Alveolar macrophages (AMs), the resident immune cells of the lung, play a critical role in maintaining pulmonary homeostasis, in part through the secretion of suppressor of cytokine signaling 3 (SOCS3)—a recognized tumor suppressor—within extracellular vesicles (EVs). While we have previously observed that SOCS3 secretion by AMs is diminished in tumor-bearing lungs, the mechanisms underlying this impairment remain unclear. Here, we investigated whether increased glycolytic metabolism in AMs contributes to this defect within the tumor microenvironment. The analysis of published single-cell RNA-sequencing datasets from an orthotopic Lewis lung cancer (LLC) model of adenocarcinoma and non-small cell lung cancer (NSCLC) patients revealed distinct AM clusters in tumor-bearing lungs enriched for glycolysis-associated genes. In a KrasG12D mutant mouse model of lung cancer, we found that AMs isolated from tumor-bearing lungs exhibited increased glucose uptake, which inversely correlated with SOCS3 secretion. Importantly, the pharmacologic inhibition of glycolysis with 2-deoxy-d-glucose restored SOCS3 secretion in these AMs. Together, our findings demonstrate that lung tumor-associated AMs undergo a time-dependent metabolic shift toward glycolysis, resulting in impaired SOCS3 secretion—a phenotype that can be reversed by targeting glycolytic flux. These results highlight a potential therapeutic approach for modulating immune suppression in the tumor microenvironment.

Introduction: Idiopathic pulmonary fibrosis (IPF) is hallmarked by an increase in aberrant, apoptosis-resistant myofibroblasts which carry out enhanced collagen I and extracellular matrix deposition. Resolution of fibrosis requires myofibroblasts to undergo phenotypic dedifferentiation, a process marked by down-regulation of fibrotic and extracellular matrix genes and reacquisition of their capacity for apoptosis. Recent reports highlight the underappreciated role of myofibroblasts in uptake of collagen fragments, a necessary step for fibrosis resolution in vivo previously attributed exclusively to phagocytic cell populations. We have previously reported that enhanced collagen uptake occurs in response to the dedifferentiating agent PGE2 in both IPF and TGF-β-elicited myofibroblasts, but the role of the collagen binding receptor MRC2 during this phenomenon is unknown. Methods: Human fibroblasts from normal and IPF lung were studied. Normal lung fibroblasts were infected with lentiviral particles containing lentiCRISPR plasmid TLCV2 with non-targeting or MRC2 targeting guide RNAs. Fibroblasts were treated with TGF-β (2 ng/mL) for 48 h to establish myofibroblasts with concurrent treatment of 1 μg/mL doxycycline to elicit MRC2 knockdown. Cells were treated for 3 h with previously established dedifferentiating agents PGE2 (500 nM), forskolin (20 μM), or bortezomib (100 nM), and collagen I uptake was assessed utilizing Oregon green-488-conjugated gelatin (5 μg/mL) over 1 h at 37°C followed by quenching and subsequent flow cytometric analysis. MRC2 expression was evaluated by Western blotting.Results: IPF fibroblasts demonstrated a reduction in both gelatin uptake capability as well as MRC2 expression as compared to normal lung fibroblasts. TGF-β-elicited myofibroblasts treated with doxycycline demonstrated 95% reduction in MRC2 protein expression compared to non-targeting control, confirming knockdown. Control myofibroblasts demonstrated a 1.5-fold increase in collagen uptake during dedifferentiation in response to PGE2, forskolin, and bortezomib, which was completely abrogated in MRC2 silenced myofibroblasts, suggesting the requirement for MRC2 in the enhanced collagen clearance associated with dedifferentiation. MRC2-mediated collagen uptake is known to depend upon clathrin coated vesicle formation and function. Treatment of IPF fibroblasts with the clathrin endocytosis inhibitor chlorpromazine completely blocked the enhanced collagen uptake seen in response to PGE2 dedifferentiation, consistent with a clathrin/MRC2 mediated requirement for this process. Conclusions: Enhanced collagen clearance during the process of elicited myofibroblast dedifferentiation requires collagen I binding to MRC2 and clathrin-mediated endocytosis. Further, MRC2-mediated collagen clearance is impaired in IPF fibroblasts owing to reduced MRC2 expression. The in vivo significance of these findings awaits further studies.

Rationale: Following lung injury, timely clearance of pathologic fibroblasts, immune cells, and epithelial populations is necessary to restore lung homeostasis and prevent progressive scarring. Although the molecular mechanisms and cellular crosstalk that promote clearance of these cells remains a critical gap in knowledge, spontaneously resolving models of pulmonary fibrosis – such as single-dose bleomycin in young mice – provide a unique opportunity to identify endogenous molecular brakes responsible for orchestrating these crucial events. The second messenger cyclic AMP (cAMP) has garnered renewed attention with nerandomilast (a PDE4B inhibitor that increases intracellular cAMP) recently achieving its primary endpoint in a phase 3 trial. Indeed, cAMP signaling elicits antifibrotic effects within all of the aforementioned cell types in vitro, and we have reported that its intracellular generation in lung fibroblasts is necessary for spontaneous fibrosis resolution in the bleomycin model. Here we extend this important finding by characterizing the epithelial, mesenchymal, and immune cell populations during and after peak fibrosis in WT and transgenic mice whose lung fibroblasts cannot generate intracellular cAMP due to genetic deletion of Gαs/Gnas (cAMP-stimulatory G-protein subunit). Methods: Young Col1a2CreER;Gnasf/f C57BL/6 mice were treated with single-dose intrapulmonary bleomycin (1.0 U/kg). Tamoxifen chow was introduced to abolish Gαs expression in fibroblasts at day 21 (peak fibrosis). scRNA-seq of mouse lung digests, hydroxyproline quantitation, and trichrome staining/immunofluorescence microscopy were performed at days 21, 42, and 63 post-bleomycin. In vitro experiments utilizing CRISPR/Cas9-mediated deletion of Gαs in human lung fibroblasts were also performed. Results: Deletion of lung fibroblast Gnas in vitro induced higher collagen/αSMA expression and increased apoptosis resistance, while Cre-mediated Gnas deletion in vivo resulted in persistence of increased lung collagen content and histopathologic evidence of alveolar damage through day 63, compared to resolved fibrosis in WT mice. scRNA-seq of mouse lungs containing Gnas-null fibroblasts revealed reduced type 1 alveolar epithelial cells at day 63 (compared to WT) and persistence of day 42 injury-associated transitional epithelial cells (Cldn4+/Sfn+/Krt8hi) and day 42/63 pathologic macrophages (CD11c+/C1qb+/Siglecf-). Differences in fibroblast subtypes between the two genotypes were relatively inconspicuous by scRNA-seq, despite substantial differences in lung collagen content in Col1a2;Gnas-null mice. Conclusion: Lung fibroblast-specific cAMP generation is a crucial endogenous brake that promotes fibrosis resolution through fibroblast deactivation/apoptosis and timely clearance of aberrant macrophages and epithelial cells. These findings highlight the importance of fibroblast-mediated cellular crosstalk in fibrosis resolution and expand the relevance of G-protein-mediated cAMP generation as a tractable therapy in lung fibrosis.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease characterized by scarring. Myofibroblasts play a crucial role in the development of IPF through their excessive production of matrix proteins and resistance to cell death. We previously reported the in vitro capacity of alveolar macrophage (AM) conditioned medium (CM) to dedifferentiate myofibroblasts from normal or IPF lung, but the mechanism behind this activity remains unknown. Because activation of Gαs (Gnas)-coupled GPCRs by cyclooxygenase (COX)-derived prostanoids (including prostaglandins E2 and I2) has been demonstrated to have potent antifibrotic activity, we sought to interrogate the role of this pathway in AM CM's dedifferentiation action. Methods: AMs were isolated from the lungs of naive 6-8 week old C57BL/6 mice via collagenase digestion and MACS separation. AMs were adhered for 1 h, washed, then cultured overnight in serum-free DMEM. AM CM was collected after 24 h and was added to TGFβ (2 ng/mL x 48 h)-differentiated CCL210 normal human lung fibroblasts or primary mouse fibroblasts isolated from tamoxifen-treated Col1a1-cre Gnas fl/fl mouse lungs for 48 h. To further delineate the role of Gnas signaling in the bioactivity, CCL210 myofibroblasts were cultured with a pharmacological inhibitor of adenylyl cyclase (SQ22536; 10 µM) prior to addition of AM CM. To assess the contribution of prostanoids contained within AM CM, AMs were treated with a cyclooxygenase (COX) inhibitor (aspirin; 200 µM) for 1 h, washed, and cultured overnight before CM collection. Myofibroblast dedifferentiation was determined via qRT-PCR and western blot for alpha-smooth muscle actin (α-sma) and collagen 1a1 (Col1a1). Results: Gnas deletion as well as pharmacologic inhibition of adenylyl cyclase in mouse myofibroblasts abrogated the ability of AM CM to induce dedifferentiation as was observed in wild-type myofibroblasts. In addition, inhibition of COX in the AMs partially abrogated AM CM's ability to dedifferentiate CCL210 myofibroblasts as compared to control. Conclusion: The ability of AM CM to elicit dedifferentiation in myofibroblasts appears to depend on elaboration of some COX-derived prostanoid. Moreover, sensitivity of the myofibroblast to such dedifferentiation depends on intracellular signaling via Gnas and adenylyl cyclase. The identity of the specific prostanoid involved and the in vivo significance of this phenomenon in fibrosis inhibition or resolution require additional study.

Supplementary Data 2 shows Supplementary Methods, Supplementary Reference, Supplementary Figure S1 and Figure Legends, Supplementary Figure S2 and Figure Legends, Supplementary Figure S3 and Figure Legends, Supplementary Figure S4 and Figure Legends, Supplementary Figure S5 and Figure Legends, Supplementary Figure S6 and Figure Legends, and Supplementary Figure S7 and Figure Legends.

Supplementary Table S1 shows Supplementary Table S1 and relative references.

How cell metabolism regulates DNA repair is incompletely understood. Here, we define a GTP-mediated signaling cascade that links metabolism to DNA repair and has significant therapeutic implications. GTP, but not other nucleotides, regulates the activity of Rac1, a guanine nucleotide-binding protein, which promotes the dephosphorylation of serine 323 on Abl-interactor 1 (Abi-1) by protein phosphatase 5 (PP5). Dephosphorylated Abi-1, a protein previously not known to activate DNA repair, promotes nonhomologous end joining. In patients and mouse models of glioblastoma, Rac1 and dephosphorylated Abi-1 mediate DNA repair and resistance to standard-of-care genotoxic treatments. The GTP-Rac1-PP5-Abi-1 signaling axis is not limited to brain cancer, as GTP supplementation promotes DNA repair and Abi-1-S323 dephosphorylation in nonmalignant cells and protects mouse tissues from genotoxic insult. This unexpected ability of GTP to regulate DNA repair independently of deoxynucleotide pools has important implications for normal physiology and cancer treatment.

Fibrosis following tissue injury is distinguished from normal repair by the accumulation of pathogenic and apoptosis-resistant myofibroblasts (MFs), which arise primarily by differentiation from resident fibroblasts. Endogenous molecular brakes that promote MF dedifferentiation and clearance during spontaneous resolution of experimental lung fibrosis may provide insights that could inform and improve the treatment of progressive pulmonary fibrosis in patients. MAPK phosphatase 1 (MKP1) influences the cellular phenotype and fate through precise and timely regulation of MAPK activity within various cell types and tissues, yet its role in lung fibroblasts and pulmonary fibrosis has not been explored. Using gain- and loss-of-function studies, we found that MKP1 promoted lung MF dedifferentiation and restored the sensitivity of these cells to apoptosis — effects determined to be mainly dependent on MKP1’s dephosphorylation of p38α MAPK (p38α). Fibroblast-specific deletion of MKP1 following peak bleomycin-induced lung fibrosis largely abrogated its subsequent spontaneous resolution. Such resolution was restored by treating these transgenic mice with the p38α inhibitor VX-702. We conclude that MKP1 is a critical antifibrotic brake whose inhibition of pathogenic p38α in lung fibroblasts is necessary for fibrosis resolution following lung injury.

Abstract OBJECTIVE: We sought to define the mechanisms by which purines regulate DNA repair and therapy response. METHODS: Phosphoproteomics was used to identify GTP-dependent (de)phosphorylation events after radiation (RT) and antibodies generated against novel sites. Animal models of glioblastoma (GBM) and normal tissues were used to assess DNA repair and treatment responses in vivo. RESULTS: Pharmacogenomic inhibition of GTP (but not ATP) synthesis sensitized GBM cells to RT by inhibiting the activity of non-homologous end joining, but not homologous recombination. We found a GTP-dependent RT-induced dephosphorylation event on Abl interactor 1 (Abi-1) serine 323 (S323) using phosphoproteomics. We generated a new antibody for p-Abi-1 (S323), validated its specificity, and confirmed that RT causes a GTP-dependent dephosphorylation of Abi-1 (S323). Knockout of Abi-1 slowed RT-induced double-strand break (DSB) repair, and this was rescued by re-expression of dephosphomimetic Abi-1 (S323A) but not phosphomimetic Abi-1 (S323D). Abi-1 canonically binds to G protein Rac1. Expression of constitutively active Rac1 promoted but dominant negative Rac1 blocked the dephosphorylation of Abi-1 (S323) and DSB repair. Knock-down or inhibition of protein phosphatase 5 reversed the GTP- and Rac1-mediated dephosphorylation of Abi-1 and DSB repair. In GBM PDX samples, p-Abi-1 (S323) levels negatively correlated with Rac1 activity and predicted favorable efficacy of genotoxic treatments. In orthotopic GBM mouse models, inhibiting Rac1 enhanced RT responses and suppressed Abi-1 (S323) dephosphorylation. Abi-1 knockout enhanced efficacy of genotoxic treatments and could be rescued by Abi-1 S323A (but not Abi-1 S323D) re-expression. This regulation is generalizable beyond brain cancer, as GTP supplementation promoted DNA repair and p-Abi1 (S323) dephosphorylation in non-malignant cells and protected mice from RT-mediated gastrointestinal injury and bleomycin-induced pulmonary fibrosis. CONCLUSION: The GTP-Rac1-PP5-Abi-1 signaling axis links metabolism and DNA repair. Disrupting this pathway can overcome cancer resistance to genotoxic therapy while augmenting it can mitigate genotoxic injury of normal tissues. Citation Format: Weihua Zhou, Zitong Zhao, Angelica Lin, John Yang, Jie Xu, Kari Wilder-Romans, Annabel Yang, Andrew J. Scott, Jing Li, Sumeet Solanki, Jennifer Speth, Natalie Walker, Ayesha U. Kothari, Yangyang Yao, Erik R. Peterson, Navyateja Korimerla, Christian K. Werner, Jessica Liang, Janna Jacobson, Sravya Palavalasa, Alexandra M Obrien, Ameer L Elaimy, Sean P. Ferris, Shuang G. Zhao, Jann N. Sarkaria, Balázs Győrffy, Shuqun Zhang, Wajd N. Al-Holou, Yoshie Umemura, Meredith A Morgan, Theodore S. Lawrence, Costas A. Lyssiotis, Marc Peters-Golden, Yatrik M. Shah, Daniel R. Wahl. GTP signaling links metabolism, DNA repair, and responses to genotoxic stress [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr A004.

Supplementary Data 1 shows the original data of phosphoproteomic assay in Figure 2.

ABSTRACT How cell metabolism regulates DNA repair is incompletely understood. Here, we define a GTP-mediated signaling cascade that links metabolism to DNA repair and has significant therapeutic implications. GTP, but not other nucleotides, regulates the activity of Rac1, a guanine nucleotide-binding protein, which promotes the dephosphorylation of serine 323 on Abl-interactor 1 (Abi-1) by protein phosphatase 5 (PP5). Dephosphorylated Abi-1, a protein previously not known to activate DNA repair, promotes nonhomologous end joining. In patients and mouse models of glioblastoma, Rac1 and dephosphorylated Abi-1 mediate DNA repair and resistance to standard-of-care genotoxic treatments. The GTP–Rac1–PP5–Abi-1 signaling axis is not limited to brain cancer, as GTP supplementation promotes DNA repair and Abi-1-S323 dephosphorylation in nonmalignant cells and protects mouse tissues from genotoxic insult. This unexpected ability of GTP to regulate DNA repair independently of deoxynucleotide pools has important implications for normal physiology and cancer treatment. SIGNIFICANCE: A newly described GTP-dependent signaling axis is an unexpected link between nucleotide metabolism and DNA repair. Disrupting this pathway can overcome cancer resistance to genotoxic therapy while augmenting it can mitigate genotoxic injury of normal tissues. This article is featured in Selected Articles from This Issue, p. 5

Abstract OBJECTIVE We sought to define the mechanisms by which purines regulate DNA repair and therapy response. METHODS Phosphoproteomics was used to identify GTP-dependent (de)phosphorylation events after radiation (RT) and antibodies generated against novel sites. Animal models of glioblastoma (GBM) and normal tissues were used to assess DNA repair and treatment responses in vivo. RESULTS Pharmacogenomic inhibition of GTP (but not ATP) synthesis sensitized GBM cells to RT by inhibiting the activity of non-homologous end joining, but not homologous recombination. We found a GTP-dependent RT-induced dephosphorylation event on Abl interactor 1 (Abi-1) serine 323 (S323) using phosphoproteomics. We generated a new antibody for p-Abi-1 (S323), validated its specificity, and confirmed that RT causes a GTP-dependent dephosphorylation of Abi-1 (S323). Knockout of Abi-1 slowed RT-induced double-strand break (DSB) repair, and this was rescued by re-expression of dephosphomimetic Abi-1 (S323A) but not phosphomimetic Abi-1 (S323D). Abi-1 canonically binds to G protein Rac1. Expression of constitutively active Rac1 promoted but dominant negative Rac1 blocked the dephosphorylation of Abi-1 (S323) and DSB repair. Knock-down or inhibition of protein phosphatase 5 reversed the GTP- and Rac1-mediated dephosphorylation of Abi-1 and DSB repair. In GBM PDX samples, p-Abi-1 (S323) levels negatively correlated with Rac1 activity and predicted favorable efficacy of genotoxic treatments. In orthotopic GBM mouse models, inhibiting Rac1 enhanced RT responses and suppressed Abi-1 (S323) dephosphorylation. Abi-1 knockout enhanced efficacy of genotoxic treatments and could be rescued by Abi-1 S323A (but not Abi-1 S323D) re-expression. This regulation is generalizable beyond brain cancer, as GTP supplementation promoted DNA repair and p-Abi1 (S323) dephosphorylation in non-malignant cells and protected mice from RT-mediated gastrointestinal injury and bleomycin-induced pulmonary fibrosis. CONCLUSION The GTP-Rac1-PP5-Abi-1 signaling axis links metabolism and DNA repair. Disrupting this pathway can overcome GBM resistance to genotoxic therapy while augmenting it can mitigate genotoxic injury of normal tissues.

There is a paucity of information about potential molecular brakes on the activation of fibroblasts that drive tissue fibrosis. The transcription factor Krüppel-like factor 4 (KLF4) is best known as a determinant of cell stemness and a tumor suppressor. We found that its expression was diminished in fibroblasts from fibrotic lung. Gain- and loss-of-function studies showed that KLF4 inhibited fibroblast proliferation, collagen synthesis, and differentiation to myofibroblasts, while restoring their sensitivity to apoptosis. Conditional deletion of KLF4 from fibroblasts potentiated the peak degree of pulmonary fibrosis and abrogated the subsequent spontaneous resolution in a model of transient fibrosis. A small molecule inducer of KLF4 was able to restore its expression in fibrotic fibroblasts and elicit resolution in an experimental model characterized by more clinically relevant persistent pulmonary fibrosis. These data identify KLF4 as a pivotal brake on fibroblast activation whose induction represents a therapeutic approach in fibrosis of the lung and perhaps other organs.

Abstract There is a paucity of information about potential molecular brakes on the activation of fibroblasts that drive tissue fibrosis. The transcription factor Kruppel-like factor 4 (KLF4) is best known as a determinant of cell stemness and a tumor suppressor. We found that its expression was diminished in fibroblasts from fibrotic lung. Gain- and loss-of-function studies showed that KLF4 inhibits fibroblast proliferation, collagen synthesis, and differentiation to myofibroblasts, while restoring their sensitivity to apoptosis. Conditional deletion of KLF4 from fibroblasts potentiated the peak degree of pulmonary fibrosis and abrogated the subsequent spontaneous resolution that follows in a model of transient fibrosis. A small molecule inducer of KLF4 was able to restore its expression in fibrotic fibroblasts and elicit resolution in an experimental model characterized by more clinically relevant persistent pulmonary fibrosis. These data identify KLF4 as a pivotal brake on fibroblast activation whose induction represents a new therapeutic approach in fibrosis of the lung, and perhaps other organs.