Journal: Cell reports

Article Title: Mutant KRAS-driven selective mRNA translation reveals mechanisms and therapeutic vulnerabilities in cancer

doi: 10.1016/j.celrep.2026.117520

Figure Lengend Snippet: (A) Schematic of ribosome footprinting strategy from MiaPaca-2 (KRAS G12C) or Panc10.05 (KRAS G12D) cells treated with 10 μM sotorasib or MRTX1133 for 2 h, respectively. Comparison of ribosome-protected RNA (RF) and total mRNA abundance isolates the translational efficiency (TE) for each mRNA genome-wide. (B and C) Changes in TE following sotorasib treatment in MiaPaca-2 and MRTX1133 treatment in Panc10.05 cells. We identify mRNAs with decreased (TE down, red) and increased (TE up, blue) and unchanged translation (background, gray); three biological replicates; the most significantly affected genes are indicated on each side using a q value cutoff of 0.0001 and 0.1 for MiaPaca-2 and Panc10.05, respectively. (D and E) Metagene plot showing the reduction in ribosome coverage in TE down transcripts. KRAS G12C inhibition reduced overall ribosome coverage, while KRAS G12D inhibition significantly reduced the ribosome coverage in the 5′UTR region of the TE down transcripts. Paired two-sided Welch’s test across the coverage in 5′UTR, CDS, and 3′UTR showed a p value of = 7.50 × 10 −9 and 4.05 × 10 −4 for sotorasib and MRTX1133, respectively.

Article Snippet: The KRAS G12D specific inhibitor, MRTX1133, was purchased from MedChemExpress (HY-134813).

Techniques: Footprinting, Comparison, Genome Wide, Inhibition

Journal: Cell reports

Article Title: Mutant KRAS-driven selective mRNA translation reveals mechanisms and therapeutic vulnerabilities in cancer

doi: 10.1016/j.celrep.2026.117520

Figure Lengend Snippet: (A and B) Ribosome coverage on all ORFs showed increased ribosomes at the initiation site following sotorasib and MRTX1133 treatment in MiaPaca-2 and Panc10.05 cells, respectively ( p = 2.73 × 10 −2 ). No significant effect on the CDS in MRTX1133-treated Panc10.05 cells ( p = 0.414). (C) Ribosome coverage on TE down transcripts showed the accumulation of ribosomes upstream and downstream of the start site following sotorasib treatment in MiaPaca-2 ( p = 4.00 × 10 𢈒2 ), suggesting a slowdown in translation. (D) Ribosome coverage on TE down transcripts showed the accumulation of ribosomes upstream of the stop site following sotorasib treatment in MiaPaca-2 ( p = 1.66 × 10 −4 ), suggesting a block in translation elongation. (E) An increase of ribosome coverage between 70 and 90 nt region was observed in TE up transcripts following sotorasib (blue) treatment over control (black) in the 5′UTR and CDS around the start site ( p = 1.73 × 10 −2 ). (F) Fraction of pause-site codons (RxK, KKK, PP, PD, and PG) across the detected ORFs for the first 50 codons. (G, H, and I) Sucrose gradient fractionation of RNAse I digested polysome showing the accumulation of disome in MiaPaca-2 cells treated with sotorasib and quantification of the disome peak relative to DMSO ( p < 0.05). Immunoblot shows RPL5 and RPS10 in the indicated fractions collected from RNase I-digested polysome following DMSO vs. sotorasib treatment in MiaPaca-2. (J, K, and L) Immunoblot shows the induction of ZNF598, EDF1, p-ZAKα, ZAKα, RPS10-Ub following sotorasib treatment in MiaPaca-2. Anisomycin (ANS) treatment is included as a positive control for ribosome stalling and collision-induced stress signaling. β-actin is used as a loading control. (M) Mutant KRAS G12C inhibitor sotorasib affects translation initiation and elongation, resulting in ribosome stalling, while mutant KRAS G12D inhibitor MRTX1133 predominantly reduces translation initiation.

Article Snippet: The KRAS G12D specific inhibitor, MRTX1133, was purchased from MedChemExpress (HY-134813).

Techniques: Blocking Assay, Control, Fractionation, Western Blot, Positive Control, Mutagenesis

Journal: Cell reports

Article Title: Mutant KRAS-driven selective mRNA translation reveals mechanisms and therapeutic vulnerabilities in cancer

doi: 10.1016/j.celrep.2026.117520

Figure Lengend Snippet: (A and B) TE targets are downregulated in both sotorasib and MRTX1133 or only with sotorasib-treated MiaPaca-2 and Panc10.05 cells, respectively. (C) Translation efficiency of translation factors is affected by sotorasib, EEF1A1 being downregulated by the drug. (D–H) Immunoblot analyses show the downregulation of RPS6, EIF4E, EEF1A, and EEF2 following sotorasib treatment in MiaPaca-2 cells, SW837 cells, and RMC-7977 treatment in MiaPaca-2 cells and MRTX1133 treatment in Panc10.05 cells. β-actin is used as a loading control. (I) Intracellular immunostaining and FACS analysis show the downregulation of EEF1A in human primary PDAC-derived organoids treated with RMC-7977 (* p < 0.05, ** p < 0.01). (J–L) In vivo MiaPaca-2 xenograft studies show reduced tumor volume upon RMC-7977 treatment compared to vehicle, and immunoblots show the downregulation of RPS6, EIF4E, and EEF2 protein levels in xenograft tissues (* p < 0.05). (M and N) Western blot analysis and quantification of EEF1A1, EEF2, and RPS6 proteins show upregulation in KRAS G12D, or KRAS G12C compared to isogenic KRAS WT HEK293 cells (** p < 0.01 and *** p < 0.001). β-actin is used as a loading control. (O and P) Analysis of pancreatic, lung, and colorectal cancer cell lines with distinct KRAS genotypes [WT: H6C7], [G12D: Panc10.05, PANC-1, AsPC-1], [G12C: MiaPaca-2, H358, SW837] shows that KRAS G12D and KRAS G12C cells exhibit elevated protein expression of EEF1A1, EEF2, RPS6, and EIF4E compared to KRAS WT (** p < 0.01 and *** p < 0.001). β-actin is used as a loading control. (Q–S) Comparison of sotorasib TE targets with total proteomics data includes all eight trajectory clusters (C1-C8). Most of the TE down and TE up targets, which include cytoplasmic ribosomal proteins and translation factors, are downregulated at the protein level (clusters C1, C2, C5, C6, and C7). TE down mitochondrial ribosomal proteins are upregulated at the protein level (cluster C3, C4, C8).

Article Snippet: The KRAS G12D specific inhibitor, MRTX1133, was purchased from MedChemExpress (HY-134813).

Techniques: Western Blot, Control, Immunostaining, Derivative Assay, In Vivo, Expressing, Comparison

Journal: Cell reports

Article Title: Mutant KRAS-driven selective mRNA translation reveals mechanisms and therapeutic vulnerabilities in cancer

doi: 10.1016/j.celrep.2026.117520

Figure Lengend Snippet: (A and B) TE fold change analysis of common transcripts affected by sotorasib and CR-1-31B shows that mutant KRAS and EIF4A oppositely regulate the translation of these mRNAs. Most of the sotorasib TE down targets are TE upregulated following EIF4A inhibition with CR-1-31B. (C) TE fold change analysis of common translation factors between sotorasib and CR-1-31B reveals the upregulation of all the translation factors following CR-1-31B treatment, except for four proteins that are downregulated by CR-1-31B. (D and E) Relative luciferase activity driven by G-quadruplex (GQ) and GC-rich motifs in response to sotorasib, RMC-7977, or MRTX1133 treatment demonstrates that sotorasib induces GQ and GC-rich mRNA translation ( * p < 0.05 and *** p < 0.001). (F and G) Luciferase reporter assays using the full-length or mutated 5′UTR of EEF1A1 show altered luciferase activity upon CR-1-31B and Torin1 treatment in MiaPaca-2 cells (* p < 0.05 and *** p < 0.001). (H) Combination treatment of CR-1-31B and Torin1 with mutant KRAS inhibitors in a panel of cancer cell lines harboring either G12C or G12D KRAS mutations leads to an additive enhancement of cell death, based on the zero interaction potency (ZIP) model. (I) Summary of our findings shows that the mutant KRAS-dependent mechanism of translational control uncouples the regulation of translation machinery from the regulation of the rest of the mRNA translation. Mutant KRAS-dependent mechanism is distinct from the mTOR and EIF4A-dependent mechanism that depends on longer and structured 5′UTR features and controls the translation of a distinct subset of mRNAs.

Article Snippet: The KRAS G12D specific inhibitor, MRTX1133, was purchased from MedChemExpress (HY-134813).

Techniques: Mutagenesis, Inhibition, Luciferase, Activity Assay, Control

Journal: bioRxiv

Article Title: Programming Biomolecular Interactions with All-Atom Generative Model

doi: 10.64898/2026.03.12.711044

Figure Lengend Snippet: Small molecules, peptides, and nanobodies were designed to bind the same switch II binding site of KRAS G12D. a , Representative visualizations of the designed small molecules, peptides, and nanobodies, together with the best half-maximal inhibitory concentration (IC 50 ) relative to a reference ligand for this site or the dissociation constant (K d ) measured across tested generations. b , Chemical structure and three-dimensional binding geometry of KRAS G12D–compound-3 generated by our AnewOmni. c , IC 50 of KRAS G12D–compound-3 measured by the homogeneous time-resolved fluorescence (HTRF) assay. d-g , Structures of KRAS G12D-linpep-12, -5 and KRAS G12D-cycpep-5, -1, as well as their IC 50 to a reference ligand determined by HTRF. h-i , Structure of KRAS G12D-nanobody-7 and -1, as well as their binding affinities to KRAS G12D determined by BLI.

Article Snippet: The KRAS G12D proteins (SinoBiological, Cat#12259-H56E-B) were immobilized on a CM5 chip (GE Healthcare) through amide coupling in 10 mM NaOAc (pH 4.5) for 420 s at a flow rate of 10 μl/min.

Techniques: Binding Assay, Concentration Assay, Generated, Fluorescence, HTRF Assay

Journal: bioRxiv

Article Title: Fatty acid scavenging enables cancer escape from KRAS inhibition

doi: 10.64898/2026.04.01.715565

Figure Lengend Snippet: a-e, The indicated KRAS G12D -mutant cells ( a , c , e ) and organoids ( b , d ) were treated with increasing concentration of MRTX1133 (MRTX) or HRS4642 (HRS) for 72 h (cells) or 7 days (organoids). Total viable cells are presented relative to vehicle (Veh). IC 50 values are provided in Source Data. Parental HPAC, AsPC-1, SW1990, and No.6 cells are denoted as KS HPAC, AsPC-1, SW1990, and No.6, respectively. Acquired KRASi resistant HPAC, AsPC-1, SW1990, No.6 cells are labeled as AR. f - i , IB analysis of indicated proteins in human PDAC cells treated -/+ 100 nM MRTX for 2 h ( f , g ), or -/+ HRS at the indicated concentrations for 2 h ( h , i ). j , k , IB analysis of indicated proteins in human PDAC organoids treated -/+ 100 nM MRTX or HRS for 24 h. l , Viability of WT and KRAS-ablated (KRAS Δ ) KS and KR/AR cells is presented relative to day 0. IB analysis of KRAS in the same cells is shown below. Data in ( a - e and l) (n=3 independent experiments) are mean ± s.e.m. Statistical significance was determined using one-way ANOVA with Tukey post-hoc tests (SW1990, HPAC, and AsPC-1 in l ) or two-sided unpaired t-test (KP-4, PANC-1 in l ) based on data normality distribution. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. NS, not significant. Exact P values are shown in Source Data.

Article Snippet: 42 human PDAC specimens bearing KRAS G12D mutation were acquired from patients who were diagnosed with PDAC between January 2017 and May 2023 at The First Affiliated Hospital of Anhui Medical University (Hefei, Anhui, China).

Techniques: Mutagenesis, Concentration Assay, Labeling

Journal: bioRxiv

Article Title: Fatty acid scavenging enables cancer escape from KRAS inhibition

doi: 10.64898/2026.04.01.715565

Figure Lengend Snippet: a - c , RNA-seq analysis was performed on KS (AsPC1) and KR (PANC-1) PDAC cells treated -/+ MRTX for 24 h. ( a ) Venn diagram showing the overlap of differentially expressed genes between MRTX- and Veh-treated cells and between MRTX-treated AsPC1 and MRTX-treated PANC-1 cells. ( b ) KEGG enrichment analysis of selected overlapping genes (n = 1,339 genes) from ( a ), representing genes whose expression is specifically altered in intrinsic resistant cells upon MRTX treatment. ( c ) Heatmap showing mRNA expression of lipid metabolism-related genes. Blue, replicates with low expression; red, replicates with high expression. d , e , qPCR analysis of indicated mRNAs in the indicated cell lines or organoids treated -/+ MRTX for 24 h. f , LC-MS quantification of intracellular acyl-coenzyme A levels in PANC-1 cells treated -/+ MRTX for 24 h. g , Fractional labelling of TCA cycle intermediates in PANC-1 cells cultured with [U- 13 C]-PA, -/+ MRTX for 12 h. h , OCR of KS and AR AsPC-1 cells treated -/+ MRTX for 24 h before and after treatment with Omy, FCCP, and rotenone/antimycin A (ROT/AA). i , Total cellular ATP in indicated KS and KR/AR cells treated -/+ MRTX for 48 h. Data are presented relative to untreated cells. KP-4 is intrinsically KRASi resistant (KR). j , OCR of KR PANC-1 cells expressing DOX ACSBG1Δ treated -/+ DOX for 48h, followed with -/+ MRTX for 24 h before and after treatment with Omy, FCCP, and rotenone/antimycin A. k , Total cellular ATP in the indicated cells treated -/+ DOX for 48 h, followed with -/+ MRTX or HRS for 48 h. Data are presented relative to untreated cells. l , IB analysis of the indicated proteins in the indicated cells treated -/+ DOX for 48 h. Data in ( f , h - k ) (n=3 independent experiments) are mean ± s.e.m. Statistical significance was determined using two-sided unpaired t -test ( f , i KP-4) or one-way ANOVA with Tukey post-hoc tests ( i , k ) based on data normality distribution. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant. Exact P values are shown in Source Data.

Article Snippet: 42 human PDAC specimens bearing KRAS G12D mutation were acquired from patients who were diagnosed with PDAC between January 2017 and May 2023 at The First Affiliated Hospital of Anhui Medical University (Hefei, Anhui, China).

Techniques: RNA Sequencing, Expressing, Liquid Chromatography with Mass Spectroscopy, Cell Culture

Journal: bioRxiv

Article Title: Fatty acid scavenging enables cancer escape from KRAS inhibition

doi: 10.64898/2026.04.01.715565

Figure Lengend Snippet: a , LC-MS analysis of intracellular acyl-coenzyme A species in sensitive (KS) and acquired (AR) AsPC-1 cells -/+ 100 nM MRTX1133 (MRTX) for 24 h. Veh, Vehicle. b , Right: Fractional labelling of TCA cycle intermediates in KS/AR AsPC-1 cells incubated with [U- 13 C]-PA and -/+ MRTX for 12 h. α-KG, α-ketoglutarate. Left: A schematic illustration of PA-derived acetyl-CoA fuels the TCA cycle. Blue, replicates with low expression; red, replicates with high expression. c , Oxygen consumption rate (OCR) of KS and AR AsPC-1 cells incubated -/+ PA and 100 nM HRS-4642 (HRS) for 24 h before and after addition of 40 μM Etomoxir (Eto), followed by oligomycin (Omy), FCCP, and rotenone/antimycin A (ROT/AA) treatments. d , KS/AR HPAC cells expressing doxycycline (DOX)-inducible CPT1 deletion (DOX CPT1Δ ) were treated -/+ 0.2 μg/mL DOX for 48 h, and then -/+ HRS for 48 h. Total cellular ATP is relative to untreated KS cells. e , Representative images of mitochondria (TIM23) and BODIPY-labelled lipid droplets (LD) in parental KS No.6 and AR No.6 human PDAC organoids treated -/+ MRTX for 24 h. f , Quantification of mitochondria, LD, and their co-localization in ( e ). g , Pancreas morphology 3 weeks after orthotopic transplantation of KS and AR KC6141 (KC) cells expressing DOX CPT1Δ and host treatment at 72 h post-transplantation -/+ 30 mg/kg MRTX (i.p.), 0.2 mg/mL DOX (drinking water), or DOX + MRTX. h , Pancreas weight relative to body weight (P/B weight) of mice in ( g ). i , Representative H&E and IHC staining for CK19 in pancreata from ( g ). Boxed areas are further magnified. Tumor areas are at the bottom. j , Representative AFADESI MSI images and quantification of endogenous C16:0 L-carnitine (m/z 400.3436) and C18:1 L-carnitine (m/z 426.3559) distribution in pancreata two weeks after orthotopic transplantation of KS/AR KC cells expressing DOX CPT1Δ . Host were treated starting 10 days post-transplantation with MRTX, DOX, or DOX + MRTX. AR tumors exhibit elevated FAO and increased sensitivity to CPT1 ablation compared to KS tumors. Boxed regions indicate tumor areas. k , Pancreata from mice in ( j ) were sectioned and cultured with 100 μM [U- 13 C]-PA for 4 h. Representative MALDI MSI images and quantification of unlabeled citrate (M + 0, m/z 191.019) and 13 C-citrate (M + 2, m/z 193.026) distribution in pancreata, showing a similar trend to ( j ). Data in ( a , c , d , k ) (n=3 independent experiments), ( f ) (n=25 fields), ( h ) (n = 5 mice), ( i ) (n=7 fields), and ( j ) (n=6 fields) are mean ± s.e.m. Statistical significance was determined using one-way analysis of variance (ANOVA) with Tukey post-hoc tests ( a , d , f , h - k ) based on data normality distribution. Exact P values are shown in the Source Data. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. NS, not significant. Scale bars ( e ) 40 μm, ( g ) 1 cm, ( i ) 100 μm, ( j , k ) 500 μm.

Article Snippet: 42 human PDAC specimens bearing KRAS G12D mutation were acquired from patients who were diagnosed with PDAC between January 2017 and May 2023 at The First Affiliated Hospital of Anhui Medical University (Hefei, Anhui, China).

Techniques: Liquid Chromatography with Mass Spectroscopy, Incubation, Derivative Assay, Expressing, Transplantation Assay, Immunohistochemistry, Cell Culture

Journal: bioRxiv

Article Title: Fatty acid scavenging enables cancer escape from KRAS inhibition

doi: 10.64898/2026.04.01.715565

Figure Lengend Snippet: a , b , Genes differentially expressed between KS (AsPC-1, SW1990) and KR/AR (PANC-1, SW1990) cells treated -/+ MRTX for 24 h. Blue, replicates with low expression; red, replicates with high expression. c , d , IB analysis of ADGRB1 in indicated organoids or cell lines treated -/+ HRS for 24 h ( c ) or -/+ DOX for 48 h ( d ). e , KS and AR cells expressing DOX ADGRB1Δ , SRF-responsive luciferase reporter (SRF-RE), and a Renilla-luciferase control were treated -/+ DOX for 48 h, followed by incubation with D-Luciferin. Normalized Firefly/Renilla luciferase ratios, reflecting ADGRB1 (Gα12/13) activity, are shown relative to untreated KS cells. f , Fractional labelling of TCA cycle intermediates in KS and AR HPAC cells expressing DOX ADGRB1Δ treated -/+ DOX for 48 h, and then incubated with [U- 13 C]-PA and MRTX for 12 h. Blue, replicates with low expression; red, replicates with high expression. g , Representative IHC of resected ADGRB1 hi (#556) and ADGRB1 lo (# 835) human PDAC tissues. Boxed areas are further magnified. h , Numbers of human PDAC specimens (n = 42) positive for the indicated proteins, indicated as low and high expression. i , Correlation between expression levels of the indicated proteins in human specimens examined by a two-tailed Chi-square test. j , Comparisons of overall survival between patients with PDAC stratified according to ADGRB1 and pS144-PAK1. Significance was determined by log-rank test. Data in ( e ) (n=3 independent experiments) are mean ± s.e.m. Statistical significance was determined using one-way ANOVA with Tukey post-hoc tests ( e ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Exact P values are shown in Source Data.

Article Snippet: 42 human PDAC specimens bearing KRAS G12D mutation were acquired from patients who were diagnosed with PDAC between January 2017 and May 2023 at The First Affiliated Hospital of Anhui Medical University (Hefei, Anhui, China).

Techniques: Expressing, Luciferase, Control, Incubation, Activity Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Fatty acid scavenging enables cancer escape from KRAS inhibition

doi: 10.64898/2026.04.01.715565

Figure Lengend Snippet: a , Immunoblot (IB) analysis of indicated proteins in acquired KRASi-resistant SW1990 cells (AR) -/+ knock down (KD) of indicated GPCRs and -/+ MRTX for 2 h. b , AR and KS KC cells expressing DOX ADGRB1Δ were orthotopically transplanted into mice -/+ 7 days MRTX, DOX, or DOX + MRTX, initiated 72 h post-transplantation. Then the BODIPY signal in PDAC cells was analyzed by flow cytometry 24 h after i.p. of BODIPY-PA loaded adipocytes (3T3-L1). The experimental scheme is shown to the left. c , IB analysis of indicated proteins in the indicated KS and AR AsPC-1 cells -/+ Flag-tagged PI3Kγ R1021C expression and -/+ DOX treatment for 48 h, followed by -/+ MRTX for 2 h. d , Representative images and quantification of MP in TMR-DEX-incubated cells from ( c ). e , OCR of cells in ( c ) treated -/+ DOX for 48 h, followed by -/+ MRTX or EIPA for 24 h before and after -/+ PA incubation, and treatment with Omy, FCCP, and rotenone/antimycin A. f , Representative H&E and IHC staining of resected human PDAC tissues classified as KS or KR based on organoid sensitivity to KRASi . Boxed areas are further magnified. g , Correlations between the indicated proteins and KRASi sensitivity in ( f ) were analyzed by a two-tailed Chi-square test. Data in b (n=3 mice), d (n=20 fields) and e (n=3 independent experiments) are mean ± s.e.m. Statistical significance was determined using Brown-Forsythe and Welch ANOVA tests with Dunnett T3 test ( d ) based on data normality distribution. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Exact P values are shown in Source Data. Scale bars ( d ) 10 μm, ( g ) 100 μm.

Article Snippet: 42 human PDAC specimens bearing KRAS G12D mutation were acquired from patients who were diagnosed with PDAC between January 2017 and May 2023 at The First Affiliated Hospital of Anhui Medical University (Hefei, Anhui, China).

Techniques: Western Blot, Knockdown, Expressing, Transplantation Assay, Flow Cytometry, Incubation, Immunohistochemistry, Two Tailed Test

Journal: bioRxiv

Article Title: Fatty acid scavenging enables cancer escape from KRAS inhibition

doi: 10.64898/2026.04.01.715565

Figure Lengend Snippet: a , IB analysis of indicated proteins in KRAS G12C mutated KS and AR MIA PaCa-2 cells (MIA) or KRAS G12D mutated GP2D cells -/+ 100 nM of the KRAS G12C i AMG510 or the KRAS G12D i HRS for 24 h. b , Representative images and quantification of MP in KS/AR GP2D cells expressing DOX ADGRB1Δ treated -/+ DOX for 48h, followed by treatment -/+ 1 μM PI3Kγi IPI549 (IPI), 1 μM PI3Kαi LY494002 (LY), HRS, HRS + IPI, or HRS + LY for 24 h. c , OCR of indicated cells -/+ DOX for 48h, followed by -/+ AMG510 (MIA) or HRS (GP2D) for 24 h, before and after -/+ PA, followed by Omy, FCCP, and rotenone/antimycin A treatments. d , e , Total cellular ATP in KS and AR MIA PaCa-2 or GP2D cells expressing DOX ADGRB1Δ treated with the indicated compounds for 48h. f , Representative IHC and quantification in resected human colon adenocarcinoma tissues before and after KRAS G12D i QLC1101 treatment. Data in ( b ) (n=15 fields), ( c , d , e ) (n=3 independent experiments), and ( f ) (n=6 fields) are mean ± s.e.m. Statistical significance was determined using one-way ANOVA with Brown-Forsythe and Welch corrections followed by Dunnett T3 test ( b ), or with Tukey post-hoc tests ( d , e ), or two-sided unpaired t -test or Mann-Whitney U -test ( f ) based on data normality distribution. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Exact P values are shown in Source Data. Scale bars ( b ) 20 μm, ( f ) 100 μm.

Article Snippet: 42 human PDAC specimens bearing KRAS G12D mutation were acquired from patients who were diagnosed with PDAC between January 2017 and May 2023 at The First Affiliated Hospital of Anhui Medical University (Hefei, Anhui, China).

Techniques: Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Fatty acid scavenging enables cancer escape from KRAS inhibition

doi: 10.64898/2026.04.01.715565

Figure Lengend Snippet: a , KS/AR No.6 organoids were treated with IPI and/or HRS at the indicated concentrations for 7 days, and cell viability was assessed with CellTiter-Glo (CTG). Upper: Dose-response matrix (viability) for IPI and HRS. Blue intensity indicates the degree of inhibition. Lower: 3D synergy plots generated by SynergyFinder + . The synergy score is shown by red (>0) and green (<0). The synergy score > 10 indicates strong synergic effect. b , Akaluciferase bioluminescence imaging of mice 3 weeks after orthotopic transplantation of KS or AR KC cells expressing DOX ADGRB1Δ and a Ki67-akaluciferase reporter, -/+ MRTX, DOX, or DOX + MRTX treatments initiated 72 h post-transplantation. Quantification of akaluciferase activity, expressed as relative light units (RLU), is on the right. c , P/B ratio of mice from ( b ). d , Representative H&E and CK19 IHC staining of pancreata from ( b ). Boxed areas are further magnified. e , Quantification of tumor areas from ( d ). f , Pancreas morphology and P/B ratio 3 weeks after orthotopic transplantation of indicated cells into mice -/+ MRTX, 15 mg/kg IPI (p.o.), or MRTX + IPI treatments initiated 72 h post-transplantation. g , Representative H&E and CK19 staining of pancreata from ( f ). Boxed areas are further magnified. Quantification of tumor areas is shown to the right. h , Flow cytometry analysis of BODIPY signals in PDAC cells harvested 24 h after i.p. injection of BODIPY-PA-loaded adipocytes into mice from ( f ) at day 7 post-KS and AR KC cell implantation. Data in ( b ) (n=5 mice), ( c , f ) (n=5-7 mice), ( e , g ) (n=6 fields), and ( h ) (n=3 mice) are mean ± s.e.m. Statistical significance was determined using one-way ANOVA with Tukey post-hoc tests. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Exact P values are shown in Source Data. Scale bars ( d , g ) 100 μm, ( f ) 1 cm.

Article Snippet: 42 human PDAC specimens bearing KRAS G12D mutation were acquired from patients who were diagnosed with PDAC between January 2017 and May 2023 at The First Affiliated Hospital of Anhui Medical University (Hefei, Anhui, China).

Techniques: Inhibition, Generated, Imaging, Transplantation Assay, Expressing, Activity Assay, Immunohistochemistry, Staining, Flow Cytometry, Injection

Journal: bioRxiv

Article Title: Fatty acid scavenging enables cancer escape from KRAS inhibition

doi: 10.64898/2026.04.01.715565

Figure Lengend Snippet: a-e, The indicated KRAS G12D -mutant cells ( a , c , e ) and organoids ( b , d ) were treated with increasing concentration of MRTX1133 (MRTX) or HRS4642 (HRS) for 72 h (cells) or 7 days (organoids). Total viable cells are presented relative to vehicle (Veh). IC 50 values are provided in Source Data. Parental HPAC, AsPC-1, SW1990, and No.6 cells are denoted as KS HPAC, AsPC-1, SW1990, and No.6, respectively. Acquired KRASi resistant HPAC, AsPC-1, SW1990, No.6 cells are labeled as AR. f - i , IB analysis of indicated proteins in human PDAC cells treated -/+ 100 nM MRTX for 2 h ( f , g ), or -/+ HRS at the indicated concentrations for 2 h ( h , i ). j , k , IB analysis of indicated proteins in human PDAC organoids treated -/+ 100 nM MRTX or HRS for 24 h. l , Viability of WT and KRAS-ablated (KRAS Δ ) KS and KR/AR cells is presented relative to day 0. IB analysis of KRAS in the same cells is shown below. Data in ( a - e and l) (n=3 independent experiments) are mean ± s.e.m. Statistical significance was determined using one-way ANOVA with Tukey post-hoc tests (SW1990, HPAC, and AsPC-1 in l ) or two-sided unpaired t-test (KP-4, PANC-1 in l ) based on data normality distribution. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. NS, not significant. Exact P values are shown in Source Data.

Article Snippet: A total of 16 specimens of human PDAC bearing KRAS G12D mutation were acquired from patients who were diagnosed with PDAC between July 2023 and September 2025 at The First Affiliated Hospital of Anhui Medical University.

Techniques: Mutagenesis, Concentration Assay, Labeling

Journal: bioRxiv

Article Title: Fatty acid scavenging enables cancer escape from KRAS inhibition

doi: 10.64898/2026.04.01.715565

Figure Lengend Snippet: a - c , RNA-seq analysis was performed on KS (AsPC1) and KR (PANC-1) PDAC cells treated -/+ MRTX for 24 h. ( a ) Venn diagram showing the overlap of differentially expressed genes between MRTX- and Veh-treated cells and between MRTX-treated AsPC1 and MRTX-treated PANC-1 cells. ( b ) KEGG enrichment analysis of selected overlapping genes (n = 1,339 genes) from ( a ), representing genes whose expression is specifically altered in intrinsic resistant cells upon MRTX treatment. ( c ) Heatmap showing mRNA expression of lipid metabolism-related genes. Blue, replicates with low expression; red, replicates with high expression. d , e , qPCR analysis of indicated mRNAs in the indicated cell lines or organoids treated -/+ MRTX for 24 h. f , LC-MS quantification of intracellular acyl-coenzyme A levels in PANC-1 cells treated -/+ MRTX for 24 h. g , Fractional labelling of TCA cycle intermediates in PANC-1 cells cultured with [U- 13 C]-PA, -/+ MRTX for 12 h. h , OCR of KS and AR AsPC-1 cells treated -/+ MRTX for 24 h before and after treatment with Omy, FCCP, and rotenone/antimycin A (ROT/AA). i , Total cellular ATP in indicated KS and KR/AR cells treated -/+ MRTX for 48 h. Data are presented relative to untreated cells. KP-4 is intrinsically KRASi resistant (KR). j , OCR of KR PANC-1 cells expressing DOX ACSBG1Δ treated -/+ DOX for 48h, followed with -/+ MRTX for 24 h before and after treatment with Omy, FCCP, and rotenone/antimycin A. k , Total cellular ATP in the indicated cells treated -/+ DOX for 48 h, followed with -/+ MRTX or HRS for 48 h. Data are presented relative to untreated cells. l , IB analysis of the indicated proteins in the indicated cells treated -/+ DOX for 48 h. Data in ( f , h - k ) (n=3 independent experiments) are mean ± s.e.m. Statistical significance was determined using two-sided unpaired t -test ( f , i KP-4) or one-way ANOVA with Tukey post-hoc tests ( i , k ) based on data normality distribution. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant. Exact P values are shown in Source Data.

Article Snippet: A total of 16 specimens of human PDAC bearing KRAS G12D mutation were acquired from patients who were diagnosed with PDAC between July 2023 and September 2025 at The First Affiliated Hospital of Anhui Medical University.

Techniques: RNA Sequencing, Expressing, Liquid Chromatography with Mass Spectroscopy, Cell Culture

Journal: bioRxiv

Article Title: Fatty acid scavenging enables cancer escape from KRAS inhibition

doi: 10.64898/2026.04.01.715565

Figure Lengend Snippet: a , LC-MS analysis of intracellular acyl-coenzyme A species in sensitive (KS) and acquired (AR) AsPC-1 cells -/+ 100 nM MRTX1133 (MRTX) for 24 h. Veh, Vehicle. b , Right: Fractional labelling of TCA cycle intermediates in KS/AR AsPC-1 cells incubated with [U- 13 C]-PA and -/+ MRTX for 12 h. α-KG, α-ketoglutarate. Left: A schematic illustration of PA-derived acetyl-CoA fuels the TCA cycle. Blue, replicates with low expression; red, replicates with high expression. c , Oxygen consumption rate (OCR) of KS and AR AsPC-1 cells incubated -/+ PA and 100 nM HRS-4642 (HRS) for 24 h before and after addition of 40 μM Etomoxir (Eto), followed by oligomycin (Omy), FCCP, and rotenone/antimycin A (ROT/AA) treatments. d , KS/AR HPAC cells expressing doxycycline (DOX)-inducible CPT1 deletion (DOX CPT1Δ ) were treated -/+ 0.2 μg/mL DOX for 48 h, and then -/+ HRS for 48 h. Total cellular ATP is relative to untreated KS cells. e , Representative images of mitochondria (TIM23) and BODIPY-labelled lipid droplets (LD) in parental KS No.6 and AR No.6 human PDAC organoids treated -/+ MRTX for 24 h. f , Quantification of mitochondria, LD, and their co-localization in ( e ). g , Pancreas morphology 3 weeks after orthotopic transplantation of KS and AR KC6141 (KC) cells expressing DOX CPT1Δ and host treatment at 72 h post-transplantation -/+ 30 mg/kg MRTX (i.p.), 0.2 mg/mL DOX (drinking water), or DOX + MRTX. h , Pancreas weight relative to body weight (P/B weight) of mice in ( g ). i , Representative H&E and IHC staining for CK19 in pancreata from ( g ). Boxed areas are further magnified. Tumor areas are at the bottom. j , Representative AFADESI MSI images and quantification of endogenous C16:0 L-carnitine (m/z 400.3436) and C18:1 L-carnitine (m/z 426.3559) distribution in pancreata two weeks after orthotopic transplantation of KS/AR KC cells expressing DOX CPT1Δ . Host were treated starting 10 days post-transplantation with MRTX, DOX, or DOX + MRTX. AR tumors exhibit elevated FAO and increased sensitivity to CPT1 ablation compared to KS tumors. Boxed regions indicate tumor areas. k , Pancreata from mice in ( j ) were sectioned and cultured with 100 μM [U- 13 C]-PA for 4 h. Representative MALDI MSI images and quantification of unlabeled citrate (M + 0, m/z 191.019) and 13 C-citrate (M + 2, m/z 193.026) distribution in pancreata, showing a similar trend to ( j ). Data in ( a , c , d , k ) (n=3 independent experiments), ( f ) (n=25 fields), ( h ) (n = 5 mice), ( i ) (n=7 fields), and ( j ) (n=6 fields) are mean ± s.e.m. Statistical significance was determined using one-way analysis of variance (ANOVA) with Tukey post-hoc tests ( a , d , f , h - k ) based on data normality distribution. Exact P values are shown in the Source Data. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. NS, not significant. Scale bars ( e ) 40 μm, ( g ) 1 cm, ( i ) 100 μm, ( j , k ) 500 μm.

Article Snippet: A total of 16 specimens of human PDAC bearing KRAS G12D mutation were acquired from patients who were diagnosed with PDAC between July 2023 and September 2025 at The First Affiliated Hospital of Anhui Medical University.

Techniques: Liquid Chromatography with Mass Spectroscopy, Incubation, Derivative Assay, Expressing, Transplantation Assay, Immunohistochemistry, Cell Culture

Journal: bioRxiv

Article Title: Fatty acid scavenging enables cancer escape from KRAS inhibition

doi: 10.64898/2026.04.01.715565

Figure Lengend Snippet: a , b , Genes differentially expressed between KS (AsPC-1, SW1990) and KR/AR (PANC-1, SW1990) cells treated -/+ MRTX for 24 h. Blue, replicates with low expression; red, replicates with high expression. c , d , IB analysis of ADGRB1 in indicated organoids or cell lines treated -/+ HRS for 24 h ( c ) or -/+ DOX for 48 h ( d ). e , KS and AR cells expressing DOX ADGRB1Δ , SRF-responsive luciferase reporter (SRF-RE), and a Renilla-luciferase control were treated -/+ DOX for 48 h, followed by incubation with D-Luciferin. Normalized Firefly/Renilla luciferase ratios, reflecting ADGRB1 (Gα12/13) activity, are shown relative to untreated KS cells. f , Fractional labelling of TCA cycle intermediates in KS and AR HPAC cells expressing DOX ADGRB1Δ treated -/+ DOX for 48 h, and then incubated with [U- 13 C]-PA and MRTX for 12 h. Blue, replicates with low expression; red, replicates with high expression. g , Representative IHC of resected ADGRB1 hi (#556) and ADGRB1 lo (# 835) human PDAC tissues. Boxed areas are further magnified. h , Numbers of human PDAC specimens (n = 42) positive for the indicated proteins, indicated as low and high expression. i , Correlation between expression levels of the indicated proteins in human specimens examined by a two-tailed Chi-square test. j , Comparisons of overall survival between patients with PDAC stratified according to ADGRB1 and pS144-PAK1. Significance was determined by log-rank test. Data in ( e ) (n=3 independent experiments) are mean ± s.e.m. Statistical significance was determined using one-way ANOVA with Tukey post-hoc tests ( e ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Exact P values are shown in Source Data.

Article Snippet: A total of 16 specimens of human PDAC bearing KRAS G12D mutation were acquired from patients who were diagnosed with PDAC between July 2023 and September 2025 at The First Affiliated Hospital of Anhui Medical University.

Techniques: Expressing, Luciferase, Control, Incubation, Activity Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Fatty acid scavenging enables cancer escape from KRAS inhibition

doi: 10.64898/2026.04.01.715565

Figure Lengend Snippet: a , Immunoblot (IB) analysis of indicated proteins in acquired KRASi-resistant SW1990 cells (AR) -/+ knock down (KD) of indicated GPCRs and -/+ MRTX for 2 h. b , AR and KS KC cells expressing DOX ADGRB1Δ were orthotopically transplanted into mice -/+ 7 days MRTX, DOX, or DOX + MRTX, initiated 72 h post-transplantation. Then the BODIPY signal in PDAC cells was analyzed by flow cytometry 24 h after i.p. of BODIPY-PA loaded adipocytes (3T3-L1). The experimental scheme is shown to the left. c , IB analysis of indicated proteins in the indicated KS and AR AsPC-1 cells -/+ Flag-tagged PI3Kγ R1021C expression and -/+ DOX treatment for 48 h, followed by -/+ MRTX for 2 h. d , Representative images and quantification of MP in TMR-DEX-incubated cells from ( c ). e , OCR of cells in ( c ) treated -/+ DOX for 48 h, followed by -/+ MRTX or EIPA for 24 h before and after -/+ PA incubation, and treatment with Omy, FCCP, and rotenone/antimycin A. f , Representative H&E and IHC staining of resected human PDAC tissues classified as KS or KR based on organoid sensitivity to KRASi . Boxed areas are further magnified. g , Correlations between the indicated proteins and KRASi sensitivity in ( f ) were analyzed by a two-tailed Chi-square test. Data in b (n=3 mice), d (n=20 fields) and e (n=3 independent experiments) are mean ± s.e.m. Statistical significance was determined using Brown-Forsythe and Welch ANOVA tests with Dunnett T3 test ( d ) based on data normality distribution. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Exact P values are shown in Source Data. Scale bars ( d ) 10 μm, ( g ) 100 μm.

Article Snippet: A total of 16 specimens of human PDAC bearing KRAS G12D mutation were acquired from patients who were diagnosed with PDAC between July 2023 and September 2025 at The First Affiliated Hospital of Anhui Medical University.

Techniques: Western Blot, Knockdown, Expressing, Transplantation Assay, Flow Cytometry, Incubation, Immunohistochemistry, Two Tailed Test

Journal: bioRxiv

Article Title: Fatty acid scavenging enables cancer escape from KRAS inhibition

doi: 10.64898/2026.04.01.715565

Figure Lengend Snippet: a , IB analysis of indicated proteins in KRAS G12C mutated KS and AR MIA PaCa-2 cells (MIA) or KRAS G12D mutated GP2D cells -/+ 100 nM of the KRAS G12C i AMG510 or the KRAS G12D i HRS for 24 h. b , Representative images and quantification of MP in KS/AR GP2D cells expressing DOX ADGRB1Δ treated -/+ DOX for 48h, followed by treatment -/+ 1 μM PI3Kγi IPI549 (IPI), 1 μM PI3Kαi LY494002 (LY), HRS, HRS + IPI, or HRS + LY for 24 h. c , OCR of indicated cells -/+ DOX for 48h, followed by -/+ AMG510 (MIA) or HRS (GP2D) for 24 h, before and after -/+ PA, followed by Omy, FCCP, and rotenone/antimycin A treatments. d , e , Total cellular ATP in KS and AR MIA PaCa-2 or GP2D cells expressing DOX ADGRB1Δ treated with the indicated compounds for 48h. f , Representative IHC and quantification in resected human colon adenocarcinoma tissues before and after KRAS G12D i QLC1101 treatment. Data in ( b ) (n=15 fields), ( c , d , e ) (n=3 independent experiments), and ( f ) (n=6 fields) are mean ± s.e.m. Statistical significance was determined using one-way ANOVA with Brown-Forsythe and Welch corrections followed by Dunnett T3 test ( b ), or with Tukey post-hoc tests ( d , e ), or two-sided unpaired t -test or Mann-Whitney U -test ( f ) based on data normality distribution. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Exact P values are shown in Source Data. Scale bars ( b ) 20 μm, ( f ) 100 μm.

Article Snippet: A total of 16 specimens of human PDAC bearing KRAS G12D mutation were acquired from patients who were diagnosed with PDAC between July 2023 and September 2025 at The First Affiliated Hospital of Anhui Medical University.

Techniques: Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Fatty acid scavenging enables cancer escape from KRAS inhibition

doi: 10.64898/2026.04.01.715565

Figure Lengend Snippet: a , KS/AR No.6 organoids were treated with IPI and/or HRS at the indicated concentrations for 7 days, and cell viability was assessed with CellTiter-Glo (CTG). Upper: Dose-response matrix (viability) for IPI and HRS. Blue intensity indicates the degree of inhibition. Lower: 3D synergy plots generated by SynergyFinder + . The synergy score is shown by red (>0) and green (<0). The synergy score > 10 indicates strong synergic effect. b , Akaluciferase bioluminescence imaging of mice 3 weeks after orthotopic transplantation of KS or AR KC cells expressing DOX ADGRB1Δ and a Ki67-akaluciferase reporter, -/+ MRTX, DOX, or DOX + MRTX treatments initiated 72 h post-transplantation. Quantification of akaluciferase activity, expressed as relative light units (RLU), is on the right. c , P/B ratio of mice from ( b ). d , Representative H&E and CK19 IHC staining of pancreata from ( b ). Boxed areas are further magnified. e , Quantification of tumor areas from ( d ). f , Pancreas morphology and P/B ratio 3 weeks after orthotopic transplantation of indicated cells into mice -/+ MRTX, 15 mg/kg IPI (p.o.), or MRTX + IPI treatments initiated 72 h post-transplantation. g , Representative H&E and CK19 staining of pancreata from ( f ). Boxed areas are further magnified. Quantification of tumor areas is shown to the right. h , Flow cytometry analysis of BODIPY signals in PDAC cells harvested 24 h after i.p. injection of BODIPY-PA-loaded adipocytes into mice from ( f ) at day 7 post-KS and AR KC cell implantation. Data in ( b ) (n=5 mice), ( c , f ) (n=5-7 mice), ( e , g ) (n=6 fields), and ( h ) (n=3 mice) are mean ± s.e.m. Statistical significance was determined using one-way ANOVA with Tukey post-hoc tests. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Exact P values are shown in Source Data. Scale bars ( d , g ) 100 μm, ( f ) 1 cm.

Article Snippet: A total of 16 specimens of human PDAC bearing KRAS G12D mutation were acquired from patients who were diagnosed with PDAC between July 2023 and September 2025 at The First Affiliated Hospital of Anhui Medical University.

Techniques: Inhibition, Generated, Imaging, Transplantation Assay, Expressing, Activity Assay, Immunohistochemistry, Staining, Flow Cytometry, Injection

Journal: Nature Metabolism

Article Title: NADPH-producing enzymes restrict the formation of pancreatic precancerous lesions

doi: 10.1038/s42255-026-01496-x

Figure Lengend Snippet: a , Scheme of the experimental model. Pancreata were dissected from LSL- Kras G12D ; Ptf1a Cre ERTM mice ( n = 3) and acinar clusters were isolated for primary, ex vivo culture. Acinar clusters were grown free-floating in media and were treated with vehicle or 2 μM 4-OHT to induce mutant Kras . Cells were collected after 1, 2 or 3 days in culture. For each timepoint, cells from one well were divided for RNA extraction and metabolite analysis. b , Volcano plots of differentially expressed genes at day 2 and day 3 following 4-OHT treatment compared to day 1 vehicle. Significantly upregulated genes are red; significantly downregulated genes are blue. False discovery rate (FDR) < 0.05 was considered significant, log 2 (fold change (FC)) > 0.5 was considered upregulated andlog 2 (FC) < –0.5 was considered downregulated. Differential gene expression was estimated using the quasi-likelihood negative binomial generalized log-linear approach in edgeR. c , Volcano plots of differentially abundant metabolites when comparing day 2 and day 3 following 4-OHT treatment compared to day 1 vehicle. Significantly increased intracellular metabolites are red; decreased metabolites are blue. P < 0.1 was considered significant, log 2 (FC) > 0.5 was considered increased and log 2 (FC) < –0.5 was considered decreased. P values were calculated using a Student’s t -test (unpaired, two-tailed). d , Significant metabolism-related pathways at day 2, identified by gene set enrichment analysis (GSEA) from KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways. Blue bars highlight pathways of interest. e , Heatmap of detected metabolites in the TCA cycle. Scale represents log 2 (FC) of median normalized abundance relative to vehicle. f , Violin plot of the leading-edge genes in the TCA cycle KEGG pathway. The y axis represents log(FC) relative to vehicle. Lines inside the plots represent the quartiles and median. g , Violin plot of the leading-edge genes in the oxidative phosphorylation (OxPhos) KEGG pathway. The y axis represents log(FC) relative to vehicle. Lines inside the plots represent the quartiles and median. h , Heatmap of metabolites related to glycolysis and the PPP. Scale represents log 2 (FC) of median normalized abundance relative to vehicle. i , Violin plot of the leading-edge genes in the glycolysis and gluconeogenesis KEGG pathway. The y axis represents log(FC) relative to vehicle. Lines inside the plots represent the quartiles and median. j , Violin plot the leading-edge genes in the PPP KEGG pathways. The y axis represents log(FC) relative to vehicle. Lines inside the plots represent the quartiles and median. k , Heatmap of metabolites related to glutathione metabolism. Scale represents log 2 (FC) of median normalized abundance relative to vehicle. GSSG, oxidized glutathione. l , Violin plot of leading-edge genes in the glutathione metabolism KEGG pathway. The y axis represents log(FC) relative to vehicle. Lines inside the plots represent the quartiles and median. m , Transcription factor enrichment analysis generated using Enrichr from ENCODE and ChEA consensus transcription factors. The top 500 upregulated genes from day 2 were analysed. The blue bar highlights a transcription factor of interest. Enrichr was used to identify differentially enriched pathways and calculate P values with Fisher’s exact test. n , Heatmap showing log(FC) of significantly differentially expressed NRF2-target genes. Genes under investigation are indicated with blue text.

Article Snippet: LSL -Kras G12D mice (Jackson Laboratory, strain no. 008179) were crossed with Ptf1a CreERTM mice to generate LSL- Kras G12D ; Ptf1a CreERTM mice and maintained on a C57BL/6J background.

Techniques: Isolation, Ex Vivo, Mutagenesis, RNA Extraction, Gene Expression, Two Tailed Test, Phospho-proteomics, Generated

Journal: Nature Metabolism

Article Title: NADPH-producing enzymes restrict the formation of pancreatic precancerous lesions

doi: 10.1038/s42255-026-01496-x

Figure Lengend Snippet: a . Scheme of the experimental model. Pancreata were dissected from Wildtype mice and acinar clusters were isolated for primary, ex vivo culture. Cells were collected after dissociation (Day 0) or grown free-floating in media and treated with TGFα (50 ng/mL), with or without 4-hydroxytamoxifen (4-OHT; 2 μM) for 3 days. Relative expression of acinar gene, Amy ( b .) and ductal gene, Spink1 ( c .) using real-time quantitative PCR. Bars represent the mean of n = 3 biological replicates for Day 0, n = 2 biological replicates for Day 3 and Day 3 + 4-OHT. d . Scheme of the experimental model. Pancreata were dissected from LSL-Kras G12D mice (n = 3) and acinar clusters were isolated for primary, ex vivo culture. Acinar clusters were grown free-floating in media and were treated with adenovirus expressing GFP (ad-GFP) or adenovirus expressing Cre recombinase (ad-Cre) to induce mutant Kras . Cells were collected after 1, 2, or 3 days in culture. e . Heatmap of acinar and ductal gene expression. Scale represents log2FC in expression relative to ad-GFP. f . Heatmap showing log2FC of key NRF2-target genes, as referenced from Fig. . Individual datapoints for log2FC of gene expression of G6pdx ( g .) and Me1 ( h .) from ADM cultures from each mouse (n = 3) after ad-Cre for 1, 2, or 3 days (relative to ad-GFP). Bars represent the mean with standard deviation. P-values were calculated using ordinary one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: LSL -Kras G12D mice (Jackson Laboratory, strain no. 008179) were crossed with Ptf1a CreERTM mice to generate LSL- Kras G12D ; Ptf1a CreERTM mice and maintained on a C57BL/6J background.

Techniques: Isolation, Ex Vivo, Expressing, Real-time Polymerase Chain Reaction, Mutagenesis, Gene Expression, Standard Deviation

Journal: Nature Metabolism

Article Title: NADPH-producing enzymes restrict the formation of pancreatic precancerous lesions

doi: 10.1038/s42255-026-01496-x

Figure Lengend Snippet: a . Mice with mutant G6pd , that mimics human G6PD-deficiency, were bred into the KC ( LSL-Kras G12D ; Ptf1a Cre ) line. Cre was always maintained in female breeders. Experiments used both male and female G6pd mutant mice, where females were homozygous for mutant G6pd ( G6pd mut/mut ) and males were hemizygous for mutant G6pd ( G6pd mut/y ), as G6pd is an X-linked gene. G6pd wildtype mice used in experiments were obtained in the same colony and age matched littermates of G6pd mutant mice when possible. Female G6pd wildtype mice have two wildtype copies of G6pd ( G6pd wt/wt ) and males have one wildtype copy (their only copy) of G6pd ( G6pd wt/y ). In the schematics and labelling, “y” in male mice refers to the y chromosome, which does not contain a copy of G6pd . b . Schematic of 14 C-labeling experiment. [1- 14 C]glucose (blue) can be used in both the oxidative pentose phosphate pathway (ox PPP) and the TCA cycle. [6- 14 C]glucose (red) can be used in the TCA cycle. Released CO 2 represented as clouds; G6PD mutation represented as star. c . Graph depicting the relative amount of [ 14 C]-labeled CO 2 derived from glucose tracing in G6pd mut mice relative to G6pd wt mice (n = 2 biological replicates). Blue bar shows the relative [ 14 C]-labeled CO 2 derived from [1- 14 C]glucose, which is generated from either the oxidative pentose phosphate pathway (oxPPP) or the TCA cycle. Red bar shows the relative amount of [ 14 C]-labeled CO 2 derived from [6- 14 C]glucose, which is only generated from the TCA cycle. Grey bar shows the ratio of [ 14 C]-labeled CO 2 derived from [1- 14 C]O 2 to [6- 14 C]O 2 representing flux from the oxPPP. Bars represent the biological replicates’ mean. d . Hematoxylin & eosin (H&E) staining and immunostaining for amylase (AMY; acinar cells), cytokeratin 19 (CK19; ductal, metaplastic, neoplastic cells), and KI67 (proliferation) in pancreata from 8-week-old KC;G6pd wt and KC;G6pd mut mice. Scale bar = 50μm. e . Percent of Amylase+ area in pancreas as quantified from male (closed circle) and female (open circle) mice in KC;G6pd wt (grey bar) and KC;G6pd mut (blue bar) mice at 8 weeks. n = 4 mice for each genotype. Bars represent the mean with standard deviation. P-values were calculated using a Student’s t-test (unpaired, two-tailed). f . Percent of CK19+ cells and g . Ki67+ cells in the pancreas as quantified from male (closed circle) and female (open circle) KC;G6pd wt (grey bar) and KC;G6pd mut (blue bar) mice at 8 weeks. n = 5 mice for each genotype. Bars represent the mean with standard deviation. P-values were calculated using a Student’s t-test (unpaired, two-tailed). h . Immunostaining for Amylase (AMY), in 8-week and 16-week-old KC;G6pd wt and KC;G6pd mut pancreas. Alcian blue (PanIN-produced mucin) & nuclear fast red counterstain in pancreas of 26-week-old KC;G6pd wt and KC;G6pd mut mice. Scale bar = 500μm. i . Pancreas weight (PW) to body weight (BW) ratios in 8-, 16-, and 26-week-old KC;G6pd wt and KC;G6pd mut mice. Bars represent the mean with standard deviation. Ratios are not significantly different, as calculated using a Student’s t-test (unpaired, two-tailed) for each age. Sample sizes for each age are as follows: 8 week, n = 11 KC;G6pd wt and n = 8 KC;G6pd mut ; 16 week, n = 9 KC;G6pd wt and n = 8 KC;G6pd mut ; 26 week, n = 8 KC;G6pd wt and n = 8 KC;G6pd mut . j . Pathological grading of 26-week-old KC;G6pd wt and KC;G6pd mut pancreas tissues representing the % of total tissue area with acinar cells, ADM, and PanIN lesions. n = 5 KC;G6pd wt mice; n = 7 KC;G6pd mut mice. Bars represent the mean with standard deviation. P-values were calculated using a two-way ANOVA with Tukey’s multiple comparisons test. k . Pathological grading of 1-year-old KC;G6pd wt and KC;G6pd mut pancreas tissues representing the % of total tissue area with acinar cells, ADM, and PanIN lesions. n = 5 mice per genotype. Bars represent the mean with standard deviation. P-values were calculated using a two-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: LSL -Kras G12D mice (Jackson Laboratory, strain no. 008179) were crossed with Ptf1a CreERTM mice to generate LSL- Kras G12D ; Ptf1a CreERTM mice and maintained on a C57BL/6J background.

Techniques: Mutagenesis, Labeling, Derivative Assay, Generated, Staining, Immunostaining, Standard Deviation, Two Tailed Test, Produced

Journal: Nature Metabolism

Article Title: NADPH-producing enzymes restrict the formation of pancreatic precancerous lesions

doi: 10.1038/s42255-026-01496-x

Figure Lengend Snippet: a . Mice with Me1 flox/flox alleles were bred into the KC ( LSL-Kras G12D ; Ptf1a Cre ) line. Cre was always maintained in female breeders. Experiments used both male and female mice that were homozygous for the Me1 flox/flox allele and mice that were Me1 +/+ (wildtype). b . Immunostaining for ME1 in 16-week-old KC;Me1 +/+ and KC;Me1 flox/flox pancreas. Scale bar = 50μm. c . Hematoxylin & eosin (H&E) staining and immunostaining for amylase (AMY; acinar cells), cytokeratin 19 (CK19; ductal, metaplastic, neoplastic cells), and Ki67 (proliferation) in pancreas of 8-week-old KC;Me1 +/+ and KC;Me1 flox/flox mice. Scale bar = 50μm. d . Percent of Amylase+ area in pancreas as quantified from male (closed circle) and female (open circle) mice in KC;Me1 +/+ (grey bar) and KC;Me1 flox/flox (blue bar) mice at 8 weeks. n = 4 mice for each genotype. Bars represent the mean with standard deviation. P-values were calculated using a Student’s t-test (unpaired, two-tailed). e . Percent of CK19+ cells and f . Ki67+ cells in the pancreas as quantified from male (closed circle) and female (open circle) KC;Me1 +/+ (grey bar) and KC;Me1 flox/flox (blue bar) mice at 8 weeks. In CK19 plot: n = 5 KC;Me1 +/+ and n = 6 KC;Me1 flox/flox mice. In Ki67 plot: n = 6 mice for each genotype. Bars represent the mean with standard deviation. P-values were calculated using a Student’s t-test (unpaired, two-tailed). g . Immunostaining for Amylase (AMY), in 8-week and 16-week-old KC;Me1 +/+ and KC;Me1 flox/flox pancreas. Alcian blue (PanIN-produced mucin) & nuclear fast red counterstain in pancreas of 26-week- KC;Me1 +/+ and KC;Me1 flox/flox mice. Scale bar = 500μm. h . Pancreas weight (PW) to body weight (BW) ratios in 8-, 16-, and 26-week-old KC;Me1 +/+ and KC;Me1 flox/flox mice. Bars represent the mean with standard deviation. Ratios are not significantly different as calculated using a Student’s t-test (unpaired, two-tailed) for each age. Sample sizes for each age are as follows: 8 week, n = 6 KC;Me1 +/+ and n = 8 KC;Me1 flox/flox ; 16 week, n = 6 KC;Me1 +/+ and n = 8 KC;Me1 flox/flox ; 26 week, n = 5 KC;Me1 +/+ and n = 7 KC;Me1 flox/flox . i . Pathological grading of 26-week-old KC;Me1 +/+ and KC;Me1 flox/flox pancreas tissues representing the % of total tissue area with acinar cells, ADM, and PanIN lesions. n = 4 mice per genotype. Bars represent the mean with standard deviation. P-values were calculated using a two-way ANOVA with Tukey’s multiple comparisons test. j . Pathological grading of 1-year-old KC;Me1 +/+ pancreas tissues representing the % of total tissue area with acinar cells, ADM, and PanIN lesions. n = 4 mice. Bars represent the mean with standard deviation.

Article Snippet: LSL -Kras G12D mice (Jackson Laboratory, strain no. 008179) were crossed with Ptf1a CreERTM mice to generate LSL- Kras G12D ; Ptf1a CreERTM mice and maintained on a C57BL/6J background.

Techniques: Immunostaining, Staining, Standard Deviation, Two Tailed Test, Produced

Journal: bioRxiv

Article Title: Cell-of-Origin, not Oncogenic Effect, Determines Desmoplastic Immune Exclusion in KRAS-Driven Liver Cancer

doi: 10.64898/2026.03.24.711280

Figure Lengend Snippet: Cholangiocyte-derived organoids harboring Trp53 deletion and Kras G12D mutation give rise to iCCA in a syngeneic orthotopic model. (A) Illustration of the syngeneic orthotopic CCA tumor model. Isolated wild-type chol-orgs were genetically engineered to harbor Trp53 deletion and Kras G12D mutation by CRISPR/Cas9 and implanted intrahepatically. (B) Bright-field microscopic images of wildtype (WT) chol-orgs and with Trp53 deletion (P) and Kras G12D mutation (PK). The CRISPR/Cas9-induced genetic modifications in Trp53 and Kras genes are indicated below. (C) Representative stains of WT and PK chol-orgs indicating positive biliary lineage marker expression (CK19) and upregulation of CD44 in chol-PK orgs. (D) Representative histopathology images of liver tumors obtained upon orthotopic implantation of chol-PK resembling CCA. Stains as indicated. Red dotted line demarcates the boundary between non-tumor liver (N) and tumor (T). All scale bars in (B) indicate 500 μm, and in (C) and (D) 100 μm.

Article Snippet: [ ] The pBabe-KrasG12D plasmid was reconstructed from pBabe-Kras G12D -puro (Addgene #58902) by removing the puromycin resistance gene through digestion with HindIII-HF and BspDI, blunt-ended by Klenow and re-ligation to restore plasmid circularity.

Techniques: Derivative Assay, Mutagenesis, Isolation, CRISPR, Marker, Expressing, Histopathology

Journal: bioRxiv

Article Title: Cell-of-Origin, not Oncogenic Effect, Determines Desmoplastic Immune Exclusion in KRAS-Driven Liver Cancer

doi: 10.64898/2026.03.24.711280

Figure Lengend Snippet: Hepatocyte-derived organoids harboring Trp53 deletion and Kras G12D overexpression give rise to HCC in a syngeneic orthotopic model. (A) Illustration of the syngeneic orthotopic HCC tumor model. Isolated wild-type hep-orgs were genetically engineered to harbor Trp53 deletion by CRISPR/Cas9 and Kras G12D via lentiviral transduction and implanted intrahepatically. (B) Bright-field microscopic images of wildtype (WT) hep-orgs and with Trp53 deletion (P) and Kras G12D overexpression (PK). The CRISPR/Cas9-induced genetic modifications in the Trp53 gene and the sequence of the overexpressing Kras G12D transgene are indicated below. (C) Representative stains of WT and PK hep-orgs indicating positive hepatocyte marker expression (HNF4α) and upregulation of CD44 in hep-PK orgs. (D) Bar plot of tumor penetrance of chol-PK and hep-PK organoids upon orthotopic tumor implantation. (E) Representative histopathology images of liver tumors obtained upon orthotopic implantation of hep-PK organoids resembling HCC. Stains as indicated. Red dotted line demarcates the boundary between non-tumor liver (N) and tumor (T). All scale bars in (B) indicate 500 μm, and in (C) and (E) 100 μm.

Article Snippet: [ ] The pBabe-KrasG12D plasmid was reconstructed from pBabe-Kras G12D -puro (Addgene #58902) by removing the puromycin resistance gene through digestion with HindIII-HF and BspDI, blunt-ended by Klenow and re-ligation to restore plasmid circularity.

Techniques: Derivative Assay, Over Expression, Isolation, CRISPR, Transduction, Sequencing, Marker, Expressing, Tumor Implantation, Histopathology

Journal: bioRxiv

Article Title: Cell-of-Origin, not Oncogenic Effect, Determines Desmoplastic Immune Exclusion in KRAS-Driven Liver Cancer

doi: 10.64898/2026.03.24.711280

Figure Lengend Snippet: Cell-of-origin is the dominant determinant of transcriptional identity in Trp53-deleted , Kras G12D -mutant liver cancer organoids. (A) Messenger RNA expression of fibroblast activation markers in mHSCs treated for 24 hours with CCM from chol-or hep-derived organoids (WT or PK). (B) Schematic of multi-factorial transcriptome analysis in chol and hep organoids. Gene expression was modelled with lineage (chol vs hep), oncogenic effect (PK vs WT), and their interaction to identify lineage-specific PK effects. (C) Principal component analysis (PCA) plot of transcriptomes from indicated lines. Shown are PC1 (79.2% variance) versus PC3 (3.9%), which separate samples primarily by cell lineage and, to a lesser extent, oncogenic activation. (D) Distribution of variance in normalized expression of DEGs explained by lineage and oncogenic effect shown as violin plot with integrated box plot. For each gene, we fit a linear model on normalized counts with lineage, oncogenic effect, their interaction (lineage × oncogenic effect), and mouse strain as predictors. Residuals capture remaining variation. DEGs are the union across the four contrasts: chol-PK vs chol-WT, chol-PK vs hep-PK, chol-WT vs hep-WT, and hep-PK vs hep-WT. (E) Scatter plot showing upregulated DEGs in chol-PK organoids from multi-factorial analysis. Dot color indicates the significance of the interaction effect (adjusted p-value), and dot size reflects the significance of PK versus WT comparison in chol-orgs (adjusted p-value). (F) Gene Set Enrichment Analysis (GSEA) of Hallmark pathways specifically enriched in the lineage-dependent PK effect. The pathways selected for cancer-related cell cycle and oncogenic signaling, tumor-stroma crosstalk, and tumor-immune and inflammation with positive enrichment (i.e. PK effects stronger in chol-orgs) are shown.

Article Snippet: [ ] The pBabe-KrasG12D plasmid was reconstructed from pBabe-Kras G12D -puro (Addgene #58902) by removing the puromycin resistance gene through digestion with HindIII-HF and BspDI, blunt-ended by Klenow and re-ligation to restore plasmid circularity.

Techniques: Mutagenesis, RNA Expression, Activation Assay, Derivative Assay, Gene Expression, Expressing, Comparison

Journal: bioRxiv

Article Title: Cell-of-Origin, not Oncogenic Effect, Determines Desmoplastic Immune Exclusion in KRAS-Driven Liver Cancer

doi: 10.64898/2026.03.24.711280

Figure Lengend Snippet: Cholangiocyte-derived organoids harboring Trp53 deletion and Kras G12D mutation give rise to iCCA in a syngeneic orthotopic model. (A) Illustration of the syngeneic orthotopic CCA tumor model. Isolated wild-type chol-orgs were genetically engineered to harbor Trp53 deletion and Kras G12D mutation by CRISPR/Cas9 and implanted intrahepatically. (B) Bright-field microscopic images of wildtype (WT) chol-orgs and with Trp53 deletion (P) and Kras G12D mutation (PK). The CRISPR/Cas9-induced genetic modifications in Trp53 and Kras genes are indicated below. (C) Representative stains of WT and PK chol-orgs indicating positive biliary lineage marker expression (CK19) and upregulation of CD44 in chol-PK orgs. (D) Representative histopathology images of liver tumors obtained upon orthotopic implantation of chol-PK resembling CCA. Stains as indicated. Red dotted line demarcates the boundary between non-tumor liver (N) and tumor (T). All scale bars in (B) indicate 500 μm, and in (C) and (D) 100 μm.

Article Snippet: The Lenti-CRISPR-sgRNA( Trp53 ), pBabe- Kras G12D , or Lenti-luciferase-P2A-Neo plasmid (Addgene #105621) was co-transfected with packaging plasmid psPAX2 and pMD2.G (both from PlasmidFactory GmbH & Co. KG, Bielefeld, Germany) into HEK293T cells by TransIT LT1 transfection reagent according to the manufacturer’s instructions (Mirus Bio, Madison, WI, USA).

Techniques: Derivative Assay, Mutagenesis, Isolation, CRISPR, Marker, Expressing, Histopathology

Journal: bioRxiv

Article Title: Cell-of-Origin, not Oncogenic Effect, Determines Desmoplastic Immune Exclusion in KRAS-Driven Liver Cancer

doi: 10.64898/2026.03.24.711280

Figure Lengend Snippet: Hepatocyte-derived organoids harboring Trp53 deletion and Kras G12D overexpression give rise to HCC in a syngeneic orthotopic model. (A) Illustration of the syngeneic orthotopic HCC tumor model. Isolated wild-type hep-orgs were genetically engineered to harbor Trp53 deletion by CRISPR/Cas9 and Kras G12D via lentiviral transduction and implanted intrahepatically. (B) Bright-field microscopic images of wildtype (WT) hep-orgs and with Trp53 deletion (P) and Kras G12D overexpression (PK). The CRISPR/Cas9-induced genetic modifications in the Trp53 gene and the sequence of the overexpressing Kras G12D transgene are indicated below. (C) Representative stains of WT and PK hep-orgs indicating positive hepatocyte marker expression (HNF4α) and upregulation of CD44 in hep-PK orgs. (D) Bar plot of tumor penetrance of chol-PK and hep-PK organoids upon orthotopic tumor implantation. (E) Representative histopathology images of liver tumors obtained upon orthotopic implantation of hep-PK organoids resembling HCC. Stains as indicated. Red dotted line demarcates the boundary between non-tumor liver (N) and tumor (T). All scale bars in (B) indicate 500 μm, and in (C) and (E) 100 μm.

Article Snippet: The Lenti-CRISPR-sgRNA( Trp53 ), pBabe- Kras G12D , or Lenti-luciferase-P2A-Neo plasmid (Addgene #105621) was co-transfected with packaging plasmid psPAX2 and pMD2.G (both from PlasmidFactory GmbH & Co. KG, Bielefeld, Germany) into HEK293T cells by TransIT LT1 transfection reagent according to the manufacturer’s instructions (Mirus Bio, Madison, WI, USA).

Techniques: Derivative Assay, Over Expression, Isolation, CRISPR, Transduction, Sequencing, Marker, Expressing, Tumor Implantation, Histopathology

Journal: bioRxiv

Article Title: Cell-of-Origin, not Oncogenic Effect, Determines Desmoplastic Immune Exclusion in KRAS-Driven Liver Cancer

doi: 10.64898/2026.03.24.711280

Figure Lengend Snippet: Cell-of-origin is the dominant determinant of transcriptional identity in Trp53-deleted , Kras G12D -mutant liver cancer organoids. (A) Messenger RNA expression of fibroblast activation markers in mHSCs treated for 24 hours with CCM from chol-or hep-derived organoids (WT or PK). (B) Schematic of multi-factorial transcriptome analysis in chol and hep organoids. Gene expression was modelled with lineage (chol vs hep), oncogenic effect (PK vs WT), and their interaction to identify lineage-specific PK effects. (C) Principal component analysis (PCA) plot of transcriptomes from indicated lines. Shown are PC1 (79.2% variance) versus PC3 (3.9%), which separate samples primarily by cell lineage and, to a lesser extent, oncogenic activation. (D) Distribution of variance in normalized expression of DEGs explained by lineage and oncogenic effect shown as violin plot with integrated box plot. For each gene, we fit a linear model on normalized counts with lineage, oncogenic effect, their interaction (lineage × oncogenic effect), and mouse strain as predictors. Residuals capture remaining variation. DEGs are the union across the four contrasts: chol-PK vs chol-WT, chol-PK vs hep-PK, chol-WT vs hep-WT, and hep-PK vs hep-WT. (E) Scatter plot showing upregulated DEGs in chol-PK organoids from multi-factorial analysis. Dot color indicates the significance of the interaction effect (adjusted p-value), and dot size reflects the significance of PK versus WT comparison in chol-orgs (adjusted p-value). (F) Gene Set Enrichment Analysis (GSEA) of Hallmark pathways specifically enriched in the lineage-dependent PK effect. The pathways selected for cancer-related cell cycle and oncogenic signaling, tumor-stroma crosstalk, and tumor-immune and inflammation with positive enrichment (i.e. PK effects stronger in chol-orgs) are shown.

Article Snippet: The Lenti-CRISPR-sgRNA( Trp53 ), pBabe- Kras G12D , or Lenti-luciferase-P2A-Neo plasmid (Addgene #105621) was co-transfected with packaging plasmid psPAX2 and pMD2.G (both from PlasmidFactory GmbH & Co. KG, Bielefeld, Germany) into HEK293T cells by TransIT LT1 transfection reagent according to the manufacturer’s instructions (Mirus Bio, Madison, WI, USA).

Techniques: Mutagenesis, RNA Expression, Activation Assay, Derivative Assay, Gene Expression, Expressing, Comparison