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Supplementary Table 1: List of the compounds in the epigenetic inhibitor library.

This file includes Supplementary Figures S1-S8. Figure S1: Inhibition of EPZ6438 and ASO-EZH2 sensitizes enzalutamide-resistant LNCaP cells to enzalutamide or ASO-AR. Figure S2: Design and validation of sgRNA targeting EZH2. Figure S3: Growth inhibitory effect of Enzalutamide in EZH2 loss-of-function CRPC cell lines. Figure S4: Knockdown efficiency of ASO-AR and ASO-EZH2 in CRPC cell lines. Figures S5: EZH2 ASOs inhibit proliferation of LNCaP-abl cells. Figure S6: EZH2 ASOs synergize with ASO-AR to inhibit growth of prostate cancer cells in soft-agar colony formation assay and Cell-titer Glo assay. Figure S7: Loss-of-function of EZH2 alters AR cistrome. Figure S8: Tumor characterization of xenografted study.

Supplementary information with additional materials and methods, references, and the legends for all supplementary figures.

Advanced prostate cancer initially responds to androgen deprivation therapy (ADT), but the disease inevitably recurs as castration-resistant prostate cancer (CRPC). Although CRPC initially responds to abiraterone and enzalutamide, the disease invariably becomes nonresponsive to these agents. Novel approaches are required to circumvent resistance pathways and to extend survival, but the mechanisms underlying resistance remain poorly defined. Our group previously showed the histone lysine-N-methyltransferase EZH2 to be overexpressed in prostate cancer and quantitatively associated with progression and poor prognosis. In this study, we screened a library of epigenetic inhibitors for their ability to render CRPC cells sensitive to enzalutamide and found that EZH2 inhibitors specifically potentiated enzalutamide-mediated inhibition of proliferation. Moreover, we identified antisense oligonucleotides (ASO) as a novel drug strategy to ablate EZH2 and androgen receptor (AR) expression, which may have advantageous properties in certain settings. RNA-seq, chromatin immunoprecipitation sequencing, and assay for transposase-accessible chromatin using sequencing demonstrated that EZH2 inhibition altered the AR cistrome to significantly upregulate AR signaling, suggesting an enhanced dependence of CRPC cells on this pathway following inhibition of EZH2. Combination treatment with ASO targeting EZH2 and AR transcripts inhibited prostate cancer cell growth in vitro and in vivo better than single agents. In sum, this study identifies EZH2 as a critical epigenetic regulator of ADT resistance and defines ASO-based cotargeting of EZH2 and AR as a promising strategy for the treatment of CRPC.Significance: Simultaneous targeting of lysine methyltransferase EZH2 and the AR with ASO proves a novel and effective therapeutic strategy in patients with CRPC. Cancer Res; 78(20); 5731–40. ©2018 AACR.

Amyloid cross-seeding and amyloid inhibition are two different research subjects being studied separately for different pathological purposes, in which amyloid cross-seeding targets to study the co-aggregation of different amyloid proteins and potential molecular links between different neurodegenerative diseases, while amyloid inhibition aims to design different molecules for preventing amyloid aggregation. While both amyloid cross-seeding and amyloid inhibition are critical for better understanding the pathological causes of different neurodegenerative diseases including Parkinson disease (PD) and Type 2 diabetes (T2D), less efforts have been made to reconcile the two phenomena. Herein, we proposed a new preventive strategy to demonstrate (i) the cross-seeding of octapeptide TKEQVTNV from α-synuclein (associated with PD) with hIAPP (associated with T2D) and (ii) the cross-seeding-promoted hIAPP fibrillization and cross-seeding-reduced hIAPP toxicity. Collective results confirmed that TKEQVTNV can indeed cross-seed with hIAPP monomers and oligomers, not protofibrils, to form β-structure-rich fibrils and to accelerate hIAPP fibrillization. Moreover, such cross-seeding-induced promotion effect by TKEQVTNV also rescued the pancreatic cells from hIAPP-induced cytotoxicity by increasing cell viability and reducing cell apoptosis simultaneously. This work provides a new angle to discover amyloid fragments and use them as amyloid modulators (inhibitors or promotors) to interfere with amyloid aggregation of other amyloid proteins, as well as sequence/structure basis to explore the amyloid cross-seeding between different amyloid proteins that may help explain a potential molecular talk between different neurodegenerative diseases. This article is protected by copyright. All rights reserved.

Immune thrombotic thrombocytopenic purpura (iTTP) is an acquired, fatal microangiopathy if untreated. Randomized controlled trials (RCTs) demonstrated faster time to response with addition of caplacizumab to standard of care (SOC). However, concerns about RCT selection bias and the high cost of caplacizumab warrant examination of all evidence, including real-world observational studies. In this systematic review and meta-analysis, we searched for comparative studies evaluating SOC with or without caplacizumab for the treatment of iTTP. We assessed risk of bias using the Cochrane risk-of-bias-2 tool (RCTs) and the Newcastle-Ottawa Scale (observational studies). The primary efficacy and safety outcomes were all-cause mortality and treatment-emergent bleeding, respectively. Secondary outcomes included exacerbation and relapse, refractory iTTP, and time to response. We included 2 high-quality RCTs and 3 observational studies at high risk of bias comprising 632 total participants. Compared with SOC, caplacizumab was associated with a nonsignificant reduction in the relative risk [RR] of death in RCTs (RR, 0.21; 95% confidence interval [CI], 0.05-1.74) and observational studies (RR, 0.62; 95% CI, 0.07-4.41). Compared with SOC, caplacizumab was associated with an increased bleeding risk in RCTs (RR, 1.37; 95% CI, 1.06-1.77). In observational studies, bleeding risk was not significantly increased (RR, 7.10; 95% CI, 0.90-56.14). Addition of caplacizumab was associated with a significant reduction in refractory iTTP and exacerbation risks and shortened response time but increased relapse risk. Frontline addition of caplacizumab does not significantly reduce all-cause mortality compared with SOC alone, although it reduces refractory disease risk, shortens time to response, and improves exacerbation rates at the expense of increased relapse and bleeding risk.

Odontoidectomy for symptomatic irreducible ventral brainstem compression at the craniovertebral junction may result in spine instability requiring subsequent instrumentation. There is no consensus on the importance of C1 anterior arch preservation in prevention of iatrogenic instability. We conducted a systematic review of the impact of C1 anterior arch preservation on postodontoidectomy spine stability.PubMed, Embase, Scopus, Web of Science, and Cochrane were searched following the PRISMA guidelines to include studies of patients undergoing odontoidectomy. Random-effect model meta-analyses were performed to compare spine stability between C1 anterior arch preservation versus removal and posttreatment outcomes between transoral approaches (TOAs) versus endoscopic endonasal approaches (EEAs).We included 27 studies comprising 462 patients. The most common lesions were basilar invagination (73.3%) and degenerative arthritis (12.6%). Symptoms included myelopathy (72%) and neck pain (43.9%). Odontoidectomy was performed through TOA (56.1%) and EEA corridors (34.4%). The C1 anterior arch was preserved in 16.7% of cases. Postodontoidectomy stabilization was performed in 83.3% patients. Median follow-up was 27 months (range, 0.1-145). Rates of spine instability were significantly lower (P = 0.004) when the C1 anterior arch was preserved. Postoperative clinical improvement and pooled complications were reported in 78.8% and 12.6% of patients, respectively, with no significant differences between TOA and EEA (P = 0.892; P = 0.346). Patients undergoing EEA had significantly higher rates of intraoperative cerebrospinal fluid leaks (P = 0.002).Odontoidectomy is safe and effective for treating craniovertebral junction lesions. Preservation of the C1 anterior arch seems to improve maintenance of spine stability. TOA and EEA show comparable outcomes and complication rates.

Decompressive craniectomy (DC) is a surgical procedure where a portion of the skull (flap) is removed to relieve the built-up pressure from the patient’s brain due to swelling of the brain tissue after a traumatic injury to the head. Subsequently, another surgical procedure called cranioplasty is carried out to fix an implant or bone flap in patients who have undergone DC. In this paper, an automatic design methodology for additive manufacturing of a PSCI (patient-specific cranial implant) has been proposed. The input is the DICOM digital data from a CT scan and the output is the STL file geometry of the cranial implant. The proposed method has been tested and validated using real de-identified DICOM data, and the resultant implant was 3D printed and fit to the skull of a cadaver. The key contribution made in this paper is the complete automation of the design of a PSCI based on the skull’s unique geometry using a combination of image-processing and computational geometry techniques. Another important characteristic of the proposed method is that medical professionals need not have any technical expertise in additive manufacturing or part design for generating a PSCI.

Multi-tyrosine kinase inhibitors (MTKIs) have thus far had limited success in the treatment of castration-resistant prostate cancer (CRPC). Here, we report a phase I-cleared orally bioavailable MTKI, ESK981, with a novel autophagy inhibitory property that decreased tumor growth in diverse preclinical models of CRPC. The anti-tumor activity of ESK981 was maximized in immunocompetent tumor environments where it upregulated CXCL10 expression through the interferon gamma pathway and promoted functional T cell infiltration, which resulted in enhanced therapeutic response to immune checkpoint blockade. Mechanistically, we identify the lipid kinase PIKfyve as the direct target of ESK981. PIKfyve-knockdown recapitulated ESK981's anti-tumor activity and enhanced the therapeutic benefit of immune checkpoint blockade. Our study reveals that targeting PIKfyve via ESK981 turns tumors from cold into hot through inhibition of autophagy, which may prime the tumor immune microenvironment in advanced prostate cancer patients and be an effective treatment strategy alone or in combination with immunotherapies.

This study explores the emergence and productivity of word order usage in Mandarin-speaking typically-developing (TD) children and children with autism spectrum disorder (ASD), and examines how this emergence relates to frequency of use in caregiver input. Forty-two caregiver-child dyads participated in video-recorded 30-min semi-structured play sessions. Eleven children with ASD were matched with 10 20-month-old TD children and another 11 children with ASD were matched with 10 26-month-old TD children, on expressive language. We report four major findings: (1) Preschool Mandarin-speaking children with ASD produced word order structures with pervasive ellipsis at similar rates to language-matched TD children, but also displayed differences from TD children in their usage of SVt and VtO frames; (2) Grammatical productivity was observed in both TD children and children with ASD; moreover, children with ASD with higher expressive language produced less stereotyped language; (3) Both TD children and children with ASD heard a range of word orders in their caregivers’ input, with TD children’s input greater in amount and complexity; however, caregivers of both groups also showed no age/language-related changes in word order usage; (4) Few word-order-specific correlations emerged between caregivers and their children; however, strong correlations were observed for mean length of utterances (MLU) for both groups: Caregivers who produced longer/more complex utterances had children who did the same. Taken together, it seems that despite their pragmatic deficits, the early grammatical knowledge of word order in Mandarin-exposed children with ASD is well preserved and in general follows the typical developmental pattern. Moreover, caregiver input is broadly rather than finely tuned to the linguistic development of TD children and children with ASD, and plays a more important role in children’s general syntactic development than in specific word order acquisition. Thus, early word order usage in preschool Mandarin-speaking TD children and children with ASD may be influenced by both caregiver input and child abilities.

Significance RAS proteins (HRAS, NRAS, and KRAS) integrate extracellular trophic signals to promote cell proliferation. Constitutively active KRAS drives tumor initiation and progression in nonsmall cell lung cancer (NSCLC). As RAS proteins are often refractory to direct pharmacological inhibition, RAS-interacting proteins are under investigation as potential drug targets. We previously found Argonaute 2 (AGO2) to bind RAS and positively regulate RAS-dependent signaling pathways. AGO2 knockdown blunted proliferation in NSCLC cells containing a KRAS-activating mutation. Here, we demonstrate that AGO2 promotes tumor progression in multiple mouse models of KRAS-driven NSCLC. In these animals, Ago2 knockout impairs tumor growth, lowers pathologic grade, and inhibits KRAS signaling. Targeting the AGO2-KRAS interaction may hold future therapeutic promise in NSCLC and other KRAS-driven malignancies., Lung cancer is the deadliest malignancy in the United States. Non–small cell lung cancer (NSCLC) accounts for 85% of cases and is frequently driven by activating mutations in the gene encoding the KRAS GTPase (e.g., KRASG12D). Our previous work demonstrated that Argonaute 2 (AGO2)—a component of the RNA-induced silencing complex (RISC)—physically interacts with RAS and promotes its downstream signaling. We therefore hypothesized that AGO2 could promote KRASG12D-dependent NSCLC in vivo. To test the hypothesis, we evaluated the impact of Ago2 knockout in the KPC (LSL-Kras G12D/+;p53f/f;Cre) mouse model of NSCLC. In KPC mice, intratracheal delivery of adenoviral Cre drives lung-specific expression of a stop-floxed KRASG12D allele and biallelic ablation of p53. Simultaneous biallelic ablation of floxed Ago2 inhibited KPC lung nodule growth while reducing proliferative index and improving pathological grade. We next applied the KPHetC model, in which the Clara cell–specific CCSP-driven Cre activates KRASG12D and ablates a single p53 allele. In these mice, Ago2 ablation also reduced tumor size and grade. In both models, Ago2 knockout inhibited ERK phosphorylation (pERK) in tumor cells, indicating impaired KRAS signaling. RNA sequencing (RNA-seq) of KPC nodules and nodule-derived organoids demonstrated impaired canonical KRAS signaling with Ago2 ablation. Strikingly, accumulation of pERK in KPC organoids depended on physical interaction of AGO2 and KRAS. Taken together, our data demonstrate a pathogenic role for AGO2 in KRAS-dependent NSCLC. Given the prevalence of this malignancy and current difficulties in therapeutically targeting KRAS signaling, our work may have future translational relevance.

Objectives Endonasal suturing is an investigational method for dural repair that has been reported to decrease the incidence of cerebrospinal fluid fistula. This method requires handling of single-shaft instrumentation in the narrow endonasal corridor. In this study, we designed a low-cost, surgical model using three-dimensional (3D) printing technology to simulate dural repair through the endonasal corridor and subsequently assess the utility of the model for surgical training. Methods Using an Ultimaker 2+ printer, a 3D-printed replica of the cranial base and nasal cavity was fitted with tissue allograft to recapitulate the dural layer. Residents, fellows, and attending surgeons were asked to place two sutures using a 0-degree endoscope and single-shaft needle driver. Task completion time was recorded. Participants were asked to fill out a Likert scale questionnaire after the experiment. Results Twenty-six participants were separated into groups based on their prior endoscope experience: novice, intermediate, and expert. Twenty-one (95.5%) residents and fellows rated the model as “excellent” or “good” in enhancing their technical skills with endoscopic instrumentation. Three of four (75%) of attendings felt that the model was “excellent” or “good” in usefulness for training in dural suturing. Novice participants required an average of 11 minutes for task completion, as compared with 8.7 minutes for intermediates and 5.7 minutes for experts. Conclusion The proposed model appears to be highly effective in enhancing the endoscopic skills and recapitulating the task of dural repair. Such a low-cost model may be especially important in enhancing endoscopic facility in countries/regions with limited access to cadaveric specimens.

Epigenome constitutes an important layer that regulates gene expression and dynamics during development and diseases. Extensive efforts have been made to develop epigenome profiling methods using a low number of cells and with high throughput. Chromatin immunoprecipitation (ChIP) is the most important approach for profiling genome-wide epigenetic changes such as histone modifications. In this report, we demonstrate microfluidic ChIPmentation (mu-CM), a microfluidic technology that enables profiling cell samples that individually do not generate enough ChIP DNA for sequencing library preparation. We used a simple microfluidic device to allow eight samples to be processed simultaneously. The samples were indexed differently using a tagmentation-based approach (ChIPmentation) and then merged for library preparation. A histone modification profile for each individual sample was obtained by demultiplexing the sequencing reads based on the indexes. Our technology allowed profiling 20 cells and is well suited for cell-type-specific studies using low-abundance tissues.

A pediatric artificial lung (PAL) is under development as a bridge to transplantation or lung remodeling for children with end-stage lung failure (ESLF). In order to evaluate the efficiency of a PAL, a disease model mimicking the physiologic derangements of pediatric ESLF is needed. Our previous right pulmonary artery ligation (rPA-LM) ovine model achieved that goal, but caused immediate mortality in nearly half of the animals. In this study, we evaluated a new technique of gradual postoperative right pulmonary artery (rPA) occlusion using a Rummel tourniquet (rPA-RT) in seven (25–40 Kg) sheep. This technique created a stable model of ESLF pathophysiology, characterized by high alveolar dead space (58.0±3.8 %), pulmonary hypertension (38.4±2.2 mmHg), tachypnea (79±20 breaths per minute), and intermittent supplemental oxygen requirement. This improvement to our technique provides the necessary physiologic derangements for testing a PAL, while avoiding the problem of high immediate perioperative mortality.

Both KRAS and EGFR are essential mediators of pancreatic cancer development and interact with Argonaute 2 (AGO2) to perturb its function. Here, in a mouse model of mutant KRAS-driven pancreatic cancer, loss of AGO2 allows precursor lesion (PanIN) formation yet prevents progression to pancreatic ductal adenocarcinoma (PDAC). Precursor lesions with AGO2 ablation undergo oncogene-induced senescence with altered microRNA expression and EGFR/RAS signaling, bypassed by loss of p53. In mouse and human pancreatic tissues, PDAC progression is associated with increased plasma membrane localization of RAS/AGO2. Furthermore, phosphorylation of AGO2Y393 disrupts both the wild-type and oncogenic KRAS-AGO2 interaction, albeit under different conditions. ARS-1620 (G12C-specific inhibitor) disrupts the KRASG12C-AGO2 interaction, suggesting that the interaction is targetable. Altogether, our study supports a biphasic model of pancreatic cancer development: an AGO2-independent early phase of PanIN formation reliant on EGFR-RAS signaling, and an AGO2-dependent phase wherein the mutant KRAS-AGO2 interaction is critical for PDAC progression., Argonaute 2 (AGO2) binds RAS and is required for cellular transformation. Here, the authors establish a KRAS-driven mouse model of pancreatic cancer with conditional loss of AGO2 and show that the early phase of neoplastic lesion initiation is dependent on EGFR/RAS but not AGO2, while AGO2 is required for pancreatic ductal adenocarcinoma progression and metastasis.

Conventional wisdom claims that racial diversity undermines public goods provision. In this paper, I show that socioeconomic diversity, instead, generates a form of collateral cooperation for public goods. I argue that socioeconomically integrated (non-segregated) cities have a higher incidence of the negative spatial externalities of inequality (e.g., crime, contamination) that spill over from impoverished localities. Such neighborhood effects, in turn, induce the middle-class' preferences for certain public goods, while decreasing the perceived relative efficacy of private solutions (e.g., private guards). Thus, socioeconomic integration -–through the spatial externalities mechanism-– enables preference convergence on the public provision of services in place of private options. To test the argument, I propose an instrument for segregation that interacts rural-to-urban predicted migration (i.e., shift-share instrument) of the poor with the destination locality's ``urban form.'' I combine this quasi-experimental strategy with neighborhood-level measures of class- and race-based segregation and an original face-to-face survey of preferences with over 4,000 households across 408 of the total 456 neighborhoods in the megacity of Sao Paulo, Brazil. The analysis introduces self-interest in reducing externalities as a new mechanism that drives cooperation for public goods. Using embedded mechanism vignettes, I distinguish this mechanism from alternatives mechanisms –e.g., racial tolerance, social affinity-– proposed in the literature.

The politics behind common-pool resource management are not well-understood. Using detailed satellite imagery data, I examine the effects of local political competition on deforestation across municipalities in the Brazilian Amazon, 2000-2012. I use the competitive of elections at higher levels of government to construct a shift-share instrument for causally estimating municipal politics competition. The results show that although the median voter in the Amazon has a strong preference for environmental conservation, local political competition causally increases deforestation. Drawing on qualitative interviews with bureaucrats and activists, I illustrate the ways in which "activist fatigue" and bureaucratic turnover explain this effect. Political turnover induces accompanying turnover in local bureaucratic structures, discouraging activists from mobilizing for conservation. To further test this mechanism, I code an original database of local protests and show that the findings cannot be explained by prominent alternative mechanisms related to partisanship, federal monitoring of local governments, nor local agricultural development. The analysis provides systematic and causal evidence of the political origins of the "tragedy of the commons.''

Lung cancer is the deadliest malignancy in the United States. Non-small cell lung cancer (NSCLC) accounts for 85% of cases, and is frequently driven by activating mutations in the gene encoding the KRAS GTPase (e.g. KRASG12D). Our previous work demonstrated that Argonaute 2 (AGO2)—a component of the RNA induced silencing complex (RISC)—physically interacts with KRAS and stabilizes it at the protein level. In multiple cell lines, AGO2 knockdown reduces KRAS protein level and attenuates cell proliferation. We therefore hypothesized that AGO2 could promote KRASG12D-dependent NSCLC in vivo. To test the hypothesis, we evaluated the impact of Ago2 knockout in the KPC mouse model of NSCLC. In KPC mice, intratracheal delivery of adenoviral Cre drives lung-specific expression of a stop-floxed KRASG12D allele and biallelic ablation of p53. Simultaneous biallelic ablation of floxed Ago2 inhibited KPC lung nodule growth while reducing proliferative index and improving pathological grade. We next applied the KPHetC model, in which the clara cell-specific CCSP-driven Cre activates KRASG12D and ablates a single p53 allele. In these mice, Ago2 ablation also reduced tumor size and grade. In both models, Ago2 knockout inhibited ERK phosphorylation in tumor nodules, indicating impaired KRAS signaling. Furthermore, RNA-seq analysis of KPC nodules demonstrated broadly reduced expression of Kras-related genes. Taken together, our data demonstrate a novel pathogenic role for AGO2 in KRAS-dependent NSCLC. Given the prevalence of this malignancy and current difficulties in therapeutically targeting KRAS signaling, our work has future translational relevance. Citation Format: Jean C. Tien, Seema Chugh, Andrew E. Goodrum, Yunhui Cheng, Lisha Wang, Rahul Mannan, Xiaoming Wang, Vijaya L. Dommeti, Yuping Zhang, Alice Xu, Fengyu Su, Xuhong Cao, Sunita Shankar, Arul M. Chinnaiyan. Ago2 induces Kras signaling and promotes tumor progression in mouse models of 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 2936.

Instability of the craniovertebral junction (CVJ) following odontoidectomy is relatively common. Traditionally, separate stage posterior atlantoaxial ± occipitocervical fusion is used for treatment. A transmucosal approach using a clean-contaminated route is associated with hypothetical risks of infectious complications. There is a paucity of information in the literature assessing the risk of surgical site infection (SSI) using the transmucosal approach for hardware placement. The authors conducted a literature search through PubMed identifying patients with pathology requiring transmucosal (i.e., transnasal or transoral) CVJ fixation. Studies that described 1) cases requiring a transmucosal approach and 2) associated infectious complications were included. Rates of SSIs, device removal, unplanned reoperation, and hardware failures were analyzed. Descriptive statistics and odds ratios (ORs) were used to compare complications. Nine studies with a total of 431 patients were identified. There were 4 (0.93%) superficial SSIs and 4 (0.93%) deep SSIs. In total, 1.86% of patients experienced SSI. There were 18 (4.18%) cases of unplanned reoperation, 4 (0.93%) related to SSI. Five (1.16%) patients required removal of their anterior fixation device, 4 (0.93%) related to SSI. ORs comparing our results with Medvedev et al's retrospective National Surgical Quality Improvement Program study assessing the risk associated with posterior cervical fixation showed no statistical difference between postoperative infection rates (OR = 0.72, P = 0.36). An extensive review of the literature found no evidence to suggest placement of spinal hardware via transmucosal corridor is associated with an increased risk of SSI.

In 2016, we identified a direct interaction between RAS and Argonaute 2 (AGO2), a key mediator of RNA-mediated gene silencing that is required for KRAS-driven oncogenesis using pancreatic and lung cancer cell line models. Recently, we employed the genetically engineered mouse model of pancreatic cancer to define the effects of conditional loss of AGO2 in KRASG12D driven pancreatic cancer. Genetic ablation of AGO2 did not interfere with development of the normal pancreas or KRASG12D-driven early precursor pancreatic intraepithelial neoplasia (PanIN) lesions. However, AGO2 loss prevents progression from early to late PanIN lesions, development of pancreatic ductal adenocarcinoma (PDAC), and metastatic progression. This results in a dramatic increase in survival of KRASG12D mutant mice deficient in AGO2 expression. Using validated pan-RAS and AGO2 antibodies for immunofluorescence (IF) and proximity ligation assay (PLA), we observed increased RAS and AGO2 co-localization at the plasma membrane in mouse and human pancreatic tissues associated with PDAC progression. AGO2 ablation permits PanIN initiation driven by the EGFR-RAS axis; however rather than progressing to PDAC, these lesions undergo profound oncogene-induced senescence (OIS). Since PanIN development requires EGFR and is not AGO2 dependent, we probed the effects of EGF stimulation in cell lines expressing wild-type and mutant forms of KRAS (using co-IP and PLA analyses). In wild-type RAS expressing cells, grown in media containing serum, RAS-AGO2 co-localization was limited to the intracellular regions of the cells, which dramatically increased and shifted to the plasma membrane under conditions of stress (serum starvation). Interestingly, EGF stimulation disrupted this membrane RAS-AGO2 interaction and restored it to intracellular basal levels. Using phosphorylation-deficient AGO2 mutants, we demonstrate that the disruption of wild type-RAS-AGO2 interaction is due to AGO2Y393 phosphorylation, a target of EGFR. Interestingly, the mutant KRAS-AGO2 interaction is not subject to EGFR activation, suggesting that although both the wild-type and mutant RAS bind AGO2, they are differentially regulated through growth factor receptor activation. Taken together, our study supports a biphasic model of pancreatic cancer development: an AGO2-independent early phase of PanIN formation reliant on EGFR and wild-type RAS signaling, and an AGO2-dependent phase wherein the mutant KRAS-AGO2 interaction is critical for PDAC progression. In the lung cancer mouse model, we also observed a similar dependence of AGO2 in KRAS-driven lung adenocarcinoma. Along with related abstracts detailing the mechanisms of OIS mediated by AGO2 (Ronald Siebenaler) and evidence of direct interaction between oncogenic KRAS and AGO2 with an affinity of 200nM (Jessica Waninger), we present our latest studies related to the KRAS-AGO2 interaction. Citation Format: Sunita Shankar, Jean Ching-Yi Tien, Ronald F. Siebenaler, Vijaya L. Dommeti, Sylvia Zelenka-Wang, Jessica Waninger, Xiao-Ming Wang, Kristin M. Juckette, Alice Xu, Seema Chugh, Malay Mody, Sanjana Eyunni, Andrew Goodrum, Grace Tsaloff, Yuping Zhang, Ingrid J. Apel, Lisha Wang, Javed Siddiqui, Richard D. Smith, Heather A. Carlson, John Tesmer, Xuhong Cao, Jiaqi Shi, Chandan Kumar-Sinha, Arul M. Chinnaiyan. An essential role for Argonaute 2 in mouse models of KRAS driven cancers [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A20.

KRAS and EGFR are known essential mediators of pancreatic cancer development. In addition, KRAS and EGFR have both been shown to interact with and perturb the function of Argonaute 2 (AGO2), a key regulator of RNA-mediated gene silencing. Here, we employed a genetically engineered mouse model of pancreatic cancer to define the effects of conditional loss ofAGO2inKRASG12D-driven pancreatic cancer. Genetic ablation ofAGO2does not interfere with development of the normal pancreas orKRASG12D-driven early precursor pancreatic intraepithelial neoplasia (PanIN) lesions. Remarkably, however,AGO2is required for progression from early to late PanIN lesions, development of pancreatic ductal adenocarcinoma (PDAC), and metastasis.AGO2ablation permits PanIN initiation driven by the EGFR-RAS axis, but rather than progressing to PDAC, these lesions undergo profound oncogene-induced senescence (OIS). Loss ofTrp53(p53) in this model obviates the requirement ofAGO2for PDAC development. In mouse and human pancreatic tissues, increased expression of AGO2 and elevated co-localization with RAS at the plasma membrane is associated with PDAC progression. Furthermore, phosphorylation of AGO2Y393by EGFR disrupts the interaction of wild-type RAS with AGO2 at the membrane, but does not affect the interaction of mutant KRAS with AGO2. ARS-1620, a G12C-specific inhibitor, disrupts the KRASG12C-AGO2 interaction specifically in pancreatic cancer cells harboring this mutant, demonstrating that the oncogenic KRAS-AGO2 interaction can be pharmacologically targeted. Taken together, our study supports a biphasic model of pancreatic cancer development: anAGO2-independent early phase of PanIN formation reliant on EGFR-RAS signaling, and anAGO2-dependent phase wherein the mutant KRAS-AGO2 interaction is critical to prevent OIS in PanINs and allow progression to PDAC.

Compared to firewood, charcoal is a relatively clean and convenient fuel. Nevertheless, the mass production of charcoal can contribute substantially to deforestation, rendering it imperative to regulate charcoal use. This article uses nationally representative panel data on Tanzania conducted in 2008 and 2010 to examine how charcoal expenditures change over time within any given household. The focus of the analysis will be on identifying certain socio-economic factors that affect charcoal use at the household level. The framing of the analysis on variation in time within each household addresses the omitted variable bias that often undermines inference from comparisons across different households. We find that while charcoal expenditures increase with household income, the rise in charcoal use with income is relatively gradual. Household size is unrelated to charcoal expenditures, but urban–rural differences in fuel choice are large even though we include household fixed effects in all specifications. In this regard, policymakers and urban planners need to pay particular attention to the role of urbanization in predicting trends in charcoal expenditures in developing countries like Tanzania.

India is the world’s second-largest host of projects implemented under the Kyoto Protocol’s Clean Development Mechanism (CDM). There is, however, considerable variation in the distribution of CDM projects implemented across different Indian states. While a large body of the literature examines cross-national variation in the implementation of CDM projects, few studies have analyzed the determinants of sub-national variation in different national contexts. We theorize that given India’s laissez-faire approach to CDM project implementation the availability of profitable climate mitigation opportunities and the political stability are two factors that promote CDM project implementation. Using sub-national data collected from a variety of sources, we conduct systematic analysis that provides empirical support for a set of hypotheses regarding the effects of these variables on project implementation. First, we find that states with a lot of public electricity-generating capacity and industrial capital implement more CDM projects than other states. Additionally, project developers rarely propose CDM projects during election years as a result of high levels of political uncertainty associated with those years. Our findings show that India’s liberal approach prevents the central government from using the CDM to promote sustainable development in less developed states. In India and other host countries where coordinated national policies to maximize their gains from CDM projects is absent, there is a paucity of project implementation in states that need it the most.

Although cancer immunotherapy has revolutionized cancer treatment, patient response to immunotherapy remains varied. Despite progress, the mechanisms limiting cancer immunotherapy are not yet fully understood. A low number of tumor infiltrating T cells (cold tumor) is one of the limiting factors for cancer immunotherapy. Agents that enhance immunotherapy by shifting cold tumors to hot tumors will greatly benefit cancer immunotherapy. In prostate cancer, the majority of tumors are known to be cold and hence, cancer immunotherapy is not the ideal treatment option. Here, we use a prostate cancer model as an example to demonstrate that modulating the tumor microenvironment through altering autophagy will change the tumor cytokine secretion profile, which in turn attracts immune lymphocytes into the tumor microenvironment. In tandem, we have identified a candidate compound known as ESK981 for such a purpose. Method A small molecule library was used for screening autophagy activity and cytokine secretion. Various types of human cancer cell lines (prostate, renal, bladder, breast etc.) and multiple syngeneic mouse lines were examined for autophagy activity as well as an in vitro response to interferon stimulation with or without ESK981. A syngeneic mouse prostate cancer was used for the in vivo examination of autophagy as well as the anti-tumor effect by ESK981 monotherapy and/or in combination with anti-PD-1 therapy. Conclusion We have discovered a robust, novel autophagy-modulating small molecule, named ESK981, for the treatment of various cancer types as a monotherapy. In addition, we have demonstrated that autophagy has an essential role in the anti-tumor effect of immunotherapy, especially for anti-PD-1 in syngeneic prostate cancer model. Therefore, the use of a small molecule such as ESK981 to target autophagy can enhance immunological infiltration induced by cancer immunotherapy, such as immune checkpoint blockade, for non-immunogenic tumors. Conflict of Interest Statement A.M.C. is a co-founder and serves on the Scientific Advisory Board of Esanik Therapeutics, Inc. which owns to the rights to the clinical development of ESK981. Esanik Therapeutics, Inc. did not fund or approve the conduct of this study. Funding This project is supported by Prostate Cancer Foundation. Citation Format: Yuanyuan Qiao, Jae E. Choi, Josh N. Vo, Jean C. Tien, Lisha Wang, Lanbo Xiao, Stephanie A. Simko, Andrew D. Delekta, Nathan B. Hodge, Parth Desai, Kristin Juckette, Alice Xu, Fengyun Su, Rui Wang, Xuhong Cao, Xiaoju Wang, Xiaoming Wang, Javed Siddiqui, Zhen Wang, Amélie Bernard, Ester Fernandez-Salas, Nora M. Navone, Ke Ding, Eeva-Liisa Eskelinen, Elisabeth I. Heath, Daniel J. Klionsky, Weiping Zou, Arul M. Chinnaiyan. Therapeutic targeting autophagy to sensitize cancer immunotherapy in various cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4153.

In 2016, we identified a direct interaction between RAS and Argonaute 2 (AGO2), a key mediator of RNA-mediated gene silencing, that is essential for KRAS-driven oncogenesis using pancreatic and lung cancer cell line models. Recently, we employed a genetically engineered mouse model of pancreatic cancer to define the effects of conditional loss of AGO2 in KRASG12D-driven pancreatic cancer (KC model). Genetic ablation of AGO2 did not interfere with development of the normal pancreas or KRASG12D-driven early precursor pancreatic intraepithelial neoplasia (PanIN) lesions. However, AGO2 loss prevents progression from early to late PanIN lesions, development of pancreatic ductal adenocarcinoma (PDAC), and metastatic progression. This results in a dramatic increase in the survival of KRASG12D mutant mice deficient in AGO2 expression. Mechanistically, lack of PanIN to PDAC progression was due to oncogene-induced senescence (OIS) through activation of EGFR-wild type RAS-phosphoERK signaling in the absence of AGO2. Using validated pan-RAS and AGO2 antibodies for immunofluorescence (IF) and proximity ligation assay (PLA), we observed increased RAS and AGO2 co-localization at the plasma membrane in mouse and human pancreatic tissues associated with PDAC progression. While AGO2 ablation permits PanIN initiation driven by the EGFR-RAS axis, these lesions undergo OIS rather than progressing to PDAC. Further, we used co-IP and PLA analyses to probe the effects of EGF stimulation in cell lines expressing wild-type and mutant forms of KRAS. In wild-type RAS expressing cells, RAS-AGO2 co-localization and interaction were limited to the intracellular regions of the cells, and dramatically increased and shifted to the plasma membrane under conditions of stress (serum starvation). Interestingly, EGF stimulation disrupted this membrane RAS-AGO2 interaction and restored it to intracellular levels. Using phosphorylation-deficient AGO2 mutants, we further demonstrate that the disruption of wild-type RAS-AGO2 interaction is due to EGFR-mediated AGO2Y393 phosphorylation. Interestingly, mutant KRAS-AGO2 interaction is not subject to EGFR activation, suggesting that although both the wild type and mutant RAS bind AGO2, they are differentially regulated through growth factor receptor activation. We will discuss our ongoing studies evaluating the effects of AGO2 ablation in the KRASG12Ddriven lung cancer mouse model and PDAC progression with p53 loss (KPC model). Our recent in vivo work supports a biphasic model of pancreatic cancer development: an AGO2-independent early phase of PanIN formation reliant on EGFR and wild-type RAS signaling, and an AGO2-dependent phase wherein the mutant KRAS-AGO2 interaction is critical for PDAC progression. Citation Format: Sunita Shankar, Jean Ching-Yi Tien, Ronald F. Siebenaler, Vijaya L. Dommeti, Sylvia Zelenka-Wang, Jessica Waninger, Kristin M. Juckette, Alice Xu, Xiao-Ming Wang, Seema Chugh, Malay Mody, Sanjana Eyunni, Andrew Goodrum, Grace Tsaloff, Yuping Zhang, Ingrid J. Apel, Javed Siddiqui, Richard D. Smith, Heather A. Carlson, John Tesmer, Xuhong Cao, Jiaqi Shi, Chandan Kumar-Sinha, Arul M. Chinnaiyan. An essential role for Argonaute 2 in mouse models of KRAS-driven cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 957.

The Kyoto Protocol’s Clean Development Mechanism (CDM) has the capacity to incentivize the international transfer of environmentally sound technologies. Given that both countries are expected to have similar incentives when managing the distribution of technology transfer within the country, why do sub-national patterns in the allocation of projects with technology transfer differ? Using comparable political–economic data compiled for China and India, we offer an explanation for these differences. In China, where the government regards the CDM as a tool for achieving sustainable development, technology transfer is concentrated in provinces that need it the most and that are most conducive to receiving transfers (i.e., economically less developed, yet heavily industrialized provinces). In India, where the government takes on a “laissez-faire” approach to the CDM, neither level of economic development nor that of industrialization affects clean technology transfer. In this regard, although the incentives are similar, the capacity to pursue them is not comparable. We test these hypotheses using data on CDM technology transfer across Chinese provinces and Indian states during the 6-year period from 2004 to 2010.

In this work we genetically programmed the M13 virus, so that the new clone expressed multifunctional coat protein sequences that selectively bind to designated surfaces and species. Using these new clones, composite network consisting of ironphosphate nanowire (a-FePO4) and single wall carbon nanotubes were self-assembled onto aluminum surfaces. Spectroscopic and electrochemical characterizations confirmed the 3D-structure as well as the electrochemical activity of such composite nanowires.

Background: Lung cancer is the deadliest malignancy in the United States. Non-small cell lung cancer (NSCLC) accounts for 85% of cases, and is frequently driven by activating mutations in the gene encoding the KRAS GTPase (e.g. KRASG12D). Our previous work demonstrated that Argonaute 2 (AGO2)—a component of the RNA induced silencing complex (RISC)—physically interacts with KRAS and stabilizes it at the protein level. In NSCLC cell lines, AGO2 knockdown reduces KRAS protein level and attenuates cell proliferation. We hypothesized that AGO2 acts as an oncogene in KRASG12D-dependent NSCLC in vivo. Methods: To test the hypothesis, we generated a NSCLC model by intercrossing mice harboring a stop-floxed KRASG12D allele with animals containing a tamoxifen-responsive Cre recombinase gene driven by the (clara cell-specific) CCSP promoter. These animals, which frequently die within 10 weeks of tamoxifen treatment, display pulmonary lesions including hyperplasia, adenoma and frank adenocarcinoma. We evaluated whether concomitant ablation of one or both copies of (floxed) AGO2 influenced the NSCLC phenotype. After tamoxifen administration, we monitored mice daily and harvested tissues when mice were clinically moribund or reached 16 weeks of post-treatment. We harvested lungs for gross/ pathological examination and gene/ protein expression analysis. Results: While pathologically discernable adenocarcinoma was detectable in AGO2+/+ and AGO2f/+ animals, AGO2f/f mice were free of these lesions. Despite this, deletion of AGO2 had no impact on frequency or extent of hyperplastic or adenomatous lesions. Immunohistochemistry demonstrated absence of AGO2 in tumor tissue of AGO2f/f mice. Conclusion: AGO2 promotes progression but not initiation of cancerous lesions in a mouse model of KRASG12D-dependent NSCLC. Citation Format: Andrew E. Goodrum, Lisha Wang, Alice Xu, Kristin M. Juckette, Arul M. Chinnaiyan, Jean C. Tien. Evaluating Ago2 as an oncogene in Kras-dependent lung carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5099.

The RAS gene family is among the most commonly mutated genes within cancer, but little progress has been made in successfully targeting RAS mutations. Targeting binding partners of mutated RAS presents as a promising alternative therapeutic strategy. With the goal of uncovering novel interactors of RAS, we recently identified Argonaute 2 (AGO2) of the RNA-induced silencing complex (RISC) as a novel partner of the KRAS through its Switch II domain. In order to assess the role of AGO2 in KRASG12D driven disease, we developed a mouse model of pancreatic cancer with conditional loss of AGO2. While AGO2 knockout did not prevent development of early precursor pancreatic intraepithelial (PanIN) lesions, AGO2 null lesions displayed increased activation of the EGFR-RAS signaling axis during PanIN development that failed to progress to late stage PanINs, pancreatic ductal adenocarcinoma (PDAC), and metastatic disease. This resulted in a dramatic increase in the survival of mice with AGO2 ablation. Unlike the PanINs in AGO2 sufficient mice, the early PanIN lesions with AGO2 ablation showed staining for the senescence associated beta galactosidase activity, suggesting that AGO2 loss induces oncogene induced senescence. To extend these observations and explore the role of AGO2 interaction with mutant forms of HRAS and NRAS proteins, we performed co-IP of AGO2 with RAS proteins using isoform specific antibodies. Both HRAS and NRAS bound AGO2 in T24 cells (HRASG12V) and SK-MEL-2 cells (NRASQ61H), respectively. In T24 cells, AGO2 knockdown led to the senescent phenotype and was accompanied with changes in the EGFR-RAS signaling axis, similar to that observed in the PanINs of the mice with AGO2 loss. In this cell line model, AGO2 loss reduced mutant HRAS expression and increased wild type RAS activity. These signaling effects were also consistent with our observation that AGO2 loss increased RAS activation in the mouse embryonic fibroblast (MEF) model. Together with our previous work with mutant KRAS dependent cells, these data suggest that 1) AGO2-wild type RAS binding prevents RAS activation and 2) mutant RAS-AGO2 association regulates oncogenic RAS levels in cell line models. Studies on the mouse model and the close proximity of RAS and AGO2 with EGFR also furthered our understanding of the RAS-AGO2 interaction. Using a variety of cell line models, we observed that EGFR-mediated phosphorylation of AGO2Y393 disrupts the interaction between WT RAS and AGO2. However, the mutant KRAS-AGO2 interaction was recalcitrant to EGFR regulation. This provides the first instance of a nucleotide dependent association of RAS and AGO2 and sheds light on the dynamic nature of the RAS-AGO2 interaction. Citation Format: Ronald F. Siebenaler, Sunita Shankar, Jean C. Tien, Vijaya L. Dommeti, Sylvia Zelenka-Wang, Seema Chugh, Ingrid J. Apel, Malay Mody, Anudeeta Gautam, Chandan Kumar-Sinha, Arul M. Chinnaiyan. An essential role for Argonaute 2 in EGFR-KRAS signaling in pancreatic cancer development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 956.

Rescue workers and residents exposed to the environment surrounding the collapse of the World Trade Center (WTC) on September 11, 2001, have suffered a disproportionate incidence of chronic lung disease attributed to the inhalation of airborne dust. To date, the pathophysiology of this lung disease is poorly understood. The aim of this study was to examine whether airborne dust contaminants recovered from the surrounding area 24-48 h after the collapse of the WTC demonstrate direct cytotoxicity to two airway cell types that were most directly exposed to inhaled dust, airway epithelial and smooth muscle cells. It was also of interest to determine whether the presence of these dusts could modulate the effects of cigarette smoke on these cell types in that some of the individuals who responded to the collapse site were also smokers. Human cultured airway epithelial (BEAS-2B) cells were exposed to 10% cigarette smoke extract (CSE), WTC dust particles (10-53 μm; 0.01-0.5 μg/μl), or a combination of the two for 2-24 h. Cell viability was measured by determining mitochondrial integrity (MTT assays) and apoptosis (poly-ADP-ribose polymerase [PARP] immunoblotting). Conditioned cell culture media recovered from the CSE- and/or WTC dust-exposed BEAS-2B cells were then applied to cultured human airway smooth muscle cells that were subsequently assayed for mitochondrial integrity and their ability to synthesize cyclic AMP (a regulator of airway smooth muscle constriction). BEAS-2B cells underwent necrotic cell death following exposure to WTC dust or CSE for 2-24 h without evidence of apoptosis. Smooth muscle cells demonstrated cellular toxicity and enhanced cyclic AMP synthesis following exposure to conditioned media from WTC- or CSE-exposed epithelial cells. These acute toxicity assays of WTC dust and CSE offer insights into lung cell toxicity that may contribute to the pathophysiology of chronic lung disease in workers and residents exposed to WTC dust. These studies clearly showed that WTC dust (at least the supercoarse particle fraction) or CSE alone exerted direct adverse effects on airway epithelial and smooth muscle cells, and altered the signaling properties of airway smooth muscle cells. In addition the combination of CSE and WTC exerted an interactive effect on cell toxicity. It remains to be determined whether these initial cell death events might account, in part, for the chronic lung effects associated with WTC dust exposure among First Responders and others.