathways including survival mechanisms of estrogen-independent breast cancer cell growth and cancer cell resistance to immune attack. In this chapter, a detailed protocol describing the use of RNAi screening to identify factors important for the proliferation of estrogen-independent MCF7 breast cancer cells will be described. Resistance to therapies that target the estrogen pathway remains a challenge in the treatment of estrogen receptor-positive breast cancer. To address this challenge, small interfering-RNA (siRNA)-based libraries targeting an estrogen receptor (ER)- and aromatase-centered network, including 631 genes relevant to estrogen signaling, was designed and constructed for RNAi screening. This protocol will include the following parts: (1) selection of RNAi transfection reagent for specific cells; (2) optimization of RNAi screening conditions using Z'-factor; (3) procedure of ER-network gene siRNA library screening using automated machines under optimized experimental conditions; and (4) method of analysis for RNAi screening data to identify specific determinants important for cell proliferation. 46 genes were found to be selectively required for the survival of estrogen-independent MCF7-derived cells.

SpCas9-expression by tumor cells can cause T cell-dependent tumor rejection in immunocompetent mice.

The CRISPR/Cas9 system has recently emerged as a highly efficient modality in genetic engineering and has been widely considered for various therapeutic applications. However, since the effector protein, SpCas9, has a bacterial origin, its immunogenicity must be explored in further depth. Here, we found that the intact immune system, in wild-type C57BL/6J and BALB/cL mice, stimulates specific immune response against SpCas9, resulting in the rejection of SpCas9-expressing tumors. However, these tumors effectively grew in syngeneic C57BL/6J immunodeficient, T cell-depleted and Cas9-KI mice. Therefore, these observations suggest that this tumor rejection phenotype is T cell-dependent. The immunological clearance of SpCas9-expressing tumors in the immunocompetent group illustrates the possibility of misinterpreting the impact of CRISPR/Cas9-mediated gene editing on in vivo tumor biology and survival. Thus, these findings have important implications for the use of this exciting approach in in vivo studies, as well as to manipulate cancer cell biology for therapeutic applications.

A Mechanism of Resistance to Antibody-Targeted Immune Attack.

Targeted monoclonal antibody therapy is a promising therapeutic strategy for cancer, and antibody-dependent cell-mediated cytotoxicity (ADCC) represents a crucial mechanism underlying these approaches. The majority of patients have limited responses to monoclonal antibody therapy due to the development of resistance. Models of ADCC provide a system for uncovering immune-resistance mechanisms. We continuously exposed epidermal growth factor receptor (EGFR+) A431 cells to KIR-deficient NK92-CD16V effector cells and the anti-EGFR cetuximab. Persistent ADCC exposure yielded ADCC-resistant cells (ADCCR1) that, compared with control ADCC-sensitive cells (ADCCS1), exhibited reduced EGFR expression, overexpression of histone- and interferon-related genes, and a failure to activate NK cells, without evidence of epithelial-to-mesenchymal transition. These properties gradually reversed following withdrawal of ADCC selection pressure. The development of resistance was associated with lower expression of multiple cell-surface molecules that contribute to cell-cell interactions and immune synapse formation. Classic immune checkpoints did not modulate ADCC in this unique model system of immune resistance. We showed that the induction of ADCC resistance involves genetic and epigenetic changes that lead to a general loss of target cell adhesion properties that are required for the establishment of an immune synapse, killer cell activation, and target cell cytotoxicity.

Ablation of PRC1 by small interfering RNA demonstrates that cytokinetic abscission requires a central spindle bundle in mammalian cells, whereas completion of furrowing does not.

The temporal and spatial regulation of cytokinesis requires an interaction between the anaphase mitotic spindle and the cell cortex. However, the relative roles of the spindle asters or the central spindle bundle are not clear in mammalian cells. The central spindle normally serves as a platform to localize key regulators of cell cleavage, including passenger proteins. Using time-lapse and immunofluorescence analysis, we have addressed the consequences of eliminating the central spindle by ablation of PRC1, a microtubule bundling protein that is critical to the formation of the central spindle. Without a central spindle, the asters guide the equatorial cortical accumulation of anillin and actin, and of the passenger proteins, which organize into a subcortical ring in anaphase. Furrowing goes to completion, but abscission to create two daughter cells fails. We conclude the central spindle bundle is required for abscission but not for furrowing in mammalian cells.

The RanGAP1-RanBP2 complex is essential for microtubule-kinetochore interactions in vivo.

RanGAP1 is the activating protein for the Ran GTPase. Vertebrate RanGAP1 is conjugated to a small ubiquitin-like protein, SUMO-1. This modification promotes association of RanGAP1 with the interphase nuclear pore complex (NPC) through binding to the nucleoporin RanBP2, also known as Nup358. During mitosis, RanGAP1 is concentrated at kinetochores in a microtubule- (MT) and SUMO-1-dependent fashion. RanBP2 is also abundantly found on kinetochores in mitosis. Here we show that ablation of proteins required for MT-kinetochore attachment (Hec1/Ndc80, Nuf2 ) disrupts RanGAP1 and RanBP2 targeting to kinetochores. No similar disruption was observed after ablation of proteins nonessential for MT-kinetochore interactions (CENP-I, Bub1, CENP-E ). Acquisition of RanGAP1 and RanBP2 by kinetochores is temporally correlated in untreated cells with MT attachment. These patterns of accumulation suggest a loading mechanism wherein the RanGAP1-RanBP2 complex may be transferred along the MT onto the kinetochore. Depletion of RanBP2 caused mislocalization of RanGAP1, Mad1, Mad2, CENP-E, and CENP-F, as well as loss of cold-stable kinetochore-MT interactions and accumulation of mitotic cells with multipolar spindles and unaligned chromosomes. Taken together, our observations indicate that RanBP2 and RanGAP1 are targeted as a single complex that is both regulated by and essential for stable kinetochore-MT association.

RNA Interference Screening to Identify Proliferation Determinants in Breast Cancer Cells.

RNAi screening technology has revealed unknown determinants of various biological signaling pathways in biomedical studies. This protocol provided detailed information about how to use RNAi screening to identify proliferation determinants in breast tumor cells. siRNA-based libraries targeting against Estrogen receptor (ER)-network, including 631 genes relevant to estrogen signaling, was constructed for screening in breast cancer cells. Briefly, reverse transfection of siRNA induced transient gene knockdown in MCF7 cells. First, the transfection reagent for MCF7 cells was selected. Next, the Z'-score assay was used to monitor if screening conditions yielded efficiently. Then, the ER-network siRNA library screening was preceded by automatic machines under optimized experimental conditions.

Chfr regulates a mitotic stress pathway through its RING-finger domain with ubiquitin ligase activity.

Resistance to chemotherapy targeting microtubules could be partially because of the delay in chromosome condensation and segregation during mitosis. The Chfr pathway has been defined recently, and its activation causes a delay in chromosome condensation in response to mitotic stress. Because Chfr contains a RING-finger domain, we tested whether Chfr inhibits chromosome condensation through an ubiquitin (ubiquitin)-dependent pathway. In the presence of purified E1, Ubc4, or Ubc5, and ubiquitin, Chfr catalyzes its own ubiquitination in vitro, an activity requiring the RING domain. In vivo, overexpressed Chfr but not a RING domain mutant is spontaneously ubiquitinated. Our studies with DLD1 cells stably expressing wild-type Chfr and Chfr lacking the RING domain indicated that the RING-finger deletion mutant was defective in inhibiting chromosome condensation after Taxol treatment. In addition, Chfr expression increases the survival rate after Taxol treatment, an activity requiring the RING domain. Preliminary studies indicate that Chfr expression is cell cycle regulated and is dependent on its ubiquitin ligase activity. It is very likely that the Chfr-mediated ubiquitin-dependent pathway is a critical component of the response to mitotic stress.

Screening of Conditionally Reprogrammed Patient-Derived Carcinoma Cells Identifies ERCC3-MYC Interactions as a Target in Pancreatic Cancer.

PURPOSE: Even when diagnosed prior to metastasis, pancreatic ductal adenocarcinoma (PDAC) is a devastating malignancy with almost 90% lethality, emphasizing the need for new therapies optimally targeting the tumors of individual patients. EXPERIMENTAL DESIGN: We first developed a panel of new physiologic models for study of PDAC, expanding surgical PDAC tumor samples in culture using short-term culture and conditional reprogramming with the Rho kinase inhibitor Y-27632, and creating matched patient-derived xenografts (PDX). These were evaluated for sensitivity to a large panel of clinical agents, and promising leads further evaluated mechanistically. RESULTS: Only a small minority of tested agents was cytotoxic in minimally passaged PDAC cultures in vitro Drugs interfering with protein turnover and transcription were among most cytotoxic. Among transcriptional repressors, triptolide, a covalent inhibitor of ERCC3, was most consistently effective in vitro and in vivo causing prolonged complete regression in multiple PDX models resistant to standard PDAC therapies. Importantly, triptolide showed superior activity in MYC-amplified PDX models and elicited rapid and profound depletion of the oncoprotein MYC, a transcriptional regulator. Expression of ERCC3 and MYC was interdependent in PDACs, and acquired resistance to triptolide depended on elevated ERCC3 and MYC expression. The Cancer Genome Atlas analysis indicates ERCC3 expression predicts poor prognosis, particularly in CDKN2A-null, highly proliferative tumors. CONCLUSIONS: This provides initial preclinical evidence for an essential role of MYC-ERCC3 interactions in PDAC, and suggests a new mechanistic approach for disruption of critical survival signaling in MYC-dependent cancers. Clin Cancer Res; 22(24); 6153-63. ©2016 AACR.

IL4 limits the efficacy of tumor-targeted antibody therapy in a murine model.

Tumor-targeted antibody therapy has had a major impact on reducing morbidity and mortality in a wide range of cancers. Antibodies mediate their antitumor activity in part by activating immune effector cells; however, the tumor microenvironment (TME) is enriched with cellular and soluble mediators that actively suppress generation of antitumor immunity. Here, we investigate the potential of prospectively identifying and neutralizing an immunomodulatory soluble mediator within the TME to enhance therapeutic efficacy of the HER2-directed antibody trastuzumab. Using the D5-HER2 cell line and an immunocompetent human HER2 transgenic animal (hmHER2Tg) in which human HER2 is a self-antigen, we determined that IL4 was present in the TME and produced by both tumor and stromal cells. A siRNA-based screening approach identified STAT5A as a novel negative regulator of IL4 production by D5-HER2 tumor cells. Furthermore, IL4 neutralization using the anti-IL4 antibody 11B11 enhanced the efficacy of trastuzumab and modulated the TME. For example, IL4 neutralization resulted in reduced levels of myeloid chemoattractants CCL2, CCL11, and CXCL5 in the TME. Combination therapy with 11B11 and trastuzumab resulted in a reduction of tumor-infiltrating CD11b(+)CD206(+) myeloid cells compared with monotherapy. These data suggest that IL4 neutralization enhances the efficacy of trastuzumab by influencing the phenotype of myeloid cells within the TME and provide further rationale for combining tumor-targeted antibody therapy with agents that neutralize factors in the TME that suppress generation of productive antitumor immune responses.

c-Abl modulates tumor cell sensitivity to antibody-dependent cellular cytotoxicity.

Monoclonal antibodies (mAb) can modulate cancer cell signal transduction and recruit antitumor immune effector mechanisms-including antibody-dependent cellular cytotoxicity (ADCC). Although several clinically effective antibodies can promote ADCC, therapeutic resistance is common. We hypothesized that oncogenic signaling networks within tumor cells affect their sensitivity to ADCC. We developed a screening platform and targeted 60 genes derived from an EGFR gene network using RNAi in an in vitro ADCC model system. Knockdown of GRB7, PRKCE, and ABL1 enhanced ADCC by primary and secondary screens. ABL1 knockdown also reduced cell proliferation, independent of its ADCC enhancement effects. c-Abl overexpression decreased ADCC sensitivity and rescued the effects of ABL1 knockdown. Imatinib inhibition of c-Abl kinase activity also enhanced ADCC-phenocopying ABL1 knockdown-against several EGFR-expressing head-and-neck squamous cell carcinoma cell lines by ex vivo primary natural killer cells. Our findings suggest that combining c-Abl inhibition with ADCC-promoting antibodies, such as cetuximab, could translate into increased therapeutic efficacy of mAbs.