Tumor RNA Sequencing Research Articles

Abstract B007: LIN28B promotes collective cell invasion and colorectal cancer metastasis via a novel CLDN1 and NOTCH3 axis

Abstract Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second most common cause of cancer death worldwide. The 5-year survival rate is 90% in patients with localized tumors, but the survival rate drastically decreases to 14% in patients with distant metastasis. Therefore, it is imperative to elucidate the molecular mechanisms underlying CRC metastasis. The RNA-binding protein LIN28B is overexpressed in over 30% of patients with CRC and is associated with poor prognosis. Indeed, our previous work revealed that LIN28B promotes liver metastasis in a subcutaneous xenograft model of CRC as well as a portal vein injection model. In the present study, we investigated the mechanism by which LIN28B promotes colorectal tumor progression and metastasis. To assess biological changes induced by LIN28B, we established CRC cells (DLD-1 and LoVo) with high expression of LIN28B (LIN28Bhigh). LIN28B overexpression upregulated Claudin-1 (CLDN1), a protein that functions as a major constituent of the tight junction complexes. RNA immunoprecipitation revealed that LIN28B directly binds to and stabilizes CLDN1 at a post-transcriptional level. Knockdown of CLDN1 expression in LIN28Bhigh cells suppresses cell aggregation, wound healing rate, and collective invasion. Using a mouse model of metastatic CRC, we reveal that knockdown of CLDN1 inhibits liver metastasis of LIN28Bhigh CRC cells. RNA-sequencing of metastatic liver tumors from LIN28Bhigh cells showed that NOTCH3 works downstream of the LIN28B-CLDN1 axis to induce cell aggregation, collective invasion, and metastatic liver tumor formation. Inhibition of Notch signaling by DAPT, an inhibitor of the γ-secretase complex, significantly reduced metastatic liver tumors in vivo. Taken together, our results indicate that LIN28B promotes cell aggregation, invasion, and liver metastasis in CRC through post-transcriptional induction of CLDN1 and upregulation of NOTCH3. Development of new therapies that target LIN28B-CLDN1-NOTCH3 axis may provide an effective strategy for stage 4 CRC. This work was supported by 9R01CA277795-22. Citation Format: Alice E. Shin, Kensuke Sugiura, Yasunori Masuike, Kensuke Suzuki, Christopher J. Lengner, Anil K. Rustgi. LIN28B promotes collective cell invasion and colorectal cancer metastasis via a novel CLDN1 and NOTCH3 axis [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B007.

Akkermansia supplementation reverses the tumor-promoting effect of the fecal microbiota transplantation in ovarian cancer

Ovarian cancer (OC) remains a clinical challenge for its difficulty in early diagnosis and insensitivity to treatments. Gut microbiota modulate multiple carcinoma progression through immunoregulation. The relationship between OC and gut microbiota has not been fully characterized. We find that the feces of patients with OC demonstrate different characteristics from benign controls. After fecal microbiota transplantation (FMT) from patients with OC into OC-bearing mice, the tumor development accelerates. Further, an Akkermansia supplementation with FMT significantly suppresses OC progression in mice. RNA sequencing of tumors shows that Tcell activation pathways are upregulated after Akkermansia supplementation with FMT. Moreover, acetate accumulation accompanies Akkermansia abundance elevation, which is associated with enhanced interferon γ (IFNγ) secretion of CD8+ Tcells and also its tumor-killing property. This work highlights the importance of protective gut microbiome in immune surveillance of OC, which connects accumulation of acetate and the cytotoxic function of CD8+ Tcells by increasing IFNγ secretion.

Multi-Omics Immune Interaction Networks in Lung Cancer Tumorigenesis, Proliferation, and Survival.

There are currently no effective biomarkers for prognosis and optimal treatment selection to improve non-small cell lung cancer (NSCLC) survival outcomes. This study further validated a seven-gene panel for diagnosis and prognosis of NSCLC using RNA sequencing and proteomic profiles of patient tumors. Within the seven-gene panel, ZNF71 expression combined with dendritic cell activities defined NSCLC patient subgroups (n = 966) with distinct survival outcomes (p = 0.04, Kaplan-Meier analysis). ZNF71 expression was significantly associated with the activities of natural killer cells (p = 0.014) and natural killer T cells (p = 0.003) in NSCLC patient tumors (n = 1016) using Chi-squared tests. Overexpression of ZNF71 resulted in decreased expression of multiple components of the intracellular intrinsic and innate immune systems, including dsRNA and dsDNA sensors. Multi-omics networks of ZNF71 and the intracellular intrinsic and innate immune systems were computed as relevant to NSCLC tumorigenesis, proliferation, and survival using patient clinical information and in-vitro CRISPR-Cas9/RNAi screening data. From these networks, pan-sensitive and pan-resistant genes to 21 NCCN-recommended drugs for treating NSCLC were selected. Based on the gene associations with patient survival and in-vitro CRISPR-Cas9, RNAi, and drug screening data, MEK1/2 inhibitors PD-198306 and U-0126, VEGFR inhibitor ZM-306416, and IGF-1R inhibitor PQ-401 were discovered as potential targeted therapy that may also induce an immune response for treating NSCLC.

Abstract C072: Cytokine modulation can suppress an EMT-like phenotype in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with a 5-year survival rate of only 11% and approximately half of patients present with distant metastasis at the time of diagnosis. PDAC is accompanied by pronounced alterations in stromal responses and immune surveillance programs, which are now recognized as some of the major drivers in PDAC tumor evolution. The role of immune modulators in driving metastatic potential in PDAC is poorly understood. We find that low expression levels of cytokine IL-23 in patients with PDAC correlate with poor differentiation status of these tumors, and thus predict poor outcomes. Consistent with this observation, we have found that a loss of host-derived IL-23 in a mouse model of pancreatic cancer promotes an EMT-like phenotype. The tumor morphology is altered and appears to be less differentiated. Additionally, we see a significant increase in the number of tumor cells expressing Zeb1 and a decrease in E-cadherin expression. We also find an increase in metastatic capacity in the absence of IL-23 in the tumor stroma. Single cell RNA sequencing of tumors injected orthotopically into IL-23 knockout mice reveals clusters of tumor cells with increases in EMT-associated genes such as Zeb1 and Vimentin and concurrent decreases in gastric identity genes. Overall, our findings indicate that IL-23 plays a role in suppressing an EMT-like phenotype in pancreatic cancer with one mechanism being through regulation of gastric lineage genes. Citation Format: Whitney Bell, Nancy Kren, Yan Wang, Yuliya Pylayeva-Gupta. Cytokine modulation can suppress an EMT-like phenotype in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C072.

TMIC-40. TRANSCRIPTOMIC CHARACTERIZATION OF PATIENT GLIOMAS AND DERIVED MODEL SYSTEMS REVEALS ENVIRONMENTAL INFLUENCE ON NEURODEVELOPMENTAL CELLULAR STATES

Abstract Bulk tumor and single-cell RNA sequencing have revealed the remarkable molecular heterogeneity and plasticity of gliomas, leading to the definition of tumor subtypes and cellular states describing inter- and intra-tumoral levels of heterogeneity respectively. While there has been great interest in creating and revising these classifications, the combination of selective pressures from molecular alterations within the tumor cell as well as extrinsic factors within the tumor microenvironment (TME) driving these levels of heterogeneity remain to be fully described. The brain TME is a complex, regionally heterogeneous ecosystem of communicating non-malignant and malignant cell types and scavenge-able nutrients and metabolites. We hypothesized that distinct environmental contexts within and across tumors may drive the intra- and inter-tumoral heterogeneity of gliomas. To identify tumorigenic programs impacted by environmental context, we performed bulk RNA sequencing of over 40 triplets of patient glioma samples and their matched derivative models established in direct orthotopic mouse xenografts and conventional gliomasphere cultures. Comparative analyses revealed environment-associated programs including in vivo immune and neuroglial signaling as well as altered in vitro lipid metabolism and upregulated cell migration. Next, to investigate associations between tumor cellular state and environment-associated programs we performed single-cell RNA sequencing of 4 patient and model system triplets. By annotating single cell profiles with previously defined single cell atlases of the developing and adult brain, 9 major states reflecting neurodevelopmental cell types were identified. Single cell profiling further revealed cell state-specific expression of environment-associated programs. We simultaneously observed the divergence of model cellular state compositions towards states expressing programs associated with their environment. We conclude that the transcriptional evolution of cellular states is connected to their ecological role within the TME. Consequently, we find grade- and diagnosis-associated composition differences between patient tumors predictive of their ability to establish a model in vitro.

LncRNA EGFEM1P promotes thyroid cancer progression by acting as an miR-369-3p sponge and upregulating TCF4.

Thyroid cancer is the most commonly diagnosed endocrine cancer, with the incidence of 14.42 per 100,000 person-years in 2010-2013. It is important to conduct an in-depth exploration into the molecular mechanisms of thyroid cancer, providing insights into the improvements of therapy. Long non-coding RNAs (lncRNAs) act as oncogenes or tumor suppressors in thyroid cancer by sponging microRNAs (miRNAs), however, the functions of numerous lncRNAs are still unknown. In the present study, via the comprehensive analysis of microarray data derived from papillary thyroid tumors and the RNA sequencing of thyroid tumors from The Cancer Genome Atlas database, EGF like and EMI domain containing 1 (EGFEM1P) expression levels in papillary thyroid tumors and normal adjacent tissues were explored. Reverse transcription-quantitative PCR was used to detect EGFEM1P, microRNA (miR)-369-3p and T cell factor 4 (TCF4) expression levels. Western blotting was used to detect TCF4 protein and cleaved caspase-3/8 expression levels. Cell proliferative ability and apoptosis were assessed using the Cell Counting Kit-8 assay and flow cytometry, respectively. The interactions between EGFEM1P and miR-369-3p, and miR-369-3p and TCF4, were determined using the dual-luciferase reporter assay. The results demonstrated that EGFEM1P was upregulated in papillary thyroid tumors and thyroid cancer cells compared with normal adjacent tissues and human normal thyroid epithelial Nthy-ori 3-1 cell line. In the examined thyroid cancer cells, EGFEM1P was demonstrated to interact with miR-369-3p and decreased miR-369-3p expression levels. Thereafter, TCF4 was determined to be a target gene of miR-369-3p and EGFEM1P promoted TCF4 expression via regulating miR-369-3p expression levels. At last, it was found that EGFEM1P expression promoted rapid cell proliferation and inhibited cell apoptosis in thyroid cancer cells via acting as a miR-369-3p sponge. In conclusion EGFEM1P promoted thyroid cancer progression via acting as a sponge of the miR-369-3p/TCF4 axis.

Combination Radiation Therapy and Selective TLR9 Agonist Improves Local Control in a Murine Model of HPV-Related HNSCC

<h3>Purpose/Objective(s)</h3> As novel immunotherapies emerge, it has become increasingly evident that personalized treatment combinations are necessary to improve outcomes in head and neck squamous cell carcinomas (HNSCC). Toll-like receptors (TLR) play a critical role in the activation of professional antigen presenting cells (APC). Given the increasing appreciation of the role of APC and B-cells in HPV+ HNSCC, we sought to evaluate the therapeutic role of selective TLR agonists in combination with radiation therapy (RT). <h3>Materials/Methods</h3> For human tumor RNA sequencing studies, we analyzed data from the TCGA and a separate independent validation cohort of HNSCC patients, cumulatively totaling nearly 600 patients. Single cell RNA sequencing (scRNA-seq) databases were analyzed using Seurat v4.0. For murine studies, wild type C3H mice were injected subcutaneously with 5 × 10⁵ cells of AT-84-E7-OVA syngeneic squamous cell carcinoma into the right flank. TLR agonists were administered intratumorally in 25 uL per published schedules for: TLR4 (MPLA-SM, 1 mg/ml), TLR7 (1V270, 2 mg/ml), and TLR9 (ODN 1826, 2 mg/ml). Control mice received PBS. Mice receiving RT were treated with 12 Gy in 1 fraction. Cell surface markers in the tumor draining lymph node (TDLN) and tumor were analyzed by flow cytometry. <h3>Results</h3> Treatment with TLR4, TLR7, and TLR9 agonists decreased the size of the primary tumor in our murine model of HPV+ HNSCC, with a B cell selective TLR9 agonist exhibiting the greatest therapeutic benefit. When analyzing transcriptomes of HPV- tumors, we found no correlation between high TLR4/7/9 expression and outcomes, however, in HPV+ tumors, among TLRs 4/7/9, only TLR9 had a significant correlation with overall survival (HR 0.38, P = 0.008). In an independent cohort of HPV+ HNSCC patients, the correlation between OS and high TLR9 expression was maintained. scRNA-seq of human HNSCC TILs revealed heterogeneity in TLR9 expression among various immune cells. In the TDLN, TLR9 agonist were shown to decrease the relative proportion of B cells and trended towards increased proportions of CD4+ T cells, which had decreased expression of CD44. In comparison, in the tumor, we observed increased CD8+ T cell infiltration but decreased proportions of B cells with TLR9 agonists. When evaluating TLR9 agonists in conjunction with RT, we found that the combination treatment led to the largest therapeutic benefit. <h3>Conclusion</h3> In HPV+ HNSCC, transcriptional analysis revealed a significant correlation between high TLR9 expression and overall survival. Concordantly, the combination of RT and TLR9 agonists lead to a significant reduction in the primary tumor, which may be driven by dynamic changes in B cell phenotype or migration patterns and CD8+ T cell tumor infiltration. These findings support the further investigation into the use of TLR9 agonists in combination with radiation therapy for the treatment of HPV+ HNSCC.

Identification of phenocopies improves prediction of targeted therapy response over DNA mutations alone

DNA mutations in specific genes can confer preferential benefit from drugs targeting those genes. However, other molecular perturbations can “phenocopy” pathogenic mutations, but would not be identified using standard clinical sequencing, leading to missed opportunities for other patients to benefit from targeted treatments. We hypothesized that RNA phenocopy signatures of key cancer driver gene mutations could improve our ability to predict response to targeted therapies, despite not being directly trained on drug response. To test this, we built gene expression signatures in tissue samples for specific mutations and found that phenocopy signatures broadly increased accuracy of drug response predictions in-vitro compared to DNA mutation alone, and identified additional cancer cell lines that respond well with a positive/negative predictive value on par or better than DNA mutations. We further validated our results across four clinical cohorts. Our results suggest that routine RNA sequencing of tumors to identify phenocopies in addition to standard targeted DNA sequencing would improve our ability to accurately select patients for targeted therapies in the clinic.

Pentraxin 3 is an adipose tissue-related serum marker for pancreatic cancer cachexia predicting subsequent muscle mass and visceral fat loss.

Cancer cachexia, a paraneoplastic syndrome characterized by ongoing skeletal muscle mass loss, is accompanied by adipose tissue loss and strongly affects chemotherapy endurance. Our aim was to detect a serum marker reflecting pancreatic cancer cachexia and predicting subsequent loss of muscle mass and adipose tissue, focusing on adipose tissue-secreted proteins. Murine-derived pancreatic cancer cells were orthotopically injected into the mouse pancreatic tail. After 3 weeks, RNA sequencing of perigonadal fat and orthotopic tumors was carried out. We analyzed stocked sera and clinical data of metastatic pancreatic cancer patients who received chemotherapy. Perigonadal fat weight/body weight decreased in mice with orthotopic tumors compared to those without tumors. By RNA sequencing and real-time PCR validation, pentraxin 3 (PTX3) was identified as a secreted protein-encoded gene whose expression was significantly higher in the perigonadal fat of mice with orthotopic tumors than in that of mice without orthotopic tumors and was least expressed in orthotopic tumors. Serum PTX3 levels correlated with PTX3 mRNA levels in perigonadal fat and were higher in mice with orthotopic tumors than in those without tumors. In 84 patients diagnosed with metastatic pancreatic cancer, patients with high serum PTX3 levels showed a greater visceral fat loss/month and skeletal muscle mass index (SMI) decrease/month than those with low serum PTX3 levels. High serum PTX3 was an independent risk factor for visceral fat loss, decreased SMI, and poor prognosis. High serum PTX3 in pancreatic cancer patients predicts visceral fat and muscle mass loss and major clinical outcomes of cancer cachexia.

Clinical Implication of Molecular Tumor Board

Due to rapid technical advances, steeply declining sequencing costs, and the ever-increasing number of targeted therapies, it is expected that extensive tumor DNA or RNA sequencing will be applied in standard of care. Clinicians will thus be confronted with increasingly complex genetic information and multiple test-platforms to choose from. General medical training, meanwhile, can hardly keep up with the pace of innovation. Consequently, there is a rapidly growing gap between clinical knowledge and genetic potential in cancer care. Multidisciplinary Molecular Tumor Boards (MTBs) have been suggested as a means to address this disparity, as a multidisciplinary approach to keep pace with the growing knowledge of complex molecular alterations in patients with advanced solid cancer. MTBs are designated to interpret these data and provide clinical recommendations. Not all patients with cancer have access to advice of an MTB. We aimed to determine the current status, opportunities, and challenges of the organization of MTBs in the Korea.

IDHwt Glioblastomas Show Opposing Resistance Mechanisms Across Patients in Response to Standard Treatment

Abstract AIMS Glioblastoma (GBM) is the most common primary malignant brain tumour in adults. Despite aggressive treatment, a resistant tumour recurs in practically all patients. We therefore aimed to better understand the mechanisms driving this treatment resistance through investigating changes in gene expression across pairs of primary and recurrent GBM tumours. METHOD We generated or acquired bulk tumour RNA sequencing data for primary and first recurrent tumours from 107 patients who received standard treatment. Differential expression analysis between primary and recurrent samples found that the most dysregulated genes were involved in neurodevelopment and neurodifferentiation. We therefore used a publicly available ChIP-seq database to identify DNA binding factors for which binding sites are enriched in the promotors of genes with the largest expression changes from primary to recurrent. RESULTS Jumonji and AT-Rich Interacting Domain 2 (JARID2) was the most strongly enriched for binding to promotors of dysregulated genes. 65 patients showed an up-regulation and 42 showed a down-regulation of genes bound by this protein. The same set of JARID2 bound genes were found to be dysregulated in each direction, and correlated with the largest source of variation between samples in their response to treatment. Further enrichment analyses indicated that ‘Up’ responders may resist treatment through reduced proliferation and increased interaction with the tumour microenvironment, whereas ‘Down’ responders instead rely on a shift to mesenchymal cell states. CONCLUSION These results indicate that GBM tumours can be split into two subtypes that transcriptionally reprogramme in different directions through treatment and may benefit from different treatment approaches.

Carbon ion irradiation plus CTLA4 blockade elicits therapeutic immune responses in a murine tumor model

Radiotherapy can act as an in situ vaccine, activating preventive tumor-specific immune responses in patients. Although carbon ion radiotherapy has superior biophysical properties over conventional photon irradiation, the immunological effects induced by this radiation type are poorly understood. Multiple strategies combining radiotherapy with immune checkpoint inhibition (radioimmunotherapy) to enhance antitumor immunity have been described; however, immune cell composition in tumors following radioimmunotherapy with carbon ions remains poorly explored. We developed a bilateral tumor model based on time-shifted subcutaneous injection of murine Her2+ EO771 tumor cells into immune-competent mice followed by selective irradiation of the primary tumor. αCTLA4-, but not αPD-L1-based radioimmunotherapy, induced complete tumor rejection and mediated the eradication of even non-irradiated, distant tumors. Cured mice were protected against the EO771 rechallenge, indicating long-lasting, tumor-specific immunological memory. Single-cell RNA sequencing and flow cytometric analyses of irradiated tumors revealed activation of NK cells and distinct tumor-associated macrophage clusters with upregulated expression of TNF and IL1 responsive genes. Distant tumors in the irradiated mice showed higher frequencies of naïve T cells activated upon the combination with CTLA4 blockade. Thus, radioimmunotherapy with carbon ions plus CTLA4 inhibition reshapes the tumor-infiltrating immune cell composition and can induce complete rejection even of non-irradiated tumors. Our data suggest combining radiotherapy approaches with CTLA4 blockade to achieve durable antitumor immunity. Evaluation of future radioimmunotherapy approaches should not be restricted to immunological impact at the irradiation site but should also consider systemic immunological effects on non-irradiated tumors.

Abstract IA09: Role of liquid biopsy in early detection and assessment of response in hepatocellular carcinoma

Abstract Early hepatocellular carcinoma detection is suboptimal with less than 25% of eligible patients enrolled in surveillance. In addition, recommended tools (biannual abdominal ultrasound with or without AFP) have poor sensitivity (63% for early-stage tumors). This has prompted the development of liquid biopsy approaches to improve performance and increase implementation. Methylation analysis of circulating tumor DNA and RNA sequencing in extracellular vesicles have shown promising performance in case control studies. This technology has also been applied to identify predictor biomarkers of response to systemic therapies. With 9 agents approved and without a clear strategy on how to select patients for therapies, the need to develop minimally-invasive biomarkers in this space cannot be overemphasized. Citation Format: Augusto Villanueva-Rodriguez. Role of liquid biopsy in early detection and assessment of response in hepatocellular carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr IA09.

An international report on bacterial communities in esophageal squamous cell carcinoma.

The incidence of esophageal squamous cell carcinoma (ESCC) is disproportionately high in the eastern corridor of Africa and parts of Asia. Emerging research has identified a potential association between poor oral health and ESCC. One possible link between poor oral health and ESCC involves the alteration of the microbiome. We performed an integrated analysis of four independent sequencing efforts of ESCC tumors from patients from high- and low-incidence regions of the world. Using whole genome sequencing (WGS) and RNA sequencing (RNAseq) of ESCC tumors from 61 patients in Tanzania, we identified a community of bacteria, including members of the genera Fusobacterium, Selenomonas, Prevotella, Streptococcus, Porphyromonas, Veillonella and Campylobacter, present at high abundance in ESCC tumors. We then characterized the microbiome of 238 ESCC tumor specimens collected in two additional independent sequencing efforts consisting of patients from other high-ESCC incidence regions (Tanzania, Malawi, Kenya, Iran, China). This analysis revealed similar ESCC-associated bacterial communities in these cancers. Because these genera are traditionally considered members of the oral microbiota, we next explored whether there was a relationship between the synchronous saliva and tumor microbiomes of ESCC patients in Tanzania. Comparative analyses revealed that paired saliva and tumor microbiomes were significantly similar with a specific enrichment of Fusobacterium and Prevotella in the tumor microbiome. Together, these data indicate that cancer-associated oral bacteria are associated with ESCC tumors at the time of diagnosis and support a model in which oral bacteria are present in high abundance in both saliva and tumors of some ESCC patients.

A genome-wide CRISPR-Cas9 knockout screen identifies novel PARP inhibitor resistance genes in prostate cancer.

DNA repair gene mutations are frequent in castration-resistant prostate cancer (CRPC), suggesting eligibility for poly(ADP-ribose) polymerase inhibitor (PARPi) treatment. However, therapy resistance is a major clinical challenge and genes contributing to PARPi resistance are poorly understood. Using a genome-wide CRISPR-Cas9 knockout screen, this study aimed at identifying genes involved in PARPi resistance in CRPC. Based on the screen, we identified PARP1, and six novel candidates associated with olaparib resistance upon knockout. For validation, we generated multiple knockout populations/clones per gene in C4 and/or LNCaP CRPC cells, which confirmed that loss of PARP1, ARH3, YWHAE, or UBR5 caused olaparib resistance. PARP1 or ARH3 knockout caused cross-resistance to other PARPis (veliparib and niraparib). Furthermore, PARP1 or ARH3 knockout led to reduced autophagy, while pharmacological induction of autophagy partially reverted their PARPi resistant phenotype. Tumor RNA sequencing of 126 prostate cancer patients identified low ARH3 expression as an independent predictor of recurrence. Our results advance the understanding of PARPi response by identifying four novel genes that contribute to PARPi sensitivity in CRPC and suggest a new model of PARPi resistance through decreased autophagy.

Targeting Glutamine Metabolism to Enhance Immunoprevention of EGFR-Driven Lung Cancer.

Lung cancer is the leading cause of cancer death worldwide. Vaccination against EGFR can be one of the venues to prevent lung cancer. Blocking glutamine metabolism has been shown to improve anticancer immunity. Here, the authors report that JHU083, an orally active glutamine antagonist prodrug designed to be preferentially activated in the tumor microenvironment, has potent anticancer effects on EGFR‐driven mouse lung tumorigenesis. Lung tumor development is significantly suppressed when treatment with JHU083 is combined with an EGFR peptide vaccine (EVax) than either single treatment. Flow cytometry and single‐cell RNA sequencing of the lung tumors reveal that JHU083 increases CD8+ T cell and CD4+ Th1 cell infiltration, while EVax elicits robust Th1 cell‐mediated immune responses and protects mice against EGFRL858R mutation‐driven lung tumorigenesis. JHU083 treatment decreases immune suppressive cells, including both monocytic‐ and granulocytic‐myeloid‐derived suppressor cells, regulatory T cells, and pro‐tumor CD4+ Th17 cells in mouse models. Interestingly, Th1 cells are found to robustly upregulate oxidative metabolism and adopt a highly activated and memory‐like phenotype upon glutamine inhibition. These results suggest that JHU083 is highly effective against EGFR‐driven lung tumorigenesis and promotes an adaptive T cell‐mediated tumor‐specific immune response that enhances the efficacy of EVax.

Molecular Heterogeneity in Pediatric Malignant Rhabdoid Tumors in Patients With Multi-Organ Involvement

Rhabdoid tumors (RTs) of the brain (atypical teratoid/rhabdoid tumor; AT/RT) and extracranial sites (most often the kidney; RTK) are malignant tumors predominantly occurring in children, frequently those with SMARCB1 germline alterations. Here we present data from seven RTs from three pediatric patients who all had multi-organ involvement. The tumors were analyzed using a multimodal molecular approach, which included exome sequencing of tumor and germline comparator and RNA sequencing and DNA array-based methylation profiling of tumors. SMARCB1 germline alterations were identified in all patients and in all tumors. We observed a second hit in SMARCB1 via chr22 loss of heterozygosity. By methylation profiling, all tumors were classified as rhabdoid tumors with a corresponding subclassification within the MYC, TYR, or SHH AT/RT subgroups. Using RNA-seq gene expression clustering, we recapitulated the classification of known AT/RT subgroups. Synchronous brain and kidney tumors from the same patient showed different patterns of either copy number variants, single-nucleotide variants, and/or genome-wide DNA methylation, suggestive of non-clonal origin. Furthermore, we demonstrated that a lung and abdominal metastasis from two patients shared overlapping molecular features with the patient’s primary kidney tumor, indicating the likely origin of the metastasis. In addition to the SMARCB1 events, we identified other whole-chromosome events and single-nucleotide variants in tumors, but none were found to be prognostic, diagnostic, or offer therapeutic potential for rhabdoid tumors. While our findings are of biological interest, there may also be clinical value in comprehensive molecular profiling in patients with multiple rhabdoid tumors, particularly given the potential prognostic and therapeutic implications for different rhabdoid tumor subgroups demonstrated in recent clinical trials and other large cohort studies.

Abstract 1681: Single cell RNA sequencing of primary Ewing sarcoma tumors and identification of circulating tumor cells in patient-matched peripheral blood samples

Abstract Ewing sarcoma (ES) is the second most common bone cancer in children, accounting for 2% of pediatric cancer diagnoses. Patients who present with metastatic disease at the time of diagnosis have a dismal prognosis, compared to the &amp;gt;70% 5-year survival of those with localized disease. Here, we utilized single-cell RNA sequencing (scRNA-seq) to characterize the transcriptional landscape of primary ES tumors, and to identify circulating tumor cells (CTCs) in peripheral blood at the time of diagnosis in order to further understand ES transcriptional heterogeneity and factors that drive metastasis. Methods: Viably frozen primary tumor and peripheral blood samples were obtained from 7 ES patients at the time of diagnosis and prior to the initiation of treatment. Tumors were dissociated into a single cell suspension and sorted for viability using FACS (fluorescence activated cell sorting) whereas peripheral blood samples were subjected to a size-based selection with the CellSieve microfiltration system. ScRNA-seq was performed using the 10xChromium platform and count matrices were generated using 10x Genomics Cell Ranger software. Quality control, integration, and cluster analysis was performed with Seurat. Results: Cluster analysis of integrated, primary tumor samples demonstrated that candidate ES cell clusters express FLI1 and ES marker NKX2-2 and cluster separately from immune cell populations. Additionally, using inferCNV, ES clusters were demonstrated to harbor chromosomal copy number alterations known to be associated with ES and that were previously identified clinically on preliminary pathology reports for each patient; further GSEA analysis showed significant overlap between published ES gene sets and genes upregulated in ES clusters. Further analysis of ES cells demonstrated that cell-cycle phase determined a cluster enriched in pro-proliferation gene signatures, and overall heterogeneity of expression of previously known therapeutic targets. Candidate ES CTCs were identified among the peripheral blood samples among clusters which corresponded with distinct immune cell populations. The candidate ES CTCs expressed NKX2-2 and demonstrated enrichment in oncogenic gene signatures. Conclusion: ScRNA-seq of primary ES tumors is feasible and demonstrates that ES tumor cells are largely homogeneous in nature; and candidate ES CTCs can be identified in peripheral blood at the time of diagnosis in ES patient and warrant further investigation as to their utility as a biomarker of metastatic disease. Citation Format: Sarah K. Nelson-Taylor, Avery Bodlack, Andrew Goodspeed, Amy Treece, Nathan Donaldson, Carrye Cost, Tim Garrington, Brian Greffe, Sandra Luna-Fineman, Jenna Sopfe, Masanori Hayashi. Single cell RNA sequencing of primary Ewing sarcoma tumors and identification of circulating tumor cells in patient-matched peripheral blood samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1681.

Abstract 1330: Role of ENPP1 mediated extracellular cGAMP hydrolysis in cancer metastasis and immune evasion

Abstract Chromosome instability (CIN) promotes metastasis through sustained tumor-cell autonomous response to cytosolic DNA. Chromosome mis-segregation engenders the formation of micronuclei, which upon rupture chronically activates the cGAS-STING DNA sensing pathway. Tumor cGAS recognizes cytosolic DNA to generate cGAMP, a potent immune-stimulatory molecule that is readily exported into the extracellular space to enforce a type 1 IFN-mediated anti-tumor response in a host STING dependent manner. However, it is unclear how highly aggressive, unstable tumors have evolved to co-opt this chronic inflammatory signaling to drive tumorigenic behaviors and immune evasion. Here, we show that ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) may drive tumor metastasis through degradation of extracellular cGAMP. Using RNA sequencing of isogenic tumors, we uncovered a link between ENPP1 and CIN. We leveraged syngeneic mouse models to demonstrate that genetic perturbation of tumor ENPP1 and overexpression of a catalytically inactive ENPP1 mutant suppress metastasis. Furthermore, loss of ENPP1 increases tumor immune infiltration, reduces extracellular adenosine, and enhances response to immune checkpoint blockade in a manner dependent on tumor cGAS and host STING. In line with these findings, ENPP1 expression correlates with immune suppression in human cancers. These results suggest that ENPP1-mediated hydrolysis of cGAMP may facilitate metastasis through evasion of immune surveillance, perhaps in part through cGAMP breakdown into immune-suppressive adenosine, in which the contributions of tumor and host adenosine can be further explored. Taken together, our study demonstrates how ENPP1-mediated hydrolysis of cGAMP can transform an otherwise inflammatory pathway into an immune-suppressive mechanism to promote tumor progression and metastasis. Citation Format: John Young Ho Kwon, Jun Li, Mercedes Duran, Ninjit Dhanota, Samuel Bakhoum. Role of ENPP1 mediated extracellular cGAMP hydrolysis in cancer metastasis and immune evasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1330.

Abstract 1129: Interrogating JAK/STAT signaling in ovarian cancer as a potential oncogenic driver and therapeutic target

Abstract Ovarian cancer is the 7th most common cancer in women. It is the most fatal of all female reproductive cancers, due to the 5-year overall survival of 10-30% and over half of patients being diagnosed with late-stage disease. Despite initial response to front line platinum- and taxol-based chemotherapies, over 70% of patients develop recurrent and chemotherapy resistant disease and nearly all of these women die of their disease. Recently, the use of targeted therapies such as bevacizumab and PARP inhibitors have been shown to improve progression-free survival. However, these regimens have thus far failed to improve overall survival in patients without specific genetic signatures, highlighting the urgent need for alternative therapies. We have identified lestaurtinib as a potent inhibitor of many ovarian cancer cell lines, including platinum and PARP inhibitor resistant models, and patient derived organoid models. RNA-sequencing following lestaurtinib treatment identified JAK/STAT signaling as one of the most profoundly downregulated pathways. Additionally, it was recently reported via single cell RNA sequencing of patient tumors that downstream mediators of the JAK/STAT pathway are among the most highly expressed genes in ovarian tumor cells. Further, we have discovered that STAT1 and STAT3 are constitutively activated in chemotherapy and PARP inhibitor resistant cell lines as reflected by high levels of Tyr701/705 and Ser727 phosphorylation suggesting induction of this pathway may drive resistance. Selective siRNA-mediated knockdown of STAT1/3 resulted in significant growth inhibition confirming their importance in maintaining cell viability. Surprisingly, we have identified profound differences in the ability of various JAK/STAT inhibitors to suppress ovarian cancer cell growth. For example, ruxolitinib, the only JAK/STAT inhibitor currently in clinical trials for ovarian cancer, failed to inhibit the growth sensitive, platinum resistant or PARP inhibitor resistant cell lines. Our preliminary data indicate that JAK/STAT inhibitor efficacy in preventing ovarian cancer cell growth is related to their ability to block specific phosphorylation events on STAT1/3 proteins. We believe that precise and selective inhibition of STAT1 and STAT3 signaling represents a robust, durable, and novel approach for the treatment of highly aggressive and resistant forms of ovarian cancer. Further we plan to elucidate the molecular mechanisms by which STAT1 and STAT3 function to support ovarian cancer cell viability and growth, in order to substantially advance our understanding of JAK/STAT signaling in ovarian cancer and to identify the most effective ways to pharmacologically inhibit JAK/STAT signaling in ovarian cancer cells. Outcomes from this work will have both immediate and long-term impacts for our patients and will lay the foundation for future biomarker-driven clinical trials. Citation Format: Esther Rodman, Michael Emch, Elizabeth Bruinsma, Xiaonan Hou, John Weroha, John Hawse. Interrogating JAK/STAT signaling in ovarian cancer as a potential oncogenic driver and therapeutic target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1129.