Potential Therapeutic Vulnerabilities Research Articles

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.

Pancreatic STAT5 activation promotes KrasG12D-induced and inflammation-induced acinar-to-ductal metaplasia and pancreatic cancer

ObjectivePancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy because it is often diagnosed at a late-stage. Signal transducer and activator of transcription 5 (STAT5) is a transcription factor implicated...

Genomic landscape and preclinical models of angiosarcoma.

Angiosarcoma is a cancer that develops in blood or lymphatic vessels that presents a significant clinical challenge due to its rarity and aggressive features. Clinical outcomes have not improved in decades, highlighting a need for innovative therapeutic strategies to treat the disease. Genetically, angiosarcomas exhibit high heterogeneity and complexity with many recurrent mutations. However, recent studies have identified some common features within anatomic and molecular subgroups. To identify potential therapeutic vulnerabilities, it is essential to understand and integrate the mutational landscape of angiosarcoma with the models that exist to study the disease. In this review, we will summarize the insights gained from reported genomic alterations in molecular and anatomic subtypes of angiosarcoma, discuss several potential actionable targets, and highlight the preclinical disease models available in the field.

Cohesin mutations in acute myeloid leukemia.

The cohesin complex, encoded by SMC3, SMC1A, RAD21, and STAG2, is a critical regulator of DNA-looping and gene expression. Over a decade has passed since recurrent mutations affecting cohesin subunits were first identified in myeloid malignancies such as Acute Myeloid Leukemia (AML). Since that time there has been tremendous progress in our understanding of chromatin structure and cohesin biology, but critical questions remain because of the multiple critical functions the cohesin complex is responsible for. Recent findings have been particularly noteworthy with the identification of crosstalk between DNA-looping and chromatin domains, a deeper understanding of how cohesin establishes sister chromatid cohesion, a renewed interest in cohesin's role for DNA damage response, and work demonstrating cohesin's importance for Polycomb repression. Despite these exciting findings, the role of cohesin in normal hematopoiesis, and the precise mechanisms by which cohesin mutations promote cancer, remain poorly understood. This review discusses what is known about the role of cohesin in normal hematopoiesis, and how recent findings could shed light on the mechanisms through which cohesin mutations promote leukemic transformation. Important unanswered questions in the field, such as whether cohesin plays a role in HSC heterogeneity, and the mechanisms by which it regulates gene expression at a molecular level, will also be discussed. Particular attention will be given to the potential therapeutic vulnerabilities of leukemic cells with cohesin subunit mutations.

Mutational Signatures in Colorectal Cancer: Translational Insights, Clinical Applications, and Limitations.

A multitude of exogenous and endogenous processes have the potential to result in DNA damage. While the repair mechanisms are typically capable of correcting this damage, errors in the repair process can result in mutations. The findings of research conducted in 2012 indicate that mutations do not occur randomly but rather follow specific patterns that can be attributed to known or inferred mutational processes. The process of mutational signature analysis allows for the inference of the predominant mutational process for a given cancer sample, with significant potential for clinical applications. A deeper comprehension of these mutational signatures in CRC could facilitate enhanced prevention strategies, facilitate the comprehension of genotoxic drug activity, predict responses to personalized treatments, and, in the future, inform the development of targeted therapies in the context of precision oncology. The efforts of numerous researchers have led to the identification of several mutational signatures, which can be categorized into different mutational signature references. In CRC, distinct mutational signatures are identified as correlating with mismatch repair deficiency, polymerase mutations, and chemotherapy treatment. In this context, a mutational signature analysis offers considerable potential for enhancing minimal residual disease (MRD) tests in stage II (high-risk) and stage III CRC post-surgery, stratifying CRC based on the impacts of genetic and epigenetic alterations for precision oncology, identifying potential therapeutic vulnerabilities, and evaluating drug efficacy and guiding therapy, as illustrated in a proof-of-concept clinical trial.

Evolution of transcriptomic profiles in relapsed inv(16) acute myeloid leukemia

Acute myeloid leukemia (AML) with inv(16) is typically associated with a favourable prognosis. However, up to 40 % of patients will eventually experience disease relapse. Herein, we dissected the genomic and transcriptomic profile of inv(16) AML to identify potential prognostic markers and therapeutic vulnerabilities. Sequencing data from 222 diagnostic samples, including 44 relapse/refractory patients, revealed a median of 1 concomitant additional mutation, cooperating with inv(16) in leukemogenesis. Notably, the mutational landscape at diagnosis did not differ significantly between patients experiencing primary induction failure or relapse when compared to the rest of the cohort, except for an increase in the mutational burden in the relapse/refractory group. RNA-Seq of unpaired diagnostic(n=7) and relapse(n=6) samples allowed the identification of oxidative phosphorylation (OXPHOS) as one of the most significantly downregulated pathways at relapse. Considering that OXPHOS could be targeted by Venetoclax/Azacitidine combination, we explored its biological effects on an inv(16) cell-line ME-1, but there was no additional advantage in terms of cell death over Azacitidine alone. To enhance Venetoclax efficacy, we tested in vitro effects of Metformin as a potential drug able to enhance chemosensitivity of AML cells by inhibiting the mitochondrial transfer. By challenging ME-1 with this combination, we observed a significant synergistic interaction at least similar to that of Venetoclax/Azacitidine. In conclusions, we identified a downregulated expression of oxidative phosphorylation (OXPHOS) at relapse in AML with inv(16), and explored the in vitro effects of metformin as a potential drug to enhance chemosensitivity in this setting.

60 Dissecting the Cellular and Epigenetic Landscape of Clear Cell Renal Cell Carcinoma Through Paired snRNA/ATAC-Seq Analysis

Abstract Background Clear cell renal cell carcinoma (ccRCC) stands as one of the most prevalent and aggressive forms of renal malignancies, accounting for a substantial portion of kidney cancer-related mortality worldwide. The heterogeneity within ccRCC poses a significant challenge to understanding its molecular mechanisms and devising effective therapeutic strategies. [1] Previous studies utilizing single nucleus RNA sequencing (snRNA-seq) have revealed transcriptional contributions to cell-type specificity in both mature human and mouse kidneys. Moreover, recent advancements have expanded this approach to single-cell profiling of chromatin accessibility. Single-nuclei assay for transposase-accessible chromatin using sequencing (snATAC-seq) has enabled the measurement of chromatin accessibility in thousands of individual cells, providing insights into the dynamic process that drives nephron development and differentiation. Integration and analysis of multimodal single-cell datasets, such as snRNA-seq and snATAC-seq, allows for the prediction of cell-type-specific cis-regulatory DNA interactions and transcription factor activity, complementing the transcriptional information obtained by snRNA-seq.[2] [3] [1] Yang, Wang, and Yang, “Treatment Strategies for Clear Cell Renal Cell Carcinoma.” [2] Muto et al., “Single Cell Transcriptional and Chromatin Accessibility Profiling Redefine Cellular Heterogeneity in the Adult Human Kidney.” [3] Monteagudo-Sánchez, Noordermeer, and Greenberg, “The Impact of DNA Methylation on CTCF-Mediated 3D Genome Organization.” Methods This study leveraged paired snRNA/ATAC-seq in tumor and normal-adjacent samples from the Renal Tumor Biobank at Dartmouth (n= 63) to explore the ccRCC landscape at single-cell resolution using 10x multiome technology. The snRNA-seq dataset was processed with Seurat v5.0 to remove low-quality nuclei. Doublets were removed with DoubletFinder v2.02, and ambient DNA was removed using decontX from celda. Normalization was conducted via SCTransform, and missing data was imputed with ALRA. Clustering was performed by constructing a KNN graph and running the Louvain algorithm. Cell type assignments were predicted using Azimuth with the human kidney reference dataset. The snATAC-seq data was processed with ArchR v1.0.1, which performed dimensionality reduction and clustering. Cluster identities defined in the Seurat workflow were cross-annotated to the ATAT-seq data, and open peaks were identified within these cell-type clusters using MACS3 v3.0.1. Identification of marker peaks, motif enrichment, and motif enrichment deviations were inferred utilizing ArchR’s getmarkerFeatures, peakAnnoEnrichment, and addMotifAnnotations functions, respectively. Results Motif enrichment analysis revealed canonical cancer-associated transcription factor (TF) dysregulation in tumor cells (nephron loop-like), including enrichment of HNF1B, SMARCC1, FOSL1, and JUND. Interestingly, HNF and SMARCC were also enriched in the lymphoid compartment, indicating possible crosstalk between these two cell types. Previous work has investigated the involvement of the HNF1 in ccRCC and in chromophobe RCC for prognosis,and we plan on investigating the relationship of these enrichments to tumor grade, stage and outcome.[1] Additional TF enrichments included BCL families and ELF within the myeloid compartment, and vascularization related TF families in endothelial cell types including enrichment of various ETS TFs. Investigation of cell-cell interactions between immune and endothelial cell populations as well as between tumor and normal adjacent tissues will be performed to further disentangle the role differentially accessable chromatin plays in ccRCC. [1] Buchner et al., “Downregulation of HNF-1B in Renal Cell Carcinoma Is Associated With Tumor Progression and Poor Prognosis.” Conclusions Our multi-omic approach has several advantages. First, it enables the characterization of cellular heterogeneity within the tumor microenvironment, identifying cell populations and their contributions to disease progression. Secondly, by integrating transcriptomic and epigenomic data, we gain insights into the dynamic interplay between gene expression and chromatin accessibility, uncovering potential therapeutic vulnerabilities and biomarkers for personalized treatment strategies. DOD CDMRP Funding: yes

GSTO1 aggravates EGFR-TKIs resistance and tumor metastasis via deglutathionylation of NPM1 in lung adenocarcinoma.

Despite significantly improved clinical outcomes in EGFR-mutant lung adenocarcinoma, all patients develop acquired resistance and malignancy on the treatment of EGFR tyrosine kinase inhibitors (EGFR-TKIs). Understanding the resistance mechanisms is crucial to uncover novel therapeutic targets to improve the efficacy of EGFR-TKI treatment. Here, integrated analysis using RNA-Seq and shRNAs metabolic screening reveals glutathione S-transferase omega 1 (GSTO1) as one of the key metabolic enzymes that is required for EGFR-TKIs resistance in lung adenocarcinoma cells. Aberrant upregulation of GSTO1 confers EGFR-TKIs resistance and tumor metastasis in vitro and in vivo dependent on its active-site cysteine 32 (C32). Pharmacological inhibition or knockdown of GSTO1 restores sensitivity to EGFR-TKIs and synergistically enhances tumoricidal effects. Importantly, nucleophosmin 1 (NPM1) cysteine 104 is deglutathionylated by GSTO1 through its active C32 site, which leads to activation of the AKT/NF-κB signaling pathway. In addition, clinical data illustrates that GSTO1 level is positively correlated with NPM1 level, NF-κB-mediated transcriptions and progression of human lung adenocarcinoma. Overall, our study highlights a novel mechanism of GSTO1 mediating EGFR-TKIs resistance and malignant progression via protein deglutathionylation, and GSTO1/NPM1/AKT/NF-κB axis as a potential therapeutic vulnerability in lung adenocarcinoma.

Timing of whole genome duplication is associated with tumor-specific MHC-II depletion in serous ovarian cancer

Whole genome duplication is frequently observed in cancer, and its prevalence in our prior analysis of end-stage, homologous recombination deficient high grade serous ovarian cancer (almost 80% of samples) supports the notion that whole genome duplication provides a fitness advantage under the selection pressure of therapy. Here, we therefore aim to identify potential therapeutic vulnerabilities in primary high grade serous ovarian cancer with whole genome duplication by assessing differentially expressed genes and pathways in 79 samples. We observe that MHC-II expression is lowest in tumors which have acquired whole genome duplication early in tumor evolution, and further demonstrate that reduced MHC-II expression occurs in subsets of tumor cells rather than in canonical antigen-presenting cells. Early whole genome duplication is also associated with worse patient survival outcomes. Our results suggest an association between the timing of whole genome duplication, MHC-II expression and clinical outcome in high grade serous ovarian cancer that warrants further investigation for therapeutic targeting.

DIPG-06. FUNCTIONAL MULTI-OMIC PROFILING OF THE LANDSCAPE OF DIFFUSE MIDLINE GLIOMAS TO IDENTIFY THERAPEUTIC VULNERABILITIES

Abstract BACKGROUND Diffuse midline glioma (DMG) is a devastating pediatric high-grade glioma (pHGG) located along the midline structures of the brain and spine. Palliative radiotherapy is the only approved treatment outside of clinical trials, with patients given 9–11-months survival post-diagnosis. Over the last 10-years, it has become common for patients to be prescribed precision therapies targeting genetic alterations, however largely, DMG harbor genetic alterations that are untargetable. METHODS To improve DMG patient outcomes, we are performing multi-omic analysis on 210 DMG, normal controls and other pHGG samples to functionally characterize the genomic landscape of these tumors and understand the protein-controlled functional consequences of recurring somatic alterations. RESULTS Genomic sequencing has been performed on 40/210 pHGG samples, identifying 290 unique mutations and several recurrent chromosomal alterations. Additionally, proteomic/phosphoproteomic profiling identified both unique and similar features of post-translational landscapes across genotypes. Examining H3.3K27M DMG cell-lines, SU-DIPG-XIII and UON-VIBE5, genomic sequencing identified 11 and 6 mutations respectively, however, only UON-VIBE5 possessed a genomically-predicted target, a PDGFRA-D842V mutation, potentially targetable by Avapritinib. Conversely, investigation of proteomes/phosphoproteomes revealed several potential therapeutic vulnerabilities. SU-DIPG-XIII possessed high enrichment of HDAC/BRD and CDK pathways and UON-VIBE5, ATM-centric pathways. In vitro proliferation assays of Avapritinib treated UON-VIBE5 failed to elicit a response at clinically relevant doses, while all proteomically-predicted therapies (HDAC – Fimepinostat, BRD – JQ1, CDK – Ribociclib and ATM – WSD-0628) successfully influenced tumor cell growth whilst leaving microglia controls cells unaffected. Avapritinib and WSD-0628 (CNS-active ATM inhibitor) drug predictions were further validated in the RA055 DMG model, which possesses similar genomic and proteomic features to UON-VIBE5. Avapritinib alone failed to influence tumor growth, while WSD-0628 notably reduced cell proliferation. The combination of these drugs, however, synergistically inhibited RA055 proliferation. CONCLUSIONS These findings thus provide preliminary evidence supporting the need to take a multi-omic approach in treating DMGs.

HGG-07. INVESTIGATING THE ROLE OF SENESCENCE IN PEDIATRIC HIGH-GRADE GLIOMAS

Abstract Cellular senescence has expanded well past the original definition of just proliferative exhaustion and tumor suppression. Today, senescence is considered a “Hallmark of Cancer” and the senescence-associated secretory phenotype (SASP) secretes molecules that can promote oncogenesis, angiogenesis, and an immunosuppressive tumor microenvironment (TME). Pediatric high-grade gliomas (pHGGs) are aggressive brain tumors with dismal outcomes and limited therapeutic options. Accumulating evidence suggests potential therapeutic vulnerabilities in targeting senescence in adult solid and brain tumors. Furthermore, senescent immune cells in the TME appear to play an important role in the initiation and progression in adult solid tumors. The role of senescence in pHGGs, however, has not been well described. Here, we show evidence of senescent glial and immune cells in pHGG tumors. Using patient-derived cell lines, we also investigate triggers of senescence and further characterize SASP in pHGGs. By understanding the role of senescence in gliomagenesis, we hope to open new approaches in treating pHGGs.

EPEN-04. OVERWHELMING ZFTA-RELA NUCLEAR LOCALIZATION AS A THERAPEUTIC STRATEGY AGAINST EPENDYMOMA

Abstract BACKGROUND Pediatric ependymoma is a malignant brain tumor without targeted therapies. The 10-year survival is close to 50%, as this relentless tumor often relapses years following initial diagnosis. Over two thirds of supratentorial ependymomas harbor a gene fusion between ZFTA and RELA (ZFTA-RELA). Despite evidence that ZFTA-RELA drives tumorigenicity, mechanistic details remain elusive, hindering efforts to uncover therapeutic vulnerabilities. Since direct targeting of ZFTA-RELA is not currently possible, we hypothesized that ZFTA-RELA fusion binding partners would represent vulnerabilities amenable to therapeutic targeting. METHODS We investigated the biochemical basis of ZFTA-RELA, with the goal of identifying key members of the protein interaction landscape. This was performed in our in utero electroporation mouse model of ZFTA-RELA ependymoma. A series of sucrose gradient and immuno-precipitation experiments were performed, coupled with mass spectrometry. ZFTA-RELA interaction proteins were functionalized using focused CRIPSR-Cas9 library screens in cell cultures. Critically, these revealed novel dependencies and druggable targets, with XPO1 emerging as a lead candidate. In vitro and in vivo studies were conducted to evaluate targeting XPO1 in ZFTA-RELA ependymoma. RESULTS ZFTA-RELA regulates XPO1 gene transcription, and elevated gene expression is associated with poor survival in these patients. While expression of ZFTA-RELA is necessary for oncogenic transcription, extremely elevated nuclear localization is associated with reduced growth. This suggests a ‘goldilocks’ model in which XPO1 titrates precise levels of nuclear ZFTA-RELA protein. We reveal that a potential therapeutic vulnerability emerges through modulation of ZFTA-RELA nuclear localization. Supporting this, chemical inhibition of XPO1 using Selinexor, has a direct effect on ZFTA-RELA cellular localization patterns. Critically, in patient derived orthotopic xenograft models, XPO1 inhibition exerts anti-tumor effects both in vitro and in vivo. CONCLUSION Our findings reveal a novel therapeutic strategy in ependymoma, and provide the mechanistic and pre-clinical data needed to develop a clinical trial for patients with ZFTA-RELA ependymoma.

The SMC5/6 complex prevents genotoxicity upon APOBEC3A-mediated replication stress

Mutational patterns caused by APOBEC3 cytidine deaminase activity are evident throughout human cancer genomes. In particular, the APOBEC3A family member is a potent genotoxin that causes substantial DNA damage in experimental systems and human tumors. However, the mechanisms that ensure genome stability in cells with active APOBEC3A are unknown. Through an unbiased genome-wide screen, we define the Structural Maintenance of Chromosomes 5/6 (SMC5/6) complex as essential for cell viability when APOBEC3A is active. We observe an absence of APOBEC3A mutagenesis in human tumors with SMC5/6 dysfunction, consistent with synthetic lethality. Cancer cells depleted of SMC5/6 incur substantial genome damage from APOBEC3A activity during DNA replication. Further, APOBEC3A activity results in replication tract lengthening which is dependent on PrimPol, consistent with re-initiation of DNA synthesis downstream of APOBEC3A-induced lesions. Loss of SMC5/6 abrogates elongated replication tracts and increases DNA breaks upon APOBEC3A activity. Our findings indicate that replication fork lengthening reflects a DNA damage response to APOBEC3A activity that promotes genome stability in an SMC5/6-dependent manner. Therefore, SMC5/6 presents a potential therapeutic vulnerability in tumors with active APOBEC3A.

RICTOR/mTORC2 downregulation in BRAFV600E melanoma cells promotes resistance to BRAF/MEK inhibition.

The main drawback of BRAF/MEK inhibitors (BRAF/MEKi)-based targeted therapy in the management of BRAF-mutated cutaneous metastatic melanoma (MM) is the development of therapeutic resistance. We aimed to assess in this context the role of mTORC2, a signaling complex defined by the presence of the essential RICTOR subunit, regarded as an oncogenic driver in several tumor types, including MM. After analyzing The Cancer Genome Atlas MM patients' database to explore both overall survival and molecular signatures as a function of intra-tumor RICTOR levels, we investigated the effects of RICTOR downregulation in BRAFV600E MM cell lines on their response to BRAF/MEKi. We performed proteomic screening to identify proteins modulated by changes in RICTOR expression, and Seahorse analysis to evaluate the effects of RICTOR depletion on mitochondrial respiration. The combination of BRAFi with drugs targeting proteins and processes emerged in the proteomic screening was carried out on RICTOR-deficient cells in vitro and in a xenograft setting in vivo. Low RICTOR levels in BRAF-mutated MM correlate with a worse clinical outcome. Gene Set Enrichment Analysis of low-RICTOR tumors display gene signatures suggestive of activation of the mitochondrial Electron Transport Chain (ETC) energy production. RICTOR-deficient BRAFV600E cells are intrinsically tolerant to BRAF/MEKi and anticipate the onset of resistance to BRAFi upon prolonged drug exposure. Moreover, in drug-naïve cells we observed a decline in RICTOR expression shortly after BRAFi exposure. In RICTOR-depleted cells, both mitochondrial respiration and expression of nicotinamide phosphoribosyltransferase (NAMPT) are enhanced, and their pharmacological inhibition restores sensitivity to BRAFi. Our work unveils an unforeseen tumor-suppressing role for mTORC2 in the early adaptation phase of BRAFV600E melanoma cells to targeted therapy and identifies the NAMPT-ETC axis as a potential therapeutic vulnerability of low RICTOR tumors. Importantly, our findings indicate that the evaluation of intra-tumor RICTOR levels has a prognostic value in metastatic melanoma and may help to guide therapeutic strategies in a personalized manner.

Abstract ED02-03: Innovative approaches at the diagnosis and treatment of invasive lobular carcinoma

Abstract Invasive Lobular Carcinoma (ILC) represents a unique subtype of breast cancer characterized by distinctive clinical and molecular features. This presentation aims to delve into innovative approaches in the diagnosis and treatment of ILC, focusing on novel techniques in imaging, unique molecular findings, signaling pathway alterations, targetable biomarkers, and microenvironment profiling that can pave the way for the development of novel therapies. Imaging Techniques in ILC: Accurate diagnosis is the foundation for effective treatment. ILC often presents diagnostic challenges due to the fact that lobular cells tends to infiltrate the affected organs in a diffuse manner instead of forming a discrete tumor mass making diagnostic imaging such as screening mammograms and CT scans less precise. This presentation will explore cutting-edge imaging techniques such as contrast-enhanced mammography, novel radioisotopes for positron emission tomography (PET) such as Fluoroestradiol F18 (FES), Fluciclovine and prostate-specific membrane antigen (PSMA) for improved detection and characterization of ILC lesions. Furthermore, we will discuss emerging modalities like radiomics and artificial intelligence, which hold promise in enhancing early diagnosis and risk assessment. Molecular Insights into ILC: ILC is distinguished by its unique molecular profile, notably the loss of E-cadherin expression. We will delve into recent advancements in understanding the molecular landscape of ILC, including genomic alterations, epigenetic changes, and the role of signaling pathways providing insights into potential therapeutic vulnerabilities. Targetable Biomarkers and Pathways: Identification of targetable biomarkers is essential for the development of precision therapies in ILC. This presentation will highlight promising targets, including ERBB2 (HER2), ROS1, TROP2, AKT1 along with other novel biomarkers. Microenvironment Profiling: The tumor microenvironment plays a pivotal role in cancer progression and therapy response. We will discuss recent advancements in characterizing the ILC microenvironment, including the immune cell infiltrate and stromal components. Understanding the dynamic interplay between the tumor and its microenvironment can inform the development of immunotherapies and combination strategies tailored to ILC. In conclusion, this presentation will provide a comprehensive overview of the latest innovations in ILC diagnosis and treatment. Our exploration of advanced imaging techniques, molecular findings, targetable pathways, biomarkers, and microenvironment profiling will shed light on the exciting potential for personalized therapeutic approaches in the management of ILC. By staying at the forefront of these developments, we can pave the way for improved outcomes and enhanced quality of life for patients with ILC. Citation Format: J. Mouabbi. Innovative approaches at the diagnosis and treatment of invasive lobular carcinoma [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr ED02-03.

The impact of ribosome biogenesis in cancer: from proliferation to metastasis.

The dysregulation of ribosome biogenesis is a hallmark of cancer, facilitating the adaptation to altered translational demands essential for various aspects of tumor progression. This review explores the intricate interplay between ribosome biogenesis and cancer development, highlighting dynamic regulation orchestrated by key oncogenic signaling pathways. Recent studies reveal the multifaceted roles of ribosomes, extending beyond protein factories to include regulatory functions in mRNA translation. Dysregulated ribosome biogenesis not only hampers precise control of global protein production and proliferation but also influences processes such as the maintenance of stem cell-like properties and epithelial-mesenchymal transition, contributing to cancer progression. Interference with ribosome biogenesis, notably through RNA Pol I inhibition, elicits a stress response marked by nucleolar integrity loss, and subsequent G1-cell cycle arrest or cell death. These findings suggest that cancer cells may rely on heightened RNA Pol I transcription, rendering ribosomal RNA synthesis a potential therapeutic vulnerability. The review further explores targeting ribosome biogenesis vulnerabilities as a promising strategy to disrupt global ribosome production, presenting therapeutic opportunities for cancer treatment.

Abstract 5105: Aberrant CLK1 splicing is a key dependency factor in pediatric high-grade gliomas

Abstract Pediatric brain cancer is the leading cause of disease-related mortality in children. With a paucity of therapeutically targetable alterations, there is a pressing need to identify effective strategies for many of these tumors. In this study, we systematically examine aberrant alternative splicing in pediatric brain tumors from the Open Pediatric Brain Tumor Atlas, and additional tumors from the Children's Brain Tumor Network and the Pacific Pediatric Neuro-Oncology Consortium (n = 1831). Notably, high-grade gliomas (HGGs), including those with and without H3 histone mutations, were enriched in the upper quartile of all tumors with high splicing burden (SB). HGGs demonstrated the greatest SB heterogeneity of all tumor types and we identified a total of 19,229 aberrant splicing events in 8,577 genes in midline tumors compared to non-tumor brainstem controls (ΔPSI >= |.20|, p < 0.05). We next integrated protein annotations from the UniProt Knowledgebase to assess and prioritize differential alternative splicing events affecting protein function. This approach led to the discovery of 3,754 splice variants in 5,036 genes resulting in the gain or ablation of functional sites. The protein kinase CDC-like kinase 1 (CLK1), a recognized master splicing factor and cell-cycle modulator, exhibited aberrant splicing of exon 4, whereby inclusion promoted the gain of two known phosphorylation sites. Additionally, we observed a strong correlation between CLK1 splicing, its own RNA expression and proteomic activity of the Serine/Arginine-rich (SR) family of splicing factors. The distribution of exon 4 splicing was significantly different (p = 0.019) between tumors with low vs high splicing burden; tumors with high splicing burden had increased exon 4 inclusion. Further, we performed experimental modulation of CLK1 exon 4 aberrant splicing in the brain tumor cell line KNS42 and demonstrated that forced exon 4 skipping leads to reduced CLK1 protein abundance and significantly reduces cell proliferation (p < 0.01 vs. control). Following bulk RNA-Seq analysis of these cell lines, we identified that forced CLK1 exon 4 skipping resulted in 4,518 dysregulated genes enriched for known cancer pathways such as mitotic spindle, E2F targets, and G2M checkpoint. Our data suggest a role for CLK1 in driving transcriptional dysregulation in pediatric high-grade gliomas and uncover a potential therapeutic vulnerability. Citation Format: Ammar S. Naqvi, Priyanka Seghal, Ryan Corbett, Komal Rathi, Brian Ennis, Bo Zhang, Miguel Brown, Daniel Miller, Adam Kraya, Joseph Dybas, Katharina Hayer, Shehbeel Arif, Zhuangzhuang Geng, Antonia Chroni, Aditya Lahiri, Karina Conkrite, Madison Hollawell, Sharon Diskin, Peter Madsen, Jessica B. Foster, Mateusz P. Koptyra, Andrei Thomas-Tikhonenko, Adam Resnick, Phillip J. Storm, Jo Lynne Rokita. Aberrant CLK1 splicing is a key dependency factor in pediatric high-grade gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5105.

Abstract 1587: Multimodal spatial transcriptomics uncover distinct tumor microenvironment states and cell-cell communication networks in molecular pancreatic cancer subtypes

Abstract The tumor microenvironment (TME) presents a complex ecosystem comprising a diversity of immune and stromal cell populations that influence tumor progression, drug delivery and therapy outcome. High levels of inter- and intra-tumor heterogeneity in TME state are driven by molecular tumor phenotypes. Pancreatic ductal adenocarcinoma (PDAC), one of the most lethal cancer types, displays a high genetic heterogeneity along with an immunosuppressive TME. However, it remains largely elusive how distinct TME states are mechanistically influenced, and which molecular processes dictate the emergence of different modes of immunosuppression within the TME of molecular PDAC subtypes.Here, we performed a systematic analysis of functional associations between the major molecular PDAC subtypes, the classical and mesenchymal subtype, and their TME states. We describe the TME composition and complex cell-cell communication networks within PDAC subtypes using a multimodal integration of spatial transcriptomics (ST) with complementary approaches, such as scRNA-seq, MS-based Secretomics and multiplexed histocytometry. We generated ST data sets (Visium) of a tumor cohort derived from a Kras-driven PDAC mouse model. To analyze TME composition as well as spatial niches and cell type communities, we computationally enhanced the spot-resolution of ST datasets, followed by cell type deconvolution and cell-cell communication analysis to identify subtype-specific TME communities and communication networks.To this end, we generated a large resource of PDAC mouse models which represent molecular subtypes of the disease and mimic the heterogeneity of TME states found in human PDAC cohorts. This analysis revealed that a set of subtype-specific secreted factors shape the immunosuppressive PDAC TME via direct and indirect communication networks with immunosuppressive myeloid and T cells in molecular PDAC subtypes. Multimodal ST analysis delineated spatial subtype-specific TME communities as well as spatial communication patterns. We functionally investigate the tumor cell intrinsic regulation of the identified subtype-specific secreted factors, providing potential therapeutic vulnerabilities for more effective combinatorial subtype-specific therapies including immunotherapeutic approaches. Citation Format: Stefanie Baerthel, Chiara Falcomatà, Vanessa Goelling, Daniele Lucarelli, Rushin Gindra, Constantin Schmitt, Jonathan Swietlik, Felix Meissner, Marc Schmidt-Supprian, Dieter Saur. Multimodal spatial transcriptomics uncover distinct tumor microenvironment states and cell-cell communication networks in molecular pancreatic cancer subtypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1587.

Abstract 6602: Targeting the SWI/SNF complex in POU2F-POU2AF transcription factor-driven malignancies

Abstract Background: Small cell lung cancer (SCLC) is a rapidly progressing subtype of lung cancer with a high growth rate and early metastasis propensity, often resulting in a more advanced disease stage at diagnosis. A recent transcriptome analysis of human SCLC tumors revealed that SCLC could be characterized by the expression pattern of certain transcription factors (TFs), including ASCL1 (achaete-scute family bHLH transcription factor 1) and POU2F3 (POU domain class 2 transcription factor 3), exemplifying SCLC as a TF-driven malignancy. ASCL1-driven SCLC (SCLC-A) manifests a neuroendocrine phenotype, while POU2F3-driven SCLC (SCLC-P) is characterized as a tuft-cell-like variant. Recent studies revealed that POU domain class 2 transcription factors uniquely rely on coactivators to achieve their lineage-defining functions in the tuft cell and B cell lineages. In tuft-cell-like SCLC cells, the coactivators of POU2F3 (POU2AF2 and POU2AF3) endow POU2F3 with a critical transactivation domain by forming a master regulator complex, which supports enhancer-mediated cancer-promoting gene activation in SCLC-P cells. This indicates a potential therapeutic vulnerability of tuft-cell-like SCLC whereby strategies aimed at blocking POU2F3-POU2AF2/3 function may lead to clinical benefit. Methods: We conducted a domain-targeted CRISPR screen to identify druggable targets for SCLC-P, followed by pharmacological validation in preclinical SCLC models. Multi-omics techniques, including ATAC-seq, ChIP-seq, RNA-seq, and RIME, were employed to elucidate the SWI/SNF complex's regulatory influence on the POU2F-POU2AF axis. Results: Here we identified that the POU2F3 molecular subtype of SCLC (SCLC-P) exhibits an exquisite dependence on the activity of the SWI/SNF epigenetic complex. SCLC-P cell lines were sensitive to low nanomolar levels of a SWI/SNF ATPase degrader when compared to other molecular subtypes of SCLC. Co-factors of POU2F were found to interact with components of the SWI/SNF complex. The POU2F3 transcription factor complex is evicted from chromatin upon SWI/SNF ATPase degradation. A novel, orally bioavailable SWI/SNF ATPase PROTAC degrader demonstrated preferential efficacy in SCLC-P relative to the SCLC-A subtype. The SWI/SNF ATPase PROTAC degrader did not alter normal tuft cell numbers in lung or colon, nor did it exhibit toxicity in mice. B cell malignancies which displayed a dependency on the POU2F co-factor, POU2AF1, were also remarkably sensitive to SWI/SNF ATPase degradation. In a POU2AF1-dependent, disseminated murine model of multiple myeloma, AU-24118 had an enhanced survival benefit as compared to pomalidomide, an approved treatment for multiple myeloma. Conclusions: Taken together, our studies suggest that POU2F-POU2AF driven malignancies have an intrinsic dependence on the SWI/SNF complex representing a striking therapeutic vulnerability. Citation Format: Lanbo Xiao, Tongchen He, Olaf Klingbeil, Young Eleanor, Xiaoli Wu, Yuanyuan Qiao, Abhijit Parolia, Sanjana Eyunni, Rahul Mannan, Somnath Mahapatra, NamHoon Kim, Heng Zheng, Fengyun Su, Xuhong Cao, Susanta Samajdar, Murali Ramachandra, Christopher R. Vakoc, Arul M. Chinnaiyan. Targeting the SWI/SNF complex in POU2F-POU2AF transcription factor-driven malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6602.

Abstract 278: Investigating the functional role of cancer-associated fibroblasts in the emergence of drug-tolerant persisters in ovarian cancer

Abstract Chemoresistance remains a major clinical challenge for the treatment of high-grade serous ovarian cancer (HGSC), the most lethal gynecologic malignancy. The majority of patients who initially respond to first-line platinum/taxane-based chemotherapy eventually relapse with chemoresistant disease, leading to poor survival outcomes. It is increasingly recognized that cancer cells can enter a reversible drug-tolerant persister (DTP) state through non-genetic mechanisms to evade chemotherapy-induced cytotoxicity prior to the development of chemoresistance. However, how environmental signals and extracellular factors from the tumor microenvironment drive the process remains poorly understood. Cancer-associated fibroblasts (CAFs) are a functionally heterogeneous population of activated fibroblasts. CAFs play a critical role in shaping the tumor microenvironment and tumor behaviors, including promoting tumor growth and mediating therapy resistance. Here, we demonstrated that HGSC cell lines co-cultured with patient-derived FAP-high CAFs, a subpopulation of CAFs previously identified in our lab to be tumor-promoting, significantly increase the formation of DTPs upon prolonged carboplatin treatment compared to the mono-cultured condition. Interestingly, the extracellular matrix (ECM) organization signature is enriched in FAP-high CAFs compared to their FAP-low counterparts, and we found that ECM deposited by FAP-high CAFs also facilitates the emergence of DTPs in HGSC cells. Future work will focus on elucidating the mechanisms by which FAP-high CAFs and the associated ECM promote DTP formation in HGSC cells through transcriptomic and proteomic profiling. This will aid the identification of potential therapeutic vulnerabilities to target cancer cells in the DTP state in FAP-high HGSC patients. Citation Format: Angel S. Ng, Ngoc Bui, Laurie Ailles. Investigating the functional role of cancer-associated fibroblasts in the emergence of drug-tolerant persisters in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 278.