Recent data establish a logarithmic expansion of leucine rich repeat containing G protein coupled receptor 5–positive (Lgr5+) colonic epithelial stem cells (CESCs) in human colorectal cancer (CRC). Complementary studies using the murine 2-stage azoxymethane–dextran sulfate sodium (AOM-DSS) colitis-associated tumor model indicate early acquisition of Wnt pathway mutations drives CESC expansion during adenoma progression. Here, subdivision of the AOM-DSS model into in vivo and in vitro stages revealed DSS induced physical separation of CESCs from stem cell niche cells and basal lamina, a source of Wnt signals, within hours, disabling the stem cell program. While AOM delivery in vivo under non-adenoma-forming conditions yielded phenotypically normal mucosa and organoids derived thereof, niche injury ex vivo by progressive DSS dose escalation facilitated outgrowth of Wnt-independent dysplastic organoids. These organoids contained 10-fold increased Lgr5+ CESCs with gain-of-function Wnt mutations orthologous to human CRC driver mutations. We posit CRC originates by niche injury–induced outgrowth of normally suppressed mutated stem cells, consistent with models of adaptive oncogenesis.
Stefan Klingler, Kuo-Shun Hsu, Guoqiang Hua, Maria Laura Martin, Mohammad Adileh, Timour Baslan, Zhigang Zhang, Philip B. Paty, Zvi Fuks, Anthony M.C. Brown, Richard Kolesnick
Chronic myeloproliferative neoplasms (MPN) frequently evolve to a blast phase (BP) that is almost uniformly resistant to induction chemotherapy or hypomethylating agents. We explored the functional properties, genomic architecture, and cell of origin of MPN-BP initiating cells (IC) using a serial NSG mouse xenograft transplantation model. Transplantation of peripheral blood mononuclear cells (MNC) from 7 of 18 patients resulted in a high degree of leukemic cell chimerism and re-created clinical characteristics of human MPN-BP. The function of MPN-BP ICs was not dependent on the presence of JAK2V617F, a driver mutation associated with the initial underlying MPN. By contrast, multiple MPN-BP IC subclones co-existed within MPN-BP MNCs characterized by different myeloid malignancy gene mutations and cytogenetic abnormalities. MPN-BP ICs in 4 patients exhibited extensive proliferative and self-renewal capacity as demonstrated by their ability to recapitulate human MPN-BP in serial recipients. These MPN-BP IC subclones underwent extensive continuous clonal competition within individual xenografts and across multiple generations and their subclonal dynamics were consistent with functional evolution of MPN-BP IC. Finally, we show that MPN-BP ICs originate from not only phenotypically identified hematopoietic stem cells but also lymphoid-myeloid progenitor cells which were each characterized by differences in MPN-BP initiating activity and self-renewal capacity.
Xiaoli Wang, Raajit K. Rampal, Cing Siang Hu, Joseph Tripodi, Noushin Farnoud, Bruce Petersen, Michael R. Rossi, Minal Patel, Erin McGovern, Vesna Najfeld, Camelia Iancu-Rubin, Min Lu, Andrew Davis, Marina Kremyanskaya, Rona Singer Weinberg, John Mascarenhas, Ronald Hoffman
Standard-of-care treatment for advanced HER2+ breast cancers (BC) is comprised of two HER2-specific monoclonal antibodies (mAb), Trastuzumab (T) and Pertuzumab (P) with chemotherapy. While this combination (T+P) is highly effective, its synergistic mechanism of action (MOA) is not completely known. Initial studies had demonstrated that Pertuzumab suppressed HER2 hetero-dimerization as the potential therapeutic MOA, thus the improved outcome associated with the T+P combination MOA compared to Trastuzumab alone has been widely reported as being due to Pertuzumab-mediated suppression of HER2 signaling in combination with Trastuzumab-mediated induction of anti-tumor immunity. Unraveling this MOA may be critical to extend this combination strategy to other antigens or other cancers, as well as improving this current treatment modality. Using novel murine and human versions of Pertuzumab, we found it induced both Antibody-Dependent-Cellular-Phagocytosis (ADCP) by tumor-associated macrophages and suppression of HER2 oncogenic signaling. Most significantly, we identified that only T+P combination therapy, but not when either antibody used in isolation, allows for the activation of the classical complement pathway, resulting in both direct complement-dependent cytotoxicity (CDC) as well as complement-dependent cellular phagocytosis (CDCP) of HER2+ BC cells. Notably, we show that tumor expression of C1q was positively associated with survival outcome in HER2+ BC patients, whereas expression of complement regulators CD55 and CD59 were inversely correlated, suggesting the importance of complement activity in clinical outcomes. Accordingly, inhibition of C1 activity in mice abolished the synergistic therapeutic activity of T+P therapy, whereas knockdown of CD55 and CD59 expression enhanced T+P efficacy. In summary, our study identifies classical complement activation as a significant anti-tumor MOA for T+P therapy that may be functionally enhanced to augment therapeutic efficacy in the clinic.
Li-Chung Tsao, Erika J. Crosby, Timothy N. Trotter, Junping Wei, Tao Wang, Xiao Yang, Amanda N. Summers, Gangjun Lei, Christopher A. Rabiola, Lewis A. Chodosh, William J. Muller, Herbert Kim Lyerly, Zachary C. Hartman
CD8+ tumor-infiltrating lymphocytes (TILs) are associated with improved survival in triple-negative breast cancer (TNBC) yet have no association with survival in estrogen receptor–positive (ER+) BC. The basis for these contrasting findings remains elusive. We identified subsets of BC tumors infiltrated by CD8+ T cells with characteristic features of exhausted T cells (TEX). Tumors with abundant CD8+ TEX exhibited a distinct tumor microenvironment marked by amplified interferon-γ signaling–related pathways and higher programmed death ligand 1 expression. Paradoxically, higher levels of tumor-infiltrating CD8+ TEX associated with decreased overall survival of patients with ER+ BC but not patients with TNBC. Moreover, high tumor expression of a CD8+ TEX signature identified dramatically reduced survival in premenopausal, but not postmenopausal, patients with ER+ BC. Finally, we demonstrated the value of a tumor TEX signature score in identifying high-risk premenopausal ER+ BC patients among those with intermediate Oncotype DX Breast Recurrence Scores. Our data highlight the complex relationship between CD8+ TILs, interferon-γ signaling, and ER status in BC patient survival. This work identifies tumor-infiltrating CD8+ TEX as a key feature of reduced survival outcomes in premenopausal patients with early-stage ER+ BC.
Colt A. Egelston, Weihua Guo, Jiayi Tan, Christian Avalos, Diana L. Simons, Min Hui Lim, Yinghui J. Huang, Michael S. Nelson, Arnab Chowdhury, Daniel B. Schmolze, John H. Yim, Laura Kruper, Laleh Melstrom, Kim Margolin, Joanne E. Mortimer, Yuan Yuan, James R. Waisman, Peter P. Lee
Treatment with anti–PD-1 and anti-PD-L1 therapies has shown durable clinical benefit in non–small cell lung cancer (NSCLC). However, patients with NSCLC with epidermal growth factor receptor (EGFR) mutations do not respond as well to treatment as patients without an EGFR mutation. We show that EGFR-mutated NSCLC expressed higher levels of CD73 compared with EGFR WT tumors and that CD73 expression was regulated by EGFR signaling. EGFR-mutated cell lines were significantly more resistant to T cell killing compared with WT cell lines through suppression of T cell proliferation and function. In a xenograft mouse model of EGFR-mutated NSCLC, neither anti–PD-L1 nor anti-CD73 antibody alone inhibited tumor growth compared with the isotype control. In contrast, the combination of both antibodies significantly inhibited tumor growth, increased the number of tumor-infiltrating CD8+ T cells, and enhanced IFN-γ and TNF-α production of these T cells. Consistently, there were increases in gene expression that corresponded to inflammation and T cell function in tumors treated with the combination of anti–PD-L1 and anti-CD73. Together, these results further support the combination of anti-CD73 and anti–PD-L1 therapies in treating EGFR-mutated NSCLC, while suggesting that increased T cell activity may play a role in response to therapy.
Eric Tu, Kelly McGlinchey, Jixin Wang, Philip Martin, Steven L.K. Ching, Nicolas Floc’h, James Kurasawa, Jacqueline H. Starrett, Yelena Lazdun, Leslie Wetzel, Barrett Nuttall, Felicia S.L. Ng, Karen T. Coffman, Paul D. Smith, Katerina Politi, Zachary A. Cooper, Katie Streicher
Cancer inflicts damage to surrounding normal tissues, which can culminate in fatal organ failure. Here, we demonstrate that cell death in organs affected by cancer can be detected by tissue-specific methylation patterns of circulating cell-free DNA (cfDNA). We detected elevated levels of hepatocyte-derived cfDNA in the plasma of patients with liver metastases originating from different primary tumors, compared with cancer patients without liver metastases. In addition, patients with localized pancreatic or colon cancer showed elevated hepatocyte cfDNA, suggesting liver damage inflicted by micrometastatic disease, by primary pancreatic tumor pressing the bile duct, or by a systemic response to the primary tumor. We also identified elevated neuron-, oligodendrocyte-, and astrocyte-derived cfDNA in a subpopulation of patients with brain metastases compared with cancer patients without brain metastasis. Cell type–specific cfDNA methylation markers enabled the identification of collateral tissue damage in cancer, revealing the presence of metastases in specific locations and potentially assisting in early cancer detection.
Asael Lubotzky, Hai Zemmour, Daniel Neiman, Marc Gotkine, Netanel Loyfer, Sheina Piyanzin, Bracha-Lea Ochana, Roni Lehmann-Werman, Daniel Cohen, Joshua Moss, Judith Magenheim, Maureen F. Loftus, Lauren Brais, Kimmie Ng, Raul Mostoslavsky, Brian M. Wolpin, Aviad Zick, Myriam Maoz, Albert Grinshpun, Anatoli Kustanovich, Chen Makranz, Jonathan E. Cohen, Tamar Peretz, Ayala Hubert, Mark Temper, Azzam Salah, Shani Avniel-Polak, Simona Grozinsky-Glasberg, Kirsty L. Spalding, Ariel Rokach, Tommy Kaplan, Benjamin Glaser, Ruth Shemer, Yuval Dor
BACKGROUND. Prostate cancer is multifocal with distinct molecular subtypes. The utility of genomic subtyping has been challenged due to inter- and intra-focal heterogeneity. We sought to characterize the subtype-defining molecular alterations of primary prostate cancer across all tumor foci within radical prostatectomy (RP) specimens and determine the prevalence of collision tumors. METHODS. From the Early Detection Research Network cohort, we identified 333 prospectively collected RPs from 2010 to 2014 and assessed ERG, SPINK1, PTEN, and SPOP molecular status. We utilized dual ERG/SPINK1 immunohistochemistry, fluorescence in situ hybridization to confirm ERG rearrangements and characterize PTEN deletion, and high-resolution melting curve analysis and Sanger sequencing to determine SPOP mutation status. Analysis of biochemical recurrence-free of patients with collision tumors was conducted using Kaplan-Meier method. RESULTS. Based on index focus alone, ERG, SPINK1, PTEN, and SPOP alterations were identified in 47.5%, 10.8%, 14.3%, and 5.1% of RP specimens, respectively. In 233 multifocal RPs with ERG/SPINK1 status in all foci, 139 (59.7%) had discordant molecular alterations between foci. Collision tumors, as defined by discrepant ERG/SPINK1 status within a single focus, were identified in 29 (9.4%) RP specimens. CONCLUSION. Interfocal molecular heterogeneity was identified in ~60% of multifocal RP specimens and collision tumors were present in ~10%. We present this phenomenon as a model for the intra-focal heterogeneity observed in previous studies and propose future genomic studies screen for collision tumors to better characterize molecular heterogeneity.
Jacqueline Fontugne, Peter Y. Cai, Hussein Alnajar, Bhavneet Bhinder, Kyung Park, Huihui Ye, Shaham Beg, Verena Sailer, Javed Siddiqui, Mirjam Blattner-Johnson, Jaclyn A. Croyle, Zohal Noorzad, Carla Calagua, Theresa Y. MacDonald, Ulrika Axcrona, Mari Bogaard, Karol Axcrona, Douglas S. Scherr, Martin G. Sanda, Bjarne Johannessen, Arul M. Chinnaiyan, Olivier Elemento, Rolf I. Skotheim, Mark A. Rubin, Christopher E. Barbieri, Juan M. Mosquera
Herein, we characterize the landscape and prognostic significance of the T cell receptor (TCR) repertoire of early-stage non–small cell lung cancer (NSCLC) for patients with an epidermal growth factor receptor (EGFR) mutation. β Chain TCR sequencing was used to characterize the TCR repertoires of paraffin-preserved pretreatment tumor and tumor-adjacent tissues from 57 and 44 patients with stage II/III NSCLC with an EGFR mutation treated with gefitinib or chemotherapy in the ADJUVANT-CTONG 1104 trial. The TCR diversity was significantly decreased in patients with an EGFR mutation, and patients with high TCR diversity had a favorable overall survival (OS). A total of 10 TCR Vβ-Jβ rearrangements were significantly associated with OS. Patients with a higher frequency of Vβ5-6Jβ2-1, Vβ20-1Jβ2-1, Vβ24-1Jβ2-1, and Vβ29-1Jβ2-7 had significantly longer OS. Weighted combinations of the 4 TCRs were significantly associated with OS and disease-free survival (DFS) of patients, which could further stratify the high and low TCR diversity groups. Importantly, Vβ5-6Jβ2-1, Vβ20-1Jβ2-1, and Vβ24-1Jβ2-1 had a significant relationship with gefitinib treatment, while Vβ29-1Jβ2-7 was associated with chemotherapy. Four TCR Vβ-Jβ rearrangements related to favorable OS and DFS for adjuvant gefitinib and chemotherapy in patients with an EGFR mutation with stage II/III NSCLC; this may provide a novel perspective for the adjuvant setting for resectable NSCLC.
Cunte Chen, Si-Yang Maggie Liu, Yedan Chen, Qiuxiang Ou, Hua Bao, Ling Xu, Yikai Zhang, Wenzhao Zhong, Qing Zhou, Xue-Ning Yang, Yang Shao, Yi-Long Wu, Si-Yang Liu, Yangqiu Li
Sustained proliferative signaling and resisting cell death are hallmarks of cancer. Zinc finger protein 277 (ZNF277; murine Zfp277), a transcription factor regulating cellular senescence, is overexpressed in colon cancer but its actions in intestinal homeostasis and neoplasia are unclear. Using human and murine intestine, human colon cancer cells, and ApcMin/+ mice with dysregulated β-catenin signaling and exuberant intestinal neoplasia, we explored the actions of ZNF/Zfp277 and defined the underlying mechanisms. In normal human and murine intestine, ZNF/Zfp277 was expressed uniquely in early stem cell progenitors, undifferentiated transit-amplifying cells (TACs). Zfp277 was overexpressed in the ApcMin/+ mouse colon, implicating ZNF/Zfp277 as a transcriptional target of β-catenin signaling. We confirmed this by showing β-catenin knockdown reduced ZNF277 expression and, using chromatin immunoprecipitation, identified two β-catenin binding sites in the ZNF277 promoter. Zfp277 deficiency attenuated intestinal epithelial cell proliferation and tumor formation, and strikingly prolonged ApcMin/+ mouse survival. RNA-Seq and PCR analyses revealed Zfp277 modulates expression of genes in key cancer pathways including β-catenin signaling, the HOXD family that regulates development, and p21WAF1, a cell cycle inhibitor and tumor suppressor. In both human colon cancer cells and the murine colon, ZNF/Zfp277 deficiency induced p21WAF1 expression and promoted senescence. Our findings identify ZNF/Zfp277 as both a TAC marker and colon cancer oncogene that regulates cellular proliferation and senescence, in part by repressing p21WAF1 expression.
Guofeng Xie, Zhongsheng Peng, Jinqing Liang, Shannon M. Larabee, Cinthia B. Drachenberg, Harris Yfantis, Jean-Pierre Raufman
The discovery of the oncometabolite 2-hydroxyglutarate in isocitrate dehydrogenase (IDH)1-mutated tumor entities affirmed the role of metabolism in cancer. Yet, large databases with tissue metabolites that are modulated by IDH1 mutation remain an area of development. Here, we present an unprecedented and valuable resource for tissue metabolites in diffuse glioma and their modulations by IDH1 mutation, histology and tumor treatments in 101 tissue samples from 73 diffuse glioma patients (24 astrocytoma, 17 oligodendroglioma, 32 glioblastoma), investigated by NMR-based metabolomics and supported by RNA sequencing. We discovered comparison-specific metabolites and pathways modulated by IDH1 (“IDH1 mutation status cohort”) and tumor entity. The “Longitudinal investigation cohort” provides metabolic profiles of untreated and corresponding treated glioma samples at first progression. Most interestingly, univariate and multivariate cox regressions and Kaplan Meier analyses revealed that tissue metabolites correlate with progression-free and overall survival. Thus, this study introduces novel candidate prognostic and surrogate metabolite biomarkers for future prospective clinical studies aiming at further refining patient stratification in diffuse glioma. Furthermore, our data will facilitate the generation of so far unanticipated hypotheses for experimental studies to advance our molecular understanding of glioma biology.
Christoph Trautwein, Laimdota Zizmare, Irina Mäurer, Benjamin Bender, Björn Bayer, Ulrike Ernemann, Marcos Tatagiba, Stefan J. Grau, Bernd J. Pichler, Marco Skardelly, Ghazaleh Tabatabai
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