Molecular Vulnerabilities Research Articles

Cell fitness screens reveal a conflict between LINE-1 retrotransposition and DNA replication.

LINE-1 retrotransposons are overexpressed in more than half of human cancers. We identified a colorectal cancer wherein a fast-growing tumor subclone downregulated LINE-1, prompting us to examine how LINE-1 expression affects cell growth. We find that non-transformed cells undergo a TP53-dependent growth arrest and activate interferon signaling in response to LINE-1. TP53 inhibition allows LINE-1(+) cells to grow, and genome wide knockout screens show that these cells require replication-coupled DNA repair pathways, replication stress signaling, and replication fork restart factors. Our findings demonstrate that LINE-1 expression creates specific molecular vulnerabilities and reveal a retrotransposition-replication conflict that may be an important determinant of cancer growth.

Systematic Characterization of Genes Representing Preferential Molecular Vulnerabilities for Myeloma Cells Compared to Other Neoplasias - Implications for the Biology and Therapeutic Targeting of Myeloma

In the last 2 decades, the improved clinical outcomes for multiple myeloma (MM) patients have been driven predominantly by therapeutics which exhibit limited activity outside plasma cell (PC) dyscrasias; do not target specific oncogenic mutations in MM cells, but rather pathways which are critical for PCs and dispensable for normal or malignant cells of most other lineages. We reasoned that identification of genes that are more potently / recurrently essential for MM cells, but less so for other neoplasias, would allow us to "re-identify" targets of currently used "PC-selective" anti-MM therapies. We also reasoned that systematic identification of MM-preferential dependencies could also uncover additional, previously underappreciated, genes which can serve as targets for potential future therapies and hopefully contribute to additional improvements in the therapeutic outcome for MM. To this end, we performed genome-scale CRISPR gene-editing studies to systematically characterize the molecular vulnerabilities of 20 MM cell lines and define which of these genes are more pronounced and/or recurrent dependencies for MM vs. cell lines (n=679) from other blood cancers and solid tumors. We identified 90+ genes whose function was significantly more essential for MM lines than other neoplasias. These MM-preferential dependencies included a large collection of transcription factors (e.g. IRF4, PRDM1, MAF, NFKB1, RELB, IKZF3, IKZF1, TCF3, CCND2, CBFB, MEF2C); transcriptional cofactors (e.g. POU2AF1); epigenetic regulators (e.g. EP300, DOT1L, HDAC1,ARID1A, CARM1); kinases such as IKBKB and CHUK/IKKa (both upstream of NF-κB), PIM2, IGF1R, SIK3,STK11; genes related to endoplasmic reticulum (ER) or Golgi function (e.g. HERPUD1, SYVN1,UBE2J1, SEC23B); as well as BCL2 and SMAD7. Results for several of these genes were further supported by in vitro studies with individual sgRNAs for CRISPR-based gene editing or activation of the respective genes; "addback" experiments with CRISPR-resistant cDNAs; shRNA studies in MM lines; use of small molecule inhibitors (e.g. against PIM kinases, CBFB, CARM1); and a focused in vivo CRISPR screen with MM.1S cells implanted in mice with BM-like scaffolds harboring a "humanized" stromal compartment: this latter in vivo study examined 46 MM-preferential dependencies which are also essential for MM.1S cells in vitro and observed that 41 of these genes were also essential for MM.1Scells in the "humanized" BM-like in vivo system. Some MM-preferential dependencies are essential for subsets of leukemia or lymphoma lines, but most have more pronounced/recurrent essentiality in MM vs. other blood cancers. In terms of overexpression (in high- vs. standard-risk MM; MM vs. normal PCs; or MM vs other cancers); frequency of mutations, DNA copy number gain or proximity to superenhancers, most of the MM-preferential dependencies do not exhibit such alterations or are not ranked in the top-100 genes in terms of the magnitude or frequency of these alterations. Notably, among the MM-preferential dependencies identified in our study, the majority are universally expressed in MM patient samples, while >80% and >75% of these genes have detectable transcript levels (RPKM>1) in CD138+ cells from at least 50% or 80%, respectively, of newly-diagnosed MM patients (MMRF CoMMpass study), suggesting broad expression of these dependencies across MM patients, including individuals with high-risk disease. It was reassuring to observe that MM-preferential dependencies identified in our study include prominent known targets for therapeutics with relatively MM-selective clinical activity (e.g. thalidomide derivatives [IKZF1/IKZF3], proteasome inhibitors [NF-kappaB genes and ER function] or panobinostat [HDAC1]). The identification of these known genes as preferential MM dependencies provides a mechanistic explanation for the relatively selective clinical activity of the respective therapies in MM/PC dyscrasias and also underscores the promising therapeutic implications of the large number of additional and previously underappreciated / understudied MM-preferential dependencies identified in our CRISPR-based functional studies. Disclosures Boise: Genentech Inc.: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Research Funding. Gray:Gatekeeper, Syros, Petra, C4, B2S and Soltego.: Equity Ownership; Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Her2llc, Deerfield and Sanofi.: Equity Ownership, Research Funding. Tsherniak:Tango Therapeutics: Consultancy. Mitsiades:Takeda: Other: employment of a relative ; Ionis Pharmaceuticals: Honoraria; Fate Therapeutics: Honoraria; Arch Oncology: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding; Abbvie: Research Funding; TEVA: Research Funding; EMD Serono: Research Funding; Janssen/Johnson & Johnson: Research Funding.

Conventional Chemotherapy Treatment Induces Unique Patterns of Clonal Evolution in a North American Adult T Cell Leukemia/Lymphoma Cohort

Adult T cell leukemia / lymphoma is a rare neoplasm with dismal cure rates and poor response to chemotherapy. The genetic basis of refractoriness against conventional agents is not known. We follow one of the largest cohorts of ATLL in the US and recently showed that North American ATLL (NA-ATLL) has a distinct mutational profile from Japanese ATLL (J-ATLL) with a higher incidence of epigenetic mutations (Shah et al, Blood, 2018; 132(14):1507-1518). Here, we analyze the mutational landscape of ATLL clones at diagnosis and at relapse in a pilot study of 7 NA-ATLL patients using deep targeted sequencing of 236 recurrently mutated genes. Our goal was to examine clonal evolution patterns that may result as a consequence of the selective pressure of chemotherapy by determining changes in mutational profiles as well as clonal abundance on patients with aggressive ATLL [acute and lymphomatous] (Table 1). All patients were treated with EPOCH (Etoposide, Prednisone, Vincristine, Cyclophosphamide and Doxorubicin) chemotherapy after initial diagnosis except one patient who received interferon therapy. Distinct patterns of clonal evolution were observed in patients with primary refractory disease when compared to those who relapsed after an initial remission. Group 1 has 3 cases (Pts 1 to 3), all of which experienced early relapse and showed mutation patterns suggestive of clonal replacement by a new emerging clone at relapse. Group 2 has 3 patients (Pts 4-6) that are characterized by worsening of primary refractory disease. Mutation patterns in the before-and-after sample pairs suggest linear progression from a previously existing clone during therapy. The clonal evolution status was not resolved in our third group (Pt#7) indicating that mutations outside the 236 genes evaluated might have been present. Frequent epigenetic mutations were found in both diagnostic and refractory/relapsed samples. Surprisingly, 4 epigenetic mutations (KMT2D, EP300, SPEN, SETBP1) identified in 4 cases were confirmed or suspected of germline origin. This suggests that ancestral genomic differences between the Caribbean and Japanese populations could contribute to the mutational and clinical disparities between NA- and J-ATLL. Multiple mutations with similar variant allele frequencies (VAF) at the time of relapse were identified, suggesting simultaneous acquisition of several mutations during clonal evolution. Clonal replacement by an emerging clone (Group 1) such as GATA3 (Pt#3) or KMT2D (Pt#1) (either germline or founding mutation) was observed in patients who achieved an initial response. On the other hand, in primary refractory disease (Group 2), relapse was driven by dominant TBL1XR1 mutations in 2 out of the 3 cases either alone (Pt#6) or in combination with mutated SMARCB1 (Pt#5). The protein encoded by TBL1XR1 is a negative regulator of the nuclear receptor corepressor (NCoR) /histone deacetylase 3 (HDAC3) complex. Therefore, TBL1XR1 inactivation is expected to result in hyper-activity of NCoR/HDAC3, an epigenetic abnormality targetable by HDAC inhibitors. SMARCB1 is also an epigenetic regulator associated with several malignancies and has been targeted with alisertib. A subclonal expansion of EP300 (a histone acetyltransferase) appears to drive relapse in the other case of this group (Pt#4). Therefore, our primary refractory cases show that epigenetic mutations seem to be of utmost importance not only at diagnosis as we have previously shown, but as the driving force behind chemotherapy refractoriness and subsequent relapse. Conclusion: To our knowledge, this is the first mutation-based clonal evolution study aimed to examine dynamic changes induced by chemotherapy among NA-ATLL patients. Early relapse appears to be driven by the emergence of new mutant clones. On the other hand, primary refractory disease appears to have epigenetic drivers that convey chemotherapy refractoriness and evolve from linear progression of a previously existing clone. Serial mutational testing can provide critical information on clonal architecture and identify actionable molecular vulnerabilities in a cohort without effective therapeutic options and a dismal prognosis. Disclosures Sica: Physician's Education Resource (PER): Honoraria. Shah:Physicians' Education Resource: Honoraria. Steidl:Aileron Therapeutics: Consultancy, Research Funding; Stelexis Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Scientific Co-Founder; Pieries Pharmaceuticals: Consultancy; BayerHealthcare: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; GlaxoSmithKline: Research Funding; Celgene: Consultancy. Verma:Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria; Celgene: Honoraria; BMS: Research Funding; Janssen: Research Funding.

COMP-04. MODELING PRECISION ONCOLOGY FOR GLIOBLASTOMA THROUGH INTEGRATION OF DESCRIPTIVE, FUNCTIONAL, AND NETWORK-BASED GENOMICS

Abstract Precision oncology is largely based on the notion that identification and targeting of oncogenic drivers will lead to improved clinical outcomes. However, the promise of precision oncology awaits to be fulfilled for many cancers, including Glioblastoma (GBM), where identification of oncogenic drivers has yet to improve survival rates. Here, we have attempted to systematically identify GBM vulnerabilities by performing genome-wide CRISRP-Cas9 lethality screens in patient-derived GBM stem-like cells (GSCs). In validation studies, we comprehensively retested GSC-specific hits in multiple GSC isolates, which were also genomically profiled (e.g. RNA-seq, exome-seq, CNV), and further integrated these data with CRISPR-Cas9 lethality screens from over 500 human cell lines from the Broad Institute’s CRISPR Avana dataset. As a result, we have begun making GBM dependency predictions and functional associations for top scoring hits, including: tumor developmental subtype; loss of functional redundancy with other genes/proteins; cancer-specific subnetworks of genes involved in mitochondrial protein turnover and membrane trafficking; and genes of unknown function essential for subset of GBMs. A few examples of these categories include the following scenarios. We find ADAR (Adenosine Deaminase RNA Specific) gene dependency is associated with the mesenchymal GBM subtype. The EFR3Agene, which has roles in maintaining active pools of phosphatidylinositol 4-kinase, appears required when the expression of its paralog EFR3Bis low or absent in tumor cells. The F-box protein-encoding gene FBXO42appears non-essential to most human cells lines and neural stem cells, but when knocked out in sensitive GSCs causes mitotic arrest, mitotic catastrophe, and cell death. While still a work in progress, we hope to use these results as a foundation for exploring and illuminating patient-specific molecular vulnerabilities for brain tumors. The results also underscore the need for integration of functional genetic approaches, where gene activities are inhibited, into precision oncology paradigms.

Genomic Profiling of Biliary Tract Cancer Cell Lines Reveals Molecular Subtypes and Actionable Drug Targets.

SummaryBiliary tract cancers (BTCs) currently have no approved targeted therapies. Although genomic profiling of primary BTCs has identified multiple potential drug targets, accurate models are needed for their evaluation. Genomic profiling of 22 BTC cell lines revealed they harbor similar mutational signatures, recurrently mutated genes, and genomic alterations to primary tumors. Transcriptomic profiling identified two major subtypes, enriched for epithelial and mesenchymal genes, which were also evident in patient-derived organoids and primary tumors. Interrogating these models revealed multiple mechanisms of MAPK signaling activation in BTC, including co-occurrence of low-activity BRAF and MEK mutations with receptor tyrosine kinase overexpression. Finally, BTC cell lines with altered ERBB2 or FGFRs were exquisitely sensitive to specific targeted agents, whereas surprisingly, IDH1-mutant lines did not respond to IDH1 inhibitors in vitro. These findings establish BTC cell lines as robust models of primary disease, reveal specific molecular disease subsets, and highlight specific molecular vulnerabilities in these cancers.

CRISPR studies identify genes preferentially essential for myeloma cells vs. other neoplasias: implications for future therapies selective against MM

Improved clinical outcomes for MM in the last 2 decades have been driven by therapeutics which have limited activity in non-MM neoplasias; do not target specific oncogenic mutations in MM cells, but rather pathways which are critical for plasma cells (PCs), yet dispensable for normal or malignant cells of most other lineages. We performed genome-scale CRISPR gene-editing studies to systematically characterize the molecular vulnerabilities of 19 MM cell lines and define which genes are more pronounced and/or recurrent dependencies for MM vs.

Molecular characteristics and therapeutic vulnerabilities across paediatric solid tumours.

The spectrum of tumours arising in childhood is fundamentally different from that seen in adults, and they are known to be divergent from adult malignancies in terms of cellular origins, epidemiology, genetic complexity, driver mutations and underlying mutational processes. Despite the immense knowledge generated through sequencing efforts and functional characterization of identified (epi-)genetic alterations over the past decade, the clinical implications of this knowledge have so far been limited. Novel preclinical platforms such as the European Innovative Therapies for Children with Cancer-Paediatric Preclinical Proof-of-Concept Platform and the US-based Pediatric Preclinical Testing Consortium are being developed to try to change this by aiming to recapitulate the extensive heterogeneity of paediatric tumours and thereby, hopefully, improve the ability to predict clinical benefit. Numerous studies have also been established worldwide to provide patients with access to real-time molecular profiling and the possibility of more precise mechanism-of-action-based treatments. In addition to tumour-intrinsic findings and mechanisms, ongoing studies are investigating features such as the immune microenvironment of paediatric tumours in comparison with adult cancers - currently of very timely clinical relevance. However, there is an ongoing need for rigorous preclinical biomarker and target validation to feed into the next generation of molecularly stratified clinical trials. This Review aims to provide a comprehensive state-of-the-art overview of the molecular landscape of paediatric solid tumours, including their underlying genomic alterations and interactions with the microenvironment, complemented with our current understanding of potential therapeutic vulnerabilities and how these can be preclinically tested using more accurate predictive methods. Finally, we provide an outlook on the challenges and opportunities associated with translating this overwhelming scientific progress into real clinical benefit.

Abstract 277: Inhibition of protein arginine methylation alters RNA metabolism and DNA damage response providing a new therapeutic strategy in pancreatic ductal adenocarcinoma

Abstract With limited therapeutic options, poor overall 5-year survival rates, and increasing incidence, pancreas cancer is estimated to become the second leading cause of cancer deaths by 2030. Recognizing the need for transformative advances in pancreas cancer management, we developed an in vivo target discovery platform to uncover molecular vulnerabilities in patient-derived pancreatic ductal adenocarcinoma (PDAC) xenografts to identify and rapidly translate novel therapeutic concepts to the clinic. We identified protein arginine methyltransferase 1 (PRMT1) as a dependency in PDAC required for disease maintenance and progression. Extensive genetic and pharmacological studies support PRMT1 as a novel vulnerability, which prompted our design and synthesis of proprietary series of potent, selective PRMT Type I inhibitors (PRMTi) with compelling in vivo activity. While advancing the project in drug discovery, we deployed a comprehensive approach to elucidate the mechanism of action of PRMTi. We characterized the PRMT1 interactome via PRMT1 immunoprecipitation followed by LC/MS and observed that PRMT1 binding partners were significantly enriched in RNA-binding and -processing genes. In addition, because methylation of arginine residues is a common post-translational modification regulating protein function, we identified substrates differentially methylated upon PRMT inhibition. Integrating these results with the PRMT1 interactome confirmed a strong correlation between PRMT1 substrates and complexes that are physically associated and linked to RNA metabolism. Transcriptome assays demonstrated that PRMT inhibition globally impaired RNA metabolism, including but not limited to RNA splicing, transcription termination, and R-loop formation. In addition, PRMTi caused a profound down-regulation of multiple pathways involved in the DNA damage response (DDR) promoting genomic instability. Taken together, these data support PRMT1 as a compelling target in an area of high unmet medical need and inform a mechanism-based translational strategy for future clinical development. Citation Format: Virginia Giuliani, Alessandro Carugo, Meredith Miller, Lionel Sanz, Chiu-Yi Liu, Christopher A Bristow, Erika Suzuki, Caleb A Class, Stella R. Hartono, Guang Gao, Ningping Feng, Jason P Gay, Bhavatarini Vangamudi, Joseph R Marszalek, Jeffrey Kovacs, Maria Emilia Di Francesco, Frederic Chedin, Philip Jones, Giulio Draetta, Timothy Heffernan. Inhibition of protein arginine methylation alters RNA metabolism and DNA damage response providing a new therapeutic strategy in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 277.

PDK1 Mediates NOTCH1-Mutated Head and Neck Squamous Carcinoma Vulnerability to Therapeutic PI3K/mTOR Inhibition.

Head and neck squamous cell carcinoma (HNSCC) is driven largely by the loss of tumor suppressor genes, including NOTCH1, but lacks a biomarker-driven targeted therapy. Although the PI3K/mTOR pathway is frequently altered in HNSCC, the disease has modest clinical response rates to PI3K/mTOR inhibitors and lacks validated biomarkers of response. We tested the hypothesis that an unbiased pharmacogenomics approach to PI3K/mTOR pathway inhibitors would identify novel, clinically relevant molecular vulnerabilities in HNSCC with loss of tumor suppressor function.Experimental Design: We assessed the degree to which responses to PI3K/mTOR inhibitors are associated with gene mutations in 59 HNSCC cell lines. Apoptosis in drug-sensitive cell lines was confirmed in vitro and in vivo. NOTCH1 pathway components and PDK1 were manipulated with drugs, gene editing, knockdown, and overexpression. PI3K/mTOR inhibition caused apoptosis and decreased colony numbers in HNSCC cell lines harboring NOTCH1 loss-of-function mutations (NOTCH1 MUT) and reduced tumor size in subcutaneous and orthotopic xenograft models. In all cell lines, NOTCH1 MUT was strongly associated with sensitivity to six PI3K/mTOR inhibitors. NOTCH1 inhibition or knockout increased NOTCH1 WT HNSCC sensitivity to PI3K/mTOR inhibition. PDK1 levels dropped following PI3K/mTOR inhibition in NOTCH1 MUT but not NOTCH1 WT HNSCC, and PDK1 overexpression rescued apoptosis in NOTCH1 MUT cells. PDK1 and AKT inhibitors together caused apoptosis in NOTCH1 WT HNSCC but had little effect as single agents. Our findings suggest that NOTCH1 MUT predicts response to PI3K/mTOR inhibitors, which may lead to the first biomarker-driven targeted therapy for HNSCC, and that targeting PDK1 sensitizes NOTCH1 WT HNSCC to PI3K/mTOR pathway inhibitors.

Integrated omics-based pathway analyses uncover CYP epoxygenase-associated networks as theranostic targets for metastatic triple negative breast cancer

BackgroundCurrent prognostic tools and targeted therapeutic approaches have limited value for metastatic triple negative breast cancer (TNBC). Building upon current knowledge, we hypothesized that epoxyeicosatrienoic acids (EETs) and related CYP450 epoxygenases may have differential roles in breast cancer signaling, and better understanding of which may uncover potential directions for molecular stratification and personalized therapy for TNBC patients.MethodsWe analyzed the oxylipin metabolome of paired tumors and adjacent normal mammary tissues from patients with pathologically confirmed breast cancer (N = 62). We used multivariate statistical analysis to identify important metabolite contributors and to determine the predictive power of tumor tissue metabolite clustering. In vitro functional assays using a panel of breast cancer cell lines were carried out to further confirm the crucial roles of endogenous and exogenous EETs in the metastasis transformation of TNBC cells. Deregulation of associated downstream signaling networks associated with EETs/CYPs was established using transcriptomics datasets from The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC). Comparative TNBC proteomics using the same tissue specimens subjected to oxylipin metabolomics analysis was used as validation set.ResultsMetabolite-by-metabolite comparison, tumor immunoreactivity, and gene expression analyses showed that CYP epoxygenases and arachidonic acid-epoxygenation products, EET metabolites, are strongly associated with TNBC metastasis. Notably, all the 4 EET isomers (5,6-, 8,9-, 11,12-, and 14,15-EET) was observed to profoundly drive the metastasis transformation of mesenchymal-like TNBC cells among the TNBC (basal- and mesenchymal-like), HER2-overexpressing and luminal breast cancer cell lines examined. Our pathway analysis revealed that, in hormone-positive breast cancer subtype, CYP epoxygenase overexpression is more related to immune cell-associated signaling, while EET-mediated Myc, Ras, MAPK, EGFR, HIF-1α, and NOD1/2 signaling are the molecular vulnerabilities of metastatic CYP epoxygenase-overexpressing TNBC tumors.ConclusionsThis study suggests that categorizing breast tumors according to their EET metabolite ratio classifiers and CYP epoxygenase profiles may be useful for prognostic and therapeutic assessment. Modulation of CYP epoxygenase and EET-mediated signaling networks may offer an effective approach for personalized treatment of breast cancer, and may be an effective intervention option for metastatic TNBC patients.

Pregnancy drugs, fetal germline epigenome, and risks for next-generation pathology: A call to action.

Drugs taken during pregnancy can affect three generations at once: the gestating woman (F0), her exposed fetus (F1), and the fetal germ cells that confer heritable information for the grandchildren (F2). Unfortunately, despite growing evidence for connections between F0 drug exposures and F2 pathology, current approaches to risk assessment overlook this important dimension of risk. In this commentary, we argue that the unique molecular vulnerabilities of the fetal germline, particularly with regard to global epigenomic reprogramming, combined with empirical evidence for F2 effects of F1 in utero drug and other exposures, should change the way we consider potential long-term consequences of pregnancy drugs and alter toxicology's standard somatic paradigm. Specifically, we (1) suggest that pregnancy drugs common in the postwar decades should be investigated as potential contributors to the "missing heritability" of many pathologies now surging in prevalence; (2) call for inclusion of fetal germline risks in pregnancy drug safety assessment; and (3) highlight the need for intensified research to ascertain generational impacts of diethylstilbestrol, a vanguard question of human germline toxicity. Only by fully addressing this important dimension of transplacental exposure can we responsibly evaluate safety of drug exposures during pregnancy and convey the full scope of risks, while also retrospectively comprehending the generational legacy of recent history's unprecedented glut of evolutionarily novel intrauterine exposures. Environ. Mol. Mutagen. 60:445-454, 2019. © 2019 Wiley Periodicals, Inc.

Developmental origins and oncogenic pathways in malignant brain tumors.

Brain tumors such as adult glioblastomas and pediatric high-grade gliomas or medulloblastomas are among the leading causes of cancer-related deaths, exhibiting poor prognoses with little improvement in outcomes in the past several decades. These tumors are heterogeneous and can be initiated from various neural cell types, contributing to therapy resistance. How such heterogeneity arises is linked to the tumor cell of origin and their genetic alterations. Brain tumorigenesis and progression recapitulate key features associated with normal neurogenesis; however, the underlying mechanisms are quite dysregulated as tumor cells grow and divide in an uncontrolled manner. Recent comprehensive genomic, transcriptomic, and epigenomic studies at single-cell resolution have shed new light onto diverse tumor-driving events, cellular heterogeneity, and cells of origin in different brain tumors. Primary and secondary glioblastomas develop through different genetic alterations and pathways, such as EGFR amplification and IDH1/2 or TP53 mutation, respectively. Mutations such as histone H3K27M impacting epigenetic modifications define a distinct group of pediatric high-grade gliomas such as diffuse intrinsic pontine glioma. The identification of distinct genetic, epigenomic profiles and cellular heterogeneity has led to new classifications of adult and pediatric brain tumor subtypes, affording insights into molecular and lineage-specific vulnerabilities for treatment stratification. This review discusses our current understanding of tumor cells of origin, heterogeneity, recurring genetic and epigenetic alterations, oncogenic drivers and signaling pathways for adult glioblastomas, pediatric high-grade gliomas, and medulloblastomas, the genetically heterogeneous groups of malignant brain tumors. This article is categorized under: Gene Expression and Transcriptional Hierarchies > Gene Networks and Genomics Adult Stem Cells, Tissue Renewal, and Regeneration > Stem Cell Differentiation and Reversion Signaling Pathways > Cell Fate Signaling.

Identifying novel molecular vulnerabilities to PTK2/FAK inhibition in G α q‐driven uveal melanoma using a kinome‐wide CRISPR/Cas9 screen

Uveal melanoma (UM) is the most common primary cancer of the eye in adults. It is diagnosed in about 2,500 adults in the United States every year. Approximately 50% of UM patients develop liver metastasis within 5–10 years after diagnosis, independently of successful treatment of the primary lesions. Activating mutations in GNAQ and GNA11 (encoding GTPase deficient/constitutively active Gαq proteins) were identified in ~90% of UM; where they act as driver oncogenes. MEK inhibition with selumetinib or trametinib has nearly no impact on the overall survival of UM patients. Therefore, there is an urgent need to understand mechanistically how Gαq promotes UM progression in order to develop new therapeutics. In a recent study, we have performed an integrated bioinformatics analysis of gene interaction networks and identified PTK2, encoding the tyrosine kinase: Focal Adhesion Kinase (FAK) as a candidate gene highly predicted to contribute to Gαq‐driven UM progression. Using a variety of biochemical and cell‐based assays, we established that FAK is indeed a pivotal signaling node downstream of oncogenic Gαq that mediates UM proliferation through the activity of the growth‐promoting transcription factor Yap. Notably, our studies with UM xenograft mouse models demonstrated that FAK blockade by VS‐4718, a new generation orally‐bioavailable FAK inhibitor, dramatically reduced tumor volume. The major objective of the current study is to further delineate the Gαq‐FAK signaling axis with interest in identifying novel synthetic lethal interactions to FAK inhibition in UM. To this end, we conducted a kinome‐wide CRISPR/Cas9 screen using VS‐4718 in UM cell line OMM1.5 to pinpoint molecular vulnerabilities in the Gαq‐FAK onco‐circuitry. Positive hits from our gene‐drug screen will be presented, which shed light on the Gαq‐FAK network and may serve as potential co‐targets to FAK inhibition in UM clinical therapy.Support or Funding InformationCancer Biology, Informatics & Omics Training ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Temozolomide Induced Hypermutation in Glioma: Evolutionary Mechanisms and Therapeutic Opportunities.

Glioma are the most common type of malignant brain tumor, with glioblastoma (GBM) representing the most common and most lethal type of glioma. Surgical resection followed by radiotherapy and chemotherapy using the alkylating agent Temozolomide (TMZ) remain the mainstay of treatment for glioma. While this multimodal regimen is sufficient to temporarily eliminate the bulk of the tumor mass, recurrence is inevitable and often poses major challenges for clinical management due to treatment resistance and failure to respond to targeted therapies. Improved tumor profiling capacity has enabled characterization of the genomic landscape of gliomas with the overarching goal to identify clinically relevant subtypes and inform treatment decisions. Increased tumor mutational load has been shown to correlate with higher levels of neoantigens and is indicative of the potential to induce a durable response to immunotherapy. Following treatment with TMZ, a subset of glioma has been identified to recur with increased tumor mutational load. These hypermutant recurrent glioma represent a subtype of recurrence with unique molecular vulnerabilities. In this review, we will elaborate on the current knowledge regarding the evolution of hypermutation in gliomas and the potential therapeutic opportunities that arise with TMZ-induced hypermutation in gliomas.

PARP14 Is a Novel Therapeutic Target in STAT6 mutant Follicular Lymphoma

The highly variable clinical course of follicular lymphoma (FL) is determined by the molecular heterogeneity of the tumor cells and complex interactions with the microenvironment. The underlying molecular mechanisms and therapeutic vulnerabilities are not well understood.IL-4 producing follicular helper T cells (TFH) have been identified as a key component of the malignant B-cell niche. IL-4 activates paracrine signaling via STAT6. In a cohort of 258 patients with advanced stage FL, we identified STAT6 mutations in 13% of diagnostic biopsies (n=33). All mutations were clustered within the DNA binding domain, mostly at D419, and included a polymorphic variant (rs11172102). Gene set enrichment analysis (GSEA) revealed that STAT6 mutant cases were significantly enriched for two distinct IL-4 gene expression signatures. Gene expression data and immunohistochemistry of primary FL samples showed significant up-regulation of IL-4/STAT6 target genes in STAT6 mutant cases, including FCER2, which encodes for CD23.We stably expressed wild type STAT6 or mutant STAT6 (D419G, N421K, and D519V) in two B-cell lymphoma lines (OCI-Ly1, OCI-Ly8), both harboring the FL hallmark translocation t(14;18). Upon IL-4 stimulation, cells expressing mutant STAT6 had significantly increased FCER2 transcript levels. Similarly, IL-4 induced expression of membrane-bound as well as soluble CD23 was significantly increased in STAT6 mutant cells. Cells expressing mutant STAT6 showed significantly increased nuclear accumulation of pSTAT6 following IL-4 stimulation. Of note, we did not observe any effect of STAT6 mutations in the absence of IL-4.RNA sequencing of IL-4-stimulated lymphoma cell lines (STAT6 mutant versus wild type) identified PARP14 -a known transcriptional co-activator of STAT6- among the top differentially expressed genes. Bioinformatics and functional experiments demonstrated that PARP14 per se is a novel STAT6 target gene. Furthermore, reporter assays showed increased transactivation activity of mutant STAT6 at the PARP14 promotor, suggesting a regulatory feed-forward loop. Pharmacological inhibition of PARP and knock-down of PARP14 completely abrogated the mutant STAT6 gain-of-function phenotype.In summary, our results suggest that PARP14 is a novel target in STAT6 mutant FL. Our data also imply that the biological and clinical impact of STAT6 mutations will heavily depend on the (targetable) upstream activation of the IL-4 signaling cascade, including the abundance of IL-4 / TFH cells in the microenvironment of FL. DisclosuresRichter:HTG Molecular Diagnostics, Inc.: Research Funding. Klapper:Amgen: Honoraria, Research Funding; F.Hoffman-La Roche: Honoraria, Research Funding; HTG Molecular Diagnostics, Inc.: Research Funding; Takeda: Honoraria, Research Funding; Regeneron: Honoraria, Research Funding. Hiddemann:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; F. Hoffman-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Research Funding. Weigert:Novartis: Research Funding; Roche: Research Funding.

Myeloma Patient-Derived MCL1 Point Mutations Can Influence MCL1-Inhibitor Function

Multiple myeloma is a malignancy of long-lived plasma cells of the bone marrow that is rarely curable. Thus, despite recent advances in the development of new therapies, additional approaches are required. We investigated potential molecular vulnerabilities in the BCL2 family. Using the MMRF CoMMpass (NCT0145429) study (IA13), we determined the frequency of nonsynonymous coding mutations in the BCL2 family. Analysis of baseline samples from 982 patients revealed that mutations in the BCL2 family are relatively rare events. No mutations were observed in the 3 pro-apoptotic effector genes BAX, BAK1 and BOK. Similarly, in the BH3-only genes, mutations were rare with no mutations in BCL2L11 (BIM), BAD, BID, HRK and BMF and only single mutations in BBC3 (PUMA) and PMAIP1 (NOXA). In the anti-apoptotic BCL2 genes, mutations were also rare with no mutations in BCL2, BCL2L1 (BCLX) and BCL2L10 (BCLB). A single sample had a mutation in BCL2A1 (A1) while 2 samples had mutations in BCL2L2 (BCLW). Interestingly MCL1 was mutated in 10 baseline samples (1.02%) and the frequency of the mutations in these samples was high (median 0.391, range 0.066-0.531). Therefore, we further investigated the functional consequences of the MCL1 mutant alleles.Of the 10 mutations detected, 1 was in the N-terminal region (G32R) and 4 were in the PEST domain in the N-terminal half of MCL1 that is associated with regulating protein stability (V140I, P142S, E149Q and E173K). An additional mutation was found in an uncharacterized region between the PEST and BH1 domains (L186F). We focused on the 4 mutations that lie within or near the functional BH1 (V249L and L267V) and within the BH3 (N223S, and R214Q) domains. Wild type (WT) MCL1 and the four mutant MCL1 constructs were introduced into a murine B-ALL cell line that has endogenous murine MCL1 flanked with LoxP sites and confirmed expression by western blot analysis.Human MCL1 can replace murine MCL1 in this cell model, therefore we are determining if the myeloma-derived mutants of MCL1 can complement loss of mouse Mcl1 and will report on these findings. However, we began to functionally characterize these mutations by taking advantage of an anomaly in the development of inhibitors of human MCL1. To date, no inhibitor developed against human MCL1 is as effective against murine Mcl1. Thus, differences in the activity of these inhibitors reflects changes in dependence on the introduced human MCL1. We treated cells with increasing concentrations of the MCL1 inhibitor S63845 and measured cell death (Annexin V/PI) at 24 hours. As expected, cells where the empty vector was introduced were highly resistant to S63845-induced cell death (less than 20% at 1000 nM) while cells expressing the human WT MCL1 were significantly more susceptible to the MCL1 inhibitor across a concentration range of 100 to 1000 nM. The V249L, N223S and R214Q mutations mimicked the sensitivity of the WT MCL1 suggesting they did not alter MCL1 function in the cells. In contrast the L267V mutation resulted in a dose curve that was more similar to the empty vector control suggesting this mutation either resulted in loss of function or in an MCL1 molecule that could not be inhibited by the drug. Since drug binding can stabilize MCL1 by competing for E3 ligase binding, we determined the effect of S63845 on MCL1 protein levels. We found that with all the mutants, S63845 dramatically increased human MCL1 protein expression ruling out lack of drug binding. We next performed MCL1 co-immunoprecipitation assays, and found that BIM release correlated with S63845 sensitivity. In addition to not releasing BIM, the L267V did not effectively release NOXA and BAK after S63845 treatment. Taken together, the L267V mutation does not prevent the binding of S63845 to free MCL1, rather it blocks the ability of drug to displace pro-apoptotic proteins required to induce cell death.We then tested another MCL-1 inhibitor in clinical trials, AZD-5991. The L267V mutation was completely resistant to AZD-5991-induced apoptosis despite evidence of drug binding. Interestingly the other 3 mutations also resulted in diminished killing activity when compared to cells expressing the WT MCL1, suggesting that these mutations may also influence drug function. Together these data suggest that myeloma-derived mutations in MCL1 may not necessarily influence MCL1 function, however they could alter responses to an emerging class of inhibitors where 3 drugs are currently in clinical trials. DisclosuresSecrist:AstraZeneca: Employment. Cidado:AstraZeneca: Employment, Equity Ownership. Tron:AstraZeneca: Employment. Lonial:Amgen: Research Funding. Boise:Abbvie: Consultancy; AstraZeneca: Honoraria.

Abstract 2408: LKB1 and KEAP1/NRF2 pathways cooperatively promote glutamine dependence and vulnerability to glutaminase inhibitors in KRAS-mutant lung adenocarcinoma

Abstract KRAS is the most commonly mutated oncogenic driver in non-small cell lung cancer (NSCLC) and other solid tumors. Recently we conducted an integrative analysis and found three major subgroups of KRAS-mutated cancer defined by co-occurring genomic events with distinct biology, molecular vulnerabilities, and therapeutic sensitivities. One of these genes, the serine/threonine kinase STK11 (LKB1), represents the second most commonly altered tumor suppressor in NSCLC and there are currently no treatment strategies tailored for LKB1-deficient NSCLC. KRAS-mutant/LKB1-deficient (KL) tumors are characterized by high co-occurrence of KEAP1 mutational inactivation. Inactivation of KEAP1 protects cells against REDOX stress via upregulation of NRF2 target genes, in part by production of glutathione. We evaluated the effects of blocking glutamine metabolism using an isogenic series of NSCLC cell lines harboring mutations in STK11 and KEAP1. Through sequential silencing or overexpression of LKB1, KEAP1, or NRF2 we demonstrated that glutaminase inhibitors (GLSi) can block cell proliferation while increasing energetic and REDOX stress specifically in LKB1 deficient cells with hyperactivation of the KEAP1/NRF2 pathway driven by KEAP1 mutations (KLK subtype). In KLK models, overexpression of LKB1 or KEAP1 partially reduced GLSi sensitivity, while siRNA-mediated down-regulation of NRF2 showed a similar effect. Furthermore, the combination of LKB1 add back coupled with down-regulation of NRF2 conferred even greater resistance to GLSi. To confirm the LKB1/KEAP1-driven response to GLSi, we performed in vivo experiments examining the response of subcutaneous xenografts of an A549 isogenic series; A549 (KLK), A549 LKB1 add back (KK) or A549 KEAP1 add back (KL); to GLS inhibition. These experiments demonstrated that GLSi impaired tumor growth in A549 (KLK) tumors, exhibiting significant statistical differences compared with the vehicle group from 18 days of treatment. Conversely, GLS inhibition did not significantly affect the growth of A549/LKB1 (KK) or A549/KEAP1 (KL) tumors. Collectively, our data indicate that in KLK tumors both pathways, LKB1 and KEAP1/NRF2, cooperatively drive a glutamine-addicted metabolic program, making KLK tumors selectively vulnerable to GLSi treatment. These findings have immediate clinical implications and support the future clinical testing of GLS inhibitors in KLK NSCLC. Citation Format: Ana Galan-Cobo, Piyada Sitthideatphaiboon, Xiao Qu, Jeffrey J. Kovacs, Alissa Poteete, Pan Tong, Sungnam Cho, Varsha V. Gandhi, Ferdinandos Skoulidis, Jing Wang, Timothy P. Heffernan, John V. Heymach. LKB1 and KEAP1/NRF2 pathways cooperatively promote glutamine dependence and vulnerability to glutaminase inhibitors in KRAS-mutant lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2408.

DNA damage pathways and B-cell lymphomagenesis.

Recent lymphoma genome sequencing projects have shed light on the genomic landscape of indolent and aggressive lymphomas, as well as some of the molecular mechanisms underlying recurrent mutations and translocations in these entities. Here, we review these recent genomic discoveries, focusing on acquired DNA repair defects in lymphoma. In addition, we highlight recently identified actionable molecular vulnerabilities associated with recurrent mutations in chronic lymphocytic leukemia (CLL), which serves as a model entity. The results of several large lymphoma genome sequencing projects have recently been reported, including CLL, T-PLL and DLBCL. We align these discoveries with proposed mechanisms of mutation acquisition in B-cell lymphomas. Moreover, novel autochthonous mouse models of CLL have recently been generated and we discuss how these models serve as preclinical tools to drive the development of novel targeted therapeutic interventions. Lastly, we highlight the results of early clinical data on novel compounds targeting defects in the DNA damage response of CLL with a particular focus on deleterious ATM mutations. Defects in DNA repair pathways are selected events in cancer, including lymphomas. Specifically, ATM deficiency is associated with PARP1- and DNA-PKcs inhibitor sensitivity in vitro and in vivo.

31IN - AKT inhibitors and other new kids on the block

AKT mutations are uncommon in cancers with the AACR GENIE Project cohort of 27,290 patients demonstrating AKT1 alterations in 1.9%, AKT2 alterations in 1.6%, and AKT3 alterations in 1.3% of cases (Courtesy AACR GENIE). The development of first generation AKT inhibitors has been limited by poor therapeutic index and the unselected patient populations being treated with these compounds. AKT1 E17K is the predominant functionally activating mutation most frequently found in breast cancer, gynecological cancer, bladder, prostate and lung cancer. A recently published basket study of the pan-AKT kinase inhibitor AZD5363, demonstrated objective responses in tumors harboring this oncogenic mutation (Hyman et al. J Clin Oncol 2106). The first-in-human phase I study of ipatasertib (GDC-0068) was also reported in solid tumor patients with or without AKT pathway aberrations, and demonstrated stabilization in about 30% of patients whose disease were progressing on prior therapies (Saura et al. Cancer Dis 2017). Paired tumor biopsies demonstrated inhibition of multiple on-target effectors but upregulation of pERK and pHER3 were observed suggesting compensatory feedback following ipatasertib administration. The path forward for AKT inhibitors will involve selection of patients with molecular vulnerabilities for this target and also combination studies with inhibitors of other signaling pathways such as the RAS/MEK pathway. Catalytic mTOR inhibitors such as MLN0128 and AZD2014 are also undergoing clinical evaluation in multiple tumor types. Challenges associated with the development of these classes of compounds will be discussed. Legal entity responsible for the study: N/A Funding: Has not received any funding Disclosure: L. Siu: Consultant for: Merck (compensated), AstraZeneca/Medimmune (compensated), Symphogen (compensated), Morphosys (compensated) Speaker’s Bureau for: None Grant/Research support from (Clinical Trials): Merck, Novartis, Bristol-Myers Squibb, Pfizer, Boerhinger-Ingelheim, GlaxoSmithKline, Roche, Karyopharm, AstraZeneca/Medimmune, Celgene, Genentech/Roche, Symphogen, Astellas. Stockholder in: None.

Abstract P2-13-01: Differential efficacies of DNA damaging agents in basal-like TNBC subtypes

Abstract New approaches are needed to improve long term survival for patients with metastatic triple negative breast cancer (TNBC). Women with metastatic TNBC have a worse 3-year survival compared to women with metastatic HER2+ or ER/PR+ disease.1 Identifying the molecular vulnerabilities of TNBC subtypes could provide for optimal treatment strategies. Gene expression analyses of TNBC patient samples by Lehmann and Bauer, et al. identified 6 subtypes of TNBC.2 Based on the analysis of laser dissected tumor samples, the number of tumor subtypes was reduced from 6 to 4.3 Both analyses identified two types of basal-like cancers, basal-like 1 (BL1) and basal-like 2 (BL2).2,3 The BL1 subtype is defined by the amplified expression of genes involved in cell division and DNA damage response, and RNA polymerase.2 BL2 cancers express high levels of genes involved in growth factor signaling, glycolysis, and gluconeogenesis.2 They show that cell lines representative of the BL1 and BL2 subtypes of TNBC are more sensitive to cisplatin than cell lines representing the other TNBC subtypes. The BL1 or BL2 categorization, however, failed to predict sensitivity to olaparib.2 BL1 and BL2 representative cell lines responded differently when treated with veliparib; BL1 cells were more sensitive than BL2 cells.2 We investigated the response of BL1 and BL2 cells to a variety of DNA damaging agents to determine if the BL1 or BL2 subtype was predictive of sensitivity to a particular drug. DNA damaging agents, including alkylating agents, anti-metabolites, topoisomerase inhibitors, and PARP inhibitors were tested in cells resenting the BL1 and BL2 subtypes. HCC1806 cells (BL2) were found to be selectively sensitive to gemcitabine when compared to HCC1937 cells (BL1). The panel of drugs and cell lines was expanded to further understand the sensitivity of BL1 and BL2 subtypes to a variety of DNA damaging agents. Our goal is to provide preclinical evidence for the informed use of specific DNA damaging agents in the treatment of patients with BL1 or BL2 subtypes of TNBCs. 1. Li, X. et al. Breast Cancer Res. Treat. 161, 279–287 (2017). 2. Lehmann, B. D. et al. J. Clin. Invest. 121, 2750–2767 (2011). 3. Lehmann, B. D. et al. PLoS One 11, e0157368 (2016). Citation Format: Shaffer CV, Robles AJ, Risinger AL, Mooberry SL. Differential efficacies of DNA damaging agents in basal-like TNBC subtypes [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-13-01.