Suppression of tumor-associated neutrophils by lorlatinib attenuates pancreatic cancer growth and improves treatment with immune checkpoint blockade.

Pancreatic ductal adenocarcinoma (PDAC) patients have a 5-year survival rate of only 8% largely due to late diagnosis and insufficient therapeutic options. Neutrophils are among the most abundant immune cell type within the PDAC tumor microenvironment (TME), and are associated with a poor clinical prognosis. However, despite recent advances in understanding neutrophil biology in cancer, therapies targeting tumor-associated neutrophils are lacking. Here, we demonstrate, using pre-clinical mouse models of PDAC, that lorlatinib attenuates PDAC progression by suppressing neutrophil development and mobilization, and by modulating tumor-promoting neutrophil functions within the TME. When combined, lorlatinib also improves the response to anti-PD-1 blockade resulting in more activated CD8 + T cells in PDAC tumors. In summary, this study identifies an effect of lorlatinib in modulating tumor-associated neutrophils, and demonstrates the potential of lorlatinib to treat PDAC

Hypoxia-inducible factor prolyl 4-hydroxylases regulating erythropoiesis, and hypoxia-inducible lysyl oxidase regulating skeletal muscle development during embryogenesis

Abstract Erythropoiesis is the process of red blood cell production. The main regulator is the erythropoietin (EPO) hormone, which is strongly upregulated in low oxygen concentration (hypoxia) in cells via the hypoxia-inducible transcription factor HIF. The stability of HIF is regulated in an oxygen-dependent manner by three HIF prolyl 4-hydroxylases, all of which are known to participate in the regulation of erythropoiesis. A role in erythropoiesis of a fourth prolyl 4-hydroxylase, P4H-TM, which possesses a transmembrane domain, is not known, but it is able to hydroxylate HIF at least in vitro and in cellulo. The role of P4H-TM in erythropoiesis was studied by administering a HIF-P4H inhibitor, FG-4497, to P4h-tm null, Hif-p4h-3 null, and Hif-p4h-2 hypomorph mouse lines. The current study suggests that P4H-TM is involved in the regulation of EPO production, hepcidin expression and erythropoiesis. P4H-TM can thus be a new target for inhibition when designing novel pharmacological treatment strategies for anemia. LOX is required for crosslink formation between lysine residues in fibrillar collagens and elastin. These crosslinks enhance the tensile strength of collagen fibers and provide elasticity to elastic fibers and thus generate important structural support for tissues. LOX is required for normal embryonic development of the cardiovascular and pulmonary systems, and its depletion leads to a generalized elastinopathy and collagenolysis leading to perinatal death of Lox null mice. The development of muscles is a delicate process, which requires coordinated signaling and a homeostatic balance between the muscle and muscle connective tissue. Based on the drastic defects that were found in the present study in the skeletal muscle of Lox null mice, lack of LOX clearly disturbs this balance and increases transforming growth factor β (TGF-β) signaling, which leads to defects in the skeletal muscles. The impaired balance can cause muscle disorders, such as Duchenne Muscular Dystrophy (DMD). Despite the clinical significance, very little is known about the mechanisms controlling this homeostatic balance. The discovery of LOX as a regulating factor during skeletal muscle development will help to clarify the role of extracellular matrix (ECM) in muscle development and in muscle related congenital diseases.Tiivistelmä Erytropoieesi on fysiologinen prosessi, jossa tuotetaan veren punasoluja ja jonka pääsäätelijänä toimii erytropoietiini (EPO) hormoni. EPO:n geeni ilmentyy voimakkaasti alhaisessa happipitoisuudessa (hypoksia) hypoksia-indusoituvan transkriptiotekijän (HIF) toimesta. HIF-tekijän stabiilisuutta säätelee kolme HIF-prolyyli-4-hydroksylaasientsyymiä (HIF-P4H) hapesta riippuvaisesti, ja niiden tiedetään siten osallistuvan myös erytropoieesin säätelyyn, HIF-P4H-2:n toimiessa pääsäätelijänä. Neljännen transmembraanisen prolyyli-4-hydroksylaasin (P4H-TM) roolia erytropoieesissa ei vielä tiedetä, mutta sen tiedetään säätelevän HIF-tekijää. Työssä käytettiin Hif-p4h-2, Hif-p4h-3 ja P4h-tm muuntogeenisiä hiirilinjoja, joiden entsymaattinen aktiivisuus on alentunut tai poistettu. P4H-TM:n osallisuutta erytropoieesin säätelyyn tutkittiin antamalla hiirilinjoille HIF-P4H-entsyymejä inhiboivaa lääkettä. Tutkimuksen tulokset osoittavat ensimmäistä kertaa P4H-TM:n säätelevän EPO-geenin ilmentymistä ja siten erytropoieesia. Ennestään tiedettyjen HIF-P4H entsyymien inhiboinnin lisäksi P4H-TM:n inhibointia voidaan pitää uutena kohteena uusien farmakologisten hoitokeinojen kehityksessä. Lysyylioksidaasi (LOX) katalysoi säikeisten kollageenien välisten sekä elastisten säikeiden välisten poikkisidosten muodostumista. Pokkisidokset antavat vetolujuutta kollageeneille ja joustavuutta elastisille säikeille ja ovat siten tärkeitä kudoksen rakenteelle. LOX:ia tarvitaan sikiön kehityksen aikana mm. hengitys-, sydän- ja verisuonielimistöjen kehityksessä. LOX:in puutos hiirillä aiheuttaa viallisia elastisia- ja kollageenisäikeitä, johtaen poikasten kuolemaan synnytyksen yhteydessä. Lihasten kehitys on tarkoin säädelty prosessi, jossa lihas ja lihaksen sidekudos säätelevät toisiansa. LOX:n suhteen poistogeenisissä Lox-/- sikiöissä löydettiin selviä ongelmia luurankolihasten kehityksessä. LOX:n puutoksen osoitettiin lisäävän transformoivan kasvutekijä beetan (TGF-β) määrää, joka estää luustolihaksia kehittymästä normaalisti. LOX kykenee sitoutumaan TGF-β:aan ja inhiboimaan sen aktiivisuutta ja LOX:n puuttuessa inhibointia ei tapahdu. Tutkimus osoittaa LOX:n olevan keskeinen tekijä lihaksen kehityksessä ja siten auttaa ymmärtämään sidekudoksen merkitystä luurankolihasten kehityksessä ja siihen liittyvissä sairauksissa

Deletion of hypoxia-inducible factor prolyl 4-hydroxylase 2 in FoxD1-lineage mesenchymal cells leads to congenital truncal alopecia

Abstract Hypoxia-inducible factors (HIFs) induce numerous genes regulating oxygen homeostasis. As oxygen sensors of the cells, the HIF prolyl 4-hydroxylases (HIF-P4Hs) regulate the stability of HIFs in an oxygen-dependent manner. During hair follicle (HF) morphogenesis and cycling, the location of dermal papilla (DP) alternates between the dermis and hypodermis and results in varying oxygen levels for the DP cells. These cells are known to express hypoxia-inducible genes, but the role of the hypoxia response pathway in HF development and homeostasis has not been studied. Using conditional gene targeting and analysis of hair morphogenesis, we show here that lack of Hif-p4h-2 in Forkhead box D1 (FoxD1)-lineage mesodermal cells interferes with the normal HF development in mice. FoxD1-lineage cells were found to be mainly mesenchymal cells located in the dermis of truncal skin, including those cells composing the DP of HFs. We found that upon Hif-p4h-2 inactivation, HF development was disturbed during the first catagen leading to formation of epithelial-lined HF cysts filled by unorganized keratins, which eventually manifested as truncal alopecia. Furthermore, the depletion of Hif-p4h-2 led to HIF stabilization and dysregulation of multiple genes involved in keratin formation, HF differentiation, and HIF, transforming growth factor β (TGF-β), and Notch signaling. We hypothesize that the failure of HF cycling is likely to be mechanistically caused by disruption of the interplay of the HIF, TGF-β, and Notch pathways. In summary, we show here for the first time that HIF-P4H-2 function in FoxD1-lineage cells is essential for the normal development and homeostasis of HFs

Contribution of HIF-P4H isoenzyme inhibition to metabolism indicates major beneficial effects being conveyed by HIF-P4H-2 antagonism

Abstract Hypoxia-inducible factor (HIF) prolyl 4-hydroxylases (HIF-P4Hs 1–3) are druggable targets in renal anemia, where pan-HIF-P4H inhibitors induce an erythropoietic response. Preclinical data suggest that HIF-P4Hs could also be therapeutic targets for treating metabolic dysfunction, although the contributions of HIF-P4H isoenzymes in various tissues to the metabolic phenotype are inadequately understood. Here, we used mouse lines that were gene-deficient for HIF-P4Hs 1 to 3 and two preclinical pan-HIF-P4H inhibitors to study the contributions of these isoenzymes to the anthropometric and metabolic outcome and HIF response. We show both inhibitors induced a HIF response in wildtype white adipose tissue (WAT), liver, and skeletal muscle and alleviated metabolic dysfunction during a 6-week treatment period, but they did not alter healthy metabolism. Our data indicate that HIF-P4H-1 contributed especially to skeletal muscle and WAT metabolism and that its loss lowered body weight and serum cholesterol levels upon aging. In addition, we found HIF-P4H-3 had effects on the liver and WAT and its loss increased body weight, adiposity, liver weight and triglyceride levels, WAT inflammation, and cholesterol levels and resulted in hyperglycemia and insulin resistance, especially during aging. Finally, we demonstrate HIF-P4H-2 affected all tissues studied; its inhibition lowered body and liver weight and serum cholesterol levels and improved glucose tolerance. We found very few HIF target metabolic mRNAs were regulated by the inhibition of three isoenzymes, thus suggesting a potential for selective therapeutic tractability. Altogether, these data provide specifications for the future development of HIF-P4H inhibitors for the treatment of metabolic diseases

Suppression of tumor-associated neutrophils by lorlatinib attenuates pancreatic cancer growth and improves treatment with immune checkpoint blockade

Pancreatic ductal adenocarcinoma (PDAC) patients have a 5-year survival rate of only 8% largely due to late diagnosis and insufficient therapeutic options. Neutrophils are among the most abundant immune cell type within the PDAC tumor microenvironment (TME), and are associated with a poor clinical prognosis. However, despite recent advances in understanding neutrophil biology in cancer, therapies targeting tumor-associated neutrophils are lacking. Here, we demonstrate, using pre-clinical mouse models of PDAC, that lorlatinib attenuates PDAC progression by suppressing neutrophil development and mobilization, and by modulating tumor-promoting neutrophil functions within the TME. When combined, lorlatinib also improves the response to anti-PD-1 blockade resulting in more activated CD8 + T cells in PDAC tumors. In summary, this study identifies an effect of lorlatinib in modulating tumor-associated neutrophils, and demonstrates the potential of lorlatinib to treat PDAC