Broad Institute Research Articles

Neumora launches with $500 million

In one of the largest biotech debuts this year, Neumora Therapeutics has raised $400 million in series A financing from more than a dozen venture capital firms, plus $100 million from Amgen. The firm’s goal: precision small-molecule therapies for a multitude of brain diseases. The start-up was quietly founded in late 2019 by several people at Arch Venture Partners, neurobiologist Morgan Sheng of Broad Institute of MIT and Harvard, and others. The company has since grown to a team of 90 people based at sites just outside Boston and San Francisco. Neumora has acquired four smaller start-ups that will help it hit the ground running. It has eight drug programs, plus a research collaboration with Amgen on additional programs for brain diseases including amyotrophic lateral sclerosis and schizophrenia. CEO and cofounder Paul L. Berns says the company is based on advances in neuroscience disease biology and computational data science. Neumora

Biden chooses science advisers

US president Joe Biden announced Sept. 22 the new members of his scientific advisory committee, including several leaders in the chemistry enterprise. The President’s Council of Advisors on Science and Technology (PCAST) will consist of 30 members from a variety of disciplines, including agriculture, engineering, neuroscience, and cybersecurity. Biden had announced the three cochairs for PCAST in January. They are Biden’s science adviser and Broad Institute mathematician and geneticist Eric Lander ; Chemistry Nobel Laureate and California Institute of Technology chemical engineer Frances Arnold; and Massachusetts Institute of Technology geophysicist and vice president for research Maria Zuber. The full council is the most diverse ever, the administration says. More than half the members are women and more than a third are people of color or immigrants. This is also the first time women have served as PCAST cochairs. “We are thrilled that some of our most accomplished Americans are willing

Neuroprotection: Why, when and how

Francisco J. Quintana. Harvard Medical School and The Broad Institute of MIT and Harvard Astrocytes play important roles in the central nervous system (CNS) during health and disease. In particular, it is now know that astrocytes play important roles in CNS inflammation and neurodegeneration in MS and other neurologic diseases. Thus, the identification of factors that regulate astrocyte activity may shed light on CNS physiology and guide new therapies for human neurologic disorders. In this presentation we will discuss mechanisms used by astrocytes to control CNS pathology. In addition, we will discuss molecular pathways involved in the control of astrocyte function. For example, we recently found that microbial metabolites limit astrocyte pathogenic activities in the context of CNS inflammation, while environmental factors can boost these disease promoting activities. Finally, we will review ongoing efforts on the characterization of astrocyte heterogeneity in MS, and novel findings on how specific subsets and their interactions participate in MS pathology.

A Missense Variant in the IL-6 Receptor and Protection From Peripheral Artery Disease

A Missense Variant in the IL-6 Receptor and Protection From Peripheral Artery Disease

MP39-14 IDENTIFICATION OF RECURRENT SOMATIC SPLICE SITE VARIANTS ACROSS MULTIPLE CLEAR CELL RENAL CELL CARCINOMA COHORTS

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology I (MP39)1 Sep 2021MP39-14 IDENTIFICATION OF RECURRENT SOMATIC SPLICE SITE VARIANTS ACROSS MULTIPLE CLEAR CELL RENAL CELL CARCINOMA COHORTS Nicholas Chakiryan, Timothy Robinson, Dongliang Du, Ali Hajiran, Logan Zemp, Youngchul Kim, Jad Chahoud, Philippe Spiess, Liang Wang, James Mule, and Brandon Manley Nicholas ChakiryanNicholas Chakiryan More articles by this author , Timothy RobinsonTimothy Robinson More articles by this author , Dongliang DuDongliang Du More articles by this author , Ali HajiranAli Hajiran More articles by this author , Logan ZempLogan Zemp More articles by this author , Youngchul KimYoungchul Kim More articles by this author , Jad ChahoudJad Chahoud More articles by this author , Philippe SpiessPhilippe Spiess More articles by this author , Liang WangLiang Wang More articles by this author , James MuleJames Mule More articles by this author , and Brandon ManleyBrandon Manley More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002054.14AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Splice-site variants (SV) are DNA mutations that ultimately result in abnormal mRNA splicing and a dramatically altered protein-coding sequence. SVs in clear cell renal cell carcinoma (ccRCC) have been largely unexplored. Our primary objective was to identify the most prevalent SVs in ccRCC, and determine their prevalence in other malignancies and adjacent normal kidney tissue. METHODS: Specific SVs were identified in RCC cell lines from the Broad Institute Cancer Cell Line Encyclopedia. Patients with a diagnosis of ccRCC were then identified in the Moffitt Cancer Center Total Cancer Care (TCC) cohort and RNA-seq data was utilized in a bioinformatics pipeline to identify the SVs among the TCC cohort. SVs identified in the TCC ccRCC cohort were then identified in TCC patients with non-ccRCC malignancies. This process was then repeated for the ccRCC RNA-seq data from CPTAC and TCGA, as well as from adjacent normal kidney tissue from these data sets. The most prevalent SVs among the ccRCC were selected for analysis, using a minimum SV read count threshold of 10. RESULTS: Among the three ccRCC datasets, 756 patients with ccRCC tumor samples were identified (N: TCC 104, CPTAC 111, TCGA 541). Eight distinct SVs were found to have high ccRCC prevalence and low prevalence in adjacent normal kidney tissue and non-ccRCC malignancies (Figure 1A). Genes with multiple distinct SVs were classified separately. The GAL3ST1-a and GAL3ST1-b SVs had the highest prevalence in ccRCC, as well as the highest read count per patient (Figure 1B). In the TCC ccRCC cohort, low read counts of SVs from the GAL3ST1 gene were associated with stage cM1. CONCLUSIONS: We identified eight novel SVs that are highly prevalent in ccRCC tumors and minimally prevalent in adjacent normal kidney tissue and other malignancies. Particularly striking were four distinct SVs affecting the the GAL3ST1 gene. These novel SVs warrant further investigation as potential biomarkers in ccRCC. Source of Funding: N/A © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e709-e710 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Nicholas Chakiryan More articles by this author Timothy Robinson More articles by this author Dongliang Du More articles by this author Ali Hajiran More articles by this author Logan Zemp More articles by this author Youngchul Kim More articles by this author Jad Chahoud More articles by this author Philippe Spiess More articles by this author Liang Wang More articles by this author James Mule More articles by this author Brandon Manley More articles by this author Expand All Advertisement Loading ...

Neuropsychiatric Genetics of Psychosis in the Mexican Population: A Genome-Wide Association Study Protocol for Schizophrenia, Schizoaffective, and Bipolar Disorder Patients and Controls

No large-scale genome-wide association studies (GWASs) of psychosis have been conducted in Mexico or Latin America to date. Schizophrenia and bipolar disorder in particular have been found to be highly heritable and genetically influenced. However, understanding of the biological basis of psychosis in Latin American populations is limited as previous genomic studies have almost exclusively relied on participants of Northern European ancestry. With the goal of expanding knowledge on the genomic basis of psychotic disorders within the Mexican population, the National Institute of Psychiatry Ramón de la Fuente Muñiz (INPRFM), the Harvard T.H. Chan School of Public Health, and the Broad Institute’s Stanley Center for Psychiatric Research launched the Neuropsychiatric Genetics Research of Psychosis in Mexican Populations (NeuroMex) project to collect and analyze case-control psychosis samples from 5 states across Mexico. This article describes the planned sample collection and GWAS protocol for the NeuroMex study. The 4-year study will span from April 2018 to 2022 and aims to recruit 9,208 participants: 4,604 cases and 4,604 controls. Study sites across Mexico were selected to ensure collected samples capture the genomic diversity within the Mexican population. Blood samples and phenotypic data will be collected during the participant interview process and will contribute to the development of a local biobank in Mexico. DNA extraction will be done locally and genetic analysis will take place at the Broad Institute in Cambridge, MA. We will collect extensive phenotypic information using several clinical scales. All study materials including phenotypic instruments utilized are openly available in Spanish and English. The described study represents a long-term collaboration of a number of institutions from across Mexico and the Boston area, including clinical psychiatrists, clinical researchers, computational biologists, and managers at the 3 collaborating institutions. The development of relevant data management, quality assurance, and analysis plans are the primary considerations in this protocol article. Extensive management and analysis processes were developed for both the phenotypic and genetic data collected. Capacity building, partnerships, and training between and among the collaborating institutions are intrinsic components to this study and its long-term success.

N6-Methyladenosine Writer Gene ZC3H13 Predicts Immune Phenotype and Therapeutic Opportunities in Kidney Renal Clear Cell Carcinoma.

BackgroundAlthough the RNA modification N6-methyladenosine ZC3H13 has been found to play vital regulatory roles in many types of cancers, its role in predicting the tumor immune microenvironment (TME) and response to immune checkpoint blockade (ICB) in kidney renal clear cell carcinoma (KIRC) remains unclear.MethodsWe comprehensively analyzed the expression, prognostic significance and immunological role of ZC3H13 in pan-cancers and systematically correlated ZC3H13 with TME cell-infiltration, ICB response and targeted therapy in KIRC. The data were collected from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), Genotype-Tissue Expression (GTEx), Broad Institute Cancer Cell Line Encyclopedia (CCLE) and DrugBank database. Also, we performed RNA sequencing (RNA-seq) of 46 renal cell carcinoma tissues and 11 adjacent normal tissues to validate our result. All analyses were implemented using R software, version 3.6.3.ResultsZC3H13 was significantly differentially expressed in most tumors. However, its expression profiles and prognostic significance were consistent only in KIRC, regardless of overall survival, progression-free survival and cancer-specific survival. Additionally, ZC3H13 expression was correlated with clinicopathological factors in KIRC. Furthermore, we found that ZC3H13 might shape a noninflamed phenotype and could predict a lower response to ICB in KIRC. These results could be validated in our own RNA-seq data. Tumor mutation burden (TMB) was significantly higher in the low ZC3H13 group. Finally, we found that ZC3H13 could predict the sensitivity of targeted therapy for KIRC.ConclusionsZC3H13 might shape a noninflamed phenotype in KIRC. Moreover, ZC3H13 could predict the prognosis and clinical response of ICB and the sensitivity to targeted therapies in KIRC.

Shifting Perceptions of CRISPR

Shifting Perceptions of CRISPR

FOXL2 mutation is prevalent in metastatic adult granulosa cell tumors and is associated with improved survival

<h3>Objectives:</h3> Adult granulosa cell tumors (AGCT) of the ovary represent less than 5% of all ovarian tumors. Despite having a favorable prognosis, nearly one-third of women with this diagnosis will recur, and therapeutic options are limited. We aim to describe a molecular profile of AGCT to improve prognostication and elucidate actionable molecular targets. <h3>Methods:</h3> AGCT samples were analyzed using a combination of next-generation sequencing (NGS), immunohistochemistry (IHC) and whole transcriptome sequencing (NovaSeq) (Caris Life Sciences, Phoenix, AZ). PD-L1 expression was tested by SP-142 (Ventana; positive cut-off ≥1%). Microsatellite instability (MSI) was tested by fragment analysis, IHC and NGS. Tumor mutational burden (TMB) was measured by counting all somatic mutations per tumor (TMB-high cut-off ≥10 mutations per MB). Immune cell fraction was calculated by QuantiSeq (Finotello 2019, Genome Medicine). Gene Set Enrichment Analysis (GSEA) was used to determine enrichment of cancer hallmark pathways (Broad Institute). Survival analyses were performed using the Kaplan-Meier estimate. <h3>Results:</h3> Of the 319 patients, the median age was 55, and most patients had metastatic disease (82.4 %). A majority of the tumors carried FOXL2 mutations (83.6%), of which 90% demonstrated a <i>C134W</i> missense mutation. Patients with FOXL2 mutations were significantly older (56 versus 47 years, p=0.003) and more likely to have metastatic disease than patients with wild-type FOXL2 (p=0.045). Other frequent mutations included KMT2D (14%) and PIK3CA (8%) mutations. There were no pathogenic <i>BRCA1</i> or <i>BRCA2</i> mutations identified in this cohort; however, a <i>BRCA2</i> mutation variant of unknown significance was noted in three tumors. Pathway analyses demonstrated that alterations in chromatin remodeling were significantly more common in tumors with FOXL2 mutations compared to wild-type status (16.1% versus 9.7%, p<0.05), as were alterations in the PI3K pathway (13.9% versus 0%, p<0.05). There was a low frequency of PD-L1 high expression (2.7%), and there were no tumors with high TMB or MSI. Patients with tumors with FOXL2 mutations had improved overall survival compared to patients with wild-type FOXL2 tumors (HR 2.07, CI 1.01-4.25, p=0.043). KMT2D and PI3KCA mutations were not associated with differences in overall survival when compared to wild-type tumors. Copy number alterations in the genes GPHN, HOOK3, SPEN, CAMTA1 and BCL2L11 were associated with survival differences in patients treated with bevacizumab. <h3>Conclusions:</h3> A majority of AGCT carry FOXL2 mutations. The next most common mutations were KMT2D, PIK3CA, and alterations in chromatin remodeling. In this cohort of patients, the FOXL2 mutation was associated with improved overall survival. Copy number alterations in specific genes were associated with differences in survival in patients treated with bevacizumab. These results support molecular profiling of AGCT to improve prognostication for these patients. These findings provide groundwork to direct further investigation into novel treatment strategies and targeted agents in AGCT.

Editing Humanity

Kevin Davies’s Editing Humanity takes you on a dizzying tour of CRISPR, from its discovery and naming by little-known Spanish microbiologist Francisco Mojica 25 years ago to the births of Lulu and Nana, the world’s first genetically edited humans. It took 13 years for the Human Genome Project to sequence the 3 billion bases in the human genome for the first time. Using a technique called micropore sequencing, you can now have yours done in less than 24 hours.Along the ride, you learn first about how CRISPR (clustered regularly interspersed short palindromic repeats) and its associated enzyme Cas-9 can be used to insert, modify, or delete DNA sequences in almost any organism. Although the system is an antiviral memory-enhanced immune system in bacteria, it can be used in almost any organism, since we all share the same basic DNA systems.The best-known researchers in this field include Nobel laureates Jennifer Doudna and Emmanuelle Charpentier, and George Church and Feng Zhang of the Broad Institute of MIT and Harvard, who currently hold the patents for most applications of the technique. A PhD in molecular genetics, Davies clearly explains the workings of the CRISPR-Cas9 system, using several colored diagrams, as well as the ongoing legal battles that will determine who will ultimately control the multibillion-dollar rights to a prokaryotic immune system.And the ride also goes worldwide. Davies brings you into interviews he does in Boston, Banff, Oslo, Singapore, Alicante, Vilnius, and Toronto, among many more. There are several reasons why I liked this book more than the four others I’ve read about CRISPR in the past two years. He explains the science without talking down to the reader. Yet he also explains the people. While in Toronto covering the Gairdner Awards (the “Nobel Prize” of Canada), Doudna and Charpentier share the award with a certain American epidemiologist named Anthony Fauci.Although most researchers (and legally some countries, including the USA) in this rapidly changing field maintain a moratorium on editing the human germ line, Chinese researcher He Jiankui, educated at Rice and Harvard, was the first to breach this barrier in 2018 with CRISPRd twin girls. He modified the CCR5 co-receptor for HIV, with the goal of making them immune to AIDS. Whether it really worked and whether there were additional off-site edits is still unknown. The uproar in the scientific world embarrassed the Chinese government, and Jiankui is currently in what amounts to house arrest, even though it is widely believed that the government knew exactly what was going on, turning a mostly blind eye. Many also believe that the Chinese will not allow such a gifted man to stay on the shelf for long, and in exchange for not naming officials in China who were complicit, Jiankui will be rehabilitated in the next few years.In the last few chapters, we learn about additional techniques, possibly more precise than the find-and-replace-all CRISPR, such as TALENs, ZFN, and single base editing. As microbiologist Fyodor Urnov wrote, “We live in an age where there is such a thing as Microsoft Word for human DNA. The cat is out of the bag, the genie is out of the bottle.

Collaboration Takes Aim at Rare Cancers

A collaboration between The University of Texas MD Anderson Cancer Center in Houston and the Broad Institute of MIT and Harvard in Cambridge, MA, is tackling rare cancers. The institutions have created a research platform to generate rare cancer models based on patients' tumor samples, which researchers will sequence to reveal the genomics of the diseases and identify potential therapeutic targets.

Traces of SARS-CoV-2 RNA in Peripheral Blood Cells of Patients with COVID-19.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third virus that caused coronavirus-related outbreaks over the past 20 years. The outbreak was first reported in December 2019 in Wuhan, China, but rapidly progressed into a pandemic of an unprecedented scale since the 1918 flu pandemic. Besides respiratory complications in patients with COVID-19, clinical characterization of severe infection cases showed several other comorbidities, including multiple organ failure, and septic shock. To better understand the systemic pathogenesis of COVID-19, we interrogated the virus's presence in the peripheral blood cells, which might provide a form of trafficking or hiding to the virus. By analyzing >2 billion sequence reads of high-throughput transcriptome sequence data from 180 samples of patients with active SARS-CoV-2 infection or healthy controls collected from 6 studies, we found evidence of traces of SARS-CoV-2 RNA in peripheral blood mononuclear cells in two samples from two independent studies. In contrast, the viral RNA was abundant in bronchoalveolar lavage specimens from the same patients. We also devised a “viral spike-to-actin” RNA normalization as a metric to compare across various samples and minimize errors caused by intersample variability in total human RNA abundance. Our observation suggests immune presentation and discounts the possibility of extensive viral infection of lymphocytes or monocytes.

Prime Medicine raises $315 million for search-and-replace gene editing

Prime Medicine has raised $315 million to commercialize an invention from David Liu’s lab at Broad Institute of MIT and Harvard. The start-up is developing therapies based on a new class of CRISPR gene-editing tools called prime editors, which some scientists say could be the most versatile form of gene editing yet . Liu and his postdoc Andrew Anzalone quietly founded the firm in 2019 before publishing their work on prime editors that October ( Nature 2019, DOI: 10.1038/s41586-019-1711-4 ). The pandemic largely delayed Prime’s operations until the second half of 2020, CEO Keith Gottesdiener says. In the months since, Prime has grown to about 50 employees—most of them scientists—with plans to expand to about 100 by the end of 2021, he adds, buoyed by its newly disclosed $115 million series A and $200 million series B financings. The earliest versions of CRISPR gene editing relied on using the enzyme

Development Of a 47 Gene Sequencing Panel For Common Mutations Present In Multiple Myeloma : Preliminary Results In 77 p53 Deleted Patients

As a consequence of the Multiple Myeloma Research Consortia (MMRC) genomics initiative, whole exome sequencing data has now been obtained on 213 primary MM samples essentially defining the exome mutation landscape for this disease. Using early access information from the MMRC we selected a panel of 39 genes, representing all expressed genes with nonsynonymous mutations found in ≥3% of MM cases, to which we then added eight additional genes targeted by the most commonly used therapies in MM, to help identify mutations associated with resistance to IMiDs (CRBN, CUL4A, CUL4B, DDB1 and IRF4), proteasome inhibitors (PSMG2, PSMB5) or glucocorticoid therapies (NR3C1). We tested this Multiple Myeloma Mutation Panel (M3P) in CD138+ bone marrow cells from 77 newly diagnosed, high risk, del17p MM patients with known clinical outcomes, obtained from the German DSMM study group. 43 of which also had a germline sample to analyze and 3 patients had a second subsequent sample from a later timepoint Methods The 47 M3P genes were sequenced using semiconductor technology (IonTorrent PGM, Life Technologies). The coding regions of the 47 genes were amplified in 200 bp libraries using customized oligos (Ion Ampliseq designer). Overall 2875 amplicons were analyzed per sample, multiplexed in two library preparations. Templating and Enrichment of DNA libraries was done using the IonOneTouch2 and IonOneTouch ES automated systems. Batches of 4-8 samples were barcoded using the Ion Xpress™ Barcode Adapters (Life Technologies), pooled and sequenced using the 318TMv2 chip (Life technologies). Data were visualized using IGV software (Broad Institute) and analyzed using IonReporter software (Life Technologies). Damaging mutations were collated using SIFT and PolyPhen. Publicly available databases including COSMIC and Oncomine were used to identify genes predicted to be cancer related. Results To validate the technology we first compared results using M3P to a previously sequenced MM whole genome. All genes in the M3P appeared adequately amplified by the designed primers. All mutations identified by the previously run WGS were successfully identified using the M3P sequencing panel. Subsequently, 33 tumor-germline pair samples, 43 single tumor samples and 1 temporally distinct paired sample for a total cohort of 77 patients have been analyzed. Under current conditions M3P achieved a mean 244X depth sequencing coverage per nucleotide for the tumor and a mean 179X depth sequencing coverage for the germline samples, respectively. Data on 33 tumor-germline comparisons so far show nonsynonymous mutations in 30 of the 47 genes with a mean of 2.7 mutations per sample (range 0-9) and 50.0% of mutations are predicted damaging by PolyPhen or SIFT. Ten genes were found to be mutated in more than 10% of the samples including genes previously known to be commonly mutated in MM: TP53, KRAS, NRAS and BRAF but also in NEB, ZFHX4, ANK2, MLL3, FAT3 and SP140. Interestingly, 46.6% of the mutations identified were present in less than 10% of reads, suggesting a high frequency of minor clones already present at the time of diagnosis. Of note, mutations once thought to be mutually exclusive such as KRAS and NRAS, were simultaneously detected in 2 patients. Analysis of the first sequential paired sample revealed nonsynonymous mutation changes over time with loss of a KRAS mutation, clonal expansion of a TP53 mutation and gain of additional FAT3, FAT1, SPEN, VCAN and FAM46C mutations, indicating the value of the panel in tracking clonal evolution over time. No further FAM46C mutations were detected in any of our newly diagnosed high risk cohort samples, which is significantly different from results in the MMRC genomics initiative, where FAM46C mutations were the fifth most common mutations (10.8%), suggesting that FAM46C mutation may be more frequent in lower risk patients or in relapse samples. Conclusion The M3P gene panel represents all genes with nonsynonymous mutations found in ≥3% of MM cases from the MMRC genomics initiative and incorporates additional genes that are known to be involved in the development of drug resistance. Thus this panel may serve as a tool to further improve MM classification, more precisely predict prognosis track clonal evolution and better guide treatment decisions. Our ongoing analysis will provide a comprehensive picture of the genetic landscape of a defined high risk MM cohort of del17p patients. Disclosures: Einsele: Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Membership on an entity's Board of Directors or advisory committees. Stewart: Onyx: Consultancy, Research Funding; Millennium: Honoraria, Research Funding; Celgene: Honoraria; BMS: Honoraria.

Abstract 1225: LNS8801: A novel, enantiomerically pure, small molecule agonist of the G protein-coupled estrogen receptor (GPER) for the treatment of cancer

Abstract Women have a lower incidence of and improved clinical outcomes for most cancer types, but the mechanistic basis for this sex difference is unknown. We hypothesized that estrogen signaling may be responsible, despite the fact that many cancer types lack classic nuclear estrogen receptors. The GPER agonist G-1, a racemate, has been demonstrated to have anticancer activities by many independent laboratories, though there are some contradictory findings in the literature. We resolved the enantiomers that compose G-1 and determined that LNS8801 is the sole enantiomer responsible for G-1's anticancer effects. Using CRISPR to deplete GPER, we determined that the anticancer activity of LNS8801 is dependent upon GPER expression within the tumor cells. Previously, we determined that the anticancer mechanism of GPER activation is through a rapid proteasomal degradation of c-Myc. The PRISM screen was performed on LNS8801 at the Broad Institute, and it was determined that this compound has universal anticancer activity across multiple cancer types, consistent with our inhouse data. Sensitivity to LNS8801 was highly correlated with expression of c-Myc target genes, which is consistent with the known mechanism of action. Lineage analysis identified two specific cancer types that were uniformly more sensitive to LNS8801 than other cancer types: diffuse large B-cell lymphoma (DLBCL) and anaplastic large cell lymphoma (ALCL). DLBCL has historically been considered to be highly dependent on c-Myc, which is consistent with the known mechanism of action of LNS8801; however, the identification of ALCL was unexpected. Deeper analysis determined that high expression of anaplastic lymphoma kinase (ALK) fusion genes tightly correlated with sensitivity to LNS8801 in ALCL and other ALK+ cancer types. Using non-small cell lung cancer cell lines engineered to express EML4-ALK, we validated that ALK fusion expression results in increased sensitivity to LNS8801 relative to isogenic controls. This work has identified an enantiomerically pure GPER agonist that has potent activity across many cancer types, with c-Myc-dependent and ALK+ cancer types being enriched for heightened sensitivity. Citation Format: Chris Natale, Tina Garyantes, Todd Ridky. LNS8801: A novel, enantiomerically pure, small molecule agonist of the G protein-coupled estrogen receptor (GPER) for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1225.

Abstract 309: Development of a multi-omic, pooled cancer cell screen for nanoparticle delivery

Abstract Background: Nanoparticles (NPs) hold enormous promise for the targeted delivery of therapeutics for cancer, but clinical translation is lacking largely due to limited tumor accumulation. Tumor heterogeneity and NP complexity make it challenging to deconvolute individual factors that contribute to NP-cell interactions. To address this, we developed a competition assay leveraging 500 stably DNA-barcoded adherent cancer cell lines annotated with multi-omic data from the Broad Institute (PRISM cells) to investigate cell association patterns across a library of NPs. We hypothesize that simultaneous screening of hundreds of cancer cell lines will identify factors underlying differential NP-cancer cell interactions. Methods: We synthesized a library of 40 fluorescently-labeled NPs comprising clinical and experimental formulations. Clinical formulations included liposomal doxorubicin and irinotecan analogs and liposomal or poly(lactide-co-glycolide, PLGA) NPs with and without polyethylene glycol (PEG); these are either FDA-approved or in clinical trials. Experimental formulations included liposomal and PLGA cores electrostatically coated with a range of native and synthetic polymers as well as polystyrene NPs of varying sizes and surface chemistries. Fluorescent antibodies -in free form or NP-conjugated—were included as validation compounds. PRISM cells were pooled and incubated with NPs prior to fluorescence-activated cell sorting (FACS) to bin cells based on strength of NP association. After cell lysis, DNA barcodes were amplified and sequenced. Using appropriate controls to adjust for baseline barcode abundance, we generated an association score for each NP-cell line pair. Next, we performed multi-omic univariate analyses and applied a random forest algorithm to identify factors predictive of NP-cancer cell association. Results: After pooled screening of PRISM cells, we consistently identified cancer cell lines based on strength of NP-association across technical and biologic replicates. Using antibodies and antibody-conjugated NPs targeting epidermal growth factor receptor (EGFR), we identified EGFR gene and protein expression as highly significant hits, validating our ability to robustly identify relevant biomarkers. Additional hits were evaluated based on strength and direction of association to identify predictive biomarkers by formulation. We also employed k-means clustering to investigate hits across NP formulations, identifying highly interconnected protein association networks that elucidate likely mechanisms of NP-cancer cell association. Conclusions: We report a new pooled screening platform to investigate factors influencing NP-cancer cell interactions. We validated the screen by identifying known biomarkers, and also identified new predictive biomarkers that may pave the way for more effective nanotherapeutics. Citation Format: Joelle P. Straehla, Natalie Boehnke, Mustafa Kocak, Melissa Ronan, Hannah Safford, Matthew G. Rees, Jennifer A. Roth, Angela N. Koehler, Paula T. Hammond. Development of a multi-omic, pooled cancer cell screen for nanoparticle delivery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 309.

Abstract 212: Sarcoma-CellMiner: An extensive resource for patient-derived sarcoma cell line epigenetics, genomics and pharmacology

Abstract Sarcomas represent a heterogeneous group of cancers with many histological subtypes. Their prognosis remains poor and treatment is mainly based on palliative chemotherapy at metastatic stage. Tumor genome sequencing failed to identify recurrent somatic drivers while several oncogenic gene fusion-translocations have been identified in specific sarcoma subtypes. Because of the rarity and heterogeneity of sarcomas, only few sarcoma patients are enrolled in clinicals trials according their subtype. Increased translational research according subtypes of sarcoma patients is needed to improve patient management. To characterize the differences between subgroups and build translational hypotheses, we built a novel resource, Sarcoma-Cellminer, which integrates drug sensitivity and genomic data from 112 patient-derived sarcoma cell lines. These data will be available from a web-based tool (https://discover.nci.nih.gov/SclcCellMinerCDB/) derived from our CellMiner cross-database web application (https://discover.nci.nih.gov/cellminercdb). Among the 112 cell lines, 65 are bone sarcomas (including 38 Ewing sarcomas and 22 osteosarcomas), 45 are soft tissue sarcomas (including 21 rhabdomyosarcomas). Transcriptome (RNAseq and microarray), copy number, microRNA, genome-wide methylation, and drug sensitivity data are included and made publicly available. We also generated new genomic data including copy number and methylation (850 k) for 79 cell lines from the NCI in addition to the 42 cell lines from Broad Institute (CCLE) and the 40 cell lines from the MGH-Sanger (GDSC). We created the “SCLC-Global” expression set by regrouping all datasets by Z-score normalization, which enables cross-database analyses of gene expression and molecular pathways. Hierarchical clustering based on expression and methylation data identifies subtypes of sarcomas. Histone genes stand out suggesting that epigenetic regulation of canonical histones is a feature of sarcoma genesis. Sarcoma-CellMiner includes drug sensitivity data for over 500 different drugs tested in the NCI, CCLE and GDSC databases. They show two subgroups of Ewing sarcomas: one sensitive to PARP inhibitors and one resistant. Similarly, profile of response to dasatinib is different when comparing alveolar and embryonal rhabdomyosarcomas. Sarcoma-CellMiner is a powerful tool demonstrating the value of patient-derived cancer cell line databases. It provides hypothesis-driven rationale for using omics, especially transcriptome and epigenetic data to better understand sarcoma heterogeneity and select personalized treatments for clinical trials. Citation Format: Camille Tlemsani, Lorinc Pongor, Javed Khan, Fathi Elloumi, Sudhir Varma, Augustin Luna, Vinodh Rajapakse, Kurt Kohn, Julia Krushkal, Mirit Aladjem, Beverly Teicher, Paul Meltzer, William Reinhold, Christine Heske, Yves Pommier. Sarcoma-CellMiner: An extensive resource for patient-derived sarcoma cell line epigenetics, genomics and pharmacology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 212.

Impact of Tyrosine Kinase Inhibitors Used in Chronic Myeloid Leukemia on Signaling and Epigenetics in Endothelial Cells

Background: Tyrosine kinase inhibitors (TKIs) have revolutionized treatment of chronic myeloid leukemia (CML) resulting in long term survival with chronic therapy. Vascular toxicities have emerged as complications of the second generation CML TKIs; dasatinib, ponatinib and nilotinib. Dasatinib is associated with increased risk of pulmonary hypertension, ponatinib with arterial ischemic events and nilotinib with peripheral arterial occlusive events and cardiovascular ischemia by unknown mechanisms. The first generation CML TKI imatinib is not associated with vascular toxicity.Hypothesis: We hypothesize that the CML TKIs- ponatinib, dasatinib and nilotinib, but not imatinib, cause vascular toxicity by affecting endothelial cell (EC) function in association with alterations in phospho-signaling and histone modifications.Methods To test this hypothesis, we studied the effect of CML TKIs imatinib, nilotinib, dasatinib and ponatinib on EC function using three in-vitro assays measuring EC survival, EC-leucocyte adhesion (a measure of vascular inflammation) and scratch wound closure (a measure of wound healing capacity). Cultured human umbilical vein ECs (HUVECs) were treated with vehicle, imatinib (4uM), dasatinib (200nM), nilotinib (3 uM) or ponatinib (100nM). Cell viability was measured at 18 hours using a Cell TiterGlo Luminescent Cell Viability assay (Promega). For the leucocyte adhesion assay, fluorescent labeled leucocytes (U937) were incubated with the ECs and adherent fluorescent cells were quantified. For the wound healing assay, a scratch “wound” was made with a plastic P1000 pipette tip and cells that migrated into the wound area were counted at 18 hours. To assess signaling, two novel, global, unbiased, high throughput proteomic assays developed at the Broad Institute were used. The P100 assay is a mass spectrometry-based targeted proteomics assay that quantifies 100 representative phosphopeptides. The global chromatin profiling (GCP) assay uses mass spectrometry to simultaneously quantify over 70 bulk histone modifications including phosphorylation, ubiquitination, methylation and acetylation. HUVECs were treated in triplicate with each drug for 3 hours for the P100 assay and for24 hours for the GCP assay. For each assay, the peptides were mixed with isotopically labeled internal standards and mass spectrometry was performed to quantify each phospho-peptide or modified histone peptide relative to the universal standard.Results: The CML TKIs that are associated with vascular toxicity (nilotinib, ponatinib and dasatinib) negatively impacted all the EC functions measured. Specifically, nilotinib, ponatinib and dasatinib significantly decreased endothelial cell viability and endothelial cell migration in response to a scratch wound and increased leucocyte adhesion to ECs. Imatinib did not have a negative effect on ECs in any of those assays. Using the mass spectrometry based P100 and GCP assays, we found that nilotinib affects histone epigenetics and protein phosphorylation in HUVECs. In particular, nilotinib treatment modulates ABL1, MAP4, BRD4 and JUND phosphorylation and also increases histone H3K9 trimethylation with H3S10 phosphorylation in HUVECS. We also compared the nilotinib profiles obtained in HUVECs to that of primary human aortic smooth muscle cells (HAoSMC) and several cancer cell lines including MCF7 (breast), A549 (lung), A375 (skin) and PC3 (prostate) (Figures 1a and 1b). While some changes are unique to ECs, others are similar to the effects in cancer cells and smooth muscle cells. GCP assay and P100 assays comparing the effects of all the CML TKIs in endothelial cells are in process.Conclusions: CML TKIs associated with vascular toxicity affect human EC function in vitro resulting in impaired endothelial survival and wound healing and increase inflammation. These changes are associated with alterations in signaling and epigenetic pathways in ECs in response to treatment with nilotinib. Increased vascular inflammation and impaired vascular healing are associated with cardiovascular thrombosis and ischemia. Further studies comparing the effects of all CML TKIs on ECs will be used to elucidate molecular mechanisms of vascular toxicity and help devise strategies to overcome these toxicities while maintaining anti-cancer efficacy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Programmed Necrosis Correlates with Development and Progression of Myelodysplastic Syndromes

Myelodysplastic syndrome (MDS) is characterized by persistent cytopenias and evidence of morphologic dysplasia in the bone marrow. Lower-grade MDS (bone marrow blasts <5%) is challenging to diagnose, as the morphologic dysplasia may be subtle and many patients lack characteristic cytogenetic abnormalities.Multiple studies of the bone marrow in MDS have demonstrated excessive hematopoietic cell death, and this increased cell death is thought to play an important role in the pathogenesis of the disease. Early studies of MDS revealed increased in situ end labeling and increased TUNEL staining which led to the conclusion of apoptotic cell death, but these methods cannot properly distinguish between necroptosis and apoptosis. Apoptosis is driven by caspases that break down the cytoskeleton to cause collapse of the cell in a predominantly immune silent process. Recently, necroptosis has been identified as a new pathway of programmed cell death, which is driven by Rip kinases and is characterized by premature rupture of the plasma membrane, resulting in an immune response. Necroptosis has been shown to play a role in inflammatory diseases as well as host response to infection, and tumors.Methods: To assess cell death status in normal bone marrow as compared to lower-grade MDS (RCUD, RA, RARS, RCMD) and higher-grade MDS (RAEBI/RAEBII, RAEB-t), we evaluated a total of 37 de novo MDS cases and 36 normal control cases. The MDS specimens included 20 cases of lower-grade MDS and 17 cases of higher-grade MDS. All cases were reviewed and the diagnosis confirmed by board-certified hematopathologists. Necroptosis was assessed by measuring Rip1 and phosphorylation of Mixed Lineage Kinase domain-like protein (MLKL) and apoptosis was measured by cleaved-caspase 3.Formalin fixed paraffin embedded bone marrow core biopsies were immunostained with the apoptosis marker (caspase 3) and necroptosis markers (Rip1, MLKL) and visualized by immunofluorescence. Whole slide images were obtained using Aperio Versa 200 (Leica Microsystems). We developed a pipeline for statistical analysis to evaluate the immunofluorescence of stained and scanned images using CellProfiler (Broad Institute) image analysis software. For each slide over 10,000 cells were analyzed.Results: We found that the proportion of RIP1 positive cells was significantly different between normal controls (6.91±1.32%) and lower-grade MDS (32.80±6.19%, p value 0.0042). Likewise, RIP1 staining in higher-grade MDS was less common than in lower-grade MDS (15.99±4.76% vs 32.90±6.19%, p value 0.0506). There was no significant difference between normal controls and high-grade MDS. In lower-grade MDS samples, RIP1 positive cells were mononuclear cells arranged in clusters, morphologically consistent with erythroid progenitors. Similar trends were seen with MLKL staining.To evaluate apoptosis, we performed cleaved caspase 3 staining on the same patient samples. There was no difference in the proportion of caspase 3 positive cells among the groups: normal controls (2.02±1.08%), lower-grade MDS (2.77±0.86%) or higher-grade MDS (1.99±0.71%). As apoptotic and necroptotic cells have markedly different cellular ultrastructural morphologies, we performed electron microscopy on fresh bone marrow cells to define cell death. After scoring cells with a nucleus as either being live, apoptotic (shrinkage of cell membrane and pyknotic nucleus), or necrotic (swelling of cell membrane and nucleus), we found that there was a significant increase in the number of necrotic cells in the bone marrow of the lower-grade MDS patients relative to the normal control or higher-grade MDS patients.We then performed over-representation analysis of the publicly available MDS microarray data (GEO GSE15061) from the international multi-institutional Microarray Innovations in Leukemia (MILE) research program. Although the study was underpowered to identify differences between normal controls and early MDS, we found that RIP1, RIP3 and MLKL had significantly increased expression in the MDS group vs the de novo AML group, suggesting that genes in the necroptotic pathway may be upregulated in MDS as compared to AML.In summary, our findings reveal that the necroptosis cell death pathway is upregulated in early MDS relative to normal controls and warrants further study to define its role in the pathogenesis of MDS and as a potential biomarker for the diagnosis of lower-grade MDS. [Display omitted] DisclosuresSavona:Astex: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Research Funding; Incyte Corporation: Consultancy, Research Funding; Karyopharm: Consultancy, Equity Ownership; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees.

Targeted RNA-Seq Identify a Subset of Adolescent and Adult Patients with Acute Lymphoblastic Leukemia with BCR-ABL1-like Characteristics

Background: BCR-ABL1-like (Ph-like) B-precursor acute lymphoblastic leukemia (ALL) shows a gene expression profile closely related to B-precursor ALL with Philadelphia (Ph) chromosome rearrangement. In addition, Ph-like ALL patients are characterized by common genetic alterations and inferior prognosis, as has been shown in pediatric and adult studies. The aim of this study was the genetic and clinical characterization of Ph-like ALL by RNAseq in a series of adolescent and adult ALL patients included in risk-adapted trials from the Spanish PETHEMA group.Methods:Fourty-nine adolescents and adults with Ph-negative B-ALL (median age 38 years, range 11-74) were included in this study. RNA-Seq custom panel of 42 genes related to the Ph-like signature (AmpliSeq, Thermo Fisher) was carried out in a S5 sequencer (Thermo Fisher). The total reads were employed to build a hierarchical clusterized heatmap with Morpheus software (Broad Institute). Mutations related to the Ph-like genotype were studied after sequencing 31 patients with a custom panel formed by 35 genes. All samples were sequenced in S5 Ion Torrent equipment. CRLF2 overexpression was analyzed by real time PCR.Contingency tables were used to analyze associations between categorical variables (χ2). Median test was used to compare medians of continuous variables between groups. Complete remission (CR) duration, disease-free survival (DFS) and overall survival (OS) curves were estimated using the Kaplan-Meier method and compared between patients using the log-rank test.Results: Thirteen out of 49 patients were classified as Ph-like (26%) based on heatmap aggrupation. There were no significant differences in baseline characteristics like age, gender, ALL sub-type, and cytogenetics, in comparison with B-ALL other subgroup (n=6, 74%). Additionally, most patients with high CRLF2 expression were classified into the Ph-like ALL subgroup (11/13, 85% vs 7/36, 19% of no Ph-like. Complete remission (CR) was attained in 42 patients (86%). At 5 years, the Ph-like ALL subgroup showed a trend to lower probability of CR duration compared to non Ph-like group (95% CI): 27% (0-58%) vs. 58% (38-78%), p=0.086. No significant differences were observed in OS (95% CI): 44% (16-72%) vs. 52% (35-69%), p=0.197), but Ph-like patients showed significantly shorter DFS probability at 5 years (95% CI): 20% (0-45%) vs. 54% (35-73%), p=0.047, (Figures 1a and 1b). A summary of the main characteristics and outcomes of the patients is presented in the Table 1. Mutations of IKZF1, KRAS, JAK2, CRLF2, NRAS, JAK1, TYK2 and PAX5 genes related to Ph-like genotype represented 7/11 Ph-like patients (64%) vs 3/20 no Ph-like (15%). Data of a validation cohort of patients will be presented.Conclusion: Targeted RNA-Seq could identify a Ph-like B-ALL subgroup within adolescents and adults with Ph-negative B-ALL homogeneously treated with risk-adapted protocols. This Ph-like B-ALL subgroup showed lower DFS probability than the remaining Ph-negative B-ALL patients. [Display omitted] DisclosuresRibera:Gilead: Honoraria; Janssen: Honoraria; Celgene: Honoraria; Incyte: Research Funding, Speakers Bureau; Pfizer: Research Funding, Speakers Bureau; Amgen Inc.: Research Funding, Speakers Bureau; Roche: Honoraria; ARIAD: Research Funding, Speakers Bureau.