Malignant Cell Populations Research Articles

Targeting glioblastoma with a brain-penetrant drug that impairs brain tumor stem cells via NLE1-Notch1 complex.

Brain tumor stem cells (BTSCs) are a population of self-renewing malignant stem cells that play an important role in glioblastoma tumor hierarchy and contribute to tumor growth, therapeutic resistance, and tumor relapse. Thus, targeting of BTSCs within the bulk of tumors represents a crucial therapeutic strategy. Here, we report that edaravone is a potent drug that impairs BTSCs in glioblastoma. We show that edaravone inhibits the self-renewal and growth of BTSCs harboring a diverse range of oncogenic mutations without affecting non-oncogenic neural stem cells. Global gene expression analysis revealed that edaravone significantly alters BTSC transcriptome and attenuates the expression of a large panel of genes involved in cell cycle progression, stemness, and DNA repair mechanisms. Mechanistically, we discovered that edaravone directly targets Notchless homolog 1 (NLE1) and impairs Notch signaling pathway, alters the expression of stem cell markers, and sensitizes BTSC response to ionizing radiation (IR)-induced cell death. Importantly, we show that edaravone treatment in preclinical models delays glioblastoma tumorigenesis, sensitizes their response to IR, and prolongs the lifespan of animals. Our data suggest that repurposing of edaravone is a promising therapeutic strategy for patients with glioblastoma.

Abstract C045: Targeting metabolic heterogeneity in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death in the US. This poor prognosis is related to the complex tumor microenvironment and tumor heterogeneity that drives resistance to chemotherapy. Here the heterogeneity of the stromal, immune, and malignant epithelial cell populations enables signaling and metabolic crosstalk mechanisms that drive immune suppression and resistance to therapy. Recently, our group unveiled that multiple distinct metabolic subclasses of cancer cells coexist within individual PDAC tumors. Among these, two PDAC clonal populations can engage in metabolic symbiosis that supports mitochondrial metabolism. One subtype has a constitutively high integrated stress response (ISR), enabling resistance to mitochondrial inhibitors. Further, these cells release asparagine that supports the mitochondrial metabolism of the other subtype that are more sensitive to mitochondrial inhibition. Given these observations, we are developing approaches to disrupt this metabolic symbiosis. Clonal PDAC cells with constitutively active ISR exhibit increased sensitivity to inhibition of the redox activity of Redox factor 1/Apurinic-apyrimidinic endonuclease 1 (Ref-1). Importantly, this can be leveraged to disrupt the support of the mitochondrial metabolism of clonal symbiotic pairs. Moreover, polyclonal PDAC cell lines representative of the heterogeneity seen in PDAC patients show a highly synergistic response to the combination of Ref-1 redox inhibitors with mitochondrial inhibitors. These data suggest this treatment combination will be synergistic in polyclonal tumors that exist in most PDAC patients. Overall, these data have the potential to lead to new avenues to directly target heterogenous populations of cancer cells to treat pancreatic cancer- an urgent clinical need. Citation Format: Taryn Morningstar, Cecily Anaraki, Lorenzo Scipioni, Giulia Tedeschi, Michelle Digman, Aimee Edinger, Claus Jorgensen, Melissa Fishel, Christopher J Halbrook. Targeting metabolic heterogeneity in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C045.

Angiogenesis related genes based prognostic model of glioma patients developed by multi-omics approach

IntroductionGlioma, particularly glioblastoma (GBM), is a highly malignant brain tumor with poor prognosis despite current therapeutic approaches. The tumor microenvironment (TME), plays a crucial role in glioma progression by promoting invasion and drug resistance. Angiogenesis, the formation of new blood vessels, is a tightly regulated process involving endothelial cell activation, proliferation, and migration. In cancer, angiogenesis becomes dysregulated, leading to excessive blood vessel formation.MethodsWe enrolled bulk data of TCGA-LGG/GBM, CGGA-693, and CGGA-325 cohorts, scRNA data of GSE162631, GSE84465, and GSE138794 cohorts. Identification of malignant cells was conducted by “copycat” R package. The “AUCell” R package scored the activity of target gene set of each single cell. Consensus clustering was applied using the “ConsensusClusterPlus” R package, while tumor-infiltrating immune cells were determined using “IOBR” R package. To construct a prognostic model, we used LASSO and multiCOX algorithms based on the expression levels of the 15 hub genes, the efficacy of which was verified by KM and ROC analysis.ResultsWe identified 4 different malignant cell subclusters in glioma and disclosed their distinct gene expression patterns and interactions within TME. We identified differentially expressed immune-related genes (DE-ARGs) in glioma and found 15 genes that were specifically expressed in the malignant glioma cell populations. Glioma cells with higher expression of these DE-ARGs were associated with gliogenesis, glial cell development, and vasculature development. We found that tumor-infiltrating monocytes were the main interacting cell type within glioma TME. Using the expression patterns of the 15 screened DE-ARGs, we categorized glioma samples into 2 molecular clusters with distinct immune features, suggesting a possible relationship between angiogenesis and immune activation and recruitment. We constructed a prognostic model based on the expression levels of the 15 DE-ARGs and evaluated its predictive ability for glioma patient outcomes, which displayed exceedingly high efficacy.ConclusionWe characterized different malignant cell subclusters in glioma and investigate their gene expression patterns and interactions within TME. We constructed a prognostic model based on the expression levels of the 15 DE-ARGs and evaluated its predictive ability for glioma patient outcomes, which displayed exceedingly high efficacy.

Single nucleus transcriptomics, pharmacokinetics, and pharmacodynamics of combined CDK4/6 and mTOR inhibition in a phase 0/1 trial of recurrent high-grade glioma.

Outcomes for adult patients with a high-grade glioma continue to be dismal and new treatment paradigms are urgently needed. To optimize the opportunity for discovery, we performed a phase 0/1 dose-escalation clinical trial that investigated tumor pharmacokinetics, pharmacodynamics, and single nucleus transcriptomics following combined ribociclib (CDK4/6 inhibitor) and everolimus (mTOR inhibitor) treatment in recurrent high-grade glioma. Patients with a recurrent high-grade glioma (n = 24) harboring 1) CDKN2A / B deletion or CDK4 / 6 amplification, 2) PTEN loss or PIK3CA mutations, and 3) wild-type retinoblastoma protein (Rb) were enrolled. Patients received neoadjuvant ribociclib and everolimus treatment and no dose-limiting toxicities were observed. The median unbound ribociclib concentrations in Gadolinium non-enhancing tumor regions were 170 nM (range, 65 - 1770 nM) and 634 nM (range, 68 - 2345 nM) in patients receiving 5 days treatment at the daily dose of 400 and 600 mg, respectively. Unbound everolimus concentrations were below the limit of detection (< 0.1 nM) in both enhancing and non-enhancing tumor regions at all dose levels. We identified a significant decrease in MIB1 positive cells suggesting ribociclib-associated cell cycle inhibition. Single nuclei RNAseq (snRNA) based comparisons of 17 IDH-wild-type on-trial recurrences to 31 IDH-wild-type standard of care treated recurrences data demonstrated a significantly lower fraction of cycling and neural progenitor-like (NPC-like) malignant cell populations. We validated the CDK4/6 inhibitor-directed malignant cell state shifts using three patient-derived cell lines. The presented clinical trial highlights the value of integrating pharmacokinetics, pharmacodynamics, and single nucleus transcriptomics to assess treatment effects in phase 0/1 surgical tissues, including malignant cell state shifts. ClinicalTrials.gov identifier: NCT03834740 .

Incidence and demographics of bile duct cystadenocarcinoma from the National Cancer Database.

e16231 Background: Bile duct cystadenocarcinoma, a rare tumor of the biliary tree, manifests itself within both multilocular and unilocular cysts composed of mucous-secreting, cytologically malignant epithelial cell populations. While similar in presentation to other biliary tree malignancies, bile duct cystadenocarcinoma has unique morphological variations between sexes. This contrast in disease manifestation based on sex could potentially impact diagnostic imaging, testing approaches, and treatment strategies for both female and male patients. The potential impact of demographics on the prognosis of bile duct cystadenocarcinoma, along with limited literature on the subject, make it a compelling subject for hypothesis generating epidemiological inquiry. The National Cancer Database will be used to illuminate the demographic intricacies. In this pursuit, we aim to provide a demographic and incidence background to prompt future research dedicated to the understanding of bile duct cystadenocarcinoma. Methods: The National Cancer Database (NCDB) was used to complete a retrospective cohort analysis of all patients diagnosed with histologically confirmed bile duct cystadenocarcinoma from the years 2004 to 2020. Demographic factors were subjected to descriptive statistical techniques, while a comprehensive regression analysis was employed to analyze the patterns in disease incidence. Results: Between 2004 and 2020, 64 patients were diagnosed with bile duct cystadenocarcinoma in the NCDB. There was a decline of incidence per year (R2 = 0.0392). The majority of those diagnosed with this disease were female (70%). Most of the patients were from Caucasian ethnicity (73%). The average age of diagnosis was 65 years old ( SD = 12.5, range = 33-88). The highest percentage of patients were on Medicare (55%). The primary site of the cancer was in the intrahepatic bile duct (63%). 70% of patients had a Charlson-Deyo Score of 0. Stage 1 was the most detected stage (42%). The average size of the tumor when detected was 65 mm (SD = 35.5, range = 9-144 mm). Most patients (72%) received surgical treatment and the remainder received radiation or chemotherapy alone. Regarding margins post-surgery, most of the patients (56%) had no residual tumor. Almost all patients (92%) received treatment designed for cure/remission. Mean survival after diagnosis was 8 years. Conclusions: Consistent with a vast majority of case reports, our results demonstrated that the majority of cases of bile duct cystadenocarcinoma were found in women (70%). Moreover, given that a significant proportion of patients affected by this disease are covered by Medicare (55%), we contend that investigating the precise influence of demographic factors and social determinants of health in this context presents a compelling avenue for future research.

CD20-bispecific antibodies improve response to CD19-CAR T cells in lymphoma in vitro and CLL in vivo models

AbstractRelapse after anti-CD19 chimeric antigen receptor (CD19-CAR) occurs in a substantial proportion of patients with lymphoid malignancies. We assessed the potential benefits of co-administering CD20-targeting bispecific antibodies (CD20-BsAbs) with CD19-CAR T cells with the aim of enhancing immunotherapeutic efficacy. Addition of CD20-BsAbs to cocultures of CD19-CARs and primary samples of B-cell malignancies, comprising malignant B cells and endogenous T cells, significantly improved killing of malignant cells and enhanced the expansion of both endogenous T cells and CD19-CAR T cells. In an immunocompetent mouse model of chronic lymphocytic leukemia, relapse after initial treatment response frequently occurred after CD19-CAR T-cell monotherapy. Additional treatment with CD20-BsAbs significantly enhanced the treatment response and led to improved eradication of malignant cells. Higher efficacy was accompanied by improved T-cell expansion with CD20-BsAb administration and led to longer survival with 80% of the mice being cured with no detectable malignant cell population within 8 weeks of therapy initiation. Collectively, our in vitro and in vivo data demonstrate enhanced therapeutic efficacy of CD19-CAR T cells when combined with CD20-BsAbs in B-cell malignancies. Activation and proliferation of both infused CAR T cells and endogenous T cells may contribute to improved disease control.

Metabolic pathway-based subtypes associate glycan biosynthesis and treatment response in head and neck cancer

Head and Neck Squamous Cell Carcinoma (HNSCC) is a heterogeneous malignancy that remains a significant challenge in clinical management due to frequent treatment failures and pronounced therapy resistance. While metabolic dysregulation appears to be a critical factor in this scenario, comprehensive analyses of the metabolic HNSCC landscape and its impact on clinical outcomes are lacking. This study utilized transcriptomic data from four independent clinical cohorts to investigate metabolic heterogeneity in HNSCC and define metabolic pathway-based subtypes (MPS). In HPV-negative HNSCCs, MPS1 and MPS2 were identified, while MPS3 was enriched in HPV-positive cases. MPS classification was associated with clinical outcome post adjuvant radio(chemo)therapy, with MPS1 consistently exhibiting the highest risk of therapeutic failure. MPS1 was uniquely characterized by upregulation of glycan (particularly chondroitin/dermatan sulfate) metabolism genes. Immunohistochemistry and pilot mass spectrometry imaging analyses confirmed this at metabolite level. The histological context and single-cell RNA sequencing data identified the malignant cells as key contributors. Globally, MPS1 was distinguished by a unique transcriptomic landscape associated with increased disease aggressiveness, featuring motifs related to epithelial-mesenchymal transition, immune signaling, cancer stemness, tumor microenvironment assembly, and oncogenic signaling. This translated into a distinct histological appearance marked by extensive extracellular matrix remodeling, abundant spindle-shaped cancer-associated fibroblasts, and intimately intertwined populations of malignant and stromal cells. Proof-of-concept data from orthotopic xenotransplants replicated the MPS phenotypes on the histological and transcriptome levels. In summary, this study introduces a metabolic pathway-based classification of HNSCC, pinpointing glycan metabolism-enriched MPS1 as the most challenging subgroup that necessitates alternative therapeutic strategies.

A Raman topography imaging method toward assisting surgical tumor resection

AbstractAchieving complete tumor resection upon initial surgical intervention can lead to better patient outcomes by making adjuvant treatments more efficacious and reducing the strain of repeat surgeries. Complete tumor resection can be difficult to confirm intraoperatively. Methods like touch preparation (TP) have been inconsistent for detecting residual malignant cell populations, and fatty specimens like breast cancer lumpectomies are too fatty to process for rapid histology. We propose a novel workflow of immunostaining and topographic surface imaging of freshly excised tissue to ensure complete resection using highly sensitive and spectrally separable surface-enhanced Raman scattering nanoparticles (SERS NPs) as the targeted contrast agent. Biomarker-targeting SERS NPs are ideal contrast agents for this application because their sensitivity enables rapid detection, and their narrow bands enable extensive intra-pixel multiplexing. The adaptive focus capabilities of an advanced Raman instrument, combined with our rotational accessory device for exposing each surface of the stained specimen to the objective lens, enable topographic mapping of complete excised specimen surfaces. A USB-controlled accessory for a Raman microscope was designed and fabricated to enable programmatic and precise angular manipulation of specimens in concert with instrument stage motions during whole-surface imaging. Specimens are affixed to the accessory on an anti-slip, sterilizable rod, and the tissue surface exposed to the instrument is adjusted on demand using a programmed rotating stepper motor. We demonstrate this topographic imaging strategy on a variety of phantoms and preclinical tissue specimens. The results show detail and texture in specimen surface topography, orientation of findings and navigability across surfaces, and extensive SERS NP multiplexing and linear quantitation capabilities under this new Raman topography imaging method. We demonstrate successful surface mapping and recognition of all 26 of our distinct SERS NP types along with effective deconvolution and localization of randomly assigned NP mixtures. Increasing NP concentrations were also quantitatively assessed and showed a linear correlation with Raman signal with an R2 coefficient of determination of 0.97. Detailed surface renderings color-encoded by unmixed SERS NP abundances show a path forward for content-rich, interactive surgical margin assessment.

Dependence of human cell survival and proliferation on the CASP3 prodomain

Mechanisms that regulate cell survival and proliferation are important for both the development and homeostasis of normal tissue, and as well as for the emergence and expansion of malignant cell populations. Caspase-3 (CASP3) has long been recognized for its proteolytic role in orchestrating cell death-initiated pathways and related processes; however, whether CASP3 has other functions in mammalian cells that do not depend on its known catalytic activity have remained unknown. To investigate this possibility, we examined the biological and molecular consequences of reducing CASP3 levels in normal and transformed human cells using lentiviral-mediated short hairpin-based knockdown experiments in combination with approaches designed to test the potential rescue capability of different components of the CASP3 protein. The results showed that a ≥50% reduction in CASP3 levels rapidly and consistently arrested cell cycle progression and survival in all cell types tested. Mass spectrometry-based proteomic analyses and more specific flow cytometric measurements strongly implicated CASP3 as playing an essential role in regulating intracellular protein aggregate clearance. Intriguingly, the rescue experiments utilizing different forms of the CASP3 protein showed its prosurvival function and effective removal of protein aggregates did not require its well-known catalytic capability, and pinpointed the N-terminal prodomain of CASP3 as the exclusive component needed in a diversity of human cell types. These findings identify a new mechanism that regulates human cell survival and proliferation and thus expands the complexity of how these processes can be controlled.The graphical abstract illustrates the critical role of CASP3 for sustained proliferation and survival of human cells through the clearance of protein aggregates.

Ergodicity Breaking and Self-Destruction of Cancer Cells by Induced Genome Chaos.

During the progression of some cancer cells, the degree of genome instability may increase, leading to genome chaos in populations of malignant cells. While normally chaos is associated with ergodicity, i.e., the state when the time averages of relevant parameters are equal to their phase space averages, the situation with cancer propagation is more complex. Chromothripsis, a catastrophic massive genomic rearrangement, is observed in many types of cancer, leading to increased mutation rates. We present an entropic model of genome chaos and ergodicity and experimental evidence that increasing the degree of chaos beyond the non-ergodic threshold may lead to the self-destruction of some tumor cells. We study time and population averages of chromothripsis frequency in cloned rhabdomyosarcomas from rat stem cells. Clones with frequency above 10% result in cell apoptosis, possibly due to mutations in the BCL2 gene. Potentially, this can be used for suppressing cancer cells by shifting them into a non-ergodic proliferation regime.

Efficient Radial-Shell Model for 3D Tumor Spheroid Dynamics with Radiotherapy

Understanding the complex dynamics of tumor growth to develop more efficient therapeutic strategies is one of the most challenging problems in biomedicine. Three-dimensional (3D) tumor spheroids, reflecting avascular microregions within a tumor, are an advanced in vitro model system to assess the curative effect of combinatorial radio(chemo)therapy. Tumor spheroids exhibit particular crucial pathophysiological characteristics such as a radial oxygen gradient that critically affect the sensitivity of the malignant cell population to treatment. However, spheroid experiments remain laborious, and determining long-term radio(chemo)therapy outcomes is challenging. Mathematical models of spheroid dynamics have the potential to enhance the informative value of experimental data, and can support study design; however, they typically face one of two limitations: while non-spatial models are computationally cheap, they lack the spatial resolution to predict oxygen-dependent radioresponse, whereas models that describe spatial cell dynamics are computationally expensive and often heavily parameterized, impeding the required calibration to experimental data. Here, we present an effectively one-dimensional mathematical model based on the cell dynamics within and across radial spheres which fully incorporates the 3D dynamics of tumor spheroids by exploiting their approximate rotational symmetry. We demonstrate that this radial-shell (RS) model reproduces experimental spheroid growth curves of several cell lines with and without radiotherapy, showing equal or better performance than published models such as 3D agent-based models. Notably, the RS model is sufficiently efficient to enable multi-parametric optimization within previously reported and/or physiologically reasonable ranges based on experimental data. Analysis of the model reveals that the characteristic change of dynamics observed in experiments at small spheroid volume originates from the spatial scale of cell interactions. Based on the calibrated parameters, we predict the spheroid volumes at which this behavior should be observable. Finally, we demonstrate how the generic parameterization of the model allows direct parameter transfer to 3D agent-based models.

R-ISS Stage-Dependent Single-Cell Sequencing Analysis Uncovers Oncogenes and Potential Immunotherapeutic Targets in Multiple Myeloma

Multiple myeloma (MM) accounts for ~10% of all haematologic malignancies. Little is known about high intratumour heterogeneities in patients stratified by the Revised International Staging System (R-ISS). Herein, we constructed a single-cell transcriptome atlas to compare differential expression patterns among stages. We found that a novel cytotoxic plasma cell (PC) population exhibited with NKG7 positive was obviously enriched in stage II patients. Additionally, a malignant plasma cell population with significantly elevated expression of MKI67 and PCNA was associated with unfavourable prognosis and Epstein-Barr virus (EBV) infection in our collected samples. Moreover, Ribonucleotide Reductase Regulatory Subunit M2 (RRM2) was found and verified to promote proliferation of MM cell lines, suggesting RRM2 may serve as a detrimental marker in MM. The percentages of CD8+ T cells and NKT cells decreased along with R-ISS stages, reflecting the plasticity of the tumour immune microenvironment. Importantly, their crosstalks with myeloid cells and PC identified several potential immunotargets such as SIRPA-CD47, and CD74-MIF, respectively. Collectively, this study provided an R-ISS-related single-cell MM atlas and revealed the clinical significance of novel PC clusters, as well as potential immunotargets in MM progression.

Anti CD161 Antibody IMT-009 Is a Novel Immunotherapeutic Agent That Effectively Blocks the Inhibitory CLEC2D/CD161 Axis in CLEC2D+ B Cell Hematological Malignancies Reinvigorating T and NK Cell Function Leading to Anti-Tumor Benefit

Background: The CLEC2D/CD161 axis is a ligand-receptor pathway that has been shown to inhibit both T and NK cell function 1,2. CD161 is a C-type lectin-like receptor, which is expressed on NK cells and subsets of both CD4 + and CD8 + T cells. The tumor infiltrating CD161+ T cells have high cytotoxic potential and display an NK cell-like signature 2. Its ligand CLEC2D is expressed on the surface of both malignant cells and immune cells including germinal center B cells, activated T cells and tumor associated macrophages. Owing to its expression in germinal center B cells, CLEC2D is highly expressed in multiple types of B cell lymphomas including Diffuse Large B Cell Lymphoma (DLBCL), Follicular Lymphoma and Burkitt lymphoma 3. Furthermore, subset of infiltrated T cells in these lymphomas are CD161+ making this axis a promising novel target for immunotherapeutic intervention in hematological malignancies (Fig1). Immunitas has developed IMT-009, a monoclonal, Fc silent, fully human IgG1, high affinity antibody to CD161 that selectively blocks its interaction with CLEC2D. We show that IMT-009 effectively reverses CLEC2D-mediated inhibition and restores NK cell and T cell functions, including antigen specific T cell mediated cytotoxicity to lymphoma cell lines. Furthermore, IMT-009 shows monotherapy benefit in reducing tumor burden in a human B cell lymphoma model engrafted in humanized mice in vivo. IMT-009 is currently being evaluated in a Phase 1/2a clinical trial in both solid tumors and heme malignancies. Results: IMT-009 is a highly selective antibody to CD161 that strongly blocks its interaction with CLEC2D. In presence of CLEC2D-expressing target cells, NK cell degranulation, cytokine production and cellular cytotoxicity towards lymphoma tumor targets is highly suppressed which is overcome by treatment with IMT-009. Similarly, IMT-009 reversed CLEC2D-mediated inhibition and restored T cell activity (TCR signaling, cytokine production) in a Jurkat cell reporter system, and enhanced polyfunctionality of primary antigen-specific human T cells shown by secretion of TNF-α, IL-2, and IFN-γ, and direct T cell mediated cytotoxicity. IMT-009 also released CD161-mediated suppression of antigen recall response by PBMC derived effector memory CD161+ CD4+ T cells, resulting in an increased frequency of CD161+ IFN-γ+ cells and an increase in their proliferation. These data together show that IMT-009 is highly effective in overcoming CLEC2D mediated suppression of both T and NK cell functions. To prioritize indications that will likely benefit from IMT-009, we performed IHC for CLEC2D in a diverse set of tumor indications and found that B cell lymphomas of various subtypes have high CLEC2D protein expression by H score. Also, DLBCL was found to have the second highest median RNA expression of CLEC2D in TCGA. Furthermore, analysis of publicly available single-cell RNA-sequencing (scRNA-seq) data from B cell lymphoma including CNS lymphoma revealed CLEC2D to be highly expressed within the B lineage cells including the abundant malignant cell population (Fig2). KLRB1 (gene encoding for CD161) was also found to be expressed on a subset of tumor infiltrating CD4 and CD8 T cells in these datasets (Fig2). To confirm this, we tested CD161 expression in B cell lymphomas by immunofluorescence. Results showed CD161+ cells infiltrated in Hodgkin Lymphoma, and post-R-CHOP treated DLBCL, highlighting the potential sensitivity of these malignancies to IMT-009 treatment. Together, these results indicate that B cell lymphomas may exhibit an immunosuppressive environment mediated by the CLEC2D/CD161 axis. To test the anti-tumor benefit of IMT-009 in a human B cell lymphoma model, we treated CD34+ humanized mice implanted with Raji, a Burkitt lymphoma cell line with IMT-009. The group of mice treated with IMT-009 had a mean tumor growth inhibition of 35% and the anti-tumor response correlated with increased frequency of CD161+ T and NK cells. We further show that IMT-009 can synergize with Rituximab in NK cell mediated killing of B cell lymphoma cell lines in vitro. Conclusion: B cell malignancies tend to respond poorly to anti-PD-1 therapy 4,5. This lack of efficacy could be driven by a highly immunosuppressive environment mediated by the CLEC2D/CD161 axis. Our results support evaluation of IMT-009 as a novel cancer immunotherapy to disrupt CLEC2D/CD161 signaling, with strong therapeutic potential in lymphomas.

EPCO-45. CELLULAR HIERARCHIES OF ETMR ARE SHAPED BY ONCOGENIC MICRORNAS AND RECEPTOR-LIGAND INTERACTIONS

Abstract Embryonal tumor with multilayered rosettes (ETMR) is a highly aggressive brain tumor that predominantly occurs in children under the age of four years. Most children succumb within 10 months of diagnosis, typically due to invasive tumor growth and therapy-refractory behavior. 95% of ETMR harbor activating genetic alterations of the chromosome 19 microRNA cluster (C19MC). Despite the identification of this presumed oncogenic driving event, its biological consequences remain understudied and targeted therapy options are lacking. Here, we set out to further explore the intracellular heterogeneity of ETMR and shed light onto its oncogenic mechanisms with the goal to identify novel therapeutic targets for this dismal disease. We have compiled and analyzed a cohort of 11 ETMR tumors using single-cell RNA sequencing and multiplexed spatial imaging. We reveal a common, spatially distinct cellular hierarchy, closely resembling physiological brain development. This hierarchy spans a highly proliferative neural stem cell-like population that gives rise to more differentiated neuron-like cells. C19MC, predominantly expressed in the malignant stem-like population, controls a transcriptional network governing stemness and lineage commitment, as resolved by genome-wide analysis of microRNA-mRNA interactions. Targeting specific C19MC members by antisense oligonucleotides results in reduced cell proliferation, suggesting potent new avenues for future therapeutic approaches. Seeking more immediate therapeutic targets, we performed comprehensive cell-cell interaction analyses and reveal that FGF-FGFR and Delta-Notch interactions are important to maintain oncogenic signaling across malignant cell populations. These interactions were successfully targeted using small molecule inhibitors in preclinical models and in one ETMR patient. Taken together, we identify different malignant ETMR cell populations that follow a common developmental hierarchy, explore the regulatory reach of C19MC activation, and identify an oncogenic receptor-ligand interaction network. Targeting this interaction network opens a powerful new rationale for more effective ETMR therapies.

STEM-12. TARGETING GLIOBLASTOMA WITH A BRAIN PENETRANT DRUG THAT IMPAIRS BRAIN TUMOUR STEM CELLS

Abstract Brain tumour stem cells (BTSCs) are a population of self-renewing malignant stem cells that play an important role in glioblastoma tumour hierarchy and contribute to tumour growth, therapeutic resistance, and tumour relapse. Thus, targeting of BTSCs within the bulk of tumours represents a crucial therapeutic strategy. Here, we report a potent brain penetrant drug (AJ5939) that impairs BTSCs and impedes glioblastoma progression. We show that AJ5939 inhibits the self-renewal and growth of BTSCs harbouring a diverse range of oncogenic mutations without affecting non-oncogenic neural stem cells. Global gene expression profiling revealed significant alterations in BTSC transcriptome and attenuation of the expression of a large panel of genes involved in cell cycle progression, stemness, and DNA repair mechanisms. Mechanistically, we show that AJ5939 impairs Notch signalling pathway via directly targeting this pathway and altering the stemness signature. In combination with ionizing radiation (IR), however, AJ5939 suppresses the DNA repair mechanisms and facilitate the response of the BTSC to ionizing radiation induced cell death. Finally, we present data that AJ5939 treatment of brain tumours in preclinical models significantly prolongs lifespan.

Unraveling intratumoral complexity in metastatic dermatofibrosarcoma protuberans through single-cell RNA sequencing analysis.

Dermatofibrosarcoma protuberans (DFSP) stands as a rare and locally aggressive soft tissue tumor, characterized by intricated molecular alterations. The imperative to unravel the complexities of intratumor heterogeneity underscores effective clinical management. Herein, we harnessed single-cell RNA sequencing (scRNA-seq) to conduct a comprehensive analysis encompassing samples from primary sites, satellite foci, and lymph node metastases. Rigorous preprocessing of raw scRNA-seq data ensued, and employing t-distributed stochastic neighbor embedding (tSNE) analysis, we unveiled seven major cell populations and fifteen distinct subpopulations. Malignant cell subpopulations were delineated using infercnv for copy number variation calculations. Functional and metabolic variations of diverse malignant cell populations across samples were deciphered utilizing GSVA and the scMetabolism R packages. Additionally, the exploration of differentiation trajectories within diverse fibroblast subpopulations was orchestrated through pseudotime trajectory analyses employing CytoTRACE and Monocle2, and further bolstered by GO analyses to elucidate the functional disparities across distinct differentiation states. In parallel, we segmented the cellular components of the immune microenvironment and verified the presence of SPP1+ macrophage, which constituted the major constituent in lymph node metastases. Remarkably, the CellChat facilitated a comprehensive intercellular communication analysis. This study culminates in an all-encompassing single-cell transcriptome atlas, propounding novel insights into the multifaceted nature of intratumor heterogeneity and fundamental molecular mechanisms propelling metastatic DFSP.

Resistance to CD38 Antigen Targeted Therapy in Multiple Myeloma May Result from Underrecognized Chromosome 4 Gene Deletions

Anti-CD38 monoclonal antibodies (mAbs), daratumumab (dara) and isatuximab (isa), are approved to treat multiple myeloma (MM) in the first line and relapsed/refractory settings. Resistance portends a poor prognosis, yet underlying mechanisms have not been well characterized. Despite generally high CD38 expression on most malignant plasma cells (PCs), antigen density may impact the clinical efficacy of targeted therapy. Unfortunately, therapeutic anti-CD38 mAbs interfere with detection and quantification of CD38 surface level by most conventional clinical flow cytometry assays, limiting characterization of CD38 antigen escape. Tumor cell evasion of antigen specific therapeutics can occur with target loss or downmodulation, a phenomenon conventionally referred to as antigen escape. To elucidate mechanisms of CD38 antigen escape, we evaluated MM patients treated with anti-CD38 mAbs who underwent a marrow evaluation between 1/2017 and 10/2022 at a single institution. Eighty-one of 161 (50%) patients had become clinically refractory over their course. Among 82 (51%) patients with ≥1 chromosomal genomic array test (CGAT) performed, 10 (12%) were found to harbor a CD38 deletion ( Table). With a CGAT coverage of 31%, the estimated prevalence of CD38 deletions was 39%. The median interval from diagnosis to CD38 deletion was calculated as 6.7 years (IQR 2.8 to 11.6) for CD38-deleted patients and estimated as 12.9 years (95% CI 12 to NA; Figure) for all patients. The presence of a CD38 deletion was associated with an increasing number of chromosomal abnormalities on concurrent FISH (OR 2.00, 95% CI 1.33 to 3.29, p=0.002). Specific FISH and flow cytometry findings occurred in conjunction with a CD38 deletion. In 50% of patients, flow cytometry detected a bimodal distribution of CD38 fluorescence intensity, reflecting two populations of malignant plasma cells with distinct CD38 surface levels. No patient showed a uniform malignant plasma cell population with a normal surface CD38 level. When the CD38 (4p15.32) deletion was identified in the context of a large 4p deletion spanning the FGFR3 locus, the FGFR3 (4p16) probe could detect the relevant loss of 4p. This was seen in 6 of 8 patients with a concurrent FGFR3 deletion by CGAT. The proportion of clones harboring a CD38 deletion could be quantified by FISH over time when testing was performed on a CD138-enriched sample. Low-level CD38 deletions (≤25%) did not confer resistance to dara-containing multi-drug regimens. In two cases where such deletions were present at diagnosis, disease control is ongoing in response to dara-Pd and dara-Rd. We suggest that acquired CD38 gene deletions appear to be common, the proportion of patients harboring them increases over time irrespective of prior exposure to CD38 mAbs, and their presence facilitates tumor antigen escape. We demonstrate that CGAT can identify CD38 deletions. Conventional clinical flow cytometry assays are unable to quantify the CD38 surface level due to interference from anti-CD38 mAbs. However, we propose that the finding of a bimodal distribution of CD38 fluorescence by flow cytometry could serve as an initial screen to identify at-risk patients warranting confirmatory CD38 mutational analysis. Following characterization of a deletion, FISH could be used to monitor the clonal level longitudinally. Our suggestion that the presence of a CD38 deletion in &amp;gt;25% of malignant PC clones predicts for reduced CD38 mAb efficacy requires prospective validation.

Single Cell Multi Omics and Spatial Analysis Reveals Plasmablast Signature Malignant Cells As a Key of Intratumor Heterogeneity in Primary Central Nervous System Lymphoma

Background: Primary central nervous system lymphoma (PCNSL) constitutes approximately 5% of extranodal lymphomas. More than 90% of PCNSL are diagnosed as diffuse large B-cell lymphomas (DLBCL), mainly classified as the activated B cell (ABC) subtype. Recent genetic analyses have identified key molecular drivers of PCNSL, including MYD88, CARD11 and CD79B. However, molecular analyses using bulk samples have provided limited insights into the heterogeneity in lymphoma cells of PCNSL. To improve our understanding in the biology of PCNSL, we conducted a multimodal single-cell analyses and integrated single-cell and spatial analyses to characterize the molecular properties of PCNSL tumors. Methods:Our study included seven PCNSL patients and two patients with reactive lymph nodes (RLN). We performed Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) combined with B-cell and T-cell receptor (BCR and TCR) analysis on samples from 3 PCNSL and 2 RLN patients, while the remaining PCNSL patients' samples were subjected to single-cell transcriptomic sequencing. Additionally, six formalin-fixed and paraffin-embedded (FFPE) tissues from the patients analyzed in the single-cell sequencing (except for one patient) were performed with GeoMx digital spatial profiling (DSP). Result: To ensure data quality, we filtered out low-quality cells and doublets, resulting in a dataset of 41,556 single cells. B cells represented the most abundant cluster, followed by T cells and monocytes. Copy number alterations (CNAs) data derived from InferCNV analysis showed distinct CNAs patterns in lymphoma cells of each patient. UMAP plot projection analysis revealed the presence of a distinct subtype of malignant cells within the B-cell population, which was characterized as high expression of plasmablast (PBL)-associated surface proteins, including SLAMF7, CD39, and CD38. Enrichment analysis based on germinal center (GC) gene expression signatures (Holmes AB et al. J Exp Med. 2020) revealed a broader spectrum of B-cell differentiation states in malignant B cells. Single-cell BCR sequencing further demonstrated that different GC signature state cells harbored the same BCR repertoire within each patient, suggesting a common origin of malignant cells. Non-negative matrix factorization (NMF) analysis confirmed the enrichment of PBL signature scores in the distinct malignant cell population observed in the UMAP plot, consistent with DPE analysis (Figure A). Interestingly, the proportion of PBL signature cells varied among patients. Signaling interaction analysis highlighted CD70-CD27 and SPP1-CD44 pathways as enriched in tumor cells. PBL signature cells received TNFSF13B and TNFSF13 signaling through TNFRSF17 (BCMA), whereas other tumor cells expressed TNFRSF13C (BAFF-R) and received TNFSF13B signaling alone. We further analyzed two public bulk RNA-seq data (Bruno A et al. Neuro Oncol 2018, n = 6; Radke J et al. Nat Commun 2022, n=38) in PCNSL to decompose tumor components. Tumor PBL components were identified in most PCNSL cases, although several cases exhibited a low presence of tumor PBL population. Of note, analysis including these public datasets with our single-cell cohort showed that PCNSL samples with high tumor PBL proportion had unfavorable prognosis . Additionally, we performed DSP to integrate the single-cell and spatial multi-omics analyses. We deconvoluted the cell composition by using transcriptomic data derived from each region of interest (ROI) and found that the cell proportions in ROI were consistent with the corresponding single-cell results. However, there were differential profiles of cell composition according to patients rather than ROIs, suggesting that PCNSL tumors may display spatial homogeneity (Figure B). Conclusion: Through the application of single-cell and spatial transcriptomic analysis, we identified a distinct subtype expressing the PBL signature gene. These findings have important therapeutic implications and provide new insights into the mechanisms of relapse in PCNSL.

Flow Sorting, Whole Genome Amplification and Next-Generation Sequencing as Combined Tools to Study Heterogeneous Acute Lymphoblastic Leukemia.

Next-generation sequencing (NGS) methods have been introduced for immunoglobulin (IG)/T-cell receptor (TR) gene rearrangement analysis in acute lymphoblastic leukemia (ALL) and lymphoma (LBL). These methods likely constitute faster and more sensitive approaches to analyze heterogenous cases of ALL/LBL, yet it is not known whether gene rearrangements constituting low percentages of the total sequence reads represent minor subpopulations of malignant cells or background IG/TR gene rearrangements in normal B-and T-cells. In a comparison of eight cases of B-cell precursor ALL (BCP-ALL) using both the EuroClonality NGS method and the IdentiClone multiplex-PCR/gene-scanning method, the NGS method identified between 29% and 139% more markers than the gene-scanning method, depending on whether the NGS data analysis used a threshold of 5% or 1%, respectively. As an alternative to using low thresholds, we show that IG/TR gene rearrangements in subpopulations of cancer cells can be discriminated from background IG/TR gene rearrangements in normal B-and T-cells through a combination of flow cytometry cell sorting and multiple displacement amplification (MDA)-based whole genome amplification (WGA) prior to the NGS. Using this approach to investigate the clonal evolution in a BCP-ALL patient with double relapse, clonal TR rearrangements were found in sorted leukemic cells at the time of second relapse that could be identified at the time of diagnosis, below 1% of the total sequence reads. These data emphasize that caution should be exerted when interpreting rare sequences in NGS experiments and show the advantage of employing the flow sorting of malignant cell populations in NGS clonality assessments.

Single-cell transcriptomic analysis of gingivo-buccal oral cancer reveals two dominant cellular programs.

Oral squamous cell carcinoma of the gingivo-buccal region (OSCC-GB) is the most common cancer among men in India, and is associated with poor prognosis and frequent recurrence. Cellular heterogeneity in OSCC-GB was investigated by single-cell RNA sequencing of tumors derived from the oral cavity of 12 OSCC-GB patients, 3 of whom had concomitant presence of a precancerous lesion (oral submucous fibrosis [OSMF]). Unique malignant cell types, features, and phenotypic shifts in the stromal cell population were identified in oral tumors with associated submucous fibrosis. Expression levels of FOS, ATP1A, and DUSP1 provided robust discrimination between tumors with or without the concomitant presence of OSMF. Malignant cell populations shared between tumors with and without OSMF were enriched with the expression of partial epithelial-mesenchymal transition (pEMT) or fetal cell type signatures indicative of two dominant cellular programs in OSCC-GB-pEMT and fetal cellular reprogramming. Malignant cells exhibiting fetal cellular and pEMT programs were enriched with the expression of immune-related pathway genes known to be involved in antitumor immune response. In the tumor microenvironment, higher infiltration of immune cells than the stromal cells was observed. The T cell population was large in tumors and diverse subtypes of T cells with varying levels of infiltration were found. We also detected double-negative PLCG2+ T cells and cells with intermediate M1-M2 macrophage polarization. Our findings shed light on unique aspects of cellular heterogeneity and cell states in OSCC-GB.