Journal: bioRxiv

Article Title: Transcriptional Dosage of Oncogenic KRAS Drives Lung Adenocarcinoma Cell States, Progression and Metastasis

doi: 10.1101/2024.12.29.630643

Figure Lengend Snippet: In vivo gain-of-function and fate mapping of Kras-driven lung cancer cells by CRISPRa-CROP-seq. A) Experimental outline: Kras G12D ;Trp53 -/- ;Eed -/- ;Tet ON -dCas9-VPR (KPE-VPR) were low-MOI- infected with a lentiviral library of gRNAs targeting Kras-associated inflammatory mediators (from Serresi et al., 2018) and non-targeting gRNAs serving as barcodes for neutral evolution. KPE-VPR; CROP-mCherry were orthotopically transplanted into recipient mouse lungs and gRNA expression was activated in vivo by doxycycline upon grafting was verified (see Fig.S1). Plasmid library, input and in vitro expanded cells served as control for stochastic gRNAs drift. B) Representative lightsheet microscopy of lung, heart and kidney from mice transplanted with the cells indicated background. C) Violin plot of bioluminescence (BLI) emission at the humane-end-point from ex-vivo isolated organs from mice injected with the indicated KPE cells. Regions of interest (ROIs) were guided by BLI signal and normalized over background-positioned ROIs. D) Graphical summary of the tissue samples collected in the screenings. E) Bar plot of sgRNA detection rate from all the samples in the screen. Color code denotes cells or tissue of origin. F) Sankey plots showing the tumor evolution in animals treated with doxycycline-inducing gRNA library activation, or sham (no gRNA activation). G) Bar plot showing the linear counts of non-targeting and targeting sgRNA value in primary lung tumors and in representative distant organs. H) Representative images of isolated primary passage 0 cancer cells from different organs. mCherry marks gRNAs containing cancer cells.

Article Snippet: Primary antibodies used against the following antigens were as follows: anti-vimentin D21H3 rabbit monoclonal antibody (mAb); Cell Signaling Technology, #5741], anti–E-cadherin (24E10 rabbit mAb; Cell Signaling Technology, #3195), anti–N-cadherin (rabbit mAb; Cell Signaling Technology, #4061), anti–Sox2 (rabbit polycolonal Abcam, #15830),anti-slug(rabbit mAb; Cell Signaling Technology, #4933) anti-snail (rabbit mAb; Cell Signaling Technology, #4933) anti-ZO1 (rabbit mAb; Cell Signaling Technology, D7D12 #8193) anti-RAS(rabbit mAb; Cell Signaling Technology, #67648)anti-slug(rabbit mAb; Cell Signaling Technology, #4933) anti-vinculin (mouse clone h-VIN1; Sigma-Aldrich, #V9131), anti–FOXA1 (Abcam AB23738 rabbit mAb; anti-FOXP1 (rabbit mAb; Cell Signaling Technology, #2005), anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (rabbit mAb; Santa Cruz Biotechnology, #sc-25778), anti-H3K273me [rabbit polyclonal antibody (pAb); Cell Signaling Technology, #9733], anti-total H3 (rabbit mAb; Cell Signaling Technology, #AB1791), anti-CAS9 polyclonal Diagenode #C15310258), anti–c-JUN (60A8 rabbit mAb; Cell Signaling Technology, #9165), anti–Ras (G12D Mutant) Recombinant Rabbit Monoclonal Antibody (HL10)Thermofisher MA5-36256, anti tubulin monoclonal Sigma #T5168

Techniques: In Vivo, Infection, Expressing, Plasmid Preparation, In Vitro, Control, Microscopy, Ex Vivo, Isolation, Injection, Activation Assay

Journal: Frontiers in Oncology

Article Title: Targeting the Mutational Landscape of Bystander Cells: Drug-Promoted Blood Cancer From High-Prevalence Pre-neoplasias in Patients on BRAF Inhibitors

doi: 10.3389/fonc.2020.540030

Figure Lengend Snippet: Patient with BRAF V600E-mutant hairy cell leukemia (HCL) and concomitant RAS -mutant B cell lymphocytosis (MBL) experiencing progression to chronic lymphocytic leukemia (CLL). (A) Computed tomography scans of cervical lymph nodes of patient with HCL and concomitant MBL/CLL. On the left, nodal progression on dual BRAFi/MEKi. On the right, nodal remission 3 months after BRAFi withdrawal. (B) Absolute MBL/CLL and HCL cell counts over time during combined BRAFi/MEKi and after BRAFi withdrawal. Cell counts were deduced from flow cytometry, IGH next-generation sequencing and KRAS G12D liquid biopsy analyses of peripheral blood. Clone CARSDFWGDAFDIW (green line) represents the MBL/CLL clone and clone CAKDPPLNHFWGGYPFSFDNW (red line) represents HCL clone. IGH , Immune globulin heavy locus. Arrows indicate cervical lymphnode.

Article Snippet: This treatment resulted in the development of chronic lymphocytic leukemia (CLL) from a preexisting KRAS G12D mutant monoclonal B-cell lymphocytosis (MBL).

Techniques: Mutagenesis, Computed Tomography, Flow Cytometry, Next-Generation Sequencing

Journal: Neoplasia (New York, N.Y.)

Article Title: The GSK3 kinase and LZTR1 protein regulate the stability of Ras family proteins and the proliferation of pancreatic cancer cells

doi: 10.1016/j.neo.2022.01.002

Figure Lengend Snippet: GSK3 inhibition reduces the level of Ras family proteins. (A) GSK3 inhibition reduces Ras signaling and the level of Ras proteins. AsPC1 cells were harvested at the indicated time points after the addition of CHIR98014 (10 µM). Samples were Western blotted with the indicated antibodies, including Ras family proteins (pan Ras) and their G12D mutant proteins (Ras G12D ). Phosphorylated and total cMyc proteins were used as surrogate markers of GSK3 kinase activity. Positions of Bio-Rad dual color standards are shown in KDa. The experiment was done twice with the same outcome. (B) Abundance of the KRAS mRNA is unchanged after GSK3 inhibition. AsPC1 cells were harvested at the indicated time points after CHIR98014 (10 µM). Real-time RT-PCR was used to quantify the abundance of KRAS and GAPDH mRNA. KRAS/GAPDH mRNA ratio is shown as the mean ± S.D. of triplicate samples ( n = 3). (C) Insulin induces the phosphorylation and inhibition of GSK3. Top panel: Insulin/IGF signaling has been shown to promote the T308- and S473-phosphorylation and activation of the Akt kinase. Akt can then phosphorylate GSK3α (at S21) and GSK3β (at S9), thereby inhibiting the two kinases. This inhibition of GSK3 allows for the activation of glycogen synthase, which otherwise is kept inhibited by the phosphorylation of its S641 residue by GSK3. Bottom panel: HPAF/CD18 cells were exposed to Insulin Aspart (0.04 U/ml). Three hours later, cells were examined for changes in Akt and GSK3 phosphorylation. The experiments was repeated 3 times with the same outcome. (D) Insulin reduces the level of Ras family proteins. The levels of Ras proteins (pan Ras) and S641-phosphorylated glycogen synthase (p-GS) were monitored in HPAF/CD18 cells after the addition of Insulin Aspart (0.04 U/ml). The experiment was repeated twice with the same results. (E) KRAS mRNA is unchanged after the addition of Insulin Aspart (0.04 U/ml). AsPC1 cells were harvested at the indicated time points after insulin. KRAS/GAPDH mRNA ratio is shown as the mean ± S.D of triplicate samples ( n = 3).

Article Snippet: An antibody that binds selectively to the G12D mutants of Ras proteins (Ras G12D ) was also used (cat# 26036; NewEast Biosciences, King of Prussia, PA).

Techniques: Inhibition, Western Blot, Mutagenesis, Activity Assay, Quantitative RT-PCR, Phospho-proteomics, Activation Assay, Residue