WO2024112656A1 - Use of a tyrosine kinase inhibitor for the treatment of hereditary hemorrhagic telangiectasia and pulmonary arterial hypertension - Google Patents

Use of a tyrosine kinase inhibitor for the treatment of hereditary hemorrhagic telangiectasia and pulmonary arterial hypertension Download PDF

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WO2024112656A1
WO2024112656A1 PCT/US2023/080515 US2023080515W WO2024112656A1 WO 2024112656 A1 WO2024112656 A1 WO 2024112656A1 US 2023080515 W US2023080515 W US 2023080515W WO 2024112656 A1 WO2024112656 A1 WO 2024112656A1
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genes
hht
expression
cells
gene
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Md Khadem ALI
Adam M. ANDRUSKA
Edda SPIEKERKOETTER
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Leland Stanford Junior University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4985—Pyrazines or piperazines ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/53—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with three nitrogens as the only ring hetero atoms, e.g. chlorazanil, melamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems

Definitions

  • PTKs Protein Tyrosine Kinases
  • VEGFR receptor
  • cytoplasmic e.g. SRC
  • Receptor-PTKs enable intercellular ligand-based communication whereas cytoplasmic-PTKs amplify external and internal signals and control functions like proliferation and motility.
  • Many non-receptor PTKs are oncogenes (e.g. BCR-ABL) which has driven the development of Tyrosine Kinase Inhibitors (TKIs).
  • TKIs are now approved for conditions ranging from cancer to pulmonary fibrosis. Though successful in treating disease, TKIs have mixed effects in pulmonary vascular biology.
  • Pulmonary arterial hypertension is a rare disease characterized by progressive and fatal obliterative vasculopathy of the small pulmonary due to endothelial cell dysfunction (ECD) and apoptosis in concert with smooth muscle cell proliferation and perivascular inflammation.
  • ECD endothelial cell dysfunction
  • This occlusive vasculopathy ultimately leads to an increased pulmonary vascular resistance, right heart failure and death.
  • the TKI Imatinib suppresses smooth muscle cell proliferation and improves clinical PAH, likely by acting on the PTKs PDGFR and c-Kit.
  • TKIs like Dasatinib, Bosutinib, Ponatinib, and Lorlatinib are either known causes of PAH or are implicated in PAH development.
  • Dasatinib may achieve this by acting on additional PTKs outside of PDGFR and c-Kit, induction of reactive oxygen species (ROS) in pulmonary artery endothelial cells (PAECs), or impairing endothelial cell (EC) barrier permeability in a Rho-kinase dependent manner.
  • ROS reactive oxygen species
  • PAECs pulmonary artery endothelial cells
  • EC impairing endothelial cell
  • HHT Hereditary Hemorrhagic Telangiectasia
  • ENG ENG
  • ACVRL1/ALK1 or SMAD4 a rare disease characterized by loss of function mutations in ENG, ACVRL1/ALK1 or SMAD4 resulting in the formation of arteriovenous malformations in the lung, brain, liver, gastrointestinal tract, nasal mucosa, and skin putting patients at risk for paradoxic emboli and stroke, pulmonary hemorrhage, cerebral bleeding, high output cardiac failure, severe anemia and nosebleeds as well as telangiectasia.
  • a goal is to identify drugs that correct the downstream signaling abnormalities of these loss of function mutations present in HHT and PAH and thereby prevent, halt or reverse the disease.
  • compositions and methods are provided for the treatment of Hereditary Hemorrhagic Telangiectasia (HHT) and Pulmonary Arterial Aypertension (PAH) by administration of an effective dose of a protein tyrosine kinase (PTK) inhibitor.
  • PTK protein tyrosine kinase
  • the inhibitor acts on vascular endothelial growth factor receptor-2 (VEGFR-2).
  • the inhibitor acts of VEGFR-2 and fibroblast growth factor receptor-1 (FGFR-1 ).
  • the inhibitor acts on FLT3, PDGFR, and VEGFR2.
  • the inhibitor is Brivanib.
  • the inhibitor is Dabrafenib or Linsitinib.
  • an individual with HHT is treated by administration of an effective dose of an agent that inhibits at least VEGFR-2, and may additionally inhibit FGFR-1 , where the treatment improves clinical indicia of the disease.
  • the agent is Brivanib. It is shown herein that Brivanib can reversed an HHT disease signature of gene expression, and can inhibit othere disease features.
  • the agent is dabrafenib.
  • the agent is linsitinib.
  • the treatment of HHT can normalize gene expression in endothelial cells in which HHT causing genes are knocked down, e.g. the treatment is capable of reducing expression of one or more of the genes LYVE1 , GPNMB, RRAGD and ANKRD33; and/or increasing expression of one or more of the genes MC5R, SLC25A47, FGF19, SHISA9, FRG2C, and HS3ST2.
  • the treatment can inhibit VEGF-induced ERK1/2 phosphorylation, proliferation, and angiogenesis in the targeted cells, e.g.
  • lung microvessel endothelial cells and also inhibit the expression of one or more of the genes upregulated in the HHT disease condition: LYVE1 , GPNMB, and HS3ST2 in the targeted tissues, e.g. lung tissue.
  • the PTK can inhibit an inflammatory gene expression signature disclosed herein.
  • the individual is monitored for efficacy of treatment, e.g. by determining an inflammatory signature change, determining changes in an HHT disease signature, changes in gene expression, and the like.
  • an individual with PAH is treated by administration of an effective dose of an agent that increases ID1 expression, a common downstream target of the BMPR2 pathway, where the treatment improves clinical indicia of the disease.
  • the agent is Brivanib.
  • the inhibitor is Dabrafenib or Linsitinib.
  • the treatment can provide for an pro-endothelial-like pattern of gene expression in targeted cells, in contrast to an inflammatory pattern of gene expression.
  • the individual is monitored for efficacy of treatment, e.g. by determining a pro-endothelial expression change, changes in Id1 gene expression, and the like. .
  • the inhibitor increases Id1 expression.
  • the inhibitor activates the endothelial transcriptomic signature.
  • FIG. 1 A-1 is not a common downstream target of ENG in PMVECs.
  • FIG. 2A-2E Common downstream gene signatures of ALK1 , ENG and SMAD4 mutations enriched for AVM and HHT related biological processes and cell signaling pathways.
  • a and B Venn diagram of common upregulated or downregulated genes following knockdown of ALK1 , ENG and SMAD4 in PMVECs stimulated with BMP9 (20ng/ml) for 2 or 24hrs (RNAseq).
  • C Heatmap of the common upregulated and downregulated genes following knockdown of ALK1 , ENG and SMAD4 in PMVECs stimulated with BMP9 for 2 and 24hrs.
  • FIG. 3A-3C Identifying common persistently dysregulated downstream gene signatures of the HHT gene knockdowns in PMVECs.
  • FIG. 4A-4K Drug prediction based on the common upregulated downstream targets after ALK1 , ENG and SMAD4 knockdown and experimental validation in PMVECs.
  • C-K Effect of Brivanib on the expression of the common persistent downstream targets after ALK1 , ENG and SMAD4 knockdown was assessed by qRT-PCR in PMVECs.
  • FIG. 5A-5F Effect of Brivanib on expression of upregulated disease signature genes in HHT in ex vivo healthy human PCLS.
  • FIG. 6A-6D Brivanib inhibits VEGF-induced pERK1/2, -proliferation, and angiogenesis in PMVECs.
  • B) The effect of Brivanib on VEGF-induced cell proliferation was assessed by MTT assay in PMVECs.
  • Angiogenesis was assessed in a Matrigel tube formation assay using PMVECs treated with either Brivanib 1 , 10, 50 uM or DMSO.
  • Figure 7A-7C Functional consequence of ALK1 , ENG, and SMAD4 knockdown in PMVECs (proliferation, apoptosis and tube formation).
  • Figure 8A-8B STRING network analysis of the common upregulated and downregulated genes following knockdown of ALK1 , ENG and SMAD4 in PMVECs stimulated
  • Figure 9A-9G Validation of common upregulated or downregulated genes signatures with BMP9 stimulation at 2 (A and B) or 24 hrs (C and D) and functional consequences of the knockdown of LYVE1 , GPNMB and MC5R in PMVECs. After 72 hrs knockdown of LYVE1 , MC5R, and GPNMB PMVECs proliferation (E), apoptosis (F), tube formation (G) were assessed.
  • FIG. 10A-10C Effect of Brivanib on the expression of the common persistent downstream targets after ALK1 , ENG and SMAD4 knockdown were assessed by qRT-PCR in PMVECs (A-C).
  • FIG. 11 A-1 1 E A High-Throughput Screen (HTS) using a C2C12 mouse myoblastoma ZcZ1-Luciferase Reporter cell lines finds Src-Family A PTKs are inherently BMPR2-Repressive, and Src-Family B PTKs are inherently BMPR2-Activating.
  • A HTS treatment and assessment strategy.
  • Figure 12A-12E Lek is expressed in the endothelium of PAECs and correlates with Id1 expression. SC-RNAseq of human PBMCs suggests Bmpr2 and Id1 is low in lymphocytes that have high expression of Lek or Fyn.
  • A smFISH in human lung for Lek mRNA (white), VE-Cadherin (magenta, EC Marker), Acta2 (green, Smooth Muscle Marker), and DAPI (blue, nuclear marker).
  • (B) Perinuclear Lek expression in human PAECs. Bar 50 gm.
  • C-D Immunoblotting of protein lysate from human PAECs from explanted lungs of donor controls and PAH patients showing correlation between Lek and Id 1 expression.
  • E SC-RNAseq analysis of 2700 PBMCs from a healthy donor (full analysis Sup Figure 1 ) using the Seurat SC-RNA seq pipeline. Lek and Fyn are highly expressed in Cd4+ and Cd8+ T-Cells, and NK Cells. However, BMPR2 and Id1 are highly expressed in Dendritic Cells.
  • Id2 rather than Id1 or Id3 is highly expressed in lymphocytes, NK cells, and dendritic cells. Dot size indicates percent of cells expressing gene of interest (cutoff of >1 count per cell). Average Expression is log-scaled Z-score of counts for each gene in each cluster.
  • FIG. 3A-13E Silencing of the SrcB LCK in human PAECs suppresses canonical BMPR2 signaling, whereas silencing the SrcA FYN supports it.
  • (A) Human PAECs transfected with LCK targeting (siLCK) or control (siNT) siRNA and lysed at 72 hours in full media (2% FBS) have decreased Lek, ld1, and Gapdh by RT-qPCR using the relative standard method. Snail was significantly increased. 1 -Way ANOVA with Tukey’s Rage Test, n 4.
  • siLCK PAECs have lower LCK expression at 72h.
  • SiLCK PAECs have significantly decreased BMP9 mediated phosopho-SMAD1 and Id1 expression.
  • N 6, 2-way ANOVA for treatment (PBS, TGF-pi and BMP) and transfection (siNT vs. siLCK) by Tukey’s Range Test.
  • siFYN PAECs maintained in full media (2% FBS), changed to starvation media (0.2% FBS) 24 hours after transfection, or changed to starvation media then given BMP9 or PBS 1 .5 hours prior to cell lysis. Fyn was significantly reduced at 72 hours. There was no change in Phospho- SMAD1 activity (pSMADI ). There was significant reduction in phospho-SMAD3 in siFYN, BMP9-treated cells and a significant increase in Id 1 . 2-way ANOVA examining treatment (2% FBS, 0.2% FBS + PBS, 0.2% FBS+BMP9) and transfection (si-NT vs. si-Fyn) followed by Tukey’s Range Test. For all above ANOVA tests, normality checks for ANOVA residuals and Levene’s test for homogeneity were carried out and assumptions are met.
  • FIG. 14A-14F Inhibition of the SrcB Lek promotes EC dysfunction, whereas inhibition of the SrcA Fyn does not.
  • siLCK and siFYN PAECs are compared to matching control conditions (termed si-NT-L or si-NT-F). Cells were passaged to Matrigel in full media (2% FBS) 48 hours after transfection and imaged 3 hours later.
  • A Raw (left) and analyzed (right) brightfield images are shown.
  • C siLCK PAEC cell counts in culture decrease over time.
  • Figure 15A-15H Whole-transcriptome analysis finds Lek knockout suppresses and Fyn knockout promotes an “endothelial” gene signature (including the BMPR2 pathway). Conversely, Lek knockout increases and Fyn knockout suppresses an “inflammatory” gene program centered on NF-KB / RelA signaling.
  • A DEGs in siLCK and siFYN PAECs.
  • B Normalization of raw counts by variance stabilizing transformation with unsupervised hierarchical clustering and heatmap generation. Genes increased in siFYN and BMP9 and decreased in siLCK correlate with canonical BMPR2 signaling (e.g. Bmpr2 and Id1).
  • Genes increased by siLCK and decreased in siFYN or BMP9 include inflammatory genes Cxcl3, 11-6, Ccl8, Vcaml, and Icaml.
  • C Selected genes important in EC biology. siLCK PAECs have reduced markers of EC identity, increased markers of EC dysfunction, and favor NF-KB over BMPR2 canonical signaling.
  • D DEGs co-regulated in siLCK and siFYN. 671 genes increase in siLCK and decrease in siFYN (Group E, “Inflammatory” signature).
  • FIG. 16A-16G Using endothelial and inflammatory signatures, CMap searches validate ligands and enable drug prediction in specific cell lines.
  • A Search strategy for perturbagens in the CMap data.
  • FDR false discovery rate
  • CS Ligands with connectivity scores (CS) matching the inflammatory and endothelial signatures across select cell lines are shown. Red indicates ligands of interest.
  • FIG. 17A-17D Brivanib and Quizartinib increase and decrease PAEC Id1 expression, whereas Regorafanib increases PAEC NF-KB activity.
  • A PAECs treated with drug or DMSO for 24 hours then given BMP9 or PBS 2 hours prior to harvest significantly increase Id1 expression with Brivanib alone or Brivanib plus BMP9 versus DMSO.
  • Quizartinib decreases Id1 in all cases.
  • Statistics by 2-way ANOVA investigating the interaction between drug and BMP9 treatment with Tukey’s Range Test, n 3.
  • Figures 18A-18C (A) UMAP plot of all 2700 human PBCMs after unsupervised clustering by the Seurat single cell RNA seq pipeline. Nine distinct clusters are identified. Clusters have been annotated for cell identity based on the gene expression detailed in (2 B- C). (B) Dot plots showing expression of canonical genes known to be expressed in specific cell types which informs cluster identity.
  • Cd3 denotes T-cells
  • Cd8 indicates CD8+ T-Cells
  • Klrdl denotes natural killer cells
  • Cd19 and Ms4a1 denotes B-Cells
  • Itgae, HLA-CQA1, and Itgax denote dendritic cells
  • Fcgr3a denotes monocytes
  • Pf4 indicates platelets
  • Cd14 denotes monocytes.
  • Dot size indicates percent expression of the gene (the number of cells in the cluster that have > 1 counts of a given gene). Average expression is the Z-score of the log-normalized counts of a gen gene within the cluster.
  • C Feature plots of all genes of interested examined in PBMCs for correlation with the clusters of A.
  • FIGS 19A-19J (A) Knockout of Lek in PAECs suppresses canonical BMPR2 signaling in different media conditions.
  • PAECs were transfected with siRNA targeting Lek (si- Lck) or a nontargeting siRNA (siNT) for 6 hours. 24 hours after transfection, the cells were either starved (0.2% FBS) or kept in full media (2% FBS). 1.5 hours prior to cell lysis, the starvation cells were either treated with PBS or 20 ng/mL of BMP9. Cell lysate was run on SDS-PAGE.
  • B-D Phospho-SMAD1 was significantly upregulated by BMP9 treatment and was significantly reduced in the si-Lck group.
  • Id1 was significantly reduced by siLck in both the full media condition (2% FBS) and the BMP9 case.
  • 2-Way ANOVA for the interaction between treatment (Full Media, Starvation Media, Starvation Media + BMP9) and transfection (si-Lck and si-NT) with Tukey’s Range Test was used to determine significance. Normality checks for ANOVA residuals and Levene’s test for homogeneity were carried out and assumptions met.
  • E Knockout of FYN in full media conditions increases phospho-SMAD 1 activity even in the setting of TGF-p treatment in full media conditions.
  • PAECs were transfected with siRNA targeting either Fyn (si-Fyn) or a nontargeting siRNA (siNT) for six hours.
  • FIGS. 20A-20B Quality control metrics for the RNA seq analysis detailed in Figure 5.
  • A Shows the distribution of RIN, Total Raw Reads, Clean/Raw Reads [%], GC Content [%], Q30 [%], and % of Reads Mapped by STAR.
  • B Principal component analysis of all samples used in analysis shows that all conditions separate with replicates clustering together
  • Figures 21 A-21 C Detailed volcano plot of differentially expressed genes in the (A) siLCK, (B) siFYN, and (C) siNT+BMP9 cases relative to the siNT case. Note that Eln is not depicted to allow a better view of all genes on the si-LCK plot.
  • Figure 22 “Inflammatory” and “Endothelial” gene signatures. Extended list of genes co-differentially expressed and are increased by si-Lck and decreased si-Fyn. Because si-Lck imparts a dysfunctional EC phenotype, genes in this list are implicated in EC dysfunction. Genes in this list are increased by Le suppression and decreased by Fyn suppression. The accompanying gene ontology analysis shows that multiple pathways are implicated that represent a response to interferon signaling, lipopolysaccharide, NF-KB signaling, or neutrophil transmigration.
  • FIG. 23 Full results for Connectivity Map (CMap) analysis detailing ligands which recapitulate the inflammatory and endothelial signatures. Data for all cell lines present in CMap data is shown. Y axis indicates significance by -Logw(False Discovery Rate) and X axis indicates the Connectivity Score.
  • Figure 24 Extended data of CMap search results.
  • the methods disclosed herein provide for treatment of HHT or PAH. Dosage and frequency may vary depending on the half-life of the agent in the patient. It will be understood by one of skill in the art that such guidelines will be adjusted for the molecular weight of the active agent, the clearance from the blood, the mode of administration, and other pharmacokinetic parameters.
  • the dosage may also be varied for localized administration, e.g. intranasal, inhalation, etc., or for systemic administration, e.g. i.m., i.p., i.v., oral, and the like.
  • An active agent can be administered by any suitable means, including topical, oral, parenteral, intrapulmonary, and intranasal. Oral is peferred. Parenteral infusions include intramuscular, intravenous (bolus or slow drip), intraarterial, intraperitoneal, intrathecal or subcutaneous administration. An agent can be administered in any manner which is medically acceptable. This may include injections, by parenteral routes such as intravenous, intravascular, intraarterial, subcutaneous, intramuscular, intratumor, intraperitoneal, intraventricular, intraepidural, or others as well as oral, nasal, ophthalmic, rectal, or topical. Sustained release administration is also specifically included in the disclosure, by such means as depot injections or erodible implants.
  • an agent can be formulated with an a pharmaceutically acceptable carrier (one or more organic or inorganic ingredients, natural or synthetic, with which a subject agent is combined to facilitate its application).
  • a suitable carrier includes sterile saline although other aqueous and non-aqueous isotonic sterile solutions and sterile suspensions known to be pharmaceutically acceptable are known to those of ordinary skill in the art.
  • An "effective amount” refers to that amount which is capable of ameliorating or delaying progression of the diseased, degenerative or damaged condition. An effective amount can be determined on an individual basis and will be based, in part, on consideration of the symptoms to be treated and results sought. An effective amount can be determined by one of ordinary skill in the art employing such factors and using no more than routine experimentation.
  • compositions comprising a pharmaceutically acceptable excipient.
  • the preferred form depends on the intended mode of administration and therapeutic application.
  • the compositions can also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration.
  • the diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution.
  • the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.
  • compounds which are "commercially available” may be obtained from commercial sources including but not limited to Acros Organics (Pittsburgh PA), Aldrich Chemical (Milwaukee Wl, including Sigma Chemical and Fluka), Apin Chemicals Ltd. (Milton Park UK), Avocado Research (Lancashire U.K.), BDH Inc. (Toronto, Canada), Bionet (Cornwall, U.K.), Chemservice Inc. (West Chester PA), Crescent Chemical Co. (Hauppauge NY), Eastman Organic Chemicals, Eastman Kodak Company (Rochester NY), Fisher Scientific Co. (Pittsburgh PA), Fisons Chemicals (Leicestershire UK), Frontier Scientific (Logan UT), ICN Biomedicals, Inc.
  • the active agents of the invention and/or the compounds administered therewith are incorporated into a variety of formulations for therapeutic administration.
  • the agents are formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and are formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols.
  • administration of the active agents and/or other compounds can be achieved in various ways, usually by oral administration.
  • the active agents and/or other compounds may be systemic after administration or may be localized by virtue of the formulation, or by the use of an implant that acts to retain the active dose at the site of implantation.
  • the active agents and/or other compounds may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds.
  • the agents may be combined, as previously described, to provide a cocktail of activities.
  • the following methods and excipients are exemplary and are not to be construed as limiting the invention.
  • the agents can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
  • conventional additives such as lactose, mannitol, corn starch or potato starch
  • binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins
  • disintegrators such as corn starch, potato starch or sodium carboxymethylcellulose
  • lubricants such as talc or magnesium stearate
  • Formulations are typically provided in a unit dosage form, where the term "unit dosage form,” refers to physically discrete units suitable as unitary dosages for human subjects, each unit containing a predetermined quantity of active agent in an amount calculated sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human subjects, each unit containing a predetermined quantity of active agent in an amount calculated sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle.
  • the specifications for the unit dosage forms of the present invention depend on the particular complex employed and the effect to be achieved, and the pharmacodynamics associated with each complex in the host.
  • the pharmaceutically acceptable excipients such as vehicles, adjuvants, carriers or diluents, are commercially available.
  • pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are commercially available.
  • Any compound useful in the methods and compositions of the invention can be provided as a pharmaceutically acceptable base addition salt.
  • “Pharmaceutically acceptable base addition salt” refers to those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid.
  • Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like.
  • Preferred inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts.
  • Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like.
  • Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.
  • the active agent may be administered in dosages of 0.01 mg to 500 mg /kg body weight per day, e.g. about 20 mg/day for an average person. Dosages will be appropriately adjusted for pediatric formulation.
  • the dose of brivanib for an adult may be a flat dose of from about 50 to about 1000 mg/day, from about 100 to about 900 mg/day, from about 200 to about 800 mg/day, from about 400 to about 800 mg/day, from about 600 to about 800 mg/day.
  • Agents approved for clinical use may be administered at conventional doses, or at less than conventional doses, e.g. reduced by 25%, by 50%, by 75%, by 90%, or more, relative to, for example, doses administered for cancer treatment.
  • compositions can also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized SepharoseTM, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).
  • a carrier may bear the agents in a variety of ways, including covalent bonding either directly or via a linker group, and non-covalent associations.
  • Suitable covalent-bond carriers include proteins such as albumins, peptides, and polysaccharides such as aminodextran, each of which have multiple sites for the attachment of moieties.
  • the nature of the carrier can be either soluble or insoluble for purposes of the invention.
  • Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyidimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine,
  • the active ingredients may also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared.
  • the preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-1 19, 1997.
  • the agents of this invention can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient.
  • the pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.
  • Toxicity of the active agents can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index.
  • the data obtained from these cell culture assays and animal studies can be used in further optimizing and/or defining a therapeutic dosage range and/or a sub-therapeutic dosage range (e.g., for use in humans).
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.
  • polypeptide peptide
  • protein protein
  • amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymer.
  • nucleic acid refers to polynucleotides such as deoxyribonucleic acid (DNA) and, where appropriate, ribonucleic acid (RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • Chromosomes, cDNAs, mRNAs, rRNAs, and ESTs are representative examples of molecules that may be referred to as nucleic acids.
  • the term “gene” has its meaning as understood in the art.
  • the term “gene” may include gene regulatory sequences, where examples of regulatory sequences include but are not limited to promoter, enhancers, introns etc., and may refer to genomic sequences, RNA or cDNA.
  • the term “gene” encompasses nucleic acids that can code for a polypeptide (mRNA), as well as nonpolypeptide coding RNAs.
  • Non-coding RNAs encoded by the genes relevant to the current disclosure include, but are not limited to, transfer RNA (tRNA), rRNA, microRNA (miRNA), long non- coding RNAs (lincRNAs), circular RNAs (circRNAs) or any other kind of RNA.
  • tRNA transfer RNA
  • miRNA microRNA
  • lincRNAs long non- coding RNAs
  • circRNAs circular RNAs
  • level of expression refers to the level of mRNA, as well as pre-mRNA nascent transcript(s), transcript processing intermediates, mature mRNA(s), and degradation products, encoded by a gene in the cell.
  • level of expression also refers to the level of protein or polypeptide in a cell.
  • sequence identity refers to the subunit sequence identity between two molecules. When a subunit position in both of the molecules is occupied by the same monomeric subunit (e.g., the same amino acid residue or nucleotide), then the molecules are identical at that position. The similarity between two amino acid or two nucleotide sequences is a direct function of the number of identical positions. In general, the sequences are aligned so that the highest order match is obtained. If necessary, identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux et al., Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, FASTA (Atschul et al., J. Molecular Biol. 215:403, 1990).
  • isolated refers to a molecule that is substantially free of its natural environment.
  • an isolated protein is substantially free of cellular material or other proteins from the cell or tissue source from which it is derived.
  • the term refers to preparations where the isolated protein is sufficiently pure to be administered as a therapeutic composition, or at least 70% to 80% (w/w) pure, more preferably, at least 80%-90% (w/w) pure, even more preferably, 90-95% pure; and, most preferably, at least 95%, 96%, 97%, 98%, 99%, or 100% (w/w) pure.
  • a “separated” compound refers to a compound that is removed from at least 90% of at least one component of a sample from which the compound was obtained. Any compound described herein can be provided as an isolated or separated compound.
  • subject is used interchangeably herein to refer to a mammal being assessed for treatment and/or being treated.
  • the mammal is a human.
  • subject encompass, without limitation, individuals having a disease.
  • Subjects may be human, but also include other mammals, particularly those mammals useful as laboratory models for human disease, e.g., mice, rats, etc.
  • sample with reference to a patient encompasses blood and other liquid samples of biological origin, solid tissue samples, such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof.
  • the term also encompasses samples that have been manipulated in any way after their procurement, such as by treatment with reagents; washed; or enrichment for certain cell populations, such as diseased cells.
  • the definition also includes samples that have been enriched for particular types of molecules, e.g., nucleic acids, polypeptides, etc.
  • biological sample encompasses a clinical sample, and also includes tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, and the like.
  • a “biological sample” includes a sample obtained from a patient's diseased cell, e.g., a sample comprising polynucleotides and/or polypeptides that is obtained from a patient’s diseased cell (e.g., a cell lysate or other cell extract comprising polynucleotides and/or polypeptides); and a sample comprising diseased cells from a patient.
  • a biological sample comprising a diseased cell from a patient can also include non-diseased cells.
  • diagnosis is used herein to refer to the identification of a molecular or pathological state, disease or condition in a subject, individual, or patient.
  • prognosis is used herein to refer to the prediction of the likelihood of death or disease progression, including recurrence, spread, and drug resistance, in a subject, individual, or patient.
  • prediction is used herein to refer to the act of foretelling or estimating, based on observation, experience, or scientific reasoning, the likelihood of a subject, individual, or patient experiencing a particular event or clinical outcome. In one example, a physician may attempt to predict the likelihood that a patient will survive.
  • treatment refers to administering an agent, or carrying out a procedure, for the purposes of obtaining an effect on or in a subject, individual, or patient.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of effecting a partial or complete cure for a disease and/or symptoms of the disease.
  • Treatment may include treatment of cancer in a mammal, particularly in a human, and includes: (a) inhibiting the disease, i.e., arresting its development; and (b) relieving the disease or its symptoms, i.e., causing regression of the disease or its symptoms.
  • Treating may refer to any indicia of success in the treatment or amelioration or prevention of a disease, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating.
  • the treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of an examination by a physician.
  • treating includes the administration of engineered cells to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with disease or other diseases.
  • therapeutic effect refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject.
  • a "therapeutically effective amount” refers to that amount of the therapeutic agent sufficient to treat or manage a disease or disorder.
  • a therapeutically effective amount may refer to the amount of therapeutic agent sufficient to delay or minimize the onset of disease, e.g., to delay or minimize the growth and spread of cancer.
  • a therapeutically effective amount may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease.
  • a therapeutically effective amount with respect to a therapeutic agent of the invention means the amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of a disease.
  • the term “dosing regimen” refers to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time.
  • a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses.
  • a dosing regimen comprises a plurality of doses each of which are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses.
  • all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts.
  • a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).
  • each component can be administered at the same time or sequentially in any order at different points in time. Thus, each component can be administered separately but sufficiently closely in time so as to provide the desired therapeutic effect.
  • Concomitant administration means administration of one or more components, such as engineered proteins and cells, known therapeutic agents, etc. at such time that the combination will have a therapeutic effect. Such concomitant administration may involve concurrent (i.e. at the same time), prior, or subsequent administration of components. A person of ordinary skill in the art would have no difficulty determining the appropriate timing, sequence and dosages of administration.
  • a first prophylactic or therapeutic agent can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second prophylactic or therapeutic agent to a subject with a disorder.
  • ID1 DNA-binding protein inhibitor
  • HHLH helix-loop-helix
  • the encoded protein has no DNA binding activity and therefore can inhibit the DNA binding and transcriptional activation ability of basic HLH proteins with which it interacts.
  • ID1 has been shown to interact weakly with MyoD but very tightly with ubiquitously expressed E proteins. E proteins heterodimerize with tissue restricted bHLH proteins such as Myod, NeuroD, etc. to form active transcription complexes so by sequestering E proteins, Id proteins can inhibit tissue restricted gene expression in multiple cell lineages using the same biochemical mechanism.
  • the human reference protein sequence can be accessed at Genbank NP 002156 and NP_851998.
  • Tyrosine Kinase Inhibitors are a group of pharmacologic agents that disrupt the signal transduction pathways of protein kinases by several modes of inhibition. This activity will review the currently available drugs, their mechanism of action, routes of administration, indications, contraindications, and adverse effects.
  • TKs Tyrosine kinase enzymes
  • RTKs receptor tyrosine kinases
  • NRTKs non-receptor tyrosine kinases
  • DSK dual-specificity kinases
  • RTKs are transmembrane receptor that includes vascular endothelial growth factor receptors (VEGFR), platelet-derived growth factor receptors (PDGFR), insulin receptor (InsR) family, and the ErbB receptor family, which includes epidermal growth factor receptors (EGFR) and the human epidermal growth factor receptor-2 (HER2).
  • VEGFR vascular endothelial growth factor receptors
  • PDGFR platelet-derived growth factor receptors
  • InsR insulin receptor
  • ErbB receptor family which includes epidermal growth factor receptors (EGFR) and the human epidermal growth factor receptor-2 (HER2).
  • tyrosine kinases phosphorylate specific amino acids on substrate enzymes, which subsequently alter signal transduction leading to downstream changes in cellular biology.
  • the downstream signal transduction set off by TKs can modify cell growth, migration, differentiation, apoptosis, and death.
  • Constitutive activation or inhibition, either by mutations or other means, can lead to dysregulated signal cascades, potentially resulting in malignancy and other pathologies. Therefore, blocking these initial signals via TKIs can prevent the aberrant action of the mutated or dysfunctional TKs.
  • Nearly all TKIs are effective when taken orally. Therapeutic loading and maintenance dosages are unique to each drug and should require unique dosing for each patient.
  • VEGFR-2 inhibitor is a 210-230 kDa glycoprotein expressed in vascular endothelial cells and in hematopoietic stem cells and binds VEGF-A.
  • VEGFR-2 is closely related to VEGFR-1 for they have common and specific ligands but VEGFR-2 is a highly active kinase while VEGFR1 is an impaired receptor tyrosine kinase. This receptor is a regulator in responses in the endothelial cells of VEGF. These regulations include permeability, proliferation, invasion and migration.
  • the signaling pathways, Y1 175 and Y1214, are the main autophosphorylation sites of the human VEGFR-2 followed by VEGF binding.
  • Inhibitors of VEGFR2 include, for example, Sorafenib, Sunitinib, Lenvatinib, Vandetanib, Pazopanib and Axitinib, Cabozantinib, Regorafenib, Nintedanib, Apatinib, etc.
  • Brivanib alaninate is an orally administered drug with the structure:
  • Brivanib is a multitargeted tyrosine kinase inhibitor, which inhibits VEGFR and fibroblast growth factor receptors.
  • Brivanib is the alanine ester of a VEGFR-2 inhibitor BMS- 540215 and is hydrolyzed to the active moiety BMS-540215 in vivo.
  • BMS-540215 a dual tyrosine kinase inhibitor, shows potent and selective inhibition of VEGFR and fibroblast growth factor receptor (FGFR) tyrosine kinases.
  • BMS-540215 is an ATP-competitive inhibitor of human VEGFR-2, with an IC50 of 25 nmol/L and Ki of 26 nmol/L.
  • BMS-540215 has been shown to selectively inhibit the proliferation of endothelial cells stimulated by VEGF and FGF in vitro with IC50 values of 40 and 276 nmol/L, respectively.
  • the dosing of brivanib may be similar to that for cancer treatment, e.g.
  • the dose may be modified, e.g. using a lower dose of from about 10, from about 50, from about 100, from about 250 mg/day, up to about 1000, up to about 750, up to about 500 mg/day.
  • Dabrafenib is an orally administered drug with the structure:
  • Dabrafenib sold under the brand name Tafinlar among others, is an anti-cancer medication used for the treatment of cancers associated with a mutated version of the gene BRAF.
  • the dosing of dabrafenib may be similar to that for cancer treatment, e.g. from about 150 mg/day to about 600 mg/day, from about 150 to about 300 mg/day. Administration may be about 150 mg, twice daily. Alternatively the dose may be modified, e.g. using a lower dose of from about 5, from about 10, from about 25, from about 50 mg/day, from about 100 mg/day up to about 300, up to about 200, up to about 150 mg/day.
  • Linsitinib is an orally administered drug with the structure:
  • Linsitinib is an inhibitor of the insulin receptor and of the insulin-like growth factor 1 receptor (IGF-1 ).
  • the dosing of linsitinib may be similar to that for clinical trials in cancer treatment, e.g. from about 150 mg/day to about 600 mg/day, from about 150 to about 300 mg/day. Administration may be about 150 mg, twice daily. Alternatively the dose may be modified, e.g. using a lower dose of from about 5, from about 10, from about 25, from about 50 mg/day, from about 100 mg/day up to about 300, up to about 200, up to about 150 mg/day.
  • HHT Hereditary haemorrhagic telangiectasia
  • Rendu-Osler-Weber syndrome is a complex genetic disease that causes abnormalities of blood vessels formation in the liver, lung, brain, skin, nasal mucosa, and gastrointestinal tract.
  • HHT is a rare disease, affecting approximately 1 in 5000-10000 peoples in the world.
  • Clinical indicia manifestations of the diasease include epistaxis (nose bleeding, present in 90% cases), gastrointestinal haemorrhage, mucocutaneous telangiectases (blood vessel dilations), and visceral arteriovenous malformations (AVMs).
  • Treatment with the agents disclosed herein may reduce one or more clinical indicia, for example AVMs, by at least about 10%, at least abou 20%, at least about 30%, at least about 50%, at least about 75%, at least about 90% or more relative to an untreated individual.
  • AVMs clinical indicia
  • HHT can be diagnosed based on the four established Curacao Criteria; recurrent epistaxis, telangiectasia, family history, and arteriovenous malformations (AVMs).
  • the diagnosisof HHT is confirmed when three or more criteria are met, while it is suspected if just two criteria are fulfilled.
  • the AVMs are abnormal large connections between arteries and veins bypassing the capillaries and result in severe bleeding when present in the nose, Gl- tract and brain or cause paradoxic emboli and stroke when present in the lungs.
  • AVMs are found in the lung (15-45%), liver (>70%), and cerebral (10-23%) of HHT patients.
  • HHT therapies aim to lessen the disease's symptoms. However, there is currently no mechanism-based targeted therapy available.
  • ID proteins are thought to inhibit differentiation and promote cell cycle progression and therefore could represent possible candidates to explain the aberrant proliferation observed during AVM pathogenesis in HHT.
  • mice with a combined loss of both Id1 and Id3 display cranial hemorrhage secondary to the formation of an anastomosing network of dilated capillaries in the brain, suggesting that ID1/3 may play a role in AVM formation.
  • PH Pulmonary hypertension
  • Pulmonary arterial hypertension (Group 1 )
  • PH due to left heart disease (Group 2)
  • PH due to underlying lung disease or chronic hypoxemia (Group 3)
  • PH attributable to chronic thromboembolic pulmonary hypertension (Group 4)
  • PH due to unknown, or overlapping, mechanisms (Group 5).
  • Pulmonary arterial hypertension (PAH, Group I) is termed “pre-capillary” disease because of the pathological vascular changes that occur prior to the pulmonary capillary bed as it delivers blood to the pulmonary venous system. It is a fatal condition characterized by aberrant proliferation of cells constituting the pulmonary vessels, resulting in vascular wall thickening and luminal obliteration to produce elevated pulmonary vascular resistance (PVR) and subsequent right ventricular (RV) failure.
  • PVR pulmonary vascular resistance
  • RV right ventricular
  • PAH has been defined by clinical indicia of hemodynamic indices measured with right heart catheterization (RHC) while the patient is at rest.
  • PAH resting RHC diagnostic criteria include increased mean pulmonary artery pressure (mPAP) > 20 mmHg and elevated PVR (>3 Wood units), along with pulmonary arterial wedge pressure (PAWP) ⁇ 15 mmHg indicative of a normal left ventricular filling pressure.
  • Treatment with the agents disclosed herein may reduce one or more clinical indicia, by at least about 10%, at least abou 20%, at least about 30%, at least about 50%, at least about 75%, at least about 90% or more relative to an untreated individual
  • PAH cardiovascular disease
  • PAH-targeted pharmacologic modalities predominantly pulmonary vasodilators
  • gene expression signatures areused to determine the efficacy of a treatment, for drug screening to determine the suitability of an agent, in the selection of cell lines as in vitro models and in drug screening, and the like.
  • the treatment of HHT can normalize gene expression in endothelial cells in which HHT causing genes are knocked down, e.g. the treatment is capable of reducing expression of one or more of the genes LYVE1 , GPNMB, RRAGD and ANKRD33; and/or increasing expression of one or more of the genes MC5R, SLC25A47, FGF19, SHISA9, FRG2C, and HS3ST2.
  • This pattern of expression may be referred to as an “HHT disease signature”.
  • An expanded HHT disease signature may comprise 5 upregulated (LYVE1 , GPNMB, RRAGD, ANKRD33, HS3ST2) and 7 downregulated (MC5R, SLC25A47, FGF19, SHISA9, FRG2C, HIST1 H2BE, CPA4) genes.
  • Gene signatures associated with PAH include the set of genes decreased by siLCK and increased by siFYN, which are termed an “endothelial signature”.
  • the “siLCK Down/siFYN Up” group comprise of EC artery and capillary specific genes (Gja5, Lyvel , Gphibpl ).
  • Upregulated genes for siLCK include Il6st, Ccl7, Wnt5a, and 111 b.
  • Top downregulated genes for siLCK are Ace, Lyvel , Gja5, and Dysf.
  • siFyn upregulated genes included Bmp4, Gdf7, Gdf1 / (Bmp11 ), Gja5, Lyvel, Id1, and Tgfb 1.
  • Genes downregulated in siFYN include Tlr3, C1s, C3, Ccl8, and Vcaml.
  • the most co-differentially regulated genes were associated with type-1 interferon signaling: Tnfaip6 and 111 B.
  • Gene ontology (GO) analysis converged on pathways elicited by interferon gamma or lipopolysaccharide and were associated with increased interferon cx/p and IL-6 production, NF- KB activation, and MHC-1 activation.
  • transcription factor prediction by the three methods found the canonical NF-KB transcription factor RelA was tied to the majority of co- DEGs.
  • the set of co-DEGs both increased in siLCK PAECs and decreased in siFYN PAECs may be referred to as an “inflammatory signature”, linked by canonical NF-KB activity.
  • gating strategies can be used in order to analyze a specific cell population in a sample of mixed cell population, e.g. to focus on endothelial cells, etc.. These gating strategies can be based on the presence of one or more specific surface markers.
  • the following gate can differentiate between dead cells and live cells and the subsequent gating of live cells classifies.
  • a clear comparison can be carried out by using two-dimensional contour plot representations, two-dimensional dot plot representations, and/or histograms.
  • Samples may be obtained at one or more time points. Where a sample at a single time point is used, comparison is made to a reference “base line” level for the expression of the gene of interest, which may be obtained from a normal control, a pre-determined level obtained from one or a population of individuals, from a negative control, and the like.
  • a reference “base line” level for the expression of the gene of interest which may be obtained from a normal control, a pre-determined level obtained from one or a population of individuals, from a negative control, and the like.
  • cells are dispersed into a single cell suspension, e.g. by enzymatic digestion with a suitable protease, e.g. collagenase, dispase, etc; and the like.
  • An appropriate solution is used for dispersion or suspension.
  • Such solution will generally be a balanced salt solution, e.g. normal saline, PBS, Hanks balanced salt solution, etc., conveniently supplemented with fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, generally from 5-25 mM.
  • Convenient buffers include HEPES1 phosphate buffers, lactate buffers, etc.
  • the cells can be fixed, e.g.
  • one or more cells are contained in a well of a 96 well plate or other commercially available multiwell plate.
  • the reaction mixture or cells are in a cytometric measurement device.
  • Other multiwell plates useful in the present invention include, but are not limited to 384 well plates and 1536 well plates. Still other vessels for containing the reaction mixture or cells and useful in the present invention will be apparent to the skilled artisan.
  • the methods of the invention include the use of liquid handling components.
  • the liquid handling systems can include robotic systems comprising any number of components.
  • any or all of the steps outlined herein can be automated; thus, for example, the systems can be completely or partially automated. See USSN 61/048,657.
  • Fully robotic or microfluidic systems include automated liquid-, particle-, cell- and organism-handling including high throughput pipetting to perform all steps of screening applications.
  • This includes liquid, particle, cell, and organism manipulations such as aspiration, dispensing, mixing, diluting, washing, accurate volumetric transfers; retrieving, and discarding of pipet tips; and repetitive pipetting of identical volumes for multiple deliveries from a single sample aspiration.
  • These manipulations are cross-contamination- free liquid, particle, cell, and organism transfers.
  • This instrument performs automated replication of microplate samples to filters, membranes, and/or daughter plates, high-density transfers, full-plate serial dilutions, and high capacity operation.
  • platforms for multi-well plates, multi-tubes, holders, cartridges, minitubes, deep-well plates, microfuge tubes, cryovials, square well plates, filters, chips, optic fibers, beads, and other solid-phase matrices or platform with various volumes are accommodated on an upgradable modular platform for additional capacity.
  • This modular platform includes a variable speed orbital shaker, and multi-position work decks for source samples, sample and reagent dilution, assay plates, sample and reagent reservoirs, pipette tips, and an active wash station.
  • the methods of the invention include the use of a plate reader.
  • interchangeable pipet heads with single or multiple magnetic probes, affinity probes, or pipetters robotically manipulate the liquid, particles, cells, and organisms.
  • Multi-well or multi-tube magnetic separators or platforms manipulate liquid, particles, cells, and organisms in single or multiple sample formats.
  • the instrumentation will include a detector, which can be a wide variety of different detectors, depending on the labels and assay.
  • useful detectors include a microscope(s) with multiple channels of fluorescence; plate readers to provide fluorescent, ultraviolet and visible spectrophotometric detection with single and dual wavelength endpoint and kinetics capability, fluorescence resonance energy transfer (FRET), luminescence, quenching, two-photon excitation, and intensity redistribution; CCD cameras to capture and transform data and images into quantifiable formats; and a computer workstation.
  • the robotic apparatus includes a central processing unit which communicates with a memory and a set of input/output devices (e.g., keyboard, mouse, monitor, printer, etc.) through a bus. Again, as outlined below, this can be in addition to or in place of the CPU for the multiplexing devices of the invention.
  • a central processing unit which communicates with a memory and a set of input/output devices (e.g., keyboard, mouse, monitor, printer, etc.) through a bus.
  • input/output devices e.g., keyboard, mouse, monitor, printer, etc.
  • this can be in addition to or in place of the CPU for the multiplexing devices of the invention.
  • the general interaction between a central processing unit, a memory, input/output devices, and a bus is known in the art. Thus, a variety of different procedures, depending on the experiments to be run, are stored in the CPU memory.
  • a signature pattern can be generated from a biological sample using any convenient protocol, for example as described below.
  • the readout can be a mean, average, median or the variance or other statistically or mathematically-derived value associated with the measurement.
  • the marker readout information can be further refined by direct comparison with the corresponding reference or control pattern.
  • a pattern can be evaluated on a number of points: to determine if there is a statistically significant change at any point in the data matrix relative to a reference value; whether the change is an increase or decrease in the binding; whether the change is specific for one or more physiological states, and the like.
  • the absolute values obtained for each marker under identical conditions will display a variability that is inherent in live biological systems and also reflects the variability inherent between individuals.
  • a reference or control signature pattern can be a signature pattern that is obtained from a sample of a patient known to have a normal pregnancy.
  • the obtained signature pattern is compared to a single reference/control profile to obtain information regarding the phenotype of the patient being assayed.
  • the obtained signature pattern is compared to two or more different reference/control profiles to obtain more in depth information regarding the phenotype of the patient.
  • the obtained signature pattern can be compared to a positive and negative reference profile to obtain confirmed information regarding whether the patient has the phenotype of interest.
  • Samples can be obtained from the tissues or fluids of an individual.
  • samples can be obtained from whole blood, tissue biopsy, serum, etc.
  • body fluids such as lymph, cerebrospinal fluid, and the like.
  • derivatives and fractions of such cells and fluids are also included in the term.
  • a statistical test can provide a confidence level for a change in the level of markers between the test and reference profiles to be considered significant.
  • the raw data can be initially analyzed by measuring the values for each marker, usually in duplicate, triplicate, quadruplicate or in 5-10 replicate features per marker.
  • a test dataset is considered to be different than a reference dataset if one or more of the parameter values of the profile exceeds the limits that correspond to a predefined level of significance.
  • the false discovery rate can be determined.
  • a set of null distributions of dissimilarity values is generated.
  • the values of observed profiles are permuted to create a sequence of distributions of correlation coefficients obtained out of chance, thereby creating an appropriate set of null distributions of correlation coefficients (see Tusher et al. (2001 ) PNAS 98, 5116-21 , herein incorporated by reference).
  • This analysis algorithm is currently available as a software “plug-in” for Microsoft Excel know as Significance Analysis of Microarrays (SAM).
  • the set of null distribution is obtained by: permuting the values of each profile for all available profiles; calculating the pair- wise correlation coefficients for all profile; calculating the probability density function of the correlation coefficients for this permutation; and repeating the procedure for N times, where N is a large number, usually 300.
  • N is a large number, usually 300.
  • the FDR is the ratio of the number of the expected falsely significant correlations (estimated from the correlations greater than this selected Pearson correlation in the set of randomized data) to the number of correlations greater than this selected Pearson correlation in the empirical data (significant correlations). This cut-off correlation value can be applied to the correlations between experimental profiles.
  • Z-scores represent another measure of variance in a dataset, and are equal to a value of X minus the mean of X, divided by the standard deviation.
  • a Z-Score tells how a single data point compares to the normal data distribution.
  • a Z-score demonstrates not only whether a datapoint lies above or below average, but how unusual the measurement is.
  • the standard deviation is the average distance between each value in the dataset and the mean of the values in the dataset.
  • a level of confidence is chosen for significance. This is used to determine the lowest value of the correlation coefficient that exceeds the result that would have obtained by chance.
  • this method one obtains thresholds for positive correlation, negative correlation or both. Using this threshold(s), the user can filter the observed values of the pairwise correlation coefficients and eliminate those that do not exceed the threshold(s). Furthermore, an estimate of the false positive rate can be obtained for a given threshold. For each of the individual “random correlation” distributions, one can find how many observations fall outside the threshold range. This procedure provides a sequence of counts. The mean and the standard deviation of the sequence provide the average number of potential false positives and its standard deviation. Alternatively, any convenient method of statistical validation can be used.
  • the data can be subjected to non-supervised hierarchical clustering to reveal relationships among profiles.
  • hierarchical clustering can be performed, where the Pearson correlation is employed as the clustering metric.
  • One approach is to consider a patient disease dataset as a “learning sample” in a problem of “supervised learning”.
  • CART is a standard in applications to medicine (Singer (1999) Recursive Partitioning in the Health Sciences, Springer), which can be modified by transforming any qualitative features to quantitative features; sorting them by attained significance levels, evaluated by sample reuse methods for Hotelling's T 2 statistic; and suitable application of the lasso method.
  • Problems in prediction are turned into problems in regression without losing sight of prediction, indeed by making suitable use of the Gini criterion for classification in evaluating the quality of regressions.
  • Cox models can be used, especially since reductions of numbers of covariates to manageable size with the lasso will significantly simplify the analysis, allowing the possibility of an entirely nonparametric approach to survival.
  • the analysis and database storage can be implemented in hardware or software, or a combination of both.
  • a machine-readable storage medium comprising a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying a any of the datasets and data comparisons of this invention.
  • Such data can be used for a variety of purposes, such as patient monitoring, initial diagnosis, and the like.
  • the invention is implemented in computer programs executing on programmable computers, comprising a processor, a data storage system (including volatile and non-volatile memory and/or storage elements), at least one input device, and at least one output device.
  • Program code is applied to input data to perform the functions described above and generate output information.
  • the output information is applied to one or more output devices, in known fashion.
  • the computer can be, for example, a personal computer, microcomputer, or workstation of conventional design.
  • Each program is preferably implemented in a high level procedural or object oriented programming language to communicate with a computer system.
  • the programs can be implemented in assembly or machine language, if desired. In any case, the language can be a compiled or interpreted language.
  • Each such computer program is preferably stored on a storage media or device (e.g., ROM or magnetic diskette) readable by a general or special purpose programmable computer, for configuring and operating the computer when the storage media or device is read by the computer to perform the procedures described herein.
  • the system can also be considered to be implemented as a computer-readable storage medium, configured with a computer program, where the storage medium so configured causes a computer to operate in a specific and predefined manner to perform the functions described herein.
  • a variety of structural formats for the input and output means can be used to input and output the information in the computer-based systems of the present invention.
  • One format for an output means test datasets possessing varying degrees of similarity to a trusted profile. Such presentation provides a skilled artisan with a ranking of similarities and identifies the degree of similarity contained in the test pattern.
  • the signature patterns and databases thereof can be provided in a variety of media to facilitate their use.
  • Media refers to a manufacture that contains the signature pattern information of the present invention.
  • the databases of the present invention can be recorded on computer readable media, e.g. any medium that can be read and accessed directly by a computer.
  • Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media.
  • magnetic storage media such as floppy discs, hard disc storage medium, and magnetic tape
  • optical storage media such as CD-ROM
  • electrical storage media such as RAM and ROM
  • hybrids of these categories such as magnetic/optical storage media.
  • Recorded refers to a process for storing information on computer readable medium, using any such methods as known in the art. Any convenient data storage structure can be chosen, based on the means used to access the stored information. A variety of data processor programs and formats can be used for storage, e.g. word processing text file, database format, etc.
  • an individual with HHT is treated by administration of an effective dose of an agent that inhibits at least VEGFR-2, and may additionally inhibit FGFR-1 , where the treatment improves clinical indicia of the disease.
  • the agent is Brivanib. It is shown herein that Brivanib can reversed an HHT disease signature of gene expression, and can inhibit othere disease features.
  • the agent is dabrafenib.
  • the agent is linsitinib.
  • Brivanib is a pyrrolotriazine-based compound and an inhibitor of vascular endothelial growth factor receptor-2 (VEGFR-2). It specifically targets and binds strongly to human VEGFR-2, a tyrosine kinase receptor and pro-angiogenic growth factor expressed almost exclusively on vascular endothelial cells. It has a role as an antineoplastic agent, an EC 2.7.10.1 (receptor protein-tyrosine kinase) inhibitor, an angiogenesis inhibitor, an apoptosis inducer, a fibroblast growth factor receptor antagonist and a drug metabolite.
  • VEGFR-2 vascular endothelial growth factor receptor-2
  • the treatment of HHT can normalize gene expression in endothelial cells in which HHT causing genes are knocked down, e.g. the treatment is capable of reducing expression of one or more of the genes LYVE1 , GPNMB, RRAGD and ANKRD33; and/or increasing expression of one or more of the genes MC5R, SLC25A47, FGF19, SHISA9, FRG2C, and HS3ST2.
  • the treatment can inhibit VEGF-induced ERK1/2 phosphorylation, proliferation, and angiogenesis in the targeted cells, e.g.
  • lung microvessel endothelial cells and also inhibit the expression of one or more of the genes upregulated in the HHT disease condition: LYVE1 , GPNMB, and HS3ST2 in the targeted tissues, e.g. lung tissue.
  • the PTK can inhibit an inflammatory gene expression signature disclosed herein.
  • the individual is monitored for efficacy of treatment, e.g. by determining an inflammatory signature change, determining changes in an HHT disease signature, changes in gene expression, and the like.
  • an individual with PAH is treated by administration of an effective dose of an agent that increases ID1 expression, a common downstream target of the BMPR2 pathway, where the treatment improves clinical indicia of the disease.
  • the agent is Brivanib.
  • the inhibitor is Dabrafenib or Linsitinib.
  • the treatment can provide for an pro-endothelial-like pattern of gene expression in targeted cells, in contrast to an inflammatory pattern of gene expression.
  • the individual is monitored for efficacy of treatment, e.g. by determining a pro-endothelial expression change, changes in Id1 gene expression, and the like. .
  • the inhibitor increases Id1 expression.
  • the inhibitor activates the endothelial transcriptomic signature.
  • the methods comprises administering an effective amount of an agent, e.g. brivanib, of the invention to a patient determined to be likely to benefit from, in need of, or likely to respond to, a therapy of the invention, thereby treating or preventing HHT or PAH, in the patient.
  • agent e.g. brivanib
  • Dosing guidelines for agents are as disclosed herein.
  • Average survival rate can be used in the methods as a parameter to determine efficacy in a population of one or more subjects.
  • a reference average survival rate is 95% or lower, 93% or lower, 90% or lower, 86% or lower, 82% or lower, or 78% or lower.
  • the average survival rate can be an average 1 -year survival rate.
  • a treatment provided in the invention is efficacious in a population of one or more subjects if, after treatment has started, the average survival rate increases. For example, the average survival rate can increase from the reference average survival rate by 1 % or more, 2% or more, 5% or more, 10% or more, or 20% or more.
  • the methods can further comprise continuing the therapy if the therapy is determined to be efficacious.
  • the methods can comprise maintaining, tapering, reducing, or stopping the administered amount of a compound or compounds in the therapy if the therapy is determined to be efficacious.
  • the methods can comprise increasing the administered amount of a compound or compounds in the therapy if it is determined not to be efficacious.
  • the methods can comprise stopping therapy if it is determined not to be efficacious.
  • the method of treating the patient may involve administering at least one additional active agent, i.e., in addition to the RTK inhibitor.
  • the additional active agent may be, for example, a vasodilator such as prostacyclin, epoprostenol, and sildenafil; an endothelin receptor antagonist such as bosentan; a calcium channel blocker such as amlodipine, diltiazem, and nifedipine; an anticoagulant such as warfarin; a diuretic, a prostanoid (e.g., prostacyclin or PGI.sub.2), drugs for treating diseases associated with overactive B cells or dysfunctional B cells such as Rituximab, and/or a Type V phosphodiesterase (PDE5) inhibitor.
  • a vasodilator such as prostacyclin, epoprostenol, and sildenafil
  • an endothelin receptor antagonist such as bosentan
  • a calcium channel blocker such
  • the agents may be administered separately, at the same, or at different times of the day, or they may be administered in a single composition.
  • the present invention provides novel pharmaceutical formulations in which a RTK inhibitor is combined with one of the active agents discussed above and unit dose forms of those formulations.
  • each agent can be administered in an "immediate release” manner or in a "controlled release manner.”
  • the additional active agent is a vasodilator
  • any dosage form containing both active agents i.e., both the RTK inhibitor and the vasodilator, can provide for immediate release or controlled release of the vasodilator, and either immediate release or controlled release of the RTK inhibitor.
  • a pharmaceutical composition of the invention is a pharmaceutical formulation containing an active agent formulated in a manner compatible with its intended route of administration.
  • routes including but not limited to, oral, pulmonary, inhalational, sublingual, intranasal, parenteral, intradermal, transdermal, topical, transmucosal, subcutaneous, intravenous, intramuscular, intraperitoneal, buccal, rectal, and the like.
  • parenteral as used herein is intended to include subcutaneous, intravenous, and intramuscular injection.
  • compositions of the invention are prepared for oral administration and in an immediate release form suitable for once per day (QD) administration. Certain formulations are suitable for intranasal administration to a patient.
  • the pharmaceutical formulations of a RTK inhibitor useful in the methods of the invention can be prepared as a liquid or in a solid form such as a powder, tablet, pill or capsule for oral administration.
  • Liquid formulations of the invention may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the formulation is an aqueous solution.
  • the final formulation is lyophilized.
  • HHT Hereditary Hemorrhagic Telangiectasia
  • AVM arteriovenous vascular malformations
  • ENG encodedoglin
  • ACVRL1 ACVRL1
  • SMAD4 all members of the TGF- P/BMPR2 signaling pathway
  • BMPR2-signaling downstream target ID1 is not a common downstream target of all the three HHT genes (ALK1 , ENG, and SMAD4) knockdown in human pulmonary microvascular endothelial cells (PMVECs).
  • PMVECs human pulmonary microvascular endothelial cells
  • novel common downstream targets of all the three HHT-causing genes that were enriched for HHT-related biological process and signaling pathways.
  • LYVE1 , GPNMB, and MC5R were strong downstream targets that could serve as a better common downstream target than ID1 .
  • HHT disease signature a small molecule drug, Brivanib, that reversed the HHT disease signature, and inhibited VEGF-induced ERK1/2 phosphorylation, proliferation, and angiogenesis in PMVECs and inhibited some of the upregulated HHT disease genes in POLS.
  • PMVECs Human healthy control pulmonary microvascular endothelial cells (PMVECs) (Cat # C12281 , PromoCell GmbH, Heidelberg, Germany) were cultured in microvascular endothelial cell basal media (Cat # C-22120; PromoCell GmbH) supplemented with growth factors (Growth Medium MV SupplementPack, Cat # C-39220, PromoCell GmbH) and 100 U/mL Penicillin-Streptomycin Solution (Gibco) and used between passages 4 to 8. PMVECs were cultured under standard conditions (37°C, 5% CO 2 , 21 % O 2 , 90% humidity).
  • RNAi Human pulmonary microvascular endothelial cells of passages 4-6 were seeded at 150K cells/well onto 6-well plates and incubated at 37 Q C in a humidified 5% CO2 atmosphere. The next day, cells were washed with PBS and transfected with 50nM siRNAs against non-target controls, ACVRL1 (ALK1 ), ENG, SMAD4, LYVE1 , GPNMB or MC5R (Thermo Fisher Scientific, Waltham, MA), and 2ul of Lipofectamine RNAiMAX in a total 1 ml of OPTIMEM media. After 5 hrs of transfection, the medium was replaced with regular complete growth media.
  • a starvation medium (0.2% FCS media) was added and incubated for 16 hrs. Cells were then stimulated with 20ng/ml of BMP9 for 2 or 24 hrs, harvested for RNA isolation, and performed RNAseq analysis.
  • RNA sequencing (RNAseq). RNA was isolated using RNeasy Plus Kits (Qiagen, Gaithersburg, MD) as per the manufacturer's instructions. RNA samples were sent to the Novogene Corporation (Sacramento, CA), where the following steps were carried out: Quality control: Quality and integrity of total RNA were controlled on Agilent Technologies 2100 Bioanalyzer (Agilent Technologies; Waldbronn, Germany). Library construction: The RNA sequencing library was constructed using NEBNext® Ultra II RNA Library Prep Kit (New England Biolabs) according to the manufacturer's protocols.
  • Library quality control Library concentration was quantified using a Qubit 2.0 fluorometer (Life Technologies) and then diluted to 1 ng/ul before checking insert size on an Agilent Technologies 2100 Bioanalyzer (Agilent Technologies; Waldbronn, Germany). The library was then quantified to greater accuracy by quantitative PGR (qPCR). Sequencing: 30 million paired reads for each sample were acquired with the Illumina NovaSeq 6000 system. Data analysis. The quality of the RNA- seq data was examined by base sequence quality plots using FastQC. TrimGalore was used to trim the sequence reads. Then, the RNA-seq reads were aligned to the human genome (hg19) using the STAR software, and a gene database was constructed from Genecode v19. Differentially expressed genes (DEG) between groups were quantified using the DESeq2 R package.
  • DEG Differentially expressed genes
  • Biological process, pathway enrichment, and protein-protein network analysis Differentially expressed downstream genes of HHT knock-down (KD) conditions at 2 and 24h of BMP9 stimulations were uploaded on the ShinyGO 0.76 platform to analyze GO biological process enrichment. The same data gene signatures were uploaded separately onto the DAVID Bioinformatics Resources 6.8 server or GO pathway enrichment analysis. The identifier was set to gene symbol, and Homo sapiens was selected to limit annotations in the gene list and background list. A significant value of p ⁇ 0.05 was set as the cutoff criterion. STRING Network analysis was performed using the SinyGO 0.76 platform.
  • MTT assay (Cat # V13154, Invitrogen, Waltham, MA), and caspase-3/7 assay (Cat # G8090, Promega, Madison, Wl) were performed using commercially available kits as per the instructions to assess proliferation and apoptosis, respectively.
  • the bottom of a 96-well plate was coated with 50ul per well of Matrigel and incubated at 37 °C for 1 h.
  • PMVECs (1 x 10 4 cells/100 pl per well) suspended in a starvation medium were added to the Matrigel and cultured at 37 °C and 5% CO2 for 5 h.
  • Vessel tube-like structures were observed and photographed under a microscope with a digital imaging system. The data were analyzed using Imaged with an angiogenesis analyzer tool.
  • RNA isolation, RT-PCR, and qRT-PCR Total RNA was isolated from cells using a commercially available RNeasy® Plus Mini Kit (Cat # 74134, Qiagen, Hilden, Germany). For precision cut lung slices (PCLS), total RNA was extracted and purified using the TRizol RNA extraction protoco. The total RNAs were then converted to cDNA using a commercially available high-capacity cDNA Reverse Transcription Kit (Cat # 4368813, Applied BiosystemsTM, Foster City, CA) according to the manufacturer’s instructions.
  • the expression levels of mRNAs were quantified using TaqManTM 2x Universal PCR Master Mix (Cat # 4304437) and targeted Taqman probes (ALK1 , ENG, SMAD4, GAPDH, Hs02786624_g1 ; ID1 , Hs03676575_s1 ; HS3ST2, Hs00428644_m1 ; GPNMB, Hs01095679_m1 ; ANKRD33, Hs05002807_s1 ; RRAGD, Hs00222001_m1 ; LYVE1 , Hs00272659_m1 ; CPA4, Hs01040939_g1 ; SLC25A47, Hs01584239_m1 ; SHISA9, Hs04188640_m1 ; HIST1 H2BE, Hs00543841_s1 ; MC5R, Hs00271882_s1 ; FGF19, Hs00192780_m1 ; F
  • the lung slices were then cultured in 1 x DMEM GlutaMAX medium containing 10% fetal calf serum, penicillin/streptomycin (1 %), and amphotericin B (0.1 %) in the presence and absence of Brivanib at 10 or 50 pM in 1 mL of media in a 12-well tissue culture plate. After 24 hrs, the slices were washed with 1x PBS two times and then harvested for RNA isolation.
  • Id 1 is not a common downstream target of all three HHT genes but is specific for ALK1 and SMAD4 mediated signaling. This is a very important finding when it comes to identifying common downstream targets and subsequently predicting repurposed drugs that might increase the signaling for all three gene mutations.
  • HHT1 and HHT2 patients show similar clinical symptoms, a previous study had identified 277 downregulated and 62 upregulated common downstream targets genes in blood outgrowth endothelial cells isolated from HHT patients carrying ENG (HHT1 ), ALK1 missense or ALK1 non-sense mutations (HHT2) compared to healthy endothelial cells, which they called a “gene expression fingerprinting of HHT’’.
  • HHT1 blood outgrowth endothelial cells isolated from HHT patients carrying ENG
  • HHT2 ALK1 missense or ALK1 non-sense mutations
  • PMVECs no studies have identified common downstream genes of the three key HHT genes in PMVECs.
  • This transcriptomic gene expression signature served as blueprint for predicting potentially beneficial drugs that mimic the complementary “antisignature” using the Broad Institute’s database of transcriptional drug signatures, a part of the NIH LINCS program.
  • This webbased clue query app allows queries with external gene sets to identify compounds that either mimic our gene sets' transcriptional signatures or reverse the signature (anti-signatures) in different cell lines.
  • We narrowed down the drug list based on the findings from the HUVECs data sets as this is the closest cell line to our data sets acquired in PMVECs.
  • the drugs were ranked following the criteria of samples >3, tas values >0.20, raw connectivity score, and FDR values on the query app.
  • Top scoring 5 HHT and anti-HHT drugs are shown in Figure 4B. From the top anti-HHT drug candidates (negative values), we concentrated on Brivanib as it is well-known for its anti-FGF/VEGF activities. We hypothesized that Brivanib would on the one hand, reverse the dysfunctional downstream targets related to BMP signaling and on the other hand could inhibit the overactivated VEGF pathway in HHT.
  • Brivanib rescued the expression of the MC5R, SLC25A47, FGF19, SHISA9, and FRG2C that were downregulated in HHT gene KD conditions ( Figures 4G-K and figures 10A-C). These results demonstrated that Brivanib could improve the dysfunctional ALK1/ENG/SMAD4 signaling in HHT.
  • Brivanib inhibited the commonly upregulated downstream gene expression signatures of HHT-causing genes in ex vivo PCLS.
  • PCLS were prepared from a healthy human donor obtained from Donor Network West, California. PCLS were treated with 50 uM Brivanib or DMSO for 24 hrs and then harvested for RNA isolation (Figure 5A). Brivanib inhibited mRNA expression of LYVE1 .
  • GPNMB GPNMB
  • HS3ST2 in PCLS
  • Figures 5B-F This inhibition in GPNMB, LYVEI and HS3ST2 expression ex vivo was similar to the inhibition of those genes in PMVECs after HHT gene knockout and Brivanib treatment in vitro. Brivanib did not change expression of RRAGD and ANKRD33 in PCLS.
  • Brivanib inhibited VEGF-induced ERK1/2 MAPK, -VEGF-induced PMVECs proliferation and tube formation in vitro.
  • pro-angiogenic pathways such as VEGF signaling
  • PMVECs were cultured in the presence and absence of 10 pM Brivanib or DMSO in serum starvation media (0.2% FCS) for 24 hrs and then stimulated with 40 ng/mL VEGF for 10 mins.
  • VEGF vascular endothelial growth factor
  • ID1 a major downstream target of the TGF3/BMP signaling pathway, is not a common downstream target of all the HHT gene knockdown (ALK1 , ENG, or SMAD4) conditions, while other downstream targets, such as LYVE1 , GPNMB, PLXDC2, and MC5R, are downstream of all three HHT genes.
  • LYVE1 a small molecule drug
  • GPNMB a small molecule drug
  • PLXDC2 a small molecule drug
  • PTK Protein tyrosine kinase
  • EC endothelial
  • BMPR2 Bone Morphogenetic Protein receptor 2
  • BMPR2 Bone Morphogenetic Protein Receptor 2
  • BMPR2 Bone Morphogenetic Protein Receptor 2
  • PTKs PTKs with BMPR2 signaling
  • lymphocyte specific protein tyrosine kinase LOK
  • Src-family tyrosine kinase LCK Src-family tyrosine kinase LCK
  • the SrcB PTK LCK is the most BMPR2 activating kinase while the SrcA PTK FYN is the most BMPR2 repressive.
  • a High-Throughput screen demonstrates that Src-Family A PTKs are BMPR2- Repressive, and Src- Family B PTKs are BMPR2-Activating.
  • Id 1 as a transcription factor read-out for BMPR2 activation.
  • C2C12 mouse myoblastoma cell line stably transfected with a BMP response element Luciferase Id1 (BRE-Luc-ld1 ) reporter. Cells were systematically transfected with a murine-wide siRNA library targeting 22,124 genes and then treated with BMP4 in the Stanford High-Throughput Bioscience Center.
  • BMPR2 activity by luciferase expression was quantified by luminescence and (Figure 11 A) cell viability was assessed with a tryptan-blue stain. The mean change in Id1 expression and cell viability in response to knockout of one of the 22,124 genes across three replicates was calculated and presented in aggregate in Figure 1 1 B. Genes left of 0 on the x-axis reduce Id1 expression when targeted by si-RNA and genes to the right of 0 on the x-axis increase Id1 expression after transfection.
  • LCK is expressed at a mRNA and Protein level in endothelial cells of human pulmonary arteries and correlates with Id1 expression both in health and in disease.
  • LCK we decided to use LCK as the representative SrcB and FYN as the representative SrcA kinase.
  • SrcA kinases are widely expressed while SrcB kinases are restricted to specific cell lineages.
  • FYN has been studied in ECs and smooth muscle cells while LCK is traditionally described in T-Cells and NK-Cells.
  • LCK is expressed in cultured human PAECs and HUVECs. It remained unknown where LCK was expressed in the whole lung and if it was altered in disease.
  • Single molecule RNA in situ hybridization sm-FISH
  • Lek mRNA was expressed at low levels in the endothelium compared to higher expression in sparse Acta2/Cdh5 negative cells presumed to be lymphocytes ( Figure 12A).
  • LCK protein was identified in cultured human PAECs with perinuclear localization (Figure 12B). Human PAECs obtained from both PAH patients and donor controls expressed LCK at a protein level, but in a heterogeneous fashion (Figure 12C).
  • PAH Pulmonary Arterial Hypertension
  • HPAH Hereditary Pulmonary Arterial Hypertension
  • IPAH Idiopathic Pulmonary Arterial Hypertension
  • PDE5i Phosphodiesterase type 5
  • Inhibitor ERA Endothelin receptor antagonist
  • PC Prostacyclin
  • BMPR2 and Id1 was expressed in few PBMC cells ( ⁇ 20%), with low expression in all cell lineages save for dendritic cells. Due to poor co-expression of Lek, Id1, and BMPR2 in lymphocytes, we concluded that PAECs would be the best first lineage to study an interaction between BMPR2, Id1, Lek, and Fyn in the human lung. Finally, efforts were undertaken to investigate the expression of Lek protein in both mouse and rat ECs. We were able to recapitulate robust LCK staining in lymphocytes, but not in the ECs of mice or rats, as opposed to human ECs.
  • Lek knockout decreases SMAD1 phosphorylation and Id1 expression
  • Fyn knockout promotes BMP9 mediated SMAD1 phosphorylation and Id1 expression.
  • RNA sequencing was conducted in four groups of PAECs: siLCK, siFYN, a scrambled siRNA control group (NT), and a scrambled siRNA group subjected to BMP9 stimulation (NT+ 50 ng/mL BMP9 for 2 hours prior to harvest).
  • siLCK siLCK
  • siFYN siFYN
  • NT scrambled siRNA control group
  • BMP9 stimulation NT+ 50 ng/mL BMP9 for 2 hours prior to harvest.
  • siRNA and lipofectamine was used the same concentration of siRNA and lipofectamine to control for gene expression variability induced by the transfection process.
  • This transfection protocol resulted in a less stringent Lek knockdown relative to Fyn and NT and was less effective at reducing LCK protein relative to FYN.
  • RNA sequencing would have the sensitivity to detect major gene expression changes in the LCK condition. Knockout of target genes was verified by RT-qPCR.
  • RNA-seq analysis Quality control metrics for the RNA-seq analysis are shown.
  • Principal component analysis demonstrates tight clustering of within-group samples with separation between experimental groups.
  • Volcano plots of differentially expressed genes (DEGs) for siLCK and siFYN conditions are shown in Figure 15A.
  • Fyn was identified in the DESEQ2 analysis as a significantly downregulated gene in the siFyn case, Lek was not found to be significantly downregulated in the siLCK case due to dropout owing to a low number of raw counts for Lek (between 10-15) in each sample.
  • a volcano plot for the siNT+BMP9 condition is shown in Figure 21 .
  • Top upregulated genes for siLCK include Il6st, Ccl7, Wnt5a, and 111 b.
  • Top downregulated genes for siLCK are Ace, Lyvel , Gja5, and Dysf.
  • siFyn upregulated genes included Bmp4, Gdf7, Gdf11 (Bmp11 ), Gja5, Lyvel, Id1, and Tgfbl.
  • Genes downregulated in siFYN include Tlr3, C1s, C3, Ccl8, and Vcaml.
  • transcriptomic analysis of siLCK and siFYN PAECs finds that Lek and Fyn can both co-regulate canonical BMPR2 signaling and canonical NF-KB signaling.
  • the set of co-DEGs regulated by Lek and Fyn can be divided into an “endothelial” and “inflammatory” signature.
  • Figure 16A details the search strategy. Genes in the endothelial and inflammatory signatures were restricted to “Best INFerrred” or BING genes and ranked on the differential magnitude of change between siFYN and siLCK conditions. As it was unclear what number of genes would produce the best search results (between 10 and 150 genes can be included in a CMap search) we performed 18 total searches using an array (10, 15, 20, 25, 50, 75, 100, 125, and 150) of BING genes each for both endothelial and inflammatory signatures.
  • CMap data includes transcriptomic data from cells subject to either gene knockdown by short hairpin (SH) RNA or gene overexpression (OE) by plasmid.
  • SH short hairpin
  • OE gene overexpression
  • the heatmap body indicates how well the CMap gene signature connects with our si-signature in PAECs.
  • the vertical axis indicates the number of genes used in the CMap search ( Figure 16B).
  • the horizontal axis shows each CMap sh- or OE-experiment, grouped by cell type.
  • the “modulation” color coding indicates if the CMap experiment was a sh (blue) or an OE (yellow) manipulation.
  • “Acceptability” color coding is green when the CMap sh signature matches the PAEC si- signature or if the PAEC si-anti-signature matches the CMap OE signature.
  • HUVEC cells would behave similarly to PAECs and were included in drug prediction analysis.
  • two observations were made. First, many of the highest connection scores occurred in HUVECs, as with Regorafanib, Gsk- 2334470, and Bx-795. Second, it was noted that most drug influenced both signatures. For instance, Regorafanib has high positive connection with both the endothelial and inflammatory signatures. This finding was interesting and led us to wonder if compound selection should be informed by the endothelial and inflammatory signatures simultaneously. To do, we required a cell line where candidate compounds had both inflammatory and endothelial signatures.
  • AGS, and HEK293T cells met the requirements of Figure 16D and 16E. However, their shared compounds had low connectivity scores. Only the HUVEC line had compounds with high inflammatory and endothelial connectivity scores; compounds are shown in Figure 16G where distance from origin indicates strength of connection to both signatures. FDR and transcriptional activation score (TAS) are color coded to indicate statistical significance and repeatability across replicates respectively. Compounds in the top right corner are connected positively with the endothelial and inflammatory signatures, whereas compounds in the bottom left corner connect negatively to both signatures. Of the options, the top left corner is theorized to produce a desired “pro-endothelial and antiinflammatory” phenotype. It is notable that TKIs (denoted in blue) appeared to localize most to the top right corner.
  • the amino acid sequence of the kinase pocket also obeys the SrcA and SrcB phylogenic division and confers kinase-substrate specificity. This is suspected to prevent kinase cross-talk in regions where PTKs are in close spatial proximity, such as with LCK and FYN in the T-Cell Receptor.
  • SrcA and B kinases can be distinguished by genetic sequence, by kinase pocket amino acid sequence, and by how they support or inhibit cellular signaling pathways. It is notable that our agnostic high throughput screen shows that all SrcA and SrcB kinases modulate canonical BMPR2 signaling in the same way, albeit at different levels.
  • hypotheses include FYN and LCK kinases potentiating conformational changes in SMAD proteins or SMAD inhibitor proteins, FYN and LCK acting as a scaffolding proteins for membrane receptors, or FYN and LCK modulating the expression of BMP ligands within culture (due to our observation of increased BMP12 and BMP13 mRNA by RNA seq).
  • LCK and FYN are present in PAECs. Selective inhibition of the SrcB LCK can drive EC dysfunction. Studies have investigated the role of Src kinases in the pulmonary vasculature, emphasizing smooth muscle cells (SMCs). Notably, LCK is not found at high levels in SMCs and few studies have investigated LCK in ECs. Here, we show LCK is not only expressed at a message and protein level in PAECs, but knockout of Lek has functional consequences in human PAECs. Notably, we were unable to detect LCK protein or mRNA in the ECs of mice or rats, by staining or from public RNA seq data sets.
  • LCK and FYN are present in lymphocytes and LCK levels are reduced in PBMCs of patients with interstitial lung disease, lupus, and, as we have shown, pulmonary arterial hypertension.
  • PBMCs lymphocytes
  • lupus interstitial lung disease
  • pulmonary arterial hypertension pulmonary arterial hypertension.
  • LCK and Id 1 expression did not translate to the lung endothelium in PAH patients.
  • LYN is expressed in SMCs, future studies could use LYN as representative SrcB kinases, potentially comparing to the known effect of SRC.
  • CMap query output was dependent on the number of BING genes used in the search, with a unique number of search genes retrieving a maximal number of perturbagens across all cell lines - on the order of 5x10 5 .
  • endothelial and inflammatory gene signatures derived from PAECs could be both induced or suppressed by the same ligand, compound, or gene manipulation depending on the CMap cell line the perturbagen was used in. It is noted that many cell lines in CMap (and LINCS) are cancer lines whose genetic programs may be very different from healthy cell lines.
  • Brivanib, Quizartinib, and Regorafanib are used to validate our search strategy.
  • Brivanib interestingly targets VEGFR1 ,2 and FGFR1 , inhibiting angiogenesis in cancer models.
  • Quizartinib is a selective FLT3 inhibitor that also targets PDGFR, and VEGFR2 to a lesser degree.
  • Regorafanib is a multi-kinase inhibitor targeting VEGFR1 ,2, KIT, BRAF, and TIE1 among others.
  • Observed effects on canonical BMPR2 expression in quiescent large vessel PAECs by Brivanib show a role in the treatment of human pulmonary vascular diseases.
  • Lisitinib and Dabrafenib match the anti-inflammatory signature similar to the TKI Pazopanib already in clinical trial for HHT.
  • High Throughput siRNA Screerr High throughput siRNA screening of > 22,000 genes using an Id1 -BRE luciferase reporter assay in a C2C12 mouse myoblastoma cell line treated with or without 250 pM BMP4 was conducted in the Stanford High- Throughput Bioscience Center, as previously described [60]. Briefly, the C2C12 myoblastoma cells, a generous gift from Dr. Peter ten Dijke, were stably transfected with BRE- Id 1 linked to luciferase and used as a reporter cell line. Cells were screened on 384-well siRNA plates. The transfection conditions were optimized with BMP4 as stimulus, siBMPR2 and a toxic (siTox) control.
  • Luminescence and viability stain intensity was normalized to the overall average luminescence and viability stain intensity of the plate to calculate change in Id1 expression and change in viability. This assumes that there are few “hits” (e.g.
  • RNA Interference Both Lek and Fyn expression was modulated by RNAi in PA endothelial cells (PAEC).
  • PAEC PA endothelial cells
  • the Lipofectamine and siRNA containing OptiMem was combined. After letting stand for 20 minutes, this 500 yL was added existing OptiMem in 6 well plates. This resulted in a final concentration siRNA concentration of 80 nM.
  • si-Fyn the procedure was the same except 2 yL of Lipofectamine in 248 yL OptiMem and 2.5 L of siRNA (either Fyn or NT) in 247.5 yL OptiMem was used to make a final siRNA oncentration of 50 nM. All analyses for each gene were done by comparing to the same concentration of NT siRNA.
  • RNAseq experiments to control for transfection mediated gene effects, the same conditions used for si FYN above were used for the transfection of Fyn, Lok, and NT siRNA. 50 nM of siRNA and 2 yL Lipofectamine in 1000 yL OptiMem was used for all experimental arms.
  • RNA quality and concentration was measured using the Nanodrop system (Thermo-Fisher). The level of mRNA expression was quantified using Taqman primer/probe sets (Table 2) for the target and normalized to a housekeeping control (18s).
  • Apoptosis and Matrigel Tube Formation Assays Assays were conducted according to the manufacturer’s instructions and as previously described. Tube formation was assessed with the Angiogenesis Analyzer package in Imaged.
  • RNA Sequencing Human PAECs were grown to 80% confluency on gel coated plates and transfected with either Fyn, Lek, or nontargeted siRNA. At 48 hours, cells were placed in 0.2% FBS starvation media. Two hours prior to harvest all cells were treated with either PBS in 0.2% FBS media or, in one case, 20 ng/mL of BMP9 in 0.2% FBS starvation media. Cells were harvested and mRNA was extracted via Qiagen mini columns. RNA quality was assessed with the Agilent 2100 Bioanalyzer system and found to have a RIN of 9.8-9.9.
  • cDNA libraries were generated (Illumina), barcoded, and sequenced via a paired-end 150 bp sequencing strategy to a depth of between 38 and 46 million paired-end reads on the Illumina platform.
  • Raw reads were mapped to the human genome using STAR.
  • Quality control metrics regarding sequencing and alignment are given in Supplemental Figure 5-A.
  • Differential gene expression analysis was performed using the R package DEseq2. Briefly, differentially expressed genes were calculated via the “DEseq” command after setting transfection condition (FYN, LCK, NT) and treatment (PBS, BMP9) as factors. Conditions were contrasted against the nontargeted PBS condition to determine the fold change and adjusted p-value for each gene in each condition.
  • a variance stabilizing transformation of the raw count data was used to generate the gene expression heat map.
  • differentially expressed genes present in each condition were found using a cutoff l_og 2 fold change of 0.3 (fold change of less than 0.8 or greater than 1.25) with a false discovery rate of 5% and the R function “venn.diagram”.
  • Lists of significant differentially expressed genes that were present in select conditions were used for gene ontology analysis.
  • Gene ontology and transcription factor analysis was facilitated using the Enrichr GSEA web server.
  • the packages TRRUST, BART, and oPOSSUM-3 were used to predict transcription factors co-regulating the genes in query set. Results were tailed to show which transcription factors were predicted by different methods.
  • sm-FISH Single Molecule mRNA Fluorescent in situ Hybridization
  • Sm-FISH for Lek was performed using the RNA-scope protocol (Advanced Cell Diagnostics, Inc.) using the Channel 1 probe to Lek (Cat No. 440201 ).
  • tissue was counter stained for endothelial (VE-Cadherin, R&D AF938, 1 :300) and smooth muscle (Acta2, Direct a488 Conjugation, 1 A4 Clone, 1 :200). Tissue was imaged by confocal microscopy and analyzed with Imaged.
  • CMap The Connectivity Map (CMap) portal was used to perform all searches.
  • the genes returned by RNA seq were narrowed to only include CMap “Best INFerred” or BING genes. These are genes used in CMap that are computationally inferred with high confidence from the 978 genes actively profiled by the L1000 platform.
  • the differentially expressed genes were ranked based on the overall magnitude of change. Genes were ranked based on ⁇ #$». Gene with a positive A #j %& (e.g. increased with si-Lck, decreased with si-Fyn) comprised the “Inflammatory” signature whereas genes with a negative il ntai comprised the “Angiogenic” signature.
  • the search results were opened online in CMap Morpheus, and then downloaded as a GCT version 1 .3 file from the “query_result” tab.
  • a custom R script was used to open the 18 GCT files and combine them into a single data structure that can be subset by gene signature, gene search length, perturbagen class, search query correlation score, cell type, transcriptional activation score (TAS), and false discovery rate. This data set was used to generate Figures 8B-8G.
  • BMP9 for Brivanib and Quizartinib, either BMP9 at 10 ng/mL or PBS was added 2 hours prior to harvesting the cells. Cells were then harvested, run on SDS-Page, and immunoblotted for Id1 , pSMADI , pSMAD3 and eNOS.
  • Regorafenib and Pazopanib cells were grown in gelatin coated slide well chambers then treated with drug for 24 hours. Each slide contained a drug replicate and a DMSO only replicate.
  • slides were fixed in 10% NBF for 25 minutes, then blocked with 5% goat serum, 1 % BSA, and 0.3% Trition in PBS for 1 hour prior to incubating with p65-RelA primary antibody and Alexa-488 conjugated phalloidin overnight in the same buffer. Finally, cells were treated with DAPI and a Alexa-633 conjugated goat anti-rabbit secondary antibody at a dilution of 1 :250 for 1 hour. Slides were then imaged by confocal microscopy, collecting z-stacks at Nyquist spacing through the entire nuclear diameter. Between 4 and 6 images were collected of 4 experiments each. Images were analyzed with Imaris 3D processing software.
  • DAPI staining was used to define a 3D volume representing the nucleus.
  • the DAPI intensity threshold at which to call a given voxel intranuclear was adjusted on a per-experiment basis. This ensured that the average nuclear diameter for all baseline DMSO experiments were the same.
  • P65 RelA signal intensity was quantified on a per voxel basis for each voxel contained in the DAPI nucleus. This data was used to compute the average p65-RelA expression for the nuclear volume for each intact nucleus imaged. Average p65-RelA intensity and overall nuclear size for Pazopanib and Regorafenib treated cells were compared relative to their DMSO counterparts which were stained on the same slide with the same primary and secondary antibody buffers.

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Abstract

Compositions and methods are provided for the treatment of Hereditary Hemorrhagic Telangiectasia (HHT) and pulmonary arterial hypertension (PAH) by administration of an effective dose of a protein tyrosine kinase (PTK) inhibitor that inhibits VEGFR-2, reverses the HHT disease signature and improves endothelial dysfunction.

Description

USE OF A TYROSINE KINASE INHIBITOR FOR THE TREATMENT OF HEREDITARY HEMORRHAGIC TELANGIECTASIA AND PULMONARY ARTERIAL HYPERTENSION
CROSS REFERENCE TO OTHER APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application No. 63/427,015, filed November 21 , 2022, the contents of which are hereby incorporated by reference in its entirety.
GOVERNMENT RIGHTS
[0002] This invention was made with Government support under contract W81 XWH-19-1 - 0505 awarded by the Department of Defense and under contract HL128734 awarded by the National Institutes of Health. The Government has certain rights in the invention.
BACKGROUND
[0003] Cells have evolved protein kinases to facilitate signal transduction. 518 protein kinases are present in humans of which 90 are Protein Tyrosine Kinases (PTKs). PTKs subdivide into receptor (e.g. VEGFR) and non-receptor, “cytoplasmic” (e.g. SRC) families. Receptor-PTKs enable intercellular ligand-based communication whereas cytoplasmic-PTKs amplify external and internal signals and control functions like proliferation and motility. Many non-receptor PTKs are oncogenes (e.g. BCR-ABL) which has driven the development of Tyrosine Kinase Inhibitors (TKIs). TKIs are now approved for conditions ranging from cancer to pulmonary fibrosis. Though successful in treating disease, TKIs have mixed effects in pulmonary vascular biology.
[0004] Pulmonary arterial hypertension (PAH) is a rare disease characterized by progressive and fatal obliterative vasculopathy of the small pulmonary due to endothelial cell dysfunction (ECD) and apoptosis in concert with smooth muscle cell proliferation and perivascular inflammation. This occlusive vasculopathy ultimately leads to an increased pulmonary vascular resistance, right heart failure and death. Though limited by adverse reactions, the TKI Imatinib suppresses smooth muscle cell proliferation and improves clinical PAH, likely by acting on the PTKs PDGFR and c-Kit. Paradoxically, TKIs like Dasatinib, Bosutinib, Ponatinib, and Lorlatinib are either known causes of PAH or are implicated in PAH development. Dasatinib may achieve this by acting on additional PTKs outside of PDGFR and c-Kit, induction of reactive oxygen species (ROS) in pulmonary artery endothelial cells (PAECs), or impairing endothelial cell (EC) barrier permeability in a Rho-kinase dependent manner. In support of Dasatinib acting on a broader set of PTKs, a recent pharmacovigilance study showed the SRC family PTKs LCK, LYN, and YES are disproportionately associated with the development of PAH. One way to distinguish beneficial from deleterious PTKs in PAH is by comparing their transcriptomic profile with regards to endothelial dystfunction and inflammation.
[0005] Hereditary Hemorrhagic Telangiectasia (HHT) is a rare disease characterized by loss of function mutations in ENG, ACVRL1/ALK1 or SMAD4 resulting in the formation of arteriovenous malformations in the lung, brain, liver, gastrointestinal tract, nasal mucosa, and skin putting patients at risk for paradoxic emboli and stroke, pulmonary hemorrhage, cerebral bleeding, high output cardiac failure, severe anemia and nosebleeds as well as telangiectasia. Currently no FDA approved therapy exists that would correct the downstream signaling abnormalities of the HHT loss of function mutations and therefore might prevent the occurrence or growth of AVMs, or might initiate regression of disease.
[0006] Both diseases, HHT and PAH, have in common that they are caused by loss of function gene mutations of members of the TGF[3/BMPR2 signaling pathway. In HHT, mutations in ALK1 , ENG or SMAD4 are responsible for 85% of patients, while in PAH, mutations in BMPR2 but also occasionally in ALK1 or ENG are found. This suggests that the pathomechanisms of both diseases might be related which has implications for therapeutic approaches.
[0007] A goal is to identify drugs that correct the downstream signaling abnormalities of these loss of function mutations present in HHT and PAH and thereby prevent, halt or reverse the disease.
SUMMARY
[0008] Compositions and methods are provided for the treatment of Hereditary Hemorrhagic Telangiectasia (HHT) and Pulmonary Arterial Aypertension (PAH) by administration of an effective dose of a protein tyrosine kinase (PTK) inhibitor. In some embodiments the inhibitor acts on vascular endothelial growth factor receptor-2 (VEGFR-2). In some embodiments the inhibitor acts of VEGFR-2 and fibroblast growth factor receptor-1 (FGFR-1 ). In some embodiments the inhibitor acts on FLT3, PDGFR, and VEGFR2. In some embodiments the inhibitor is Brivanib. In some embodiments the inhibitor is Dabrafenib or Linsitinib.
[0009] In an embodiment, an individual with HHT is treated by administration of an effective dose of an agent that inhibits at least VEGFR-2, and may additionally inhibit FGFR-1 , where the treatment improves clinical indicia of the disease. In some embodiments the agent is Brivanib. It is shown herein that Brivanib can reversed an HHT disease signature of gene expression, and can inhibit othere disease features. In another embodiment the agent is dabrafenib. In another ambodiment the agent is linsitinib.
[0010] The treatment of HHT can normalize gene expression in endothelial cells in which HHT causing genes are knocked down, e.g. the treatment is capable of reducing expression of one or more of the genes LYVE1 , GPNMB, RRAGD and ANKRD33; and/or increasing expression of one or more of the genes MC5R, SLC25A47, FGF19, SHISA9, FRG2C, and HS3ST2. The treatment can inhibit VEGF-induced ERK1/2 phosphorylation, proliferation, and angiogenesis in the targeted cells, e.g. lung microvessel endothelial cells, and also inhibit the expression of one or more of the genes upregulated in the HHT disease condition: LYVE1 , GPNMB, and HS3ST2 in the targeted tissues, e.g. lung tissue. In some embodiments the PTK can inhibit an inflammatory gene expression signature disclosed herein. In some embodiments the individual is monitored for efficacy of treatment, e.g. by determining an inflammatory signature change, determining changes in an HHT disease signature, changes in gene expression, and the like.
[001 1] In an embodiment, an individual with PAH is treated by administration of an effective dose of an agent that increases ID1 expression, a common downstream target of the BMPR2 pathway, where the treatment improves clinical indicia of the disease. In some embodiments the agent is Brivanib. . In some embodiments the inhibitor is Dabrafenib or Linsitinib. The treatment can provide for an pro-endothelial-like pattern of gene expression in targeted cells, in contrast to an inflammatory pattern of gene expression. In some embodiments the individual is monitored for efficacy of treatment, e.g. by determining a pro-endothelial expression change, changes in Id1 gene expression, and the like. . In some embodiments the inhibitor increases Id1 expression. In some embodiments the inhibitor activates the endothelial transcriptomic signature.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.
[0013] Figure 1 A-1 G. ID1 is not a common downstream target of ENG in PMVECs. A) Cartoon of BMP9 signaling in HHT. B) Western blot verification of BMP9 in PMVECs. C-E) siRNA- mediated knockdown verification of ALK1 , ENG, and SMAD4 by qPCR. F and G) Validation of BMP9 signaling in ALK1 , ENG, and SMAD4 knockdown conditions in PMVECs by qPCR (read out I D1 ).
[0014] Figure 2A-2E. Common downstream gene signatures of ALK1 , ENG and SMAD4 mutations enriched for AVM and HHT related biological processes and cell signaling pathways. A and B) Venn diagram of common upregulated or downregulated genes following knockdown of ALK1 , ENG and SMAD4 in PMVECs stimulated with BMP9 (20ng/ml) for 2 or 24hrs (RNAseq). C) Heatmap of the common upregulated and downregulated genes following knockdown of ALK1 , ENG and SMAD4 in PMVECs stimulated with BMP9 for 2 and 24hrs. D) SinyGo biological processes analysis of the common upregulated and downregulated genes following knockdown of ALK1 , ENG and SMAD4 in PMVECs stimulated with BMP9 for 2 and 24hrs. E) Panther pathway analysis of the common upregulated and downregulated genes following knockdown of ALK1 , ENG and SMAD4 in PMVECs stimulated BMP9 for 2 and 24hrs.
[0015] Figure 3A-3C. Identifying common persistently dysregulated downstream gene signatures of the HHT gene knockdowns in PMVECs. A) Venn diagram of the common persistent upregulated and downregulated downstream gene signatures between 2 and 24 hrs of BMP9 stimulation following HHT gene knockdown. B) qRT-PCR validation of the common persistent upregulated and downregulated downstream gene signatures at 2 hrs of BMP9 stimulation following HHT gene knockdown. C) qRT-PCR validation of the common persistent upregulated and downregulated downstream gene signatures at 24 hrs of BMP9 stimulation following HHT gene knockdown.
[0016] Figure 4A-4K. Drug prediction based on the common upregulated downstream targets after ALK1 , ENG and SMAD4 knockdown and experimental validation in PMVECs. A) Experimental strategy: Clue database drug prediction based on upregulated genes after knockdown of the three HHT genes and stimulation with BMP9 for 2 and 24hrs (HHT disease signature). B) top scoring 5 HHT (positive values) and anti-HHT drugs (negative values) are represented. (C-K) Effect of Brivanib on the expression of the common persistent downstream targets after ALK1 , ENG and SMAD4 knockdown was assessed by qRT-PCR in PMVECs.
[0017] Figure 5A-5F. Effect of Brivanib on expression of upregulated disease signature genes in HHT in ex vivo healthy human PCLS. A) PCLS protocol. B) Expression of LYVE1 , GPNMB, HS3ST2, RRAGD and ANKRD33 were measured by qRT-PCR following 24hrs treatment of Brivanib in PCLS (B-F).
[0018] Figure 6A-6D. Brivanib inhibits VEGF-induced pERK1/2, -proliferation, and angiogenesis in PMVECs. A) PMVECs were treated with 10uM Brivanib or DMSO for 24 hrs followed by 20ng/mL VEGF or PBS for 10 mins. Protein was harvested and the effect of Brivanib on VEGF-induced phosphorylation of ERK1/2 was assessed in PMVECs by western blotting. B) The effect of Brivanib on VEGF-induced cell proliferation was assessed by MTT assay in PMVECs. C) Angiogenesis was assessed in a Matrigel tube formation assay using PMVECs treated with either Brivanib 1 , 10, 50 uM or DMSO. D) Proposed model for the mechanism by which Brivanib might influence AVM and HHT pathogenesis.
[0019] Figure 7A-7C. Functional consequence of ALK1 , ENG, and SMAD4 knockdown in PMVECs (proliferation, apoptosis and tube formation).
[0020] Figure 8A-8B. STRING network analysis of the common upregulated and downregulated genes following knockdown of ALK1 , ENG and SMAD4 in PMVECs stimulated [0021] Figure 9A-9G. Validation of common upregulated or downregulated genes signatures with BMP9 stimulation at 2 (A and B) or 24 hrs (C and D) and functional consequences of the knockdown of LYVE1 , GPNMB and MC5R in PMVECs. After 72 hrs knockdown of LYVE1 , MC5R, and GPNMB PMVECs proliferation (E), apoptosis (F), tube formation (G) were assessed.
[0022] Figure 10A-10C. Effect of Brivanib on the expression of the common persistent downstream targets after ALK1 , ENG and SMAD4 knockdown were assessed by qRT-PCR in PMVECs (A-C).
[0023] Figure 11 A-1 1 E A High-Throughput Screen (HTS) using a C2C12 mouse myoblastoma ZcZ1-Luciferase Reporter cell lines finds Src-Family A PTKs are inherently BMPR2-Repressive, and Src-Family B PTKs are inherently BMPR2-Activating. (A) HTS treatment and assessment strategy. (B) Change in ZdZ-linked luciferin expression and cell viability after gene knockout across 22,124 gene. Red dots = major PTK families. (C) Change in ZdZ-linked luciferin expression in selected receptor and non-receptor PTKs. Bars = mean ± standard deviation (SD). Fyn and Lek generate the greatest change in Id1 expression. (D) Change in Zc/7-linked luciferin expression for Src-Family PTKs. Bars: mean ± SD . Overall magnitude of Id1 change correlates to the PTK’s sub-family (shown in (E), reconstructed from data in Src-A PTK knockout increases and Src-B PTK knockout decreases Id1 expression.
[0024] Figure 12A-12E: Lek is expressed in the endothelium of PAECs and correlates with Id1 expression. SC-RNAseq of human PBMCs suggests Bmpr2 and Id1 is low in lymphocytes that have high expression of Lek or Fyn. (A) smFISH in human lung for Lek mRNA (white), VE-Cadherin (magenta, EC Marker), Acta2 (green, Smooth Muscle Marker), and DAPI (blue, nuclear marker). LcA/VE-Cadherin colocalizes in pulmonary arteries. Lek- Hi, VE- Cadherin/ Acta2 negative cells (arrow) likely represent lymphocytes. Bars = 20 jim. (B) Perinuclear Lek expression in human PAECs. Bar = 50 gm. (C-D) Immunoblotting of protein lysate from human PAECs from explanted lungs of donor controls and PAH patients showing correlation between Lek and Id 1 expression. (E) SC-RNAseq analysis of 2700 PBMCs from a healthy donor (full analysis Sup Figure 1 ) using the Seurat SC-RNA seq pipeline. Lek and Fyn are highly expressed in Cd4+ and Cd8+ T-Cells, and NK Cells. However, BMPR2 and Id1 are highly expressed in Dendritic Cells. Id2 rather than Id1 or Id3 is highly expressed in lymphocytes, NK cells, and dendritic cells. Dot size indicates percent of cells expressing gene of interest (cutoff of >1 count per cell). Average Expression is log-scaled Z-score of counts for each gene in each cluster.
[0025] Figure 3A-13E: Silencing of the SrcB LCK in human PAECs suppresses canonical BMPR2 signaling, whereas silencing the SrcA FYN supports it. (A) Human PAECs transfected with LCK targeting (siLCK) or control (siNT) siRNA and lysed at 72 hours in full media (2% FBS) have decreased Lek, ld1, and Gapdh by RT-qPCR using the relative standard method. Snail was significantly increased. 1 -Way ANOVA with Tukey’s Rage Test, n = 4. (B). siLCK PAECs have lower LCK expression at 72h. (C). SiLCK PAECs have significantly decreased BMP9 mediated phosopho-SMAD1 and Id1 expression. N = 6, 2-way ANOVA for treatment (PBS, TGF-pi and BMP) and transfection (siNT vs. siLCK) by Tukey’s Range Test. (D) PAECs transfected with siFYN or siNT for 6 hours, starved at 24 hours in 0.2% FBS, harvested at 72 hours, have significantly decreased Fyn, but increased Id1 and Bmpr2 using RT-qPCR by relative standard method with normalization to 18s RNA. Analysis by 1 -way ANOVA, n = 4. (E) siFYN PAECs maintained in full media (2% FBS), changed to starvation media (0.2% FBS) 24 hours after transfection, or changed to starvation media then given BMP9 or PBS 1 .5 hours prior to cell lysis. Fyn was significantly reduced at 72 hours. There was no change in Phospho- SMAD1 activity (pSMADI ). There was significant reduction in phospho-SMAD3 in siFYN, BMP9-treated cells and a significant increase in Id 1 . 2-way ANOVA examining treatment (2% FBS, 0.2% FBS + PBS, 0.2% FBS+BMP9) and transfection (si-NT vs. si-Fyn) followed by Tukey’s Range Test. For all above ANOVA tests, normality checks for ANOVA residuals and Levene’s test for homogeneity were carried out and assumptions are met.
[0026] Figure 14A-14F: Inhibition of the SrcB Lek promotes EC dysfunction, whereas inhibition of the SrcA Fyn does not. siLCK and siFYN PAECs are compared to matching control conditions (termed si-NT-L or si-NT-F). Cells were passaged to Matrigel in full media (2% FBS) 48 hours after transfection and imaged 3 hours later. (A) Raw (left) and analyzed (right) brightfield images are shown. (B) Total tube length and junctions are decreased in siLCK PAECs. Conversely, siFYN PAECs have increased total tube length and junctions. N = 4 experiments. (C) siLCK PAEC cell counts in culture decrease over time. (D) RT-qPCR by relative standard methods in siLCK and siFYN PAECs in starvation media (0.2% FBS) at 72 hours shows a significant increase for Bax and Bel mRNA relative to 18s mRNA. 1 -Way ANOVA with Tukey’s range test. N = 4. (E) Increased (siLCK vs. siNT-L) PAECs and decreased (siFYN vs. siNT-F) Caspase 3/7 activity at 48 hours post transfection in full (2% FBS) media. (F) siLCK PAECs had increased expression of VCAM1 despite minimal reduction in Lek protein 72 hours after transfection in full (2% FBS) media. Statistical comparison with Student’s T-Test.
[0027] Figure 15A-15H: Whole-transcriptome analysis finds Lek knockout suppresses and Fyn knockout promotes an “endothelial” gene signature (including the BMPR2 pathway). Conversely, Lek knockout increases and Fyn knockout suppresses an “inflammatory” gene program centered on NF-KB / RelA signaling. (A) DEGs in siLCK and siFYN PAECs. (B) Normalization of raw counts by variance stabilizing transformation with unsupervised hierarchical clustering and heatmap generation. Genes increased in siFYN and BMP9 and decreased in siLCK correlate with canonical BMPR2 signaling (e.g. Bmpr2 and Id1). Genes increased by siLCK and decreased in siFYN or BMP9 include inflammatory genes Cxcl3, 11-6, Ccl8, Vcaml, and Icaml. (C) Selected genes important in EC biology. siLCK PAECs have reduced markers of EC identity, increased markers of EC dysfunction, and favor NF-KB over BMPR2 canonical signaling. (D) DEGs co-regulated in siLCK and siFYN. 671 genes increase in siLCK and decrease in siFYN (Group E, “Inflammatory” signature). 481 decrease in siLCK and increase in siFYN (Group F, “Endothelial” signature.) (E) “Inflammatory” signature GO analysis points to interferon-p, interferon-y, and lipopolysaccharide pathways. Transcription factor (TF) analysis shows DEGs center on canonical NF-KB/p65 RelA signaling. (F) “Angiogenic” GO analysis points to EC migration, adherence, junction organization, and sprouting angiogenesis. Multiple TFs are implicated. (G,H) IF staining of siLCK, siNT-L, siFYN, and siNT-F PAECs verifies increased pp65 RelA nuclear co-localization in siLCK PAECs. Statistical analysis with 1 -Way ANOVA.
[0028] Figure 16A-16G: Using endothelial and inflammatory signatures, CMap searches validate ligands and enable drug prediction in specific cell lines. (A) Search strategy for perturbagens in the CMap data. (B) Results for n = 18 searches. Increasing BING genes increases the number of perturbagens found with a false discovery rate (FDR) of < 0.05. Perturbagens with an FDR of < 0.05 linearly correlates with number of inflammatory genes, but peak at a gene list length of 75 for the endothelial signature. (C) Ligands with connectivity scores (CS) matching the inflammatory and endothelial signatures across select cell lines are shown. Red indicates ligands of interest. (D) CMap experiments (heatmap columns) in cell lines (column groupings) which altered Fyn expression by sh-RNA (blue in “CMap Fyn Modulation”) or overexpression (gold in “CMap Fyn Modulation”). In heatmap body, blue indicates positive CS with the si-Fyn signature and gold indicates negative CS with si-Fyn. Heatmap column colors matching “CMap Fyn Modulation” color indicate behavior similar to siFYN PAECs, as with SW480 cells. HCC515 cells indicate opposite behavior. Red asterisk indicates cells matching the PAEC siFYN signature. (E) Similar analysis as D but instead using Lek. ES2 with sh-Lck have a similar gene signature to siLCK PAECs, VCAP have an opposite signature. (F) Top compounds with high connectivity to endothelial (top) and inflammatory (bottom) signature/anti-signature in appropriate cell lines. (G) Compounds with high CS in both the endothelial and inflammatory signatures. TKIs are denoted with blue text.
[0029] Figure 17A-17D: Brivanib and Quizartinib increase and decrease PAEC Id1 expression, whereas Regorafanib increases PAEC NF-KB activity. (A) PAECs treated with drug or DMSO for 24 hours then given BMP9 or PBS 2 hours prior to harvest significantly increase Id1 expression with Brivanib alone or Brivanib plus BMP9 versus DMSO. Quizartinib decreases Id1 in all cases. Statistics by 2-way ANOVA investigating the interaction between drug and BMP9 treatment with Tukey’s Range Test, n = 3. (B) PAECs treated with Regorafenib, Pazopanib, or DMSO for 24 hours in full (2% FBS) media were fixed and stained for p65 RelA, F-Actin, and DAPI. DMSO controls were paired on the same slide to control for irregularities in the staining. Cell barrier integrity was compromised in Regorafanib treated cells. (C) The average (average of p65 intensity across each voxel contained in the nucleus) and maximum nuclear p65-RelA expression was increased in Regorafenib treated PAECs relative to DMSO. Nuclear size was significantly decreased in Regorafenib treated cells relative to DMSO treated cells. Statistics by Student’s two-tailed T-test. Supplementary Figure 2: STRING network analysis of the common upregulated and downregulated genes following knockdown of ALK1 , ENG and SMAD4 in PMVECs stimulated with 2h and 24h of BMP9.
[0030] Figures 18A-18C: (A) UMAP plot of all 2700 human PBCMs after unsupervised clustering by the Seurat single cell RNA seq pipeline. Nine distinct clusters are identified. Clusters have been annotated for cell identity based on the gene expression detailed in (2 B- C). (B) Dot plots showing expression of canonical genes known to be expressed in specific cell types which informs cluster identity. Cd3 denotes T-cells, Cd8 indicates CD8+ T-Cells, Klrdl denotes natural killer cells, Cd19 and Ms4a1 denotes B-Cells, Itgae, HLA-CQA1, and Itgax denote dendritic cells, Fcgr3a denotes monocytes, Pf4 indicates platelets, and Cd14 denotes monocytes. Dot size indicates percent expression of the gene (the number of cells in the cluster that have > 1 counts of a given gene). Average expression is the Z-score of the log-normalized counts of a gen gene within the cluster. (C) Feature plots of all genes of interested examined in PBMCs for correlation with the clusters of A.
[0031] Figures 19A-19J: (A) Knockout of Lek in PAECs suppresses canonical BMPR2 signaling in different media conditions. PAECs were transfected with siRNA targeting Lek (si- Lck) or a nontargeting siRNA (siNT) for 6 hours. 24 hours after transfection, the cells were either starved (0.2% FBS) or kept in full media (2% FBS). 1.5 hours prior to cell lysis, the starvation cells were either treated with PBS or 20 ng/mL of BMP9. Cell lysate was run on SDS-PAGE. (B-D) Phospho-SMAD1 was significantly upregulated by BMP9 treatment and was significantly reduced in the si-Lck group. Id1 was significantly reduced by siLck in both the full media condition (2% FBS) and the BMP9 case. 2-Way ANOVA for the interaction between treatment (Full Media, Starvation Media, Starvation Media + BMP9) and transfection (si-Lck and si-NT) with Tukey’s Range Test was used to determine significance. Normality checks for ANOVA residuals and Levene’s test for homogeneity were carried out and assumptions met. (E) Knockout of FYN in full media conditions increases phospho-SMAD 1 activity even in the setting of TGF-p treatment in full media conditions. PAECs were transfected with siRNA targeting either Fyn (si-Fyn) or a nontargeting siRNA (siNT) for six hours. These cells were maintained in full media (2% FBS) for 72 hours prior to cell lysis. At 1.5 hours prior to cell lysis, cells were treated with either PBS or 10 ng/mL of TGF-p. (F-J) There were significant reduction in Fyn for all the si-Fyn cases. Treatment with si-Fyn significantly increased phospho-SMAD1 in both the PBS and TGF-p conditions. There was no significant change in phospho-SMAD3. [0032] Figures 20A-20B: Quality control metrics for the RNA seq analysis detailed in Figure 5. (A) Shows the distribution of RIN, Total Raw Reads, Clean/Raw Reads [%], GC Content [%], Q30 [%], and % of Reads Mapped by STAR. (B) Principal component analysis of all samples used in analysis shows that all conditions separate with replicates clustering together
[0033] Figures 21 A-21 C: Detailed volcano plot of differentially expressed genes in the (A) siLCK, (B) siFYN, and (C) siNT+BMP9 cases relative to the siNT case. Note that Eln is not depicted to allow a better view of all genes on the si-LCK plot.
[0034] Figure 22: “Inflammatory” and “Endothelial” gene signatures. Extended list of genes co-differentially expressed and are increased by si-Lck and decreased si-Fyn. Because si-Lck imparts a dysfunctional EC phenotype, genes in this list are implicated in EC dysfunction. Genes in this list are increased by Le suppression and decreased by Fyn suppression. The accompanying gene ontology analysis shows that multiple pathways are implicated that represent a response to interferon signaling, lipopolysaccharide, NF-KB signaling, or neutrophil transmigration.
[0035] Figure 23: Full results for Connectivity Map (CMap) analysis detailing ligands which recapitulate the inflammatory and endothelial signatures. Data for all cell lines present in CMap data is shown. Y axis indicates significance by -Logw(False Discovery Rate) and X axis indicates the Connectivity Score.
[0036] Figure 24. Extended data of CMap search results.
DETAILED DESCRIPTION
[0037] Before the present methods and compositions are described, it is to be understood that this invention is not limited to particular method or composition described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
[0038] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. [0039] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It is understood that the present disclosure supercedes any disclosure of an incorporated publication to the extent there is a contradiction.
[0040] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the peptide" includes reference to one or more peptides and equivalents thereof, e.g. polypeptides, known to those skilled in the art, and so forth.
[0041] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present patent application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
[0042] The methods disclosed herein provide for treatment of HHT or PAH. Dosage and frequency may vary depending on the half-life of the agent in the patient. It will be understood by one of skill in the art that such guidelines will be adjusted for the molecular weight of the active agent, the clearance from the blood, the mode of administration, and other pharmacokinetic parameters. The dosage may also be varied for localized administration, e.g. intranasal, inhalation, etc., or for systemic administration, e.g. i.m., i.p., i.v., oral, and the like.
[0043] An active agent can be administered by any suitable means, including topical, oral, parenteral, intrapulmonary, and intranasal. Oral is peferred. Parenteral infusions include intramuscular, intravenous (bolus or slow drip), intraarterial, intraperitoneal, intrathecal or subcutaneous administration. An agent can be administered in any manner which is medically acceptable. This may include injections, by parenteral routes such as intravenous, intravascular, intraarterial, subcutaneous, intramuscular, intratumor, intraperitoneal, intraventricular, intraepidural, or others as well as oral, nasal, ophthalmic, rectal, or topical. Sustained release administration is also specifically included in the disclosure, by such means as depot injections or erodible implants.
[0044] As noted above, an agent can be formulated with an a pharmaceutically acceptable carrier (one or more organic or inorganic ingredients, natural or synthetic, with which a subject agent is combined to facilitate its application). A suitable carrier includes sterile saline although other aqueous and non-aqueous isotonic sterile solutions and sterile suspensions known to be pharmaceutically acceptable are known to those of ordinary skill in the art. An "effective amount" refers to that amount which is capable of ameliorating or delaying progression of the diseased, degenerative or damaged condition. An effective amount can be determined on an individual basis and will be based, in part, on consideration of the symptoms to be treated and results sought. An effective amount can be determined by one of ordinary skill in the art employing such factors and using no more than routine experimentation.
[0045] An agent can be administered as a pharmaceutical composition comprising a pharmaceutically acceptable excipient. The preferred form depends on the intended mode of administration and therapeutic application. The compositions can also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.
[0046] As used herein, compounds which are "commercially available" may be obtained from commercial sources including but not limited to Acros Organics (Pittsburgh PA), Aldrich Chemical (Milwaukee Wl, including Sigma Chemical and Fluka), Apin Chemicals Ltd. (Milton Park UK), Avocado Research (Lancashire U.K.), BDH Inc. (Toronto, Canada), Bionet (Cornwall, U.K.), Chemservice Inc. (West Chester PA), Crescent Chemical Co. (Hauppauge NY), Eastman Organic Chemicals, Eastman Kodak Company (Rochester NY), Fisher Scientific Co. (Pittsburgh PA), Fisons Chemicals (Leicestershire UK), Frontier Scientific (Logan UT), ICN Biomedicals, Inc. (Costa Mesa CA), Key Organics (Cornwall U.K.), Lancaster Synthesis (Windham NH), Maybridge Chemical Co. Ltd. (Cornwall U.K.), Parish Chemical Co. (Orem UT), Pfaltz & Bauer, Inc. (Waterbury CN), Polyorganix (Houston TX), Pierce Chemical Co. (Rockford IL), Riedel de Haen AG (Hannover, Germany), Spectrum Quality Product, Inc. (New Brunswick, NJ), TCI America (Portland OR), Trans World Chemicals, Inc. (Rockville MD), Wako Chemicals USA, Inc. (Richmond VA), Novabiochem and Argonaut Technology.
[0047] Compounds can also be made by methods known to one of ordinary skill in the art. As used herein, "methods known to one of ordinary skill in the art" may be identified though various reference books and databases. Suitable reference books and treatises that detail the synthesis of reactants useful in the preparation of compounds of the present invention, or provide references to articles that describe the preparation, include for example, "Synthetic Organic Chemistry", John Wiley & Sons, Inc., New York; S. R. Sandler et al., "Organic Functional Group Preparations," 2nd Ed., Academic Press, New York, 1983; H. O. House, "Modern Synthetic Reactions", 2nd Ed., W. A. Benjamin, Inc. Menlo Park, Calif. 1972; T. L. Gilchrist, “Heterocyclic Chemistry”, 2nd Ed., John Wiley & Sons, New York, 1992; J. March, “Advanced Organic Chemistry: Reactions, Mechanisms and Structure”, 4th Ed., Wiley-lnterscience, New York, 1992. Specific and analogous reactants may also be identified through the indices of known chemicals prepared by the Chemical Abstract Service of the American Chemical Society, which are available in most public and university libraries, as well as through on-line databases (the American Chemical Society, Washington, D.C. may be contacted for more details). Chemicals that are known but not commercially available in catalogs may be prepared by custom chemical synthesis houses, where many of the standard chemical supply houses (e.g., those listed above) provide custom synthesis services.
[0048] The active agents of the invention and/or the compounds administered therewith are incorporated into a variety of formulations for therapeutic administration. In one aspect, the agents are formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and are formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. As such, administration of the active agents and/or other compounds can be achieved in various ways, usually by oral administration. The active agents and/or other compounds may be systemic after administration or may be localized by virtue of the formulation, or by the use of an implant that acts to retain the active dose at the site of implantation.
[0049] In pharmaceutical dosage forms, the active agents and/or other compounds may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds. The agents may be combined, as previously described, to provide a cocktail of activities. The following methods and excipients are exemplary and are not to be construed as limiting the invention.
[0050] For oral preparations, the agents can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
[0051] Formulations are typically provided in a unit dosage form, where the term "unit dosage form," refers to physically discrete units suitable as unitary dosages for human subjects, each unit containing a predetermined quantity of active agent in an amount calculated sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the unit dosage forms of the present invention depend on the particular complex employed and the effect to be achieved, and the pharmacodynamics associated with each complex in the host.
[0052] The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are commercially available. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are commercially available. Any compound useful in the methods and compositions of the invention can be provided as a pharmaceutically acceptable base addition salt. "Pharmaceutically acceptable base addition salt" refers to those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Preferred inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.
[0053] Depending on the patient and condition being treated and on the administration route, the active agent may be administered in dosages of 0.01 mg to 500 mg /kg body weight per day, e.g. about 20 mg/day for an average person. Dosages will be appropriately adjusted for pediatric formulation. For example, the dose of brivanib for an adult may be a flat dose of from about 50 to about 1000 mg/day, from about 100 to about 900 mg/day, from about 200 to about 800 mg/day, from about 400 to about 800 mg/day, from about 600 to about 800 mg/day. Agents approved for clinical use may be administered at conventional doses, or at less than conventional doses, e.g. reduced by 25%, by 50%, by 75%, by 90%, or more, relative to, for example, doses administered for cancer treatment.
[0054] In some embodiments, pharmaceutical compositions can also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized SepharoseTM, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes). [0055] A carrier may bear the agents in a variety of ways, including covalent bonding either directly or via a linker group, and non-covalent associations. Suitable covalent-bond carriers include proteins such as albumins, peptides, and polysaccharides such as aminodextran, each of which have multiple sites for the attachment of moieties. The nature of the carrier can be either soluble or insoluble for purposes of the invention.
[0056] Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyidimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions, such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants, such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG). Formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
[0057] The active ingredients may also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
[0058] Compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-1 19, 1997. The agents of this invention can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient. The pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration. [0059] Toxicity of the active agents can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used in further optimizing and/or defining a therapeutic dosage range and/or a sub-therapeutic dosage range (e.g., for use in humans). The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.
[0060] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymer.
[0061] As used herein, the term "nucleic acid" refers to polynucleotides such as deoxyribonucleic acid (DNA) and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and, as applicable to the embodiment being described, single-stranded (sense or antisense) and double- stranded polynucleotides. Chromosomes, cDNAs, mRNAs, rRNAs, and ESTs are representative examples of molecules that may be referred to as nucleic acids.
[0062] As used herein, the term "gene" has its meaning as understood in the art. The term "gene" may include gene regulatory sequences, where examples of regulatory sequences include but are not limited to promoter, enhancers, introns etc., and may refer to genomic sequences, RNA or cDNA. For the purposes of the current disclosure the term "gene" encompasses nucleic acids that can code for a polypeptide (mRNA), as well as nonpolypeptide coding RNAs. Examples of non-coding RNAs encoded by the genes relevant to the current disclosure include, but are not limited to, transfer RNA (tRNA), rRNA, microRNA (miRNA), long non- coding RNAs (lincRNAs), circular RNAs (circRNAs) or any other kind of RNA.
[0063] The phrase "level of expression" refers to the level of mRNA, as well as pre-mRNA nascent transcript(s), transcript processing intermediates, mature mRNA(s), and degradation products, encoded by a gene in the cell. The phrase "level of expression" also refers to the level of protein or polypeptide in a cell.
[0064] The term "sequence identity," as used herein in reference to polypeptide or DNA sequences, refers to the subunit sequence identity between two molecules. When a subunit position in both of the molecules is occupied by the same monomeric subunit (e.g., the same amino acid residue or nucleotide), then the molecules are identical at that position. The similarity between two amino acid or two nucleotide sequences is a direct function of the number of identical positions. In general, the sequences are aligned so that the highest order match is obtained. If necessary, identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux et al., Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, FASTA (Atschul et al., J. Molecular Biol. 215:403, 1990).
[0065] The term “isolated” refers to a molecule that is substantially free of its natural environment. For instance, an isolated protein is substantially free of cellular material or other proteins from the cell or tissue source from which it is derived. The term refers to preparations where the isolated protein is sufficiently pure to be administered as a therapeutic composition, or at least 70% to 80% (w/w) pure, more preferably, at least 80%-90% (w/w) pure, even more preferably, 90-95% pure; and, most preferably, at least 95%, 96%, 97%, 98%, 99%, or 100% (w/w) pure. A “separated” compound refers to a compound that is removed from at least 90% of at least one component of a sample from which the compound was obtained. Any compound described herein can be provided as an isolated or separated compound.
[0066] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a mammal being assessed for treatment and/or being treated. In some embodiments, the mammal is a human. The terms “subject,” “individual,” and “patient” encompass, without limitation, individuals having a disease. Subjects may be human, but also include other mammals, particularly those mammals useful as laboratory models for human disease, e.g., mice, rats, etc.
[0067] The term “sample” with reference to a patient encompasses blood and other liquid samples of biological origin, solid tissue samples, such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. The term also encompasses samples that have been manipulated in any way after their procurement, such as by treatment with reagents; washed; or enrichment for certain cell populations, such as diseased cells. The definition also includes samples that have been enriched for particular types of molecules, e.g., nucleic acids, polypeptides, etc. The term “biological sample” encompasses a clinical sample, and also includes tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, and the like. A “biological sample” includes a sample obtained from a patient's diseased cell, e.g., a sample comprising polynucleotides and/or polypeptides that is obtained from a patient’s diseased cell (e.g., a cell lysate or other cell extract comprising polynucleotides and/or polypeptides); and a sample comprising diseased cells from a patient. A biological sample comprising a diseased cell from a patient can also include non-diseased cells. [0068] The term “diagnosis” is used herein to refer to the identification of a molecular or pathological state, disease or condition in a subject, individual, or patient.
[0069] The term “prognosis” is used herein to refer to the prediction of the likelihood of death or disease progression, including recurrence, spread, and drug resistance, in a subject, individual, or patient. The term “prediction” is used herein to refer to the act of foretelling or estimating, based on observation, experience, or scientific reasoning, the likelihood of a subject, individual, or patient experiencing a particular event or clinical outcome. In one example, a physician may attempt to predict the likelihood that a patient will survive.
[0070] As used herein, the terms “treatment,” “treating,” and the like, refer to administering an agent, or carrying out a procedure, for the purposes of obtaining an effect on or in a subject, individual, or patient. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of effecting a partial or complete cure for a disease and/or symptoms of the disease. “Treatment,” as used herein, may include treatment of cancer in a mammal, particularly in a human, and includes: (a) inhibiting the disease, i.e., arresting its development; and (b) relieving the disease or its symptoms, i.e., causing regression of the disease or its symptoms.
[0071] Treating may refer to any indicia of success in the treatment or amelioration or prevention of a disease, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of an examination by a physician. Accordingly, the term "treating" includes the administration of engineered cells to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with disease or other diseases. The term "therapeutic effect" refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject.
[0072] As used herein, a "therapeutically effective amount" refers to that amount of the therapeutic agent sufficient to treat or manage a disease or disorder. A therapeutically effective amount may refer to the amount of therapeutic agent sufficient to delay or minimize the onset of disease, e.g., to delay or minimize the growth and spread of cancer. A therapeutically effective amount may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease. Furthermore, a therapeutically effective amount with respect to a therapeutic agent of the invention means the amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of a disease. [0073] As used herein, the term “dosing regimen” refers to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).
[0074] "In combination with", "combination therapy" and "combination products" refer, in certain embodiments, to the concurrent administration to a patient of the engineered proteins and cells described herein in combination with additional therapies, e.g. surgery, radiation, chemotherapy, and the like. When administered in combination, each component can be administered at the same time or sequentially in any order at different points in time. Thus, each component can be administered separately but sufficiently closely in time so as to provide the desired therapeutic effect.
[0075] "Concomitant administration" means administration of one or more components, such as engineered proteins and cells, known therapeutic agents, etc. at such time that the combination will have a therapeutic effect. Such concomitant administration may involve concurrent (i.e. at the same time), prior, or subsequent administration of components. A person of ordinary skill in the art would have no difficulty determining the appropriate timing, sequence and dosages of administration.
[0076] The use of the term "in combination" does not restrict the order in which prophylactic and/or therapeutic agents are administered to a subject with a disorder. A first prophylactic or therapeutic agent can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second prophylactic or therapeutic agent to a subject with a disorder.
[0077] ID1 (DNA-binding protein inhibitor) is a helix-loop-helix (HLH) protein that can form heterodimers with members of the basic HLH family of transcription factors. The encoded protein has no DNA binding activity and therefore can inhibit the DNA binding and transcriptional activation ability of basic HLH proteins with which it interacts. ID1 has been shown to interact weakly with MyoD but very tightly with ubiquitously expressed E proteins. E proteins heterodimerize with tissue restricted bHLH proteins such as Myod, NeuroD, etc. to form active transcription complexes so by sequestering E proteins, Id proteins can inhibit tissue restricted gene expression in multiple cell lineages using the same biochemical mechanism. The human reference protein sequence can be accessed at Genbank NP 002156 and NP_851998.
[0078] Tyrosine Kinase Inhibitors. Tyrosine kinase inhibitors (TKI) are a group of pharmacologic agents that disrupt the signal transduction pathways of protein kinases by several modes of inhibition. This activity will review the currently available drugs, their mechanism of action, routes of administration, indications, contraindications, and adverse effects.
[0079] Mutations, dysregulation, and overexpression of protein kinases are involved in a multitude of disease processes. Around 1 in every 40 human genes codes for a protein kinase and nearly half of those genes map to either disease loci or cancer amplicons. Tyrosine kinase enzymes (TKs) can be categorized into receptor tyrosine kinases (RTKs), non-receptor tyrosine kinases (NRTKs), and a small group of dual-specificity kinases (DSK) which can phosphorylate serine, threonine, and tyrosine residues. RTKs are transmembrane receptor that includes vascular endothelial growth factor receptors (VEGFR), platelet-derived growth factor receptors (PDGFR), insulin receptor (InsR) family, and the ErbB receptor family, which includes epidermal growth factor receptors (EGFR) and the human epidermal growth factor receptor-2 (HER2).
[0080] As a whole, tyrosine kinases phosphorylate specific amino acids on substrate enzymes, which subsequently alter signal transduction leading to downstream changes in cellular biology. The downstream signal transduction set off by TKs can modify cell growth, migration, differentiation, apoptosis, and death. Constitutive activation or inhibition, either by mutations or other means, can lead to dysregulated signal cascades, potentially resulting in malignancy and other pathologies. Therefore, blocking these initial signals via TKIs can prevent the aberrant action of the mutated or dysfunctional TKs. [0081] Nearly all TKIs are effective when taken orally. Therapeutic loading and maintenance dosages are unique to each drug and should require unique dosing for each patient. When administering specific TKIs, many factors can contribute to reduced potency and the development of acquired resistance. These factors include such as whether or not food intake affects bioavailability, the mechanism of drug metabolism and elimination, liver and kidney function, drug-drug interactions, the presence of other medications that alter stomach pH, and patient demographics.
[0082] VEGFR-2 inhibitor. VEGFR-2 is a 210-230 kDa glycoprotein expressed in vascular endothelial cells and in hematopoietic stem cells and binds VEGF-A. VEGFR-2 is closely related to VEGFR-1 for they have common and specific ligands but VEGFR-2 is a highly active kinase while VEGFR1 is an impaired receptor tyrosine kinase. This receptor is a regulator in responses in the endothelial cells of VEGF. These regulations include permeability, proliferation, invasion and migration. The signaling pathways, Y1 175 and Y1214, are the main autophosphorylation sites of the human VEGFR-2 followed by VEGF binding. Autophosphorylated VEGFR-2 is required for activation of several downstream pathways. Inhibitors of VEGFR2 include, for example, Sorafenib, Sunitinib, Lenvatinib, Vandetanib, Pazopanib and Axitinib, Cabozantinib, Regorafenib, Nintedanib, Apatinib, etc.
[0083] Brivanib alaninate is an orally administered drug with the structure:
Figure imgf000021_0001
[0084] Brivanib is a multitargeted tyrosine kinase inhibitor, which inhibits VEGFR and fibroblast growth factor receptors. Brivanib is the alanine ester of a VEGFR-2 inhibitor BMS- 540215 and is hydrolyzed to the active moiety BMS-540215 in vivo. BMS-540215, a dual tyrosine kinase inhibitor, shows potent and selective inhibition of VEGFR and fibroblast growth factor receptor (FGFR) tyrosine kinases. BMS-540215 is an ATP-competitive inhibitor of human VEGFR-2, with an IC50 of 25 nmol/L and Ki of 26 nmol/L. In addition, it inhibits VEGFR- 1 (IC50 = 380 nmol/L) and VEGFR-3 (IC50 = 10 nmol/L). BMS-540215 also showed good selectivity for FGFR-1 (IC50 = 148 nmol/L), FGFR-2 (IC50 = 125 nmol/L), and FGFR-3 (IC50 = 68 nmol/L). Furthermore, BMS-540215 has been shown to selectively inhibit the proliferation of endothelial cells stimulated by VEGF and FGF in vitro with IC50 values of 40 and 276 nmol/L, respectively. [0085] The dosing of brivanib may be similar to that for cancer treatment, e.g. from about 500 to about 1000 mg/day, from about 600 to about 800 mg/day. Alternatively the dose may be modified, e.g. using a lower dose of from about 10, from about 50, from about 100, from about 250 mg/day, up to about 1000, up to about 750, up to about 500 mg/day.
[0086] Dabrafenib is an orally administered drug with the structure:
Figure imgf000022_0001
[0087] Dabrafenib, sold under the brand name Tafinlar among others, is an anti-cancer medication used for the treatment of cancers associated with a mutated version of the gene BRAF.
[0088] The dosing of dabrafenib may be similar to that for cancer treatment, e.g. from about 150 mg/day to about 600 mg/day, from about 150 to about 300 mg/day. Administration may be about 150 mg, twice daily. Alternatively the dose may be modified, e.g. using a lower dose of from about 5, from about 10, from about 25, from about 50 mg/day, from about 100 mg/day up to about 300, up to about 200, up to about 150 mg/day.
[0089] Linsitinib is an orally administered drug with the structure:
Figure imgf000022_0002
[0090] Linsitinib is an inhibitor of the insulin receptor and of the insulin-like growth factor 1 receptor (IGF-1 ).
[0091] The dosing of linsitinib may be similar to that for clinical trials in cancer treatment, e.g. from about 150 mg/day to about 600 mg/day, from about 150 to about 300 mg/day. Administration may be about 150 mg, twice daily. Alternatively the dose may be modified, e.g. using a lower dose of from about 5, from about 10, from about 25, from about 50 mg/day, from about 100 mg/day up to about 300, up to about 200, up to about 150 mg/day. [0092] Hereditary haemorrhagic telangiectasia (HHT), also known as Rendu-Osler-Weber syndrome, is a complex genetic disease that causes abnormalities of blood vessels formation in the liver, lung, brain, skin, nasal mucosa, and gastrointestinal tract. HHT is a rare disease, affecting approximately 1 in 5000-10000 peoples in the world. Clinical indicia manifestations of the diasease include epistaxis (nose bleeding, present in 90% cases), gastrointestinal haemorrhage, mucocutaneous telangiectases (blood vessel dilations), and visceral arteriovenous malformations (AVMs). Treatment with the agents disclosed herein may reduce one or more clinical indicia, for example AVMs, by at least about 10%, at least abou 20%, at least about 30%, at least about 50%, at least about 75%, at least about 90% or more relative to an untreated individual.
[0093] Clinically, HHT can be diagnosed based on the four established Curacao Criteria; recurrent epistaxis, telangiectasia, family history, and arteriovenous malformations (AVMs). The diagnosisof HHT is confirmed when three or more criteria are met, while it is suspected if just two criteria are fulfilled. The AVMs are abnormal large connections between arteries and veins bypassing the capillaries and result in severe bleeding when present in the nose, Gl- tract and brain or cause paradoxic emboli and stroke when present in the lungs. AVMs are found in the lung (15-45%), liver (>70%), and cerebral (10-23%) of HHT patients. Currently, HHT therapies aim to lessen the disease's symptoms. However, there is currently no mechanism-based targeted therapy available.
[0094] The majority (about >85%) of the HHT patients have loss of function mutations in either ENG (HHT type 1 ) or ACVRL1 (HHT type 2), but a small percentage of them (approximately <1 %) have mutations in the SMAD4 (juvenile polyposis/HHT combined syndrome). Interestingly, the three HHT-causing genes encode proteins that belong to same TGFB superfamily of proteins. All three HHT gene products function in SMAD-dependent signaling pathways in which the activated Smad complex enters into the cell nucleus and regulates transcriptional programs. Previous in vivo studies showed that any of the three HHT gene mutations can cause vascular malformation since knockout alleles for ENG, ACVRL1 , and SMAD4 all result in HHT phenotypes in mice. Mechanistically, in addition to the TGFB/BMP pathway, several other pathways, such as VEGF, mTOR, PI3K/AKT signaling pathways have been shown to be associated with AVM formation and HHT pathogenesis. Recently, pharmacologically combined treatment with sirolimus and nintedanib has been shown to correct Smad1/5/8 reduction, and decrease mTOR and VEGFR2 activation thereby reversing and preventing the vascular abnormalities, and improving anemia and bleeding in two HHT mouse models.
[0095] One of the known downstream targets of BMPR2/ALK1 signaling is the inhibitors of DNA- binding/differentiation proteins (IDs). ID proteins are thought to inhibit differentiation and promote cell cycle progression and therefore could represent possible candidates to explain the aberrant proliferation observed during AVM pathogenesis in HHT. In support of this, mice with a combined loss of both Id1 and Id3 display cranial hemorrhage secondary to the formation of an anastomosing network of dilated capillaries in the brain, suggesting that ID1/3 may play a role in AVM formation.
[0096] Current therapies for HHT are limited and consist of treating large AVMs with catheter- directed embolization, but no treatment exists that corrects the dysfunctional signaling pathways. Drugs applied systemically that restore normal signaling could prevent the development and growth of existing AVMs, an approach particular of importance for children with HHT whose small AVMs grow with age. Furthermore, this approach might be beneficial for patients with multiple smaller AVMs (in lungs, nose, Gl tract) and severe hypoxia, bleeding and subsequent anemia, whose AVMs are not amenable to embolization because of their size and number but might respond to medical therapy.
[0097] “Pulmonary hypertension” (PH, or PAH) is an umbrella term that describes patients with pulmonary artery pressures exceeding 20mmHg on right heart catheterization. PH is a disease highlighted by an increased resistance to blood flow through the pulmonary arterial system. Though the factors responsible for the development of this vasculopathy are unknown, it is clear that over time hypertrophy of the medial layer of the vascular bed, thickening of the adventitia, perivascular inflammation and fibrosis of the intima leads to dysregulation of the signaling pathways responsible for vasoreactivity and autoregulation of blood flow through the pulmonary artery.
[0098] The World Symposium on Pulmonary Hypertension in February 2018 organized PH into five groups: Pulmonary arterial hypertension (Group 1 ), PH due to left heart disease (Group 2), PH due to underlying lung disease or chronic hypoxemia (Group 3), PH attributable to chronic thromboembolic pulmonary hypertension (Group 4), and PH due to unknown, or overlapping, mechanisms (Group 5).
[0099] Pulmonary arterial hypertension (PAH, Group I) is termed “pre-capillary” disease because of the pathological vascular changes that occur prior to the pulmonary capillary bed as it delivers blood to the pulmonary venous system. It is a fatal condition characterized by aberrant proliferation of cells constituting the pulmonary vessels, resulting in vascular wall thickening and luminal obliteration to produce elevated pulmonary vascular resistance (PVR) and subsequent right ventricular (RV) failure.
[00100] Historically, PAH has been defined by clinical indicia of hemodynamic indices measured with right heart catheterization (RHC) while the patient is at rest. PAH resting RHC diagnostic criteria include increased mean pulmonary artery pressure (mPAP) > 20 mmHg and elevated PVR (>3 Wood units), along with pulmonary arterial wedge pressure (PAWP) < 15 mmHg indicative of a normal left ventricular filling pressure. Treatment with the agents disclosed herein may reduce one or more clinical indicia, by at least about 10%, at least abou 20%, at least about 30%, at least about 50%, at least about 75%, at least about 90% or more relative to an untreated individual
[00101] It has been shown that the main factors contributing to increased pulmonary vascular resistance during the development of PAH are remodeling and narrowing of the pulmonary arteries. An increase in cytoplasmic free Ca2+ concentration ([Ca2+] cyt) in pulmonary arterial smooth muscle cells (PASMCs) is the main cause of pulmonary vasoconstriction, while an imbalance in intracellular calcium (Ca2+) homeostasis stimulates PASMCs to proliferate and inhibits their apoptosis, leading to pulmonary vascular remodeling. The imbalance in intracellular calcium (Ca2+) homeostasis stimulates PASMCs to proliferate and inhibits their apoptosis, leading to pulmonary vascular remodeling and driving PAH disease progression.
[00102] Despite introduction of new therapies and improved survival, PAH remains incurable, and the anticipated 5-years survival of PAH patients can be as low as 50%. Notably, significant pulmonary vascular remodeling persists in the pulmonary arteries of PAH patients treated with current PAH-targeted pharmacologic modalities, predominantly pulmonary vasodilators, underscoring shortcomings of diagnostic and therapeutic approaches in detecting PAH at its initial stages and achieving reversal of pulmonary vascular remodeling.
Gene expression signatures
[00103] In some embodiments, gene expression signatures areused to determine the efficacy of a treatment, for drug screening to determine the suitability of an agent, in the selection of cell lines as in vitro models and in drug screening, and the like.
[00104] The treatment of HHT can normalize gene expression in endothelial cells in which HHT causing genes are knocked down, e.g. the treatment is capable of reducing expression of one or more of the genes LYVE1 , GPNMB, RRAGD and ANKRD33; and/or increasing expression of one or more of the genes MC5R, SLC25A47, FGF19, SHISA9, FRG2C, and HS3ST2. This pattern of expression may be referred to as an “HHT disease signature”. An expanded HHT disease signature may comprise 5 upregulated (LYVE1 , GPNMB, RRAGD, ANKRD33, HS3ST2) and 7 downregulated (MC5R, SLC25A47, FGF19, SHISA9, FRG2C, HIST1 H2BE, CPA4) genes.
[00105] Gene signatures associated with PAH include the set of genes decreased by siLCK and increased by siFYN, which are termed an “endothelial signature”. The “siLCK Down/siFYN Up” group comprise of EC artery and capillary specific genes (Gja5, Lyvel , Gphibpl ). Upregulated genes for siLCK include Il6st, Ccl7, Wnt5a, and 111 b. Top downregulated genes for siLCK are Ace, Lyvel , Gja5, and Dysf. For siFyn upregulated genes included Bmp4, Gdf7, Gdf1 / (Bmp11 ), Gja5, Lyvel, Id1, and Tgfb 1. Genes downregulated in siFYN include Tlr3, C1s, C3, Ccl8, and Vcaml. In the “siLCK up/siFYN down”, inflammation signature genes, the most co-differentially regulated genes were associated with type-1 interferon signaling: Tnfaip6 and 111 B. Gene ontology (GO) analysis converged on pathways elicited by interferon gamma or lipopolysaccharide and were associated with increased interferon cx/p and IL-6 production, NF- KB activation, and MHC-1 activation. Indeed, transcription factor prediction by the three methods found the canonical NF-KB transcription factor RelA was tied to the majority of co- DEGs. The set of co-DEGs both increased in siLCK PAECs and decreased in siFYN PAECs may be referred to as an “inflammatory signature”, linked by canonical NF-KB activity.
[00106] In some embodiments of the invention, gating strategies can be used in order to analyze a specific cell population in a sample of mixed cell population, e.g. to focus on endothelial cells, etc.. These gating strategies can be based on the presence of one or more specific surface markers. The following gate can differentiate between dead cells and live cells and the subsequent gating of live cells classifies. A clear comparison can be carried out by using two-dimensional contour plot representations, two-dimensional dot plot representations, and/or histograms.
[00107] Samples may be obtained at one or more time points. Where a sample at a single time point is used, comparison is made to a reference “base line” level for the expression of the gene of interest, which may be obtained from a normal control, a pre-determined level obtained from one or a population of individuals, from a negative control, and the like.
[00108] If necessary, cells are dispersed into a single cell suspension, e.g. by enzymatic digestion with a suitable protease, e.g. collagenase, dispase, etc; and the like. An appropriate solution is used for dispersion or suspension. Such solution will generally be a balanced salt solution, e.g. normal saline, PBS, Hanks balanced salt solution, etc., conveniently supplemented with fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, generally from 5-25 mM. Convenient buffers include HEPES1 phosphate buffers, lactate buffers, etc. The cells can be fixed, e.g. with 3% paraformaldehyde, and are usually permeabilized, e.g. with ice cold methanol; HEPES- buffered PBS containing 0.1 % saponin, 3% BSA; covering for 2 min in acetone at -200C; and the like as known in the art and according to the methods described herein.
[00109] In some embodiments, one or more cells are contained in a well of a 96 well plate or other commercially available multiwell plate. In an alternate embodiment, the reaction mixture or cells are in a cytometric measurement device. Other multiwell plates useful in the present invention include, but are not limited to 384 well plates and 1536 well plates. Still other vessels for containing the reaction mixture or cells and useful in the present invention will be apparent to the skilled artisan. [001 10] In some embodiment, the methods of the invention include the use of liquid handling components. The liquid handling systems can include robotic systems comprising any number of components. In addition, any or all of the steps outlined herein can be automated; thus, for example, the systems can be completely or partially automated. See USSN 61/048,657. As will be appreciated by those in the art, there are a wide variety of components which can be used, including, but not limited to, one or more robotic arms; plate handlers for the positioning of microplates; automated lid or cap handlers to remove and replace lids for wells on noncross contamination plates; tip assemblies for sample distribution with disposable tips; washable tip assemblies for sample distribution; 96 well loading blocks; cooled reagent racks; microtiter plate pipette positions (optionally cooled); stacking towers for plates and tips; and computer systems.
[001 11 ] Fully robotic or microfluidic systems include automated liquid-, particle-, cell- and organism-handling including high throughput pipetting to perform all steps of screening applications. This includes liquid, particle, cell, and organism manipulations such as aspiration, dispensing, mixing, diluting, washing, accurate volumetric transfers; retrieving, and discarding of pipet tips; and repetitive pipetting of identical volumes for multiple deliveries from a single sample aspiration. These manipulations are cross-contamination- free liquid, particle, cell, and organism transfers. This instrument performs automated replication of microplate samples to filters, membranes, and/or daughter plates, high-density transfers, full-plate serial dilutions, and high capacity operation.
[001 12] In some embodiments, platforms for multi-well plates, multi-tubes, holders, cartridges, minitubes, deep-well plates, microfuge tubes, cryovials, square well plates, filters, chips, optic fibers, beads, and other solid-phase matrices or platform with various volumes are accommodated on an upgradable modular platform for additional capacity. This modular platform includes a variable speed orbital shaker, and multi-position work decks for source samples, sample and reagent dilution, assay plates, sample and reagent reservoirs, pipette tips, and an active wash station. In some embodiments, the methods of the invention include the use of a plate reader.
[001 13] In some embodiments, interchangeable pipet heads (single or multi-channel) with single or multiple magnetic probes, affinity probes, or pipetters robotically manipulate the liquid, particles, cells, and organisms. Multi-well or multi-tube magnetic separators or platforms manipulate liquid, particles, cells, and organisms in single or multiple sample formats.
[001 14] In some embodiments, the instrumentation will include a detector, which can be a wide variety of different detectors, depending on the labels and assay. In some embodiments, useful detectors include a microscope(s) with multiple channels of fluorescence; plate readers to provide fluorescent, ultraviolet and visible spectrophotometric detection with single and dual wavelength endpoint and kinetics capability, fluorescence resonance energy transfer (FRET), luminescence, quenching, two-photon excitation, and intensity redistribution; CCD cameras to capture and transform data and images into quantifiable formats; and a computer workstation.
[001 15] In some embodiments, the robotic apparatus includes a central processing unit which communicates with a memory and a set of input/output devices (e.g., keyboard, mouse, monitor, printer, etc.) through a bus. Again, as outlined below, this can be in addition to or in place of the CPU for the multiplexing devices of the invention. The general interaction between a central processing unit, a memory, input/output devices, and a bus is known in the art. Thus, a variety of different procedures, depending on the experiments to be run, are stored in the CPU memory.
[001 16] Any convenient method as kown in the art may be used for determining gene expression, e.g. RT-PCR, RNA-seq, hybridization, sequencing, etc.
[001 17] A signature pattern can be generated from a biological sample using any convenient protocol, for example as described below. The readout can be a mean, average, median or the variance or other statistically or mathematically-derived value associated with the measurement. The marker readout information can be further refined by direct comparison with the corresponding reference or control pattern. A pattern can be evaluated on a number of points: to determine if there is a statistically significant change at any point in the data matrix relative to a reference value; whether the change is an increase or decrease in the binding; whether the change is specific for one or more physiological states, and the like. The absolute values obtained for each marker under identical conditions will display a variability that is inherent in live biological systems and also reflects the variability inherent between individuals.
[001 18] Following obtainment of the signature pattern from the sample being assayed, the signature pattern can be compared with a reference or base line profile to make a prognosis regarding the phenotype of the patient from which the sample was obtained/derived. Additionally, a reference or control signature pattern can be a signature pattern that is obtained from a sample of a patient known to have a normal pregnancy.
[001 19] In certain embodiments, the obtained signature pattern is compared to a single reference/control profile to obtain information regarding the phenotype of the patient being assayed. In yet other embodiments, the obtained signature pattern is compared to two or more different reference/control profiles to obtain more in depth information regarding the phenotype of the patient. For example, the obtained signature pattern can be compared to a positive and negative reference profile to obtain confirmed information regarding whether the patient has the phenotype of interest.
[00120] Samples can be obtained from the tissues or fluids of an individual. For example, samples can be obtained from whole blood, tissue biopsy, serum, etc. Other sources of samples are body fluids such as lymph, cerebrospinal fluid, and the like. Also included in the term are derivatives and fractions of such cells and fluids
[00121 ] In order to identify profiles that are indicative of responsiveness, a statistical test can provide a confidence level for a change in the level of markers between the test and reference profiles to be considered significant. The raw data can be initially analyzed by measuring the values for each marker, usually in duplicate, triplicate, quadruplicate or in 5-10 replicate features per marker. A test dataset is considered to be different than a reference dataset if one or more of the parameter values of the profile exceeds the limits that correspond to a predefined level of significance.
[00122] To provide significance ordering, the false discovery rate (FDR) can be determined. First, a set of null distributions of dissimilarity values is generated. In one embodiment, the values of observed profiles are permuted to create a sequence of distributions of correlation coefficients obtained out of chance, thereby creating an appropriate set of null distributions of correlation coefficients (see Tusher et al. (2001 ) PNAS 98, 5116-21 , herein incorporated by reference). This analysis algorithm is currently available as a software “plug-in” for Microsoft Excel know as Significance Analysis of Microarrays (SAM). The set of null distribution is obtained by: permuting the values of each profile for all available profiles; calculating the pair- wise correlation coefficients for all profile; calculating the probability density function of the correlation coefficients for this permutation; and repeating the procedure for N times, where N is a large number, usually 300. Using the N distributions, one calculates an appropriate measure (mean, median, etc.) of the count of correlation coefficient values that their values exceed the value (of similarity) that is obtained from the distribution of experimentally observed similarity values at given significance level.
[00123] The FDR is the ratio of the number of the expected falsely significant correlations (estimated from the correlations greater than this selected Pearson correlation in the set of randomized data) to the number of correlations greater than this selected Pearson correlation in the empirical data (significant correlations). This cut-off correlation value can be applied to the correlations between experimental profiles.
[00124] For SAM, Z-scores represent another measure of variance in a dataset, and are equal to a value of X minus the mean of X, divided by the standard deviation. A Z-Score tells how a single data point compares to the normal data distribution. A Z-score demonstrates not only whether a datapoint lies above or below average, but how unusual the measurement is. The standard deviation is the average distance between each value in the dataset and the mean of the values in the dataset.
[00125] Using the aforementioned distribution, a level of confidence is chosen for significance. This is used to determine the lowest value of the correlation coefficient that exceeds the result that would have obtained by chance. Using this method, one obtains thresholds for positive correlation, negative correlation or both. Using this threshold(s), the user can filter the observed values of the pairwise correlation coefficients and eliminate those that do not exceed the threshold(s). Furthermore, an estimate of the false positive rate can be obtained for a given threshold. For each of the individual “random correlation” distributions, one can find how many observations fall outside the threshold range. This procedure provides a sequence of counts. The mean and the standard deviation of the sequence provide the average number of potential false positives and its standard deviation. Alternatively, any convenient method of statistical validation can be used.
[00126] The data can be subjected to non-supervised hierarchical clustering to reveal relationships among profiles. For example, hierarchical clustering can be performed, where the Pearson correlation is employed as the clustering metric. One approach is to consider a patient disease dataset as a “learning sample” in a problem of “supervised learning”. CART is a standard in applications to medicine (Singer (1999) Recursive Partitioning in the Health Sciences, Springer), which can be modified by transforming any qualitative features to quantitative features; sorting them by attained significance levels, evaluated by sample reuse methods for Hotelling's T2 statistic; and suitable application of the lasso method. Problems in prediction are turned into problems in regression without losing sight of prediction, indeed by making suitable use of the Gini criterion for classification in evaluating the quality of regressions.
[00127] Other methods of analysis that can be used include logistic regression. One method of logic regression Ruczinski (2003) Journal of Computational and Graphical Statistics 12:475- 512. Logic regression resembles CART in that its classifier can be displayed as a binary tree. It is different in that each node has Boolean statements about features that are more general than the simple “and” statements produced by CART.
[00128] Another approach is that of nearest shrunken centroids (Tibshirani (2002) PNAS 99:6567-72). The technology is k-means-like, but has the advantage that by shrinking cluster centers, one automatically selects features (as in the lasso) so as to focus attention on small numbers of those that are informative. The approach is available as Prediction Analysis of Microarrays (PAM) software, a software “plug-in” for Microsoft Excel, and is widely used. Two further sets of algorithms are random forests (Breiman (2001 ) Machine Learning 45:5-32 and MART (Hastie (2001 ) The Elements of Statistical Learning, Springer). These two methods are already “committee methods.” Thus, they involve predictors that “vote” on outcome. Several of these methods are based on the “R” software, developed at Stanford University, which provides a statistical framework that is continuously being improved and updated in an ongoing basis.
[00129] Other statistical analysis approaches including principle components analysis, recursive partitioning, predictive algorithms, Bayesian networks, and neural networks. [00130] These tools and methods can be applied to several classification problems. For example, methods can be developed from the following comparisons: i) all cases versus all controls, ii) all cases versus nonresponsive controls, Hi) all cases versus responsive controls.
[00131 ] In a second analytical approach, variables chosen in the cross-sectional analysis are separately employed as predictors. Given the specific outcome, the random lengths of time each patient will be observed, and selection of proteomic and other features, a parametric approach to analyzing responsiveness can be better than the widely applied semi-parametric Cox model. A Weibull parametric fit of survival permits the hazard rate to be monotonically increasing, decreasing, or constant, and also has a proportional hazards representation (as does the Cox model) and an accelerated failure-time representation. All the standard tools available in obtaining approximate maximum likelihood estimators of regression coefficients and functions of them are available with this model.
[00132] In addition the Cox models can be used, especially since reductions of numbers of covariates to manageable size with the lasso will significantly simplify the analysis, allowing the possibility of an entirely nonparametric approach to survival.
[00133] The analysis and database storage can be implemented in hardware or software, or a combination of both. In one embodiment of the invention, a machine-readable storage medium is provided, the medium comprising a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying a any of the datasets and data comparisons of this invention. Such data can be used for a variety of purposes, such as patient monitoring, initial diagnosis, and the like. Preferably, the invention is implemented in computer programs executing on programmable computers, comprising a processor, a data storage system (including volatile and non-volatile memory and/or storage elements), at least one input device, and at least one output device. Program code is applied to input data to perform the functions described above and generate output information. The output information is applied to one or more output devices, in known fashion. The computer can be, for example, a personal computer, microcomputer, or workstation of conventional design.
[00134] Each program is preferably implemented in a high level procedural or object oriented programming language to communicate with a computer system. However, the programs can be implemented in assembly or machine language, if desired. In any case, the language can be a compiled or interpreted language. Each such computer program is preferably stored on a storage media or device (e.g., ROM or magnetic diskette) readable by a general or special purpose programmable computer, for configuring and operating the computer when the storage media or device is read by the computer to perform the procedures described herein. The system can also be considered to be implemented as a computer-readable storage medium, configured with a computer program, where the storage medium so configured causes a computer to operate in a specific and predefined manner to perform the functions described herein.
[00135] A variety of structural formats for the input and output means can be used to input and output the information in the computer-based systems of the present invention. One format for an output means test datasets possessing varying degrees of similarity to a trusted profile. Such presentation provides a skilled artisan with a ranking of similarities and identifies the degree of similarity contained in the test pattern.
[00136] The signature patterns and databases thereof can be provided in a variety of media to facilitate their use. “Media” refers to a manufacture that contains the signature pattern information of the present invention. The databases of the present invention can be recorded on computer readable media, e.g. any medium that can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media. One of skill in the art can readily appreciate how any of the presently known computer readable mediums can be used to create a manufacture comprising a recording of the present database information. "Recorded" refers to a process for storing information on computer readable medium, using any such methods as known in the art. Any convenient data storage structure can be chosen, based on the means used to access the stored information. A variety of data processor programs and formats can be used for storage, e.g. word processing text file, database format, etc.
Methods of Treatment
[00137] In an embodiment, an individual with HHT is treated by administration of an effective dose of an agent that inhibits at least VEGFR-2, and may additionally inhibit FGFR-1 , where the treatment improves clinical indicia of the disease. In some embodiments the agent is Brivanib. It is shown herein that Brivanib can reversed an HHT disease signature of gene expression, and can inhibit othere disease features. In another embodiment the agent is dabrafenib. In another ambodiment the agent is linsitinib.
[00138] Brivanib is a pyrrolotriazine-based compound and an inhibitor of vascular endothelial growth factor receptor-2 (VEGFR-2). It specifically targets and binds strongly to human VEGFR-2, a tyrosine kinase receptor and pro-angiogenic growth factor expressed almost exclusively on vascular endothelial cells. It has a role as an antineoplastic agent, an EC 2.7.10.1 (receptor protein-tyrosine kinase) inhibitor, an angiogenesis inhibitor, an apoptosis inducer, a fibroblast growth factor receptor antagonist and a drug metabolite. It is a pyrrolotriazine, a fluoroindole, a diether, an aromatic ether and a secondary alcohol. It is an ATP-competitive inhibitor against VEGFR2 with IC50 of 25 nM, moderate potency against VEGFR-1 and FGFR-1 , but >240-fold against PDGFR-p.
[00139] The treatment of HHT can normalize gene expression in endothelial cells in which HHT causing genes are knocked down, e.g. the treatment is capable of reducing expression of one or more of the genes LYVE1 , GPNMB, RRAGD and ANKRD33; and/or increasing expression of one or more of the genes MC5R, SLC25A47, FGF19, SHISA9, FRG2C, and HS3ST2. The treatment can inhibit VEGF-induced ERK1/2 phosphorylation, proliferation, and angiogenesis in the targeted cells, e.g. lung microvessel endothelial cells, and also inhibit the expression of one or more of the genes upregulated in the HHT disease condition: LYVE1 , GPNMB, and HS3ST2 in the targeted tissues, e.g. lung tissue. In some embodiments the PTK can inhibit an inflammatory gene expression signature disclosed herein. In some embodiments the individual is monitored for efficacy of treatment, e.g. by determining an inflammatory signature change, determining changes in an HHT disease signature, changes in gene expression, and the like.
[00140] In an embodiment, an individual with PAH is treated by administration of an effective dose of an agent that increases ID1 expression, a common downstream target of the BMPR2 pathway, where the treatment improves clinical indicia of the disease. In some embodiments the agent is Brivanib. . In some embodiments the inhibitor is Dabrafenib or Linsitinib. The treatment can provide for an pro-endothelial-like pattern of gene expression in targeted cells, in contrast to an inflammatory pattern of gene expression. In some embodiments the individual is monitored for efficacy of treatment, e.g. by determining a pro-endothelial expression change, changes in Id1 gene expression, and the like. . In some embodiments the inhibitor increases Id1 expression. In some embodiments the inhibitor activates the endothelial transcriptomic signature.
[00141] The methods comprises administering an effective amount of an agent, e.g. brivanib, of the invention to a patient determined to be likely to benefit from, in need of, or likely to respond to, a therapy of the invention, thereby treating or preventing HHT or PAH, in the patient. Dosing guidelines for agents are as disclosed herein.
[00142] Various methods can be used to monitor efficacy of treatment, including measuring clinical indidicia, monitored gene expression signatures, measuring average survival rate, etc. Average survival rate can be used in the methods as a parameter to determine efficacy in a population of one or more subjects. A reference average survival rate is 95% or lower, 93% or lower, 90% or lower, 86% or lower, 82% or lower, or 78% or lower. The average survival rate can be an average 1 -year survival rate. A treatment provided in the invention is efficacious in a population of one or more subjects if, after treatment has started, the average survival rate increases. For example, the average survival rate can increase from the reference average survival rate by 1 % or more, 2% or more, 5% or more, 10% or more, or 20% or more. [00143] The methods can further comprise continuing the therapy if the therapy is determined to be efficacious. The methods can comprise maintaining, tapering, reducing, or stopping the administered amount of a compound or compounds in the therapy if the therapy is determined to be efficacious. The methods can comprise increasing the administered amount of a compound or compounds in the therapy if it is determined not to be efficacious. Alternatively, the methods can comprise stopping therapy if it is determined not to be efficacious.
[00144] The method of treating the patient may involve administering at least one additional active agent, i.e., in addition to the RTK inhibitor. The additional active agent may be, for example, a vasodilator such as prostacyclin, epoprostenol, and sildenafil; an endothelin receptor antagonist such as bosentan; a calcium channel blocker such as amlodipine, diltiazem, and nifedipine; an anticoagulant such as warfarin; a diuretic, a prostanoid (e.g., prostacyclin or PGI.sub.2), drugs for treating diseases associated with overactive B cells or dysfunctional B cells such as Rituximab, and/or a Type V phosphodiesterase (PDE5) inhibitor.
[00145] When the method of the invention involves combination therapy, the agents may be administered separately, at the same, or at different times of the day, or they may be administered in a single composition. Thus, the present invention provides novel pharmaceutical formulations in which a RTK inhibitor is combined with one of the active agents discussed above and unit dose forms of those formulations.
[00146] In the combination therapies of the invention, each agent can be administered in an "immediate release" manner or in a "controlled release manner." When the additional active agent is a vasodilator, for instance, any dosage form containing both active agents, i.e., both the RTK inhibitor and the vasodilator, can provide for immediate release or controlled release of the vasodilator, and either immediate release or controlled release of the RTK inhibitor.
[00147] A pharmaceutical composition of the invention is a pharmaceutical formulation containing an active agent formulated in a manner compatible with its intended route of administration. A variety of routes are contemplated, including but not limited to, oral, pulmonary, inhalational, sublingual, intranasal, parenteral, intradermal, transdermal, topical, transmucosal, subcutaneous, intravenous, intramuscular, intraperitoneal, buccal, rectal, and the like. The term "parenteral" as used herein is intended to include subcutaneous, intravenous, and intramuscular injection.
[00148] Generally, pharmaceutical formulations of the invention are prepared for oral administration and in an immediate release form suitable for once per day (QD) administration. Certain formulations are suitable for intranasal administration to a patient.
[00149] The pharmaceutical formulations of a RTK inhibitor useful in the methods of the invention can be prepared as a liquid or in a solid form such as a powder, tablet, pill or capsule for oral administration. Liquid formulations of the invention may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. In one embodiment, the formulation is an aqueous solution. In another embodiment, the final formulation is lyophilized.
EXPERIMENTAL
[00150] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
EXAMPLE 1 HHT
[00151] Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant disease that causes arteriovenous vascular malformations (AVMs) in different organs, including the lung. Three genes, ENG (endoglin), ACVRL1 (ALK1 ) and SMAD4, all members of the TGF- P/BMPR2 signaling pathway, are responsible for over 85% of all HHT cases. However, how these loss-of-function gene mutations lead to AVMs formation and what common downstream signaling they target is unknown. Current therapies for HHT are limited and consist of treating large AVMs with catheter-directed embolization, but no treatment exists that restores normal signaling.
[00152] Using a combination of siRNA-mediated gene silencing, whole transcriptomic RNA sequencing, bioinformatic analysis, transcriptomic-based drug discovery, endothelial cells functional assays and VEGF signaling analysis, and ex vivo precision cut lung slice (PCLS) cultures approach, we uncovered common downstream transcriptomic gene signatures of HHT-casing genes and identified promising drug for HHT
[00153] We found the commonly used BMPR2-signaling downstream target ID1 is not a common downstream target of all the three HHT genes (ALK1 , ENG, and SMAD4) knockdown in human pulmonary microvascular endothelial cells (PMVECs). We identified novel common downstream targets of all the three HHT-causing genes that were enriched for HHT-related biological process and signaling pathways. Among those downstream genes, LYVE1 , GPNMB, and MC5R were strong downstream targets that could serve as a better common downstream target than ID1 . Furthermore, using the common downstream upregulated genes (HHT disease signature) following HHT gene knockdown, we identified a small molecule drug, Brivanib, that reversed the HHT disease signature, and inhibited VEGF-induced ERK1/2 phosphorylation, proliferation, and angiogenesis in PMVECs and inhibited some of the upregulated HHT disease genes in POLS.
[00154] Our data show that our identified novel downstream targets of the commonly mutated HHT genes could serve as a better readout of the dysfunctional BMP downstream signaling than ID1 in HHT and that Brivanib is a new drug in treating HHT as it can correct two important signaling pathways BMP and VEGF, and improve endothelial cell behaviors.
[00155] Our study had two aims: First, to identify common downstream genes and signaling pathways of the three known HHT-causing gene mutations (ALK1 , ENG, SMAD4) using siRNA mediated gene downregulation, thereby mimicking complete local loss of function as seen in AVMs in HHT patients. Second, to identify repurposed/novel drugs that reverse the dysfunctional transcriptomic gene expression signature and normalize downstream signaling. We found that ID1 is not a common downstream target of all the three HHT gene mutations in PMVECs, and identified LYVE1 , GPNMB, MC5R, and PLXDC2 as downstream targets of all three HHT genes. Importantly, we discovered a small molecule drug, Brivanib, that can activate downstream targets of ALK1/ENG/SMAD4 signaling, inhibit VEGF signaling pathways, and improve PMVECs functions.
Methods:
[00156] Cell culture: Human healthy control pulmonary microvascular endothelial cells (PMVECs) (Cat # C12281 , PromoCell GmbH, Heidelberg, Germany) were cultured in microvascular endothelial cell basal media (Cat # C-22120; PromoCell GmbH) supplemented with growth factors (Growth Medium MV SupplementPack, Cat # C-39220, PromoCell GmbH) and 100 U/mL Penicillin-Streptomycin Solution (Gibco) and used between passages 4 to 8. PMVECs were cultured under standard conditions (37°C, 5% CO2, 21 % O2, 90% humidity).
[00157] RNAi. Human pulmonary microvascular endothelial cells of passages 4-6 were seeded at 150K cells/well onto 6-well plates and incubated at 37QC in a humidified 5% CO2 atmosphere. The next day, cells were washed with PBS and transfected with 50nM siRNAs against non-target controls, ACVRL1 (ALK1 ), ENG, SMAD4, LYVE1 , GPNMB or MC5R (Thermo Fisher Scientific, Waltham, MA), and 2ul of Lipofectamine RNAiMAX in a total 1 ml of OPTIMEM media. After 5 hrs of transfection, the medium was replaced with regular complete growth media. The following day, a starvation medium (0.2% FCS media) was added and incubated for 16 hrs. Cells were then stimulated with 20ng/ml of BMP9 for 2 or 24 hrs, harvested for RNA isolation, and performed RNAseq analysis.
[00158] RNA sequencing (RNAseq). RNA was isolated using RNeasy Plus Kits (Qiagen, Gaithersburg, MD) as per the manufacturer's instructions. RNA samples were sent to the Novogene Corporation (Sacramento, CA), where the following steps were carried out: Quality control: Quality and integrity of total RNA were controlled on Agilent Technologies 2100 Bioanalyzer (Agilent Technologies; Waldbronn, Germany). Library construction: The RNA sequencing library was constructed using NEBNext® Ultra II RNA Library Prep Kit (New England Biolabs) according to the manufacturer's protocols. Library quality control: Library concentration was quantified using a Qubit 2.0 fluorometer (Life Technologies) and then diluted to 1 ng/ul before checking insert size on an Agilent Technologies 2100 Bioanalyzer (Agilent Technologies; Waldbronn, Germany). The library was then quantified to greater accuracy by quantitative PGR (qPCR). Sequencing: 30 million paired reads for each sample were acquired with the Illumina NovaSeq 6000 system. Data analysis. The quality of the RNA- seq data was examined by base sequence quality plots using FastQC. TrimGalore was used to trim the sequence reads. Then, the RNA-seq reads were aligned to the human genome (hg19) using the STAR software, and a gene database was constructed from Genecode v19. Differentially expressed genes (DEG) between groups were quantified using the DESeq2 R package.
[00159] Biological process, pathway enrichment, and protein-protein network analysis: Differentially expressed downstream genes of HHT knock-down (KD) conditions at 2 and 24h of BMP9 stimulations were uploaded on the ShinyGO 0.76 platform to analyze GO biological process enrichment. The same data gene signatures were uploaded separately onto the DAVID Bioinformatics Resources 6.8 server or GO pathway enrichment analysis. The identifier was set to gene symbol, and Homo sapiens was selected to limit annotations in the gene list and background list. A significant value of p < 0.05 was set as the cutoff criterion. STRING Network analysis was performed using the SinyGO 0.76 platform.
[00160] In silico Drug prediction: Following knockdown of the HHT-causing genes (ALK1 , ENG, SMAD4) in PMVECs, we performed RNAseq and defined gene expression changes common to silencing of the three HHT-causing genes as “disease signature”. We then used the common downstream gene signature of all up-regulated genes (n=229) following HHT gene knockdown at 2 and 24 h for drug prediction. Using the Broad Institute’s database of transcriptional drug signatures, also called Connectivity map (CMAP), part of the NIH LINCS program, the goal was to identify compounds that reverse the common HHT disease signature of up-regulated genes. The list of up-regulated genes following RNAseq was uploaded on the web-based application database https://clue.io/query, selecting with the gene expression L1000 query parameter. This web-based clue query app finds perturbagens that give rise to similar (or opposing) expression signatures. After running this query, we identified compounds that either mimic the transcriptional disease signature (compounds that could potentially worsen disease) or mimic the anti-signature (compounds that could potentially improve disease) in different cell lines. We only focused on findings from the human umbilical vein endothelial cells (HUVECs) data sets as this was the only dataset performed in endothelial cells, which are the critical cell type for AVM formation. Top-ranked 5 CMAP compounds/drugs induced transcriptome alterations oppositional to (indicated by negative similarity mean) or overlapping with (indicated by positive similarity mean) caused by HHT causing gene knockdowns. Rank was determined by samples n>=3, tas value >=0.20, raw connectivity score, and FDR value. Next, we narrowed down the top scoring drug list based on their relevance to VEGF inhibition as VEGF signaling is overactivated in HHT.
[00161] Cell proliferation, apoptosis, and tube formation assay. MTT assay (Cat # V13154, Invitrogen, Waltham, MA), and caspase-3/7 assay (Cat # G8090, Promega, Madison, Wl) were performed using commercially available kits as per the instructions to assess proliferation and apoptosis, respectively.
[00162] Matrigel tube formation assay. The bottom of a 96-well plate was coated with 50ul per well of Matrigel and incubated at 37 °C for 1 h. PMVECs (1 x 104 cells/100 pl per well) suspended in a starvation medium were added to the Matrigel and cultured at 37 °C and 5% CO2 for 5 h. Vessel tube-like structures were observed and photographed under a microscope with a digital imaging system. The data were analyzed using Imaged with an angiogenesis analyzer tool.
[00163] RNA isolation, RT-PCR, and qRT-PCR: Total RNA was isolated from cells using a commercially available RNeasy® Plus Mini Kit (Cat # 74134, Qiagen, Hilden, Germany). For precision cut lung slices (PCLS), total RNA was extracted and purified using the TRizol RNA extraction protoco. The total RNAs were then converted to cDNA using a commercially available high-capacity cDNA Reverse Transcription Kit (Cat # 4368813, Applied Biosystems™, Foster City, CA) according to the manufacturer’s instructions. The expression levels of mRNAs were quantified using TaqMan™ 2x Universal PCR Master Mix (Cat # 4304437) and targeted Taqman probes (ALK1 , ENG, SMAD4, GAPDH, Hs02786624_g1 ; ID1 , Hs03676575_s1 ; HS3ST2, Hs00428644_m1 ; GPNMB, Hs01095679_m1 ; ANKRD33, Hs05002807_s1 ; RRAGD, Hs00222001_m1 ; LYVE1 , Hs00272659_m1 ; CPA4, Hs01040939_g1 ; SLC25A47, Hs01584239_m1 ; SHISA9, Hs04188640_m1 ; HIST1 H2BE, Hs00543841_s1 ; MC5R, Hs00271882_s1 ; FGF19, Hs00192780_m1 ; FRG2C, Hs01695863_sH; PLXDC2, Hs00262350_m1 ; KCNK5, Hs01123564_m1 ; and CACNA1 G, Hs00367969_m1 , Thermo Fisher Scientific, Waltham, MA) and normalized to a housekeeping control GAPDH.
[00164] Western blot. Western blotting was carried out following the standard protocol. The following antibodies were used: P-Smad1/5/9 (Cat # CST13820S, 1 :1000), p44/42 MAPK (Erk1/2) (137F5) (ERK1/2 MAPK) (Cat # CST4695S, 1 :1000), phosphorylated ERK1/2 (Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (E10), Cat # CST9106S, 1 :1000), Id1 (sc133104, monoclonal, Santa Cruz Biotechnology, 1 :100) and an HRP-conjugated secondary antibodies (Cat # ab205719 and ab6721 , Abeam, 1 :5000). The western blot band densitometric analysis was performed with Imaged.
[00165] Precision-cut lung slices (PCLS) culture. Lung tissue pieces were obtained from a healthy donor collected from the Donor Network West, San Ramon, CA. First, the tissue pieces were inflated with 2% low melting agarose prepared in 1 x PBS, and kept on ice for 15mins. Then the solidified pieces were placed onto a petri dish plate and 8mm punch biopsies were carefully made, creating cylinders of lung tissue. 6% agarose was poured into the compress tome mold and the cylinder of lung tissue was quickly placed into the mold, ensuring that 6% agarose surrounded the cylinder on all sides. After being solidified, the tissue pieces were sliced using the compress tome at 400 pm thick slices. The lung slices were then cultured in 1 x DMEM GlutaMAX medium containing 10% fetal calf serum, penicillin/streptomycin (1 %), and amphotericin B (0.1 %) in the presence and absence of Brivanib at 10 or 50 pM in 1 mL of media in a 12-well tissue culture plate. After 24 hrs, the slices were washed with 1x PBS two times and then harvested for RNA isolation.
[00166] Statistics analysis. All data analyses were performed using GraphPad Prism V9. All data are represented as the mean ± standard error of the mean. An unpaired Student's t-test was used to compare two groups, and one-way ANOVA was performed for comparing data with more than two groups, followed by an appropriate post-hoc test for multiple comparisons. The statistically significant differences were considered at p < 0.05.
Results
[00167] Identifying common downstream gene signatures of HHT-causing genes mutations in human PMVECs. As loss of function mutations in ALK1 , ENG and SMAD4 are causative of HHT, and as all three genes belong to the TGFp/BMPR2 super family signaling pathway (Figure 1A), we sought to identify common downstream signatures of all three HHT-causing gene mutations in PMVECs by RNAseq following siRNA-mediated silencing of the three genes. We used pulmonary microvascular endothelial cells, PMVECs, as endothelial cells are believed to be the critical cell type for AVM formation, and pulmonary cells, as pulmonary AVMs are one of the most common visceral manifestations in HHT. We first confirmed that following adding BMP ligand, BMP9, BMP signaling is activated in PMVECs, as evidenced by increased pSMAD1/5/9 and Id1 levels measured by western blot (Figure 1 B). Next, we silenced ALK1 , ENG, and SMAD4 with siRNA in PMVECs. As seen in Figures 1 C-E, we achieved a knockdown of over 80% for all three genes, ALK1 , ENG, and SMAD4, measured by qRT-PCR. We activated the pathway with 20ng/mL BMP9 for 2 and 24 hrs in the HHT genes knocked down cells. Stimulation with the ligand BMP9 had no effect on the expression of ALK1 , ENG, and SMAD4 (Figures 1 C-E). We then looked at one of the known downstream targets of the BMPR2/ALK1/ENG/SMAD4 pathway, Id1 (Figure 1 F and G), and whether the expression of Id1 is reduced by knocking down all three HHT genes. Interestingly, while BMP9-induced Id1 expression is decreased by knocking down ALK1 and SMAD4, a knockdown of ENG did not affect BMP9-induced Id 1 expression. In fact, Id 1 expression was rather slightly increased after ENG knockdown compared to the NT siRNA condition. These findings indicated that Id 1 is not a common downstream target of all three HHT genes but is specific for ALK1 and SMAD4 mediated signaling. This is a very important finding when it comes to identifying common downstream targets and subsequently predicting repurposed drugs that might increase the signaling for all three gene mutations.
[00168] We also carried out experiments to understand how knocking down ALK1 , ENG, and SMAD4 effected the function of PMVECs in vitro. We assessed proliferation, apoptosis, and tube formation following the silencing of the three HHT genes with siRNA. The MTT assay showed that knockdown of the HHT genes did not significantly change cell proliferation. Silencing of ENG induced apoptosis as evidenced by increased caspase 3/7 activity and decreased angiogenesis, as evidenced by reduced numbers of nodes, junctions and tube length compared to controls at baseline. ALK1 and SMAD4 knockdown had no significant effect on apoptosis and tube formation. These results suggested that while ALK1 , ENG, and SMAD4 deficiency are linked with HHT, at baseline, only ENG deficiency induced PMVECs dysfunction in vitro.
[00169] Since HHT1 and HHT2 patients show similar clinical symptoms, a previous study had identified 277 downregulated and 62 upregulated common downstream targets genes in blood outgrowth endothelial cells isolated from HHT patients carrying ENG (HHT1 ), ALK1 missense or ALK1 non-sense mutations (HHT2) compared to healthy endothelial cells, which they called a “gene expression fingerprinting of HHT’’. However, to date, no studies have identified common downstream genes of the three key HHT genes in PMVECs. Thus, we performed high throughput RNA sequencing of PMVECs silenced to either ALK1 , ENG, or SMAD4 and treated with BMP920ng/ml_ for 2 or 24 hrs. The list of significant differentially expressed genes was identified by employing the criteria of changes in gene expression of Iog2-fold changes value =>2 and p adjusted value =<0.05 between the groups. We identified 1 17 upregulated and 125 downregulated common genes 2hrs after BMP9 stimulation in PMVECs silenced for all three HHT genes (Figures 2A and C) and 1 12 upregulated and 132 downregulated common downstream genes 24 hrs after BMP9 stimulation in PMVECs silenced for all three HHT genes (Figures 2B and C). Consistent with previously identified genes associated with AVM/HHT, we found a significant upregulation of ANGPT2 and APLN and downregulation of TMEM100 after 2hrs and 24hrs BMP9 stimulation in PMVECs knockdown cells (KD) , respectively, along with other important vascular dysfunction related gene signature changes (Figures 2A-C).
[00170] To determine whether the commonly identified downstream targets (“UP and Down genes”) of the HHT genes after 2hrs or 24hrs BMP9 stimulation could potentially associate with biological processes and signaling pathways relevant to HHT, we carried out an in silica analysis of the common downstream genes using the GO biological process enrichment analysis tool, the DAVID panther pathway analysis tool, and the STRING protein-protein interaction database. GO enrichment analysis of the commonly dysregulated downstream targets uncovered a significant enrichment of HHT-related biological processes, including cell migration, adhesion, tube and vascular development and angiogenesis, extracellular matrix organization, and ion transport after 2 and 24hrs of BMP9 stimulation following HHT gene KD (Figure 2D). Moreover, we also observed enrichment of signaling pathways that are thought to regulate AVM formation in HHT, including Wnt signaling, cadherin signaling, integrin signaling, inflammation-mediated cytokines and chemokine signaling, blood coagulation, angiogenesis and TGFp-signaling pathways (Figure 2E). STRING network analysis identified some hub genes, such as PDGFRB, and VCAM that could play roles in AVM/HHT (Figure 8A- 8B)
[00171] Next, we identified the common persistently and consistently dysregulated gene signatures after 2 and 24 hrs of BMP9 stimulation in the common HHT gene KD conditions. We found 5 upregulated genes (Heparan Sulfate-Glucosamine 3-Sulfotransferase 2 (HS3ST2), Glycoprotein Nmb (GPNMB), Ankyrin Repeat Domain 33 (ANKRD33), Ras-related GTP-binding protein D (RRAGD), Lymphatic Vessel Endothelial Hyaluronan Receptor (LYVE1 )) and 7 downregulated (Carboxypeptidase A4 (CPA4), Solute Carrier Family 25 Member 47 (SLC25A47), Shisa Family Member 9 (SHISA9), Histone cluster 1 H2B family member e (HIST1 H2BE), Melanocortin 5 Receptor (MC5R), Fibroblast Growth Factor 19 (FGF19), FSHD Region Gene 2 Family Member C (FRG2C)) genes (Figure 3A). qRT-PCR validation of the common persistently dysregulated genes in PMVECs further confirmed upregulation of GPNMB and LYVE1 and downregulation of MC5R in the common HHT KD conditions after BMP9 stimulation. As further validation, those genes were regulated in the opposite direction when stimulated with BMP9 alone (Figures 3B and C and Figures 9A-D). We therefore concentrated on MC5R, GPNMB, and LYVE1 to investigate further their known importance related to HHT. LYVE1 expression was positively correlated with preoperative edema in brain AVM, while the role of MC5R and GPNMB in AVM formation and HHT is unknown. Our in vitro functional assays did not exhibit significant changes in cell proliferation, apoptosis, and tube formation in PMVECs silenced to either MC5R, GPNMB or LYVE1 at baseline (Figures 9E-G).
[00172] In silica screening of drugs that can reverse common downstream gene signatures of HHT causing gene mutations. We hypothesized that novel/re-purposed drugs could reverse the HHT/PAVM pathological downstream targets/signaling and thereby reverse abnormal endothelial cell functions (tube formation, migration, proliferation) in PMVECs. A powerful strategy to predict novel drugs that might be beneficial in HHT is to use bioinformatics approaches to match in silico drug gene expression profiles with disease or anti-disease signature profiles, as previously shown by our group. Here, we used the list of commonly upregulated genes that were differentially expressed in PMVEC silenced to all three HHT genes and stimulated with BMP9 for 2 and 24hrs, 1 17 and 1 12, respectively, which we labelled as "HHT disease signature". This transcriptomic gene expression signature served as blueprint for predicting potentially beneficial drugs that mimic the complementary “antisignature” using the Broad Institute’s database of transcriptional drug signatures, a part of the NIH LINCS program. We uploaded the genes on the web-based application database https://clue.io/query and selected with the gene expression L1000 query parameter. This webbased clue query app allows queries with external gene sets to identify compounds that either mimic our gene sets' transcriptional signatures or reverse the signature (anti-signatures) in different cell lines. We narrowed down the drug list based on the findings from the HUVECs data sets as this is the closest cell line to our data sets acquired in PMVECs. The drugs were ranked following the criteria of samples >3, tas values >0.20, raw connectivity score, and FDR values on the query app. Top scoring 5 HHT and anti-HHT drugs are shown in Figure 4B. From the top anti-HHT drug candidates (negative values), we concentrated on Brivanib as it is well-known for its anti-FGF/VEGF activities. We hypothesized that Brivanib would on the one hand, reverse the dysfunctional downstream targets related to BMP signaling and on the other hand could inhibit the overactivated VEGF pathway in HHT. To test the effect Brivanib had on restoring members of dysfunctional BMP signaling, we determined whether Brivanib reversed the 5 upregulated (LYVE1 , GPNMB, RRAGD, ANKRD33, HS3ST2) and 7 downregulated (MC5R, SLC25A47, FGF19, SHISA9, FRG2C, HIST1 H2BE, CPA4) genes, we had previously defined a being consistently dysregulated after silencing of all three HHT genes and stimulation with BMP9 for 2 and 24hrs. Importantly, we observed that Brivanib inhibited the expression of LYVE1 , GPNMB, RRAGD, and ANKRD33 induced in HHT gene KD conditions (Figures 4C-F and figures 10A-C). Furthermore, Brivanib rescued the expression of the MC5R, SLC25A47, FGF19, SHISA9, and FRG2C that were downregulated in HHT gene KD conditions (Figures 4G-K and figures 10A-C). These results demonstrated that Brivanib could improve the dysfunctional ALK1/ENG/SMAD4 signaling in HHT.
[00173] Brivanib inhibited the commonly upregulated downstream gene expression signatures of HHT-causing genes in ex vivo PCLS. To investigate whether Brivanib also inhibited the commonly upregulated signatures of HHT-causing genes in human lung tissue, we measured expression of LYVE1 , GPNMB, HS3ST2, RRAGD, and ANKRD33 using qRT-PCR in human PCLS treated with Brivanib for 24 hrs. PCLS were prepared from a healthy human donor obtained from Donor Network West, California. PCLS were treated with 50 uM Brivanib or DMSO for 24 hrs and then harvested for RNA isolation (Figure 5A). Brivanib inhibited mRNA expression of LYVE1 . GPNMB, and HS3ST2 in PCLS (Figures 5B-F). This inhibition in GPNMB, LYVEI and HS3ST2 expression ex vivo was similar to the inhibition of those genes in PMVECs after HHT gene knockout and Brivanib treatment in vitro. Brivanib did not change expression of RRAGD and ANKRD33 in PCLS.
[00174] Brivanib inhibited VEGF-induced ERK1/2 MAPK, -VEGF-induced PMVECs proliferation and tube formation in vitro. As over-activation of pro-angiogenic pathways, such as VEGF signaling, is strongly linked with the development of vascular dysfunction in HHT, and as Brivanib was previously shown to exhibit anti-VEGF effects both in vitro and in vivo in cancer and liver fibrosis studies, we carried out cell culture experiments to determine whether Brivanib can also inhibit the VEGF signaling pathway in PMVECs. PMVECs were cultured in the presence and absence of 10 pM Brivanib or DMSO in serum starvation media (0.2% FCS) for 24 hrs and then stimulated with 40 ng/mL VEGF for 10 mins. We measured phospho- ERK1/2 MAPK, total ERK1/2 MAPK (a downstream target of VEGF signaling) levels by western blotting. Notably, our results showed that Brivanib completely blocked VEGF-induced phospho-ERK1/2 levels in PMVECs (Figure 6A).
[00175] We next assessed functional properties and endothelial behaviors with regards to tube formation and proliferation with and without Brivanib treatment. T o determine whether Brivanib inhibits the VEGF-induced proliferation of PMVECs, we performed an MTT assay to measure the viability of PMVECs treated with VEGF (20 ng/mL) in the presence and absence of Brivanib (10 pM) in serum-starved media (0.2% FCS). As expected, VEGF treatment significantly increased PMVECs proliferation (Figure 6B). Importantly, Brivanib completely blocked the VEGF-induced proliferation of PMVECs (Figure 6B). Brivanib alone, without ligand stimulation, did not change proliferation of PMVECs. These findings suggested that Brivanib attenuated VEGF-induced PMVECs proliferation in vitro. To further explore the anti- angiogenic role of Brivanib on PMVECs in vitro, a matrigel based tube formation assay was performed. Results revealed that Brivanib inhibited tube formation in PMVECs as evidenced by the decreased number of junctions and total tube lengths compared to DMSO-treated control cells (Figure 6C).
[00176] Loss of function mutations in ALK1 , ENG or SMAD4 are linked with the development of AVM and HHT and the penetration rate of the gene mutations to the disease phenotypes is high (-100%). Thus, we identified common downstream targets/pathways of the three HHT- causing genes, ALK1 , ENG, and SMAD4 using whole genome RNAseq following knock down of the HHT genes in PMVECs in vitro. VJe also investigated whether we could identify a drug that can reverse the dysregulated downstream gene signatures and improve the common downstream dysfunctional pathways and HHT-related dysfunction of cellular phenotypes. Here, we found that ID1 , a major downstream target of the TGF3/BMP signaling pathway, is not a common downstream target of all the HHT gene knockdown (ALK1 , ENG, or SMAD4) conditions, while other downstream targets, such as LYVE1 , GPNMB, PLXDC2, and MC5R, are downstream of all three HHT genes. Furthermore, we identified a small molecule drug, Brivanib, that could, on the one hand, reverse the downstream gene signatures following knockdown of all three HHT genes and, on the other hand, inhibit the VEGF signaling pathway, improve proliferation and tube formation in PMVECs. These findings suggest that Brivanib might be effective in treating AVMs and HHT by normalizing dysfunctional downstream signaling.
EXAMPLE 2 PAH
[00177] Protein tyrosine kinase (PTK) inhibition has mixed effects on the pulmonary vasculature. We hypothesized subsets of PTKs are required for endothelial (EC) homeostasis by supporting pathways like Bone Morphogenetic Protein receptor 2 (BMPR2) signaling. In a high-throughput siRNA screen we identified that SRC-Family B (SrcB) PTKs activated whereas SRC-Family A (SrcA) PTKs suppressed BMPR2 signaling, measured by inhibitor of differentiation 1 (Id1 ). Inhibiting the strongest activator Lek (SrcB) in human pulmonary artery endothelial cells (PAECs) suppressed BMPR2 signaling and tube formation and increased apoptosis whereas inhibiting the strongest inhibitor, Fyn (SrcA) did the opposite. Whole- transcriptome analysis identified an “endothelial” and “inflammatory” gene signature coregulated by siLck and siFyn. Using Connectivity Map, we predicted compounds that recapitulated these signatures identifying Quizartinib as an inhibitor and Brivanib as an activator of Id 1 in PAECs. This SrcA/SrcB relationship can guide transcriptionally based drug discovery to identify compounds that promote or suppress EC dysfunction.
[00178] Despite an association between TKIs, PTKs, and PAH development, knowledge of how PTKs act on specific cell types and signaling pathways continues to evolve. Since it is observed that (1 ) different TKIs generate widely different clinical phenotypes and (2) specific PTKs are associated with human disease development, we theorized that individual PTKs may specifically target signaling pathways critical for maintaining pulmonary vascular integrity.
[00179] One model pathway is the Bone Morphogenetic Protein Receptor 2 (BMPR2) pathway. Heterozygous mutations in BMPR2 are the most common cause of hereditary PAH. In vitro silencing of BMPR2 recapitulates many features of ECD. However, because BMPR2 disease penetrance is 20%, other genetic and environmental factors are required to develop PAH. Work investigating the interaction of PTKs with BMPR2 signaling shows that inhibition of the PTK SRC can improve BMPR2 signaling via improving BMPR2 receptor plasma membrane localization. Conversely, we recently identified that lymphocyte specific protein tyrosine kinase (LOK), a gene coding for the Src-family tyrosine kinase LCK, has the potential to support BMPR2 signaling. Given the opposite effects on BMPR2 signaling by LCK and SRC, we hypothesized subsets of non-receptor PTKs may differentially affect BMPR2 signaling and EC health.
[00180] To test this, we examined a broad group of PTKs in a high-throughput siRNA screen and found that Src-family non-receptor PTKs divide into BMPR2-supportive and -repressive groups when using a canonical BMPR2 responsive transcription factor, Inhibitor of Differentiation 1 (Id1 ) as a readout for BMPR2 signaling. Further, PTKs modulate BMPR2 in a way that respects an evolutionarily defined boundary separating Src Family A (SrcA) from Src Family B (SrcB) PTKs. Within these groups, the SrcB PTK LCK is the most BMPR2 activating kinase while the SrcA PTK FYN is the most BMPR2 repressive. We also find this divided effect on cell signaling pathways also extends to canonical NF-KB signaling. We use the transcriptom ic signature of SrcA FYN and SrcB LCK knockout in PAECs to search the publicly available Connectivity Map (CMap) database and find drugs that mirror the shared effects of Fyn and Lek knockout. This leads us to two TKIs, Brivanib and Quizartinib, that potentiate and suppress canonical BMPR2 signaling in PAECs.
[00181 ] We demonstrate for the first time that in PAECs the evolutionary divide between SrcA and SrcB PTKs also extends to the differential control of cell signaling pathways. In PAECs, Src PTKs split into two groups: a pro- BMPR2, pro-endothelial and anti-inflammatory SrcB, and an anti-BMPR2, anti-endothelial and pro-inflammatory SrcA. We use this knowledge to find compounds that differentially modulate canonical BMPR2 signaling. This guides the selection of TKIs that achieve therapeutic goals and avoid ECD.
Results:
[00182] A High-Throughput screen demonstrates that Src-Family A PTKs are BMPR2- Repressive, and Src- Family B PTKs are BMPR2-Activating. To assess canonical BMPR2 signaling, we used Id 1 as a transcription factor read-out for BMPR2 activation. As previously described, we employed a C2C12 mouse myoblastoma cell line stably transfected with a BMP response element Luciferase Id1 (BRE-Luc-ld1 ) reporter. Cells were systematically transfected with a murine-wide siRNA library targeting 22,124 genes and then treated with BMP4 in the Stanford High-Throughput Bioscience Center. BMPR2 activity by luciferase expression was quantified by luminescence and (Figure 11 A) cell viability was assessed with a tryptan-blue stain. The mean change in Id1 expression and cell viability in response to knockout of one of the 22,124 genes across three replicates was calculated and presented in aggregate in Figure 1 1 B. Genes left of 0 on the x-axis reduce Id1 expression when targeted by si-RNA and genes to the right of 0 on the x-axis increase Id1 expression after transfection. Using published phylogenetic classifications of receptor- and non-receptor-PTKs, representative PKTs from each major family were chosen and highlighted within the HTS with an even subdivision of PTKs that activated or repressed Id1 expression (Figure 7B, red dots). Change in Id1 expression relative to major representative PTKs repeated this heterogeneity but showed the largest magnitude of change was seen with LCK and FYN, both members of the Src-Family of protein tyrosine kinases (Figure 1 1 C). Focusing specifically on the Src-Family of, we found the magnitude of change in Id1 expression (an increase versus decrease) induced by PTK knockout in our HTS data matched exactly the phylogenetic division between SrcA and SrcB kinases (Figure 1 1 D and 1 1 E). In summary, our agnostic, high throughput screen shows Id1 modulation by Src-family kinases respects the phylogenetic division between SrcA and SrcB kinases.
[00183] LCK is expressed at a mRNA and Protein level in endothelial cells of human pulmonary arteries and correlates with Id1 expression both in health and in disease. We next aimed to find a human cell lineage suitable to study the relationship between SrcA, SrcB and BMPR2 pathway members. For simplicity, we decided to use LCK as the representative SrcB and FYN as the representative SrcA kinase. Dogmatically, SrcA kinases are widely expressed while SrcB kinases are restricted to specific cell lineages. FYN has been studied in ECs and smooth muscle cells while LCK is traditionally described in T-Cells and NK-Cells. Recently have we and other have shown LCK is expressed in cultured human PAECs and HUVECs. It remained unknown where LCK was expressed in the whole lung and if it was altered in disease. Single molecule RNA in situ hybridization (sm-FISH) showed that Lek mRNA was expressed at low levels in the endothelium compared to higher expression in sparse Acta2/Cdh5 negative cells presumed to be lymphocytes (Figure 12A). LCK protein was identified in cultured human PAECs with perinuclear localization (Figure 12B). Human PAECs obtained from both PAH patients and donor controls expressed LCK at a protein level, but in a heterogeneous fashion (Figure 12C).
[00184] Table 1 : Patient Demographics
Figure imgf000046_0001
Figure imgf000047_0001
PAH = Pulmonary Arterial Hypertension
HPAH = Hereditary Pulmonary Arterial Hypertension IPAH = Idiopathic Pulmonary Arterial Hypertension PDE5i = Phosphodiesterase type 5 Inhibitor ERA = Endothelin receptor antagonist PC = Prostacyclin
[00185] Assessing for Id1 and Snail/Slug expression as surrogates for active BMP and TGF-p signaling in the PAH and donor cell lines found a significant correlation between Id 1 and LCK irrespective of whether the sample was obtained from control or PAH patients (r = 0.68, p = 0.043, Figure 12D). Publicly available single cell RNA sequencing data of Peripheral Blood Mononuclear Cells (PBMCs) from healthy controls was used to quantify Lek and BMPR2 pathway member genes in lymphocytes. (Figure 12E). High Le expression was verified in T- Cells and NK-Cells. However, BMPR2 and Id1 was expressed in few PBMC cells (<20%), with low expression in all cell lineages save for dendritic cells. Due to poor co-expression of Lek, Id1, and BMPR2 in lymphocytes, we concluded that PAECs would be the best first lineage to study an interaction between BMPR2, Id1, Lek, and Fyn in the human lung. Finally, efforts were undertaken to investigate the expression of Lek protein in both mouse and rat ECs. We were able to recapitulate robust LCK staining in lymphocytes, but not in the ECs of mice or rats, as opposed to human ECs.
[00186] Silencing Lek significantly impairs basal and BMP9-mediated canonical BMPR2 signaling whereas silencing Fyn does the opposite. It was recently found that the kinase pockets of SrcA and SrcB kinases are structured such that their preference of tyrosine substrate is influenced by the net charge of nearby aminio acids. Because studies have demonstrated a link between the SH3 region of LCK and SMAD proteins through immunoprecipitation, we wondered if the discordant response of LCK and FYN on Id1 expression might be due different interactions with second messenger SMAD proteins. First, itwas verified via RT-aPCR that siRNA silenced LCK in PAECs (Figure 13A). In PAECs subject to Lek siRNA knockdown (siLCK PAECs), Id1 expression by RT-qPCR significantly decreased in non-serum reduced (2% FBS) media whereas Snail expression was upregulated (Figure 13A). A decrease in Id1 in siLCK PAECs in basal media (0.2% FBS) was also observed. When starved PAECs were stimulated with BMP9 for 1 .5 hours, phosphorylated SMAD-1 (pSMADI ) and Id1 expectedly increased (Figure 13C). SiLCK PAECs exhibited a significant impairment in BMP9-induced Id1 expression (Figure 13C) to nearly basal levels. Similarly, pSMADI was reduced but not fully to baseline. Though there is a trend towards an increase in Snail/Slug expression in siLCK cells, the effect was variable and not significant at a protein level. There was no change in BMPR2 or total SMAD1 expression across all conditions. Finally, pSMAD3 levels mildly, but not significantly, increased with either siLCK or TGF-fS1 . This may be attributable to a short treatment duration in the TGF- 1 case (Both TGF-pi and BMP9 were given 1 .5 hours prior to cell harvest).
[00187] Next, we investigated the effect of silencing the SrcA Fyn (siFyn) in PAECs. In nonreduced serum media, there was no effect on the expression of Id1 expression by RT-qPCR. However, under starvation conditions (0.2% FBS) for 24 hours, siFYN cells significantly increased Id1 expression and BMPR2 at 48 hours (Figure 13D). We did not observe a significant change in pSMADI or Id1 induced by siFYN in PAECs grown in starvation (0.2% FBS) conditions by 72 hours. Also, the effect of siFYN on Id 1 was variable in PAECs grown in full media (2% FBS). However, there was a significant increase in BMP9 mediated Id1 expression in siFYN cells versus siNT cells (Figure 13E). BMP-9 treated siFYN PAECs had reduced pSMAD3 expression relative to nontargeted controls (Figure 13E). We were unable to detect a change in SNAIL/Slug expression at 72 hours in si-Fyn cells regardless of media conditions
[00188] Taken together, this shows that Lek and Fyn have opposing effects in the pulmonary endothelium with respect to canonical BMPR2 signaling. Lek knockout decreases SMAD1 phosphorylation and Id1 expression, while Fyn knockout promotes BMP9 mediated SMAD1 phosphorylation and Id1 expression.
[00189] Lek but not Fyn suppression in human PAECs is associated with EC dysfunction. Next, we sought to determine if the changes in BMPR2 signaling due to Lek or Fyn knockout associate with phenotypic changes in PAECs. siLCK and siFYN PAECs were examined in tube formation angiogenesis assays alongside non-targeted siRNA treated (siNT) PAECs. Because the optimal concentrations of siRNA and lipofectamine required to achieve an effective knockdown of Lek in PAECs is different than the optimal concentrations for Fyn, siLCK and siFYN cells are compared to siNT controls termed siNT-F and siNT-L that have matched siRNA and lipofectamine concentrations. Viable cells were seeded at a constant density on Matrigel 48-hours post transfection. As hypothesized given its beneficial effect on BMPR2 signaling, siFYN cells had increased tube formation with a higher total tube length and more tube junctions relative to siNT-F (Figure 14A,B). Conversely, siLCK PAECs sprouted few, short tubes with shorter total tube length and decreased tube junctions compared to siNT- L (Figure 14A,B). Notably, cell death was accelerated after si-Lck versus si-NT-L (Figure 14C). We hypothesized this correlated to an increase in PAEC apoptosis, and indeed, we found higher Bax and Bel levels in siLck cells versus siFyn cells relative to their respective controls (Figure 14D). [00190] Increased apoptosis was confirmed by assessing Caspase 3/7 activity (Figure 14E). Because EC dysfunction includes upregulation of integrins along with other pro-inflammatory cues, we finally assessed VCAM1 expression 72 hours after siLCK and siFYN transfection in full (2% FBS) media. We found increased expression of VCAM1 in siLCK cells but not siFYN cells (Figure 14D). Taken together, this suggests that suppression of the SrcB LCK generates a dysfunctional EC phenotype as opposed to the SrcA FYN.
[00191 ] Whole transcriptome analysis of siLCK and siFYN PAECs validates a differential effect on BMPR2 signaling and finds siLCK cells adopt a pro-inflammatory signature centered on NF-KB canonical signaling. To this point, we have taken a narrow view examining canonical BMPR2 signaling in response to Lek and Fyn knockout in PAECs. As each PTK engages thousands of tyrosine phospho-targets in eukaryotic cells, it is unlikely that LCK and FYN only act on BMPR2 signaling. To determine if additional signaling pathways outside of the canonical BMPR2 signaling pathway are differentially regulated by LCK and FYN we performed a transcriptome-wide assessment and curated sets of genes differentially regulated by LCK and FYN.
[00192] Bulk-cell RNA sequencing was conducted in four groups of PAECs: siLCK, siFYN, a scrambled siRNA control group (NT), and a scrambled siRNA group subjected to BMP9 stimulation (NT+ 50 ng/mL BMP9 for 2 hours prior to harvest). For all arms, we used the same concentration of siRNA and lipofectamine to control for gene expression variability induced by the transfection process. This transfection protocol resulted in a less stringent Lek knockdown relative to Fyn and NT and was less effective at reducing LCK protein relative to FYN. However, we felt that RNA sequencing would have the sensitivity to detect major gene expression changes in the LCK condition. Knockout of target genes was verified by RT-qPCR. Quality control metrics for the RNA-seq analysis are shown. Principal component analysis demonstrates tight clustering of within-group samples with separation between experimental groups. Volcano plots of differentially expressed genes (DEGs) for siLCK and siFYN conditions are shown in Figure 15A. Although Fyn was identified in the DESEQ2 analysis as a significantly downregulated gene in the siFyn case, Lek was not found to be significantly downregulated in the siLCK case due to dropout owing to a low number of raw counts for Lek (between 10-15) in each sample. A volcano plot for the siNT+BMP9 condition is shown in Figure 21 . Top upregulated genes for siLCK include Il6st, Ccl7, Wnt5a, and 111 b. Top downregulated genes for siLCK are Ace, Lyvel , Gja5, and Dysf. For siFyn upregulated genes included Bmp4, Gdf7, Gdf11 (Bmp11 ), Gja5, Lyvel, Id1, and Tgfbl. Genes downregulated in siFYN include Tlr3, C1s, C3, Ccl8, and Vcaml.
[00193] Cluster of genes upregulated in siFYN and siNT+BMP9 conditions and downregulated in siLCK were identified (Figure 15B) and included known BMP9 responsive genes Id1 , SMAD6, Hey2, and BMPR2. Genes upregulated in siLCK and downregulated in siFYN and siNT+BMP9 conditions correlated with inflammation and ECD, and included Vcaml , Icami , IL-1 b, IL-6, and Ccl8. We specifically examined genes known to be important in EC biology and found a theme is the downregulation of EC specific marker genes (Pecaml , Gja5, Cdh5, Cldn5) in siLCK with an upregulation of the same genes in siFYN (Figure 15C). This extends to genes implicated in endothelial to mesenchymal transition (EndMT - MMP9, COL1 A1 increased in siLCK, Twistl down in siFYN), ECD (Ccl8, Cx3cl1 , IL-1 B, HLA-A, Vcaml , Icami up in siLCK), genes involved in prostaglandin synthesis (Ptgsl , Ptgis down in siLCK), nitric oxide synthase (Nos3 down in siLCK), and apoptosis (Bak, Fas up in siLCK). Taken together, this suggests that siLCK and siFYN cells have significant and opposing changes in gene expression correlating with EC identity and function.
[00194] To better understand the gene set under control of both FYN and LCK in PAECs, we focused on DEGs inversely regulated by Fyn and Lek - genes both significantly upregulated in siLCK cells and significantly downregulated in siFYN cells or vice versa. A lower fold change of greater than 1 .25 or less than 0.8 with a false discovery rate of less than 5% was used because some genes may not produce as large a fold change in the si-Lck condition versus si-Fyn condition due to transfection conditions. In Figure 15D the Venn diagram shows 671 genes increased in siLCK and decreased in siFYN conditions (Figure 15E). 481 genes are decreased in siLCK and increased in siFYN conditions (Figure 15F). Gene set enrichment analysis and transcription factor prediction was performed on these two gene sets.
[00195] In the “siLCK up/siFYN down” group (Figure 15E), the most co-differentially regulated genes were associated with type-1 interferon signaling: Tnfaip6 and 111 B. Gene ontology (GO) analysis converged on pathways elicited by interferon gamma or lipopolysaccharide and were associated with increased interferon oc/p and IL-6 production, NF-KB activation, and MHC-1 activation. Indeed, transcription factor prediction by the three methods found the canonical NF-KB transcription factor RelA was tied to the majority of co-DEGs (Figure 15E). Returning to cell culture, it was verified that phosphorylated p65-RelA was significantly increased in the nuclei of siLCK PAECs versus siFYN or siNT conditions (Figure 15G). Given this, we refer to the set of co-DEGs both increased in siLCK PAECs and decreased in siFYN PAECs as an “inflammatory signature” linked by canonical NF-KB activity.
[00196] In the “siLCK Down/siFYN Up” group (Figure 15F), co-DEGs were comprised of EC artery and capillary specific genes (Gja5, Lyvel , Gphibpl ). GO analysis included sprouting angiogenesis and endothelial cell migration. Transcription factor analysis did not predict one single transcription factor, but did implicate Sp1 , Erg, Runx2, and Snail (Figure 15F). Erg is decreased in PAH and exerts control over Notch signaling to promote vascular stability. Sp1 activates VEGF expression in tumor derived ECs and acts as a promoter for the gene Endoglin. Sp1 loss can contribute to HHT and PAH via Endoglin suppression. Based on this, the set of genes decreased by siLCK and increased by siFYN are termed an “endothelial signature” and incorporate genes associated with EC identity, BMPR2 signaling, and migration.
[00197] In summary, transcriptomic analysis of siLCK and siFYN PAECs finds that Lek and Fyn can both co-regulate canonical BMPR2 signaling and canonical NF-KB signaling. The set of co-DEGs regulated by Lek and Fyn can be divided into an “endothelial” and “inflammatory” signature.
[00198] Transcriptomic gene signatures combined with searches in the CLUE Connectivity Map (CMap) database predict compounds that mirror the differential effects of Lek and Fyn knockout. To this point we have manipulated Lek and Fyn through transfection of siRNA. We theorized that there may exist drugs, possibly TKIs, mirroring the effects of Lek or Fyn knockout on BMPR2 and NF-KB in ECs. A major challenge in selecting a TKI based on its kinase avidity is the fact that TKIs are promiscuous with respect to their PTK targets and seldom specifically targeting a single kinase. This is further complicated by the fact that the number of kinase targets exceeds PTKs by a factor of 100. Thus, each PTK has the potential to unpredictably influence multiple cellular signaling pathways.
[00199] The availability of Broad Institute CLUE Connectivity Map (CMap) database and search tools (a superset of gene signatures which includes NIH LINCS/L1000 data) facilitates linkage of genes, ligands, and compounds (termed perturbagens) to a desired DEG signature. We hypothesized that using higher resolution transcriptomic data that represents the cell states described by the endothelial and inflammatory signatures could circumvent the challenges associated with drug prediction at a protein kinase level.
[00200] Figure 16A details the search strategy. Genes in the endothelial and inflammatory signatures were restricted to “Best INFerrred” or BING genes and ranked on the differential magnitude of change between siFYN and siLCK conditions. As it was unclear what number of genes would produce the best search results (between 10 and 150 genes can be included in a CMap search) we performed 18 total searches using an array (10, 15, 20, 25, 50, 75, 100, 125, and 150) of BING genes each for both endothelial and inflammatory signatures.
[00201] Aggregate search results are shown in Figure 16B. For the inflammatory signature, there was a linear relationship between the number of genes returned by the search and the number of significant perturbagens (defined as having a false discovery rate of < 5%) returned. The endothelial signature returned the most perturbagens when 75 genes were used, with numbers trailing off after. Given our gene ontology above, it may be that the endothelial signature represents a more heterogeneous set of pathways, with higher numbers of genes distracting the search algorithm after a certain point. Due to evolving methods in the usage CMap data, we used the 150-gene inflammatory signature search and the 75-gene endothelial signature search because they returned the most perturbagens.
[00202] To validate if the perturbagens returned in our query matched our prior experimental results, we focused on the ligand subset of perturbagens. Figure 16C, the x axis shows the strength of correlation between our endothelial and inflammatory gene signature and the CMap gene signature generated by a specific ligand. Negative values indicate inverse connection, or the “anti-signature.” The y-axis indicates significance, with higher values having a lower false discovery rate. Results are divided by cell line. In CMap, the ligands TNF, 111 a, and Interferon-x]/ consistently connected with the inflammatory signature in multiple cell lines (A375, A549, HT29, MCF10A, and SKBR3), agreeing with the notion of upregulated canonical NF-KB signaling. Ligands that generated the anti-inflammatory signature are varied and included Bmp1 1 and Flt3. For the endothelial signature, there was a cell specific effect. Top ligands and cell lines connecting with the endothelial signature were Apelin (Apln A375), Gdf3 (A549), Bmp4 (HCC515), Bmp2 (HA1 E, PC3), and Vegf-a (PC3). Interestingly, using the same ligand to treat a different cell line could induce the endothelial anti-signature, as with Bmp2 in MCF7 cell lines, Bmp1 1 in HCC515 cells, or Bmp2 or Gdf3 in A375 cells. Thus, although there was agreement between the CMap ligand data and our prior PAEC RNA-seq gene ontology data (e.g. Tnf, 111 a, Bmp4, and Apln), the choice of CMap cell line in which to select a potential compound is important because the same ligand could positively connect with a gene signature in one cell line while negatively connecting in another.
[00203] We attempted to determine the cell lines in CMap that would make the best surrogates for PAECs. CMap data includes transcriptomic data from cells subject to either gene knockdown by short hairpin (SH) RNA or gene overexpression (OE) by plasmid. Importantly, Lek and Fyn SH and OE experiments were conducted in multiple cell lines in Cmap. Therefore, we reasoned that cell lines in which the CMap sh-Lck or sh- Fyn signature matched that of our own PAEC siLCK or siFYN gene signature would be a suitable substrate for drug prediction. [00204] Results of this analysis are shown in Figure 16D for siFYN and Figure 16E for siLCK.
The heatmap body indicates how well the CMap gene signature connects with our si-signature in PAECs. The vertical axis indicates the number of genes used in the CMap search (Figure 16B). The horizontal axis shows each CMap sh- or OE-experiment, grouped by cell type. The “modulation” color coding indicates if the CMap experiment was a sh (blue) or an OE (yellow) manipulation. “Acceptability” color coding is green when the CMap sh signature matches the PAEC si- signature or if the PAEC si-anti-signature matches the CMap OE signature. In Figure 16D, only the AGS, HEK293T, and SW480 cell lines shared similarity between the CMap sh/OE-Fyn signature and our own siFYN PAEC signature. HA1 E and PC3 cell lines have near inverse signatures with respect to PAECs, whereas the A375 cell line had inconsistent correlation. In Figure 12E, HA1 D, HEK293T, AGS, and ES2 cell lines had similarity between CMap sh/OE- Lek signatures and our own siLCK PAEC signature. PC3 and HT29 cell lines have poor correlation. From this, we conclude that only a subset of appropriate cell lines can be used for drug prediction. Using all cell lines in aggregate for drug prediction may find compounds with good connection scores that produce an effect opposite to what is intended.
[00205] We examined the list of compounds and small molecules in CMap with strong connection to the endothelial and inflammatory gene signatures, restricting to AGS, HEK293T, and SW480 cell lines for the endothelial (siFYN) signature and the A375, AGS, ES2, HEK293T, and YAPC cell lines for the inflammatory signature (siLCK), Figure 16F. Importantly, the CMap data set has transcriptomic data compiled from HUVEC cells treated with small molecules. However, we could not perform the analysis of Figure 16C-16E to validate the HUVEC line sbecause at the time of this study ligand, sh and OE data from HUVECs was not included in the CMap. It was assumed HUVEC cells would behave similarly to PAECs and were included in drug prediction analysis. In Figure 16F, two observations were made. First, many of the highest connection scores occurred in HUVECs, as with Regorafanib, Gsk- 2334470, and Bx-795. Second, it was noted that most drug influenced both signatures. For instance, Regorafanib has high positive connection with both the endothelial and inflammatory signatures. This finding was intriguing and led us to wonder if compound selection should be informed by the endothelial and inflammatory signatures simultaneously. To do, we required a cell line where candidate compounds had both inflammatory and endothelial signatures. Of the options available, AGS, and HEK293T cells met the requirements of Figure 16D and 16E. However, their shared compounds had low connectivity scores. Only the HUVEC line had compounds with high inflammatory and endothelial connectivity scores; compounds are shown in Figure 16G where distance from origin indicates strength of connection to both signatures. FDR and transcriptional activation score (TAS) are color coded to indicate statistical significance and repeatability across replicates respectively. Compounds in the top right corner are connected positively with the endothelial and inflammatory signatures, whereas compounds in the bottom left corner connect negatively to both signatures. Of the options, the top left corner is theorized to produce a desired “pro-endothelial and antiinflammatory” phenotype. It is notable that TKIs (denoted in blue) appeared to localize most to the top right corner.
[00206] Ten compounds underwent a preliminary screen in PAECs. Selection criteria were that compounds are (1 ) a TKI, (2) located in the periphery of Figure 16G (highest magnitude of connectivity scores), (3) have low FDR and high TAS scores, and (4) are commercial availability. The most promising candidates from preliminary screening were selected from each quadrant: Regorafenib (top right), Brivanib (top left), Pazopanib (bottom left) and Quizartinib (bottom right). [00207] In Figure 17A, we examine if Quizartinib and Brivanib generate the anticipated endothelial signature effect by examining SMAD phosphorylation and Id1 expression in PAECs, with or without BMP9 stimulation. Pre- treatment with Quizartinib in DMSO versus DMSO alone for 24 hours fully suppressed expression and significantly decreased p- SMAD1 expression 2 hours after treatment with BMP9. Conversely, Brivanib increased basal expression of Id 1 BMP9 mediated expression of Id 1 but did not have a significant effect on p- SMAD1 expression. Thus, Quizartanib and Brivanib did exhibit the anticipated effect on Id1 as predicted by the endothelial signature, but Brivanib may work via a mechanism independent of canonical SMAD signaling.
[00208] In Figure 17B-C, we examine how Regorafenib and Pazopanib modulate NK-KB signaling. Regorafenib treatment resulted in disruption of the EC barrier with increased nuclear p65 RelA expression (Figure 17B) and an overall smaller nuclear size. Thus Regorafanib increases canonical NK-KB signaling as predicted by our CMap searches. Pazopanib had little effect on NK-KB signaling or the EC barrier, possibly due to low baseline activity or lack of direct NK-KB stimulation. It is also notable that despite the predicted endothelial effect of Regorafanib, the inflammatory signature appears to “win out”, negating the endothelial effect. While Brivanib is found in the left upper quandant, representing a drug with a pro-endothelial, anti-inflammatory signature, three other TKIs were found in the left lower quandrant, representing the anti-inflammatory signature. Pazopanib is already tested for HHT in clinical trials, while the use of Linsitinib as well as Dabrafenib have not been described in HHT or PAH before.
[00209] This study is significant for several reasons. First, it demonstrates Src-A and Src-B protein tyrosine kinases differ not only by their phylogenetic classification, but also in how they modulate cellular signaling pathways. When granularly focusing on example SrcA and SrcB kinases, we show differential regulation of both the BMPR2 and the NF-KB pathways occurs in PAECs. Second, although prior studies have investigated the effect of Src-Family PTKs in the pulmonary vasculature, we specifically investigate FYN and LCK in PAECs, a lineage implicated at the inception of lung diseases such as PAH. By showing selective SrcB (LCK) PTK knockout both blocks BMPR2 and increase NF-KB signaling we provide an additional rational to link TKIs to the development of EC dysfunction through inhibition of SrcB kinases. Third, we use CMap data to predict drugs mimicking the effects of FYN and LCK inhibition in PAECs. Though we successfully predict drugs, we also reach several conclusions about the drug prediction methodology. We find a transcriptomic signature can be both upregulated or downregulated by the same perturbagen in different CMap cell lines. This argues against indiscriminately using all available cell lines for drug prediction. Instead, by tailoring the search to include biologically congruent cell lines, we recover ligands and drugs matching our predicted outcome. Finally, as expected, we find that the same drug can manipulate two gene signatures, and one signature (e.g. inflammatory) can “win out” over another (e.g. endothelial) in testing. Thus, using multiple gene signatures can more efficiently find compounds with a desired outcome.
[00210] SrcA and SrcB Kinases Differentially Regulate Cell Signaling Pathways: Because 518 kinases phosphorylate an estimated 119,809 unique substrates, PTKs must be regulated to avoid unintended signal activation via crosstalk. For Src kinases, it was believed this was achieved by cell-specific expression, with SrcA kinases expressed in diverse cell types and SrcB kinases restricted to hematopoietic lineages. Phylogenetic analyses now show that SrcA and SrcB kinases are genetically distinct. Further, the amino acid sequence of the kinase pocket also obeys the SrcA and SrcB phylogenic division and confers kinase-substrate specificity. This is suspected to prevent kinase cross-talk in regions where PTKs are in close spatial proximity, such as with LCK and FYN in the T-Cell Receptor. To our knowledge, we are the first to demonstrate of cellular signaling pathways react in a different ways to SrcA and SrcB knockout. Thus, SrcA and B kinases can be distinguished by genetic sequence, by kinase pocket amino acid sequence, and by how they support or inhibit cellular signaling pathways. It is notable that our agnostic high throughput screen shows that all SrcA and SrcB kinases modulate canonical BMPR2 signaling in the same way, albeit at different levels.
[00211] For granularity, we narrow to two example PTKs and show this effect holds in human PAECs and extends to the NF-KB pathway. It is likely that other BMP signaling pathways comprised of different type 1 and type 2 TGF-p receptors are differentially regulated by SrcA and SrcB family members. A natural next step would be examination of LCK and FYN on BMP signaling in lymphocytes. From the perspective of our public sc-RNA seq PBMC analysis, Id2 may be a better BMPR2-responsive transcription factor in lymphocytes versus Id1. Finally, the exact mechanism for FYN and LCK regulation of BMPR2 and NF-KB are not fully explored. Hypotheses include FYN and LCK kinases potentiating conformational changes in SMAD proteins or SMAD inhibitor proteins, FYN and LCK acting as a scaffolding proteins for membrane receptors, or FYN and LCK modulating the expression of BMP ligands within culture (due to our observation of increased BMP12 and BMP13 mRNA by RNA seq).
[00212] LCK and FYN are present in PAECs. Selective inhibition of the SrcB LCK can drive EC dysfunction. Studies have investigated the role of Src kinases in the pulmonary vasculature, emphasizing smooth muscle cells (SMCs). Notably, LCK is not found at high levels in SMCs and few studies have investigated LCK in ECs. Here, we show LCK is not only expressed at a message and protein level in PAECs, but knockout of Lek has functional consequences in human PAECs. Notably, we were unable to detect LCK protein or mRNA in the ECs of mice or rats, by staining or from public RNA seq data sets. LCK and FYN are present in lymphocytes and LCK levels are reduced in PBMCs of patients with interstitial lung disease, lupus, and, as we have shown, pulmonary arterial hypertension. However, as shown here, the more ubiquitous peripheral reduction of LCK in PAH patients does not translate to the lung endothelium in PAH patients. We did however observe a strong correlation between LCK and Id 1 expression in cultured PAECs, implying LCK may positively facilitate BMP signaling ECs. As LYN is expressed in SMCs, future studies could use LYN as representative SrcB kinases, potentially comparing to the known effect of SRC.
[00213] Using endothelial and inflammatory gene signatures, we use a biologically tailored methodology of drug prediction to manipulate canonical BMPR2 and NF-KB signaling in PAECs. Drugs are inherently dirty and can modulate several signaling pathways simultaneously. TKI-PTK kinome analyses show that the ATP-kinase-pockets of PTKs are highly conserved resulting in promiscuous PTK inhibition by TKIs and few TKIs that are truly specific for a single PTK target. The kinases pockets of SrcA and B kinases are distinct but still share homology. It is therefore reasonable to assume a candidate TKI will bind many PTKs simultaneously in a cell with varying affinity, influencing several pathways at once. To manage this, we hypothesized that instead of focusing on protein-TKI interactions we could instead find TKIs generating a desired transcriptomic cell state. By using our FYN- and LCK- derived endothelial and inflammatory signatures, we may find TKIs with inherent affinities for SrcA or SrcB PTKs.
[00214] We leveraged the Broad institute's CLUE CMap database noting that methodology around the usage of CMap is evolving. We note several observations. First, CMap query output was dependent on the number of BING genes used in the search, with a unique number of search genes retrieving a maximal number of perturbagens across all cell lines - on the order of 5x105. We found the endothelial and inflammatory gene signatures derived from PAECs could be both induced or suppressed by the same ligand, compound, or gene manipulation depending on the CMap cell line the perturbagen was used in. It is noted that many cell lines in CMap (and LINCS) are cancer lines whose genetic programs may be very different from healthy cell lines. To date, studies using LINCS to predict drugs from, for example, PAH or Ulcerative Colitis gene signatures have not restricted by cell line. Our data suggests cell line choice is important. A biologically tailored search approach to match substrate cell line with the experimental or disease cell line may provide focused results.
[00215] Brivanib, Quizartinib, and Regorafanib are used to validate our search strategy. Brivanib interestingly targets VEGFR1 ,2 and FGFR1 , inhibiting angiogenesis in cancer models. Quizartinib is a selective FLT3 inhibitor that also targets PDGFR, and VEGFR2 to a lesser degree. Regorafanib is a multi-kinase inhibitor targeting VEGFR1 ,2, KIT, BRAF, and TIE1 among others. Observed effects on canonical BMPR2 expression in quiescent large vessel PAECs by Brivanib show a role in the treatment of human pulmonary vascular diseases. Lisitinib and Dabrafenib match the anti-inflammatory signature similar to the TKI Pazopanib already in clinical trial for HHT.
Methods:
[00216] High Throughput siRNA Screerr. High throughput siRNA screening of > 22,000 genes using an Id1 -BRE luciferase reporter assay in a C2C12 mouse myoblastoma cell line treated with or without 250 pM BMP4 was conducted in the Stanford High- Throughput Bioscience Center, as previously described [60]. Briefly, the C2C12 myoblastoma cells, a generous gift from Dr. Peter ten Dijke, were stably transfected with BRE- Id 1 linked to luciferase and used as a reporter cell line. Cells were screened on 384-well siRNA plates. The transfection conditions were optimized with BMP4 as stimulus, siBMPR2 and a toxic (siTox) control. Cells were seeded in wells at a density of 1500 cells/well. Next, siRNA was transfected at an optimized concentration of 25 nM using DharmaFect3. After 48 hours, cells were stimulated with 250 pM of BMP4. Two hours after stimulation, the change in luminescence of an individual well was measured relative to the average luminescence of all wells in the plate. After this, a tryptan-blue cell viability stain was automatically performed and quantified. Luminescence and viability stain intensity was normalized to the overall average luminescence and viability stain intensity of the plate to calculate change in Id1 expression and change in viability. This assumes that there are few “hits” (e.g. few significant deviations in Id1 expression or cell viability from the norm) per 384 well plate and is a standard method for batch effect normalization in multi-plate high throughput assays. Transfections were performed in triplicate for each gene. Change in Id1 linked luminescence relative to baseline and change in cell viability relative to baseline was averaged for three samples across all 22,124 genes used in the high throughput murine wide si-RNA screen.
[00217] Cell Culture. Human large vessel PAEC (Promocell) were grown as monolayers in 0.2% gelatin in PBS coated dishes in a commercial EC (Promocell) media. Cells were passaged at 1 :3 ratios and used for experiments at passages of between 3-6. For all basal media experiments, fetal bovine serum (FBS) was used at a 2% concentration. For starvation, FBS levels were reduced to 0.2%.
[00218] Human PAH Tissue'. Isolation of Cells lung tissue. PAEC of IPAH and FPAH patients at time of lung transplant were obtained from digested whole lung tissue, using CD31 -AB pulldown beads (Dynabeads; Invitrogen), as previously described [60]. Experiments involving human tissue or derived primary cells were approved by the Stanford University Institutional Review Board and the Administrative Panel on Human Subject Research.
[00219] RNA Interference. Both Lek and Fyn expression was modulated by RNAi in PA endothelial cells (PAEC). A pool of 4 siRNAs for BMPR2, FHIT, LCK or a non-targeting control pool (Ambion, Table. 2) were transfected into PAECs using the RNAi Max kit (Invitrogen) for 48 hours. mRNA knockdown efficiency was determined by qPCR.
Table 2:
Target Gene Cat. # Sequence (Sense)
Lek Ambion 4392440 GGAAUUAUAUUCAUCGUGAtt
Fyn Ambion 4390824 GAUUGAUAGAAGACAAUGAtt
Non-Targeted (NT) Ambion 4390843 Not published
[00220] All transfections were carried out in 6 well plate format. Transfection conditions were varied to independently optimize transfection efficiencies for Lek and Fyn, resulting in different concentrations of siRNA and Lipofectamine for each gene. First, cell media was changed to 500 j L of OptiMem Media (Thermo Fisher 31985062) 30 minutes prior to transfection. For si-Lck conditions, 4 jxL of Lipofectamine (RNA iMax, Invitrogen 13778) was added to 246 yL of OptiMem and 4 yL of 20 nM siRNA (either Lek or nontargeting, NT) was added to 246 yL of OptiMem. The Lipofectamine and siRNA containing OptiMem was combined. After letting stand for 20 minutes, this 500 yL was added existing OptiMem in 6 well plates. This resulted in a final concentration siRNA concentration of 80 nM. For si-Fyn, the procedure was the same except 2 yL of Lipofectamine in 248 yL OptiMem and 2.5 L of siRNA (either Fyn or NT) in 247.5 yL OptiMem was used to make a final siRNA oncentration of 50 nM. All analyses for each gene were done by comparing to the same concentration of NT siRNA. For the RNAseq experiments, to control for transfection mediated gene effects, the same conditions used for si FYN above were used for the transfection of Fyn, Lok, and NT siRNA. 50 nM of siRNA and 2 yL Lipofectamine in 1000 yL OptiMem was used for all experimental arms.
[00221] qPCR Assay to Detect mRNA Expression. For mRNA, total RNA was extracted from adherent cell layers using the RNAeasy Plus Kit (Qiagen) and reverse transcribed into cDNA using random primers with the Taqman cDNA reverse transcription Kit (Applied Biosciences) according to the manufacturer’s instructions. Prior to the generation of cDNA, RNA quality and concentration was measured using the Nanodrop system (Thermo-Fisher). The level of mRNA expression was quantified using Taqman primer/probe sets (Table 2) for the target and normalized to a housekeeping control (18s). To validate new PCR probes, standard curves were calculated from serial 1 :2 dilutions of cDNA across 6 different concentrations, and PCR probe efficiency (E) was calculated. From the standard curve, the relative standard method was used to determine relative concentrations of mRNA in lysates.
Table 3:
Figure imgf000059_0001
[00222] Western Blotting: Western blotting was performed as previously described. Antibodies were used as described in Table 4:
Target Manufacturer Cat. # Clone Species Dilution
P-Actin Santa Cruz SC4778 C4 Mouse 1 :600
BMPR2 Invitrogen MA5-15827 3F6F8 Mouse 1 :800
Lek Abeam Ab227975 EPR20798-107 Rabbit 1 :300 pY-394 Lek Abeam Ab201567 MM0795-71W27 Mouse 1 :500
Id1 Santa Cruz sc-133104 B8 Mouse 1 :100
Snail/Slug Abeam Ab180714 Polyclonal Rabbit 1 :500
VCAM1 R&D Systems BB16-V1 BBIG-V1 Mouse 1 :500
Fyn BD Biosciences BD61063 25/Fyn Mouse 1 :500 pSMAD-1/3 Abeam ab52903 EP823Y Rabbit 1 :1000
SMAD1 Cell Signaling CST 9743 Polyclonal Rabbit 1 :500
RelA Cell Signaling CST 8242 D14E12 Rabbit 1 :1000
[00223] Apoptosis and Matrigel Tube Formation Assays: Assays were conducted according to the manufacturer’s instructions and as previously described. Tube formation was assessed with the Angiogenesis Analyzer package in Imaged.
[00224] RNA Sequencing: Human PAECs were grown to 80% confluency on gel coated plates and transfected with either Fyn, Lek, or nontargeted siRNA. At 48 hours, cells were placed in 0.2% FBS starvation media. Two hours prior to harvest all cells were treated with either PBS in 0.2% FBS media or, in one case, 20 ng/mL of BMP9 in 0.2% FBS starvation media. Cells were harvested and mRNA was extracted via Qiagen mini columns. RNA quality was assessed with the Agilent 2100 Bioanalyzer system and found to have a RIN of 9.8-9.9. cDNA libraries were generated (Illumina), barcoded, and sequenced via a paired-end 150 bp sequencing strategy to a depth of between 38 and 46 million paired-end reads on the Illumina platform. Raw reads were mapped to the human genome using STAR. Quality control metrics regarding sequencing and alignment are given in Supplemental Figure 5-A. Differential gene expression analysis was performed using the R package DEseq2. Briefly, differentially expressed genes were calculated via the “DEseq” command after setting transfection condition (FYN, LCK, NT) and treatment (PBS, BMP9) as factors. Conditions were contrasted against the nontargeted PBS condition to determine the fold change and adjusted p-value for each gene in each condition. A variance stabilizing transformation of the raw count data was used to generate the gene expression heat map. Next, differentially expressed genes present in each condition were found using a cutoff l_og2 fold change of 0.3 (fold change of less than 0.8 or greater than 1.25) with a false discovery rate of 5% and the R function “venn.diagram”. Lists of significant differentially expressed genes that were present in select conditions (IE increased in the LCK case and decreased in the FYN case) were used for gene ontology analysis. Gene ontology and transcription factor analysis was facilitated using the Enrichr GSEA web server. For transcription factor analysis, the packages TRRUST, BART, and oPOSSUM-3 were used to predict transcription factors co-regulating the genes in query set. Results were tailed to show which transcription factors were predicted by different methods.
[00225] Analysis of Public PBMC Single Cell RNA Seq Data: Raw count data of 2700 PBMCs (PBMCs from a health human donor, single cell immune profiling dataset by Cell Ranger 1 .1 .0 on Illumina NextSeq 500 with -69,000 reads per cell, 10x Genomics, 2016, May 26) were analyzed using the Seurat V 3.0 sc-RNA seq pipeline to perform unsupervised hierarchical clustering. Average gene expression was calculated on a per-cluster basis. Cell identity was determined based on differentially upregulated genes in a cluster of interest relative to all the other clusters in the dataset (Supplemental Figure 2). After clusters were developed and identified, the average expression of genes of interest (Bmpr2, Id1, Id2, Id3, Lek) was determined. The percentof cells expressing the gene of interest (defined as a cell with > 1 count) was also calculated. Both the magnitude of expression (presented as a Z-score relative to all cells in the sample) and the percent expression are presented in dot plot format.
[00226] Immunofluorescence Staining of Adherent Cells and Human Lung Tissues: For confocal localization of proteins in cells, cells were washed in warm PBS and then fixed in 10% normal buffered formalin for 20 minutes at room temperature. Cells were washed with PBS, then permeabilized and blocked with 0.3% Triton X-100 and 5% Serum for 1 -3 hours at room temperature. Primary antibodies were incubated overnight at 4C at the following dilutions in a 5% serum buffer in PBS: LCK (ABCAM Ab227975, 1 :200), FYN (BD Biosciences BD61063 1 :150), phopho-P65 RelA (Cell Signaling Technologies 93H1 , 1 :200). Appropriate secondary antibodies were used at a 1 :250 dilution in a 5% serum PBS buffer for 1 hour. Cells were washed in PBS + 0.5% Tween-20 x 3. In all cases DAPI counter stains or F-Actin counter stains (fluorescently labeled phalloidin) were used per published protocols. Cells were imaged by confocal microscopy. Quantification of signal was performed in Imaged.
[00227] Single Molecule mRNA Fluorescent in situ Hybridization (sm-FISH): Sm-FISH for Lek was performed using the RNA-scope protocol (Advanced Cell Diagnostics, Inc.) using the Channel 1 probe to Lek (Cat No. 440201 ). After completion of the sm-FISH protocol, tissue was counter stained for endothelial (VE-Cadherin, R&D AF938, 1 :300) and smooth muscle (Acta2, Direct a488 Conjugation, 1 A4 Clone, 1 :200). Tissue was imaged by confocal microscopy and analyzed with Imaged.
[00228] Statistical Analysis: Data were analyzed using R/R-Studio version 1 .4.1 106. Statistical tests were performed as appropriate and included the following: Student’s t-test, One-Way ANOVA and Two-way ANOVA, followed by the appropriate post-hoc test, as indicated. Bars show mean ± Standard Deviation. Differences are considered statistically significant for p values less than 0.05. All significant p values are reported on graphs.
[00229] Drug Prediction by Broad Institute Connectivity Map: The Connectivity Map (CMap) portal was used to perform all searches. First, the genes returned by RNA seq were narrowed to only include CMap “Best INFerred” or BING genes. These are genes used in CMap that are computationally inferred with high confidence from the 978 genes actively profiled by the L1000 platform. Next, the differentially expressed genes were ranked based on the overall magnitude of change. Genes were ranked based on ■■#$». Gene with a positive A #j%& (e.g. increased with si-Lck, decreased with si-Fyn) comprised the “Inflammatory” signature whereas genes with a negative il ntai comprised the “Angiogenic” signature. Genes with the highest |A"#$%S| were filtered against the list of available CMap BING genes. The remaining BING gene-based angiogenic and inflammatory signatures were truncated to lists of lengths 10, 15, 20, 25, 50, 75, 100, 125 and 150 genes long. Each gene list (both angiogenic and inflammatory, n = 18) was then submitted in the CMap “Query” program as an Up- Regulated gene. Query parameters are “Gene expression (L1000)”, “Touchstone”, “Individual Query”, and “Latest.” All searches were performed between 11/2021 and 12/2021 . The search results were opened online in CMap Morpheus, and then downloaded as a GCT version 1 .3 file from the “query_result” tab. A custom R script was used to open the 18 GCT files and combine them into a single data structure that can be subset by gene signature, gene search length, perturbagen class, search query correlation score, cell type, transcriptional activation score (TAS), and false discovery rate. This data set was used to generate Figures 8B-8G.
[00230] Drug Treatment in Cell Lines: Human PAECs were grown to near confluency (90%) and used between passages 4-6. All drugs were diluted in DMSO at concentrations similar to those used in the CMap/L1000 experiments in HUVEC cell lines. Drug dilutions were prepared in DMSO and added to full (2% FBS) media. Final concentrations were: Brivanib (10 |iM), Quizartinib (10 |1M), Regorafanib (37 jiM), and Pazopanib (37 uM). Cells were starved for 6 hours in reduced serum media (0.2% FBS) prior to giving drug or DMSO. Drug/DMSO in full media (2% FBS) was added to cells for a total treatment time of 24 hours. For Brivanib and Quizartinib, either BMP9 at 10 ng/mL or PBS was added 2 hours prior to harvesting the cells. Cells were then harvested, run on SDS-Page, and immunoblotted for Id1 , pSMADI , pSMAD3 and eNOS. For Regorafenib and Pazopanib, cells were grown in gelatin coated slide well chambers then treated with drug for 24 hours. Each slide contained a drug replicate and a DMSO only replicate. After 24 hours, slides were fixed in 10% NBF for 25 minutes, then blocked with 5% goat serum, 1 % BSA, and 0.3% Trition in PBS for 1 hour prior to incubating with p65-RelA primary antibody and Alexa-488 conjugated phalloidin overnight in the same buffer. Finally, cells were treated with DAPI and a Alexa-633 conjugated goat anti-rabbit secondary antibody at a dilution of 1 :250 for 1 hour. Slides were then imaged by confocal microscopy, collecting z-stacks at Nyquist spacing through the entire nuclear diameter. Between 4 and 6 images were collected of 4 experiments each. Images were analyzed with Imaris 3D processing software. DAPI staining was used to define a 3D volume representing the nucleus. To control for variations in staining, the DAPI intensity threshold at which to call a given voxel intranuclear was adjusted on a per-experiment basis. This ensured that the average nuclear diameter for all baseline DMSO experiments were the same. P65 RelA signal intensity was quantified on a per voxel basis for each voxel contained in the DAPI nucleus. This data was used to compute the average p65-RelA expression for the nuclear volume for each intact nucleus imaged. Average p65-RelA intensity and overall nuclear size for Pazopanib and Regorafenib treated cells were compared relative to their DMSO counterparts which were stained on the same slide with the same primary and secondary antibody buffers.
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[00255] The preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims.

Claims

T HAT WHICH IS CLAIMED IS:
1 . A method for treating Hereditary Hemorrhagic Telangiectasia (HHT) in a human subject, the method comprising: administering an effective dose of an agent that inhibits VEGF signaling, to improve clinical indicia of HHT.
2. The method of claim 1 , wherein the agent inhibits VEGFR-2.
3. The method of claim 1 or claim 2, wherein the agent is Brivanib.
4. The method of any of claims 1 -3, wherein expression is reduced of one or more of genes LYVE1 , GPNMB, RRAGD and ANKRD33.
5. The method of any of claims 1 -4, wherein expression is increased of one or more of genes MC5R, SLC25A47, FGF19, SHISA9, FRG2C; and HS3ST2.
6. The method of any of claims 1 -5, wherein the agent inhibits VEGF-induced ERK1/2 phosphorylation, proliferation and angiogenesis in the targeted cells
7. The method of any of claims 1 -6, wherein the agent inhibits the upregulated HHT- related disease gene signatures in the targeted tissues.
8. The method of any of claims 1 -7, where the agent inhibits the inflammatory signature.
9. The method of any of claims 1 -8, wherein the agent is Linsitinib or Dabrafenib.
10. A method for treating pulmonary artery hypertension (PAH) in a human subject, the method comprising: administering an effective dose of an agent that inhibits VEGF signaling, to improve clinical indicia of PAH.
1 1 . The method of claim 9, wherein the agent inhibits VEGFR-2.
12. The method of claim 9 or claim 10, wherein the agent is Brivanib.
13. The method of any of claims 10-12, wherein the agent increases Id1 expression in pulmonary artery endothelial cells.
PCT/US2023/080515 2022-11-21 2023-11-20 Use of a tyrosine kinase inhibitor for the treatment of hereditary hemorrhagic telangiectasia and pulmonary arterial hypertension Ceased WO2024112656A1 (en)

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