Chimeric Fusion Gene Research Articles

RNA Sequencing for Solid Tumor Fusion Gene Detection: Proficiency Testing Practice and Performance Comparison.

Next-generation sequencing-based approaches using RNA have increasingly been used by clinical laboratories for the detection of fusion genes, intragenic rearrangements, and exon-skipping events. Correspondingly, the College of American Pathologists (CAP) has advanced RNA sequencing proficiency testing (PT) to ensure optimal performance of these assays. To report on laboratory performance and practices of RNA sequencing for the detection of fusion genes, intragenic rearrangements, and exon-skipping events using CAP PT data from 8 mailings (2018-A through 2021-B). CAP PT RNA sequencing program results from 153 laboratories across 24 proficiency test specimens, interrogating 22 distinct engineered fusion transcripts, were analyzed for correct identification of the fusion event, associated performance variables, and laboratory practices. Overall, the 4-year program detection rate (sensitivity) was 95.5% (1486 of 1556 results). False-negative rates were 3.6% (53 of 1463) and 18.3% (17 of 93) for fusion gene and intragenic rearrangement/exon-skipping events, respectively. Only 19 false-positive results were reported among the 8 PT mailings, and most were likely the result of preanalytical or postanalytical errors. There were no practice characteristics (eg, instrumentation, sequencing method) significantly associated with the fusion detection results. These data reveal a high overall sensitivity and specificity for fusion gene detection by participating laboratories using clinical RNA sequencing. Performance was comparable across all laboratories, regardless of methodology. The fraction of false-negative results for intragenic rearrangement/exon-skipping events was greater than that for the chimeric fusion genes. False-negative results could not be attributed to any specific practice characteristics.

Advances in molecular characterization of pediatric acute megakaryoblastic leukemia not associated with Down syndrome; impact on therapy development.

Acute megakaryoblastic leukemia (AMKL) is a rare subtype of acute myeloid leukemia (AML) in which leukemic blasts have megakaryocytic features. AMKL makes up 4%-15% of newly diagnosed pediatric AML, typically affecting young children (less than 2years old). AMKL associated with Down syndrome (DS) shows GATA1 mutations and has a favorable prognosis. In contrast, AMKL in children without DS is often associated with recurrent and mutually exclusive chimeric fusion genes and has an unfavorable prognosis. This review mainly summarizes the unique features of pediatric non-DS AMKL and highlights the development of novel therapies for high-risk patients. Due to the rarity of pediatric AMKL, large-scale multi-center studies are needed to progress molecular characterization of this disease. Better disease models are also required to test leukemogenic mechanisms and emerging therapies.

'Evaluation of adverse prognostic gene alterations & MRD positivity in BCR::ABL1-like B-lineage acute lymphoblastic leukaemia patients, in a resource-constrained setting.

Early detection of BCR::ABL1-like ALL could impact treatment management and improve the overall survival/outcome. BCR::ABL1-like ALL cases are characterised by diverse genetic alterations activating cytokine receptors and kinase signalling. Its detection is still an unmet need in low-middle-income countries due to the unavailability of a patented TLDA assay. This study's rationale is to identify BCR::ABL1-like ALLs using the PHi-RACE classifier, followed by the characterisation of underlying adverse genetic alterations in recurrent gene abnormalities negative (RGAneg) B-ALLs (n = 108). We identified 34.25% (37/108) BCR::ABL1-like ALLs using PHi-RACE classifier, characterised by TSLPR/CRLF2 expression (11.58%), IKZF1 (Δ4-7) deletion (18.9%) and chimeric gene fusions (34.61%). In overexpressed TSLPR/CRLF2 BCR::ABL1-like ALLs, we identified 33.33% (1/3) CRLF2::IGH and 33.33% (1/3) EPOR::IGH rearrangements with concomitant JAK2 mutation R683S (50%). We identified 18.91% CD13 (P = 0.02) and 27.02% CD33 (P = 0.05) aberrant myeloid markers positivity, which was significantly higher in BCR::ABL1-like ALLs compared to non-BCR::ABL1-like ALLs. MRD positivity was considerably higher (40% in BCR::ABL1-like vs. 19.29% in non-BCR::ABL1-like ALLs). With this practical approach, we reported a high incidence of BCR::ABL1-like ALLs, and a lower frequency of CRLF2 alteration & associated CGFs. Recognising this entity, early at diagnosis is crucial to optimise personalised treatment strategies.

Analysis of breast cancer next-generation sequencing datasets for identifying fusion genes responsible for the cancer progression

Structural Variants (SVs) and Copy Number Variations (CNVs) at the genomic level are responsible for forming chimeric genes at the transcriptome level, posing significant implications in cancers. In this study, we performed an integrated analysis of the SVs, CNVs and fusion genes in breast cancer sequencing datasets to predict driver fusion genes using paired RNA and WGS (Whole Genome Sequencing) datasets. The combined effect of genomic fusions, chromosomal rearrangements and CNV can help analyze the highly perturbed cancer genome and how they work together, leading to cancer progression. Our analysis yielded chimeric fusion genes supported by multiple events, such as trans-splicing at the transcriptome level, formed due to CNV variation and SVs at the genomic level. We discovered novel gene fusions, such as RARA-PKIA in the SK-BR3 cell line and PLA2R1-RBMS1 in the HCC1395BL cell line. The analysis pipeline of the study can be utilized to explore the other less investigated cancer genomes, which may also help develop diagnostics and better cancer therapeutics, especially for personalized treatments.

AML-336 M5b AML With t(8;16) (p11.2; p13.3) KAT6A/CREBBP Fusion and FLT3-TKD Mutation: Analysis of Mitelman Database

t(8;16) (p11.2; p13.3)/KAT6A-CREBBP fusion is a rare cytogenetic abnormality usually associated with treatment-related AML (t-AML) that usually exhibits unique characteristics while accounting for <0.5% cases of AML and being more common in female patients. Reported overall survival (OS) in these patients is poor and varies from 4.7 to 18.2 months. Patients with de novo AML with t(8;16) or t-AML with t(8;16) without adverse prognostic factors (e.g., complex karyotype) have a good outcome. We present a case of AML with a rare translocation abnormality, t(8;16), which is usually associated with cytotoxic therapies, but there was no prior history in our patient. The translocation results in KAT6A/CREBBP fusion. Immunophenotyping revealed expected myeloid and monocytic markers with CD45 (dim), cMPO, CD56 (partial), CD71, and CD38. The patient was started on a 7+3 induction regimen with cytarabine and daunorubicin. Midostaurin was added due to FLT3-TKD mutation. She was found to be disease free on a follow-up marrow biopsy, and she received a single cycle of HiDAC for maintenance. On a subsequent visit, she was found to have relapsed, so a second-line therapy with FLAG-IDA was considered. Day 17 bone marrow biopsy was performed with no flow cytometric evidence of leukemic cells, with a CR+MRD negative status, so she was discharged with follow-up at the BMT clinic for allogeneic stem cell transplant. KAT6A and CREBBP genes produce transcripts that are involved in histone acetylation. It is unknown whether the chimeric fusion gene produces a functional transcript that could contribute to leukemogenesis. Our Mitelman database analysis reveals only 175 reported cases; furthermore, this is the first patient who had an FLT3-TKD mutation. A vast majority of these are either M4 or M5 types (132/175, 75.4%). OS is poor, ranging from 4.7 to 18.2 months. This AML type exhibits unique characteristics such as hemophagocytosis, DIC, leukemia cutis, and monoblastic or myelomonocytic differentiation. Mitelman database analysis reveals that a vast majority of cases with t(8;16) are either M4 or M5 AML. Careful consideration of treatment options would be recommended based on age as chromosomal abnormalities play a major role in prognosis, especially with t(8;16).

ALL-061 Delineating the Genomic and Proteomic Landscape of BCR-ABL1-like Acute Lymphoblastic Leukemia Subtype: Bench-to-Bedside Translational Research

Baseline detection of Ph/BCR-ABL1-like-ALL cases is extremely beneficial for hematologists/physicians to design personalized medicine and to treat these patients efficiently. Worldwide, researchers use patented TaqMan low-density array (TLDA) gene expression profiling (GEP) to identify these cases. In developing countries, its detection is very challenging and expensive due to the unavailability of patented TLDA and there is a paucity of literature on the identification and characterization of Ph-like ALL cases. Stepwise identification followed by genomic and proteomic characterization of Ph-like ALL cases. This study was conducted at PGIMER, India (2016-2021). Flowcytometric-immunophenotyping and multiplex RT-PCR was performed to detect four recurrent fusion transcripts (n=1021). Transcriptome GEP was performed on 12 Ph-positive ALL cases. Ph-like-ALL cases identified using nCounter Platform (n=96) and RQ-PCR (n=536) were characterized using FISH, DD-PCR, RQ-PCR and MRD-positivity. Nine HKGs' expression was evaluated using 6 algorithms. Furthermore, we built and validated PHi-RACE classifier (n=629 cases) for its rapid detection. Lastly, we delineated the proteomic landscape of Ph-like ALL cases using LC-MS/MS. We studied 1021 treatment-naive and newly diagnosed B-ALL cases in this study cohort. Multiplex assay revealed 18.66% BCR-ABL1, 3.85% TCF3-PBX1, 5.27% ETV6-RUNX1, and 1.21% KMT2A-AFF1. Using transcriptome GEP, we identified 32.05% Ph-like-ALL cases using NanoString and 26.67% using RQ-PCR. These cases were substantially older and represented higher MRD positivity. In CRLF2 subgroup of Ph-like ALLs, we identified chimeric gene fusions including 10% JAK2 rearrangement, 8.33% CRLF2-IGH and CRLF2-P2RY8 translocation with concomitant R683S mutation. We found 8.33% IKZF1 (Δ4-7) deletion using DD-PCR. Furthermore, we developed an online logistic classifier entitled PHi-RACE (sensitivity=95.20%, specificity=83.70%, cut-off >0.28) for its detection using logistic regression. We have validated PHi-RACE classifier (n=93) and identified 56 Ph-like ALL cases. Six algorithms' findings revealed that EEF2, GAPDH and PGK1 represent the best HKGs for its detection. Lastly, from LC-MS/MS findings, we identified 3079 proteins, spliceosomal mediated mRNA splicing pathway and four miRNAs were significantly expressed in Ph-like ALL cases. The authors created and published the PHi-RACE classifier to detect Ph-like ALL cases, which has direct clinical implications. Further, we have delineated the genomic and proteomic landscape of Ph-like-ALL for the first time from developing countries.

Abstract A027: Characterization of WEE1 kinase activity in myxoid liposarcoma

Abstract Introduction: Myxoid liposarcomas (MLS), malignant soft tissue tumors of adipocyte origin, are genetically characterized by a chromosomal t(12;16)(q13;p11) translocation encoding the chimeric FUS-DDIT3 fusion gene. The resulting fusion protein drives MLS pathogenesis via (dys-)regulation of oncogenic signaling pathways. Since FUS-DDIT3 is not selectively antagonizable, counteracting the oncogenic effects of FUS-DDIT3 fusion protein represents the most promising strategy to target MLS cells. In this study, we identified cell cycle checkpoint kinase WEE1 as FUS-DDIT3 depending effector and investigated the functional requirement for WEE1 kinase activity in MLS pathogenesis. Experimental Procedures: Characterization of WEE1 expression and kinase activity was performed in multiple MLS cell lines, cell lines derived from other liposarcoma subtypes and a mesenchymal stem cell system. Modulation of WEE1 signaling was carried out by means of small-molecule inhibitor Adavosertib (MK-1775) and RNA interference (RNAi)-mediated depletion, and effects were analyzed in immunoblots, cell proliferation assays and caspase 3/7 activity-based apoptosis assays in vitro. Results: Functional genomic RNAi screening uncovered dependence of FUS-DDIT3-expressing mesenchymal stem cells and MLS cell lines on WEE1 kinase activity. Additional expression analysis revealed increased WEE1 protein levels in MLS cell lines. Moreover, pharmacologic inhibition of WEE1 demonstrated significant reduction of MLS cell viability. Accordingly, functional loss of WEE1 by inhibition or RNAi-mediated depletion was found to induce DNA damage accompanied by unscheduled mitotic entry and cell death via activation of the apoptotic program in MLS cells. Conclusions: Our results identify WEE1 kinase activity as functional liability of FUS-DDIT3 expressing MLS cells and provide first evidence that overactive WEE1 signaling represents a promising target for therapeutic intervention in MLS. Citation Format: Lorena Heinst, Ruth Berthold, Ilka Isfort, Svenja Wosnig, Thomas Kindler, Pierre Åman, Eva Wardelmann, Claudia Scholl, Stefan Fröhling, Wolfgang Hartmann, Marcel Trautmann. Characterization of WEE1 kinase activity in myxoid liposarcoma [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 A027.

Abstract PR004: Fusion protein-driven IGF-IR signals deregulate hippo pathway promoting oncogenic cooperation of YAP1 and FUS-DDIT3

Abstract Introduction: Myxoid liposarcoma (MLS) is molecularly characterized by a recurrent chromosomal translocation which generates a chimeric FUS-DDIT3 fusion gene. The FUS-DDIT3 oncoprotein, acting as a transcriptional dysregulator, has been shown to be essential in MLS pathogenesis, among others through deregulation of IGF-IR/PI3K/AKT signaling, but its exact mode of function remains incompletely understood. Recently, a particular reliance on the Hippo pathway effector and transcriptional co-regulator YAP1 was found in MLS; however, the molecular mechanism of FUS-DDIT3-dependent YAP1 activation and its contribution to MLS pathogenesis remain unclear. Experimental Procedures: The expression of IGF-IR and YAP1 was analyzed in a large cohort of MLS specimens by immunohistochemistry. In vitro analyses were performed employing a human mesenchymal stem cell system stably expressing FUS-DDIT3 and human MLS cell lines. RNA interference-based approaches, experiments with small-molecule kinase inhibitors, co-immunoprecipitation, proximity ligation assays, transcriptome sequencing and adipogenic differentiation assays were performed to determine the interplay of FUS-DDIT3, IGF-IR-dependent signals, and YAP1 in MLS cells. Results: Immunohistochemically, a significant subset of MLS samples showed concurrent expression of IGF-IR and nuclear YAP1. In vitro, FUS-DDIT3-driven IGF-IR signaling was found to promote stability and nuclear accumulation of YAP1 via deregulation of the Hippo pathway. Co-immunoprecipitation and proximity ligation assays revealed nuclear co-localization of FUS-DDIT3 and YAP1 in FUS-DDIT3-expressing mesenchymal stem cells and MLS cell lines. Transcriptome sequencing of MLS cells demonstrated that FUS-DDIT3 and YAP1 co-regulate specific oncogenic gene signatures related to proliferation, cell cycle progression, apoptosis, and adipogenesis. In differentiation assays, FUS-DDIT3 and YAP1 were found to cooperate in adipogenic differentiation arrest. Conclusions: Our study provides molecular insights into a complex FUS-DDIT3-driven network involving IGF-IR signals acting on Hippo/YAP1, and uncovers cooperative effects of YAP1 and FUS-DDIT3 in the pathogenesis of MLS. Citation Format: Ruth Berthold, Ilka Isfort, Cihan Erkut, Lorena Heinst, Inga Grünewald, Eva Wardelmann, Thomas Kindler, Pierre Åman, Thomas G.P. Grünewald, Florencia Cidre-Aranaz, Marcel Trautmann, Stefan Fröhling, Claudia Scholl, Wolfgang Hartmann. Fusion protein-driven IGF-IR signals deregulate hippo pathway promoting oncogenic cooperation of YAP1 and FUS-DDIT3 [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 PR004.

Fusion protein-driven IGF-IR/PI3K/AKT signals deregulate Hippo pathway promoting oncogenic cooperation of YAP1 and FUS-DDIT3 in myxoid liposarcoma

Myxoid liposarcoma (MLS) represents a common subtype of liposarcoma molecularly characterized by a recurrent chromosomal translocation that generates a chimeric FUS-DDIT3 fusion gene. The FUS-DDIT3 oncoprotein has been shown to be crucial in MLS pathogenesis. Acting as a transcriptional dysregulator, FUS-DDIT3 stimulates proliferation and interferes with adipogenic differentiation. As the fusion protein represents a therapeutically challenging target, a profound understanding of MLS biology is elementary to uncover FUS-DDIT3-dependent molecular vulnerabilities. Recently, a specific reliance on the Hippo pathway effector and transcriptional co-regulator YAP1 was detected in MLS; however, details on the molecular mechanism of FUS-DDIT3-dependent YAP1 activation, and YAP1´s precise mode of action remain unclear. In elaborate in vitro studies, employing RNA interference-based approaches, small-molecule inhibitors, and stimulation experiments with IGF-II, we show that FUS-DDIT3-driven IGF-IR/PI3K/AKT signaling promotes stability and nuclear accumulation of YAP1 via deregulation of the Hippo pathway. Co-immunoprecipitation and proximity ligation assays revealed nuclear co-localization of FUS-DDIT3 and YAP1/TEAD in FUS-DDIT3-expressing mesenchymal stem cells and MLS cell lines. Transcriptome sequencing of MLS cells demonstrated that FUS-DDIT3 and YAP1 co-regulate oncogenic gene signatures related to proliferation, cell cycle progression, apoptosis, and adipogenesis. In adipogenic differentiation assays, we show that YAP1 critically contributes to FUS-DDIT3-mediated adipogenic differentiation arrest. Taken together, our study provides mechanistic insights into a complex FUS-DDIT3-driven network involving IGF-IR/PI3K/AKT signals acting on Hippo/YAP1, and uncovers substantial cooperative effects of YAP1 and FUS-DDIT3 in the pathogenesis of MLS.

Identification of EWSR1 rearrangements in patients with immature hematopoietic neoplasms: A case series and review of literature

Rearrangement of the EWSR1 gene (22q12.2) is a well-recognized genetic lesion in bone and soft tissue tumors. However, few reports have suggested that EWSR1 rearrangements may also occur in the setting of hematopoietic tumors. We herein describe two cases of immature hematopoietic neoplasms presenting with EWSR1 rearrangements. The first occurred in a 41-year-old female diagnosed with mixed-phenotype acute leukemia, B/T/myeloid, in which conventional chromosome analysis revealed a t(2;22)(q35;q12). Further analysis with whole genome sequencing revealed that this rearrangement led to an EWSR1::FEV gene fusion. The second case was identified in an 18-year-old male with a high-grade B-cell lineage malignant neoplasm with immature features in which conventional chromosome analysis revealed a t(17;22)(q25;q12). Mate-pair sequencing, a next generation sequencing-based assay, was performed and revealed three in-frame chimeric gene fusions involving the EWSR1, TEF and STRADA gene regions. This report further expands the repertoire of hematopoietic neoplasms with EWSR1 fusions and partner genes involved in these rearrangements.

Chimeric RNA Design Principles for RNA-Mediated Gene Fusion

One common genetic alteration in cancer is gene fusion resulting from chromosomal translocations. The mechanisms that create such oncogenic fusion genes are not well understood. Previously, we provided the direct evidence that expression of a designed chimeric RNA can drive the formation of TMPRSS2-ERG gene fusion. Central to this RNA-mediated gene fusion mechanism is a proposed three-way junction formed by RNA/DNA hybrid and the intergenic DNA stem formed by target genes. In this study, we determined the important parameters for chimeric RNA-mediated gene fusion using TMPRSS2-ERG fusion gene as the model. Our results indicate that both the chimeric RNA lengths and the sizes of unpaired bulges play important roles in inducing TMPRSS2-ERG gene fusion. The optimal length of unpaired bulges was about 35 nt, while the optimal chimeric RNA length was about 50 nt for targeting. These observations were consistent regardless of the target locations within TMPRSS2 and ERG genes. These empirically determined parameters provide important insight for searching cellular RNAs that may initiate oncogenic fusion genes. The knowledge could also facilitate the development of useful genomic technology for manipulating mammalian genomes.

3173 – HUMAN MODELS OF PEDIATRIC CBFA2T3-GLIS2 ACUTE MEGAKARYOBLASTIC LEUKEMIA CONTRIBUTE TO UNCOVER THERAPEUTIC VULNERABILITIES

Acute megakaryoblastic leukemia (AMKL) is a poor prognostic subtype of AML afflicting children mostly under 3 years of age. Chromosomal translocations generating chimeric fusion genes are considered critical transforming events and are present in a high proportion of cases. Among them, the CBFA2T3-GLIS2 (CG2) fusion is associated with a high risk of relapse and accounts for ∼15% of pediatric AMKL cases. Using overexpression of CG2 in human cord blood CD34+ stem /progenitor cells (hHSPC) followed by xenotransplantation in immunodeficient mice, we are reporting the generation of multiple synthetic AMKL models that phenocopy the human disease in term of immunophenotype, morphology and gene expression. CG2 models revealed a stable genomic landscape with few copy number anomalies and lack of recurrent cooperating mutations. Leveraging from local and published transcriptomic datasets of CG2 AMKL samples (14 patients, 10 models), non-CG2 pediatric AMKL (n=66), NUP98-KDM5A synthetic models (n=5) and normal hHSPC (n=4), we have determined a robust gene expression signature defining CG2 AMKL. Gene set enrichment analysis of CG2 AMKL expression profile identified the JAK2-STAT5 axis as a potential pathway that is upregulated in CG2 leukemia, confirmed by downstream STAT5 phosphorylation in CG2 cells. Cell surface proteomic analysis by LC-MS identified multiple membrane proteins upstream of JAK-STAT signaling in CG2 model leukemia compared to normal hHSPC. Single-agent dose-response curves conducted with multiple FDA-approved drugs targeting JAK2 showed specific grow inhibition of CG2 AMKL models compared to normal hHSPC and non-AMKL cell line. Progress in leukemia research is hampered by the paucity of patient samples and the generated AMKL models pave the way toward a better understanding of leukemogenesis and development of targeted therapy. Acute megakaryoblastic leukemia (AMKL) is a poor prognostic subtype of AML afflicting children mostly under 3 years of age. Chromosomal translocations generating chimeric fusion genes are considered critical transforming events and are present in a high proportion of cases. Among them, the CBFA2T3-GLIS2 (CG2) fusion is associated with a high risk of relapse and accounts for ∼15% of pediatric AMKL cases. Using overexpression of CG2 in human cord blood CD34+ stem /progenitor cells (hHSPC) followed by xenotransplantation in immunodeficient mice, we are reporting the generation of multiple synthetic AMKL models that phenocopy the human disease in term of immunophenotype, morphology and gene expression. CG2 models revealed a stable genomic landscape with few copy number anomalies and lack of recurrent cooperating mutations. Leveraging from local and published transcriptomic datasets of CG2 AMKL samples (14 patients, 10 models), non-CG2 pediatric AMKL (n=66), NUP98-KDM5A synthetic models (n=5) and normal hHSPC (n=4), we have determined a robust gene expression signature defining CG2 AMKL. Gene set enrichment analysis of CG2 AMKL expression profile identified the JAK2-STAT5 axis as a potential pathway that is upregulated in CG2 leukemia, confirmed by downstream STAT5 phosphorylation in CG2 cells. Cell surface proteomic analysis by LC-MS identified multiple membrane proteins upstream of JAK-STAT signaling in CG2 model leukemia compared to normal hHSPC. Single-agent dose-response curves conducted with multiple FDA-approved drugs targeting JAK2 showed specific grow inhibition of CG2 AMKL models compared to normal hHSPC and non-AMKL cell line. Progress in leukemia research is hampered by the paucity of patient samples and the generated AMKL models pave the way toward a better understanding of leukemogenesis and development of targeted therapy.

Abstract 2003: Oncogenic interplay of FUS-DDIT3 and YAP1 in myxoid liposarcoma

Abstract Introduction: Myxoid liposarcoma (MLS) represents the second most common subtype of liposarcoma. MLS is characterized by a chromosomal translocation t(12;16)(q13;p11) encoding a chimeric FUS-DDIT3 fusion gene. The resulting FUS-DDIT3 oncoprotein acts as a transcriptional dysregulator that was recently shown to mediate (i) IGF-IR/PI3K/AKT signaling and (ii) aberrant activation of the Hippo pathway effector YAP1 in MLS. This study was performed to analyze the functional interplay between IGF-IR/PI3K/AKT signals and aberrant YAP1 activity in MLS, aiming at a better functional understanding of MLS and the identification of specific molecular vulnerabilities. Experimental procedures: Immunohistochemical stainings of IGF-IR, IGF-II and YAP1 were performed in a cohort of MLS specimens (n=45). To study FUS-DDIT3-dependency in vitro, SCP-1 mesenchymal stem cells stably expressing FUS-DDIT3, and MLS cell lines expressing a doxycycline-inducible shRNA against FUS-DDIT3 were employed. Interactions between the IGF-IR/PI3K/AKT and Hippo/YAP1 pathways were investigated using RNAi approaches as well as the small molecule compounds BMS-754807 and BKM120; effects were analyzed by immunoblotting and TEAD luciferase reporter assays. To determine the impact of YAP1 in FUS-DDIT3-mediated oncogenic effects, qPCR analysis and adipogenic differentiation assays were performed. Results: Immunohistochemical analysis of human MLS tissue specimens demonstrated that expression of IGF-II and IGF-IR is associated with concomitant nuclear expression of YAP1 in a significant subset of MLS. Both, IGF-IR-dependent signals and YAP1 expression were shown to be functionally dependent on FUS-DDIT3. In MLS cell lines, inhibition of the IGF-IR/PI3K/AKT signaling cascade promoted downregulation of YAP1, accompanied by reduced TEAD transcriptional activity. Employing qPCR analyses, YAP1 was shown to co-regulate FUS-DDIT3 transcriptional targets and to be functionally involved in FUS-DDIT3-driven disruption of normal adipocytic differentiation. Conclusions: Our study provides evidence of a complex regulatory interplay in MLS with FUS-DDIT3-driven IGF-IR/PI3K/AKT signals acting as activators of nuclear YAP1 expression. Conversely, YAP1 contributes to shape FUS-DDIT3 effects on the MLS transcriptional landscape and functionally adds to an immature non-lipogenic phenotype. Our data contribute to the understanding of MLS biology and reveal specific molecular liabilities to be considered in therapeutic approaches of MLS. Citation Format: Ruth Berthold, Ilka Isfort, Jonas Breuer, Lorena Heinst, Thomas Kindler, Pierre Åman, Inga Grünewald, Eva Wardelmann, Thomas G. Grünewald, Florencia Cidre-Aranaz, Claudia Scholl, Stefan Fröhling, Marcel Trautmann, Wolfgang Hartmann. Oncogenic interplay of FUS-DDIT3 and YAP1 in myxoid liposarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2003.

Overcoming Tyrosine Kinase Inhibitor Resistance to SEPT9-ABL1 Chimeric Fusion Protein Using CRM1 Inhibitors

ABL1 fusion genes, which have been reported in various hematological malignancies, are thought to contribute to leukemogenesis through aberrant ABL1 signaling and activating multiple downstream signals. Tyrosine kinase inhibitors (TKIs), such as imatinib and dasatinib, have been developed to inhibit the function of the most- characterized ABL1 fusion product, BCR-ABL1. Because TKIs target the ABL1 kinase domain, ABL1 fusion products are usually sensitive to TKIs both in vitro and in vivo .We previously identified the chimeric fusion gene SEPT9 - ABL1 in a case of T-prolymphocytic leukemia that was unexpectedly resistant to imatinib and dasatinib without mutations in the domains derived from ABL1. We proved that SEPT9-ABL1 exhibited TKI resistance in vitro and in vivo, as seen in theclinical course (Leuk Res 2014). The purpose of this study was to reveal the mechanism underlying TKI resistance and to overcome it in a specific way.SEPT9f-ABL1, which has the most typical characteristics among SEPT9 isoforms, was retrovirally transduced into interleukin-3 (IL-3)-dependent murine hematopoietic cell lines 32D and BaF3 cells to generate 32D/SEPT9-ABL1 and BaF3/SEPT9-ABL1 cells. 32D/BCR-ABL1 and BaF3/BCR-ABL1 cells were also generated as references. The 50% inhibitory concentration (IC50) of imatinib was higher with SEPT9-ABL1 than with BCR-ABL1 (in 32D cells and BaF3 cells, respectively), confirming that SEPT9-ABL1 contributed to TKI resistance.Because TP53 has been suggested to involved in TKI resistance, we first analyzed the TP53 expression and phosphorylation in these cells. Western blot revealed that TP53 was downregulated and less phosphorylated in the cells expressing SEPT9-ABL1 than in those with BCR-ABL1. These findings were consistent with the analysis using primary leukemic cells. Imatinib treatment reduced the TP53 expression in the cells expressing SEPT9-ABL1. When the cell pellets were fractioned, the TP53 expression in the cytoplasm was comparable between cells expressing SEPT9-ABL1 and those with BCR-ABL1 under imatinib treatment. However, the TP53 expression remained higher in the nucleus of the cells with BCR-ABL1 than in those with SEPT9-ABL1, suggesting that the decreased nuclear TP53 expression prevented the cells expressing SEPT9-ABL1 from undergoing apoptosis induced by imatinib.Because the distribution of TP53 in the nucleus and the cytoplasm is controlled by chromosomal region maintenance 1 (CRM1), a nuclear export receptor, we next evaluated the effects of CRM1 inhibition. When the cells were cultured with the combination of imatinib (1 µM) and KPT-185 (1 µM), TP53 was markedly accumulated in the cells expressing SEPT9-ABL1; however, no such accumulation was noted when cells were cultured with imatinib alone, suggesting that KPT-185 treatment enhanced the effect of imatinib in the cells expressing SEPT9-ABL1 through the inhibition of TP53 export from the nucleus. This combination dramatically increased the apoptotic cells with SEPT9-ABL1. To evaluate CRM1 inhibition in vivo, we utilized a subcutaneous tumor model with BaF3/SEPT9-ABL1 cells. Treatment with imatinib 20 mg/kg plus another CRM1 inhibitor (KPT-330, 20 mg/kg) led to the significant regression of tumors composed of BaF3/SEPT9-ABL1 cells compared to imatinib alone (p=0.029).In conclusion, decreased nuclear expression of TP53 is a possible mechanism underlying TKI resistance in cells harboring SEPT9-ABL1. CRM1 inhibition is an effective method of overcoming TKI resistance in cells with SEPT9-ABL1 through induction of the nuclear accumulation of TP53 and apoptosis. [Display omitted] DisclosuresAndo:NOVARTIS: Other: Donation to institute; Meiji Seika Pharma: Other: Donation to institute; CHUGAI PHARMACEUTICAL: Other: Donation to institute; TOYAMA CHEMICAL: Other: Donation to institute; MOCHIDA PHARMACEUTICAL: Other: Donation to institute; Sumitomo Dainippon Pharma: Other: Donation to institute; Eisai: Other: Donation to institute; Bristol-Myers Squibb: Other: Donation to institute; MSD: Other: Donation to institute; Kyowa Hakko Kirin: Other: Donation to institute; ALEXION: Other: Donation to institute; Takeda: Other: Donation to institute; Japan Blood Products Organization: Other: Donation to institute; Nippon Shinyaku: Other: Donation to institute; NIHON PHARMACEUTICAL: Other: Donation to institute; TAIHO: Other: Donation to institute; Asahi KASEI: Other: Donation to institute.

Late Events in Adult Patients with ALK-Positive Anaplastic Large Cell Lymphoma

ALK-positive anaplastic large cell lymphoma is a lymphoproliferative disorder of “young age”; its incidence among all non-Hodgkin lymphomas in adult patient cohort is 2-15%. Cytogenetic aberrations associated with ALK rearrangements mainly include t(2;5)(p23;q32) and the chimeric fusion gene NPM-ALK, while less than 10-15% of cases are associated with variant translocations involving 2p23 and resulting in formation of rare oncogenes.Favorable prognosis in patients with this type of the disorder who receive high-dose chemotherapy regimens (NHL BFM-90) is associated with 75%-92% overall and event-free survival rates, including in adult patients. Therapy failures are generally diagnosed during the first 1.5-2 years of follow-up. The risk of development of secondary tumor lesions following high-dose chemotherapy does not exceed 10% (in children).The goal of the study was to evaluate the development of late events (relapse, secondary tumors) in 52 adult patients (median age 33 years) with ALK-positive anaplastic large cell lymphoma who had received unified program of high-dose chemotherapy according to NHL BFM-90 protocol.The results: this therapy program was associated with 83% and 79% overall and event-free survival rates, respectively (median follow-up - 83 months), early relapses (median time to development 12 months) were verified in 3 patients (6%), disease progression - in 3 (6%) cases. Three patients developed late events (>5.5 years following the end of treatment). In two cases, a tumor relapse was diagnosed: in one patient who initially had stage IV disease and extranodal lesions in the skin and soft tissues - after 5.5 years of follow-up; and in another patient - after 7 years of follow-up. Peculiar features of the first clinical case - minimal disseminated disease was revealed at the moment of diagnosis verification, with the ATIC-ALK chimeric transcript detected in peripheral blood and bone marrow (which was consistent with a variant translocation, inv (2)(p23;q35)); no chimeric oncogene has been detected after the courses of induction therapy and during subsequent years of follow-up. In the second case, at the moment of the relapse verification, the ATIC-ALK transcript was detected again in the specimens of bone marrow and peripheral blood, indicating the development of a distant relapse from the identical primary tumor clone.The third late event (diagnosed in >11 years after the end of chemotherapy) was the development of epithelioid sarcoma with primary spleen involvement.Thus, high-dose first-line chemotherapy in adult patients with ALK+ ALGL has proven to be highly effective. Early relapses and progressions were verified not more than in 6% of cases. The characteristics of these late events are of special interest: a relapse in the form of development of new skin lesions (as was observed at the time of the disease manifestation); in the second case - relapse from the identical tumor cell clone in 7 years, and, finally, the development of a rare secondary tumor (epithelioid sarcoma) together with non-conventional clinical course (tumor nodes in the spleen and retroperitoneal lymphadenopathy). DisclosuresNo relevant conflicts of interest to declare.

SETBP1 overexpression acts in the place of class-defining mutations to drive FLT3-ITD–mutant AML

Advances in cancer genomics have revealed genomic classes of acute myeloid leukemia (AML) characterized by class-defining mutations, such as chimeric fusion genes or in genes such as NPM1, MLL, and CEBPA. These class-defining mutations frequently synergize with internal tandem duplications in FLT3 (FLT3-ITDs) to drive leukemogenesis. However, ∼20% of FLT3-ITD–positive AMLs bare no class-defining mutations, and mechanisms of leukemic transformation in these cases are unknown. To identify pathways that drive FLT3-ITD mutant AML in the absence of class-defining mutations, we performed an insertional mutagenesis (IM) screening in Flt3-ITD mice, using Sleeping Beauty transposons. All mice developed acute leukemia (predominantly AML) after a median of 73 days. Analysis of transposon insertions in 38 samples from Flt3-ITD/IM leukemic mice identified recurrent integrations at 22 loci, including Setbp1 (20/38), Ets1 (11/38), Ash1l (8/38), Notch1 (8/38), Erg (7/38), and Runx1 (5/38). Insertions at Setbp1 led exclusively to AML and activated a transcriptional program similar, but not identical, to those of NPM1-mutant and MLL-rearranged AMLs. Guide RNA targeting of Setbp1 was highly detrimental to Flt3ITD/+/Setbp1IM+, but not to Flt3ITD/+/Npm1cA/+, AMLs. Also, analysis of RNA-sequencing data from hundreds of human AMLs revealed that SETBP1 expression is significantly higher in FLT3-ITD AMLs lacking class-defining mutations. These findings propose that SETBP1 overexpression collaborates with FLT3-ITD to drive a subtype of human AML. To identify genetic vulnerabilities of these AMLs, we performed genome-wide CRISPR-Cas9 screening in Flt3ITD/+/Setbp1IM+ AMLs and identified potential therapeutic targets, including Kdm1a, Brd3, Ezh2, and Hmgcr. Our study gives new insights into epigenetic pathways that can drive AMLs lacking class-defining mutations and proposes therapeutic approaches against such cases.

Abstract PO-009: Disrupting chromatin architecture: The NuRD subunit and ATPase CHD4 as a new therapeutic target in pediatric sarcoma

Abstract Cancer-specific chromosomal aberrations producing chimeric fusion genes are recurrently found in pediatric sarcomas. Fusion positive rhabdomyosarcoma (FP-RMS) and Ewing sarcoma (ES) are two rare but lethal pediatric malignancies driven by such chromosomal translocations. PAX3-FOXO1 and EWS-FLI1 are the most common products of the fusion genes found in FP-RMS and ES, respectively, and they are commonly perceived as the founding genetic abnormality driving the development of these malignancies by changing gene expression. Since direct targeting of transcription factors is still very challenging, acting on the activity of these oncogenic transcription factors at the chromatin level presents a robust alternative for targeted therapy. The Nucleosome Remodeling and Deacetylase (NuRD) complex subunit CHD4 has been previously identified as an interactor of both PAX3-FOXO1 and EWS-FLI1. Hence, we decided here to further characterize the role of this chromatin remodeler in the regulation of fusion protein-mediated gene expression in both FP-RMS and ES. Our NuRD-centered CRISPR/Cas9 screen demonstrated that both these malignancies are especially dependent on CHD4 amongst all other complex members. In fact, CHD4 silencing in both tumors through shRNA knockdown or CRISPR knockout drastically reduces tumor cell proliferation and induces cell death. In vivo, CHD4 knockdown also impaired tumour growth in both FP-RMS and ES. Mechanistically, our RNA-seq assays demonstrated that silencing of the nucleosome remodeller CHD4 alters gene expression in both tumours and our ChIP-seq experiments show that CHD4 binding sites are highly enriched for the binding motif of PAX3-FOXO1 in FP-RMS and EWS-FLI1 in ES. In FP-RMS, we observed that CHD4 particularly regulates super-enhancer accessibility creating a chromatin architecture permissive to the binding of PAX3-FOXO1 and allowing the expression of the fusion gene signature. Similar studies in ES to further investigate CHD4 as a regulator of gene expression are currently ongoing. Finally, our analysis of genome-wide cancer dependency databases identified CHD4 as general novel cancer vulnerability amongst NuRD subunits and other SNF2-like ATPases. In summary, we have unravelled the prominent role of CHD4 in regulation of super-enhancer driven gene expression in FP-RMS and exposed this chromatin remodeler as novel potential drug target for pediatric sarcoma therapy. Our work has motivated us to establish several collaborations with computational and biophysics experts and we are now currently working to identify the first CHD4 specific small molecule inhibitor. Citation Format: Joana G. Marques, Berkley Gryder, Blaz Pavlovic, Yeonjoo Chung, Quy Ngo, Marco Wachtel, Javed Khan, Beat Schäfer. Disrupting chromatin architecture: The NuRD subunit and ATPase CHD4 as a new therapeutic target in pediatric sarcoma [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-009.

Setbp1 Overexpression Acts in the Place of Class-Defining Somatic Mutations to Drive Mouse and Human FLT3-ITD-Mutant AMLs

Setbp1 overexpression acts in the place of class-defining somatic mutations to drive mouse and human FLT3-ITD-mutant AMLs Suruchi Pacharne,1,2 Oliver M. Dovey,1 Jonathan L. Cooper,1 Muxin Gu,1,2 MS Vijaybaskar,1,2 Mathias J. Friedrich,1,5 Malgorzata Gozdecka,1,2 Sandeep S. Rajan,1,4, Etienne De Braekeleer,1,2 Maxim Barenboim,5,6 Grace Collord,1,2 Hannes Ponstingl,1 Ruben Bautista,1 Milena Mazan,1,8 Roland Rad,5,6 Konstantinos Tzelepis,1,7 Penny Wright,3 and George S. Vassiliou1,2,9* Abstract Internal tandem duplications in FLT3 (FLT3-ITD) are found in 30% of acute myeloid leukemia (AML) cases and impart a poor prognosis. FLT3-ITD commonly synergizes with class-defining mutations such as chimeric fusion genes or mutations in NPM1, RUNX1, CEBPA or MLL to drive AML. However, 20% of FLT3-ITD-mutant AMLs bare no class-defining mutations and the mechanisms of acute leukemic transformation in these cases are unknown. To identify pathways that can drive FLT3-ITD-mutant AML in the absence of class-defining mutations, we performed an insertional mutagenesis (IM) screen in Flt3-ITD mice using the Sleeping Beauty transposon system, activated by the Mx1-Cre recombinase in hematopoietic stem cells. All mice developed acute leukemia, predominantly AML, after a median latency of 73 days (Figure A). Analysis of transposon insertions in 38 Flt3-ITD/IM leukemias identified common integration sites (CISs) in 22 loci (Figure B). The most commonly "hit" genes were Setbp1 (20/38), Ets1 (11/38), Ash1l (8/38), Notch1 (8/38), Erg (7/38), Flt3 (6/38) and Runx1 (5/38) (Figure B). Of these, Setbp1 and Runx1 were unique to Flt3-ITD and not identified as CISs in insertional mutagenesis screens of wild type, Npm1c or BCR-ABL-expressing mice. Transposon insertions in Setbp1, primarily located upstream of its first coding exon, were associated with Setbp1 and Hoxa mRNA overexpression and were invariably associated with AML development (Figure B). These findings propose that overexpression of wild type SETBP1 may collaborate with FLT3-ITD to drive leukemogenesis in human AMLs lacking mutations known to collaborate with mutant FLT3. Corroborating this, we found that SETBP1 expression was higher in human FLT3-ITD-mutant AMLs lacking class-defining mutations and in those with RUNX1 mutations (Figure C). We go on to show that Setbp1 insertions activate a Hoxa gene signature such that shares significant similarities, but also specific differences to those driven by mutant Npm1 and MLL fusion genes. We go on to show, using CRISPR-gRNA, that whilst Flt3ITD/+/SETBP1IM+AMLs are entirely dependent on Setbp1 expression, Flt3ITD/+/Npm1cA/+AMLs are not, but do depend on the expression of the homebox gene Nkx2.3. Our findings propose that SETBP1 overexpression activates a gene expression pattern that collaborates with FLT3-ITD to drive many human AMLs and that this combination represents a specific subtype of AML amongst AMLs lacking class-defining mutations. To identify genetic vulnerabilities of this AML subtype, we performed genome-wide CRISPR-Cas9 recessive screens in primary murine Flt3ITD/+SETBP1IM+AMLs and identified more than 2000 genetic vulnerabilities, of which 677 were not required for the survival of HPC7 non-leukemic hematopoietic cells including &amp;gt;100 "druggable" genes such as Brd3, Ezh2 and Hmgcr (Figure D). Collectively our study: i) identifies SETBP1overexpression as a non-genetic alteration driving a subgroup of FLT3-ITD mutant AMLs lacking class-defining somatic mutations and ii) goes on to define the genetic vulnerabilities of such AMLs as a starting point for the development of targeted therapies. Figure Disclosures Vassiliou: Kymab Ltd - Monoclonal antibody company. Currently not working in myeloid cancers or clonal haematopoiesis.: Consultancy.

The Diverse Landscape of Fusion Transcripts in 25 Different Hematological Entities

Background: Genomic alterations are a hallmark of hematological malignancies and comprise small nucleotide variants, copy number alterations and structural variants (SV). SV lead to the co-localization of remote genomic material resulting in 2 different scenarios: 1. breakpoints are located within 2 genes leading to a chimeric fusion gene and a fusion transcript, 2. breakpoints are located outside of genes, frequently placing one nearby gene under the influence of the regulatory sequences of the partner, leading to a deregulated - usually increased - transcription. Aim: The frequency of fusion transcripts was determined across hematological entities in order to 1) identify recurrent partner genes across entities, 2) evaluate the specificity of fusion transcripts and genes involved in fusions for distinct entities. Cohort and Methods: Whole transcriptome sequencing (WTS) was performed in 3,549 patients in 25 different hematological entities (table). 101 bp paired-end reads were produced on a NovaSeq 6000 system (Illumina, San Diego, CA) with a yield between 35 and 125 million paired reads per sample. Potential fusions were called using 3 different callers (Arriba, STAR-Fusion, Manta), only fusions called by at least 2 callers, validated by whole genome sequencing (data available for all cases) and with at least one protein coding partner were kept for further analyses. Reciprocal fusion transcripts were counted as one fusion event. Results: In total 1,309 fusion transcripts were identified in 932 of 3,549 (26.3%) patients. 221 patients showed &amp;gt; 1 fusion (2 fusions: 150, 3: 36, &amp;gt;3: 35). 806 distinct fusion transcripts were divided into recurrent fusions (n=50) and unique fusions, i.e. found only in 1 case (n=756). Out of 932 patients with at least 1 fusion, 541 (58%) patients harbored a minimum of one recurrent fusion. The proportion of patients harboring any or a recurrent fusion varied substantially between different entities with high frequencies for both in CML (96.5%/96.5%), B-lineage ALL (53.1%/41.3%), AML (42.8%/31.2%), and T-lineage ALL (35.3%/12.6%). In several myeloid entities low fusion frequencies were observed (e.g. PMF, MDS/MPN-U, MDS, figure A). No fusion transcripts were detected in ET. Strikingly, fusions were detected in a substantial proportion of cases with lymphoid neoplasms but only very few occurred recurrently (e.g. T-PLL: 47.8%/4.3%, FL: 39.3%/4.9%, figure A). With regard to age, only patients with AML and T-ALL harboring recurrent fusions were significantly younger than corresponding cases without recurrent fusions (59 vs 71 yrs, p&amp;lt;0.0001; 35 vs 38 yrs, p=0.02). Only in AML patients with unique fusions were older (70 vs 66 yrs, p=0.02), while no age differences were observed between cases with and without unique fusions in other entities. 23/50 (46%) of the recurrent fusions were specific for one entity (12 in myeloid, 11 in lymphatic entities), while the other 54% (27/50) were observed in 2 to 7 different entities. Of these 27 recurrent fusions, only 16 fusions were shared between myeloid and lymphatic entities, while 10 were restricted to myeloid and one fusion to lymphatic entities (figure B). In total 1,270 different genes were involved in the 806 distinct fusions, indicating a broad spectrum of potential functional impact. 54 genes were involved only in recurrent fusions, 27 genes in both recurrent and unique fusions, while 1,189 genes were solely involved in unique fusions. Four genes involved in recurrent fusions and 32 genes involved in unique fusions are FDA approved drug targets (Human Protein Atlas). Only 16% (199/1270) of the genes were involved in more than one fusion: 3 genes (ETV6, KMT2A, RUNX1) in 14 fusions, 2 genes (ABL1, BCR) in 11 fusions, 16 genes in 4 to 10 fusions, 38 genes in 3 fusions, 140 in 2 fusions. Several genes frequently involved in fusions in hematological malignancies (e.g. ABL1, ETV6, KMT2A) and 78/1189 genes only involved in unique fusions were also reported to be partners in fusions in non-hematological malignancies. Conclusions: As known, in CML and acute several leukemias a high proportion of patients harbor fusions of which many occur recurrently, suggesting a substantial pathogenic impact and, thus, requiring detection in a diagnostic work-up. In BCR-ABL1 negative chronic myeloid malignancies few fusions were observed while lymphoma patients carry frequently non-recurrent fusions with so far unknown impact on pathogenesis and prognosis. Disclosures No relevant conflicts of interest to declare.

A Rare Case of Chronic Myeloid Leukemia with t(3;9;22) 3-way Translocation

Abstract Introduction/Objective Chronic Myeloid Leukemia (CML) is a myeloproliferative neoplasm originating from malignant clonal proliferation of a pluripotent hematopoietic stem cell. CML is characterized by a reciprocal translocation between chromosomes 9 and 22, t(9;22)(q34;q11), that gives rise to an abnormal chromosome 22 called the Philadelphia (Ph) chromosome. The translocation results in the formation of a chimeric BCR-ABL1 fusion gene, which is the molecular hallmark of the disease. However, 5-10% of CML patients present with additional chromosomal abnormalities which is often considered a sign of clonal evolution, genetic instability, and is generally thought to portend a poor prognosis. Methods We present a case of CML with a rare 3- way translocation, t(3;9;22)(q21;q34;q11.2), who achieved a major molecular response on imatinib for 18 months. A review of the literature and Mitelman database search is presented focusing on the prognostic implications of this 3 way translocation in the era of tyrosine kinase inhibitors starting in 2001 till now. Results Twenty seven cases were reported, but the patient therapeutic response to imatinib and clinical outcome were only reported in 11 cases. Nine cases achieved a cytogenetic remission while the remaining two cases had an adverse outcome. Conclusion Taken in conjunction with the favorable outcome in our patient, we suggest that t(3;9;22) is not an adverse prognostic factor in the era of tyrosine kinase inhibitors.