Lou, Zhenkun

AbstractDNA damage plays a significant role in the tumorigenesis and progression of the disease. Abnormal DNA repair affects the therapy and prognosis of cancer. In this study, it is demonstrated that the deubiquitinase USP25 promotes non‐homologous end joining (NHEJ), which in turn contributes to chemoresistance in cancer. It is shown that USP25 deubiquitinates SHLD2 at the K64 site, which enhances its binding with REV7 and promotes NHEJ. Furthermore, USP25 deficiency impairs NHEJ‐mediated DNA repair and reduces class switch recombination (CSR) in USP25‐deficient mice. USP25 is overexpressed in a subset of colon cancers. Depletion of USP25 sensitizes colon cancer cells to IR, 5‐Fu, and cisplatin. TRIM25 is also identified, an E3 ligase, as the enzyme responsible for degrading USP25. Downregulation of TRIM25 leads to an increase in USP25 levels, which in turn induces chemoresistance in colon cancer cells. Finally, a peptide that disrupts the USP25‐SHLD2 interaction is successfully identified, impairing NHEJ and increasing sensitivity to chemotherapy in PDX model. Overall, these findings reveal USP25 as a critical effector of SHLD2 in regulating the NHEJ repair pathway and suggest its potential as a therapeutic target for cancer therapy.

AbstractObjectiveTo elucidate the relationship between USP19 and O(6)‐methylguanine‐DNA methyltransferase (MGMT) after temozolomide treatment in glioblastoma (GBM) patients with chemotherapy resistance.MethodsScreening the deubiquitinase pannel and identifying the deubiquitinase directly interacts with and deubiquitination MGMT. Deubiquitination assay to confirm USP19 deubiquitinates MGMT. The colony formation and tumor growth study in xenograft assess USP19 affects the GBM sensitive to TMZ was performed by T98G, LN18, U251, and U87 cell lines. Immunohistochemistry staining and survival analysis were performed to explore how USP19 is correlated to MGMT in GBM clinical management.ResultsUSP19 removes the ubiquitination of MGMT to facilitate the DNA methylation damage repair. Depletion of USP19 results in the glioblastoma cell sensitivity to temozolomide, which can be rescued by overexpressing MGMT. USP19 is overexpressed in glioblastoma patient samples, which positively correlates with the level of MGMT protein and poor prognosis in these patients.ConclusionThe regulation of MGMT ubiquitination by USP19 plays a critical role in DNA methylation damage repair and GBM patients’ temozolomide chemotherapy response.

Abstract The stiffening of the extracellular matrix (ECM), a phenomenon that often promotes metastasis and immunosuppression in many cancers, is a process that is not yet fully understood. Our study reveals that ATR, a molecule primarily recognized for its role in maintaining the genome, plays a crucial role in ECM stiffness-induced immune evasion and metastasis in triple negative breast cancer (TNBC) through DNA repair independent manner. In response to ECM stiffness, ATR activates SUN2, a component of the linker of nucleoskeleton and cytoskeleton (LINC) complex. This activation enhances the interaction of SUN2 with outer nuclear membrane proteins KASH1 and KASH2. The interaction between SUN2 and KASH1/2 facilitates the nuclear localization of β-catenin, which in turn promotes epithelial mesenchymal transition (EMT) and reduces the expression of E-cadherin. E-cadherin is essential for attracting CD103+ immune cells that perform anti-tumor immunity. Interestingly, inhibiting ATR reduces EMT in tumor cells, leading to an increase in CD103+ dendritic cell infiltration while decreasing neutrophil and polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) infiltration. Further, we found that ATR levels are inversely correlated with immunotherapy response rates in patients. The alteration in immune cell composition sensitizes tumors to immunotherapy, thereby suppressing tumor growth and metastasis. In conclusion, the role of ATR in remodeling the immune environment could be crucial for optimizing the clinical use of ATR inhibitors. This makes ATR a promising target for overcoming immunotherapy resistance. We suggest early or prolonged treatment with an ATR inhibitor to potentially reduce metastasis rates and improve the prognosis of TNBC patients. Furthermore, we suggest using ATR levels as a marker to predict response rates and prognosis in TNBC patients. For patients with higher ATR expression levels, combination treatment with ATR inhibitors and immunotherapy may yield better outcomes. Enhanced anti-tumor immunity could increase the chances for patients to eliminate cancer cells and help end breast cancer. Finally, our research suggests the combined treatment of ATR inhibition and immunotherapy for patients without necessarily applying DNA damage stimulation. This approach could potentially reduce toxicity while still benefiting patients. Citation Format: Xinyi Tu, Zhenkun Lou, Robert Mutter. ATR promotes extracellular matrix stiffness mediated cytoskeleton remodeling and immunosuppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5366.

Abstract BACKGROUND: Cancer is typically treated with a combination of drugs rather than monotherapy stratagems, but identification of synergistic drug combinations for the treatment of cancers is challenging. Here we present the combination of PARP and Aurora Kinase inhibitors synergize through induction of the ferroptosis pathway of cell death. METHODS: We identified these drugs through an unbiased screen of enriched drug class response correlations. We then validated a synergistic cell killing effect of multiple drugs of each class by in vitro drug screening. Next, we performed reversal experiments using a ferroptosis inhibitor and lipid peroxide staining to confirm ferroptosis is a relevant cell death pathway. We performed Western immunoblot analysis in cells overexpressing putative Aurora Kinase phospho-substrates in the relevant pathway of interest, and performed site directed mutagenesis experiments to show the relevant amino acid altered by kinase activity. We also performed a series of qPCR and co-IP experiments to show PARPi effect on the antioxidant signaling pathway. RESULTS: We validated and identified synergy in a prostate cancer specific context. Next, we found Talazoparib and Alisertib induced ferroptosis after synergy was reduced with a ferroptosis inhibitor and lipid peroxides increased accumulation. We found a novel mechanism action for Alisertib, wherein Aurora Kinase inhibitors can alter the phospho-status and activity of the iron reduction enzyme Steap3.We also found that PARP inhibitors can affect the Nrf2-driven antioxidant signaling pathway on a transcriptional level. CONCLUSION: Aurora Kinase inhibitors and PARP inhibitors for the treatment of prostate cancer was identified and was validated as synergistic, which revealed new functional roles of both drugs in the context of cell killing. These findings represent a new clinical combination of drugs in prostate cancer and reveal a novel role for this drug class in ferroptosis. Citation Format: August John, Kuntian Luo, Zhenkun Lou, Liewei Wang. Ferroptotic mechanism of drug induced toxicity of PARPi and AurKi in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4563.

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 PD-L1/PD-1 receptor pathway represents an important way of cancer cells to escape immune surveillance, yet only small portions of patients can benefit from PD-1/PD-L1 inhibitors. This is at least partially due to incomplete understanding of PD-1/PD-L1 biology in cancer. Although the interaction of extracellular domains between PD-L1 and PD-1 on cancer cells and immune cells is well studied, the function of PD-L1 inside cells, however, has not been well established. In our previous data, we found that intracellular PD-L1 acts as an RNA binding protein and interacts with NBS1 and BRCA1 mRNAs. Furthermore, intracellular PD-L1 protects targeted RNAs from degradation by RNA exosome, therefore increasing cellular resistance to DNA damage. Since Clinical approved PD-1/PD-L1 inhibitors have little effect on intracellular PD-L1, we sought to find new drugs to target intracellular PD-L1. Here we developed a new PD-L1 antibody, H1A, which has a different effect on PD-L1. Instead of blocking of PD-1/PD-L1 interaction, we found that this antibody disrupts the interaction of PD-L1 and CMTM6, which leads to degradation of PD-L1 by the lysosome. Therefore, H1A can unleash host antitumor activity and enhance the effects of DNA damaging therapy in cancer cells at the same time. Indeed, applying H1A in vivo significantly sensitized tumors to radiation and chemotherapy in a PDX models. Moreover, in a humanized immune competent TNBC PDX model, H1A monotherapy exhibited better therapeutic efficacy compared with clinically available PD-L1 inhibitor, suggesting that H1A activates anti-tumor immunity through PD-L1 degradation. Therefore, H1A may be a potential new drug to target both intracellular and extracellular PD-L1 to improve cancer therapy.

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.

Abstract Enhanced DNA repair is an important mechanism of inherent and acquired resistance to DNA targeted therapies, including poly ADP ribose polymerase inhibition. Spleen associated tyrosine kinase (Syk) is a non-receptor tyrosine kinase known to regulate immune cell function, cell adhesion, and vascular development. Here, we report that Syk can be expressed in high grade serous ovarian cancer and triple negative breast cancers and promotes DNA double strand break resection, homologous recombination (HR) and therapeutic resistance. We found that Syk is activated by ATM following DNA damage and is recruited to DNA double strand breaks by NBS1. Once at the break site, Syk phosphorylates CtIP, a key mediator of resection and HR, at Thr-847 to promote repair activity, specifically in Syk expressing cancer cells. Syk inhibition or genetic deletion abolished CtIP Thr-847 phosphorylation and overcame the resistant phenotype. Collectively, our findings suggest that Syk drives therapeutic resistance by promoting DNA resection and HR through a novel ATM-Syk-CtIP pathway, and that Syk is a new tumor-specific target to sensitize Syk-expressing tumors to PARPi and other DNA targeted therapy.