Triple-negative breast cancers (TNBCs) are highly heterogeneous and aggressive, with high mortality rates. Although TNBC is typically more responsive to chemotherapy than other breast cancer subtypes, many patients develop chemo-resistance. The molecular processes contributing to chemo-resistance, and the roles of tumor cell-stromal crosstalk in establishing chemo-resistance are complex and largely unclear. Here we report molecular studies of paired TNBC patient-derived xenografts (PDX) established from patient biopsies before and after the development of chemo-resistance. Interestingly, the chemo-resistant model acquired a distinct KRASQ61R mutation that activates K-Ras. The chemo-resistant KRAS-mutant model showed gene expression and proteomic changes indicative of altered tumor cell metabolism. Specifically, KRAS-mutant PDXs exhibit increased redox ratios and decreased activation of AMPK, a protein involved in responding to metabolic homeostasis. Additionally, the chemo-resistant model exhibited increased immunosuppression including expression of CXCL1 and CXCL2, cytokines responsible for recruiting immunosuppressive leukocytes to tumors. Notably, chemo-resistant KRAS-mutant tumors harbored increased numbers of granulocytic myeloid-derived suppressor cells (gMDSCs). Interestingly, previously established gene expression signatures of Ras/MAPK activity correlated with myeloid/neutrophil-recruiting CXCL1/2 expression and negatively with T-cell recruiting chemokines (CXCL9/10/11) across TNBC patients, even in the absence of KRAS mutations. Importantly, MEK inhibition induced tumor suppression in mice while simultaneously reversing metabolic and immunosuppressive phenotypes including chemokine production and gMDSC tumor recruitment in the chemo-resistant KRAS mutant tumors. These results suggest that Ras/MAPK pathway inhibitors may be effective in some breast cancer patients to reverse Ras/MAPK-driven tumor metabolism and immunosuppression, particularly in the setting of chemo-resistance.
Derek A. Franklin, Joe T. Sharick, Paula I. Gonzalez-Ericsson, Violeta Sanchez, Phillip Dean, Susan R. Opalenik, Stefano Cairo, Jean-Gabriel Judde, Michael T. Lewis, Jenny C. Chang, Melinda E. Sanders, Rebecca S. Cook, Melissa C. Skala, Jennifer Bordeaux, Jehovana Orozco Bender, Christine A. Vaupel, Gary Geiss, Douglas Hinerfeld, Justin M. Balko
One of the major challenges in using pancreatic cancer patient-derived organoids (PDOs) in precision oncology is the time from biopsy to functional characterization. This is particularly true for biopsy specimen with limited tumor cell yield, typical characteristics of biopsies from endoscopic ultrasound-guided fine needle aspirations (EUS-FNAs).Here, we tested conditioned media of individual PDOs for cell-free tumor DNA (cfDNA) to detect driver mutations already early on during the expansion process in order to accelerate the genetic characterization of PDOs as well as subsequent functional testing. Importantly, genetic alterations detected in the PDO supernatant, collected as early as 72h after biopsy, recapitulate the mutational profile of the primary tumor indicating suitability of this approach to subject PDOs to drug testing in a reduced timeframe. In addition, we demonstrate that this workflow is practicable even in patients of whom the amount of tumor material was not sufficient for molecular characterization by established means.Our findings demonstrate that generating PDOs from very limited biopsy material permits molecular profiling and drug testing. With our approach this can be achieved in a rapid and feasible fashion with broad implications in clinical practice.
Zahra Dantes, Hsi-Yu Yen, Nicole Pfarr, Christof Winter, Katja Steiger, Alexander Muckenhuber, Alexander Hennig, Sebastian Lange, Thomas Engleitner, Rupert Öllinger, Roman Maresch, Felix Orben, Irina Heid, Georgios A. Kaissis, Kuangyu Shi, Geoffrey J. Topping, Fabian Stögbauer, Matthias Wirth, Katja Peschke, Aristeidis Papargyriou, Massoud Rezaee-Oghazi, Karin Feldmann, Arlett P. G. Schäfer, Raphela Ranjan, Clara Lubeseder-Martellato, Daniel E. Stange, Thilo Welsch, Marc E. Martignoni, Güralp Onur Ceyhan, Helmut Friess, Alexander Herner, Lucia Liotta, Matthias Treiber, Guido von Figura, Mohamed Abdelhafez, Peter Klare, Christoph Schlag, Hana Algül, Jens T. Siveke, Rickmer F. Braren, Gregor Weirich, Wilko Weichert, Dieter Saur, Roland Rad, Roland Schmid, Günter Schneider, Maximilian Reichert
Dendritic cells (DCs) are critical component of immune responses in cancer primarily due to their ability to cross-present tumor associated antigens. Cross-presentation by DCs in cancer is impaired, which may represent one of the obstacles for the success of cancer immunotherapies. Here, we report that polymorphonuclear myeloid derived suppressor cells (PMN-MDSC) blocked cross-presentation by DCs without affecting direct presentation of antigens by these cells. This effect did not require direct cell-cell contact and was associated with transfer of lipids. Neutrophils (PMN) and PMN-MDSC transferred lipid to DCs equally well, however PMN did not affect DC cross-presentation. PMN-MDSC generate oxidatively truncated lipids previously shown to be involved in impaired cross-presentation by DCs. Accumulation of oxidized lipids in PMN-MDSC was dependent on myeloperoxidase (MPO). MPO deficient PMN-MDSC did not affect cross-presentation by DCs. Cross-presentation of tumor associated antigens in vivo by DCs was improved in MDSC depleted or tumor-bearing MPO KO mice. Pharmacological inhibition of MPO in combination with checkpoint blockade reduced tumor progression in different tumor models. These data suggest MPO-driven lipid peroxidation in PMN-MDSC as a possible non-cell autonomous mechanism of inhibition of antigen cross-presentation by DCs and propose MPO as potential therapeutic target to enhance the efficacy of current immunotherapies for cancer patients.
Alessio Ugolini, Vladimir Tyurin, Yulia Tyurina, Evgenii Tsyganov, Laxminarasimha Donthireddy, Valerian E Kagan, Dmitry I. Gabrilovich, Filippo Veglia
Expression of immune checkpoint ligands (ICLs) is necessary to trigger the inhibitory signal via immune checkpoint receptors (ICRs) in exhausted T cells under tumor immune microenvironment. Nevertheless, ICL expression profile in cancer patients has not been investigated. Using previously reported RNA-seq datasets, we found that expression of ICLs was patient-specific but their co-expression can be patterned in non-small-cell lung cancers (NSCLCs). Since the expressions of PD-L1 and PVR among various ICLs were independently regulated, we could stratify the patients, who were treated with anti-PD-1 later, into four groups according to the expression level of PD-L1 and PVR. Of interest, high PVR and low PVR expressions in PD-L1-expressing patients enriched non-responders and responders to PD-1 blockade, respectively, helping in further selection of responders. Using genetically engineered cancer model, we also found that PVR-deficient and PD-L1-sufficient tumor-bearing mice were highly sensitive to anti-PD-1 therapy, whereas PVR-sufficient and PD-L1-deficient tumor-bearing mice were resistant to anti-PD-1 therapy. Taken together, our study provides a concept that combinatorial expression patterns of PVR and PD-L1 are a key determinant for PD-1 blockade and furthermore suggest a better therapeutic usage of immune checkpoint blockades (ICBs).
Bo Ryeong Lee, Sehyun Chae, Jihyun Moon, Myeong Joon Kim, Hankyu Lee, Hyuk Wan Ko, Byoung Chul Cho, Hyo Sup Shim, Daehee Hwang, Hye Ryun Kim, Sang-Jun Ha
Oxidative stress and inadequate redox homeostasis is crucial for tumor initiation and progression. MTH1 (NUDT1) enzyme prevents incorporation of oxidized dNTPs by sanitizing the deoxynucleoside triphosphate (dNTP) pool and is therefore vital for the survival of tumor cells. MTH1 inhibition has been found to inhibit the growth of several experimental tumors, but its role in mesothelioma progression remained elusive. Moreover, although MTH1 is nonessential to normal cells, its role in survival of host cells in tumor milieu, especially tumor endothelium, is unclear. We validated a clinically relevant MTH1 inhibitor (Karonudib) in mesothelioma treatment using human xenografts and syngeneic murine models. We show that MTH1 inhibition impedes mesothelioma progression and that inherent tumoral MTH1 levels are associated with a tumor’s response. We also identified tumor endothelial cells as selective targets of Karonudib and propose a model of intercellular signaling among tumor cells and bystander tumor endothelium. We finally determined the major biological processes associated with elevated MTH1 gene expression in human mesotheliomas.
Sophia F. Magkouta, Apostolos G. Pappas, Photene C. Vaitsi, Panagiotis C. Agioutantis, Ioannis S. Pateras, Charalampos A. Moschos, Marianthi P. Iliopoulou, Chrysavgi N. Kosti, Heleni V. Loutrari, Vassilis G. Gorgoulis, Ioannis T. Kalomenidis
Interferon regulatory factor 1 (IRF1) regulates diverse biological functions, including modulation of cellular responses involved in tumorigenesis. Genetic mutations and altered IRF1 function are associated with several cancers. Although the function of IRF1 in the immunobiology of cancer is emerging, IRF1-specific mechanisms regulating tumorigenesis and tissue homeostasis in vivo are not clear. Here, we found that mice lacking IRF1 were hypersusceptible to colorectal tumorigenesis. IRF1 functions in both the myeloid and epithelial compartments to confer protection against AOM/DSS-induced colorectal tumorigenesis. We further found that IRF1 also prevents tumorigenesis in a spontaneous mouse model of colorectal cancer. The attenuated cell death in the colons of Irf1–/– mice was due to defective pyroptosis, apoptosis, and necroptosis (PANoptosis). IRF1 does not regulate inflammation and the inflammasome in the colon. Overall, our study identified IRF1 as an upstream regulator of PANoptosis to induce cell death during colitis-associated tumorigenesis.
Rajendra Karki, Bhesh Raj Sharma, Ein Lee, Balaji Banoth, R.K. Subbarao Malireddi, Parimal Samir, Shraddha Tuladhar, Harisankeerth Mummareddy, Amanda R. Burton, Peter Vogel, Thirumala-Devi Kanneganti
Wnt/β-catenin signaling is active in small subpopulations of Ewing sarcoma cells and these cells display a more metastatic phenotype, in part due to antagonism of EWS-FLI1-dependent transcriptional activity. Importantly, these β-catenin-activated Ewing cells also alter secretion of extracellular matrix (ECM) proteins. We thus hypothesized that, in addition to cell autonomous mechanisms, Wnt/β-catenin-active tumor cells might contribute to disease progression by altering the tumor microenvironment (TME). Analysis of transcriptomic data from primary patient biopsies and from β-catenin-active versus non-active tumor cells identified angiogenic switch genes as being highly and reproducibly upregulated in the context of β-catenin activation. In addition, in silico and in vitro analyses, along with chorioallantoic membrane assays, demonstrated that β-catenin-activated Ewing cells secrete factors that promote angiogenesis. In particular, activation of canonical Wnt signaling leads Ewing sarcoma cells to upregulate expression and secretion of pro-angiogenic ECM proteins, collectively termed the angiomatrix. Significantly, our data show that induction of the angiomatrix by Wnt-responsive tumor cells is indirect and is mediated by TGF-β. Mechanistically, Wnt/β-catenin signaling antagonizes EWS-FLI1-dependent repression of TGFBR2, thereby sensitizing tumor cells to TGF-β ligands. Together these findings suggest that Wnt/β-catenin active tumor cells can contribute to Ewing sarcoma progression by promoting angiogenesis in the local TME.
Allegra G. Hawkins, Elisabeth A. Pedersen, Sydney Treichel, Kelsey Temprine, Colin Sperring, Jay A. Read, Brian Magnuson, Rashmi Chugh, Elizabeth R. Lawlor
Next-generation sequencing (NGS) has not revealed all the mechanisms underlying resistance to genomically matched drugs. Here, we performed in 1417 tumors whole-exome tumor (somatic)/normal (germline) NGS and whole-transcriptome sequencing, the latter focusing on a clinically oriented 50-gene panel in order to examine transcriptomic silencing of putative driver alterations. In this large-scale study, approximately 13% of the somatic single nucleotide variants (SNVs) were unexpectedly not expressed as RNA; 23% of patients had ≥1 nonexpressed SNV. SNV-bearing genes consistently transcribed were TP53, PIK3CA, and KRAS; those with lower transcription rates were ALK, CSF1R, ERBB4, FLT3, GNAS, HNF1A, KDR, PDGFRA, RET, and SMO. We also determined the frequency of tumor mutations being germline, rather than somatic, in these and an additional 462 tumors with tumor/normal exomes; 33.8% of germline SNVs within the gene panel were rare (not found after filtering through variant information domains) and at risk of being falsely reported as somatic. Both the frequency of silenced variant transcription and the risk of falsely identifying germline mutations as somatic/tumor related are important phenomena. Therefore, transcriptomics is a critical adjunct to genomics when interrogating patient tumors for actionable alterations, because, without expression of the target aberrations, there will likely be therapeutic resistance.
Jacob J. Adashek, Shumei Kato, Rahul Parulkar, Christopher W. Szeto, J. Zachary Sanborn, Charles J. Vaske, Stephen C. Benz, Sandeep K. Reddy, Razelle Kurzrock
Recently, we reported that expression of endogenous retroviruses (ERVs) is associated with response to immune checkpoint blockade (ICB) in renal cell carcinoma (RCC). We show that decitabine, a DNA hypomethylating agent, activates transposable element (TE) expression (LINE1 and ERVs ERV3-2 and ERV4700) and antiviral signaling to potentially enhance response to ICB in kidney cancer cell lines and primary cells. KO of RIGI and MDA5 dsRNA sensors attenuated activation of antiviral signaling associated with DNA hypomethylation, and RIGI and MDA5 IPs showed increased ERV binding with decitabine treatment. Bioinformatic analyses showed the decitabine-induced signature could be associated with increased immune infiltration and response to ICB. Cytokine secretion induced by decitabine could modestly improve T cell activation and robustly enhanced T cell migration. In a small retrospective cohort of metastatic clear cell RCC (ccRCC) patients treated with anti-PD1/PDL1 blockade, activation of some antiviral genes was significantly higher in responders. Thus, we identified a potential strategy to induce TE expression through inhibition of DNA methylation in modulating T cell action via regulation of the innate antiviral pathway.
Aguirre A. de Cubas, William Dunker, Andrew Zaninovich, Rachel A. Hongo, Anuj Bhatia, Anshuman Panda, Kathryn E. Beckermann, Gyan Bhanot, Shridar Ganesan, John Karijolich, W. Kimryn Rathmell
The mechanisms of CAR-T cell mediated anti-tumor immunity and toxicity remain poorly characterized due to few studies examining the intact tumor microenvironment (TME) following CAR T-cell infusion. Axicabtagene ciloleucel is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy approved for patients with large B-cell lymphoma. We devised multiplex immunostaining and in-situ hybridization assays to interrogate CAR T cells and other immune cell infiltrates in biopsies of diffuse large B-cell lymphoma following axicabtagene ciloleucel infusion. We found a majority of intratumoral CAR T cells expressed markers of T-cell activation but, unexpectedly, comprised ≤ 5% of all T cells within the TME five days or more after therapy. T cells lacking CAR (non-CAR T cells) were also activated within the TME after axicabtagene ciloleucel infusion, being positive for Ki-67, interferon-γ, granzyme B and/or PD-1, and highest in biopsies with CAR T cells. Additionally, non-CAR immune cells were the exclusive source of IL-6, a cytokine associated with cytokine release syndrome, and highest in biopsies with CAR T cells. These data indicate that intratumoral CAR T cells are associated with generalized immune cell activation within the TME with both beneficial and pathological effects.
Pei-Hsuan Chen, Mikel Lipschitz, Jason L. Weirather, Caron Jacobson, Philippe Armand, Kyle Wright, F. Stephen Hodi, Zachary J. Roberts, Stuart A. Sievers, John Rossi, Adrian Bot, William Y. Go, Scott J. Rodig
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