Chimeric antigen receptor (CAR) designs that incorporate pharmacologic control are desirable, however designs suitable for clinical translation are needed. We designed a fully human, rapamycin-regulated, drug product for targeting CD33+ tumors called dimerization agent regulated immunoreceptor complex (DARIC33). T cell products demonstrated target specific and rapamycin-dependent cytokine release, transcriptional responses, cytotoxicity, and in vivo antileukemic activity in the presence of as little as 1nM rapamycin. Rapamycin withdrawal paused DARIC33-stimulated T cell effector functions, which were restored following re-exposure to rapamycin, demonstrating reversible effector function control. While rapamycin-regulated DARIC33 T cells were highly sensitive to target antigen, CD34+ stem cell colony forming capacity was not impacted. We benchmarked DARIC33 potency relative to CD19 CAR T cells to estimate a T cell dose for clinical testing. In addition, we integrated in vitro and preclinical in vivo drug concentration thresholds for OFF-ON state transitions, as well as murine and human rapamycin pharmacokinetics, to estimate a clinically applicable rapamycin dosing schedule. A phase 1 DARIC33 trial has been initiated (PLAT-08, NCT05105152), with initial evidence of rapamycin-regulated T cell activation and anti-tumor impact. Our findings provide evidence that the DARIC platform exhibits sensitive regulation and potency needed for clinical application to other important immunotherapy targets.
Jacob Appelbaum, April E. Price, Kaori Oda, Joy Zhang, Wai-Hang Leung, Giacomo Tampella, Dong Xia, Pauline P.L. So, Sarah K. Hilton, Claudya Evandy, Semanti Sarkar, Unja Martin, Anne-Rachel Krostag, Marissa Leonardi, Daniel E. Zak, Rachael Logan, Paula Lewis, Secil Franke-Welch, Njabulo Ngwenyama, Michael Fitzgerald, Niklas Tulberg, Stephanie Rawlings-Rhea, Rebecca A. Gardner, Kyle Jones, Angelica Sanabria, William Crago, John Timmer, Andrew Hollands, Brendan Eckelman, Sanela Bilic, Jim Woodworth, Adam Lamble, Philip D. Gregory, Jordan Jarjour, Mark Pogson, Joshua A. Gustafson, Alexander Astrakhan, Michael C. Jensen
Neurofibromatosis Type 1 (NF1) is caused by mutations in the NF1 gene that encodes neurofibromin, a RAS GTPase-Activating Protein. Inactivating NF1 mutations cause hyperactivation of RAS-mediated signaling, resulting in development of multiple neoplasms, including Malignant Peripheral Nerve Sheath Tumors (MPNSTs). MPNSTs are an aggressive tumor and the main cause of mortality in NF1 patients. MPNSTs are difficult to resect and refractory to chemo- and radiotherapy, and no molecular therapies currently exist. Immune Checkpoint Blockade (ICB) is an approach to treat inoperable, undruggable cancers like MPNST, but successful outcomes require an immune cell-rich tumor microenvironment (TME). While MPNSTs are non-inflamed “cold” tumors, here, we turned MPNSTs into T cell-inflamed “hot” tumors by activating “stimulator of interferon genes” (STING) signaling. Mouse genetic and human xenograft MPNST models treated with STING agonist plus ICB exhibited growth delay via increased apoptotic cell death. This strategy offers a potential treatment regimen for MPNST.
Bandarigoda N. Somatilaka, Laasya Madana, Ali Sadek, Zhiguo Chen, Sanjay Chandrasekaran, Renee M. McKay, Lu Q. Le
SARS-CoV-2 infection of the upper airway and the subsequent immune response are early, critical factors in COVID-19 pathogenesis. By studying infection of human biopsies in vitro and in a hamster model in vivo, we demonstrated a transition in nasal tropism from olfactory to respiratory epithelium as the virus evolved. Analyzing each variant revealed that SARS-CoV-2 WA1 or Delta infect a proportion of olfactory neurons in addition to the primary target sustentacular cells. The Delta variant possessed broader cellular invasion capacity into the submucosa, while Omicron displayed enhanced nasal respiratory infection and longer retention in the sinonasal epithelium. The olfactory neuronal infection by WA1 and the subsequent olfactory bulb transport via axon were more pronounced in younger hosts. In addition, the observed viral clearance delay and phagocytic dysfunction in aged olfactory mucosa were accompanied by a decline of phagocytosis related genes. Furthermore, robust basal stem cell activation contributed to neuroepithelial regeneration and restores ACE2 expression post-infection. Together, our study characterized the nasal tropism of SARS-CoV-2 strains, immune clearance, and regeneration post infection. The shifting characteristics of viral infection at the airway portal provides insight into the variability of COVID-19 clinical features, particularly long COVID, and may suggest differing strategies for early local intervention.
Mengfei Chen, Andrew Pekosz, Jason S. Villano, Wenjuan Shen, Ruifeng Zhou, Heather Kulaga, Zhexuan Li, Amy Smith, Asiana Gurung, Sarah E. Beck, Kenneth W. Witwer, Joseph L. Mankowski, Murugappan Ramanathan Jr., Nicholas R. Rowan, Andrew P. Lane
In lung, thromboxane A2 (TXA2) activates the TP receptor to induce pro-inflammatory and bronchoconstrictor effects. Thus, TP receptor antagonists and TXA2 synthase inhibitors have been tested as potential asthma therapeutics in humans. Th9 cells play key roles in asthma and regulate the lung immune response to allergens. Herein, we found that TXA2 reduces Th9 cell differentiation during allergic lung inflammation. Th9 cells were decreased ~2-fold and airway hyperresponsiveness was attenuated in lungs of allergic mice treated with TXA2. Naïve CD4+ T cell differentiation to Th9 cells and IL-9 production was inhibited dose-dependently by TXA2 in vitro. TP receptor deficient mice had a ~2-fold increase in numbers of Th9 cells in lungs in vivo after OVA exposure compared to wild type (WT) mice. Naïve CD4+ T cells from TP deficient mice exhibited increased Th9 cell differentiation and IL-9 production in vitro compared to CD4+ T cells from WT mice. TXA2 also suppressed Th2 and enhanced Treg differentiation both in vitro and in vivo. Thus, in contrast to its acute, pro-inflammatory effects, TXA2 also has longer-lasting immunosuppressive effects that attenuate the Th9 differentiation that drives asthma progression. These findings may explain the paradoxical failure of anti-thromboxane therapies in the treatment of asthma.
Hong Li, J. Alyce Bradbury, Matthew L. Edin, Artiom Gruzdev, Huiling Li, Joan P. Graves, Laura M. DeGraff, Fred B. Lih, Chiguang Feng, Erin R. Wolf, Carl D. Bortner, Stephanie J. London, Matthew A. Sparks, Thomas M. Coffman, Darryl C. Zeldin
Macrophage immune checkpoint inhibitors, such as anti-CD47 antibodies, show promise in clinical trials for solid and hematologic malignancies. However, the best strategies to use these therapies remain unknown, and ongoing studies suggest they may be most effective when used in combination with other anticancer agents. Here, we developed a novel screening platform to identify drugs that render lung cancer cells more vulnerable to macrophage attack, and we identified therapeutic synergy exists between genotype-directed therapies and anti-CD47 antibodies. In validation studies, we found the combination of genotype-directed therapies and CD47 blockade elicited robust phagocytosis and eliminated persister cells in vitro and maximized anti-tumor responses in vivo. Importantly, these findings broadly applied to lung cancers with various RTK/MAPK pathway alterations—including EGFR mutations, ALK fusions, or KRASG12C mutations. We observed downregulation of β2-microglobulin and CD73 as molecular mechanisms contributing to enhanced sensitivity to macrophage attack. Our findings demonstrate that dual inhibition of the RTK/MAPK pathway and the CD47/SIRPa axis is a promising immunotherapeutic strategy. Our study provides strong rationale for testing this therapeutic combination in patients with lung cancers bearing driver mutations.
Kyle Vaccaro, Juliet Allen, Troy W. Whitfield, Asaf Maoz, Sarah Reeves, José Velarde, Dian Yang, Anna Meglan, Juliano Ribeiro, Jasmine Blandin, Nicole Phan, George W. Bell, Aaron Hata, Kipp Weiskopf
CD4 T cells survey and maintain immune homeostasis in the brain, yet their differentiation states and functional capabilities remain unclear. Our approach, combining single-cell transcriptomic analysis, ATAC-seq, spatial transcriptomics, and flow cytometry, revealed a distinct subset of CCR7+ CD4 T cells resembling lymph node central memory (TCM) cells. We observed chromatin accessibility at the CCR7, CD28, and BCL-6 loci, defining molecular features of TCM. Brain CCR7+ CD4 T cells exhibited recall proliferation and interleukin-2 production ex vivo, showcasing their functional competence. We identified the skull bone marrow as a local niche for these cells alongside CNS border tissues. Sequestering TCM cells in lymph nodes using FTY720 led to reduced CCR7+ CD4 T cell frequencies in the cerebrospinal fluid, accompanied by increased monocyte levels and soluble markers indicating immune activation. In macaques chronically infected with SIVCL757 and experiencing viral rebound due to cessation of antiretroviral therapy, a decrease in brain CCR7+ CD4 T cells was observed, along with increased microglial activation and initiation of neurodegenerative pathways. Our findings highlight a role for CCR7+ CD4 T cells in CNS immune surveillance and their decline during chronic SIV highlights their responsiveness to neuroinflammation.
Sonny R. Elizaldi, Chase E. Hawes, Anil Verma, Yashavanth Shaan Lakshmanappa, Ashok R. Dinasarapu, Brent T. Schlegel, Dhivyaa Rajasundaram, Jie Li, Blythe P. Durbin-Johnson, Zhong-Min Ma, Pabitra B. Pal, Danielle Beckman, Sean Ott, Reben Raeman, Jeffrey Lifson, John H. Morrison, Smita S. Iyer
Craniofacial anomalies, especially midline facial defects, are among the most common birth defects in patients associated with increased mortality or require lifelong treatment. During mammalian embryogenesis, specific instructions arising at genetic, signaling, and metabolic levels are important for stem cell behaviors and fate determination, but how these functionally relevant mechanisms are coordinated to regulate craniofacial morphogenesis remain unknown. Here, we report that BMP signaling in cranial neural crest cells (CNCCs) is critical for glycolytic lactate production and subsequent epigenetic histone lactylation, thereby dictating craniofacial morphogenesis. Elevated BMP signaling in CNCCs through constitutively activated ACVR1 (ca-ACVR1) suppressed glycolytic activity and blocked lactate production via a p53-dependent process that resulted in severe midline facial defects. By modulating epigenetic remodeling, BMP signaling-dependent lactate generation drived histone lactylation levels to alter essential genes of Pdgfra thus regulating CNCC behavior in vitro as well as in vivo. These findings define an axis wherein the BMP signaling controls a metabolic-epigenetic cascade to direct craniofacial morphogenesis, thus providing a conceptual framework for understanding the interaction between genetic and metabolic cues operative during embryonic development. These findings indicate potential preventive strategies of congenital craniofacial birth defects via modulating metabolic-driven histone lactylation.
Jingwen Yang, Lingxin Zhu, Haichun Pan, Hiroki Ueharu, Masako Toda, Qian Yang, Shawn A. Hallett, Lorin E. Olson, Yuji Mishina
Development of effective strategies to manage the inevitable acquired resistance to osimertinib, an approved 3rd generation EGFR inhibitor for the treatment of EGFR mutant (EGFRm) non-small cell lung cancer (NSCLC), is urgently needed. This study reported that the DNA topoisomerase II (Topo II) inhibitors, doxorubicin and etoposide (VP-16) synergistically decreased cell survival with enhanced induction of DNA damage and apoptosis in osimertinib-resistant cells, suppressed the growth of osimertinib-resistant tumors, and delayed the emergence of osimertinib acquired resistance. Mechanistically, osimertinib decreased Topo IIα levels in EGFRm NSCLC cells by facilitating FBXW7-mediated proteasomal degradation, resulting in induction of DNA damage; these effects were lost in osimertinib-resistant cell lines possessing elevated levels of Topo IIα. Topo IIα elevation was also detected in the majority of EGFRm NSCLC tissues relapsed from EGFR-TKI treatment. Enforced expression of an ectopic TOP2A gene in sensitive EGFRm NSCLC cells conferred resistance to osimertinib, whereas knockdown of TOP2A in osimertinib-resistant cell lines restored their response to undergo osimertinib-induced DNA damage and apoptosis. Together, these results reveal an essential role of Topo IIα inhibition in mediating the therapeutic efficacy of osimertinib against EGFRm NSCLC, providing scientific rationale for targeting Topo II to manage acquired resistance to osimertinib.
Zhen Chen, Karin A. Vallega, Dongsheng Wang, Zihan Quan, Songqing Fan, Qiming Wang, Ticiana Leal, Suresh S. Ramalingam, Shi-Yong Sun
The appearance of senescent cells in age-related diseases has spurred the search for compounds that can target senescent cells in tissues (“senolytics”). However, a major caveat with current senolytic screens is the use of cell lines as targets where senescence is induced in vitro, which does not necessarily reflect the identity and function of pathogenic senescent cells in vivo. Here, we developed a new pipeline leveraging a fluorescent murine reporter that allows for isolation and quantification of p16Ink4a+ cells in diseased tissues. By high-throughput screening in vitro, precision cut lung slice (PCLS) screening ex vivo, and phenotypic screening in vivo, we identified a HSP90 inhibitor (XL888) as a potent senolytic in tissue fibrosis. XL888 treatment eliminated pathogenic p16Ink4a+ fibroblasts in a murine model of lung fibrosis and reduced fibrotic burden. Finally, XL888 preferentially targeted p16INK4a-high human lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis (IPF), and reduced p16INK4a+ fibroblasts from IPF PCLS ex vivo. This study provides proof of concept for a platform where p16INK4a+ cells are directly isolated from diseased tissues to identify compounds with in vivo and ex vivo efficacy in mouse and human respectively and provides a senolytic screening platform for other age-related diseases.
Jin Young Lee, Nabora S. Reyes, Supriya Ravishankar, Minqi Zhou, Maria Krasilnikov, Christian Ringler, Grace Pohan, Chris Wilson, Kenny Kean-Hooi Ang, Paul J. Wolters, Tatsuya Tsukui, Dean Sheppard, Michelle R. Arkin, Tien Peng
Antitumor responses of CD8+ T cells are tightly regulated by distinct metabolic fitness. High levels of glutathione (GSH) are observed in the majority of tumors contributing to cancer progression and treatment resistance in part by preventing glutathione peroxidase 4 (GPX4) dependent ferroptosis. Here, we show the necessity of the adenosine A2A receptor (A2AR) signaling and the glutathione (GSH)-GPX4 axis in orchestrating metabolic fitness and survival of functionally competent CD8+ T cells. Activated CD8+ T cells treated ex vivo with simultaneous inhibition of A2AR and lipid peroxidation acquire a superior capacity to proliferate and persist in vivo, demonstrating a translatable means to prevent ferroptosis in adoptive cell therapy (ACT). Additionally, we identify a particular cluster of intratumoral CD8+ T cells expressing a putative gene signature of GSH metabolism (GMGS) in association with clinical response and survival across several human cancers. Our study addresses a key role of GSH-GPX4 and adenosinergic pathways in fine-tuning the metabolic fitness of antitumor CD8+ T cells.
Siqi Chen, Jie Fan, Ping Xie, Jihae Ahn, Michelle Fernandez, Leah K. Billingham, Jason Miska, Jennifer D. Wu, Derek A. Wainwright, Deyu Fang, Jeffrey A. Sosman, Yong Wan, Yi Zhang, Navdeep S. Chandel, Bin Zhang
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