Breast cancer stem cells (BCSCs) mitigate oxidative stress to maintain their viability and plasticity. However, the regulatory mechanism of oxidative stress in BCSCs remains unclear. We recently found that the histone reader ZMYND8 was upregulated in BCSCs. Here, we showed that ZMYND8 reduced ROS and iron to inhibit ferroptosis in aldehyde dehydrogenase–high (ALDHhi) BCSCs, leading to BCSC expansion and tumor initiation in mice. The underlying mechanism involved a two-fold posttranslational regulation of nuclear factor erythroid 2–related factor 2 (NRF2). ZMYND8 increased stability of NRF2 protein through KEAP1 silencing. On the other hand, ZMYND8 interacted with and recruited NRF2 to the promoters of antioxidant genes to enhance gene transcription in mammospheres. NRF2 phenocopied ZMYND8 to enhance BCSC stemness and tumor initiation by inhibiting ROS and ferroptosis. Loss of NRF2 counteracted ZMYND8’s effects on antioxidant genes and ROS in mammospheres. Interestingly, ZMYND8 expression was directly controlled by NRF2 in mammospheres. Collectively, these findings uncover a positive feedback loop that amplifies the antioxidant defense mechanism sustaining BCSC survival and stemness.
Maowu Luo, Lei Bao, Yuanyuan Xue, Ming Zhu, Ashwani Kumar, Chao Xing, Jennifer E. Wang, Yingfei Wang, Weibo Luo
Wnts, cholesterol, and MAPK signaling are essential for development and adult homeostasis. Here, we report that fatty acid hydroxylase domain containing 2 (FAXDC2), a previously uncharacterized enzyme, functions as a methyl sterol oxidase catalyzing C4 demethylation in the Kandutsch-Russell branch of the cholesterol biosynthesis pathway. FAXDC2, a paralog of MSMO1, regulated the abundance of the specific C4-methyl sterols lophenol and dihydro-T-MAS. Highlighting its clinical relevance, FAXDC2 was repressed in Wnt/β-catenin–high cancer xenografts, in a mouse genetic model of Wnt activation, and in human colorectal cancers. Moreover, in primary human colorectal cancers, the sterol lophenol, regulated by FAXDC2, accumulated in the cancerous tissues and not in adjacent normal tissues. FAXDC2 linked Wnts to RTK/MAPK signaling. Wnt inhibition drove increased recycling of RTKs and activation of the MAPK pathway, and this required FAXDC2. Blocking Wnt signaling in Wnt-high cancers caused both differentiation and senescence; and this was prevented by knockout of FAXDC2. Our data show the integration of 3 ancient pathways, Wnts, cholesterol synthesis, and RTK/MAPK signaling, in cellular proliferation and differentiation.
Babita Madan, Shawn R. Wadia, Siddhi Patnaik, Nathan Harmston, Emile Tan, Iain Bee Huat Tan, W. David Nes, Enrico Petretto, David M. Virshup
Virtually all patients with BRAF-mutant melanoma develop resistance to MAPK inhibitors largely through nonmutational 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. Here, we evaluated the corepressor for element 1–silencing transcription factor (CoREST) epigenetic repressor complex and the recently developed bivalent inhibitor corin within the context of melanoma phenotype plasticity and therapeutic resistance. We found that CoREST was a critical mediator of the major distinct melanoma phenotypes and that corin treatment of melanoma cells led to phenotype reprogramming. Global assessment of transcript and chromatin changes conferred by corin revealed specific effects on histone marks connected to epithelial-mesenchymal transition–associated (EMT-associated) transcription factors and the dual-specificity phosphatases (DUSPs). Remarkably, treatment of BRAF inhibitor–resistant (BRAFi-R) melanomas with corin promoted resensitization to BRAFi therapy. DUSP1 was consistently downregulated in BRAFi-R melanomas, which was reversed by corin treatment and associated with inhibition of p38 MAPK activity and resensitization to BRAFi therapies. Moreover, this activity was 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 for 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
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 patient derived models of DIPG, 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 while maintaining compliance and therapeutic benefit, we combined paxalisib with the antihyperglycemic 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-Seq, identifying changes in myelination and tumor immune microenvironment crosstalk. Collectively, this study has identified what we believe to be a clinically relevant DIPG therapeutic combinational strategy.
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 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
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 enhanced spontaneous priming of thymus-derived (FOXP3+Helios+) Tregs by the tumor. These Tregs acquired an effector phenotype, populated the tumor, and impeded 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, the CD28 ligands CD86 and CD80 differentially affected the Treg response. CD86, but not CD80, blockade prevented the effector Treg response, enriched the tumor-draining lymph node migratory conventional DCs that were positive for PD-L1 and CD80 (PD-L1+CD80+), and promoted CTL priming. Blockade of CD86 alone or in combination with PD-1 enhanced intratumoral CTL accumulation, and the combination significantly increased RT-induced tumor regression and OS. We advise that combining RT with PD-1 and/or CTLA-4 blockade may be counterproductive in lymphocyte-depleted cancers, since these interventions drive Treg responses in this context. However, combining RT with CD86 blockade may promote the 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
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-like 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 CINP in a DBP/METTL14-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
Cancer cell plasticity contributes to therapy resistance and metastasis, which represent the main causes of cancer-related death, including in breast cancer. The tumor microenvironment drives cancer cell plasticity and metastasis, and unraveling the underlying cues may provide novel strategies for managing metastatic disease. Using breast cancer experimental models and transcriptomic analyses, we show that stem cell antigen-1 positive (SCA1+) murine breast cancer cells enriched during tumor progression and metastasis had higher in vitro cancer stem cell–like properties, enhanced in vivo metastatic ability, and generated tumors rich in Gr1hiLy6G+CD11b+ cells. In turn, tumor-educated Gr1+CD11b+ (Tu-Gr1+CD11b+) cells rapidly and transiently converted low metastatic SCA1– cells into highly metastatic SCA1+ cells via secreted oncostatin M (OSM) and IL-6. JAK inhibition prevented OSM/IL-6–induced SCA1+ population enrichment, while OSM/IL-6 depletion suppressed Tu-Gr1+CD11b+–induced SCA1+ population enrichment in vitro and metastasis in vivo. Moreover, chemotherapy-selected highly metastatic 4T1 cells maintained high SCA1+ positivity through autocrine IL-6 production, and in vitro JAK inhibition blunted SCA1 positivity and metastatic capacity. Importantly, Tu-Gr1+CD11b+ cells invoked a gene signature in tumor cells predicting shorter overall survival (OS), relapse-free survival (RFS), and lung metastasis in breast cancer patients. Collectively, our data identified OSM/IL-6/JAK as a clinically relevant paracrine/autocrine axis instigating breast cancer cell plasticity and triggering metastasis.
Sanam Peyvandi, Manon Bulliard, Alev Yilmaz, Annamaria Kauzlaric, Rachel Marcone, Lisa Haerri, Oriana Coquoz, Yu-Ting Huang, Nathalie Duffey, Laetitia Gafner, Girieca Lorusso, Nadine Fournier, Qiang Lan, Curzio Rüegg
Aneuploidy, a deviation from the normal chromosome copy number, is common in human embryos and is considered a primary cause of implantation failure and early pregnancy loss. Meiotic errors lead to uniformly abnormal karyotypes, while mitotic errors lead to chromosomal mosaicism: the presence of cells with at least 2 different karyotypes within an embryo. Knowledge about mosaicism in blastocysts mainly derives from bulk DNA sequencing (DNA-Seq) of multicellular trophectoderm (TE) and/or inner cell mass (ICM) samples. However, this can only detect an average net gain or loss of DNA above a detection threshold of 20%–30%. To accurately assess mosaicism, we separated the TE and ICM of 55 good-quality surplus blastocysts and successfully applied single-cell whole-genome sequencing (scKaryo-Seq) on 1,057 cells. Mosaicism involving numerical and structural chromosome abnormalities was detected in 82% of the embryos, in which most abnormalities affected less than 20% of the cells. Structural abnormalities, potentially caused by replication stress and DNA damage, were observed in 69% of the embryos. In conclusion, our findings indicated that mosaicism was prevalent in good-quality blastocysts, whereas these blastocysts would likely be identified as normal with current bulk DNA-Seq techniques used for preimplantation genetic testing for aneuploidy.
Effrosyni A. Chavli, Sjoerd J. Klaasen, Diane Van Opstal, Joop S.E. Laven, Geert J.P.L. Kops, Esther B. Baart
Ischemia/reperfusion injury–mediated (IRI-mediated) primary graft dysfunction (PGD) adversely affects both short- and long-term outcomes after lung transplantation, a procedure that remains the only treatment option for patients suffering from end-stage respiratory failure. While B cells are known to regulate adaptive immune responses, their role in lung IRI is not well understood. Here, we demonstrated by intravital imaging that B cells are rapidly recruited to injured lungs, where they extravasate into the parenchyma. Using hilar clamping and transplant models, we observed that lung-infiltrating B cells produce the monocyte chemokine CCL7 in a TLR4-TRIF–dependent fashion, a critical step contributing to classical monocyte (CM) recruitment and subsequent neutrophil extravasation, resulting in worse lung function. We found that synergistic BCR-TLR4 activation on B cells is required for the recruitment of CMs to the injured lung. Finally, we corroborated our findings in reperfused human lungs, in which we observed a correlation between B cell infiltration and CM recruitment after transplantation. This study describes a role for B cells as critical orchestrators of lung IRI. As B cells can be depleted with currently available agents, our study provides a rationale for clinical trials investigating B cell–targeting therapies.
Khashayar Farahnak, Yun Zhu Bai, Yuhei Yokoyama, Deniz B. Morkan, Zhiyi Liu, Junedh M. Amrute, Alejandro De Filippis Falcon, Yuriko Terada, Fuyi Liao, Wenjun Li, Hailey M. Shepherd, Ramsey R. Hachem, Varun Puri, Kory J. Lavine, Andrew E. Gelman, Ankit Bharat, Daniel Kreisel, Ruben G. Nava
Virus-induced memory T cells often express functional cross-reactivity, or heterologous immunity, to other viruses and to allogeneic MHC molecules that is an important component of pathogenic responses to allogeneic transplants. During immune responses, antigen-reactive naive and central memory T cells proliferate in secondary lymphoid organs to achieve sufficient cell numbers to effectively respond, whereas effector memory T cell proliferation occurs directly within the peripheral inflammatory microenvironment. Mechanisms driving heterologous memory T cell proliferation and effector function expression within peripheral tissues remain poorly understood. Here, we dissected proliferation of heterologous donor-reactive memory CD8+ T cells and their effector functions following infiltration into heart allografts with low or high intensities of ischemic inflammation. Proliferation within both ischemic conditions required p40 homodimer–induced IL-15 transpresentation by graft DCs, but expression of effector functions mediating acute allograft injury occurred only in high-ischemic allografts. Transcriptional responses of heterologous donor-reactive memory CD8+ T cells were distinct from donor antigen–primed memory CD8+ T cells during early activation in allografts and at graft rejection. Overall, the results provide insights into mechanisms driving heterologous effector memory CD8+ T cell proliferation and the separation between proliferation and effector function that is dependent on the intensity of inflammation within the tissue microenvironment.
Hidetoshi Tsuda, Karen S. Keslar, William M. Baldwin III, Peter S. Heeger, Anna Valujskikh, Robert L. Fairchild
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