Journal: bioRxiv

Article Title: Phosphoinositide 3-kinase regulates wild-type RAS signaling to confer resistance to KRAS inhibition

doi: 10.1101/2025.06.20.660715

Figure Lengend Snippet: (A) Immunoblots of phosphorylated ERK1/2 (pERK T202/Y204 ) and phosphorylated AKT (pAKT S473 ) in 8988T-KO cells transduced with HA-tagged SOS1 and treated with GDC-0941 (2 μM) or ZSTK474 (2 μM) for 4 hours. HSP90 is loading control. (B) Quantification of knockdown of GAPs NF1, RASA1, and RASAL1 by quantitative RT-PCR. Bar graph shows relative mRNA levels normalized to control (mean ± SD, n = 3 biologic replicates). ** p < 0.01, *** p < 0.001, repeated measures ANOVA (rmANOVA) with Dunnett’s test. (C) Immunoblots of pERK1/2 and pAKT in 293HEK and 8988T-KO cells subject to stable knockdown of NF1 using two independent shRNAs and treated with GDC-0941 or ZSTK474 (2 μM, 4 hours). (D) Immunoblots of pERK1/2 and pAKT in 293HEK cells subject to stable knockdown of RASA1 or RASAL1 using two independent shRNAs and treated with GDC-0941 (2 μM, 4 hours).

Article Snippet: Quantitative PCR was performed using TaqMan™ Universal PCR Master Mix (Thermo Fisher, Scientific, 4304437) and gene-specific FAM probes (Thermo Fisher Scientific) on a CFX Opus 384 Real-Time PCR System (Bio-Rad) with the following probes: GAPDH(Hs99999905_m1), RASA1(Hs00243115_m1), RASAL1(Hs00183013_m1), NF1(Hs01035108_m1).

Techniques: Western Blot, Transduction, Control, Knockdown, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Rasal1 impairment unleashes anticancer immunity - a focus on T cells

doi: 10.64898/2026.01.19.700452

Figure Lengend Snippet: Percentage of viable CD45 + TILs detected in the tumors ( A ). Percentage of TCRβ + T cells within CD45 + TIL population ( B ). Percentage of CD8 + T cells within TCRβ + T population ( C ). Percentage of CD4 + T cells within TCRβ + T cell population ( D ). Percent CD44 + cells within CD8 + TILs in melanoma ( E ). Percentage of Ki67 + cells within CD8 + TILs in melanoma ( F ). Analysis conducted on Cytobank (aka viSNE) showing the T cell compartment in the context of B16F10 melanoma ( G ). Cartoon showing the essential sequential activations occurring in T cells ( H ). Expression of Rasal1 in murine hematopoietic lineages according to the European Bioinformatics Institute database ( https://ebi.ac.uk/gxa/home ) ( I ). For statistical analyses, parametric unpaired two-tailed t-tests were used. *p < 0.05, **p < 0.01 and ***p < 0.001 .

Article Snippet: The Rasal1 impaired (Rasal1i) mouse model was purchased from the mouse biology program in California ( https://www.mmrrc.org/ ).

Techniques: Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Rasal1 impairment unleashes anticancer immunity - a focus on T cells

doi: 10.64898/2026.01.19.700452

Figure Lengend Snippet: Percentage of Tbet + , GZB + , CXCR5 + and PD1 + cells within CD8 + TILs in B16F10 melanoma ( A ). Percentage of Tbet + , CXCR5 + and GZB + cells within helper CD4 + TILs (Foxp3 - cells) in B16F10 melanoma ( B ). A viSNE analysis conducted on Cytobank showing the colocalization of Tbet, GZB, CXCR5 and PD1 within the CD8 + TIL compartment in the context of B16F10 melanoma ( C ). Comparison of relative expression values between Rasal1 expression versus T-bet (left) and PD1 (right) in murine CD4 + T cell lineages (data collected from the the European Bioinformatics Institute database; https://ebi.ac.uk/gxa/home ) ( D ). For statistical analyses, parametric unpaired two-tailed t-tests were used. *p < 0.05, **p < 0.01 and ***p < 0.001 .

Article Snippet: The Rasal1 impaired (Rasal1i) mouse model was purchased from the mouse biology program in California ( https://www.mmrrc.org/ ).

Techniques: Comparison, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Rasal1 impairment unleashes anticancer immunity - a focus on T cells

doi: 10.64898/2026.01.19.700452

Figure Lengend Snippet: ( A ) Boxplots of log₂(FPKM+1) RASAL1 expression in tumor vs normal tissue. Top: melanoma (TCGA-SKCM; n =103 tumors, n =1 normal; Wilcoxon p =0.97; note: severely limited normal samples preclude robust comparison). Middle: colorectal adenocarcinoma (TCGA-COAD/READ; n =647 tumors, n =51 normals; p =0.0004). Bottom: non-small cell lung cancer (TCGA-LUAD/LUSC; n =1051 tumors, n =110 normals; p <2.22e-16). Red = tumor, blue = normal. Horizontal lines: median; whiskers: 1.5× interquartile range; points: individual samples. ( B ) Representative immunohistochemistry images of RASAL1 protein expression in punch biopsies from the Human Protein Atlas (HPA). Top: malignant melanoma (metastatic site) showing low staining, moderate to weak intensity, <25% quantity, cytoplasmic/membranous/nuclear localization. Middle: colon adenocarcinoma showing medium staining, moderate intensity, >75% quantity, cytoplasmic/membranous. Bottom: lung adenocarcinoma showing medium staining, moderate intensity, >75% quantity, cytoplasmic/membranous. Images selected as representative of the most common/average patterns reported in HPA for each cancer type. ( C ) Kaplan–Meier survival curves stratified by median RASAL1 expression (log₂(FPKM+1)). Top: melanoma (TCGA-SKCM; High n =156 red vs Low n =157 blue; log-rank p =0.8). Middle: colorectal (TCGA-COAD/READ; High n =298 red vs Low n =298 blue; p =0.97). Bottom: NSCLC (TCGA-LUAD/LUSC; High n =519 red vs Low n =519 blue; p =0.085). Shaded areas: 95% CI. Risk tables below each plot show patients at risk over time. ( D ) Forest plots from univariate Cox regression. HR compares high vs low RASAL1 (low = reference). Top: Melanoma ( n =313; HR=0.97 [0.74–1.3]). Middle: Colorectal ( n =596; HR=1 [0.72–1.4]). Bottom: NSCLC ( n =1096; HR=1.2 [0.98–1.4]). Error bars: 95% CI.

Article Snippet: The Rasal1 impaired (Rasal1i) mouse model was purchased from the mouse biology program in California ( https://www.mmrrc.org/ ).

Techniques: Expressing, Comparison, Immunohistochemistry, Staining

Journal: Molecular Oncology

Article Title: The genetic duet of concurrent RASAL1 and PTEN alterations promotes cancer aggressiveness by cooperatively activating the PI3K – AKT pathway

doi: 10.1002/1878-0261.13701

Figure Lengend Snippet: Summary of the cancer types and frequencies of the genetic alterations of RASAL1 in 33 types of cancer. (A) Cancer types studied from TCGA database and their full names and corresponding abbreviations. The value in the parentheses represents the number of cases. (B) Presentation of the frequencies of genetic alterations of RASAL1 . For each cancer type, yellow bar represents the collective frequency of mutation and copy number loss of RASAL1 , the blue bar represents the frequency of copy number loss, and the green bar represents the frequency of mutations. The first group, labeled as “Total,” shows the overall frequencies of genetic alterations of RASAL1 among the 33 types of cancer of the entire cohort of the patients. (C) Genes whose mutations showed concurrence with RASAL1 alterations. Shown are 11 genes whose mutations frequencies were > 5% and concurred with RASAL1 alterations in all cancer samples. The width of the line linking each gene pair is calculated by the −log ( P ) ( P is the adjusted P value calculated for the strength of concurrence between the pair of genes by fisher exact test). The more significant the adjusted P value is, the wider the line linking the pair of genes is. The abbreviations of the cancer names are as defined in Panel A and Table .

Article Snippet: A Rasal1 knockout (KO) mouse model was created using CRISPR‐CAS technology (Applied StemCell, Inc.).

Techniques: Mutagenesis, Labeling

Journal: Molecular Oncology

Article Title: The genetic duet of concurrent RASAL1 and PTEN alterations promotes cancer aggressiveness by cooperatively activating the PI3K – AKT pathway

doi: 10.1002/1878-0261.13701

Figure Lengend Snippet: Association between RASAL1 alterations and the PI3K/AKT pathway activation and poor clinical outcomes of cancer. (A) Association between RASAL1 alterations and AKT phosphorylation (two‐sided Student's t ‐test). Shown are the results in breast cancer, prostate adenocarcinoma and thymoma. The thick black bar in the middle represents the quartile range and the thin black line extending from it represents the 95% confidence interval. (B) Association between RASAL1 alterations and AKT phosphorylation in patients without PTEN and TP53 mutations in breast cancer and lung adenocarcinoma (two‐sided Student's t ‐test). (C) Association between RASAL1 alterations and disease‐specific mortality on the analyses of all patients or patients without PTEN and TP53 alterations (chi‐squared test). (D) Effects of RASAL1 alterations on Kaplan–Meier disease‐specific survival curves on the analysis of all patients. (E) Effects of RASAL1 alterations on Kaplan–Meier disease‐specific survival curves on the analysis of patients without PTEN and TP53 alterations. (F) Effects of RASAL1 alterations on disease progression rates on the analyses of all patients or patients without PTEN and TP53 alterations (chi‐squared test). (G) Effects of RASAL1 alterations on Kaplan–Meier disease progression‐free survival curves on the analysis of all patients. (H) Effects of RASAL1 alterations on Kaplan–Meier disease progression‐free survival curves on the analysis of patients without PTEN and TP53 alterations. Definitions of genotypes: no RASAL1 , no RASAL1 alterations; RASAL1 , there were RASAL1 alterations; no PTEN , no PTEN alterations; no TP53 , no TP53 alterations; no PTEN & TP53 , no PTEN and TP53 alterations; no RASAL1 _no PTEN _no TP53 , no alterations in RASAL1 , PTEN and TP53 ; RASAL1 _no PTEN _no TP53 , there were RASAL1 alterations, but no PTEN and TP5 3 alterations. The name abbreviations for various cancer types are as defined in Fig. and Table .

Article Snippet: A Rasal1 knockout (KO) mouse model was created using CRISPR‐CAS technology (Applied StemCell, Inc.).

Techniques: Activation Assay, Phospho-proteomics, Biomarker Discovery

Journal: Molecular Oncology

Article Title: The genetic duet of concurrent RASAL1 and PTEN alterations promotes cancer aggressiveness by cooperatively activating the PI3K – AKT pathway

doi: 10.1002/1878-0261.13701

Figure Lengend Snippet: Cooperation of concurrent RASAL1 and PTEN alterations in synergistically activating the PI3K pathway (AKT phosphorylation) and promoting aggressive clinical outcomes. (A) Cooperative effects of concurrent RASAL1 and PTEN alterations on AKT phosphorylation in breast cancer, cervical cancer, and low‐grade glioma patients without TP53 mutations (two‐sided Student's t ‐test). The thick black bar in the middle represents the quartile range and the thin black line extending from it represents the 95% confidence interval. (B) Cooperative effects of concurrent RASAL1 and PTEN alterations on disease‐specific mortality rates and disease progression rates on the analyses of all cancer patients (chi‐squared test). (C) Cooperative effects of concurrent RASAL1 and PTEN alterations on Kaplan–Meier disease‐specific survival curves in patients with breast cancer or cervical cancer patients without TP53 mutations. (D) Cooperative effects of concurrent RASAL1 and PTEN alterations on Kaplan–Meier disease‐specific survival curves and disease progression‐free survival curves on the analyses of all cancer patients. (E) Status of ER, PR, and HER2 expression with various genotypes of RASAL1 , PTEN , and TP53 alterations in breast cancer (chi‐squared test). (F) Distribution of breast cancer subtypes among various genotypes of RASAL1 , PTEN and TP53 alterations (chi‐squared test). (G) Kaplan–Meier disease‐specific survival curves and disease progression‐free survival curves with various genotypes of RASAL1 , PTEN , and TP53 alterations on the analyses of all cancer patients. (H) Kaplan–Meier disease‐specific survival curves in various genotypes of RASAL1 , PTEN and TP53 alterations in patients with chromophobe renal cell carcinoma or uterine corpus endometrial carcinoma. The definitions of various genotypes of RASAL1 , PTEN , and TP53 alterations are as defined in Fig. .

Article Snippet: A Rasal1 knockout (KO) mouse model was created using CRISPR‐CAS technology (Applied StemCell, Inc.).

Techniques: Phospho-proteomics, Biomarker Discovery, Expressing

Journal: Molecular Oncology

Article Title: The genetic duet of concurrent RASAL1 and PTEN alterations promotes cancer aggressiveness by cooperatively activating the PI3K – AKT pathway

doi: 10.1002/1878-0261.13701

Figure Lengend Snippet: Genetic knockout mice show the tumor suppressor role of RASAL1 and the cooperation between RASAL1 and PTEN defects in driving oncogenesis and cancer aggressiveness, as found in humans. (A) Generation of the global Rasal1 ‐knockout mouse model and colony development. (B) Illustration of the location of two candidate gRNAs within Exon 2 of mRasal1 gene. (C) All mice ( n = 150) were genotyped for the Rasal1 gene using PCR sequencing. Representative PCR amplification patterns demonstrate a single upper band (398 bp) in wild‐type Rasal1 +/+ mice, a single lower band (351 bp) in nullizygous Rasal1 −/− mice, and both bands in hemizygous Rasal1 +/− mice. (D) Western blotting analyses ( n = 9) of the expression levels of several signaling proteins in various Rasal1 genotypes. (E) Pie chart summary ( n = 40, 74, 15 and 21, respectively) of the frequencies of pathological conditions in various knockout genotype mice. (F) Representative microscopic images of pathological conditions (thyroid hyperplasia ( n = 17), thyroid cancer ( n = 11), metastatic thyroid cancer in lungs ( n = 6), and uterine adenocarcinoma ( n = 2)) that developed in knockout mice. The scale bars represent 50 μm. (G) Schematic illustration of the mechanistic model in which concurrent RASAL1 and PTEN alterations can cooperatively activate the PI3K pathway to promote oncogenesis and cancer aggressiveness. In this mechanism, the defect of RASAL1 results in retention of RAS‐GTP, thus maintaining constitutive RAS activation; this initiates the signaling of the PI3K‐AKT pathway, which cannot be terminated in the presence of PTEN defects.

Article Snippet: A Rasal1 knockout (KO) mouse model was created using CRISPR‐CAS technology (Applied StemCell, Inc.).

Techniques: Knock-Out, Sequencing, Amplification, Western Blot, Expressing, Activation Assay