Lou, Zhenkun

Abstract Pancreatic adenocarcinoma remains one of the most lethal malignancies, primarily due to its robust resistance to genotoxic therapies, such as chemotherapy and radiotherapy. Understanding the mechanisms underlying this resistance is essential to improve clinical outcomes. In this study, we identified trimethylguanosine synthase 1 (TGS1), previously known for its role in RNA modification, as a critical mediator of homologous recombination (HR) repair that specifically contributes to resistance in pancreatic adenocarcinoma. TGS1 was significantly overexpressed in pancreatic adenocarcinoma tissues, correlating strongly with advanced disease stages, therapy resistance, and poor patient prognosis. Following DNA damage, ATM kinase phosphorylated TGS1 at serine residues S389 and S531, which mediated its direct interaction with BRCA1 and subsequent recruitment of BRCA1 to DNA damage sites. The phosphorylation-dependent interaction enhanced HR repair efficiency, enabling cancer cells to survive genotoxic stress. Depletion or pharmacologic inhibition of TGS1 induced HR deficiency and markedly enhanced sensitivity to DNA-damaging agents, particularly PARP inhibitors, in pancreatic adenocarcinoma cell lines in vitro and in both cell line– and patient-derived xenograft models in vivo. Collectively, these findings uncover an ATM–TGS1–BRCA1 signaling axis that promotes DNA repair and resistance to genotoxic therapies in pancreatic cancer, positioning TGS1 as a promising predictive biomarker and therapeutic target to enhance treatment efficacy. Significance: The ATM–TGS1–BRCA1 signaling axis is a critical regulator of DNA repair that can be targeted to overcome genotoxic therapy resistance, providing a promising therapeutic strategy that could transform pancreatic cancer treatment.

Metastatic castration-resistant prostate cancer (mCRPC) progresses aggressively and resists existing therapies. Although poly(ADP-ribose) polymerase inhibitors (PARPis) benefit a subset of patients with mCRPC and BRCA1/2 deficiencies, therapeutic options remain limited for those without such mutations. Here, we uncover a critical role for the ATM-TRMT10A-BRCA1 signaling axis in regulating homologous recombination (HR) repair and PARPi sensitivity. We demonstrate that ATM phosphorylates TRMT10A at serine-28 after DNA damage, promoting BRCA1 recruitment and efficient HR repair. TRMT10A deletion disrupts HR repair, sensitizing cells to PARPis. Moreover, TRMT10A is up-regulated in mCRPC through stabilization by USP10. Targeting USP10 with spautin-1 induces TRMT10A degradation and enhances tumor sensitivity to PARPis in cell-derived xenografts and patient-derived xenograft models. These findings identify TRMT10A as a therapeutic vulnerability in mCRPC and demonstrate that combined inhibition of PARP and USP10 offers a promising synthetic lethal strategy for a broader group of patients lacking classical BRCA mutations.

CDK12 primarily functions as a transcription regulatory cyclin-dependent kinase (CDK) that controls mRNA elongation, splicing, and polyadenylation. The CDK12 gene is implicated in human cancers since it is frequently mutated and/or deleted in prostate and ovarian cancer but paradoxically amplified in breast cancer. Here, we demonstrate that CDK12 promotes serine-933 phosphorylation of DNA2, a nuclease/helicase critical for replication fork stress regulation, and the phosphorylation subsequently facilitates DNA2 polyubiquitination and degradation mediated by the APC/C CDC20 E3 ubiquitin ligase. CDK12 inactivation induces but amplification suppresses genome-wide expression of interferon response and antigen processing and presentation machinery genes in ovarian and breast cancer cells, respectively. Besides causing aberrant DNA2 stabilization, replication stress, genomic instability, and cytosolic double-stranded DNA (dsDNA) accumulation, CDK12 loss also triggers cGAS–STING activation and innate immune response, which can be reversed by forced expression of replication protein A (RPA) subunits or DNA2 depletion. Our findings identify DNA2 as a phosphorylation substrate of CDK12, connecting CDK12 to cell cycle regulation. These data also reveal DNA2 protein destruction as a critical mechanism that dictates genomic instability, cGAS–STING signaling activation, and innate immune response in CDK12-deregulated cancers.

Abstract The TopBP1-ATR axis is critical for maintaining genomic stability during DNA replication stress, yet the precise regulation of TopBP1 in replication stress responses remains poorly understood. In this study, we identified PHD and Ring Finger Domains 1 (PHRF1) as an important ATR activator through its interaction with TopBP1. Our analysis revealed a correlation between PHRF1 and genomic stability in cancer patients. Mechanistically, PHRF1 is recruited to DNA lesions in a manner dependent on its PHD domain and histone methylation. Subsequently, PHRF1 mono-ubiquitinates TopBP1 at lysine 73, which enhances the TopBP1-ATR interaction and activates ATR. Depletion of PHRF1 disrupts ATR activation and sensitizes cells to replication stress-inducing agents. Furthermore, conditional knockout of Phrf1 in mice leads to early lethality and impaired ATR-Chk1 axis signaling. Collectively, our findings establish PHRF1 as a novel E3 ligase for TopBP1, coordinating the replication stress response by enhancing TopBP1-ATR signaling.

Abstract The phosphorylation of histone H2A.X into γH2A.X is a crucial early event in the DNA damage response, marking DNA damage sites and initiating repair processes. While ATM kinase is traditionally recognized as the primary mediator of H2A.X phosphorylation, our study identifies serine/threonine kinase 39 (STK39) as a novel enhancer of this critical signaling pathway. We demonstrate that after DNA damage, STK39 undergoes phosphorylation by the ATM kinase, facilitating its interaction with the Mre11-Rad50-Nbs1 complex and subsequent recruitment to chromatin. This recruitment enables STK39 to further phosphorylate H2A.X, thus amplifying γH2A.X production and promoting homologous recombination repair. Notably, we observe a significant upregulation of STK39 in pancreatic adenocarcinoma (PAAD) tissues, correlating with heightened resistance to PARPi therapy. Furthermore, we demonstrate the synergistic efficacy of combining STK39 inhibition with PARP inhibitors in suppressing and reversing PAAD growth. This study not only provides new insights into the molecular dynamics of H2A.X phosphorylation but also highlights the therapeutic potential of targeting STK39 to enhance PARPi sensitivity in PAAD (created with BioRender).

ATM is a key mediator of radiation response, and pharmacological inhibition of ATM is a rational strategy to radiosensitize tumors. AZD1390 is a brain-penetrant ATM inhibitor and a potent radiosensitizer. This study evaluated the spectrum of radiosensitizing effects and the impact of TP53 mutation status in a panel of IDH1 wild-type (WT) glioblastoma (GBM) patient-derived xenografts (PDXs). AZD1390 suppressed radiation-induced ATM signaling, abrogated G 0 -G 1 arrest, and promoted a proapoptotic response specifically in p53-mutant GBM in vitro. In a preclinical trial using 10 orthotopic GBM models, AZD1390/RT afforded benefit in a cohort of TP53 -mutant tumors but not in TP53 -WT PDXs. In mechanistic studies, increased endogenous DNA damage and constitutive ATM signaling were observed in TP53 -mutant, but not in TP53 -WT, PDXs. In plasmid-based reporter assays, GBM43 ( TP53 -mutant) showed elevated DNA repair capacity compared with that in GBM14 (p53-WT), whereas treatment with AZD1390 specifically suppressed homologous recombination (HR) efficiency, in part, by stalling RAD51 unloading. Furthermore, overexpression of a dominant-negative TP53 (p53DD) construct resulted in enhanced basal ATM signaling, HR activity, and AZD1390-mediated radiosensitization in GBM14. Analyzing RNA-seq data from TCGA showed up-regulation of HR pathway genes in TP53 -mutant human GBM. Together, our results imply that increased basal ATM signaling and enhanced dependence on HR represent a unique susceptibility of TP53 -mutant cells to ATM inhibitor–mediated radiosensitization.

Abstract Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer with limited therapeutic strategy and unfavorable prognosis, especially for patients bearing brain metastasis. Here, we demonstrate that MTAP gene deletion or MTAP inhibition by MTAP inhibitor, MTDIA, sensitizes breast cancer cells to poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi). Mechanistically, PARPi suppresses the expression of methionine adenosyltransferase 2A (MAT2A) by inducing METTL16 auto-inhibition mediated intron retention. Given that MAT2A and MTAP are key enzymes in methionine metabolism, the dual inhibition of MAT2A and MTAP disrupts methionine metabolism and leads to reduced s-adenosylmethionine. Dietary methionine restriction further boosts the combined anti-tumor efficacy of MTAP depletion/inhibition and PARPi. Most importantly, in brain metastatic TNBC, due to the natural low methionine environment in the brain, the combination of MTAP depletion/inhibition and PARPi confers significant therapeutic efficacy. Therefore, our findings provide a promising strategy for inhibiting primary or brain metastatic TNBC.

Abstract Imatinib mesylate (IM) has revolutionized the treatment of gastrointestinal stromal tumor (GIST). However, most patients inevitably acquire IM resistance. Second- and third-line treatments exhibit modest clinical benefits with a median time to disease progression of 4 to 6 months, highlighting the urgency for novel therapeutic approaches. Here, we report that the expression of BCL6, a known oncogenic driver and transcriptional repressor, was significantly induced in GIST cells following IM treatment. Elevated BCL6 levels suppressed apoptosis and contributed to IM resistance. Mechanistically, BCL6 recruited SIRT1 to the TP53 promoter to modulate histone acetylation and transcriptionally repress TP53 expression. The reduction in p53 subsequently attenuated cell apoptosis and promoted tolerance of GIST cells to IM. Concordantly, treatment of GIST cells showing high BCL6 expression with a BCL6 inhibitor, BI-3802, conferred IM sensitivity. Furthermore, BI-3802 showed striking synergy with IM in IM-responsive and IM-resistant GIST cells in vitro and in vivo. Thus, these findings reveal a role for BCL6 in IM resistance and suggest that a combination of BCL6 inhibitors and IM could be a potentially effective treatment for GIST. Significance: BCL6 drives resistance to imatinib by inhibiting p53-mediated apoptosis and can be targeted in combination with imatinib to synergistically suppress tumor growth, providing a therapeutic strategy for treating gastrointestinal stromal tumor.

The EGFR-RAS-ERK pathway is one of the most important signaling cascades in cell survival, growth, and proliferation. Aberrant activation of this pathway is a common mechanism in various cancers. Here, we report that CDK2 is a novel regulator of the ERK pathway via USP37 deubiquitinase (DUB). Mechanistically, CDK2 phosphorylates USP37, which is required for USP37 DUB activity. Further, USP37 deubiquitinates and stabilizes ERK1/2, thereby enhancing cancer cell proliferation. Thus, CDK2 is able to promote cell proliferation by activating USP37 and, in turn, stabilizing ERK1/2. Importantly, combined CDK1/2 and EGFR inhibitors have a synergetic anticancer effect through the downregulation of ERK1/2 stability and activity. Indeed, our patient-derived xenograft (PDX) results suggest that targeting both ERK1/2 stability and activity kills cancer cells more efficiently even at lower doses of these two inhibitors, which may reduce their associated side effects and indicate a potential new combination strategy for cancer therapy.