Journal: Molecular Oncology

Article Title: Dual targeting of RET and SRC synergizes in RET fusion‐positive cancer cells

doi: 10.1002/1878-0261.70155

Figure Lengend Snippet: Signaling analysis of pralsetinib and dasatinib combination effects in RET + non‐small cell lung cancer (NSCLC) cells. (A) (Left) Western blot analysis of indicated signaling proteins in CUTO32 cells after treatment with pralsetinib (1 μ m ), dasatinib (0.1 μ m ) or their combination for 3 h. (Right) Quantification of pRET ( n = 4), pSRC ( n = 3), pPAK1/2 ( n = 4), pERK1/2 ( n = 3), pAKT ( n = 3), and pS6 ( n = 3) blots, normalized by loading controls (Actin or vinculin). (B) (Left) Western blot analysis of indicated signaling proteins after treatment with pralsetinib (1 μ m ), dasatinib (0.1 μ m ) or their combination for 3 h in CUTO32 cells expressing SRC wild‐type (WT) or SRC T341I gatekeeper. (Right) Quantification of pRET ( n = 3), pSRC ( n = 3), pPAK1/2 ( n = 4), pERK1/2 ( n = 3), pAKT ( n = 3), and pS6 ( n = 3) blots, normalized by loading controls (Actin or vinculin). Statistical significance was determined by one‐way ANOVA with Tukey's multiple comparisons; n.s., not significant. Results are shown as mean ± SD.

Article Snippet: Primary antibodies against PARP1 (9542), cleaved Caspase‐3 (9661), p27 (2552), p21 (2947), p‐RET (Y905, 3221), RET (3223) p‐SRC (Y419, 2101), SRC (2108), p‐PAK1/2 (S144/S141, 2606), PAK1/2/3 (2604), p‐ERK1/2 (Y204/Y187, 4370), p‐AKT (S473, 9271), AKT (9272), p‐S6 (S235/S236, 4858), S6 (2217), and Vinculin (13901) were acquired from Cell Signaling Technology.

Techniques: Western Blot, Expressing

Journal: Molecular Oncology

Article Title: Dual targeting of RET and SRC synergizes in RET fusion‐positive cancer cells

doi: 10.1002/1878-0261.70155

Figure Lengend Snippet: Combination effects of the next‐generation SRC tyrosine kinase inhibitor (TKI) eCF506 with pralsetinib in RET + non‐small cell lung cancer (NSCLC) cells. (A) (Left) Western blotting of indicated signaling proteins in CUTO32 cells after treatment with pralsetinib (1 μ m ), eCF506 (0.1 μ m ) or their combination for 3 h. (Right) Quantification of p‐RET ( n = 3), p‐SRC ( n = 3), pPAK1/2 ( n = 3), pERK1/2 ( n = 3), pAKT ( n = 3), and p‐S6 ( n = 3) blots, normalized by loading controls (Actin or vinculin). (B) (Top) Cell viability of CUTO32 ( n = 3) and LC‐2/Ad ( n = 3) cells upon treatment with RET TKIs alone and in combination with eCF506 (0.1 μ m ) for 3 days. (Bottom) Cell viability and Bliss synergy analysis of RET + NSCLC cells at single concentrations of RET TKIs (0.625 μ m , see gray arrows above) and eCF506 (0.1 μ m ). (C) Clonogenic survival, quantification and Bliss synergy analysis of RET + NSCLC cells ( n = 4 for CUTO32 and n = 3 for LC‐2/Ad) after treatment with pralsetinib (1 μ m ), eCF506 (0.1 μ m ) or their combination for 7 days. (D) Western blotting and quantification of apoptosis markers upon treatment of CUTO32 cells with pralsetinib (1 μ m ), eCF506 (0.1 μ m ) and their combination for 3 days ( n = 3). Statistical significance was determined by one‐way ANOVA with Tukey's multiple comparisons; n.s., not significant. Results are shown as mean ± SD.

Article Snippet: Primary antibodies against PARP1 (9542), cleaved Caspase‐3 (9661), p27 (2552), p21 (2947), p‐RET (Y905, 3221), RET (3223) p‐SRC (Y419, 2101), SRC (2108), p‐PAK1/2 (S144/S141, 2606), PAK1/2/3 (2604), p‐ERK1/2 (Y204/Y187, 4370), p‐AKT (S473, 9271), AKT (9272), p‐S6 (S235/S236, 4858), S6 (2217), and Vinculin (13901) were acquired from Cell Signaling Technology.

Techniques: Western Blot

Journal: bioRxiv

Article Title: RET Receptor Tyrosine Kinase Promotes Breast Cancer Metastasis to the Brain and RET Inhibitors Pralsetinib and Selpercatinib Suppress Breast Cancer Brain Metastases

doi: 10.1101/2025.10.07.680986

Figure Lengend Snippet: (A) RET pathway activation scores between breast cancer patients with no metastases vs. brain metastases using GEO breast cancer datasets (GSE2034, GSE2603, GSE5327, GSE12276, GSE14020). An unpaired, two-tailed t-test was used in panel (A). (B) Pearson correlation analysis using GEO correlating RET activation signatures to a breast-to-brain metastasis signature. (C) Using the Kaplan-Meier analysis, log-rank analyses, GEO datasets, and the RET activation signatures, 672 breast cancer patients were stratified into two groups, either a high or low RET pathway activation for overall metastasis-free survival. Median of overall metastasis-free survival times are indicated in months (N=672). (D) High versus low RET activation scores were analyzed for brain metastasis-free survival (N = 318). (E) IHC staining of p-RET (Y905) expression levels in 15 matched pairs of primary breast tumors and their corresponding BCBM samples either luminal, HER2-enriched, or TNBC subtypes (20X images presented). (F) H-score quantification of p-RET (Y905) positivity, presented as a ladder plot (N=15). A paired, two-tailed t-test was used.

Article Snippet: The following antibodies were used: p-RET (Y905) (CST 3221s, 1:500); p-AKT (5473) (CST 9271s, 1:1000); p-ERK (T202/Y204) (CST 91015, 1:1000); p-STAT3 (CST 9131L, 1:1000); Vinculin (CST 13901s, 1:5000); RET (CST 3220s, 1:500); RET (CST 3223s, 1:1000); AKT (CST 9272s, 1:1000); ERK (CST 4695s, 1:1000); STAT3 (CST 9139, 1:1000); β-actin (CST 3700s, 1:5000); α-tubulin (Sigma-Aldrich T6074, 1:5000–10,000).

Techniques: Activation Assay, Two Tailed Test, Immunohistochemistry, Expressing

Journal: bioRxiv

Article Title: RET Receptor Tyrosine Kinase Promotes Breast Cancer Metastasis to the Brain and RET Inhibitors Pralsetinib and Selpercatinib Suppress Breast Cancer Brain Metastases

doi: 10.1101/2025.10.07.680986

Figure Lengend Snippet: (A) Schematic of intracranial mouse model. 6–7-week-old athymic nude mice were intracranially inoculated with luciferase-expressing MDA-MB-231-RET cells (N=12) or MDA-MB-231-Ctrl cells (N=11). Mice were imaged twice weekly to monitor tumor formation and growth. At the study endpoint (Day 28), mice were euthanized, and organs were collected for ex vivo analyses. (B) Bi-weekly average brain tumor burden per group throughout the study, assessed by BLI (top). Representative BLI images from Day 28 (bottom). (C) Quantification of ex vivo BLI of brains (top). Representative BLI images of ex vivo brain (bottom). (D) IHC staining of p-RET (Y905) and Ki-67 expression levels in brain tumors (scale bar 100µm). (E) H-score quantification of p-RET (Y905) positivity and Ki-67 positivity. (F) Tumor retention rate following inoculation. (H) Average weight of mice in each group. Two-way ANOVAs were used to calculate p-values with a mixed-effects analysis for panels (B and G). An unpaired, two-tailed t-test was used in panels (C and E).

Article Snippet: The following antibodies were used: p-RET (Y905) (CST 3221s, 1:500); p-AKT (5473) (CST 9271s, 1:1000); p-ERK (T202/Y204) (CST 91015, 1:1000); p-STAT3 (CST 9131L, 1:1000); Vinculin (CST 13901s, 1:5000); RET (CST 3220s, 1:500); RET (CST 3223s, 1:1000); AKT (CST 9272s, 1:1000); ERK (CST 4695s, 1:1000); STAT3 (CST 9139, 1:1000); β-actin (CST 3700s, 1:5000); α-tubulin (Sigma-Aldrich T6074, 1:5000–10,000).

Techniques: Luciferase, Expressing, Ex Vivo, Immunohistochemistry, Two Tailed Test

Journal: bioRxiv

Article Title: RET Receptor Tyrosine Kinase Promotes Breast Cancer Metastasis to the Brain and RET Inhibitors Pralsetinib and Selpercatinib Suppress Breast Cancer Brain Metastases

doi: 10.1101/2025.10.07.680986

Figure Lengend Snippet: RET inhibitors reduce cell viability, suppress downstream RET signaling, and induce apoptosis in brain-tropic breast cancer cells in vitro . The effect of treatment with (A) pralsetinib or (B) selpercatinib on cell viability of brain-tropic breast cancer cell lines (N=5). (C) Western blot analysis to determine protein expression of p-RET (Y905), p-STAT3 (Y705), total RET, total STAT3, p-ERK (T202/Y204), and total ERK ½ following treatment with prasetinib (left) or selpercatinib (right). Vinculin serves as high molecular weight loading control, and β-actin serves as a low molecular weight loading control. Annexin V/PI flow cytometry co-staining of (D) SKBRM cells, (E) CN34-BrM cells, and (F) MDA-MB-231-BrM cells treated with pralsetinib or selpercatinib (quantification on the left and representative images on the right) (N=5). Western blot analysis of cleaved PARP and full-length PARP in brain-tropic breast cancer cells following treatment with (G) pralsetinib, or (H) selpercatinib. An unpaired, two-tailed t-test between vehicle and treatment was used in panels (A,B,D,E and F).

Article Snippet: The following antibodies were used: p-RET (Y905) (CST 3221s, 1:500); p-AKT (5473) (CST 9271s, 1:1000); p-ERK (T202/Y204) (CST 91015, 1:1000); p-STAT3 (CST 9131L, 1:1000); Vinculin (CST 13901s, 1:5000); RET (CST 3220s, 1:500); RET (CST 3223s, 1:1000); AKT (CST 9272s, 1:1000); ERK (CST 4695s, 1:1000); STAT3 (CST 9139, 1:1000); β-actin (CST 3700s, 1:5000); α-tubulin (Sigma-Aldrich T6074, 1:5000–10,000).

Techniques: In Vitro, Western Blot, Expressing, High Molecular Weight, Control, Molecular Weight, Flow Cytometry, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: RET Receptor Tyrosine Kinase Promotes Breast Cancer Metastasis to the Brain and RET Inhibitors Pralsetinib and Selpercatinib Suppress Breast Cancer Brain Metastases

doi: 10.1101/2025.10.07.680986

Figure Lengend Snippet: Systemic administration of pralsetinib selectively reduces established brain metastatic tumor growth and proliferation in vivo . (A) Schematic of intracranial inoculation mouse model. 6–7-week-old athymic nude mice (N=10/group) were inoculated with luciferase-expressing MDA-MB-231-BrM cells. Mice received their first treatment 2 days after inoculation and were treated 5 times a week and imaged twice a week to monitor tumor growth. At the study endpoint (Day 24), mice were euthanized, and brains were collected for ex vivo analyses. (B) Weekly average metastatic burden per group throughout the study, assessed by BLI (bottom), and representative BLI images from Day 25(top), vehicle N = 9 and Pralsetinib N = 10. (C) Representative BLI images of ex vivo brain (top). Quantification of ex vivo BLI of brains (bottom). (D) Circulating ALT levels between treatment groups. (E) The average weight of mice throughout the study. (F) IHC staining of p-RET (Y905) and Ki-67 expression levels in brain tumors (left) (scale bar = 100µm), and H-score quantification of p-RET (Y905) positivity and Ki-67 positivity (right). (G) Fluorescent TUNEL assay on mouse brain tumors. Images of representative fields show nuclei from control and treated tumors (left) (scale bar = 100µm). Two-way ANOVAs were used to calculate p-values with a mixed-effects analysis for panels (B and E). An unpaired, two-tailed t-test was used in panels (C, D, F and G).

Article Snippet: The following antibodies were used: p-RET (Y905) (CST 3221s, 1:500); p-AKT (5473) (CST 9271s, 1:1000); p-ERK (T202/Y204) (CST 91015, 1:1000); p-STAT3 (CST 9131L, 1:1000); Vinculin (CST 13901s, 1:5000); RET (CST 3220s, 1:500); RET (CST 3223s, 1:1000); AKT (CST 9272s, 1:1000); ERK (CST 4695s, 1:1000); STAT3 (CST 9139, 1:1000); β-actin (CST 3700s, 1:5000); α-tubulin (Sigma-Aldrich T6074, 1:5000–10,000).

Techniques: In Vivo, Luciferase, Expressing, Ex Vivo, Immunohistochemistry, TUNEL Assay, Control, Two Tailed Test