Cancer Biology

Inhibition of ULK1/2 and KRAS^G12C controls tumor growth in preclinical models of lung cancer

Phaedra C Ghazi
Kayla T O’Toole
Sanjana Srinivas Boggaram
Michael T Scherzer
Mark R Silvis
Yun Zhang
Madhumita Bogdan
Bryan D Smith
Guillermina Lozano
Daniel L Flynn
Eric L Snyder
Conan G Kinsey
Martin McMahon author has email address

Department of Oncological Sciences, University of Utah, Salt Lake City, UT
Huntsman Cancer Institute, University of Utah, Salt Lake City, UT
Department of Genetics, University of Texas MD Anderson Cancer Center, Houston, TX
Deciphera Pharmaceuticals LLC, 643 Massachusetts St, Lawrence, Kansas
Department of Pathology, University of Utah, Salt Lake City, UT
Department of Internal Medicine, Division of Medical Oncology, University of Utah, Salt Lake City, UT
Department of Dermatology, University of Utah, Salt Lake City, UT

https://doi.org/10.7554/eLife.96992.2

Open access
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Figures and data

Human KRAS^G12C-driven lung cancer cells are sensitive to co-inhibition of KRAS^G12C and ULK1/2.
A-C, Human KRAS^G12C-driven cell lines NCI-H2122 (A), Calu-1 (B) and NCI-H358 (C) increase autophagy as assessed by mCherry-EGFP-LC3 reporter after 48 hours of sotorasib treatment and decrease autophagy after 48h hours of DCC-3116 treatment. Red=high autophagy, Yellow= medium autophagy, Green= low autophagy. Statistical significance was determined by comparing autophagic flux levels to DMSO control and an ordinary one-way ANOVA with Dunnett’s multiple comparisons was used. Ns= not significant, *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001. N=9
D-F, Quantification of percent confluence of human KRAS^G12C-driven cell lines at 72 hours post-drug treatment. Statistical significance was determined by an ordinary one-way ANOVA. Ns= not significant,*p<0.05 **p<0.01 ***p<0.001 ****p<0.0001.N=3
G- I, In vitro synergy assay of human KRAS^G12C-driven cell lines using the Loewe method after 72 hours of treatment. N=3
J-L, Tumor volume measured over 28 days of treatment in mice inoculated with NCI-H2122 (J), Calu-1
(K) and NCI-H358 (L) cells. Vehicle and sotorasib were administered once daily via oral gavage and DCC-3116 was formulated in the chow. Statistical significance was determined by an ordinary one-way
ANOVA compared to vehicle treated tumors. Ns= not significant, *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001. N=4-5 mice per treatment.

Genetic inhibition of ULK1 decreases autophagy and cooperates with sotorasib to reduce cell viability.
A, Immunoblot of NCI-H2122:ULK1^K46N cells after 24 hours of doxycycline treatment.
B, Immunoblot of NCI-H2122:ULK1^K46N cells treated with 1 ug/mL doxycycline over time (hours).
C, ELISA assay of pS318-ATG13 expression after 16 hours of doxycycline treatment. Statistical significance was determined by an ordinary one-way ANOVA. Ns= not significant, *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001. N=3.
D, NCI-H2122:ULK1^K46N cells were engineered to express the mCherry-EGFP-LC3 reporter, and a decrease in autophagy was demonstrated after 48 hours of doxycycline treatment. N=3. All statistical significance was measured using an ordinary one-way ANOVA with Dunnett’s multiple comparisons test. *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001
E, In vitro synergy assay of NCI-H2122:ULK1^K46N cells treated with DMSO control, sotorasib and/or doxycycline over 48 hours using the Loewe method. N=3.

Either LKB1 silencing or expression of dominant-negative TP53^R172H cooperates with KRAS^G12C in GEM models of lung cancer.
A, Schematic of genotypes of GEM models and abbreviations.
B, Representative images of lung lobes from GEM models at indicated time points post-initiation of lung tumorigenesis.
C, Quantification of lung tumor burdens from GEM models 14 weeks post-initiation of tumorigenesis. Statistical analysis was performed using an ordinary one-way ANOVA. Ns= not significant, *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001. N=4.
D, Hematoxylin and eosin (H&E) and immunohistochemical analysis of representative lung sections from GEM models 14 weeks post-initiation of tumorigenesis. p.i.=post initiation.
Any parts of this image created with BioRender are not made available under the same license as the Reviewed Preprint, and are © 2024, BioRender Inc.

Reagent Table

Reagent Table

Combined inhibition of KRAS^G12C and ULK1/2 decreases tumor initiation and increases survival in KL GEMMs.
A, Schematic of lung tumor prevention dosing strategy of KL GEMMs. DCC-3116 was administered in drug-formulated chow. N=4
B, Representative images of lung lobes from GEM models 12 weeks post-initiation of tumorigenesis and DCC-3116 treatment.
C, Quantification of tumor burden of (B). Statistical analysis was measured by an unpaired student’s t-test. Ns= not significant, *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001. N=4
D, Schematic of treating tumor-bearing KL mice with vehicle control, 30 mg/kg sotorasib, chow containing DCC-3116 or the combination. DCC-3116 was administered in drug-formulated chow (Table 1). Mice were treated daily for 56 days or until termination criteria were reached, whichever was reached first. N=4-6.
E, Kaplan-Meyer survival curve of survival on treatment of KL mice treated as indicated. Statistical analysis was performed using a Log-Rank test. **p<.01 N=4-6
F, Quantification of tumor burden of (E).
G, H&E analysis of representative lung sections from KL mice after treatment. AB/PAS= Alcian Blue Periodic Acid Schiff, for staining mucins.
H- I, Quantification of immunohistochemical staining of treated mice pERK (H) and pAKT (I) as described in the methods. Statistical analysis was measured with an ordinary one-way ANOVA *p<0.05 **p<0.01 ns= not significant. N= 4-5
Any parts of this image created with BioRender are not made available under the same license as the Reviewed Preprint, and are © 2024, BioRender Inc.

Inhibition of KRAS^G12C and ULK1/2 reduces tumor burden in a KP GEM model
A, Schematic of the treatment of KP GEM models. Mice were administered vehicle control or 30 mg/kg sotorasib once daily via oral gavage. DCC-3116 was administered in drug-formulated chow. N=4-5
B, Quantification of tumor burden of mice after 4 weeks of treatment. Statistical analysis was performed using an ordinary one-way ANOVA *p<0.05 ns=not significant. N=4-5
C, Percent change in the body weight of mice on treatment over 4 weeks. Each line depicts an individual mouse.
D, Representative images of histological analysis of lung lobes from KP mice 4 weeks after treatment.
E-F, Quantification of immunohistochemical staining of treated mice pERK1/2 (E) and pAKT1-3 (F) as described in the methods. Statistical analysis was performed using an ordinary one-way ANOVA *p<0.05 **p<0.01 ns= not significant
Any parts of this image created with BioRender are not made available under the same license as the Reviewed Preprint, and are © 2024, BioRender Inc.

KL lung-cancer-derived cells that acquire resistance to sotorasib increase RAS and pERK1/2 expression and do not increase autophagy after sotorasib treatment.
A, Immunoblot analysis of KL.70 and KL.70^R cells treated with 100nM sotorasib or 100nM trametinib after 48 hours of treatment.
B-C, Quantification of signal from A normalized to b-actin.
D, Live cell imaging of percent confluence of KL.70 cells over time treated with DMSO, 100nm sotorasib of 100nM trametinib.
E, Autophagy measurement with FAR reporter in cells assessed by mCherry-eGFP-LC3 reporter after 48 hours of 100nM sotorasib or 100nM trametinib treatment. Red=high autophagy, Yellow= medium autophagy, Green= low autophagy. Statistical significance was determined by comparing autophagic flux levels to DMSO control and an ordinary one-way ANOVA with Dunnett’s multiple comparisons was used. Ns= not significant, *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001. N=9
F-G, In vitro synergy assay of KL70 cells treated with indicated doses of sotorasib and/or DCC-3116 using the Loewe method after 72 hours of treatment. N=3
H-I, In vitro synergy assay of KL70^SR cells treated with indicated doses of sotorasib and/or DCC-3116 using the Loewe method after 72 hours of treatment. N=3

Human KRAS-driven lung cancer cells increase autophagy after KRAS^G12C or MEK inhibition.
A-E, Autophagy levels in human KRAS^G12C-driven lung cancer cells Calu-1 (A), NCI-H2122 (B), NCI-H23 (C), NCI-H358 (D) and the KRAS^G12V-driven human lung cancer cell line Cor-L23 (E) after 48 hours of sotorasib at indicated concentrations with the FAR reporter. Red=high autophagy, Yellow= medium autophagy, Green= low autophagy. Statistical significance was determined by comparing autophagic flux levels to DMSO control and an ordinary one-way ANOVA with Dunnett’s multiple comparisons was used. ns= not significant, *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001. N=9
F-J, Autophagy levels in human KRAS^G12C-driven lung cancer cells Calu-1 (F), NCI-H2122 (G), NCI-H23 (H), NCI-H358 (I) and the KRAS^G12V-driven human lung cancer cell line Cor-L23 (J) after 48 hours of trametinib at indicated concentrations with the FAR reporter. Red=high autophagy, Yellow= medium autophagy, Green= low autophagy. Statistical significance was determined by comparing autophagic flux levels to DMSO control and an ordinary one-way ANOVA with Dunnett’s multiple comparisons was used. ns= not significant, *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001. N=9

Human KRAS^G12C-driven lung cancer cell lines increase autophagy and decrease cellular proliferation after sotorasib treatment.
A-C, ELISA measurement of pS318-ATG13 signal after 16 hours of drug treatment in NCI-H2122 (A), Calu-1 (B) and NCI-H358 cell lines (C). N=3. Statistical analysis was performed using an ordinary one-way ANOVA with Tukey’s multiple comparisons test. ns= not significant, *p<0.05 ****p<0.0001.
D-F, Percent confluence over time of NCI-H2122 (D), Calu-1 (E) and NCI-H358 (F) cell lines treated with indicated compounds over time. Statistical analysis was performed using an ordinary one-way ANOVA with Tukey’s multiple comparisons test. ns= not significant, *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001. N=3-5
G-I, Immunoblotting analysis of NCI-H2122 (G), Calu-1 (H), and NCI-H358 (I) cell lines after 100nM sotorasib treatment over 24 hours.

Loss of LKB1 expression leads to mixed ASC and mucinous ADC lung tumors in KRAS^G12C-driven GEMMs.
A, Schematic of allele, structure, and protein changes in GEM models used.
B, Representative hematoxylin and eosin stains of K, KL and KP GEM models 14 weeks post-initiation. N.O.= not observed, AAH= atypical adenomatous hyperplasia, ADC= adenocarcinoma, ASC= adenosquamous cell carcinoma, mucinous ADC= mucinous adenocarcinoma.
C-D, Representative immunohistochemical stains of pERK1/2 (C) and pAKT1-3 (D) signal in K, KL and KP GEM models.
E, Representative images of hematoxylin and eosin (H&E) staining and immunohistochemistry of indicated proteins in KL mice.

MicroCT, body weight and pathological analysis of treated KL mice.
A, Representative microCT images of KL mice pre-treatment (Day 0) and 14 days, 28 days and 56 days post-treatment. Vehicle-treated mice and most DCC-3116 treated mice reached termination criteria before the 56-day endpoint. Red arrows indicate lung tumors.
B, Percent change in the body weight of mice over the course of the treatment period. Each line depicts an individual mouse.
C, Representative images of hematoxylin and eosin staining of lung sections of KL mice at the end of treatment. N.O.= not observed, AH= alveolar hyperplasia, ADC= adenocarcinoma, ASC= adenosquamous cell carcinoma, mucinous ADC= mucinous adenocarcinoma.

MicroCT images of KP mice on treatment.
Representative microCT images of KP mice pre-treatment (Day 0) and 7 days, 14 days and 28 days post-treatment. Red arrows indicate lung tumors.

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