Recent transcriptomic-based analysis of diffuse large B cell lymphoma (DLBCL) has highlighted the clinical relevance of lymph node (LN) fibroblast and tumor-infiltrating lymphocyte (TIL) signatures within the tumor microenvironment (TME). However, the immunomodulatory role of fibroblasts in lymphoma remains unclear. Here, by studying human and mouse DLBCL-LNs, we identify the presence of an aberrantly remodeled fibroblastic reticular cell (FRC) network, expressing elevated fibroblast activated protein (FAP). RNA-sequencing analyses reveal that exposure to DLBCL reprograms key immunoregulatory pathways in FRCs, including a switch from homeostatic to inflammatory chemokine expression and elevated antigen presentation molecules. Functional assays show that DLBCL-activated FRCs (DLBCL-FRCs) hinder optimal TIL and chimeric antigen receptor T cell (CAR-T) migration. Moreover, DLBCL-FRCs inhibited CD8+ TIL cytotoxicity in an antigen-specific manner. Notably, the interrogation of patient LNs with imaging mass cytometry identified distinct environments differing in their CD8+ TIL-FRC composition and spatial organization that associated with survival outcomes. We further demonstrate the potential to target inhibitory FRCs to rejuvenate interacting TILs. Co-treating organotypic cultures with FAP-targeted immunostimulatory drugs and a bispecific antibody (glofitamab) augmented anti-lymphoma TIL cytotoxicity. Together, our study reveals an immunosuppressive role of FRCs in DLBCL, with implications for immune evasion, disease pathogenesis and optimizing immunotherapy for patients.
Benedetta Apollonio, Filomena Spada, Nedyalko Petrov, Domenico Cozzetto, Despoina Papazoglou, Peter Jarvis, Shichina Kannambath, Manuela Terranova-Barberio, Rose-Marie Amini, Gunilla Enblad, Charlotte E. Graham, Reuben Benjamin, Elizabeth H. Phillips, Richard J. Ellis, Rosamond Nuamah, Mansoor Saqi, Dinis P. Calado, Richard Rosenquist, Lesley A. Sutton, Jonathan R. Salisbury, Georgios Zacharioudakis, Anna Vardi, Patrick R. Hagner, Anita K. Gandhi, Marina Bacac, Christina Claus, Pablo Umana, Ruth F. Jarrett, Christian Klein, Alexander J.A. Deutsch, Alan G. Ramsay
ATRX is one of the most frequently altered genes in solid tumors, and mutation is especially frequent in soft tissue sarcomas. However, the role of ATRX in tumor development and response to cancer therapies remains poorly understood. Here, we developed a primary mouse model of soft tissue sarcoma and showed that Atrx deleted tumors were more sensitive to radiation therapy and to oncolytic herpesvirus. In the absence of Atrx, irradiated sarcomas had increased persistent DNA damage, telomere dysfunction, and mitotic catastrophe. Our work also showed that Atrx deletion resulted in downregulation of the CGAS/STING signaling pathway at multiple points in the pathway, and was not driven by mutations or by transcriptional downregulation of the CGAS/STING pathway components. We found that both human and mouse models of Atrx deleted sarcoma had a reduced adaptive immune response, markedly impaired CGAS/STING signaling, and increased sensitivity to TVEC, an oncolytic herpesvirus that is currently FDA approved for the treatment of aggressive melanomas. Translation of these results to patients with ATRX mutant cancers could enable genomically-guided cancer therapeutic approaches that improve patient outcomes.
Warren Floyd, Matthew Pierpoint, Chang Su, Rutulkumar Patel, Lixia Luo, Katherine Deland, Amy J. Wisdom, Daniel Zhu, Yan Ma, Suzanne Bartholf DeWitt, Nerissa T. Williams, Alexander L. Lazarides, Jason A. Somarelli, David L. Corcoran, William C. Eward, Diana M. Cardona, David G. Kirsch
Increased levels and diversity of human endogenous retrovirus (HERV) transcription characterizes most cancer types, linked with disease outcomes. However, the underlying processes are incompletely understood. We show that elevated transcription of HERVH proviruses predicts survival of lung squamous cell carcinoma (LUSC) and identify an isoform of CALB1, encoding Calbindin, ectopically driven by an upstream HERVH provirus under the control of KLF5, as the mediator of this effect. HERVH-CALB1 expression initiates in pre-invasive lesions and associates with their progression. Calbindin loss in LUSC cell lines impairs in vitro and in vivo growth and triggers senescence, consistent with a pro-tumor effect. However, Calbindin also directly controls the senescence-associated secretory phenotype (SASP), marked by secretion of CXCL8 and other neutrophil chemoattractants. In established carcinomas, CALB1-negative cancer cells become the dominant source of CXCL8, correlating with neutrophil infiltration and worse prognosis. Thus, HERVH-CALB1 expression in LUSC may display antagonistic pleiotropy, whereby the benefits of escaping senescence early during cancer initiation and clonal competition are offset by the prevention of SASP and pro-tumor inflammation at later stages.
Jan Attig, Judith Pape, Laura Doglio, Anastasiya Kazachenka, Eleonora Ottina, George R. Young, Katey S.S. Enfield, Iker Valle Aramburu, Kevin W. Ng, Nikhil Faulkner, William Bolland, Venizelos Papayannopoulos, Charles Swanton, George Kassiotis
BACKGROUND We previously demonstrated the safety of stereotactic body radiotherapy followed by pembrolizumab (SBRT+P) in patients with advanced solid tumors. This phase I clinical trial was expanded to study the safety of partial tumor irradiation (partial-Rx). We assessed irradiated local failure (LF) and clinical outcomes with correlations to biomarkers including CD8+ T cell radiomics score (RS) and circulating cytokines.METHODS Patients received SBRT to 2–4 metastases and pembrolizumab for up to 7 days after SBRT. Tumors measuring up to 65 cc received the full radiation dose (complete-Rx), whereas tumors measuring more than 65 cc received partial-Rx. Landmark analysis was used to assess the relationship between tumor response and overall survival (OS). Multivariable analysis was performed for RS and circulating cytokines.RESULTS In the combined (expansion plus original) cohort, 97 patients (219 metastases) were analyzed and received SBRT+P. Forty-six (47%) patients received at least 1 partial-Rx treatment. There were 7 (7.2%)dose-limiting toxicities (DLTs). 1-year LF was 7.6% overall, and 13.3% and 5.4% for partial-Rx and complete-Rx tumors, respectively (HR 2.32, 95% CI 0.90–5.97, P = 0.08). The overall, unirradiated, and irradiated objective response rates were 22%, 12%, and 34%, respectively. Irradiated tumor response to SBRT+P was associated with prolonged OS; 1-year OS was 71% (responders), 42% (mixed-responders), and 0% (nonresponders) (P < 0.01). High-RS was significantly associated with improved LF, progression-free survival (PFS), and OS. Elevated circulating IL-8 was independently associated with inferior PFS and OS.CONCLUSION SBRT+P is safe in patients with large, advanced solid tumors. Additional studies are warranted to assess noninferiority of complete versus partial irradiation of tumors in the setting of immunotherapy.TRIAL REGISTRATION Clinicaltrials.gov NCT02608385FUNDING Merck Investigator Studies Program; Hillman Fellows for Innovative Cancer Research Program; NIH grants UM1CA186690-06, P50CA254865-01A1, P30CA047904-32, and R01DE031729-01A1.
Mark C. Korpics, Benjamin E. Onderdonk, Rebekah E. Dadey, Jared H. Hara, Lilit Karapetyan, Yuanyuan Zha, Theodore G. Karrison, Adam C. Olson, Gini F. Fleming, Ralph R. Weichselbaum, Riyue Bao, Steven J. Chmura, Jason J. Luke
Childhood neuroblastomas exhibit plasticity between an undifferentiated neural crest–like mesenchymal cell state and a more differentiated sympathetic adrenergic cell state. These cell states are governed by autoregulatory transcriptional loops called core regulatory circuitries (CRCs), which drive the early development of sympathetic neuronal progenitors from migratory neural crest cells during embryogenesis. The adrenergic cell identity of neuroblastoma requires LMO1 as a transcriptional cofactor. Both LMO1 expression levels and the risk of developing neuroblastoma in children are associated with a single nucleotide polymorphism, G/T, that affects a GATA motif in the first intron of LMO1. Here, we showed that WT zebrafish with the GATA genotype developed adrenergic neuroblastoma, while knock-in of the protective TATA allele at this locus reduced the penetrance of MYCN-driven tumors, which were restricted to the mesenchymal cell state. Whole genome sequencing of childhood neuroblastomas demonstrated that TATA/TATA tumors also exhibited a mesenchymal cell state and were low risk at diagnosis. Thus, conversion of the regulatory GATA to a TATA allele in the first intron of LMO1 reduced the neuroblastoma-initiation rate by preventing formation of the adrenergic cell state. This mechanism was conserved over 400 million years of evolution, separating zebrafish and humans.
Nina Weichert-Leahey, Hui Shi, Ting Tao, Derek A. Oldridge, Adam D. Durbin, Brian J. Abraham, Mark W. Zimmerman, Shizhen Zhu, Andrew C. Wood, Deepak Reyon, J. Keith Joung, Richard A. Young, Sharon J. Diskin, John M. Maris, A. Thomas Look
Deciphering the crosstalk between metabolic reprogramming and epigenetic regulation is a promising strategy for cancer therapy. In this study, we discovered that the gluconeogenic enzyme PCK1 fueled the generation of S-adenosylmethionine (SAM) through the serine synthesis pathway. The methyltransferase SUV39H1 catalyzed SAM, which served as a methyl donor to support H3K9me3 modification, leading to the suppression of the oncogene S100A11. Mechanistically, PCK1 deficiency-induced oncogenic activation of S100A11 was due to its interaction with AKT1, which upregulated PI3K/AKT signaling. Intriguingly, the progression of hepatocellular carcinoma (HCC) driven by PCK1 deficiency was suppressed by SAM supplement or S100A11 knockout in vivo and in vitro. These findings reveal the availability of key metabolite SAM as a bridge connecting the gluconeogenic enzyme PCK1 and H3K9 trimethylation in attenuating HCC progression, thus suggesting a potential therapeutic strategy against HCC.
Dongmei Gou, Rui Liu, Xiaoqun Shan, Haijun Deng, Chang Chen, Jin Xiang, Yi Liu, Qingzhu Gao, Zhi Li, Ailong Huang, Kai Wang, Ni Tang
Targeted therapies such as venetoclax (Bcl-2 inhibitor) have revolutionized the treatment of chronic lymphocytic leukemia (CLL). We previously reported that persister CLL cells in treated patients overexpress multiple anti-apoptotic proteins and display resistance to pro-apoptotic agents. Here, we demonstrated that multidrug resistant CLL cells in vivo exhibit apoptosis restriction at a premitochondrial level due to insufficient activation of the Bax and Bak proteins. Co-immunoprecipitation analyses with selective BH-domain antagonists revealed that the pleotropic pro-apoptotic protein (Bim) is prevented from activating Bax/Bak by “switching” interactions to other upregulated anti-apoptotic proteins (Mcl-1/Bcl-xL/Bcl-2). Hence, treatments that bypass Bax/Bak restriction are required to deplete these resistant cells in patients. Protein Phosphatase 2A (PP2A) contributes to oncogenesis and treatment resistance. We observed that a small molecule activator of PP2A (SMAP) induced cytotoxicity in multiple cancer cell lines and CLL samples, including multidrug resistant leukemia/lymphoma cells. The SMAP (DT-061) activated apoptosis in multidrug resistant CLL cells through induction of mitochondrial permeability transition pores (mPTP), independent of Bax/Bak. DT-061 inhibited the growth of wild type and Bax/Bak double knockout multidrug resistant CLL cells in a xenograft mouse model. Collectively, we discovered multidrug resistant CLL cells in patients, and validated a pharmacologically tractable pathway to deplete this reservoir.
Kallesh D. Jayappa, Brian Tran, Vicki L. Gordon, Christopher G. Morris, Shekhar Saha, Caroline C. Farrington, Caitlin M. O’Connor, Kaitin P. Zawacki, Krista M. Isaac, Mark Kester, Timothy P. Bender, Michael E. Williams, Craig A. Portell, Michael J. Weber, Goutham Narla
Neurofibromatosis Type 1 (NF1) is one of the most common tumor-predisposing genetic disorders. Neurofibromas are NF1-associated benign tumors. A hallmark feature of neurofibromas is an abundant collagen-rich extracellular matrix (ECM) that constitutes >50% of the tumor dry weight. However, little is known about the mechanism underlying ECM deposition during neurofibroma development and treatment response. We performed a systematic investigation of ECM enrichment during plexiform neurofibroma (pNF) development, and identified basement membrane (BM) proteins, rather than major collagen isoforms, as the most upregulated ECM component. Following MEK inhibitor treatment, the ECM profile displayed an overall down-regulation signature, suggesting ECM reduction as a therapeutic benefit of MEK inhibition. Through these proteomic studies, TGF-β1 signaling was identified as playing a role in ECM dynamics. Indeed, TGF-β1 overexpression promoted pNF progression in vivo. Furthermore, by integrating single-cell RNA-sequencing, we found that immune cells including macrophages and T cells produce TGF-β1 to induce Schwann cells to produce and deposit BM proteins for ECM remodeling. Following Nf1 loss, neoplastic Schwann cells further increased BM protein deposition in response to TGF-β1. Our data delineate the regulation governing ECM dynamics in pNF and suggest that BM proteins could serve as markers for disease diagnosis and treatment response.
Chunhui Jiang, Ashwani Kumar, Ze Yu, Tracey Shipman, Yong Wang, Renee M. McKay, Chao Xing, Lu Q. Le
Seizures are a frequent complication of adult-type diffuse gliomas, and are often difficult to control with medications. Gliomas with mutations in isocitrate dehydrogenase 1 or 2 (IDHmut) are more likely than IDH wild-type (IDHwt) gliomas to cause seizures as part of their initial clinical presentation. However, whether IDHmut is also associated with seizures during the remaining disease course, and whether IDHmut inhibitors can reduce seizure risk, are unclear. Clinical multivariable analyses showed that preoperative seizures, glioma location, extent of resection, and glioma molecular subtype (including IDHmut status) all contribute to postoperative seizure risk in adult-type diffuse glioma patients, and that postoperative seizures are often associated with tumor recurrence. Experimentally, the metabolic product of IDHmut, D-2-hydroxyglutarate, rapidly synchronizes neuronal spike firing in a seizure-like manner, but only when nonneoplastic glial cells are present. In vitro and in vivo models can recapitulate IDHmut glioma-associated seizures, and IDHmut inhibitors currently being evaluated in glioma clinical trials inhibit seizures in those models, independent of their effects on glioma growth. These data show that postoperative seizure risk in adult-type diffuse gliomas varies in large part by molecular subtype, and that IDHmut inhibitors could play a key role in mitigating such risk in IDHmut glioma patients.
Michael Drumm, Wenxia Wang, Thomas K. Sears, Kirsten Bell-Burdett, Rodrigo Javier, Kristen Y. Cotton, Brynna T. Webb, Kayla T. Byrne, Dusten Unruh, Vineeth Thirunavu, Jordain Walshon, Alicia Steffens, Kathleen McCortney, Rimas V. Lukas, Joanna J. Phillips, Esraa Mohamed, John D. Finan, Lucas Santana-Santos, Amy B. Heimberger, Colin K. Franz, Jonathan E. Kurz, Jessica W. Templer, Geoffrey T. Swanson, Craig Horbinski
The deadliest anaplastic thyroid cancer (ATC) often transforms from indolent differentiated thyroid cancer (DTC); however, the complex intra-tumor transformation process is poorly understood. We investigated an anaplastic transformation model by dissecting both cell lineage and cell fate transitions using single cell transcriptomes and genetic alteration data from patients with different subtypes of thyroid cancer. The resulting spectrum of ATC transformation included stress-responsive DTC cells, inflammatory ATC cells (iATCs), mitotic-defective ATC cells and extended all the way to mesenchymal ATC cells (mATCs). Further, our analysis identified two important milestones: 1) a diploid stage, where iATC cells were diploids with inflammatory phenotypes, and 2) an aneuploid stage, where mATCs gained aneuploid genomes and mesenchymal phenotypes producing excessive collagens and collagen-interacting receptors. In parallel, cancer-associated-fibroblasts showed strong interactions among mesenchymal cell-types, macrophages shifted from M1 to M2 states, and T cells reprogrammed from cytotoxic to exhausted states, highlighting new therapeutic opportunities for ATC.
Lina Lu, Jennifer Rui Wang, Ying C. Henderson, Shanshan Bai, Jie Yang, Min Hu, Cheng-Kai Shiau, Timothy Y. Pan, Yuanqing Yan, Tuan M. Tran, Jianzhuo Li, Rachel Kieser, Xiao Zhao, Jiping Wang, Roza Nurieva, Michelle D. Williams, Maria E. Cabanillas, Ramona Dadu, Naifa Busaidy, Mark Zafereo, Nicholas Navin, Stephen Y. Lai, Ruli Gao