Promote Cancer Initiation Research Articles

PARD6A promotes lung adenocarcinoma cell proliferation and invasion through Serpina3.

Par6α encoded by PARD6A is a member of the PAR6 family and is reported to promote cancer initiation and progression. PARD6A is frequently upregulated in different types of cancers, but its regulatory role in lung cancer progression is yet to be established. In this study, we analyzed the PARD6A expression in biopsies from lung adenocarcinoma (LUAD) patients, and the survival probability using LUAD tissue microarray (TMA) and online datasets from TCGA and GEO. We conducted in vitro and in vivo assays to assess the role of PARD6A in regulating lung cancer progression, including proliferation, wound healing, transwell, RNA-seq, and subcutaneous tumor mice models. Our findings revealed that PARD6A is highly expressed in cancer tissues from LUAD patients and is associated with poor prognosis in LUAD patients. In vitro assays showed that PARD6A promoted cell proliferation, migration, and invasion. The transcriptome sequencing identified Serpina3 as one of the key downstream molecules of PARD6A. Ectopic expression of Serpina3 rescued impaired proliferation, migration, and invasion in PARD6A-knocking down H1299 cells, whereas silencing Serpina3 impeded enhanced proliferation, migration, and invasion in PARD6A-overexpressing H1975 cells. Our findings suggest that PARD6A promotes lung cancer progression by inducing Serpina3, which may be a promising therapeutic target.

Spatial resolved transcriptomics reveals distinct cross-talk between cancer cells and tumor-associated macrophages in intrahepatic cholangiocarcinoma

BackgroundMultiple studies have shown that tumor-associated macrophages (TAMs) promote cancer initiation and progression. However, the reprogramming of macrophages in the tumor microenvironment (TME) and the cross-talk between TAMs and malignant subclones in intrahepatic cholangiocarcinoma (iCCA) has not been fully characterized, especially in a spatially resolved manner. Deciphering the spatial architecture of variable tissue cellular components in iCCA could contribute to the positional context of gene expression containing information pathological changes and cellular variability.MethodsHere, we applied spatial transcriptomics (ST) and digital spatial profiler (DSP) technologies with tumor sections from patients with iCCA.ResultsThe results reveal that spatial inter- and intra-tumor heterogeneities feature iCCA malignancy, and tumor subclones are mainly driven by physical proximity. Tumor cells with TME components shaped the intra-sectional heterogenetic spatial architecture. Macrophages are the most infiltrated TME component in iCCA. The protein trefoil factor 3 (TFF3) secreted by the malignant subclone can induce macrophages to reprogram to a tumor-promoting state, which in turn contributes to an immune-suppressive environment and boosts tumor progression.ConclusionsIn conclusion, our description of the iCCA ecosystem in a spatially resolved manner provides novel insights into the spatial features and the immune suppressive landscapes of TME for iCCA.

Stem cells, Notch-1 signaling, and oxidative stress: a hellish trio in cancer development and progression within the airways. Is there a role for natural compounds?

Notch-1 signaling plays a crucial role in stem cell maintenance and in repair mechanisms in various mucosal surfaces, including airway mucosa. Persistent injury can induce an aberrant activation of Notch-1 signaling in stem cells leading to an increased risk of cancer initiation and progression. Chronic inflammatory respiratory disorders, including chronic obstructive pulmonary disease (COPD) is associated with both overactivation of Notch-1 signaling and increased lung cancer risk. Increased oxidative stress, also due to cigarette smoke, can further contribute to promote cancer initiation and progression by amplifying inflammatory responses, by activating the Notch-1 signaling, and by blocking regulatory mechanisms that inhibit the growth capacity of stem cells. This review offers a comprehensive overview of the effects of aberrant Notch-1 signaling activation in stem cells and of increased oxidative stress in lung cancer. The putative role of natural compounds with antioxidant properties is also described.

Dual roles of myeloid-derived suppressor cells in various diseases: a review.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that originate from bone marrow stem cells. In pathological conditions, such as autoimmune disorders, allergies, infections, and cancer, normal myelopoiesis is altered to facilitate the formation of MDSCs. MDSCs were first shown to promote cancer initiation and progression by immunosuppression with the assistance of various chemokines and cytokines. Recently, various studies have demonstrated that MDSCs play two distinct roles depending on the physiological and pathological conditions. MDSCs have protective roles in autoimmune disorders (such as uveoretinitis, multiple sclerosis, rheumatoid arthritis, ankylosing spondylitis, type 1 diabetes, autoimmune hepatitis, inflammatory bowel disease, alopecia areata, and systemic lupus erythematosus), allergies, and organ transplantation. However, they play negative roles in infections and various cancers. Several immunosuppressive functions and mechanisms of MDSCs have been determined in different disease conditions. This review comprehensively discusses the associations between MDSCs and various pathological conditions and briefly describes therapeutic approaches.

Liver cancer initiation requires translational activation by an oncofetal regulon involving LIN28 proteins.

It is unknown which posttranscriptional regulatory mechanisms are required for oncogenic competence. Here, we show that the LIN28 family of RNA-binding proteins (RBPs), which facilitate posttranscriptional RNA metabolism within ribonucleoprotein networks, is essential for the initiation of diverse oncotypes of hepatocellular carcinoma (HCC). In HCC models driven by NRASG12V/Tp53, CTNNB1/YAP/Tp53, or AKT/Tp53, mice without Lin28a and Lin28b were markedly impaired in cancer initiation. We biochemically defined an oncofetal regulon of 15 factors connected to LIN28 through direct mRNA and protein interactions. Interestingly, all were RBPs and only 1 of 15 was a Let-7 target. Polysome profiling and reporter assays showed that LIN28B directly increased the translation of 8 of these 15 RBPs. As expected, overexpression of LIN28B and IGFBP1-3 was able to genetically rescue cancer initiation. Using this platform to probe components downstream of LIN28, we found that 8 target RBPs were able to restore NRASG12V/Tp53 cancer formation in Lin28a/Lin28b-deficient mice. Furthermore, these LIN28B targets promote cancer initiation through an increase in protein synthesis. LIN28B, central to an RNP regulon that increases translation of RBPs, is important for tumor initiation in the liver.

M2 macrophage-derived exosomal circTMCO3 acts through miR-515-5p and ITGA8 to enhance malignancy in ovarian cancer

Tumor-associated macrophages of the M2 phenotype promote cancer initiation and progression. Importantly, M2 macrophage-derived exosomes play key roles in the malignancy of cancer cells. Here, we report that circTMCO3 is upregulated in ovarian cancer patients, and its high expression indicates poor survival. M2-derived exosomes promote proliferation, migration, and invasion in ovarian cancer, but these effects are abolished by knockdown of circTMCO3. Furthermore, circTMCO3 functions as a competing endogenous RNA for miR-515-5p to reduce its abundance, thus upregulating ITGA8 in ovarian cancer. miR-515-5p inhibits ovarian cancer malignancy via directly downregulating ITGA8. The decreased oncogenic activity of circTMCO3-silencing exosomes is reversed by miR-515-5p knockdown or ITGA8 overexpression. Exosomal circTMCO3 promotes ovarian cancer progression in nude mice. Thus, M2 macrophage-derived exosomes promote malignancy by delivering circTMCO3 and targeting the miR-515-5p/ITGA8 axis in ovarian cancer. Our findings not only provide mechanistic insights into ovarian cancer progression, but also suggest potential therapeutic targets.

Abstract 1485: Hallmarks of APOBEC3 mutagenesis in normal, pre-leukemic, and myeloproliferative neoplasm cell populations

Abstract Changes in gene expression and subsequent editing patterns of APOBEC3 (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like) cytosine deaminases have been shown to be present in solid tumor cancer evolution, however, the context specificity and mechanisms by which APOBEC3 enzymes promote cancer initiation and progression require further elucidation, especially in the hematopoietic niche. Building on advanced sequencing data of healthy, pre-leukemic, and myeloproliferative neoplasm patient samples, we seek to uncover normal and malignant patterns of APOBEC3C overexpression in hematopoietic cells. Overexpression of lentiviral APOBEC3C and an editase-mutated APOBEC3C in normal cord blood, healthy bone marrow, and MPN patient hematopoietic stem/progenitor cells (HSPCs) allows us to study the effects of deaminase dysregulation in hematopoietic cells. In addition to APOBEC3, we are exploring the upregulation of adenosine deaminase acting on RNA1 (ADAR1), as we have previously shown that these two innate immune deaminases are synchronously upregulated in the high-risk myelofibrosis (MF) stem cell population compared to normal aged bone marrow. These overexpression studies show novel differential gene expression changes, RNA hyper-editing sites, and DNA mutation signatures induced by APOBEC3 mutagenesis, which can be cross-referenced to gene expression and mutation signatures seen in hematopoietic malignancies and solid tumor cancers. In addition to mutation patterns, the regulation of LINE-1 elements has been shown to be another important function of APOBEC3 enzymes. In these studies, we examine the effects of APOBEC3 overexpression on repetitive element expression in hematopoietic cell populations and have found that there are strong cell type and context patterns across the APOBEC3 family and between cell types within the hematopoietic system. We will use this comprehensive study of APOBEC3 deregulation to uncover predictive biomarkers of malignant transformation. Citation Format: Jane Marie Isquith, Thomas Whisenant, Jessica Pham, Ludmil Alexandrov, Catriona Jamieson. Hallmarks of APOBEC3 mutagenesis in normal, pre-leukemic, and myeloproliferative neoplasm cell populations [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 1485.

Neutrophils in cancer: dual roles through intercellular interactions.

Neutrophils, the most abundant immune cells in human blood, play crucial and diverse roles in tumor development. In the tumor microenvironment (TME), cancer cells regulate the recruitment and behaviors of neutrophils, transforming some of them into a pro-tumor phenotype. Pro-tumor neutrophils interact with cancer cells in various ways to promote cancer initiation, growth, and metastasis, while anti-tumor neutrophils interact with cancer cells to induce senescence and death. Neutrophils can also interact with other cells in TME, including T cells, macrophages, stromal cells, etc. to exert anti- or pro-tumor functions. In this review, we will analyze the anti- and pro-tumor intercellular interactions mediated by neutrophils, with a focus on generalizing the mechanisms underlying the interaction of neutrophils with tumor cells and T cells. Furthermore, we will provide an overview of cancer treatment strategies targeting neutrophil-mediated cellular interactions.

Microbiota enterotoxigenic Bacteroides fragilis-secreted BFT-1 promotes breast cancer cell stemness and chemoresistance through its functional receptor NOD1.

Tumor-resident microbiota in breast cancer promotes cancer initiation and malignant progression. However, targeting microbiota to improve the effects of breast cancer therapy has not been investigated in detail. Here, we evaluated the microbiota composition of breast tumors and found that enterotoxigenic Bacteroides fragilis (ETBF) was highly enriched in the tumors of patients who did not respond to taxane-based neoadjuvant chemotherapy. ETBF, albeit at low biomass, secreted the toxic protein BFT-1 to promote breast cancer cell stemness and chemoresistance. Mechanistic studies showed that BFT-1 directly bound to NOD1 and stabilized NOD1 protein. NOD1 was highly expressed on ALDH+ breast cancer stem cells (BCSCs) and cooperated with GAK to phosphorylate NUMB and promote its lysosomal degradation, thereby activating the NOTCH1-HEY1 signaling pathway to increase BCSCs. NOD1 inhibition and ETBF clearance increase the chemosensitivity of breast cancer by impairing BCSCs.

Novel therapeutic strategies targeting myeloid-derived suppressor cell immunosuppressive mechanisms for cancer treatment.

Cancer is the leading cause of death globally superseded only by cardiovascular diseases, and novel strategies to overcome therapeutic resistance against existing cancer treatments are urgently required. Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells with potent immunosuppressive capacity against well-established anti-tumour effectors such as natural killer cells (NK cells) and T cells thereby promoting cancer initiation and progression. Critically, MDSCs are readily identified in almost all tumour types and human cancer patients, and numerous studies in the past decade have recognised their role in contributing to therapeutic resistance against all four pillars of modern cancer treatment, namely surgery, chemotherapy, radiotherapy and immunotherapy. MDSCs suppress anti-tumour immunity through a plethora of mechanisms including the well-characterised arginase 1 (Arg1), inducible nitric oxide synthase (iNOS) and reactive oxygen species (ROS)-mediated pathways, along with several other more recently discovered. MDSCs are largely absent in healthy homeostatic states and predominantly exist in pathological conditions, making them attractive therapeutic targets. However, the lack of specific markers identified for MDSCs to date greatly hindered therapeutic development, and currently there are no clinically approved drugs that specifically target MDSCs. Methods to deplete MDSCs clinically and inhibit their immunosuppressive function will be crucial in advancing cancer treatment and to overcome treatment resistance. This review provides a detailed overview of the current understandings behind the mechanisms of MDSC-mediated suppression of anti-tumour immunity, and discusses potential strategies to target MDSC immunosuppressive mechanisms to overcome therapeutic resistance.

NonB Cell-Derived Immunoglobulins in Intestinal Tract.

Intestinal epithelium constitutes a barrier to the unrestricted movement of pathogens, and other detrimental substances from the external world (gut lumen) into the interstitial environment. Intestinal epithelial cells obstruct harmful substances passing through the epithelium as a physical and chemical barrier; Moreover, the epithelial cells can express Toll-like receptors (TLRs) and cytokines to exert innate immune function. In addition, high levels of immunoglobulin A (IgA) and other antibodies exist in the intestinal mucosa, maintaining intestinal immune homeostasis in conjunction with intestinal probiotics. Traditionally, these antibodies have been deemed to be secreted by submucosal plasma cells. Nonetheless, in recent years, it has been demonstrated that intestinal epithelial cells produce a substantial amount of Igs, especially IgA or free Ig light chains, which are involved in intestinal immune homeostasis and the survival of normal epithelial cells. Furthermore, mounting evidence affirms that many human carcinoma cells, including colorectal cancer (CRC), can overexpress Igs, particularly IgG. Cancer-derived Igs exhibit a unique V(D)J rearrangement pattern distinct from B cell-derived Ig; moreover, this cancer cell-derived IgG also has a unique sialic acid modification on the 162 site of CH1 domain (SIA-IgG). The SIA-IgG plays a crucial role in promoting cancer initiation, progression, metastasis, and tumour immune escape. Simultaneously, CRC cells can also express free Ig light chains, which promote colitis, colitis-associated colon carcinogenesis, and CRC progression. Therefore, Igs expressed by CRC cells could be a potential target for diagnosing and preventing the transformation of inflammation into cancer, as well as treating CRC.

DPP9 Stabilizes NRF2 to Suppress Ferroptosis and Induce Sorafenib Resistance in Clear Cell Renal Cell Carcinoma.

DPP9 overcomes oxidative stress and suppresses ferroptosis in ccRCC by binding to KEAP1 and promoting NRF2 stability, which drives tumor development and sorafenib resistance.

Abstract PO-088: TRPV1+ sensory neurons provide a tumor supportive environment through recruitment of MDSCs

Abstract Cancer neuroscience has become an increasing area of focus in cancer research as our understanding of the presence and function of intra-tumoral neurons continues to emerge, with studies indicating that tumor infiltrating neurons impact components of the tumor microenvironment promoting cancer initiation and progression. We have previously published that head and neck squamous cell carcinomas are innervated. We now focus on defining the contributions of intra-tumoral neurons to disease progression in HNSCC. Tumor implantation in a double transgenic mouse (TRPV1cre::DTAfl/wt) lacking the cancer-recruited TRPV1-expressing sensory neurons allows us to study the consequences of this neuronal loss on the local immune response. Importantly, comparison between wild type and TRPV1cre::DTAfl/wt mice demonstrated no significant immune population differences at baseline. Thus, wildtype and TRPV1cre::DTAfl/wt mice were orthotopically implanted with tumor and tumoral immune cell populations identified by flow cytometry. Cytokine and peptide concentrations were measured by ELISA. In vitro assays were preformed to molecularly define the neuronal signals and immune responses. Here, co-culture experiments of immortalized head and neck cancer cell lines and dorsal root ganglia (DRG) from wildtype mice were used to model tumor innervation in vitro. The media generated was applied to bone marrow derived (BMD) immune cells and the impact on immune cell phenotypes assayed by flow cytometry. Our orthotopic model demonstrates that the presence of HNSCC promotes a nerve-injury phenotype in ipsilateral trigeminal ganglion, demonstrated by increased expression of activating transcription factor 3 (ATF3). Utilizing a 12-color flow cytometry panel we show that tumors from TRPV1cre::DTAfl/wt mice harbor a significantly smaller population of myeloid derived suppressor cells (MDSCs) compared to those from wildtype animals. Further analysis demonstrates this population shift occurs primarily due to a decrease in the CD11b+ Ly6G+/Ly6Clo population. In vitro studies using BMD immune cells show that tumor cell-DRG co-culture conditioned media induces an expansion of the MDSC population compared to condition media from DRG or tumor cells alone. Additionally, transwell migration assays show that co-culture condition media also increases cellular migration compared to condition media from DRG or tumor cells alone. Moreover, ELISA analysis indicates that co-culture of tumor cells and DRG increases neuronal release of the neuropeptide, Substance P (SP), potentiating IL-6 release from tumor cells. In summary, we identified several mechanisms by which TRPV1-expressing intra-tumoral neurons promote MDSC infiltration at the tumor bed and promote disease progression. These mechanisms define targets for therapeutic intervention including SP and IL-6 signaling. Future studies will test the clinical utility of these targets. Together, our data suggests that intra-tumoral neurons contribute to cancer progression through generation of a tumor supportive microenvironment. Citation Format: Anthony C. Restaino, Maryam Ahmadi, Sebastien Talbot, Paola D. Vermeer. TRPV1+ sensory neurons provide a tumor supportive environment through recruitment of MDSCs [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-088.

RUNX3 inactivates oncogenic MYC through disruption of MYC/MAX complex and subsequent recruitment of GSK3β-FBXW7 cascade

MYC is one of the most commonly dysregulated proto-oncogenes in cancer. MYC promotes cancer initiation and maintenance by regulating multiple biological processes, such as proliferation and stem cell function. Here, we show that developmental regulator RUNX3 targets MYC protein for rapid degradation through the glycogen synthase kinase-3 beta-F-box/WD repeat-containing protein 7 (GSK3β-FBXW7) proteolytic pathway. The evolutionarily conserved Runt domain of RUNX3 interacts directly with the basic helix–loop–helix leucine zipper of MYC, resulting in the disruption of MYC/MAX and MYC/MIZ-1 interactions, enhanced GSK3β-mediated phosphorylation of MYC protein at threonine-58 and its subsequent degradation via the ubiquitin-proteasomal pathway. We therefore uncover a previously unknown mode of MYC destabilization by RUNX3 and provide an explanation as to why RUNX3 inhibits early-stage cancer development in gastrointestinal and lung mouse cancer models.

The neural addiction of cancer.

The recently uncovered key role of the peripheral and central nervous systems in controlling tumorigenesis and metastasis has opened a new area of research to identify innovative approaches against cancer. Although the 'neural addiction' of cancer is only partially understood, in this Perspective we discuss the current knowledge and perspectives on peripheral and central nerve circuitries and brain areas that can support tumorigenesis and metastasis and the possible reciprocal influence that the brain and peripheral tumours exert on one another. Tumours can build up local autonomic and sensory nerve networks and are able to develop a long-distance relationship with the brain through circulating adipokines, inflammatory cytokines, neurotrophic factors or afferent nerve inputs, to promote cancer initiation, growth and dissemination. In turn, the central nervous system can affect tumour development and metastasis through the activation or dysregulation of specific central neural areas or circuits, as well as neuroendocrine, neuroimmune or neurovascular systems. Studying neural circuitries in the brain and tumours, as well as understanding how the brain communicates with the tumour or how intratumour nerves interplay with the tumour microenvironment, can reveal unrecognized mechanisms that promote cancer development and progression and open up opportunities for the development of novel therapeutic strategies. Targeting the dysregulated peripheral and central nervous systems might represent a novel strategy for next-generation cancer treatment that could, in part, be achieved through the repurposing of neuropsychiatric drugs in oncology.

Abstract 123: Molecular mechanisms of RNA and DNA editing in leukemic transformation of hemopoietic stem and progenitor cells

Abstract Dysregulation of inflammatory cytokine responsive APOBEC3 cytosine deaminases has been shown to be a contributing factor in cancer evolution, presenting as gene expression changes and inclusion of distinct C-to-T mutation patterns. However, the context specificity and mechanisms by which APOBEC3 enzymes promote cancer initiation and progression require further elucidation. Lentiviral overexpression of APOBEC3C and an editase deficient APOBEC3C mutant in healthy cord blood, bone marrow and MPN patient hematopoietic stem/progenitor cells (HSPCs) allows us to study the effects of innate immune deaminase dysregulation in the hematopoietic niche. We are focusing on the upregulation of APOBEC3C and adenosine deaminase acting on RNA1 (ADAR1), as we have previously shown them to be contemporaneously upregulated in the high-risk myelofibrosis (MF) stem cell population compared to normal aged bone marrow. We can compare these novel differential gene expression changes, RNA hyper-editing sites, and DNA mutation signatures induced by APOBEC3 mutagenesis to abnormalities seen in both hematopoietic malignancies and solid tumor cancers. Gene set enrichment analysis (GSEA) performed on this dataset has exposed numerous deregulated pathways brought on by exaggerated levels of APOBEC3, including changes in splicing pathways. To further investigate the complex relationship between splicing and deaminase deregulation, we treated myeloproliferative neoplasm patient samples and normal HPSCs with Rebecsinib (also known as 17S-FD-895), a pharmacologically stable, potent, and selective small molecule splicing modulator, which interestingly caused significant downregulation of APOBEC3C. We will continue to investigate these findings as a potential target to correct the dysregulation seen in MPN progression. Consequently, we aim to use these findings to identify predictive biomarkers and druggable targets of leukemic initiation progression. Citation Format: Jane Marie Isquith, Jessica Pham, Thomas Whisenant, Larisa Balaian, Catriona Jamieson. Molecular mechanisms of RNA and DNA editing in leukemic transformation of hemopoietic stem and progenitor cells [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 123.

Abstract 482: Discovery of a novel and oral CHK1 inhibitor for the treatment of solid tumors

Abstract Genomic instability is a key hallmark of cancer that arises owing to defects in the DNA damage response (DDR), a complex network of genes responsible for sensing and responding to specific types of DNA damage, in which numerous interdependent signaling pathways and machineries are involved. Although deficiencies in these responses promote cancer initiation and progression via accumulated mutations, they also provide targetable vulnerabilities relatively specific to cancer cells that can be exploited with the use of DDR inhibitors. The cell cycle checkpoint kinases CHK1 and CHK2 are prime targets of DDR inhibitors, working with the key upstream regulators of the DDR signaling cascade, ATR and ATM, respectively, to halt cell cycle progression and initiate DNA repair via distinct but overlapping pathways. CHK1 and/or CHK2 inhibitors have been developed in the past. However, many of them were discontinued before making to phase III clinical trials, in most cases owing to severe side effects induced by their non-specific nature. Others are related to their limited clinical efficacy, such as SRA737 which selectively inhibits CHK1 but not CHK2. Prexasertib (LY2606368), one of the few agents to undergo clinical trials, specifically inhibits both CHK1 and CHK2, displaying obvious therapeutic effects but also raising inevitable target-associated drug toxicities and a potential compliance issue due to intravenous administration. Given the fact that CHK2 aids in the coordination with CHK1 pathway, adjustment in the intensity of inhibition to both kinases could provide a suitable balance between efficacy and toxicity. Here, we describe the characterization of XS-02, a novel and orally bioavailable CHK1 inhibitor with moderate potency against CHK2, which shows manageable side effects and broad antitumor activity in various solid tumors, including tumors resistant to PARP inhibitors. XS-02 specifically inhibited both CHK1 and CHK2 kinases in different degree with IC50 values of 2 and 282 nM, respectively. In vitro western blotting assays demonstrated that XS-02 decreased phosphorylation of CHK1 completely but partially for CHK2 in OVCAR3 cancer cells. Its potency against proliferation of cells with low nM IC50 was exhibited in multiple cancer cell lines instead of normal cells. Oral dosing of XS-02 as a single agent induced dose-dependent tumor growth inhibition of OVCAR3 and MD-MBA-436 xenografts. Moreover, the combination of XS-02 and olaparib, a PARP inhibitor, accelerated tumor regression, which synergy was confirmed in a patient-derived tumor xenograft animal model of acquired resistance to olaparib, without significant body weight changes. In vivo assays showed that XS-02 was generally well tolerated with an acceptable oral bioavailability across multiple non-clinical species. These results support the agent as a clinical candidate for the treatment of solid tumors. An Investigational New Drug application is planned for 2023. Citation Format: Xin Gao, Yonggang Liao, Qi Li, Ling Yang, Chunyan Zhao, Yanfen Teng, Lei Liu, Wenyuan Qian, Xiaohong Yu. Discovery of a novel and oral CHK1 inhibitor for the treatment of solid tumors [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 482.

Abstract 6211: Development and validation of a novel sSTRIDE-MMR functional assay to study the efficiency of MMR inhibitors

Abstract Mismatch repair (MMR) is considered to be one of the fundamental pathways that guards genome stability and mutations in genes encoding for crucial MMR proteins have been shown to promote cancer initiation. On the other hand, inactivation of MMR components in cancer cells leads to increased neoantigen formation and in consequence, exposes the tumor to the immune system. Therapeutic strategies aiming at inhibiting MMR are thus under development, yet methods reporting on the status of MMR are lacking. We report here the development, optimization and validation of a first MMR-specific functional assay. The assay, which is based on the STRIDE platform technology, detects DNA nicks localized in close proximity to PMS2 protein and can thus be utilized as a direct reporter of inhibition of PSM2 itself or upstream acting proteins. The optimization phase of assay development was performed in U2OS cells. First, in untreated cells we have shown that the sSTRIDE-MMR signals constitute ca. 10-15% of total single-strand DNA breaks foci detected by the classic sSTRIDE assay variant. In a series of technical negative controls we have then shown that the number of false-positive foci never exceeds 10% of the total number of signals, with as little as 2% for the isotype controls. The assay was further validated in HAP1 wild-type and PMS2-KO isogenic cell line pair. As expected, the number of sSTRIDE-MMR foci in the PMS2-KO cell line was significantly lower than in wild-type cells and additionally, no response to the treatment with 6TG, a known inducer of MMR, was detected in PMS2 null cells. Finally, the assay was used to verify the efficiency of first-in-class small-molecule MMR inhibitors. After treatment with the compounds, the number of detected sSTRIDE-MMR foci was significantly lower when compared to the number of foci in untreated cells. In conclusion, we show here that sSTRIDE-MMR is an assay characterized by high specificity, low false positives rate and a high-dynamic range. We believe that sSTRIDE-MMR can prove to be a very useful cell-based solution to study the efficiency of MMR inhibitors. Citation Format: Kamil Solarczyk, Karolina Uznańska, Olga Wójcikowska, Maja Białecka, Agnieszka Waligórska, Szymon Koman, Magdalena Kordon-Kiszala. Development and validation of a novel sSTRIDE-MMR functional assay to study the efficiency of MMR inhibitors. [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 6211.

Single-cell and spatial dissection of precancerous lesions underlying the initiation process of oral squamous cell carcinoma

Precancerous lesions of the oral mucosa, especially those accompanied by moderate to severe dysplasia, contribute to the initiation of oral squamous cell carcinoma (OSCC). However, the cellular compositions and spatial organization of the precancerous stage and how these factors promote human OSCC initiation remain unclear. Here, we built a single-cell transcriptome atlas and a spatial transcriptome map after obtaining data from pairwise human oral mucosal biopsies of 9 individuals consisting of very early-stage OSCC, adjacent precancerous lesions with moderate to severe dysplasia, as well as a matched normal region. An altered epithelial gene-expression profile was identified which favored OSCC initiation. This observation was coupled with distinct fibroblast, monocytic, and regulatory T-cell subclusters involved in reshaping the microenvironment. In particular, a unique immune-inhibitory monocyte subtype and spatial-switching regulation of VEGF signaling were observed surrounding precancerous lesions, concertedly strengthening activities in promoting cancer initiation. Collectively, our work elucidated the cellular landscapes and roles of precancerous lesions underlying OSCC initiation, which is essential for understanding the entire OSCC initiation process and helps inform therapeutic strategies for cancer intervention.

Abstract B007: Remodeling the hyaluronan-rich tumor extracellular matrix using hyaluronidase-expressing genetically engineered MSCs for tumor regression and metastasis prevention in sarcomas

Abstract Sarcomas are a diverse group of rare malignant tumors the develop in the bone and connective tissue and are often aggressive with ~50% of patients developing metastases. Due to their rarity and diversity, there is a great lack of standard therapies resulting in a 65% and 15% 5-year survival rate for regional and metastatic disease, respectively. Tumorigenesis is largely influenced by communication between the tumor microenvironment (TME), cancer cells, and the extracellular matrix (ECM). The ECM promotes cancer initiation and dissemination from the primary tumor and fosters a favorable microenvironment for metastatic colonization of tumor cells. Due to their mesenchymal origin, sarcomas are thought to produce and deposit large quantities of ECM. One component of the tumor-ECM known to promote an aggressive cancer phenotype is hyaluronan (HA). Abundant HA in the TME arises from overactive HA synthases that produce HA or downregulation of hyaluronidases (Hyals) that catalyze HA degradation. HA-rich tumor ECM promote cancer progression by regulating chemokine and cytokine trafficking, increasing hypoxia and interstitial pressure, and limiting delivery of antitumor therapies. In-silico profiling of sarcomas revealed an increase in HA synthase mRNA expression in 50.78% and correlated with 35% decrease in overall patient survival (P = 0.0027), compared to sarcomas with low HA synthase expression. To target this HA-rich tumor ECM, we sought to develop a cell-based approach to locally deliver HA-remodeling enzymes to minimize the drawbacks of systemic therapy and enhance anti-tumor responses. We developed genetically engineered mesenchymal stromal cells (GEMesys) that express Hyal, using mesenchymal stem cells derived from mouse lungs. We hypothesized that the Hyal-GEMesys would home to and degrade the HA-rich ECM preventing cancer progression. The therapeutic efficacy of Hyal-GEMesys was tested in osteosarcoma (F42010) and rhabdomyosarcoma (M-3-9M) syngeneic murine models. Preliminary data demonstrated encouraging efficacy of Hyal-GEMesys in reducing tumor volume in the osteosarcoma (up to 1.4-fold, p=0.0035-0.0172) and rhabdomyosarcoma M-3-9M model (up to 2-fold, P= 0.0428). Further, analysis of osteosarcoma tumors revealed reduced HA (up to 2.3-fold, P=0.0078) and collagen-I content (up to 3-fold, P= 0.0025) indicating our Hyal-GEMesys functionally remodel the ECM in vivo. Changes in ECM in GEMesy treated group were associated with changes in immune infiltration with an increase in CD4+ (P=0.0221) and CD8+ (P=0.0087) T cells and a decrease in IBA1+ (P=0.0144) macrophages. Analysis of rhabdomyosarcoma tumors revealed reduced HA content (up to 2-fold, P-0.00206). Future studies will more deeply explore the mechanisms of ECM-mediated immune changes and impact on metastatic progression. Our cell-based approach to deliver hyaluronidases holds promise of effectively degrading and remodel the HA-rich tumor ECM to enhance anti-tumor responses, prevent metastases and improve clinical outcomes for patients with high-risk sarcoma malignancies. Citation Format: Alice Browne, Miranda Clements, Wei Ju, Daniel Brock, Rosandra Kaplan. Remodeling the hyaluronan-rich tumor extracellular matrix using hyaluronidase-expressing genetically engineered MSCs for tumor regression and metastasis prevention in sarcomas [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B007.