Chimeric Transcripts Research Articles

The Landscape of Expressed Chimeric Transcripts in the Blood of Severe COVID-19 Infected Patients.

The ongoing COVID-19 pandemic caused by SARS-CoV-2 infections has quickly developed into a global public health threat. COVID-19 patients show distinct clinical features, and in some cases, during the severe stage of the condition, the disease severity leads to an acute respiratory disorder. In spite of several pieces of research in this area, the molecular mechanisms behind the development of disease severity are still not clearly understood. Recent studies demonstrated that SARS-CoV-2 alters the host cell splicing and transcriptional response to overcome the host immune response that provides the virus with favorable conditions to replicate efficiently within the host cells. In several disease conditions, aberrant splicing could lead to the development of novel chimeric transcripts that could promote the functional alternations of the cell. As severe SARS-CoV-2 infection was reported to cause abnormal splicing in the infected cells, we could expect the generation and expression of novel chimeric transcripts. However, no study so far has attempted to check whether novel chimeric transcripts are expressed in severe SARS-CoV-2 infections. In this study, we analyzed several publicly available blood transcriptome datasets of severe COVID-19, mild COVID-19, other severe respiratory viral infected patients, and healthy individuals. We identified 424 severe COVID-19 -specific chimeric transcripts, 42 of which were recurrent. Further, we detected 189 chimeric transcripts common to severe COVID-19 and multiple severe respiratory viral infections. Pathway and gene enrichment analysis of the parental genes of these two subsets of chimeric transcripts reveals that these are potentially involved in immune-related processes, interferon signaling, and inflammatory responses, which signify their potential association with immune dysfunction leading to the development of disease severity. Our study provides the first detailed expression landscape of chimeric transcripts in severe COVID-19 and other severe respiratory viral infections.

BET Bromodomain Degradation Disrupts Function but Not 3D Formation of RNA Pol2 Clusters.

Fusion-positive rhabdomyosarcoma (FP-RMS) is driven by a translocation that creates the chimeric transcription factor PAX3-FOXO1 (P3F), which assembles de novo super enhancers to drive high levels of transcription of other core regulatory transcription factors (CRTFs). P3F recruits co-regulatory factors to super enhancers such as BRD4, which recognizes acetylated lysines via BET bromodomains. In this study, we demonstrate that inhibition or degradation of BRD4 leads to global decreases in transcription, and selective downregulation of CRTFs. We also show that the BRD4 degrader ARV-771 halts transcription while preserving RNA Polymerase II (Pol2) loops between super enhancers and their target genes, and causes the removal of Pol2 only past the transcriptional end site of CRTF genes, suggesting a novel effect of BRD4 on Pol2 looping. We finally test the most potent molecule, inhibitor BMS-986158, in an orthotopic PDX mouse model of FP-RMS with additional high-risk mutations, and find that it is well tolerated in vivo and leads to an average decrease in tumor size. This effort represents a partnership with an FP-RMS patient and family advocates to make preclinical data rapidly accessible to the family, and to generate data to inform future patients who develop this disease.

The landscape of differential splicing and transcript alternations in severe COVID-19 infection.

Viral infections can modulate the widespread alternations of cellular splicing, favouring viral replication within the host cells by overcoming host immune responses. However, how SARS-CoV-2 induces host cell differential splicing and affects the landscape of transcript alternation in severe COVID-19 infection remains elusive. Understanding the differential splicing and transcript alternations in severe COVID-19 infection may improve our molecular insights into the SARS-CoV-2 pathogenesis. In this study, we analysed the publicly available blood and lung transcriptome data of severe COVID-19 patients, blood transcriptome data of recovered COVID-19 patients at 12-, 16- and 24-week postinfection and healthy controls. We identified a significant transcript isoform switching in the individual blood and lung RNA-seq data of severe COVID-19-infected patients and 25 common genes that alter their transcript isoform in both blood and lung samples. Altered transcripts show significant loss of the open reading frame, functional domains and switch from coding to noncoding transcript, impacting normal cellular functions. Furthermore, we identified the expression of several novel recurrent chimeric transcripts in the blood samples from severe COVID-19 patients. Moreover, the analysis of the isoform switching into blood samples from recovered COVID-19 patients highlights that there is no significant isoform switching in 16- and 24-week postinfection, and the levels of expressed chimeric transcripts are reduced. This finding emphasizes that SARS-CoV-2 severe infection induces widespread splicing in the host cells, which could help the virus alter the host immune responses and facilitate the viral replication within the host and the efficient translation of viral proteins.

CircPVT1 and PVT1/AKT3 show a role in cell proliferation, apoptosis, and tumor subtype-definition in small cell lung cancer.

Small cell lung cancer (SCLC) is treated as a homogeneous disease, although the expression of NEUROD1, ASCL1, POU2F3, and YAP1 identifies distinct molecular subtypes. The MYC oncogene, amplified in SCLC, was recently shown to act as a lineage-specific factor to associate subtypes with histological classes. Indeed, MYC-driven SCLCs show a distinct metabolic profile and drug sensitivity. To disentangle their molecular features, we focused on the co-amplified PVT1, frequently overexpressed and originating circular (circRNA) and chimeric RNAs. We analyzed hsa_circ_0001821 (circPVT1) and PVT1/AKT3 (chimPVT1) as examples of such transcripts, respectively, to unveil their tumorigenic contribution to SCLC. In detail, circPVT1 activated a pro-proliferative and anti-apoptotic program when over-expressed in lung cells, and knockdown of chimPVT1 induced a decrease in cell growth and an increase of apoptosis in SCLC in vitro. Moreover, the investigated PVT1 transcripts underlined a functional connection between MYC and YAP1/POU2F3, suggesting that they contribute to the transcriptional landscape associated with MYC amplification. In conclusion, we have uncovered a functional role of circular and chimeric PVT1 transcripts in SCLC; these entities may prove useful as novel biomarkers in MYC-amplified tumors.

Synthetic sub-genomic transcript promoter from Horseradish Latent Virus (HRLV).

We characterized an efficient chimeric sub-genomic transcript promoter from Horseradish Latent Virus, FHS4, active in both dicot and monocot plants, and it could be a potential tool for plant biotechnology. Plant pararetroviruses are a rich source of novel plant promoters widely used for biotechnological applications. Here, we comprehensively characterized a unique sub-genomic transcript (Sgt) promoter of Horseradish Latent Virus (HRLV) and identified a fragment (HS4; -340 to + 10; 351bp) that showed the highest expression of reporter genes in both transient and transgenic assays as evidenced by biochemical, histochemical GUS reporter assay and transcript analysis of uidA gene by qRT-PCR. Phylogenetic analysis showed that the HSgt promoter was closely related to the sub-genomic promoter of the Cauliflower Mosaic Virus (CaMV19S). We found that the as-1 element and W-box played an important role in the transcriptional activity of the HS4 promoter. Furthermore, the HS4 promoter was also induced by salicylic acid. Alongside, we enhanced the activity of the HS4 promoter by coupling the enhancer region from Figwort Mosaic Virus (FMV) promoter to the upstream region of it. This hybrid promoter FHS4 was around 1.1 times stronger than the most commonly used promoter, 35S (Cauliflower Mosaic Virus full-length transcript promoter), and was efficient in driving reporter genes in both dicot and monocot plants. Subsequently, transgenic tobacco plants expressing an anti-microbial peptide BrLTP2.1 (Brassica rapa lipid transport protein 2.1), under the control of the FHS4 promoter, were developed. The in vitro anti-fungal assay revealed that the plant-derived BrLTP2.1 protein driven by an FHS4 promoter manifested increased resistance against an important plant fungal pathogen, Alternaria alternata. Finally, we concluded that the FHS4 promoter can be used as an alternative to the 35S promoter and has a high potential to become an efficient tool in plant biotechnology.

Chromatin and Cancer: Implications of Disrupted Chromatin Organization in Tumorigenesis and Its Diversification.

A hallmark of cancers is uncontrolled cell proliferation, frequently associated with an underlying imbalance in gene expression. This transcriptional dysregulation observed in cancers is multifaceted and involves chromosomal rearrangements, chimeric transcription factors, or altered epigenetic marks. Traditionally, chromatin dysregulation in cancers has been considered a downstream effect of driver mutations. However, here we present a broader perspective on the alteration of chromatin organization in the establishment, diversification, and therapeutic resistance of cancers. We hypothesize that the chromatin organization controls the accessibility of the transcriptional machinery to regulate gene expression in cancerous cells and preserves the structural integrity of the nucleus by regulating nuclear volume. Disruption of this large-scale chromatin in proliferating cancerous cells in conventional chemotherapies induces DNA damage and provides a positive feedback loop for chromatin rearrangements and tumor diversification. Consequently, the surviving cells from these chemotherapies become tolerant to higher doses of the therapeutic reagents, which are significantly toxic to normal cells. Furthermore, the disorganization of chromatin induced by these therapies accentuates nuclear fragility, thereby increasing the invasive potential of these tumors. Therefore, we believe that understanding the changes in chromatin organization in cancerous cells is expected to deliver more effective pharmacological interventions with minimal effects on non-cancerous cells.

A t(4;13)(q21;q14) translocation in B-cell chronic lymphocytic leukemia causing concomitant homozygous DLEU2/miR15a/miR16-1 and heterozygous ARHGAP24 deletions

13q14 deletion is the most recurrent chromosomal aberration reported in B-CLL, having a favorable prognostic significance when occurring as the sole cytogenetic alteration. However, its clinical outcome is also related to the deletion size and number of cells with the del(13)(q14) deletion. In 10% of cases, 13q14 deletion arises following a translocation event with multiple partner chromosomes, whose oncogenic impact has not been investigated so far due to the assumption of a possible role as a passenger mutation.Here, we describe a t(4;13)(q21;q14) translocation occurring in a B-CLL case from the diagnosis to spontaneous regression. FISH and SNP-array analyses revealed a heterozygous deletion at 4q21, leading to the loss of the Rho GTPase Activating Protein 24 (ARHGAP24) tumor suppressor gene, down-regulated in the patient RNA, in addition to the homozygous deletion at 13q14 involving DLEU2/miR15a/miR16–1 genes. Interestingly, targeted Next Generation Sequencing analysis of 54 genes related to B-CLL indicated no additional somatic mutation in the patient, underlining the relevance of this t(4;13)(q21;q14) aberration in the leukemogenic process.In all tested RNA samples, RT-qPCR experiments assessed the downregulation of the PCNA, MKI67, and TOP2A proliferation factor genes, and the BCL2 anti-apoptotic gene as well as the up-regulation of TP53 and CDKN1A tumor suppressors, indicating a low proliferation potential of the cells harboring the aberration. In addition, RNA-seq analyses identified four chimeric transcripts (ATG4B::PTMA, OAZ1::PTMA, ZFP36::PTMA, and PIM3::BRD1), two of which (ATG4B::PTMA and ZFP36::PTMA) failed to be detected at the remission, suggesting a possible transcriptional remodeling during the disease course. Overall, our results indicate a favorable prognostic impact of the described chromosomal aberration, as it arises a permissive molecular landscape to the spontaneous B-CLL regression in the patient, highlighting ARHGAP24 as a potentially relevant concurrent alteration to the 13q14 deletion in delineating B-CLL disease evolution.

ESR1 fusions and therapeutic resistance in metastatic breast cancer.

Breast cancer is the most frequent female malignant tumor, and the leading cause of cancer death in women worldwide. The most common subtype of breast cancer is hormone receptor positive that expresses the estrogen receptor (ER). Targeting ER with endocrine therapy (ET) is the current standard of care for ER positive (ER+) breast cancer, reducing mortality by up to 40% in early- stage disease. However, resistance to ET represents a major clinical challenge for ER+ breast cancer patients leading to disease recurrence or progression of metastatic disease. Salient drivers of ET resistance are missense mutations in the ER gene (ESR1) leading to constitutive transcriptionalactivity and reduced ET sensitivity. These mutations are particularly prominent and deleterious in metastatic breast cancer (MBC). In addition to activating ESR1 point mutations, emerging evidence imposes that chromosomal translocation involving the ESR1 gene can also drive ET resistance through the formation of chimeric transcription factors with constitutive transcriptional activity. Although these ESR1 gene fusions are relatively rare, they are enriched in ET resistant metastatic disease. This review discusses the characteristics of ER fusion proteins and their association with clinical outcomes in more aggressive and metastatic breast cancer. The structure and classification of ER fusion proteins based on function and clinical significance are also addressed. Finally, this review summarizes the metastatic phenotypes exhibited by the ER fusion proteins and their role in intrinsic ET resistance.

Pattern recognition in the landscape of seemingly random chimeric transcripts

The molecular and functional diversity generated by chimeric transcripts (CTs) that are derived from two genes is indicated to contribute to tumor cell survival. Several gaps yet exist. The present research is a systematic study of the spectrum of CTs identified in RNA sequencing datasets of 160 ovarian cancer samples in the The Cancer Genome Atlas (TCGA) (https://portal.gdc.cancer.gov). Structural annotation revealed complexities emerging from chromosomal localization of partner genes, differential splicing and inclusion of regulatory, untranslated regions. Identification of phenotype-specific associations further resolved a dynamically modulated mesenchymal signature during transformation. On an evolutionary background, protein-coding CTs were indicated to be highly conserved, while non-coding CTs may have evolved more recently. We also realized that the current premise postulating structural alterations or neighbouring gene readthrough generating CTs is not valid in instances wherein the parental genes are genomically distanced. In addressing this lacuna, we identified the essentiality of specific spatiotemporal arrangements mediated gene proximities in 3D space for the generation of CTs. All these features together suggest non-random mechanisms towards increasing the molecular diversity in a cell through chimera formation either in parallel or with cross-talks with the indigenous regulatory network.

IFT46 gene promoter-driven ciliopathy disease model in zebrafish.

Ciliopathies are human genetic disorders caused by abnormal formation and dysfunction of cellular cilia. Cilia are microtubule-based organelles that project into the extracellular space and transduce molecular and chemical signals from the extracellular environment or neighboring cells. Intraflagellar transport (IFT) proteins are required for the assembly and maintenance of cilia by transporting proteins along the axoneme which consists of complexes A and B. IFT46, a core IFT-B protein complex, is required for cilium formation and maintenance during vertebrate embryonic development. Here, we introduce transgenic zebrafish lines under the control of ciliated cell-specific IFT46 promoter to recapitulate human ciliopathy-like phenotypes. We generated a Tg(IFT46:GAL4-VP16) line to temporo-spatially control the expression of effectors including fluorescent reporters or nitroreductase based on the GAL4/UAS system, which expresses GAL4-VP16 chimeric transcription factors in most ciliated tissues during embryonic development. To analyze the function of IFT46-expressing ciliated cells during zebrafish development, we generated the Tg(IFT46:GAL4-VP16;UAS;nfsb-mCherry) line, a ciliated cell-specific injury model induced by nitroreductase (NTR)/metrodinazole (MTZ). Conditionally, controlled ablation of ciliated cells in transgenic animals exhibited ciliopathy-like phenotypes including cystic kidneys and pericardial and periorbital edema. Altogether, we established a zebrafish NTR/MTZ-mediated ciliated cell injury model that recapitulates ciliopathy-like phenotypes and may be a vertebrate animal model to further investigate the etiology and therapeutic approaches to human ciliopathies.

Genome-wide Detection of Chimeric Transcripts in Early-stage Non-small Cell Lung Cancer.

Lung cancer remains the main culprit in cancer-related mortality worldwide. Transcript fusions play a critical role in the initiation and progression of multiple cancers. Treatment approaches based on specific targeting of discovered driver events, such as mutations in EGFR, and fusions in NTRK, ROS1, and ALK genes led to profound improvements in clinical outcomes. The formation of chimeric proteins due to genomic rearrangements or at the post-transcriptional level is widespread and plays a critical role in tumor initiation and progression. Yet, the fusion landscape of lung cancer remains underexplored. We used the JAFFA pipeline to discover transcript fusions in early-stage non-small cell lung cancer (NSCLC). The set of detected fusions was further analyzed to identify recurrent events, genes with multiple partners and fusions with high predicted oncogenic potential. Finally, we used a generalized linear model (GLM) to establish statistical associations between fusion occurrences and clinicopathological variables. RNA sequencing was used to discover and characterize transcript fusions in 270 NSCLC samples selected from the Glans-Look specimen repository. The samples were obtained during the early stages of disease prior to the initiation of chemo- or radiotherapy. We identified a set of 792 fusions where 751 were novel, and 33 were recurrent. Four of the 33 recurrent fusions were significantly associated with clinicopathological variables. Several of the fusion partners were represented by well-established oncogenes ERBB4, BRAF, FGFR2, and MET. The data presented in this study allow researchers to identify, select, and validate promising candidates for targeted clinical interventions.

Discovery of fusion circular RNAs in leukemia with KMT2A::AFF1 rearrangements by the new software CircFusion.

Chromosomal translocations in cancer genomes, key players in many types of cancers, generate chimeric proteins that drive oncogenesis. Genomes with chromosomal rearrangements can also produce fusion circular RNAs (f-circRNAs) by backsplicing of chimeric transcripts, as first shown in leukemias with PML::RARα and KMT2A::MLLT3 translocations and later in solid cancers. F-circRNAs contribute to the oncogenic processes and reinforce the oncogenic activity of chimeric proteins. In leukemia with KMT2A::AFF1 (MLL::AF4) fusions, we previously reported specific alterations of circRNA expression, but nothing was known about f-circRNAs. Due to the presence of two chimeric sequences, fusion and backsplice junctions, the identification of f-circRNAs with available tools is challenging, possibly resulting in the underestimation of this RNA species, especially when the breakpoint is not known. We developed CircFusion, a new software tool to detect linear fusion transcripts and f-circRNAs from RNA-seq data, both in samples for which the breakpoints are known and when the information about the joined exons is missing. CircFusion can detect linear and circular chimeric transcripts deriving from the main and reciprocal translocations also in the presence of multiple breakpoints, which are common in malignant cells. Benchmarking tests on simulated and real datasets of cancer samples with previously experimentally determined f-circRNAs showed that CircFusion provides reliable predictions and outperforms available methods for f-circRNA detection. We discovered and validated novel f-circRNAs in acute leukemia harboring KMT2A::AFF1 rearrangements, leading the way to future functional studies aimed to unveil their role in this malignancy.

Engineering a two-gene system to operate as a highly sensitive biosensor or a sharp switch upon induction with β-estradiol

The human estrogen receptor has been used for about thirty years, in the yeast S. cerevisiae, as a component of chimeric transcription factors. Its ligand, β-estradiol, permits to control the protein translocation into the nucleus and, as a consequence, the expression of the gene(s) targeted by the synthetic transcription factor. Activators that are orthogonal to the yeast genome have been realized by fusing the human estrogen receptor to an activation and a DNA-binding domain from bacteria, viruses, or higher eukaryotes. In this work, we optimized the working of a β-estradiol-sensing device—in terms of detection range and maximal output signal—where the human estrogen receptor is flanked by the bacterial protein LexA and either the strong VP64 (from herpes simplex virus) or the weaker B42 (from E. coli) activation domain. We enhanced the biosensor performance by thoroughly engineering both the chimeric activator and the reporter protein expression cassette. In particular, we constructed a synthetic promoter—where transcription is induced by the chimeric activators—based on the core sequence of the yeast CYC1 promoter, by tuning parameters such as the length of the 5′ UTR, the distance between adjacent LexA binding sites (operators), and the spacing between the whole operator region and the main promoter TATA box. We found a configuration that works both as a highly sensitive biosensor and a sharp switch depending on the concentration of the chimeric activator and the strength of its activation domain.

Systematic multi-omics cell line profiling uncovers principles of Ewing sarcoma fusion oncogene-mediated gene regulation.

Ewing sarcoma (EwS) is characterized by EWSR1-ETS fusion transcription factors converting polymorphic GGAA microsatellites (mSats) into potent neo-enhancers. Although the paucity of additional mutations makes EwS a genuine model to study principles of cooperation between dominant fusion oncogenes and neo-enhancers, this is impeded by the limited number of well-characterized models. Here we present the Ewing Sarcoma Cell Line Atlas (ESCLA), comprising whole-genome, DNA methylation, transcriptome, proteome, and chromatin immunoprecipitation sequencing (ChIP-seq) data of 18 cell lines with inducible EWSR1-ETS knockdown. The ESCLA shows hundreds of EWSR1-ETS-targets, the nature of EWSR1-ETS-preferred GGAA mSats, and putative indirect modes of EWSR1-ETS-mediated gene regulation, converging in the duality of a specific but plastic EwS signature. We identify heterogeneously regulated EWSR1-ETS-targets as potential prognostic EwS biomarkers. Our freely available ESCLA (http://r2platform.com/escla/) is a rich resource for EwS research and highlights the power of comprehensive datasets to unravel principles of heterogeneous gene regulation by chimeric transcription factors.

Recurrent DNMT3B Alterations Are Associated with Unfavorable Outcome in Dicentric (9;20)-Positive Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia

The dicentric chromosome dic(9;20)(p11∼13;q11), occurs in 2% of the cases with pediatric acute lymphoblastic leukemia (ALL), almost exclusively of the B-cell lineage. Although previous cytogenetic studies have provided first insights into the genetic alterations in cases with dic(9;20), they were limited by the lack of sensitive genome-wide and next generation sequencing-based methods. Furthermore, the prognostic relevance of the dic(9;20) remains unclear, as previous studies have reported variable outcomes among these cases. Therefore, a better understanding of the (epi)genomic landscape and prognostic relevance of dic(9;20) ALL is essential to further optimize treatment of children with these alterations. The aim of this study was to unravel the demographic, clinical, prognostic and molecular characteristics of dic(9;20) in pediatric ALL. The present study investigates a cohort of 57 children with B-ALL, enrolled in the German ALL-BFM 2000, 2009, or 2017 trials, which underwent cytogenetic diagnostics in our center. Detection of dic(9;20) by conventional cytogenetic and molecular methods was the prerequisite for inclusion into the study. All 57 dic(9;20) cases, and 56 cases for which RNA samples were available, were subjected to genome-wide CGH+SNP array and targeted RNA-sequencing analysis. In addition, we performed whole transcriptome, whole genome and whole genome bisulfite sequencing for a selected number of cases. Event-free survival (EFS) was estimated according to Kaplan-Meier, and the curves were compared by log-rank test. Overall, the cohort consists of 22 male and 35 female children, with a median age at diagnosis of 3 years (range: 1-17 years). Patients were treated according to the AIEOP-BFM treatment protocols and stratified in the standard risk (n=15), medium risk (n=37) or high risk (n=5) treatment arms. All 57 patients achieved remission, 11 experienced relapses, while two patients in remission were lost during follow-up. Integrative analysis of RNA-seq and CGH+SNP array revealed recurrent genomic rearrangements of the DNMT3B gene with PAX5 or adjacent ZCCHC7 gene (n=3, each). DNMT3B is a de novo DNA methyltransferase and deletions of this gene have been previously reported in various solid malignancies and AML. The rearrangement of DNMT3B and ZCCHC7 resulted in an in-frame chimeric fusion transcript, containing the 5’ end of the open reading frame of DNMT3B and the 3’ end of ZCCHC7. In contrast, due to opposing orientation of the DNMT3B and PAX5 genes, the resulting chimeric transcript contained the 5’ end of the open reading frame of DNMT3B or PAX5 and a run-through transcript of the antisense strand of the partner gene. All genomic rearrangements with their breakpoints and fusion transcripts were validated using whole genome and Sanger sequencing. In the total group of six cases with rearrangements in the DNMT3B and PAX5/ZCCHC7 genes, four experienced relapses, resulting in a 5-year EFS of 25% (SE=20.4), compared to 75.4% (SE=8.2) for the remaining cohort (p=0.015). All six patients were treated according to the medium risk arm of the respective AIEOP-BFM 2000 or 2009 protocols (n=1 and n=5, respectively), and all showed good initial response to prednisone. Three patients had non-quantifiable levels of minimal residual disease after induction, while one of each had measurable levels ranging from 10-3-10-5. The unfavorable prognostic marker IKZF1plus was detected in four patients (66%), two of which relapsed. Since the DNMT3B gene functions as de novo methyltransferase, we examined whether genomic rearrangements in these cases can lead to global changes in methylation, but we did not observe either global hypermethylation, or global hypomethylation in dic(9;20) compared to cases of other B-ALL subtypes. In this study we describe novel recurrent genomic rearrangements involving the DNMT3B and PAX5/ZCCHC7 genes in pediatric ALL with dic(9;20). Although our observations should be confirmed in an independent cohort and larger series of patients, cases with rearrangements involving DNMT3B and PAX5/ZCCHC7 genes appear to have very poor outcome compared to other cases with dic(9;20). Once confirmed in an independent cohort, dic(9;20)-positive cases with rearrangements involving DNMT3B and PAX5/ZCCHC7 genes should be considered as high risk for relapse and treated accordingly.

Bone Marrow WT1 mRNA Upregulation in Acute Myeloid Leukemia (AML) in Complete Molecular Remission (mCR): Understanding the Limitations and Advantages of WT1 As a Measurable Residual Disease (MRD) Target

Introduction WT1 mRNA levels assessed by q-PCR have been employed as a marker of remission in AML patients undergoing intensive chemotherapy (Nomdedeu J. et al. Leukemia 2013;27:2157) and hematopoietic cell transplantation (HCT) (Nomdedeu J et al. Biol Blood Marrow Transplant 2018;24:55). Patients with adjusted bone marrow WT1 mRNA levels greater than 100 copies showed a high probability of relapse. Despite the usefulness of WT1 quantification in this and other settings, this marker is scarcely employed as an MRD target because levels greater than >100 copies can also be observed in normal bone marrows. Furthermore, 10-20% of AML at diagnosis and most chronic myelomonocytic leukemia (CMML) cases lack WT1 upregulation. The high levels of bone marrow WT1 copy numbers observed in most AML, decline in responding patients, although the utility, if any, of WT1 levels in long-term survivors of AML is currently unknown. We investigated the frequency and biological findings of mCR AML samples showing WT1 upregulation(>100 copies) compared to that mCR AML with concordant low WT1 levels. Patients and methods The study included 717 bone marrow samples from 80 AML patients (23 patients with RUNX1-RUNX1T1, 18 pts with CBFb-MYH11, four pts with NPM1 mutations, and 35 pts with PML-RARa). Samples were obtained at diagnosis (58 samples) and the follow-up (659 samples, all in mCR and without relapses) from one month to 219 months after diagnosis. Chimeric transcripts and WT1 levels were investigated by standardized real-time methods (ELN). Hemoglobin and leukocyte counts were also recorded. Flow cytometry immunophenotyping was also performed using four-color flow cytometry. We analyzed the percentage of CD34+, CD117+, CD123+ cells, and the Myeloid/Lymphoid CD34 ratio. nCounter Stem cell panel (Nanostring) was used to investigate the differences between that AML in mCR, showing high (>100) and low (<100) WT1 levels. In an additional cohort (11 samples: 4 with WT1>100 and 7 with WT1 <100), we performed bulk RNAseq (Novogene) to investigate enriched genes in samples with high WT1 levels. Results Thirty-five patients had at least one sample with WT1 levels greater than 100 copies (44%) despite being in mCR. Twelve samples had WT1 levels greater than 300 copies. In one case, this level reached 770 copies. These WT1 "peak" samples were distributed as follows: 49/306 PML-RARa, 25/161 RUNX1-RUNX1T1, 10/174 CBFb-MYH11, and 1/18 NPM1. Patients showing WT1 peaks were older than those without elevations (47 years vs. 36 years, p:0.003) For PML-RARa samples, "peaking" was associated with low levels of hemoglobin (133 g/L for <100 copies vs. 125 g/L >100 copies, p:0.009). For RUNX1-RUNX1T1, "peaking" was linked to less platelets (132x109 vs. 197x109 p:0.05) , higher CD34 (1.5% vs. 1% p:0.05) and also higher CD117+ cells (1.6 vs.1%, p:0.01). Lastly, CBFb-MYH11+ samples with WT1 peaks had higher leukocyte counts that those showing low WT1 levels (4.5x109 vs. 2.9x109, p:0.006). Nanostring comparisons between mCR WT1 high vs. low samples showed upregulation of TOR1A, PIK3CB, PTPN6, and PGM1 with downregulation of HMGA1, HOXB2 and CDC6. RNA seq analysis detected an enrichment of SUZ12 targets in those samples with high WT1 levels. Conclusions Bone marrow mRNA WT1 levels commonly exceed the clinical relevant value of 100 copies in AML samples in mCR (13%). Peak samples were related to peripheral blood values and/or raised numbers of immature bone marrow cells (CD34, CD117). Bone marrow WT1 although unable to accurately predict relapses in long-term survivors of AML may be a non-invasive sensor of a yet undefined myeloid state.

SRF Rearrangements in Soft Tissue Tumors with Muscle Differentiation.

The Serum Response Factor (SRF) is a transcription factor that regulates the expression of a wide set of genes involved in cell proliferation, migration, cytoskeletal organization and myogenesis. Accumulating evidence suggests that SRF may play a role in carcinogenesis and tumor progression in various neoplasms, where it is often involved in different fusion events. Here we investigated SRF rearrangements in soft tissue tumors, along with a gene expression profile analysis to gain insight into the oncogenic mechanism driven by SRF fusion. Whole transcriptome analysis of cell lines transiently overexpressing the SRF::E2F1 chimeric transcript uncovered the specific gene expression profile driven by the aberrant gene fusion, including overexpression of SRF-dependent target genes and of signatures related to myogenic commitment, inflammation and immune activation. This result was confirmed by the analysis of two cases of myoepitheliomas harboring SRF::E2F1 fusion with respect to EWSR1-fusion positive tumors. The recognition of the specific gene signature driven by SRF rearrangement in soft tissue tumors could aid the molecular classification of this rare tumor entity and support therapeutic decisions.

Sequence characterization of extracellular HBV RNA in patient plasma.

Antiviral nucleos(t)ide analogue therapies inhibit HBV replication and suppress the HBV DNA levels in patients with chronic HBV infection. Since HBV RNAs are expressed from cccDNA or HBV integrated sequences, independently of viral genome replication, levels of HBV RNAs in plasma may remain high following treatment with nucleos(t)ide analogue. Thus, HBV RNAs have been proposed to be used as a viral biomarker for treatment outcome and disease progression. Recent investigations of plasma HBV RNAs described the presence of full length as well as subgenomic forms of RNA. To support the usage of plasma HBV RNAs as a viral biomarker, further understanding of HBV RNA composition in clinical samples is needed. Here, sequence of extracellular HBV RNAs was characterized in plasma samples of patients with chronic HBV infection using two independent RNA amplification methods that do not use HBV-specific primers for amplification: total RNA (NuGEN RNAseq) and mRNA (TruSeq RNAseq). Sequencing coverage was obtained across the full length of HBV genome for both methods, confirming the presence of full-length HBV RNA in plasma. The sequence of HBV RNA was nearly identical to plasma HBV DNA sequence in each sample with only 0-14 (median 4) mismatches over 3kb. Thus, sequence of HBV RNA plasma reflects the intrahepatic viral reservoir and can be used for monitoring of sequence variants such as resistance in clinical trials. Additionally, RNA splice forms, different polyA tails start positions and presence of HBV-human chimeric transcript were identified.

Neomorphic DNA-binding enables tumor-specific therapeutic gene expression in fusion-addicted childhood sarcoma

Chimeric fusion transcription factors are oncogenic hallmarks of several devastating cancer entities including pediatric sarcomas, such as Ewing sarcoma (EwS) and alveolar rhabdomyosarcoma (ARMS). Despite their exquisite specificity, these driver oncogenes have been considered largely undruggable due to their lack of enzymatic activity.Here, we show in the EwS model that – capitalizing on neomorphic DNA-binding preferences – the addiction to the respective fusion transcription factor EWSR1-FLI1 can be leveraged to express therapeutic genes.We genetically engineered a de novo enhancer-based, synthetic and highly potent expression cassette that can elicit EWSR1-FLI1-dependent expression of a therapeutic payload as evidenced by episomal and CRISPR-edited genomic reporter assays. Combining in silico screens and immunohistochemistry, we identified GPR64 as a highly specific cell surface antigen for targeted transduction strategies in EwS. Functional experiments demonstrated that anti-GPR64-pseudotyped lentivirus harboring our expression cassette can specifically transduce EwS cells to promote the expression of viral thymidine kinase sensitizing EwS for treatment to otherwise relatively non-toxic (Val)ganciclovir and leading to strong anti-tumorigenic, but no adverse effects in vivo. Further, we prove that similar vector designs can be applied in PAX3-FOXO1-driven ARMS, and to express immunomodulatory cytokines, such as IL-15 and XCL1, in tumor entities typically considered to be immunologically ‘cold’.Collectively, these results generated in pediatric sarcomas indicate that exploiting, rather than suppressing, the neomorphic functions of chimeric transcription factors may open inroads to innovative and personalized therapies, and that our highly versatile approach may be translatable to other cancers addicted to oncogenic transcription factors with unique DNA-binding properties.

Abstract IA009: Oncogenic transcription factors drive highly tumor-specific LincRNA

Abstract Many cancers are characterized by gene fusions encoding oncogenic chimeric transcription factors (TFs) such as EWS::FLI1 in Ewing sarcoma (EwS). EWS::FLI1 induces a complete reprogramming of mesenchymal stem cells, the putative cell of origin of Ewing sarcoma, through binding and subsequent generation of thousands of neoenhancers at GGAA microsatellite repeats. We show that EWS::FLI1 induces the robust expression of a specific set of novel spliced and polyadenylated transcripts within otherwise transcriptionally silent regions of the genome. These neo-genes (NGs) are virtually undetectable in large collections of normal tissues or non-EwS tumors and can be silenced by CRISPR interference at regulatory EWS::FLI1-bound microsatellites. Ribosome profiling and proteomics further show that some NGs are translated into highly EwS-specific peptides. More generally, hundreds of NGs can be detected in diverse cancers characterized by chimeric TFs. Altogether, we identify the transcription, processing, and translation of novel, specific, highly expressed multi-exonic transcripts from otherwise silent regions of the genome as a new activity of aberrant TFs in cancer. These neotranscripts, which can be classified as LincRNAs, or neopeptides may have specific functions in the tumor cells. The latter may also constitute ideal neoepitopes as they are tumor-specific, clonally expressed in sarcoma cells, induced by the driver genetic alterations and public for a given sarcoma. Citation Format: Olivier Delattre, Julien Vibert, Olivier Saulnier, Joshua Waterfall. Oncogenic transcription factors drive highly tumor-specific LincRNA [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr IA009.