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
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
Merkel cell carcinoma (MCC) is a highly immunogenic skin cancer primarily induced by Merkel Cell Polyomavirus, driven by the expression of the oncogenic T antigens (T-Ags). Blockade of the programmed cell death protein-1 (PD-1) pathway has shown remarkable response rates, but evidence for therapy-associated T-Ag-specific immune response and therapeutic strategies for the non-responding fraction are both limited. We tracked T-Ag-reactive CD8+ T cells in peripheral blood of 26 MCC patients under anti-PD1 therapy, using DNA-barcoded pMHC multimers, displaying all peptides from the predicted HLA ligandome of the oncoproteins, covering 33 class-I haplotypes. We observed a broad T-cell recognition of T-Ags, including identification of 20 novel T-Ag-derived epitopes. Broadening of the T-Ag recognition profile and increased T-cell frequencies during therapy were strongly associated with clinical response and prolonged progression-free survival. T-Ag-specific T cells could be further boosted and expanded directly from peripheral blood using artificial antigen-presenting scaffolds, even in patients with no detectable T-Ag-specific T cells. These T cells provided strong tumor rejection capacity while retaining a favorable phenotype for adoptive cell transfer. These findings demonstrate that T-Ag-specific T cells are associated with the clinical outcome to PD-1 blockade and that Ag-presenting scaffolds can be used to boost such responses.
Ulla Kring Hansen, Candice D. Church, Ana Micaela Carnaz Simões, Marcus Svensson Frej, Amalie Kai Bentzen, Siri A. Tvingsholm, Jürgen C. Becker, Steven P. Fling, Nirasha Ramchurren, Suzanne L. Topalian, Paul T. Nghiem, Sine Reker Hadrup
Wnts, cholesterol, and MAPK signaling are essential for development and adult homeostasis. Here we report for the first time 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, regulates the abundance of specific C4-methyl sterols lophenol and dihydro-TMAS. Highlighting its clinical relevance, FAXDC2 is 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, accumulates in the cancerous tissues and not in adjacent normal tissues. FAXDC2 links Wnts to RTK/MAPK signaling. Wnt inhibition drives increased recycling of RTKs and activation of the MAPK pathway, and this requires FAXDC2. Blocking Wnt signaling in Wnt-high cancers causes both differentiation and senescence; and this is prevented by knockout of FAXDC2. Our data shows the integration of three ancient pathways, Wnts, cholesterol synthesis, and RTK/MAPK signaling, in cellular proliferation and differentiation.
Babita Madan, Shawn R. Wadia, Siddhi Patnaik, Nathan Harmston, Emile K.W. Tan, Iain Bee Huat Tan, W. David Nes, Enrico Petretto, David M. Virshup
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 unravelling the underlying cues may provide novel strategies to manage metastatic disease. Using breast cancer experimental models and transcriptomic analyses, we showed 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 Gr1high Ly6G+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 OSM and IL6. JAK inhibition prevented OSM/IL6-induced SCA1+ population enrichment while OSM/IL6 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 IL6 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 OS, RFS and lung metastasis in breast cancer patients. Collectively, our data identified OSM/IL6-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
The combination of targeted therapy with immune checkpoint inhibition (ICI) is an area of intense interest. We studied the interaction of fibroblast growth factor receptor (FGFR) inhibition with ICI in urothelial carcinoma (UC) of the bladder, in which FGFR3 is altered in 50% of cases. Using an FGFR3-driven, Trp53-mutant genetically engineered murine model (UPFL), we demonstrate that UPFL tumors recapitulate the histology and molecular subtype of their FGFR3-altered human counterparts. Additionally, UPFL1 allografts exhibit hyperprogression to ICI associated with an expansion of T regulatory cells (Tregs). Erdafitinib blocked Treg proliferation in vitro, while in vivo ICI-induced Treg expansion was fully abrogated by FGFR inhibition. Combined erdafitinib and ICI resulted in high therapeutic efficacy. In aggregate, our work establishes that, in mice, co-alteration of FGFR3 and Trp53 results in high-grade, non–muscle-invasive UC and presents a previously underappreciated role for FGFR inhibition in blocking ICI-induced Treg expansion.
Atsushi Okato, Takanobu Utsumi, Michela Ranieri, Xingnan Zheng, Mi Zhou, Luiza D. Pereira, Ting Chen, Yuki Kita, Di Wu, Hyesun Hyun, Hyojin Lee, Andrew S. Gdowski, John D. Raupp, Sean Clark-Garvey, Ujjawal Manocha, Alison Chafitz, Fiona Sherman, Janaye Stephens, Tracy L. Rose, Matthew I. Milowsky, Sara E. Wobker, Jonathan S. Serody, Jeffrey S. Damrauer, Kwok-Kin Wong, William Y. Kim
Neutrophil Extracellular Traps (NETs), a web-like structure of cytosolic and granule proteins assembled on decondensed chromatin, kill pathogens and causes tissue damage in diseases. Whether NETs can kill cancer cells is unexplored. Here, we report that a combination of glutaminase inhibitor CB-839 and 5-FU inhibits the growth of PIK3CA mutant colorectal cancers (CRCs) in xenograft, syngeneic, and genetically engineered mouse models in part through NETs. Disruption of NETs by either DNase I treatment or depletion of neutrophils in CRCs attenuated the efficacy of the drug combination. Moreover, NETs were present in tumor biopsies taken from patients treated with the drug combination in a phase II clinical trial. Increased NET levels in tumors are associated with longer progression-free survival. Mechanistically, the drug combination induced the expression of IL-8 preferentially in PIK3CA mutant CRCs to attract neutrophils into the tumors. Further, the drug combination increased the levels of reactive oxygen species in neutrophils, thereby inducing NETs. Cathepsin G (CTSG), a serine protease localized in NETs, enters CRC cells through the RAGE cell surface protein. The internalized CTSG cleaves 14-3-3 proteins, releases Bax, and triggers apoptosis in CRC cells. Thus, our studies illuminate a previously unrecognized mechanism by which chemotherapy-induced NETs kill cancer cells.
Yamu Li, Sulin Wu, Yiqing Zhao, Trang Dinh, Dongxu Jiang, J. Eva Selfridge, George Myers, Yuxiang Wang, Xuan Zhao, Suzanne L. Tomchuck, George Dubyak, Richard T. Lee, Bassam Estfan, Marc Shapiro, Suneel D. Kamath, Amr Mohamed, Stanley C.-C. Huang, Alex Y. Huang, Ronald A. Conlon, Smitha S. Krishnamurthi, Jennifer R. Eads, Joseph E. Willis, Alok A. Khorana, David L. Bajor, Zhenghe Wang
BACKGROUND. Improving and predicting tumor response to immunotherapy remains challenging. Combination therapy with a transforming growth factor-β receptor (TGF-βR) inhibitor that targets cancer associated fibroblasts (CAFs) is promising to enhance efficacy of immunotherapies. However, the effect of this approach in clinical trials is limited, requiring in vivo methods to better assess tumor responses to combination therapy. METHODS. We measure CAFs in vivo using 68Ga-labeled fibroblast activation protein inhibitor (68Ga-FAPI)-04 for PET/CT imaging to guide combination of TGF-β inhibition and immunotherapy. 131 patients with metastatic colorectal cancer (CRC) underwent 68Ga-FAPI and 18F-fludeoxyglucose (18F-FDG) PET/CT imaging. Relationship between uptake of 68Ga-FAPI and tumor immunity was analyzed in patients. Mouse cohorts of metastatic CRC were treated with TGF-βR inhibitor combined with KN046 which blocks PD-L1 and CTLA4, followed with 68Ga-FAPI and 18F-FDG micro-PET/CT imaging to assess tumor responses. RESULTS. Patients with metastatic CRC demonstrated high uptakes of 68Ga-FAPI, along with suppressive tumor immunity and poor prognosis. TGF-βR inhibitor enhanced tumor infiltrating T cells and significantly sensitized metastatic CRC to KN046. 68Ga-FAPI PET/CT imaging accurately monitored the dynamical changes of CAFs and tumor response to combined TGF-βR inhibitor with immunotherapy. CONCLUSION. 68Ga-FAPI PET/CT imaging is powerful in assessing tumor immunity and response to immunotherapy in metastatic CRC. This study supports future clinical application of 68Ga-FAPI PET/CT to guide CRC patients for precise TGF-β inhibition plus immunotherapy, recommending 68Ga-FAPI and 18F-FDG dual PET/CT for CRC management. TRIAL REGISTRATION. CFFSTS Trial, ChiCTR2100053984, Chinese Clinical Trial Registry. FUNDING. National Natural Science Foundation of China (82072695, 32270767, 82272035,81972260).
Ke Li, Wei Liu, Hang Yu, Jiwei Chen, Wenxuan Tang, Jianpeng Wang, Ming Qi, Yuyun Sun, Xiaoping Xu, Ji Zhang, Xinxiang Li, Weijian Guo, Xiaoling Li, Shaoli Song, Shuang Tang