Chemoradiation Resistance Research Articles

BUB1 Inhibition Overcomes Radio- and Chemoradiation Resistance in Lung Cancer.

Background: Despite advances in targeted therapies and immunotherapies, traditional treatments like microtubule stabilizers (paclitaxel, docetaxel), DNA-intercalating platinum drugs (cisplatin), and radiation therapy remain essential for managing locally advanced and metastatic lung cancer. Identifying novel molecular targets could enhance the efficacy of these treatments. Hypothesis: We hypothesize that BUB1 (Ser/Thr kinase) is overexpressed in lung cancers and its inhibition will sensitize lung cancers to chemoradiation. Methods: BUB1 inhibitor (BAY1816032) was combined with cisplatin, paclitaxel, a PARP inhibitor olaparib, and radiation in cell proliferation and radiation-sensitization assays. Biochemical and molecular assays evaluated the impact on DNA damage signaling and cell death. Results: Immunostaining of lung tumor microarrays (TMAs) confirmed higher BUB1 expression in non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) compared to normal tissues. In NSCLC, BUB1 overexpression correlated directly with the expression of TP53 mutations and poorer overall survival in NSCLC and SCLC patients. BAY1816032 synergistically sensitized lung cancer cell lines to paclitaxel and olaparib and enhanced cell killing by radiation in both NSCLC and SCLC. Molecular analysis indicated a shift towards pro-apoptotic and anti-proliferative states, evidenced by altered BAX, BCL2, PCNA, and Caspases-9 and -3 expressions. Conclusions: Elevated BUB1 expression is associated with poorer survival in lung cancer. Inhibiting BUB1 sensitizes NSCLC and SCLC to chemotherapies (cisplatin, paclitaxel), targeted therapy (olaparib), and radiation. Furthermore, we present the novel finding that BUB1 inhibition sensitized both NSCLC and SCLC to radiotherapy and chemoradiation. Our results demonstrate BUB1 inhibition as a promising strategy to sensitize lung cancers to radiation and chemoradiation therapies.

AB036. Targeting glioblastoma de-novo purine metabolism to overcome chemoradiation resistance: an interim result of phase 0/1 clinical trial in newly diagnosed and recurrent glioblastoma.

Glioblastoma cells preferentially use de-novo purine synthesis pathway, whereas normal brain prefers salvage pathway. Mycophenolate mofetil (MMF), a commonly used oral immunosuppressant that inhibits inosine-5'-monophosphate dehydrogenase (IMPDH), a key enzyme in the de-novo purine pathway. Pre-clinical suggested MMF can improve radiation and temozolomide efficacy in glioblastoma which led to this phase 0/1 trial (NCT04477200) to assess MMF's tolerability with chemoradiation in glioblastoma, mycophenolic acid accumulation, and purine synthesis inhibition in tumor. In the phase 0 study, eight recurrent glioblastoma patients received MMF at doses ranging 500-2,000 mg BID for 1-week before surgery. The tissues were analyzed using mass spectrometry for drug accumulation and purine synthesis inhibition. In the phase 1 study, adult patients were given MMF starting at 1,000 mg orally (PO) twice daily (BID), with the possible dose ranging 500-2,000 PO BID. Nineteen recurrent glioblastoma patients (target N=30) received MMF 1-week prior to and concurrently with re-irradiation (40.5 Gy). Thirty newly diagnosed glioblastoma patients received MMF 1-week prior to and concurrently with chemoradiation, followed by MMF 1-day before and during 5 days of each adjuvant temozolomide cycle. Both enhancing and non-enhancing tumors from phase 0 subjects yielded >1 µM active drug metabolite, and the guanosine triphosphate: inosine monophosphate ratio was decreased by 75% in enhancing tumors in MMF-treated patients compared to untreated controls (P=0.009), indicating effective target engagement and inhibition of purine synthesis. In the phase 1 study, no dose-limiting toxicities (DLTs) were observed at the interim analysis at MMF 1,000-1,500 mg BID combined with chemoradiation. At 2,000 mg BID, there was no DLT combined with temozolomide alone, however, there were four DLTs noted (hemiparesis, cognitive disturbance, fatigue, thrombocytopenia) when combined with radiotherapy and temozolomide together, though all were reversible. Interim median overall survival in recurrent phase 1 is 15.6 months, and not reached yet in newly diagnosed phase 1. MMF with chemoradiation has been reasonably well tolerated and showed promising evidence of brain tumor target engagement and drug accumulation. This study led to a recommended phase 2 dose of MMF 1,500 mg BID and will provide a preliminary efficacy estimate for a randomized phase 2/3 trial through the Alliance for Clinical Trials in Oncology.

Vaginal microbial profile of cervical cancer patients receiving chemoradiotherapy: the potential involvement of Lactobacillus iners in recurrence

The vaginal microbiome is an immune defense against reproductive diseases and can serve as an important biomarker for cervical cancer. However, the intrinsic relationship between the recurrence and the vaginal microbiome in patients with cervical cancer before and after concurrent chemoradiotherapy is poorly understood. Here, we analyzed 125 vaginal microbial profiles from a patient cohort of stage IB–IVB cervical cancer using 16S metagenomic sequencing and deciphered the microbial composition and functional characteristics of the recurrent and non-recurrent both before and after chemoradiotherapy. We demonstrated that the abundance of beneficial bacteria and stability of the microbial community in the vagina decreased in the recurrence group, implying the unique characteristics of the vaginal microbiome for recurrent cervical cancer. Moreover, using machine learning, we identified Lactobacillus iners as the most important biomarker, combined with age and other biomarkers (such as Ndongobacter massiliensis, Corynebacterium pyruviciproducens ATCC BAA-1742, and Prevotella buccalis), and could predict cancer recurrence phenotype before chemoradiotherapy. This study prospectively employed rigorous bioinformatics analysis and highlights the critical role of vaginal microbiota in post-treatment cervical cancer recurrence, identifying promising biomarkers with prognostic significance in the context of concurrent chemoradiotherapy for cervical cancer. The role of L. iners in determining chemoradiation resistance in cervical cancer warrants further detailed investigation. Our results expand our understanding of cervical cancer recurrence and help develop better strategies for prognosis prediction and personalized therapy.

VC-resist glioblastoma cell state: vessel co-option as a key driver of chemoradiation resistance

Glioblastoma (GBM) is a highly lethal type of cancer. GBM recurrence following chemoradiation is typically attributed to the regrowth of invasive and resistant cells. Therefore, there is a pressing need to gain a deeper understanding of the mechanisms underlying GBM resistance to chemoradiation and its ability to infiltrate. Using a combination of transcriptomic, proteomic, and phosphoproteomic analyses, longitudinal imaging, organotypic cultures, functional assays, animal studies, and clinical data analyses, we demonstrate that chemoradiation and brain vasculature induce cell transition to a functional state named VC-Resist (vessel co-opting and resistant cell state). This cell state is midway along the transcriptomic axis between proneural and mesenchymal GBM cells and is closer to the AC/MES1-like state. VC-Resist GBM cells are highly vessel co-opting, allowing significant infiltration into the surrounding brain tissue and homing to the perivascular niche, which in turn induces even more VC-Resist transition. The molecular and functional characteristics of this FGFR1-YAP1-dependent GBM cell state, including resistance to DNA damage, enrichment in the G2M phase, and induction of senescence/stemness pathways, contribute to its enhanced resistance to chemoradiation. These findings demonstrate how vessel co-option, perivascular niche, and GBM cell plasticity jointly drive resistance to therapy during GBM recurrence.

Abstract 1438: Investigating the roles of EMT in small cell lung cancer tumorigenesis and chemoradiation response

Abstract In this study, we investigated the contributions of the epithelial-mesenchymal transition (EMT) program to small cell lung cancer (SCLC) tumorigenesis and chemoradiation (CRT) response. SCLC is a highly aggressive and deadly neuroendocrine malignancy. Two major factors that contribute to the high mortality of SCLC are (1) early metastasis and (2) rapid development of therapy resistance. Recent research suggests upregulation of EMT and the EMT transcription factor Twist1 correlated with accelerated tumor progression and CRT resistance in SCLC. Due to the lack of suitable animal models, the causal relationship between the EMT program and SCLC tumorigenesis and therapy response has not been rigorously investigated. To this end, we have generated a novel genetically engineered mouse model (GEMM) termed RPGT (Rb1Flox; Trp53Flox; ROSA26LSL-rtTA-IRES-EGFP; Twist1-TetO7-Luc). The RPGT GEMM enables the generation of autochthonous SCLC tumors after induction with Cre recombinase adenovirus. By withdrawing or providing doxycycline to mice, we can control Twist1 expression to activate or inactivate EMT in tumors. Upon characterizing mouse tumor samples with histological and molecular analyses, we observed that both control (no Twist1 overexpression) and Twist1-overexpressing RPGT mice developed neuroendocrine SCLC tumors. While SCLC-A was the predominant subtype in both cohorts, RPGT tumors exhibited more heterogeneity. Importantly, although no difference in tumorigenesis was observed, Twist1 overexpression dramatically increased the metastatic incidence in RPGT (87-100%) compared to control (25-50%) animals, indicating an important role of EMT in SCLC dissemination. Furthermore, transcriptomic profiling of primary tumors and matching metastases in RPGT mice revealed downregulation of Twist1 and EMT in metastases, suggesting that EMT suppression was necessary for metastatic outgrowth. To evaluate the impact of EMT activation on SCLC sensitivity to CRT, we performed in vitro viability assays on primary tumor cell lines established from RPGT tumors. We found that repressing Twist1 expression enhanced SCLC susceptibility to CRT. To validate these results in vivo, we treated RPGT mice with vs. without Twist1 overexpression with CRT. Consistent with our in vitro results, our in vivo data indicated that Twist1 overexpression promoted CRT resistance in SCLC tumors. Overall, our data suggest that the EMT program plays an important role in SCLC differentiation, metastasis, and resistance against chemo-radiotherapy. Citation Format: Triet Nguyen, Jinhee Chang, Kathleen Gabrielson, Amol Shetty, Yang Song, Audrey Lafargue, Shreya Jagtap, Danielle Council, Aaron Chan, Dipanwita Dutta Chowdhury, Muhammad Ajmal Khan, Nick Connis, Francesca Carrieri, Eddie Imada, Daniel Sforza, Eric Gardner, Christopher McFarland, Luigi Marchionni, Mohammad Rezaee, Christine Hann, Phuoc Tran. Investigating the roles of EMT in small cell lung cancer tumorigenesis and chemoradiation response [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 1438.

Abstract 1749: Esophageal adenocarcinoma single-cell resolution ecotypes and subtypes mediate chemoradiation resistance

Abstract Esophageal adenocarcinoma (EAC) is an aggressive and lethal disease with a 5-year survival rate less than 20%. The current standard of care for locally advanced EAC patients is neoadjuvant chemoradiation (CRT) followed by surgery and the pathological response rate is low in clinic. An unresolved question is how the cancer cells and their tumor microenvironment (TME) mediate CRT resistance in EAC. To address this question, we performed single-cell RNA sequencing on 84 longitudinal samples from a cohort of patients (N=47) containing 20 good responders (GR) and 27 non-responders (NR). In total, we obtained 302,918 single cells which enabled us to delineate the phenotypic heterogeneity landscape in EAC at baseline, and identify reprogramming in both the immune and tumor cells during treatment that lead to CRT resistance. We identified 4 major cell type compartments in the EAC TME: lymphoid cells (T cells, NK cells, B cells, and plasma cells), myeloid cells (macrophages, neutrophils, dendritic cells, and mast cells), stromal cells (fibroblasts and endothelial cells) and epithelial cells. Our data shows that the TME enriched in lymphoid cells, including resident and effector memory CD8 T cells, follicular helper CD4 T cells, and B cells was associated with better CRT responses. Moreover, a significant myeloid cell infiltration was observed across most patients during CRT and the infiltrated macrophages from GR harbored a more pro-inflammation phenotype. In addition, we identified 4 major ecotypes of the TME cells and 4 major expression subtypes of the cancer cells. Notably, the interactions between TME ecotypes and cancer subtypes are significantly associated with CRT responsiveness. In summary, this study has provided valuable insights into the biology of EAC CRT response, and identified novel predictive biomarkers and actionable targets to enhance CRT efficacy. Citation Format: Rui Ye, Yawei Qiao, Shanshan Bai, Emi Sei, Min Hu, Jianzhong He, Nan Li, Yifan Wang, Pablo Lopez, Wei Zhang, Fei Ye, Brian Weston, Emmanuel Coronel, William Ross, Phillip Ge, Bhutani Manoop, Nicholas Navin, Steven Lin. Esophageal adenocarcinoma single-cell resolution ecotypes and subtypes mediate chemoradiation resistance [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 1749.

Abstract 1125: TNIK induced chemoradiation resistance in small cell lung cancer

Abstract Chemoradiation (CRT) is the standard first-line therapy for limited stage small cell lung cancer (LS-SCLC), which is characterized by early metastasis, intrinsic-acquired CRT resistance and tumor recurrence. CRT confers a 20-25% 5-year overall survival in patients with LS-SCLC. In this study, we utilized patient-derived xenograft (PDX) models to demonstrate CRT resistance of SCLC and identified CRT resistance candidate genes from various SCLC PDX that represent the majority of molecular subtypes (n=4 that include SCLC-A and SCLC-N subtypes). RNA-seq data identified TNIK (Traf2- and Nck-interacting kinase) as one such gene that is consistently upregulated in SCLC PDXs exposed to CRT compared to single modality treatments. Copy number gains of TNIK were also present in human SCLC samples. Overcoming this CRT resistance is crucial in improving treatment outcomes and patient survival. Genetic depletion or pharmacological inhibition of TNIK reduced the in vitro clonogenic survival of TNIKhigh SCLC cells, NCI-H446 and makes them increasingly sensitive to CRT. In vivo, pharmacological inhibition of TNIK with the inhibitor NCB-0846, enhances the CRT sensitivity of NCI-H446 cell line-derived xenografts (CDX) in NOD SCID immunodeficient mice. Furthermore, pharmacological inhibition of TNIK in vivo demonstrated sensitivity to CRT in LX33 PDX. In conclusion, our results indicate that TNIK plays a role in conferring resistance to CRT in vitro in SCLC cell lines and in vivo in SCLC CDX and PDX models and therefore, can be a potential therapeutic target in limited stage SCLC. Citation Format: Dipanwita Dutta Chowdhury, Eddie Imada, Nick Connis, Triet Nguyen, Jinhee Chang, Danielle Council, Audrey Lafargue, Mohammad Rezaee, Phuoc T. Tran, Luigi Marchionni, Christine L. Hann. TNIK induced chemoradiation resistance in small cell lung 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 1125.

Review: therapeutic approaches for circadian modulation of the glioma microenvironment.

High-grade gliomas are malignant brain tumors that are characteristically hard to treat because of their nature; they grow quickly and invasively through the brain tissue and develop chemoradiation resistance in adults. There is also a distinct lack of targeted treatment options in the pediatric population for this tumor type to date. Several approaches to overcome therapeutic resistance have been explored, including targeted therapy to growth pathways (ie. EGFR and VEGF inhibitors), epigenetic modulators, and immunotherapies such as Chimeric Antigen Receptor T-cell and vaccine therapies. One new promising approach relies on the timing of chemotherapy administration based on intrinsic circadian rhythms. Recent work in glioblastoma has demonstrated temporal variations in chemosensitivity and, thus, improved survival based on treatment time of day. This may be due to intrinsic rhythms of the glioma cells, permeability of the blood brain barrier to chemotherapy agents, the tumor immune microenvironment, or another unknown mechanism. We review the literature to discuss chronotherapeutic approaches to high-grade glioma treatment, circadian regulation of the immune system and tumor microenvironment in gliomas. We further discuss how these two areas may be combined to temporally regulate and/or improve the effectiveness of immunotherapies.

Glioblastoma may evade immune surveillance through primary cilia-dependent signaling in an IL-6 dependent manner.

Glioblastoma is the most common, malignant primary brain tumor in adults and remains universally fatal. While immunotherapy has vastly improved the treatment of several solid cancers, efficacy in glioblastoma is limited. These challenges are due in part to the propensity of glioblastoma to recruit tumor-suppressive immune cells, which act in conjunction with tumor cells to create a pro-tumor immune microenvironment through secretion of several soluble factors. Glioblastoma-derived EVs induce myeloid-derived suppressor cells (MDSCs) and non-classical monocytes (NCMs) from myeloid precursors leading to systemic and local immunosuppression. This process is mediated by IL-6 which contributes to the recruitment of tumor-associated macrophages of the M2 immunosuppressive subtype, which in turn, upregulates anti-inflammatory cytokines including IL-10 and TGF-β. Primary cilia are highly conserved organelles involved in signal transduction and play critical roles in glioblastoma proliferation, invasion, angiogenesis, and chemoradiation resistance. In this perspectives article, we provide preliminary evidence that primary cilia regulate intracellular release of IL-6. This ties primary cilia mechanistically to tumor-mediated immunosuppression in glioblastomas and potentially, in additional neoplasms which have a shared mechanism for cancer-mediated immunosuppression. We propose potentially testable hypotheses of the cellular mechanisms behind this finding.

EXTH-79. A NOVEL ANTI-GALECTIN-3 ANTIBODY THERAPEUTIC SYNERGIZES WITH TEMOZOLOMIDE IN PRECLINICAL MODELS TO DIMINISH TUMOR GROWTH, DECREASE MIGRATION, AND PROLONG SURVIVAL

Abstract Glioblastoma (GBM) is the most aggressive, invasive, and malignant primary brain cancer whose standard of care for nearly 20 years has been surgical resection followed by radiotherapy and temozolomide (TMZ) treatment, resulting in a median survival of 15 months following diagnosis. Heterogenous tumor cell populations that evade resection and develop chemoradiation resistance highlight the need for novel therapies that can target both the migratory and proliferative nature of GBM cells. Galectin-3, a carbohydrate-binding protein overexpressed in the GBM parenchyma and stromal astrocytes, is a potent modulator of cell proliferation, migration, angiogenesis, T-cell inhibition, and M2 macrophage polarization, and is seen to increase following TMZ and radiation treatment. Heightened Galectin-3 expression levels correlate to lower survival in glioma patients, thus greatly underscoring the need for targeting Galectin-3 in conjunction with initial standard-of-care treatment. Here, we characterize the therapeutic effects of a brain-penetrant monoclonal anti-Galectin-3 antibody (Gal3 Ab) we have developed for its ability to inhibit GBM growth and migration in several in vitro assays and in vivo heterotopic and orthotopic mouse models. Our Gal3 Ab significantly decreases cell viability, neurosphere migration, and cell invasion in vitro in the presence of full length or truncated Gal3, whose expression and secretion in GBM cells is enhanced following short and long-term TMZ stimulation. We find that Galectin-3 directly binds to EGFR, amplified in ~40% of GBM patients, as well as its most commonly mutated form, EGFRvIII, and our Gal3 Ab can block these bindings, decrease EGFR activation, and alter EGFR localization. In vivo, our Gal3 Ab decreases tumor burden and improves survival in combination with temozolomide, compared to standard-of-care treatment alone in multiple cell-line models. Our pre-clinical studies highlight the potential for our novel Gal3 antibody to further improve TMZ efficacy and benefit GBM patients by diminishing tumor cell migration and slowing tumor growth.

PATH-09. THE IMPACT OF MISMATCH REPAIR DEFICIENCY ON GLIOMAS IN CHILDREN, ADOLESCENTS, AND YOUNG ADULTS; A MULTI-CENTRIC, COLLABORATIVE STUDY LED BY THE IRRDC AND GLIOMA TASKFORCE

Abstract BACKGROUND Primary mismatch repair deficiency (pMMRD) is a pan-cancer mechanism caused by somatic mutations or inherited as part of Lynch Syndrome (LS) or constitutional mismatch repair deficiency (CMMRD). PMMRD results in universal hypermutation and microsatellite instability leading to chemoradiation resistance but sensitivity to immunotherapy. The prevalence of pMMRD in gliomas of children, adolescents, and young adults (CAYA), and the impact of germline inheritance is unknown. METHODS We harnessed functional genomic tools across population-based (Toronto), and multiple institutional (SJ, CBTN) cancer databases (n=1276) to determine the prevalence, subgroups, and impact of germline mutations in pMMRD gliomas. RESULTS Data from Toronto, SJ and CBTN reveals prevalence of 10%, 5% and 5% in pediatric high-grade gliomas (HGG). Molecularly, pMMRD is absent in gliomas harboring pediatric-type fusions, BRAF-V600E, and histone mutations, but enriched in HGG harboring IDH and TP53 mutations. In AYA (n=660), pMMRD was detected in 5% of IDH-WT and IDH-mutant high-grade astrocytomas, but notably absent in all oligodendrogliomas and other low-grade gliomas. Across this entire CAYA dataset, all except one pMMRD gliomas harbored germline MMR mutations (97%). Strikingly, LS predominated over CMMRD with significant difference in age of glioma onset (LS: 29-years versus CMMRD: 11-years; p< 0.001, IRRDC dataset). Survival analysis (Kaplan-Meier) confirmed the poor overall survival (OS) for pMMRD and other pediatric subtypes of HGG (3-year OS < 25%). In contrast, IDH-mutant pMMRD HGG have significantly worse OS than IDH-mutant non-pMMRD counterparts. Remarkably, immunotherapy significantly improves survival not only in children, but also for AYA patients with refractory pMMRD HGG (3-year OS > 50%). CONCLUSION This large study demonstrates that pMMRD drives a significant proportion of HGG across CAYA with an alarming impact of germline predisposition. Specifically, unrecognized AYA LS patients with MMRD HGG. These findings support universal screening for MMRD in HGG to identify patients for surveillance and immunotherapy.

Tumor-resident Lactobacillus iners confer chemoradiation resistance through lactate-induced metabolic rewiring

Tumor microbiota can produce active metabolites that affect cancer and immune cell signaling, metabolism, and proliferation. Here, we explore tumor and gut microbiome features that affect chemoradiation response in patients with cervical cancer using a combined approach of deep microbiome sequencing, targeted bacterial culture, and invitro assays. We identify that an obligate L-lactate-producing lactic acid bacterium found in tumors, Lactobacillus iners, is associated with decreased survival in patients, induces chemotherapy and radiation resistance in cervical cancer cells, and leads to metabolic rewiring, or alterations in multiple metabolic pathways, in tumors. Genomically similar L-lactate-producing lactic acid bacteria commensal to other body sites are also significantly associated with survival in colorectal, lung, head and neck, and skin cancers. Our findings demonstrate that lactic acid bacteria in the tumor microenvironment can alter tumor metabolism and lactate signaling pathways, causing therapeutic resistance. Lactic acid bacteria could be promising therapeutic targets across cancer types.

Targeted Proteomic Quantitation of NRF2 Signaling and Predictive Biomarkers in HNSCC

The NFE2L2 (NRF2) oncogene and transcription factor drives a gene expression program that promotes cancer progression, metabolic reprogramming, immune evasion, and chemoradiation resistance. Patient stratification by NRF2 activity may guide treatment decisions to improve outcome. Here, we developed a mass spectrometry-based targeted proteomics assay based on internal standard-triggered parallel reaction monitoring to quantify 69 NRF2 pathway components and targets, as well as 21 proteins of broad clinical significance in head and neck squamous cell carcinoma (HNSCC). We improved an existing internal standard-triggered parallel reaction monitoring acquisition algorithm, called SureQuant, to increase throughput, sensitivity, and precision. Testing the optimized platform on 27 lung and upper aerodigestive cancer cell models revealed 35 NRF2 responsive proteins. In formalin-fixed paraffin-embedded HNSCCs, NRF2 signaling intensity positively correlated with NRF2-activating mutations and with SOX2 protein expression. Protein markers of T-cell infiltration correlated positively with one another and with human papilloma virus infection status. CDKN2A (p16) protein expression positively correlated with the human papilloma virus oncogenic E7 protein and confirmed the presence of translationally active virus. This work establishes a clinically actionable HNSCC protein biomarker assay capable of quantifying over 600 peptides from frozen or formalin-fixed paraffin-embedded archived tissues in under 90min.

OS07.5.A GLIOBLASTOMA VESSEL CO-OPTION AND TRANSITION TO A RESISTANT CELL STATE ARE INDUCED BY CHEMORADIATION

Abstract BACKGROUND Glioblastoma (GB) is one of the deadliest types of human cancer. Despite a very aggressive treatment regime, including resection of the tumor, radiation, and chemotherapy, the recurrence rate is more than 90%. Recurrence is mostly caused by the regrowth of highly invasive and resistant cells that have spread from the tumor bulk and are not removed by resection. To develop an effective therapeutic approach, we need to better understand the underlying molecular and cellular mechanisms of GB chemoradiation resistance and tumor spreading. MATERIAL AND METHODS To dynamically follow the changes occurring in GB post-therapy and investigate its relationship with vascular microenvironment, we employed multiple bulk and single-cell RNA-Seq analyses, phosphoproteome, in vitro and in vivo real-time imaging, organotypic cultures and functional assays, digital pathology, and spatial transcriptomics on patient material or preclinical models of GB. RESULTS We demonstrated that chemoradiation and the brain vasculature induce a transition to a functional cell state, which we named VC-Resist. This cell state is midway through the transcriptomic axis between proneural and mesenchymal GB cells and is closer to the AC/MES-like state. Better cell survival, G2M-arrest, activation of senescence/stemness pathways make this GB cell state more resistant to therapy. Notably, these persister GB cells are highly vessel co-opting, allowing homing to the perivascular niche, which, in turn, increases their transition to this cell state and resistance to therapy. Molecularly, the transition to the VC-Resist cell state is driven by FGF-FGFR1 signaling, which leads to the activation of DNA damage repair and YAP1 pathways. CONCLUSION These findings demonstrate that the perivascular niche and GB cell plasticity jointly generate a vicious loop that leads to resistance to therapy and brain infiltration during GB recurrence. SUPPORT This work was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 (Grant Agreement No. 805225), the INSERM-CNRS ATIP-Avenir grant, the NanoTheRad grant from Paris-Saclay University, Fondation ARC pour la recherche sur le cancer, Campus France and Canceropole Ile-de-France (2022-1-EMERG-06-ICR-1).

Predictive molecular markers of chemoradiation resistance in cervical cancer.

e17505 Background: Cisplatin based concurrent chemoradiation therapy (CCRT) has been recognized as the standard treatment for locally advanced cervical cancer. Although patients treated with CCRT have better chances of survival and prognosis, over 30–40% of patients do not achieve a complete response and may develop locoregional recurrence. Hence predictive biomarkers-based treatment selection of right cohort could be a strong approach for personalized treatment. Therapy resistance occur due to multifactorial events and targeting key molecules involved in treatment resistance could combat treatment resistance. Herein, we profiled both the global proteomic and phosphoproteomic landscape of the concurrent chemotherapy resistant cervical cancer to identify potential predictive biomarkers as well as therapeutic targets. Methods: We employed mass spectrometry based global proteomic and phosphoproteomic strategies to identify proteomic signature and dysregulated kinase involved in CCRT resistance. The biopsy tissue samples were collected before the start of the therapy and the protein was extracted, digested from each sample and labelled with 10 plex TMT (Tandem Mass Tags). The pooled peptides were fractionated by basic pH liquid chromatography analyzed on Orbitrap-Fusion-Tribrid mass spectrometer (ThermoFisher Scientific). For the phosphoproteomic experiment, the peptides were enriched using IMAC based phosphopeptide enrichment method. Liquid chromatography-mass spectrometry/ experiments together with thorough bioinformatics analysis were carried out to characterize the global proteome and phosphoproteome. Results: Analysis of protein expression and phosphorylation led to the identification of dysregulated protein expression and phosphorylation between treatment sensitive and resistant patient cohorts. Further analysis and validation experiment revealed the potential of differentially expressed protein syntaxin 3 (STX3) as a predictive biomarker for treatment resistance. Phosphoproteomic analysis revealed the enrichment of DNA repair pathway in treatment-resistant cohort and the phosphorylation of the proteins involved in DNA repair such as SMC1A, HMGN1, MGMT, DDB2, MSH6 are dysregulated between the cohort suggesting role of DNA repair pathway in the treatment resistance. Kinase-substrate enrichment analysis revealed the activation of kinases such as CSNK2A1, PRKDC, PLK1, NEK2 and ATM in non-responders. Conclusions: We have identified an accurate and reliable biomarker STX3 to predict CCRT resistance in cervical cancer. Further, the phosphoproteomic profiling revealed dysregulation of DNA repair pathways which may be involved in treatment resistance. Targeting the activated kinases such as CSNK2A1, PRKDC, PLK1, NEK2 and ATM could combat treatment resistance in cervical cancer patients.

PO-2220 Single cell sequencing is feasible in delineating chemoradiation resistance in rectal cancer

PO-2220 Single cell sequencing is feasible in delineating chemoradiation resistance in rectal cancer

Abstract 3677: DNA damage signaling activates de novo GTP synthesis to promote chemoradiation resistance in glioblastoma

Abstract Glioblastoma (GBM) is uniformly fatal due to inherent radiation (RT) and chemotherapy resistance. We have found this therapeutic resistance is mediated by alterations in tumor cellular metabolic activity. Our group and others have found that metabolites can regulate DNA repair and RT resistance in brain tumors, but little is known about how DNA damage regulates metabolic pathway activity in cancer. Here, we show that DNA damage acutely increases guanine-containing purine metabolites in multiple in vitro and intracranial GBM models. By interrogating metabolic fluxes in vitro using a variety of stable isotope tracers, we confirmed RT-induced elevation in guanylates was due to increased de novo purine synthesis (DNPS) rather than activation of purine salvage. By developing and using novel stable isotope tracing methods to directly measure DNPS in awake, unrestrained mice, we confirmed that orthotopic GBMs have higher DNPS rates than adjacent cortical tissue that further increase after treatment with RT. Neither salvage synthesis of purines nor pyrimidine synthesis were impacted by RT in any intracranial tissues. With these findings, we opened a clinical study to directly measure purine synthesis in patients, and we found that human GBMs have similarly high purine synthesis rates compared to normal brain tissue. Because DNA damage activated DNPS without affecting purine salvage or pyrimidine synthesis, we reasoned that active signaling may be involved. Indeed, therapy-induced DNPS increases are lost in vitro and in vivo upon pharmacological or genomic inhibition of the DNA-damage sensing kinase DNA-PK. Moreover, RT and DNA-PK have direct influence over the spatial organization of DNPS enzymes, including IMPDH, the rate-limiting step in guanylate synthesis. Because purines can promote DNA repair, these findings suggest that DNA-PK signaling helps promote DNA repair in part by causing the spatial reorganization of DNPS enzymes, thereby activating purine synthesis. To determine if disrupting this regulation can augment GBM treatment efficacy, we combined an FDA-approved inhibitor of purine synthesis with chemoradiation in a variety of mouse models of GBM. Critically, targeting GTP synthesis improved the efficacy of both RT alone and chemoradiation in multiple patient-derived and syngeneic intracranial models, suggesting a potential therapeutic targeting opportunity in patients. In this study, we have developed novel methodology to directly measure purine synthesis in brain tumors in mice and humans. With these tools, we discovered that after DNA damage, DNA-PK mediates a novel pathway controlling the spatial reorganization of purine synthesis enzymes and subsequent DNPS increases. The resulting elevation of GTP levels promotes therapy resistance in tumors, and we are now directly measuring and inhibiting this molecular activity in patients with GBM in an effort to improve standard therapy. Citation Format: Andrew J. Scott, Alexandra M. O'Brien, Weihua Zhou, Vidhi Pareek, Zhou Sha, Sravya Palavalasa, Ayesha U. Kothari, Kari Wilder-Romans, Li Zhang, Anthony C. Andren, Sriram Chandrasekaran, Jason Heth, Yoshie Umemura, Nathan Qi, John Woulfe, Sriram Venneti, Meredith A. Morgan, Theodore S. Lawrence, Wajd N. Al-Holou, Costas A. Lyssiotis, Daniel R. Wahl. DNA damage signaling activates de novo GTP synthesis to promote chemoradiation resistance in glioblastoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3677.

520 Glioblastoma Susceptibility to SARS-CoV-2: Analysis of Cancer Stem Cells and Immune Tumor Microenvironment on Human Brain, Patient-Derived Tumors, and Organoid Models

INTRODUCTION: Glioblastoma (GBM) is the most common and deadliest primary brain tumor, characterized by chemoradiation resistance and an immunosuppressive tumor microenvironment (TME). SARS-CoV-2, the COVID-19 virus, produces a significant pro-inflammatory response and a spectrum of clinical presentations after central nervous system infection. METHODS: Patient-derived GBM tissue, primary cell lines, and organoids were analyzed with immunohistochemistry and pixel-line intensity quantification. Data from tumor-bulk and single-cell transcriptomics served to describe the cell-specific expression of SARS-CoV-2 receptors in GBM and its association with the immune TME phenotype. Normal brain and iPSC-derived organoids served as controls. RESULTS: We demonstrate that patient-derived GBM tissue and cell cultures express SARS-CoV2 entry factors such as ACE2, TMPRSS2, and NRP1. NRP1 expression was higher in GBM than in normal brains (p<0.05), where it plays a crucial role in SARS-CoV-2 infection. NRP1 was expressed in a cell-type and phenotype-specific manner and correlated with TME infiltration of immunosuppressive cells: M2 macrophages (r = 0.229), regulatory T cells (r = 0.459), NK cells (r = -0.346), and endothelial cells (r = 0.288) (p < 0.05). Furthermore, gene ontology enrichment analysis showed that leukocyte migration and chemotaxis are among the top 5 biological functions mediated by NRP1 (p < 0.05). We found our GBM organoids recapitulate tumoral expression of SARS-CoV-2 entry factors, which varies based on distance from surface as surrogate of TME oxygenation (p < 0.05). CONCLUSIONS: GBM cancer cells and immune TME cells express SARS-CoV-2 entry factors. Glioblastoma organoids recapitulate this expression and allow for currently undergoing studies analyzing the effect of SARS-CoV-2 infection in GBM. Our findings suggest that SARS-CoV-2 could potentially target GBM, opening the door to future studies evaluating SARS-CoV-2-driven immune modulation.

1H, 13C, and 15N assignments of the mRNA binding protein hnRNP A18

Heterogeneous ribonuclear protein A18 (hnRNP A18) is an RNA binding protein (RBP) involved in the hypoxic cellular stress response and regulation of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) expression in melanoma, breast cancer, prostate cancer, and colon cancer solid tumors. hnRNP A18 is comprised of an N-terminal structured RNA recognition motif (RMM) and a C-terminal intrinsically disordered domain (IDD). Upon cellar stressors, such as UV and hypoxia, hnRNP A18 is phosphorylated by casein kinase 2 (CK2) and glycogen synthase kinase 3β (GSK-3β). After phosphorylation, hnRNP A18 translocates from the nucleus to the cytosol where it interacts with pro-survival mRNA transcripts for proteins such as hypoxia inducible factor 1α and CTLA-4. Both the hypoxic cellular response and modulation of immune checkpoints by cancer cells promote chemoradiation resistance and metastasis. In this study, the 1 H, 13 C, and 15 N backbone and sidechain resonances of the 172 amino acid hnRNP A18 were assigned sequence-specifically and provide a framework for future NMR-based drug discovery studies toward targeting hnRNP A18. These data will also enable the investigation of the dynamic structural changes within the IDD of hnRNP A18 upon phosphorylation by CK2 and GSK-3β to provide critical insight into the structure and function of IDDs.

Exploiting Pretreatment Nodal Involvement to Identify Drivers of Chemoradiation Resistance in Locally Advanced Cervical Cancer

<h3>Purpose/Objective(s)</h3> Following standard-of-care chemoradiation therapy (CRT), 30-50% of locally advanced cervical cancer (LACC) patients experience recurrence. Thus, more sophisticated prognostic markers and targeted therapeutic options are needed. The Cancer Genome Atlas (TCGA) molecular classification of cervical cancer did not identify clinically significant patient strata for overall prognosis or risk of recurrence. Notably, the TCGA study did not collect data on clinical factors such as tumor size and nodal status and thus did not incorporate these into their clustering strategies. We hypothesized that at different stages of initial presentation (no nodal disease, locoregional nodal disease, distant nodal disease), different gene programs may be important for progression and resistance to standard of care therapy. <h3>Materials/Methods</h3> We utilized institutional PET and RNA-seq data (<i>N</i>=99) to separate the gene expression profiles of patients with LACC into three groups (Negative nodal (NN) disease, pelvic/locoregional nodal (LRN) disease, aortic/distant (DN) nodal disease) as assessed by pre-treatment PET imaging. Next, to globally assess gene contribution to treatment resistance, we constructed an Rscript to perform univariate Kaplan Meier (KM) analysis for disease recurrence, for all genes in each pre-treatment PET nodal group. FDR multiple hypothesis correction was employed. Lastly, the lists of genes significantly associated with treatment resistance in each PET nodal group were compared. Pathway Enrichment Analysis (PAE) was performed using EnrichR. <h3>Results</h3> When combining all genes statistically associated with recurrence following CRT in at least one nodal cohort, only 7% were shared between two groups (3% LRN-NN, 2% DN-NN, 2% LRN-DN), and <1% was shared by all three groups. Thus, the genes shown to be significant predictors of recurrence following standard of care chemoradiation therapy are largely different depending on the degree of spread of cervical cancer at the time of initiation of therapy (21% NN, 24% DN, 49% LRN). Concordantly, PAE shows striking differences in pathways associated with recurrence. The Immune Response, (p=3.9*10⁻⁶), Epithelial to Mesenchymal Transition (p=1.6*10⁻¹⁰), and MYC/E2F targets (p=5.3*10⁻⁸) were most enriched in NN, LRN, and DN, respectively. Lastly, expression levels of the top 5 genes associated with recurrence within nodal groups identifies high and low risk clusters of patients' prognostic for recurrence (p=0.00304, p=0.00137, and p=0.019 for NN, LRN, DN, respectively). <h3>Conclusion</h3> Our novel findings suggest that different genes drive cervical cancer progression following CRT depending on the degree of nodal spread at presentation. Utilizing these gene clusters identifies high and low risk groups of patients in each presenting nodal strata.