Partially Differentiated Ileal and Rectal Human CFTR-F508del Enteroids Secrete Fluid in Response to cAMP and cGMP: Targeted Preclinical Studies of Pharmaco-Therapy of CF Constipation

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Introduction: Treatment of Cystic Fibrosis (CF) patients with Trikafta is prolonging life. This has allowed the importance of other causes of morbidity in these patients to become clearer and to emphasize the need to improve their quality of life by developing additional treatments. Chronic constipation is one such continuing unsolved medical issue for patients with CF even on Trikafta. In general, approaches to treat chronic constipation include increasing intestinal fluid secretion that involves inhibiting intestinal NaCl absorption and/or stimulating intestinal Cl secretion, with inhibition of the latter being the primary defect in CF. Drug development efforts have mostly aimed to improve CFTR function and control pancreatic insuffciency. We have recently reported an enterocyte population in the human ileal and colon that is present between the crypt Cl− secretory cells and the mature small intestinal villus or colonic surface Na+ absorptive cells that contain both DRA and CFTR. This population is called partially differentiated enterocytes. This study was undertaken to derive a strategy to stimulate intestinal fluid secretion from partially differentiated enterocytes in the F508del-CF-patient population by enhancing residual CFTR F508del activity using drugs that stimulate cAMP and cGMP. Methods: Ileal and rectal enteroids from F508del CF patients and healthy subjects (HS/normal) were studied. Enteroids were maintained in growth media containing Wnt3A, R-spondin, and Noggin (undifferentiated/UD); induced to partially differentiate (3d-DF) or fully differentiate (6d-DF) by removal of growth factors (Wnt3A and R-spondin) for 3 or 6 days, respectively. Forskolin-induced swelling (FIS) assay and live cell microscopy were used to determine fluid secretion in enteroids. A relative increase in organoid surface area over a time series of 60 min was measured to quantify FIS response. mRNA expression was determined using qRT-PCR. Results: Compared to HS enteroids, F508del enteroids had significantly higher mRNA expression of LGR5, and Ki67 in the UD state. Expression of goblet cell marker MUC2 and enteroendocrine (EEC) marker ChgA increased with differentiation in both HS and F508del, while 6d-DF F508del enteroids had a significantly higher ChgA compared to 6d-DF enteroids from HS. UD F508del enteroids had significantly higher expression of phosphodiesterase-3a (PDE3a) than HS enteroids, which further increased with differentiation, but did not change in HS. While UD and 6d-DF F508del rectal and ileal enteroids did not swell when exposed to cAMP (forskolin) and cGMP (linaclotide), 3d-DF F508del enteroids showed a swelling response when exposed to cAMP (forskolin) and cGMP (linaclotide). Fluid secretion from 3d-DF F508del enteroids was ~50% (cAMP) and ~67% (cGMP) of the response of intestinal enteroids from HS enteroids. Combinations of CFTR correctors and potentiators mimicking the makeup of Trikafta only partially enhanced fluid secretion in F508del CF enteroids as compared to HS. However, cGMP (linaclotide) additively enhanced Trikafta-induced fluid secretion from 3d-DF F508del enteroids. Conclusions: CFTR F508del can be stimulated by cAMP and cGMP in partially differentiated enterocyte populations. Our findings identify a novel role of the partially differentiated ileal and rectal enterocyte populations which behave in a way that offers the potential to improve treatment for CF constipation. Funding: This study is partly supported by Cystic Fibrosis Foundation grant CF-SINGH20G0, National Institutes of Health grants R01-DK-116352 and P30-DK-89502 (the Hopkins Basic and Translational Research Digestive Diseases Research Core Center). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.