Compositions and methods of using tyrosine kinase inhibitors

Abstract

To provide a method of treating a cardiovascular disease or condition having aberrant protein tyrosine phosphorylation.SOLUTION: The method comprises administering a low-dosage of a tyrosine kinase inhibitor to a subject, where the tyrosine kinase inhibitor decreases aberrant levels of tyrosine phosphorylation and improves at least one cardiac function in the subject. Preferably, the tyrosine kinase inhibitor is selected from the group consisting of afatinib, axitinib, bosutinib, cabozantinib, cediranib, ceritinib, crizotinib, dabrafenib, dasatinib, and the like.SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 250,052, filed November 3, 2015, and U.S. Provisional Application No. 62 / 107,553, filed January 26, 2015, the contents of which are incorporated by reference in their entireties.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under GM099801 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0003] 2. Background of the Invention Despite significant progress, cardiovascular disease remains the leading cause of death for both men and women worldwide. According to a report by the World Health Organization (WHO), it is estimated that by 2030, 23.6 million people will die from cardiovascular disease each year.

[0004] The RAS-MAPK pathway has critical implications for human growth and development. Abnormalities in various steps of this signaling cascade result in neuro-cardio-facio-cutaneous syndromes or RASopathies, a group of disorders with overlapping but distinct phenotypes. RASopathic patients exhibit varying degrees of intellectual disability, poor growth, relative macrocephaly, ectodermal abnormalities, dysmorphic features, and an increased risk of certain malignancies. Significant locus heterogeneity is observed in many of the RASopathies.

[0005] Congenital heart disease (CHD) is the most common defect found in newborns, occurring in approximately 1% of live births. Over one million people in the United States have some form of CHD, and the majority require ongoing monitoring and treatment to prevent deterioration of cardiac function. AVCD includes various abnormalities of the atrioventricular valves and the atrial and ventricular septum. In the complete form, there is one common atrioventricular valve and an atrial septal defect (ostium primum) that merges with the posterior ventricular septal defect at the inlet of the ventricular septum. In the incomplete form, there are two separate right and left atrioventricular valves, a clefted mitral valve, an atrial septal defect (ostium primum), and no ventricular septal communication. A clefted mitral valve is considered a less severe form of AVCD. AVCD is also the most common CHD found in children with Down's syndrome and is one of the structural heart defects most frequently associated with extracardiac anomalies in the context of chromosomal and Mendelian genetic disorders. Unique anatomical features have been found in AVCD associated with NS. In fact, the defect is usually of the incomplete type, resulting from abnormal insertion of the mitral valve by accessory fibrous tissue and / or abnormal papillary muscles of the left ventricle, ultimately resulting in subaortic stenosis.

[0006] Congenital heart disease (CHD) occurs in approximately 60-86% of patients affected by RASopathies, a group of disorders that harbor defects in the RAS-MAPK pathway. Pulmonary valve stenosis (PVS) and hypertrophic cardiomyopathy are the most common defects that show a clear association with RASopathies. The spectrum of CHD in Noonan syndrome with multiple lentigines (NSML) is even broader, with the family of atrioventricular canal defects (AVCD) being the third most common cardiac defect.

[0007] Most patients with cardiovascular disease and congenital heart disease associated with RASopathy require treatment for many years. In particular, congenital heart disease associated with RASopathy is usually associated with a low mortality rate. Therefore, it is necessary to treat the cardiovascular disease of patients with a low-risk treatment that has the greatest effect on heart disease. Summary of the Invention

[0008] As described below, the present invention includes compositions and methods for inhibiting aberrant protein tyrosine phosphorylation, such as the phosphorylation of Src family tyrosine kinases and their substrates.

[0009] In one aspect, the present invention includes a method of treating a cardiovascular disease or condition having abnormal protein tyrosine phosphorylation in a subject, comprising administering to a subject in need thereof a low dosage of a tyrosine kinase inhibitor, wherein the tyrosine kinase inhibitor reduces the abnormal level of tyrosine phosphorylation and improves at least one cardiac function of the subject.

[0010] In another aspect, the present invention includes a method of treating a congenital heart disease comprising administering to a subject in need thereof a low dose of a tyrosine kinase inhibitor, wherein the tyrosine kinase inhibitor reduces abnormal levels of tyrosine phosphorylation and improves at least one cardiac function of the subject.

[0011] In yet another aspect, the present invention includes a method of treating a cardiovascular disease or condition associated with a RASopathy having abnormal protein tyrosine phosphorylation, comprising administering to a subject in need thereof a low dosage of a tyrosine kinase inhibitor, wherein the tyrosine kinase inhibitor reduces the abnormal level of tyrosine phosphorylation and improves at least one cardiac function of the subject.

[0012] In yet another aspect, the present invention includes a composition comprising a low-dose of a tyrosine kinase inhibitor, wherein the low-dose of the tyrosine kinase inhibitor can reduce tyrosine phosphorylation and improve at least one cardiac function in a subject in need thereof.

[0013] In another aspect, the invention includes a pharmaceutical composition comprising a composition described herein and a pharma- ceutically acceptable carrier.

[0014] In yet another aspect, the invention includes the use of a composition described herein in the manufacture of a medicament for treating a cardiovascular disease or condition in a subject.

[0015] In various embodiments of the above or any other aspects of the invention detailed herein, the congenital heart disease is associated with a RASopathy, e.g., a RASopathy selected from the group consisting of neurofibromatosis type 1, Noonan syndrome, Noonan syndrome with multiple lentigines (Leopard syndrome), capillary malformation-arteriovenous malformation syndrome, Costello syndrome, cardio-facio-cutaneous syndrome, and Regius syndrome. In one embodiment, the cardiovascular disease or condition is a congenital heart disease.

[0016] In another embodiment, the low dose ranges from about 175 to about 250 times lower than the chemotherapeutic dose of the tyrosine kinase inhibitor.

[0017] In another embodiment, the cardiac function is selected from the group consisting of myofibrillar organization, cardiomyocyte contractility, SERCA2A expression, and cardiac fibrosis.

[0018] In another embodiment, the tyrosine kinase inhibitor is selected from the group consisting of afatinib, axitinib, bosutinib, cabozantinib, cediranib, ceritinib, crizotinib, dabrafenib, dasatinib, erlotinib, everolimus, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, lestaurtinib, nilotinib, nintedanib, palbociclib, pazopanib, ponatinib, regorafenib, ruxolitinib, semananib, sirolimus, sorafenib, sunitinib, temsirolimus, tofacitinib, trametinib, vandetanib, and vemurafenib. In yet another embodiment, the tyrosine kinase inhibitor is a Src family tyrosine kinase inhibitor, e.g., A419259, AP23451, AP23464, AP23485, AP23588, AZD0424, AZM475271, BMS354825, CGP77675, CU201, ENMD2076, KB SRC 4, KX2361, KX2-391, MLR 1023, MNS, PCI-32765, PD166285, PD180970, PKC-412, PKI166, PP1, PP2, SRN 004, SU6656, TC-S7003, TG100435, TG100948, TX-1123, VAL 201, WH-4-023, XL 228, an Src family tyrosine kinase inhibitor selected from the group consisting of altenusin, bosutinib, damnacanthal, dasatinib, herbimycin A, indirubin, neratinib, lavendustin A, pelitinib, piceatannol, saracatinib, SrcI1, and analogs thereof.

[0019] In another embodiment, the subject is a pediatric patient, such as a pediatric subject under the age of 12. In yet another embodiment, the subject is over 18 years of age.

[0020] In another embodiment, the abnormal level of tyrosine phosphorylation comprises an abnormal level of tyrosine phosphorylation Protein Zero-Related (PZR). In such an embodiment, the low-dose tyrosine kinase inhibitor reduces PZR tyrosine phosphorylation. In yet another embodiment, the low-dose tyrosine kinase inhibitor provides an anti-fibrotic effect in cardiac tissue to the subject. In yet another embodiment, the low-dose tyrosine kinase inhibitor reduces the abnormal tyrosine phosphorylation of transmembrane glycoproteins, such as the transmembrane glycoprotein Protein Zero-Related (PZR). In another embodiment, the low-dose tyrosine kinase inhibitor provides an anti-fibrotic effect in cardiac tissue. [The present invention 1001] A method for treating a cardiovascular disease or condition having abnormal protein tyrosine phosphorylation in a subject, comprising administering to a subject in need thereof a low dose of a tyrosine kinase inhibitor, wherein the tyrosine kinase inhibitor reduces the abnormal level of tyrosine phosphorylation and improves at least one cardiac function of the subject. [The present invention 1002] A method for treating a congenital heart disease, comprising administering to a subject in need thereof a low dose of a tyrosine kinase inhibitor, wherein the tyrosine kinase inhibitor reduces abnormal levels of tyrosine phosphorylation and improves at least one cardiac function of the subject. [The present invention 1003] A method for treating a cardiovascular disease or condition associated with a RASopathy having abnormal protein tyrosine phosphorylation, comprising administering to a subject in need thereof a low dose of a tyrosine kinase inhibitor, wherein the tyrosine kinase inhibitor reduces the abnormal level of tyrosine phosphorylation and improves at least one cardiac function of the subject. [The present invention 1004] The method of the present invention 1002, wherein the congenital heart disease is associated with a RASopathy. [The present invention 1005] The method of claim 1003, wherein the cardiovascular disease or condition is congenital heart disease. [The present invention 1006] The method of any of claims 1003 or 1004, wherein the RASopathy is selected from the group consisting of neurofibromatosis type 1, Noonan syndrome, Noonan syndrome with multiple lentigines (Leopard syndrome), capillary malformation-arteriovenous malformation syndrome, Costello syndrome, cardio-facio-cutaneous syndrome, and Regius syndrome. [The present invention 1007] The method according to any one of claims 1001 to 1003, wherein the low dose is in the range of about 1 / 175 to about 1 / 250 of the chemotherapy dose of the tyrosine kinase inhibitor. [The present invention 1008] The method according to any one of claims 1001 to 1003, wherein the cardiac function is selected from the group consisting of myofibrillar organization, cardiomyocyte contractility, SERCA2A expression, and cardiac fibrosis. [The present invention 1009] The method of any one of 1001 to 1003, wherein the tyrosine kinase inhibitor is selected from the group consisting of afatinib, axitinib, bosutinib, cabozantinib, cediranib, ceritinib, crizotinib, dabrafenib, dasatinib, erlotinib, everolimus, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, lestaurtinib, nilotinib, nintedanib, palbociclib, pazopanib, ponatinib, regorafenib, ruxolitinib, semananib, sirolimus, sorafenib, sunitinib, temsirolimus, tofacitinib, trametinib, vandetanib, and vemurafenib. [The present invention 1010] The method according to any one of claims 1001 to 1003, wherein the tyrosine kinase inhibitor is a Src family tyrosine kinase inhibitor. [The present invention 1011] Src family tyrosine kinase inhibitors include A419259, AP23451, AP23464, AP23485, AP23588, AZD0424, AZM475271, BMS354825, CGP77675, CU201, ENMD2076, KB SRC 4, KX2361, KX2-391, MLR 1023, MNS, PCI-32765, PD166285, PD180970, PKC-412, PKI166, PP1, PP2, SRN 004, SU6656, TC-S7003, TG100435, TG100948, TX-1123, VAL 201, WH-4-023, XL 228, altenusin, bosutinib, damnacanthal, dasatinib, herbimycin A, indirubin, neratinib, lavendustin A, pelitinib, piceatannol, saracatinib, SrcI1, and analogs thereof. [The present invention 1012] The method according to any one of claims 1001 to 1003, wherein the subject is a pediatric patient. [The present invention 1013] The method of claim 1012, wherein the pediatric subject is under 12 years of age. [The present invention 1014] Any of the methods of 1001 to 1003, wherein the subject is older than 18 years of age. [The present invention 1015] The method of any of claims 1001 to 1003, wherein the abnormal level of tyrosine phosphorylation comprises an abnormal level of tyrosine phosphorylation protein zero-related (PZR). [The present invention 1016] The method of the present invention, wherein a low dose of a tyrosine kinase inhibitor reduces PZR tyrosine phosphorylation. [The present invention 1017] The method according to any one of claims 1001 to 1003, wherein a low dose of a tyrosine kinase inhibitor provides the subject with an anti-fibrotic effect in cardiac tissue. [The present invention 1018] A composition comprising a low dose of a tyrosine kinase inhibitor capable of decreasing tyrosine phosphorylation and improving at least one cardiac function in a subject in need thereof. [The present invention 1019] The composition of the present invention, wherein a low dose of a tyrosine kinase inhibitor reduces aberrant tyrosine phosphorylation of a transmembrane glycoprotein. [The present invention 1020] The composition of the present invention 1019, wherein the transmembrane glycoprotein is protein zero related (PZR). [The present invention 1021] The composition of the present invention, wherein the low dose of the tyrosine kinase inhibitor is in the range of about 1 / 175 to about 1 / 250 of the chemotherapeutic dose of said tyrosine kinase inhibitor. [The present invention 1022] The composition of claim 1018, wherein the cardiac function is selected from the group consisting of myofibrillar organization, cardiomyocyte contractility, SERCA2A expression, and cardiac fibrosis. [The present invention 1023] The composition of the present invention 1018, wherein the low-dose tyrosine kinase inhibitor is selected from the group consisting of afatinib, axitinib, bosutinib, cabozantinib, cediranib, ceritinib, crizotinib, dabrafenib, dasatinib, erlotinib, everolimus, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, lestaurtinib, nilotinib, nintedanib, palbociclib, pazopanib, ponatinib, regorafenib, ruxolitinib, semananib, sirolimus, sorafenib, sunitinib, temsirolimus, tofacitinib, trametinib, vandetanib, and vemurafenib. [The present invention 1024] The composition of the present invention, wherein the tyrosine kinase inhibitor is a Src family tyrosine kinase inhibitor. [The present invention 1025] Src family tyrosine kinase inhibitors include A419259, AP23451, AP23464, AP23485, AP23588, AZD0424, AZM475271, BMS354825, CGP77675, CU201, ENMD2076, KB SRC 4, KX2361, KX2-391, MLR 1023, MNS, PCI-32765, PD166285, PD180970, PKC-412, PKI166, PP1, PP2, SRN 004, SU6656, TC-S7003, TG100435, TG100948, TX-1123, VAL 201, WH-4-023, XL 228, the composition of the present invention 1024 selected from the group consisting of altenusin, bosutinib, damnacanthal, dasatinib, herbimycin A, indirubin, neratinib, lavendustin A, pelitinib, piceatannol, saracatinib, SrcI1, and analogs thereof. [The present invention 1026] The composition of the present invention, wherein low doses of a tyrosine kinase inhibitor produce an anti-fibrotic effect in cardiac tissue. [The present invention 1027] A pharmaceutical composition comprising the composition of the present invention and a pharma- ceutically acceptable carrier. [The present invention 1028] Use of a composition of the invention 1018 in the manufacture of a medicament for treating a cardiovascular disease or condition in a subject. [Brief description of the drawings]

[0021] The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the accompanying drawings, in which: For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred, but it should be understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0022] [Figure 1A]Figure 1. Proteomic analysis of differentially tyrosyl phosphorylated proteins in the hearts of Ptpn11D61G / + mice. Classification of hypo- and hyper-tyrosyl phosphorylated proteins in the hearts of Ptpn11D61G / + mice. [Figure 1B] 1 is a graph showing the log2-transformed ratios of each peptide containing phosphotyrosine in wild-type and Ptpn11D61G / + mouse hearts. [Figure 1C] Heatmap of differentially hypertyrosyl phosphorylated peptides (phosphorylation sites identified by MS and listed in brackets). [Figure 1D] Panel of extracted ion chromatogram images and peptide sequences of PZR containing tyrosine 242 (upper panel) and PZR containing tyrosine 264 (lower panel) by differential proteomics. [Figure 1E] C-terminal amino acid sequences of protein zero-related (PZR) from various vertebrates are shown. The consensus sequence for immunoreceptor tyrosine-based inhibitory motifs (ITIMs; S / I / V / LXYXXI / V / L) is shown in bold, with tyrosine residues in red and the appropriate amino acid numbering. C-terminal sequences of PZR from human (Homo sapiens), mouse (Mus musculus), rat (Rattus norvegicus), cow (Bos taurus), dog (Canis lupus familiaris), zebrafish (Danio rerio), and chicken (Gallus gallus) are shown. [Figure 2A]Panel of blots showing characterization of PZR tyrosyl phosphorylation. C2C12 cells were cotransfected with empty vector or activated glutathione S-transferase (GST)-Shp2E76A and either empty vector (Vector), wild-type human PZR (WT), or PZR mutated at tyrosine 241 (Y241F), PZR mutated at tyrosine 263 (Y263F), or PZR mutated at both tyrosines (2YF). Cell lysates were immunoblotted with anti-pPZR (Y241 or Y263), anti-PZR, or anti-Shp2 antibodies. [Figure 2B] Panel of blots showing characterization of PZR tyrosyl phosphorylation. HEK-293 cells were co-transfected with empty vector (Vector) or activated Shp2E76A and either empty vector (Vector), wild-type zebrafish PZR (WT), or PZR mutated at tyrosine 236 (Y241F), PZR mutated at tyrosine 258 (Y263F), or PZR mutated at both (2YF). Cell lysates were immunoblotted with anti-pPZR (Y241 or Y263), anti-PZR, or anti-Shp2 antibodies. ERK1 / 2 were used as loading controls. [Figure 2C] (A) is a panel of blots showing characterization of PZR tyrosyl phosphorylation. HUVECs were infected with adenovirus expressing either GFP as a control, wild-type Shp2, or Shp2E76A. Cell lysates were immunoblotted with anti-pPZR (Y241 or Y263), anti-total PZR, and anti-Shp2 antibodies. [Figure 2D]Panel of blots showing characterization of PZR tyrosyl phosphorylation. HEK-293 cells were transiently transfected with empty vector, wild-type Shp2 (WT), or the indicated Shp2 mutants (activated Shp2, E76A; Noonan syndrome (NS) mutant, N308D; or Noonan syndrome with multiple lentigines (NSML) mutants, Y279C and T468M). Cell lysates were immunoblotted with anti-pPZR (Y241 or Y263), anti-PZR, and anti-Shp2 antibodies. ERK1 / 2 were used as loading controls. [Figure 2E] Panel of blots showing characterization of PZR tyrosyl phosphorylation. HEK-293T cells were transfected with HA-tagged zebrafish PZR along with empty vector, wild-type Shp2, Shp2D61G (NS mutant), or Shp2A462T (NSML mutant). Cell lysates were immunoprecipitated with anti-HA antibody, and immune complexes were immunoblotted with anti-Shp2 and anti-HA antibodies. Whole cell lysates (WCL) were blotted with anti-pPZR (Y241 and Y263), anti-Shp2, and anti-HA antibodies. [Figure 3A] Figure 1 shows PZR tyrosyl phosphorylation in hearts of Ptpn11D61G / + mice. Hearts were isolated from 5-week-old WT and Ptpn11D61G / + mice. Tissue lysates were immunoblotted with pPZR(Y263) and total PZR antibodies. Phosphorylation of tyrosine 264 in PZR, n=5 per genotype. All data represent mean±standard error of the mean (SEM). **, P<0.01. [Figure 3B] Figure 1 shows PZR tyrosyl phosphorylation in the cortex of Ptpn11D61G / + mice. Cortex was isolated from 5-week-old WT and Ptpn11D61G / + mice. Tissue lysates were immunoblotted with pPZR(Y263) and total PZR antibodies. Phosphorylation of tyrosine 264 in PZR, n=5 per genotype. All data represent mean±standard error of the mean (SEM). ***, P<0.001. [Figure 3C]PZR tyrosyl phosphorylation in Ptpn11Y279C / + hearts. Hearts were isolated from 8-week-old WT and Ptpn11Y279C / + mice. Tissue lysates were immunoblotted with pPZR(Y263) and total PZR antibodies. Phosphorylation of tyrosine 264 in PZR, n=5 per genotype. All data are means ± standard error of the mean (SEM). *, P<0.05. [Figure 3D] Figure 1 shows PZR tyrosyl phosphorylation in the cortex of Ptpn11 Y279C / + mice. Cortex was isolated from 8-week-old WT and Ptpn11 Y279C / + mice. Tissue lysates were immunoblotted with pPZR(Y263) and total PZR antibodies. Phosphorylation of tyrosine 264 in PZR, n=5 per genotype. All data are means ± standard error of the mean (SEM). ***, P<0.001. [Figure 4A] Figure 1 shows PZR tyrosyl phosphorylation in livers of Ptpn11D61G / + mice. Livers were isolated from 5-week-old wild-type and Ptpn11D61G / + mice. Tissue lysates were immunoblotted with anti-pPZR(Y263) and anti-total PZR antibodies. Densitometric analysis of phosphorylation levels of tyrosine 263 in PZR was performed. Results represent the mean ± SEM from five mice per genotype. [Figure 4B] Images and graphs showing PZR tyrosyl phosphorylation in kidneys of Ptpn11D61G / + mice. Kidneys were isolated from 5-week-old wild-type and Ptpn11D61G / + mice. Tissue lysates were immunoblotted with anti-pPZR(Y263) and anti-total PZR antibodies. Densitometric analysis of tyrosine 263 phosphorylation levels in PZR was performed. Results represent the mean±SEM from five mice per genotype. **, P<0.01 (WT vs. NS). [Figure 4C]Images and graphs showing PZR tyrosyl phosphorylation in spleens of Ptpn11D61G / + mice. Spleens were isolated from 5-week-old wild-type and Ptpn11D61G / + mice. Tissue lysates were immunoblotted with anti-pPZR(Y263) and anti-total PZR antibodies. Densitometric analysis of phosphorylation levels of tyrosine 263 in PZR was performed. Results represent the mean±SEM from five mice per genotype. ***, P<0.001 (WT vs. NS). [Figure 5A] Panel of immunoblots showing ERK and Akt phosphorylation in hearts of Ptpn11D61G / + mice. Hearts were isolated from 5-week-old wild-type and Ptpn11D61G / + mice (A and B). Tissue lysates were subjected to immunoblotting with anti-Shp2, anti-pERK1 / 2, anti-total ERK1 / 2, anti-pAkt, and anti-Akt antibodies. Results represent the mean ± SEM of densitometric analysis for pERK1 / 2 and pAkt from 5 mice per genotype. [Figure 5B] FIG. 13 is a panel of images showing ERK and Akt phosphorylation in the cortex of Ptpn11D61G / + mice. Cortex was isolated from 5-week-old wild-type and Ptpn11D61G / + mice. Tissue lysates were subjected to immunoblotting with anti-Shp2, anti-pERK1 / 2, anti-total ERK1 / 2, anti-pAkt, and anti-Akt antibodies. Results represent the mean±SEM of densitometric analysis for pERK1 / 2 and pAkt from 5 mice per genotype. [Figure 5C] FIG. 13 is a panel of images showing ERK and Akt phosphorylation in the hearts of Ptpn11Y279C / + mice. Hearts were isolated from 8-week-old wild-type and Ptpn11Y279C / + mice. Tissue lysates were subjected to immunoblotting with anti-Shp2, anti-pERK1 / 2, anti-total ERK1 / 2, anti-pAkt, and anti-Akt antibodies. Results represent the mean±SEM of densitometric analysis for pERK1 / 2 and pAkt from five mice per genotype. [Figure 5D] FIG. 13 is a panel of images showing ERK and Akt phosphorylation in the cortex of Ptpn11Y279C / + mice. Cortex was isolated from 8-week-old wild-type and Ptpn11Y279C / + mice. Tissue lysates were subjected to immunoblotting with anti-Shp2, anti-pERK1 / 2, anti-total ERK1 / 2, anti-pAkt, and anti-Akt antibodies. Results represent the mean±SEM of densitometric analysis for pERK1 / 2 and pAkt from five mice per genotype. [Figure 6A] Figure 1 shows a panel of blots showing the effect of Src family kinases on phosphorylation of PZR at Y241 and Y263. HEK-293 cells were transiently transfected with the indicated Shp2 mutants and treated with either dimethyl sulfoxide (DMSO) as a control or 5 μM SU6656. Cell lysates were immunoblotted with anti-Shp2, anti-pSrc (Y416), anti-Src, anti-pPZR (Y241 or Y263), and anti-total PZR antibodies. ERK1 / 2 were used as loading controls. [Figure 6B] Figure 1 shows a panel of blots showing the effect of Src family kinases on phosphorylation of PZR at Y241 and Y263. NIH 3T3 cells were infected with adenovirus expressing either GFP as a control or constitutively active Shp2E76A in the presence of DMSO, PP2, or SU6656 at the concentrations indicated. Cell lysates were immunoblotted with anti-Shp2, anti-pSrc(Y416), anti-Src, anti-pPZR(Y241 or Y263), and anti-total PZR antibodies. ERK1 / 2 were used as loading controls. [Figure 7A]Blots showing that Src kinase mediated NS-Shp2- or NSML-Shp2-induced PZR hypertyrosyl phosphorylation. SYF cells (Src- / -Fyn- / -Yes- / - MEFs) and Src+ / + cells (SYF cells expressing wild-type Src) were infected with adenovirus expressing either GFP or Shp2E76A. Cell lysates were immunoblotted with anti-Shp2, anti-pSrc (Y416), anti-pPZR (Y241 or Y263), anti-PZR, and anti-Src antibodies. ERK1 / 2 was used as a loading control. [Figure 7B] Blots showing that Src kinase mediated NS-Shp2- or NSML-Shp2-induced PZR hypertyrosyl phosphorylation. SYF cells were transiently transfected with wild-type c-Src or kinase-dead c-SrcK295R / Y527F (KR / YF) and infected with adenovirus expressing either GFP, wild-type Shp2, or Shp2E76A. Cell lysates were immunoblotted with anti-Shp2, anti-pSrc(Y416), anti-pPZR(Y241 or Y263), anti-PZR, and anti-Src antibodies. ERK1 / 2 was used as a loading control. [Figure 7C] Blots showing that Src kinase induced PZR hypertyrosyl phosphorylation. HEK-293 cells were transfected with Flag-tagged human PZR. Cell lysates were immunoprecipitated with anti-Flag antibody. These immunoprecipitates were subjected to an in vitro Src kinase assay with Src recombinant protein. The reaction products were immunoblotted with anti-pPZR (Y241 or Y263) antibody. [Figure 7D]Blots showing Src family kinases induced PZR hypertyrosyl phosphorylation. HEK-293 cells were co-transfected with constitutively active Src mutants and either HA-tagged wild-type zebrafish PZR (WT), PZR mutated at tyrosine 236 (Y241F), PZR mutated at tyrosine 258 (Y263F), or PZR mutated at both sites (2YF). Cell lysates were immunoblotted with anti-pPZR (Y241 or Y263) and anti-HA antibodies. [Figure 8] FIG. 8A is a blot and graph showing increased Src complex formation with Shp2 mutants associated with NS / NSML. HEK-293 cells were transiently transfected with either Shp2 WT or the indicated Shp2 mutants. Cell lysates were immunoprecipitated (IP) with anti-c-Src antibodies, and immune complexes were immunoblotted (IB) with anti-Shp2 and anti-Src antibodies. Graphs represent mean ± SEM of densitometric analysis from three independent experiments. Statistical significance was derived using Dunnett's test comparing Shp2 mutants to WT. *, P<0.05; **, P<0.01. FIG. 8B is an image of a blot showing increased Src complex formation with Shp2 mutants and PZR associated with NS / NSML. HEK-293 cells were co-transfected with Shp2 WT or E76A or Y279C mutants and either empty vector (vector), WT human PZR, or PZR 2YF mutant. Cell lysates were immunoblotted with anti-pPZR (Y241 or Y263) or anti-Shp2 antibodies. ERK1 / 2 was used as a loading control. Immune complexes were immunoblotted with anti-Src, anti-Shp2, and anti-PZR antibodies. [Figure 9] Illustrative model of the effect of NS and NSML mutants on PZR tyrosyl phosphorylation. [Figure 10]Panel of images showing administration of dasatinib. Male Ptpn11D61G / + mice were injected intraperitoneally (ip) with the indicated doses of dasatinib or DMSO control, and 24 hours later mice were sacrificed and cardiac tissue was harvested and immunoblotted with the indicated antibodies. [Figure 11] FIG. 11A shows the dasatinib prenatal dosing schedule in the NS mouse model. Pregnant mothers were given daily dasatinib from when the animals were in utero at E7.5 until postnatal day 9 (P9). Starting at postnatal day 10 (P10), mice were given dasatinib directly by daily injection for 46 days (P56). Cardiac function of mice was evaluated at P42 and P56. FIG. 11B shows the dasatinib postnatal dosing schedule in the NS mouse model. Dasatinib was given daily to mice starting at P10 for 32 days (P42) and discontinued for 14 days (P56). Cardiac function of mice was evaluated at P42 and P56. [Figure 12A] Figure 1 is a panel of graphs showing that prenatal treatment of NS mice with dasatinib improves cardiac function. Pregnant mice were injected intraperitoneally with dasatinib (0.1 mg / kg) according to the protocol described herein. The number of mice in each group is indicated in the figure. Statistical significance is indicated by *; P<0.05, **; P<0.01, ***; P<0.001 based on two-way ANOVA test. LV vol;s - Left ventricular volume in systole, LV vol;d - Left ventricular volume in diastole, FS - Fractional shortening, and EF - Ejection fraction. [Figure 12B]FIG. 1 is a panel of graphs showing that postnatal NS mice treated with dasatinib improves cardiac function. NS mice were intraperitoneally injected with dasatinib (0.1 mg / kg) starting at P10 according to the protocol described herein. After 32 days of treatment, mice were evaluated for cardiac function. The number of mice in each group is shown in the figure. Statistical significance is indicated by *; P<0.05, **; P<0.01, ***; P<0.001 based on two-way ANOVA test. LV vol;s - Left ventricular volume in systole, LV vol;d - Left ventricular volume in diastole, FS - Fractional shortening, and EF - Ejection fraction. [Figure 12C] Figure 1B is a panel of graphs showing that cardiac function improvement was maintained after cessation of dasatinib treatment. NS mice were intraperitoneally injected with dasatinib (0.1 mg / kg) starting at P10 according to the protocol shown in Figure 1B. Cardiac function of mice given dasatinib for 32 days was reassessed after 2 weeks. The number of mice in each group is shown in the figure. Statistical significance is indicated by *; P<0.05, **; P<0.01, ***; P<0.001 based on two-way ANOVA test. LV vol;s - Left ventricular volume in systole, LV vol;d - Left ventricular volume in diastole, FS - Fractional shortening, and EF - Ejection fraction. [Figure 13-1]Panel of images showing that c-Src kinase is a putative target for Noonan syndrome. Figure 13a is a schematic diagram of wild-type full-length, N+C-SH2, and PTP domain constructs of human Shp2. Figure 13b is an image showing detection of Myc-Src full-length co-transfected with Flag-Shp2 full-length, N+C, or PTP domain in HEK-293T cells. Protein interactions were revealed by immunoprecipitation. Figure 13c is an image showing detection of GST-SH3 domain of purified c-Src incubated overnight at 4°C with His-tagged PTP domain of purified Shp2. Proteins were immobilized with GST sepharose beads and separated by SDS-PAGE. His-PTP domain in GST complex was detected by immunoblotting with anti-His antibody. WCL: whole cell lysate, IP: immunoprecipitation, IB: immunoblotting. [Figure 13-2]A panel of images showing that c-Src kinase is a putative target for Noonan syndrome. Fig. 13d is an image showing the phosphorylation level of Src (Y416). Mouse embryonic fibroblasts (MEFs) derived from Ptpn11D61G / + mice incubated for 18 hours with dasatinib at the concentrations shown in the figure. Total cell lysates were immunoblotted with anti-p-Src (Y416), anti-Src, anti-p-ERK1 / 2, and anti-ERK1 / 2 antibodies. Tyrosyl phosphorylation of PZR was measured using a phospho-specific PZR antibody, and the molecular interaction of PZR with Shp2 was revealed by immunoprecipitation. Fig. 13e is a graph showing the phosphorylation level of Src (Y416). Fig. 13f is a graph showing the phosphorylation level of ERK1 / 2. Fig. 13g is a graph showing the amount of PZR phosphorylation at tyrosine 241. Figure 13h is a graph showing the amount of tyrosine 263 analyzed by densitometry. All data show mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001 indicate significance compared to vehicle-treated Ptpn11D61G / + MEFs (n=3 for each condition; one-way ANOVA test). [Figure 13-3]Panel of images showing c-Src kinase as a putative target for Noonan syndrome. Figure 13i is an image showing cardiac tissue immunoblotted with anti-Shp2, anti-p-ERK1 / 2, anti-ERK1 / 2, anti-p-Src (Y416), anti-Src, anti-p-PZR (Y263), and anti-PZR antibodies. Three-week-old WT and Ptpn11D61G / + mice were intraperitoneally injected with vehicle or dasatinib (0.1, 0.5, or 1.0 mg / kg). 18 hours later, cardiac tissue was isolated and tissue lysates were immunoblotted. Figure 13j is a graph showing tyrosyl phosphorylation of ERK1 / 2. Figure 13k is a graph showing tyrosyl phosphorylation of Src. Figure 13l is a graph showing tyrosyl phosphorylation of PZR. All data show mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001 indicate significance compared to vehicle-treated Ptpn11D61G / + cardiac tissue (n=3 for each condition; one-way ANOVA test). [Figure 14-1] Figure 14 is a panel of images showing that dasatinib improves cardiac function in Ptpn11D61G / + mice. Figure 14a is a schematic diagram of prenatal administration of dasatinib to Ptpn11D61G / + mice. Figure 14b is a panel of images showing representative echocardiographic images of WT and Ptpn11D61G / + mice treated with vehicle or dasatinib at P42. Figure 14c is a graph showing the ejection fraction (%) (EF) measured from echocardiograms at P42. Figure 14d is a panel of images showing representative echocardiographic images of WT and Ptpn11D61G / + mice treated with vehicle or dasatinib at P56. Figure 14e is a graph showing the ejection fraction (%) (EF) measured from echocardiograms at P56. All data are shown as mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001. (n=10-16 for each group; two-way ANOVA test). [Figure 14-2]Figure 14f is a schematic diagram of prenatal administration of dasatinib to Ptpn11D61G / + mice. Figure 14g is a panel of images showing representative echocardiographic images of WT and Ptpn11D61G / + mice treated with vehicle or dasatinib at P42. Figure 14h is a graph showing the ejection fraction (%) (EF) measured from echocardiograms at P42. Figure 14i is a panel of images showing representative echocardiographic images of WT and Ptpn11D61G / + mice treated with vehicle or dasatinib at P56. Figure 14j is a graph showing the ejection fraction (%) (EF) measured from echocardiograms at P56. All data are shown as mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001. (n=10-16 for each group; two-way ANOVA test). [Figure 14-3] A panel of images showing that dasatinib improves cardiac function in Ptpn11D61G / + mice. Figure 14k is a graph showing arterial systolic pressure at P56 from invasive hemodynamic studies of WT and Ptpn11D61G / + mice treated with vehicle or dasatinib after birth. Figure 14l is a graph showing diastolic pressure at P56 in WT and Ptpn11D61G / + mice treated with vehicle or dasatinib after birth. Figure 14m is a graph showing mean arterial pressure (MAP) at P56 in WT and Ptpn11D61G / + mice treated with vehicle or dasatinib after birth. Figure 14n is a graph showing left ventricular blood pressure (LV pressure) at P56 in WT and Ptpn11D61G / + mice treated with vehicle or dasatinib after birth. All data are means ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001. (n=10-16 for each group; two-way ANOVA test). [Figure 15-1]Panel of images showing cardiomyopathy and cardiac fibrosis in Ptpn11D61G / + mice rescued by dasatinib. Figure 15a is a graph showing heart weight at P56 measured in WT and Ptpn11D61G / + mice treated postnatally with vehicle or dasatinib (n=8-9 for each group). Figure 15b is a graph showing the ratio of heart weight (HW) to body weight (BW) at P56 measured in WT and Ptpn11D61G / + mice treated postnatally with vehicle or dasatinib (n=8-9 for each group). Figure 15c is a panel of images showing representative images of longitudinal sections stained with Masson's trichrome of hearts at P56 from WT and Ptpn11D61G / + mice treated postnatally with vehicle or dasatinib (scale bar=2 mm). All data are means ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001. (Two-way ANOVA test). [Figure 15-2] Figure 15d is a panel of images showing cardiomyopathy and cardiac fibrosis in Ptpn11D61G / + mice rescued by dasatinib. Figure 15d is a panel of images showing Masson's trichrome staining of left ventricles at P56 from WT and Ptpn11D61G / + mice treated postnatally with vehicle or dasatinib (scale bar = 200 μm). Figure 15e is a graph showing the relative expression of the fibrosis marker gene Col1a2. Total cardiac RNA was isolated from WT and Ptpn11D61G / + mice (P56) treated postnatally with vehicle or dasatinib. Figure 15f is a graph showing the relative expression of the fibrosis marker gene Col3a1. All data show mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001. (Two-way ANOVA test). [Figure 15-3]Panel of images showing cardiomyopathy and cardiac fibrosis in Ptpn11D61G / + mice rescued by dasatinib. Figure 15g is a graph showing the relative expression of cardiac fetal gene Myh6 (αMHC). Figure 15h is a graph showing the relative expression of cardiac fetal gene Myh7 (βMHC). Figure 15i is a graph showing the relative expression of cardiac fetal gene Anf. Figure 15j is a graph showing the relative expression of cardiac fetal gene Bnp. These genes were measured by quantitative RT-PCR (n=6 for each group). All data are mean±SEM. *, p<0.05; **, p<0.01; ***, p<0.001. (Two-way ANOVA test). [Figure 16-1] Panel of images showing that cardiomyocytes from Ptpn11D61G / + mice treated with dasatinib showed normal Ca2+ signaling during excitation-contraction coupling. Figure 16a is an image showing Ca2+ excitation-contraction coupling measured in cardiomyocytes isolated from WT and Ptpn11D61G / + mice treated postnatally with vehicle or dasatinib. Representative traces of cardiomyocyte kinetics for calcium transient traces (top) and corresponding sarcomere length (leng) shortening traces (bottom). Figure 16b is a graph showing a summary of the relative calcium release (Rmag Ca2+) data. Figure 16c is a graph showing the percentage of sarcomere shortening (n=111-162 cells from 3 mice per group). All data show mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001 (2-way ANOVA test). [Figure 16-2]FIG. 16d is a panel of images showing that cardiomyocytes from Ptpn11D61G / + mice treated with dasatinib exhibited normal Ca2+ signaling during excitation-contraction coupling. FIG. 16d is an image showing cardiac tissue immunoblotted with anti-SERCA2A, anti-troponin I (tTnI), anti-troponin T (tTnT), and anti-tubulin antibodies. Cardiac tissue was isolated from WT and Ptpn11D61G / + mice treated with vehicle or dasatinib after birth, and tissue lysates were immunoblotted. FIG. 16e is a graph showing SERCA2A expression. FIG. 16f is a graph showing troponin I expression. FIG. 16g is a graph showing troponin T expression. Expression was statistically evaluated after normalization with tubulin (n=6 for each group). All data show mean±SEM. *, p<0.05; **, p<0.01; ***, p<0.001 (two-way ANOVA test). [Figure 17-1] FIG. 17A is a panel of images showing the effect of dasatinib on NS signaling in vitro. Mouse embryonic fibroblasts (MEFs) derived from Ptpn11D61G / + mice were incubated with dasatinib for 18 hours. Total cell lysates were immunoblotted with anti-Shp2, anti-p-Src (Y416), anti-Src, anti-p-Raf1 (Y341), anti-Raf1, anti-p-MEK1 / 2, anti-MEK1 / 2, anti-p-ERK1 / 2, anti-ERK1 / 2, anti-p-JNK, anti-JNK, anti-p-p38, anti-p38, anti-p-Akt, and anti-Akt antibodies (FIG. 17A). The phosphorylation levels of Raf1 (Y341) (FIG. 17B) and MEK1 / 2 (FIG. 17C) were statistically evaluated. All data show the mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001 indicate significance compared to vehicle-treated Ptpn11D61G / + MEFs (n=3 for each condition; one-way ANOVA test). [Figure 17-2]Panel of images showing the effect of dasatinib on NS signaling in vitro. Mouse embryonic fibroblasts (MEFs) derived from Ptpn11D61G / + mice were incubated with dasatinib for 18 hours. Phosphorylation levels of JNK (FIG. 17d) and Akt (S473) (FIG. 17e) were statistically evaluated. All data show mean±SEM. *, p<0.05; **, p<0.01; ***, p<0.001 indicate significance compared to vehicle-treated Ptpn11D61G / + MEFs (n=3 for each condition; one-way ANOVA test). Mouse embryonic fibroblasts (MEFs) derived from Ptpn11D61G / + mice were incubated with STI-571 (FIG. 17f) or Shp2 inhibitor (FIG. 17g) for 18 hours. Tyrosyl phosphorylation of PZR was measured using a phospho-specific PZR antibody. [Figure 18-1] Figure 18a is a panel of images showing the effect of dasatinib on NS signaling in vivo. Three-week-old WT and Ptpn11D61G / + mice were intraperitoneally injected with vehicle or dasatinib (0.1, 0.5, or 1.0 mg / kg). Figure 18a is an image showing cardiac tissue immunoblotted with p-Raf1 (Y341), Raf1, p-MEK1 / 2, MEK1 / 2, p-JNK, JNK, p-p38, p38, p-Akt (S473), and Akt antibodies. After 18 hours, cardiac tissue was isolated and tissue lysates were immunoblotted. [Figure 18-2]Figure 18 is a panel of images showing the effect of dasatinib on NS signaling in vivo. Three-week-old WT and Ptpn11D61G / + mice were intraperitoneally injected with vehicle or dasatinib (0.1, 0.5, or 1.0 mg / kg). Figure 18b is a graph showing phosphorylation levels of Raf1 (Y341). Figure 18c is a graph showing phosphorylation levels of MEK1 / 2. Figure 18d is a graph showing phosphorylation levels of JNK. Figure 18e is a graph showing phosphorylation levels of p38. Figure 18f is a graph showing phosphorylation levels of Akt (S473). All data show mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001 indicate significance compared to vehicle-treated Ptpn11D61G / + mice (n=3 for each condition; one-way ANOVA test). [Figure 19] Panel of images showing that postnatal dasatinib administration did not improve the whole body growth of Ptpn11D61G / + mice. Figure 19a is a graph showing the growth curves of WT and Ptpn11D61G / + mice treated with vehicle or dasatinib postnatally. The difference between the treatment groups was significant from 3 to 8 weeks of age (p<0.001, two-way ANOVA test). Figure 19b is a graph showing the body weight measured at P42. Figure 19c is a graph showing the body length measured at P42. Figure 19d is a graph showing the body weight measured at P56. Figure 19c is a graph showing the body length measured at P42. Figure 19e is a graph showing the body length measured at P56. All data are shown as mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001. (n = 10–16 for each group; two-way ANOVA test). [Figure 20-1] Figure 20a is a panel of representative images of the skull and mandible at P56 in WT and Ptpn11D61G / + mice treated with vehicle or dasatinib after birth. [Figure 20-2]Panel of images showing that facial dysmorphic features were not changed in Ptpn11D61G / + mice treated with dasatinib. Figure 20b is a graph showing measurements of skull length at P42 in WT and Ptpn11D61G / + mice treated with vehicle or dasatinib. Figure 20c is a graph showing measurements of skull width at P42 in WT and Ptpn11D61G / + mice treated with vehicle or dasatinib. Figure 20d is a graph showing measurements of skull length to skull width ratio at P42 in WT and Ptpn11D61G / + mice treated with vehicle or dasatinib. Figure 20e is a graph showing measurements taken from intercanthal distance (ICD) at P42 for WT and Ptpn11D61G / + mice treated with vehicle or dasatinib. Figure 20f is a graph showing measurements taken from mandible length at P42 for WT and Ptpn11D61G / + mice treated with vehicle or dasatinib. Figure 20g is a graph showing measurements taken from skull length at P56 for WT and Ptpn11D61G / + mice treated with vehicle or dasatinib. Figure 20h is a graph showing measurements taken from skull width at P56 for WT and Ptpn11D61G / + mice treated with vehicle or dasatinib. Figure 20i shows measurements of skull length to skull width ratios in WT and Ptpn11D61G / + mice treated with vehicle or dasatinib at P56. Figure 20j shows measurements of intercanthal distance (ICD) in WT and Ptpn11D61G / + mice treated with vehicle or dasatinib at P56. Figure 20k shows measurements of mandibular length in WT and Ptpn11D61G / + mice treated with vehicle or dasatinib at P56. All data are mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001. (n=10-16 for each group; two-way ANOVA test). [Figure 21-1]Figure 21a is a panel of representative histological images of H&E stained spleens at P56 from 8-week-old WT and Ptpn11D61G / + mice treated with vehicle or dasatinib after birth (scale bar=200 μm). [Figure 21-2] A panel of images showing that postnatal dasatinib treatment did not eliminate the splenomegaly phenotype of Ptpn11D61G / + mice. Figure 21b is a graph showing spleen weights measured in WT and Ptpn11D61G / + mice treated with vehicle or dasatinib at P42. Figure 21c is a graph showing the ratio of spleen weights to body weights measured in WT and Ptpn11D61G / + mice treated with vehicle or dasatinib at P42. Figure 21d is a graph showing spleen weights measured in WT and Ptpn11D61G / + mice treated with vehicle or dasatinib at P56. Figure 21e is a graph showing the ratio of spleen weights to body weights measured in WT and Ptpn11D61G / + mice treated with vehicle or dasatinib at P56. All data represent the mean ± SEM. *, p<0.05; ***, p<0.001. (n=7-10 for each group; two-way ANOVA test). [Figure 22]Figure 22a is a panel of images showing that dasatinib does not induce liver injury. Figure 22a is a panel of representative histological images of H&E stained livers from 8-week-old WT and Ptpn11D61G / + mice treated with vehicle or dasatinib after birth at P56 (scale bar=200 μm). Figure 22b is a graph showing serum alanine aminotransferase (ALT) enzyme activity measured in WT and Ptpn11D61G / + mice treated with vehicle or dasatinib at P42. Figure 22c is a graph showing serum alanine aminotransferase (ALT) enzyme activity measured in WT and Ptpn11D61G / + mice treated with vehicle or dasatinib at P56. All data show mean±SEM. *, p<0.05; ***, p<0.001. (n = 7–10 for each group; two-way ANOVA test). [Figure 23] Image showing the molecular interaction between the SH3 domain of Src and the PTP domain of Shp2. The GST-tagged SH3 domain of purified c-Src was incubated with the His-tagged PTP domain of purified Shp2 overnight at 4° C. Proteins were immobilized with GST-Sepharose beads and separated by SDS-PAGE. The His-PTP domain in the GST complex was detected by immunoblotting with an anti-His antibody. [Figure 24] 24a-b are panels of images showing that PZR hyperphosphorylation induced by NS-Shp2 is independent of Shp2 phosphatase activity or c-Abl kinase activity. Mouse embryonic fibroblasts (MEFs) derived from Ptpn11D61G / + mice were incubated with STI-571 (FIG. 24a) or Shp2 inhibitors (FIG. 24b) for 18 hours. Tyrosyl phosphorylation of PZR was measured using a phospho-specific PZR antibody. [Diagram 25]FIG. 25 is a panel of graphs showing that molecular markers of cardiomyopathy and fibrosis improved in NSML (Y279C / +) mice after dasatinib treatment. Total cardiac RNA was isolated from WT and Ptpn11 Y279C / + mice (P42) treated postnatally with vehicle or dasatinib (0.1 mg / kg / day). Fibrosis marker genes Col1a2 (FIG. 25a) and Col3a1 (FIG. 25b), cardiomyopathy marker ANP (FIG. 25c), and cardiac fetal genes Myh6 (aMHC) (FIG. 25d), Myh7 (bMHC) (FIG. 25e), and Myh6 / Myh7 ratio (FIG. 25f) were measured by quantitative RT-PCR (n=6 for each group). All data represent mean±SEM. *, p<0.05; **, p<0.01; ***, p<0.001. (Two-way ANOVA test). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Detailed Description of the Invention definition Unless otherwise defined, 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 pertains. Although any methods and materials similar or equivalent to those described herein may be used in the practice to test the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology is used.

[0024] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting.

[0025] As used herein, the articles "a" and "an" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0026] As used herein, when referring to measurable values ​​such as amounts and durations of time, the term "about" is intended to encompass variations within ±20% or 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the specified value, where such variations are appropriate for carrying out the disclosed methods. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term about.

[0027] The phrase "abnormal protein tyrosine phosphorylation" refers to hyperphosphorylation or hypophosphorylation of one or more target proteins and / or abnormal protein kinase activity. In one embodiment, the abnormal protein tyrosine phosphorylation is compared to a control.

[0028] The term "cardiovascular disease or condition" refers to any disease or condition that affects the cardiovascular system, including, but not limited to, nerve conduction disorder, thrombosis tendency, atherosclerosis, angina pectoris, hypertension, arteriosclerosis, myocardial infarction, congestive heart failure, cardiomyopathy, hypertension, arterial and venous stenosis, valvular disease, myocarditis, and arrhythmia.Cardiovascular disease conditions also include, but are not limited to, any clinical symptoms of disease states related to the heart and central or peripheral arterial and venous vascular systems.For example, the clinical symptoms include, but are not limited to, pain, weakness, hypertension, elevated plasma cholesterol, elevated plasma fatty acid, tachycardia, bradycardia, abnormal electrocardiogram, external or internal bleeding, headache, dizziness, nausea, and vomiting.

[0029] The term "cardiac function" refers to the activity of the heart or the interaction of cardiac cells or tissues to perform an activity. Examples of cardiac function include, but are not limited to, myofibrillar organization, myocardial cell contractility, and proper delivery of blood and nutrients to tissues that need it. Abnormal cardiac function (improper delivery of blood and nutrients to tissues) can lead to problems such as, but are not limited to, blood pressure changes, thrombosis, electrocardiogram changes, arrhythmia, myocarditis, pericarditis, myocardial infarction, cardiomyopathy, hypertrophy, growth retardation, heart failure (ventricular failure (left or right)), congestive heart failure, and cardiac arrest. Improvement of cardiac function can include the improvement, elimination, or prevention of at least one abnormal cardiac function, such as, but are not limited to, myofibrillar disorganization, abnormal myocardial cell contractility, cardiac fibrosis, abnormal blood pressure, excessive blood pressure changes, thrombosis, electrocardiogram changes, arrhythmia, myocarditis, pericarditis, myocardial infarction, cardiomyopathy, and congestive heart failure.

[0030] In this disclosure, "comprises," "comprising," "containing," "having," and the like can have the meaning given them in United States Patent Law and can mean "includes," "including," and the like. Similarly, "consisting essentially of" or "consists essentially" has the meaning given to it in United States Patent Law and is open-ended, permitting the presence of more than what is recited, but excluding prior art aspects, so long as the basic or novel characteristics of that are not altered by the presence of more than what is recited.

[0031] "Congenital heart disease" refers to a cardiovascular category that includes abnormalities in cardiovascular structures that occur before birth.

[0032] By "effective amount" is meant the amount required to reduce or ameliorate at least one symptom of a disease compared to an untreated patient. The effective amount of an active compound used in the therapeutic treatment of a disease varies depending on the mode of administration, the age, weight, and general health of the subject.

[0033] As used herein, the term "expression" is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0034] "Fragment" refers to a portion of a polynucleotide or nucleic acid molecule. Preferably, the portion comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total length of the reference nucleic acid. A fragment may comprise 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, or 2500 nucleotides (and any integer value in between). When applied to nucleic acid molecules, a fragment refers to a subsequence of a larger nucleic acid. A "fragment" of a nucleic acid molecule can be at least about 15 nucleotides in length; e.g., at least about 50 nucleotides to about 100 nucleotides; at least about 100 to about 500 nucleotides, at least about 500 to about 1000 nucleotides, at least about 1000 nucleotides to about 1500 nucleotides; or about 1500 nucleotides to about 2500 nucleotides; or about 2500 nucleotides (and any integer value in between).

[0035] "Low dose" refers to a therapeutically effective dose that is less than the dose typically prescribed for indications other than cardiac disease, congenital heart disease, heart failure, or similar conditions. In one embodiment, the low dose is less than a chemotherapy dose. In another embodiment, the low dose is less than, in the range of about 200-fold, a chemotherapy dose of a tyrosine kinase inhibitor. In another embodiment, the low dose of a tyrosine kinase inhibitor improves at least one cardiac function. Dasatinib has been shown to be effective in preventing tumor development in mice at a dose of about 20 mg / kg (Kantarjian, H. et al. Dasatinib versus imatinib in newly diagnosed chronic-phase chronic myeloid leukemia. N Engl J Med 362, 2260-2270 (2010)). The therapeutic effect of dasatinib in humans has been reported to be about 2 mg / kg, a dose equivalent to about 24 mg / kg in mice (Yu, EY et al. Phase II study of dasatinib in patients with metastatic castration-resistant prostate cancer. Clinical cancer research: an official journal of the American Association for Cancer Research 15, 7421-7428 (2009) and Apperley, JF et al. Dasatinib in the treatment of chronic myeloid leukemia in accelerated phase after imatinib failure: the START a trial. J Clin Oncol 27, 3472-3479, doi:10.1200 / JCO.2007.14.3339 (2009)). Doses as low as 0.1 mg / kg of dasatinib (about 200 times lower than the therapeutic dose) were sufficient to treat cardiac disease associated with CHD.

[0036] The terms "isolated," "purified," or "biologically pure" refer to a substance that is free to varying degrees from components that normally accompany the substance when present in the natural state. "Isolate" refers to a degree of separation from the original source or environment. "Purify" refers to a degree of separation greater than isolation. A "purified" or "biologically pure" protein has been sufficiently free of other substances such that any impurities do not specifically affect the biological properties of the protein or cause other deleterious consequences. That is, a nucleic acid or peptide is purified when it is substantially free of cellular material, viral material, and culture medium when produced by recombinant DNA techniques, as well as chemical precursors and other chemicals when chemically synthesized. Purity and homogeneity are typically measured using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" may indicate that the nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. In the case of proteins that can be subject to modifications, such as phosphorylation or glycosylation, different modifications can result in different isolated proteins that can be purified separately.

[0037] "Pharmaceutically acceptable" refers to those properties and / or substances that are acceptable to the patient from a pharmacological / toxicological standpoint and acceptable to the pharmaceutical pharmacist from a physical / chemical standpoint with regard to composition, formulation, stability, patient acceptability, and bioavailability. "Pharmaceutically acceptable carrier" means a medium that does not interfere with the effectiveness of the biological activity of the active ingredient and that is not toxic to the host to which it is administered.

[0038] As used herein, the term "pharmaceutical composition" or "pharmaceutical acceptable composition" refers to a mixture of at least one compound or molecule useful within the scope of the present invention and a pharmaceutical acceptable carrier. Pharmaceutical compositions facilitate the administration of compounds or molecules to patients. There are numerous techniques in the art for administering compounds or molecules, including but not limited to intravenous administration, oral administration, aerosol administration, parenteral administration, ocular administration, pulmonary administration, and topical administration.

[0039] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable substance, composition, or carrier, such as a liquid or solid bulking agent, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickener, solvent, or encapsulating material, that is involved in carrying or transporting a compound or molecule useful within the scope of the present invention into or to a patient so that it can perform its intended function. Typically, such constructs are carried or transported from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense that it is compatible with the other components of the formulation, including the compound useful within the scope of the present invention, and is not harmful to the patient. Some examples of substances which may serve as pharma- ceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyhydric alcohols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; surfactants; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffers; and other non-toxic, compatible substances used in pharmaceutical preparations. As used herein, "pharmaceutically acceptable carrier" also includes any and all coating agents, antibacterial and antifungal agents, absorption delaying agents, and the like, which are compatible with the activity of the compounds useful within the scope of the present invention and are physiologically acceptable to the patient. Supplementary active compounds may also be mixed into the composition. "Pharmaceutically acceptable carrier" may further include pharmaceutically acceptable salts of compounds or molecules useful within the scope of the present invention.Other additional ingredients that may be included in pharmaceutical compositions used in the practice of the present invention are known in the art and described, for example, in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, Pa.), which is incorporated herein by reference.

[0040] "Protein Zero Related" or "PZR", also known as "Myelin Protein Zero-Like Protein 1" or "MPZL1", refers to a protein that is an immunoglobulin superfamily cell surface protein. PZR contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that are responsible for binding to Shp2. When phosphorylated, PZR can specifically bind to Shp2 and thus activate the tyrosine phosphatase activity of Shp2. Once activated, the tyrosine phosphatase activity of Shp2 serves to dephosphorylate downstream substrates that transduce cellular signals. Shp2 can also transduce signals by acting as a scaffold or adaptor protein that recruits other molecules / activities to specific complexes. Shp2 can control signal transduction in both catalytically dependent and catalytically independent manners.

[0041] One isoform of PZR, called PZR1b, lacks ITIM and has a dominant negative effect on full-length PZR and its Shp2 recruitment.Exemplary PZR sequences include the human PZR or fragments thereof found in GenBank accession numbers NM_001146191 and NP_001139663, and the mouse PZR or fragments thereof found in NM_001001880 or NP_001001880.Much of the information known about PZR is related to its role in adhesion-mediated cell signaling and cell migration.However, it remains unclear whether PZR is involved in pathophysiological cell signaling, and the relevance of PZR as a target for any human disease is still not understood.

[0042] "RASopathy" refers to a group of genetic syndromes caused by germline mutations in genes encoding components or regulators of the Ras / mitogen-activated protein kinase (MAPK) pathway. These syndromes include neurofibromatosis type 1, Noonan syndrome, Noonan syndrome with multiple lentigines, capillary malformation-arteriovenous malformation syndrome, Costello syndrome, cardio-facio-cutaneous syndrome, and Regius syndrome. The Ras / MAPK pathway plays a vital role in regulating the cell cycle as well as cell proliferation, differentiation, and senescence, all of which are essential for normal development. Due to a common underlying Ras / MAPK pathway dysregulation, RASopathies display a number of overlapping phenotypic features. In some cases, these overlapping phenotypes may exist or be caused by mechanisms that operate independently of MAPK itself. The PZR / Shp2 complex described herein is located upstream of Ras.

[0043] Noonan syndrome (NS) is an autosomal dominant disorder occurring with an incidence of approximately 1:1,000-2,500 live births in the United States. The most common cardiac defects in NS are pulmonary valve stenosis, atrial septal defect, and hypertrophic cardiomyopathy, each ranging in severity from mild to life-threatening. Noonan syndrome with multiple lentigines (NSML) is a rare autosomal dominant disorder with a "Noonan-like" appearance and a phenotype similar to NS, including multiple lentigines, electrical conduction abnormalities, hypertelorism, pulmonary valve stenosis, abnormal genitalia, developmental delay, and deafness. Mutations associated with NS result in increased phosphatase activity. Mutations associated with NSML result in decreased phosphatase activity.

[0044] "Reference" means a standard or control. A "reference" is a defined standard or control used as a standard for comparison.

[0045] As used herein, "sample" or "biological sample" means anything that may contain cells of interest (e.g., cancer or tumor cells) for which screening method or treatment is desired. The sample may be a biological sample, such as a biological fluid or biological tissue. In one embodiment, the biological sample is a tissue sample that contains pulmonary artery endothelial cells. Such samples may contain a variety of cells, proteins, and genetic materials. Examples of biological tissues include organs, tumors, lymph nodes, arteries, and individual cells. Examples of biological fluids include urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebrospinal fluid, tears, mucus, or amniotic fluid, etc.

[0046] "Src family tyrosine kinases" or "SFKs" are a family of enzymes that catalyze the addition of phosphate groups to tyrosine residues of protein substrates. c-Src represents one member of the SFK family.

[0047] "Src family tyrosine kinase inhibitor" refers to a molecule that reduces or prevents phosphorylation of tyrosine residues on Src family protein substrates. Src family tyrosine kinase inhibitors can interfere with tyrosyl phosphorylation, possibly bind to tyrosine kinases or tyrosine residues with higher binding efficiency than tyrosine kinases or phosphate groups, and / or prevent effective binding of phosphate groups to tyrosine residues, thereby reducing or preventing phosphorylation. Src family tyrosine kinase inhibitors include, but are not limited to, small molecule Src family tyrosine kinase inhibitors, Src family tyrosine kinase antagonists, neutralizing antibodies, and inhibitory peptides and / or oligonucleotides. Examples of small molecule Src family tyrosine kinase inhibitors include A419259, AP23451, AP23464, AP23485, AP23588, AZD0424, AZM475271, BMS354825, CGP77675, CU201, ENMD2076, KB SRC 4, KX2361, KX2-391, MLR 1023, MNS, PCI-32765, PD166285, PD180970, PKC-412, PKI166, PP1, PP2, SRN 004, SU6656, TC-S7003, TG100435, TG100948, TX-1123, VAL 201, WH-4-023, XL 228, altenusin, bosutinib, damnacanthal, dasatinib, herbimycin A, indirubin, neratinib, lavendustin A, pelitinib, piceatannol, saracatinib, SrcI1, and analogs thereof.

[0048] "Src homology 2 (SH2) domain-containing (SH2) protein tyrosine phosphatase-2" or "Shp2" is a member of the tyrosine-specific family of protein tyrosine phosphatases (PTPs). Shp2 is a tyrosine phosphatase that catalyzes the tyrosine dephosphorylation of proteins. Mutations in the human gene PTPN11 have been found to be responsible for approximately half of Noonan syndrome cases and approximately one-tenth of NSML cases.

[0049] A "subject" or "patient" as used herein may be a human or non-human mammal. Non-human mammals include, for example, livestock and pets such as ovine, bovine, porcine, canine, feline, and murine mammals. Preferably, the subject is a human.

[0050] The term "transmembrane glycoprotein" refers to a membrane protein that spans the cell membrane. In one embodiment, the transmembrane glycoprotein comprises an immunoglobulin superfamily cell surface protein such as PZR.

[0051] As used herein, terms such as "treat," "treating," and "treatment" refer to the alleviation or amelioration of a disorder and / or its associated symptoms. It will be recognized that treating a disorder or condition does not require, but does not preclude, that the disorder, condition, or its associated symptoms be completely improved or eliminated.

[0052] "Tyrosine kinase inhibitor" refers to a molecule that reduces or prevents the phosphorylation of tyrosine residues on protein substrates. Tyrosine kinase inhibitors can interfere with tyrosyl phosphorylation, possibly bind to tyrosine kinases or tyrosine residues with higher binding efficiency than tyrosine kinases or phosphate groups, and / or prevent the effective binding of phosphate groups to tyrosine residues, thereby reducing or preventing phosphorylation. Tyrosine kinase inhibitors include, but are not limited to, small molecule tyrosine kinase inhibitors, tyrosine kinase antagonists, neutralizing antibodies, and inhibitory peptides and / or oligonucleotides. Examples of small molecule tyrosine kinase inhibitors include, but are not limited to, afatinib, axitinib, bosutinib, cabozantinib, cediranib, ceritinib, crizotinib, dabrafenib, dasatinib, erlotinib, everolimus, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, lestaurtinib, nilotinib, nintedanib, palbociclib, pazopanib, ponatinib, regorafenib, ruxolitinib, semananib, sirolimus, sorafenib, sunitinib, temsirolimus, tofacitinib, trametinib, vandetanib, and vemurafenib.

[0053] It is understood that the ranges provided herein are shorthand for all values ​​within the range. For example, the range of 1 to 50 is understood to include any number, combination of numbers, or subranges from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0054] The description of an embodiment with respect to a variable or aspect herein includes that embodiment in any single embodiment or in combination with any other embodiment or portion thereof.

[0055] Any composition or method provided herein may be combined with any one or more of the other compositions and methods provided herein.

[0056] composition Abnormal protein tyrosine phosphorylation, such as phosphorylation of Src family tyrosine kinases and their substrates, has been found to be altered in subjects suffering from cardiovascular disease. Abnormal protein tyrosine phosphorylation has also been found to be altered in subjects suffering from certain diseases, such as RASopathy. Inhibiting tyrosine kinase activity treats cardiac disease and improves at least one cardiac function. Inhibition also improves cardiovascular function in subjects with congenital heart defects associated with RASopathy. The present invention includes compositions that inhibit tyrosine kinases, such as Src family tyrosine kinases, to improve at least one cardiac function, thereby preventing or reducing tyrosine phosphorylation. In one aspect, the present invention includes compositions that include a low dose of a tyrosine kinase inhibitor that reduces tyrosine phosphorylation and improves at least one cardiac function in subjects in need thereof.

[0057] In one embodiment, a low-dose tyrosine kinase inhibitor reduces aberrant tyrosine phosphorylation of transmembrane glycoproteins, such as Src family tyrosine kinases and protein zero-related (PZR).

[0058] In one embodiment, the amount of the low-dose tyrosine kinase inhibitor is in the range of about 1 / 25 to about 1 / 500 of the chemotherapy dose of the tyrosine kinase inhibitor. In another embodiment, the amount of the low-dose tyrosine kinase inhibitor is in the range of about 1 / 25 to about 1 / 400 of the chemotherapy dose of the tyrosine kinase inhibitor. In another embodiment, the amount of the low-dose tyrosine kinase inhibitor is in the range of about 1 / 25 to about 1 / 300 of the chemotherapy dose of the tyrosine kinase inhibitor. In another embodiment, the amount of the low-dose tyrosine kinase inhibitor is in the range of about 1 / 35 to about 1 / 300 of the chemotherapy dose of the tyrosine kinase inhibitor. In another embodiment, the amount of the low-dose tyrosine kinase inhibitor is in the range of about 1 / 50 to about 1 / 300 of the chemotherapy dose of the tyrosine kinase inhibitor. In another embodiment, the amount of the low-dose tyrosine kinase inhibitor is in the range of about 1 / 100 to about 1 / 300 of the chemotherapy dose of the tyrosine kinase inhibitor. In another embodiment, the amount of the low-dose tyrosine kinase inhibitor is in the range of about 1 / 150 to about 1 / 250 of the chemotherapy dose of the tyrosine kinase inhibitor. In another embodiment, the amount of the low-dose tyrosine kinase inhibitor is in the range of about 1 / 175 to about 1 / 250 of the chemotherapy dose of the tyrosine kinase inhibitor.Low-dose tyrosine kinase inhibitors are approximately 25th, 30th, 35th, 40th, 45th, 50th, 55th, 60th, 65th, 70th, 75th, 80th, 85th, 90th, 95th, 100th, 105th, 110th, 115th, 120th, 125th, 130th, 135th, 140th, 145th, 150th, 155th, or 160th the chemotherapy dose of tyrosine kinase inhibitors. 1, 1 / 165, 1 / 170, 1 / 175, 1 / 180, 1 / 185, 1 / 190, 1 / 195, 1 / 200, 1 / 205, 1 / 210, 1 / 215, 1 / 220, 1 / 225, 1 / 230, 1 / 235, 1 / 240, 1 / 245, 1 / 250, 1 / 255, 1 / 260, 1 / 265, 1 / 270, 1 / 275, 1 / 280, 1 / 285, 1 / 290, 1 / 295, 1 / 300, and any fold change therebetween. In some embodiments, the chemotherapy dosage of the tyrosine kinase inhibitor ranges from about 75 to about 170 mg / day or about 1.1 to about 2.4 for a 70 kg adult.

[0059] In another embodiment, the low-dose tyrosine kinase inhibitor is selected from the group consisting of afatinib, axitinib, bosutinib, cabozantinib, cediranib, ceritinib, crizotinib, dabrafenib, dasatinib, erlotinib, everolimus, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, lestaurtinib, nilotinib, nintedanib, palbociclib, pazopanib, ponatinib, regorafenib, ruxolitinib, semananib, sirolimus, sorafenib, sunitinib, temsirolimus, tofacitinib, trametinib, vandetanib, and vemurafenib. In another embodiment, the composition comprises more than one of the tyrosine kinase inhibitors disclosed herein.

[0060] In yet another embodiment, the low dose tyrosine kinase inhibitor is a Src family tyrosine kinase inhibitor, such as, but not limited to, A419259, AP23451, AP23464, AP23485, AP23588, AZD0424, AZM475271, BMS354825, CGP77675, CU201, ENMD2076, KB SRC 4, KX2361, KX2-391, MLR 1023, MNS, PCI-32765, PD166285, PD180970, PKC-412, PKI166, PP1, PP2, SRN 004, SU6656, TC-S7003, TG100435, TG100948, TX-1123, VAL 201, WH-4-023, XL 228, altenusin, bosutinib, damnacanthal, dasatinib, herbimycin A, indirubin, neratinib, lavendustin A, pelitinib, piceatannol, saracatinib, SrcI1, and analogs thereof. In another embodiment, the composition comprises at least one Src family tyrosine kinase inhibitor.

[0061] In another embodiment, low dose tyrosine kinase inhibitor improves at least one cardiac function.Cardiac function can include, but is not limited to, myofibrillar organization, myocardial cell contractility, SERCA2A expression, and cardiac fibrosis.Abnormal cardiac function can lead to problems such as, but is not limited to, blood pressure change, thrombosis, electrocardiogram change, arrhythmia, myocarditis, pericarditis, myocardial infarction, cardiomyopathy, heart failure (ventricular failure), congestive heart failure, and cardiac arrest.Improvement of cardiac function can include, but is not limited to, improving, eliminating, or preventing at least one abnormal cardiac function such as, but is not limited to, myofibrillar organization disorganization, abnormal myocardial cell contractility, abnormally regulated SERCA2A expression, cardiac fibrosis, abnormal blood pressure, excessive blood pressure change, thrombosis, electrocardiogram change, arrhythmia, myocarditis, pericarditis, myocardial infarction, cardiomyopathy, and congestive heart failure.

[0062] In yet another embodiment, low doses of tyrosine kinase inhibitors produce anti-fibrotic effects. Elevated levels of fibrotic components in myocardium are associated with the progression of heart failure. Low doses of tyrosine kinase inhibitors reduce the accumulation of fibrotic components in myocardium.

[0063] Compositions that reduce abnormal protein tyrosine phosphorylation are also included in the present invention. Certain diseases, such as cardiovascular diseases or conditions, such as congenital heart disease, are characterized by abnormal protein tyrosine phosphorylation. Therefore, treatments that prevent or reduce the tyrosine phosphorylation of one or more transmembrane glycoproteins, such as protein zero-related or PZR, are included in the present invention. In another aspect, the present invention includes compositions that can reduce abnormal protein tyrosine phosphorylation associated with cardiovascular diseases or conditions. In yet another aspect, the present invention includes compositions that can reduce abnormal protein tyrosine phosphorylation associated with congenital heart disease. In yet another aspect, the present invention includes compositions that can reduce abnormal protein tyrosine phosphorylation associated with cardiovascular diseases or conditions associated with RASopathy.

[0064] method The present invention also includes a method for preventing or treating cardiovascular disease or pathology in a subject in need thereof.As described herein, suppressing abnormal tyrosine phosphorylation prevents and / or treats cardiovascular disease or pathology.A composition comprising a low dose of a tyrosine kinase inhibitor is administered to a subject in need thereof, such as a pediatric subject, to reduce abnormal levels of tyrosine phosphorylation to prevent or treat cardiovascular disease or pathology.

[0065] In one aspect, the present invention includes a method of treating a cardiovascular disease or condition having abnormal protein tyrosine phosphorylation in a subject, comprising administering to a subject in need thereof a low dosage of a tyrosine kinase inhibitor, wherein the tyrosine kinase inhibitor reduces the abnormal level of tyrosine phosphorylation and improves at least one cardiac function of the subject.

[0066] In another aspect, the present invention includes a method of treating a congenital heart disease comprising administering to a subject in need thereof a low dose of a tyrosine kinase inhibitor, wherein the tyrosine kinase inhibitor reduces abnormal levels of tyrosine phosphorylation and improves at least one cardiac function of the subject.

[0067] In yet another aspect, the present invention includes a method of treating a cardiovascular disease or condition associated with a RASopathy having abnormal protein tyrosine phosphorylation, comprising administering to a subject in need thereof a low dose of a tyrosine kinase inhibitor, wherein the tyrosine kinase inhibitor reduces the abnormal level of protein zero-related (PZR) tyrosyl phosphorylation and improves at least one cardiac function of the subject.

[0068] In one embodiment, the cardiovascular disease or condition in the methods described herein is a congenital heart disease or a cardiovascular disease or condition associated with a RASopathy, for example, but not limited to, a RASopathy selected from the group consisting of neurofibromatosis type 1, Noonan syndrome, Noonan syndrome with multiple lentigines (Leopard syndrome), capillary malformation-arteriovenous malformation syndrome, Costello syndrome, cardio-facio-cutaneous syndrome, and Regius syndrome.

[0069] In another embodiment, the method comprises administering a tyrosine kinase inhibitor to a subject that is a pediatric patient. The pediatric subject can be under 18 years old. The pediatric subject can be under 12 years old. The pediatric subject can be under 10, 9, 8, 7, 6, 5, 4, 3, 2, and 1 years old. In another embodiment, the subject is a pediatric patient under 12 years old. In an alternative embodiment, the method comprises administering a tyrosine kinase inhibitor to a subject that is over 18 years old.

[0070] In one embodiment, the method comprises administering a low dose of a tyrosine kinase inhibitor that reduces aberrant tyrosine phosphorylation of transmembrane glycoproteins, such as Src family tyrosine kinases and protein zero-related (PZR). In one embodiment, the abnormal level of tyrosine phosphorylation comprises an abnormal level of tyrosine phosphorylation protein zero-related (PZR).

[0071] In another embodiment, administering a low dose of tyrosine kinase inhibitor improves cardiac function, including but not limited to myofibrillar organization, cardiomyocyte contractility, SERCA2A expression, and cardiac fibrosis.In another embodiment, improving cardiac function can include but not limited to improving, eliminating, or preventing at least one abnormal cardiac function, including but not limited to myofibrillar disorganization, abnormal cardiomyocyte contractility, non-regulatory SERCA2A expression, cardiac fibrosis, abnormal blood pressure, excessive blood pressure change, thrombosis, electrocardiogram change, arrhythmia, myocarditis, pericarditis, myocardial infarction, cardiomyopathy, and congestive heart failure.In yet another embodiment, administering a low dose of tyrosine kinase inhibitor brings anti-fibrosis effect to the subject.

[0072] In one embodiment, the dose of the low-dose tyrosine kinase inhibitor used in the methods described herein ranges from about 1 / 25 to about 1 / 500 of the chemotherapeutic dose of the tyrosine kinase inhibitor. In another embodiment, the amount of the low-dose tyrosine kinase inhibitor ranges from about 1 / 25 to about 1 / 400 of the chemotherapeutic dose of the tyrosine kinase inhibitor. In another embodiment, the amount of the low-dose tyrosine kinase inhibitor ranges from about 1 / 25 to about 1 / 300 of the chemotherapeutic dose of the tyrosine kinase inhibitor. In another embodiment, the amount of the low-dose tyrosine kinase inhibitor ranges from about 1 / 35 to about 1 / 300 of the chemotherapeutic dose of the tyrosine kinase inhibitor. In another embodiment, the amount of the low-dose tyrosine kinase inhibitor ranges from about 1 / 50 to about 1 / 300 of the chemotherapeutic dose of the tyrosine kinase inhibitor. In another embodiment, the amount of the low-dose tyrosine kinase inhibitor ranges from about 1 / 100 to about 1 / 300 of the chemotherapeutic dose of the tyrosine kinase inhibitor. In another embodiment, the amount of the low-dose tyrosine kinase inhibitor is in the range of about 1 / 150 to about 1 / 250 of the chemotherapy dose of the tyrosine kinase inhibitor. In another embodiment, the amount of the low-dose tyrosine kinase inhibitor is in the range of about 1 / 175 to about 1 / 250 of the chemotherapy dose of the tyrosine kinase inhibitor.Low-dose tyrosine kinase inhibitors are approximately 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, or 160 times lower than the chemotherapy dose of the tyrosine kinase inhibitor. , 1 / 165, 1 / 170, 1 / 175, 1 / 180, 1 / 185, 1 / 190, 1 / 195, 1 / 200, 1 / 205, 1 / 210, 1 / 215, 1 / 220, 1 / 225, 1 / 230, 1 / 235, 1 / 240, 1 / 245, 1 / 250, 1 / 255, 1 / 260, 1 / 265, 1 / 270, 1 / 275, 1 / 280, 1 / 285, 1 / 290, 1 / 295, 1 / 300, and any fold change therebetween. In some embodiments, the chemotherapy dosage of the tyrosine kinase inhibitor ranges from about 75 to about 170 mg / day or about 1.1 to about 2.4 for a 70 kg adult.

[0073] In yet another embodiment, the low-dose tyrosine kinase inhibitor is selected from the group consisting of afatinib, axitinib, bosutinib, cabozantinib, cediranib, ceritinib, crizotinib, dabrafenib, dasatinib, erlotinib, everolimus, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, lestaurtinib, nilotinib, nintedanib, palbociclib, pazopanib, ponatinib, regorafenib, ruxolitinib, semananib, sirolimus, sorafenib, sunitinib, temsirolimus, tofacitinib, trametinib, vandetanib, and vemurafenib. In another embodiment, the tyrosine kinase inhibitor is a Src family tyrosine kinase inhibitor, such as, but not limited to, A419259, AP23451, AP23464, AP23485, AP23588, AZD0424, AZM475271, BMS354825, CGP77675, CU201, ENMD2076, KB SRC 4, KX2361, KX2-391, MLR 1023, MNS, PCI-32765, PD166285, PD180970, PKC-412, PKI166, PP1, PP2, SRN 004, SU6656, TC-S7003, TG100435, TG100948, TX-1123, VAL 201, WH-4-023, XL 228, altenusin, bosutinib, damnacanthal, dasatinib, herbimycin A, indirubin, neratinib, lavendustin A, pelitinib, piceatannol, saracatinib, SrcI1, and their analogs.In another embodiment, the composition comprises more than one of the tyrosine kinase inhibitors disclosed herein.In yet another embodiment, the composition comprises at least one Src family tyrosine kinase inhibitor.In such an embodiment, the tyrosine kinase inhibitors can be administered together or sequentially, by different administration routes, or in the same pharmaceutical composition or in different pharmaceutical compositions.

[0074] The methods and compositions disclosed herein are also useful as treatments for cardiovascular diseases or conditions in a subject having a cardiovascular disease or condition characterized by aberrant protein tyrosine phosphorylation.

[0075] Pharmaceutical Compositions The present invention also encompasses the use of the pharmaceutical composition of the present invention to practice the method of the present invention. In one aspect, the present invention includes a pharmaceutical composition comprising the composition described herein and a pharma- ceutically acceptable carrier. In another aspect, the composition described herein is used in the manufacture of a medicament for treating a cardiovascular disease or condition in a subject in need thereof. In yet another aspect, the present invention includes a pharmaceutical composition comprising the composition described herein in combination with another therapeutic agent used in the treatment of a cardiovascular disease or condition. Such a pharmaceutical composition may be provided in a form suitable for administration to a subject, and may include one or more pharma- ceutically acceptable carriers, one or more additional components, or some combination thereof. The compositions described herein may include the compounds contemplated in the present invention in combination with physiologically acceptable salts, e.g., physiologically acceptable cations or anions, as is well known in the art.

[0076] Pharmaceutical compositions useful in the methods of the present invention may be suitably developed for inhalation, oral, rectal, vaginal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ocular, intrathecal, intravenous, or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal formulations, resealed red blood cells containing the active ingredient, and immunologically-based formulations. The route of administration will be readily apparent to those skilled in the art and will vary depending on any number of factors, including the type and severity of the disease being treated, and the type and age of the animal or human patient being treated.

[0077] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. Typically, such preparatory methods include mixing the active ingredient with a carrier or one or more other accessory ingredients, and then, if necessary or desired, shaping or packaging the product into the desired single- or multi-dosage unit.

[0078] In one embodiment, the composition of the present invention is formulated with one or more pharma- ceutically acceptable excipients or carriers.In one embodiment, the pharmaceutical composition of the present invention comprises a therapeutically effective amount of at least one compound of the present invention and a pharma- ceutically acceptable carrier.Useful pharma-ceutically acceptable carriers include, but are not limited to, glycerol, water, saline, ethanol, and other pharma-ceutically acceptable salt solutions, such as phosphoric acid and organic acid salts.These and other pharma-ceutically acceptable carrier examples are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

[0079] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are well within the skill of those in the art. Such techniques are fully described in such publications as "Molecular Cloning: A Laboratory Manual", fourth edition (Sambrook, 2012); "Oligonucleotide Synthesis" (Gait, 1984); "Culture of Animal Cells" (Freshney, 2010); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1997); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Short Protocols in Molecular Biology" (Ausubel, 2002); "Polymerase Chain Reaction: Principles, Applications and Troubleshooting", (Babar, 2011); "Current Protocols in Immunology" (Coligan, 2002). These techniques are applicable to the production of the polynucleotides and polypeptides of the present invention and may therefore be considered in making and practicing the present invention. Techniques that are particularly useful for particular embodiments are discussed in the following sections. EXAMPLES

[0080] The present invention will be further described in detail by referring to the following experimental examples.These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified.Therefore, the present invention should in no way be construed as being limited to the following examples, but rather should be construed as embracing any and all modifications that become evident as a result of the teachings provided herein.

[0081] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the methods of the claims. Accordingly, the following examples illustrate aspects of the present invention and are not to be construed as limiting in any way.

[0082] Noonan syndrome (NS) is an autosomal dominant disorder caused by activating mutations in the PTPN11 gene encoding Shp2, and manifests as congenital heart disease, short stature, and facial dysmorphism. The complexity of Shp2 signaling is exemplified by the observation that patients with Noonan syndrome with multiple lentigines (NSML) have symptoms similar to NS, despite carrying inactivating PTPN11 mutations. "Protein Zero-Related" (PZR), a transmembrane glycoprotein that links with the extracellular matrix to promote cell migration, was identified as the major hyper-tyrosyl-phosphorylated protein in mouse models of NS and NSML. PZR hyper-tyrosyl phosphorylation was promoted by enhanced Src recruitment to Shp2 in NS and NSML in a phosphatase-independent manner. Thus, PZR was identified as a target of NS and NSML. PZR-mediated enhanced membrane recruitment of Shp2 served as a common mechanism to direct the overlapping pathophysiological features of these PTPN11 mutations.

[0083] The materials and methods used in carrying out the experiments disclosed herein are described below.

[0084] Antibodies, chemicals, cell lines, and expression reagents. Rabbit monoclonal phospho-PZR (Y241) and rabbit monoclonal phospho-PZR (Y263) antibodies were produced in collaboration with Cell Signaling. Mouse monoclonal Src antibody, rabbit polyclonal Src antibody, rabbit polyclonal phospho-ERK1 / 2 (T202 Y204), mouse monoclonal ERK1 / 2 antibody, rabbit polyclonal phospho-Akt (S473), and mouse monoclonal Akt antibody were purchased from Cell Signaling. Rabbit polyclonal Shp2 and rabbit polyclonal ERK1 / 2 antibodies were purchased from Santa Cruz Biotechnology. Mouse monoclonal Shp2 antibody was purchased from BD Bioscience. Mouse anti-phosphotyrosine antibody 4G10 (05-321) was from Merck Millipore, rabbit anti-GFP (TP401) from Acris, and mouse anti-HA.11 clone 16B12 from Covance. Rabbit polyclonal PZR (105-6) was generously provided by ZJ Zhao. Src family kinase inhibitors PP2 and SU6656 were purchased from Calbiochem. HEK-293 cells, NIH 3T3 cells, SYF (Src - / - Yes - / - Fyn - / - Mouse embryonic fibroblast (MEF) cells, and Src ++(SYF overexpressing Src) cells were purchased from ATCC and grown in growth medium (Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum) at 37°C in a 5% CO2 incubator. Replication-deficient adenovirus (Ad) constructs containing wild-type Shp2 (Ad-Shp2 WT), E76A gain-of-function Shp2 mutant (Ad-Shp2E76A), and green fluorescent protein (GFP) were prepared as previously described (Eminaga, S., et al., J. Biol. Chem., 283:15328-15338). NIH 3T3 and SYF cells were infected with adenovirus at a multiplicity of infection (MOI) of 50. The pJ3Ω vectors containing SrcWT and the dominant negative Src mutant K295R / Y527F (SrcK295R / Y527F) were previously described (Fornaro, M., et al., J. Cell Biol., 175:87-97). The pIRES-GFP plasmids encoding Shp2 WT, gain-of-function / Noonan syndrome Shp2 mutants (Shp2E76A and Shp2N308D), and Noonan syndrome with multiple lentigines Shp2 mutants (Shp2Y279C and Shp2T468M) were previously described (Kontaridis, M. I, et al., J. Biol. Chem., 281:6785-6792). Zebrafish Shp2 mutants have been cloned previously (Jopling, C., et al., PLoS Genet., 3:e225). Zebrafish PZR (zPZR) was cloned by nested PCR from zebrafish embryo cDNA (from the bud stage to 48 hours postfertilization (hpf)). Y236F, Y258F, and Y236F Y258F mutants of the zPZR ITIM were generated using site-directed mutagenesis. The RPTPa signal sequence and hemagglutinin (HA) tag were incorporated at the N-terminus of zPZR.DNA transfection into HEK-293 and SYF cells was performed using Lipofectamine 2000 according to the manufacturer's protocol.

[0085] MS Analysis. PhosphoScan was performed as previously described (Rikova, K., et al., Cell, 131:1190-1203). Wild-type and Shp2 mutant (Noonan syndrome) mouse hearts were homogenized, sonicated, and centrifuged to remove cellular debris. ProteinPlus Coomassie Reagent (Pierce) was used to normalize total protein content for each tissue, and proteins were reduced, alkylated, and digested overnight with trypsin (Worthington). Sep-Pak Classic C 18 The resulting peptides were separated from non-peptide material by solid-phase extraction using cartridges (Waters). Lyophilized peptides were reconstituted and phosphopeptides were enriched by immunoaffinity purification using the pY-100 phosphotyrosine antibody (9411; Cell Signaling Technology). Peptides were eluted with 0.15% trifluoroacetic acid (TFA) and washed with C immediately prior to liquid chromatography-mass spectrometry (LC-MS) analysis. 18 Spin tips were used for enrichment. Duplicate injections were performed for each sample to generate analytical replicates, increasing the number of tandem MS (MS / MS) identifications from each sample. 18Peptides were loaded directly onto a 10 cm x 75 μm PicoFrit capillary column packed with AQ reversed-phase resin. The column was developed using a 45 min linear gradient of acetonitrile in 0.125% formic acid delivered at 280 nl / min. Tandem mass spectra were collected using an LTQ-Orbitrap XL mass spectrometer running XCalibur using the Top 10 method, dynamic exclusion iteration number of 1, and iteration period of 30 seconds. MS spectra were collected in the Orbitrap component of the mass spectrometer, and MS / MS spectra were collected in the LTQ portion. MS / MS spectra were processed using the SEQUEST and Core platforms (Gygi Lab, Harvard University). Searches were performed against the mouse NCBI database, including a reverse decoy database for all searches to estimate false positive rates. Peptide assignments were obtained using a precision cutoff of 0.98 in the linear discriminant analysis module of Core. Cysteine ​​carboxamidomethylation was designated as a static modification, while methionine oxidation and phosphorylation of serine, threonine, and tyrosine were allowed. Results were further refined using a mass accuracy (5 ppm) filter and the presence of phosphotyrosine in peptides. Label-free quantification was performed using Progenesis v4.1 (Nonlinear Dynamics). To ensure accuracy of results, peptide abundance data was manually reviewed in Progenesis for all peptides that changed at least 2.0-fold.

[0086] Handling Animals - Ptpn11 D61G / +Mice were provided by Dr. Benjamin Neel (University of Toronto, Toronto) and genotyped as previously described (Araki T, Mohi MG, Ismat FA, Bronson RT, Williams IR, Kutok JL, Yang W, Pao LI, Gilliland DG, Epstein JA, Neel BG. 2004. Mouse model of Noonan syndrome reveals cell type- and gene dosage-dependent effects of Ptpn11 mutation. Nat Med 10:849-857). Briefly, Ptpn11 D61G / + Male mice were crossed with WT C75BL / 6 x SV129 female mice and their progeny were genotyped by PCR and digestion with AgeI for the D61G allele.

[0087] Dasatinib treatment - N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1-piperazinyl]-2-methyl-4-pyrimidinyl]amino]-5-thiazolecarboxamide monohydrate (dasatinib, BMS-354825) was purchased from Biovision. Dasatinib was dissolved in DMSO at a concentration of 10 mg / ml and then resuspended in vehicle (1x Dulbecco's PBS) at a concentration of 200 μg / ml. WT male and Ptpn11 mice were treated with DA10 and DA210 at postnatal day 10 and postnatal week 6 (P42). D61G / + Male mice were injected daily with dasatinib (0.1 mg / kg, i.p.). Injections were then continued or discontinued for 2 weeks. Mice injected with vehicle served as controls. Body weights were measured weekly and echocardiograms were performed at P42 (week 6) and P56 (week 8). Animal procedures were approved by The Yale University Institutional Animal Care and Use Committee.

[0088] Echocardiographic studies - Cardiac size and function were analyzed by echocardiography using a Vevo770 console. Mice were lightly anesthetized with inhaled isoflurane (0.2% in O2). All measurements were taken from 3-6 consecutive cardiac cycles, and their average values ​​were used for analysis. Interventricular septum (IVS), left ventricular internal diameter (LVID), and left ventricular posterior wall thickness (LVPW) at both end diastole (d) and end systole (s) were measured from T-mode and M-mode tracings. Diastolic measurements were performed using the state-of-the-art method of the American Society of Echocardiography. For TM-mode measurements, left ventricular end diastolic volume (LV vol,d) and left ventricular end systolic volume (LV vol,s) were calculated. Ejection fraction (EF) (%) was calculated by [(LVvol,d-LVvol,s) / VLvol,d] × 100, and fractional shortening (%) was calculated by [(LVID,d-LVID,s) / LVID,d] × 100.

[0089] Statistical analysis - Statistics are presented as mean ± sem. P values ​​were calculated using two-way ANOVA (Tukey's multiple comparisons) test. All statistical analyses were performed using GraphPad Prism 5. For all studies, a P value of less than 0.05 was considered significant.

[0090] The materials and methods used in carrying out the experiments of Example 2 disclosed herein are described below.

[0091] Antibodies, Chemicals, Cell Lines, and Plasmids - The following antibodies were used for either immunoblotting (IB) or immunoprecipitation (IP) as indicated: Mouse monoclonal Flag antibody (F1804, IP-1:100, IB-1:1,000) and mouse monoclonal biotin-labeled Flag antibody (F9291, IB-1:1,000) were obtained from Sigma. Mouse monoclonal Myc antibody (sc-40, IP-1:100, IB-1:1,000), mouse monoclonal biotin-labeled Myc antibody (sc-40B, IB-1:1,000), rabbit polyclonal Shp2 antibody (sc-280, IB-1:1,000), mouse monoclonal p38 antibody (sc-535, IB-1:1,000), and mouse monoclonal GST antibody (sc-138, IB-1:1,000) were obtained from Santa Cruz Biotechnology.Rabbit monoclonal phospho-PZR antibody (Y241; no. 8181, IB-1:1,000), rabbit monoclonal phospho-PZR antibody (Y263; no. 8088, IB-1:1,000), rabbit polyclonal phospho-Src antibody (Y416; no. 2101, IB-1:1,000), mouse monoclonal Src antibody (no. 2110, IB-1:1,000), mouse polyclonal Mouse monoclonal Raf1 antibody (no. 12552, IB-1:1,000), rabbit polyclonal phospho-MEK1 / 2 antibody (S217 / 221; no. 9154, IB-1:1,000), mouse monoclonal MEK1 / 2 antibody (no. 4694, IB-1:1,000), rabbit polyclonal phospho-ERK1 / 2 antibody (T202 / Y204; no. 9101, IB-1:1,000). :1,000), mouse monoclonal ERK antibody (no. 9107, IB-1:1,000), rabbit polyclonal phospho-p38 antibody (T180 / Y182; no. 9215, IB-1:1,000), rabbit polyclonal phospho-JNK antibody (T183 / Y185; no. 4668, IB-1:1,000), mouse monoclonal JNK antibody (no. 3708, IB-1 :1,000), rabbit polyclonal phospho-Akt antibody (S473; no. 9271, IB-1:1,000), mouse monoclonal Akt antibody (no. 2967, IB-1:1,000), rabbit polyclonal SERCA2A antibody (no. 9580, IB-1:1,000), rabbit polyclonal troponin I antibody (no. 4002, IB-1:1,000) were purchased from Cell Signaling. Rabbit polyclonal phospho-Raf1 antibody (Y341; ab192820, IB-1:1,000) and rabbit polyclonal α-tubulin antibody (ab4074, IB-1:1,000) were obtained from Abcam. Mouse monoclonal Shp2 antibody (no. 610622, IB-1:1,000) was purchased from BD Bioscience. Mouse monoclonal His antibody (#11922416, IB-1:1,000) was obtained from Roche. Rabbit polyclonal Troponin T (MS-295, IB-1:1,000) was obtained from Thermo Scientific. Rabbit polyclonal PZR antibody (IB-1:1,000) was generously provided by ZJ Zhao.Dasatinib was purchased from Biovision and STI-571 was obtained from LKT laboratories. Shp2 phosphatase inhibitor was generously provided by Z.-Y. Zhang (Indiana University). HEK-293T cells were purchased from ATCC and mouse embryonic fibroblasts (MEFs) were from WT mice and Ptpn11. D61G / + The human Src constructs, as well as the Ptpn11 full-length construct, N+C construct, and PTP construct were generated by PCR and cloned into pCMV-3Tag4a and pCMV-Tag2b vectors (Clontech laboratories). DNA transfection into HEK-293T cells was performed using Lipofectamine 3000 (Invitrogen) according to the manufacturer's protocol.

[0092] Immunoprecipitation and immunoblotting - Cells or cardiac tissues were lysed on ice in lysis buffer (25 mM Tris-HCl, pH 7.4, 136 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 1% Nonidet P-40, 1 mM Na3VO4, 10 mM NaF, 1 mM benzamidine, 1 mM PMSF, 1 μg / ml pepstatin A, 5 μg / ml aprotinin, and 5 μg / ml leupeptin). Cell or tissue lysates were incubated at 4°C for 30 min and clarified by centrifugation at 14,000 rpm for 10 min at 4°C. Protein concentration was measured using BCA reagent according to the manufacturer's instructions (Pierce). For immunoprecipitation, 500 μg of lysate was incubated with 1 μg of the indicated antibody overnight at 4°C. Immune complexes were collected on either Protein A or Protein G Sepharose beads for 4 h at 4°C, washed three times with the same lysis buffer, and then heated to 95°C for 5 min in sample buffer. Total lysates or immune complexes were subjected to SDS-PAGE and immunoblotting. Antibody binding sites were visualized using sensitive chemiluminescence detection or the Odyssey Imaging System.

[0093] In vitro GST pull-down assay - Bacterial purified GST-Src SH3 and His-Shp2 PTP were provided by T. Boggon (Yale University). Pull-down assays were performed overnight at 4°C in 1 ml of lysis buffer containing GST-Src protein SH3 together with either His-Shp2 PTP or HEK-293 cell lysates overexpressing Flag-tagged Shp2. SH3-bound Shp2 protein was affinity purified with BSA-coated GST-Sepharose beads for 1 h at 4°C. The interaction of Src protein SH3 with Shp2 protein was examined by immunoblotting with anti-His or anti-Flag and anti-GST antibodies.

[0094] Handling Animals - Ptpn11 D61G / +Mice were provided by Dr. Benjamin Neel (University of Toronto, Toronto) and genotyped as previously described. 9 In brief, Ptpn11 D61G / + Male mice were crossed with wild-type C75BL / 6 x SV129 female mice, and their offspring were genotyped by PCR and digestion with AgeI for the D61G allele. Dasatinib (Biovision) was suspended in vehicle (1% DMSO in phosphate-buffered saline). For prenatal treatment, pregnant mice were injected intraperitoneally with dasatinib (0.1 mg / kg body weight) daily, beginning on gestational day 7.5 (E7.5) and continuing until postnatal day 9 (in lactating females). Mice injected with vehicle served as controls. Starting on P10, pups were directly (i.p.) injected daily with dasatinib or vehicle alone until postnatal week 8. For postnatal treatment, pups were injected (i.p.) with dasatinib from P10 until postnatal week 6; injections were discontinued for 2 weeks. Animal procedures were approved by the Yale University Institutional Animal Care and Use Committee.

[0095] Histological examination - Heart, liver, and spleen were isolated from wild-type and NS mice treated with vehicle or dasatinib. Tissues were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), processed for paraffin sectioning, and stained with hematoxylin and eosin (H&E) or Masson's trichrome staining. Tissue images were obtained under a bright-field microscope (Olympus BX51, Yale Liver Center).

[0096] Echocardiography - Mice were anesthetized in a sealed plastic container with 1% isofurane in oxygen until immobile, then transferred onto a warmed procedure board (37°C). Animals were kept anesthetized throughout the procedure with 1% isoflurane delivered by a nose cone connected to an isoflurane vaporizer. A scan head was placed on the mouse's chest to acquire stable image signals (both B-mode and M-mode), and data were analyzed using a Vevo 770 (VisualSonics). M-mode images were used to measure systolic and distolic left ventricular peripheral wall thickness, chamber diameters, and interventricular wall thickness. Ejection fraction (EF) (%) and fractional shortening (FS) (%) were calculated.

[0097] Hemodynamic studies - Anesthesia was induced by intraperitoneal injection of ketamine (100 mg / kg) and xylazine (5 mg / kg). The animal was placed on a warm pad and an incision was made in the neck. The right carotid artery was exposed and a 1.9 French transducer-tipped catheter (Millar Inc., Houston, TX) was inserted into the artery and then advanced into the left ventricle. Left ventricular pressure and heart rate, including high fidelity positive and negative dp / dt, were measured under basal conditions. Data were recorded and analyzed using LabChart software.

[0098] RNA extraction and quantitative real-time PCR analysis - RNA was isolated from mouse hearts using the RNeasy kit (Qiagen, CA) according to the manufacturer's instructions. 1 μg total RNA was reverse transcribed to cDNA using a reverse transcriptase PCR kit (Applied Biosystems, CA). Real-time quantitative PCR was performed in triplicate using an Applied Biosytems 7500 Fast real-time PCR system and SYBR Green Gene Expression Master Mix with the following primer pairs: TIFF2025061369000002.tif100144

[0099] All relative gene expression levels are expressed as ΔC T The results were analyzed using the ELISA method and normalized to 18S rRNA expression.

[0100] Cardiomyocytes were isolated from 8-week-old mice by a modified Langendorff procedure for enzymatic digestion of cardiac tissue for single-cell analysis - Xianghua Xu, et al. J Vis Exp. 2009; (28):1308. Briefly, hearts were rapidly excised, cannulated, and mounted in a Langendorff apparatus. In the Langendorff apparatus, the hearts were incubated at 37°C in Ca 2+ The heart was perfused with perfusion buffer (118 mM NaCl, 4.8 mM KCl, 2.0 mM KH2PO4, 2.55 mM MgSO4, 10 mM BDM, and 10 mM glucose in 25 mM HEPES) without ATP. To digest the tissue, the heart was perfused with buffer containing 0.5 mg / mL Liberase TH (Roche Applied Science, Penzberg, Germany). After approximately 10 min, the heart was removed from the Langendorff apparatus and the right ventricle and atrium were removed. The left ventricle was isolated, cut into small pieces, and further digested in digestion solution at 37 °C for 5–10 min with mechanical agitation, then gently triturated to liberate individual cells. Any large chunks of remaining tissue were transferred to fresh digestion buffer and the above process was repeated up to six times or until the entire tissue was digested. Cells were cleared from collagenase by gentle centrifugation, resuspended in FBS-containing buffer for several washing steps, and calcium (0.05-1.1 mM) was gradually reintroduced by the stepwise addition of high-concentration CaCl2 solutions. Cells were allowed to rest for at least 1 h before imaging.

[0101] Characterization of cardiomyocyte function - Cardiomyocytes were imaged in Tyrode's solution (150 mM: NaCl: 140, KCl: 5.4, CaCl2: 1.8, MgCl2: 1, HEPES: 25 mM, glucose: 10 mM). For calcium fluorescence imaging, cell pellets were loaded with Tyrode's supplemented with 2.5 μM Fura-2 AM and complimented with pluronic acid (20% w / v) for 15 min in the dark. After 15 min loading, cells were resuspended in fresh Tyrode's solution and allowed to stabilize until imaging. Cardiomyocyte Ca 2+ Transient and unloaded shortening contractions were measured under constant perfusion of 37°C Tyrode's solution using an inverted microscope (Nikon Eclipse, Chiyoda, Tokyo) equipped with a thermostatic perfusion bath (Cell MicroControls, Norfolk, VA). Cells were electric-field stimulated at 1 Hz. Contractile events were imaged in real time using a sarcomere length camera system (HVSL, Aurora Scientific, Ontario, Canada). Only rod-shaped cells with distinct sarcomere striations that contracted when stimulated were selected for measurements. Sarcomere length was measured and recorded for 10 consecutive beats, and all beats were subsequently averaged to generate a single waveform. Calcium transient measurements were recorded simultaneously using alternating excitation wavelengths of 340 and 380 nm generated by a RatioMaster fluorescence system (PTI, Birmingham, NJ) at an overall rate of 100 Hz. Fluorescence emission was filtered at a central wavelength of 510 nm and quantified to obtain responses to alternating excitation wavelengths (F 340 and F 380) Ca 2+ The transients were calculated as the interpolated ratio of the two fluorescence intensities at each time point (F 340 / F 380) were reported. Data were recorded using a DAP5216a data acquisition system (Microstar Laboratories, Bellevue, WA) and processed using custom software written in MATLAB (MathWorks, Natick, MA). Peak sarcomere length shortening (Peak SL shortening), time to peak shortening (TTP), time to 50% lengthening again (RT50), calcium transient magnitude (Ca 2+ R mag :Maximum F 340 / F 380 -Minimum F 340 / F 380 ), and the calcium decay rate Tau (Tau Ca2+ ) was calculated by computer.

[0102] Statistical Analysis - All data represent the mean ± standard error of the mean (SEM). Differences between groups were evaluated using analysis of variance (ANOVA) with Tukey's multiple comparisons using the GraphPad Prism 6 statistical software program.

[0103] The results of the experiments disclosed herein are set forth below.

[0104] Example 1: Targeting RASopathy-Induced Heart Disease with Tyrosine Kinase Intervention Figure 1A shows Ptpn11 D61G / + FIG. 1. Proteomic analysis of differentially tyrosyl phosphorylated proteins in mouse heart.

[0105] Figure 1B shows wild-type mouse heart and Ptpn11 D61G / +Figure 1C is a graph showing the log2-transformed ratios of peptides containing phosphotyrosine in mouse heart. Figure 1D is a panel of extracted ion chromatograms and peptide sequences from differential proteomics of PZR containing tyrosine 242 (upper panel) and PZR containing tyrosine 264 (lower panel). Figure 1E shows the amino acid sequences of the C-terminus of PZR in various vertebrates. Noonan syndrome mutant (Shp2 D61G / + Global phosphotyrosyl proteomics of mouse hearts carrying a knock-in mutation of Shp2 reveals altered regulation of tyrosyl phosphorylated proteins. MS analysis shows that the most abundantly hypertyrosyl phosphorylated protein in these mice is PZR. Tyrosyl residues 264 and 242 are involved in the regulation of Shp2 in Noonan syndrome (Shp2) D61G / + ) mice hearts. Given that PZR tyrosines 242 and 264 are highly conserved during evolution, they are likely important for PZR function. These results suggest that increased PZR tyrosyl phosphorylation may play a role in the pathogenesis of cardiac disease associated with Noonan syndrome. These results confirm that Y242 and Y264 are PZR hypertyrosyl phosphorylation sites in this NS mouse model.

[0106] Figures 2A-2E show immunoblots showing the characterization of PZR tyrosyl phosphorylation. Conformation of PZR hypertyrosyl phosphorylation sites by NS and NSML mutants. Expression of phosphorylation-resistant mutants of PZR at tyrosyl phosphorylation sites present in mice impairs their ability to be phosphorylated, even in cultured cells, as detected with phospho-specific anti-PZR(Y242) and anti-PZR(Y263). Shp2 mutants corresponding to those found in either NS or NSML patients can induce PZR hypertyrosyl phosphorylation at Y241 and Y263. Similarly, zebrafish PZR also shows the same property of being able to be tyrosyl phosphorylated at the equivalent residues. These results suggest that Shp2 mutants associated with NS or Leopard syndrome induce PZR hyperphosphorylation in various cell lines.

[0107] 3A to 3D show Ptpn11 D61G / + Mice and Ptpn11 Y279C / + Showing PZR tyrosyl phosphorylation in mouse heart and cortex. Using a site-specific phospho-PZR antibody, we identified Shp2 D61G / + We show that mice expressing knock-in mutations of the Shp2 allele show increased PZR tyrosyl phosphorylation in the heart and cortex. Y279C / + Mice expressing allelic knock-in mutations also show increased PZR tyrosyl phosphorylation in the heart and cortex. These results demonstrate that both NS and NSML mutations, which have enhanced and reduced phosphatase catalytic activity, respectively, can increase PZR tyrosyl phosphorylation. These results demonstrate that PZR is a target for both NS and NSML and suggest that PZR represents a novel common signaling component of these RASopathies. These results show that PZR hypertyrosyl phosphorylation in the heart and cortex of NS and NSML model mice. These in vivo data corroborated the in silico (Figures 1A-1E) and in vitro (Figures 2A-2E) experiments.

[0108] 4A to 4C show Ptpn11 D61G / + Images and graphs of PZR tyrosyl phosphorylation in mouse liver, kidney, and spleen. D61G / + Hypertyrosyl phosphorylated in the liver, kidney, and spleen of mice. These results demonstrated hypertyrosyl phosphorylation of PZR in various tissues of NS mice.

[0109] 5A to 5D show Ptpn11 D61G / + Mice and Ptpn11 Y279C / + Images of ERK and Akt phosphorylation in mouse heart and cortex. D61G / + Despite the phosphorylation status of ERK and AKT in mouse heart and cortex showing no substantial difference from that of wild type, under similar conditions, PZR is hypertyrosyl phosphorylated (see Figures 3A-3D). These results implicated that no obvious differences were observed in the basal levels of Shp2, phospho-ERK1 / 2, and phospho-Akt between NS or NSML mice. Furthermore, these results indicated that the effects of these RAS opacities on MAPK and AKT signaling in heart and cortex are distinct from those driving PZR hypertyrosyl phosphorylation.

[0110] Figures 6A-6B are blots showing the effect of Src family kinases on NS / NMLS-Shp2-mediated PZR Y241 and Y263 phosphorylation. NS- and NMLS-associated mutants induced PZR hypertyrosylation, which could be inhibited by pretreating cells with the SFK inhibitor SU6656. These results suggested that SFKs could phosphorylate both Y241 and Y263 of PZR. Furthermore, these results demonstrated that PZR hypertyrosyl phosphorylation induced by NS-Shp2 and NSML-Shp2 mutants was dependent on Src family kinases.

[0111] Figures 7A-7B are blots showing that Src kinase mediated NS / LS-Shp2-induced PZR hypertyrosyl phosphorylation. Figures 7C-7D are blots showing that Src kinase mediated PZR hypertyrosyl phosphorylation. These figures show a comparison of the effect of tyrosine kinase inhibitory activity on PZR tyrosyl phosphorylation. Cells expressing an activated Shp2 (Shp2-E76A) mutant were treated with tyrosine kinase inhibitors PP2 and SU6656. Both PP2 and SU6656 can inhibit Shp2-E76A-induced PZR hypertyrosyl phosphorylation, but SU6656 was more effective. PZR hypertyrosyl phosphorylation was completely inhibited by 1 μM SU6656, whereas PP2 was inhibited by 5 μM. These results support the idea that Src family kinases are responsible for PZR phosphorylation. Importantly, PZR phosphorylation by Src created a binding site (pY241 / pY263) for Shp2 to interact with PZR, implying that c-Src directly phosphorylates the Shp2-binding site on PZR.

[0112] In Noonan syndrome, increased phosphorylation of Y241 and Y263 by Src results in high levels of the deleterious PZR / Shp2 complex, which has been proposed to be a mechanism promoting the development of congeneital heart disease in these patients.

[0113] Figures 8A-8B show increased Src complex formation with Shp2 mutants and PZR associated with NS / NSML. Figure 8A shows that mutant forms of Shp2 known to cause either NS or NSML can bind c-Src with higher affinity than wild-type Shp2. Furthermore, these results suggest that c-Src directly phosphorylates the Shp2-binding site of PZR.

[0114] Figure 9 illustrates a model for the effect of NS-Shp2 and NSML-Shp2 mutants on PZR tyrosyl phosphorylation. The model is based on experimental data in which the Shp2 binding site of PZR was observed to be hypertyrosyl phosphorylated in the hearts of both NS and NSML mouse models. Increased PZR tyrosyl phosphorylation promoted increased Shp2 binding to PZR. The diagram proposes that, as with other potential Src substrates in close proximity, increased PZR tyrosyl phosphorylation increases Shp2 recruitment to PZR, further promoting PZR tyrosyl phosphorylation.

[0115] Furthermore, NS and NSML mutants also interacted with the tyrosine kinase Src with high affinity. Collectively, these promiscuous interactions resulted in dysfunctional downstream signaling from PZR, which contributes to the development of congenital heart disease. It is proposed that intervening in Src tyrosine kinase activity reduces the PZR / Shp2 complex and corrects the altered signaling from PZR and possibly other targets.

[0116] Figure 10 is a panel of images showing administration of dasatinib. Male Ptpn11D61G / + mice were intraperitoneally injected with the indicated doses of dasatinib or DMSO control. After 24 hours, mice were sacrificed and cardiac tissue was harvested and immunoblotted with total PZR and pY(263)-PZR antibodies. These results showed that dasatinib injection into NS mice was effective in reducing PZR tyrosyl phosphorylation.

[0117] Figure 11A shows the dasatinib prenatal dosing schedule in the NS mouse model. Pregnant mothers were given daily dasatinib from the time the animals were in utero at E7.5 until postnatal day 9 (P9). Beginning at postnatal day 10 (P10), NS mice were given dasatinib directly by daily injection (intraperitoneal) for 6 weeks (P42) and 8 weeks (P56).

[0118] Figure 11B shows the postnatal dosing schedule of dasatinib in the NS mouse model. Dasatinib was administered daily to NS mice starting on postnatal day 10 (P10) for 6 weeks (P42), after which treatment was stopped and cardiac function was measured. This same group of mice was then evaluated after 2 weeks of cessation of dasatinib treatment.

[0119] In Figures 11A and 11B, cardiac function of these mice was evaluated at 6 and 8 weeks. These figures illustrate prenatal or postnatal Dasatinb treatment strategies for NS mice. The dosing regimens described herein were designed to test three aspects of the efficacy of Dasatinib to therapeutically intervene in cardiac disease associated with NS. Because NS is a developmental disorder, the first dosing regimen shown in Figure 11A tested the efficacy of Dasatinib in exerting a therapeutic effect when administered to the developing embryo. The second evaluation revealed the efficacy of Dasatinib in treating cardiac disease associated with NS when administered postnatally. This strategy was more closely related to cardiac disease outcomes, as it is understood that therapeutic doses of Dasatinib can be administered to patients after birth. This dosing strategy would reduce the risk of intrauterine complications. Finally, the third study was intended to confirm that if cardiac function improved with treatment, Dasatinib administration was necessary to continue treatment.

[0120] Figure 12A is a panel of graphs showing that prenatal treatment of NS mice with dasatinib improves cardiac function (P42, seen in Figure 11A). Figure 12B is a panel of graphs showing that postnatal treatment of NS mice with dasatinib improves cardiac function (P42). Figure 12C is a panel of graphs showing that the improvement in cardiac function was maintained after cessation of dasatinib treatment (P56, seen in Figure 11B). These results provided evidence for the involvement of Src signaling in the pathogenesis of NS.

[0121] The results of these experiments demonstrated that low doses of dasatinib (defined herein as doses lower than those found to be effective in treating cancer) were effective in improving cardiac function in NS mice. In FIG. 12A, the results demonstrated that cardiac function was fully restored to wild-type parameters when pregnant mice were injected with dasatinib, as assessed by ejection fraction (EF) and fractional shortening (FS). In FIG. 11B, dasatinib was shown to be effective even when administered post-emergence. The treatment was still functional to completely restore cardiac function in NS mice. These results demonstrated that therapeutic administration of dasatinib to treat congenital heart disease in RASopathy patients can be performed with substantially lower risk by post-emergence administration. Finally, FIG. 11C shows that cardiac function was maintained when dasatinib was withdrawn for 2 weeks after effective cardiac function was achieved. These results demonstrate that once effective therapy is achieved and cardiac functionality is restored, continued exposure to dasatinib is unnecessary.

[0122] Example 2: Selective rescue of cardiac defects in a mouse model of Noonan syndrome (NS) by dasatinib Shp2 is composed of two Src homology 2 (SH2) domains, a protein tyrosine phosphatase (PTP) domain and a carboxy-terminal tail. NS-associated Shp2 (NS-Shp2) mutations frequently occur in amino acid residues occupying the interface between the amino-terminal SH2 and PTP domains. The resulting mutations disrupt the autoinhibitory "closed" conformation that exists between the SH2 and PTP domains in favor of a more "open" conformation that promotes catalysis.

[0123] Protein zero-related protein (PZR), a transmembrane glycoprotein containing two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) at its C-terminus, serves as a c-Src substrate and is the major highly tyrosyl phosphorylated protein and Shp2 binding target in the heart of NS mouse models. NS-Shp2 mutants interact with c-Src with high affinity, endowing them with the ability to indiscriminately target c-Src through PZR complex formation. Using zebrafish as a model of NS-induced CHD, it was proposed that PZR-Shp2-Src complex formation promotes aberrant signaling in NS-induced CHD.

[0124] Critical to this hypothesis is the ability of the NS-Shp2 mutant to exhibit enhanced interaction with c-Src, leading to increased signaling through c-Src. The nature of the enhanced interaction between Shp2 and c-Src likely results from increased exposure of a binding surface within the PTP domain of Shp2 that is not otherwise exposed in the "closed" conformation. Shp2 has been shown to form a complex with c-Src through the SH3 domain of c-Src, but the region of Shp2 with which c-Src interacts has yet to be defined.

[0125] To address this, we designed a series of Shp2 deletion mutants (Fig. S13a), co-transfected them into HEK-293T cells, and examined complex formation by co-immunoprecipitation (Fig. S23). As expected, full-length Shp2 was detected in complex with c-Src, whereas deletion mutants of Shp2 lacking the PTP domain failed to interact (Fig. S13b). Furthermore, in vitro binding assays confirmed that the PTP domain of Shp2 and the SH3 domain of c-Src directly interacted with each other (Fig. S13c).

[0126] Since this interaction occurs within the PTP domain of Shp2, the "open" conformation of the NS-Shp2 mutant is thought to be primed to establish a more stable interaction with the SH3 domain of c-Src compared to wild-type Shp2. Thus, the NS-Shp2 mutant forms a more stable complex with c-Src at the membrane via PZR, which has previously been proposed as a putative mechanism for aberrant signaling through c-Src. Importantly, these observations implicate c-Src or Src family kinase (SFK) members as candidates in Shp2-mediated NS pathogenesis.

[0127] To test whether SFKs are involved in NS pathogenesis, we pharmacologically inhibited c-Src to test whether inhibition of c-Src improves Shp2-NS signaling. To inhibit c-Src, we used dasatinib (Sprycel©), a dual Abl-Src kinase inhibitor approved for the treatment of chronic myeloid leukemia. Treatment of mouse embryonic fibroblasts (MEFs) isolated from NS mice with dasatinib disrupted tyrosyl phosphorylation of c-Src, ERK1 / 2, and PZR (Figures 13d-13h and 24).

[0128] Inhibition of PZR tyrosyl phosphorylation by dasatinib also disrupted PZR / Shp2 complex formation (Fig. S4d).Furthermore, Raf-1, MEK1, JNK, and Akt were also inhibited by dasatinib in NS-derived MEFs (Fig. S5c).

[0129] The BCR-Abl kinase inhibitor STI-571 (Gleevec©) was ineffective in reducing PZR tyrosyl phosphorylation, suggesting that inhibition of PZR tyrosyl phosphorylation and disruption of the PZR / Shp2 complex are likely the result of dasatinib's effects on c-Src rather than Abl (Figure 17). Furthermore, Shp2 inhibitors did not disrupt NS-Shp2-mediated PZR hypertyrosyl phosphorylation (Figure 17), suggesting that NS-Shp2-mediated c-Src PZR tyrosyl phosphorylation occurs independently of Shp2 phosphatase activity.

[0130] To investigate the effect of dasatinib on NS-induced tyrosyl phosphorylation of c-Src and PZR in vivo, we injected dasatinib into mice containing a knock-in mutation (D61G) in Shp2 that replaces Asp61 with Gly61 (Araki et al. Nat Med 10, 849-857 (2004)). D61G / + Mice heterozygous for recapitulate many features of the human disease, including short stature, craniofacial abnormalities, myeloproliferative disorder, and CHD.

[0131] Dasatinib has been shown to be effective at a dose of about 20 mg / kg in preventing tumor development in mice (Shah et al., Science 305, 399-401(2004)). The therapeutic effect of dasatinib in humans has been reported to be about 2 mg / kg, a dose equivalent to about 24 mg / kg in mice (Kantarjian et al., N Engl J Med 362, 2260-2270 (2010); Yu et al., Clinical Cancer Research 15, 7421-7428 (2009); Apperley J Clin Oncol 27, 3472-3479 (2009)). A dose of dasatinib as low as 0.5 mg / kg was sufficient to significantly inhibit both c-Src and PZR tyrosyl phosphorylation in the hearts of 3-week-old NS mice (Fig. 13i-13l). Notably, these doses of dasatinib (0.1-0.5 mg / kg) did not affect ERK1 / 2 phosphorylation (Fig. 13i-13l), Raf-1, MEK1, p38MAPK, or JNK in the hearts of 3-week-old NS mice (Fig. 18). These results indicate that a dose of dasatinib as low as 250-fold lower than the effective chemotherapy dose (approximately 100-140 mg / day or approximately 1.4-2.0 mg / kg / day for adult patients with chronic myelogous leukemia) can inhibit PZR tyrosyl phosphorylation in the hearts of NS mice.

[0132] Furthermore, in the hearts of NS mice, inhibition of PZR tyrosyl phosphorylation by dasatinib was not coupled to inhibition of ERK1 / 2 phosphorylation (Fig. S13). Thus, low doses of dasatinib interfered with NS-Shp2 signaling independently of the ERK1 / 2 pathway.

[0133] Dasatinib was administered to pregnant dams to assess amelioration of cardiac defects in NS mice. Starting on embryonic day 7, wild-type pregnant mice crossed with NS mice were administered 0.1, 0.5, or 1.0 mg / kg dasatinib daily interperitoneally until postnatal day 9 (to lactating females). From postnatal day 10 (P10) onwards, direct daily dasatinib injections were resumed into individual pups until postnatal week 8 (P56) (Figure 14a). Dasatinib treatment at 0.5 mg / kg / day and 1.0 mg / kg / day was embryonic lethal, whereas dasatinib treatment at 0.1 mg / kg / day had no observable adverse effects (Table 1).

[0134] Cardiac function of NS mice was examined at 6 and 8 weeks based on echocardiography and invasive hemodynamics. The ejection fraction (EF) and fractional shortening (FS) of untreated NS mice were significantly decreased by 35% compared to vehicle-treated wild-type mice (P<0.01). However, dasatinib-treated NS mice showed complete recovery of cardiac function at P42 compared to vehicle-treated NS mice (Figures 14b and 14c and Table 2). However, continued administration of dasatinib for an additional 2 weeks induced heart failure in both wild-type and dasatinib-treated NS mice (Figures 14d-14e and Table 3). These data suggest that in utero dasatinib treatment can rescue the impaired cardiac function observed in NS mice. Thus, c-Src activity contributes to the manifestation of Shp2-NS CHD symptoms.

[0135] Table 1. Ptpn11 treated prenatally with dasatinib D61G / + × Progeny from WT breeders TIFF2025061369000003.tif24151

[0136] Table 2. WT and Ptpn11D mice prenatally treated with vehicle or dasatinib 61G / + Echocardiographic parameters at P42 in mice TIFF2025061369000004.tif82150 Data represent mean ± SEM. * , p < 0.05; ** , p<0.01 indicates significance compared to vehicle-treated WT mice. ††, p<0.01 indicates significance compared to vehicle-treated Ptpn11 D61G / + Significance compared to mice is indicated. All p values ​​were derived using two-way ANOVA (Tukey's multiple comparisons). IVS, intraventricular septum wall thickness; LVID, left ventricular internal dimension; LVPW, left ventricular posterior wall thickness; LV vol, left ventricle volume; EF, ejection fraction; FS, fractional shortening; d, diastole; s, systole.

[0137] Table 3. WT and Ptpn11D mice prenatally treated with vehicle or dasatinib 61G / + Echocardiographic parameters at P56 in mice TIFF2025061369000005.tif78151 Data represent mean ± SEM. * , p < 0.05; ***, p<0.001 indicates significance compared to vehicle-treated WT mice. All p values ​​were derived using two-way ANOVA (Tukey's multiple comparisons). IVS, intraventricular septum wall thickness; LVID, left ventricular internal dimension; LVPW, left ventricular posterior wall thickness; LV vol, left ventricle volume; EF, ejection fraction; FS, fractional shortening; d, diastole; s, systole.

[0138] Dasatinib was effective in reducing the impact of NS-Shp2 on CHD after development. NS mice were treated with dasatinib (0.1 mg / kg / day) from P10 to P42 (Figure 14f). In addition to exhibiting CHD, NS humans and mice exhibit growth retardation, facial dysmorphism, and splenomegaly resembling those of the human disease. NS mice were found to exhibit growth retardation, facial dysmorphism, and splenomegaly, but dasatinib treatment did not ameliorate any of these NS-associated pathologies (Figures 19-21). Furthermore, no evidence of liver injury was observed in either wild-type or NS mice treated with dasatinib (Figure 22).

[0139] However, when cardiac parameters were examined, NS mice (P42) treated with dasatinib had fully restored cardiac functionality, as determined based on indices such as EF and FS (Figures 14g and 14h and Tables 4-5). Remarkably, when cardiac function of NS mice was evaluated at a later time point, i.e., after 2 weeks of cessation of dasatinib treatment, a similar level of cardiac functional improvement was observed compared to vehicle-treated wild-type controls (Figures 14i and 14j). Other cardiac parameters were also assessed based on invasive hemodynamics, and these results showed that aortic and left ventricular pressures were significantly restored in NS mice treated with dasatinib (Figures 14k-14n and Table 6).

[0140] Taken together, these data demonstrate that dasatinib provides selective efficacy for preventing heart failure in NS mice when administered post-onset to these mice at sub-therapeutic doses used to treat CML. Interestingly, removal of dasatinib did not reverse the recovery of cardiac function in NS mice, suggesting that the improvement in cardiac function is not temporary.

[0141] Table 4. WT and Ptpn11D mice postnatally treated with vehicle or dasatinib 61G / + Echocardiographic parameters at P42 in mice TIFF2025061369000006.tif71149Data represent mean ± SEM. * , p < 0.05; ** , p<0.01 indicates significance compared to vehicle-treated WT mice. †, p<0.05 indicates significance compared to vehicle-treated Ptpn11 mice. D61G / +Significance compared to mice is indicated. All p values ​​were derived using two-way ANOVA (Tukey's multiple comparisons). IVS, intraventricular septum wall thickness; LVID, left ventricular internal dimension; LVPW, left ventricular posterior wall thickness; LV vol, left ventricle volume; EF, ejection fraction; FS, fractional shortening; d, diastole; s, systole.

[0142] Table 5. WT and Ptpn11D mice postnatally treated with vehicle or dasatinib 61G / + Echocardiographic parameters at P56 in mice TIFF2025061369000007.tif70150 Data represent mean ± SEM. *** , p<0.001 indicates significance compared to vehicle-treated WT mice. †, p<0.05; †††, p<0.001 indicates significance compared to vehicle-treated Ptpn11 D61G / + Significance compared to mice is indicated. All p values ​​were derived using two-way ANOVA (Tukey's multiple comparisons). IVS, intraventricular septum wall thickness; LVID, left ventricular internal dimension; LVPW, left ventricular posterior wall thickness; LV vol, left ventricle volume; EF, ejection fraction; FS, fractional shortening; d, diastole; s, systole.

[0143] Table 6. WT and Ptpn11D mice treated postnatally with vehicle or dasatinib61G / + Hemodynamic parameters at P56 in mice TIFF2025061369000008.tif57150 Data represent mean ± SEM. ** , p < 0.01; *** , p<0.001 indicates significance compared to vehicle-treated WT mice. †, p<0.05; ††, p<0.01 indicates significance compared to vehicle-treated Ptpn11 D61G / + Significance compared to mice is shown. All p values ​​were derived using two-way ANOVA (Tukey's multiple comparisons).

[0144] To gain a more precise understanding of the nature of the cardiac phenotype exhibited by dasatinib-treated NS mice, we performed gross morphological and histological examination of these hearts. NS mice had smaller heart weights compared to wild-type mice (Figure 15a). The ratio of heart weight to body weight was significantly elevated in NS mice (Figure 15b). Histological analysis also revealed that NS mice suffered from dilated cardiomyopathy (DCM). This was indicated by significantly decreased left ventricular septal wall thickness and increased left ventricular chamber diameter during systole (Figure 15c and Table 4).

[0145] As expected, histological examination of cardiac tissue revealed disorganized myofibrillar structures in the left ventricular wall of vehicle-treated NS mice (Figure 15d). In contrast, dasatinib-treated NS mice showed a profound reversal of all of these pathological cardiac phenotypes to levels essentially similar to those of vehicle-treated wild-type mice (Figures 15a-15d).

[0146] Another hallmark of the failing heart is the development of cardiac fibrosis. Consistent with the notion that dasatinib treatment prevents cardiac failure in NS hearts, fibrosis in dasatinib-treated NS mouse hearts was significantly attenuated at the tissue level compared to vehicle-treated wild-type mice, coinciding with reduced mRNA expression levels of the fibrosis genes Col1a2 and Col3a1 (Figures 15d-15f). Deposition of fibrotic components such as collagen, encoded by the Col1a2 and Col3a1 genes, is associated with cardiac failure. Thus, the reduced expression of Col1a2 and Col3a1 is consistent with the rescue of cardiac failure by low-dose dasatinib treatment.

[0147] Re-expression of cardiac structural proteins, such as α-myosin heavy chain (MYH6) and β-myosin heavy chain (MYH7) genes, is a sign of cardiomyopathy. In particular, inactivation of MYH6 and activation of MYH7 represent features of cardiac reprogramming that support the development of cardiomyopathy (Morita et al., J Clin Invest 115 (2005)). MYH6 expression in vehicle-treated NS mice was significantly downregulated compared to wild-type mice. Dasatinib treatment resulted in comparable levels of MYH6 expression in wild-type and NS mice (Figure 15g). MYH7 was significantly re-expressed in vehicle-treated NS mice, which was completely normalized back to vehicle-treated wild-type levels after dasatinib treatment (Figure 15h).

[0148] We further strengthened the efficacy of dasatinib in improving heart failure in NS mice by evaluating atrial naturietic peptide (Anp) and brain natriuretic peptide (Bnp). Both Anp and Bnp mRNA expression levels were significantly upregulated in NS mice compared to vehicle-treated wild-type controls (Figures 15i and 15j). In contrast, NS mice treated with dasatinib were completely rescued from the elevation of both Anp and Bnp mRNA expression levels (Figures 15i and 15j). Collectively, these results support the conclusion that Src family kinase activity plays a crucial role in the development of NS-associated CHD.

[0149] To determine whether the effects of dasatinib on NS cardiac function are intrinsic to the myocardium, calcium (Ca) levels were measured in cardiomyocytes isolated from vehicle- and dasatinib-treated wild-type and NS mice. 2+ The force dynamics mediated by Ca was measured in isolated cardiac myocytes under electrical pacing. 2+ The handling and contraction kinetics were characterized simultaneously. The relative calcium release rate (R mag Ca 2+ ) was 55% higher compared to wild-type cardiomyocytes, and this difference was substantially ameliorated in NS mice treated with dasatinib (Figures 16a and 16b).

[0150] Cardiomyocytes from vehicle-treated NS mice showed a contractility deficit, with peak shortening 22% less than that of vehicle-treated wild-type mice (Figures 16a and 16c). This result indicates that in these same cardiomyocytes, there was a marked increase in Ca2+ upregulation compared to wild-type. 2+ This is striking in view of the increased myofilament Ca release observed in vehicle-treated NS cells. 2+However, sarcomere shortening fraction in NS cardiomyocytes was significantly reduced, 22% lower than that in wild-type cardiomyocytes (Fig. 16a-16c and Table 7). Importantly, these differences were completely restored in cardiomyocytes isolated from dasatinib-treated NS mice (Fig. 16a-16c and Table 7).

[0151] The molecular mechanisms of calcium handling and contractility changes were investigated using sarco(endo)plasmic reticulum Ca 2+ In cardiac muscle, SERCA2A is the major isoform responsible for calcium delivery to the contractile machinery. A hallmark of heart failure is a decrease in SERCA2A expression, which reduces the delivery of Ca to contractile proteins. 2+ This results in impaired delivery and therefore reduced contractile force.

[0152] Strikingly, hearts from vehicle-treated NS mice showed significantly reduced SERCA2A protein expression and increased TnI and TnT expression compared to wild-type (Figures 16d-16g). Consistent with the recovery of cardiac function in dasatinib-treated NS mice, cardiac tissue isolated from dasatinib-treated NS mice showed completely normalized SERCA2A expression levels (Figures 16d and 16e).

[0153] Continuous remodeling during heart failure as a result of compensatory mechanisms to maintain contractility results in upregulation of the contractile proteins troponin T (TnT) and troponin I (TnI). Both TnT and TnI were significantly increased in vehicle-treated NS mice compared to vehicle-treated wild-type mice (Fig. 16d, 16f, and 16g). In NS mice treated with dasatinib, both TnT and TnI expression levels returned to levels comparable to those in vehicle-treated wild-type mice, indicating complete recovery of the failing cardiac phenotype (Fig. 16d, 16f, and 16g). Collectively, these findings clearly demonstrate that post-developmental treatment with dasatinib attenuates myocardial contractile dysfunction in the NS mouse model.

[0154] Table 7. WT and Ptpn11D mice postnatally treated with vehicle or dasatinib 61G / + Ca2+ excitation-contraction coupling parameters in isolated cardiomyocytes from mouse hearts at P56 TIFF2025061369000009.tif71149Data represent mean ± SEM. * , p < 0.05; *** , p<0.001 indicates significance compared to vehicle-treated WT cardiomyocytes. †, p<0.05; †††, p<0.001 indicates significance compared to vehicle-treated Ptpn11 cardiomyocytes. D61G / + Significance compared to cardiomyocytes is indicated. All p values ​​were derived using two-way ANOVA (Tukey's multiple comparisons). TTP, time to peak; RT50, time from peak tension to 50% relaxation; RT90, time from peak tension to 90% relaxation.

[0155] It was previously shown that the transmembrane glycoprotein PZR was the most abnormally hypertyrosyl phosphorylated protein in NS mouse hearts (Eminaga et al., J Biol Chem 283, 15328-15338 (2008)). PZR is a Shp2-binding protein and an SFK substrate. PZR hypertyrosyl phosphorylation is a direct consequence of enhanced Src signaling through NS. These data suggest that c-Src functions in the propagation of NS-associated CHD.

[0156] Data shown in Figures 25a-25f are from wild-type (WT) mice and NSML (Ptpn11 Y279C / + ) corresponds to 6 weeks (P42) of dasatinib treatment starting on postnatal day 10 (P10) in mice. Y279C / + were obtained from Dr. Kontaridis (Beth Israel Deaconess Hospital, Boston, MA) and bred as described (Marin, et al, J Clin. Invest., 121:1026-1043(2011)). Mice were treated with either vehicle or dasatinib at a dose of 0.1 mg / kg / day for 6 weeks, after which dasatinib treatment was discontinued and mice were sacrificed 2 weeks later. Upon completion of the study, wild-type and Ptpn11 mice were Y279C / + Mice were sacrificed, total RNA from the heart was isolated, and qPCR was performed to detect mRNA expression for genes Myh6 and Myh7, which are involved in the development of cardiomyopathy, and for genes col1a2 and Col3a1, which are involved in the development of cardiac fibrosis.

[0157] As shown in the figure, Ptpn11 treated with vehicle Y279C / + By 6 weeks of age, mice began to show signs of tissue cardiomyopathy, as evidenced by increased expression of ANP, Myh6, and Myh7 (Figures 25d and 25e). Furthermore, Ptpn11 was significantly increased in mice treated with Ptpn11 compared to vehicle-treated wild-type mice. Y279C / +There was a concomitant increase in cardiac fibrosis in mice (Figures 25a and 25b). However, Ptpn11 mice treated with dasatinib showed Y279C / + Mice showed complete restoration of ANP, Myh6, and Myh7 expression to wild-type levels. Significantly, Ptpn11 mice treated with dasatinib showed a significant improvement in expression of ANP, Myh6, and Myh7. Y279C / + In mice, the Myh6 / Myh7 ratio, which represents a switch in fetal / adult myosin contractility genes, was also restored to wild-type levels (Figure 25f). Collectively, these data demonstrate the efficacy of low-dose dasatinib treatment in correcting the cardiomyopathy associated with Noonan syndrome with multiple lentigines (NSML) at the molecular level.

[0158] This is the first evidence for the involvement of c-Src signaling in the pathogenesis of NS. Other groups have reported that Shp2 is located upstream of SFKs in a phosphatase-dependent manner (Zhang et al., Mol Cell 13, 341-355 (2004)). Herein, we demonstrate a unique mechanism that causes an "open" conformation of NS-Shp2 mutants that achieves increased PTP-Shp2 / Src-SH3 binding and localization to promote c-Src signaling. Subtherapeutic doses of dasatinib required for CML treatment were sufficient to restore cardiac contractility and function after onset. These data strongly suggest that c-Src is a central mediator of NS pathogenesis. At low doses of dasatinib, the Src pathway appears to be selectively affected, and ERK1 / 2 signaling was negligibly inhibited, at least in the myocardium. However, it is conceivable that ERK1 / 2 exists in a specific subset of cells in the heart that are affected by dasatinib treatment.

[0159] Importantly, analysis of calcium-mediated contractile coupling in cardiomyocytes isolated from dasatinib-treated NS mice clearly demonstrated that these cells are the site of action through which c-Src inhibition by dasatinib exerts its effects on the contractile machinery. Consistent with previous observations, NS-Shp2 mutants upregulate Ca in the myocardium.2+ (Uhlen, et al. PNAS 103, 2160-2165 (2006)). Interestingly, cardiac muscle from NS mice showed reduced contractility, suggesting that Ca 2+ These results suggest a decreased sensitivity at the level of force contraction mediated by SERCA2A, which can be explained, at least in part, by a decrease in SERCA2A expression levels.

[0160] In summary, a novel and unexpected therapeutic strategy for treating PTPN11-mediated CHD is described herein.These data identify Src family kinases as a target class that leads to PTPN11-related CHD.The therapeutic strategy of "low-dose" dasatinib or other c-Src inhibitors may open up new avenues for treating heart disease.

[0161] Other Aspects The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of the listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment, or that embodiment in combination with any other embodiment or portion thereof.

[0162] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated by reference in their entirety. Although the present invention has been disclosed with respect to certain embodiments, it is apparent that other embodiments and variations of the present invention may be devised by those skilled in the art without departing from the true spirit and scope of the present invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

[0163] Sequence information SEQUENCE LISTING <110> Yale University <120> COMPOSITIONS AND METHODS OF USING TYROSINE KINASE INHIBITORS <150> US 62 / 250,052 <151> 2015-11-03 <150> US 62 / 107,553 <151> 2015-01-26 <160> 14 <170> PatentIn version 3.5 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <400> 1 accgcagcta ggaataatgg a 21 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <400> 2 accaaaagcc ttgactccg 19 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <400> 3 cctggaggag aagatgccgg tagaa 25 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <400> 4 ccccagtcca gggaggcacc tcgg 24 <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <400> 5 cacttcaaag gtggtcccag agctgc 26 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <400> 6 gaccggatcg gatccgtcag tcg 23 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <400> 7 gtcccggaca ctggaccagg cc 22 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <400> 8 ctccttttct tccagttgcc tagccaa 27 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <400> 9 gagcaaggcc gaggagacgc agcgt 25 <210> 10 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <400> 10 gagcctcctt ctcgtccagc tgccgg 26 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <400> 11 aggtcttcct ggagctgatg 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <400> 12 acccacaggg ccttctttac 20 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <400> 13 acagcaaatt cacttacaca gttc 24 <210> 14 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <400> 14 ctcattgcct tgcgtgttt 19

Claims

1. A method for treating a cardiovascular disease or condition having abnormal protein tyrosine phosphorylation in a subject, comprising administering to a subject in need thereof a low dose of a tyrosine kinase inhibitor, wherein the tyrosine kinase inhibitor reduces the abnormal level of tyrosine phosphorylation and improves at least one cardiac function of the subject.

2. A method for treating a congenital heart disease, comprising administering to a subject in need thereof a low dose of a tyrosine kinase inhibitor, wherein the tyrosine kinase inhibitor reduces abnormal levels of tyrosine phosphorylation and improves at least one cardiac function of the subject.

3. A method for treating a cardiovascular disease or condition associated with a RASopathy having abnormal protein tyrosine phosphorylation, comprising administering to a subject in need thereof a low dose of a tyrosine kinase inhibitor, wherein the tyrosine kinase inhibitor reduces the abnormal level of tyrosine phosphorylation and improves at least one cardiac function of the subject.

4. 3. The method of claim 2, wherein the congenital heart disease is associated with a RASopathy.

5. 4. The method of claim 3, wherein the cardiovascular disease or condition is congenital heart disease.

6. 5. The method of claim 3, wherein the RASopathy is selected from the group consisting of neurofibromatosis type 1, Noonan syndrome, Noonan syndrome with multiple lentigines (Leopard syndrome), capillary malformation-arteriovenous malformation syndrome, Costello syndrome, cardio-facio-cutaneous syndrome, and Regius syndrome.

7. The method of any one of claims 1 to 3, wherein the low dosage is in the range of about 175 to about 250 times lower than the chemotherapy dosage of the tyrosine kinase inhibitor.

8. The method of any one of claims 1 to 3, wherein the cardiac function is selected from the group consisting of myofibrillar organization, cardiomyocyte contractility, SERCA2A expression, and cardiac fibrosis.

9. The method of any one of claims 1 to 3, wherein the tyrosine kinase inhibitor is selected from the group consisting of afatinib, axitinib, bosutinib, cabozantinib, cediranib, ceritinib, crizotinib, dabrafenib, dasatinib, erlotinib, everolimus, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, lestaurtinib, nilotinib, nintedanib, palbociclib, pazopanib, ponatinib, regorafenib, ruxolitinib, semananib, sirolimus, sorafenib, sunitinib, temsirolimus, tofacitinib, trametinib, vandetanib, and vemurafenib.

10. The method according to any one of claims 1 to 3, wherein the tyrosine kinase inhibitor is a Src family tyrosine kinase inhibitor.

11. Src family tyrosine kinase inhibitors include A419259, AP23451, AP23464, AP23485, AP23588, AZD0424, AZM475271, BMS354825, CGP77675, CU201, ENMD2076, KB SRC 4, KX2361, KX2-391, MLR 1023, MNS, PCI-32765, PD166285, PD180970, PKC-412, PKI166, PP1, PP2, SRN 004, SU6656, TC-S7003, TG100435, TG100948, TX-1123, VAL 201, WH-4-023, XL 228, altenusin, bosutinib, damnacanthal, dasatinib, herbimycin A, indirubin, neratinib, lavendustin A, pelitinib, piceatannol, saracatinib, SrcI1, and analogs thereof.

12. The method of any one of claims 1 to 3, wherein the subject is a pediatric patient.

13. 13. The method of claim 12, wherein the pediatric subject is under 12 years of age.

14. The method of any one of claims 1 to 3, wherein the subject is older than 18 years of age.

15. The method of any one of claims 1 to 3, wherein the abnormal level of tyrosine phosphorylation comprises an abnormal level of tyrosine phosphorylation protein zero-related (PZR).

16. The method of claim 15, wherein the low-dose tyrosine kinase inhibitor reduces PZR tyrosine phosphorylation.

17. The method of any one of claims 1 to 3, wherein a low dose of a tyrosine kinase inhibitor provides an anti-fibrotic effect in cardiac tissue to the subject.

18. A composition comprising a low dose of a tyrosine kinase inhibitor capable of decreasing tyrosine phosphorylation and improving at least one cardiac function in a subject in need thereof.

19. The composition of claim 18, wherein the low-dose tyrosine kinase inhibitor reduces aberrant tyrosine phosphorylation of transmembrane glycoproteins.

20. 20. The composition of claim 19, wherein the transmembrane glycoprotein is Protein Zero Related (PZR).

21. 20. The composition of claim 18, wherein the low dose of the tyrosine kinase inhibitor is in the range of about 175-fold to about 250-fold lower than the chemotherapeutic dose of the tyrosine kinase inhibitor.

22. 19. The composition of claim 18, wherein the cardiac function is selected from the group consisting of myofibrillar organization, cardiomyocyte contractility, SERCA2A expression, and cardiac fibrosis.

23. 19. The composition of claim 18, wherein the low-dose tyrosine kinase inhibitor is selected from the group consisting of afatinib, axitinib, bosutinib, cabozantinib, cediranib, ceritinib, crizotinib, dabrafenib, dasatinib, erlotinib, everolimus, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, lestaurtinib, nilotinib, nintedanib, palbociclib, pazopanib, ponatinib, regorafenib, ruxolitinib, semananib, sirolimus, sorafenib, sunitinib, temsirolimus, tofacitinib, trametinib, vandetanib, and vemurafenib.

24. 19. The composition of claim 18, wherein the tyrosine kinase inhibitor is a Src family tyrosine kinase inhibitor.

25. Src family tyrosine kinase inhibitors include A419259, AP23451, AP23464, AP23485, AP23588, AZD0424, AZM475271, BMS354825, CGP77675, CU201, ENMD2076, KB SRC 4, KX2361, KX2-391, MLR 1023, MNS, PCI-32765, PD166285, PD180970, PKC-412, PKI166, PP1, PP2, SRN 004, SU6656, TC-S7003, TG100435, TG100948, TX-1123, VAL 201, WH-4-023, XL 228, altenusin, bosutinib, damnacanthal, dasatinib, herbimycin A, indirubin, neratinib, lavendustin A, pelitinib, piceatannol, saracatinib, SrcI1, and analogs thereof.

26. The composition of claim 18, wherein a low dose of the tyrosine kinase inhibitor provides an anti-fibrotic effect in cardiac tissue.

27. 20. A pharmaceutical composition comprising the composition of claim 18 and a pharma- ceutically acceptable carrier.

28. 20. Use of the composition of claim 18 in the manufacture of a medicament for treating a cardiovascular disease or condition in a subject.

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