Human KRASG12C-driven lung cancer cells are sensitive to co-inhibition of KRASG12C and ULK1/2.

A-C, Human KRASG12C-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. Ns= not significant, *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001.

D-F, Quantification of percent confluence of human KRASG12C-driven cell lines at 72 hours post-drug treatment.

G-I, In vitro synergy assay of human KRASG12C-driven cell lines using the Loewe method after 72 hours of treatment.

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.

Genetic inhibition of ULK1 decreases autophagy and cooperates with sotorasib to reduce cell viability.

A, Immunoblot of NCI-H2122:ULK1K46N cells after 24 hours of doxycycline treatment.

B, Immunoblot of NCI-H2122:ULK1K46N cells treated with 1 ug/mL doxycycline over time (hours).

C, ELISA assay of pS318-ATG13 expression after 16 hours of doxycycline treatment. N=3.

D, NCI-H2122:ULK1K46N 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 performed with an ordinary one-way ANOVA with Dunnett’s multiple comparisons test. *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001

E, Synergy analysis of NCI-H2122:ULK1K46N cells treated with DMSO control, sotorasib and/or doxycycline over 48 hours.

Reagent Table

Either LKB1 silencing or expression of dominant-negative TP53R172H cooperates with KRASG12C 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. N= 3-4

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.

Combined inhibition of KRASG12C 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 GEMMs 12 weeks post-initiation of tumorigenesis and DCC-3116 treatment.

C, Quantification of tumor burden of (B).

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. Mice were treated every day 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

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) *p<0.05 **p<0.01 ns= not significant

Inhibition of KRASG12C and ULK1/2 reduces tumor burden in a KP GEM models

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. *p<0.05 ns=not significant.

C, Percent change in the body weight of mice on treatment over 4 weeks.

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) *p<0.05 **p<0.01 ns= not significant

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.70R 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. Ns= not significant, *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001.

F-G, Cell viability assay of KL.70 cells treated with indicated compounds at indicated doses after 72 hours of treatment.

H-I, Cell viability assay of KL.70R cells treated with indicated compounds at indicated doses after 72 hours of treatment.

Human KRAS-driven lung cancer cells increase autophagy after KRASG12C or MEK inhibition.

A-E, Autophagy levels in human KRASG12C-driven lung cancer cells Calu-1 (A), NCI-H2122 (B), NCI-H23 (C), NCI-H358 (D) and the KRASG12V-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. ns= not significant, *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001.

F-J, Autophagy levels in human KRASG12C-driven lung cancer cells Calu-1 (F), NCI-H2122 (G), NCI-H23 (H), NCI-H358 (I) and the KRASG12V-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. ns= not significant, *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001.

Human KRASG12C-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

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 KRASG12C-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= adeno/squamous 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 GEMMs.

E, Representative images of hematoxylin and eosin (H&E) staining and immunohistochemistry of indicated proteins.

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.

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= adenocarcinoma/squamous 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.