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

Reviewer #1 (Public Review):

Summary:

Given that KRAS inhibition approaches are a relatively new innovation and that resistance is now being observed to such therapies in patients with NSCLC, investigation of combination therapies is valuable. The manuscript furthers our understanding of combination therapy for KRAS mutant non-small cell lung cancer by providing evidence that combined inhibition of ULK1/2 (and therefore autophagy) and KRAS can inhibit KRAS-mutant lung cancer growth. The manuscript will be of interest to the lung cancer community but also to researchers in other cancer types where KRAS inhibition is relevant.

Strengths:

The manuscript combines cell line, cell line-derived xenograft, and genetically-engineered mouse model data to provide solid evidence for the proposed combination therapy.

The manuscript is well written, and experiments are broadly well performed and presented.

Weaknesses:

With 3-4 mice per group in many experiments, experimental power is a concern and some comparisons (e.g. mono vs combination therapy) seem to be underpowered to detect a difference. Both male and female mice are used in experiments which may increase variability.

Reviewer #2 (Public Review):

Summary:

In this manuscript, Ghazi et reported that inhibition of KRASG12C signaling increases autophagy in KRASG12C-expressing lung cancer cells. Moreover, the combination of DCC 3116, a selective ULK1/2 inhibitor, plus sotorasib displays cooperative/synergistic suppression of human KRASG12C-driven lung cancer cell proliferation in vitro and tumor growth in vivo. Additionally, in genetically engineered mouse models of KRASG12C-driven NSCLC, inhibition of either KRASG12C or ULK1/2 decreases tumor burden and increases mouse survival. Additionally, this study found that LKB1 deficiency diminishes the sensitivity of KRASG12C/LKB1Null-driven lung cancer to the combination treatment, perhaps through the emergence of mixed adeno/squamous cell carcinomas and mucinous adenocarcinomas.

Strengths:

Both human cancer cells and mouse models were employed in this study to illustrate that inhibiting ULK1/2 could enhance the responsiveness of KRASG12C lung cancer to sotorasib. This research holds translational importance.

Weaknesses:

Additional validation of certain data is necessary.

(1) mCherry-EGFP-LC3 reporter was used to assess autophagy flux in Figure 1A. Please explain how autophagy status (high, medium, and low) was defined. It's also suggested to show WB of LC3 processing in different treatments as in Figure 1A at 48 hours.

(2) For Figures 1J, K, and L, please provide immunohistochemistry (IHC) images demonstrating RAS downstream signaling blockade by sotorasib and autophagy blockade by DCC 3116 in tumors.

(3) Given that both DCC 3116 and ULK1K46N exhibit the ability to inhibit autophagy and synergize with sotorasib in inhibiting cell proliferation, in addition to demonstrating decreased levels of pATG13 via ELISA assay, please include Western blot analyses of LC3 or p62 to confirm the blockade of autophagy by DCC 3116 and ULK1K46N in Figure 1 & Figure 2.

(4) Since adenocarcinomas, adenosquamous carcinomas (ASC), and mucinous adenocarcinomas were detected in KL lung tumors, please conduct immunohistochemistry (IHC) to detect these tumors, including markers such as p63, SOX2, Katrine 5.

(5) Please provide the sample size (n) for each treatment group in the survival study (Figure 4E). It appears that all mice were sacrificed for tumor burden analysis in Figure 4F. However, there doesn't seem to be a significant difference among the treatment groups in Figure 4F, which contrasts with the survival analysis in Figure 4E. It is suggested to increase the sample size in each treatment group to reduce variation.

(6) In KP mice (Figure 5), it seems that a single treatment alone is sufficient to inhibit established KP lung tumor growth. Combination treatment does not further enhance anti-tumor efficacy. Therefore, this result doesn't support the conclusion generated from human cancer cell lines. Please discuss.