Journal: bioRxiv

Article Title: Sustained interferon exposure creates a hyper-metastatic subset of melanoma cells

doi: 10.64898/2026.04.01.715921

Figure Lengend Snippet: Higher interferon-regulated gene expression in metastasizing melanoma cells and increased formation of metastatic tumors after interferon treatment. a, We performed RNA sequencing on patient-derived xenograft cells (M405 and M481) isolated by flow cytometry from subcutaneous tumors, the blood, and metastatic tumors in NSG mice. After eliminating cell cycle-related genes, the 10 most significantly enriched gene sets in melanoma cells from the blood included ‘viral genome replication’ (red), which contains interferon-regulated genes. b, Interferon-regulated genes were more highly expressed by circulating (CMC) and metastatic as compared to primary subcutaneous (SQ) melanoma cells by gene set variation analysis (“Response to type I interferon” gene set). c-e, By qRT-PCR, transcript levels for the interferon-regulated genes ISG15, IFI27 and IFITM3 were higher in melanoma cells isolated from the blood as compared to subcutaneous or metastatic tumors of xenografted mice (two to three independent experiments per melanoma with a total of 3-5 mice per melanoma). f, Luciferase-expressing human melanoma cells were cultured overnight in 10 ng/mL hIFNa2 or vehicle and then intravenously injected into NSG mice. Metastatic disease burden was assessed five to nine weeks later by bioluminescence imaging of visceral organs and normalized to controls (two experiments per melanoma with a total of nine to ten mice per melanoma). g-j, Luciferase-expressing YUMM1.7, YUMM3.3, or YUMM5.2 mouse melanoma cells were cultured overnight in 10 ng/mL mIFN51, mIFNa2, mIFNy , or vehicle control and then injected subcutaneously (g, i) or intravenously (h, j) into NSG (g-h) or C57BL mice (i-j). We observed no differences in subcutaneous tumor growth (representative data are shown for YUMM1.7 cells; g, i). Interferon-treated mice gave rise to significantly higher tumor burden after intravenous injection into C57BL but not NSG mice (two independent experiments with a total of 9-20 mice per melanoma). k-n, IFNAR mutant or control mouse melanoma cells were injected subcutaneously (k, m) or intravenously (l, n) into NSG (k, l) or C57BL mice (m, n). IFNAR mutant and control cells did not differ in the size of the subcutaneous tumors they formed in NSG (k) or C57BL (m) mice (one experiment using YUMM1.7 cells is shown for each mouse strain, representative of 2 to 3 independent experiments for each of 3 melanomas). IFNAR mutant cells generally gave rise to fewer tumors in visceral organs than control cells after intravenous injection in NSG (l) and C57BL (n) mice, though the difference was much greater in C57BL mice. For each melanoma, two control clones and three independently-targeted IFNAR1 mutant clones were studied in two to four independent experiments per melanoma with a total of 10-25 mice per melanoma. Each dot represents a different mouse and all data represent mean ± s.d. Statistical significance was assessed using repeated measures one-way (c) or two-way ANOVAs (d-e) with Dunnett’s multiple comparisons adjustments (c-e), Mann-Whitney tests followed by Holm-Sidak’s multiple comparisons adjustments (f, Y3.3 and Y5.2 of h and j, l, and n), nparLD tests (g, i, k, and m) followed by FDR multiple comparisons adjustments (g and i), or Kruskal-Wallis tests with Dunn’s multiple comparisons adjustments (for Y1.7 of h and j). All statistical tests were two-sided. No statistically significant differences were observed in f, g-i, k, or m.

Article Snippet: YUMM 1.7 ( Braf V600E/+ ; PTEN -/- ; Cdkn2 -/- ), YUMM3.3 ( Braf V600E/+ ; Cdkn2 -/- ), and YUMM5.2 (Braf V600E/+ p53 -/- ) mouse melanomas were obtained from, and authenticated by, the American Type Culture Collection (ATCC).

Techniques: Gene Expression, RNA Sequencing, Derivative Assay, Isolation, Flow Cytometry, Quantitative RT-PCR, Luciferase, Expressing, Cell Culture, Injection, Imaging, Control, Mutagenesis, Clone Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Dual targeting of PDPK1 and BRAF V600E is synthetically lethal

doi: 10.64898/2026.03.15.711663

Figure Lengend Snippet: (A) Immunobloting of pPDPK1, PDPK1, pMEK, and MEK in thyroid cancer lines: papillary thyroid cancer (PTC, brown color) (TPC1), follicular thyroid cancer (FTC, blue color) (FTC-133 and FTC-236), poorly differentiated thyroid cancer (PDTC, green color) (BCPAP), BRAF wild type anaplastic thyroid cancer (ATC) (red color) (C643 and THJ29T), BRAF V600E–mutant ATC (8505C, SW1736, and THJ16T), and HEK293 cells using β-actin as a loading control. The relative ratio of pPDPK1 to total PDPK1 and pMEK to total MEK is listed below each phospho-protein compared to HEK293 cells. (B) Immunoblotting of phosphoPDPK1, total PDPK1 in untreated and dabrafenib (Dab) and trametinib–treated patient derived ATC and normal thyroid tissues using β-actin as a loading control. The relative ratio of pPDPK1 to total PDPK1 and pMEK to total MEK is listed below each phospho-protein compared to normal thyroid tissue. (C–D) Dose-dependent inhibition of cell proliferation with BX795 treatment in 8505C and SW1736 cells after 48 h. (E–F) Dose-dependent inhibition of cell proliferation with Dab treatment in 8505C and SW1736 ATC cells after 48 h. (G–H) Analysis of the combination index (CI) using the CompuSyn software for BX795, Dab, and their combination in BRAF V600E–mutant ATC cell lines. CI < 1 indicates synergism, and fa denotes fraction affected. (I–J) The effect of BX795 (2.5 µM), Dab (2.5 µM), and their combination on cellular proliferation in 8505C and SW1736 cells after 48 h. (K–L) Colony-formation assay in 8505C and SW1736 cells treated with BX795 (2.5 µM), Dab (2.5 µM), or their combination. Quantification was performed using ImageJ. (M–N) Cellular migration as measured with a wound-healing assay in 8505C and SW1736 cells treated with BX795 (2.5 µM), Dab (2.5 µM), or their combination. Quantification was performed using the ImageJ software. (O–P) Concentration-dependent effects of BX795 treatment on proliferation of patient-derived ATC cells (ATC01 and ATC02). ATC01 was derived from a treatment-naïve patient tumor positive for BRAF V600E mutation, and ATC02 was derived from a residual tumor in a patient with an exceptional treatment response to Dab and trametinib, and the tumor was positive for BRAF V600E mutation. (Q–R) The effects of treatment with BX795 (2.5 µM), Dab (2.5 µM), or their combination on the proliferation of patient-derived ATC cells (ATC01 and ATC02). (S) The effect of treatment with BX795 (2.5 µM), Dab (2.5 µM), or their combination on ATC spheroids in BRAF V600E–mutant in the 8505C and SW1736 cell lines. (T) The effect of treatment with BX795 (2.5 µM), Dab (2.5 µM), or their combination on patient-derived ATC spheroids (ATC01 and ATC02). All data are presented as the mean ± standard deviation. Statistical significance is indicated as ns = nonsignificant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: The BCPAP (homozygous mutated BRAF V600E) cell line was purchased from Leibniz Institute DSMZ (Lower Saxony, Germany).

Techniques: Western Blot, Mutagenesis, Control, Derivative Assay, Inhibition, Software, Colony Assay, Migration, Wound Healing Assay, Concentration Assay, Standard Deviation

Journal: bioRxiv

Article Title: Dual targeting of PDPK1 and BRAF V600E is synthetically lethal

doi: 10.64898/2026.03.15.711663

Figure Lengend Snippet: (A) Mutated BRAF V600E ATC cells (8505C) were treated for 48 h with BX795 (2.5 µM), dabrafenib (Dab) (2.5 µM), or their combination. Then, total and phosphorylated protein levels were measured using mass spectrometry. (A–C) The volcano plots show significantly upregulated proteins in red, significantly downregulated proteins in blue (p < 0.05), and non-significant proteins in gray. The x-axis represents log 2 fold change, and the y-axis shows –log 10 (p-value). The dashed lines indicate thresholds for statistical significance and fold change. (D) The Venn diagrams shows shared and treatment-specific differentially expressed proteins and phosphorylated proteins across the treatment groups (p < 0.05). The overlapping regions represent a conserved core proteomic response, while the non-overlapping regions indicate pathway-specific effects of PDPK1 or BRAF inhibition. (E–G) Gene set enrichment analysis (GSEA) of ranked phosphorylated site changes following BX795 (E), Dab (F), or combination (G) treatment. The bar plots show normalized enrichment scores (NES) for Hallmark Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways. (H) Western blot analysis of the effect of BX795 (2.5 µM), Dab (2.5 µM), or combination treatment on BRAF V600E–mutant ATC cell lines (8505C and SW1736) after 48 h. pMEK, total MEK, pAKT 308 , total AKT, pPDPK1 S241 , PARP-1, BCL2, and caspase-3 protein levels are shown, with β-actin used as a loading control. (I) Flow cytometry results of Annexin V/PI staining with BX795 (2.5 µM), Dab (2.5 µM), or combination treatment. (J) Western blot analysis of apoptosis regulatory proteins 40 h after treatment. pBAD S112 , BAD, BCL2, pGSK3β Ser9 , GSK3β, and the double-stranded DNA damage marker pH2AX S139 are shown, with β-actin used as a loading control. Protein band density was measured and normalized to β-actin. The phosphorylated to total protein ratios were measured, and the values are listed above the phosphoprotein band. Protein band density of BCL-xL and pH2AX S139 were also measured and normalized to β-actin. (K) Immunoprecipitation of BCL-xL and BCL2 with BAD 30 h after treatment with BX795 and Dab in BRAF V600E–mutant ATC cell lines. Protein band density was measured and normalized to the input protein band. The ratio of immunoprecipitated to total input protein was measured and is listed below the protein blot. P = phosphorylation. The superscript indicates the amino acid phosphorylation site. (L) The heatmap shows z-scored log 2 -transformed abundance of apoptosis-related proteins for the BX795, Dab, and combination treatment groups in 8505C ATC cells. The proteins are displayed in sequential order—BAD, TNFRSF10A, PARP1, FAS, BID, CASP3, CASP8, BAX, CASP9, CASP7, BCL2L1, and BAK1—with hierarchical clustering applied to the rows. (M) The effect of BX795 (2.5 µM), Dab (2.5 µM), or combination treatment for 16 h on γH2AX foci formation in the 8505C and SW1736 ATC cell lines using immunofluorescence. γH2AX was labelled with Alexa Fluor™ 546 secondary antibody, with DAPI used for nuclear staining. A Zeiss LSM 800 confocal microscope was used to examine the cells (400× magnification). The number of foci per cell was quantified using ImageJ. All data are presented as the mean ± standard error of the mean of 25 cells. Statistical significance is indicated as ns = nonsignificant; *p < 0.05; **p < 0.01; ***p<0.001; ****p < 0.0001. (N) Western blot analysis of the effect of BX795 (2.5 µM), Dab (2.5 µM), or combination treatment for 16 h on DNA damage–dependent proteins (pATM, total ATM, pCHK2, Total CHK2, and pCHK1). β-Actin was used as a loading control. (O) Cell cycle analysis after BX795 (2.5 µM), Dab (2.5 µM), or combination treatment for 16 h in BRAF V600E–mutant ATC cell lines (8505C and SW1736). Cells were stained with propidium iodide (PI) and analyzed by fluorescence-activated cell sorting. The DNA content was measured, 2N (diploid) and 4N (tetraploid), based on the PI-stained DNA content. (P) The effect of BX795 (2.5 µM), Dab (2.5 µM), or combination treatment for 16 h on DNA repair proteins—cyclin D, cyclin B, pcdc25c, pCDK1, total CDK1, pH3A, H3A, and cyclin A2—by immunoblotting. β-actin was used as a loading control. The ratio of phospho-H3A to total H3A was measured and is listed below the phospho-H3A protein band. All data are presented as the mean ± standard deviation. Statistical significance is indicated as ns = nonsignificant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (Q) Analysis of the effect of combination BX795 (2.5 µM) and Dab (2.5 µM) treatment for 16 h followed by proteasomal degradation inhibition for 6 h (MG132, 10 µM). Protein band density was measured and normalized to β-actin. (R) The heatmap shows z-scored log2-transformed phosphorylation levels of DNA damage response, DNA repair, and cell-cycle regulatory proteins for the BX795, Dab, and combination treatment groups in 8505C ATC cell line. Proteins are displayed in sequential order as RAD50, CDK1, MCM2, PRKDC, RB1, TP53BP1, TP53, XRCC1, XRCC5, LIG1, MDC1, and MSH6, with supervised hierarchical clustering applied to the rows. The color scale represents relative protein levels (red = higher; blue = lower).

Article Snippet: The BCPAP (homozygous mutated BRAF V600E) cell line was purchased from Leibniz Institute DSMZ (Lower Saxony, Germany).

Techniques: Mass Spectrometry, Inhibition, Protein-Protein interactions, Western Blot, Mutagenesis, Control, Flow Cytometry, Staining, Marker, Immunoprecipitation, Phospho-proteomics, Transformation Assay, Immunofluorescence, Microscopy, Cell Cycle Assay, Fluorescence, FACS, Standard Deviation

Journal: bioRxiv

Article Title: Dual targeting of PDPK1 and BRAF V600E is synthetically lethal

doi: 10.64898/2026.03.15.711663

Figure Lengend Snippet: Mutated BRAF V600E cell were treated with BX795 (2.5 µM), Dab (2.5 µM), or their combination. (A) Total cellular ROS was measured using the DCFH-DA fluorescent dye and quantified with flow cytometry. (B) Mitochondrial superoxide was measured based on MitoSOX red fluorescence, quantified with flow cytometry. (C–D) The mitochondrial content was assessed by MitoTracker green fluorescence, as quantified by flow cytometry and visualized with live-cell confocal microscopy (Zeiss LSM 800, 400× magnification). (E) The mitochondrial membrane potential was evaluated by TMRM (100 nM) fluorescence and analyzed by flow cytometry. (F) The oxygen consumption rate (OCR) was measured using a Seahorse analyzer with sequential injections of oligomycin (Oligo), 2,4-dinitrophenol (DNP), and antimycin A plus rotenone (Rot+AA). The data were normalized to the total protein content, and maximal respiration and adenosine triphosphate (ATP)-linked respiration were quantified accordingly. (G) The heatmap shows z-scored log 2 -transformed abundance of proteins associated with mitochondrial oxidative phosphorylation, the tricarboxylic acid (TCA) cycle, mitochondrial ribosomal, transport, and mitochondrial dynamics–associated proteins in 8505C ATC cells treated with BX795 (2.5 µM), Dab (2.5 µM), or their combination. The proteins include representatives of electron transport chain complexes I–V (NDUF subunits, SDHA–D, UQCRC1/2, COX subunits, and ATP5 subunits), TCA cycle enzymes (IDH1, IDH2, IDH3, FH, OGDH, DLST, CS, MDH2, and PDHA1/PDHB), mitochondrial transport and structure (TOMM20, TIMM23, VDAC1–3, MFN1, OPA1, DNM1L), and mitochondrial genome maintenance (TFAM). Hierarchical clustering was applied to the rows. The color scale represents relative protein abundance (red = higher; blue = lower). All data are expressed as the mean ± standard deviation. Statistical significance is indicated as ns = nonsignificant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: The BCPAP (homozygous mutated BRAF V600E) cell line was purchased from Leibniz Institute DSMZ (Lower Saxony, Germany).

Techniques: Flow Cytometry, Fluorescence, Confocal Microscopy, Membrane, Transformation Assay, Phospho-proteomics, Quantitative Proteomics, Standard Deviation

Journal: bioRxiv

Article Title: Dual targeting of PDPK1 and BRAF V600E is synthetically lethal

doi: 10.64898/2026.03.15.711663

Figure Lengend Snippet: (A) Time-dependent effect of combination BX795 and Dab treatment based on propidium iodide (PI) staining, showing the proportion of cells arrested in the G2/M phase. Cells were stained with PI and analyzed by fluorescence-activated cell sorting. The results are presented as the mean ± standard deviation. Statistical significance is indicated as ns = nonsignificant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (B) Intracellular ROS levels were measured using DCFH-DA staining and analyzed by flow cytometry. The results are presented as the mean ± standard deviation. (C–E) The effects of N -acetyl cysteine (NAC) (5 mM) on combination treatment–induced responses in BRAF V600E–mutant ATC cell lines. (C) ROS generation was analyzed by DCFH-DA staining and is presented as the mean ± standard deviation. (D) Apoptosis was assessed based on Annexin V/PI staining. (E) Immunoblot analysis of PARP-1 cleavage using β-actin as a loading control. The densitometric quantification of cleaved vs pro-PARP cleavage in relation to loading control is shown below the bands. (F) The effect of the mitochondrial ROS scavenger MitoQ on combination treatment–induced G2/M arrest in BRAF V600E–mutant ATC cell lines. The DNA content was measured, 2N (diploid) and 4N (tetraploid), based on PI staining. The results are presented as the mean ± standard deviation. Statistical significance is indicated as ns = nonsignificant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (G–H) The impact of CHK2 phosphorylation inhibitor BML-277 (ML277) on combination BX795 and Dab treatment-induced. (G) Cell cycle arrest was analyzed by PI staining and flow cytometry. (H) Antiproliferative effect in BRAF V600E–mutant ATC cells. The DNA content was measured, 2N (diploid) and 4N (tetraploid), based on PI staining. The results are presented as the mean ± standard deviation. Statistical significance is indicated as ns = nonsignificant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (I) Apoptosis was measured with Annexin V/PI staining and flow cytometry. The results are presented as mean ± standard deviation. Statistical significance is indicated as ns = nonsignificant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: The BCPAP (homozygous mutated BRAF V600E) cell line was purchased from Leibniz Institute DSMZ (Lower Saxony, Germany).

Techniques: Staining, Fluorescence, FACS, Standard Deviation, Flow Cytometry, Mutagenesis, Western Blot, Control, Phospho-proteomics

Journal: bioRxiv

Article Title: A Recombinant Antibody Against Human DRP1 Serine 616 Phosphorylation Enables Detection of BRAF V600E -Associated Mitochondrial Division in Cancer

doi: 10.64898/2026.01.16.699897

Figure Lengend Snippet: (A) Representative images of recombinant 3G11 staining in normal skin, benign nevi, atypical nevi, and primary melanoma lesions. Normal skin serves as the control tissue and defines negative staining. Benign nevi show limited and diffuse staining. Atypical nevi show increased staining relative to benign nevi. Primary melanoma lesions show strong and diffuse staining. (B) Representative images of BRAF V600E staining in normal skin, benign nevi, atypical nevi, and primary melanoma lesions. Normal skin serves as the control tissue and defines negative staining. Benign nevi showed broad BRAF V600E positivity patterns including no staining, diffuse, and/or strong intensity; we presume due to the heterogeneous nature of benign nevi and a combination of lesions that were stable versus a set that potentially progressed to dysplastic and/or primary disease. Atypical nevi show increased staining compared to benign nevi. Primary melanoma lesions show robust cytosolic staining.

Article Snippet: For immunohistochemistry, tissues were stained with recombinant 3G11 and BRAF V600E (Cell Signaling Technology, Cat. No. 2900S).

Techniques: Recombinant, Staining, Control, Negative Staining

Journal: bioRxiv

Article Title: A Recombinant Antibody Against Human DRP1 Serine 616 Phosphorylation Enables Detection of BRAF V600E -Associated Mitochondrial Division in Cancer

doi: 10.64898/2026.01.16.699897

Figure Lengend Snippet:

Article Snippet: For immunohistochemistry, tissues were stained with recombinant 3G11 and BRAF V600E (Cell Signaling Technology, Cat. No. 2900S).

Techniques: Recombinant, Staining, Immunohistochemistry