Oncogenic Mechanisms Research Articles

Beta-catenin activation and immunotherapy resistance in hepatocellular carcinoma: mechanisms and biomarkers.

Mutations involving CTNNB1, the gene encoding beta-catenin, and other molecular alterations that affect the Wnt/beta-catenin signaling pathway are exceptionally common in hepatocellular carcinoma. Several of these alterations have also been associated with scarcity of immune cells in the tumor microenvironment and poor clinical response to immune checkpoint inhibitor therapy. In light of these associations, tumor biomarkers of beta-catenin status could have the potential to serve as clinical predictors of immunotherapy outcome. This editorial review article summarizes recent pre-clinical and clinical research pertaining to associations between beta-catenin activation and diminished anti-tumor immunity. Potential non-invasive biomarkers that may provide a window into this oncogenic mechanism of immune evasion are also presented and discussed.

Genetic characterization of B-cell prolymphocytic leukemia: a prognostic model involving MYC and TP53

AbstractB-cell prolymphocytic leukemia (B-PLL) is a rare hematological disorder whose underlying oncogenic mechanisms are poorly understood. Our cytogenetic and molecular assessments of 34 patients with B-PLL revealed several disease-specific features and potential therapeutic targets. The karyotype was complex (≥3 abnormalities) in 73% of the patients and highly complex (≥5 abnormalities) in 45%. The most frequent chromosomal aberrations were translocations involving MYC [t(MYC)] (62%), deletion (del)17p (38%), trisomy (tri)18 (30%), del13q (29%), tri3 (24%), tri12 (24%), and del8p (23%). Twenty-six (76%) of the 34 patients exhibited an MYC aberration, resulting from mutually exclusive translocations or gains. Whole-exome sequencing revealed frequent mutations in TP53, MYD88, BCOR, MYC, SF3B1, SETD2, CHD2, CXCR4, and BCLAF1. The majority of B-PLL used the IGHV3 or IGHV4 subgroups (89%) and displayed significantly mutated IGHV genes (79%). We identified 3 distinct cytogenetic risk groups: low risk (no MYC aberration), intermediate risk (MYC aberration but no del17p), and high risk (MYC aberration and del17p) (P = .0006). In vitro drug response profiling revealed that the combination of a B-cell receptor or BCL2 inhibitor with OTX015 (a bromodomain and extra-terminal motif inhibitor targeting MYC) was associated with significantly lower viability of B-PLL cells harboring a t(MYC). We concluded that cytogenetic analysis is a useful diagnostic and prognostic tool in B-PLL. Targeting MYC may be a useful treatment option in this disease.

Mutations and Copy Number Alterations in IDH Wild-Type Glioblastomas Are Shaped by Different Oncogenic Mechanisms.

Little is known about the mutational processes that shape the genetic landscape of gliomas. Numerous mutational processes leave marks on the genome in the form of mutations, copy number alterations, rearrangements or their combinations. To explore gliomagenesis, we hypothesized that gliomas with different underlying oncogenic mechanisms would have differences in the burden of various forms of these genomic alterations. This was an analysis on adult diffuse gliomas, but IDH-mutant gliomas as well as diffuse midline gliomas H3-K27M were excluded to search for the possible presence of new entities among the very heterogenous group of IDH-WT glioblastomas. The cohort was divided into two molecular subsets: (1) Molecularly-defined GBM (mGBM) as those that carried molecular features of glioblastomas (including TERT promoter mutations, 7/10 pattern, or EGFR-amplification), and (2) those who did not (others). Whole exome sequencing was performed for 37 primary tumors and matched blood samples as well as 8 recurrences. Single nucleotide variations (SNV), short insertion or deletions (indels) and copy number alterations (CNA) were quantified using 5 quantitative metrics (SNV burden, indel burden, copy number alteration frequency-wGII, chromosomal arm event ratio-CAER, copy number amplitude) as well as 4 parameters that explored underlying oncogenic mechanisms (chromothripsis, double minutes, microsatellite instability and mutational signatures). Findings were validated in the TCGA pan-glioma cohort. mGBM and “Others” differed significantly in their SNV (only in the TCGA cohort) and CNA metrics but not indel burden. SNV burden increased with increasing age at diagnosis and at recurrences and was driven by mismatch repair deficiency. On the contrary, indel and CNA metrics remained stable over increasing age at diagnosis and with recurrences. Copy number alteration frequency (wGII) correlated significantly with chromothripsis while CAER and CN amplitude correlated significantly with the presence of double minutes, suggesting separate underlying mechanisms for different forms of CNA.

MODL-23. DNA METHYLATION AND COPY NUMBER VARIATION PROFILE FOR CHARACTERIZATION OF PEDIATRIC BRAIN TUMOR PRIMARY CELL LINES

BACKGROUNDIn vitro models of pediatric brain tumors (pBT) are instrumental for both understanding the oncogenic molecular mechanisms and identifying/testing new therapeutic strategies. DNA methylation (DM) is a stable epigenetic modification recently used to classify tumors. We aim to apply DM and Copy Number Variation (CNV) profiling to characterize pBT primary cell lines and tumors.METHODSWe included 36 pBT tissues from different histology (13 LGG, 9 DIPG, 9 HGG, 3 MB, and 2 Ependymomas), paired to their derived primary cultures. Cultures were established in two-dimensional (2D) or three-dimensional (3D) condition, as stem-cell or in serum-supplemented medium. For 9 cultures, both early (P2-P3) and long-term passages (>P4) were considered. Samples were analyzed for DM and CNV profiles using Illumina EPIC arrays and data compared with those of the brain tumor classifier.RESULTSAt early passages all cells retained the same DM and genetic patterns of original tumors, with no differences related to 2D/3D methods or presence of serum in media. Primary cell lines analyzed at > P4 and cultured in serum diverged from the primary tumor.CONCLUSIONSDM profiles and CNV are useful tools to detect the recapitulation of pBT-derived primary cell-lines from the original tumor. Whatever subgroups tested, results suggest that in vitro models should be passaged as little as possible to retain the epigenetic and genetic alterations of the tumors and thus to be considered relevant for basic and translational biology. Ongoing experiments are aimed to determine how stable DM and CNV are in other conditions/tumor subgroups.

EPEN-18. CROSS-SPECIES GENOMICS IDENTIFIES GLI2 AS AN ONCOGENE OF C11orf95 FUSION-POSITIVE SUPRATENTORIAL EPENDYMOMA

The majority of supratentorial ependymomas (ST-EPN) are driven by fusions between RELA and a zinc finger containing gene, C11orf95. Apart from fusions to the Hippo effector YAP1, which affects a small group of infant patients, the oncogenic mechanism of remaining ST-EPNs is unclear. Aiming at refining the molecular classification of ST-EPNs, we analyzed methylation profiles, RNA and DNA sequencing results as well as clinical data in a cohort of 617 ST-EPNs. Unsupervised clustering analysis of DNA methylation data revealed four distinct clusters that formed in addition to the known molecular groups ST-EPN-RELA and –YAP1. Tumors within these additional clusters were characterized by fusions of C11orf95 to numerous fusion partners different from RELA, e.g. MAML2, MAML3, NCOA2 and SS18, suggesting a general role of C11orf95 in tumorigenesis of ST-EPN. Transforming capacity of newly identified fusion genes was validated using an electroporation-based in vivo gene transfer technology. All fusion genes were sufficient to drive malignant transformation in the cerebral cortex of mice and resulting tumors faithfully recapitulated molecular characteristics of their human counterparts. We found that both, the partner gene and the zinc finger DNA binding domain of C11orf95, were essential to exert tumorigenesis. When exploring genes commonly upregulated in C11orf95 fusion-expressing tumors of human and murine origin, the Sonic Hedgehog effector gene Gli2 was identified as a promising downstream target. Subsequent co-expression of C11orf95:RELA and a dominant negative form of Gli2 indeed hampered tumorigenesis. We thus propose GLI2 as a potential therapeutic downstream target of C11orf95 fusion-dependent oncogenic signaling in ST-EPN.

HGG-57. WHOLE-GENOME SEQUENCING, METHYLATION ANALYSIS, AND SINGLE-CELL RNA-SEQ DEFINE UNIQUE CHARACTERISTICS OF PEDIATRIC TREATMENT-INDUCED HIGH-GRADE GLIOMA AND SUGGEST ONCOGENIC MECHANISMS

BACKGROUNDPediatric treatment-induced high-grade glioma (TIHGG) is among the most severe late effects observed in childhood cancer survivors and is uniformly fatal. We previously showed that TIHGG are divergent from de novo pediatric high-grade glioma (pHGG) and cluster into two gene expression subgroups, one stemlike and the other inflammatory. Here we systematically compared TIHGG molecular profiles to pHGG and evaluated expression and single cell sequencing profiles in order to identify oncogenic mechanisms and the cellular basis for the observed TIHGG gene expression subgroups. MATERIALS/METHODS450/850K methylation and mutational signature analysis was conducted in 36 TIHGG samples. Resultant data were analyzed for the presence of chromothripsis, distinct molecular alterations, and mutational signatures in a subset of 10 samples with whole genome sequencing data. Five TIHGGs underwent single-cell RNA-Seq analysis (scRNAseq).RESULTS26/36 TIHGG clustered with the pedRTK1 methylation class. TIHGG were characterized by an increased frequency of chromothripsis relative to pHGG (67% vs. 31%, p=0.036). FISH and WGS revealed frequent PDGFRA amplification secondary to enrichment in ecDNA. TIHGG were enriched for COSMIC mutational signatures 5 and 19 (p=0.0003) relative to pHGG. scRNAseq data showed that TIHGG tumors are composed of stem-like, neuronal, and inflammatory cell populations which may contribute to the previously described dominant expression profiles.CONCLUSIONSTIHGG represents a distinct molecular subtype of pHGG. Chromothripsis, leading to enriched expression of genes in extrachromosomal DNA, likely contribute to TIHGG oncogenesis. The dominant cell type (stem-like vs. inflammatory) may define the expression subgroup derived from bulk RNA-seq in heterogeneous tumors.

HumanaFly: high-throughput transgenesis and expression of breast cancer transcripts in Drosophila eye discovers the RPS12-Wingless signaling axis

Drosophila melanogaster has been a model for multiple human disease conditions, including cancer. Among Drosophila tissues, the eye development is particularly sensitive to perturbations of the embryonic signaling pathways, whose improper activation in humans underlies various forms of cancer. We have launched the HumanaFly project, whereas human genes expressed in breast cancer patients are screened for their ability to aberrate development of the Drosophila eye, hoping to thus identify novel oncogenes. Here we report identification of a breast cancer transgene, which upon expression in Drosophila produces eye malformation similar to the famous Glazed phenotype discovered by Thomas Morgan and decades later dissected to originate from mis-expression of Wingless (Wg). Wg is the ortholog of human Wnt proteins serving as ligands to initiate the developmental/oncogenic Wnt signaling pathway. Through genetic experiments we identified that this transgene interacted with the Wg production machinery, rather than with Wg signal transduction. In Drosophila imaginal discs, we directly show that the transgene promoted long-range diffusion of Wg, affecting expression of the Wg target genes. The transgene emerged to encode RPS12—a protein of the small ribosomal subunit overexpressed in several cancer types and known to also possess extra-ribosomal functions. Our work identifies RPS12 as an unexpected regulator of secretion and activity of Wnts. As Wnt signaling is particularly important in the context of breast cancer initiation and progression, RPS12 might be implicated in tumorigenesis in this and other Wnt-dependent cancers. Continuation of our HumanaFly project may bring further discoveries on oncogenic mechanisms.

Calcium cytotoxicity sensitizes prostate cancer cells to standard-of-care treatments for locally advanced tumors

Therapy resistance is a major roadblock in oncology. Exacerbation of molecular dysfunctions typical of cancer cells have proven effective in twisting oncogenic mechanisms to lethal conditions, thus offering new therapeutic avenues for cancer treatment. Here, we demonstrate that selective agonists of Transient Receptor Potential cation channel subfamily M member 8 (TRPM8), a cation channel characteristic of the prostate epithelium frequently overexpressed in advanced stage III/IV prostate cancers (PCa), sensitize therapy refractory models of PCa to radio, chemo or hormonal treatment. Overall, our study demonstrates that pharmacological-induced Ca2+ cytotoxicity is an actionable strategy to sensitize cancer cells to standard therapies.

Extracellular MicroRNAs as Intercellular Mediators and Noninvasive Biomarkers of Cancer.

Simple SummaryThere are an extensive number of publications regarding the role of endogenous miRNAs as regulators of gene expression in cancer. However, extracellular miRNAs have emerged as a novel mechanism of cell-to-cell communication in normal conditions and disease and have drawn a large amount of interest as regulators of gene expression and as potential non-invasive biomarkers in cancer. Despite this high interest and the abundance of research on the biology and role of extracellular miRNAs in cancer, they are not yet completely understood. The aim of this review is to highlight the relevant biological characteristics of extracellular miRNAs that enable them to function as intercellular mediators of gene expression regulation and provide the recently published evidence of the specific role of extracellular miRNAs in tumor development and progression.MicroRNAs (miRNAs) are released by different types of cells through highly regulated mechanisms under normal and pathological conditions. These extracellular miRNAs can be delivered into recipient cells for functional purposes, acting as cell-to-cell signaling mediators. It has been discovered that cancer cells release miRNAs into their surroundings, targeting normal cells or other cancer cells, presumably to promote tumor development and progression. These extracellular miRNAs are associated with oncogenic mechanisms and, because they can be quantified in blood and other bodily fluids, may be suitable noninvasive biomarkers for cancer detection. This review summarizes recent evidence of the role of extracellular miRNAs as intercellular mediators, with an emphasis on their role in the mechanisms of tumor development and progression and their potential value as biomarkers in solid tumors. It also highlights the biological characteristics of extracellular miRNAs that enable them to function as regulators of gene expression, such as biogenesis, gene silencing mechanisms, subcellular compartmentalization, and the functions and mechanisms of release.

Molecular Features of Cancers Exhibiting Exceptional Responses to Treatment

A small fraction of cancer patients with advanced disease survive significantly longer than patients with clinically comparable tumors. Molecular mechanisms for exceptional responses to therapy have been identified by genomic analysis of tumor biopsies from individual patients. Here, we analyzed tumor biopsies from an unbiased cohort of 111 exceptional responder patients using multiple platforms to profile genetic and epigenetic aberrations as well as the tumor microenvironment. Integrative analysis uncovered plausible mechanisms for the therapeutic response in nearly a quarter of the patients. The mechanisms were assigned to four broad categories-DNA damage response, intracellular signaling, immune engagement, and genetic alterations characteristic of favorable prognosis-with many tumors falling into multiple categories. These analyses revealed synthetic lethal relationships that may be exploited therapeutically and rare genetic lesions that favor therapeutic success, while also providing a wealth of testable hypotheses regarding oncogenic mechanisms that may influence the response to cancer therapy.

TP53 mutations and CDKN2A mutations/deletions are highly recurrent molecular alterations in the malignant progression of sinonasal papillomas.

Sinonasal papillomas are benign epithelial tumors of the sinonasal tract that are associated with a synchronous or metachronous sinonasal carcinoma in a subset of cases. Our group recently identified mutually exclusive EGFR mutations and human papillomavirus (HPV) infection in inverted sinonasal papillomas and frequent KRAS mutations in oncocytic sinonasal papillomas. We also demonstrated concordant mutational and HPV infection status in sinonasal papilloma-associated sinonasal carcinomas, confirming a clonal relationship between these tumors. Despite our emerging understanding of the oncogenic mechanisms driving formation of sinonasal papillomas, little is currently known about the molecular mechanisms of malignant progression to sinonasal carcinoma. In the present study, we utilized targeted next-generation DNA sequencing to characterize the molecular landscape of a large cohort of sinonasal papilloma-associated sinonasal carcinomas. As expected, EGFR or KRAS mutations were present in the vast majority of tumors. In addition, highly recurrent TP53 mutations, CDKN2A mutations, and/or CDKN2A copy number losses were detected; overall, nearly all tumors (n = 28/29; 96.6%) harbored at least one TP53 or CDKN2A alteration. TERT copy number gains also occurred frequently (27.6%); however, no TERT promoter mutations were identified. Other recurrent molecular alterations included NFE2L2 and PIK3CA mutations and SOX2, CCND1, MYC, FGFR1, and EGFR copy number gains. Importantly, TP53 mutations and CDKN2A alterations were not detected in matched sinonasal papillomas, suggesting that these molecular events are associated with malignant transformation. Compared to aerodigestive tract squamous cell carcinomas from The Cancer Genome Atlas (TCGA) project, sinonasal papilloma-associated sinonasal carcinomas have a distinct molecular phenotype, including more frequent EGFR, KRAS, and CDKN2A mutations, TERT copy number gains, and low-risk human papillomavirus (HPV) infection. These findings shed light on the molecular mechanisms of malignant progression of sinonasal papillomas and may have important diagnostic and therapeutic implications for patients with advanced sinonasal cancer.

Oncogenic Viruses and Mechanisms in Oral Squamous Cell Carcinoma: A Mini Review

Oral squamous cell carcinoma (OSCC) is a common epithelial malignancy with varied etiology. The traditional risk factors are the usage of tobacco and tobacco products. About 15-20% of the population exists with oral squamous cell carcinoma without being exposed to these carcinogenic products. The other factors which may influence the disease process are environmental factors, genetic alterations and viruses. Some oncogenic DNA viruses are strongly associated with OSCC while other viruses are little expressed. While diagnosing OSCC one should always consider the possible role of viruses in their etiopathogenesis as it is crucial for diagnosis and successful treatment. Screening of the oncogenic viruses at an early stage may also aid in the prognosis of oral squamous cell carcinoma. The current review focuses on the possible mechanisms of the oncogenic viruses in the etiopathogenesis of OSCC.

PBK promotes aggressive phenotypes of cervical cancer through ERK/c-Myc signaling pathway.

Cervical cancer is the fourth most frequent cancer in women worldwide. PDZ-binding kinase (PBK) is proven to promote the malignant behaviors of various carcinomas. However, its functional roles and oncogenic mechanisms in cervical cancer are poorly understood. In this study, we reported that PBK was highly expressed in cervical cancer tissues. PBK promoted the proliferation, metastasis, and cisplatin resistance of cervical cancer cells. OTS514, a specific PBK inhibitor, could significantly suppress proliferation and metastasis of cervical cancer cells in vitro and in a xenograft model. Besides, OTS514 could enhance cisplatin-based chemosensitivity in cervical cancer cells. Mechanistically, PBK promoted the expression and stabilization of c-Myc through phosphorylating ERK1/2. OTS514 suppressed the phosphorylation of ERK1/2 and the transcriptional activity of c-Myc. Furthermore, inhibition of the ERK signal pathway by U0126 reversed the increased proliferation and metastasis induced by overexpression of PBK. Exogenous expression of c-Myc counteracted the decreased proliferation and metastasis evoked by knockdown of PBK. In conclusion, PBK promoted the malignant progression of cervical cancer through ERK/c-Myc signal pathway. PBK might be a promising molecular target for cervical cancer treatment.

A novel model of alternative NF-κB pathway activation in anaplastic large cell lymphoma.

Aberrant activation of NF-κB is the most striking oncogenic mechanism in B-cell lymphoma; however, its role in anaplastic large cell lymphomas (ALCL) has not been fully established and its activation mechanism(s) remain unclear. Using ALCL cell line models, we revealed the supporting roles for NFKB2 and the NIK pathway in some ALCL lines. To investigate the detailed activation mechanisms for this oncogenic pathway, we performed specifically designed alternative NF-κB reporter CRISPR screens followed by the RNA-seq analysis, which led us to identify STAT3 as the major mediator for NIK-dependent NF-κB activation in ALCL. Consistently, p-STAT3 level was correlated with NFKB2 nuclear accumulation in primary clinical samples. Mechanistically, we found that in NIK-positive ALK- ALCL cells, common JAK/STAT3 mutations promote transcriptional activity of STAT3 which directly regulates NFKB2 and CD30 expression. Endogenous expression of CD30 induces constitutive NF-κB activation through binding and degrading of TRAF3. In ALK+ ALCL, the CD30 pathway is blocked by the NPM-ALK oncoprotein, but STAT3 activity and resultant NFKB2 expression can still be induced by NPM-ALK, leading to minimal alternative NF-κB activation. Our data suggest combined NIK and JAK inhibitor therapy could benefit patients with NIK-positive ALK- ALCL carrying JAK/STAT3 somatic mutations.

Deranged Transcription and Replication in MYC-Induced B and T Cell Leukemias

MYCis a key oncogene overexpressed by many cancers, however, its oncogenic mechanisms are poorly understood. MYC is also central to acute lymphoblastic leukemia (ALL), the most common and second most lethal pediatric malignancy. Much of MYC's oncogenicity has been attributed to its transcription factor function, but data suggest MYC also deregulates replication in transcription-independent fashion. As a known master regulator of cancer transcriptomes and epigenomes, we hypothesize that MYC dramatically alters both gene expression and replication timing (non-random spatiotemporal process where some part of the genome replicates early, and other late) in both types of ALL - B-ALL and T-ALL. Conceivably, MYC exerts oncogenic effects upon the ALL transcription and replication programs, with some changes shared by B- and T-ALL, and others unique to only one. We aim to address two novel questions not been investigated before. First, in ALL, do the same genetic loci show aberrant RNA transcriptionandDNA replication? Second, how similar are the affected loci in two closely-related, yet distinct, ALL types driven by the same oncogene? The basis of our project is a unique double-transgenicrag2:hMYC,lck:GFPzebrafish pre-clinical model we established, which is the only animal model proven to develop both B-ALL and T-ALL. We previously showed that gene expression profiles (GEP) differentiating zebrafish B- and T-ALL also distinguish human B- and T-ALL, making this an ideal model system to study human ALL. In this model, B-ALL and T-ALL are induced by human MYC(hMYC) regulated by aD.rerio(zebrafish)rag2promoter.Since B and T lymphoblasts both expressrag2, both lineages over-express MYC, causing highly-penetrant B- and T-ALL. Differential activity of aD. rerio lckpromoter causes B cells to fluoresce dimly and T cells to fluoresce brightly, allowing us to identify and purify B-ALL and T-ALL by fluorescent microscopy and fluorescence-based flow cytometry, respectively. This unique model enables comparing B- and T-ALL in one genetic background. We have purified >20 zebrafish ALL (both T-ALL and B-ALL) and isolated their RNA and DNA. We are now analyzing RNA-seq gene expression profiles (GEP) and replication timing (RT) profiles via next generation sequencing (NGS). We will compare both ALL types to identify mRNA signatures that are unique to, or shared by, both types. We seek loci that shift DNA replication from early-to-late, or late-to-early, to define the regions that replicate at the same time in both ALL types, versus loci that vary by ALL type. We will also interrogate these data to determine whether GEP and RT profiles correlate with each other, and with known MYC target genes. In conclusion, GEP and RT have never been analyzed in the same cancer sample, or in related cancers driven by the same oncogene. Exploiting our expertise with thehMYCzebrafish model, we are delineating how MYC alters transcription and replication, to ascertain if these affect the same loci and define which loci are unique to one ALL type or shared by both. MYC hyper-activity is seen ~70% of human cancers - making MYC a crucial oncogene in human cancer biology, so our findings are likely to inform not only mechanisms operative in ALL, but also other MYC-driven cancers. Disclosures No relevant conflicts of interest to declare.

The Genomic and Transcriptomic Landscape of Plasma Cell Leukemia

Background: Primary plasma cell leukemia (pPCL) is a rare and aggressive form of multiple myeloma (MM) with an extremely poor prognosis and distinct biological and clinical features. Because of its low incidence and its heterogeneity, biological knowledge about pPCL is lacking especially molecular process responsible for its aggressiveness. Here, we took advantage of a large series of pPCL to describe the genomic and transcriptomic landscape of pPCL, to identify potential driver mutations and pathways, and to determine their clinical impacts. Methods: To address these issues, we performed a targeted DNA sequencing and a RNA sequencing of sorted bone marrow plasma cells collected at the time of diagnosis from 96 patients with pPCL between 2014 and 2020. We compared their genomic profiles with those of 907 MM at diagnosis previously obtained in our laboratory and their transcriptomic profiles with those of 300 MM at diagnosis obtained from the IFM2009/DFCI trial (NCT01191060). Copy number aberrations (CNA), single nucleotide variants (SNV), translocations, mutations, gene expression (GE) and gene set enrichment were analyzed and correlated with clinical information (overall survival and progression-free survival). Results: Genome analysis highlighted a specific genomic profile of pPCL. Indeed, hyperdiploid karyotypes were less frequent in pPCL compared with MM (20% vs 57%, p<0,001). We found a high prevalence of translocations involving the heavy chain locus (IGH) in pPCL with higher incidences of t(11;14) (51% vs 23%, p<0,001) and t(14;16) (14% vs 3%, p<0,001), but an identical incidence of t(4;14) (11% vs 10%, p=0,7). pPCL presented more adverse cytogenetic abnormalities such as del(17p) (30% vs 9,5%, p<0,001), 1q gain (53% vs 32%, p<0,001) and del(1p32) (24% vs 9%, p<0,001). Among the 246 recurrently mutated genes in MM, mutations of TP53 (21% vs 5%, p<0,001) and IRF4 (11% vs 4%, p<0,005) were significantly more frequent in pPCL. Furthermore, pPCL presented high-risk genomic features with an increased proportion of Double Hit profiles (27% vs 5%, p<0,001) with more bi-allelic inactivation of TP53 (17% vs 3%, p<0,001) and more amp1q on the background of International Staging System III (11% vs 5%, p<0,005). Interestingly, by comparing genomic profiles from pPCL with and without t(11;14) we found two distinctive patterns. Indeed pPCL with t(11;14) showed more TP53 mutations and more bi-allelic inactivation of TP53. While pPCL without t(11;14) showed more adverse cytogenetic abnormalities such as trisomy 21, 1q gains and del(1p32). These results suggest two distinctive oncogenic mechanisms. RNA-seq analysis showed also a specific transcriptional landscape of pPCL. Indeed, unsupervised hierarchical clustering of gene expression profiles demonstrated two distinct clusters between pPCL and MM. Gene set enrichment analysis identified a significantly higher expression of genes involved in MYC Targets and G2M checkpoint, and a significantly lower expression of genes involved in P53 pathway, hypoxia and TNF alpha signaling via NF-κB. Furthermore, pPCL with and without t(11;14) presented two distinct transcriptomic patterns, in particular for genes implicated in the apoptotic machinery. Three members of the BCL2 family were differentially expressed with BCL2 and PMAIP1 [NOXA] significantly overexpressed and BCL2L1 significantly underexpressed in pPCL with t(11;14). Median PFS and OS of patients with pPCL were respectively at 11 and 15 months. Presence of TP53 mutations was associated with a significantly lower PFS (4 months, p<0,05) and OS (5 months, p<0,05). Neither the IgH translocations nor the ploidy status predicted for survival. Conclusion: To our knowledge, we present the study on the largest series of patients with pPCL. Our results provide new information on both genomic and transcriptomic landscape of pPCL. Despite their heterogeneity, pPCL present a specific mutational landscape with high prevalence of t(11;14) and high-risk genomic features. These results help to better understand oncogenicity and the aggressive behavior of pPCL and support the use of new treatment strategies such as BCL2 inhibitor (Venetoclax) for pPCL with t(11;14). Disclosures Perrot: Amgen, BMS/Celgene, Janssen, Sanofi, Takeda: Consultancy, Honoraria, Research Funding. Hulin:Celgene/Bristol-Myers Squibb, Janssen, GlaxoSmithKline, and Takeda: Honoraria.

Multidisciplinary Treatment, Including Locoregional Chemotherapy, for Merkel-Polyomavirus-Positive Merkel Cell Carcinomas: Perspectives for Patients Exhibiting Oncogenic Alternative Δ exon 6-7 TrkAIII Splicing of Neurotrophin Receptor Tropomyosin-Related Kinase A.

Merkel cell carcinomas (MCCs) are rare, aggressive, cutaneous neuroendocrine tumours, approximately 80% of which are caused by the genomic integration of Merkel cell polyomavirus (MCPyV). MCPyV-positive MCCs carry poor prognosis in approximately 70% of cases, highlighting the need for greater understanding of the oncogenic mechanisms involved in pathogenesis, progression and post-therapeutic relapse, and translation into novel therapeutic strategies. In a previous pilot study, we reported a potential relationship between MCPyV gene expression and oncogenic alternative Δ exon 6–7 TrkAIII splicing in formalin-fixed paraffin-embedded (FFPE) MCC tissues from a 12-patient cohort of >90% MCPyV-positive MCCs, diagnosed at San Salvatore Hospital, L’Aquila, Italy, characterising a new MCC subgroup and unveiling a novel potential MCPyV oncogenic mechanism and therapeutic target. This, however, could not be fully verified due to poor RNA quality and difficulty in protein extraction from FFPE tissues. Here, therefore, we extend our previous observations to confirm the relationship between MCPyV and oncogenic alternative Δ exon 6–7 TrkAIII splicing in fresh, nonfixed, MCPyV-positive MCC metastasis by detecting sequence-verified RT-PCR products, including full-length Δ exon 6–7 TrkAIII, and by Western blot detection of a 100 kDa TrkA protein isoform of identical size to 100 kDa Δ exon 6–7 TrkAIII expressed by stable transfected SH-SY5Y cells. We also report that in three MCC patients submitted for multidisciplinary treatment, including locoregional chemotherapy, MCPyV large T-antigen mRNA expression, Δ exon 6–7 TrkAIII mRNA expression and intracellular indirect immunofluorescence (IF) TrkA and phosphorylation protein isoform(s) immunoreactivity in FFPE tissues were not reduced in postchemotherapeutic-relapsed MCCs compared to pretherapeutic MCCs, extending the possible roles of this novel potential MCPyV oncogenic mechanism from MCC pathogenesis to post-therapeutic relapse and progression. Detection of alternative Δ exon 6–7 TrkAIII splicing in MCC, therefore, not only characterises a new MCPyV-positive MCC subgroup and unveils a novel potential MCPyV oncogenic mechanism but also identifies patients who may benefit from inhibitors of MCPyV T-antigen and/or TrkAIII expression or clinically approved Trk kinase inhibitors such as larotrectinib or entrectinib, which are known to inhibit activated TrkA oncogenes and to elicit durable responses in TrkA-fusion oncogene-driven cancers, supporting the call for a large-scale multicentre clinical study.

Abstract PO-04: Noncoding mutations in mantle cell lymphoma disrupt regulation of HNRNPH1 by alternative splicing

Abstract Non-Hodgkin lymphomas (NHL) are a collection of cancers with each malignancy having distinct clinical management and prognosis. Mantle cell lymphoma (MCL) is particularly genetically heterogeneous and is considered incurable. Through a combination of exome, genome, and targeted sequencing of MCL tumors, we identified recurrent mutations in HNRNPH1 (heterogeneous nuclear ribonucleoprotein H1). These mutations are largely intronic or silent and are associated with a putative cis regulatory region involving a single exon. In RNA-seq data from matched cases, we identified variable representation of two distinct HNRNPH1 isoforms. Based on the reading frame of the affected exons, canonical splicing is predicted to produce a functional protein, while alternative splicing introduces a premature termination codon leading to nonsense-mediated decay. We observed a significantly higher proportion of canonical transcripts in MCL tumors bearing HNRNPH1 mutations, leading us to conclude that mutations in HNRNPH1 significantly alter its splicing. Furthermore, increased canonical splicing results in higher HNRNPH1 protein abundance as determined by immunohistochemical analysis of an MCL tissue microarray. HNRNPH1 mutation status and splicing ratio are associated with shorter survival of MCL patients. We developed an in vitro reporter minigene and introduced three specific mutations corresponding to the patient-identified mutations in HNRNPH1. These mutations lead to an increase in canonical splicing and translation of the HNRNPH1 minigene-derived peptide. Additionally, when HNRNPH1 is overexpressed, we observe a decrease of this peptide, implicating HNRNPH1 in the regulation of its own splicing. This is supported by preliminary data indicating that HNRNPH1 binds its own RNA. Beyond its own splicing, HNRNPH1 is likely also involved in the splicing of additional splicing factors. Overexpression of HNRNPH1 affects the splicing of SRSF3 and HNRNPDL, and many other targets are likely to be identified. This work elucidates a functional role for recurrent noncoding HNRNPH1 mutations and implicates HNRNPH1 expression and splicing of its downstream targets in lymphomagenesis. We continue to explore trans regulatory targets of HNRNPH1 using in vitro and cell-based models. While splicing is a growing field of interest in lymphoma biology, the unique pattern and consequences of these largely silent mutations specifically implicate alternative splicing as an oncogenic mechanism in MCL. Citation Format: Krysta M. Coyle, Quratulain Qureshi, Prasath Pararajalingam, Nicole Thomas, Timothy E. Audas, Ryan D. Morin. Noncoding mutations in mantle cell lymphoma disrupt regulation of HNRNPH1 by alternative splicing [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-04.

Autophagy-related gene 7 deficiency caused by miR-154-5p overexpression suppresses the cell viability and tumorigenesis of retinoblastoma by increasing cell apoptosis.

BackgroundRetinoblastoma is a rare cancer of the retina that accounts for 3% of all childhood cancers. The aim of this study was to illuminate the oncogenic role and potential molecular mechanisms of the microRNA miR-154-5p and autophagy-related gene 7 (ATG7) in retinoblastoma, and to establish a nude mouse model in order to explore new therapeutic horizons for the disease.MethodsQuantitative reverse transcription-polymerase chain reaction and western blot were performed to detect the expression levels of miR-154-5p and ATG7. The targeting relationship between miR-154-5p and ATG7 was analyzed by employing the luciferase reporter assay. MiR-154-5p mimic and pcDNA-ATG7 were transfected, either alone or in combination, into Y79 cells. The subsequent in vitro experiments involved four groups: the control group, miR-154-5p group, ATG7 group, and miR-154-5p + ATG7 group. Orthotopic xenograft models were established by injecting BALB/c athymic nude mice with treated and untreated Y79 cells.ResultsY79 cells were transfected with miR-NC or miR-154-5p. Compared to those in the control group, the mRNA expression levels of miR-154-5p were increased in the miR-154-5p mimic group; in contrast, decreases were observed in the mRNA and protein expression levels of ATG7. Y79 cells were transfected with PcDNA or pcDNA-ATG7. The mRNA expression level of ATG7 was increased in pcDNA-ATG7 group. MiR-154-5p was found to have an element complementary to the three prime untranslated region of ATG7. Overexpression of miR-154-5p inhibited Y79 cells proliferation and migration, and promoted Y79 cells apoptosis via targeting of ATG7. In the in vivo experiment, the tumors of the miR-154-5p group of mice were significantly reduced in weight. Tumor growth and the protein levels of Survivin were both suppressed when miR-154-5p was overexpressed in vivo; however, cell apoptosis and the protein levels of p21 were promoted. In the miR-154-5p group, the expression levels of miR-154-5p were upregulated compared to those in the control group, but the ATG7 expression level was downregulated.ConclusionsMiR-154-5p overexpression downregulated ATG7, which inhibited cell proliferation and apoptosis in vitro, as well as tumor formation in vivo.

Abstract PO-08: Understanding the properties and oncogenic mechanisms of EZH2 Y641 mutations in lymphoma

Abstract EZH2 (Enhancer of Zeste Homolog 2) is the catalytic component of the Polycomb Repressive Complex group 2 (PRC2), which establishes the repressive epigenetic mark histone 3 lysine 27 trimethylation (H3K27me3), resulting in silencing of gene expression. EZH2 was originally identified as a tumor suppressor since loss-of-function events were observed in myelodysplastic syndrome, acute myeloid, and T-cell leukemias. However, next-generation sequencing identified recurrent activating point mutations at tyrosine residue 641 (Y641) in 25% of germinal center diffuse large B-cell lymphoma and 15% of follicular lymphomas, suggesting that EZH2 also functions as an oncogene in a cell type-dependent manner. Using a faithful genetically engineered mouse model, we previously demonstrated that the Ezh2Y641F mutation drives formation of B-cell lymphoma as a single event and in cooperation with Bcl2 amplification or p53 loss. Unexpectedly, at the chromatin level, expression of Ezh2Y641F did not monotonically increase abundance of global H3K27me3 but rather resulted in redistribution of this mark across the genome, exhibiting loss of H3K27me3 at many loci, with direct effects on transcription. Others have shown that EZH2 mediates germinal center proliferation via repression of p21 and cooperation with Bcl6; however, given the global effect of Ezh2Y641F on chromatin, the oncogenic mechanisms of these mutations remain underexplored. These include the timing of the mutation during hematopoietic development, its role during differentiation, and the oncogenic activity of downstream targets. In order to investigate the effect of Ezh2Y641 mutations on hematopoietic development and differentiation, we used Cre alleles to drive expression of the mutant protein at different stages of hematopoietic development. Our data demonstrate that expression of Ezh2Y641 in early B cells is sufficient to drive oncogenic transformation. However, expression of Ezh2Y641 in hematopoietic stem cell (HSC) is not sufficient to drive oncogenic transformation despite the presence of mature healthy B cells. Additionally, expression of Ezh2Y641F in HSCs results in loss of self-renewal, differentiation bias towards the lymphoid lineages, and a partial block at the pre-pro B-cell stage. With regards to direct downstream targets, we identified upregulation of several HoxC cluster genes in Ezh2Y641F-mutant B cells prior to transformation. Upregulation of these genes is mediated via loss of H3K27me3 at the HoxC cluster and focal gains of H3K27 acetylation (H3K27ac). Overall, our findings underline the complicated biology and oncogenic activity of EZH2Y641 mutations in lymphoma, shed light on the effect of these mutations during hematopoietic development, and underscore some of the consequences of the paradoxical loss of H3K27me3 at certain loci. Citation Format: Samantha J. Nixon, Sarah Zimmerman, Jeremy M. Simon, George P. Souroullas. Understanding the properties and oncogenic mechanisms of EZH2 Y641 mutations in lymphoma [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-08.