Excellence in Science

A Genetic Mouse Model Recapitulates Immune Checkpoint Inhibitor–Associated Myocarditis and Supports a Mechanism-Based Therapeutic Intervention

Immune checkpoint inhibitors (ICI) targeting CTLA4 or PD-1/PD-L1 have transformed cancer therapy but are associated with immune-related adverse events, including myocarditis. Here, we report a robust preclinical mouse model of ICI-associated myocarditis in which monoallelic loss of Ctla4 in the context of complete genetic absence of Pdcd1 leads to premature death in approximately half of mice. Premature death results from myocardial infiltration by T cells and macrophages and severe ECG abnormalities, closely recapitulating the clinical and pathologic hallmarks of ICI-associated myocarditis observed in patients. Using this model, we show that Ctla4 and Pdcd1 functionally interact in a gene dosage-dependent manner, providing a mechanism by which myocarditis arises with increased frequency in the setting of combination ICI therapy. We demonstrate that intervention with CTLA4–Ig (abatacept) is sufficient to ameliorate disease progression and additionally provide a case series of patients in which abatacept mitigates the fulminant course of ICI myocarditis.

SIGNIFICANCE: We provide a preclinical model of ICI-associated myocarditis which recapitulates this clinical syndrome. Using this model, we demonstrate that CTLA4 and PD-1 (ICI targets) functionally interact for myocarditis development and that intervention with CTLA4–Ig (abatacept) attenuates myocarditis, providing mechanistic rationale and preclinical support for therapeutic clinical studies.

Spencer C. Wei, Wouter C. Meijers, Margaret L. Axelrod, Nana-Ama A.S. Anang, Elles M. Screever, Elizabeth C. Wescott, Douglas B. Johnson, Elizabeth Whitley, Lorenz Lehmann, Pierre-Yves Courand, James J. Mancuso, Lauren E. Himmel, Benedicte Lebrun-Vignes, Matthew J. Wleklinski, Bjorn C. Knollmann, Jayashree Srinivasan, Yu Li, Oluwatomisin T. Atolagbe, Xiayu Rao, Yang Zhao, Jing Wang, Lauren I.R. Ehrlich, Padmanee Sharma, Joe-Elie Salem, Justin M. Balko, Javid J. Moslehi, and James P. Allison

Read more in Cancer Discovery

Elevated NSD3 histone methylation activity drives squamous cell lung cancer

Amplification of chromosomal region 8p11–12 is a common genetic alteration that has been implicated in the aetiology of lung squamous cell carcinoma (LUSC) The FGFR1 gene is the main candidate driver of tumorigenesis within this region. However, clinical trials evaluating FGFR1 inhibition as a targeted therapy have been unsuccessful. Here we identify the histone H3 lysine 36 (H3K36) methyltransferase NSD3, the gene for which is located in the 8p11–12 amplicon, as a key regulator of LUSC tumorigenesis. In contrast to other 8p11–12 candidate LUSC drivers, increased expression of NSD3 correlated strongly with its gene amplification. Ablation of NSD3, but not of FGFR1, attenuated tumour growth and extended survival in a mouse model of LUSC. We identify an LUSC-associated variant NSD3(T1232A) that shows increased catalytic activity for dimethylation of H3K36 (H3K36me2) in vitro and in vivo. Structural dynamic analyses revealed that the T1232A substitution elicited localized mobility changes throughout the catalytic domain of NSD3 to relieve auto-inhibition and to increase accessibility of the H3 substrate. Expression of NSD3(T1232A) in vivo accelerated tumorigenesis and decreased overall survival in mouse models of LUSC. Pathological generation of H3K36me2 by NSD3(T1232A) reprograms the chromatin landscape to promote oncogenic gene expression signatures. Furthermore, NSD3, in a manner dependent on its catalytic activity, promoted transformation in human tracheobronchial cells and growth of xenografted human LUSC cell lines with amplification of 8p11–12. Depletion of NSD3 in patient-derived xenografts from primary LUSCs containing NSD3 amplification or the NSD3(T1232A)-encoding variant attenuated neoplastic growth in mice. Finally, NSD3-regulated LUSC-derived xenografts were hypersensitive to bromodomain inhibition. Thus, our work identifies NSD3 as a principal 8p11–12 amplicon-associated oncogenic driver in LUSC, and suggests that NSD3-dependency renders LUSC therapeutically vulnerable to bromodomain inhibition.

Gang Yuan, Natasha M. Flores, Simone Hausmann, Shane M. Lofgren, Vladlena Kharchenko, Maria Angulo-Ibanez, Deepanwita Sengupta, Xiaoyin Lu, Iwona Czaban, Dulat Azhibek, Silvestre Vicent, Wolfgang Fischle, Mariusz Jaremko, Bingliang Fang, Ignacio I. Wistuba, Katrin F. Chua, Jack A. Roth, John D. Minna, Ning-Yi Shao, Łukasz Jaremko, Pawel K. Mazur, and Or Gozani

Read more in Nature

Oncogenic KRAS Recruits an Expansive Transcriptional Network through Mutant p53 to Drive Pancreatic Cancer Metastasis

Pancreatic ductal adenocarcinoma (PDAC) is almost uniformly fatal and characterized by early metastasis. Oncogenic KRAS mutations prevail in 95% of PDAC tumors and co-occur with genetic alterations in the TP53 tumor suppressor in nearly 70% of patients. Most TP53 alterations are missense mutations that exhibit gain-of-function phenotypes that include increased invasiveness and metastasis, yet the extent of direct cooperation between KRAS effectors and mutant p53 remains largely undefined. We show that oncogenic KRAS effectors activate CREB1 to allow physical interactions with mutant p53 that hyperactivate multiple prometastatic transcriptional networks. Specifically, mutant p53 and CREB1 upregulate the prometastatic, pioneer transcription factor FOXA1, activating its transcriptional network while promoting WNT/β-catenin signaling, together driving PDAC metastasis. Pharmacologic CREB1 inhibition dramatically reduced FOXA1 and β-catenin expression and dampened PDAC metastasis, identifying a new therapeutic strategy to disrupt cooperation between oncogenic KRAS and mutant p53 to mitigate metastasis.

SIGNIFICANCE: Oncogenic KRAS and mutant p53 are the most commonly mutated oncogene and tumor suppressor gene in human cancers, yet direct interactions between these genetic drivers remain undefined. We identified a cooperative node between oncogenic KRAS effectors and mutant p53 that can be therapeutically targeted to undermine cooperation and mitigate metastasis.

Michael P. Kim, Xinqun Li, Jenying Deng, Yun Zhang, Bingbing Dai, Kendra L. Allton, Tara G. Hughes, Christian Siangco, Jithesh J. Augustine, Ya'an Kang, Joy M. McDaniel, Shunbin Xiong, Eugene J. Koay, Florencia McAllister, Christopher A. Bristow, Timothy P. Heffernan, Anirban Maitra, Bin Liu, Michelle C. Barton, Amanda R. Wasylishen, Jason B. Fleming, and Guillermina Lozano

Read more in Cancer Discovery

DHODH-mediated ferroptosis defence is a targetable vulnerability in cancer

Ferroptosis, a form of regulated cell death that is induced by excessive lipid peroxidation, is a key tumour suppression mechanism. Glutathione peroxidase 4 (GPX4) and ferroptosis suppressor protein 1 (FSP1) constitute two major ferroptosis defence systems. Here we show that treatment of cancer cells with GPX4 inhibitors results in acute depletion of N-carbamoyl-L-aspartate, a pyrimidine biosynthesis intermediate, with concomitant accumulation of uridine. Supplementation with dihydroorotate or orotate—the substrate and product of dihydroorotate dehydrogenase (DHODH)—attenuates or potentiates ferroptosis induced by inhibition of GPX4, respectively, and these effects are particularly pronounced in cancer cells with low expression of GPX4 (GPX4^low). Inactivation of DHODH induces extensive mitochondrial lipid peroxidation and ferroptosis in GPX4^low cancer cells, and synergizes with ferroptosis inducers to induce these effects in GPX4^high cancer cells. Mechanistically, DHODH operates in parallel to mitochondrial GPX4 (but independently of cytosolic GPX4 or FSP1) to inhibit ferroptosis in the mitochondrial inner membrane by reducing ubiquinone to ubiquinol (a radical-trapping antioxidant with anti-ferroptosis activity). The DHODH inhibitor brequinar selectively suppresses GPX4^low tumour growth by inducing ferroptosis, whereas combined treatment with brequinar and sulfasalazine, an FDA-approved drug with ferroptosis-inducing activity, synergistically induces ferroptosis and suppresses GPX4^high tumour growth. Our results identify a DHODH-mediated ferroptosis defence mechanism in mitochondria and suggest a therapeutic strategy of targeting ferroptosis in cancer treatment.

Chao Mao, Xiaoguang Liu, Yilei Zhang, Guang Lei, Yuelong Yan, Hyemin Lee, Pranavi Koppula, Shiqi Wu, Li Zhuang, Bingliang Fang, Masha V. Poyurovsky, Kellen Olszewski, and Boyi Gan

Read more in Nature

Characterizing genetic intra-tumor heterogeneity across 2,658 human cancer genomes

Intra-tumor heterogeneity (ITH) is a mechanism of therapeutic resistance and therefore an important clinical challenge. However, the extent, origin, and drivers of ITH across cancer types are poorly understood. To address this, we extensively characterize ITH across whole-genome sequences of 2,658 cancer samples spanning 38 cancer types. Nearly all informative samples (95.1%) contain evidence of distinct subclonal expansions with frequent branching relationships between subclones. We observe positive selection of subclonal driver mutations across most cancer types and identify cancer type-specific subclonal patterns of driver gene mutations, fusions, structural variants, and copy number alterations as well as dynamic changes in mutational processes between subclonal expansions. Our results underline the importance of ITH and its drivers in tumor evolution and provide a pan-cancer resource of comprehensively annotated subclonal events from whole-genome sequencing data.

SC Dentro, I Leshchiner, K Haase, M Tarabichi, J Wintersinger, AG Deshwar, Kaixian Yu, Y Rubanova, G Macintyre, J Demeulemeester, I Vazquez-Garcıa, K Kleinheinz, DG Livitz, Malikic, N Donmez, S Sengupta, P Anur, C Jolly, M Cmero, D Rosebrock, SE Schumacher, Yu Fan, M Fittall, RM Drews, X Yao, TBK Watkins, J Lee, M Schlesner, H Zhu, DJ Adams, N McGranahan, C Swanton, G Getz, PC Boutros, M Imielinski, R Beroukhim, SC Sahinalp,15 Y Ji, M Peifer, I Martincorena, F Markowetz, V Mustonen, K Yuan, M Gerstung PT Spellman, Wenyi Wang, QD Morris, DC Wedge, P Van Loo, and on behalf of the PCAWG Evolution and Heterogeneity Working Group and the PCAWG Consortium

Read more in Cell

A High-Resolution Map of Human Enhancer RNA Loci Characterizes Super-enhancer Activities in Cancer

Although enhancers play critical roles in cancer, quantifying en­hancer activities in clinical samples remains challenging, especially for super-enhancers. Enhancer activities can be inferred from en­hancer RNA (eRNA) signals, which requires enhancer transcription loci definition. Only a small pro­portion of human eRNA loci has been precisely identified, limiting investigations of enhancer-me­diated oncogenic mechanisms. Here, we characterize super-en­hancer regions using aggregated RNA sequencing (RNA-seq) data from large cohorts. Super-en­hancers usually contain discrete loci featuring sharp eRNA expres­sion peaks. We identify >300,000 eRNA loci in ~377 Mb super-en­hancer regions that are regulat­ed by evolutionarily conserved, well-positioned nucleosomes and are frequently dysregulated in can­cer. The eRNAs provide explana­tory power for cancer phenotypes beyond that provided by mRNA expression through resolving in­tratumoral heterogeneity with en­hancer cell-type specificity. Our study provides a high-resolution map of eRNA loci through which super-enhancer activities can be quantified by RNA-seq and a us­er-friendly data portal, enabling a broad range of biomedical investi­gations.

Han Chen, Ph.D. and Han Liang, Ph.D.

Read more in Cancer Cell

Large-Scale Characterization of Drug Responses of Clinically Relevant Proteins in Cancer Cell Lines

Perturbation biology is a powerful approach to modeling quantitative cellular behaviors and understanding detailed disease mechanisms. However, largescale protein response resources of cancer cell lines to perturbations are not available, resulting in a critical knowledge gap. Here we generated and compiled perturbed expression profiles of ~210 clinically relevant proteins in >12,000 cancer cell line samples in response to ~170 drug compounds using reverse-phase protein arrays. We show that integrating perturbed protein response signals provides mechanistic insights into drug resistance, increases the predictive power for drug sensitivity, and helps identify effective drug combinations. We build a systematic map of "protein-drug" connectivity and develop a user-friendly data portal for community use. Our study provides a rich resource to investigate the behaviors of cancer cells and the dependencies of treatment responses, thereby enabling a broad range of biomedical applications.

W Zhao, J Li, MJM Chen, Y Luo, Z Ju, NK Nesser, K Johnson-Camacho, CT Boniface, Y Lawrence, NT Pande, MA Davies, M Herlyn, T Muranen, IK Zervantonakis, E von Euw, A Schultz, SV Kumar, A Korkut, PT Spellman, R Akbani, DJ Slamon, JW Gray, JS Brugge, Y Lu, GB Mills, and H Liang

Read more in Cancer Cell

NF-κB-inducing kinase maintains T cell metabolic fitness in antitumor immunity

Metabolic reprograming toward aerobic glycolysis is a pivotal mechanism shaping immune responses. Here we show that deficiency in NF-κB-inducing kinase (NIK) impairs glycolysis induction, rendering CD8⁺ effector T cells hypofunctional in the tumor microenvironment. Conversely, ectopic expression of NIK promotes CD8⁺ T cell metabolism and effector function, thereby profoundly enhancing antitumor immunity and improving the efficacy of T cell adoptive therapy. NIK regulates T cell metabolism via a NF-κB-independent mechanism that involves stabilization of hexokinase 2 (HK2), a rate-limiting enzyme of the glycolytic pathway. NIK prevents autophagic degradation of HK2 through controlling cellular reactive oxygen species levels, which in turn involves modulation of glucose-6-phosphate dehydrogenase (G6PD), an enzyme that mediates production of the antioxidant NADPH. We show that the G6PD–NADPH redox system is important for HK2 stability and metabolism in activated T cells. These findings establish NIK as a pivotal regulator of T cell metabolism and highlight a post-translational mechanism of metabolic regulation.

Meidi Gu, Xiaofei Zhou, Jee Hyung Sohn, Lele Zhu, Zuliang Jie, Jin-Young Yang, Xiaofeng Zheng, Xiaoping Xie, Jie Yang, Yaoyao Shi, Hans D. Brightbill, Jae Bum Kim, Jing Wang, Xuhong Cheng, and Shao-Cong Sun

Read more in Nature Immunology

Frequency of and Factors Associated With Nonmedical Opioid Use Behavior Among Patients With Cancer Receiving Opioids for Cancer Pain

Importance One of the main aims of re­search on nonmedical opioid use (NMOU) is to reduce the frequency of NMOU behav­iors through interventions such as univer­sal screening, reduced opioid exposure, and more intense follow-up of patients with elevated risk. The absence of data on the frequency of NMOU behavior is the major barrier to conducting research on NMOU.

Results A total of 1554 patients (median [interquartile range (IQR)] age, 61 [IQR, 52-69] years; 816 women [52.5%]; 1124 White patients [72.3%]) were evaluable for the study, and 299 patients (19.2%) had 1 or more NMOU behaviors. The medi­an (IQR) number of NMOU behaviors per patient was 1 (IQR, 1-3). A total of 576 of 745 NMOU behaviors (77%) occurred by the first 2 follow-up visits. The most fre­quent NMOU behavior was unscheduled clinic visits for inappropriate refills (218 of 745 [29%]). Eighty-eight of 299 patients (29.4%) scored 7 or higher on SOAPP, and 48 (16.6%) scored at least 2 out of 4 points on the CAGE-AID survey. Results from the multivariate model suggest that marital sta­tus (single, hazard ratio [HR], 1.58; 95% CI, 1.15-2.18; P = .005; divorced, HR, 1.43; 95% CI, 1.01-2.03; P = .04), SOAPP score (pos­itive vs negative, HR, 1.35; 95% CI, 1.04-1.74; P = .02), morphine equivalent daily dose (MEDD) (HR, 1.003; 95% CI, 1.002-1.004; P < .001), and Edmonton Symptom Assessment Scale pain level (HR, 1.11; 95% CI, 1.06-1.16; P < .001) were inde­pendently associated with the presence of NMOU behavior. In recursive partition anal­ysis, single marital status, MEDD greater than 50 mg, and SOAPP scores greater than 7 were associated with a higher risk (56%) for the presence of NMOU behavior.

Conclusions and Relevance This prog­nostic study of patients with cancer taking opioids for cancer pain found that 19% of patients developed NMOU behavior within a median duration of 8 weeks after initial supportive care clinic consultation. Marital status (single or divorced), SOAPP score greater than 7, higher levels of pain severity, and MEDD level were independently asso­ciated with NMOU behavior. This informa­tion will assist clinicians and investigators designing clinical and research programs in this important field.

Sriram Yennurajalingam, MD, MS; Joseph Arthur, MD; Suresh Reddy, MD; Tonya Edwards, MS, MSN, FNP-C; Zhanni Lu, Dr Ph; Aline Rozman de Moraes, MD; Susamma M. Wilson, RN, CDN; Elif Erdogan, MD; Manju P. Joy, MSN, BSN, RN, CMSRN; Shirley Darlene Ethridge, BSN, RN, CHPN; Leela Kuriakose, BSN, CHPN; Jimi S. Malik, MD; John M. Najera, MA, LPC; Saima Rashid, MD; Yu Qian, MD; Michal J. Kubiak, MD; Kristy Nguyen;  PharmD; Jimin Wu, MS; David Hui, MD; Eduardo Bruera, MD

Read more in JAMA Oncology

Characteristics of anti-CD19 CAR T cell infusion products associated with efficacy and toxicity in patients with large B cell lymphomas

Autologous chimeric antigen receptor (CAR) T cell therapies targeting CD19 have high efficacy in large B cell lymphomas (LBCLs), but long-term remissions are observed in less than half of patients, and treatment-associated adverse events, such as immune effector cell-associated neurotoxicity syndrome (ICANS), are a clinical challenge. We performed single-cell RNA sequencing with capture-based cell identification on autologous axicabtagene ciloleucel (axi-cel) anti-CD19 CAR T cell infusion products to identify transcriptomic features associated with efficacy and toxicity in 24 patients with LBCL. Patients who achieved a complete response by positron emission tomography/computed tomography at their 3-month follow-up had three-fold higher frequencies of CD8 T cells expressing memory signatures than patients with partial response or progressive disease. Molecular response measured by cell-free DNA sequencing at day 7 after infusion was significantly associated with clinical response (P = 0.008), and a signature of CD8 T cell exhaustion was associated (q = 2.8 × 10⁻¹⁴⁹) with a poor molecular response. Furthermore, a rare cell population with monocyte-like transcriptional features was associated (P = 0.0002) with high-grade ICANS. Our results suggest that heterogeneity in the cellular and molecular features of CAR T cell infusion products contributes to variation in efficacy and toxicity after axi-cel therapy in LBCL, and that day 7 molecular response might serve as an early predictor of CAR T cell efficacy.

Qing Deng, Guangchun Han, Nahum Puebla-Osorio, Man Chun John Ma, Paolo Strati, Beth Chasen, Enyu Dai, Minghao Dang, Neeraj Jain, Haopeng Yang, Yuanxin Wang, Shaojun Zhang, Ruiping Wang, Runzhe Chen, Jordan Showell, Sreejoyee Ghosh, Sridevi Patchva, Qi Zhang, Ryan Sun, Frederick Hagemeister, Luis Fayad, Felipe Samaniego, Hans C. Lee, Loretta J. Nastoupil, Nathan Fowler, R. Eric Davis, Jason Westin, Sattva S. Neelapu, Linghua Wang & Michael R. Green

Read more in Nature Medicine

Neoadjuvant PD-L1 plus CTLA-4 blockade in patients with cisplatin-ineligible operable high-risk urothelial carcinoma

Immune checkpoint therapy is being tested in the neoadjuvant setting for patients with localized urothelial carcinoma, with one study reporting data in cisplatin-ineligible patients who received anti-PD-L1 monotherapy. The study reported that patients with bulky tumors, a known high-risk feature defined as greater than clinical T2 disease, had fewer responses, with pathological complete response rate of 17%. Here we report on the first pilot combination neoadjuvant trial (NCT02812420) with anti-PD-L1 (durvalumab) plus anti-CTLA-4 (tremelimumab) in cisplatin-ineligible patients, with all tumors identified as having high-risk features (n = 28). High-risk features were defined by bulky tumors, variant histology, lymphovascular invasion, hydronephrosis and/or high-grade upper tract disease. The primary endpoint was safety and we observed 6 of 28 patients (21%) with grade ≥3 immune-related adverse events, consisting of asymptomatic laboratory abnormalities (n = 4), hepatitis and colitis (n = 2). We also observed pathological complete response of 37.5% and downstaging to pT1 or less in 58% of patients who completed surgery (n = 24). In summary, we provide initial safety, efficacy and biomarker data with neoadjuvant combination anti-PD-L1 plus anti-CTLA-4, which warrants further development for patients with localized urothelial carcinoma, especially cisplatin-ineligible patients with high-risk features who do not currently have an established standard-of-care neoadjuvant treatment.

Jianjun Gao, Neema Navai, Omar Alhalabi, Arlene Siefker-Radtke, Matthew T. Campbell, Rebecca Slack Tidwell, Charles C. Guo, Ashish M. Kamat, Surena F. Matin, John C. Araujo, Amishi Y. Shah, Pavlos Msaouel, Paul Corn, Jianbo Wang, John N. Papadopoulos, Shalini S. Yadav, Jorge M. Blando, Fei Duan, Sreyashi Basu, Wenbin Liu, Yu Shen, Yuwei Zhang, Marc Daniel Macaluso, Ying Wang, Jianfeng Chen, Jianhua Zhang, Andrew Futreal, Colin Dinney, James P. Allison, Sangeeta Goswami & Padmanee Sharma

Read more in Nature Medicine

The lncRNA H19 alleviates muscular dystrophy by stabilizing dystrophin

Dystrophin proteomic regulation in muscular dystrophies (MDs) remains unclear. We report that a long noncoding RNA (lncRNA), H19, associates with dystrophin and inhibits E3-ligase-dependent polyubiquitination at Lys 3584 (referred to as Ub-DMD) and its subsequent protein degradation. In-frame deletions in BMD and a DMD non-silent mutation (C3340Y) resulted in defects in the ability of the protein to interact with H19, which caused elevated Ub-DMD levels and dystrophin degradation. Dmd C3333Y mice exhibited progressive MD, elevated serum creatine kinase, heart dilation, blood vessel irregularity and respiratory failure with concurrently reduced dystrophin and increased Ub-DMD status. H19 RNA oligonucleotides conjugated with agrin (AGR–H19) and nifenazone competed with or inhibited TRIM63. Dmd C3333Y animals, induced-pluripotent-stem-cell-derived skeletal muscle cells from patients with Becker MD and mdx mice subjected to exon skipping exhibited inhibited dystrophin degradation, preserved skeletal and cardiac muscle histology, and improved strength and heart function following AGR–H19 or nifenazone treatment. Our study paves the way for meaningful targeted therapeutics for Becker MD and for certain patients with Duchenne MD.

Yaohua Zhang, Yajuan Li, Qingsong Hu, Yutao Xi, Zhen Xing, Zhao Zhang, Lisa Huang, Jianbo Wu, Ke Liang, Tina K. Nguyen, Sergey D. Egranov, Chengcao Sun, Zilong Zhao, David H. Hawke, Jin Li, Deqiang Sun, Jean J. Kim, Ping Zhang, Jie Cheng, Abid Farida, Mien-Chie Hung, Leng Han, Radbod Darabi, Chunru Lin & Liuqing Yang

Read more in Nature Cell Biology