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MT-EGFR expression in HBEC cells is associated with dramatically elevated levels of replication factors.

Supplementary Figure Legends.

Supplementary Figure S3. Pathway annotation for HNSCC super-groups. Each horizontal bar graph represents enriched pathways in different subtypes. The number at the end of bar is the total number of genes in a pathway. Percentage of genes in the dataset that overlap with the pathway are shown in grey colour of the bar. The trend line represents the -log(p-value).

Purpose: Current classification of head and neck squamous cell carcinomas (HNSCC) based on anatomic site and stage fails to capture biologic heterogeneity or adequately inform treatment.Experimental Design: Here, we use gene expression-based consensus clustering, copy number profiling, and human papillomavirus (HPV) status on a clinically homogenous cohort of 134 locoregionally advanced HNSCCs with 44% HPV+ tumors together with additional cohorts, which in total comprise 938 tumors, to identify HNSCC subtypes and discover several subtype-specific, translationally relevant characteristics.Results: We identified five subtypes of HNSCC, including two biologically distinct HPV subtypes. One HPV+ and one HPV− subtype show a prominent immune and mesenchymal phenotype. Prominent tumor infiltration with CD8+ lymphocytes characterizes this inflamed/mesenchymal subtype, independent of HPV status. Compared with other subtypes, the two HPV subtypes show low expression and no copy number events for EGFR/HER ligands. In contrast, the basal subtype is uniquely characterized by a prominent EGFR/HER signaling phenotype, negative HPV-status, as well as strong hypoxic differentiation not seen in other subtypes.Conclusion: Our five-subtype classification provides a comprehensive overview of HPV+ as well as HPV− HNSCC biology with significant translational implications for biomarker development and personalized care for patients with HNSCC. Clin Cancer Res; 21(4); 870–81. ©2014 AACR.

Purpose: Carcinogenesis is an adaptive process between nascent tumor cells and their microenvironment, including the modification of inflammatory responses from antitumorigenic to protumorigenic. Radiation exposure can stimulate inflammatory responses that inhibit or promote carcinogenesis. The purpose of this study is to determine the impact of radiation exposure on lung cancer progression in vivo and assess the relevance of this knowledge to human carcinogenesis.Experimental Design: K-rasLA1 mice were irradiated with various doses and dose regimens and then monitored until death. Microarray analyses were performed using Illumina BeadChips on whole lung tissue 70 days after irradiation with a fractionated or acute dose of radiation and compared with age-matched unirradiated controls. Unique group classifiers were derived by comparative genomic analysis of three experimental cohorts. Survival analyses were performed using principal component analysis and k-means clustering on three lung adenocarcinoma, three breast adenocarcinoma, and two lung squamous carcinoma annotated microarray datasets.Results: Radiation exposure accelerates lung cancer progression in the K-rasLA1 lung cancer mouse model with dose fractionation being more permissive for cancer progression. A nonrandom inflammatory signature associated with this progression was elicited from whole lung tissue containing only benign lesions and predicts human lung and breast cancer patient survival across multiple datasets. Immunohistochemical analyses suggest that tumor cells drive predictive signature.Conclusions: These results demonstrate that radiation exposure can cooperate with benign lesions in a transgenic model of cancer by affecting inflammatory pathways, and that clinically relevant similarities exist between human lung and breast carcinogenesis. Clin Cancer Res; 20(6); 1610–22. ©2014 AACR.

Glioblastomas are lethal brain tumors that are treated with conventional radiation (X-rays and gamma rays) or particle radiation (protons and carbon ions). Paradoxically, radiation is also a risk factor for GBM development, raising the possibility that radiotherapy of brain tumors could promote tumor recurrence or trigger secondary gliomas. In this study, we determined whether tumor suppressor losses commonly displayed by patients with GBM confer susceptibility to radiation-induced glioma. Mice with Nestin-Cre-driven deletions of Trp53 and Pten alleles were intracranially irradiated with X-rays or charged particles of increasing atomic number and linear energy transfer (LET). Mice with loss of one allele each of Trp53 and Pten did not develop spontaneous gliomas, but were highly susceptible to radiation-induced gliomagenesis. Tumor development frequency after exposure to high-LET particle radiation was significantly higher compared with X-rays, in accordance with the irreparability of DNA double-strand breaks (DSB) induced by high-LET radiation. All resultant gliomas, regardless of radiation quality, presented histopathologic features of grade IV lesions and harbored populations of cancer stem-like cells with tumor-propagating properties. Furthermore, all tumors displayed concomitant loss of heterozygosity of Trp53 and Pten along with frequent amplification of the Met receptor tyrosine kinase, which conferred a stem cell phenotype to tumor cells. Our results demonstrate that radiation-induced DSBs cooperate with preexisting tumor suppressor losses to generate high-grade gliomas. Moreover, our mouse model can be used for studies on radiation-induced development of GBM and therapeutic strategies.Significance:This study uncovers mechanisms by which ionizing radiation, especially particle radiation, promote GBM development or recurrence.

PDF file - 1617KB, Figure S1. Malignant Characteristics of Invasive Adenocarcinoma in K-rasLA1 Mice; Figure S2. Radiation Effects on the Incidence of Various Endpoints in K-rasLA1 Mice; Figure S3. Comparative Genomic Analyses and Classifier Isolation from Irradiated Versus Unirradiated Control K-rasLA1 Mice; Figure S4. Comparative Genomic Analysis of Whole Lungs Reveals Unique Gene Classifiers Capable of Specifying Individual Experimental Cohorts; Figure S5. Only "Fractionated" Classifier Demonstrates Clinical Relevance for Lung Cancer Patient Survival; Figure S6. "Fractionated" Classifier Capable of Predicting Overall Survival in Patients with Breast, but not Lung Squamous Cell Cancer; Supplementary Figure S7. Cox Regression Analysis Exposes 6 Genes Within "Fractionated" Classifier Which Retain Predictive Capacity; Table S1. Logistic Regression Analysis of Unirradiated K-rasLA1 Mice for Gender and Strain Effects on Various Endpoints; Table S2. Logistic Regression Analysis for Gender and Strain Effects on Various Endpoints Controlling for Experiment; Table S3. Logistic Regression Analysis of Radiation Effects on the Incidence of Invasive Adenocarcinoma Controlling for Gender and Strain; Table S4. Multivariate Cox Analysis for Gender and Strain Effects on Overall Survival Controlling for Experiment; Table S5. Multivariate Cox Analysis of Radiation Effects on Overall Survival Controlling for Gender and Strain Effects; Table S6. IPA Network Annotations Associated with Corresponding Gene Lists.

Purpose: The primary cause of death due to head and neck squamous cell carcinoma (HNSCC) is local treatment failure. The goal of this study was to examine this phenomenon using an unbiased approach.Experimental Design: We utilized human papilloma virus (HPV)-negative cell lines rendered radiation-resistant (RR) via repeated exposure to radiation, a panel of HPV-negative HNSCC cell lines and three cohorts of HPV-negative HNSCC tumors (n = 68, 97, and 114) from patients treated with radiotherapy and subjected to genomic, transcriptomic, and proteomic analysis.Results: RR cell lines exhibited upregulation of several proteins compared with controls, including increased activation of Axl and PI3 kinase signaling as well as increased expression of PD-L1. Additionally, inhibition of either Axl or PI3 kinase led to decreased PD-L1 expression. When clinical samples were subjected to RPPA and mRNA expression analysis, PD-L1 was correlated with both Axl and PI3K signaling as well as dramatically associated with local failure following radiotherapy. This finding was confirmed examining a third cohort using immunohistochemistry. Indeed, tumors with high expression of PD-L1 had failure rates following radiotherapy of 60%, 70%, and 50% compared with 20%, 25%, and 20% in the PD-L1–low expression group (P = 0.01, 1.9 × 10−3, and 9 × 10−4, respectively). This finding remained significant on multivariate analysis in all groups. Additionally, patients with PD-L1 low/CD8+ tumor-infiltrating lymphocytes high had no local failure or death due to disease (P = 5 × 10−4 and P = 4 × 10−4, respectively).Conclusions: Taken together, our data point to a targetable Axl–PI3 kinase–PD-L1 axis that is highly associated with radiation resistance. Clin Cancer Res; 23(11); 2713–22. ©2017 AACR.

Supplementary Table S2. Centroids of the 821 gene signature for the three super-groups

Supplemental data

Purpose: Head and neck squamous cell carcinoma (HNSCC) is commonly treated with radiotherapy, and local failure after treatment remains the major cause of disease-related mortality. To date, human papillomavirus (HPV) is the only known clinically validated, targetable biomarkers of response to radiation in HNSCC.Experimental Design: We performed proteomic and transcriptomic analysis of targetable biomarkers of radioresistance in HPV-negative HNSCC cell lines in vitro, and tested whether pharmacologic blockade of candidate biomarkers sensitized cells to radiotherapy. Candidate biomarkers were then investigated in several independent cohorts of patients with HNSCC.Results: Increased expression of several targets was associated with radioresistance, including FGFR, ERK1, EGFR, and focal adhesion kinase (FAK), also known as PTK2. Chemical inhibition of PTK2/FAK, but not FGFR, led to significant radiosensitization with increased G2–M arrest and potentiated DNA damage. PTK2/FAK overexpression was associated with gene amplification in HPV-negative HNSCC cell lines and clinical tumors. In two independent cohorts of patients with locally advanced HPV-negative HNSCC, PTK2/FAK amplification was highly associated with poorer disease-free survival (DFS; P = 0.012 and 0.034). PTK2/FAK mRNA expression was also associated with worse DFS (P = 0.03). Moreover, both PTK2/FAK mRNA (P = 0.021) and copy number (P = 0.063) were associated with DFS in the Head and Neck Cancer subgroup of The Cancer Genome Atlas.Conclusions: Proteomic analysis identified PTK2/FAK overexpression is a biomarker of radioresistance in locally advanced HNSCC, and PTK2/FAK inhibition radiosensitized HNSCC cells. Combinations of PTK2/FAK inhibition with radiotherapy merit further evaluation as a therapeutic strategy for improving local control in HPV-negative HNSCC. Clin Cancer Res; 22(18); 4643–50. ©2016 AACR.

Figure S1: Cre-mediated excision of floxed alleles and induction of double strand breaks in murine brains. Figure S2: DNA damage in primary astrocytes and murine brains induced by different radiation types. Figure S3: Histopathology and cell lineage markers in radiation-induced gliomas (RIG). Figure S4: Sox2 expression in RIG-derived cell lines and tumor growth kinetics. Figure S5: Loss of p53 and Pten in RIG-derived cell lines. Figure S6: Non-Met amplified RIG. Table S1: List of PCR and RT-PCR primers. Table S2: RIG-derived lines. Table S3: Subcutaneous and orthotopic tumors generated from RIG-derived cell lines. Table S4: A panel of 33 RIGs used for immunohistochemical analysis. Table S5. List of GISTIC hits.

Purpose: Radiotherapy plays an integral role in the treatment of head and neck squamous cell carcinoma (HNSCC). Although proteins involved in DNA repair may predict HNSCC response to radiotherapy, none has been validated in this context. We examined whether differential expression of double-strand DNA break (DSB) repair proteins in HNSCC, the chief mediators of DNA repair following irradiation, predict for treatment outcomes.Experimental Design: Archival HNSCC tumor specimens (n = 89) were assembled onto a tissue microarray and stained with antibodies raised against 38 biomarkers. The biomarker set was enriched for proteins involved in DSB repair, in addition to established mechanistic markers of radioresistance. Staining was correlated with treatment response and survival alongside established clinical and pathologic covariates. Results were validated in an independent intramural cohort (n = 34).Results: Ku80, a key mediator of DSB repair, correlated most closely with clinical outcomes. Ku80 was overexpressed in half of all tumors, and its expression was independent of all other covariates examined. Ku80 overexpression was an independent predictor for both locoregional failure and mortality following radiotherapy (P < 0.01). The predictive power of Ku80 overexpression was confined largely to HPV-negative HNSCC, where it conferred a nine-fold greater risk of death at two years.Conclusions: Ku80 overexpression is a common feature of HNSCC, and is a candidate DNA repair-related biomarker for radiation treatment failure and death, particularly in patients with high-risk HPV-negative disease. It is a promising, mechanistically rational biomarker to select individual HPV-negative HNSCC patients for strategies to intensify treatment. Clin Cancer Res; 17(7); 2035–43. ©2011 AACR.

Supplementary Figures S1-S3; Supplementary Tables S1-S4.

Supplemental Materials and Legend. Supplemental Experimental Procedures: Determination of HPV status; Gene expression profiling; Copy number aberration (CNA) experiment; Copy number aberration (CNA) analysis; Published Data Set Collection and Integration; Unsupervised Discovery of Gene Expression-Based Subtypes and Derivation of Gene Signatures; Validation of HNSCC Classification; Single Sample Gene Set Enrichment Analysis (ssGSEA); Pathway Analysis; Survival Analysis; Immunohistochemistry and Immunofluorescence. Supplementary Figure Legends. Supplementary Table S1. Data set source and group allocation Supplementary Table S3. Significant copy number gain and loss in the discovery set (101 samples and 75 genes). Supplementary Table S4. Significant copy number gain and loss in the validation set (55 samples and 72 genes) Supplementary Table S5. Significant copy number alterations per subtype.

Supplemental methods Supplementary Figure 1. Total and phospho-PTK2/FAK is overexpressed in selected radioresistant HNSCC cells compared to radiosensitive cell lines via immunoblot. Supplementary Figure 2. A.) Inhibition of PTK2/FAK expression via stable expression of shRNA to PTK2/FAK in HN31 HNSCC cells. B.) Stable inhibition of PTK2/FAK leads to radiosensitization in vitro. C & D.) The combination of PF562271 and radiation leads to C.) increased γ-H2AX expression and D.) higher levels of G2/M arrest and sub-G1 staining in Fadu HNSCC cells. Supplementary Figure 3. A & B.) PTK2/FAK is highly expressed in a large public database of HNSCC cell lines (www.oncomine.com) (A.) and tumor specimens (TCGA) (B.) compared to other tumor types. Supplementary Table 1. HNSCC cell lines included in RPPA. Supplementary Table 2. Tumor characteristics of the institutional cohorts. Supplementary Table 3. Univariate and multivariate analysis of PTK2/FAK copy number and disease free survival (DFS) in the two institutional cohorts and the Head and Neck Cancer TCGA cohort. NA - not included in the model. HR - Hazard ratio. 95% CI- 95% Confidence Interval. Supplementary Table 4. Tumor characteristics of the HNSCC TCGA cohort.

Supplementary Figure 1 from Non–Small Cell Lung Cancers with Kinase Domain Mutations in the Epidermal Growth Factor Receptor Are Sensitive to Ionizing Radiation

Supplemental Methods. Description of additional methods and procedures used in the study.

Non–small cell lung cancers (NSCLCs) bearing mutations in the tyrosine kinase domain (TKD) of the epidermal growth factor receptor (EGFR) often exhibit dramatic sensitivity to the EGFR tyrosine kinase inhibitors gefitinib and erlotinib. Ionizing radiation (IR) is frequently used in the treatment of NSCLC, but little is known how lung tumor–acquired EGFR mutations affect responses to IR. Because this is of great clinical importance, we investigated and found that clonogenic survival of mutant EGFR NSCLCs in response to IR was reduced 500- to 1,000-fold compared with wild-type (WT) EGFR NSCLCs. Exogenous expression of either the L858R point mutant or the ΔE746-E750 deletion mutant form of EGFR in immortalized human bronchial epithelial cells, p53 WT NSCLC (A549), or p53-null NSCLC (NCI-H1299) resulted in dramatically increased sensitivity to IR. We show that the majority of mutant EGFR NSCLCs, including those that contain the secondary gefitinib resistance T790M mutation, exhibit characteristics consistent with a radiosensitive phenotype, which include delayed DNA repair kinetics, defective IR-induced arrest in DNA synthesis or mitosis, and pronounced increases in apoptosis or micronuclei. Thus, understanding how activating mutations in the TKD domain of EGFR contribute to radiosensitivity should provide new insight into effective treatment of NSCLC with radiotherapy and perhaps avoid emergence of single agent drug resistance. (Cancer Res 2006; 66(19): 9601-07)

Supplemental Table S1. Summary of patient data and prognostic data for both the test and validation datasets.

The epidermal growth factor receptor (EGFR) is an important determinant of radioresponse, whose elevated expression and activity frequently correlates with radioresistance in several cancers, including non–small cell lung carcinoma (NSCLC). We reported recently that NSCLC cell lines harboring somatic, activating mutations in the tyrosine kinase domain (TKD) of the EGFR exhibit significant delays in the repair of DNA double-strand breaks (DSB) and poor clonogenic survival in response to radiation. Here, we explore the mechanisms underlying mutant EGFR-associated radiosensitivity. In three representative NSCLC cell lines, we show that, unlike wild-type (WT) EGFR, receptors with common oncogenic TKD mutations, L858R or ΔE746-E750, are defective in radiation-induced translocation to the nucleus and fail to bind the catalytic and regulatory subunits of the DNA-dependent protein kinase (DNA-PK), a key enzyme in the nonhomologous end-joining repair pathway. Moreover, despite the presence of WT EGFR, stable exogenous expression of either the L858R or the ΔE746-E750 mutant forms of EGFR in human bronchial epithelial cells significantly delays repair of ionizing radiation (IR)–induced DSBs, blocks the resolution of frank or microhomologous DNA ends, and abrogates IR-induced nuclear EGFR translocation or binding to DNA-PK catalytic subunit. Our study has identified a subset of naturally occurring EGFR mutations that lack a critical radioprotective function of EGFR, providing valuable insights on how the EGFR mediates cell survival in response to radiation in NSCLC cell lines. [Cancer Res 2007;67(11):5267–74]

Therapeutic inhibition of mammalian target of rapamycin (mTOR) in cancer is complicated by the existence of a negative feedback loop linking mTOR to the phosphatidylinositol 3-kinase (PI3K)-Akt pathway. Thus, mTOR inhibition by rapamycin or TSC1/2 results in increased PI3K-Akt activation. The death domain kinase receptor interacting protein 1 (RIP1) plays a key role in nuclear factor-κB (NF-κB) activation and also activates the PI3K-Akt pathway through unknown mechanisms. RIP1 has recently been found to be overexpressed in glioblastoma multiforme, the most common adult primary malignant brain tumor, but not in grade II to III glioma. Our data suggest that RIP1 activates PI3K-Akt using dual mechanisms by removing the two major brakes on PI3K-Akt activity. First, increased expression of RIP1 activates PI3K-Akt by interrupting the mTOR negative feedback loop. However, unlike other signals that regulate mTOR activity without affecting its level, RIP1 negatively regulates mTOR transcription via a NF-κB–dependent mechanism. The second mechanism used by RIP1 to activate PI3K-Akt is down-regulation of cellular PTEN levels, which appears to be independent of NF-κB activation. The clinical relevance of these findings is highlighted by the demonstration that RIP1 levels correlate with activation of Akt in glioblastoma multiforme. Thus, our study shows that RIP1 regulates key components of the PTEN-PI3K-Akt-mTOR pathway and elucidates a novel negative regulation of mTOR signaling at the transcriptional level by the NF-κB pathway. Our data suggest that the RIP1-NF-κB status of tumors may influence response to treatments targeting the PTEN-PI3K-mTOR signaling axis. [Cancer Res 2009;69(10):4107–11]

Supplementary Figure 2 from Non–Small Cell Lung Cancers with Kinase Domain Mutations in the Epidermal Growth Factor Receptor Are Sensitive to Ionizing Radiation

Supplemental Figure S2. Cellular proliferation is independent of TP53 status.

Supplementary Figure Legends 1-3 from Non–Small Cell Lung Cancers with Kinase Domain Mutations in the Epidermal Growth Factor Receptor Are Sensitive to Ionizing Radiation

Supplementary Figures 1-5 from RIP1 Activates PI3K-Akt via a Dual Mechanism Involving NF-κB–Mediated Inhibition of the mTOR-S6K-IRS1 Negative Feedback Loop and Down-regulation of PTEN

Legends for Supplemental Figures S1-S4.

Supplementary Figures S1-S2 from Somatic Mutations in the Tyrosine Kinase Domain of Epidermal Growth Factor Receptor (EGFR) Abrogate EGFR-Mediated Radioprotection in Non–Small Cell Lung Carcinoma

Determining the mechanistic causes of lung diseases, developing new treatments thereof, and assessing toxicity whether from chemical exposures or engineered nanomaterials would benefit significantly from a preclinical human lung alveolar interstitium model of physiological relevance. The existing preclinical models have limitations because they fail to replicate the key anatomical and physiological characteristics of human alveoli. Thus, a human lung alveolar interstitium chip was developed to imitate key alveolar microenvironmental factors including: an electrospun nanofibrous membrane as the analogue of the basement membrane for co-culture of epithelial cells with fibroblasts embedded in 3D collagenous gels; physiologically relevant interstitial matrix stiffness; interstitial fluid flow; and 3D breathing-like mechanical stretch. The biomimetic chip substantially improved epithelial barrier function compared to transwell models. Moreover, the chip having a gel made of a collagen I-fibrin blend as the interstitial matrix sustained the interstitium integrity and further enhanced the epithelial barrier resulting in a longevity that extended beyond eight weeks. The assessment of multiwalled carbon nanotube toxicity on the chip was in line with the animal study.

Chromosomal instability (CIN) is a driving force for cancer development. The most common causes of CIN include the dysregulation of the spindle assembly checkpoint (SAC), which is a surveillance mechanism that prevents premature chromosome separation during mitosis by targeting anaphase-promoting complex/cyclosome (APC/C). DAB2IP is frequently silenced in advanced prostate cancer (PCa) and is associated with aggressive phenotypes of PCa. Our previous study showed that DAB2IP activates PLK1 and functions in mitotic regulation. Here, we report the novel mitotic phosphorylation of DAB2IP by Cdks, which mediates DAB2IP’s interaction with PLK1 and the activation of the PLK1-Mps1 pathway. DAB2IP interacts with Cdc20 in a phosphorylation-independent manner. However, the phosphorylation of DAB2IP inhibits the ubiquitylation of Cdc20 in response to SAC, and blocks the premature release of the APC/C-MCC. The PLK1-Mps1 pathway plays an important role in mitotic checkpoint complex (MCC) assembly. It is likely that DAB2IP acts as a scaffold to aid PLK1-Mps1 in targeting Cdc20. Depletion or loss of the Cdks-mediated phosphorylation of DAB2IP destabilizes the MCC, impairs the SAC, and increases chromosome missegregation and subsequent CIN, thus contributing to tumorigenesis. Collectively, these results demonstrate the mechanism of DAB2IP in SAC regulation and provide a rationale for targeting the SAC to cause lethal CIN against DAB2IP-deficient aggressive PCa, which exhibits a weak SAC.

The space radiation environment consists of multiple species of charged particles, including 28Si ions, that may impact brain function during and following missions. To develop biomarkers of the space radiation response, BALB/c and C3H female and male mice and their F2 hybrid progeny were irradiated with 28Si ions (350 MeV/n, 0.2 Gy) and tested for behavioral and cognitive performance 1, 6, and 12 months following irradiation. The plasma of the mice was collected for analysis of miRNA levels. Select pertinent brain regions were dissected for lipidomic analyses and analyses of levels of select biomarkers shown to be sensitive to effects of space radiation in previous studies. There were associations between lipids in select brain regions, plasma miRNA, and cognitive measures and behavioral following 28Si ion irradiation. Different but overlapping sets of miRNAs in plasma were found to be associated with cognitive measures and behavioral in sham and irradiated mice at the three time points. The radiation condition revealed pathways involved in neurodegenerative conditions and cancers. Levels of the dendritic marker MAP2 in the cortex were higher in irradiated than sham-irradiated mice at middle age, which might be part of a compensatory response. Relationships were also revealed with CD68 in miRNAs in an anatomical distinct fashion, suggesting that distinct miRNAs modulate neuroinflammation in different brain regions. The associations between lipids in selected brain regions, plasma miRNA, and behavioral and cognitive measures following 28Si ion irradiation could be used for the development of biomarker of the space radiation response.

Tumor Treating Fields (TTFields) is a new mode of physical cancer treatment that delivers low-intensity, intermediate frequency, alternating electric fields to tumors in a non-invasive manner. A number of in vitro studies have demonstrated that TTFields play an important role in suppressing the DNA damage pathways and increasing replication stress, in addition to interfering with mitosis. During interphase TTFields creates a conditional vulnerability environment that would make cells more susceptible to radiation and chemotherapy. The PULSAR concept relies on ablative doses of radiation spaced at least one week apart to allow a tumor to adapt to the first radiation dose before a second dose of radiation is applied. As a result, the therapy should be less toxic and be adaptable according to changes in tumor morphology, location, and response to radiation. TTFields seems well suited to the PULSAR paradigm given the time between radiation doses where TTFields application would have the potential to enhance tumor response to a subsequent radiation exposure and this is described here. First, in in vitro studies, a greater increase in radiation susceptibility was observed when cells were exposed to TTFields before radiation (IR) treatment compared to IR treatment followed by TTFields. In the present study, TTFields were combined with IR or IR+immune checkpoint inhibitor (ICI) to evaluate the combinatorial effectiveness in mouse tumor models. Three syngeneic mouse tumor models, LLC (Murine Lewis Lung Carcinoma), KPC63 (Pancreatic Cancer) and MC38 (Colon Cancer), were used in the initial tumor growth delay (TGD) studies. TTFields exposure increased TGD in all three mouse models, but the difference in tumor volume over time was not statistically significant due to small sample sizes. Combination studies were then initiated using the MC38 mouse model since TTFields resulted in a greater TGD effect in this model. The first in vivo experiment combined one round of TTFields with radiation therapy, which significantly delayed tumor growth. The combined efficacy of two rounds of TTFields and IR treatment was further delayed compared to radiation or TTFields alone. The combination of TTFields prior to each IR dose, two rounds of IR spaced one week apart, and an ICI (PD-L1 Ab) given sequentially inhibited tumor growth far more than either TTFields plus the ICI or TTFields plus IR or IR plus the ICI alone. To explain this increased efficacy immune-related changes in the tumor microenvironment is being evaluated. In addition, gene expression analysis is also ongoing to confirm the changes in gene expression underlying the in vitro TTFields effect on DNA repair and replication stress. In conclusion, the PULSAR paradigm for radiotherapy seems well suited for the application of TTFields prior to each radiation dose and that effect is enhanced when an ICI is also introduced. Citation Format: Narasimha K. Karanam, Zengfu Shang, Robert Timmerman, Michael D. Story, Debabrata Saha. Tumor treating fields combined with the PULSAR paradigm for radiotherapy and an immune checkpoint inhibitor enhances antitumor efficacy in vivo [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 3272.

Research examining the potential for circulating miRNA as serve as markers for preneoplastic lesions or early-stage hepatocellular carcinoma (HCC) is hindered by the difficulties of obtaining samples from asymptomatic individuals. As a surrogate for human samples, we identified hub miRNAs in gene co-expression networks using HCC-bearing C3H mice. We confirmed 38 hub miRNAs as associated with HCC in F2 hybrid mice derived from radiogenic HCC susceptible and resistant founders. When compared to a panel of 12 circulating miRNAs associated with human HCC, two had no mouse ortholog and 7 of the remaining 10 miRNAs overlapped with the 38 mouse HCC hub miRNAs. Using small RNA sequencing data generated from serially collected plasma samples in F2 mice, we examined the temporal levels of these 7 circulating miRNAs and found that the levels of 4 human circulating markers, miR-122-5p, miR-100-5p, miR-34a-5p and miR-365-3p increased linearly as the time approaching HCC detection neared, suggesting a correlation of miRNA levels with oncogenic progression. Estimation of change points in the kinetics of the 4 circulating miRNAs suggested the changes started 17.5 to 6.8 months prior to HCC detection. These data establish these 4 circulating miRNAs as potential sentinels for preneoplastic lesions or early-stage HCC.

Traditional cancer therapy choices for clinicians are surgery, chemotherapy, radiation and immune therapy which are used either standalone therapies or in various combinations. Other physical modalities beyond ionizing radiation include photodynamic therapy and heating and the more recent approach referred to as Tumor Treating Fields (TTFields). TTFields are intermediate frequency, low-intensity, alternating electric fields that are applied to tumor regions and cells using noninvasive arrays. TTFields have revolutionized the treatment of newly diagnosed and recurrent glioblastoma (GBM) and unresectable and locally advanced malignant pleural mesothelioma (MPM). TTFields are thought to kill tumor cells predominantly by disrupting mitosis; however it has been shown that TTFields increase efficacy of different classes of drugs, which directly target mitosis, replication stress and DNA damage pathways. Hence, a detailed understanding of TTFields' mechanisms of action is needed to use this therapy effectively in the clinic. Recent findings implicate TTFields' role in different important pathways such as DNA damage response and replication stress, ER stress, membrane permeability, autophagy, and immune response. This review focuses on potentially novel mechanisms of TTFields anti-tumor action and their implications in completed and ongoing clinical trials and pre-clinical studies. Moreover, the review discusses advantages and strategies using chemotherapy agents and radiation therapy in combination with TTFields for future clinical use.

Tumor treating fields (TTFields) is a noninvasive physical modality of cancer therapy that applies low-intensity, intermediate frequency, and alternating electric fields to a tumor. Interference with mitosis was the first mechanism describing the effects of TTFields on cancer cells; however, TTFields was shown to not only reduce the rejoining of radiation-induced DNA double-strand breaks (DSBs), but to also induce DNA DSBs. The mechanism(s) by which TTFields generates DNA DSBs is related to the generation of replication stress including reduced expression of the DNA replication complex genes MCM6 and MCM10 and the Fanconi's Anemia pathway genes. When markers of DNA replication stress as a result of TTFields exposure were examined, newly replicated DNA length was reduced with TTFields exposure time and there was increased R-loop formation. Furthermore, as cells were exposed to TTFields a conditional vulnerability environment developed which rendered cells more susceptible to DNA damaging agents or agents that interfere with DNA repair or replication fork maintenance. The effect of TTFields exposure with concomitant exposure to cisplatin or PARP inhibition, the combination of TTFields plus concomitant PARP inhibition followed by radiation, or radiation alone at the end of a TTFields exposure were all synergistic. Finally, gene expression analysis of 47 key mitosis regulator genes suggested that TTFields-induced mitotic aberrations and DNA damage/replication stress events, although intimately linked to one another, are likely initiated independently of one another. This suggests that enhanced replication stress and reduced DNA repair capacity are also major mechanisms of TTFields effects, effects for which there are therapeutic implications.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide. Thirty percent of patients will experience locoregional recurrence for which median survival is less than 1 year. Factors contributing to treatment failure include inherent resistance to X-rays and chemotherapy, hypoxia, epithelial to mesenchymal transition, and immune suppression. The unique properties of

High-charge, high-energy ion particle (HZE) radiations are extraterrestrial in origin and characterized by high linear energy transfer (high-LET), which causes more severe cell damage than low-LET radiations like γ-rays or photons. High-LET radiation poses potential cancer risks for astronauts on deep space missions, but the studies of its carcinogenic effects have relied heavily on animal models. It remains uncertain whether such data are applicable to human disease. Here, we used genomics approaches to directly compare high-LET radiation-induced, low-LET radiation-induced and spontaneous hepatocellular carcinoma (HCC) in mice with a human HCC cohort from The Cancer Genome Atlas (TCGA). We identified common molecular pathways between mouse and human HCC and discovered a subset of orthologous genes (mR-HCC) that associated high-LET radiation-induced mouse HCC with a subgroup (mrHCC2) of the TCGA cohort. The mrHCC2 TCGA cohort was more enriched with tumor-suppressing immune cells and showed a better prognostic outcome than other patient subgroups.

The clinical success of immune checkpoint inhibitors in treating metastatic and refractory cancers has generated significant interest in investigating their role in treating locally advanced diseases, thus requiring them to be combined with standard treatments in the hope of producing synergistic antitumor responses. Radiotherapy, in particular, has long been hypothesized to have actions complementary to those of immune checkpoint blockade, and a growing body of evidence indicates that cancer immunotherapy may also have radiosensitizing effects, which would provide unique benefit for locoregional treatments. Recent studies have demonstrated that when immune cells are activated by immunotherapeutics, they can reprogram the tumor microenvironment in ways that may potentially increase the radiosensitivity of the tumor. In this review, we highlight the evidence that supports reciprocal interactions between cancer immunotherapy and radiotherapy, where in addition to the traditional notion that radiation serves to enhance the activation of antitumor immunity, an alternative scenario also exists in which T-cell activation by cancer immunotherapy may sensitize tumors to radiation treatment through mechanisms that include normalization of the tumor vasculature and tissue hypoxia. We describe the empirical observations from preclinical models that support such effects and discuss their implications for future research and trial design.

New physical cancer treatment modality called Tumor Treating Fields (TTFields) deliver low-intensity, intermediate frequency, alternating electric fields non-invasively to the tumor. TTFields is believed to inhibit mitosis as its primary mechanism of action. We previously showed that TTFields exposure decreased the expression of FANC/BRCA1 pathway genes, which play an essential role in DNA double strand break (DSB) repair. As a result, DNA DSB repair under IR exposure is downregulated, and most importantly, the TTFields alone increased gH2AX foci and chromatid aberrations as a function of TTFields time exposure. The length of newly replicated DNA decreased and R-loop formation increased after TTFields treatment, suggesting replication stress is induced by TTFields. Our research revealed that TTFields are involved in DNA damage and replication stress pathways besides mitosis, thus, we predicted that, by applying TTFields first, a conditional vulnerability environment would be created, rendering cells more susceptible to agents like radiation and chemotherapy. In accordance with our hypothesis, radiation susceptibility increased when cells were exposed to TTFields prior to IR treatment compared to IR treatment followed by TTFields. The combination of TTFields together with Radiation has been tested in-vivo with the newly designed Inovivo system. To measure tumor growth delay caused by TTFields treatment, three distinct syngenic tumor mouse models were utilized, namely LLC (Murine Lewis Lung Carcinoma), KPC63 (Pancreatic Cancer) and MC38 (Colon Cancer). Although TTField exposure decreased tumor volume in the three mouse models tested, there was no statistically significant difference in tumor growth between the heat and TTField groups. In IR combination experiments, the MC38 mouse model was chosen since TTFields induced a greater TGD effect in this model. In the first Inovivo experiment, we combined TTFields and radiation therapy, which delayed tumor growth significantly. Following that, we tested the combined efficacy of two rounds of TTFields and radiation treatment. The tumor growth delay was more pronounced after two rounds of TTFields treatment compared to one, which confirms the hypothesis that TTFields treatment induces conditional vulnerability. The results are consistent with observations made by the researchers in the clinic that high compliance patients (who have received more exposure to TTFields) have better prognosis compared to low compliance patients. Our inovivo experiment results not only confirm our earlier in vitro results, but also support the use of TTFields in IR combination therapies. Citation Format: Narasimha Kumar Karanam, Zengfu Shang, Michael D. Story, Debabrata Saha. Tumor Treating Fields in combination with radiation cause significant delay in tumor growth in in-vivo mice modelsignificant delay in tumor growth in in-vivo mice model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3316.

Introduction: Superoxide dismutase mimetics (SODm) synergize with radiation (IR) by converting radiolytically produced superoxide to hydrogen peroxide. Cancer cells lacking catalase function fail to clear H2O2 resulting in increased tumoricidal load. In addition, complex interactions through redox signaling and immune interplay are suggested mechanisms of action. Our pre-clinical studies with the SODm GC4419 (AVA) indicated that increased inflammatory, TNFα, and apoptosis signaling contribute to treatment synergy that was most effective at hypofractionation (Sci Transl Med. 13:593). The analogue GC4711 (GC) is currently in clinical trials for SBRT against NSCLC and pancreatic cancer. However, optimal combination schedules and molecular mechanisms are largely unknown. Here, we present optimized combination schedules using human lung tumor xenografts regarding drug administration timing and dosage, as well as local and abscopal tumor control in a syngeneic lung tumor model. Further, we tested various cell stress and immune pathways to identify targets of synergistic IR-drug interaction. Methods: Female athymic nude and C57BL/6 mice were inoculated with H1299 and LLC, respectively. H1299 tumors were irradiated with 10-15 Gy and GC was given once or twice daily (24 or 2x20 mg/kg, respectively) over 5 days, including untreated controls. LLC tumors were set up as an abscopal model and mice received combination treatment with GC and 15 Gy, 15 Gy only, or GC only, including untreated controls. In vitro, lung cancer cell lines were pre-treated with AVA or GC 30 min prior to IR and assayed for various immune or DNA damage-related endpoints. Results: The anti-tumor efficacy of single or double GC administration showed nonlinear dose-dependence indicating complex drug-IR interplay. Optimal drug administration appears to be around 3 hours prior to irradiation while time-of-day of treatment has no observable effect in this model. In the LLC double tumor model, GC in combination with IR induced significant and pronounced abscopal tumor control. In vitro, analysis revealed GC action related to e.g. 53BP1, yH2AX, Akt, R-loop formation and replication stress, and interaction with various immune pathways. Discussion and Conclusions: The radiotherapeutic effect can be vastly increased with superoxide dismutase mimetics, both within and outside the treatment field. Combination therapy requires screening of schedules regarding timing, drug dosage, and IR dose and fractionation to achieve optimal tumoricidal effects. SODm synergize with IR acting on various molecular targets that can be harnessed to overcome treatment resistance. Secondary tumor control is a promising new venue for IR-SODm combination therapy. Moreover, redox-immune interaction opens new therapeutic applications with combined immune checkpoint inhibitors for unprecedented tumor control and cure rate. Citation Format: Britta Langen, Laurentiu Pop, Zengfu Shang, Moito Iijima, James Nicholson, Mingming Yang, Michael D. Story. Novel SOD mimetics GC4419 & GC4711 induce synergistic tumoricidal effects combined with radiotherapy in lung cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 218.