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The NCCN Guidelines for Non–Small Cell Lung Cancer (NSCLC) provide recommendations for management of disease in patients with NSCLC. These NCCN Guidelines Insights focus on neoadjuvant and adjuvant (also known as perioperative) systemic therapy options for eligible patients with resectable NSCLC.

The retrovirus human immunodeficiency virus-1 (HIV-1) is the causative agent of AIDS. Although treatment of HIV/AIDS with antiretroviral therapy provides suppression of viremia, latent reservoirs of integrated proviruses preclude cure by current antiviral treatments. Understanding the mechanisms of host–viral interactions may elucidate new treatment strategies. Here, we performed a CRISPR/Cas9 transcriptional activation screen using a high-complexity, genome-wide sgRNA library to identify cellular factors that inhibit HIV-1 infection of human CD4+ T cells. MT4 cells were transduced with a CRISPR/Cas9 sgRNA library and infected with nef-deficient HIV-1NL4-3 expressing ganciclovir-sensitive thymidine kinase, thus enabling selection of HIV-1-resistant cells for analysis of enriched sgRNAs. After validation of screen hits, multiple host factors essential for HIV-1 infection were identified, including SET (SET nuclear proto-oncogene) and ANP32A (acidic nuclear phosphoprotein 32A, PP32A), which together form a histone acetylase inhibitor complex. Using multiple human cell lines and peripheral blood mononuclear cells (PBMCs) from healthy donors and HIV-1-infected individuals, we demonstrate that SET depletion increased HIV-1 infectivity by augmenting DNA integration without significantly changing sites of integration. Conversely, SET overexpression decreased HIV-1 integration and infectivity. SET protein expression was significantly reduced in PBMCs from HIV-1-infected individuals and was downregulated by HIV-1 infection of healthy donor cells in vitro. Notably, HIV-1-induced downregulation of SET could be alleviated by inhibition of the protease granzyme A. Altogether, we have identified cellular inhibitors of HIV-1 infection on a genome-wide scale, which affords new insight into host–virus interactions and may provide new strategies for HIV-1 treatment.

Legend for Supplementary Figures S1-S8

Mutation Background of Non-Small Cell Lung Cancer Lines.

Global miRNA functional screens can offer a strategy to identify synthetic lethal interactions in cancer cells that might be exploited therapeutically. In this study, we applied this strategy to identify novel gene interactions in KRAS-mutant cancer cells. In this manner, we discovered miR-1298, a novel miRNA that inhibited the growth of KRAS-driven cells both in vitro and in vivo. Using miR-TRAP affinity purification technology, we identified the tyrosine kinase FAK and the laminin subunit LAMB3 as functional targets of miR-1298. Silencing of FAK or LAMB3 recapitulated the synthetic lethal effects of miR-1298 expression in KRAS-driven cancer cells, whereas coexpression of both proteins was critical to rescue miR-1298–induced cell death. Expression of LAMB3 but not FAK was upregulated by mutant KRAS. In clinical specimens, elevated LAMB3 expression correlated with poorer survival in lung cancer patients with an oncogenic KRAS gene signature, suggesting a novel candidate biomarker in this disease setting. Our results define a novel regulatory pathway in KRAS-driven cancers, which offers a potential therapeutic target for their eradication. Cancer Res; 76(19); 5777–87. ©2016 AACR.

NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Non–Small Cell Lung Cancer (NSCLC) provide recommended management for patients with NSCLC, including diagnosis, primary treatment, surveillance for relapse, and subsequent treatment. Patients with metastatic lung cancer who are eligible for targeted therapies or immunotherapies are now surviving longer. This selection from the NCCN Guidelines for NSCLC focuses on targeted therapies for patients with metastatic NSCLC and actionable mutations.

IMPORTANCE: Few clinical trials have been specifically designed for elderly patients with advanced non–small cell lung cancer (NSCLC), and the anticipated increase in the number of such patients has prompted a search for new treatment options that provide a greater palliative benefit. OBJECTIVE: To determine whether treatment with carboplatin plus pemetrexed followed by pemetrexed maintenance is noninferior compared with docetaxel monotherapy with regard to overall survival (OS) for elderly patients with advanced nonsquamous NSCLC. DESIGN, SETTING, AND PARTICIPANTS: This open-label, multicenter, noninferiority phase 3 randomized clinical trial was conducted at 79 institutions in Japan. Cytotoxic chemotherapy–naive patients with advanced nonsquamous NSCLC, an Eastern Cooperative Oncology Group performance status of 0 or 1, and age of 75 years or older were enrolled between August 2013 and February 2017. Data were analyzed from November 2018 to February 2019. INTERVENTIONS: Patients were randomized to receive either docetaxel monotherapy (60 mg/m(2)) every 3 weeks or 4 cycles of carboplatin (area under the curve of 5) plus pemetrexed (500 mg/m(2)) administered every 3 weeks followed by maintenance therapy with the same dose of pemetrexed for 3 weeks. MAIN OUTCOMES AND MEASURES: The primary end point was OS analyzed on an intention-to-treat basis with a noninferiority margin of 1.154 for the upper limit of the 95% CI of the hazard ratio (HR) estimated with a stratified Cox regression model. RESULTS: Of the 433 enrolled patients, 250 (57.7%) were male, and the median (range) age was 78 (75-88) years. The median OS was 15.5 months (95% CI, 13.6-18.4) in the docetaxel group (n = 217) and 18.7 months (95% CI, 16.0-21.9) in the carboplatin-pemetrexed group (n = 216), with a stratified HR for OS of 0.850 (95% CI, 0.684-1.056; P for noninferiority = .003). Progression-free survival was also longer in the carboplatin-pemetrexed group (unstratified HR, 0.739; 95% CI, 0.609-0.896). Compared with those in the docetaxel group, those in the carboplatin-pemetrexed had lower rates of leukopenia (60 of 214 [28.0%] vs 147 of 214 [68.7%]) and neutropenia (99 of 214 [46.3%] vs 184 of 214 [86.0%]) of grade 3 or 4 and of febrile neutropenia (9 of 214 [4.2%] vs 38 of 214 [17.8%]) and higher rates of thrombocytopenia (55 of 214 [25.7%] vs 3 of 214 [1.4%]) and anemia (63 of 214 [29.4%] vs 4 of 214 [1.9%]) of grade 3 or 4. Dose reductions were less frequent with carboplatin-pemetrexed. CONCLUSION AND RELEVANCE: Carboplatin-pemetrexed treatment followed by pemetrexed maintenance is a valid option for first-line treatment of elderly patients with advanced nonsquamous NSCLC. TRIAL REGISTRATION: University Hospital Medical Information Network Clinical Trials Registry Identifier: UMIN000011460

SUMMARY We perform a CRISPR-Cas9 genome-wide screen in glioblastoma stem cells and identify integrin αvβ5 as an internalization factor for Zika virus (ZIKV). Expression of αvβ5 is correlated with ZIKV susceptibility in various cells and tropism in developing human cerebral cortex. A blocking antibody against integrin αvβ5, but not αvβ3, efficiently inhibits ZIKV infection. ZIKV binds to cells but fails to internalize when treated with integrin αvβ5-blocking antibody. αvβ5 directly binds to ZIKV virions and activates focal adhesion kinase, which is required for ZIKV infection. Finally, αvβ5 blocking antibody or two inhibitors, SB273005 and cilengitide, reduces ZIKV infection and alleviates ZIKV-induced pathology in human neural stem cells and in mouse brain. Altogether, our findings identify integrin αvβ5 as an internalization factor for ZIKV, providing a promising therapeutic target, as well as two drug candidates for prophylactic use or treatments for ZIKV infections., Graphical Abstract, In Brief Wang et al. show that Zika virus (ZIKV) uses integrin αvβ5 to infect neural stem cells. ZIKV infection can be inhibited by αvβ5 blocking antibody or inhibitors, SB273005 and cilengitide, in human neural stem cells and in mouse brain, providing drug candidates for prophylactic use or treatments for ZIKV infections.

Background: Neoadjuvant anti-PD-(L)1 therapy may offer clinical benefit in resectable, early-stage NSCLC. NeoCOAST (NCT03794544) is a global, randomized phase 2 study of the anti-PD-L1 monoclonal antibody (mAb) durvalumab (D) alone or combined with the anti-CD73 mAb oleclumab (O), the anti-NKG2A mAb monalizumab (M), or the anti-STAT3 antisense oligonucleotide danvatirsen (Da) as neoadjuvant therapy. Methods: Pts with previously untreated, cytologically/histologically documented, resectable, Stage I [>2 cm]-IIIA NSCLC and ECOG PS 0-1 were randomized 1:1:1:1 (stratified by lymph node involvement) to receive D 1500 mg IV alone Q4W or combined with O 3000 mg IV Q2W, M 750 mg IV Q2W, or Da 200 mg IV QW, for one 28-day cycle, followed by surgery. Da monotherapy was also given on days 1, 3 and 5 of the week prior to combination treatment. The primary endpoint was investigator-assessed major pathological response (MPR; ≤10% residual viable tumor cells at tumor site and nodes, at surgery). Secondary endpoints were pathological complete response (pCR; no viable tumor cells), safety and tolerability, feasibility of surgery, PK and immunogenicity. Exploratory endpoints included tumor and microbiome biomarkers and blood mRNA signatures. Results: From March 2019 to Sept 2020, 84 pts were randomized and 83 received D (n=26), D+O (n=21), D+M (n=20), or D+Da (n=16). MPR occurred in 11.1% (95% CI, 2.4-29.2), 19.0% (95% CI, 5.4-41.9), 30.0% (95% CI, 11.9-54.3), and 31.3% (95% CI, 11.0-58.7%), respectively. pCR occurred in 3.7% (95% CI, 0.1-19.0), 9.5% (95% CI, 1.2-30.4), 10.0% (95% CI, 1.2-31.7%) and 12.5% (95% CI, 1.6-38.3), respectively. Rates of treatment-related AEs were 34.6% with D, 57.1% with D+O, 50.0% with D+M, and 43.8% with D+Da (grade ≥3 in 0%, 4.8%, 0% and 6.3%, respectively). Most pts (76/83; 91.6%) completed surgery with no significant delay; of the 7 pts unable to, 5 had progressive or stage IV disease. Across all arms, MPR was more common in pts with baseline tumor PD-L1 expression ≥1% vs those with Conclusions: One cycle of D + O, M or Da improved MPR and pCR rates vs D alone, with no new safety signals. Responses were associated with baseline tumor PD-L1 and CD73 expression levels. Pts with MPR receiving D+O or D+M had peripheral transcriptomic signatures related to immune cell function. These data warrant further investigation of these agents in resectable NSCLC. Citation Format: Tina Cascone, Rosario García-Campelo, Jonathan Spicer, Walter Weder, Davey Daniel, David Spigel, Maen Hussein, Julien Mazieres, Julio Oliveira, Edwin Yau, Alexander Spira, Raymond Mager, Oday Hamid, Lin-Yang Cheng, Ying Zheng, Jorge Blando, Lara McGrath, Italia Grenga, Yee Soo-Hoo, Rakesh Kumar, Patrick Forde. NeoCOAST: open-label, randomized, phase 2, multidrug platform study of neoadjuvant durvalumab alone or combined with novel agents in patients (pts) with resectable, early-stage non-small-cell lung cancer (NSCLC) [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 CT011.

8520 Background: There are clinical controversies surrounding the US FDA approval of Osimertinib in December 2020 as adjuvant therapy, based on disease-free survival (DFS) improvement in patients (pts) with surgically resected stage IB-IIIA EGFRm NSCLC. We initiated a survey study to investigate our hypothesis that DFS benefit alone even without significant OS maybe deemed a valuable endpoint to pts after considering trade-offs. Methods: Participants were recruited from pts seen at the RP Thoracic Clinic from 01/21 to 12/21. Eligible pts who were being evaluated for adjuvant systemic therapy following surgical resection were given a self-administered survey based on the validated questionnaire by Blinman et al, which was modified to provide explanation of the differences between OS and DFS and the ADAURA trial results. Survey responses were collected in an online repository. Associations between survey responses and demographics were assessed using Fisher’s exact test. Changes in preference responses were assessed using McNemar’s test. Results: A total of 524 pts with NSCLC were screened, of which 101 pts were eligible to receive the survey. 51 pts (50%) responded to the survey. Median age of respondents was 69yrs (37-83), majority were female (69%, n = 35,), married (61%, n = 31), retired ( 63%, n = 32), had at least some college or higher education level(54%, n = 28), with history of smoking (84%, n = 43) and with stage IIIA (43%, n = 22) adenocarcinoma (80%, n = 41). To evaluate toxicity-related tradeoffs (Q1), a ≥12 mo. improvement in OS benefit was needed for 66% of pts to consider adjuvant Osi. However, an increase of ≥ 6 mo. of DFS was enough for 66% of pts to justify taking a daily medication (Q2). One mo. increase in DFS or OS was not enough for 60% and 78% of pts respectively to justify taking the medication. A threshold 1% increase in 5-year OS was sufficient to persuade patients to take Osi for three years, even with respect to toxicity side effects (p = 0.023). (Q3). Finally, in the hypothetical cost-based scenario (Q4), there was no indication that pts were willing to pay more for each incremental increase in OS. There appears to be some association between employment status (p =.033) or educational degree (p =.049) for tolerance of side effects if there is at least 1 additional year of DFS or OS. Conclusions: We observed that the value patients ascribe to adjuvant Osimertinib is influenced by factors besides efficacy. Knowing pts' preferences for cancer treatments can better inform regulatory bodies in formulating cost-sharing structure for cancer therapies. Our study highlights the importance of shared decision making based on individual pts' preferences.

9002 Background: KRAS is a key mediator of the RAS/MAPK signaling cascade that promotes cellular growth and proliferation. KRASG12C mutation occurs in ̃14% of NSCLC. Adagrasib, an investigational agent, is a KRASG12C inhibitor that irreversibly and selectively binds KRASG12C, locking it in its inactive state. Adagrasib is optimized for favorable pharmacokinetic (PK) properties, including long half-life (̃24 h), dose-dependent PK, and central nervous system penetration; it has demonstrated objective response and favorable tolerability in the Phase 1/1b setting. Methods: KRYSTAL-1 (NCT03785249) is a multicohort Phase 1/2 study evaluating adagrasib as monotherapy or in combination regimens in patients with advanced solid tumors harboring a KRASG12C mutation. Here we report the first disclosure from all patients enrolled in Cohort A, a Phase 2 cohort with registrational intent, evaluating adagrasib given 600 mg orally BID in patients with NSCLC previously treated with platinum-based chemotherapy and anti-PD-1/L1 therapy. Study objectives include evaluating efficacy (objective response rate [ORR], duration of response [DOR], progression-free survival [PFS], overall survival [OS]), safety, PK, and exploratory correlative analyses. Objective tumor response was assessed per RECIST v1.1 by blinded independent central review (BICR). Results: As of the 15 October 2021 data cutoff, 116 patients with NSCLC harboring a KRASG12C mutation were enrolled and treated, with a median follow-up of 12.5 months. Baseline characteristics include median age 64 years, 65% female, and 15.5%/83.6% with ECOG PS 0/1; 98.3% of patients received adagrasib following prior treatment with immunotherapy and chemotherapy, with a median of 2 prior systemic therapies. The ORR (by BICR) was 42.9% (48/112) and the disease control rate was 79.5% (89/112); 31 patients remain on treatment. Median DOR was 8.5 months (95% CI 6.2–13.8), median PFS was 6.5 months (95% CI 4.7–8.4), median OS was 12.6 months (95% CI 9.2–NE). Treatment-related AEs (TRAEs) of any grade occurred in 97.4% of patients, grade ≥3 TRAEs in 45.7%, 2 grade 5 TRAEs, and 8 (6.9%) TRAEs led to discontinuation. The most commonly reported (≥25%) TRAEs (any grade) were diarrhea (62.9%), nausea (62.1%), vomiting (47.4%), fatigue (40.5%), ALT/AST increased (27.6%/25%), blood creatinine increased (25.9%); the most commonly reported (≥5%) TRAEs (grade 3/4) were lipase increased (6%) and anemia (5.2%). Additional subgroup analyses will be presented, including selected demographics, molecular markers and sites of metastases. Conclusions: Adagrasib is well tolerated and demonstrates promising efficacy in pretreated patients with NSCLC harboring a KRASG12C mutation. A Phase 3 trial evaluating adagrasib monotherapy versus docetaxel in previously treated patients with KRASG12C-mutant NSCLC is ongoing (NCT04685135). Clinical trial information: NCT03785249.

This cohort study investigates the association between nonsteroidal anti-inflammatory drug (NSAID) use and survival in patients with head and neck squamous cell carcinoma treated with chemoradiation therapy (CRT) at a single institution between 2005 and 2017., Key Points Question Is nonsteroidal anti-inflammatory drug (NSAID) use associated with a survival advantage in patients with head and neck squamous cell carcinoma who are treated with definitive chemoradiation therapy? Findings In this cohort study of 460 patients, NSAID use was associated with significantly better overall survival at 5 years compared with no use of concurrent NSAIDs. Meaning These findings suggest there may be an overall survival advantage for patients with head and neck squamous cell carcinoma who take NSAIDs during chemoradiation., Importance Nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin, are commonly prescribed medications with anti-inflammatory and antiplatelet properties used long term to decrease the risk of cardiovascular events. A recent study showed that aspirin was associated with improved survival in patients with head and neck squamous cell carcinoma (HNSCC) who were treated with surgery. Objective To examine whether use of NSAIDs during definitive chemoradiation therapy (CRT) was associated with improved outcomes in patients with HNSCC. Design, Setting, and Participants This cohort study analyzed patients with HNSCC who were treated with CRT at a single institution between January 1, 2005, and August 1, 2017. Patient and tumor characteristics included age, race/ethnicity, smoking status, alcohol use, comorbidities (respiratory, cardiovascular, immune, renal, endocrine), disease stage, human papillomavirus status, and treatment duration. Data were analyzed from May 1, 2019, to March 17, 2020. Exposures Patients were dichotomized by NSAID use during treatment. Main Outcomes and Measures The association of NSAID use with patterns of failure, disease-specific survival (DSS), and overall survival (OS) was examined using multivariate Cox proportional hazard regression models. Survival estimates for OS and DSS were generated using Kaplan-Meier survival curves. Results A total of 460 patients (median [interquartile range] age, 60 [53.9-65.6] years; 377 [82.0%] men) were included in the analysis. Among these patients, 201 (43.7%) were taking NSAIDs during treatment. On univariate analysis, NSAID use (hazard ratio [HR], 0.63; 95% CI, 0.43-0.92; P = .02) was associated with better OS. On Cox regression analysis, after backward selection adjustment for potentially confounding factors (age, smoking status, primary tumor site, human papillomavirus status, diabetes, stroke, hyperlipidemia), NSAID use remained significantly associated with better OS (HR, 0.59; 95% CI, 0.38-0.90; P = .02). NSAID use was associated with significantly better OS at 5 years compared with patients who did not take concurrent NSAIDs (63.6% [56 of 88 patients]; 95% CI, 58%-73% vs 56.1% [83 of 148 patients]; 95% CI, 50%-63%; P = .03). NSAID use was not associated with better DSS in univariate (HR, 0.82; 95% CI, 0.48-1.41; P = .47) or multivariate (HR, 0.98; 95% CI, 0.57-1.70; P = .44) analysis. NSAID use was not associated with better response to treatment (HR, 1.44; 95% CI, 0.91-2.27; P = .12) or distant failure (HR, 1.12; 95% CI, 0.68-1.84; P = .65). Change in local control with NSAID use was not statistically significant (HR, 0.59; 95% CI, 0.31-1.10; P = .10). Conclusions and Relevance This cohort study suggests a possible OS advantage for patients taking NSAIDs during chemoradiation for HNSCC. Further studies examining this association are warranted.

Background Resistance to immune checkpoint inhibitors (ICIs) has been linked to local immunosuppression independent of major ICI targets (e.g., PD-1). Clinical experience with response prediction based on PD-L1 expression suggests that other factors influence sensitivity to ICIs in non-small cell lung cancer (NSCLC) patients. Methods Tumor specimens from 120 NSCLC patients from 10 institutions were evaluated for PD-L1 expression by immunohistochemistry, and global proliferative profile by targeted RNA-seq. Results Cell proliferation, derived from the mean expression of 10 proliferation-associated genes (namely BUB1, CCNB2, CDK1, CDKN3, FOXM1, KIAA0101, MAD2L1, MELK, MKI67, and TOP2A), was identified as a marker of response to ICIs in NSCLC. Poorly, moderately, and highly proliferative tumors were somewhat equally represented in NSCLC, with tumors with the highest PD-L1 expression being more frequently moderately proliferative as compared to lesser levels of PD-L1 expression. Proliferation status had an impact on survival in patients with both PD-L1 positive and negative tumors. There was a significant survival advantage for moderately proliferative tumors compared to their combined highly/poorly counterparts (p = 0.021). Moderately proliferative PD-L1 positive tumors had a median survival of 14.6 months that was almost twice that of PD-L1 negative highly/poorly proliferative at 7.6 months (p = 0.028). Median survival in moderately proliferative PD-L1 negative tumors at 12.6 months was comparable to that of highly/poorly proliferative PD-L1 positive tumors at 11.5 months, but in both instances less than that of moderately proliferative PD-L1 positive tumors. Similar to survival, proliferation status has impact on disease control (DC) in patients with both PD-L1 positive and negative tumors. Patients with moderately versus those with poorly or highly proliferative tumors have a superior DC rate when combined with any classification schema used to score PD-L1 as a positive result (i.e., TPS ≥ 50% or ≥ 1%), and best displayed by a DC rate for moderately proliferative tumors of no less than 40% for any classification of PD-L1 as a negative result. While there is an over representation of moderately proliferative tumors as PD-L1 expression increases this does not account for the improved survival or higher disease control rates seen in PD-L1 negative tumors. Conclusions Cell proliferation is potentially a new biomarker of response to ICIs in NSCLC and is applicable to PD-L1 negative tumors. Electronic supplementary material The online version of this article (10.1186/s40425-019-0506-3) contains supplementary material, which is available to authorized users.

Background: The use of immune PD-1/PD-L1 checkpoint inhibitors (ICI) has dramatically altered the treatment of advanced Non-Small Cell Lung Cancer (NSCLC). However, a large proportion of patients with NSCLC do not derive clinical benefit from ICI treatment. Recent studies have identified STK11 mutations leading to loss of LKB1 in NSCLC as drivers of primary resistance to ICI treatment. Using a clinical cohort of patients whose tumors underwent comprehensive genomic and immune transcriptomic analysis, we characterized the immune gene expression profile of STK11 mutant non-squamous NSCLC tumors and identified TRIM29 as a potential therapeutic target in STK11 mutant non-squamous NSCLC. Methods: We performed comprehensive analysis of the genomic and immunological landscape of 199 formalin-fixed, paraffin-embedded tumor samples (obtained prior to ICI treatment) from advanced stage non-squamous NSCLC patients treated at Roswell Park Comprehensive Cancer Center using a CLIA-certified laboratory test that included targeted NGS genomic sequencing, gene fusion analysis, and RNA-seq of 394 immune transcripts. Differential gene expression analysis was performed using R/Bioconductor package limma with Benjamini-Hochberg adjusted p-values reported. This study was approved by Roswell Park internal review board review (protocol BDR 091817) according to institutional policy for nonhuman subjects research. Results: Among the cohort of 199 cases, 32 contained truncating STK11 mutations. STK11 mutant cases showed an immunosuppressive environment with decreased PD-L1 expression (p=0.01) and decreased T-cell inflammation gene expression signature (p=0.0029) consistent with prior published cohorts. Analysis of differentially expressed immune genes signatures showed significant decreases in antigen presentation (p=0.015) and processing (p=0.044), NK cell activation (p=0.039), chemokine and cytokine signaling (p=0.044), immune checkpoint pathways (p=0.015), innate immune response (p=0.04), and interferon signaling (p=0.039). The top upregulated gene in STK11 mutant cases was TRIM29 (p=0.03). Other overexpressed targets included ID3 (p=0.009), KRT7 (p=0.02), and NOTCH3 (p=0.05). Interrogation of TCGA LUAD dataset also demonstrated elevated TRIM29 expression (p=0.006) in STK11-mutant compared to STK11-wild-type samples and we also confirmed elevated TRIM29 RNA and protein expression in a panel of STK11 mutant NSCLC human cell lines. Immunohistochemical staining of a cohort of 68 KRAS mutant NSLCLC patient samples also showed higher levels of TRIM29 protein staining in STK11 mutant samples. Knockdown of TRIM29 using shRNA inhibited growth of STK11 mutant tumor xenografts. TRIM29 is a E3 ubiquitin ligase and we evaluated known targets of TRIM29 and found decreased TAB2 expression in STK11 mutant NSCLC. Notably, TAB2 is also regulated by SIK1/3, which have been recently identified as signaling partners with STK11. TRIM29 has been implicated as a mediator of processes also involved in STK11 tumorigenesis such as cellular metabolism, DNA damage repair, innate immunity, and squamous differentiation. Conclusion: Using a large clinical cohort of non-squamous NSCLC patients we characterized the immunosuppressive environment of STK11 mutant tumors and identified the E3 ubiquitin ligase TRIM29 as a potential therapeutic target. Citation Format: Edwin Yau, Sarabjot Pabla, Sean Glenn, Antonios Papanicolau-Sengos, Mary Nesline, Grace Dy, Carl Morrison. Clinical immune and genomic profiling of STK11 mutant non-squamous non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5903.

Genome-wide functional genomic screens utilizing the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system have proven to be a powerful tool for systematic genomic perturbation in mammalian cells and provide an alternative to previous screens utilizing RNA interference technology. The wide availability of these libraries through public plasmid repositories as well as the decreasing cost and speed in quantifying these screens using high-throughput next-generation sequencing (NGS) allows for the adoption of the technology in a variety of laboratories interested in diverse biologic questions. Here, we describe the protocol to generate next-generation sequencing libraries from genome-wide CRISPR genomic screens.

62 Background: Cell proliferation is an important marker of survival in many tumors, and we hypothesized that this attribute could be related to response to immune checkpoint inhibitors (ICIs) in RCC. Previously we reported (SITC 2018) that moderately proliferative lung cancer have a much higher response rate than either poorly or highly proliferative tumors. Methods: 69 FFPE RCC tumor samples were evaluated by RNA-seq to measure transcript levels of 394 immune related genes. Cell proliferation was defined as the mean mRNA expression of 10 genes (BUB1, CCNB2, CDK1, CDKN3, FOXM1, KIAA0101, MAD2L1, MELK, MKI67, TOP2A) which was evaluated for association with ORR to ICIs by RECIST v1.1 criteria for both PD-L1 IHC positive and negative cases. Cell proliferation for each case was split into 3 tertiles of poorly ( < 33), moderately (33-66) and highly ( > 66) proliferative versus a reference population. Poorly and highly proliferative were grouped for comparison to moderately proliferative tumors. PD-L1 IHC was performed using DAKO 22C3 antibody and scored by FDA guidelines and considered positive if TPS ≥1% and negative if < 1%. Results: In our cohort of 69 patients, the overall ORR was 18.8. The majority, 91% of tumors were PD-L1 negative, with ORR of 14.8%, as opposed to ORR of 50% in PD-L1 positive cases. 62.2% of tumors were poorly proliferative, 8.7% were highly proliferative, and 29% were moderately proliferative. ORR in moderately proliferative tumors was 30% and 14.2% in poorly/highly proliferative tumors. In PD-L1 negative tumors the ORR in moderately proliferative tumors was 20% and 13% in poorly/highly proliferative tumors. For the 5 moderately proliferative, PD-L1 positive tumors, there were 2 PR and 1 CR, and in the 3 poorly/highly proliferative tumors there was 1 PR. Conclusions: Cell proliferation may play a crucial role in distinguishing RCC patients who may have a clinical benefit to ICI, including the important subgroup of PD-L1 negative tumors. Moderately proliferative tumors have a higher ORR than their poorly/highly counterparts.

61 Background: Cell proliferation is an important marker of survival in many tumors and we hypothesized that this attribute could be related to response to immune checkpoint inhibitors in RCC. Previously we reported (SITC 2018) moderately proliferative lung cancer has a much higher response rate than either poorly or highly proliferative tumors. Methods: 69 FFPE tumor samples of RCC were evaluated by RNA-seq to measure transcript levels of 394 immune related genes, including 10 related to cell proliferation (BUB1, CCNB2, CDK1, CDKN3, FOXM1, KIAA0101, MAD2L1, MELK, MKI67, TOP2A). Cell proliferation, defined as the mean mRNA expression of these 10 genes was evaluated for association with ORR to ICIs by RECIST v1.1 criteria for both PD-L1 IHC positive and negative cases. Cell proliferation for each case was split into 3 tertiles of poorly ( < 33), moderately (33-66) and highly ( > 66) proliferative compared to a reference population. Poorly and highly proliferative were grouped for comparison to moderately proliferative tumors. Tumors were inflamed or non-inflamed based upon RNA‐seq analysis of CD8 compared to a reference population of more than 500 cases of multiple tumors. Non-inflamed, or immune desert tumors, defined as the lower 25th percentile of rank for CD8+ T-cells, and greater than 75th percentile of rank as inflamed. Results: In our cohort of 69 patient the overall ORR was 18.8%. 15.9% of tumors were non-inflamed with an ORR of 9.1%. For 36.2% inflamed tumor the ORR was 32%. For cell proliferation 62.2% were poorly proliferative, 8.7% were highly proliferative, and 29% were moderately. ORR in moderately proliferative tumors was 30% versus 14.2% in poorly/highly proliferative tumors. In inflamed tumors, ORR in moderately proliferative tumors was 37.5% as opposed to 17.6% in poorly/highly proliferative tumors. In 11 non-inflamed tumors, there was only one responder, which was a poorly/highly proliferative tumor. Conclusions: Cell proliferation may play a crucial role in distinguishing RCC patients who may have a clinical benefit to ICI, including the important subgroup of inflamed tumors. Moderately proliferative tumors have a higher ORR than their poorly/highly counterparts.

Global miRNA functional screens can offer a strategy to identify synthetic lethal interactions in cancer cells that might be exploited therapeutically. In this study, we applied this strategy to identify novel gene interactions in KRAS-mutant cancer cells. In this manner, we discovered miR-1298, a novel miRNA that inhibited the growth of KRAS-driven cells both in vitro and in vivo. Using miR-TRAP affinity purification technology, we identified the tyrosine kinase FAK and the laminin subunit LAMB3 as functional targets of miR-1298. Silencing of FAK or LAMB3 recapitulated the synthetic lethal effects of miR-1298 expression in KRAS-driven cancer cells, whereas coexpression of both proteins was critical to rescue miR-1298–induced cell death. Expression of LAMB3 but not FAK was upregulated by mutant KRAS. In clinical specimens, elevated LAMB3 expression correlated with poorer survival in lung cancer patients with an oncogenic KRAS gene signature, suggesting a novel candidate biomarker in this disease setting. Our results define a novel regulatory pathway in KRAS-driven cancers, which offers a potential therapeutic target for their eradication. Cancer Res; 76(19); 5777–87. ©2016 AACR.

The purpose of this investigation was to determine the cytotoxicity of amphotericin B (AMB; trade name Fungizone [FZ]) following the administration of FZ and a heat-treated form of FZ (HFZ) to LLC-PK 1 pig kidney cells and Cryptococcus neoformans var. gattii cells. HFZ was significantly less toxic to kidney cells than FZ at all concentrations tested. For both FZ and HFZ, the concentration range which resulted in a 50% reduction of the growth of fungal cells was 0.125 to 1 mg/ml. These findings suggest that heat treatment decreases AMB's renal cytotoxicity without modifying its antifungal activity.

The KRAS oncogene is frequently mutated in many of the most lethal human cancers and has been resistant to targeted therapies, leading to efforts to identify synthetic lethal genetic interactions in large-scale screens. These screens have been carried out largely in in-vitro cell culture systems using RNA interference (RNAi). Functional genomic screening using the bacterial type II clustered regularly interspaced short palindrome repeats and their associated proteins (CRISPR-Cas9) system has proven to be more powerful than RNAi for systematic genomic perturbation due to its simplicity, decreased off-target effects, and the ability to create knockout rather than knockdown phenotypes. We have conducted a genome-wide CRISPR-Cas9 loss-of-function screen with the human GeCKO v2 library directly in-vivo utilizing a paired isogenic human colorectal cell line (HCT116) with and without a KRAS oncogenic mutation (G13D) to identify genes whose knockout results in synthetic lethal interactions in tumor xenografts with mutated KRAS. Compared to cell culture models, tumor xenografts better recapitulate obstacles tumor cells must overcome for continued proliferation including limited accessibility to nutrients and oxygen. Many recent studies have also highlighted the effect of KRAS oncogenic mutations on the rewiring of metabolic pathways to fuel increased tumor growth. Tumor xenografts offer a better screening platform in identifying factors that might exploit these metabolic pathways. Pathway analysis of our genome-wide CRISPR screen indeed revealed candidate synthetic lethal genes involved in metabolic and nucleic acid synthesis pathways in KRAS mutant tumor xenografts. We individually validated some of the candidate genes in these metabolic pathways (including the TCA cycle, pentose phosphate pathway, and fructose metabolism) with individual CRISPR-Cas9 knockouts and confirmed that knockout of these genes resulted in decreased tumor xenograft growth in KRAS mutant tumors. One of the KRAS synthetic lethal genes that we identified was the gene NADK, which encodes NAD+ kinase that converts NAD+ into NADP+. Low-frequency activating mutations in NADK were also recently discovered by another group to harbor oncogenic potential in pancreatic adenocarcinoma. Given the high frequency of KRAS mutations in pancreatic adenocarcinoma, NADK may represent a therapeutic target in KRAS mutant tumors in general. While more sensitive than RNAi screens, there was still significant noise in our CRISPR-Cas9 genome-wide screen as expected in an in-vivo dropout screen. To overcome this noise, we generated a smaller pooled lentiviral CRISPR library (targeting ~250 genes) based on candidate hits in the genome-wide screen and again directly screened this smaller but deeper pooled library in tumor xenografts with higher coverage and more replicates. This strategy allowed for better discrimination of novel KRAS dependent synthetic lethal genes. Citation Format: Edwin H. Yau, Tariq Rana. Genome-wide in-vivo tumor xenograft CRISPR knockout screening for identifying KRAS mutant synthetic lethal interactions [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr PR12.

Eritoran, a structural analogue of the lipid A portion of lipopolysaccharide (LPS), is an antagonist of LPS in animal and human endotoxemia models. Previous studies have shown that low doses (350 to 3,500 μg) of eritoran have demonstrated a long pharmacokinetic half-life but a short pharmacodynamic half-life. The present study describes the safety, pharmacokinetics and pharmacodynamics, and lipid distribution profile of eritoran during and after a 72-h intravenous infusion of 500, 2,000, or 3,500 μg/h into healthy volunteers. Except for the occurrence of phlebitis, eritoran administration over 72 h was safe and well tolerated. Eritoran demonstrated a slow plasma clearance (0.679 to 0.930 ml/h/kg of body weight), a small volume of distribution (45.6 to 49.8 ml/kg), and a relatively long half-life (50.4 to 62.7 h). In plasma, the majority (∼55%) of eritoran was bound to high-density lipoproteins. During infusion and for up to 72 h thereafter, ex vivo response of blood to 1- or 10-ng/ml LPS was inhibited by ≥85%, even when the lowest dose of eritoran (500 μg/h) was infused. Inhibition of response was dependent on eritoran dose and the concentration of LPS used as an agonist. Finally, in vitro analysis with purified lipoprotein and protein fractions from plasma obtained from healthy volunteers indicated that eritoran is inactivated by high-density but not low-density lipoproteins, very-low-density lipoproteins, or albumin. From these results, we conclude that up to 252 mg of eritoran can be safely infused into normal volunteers over 72 h and even though it associates extensively with high-density lipoproteins, antagonistic activity is maintained, even after infusion ceases.

The objective of this study was to determine whether FM-VP4, a novel compound derived from plant sterols, can effectively reduce cholesterol accumulation within rat intestinal epithelial crypt (IEC-6) cells. EC-6 cells were cultured in Dulbecco's minimal essential medium (DMEM) containing 5% fetal bovine serum, 100 U/mL penicillin, 100 micro g/mL streptomycin, and 0.1 units/mL insulin at 37 degrees C under a humidified 5% CO2 atmosphere and seeded at 6.4 x 10(4) cells/well in 48-well plates. Experiments were initiated 14 days postconfluence. IEC-6 cells were exposed to [3H]cholesterol micelles (containing oleic and taurcholic acids), co-incubated with FM-VP4 (0, 10, 50, and 100 micro M) in Hepes Buffered Sterile Saline (HBSS). Cells were also preincubated with FM-VP4 prior to [3H]cholesterol micelle incubation to determine whether its effects are elicited intracellularly. The cellular localization of cholesterol was determined using digitonin. To determine the effects of cholesterol on the extent of FM-VP4 accumulation within IEC-6 cells, [3H]FM-VP4 was incubated with IEC-6 cells in the presence of unlabeled cholesterol micelles (0, 10, and 50 micro M). The extent of [3H]cholesterol or [3H]FM-VP4 associated with cell monolayers was determined after cell lysis using liquid scintillation counting in a Beckman LS6500 Scintillation Counter. Dose-response and time course studies were performed in which control (no FM-VP4 treatment) and FM-VP4 (10-100 micro M) were co-incubated with 50- micro M [3H]cholesterol micelles from 1 minute to 24 hours. Incubation with only 50- micro M FM-VP4 for less than 24 hours resulted in a 50% to 60% reduction (n = 6, P.05) in [3H]cholesterol associated with the monolayer compared with control (n = 6). Preincubation of FM-VP4 did not elicit a significant reduction in cholesterol accumulation compared with control (n = 6). Approximately 25% of the total [3H]cholesterol associated with the cells was determined to be cytosolic, while 75% was noncytosolic in the presence and/or absence of FM-VP4. [3H]FM-VP4 was also shown to associate with IEC-6 cells at similar concentrations to cholesterol with the most pronounced inhibition of FM-VP4 accumulation occurring at a cholesterol concentration of 50 micro M. However, cholesterol-induced inhibition was detectable only after 1 hour of incubation. FM-VP4 inhibits cholesterol accumulation within IEC-6 cells and is most effective at equimolar concentrations with cholesterol. Our findings further suggest that the action of FM-VP4 is likely at the cell surface and not elicited intracellularly.

The objective of this study was to determine how a novel hydrophilic phytostanol (FM-VP4) affects the cellular accumulation of [3H]cholesterol in human colon carcinoma (Caco-2) cell monolayers grown in Transwell chambers.To determine cellular accumulation of cholesterol and FM-VP4, [3H]cholesterol- containing micelles (50 microM cholesterol containing 1.27x10 (-4)% [3H]cholesterol) or [3H]FM-VP4 (50 microM) was incubated on the apical side of differentiated Caco-2 cell monolayers for 1 to 4 h at 37 degrees C in the absence or presence of increasing concentrations (10-200 microM) of unlabeled FM-VP4 or cholesterol, respectively.The accumulation of [3H]cholesterol (presented in micelles) into Caco-2 cell monolayers in the presence of 50 microM FM-VP4 was significantly lower (33.7 +/- 7.0%) compared to control (59.8 +/- 5.2%, p0.05) following 4 h of incubation. Conversely, cholesterol inhibited the accumulation of [3H]FM-VP4, although to a lesser extent, suggesting competition for binding sites. The inhibitory effects of FM-VP4 and cholesterol on each other were detectable after 1 h of incubation and increased with time. The extent of FM-VP4 inhibition of [3H]cholesterol accumulation was consistent whether FM-VP4 was co-incorporated into micelles or added separately in solution, suggesting that FM-VP4 does not elicit its effects through inhibition of cholesterol incorporation into micelles. In addition, pancreatic lipase activity ([3H]triolein hydrolysis) and p-glycoprotein (rhodamine 123 fluorescence) activity, were not affected by FM-VP4.In conclusion, FM-VP4 rapidly inhibits cholesterol accumulation within Caco-2 cell monolayers in a mode independent of pancreatic lipase activity, p-glycoprotein activity or cholesterol incorporation in micelles.