Publications

Abstract The vascular endothelium is characterized by a remarkable level of plasticity, which is the driving force not only of physiological repair/remodeling of adult tissues but also of pathological angiogenesis. The resulting heterogeneity of endothelial cells (ECs) makes targeting the endothelium challenging, no less because many EC phenotypes are yet to be identified and functionally inventorized. Efforts to map the vasculature at the single-cell level have been instrumental to capture the diversity of EC types and states at a remarkable depth in both normal and pathological states. Here, we discuss new EC subtypes and functions emerging from recent single-cell studies in health and disease. Interestingly, such studies revealed distinct metabolic gene signatures in different EC phenotypes, which deserve further consideration for therapy. We highlight how this metabolic targeting strategy could potentially be used to promote (for tissue repair) or block (in tumor) angiogenesis in a tissue or even vascular bed-specific manner.

<p>Antibodies used in this study.</p>

Abstract BCR-ABL1 inhibitors have revolutionized the treatment of CML patients. However several drawbacks remain, including clinical resistance of T315I-mutated CML. Further, the clinical success of ponatinib, a selective inhibitor of T315I-mutated BCR-ABL1 is hampered by vascular side effects. Therefore, novel treatment strategies are warranted especially in T315I-mutated CML. We investigated the relevance of the Gas6-Axl axis in CML patients and the therapeutic potential of the clinically applicable small molecule Axl inhibitor BGB324 in primary CML (stem cell) samples, cell lines and preclinical models. We previously found that Gas6 and Axl represent potential novel targets in this disease and that BGB324 inhibits CML growth in vitro and in vivo (Erdmann et al., ASH meeting 2013, New Orleans, #1469). We next wished to confirm Axl as specific therapeutic target and therefore down regulated its expression in KCL-22 and K562 cells by means of shRNA. In these experiments blockade of Axl inhibited CML cell proliferation in comparison to control-transduced cells, thereby confirming that Axl promotes CML growth. Subsequently, we added BGB324 to shAxl- and shcontrol-transduced cells. Surprisingly BGB324 inhibited cell growth significantly more in shAxl-transduced cells in comparison to control-treated shAxl-transduced cells (p<0.05). These experiments indicated that BGB324 was inhibiting an additional target which supported CML cell proliferation, besides Axl. In order to identify this target we carried out a Kinome Scan revealing that BGB324 binds to native and mutated ABL1. Interestingly, the affinity of BGB324 for ABL1 carrying different mutations including T315I was 5 to 50 fold higher compared to unmutated ABL1 (Table 1). Next, we incubated BaF3 cells stably transfected with BCR-ABL1p210, BCR-ABL1T315I, BCR-ABL1M351T and BCR-ABL1E255K with various concentrations of BGB324 in order to determine its IC50 in the different cell lines. These experiments showed in concordance with the Kinome Scan that BGB324 was more potently inhibiting growth of mutated BCR-ABL1 compared to BCR-ABL1p210 (IC50 BCR-ABL1p210 1266 ± 126 nM; BCR-ABL1T315I 726 ± 194 nM; BCR-ABL1M351T 847 ± 10 nM and BCR-ABL1E255K 794 ± 39 nM; n=2-3; p<0.05 compared to BCR-ABL1p210). Notably, further experiments revealed that BGB324 inhibited KCL-22 cells and K526 cells to a similar extent compared to the combination of imatinib (IM) and shAxl. Thus, BGB324 is a dual inhibitor of BCR-ABL1 and Axl. As the inhibition of BCR-ABL1T315I is of special clinical interest we wished to confirm this finding further in vivo. Therefore we inoculated BCR-ABL1p210 and BCR-ABL1T315I cells subcutaneously into NSG mice. After the tumors reached a size of 80-100 mm3 mice were randomized to receive either placebo control or 50 mg/kg BGB324 delivered twice daily by oral gavage. This experiment showed potent inhibition of tumor growth after 12 days with higher activity of BGB324 in mice bearing BCR-ABL1T315I tumors (placebo: 1751 ± 606 mm3, BGB324: 614 ± 224 mm3; p=0.001) compared to mice bearing BCR-ABL1p210 tumors (placebo: 1432 ± 403 mm3; BGB324: 632 ± 229 mm3; p=0.05) (Figure 1). Subsequently, tissue harvested at end-stage was subjected to immunohistochemical staining for the proliferation marker phospho-histone H3 and Western Blot analyses of cleaved caspase 3 in order to determine whether reduced proliferation and/or increased apoptosis was responsible for reduced growth of BCR-ABL1T315I tumors upon treatment with BGB324. These analyses revealed that proliferation as determined by histomorphometric analysis of phosho-histone H3 was reduced while cleaved caspase 3 levels were unchanged. These data were further corroborated by the finding that treatment with BGB324 reduced the level of phosphorylated MapK as determined by immunoblotting and densitometry. Thus, BGB324 inhibits proliferation of BCR-ABL1T315I cells in vivo. Altogether, our findings show that BGB324 represents a dual inhibitor of Axl and ABL kinase with therapeutic potential in CML, in particular in BCR-ABL1T315I disease. As BGB324 was shown to be well tolerated in healthy volunteers (Wnuk-Lipinska et al., AACR meeting 2013 San Diego #1747), our findings pave the way for clinical investigation of BGB324 in (T315I-mutated) CML. Table 1 Gene KinomeScan Kd (nM) KinaseProfiler IC50 (nM) Axl 0.4 3 ABL1 51.88 51 ABL1(E255K) 1.15 n/a ABL1(T315I) 10.13 4 ABL1(Y253F) 18.19 26 Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Loges: BerGenBio: Research Funding, travel support, advisory boards Other.

Summary Mice with combined homozygous deficiency of tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) (T‾U‾), of t-PA and plasminogen activator inhibitor-1 (PAI-1) (T‾P‾), of u-PA and PAI-1 (U‾P‾) or of t-PA, u-PA, and PAI-1 (T‾U‾P‾) were generated by inbreeding of mice with the respective deficiencies. Homologous recombination at the t-PA, u-PA and PAI-1 locus was verified by Southern blot analysis of genomic tail tip DNA, and confirmed by measurement of antigen levels in plasma or urine. T‾P‾ and U‾P‾ mice were apparently healthy and fertile. T‾U‾ mice showed extensive fibrin deposition with calcification in the liver, whereas T‾U‾P‾ mice were significantly (p <0.001) less affected. Spontaneous in vivo clot lysis measured 4 h after injection of a 125I-fibrin-labeled clot prepared from plasma of wild-type (WT) mice into the jugular vein, was (mean ± SEM of n experiments) 2 ± 1% (n = 8) for T P , 49 ± 6% (n = 9) for U‾P‾, 1 ± 1% (n = 4) for T‾U‾ and 3 ± 3% (n = 3) for TU‾P‾ mice, as compared to 32 ± 4% (n = 10) for WT, 1 ± 0% (n = 7) for T‾, 30 ± 5% (n = 5) for U‾ and 58 ± 10% (n = 6) for P‾ mice. Plasminogen-dependent lysis of 125I-fibrin-labeled matrix and of 3H-proline-labeled subendothelial matrix (mean ± SEM; n = 4 to 6) was lower with thioglycollate-stimulated macrophages obtained from U‾P‾ mice (22 ± 7% and 5 ± 1%, respectively), as compared to WT mice (57 ± 14% and 18 ± 5%, respectively) and T‾P‾ mice (87 ± 6% and 27 ± 4%, respectively). A similar decrease was previously observed with U‾ mice, but not with T‾ or P‾ mice. Thus, the phenotype of mice with combined deficiency of t-PA and PAI-1 or of u-PA and PAI-1 is similar to the phenotype observed in mice with single deficiency of the plasminogen activator. Additional deletion of PAI-1 does not affect viability, fertility, macrophage function or thrombolytic potential of the single deficient mice. Additional deletion of PAI in mice with combined deficiency of t-PA and u-PA does not restore the deficient in vivo fibrinolytic capacity, but significantly reduces the thrombotic phenotype, as revealed by fewer, smaller and less calcified fibrin deposits in the liver.

A new diabetic mouse strain, the Akita.PlGF knockout (−/−), was generated to study the role of placental growth factor (PlGF) in the pathogenesis of diabetic retinopathy (DR). PlGF deletion did not affect blood glucose but reduced the body weight of Akita.PlGF−/− mice. Diabetes-induced retinal cell death, capillary degeneration, pericyte loss, and blood-retinal barrier breakdown were prevented in these mice. Protein expression of PlGF was upregulated by diabetes, particularly in vascular cells. Diabetes-induced degradation of ZO-1 and VE-cadherin was reversed due to PlGF deficiency; their expression was correlated with that of sonic hedgehog and angiopoietin-1. PlGF deletion in Akita mice resulted in an increased Akt phosphorylation. Diabetes-activated hypoxia-inducible factor (HIF)1α–vascular endothelial growth factor (VEGF) pathway, including expression of HIF1α, VEGF, VEGFR1–3, and the extent of phospho (p)-VEGFR1, p-VEGFR2, and p–endothelial nitric oxide synthase, was inhibited in the retinas of diabetic PlGF−/− mice. However, expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, CD11b, and CD18 was not inhibited by PlGF deletion, nor was retinal leukostasis. These results suggest that PlGF is critical for the development of DR, and its genetic deletion protects the retina from diabetic damage. Protective mechanisms are associated with Akt activation and HIF1α-VEGF pathway inhibition, but independent of retinal leukostasis in the retinas of diabetic PlGF−/− mice.