This study presents the first proteome-wide comparison of pharmacological ULK1 activation and inhibition in TNBC cells. Our findings underscore the multifaceted role of ULK1 in regulating autophagy, transcription, immune signaling, and protein homeostasis, offering valuable insights into potential therapeutic strategies for this aggressive breast cancer subtype.

We observed that both ULK1 activation via LYN-1604 and inhibition via MRT68921 led to increased autophagic vesicle formation and LC3B accumulation. These results may initially appear paradoxical, as ULK1 is canonically required for autophagy initiation. However, prior studies have shown that ULK1 inhibition does not uniformly suppress autophagic flux; rather, it can activate compensatory or alternative pathways that maintain autophagosome formation under stress conditions [19, 20]. For instance, AMPK, a major energy sensor, can become hyperactivated upon ULK1 inhibition, potentially driving autophagy through ULK1-independent mechanisms such as Beclin 1 phosphorylation or activation of ULK2 [12, 21]. Our results support this model, suggesting that ULK1 inhibition may uncouple autophagosome initiation from flux, thereby altering the qualitative nature of the autophagic response.

Proteomic profiling revealed distinct functional outcomes depending on the direction of ULK1 modulation. ULK1 activation led to upregulation of RNA polymerase II transcription termination and mRNA 3′-end processing pathways, coupled with downregulation of translational machinery. This dichotomy suggests that ULK1 activation promotes transcriptional remodeling while conserving energy by attenuating protein synthesis -features commonly associated with integrated stress responses and autophagy-mediated survival mechanisms [22, 23]. These findings align with prior evidence linking ULK1 to nuclear functions beyond its canonical cytoplasmic role, including regulation of mRNA dynamics and epigenetic signaling [11, 22, 23].

Conversely, ULK1 inhibition reprogrammed the proteome toward immunological engagement. We observed increased abundance of proteins annotated to complement-related terms, vesicle-mediated transport, and platelet degranulation, a processes frequently associated with immune signaling and antigen presentation. Although ULK1 is a canonical initiator of autophagy, our findings demonstrate that its inhibition with MRT68921 does not fully suppress autophagic activity; instead, it appears to induce compensatory autophagy through ULK1-independent mechanisms. This autophagy-associated phenotype (LC3B/vesicle signal accumulation) may reflect compensatory stress responses and/or altered autophagosome maturation/clearance; although increased autophagic flux was not directly demonstrated in this study, autophagy-related processes could still intersect with immune signaling by facilitating intracellular antigen presentation via MHC class I, promoting DAMP release, and modulating inflammatory responses [24]. Supporting this, Li Jin et al. revealed that LPS-induced activation of the MAPK p38/ULK1 pathway inhibits autophagy and induces IL-1β expression in hepatic stellate cells, highlighting the interplay between autophagy inhibition and immune activation [25]. Similarly, He Y et al. found that p38 MAPK inhibits autophagy and promotes microglial inflammatory responses by phosphorylating ULK1, suggesting that ULK1 activity modulates immune responses through autophagy regulation [26]. Notably, MRT68921 has been shown to increase immunoproteasome function and reverse immune evasion in LKB1-deficient cancers, suggesting a broader application of ULK1 inhibitors in immunotherapy [27]. Thus, rather than resulting from autophagy blockade, the immune-related enrichment signature observed in the proteomic data may reflect a distinct stress phenotype in which compensatory autophagy and disrupted proteostasis collectively enhance tumor immunogenicity.

Importantly, a subset of proteins was consistently regulated under both ULK1 activation and inhibition, suggesting that specific cellular functions are highly sensitive to ULK1 perturbation regardless of directionality. Protein–protein interaction network analysis revealed five common hub proteins: PSIP1 (LEDGF/p75), AGO2, MORF4L1, HNRNPC, and SETD2. Functional enrichment analysis indicated their involvement in autophagy-associated pathways such as mitochondrial calcium ion transport, MET–PTK2 (c-Met–FAK) signaling, extracellular matrix (ECM) remodeling, FasL/CD95 death receptor signaling, and cholesterol metabolism.

These pathways have been implicated in autophagy regulation and TNBC biology in prior studies; however, our proteomic data provide association-level evidence rather than functional confirmation. For example, autophagy can promote lysosomal degradation of the MET receptor, dampening downstream MET–FAK signaling and limiting invasive behavior [28]. Similarly, the ULK1-FIP200 complex facilitates turnover of focal adhesion components, thereby regulating cell adhesion and motility through FAK modulation [29]. Mitochondrial Ca²⁺ flux, a central node in autophagy signaling, governs cross-talk with apoptotic programs [30], while autophagy has also been shown to modulate death receptor pathways (e.g., Fas/CD95) and promote lipid droplet degradation and cholesterol efflux via HDL [31].

Our findings extend this knowledge by suggest that ULK1 modulation is associated with coordinated changes in hub proteins and pathway signatures implicated in TNBC biology; the functional consequences remain to be tested. We propose that these signatures represent testable hypotheses; whether they translate into tumor-suppressive phenotypes requires proliferation/migration/invasion assays.

Indeed, previous studies support this rationale. PSIP1 (LEDGF/p75), a chromatin-bound transcription coactivator, has been implicated in chemoresistance and metastasis in TNBC. Its silencing significantly impaired tumorigenicity and metastasis in vivo, highlighting its role in cancer aggressiveness [32]. AGO2, an essential component of the RNA-induced silencing complex (RISC), facilitates microRNA-mediated post-transcriptional repression. Overexpression of AGO2 has been associated with enhanced proliferation, therapy resistance, and epithelial-to-mesenchymal transition (EMT) in breast cancer, including TNBC [33]. MORF4L1, a chromatin remodeling factor involved in histone acetylation, has been linked to stemness, metastatic potential, and altered transcriptional landscapes in breast cancer. Its role in epigenetic regulation suggests potential as a therapeutic target in aggressive subtypes [34]. HNRNPC, a heterogeneous nuclear ribonucleoprotein, governs alternative splicing and mRNA stability. In basal-like and triple-negative breast cancer, its overexpression promotes tumor growth, and correlates with poor survival outcomes [35, 36]. SETD2, the sole H3K36 trimethyltransferase, plays a critical role in maintaining genomic integrity, DNA repair, and transcription fidelity. Mutations or reduced expression of SETD2 contribute to tumorigenesis and therapy sensitivity in TNBC, particularly in relation to PARP inhibitor response [37, 38].

These findings support the hypothesis that ULK1 modulation may exert anti-tumor effects in TNBC via coordinated regulation of these hub proteins. Targeting PSIP1, AGO2, MORF4L1, HNRNPC, and SETD2 offers a novel strategy to disrupt interconnected networks of transcriptional control, RNA processing, and chromatin remodeling—all of which are critical to the aggressive phenotype and therapy resistance of TNBC.

Collectively, our findings highlight the potential of modulating autophagy-associated networks in TNBC as a therapeutic strategy. While ULK1 remains a central regulator of autophagy, the identification of downstream targets -particularly PSIP1 (LEDGF/p75), AGO2, MORF4L1, HNRNPC, and SETD2- opens the door to more precise interventions. These proteins, which may act as intrinsic barriers to autophagy-linked tumor suppression, represent promising molecular entry points for future combination therapies. Ultimately, therapeutically targeting this specific set of hub proteins may provide a means to replicate the multi-faceted anti-tumor benefits of autophagy, while circumventing the challenges of direct ULK1 modulation.

This study has several limitations that should be considered when interpreting the findings. First, our autophagy readouts were based on an autophagy detection dye and LC3B immunoblotting, and we did not perform lysosomal inhibitor–based turnover assays to directly assess autophagic flux; therefore, the observed increases in vesicle-associated fluorescence and LC3B signal may reflect enhanced autophagosome formation and/or altered autophagosome maturation with impaired lysosomal clearance rather than confirmed increases in flux. Second, the experiments were conducted in a single TNBC cell line (MDA-MB-231), without additional TNBC models or non-malignant breast epithelial controls, which limits the generalizability of the proteomic signatures across TNBC subtypes and normal tissues. Third, while ULK1 was pharmacologically modulated using LYN-1604 and MRT68921, we did not perform genetic perturbation or orthogonal compound validation to confirm ULK1 dependency and to exclude potential off-target contributions; thus, conclusions should be interpreted as reflecting pharmacological ULK1 pathway modulation under the tested conditions. Fourth, the proteomic analyses are primarily hypothesis-generating and were not systematically corroborated by independent assays for key candidates, and no functional phenotypic assays (proliferation, migration, invasion) or in vivo validation were included. In addition, candidate proteins were prioritized using a ≥ 2-fold screening threshold; therefore, these candidate lists should be interpreted as hypothesis-generating and may include false positives in the absence of formal differential-abundance hypothesis testing with multiple-testing correction. Accordingly, enrichment results should be interpreted as pathway-level associations rather than direct evidence of functional phenotypes, and the highlighted pathways and hub proteins should be viewed as prioritized candidates for future mechanistic and translational studies.