Understanding the tumor immune microenvironment (TIME) promises to be key for optimal cancer therapy, especially in triple-negative breast cancer (TNBC). Integrating spatial resolution of immune cells with laser capture microdissection gene expression profiles, we defined distinct TIME stratification in TNBC with implications for current therapies, including immune checkpoint blockade. TNBCs with an immunoreactive microenvironment exhibited tumoral infiltration of granzyme B+ CD8+ T cells, a type I interferon signature, elevated expression of multiple immune inhibitory molecules, including IDO, PD-L1, and good outcome. An “immune-cold” microenvironment with absence of tumoral CD8+ T cells was defined by elevated expression of the immunosuppressive marker B7-H4, signatures of fibrotic stroma and poor outcome. A distinct poor outcome immunomodulatory microenvironment, hitherto poorly characterized, exhibited stromal restriction of CD8+ T cells, stromal expression of PD-L1 and enrichment for signatures of cholesterol biosynthesis. Metasignatures defining these TIME subtypes stratified TNBC, predicting outcome and identifying potential therapeutic targets for TNBC.
Tina Gruosso, Mathieu Gigoux, Venkata Satya Kumar Manem, Nicholas Bertos, Dongmei Zuo, Irina Perlitch, Sadiq Mehdi Ismail Saleh, Hong Zhao, Margarita Souleimanova, Radia Marie Johnson, Anne Monette, Valentina Munoz Ramos, Michael Trevor Hallett, John Stagg, Réjean Lapointe, Atilla Omeroglu, Sarkis Meterissian, Laurence Buisseret, Gert Van den Eynden, Roberto Salgado, Marie-Christine Guiot, Benjamin Haibe-Kains, Morag Park
Constitutive JAK2 signaling is central to myeloproliferative neoplasm (MPN) pathogenesis and results in activation of STAT, PI3K/AKT and MEK/ERK signaling. However, the therapeutic efficacy of current JAK2 inhibitors is limited. We investigated the role of MEK/ERK signaling in MPN cell survival in the setting of JAK kinase inhibition. Type I and II JAK2 inhibition suppressed MEK/ERK activation in MPN cell lines in vitro, but not in Jak2V617F and MPLW515L mouse models in vivo. JAK2 inhibition ex vivo inhibited MEK/ERK signaling suggesting cell extrinsic factors maintain ERK activation in vivo. We identified PDGFRα as an activated kinase that remains activated upon JAK2 inhibition in vivo, and PDGF-AA/PDGF-BB production persisted in the setting of JAK kinase inhibition. PDGF-BB maintained ERK activation in presence of ruxolitinib consistent with its function as a ligand-induced bypass for ERK activation. Combined JAK/MEK inhibition suppressed MEK/ERK activation in Jak2V617F and MPLW515L mice with increased efficacy and reversal of fibrosis to an extent not seen with JAK inhibitors. This demonstrates that compensatory ERK activation limits the efficacy of JAK2 inhibition and dual JAK/MEK inhibition provides an opportunity for improved therapeutic efficacy in MPNs and in other malignancies driven by aberrant JAK-STAT signaling.
Simona Stivala, Tamara Codilupi, Sime Brkic, Anne Baerenwaldt, Nilabh Ghosh, Hui Hao-Shen, Stephan Dirnhofer, Matthias S. Dettmer, Cedric Simillion, Beat A. Kaufmann, Sophia Chiu, Matthew D. Keller, Maria Kleppe, Morgane Hilpert, Andreas S. Buser, Jakob R. Passweg, Thomas Radimerski, Radek C. Skoda, Ross L. Levine, Sara C. Meyer
The discovery of recurrent mutations in subunits of the vacuolar-type H+-translocating ATPase (v-ATPase) in follicular lymphoma (FL) highlights a role for the amino acid- and energy-sensing pathway to MTOR in the pathogenesis of this disease. Here, through the use of complementary experimental approaches involving mammalian cells and Saccharomyces cerevisiae, we have demonstrated that mutations in the v-ATPase subunit ATP6V1B2/Vma2 activate autophagic flux and maintain MTOR/Tor in an active state. Engineered lymphoma cell lines and primary follicular lymphoma B cells (FL B cells) carrying mutated ATP6V1B2 demonstrated a remarkable ability to survive low leucine concentrations. The treatment of primary FL B cells with inhibitors of autophagy uncovered an addiction for survival for FL B cells harboring ATP6V1B2 mutants. These data support mutational activation of autophagic flux by recurrent hotspot mutations in ATP6V1B2 as an adaptive mechanism in FL pathogenesis and as a new possible therapeutically targetable pathway.
Fangyang Wang, Damián Gatica, Zhang Xiao Ying, Luke F. Peterson, Peter K. Kim, Denzil Bernard, Kamlai Saiya-Cork, Shaomeng Wang, Mark S. Kaminski, Alfred E. Chang, Tycel Phillips, Daniel J. Klionsky, Sami N. Malek
Hyperactivated AKT/mTOR signaling is a hallmark of pancreatic neuroendocrine tumors (PNETs). Drugs targeting this pathway are used clinically but tumor resistance invariably develops. A better understanding of factors regulating AKT/mTOR signaling and PNET pathogenesis is needed to improve current therapies. We discovered that RABL6A, a new oncogenic driver of PNET proliferation, is required for AKT activity. Silencing RABL6A caused PNET cell cycle arrest that coincided with selective loss of AKT-S473 (not T308) phosphorylation and AKT/mTOR inactivation. Restoration of AKT phosphorylation rescued the G1 phase block triggered by RABL6A silencing. Mechanistically, loss of AKT-S473 phosphorylation in RABL6A depleted cells resulted from increased protein phosphatase 2A (PP2A) activity. Inhibition of PP2A restored phosphorylation of AKT-S473 in RABL6A depleted cells whereas PP2A reactivation using a specific small molecule activator of PP2A (SMAP) abolished that phosphorylation. Moreover, SMAP treatment effectively killed PNET cells in a RABL6A-dependent manner and suppressed PNET growth in vivo. This work identifies RABL6A as a new inhibitor of the PP2A tumor suppressor and essential activator of AKT in PNET cells. Our findings offer what we believe is a novel strategy of PP2A reactivation for treatment of PNETs as well as other human cancers driven by RABL6A overexpression and PP2A inactivation.
Shaikamjad Umesalma, Courtney A. Kaemmer, Jordan L. Kohlmeyer, Blake L. Letney, Angela M. Schab, Jacqueline A. Reilly, Ryan M. Sheehy, Jussara Hagen, Nitija Tiwari, Fenghuang Zhan, Mariah R. Leidinger, Thomas M. O'Dorisio, Joseph S. Dillon, Ronald A. Merrill, David K. Meyerholz, Abbey L. Perl, Bart J. Brown, Terry A. Braun, Aaron T. Scott, Timothy Ginader, Agshin F. Taghiyev, Gideon K. Zamba, James R. Howe, Stefan Strack, Andrew M. Bellizzi, Goutham Narla, Benjamin W. Darbro, Frederick W. Quelle, Dawn E. Quelle
Parisa Malekzadeh, Anna Pasetto, Paul F. Robbins, Maria R. Parkhurst, Biman C. Paria, Li Jia, Jared J. Gartner, Victoria Hill, Zhiya Yu, Nicholas P. Restifo, Abraham Sachs, Eric Tran, Winifred Lo, Robert P.T. Somerville, Steven A. Rosenberg, Drew C. Deniger
Although ccRCC has been shown to have widespread aberrant cytosine methylation and loss of hydroxymethylation (5hmC), the prognostic impact and therapeutic targeting of this epigenetic aberrancy has not been fully explored. Analysis of 576 primary ccRCC samples demonstrated that loss of 5hmC was significantly associated with aggressive clinicopathologic features and was an independent adverse prognostic factor. Loss of 5hmC also predicted reduced progression free survival after resection of non-metastatic disease. The loss of 5hmC in ccRCC was not due to mutational or transcriptional inactivation of TET enzymes, but by their functional inactivation by l-2-hydroxyglutarate (L2HG) that was overexpressed due to the deletion and under-expression of l-2-hydroxyglutarate dehydrogenase (L2HGDH). Ascorbic acid (AA) reduced methylation and restored genome wide 5hmC levels via TET activation. Fluorescence quenching of the recombinant TET-2 protein was unaffected by L2HG in the presence of AA. Pharmacologic AA treatment led to reduced growth of ccRCC in vitro and reduced tumor growth in vivo, with increased intratumoral 5hmC. These data demonstrate that reduced 5hmC is associated with reduced survival in ccRCC and provide a preclinical rationale for exploring the therapeutic potential of high dose AA in ccRCC.
Niraj Shenoy, Tushar D. Bhagat, John C. Cheville, Christine Lohse, Sanchari Bhattacharyya, Alexander Tischer, Venkata Machha, Shanisha Gordon-Mitchell, Gaurav S. Choudhary, Li-Fan Wong, LouAnn Gross, Emily Ressegue, Bradley C. Leibovich, Stephen A. Boorjian, Ulrich Steidl, Xiaosheng Wu, Kith Pradhan, Benjamin Gartrell, Beamon Agarwal, Lance Pagliaro, Masako Suzuki, John M. Greally, Dinesh Rakheja, R. Houston Thompson, Katalin Susztak, Thomas Witzig, Yiyu Zou, Amit Verma
Across clinical trials, T cell expansion and persistence following adoptive cell transfer (ACT) have correlated with superior patient outcomes. Herein, we undertook a pan-cancer analysis to identify actionable ligand/receptor pairs capable of compromising T cell durability following ACT. We discovered that FASLG, the gene encoding the apoptosis-inducing ligand FasL, is overexpressed within the majority of human tumor microenvironments (TMEs). Further, we uncovered that Fas, the receptor for FasL, is highly expressed on patient-derived T cells used for clinical ACT. We hypothesized that a cognate Fas-FasL interaction within the TME might limit both T cell persistence and anti-tumor efficacy. We discovered that genetic engineering of Fas variants impaired in the ability to bind FADD functioned as dominant negative receptors (DNRs), preventing FasL-induced apoptosis in Fas-competent T cells. T cells co-engineered with a Fas DNR and either a T cell receptor or chimeric antigen receptor exhibited enhanced persistence following ACT, resulting in superior anti-tumor efficacy against established solid and hematologic cancers. Despite increased longevity, Fas DNR-engineered T cells did not undergo aberrant expansion or mediate autoimmunity. Thus, T cell-intrinsic disruption of Fas signaling through genetic engineering represents a potentially universal strategy to enhance ACT efficacy across a broad range of human malignancies.
Tori N. Yamamoto, Ping-Hsien Lee, Suman K. Vodnala, Devikala Gurusamy, Rigel J. Kishton, Zhiya Yu, Arash Eidizadeh, Robert Eil, Jessica Fioravanti, Luca Gattinoni, James N. Kochenderfer, Terry J. Fry, Bulent Arman Aksoy, Jeffrey Hammerbacher, Anthony C. Cruz, Richard M. Siegel, Nicholas P. Restifo, Christopher A. Klebanoff
Metastasis is the dominant cause of patient death in small-cell lung cancer (SCLC), and a better understanding of the molecular mechanisms underlying SCLC metastasis may potentially improve clinical treatment. Through genome-scale screening for key regulators of mouse Rb1–/– Trp53–/– SCLC metastasis using the pooled CRISPR/Cas9 library, we identified Cullin5 (CUL5) and suppressor of cytokine signaling 3 (SOCS3), two components of the Cullin-RING E3 ubiquitin ligase complex, as top candidates. Mechanistically, the deficiency of CUL5 or SOCS3 disrupted the functional formation of the E3 ligase complex and prevented the degradation of integrin β1, which stabilized integrin β1 and activated downstream focal adhesion kinase/SRC (FAK/SRC) signaling and eventually drove SCLC metastasis. Low expression levels of CUL5 and SOCS3 were significantly associated with high integrin β1 levels and poor prognosis in a large cohort of 128 clinical patients with SCLC. Moreover, the CUL5-deficient SCLCs were vulnerable to the treatment of the FDA-approved SRC inhibitor dasatinib. Collectively, this work identifies the essential role of CUL5- and SOCS3-mediated integrin β1 turnover in controlling SCLC metastasis, which might have therapeutic implications.
Gaoxiang Zhao, Liyan Gong, Dan Su, Yujuan Jin, Chenchen Guo, Meiting Yue, Shun Yao, Zhen Qin, Yi Ye, Ying Tang, Qibiao Wu, Jian Zhang, Binghai Cui, Qiurong Ding, Hsinyi Huang, Liang Hu, Yuting Chen, Peiyuan Zhang, Guohong Hu, Luonan Chen, Kwok-Kin Wong, Daming Gao, Hongbin Ji
MAPK4 is an atypical MAPK. Currently, little is known about its physiological function and involvement in diseases, including cancer. A comprehensive analysis of 8887 gene expression profiles in The Cancer Genome Atlas (TCGA) revealed that MAPK4 overexpression correlates with decreased overall survival, with particularly marked survival effects in patients with lung adenocarcinoma, bladder cancer, low-grade glioma, and thyroid carcinoma. Interestingly, human tumor MAPK4 overexpression also correlated with phosphorylation of AKT, 4E-BP1, and p70S6K, independent of the loss of PTEN or mutation of PIK3CA. This led us to examine whether MAPK4 activates the key metabolic, prosurvival, and proliferative kinase AKT and mTORC1 signaling, independent of the canonical PI3K pathway. We found that MAPK4 activated AKT via a novel, concerted mechanism independent of PI3K. Mechanistically, MAPK4 directly bound and activated AKT by phosphorylation of the activation loop at threonine 308. It also activated mTORC2 to phosphorylate AKT at serine 473 for full activation. MAPK4 overexpression induced oncogenic outcomes, including transforming prostate epithelial cells into anchorage-independent growth, and MAPK4 knockdown inhibited cancer cell proliferation, anchorage-independent growth, and xenograft growth. We concluded that MAPK4 can promote cancer by activating the AKT/mTOR signaling pathway and that targeting MAPK4 may provide a novel therapeutic approach for cancer.
Wei Wang, Tao Shen, Bingning Dong, Chad J. Creighton, Yanling Meng, Wolong Zhou, Qing Shi, Hao Zhou, Yinjie Zhang, David D. Moore, Feng Yang
Chronic stress triggers activation of the sympathetic nervous system and drives malignancy. Using an immunodeficient murine system, we showed that chronic stress–induced epinephrine promoted breast cancer stem-like properties via lactate dehydrogenase A–dependent (LDHA-dependent) metabolic rewiring. Chronic stress–induced epinephrine activated LDHA to generate lactate, and the adjusted pH directed USP28-mediated deubiquitination and stabilization of MYC. The SLUG promoter was then activated by MYC, which promoted development of breast cancer stem-like traits. Using a drug screen that targeted LDHA, we found that a chronic stress–induced cancer stem-like phenotype could be reversed by vitamin C. These findings demonstrated the critical importance of psychological factors in promoting stem-like properties in breast cancer cells. Thus, the LDHA-lowering agent vitamin C can be a potential approach for combating stress-associated breast cancer.
Bai Cui, Yuanyuan Luo, Pengfei Tian, Fei Peng, Jinxin Lu, Yongliang Yang, Qitong Su, Bing Liu, Jiachuan Yu, Xi Luo, Liu Yin, Wei Cheng, Fan An, Bin He, Dapeng Liang, Sijin Wu, Peng Chu, Luyao Song, Xinyu Liu, Huandong Luo, Jie Xu, Yujia Pan, Yang Wang, Dangsheng Li, Peng Huang, Qingkai Yang, Lingqiang Zhang, Binhua P. Zhou, Suling Liu, Guowang Xu, Eric W.-F. Lam, Keith W. Kelley, Quentin Liu