The non-physiological nutrient levels found in traditional culture media have been shown to affect numerous aspects of cancer cell physiology, including how cells respond to certain therapeutic agents. Here, we comprehensively evaluated how physiological nutrient levels impact therapeutic response by performing drug screening in human plasma-like medium (HPLM). We observed dramatic nutrient-dependent changes in sensitivity to a variety of FDA-approved and clinically trialed compounds including rigosertib, an experimental cancer therapeutic that has recently failed in phase 3 clinical trials. Mechanistically, we found that the ability of rigosertib to destabilize microtubules is strongly inhibited by the purine metabolism end product uric acid, which is uniquely abundant in humans relative to traditional in vitro and in vivo cancer models. These results demonstrate the broad and dramatic effects nutrient levels can have on drug response, and how incorporation of human-specific physiological nutrient media might help to identify compounds whose efficacy could be impacted in humans.
Vipin Rawat, Patrick DeLear, Prarthana Prashanth, Mete Emir Ozgurses, Anteneh Tebeje, Philippa A. Burns, Kelly O. Conger, Christopher Solís, Yasir Hasnain, Anna Novikova, Jennifer E. Endress, Paloma González-Sánchez, Wentao Dong, Greg Stephanopoulos, Gina M. DeNicola, Isaac Harris, David Sept, Frank M. Mason, Jonathan L. Coloff
Tumor evolution is driven by genetic variation; however, it is the tumor microenvironment (TME) that provides the selective pressure contributing to evolution in cancer. Despite high histopathological heterogeneity within glioblastoma (GBM), the most aggressive brain tumor, the interactions between the genetically distinct GBM cells and the surrounding TME are not fully understood. To address this, we analyzed matched primary and recurrent GBM archival tumor tissues with imaging-based techniques aimed to simultaneously evaluate tumor tissues for presence of hypoxic, angiogenic, and inflammatory niches, extracellular matrix organization, TERT promoter mutational status, and several oncogenic amplifications on the same slide and location. We found that the relationships between genetic and TME diversity are different in primary and matched recurrent tumors. Interestingly, the texture of the extracellular matrix (ECM), identified by label-free reflectance imaging, was predictive of single-cell genetic traits present in the tissue. Moreover, reflectance of ECM revealed structured organization of the perivascular niche in recurrent GBM, enriched in immunosuppressive macrophages. Single-cell spatial transcriptomics further confirmed the presence of the niche-specific macrophage populations and identified interactions between endothelial cells, perivascular fibroblasts, and immunosuppressive macrophages. Our results underscore the importance of GBM tissue organization in tumor evolution and highlight novel genetic and spatial dependencies.
Ugoma Onubogu, Chandler D. Gatenbee, Sandhya Prabhakaran, Kelsey Wolfe, Benjamin Oakes, Roberto Salatino, Rachael Vaubel, Oszkar Szentirmai, Alexander R. A. Anderson, Michalina Janiszewska
Because cancer cells have a genetically unstable nature, they give rise to genetically different variant subclones inside a single tumor. Understanding cancer heterogeneity and subclone characteristics is crucial for developing more efficacious therapies. Oral squamous cell carcinoma (OSCC) is characterized by high heterogeneity and plasticity. On the other hand, CX3C motif ligand 1 (CX3CL1) is a double-faced chemokine with anti- and pro-tumor functions. Our study reported that CX3CL1 functioned differently in tumors with different cancer phenotypes, both in vivo and in vitro. Mouse OSCC 1 (MOC1) and MOC2 cells responded similarly to CX3CL1 in vitro. However, in vivo, CX3CL1 increased keratinization in indolent MOC1 cancer, while CX3CL1 promoted cervical lymphatic metastasis in aggressive MOC2 cancer. These outcomes were due to double-faced CX3CL1 effects on different immune microenvironments indolent and aggressive cancer created. Furthermore, we established that CX3CL1 promoted cancer metastasis via the lymphatic pathway by stimulating lymphangiogenesis and transendothelial migration of lymph-circulating tumor cells. CX3CL1 enrichment in lymphatic metastasis tissues was observed in aggressive murine and human cell lines. OSCC patient samples with CX3CL1 enrichment exhibited a strong correlation with lower overall survival rates and higher recurrence and distant metastasis rates. In conclusion, CX3CL1 is a pivotal factor that stimulates the metastasis of aggressive cancer subclones within the heterogeneous tumors to metastasize, and our study demonstrates the prognostic value of CX3CL1 enrichment in long-term monitoring in OSCC.
Htoo Shwe Eain, Hotaka Kawai, Masaaki Nakayama, May Wathone Oo, Toshiaki Ohara, Yoko Fukuhara, Kiyofumi Takabatake, Quisheng Shan, Yamin Soe, Kisho Ono, Keisuke Nakano, Nobuyoshi Mizukawa, Seiji Iida, Hitoshi Nagatsuka
Small cell lung cancer (SCLC) is the most aggressive lung cancer entity with an extremely limited therapeutic outcome. Most patients are diagnosed at an extensive stage. However, the molecular mechanisms driving SCLC invasion and metastasis remain largely elusive. We used an autochthonous SCLC mouse model and matched samples from patients with primary and metastatic SCLC to investigate the molecular characteristics of tumor metastasis. We demonstrate that tumor cell invasion and liver metastasis in SCLC are triggered by an Angiopoietin-2 (ANG-2)/Integrin β-1–dependent pathway in tumor cells, mediated by focal adhesion kinase/Src kinase signaling. Strikingly, CRISPR-Cas9 KO of Integrin β-1 or blocking Integrin β-1 signaling by an anti–ANG-2 treatment abrogates liver metastasis formation in vivo. Interestingly, analysis of a unique collection of matched samples from patients with primary and metastatic SCLC confirmed a strong increase of Integrin β-1 in liver metastasis in comparison with the primary tumor. We further show that ANG-2 blockade combined with PD-1–targeted by anti-PD-1 treatment displays synergistic treatment effects in SCLC. Together, our data demonstrate a fundamental role of ANG-2/Integrin β-1 signaling in SCLC cells for tumor cell invasion and liver metastasis and provide a potentially new effective treatment strategy for patients with SCLC.
Lydia Meder, Charlotte Isabelle Orschel, Christoph Julius Otto, Mirjam Koker, Johannes Brägelmann, Meryem S. Ercanoglu, Sabrina Dähling, Anik Compes, Carolin Selenz, Marieke Nill, Felix Dietlein, Alexandra Florin, Marie-Lisa Eich, Sven Borchmann, Margarete Odenthal, Raquel Blazquez, Frank Hilberg, Florian Klein, Michael Hallek, Reinhard Büttner, H. Christian Reinhardt, Roland T. Ullrich
Redundant tumor microenvironment (TME) immunosuppressive mechanisms and epigenetic maintenance of terminal T cell exhaustion greatly hinder functional antitumor immune responses in chronic lymphocytic leukemia (CLL). Bromodomain and extraterminal (BET) proteins regulate key pathways contributing to CLL pathogenesis and TME interactions, including T cell function and differentiation. Herein, we report that blocking BET protein function alleviates immunosuppressive networks in the CLL TME and repairs inherent CLL T cell defects. The pan-BET inhibitor OPN-51107 reduced exhaustion-associated cell signatures resulting in improved T cell proliferation and effector function in the Eμ-TCL1 splenic TME. Following BET inhibition (BET-i), TME T cells coexpressed significantly fewer inhibitory receptors (IRs) (e.g., PD-1, CD160, CD244, LAG3, VISTA). Complementary results were witnessed in primary CLL cultures, wherein OPN-51107 exerted proinflammatory effects on T cells, regardless of leukemic cell burden. BET-i additionally promotes a progenitor T cell phenotype through reduced expression of transcription factors that maintain terminal differentiation and increased expression of TCF-1, at least in part through altered chromatin accessibility. Moreover, direct T cell effects of BET-i were unmatched by common targeted therapies in CLL. This study demonstrates the immunomodulatory action of BET-i on CLL T cells and supports the inclusion of BET inhibitors in the management of CLL to alleviate terminal T cell dysfunction and potentially enhance tumoricidal T cell activity.
Audrey L. Smith, Sydney A. Skupa, Alexandria P. Eiken, Timothy E. Reznicek, Elizabeth Schmitz, Nolan Williams, Dalia Y. Moore, Christopher R. D’Angelo, Avyakta Kallam, Matthew A. Lunning, R. Gregory Bociek, Julie M. Vose, Eslam Mohamed, Anna R. Mahr, Paul W. Denton, Ben Powell, Gideon Bollag, M. Jordan Rowley, Dalia El-Gamal
Sarcomatoid dedifferentiation is common to multiple renal cell carcinoma (RCC) subtypes, including chromophobe RCC (ChRCC), and is associated with increased aggressiveness, resistance to targeted therapies, and heightened sensitivity to immunotherapy. To study ChRCC dedifferentiation, we performed multiregion integrated paired pathological and genomic analyses. Interestingly, ChRCC dedifferentiates not only into sarcomatoid but also into anaplastic and glandular subtypes, which are similarly associated with increased aggressiveness and metastases. Dedifferentiated ChRCC shows loss of epithelial markers, convergent gene expression, and whole genome duplication from a hypodiploid state characteristic of classic ChRCC. We identified an intermediate state with atypia and increased mitosis but preserved epithelial markers. Our data suggest that dedifferentiation is initiated by hemizygous mutation of TP53, which can be observed in differentiated areas, as well as mutation of PTEN. Notably, these mutations become homozygous with duplication of preexisting monosomes (i.e., chromosomes 17 and 10), which characterizes the transition to dedifferentiated ChRCC. Serving as potential biomarkers, dedifferentiated areas become accentuated by mTORC1 activation (phospho-S6) and p53 stabilization. Notably, dedifferentiated ChRCC share gene enrichment and pathway activation features with other sarcomatoid RCC, suggesting convergent evolutionary trajectories. This study expands our understanding of aggressive ChRCC, provides insight into molecular mechanisms of tumor progression, and informs pathologic classification and diagnostics.
Payal Kapur, Hua Zhong, Daniel Le, Ratna Mukhopadhyay, Jeffrey Miyata, Deyssy Carrillo, Dinesh Rakheja, Satwik Rajaram, Steffen Durinck, Zora Modrusan, James Brugarolas
Pancreatic ductal adenocarcinoma (PDA) is a lethal cancer characterized by a poor outcome and an increasing incidence. A significant majority (>80%) of newly diagnosed cases are deemed unresectable, leaving chemotherapy as the sole viable option, though with only moderate success. This necessitates the identification of improved therapeutic options for PDA. We hypothesized that there are temporal variations in cancer-relevant processes within PDA tumors, offering insights into the optimal timing of drug administration — a concept termed chronotherapy. In this study, we explored the presence of the circadian transcriptome in PDA using patient-derived organoids and validated these findings by comparing PDA data from The Cancer Genome Atlas with noncancerous healthy pancreas data from GTEx. Several PDA-associated pathways (cell cycle, stress response, Rho GTPase signaling) and cancer driver hub genes (EGFR and JUN) exhibited a cancer-specific rhythmic pattern intricately linked to the circadian clock. Through the integration of multiple functional measurements for rhythmic cancer driver genes, we identified top chronotherapy targets and validated key findings in molecularly divergent pancreatic cancer cell lines. Testing the chemotherapeutic efficacy of clinically relevant drugs further revealed temporal variations that correlated with drug-target cycling. Collectively, our study unravels the PDA circadian transcriptome and highlights a potential approach for optimizing chrono-chemotherapeutic efficacy.
Deepak Sharma, Darbaz Adnan, Mostafa K. Abdel-Reheem, Ron C. Anafi, Daniel D. Leary, Faraz Bishehsari
Lung cancer is the leading cause of cancer-related deaths in the world, and non–small cell lung cancer (NSCLC) is the most common subset. We previously found that infiltration of tumor inflammatory monocytes (TIMs) into lung squamous carcinoma (LUSC) tumors is associated with increased metastases and poor survival. To further understand how TIMs promote metastases, we compared RNA-Seq profiles of TIMs from several LUSC metastatic models with inflammatory monocytes (IMs) of non–tumor-bearing controls. We identified Spon1 as upregulated in TIMs and found that Spon1 expression in LUSC tumors corresponded with poor survival and enrichment of collagen extracellular matrix signatures. We observed SPON1+ TIMs mediate their effects directly through LRP8 on NSCLC cells, which resulted in TGF-β1 activation and robust production of fibrillar collagens. Using several orthogonal approaches, we demonstrated that SPON1+ TIMs were sufficient to promote NSCLC metastases. Additionally, we found that Spon1 loss in the host, or Lrp8 loss in cancer cells, resulted in a significant decrease of both high-density collagen matrices and metastases. Finally, we confirmed the relevance of the SPON1/LRP8/TGF-β1 axis with collagen production and survival in patients with NSCLC. Taken together, our study describes how SPON1+ TIMs promote collagen remodeling and NSCLC metastases through an LRP8/TGF-β1 signaling axis.
Kristina M. Whately, Nisitha Sengottuvel, Lincy Edatt, Sonal Srivastava, Allison T. Woods, Yihsuan S. Tsai, Alessandro Porrello, Matthew P. Zimmerman, Aaron C. Chack, Stuart R. Jefferys, Gabriella Yacovone, Dae Joong Kim, Andrew C. Dudley, Antonio L. Amelio, Chad V. Pecot
Esophageal squamous cell carcinoma (ESCC) is the predominant form of esophageal cancer and is characterized by an unfavorable prognosis. To elucidate the distinct molecular alterations in ESCC and investigate therapeutic targets, we performed a comprehensive analysis of transcriptomic, proteomic, and phosphoproteomic data derived from 60 paired treatment-naive ESCC and adjacent non-tumor tissue samples. Additionally, we conducted a correlation analysis to describe the regulatory relationship between transcriptomic and proteomic processes, revealing alterations in key metabolic pathways. Unsupervised clustering analysis of the proteomic data stratified ESCC patients into three subtypes with different molecular characteristics and clinical outcomes. Notably, subtype III exhibited the worst prognosis and enrichment in proteins associated with malignant processes, including glycolysis and DNA repair pathways. Furthermore, translocase of inner mitochondrial membrane domain containing 1 (TIMMDC1) was validated as a potential prognostic molecule for ESCC. Moreover, integrated kinase-substrate network analysis using the phosphoproteome nominated candidate kinases as potential targets. In vitro and in vivo experiments further confirmed casein kinase II subunit alpha (CSNK2A1) as a potential kinase target for ESCC. These underlying data represent a valuable resource for researchers, which may provide better insights into the biology and treatment of ESCC.
Dengyun Zhao, Yaping Guo, Huifang Wei, Xuechao Jia, Yafei Zhi, Guiliang He, Wenna Nie, Limeng Huang, Penglei Wang, Kyle Vaughn Laster, Zhicai Liu, Jinwu Wang, Mee-Hyun Lee, Zigang Dong, Kangdong Liu
Acute myeloid leukemia (AML) is a fatal disease characterized by the accumulation of undifferentiated myeloblasts, and agents that promote differentiation have been effective in this disease but are not curative. Dihydroorotate dehydrogenase inhibitors (DHODHi) have the ability to promote AML differentiation and target aberrant malignant myelopoiesis. We introduce HOSU-53, a DHODHi with significant monotherapy activity, which is further enhanced when combined with other standard-of-care therapeutics. We further discovered that DHODHi modulated surface expression of CD38 and CD47, prompting the evaluation of HOSU-53 combined with anti-CD38 and anti-CD47 therapies, where we identified a compelling curative potential in an aggressive AML model with CD47 targeting. Finally, we explored using plasma dihydroorotate (DHO) levels to monitor HOSU-53 safety and found that the level of DHO accumulation could predict HOSU-53 intolerability, suggesting the clinical use of plasma DHO to determine safe DHODHi doses. Collectively, our data support the clinical translation of HOSU-53 in AML, particularly to augment immune therapies. Potent DHODHi to date have been limited by their therapeutic index; however, we introduce pharmacodynamic monitoring to predict tolerability while preserving antitumor activity. We additionally suggest that DHODHi is effective at lower doses with select immune therapies, widening the therapeutic index.
Ola A. Elgamal, Sydney Fobare, Sandip Vibhute, Abeera Mehmood, Dennis C. Vroom, Mariah L. Johnson, Blaise Stearns, James R. Lerma, Jean Truxall, Emily Stahl, Bridget Carmichael, Shelley J. Orwick, Alice S. Mims, Emily Curran, Ramasamy Santhanam, Susheela Tridandapani, Mitch A. Phelps, Zhiliang Xie, Christopher C. Coss, Sharyn D. Baker, Jeffrey Patrick, Janel K. Ezzell, Jayesh Rai, Jianmin Pan, Shesh N. Rai, Cody Stillwell, Mark Wunderlich, Mouad Abdulrahim, Thomas E. Goodwin, Gerard Hilinski, Chad E. Bennett, Erin Hertlein, John C. Byrd
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