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Purpose: Preclinical evidence supporting immunomodulatory effect of MEK inhibition that augments anti-tumor activity of PD-1 inhibitors is a compelling rationale for investigating combined MEK and PD-L1 inhibition in advanced microsatellite-stable (MSS) SBA wherein immune checkpoint inhibitors (ICIs) have little clinical benefit. We conducted a prospective, phase 2 trial evaluating efficacy and safety of COTEZO regimen in treatment refractory SBA. Procedures: Patients aged ≥ 18 years with metastatic MSS SBA, ECOG PS 0 - 2, and disease progression on prior systemic chemotherapy were enrolled and treated with cobimetinib (60 mg orally once daily for days 1 - 21) and atezolizumab (840 mg intravenously every 2 weeks) every 28-day cycle. The primary endpoint was objective response rate (ORR) per Response Evaluation Criteria in Solid Tumors (RECIST) Version 1.1. by independent radiology review. Key pre-specified secondary endpoints were safety, disease control rate (DCR), progression-free (PFS) and overall (OS) survival. Pre/on-treatment biopsies were examined for response biomarkers. Results: Between 4/2017 and 7/2020, 20 patients were enrolled. The confirmed ORR per RECISTv1.1 was 10% [2/20; 95%CI: 1.2 - 31.7] (1 complete response and 1 partial response). Seven patients had stable disease for a DCR of 45% (95%CI: 23.1 - 68.5). Median PFS and OS were 2.4 (95% CI 1.3-3.5) and 8.8 (95% CI 5.6 - 12) months, respectively. Grade 3 treatment-emergent adverse events (no grade 4/5) occurred in 7 (35%) patients; most common being elevated CPK (15%) and vomiting (10%). One (5%) patient had grade 3 vomiting, necessitating treatment discontinuation. There appeared to be a marked difference in OS between patients with increased pre-treatment tumor immune infiltration as compared to those lacking immune infiltration amongst patients with RNASeq data available (n=13). Specifically, increased immune infiltration as assessed by computational deconvolution of RNASeq data pointed to increased macrophage and monocyte infiltration as prognostic factors. This was additionally assessed via application of a global “immune score” algorithm, ESTIMATE. In order to generalize our findings, we expanded our computational deconvolution analysis to include additional small bowel adenocarcinoma patients who received standard-of-care treatment (n=7). For the combined standard-of-care and experimentally treated patients we saw that those with immune infiltration had significantly better OS (9 months vs greater than 3 years, HR 5.7, Padj = 0.017), suggesting that immune infiltration is a prognostic, rather than predictive biomarker. Conclusions: Although our study did not meet its primary endpoint of ORR, we identified tumor immune infiltration as a prognostic biomarker in SBA, which could select patients with better outcomes on immunotherapy. Citation Format: Nicholas Hornstein, John P. Shen, Andrew J. Pellatt, Suyu Liu, Mark Knafl, Anneleis F. Willett, Haifeng Zhu, Dipen M. Maru, Walter Darbonne, Ignacio I. Wistuba, Andy Futreal, James C. Yao, Scott E. Woodman, Michael J. Overman, Kanwal P. Raghav. Efficacy, safety, and biomarker analysis of combined MEK (Cobimetinib) and PD-L1 (Atezolizumab) inhibition (COTEZO) in advanced small bowel adenocarcinoma (SBA) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT148.

Cancer stem cells (CSCs) play a critical role in metastasis, relapse and therapy resistance in colorectal cancer (CRC). Although the normal lineage of cell development in the intestine has identified many of the genes involved in the induction and maintenance of pluripotency, recent studies of CSCs suggest significant heterogeneity and lack of common surface markers for identification. In this study, we perform single cell RNA sequencing (scRNA-seq) to extract a single cell stemness signature (SCS_sig) for colorectal CSCs identification and as a potential predictor of relapse after surgical resection. A total of 26 fresh surgery specimens (6 colon tumor, 3 colon matched normal, 9 liver metastasis, 8 liver matched normal) from neoadjuvant chemotherapy (NAC) treated patients with single-cell transcriptome generated using 10X Chromium scRNA-seq and Illumina platforms were analyzed. Following extensive quality control and batch effect correction, we identified 20k epithelial cells using canonical epithelial cell marker genes (EPCAM, KRT8 and KRT18) from a total of 111,200 cells, with mean 1113 epithelial cell per sample (excluding liver normal samples).To obtain the CSCs profiling signature, we first annotated a ‘gold-standard’ set of CSC that expressed all canonical colon CSC marker genes (LGR5, ASCL2, EPHB2, PROM1, AXIN2, LEFTY1, RNF43, CD44 and SLC12A2), identifying 346 CSCs (1.71% of epithelial cells). Published stemness signatures were significantly enriched in the gold standard stem cells (false discovery rate (FDR) < 0.05). Then we selected the 50 most significantly up-regulated genes in this gold standard set (relative to other epithelial cells) as a single-cell stemness signature (named SCS_sig hereafter). Interesting, we saw that in every tumor there was a continuum of stemness, rather than distinct stem and differentiated populations. The stemness state by SCS_sig was positively correlated with less differentiated state calculated by CytoTRACE score (r = 0.76, p< 0.001), a computational method that predicts the differentiation state of cells from scRNA-seq data. The SCS_sig was significantly higher in tumor cells compared to normal cells in our cohort as well as the previously published SMC cohort (fold change = 2.52 and p < 0.001, fold change = 1.44 and p < 0.001, respectively). There was notable heterogeneity in the fraction of stem-like tumor cells with in 15 tumor samples, with the tumor with greatest CSC percentage having the shortest time to relapse. The SCS_sig generated from stem-like cells can identify the stemness state of epithelial cells in scRNAseq data. A signature score is more simple and robust than using few or panel of CSCs marker genes to identify single CSC because of the reads dropout in scRNA-seq. Further investigation of the SCS_sig as a predictive and prognostic biomarker in CRC is ongoing in larger patient cohorts. Citation Format: Kangyu Lin, Mohammad Zeineddine, Saikat Chowdhury, Nicholas Hornstein, Sendurai Mani, Scott Kopetz, John Paul Shen. Identification of colorectal cancer stem cells from single cell RNA sequencing [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 2454.

BackgroundThe SARS-CoV-2 virus has infected and killed millions of people worldwide. Breast cancer is the most prevalent cancer in women and few studies have investigated the outcomes of patients with a history of breast cancer and COVID-19. We report the clinical outcomes of patients with invasive breast cancer who tested positive for SARS-CoV-2, including hospitalization and death, and evaluate demographic and cancer-related factors associated with these outcomes.PatientsPatients with a history of invasive breast cancer and positive SARS-CoV-2 test from January 1 to December 31, 2020 at two large, academic Los Angeles health systems were included.MethodsRetrospective chart review of the electronic medical record was performed. Data for demographic and cancer-related factors were manually abstracted. Relationships between outcomes and clinical variables were evaluated using Fisher's exact test and linear regression analysis.ResultsAmong a total of 132 patients, 40 (30.3%) were hospitalized, while 11 (8.3%) required intensive care support, and 8 patients (6.1%) died. Older age and presence of one or more additional comorbidities were associated with hospitalization and death (P=.010, P=.003, P=.034, P < .001). Hispanic/Latinx ethnicity was associated with hospitalization (P=.047). Cancer treatment was not associated with hospitalization or death.ConclusionIn our diverse, multi-center, breast cancer cohort, Hispanic/Latinx ethnicity, older age and presence of other comorbidities were associated with worse outcomes from COVID-19. Breast cancer treatment, including surgery, radiation, systemic therapy, and endocrine therapy, was not associated with hospitalization in our cohort. Further studies are needed to explore the relationship between breast cancer and COVID-19 outcomes.

Human leukocyte antigen (HLA)-B has been recognized as a major determinant of discrepancies in disease outcomes, and recent evidence indicates a role in immune checkpoint blockade (ICB) efficacy. The B44 supertype, which features an electropositive binding pocket that preferentially displays peptides with negatively charged amino acid anchors, is associated with improved survival in ICB-treated melanoma. Yet this effect was not seen in ICB-treated non-small-cell lung cancer (NSCLC). Here we show that mutations leading to glutamic acid substitutions occur more often in melanoma than NSCLC based on mutational landscape. We additionally show stratifying B44 based on the presence of somatic mutations that lead to negatively charged glutamic acid anchors identifies patients with NSCLC with an ICB benefit similar to that seen in melanoma. We anticipate these findings could improve assessment of HLA-related outcomes and prediction of ICB benefit in those with B44, representing approximately half of the world’s population. Garon and colleagues demonstrate that the association of HLA supertype B44 with response to immune checkpoint blockade in melanoma but not NSCLC is related to differential mutational features that influence HLA binding of neoepitopes.

Protein synthesis is dysregulated in many diseases, but we lack a systems-level picture of how signaling molecules and RNA binding proteins interact with the translational machinery, largely due to technological limitations. Here we present riboPLATE-seq, a scalable method for generating paired libraries of ribosome-associated and total mRNA. As an extension of the PLATE-seq protocol, riboPLATE-seq utilizes barcoded primers for pooled library preparation, but additionally leverages anti-rRNA ribosome immunoprecipitation on whole polysomes to measure ribosome association (RA). We compare RA to its analogue in ribosome profiling and RNA sequencing, translation efficiency, and demonstrate both the performance of riboPLATE-seq and its utility in detecting translational alterations induced by specific inhibitors of protein kinases.

Cardiovascular disease (CVD) is the leading cause of death worldwide. Human embryonic stem cell (hESC)-derived cardiovascular progenitors (CVPs) or cardiomyocytes (CMs) represent a promising candidate for cell-based therapies to treat CVD. Myocardial infarction leads to extensive CM death mainly within the left ventricle, which is predominantly derived from the first heart field (FHF) during embryonic development. We postulate that the generation of chamber-specific CMs may play a key role in the development of safe and efficacious regenerative therapy. As a first step, we generated and characterized a FHF-specific TBX5-TdTomato +/W hESC reporter line. We show that TBX5 + cells represent an enriched population of FHF CVPs that can give rise to CMs, endothelial, and smooth muscle cells in vitro . Interestingly, we observed that TBX5 - cells can also generate contractile CMs. Bulk RNA-sequencing analysis at different stages of development suggested that TBX5 - cells are enriched for second heart field (SHF) CMs. To enable prospective isolation of FHF and SHF CMs, we generated a double transgenic TBX5-TdTomato +/W /NKX2-5 eGFP/W hESC reporter line. We performed detailed electrophysiological, functional, and transcriptional studies to characterize the heart-field specificity of these hESC-derived CMs. Electrophysiological studies revealed that, despite the presence of atrial and ventricular action potentials (APs) in both FHF and SHF, there are significant differences in their AP duration and cycle length. In addition, both FHF and SHF CMs responded appropriately to adrenergic stimuli. Single-cell RNA sequencing analysis confirmed the absence of nodal genes within these populations and provided evidence of unique molecular signatures for isolating FHF- and SHF-like CMs. Finally, we identified CORIN as a novel cell surface marker for FHF CMs. Our studies provide a platform for investigating in vitro cardiovascular development, drug screening, and may facilitate a safe approach for cell therapy in heart disease.

Summary: FMRP (fragile X mental retardation protein) is a polysome-associated RNA-binding protein encoded by Fmr1 that is lost in fragile X syndrome. Increasing evidence suggests that FMRP regulates both translation initiation and elongation, but the gene specificity of these effects is unclear. To elucidate the impact of Fmr1 loss on translation, we utilize ribosome profiling for genome-wide measurements of ribosomal occupancy and positioning in the cortex of 24-day-old Fmr1 knockout mice. We find a remarkably coherent reduction in ribosome footprint abundance per mRNA for previously identified, high-affinity mRNA binding partners of FMRP and an increase for terminal oligopyrimidine (TOP) motif-containing genes canonically controlled by mammalian target of rapamycin-eIF4E-binding protein-eIF4E binding protein-eukaryotic initiation factor 4E (mTOR-4E-BP-eIF4E) signaling. Amino acid motif- and gene-level analyses both show a widespread reduction of translational pausing in Fmr1 knockout mice. Our findings are consistent with a model of FMRP-mediated regulation of both translation initiation through eIF4E and elongation that is disrupted in fragile X syndrome. : Silencing of Fmr1, the gene that encodes FMRP, causes fragile X syndrome. Das Sharma et al. used ribosome profiling in the cortex of 24-day-old Fmr1 knockout mice to dissect FMRP-mediated translational regulation. Fmr1 loss leads to a relief of translational pausing across a large number of genes. Keywords: fragile X syndrome, translational regulation, ribosome profiling

Here, we describe the case of a 74-year-old man who was incidentally found to have a hepatic lesion during routine screening. Additional diagnostic studies demonstrated elevated IgG4 levels, IgG4 plasma cell-predominant lymphadenopathy and an inflammatory retroperitoneal mass encasing the bilateral ureters and the aorta. Given the concurrence of a lymphomatous process and IgG4-related disease (IgG4-RD), a multidisciplinary approach was required to determine whether targeting the lymphoma or IgG4-RD would be most efficacious. Discussions led to the decision to target treatment against IgG4-RD with systemic glucocorticoids, and subsequent imaging showed resolution of all lesions. To date, the patient remains symptom-free and has not experienced recurrence of his disease. This case highlights the importance of multidisciplinary care and the challenge inherent in targeting treatment between IgG4-RD and a concomitant lymphomatous process.

Purpose: Early detection of pulmonary malignancies is critical to achieving positive treatment outcomes. However, due to the minimal symptom burden often associated with early-stage neoplasms, in addition to the cost and limitations of advanced imaging modalities, detection of disease is often delayed until nodal or metastatic spread has occurred. Ongoing cellular turnover releases DNA into systemic circulation where it is able to achieve a steady state level. We postulated that we could identify methylation sites specific to pulmonary Squamous Cell Carcinoma (SCC) and Adenocarcinoma (AD) and then computationally determine the presence or absence of neoplastic signatures from a heterogeneous tissue sample, such as is found in cell free DNA isolated from plasma. Methods: We designed a computational framework for the detection of informative methylation sites based on an iterative Euclidean Distance algorithm. After generation of a reference matrix comprised of 25 cells of origin, SCC and AD (via 444 biopsy-derived methylomes), we sought informative methylation sites which had unique expression patterns among the tested cell types. To quantify the reliability of this approach, we generated synthetic In sillico “plasma” samples comprised of varying compositions of SCC, AD and whole blood methylation data which we then deconvolved using a NNLS algorithm. Results: We identified 200 informative sites out of 390,000 tested. Based on the 200 identified informative sites, we were able to define a Euclidean Space with robust separation of individual cell types (L2>5). Pairings of CD4+/CD8+ T-Cells and SCC/AD had the closest Euclidean distances, likely related to cellular origin and biologic similarities. However, we accurately distinguished between these cell type pairs in >95% of In silico experiments. Additionally, we demonstrated accurate detection of neoplastic signature within synthetic heterogeneous mixtures ranging from .1 to 100% of neoplastic “spike-in” (PearsonR =.92, p=1.5e-42). Going forward, we hope to further define the operating characteristics of this approach In vivo and develop its capability as we look to recapitulate our results with plasma samples from patients with and without pulmonary malignancies. Conclusion: Here, we describe a computational framework for the detection of pulmonary malignancy via methylation signatures. Using this framework we identified 200 informative methylation sites which allow for In silico deconvolution and detection of NSCLC within heterogeneously complex samples. Citation Format: Nicholas J. Hornstein, Zorawar S. Noor, Matteo Pellegrini, Edward Garon. Non-small cell lung cancer (NSCLC) detection by DNA methylation profiling and computational deconvolution [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 149.

Purpose: Immune checkpoint blockade (ICB) has revolutionized treatment of melanoma and lung cancer. Recent evidence suggests that human leukocyte antigen (HLA) B supertype influences efficacy of ICB. HLA-B27 and B44 supertypes have electronegative and electropositive binding pockets, respectively, and preferentially bind and display oppositely charged neoepitopes. We sought to evaluate the relationship between baseline immune activation and charged neoepitopes in the context of oppositely charged HLA-B binding pockets. Methods: Datasets from a total of 466 MEL and 513 LUAD patients included in the TCGA were used. HLA type was obtained with OptiType. Nonsynonymous mutations were annotated with SnpEff, Ensembl VEP, and VAtools. Only transcribed variants were filtered for analysis. pVAC-Seq using NetMHCpan algorithm predicted nonamer neoepitopes. Favorable B27 neoepitopes were defined as having new positively charged amino acid (AA) substitutions (H/K/R) from oppositely charged or uncharged wildtype AA, while neoepitopes with new negatively charged AAs (D/E) were considered favorable for B44. Three gene signatures were evaluated with normalized RNA expression values from 14 antigen presentation, 15 immunostimulator, and 11 immunoinhibitor genes. Linear regression tests were performed between gene expression values and fraction of nonsynonymous mutations resulting in neoepitopes with new positively and negatively charged AAs. Paired t-tests were performed between the mean of Pearson's r for fraction of new positively versus negatively charged AAs compared to gene expression values, and subsetted to B27/B44. Heterozygotes for B27/B44 were excluded from analysis to avoid competing charges. Results: 26.4% and 48.7% of MEL patients had B27 and B44, respectively. In LUAD, 28.6% had B27 and 47.2% had B44. Of new charged AAs resulting from nonsynonymous mutations, 72.6% in MEL and 75.0% in LUAD were positively charged (p=0.0053). In MEL, difference in Pearson's r values between fraction of new positively and negatively charged AAs as compared to immunostimulator (B27 p=.016; B44 p Conclusion: Immune gene signatures showed association with neoepitopes that have oppositely charged AAs than their HLA-B binding pocket in both MEL and LUAD, indicating that presentation of favorable neoepitopes stimulates an immune response. The association was limited to B27 in LUAD and strongest for B44 in MEL. Citation Format: Charlene M. Fares, Amy L. Cummings, Matthew K. Theisen, Nicholas Hornstein, Jaklin Gukasyan, Wisdom Akingbemi, Debory Li, Paige Brodrick, Aaron Lisberg, Edward B. Garon. Association between immune gene signatures and neoepitopes with electrostatic charge opposite to their HLA-B binding pocket in melanoma (MEL) and lung adenocarcinoma (LUAD) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6614.

SummaryFMRP is a polysome-associated RNA-binding protein encoded by Fmr1 that is lost in Fragile X syndrome. Increasing evidence suggests that FMRP regulates both translation initiation and elongation, but the gene-specificity of these effects is unclear. To elucidate the impact of Fmr1 loss on translation, we used ribosome profiling for genome-wide measurements of ribosomal occupancy and positioning in the cortex of 24 day-old Fmr1 knock-out mice. We found a remarkably coherent reduction in ribosome footprint abundance per mRNA for previously identified, high-affinity mRNA binding partners of FMRP, and an increase for terminal oligo-pyrimidine (TOP) motif-containing genes canonically controlled by mTOR-4EBP-eIF4E signaling. Amino acid motif- and gene-level analyses both showed a widespread reduction of translational pausing in Fmr1 knock-out mice. Our findings are consistent with a model of FMRP-mediated regulation of both translation initiation through eIF4E and elongation that is disrupted in Fragile X syndrome.

Ribosome profiling has emerged as a powerful tool for genome-wide measurements of translation, but library construction requires multiple ligation steps and remains cumbersome relative to more conventional deep-sequencing experiments. We report a new, ligation-free approach to ribosome profiling that does not require ligation. Library construction for ligation-free ribosome profiling can be completed in one day with as little as 1 ng of purified RNA footprints. We apply ligation-free ribosome profiling to mouse brain tissue to identify new patterns of cell type-specific translation and test its ability to identify translational targets of mTOR signaling in the brain. Electronic supplementary material The online version of this article (doi:10.1186/s13059-016-1005-1) contains supplementary material, which is available to authorized users.

Diffusely infiltrating gliomas are the most common type of primary brain tumor seen in adults. Patients with low grade glioma (LGG) can survive for many years, but often suffer from seizures and cognitive impairment, and these symptoms are associated with an unfavorable prognosis. Glioma cells diffusely infiltrate cortical tissue, where tumor cells aggregate around neuronal cell bodies, a growth pattern referred to as perineuronal satellitosis, however the effects of tumor cells on neurons has not been well defined. To address this important issue we generated a mouse model that recapitulates the genetic and histological features of diffusely infiltrating LGG. In our model tumor growth is driven by PDGFA overexpression and p53 deletion in a RiboTag-Camk2a-Cre transgenic mouse. The RiboTag system allows for the isolation and sequencing of ribosome-bound transcripts from Camk2a neurons to obtain a measure of transcription and translation. Our analysis in non-tumor bearing brains identified neuron-specific genes that are highly regulated at the level of translation. mTOR signaling regulates cell growth, proliferation, and translation in response to diverse stimuli. To characterize mTOR signaling in our model we used immunohistochemical staining of pS6 as a marker of mTOR activity and found that neurons within the glioma infiltrated cortex have lower levels of pS6 staining compared to neurons in the surrounding cortex. To further assess these results we are using the RiboTag system to identify alterations in neuronal transcription and translation that occur in our model during glioma formation and in response to mTOR targeted treatments. These studies will provide new insights into the neuronal alterations contributing to seizures and cognitive impairment in glioma and enable the identification of potential molecular targets for novel therapies to treat the devastating consequences of cortical dysfunctions associated with glioma. Citation Format: Daniela Torres, Angeliki Mela, Sohani Das Sharma, Nicholas Hornstein, Peter Sims, Peter Canoll. Characterization of neuronal alterations in a new mouse model of low grade glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1841. doi:10.1158/1538-7445.AM2017-1841

Glioma growth is driven by signaling that ultimately regulates protein synthesis. Gliomas are also complex at the cellular level and involve multiple cell types, including transformed and reactive cells in the brain tumor microenvironment. The distinct functions of the various cell types likely lead to different requirements and regulatory paradigms for protein synthesis. Proneural gliomas can arise from transformation of glial progenitors that are driven to proliferate via mitogenic signaling that affects translation. To investigate translational regulation in this system, we developed a RiboTag glioma mouse model that enables cell-type-specific, genome-wide ribosome profiling of tumor tissue. Infecting glial progenitors with Cre-recombinant retrovirus simultaneously activates expression of tagged ribosomes and delivers a tumor-initiating mutation. Remarkably, we find that although genes specific to transformed cells are highly translated, their translation efficiencies are low compared with normal brain. Ribosome positioning reveals sequence-dependent regulation of ribosomal activity in 5′-leaders upstream of annotated start codons, leading to differential translation in glioma compared with normal brain. Additionally, although transformed cells express a proneural signature, untransformed tumor-associated cells, including reactive astrocytes and microglia, express a mesenchymal signature. Finally, we observe the same phenomena in human disease by combining ribosome profiling of human proneural tumor and non-neoplastic brain tissue with computational deconvolution to assess cell-type-specific translational regulation.