FTO inhibition enhances the therapeutic index of radiation therapy in head and neck cancer

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Abstract

Despite aggressive chemoradiation treatment, the overall survival rate for patients with HPV– head and neck squamous cell carcinoma (HNSCC) remains poor, highlighting the urgent need for more effective drug-radiotherapy combinations to improve the therapeutic index of radiation therapy (RT). The fat mass and obesity-related gene (FTO) is emerging as a promising cancer therapeutic target; however, its role in the RT response has been underexplored. In our study, we found that both genetic and pharmacologic inhibition of FTO enhanced the efficacy of RT in human and mouse HNSCC tumor xenografts. Mechanistically, inhibition of FTO improved the RT response in HPV– HNSCC cells, which was associated with increased DNA damage, reduced efficiency of homology directed repair, and decreased formation of RAD51 homolog 1 (RAD51) foci. Importantly, pharmacologic inhibition of FTO did not exacerbate radiation-induced oral mucositis, a significant normal-tissue toxicity associated with HNSCC RT. In summary, our results indicate a role for FTO in regulating homologous recombination while identifying FTO as a potential therapeutic target to enhance the therapeutic index of RT in HPV– HNSCC treatment.

Authors

Lu Ji, Leighton Pu, Jinglong Wang, Hongbin Cao, Stavros Melemenidis, Subarna Sinha, Li Guan, Eyiwunmi E. Laseinde, Rie von Eyben, Sara A. Richter, Jin-Min Nam, Christina Kong, Kerriann M. Casey, Edward E. Graves, Richard L. Frock, Quynh Thu Le, Erinn B. Rankin

Label-free single cell phenotyping to determine tumor cell heterogeneity in pancreatic cancer in real-time

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Abstract

Resistance to chemotherapy of pancreatic ductal adenocarcinoma (PDAC) is largely driven by intratumoral heterogeneity (ITH) due to tumor cell plasticity and clonal diversity. In order to develop novel strategies to overcome this defined mechanism of resistance, tools to monitor and quantify ITH in a rapid and scalable fashion are needed urgently. Here, we employed label-free digital holographic microscopy (DHM) to characterize ITH in PDAC. We established a robust experimental and machine learning analysis pipeline to perform single cell phenotyping based on DHM-derived phase images of PDAC cells in suspension. Importantly, we are able to detect dynamic changes in tumor cell differentiation and heterogeneity of distinct PDAC subtypes upon induction of epithelial-to-mesenchymal transition and under treatment-imposed pressure in murine and patient-derived model systems. This platform allows us to assess phenotypic ITH in PDAC on a single cell level in real-time. Implementing this technology into the clinical workflow has the potential to fundamentally increase our understanding of tumor heterogeneity during evolution and treatment response.

Authors

Katja Wittenzellner, Manuel Lengl, Stefan Röhrl, Carlo Maurer, Christian Klenk, Aristeidis Papargyriou, Laura Schmidleitner, Nicole Kabella, Akul Shastri, David E. Fresacher, Farid Harb, Nawal Hafez, Stefanie Bärthel, Daniele Lucarelli, Carmen Escorial-Iriarte, Felix Orben, Rupert Öllinger, Ellen Emken, Lisa Fricke, Joanna Madej, Patrick Wustrow, I. Ekin Demir, Helmut Friess, Tobias Lahmer, Roland M. Schmid, Roland Rad, Günter Schneider, Bernhard Kuster, Dieter Saur, Oliver Hayden, Klaus Diepold, Maximilian Reichert

DAZAP1 maintains gastric cancer stemness by inducing mitophagy

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Abstract

Stem cells play a pivotal role in the malignant behavior of gastric cancer (GC), complicating its treatment and prognosis. However, the regulatory mechanisms of GC stem cells (GCSCs) remain poorly understood. DAZ-associated protein 1 (DAZAP1), a splicing regulator linked to various malignancies, has an unclear role in GC. This study investigated DAZAP1’s impact on GC stemness and its mechanisms. DAZAP1 promoted tumor progression in GCSCs, as shown by sphere formation assays and stemness marker analysis. Functional enrichment analysis suggested that DAZAP1 enhanced tumor stemness by promoting oxidative phosphorylation (OXPHOS), which was validated through Seahorse assays and measurements of mitochondrial potential. Transmission electron microscopy and immunofluorescence analyses demonstrated that DAZAP1 promoted mitophagy. RNA immunoprecipitation and PCR analysis revealed that DAZAP1 regulated the splicing and expression of the mitophagy-related gene ULK1 through nonsense-mediated mRNA decay. Rescue experiments showed that overexpression of ULK1 reversed the suppression of GC stemness and OXPHOS levels induced by DAZAP1 silencing. Our findings indicate that DAZAP1 reduces ULK1 decay, thereby activating mitophagy and enhancing OXPHOS to fulfill the metabolic demands of cancer stem cells. These findings highlight the therapeutic potential of DAZAP1 as a target for treating GC.

Authors

Peiling Zhang, Wei Wang, Hong Xiang, Yun Zhou, Qian Peng, Guolong Liu, Zhi-Xiang Xu, Lin Lu

Radiosensitizing the SUMO Stress Response Intensifies Single Dose Radiotherapy Tumor Cure

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Abstract

Single dose radiotherapy (SDRT) is a highly-curative modality that may transform radiotherapy practice. Unfortunately, only ~50% of oligometastatic lesions are SDRT treatable due to adjacent radiosensitive normal organs at risk. Here we address extent to which an anti-angiogenic drug, VEGFR2-antagonist DC101, radiosensitizes SDRT using murine MCA/129 fibrosarcomas and Lewis Lung Carcinomas, which display a dose range for SDRT lesional eradication virtually identical to that employed clinically (10-30Gy). SDRT induces unique tumor cure, stimulating rapid endothelial acid sphingomyelinase (ASMase)/ceramide signaling that yields marked vasoconstriction and perfusion defects in tumor xenografts and human oligometastases. Ensuing tumor parenchymal oxidative damage initiates a SUMO Stress Response (SSR), which inactivates multiple homologous recombination repair enzymes, radiosensitizing all tumor types. While VEGF inhibits neo-angiogenic ASMase, optimal radiosensitization occurs only upon anti-angiogenic drug delivery at ~1h preceding SDRT. Obeying these principles, we find DC101 radiosensitizes SSR, DNA double strand break unrepair and tumor cure by 4-8Gy at all clinically-relevant doses. Critically, DC101 fails to sensitize small intestinal endothelial injury or lethality from the gastrointestinal-acute radiation syndrome.

Authors

Jin Cheng, Liyang Zhao, Sahra Bodo, Prashanth K. B. Nagesh, Rajvir Singh, Adam O. Michel, Regina Feldman, Zhigang Zhang, Simon N. Powell, Zvi Fuks, Richard Kolesnick

Chemotherapy and the somatic mutation burden of sperm

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Abstract

Many chemotherapeutic agents impair cancer growth by inducing DNA damage. The impact of these agents on mutagenesis in normal cells, including sperm, is largely unknown. Here, we applied high-fidelity duplex sequencing to 94 samples from 36 individuals exposed to diverse chemotherapies and 32 controls. We found that many of the sperm samples from men exposed to chemotherapy, the mutation burden was elevated as compared to controls and the expected burden based on trio studies, with one subject having >10-fold increase over expected for age. Saliva from this same individual also had a markedly higher mutation burden. We then validated this finding using other tissues, also finding an increased mutation burden in the blood and liver of many subjects exposed to chemotherapy as compared to unexposed controls. Similarly, mice treated with three cycles of cisplatin had an increased mutation burden in sperm but also in the liver, and hematopoietic progenitor cells. These results suggest an association between cancer therapies and mutation burden, with implications for counseling cancer patients considering banking sperm prior to therapy and for cancer survivors considering the tradeoffs of using banked sperm as compared to conceiving naturally.

Authors

Shany Picciotto, Camilo Arenas-Gallo, Amos Toren, Ruty Mehrian-Shai, Bryan Daly, Stephen Rhodes, Megan Prunty, Ruolin Liu, Anyull Bohorquez, Marta Grońska-Pęski, Shana Melanaphy, Pamela Callum, Emilie Lassen, Anne-Bine Skytte, Rebecca C. Obeng, Christopher Barbieri, Molly Gallogly, Brenda Cooper, Katherine Daunov, Lydia Beard, Koen Van-Besien, Joshua Halpern, Quintin Pan, Gilad D. Evrony, Viktor A. Adalsteinsson, Jonathan E. Shoag

Tumor suppressors in Sox2-mediated lung cancers promote distinct cell intrinsic and immunologic remodeling

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Abstract

Non-small cell lung cancer (NSCLC) largely consists of lung squamous (LUSC) and lung adenocarcinoma (LUAD). Alterations in the TRP53 and PTEN tumor suppressors are common in both subtypes, but their relationship with SOX2 is poorly understood. We deleted Trp53 or Pten in a C57BL/6J-Sox2hi;Nkx2-1-/-;Lkb1-/- (SNL) genetic background and generated a highly metastatic LUSC cell line (LN2A; derived from a Sox2hi mouse model, followed by Trp53, Pten, and Cdkn2a deletion). Histologic and single-cell RNAseq analyses corroborated that SNL mice developed mixed tumors with both LUAD and LUSC histopathology while SNL-Trp53 and SNL-Pten mice developed LUAD and LN2A tumors retained LUSC morphology. Compared with SNL mice, additional loss of Trp53 or Pten resulted in significantly reduced survival, increased tumor burden and altered tumor mucin composition. We identified a sub-cluster of CD38+ tumor-associated inflammatory monocytes in the LN2A model that significantly enriched for activation of the classical and alternative complement pathways. Complement Factor B (CFB) is associated with poor survival in LUSC patients, and we observed the LN2A model had significantly improved survival on a Cfb-/- background. Our findings demonstrate a cooperative role of Trp53 and Pten tumor suppressors in Sox2-mediated NSCLC tumor progression, mucin production, and remodeling of the immune tumor microenvironment.

Authors

Nisitha Sengottuvel, Kristina M. Whately, Jennifer L Modliszewski, Rani S. Sellers, William D. Green, Weida Gong, Allison T. Woods, Eric W. Livingston, Katerina D. Fagan-Solis, Gabrielle Cannon, Lincy Edatt, Hong Yuan, Aaron C. Chack, Yazmin Sanchez, Katherine Zhou, Alyaa Dawoud, Jarred M. Green, Virginia Godfrey, J Justin Milner, Gaorav P. Gupta, Chad V. Pecot

Imlunestrant a next-generation oral SERD overcomes ESR1 mutant resistance in estrogen receptor-positive breast cancer

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Abstract

Estrogen receptor alpha (ER) is a critical driver of tumorigenesis and tumor progression in most breast cancers. Endocrine therapies (ET) targeting ER are central to treating hormone receptor-positive breast cancer (BC), but resistance poses a clinical challenge. Some resistance mechanisms, particularly those involving estrogen-independent activity such as the ESR1 mutations, rely on ER signaling, supporting the need for next-generation ET. We investigated the preclinical efficacy of imlunestrant, an oral selective ER degrader, in ER-positive BC pre-clinical models, including models harboring the Y537S ESR1 mutation, an activating mutation. Imlunestrant demonstrated antagonistic activity and effective degradation of both wild-type and mutant ER, resulting in cell growth suppression. In vivo, imlunestrant outperformed fulvestrant leading to tumor regression in a patient derived xenograft harboring the Y537S ESR1 mutation. Cyclic mutiplexed immunofluorescence and transcriptomic analysis revealed enhanced cell cycle arrest and downregulation of estrogen-responsive genes with imlunestrant treatment. Additionally, a genome wide CRISPR knock-out screen identified several vulnerabilities that were either persistent or gained after imlunestrant treatment, providing a rationale for future studies of combination treatments with imlunestrant. Collectively, these results highlight the on-target and selective activity of imlunestrant, which can circumvent resistance engendered by the Y537S ESR1 mutation.

Authors

Shira Sherman, Zachary M. Sandusky, Douglas Russo, David Zak, Agostina Nardone, Delia Friel, Francisco Hermida-Prado, Capucine Heraud, Genevra Kuziel, Ana Verma, Giorgio Gaglia, Sheheryar Kabraji, Quang-De Nguyen, Sandro Santagata, Sean W. Fanning, Rinath Jeselsohn

T-cell phenotype and clonality changes in myeloma patients with short OS

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Abstract

Overall survival (OS) in multiple myeloma (MM) varies between a couple of months to more than 20 years, influenced by tumor characteristics, the tumor microenvironment (TME), and patient factors such as age and frailty. We analyzed sequential BM samples from 45 MM patients with OS < 3 years versus > 8 years using mass cytometry and bulk TCRβ sequencing. Patients with long OS demonstrated stability in the TME and T cell environments, while those with short OS had significant changes at relapse, including fewer T cells, increased Treg cells, and more activated and exhausted CD8 T cells. Notably, higher PD-1 expression in CD8 T cells at diagnosis correlated with short OS. Additionally, short-OS patients exhibited a more monoclonal T cell environment at relapse, with abundance of hyperexpanded clones. These findings reveal distinct immune cell differences between patients with short and long OS.

Authors

Alenka Djarmila Behsen, Esten Nymoen Vandsemb, Tobias Schmidt Slørdahl, Karen Dybkær, Maja Zimmer Jakobsen, Muhammad Kashif, Johan Lund, Vincent Luong, Astrid Marta Olsnes, Anders Waage, Anne Marit Sponaas, Kristine Misund

Identification and regulation of circulating tumor TCR-matched cytotoxic CD4⁺ lymphocytes by KLRG1 in bladder cancer

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Abstract

While cytotoxic CD4+ tumor-infiltrating lymphocytes have anti-cancer activity in patients, whether these can be non-invasively monitored and how these are regulated remains obscure. By matching single cells with T cell receptors (TCR) in tumor and blood of bladder cancer patients, we identified distinct pools of tumor-matching cytotoxic CD4+ T cells in the periphery directly reflecting the predominant antigenic specificities of intratumoral CD4+ TILs. On one hand, the granzyme B (GZMB)-expressing cytotoxic CD4+ subset proliferated in blood in response to PD-1 blockade, but was separately regulated by the killer cell lectin-like receptor G1 (KLRG1) which inhibited their killing by interacting with E-cadherin. Conversely, a clonally related, granzyme K (GZMK)-expressing circulating CD4+ population demonstrated basal proliferation and a memory phenotype that may result from activation of GZMB+ cells, but was not directly mobilized by PD-1 blockade. As KLRG1 marked the majority of circulating tumor TCR-matched cytotoxic CD4+ T cells, this work nominates KLRG1 as a means to isolate them from blood and provide a window into intratumoral CD4+ recognition, as well as a putative regulatory receptor to mobilize the cytolytic GZMB+ subset for therapeutic benefit. Our findings also underscore ontogenic relationships of GZMB- and GZMK-expressing populations and the distinct cues that regulate their activity.

Authors

Serena S. Kwek, Hai Yang, Tony Li, Arielle Ilano, Eric D. Chow, Li Zhang, Hewitt Chang, Diamond Luong, Averey Lea, Matthew Clark, Alec Starzinski, Yimin Shi, Elizabeth McCarthy, Sima Porten, Maxwell V. Meng, Chun Jimmie Ye, Lawrence Fong, David Y. Oh

The gut microbiome enhances breast cancer immunotherapy following bariatric surgery

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Abstract

Bariatric surgery is associated with improved breast cancer (BC) outcomes, including greater immunotherapy effectiveness in a pre-clinical BC model. A potential mechanism of bariatric surgery-associated protection is the gut microbiota. Here, we demonstrate the dependency of improved immunotherapy response on the post-bariatric surgery gut microbiome via fecal microbial transplant (FMT). Response to αPD-1 immunotherapy was significantly improved following FMT from formerly obese bariatric surgery-treated mice. When stool from post-bariatric surgery patients was transplanted into recipient mice and compared to the patients’ pre-surgery transplants, post-surgery microbes significantly reduced tumor burden and doubled immunotherapy effectiveness. Microbes impact tumor burden through microbially derived metabolites, including branched chain amino acids (BCAA). Circulating BCAAs correlated significantly with natural killer T (NKT) cell content in the tumor microenvironment in donor mice after bariatric surgery and FMT recipients of donor cecal content after bariatric surgery compared to obese controls. BCAA supplementation replicated improved αPD-1 effectiveness in two BC models, supporting the role of BCAAs in increased immunotherapy effectiveness after bariatric surgery. Ex vivo exposure increased primary NKT cell expression of anti-tumor cytokines, demonstrating direct activation of NKT cells by BCAAs. Together, findings suggest that reinvigorating anti-tumor immunity may depend upon bariatric surgery-associated microbially derived metabolites, namely BCAAs.

Authors

Margaret S. Bohm, Sydney C. Joseph, Laura M. Sipe, Minjeong Kim, Cameron T. Leathem, Tahliyah S. Mims, Nathaniel B. Willis, Ubaid A. Tanveer, Joel H. Elasy, Emily W. Grey, Madeline E. Pye, Zeid T. Mustafa, Barbara Anne Harper, Logan G. McGrath, Deidre Daria, Brenda Landvoigt Schmitt, Jelissa A. Myers, Patricia Pantoja Newman, Brandt D. Pence, Marie Van der Merwe, Matthew J. Davis, Joseph F. Pierre, Liza Makowski