Coatomer subunit α (COPA) syndrome is a rare, autosomal dominant inborn error of immunity driven by dysregulated type I interferon signalling. It typically presents in early childhood with interstitial lung disease, arthritis, and systemic inflammation. Current treatments are largely extrapolated from adult protocols, and disease management remains challenging. Recent evidence suggests that Janus kinase inhibitors (JAKi), which block interferon signalling, may offer a targeted therapeutic approach. We report on two paediatric patients with genetically confirmed COPA syndrome who demonstrated sustained clinical improvement following the initiation of baricitinib, a JAKi. Both cases exhibited early onset interstitial lung disease and systemic inflammation. Genetic testing identified pathogenic variants in the COPA gene. Conventional immunosuppressants provided only partial and temporary relief with disease flares occurring when immunosuppression was tapered. Baricitinib was initiated, and both patients showed a rapid clinical response, improved pulmonary function, reduced systemic inflammation, lung parenchymal radiographic stabilisation, and improvement of interstitial lung disease symptoms. Baricitinib was well tolerated overall and enabled the tapering and discontinuation of corticosteroids and other immunosuppressants. These cases align with the limited existing literature showing the efficacy of JAKi in COPA syndrome and further support the role of precision medicine targeting type I interferonopathies. While long-term safety data remain limited, early findings suggest that JAK inhibition represents a promising therapeutic strategy in COPA syndrome. Careful monitoring for viral reactivation and infection is essential. Larger studies and longer follow-up are needed to validate these results and to optimise the management of this complex paediatric autoimmune disease.

Quercetin is a dietary flavonoid found in apples, onions, and berries. Previous in vitro and in vivo studies have identified its pro-apoptotic, anti-angiogenic, antioxidant, and anti-inflammatory activities, in addition to its ability to sensitize cancer cells to traditional chemotherapies. However, there has not yet been a comprehensive evaluation of its anticancer properties on a large panel of cancer cell lines. We hypothesized that quercetin exhibits varied anticancer effects depending on the particular cell line and its tissue of origin. For cell survival assessments, 77 cancer cell lines from the NCI60 dataset were seeded into 96-well plates and incubated for 24 hours. DMSO control or quercetin was then added at 5 ten-fold concentrations to a maximum of 100 µM. 48 hours later, the cells were fixed and stained, and cell viability measured with CellTiter-Glo. Next, for transcriptomic profiling, the L1000 assay from The Broad Institute was used. For these experiments, 10 µM of quercetin was added to 9 core cell lines seeded in 384-well plates. Then, the Affymetrix GeneChip HG-U133 Plus 2.0 Array was used to generate a gene expression signature for quercetin, which was then compared with signatures from other compound and genetic perturbations. Connectivity scores were used to classify the L1000 signatures according to similarity to the gene expression changes produced by quercetin. The data for cell survival and transcriptomic profiling were extracted from PharmacoDB and Clue.io, respectively. The most sensitive cell line to quercetin was the glioblastoma cell line U-87/H.Fine (IC50=5.03 µM), and the least sensitive was the renal cell carcinoma cell line TK-10 (IC50 = 5780.61 µM). Overall, quercetin was most potent against myeloid and prostate cancers and least potent against ovary and fallopian tubes cancers. As indicated by high median tau scores, quercetin administration induced gene expression changes similar to NFKB2, PTK2 and EPCAM knock-downs, which are associated with reductions in inflammation, cell proliferation, and cancer growth and progression. The cellular effects of quercetin were analogous to the administration of other anticancer compounds such as CP466722, rhamnetin, and piceatannol. CP46672 can sensitize tumors to ionizing radiation, rhamnetin acts as an antioxidant, and piceatannol is a naturally occurring anticancer agent. This study provides a comprehensive in vitro examination of the impact of quercetin on the viability and gene expression profiles of cancer cells of various origins. Further research on the molecular changes induced by quercetin in animal models and human subjects would help inform the applicability of quercetin in human cancers and guide its use in cancer prevention and treatment. Adriana Goraieb, Adin Aggarwal, Kenneth W. Yip. Anticancer mechanisms of quercetin revealed via a systems biology approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6760.

Table S1: Patient Radiotherapy Information and Multivariate Analysis.

Tissue fibrosis is a common consequence of many different acute and chronic injuries, which severely impairs the function of affected organs. A significant challenge is the lack of effective strategies to treat fibrotic disorders. The metabolic dysregulation underlying fibrosis may be reversed by the small molecule caffeic acid phenethyl ester (CAPE), but there are limitations which prevent its clinical use. Following the identification of caffeic acid derivative 1 from an in-house library screen, we performed structure-activity relationship studies which led to the discovery of novel small molecule inhibitors of extracellular matrix (ECM) collagen secretion. The small molecules increased PPARG and CD36 expression (markers of fatty acid metabolism), suggesting a mechanism of action involving a metabolic shift from fibrotic-to-normal state. The compounds identified in this study provide a foundation for further development towards a novel, first-in-class therapeutic agent for fibrosis.

Vitamin D3 (also known as cholecalciferol) is an essential nutrient and hormone that is obtained through both sunlight-mediated cutaneous synthesis and direct dietary intake. Vitamin D3 functions to inhibit tumor cell growth by arresting the cell cycle at G0/G1 phases, increasing apoptosis, and reducing nuclear β-catenin levels to prevent the formation of cancer-promoting T-cell factor complexes. This study identified the cancer cell lines most responsive to in vitro treatment with vitamin D3, as well as the compounds and genetic perturbations that are most similar to treatment with vitamin D3. For cell line response analyses, the NCI60 dataset from the US National Cancer Institute was used. Human cancer cell lines were incubated for 24 hours and treated with cholecalciferol for 48 hours at 5 different ten-fold serial dilution concentrations. Sulforhodamine B was used to measure cell survival. To identify genetic and compound perturbations similar to vitamin D3, data was obtained from the L1000 gene expression assay and CLUE. In this assay, mRNA transcript abundance was measured in 978 human landmark genes and inferred for over 11, 000 others to compare gene expression levels to those induced by vitamin D3. The least sensitive cell line to vitamin D3 was the multiple myeloma cell line RPMI-8226 (IC50=56.3 µM), and the most sensitive was the colorectal cancer cell line HCT-116 (IC50 = 18.2 µM). Overall, vitamin D3 was most potent against lymphoid, renal, and melanoma cancer cell lines, and least potent against myeloma cell lines. As indicated by high median tau scores, administration of vitamin D3 induced gene expression changes similar to OTX2 and ZNF114 knockdowns; OTX2 is a transcription factor associated with neurological organ development while ZNF114 is a zinc-finger protein important for transcription regulation, and both have a biological network connection to vitamin D3. Vitamin D3 treatment has similar genetic effects to known therapeutic compounds like VX-702, nelfinavir, and calcipotriol. This study provides a comprehensive in vitro examination of the cancer cell lines most impacted by treatment with vitamin D3. Further research will help better inform the use of vitamin D3 in nutrition plans and guide its use in cancer prevention and treatment. Ashiana M. Sunderji, Shivangi Roy, Adin Aggarwal, Kenneth W. Yip. Systems biology analysis of vitamin D3 reveals new anticancer properties [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6831.

PURPOSE:Radiation-induced fibrosis (RIF) is a significant long-term complication of radiotherapy, affecting many cancer patients months to years after treatment. Characterized by progressive tissue stiffening, loss of elasticity, and impaired organ function, RIF can deleteriously impact a patient's quality of life. Commonly affected sites include the skin, lung, heart, and kidney. Advances in radiotherapy techniques, such as intensity-modulated radiation therapy (IMRT), stereotactic body radiotherapy (SBRT), and image-guided radiotherapy (IGRT), have improved the precision of radiation delivery, reducing acute damage to healthy tissues; RIF however, remains a prevalent complication despite these technological advancements. This review explores the underlying cellular and molecular mechanisms of RIF, emphasizing fibroblast proliferation, myofibroblast activation, and excessive extracellular matrix (ECM) deposition in its progression. Additionally, this review highlights in vitro and in vivo models that are instrumental in studying RIF and evaluates current therapeutic strategies aimed at mitigating RIF. CONCLUSION:Radiation-induced fibrosis continues to affect a considerable number of patients due to the chronic nature of the fibrotic processes, driven by sustained fibroblast activation, ECM accumulation, and inflammatory responses. Newly developed approaches, such as stem cell-based therapies, TGF-β inhibitors, and molecular interventions aimed at ECM regulation, offer promising avenues for mitigating or reversing RIF. Additionally, integrating computational models into clinical practice could enhance personalized treatment planning, enabling better prediction and prevention of RIF in patients. Addressing these challenges is critical for improving the quality of life of patients affected by RIF and improving their outcomes, particularly with the growing population of long-term cancer survivors in the world.

Radiation therapy (RT) is administered to ∼50% of all cancer patients yet can lead to toxicities such as radiation-induced fibrosis and/or lymphedema. Detailed understanding of the underlying pathogenesis of these post-RT complications remains to be elucidated. One potential factor might relate to clonal hematopoiesis of indeterminate potential (CHIP); an age-related clonal expansion of hematopoietic stem cells (HSCs) harboring characteristic somatic mutations (e.g., Tet2). CHIP is implicated in cardiovascular disease and leukemia progression, as well as several chronic fibrosis states. The current hypothesis is that breast cancer patients with CHIP at the time of radiation therapy (RT) are at higher risk of radiation-induced toxicities. To investigate this hypothesis, we utilized patient samples and mouse models with Tet2 mutations. Blood samples were collected from breast cancer patients undergoing adjuvant RT prior to treatment. DNA was extracted, and CHIP mutations were identified using next-generation sequencing. We then examined the association between CHIP status and clinical data related to radiation-induced fibrosis and/or lymphedema. In addition, a CHIP mouse model was generated by transplanting myelo-ablated mice with HSCs heterozygous for Tet2 null mutations. Mouse hindlimbs were irradiated at 20 Gy to induce fibrosis, evaluated through joint angle contracture (measured weekly), dermal thickness, and collagen. Smaller joint angles indicated increasing contracture/fibrosis. At 16 weeks post-RT, skin samples were collected to assay dermal thickness and collagen deposition. Amongst 138 breast cancer patients, 28 had CHIP mutations at baseline, of which only 1 patient developed radiation-induced fibrosis/lymphedema. Amongst the remaining 110 patients without CHIP, 24 developed fibrosis/lymphedema, demonstrating a significant association between toxicity outcomes with no-CHIP patients (Fisher’s exact test, p<0.05). This was reflected in the in vivo model, wherein CHIP-positive mice (carrying Tet2 mutant HSCs, n=9) exhibited reduced fibrosis, thinner skin (27% decrease), and less collagen (51% decrease) post-RT, compared to control mice with wild-type Tet2 HSCs (n=9). Our findings disproved the null hypothesis and unexpectedly demonstrated that breast cancer patients with CHIP displayed a lower risk of fibrosis/lymphedema compared to patients without CHIP. These findings need to be validated in other clinical cohorts and additional pre-clinical models to further understand the apparent protective effects of CHIP with development of radiation fibrosis. Tirthankar Ray, Madison Hossack, Pierre-Antoine Bissey, Justin Williams, Wei Shi, Sagi Abelson, Steven Chan, Kenneth W. Yip, Fei-Fei Liu. Clonal hematopoiesis of indeterminate potential may protect against radiation-induced toxicities in breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4687.

Approximately 50% of cancer patients are treated with radiation therapy; ∼60% of whom will experience significant radiation-induced fibrosis (RF) caused by excessive collagen accumulation. RF reduces tissue function and flexibility, increasing surgical complications during disease recurrence. Accurate and reproducible in vivo models are required to elucidate the mechanisms of this condition and to facilitate the development of effective therapeutic strategies. Although there have been comprehensive studies related to model development for other forms of fibrosis, the characterization and comparison of murine RF models remains limited in the literature. This work aims to assess and contrast C3H and C57Bl/6 mice as models for RF. RF was assessed in two mouse strains, C3H and C57BL/6. Mice were irradiated using the Precision SmART+ irradiator. C3H mice received 40 Gy, while C57BL/6 mice received 10 Gy, 15 Gy, or 20 Gy. Following irradiation, the tibiofemoral joint angle was measured using X-ray images acquired weekly. At 4-, 8-, 12-, and 16-weeks post-irradiation, skin samples were collected, and collagen deposition was analyzed. Collagen was measured using: a) picrosirius red staining and polarized light microscopy; b) pro-collagen ELISA; and c) the hydroxyproline assay. The pattern of RF development was unique to each mouse strain. In C3H mice, three phases were observed: an acute response with increased limb stiffness, a recovery phase with reduced stiffness, and a fibrotic phase correlating with collagen accumulation, evidenced by histology and biochemical analyses. At 16 weeks post-irradiation, collagen deposition was observed to increase by 1.48- (p<0.05), 1.62- (p<0.05), and 1.32-fold via picrosirius, ELISA, and hydroxyproline assays, respectively. In C57BL/6 mice, 20 Gy induced RF without a recovery phase, 10 Gy did not induce fibrosis, while 15 Gy induced RF in 50% of cases, revealing unexpected variability despite genetic uniformity. At 16 weeks post-20 Gy irradiation, collagen deposition was observed to increase by 4.2- (p<0.01), 1.55-, and 2.69-fold (p<0.05) via picrosirius, ELISA, and hydroxyproline assays, respectively. Skin thickness following irradiation increased by 1.66-fold (p<0.05) in C3H mice, and by 2.35-fold (p<0.01) in C57BL/6 mice, compared to non-irradiated controls. Measuring the tibiofemoral joint angle using weekly X-ray is non-invasive approach in assessing murine RF. The data reveal mouse strain-specific differences in RF. C3H mice may more accurately recapitulate the development of RF in humans. However, due to many more gain- and loss-of-function genetic models, C57BL/6 mice remain essential for investigating RF. Pierre-Antoine Bissey, Justin Williams, Wei Shi, Madison Hossack, Ken W. Yip, Fei-Fei Liu. A comparative study of C3H and C57Bl/6 murine models of radiation-induced fibrosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6326.

Glucosinolates are organic compounds found in high concentrations within cruciferous vegetables. Plant tissue damage initiates the release of myrosinase, which breaks down glucosinolates into the biologically active compounds indole-3-carbinol (I3C) and sulforaphane. In vitro studies have suggested that I3C and sulforaphane can mitigate cancer progression via hormone regulation and antioxidant mechanisms, respectively. However, there has yet to be a systematic analysis of these two glucosinolates against a wide range of human cancer cell lines. We hypothesized that I3C would differentially affect cancer cell growth depending on cell line of origin, and that sulforaphane would induce a genetic signature similar to compounds and genetic perturbations with anticancer properties. Cell survival data was obtained from the NCI-60 human tumor cell lines screen. 59 tumor cell lines were seeded into 96-well microtiter plates. 100 µL of 5 different I3C 10-fold dilutions (0.01 - 100 µM) were added to the wells and incubated for 48 hours. After staining with sulforhodamine, absorbance was measured, and percent growth was calculated relative to the no-drug control and number of cells at baseline. Genetic profiling data was obtained from the Broad Institute’s L1000 assay. Sulforaphane was added to 9 cell lines in 384-well plates. mRNA was extracted and expression levels of 978 landmark genes were measured. This provided a transcriptomic signature that was compared to signatures from other compounds and genetic perturbations. A Connectivity Score was then developed to measure the similarity of each signature to the one induced by sulforaphane. I3C was most potent in the KM12 cell line (IC50 = 14.9 µM; colon cancer) where it inhibited ∼90% of growth at a 100 µM concentration. Cancers of the colon and skin were most sensitive to I3C (median IC50 values of 28.3 µM and 36.1 µM, respectively), while breast and myeloid tissue cancers were least sensitive. Gene expression induced by sulforaphane was most similar to that induced by isoliquiritigenin, a flavonoid compound that exhibits antiproliferative and anti-inflammatory effects. Transcriptomic changes caused by sulforaphane were highly similar to NFE2L2 overexpression (tau score = +97.87), and SLC7A5 knockdown (tau score = +97.16). The NFE2L2 gene encodes a transcription factor (NRF2) that regulates antioxidant enzymes, and the SLC7A5 gene encodes an amino acid transporter that is often overexpressed in many cancer types. Glucosinolates, specifically I3C and sulforaphane, demonstrate anticancer properties by inducing cell survival and gene expression changes in several cancer cell lines. In the future, identifying the genes and proteins targeted by I3C, as well as the pathway through which sulforaphane acts, will be crucial to further elucidate the effects of glucosinolates on cancer. Katerina Carrozzi, Adin Aggarwal, Kenneth W. Yip. Glucosinolates selectively target cancer cell types and exhibit antineoplastic effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4339.

Table S1: Patient Radiotherapy Information and Multivariate Analysis. Figure S1: Workflow of evaluation of T cell receptor (TCR) rearrangement and ESTIMATE immune score from RNA-sequencing data, and the expression of tumor-infiltrating lymphocytes (TILs) in NPC patients. Figure S2: The correlation of ESTIMATE immune score with rearranged TCR reads and CD8+ TILs. Figure S3: The correlation of CIBERSORT and TIMER 2.0 with rearranged TCR reads and CD8+ TILs. Figure S4: The distribution of TILs in NPC samples. Figure S5: Clinical impact of CD8+ TILs in the combined cohort of NPC patients.

Abstract Introduction: Epigallocatechin gallate (EGCG) is a polyphenol present in green tea that is known for its anti-oxidant, anti-inflammatory, anti-angiogenic, pro-apoptotic, and anti-cancer properties. However, EGCG has not been systematically evaluated on a large panel of cancer cell lines. We hypothesized that EGCG exhibits varying effects on specific cancer cell genetic backgrounds and types. This study aimed to provide a comprehensive analysis of EGCG on both cell viability and gene expression profiles. Methods: 750 cancer cell lines were cultured and seeded in 1536-well plates for cell viability studies (by the CTD2 Center at the Broad Institute). After 24 h, cells were treated with 16 two-fold serial dilutions of EGCG. After 72 h, cell viability was assayed using CellTiter-Glo. For gene expression profiling (with the Broad Institute and NIH), 8 cell lines were treated with 10 µM EGCG for 6 and 24 h, and transcriptomes were assessed using Affymetrix GeneChip Human Genome U133 Plus 2.0 Arrays. The CMap large-scale transcriptome dataset was used for comparisons. Results: EGCG had the highest potency in the non-Hodgkins B-cell lymphoma cell line DoHH2 (IC50=0.42 µM), the myeloid leukemia cell line Ku812 (IC50=3.76 µM), and the multiple myeloma cell line KMS-28BM (IC50=4.57 µM). Tissue-of-origin analysis showed that EGCG was most potent against lymphoid cancers, and least potent against prostate cancers. Bioinformatics-based analyses found that EGCG-treated cells resembled cells overexpressing CHEK2, DDP4, CBLC, ZNF350, and TRAF6, and cells underexpressing TRIM16. EGCG-induced gene expression resembled lapatinib and sulfasalazine treatment. Conclusions: This study provides a comprehensive examination of the differential impact of EGCG treatment on cancer cells. Subsequent research on the timing and duration of the changes, along with pathway enrichment analyses, will potentially guide the translational potential of EGCG and its chemical analogs in personalized cancer treatment approaches. Citation Format: Aria Panchal, Jacqueline H. Law, Clement Lo, Kenneth W. Yip. Anticancer mechanisms of epigallocatechin gallate revealed via cellular and molecular profiling [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 521.

Abstract Introduction: Curcumin, the active ingredient of turmeric, generates reactive oxygen species, increases p53 expression, inhibits NFⲕB signaling, and can induce apoptosis. Despite these potential anticancer effects, there has not yet been a systematic analysis of the effects of curcumin on a large panel of cancer cell lines. We hypothesized that curcumin has differential effects based on the specific cell line and its tissue of origin. This study assessed curcumin’s anticancer properties via cell survival assays and gene expression analyses. Methods: Cell survival assays were performed by the CTD2 Centre at the Broad Institute. 860 human cancer cell lines were seeded into 1536-well plates, and 24 hours after plating, either DMSO control or curcumin was added to the wells at 16 different concentrations by serial dilution. 72 hours later, cellular ATP levels were measured with CellTiter-Glo as an indication of cell survival. For gene expression analysis, the L1000 assay from the Broad Institute was used. 9 core cell lines were treated with 10 µM of curcumin in 384-well plates. After 6 and 24 hours of incubation, a gene expression signature was identified with the Affymetrix GeneChip HG-U133 Plus 2.0 Array. This signature was compared against signatures from other small molecule and genetic perturbations contained in the CMap database. Afterwards, connectivity scores (incorporating an enrichment score, nominal p-value, and false discovery rate) were used to rank the L1000 signatures by similarity to the signature induced by curcumin. Results: Curcumin was most potent against SEM (IC50 = 1.00 µM; B-acute lymphoblastic leukemia), RPMI-8226 (IC50= 1.62 µM; myeloma), and SCC-4 (IC50 = 1.81 µM; squamous cell carcinoma) cell lines. Lymphoid and peripheral nervous system cancers were generally the most sensitive to curcumin, whereas prostate cancers were the least sensitive. Curcumin-induced gene expression changes resembled GPR87 and PRKRA knockdowns, which are associated with reductions in cancer cell survival, chemoresistance, and proliferation in various cancer cell lines. However, these transcriptomic changes also resembled CERS2 downregulation, which is associated with increased cell migration. In vitro, curcumin treatment mimicked 2’,5’-dideoxyadenosine, BMS-299897, and eudesmic acid administration. Conclusion: Curcumin induces changes in cell survival and gene expression that support its anticancer properties in a variety of cancer cell lines. However, separating these effects from CERS2 downregulation may be an important component of future translational research for curcumin and its analogs. Citation Format: Adin Aggarwal, Jacqueline H. Law, Clement Lo, Kenneth W. Yip. A systems biology approach uncovers the anticancer mechanisms of curcumin [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 645.

Abstract Introduction: Radiation therapy is a vital cancer treatment for nearly 50% of patients, yet it poses a significant challenge in the form of radiation-induced fibrosis (RF). RF affects about 60% of those undergoing radiation, leading to painful scarring from excessive collagen accumulation. In head and neck cancer patients, as an example, RF reduces tissue flexibility, causing limited neck mobility and impaired swallowing. Furthermore, approximately 30% of patients also face disease recurrence post-radiation, with RF increasing surgical complications. Our laboratory previously identified the role of reduced fatty acid oxidation in RF (Zhao et al; Nat Metab;1(1):147 2019), offering a promising avenue for future drug development. We described the potential therapeutic benefit of caffeic acid phenethyl ester (CAPE) in countering pro-fibrotic metabolic changes. Our current objective is to develop more effective lead compounds to mitigate RF. Methods: Collagen expression, utilized as a surrogate marker for fibrosis, was quantified using ELISA, Western blot analysis, and RT-qPCR. Metabolic changes were assessed by examining the expression levels of PPARG and CD36. CAPE-like analogs were derived from an in-house collection, a commercially available library, and structure-activity relationship (SAR) analyses. In vivo, RF was induced in the hindlimbs of mice through exposure to 40 Gy, followed by weekly X-ray evaluations of hindlimb angles for 16 weeks. During X-ray assessments, both hindlimbs were subjected to a 5 g weight to enable measurement of the joint angle between the femur and tibia (wherein higher acute angles were a measure of worsening fibrosis). Skin samples from irradiated and unirradiated limbs were collected for the evaluation of collagen deposition, utilizing Picro Sirius staining followed by polarized microscopy. Results: Amongst 200 newly synthesized analogs, approximately one-third successfully reduced collagen secretion in human primary dermal fibroblasts. The most promising compounds exhibited a half-maximum inhibition concentration (IC50) for collagen secretion from 1 to 5µM. They also decreased intracellular collagen production and gene expression, associated with upregulation of PPARG and CD36 transcripts. The joint angle measurements in vivo revealed three distinct phases in the development of dermal fibrosis: 1) inflammatory; 2) recovery; and 3) fibrotic phases. Conclusions: We have successfully synthesized novel and potent analogs of CAPE which were able to reduce secretion of collagen. Furthermore, a novel method for the in vivo assessment of fibrosis was also developed. This innovative approach will facilitate the detailed assessments of future compounds capable of reducing collagen deposition in vivo, thereby providing an innovative avenue to mitigate one of the most pressing late normal tissue toxicities of radiation therapy. Citation Format: Pierre-Antoine Bissey, Leonardo Massignan, Ross Mancini, Wei Shi, Justin Williams, Mark Reed, Kenneth W. Yip, Fei-Fei Liu. Targeting radiation-induced fibrosis: Exploring promising therapeutic avenues and innovative assessment methods [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 6383.

Advances in radiation techniques have enabled the precise delivery of higher doses of radiotherapy to tumours, while sparing surrounding healthy tissues. Consequently, the incidence of radiation toxicities has declined, and will likely continue to improve as radiotherapy further evolves. Nonetheless, ionizing radiation elicits tissue -speci fi c toxicities that gradually develop into radiation -induced fi brosis, a common long-term side -effect of radiotherapy. Radiation fi brosis is characterized by an aberrant wound repair process, which promotes the deposition of extensive scar tissue, clinically manifesting as a loss of elasticity, tissue thickening, and organ -speci fi c functional consequences. In addition to improving the existing technologies and guidelines directing the administration of radiotherapy, understanding the pathogenesis underlying radiation fi brosis is essential for the success of cancer treatments. This review integrates the principles for radiotherapy dosimetry to minimize off -target effects, the tissue -speci fi c clinical manifestations, the key cellular and molecular drivers of radiation fi brosis, and emerging therapeutic opportunities for both prevention and treatment.

Advances in radiation techniques have enabled the precise delivery of higher doses of radiotherapy to tumours, while sparing surrounding healthy tissues. Consequently, the incidence of radiation toxicities has declined, and will likely continue to improve as radiotherapy further evolves. Nonetheless, ionizing radiation elicits tissue-specific toxicities that gradually develop into radiation-induced fibrosis, a common long-term side-effect of radiotherapy. Radiation fibrosis is characterized by an aberrant wound repair process, which promotes the deposition of extensive scar tissue, clinically manifesting as a loss of elasticity, tissue thickening, and organ-specific functional consequences. In addition to improving the existing technologies and guidelines directing the administration of radiotherapy, understanding the pathogenesis underlying radiation fibrosis is essential for the success of cancer treatments. This review integrates the principles for radiotherapy dosimetry to minimize off-target effects, the tissue-specific clinical manifestations, the key cellular and molecular drivers of radiation fibrosis, and emerging therapeutic opportunities for both prevention and treatment.

Introduction: Head and neck cancer (HNC) is the seventh most common cancer worldwide with over 1 million new cases diagnosed each year. Radioresistance is a major cause of both treatment failure and poor prognosis, with locoregional recurrence after radiotherapy occurring in up to 50% of HNC patients. Despite the discovery of individual genes implicated in the ability of HNC cells to develop radioresistance, a systematic evaluation remains to be undertaken. This project is the first to conduct genome-wide CRISPR screens for regulators of radioresistance in HNC, with the aim to identify phenotype-driving genes and pathways. Experimental Procedures: Genome-wide negative selection screens were conducted on HNC cell lines using the Toronto KnockOut (TKO) CRISPR library. Monoclonal Cas9 cell lines were derived from UT-SCC-42A (42A), FaDu, and PE/CA-PJ41 (clone D2) with Cas9 editing efficiencies >80%. Library-transduced cells were treated with the minimum dose of radiation resulting in cessation of cell growth (10 Gy for 42A and FaDu; 8 Gy for PE/CA-PJ41). Genomic DNA from the resultant radioresistant populations was extracted and sequenced on the Illumina NextSeq 500. MAGeCK analysis of read counts identified gene targets of significantly depleted gRNAs after irradiation. Genes that correlated with overall survival (OS) in the TCGA Pan-Cancer database were selected for further investigation. The top candidate genes in the determination of radioresponse were validated using cell proliferation and clonogenic assays. Effects of loss-of-function on cell migration were assessed using scratch wound and transwell migration assays. Results: 117 putative radioresistance genes were identified in the 42A cell line screen; the top ranked hits were MMP14, CD44, CALR, and HHLA1. RNA expression levels of these genes had a significant correlation with OS of radiation-treated HNC patients in the TCGA Pan-Cancer Atlas. Loss-of-function of the candidate genes was confirmed to increase radiosensitivity through live cell imaging in 42A and PJ41 cells, which were further corroborated with clonogenic assays. Downregulation of at least one gene impaired the migration of 42A and PJ41 cells, suggesting a role in cellular invasion and migration. Conclusion: This study will contribute to a deeper understanding of mechanisms of radioresistance in HNC, which continues to be a leading cause of mortality in HNC patients. Future pathway elucidation through transcriptome analysis and functional characterization may reveal additional therapeutic targets that can improve the outcome for HNC patients treated with radiation. Citation Format: Jacqueline H. Law, Pierre-Antoine Bissey, Isabella Kojundzic, Kenneth W. Yip, Fei-Fei Liu. A genome-wide screen for determinants of radioresistance in head and neck cancer [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 3501.

COPYRIGHT © 2022 Zhu, Yang, Law, Khan, Yip and Sun. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. TYPE Editorial PUBLISHED 07 November 2022 DOI 10.3389/fonc.2022.1069947

Abstract Purpose: Tumor-infiltrating lymphocytes (TIL) are immune cell populations found within tumors, critical in the antigen-specific host immune response. In this study, we aimed to elucidate the prognostic significance of CD3+, CD4+, and CD8+ TILs in nasopharyngeal cancer (NPC). Experimental Design: Immune cell infiltration was quantified in NPC samples (n = 50) using RNA-sequencing (RNA-seq) data based on rearranged T-cell receptor (TCR) reads and the Estimation of Stromal and Immune cells in malignant tumors using expression data (ESTIMATE) immune score tool. The differential abundances of TIL subset populations were also characterized through IHC staining of formalin-fixed, paraffin-embedded samples from a training cohort (n = 35), which was a subset of the RNA-seq cohort (n = 50). Results: In the RNA-seq cohort, patients with higher rearranged TCR reads experienced superior 5- and 10-year overall survival (OS; P < 0.001), and disease-free survival (DFS; P < 0.001). Similarly, patients with higher ESTIMATE immune scores experienced superior 5- and 10-year OS (P = 0.024) and DFS (P = 0.007). In the training cohort, high abundances of CD8+ TILs were significantly associated with improved 5- and 10-year OS (P = 0.003) and DFS (P = 0.005). These findings were corroborated in an independent validation cohort (n = 84), and combined analysis of the training and validation cohorts [n = 119 (35+84)], which further demonstrated improved 5- and 10-year survival in terms of locoregional control (P < 0.001) and distant metastasis (P = 0.03). Conclusions: Taken together, our study highlights the prognostic value of CD8+ TILs in NPC, and the potential of future investigations into cellular-based immunotherapies employing CD8+ lymphocytes.

MicroRNAs (miRNAs) are short non-coding RNAs that have been termed “master regulators of the genome” given their significance in post-transcriptional gene regulation, and roles in a multitude of normal and disease processes. In cancer, dysregulation of miRNAs can facilitate disease progression and therapeutic resistance, affecting tumour-suppressors and oncogenes. Nasopharyngeal carcinoma (NPC) is a unique head and neck cancer that is frequently associated with the Epstein-Barr virus (EBV). Advances in miRNA profiling techniques have highlighted the significance of both EBV- and human genome-encoded miRNAs in the pathogenesis of NPC. These miRNAs have been implicated in critical NPC processes such as epithelial-to-mesenchymal transition (EMT) and metastasis, while further promoting chemoresistance and radioresistance. MiRNA signatures derived from profiling data and bioinformatics/statistical analyses may be particularly useful for the diagnosis of NPC, as well as the stratification of patients into clinically relevant groups to guide treatment selection. The identification and characterization of biologically relevant biomarkers will be crucial to improving patient outcomes and achieving greater understanding of the molecular underpinnings of NPC. This review integrates the literature on EBV-miRNAs, cellular miRNAs, and miRNA signatures to guide future research, while acknowledging challenges in the selection and clinical implementation of relevant miRNA biomarkers and signatures.