The PI3K/Akt pathway, one of the most frequently dysregulated signaling pathways in various cancers, plays a key role in tumorigenesis and resistance to chemotherapy. Recent discovery highlights an important regulatory step where Smurf1-mediated PDK1 neddylation through poly-Nedd8 chains is an early event for oncogenic Akt activation in KRAS-mutant colorectal cancer.

The phosphoinositide 3-kinase (PI3K)/Akt axis regulates diverse biological processes including cell proliferation, survival, apoptosis, and metabolism critical for tumorigenesis.1 The activation of Akt, a critical downstream effector of PI3K, is central for cancer development. While the mechanisms underlying Akt activation have been extensively characterized in literature,2 intensive ongoing research continues to advance our understanding of Akt activation. In a recent paper published in Nature Chemical Biology, Peng et al.3 identified SMAD ubiquitylation regulatory factor 1 (Smurf1)-mediated PDK1 neddylation as an early event for Akt activation upon growth factor treatment and subsequent downstream oncogenic signaling in KRAS-mutated colorectal cancer (CRC).

It is well established that PI3K activated by growth factors catalyzes the production of PIP3 at the plasma membrane (PM), which recruits cytosolic inactive PDK1 and Akt to the PM through its binding to the pleckstrin homology (PH) domain of these proteins. Subsequently, PDK1 phosphorylates Akt at Thr308 and mTORC2 at Ser473 for full activation of Akt.4 Apart from PI3K, post-translational modifications (PTMs) including methylation and ubiquitination of Akt are also critical early events for PM translocation and activation of Akt.5,6,7 Upon growth factor stimulation, SET domain bifurcated histone lysine methyltransferase 1 (SETDB1) interacts with and methylates Akt, leading to recruitment of JMJD2A along with the E3 ligases TRAF6 and/or Skp2-SCF to promote K63-linked ubiquitination of Akt for the PM translocation and activation of Akt.6,7,8 Although PIP3-driven trans-autophosphorylation of PDK1 is known for its activation, the full spectrum of regulatory mechanisms, such as PTMs, and their effects on PDK1 activation remain unknown.9 The study by Peng and his colleagues uncovered a novel mechanism underlying PDK1 and Akt activation via PDK1 neddylation, offering a promising strategy for targeting the PI3K/Akt pathway in KRAS-mutant CRC.3 By using systematic mass spectrometry (MS) analysis and cell-free system, they identified Smurf1, homologous to E6-AP C terminus (HECT) domain-type ubiquitin E3 ligase, as a new PDK1-interacting protein upon insulin stimulation in KRAS-mutant HCT116 cells. The Smurf1 knockout mouse model revealed that Smurf1 is crucial for PDK1-Akt signaling activation and displays an oncogenic activity. Interestingly, Smurf1 enhances PDK1-Akt pathway in a manner that is independent of its E3 ligase activity but dependent on its neddylation activity in response to insulin stimulation. MS analysis revealed that Smurf1 knockdown decreases PDK1-Nedd8 conjugation at K163, which is essential for Akt T308 phosphorylation and activation in response to insulin treatment. They further verified that Smurf1 directly induced PDK1 neddylation in the in vitro cell-free system. Using the PDK1 K163R mutant to explore the role of PDK1 neddylation in Akt activation, the authors unraveled that abolishment of PDK1 neddylation at K163 does not impact PDK1’s ATP binding ability. Instead, it disrupts the interaction between Akt and SETDB1, thus leading to defective Akt methylation and ubiquitylation. Subsequently, through in vitro binding assays and gel filtration chromatography, the authors found that neddylated PDK1 interacts with SETDB1, forming a cytoplasmic complex of PDK1 assembled with Smurf1 and SETDB1 (cCOMPASS). This complex helps direct the K64 methylation of Akt for T308 phosphorylation and PM translocation of Akt. As such, Smurf1 deficiency impairs CRC tumorigenesis in a manner dependent on Akt activation in a genetic model. Importantly, analysis of tumor sample specimens revealed a positive correlation of Smurf1 with PDK1 neddylation, Akt phosphorylation, and poor survival outcomes, especially in KRAS-mutated CRC samples with G12D or G13D mutations. Further study revealed that KRAS mutations in CRC induced Smurf1 expression via assembled AP-1 transcription factor complex, PDK1 neddylation and Akt phosphorylation. These data reveal the essential role of PDK1 neddylaiton in forming the cCOMPASS complex for methylation, ubiquitination and activation of Akt.

Deregulated PI3K/Akt pathway is frequently associated with KRAS mutations found in CRC, contributing to cancer progression and drug resistance.10 Combining PI3K/Akt inhibitors with mutant KRAS inhibitors has emerged as a promising strategy for cancer treatment, particularly in overcoming drug resistance.10 However, directly targeting PI3K can lead to significant toxicities because of its essential role in maintaining normal cell homeostasis. Hence, targeting the downstream effectors may potentially minimize side effects while inhibiting tumor growth. One of the downstream effectors, PDK1, has been shown to be a promising target for therapeutic intervention in KRAS-driven malignancies. However, direct target of PDK1 still remains challenging owing to its prevalent physiological role.11 To overcome this, Peng et al. utilized the NanoLuc and HiBiT technology platforms to develop SMART1, a CRBN-based PROTAC targeting Smurf1 degradation, aiming at inhibiting PDK1-Akt pathway in KRAS-mutated CRC. Remarkably, SMART1 exhibited a potent Smurf1 degradation activity at the nanomolar concentration, leading to defective PDK1 neddylation and Akt phosphorylation in KRAS-mutated CRC cell line. In CRC xenografts and patient-derived xenograft models, SMART1 alone or in combination with the PDK1 inhibitor (AR12) showed a potent tumor suppression activity. Collectively, this novel Smurf1-mediated PDK1 neddylation is considered clinically significant and can be very useful in the future for developing KRAS-mutant CRC therapeutic strategies.

The elegant work by Peng et al. provides not only invaluable insights into PDK1/Akt regulation but also presents a promising strategy for targeting KRAS-mutated CRC (Fig. 1). While this study reveals new insights into Akt activation, several key questions remain to be addressed. First, it is unclear how Smurf1 is recruited to membrane-bound PDK1 for driving neddylation of PDK1 following insulin stimulation. Are there PTMs of Smurf1 involved in this interaction? Second, it remains unclear about the relationship between PDK1 autophosphorylation and neddylation by Smurf1. Investigating whether PDK1’s trans-autophosphorylation precedes its neddylation or whether neddylation induces autophosphorylation could provide deeper insights into the activation sequence and the functional interplay between these processes. Third, how does Smurf1-mediated PDK1 neddylation occurring in PM promote cytosolic localization of PDK1 for the assembly of the cCOMPASS complex upon growth factor treatment? Finally, SETDB1-mediated methylation of Akt at K64 recruits JMJD2A along with TRAF6 and/or Skp2 for K63-linked ubiquitination of Akt. How does SETDB1 recognize neddylated PDK1 for subsequent Akt methylation? Understanding the dynamics of PDK1, SETDB1 and Smurf1’s involvement in the cCOMPASS complex, including the oscillation that allows for Akt ubiquitination and membrane localization, is of significance. We speculate that this process may involve additional regulatory steps that are yet to be uncovered. Addressing these questions will enhance our further understanding of the regulatory mechanisms governing PDK1 and Akt activation and could reveal new therapeutic targets or strategies for cancer targeting.

Peng et al. identified Smurf1-mediated PDK1 neddylation at K163 as an early event for Akt activation and subsequent downstream oncogenic signaling in KRAS-mutated CRC. This new PDK1 neddylation through poly-Nedd8 chains is critical for Akt K64 methylation and T308 phosphorylation and activation, and subsequent oncogenic signaling for tumorigenesis. How Smurf1 is recruited to membrane PDK1 to drive PDK1 neddylation for forming a cCOMPASS complex in response to growth factor stimulation is not clear. This process might involve additional regulatory steps that have yet to be elucidated. AR12, a selective PDK1 inhibitor.