Ashley Kidwell

Introduction. Pulmonary lymphangioleiomyomatosis (LAM) is a rare, lymphatic disease that affects 5-9 million women of child-bearing age.^1,2 This is the second leading cause of mortality in women with tuberous sclerosis.³ Pulmonary LAM begins with a loss of function mutation in tuberous sclerosis complex 2 (Tsc2).¹ This removes its inhibitory effect on the mammalian target of rapamycin complex 1 (mTORC1), which ultimately causes cystic airspace enlargement and respiratory failure.¹ The current treatment is rapamycin, an inhibitor of mTORC1.¹ In 84% of patients, this treatment improved lung function, but 16% of patients showed no improvement.⁴ It has been found that WNT5a is upregulated in Tsc2 knockout (KO) mutants without treatment, but with rapamycin treatment, WNT5a was decreased to wild-type expression.⁵ This finding necessitated further investigation into the role of WNT5a in refractoriness to rapamycin treatment.  Methods. Alveolar type 2 (AT2) cell lines (A549) were transfected with a β-catenin/luciferase reporter plasmid and treated with WNT5a.⁶ A double KO β-catenin and Tsc2 mouse model was made and compared to a single Tsc2 KO model using mean linear intercept (MLI).⁵ AT2 cells were sorted from whole mouse lungs by fluorescence-activated cell sorting (FACS).⁷ Cells were cultured under adherent, AT1-promoting conditions and treated with WNT5a.⁷ Single-cell RNA sequencing (scRNA-seq) was performed on Tsc2 KO mouse models.⁵ Mouse models were made with Ror2 KO in the lung epithelial cells and WNT5a KO in the lung fibroblast cells.⁸ Expression of Acta2 in an Axin2+ mesenchymal lineage (AMP) from a gain of function (GOF) β-catenin mouse model was analyzed using real-time polymerase chain reaction.⁹ Results. WNT5a treatment drove down β-catenin expression in AT2 cells.⁶ Ablation of β-catenin in Tsc2 KO mouse models restored MLI to wild-type levels.⁵ AT2 cells treated with WNT5a had lower %AT1 differentiation.⁷ scRNA-seq revealed that two unique cell types, Acta2+ fibrotically-activated mesenchymal alveolar cells (MACs) and AT1/AT2 transition state cells characterize the LAM lung.⁵  Ror2 KO in the epithelium and WNT5a KO in fibroblast cells of mouse lungs significantly increased MLI.⁸ Acta2 expression was significantly upregulated in AMP lineages in GOF β-catenin mouse models.⁹  Conclusions. WNT5a, produced by Acta2+ fibrotically-activated MACs in the LAM lung niche promotes a negative feedback mechanism through β-catenin, which inhibits cystic airspace enlargement and may promote the AT1/AT2 transition state. Rapamycin inhibits this endogenous protective mechanism through mTORC1. This suggests potential for future dual therapy with rapamycin and WNT5a pathway enhancers to address patients who are refractory to rapamycin alone.

1. Johnson SR, Taveira-DaSilva AM, Moss J. Lymphangioleiomyomatosis. Clin Chest Med. 2016;37(3):389-403. doi:10.1016/j.ccm.2016.04.002
2. Wahid S, Chiang PC, Luo HL, Huang S-C, Tsai E-M, Chiang PH. Pelvic lymphangioleiomyomatosis treated successfully with everolimus: Two case reports with literature review. Medicine (Baltimore). 2017;96(10):e4562. doi:10.1097/MD.0000000000004562
3. Zak S, Mokhallati N, Su W, et al. Lymphangioleiomyomatosis Mortality in Patients with Tuberous Sclerosis Complex. Ann Am Thorac Soc. 2019;16(4):509-512. doi:10.1513/AnnalsATS.201807-471RL
4. McCormack FX, Gupta N, Finlay GR, et al. Official American Thoracic Society/Japanese Respiratory Society Clinical Practice Guidelines: Lymphangioleiomyomatosis Diagnosis and Management. Am J Respir Crit Care Med. 2016;194(6):748-761. doi:10.1164/rccm.201607-1384ST
5. Obraztsova K, Basil MC, Rue R, et al. mTORC1 activation in lung mesenchyme drives sex- and age-dependent pulmonary structure and function decline. Nat Commun. 2020;11(1):5640. doi:10.1038/s41467-020-18979-4
6. Wu, van Dijk, Ng-Blichfeldt, et al. Mesenchymal WNT-5A/5B Signaling Represses Lung Alveolar Epithelial Progenitors. Cells. 2019;8(10):1147. doi:10.3390/cells8101147
7. Nabhan AN, Brownfield DG, Harbury PB, Krasnow MA, Desai TJ. Single-cell Wnt signaling niches maintain stemness of alveolar type 2 cells. Science. 2018;359(6380):1118-1123. doi:10.1126/science.aam6603
8. Zhang K, Yao E, Lin C, et al. A mammalian Wnt5a–Ror2–Vangl2 axis controls the cytoskeleton and confers cellular properties required for alveologenesis. eLife. 2020;9:e53688. doi:10.7554/eLife.53688
9. Zepp JA, Zacharias WJ, Frank DB, et al. Distinct Mesenchymal Lineages and Niches Promote Epithelial Self-Renewal and Myofibrogenesis in the Lung. Cell. 2017;170(6):1134-1148.e10. doi:10.1016/j.cell.2017.07.034