Targeted therapy for capillary-venous malformations

Abstract

Sporadic venous malformations are genetic conditions primarily caused by somatic gain-of-function mutation of PIK3CA or TEK, an endothelial transmembrane receptor signaling through PIK3CA. Venous malformations are associated with pain, bleedings, thrombosis, pulmonary embolism, esthetic deformities and, in severe cases, life-threatening situations. No authorized medical treatment exists for patients with venous malformations. Here, we created a genetic mouse model of PIK3CA-related capillary venous malformations that replicates patient phenotypes. We showed that these malformations only partially signal through AKT proteins. We compared the efficacy of different drugs, including rapamycin, a mTORC1 inhibitor, miransertib, an AKT inhibitor and alpelisib, a PI3Kα inhibitor at improving the lesions seen in the mouse model. We demonstrated the effectiveness of alpelisib in preventing vascular malformations' occurrence, improving the already established ones, and prolonging survival. Considering these findings, we were authorized to treat 25 patients with alpelisib, including 7 children displaying PIK3CA (n = 16) or TEK (n = 9)-related capillary venous malformations resistant to usual therapies including sirolimus, debulking surgical procedures or percutaneous sclerotherapies. We assessed the volume of vascular malformations using magnetic resonance imaging (MRI) for each patient. Alpelisib demonstrated improvement in all 25 patients. Vascular malformations previously considered intractable were reduced and clinical symptoms were attenuated. MRI showed a decrease of 33.4% and 27.8% in the median volume of PIK3CA and TEK malformations respectively, over 6 months on alpelisib. In conclusion, this study supports PI3Kα inhibition as a promising therapeutic strategy in patients with PIK3CA or TEK-related capillary venous malformations.

Fig. 1

A mouse model of PIK3CA-related capillary venous malformations. a Representative photography of PIK3CA^WT and PIK3CA^Tie2-CreER mice 4 weeks after Cre recombination. b Kaplan–Meier survival curves of PIK3CA^WT and PIK3CA^Tie2-CreER mice (n = 25 per group). c Coronal whole-body T2-weighted fat saturated magnetic resonance images (MRI) of PIK3CA^WT and PIK3CA^Tie2-CreER mice (n = 4–6 mice per group) 5 weeks after Cre recombination. Volumetric quantification of the vascular malformations. d Representative hematoxylin and eosin (H&E) staining of the skin of PIK3CA^WT and PIK3CA^Tie2-CreER mice and patient with venous malformation (VM). Large irregular and dilated vessels filled with red blood cells were visible in the skin of PIK3CA^Tie2-CreER mice. Scale bar: 40 μm. e Representative coimmunofluorescence staining of Tie2 and GFP in the skin of PIK3CA^WT and PIK3CA^Tie2-CreER mice. Scale bar: 10 μm. f GFP is express in CD31+ cells as assessed by flow cytometry experiments of cells isolated from the skin of PIK3CA^WT (n = 4 mice). g GFP and podoplanin staining using flow cytometry experiments of cells isolated from the skin of PIK3CA^WT (n = 4 mice). h Representative immunofluorescence staining of P-AKT^Thr308 and P-S6RP in the skin of PIK3CA^WT and PIK3CA^Tie2-CreER mice. Scale bar: 10 μm. i Western blot and j quantification of P-AKT^Ser473 and P-S6RP in the skin of PIK3CA^WT and PIK3CA^Tie2-CreER mice (n = 5–8 mice per group). k Flow cytometry experiments showing the percentage of GFP + CD31+ cells isolated from the skin of PIK3CA^WT and PIK3CA^Tie2-CreER mice expressing P-AKT^Ser473 (n = 6–7 mice per group). l Representative immunofluorescence staining of KI67 in the skin of PIK3CA^WT and PIK3CA^Tie2-CreER mice. Scale bar: 10 μm and m quantification (n = 4 mice per group). n Flow cytometry experiments showing the percentage of GFP + CD31+ cells isolated from the skin of PIK3CA^WT and PIK3CA^Tie2-CreER mice expressing KI67 (n = 3–4 mice per group). o Flow cytometry experiments showing the percentage of Tomato+ cells isolated from the skin of PIK3CA^WT and PIK3CA^Tie2-CreER mice expressing KI67 (n = 3–4 mice per group). p Quantification of GFP positive cell surface isolated from PIK3CA^WT and PIK3CA^Tie2-CreER mice (n = 5 mice per group). q Complete blood count and D-Dimers measurement in PIK3CA^WT and PIK3CA^Tie2-CreER mice (n = 11 per group)

Fig. 2

PIK3CA-related capillary venous malformations in PIK3CA^Tie2-CreER mice only partially signal through AKT proteins. a Kaplan–Meier survival curves of PIK3CA^WT, PIK3CA^Tie2-CreER, PIK3CA^AKT1KO, PIK3CA^AKT2KO and PIK3CA^AKT1AKT2-KO mice (n = 20 per group). b Coronal whole-body T2 weighted magnetic resonance images (MRI) of PIK3CA^WT, PIK3CA^Tie2-CreER, PIK3CA^AKT1KO, PIK3CA^AKT2KO and PIK3CA^AKT1AKT2-KO mice 6 weeks after Cre recombination. Volumetric quantification of the vascular malformations (n = 4–6 mice per group). c Representative hematoxylin and eosin (H&E) staining of the skin of PIK3CA^WT, PIK3CA^Tie2-CreER, PIK3CA^AKT1KO, PIK3CA^AKT2KO and PIK3CA^AKT1AKT2-KO mice. Scale bar: 10 μm. d Representative P-AKT^Ser473 in the skin of PIK3CA^WT, PIK3CA^Tie2-CreER, PIK3CA^AKT1KO, PIK3CA^AKT2KO and PIK3CA^AKT1AKT2-KO mice. Scale bar: 10μm.*: Vascular malformation. e Western blot and quantification of P-AKT^Ser473 and P-S6RP in the skin of PIK3CA^WT, PIK3CA^Tie2-CreER, PIK3CA^AKT1KO, PIK3CA^AKT2KO and PIK3CA^AKT1AKT2-KO mice (n = 4–5 mice per group). f Representative immunofluorescence staining of KI67 in the skin of PIK3CA^WT, PIK3CA^Tie2-CreER, PIK3CA^AKT1KO, PIK3CA^AKT2KO and PIK3CA^AKT1AKT2-KO mice. Scale bar: 10 μm. g Flow cytometry experiments showing the percentage of GFP + CD31+ cells isolated from the skin of the different mouse models expressing KI67 (n = 3–4 mice per group). (n = 3–7 mice per group). h Western blot and quantification of AKT1 and AKT2 in vessels of PIK3CA^WT, PIK3CA^Tie2-CreER and PIK3CA^AKT1AKT2-KO mice (n = 3–4 mice per group)

Fig. 3

Targeted therapies for capillary venous malformations in PIK3CA^Tie2-CreER mice. a Kaplan–Meier survival curves of PIK3CA^WT and PIK3CA^Tie2-CreER mice treated with either vehicle, rapamycin, miransertib or alpelisib (n = 12 per group). b Coronal whole-body T2-weighted magnetic resonance images (MRI) of PIK3CA^WT and PIK3CA^Tie2-CreER mice 6 weeks after Cre recombination treated with either vehicle, rapamycin, miransertib or alpelisib. Volumetric quantification of the vascular malformations (n = 3-4 mice per group). c Complete blood count in PIK3CA^WT and PIK3CA^Tie2-CreER mice treated with either vehicle, rapamycin, miransertib or alpelisib (n = 3–5 mice per group). d Representative hematoxylin and eosin (H&E) staining of the skin of PIK3CA^WT and PIK3CA^Tie2-CreER mice treated with either vehicle, rapamycin, miransertib or alpelisib. Scale bar: 10 μm. e Representative P-AKT^Thr308 and P-S6RP immunostaining in the skin of PIK3CA^WTand PIK3CA^Tie2-CreER mice treated with either vehicle, rapamycin, miransertib or alpelisib. Scale bar: 10 μm. f Western blot and quantification of P-AKT^Ser473 and P-S6RP of the skin of PIK3CA^WTand PIK3CA^Tie2-CreER mice treated with either vehicle, rapamycin, miransertib or alpelisib (n = 3–4 per group). g Representative immunofluorescence staining of KI67 in the skin of PIK3CA^WTand PIK3CA^Tie2-CreER mice treated with either vehicle, rapamycin, miransertib or alpelisib. Scale bar: 10 μm. h Proliferation index quantification (n = 3–4 mice per group)

Fig. 4

Alpelisib improves and prevent capillary venous malformations in PIK3CA^Tie2-CreER mice. a Kaplan–Meier survival curves of PIK3CA^WT and PIK3CA^Tie2-CreER mice treated with either vehicle or preventive alpelisib (n = 12 per group). b Coronal whole-body T2-weighted magnetic resonance images (MRI) of PIK3CA^WT and PIK3CA^Tie2-CreER mice 6 weeks after Cre recombination treated with either vehicle, preventive or therapeutic alpelisib (n = 4–6 mice per group). Volumetric quantification of the vascular malformations. c Representative hematoxylin and eosin (H&E) staining of the skin of PIK3CA^WT and PIK3CA^Tie2-CreER mice treated with either vehicle, preventive or therapeutic alpelisib. Scale bar: 10 μm. d Representative P-AKT^Thr308 and P-S6RP immunostaining in the skin of PIK3CA^WTand PIK3CA^Tie2-CreER mice treated with either vehicle, preventive or therapeutic alpelisib. Scale bar: 10 μm. e Western blot and quantification of P-AKT^Ser473 and P-S6RP in skin of PIK3CA^WTand PIK3CA^Tie2-CreER mice treated with either vehicle, preventive or therapeutic alpelisib (n = 6–7 mice per group). f Complete blood count of PIK3CA^WTand PIK3CA^Tie2-CreER mice treated with either vehicle, preventive or therapeutic alpelisib (n = 5–10 mice). g Representative photography of PIK3CA^Tie2-CreER mice before and two weeks after alpelisib initiation. h Kaplan–Meier survival curves of PIK3CA^WT and PIK3CA^Tie2-CreER mice treated with either vehicle or therapeutic alpelisib (n = 12 per group)

Fig. 5

Alpelisib improves patients with PIK3CA or TEK-related capillary venous malformations. a Representative Hematoxylin and eosin (H&E) staining and b Immunofluorescence of P-AKT^Thr308 and P-S6RP in skin biopsies performed in controls and in patients with PIK3CA-related capillary venous malformation. Scale bar: 10 μm. c Immunofluorescence quantification (n = 6 controls and 6 patients). AU Arbitrary units. d Representative photographs of the morphological changes observed in patients with PIK3CA-related capillary venous malformations receiving alpelisib for 6 months. e Representative photographs of the morphological changes observed in patients with TEK-related capillary venous malformations receiving alpelisib for 6 months. f Transversal (upper panel) and coronal (lower panel) T2-weighted fat saturated MRI sequence of patient 1 and 5 before and after alpelisib introduction. In red, segmentation in 2D (left panel) and 3D (right panel). g Percentage change of the volume of the preselected lesion in patients with PIK3CA-related capillary venous malformations (left panel) and TEK-related capillary venous malformations (right panel)