Primary Samples Research Articles

Abstract 1646: IL-23R is a regulator of the mitotic spindle and is critical for cell viability in AML

Abstract To identify novel biological vulnerabilities in AML, we searched for gene ontologies that were upregulated in AML compared to normal hematopoietic cells. Of the upregulated processes, the mitotic spindle ontology was a top hit. Unexpectedly, expression of the IL-23 receptor (IL-23R) showed a strong positive correlation with the mitotic spindle ontology. IL-23R is a cell surface cytokine receptor that is canonically expressed on T cells, but not known to have a role in AML. In 7 of 7 tested AML cell lines and 15 of 20 primary AML patient samples, IL-23R protein was expressed >2 fold higher compared to mean IL-23R in normal mononuclear hematopoietic cells (n=5) and CD34+ cells (n=3). IL-23R is classically a T cell surface receptor and we confirmed localization to the cell surface in T cells. However, only small amounts of IL-23R were present on the cell surface of AML cell lines and primary AML samples. Rather, in AML cells, primary AML samples, and AML stem cells, IL-23R was located intracellularly as determined by 4 different modalities and 4 different antibodies directed against 4 different epitopes of the receptor. Of note, only the full-length IL-23R protein was detected by immunoblotting. We also detected IL-23R’s heterodimeric partner, IL12Rβ1, in AML cells. To understand the function of intracellular IL-23R, we used BioID mass spectrometry to identify proteins that interacted with IL-23R. Pathway analysis of interacting proteins identified mitotic spindle formation as the top pathway. Using Proximity Ligation Assays, we validated interactions with mitotic spindle proteins, and BioID hits, NUMA, TMEM201, TACC1, and BAG6 in AML cells. By confocal microscopy, we demonstrated that IL-23R colocalized with the mitotic spindle and centrosomes in AML cells and primary AML samples. IL-23R interacted with the mitotic spindle via its (S/T)x(I/L)P motif (amino acids 588-591). IL-12Rβ1 was also found in complex with IL-23R at the mitotic spindle in AML cells. Additionally, it was discovered that the cytokine, IL-23, promoted translocation of IL-23R to the mitotic spindle through clathrin-mediated endocytosis. Knockdown and knockout of IL-23R in AML cells led to dysregulation of the mitotic spindle with multipolarity, lagging chromosomes, and other spindle defects. Knockdown of IL-23R also reduced proliferation, clonogenic growth, and marrow engraftment of AML cells and primary AML samples. In contrast, knockdown of IL-23R in normal CD34+ cells did not impair engraftment. Likewise, constitutive homozygote IL-23R knockout mice had no difference in complete blood counts and stem cell number/function compared to wild type. In summary, IL-23R interacts with the mitotic spindle and centrosome where it regulates mitotic spindle formation and is critical for AML cell viability. Thus, we discover a novel function for IL-23R and a potential therapeutic target for this disease. Citation Format: Nathan Duong, Dilshad H. Khan, Geethu E. Thomas, Rose Hurren, Jong Bok Lee, Jonathan St-Germain, Lily Drimmer, Yongran Yan, Neil Maclean, Marcela Gronda, Vincent Rondeau, Brandon D. Brown, Courtney L. Jones, Hong Chang, Andrea Arruda, Mark D. Minden, Li Zhang, Steven M. Kornblau, Brian Raught, Vito Spadavecchio, Aaron D. Schimmer. IL-23R is a regulator of the mitotic spindle and is critical for cell viability in AML [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 1646.

Abstract 6566: Clinical evaluation of functional combinatorial precision medicine platform to predict treatment outcomes and enhance combination therapy design in soft tissue sarcomas

Abstract Purpose: Soft tissue sarcomas (STS) are a collection of heterogenous cancers with limited range of treatment options and poor outcomes. First line treatment for most STS is anthracycline-based, with gemcitabine-based combinations, pazopanib or ifosfamide as treatment options in refractory/relapsed STS. There is a clinical unmet need to improve treatment options for STS as median time to progression following second- and third-line treatments is less than 4 months. We have previously demonstrated in a 71-patient relapsed/refractory non-Hodgkin’s lymphoma clinical study that a functional precision medicine platform can effectively guide treatment, with several exceptional responders (Science Translational Medicine, 2022). This ex vivo drug sensitivity platform, quadratic phenotypic optimization platform (QPOP), analyses a predesigned array of 155 test combinations performed on a primary patient sample to rank and compare all possible therapeutic combinations from a 12-drug set. In this study, we evaluated clinical concordance of QPOP to predict treatment outcomes in an STS cohort. We also explored QPOP’s combination therapy ranking function to identify novel combinations that may benefit larger groups of STS patients when appropriately guided. Experimental Detail: A 12-drug set comprised of STS standard of care, FDA approved drugs and promising investigational drugs was evaluated in QPOP analysis of primary STS tumor samples. Ex vivo cultures of primary STS samples were treated with a predesigned array of 155 test combinations to provide a quantitative phenotypic output for each test combination. This dataset was analyzed by QPOP to rank all possible drug combinations towards a predicted treatment outcome. Concordance analysis was then performed comparing patient sample results with either parallel treatment initiated at time of sample collection or physician choice to use QPOP-guided treatment. Validation of QPOP-derived top-ranked drug combinations was also performed. Results: 12-drug, 3-level QPOP was successfully performed on 45 of 51 primary patient samples. Clinical concordance analysis showed QPOP had a total predictive value (TPV) of 76.9% and an AUCROC of 85.7%. Exceptional responders to guided treatment with pazopanib or eribulin were observed. Additionally, top-ranked epigenetic-based combinations were identified as effective across multiple STS subtypes and validated preclinically. Conclusion: The results show the potential for QPOP to predict clinical response in solid cancers, such as STS. Furthermore, QPOP can improve drug development pipelines through patient samples-based design of effective combinations with investigational drugs or underutilized drugs that may benefit larger populations of patients or specific subgroups of patients. Citation Format: Edward Kai-Hua Chow, Sharon P. Chan, Tan Boon Toh, Masturah Rashid, Valerie S. Yang. Clinical evaluation of functional combinatorial precision medicine platform to predict treatment outcomes and enhance combination therapy design in soft tissue sarcomas [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 6566.

Abstract 3886: Analysis of clonal heterogeneity within paired primary and metastatic tumor samples of patients with solid tumors and implications for neoantigen-based personalized cancer vaccines

Abstract Background: Neoantigen-based personalized cancer vaccines carry significant promise in treating solid malignancies. However, there are uncertainties regarding the choice between the primary or the metastatic tumor for neoantigen prediction in individual patients. Here, we conducted a thorough examination of somatic variations in 676 patients who had paired primary and metastatic solid tumors. Methods: Patients were enrolled in the Total Cancer Care protocol (NCT03977402) to which patients provided an IRB-approved written informed consent within the Oncology Research Information Exchange Network (ORIEN). Whole-exome sequencing of 756 primary and metastatic tumor pairs was performed (N = 676 patients). These included Genitourinary (n=83), Gynecological (n=97), Gastrointestinal (n=213), Thoracic (n=33), Cutaneous (n=24), Breast (n=108), Endometrial (n=49), Sarcoma (n=35), Head-and-Neck (n=106) and others (n=8). The data was analyzed through the ORIEN AVATAR Molecular Analysis Pipeline for somatic mutation variant detection and variant annotation. In this analysis, we focused on somatic events that result in an in-frame alteration (such as missense, in-frame deletion and in-frame insertion) and out-of-frame protein-altering mutations (such as frameshifts, de novo start, out-of-frame, and nonstop gain). Clonal population structure was determined based on pyclone-vi. Results: For in-frame events, bladder cancer, melanoma, and gynecological cancers shared close to 75% of the mutations between paired primary and metastatic cases. In contrast, sarcoma and thyroid cancer had a low overlap (~ 25%) of variants. For out-of-frame events, these events tend to have a lower proportion of shared somatic variants between primary and metastasis than in-frame variants. Oncogenic drivers (e.g., BRAF V600E, KRAS G12A, and TP53 loss-of-function) were highly likely to be present in both paired primary and metastatic tumors. Next, we performed additional analysis on evolutionary selection of protein-coding variants via dN/dS calculation. We found no significant global shift in dN/dS ratio between paired primary and metastatic tumors across malignancies. However, we found increased selection of protein-coding variants in brain and liver metastatic sites, which correlated with increased homologous recombination deficiency within these sites. Conclusions: Our analysis demonstrates genetic variations that exist when comparing paired primary and metastatic tumors that appear to vary by histology. Variants are potentially undergoing negative selection supported by the preferential loss of out-of-frame events in metastatic tumors and positive selection in specific metastatic sites. Overall, understanding the clonal structure will be key to neoantigen prediction for effective neoantigen-based vaccines. Citation Format: Alyssa Obermayer, Timothy Shaw, Darwin Chang, Joshua Davis, Jamie K. Teer, Xiaoqing Yu, Xuefeng Wang, Dale Hedges, Aik Choon Tan, Robert Rounbehler, Abdul Rafeh Naqash, Margaret Gatti-Mays, Aakrosh Ratan, Martin McCarter, Howard Colman, Igor Puzanov, Susanne Arnold, Michelle Churchman, Patrick Hwu, William Dalton, George Weiner, Jose Conejo-Garcia, Paulo C. Rodriguez, Bodour Salhia, Ahmad A. Tarhini. Analysis of clonal heterogeneity within paired primary and metastatic tumor samples of patients with solid tumors and implications for neoantigen-based personalized cancer vaccines [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 3886.

Abstract 947: A nanodroplet cell processing platform for identifying synergistic drug combinations for neuroblastoma treatment in TH-MYCN transgenic mice

Abstract Neuroblastoma is the most common pediatric extracranial solid tumor and contributes to more than 15% of child mortality. It is characterized by extreme heterogeneity and high-risk tumors often resulting in poor prognosis. Currently, chemotherapy is a prevailing standard treatment for neuroblastoma. However, it is plagued by multiple challenges such as drug resistance and debilitating side effects. Synergistic drug combination is proposed as an innovative approach to potentially improve clinical outcomes of single-agent chemotherapy. We propose a novel approach to screen for effective chemo-synergistic drug combinations using Bioinspired nanodroplet processing (BioNDP) screening platform. This platform overcomes the limitations of conventional drug screening approaches, including the requirement for large cell numbers and the low translation rate from bench to bedside. In this study, we utilize TH-MYCN transgenic mice, a well-known genetically engineered mouse model of neuroblastoma that recapitulates critical genetic and clinical aspects of high-risk MYCN-amplified neuroblastoma. With its genetically homogeneous background and spontaneous tumor development, TH-MYCN mice offer a valuable comparison to traditional xenograft mouse models. We have also developed an optimal procedure of primary murine tumor isolation using both negative and positive selection approaches. The purity of the isolated cells was verified with GD2 expression, one of well-known marker of neuroblastoma. In addition, we have successfully identified an effective drug combination of cyclophosphamide, doxorubicin, and vincristine, using primary neuroblastoma tumor samples derived from TH-MYCN transgenic mice as a target. The in vitro synergistic effect of primary mouse tumor cell cytotoxicity was confirmed by using luminescent cell viability assay. Further in vivo experiments have demonstrated the therapeutic efficacy of this selected drug combination in suppressing tumor growth and improving survival rate in all TH-MYCN transgenic mice treated. To sum up, we aim to establish a future patient-centric paradigm by which minimal primary cell samples from a personalized biopsy of patients may undergo drug screening, leading to the identification of a tailored drug combination. The BioNDP platform we developed may become a pivotal resource for personalized therapy to ensure a faster and more efficient treatment strategy for patients. Citation Format: Yen-Tzu Liao, Zhi-Kai Yu, Ching-Te Kuo, Wen-Ming Hsu, Yi-Xun Huang, Hsinyu Lee. A nanodroplet cell processing platform for identifying synergistic drug combinations for neuroblastoma treatment in TH-MYCN transgenic mice [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 947.

Abstract 2305: Label-free high dimensional single cell morphological profiling of different hematological malignancies by ghost cytometry

Abstract Characterizing the heterogeneity in human leukemias is critical for understanding mechanisms of disease onset, uncovering biomarkers for disease diagnosis, and guiding research into novel treatment approaches. While extensive research focus has been placed on characterizing the molecular heterogeneity in leukemia by modern omics technologies, comparatively less emphasis has been placed on studying the diversity in holistic morphological changes that occur in single cells in the context of disease. Here, we report on a novel approach to understanding and characterizing different hematological malignancies using ghost cytometry, a recently developed high-content flow cytometric method that leverages high-speed, artificial intelligence (AI) driven morphological characterization and analysis of single cells. We performed morphometric profiling on human bone marrow samples from patients with T and B-cell acute lymphoblastic leukemia (ALL, n=5), acute myeloid leukemia (AML, n=5), multiple myeloma (MM, n=5) and healthy bone marrow mononuclear cell (BM-MNC) controls (n=6). Morphological profiles from individual cells were analyzed by unsupervised machine learning (universal manifold approximation and projection, UMAP) and compared within disease subsets and against controls. We identified disease-specific cell populations from a mixture of healthy control and disease samples without any labeling, based on completely label-free information. For validation of the specific cell subsets, we overlaid known cell surface markers (e.g. CD45 for blast cells, CD10/CD19 for B-ALL, CD33/CD34 for AML, CD38/CD138 for MM). The intensity of CD45 was dimmer and disease-specific markers were highly expressed in the disease-specific cell population. We also identified a unique cell subpopulation in the disease-specific cell population that could not be distinguished by existing markers. We also analyzed three human myeloma cell lines (AMO1, KMS, and L363) for comparison to the primary MM samples. In the UMAP analysis using LF-GC data, in all three types of cell lines, Bortezomib-resistant cell lines exhibited different distributions from the control parental cell lines. In addition, we observed distinct morphological profiles between primary myeloma samples and myeloma cell lines. Here we present a novel cell characterization approach for human hematological diseases that leverages AI-based, label-free, high-speed morphological characterization of single cells. We demonstrate that the approach can be used to identify subtle morphological differences in blast cells from a range of blood cancer subtypes. Application of morphological profiling and AI can be used to identify measurable, disease-related changes in these diseases that have diagnostic, drug screening, and therapeutic monitoring potential. Citation Format: Asako Tsubouchi, Juho J. Miettinen, Keisuke Wagatsuma, Satoru Akai, Yuri An, Mika Uematsu, Philipp Sergeev, Dimitrios Tsallos, Markus J. Vähä-Koskela, Sadao Ota, Caroline A. Heckman. Label-free high dimensional single cell morphological profiling of different hematological malignancies by ghost cytometry [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 2305.

Abstract 3928: Genomic landscape and estimation of immune infiltration of soft tissue sarcoma histology subtypes from the ORIEN network

Abstract Sarcomas encompass a group of malignant diseases arising from mesenchymal origins. Given their rarity and diversity, a fundamental understanding of the genomic underpinnings for many sarcoma subtypes is still lacking. Studies are often limited to one or several of the more common subtypes or a narrow evaluation of a broader sampling. We therefore report on one of the largest comprehensive omics evaluation in sarcomas to date, including whole exome sequencing (WES; n = 1170) and RNA-sequencing (n = 983) of tissues from 29 different sarcoma histologic subtypes collected at 13 institutions in the US as part of the Oncology Research Information Exchange Network (ORIEN). We identified recurrent somatic mutations previously identified in sarcomas (e.g. TP53, KIT) as well as other cancer types (e.g. BRCA1). The burden of putatively pathogenic driver point mutations was higher in metastatic samples (median = 3) as compared to primary tumor samples (median = 2; p < 0.001). We observed frequent copy number alterations including whole genome doubling more commonly in metastatic compared to primary tumors (23.4% vs. 16.9%; p = 0.0.25). Inspection of gene expression dimensionality reduction (UMAP) showed separation of gastrointestinal stromal tumors (GISTs), leiomyosarcomas, myxoid liposarcomas, and well/de-differentiated liposarcomas from the other histologies. Differential expression analysis for these four histologies with gene set enrichment analysis highlights the diversity of disease-specific pathways and need for sarcoma subtype-specific translational focus. Estimation of immune cell abundances based on RNA-seq followed by hierarchical clustering identified five immune subtypes. The subtypes ranged from low (clusters A, B) to high (clusters D, E) immune infiltration with higher abundance of T, B, Natural Killer (NK), and myeloid cells (FDR < 0.01). Intermediate immune group C was predominantly composed by GISTs and marked by an enrichment for NK cells (FDR < 0.01) compared to all groups except the immune “hot” group E; however, this immune group exhibited modest infiltration by other immune cell types. Notably, we observed significant differences in the overall survival of patients with sarcomas in immune enriched (C, D, E) compared to immune depleted clusters (A, B; p = 0.002). In summary, we report the genomic and expressional landscape of over 1000 sarcomas, representing one of the largest comprehensive profiling efforts in this disease. We identify the mutational and copy number variation landscape and observe differences between primary and metastatic samples. We highlight expression pathways that are enriched in histologic subtypes that cluster most distinctly from others, providing a subtype-specific roadmap for further translational efforts. Finally, we define immune enriched or depleted sarcoma subgroupings that carry a prognostic impact. Citation Format: Alex C. Soupir, Oscar E. Ospina, Dale Hedges, Jamie K. Teer, Michael D. Radmacher, David M. McKean, Nathan Seligson, Martin McCarter, Breelyn Wilkey, Greg Riedlinger, John Groundland, Benjamin J. Miller, Bryan Schneider, Reema Patel, Abdul Rafeh-Naqash, Stephen Edge, Bodour Salhia, Chris Moskaluk, Maggy Johns, Michelle L. Churchman, Oliver Hampton, David Liebner, Brooke L. Fridley, Andrew S. Brohl. Genomic landscape and estimation of immune infiltration of soft tissue sarcoma histology subtypes from the ORIEN network [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 3928.

Abstract 1373: Gene expression correlation of immune checkpoint molecules Siglec-15 and PD-L1 varies widely by cancer indication

Abstract Siglec-15 is a novel target for immunotherapy in cancer and exhibits low mRNA expression in normal tissues but broad expression across cancer indications, specifically on tumor-associated macrophages and tumor cells [1]. Expression of Siglec-15 and the immune checkpoint molecule PD-L1 have previously been reported to be mutually exclusive [1][2]. However, a large-scale meta-analysis of this claim has not been undertaken across cancer indications. Here, RNA-Seq analyses were performed across single cells, cell lines, and bulk tumor samples for Siglec-15 and PD-L1 mRNA expression. Data and meta-data from TCGA, CCLE, and primary tumor samples from 62 studies were obtained through OmicSoft OncoLand and single cell analysis was performed on the BioTuring Talk2Data platform. Analyses were limited to non-small cell lung cancer (NSCLC), cholangiocarcinoma, breast, thyroid, head and neck, colon, rectal, bladder, kidney, and endometrial cancers. Expression values were binned separately into percentiles across all indications and samples in increments of 10%. These analyses revealed individual gene expression levels of Siglec-15 and PD-L1 in bulk tumor samples varied substantially by indication, while co-expression of Siglec-15 and PD-L1 varied across indications. Analysis of gene expression of Siglec-15 and PD-L1 from the TCGA database showed a positive correlation in several indications including NSCLC, head and neck, kidney, and thyroid cancers. Analyses from additional studies in primary tumor samples confirmed co-expression of Siglec-15 and PD-L1 in NSCLC and head and neck cancer. Cell lines showed a similar trend of positive correlation with varying levels of Siglec-15 and PD-L1 expression. However, when examining expression at the single cell level (not bulk tumor), in 30 single cell studies spanning >1.3M cells, only 731 cells in 20 studies were found to co-express Siglec-15 and PD-L1 on the same cell while each gene was expressed individually on other cells. The majority of the cells co-expressing the genes were macrophages (n=~350), followed by CD4 (n=~115) and CD8 T cells (n=~50). This study demonstrates that at the bulk tumor level, Siglec-15 and PD-L1 mRNA expression are not broadly mutually exclusive across cancer indications and instead expression of each gene varies broadly within a given indication. Additionally, their expression is frequently positively correlated, including in NSCLC, head and neck, kidney, and thyroid cancers. These results also demonstrate that Siglec-15 and PD-L1 are rarely found co-expressed on same cell. These associations enable a better understanding of the landscape of target expression in patients across a wide variety of cancer indications and can inform combination strategies with anti-Siglec-15 therapies. [1] Sun J, et al. Clin Cancer Res, 2021;27(3):680-688. [2] Wang J, et al. Nat Med, 2019;25(4):656-666. Citation Format: Krystal Watkins, Liyang Diao, Marsha Crochiere, Jan Pinkas. Gene expression correlation of immune checkpoint molecules Siglec-15 and PD-L1 varies widely by cancer indication [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 1373.

Abstract 5676: Spatial 3D genome organization reveals intratumor heterogeneity in primary glioblastoma samples

Abstract Glioblastoma (GBM) represents the most prevalent malignant primary brain tumor with highly unfavorable prognosis. Currently, most genomic studies are conducted at a single site of a tumor, which do not reflect the complete genetic or epigenetic information across the whole tumor. Furthermore, the intra-tumoral heterogeneity (ITH) of 3D genome organization has also not been studied yet. To address these gaps, we performed Hi-C experiments in 21 samples obtained from 9 GBM patients, with 15 of them being spatially mapped based on their 3D coordinates from the same patients. We identified extensive inter-tumoral and intra-tumoral heterogeneity in genome compartmentalization and chromatin interactions. Notably, in a patient with 9 spatially mapped samples from both temporal and frontal regions, we accumulated over 6 billion reads and defined high-resolution region-specific chromatin interactions, regulatory networks, and key regulators within the same patient. We detected structural variation (SV) and enhancer hijacking across all the samples, and identified recurrent events that affect known cancer-related genes such as CDKN2A/B. Finally, we introduce the concept of 'enhancer amputation', defined as the loss of enhancers due to SVs which lead to decreased expression of their original target genes. To our knowledge, this study represents the first large-scale exploration of the 3D genome in primary GBM patients and the first investigation of 3D genome organization in multiple regions of the same tumor. Our findings provide unprecedented insights into the ITH of GBM at the 3D genomic level, opening new avenues for understanding and potentially targeting this devastating disease. Citation Format: Qixuan Wang, Juan Wang, Radhika Mathur, Mark W. Youngblood, Qiushi Jin, Ye Hou, Lena A. Stasiak, Yu Luan, Joseph F. Costello, Feng Yue. Spatial 3D genome organization reveals intratumor heterogeneity in primary glioblastoma samples [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 5676.

Abstract 2466: tRNA-derived fragments as immunotherapy response targets in head and neck squamous cell carcinoma

Abstract Background: Despite significant advances in immunotherapy-based treatment options for Head and Neck Squamous Cell Carcinoma (HNSCC), patient prognosis and survival remains poor. To enhance response to immune-based therapies, specific targeting of immunomodulatory pathways has been investigated through co-stimulatory gene targets, the microbiome, and more. An emerging biomolecule is Transfer RNAs (tRNAs), which are a class of RNAs that transport amino acids from the cytoplasm to ribosomes, and synthesize new proteins encoded by mRNA transcripts. tRNA-derived fragments (tRFs) are a category of cleaved, mature tRNA that participate in post-transcriptional regulation and have been implicated in the modulation of cancer and other immune-associated diseases. While tRFs are primarily implicated in diagnostic signatures for various cancers, few studies have investigated their role in modulating the immunotherapy-based response in HNSCC. In this study, we characterized the role of tRFs in modulating immunotherapy gene targets pertinent to HNSCC. Methods: We extracted tRF read counts from MINTbase 2.0 for 455 primary HNSCC tumor samples and 44 adjacent normals. Differential expression analysis was first performed to identify tRFs implicated in HNSCC tumors, with respect to adjacent normal tissue. Next, these tRFs were associated with immunotherapy target genes and response markers with Spearman’s correlation. Further characterization of these tRFs was performed by correlating their abundance to infiltrating immune cells, pathway enrichment of immunotherapy targets, and clinical variables. Results: Significant differential expression of tRFs were found between HNSCC primary tumor samples and adjacent normal tissue, including 7 tRFs (FDR < 0.05, |log fold change| > 0.5). All were enriched in normal samples. AlaCGC5tRF appeared to be an important regulator of immunotherapy target genes through increased co-expression of PTPN11, STAT3, and PTEN (p-value < 0.05, R > 0) and decreased co-expression of CD58 and TNFRSF14 (p-value < 0.05, R < 0). Immune Checkpoint Inhibitor (ICI) genes were significantly correlated with several tRFs. Among several pertinent tRF markers, AlaCGC5tRF was strongly associated with negatively correlated with infiltrating activated NK cells naïve CD4 T cells (p-value < 0.05, R < 0). Conclusion: Our study is the most comprehensive to date to characterize the role of tRF elements in modulating HNSCC immunotherapy targets. We found several potential tRF biomarkers that may be indicative of enhanced immunotherapy treatment susceptibility, including AlaCGC5tRF and ArgTCT3tRf. Further studies are needed to investigate how these tRFs may be targeted alongside immunotherapies to enhance response to treatment. Citation Format: Rishabh Yalamarty, Daniel John, Annie Do, Tianyi Chen, Megan Ngo, Pranava Gande, Matthew Uzelac, Wei Tse Li, Weg M. Ongkeko. tRNA-derived fragments as immunotherapy response targets in head and neck squamous cell carcinoma [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 2466.

Abstract 2407: Detection of hotspot PIK3CA mutations in primary tumors and plasma-cfDNA of breast cancer patients using a novel highly sensitive CE-IVD kit

Abstract Introduction: Detection of PIK3CA mutations in primary tumors (FFPEs) and liquid biopsy samples is of increasing importance for treatment decisions in breast cancer. Oncolipsy PIK3CA kit (Pharmassist Ltd, Greece) is based on the combination of allele-specific priming, asymmetric PCR, and melting curve analysis. The CE-IVD kit can detect the presence of four PIK3CA hotspot mutations (E542K, E545K, E545Q, H1047R). The aim of the present study was to: a) evaluate the performance of this novel CE-IVD kit both in FFPEs and plasma-cfDNA samples from patients with metastatic breast cancer (MBC) and b) directly compare the derived results using the Oncolipsy PIK3CA kit with the cobas® PIK3CA mutation test (CE-IVD, Roche Diagnostics, Germany) in identical gDNAs isolated from FFPEs samples. Patients and methods: We analyzed 42 primary tumors (FFPEs) and 29 plasma-cfDNA samples from patients with estrogen receptor (ER) positive metastatic breast cancer. Genomic DNA was isolated from FFPEs using the QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany), while cfDNA was extracted from 2.00 mL of plasma using the QIAamp DSP cNA Kit (Qiagen, Hilden, Germany). PIK3CA hotspot mutations were detected in the LightCycler® 2.0 (IVD instrument, Roche Diagnostics, Germany) using the highly sensitive Oncolipsy PIK3CA kit (analytical sensitivity 0.05%). A direct comparison study was performed using the cobas® PIK3CA Mutation Test on the cobas z 480 analyzer (IVD instrument, Roche Diagnostics, Germany). Results: In primary tumors the E545K mutation was detected in 22/42 (52.4%), the E542K and H1047R mutations were detected in 11/42 (26.2%) and 4/42 (9.5%) of primary tumor samples, respectively, while the E545Q mutation was not detected in any FFPE sample tested. At least one PIK3CA mutation was detected in 14/29 (48.3%) plasma-cfDNA samples. More specifically the E545K mutation was detected in 11/29 (38%), the H1047R mutation in 3/29 (10.4%) and the E542K mutation in 2/29 (6.9%) while the E545Q mutation was not detected in any plasma-cfDNA sample tested. In FFPEs, as expected, PIK3CA mutation frequency was higher compared to plasma-cfDNA. Direct comparison of the results derived using these two commercially available kits, the Oncolipsy CE-IVD kit and the cobas PIK3CA mutation test, using the same gDNA isolated from 42 FFPEs revealed a concordance of 34/42 (81%). Conclusions: The commercially available Oncolipsy PIK3CA CE-IVD kit is highly sensitive and specific for the detection of four PIK3CA hotspot mutations in primary tumors and plasma-cfDNA. Acknowledgement: This study has been financially supported by the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation, under the call RESEARCH - CREATE - INNOVATE (project code: T1RCI-02935). Citation Format: Stavroula Smilkou, Dimitra Stergiopoulou, Eleni Tzanikou, Loukas Kaklamanis, Ioanna Balgkouranidou, Helena Linardou, Flora Zagouri, Evangelia Razi, Stylianos Kakolyris, Amanda Psyrri, Christos Papadimitriou, Ioanna Koukli, Athina Markou, Evi Lianidou. Detection of hotspot PIK3CA mutations in primary tumors and plasma-cfDNA of breast cancer patients using a novel highly sensitive CE-IVD kit [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 2407.

Abstract 4869: Efficient study design for the discovery of a gene expression signature predicting metastasis in cutaneous squamous cell carcinoma

Abstract Background: The StepIdent study aims to develop a gene signature predicting metastasis in patients with cutaneous squamous cell carcinoma (cSCC) to improve risk stratification, thus enabling personalized decisions about follow-up schedules and treatment options. Here we describe the unique characteristics, challenges, and best practices for an efficient design of a discovery cohort for a rare outcome (metastasis prevalence: 2-5%); for retrieving, curating, and linking the clinical and pathological data through nationwide databases; and for measuring gene expression through sequencing of archived Formalin-Fixed Paraffin-Embedded (FFPE) primary tumor samples. Methods: Following a predefined protocol, we identified a nested-case control cohort (NCC) of 305 cases and 305 controls from a nationwide cohort of 19,120 patients with a first cSCC in the Netherlands from 2007 to 2009, followed up until 2020. We chose an NCC design since it is an efficient study design in a rare outcome setting (weighting is needed to accommodate the under-sampling of the controls). Patients were identified from the Dutch National Cancer Registry (NCR) and the clinical information was retrieved from the NCR which is linked to the nationwide registry of histo- and cytopathology (PALGA). Tumor blocks were requested from PALGA, and pathological characteristics were assessed by dermatopathologists. We matched controls to cases, based on a risk score estimated by a clinicopathological model. Gene expression was measured using the Illumina RNA Prep with Enrichment kit combined with the whole exome panel and paired-end sequenced on the NextSeq 550. Results: Tissue slides for 541 samples were retrieved for sequencing. 151 samples were excluded after pathology review or due to low pre-library concentration. The final cohort includes 195 case-control pairs (n=390). The median sequencing depth was 43M (Q1-Q3: 35-52M); the median Q30 was 85% (Q1-Q3: 83-87%); the median GC content was 51% (Q1-Q3: 50-52%); a median of 1.8% of base pairs (Q1-Q3: 1.4-2.1%) was trimmed prior to the mapping/alignment; a median of 69% (Q1-Q3: 65-74%) of reads were aligned as protein-coding and a median of 7% (Q1-Q3: 6-10%) as rRNA; a median of 95% (Q1-Q3: 93-96%) of reads were aligned by STAR. Two samples were excluded based on quality control. Conclusion: We described an efficient design and implementation of a nationwide discovery study in cSCC, involving the retrieval of clinicopathological data, the collection of FFPE materials, and the execution of omics measurements. This study presents the largest cohort to date, incorporating omics measurements of primary cSCC samples, combined with simultaneous access to well-curated clinical and pathological information and follow-up data. Our findings can provide guidance for similar studies involving a rare clinical endpoint, where an efficient study design is a necessity. Citation Format: Barbara Rentroia-Pacheco, Lara Pozza, Yan Ting Chen, Daphne Huigh, Celeste J. Eggermont, Olivia FM Steijlen, Sheril Alex, Jvalini Dwarkasing, Domenico Bellomo, Harmen JG van de Werken, Antien L. Mooyaart, Marlies Wakkee, Loes M. Hollestein. Efficient study design for the discovery of a gene expression signature predicting metastasis in cutaneous squamous cell carcinoma [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 4869.

Abstract 3201: Preclinical development of a novel Romidepsin nanoparticle for the treatment of T-Cell lymphoid malignancies

Abstract The histone deacetylase inhibitors (HDACi) have long been regarded as an important class of drugs for treating T-cell lymphoid malignancies (TCL). Romidepsin (Romi), a bicyclic peptide, was approved for patients with relapsed or refractory cutaneous (CTCL) and peripheral T-cell lymphomas (PTCL). Unfortunately, the approval of Romi for relapsed/refractory PTCL was withdrawn due to a negative Phase 4 post-marketing requirement. Given its potent activity against Class I HDACs, Romi is considered one of the most effective HDAC inhibitors studied and used in patients with TCL. Herein, we report on a first-in-class nanoparticle of Romi (NanoRomi) with a superior safety and efficacy profile compared to the free drug in preclinical models of PTCL and large granular lymphocyte leukemia (LGLL). We developed a first-in-class NanoRomi using our proprietary amphiphilic di-block copolymer-based nanochemistry platform. NanoRomi was characterized in cell viability, flow cytometry, and western blotting (WB) assays to determine the concentration-effect relationships with regard to the induction of apoptosis and histone acetylation in cell lines representative of TCL and LGLL, as well as primary LGLL patient PBMC samples. In addition, we performed extensive in vivo modeling using murine xenografts of CTCL, including repeated dose toxicity, biodistribution, efficacy, and survival studies. Cytotoxicity assays demonstrated superior/comparable activity of NanoRomi (IC50: 1.1-2.3 nM) compared to free Romi (IC50: 4.8-10.6 nM) across all PTCL and LGLL cell lines studied, while in primary LGLL samples, NanoRomi exhibited superior cytotoxicity (2-fold) compared to Romi. WB analysis showed that NanoRomi induced apoptosis and increased acetylation of histone H3 and H4 in a time and concentration-dependent manner in cell lines representing CTCL, ALK+-ALCL, and LGLL. Repeat dose toxicity studies in CTCL xenografts confirmed that the overall tolerability of NanoRomi was substantially better than Romi. Tracking studies with a fluorescent dye (DiO) in the NanoRomi demonstrated preferential localization of NanoRomi in tumors of mice compared to the normal organs after 6 hours of drug administration. NanoRomi exhibited superior efficacy in the murine CTCL xenograft models, including a significant improvement in overall survival compared to Romi. Collectively, these data show that NanoRomi exhibits markedly superior pharmacokinetics, tolerability, and efficacy in models of TCL. We anticipate that the improved activity and tolerability of NanoRomi relate to the altered pharmacologic profile and superior bioavailability of the drug, which optimizes the effects of the drug for its influence on epigenetic biology. This unique platform offers the prospect of improving the tolerability and efficacy of epigenetic treatment strategies in these challenging diseases. Citation Format: Ipsita Pal, Anuradha Illendula, Andrea Joyner, Tess M. Deddens, Deepthi P. Damera, Todd E. Fox, Marya E. Dunlap-Brown, Kallesh D. Jayappa, Thomas P. Loughran, David J. Feith, Owen A. O'Connor. Preclinical development of a novel Romidepsin nanoparticle for the treatment of T-Cell lymphoid 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 3201.

Abstract 2500: Technical feasibility of CRISPR-based functional profiling for solid tumor precision medicine

Abstract Introduction: Next generation sequencing (NGS) has revolutionized precision oncology by enabling identification of cancer patients harboring actionable mutations who may benefit from corresponding targeted therapies. However, sequencing-based biomarkers are not detected for most patients and imperfectly predict response to therapy even when present. To address this unmet need, we have developed a CRISPR-based diagnostics platform for ‘personalized functional genomics’, allowing identification of patient-specific therapeutic vulnerabilities irrespective of mutation status. We have previously applied this approach to primary hematologic malignancies, and here report application to solid tumor-derived samples. Methods: Patient-derived solid tumor samples from several tumor types including breast, ovarian, colon and lung were obtained from 3 sources: fresh tissue (obtained from patients via core biopsy, surgical resection, or malignant fluid sampling), primary Patient-Derived Cell Lines (PDCLs, generated from surgical material and adapted for ex vivo culture), and commercially obtained viably frozen dissociated tumor cells (DTCs). Samples were dissociated to single cell suspensions and transduced with a lentivirus encoding Cas9 and an sgRNA library covering a custom targeted oncology gene panel. The panel consists of genes encoding targets of FDA-approved oncology drugs, as well as reference genes. After a short culture period, transduced cells were harvested and relative sgRNA distribution was measured by amplicon NGS. Changes in sgRNA abundance were collapsed into gene-level log2-fold changes, and significantly depleted genes scored as functional dependencies. For select PDCLs, cells were treated with cognate small molecule inhibitors over the course of 4 days. Cell viability was read out using CTG assay and a calculated IC50 lower than 500nM inferred as inhibitor sensitive. Results: In a double-blind study using primary PDCLs, functional dependencies identified by CRISPR perfectly discriminated alpelisib sensitive (2/2) from resistant samples (6/6). Using fresh primary samples from varying cancer types and specimen sources, we achieve a technical success rate >70%. Among gene dependencies identified, targets of FDA-approved oncology drugs and those under late-stage clinical investigation were both observed including BRAF, CDK4/6, VEGFR1/2 and PARP1/2. Conclusion: We demonstrate technical proof of concept of the FX platform for CRISPR-based personalized functional genomics. This approach is feasible when applied to primary patient samples from a variety of tumor types and tissue sources, and results are validated by comparison with orthogonal methods. CRISPR-based functional profiling represents a powerful adjunct to traditional genomic profiling, for the first time allowing drug target dependency mapping in a patient-specific and mutation-agnostic manner. Citation Format: Karl Hodel, Andreas Varkaris, Nicholas Chevalier, Maci Meyers, Allison Kehlmann, Stephane Corbel, Anastasia Stavridi, Peter Kilfeather, Leonardo Hagmann, Kathleen Tran, Yali Li, Christian Schmedt, Ioannis Sanidas, Srinath Sampath, Srihari Sampath, Dejan Juric. Technical feasibility of CRISPR-based functional profiling for solid tumor precision medicine [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 2500.

Abstract 350: Targeting prohibitins by fluorizoline leads to the activation of ISR and apoptosis through DELE1 and HRI by an impairment of the mitochondrial protein import machinery

Abstract Prohibitins are ubiquitously expressed within the cell, but they are mainly located at the inner mitochondrial membrane. These proteins are closely associated with tumour progression, invasion, and apoptotic resistance, being overexpressed in various primary tumours such as lymphoma and endometrial cancer. Notably, we have developed a novel synthetic molecule, fluorizoline, which induces apoptosis by selectively targeting PHBs in different cancer cell lines and primary samples from various haematological neoplasias. We have previously described that fluorizoline-induced apoptosis is mediated through the activation of the pro-apoptotic branch of the integrated stress response (ISR) pathway in HeLa and HAP1 cells, concretely, by the ATF4-CHOP-NOXA axis. In a recent study, we identified compensatory mechanisms for four ISR-related kinases. Importantly, HRI emerged as the primary kinase responsible for activating this pathway and promoting apoptosis, suggesting that mitochondrial stress could lead to this activation of the ISR.Here, we investigate the OMA1-DELE1-HRI molecular pathway responsible for sensing mitochondrial stress and activating the ISR cancer cell lines. Firstly, we demonstrate that fluorizoline treatment triggers the cleavage of large DELE1 (L-DELE1) to its cleaved form (S-DELE1) through western blot analysis, and its subsequent sorting to the cytosol as confirmed by confocal analysis. Using DELE1 KO lines generated by CRISPR/Cas9 technology, we demonstrate that DELE1 is involved in the activation of the ISR and apoptosis induced by fluorizoline. However, although we cannot rule out OMA1 involvement, fluorizoline-induced ISR activation does not strongly depend on OMA1. Interestingly, our data suggests that targeting PHBs, either by fluorizoline or by their downregulation, could result in mitochondrial protein import machinery impairment. This impairment may potentially be the cause of DELE1 accumulation in the cytosol, leading to the subsequent activation of the ISR by HRI upon fluorizoline treatment. Citation Format: Ismael Sánchez-Vera, Nekane Maritorena-Hualde, Max-Hinderk Schuler, Ana M- Cosialls, Rodolfo Lavilla, Gabriel Pons, Lucas T Jae, Daniel Iglesias-Serret, Joan Gil. Targeting prohibitins by fluorizoline leads to the activation of ISR and apoptosis through DELE1 and HRI by an impairment of the mitochondrial protein import machinery [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 350.

Abstract 1618: Highly populated phylogenetic reconstruction in patients with hormone receptor positive - HER2 non-amplified metastatic breast cancer

Abstract Background: Tumor progression still needs to be further elucidated in hormone receptor positive (HR+) breast cancer, the most common subtype of breast cancer. Here, leveraging the extensive sampling approach of our postmortem rapid tissue donation program UPTIDER (NCT04531696), we used phylogenic reconstruction to analyze disease progression. Patients and Methods: Thirteen patients with HR+/HER2 non-amplified primary breast cancer, recruited in UPTIDER between 12/2020 and 05/2022, were considered. Shallow whole-genome sequencing was performed on 495 solid samples including a matched germline DNA and archived primary tumor sample for each patient. Log2 coverage ratios were computed with CNVkit and profiles with median absolute pairwise deviation < 0.35 were co-segmented per patient using the copynumber R package. Purity and ploidy were assessed by ABSOLUTE and manually reviewed. Samples with purity > 30% were kept for downstream analysis. Phylogenetic reconstruction was performed using MEDICC2 for each patient. Metrics derived from the trees were: (i) tree length, (ii) trunk length, (iii) trunk ratio as trunk length/tree length*100. Overall survival (OS) was defined as time between primary diagnosis and death. Comparison was done using Wilcoxon test, time to event analysis using cox regression. Endocrine resistance was defined as per the ESMO guidelines (PMID:34678411). Results: 81% (399/495) of the samples passed QC. Phylogenies included a median of 28 samples per patient (range: 9-59) and 10/13 phylogenies included primary samples at diagnosis. Multi-seeding from the primary tumor was observed in 7 patients, suspected in 1, and a single seeding was observed in 2 patients. Median OS was 8.0 years. Higher trunk ratios tended to associate with poorer OS (Hazard ratio: 1.08 per 1% increase, 95% confidence interval: 0.99-1.16, p=0.06). In organs where multiple metastatic lesions were sampled (n= 73 organs, median of 3 samples/organ, range: 2-18 and median of 5 organs per patient, range: 1-10) seeding from at least 2 organs was detected in 48% (35/73) of the organs. Phylogenies from patients with secondary endocrine resistance (7 patients) tended to have a shorter trunk compared to patients with primary endocrine resistance (5 patients, median of 18 and 48 alterations respectively, p=0.15). Conclusion: Branching evolution from the primary tumor was more frequently observed than linear evolution. Phylogenies revealed extensive tumor heterogeneity, questioning treatment decisions based on a single biopsy. Additionally, evaluation of multiple metastatic samples per organ showed seeding from multiple organs in 48% of organs evaluated, indicating greater disease heterogeneity than previously thought. Phylogenetic differences were also noted according to the type of endocrine resistance (primary versus secondary). Citation Format: Francois Richard, Tatjana Geukens, Maxim De Schepper, Amena Mahdami, Karen Van Baelen, Marion Maetens, Gitte Zels, Ha-Linh Nguyen, Anirudh Pabba, Sophoa Leduc, Edoardo Isnaldi, Josephine Van Cauwenberge, Kristien Borremans, Hava Izci, Imane Bachir, Sigrid Hatse, Peter Vermeulen, Evy Vanderheyden, Bram Boeckx, Diether Lambrechts, Ann Smeets, Ines Nevelsteen, Kevin Punie, Patrick Neven, Hans Wildiers, Wouter Van Den Bogaert, Jonas Demeulemeester, Elia Biganzoli, Giuseppe Floris, Christine Desmedt. Highly populated phylogenetic reconstruction in patients with hormone receptor positive - HER2 non-amplified metastatic breast 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 1618.

Abstract 2064: Unveiling the correlation between gemtuzumab-ozogamicin efficacy and CD33+ expression in AML primary samples using the novel AML vitroscreen

Abstract Acute myeloid leukemia (AML) poses a significant therapeutic challenge, with a rising incidence in recent times. This clonal hematopoietic disorder of progenitor and stem cells exhibits aggressive behavior, and despite initial remission, recurrence is common1. The conventional shift from chemotherapy to monoclonal antibodies and antibody-drug conjugates (ADCs) has been hindered by the absence of AML-specific antigens. Notably, CD33, expressed strongly in 80-90% of AML cells, has become a target for gemtuzumab-ozogamicin (Mylotarg), the sole FDA approved ADC for AML treatment2. While advancements in understanding AML molecular mechanisms have influenced clinical approaches, the lack of clinically representative models hinders therapeutic progress. Existing models, both in vivo and ex vivo, struggle to capture the heterogeneity and complexity of AML3. Addressing this, we present our proprietary assay, Champions AML VitroScreen®4, designed for testing therapeutic candidates in primary AML samples. Our diverse bank of deeply characterized AML samples encompasses multiple subtypes, allowing evaluation of therapeutic responses through cell viability, proliferation, and clonal composition analyses. To validate our novel assay, we employed the VitroScreen platform to test Mylotarg across various AML subtypes, considering CD33 expression levels. By assessing more than 20 AML models, we determined the IC50 for Mylotarg and observed dose-dependent responses, where sensitivity to the drugh correlated with CD33 expression. Through multiple endpoints, including viability, proliferation, apoptosis, and phenotyping, we categorized primary AML models as responders, low responders, or resistant, establishing a strong positive correlation between Mylotarg sensitivity and CD33 expression. Our innovative approach, utilizing deeply characterized AML patient samples in the AML VitroScreen® platform, facilitates high-throughput drug testing. This system, serving as an ideal clinical translational setting in preclinical research, holds promise for developing tailored therapeutic strategies. Ultimately, this approach could significantly impact patient outcomes by offering improved strategies for both primary and recurrent AML cases. Citation Format: Mara Gilardi, Garima Kaushik, Brandon Walling, Paolo Schiavini, Stefano Cairo, Marianna Zipeto. Unveiling the correlation between gemtuzumab-ozogamicin efficacy and CD33+ expression in AML primary samples using the novel AML vitroscreen [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 2064.

The POKEMON Speckle Survey of Nearby M Dwarfs. III. The Stellar Multiplicity Rate of M Dwarfs within 15 pc

M dwarfs are ubiquitous in our Galaxy, and the rate at which they host stellar companions, and the properties of these companions, provide a window into the formation and evolution of the star(s), and of any planets that they may host. The Pervasive Overview of “Kompanions” of Every M dwarf in Our Neighborhood (POKEMON) speckle survey of nearby M dwarfs is volume limited from M0V through M9V out to 15 pc, with additional targets at larger distances. In total, 1125 stars were observed, and 455 of these are within the volume-limited, 15 pc sample of M-dwarf primaries. When we combine the speckle observations with known companions from the literature, we find that the stellar multiplicity rate of M dwarfs within 15 pc is 23.5% ± 2.0%, and that the companion rate is 28.8% ± 2.1%. We also find that the projected separation distribution for multiples that are known to host planets peaks at 198 au, while the distribution for multiples that are not yet known to host planets peaks at 5.57 au. This result suggests that the presence of close-in stellar companions inhibits the formation of M-dwarf planetary systems, similar to what has been found for FGK stars.

Abstract 1721: Epigenetic heterogeneity of osteosarcoma reveals distinct cellular states driven by enhancer regulation

Abstract Osteosarcoma (OS) is characterized by structural rearrangements and copy number changes which drive considerable heterogeneity. What is less clear is how these genetic rearrangements alter are in turn regulated at the epigenetic level to drive gene expression. Defining the epigenetic circuitry of osteosarcoma could help to understand the role of chromatin accessibility in determining cell plasticity. Chromatin accessibility can prime gene expression and define a cellular state. As chromatin accessibility is a major determinant of epigenetic transcriptional regulation, understanding the variability of chromatin accessibility could identify clinically-relevant subtypes and biomarkers of therapy response in Osteosarcoma, a disease that has seen few advances in therapy for over 40 years. Using ATAC-seq, we identified two epigenetic subtypes (EC1 and EC2) of OS with distinct chromatin accessibility. These studies were done in a panel of 11 PDX-derived cell lines and 9 established OS cell lines. We also performed ATACseq on the matching PDX for the 11 PDX-derived lines as well as 8 primary tumor samples. Analysis of transcription factor (TF) motifs and footprints of ATAC-seq peaks identified transcription factors distinctly activated in these two subtypes. For example, EC1 is characterized by high level of activity of developmental transcription factors including RUNX2, MEF2C and homeobox TFs. In contrast, EC2 is characterized by high activity of AP1 transcription factors such as FOSL1 and FOSL2. In order to understand the underlying transcriptional circuitry of these cell states, we performed CUT&RUN assay for the complete panel of PDX-derived cell lines. We found that the subtypes are driven by enhancer regulation (H3K27ac). We defined the specific Core Regulatory Circuitry (CRC) per subtype by defining the superenhancers by ROSE. We overlapped ATAC-seq data in the acetylated regions to demarcate TF binding regions dramatically and reduce the search space for TF cis-regulatory sites. In parallel, we used our PDX-derived cell line panel to screen cluster-specific targeted therapies and found differential responses dependent on cellular states defined by chromatin accessibility. For example, cell lines in the EC2 cluster are marked by upregulation of AP1 transcription factors, have evidence of ERK activation and are highly responsive to ERK inhibitors. In contrast cell lines in the EC1 cluster are sensitive to AURKA and AURKB inhibitors. Single-cell RNAseq analysis indicates that these subtypes may coexist within a single tumor. In summary, we discovered two epigenetically distinct cell states that are controlled by a state-specific collection of transcription factors, creating a gene signature that may aid in distinguishing osteosarcoma subclasses and predicting treatment response. Citation Format: Eunice Lopez Fuentes, Andrew Clugston, Alex Lee, Leanne Sayles, Maria Pons, Alejandro Sweet-Cordero. Epigenetic heterogeneity of osteosarcoma reveals distinct cellular states driven by enhancer regulation [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 1721.

Abstract 3305: Golcadomide (CC-99282) is a novel CELMoD® agent with antiproliferative activity and combinatorial potential in disease models of chronic lymphocytic leukemia

Abstract Chronic Lymphocytic Leukemia (CLL) is an adult leukemia characterized by the accumulation of incompetent B lymphocytes expressing CD19, CD20, CD23, and CD5. Current therapies include chemotherapy and combination with targeted therapies such as ibrutinib (Bruton’s tyrosine kinase inhibitor), venetoclax (BCL2 inhibitor), and obinutuzumab (anti-CD20). The tumor microenvironment promotes CLL development and drug resistance. Golcadomide (GOLCA, CC-99282) is an oral cereblon E3 ligase modulator (CELMoD®) agent with immunomodulatory and tumor cell-autonomous activities under clinical investigation for R/R non-Hodgkin lymphomas and CLL/SLL. Here we investigated the effect of GOLCA on inhibiting proliferation and inducing apoptosis of CLL preclinical models by degrading Ikaros and Aiolos. We also studied its combination with other anti-CLL agents. A panel of 10 CLL cell lines and 15 primary CLL patient samples were used to assess efficacy of GOLCA alone or in combination with ibrutinib (Ibru), venetoclax (Ven), and obinutuzumab (Obi). Primary CLL cells were grown ex vivo in a co-culture system with CD40L expressing fibroblasts designed to mimic the CLL lymph node microenvironment. Substrate degradation, viability, cell cycle and immunophenotyping were assessed by flow cytometry. GOLCA showed potent in vitro antiproliferative activity on 6 out of 10 CLL cell lines, including those with high-risk features, with IC50 of 1-20 nM. It also inhibited CLL stimulated cell proliferation and induced apoptosis in all evaluated patient samples in the co-culture system, with low to sub-nanomolar IC50, independent of IGHV mutation status and other chromosomal characteristics. Cell-cycle analysis confirmed inhibition of proliferation in the 5 patient samples evaluated after 3 days of treatment with GOLCA, with a dose-dependent decrease in the fraction of cells in the S phase. GOLCA also demonstrated degradation of the proximal substrates Ikaros and Aiolos in tumor and T cells from CLL patient samples. In combination with Obi, Ven, and Ibru, GOLCA led to synergistic or additive tumor cell toxicity in most patient samples evaluated, suggesting potential clinical benefit of these targeted agent combinations. Additive tumor toxicity was observed in two out of four patient samples when GOLCA was combined with Ibru. We have previously demonstrated that GOLCA induces immune activation in T cells from healthy donor PBMCs. In co-cultures of CLL PBMCs and CD40L-expressing fibroblasts, GOLCA increased T cell numbers and activation characteristics. Ven inhibited this immune activation, while Obi or Ibru maintained the immune activation. Collectively, these findings suggest that GOLCA, alone or in combination with targeted agents, may produce clinical benefit in CLL patients. Citation Format: Antonia Lopez-Girona, Maria Dolores Jimenez-Nunez, Diego Sobradillo, Soraya Carrancio, Preethi Janardhanan, Gauri Deb, Lynda Groocock, Daniel Pierce, Neil Bence, Mark Rolfe. Golcadomide (CC-99282) is a novel CELMoD® agent with antiproliferative activity and combinatorial potential in disease models of chronic lymphocytic leukemia [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 3305.

Abstract 3439: Factors affecting the performance of existing epigenetic clocks to predict chronological age in colorectal tumor and normal tissues

Abstract Epigenetic age (EA) may serve as a risk stratification biomarker for early-onset colorectal cancer (EOCRC, <50 years old), which has been rising substantially in recent decades. We utilized publicly available data in a descriptive analysis to compare epigenetic clocks in colorectal tissue and to investigate differences between chronological age (CA) and biological age, as measured by EA, in relation to demographic and clinical features. We analyzed DNA methylation (DNAm) array data on 144 participants (96 CRC cases, 48 healthy controls) from the Colonomics Study (CLX; Barcelona, Spain) and 331 CRC cases (51 EOCRC, 316 average-onset (≥50) colorectal cancer (AOCRC)) from The Cancer Genome Atlas (TCGA) as well as corresponding demographic and clinical variables. Among CLX participants, the 96 CRC cases provided colon tumor and paired normal mucosa samples, while the 48 controls provided healthy colon mucosa samples. TCGA included primary tumor samples from 331 cases with colon or rectal adenocarcinoma (TCGA-COADREAD). The 2013 Horvath clock estimated EA based on CA, while epiTOC, estimated the relative stem cell division rate from tissue biospecimens. We examined the correlations between CA and EA stratified by case/control status, sample type, sex, and age of onset (TCGA only). Tissue from CLX controls demonstrated a strong, positive correlation (r>0.9) between CA and EA for both the 2013 Horvath and epiTOC clocks. Using the Horvath clock, CA and EA correlations in normal mucosa and tumor tissue samples from CLX varied, with r = 0.74 and r = 0.41, respectively, and were weaker using the epiTOC clock, with r = 0.26 (normal) and r = 0.10 (tumor). When CLX cases were stratified by sex, the CA and EA (Horvath) correlations among female cases were r = 0.80 (normal) and r = -0.12 (tumor), while the correlations for males were stronger at r = 0.58 (normal) and r = 0.31 (tumor). This is in comparison to the epiTOC clock with females showing correlations of r = -0.02 (normal) and r = 0.41 (tumor), while males had r = 0.06 (normal) and r = 0.11 (tumor). In TCGA, correlations for EOCRC were r = -0.20 (Horvath) and r = -0.08 (epiTOC) and for AOCRC were r = 0.29 and 0.20, respectively. By sex, correlations for females with EOCRC for Horvath and epiTOC were r = -0.09 and r = -0.07, respectively, while correlations for males with EOCRC were r = -0.02 and r = -0.08, respectively. For TCGA cases with AOCRC, the correlations between CA and EA were r = 0.35 and r = 0.22 for females, and r = 0.26 and r = 0.20 for males, respectively for Horvath and epiTOC. This study revealed substantial variability in the ability of existing epigenetic clocks to accurately predict chronological age, with tissue type and sex strongly influencing the resulting correlations. Our findings highlight the importance of considering these factors when choosing an epigenetic clock. Citation Format: Christopher L. Benson, Ferran Moratalla-Navarro, Anna Diez-Villanueva, Matthew A. Devall, Victor Moreno, Stephanie L. Schmit, Fredrick R. Schumacher. Factors affecting the performance of existing epigenetic clocks to predict chronological age in colorectal tumor and normal tissues [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 3439.