Virtually all pancreatic ductal adenocarcinomas (PDACs) are initiated by activating mutations in the oncogene KRAS, which occur in multiple distinct allelic forms. Although considerable efforts have led to the development of inhibitors targeting specific mutant KRAS proteins, the only agents currently approved for clinical use selectively target the KRASG12C variant. However, KRASG12C mutations are exceedingly rare in pancreatic cancer.
Furthermore, in patients with KRASG12C-mutant pancreatic cancer, treatment with KRASG12C inhibitors has shown only modest clinical benefit, comparable to that of conventional chemotherapeutic regimens, and even in cases with an initial objective response, acquired resistance almost invariably emerges within a limited time frame.
More broadly, direct pharmacological inhibition of driver oncogenes frequently precipitates the rapid development of durable therapeutic resistance. These observations underscore a critical unmet need for therapeutic strategies that circumvent direct targeting of driver mutant proteins while achieving antitumor efficacy comparable to that of their direct inhibition.
Zhang et al. addressed how to cure PDAC patients with avoiding direct inhibition of KRAS (Zhang et al., Cell Death and Differentiation published on line, Dec 18, 2025).
Constitutively activated mutant KRAS drives transcriptional upregulation of the cyclin D1 (CCND1) gene primarily through sequential activation of the RAF–MEK–ERK signaling cascade. Cyclin D1 forms an active complex with cyclin-dependent kinase 4 or 6 (CDK4/6), which phosphorylates multiple substrate proteins. Among these substrates, the tumor suppressor RB1 is the most critical.
RB1 associates with members of the E2F family of transcription factors to restrain cell-cycle progression. Initial monophosphorylation of RB1 by the cyclin D1–CDK4/6 complex at one of its fourteen phosphorylation sites subsequently licenses further phosphorylation of the remaining sites by cyclin E–CDK2 complexes, resulting in functional inactivation of RB1 and release of E2F-driven cell-cycle entry. Thus, aberrant KRAS activation potently suppresses RB1 tumor-suppressive function.
This concept was originally inspired by seminal studies in Caenorhabditis elegans by Horvitz and colleagues, which were awarded the Nobel Prize. The biochemical and genetic evidence has been provided by Ewen and colleagues.
Newly synthesized KRAS initially resides in the cytosol and is unable to signal in this state. Throughisoprenylation, a post-translational modification, KRAS is trafficked to the Golgi apparatus, where it becomes competent for activation.
Activation of RB1 suppresses this isoprenylation process, thereby inhibiting KRAS activation. Thus, activated KRAS suppresses RB1 function, whereas activated RB1, in turn, restrains KRAS signaling, establishing a reciprocal inhibitory circuit. This mutual antagonism was previously reported by Takahashi et al. (Nature Genetics 38, 113–128, 2006; Cancer Cell 15, 255–269, 2009) (Fig. 1).
Fig. 1 The mutual antagonism between RB1 and RAS.
The reciprocal inhibitory relationship between RB1 and KRAS predicts that, in cancer, either inactivation of RB1 or activating mutation of KRAS alone may be sufficient to drive tumorigenesis. Consistent with this notion, in pancreatic cancer—where activating KRAS mutations are overwhelmingly prevalent—genetic inactivation of RB1 is exceedingly rare.
This implies that in nearly all pancreatic cancers, RB1 remains wild-type and functionally intact, thereby providing a substantial therapeutic opportunity to enhance RB1 activity. Importantly, such activation of RB1 would be expected not only to restore cell-cycle control but also to indirectly suppress oncogenic KRAS signaling.
Against this background, we focused on CDK4/6 inhibitors, which are clinically approved for the treatment of hormone receptor–positive, HER2-negative advanced breast cancer. These agents inhibit the kinase activity of CDK4/6, which forms a complex with cyclin D1 to mediate monophosphorylation of RB1, thereby prolonging the hypophosphorylated, active state of RB1. Sustained RB1 activation imposes substantial cellular stress and can ultimately trigger cell death. We therefore initially evaluated the effects of CDK4/6 inhibition as a monotherapy in pancreatic cancer.
Although CDK4/6 inhibitors efficiently induced cellular senescence, they failed to elicit cell death to an extent sufficient for therapeutic benefit. In breast cancer, CDK4/6 inhibitors are invariably administered in combination with agents that suppress estrogen receptor signaling. By analogy, these observations suggested that effective treatment of pancreatic cancer with CDK4/6 inhibitors would likewise require combination with an additional therapeutic agent.
The research team led by Takahashi established an inducible system that allows temporally controlled expression of a constitutively active RB1 mutant, thereby functionally mimicking the effects of CDK4/6 inhibition. Using this platform, we screened for compounds capable of selectively killing pancreatic cancer cells upon RB1 activation and identified ERK inhibitors as potent candidates.
As anticipated, treatment of pancreatic cancer cells with CDK4/6 inhibitors suppressed the activity of mutant KRAS. Given that ERK lies downstream of KRAS signaling, its activity was expected to decrease accordingly. Unexpectedly, however, ERK activity increased progressively over time following CDK4/6 inhibition.
This paradoxical ERK reactivation provided a mechanistic rationale for the efficacy of ERK inhibitors in this context and prompted us to investigate the underlying mechanism in depth. Importantly, this sustained elevation of ERK activity attenuated the antitumor effects of CDK4/6 inhibitors, thereby limiting their therapeutic efficacy.
By tracing ERK-activating signaling pathways upstream, we first found that activation of the epidermal growth factor receptor (EGFR), the receptor for the proliferative ligand EGF, was rapidly enhanced immediately after treatment with CDK4/6 inhibitors. We next examined the expression of EGFR-stimulating ligands and observed a marked upregulation of a subset of EGFR ligands known to be induced during cellular senescence.
Consistent with this finding, conditioned media collected from cells expressing a constitutively active RB1 mutant that phenocopies CDK4/6 inhibitor treatment contained markedly elevated levels of EGFR ligands.
This phenomenon is characteristic of the senescence-associated secretory phenotype (SASP), in which senescent cells secrete a wide array of soluble factors that exert autocrine and paracrine effects on both senescent and neighboring cells. Our data indicate that CDK4/6 inhibition promotes SASP-dependent production of EGFR ligands, leading to activation of EGFR and downstream ERK signaling, likely through a RAS-independent mechanism. This signaling cascade subsequently enhances pro-survival pathways, including BCL2 and NF-kB signaling.
Collectively, these findings suggest that pancreatic cancer cells acquire resistance to CDK4/6 inhibitor–induced cell death through SASP-mediated activation of EGFR signaling and its downstream survival pathways.
How, then, can pancreatic cancer be effectively treated in light of these findings? Although no ERK inhibitors are currently approved for routine clinical use, a wide range of EGFR-targeting agents are already available and covered by health insurance. Based on this rationale, we evaluated combination therapies consisting of CDK4/6 inhibitors together with either the EGFR tyrosine kinase inhibitor gefitinib or the anti-EGFR monoclonal antibody cetuximab.
Strikingly, these combination treatments demonstrated robust therapeutic efficacy not only in vitro but also in vivo, both in immunodeficient mice bearing human pancreatic cancer xenografts and in genetically engineered mouse models that spontaneously develop pancreatic cancer. These results indicate that pharmacological blockade of EGFR effectively overcomes CDK4/6 inhibitor–induced resistance mechanisms and provides a promising, immediately translatable therapeutic strategy for pancreatic cancer.
Furthermore, by carefully examining the cellular trajectory leading to cell death during combination therapy, we found that PDACs initially undergo CDK4/6 inhibitor–dependent cellular senescence. Notably, inhibition of EGFR in these senescent cells subsequently triggers cell death. The selective elimination of senescent cells by such an approach is referred to as senolysis (Fig. 2).
Fig. 2 The summary of findings reported in the paper.
Importantly, this effect was not observed when EGFR inhibition preceded CDK4/6 inhibitor treatment. Based on these findings, we predict that in the clinical setting, administering EGFR inhibitors or anti-EGFR antibodies prior to CDK4/6 inhibition would be unlikely to confer therapeutic benefit. Instead, our data highlight the critical importance of treatment sequencing, with EGFR blockade exerting senolytic effects specifically in CDK4/6 inhibitor–induced senescent pancreatic cancer cells.
A major concern associated with this combination therapy was the possibility that CDK4/6 inhibitor treatment might induce cellular senescence in normal tissues, thereby rendering non-malignant cells susceptible to EGFR inhibition–induced cell death. To address this issue, we administered CDK4/6 inhibitors to a reporter mouse model that enables in vivo tracking of the activity of the gene encoding p16, a protein whose expression is frequently upregulated during cellular senescence.
Notably, we did not observe an increase in p16 expression in normal tissues following CDK4/6 inhibitor treatment. These findings suggest that, under the conditions tested, CDK4/6 inhibition does not induce detectable senescence in normal cells, supporting a favorable therapeutic window for the proposed combination strategy.
EGFR tyrosine kinase inhibitors are typically administered exclusively to patients whose tumors harbor activating mutations in the EGFR gene and are therefore rarely used in patients with EGFR–wild-type cancers. In contrast, anti-EGFR monoclonal antibodies can be administered irrespective of EGFR mutational status. Based on this distinction, we propose a therapeutic strategy combining CDK4/6 inhibitors with anti-EGFR antibodies.
Although CDK4/6 inhibitors have been evaluated in clinical trials across multiple cancer types, their efficacy as monotherapy has generally been limited. In this study, however, we provide mechanistic insights and proof of concept (POC) demonstrating that rational combination therapy can markedly enhance their antitumor activity. Importantly, these findings are not restricted to pancreatic cancer but may extend the clinical applicability of CDK4/6 inhibitors to a broader spectrum of refractory malignancies.
Because this strategy relies on the combination of existing, clinically approved agents, we anticipate that it will facilitate rapid translation into investigator-initiated clinical trials.
Dec 19, 2026; Chiaki Takahashi and Zhang Yuanyuan