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Introduction Cellular metabolism drives [CD4.sup.+] T cell differentiation and stability, and [CD4.sup.+] T cell metabolic pathways are promising therapeutic targets for cancers, autoimmune diseases, and asthma (1-3). Once activated, effector [...], Female individuals have an increased prevalence of many Th17 cell-mediated diseases, including asthma. Androgen signaling decreases Th17 cell-mediated airway inflammation, and Th17 cells rely on glutaminolysis. However, it remains unclear whether androgen receptor (AR) signaling modifies glutamine metabolism to suppress Th17 cell-mediated airway inflammation. We show that Th17 cells from male humans and mice had decreased glutaminolysis compared with female individuals, and that AR signaling attenuated Th17 cell mitochondrial respiration and glutaminolysis in mice. Using allergen-induced airway inflammation mouse models, we determined that females had a selective reliance upon glutaminolysis for Th17-mediated airway inflammation, and that AR signaling attenuated glutamine uptake in [CD4.sup.+] T cells by reducing expression of glutamine transporters. In patients with asthma, circulating Th17 cells from men had minimal reliance upon glutamine uptake compared to Th17 cells from women. AR signaling thus attenuates glutaminolysis, demonstrating sexspecific metabolic regulation of Th17 cells with implications for Th17 or glutaminolysis targeted therapeutics.

Introduction Clear cell renal cell carcinoma (ccRCC) is the most abundant subtype of kidney cancer, accounting for 75% of all cases (1). In ccRCC, a deregulated hypoxia transcriptional response is [...], Clear cell renal cell carcinoma (ccRCC) is an aggressive cancer driven by VHL loss and aberrant HIF-2[alpha] signaling. Identifying means to regulate HIF-2[alpha] thus has potential therapeutic benefit. Acetyl- CoA synthetase 2 (ACSS2) converts acetate to acetyl-CoA and is associated with poor patient prognosis in ccRCC. Here we tested the effects of ACSS2 on HIF-2[alpha] and cancer cell metabolism and growth in ccRCC models and clinical samples. ACSS2 inhibition reduced HIF-2[alpha] levels and suppressed ccRCC cell line growth in vitro, in vivo, and in cultures of primary ccRCC patient tumors. This treatment reduced glycolytic signaling, cholesterol metabolism, and mitochondrial integrity, all of which are consistent with loss of HIF-2[alpha]. Mechanistically, ACSS2 inhibition decreased chromatin accessibility and HIF- 2[alpha] expression and stability. While HIF-2[alpha] protein levels are widely regulated through pVHL-dependent proteolytic degradation, we identify a potential pVHLindependent pathway of degradation via the E3 ligase MUL1. We show that MUL1 can directly interact with HIF-2[alpha] and that overexpression of MUL1 decreased HIF-2[alpha] levels in a manner partially dependent on ACSS2. These findings identify multiple mechanisms to regulate HIF-2[alpha] stability and ACSS2 inhibition as a strategy to complement HIF-2[alpha]-targeted therapies and deplete pathogenically stabilized HIF-2[alpha].

Introduction Clear cell renal cell carcinoma (ccRCC) represents the most frequent subtype of RCC, accounting for approximately 75% of adult clinical cases (1, 2). ccRCC biology is unique due to [...], Clear cell renal cell carcinoma (ccRCC) is characterized by dysregulated hypoxia signaling and a tumor microenvironment (TME) highly enriched in myeloid and lymphoid cells. Loss of the von Hippel Lindau (VHL) gene is a critical early event in ccRCC pathogenesis and promotes stabilization of HIF. Whether VHL loss in cancer cells affects immune cells in the TME remains unclear. Using Vhl WT and Vhl-KO in vivo murine kidney cancer Renca models, we found that Vhl-KO tumors were more infiltrated by immune cells. Tumor-associated macrophages (TAMs) from Vhl-deficient tumors demonstrated enhanced in vivo glucose consumption, phagocytosis, and inflammatory transcriptional signatures, whereas lymphocytes from Vhl-KO tumors showed reduced activation and a lower response to anti- programmed cell death 1 (anti-PD-1) therapy in vivo. The chemokine CX3CL1 was highly expressed in human ccRCC tumors and was associated with Vhl deficiency. Deletion of Cx3cl1 in cancer cells decreased myeloid cell infiltration associated with Vhl loss to provide a mechanism by which Vhl loss may have contributed to the altered immune landscape. Here, we identify cancer cell-specific genetic features that drove environmental reprogramming and shaped the tumor immune landscape, with therapeutic implications for the treatment of ccRCC.

The receptor tyrosine kinase AXL promotes tumor progression, metastasis, and therapy resistance through the induction of epithelial-mesenchymal transition (EMT). Here, we found that activation of AXL resulted in the phosphorylation of TANK-binding kinase 1 (TBK1) and the downstream activation of AKT3 and Snail, a transcription factor critical for EMT. Mechanistically, we showed that TBK1 directly bound to and phosphorylated AKT3 in a manner dependent on the multiprotein complex mTORC1. Upon activation, AKT3 interacted with and promoted the nuclear accumulation of Snail, which led to increased EMT as assessed by marker abundance. In human pancreatic ductal adenocarcinoma tissue, nuclear AKT3 colocalized with Snail and correlated with worse clinical outcomes. Primary mouse pancreatic cancer cells deficient in AKT3 showed reduced metastatic spread in vivo, suggesting selective AKT3 inhibition as a potential therapeutic avenue for targeting EMT in aggressive cancers. Editor's summary: Metastatic spread is associated with cancer cells undergoing epithelial-to-mesenchymal transition (EMT), which can be driven by the activity of the serine-threonine kinase AKT. Arner et al. investigated the activation and contribution of a specific AKT isoform in pancreatic cancer. TANK-binding kinase 1 (TBK1) phosphorylated and activated AKT3 in vitro downstream of the receptor tyrosine kinase AXL. In mouse pancreatic cancer cells, TBK1 was required for the increase in the abundance of EMT markers in response to AKT3 activation. Inhibiting AKT3 reduced the metastasis of pancreatic cancer in mice. Thus, the specific targeting of AKT3 may be useful in limiting pancreatic cancer metastasis. —Amy E. Baek [ABSTRACT FROM AUTHOR]

Heat is a cardinal feature of inflammation, yet its impacts on immune cells remain uncertain. We show that moderate-grade fever temperatures (39°C) increased murine CD4 T cell metabolism, proliferation, and inflammatory effector activity while decreasing regulatory T cell suppressive capacity. However, heat-exposed T helper 1 (TH1) cells selectively developed mitochondrial stress and DNA damage that activated Trp53 and stimulator of interferon genes pathways. Although many TH1 cells subjected to such temperatures died, surviving TH1 cells exhibited increased mitochondrial mass and enhanced activity. Electron transport chain complex 1 (ETC1) was rapidly impaired under fever-range temperatures, a phenomenon that was specifically detrimental to TH1 cells. TH1 cells with elevated DNA damage and ETC1 signatures were also detected in human chronic inflammation. Thus, fever-relevant temperatures disrupt ETC1 to selectively drive apoptosis or adaptation of TH1 cells to maintain genomic integrity and enhance effector functions. Editor's summary: Heat is one of the hallmarks of inflammation, but its effect on immune cells is not well understood. Heintzman et al. report that temperatures associated with moderate-grade fevers (39°C) both enhance the metabolism, proliferation, and effector function of mouse CD4 T cells and dampen regulatory T cell suppressive potential. In addition, febrile temperatures disrupt electron transport chain complex 1, which selectively drives apoptosis and elimination of a substantial number of mouse and human T helper 1 (TH1) cells. Those TH1 cells that can adapt by increasing their mitochondrial mass and DNA damage responses, however, exhibit genomic integrity and augmented effector functions. —Seth Thomas Scanlon [ABSTRACT FROM AUTHOR]

Ferroptosis is an iron-dependent form of cell death that results from excess lipid peroxidation in cellular membranes. Within the last decade, physiological and pathological roles for ferroptosis have been uncovered in autoimmune diseases, inflammatory conditions, infection, and cancer biology. Excitingly, cancer cell metabolism may be targeted to induce death by ferroptosis in cancers that are resistant to other forms of cell death. Ferroptosis sensitivity is regulated by oxidative stress, lipid metabolism, and iron metabolism, which are all influenced by the tumor microenvironment (TME). Whereas some cancer cell types have been shown to adapt to these stressors, it is not clear how immune cells regulate their sensitivities to ferroptosis. In this review, we discuss the mechanisms of ferroptosis sensitivity in different immune cell subsets, how ferroptosis influences which immune cells infiltrate the TME, and how these interactions can determine epithelial-to-mesenchymal transition (EMT) and metastasis. While much focus has been placed on inducing ferroptosis in cancer cells, these are important considerations for how ferroptosismodulating strategies impact anti-tumor immunity. From this perspective, we also discuss some promising immunotherapies in the field of ferroptosis and the challenges associated with targeting ferroptosis in specific immune cell populations. [ABSTRACT FROM AUTHOR]

Metastasis causes most cancer deaths and reflects transitions from primary tumor escape to seeding and growth at metastatic sites. Epithelial-to-mesenchymal transition (EMT) is important early in metastasis to enable cancer cells to detach from neighboring cells, become migratory, and escape the primary tumor. While different phases of metastasis expose cells to variable nutrient environments and demands, the metabolic requirements and plasticity of each step are uncertain. Here we show that EMT and primary tumor escape are stimulated by disrupted oxidative metabolism. Using Renal Cell Carcinoma (RCC) patient samples, we identified the mitochondrial electron transport inhibitor NDUFA4L2 as upregulated in cells undergoing EMT. Deletion of NDUFA4L2 enhanced oxidative metabolism and prevented EMT and metastasis while NDUFA4L2 overexpression enhanced these processes. Mechanistically, NDUFA4L2 suppressed oxidative phosphorylation and caused citric acid cycle intermediates to accumulate, which modified chromatin accessibility of EMT-related loci to drive primary tumor escape. The effect of impaired mitochondrial metabolism to drive EMT appeared general, as renal cell carcinoma patient tumors driven by fumarate hydratase mutations with disrupted oxidative phosphorylation were highly metastatic and also had robust EMT. These findings highlight the importance of dynamic shifts in metabolism for cell migration and metastasis, with mitochondrial impairment driving early phases of this process. Understanding mitochondrial dynamics may have important implications in both basic and translational efforts to prevent cancer deaths.

Lipid metabolism is fundamental to CD4+ T cell metabolism yet remains poorly understood across subsets. Therefore, we performed targeted in vivo CRISPR/Cas9 screens to identify lipid-associated genes essential for T cell subset functions. These screens established mitochondrial fatty acid synthesis (mtFAS) genes Mecr, Mcat and Oxsm as highly impactful. Of these, the inborn error of metabolism gene Mecr was most dynamically regulated. Effector and memory T cells were reduced in Mecrfl/fl; Cd4cre mice, and MECR was required for activated CD4+ T cells to efficiently proliferate, differentiate, and survive. Mecr-deficient T cells also had decreased mitochondrial respiration, reduced TCA intermediates, and accumulated intracellular iron, which contributed to cell death and sensitivity to ferroptosis. Importantly, Mecr-deficient T cells exhibited fitness disadvantages in inflammatory, tumor, and infection models. mtFAS and MECR thus play important roles in activated T cells and may provide targets to modulate immune functions in inflammatory diseases. The immunological state of MECR- and mtFAS-deficient patients may also be compromised.

Stimulator of interferon genes (STING) is a promising target for potentiating antitumor immunity, but multiple pharmacological barriers limit the clinical utility, efficacy, and/or safety of STING agonists. Here we describe a modular platform for systemic administration of STING agonists based on nanobodies engineered for in situ hitchhiking of agonist cargo on serum albumin. Using site-selective bioconjugation chemistries to produce molecularly defined products, we found that covalent conjugation of a STING agonist to anti-albumin nanobodies improved pharmacokinetics and increased cargo accumulation in tumor tissue, stimulating innate immune programs that increased the infiltration of activated natural killer cells and T cells, which potently inhibited tumor growth in multiple mouse tumor models. We also demonstrated the programmability of the platform through the recombinant integration of a second nanobody domain that targeted programmed cell death ligand-1 (PD-L1), which further increased cargo delivery to tumor sites while also blocking immunosuppressive PD-1/PD-L1 interactions. This bivalent nanobody carrier for covalently conjugated STING agonists stimulated robust antigen-specific T cell responses and long-lasting immunological memory, conferred enhanced therapeutic efficacy, and was effective as a neoadjuvant treatment for improving responses to adoptive T cell transfer therapy. Albumin-hitchhiking nanobodies thus offer an enabling, multimodal, and programmable platform for systemic delivery of STING agonists with potential to augment responses to multiple immunotherapeutic modalities., Competing Interests: Competing Interests J.T.W., K.A., and B.R.K. are inventors on U.S. Provisional Application No. 63/472,528 “NANOBODY-DRUG CONJUGATES AND METHODS OF PREPARING THEREOF” which describes nanobody conjugation and delivery technologies. W.K.R. is an employee of Vanderbilt University Medical Center and appointed to the Vanderbilt University School of Medicine. She has received research support from Incyte Corp. within the past three years, and serves on the Board of Scientific Advisors for the National Cancer Institute. J.C.R. is an employee of Vanderbilt University Medical Center and appointed to the Vanderbilt University School of Medicine. He is a founder, scientific advisory board member, and stockholder of Sitryx Therapeutics, a scientific advisory board member and stockholder of Caribou Biosciences and holds stock options for Nirogy Therapeutics. He has consulted and received speaker fees from Merck, Pfizer, and Abbie. He has received research support from Incyte Corp. within the past three years. J.M.B. receives research support from Genentech/Roche and Incyte Corporation, has received advisory board payments from AstraZeneca and Mallinckrodt and is an inventor on patents regarding immunotherapy targets and biomarkers in cancer.

A series of 2-amino-2,3-dihydro-1 H -indene-5-carboxamides were designed and synthesized as new selective discoidin domain receptor 1 (DDR1) inhibitors. One of the representative compounds, 7f , bound with DDR1 with a K d value of 5.9 nM and suppressed the kinase activity with an half-maximal (50%) inhibitory concentration value of 14.9 nM. 7f potently inhibited collagen-induced DDR1 signaling and epithelial-mesenchymal transition, dose-dependently suppressed colony formation of pancreatic cancer cells, and exhibited promising in vivo therapeutic efficacy in orthotopic mouse models of pancreatic cancer.