Translocation Renal Cell Carcinoma (tRCC) most commonly involves an ASPSCR1-TFE3 fusion, but molecular mechanisms remain elusive and animal models are lacking. Here, we show that human ASPSCR1-TFE3 driven by Pax8-Cre (a credentialed ccRCC driver) disrupted nephrogenesis and glomerular development causing neonatal death, whilst the ccRCC failed driver, Sglt2-Cre, induced aggressive tRCC (as well as ASPS) with complete penetrance and short latency. However, in both contexts, ASPSCR1-TFE3 led to characteristic morphological cellular changes, loss of epithelial markers, and an EMT program. Electron microscopy of tRCC tumors showed lysosome expansion and functional studies revealed simultaneous activation of autophagy and mTORC1 pathways. Comparative genomic analyses encompassing an institutional human tRCC cohort (including a hitherto unreported SFPQ-TFEB fusion) and a variety of tumorgraft models (ASPSCR1-TFE3, PRCC-TFE3, SFPQ-TFE3, RBM10-TFE3, and MALAT1-TFEB) disclosed significant convergence in canonical (cell cycle, lysosome and mTORC1) and less established pathways such as Myc, E2F and inflammation (IL6/JAK/STAT3, interferon-γ, TLR signaling, systemic lupus, etc). Therapeutic trials (adjusted for human drug exposures) showed anti-tumor activity of cabozantinib. Overall, this study provides insight into MiT/TFE-driven tumorigenesis including the cell of origin and characterizes diverse mouse models available for research.
Gopinath Prakasam, Akhilesh Mishra, Alana Christie, Jeffrey Miyata, Deyssy Carrillo, Vanina T. Tcheuyap, Hui Ye, Quyen N. Do, Yunguan Wang, Oscar Reig Torras, Ramesh Butti, Hua Zhong, Jeffrey Gagan, Kevin B. Jones, Thomas J. Carroll, Zora Modrusan, Steffen Durinck, Mai-Carmen Requena-Komuro, Noelle S. Williams, Ivan Pedrosa, Tao Wang, Dinesh Rakheja, Payal Kapur, James Brugarolas
Background: Persistent controllers (PC) maintain antiretroviral-free HIV-1 control indefinitely over time while transient controllers (TC) eventually lose virological control. It is essential to characterize the quality of the HIV reservoir of these phenotypes to identify the factors that lead to HIV progression and to open new avenues in HIV cure strategies. Methods: The characterization of HIV-1 reservoir, from peripheral blood mononuclear cells, was performed using next-generation sequencing techniques, such as full-length individual and matched integration site proviral sequencing (FLIP-seq; MIP-seq). Results: PC and TC before losing virological control, presented significantly lower total, intact and defective proviruses compared to participants on antiretroviral therapy (ART). No differences were found in total and defective proviruses between PC and TC. However, intact provirus levels were lower in PC compared to TC, being the intact/defective HIV-DNA ratio significantly higher in TC. Clonally expanded intact proviruses were found only in PC and located in centromeric satellite DNA or zinc-finger genes, both associated with heterochromatin features. In contrast, sampled intact proviruses were located in permissive genic euchromatic positions in TC. Conclusions: These results suggest the need for, and can give guidance to the design of, future research to identify a distinct proviral landscape that may be associated with the persistent control of HIV-1 without ART. Funding: Instituto de Salud Carlos III (FI17/00186, FI19/00083, MV20/00057 PI18/01532, PI19/01127 and PI22/01796), Consejería de Economía, Conocimiento, Empresas y Universidad, Junta de Andalucía (PI20/1276), Gilead Fellowships (GLD22/00147) and I+D+iFEDER Andalucía 2014-2020 (US-1380938).
Carmen Gasca-Capote, Xiaodong Lian, Ce Gao, Isabelle C. Roseto, María Reyes Jiménez-León, Gregory Gladkov, María Inés Camacho-Sojo, Alberto Pérez-Gómez, Isabel Gallego, Luis E. Lopez-Cortes, Sara Bachiller, Joana Vitalle, Mohammed Rafii-El-Idrissi Benhnia, Francisco J. Ostos, Antonio R. Collado-Romacho, Jesús Santos, Rosario Palacios, Cristina Gomez-Ayerbe, Leopoldo Muñoz-Medina, Andrés Ruiz-Sancho, Mario Frias, Antonio Rivero-Juarez, Cristina Roca-Oporto, Carmen Hidalgo-Tenorio, Anna Rull, Julian Olalla, Miguel A. Lopez-Ruz, Francesc Vidal, Consuelo Viladés, Andrea Mastrangelo, Matthias Cavassini, Nuria Espinosa, Matthieu Perreau, Joaquin Peraire, Antonio Rivero, Luis F. López-Cortes, Mathias Lichterfeld, Xu G. Yu, Ezequiel Ruiz-Mateos
While therapies targeting CD19 by antibodies, CAR-T cells and T cell engagers have improved the response rates in B-cell malignancies; the emergence of resistant cell populations with low CD19 expression can lead to relapsed disease. We developed an in vitro model of adaptive resistance facilitated by chronic exposure of leukemia cells to a CD19-immunotoxin. Single-cell (sc) RNAseq showed increase in transcriptionally distinct CD19low populations in resistant cells. Mass cytometry demonstrated that CD22 was also decreased in these CD19low resistant cells. ATAC-seq showed decreased chromatin accessibility at promoters of both CD19 and CD22 during development of resistance. Combined loss of both CD19 and CD22 antigens was validated in samples from pediatric and young adult patients with ALL that relapsed after CD19 CAR-T targeted therapy. Functionally, resistant cells were characterized by slower growth and lower basal levels of MEK activation. CD19low resistant cells exhibited preserved B cell receptor signaling and were more sensitive to both BTK and MEK inhibition. These data demonstrate that resistance to CD19 immunotherapies can result in decreased expression of both CD19 and CD22 and can result in dependency on BTK pathways.
Sarah Aminov, Orsi Giricz, David T. Melnekoff, R. Alejandro Sica, Veronika Polishchuk, Cristian Papazoglu, Bonnie Yates, Hao-Wei Wang, Srabani Sahu, Yanhua Wang, Shanisha Gordon-Mitchell, Violetta V. Leshchenko, Carolina Schinke, Kith Pradhan, Srinivas Aluri, Moah Sohn, Stefan K. Barta, Beamon Agarwal, Mendel Goldfinger, Ioannis Mantzaris, Aditi Shastri, William Matsui, Ulrich Steidl, Joshua D. Brody, Nirali N. Shah, Samir Parekh, Amit Verma
Corticosteroid treatment (CST) failure is associated with poor outcomes for patients with gastrointestinal graft-versus-host disease (GI GVHD). CST is intended to target the immune system, but the glucocorticoid receptor is widely expressed, including within the intestines, where its effects are poorly understood. Here, we report that corticosteroids directly target intestinal epithelium, potentially worsening immune-mediated GI damage. Corticosteroids administered to mice in vivo and intestinal organoid cultures ex vivo reduced epithelial proliferation. Following irradiation, immediate CST mitigated GI damage, but delayed treatment attenuated regeneration and exacerbated damage. In a murine steroid-refractory GVHD model, CST impaired epithelial regeneration, worsened crypt loss, and reduced intestinal stem cell (ISC) frequencies. CST also exacerbated immune-mediated damage in organoid cultures with “steroid-refractory” GR-deficient T cells or Interferon-γ. These findings correlated with corticosteroid-dependent changes in apoptosis-related gene expression and STAT3-related epithelial proliferation. Conversely, Interleukin-22 administration enhanced STAT3 activity and overcame corticosteroid-mediated attenuation of regeneration, reducing crypt loss and promoting ISC expansion in steroid-treated mice with GVHD. Therefore, CST has the potential to exacerbate GI damage if it fails to control the damage-inducing immune response, but this risk may be countered by strategies augmenting epithelial regeneration, thus providing rationale for clinical approaches combining such tissue-targeted therapies with immunosuppression.
Viktor Arnhold, Winston Y. Chang, Suze A. Jansen, Govindarajan Thangavelu, Marco Calafiore, Paola Vinci, Ya-Yuan Fu, Takahiro Ito, Shuichiro Takashima, Anastasiya Egorova, Jason Kuttiyara, Adam Perlstein, Marliek van Hoesel, Chen Liu, Bruce R. Blazar, Caroline A. Lindemans, Alan M. Hanash
Radiotherapy (RT) is considered immunogenic, but clinical data demonstrating RT-induced T-cell priming are scarce. Here, we show in a mouse tumor model representative of human lymphocyte-depleted cancer that RT enhances spontaneous priming of thymus-derived (FOXP3+ Helios+) regulatory T-cells (Tregs) by the tumor. These Tregs acquire an effector phenotype, populate the tumor and impede tumor control by a simultaneous, RT-induced CD8+ cytotoxic T-cell (CTL) response. Combination of RT with CTLA-4 or PD-1 blockade, which enables CD28 costimulation, further increased this Treg response and failed to improve tumor control. We discovered that upon RT, CD28-ligands CD86 and CD80 differentially affected the Treg response. CD86, but not CD80, blockade prevented the effector (e)Treg response, enriched the tumor-draining lymph node for PD-L1+CD80+ migratory, conventional dendritic cells (cDCs) and promoted CTL priming. Blockade of CD86 alone or in combination with PD-1, enhanced intra-tumoral CTL accumulation and the combination significantly increased RT-induced tumor regression and overall survival. We advise that combining RT with PD-1 and/or CTLA-4 blockade may be counterproductive in lymphocyte-depleted cancers, since they drive Treg responses in this context. However, combining RT with CD86 blockade may promote control of such tumors by enabling a CTL response.
Elselien Frijlink, Douwe M.T. Bosma, Julia Busselaar, Thomas W. Battaglia, Mo D. Staal, Inge Verbrugge, Jannie Borst
Neutrophil (PMN) tissue accumulation is an established feature of ulcerative colitis (UC) lesions and colorectal cancer (CRC). To assess the PMN phenotypic and functional diversification during inflammatory ulceration to CRC transition we analyzed the transcriptomic landscape of blood and tissue PMNs. Transcriptional programs effectively separated PMNs based on their localization to peripheral blood, inflamed colon, and tumors. In silico pathway overrepresentation analysis, protein-network mapping, gene signature identification, and gene-ontology scoring revealed unique enrichment of angiogenic and vasculature development pathways in tumor-associated neutrophils (TANs). Functional studies utilizing ex vivo cultures, colitis-induced murine CRC, and patient-derived xenograft models demonstrated a critical role for TANs in promoting tumor vascularization. Spp1 (OPN) and Mmp14 (MT1-MMP) were identified by unbiased -omics and mechanistic studies to be highly induced in TANs, acting to critically regulate endothelial cell chemotaxis and branching. TCGA dataset and clinical specimens confirmed enrichment of SPP1 and MMP14 in high-grade CRC but not in UC patients. Pharmacological inhibition of TAN trafficking or MMP14 activity effectively reduced tumor vascular density, leading to CRC regression. Our findings, demonstrate a niche-directed PMN functional specialization, and identify TAN contributions to tumor vascularization, delineating a new therapeutic framework for CRC treatment focused on TAN angiogenic properties.
Triet M. Bui, Lenore K. Yalom, Edward Ning, Jessica M. Urbanczyk, Xingsheng Ren, Caroline J. Herrnreiter, Jackson A. DiSario, Brian Wray, Matthew J. Schipma, Yuri S. Velichko, David P. Sullivan, Kouki Abe, Shannon M. Lauberth, Guang-Yu Yang, Parambir S. Dulai, Stephen B. Hanauer, Ronen Sumagin
Epigenetics is a biological process that modifies and regulates gene expression, affects neuronal function, and contributes to pain. However, the mechanism by which epigenetics facilitates and maintains chronic pain is poorly understood. We aimed to determine whether N6-methyladenosine (m6A) specifically modified by methyltransferase 14 (METTL14) alters neuronal activity and governs pain by sensitizing the GluN2A subunit of the N-methyl-D-aspartate receptor (NMDAR) in the dorsal root ganglion (DRG) neurons in a model of chemotherapy-induced neuropathic pain (CINP). Using dot blotting, immunofluorescence, gain/loss-of-function, and behavioral assays, we found that m6A levels were upregulated in L4–L6 DRG neurons in the CINP in a DBP/METT14-dependent manner, which was also confirmed in human DRGs. Blocking METTL14 reduced m6A methylation and attenuated pain hypersensitivity. Mechanistically, METTL14-mediated m6A modification facilitated the synaptic plasticity of DRG neurons by enhancing the GluN2A subunit of NMDAR, and inhibiting METTL14 blocked this effect. In contrast, overexpression of METTL14 upregulated m6A modifications, enhanced presynaptic NMDAR activity in DRG neurons, and facilitated pain sensation. Our findings reveal a previously unrecognized mechanism of METTL14-mediated m6A modification in DRG neurons to maintain neuropathic pain. Targeting these molecules may provide a new strategy for pain treatment.
Weicheng Lu, Xiaohua Yang, Weiqiang Zhong, Guojun Chen, Xinqi Guo, Qingqing Ye, Yixin Xu, Zhenhua Qi, Yaqi Ye, Jingyun Zhang, Yuge Wang, Xintong Wang, Shu Wang, Qiyue Zhao, Weian Zeng, Junting Huang, Huijie Ma, Jingdun Xie
Diffuse midline glioma (DMG), including tumors diagnosed in the brainstem (diffuse intrinsic pontine glioma – DIPG), are uniformly fatal brain tumors that lack effective treatment. Analysis of CRISPR-Cas9 loss-of-function gene deletion screens identified PIK3CA and MTOR as targetable molecular dependencies across DIPG patient models, highlighting the therapeutic potential of the blood-brain barrier penetrant PI3K/Akt/mTOR inhibitor, paxalisib. At the human equivalent maximum tolerated dose, mice treated with paxalisib experienced systemic glucose feedback and increased insulin levels commensurate with patients using PI3K inhibitors. To exploit genetic dependence and overcome resistance whilst maintaining compliance and therapeutic benefit, we combined paxalisib with the anti-hyperglycemic drug, metformin. Metformin restored glucose homeostasis and decreased phosphorylation of the insulin receptor in vivo, a common mechanism of PI3K-inhibitor resistance, extending survival of orthotopic models. DIPG models treated with paxalisib increased calcium-activated PKC signaling. The brain penetrant PKC inhibitor enzastaurin in combination with paxalisib, synergistically extended the survival of multiple orthotopic patient-derived and immunocompetent syngeneic allograft models; benefits potentiated in combination with metformin and standard-of-care radiotherapy. Therapeutic adaptation was assessed using spatial transcriptomics and ATAC-sequencing, identifying changes in myelination and tumor immune microenvironment crosstalk. Together, we have identified a clinically relevant DIPG therapeutic combinatorial approach.
Ryan J. Duchatel, Evangeline R. Jackson, Sarah G. Parackal, Dylan Kiltschewskij, Izac J. Findlay, Abdul Mannan, Dilana E. Staudt, Bryce C. Thomas, Zacary P. Germon, Sandra Laternser, Padraic S. Kearney, M. Fairuz B. Jamaluddin, Alicia M. Douglas, Tyrone S. Beitaki, Holly P. McEwen, Mika L. Persson, Emily A. Hocke, Vaibhav Jain, Michael Aksu, Elizabeth E. Manning, Heather C. Murray, Nicole M. Verrills, Claire Xin Sun, Paul Daniel, Ricardo E. Vilain, David A. Skerrett-Byrne, Brett Nixon, Susan Hua, Charles E. de Bock, Yolanda Colino-Sanguino, Fatima Valdes-Mora, Maria Tsoli, David S. Ziegler, Murray J. Cairns, Eric H. Raabe, Nicholas A. Vitanza, Esther Hulleman, Timothy N. Phoenix, Carl Koschmann, Frank Alvaro, Christopher V. Dayas, Christopher L. Tinkle, Helen Wheeler, James R. Whittle, David D. Eisenstat, Ron Firestein, Sabine Mueller, Santosh Valvi, Jordan R. Hansford, David M. Ashley, Simon G. Gregory, Lindsay B. Kilburn, Javad Nazarian, Jason E. Cain, Matthew D. Dun
Virtually all patients with BRAF-mutant melanoma develop resistance to MAPK inhibitors largely through non-mutational events. Although the epigenetic landscape is shown to be altered in therapy-resistant melanomas and other cancers, a specific targetable epigenetic mechanism has not been validated to date. Here, we evaluate the CoREST repressor complex and the recently developed bivalent inhibitor, corin, within the context of melanoma phenotype plasticity and therapeutic resistance. We find that CoREST is a critical mediator of the major distinct melanoma phenotypes and that corin treatment of melanoma cells leads to phenotype reprogramming. Global assessment of transcript and chromatin changes conferred by corin reveals specific effects on histone marks connected to EMT-associated transcription factors and the dual-specificity phosphatases (DUSPs). Remarkably, treatment of BRAF inhibitor (BRAFi)-resistant melanomas with corin promotes resensitization to BRAFi therapy. DUSP1 is consistently downregulated in BRAFi-resistant melanomas which is reversed by corin treatment and associated with inhibition of p38 MAPK activity and resensitization to BRAFi therapies. Moreover, this activity can be recapitulated by the p38 MAPK inhibitor, BIRB 796. These findings identify the CoREST repressor complex as a central mediator of melanoma phenotype plasticity and resistance to targeted therapy and suggest that CoREST inhibitors may prove beneficial to patients with BRAFi-resistant melanoma.
Muzhou Wu, Ailish Hanly, Frederick Gibson, Robert Fisher, Samantha Rogers, Kihyun Park, Angelina Zuger, Kevin Kuang, Jay H. Kalin, Sarah Nocco, Matthew Cole, Amy Xiao, Filisia Agus, Adam Labadorf, Samuel Beck, Marianne Collard, Philip A. Cole, Rhoda M. Alani
BACKGROUND. HER2-targeting therapies have great efficacy in HER2-positive breast cancer, but resistance in part due to HER2 heterogeneity (HET) is a significant clinical challenge. We previously described that in a phase II neoadjuvant trastuzumab emtansine (T-DM1) and pertuzumab (T-DM1+P) clinical trial in early-stage HER2-positive breast cancer, none of the patients with HER2-HET tumors had pathologic complete response (pCR). METHODS. To investigate cellular and molecular differences among tumors according to HER2 heterogeneity and pCR, we performed RNA sequencing (RNA-seq) and ERBB2 FISH of 285 pre/post-treatment tumors from 129 patients in this T-DM1+P neoadjuvant trial. A subset of cases was also subject to Nanostring spatial digital profiling. RESULTS. Pre-treatment tumors from patients with pCR had the highest level of ERBB2 mRNA and ERBB signaling. HET was associated with no pCR, basal-like features, low ERBB2 expression yet high ERBB signaling sustained by activation of downstream pathway components. Residual tumors showed decreased HER2 protein levels and ERBB2 copy number heterogeneity and increased PI3K pathway enrichment and luminal features. HET tumors showed minimal treatment-induced transcriptomic changes compared to non-HET tumors. Immune infiltration correlated with pCR and HER2-HET status. CONCLUSION. Resistance mechanisms in HET and non-HET tumors are distinct. HER2-targeting antibodies have limited efficacy in HET tumors. Our results support the stratification of patients based on HET status and the use of agents that target downstream components of the ERBB signaling pathway in patients with HET tumors. TRIAL REGISTRATION. Clinicaltrials.gov NCT02326974. FUNDING. This study was funded by Roche and the National Cancer Institute.
Zheqi Li, Otto Metzger Filho, Giuseppe Viale, Patrizia dell'Orto, Leila Russo, Marie-Anne Goyette, Avni Kamat, Denise A. Yardley, Vandana Gupta Abramson, Carlos L. Arteaga, Laura M. Spring, Kami Chiotti, Carol Halsey, Adrienne G. Waks, Tari A. King, Susan C. Lester, Jennifer R. Bellon, Eric P. Winer, Paul T. Spellman, Ian E. Krop, Kornelia Polyak
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