Oncogenic Fusion Research Articles

Abstract C008: New insights from a novel mouse model of synovial sarcoma

Abstract Mouse models are the basis of preclinical and translational research in solid tumors. Multiple methods exist to induce tumor formation in mice, including genetically engineered mouse models, chemotoxic agents, injection of tumor cells, and xenograft approaches. Synovial sarcoma, a rare highly malignant soft-tissue tumor, poses a serious threat to patients due to its aggressive nature and associated high rates of mortality and morbidity. This disease's underlying cause and development remain poorly comprehended, and existing treatment approaches have yielded unsatisfactory outcomes. In the last two decades, numerous effective animal models of soft tissue sarcoma (STS) have been created, primarily utilizing genetically engineered mice and zebrafish, however long latency of tumor development for several months in these models creates a barrier for practical in-vivo experiments for drug testing or manipulating the cellular composition of the tumor microenvironment. In studying cancer immunotherapy, it is important to consider aspects of antitumor immune responses and to produce a model that mimics the complexity of the immune system We have generated a new mouse model of synovial sarcoma of exceptionally fast tumor development. Intramuscular delivery of Cre recombinase protein in mice which drives conditional knockdown in PTEN, stabilization of β-catenin, and formation of fusion oncogenes of SS18-SSX2 was sufficient to initiate high-grade synovial sarcomas with myofibroblastic differentiation in 3 weeks. As immunotherapy is increasingly applied to sarcomas, our mouse model will serve as a great tool for preclinical data. Flow cytometry-based analysis of peripheral blood shows significant changes in immune cell populations as early as five days after Tat-Cre injection and continues throughout the development of synovial sarcoma. These results suggest a significant systemic phenotypic alteration of circulating immune cells, which may impact other major organs in this genetic mouse model of synovial sarcoma. Single- cell RNA sequencing of the CD45+ leukocytes in the tumor microenvironment reveals subsets that may promote tumor progression. Our work promises to significantly enhance our understanding of the pathogenesis of this disease and establish a reliable preclinical platform for devising and assessing therapeutic interventions. Citation Format: Hesham Mohei, Donn Van Deren, Irene S. Molina, Malay Haldar. New insights from a novel mouse model of synovial sarcoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C008.

Exploring the driver events of eccrine poromas and porocarcinomas: A retrospective, cross-institutional study of 54 cases.

We have comprehensively characterised the mutational landscape of eccrine poroma (EP) and eccine porocarcinoma (EPC), uncovering novel events and delineating different pathways of tumorigenesis underlying these tumours. EPs are driven largely by oncogenic fusion genes, whereas EPCs are driven largely by somatic mutations affecting various pathways, with a subset driven by fusion genes as the first oncogenic driver, and somatic mutations representing secondary events contributing to progression of the tumour. Fusion genes in EP predominantly involve YAP1 whereas those in EPC preferentially involve PAK gene family members.

The Capicua C1 Domain is Required for Full Activity of the CIC::DUX4 Fusion Oncoprotein.

Rearrangements between genes can yield neomorphic fusions that drive oncogenesis. Fusion oncogenes are made up of fractional segments of the partner genes that comprise them, with each partner potentially contributing some of its own function to the nascent fusion oncoprotein. Clinically, fusion oncoproteins driving one diagnostic entity are typically clustered into a single molecular subset and are often treated a similar fashion. However, knowledge of where specific fusion breakpoints occur in partner genes, and the resulting retention of functional domains in the fusion, is an important determinant of fusion oncoprotein activity and may differ between patients. This study investigates this phenomena through the example of CIC::DUX4, a fusion between the transcriptional repressor capicua (CIC) and the double homeobox 4 gene (DUX4), which drives an aggressive subset of undifferentiated round cell sarcoma. Using a harmonized dataset of over 100 patient fusion breakpoints from the literature, we show that most bona fide CIC::DUX4 fusions retain the C1 domain, which is known to contribute to DNA binding by wild type CIC. Mechanistically, deletion or mutation of the C1 domain reduces, but does not eliminate, activation of CIC target genes by CIC::DUX4. We also find that expression of C1-deleted CIC::DUX4 is capable of exerting intermediate transformation-related phenotypes compared with those imparted by full-length CIC::DUX4, but was not sufficient for tumorigenesis in a subcutaneous mouse model. In summary, our results suggest a supercharging role for the C1 domain in the activity of CIC::DUX4.

BIOM-05. FUSION TRANSCRIPTOME LANDSCAPE IN GLIOBLASTOMA

Abstract BACKGROUND For most patients with glioblastoma (GBM), no truly effective salvage therapies exist. However, there are compelling examples in oncology where gene fusions lead to functioning oncoproteins that are highly effective targets (ie BCR-ABL1 in CML). Using a large genomic database, we sought to determine the prevalence of fusion transcripts in GBMs. METHODS A total of 4392 GBMs were tested at Caris Life Sciences (Phoenix, AZ). Mutations were detected by NextGen sequencing of DNA (592-gene panel or whole exome sequencing); gene fusions were detected by Whole Transcriptome Sequencing. All tumors were wild type for IDH1/2. Significance was tested by Fisher-Exact and ChiSquare and adjusted by Benjamini-Hochberg (q <0.05). RESULTS Overall, pathogenic fusions were found in 428 (9.7%). The most prevalent were FGFR3 [N=159 (37%), highest FGFR3:TACC3, N=134], MET [N=92 (21%), highest PTPRZ1:Met, N=31; ST7:MET N=25; CAPZA2: MET, N=23), EGFR [N=87(20%), highest EGFR:SETP14, N=21 and SEC61G: EGFR, N=19], NTRK2 (N=27), PDGFRA (N=23), ROS1 (N=14) and BRAF (N=10). Other actionable fusions including ALK (N=3), NTRK3 (N=3), RAF1(N=3), RET (N=3), NTRK1(N=2) were seen. Tumors with fusions were more likely to have pathogenic gene amplifications including: MET (7.5% vs. 0.7%), FGFR3 (5% vs. 0.2%), CDK4 (17% vs. 11%) and MDM2 (12% and 7.5%) but less frequent EGFR mutation (6.1% vs. 18%), amplification (6.1% vs. 18%) and EGFRvIII mutation (11.9% vs. 22.5%), RB1 (2.9% vs. 11.5%) and TP53mutation (22% vs. 30%) (all q<0.05). EGFR fusions are enriched with EGFR amplification (92%) and EGFRvIII mutation (51%); cMET fusion with MET amplification (40%). CONCLUSIONS Comprehensive molecular profiling reveals that approximately 10% of IDH WT GBMs carry oncogenic fusions that may be therapeutic targets. Broad spectrum of observed fusions underscores the need for novel clinical trial designs to allow efficient enrollment for prospective testing of potential targeted agents.

TMET-36. INCREASED METHIONINE CYCLE DRIVES PYRIMIDINE METABOLISM IN LETHAL PEDIATRIC EPENDYMOMAS

Abstract Supratentorial ependymomas (ST-EPN) are pediatric brain tumors found in the cerebral hemisphere, driven by the oncogenic fusion of ZFTA with RELA. Other than surgery and radiotherapy, these tumors do not have effective therapeutic options. Recurrent ST-EPN tumors are highly aggressive and hard to treat, which makes these tumors lethal. To understand the disease mechanism and potentially identify or develop therapeutic targets, we established patient-derived models and interrogated the genetic and metabolic dependencies using cutting-edge, near-genome-wide genetic screening and metabolomic tools. Our integrative transcriptomics and metabolic analysis identify that the ZFTA-RELA fusion drives methionine metabolic reprogramming in the cancer stem cells to regulate permissive epigenetic status and pyrimidine nucleotide synthesis to fuel tumor growth. Limiting methionine or inhibiting MAT2A, a rate-limiting enzyme of methionine metabolism in ST-EPN cells, results in profound cell cycle arrest and make the cells vulnerable to pyrimidine inhibitors compared to other pediatric brain tumors. Thus, for treatment, along with radiotherapy, blocking the methionine cycle by using the methionine limited diet or in combination with pyrimidine metabolism inhibitors can be a novel therapeutic strategy against the aggressive paediatric ST-EPN tumors.

Genomic profiling of circulating tumor DNA for childhood cancers.

The utility of circulating tumor DNA (ctDNA) analysis has not been well-established for disease detection and monitoring of childhood cancers, especially leukemias. We developed PeCan-Seq, a deep sequencing method targeting diverse somatic genomic variants in cell-free samples in childhood cancer. Plasma samples were collected at diagnosis from 233 children with hematologic, solid and brain tumors. All children with hematologic malignancy (n = 177) had detectable ctDNA at diagnosis. The median ctDNA fraction was 0.77, and 97% of 789 expected tumor variants were identified, including sequence mutations, copy number variations, and structural variations responsible for oncogenic fusions. In contrast, ctDNA was detected in 19 of 38 solid tumor patients and 1 of 18 brain tumor patients. Somatic variants from ctDNA were correlated with minimal residual disease levels as determined by flow cytometry in serial plasma samples from patients with B-cell acute lymphoblastic leukemia (B-ALL). We showcase multi-tumor detection by ctDNA analysis for a patient with concurrent B-ALL and neuroblastoma. In conclusion, PeCan-seq sensitively identified heterogeneous ctDNA alterations from 1 mL plasma for childhood hematologic malignancies and a subset of solid tumors. PeCan-seq provides a robust, non-invasive approach to augment comprehensive genomic profiling at diagnosis and mutation-specific detection during disease monitoring.

The role of genetic and epigenetic modifications as potential biomarkers in the diagnosis and prognosis of thyroid cancer

Thyroid cancer (TC) is the most common endocrine cancer, which contributes to more than 43,600 deaths and 586,000 cases worldwide every year. Among the TC types, PTC and FTC comprise 90% of all TCs. Genetic modifications in genes are responsible for encoding proteins of mitogen-associated protein kinase cascade, which is closely related with numerous cellular mechanisms, including controlling programmed cell death, differentiation, proliferation, gene expression, as well as in genes encoding the PI3K (phosphatidylinositol 3-kinase)/protein kinase B (AKT) cascade, which has contribution in controlling cell motility, adhesion, survival, and glucose metabolism, have been associated with the TC pathogenesis. Various genetic modifications including BRAF mutations, RAS mutations, RET mutations, paired-box gene 8/peroxisome proliferator-activated receptor-gamma fusion oncogene, RET/PTC rearrangements, telomerase reverse transcriptase mutations, neurotrophic tyrosine receptor kinase fusion genes, TP53 mutations, and eukaryotic translation initiation factor 1A X-linked mutations can effectively serve as potential biomarkers in both diagnosis and prognosis of TC. On the other hand, epigenetic modifications can lead to aberrant functions or suppression of a range of signalling cascades, which can ultimately result in cancer. Various studies have observed the link between epigenetic modification and multiple cancers including TC. It has been reported that several epigenetic alterations including histone modifications, aberrant DNA methylation, and epigenetic modulations of non-coding RNAs can play significant roles as potential biomarkers in the diagnosis and prognosis of TC. Therefore, a good understanding regarding the genetic and epigenetic modifications is not only essential for the diagnosis and prognosis of TC, but also for the development of novel therapeutics. In this review, most of the major TC-related genetic and epigenetic modifications and their potential as biomarkers for TC diagnosis and prognosis have been extensively discussed.

Oncogenic EML4-ALK assemblies suppress growth factor perception and modulate drug tolerance

Drug resistance remains a challenge for targeted therapy of cancers driven by EML4-ALK and related fusion oncogenes. EML4-ALK forms cytoplasmic protein condensates, which result from networks of interactions between oncogene and adapter protein multimers. While these assemblies are associated with oncogenic signaling, their role in drug response is unclear. Here, we use optogenetics and live-cell imaging to find that EML4-ALK assemblies suppress transmembrane receptor tyrosine kinase (RTK) signaling by sequestering RTK adapter proteins including GRB2 and SOS1. Furthermore, ALK inhibition, while suppressing oncogenic signaling, simultaneously releases the sequestered adapters and thereby resensitizes RTK signaling. Resensitized RTKs promote rapid and pulsatile ERK reactivation that originates from paracrine ligands shed by dying cells. Reactivated ERK signaling promotes cell survival, which can be counteracted by combination therapies that block paracrine signaling. Our results identify a regulatory role for RTK fusion assemblies and uncover a mechanism of tolerance to targeted therapies.

MLL oncoprotein levels influence leukemia lineage identities

Chromosomal translocations involving the mixed-lineage leukemia (MLL) locus generate potent oncogenic fusion proteins (oncoproteins) that disrupt regulation of developmental gene expression. By profiling the oncoprotein-target sites of 36 broadly representative MLL-rearranged leukemia samples, including three samples that underwent a lymphoid-to-myeloid lineage-switching event in response to therapy, we find the genomic enrichment of the oncoprotein is highly variable between samples and subject to dynamic regulation. At high levels of expression, the oncoproteins preferentially activate either an acute lymphoblastic leukemia (ALL) program, enriched for pro-B-cell genes, or an acute myeloid leukemia (AML) program, enriched for hematopoietic-stem-cell genes. The fusion-partner-specific-binding patterns over these gene sets are highly correlated with the prevalence of each mutation in ALL versus AML. In lineage-switching samples the oncoprotein levels are reduced and the oncoproteins preferentially activate granulocyte-monocyte progenitor (GMP) genes. In a sample that lineage switched during treatment with the menin inhibitor revumenib, the oncoprotein and menin are reduced to undetectable levels, but ENL, a transcriptional cofactor of the oncoprotein, persists on numerous oncoprotein-target loci, including genes in the GMP-like lineage-switching program. We propose MLL oncoproteins promote lineage-switching events through dynamic chromatin binding at lineage-specific target genes, and may support resistance to menin inhibitors through similar changes in chromatin occupancy.

Condensate remodeling reorganizes innate SS18 in synovial sarcomagenesis

SS18-SSX onco-fusion protein formed through aberrant chromosomal translocation t (X, 18; p11, q11), is the hallmark and plays a critical role in synovial sarcomagenesis. The recent works indicated that both the pathological SS18-SSX tumorigenic fusion and the corresponding intrinsic physiological SS18 protein can form condensates but appear to have disparate properties. The underlying regulatory mechanism and the consequent biological significance remain largely unknown. We show that the physical properties of oncogenic fusion protein SS18-SSX condensates within cells undergo alterations compared to the proto-oncogene protein SS18. By small-molecule screening and mutant assay, we identified the recognition of H2AK119ub histone modification could account for the distinctive properties of SS18-SSX1 condensates. Notably, we show that SS18-SSX1 condensates have impact on SS18 condensates and hijack that in a phase separation manner, resulting in the relocation of protein SS18 to the H2AK119ub modification targeted by SS18-SSX1. Consequently, this leads to the downregulation of tumor suppressor genes occupied by SS18 physiologically, like CAV1 and DAB2. These results reveal the underlying mechanism of genomic disorder and tumorigenesis caused by the remodeling of oncoprotein SS18-SSX1 condensates at the macroscopic level.

Bihemispheric IDH-Wildtype Glioblastoma with Unilateral FGFR3-TACC3 Fusion in Patient with Breast Cancer History: A Case Report and Literature Review

Abstract Introduction/Objective Adult IDH-wild type glioblastomas comprise a heterogeneous spectrum of neoplasms, characterized by poor prognosis and resistance to the chemoradiotherapy. For WHO integrated diagnosis classification and to establish treatment options, molecular analysis is necessary. Herein, we present a case of a glioblastoma, IDH-wildtype with involvement of both cerebral hemispheres and an uncharacteristic in-frame fusion limited to one hemisphere. Methods/Case Report The patient is a 52-year-old woman with past medical history of metastatic breast cancer who presented with headache and worsening facial pain. Brain magnetic resonance imaging brain demonstrated a right frontal lobe lesion with vasogenic edema. The patient was treated with Gamma Knife radiosurgery. Scans revealed enlargement and a new left frontal lobe lesion. Bihemispheric tumor resection and GammaTile placement was undertaken. Results (if a Case Study enter NA) Histologically, the tumor was composed of hypercellular astrocytes, microvascular proliferation, and necrosis with no evidence of metastatic disease. Immunohistochemistically, neoplastic cells showed glial fibrillary acidic protein expression and elevated Ki-67 proliferation index. Molecular examination revealed in the right hemisphere lesion post-radiation (1) FGFR3::TACC3 (exon 17::exon 11) fusion and (2) TERT promoter, while the left hemisphere lesion which did not receive radiation showed clinically relevant variants in (1) TERT promoter, (2) TP53, (3) NF1, and (3) PIK3CA without identification of a fusion. An integrated diagnosis of glioblastoma, IDH- wildtype, CNS WHO grade 4 was established bilaterally. Conclusion The fibroblast growth factor receptor 3 (FGFR3)-transforming acidic coiled-coil 3 (TACC3) fusion is identified as a rare molecular feature in glioblastomas with an estimated prevalence of 4%. Clinical trials suggest that F3T3-positive tumors have a better prognosis than those without the translocation. The identification of the oncogenic FGFR3-TACC3 fusion highlights the possibility of identifying patients potentially responsive to targeted therapy with FGFR kinase inhibitors. The significance of the molecular differences in the right and left hemisphere lesions is uncertain; glioblastomas are heterogeneous tumors with intra-and intertumoral heterogeneity but it may suggest independent origins.

CBFA2T3-GLIS2 mediates transcriptional regulation of developmental pathways through a gene regulatory network

CBFA2T3-GLIS2 is a fusion oncogene found in pediatric acute megakaryoblastic leukemia that is associated with a poor prognosis. We establish a model of CBFA2T3-GLIS2 driven acute megakaryoblastic leukemia that allows the distinction of fusion specific changes from those that reflect the megakaryoblast lineage of this leukemia. Using this model, we map fusion genome wide binding that in turn imparts the characteristic transcriptional signature. A network of transcription factor genes bound and upregulated by the fusion are found to have downstream effects that result in dysregulated signaling of developmental pathways including NOTCH, Hedgehog, TGFβ, and WNT. Transcriptional regulation is mediated by homo-dimerization and binding of the ETO transcription factor through the nervy homology region 2. Loss of nerve homology region 2 abrogated the development of leukemia, leading to downregulation of JAK/STAT, Hedgehog, and NOTCH transcriptional signatures. These data contribute to the understanding of CBFA2T3-GLIS2 mediated leukemogenesis and identify potential therapeutic vulnerabilities for future studies.

8576 Genomic and Transcriptomic Characterization of Pediatric Thyroid Cancers

Abstract Disclosure: J.C. Ricarte-Filho: None. E.R. Reichenberger: None. K. Hinkle: None. A.R. Isaza: None. A.J. Bauer: None. A.T. Franco: None. Background: Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer in both pediatric and adult populations. Recent genomic profiling of papillary thyroid cancers, including that of the Cancer Genome Atlas, has uncovered the genetic alterations and molecular mechanisms driving thyroid cancer. However, these studies were primarily focused on adult patients. Despite their favorable prognosis, pediatric PTCs have higher rates of metastasis and recurrence than adult PTCs, but the molecular characterization of these tumors have been comparatively lacking. Methods: Here we explore the genomic and transcriptomic landscape of pediatric PTCs from 126 patients, including 107 primary tumors and 19 lymph node metastases (non-paired) using whole-exome sequencing and RNA sequencing. Thirteen primary tumors also had a paired lymph node metastasis analyzed. Results: We found driver alterations in 113 of 126 (90%) of the PTCs investigated, including the identification of a novel receptor tyrosine kinase (RTK) fusion not previously reported in thyroid cancer. Genetic drivers were predominantly kinase fusions (48%), most commonly fusions of RET (22%), NTRK3 (10%), ALK (5%) and NTRK1 (5%). The most common point mutations were BRAF p.V600E (21%), NRAS p.Q61R/K (9%) and hotspot mutations in DICER1 (4%). We found genetic alterations in 12 of the 13 paired lymph node metastasis. In all 12 cases the metastatic tissue harbored the same alterations found in the primary tumor. Eight out of 12 cases had fusions (3 RET, 3 NTRK3 and 2 NTRK1), and the additional 4 cases had point mutations (3 BRAFV600E and 1 TSHRM543T). Fourteen patients (11%) had widely invasive disease with lateral lymph node and distant metastases (N1bM1). Most of these N1bM1 tumors (13/14; 93%) were driven by RTK fusions (5 NTRK3, 3 RET, 3 NTRK1 and 2 ALK). The one case lacking a kinase fusion had a CHEK2 mutation. Conclusion: Our genomic and transcriptomic analysis allowed the detection of driver alterations in ∼90% of the cases, reducing the so-called “dark matter” in our cohort. Our data support that kinase fusions are associated with metastatic disease in pediatric PTC. Patients with RET/NTRK/ALK fusions have worse outcomes than those with BRAF p.V600E mutation and RAS-like alterations (NRAS, DICER1, non-V600 BRAF, PTEN). By using the transcriptional profiling generated in this study, we are currently investigating the molecular mechanisms underlying the metastatic behavior of pediatric PTCs harboring oncogenic fusions, including changes in MAPK transcriptional output, differentiation score and recruitment of signaling pathways potentially associated with the metastatic behavior of these tumors. Presentation: 6/3/2024

EP.06E.06 Elucidating the Interplay Between Oncogenic Fusions and the Tumor Immune Microenvironment in Lung Adenocarcinoma

EP.06E.06 Elucidating the Interplay Between Oncogenic Fusions and the Tumor Immune Microenvironment in Lung Adenocarcinoma

Type II topoisomerases shape multi-scale 3D chromatin folding in regions of positive supercoils.

Type II topoisomerases (TOP2s) resolve torsional stress accumulated during various cellular processes and are enriched at chromatin loop anchors and topologically associated domain (TAD) boundaries, where, when trapped, can lead to genomic instability promoting the formation of oncogenic fusions. Whether TOP2s relieve topological constraints at these positions and/or participate in 3D chromosome folding remains unclear. Here, we combine 3D genomics, imaging, and GapRUN, a method for the genome-wide profiling of positive supercoiling, to assess the role of TOP2s in shaping chromosome organization in human cells. Acute TOP2 depletion led to the emergence of new, large-scale contacts at the boundaries between active, positively supercoiled, and lamina-associated domains. TOP2-dependent changes at the higher-order chromatin folding were accompanied by remodeling of chromatin-nuclear lamina interactions and of gene expression, while at the chromatin loop level, TOP2 depletion predominantly remodeled transcriptionally anchored, positively supercoiled loops. We propose that TOP2s act as a fine regulator of chromosome folding at multiple scales.

Rare Oncogenic Fusions in Pediatric Central Nervous System Tumors: A Case Series and Literature Review

Background and Objectives: Central Nervous System (CNS) pediatric tumors represent the most common solid tumors in children with a wide variability in terms of survival and therapeutic response. By contrast to their adult counterpart, the mutational landscape of pediatric CNS tumors is characterized by oncogenic fusions rather than multiple mutated genes. CNS pediatric tumors associated with oncogenic fusions represent a complex landscape of tumors with wide radiological, morphological and clinical heterogeneity. In the fifth CNS WHO classification, there are few pediatric CNS tumors for which diagnosis is based on a single oncogenic fusion. This work aims to provide an overview of the impact of rare oncogenic fusions (NTRK, ROS, ALK, MET, FGFR, RAF, MN1, BCOR and CIC genes) on pathogenesis, histological phenotype, diagnostics and theranostics in pediatric CNS tumors. We report four cases of pediatric CNS tumors associated with NTRK (n = 2), ROS (n = 1) and FGFR3 (n = 1) oncogenic fusion genes as a proof of concept. Cases presentation and literature review: The literature review and the cohort that we described here underline that most of these rare oncogenic fusions are not specific to a single morpho-molecular entity. Even within tumors harboring the same oncogenic fusions, a wide range of morphological, molecular and epigenetic entities can be observed. Conclusions: These findings highlight the need for caution when applying the fifth CNS WHO classification, as the vast majority of these fusions are not yet incorporated in the diagnosis, including grade evaluation and DNA methylation classification.

Novel insight into mechanisms of ROS1 catalytic activation via loss of the extracellular domain.

The ROS1 receptor tyrosine kinase (RTK) possesses the largest extracellular amino-terminal domain (ECD) among the human RTK family, yet the mechanisms regulating its activation are not fully understood. While chimeric ROS1 fusion proteins, resulting from chromosomal rearrangements, are well-known oncogenic drivers, their activation mechanisms also remain underexplored. To elucidate the role of the ROS1 ECD in catalytic regulation, we engineered a series of amino-terminal deletion mutants. Our functional studies compared the full-length ROS1 receptor, the CD74-ROS1 oncogenic fusion, and ECD-deleted ROS1 constructs, identifying the ECD regions that inhibit ROS1 tyrosine kinase activity. Notably, we found that deletion of the ROS1 ECD alone significantly increases constitutive catalytic activation and neoplastic transformation in the absence of an amino-terminal fusion partner, challenging the presumed necessity for a dimerization domain in the activation mechanism of kinase fusions in cancer. Our data suggest that inter-genic deletions resulting in the loss of the ECD may be underappreciated oncogenic drivers in cancer. Furthermore, our studies demonstrate that RNASE7 is not a ligand for the ROS1 receptor as previously reported, confirming that ROS1 remains an orphan receptor. Thus, the discovery of a ROS1 ligand remains an important future priority. These findings highlight the potential for disease-associated somatic aberrations or splice variants that modify the ROS1 ECD to promote constitutive receptor activation, warranting further investigation.

Developmental interplay between transcriptional alterations and a targetable cytokine signaling dependency in pediatric ETO2::GLIS2 leukemia

BackgroundSeveral fusion oncogenes showing a higher incidence in pediatric acute myeloid leukemia (AML) are associated with heterogeneous megakaryoblastic and other myeloid features. Here we addressed how developmental mechanisms influence human leukemogenesis by ETO2::GLIS2, associated with dismal prognosis.MethodsWe created novel ETO2::GLIS2 models of leukemogenesis through lentiviral transduction and CRISPR-Cas9 gene editing of human fetal and post-natal hematopoietic stem/progenitor cells (HSPCs), performed in-depth characterization of ETO2::GLIS2 transformed cells through multiple omics and compared them to patient samples. This led to a preclinical assay using patient-derived-xenograft models to test a combination of two clinically-relevant molecules.ResultsWe showed that ETO2::GLIS2 expression in primary human fetal CD34+ hematopoietic cells led to more efficient in vivo leukemia development than expression in post-natal cells. Moreover, cord blood-derived leukemogenesis has a major dependency on the presence of human cytokines, including IL3 and SCF. Single cell transcriptomes revealed that this cytokine environment controlled two ETO2::GLIS2-transformed states that were also observed in primary patient cells. Importantly, this cytokine sensitivity may be therapeutically-exploited as combined MEK and BCL2 inhibition showed higher efficiency than individual molecules to reduce leukemia progression in vivo.ConclusionsOur study uncovers an interplay between the cytokine milieu and transcriptional programs that extends a developmental window of permissiveness to transformation by the ETO2::GLIS2 AML fusion oncogene, controls the intratumoral cellular heterogeneity, and offers a ground-breaking therapeutical opportunity by a targeted combination strategy.

Abstract C053: Pancreatic cancer patient-derived organoids, preclinical tools for therapeutic profiling, patient response prediction, and tumor evolution

Abstract Pancreatic cancer (PC) is characterized by an aggressive biology and an exceptionally high tumor heterogeneity that causes considerable variations in response to chemotherapies, targeted agents, and immunotherapies. Patient-derived organoids (PDOs) accurately reflect parental tumor biological and molecular features and may represent powerful preclinical avatars to predict drug response and support clinical decision-making. We generated a living organoid biobank of >100 PC PDO lines from treatment-naïve and pretreated primary tumor and metastases with a reliable efficacy (60.8%), and previously developed a pharmacotyping-guided prediction model to prognosticate patient therapy response in an initial feasibility trial (Beutel AK et al, 2021). Real-life chemotherapy responses in 46 patients (for a total of 93 therapy lines) matched to pharmacotyping-informed predictions with overall 73.1% accuracy, 85.4% sensitivity (responsiveness), and 63.5% specificity (non-responsiveness). Overall, our model allowed a successful drug-response prediction in naïve patients with an accuracy of 73.0% and 70.0% for first and second-line regimens. Prediction power was nevertheless lower in pretreated and heavily pretreated patients with a precision of 68.2% and 50.0% for subsequent chemotherapy lines, respectively. Interestingly, PDO-based pharmacotyping also precisely predicted patient clinical outcome in the neoadjuvant and adjuvant settings, with respective accuracies of 83.3% and 71.4%. Retrospective analysis of patient clinical data in palliative setting finally showed that the administration of a regimen predicted to be efficient ultimately translated into a significantly longer progression-free survival. Implementing automation and drug screening miniaturization to our workflow also significantly enhanced our process capacity, reduced time before pharmacotyping, and improved compliance to internal quality controls. Tracking clonal evolution in longitudinal biopsies (a set of seven PDO pairs derived from the same patients at two distinct timepoints) revealed lower mutational burden and therapy-driven genetic alterations upon progression. Notably, this approach revealed a CHEK2-mutated patient responded over time to PARP inhibitor maintenance therapy, aligning with our predictions and underscoring the robustness of our method. Single nucleus-resolved RNA and ATAC-seq analysis of a unique longitudinally-collected case uncovered transcriptomic and epigenetic dynamics associated with an oncogenic FGFR2 fusion and with a subsequent resistance-mediating mutation upon targeted treatment with FGFR2 inhibitors. Particularly, FGFR2 constitutive activation correlated with aberrant epigenetic trace and transcription programs of downstream targets as PI3K-AKT and RAS family members and of RNA polymerase II transcription machinery. Here, we report a robust and clinically-relevant preclinical tool for drug-response prediction, one more step towards a PDO therapeutic profiling-guided personalized medicine in clinical routine. Citation Format: Johann Gout, Yazid J Resheq, Jessica Lindenmayer, Julian D Schwab, Elodie Roger, Johann M Kraus, Thomas Ettrich, Alica K Beutel, Lukas Perkhofer, Hans A Kestler, Thomas Seufferlein, Alexander Kleger. Pancreatic cancer patient-derived organoids, preclinical tools for therapeutic profiling, patient response prediction, and tumor evolution [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C053.

Palmitoyltransferase ZDHHC3 is essential for the oncogenic activity of PML/RARα in acute promyelocytic leukemia.

The oncogenic fusion protein promyelocytic leukemia/retinoic acid receptor alpha (PML/RARα) is critical for acute promyelocytic leukemia (APL). PML/RARα initiates APL by blocking the differentiation and increasing the self-renewal of leukemic cells. The standard clinical therapies all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), which induce PML/RARα proteolysis, have dramatically improved the prognosis of APL patients. However, the emergence of mutations conferring resistance to ATRA and ATO has created challenges in the treatment of APL patients. Exploring pathways that modulate the oncogenic activity of PML/RARα could help develop novel therapeutic strategies for APL, particularly for drug-resistant APL. Herein, we demonstrated for the first time that palmitoylation of PML/RARα was a critical determinant of its oncogenic activity. PML/RARα palmitoylation was found to be catalyzed mainly by the palmitoyltransferase ZDHHC3. Mechanistically, ZDHHC3-mediated palmitoylation regulated the oncogenic transcriptional activity of PML/RARα and APL pathogenesis. The knockdown or overexpression of ZDHHC3 had respective effects on the expression of proliferation- and differentiation-related genes. Consistently, the depletion or inhibition of ZDHHC3 could significantly arrest the malignant progression of APL, particularly drug-resistant APL, whereas ZDHHC3 overexpression appeared to have a promoting effect on the malignant progression of APL. Thus, our study not only reveals palmitoylation as a novel regulatory mechanism that modulates PML/RARα oncogenic activity but also identifies ZDHHC3 as a potential therapeutic target for APL, including drug-resistant APL.