CDK4 or CDK6 upregulation induces DNA replication stress and genomic instability to cause EGFR targeted therapy resistance in lung cancer

Reviewer #1 (Public review):

Summary:

The authors used a panel of cell models to determine whether CDK4/6 overexpression resulted in resistance to the EGFR inhibitor Osimertinib, and the mechanisms underlying the resistance.

(1) Major Concerns (highest priority):

There is a lack of detail about the methodology in the results section/figure legends, which makes it difficult to interpret the data. Sometimes, adequate information is also not included in the methods themselves. For example, Figure 1A: how many doses did each mouse receive? How long after dosing were animals sacrificed? Figures 1E and 2A: is this RNA-seq analysis?

Using a second EGFR inhibitor for some of the key experiments would increase the rigor of the studies shown.

(2) Nice to have experiments:

Using CRISPR KO of CDK4 in the CDK4-amplified HCC827 and testing response to Osi and presence of replication stress would also increase the rigor of the studies.

The authors show that in their patient data, some cell cycle regulators which are amplified in NSCLC at similar rates to CDK4/6, such as CCNE1, had no increase in FGA. Overexpressing CCNE1 and testing Osi response in their cell models would be a nice test of their proposed mechanism that it is the genomic instability and FGA that are driving resistance. This wouldn't need to be done in vivo, but could be done using cell culture-based methods.

Similarly, testing the overexpression of some of the proposed target genes, such as STEAP1 and AGR2, on the therapeutic response to Osi in cell culture would also be a nice test of the mechanism proposed.

Reviewer #2 (Public review):

Summary:

In this manuscript, Gini et al. investigate the mechanisms by which CDK4 and CDK6 upregulation drives resistance to EGFR tyrosine kinase inhibitors (TKIs) in EGFR-mutant lung adenocarcinoma (LUAD). The study utilizes preclinical models, including cell line-derived xenografts (CDXs), patient-derived xenografts (PDXs), and primary organoids, alongside large-scale clinical genomic datasets. The authors demonstrate that CDK4 or CDK6 overexpression allows cancer cells to bypass EGFR TKI-induced G1/S arrest, leading to continuous cell cycle progression. This sustained proliferation during EGFR inhibition induces DNA replication stress, activates DNA damage response pathways (such as ATM and TPX2), and ultimately causes genomic instability. The authors also show that this leads in turn to the upregulation of tumor-promoting genes (e.g., AGR2, ASNS, STEAP1) and an epithelial-mesenchymal transition (EMT) phenotype. Moreover, the authors show that combinatorial treatment utilizing TKIs such as osimertinib alongside CDK4/6 inhibitors effectively suppresses proliferation, mitigates DNA damage, and restores TKI sensitivity in preclinical models.

Overall, this is a highly translational study that provides a strong mechanistic rationale for biomarker-driven clinical trials combining EGFR and CDK4/6 inhibitors. However, there are a few experimental and analytical areas that require clarification or additional data to fully support the authors' conclusions.

Major Comments:

(1) Reliance on overexpression models over loss-of-function

The mechanistic studies mainly rely on overexpression of CDK4 and CDK6 to simulate the amplified state. Although the authors argued that the level of overexpression mimics that observed in resistant tumors, a complementary study in which CDK4/CDK6 were suppressed in a model where CDK4/CDK6 is amplified (such as HCC827 or TH116), and replication stress and osimertinib sensitivity tested would greatly strengthen their observations. Indeed, there is mention of CDK4 constructs to perform knockdown studies in the methods, but those studies are not included in this submission.

(2) Mechanistic link between genomic instability and specific gene amplifications

The authors highlight that CDK4/6 activation leads to recurrent copy number gains and transcriptional upregulation of specific pro-tumor genes including AGR2, ASNS, and STEAP1. While the paper establishes that CDK4/6 overexpression causes general genomic instability (increased FGA), it does not mechanistically explain why these specific genes are consistently amplified. The authors should investigate or discuss whether these specific loci are inherently fragile under replication stress, if they are direct downstream targets of the E2F transcriptional program, or if this is a result of random genomic instability followed by strong positive selection under osimertinib pressure.

(3) Discrepancies in tumor mutational burden (TMB) reporting

There is a slight contradiction regarding the TMB data that needs to be clarified for readers. The manuscript states that in the clinical datasets, "EGFR-mutant LUAD harboring cell cycle gene alterations exhibited significantly elevated FGA and TMB relative to cell cycle-negative tumors" (Line 265-266). However, in the next section, the authors say, "Notably, no corresponding increase in TMB was observed with CDK4 or CDK6 CNA, similar to our findings in preclinical models" (Line 271-273). The authors should clarify or discuss why broad cell cycle alterations correlate with high TMB, while CDK4/6-specific alterations drive structural instability (FGA) without increasing TMB.

Author response:

Reviewer 1:

(1) Major Concerns (highest priority):

There is a lack of detail about the methodology in the results section/figure legends, which makes it difficult to interpret the data. Sometimes, adequate information is also not included in the methods themselves. For example, Figure 1A: how many doses did each mouse receive? How long after dosing were animals sacrificed? Figures 1E and 2A: is this RNA-seq analysis?

Thank you for pointing this out. We will provide additional details about the methodology in the results, figure legends, and methods to clarify our findings.

Using a second EGFR inhibitor for some of the key experiments would increase the rigor of the studies shown.

Thank you for this suggestion. We agree that a second EGFR inhibitor may increase the scientific rigor of the findings. However, at the time the experiments were completed, osimertinib was the clear standard of care first-line therapy for EGFR-mutated lung cancer and the clinical relevance of using other inhibitors in these experiments is not clear.

(2) Nice to have experiments:

Using CRISPR KO of CDK4 in the CDK4-amplified HCC827 and testing response to Osi and presence of replication stress would also increase the rigor of the studies.

Thank you for this suggestion. We agree that this would increase the rigor of the studies, but these experiments are currently beyond the scope of this manuscript.

The authors show that in their patient data, some cell cycle regulators which are amplified in NSCLC at similar rates to CDK4/6, such as CCNE1, had no increase in FGA. Overexpressing CCNE1 and testing Osi response in their cell models would be a nice test of their proposed mechanism that it is the genomic instability and FGA that are driving resistance. This wouldn't need to be done in vivo, but could be done using cell culture-based methods.

Thank you for this suggestion. For this manuscript, we have chosen to focus on the role of CDK4 and CDK6 in osimertinib resistance as they have shown the clearest correlation with decreased responsiveness to EGFR TKI treatment in other studies. We agree that assessing the role of CCNE1 in osimertinib resistance is important and will address this in future studies.

Similarly, testing the overexpression of some of the proposed target genes, such as STEAP1 and AGR2, on the therapeutic response to Osi in cell culture would also be a nice test of the mechanism proposed.

Thank you for this suggestion. We agree that these experiments are important, but they are currently beyond the scope of this study.

Reviewer 2:

Major Comments:

(1) Reliance on overexpression models over loss-of-function

The mechanistic studies mainly rely on overexpression of CDK4 and CDK6 to simulate the amplified state. Although the authors argued that the level of overexpression mimics that observed in resistant tumors, a complementary study in which CDK4/CDK6 were suppressed in a model where CDK4/CDK6 is amplified (such as HCC827 or TH116), and replication stress and osimertinib sensitivity tested would greatly strengthen their observations. Indeed, there is mention of CDK4 constructs to perform knockdown studies in the methods, but those studies are not included in this submission.

Thank you for this suggestion. We agree that loss of function studies are an important complement to over expression studies. However, we have chosen to focus on the use of pharmacologic inhibitors of CDK4/6, which are more clinically relevant than knockdown studies. We demonstrate that the CDK4/6 inhibitor palbociclib is able to prevent replication stress and restore osimertinib sensitivity in CDK6 amplified TH116 patient-derived xenografts (Figure 5 and Supplementary Figure 10).

(2) Mechanistic link between genomic instability and specific gene amplifications

The authors highlight that CDK4/6 activation leads to recurrent copy number gains and transcriptional upregulation of specific pro-tumor genes including AGR2, ASNS, and STEAP1. While the paper establishes that CDK4/6 overexpression causes general genomic instability (increased FGA), it does not mechanistically explain why these specific genes are consistently amplified. The authors should investigate or discuss whether these specific loci are inherently fragile under replication stress, if they are direct downstream targets of the E2F transcriptional program, or if this is a result of random genomic instability followed by strong positive selection under osimertinib pressure.

Thank you for this suggestion. We will provide additional discussion about whether these specific loci are likely to be inherently fragile under replication stress, if they are direct downstream targets of the E2F transcriptional program, or if it is more likely the result of random genomic instability followed by strong positive selection under osimertinib pressure.

(3) Discrepancies in tumor mutational burden (TMB) reporting

There is a slight contradiction regarding the TMB data that needs to be clarified for readers. The manuscript states that in the clinical datasets, "EGFR-mutant LUAD harboring cell cycle gene alterations exhibited significantly elevated FGA and TMB relative to cell cycle-negative tumors" (Line 265-266). However, in the next section, the authors say, "Notably, no corresponding increase in TMB was observed with CDK4 or CDK6 CNA, similar to our findings in preclinical models" (Line 271-273). The authors should clarify or discuss why broad cell cycle alterations correlate with high TMB, while CDK4/6-specific alterations drive structural instability (FGA) without increasing TMB.

Thank you for this suggestion. We will clarify our findings and provide further discussion about why there may be a difference between the effects of broad cell cycle alterations and CDK4/6-specific alterations on TMB and structural genomic instability.