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Purpose: In the proper context, radiotherapy can promote antitumor immunity. It is unknown if elective nodal irradiation (ENI), a strategy that irradiates tumor-associated draining lymph nodes (DLN), affects adaptive immune responses and combinatorial efficacy of radiotherapy with immune checkpoint blockade (ICB).Experimental Design: We developed a preclinical model to compare stereotactic radiotherapy (Tumor RT) with or without ENI to examine immunologic differences between radiotherapy techniques that spare or irradiate the DLN.Results: Tumor RT was associated with upregulation of an intratumoral T-cell chemoattractant chemokine signature (CXCR3, CCR5-related) that resulted in robust infiltration of antigen-specific CD8+ effector T cells as well as FoxP3+ regulatory T cells (Tregs). The addition of ENI attenuated chemokine expression, restrained immune infiltration, and adversely affected survival when combined with ICB, especially with anti-CLTA4 therapy. The combination of stereotactic radiotherapy and ICB led to long-term survival in a subset of mice and was associated with favorable CD8 effector-to-Treg ratios and increased intratumoral density of antigen-specific CD8+ T cells. Although radiotherapy technique (Tumor RT vs. ENI) affected initial tumor control and survival, the ability to reject tumor upon rechallenge was partially dependent upon the mechanism of action of ICB; as radiotherapy/anti-CTLA4 was superior to radiotherapy/anti-PD-1.Conclusions: Our results highlight that irradiation of the DLN restrains adaptive immune responses through altered chemokine expression and CD8+ T-cell trafficking. These data have implications for combining radiotherapy and ICB, long-term survival, and induction of immunologic memory. Clinically, the immunomodulatory effect of the radiotherapy strategy should be considered when combining stereotactic radiotherapy with immunotherapy. Clin Cancer Res; 24(20); 5058–71. ©2018 AACR.

Quantitative analysis of radiation-induced intratumoral chemokine and cytokine expression over time.

Comparison of intratumoral Treg depletion by anti-CTLA4 Ig2Ga and IgG2b isotypes.

Multi-parametric flow cytometry gating schemas

Quantitative analysis of radiation-induced changes in peripheral blood subsets over time.

Estrogen receptor–positive breast tumors, which initially respond effectively to endocrine therapy, progress due to acquired endocrine therapy resistance, including genomic alterations in estrogen receptor alpha (ESR1). A recent study has suggested that there is a sufficient number of preexisting ESR1 mutations acting as an intrinsic resistance mechanism to warrant primary screening. We determined the incidence of de novo ESR1 mutations in hormone-positive treatment-naïve primary breast tumors using 12 publicly available international datasets in the cBioPortal. The prevalence of mutation was statistically significantly lower in treatment-naïve primary tumors (n = 6 of 3682, 0.16%) than in metastatic (n = 156 of 1089, 14.3%, 2-sided P

Background Lymphedema is a frequent complication after surgical treatments of cancer involving lymph node resection. However, research of lymphedema treatments, such as vascularized lymph node transfer, is limited by the absence of an adequate lymphedema animal model. The purpose of this study was to determine if we could create sustainable lower limb lymphedema in the rat with a combination of inguinal lymphadenectomy, circumferential skin and subcutaneous tissue excision, and radiotherapy. Methods Inguinal lymphadenectomies were completed in 15 Sprague-Dawley rats. In cohort A, 5 rats received a 0.5- to 1.0-cm wide excision of proximal thigh skin and subcutaneous tissue. This step was omitted for the 10 rats in cohort B. Cohort A then received a single radiation dose of 22.7 Gy, whereas cohort B received a cumulative dose of 40.5 Gy. Bioimpedance measurements were obtained monthly to assess lymphedema progression, and lymphatic drainage at 6 months postradiation was visualized via indocyanine green (ICG) lymphangiography. Results Two rats in cohort A developed visually appreciable lymphedema in the lower limb, with bioimpedance ratios of 0.684 and 0.542 and ankle circumference ratios of 1.294 and 1.061, respectively, consistent with lymphedema. Furthermore, ICG lymphangiography in these cohort A rats revealed impaired lower limb lymphatic drainage. In cohort B, however, bioimpedance and circumference ratios, and ICG lymphangiography, did not reveal abnormal lymphatic drainage. Conclusions The combination of inguinal lymphadenectomy, circumferential skin and subcutaneous tissue excision, and radiotherapy can successfully create lower limb lymphedema in the rat. When soft tissue excision is omitted, lymphedema does not develop.

PURPOSE/OBJECTIVE(S) Combined chemotherapy and radiation is a standard therapy for locally advanced lung cancers. However, the resistance mechanisms to these therapies and how they affect further adjuvant immunotherapy are not well understood. Our previous studies have shown that radiation led to the activation of Bmi1 as a mechanism of radiation resistance, which in turn upregulated the inhibitory immune checkpoint ligand PD-L1. Our hypothesis is that inhibition of Bmi1, a target for chemoradiotherapy resistance, with concurrent PD-L1 checkpoint blockade would have a synergistic enhanced immune response after chemoradiation therapy in a preclinical model of lung cancer. MATERIALS/METHODS A small molecule inhibitor, PTC-596 (PTC Therapeutics, NJ), has been shown to inhibit the expression of Bmi1. For in vitro studies, we tested the effects of PTC-596 on the expression of both Bmi1 and PD-L1 in several lung tumor cell lines. To create a mouse model that simulates the PACIFIC trial treatment regimen, Lewis lung carcinoma cells (LLC1; 1 x 106 unsorted cells in 100 ul) derived in C57BL/6J strain were injected subcutaneously into the flank of immunocompetent C67BL/6J mice. When average tumor volume reached 100 mm3, the mice were treated with phase 1 treatment of radiation (dose 30 Gy in 10 fractions daily) delivered through SAARP platform and cisplatin (2mg/kg, every 3rd day for 10 days) and then randomized to receive phase 2 treatments: 1). Vehicle control every day; 2). Inhibitory anti-PD-L1 antibody every other day (200mg/kg, every 3 days for 4 cycles); 3). PTC-596 once a day (12.5 mg/kg, once a day); 4). Combined inhibitory anti-PD-1 antibody and PTC-596. Each treatment group had 10 mice. RESULTS In both human and mouse lung tumor cell lines, fractionated radiation treatments upregulated Bmi1 expression and also increase the frequency of Bmi1-high, PD-L1-high expressing cells. Treatment with PTC-596 downregulated expression of Bmi1 and PD-L1 in these cells. In the PACIFIC study mouse model, cisplatin chemotherapy with fractionated radiation resulted significant growth delay than compared to no treatment. In the phase 2 of adjuvant treatment, the anti-PD-L1 antibody plus PTC-596 combination group, but not in groups receiving either treatment alone, had continued significant tumor size reduction. Immunological markers are being analyzed to determine if the immune response is enhanced. CONCLUSION These results suggest that chemoradiation therapy leads to the activation of Bmi1 as a resistance mechanism, which in turn results in the activation of inhibitory immune checkpoint PD-L1 leading to immune escape and acquired resistance to immunotherapy. Treatment with PTC-596 demonstrated synergistic benefits to anti-PD-L1 immunotherapy after pre-chemoradiation treatment of preclinical mouse lung tumor model.

BACKGROUND: Treatment paradigms for large cell neuroendocrine carcinoma (LCNEC) of the lung are based largely upon small retrospective studies and smaller prospective trials. It is unclear if these tumors behave like non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC). Data are lacking with regard to the role of radiotherapy (RT). U. S. guidelines recommend that LCNEC be treated as a NSCLC. We sought to perform a cross-sectional study of LCNEC cases to understand treatment paradigms and outcomes in this disease. METHODS: The Surveillance, Epidemiology and End Results database was queried for cases of stage I–III pulmonary LCNEC diagnosed 2004–2013. Treatment groups were defined as no surgery, RT alone, surgery alone, and surgery + RT. The Cox-proportional hazards regression model was used to compare overall survival and cause-specific survival (OS/CSS), stratified by AJCC 6th Staging. Factors that were significant on univariable analysis were included in multivariable analysis. RESULTS: We identified 1,523 cases of LCNEC, with 748, 177, and 598 cases of stage I, II, and III disease, respectively. In stage I and II disease, RT was associated with improved survival for non-surgical patients, but not for those who underwent surgery. In stage I disease, the adjusted hazard ratios for OS for RT alone, surgery, and surgery + RT were 0.39, 0.21, and 0.22, respectively (P

Purpose To implement Velocity-based image fusion and adaptive deformable registration to enable treatment planning for preclinical murine models of fractionated stereotactic radiosurgery (fSRS) using the small animal radiation research platform (SARRP). Methods and Materials C57BL6 mice underwent 3 unique cone beam computed tomography (CBCT) scans: 2 in the prone position and a third supine. A single T1-weighted post-contrast magnetic resonance imaging (MRI) series of a murine metastatic brain tumor model was selected for MRI-to-CBCT registration and gross tumor volume (GTV) identification. Two arms were compared: Arm 1, where we performed 3 individual MRI-to-CBCT fusions using rigid registration, contouring GTVs on each, and Arm 2, where the authors performed MRI-to-CBCT fusion and contoured GTV on the first CBCT followed by Velocity-based adaptive registration. The first CBCT and associated GTV were exported from MuriPlan (Xstrahl Life Sciences) into Velocity (Varian Medical Systems, Inc, Palo Alto, CA). In Arm 1, the second and third CBCTs were exported similarly along with associated GTVs (Arm 1), while in Arm 2, the first (prone) CBCT was fused separately to the second (prone) and third (supine) CBCTs, performing deformable registrations on initial CBCTs and applying resulting matrices to the contoured GTV. Resulting GTVs were compared between Arms 1 and 2. Results Comparing GTV overlays using repeated MRI fusion and GTV delineation (Arm 1) versus those of Velocity-based CBCT and GTV adaptive fusion (Arm 2), mean deviations ± standard deviation in the axial, sagittal, and coronal planes were 0.46 ± 0.16, 0.46 ± 0.22, and 0.37 ± 0.22 mm for prone-to-prone and 0.52 ± 0.27, 0.52 ± 0.36, and 0.68 ± 0.31 mm for prone-to-supine adaptive fusions, respectively. Conclusions Velocity-based adaptive fusion of CBCTs and contoured volumes allows for efficient fSRS planning using a single MRI-to-CBCT fusion. This technique is immediately implementable on current SARRP systems, facilitating advanced preclinical treatment paradigms using existing clinical treatment planning software.

Purpose: In the proper context, radiotherapy can promote antitumor immunity. It is unknown if elective nodal irradiation (ENI), a strategy that irradiates tumor-associated draining lymph nodes (DLN), affects adaptive immune responses and combinatorial efficacy of radiotherapy with immune checkpoint blockade (ICB). Experimental Design: We developed a preclinical model to compare stereotactic radiotherapy (Tumor RT) with or without ENI to examine immunologic differences between radiotherapy techniques that spare or irradiate the DLN. Results: Tumor RT was associated with upregulation of an intratumoral T-cell chemoattractant chemokine signature (CXCR3, CCR5-related) that resulted in robust infiltration of antigen-specific CD8+ effector T cells as well as FoxP3+ regulatory T cells (Tregs). The addition of ENI attenuated chemokine expression, restrained immune infiltration, and adversely affected survival when combined with ICB, especially with anti-CLTA4 therapy. The combination of stereotactic radiotherapy and ICB led to long-term survival in a subset of mice and was associated with favorable CD8 effector-to-Treg ratios and increased intratumoral density of antigen-specific CD8+ T cells. Although radiotherapy technique (Tumor RT vs. ENI) affected initial tumor control and survival, the ability to reject tumor upon rechallenge was partially dependent upon the mechanism of action of ICB; as radiotherapy/anti-CTLA4 was superior to radiotherapy/anti-PD-1. Conclusions: Our results highlight that irradiation of the DLN restrains adaptive immune responses through altered chemokine expression and CD8+ T-cell trafficking. These data have implications for combining radiotherapy and ICB, long-term survival, and induction of immunologic memory. Clinically, the immunomodulatory effect of the radiotherapy strategy should be considered when combining stereotactic radiotherapy with immunotherapy. Clin Cancer Res; 24(20); 5058–71. ©2018 AACR.

Purpose/Objective(s): Lung cancer is the leading cause of cancer deaths in the United States and worldwide. There has been little improvement in survival over the past several decades and surgery is generally considered to be the only curative treatment. We, therefore, performed a genome-wide functional genomics screen on human lung adenocarcinoma cells to identify genes involved in chemo- and radiotherapy resistance. Based on these results, we hypothesized that serine hydroxymethyltransferase-2 (SHMT2), which catalyzes the conversion of serine to glycine, is a target for chemoradiotherapy resistance in preclinical models of lung adenocarcinoma. Materials/Methods: PC9 cells were treated three times with 1 uM cisplatin and 2 Gy radiation before being subjected to genomic, untargeted metabolomic, or proteomic analysis. For the genomic analysis, PC9 cells were transfected with an shRNA viral library containing ~60,000 individual shRNAs following treatment. shRNAs that were depleted were hypothesized to represent genes involved in chemo-/radioresistance. Proteomics analysis was conducted after treatment and quantified by tandem mass tags isobaric labeling, totaling ~2,100 quantifiable proteins. An untargeted metabolomics approach was conducted after treatment, using gas chromatography mass spectrometry to quantify ~95 metabolites. Protein expression following treatment was determined by Western blot. Potential drug targets were assessed via colony formation assays. The Cancer Genome Atlas (TCGA) was queried to confirm in vivo human clinical correlations. Results: Our genomic, proteomic, and metabolomics screens indicated glutathione detoxification and oxidative stress response were major compensatory pathways upregulated after cisplatin and radiation treatment. We validated three targets, SLC7A11, GPX4, and SHMT2, as genes involved in chemo-radioresistance in vitro. The respective small molecule inhibitors of these gene targets, IKE, RSL3, and SHIN1, were synergistic when combined with cisplatin and radiation. SHIN1 appeared to also induce oxidative stress through ferroptosis, although this mechanism of cell death must be interrogated further. Analysis of TCGA revealed SHMT2 expression is upregulated in human lung adenocarcinoma in vivo and that higher expression is associated with decreased overall and disease-free survival. Conclusion: SHMT2 was identified as a novel target to overcome treatment resistance in human lung adenocarcinoma, which may be regulated ferroptotic cell death. Citation Format: Kunal R. Chaudhary, Connor J. Kinslow, Ling F. Ye, Pavan S. Upadhyayula, Eun Y. Lee, Ramon C. Sun, Simon K. Cheng. Serine hydroxymethyltransferase-2 as regulator of oxidative stress and chemoradiation resistance in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1992.

Radiotherapy is commonly used to treat a variety of solid human tumors, including localized prostate cancer. However, treatment failure often ensues due to tumor intrinsic or acquired radioresistance. Here we find that the MEK5/ERK5 signaling pathway is associated with resistance to genotoxic stress in aggressive prostate cancer cells. MEK5 knockdown by RNA interference sensitizes prostate cancer cells to ionizing radiation (IR) and etoposide treatment, as assessed by clonogenic survival and short-term proliferation assays. Mechanistically, MEK5 downregulation impairs phosphorylation of the catalytic subunit of DNA-PK at serine 2056 in response to IR or etoposide treatment. Although MEK5 knockdown does not influence the initial appearance of radiation- and etoposide-induced γH2AX and 53BP1 foci, it markedly delays their resolution, indicating a DNA repair defect. A cell-based assay shows that non-homologous end joining (NHEJ) is compromised in cells with ablated MEK5 protein expression. Finally, MEK5 silencing combined with focal irradiation causes strong inhibition of tumor growth in mouse xenografts, compared with MEK5 depletion or radiation alone. These findings reveal a convergence between MEK5 signaling and DNA repair by NHEJ in conferring resistance to genotoxic stress in advanced prostate cancer and suggest targeting MEK5 as an effective therapeutic intervention in the management of this disease.

Large-cell neuroendocrine carcinoma (LCNEC) accounts for approximately 3% of lung malignancies. There are limited data on the epidemiology and best treatment practices for this malignancy. This study aimed to be the largest cohort with the most up-to-date analysis of the epidemiology of LCNEC.The Surveillance, Epidemiology, and End Results (SEER) database was queried to identify cases of LCNEC diagnosed from 2010 through 2015, reflecting years the American Joint Committee on Cancer 7th edition staging system was in use. Using these data, we compared the epidemiology, demographics, clinical characteristics, and survival times of LCNEC with small-cell lung carcinoma (SCLC) and non-SCLC (NSCLC). Trends in incidence and mortality were recorded from 2004 to 2015.A total of 195,148 cases of lung cancer, including 1681 (0.9%) cases of LCNEC, were analyzed. LCNEC was more common among male subjects, and disease usually presented at stage IV (55%). Brain metastasis occurred more frequently in LCNEC (19.2%) than SCLC (16.7%, P .001) or NSCLC (13%, P .001). Incidence increased by 0.011 people per 100,000 per year, primarily of stage IV disease. Annual mortality from LCNEC doubled over the time period studied. Survival in patients with stage I-III LCNEC mirrored survival trends of patients with NSCLC, whereas stage IV LCNEC behaved similarly to SCLC.LCNEC generally presents at more advanced stages than NSCLC but earlier than SCLC. Stage I-III LCNEC behaves similarly to NSCLC, whereas stage IV is more akin to SCLC. LCNEC incidence is increasing. Despite this, it remains poorly studied and did not demonstrate an improved prognosis in our cohort.

Although radiation is widely used to treat cancers, resistance mechanisms often develop and involve activation of DNA repair and inhibition of apoptosis. Therefore, compounds that sensitize cancer cells to radiation via alternative cell death pathways are valuable. We report here that ferroptosis, a form of non-apoptotic cell death driven by lipid peroxidation, is partly responsible for radiation-induced cancer cell death. Moreover, we found that small molecules activating ferroptosis through system x(c)(−) inhibition or GPX4 inhibition synergize with radiation to induce ferroptosis in several cancer types by enhancing cytoplasmic lipid peroxidation, but not increasing DNA damage or caspase activation. Ferroptosis inducers synergized with cytoplasmic irradiation, but not nuclear irradiation. Finally, administration of ferroptosis inducers enhanced the anti-tumor effect of radiation in a murine xenograft model and in human patient-derived models of lung adenocarcinoma and glioma. These results suggest that ferroptosis inducers may be effective radiosensitizers that can expand the efficacy and range of indications for radiation therapy.

Metastasis represents the main route of cancer progression and is responsible for approximately 90% of all cancer-related deaths ([1][1]). Metastatic disease is generally considered incurable, emphasizing the need for novel biologically targeted pharmaceutical therapies. In this issue, Li and

Objective: Investigate the temporal effects of focused ultrasound (FUS)-induced blood–brain barrier (BBB) opening in post-radiotherapy mouse brains. Methods and materials: C57B6 mice without tumors were used to simulate the scenario after gross total resection (GTR) of brain tumor. Radiation dose of 6 Gy x 5 was delivered to one-hemisphere of the mouse brain. FUS-induced BBB-opening was delivered to the irradiated and non-irradiated brain and was confirmed with MRI. Dynamic MRI was performed to evaluate blood vessel permeability. Two time points were selected: acute (2 days after radiation) and chronic (31 days after radiation). Results: BBB opening was achieved after FUS in the irradiated field as compared to the contralateral non-irradiated brain without any decrease in permeability. In the acute group, a trend for higher gadolinium concentration was observed in radiated field. Conclusion: Localized BBB-opening can be successfully achieved without loss of efficacy by FUS as early as 2 days after radiotherapy. Advances in knowledge: Adjuvant radiation after GTR is commonly used for brain tumors. Focused ultrasound facilitated BBB-opening can be achieved without loss of efficacy in the post-irradiated brain as early as 2 days after radiation therapy. This allows for further studies on early application of FUS-mediated BBB-opening.

Purpose To establish a novel preclinical model for stereotactic radiosurgery (SRS) with combined mouselike phantom quality assurance in the setting of brain metastases. Methods and Materials C57B6 mice underwent intracranial injection of B16-F10 melanoma cells. T1-weighted postcontrast magnetic resonance imaging (MRI) was performed on day 11 after injection. The MRI images were fused with cone beam computed tomography (CBCT) images using the Small Animal Radiation Research Platform (SARRP). The gross tumor volume (GTV) was contoured using the MRI. A single sagittal arc using the 3 × 3 mm2 collimator was used to deliver 18 Gy prescribed to the isocenter. MRI was performed 7 days after radiation treatment, and the dose delivered to the mice was confirmed using 2 mouselike anthropomorphic phantoms: 1 in the axial orientation and the other in the sagittal orientation. The SARRP output was measured using a PTW Farmer type ionization chamber as per the American Association of Physicists in Medicine Task Group report 61, and the H-D curve was generated up to a maximum dose of 30 Gy. Irradiated films were analyzed based on optical density distribution and H-D curve. Results The tumor volume on day 11, before intervention, was 2.48 ± 1.37 mm3 in the no-SRS arm versus 3.75 ± 1.19 mm3 in the SRS arm (NS). In the SRS arm, GTV maximum dose (Dmax) and mean dose were 2048 ± 207 and 1785 ± 14 cGy. Using the mouselike phantoms, the radiochromic film showed close precision in comparison with projected isodose lines, with a Dmax of 1903.4 and 1972.7 cGy, the axial and sagittal phantoms, respectively. Tumor volume 7 days after treatment was 7.34 ± 8.24 mm3 in the SRS arm and 60.20 ± 40.4 mm3 in the no-SRS arm (P=.009). No mice in the control group survived more than 22 days after implantation, with a median overall survival (mOS) of 19 days; mOS was not reached in the SRS group, with 1 death noted. Conclusions Single-fraction SRS of 18 Gy delivered in a single arc can be delivered accurately with MRI T1-weighted postcontrast–based treatment planning. The mouse like phantom allows for verification of dose delivery and accuracy.

Introduction: Locally advanced non-small cell lung cancer (NSCLC) is highly resistant to chemoradiotherapy, and many cancer patients experience chronic stress. Studies that suggest stimulation of &beta, adrenergic receptors (&beta, AR) promotes tumor invasion and therapy resistance. We investigated whether &beta, AR inhibition with beta-blockers acts as a chemotherapy and radiation sensitizer in vitro and in patients treated with chemoradiation for locally advanced NSCLC. Methods: We investigated the effects of the non-selective beta-blocker propranolol on two human lung adenocarcinoma cell lines (PC9, A549) treated with radiation or cisplatin. We retrospectively evaluated 77 patients with Stage IIIA NSCLC who received induction chemoradiation followed by surgery. Pathological and imaging response, metastatic rate, and survival were analyzed using SPSS v22.0 and PrismGraphpad6. Results: Propranolol combined with radiation or cisplatin decreased clonogenic survival of PC9 and A549 cells in vitro (p &lt, 0.05). Furthermore, propranolol decreased expression of phospho-protein kinase A (p-PKA), a &beta, adrenergic pathway downstream activation target, in both cell lines compared to irradiation or cisplatin alone (p &lt, 0.05). In patients treated for Stage IIIA NSCLC, 16 took beta-blockers, and 61 did not. Beta-blockade is associated with a trend to improved overall survival (OS) at 1 year (81.3% vs 57.4%, p = 0.08) and distant metastasis-free survival (DMFS) (2.6 years vs. 1.3 years, p = 0.16). Although beta-blocker use was associated with decreased distant metastases (risk ratio (RR) 0.19, p = 0.03), it did not affect primary tumor pathological response (p = 0.40) or imaging response (p = 0.36). Conclusions: &beta, AR blockade enhanced radiation and cisplatin sensitivity of human lung cancer cells in vitro. Use of beta-blockers is associated with decreased distant metastases and potentially improved OS and DMFS. Additional studies are warranted to evaluate the role of beta-blockers as a chemoradiation sensitizer in locally advanced NSCLC.

Background & Aims Receptor mediated cell death through the activation of caspases has been identified as an important mechanism to control life and death in various tissues and is thus crucial for the maintenance of liver tissue homeostasis. Here we investigated how caspase 8 (Casp8) differentially regulates immune-mediated liver injury and regeneration in distinct liver cell types during chronic liver injury. Methods Conditional knockout mice with hepatocellular ( Casp8 Δhepa ) and ubiquitous deletion of Casp8 ( Casp8 ΔMx ) were used in models of cholestatic hepatitis [(DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine) treatment, bile duct ligation (BDL) and choline deficient diet with ethionine supplementation (CDE)]. Results Mice with a hepatocellular deletion of Casp8 ( Casp8 Δhepa ) were protected after DDC-treatment. Animals with a ubiquitous conditional Casp8 knockout ( Casp8 ΔMx ) displayed a significantly enhanced liver injury in various models of cholestatic liver injury. This was associated with higher transaminases, bilirubin levels and finally more liver fibrosis. However, caspase 3 (Casp3) activity was reduced in both knockout strains, suggesting additionally mechanisms contributing to the phenotype. Casp8 ΔMx mice displayed a stronger infiltration of mononuclear immune cells and more proliferation of liver-parenchymal cells in periportal areas. Further analysis confirmed that these infiltrating immune cells are resistant against extrinsic apoptosis. Bone-marrow-transplantation (BMT) experiments demonstrated that Casp8-deficient bone marrow derived cells are responsible for increased liver injury in DDC fed mice. Conclusions Our results demonstrate that cell-type specific differences in apoptosis resistance mediated by Casp8 deletion are of significant relevance for the outcome of chronic liver injury.

Platinum-based concurrent chemoradiation therapy is the mainstay of treatment for locally advanced lung cancer. However patient outcomes remain dismal and overall survival at 5 years is only 15%. The resistance mechanisms for concurrent chemoradiation therapy are poorly studied. Cancer stem cells have been proposed to be the driver for many cancers including lung cancer and may be also responsible for therapy resistance. We sought therefore to identify therapy resistance pathways in lung cancer by using small interfering RNAs (siRNAs) to increase the sensitivity of tumor cells to therapeutic challenge. To identify novel genes that modulate the response to platinum chemotherapy and radiation therapy we have performed a genome-wide RNAi high-throughput screen via a shRNA viral library containing approx. 60,000 individual shRNAs on a human lung adenocarcinoma cell line (PC9). After two weeks of cisplatin and/or radiation treatment of PC9 cells with shRNA and controls, depleted versus enriched shRNAs were identified through library sequencing. We hypothesized that depleted shRNAs in the treated versus control cells might represent genes whose function is crucial for resistance to therapy-induced cell death. From the cisplatin and radiation screen, analysis of out of top 100 potential hits (depleted shRNAs) interestingly showed several cancer stem cells markers including Sox, Lrg6, and members of the Hedgehog signaling pathway Patched and Bmi1. We further determined the effects of Bmi1 on therapy resistance with survival assays by treating PC9 cells with Bmi1 inhibitor PTC-09. MTT cell survival and colonogenic assays was performed by treating PC9 cells with PTC-09 in triplicate and then treated with increasing dosage of cisplatin (0.1, 1 and 10 μM) or X-ray radiation (2, 4 and 6 Gy). Significantly decreased cell survival was observed in PTC-09 treated PC9 cells treated with cisplatin or radiation compared to control and cisplatin or radiation alone treated cells. Further colonogenic assay of PC9 cells treated with 2Gy+1 um cisplatin and increasing dosage of PTC-09 showed significant decrease in the ability of cells to form colonies compared to control. By performing an unbiased genome wide for therapeutic resistance, we have successfully identified and validated a target in the cancer stem cell pathways. We are further evaluating effect of Bmi1 using CRISPR-Cas9 knock out model. Citation Format: Kunal R. Chaudhary, Haiying Cheng, Balaz Halmos, Jose M. Silva, K.S. Clifford Chao, Tom K. Hei, Simon Cheng. Lung cancer stem cell targets sensitizing tumor cells to chemo therapy and radiation therapy by exploiting synthetic lethal relationships in an RNAi high-throughput screen. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4238. doi:10.1158/1538-7445.AM2015-4238