A deep learning image-based intrinsic molecular subtype classifier of breast tumors reveals tumor heterogeneity that may affect survival.

BACKGROUND: Breast cancer intrinsic molecular subtype (IMS) as classified by the expression-based PAM50 assay is considered a strong prognostic feature, even when controlled for by standard clinicopathological features such as age, grade, and nodal status, yet the molecular testing required to elucidate these subtypes is not routinely performed. Furthermore, when such bulk assays as RNA sequencing are performed, intratumoral heterogeneity that may affect prognosis and therapeutic decision-making can be missed. METHODS: As a more facile and readily available method for determining IMS in breast cancer, we developed a deep learning approach for approximating PAM50 intrinsic subtyping using only whole-slide images of H&E-stained breast biopsy tissue sections. This algorithm was trained on images from 443 tumors that had previously undergone PAM50 subtyping to classify small patches of the images into four major molecular subtypes-Basal-like, HER2-enriched, Luminal A, and Luminal B-as well as Basal vs. non-Basal. The algorithm was subsequently used for subtype classification of a held-out set of 222 tumors. RESULTS: This deep learning image-based classifier correctly subtyped the majority of samples in the held-out set of tumors. However, in many cases, significant heterogeneity was observed in assigned subtypes across patches from within a single whole-slide image. We performed further analysis of heterogeneity, focusing on contrasting Luminal A and Basal-like subtypes because classifications from our deep learning algorithm-similar to PAM50-are associated with significant differences in survival between these two subtypes. Patients with tumors classified as heterogeneous were found to have survival intermediate between Luminal A and Basal patients, as well as more varied levels of hormone receptor expression patterns. CONCLUSIONS: Here, we present a method for minimizing manual work required to identify cancer-rich patches among all multiscale patches in H&E-stained WSIs that can be generalized to any indication. These results suggest that advanced deep machine learning methods that use only routinely collected whole-slide images can approximate RNA-seq-based molecular tests such as PAM50 and, importantly, may increase detection of heterogeneous tumors that may require more detailed subtype analysis.

Targeting RPL39 and MLF2 reduces tumor initiation and metastasis in breast cancer by inhibiting nitric oxide synthase signaling.

We previously described a gene signature for breast cancer stem cells (BCSCs) derived from patient biopsies. Selective shRNA knockdown identified ribosomal protein L39 (RPL39) and myeloid leukemia factor 2 (MLF2) as the top candidates that affect BCSC self-renewal. Knockdown of RPL39 and MLF2 by specific siRNA nanoparticles in patient-derived and human cancer xenografts reduced tumor volume and lung metastases with a concomitant decrease in BCSCs. RNA deep sequencing identified damaging mutations in both genes. These mutations were confirmed in patient lung metastases (n = 53) and were statistically associated with shorter median time to pulmonary metastasis. Both genes affect the nitric oxide synthase pathway and are altered by hypoxia. These findings support that extensive tumor heterogeneity exists within primary cancers; distinct subpopulations associated with stem-like properties have increased metastatic potential.

Learning subgroup-specific regulatory interactions and regulator independence with PARADIGM.

UNLABELLED: High-dimensional '-omics' profiling provides a detailed molecular view of individual cancers; however, understanding the mechanisms by which tumors evade cellular defenses requires deep knowledge of the underlying cellular pathways within each cancer sample. We extended the PARADIGM algorithm (Vaske et al., 2010, Bioinformatics, 26, i237-i245), a pathway analysis method for combining multiple '-omics' data types, to learn the strength and direction of 9139 gene and protein interactions curated from the literature. Using genomic and mRNA expression data from 1936 samples in The Cancer Genome Atlas (TCGA) cohort, we learned interactions that provided support for and relative strength of 7138 (78%) of the curated links. Gene set enrichment found that genes involved in the strongest interactions were significantly enriched for transcriptional regulation, apoptosis, cell cycle regulation and response to tumor cells. Within the TCGA breast cancer cohort, we assessed different interaction strengths between breast cancer subtypes, and found interactions associated with the MYC pathway and the ER alpha network to be among the most differential between basal and luminal A subtypes. PARADIGM with the Naive Bayesian assumption produced gene activity predictions that, when clustered, found groups of patients with better separation in survival than both the original version of PARADIGM and a version without the assumption. We found that this Naive Bayes assumption was valid for the vast majority of co-regulators, indicating that most co-regulators act independently on their shared target. AVAILABILITY: http://paradigm.five3genomics.com. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Inferred Immune-Cell Activity Is an Independent Predictor of HER2-Negative Breast Cancer Prognosis and Response to Paclitaxel-Based Therapy in the GeparSepto Trial.

PURPOSE: Tumor microenvironment (TME) immune markers have been correlated with both response to neoadjuvant therapy and prognosis in patients with breast cancer. Here, immune-cell activity of breast cancer tumors was inferred by expression-based analysis to determine if it is prognostic and/or predictive of response to neoadjuvant paclitaxel-based therapy in the GeparSepto (G7) trial (NCT01583426). EXPERIMENTAL DESIGN: Pre-study biopsies from 279 patients with HER2-negative breast cancer in the G7 trial underwent RNA-seq-based profiling of 104 immune-cell-specific genes to assess inferred Immune Cell Activity (iICA) of 23 immune-cell types. Hierarchical clustering was used to classify tumors as iICA "hot," "warm," or "cold" by comparison of iICA in the G7 cohort relative to that of 1,467 samples from a tumor database established by Nantomics LLC. Correlations between iICA cluster, pathology-assessed tumor-infiltrating lymphocytes (TIL), and hormone receptor (HR) status for pathologic complete response (pCR), disease-free survival (DFS), and overall survival (OS) were determined. RESULTS: iICA cluster correlated with TIL levels. The highest pCR rates were observed in hot cluster tumors, and those with relatively higher TILs. Greater inferred activity of several T-cell types was significantly associated with pCR and survival. DFS and OS were prolonged in patients with hot or warm cluster tumors, the latter particularly for HR negative tumors, even if TILs were relatively low. CONCLUSIONS: Overall, TIL level better predicted pCR, but iICA cluster better predicted survival. Differences in associations between TILs, cluster, pCR, and survival were observed for HR-positive tumors versus HR-negative tumors, suggesting expanded study of the implication of these findings is warranted.

Rapid identification of neutralizing antibodies against SARS-CoV-2 variants by mRNA display.

The increasing prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with the ability to escape existing humoral protection conferred by previous infection and/or immunization necessitates the discovery of broadly reactive neutralizing antibodies (nAbs). Utilizing mRNA display, we identify a set of antibodies against SARS-CoV-2 spike (S) proteins and characterize the structures of nAbs that recognize epitopes in the S1 subunit of the S glycoprotein. These structural studies reveal distinct binding modes for several antibodies, including the targeting of rare cryptic epitopes in the receptor-binding domain (RBD) of S that interact with angiotensin-converting enzyme 2 (ACE2) to initiate infection, as well as the S1 subdomain 1. Further, we engineer a potent ACE2-blocking nAb to sustain binding to S RBD with the E484K and L452R substitutions found in multiple SARS-CoV-2 variants. We demonstrate that mRNA display is an approach for the rapid identification of nAbs that can be used in combination to combat emerging SARS-CoV-2 variants.

Safety, Feasibility, and Merits of Longitudinal Molecular Testing of Multiple Metastatic Sites to Inform mTNBC Patient Treatment in the Intensive Trial of Omics in Cancer.

PURPOSE: Patients with metastatic triple-negative breast cancer (mTNBC) have poor outcomes. The Intensive Trial of Omics in Cancer (ITOMIC) sought to determine the feasibility and potential efficacy of informing treatment decisions through multiple biopsies of mTNBC deposits longitudinally over time, accompanied by analysis using a distributed network of experts. METHODS: Thirty-one subjects were enrolled and 432 postenrollment biopsies performed (clinical and study-directed) of which 332 were study-directed. Molecular profiling included whole-genome sequencing or whole-exome sequencing, cancer-associated gene panel sequencing, RNA-sequencing, and immunohistochemistry. To afford time for analysis, subjects were initially treated with cisplatin (19 subjects), or another treatment they had not received previously. The results were discussed at a multi-institutional ITOMIC Tumor Board, and a report transmitted to the subject's oncologist who arrived at the final treatment decision in conjunction with the subject. Assistance was provided to access treatments that were predicted to be effective. RESULTS: Multiple biopsies in single settings and over time were safe, and comprehensive analysis was feasible. Two subjects were found to have lung cancer, one had carcinoma of unknown primary site, tumor samples from three subjects were estrogen receptor-positive and from two others, human epidermal growth factor receptor 2-positive. Two subjects withdrew. Thirty-four of 112 recommended treatments were accessed using approved drugs, clinical trials, and single-patient investigational new drugs. After excluding the three subjects with nonbreast cancers and the two subjects who withdrew, 22 of 26 subjects (84.6%) received at least one ITOMIC Tumor Board-recommended treatment. CONCLUSION: Further exploration of this approach in patients with mTNBC is merited.

Glycolytic expression in lower-grade glioma reveals an epigenetic association between IDH mutation status and PDL1/2 expression.

BACKGROUND: The interplay between glycolysis and immunosuppression in cancer has recently emerged as an intriguing area of research. The aim of this study was to elucidate a potential epigenetic link between glycolysis, isocitrate hydrogenase (IDH) status, and immune checkpoint expression in human lower-grade glioma (LGG). METHODS: Genomic analysis was conducted on 507 LGG samples from The Cancer Genome Atlas (TCGA). Data types analyzed included RNA-seq (IlluminaHiSeq) and DNA methylation (Methylation450K). Unsupervised clustering grouped samples according to glycolytic expression level and IDH status. Global promoter methylation patterns were examined, as well as methylation levels of LDHA/LDHB and immune checkpoint genes. Methylation data from a knock-in IDH1R132H/WT allele in HCT116 cells and ChIP-seq data from immortalized human astrocytes using an inducible IDH1R132H mutation were also assessed. RESULTS: Glycolytic expression distinguished a tumor cluster enriched for wild-type IDH and poorer overall survival (P < .0001). This cluster showed lower levels of LDHA promoter methylation and a higher LDHA/LDHB expression ratio. These samples also displayed lower PDL1/2 promoter methylation and higher PDL1/2 expression, which was more pronounced for PDL2. IDH1R132H/WT cell line data showed that induced changes in methylation were enriched for genes involved in immune regulation, and ChIP-seq data showed that promoter H3K4me3 decreased for LDHA, PDL2, and PDL1 upon induction of IDH1R132H. CONCLUSIONS: These results suggest a previously unrecognized epigenetic link between glycolysis and immune checkpoint expression in LGG. This work advances our understanding of glioma genomics and provides support for further exploration of the metabolic-immune interface in LGG.

Stable transplantation of human mitochondrial DNA by high-throughput, pressurized isolated mitochondrial delivery.

Generating mammalian cells with specific mitochondrial DNA (mtDNA)-nuclear DNA (nDNA) combinations is desirable but difficult to achieve and would be enabling for studies of mitochondrial-nuclear communication and coordination in controlling cell fates and functions. We developed 'MitoPunch', a pressure-driven mitochondrial transfer device, to deliver isolated mitochondria into numerous target mammalian cells simultaneously. MitoPunch and MitoCeption, a previously described force-based mitochondrial transfer approach, both yield stable isolated mitochondrial recipient (SIMR) cells that permanently retain exogenous mtDNA, whereas coincubation of mitochondria with cells does not yield SIMR cells. Although a typical MitoPunch or MitoCeption delivery results in dozens of immortalized SIMR clones with restored oxidative phosphorylation, only MitoPunch can produce replication-limited, non-immortal human SIMR clones. The MitoPunch device is versatile, inexpensive to assemble, and easy to use for engineering mtDNA-nDNA combinations to enable fundamental studies and potential translational applications.

Mitochondria are specialized structures within cells that generate vital energy and biological building blocks. Mitochondria have a double membrane and contain many copies of their own circular DNA (mitochondrial DNA), which include the blueprints to create just thirteen essential mitochondrial proteins. Like all genetic material, mitochondrial DNA can become damaged or mutated, and these changes can be passed on to offspring. Some of these alterations are linked to severe and debilitating diseases. Both the double membrane of the mitochondria and their high number of DNA copies make treating such diseases difficult. A successful therapy must be capable of correcting almost every copy of mitochondrial DNA. However, the multiple copies of mitochondrial DNA create a problem for genetic research as current techniques are unable to reliably introduce particular mitochondrial mutations to all types of human cells to investigate how they may alter cell function. Sercel, Patananan et al. have developed a method to deliver new mitochondria into thousands of cells at the same time. This technique, called MitoPunch, uses a pressure-driven device to propel mitochondria taken from donor cells into recipient cells without mitochondrial DNA to reestablish their function. Using human cancer cells and healthy skin cells that lack mitochondrial DNA, Sercel, Patananan et al. showed that cells that received mitochondria retained the new mitochondrial DNA. The technique uses readily accessible parts, meaning it can be performed quickly and inexpensively in any laboratory. It further only requires a small amount of donor starting material, meaning that even precious samples with limited material could be used as mitochondrial donors. This new technique has several important potential applications for mitochondrial DNA research. It could be used in the lab to create large numbers of cell lineswith known mutations in the mitochondrial DNA to establish new systems that test drugs or probe the interaction between mitochondrial and nuclear DNA. It could be used to study a broad spectrum of biological questions since mitochondrial function is essential for several processes required for life. Critically, it could also be used as a starting point to develop next-generation therapies capable of treating inherited mitochondrial genetic diseases in severely affected patients.

Quantitative Multiplexed Proteomics Could Assist Therapeutic Decision Making in Non-Small Cell Lung Cancer Patients with Ambiguous ALK Test Results.

Therapeutic guidance in non-small cell lung cancer (NSCLC) tumors that are positive for anaplastic lymphoma kinase (ALK) fluorescent in situ hybridization (FISH), but negative for ALK immunohistochemistry, is still challenging. Parallel routine screening of 4588 NSCLC cases identified 22 discordant cases. We rechecked these samples using ALK antibodies and selected reaction monitoring (SRM) quantitative multiplexed proteomics screening multiple protein targets, including ALK and MET for the ALK tyrosine kinase inhibitor (TKI), and FR-alpha, hENT1, RRM1, TUBB3, ERCC1, and XRCC1 for chemotherapy. The presence of ALK (31.8%), MET (36.4%), FR-alpha (72.7%), hENT1 (18.2%), RRM1 (31.8%), TUBB3 (72.9%), ERCC1 (4.5%), and a low level of XRCC1 (54.4%) correlated with clinical outcomes. SRM was more sensitive than the ALK D5F3 assay. Among the eight cases receiving ALK TKI, four cases with ALK or MET detected by SRM had complete or partial responses, whereas four cases without ALK or MET showed progression. Twenty-seven treatment outcomes from 20 cases were assessed and cases expressing more than half of the specific predictive proteins were sensitive to matching therapeutic agents and showed longer progression-free survival than the other cases (p < 0.001). SRM showed a potential role in therapeutic decision making in NSCLC patients with ambiguous ALK test results.

An ACE2 Triple Decoy that neutralizes SARS-CoV-2 shows enhanced affinity for virus variants.

The SARS-CoV-2 variants replacing the first wave strain pose an increased threat by their potential ability to escape pre-existing humoral protection. An angiotensin converting enzyme 2 (ACE2) decoy that competes with endogenous ACE2 for binding of the SARS-CoV-2 spike receptor binding domain (S RBD) and inhibits infection may offer a therapeutic option with sustained efficacy against variants. Here, we used Molecular Dynamics (MD) simulation to predict ACE2 sequence substitutions that might increase its affinity for S RBD and screened candidate ACE2 decoys in vitro. The lead ACE2(T27Y/H34A)-IgG1FC fusion protein with enhanced S RBD affinity shows greater live SARS-CoV-2 virus neutralization capability than wild type ACE2. MD simulation was used to predict the effects of S RBD variant mutations on decoy affinity that was then confirmed by testing of an ACE2 Triple Decoy that included an additional enzyme activity-deactivating H374N substitution against mutated S RBD. The ACE2 Triple Decoy maintains high affinity for mutated S RBD, displays enhanced affinity for S RBD N501Y or L452R, and has the highest affinity for S RBD with both E484K and N501Y mutations, making it a viable therapeutic option for the prevention or treatment of SARS-CoV-2 infection with a high likelihood of efficacy against variants.

Rapid Identification of Neutralizing Antibodies against SARS-CoV-2 Variants by mRNA Display.

The increasing prevalence of SARS-CoV-2 variants with the ability to escape existing humoral protection conferred by previous infection and/or immunization necessitates the discovery of broadly-reactive neutralizing antibodies (nAbs). Utilizing mRNA display, we identified a set of antibodies against SARS-CoV-2 spike (S) proteins and characterized the structures of nAbs that recognized epitopes in the S1 subunit of the S glycoprotein. These structural studies revealed distinct binding modes for several antibodies, including targeting of rare cryptic epitopes in the receptor-binding domain (RBD) of S that interacts with angiotensin- converting enzyme 2 (ACE2) to initiate infection, as well as the S1 subdomain 1. A potent ACE2-blocking nAb was further engineered to sustain binding to S RBD with the E484K and L452R substitutions found in multiple SARS-CoV-2 variants. We demonstrate that mRNA display is a promising approach for the rapid identification of nAbs that can be used in combination to combat emerging SARS-CoV-2 variants.

Phase I study of a multitargeted recombinant Ad5 PSA/MUC-1/brachyury-based immunotherapy vaccine in patients with metastatic castration-resistant prostate cancer (mCRPC).

BACKGROUND: Antitumor vaccines targeting tumor-associated antigens (TAAs) can generate antitumor immune response. A novel vaccine platform using adenovirus 5 (Ad5) vectors [E1-, E2b-] targeting three TAAs-prostate-specific antigen (PSA), brachyury, and MUC-1-has been developed. Both brachyury and the C-terminus of MUC-1 are overexpressed in metastatic castration-resistant prostate cancer (mCRPC) and have been shown to play an important role in resistance to chemotherapy, epithelial-mesenchymal transition, and metastasis. The transgenes for PSA, brachyury, and MUC-1 all contain epitope modifications for the expression of CD8+ T-cell enhancer agonist epitopes. We report here the first-in-human trial of this vaccine platform. METHODS: Patients with mCRPC were given concurrently three vaccines targeting PSA, brachyury, and MUC-1 at 5×1011 viral particles (VP) each, subcutaneously every 3 weeks for a maximum of three doses (dose de-escalation cohort), followed by a booster vaccine every 8 weeks for 1 year (dose-expansion cohort only). The primary objective was to determine the safety and the recommended phase II dose. Immune assays and clinical responses were evaluated. RESULTS: Eighteen patients with mCRPC were enrolled between July 2018 and September 2019 and received at least one vaccination. Median PSA was 25.58 ng/mL (range, 0.65-1006 ng/mL). The vaccine was tolerable and safe, and no grade >3 treatment-related adverse events or dose-limiting toxicities (DLTs) were observed. One patient had a partial response, while five patients had confirmed PSA decline and five had stable disease for >6 months. Median progression-free survival was 22 weeks (95% CI: 19.1 to 34). Seventeen (100%) of 17 patients mounted T-cell responses to at least one TAA, whereras 8 (47%) of 17 patients mounted immune responses to all three TAAs. Multifunctional T-cell responses to PSA, MUC-1, and brachyury were also detected after vaccination in the majority of the patients. CONCLUSIONS: Ad5 PSA/MUC-1/brachyury vaccine is well tolerated. The primary end points were met and there were no DLTs. The recommended phase II dose is 5×1011 VP. The vaccine demonstrated clinical activity, including one partial response and confirmed PSA responses in five patients. Three patients with prolonged PSA responses received palliative radiation therapy. Further research is needed to evaluate the clinical benefit and immunogenicity of this vaccine in combination with other immuno-oncology agents and/or palliative radiation therapy. TRIAL REGISTRATION NUMBER: NCT03481816.

Intranasal plus subcutaneous prime vaccination with a dual antigen COVID-19 vaccine elicits T-cell and antibody responses in mice.

We have developed a COVID-19 vaccine, hAd5 S-Fusion + N-ETSD, that expresses SARS-CoV-2 spike (S) and nucleocapsid (N) proteins with modifications to increase immune responses delivered using a human adenovirus serotype 5 (hAd5) platform. Here, we demonstrate subcutaneous (SC) prime and SC boost vaccination of CD-1 mice with this dual-antigen vaccine elicits T-helper cell 1 (Th1) biased T-cell and humoral responses to both S and N that are greater than those seen with hAd5 S wild type delivering only unmodified S. We then compared SC to intranasal (IN) prime vaccination with SC or IN boosts and show that an IN prime with an IN boost is as effective at generating Th1 biased humoral responses as the other combinations tested, but an SC prime with an IN or SC boost elicits greater T cell responses. Finally, we used a combined SC plus IN (SC + IN) prime with or without a boost and found the SC + IN prime alone to be as effective in generating humoral and T-cell responses as the SC + IN prime with a boost. The finding that SC + IN prime-only delivery has the potential to provide broad immunity-including mucosal immunity-against SARS-CoV-2 supports further testing of this vaccine and delivery approach in animal models of viral challenge.

Identification and validation of expressed HLA-binding breast cancer neoepitopes for potential use in individualized cancer therapy.

BACKGROUND: Therapeutic regimens designed to augment the immunological response of a patient with breast cancer (BC) to tumor tissue are critically informed by tumor mutational burden and the antigenicity of expressed neoepitopes. Herein we describe a neoepitope and cognate neoepitope-reactive T-cell identification and validation program that supports the development of next-generation immunotherapies. METHODS: Using GPS Cancer, NantOmics research, and The Cancer Genome Atlas databases, we developed a novel bioinformatic-based approach which assesses mutational load, neoepitope expression, human leukocyte antigen (HLA)-binding prediction, and in vitro confirmation of T-cell recognition to preferentially identify targetable neoepitopes. This program was validated by application to a BC cell line and confirmed using tumor biopsies from two patients with BC enrolled in the Tumor-Infiltrating Lymphocytes and Genomics (TILGen) study. RESULTS: The antigenicity and HLA-A2 restriction of the BC cell line predicted neoepitopes were determined by reactivity of T cells from HLA-A2-expressing healthy donors. For the TILGen subjects, tumor-infiltrating lymphocytes (TILs) recognized the predicted neoepitopes both as peptides and on retroviral expression in HLA-matched Epstein-Barr virus-lymphoblastoid cell line and BC cell line MCF-7 cells; PCR clonotyping revealed the presence of T cells in the periphery with T-cell receptors for the predicted neoepitopes. These high-avidity immune responses were polyclonal, mutation-specific and restricted to either HLA class I or II. Interestingly, we observed the persistence and expansion of polyclonal T-cell responses following neoadjuvant chemotherapy. CONCLUSIONS: We demonstrate our neoepitope prediction program allows for the successful identification of neoepitopes targeted by TILs in patients with BC, providing a means to identify tumor-specific immunogenic targets for individualized treatment, including vaccines or adoptively transferred cellular therapies.

Dual-Antigen COVID-19 Vaccine Subcutaneous Prime Delivery With Oral Boosts Protects NHP Against SARS-CoV-2 Challenge.

We have developed a dual-antigen COVID-19 vaccine incorporating genes for a modified SARS-CoV-2 spike protein (S-Fusion) and the viral nucleocapsid (N) protein with an Enhanced T-cell Stimulation Domain (N-ETSD) to increase the potential for MHC class II responses. The vaccine antigens are delivered by a human adenovirus serotype 5 platform, hAd5 [E1-, E2b-, E3-], previously demonstrated to be effective in the presence of Ad immunity. Vaccination of rhesus macaques with the hAd5 S-Fusion + N-ETSD vaccine by subcutaneous prime injection followed by two oral boosts elicited neutralizing anti-S IgG and T helper cell 1-biased T-cell responses to both S and N that protected the upper and lower respiratory tracts from high titer (1 x 106 TCID50) SARS-CoV-2 challenge. Notably, viral replication was inhibited within 24 hours of challenge in both lung and nasal passages, becoming undetectable within 7 days post-challenge.

Selective vulnerability in Alzheimer's disease: amyloid precursor protein and p75(NTR) interaction.

OBJECTIVE: Selective neuronal vulnerability in neurodegenerative diseases is poorly understood. In Alzheimer's disease, the basal forebrain cholinergic neurons are selectively vulnerable, putatively because of their expression of the cell death mediator p75(NTR) (the common neurotrophin receptor), and its interaction with proapoptotic ligands pro-nerve growth factor and amyloid-beta peptide. However, the relation between amyloid precursor protein (APP) and p75(NTR) has not been described previously. METHODS: APP and p75(NTR) were assayed for interaction by coimmunoprecipitation in vitro and in vivo, yeast two-hybrid assay, bioluminescence resonance energy transfer, and confocal microscopy. Effects on APP processing and signaling were studied using immunoblotting, enzyme-linked immunosorbent assays, and luciferase reporter assays. RESULTS: The results of this study are as follows: (1) p75(NTR) and APP interact directly; (2) this interaction is modified by ligands nerve growth factor and beta-amyloid; (3) APP and p75(NTR) colocalization in vivo is modified in Alzheimer's model transgenic mice; (4) APP processing is altered by p75(NTR), and to a lesser extent, p75(NTR) processing is altered by the presence of APP; (5) APP-dependent transcription mediated by Fe65 is blocked by p75(NTR); and (6) coexpression of APP and p75(NTR) triggers cell death. INTERPRETATION: These results provide new insight into the emerging signaling network that mediates the Alzheimer's phenotype and into the mechanism of basal forebrain cholinergic neuronal selective vulnerability. In addition, the results argue that the interaction between APP and p75(NTR) may represent a therapeutic target in Alzheimer's disease.

Large scale, robust, and accurate whole transcriptome profiling from clinical formalin-fixed paraffin-embedded samples.

Transcriptome profiling can provide information of great value in clinical decision-making, yet RNA from readily available formalin-fixed paraffin-embedded (FFPE) tissue is often too degraded for quality sequencing. To assess the clinical utility of FFPE-derived RNA, we performed ribo-deplete RNA extractions on > 3200 FFPE slide samples; 25 of these had direct FFPE vs. fresh frozen (FF) replicates, 57 were sequenced in 2 different labs, 87 underwent multiple library analyses, and 16 had direct microdissected vs. macrodissected replicates. Poly-A versus ribo-depletion RNA extraction methods were compared using transcriptomes of TCGA cohort and 3116 FFPE samples. Compared to FF, FFPE transcripts coding for nuclear/cytoplasmic proteins involved in DNA packaging, replication, and protein synthesis were detected at lower rates and zinc finger family transcripts were of poorer quality. The greatest difference in extraction methods was in histone transcripts which typically lack poly-A tails. Encouragingly, the overall sequencing success rate was 81%. Exome coverage was highly concordant in direct FFPE and FF replicates, with 98% agreement in coding exon coverage and a median correlation of whole transcriptome profiles of 0.95. We provide strong rationale for clinical use of FFPE-derived RNA based on the robustness, reproducibility, and consistency of whole transcriptome profiling.

Overcoming hypoxia-induced functional suppression of NK cells.

BACKGROUND: Natural killer (NK) cells are immune cells capable of killing virally infected cells and tumor cells without the need for antigen stimulation. Tumors, however, can create a suppressive microenvironment that decreases NK function. A feature of many tumors is hypoxia (low oxygen perfusion), which has been previously shown to decrease NK function. A high affinity NK (haNK) cell has been engineered to express a high affinity CD16 receptor as well as internal interleukin (IL)-2 for increased antibody-dependent cellular cytotoxicity (ADCC) and activation, respectively. We sought to investigate the tolerance of NK cells versus haNK cells to hypoxia. METHODS: We exposed healthy donor (HD) NK and X-irradiated haNK cells to normoxia (20% oxygen) as well as hypoxia (0% oxygen) and investigated their ability to kill prostate, breast and lung tumor cell lines after 5 hours. We also used monoclonal antibodies cetuximab (anti-EGFR) or avelumab (antiprogrammed death-ligand 1) to investigate the effects of hypoxia on NK ADCC. Genomic and proteomic analyzes were done to determine the effect of hypoxia on the expression of factors important to NK cell function. RESULTS: While HD NK cell cytolytic abilities were markedly and significantly impaired under hypoxic conditions, haNK cells maintained killing capacity under hypoxic conditions. NK killing, serial killing and ADCC were maintained under hypoxia in haNK cells. IL-2 has been previously implicated in serial killing and perforin regeneration and thus the endogenous IL-2 produced by haNK cells is likely a driver of the maintained killing capacity of haNK cells under hypoxic conditions. Activation of signal transducer and activator of transcription 3 (STAT3) is not seen in haNKs under hypoxia but is significant in HD NK cells. Pharmaceutical activation of STAT3 in haNKs led to reduced killing, implicating active STAT3 in reduced NK cell function. CONCLUSIONS: In contrast to HD NK cells, haNK cells are resistant to acute hypoxia. The potent cytolytic function of haNK cells was maintained in an environment comparable to what would be encountered in a tumor. The data presented here provide an additional mechanism of action for haNK cells that are currently being evaluated in clinical trials for several tumor types.

PD-L1 targeting high-affinity NK (t-haNK) cells induce direct antitumor effects and target suppressive MDSC populations.

BACKGROUND: Although immune checkpoint inhibitors have revolutionized cancer treatment, clinical benefit with this class of agents has been limited to a subset of patients. Hence, more effective means to target tumor cells that express immune checkpoint molecules should be developed. For the first time, we report a novel natural killer (NK) cell line, programmed death-ligand 1 (PD-L1) targeting high-affinity natural killer (t-haNK), which was derived from NK-92 and was engineered to express high-affinity CD16, endoplasmic reticulum-retained interleukin (IL)-2, and a PD-L1-specific chimeric antigen receptor (CAR). We show that PD-L1 t-haNK cells also retained the expression of native NK receptors and carried a high content of granzyme and perforin granules. METHODS: NanoString, flow cytometry, and immunofluorescence analyses were performed to characterize the phenotype of irradiated PD-L1 t-haNK cells. In vitro PD-L1 t-haNK cell activity against cancer cell lines and human peripheral blood mononuclear cells (PBMCs) was determined via flow-based and 111In-release killing assays. The antitumor effect of PD-L1 t-haNK cells in vivo was investigated using MDA-MB-231, H460, and HTB1 xenograft models in NOD-scid IL2Rgammanull (NSG) mice. Additionally, the antitumor effect of PD-L1 t-haNK cells, in combination with anti-PD-1 and N-803, an IL-15 superagonist, was evaluated using mouse oral cancer 1 syngeneic model in C57BL/6 mice. RESULTS: We show that PD-L1 t-haNK cells expressed PD-L1-targeting CAR and CD16, retained the expression of native NK receptors, and carried a high content of granzyme and perforin granules. In vitro, we demonstrate the ability of irradiated PD-L1 t-haNK cells to lyse 20 of the 20 human cancer cell lines tested, including triple negative breast cancer (TNBC) and lung, urogenital, and gastric cancer cells. The cytotoxicity of PD-L1 t-haNK cells was correlated to the PD-L1 expression of the tumor targets and can be improved by pretreating the targets with interferon (IFN)-γ. In vivo, irradiated PD-L1 t-haNK cells inhibited the growth of engrafted TNBC and lung and bladder tumors in NSG mice. The combination of PD-L1 t-haNK cells with N-803 and anti-PD-1 antibody resulted in superior tumor growth control of engrafted oral cavity squamous carcinoma tumors in C57BL/6 mice. In addition, when cocultured with human PBMCs, PD-L1 t-haNK cells preferentially lysed the myeloid-derived suppressor cell population but not other immune cell types. CONCLUSION: These studies demonstrate the antitumor efficacy of PD-L1 t-haNK cells and provide a rationale for the potential use of these cells in clinical studies.

Cooperative Immune-Mediated Mechanisms of the HDAC Inhibitor Entinostat, an IL15 Superagonist, and a Cancer Vaccine Effectively Synergize as a Novel Cancer Therapy.

PURPOSE: Immunotherapy has demonstrated clinical efficacy in subsets of patients with solid carcinomas. Multimodal therapies using agents that can affect different arms of the immune system and/or tumor microenvironment (TME) might increase clinical responses. EXPERIMENTAL DESIGN: We demonstrate that entinostat, a class I histone deacetylase inhibitor, enhances the antitumor efficacy of the IL15 superagonist N-803 plus vaccine in 4T1 triple-negative breast and MC38-CEA colon murine carcinoma models. A comprehensive immune and gene-expression analysis was performed in the periphery and/or TME of MC38-CEA tumor-bearing mice. RESULTS: Although N-803 plus vaccine induced peripheral CD8+ T-cell activation and cytokine production, there was no reduction in tumor burden and poor tumor infiltration of CD8+ T cells with minimal levels of granzyme B. For the first time, we demonstrate that the addition of entinostat to N-803 plus vaccine promoted significant tumor control, correlating with increased expression of genes associated with tumor inflammation, enhanced infiltration of activated CD8+ T cells with maximal granzyme B, T-cell responses to multiple tumor-associated antigens, increased serum IFNγ, reduction of regulatory T cells in the TME, and decreased expression of the checkpoint V-domain Ig suppressor of T-cell activation (VISTA) on multiple immune subsets. CONCLUSIONS: Collectively, these data demonstrate that the synergistic combination of entinostat, N-803, and vaccine elicits potent antitumor activity by generating a more inflamed TME. These findings thus form the rationale for the use of this combination of agents for patients harboring poorly or noninflamed solid carcinomas.