胰腺癌（Pancreatic adenocarcinoma, PAAD）是一种侵袭性强、预后极差的恶性肿瘤，其全球发病率和死亡率持续攀升，2024年已位列癌症相关死亡的第三大原因。约80%的胰腺癌患者确诊时处于晚期，无法进行手术切除治疗，主要依赖于放化疗，整体5年生存率仅约12%。

以“DNA损伤修复（DNA Damage Response, DDR）-合成致死（Synthetic Lethality, SL）”为核心理念的PARP抑制剂（PARP inhibitor, PARPi）在多种肿瘤治疗中展现出显著疗效，尤其对存在BRCA1/2或PALB2等同源重组修复缺陷（Homologous Recombination Deficiency, HRD）表型的胰腺癌患者表现出较好的临床应答。然而，胰腺癌患者中HRD相关基因突变比例较低，且部分DDR基因突变（如ATM、CDK12等）对PARPi敏感性不足，限制了PARPi在胰腺癌中的适用范围。因此，亟需探索新的分子靶点或机制，以诱导或增强胰腺癌细胞的HRD表型，从而拓宽PARPi的治疗适应症并提升其疗效。

为探索调节同源重组修复的关键基因，本研究采用DNA损伤诱导模型，结合高通量质谱技术分析磷酸化修饰谱，构建蛋白激酶信号网络。结果发现，除经典激酶ATM、ATR外，丝氨酸/苏氨酸激酶39（Serine/Threonine Kinase 39, STK39）的磷酸化水平与ATM同步动态变化，提示其可能参与DNA损伤应答。

STK39在脑和胰腺组织中高表达，既往研究集中于其调控离子转运及血压的功能。近年发现，STK39通过激活MAPK/ERK等信号通路促进肝癌、乳腺癌及骨肉瘤进展，但其在胰腺癌中的作用尚未明确。基于TCGA数据分析，STK39在胰腺癌组织中显著高表达，且与患者不良预后正相关。这一发现提示STK39可能作为胰腺癌的新型分子标志物和治疗靶点。因此，我们需要进一步探究STK39在胰腺癌中的功能。

首先我们通过免疫组化分析了PAAD组织微阵列上STK39和DNA损伤标志物γH2AX表达的相关性，发现两者之间存在明显的正相关，提示STK39参与DNA损伤反应过程。随后通过构建STK39敲低、过表达、酶活突变稳转株细胞验证DNA修复效率，我们发现STK39能促进DNA损伤后的同源重组（Homologous Recombination, HR）和非同源末端连接（Non-homologous End Joining, NHEJ）。免疫荧光检测以上稳转株中DNA修复因子foci的形成，发现STK39能增强细胞核中γH2A.X，MDC1，FK2，53BP1，BRCA1等DNA损伤修复因子foci的形成。平板克隆形成实验发现STK39敲低后能增加胰腺癌细胞对辐照、奥拉帕尼的敏感性。我们进一步研究发现，在分子机制层面上STK39可直接磷酸化组蛋白变体H2A.X。蛋白免疫印迹实验显示，STK39敲低或激酶失活导致γH2A.X水平下降，而体外激酶实验证实纯化的STK39野生型（而非D212A突变体）可直接催化H2A.X磷酸化。随后研究发现，ATM激酶在DNA损伤后通过磷酸化STK39的S401位点，促进其与MRN复合物（Rad50/Nbs1）结合并募集至染色质。

基于上述机制，我们探索了靶向STK39的临床转化潜力。在裸鼠移植瘤模型（包括患者来源的异种移植物模型和细胞衍生的异种移植物模型）中，STK39敲低联合奥拉帕尼治疗使肿瘤体积生长速度更加缓慢，肿瘤生长体积更小，其效果显著优于PARP抑制剂单药组。更关键的是，小分子抑制剂ZT-1a可模拟STK39基因敲低效果，增强PARP抑制剂疗效，为临床联合用药提供了直接依据。

综上所述，本研究不仅揭示了胰腺癌中H2A.X磷酸化的新机制，还发现靶向STK39可增强胰腺癌对PARPi治疗的敏感性，为胰腺癌的精准治疗提供了新的理论基础与临床治疗策略。

Pancreatic adenocarcinoma (PAAD) is a highly aggressive malignant tumor characterized by an extremely poor prognosis. The global incidence and mortality rates of PAAD continue to rise, and as of 2024, it has become the third leading cause of cancer-related deaths worldwide. One of the major challenges in the clinical management of PAAD is that approximately 80% of patients are diagnosed at advanced stages, at which point the tumor is no longer amenable to surgical resection. As a result, these patients primarily rely on radiotherapy and systemic treatments; however, the overall 5-year survival rate remains dismally low, at approximately 12%.

Poly (ADP-ribose) polymerase inhibitors (PARPi) are a class of targeted therapeutic agents based on the concept of "DNA Damage Response (DDR)-Synthetic Lethality." These inhibitors have demonstrated remarkable clinical efficacy in the treatment of various malignancies, particularly in patients with pancreatic adenocarcinoma (PAAD) harboring homologous recombination deficiency (HRD) phenotypes, such as mutations in BRCA1, BRCA2, or PALB2. By exploiting defects in DNA repair mechanisms, PARPi selectively induces tumor cell death while sparing normal cells, making them a promising strategy for precision oncology.

Despite their potential, the therapeutic applicability of PARPi in pancreatic cancer remains significantly limited. One major challenge is the low prevalence of HRD-associated gene mutations among pancreatic cancer patients, which restricts the proportion of individuals who can benefit from these agents. Additionally, some DDR-related gene mutations, such as those in ATM and CDK12, exhibit insufficient sensitivity to PARPi, further reducing the effectiveness of this treatment approach. Given these limitations, there is an urgent need to identify novel molecular targets or mechanisms capable of inducing or enhancing the HRD phenotype in pancreatic cancer cells. By expanding the therapeutic window of PARPi, these strategies could significantly broaden the spectrum of eligible patients and enhance the overall efficacy of PARPi-based therapies in pancreatic cancer treatment.

To investigate the key regulatory genes involved in homologous recombination repair (HRR) , this study employed a DNA damage induction model in combination with high-throughput mass spectrometry to comprehensively analyze phosphorylation modification profiles and construct a protein kinase signaling network. Through this systematic analysis, we identified dynamic changes in phosphorylation patterns associated with the DNA damage response (DDR).

The results demonstrated that, in addition to the well-established DDR kinases ATM (ataxia-telangiectasia mutated) and ATR (ataxia-telangiectasia and Rad3-related), the phosphorylation level of serine/threonine kinase 39 (STK39) exhibited synchronized dynamic changes with ATM. This observation strongly suggests that STK39 may play a previously unrecognized role in the DNA damage response, potentially contributing to the regulation of homologous recombination repair pathways. These findings provide a foundation for further mechanistic investigations into the role of STK39 in genomic stability and DNA repair.

STK39 is known to be highly expressed in both brain and pancreatic tissues. Historically, research on STK39 has primarily focused on its role in regulating ion transport and blood pressure homeostasis. However, in recent years, emerging evidence has indicated that STK39 plays a critical role in tumor progression. Specifically, studies have demonstrated that STK39 promotes the development and progression of hepatocellular carcinoma, breast cancer, and osteosarcoma by activating oncogenic signaling pathways, such as the MAPK/ERK pathway. Despite these findings, the precise biological function and oncogenic role of STK39 in pancreatic cancer remain largely unexplored.

To gain further insights into its potential involvement in pancreatic cancer, we conducted a comprehensive analysis of publicly available datasets from The Cancer Genome Atlas (TCGA). Our results revealed that STK39 is significantly overexpressed in pancreatic cancer tissues compared to normal tissues, and its elevated expression levels are positively correlated with poor patient prognosis. This observation strongly suggests that STK39 may serve as a novel molecular biomarker for pancreatic cancer and could represent a promising therapeutic target for improving treatment outcomes. Given these findings, it is essential to further investigate the functional role of STK39 in pancreatic cancer to elucidate its underlying mechanisms and therapeutic potential.

To investigate the potential role of STK39 in the DNA damage response (DDR), we first examined its correlation with the well-established DNA damage marker γH2AX in pancreatic adenocarcinoma (PAAD) tissue microarrays using immunohistochemistry (IHC). The analysis revealed a significant positive correlation between the expression levels of STK39 and γH2AX, strongly suggesting that STK39 may be actively involved in the DNA damage response process.

To further explore the functional role of STK39 in DNA repair, we constructed stable pancreatic cancer cell lines with STK39 knockdown, STK39 overexpression, and a kinase activation mutant (D212A). DNA repair efficiency was assessed in these models, and the results demonstrated that STK39 enhances both homologous recombination (HR) and non-homologous end-joining (NHEJ) repair pathways, indicating its role in maintaining genomic stability. Additionally, immunofluorescence assays were performed to assess the formation of DNA repair foci in these stable cell lines. The results showed that STK39 significantly promoted the nuclear accumulation of critical DNA damage repair factors, including γH2AX, MDC1, FK2, 53BP1, and BRCA1, further supporting its involvement in the DDR. To evaluate the potential impact of STK39 on therapeutic resistance, we conducted a colony formation assay to assess cellular sensitivity to DNA-damaging agents. The results revealed that STK39 knockdown significantly increased the sensitivity of pancreatic cancer cells to ionizing radiation and the PARP inhibitor olaparib, suggesting that STK39 may contribute to resistance against these therapies. At the molecular level, we discovered a novel mechanism by which STK39 directly phosphorylates the histone variant H2AX, a key event in the DDR. Western blot analysis revealed that STK39 knockdown or kinase inactivation led to a notable reduction in γH2AX levels, whereas in vitro kinase assays confirmed that wild-type STK39 (but not the kinase-dead D212A mutant) directly catalyzed H2AX phosphorylation, indicating its essential role in this process. Further mechanistic investigations revealed that STK39 functions downstream of ATM kinase. Specifically, following DNA damage, ATM phosphorylates STK39 at the S401 site, which in turn facilitates its interaction with the MRN (Mre11-Rad50-Nbs1) complex and promotes chromatin recruitment, thereby enhancing DNA damage repair efficiency. These findings collectively suggest that STK39 is a critical regulator of the DDR and may represent a novel therapeutic target for sensitizing pancreatic cancer cells to DNA damage-based therapies.

Building upon these mechanistic insights, we further explored the clinical translational potential of targeting STK39 as a strategy to enhance the efficacy of PARP inhibitors in PAAD. To evaluate this approach, we conducted in vivo experiments using nude mouse transplantation tumor models, including both patient-derived xenograft (PDX) models and cell line-derived xenograft (CDX) models. In these models, we assessed the effects of STK39 knockdown in combination with olaparib treatment. The results demonstrated that this combination therapy led to a significantly slower tumor growth rate and a marked reduction in overall tumor volume compared to the group treated with PARP inhibitor monotherapy, suggesting that STK39 depletion enhances the therapeutic efficacy of PARP inhibition in pancreatic cancer. More importantly, the small-molecule inhibitor ZT-1a was found to effectively mimic the functional effects of STK39 knockdown, leading to a significant enhancement in the therapeutic efficacy of PARP inhibitors. These findings provide compelling preclinical evidence supporting the potential of ZT-1a as a combinatorial therapeutic strategy with PARP inhibitors. By targeting STK39, ZT-1a may help to sensitize pancreatic cancer cells to DNA damage-based therapies, thereby offering a strong scientific rationale for its clinical application in combination with PARP inhibitors.

In conclusion, this study not only uncovers a previously unrecognized mechanism of H2A.X phosphorylation in pancreatic cancer, but also identifies STK39 as a critical regulatory factor in the DNA damage response Furthermore, our findings demonstrate that targeting STK39 significantly enhances the sensitivity of pancreatic cancer cells to PARP inhibitor treatment, thereby overcoming a key limitation of current PARPi-based therapies. These results provide a novel theoretical foundation and offer a potential clinical treatment strategy for the precision therapy of pancreatic cancer, paving the way for future translational and clinical research.