Development of a more penetrating HER2-positive Breast Cancer Tumor Model and Testing Efficacy of SHP2 Targeting with an Active-Site Inhibitor

Development of a more penetrating HER2-positive Breast Cancer Tumor Model and Testing Efficacy of SHP2 Targeting with an Active-Site Inhibitor | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Development of a more penetrating HER2-positive Breast Cancer Tumor Model and Testing Efficacy of SHP2 Targeting with an Active-Site Inhibitor Yehenew AGAZIE, James Mersch, Dhanaji Lade, Paul Lockman This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8196938/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Advanced stage HER2-positive (HER2+) breast cancer (BC) is very challenging for treatment. These premises justify the need to develop novel targeted therapies that can be used to complement existing anti-HER2 therapies. Accordingly, we investigated the potential of the Src homology phosphotyrosyl phosphatase 2 (SHP2), the master regulator of RTK signaling, for use in advanced stage HER2 + BC. To test this possibility, we first generated a more-penetrating tumor model referred to as ErbB2+;p53 −/− . This model exhibited tumor properties reminiscent of advanced stage HER2 + BC. To target SHP2, we used our previously reported active-site SHP2 inhibitor referred to as BPDA2. The results showed suppression of tumor growth in a 20 mg/kg group and complete blockade in a 40 mg/kg group when compared to the placebo. Histopathology analysis showed a locally invasive and highly vascularized tumor with metastasis to the lungs and the liver in the placebo and a non-invasive tumor with undetectable vascularization in the treated mice. Body weight measurement showed no major changes between the placebo and the treated mice, suggesting that BPDA2 is a very well tolerated SHP2 inhibitor. Biochemical analysis of tumor protein extracts showed downregulation of HER2 expression and mitogenic and cell survival signaling. Overall, the results in this study demonstrate that targeting SHP2 with the active-site inhibitor blocks tumorigenesis and metastasis in HER2-positive BC. Biological sciences/Drug discovery/Target validation Health sciences/Biomarkers Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Approximately 20% of breast cancer (BC) is caused by overexpression of the HER2 (human epidermal growth factor receptor 2) protein ( 1 – 3 ). The detection of HER2 overexpression and the discovery of its oncogenic property led to the development of anti-HER2 drugs ( 4 ). The HER2 targeting drugs currently in clinical use include the humanized monoclonal antibodies such as trastuzumab (herceptin) ( 5 ) and pertuzumab ( 6 ), antibody drug conjugates such as T-DM1 ( 7 ) and T-DXd ( 8 ), and tyrosine kinase inhibitors such as neratinib ( 9 ) and afatinib ( 10 ). These treatments provide a tremendous benefit to patients afflicted with HER2-positive (HER2+) BC by increasing progression free survival (PFS) and overall survival (OS). However, advanced stage HER2 + BC continues to pose significant clinical challenges for managing the disease ( 11 , 12 ). These premises justify the need to target other signaling proteins in the pathway that can be used for complementing current anti-HER2 therapies. In line with this need, we have embarked on investigating the potential of the Src homology 2 domain containing phosphotyrosyl phosphatase 2 (SHP2) as a novel therapeutic target in HER2 + BC. The rationale for choosing SHP2 as a potential target emanates from its master regulator role in RTK signaling, including HER2 signaling ( 13 – 17 ). Also, the SHP2 protein is co-overexpressed with HER2 in HER2 + BC ( 18 ) most probably dues due to a stochiometric need. Furthermore, reports by us and others show that SHP2 promotes the cancerous phenotypes of BC cells, including epithelial to mesenchymal transition (EMT) ( 19 ), cell growth and transformation ( 20 ), polarity and migration ( 21 ), extracellular matrix degradation and invasion ( 22 ), and oncogene expression and tumor growth ( 23 , 24 ). SHP2 is a cytoplasmic protein with two SH2 domains in the N-terminal region, a PTP (phosphotyrosine phosphatase) domain in the C-terminal region, and Tyr phosphorylation sites in the C-terminal tail ( 25 ). The uniqueness of SHP2 is that its PTPase activity promotes rather than inhibits Tyr kinase signaling ( 14 , 26 – 28 ). SHP2 assumes a closed conformation when inactive and an open conformation when active. In the wild type SHP2 protein, activation involves engagement of the SH2 domains with phosphotyrosine on interacting proteins ( 28 ). Since RTK overexpression in HER2 + BC induces hyperactive Tyr kinase signaling, most SHP2 molecules are likely to exist in an open and active conformation, which in turn suggest effective inhibition with an active site inhibitor. In this report, we describe two major accomplishments. The first is the development of a more penetrating HER2 + BC tumor model by adding a TP53 gene conditional knockout (KO) in the mammary glands of the ErbB2/Neu transgenic mice. The rational for including TP53 conditional KO is based on prior reports that showed that advanced HER2 + BC is often associated with functional loss of the p53 protein ( 29 ) and that expression of a p53 mutant protein with ErbB2/neu (double transgenic) accelerates tumorigenesis ( 30 ). We further report that targeting SHP2 with our previously developed active-site SHP2 inhibitor referred to as BPDA2 ( 31 ) effectively suppressed tumor growth and metastasis. Materials and Methods Generation of the ErbB2 + ; p53 −/− tumor model The tumor model used in this study was produced by crossing the MMTV-ErbB2/Neu (stock #002376, Jackson labs), the p53 -floxed (stock #008462, Jackson labs), and the MMTV-Cre obtained from Dr. Lane ( 32 ). Because the MMTV-ErbB2/Neu and the MMTV-Cre mice are in FVB background, and because the p53 f/f mice are in C57 background, it was necessary to first backcross the p53 f/f mice to FVB to put all three strains in similar genetic backgrounds. Accordingly, the p53 f/f mice were backcrossed 5× to wild type FVB to make them ~ 95% FVB. Next, all three strains were crossed to obtain the test strain. Briefly, the MMTV-ErbB2/Neu mice were first crossed with the MMTV-Cre to obtain bi-transgenic MMTV-ErbB2/Neu;MMTV-Cre mice. These mice were then crossed with the p53 f/f mice to obtain MMTV-ErbB2/Neu;p53 f/f , which is designated as ErbB2 + ; p53 −/− hereinafter. The primers used for genotyping are described in the Jackson Laboratory website and in our previous report ( 33 ). Finally, the test strain was expanded by inbreeding to obtain enough mice to conduct the anti-SHP2 therapeutic studies. The use of mice in this study was approved by the West Virginia University IACUC and following the guidelines set forth by the committee. Stability of BPDA2 in S9 fractions The assay buffer consisted of 100 mM potassium phosphate (pH = 7.4), 47.1 mM potassium chloride, and 11.4 mM magnesium chloride. Mouse S9 fractions (MSS9PL) (20 mg/mL) were purchased from Gibco. NADPH was dissolved to 20 mM in assay buffer, and BPDA2 and 7-ethoxycouarin (7-EC) solutions were prepared in DMSO at 1 mM. The final reaction mixture contained 2 mg/mL S9 fraction protein, 10 µM BPDA2 or 7-EC (1% DMSO), and 2 mM NADPH in assay buffer. Contents were mixed by gentle inversion and the reaction was initiated by addition of 20 mM NADPH to a final concentration of 2 mM. The reactions were incubated at 37°C in a water bath, and 50 µL samples were taken at time 0, 30 minutes, 1 hour, 3 hours, and 6 hours. Samples were mixed with 150 µL ice cold methanol and kept on ice for 30 minutes to precipitate proteins, centrifuged for 2 minutes at 10,000 RPM and the supernatants analyzed by LC-MS (SCIEX ExionLC/SCIEX Triple Quad 5500) with comparison to a standard curve. Determining Distribution Coefficient (LogD) of BPDA2 An octanol-saturated solution of PBS (pH = 7.4) was prepared by stirring 250 mL of PBS with approximately 20 mL of n-octanol for 5 hours, and a PBS-saturated solution of octanol was prepared by stirring 250 mL of n-octanol with approximately 20 mL of PBS for 5 hours. 495 µL each of presaturated PBS and n-octanol were added to 1.5 mL centrifuge tubes, and 10 µL of 10 mM stock solution of BPDA2 in DMSO was added. The mixtures were vortexed for 10 seconds and placed on a rocker at room temperature for 6 hours with intermittent vortexing for 10 seconds every 1.5 hours. Samples were then stored upright at room temperature overnight to allow layer separation. N-octanol and PBS layers were carefully pipetted into separate vials, and three tenfold serial dilutions of each were prepared and analyzed by LC-MS (SCIEX ExionLC/SCIEX Triple Quad 5500) with comparison to a standard curve. LogD was calculated as log 10 (octanol concentration/PBS concentration). Determining biodistribution of BPDA2 in mice Mice were administered 40 mg/kg BPDA2 in 0.5% methylcellulose, 0.4% tween-80, and 10% DMSO by oral gavage. Next, blood was collected from the facial vein at 1, 5, 10, 30, 60, and 180 minutes ( n = 3 mice per time point) in 1.5 mL tubes containing 4 µL 10% K 2 -EDTA to prevent clotting. 40 µL plasma was transferred to a separate 1.5 mL tube and mixed with 250 µL ice-cold methanol, vortexed briefly, kept on ice for 30 minutes to precipitate proteins, centrifuged at 10,000 RPM (~ 9400xg) for five minutes, and supernatant analyzed by LC-MS (SCIEX ExionLC/SCIEX Triple Quad 5500). Sample concentrations were determined in micromolar with comparison to a standard curve. Concentrations are presented as mean ± standard deviation. For tissue analysis, tumor, brain, heart, liver, lung, and kidney samples were washed with PBS and then snap-frozen with liquid nitrogen, pulverized with mortar and pestle, and transferred to pre-weighed tubes to determine tissue mass. Pulverized tissue was resuspended in 800 µL 80% methanol/water and sonicated twice for 10 seconds each time to extract BPDA2. After centrifugation for 5 minutes at 12,000 RPM (11,300xg), the supernatants were analyzed by LC-MS (SCIEX ExionLC/SCIEX Triple Quad 5500) to determine BPDA2 concentration by comparison to a standard curve. Concentrations are presented as nanomoles BPDA2 per gram of tissue. Values are presented as mean ± standard deviations. Determining efficacy of SHP2 targeting on tumor growth To synchronize tumor growth, primary tumors from ErbB2 + ; p53 -/- mice were dissociated and injected into the mammary fat pad of syngeneic 10 week old female mice. Upon formation of palpable tumors with an average size of approximately 120 mm 3 , mice were administered with vehicle ( n = 10), 20 mg/kg ( n = 10), or 40 mg/kg BPDA2 ( n = 10) in 0.5% methylcellulose, 0.4% tween-80, and 10% DMSO by oral gavage. Mice were treated every three days for a total of ten treatments. Tumors were imaged by ultrasound (VisualSonics Vevo F2) at the start of treatment and at day 32, and tumor growth was monitored by caliper measurements. Tumor volumes were calculated from caliper measurements using the formula L×W×( \(\:\frac{\text{L}+\text{W}}{2}\) ). After the end of the experiments, mice were euthanized and tumor, lung, and liver tissue samples were harvested for histopathology and biochemical analyses. Immunoblotting (IB) analyses For biochemical analysis of tumors, samples were collected from mice, sonicated in lysis buffer, mixed with 2× Laemmli sample buffer, boiled at 100 o C for 10 minutes, proteins separated by SDS-PAGE, transferred to nitrocellulose membranes, and blocked with 3% BSA. The membranes were then probed with primary antibodies against HER2, SHP2, p53, pERK1/2, pAkt, panAkt, and panERK1/2 overnight at 4 o C. The next day, membranes were washed three times with TBST, incubated with appropriate secondary antibodies, washed three times, and visualized and imaged by the chemiluminescence method (Pierce Inc. and the Syngene G:Box). Antibodies The anti-HER2 antibody was from BD Biosciences (610162), while the anti-SHP2 antibody was from Cell Signaling Technologies (3397). The other antibodies used were anti-phospho-ERK1/2 (9101), anti-phospho-Akt (9271), and anti-panAkt (4685) from Cell Signaling Technologies. Additionally, we have used anti-panERK1/2 (11257-1-AP) and anti-β-actin (66009) antibodies from Proteintech. Results Generating an advanced-stage HER2-positive BC tumor model The original MMTV-ErbB2/Neu transgenic HER2 + tumor model ( 34 ) exhibits a protracted tumor latency, making them very expensive for use in testing efficacy of drugs in a spontaneous preclinical context. Also, the tumors formed by these mice represent an early stage primary HER2 + disease. To test efficacy of SHP2 targeting in an advanced-stage HER2 + BC, it was necessary to generate a tumor model that better mimics the advanced-stage disease. For this, we first generated a HER2 + BC tumor model with a reduced latency and a more penetrating tumorigenesis phenotype by introducing a conditional p53 knockout (KO) in the mammary glands of the MMTV-ErbB2/Neu mice. This step was inspired by previous reports that showed that patients with HER2 + BC harboring germline TP53 mutations exhibit an early disease onset in their life ( 29 ) and that p53 functional loss (addition of R172H-p53 ) in the MMTV-ErbB2/Neu mice (bi-transgenic) accelerated tumor onset ( 30 ). Since the general p53 functional loss is known to induce tumor development in other tissues, we decided to use a mammy-specific conditional KO of p53 to represent an advanced-stage HER2 + BC. We thus crossed the MMTV-ErbB2/Neu (stock #002376, Jackson labs), the p53 -floxed (stock #008462, Jackson labs), and the MMTV-Cre ( 32 ) mice to generate the desired strain. Because the MMTV-ErbB2/Neu and the MMTV-Cre mice are in FVB background, and because the p53-floxed ( p53 f/f ) mice are in C57 background, it was necessary to first backcross the p53 f/f mice to FVB to put all three strains in similar genetic backgrounds. Accordingly, they were backcrossed 5× to wild type FVB to obtain the p53 f/f mice with an approximately ~ 95% FVB background as outlined in Fig. 1 A. Next, all three strains were crossed to obtain the test strain as outlined in Fig. 1 B. Briefly, the MMTV-ErbB2/Neu mice, hereinafter referred to as ErbB2+ , were first crossed with the MMTV-Cre to obtain bi-transgenic ErbB2+;MMTV-Cre mice. These mice were then crossed with the p53 f/f mice to obtain the test strain designated as ErbB2+;MMTV-Cre+;p53f/f , which is referred to as ErbB2 + ; p53 −/− hereinafter for simplicity. The PCR genotyping data confirmed that mouse 6, mouse 8, and mouse 9 represent the test strain (Fig. 1 C). Finally, the test strain was expanded by inbreeding. Analyzing the ErbB2 + ; p53 −/− tumor model Next, we determined the tumor development phenotype in the ErbB2 + ; p53 −/− test strain. At least 10 female mice were put under continuous pregnancy and lactation cycles to activate the MMTV promoter, which drives the expression of the ErbB2/Neu oncogene and the Cre recombinase. Tumor development was monitored by visual observation and physical palpation. The results showed formation of palpable tumors by all mice within 5–6 months. Representative images taken from three live mice are presented in Fig. 2 A. As shown, mice formed lobulated tumors and, in some cases, tumors involving more than one mammary gland. When the tumors reached a size of 1.5 cm, a size slightly below the IACUC policy, the mice were euthanized, tumors exposed, and imaged. These images confirmed the formation of highly lobulated and vascularized tumors (Supplementary Fig. 1). These results are consistent with acceleration of tumor development by p53 conditional KO and formation of tumors with malignant phenotype in 5–6 months. To determine tumor growth property, tumor, lung, and liver tissue samples were harvested after euthanasia, fixed in formalin, embedded in paraffin, sectioned, stained with hematoxylin and eosin (H&E) using a standard protocol, and imaged under microscope. The results showed locally invasive and highly vascularized properties with presence of reservoir-like blood pouches, also known as “blood lakes” (Fig. 2 B). These lesions are typical of observations in advanced-stage HER2 + BC tumors in patients that form blood pouches ( 35 ). Similar analysis of H&E-stained lung and liver sections showed presence of metastatic lesions (Fig. 2 B). These findings suggest that we have generated a highly penetrating tumor model for HER2 + BC. Finally, protein extracts from tumor samples were analyzed for expression of HER2 and p53 . Protein extracts from three MMTV-ErbB2/Neu , three ErbB2;p53 −/− tumors, and one lactating mammary gland of wild type FVB mouse were compared in this analysis (Fig. 2 C). Probing for ErbB2 showed expression in both ErbB2 + and ErbB2 + ; p53 −/− tumors, while probing for p53 showed expression in ErbB2 + tumors and the lactating mammary glands of the FVB mouse but not in the ErbB2 + ; p53 −/− tumors. Also, the expression of ErbB2 seemed to be reduced in the ErbB2 + ; p53 −/− tumors particularly when considering the increased protein level in those samples as evidenced by the beta actin signal. These results show that we have successfully knocked out p53 in the mammary gland to obtain the ErbB2 + ; p53 −/− strain. Finally, we probed these samples for SHP2 and found that its expression is elevated both in the ErbB2 + and ErbB2 + ; p53 −/− tumors when compared to the wild type FVB. These results are consistent with our previous report that showed co-overexpression of SHP2 with HER2 in patient samples ( 18 ). BPDA2 is stable in biosamples and possesses moderate lipophilicity. We have previously reported the design and chemical synthesis of an active-site SHP2 inhibitor named BPDA2 ( 36 ), but it was not characterized for in vivo use. For this, we first determined its stability in biosamples using the Liver S9 fraction (Gibco) as a surrogate. BPDA2 at 10 µM was incubated in these preparations for various time points, extracted with methanol, and the concentration determined by LC/MS. We have used the known standard, 7-ethoxycoumarin (7-EC) at 10 µM in these experiments to confirm that the S9 fractions we are using are metabolically active. Area under the curve (AUC) and concentration calculations showed that BPDA2 was stable for at least 6 hours, while the standard 7-EC was reduced to background within an hour (Table 1 ). These findings suggest that BPDA2 is metabolically stable in biosamples. Table 1 BPDA2 is stable in S9 fraction metabolic assay. Thew concentrations are entered as mean ± standard deviations. BPDA2 7-EC Time Peak Area Conc., nM Peak Area Conc., nM 0 Hour 4.99E + 06 ± 3.50E + 05 2734 ± 186 1.34E + 07 ± 6.14E + 05 1433 ± 91.3 30 Minutes 4.74E + 06 ± 2.24E + 05 2605 ± 119 5.48E + 06 ± 1.09E + 05 500 ± 10.7 1 Hour 4.51E + 06 ± 2.84E + 05 2484 ± 152 1.28E + 06 ± 3.35E + 04 113 ± 2.9 3 Hours 4.91E + 06 ± 2.52E + 05 2696 ± 134 1.33E + 04 ± 7.65E + 02 6 ± 0.1 6 Hours 4.91E + 06 ± 2.76E + 05 2697 ± 146 9.62E + 03 ± 1.59E + 03 6 ± 0.1 We also determined the lipophilicity of BPDA2 using the LogD assay in which the distribution of a compound between layers of pH 7.4 PBS and n-octanol was measured. It was important to use pH 7.4 PBS (LogD 7.4 ) rather than pure water (LogP) due to the presence of two ionizable carboxylic acid groups in the structure of BPDA2 ( 36 ). A 10 µL volume of 10 mM BPDA2 in DMSO was mixed with 495 µL each of PBS and n-octanol. The protocol for these analyses is described in the materials and methods section. Finally, the concentrations in each layer were determined by LC-MS in comparison to a standard curve. LogD 7.4 was calculated as Log 10 (octanol concentration/PBS concentration). Based on data presented in Table 2 , the LogD 7.4 value was determined to be 0.61 ± 0.03 (Log 10 of 1778.6/436.2). These findings suggest that BPDA2 possesses moderate lipophilicity at pH 7.4. Table 2 Shows concentrations of BPDA2 in PBS and octanol, from which LogD was calculated. The bottom row in bold shows averages. pH 7.4 PBS Octanol LogD AUC Concentration (nM) AUC Concentration (nM) 8.14E + 05 465.9 3.09E + 06 1719.9 0.567 7.33E + 05 420.5 3.37E + 06 1869.5 0.648 7.36E + 05 422.2 3.14E + 06 1746.4 0.617 7.61E + 05 436.2 3.20E + 06 1778.6 0.616 BPDA2 is bioavailable from the gut and tolerable in mice. Before testing the anti-tumor effect, we found it necessary to determine maximum tolerable dose (MTD) of BPDA2 in mice. We thus determined the MTD in non-tumor bearing 10-week-old female ErbB2 + ; p53 −/− mice by administering variable doses, ranging from 10 mg/kg to 200 mg/kg. These doses were formulated in 10% DMSO, 0.5% methylcellulose, and 0.4% Tween-80 as described previously ( 37 ) and administered by oral gavage ( n = 3 mice per dosage group); the placebo mice were administered with the formulation alone. Clinical observations in a timeframe of 24 hours showed absence of any acute toxicity symptoms as there was no difference between the placebo and the test groups in movement, eating and drinking activities, and general demeanor (Supplementary Table 1). To determine any acute tissue damage, we harvested livers and lungs from the three mice in the 100 mg/kg group, from three mice in the 200 mg/kg group and from three mice in the placebo group and conducted H&E staining of tissue sections. Representative images of livers and lungs from one placebo, one 100 mg/kg, and one 200 mg/kg mouse are presented in Fig. 3 . The results showed absence of any hemorrhage, edema, or detectable tissue damage when compared to the placebo group. These results suggest that BPDA2 is tolerable, at least up to 200 mg/kg BW, and does not cause acute tissue toxicity. Next, we determine whether BPDA2 could be absorbed from the gut when given orally. A single dose of BPDA2 at 40 mg/kg formulated as above was administered to 10-week-old female mice by oral gavage, and blood samples were collected from three mice at the indicated time points (Table 3 ). BPDA2 in cleared plasma was extracted with methanol and analyzed by LC-MS with comparison to a standard curve. We found that BPDA2 is rapidly absorbed from the gut into the circulation with a peak concentration of 56 µM at the 10 minutes time point (Table 3 ). Table 3 Plasma concentration of BPDA2. The results are entered as mean ± standard deviations. Time AUC Plasma, µM 0 0 0 1 minute 2.13E + 05 ± 1.9E + 05 1.6 ± 1.46 5 minutes 3.61E + 06 ± 2.0E + 05 36.4 ± 2.24 10 minutes 6.20E + 06 ± 2.6E + 06 53.8 ± 15.86 30 minutes 6.72E + 05 ± 7.7E + 05 4.4 ± 4.49 60 minutes 6.72E + 05 ± 5.0E + 05 4.8 ± 3.23 3 hours 5.50E + 05 ± 3.0E + 05 4.3 ± 2.47 6 hours 2.56E + 05 ± 5.8E + 04 1.9 ± 0.45 The rapid drop in plasma levels suggested that BPDA2 was being absorbed into tissues very quickly. To verify this point, we determined tissue concentrations of BPDA2 after administering 40 mg/kg by oral gavage into tumor-bearing mice. Based on data in Table 3 , we chose the 3-hour time point for tissue collection. BPDA2 was extracted with 80%/20% methanol/water from tumor, liver, kidney, heart, lung, and brain samples, and the concentration determined by LC-MS as described in the materials and methods. We found the highest concentration in the liver, which is expected given that orally administered drugs first reach the liver via the portal vein. Significant concentration of BPDA2 was recovered from the lungs, the heart, the kidneys, and the tumor tissue in decreasing order, while the brain tissues had the lowest amount (Table 4 ). Accumulation of quantifiable BPDA2 in the tumor tissue suggested potential anti-tumor efficacy. Table 4 Tissue concentration of BPDA2 expressed as nanomoles per gram tissue. The results are entered as mean ± standard deviations. Tissue AUC BPDA2, nmol/g Liver 2.76E + 06 ± 2.02E + 05 9.36 ± 3.75 Kidney 4.73E + 05 ± 1.72E + 05 2.63 ± 0.03 Lung 3.15E + 05 ± 2.54E + 05 2.40 ± 1.13 Heart 1.84E + 05 ± 4.47E + 04 1.74 ± 0.27 Tumor 2.36E + 05 ± 1.67E + 04 0.89 ± 0.21 Brain 2.05E + 04 ± 1.37E + 04 0.19 ± 0.06 Targeting SHP2 with BPDA2 suppresses tumor growth and metastasis Based on the MTD data (Supplementary Table 1), we chose the middle two doses, the 20 mg/kg and the 40 mg/kg dosages for testing efficacy. Since the time of spontaneous tumor development varies among individual mice, we resorted to syngeneic tumor implantation methods to synchronize tumor formation as described in the materials and methods. When tumor volumes reached approximately 100 mm 3 , mice were divided into three groups, designated as placebo ( n = 10), 20 mg/kg BPDA2 ( n = 10), and 40 mg/kg ( n = 10) BPDA2 groups. To serve as reference points for before-treatment tumor sizes, ultrasound images were collected from three representative mice (Fig. 4 A). As shown, the starting tumor volumes ranged between 98 mm 3 and 120 mm 3 . BPDA2 at 20 mg/kg or 40 mg/kg was formulated in 10% DMSO, 0.5% Methylcellulose, and 0.4% Tween-80 and administered by oral gavage every 72 hours for a total of 10 treatments. The placebo mice were given the formulation alone without BPDA2. At the end of the treatments, ultrasound images were collected from three representative mice in each group for comparing the start and the end-point tumor sizes (Fig. 4 B). The average tumor volumes calculated from the ultrasound images were 1,321.7 mm 3 , 381.7 mm 3 , and 136.3 mm 3 for the placebo, 20 mg/kg, and 40 mg/kg groups, respectively (Fig. 4 B). To complement the ultrasound images, tumor volumes of each mouse were measured with a caliper at the start and every 5 days till the end of the experiment. For simplicity, the start and the final tumor volumes were used for plotting the results. Tumor volume data from individual mice showed a linear growth in the placebo mice, a highly slowed growth in the 20 mg/kg group, and blockade of growth in the 40 mg/kg group (Fig. 4 C). When averages of tumor volumes were plotted, the placebo tumors reached ~ 1,300 mm 3 , the 20 mg/kg reached ~ 380 mm 3 , and the 40 mg/kg tumors did not grow that much (Fig. 4 D). The p value between the placebo and 20 mg/kg and between the placebo and 40 mg/kg was < 0.001 . These findings suggest that BPDA2 effectively suppressed tumor growth in the ErbB2 + ; p53 -/- mice in a concentration-dependent manner. Body weight measurement showed no significant difference between the placebo and treatment groups (Fig. 2 E), suggesting that BPDA2 is highly tolerated. Comparative analysis of placebo and BPDA2-treated tumors To complement observations in live animals, tissue samples including tumors, lungs, and livers were harvested the placebo and BPDA2-treated mice and sections analyzed by H&E staining and microscopic imaging. Representative images from one placebo and one 40 mg/kg mouse are shown in Fig. 5 A. The results showed locally invasive and highly vascularized tumors with reservoir-like blood pouches in the placebo and non-invasive tumors with unremarkable vascularization in the treatment group mouse. Similar analysis of H&E-stained lung and liver sections showed presence of metastatic lesions in the placebo and absence of metastatic lesions in the treatment group (Fig. 5 A). These findings suggest that targeting SHP2 blocks tumor vascularization, which in turn blocks tumor growth, local invasiveness, and distant metastasis. We also analyzed protein extracts of tumors from two mice each in the three experimental groups for ErbB2 expression and the state of downstream signaling. The results showed downregulation of the ErbB2 protein as well as pAkt and pERK1/2 levels (Fig. 5 B) by BPDA2 treatment in a concentration-dependent manner. These findings suggest that targeting SHP2 with BPDA2 blocks oncogene expression which in turn blocks mitogenic and cell survival signaling. Discussion Previous genetic studies by us and others showed that SHP2 plays fundamental roles in promoting cell transformation and tumorigenesis in HER2 + and triple-negative BC ( 18 , 24 , 38 ). These reports led us to investigate SHP2 as a potential drug target in a new HER2 + BC tumor model, which exhibits the traits of an advanced-stage disease in patients. This tumor model was generated by crossing the MMTV-ErbB2/Neu mice with the p53 -floxed and the MMTV-Cre mice. The objective was to add a conditional p53 knockout in the mammary gland to better represent an advanced stage HER2 + BC in patients, in which p53 functional loss is common ( 38 ). The ErbB2 + ; p53 -/- tumor model exhibited reduced tumor latency and increased metastatic potential when compared to the original MMTV-ErbB2/Neu strain (5–6 months versus 7–10 months). These findings suggested that we have effectively generated an advanced stage HER2 + tumor model for use in the testing of novel targeted therapies. We chose to use our previously reported active-site SHP2 inhibitor referred to as BPDA2 ( 36 ) for the efficacy studies since most SHP2 proteins in HER2 + BC are likely to exist in activated conformation due to hyperactive tyrosine kinase signaling that mediate SH2-pTyr interactions. Because BPDA2 was not characterized for in vivo use, it was necessary to evaluate its pharmacologic properties, including metabolic stability, LogD, and in vivo biodistribution before moving into a preclinical efficacy study. We have found that BPDA2 is stable for at least 6 hours in mouse liver S9 fractions (Table 1 ), indicating that it is not significantly degraded by phase 1 metabolic processes. We also determined a LogP value, a commonly used parameter to assess the lipophilicity of drug candidates and found that it has moderate lipophilicity at physiological pH 7.4 with a LogD 7.4 value of 0.61 ± 0.03 (Table 2 ), which is reasonably close to the ideal range of 1–3. Analyzing plasma levels of BPDA2 after oral administration showed a rapid absorption into the circulation (Table 3 ) and quickly clearing into tissues (Table 4 ). The highest concentration of BPDA2 was present in the liver, which is expected given that all absorbed materials from the gut must pass through this organ. The presence of a significant concentration of BPDA2 in the tumor tissue was a key finding, which suggested a potential anti-tumor activity and a possible adjustment in dosage to enhance efficacy in the future. The brain had the lowest concentration of BPDA2 (~ 0.19 µM), suggesting that BPDA2 needs to be modified if targeting SHP2 in brain cancers is desired in future studies. We have also assessed toxicity profiles in mice treated with a range of BPDA2 concentrations (10 mg/kg to 200 mg/kg, Supplementary Table 1) and found that BPDA2 is well tolerated, as no acute toxicity symptoms were observed at any concentration used. The histopathology analysis of the lung and liver sections showed no signs of tissue damage even at 200 mg/kg (Fig. 3 ). This indicates that BPDA2 is tolerable in mice, at least up to 200 mg/kg. These results inspired us to move forward with testing the efficacy in the ErbB2 + ; p53 -/- tumor model. Mice bearing mammary tumors were divided into three groups designated as placebo, 20 mg/kg, and 40 mg/kg. The use of two different doses of BPDA2 was necessary since no prior information on the anti-tumor effect was available. These treatments showed suppression of tumor growth at the 20 mg/kg dosage and blockade of tumor growth at the 40 mg/kg dosage (Fig. 4 C and D), which was confirmed by ultrasound imaging at the start and at the end of the treatments (Fig. 4 B). Importantly, BPDA2 treatment did not lead to significant differences in body weight as evidenced by data in Fig. 4 E. Comparative H&E analysis of placebo and BPDA2 treated tumors showed abolition of the characteristic tumor vascularization in HER2 + BC, which is likely responsible for blocking tumor growth and metastasis (Fig. 5 ). Overall, these findings clearly suggest that inhibition of SHP2 with BPDA2 can be effective in advanced stage HER2 + BC. We have not observed complete disappearance of the tumors in this study, but since the MTD studies showed no toxicity at doses up to 200 mg/kg, dose escalation or changes in dosing schedules in future studies may provide further insights on the potential of BPDA2 for targeting SHP2 in advanced HER2 + BC. Declarations Competing interests statement : The authors declare no potential conflicts of interest. This work was supported by a grant from the National Institute of Health/National Cancer Institute, USA. Acknowledgement This work was supported by a grant (CA213996) from the National Cancer Institute (NCI), a component of the National Institute of Health (NIH) to YMA. The final aspects of the manuscript were supported by bridge funding from the WVU cancer Institute. Author contribution JM is responsible for determining stability of BPDA2 in S9 fractions, LogD value of BPDA2, LC/MS analysis of BPDA2 in blood and tissue samples, mice genotyping, and ultrasound imaging. DL is responsible for chemical synthesis of BPDA2. PL was responsible for formulation of BPDA2 for oral administration and for providing advice in the efficacy studies. YMA is responsible for leading the generation of the ErbB2 + ;p53 -/- tumor model, for in vivo efficacy studies, signaling work, and for overseeing the overall project. Competing interests statement The authors declare no potential conflicts of interest. This work was supported by a grant from the National Institute of Health/National Cancer Institute, USA. Data availability statement There is no restriction on the use of the methods and data described in this report. References Dent S, Oyan B, Honig A, Mano M, Howell S. HER2-targeted therapy in breast cancer: a systematic review of neoadjuvant trials. Cancer treatment reviews. 2013;39(6):622-31. Pegram MD, Konecny G, Slamon DJ. The molecular and cellular biology of HER2/neu gene amplification/overexpression and the clinical development of herceptin (trastuzumab) therapy for breast cancer. Cancer Treat Res. 2000;103:57-75. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science. 1987;235(4785):177-82. Giordano SH, Temin S, Kirshner JJ, Chandarlapaty S, Crews JR, Davidson NE, et al. Systemic therapy for patients with advanced human epidermal growth factor receptor 2-positive breast cancer: American Society of Clinical Oncology clinical practice guideline. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2014;32(19):2078-99. Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. 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Journal of Pharmacology and Experimental Therapeutics. 2012;343(2):342-50. Gamez-Chiachio M, Sarrio D, Moreno-Bueno G. Novel Therapies and Strategies to Overcome Resistance to Anti-HER2-Targeted Drugs. Cancers. 2022;14(18). Vernieri C, Milano M, Brambilla M, Mennitto A, Maggi C, Cona MS, et al. Resistance mechanisms to anti-HER2 therapies in HER2-positive breast cancer: Current knowledge, new research directions and therapeutic perspectives. Crit Rev Oncol Hemat. 2019;139:53-66. Agazie YM, Hayman MJ. Molecular mechanism for a role of SHP2 in epidermal growth factor receptor signaling. Molecular and cellular biology. 2003;23(21):7875-86. Feng GS, Hui CC, Pawson T. SH2-containing phosphotyrosine phosphatase as a target of protein-tyrosine kinases. Science. 1993;259(5101):1607-11. Feng GS, Shen R, Heng HH, Tsui LC, Kazlauskas A, Pawson T. Receptor-binding, tyrosine phosphorylation and chromosome localization of the mouse SH2-containing phosphotyrosine phosphatase Syp. Oncogene. 1994;9(6):1545-50. Li J, Reed SA, Johnson SE. Hepatocyte growth factor (HGF) signals through SHP2 to regulate primary mouse myoblast proliferation. Experimental cell research. 2009;315(13):2284-92. Zhou XD, Agazie YM. Molecular Mechanism for SHP2 in Promoting HER2-induced Signaling and Transformation. Journal of Biological Chemistry. 2009;284(18):12226-34. Zhou XD, Agazie YM. Inhibition of SHP2 leads to mesenchymal to epithelial transition in breast cancer cells. Cell death and differentiation. 2008;15(6):988-96. Zhou X, Agazie YM. Molecular mechanism for SHP2 in promoting HER2-induced signaling and transformation. The Journal of biological chemistry. 2009;284(18):12226-34. Hartman ZR, Schaller MD, Agazie YM. The tyrosine phosphatase SHP2 regulates focal adhesion kinase to promote EGF-induced lamellipodia persistence and cell migration. Molecular cancer research : MCR. 2013;11(6):651-64. Zhao H, Agazie YM. Inhibition of SHP2 in basal-like and triple-negative breast cells induces basal-to-luminal transition, hormone dependency, and sensitivity to anti-hormone treatment. BMC cancer. 2015;15:109. Aceto N, Sausgruber N, Brinkhaus H, Gaidatzis D, Martiny-Baron G, Mazzarol G, et al. Tyrosine phosphatase SHP2 promotes breast cancer progression and maintains tumor-initiating cells via activation of key transcription factors and a positive feedback signaling loop. Nature medicine. 2012;18(4):529-37. Matalkah F, Martin E, Zhao H, Agazie YM. SHP2 acts both upstream and downstream of multiple receptor tyrosine kinases to promote basal-like and triple-negative breast cancer. Breast cancer research : BCR. 2016;18(1):2. Dardaei L, Wang HQ, Singh M, Fordjour P, Shaw KX, Yoda S, et al. SHP2 inhibition restores sensitivity in ALK-rearranged non-small-cell lung cancer resistant to ALK inhibitors. Nature medicine. 2018;24(4):512-7. Jiang L, Xu W, Chen Y, Zhang Y. SHP2 inhibitor specifically suppresses the stemness of KRAS-mutant non-small cell lung cancer cells. Artif Cells Nanomed Biotechnol. 2019;47(1):3231-8. Melhem-Bertrandt A, Bojadzieva J, Ready KJ, Obeid E, Liu DD, Gutierrez-Barrera AM, et al. Early onset HER2-positive breast cancer is associated with germline TP53 mutations (vol 118, pg 908, 2012). Cancer. 2012;118(9):2561-. Li BL, Rosen JM, McMenaminBalano J, Muller WJ, Perkins AS. neu/ERBB2 cooperates with p53-172H during mammary tumorigenesis in transgenic mice. Molecular and cellular biology. 1997;17(6):3155-63. Lade DM, Nicoletti R, Mersch J, Agazie YM. Design and synthesis of improved active-site SHP2 inhibitors with anti-breast cancer cell effects. European journal of medicinal chemistry. 2023;247:115017. Li G, Robinson GW, Lesche R, Martinez-Diaz H, Jiang Z, Rozengurt N, et al. Conditional loss of PTEN leads to precocious development and neoplasia in the mammary gland. Development. 2002;129(17):4159-70. Zhao H, Martin E, Matalkah F, Shah N, Ivanov A, Ruppert JM, et al. Conditional knockout of SHP2 in ErbB2 transgenic mice or inhibition in HER2-amplified breast cancer cell lines blocks oncogene expression and tumorigenesis. Oncogene. 2019;38(13):2275-90. Guy CT, Webster MA, Schaller M, Parsons TJ, Cardiff RD, Muller WJ. Expression of the neu protooncogene in the mammary epithelium of transgenic mice induces metastatic disease. Proceedings of the National Academy of Sciences of the United States of America. 1992;89(22):10578-82. Zhang HN, Turner BM, Katerji H, Hicks DG, Wang X. Vascular lesions of the breast: Essential pathologic features and diagnostic pitfalls. Hum Pathol Rep. 2021;26. Zhou X, Coad J, Ducatman B, Agazie YM. SHP2 is up-regulated in breast cancer cells and in infiltrating ductal carcinoma of the breast, implying its involvement in breast oncogenesis. Histopathology. 2008;53(4):389-402. Lade DM, Nicoletti R, Mersch J, Agazie YM. Design and synthesis of improved active-site SHP2 inhibitors with anti-breast cancer cell effects. European journal of medicinal chemistry. 2023;247. Singh S, Dwivedi R, Chaturvedi V. Influence of Vehicles Used for Oral Dosing of Test Molecules on the Progression of Infection in Mice (vol 56, pg 6026, 2012). Antimicrobial agents and chemotherapy. 2014;58(6):3579-. Muenst S, Obermann EC, Gao F, Oertli D, Viehl CT, Weber WP, et al. Src homology phosphotyrosyl phosphatase-2 expression is an independent negative prognostic factor in human breast cancer. Histopathology. 2013;63(1):74-82. Additional Declarations There is NO conflict of interest to disclose. Supplementary Files SupplementaryData.docx Supplementary data Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8196938","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":551760816,"identity":"713dba0d-3662-48bf-b3df-09baddd17d5e","order_by":0,"name":"Yehenew AGAZIE","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYNACAwY5AwQvgTgtxqRqYWBI3EC0FoPjxy9+/FFgk76d//ADZt62wwz87DkG+LWcySmWkDBIy905I80ArEWy5w0BLQdyEiQMDA7nbrjBw8Ccc+Ywg8ENQracf5P8I8Hgf7rB+TMQLfYEtdxIPyZxwOBAAtA6oJYKoC0SBLRI3njDZtlgkGy44UaaweE/Fek8EmeeFeDVwnc+/fHNH3/s5A3OH374cIaBtRx/e/IGvFoUDvAgnHEAiHnwKgcB+Qb2BwQVjYJRMApGwQgHACh5SlkRVXzlAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-2410-5371","institution":"West Virginia University","correspondingAuthor":true,"prefix":"","firstName":"Yehenew","middleName":"","lastName":"AGAZIE","suffix":""},{"id":551760817,"identity":"dd4f2003-0d88-467a-8d6b-2ab78ccbfdec","order_by":1,"name":"James Mersch","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"James","middleName":"","lastName":"Mersch","suffix":""},{"id":551760818,"identity":"2916c555-81df-4e1c-a0ab-77d1f116bcb0","order_by":2,"name":"Dhanaji Lade","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Dhanaji","middleName":"","lastName":"Lade","suffix":""},{"id":551760819,"identity":"1ee9b945-b88a-470a-96fa-a8d143916d26","order_by":3,"name":"Paul Lockman","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Paul","middleName":"","lastName":"Lockman","suffix":""}],"badges":[],"createdAt":"2025-11-24 21:20:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8196938/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8196938/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":97672728,"identity":"cfb9d1e4-7207-492b-966a-34903b177815","added_by":"auto","created_at":"2025-12-08 09:38:40","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":120402,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-8196938/v1/509bc87898417d45fb17c29f.docx"},{"id":97532009,"identity":"603692f7-30f7-4e47-ac6b-b578906ff0b9","added_by":"auto","created_at":"2025-12-05 13:28:47","extension":"json","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5589,"visible":true,"origin":"","legend":"","description":"","filename":"ONC202503727.json","url":"https://assets-eu.researchsquare.com/files/rs-8196938/v1/b7ea6f309f34f9930422c040.json"},{"id":97532012,"identity":"5fd3bfce-7839-4977-ba5f-a9f7482c5991","added_by":"auto","created_at":"2025-12-05 13:28:47","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1851367,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryData.docx","url":"https://assets-eu.researchsquare.com/files/rs-8196938/v1/8450146b3959f8b5d0500b1b.docx"},{"id":97532013,"identity":"ba7ab8a3-e065-4900-bc5a-870a24de5199","added_by":"auto","created_at":"2025-12-05 13:28:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":929972,"visible":true,"origin":"","legend":"\u003cp\u003eGenerating the \u003cem\u003eHER2\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e;p53\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e tumor model. \u003cstrong\u003eA\u003c/strong\u003e. Scheme showing the backcrossing of \u003cem\u003ep53\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f \u003c/em\u003e\u003c/sup\u003ewith wild type FVB. \u003cstrong\u003eB\u003c/strong\u003e. Scheme showing the crossbreeding steps of the three strains (\u003cem\u003ep53\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eMMTV-ErbB2 \u003c/em\u003eand \u003cem\u003eMMTV-Cre\u003c/em\u003e) to obtain the \u003cem\u003eErbB2\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e;p53\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003etest strain. \u003cstrong\u003eC\u003c/strong\u003e. Representative PCR genotyping data to obtain the test strain.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8196938/v1/ca9f07c79de6fc93b4bad4d7.png"},{"id":97532005,"identity":"d8fcc13c-2b23-42cf-a450-390cec5f1281","added_by":"auto","created_at":"2025-12-05 13:28:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6309004,"visible":true,"origin":"","legend":"\u003cp\u003eAnalyzing the \u003cem\u003eHER2\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e;p53\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e tumor model. \u003cstrong\u003eA\u003c/strong\u003e. Representative tumor-bearing mice images from live animals. \u003cstrong\u003eB\u003c/strong\u003e. H\u0026amp;E-stained images of tumor, lung, and liver sections from two representative mice. Arrows show blood pouches in tumor sections and metastatic lesions in the lungs and livers. White bar in the bottom right of \u003cstrong\u003eB\u003c/strong\u003e represents 100 µm. \u003cstrong\u003eC\u003c/strong\u003e. Immunoblotting analysis of tumor samples for ErbB2, p53, SHP2 and beta actin.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8196938/v1/603cd9435a1aa8f50cff866c.png"},{"id":97532007,"identity":"2acd7d70-d95f-494e-baec-e5297453331f","added_by":"auto","created_at":"2025-12-05 13:28:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3535719,"visible":true,"origin":"","legend":"\u003cp\u003eToxicity studies. Representative H\u0026amp;E stained images of liver and lung sections from one placebo, one 100 mg/kg, and one 200 mg/kg mouse in the acute toxicity studies.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8196938/v1/90b3ad598b8491a84032e5f9.png"},{"id":97532011,"identity":"90462898-b408-4b77-aac9-6b5b3119a9fb","added_by":"auto","created_at":"2025-12-05 13:28:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1678770,"visible":true,"origin":"","legend":"\u003cp\u003eEfficacy studies. \u003cstrong\u003eA\u003c/strong\u003e. Ultrasound images of three representative tumors before start of the treatments. \u003cstrong\u003eB\u003c/strong\u003e. Ultrasound images of three placebo, three 20 mg/kg, and three 40 mg/kg tumors in anesthetized live animals taken two days after end of the treatments. \u003cstrong\u003eC\u003c/strong\u003e. Individual tumor volume plot based on caliper measurement. \u003cstrong\u003eD\u003c/strong\u003e. Average tumor volume plot based on caliper measurement. The double-pointed arrows show the significance of the differences (\u003cem\u003ep\u0026lt;0.0001\u003c/em\u003e). \u003cstrong\u003eE\u003c/strong\u003e. Body weight measurement showed no significant difference between the treatment and the placebo groups.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8196938/v1/0bf4c0b9cd7b05f0cc166f2a.png"},{"id":97671598,"identity":"3cf2500c-f85b-4d88-9016-d2c34c09ce77","added_by":"auto","created_at":"2025-12-08 09:32:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4482302,"visible":true,"origin":"","legend":"\u003cp\u003eComparative analysis of placebo and treated mice. \u003cstrong\u003eA\u003c/strong\u003e. Histopathology analysis showing H\u0026amp;E-stained tumor, lung, and liver sections from representative placebo and BPDA2 treated (40 mg/kg) mice. The yellow arrows point to blood pouches in a placebo tumor, and metastatic lesions in the lungs and liver of a placebo mouse. The white horizontal bar in the bottom right of each image represents 100 µm. \u003cstrong\u003eB\u003c/strong\u003e. Immunoblotting analysis of tumor protein extracts from placebo, 20 mg/kg BPDA2, and 40 mg/kg BPDA2 groups.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8196938/v1/1e7daa683894d48de1e96c8c.png"},{"id":97678783,"identity":"0159751b-3056-4a00-a4e0-8bbb3bd9b87b","added_by":"auto","created_at":"2025-12-08 09:56:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":24010921,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8196938/v1/8e093ed2-d72e-42f0-b145-2c4dda5a4002.pdf"},{"id":97672267,"identity":"afdfd760-832e-4b17-85c8-6b4b29d51edb","added_by":"auto","created_at":"2025-12-08 09:34:59","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1851367,"visible":true,"origin":"","legend":"Supplementary data","description":"","filename":"SupplementaryData.docx","url":"https://assets-eu.researchsquare.com/files/rs-8196938/v1/68efa222fe04f60ab4a641c3.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"Development of a more penetrating HER2-positive Breast Cancer Tumor Model and Testing Efficacy of SHP2 Targeting with an Active-Site Inhibitor","fulltext":[{"header":"Introduction","content":"\u003cp\u003eApproximately 20% of breast cancer (BC) is caused by overexpression of the HER2 (human epidermal growth factor receptor 2) protein (\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). The detection of HER2 overexpression and the discovery of its oncogenic property led to the development of anti-HER2 drugs (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). The HER2 targeting drugs currently in clinical use include the humanized monoclonal antibodies such as trastuzumab (herceptin) (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) and pertuzumab (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e), antibody drug conjugates such as T-DM1 (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) and T-DXd (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), and tyrosine kinase inhibitors such as neratinib (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e) and afatinib (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). These treatments provide a tremendous benefit to patients afflicted with HER2-positive (HER2+) BC by increasing progression free survival (PFS) and overall survival (OS). However, advanced stage HER2\u0026thinsp;+\u0026thinsp;BC continues to pose significant clinical challenges for managing the disease (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). These premises justify the need to target other signaling proteins in the pathway that can be used for complementing current anti-HER2 therapies.\u003c/p\u003e\u003cp\u003eIn line with this need, we have embarked on investigating the potential of the Src homology 2 domain containing phosphotyrosyl phosphatase 2 (SHP2) as a novel therapeutic target in HER2\u0026thinsp;+\u0026thinsp;BC. The rationale for choosing SHP2 as a potential target emanates from its master regulator role in RTK signaling, including HER2 signaling (\u003cspan additionalcitationids=\"CR14 CR15 CR16\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Also, the SHP2 protein is co-overexpressed with HER2 in HER2\u0026thinsp;+\u0026thinsp;BC (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e) most probably dues due to a stochiometric need. Furthermore, reports by us and others show that SHP2 promotes the cancerous phenotypes of BC cells, including epithelial to mesenchymal transition (EMT) (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e), cell growth and transformation (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e), polarity and migration (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e), extracellular matrix degradation and invasion (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e), and oncogene expression and tumor growth (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSHP2 is a cytoplasmic protein with two SH2 domains in the N-terminal region, a PTP (phosphotyrosine phosphatase) domain in the C-terminal region, and Tyr phosphorylation sites in the C-terminal tail (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). The uniqueness of SHP2 is that its PTPase activity promotes rather than inhibits Tyr kinase signaling (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). SHP2 assumes a closed conformation when inactive and an open conformation when active. In the wild type SHP2 protein, activation involves engagement of the SH2 domains with phosphotyrosine on interacting proteins (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Since RTK overexpression in HER2\u0026thinsp;+\u0026thinsp;BC induces hyperactive Tyr kinase signaling, most SHP2 molecules are likely to exist in an open and active conformation, which in turn suggest effective inhibition with an active site inhibitor.\u003c/p\u003e\u003cp\u003eIn this report, we describe two major accomplishments. The first is the development of a more penetrating HER2\u0026thinsp;+\u0026thinsp;BC tumor model by adding a \u003cem\u003eTP53\u003c/em\u003e gene conditional knockout (KO) in the mammary glands of the \u003cem\u003eErbB2/Neu\u003c/em\u003e transgenic mice. The rational for including \u003cem\u003eTP53\u003c/em\u003e conditional KO is based on prior reports that showed that advanced HER2\u0026thinsp;+\u0026thinsp;BC is often associated with functional loss of the \u003cem\u003ep53\u003c/em\u003e protein (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e) and that expression of a \u003cem\u003ep53\u003c/em\u003e mutant protein with \u003cem\u003eErbB2/neu\u003c/em\u003e (double transgenic) accelerates tumorigenesis (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). We further report that targeting SHP2 with our previously developed active-site SHP2 inhibitor referred to as BPDA2 (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e) effectively suppressed tumor growth and metastasis.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eGeneration of the\u003c/b\u003e \u003cb\u003eErbB2\u003c/b\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e;\u003cb\u003ep53\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;/\u0026minus;\u003c/b\u003e\u003c/sup\u003e \u003cb\u003etumor model\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe tumor model used in this study was produced by crossing the \u003cem\u003eMMTV-ErbB2/Neu\u003c/em\u003e (stock #002376, Jackson labs), the \u003cem\u003ep53\u003c/em\u003e-floxed (stock #008462, Jackson labs), and the \u003cem\u003eMMTV-Cre\u003c/em\u003e obtained from Dr. Lane (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Because the \u003cem\u003eMMTV-ErbB2/Neu\u003c/em\u003e and the \u003cem\u003eMMTV-Cre\u003c/em\u003e mice are in FVB background, and because the \u003cem\u003ep53\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e mice are in C57 background, it was necessary to first backcross the \u003cem\u003ep53\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e mice to FVB to put all three strains in similar genetic backgrounds. Accordingly, the \u003cem\u003ep53\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e mice were backcrossed 5\u0026times; to wild type FVB to make them\u0026thinsp;~\u0026thinsp;95% FVB. Next, all three strains were crossed to obtain the test strain. Briefly, the \u003cem\u003eMMTV-ErbB2/Neu\u003c/em\u003e mice were first crossed with the \u003cem\u003eMMTV-Cre\u003c/em\u003e to obtain bi-transgenic \u003cem\u003eMMTV-ErbB2/Neu;MMTV-Cre\u003c/em\u003e mice. These mice were then crossed with the \u003cem\u003ep53\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e mice to obtain \u003cem\u003eMMTV-ErbB2/Neu;p53\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e, which is designated as \u003cem\u003eErbB2\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003ep53\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e hereinafter. The primers used for genotyping are described in the Jackson Laboratory website and in our previous report (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Finally, the test strain was expanded by inbreeding to obtain enough mice to conduct the anti-SHP2 therapeutic studies. The use of mice in this study was approved by the West Virginia University IACUC and following the guidelines set forth by the committee.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStability of BPDA2 in S9 fractions\u003c/h2\u003e\u003cp\u003eThe assay buffer consisted of 100 mM potassium phosphate (pH\u0026thinsp;=\u0026thinsp;7.4), 47.1 mM potassium chloride, and 11.4 mM magnesium chloride. Mouse S9 fractions (MSS9PL) (20 mg/mL) were purchased from Gibco. NADPH was dissolved to 20 mM in assay buffer, and BPDA2 and 7-ethoxycouarin (7-EC) solutions were prepared in DMSO at 1 mM. The final reaction mixture contained 2 mg/mL S9 fraction protein, 10 \u0026micro;M BPDA2 or 7-EC (1% DMSO), and 2 mM NADPH in assay buffer. Contents were mixed by gentle inversion and the reaction was initiated by addition of 20 mM NADPH to a final concentration of 2 mM. The reactions were incubated at 37\u0026deg;C in a water bath, and 50 \u0026micro;L samples were taken at time 0, 30 minutes, 1 hour, 3 hours, and 6 hours. Samples were mixed with 150 \u0026micro;L ice cold methanol and kept on ice for 30 minutes to precipitate proteins, centrifuged for 2 minutes at 10,000 RPM and the supernatants analyzed by LC-MS (SCIEX ExionLC/SCIEX Triple Quad 5500) with comparison to a standard curve.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eDetermining Distribution Coefficient (LogD) of BPDA2\u003c/h3\u003e\n\u003cp\u003eAn octanol-saturated solution of PBS (pH\u0026thinsp;=\u0026thinsp;7.4) was prepared by stirring 250 mL of PBS with approximately 20 mL of n-octanol for 5 hours, and a PBS-saturated solution of octanol was prepared by stirring 250 mL of n-octanol with approximately 20 mL of PBS for 5 hours. 495 \u0026micro;L each of presaturated PBS and n-octanol were added to 1.5 mL centrifuge tubes, and 10 \u0026micro;L of 10 mM stock solution of BPDA2 in DMSO was added. The mixtures were vortexed for 10 seconds and placed on a rocker at room temperature for 6 hours with intermittent vortexing for 10 seconds every 1.5 hours. Samples were then stored upright at room temperature overnight to allow layer separation. N-octanol and PBS layers were carefully pipetted into separate vials, and three tenfold serial dilutions of each were prepared and analyzed by LC-MS (SCIEX ExionLC/SCIEX Triple Quad 5500) with comparison to a standard curve. LogD was calculated as log\u003csub\u003e10\u003c/sub\u003e(octanol concentration/PBS concentration).\u003c/p\u003e\n\u003ch3\u003eDetermining biodistribution of BPDA2 in mice\u003c/h3\u003e\n\u003cp\u003eMice were administered 40 mg/kg BPDA2 in 0.5% methylcellulose, 0.4% tween-80, and 10% DMSO by oral gavage. Next, blood was collected from the facial vein at 1, 5, 10, 30, 60, and 180 minutes (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3 mice per time point) in 1.5 mL tubes containing 4 \u0026micro;L 10% K\u003csub\u003e2\u003c/sub\u003e-EDTA to prevent clotting. 40 \u0026micro;L plasma was transferred to a separate 1.5 mL tube and mixed with 250 \u0026micro;L ice-cold methanol, vortexed briefly, kept on ice for 30 minutes to precipitate proteins, centrifuged at 10,000 RPM (~\u0026thinsp;9400xg) for five minutes, and supernatant analyzed by LC-MS (SCIEX ExionLC/SCIEX Triple Quad 5500). Sample concentrations were determined in micromolar with comparison to a standard curve. Concentrations are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation.\u003c/p\u003e\u003cp\u003eFor tissue analysis, tumor, brain, heart, liver, lung, and kidney samples were washed with PBS and then snap-frozen with liquid nitrogen, pulverized with mortar and pestle, and transferred to pre-weighed tubes to determine tissue mass. Pulverized tissue was resuspended in 800 \u0026micro;L 80% methanol/water and sonicated twice for 10 seconds each time to extract BPDA2. After centrifugation for 5 minutes at 12,000 RPM (11,300xg), the supernatants were analyzed by LC-MS (SCIEX ExionLC/SCIEX Triple Quad 5500) to determine BPDA2 concentration by comparison to a standard curve. Concentrations are presented as nanomoles BPDA2 per gram of tissue. Values are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations.\u003c/p\u003e\n\u003ch3\u003eDetermining efficacy of SHP2 targeting on tumor growth\u003c/h3\u003e\n\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTo synchronize tumor growth, primary tumors from \u003cem\u003eErbB2\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003ep53\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice were dissociated and injected into the mammary fat pad of syngeneic 10 week old female mice. Upon formation of palpable tumors with an average size of approximately 120 mm\u003csup\u003e3\u003c/sup\u003e, mice were administered with vehicle (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10), 20 mg/kg (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10), or 40 mg/kg BPDA2 (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10) in 0.5% methylcellulose, 0.4% tween-80, and 10% DMSO by oral gavage. Mice were treated every three days for a total of ten treatments. Tumors were imaged by ultrasound (VisualSonics Vevo F2) at the start of treatment and at day 32, and tumor growth was monitored by caliper measurements. Tumor volumes were calculated from caliper measurements using the formula L\u0026times;W\u0026times;(\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\text{L}+\\text{W}}{2}\\)\u003c/span\u003e\u003c/span\u003e). After the end of the experiments, mice were euthanized and tumor, lung, and liver tissue samples were harvested for histopathology and biochemical analyses.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eImmunoblotting (IB) analyses\u003c/h3\u003e\n\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eFor biochemical analysis of tumors, samples were collected from mice, sonicated in lysis buffer, mixed with 2\u0026times; Laemmli sample buffer, boiled at 100\u003csup\u003eo\u003c/sup\u003eC for 10 minutes, proteins separated by SDS-PAGE, transferred to nitrocellulose membranes, and blocked with 3% BSA. The membranes were then probed with primary antibodies against HER2, SHP2, p53, pERK1/2, pAkt, panAkt, and panERK1/2 overnight at 4\u003csup\u003eo\u003c/sup\u003eC. The next day, membranes were washed three times with TBST, incubated with appropriate secondary antibodies, washed three times, and visualized and imaged by the chemiluminescence method (Pierce Inc. and the Syngene G:Box).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eAntibodies\u003c/h2\u003e\u003cp\u003eThe anti-HER2 antibody was from BD Biosciences (610162), while the anti-SHP2 antibody was from Cell Signaling Technologies (3397). The other antibodies used were anti-phospho-ERK1/2 (9101), anti-phospho-Akt (9271), and anti-panAkt (4685) from Cell Signaling Technologies. Additionally, we have used anti-panERK1/2 (11257-1-AP) and anti-β-actin (66009) antibodies from Proteintech.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eGenerating an advanced-stage HER2-positive BC tumor model\u003c/h2\u003e\u003cp\u003eThe original \u003cem\u003eMMTV-ErbB2/Neu\u003c/em\u003e transgenic HER2\u0026thinsp;+\u0026thinsp;tumor model (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e) exhibits a protracted tumor latency, making them very expensive for use in testing efficacy of drugs in a spontaneous preclinical context. Also, the tumors formed by these mice represent an early stage primary HER2\u0026thinsp;+\u0026thinsp;disease. To test efficacy of SHP2 targeting in an advanced-stage HER2\u0026thinsp;+\u0026thinsp;BC, it was necessary to generate a tumor model that better mimics the advanced-stage disease. For this, we first generated a HER2\u0026thinsp;+\u0026thinsp;BC tumor model with a reduced latency and a more penetrating tumorigenesis phenotype by introducing a conditional \u003cem\u003ep53\u003c/em\u003e knockout (KO) in the mammary glands of the \u003cem\u003eMMTV-ErbB2/Neu\u003c/em\u003e mice. This step was inspired by previous reports that showed that patients with HER2\u0026thinsp;+\u0026thinsp;BC harboring germline \u003cem\u003eTP53\u003c/em\u003e mutations exhibit an early disease onset in their life (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e) and that \u003cem\u003ep53\u003c/em\u003e functional loss (addition of \u003cem\u003eR172H-p53\u003c/em\u003e) in the \u003cem\u003eMMTV-ErbB2/Neu\u003c/em\u003e mice (bi-transgenic) accelerated tumor onset (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Since the general \u003cem\u003ep53\u003c/em\u003e functional loss is known to induce tumor development in other tissues, we decided to use a mammy-specific conditional KO of \u003cem\u003ep53\u003c/em\u003e to represent an advanced-stage HER2\u0026thinsp;+\u0026thinsp;BC. We thus crossed the \u003cem\u003eMMTV-ErbB2/Neu\u003c/em\u003e (stock #002376, Jackson labs), the \u003cem\u003ep53\u003c/em\u003e-floxed (stock #008462, Jackson labs), and the \u003cem\u003eMMTV-Cre\u003c/em\u003e (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e) mice to generate the desired strain. Because the \u003cem\u003eMMTV-ErbB2/Neu\u003c/em\u003e and the \u003cem\u003eMMTV-Cre\u003c/em\u003e mice are in FVB background, and because the p53-floxed (\u003cem\u003ep53\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e) mice are in C57 background, it was necessary to first backcross the \u003cem\u003ep53\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e mice to FVB to put all three strains in similar genetic backgrounds. Accordingly, they were backcrossed 5\u0026times; to wild type FVB to obtain the \u003cem\u003ep53\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e mice with an approximately\u0026thinsp;~\u0026thinsp;95% FVB background as outlined in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. Next, all three strains were crossed to obtain the test strain as outlined in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB. Briefly, the \u003cem\u003eMMTV-ErbB2/Neu\u003c/em\u003e mice, hereinafter referred to as \u003cem\u003eErbB2+\u003c/em\u003e, were first crossed with the \u003cem\u003eMMTV-Cre\u003c/em\u003e to obtain bi-transgenic \u003cem\u003eErbB2+;MMTV-Cre\u003c/em\u003e mice. These mice were then crossed with the \u003cem\u003ep53\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e mice to obtain the test strain designated as \u003cem\u003eErbB2+;MMTV-Cre+;p53f/f\u003c/em\u003e, which is referred to as \u003cem\u003eErbB2\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003ep53\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e hereinafter for simplicity. The PCR genotyping data confirmed that mouse 6, mouse 8, and mouse 9 represent the test strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Finally, the test strain was expanded by inbreeding.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnalyzing the\u003c/b\u003e \u003cb\u003eErbB2\u003c/b\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e;\u003cb\u003ep53\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;/\u0026minus;\u003c/b\u003e\u003c/sup\u003e \u003cb\u003etumor model\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNext, we determined the tumor development phenotype in the \u003cem\u003eErbB2\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003ep53\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e test strain. At least 10 female mice were put under continuous pregnancy and lactation cycles to activate the \u003cem\u003eMMTV\u003c/em\u003e promoter, which drives the expression of the \u003cem\u003eErbB2/Neu\u003c/em\u003e oncogene and the \u003cem\u003eCre\u003c/em\u003e recombinase. Tumor development was monitored by visual observation and physical palpation. The results showed formation of palpable tumors by all mice within 5\u0026ndash;6 months. Representative images taken from three live mice are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. As shown, mice formed lobulated tumors and, in some cases, tumors involving more than one mammary gland. When the tumors reached a size of 1.5 cm, a size slightly below the IACUC policy, the mice were euthanized, tumors exposed, and imaged. These images confirmed the formation of highly lobulated and vascularized tumors (Supplementary Fig.\u0026nbsp;1). These results are consistent with acceleration of tumor development by \u003cem\u003ep53\u003c/em\u003e conditional KO and formation of tumors with malignant phenotype in 5\u0026ndash;6 months.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo determine tumor growth property, tumor, lung, and liver tissue samples were harvested after euthanasia, fixed in formalin, embedded in paraffin, sectioned, stained with hematoxylin and eosin (H\u0026amp;E) using a standard protocol, and imaged under microscope. The results showed locally invasive and highly vascularized properties with presence of reservoir-like blood pouches, also known as \u0026ldquo;blood lakes\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). These lesions are typical of observations in advanced-stage HER2\u0026thinsp;+\u0026thinsp;BC tumors in patients that form blood pouches (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Similar analysis of H\u0026amp;E-stained lung and liver sections showed presence of metastatic lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). These findings suggest that we have generated a highly penetrating tumor model for HER2\u0026thinsp;+\u0026thinsp;BC.\u003c/p\u003e\u003cp\u003eFinally, protein extracts from tumor samples were analyzed for expression of HER2 and \u003cem\u003ep53\u003c/em\u003e. Protein extracts from three \u003cem\u003eMMTV-ErbB2/Neu\u003c/em\u003e, three \u003cem\u003eErbB2;p53\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e tumors, and one lactating mammary gland of wild type FVB mouse were compared in this analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Probing for \u003cem\u003eErbB2\u003c/em\u003e showed expression in both \u003cem\u003eErbB2\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eErbB2\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003ep53\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e tumors, while probing for \u003cem\u003ep53\u003c/em\u003e showed expression in \u003cem\u003eErbB2\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e tumors and the lactating mammary glands of the FVB mouse but not in the \u003cem\u003eErbB2\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003ep53\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e tumors. Also, the expression of \u003cem\u003eErbB2\u003c/em\u003e seemed to be reduced in the \u003cem\u003eErbB2\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003ep53\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e tumors particularly when considering the increased protein level in those samples as evidenced by the beta actin signal. These results show that we have successfully knocked out \u003cem\u003ep53\u003c/em\u003e in the mammary gland to obtain the \u003cem\u003eErbB2\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003ep53\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e strain. Finally, we probed these samples for SHP2 and found that its expression is elevated both in the \u003cem\u003eErbB2\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eErbB2\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003ep53\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e tumors when compared to the wild type FVB. These results are consistent with our previous report that showed co-overexpression of SHP2 with HER2 in patient samples (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eBPDA2 is stable in biosamples and possesses moderate lipophilicity.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe have previously reported the design and chemical synthesis of an active-site SHP2 inhibitor named BPDA2 (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e), but it was not characterized for \u003cem\u003ein vivo\u003c/em\u003e use. For this, we first determined its stability in biosamples using the Liver S9 fraction (Gibco) as a surrogate. BPDA2 at 10 \u0026micro;M was incubated in these preparations for various time points, extracted with methanol, and the concentration determined by LC/MS. We have used the known standard, 7-ethoxycoumarin (7-EC) at 10 \u0026micro;M in these experiments to confirm that the S9 fractions we are using are metabolically active. Area under the curve (AUC) and concentration calculations showed that BPDA2 was stable for at least 6 hours, while the standard 7-EC was reduced to background within an hour (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These findings suggest that BPDA2 is metabolically stable in biosamples.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBPDA2 is stable in S9 fraction metabolic assay. Thew concentrations are entered as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eBPDA2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e7-EC\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTime\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePeak Area\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eConc., nM\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePeak Area\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eConc., nM\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0 Hour\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.99E\u0026thinsp;+\u0026thinsp;06\u0026thinsp;\u0026plusmn;\u0026thinsp;3.50E\u0026thinsp;+\u0026thinsp;05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e2734\u0026thinsp;\u0026plusmn;\u0026thinsp;186\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.34E\u0026thinsp;+\u0026thinsp;07\u0026thinsp;\u0026plusmn;\u0026thinsp;6.14E\u0026thinsp;+\u0026thinsp;05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1433\u0026thinsp;\u0026plusmn;\u0026thinsp;91.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e30 Minutes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.74E\u0026thinsp;+\u0026thinsp;06\u0026thinsp;\u0026plusmn;\u0026thinsp;2.24E\u0026thinsp;+\u0026thinsp;05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e2605\u0026thinsp;\u0026plusmn;\u0026thinsp;119\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.48E\u0026thinsp;+\u0026thinsp;06\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09E\u0026thinsp;+\u0026thinsp;05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e500\u0026thinsp;\u0026plusmn;\u0026thinsp;10.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1 Hour\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.51E\u0026thinsp;+\u0026thinsp;06\u0026thinsp;\u0026plusmn;\u0026thinsp;2.84E\u0026thinsp;+\u0026thinsp;05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e2484\u0026thinsp;\u0026plusmn;\u0026thinsp;152\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.28E\u0026thinsp;+\u0026thinsp;06\u0026thinsp;\u0026plusmn;\u0026thinsp;3.35E\u0026thinsp;+\u0026thinsp;04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e113\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3 Hours\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.91E\u0026thinsp;+\u0026thinsp;06\u0026thinsp;\u0026plusmn;\u0026thinsp;2.52E\u0026thinsp;+\u0026thinsp;05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e2696\u0026thinsp;\u0026plusmn;\u0026thinsp;134\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.33E\u0026thinsp;+\u0026thinsp;04\u0026thinsp;\u0026plusmn;\u0026thinsp;7.65E\u0026thinsp;+\u0026thinsp;02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6 Hours\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.91E\u0026thinsp;+\u0026thinsp;06\u0026thinsp;\u0026plusmn;\u0026thinsp;2.76E\u0026thinsp;+\u0026thinsp;05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e2697\u0026thinsp;\u0026plusmn;\u0026thinsp;146\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9.62E\u0026thinsp;+\u0026thinsp;03\u0026thinsp;\u0026plusmn;\u0026thinsp;1.59E\u0026thinsp;+\u0026thinsp;03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eWe also determined the lipophilicity of BPDA2 using the LogD assay in which the distribution of a compound between layers of pH 7.4 PBS and n-octanol was measured. It was important to use pH 7.4 PBS (LogD\u003csub\u003e7.4\u003c/sub\u003e) rather than pure water (LogP) due to the presence of two ionizable carboxylic acid groups in the structure of BPDA2 (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). A 10 \u0026micro;L volume of 10 mM BPDA2 in DMSO was mixed with 495 \u0026micro;L each of PBS and n-octanol. The protocol for these analyses is described in the materials and methods section. Finally, the concentrations in each layer were determined by LC-MS in comparison to a standard curve. LogD\u003csub\u003e7.4\u003c/sub\u003e was calculated as Log\u003csub\u003e10\u003c/sub\u003e(octanol concentration/PBS concentration). Based on data presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the LogD\u003csub\u003e7.4\u003c/sub\u003e value was determined to be 0.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 (Log\u003csub\u003e10\u003c/sub\u003e of 1778.6/436.2). These findings suggest that BPDA2 possesses moderate lipophilicity at pH 7.4.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eShows concentrations of BPDA2 in PBS and octanol, from which LogD was calculated. The bottom row in bold shows averages.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003epH 7.4 PBS\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eOctanol\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLogD\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAUC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eConcentration (nM)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAUC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eConcentration (nM)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8.14E\u0026thinsp;+\u0026thinsp;05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e465.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.09E\u0026thinsp;+\u0026thinsp;06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1719.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.567\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7.33E\u0026thinsp;+\u0026thinsp;05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e420.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.37E\u0026thinsp;+\u0026thinsp;06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1869.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.648\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7.36E\u0026thinsp;+\u0026thinsp;05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e422.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.14E\u0026thinsp;+\u0026thinsp;06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1746.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.617\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7.61E\u0026thinsp;+\u0026thinsp;05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e436.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.20E\u0026thinsp;+\u0026thinsp;06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1778.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.616\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eBPDA2 is bioavailable from the gut and tolerable in mice.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBefore testing the anti-tumor effect, we found it necessary to determine maximum tolerable dose (MTD) of BPDA2 in mice. We thus determined the MTD in non-tumor bearing 10-week-old female \u003cem\u003eErbB2\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003ep53\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice by administering variable doses, ranging from 10 mg/kg to 200 mg/kg. These doses were formulated in 10% DMSO, 0.5% methylcellulose, and 0.4% Tween-80 as described previously (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) and administered by oral gavage (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3 mice per dosage group); the placebo mice were administered with the formulation alone. Clinical observations in a timeframe of 24 hours showed absence of any acute toxicity symptoms as there was no difference between the placebo and the test groups in movement, eating and drinking activities, and general demeanor (Supplementary Table\u0026nbsp;1). To determine any acute tissue damage, we harvested livers and lungs from the three mice in the 100 mg/kg group, from three mice in the 200 mg/kg group and from three mice in the placebo group and conducted H\u0026amp;E staining of tissue sections. Representative images of livers and lungs from one placebo, one 100 mg/kg, and one 200 mg/kg mouse are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The results showed absence of any hemorrhage, edema, or detectable tissue damage when compared to the placebo group. These results suggest that BPDA2 is tolerable, at least up to 200 mg/kg BW, and does not cause acute tissue toxicity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNext, we determine whether BPDA2 could be absorbed from the gut when given orally. A single dose of BPDA2 at 40 mg/kg formulated as above was administered to 10-week-old female mice by oral gavage, and blood samples were collected from three mice at the indicated time points (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). BPDA2 in cleared plasma was extracted with methanol and analyzed by LC-MS with comparison to a standard curve. We found that BPDA2 is rapidly absorbed from the gut into the circulation with a peak concentration of 56 \u0026micro;M at the 10 minutes time point (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePlasma concentration of BPDA2. The results are entered as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTime\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAUC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePlasma, \u0026micro;M\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1 minute\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.13E\u0026thinsp;+\u0026thinsp;05\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9E\u0026thinsp;+\u0026thinsp;05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.46\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5 minutes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.61E\u0026thinsp;+\u0026thinsp;06\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0E\u0026thinsp;+\u0026thinsp;05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e36.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.24\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10 minutes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.20E\u0026thinsp;+\u0026thinsp;06\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6E\u0026thinsp;+\u0026thinsp;06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e53.8\u0026thinsp;\u0026plusmn;\u0026thinsp;15.86\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e30 minutes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.72E\u0026thinsp;+\u0026thinsp;05\u0026thinsp;\u0026plusmn;\u0026thinsp;7.7E\u0026thinsp;+\u0026thinsp;05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.49\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e60 minutes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.72E\u0026thinsp;+\u0026thinsp;05\u0026thinsp;\u0026plusmn;\u0026thinsp;5.0E\u0026thinsp;+\u0026thinsp;05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.23\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3 hours\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.50E\u0026thinsp;+\u0026thinsp;05\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0E\u0026thinsp;+\u0026thinsp;05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.47\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6 hours\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.56E\u0026thinsp;+\u0026thinsp;05\u0026thinsp;\u0026plusmn;\u0026thinsp;5.8E\u0026thinsp;+\u0026thinsp;04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe rapid drop in plasma levels suggested that BPDA2 was being absorbed into tissues very quickly. To verify this point, we determined tissue concentrations of BPDA2 after administering 40 mg/kg by oral gavage into tumor-bearing mice. Based on data in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, we chose the 3-hour time point for tissue collection. BPDA2 was extracted with 80%/20% methanol/water from tumor, liver, kidney, heart, lung, and brain samples, and the concentration determined by LC-MS as described in the materials and methods. We found the highest concentration in the liver, which is expected given that orally administered drugs first reach the liver via the portal vein. Significant concentration of BPDA2 was recovered from the lungs, the heart, the kidneys, and the tumor tissue in decreasing order, while the brain tissues had the lowest amount (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Accumulation of quantifiable BPDA2 in the tumor tissue suggested potential anti-tumor efficacy.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eTissue concentration of BPDA2 expressed as nanomoles per gram tissue. The results are entered as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTissue\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAUC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBPDA2, nmol/g\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eLiver\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.76E\u0026thinsp;+\u0026thinsp;06\u0026thinsp;\u0026plusmn;\u0026thinsp;2.02E\u0026thinsp;+\u0026thinsp;05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e9.36\u0026thinsp;\u0026plusmn;\u0026thinsp;3.75\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eKidney\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.73E\u0026thinsp;+\u0026thinsp;05\u0026thinsp;\u0026plusmn;\u0026thinsp;1.72E\u0026thinsp;+\u0026thinsp;05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e2.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eLung\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.15E\u0026thinsp;+\u0026thinsp;05\u0026thinsp;\u0026plusmn;\u0026thinsp;2.54E\u0026thinsp;+\u0026thinsp;05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e2.40\u0026thinsp;\u0026plusmn;\u0026thinsp;1.13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eHeart\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.84E\u0026thinsp;+\u0026thinsp;05\u0026thinsp;\u0026plusmn;\u0026thinsp;4.47E\u0026thinsp;+\u0026thinsp;04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTumor\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.36E\u0026thinsp;+\u0026thinsp;05\u0026thinsp;\u0026plusmn;\u0026thinsp;1.67E\u0026thinsp;+\u0026thinsp;04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eBrain\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.05E\u0026thinsp;+\u0026thinsp;04\u0026thinsp;\u0026plusmn;\u0026thinsp;1.37E\u0026thinsp;+\u0026thinsp;04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eTargeting SHP2 with BPDA2 suppresses tumor growth and metastasis\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eBased on the MTD data (Supplementary Table\u0026nbsp;1), we chose the middle two doses, the 20 mg/kg and the 40 mg/kg dosages for testing efficacy. Since the time of spontaneous tumor development varies among individual mice, we resorted to syngeneic tumor implantation methods to synchronize tumor formation as described in the materials and methods. When tumor volumes reached approximately 100 mm\u003csup\u003e3\u003c/sup\u003e, mice were divided into three groups, designated as placebo (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10), 20 mg/kg BPDA2 (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10), and 40 mg/kg (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10) BPDA2 groups.\u003c/p\u003e\u003cp\u003eTo serve as reference points for before-treatment tumor sizes, ultrasound images were collected from three representative mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). As shown, the starting tumor volumes ranged between 98 mm\u003csup\u003e3\u003c/sup\u003e and 120 mm\u003csup\u003e3\u003c/sup\u003e. BPDA2 at 20 mg/kg or 40 mg/kg was formulated in 10% DMSO, 0.5% Methylcellulose, and 0.4% Tween-80 and administered by oral gavage every 72 hours for a total of 10 treatments. The placebo mice were given the formulation alone without BPDA2. At the end of the treatments, ultrasound images were collected from three representative mice in each group for comparing the start and the end-point tumor sizes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The average tumor volumes calculated from the ultrasound images were 1,321.7 mm\u003csup\u003e3\u003c/sup\u003e, 381.7 mm\u003csup\u003e3\u003c/sup\u003e, and 136.3 mm\u003csup\u003e3\u003c/sup\u003e for the placebo, 20 mg/kg, and 40 mg/kg groups, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). To complement the ultrasound images, tumor volumes of each mouse were measured with a caliper at the start and every 5 days till the end of the experiment. For simplicity, the start and the final tumor volumes were used for plotting the results. Tumor volume data from individual mice showed a linear growth in the placebo mice, a highly slowed growth in the 20 mg/kg group, and blockade of growth in the 40 mg/kg group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). When averages of tumor volumes were plotted, the placebo tumors reached\u0026thinsp;~\u0026thinsp;1,300 mm\u003csup\u003e3\u003c/sup\u003e, the 20 mg/kg reached\u0026thinsp;~\u0026thinsp;380 mm\u003csup\u003e3\u003c/sup\u003e, and the 40 mg/kg tumors did not grow that much (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). The \u003cem\u003ep\u003c/em\u003e value between the placebo and 20 mg/kg and between the placebo and 40 mg/kg was \u003cem\u003e\u0026lt;\u0026thinsp;0.001\u003c/em\u003e. These findings suggest that BPDA2 effectively suppressed tumor growth in the \u003cem\u003eErbB2\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003ep53\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice in a concentration-dependent manner. Body weight measurement showed no significant difference between the placebo and treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), suggesting that BPDA2 is highly tolerated.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eComparative analysis of placebo and BPDA2-treated tumors\u003c/h2\u003e\u003cp\u003eTo complement observations in live animals, tissue samples including tumors, lungs, and livers were harvested the placebo and BPDA2-treated mice and sections analyzed by H\u0026amp;E staining and microscopic imaging. Representative images from one placebo and one 40 mg/kg mouse are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA. The results showed locally invasive and highly vascularized tumors with reservoir-like blood pouches in the placebo and non-invasive tumors with unremarkable vascularization in the treatment group mouse. Similar analysis of H\u0026amp;E-stained lung and liver sections showed presence of metastatic lesions in the placebo and absence of metastatic lesions in the treatment group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). These findings suggest that targeting SHP2 blocks tumor vascularization, which in turn blocks tumor growth, local invasiveness, and distant metastasis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe also analyzed protein extracts of tumors from two mice each in the three experimental groups for \u003cem\u003eErbB2\u003c/em\u003e expression and the state of downstream signaling. The results showed downregulation of the \u003cem\u003eErbB2\u003c/em\u003e protein as well as pAkt and pERK1/2 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) by BPDA2 treatment in a concentration-dependent manner. These findings suggest that targeting SHP2 with BPDA2 blocks oncogene expression which in turn blocks mitogenic and cell survival signaling.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePrevious genetic studies by us and others showed that SHP2 plays fundamental roles in promoting cell transformation and tumorigenesis in HER2\u0026thinsp;+\u0026thinsp;and triple-negative BC (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). These reports led us to investigate SHP2 as a potential drug target in a new HER2\u0026thinsp;+\u0026thinsp;BC tumor model, which exhibits the traits of an advanced-stage disease in patients. This tumor model was generated by crossing the \u003cem\u003eMMTV-ErbB2/Neu\u003c/em\u003e mice with the \u003cem\u003ep53\u003c/em\u003e-floxed and the \u003cem\u003eMMTV-Cre\u003c/em\u003e mice. The objective was to add a conditional \u003cem\u003ep53\u003c/em\u003e knockout in the mammary gland to better represent an advanced stage HER2\u0026thinsp;+\u0026thinsp;BC in patients, in which \u003cem\u003ep53\u003c/em\u003e functional loss is common (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). The \u003cem\u003eErbB2\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003ep53\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e tumor model exhibited reduced tumor latency and increased metastatic potential when compared to the original \u003cem\u003eMMTV-ErbB2/Neu\u003c/em\u003e strain (5\u0026ndash;6 months versus 7\u0026ndash;10 months). These findings suggested that we have effectively generated an advanced stage HER2\u0026thinsp;+\u0026thinsp;tumor model for use in the testing of novel targeted therapies.\u003c/p\u003e\u003cp\u003eWe chose to use our previously reported active-site SHP2 inhibitor referred to as BPDA2 (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e) for the efficacy studies since most SHP2 proteins in HER2\u0026thinsp;+\u0026thinsp;BC are likely to exist in activated conformation due to hyperactive tyrosine kinase signaling that mediate SH2-pTyr interactions. Because BPDA2 was not characterized for \u003cem\u003ein vivo\u003c/em\u003e use, it was necessary to evaluate its pharmacologic properties, including metabolic stability, LogD, and \u003cem\u003ein vivo\u003c/em\u003e biodistribution before moving into a preclinical efficacy study. We have found that BPDA2 is stable for at least 6 hours in mouse liver S9 fractions (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), indicating that it is not significantly degraded by phase 1 metabolic processes. We also determined a LogP value, a commonly used parameter to assess the lipophilicity of drug candidates and found that it has moderate lipophilicity at physiological pH 7.4 with a LogD\u003csub\u003e7.4\u003c/sub\u003e value of 0.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), which is reasonably close to the ideal range of 1\u0026ndash;3.\u003c/p\u003e\u003cp\u003eAnalyzing plasma levels of BPDA2 after oral administration showed a rapid absorption into the circulation (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and quickly clearing into tissues (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The highest concentration of BPDA2 was present in the liver, which is expected given that all absorbed materials from the gut must pass through this organ. The presence of a significant concentration of BPDA2 in the tumor tissue was a key finding, which suggested a potential anti-tumor activity and a possible adjustment in dosage to enhance efficacy in the future. The brain had the lowest concentration of BPDA2 (~\u0026thinsp;0.19 \u0026micro;M), suggesting that BPDA2 needs to be modified if targeting SHP2 in brain cancers is desired in future studies.\u003c/p\u003e\u003cp\u003eWe have also assessed toxicity profiles in mice treated with a range of BPDA2 concentrations (10 mg/kg to 200 mg/kg, Supplementary Table\u0026nbsp;1) and found that BPDA2 is well tolerated, as no acute toxicity symptoms were observed at any concentration used. The histopathology analysis of the lung and liver sections showed no signs of tissue damage even at 200 mg/kg (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This indicates that BPDA2 is tolerable in mice, at least up to 200 mg/kg. These results inspired us to move forward with testing the efficacy in the \u003cem\u003eErbB2\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003ep53\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e tumor model.\u003c/p\u003e\u003cp\u003eMice bearing mammary tumors were divided into three groups designated as placebo, 20 mg/kg, and 40 mg/kg. The use of two different doses of BPDA2 was necessary since no prior information on the anti-tumor effect was available. These treatments showed suppression of tumor growth at the 20 mg/kg dosage and blockade of tumor growth at the 40 mg/kg dosage (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and D), which was confirmed by ultrasound imaging at the start and at the end of the treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Importantly, BPDA2 treatment did not lead to significant differences in body weight as evidenced by data in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE. Comparative H\u0026amp;E analysis of placebo and BPDA2 treated tumors showed abolition of the characteristic tumor vascularization in HER2\u0026thinsp;+\u0026thinsp;BC, which is likely responsible for blocking tumor growth and metastasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Overall, these findings clearly suggest that inhibition of SHP2 with BPDA2 can be effective in advanced stage HER2\u0026thinsp;+\u0026thinsp;BC. We have not observed complete disappearance of the tumors in this study, but since the MTD studies showed no toxicity at doses up to 200 mg/kg, dose escalation or changes in dosing schedules in future studies may provide further insights on the potential of BPDA2 for targeting SHP2 in advanced HER2\u0026thinsp;+\u0026thinsp;BC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interests statement\u003c/strong\u003e: The authors declare no potential conflicts of interest. This work was supported by a grant from the National Institute of Health/National Cancer Institute, USA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by a grant (CA213996) from the National Cancer Institute (NCI), a component of the National Institute of Health (NIH) to YMA. The final aspects of the manuscript were supported by bridge funding from the WVU cancer Institute. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJM is responsible for determining stability of BPDA2 in S9 fractions, LogD value of BPDA2, LC/MS analysis of BPDA2 in blood and tissue samples, mice genotyping, and ultrasound imaging. DL is responsible for chemical synthesis of BPDA2. PL was responsible for formulation of BPDA2 for oral administration and for providing advice in the efficacy studies. YMA is responsible for leading the generation of the \u003cem\u003eErbB2\u003csup\u003e+\u003c/sup\u003e;p53\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e tumor model, for \u003cem\u003ein vivo\u003c/em\u003e efficacy studies, signaling work, and for overseeing the overall project.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCompeting interests statement\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no potential conflicts of interest. This work was supported by a grant from the National Institute of Health/National Cancer Institute, USA.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere is no restriction on the use of the methods and data described in this report.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDent S, Oyan B, Honig A, Mano M, Howell S. 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Crit Rev Oncol Hemat. 2019;139:53-66.\u003c/li\u003e\n\u003cli\u003eAgazie YM, Hayman MJ. Molecular mechanism for a role of SHP2 in epidermal growth factor receptor signaling. Molecular and cellular biology. 2003;23(21):7875-86.\u003c/li\u003e\n\u003cli\u003eFeng GS, Hui CC, Pawson T. SH2-containing phosphotyrosine phosphatase as a target of protein-tyrosine kinases. Science. 1993;259(5101):1607-11.\u003c/li\u003e\n\u003cli\u003eFeng GS, Shen R, Heng HH, Tsui LC, Kazlauskas A, Pawson T. Receptor-binding, tyrosine phosphorylation and chromosome localization of the mouse SH2-containing phosphotyrosine phosphatase Syp. Oncogene. 1994;9(6):1545-50.\u003c/li\u003e\n\u003cli\u003eLi J, Reed SA, Johnson SE. Hepatocyte growth factor (HGF) signals through SHP2 to regulate primary mouse myoblast proliferation. Experimental cell research. 2009;315(13):2284-92.\u003c/li\u003e\n\u003cli\u003eZhou XD, Agazie YM. Molecular Mechanism for SHP2 in Promoting HER2-induced Signaling and Transformation. 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BMC cancer. 2015;15:109.\u003c/li\u003e\n\u003cli\u003eAceto N, Sausgruber N, Brinkhaus H, Gaidatzis D, Martiny-Baron G, Mazzarol G, et al. Tyrosine phosphatase SHP2 promotes breast cancer progression and maintains tumor-initiating cells via activation of key transcription factors and a positive feedback signaling loop. Nature medicine. 2012;18(4):529-37.\u003c/li\u003e\n\u003cli\u003eMatalkah F, Martin E, Zhao H, Agazie YM. SHP2 acts both upstream and downstream of multiple receptor tyrosine kinases to promote basal-like and triple-negative breast cancer. Breast cancer research : BCR. 2016;18(1):2.\u003c/li\u003e\n\u003cli\u003eDardaei L, Wang HQ, Singh M, Fordjour P, Shaw KX, Yoda S, et al. SHP2 inhibition restores sensitivity in ALK-rearranged non-small-cell lung cancer resistant to ALK inhibitors. Nature medicine. 2018;24(4):512-7.\u003c/li\u003e\n\u003cli\u003eJiang L, Xu W, Chen Y, Zhang Y. SHP2 inhibitor specifically suppresses the stemness of KRAS-mutant non-small cell lung cancer cells. Artif Cells Nanomed Biotechnol. 2019;47(1):3231-8.\u003c/li\u003e\n\u003cli\u003eMelhem-Bertrandt A, Bojadzieva J, Ready KJ, Obeid E, Liu DD, Gutierrez-Barrera AM, et al. Early onset HER2-positive breast cancer is associated with germline TP53 mutations (vol 118, pg 908, 2012). Cancer. 2012;118(9):2561-.\u003c/li\u003e\n\u003cli\u003eLi BL, Rosen JM, McMenaminBalano J, Muller WJ, Perkins AS. neu/ERBB2 cooperates with p53-172H during mammary tumorigenesis in transgenic mice. Molecular and cellular biology. 1997;17(6):3155-63.\u003c/li\u003e\n\u003cli\u003eLade DM, Nicoletti R, Mersch J, Agazie YM. Design and synthesis of improved active-site SHP2 inhibitors with anti-breast cancer cell effects. European journal of medicinal chemistry. 2023;247:115017.\u003c/li\u003e\n\u003cli\u003eLi G, Robinson GW, Lesche R, Martinez-Diaz H, Jiang Z, Rozengurt N, et al. 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SHP2 is up-regulated in breast cancer cells and in infiltrating ductal carcinoma of the breast, implying its involvement in breast oncogenesis. Histopathology. 2008;53(4):389-402.\u003c/li\u003e\n\u003cli\u003eLade DM, Nicoletti R, Mersch J, Agazie YM. Design and synthesis of improved active-site SHP2 inhibitors with anti-breast cancer cell effects. European journal of medicinal chemistry. 2023;247.\u003c/li\u003e\n\u003cli\u003eSingh S, Dwivedi R, Chaturvedi V. Influence of Vehicles Used for Oral Dosing of Test Molecules on the Progression of Infection in Mice (vol 56, pg 6026, 2012). Antimicrobial agents and chemotherapy. 2014;58(6):3579-.\u003c/li\u003e\n\u003cli\u003eMuenst S, Obermann EC, Gao F, Oertli D, Viehl CT, Weber WP, et al. Src homology phosphotyrosyl phosphatase-2 expression is an independent negative prognostic factor in human breast cancer. Histopathology. 2013;63(1):74-82.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cancer-gene-therapy","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cgt","sideBox":"Learn more about [Cancer Gene Therapy](http://www.nature.com/cgt/)","snPcode":"41417","submissionUrl":"https://mts-cgt.nature.com/cgi-bin/main.plex","title":"Cancer Gene Therapy","twitterHandle":"@cgtnature","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8196938/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8196938/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAdvanced stage HER2-positive (HER2+) breast cancer (BC) is very challenging for treatment. These premises justify the need to develop novel targeted therapies that can be used to complement existing anti-HER2 therapies. Accordingly, we investigated the potential of the Src homology phosphotyrosyl phosphatase 2 (SHP2), the master regulator of RTK signaling, for use in advanced stage HER2\u0026thinsp;+\u0026thinsp;BC. To test this possibility, we first generated a more-penetrating tumor model referred to as \u003cem\u003eErbB2+;p53\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e. This model exhibited tumor properties reminiscent of advanced stage HER2\u0026thinsp;+\u0026thinsp;BC. To target SHP2, we used our previously reported active-site SHP2 inhibitor referred to as BPDA2. The results showed suppression of tumor growth in a 20 mg/kg group and complete blockade in a 40 mg/kg group when compared to the placebo. Histopathology analysis showed a locally invasive and highly vascularized tumor with metastasis to the lungs and the liver in the placebo and a non-invasive tumor with undetectable vascularization in the treated mice. Body weight measurement showed no major changes between the placebo and the treated mice, suggesting that BPDA2 is a very well tolerated SHP2 inhibitor. Biochemical analysis of tumor protein extracts showed downregulation of HER2 expression and mitogenic and cell survival signaling. Overall, the results in this study demonstrate that targeting SHP2 with the active-site inhibitor blocks tumorigenesis and metastasis in HER2-positive BC.\u003c/p\u003e","manuscriptTitle":"Development of a more penetrating HER2-positive Breast Cancer Tumor Model and Testing Efficacy of SHP2 Targeting with an Active-Site Inhibitor","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-05 13:28:42","doi":"10.21203/rs.3.rs-8196938/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-gene-therapy","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cgt","sideBox":"Learn more about [Cancer Gene Therapy](http://www.nature.com/cgt/)","snPcode":"41417","submissionUrl":"https://mts-cgt.nature.com/cgi-bin/main.plex","title":"Cancer Gene Therapy","twitterHandle":"@cgtnature","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"25622148-3e1e-48ba-aff3-5081f72f96f8","owner":[],"postedDate":"December 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":58715783,"name":"Biological sciences/Drug discovery/Target validation"},{"id":58715784,"name":"Health sciences/Biomarkers"}],"tags":[],"updatedAt":"2025-12-05T13:28:42+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-05 13:28:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8196938","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8196938","identity":"rs-8196938","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}