Vertical pathway inhibition of receptor tyrosine kinases and BAD with synergistic efficacy in triple negative breast cancer

Aberrant activation of the PI3K/AKT signaling axis along with the sustained phosphorylation of downstream BAD is associated with a poor outcome of TNBC. Herein, the phosphorylated to non-phosphorylated ratio of BAD, an effector of PI3K/AKT promoting cell survival, was observed to be correlated with worse clinicopathologic indicators of outcome, including higher grade, higher proliferative index and lymph node metastasis. The structural optimization of a previously reported inhibitor of BAD-Ser99 phosphorylation was therefore achieved to generate a small molecule inhibiting the phosphorylation of BAD at Ser99 with enhanced potency and improved oral bioavailability. The molecule 2-((4-(2,3-dichlorophenyl)piperazin-1-yl)(pyridin-3-yl)methyl) phenol (NCK) displayed no toxicity at supra-therapeutic doses and was therefore assessed for utility in TNBC. NCK promoted apoptosis and G0/G1 cell cycle arrest of TNBC cell lines in vitro, concordant with gene expression analyses, and reduced in vivo xenograft growth and metastatic burden, demonstrating efficacy as a single agent. Additionally, combinatorial oncology compound library screening demonstrated that NCK synergized with tyrosine kinase inhibitors (TKIs), specifically OSI-930 or Crizotinib in reducing cell viability and promoting apoptosis of TNBC cells. The synergistic effects of NCK and TKIs were also observed in vivo with complete regression of a percentage of TNBC cell line derived xenografts and prevention of metastatic spread. In patient-derived TNBC xenograft models, NCK prolonged survival times of host animals, and in combination with TKIs generated superior survival outcomes to single agent treatment. Hence, this study provides proof of concept to further develop rational and mechanistic based therapeutic strategies to ameliorate the outcome of TNBC.


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LC chromatogram of 2-((4-(2,3-dichlorophenyl)piperazin-1-yl)(pyridin-3-yl)methyl) phenol (A) IC50 of NCK in MDA-MB-231 and BT549 cells measured by using AlamarBlue assay.Data represent means ± SD. (B) Cartoon representation of the compounds NPB (red) and NCK (black) (depicted in ball & stick model) docked to the BAD protein, occupying the same groove with the BAD Serine 99 residue (yellow) nearby.(C) 2D structure representation of compound NPB interacting with BAD protein residues.(D) Cartoon representation of docked compound and molecular electrostatic potential surfaces of NCK.The color code of the compound lies in the range of -0.121e0 to +0.121e0.Red and blue color in the MEP structure point to more electron rich and electron poor regions respectively.(E) The calculated FMOs and Global chemical reactivity descriptors.Computed EHOMO, ELUMO, frontier molecular orbital's (FMO) energy gap (∆ELUMO-HOMO), ionization potential (I), electron affinity (A), chemical hardness (ɳ), chemical softness (S), Chemical potential (μ), electronegativity (χ) and electrophilicity index (ψ) in eV of NCK.Supplementary Figure 6: (A) Sensorgrams obtained by SPR analysis of NPB with the BAD protein.The BAD protein was immobilized on the surface of a CM5 sensor chip.A solution of NPB at variable concentrations (1.25-160 μM) was injected to generate the binding responses (RU) recorded as a function of time (s).The results were analyzed using BIA evaluation 4.1.(B) Densitometric analysis of western blots in Figure 2E.MDA-MB-231 and BT549 cells were treated with 0-10 μM NCK or NPB (n=3).Densitometric analysis of protein blots was determined using ImageJ software (https://imagej.nih.gov/ij/).Statistical changes were assessed by using ANOVA.*P < 0.05, **P < 0.01, and ***P < 0.001.(C) Western blot analysis was used to assess the levels of pBADSer99, pBADSer75, pBADSer118 and BAD expression in TNBC cell lines.β-ACTIN was used as input control for cell lysate.The sizes of detected protein bands in kDa are shown on the left.(D) Dose-dependent effect of NPB and NCK in 2D and 3D culture on TNBC cell viability (in HCC1937, MDA-MB-468, SUM149PT, MDA-MB-436, SUM159PT and Hs578T cell lines) measured by using total cell number assay and AlamarBlue assay respectively (n=3).

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(A) Plasma concentration of NCK after intravenous (IV, 1 mg/kg) (red) or oral (PO, 10 mg/kg) (blue) administration in SD rats.Results represent the mean ± SD of three animals.(B) Pharmacokinetic parameters of NCK in plasma after IV injection (1 mg/kg) or oral administration (10 mg/kg).AUCinf: Area Under the Plasma Concentration-Time Curve from t=0 to infinity; AUClast: Area Under the Plasma Concentration-Time Curve from t=0 to last measurable positive concentration; AUC_%Extrap_obs: Percent Extrapolated AUC from tlast to infinity; C0: Initial concentration in plasma; Cmax: Peak concentration in plasma; Cl_obs: Plasma systemic clearance; F: Absolute oral bioavailability; MRTInf_obs: Mean residence time from t=0 to infinity ;T1/2: Elimination half-life; Tmax: Time of peak concentration in plasma; Vss_obs: Volume of Distribution at Steady-state Supplementary Figure 8: (A) GSEA analyses of gene sets for apoptosis.NES, normalized enrichment score.FDR, false discovery rate.(B) GO biological process analysis of the DEGs after 5 μM NCK treatment.Volcano map of differential expressed genes (DEGs) after NCK treatment.Advanced bubble chart shows enrichment of DEGs in GO biological processes after treatment with 5 μM NCK.(C) GO biological process analysis of the DEGs after 5 μM NPB treatment.Volcano map of differential expressed genes (DEGs) after NPB treatment.Advanced bubble chart shows enrichment of DEGs in GO biological processes after treatment with 5 μM NPB.(D) Bar charts depicting cell cycle related DEGs upregulated or downregulated by 5 μM NCK treatment.Supplementary Figure 9: (A) Representative flow cytometry plots using PI staining for DNA of MDA-MB-231 cells and BT549 cells measured after treatment with 5 μM NCK or NPB using flow cytometry analysis at 72 hours as described in materials and methods.(B) Representative flow cytometry plots of Annexin-V and propidium iodide (PI) stained apoptotic cell death of MDA-MB-231 cells and BT549 cells measured after treatment with 5 μM NCK or NPB using flow cytometry analysis.Annexin V-FITC staining is indicated on the x axis, and PI staining is indicated on the y axis.The lower left quadrants represent live cells, the lower right quadrants represent early apoptotic cells, the upper left quadrants represent necrotic cells, and the upper right quadrants display late apoptotic cells.Acquisition of Annexin V and PI data are presented as a percentage (%) in each quadrant.Early apoptotic cells are referred to as Annexin-V positive and late apoptotic cells are referred to as Annexin-V and PI double positive.

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Supplementary Figure 11: (A) Target and pathway information of 247 anti-cancer compounds of Cambridge anti-cancer compound library.(B) Schematic of anti-cancer compound library screen.247 anti-cancer agents (compound X) were combined with NCK to treat MDA-MB-231.Cell viability was determined 72-hour post-treatment with AlamarBlue viability assay.

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during the acute toxicity study.Comparison of relative lung, liver, spleen, heart, kidney weights (%, organ body index) and colon length at day 15.(E) Photographs of organs from the vehicle and NCK-treated mice during the MTD study.(F) Effects of NCK on serum hematological and biochemical parameters during the acute toxicity study.Abbreviations: Data represent means ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.Data represent means ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.Supplementary Figure 10: (A) Individual daily changes in body weight of female ICR mice administrated with NCK during the acute toxicity study.Mean body weight, food consumption, and water intake following by 14-days consecutive intraperitoneal injection at 20 and 50 mg/kg of NCK in female ICR mice were recorded (n = 8).Data represent means ± SD. (B) Individual daily changes in food consumption of female ICR mice administrated with NCK during the acute toxicity study.Mean body weight, food consumption, and water intake following by 14-Mean body weight, food consumption, and water intake following by 14-days consecutive intraperitoneal injection at 20 and 50 mg/kg of NCK in female ICR mice were recorded (n = 8).Data represent means ± SD. (D) Changes in the organs of female ICR mice administrated with NCK creatine kinase; CREA, creatinine; GLU, glucose; GSP, glycated serum protein; LDH, lactate dehydrogenase; UREA, urea.
(A) Targets of the synergistic TKIs, IC50 of the inhibitors on corresponding targets and their clinical status (SelleckChem).(B) Information of OSI-930 and Crizotinib, and associated side effects and toxicities in FDA/clinical trials.