An Overview of Advances in Rare Cancer Diagnosis and Treatment.

Cancer stands as the leading global cause of mortality, with rare cancer comprising 230 distinct subtypes characterized by infrequent incidence. Despite the inherent challenges in addressing the diagnosis and treatment of rare cancers due to their low occurrence rates, several biomedical breakthroughs have led to significant advancement in both areas. This review provides a comprehensive overview of state-of-the-art diagnostic techniques that encompass new-generation sequencing and multi-omics, coupled with the integration of artificial intelligence and machine learning, that have revolutionized rare cancer diagnosis. In addition, this review highlights the latest innovations in rare cancer therapeutic options, comprising immunotherapy, targeted therapy, transplantation, and drug combination therapy, that have undergone clinical trials and significantly contribute to the tumor remission and overall survival of rare cancer patients. In this review, we summarize recent breakthroughs and insights in the understanding of rare cancer pathophysiology, diagnosis, and therapeutic modalities, as well as the challenges faced in the development of rare cancer diagnosis data interpretation and drug development.

Targeting the DNA repair pathway for breast cancer therapy: Beyond the molecular subtypes.

DNA repair is a vital mechanism in cells that protects against DNA damage caused by internal and external factors. It involves a network of signaling pathways that monitor and transmit damage signals, activating various cellular activities to repair DNA damage and maintain genomic integrity. Dysfunctions in this repair pathway are strongly associated with the development and progression of cancer. However, they also present an opportunity for targeted therapy in breast cancer. Extensive research has focused on developing inhibitors that play a crucial role in the signaling pathway of DNA repair, particularly due to the remarkable success of PARP1 inhibitors (PARPis) in treating breast cancer patients with BRCA1/2 mutations. In this review, we summarize the current research progress and clinical implementation of BRCA and BRCAness in targeted treatments for the DNA repair pathway. Additionally, we present advancements in diverse inhibitors of DNA repair, both as individual and combined approaches, for treating breast cancer. We also discuss the clinical application of DNA repair-targeted therapy for breast cancer, including the rationale, indications, and summarized clinical data for patients with different breast cancer subtypes. We assess their influence on cancer progression, survival rates, and major adverse reactions. Last, we anticipate forthcoming advancements in targeted therapy for cancer treatment and emphasize prospective areas of development.

Maintaining Genome Integrity: Protein Kinases and Phosphatases Orchestrate the Balancing Act of DNA Double-Strand Breaks Repair in Cancer.

DNA double-strand breaks (DSBs) are the most lethal DNA damages which lead to severe genome instability. Phosphorylation is one of the most important protein post-translation modifications involved in DSBs repair regulation. Kinases and phosphatases play coordinating roles in DSB repair by phosphorylating and dephosphorylating various proteins. Recent research has shed light on the importance of maintaining a balance between kinase and phosphatase activities in DSB repair. The interplay between kinases and phosphatases plays an important role in regulating DNA-repair processes, and alterations in their activity can lead to genomic instability and disease. Therefore, study on the function of kinases and phosphatases in DSBs repair is essential for understanding their roles in cancer development and therapeutics. In this review, we summarize the current knowledge of kinases and phosphatases in DSBs repair regulation and highlight the advancements in the development of cancer therapies targeting kinases or phosphatases in DSBs repair pathways. In conclusion, understanding the balance of kinase and phosphatase activities in DSBs repair provides opportunities for the development of novel cancer therapeutics.

DNA Damage and Its Role in Cancer Therapeutics.

DNA damage is a double-edged sword in cancer cells. On the one hand, DNA damage exacerbates gene mutation frequency and cancer risk. Mutations in key DNA repair genes, such as breast cancer 1 (BRCA1) and/or breast cancer 2 (BRCA2), induce genomic instability and promote tumorigenesis. On the other hand, the induction of DNA damage using chemical reagents or radiation kills cancer cells effectively. Cancer-burdening mutations in key DNA repair-related genes imply relatively high sensitivity to chemotherapy or radiotherapy because of reduced DNA repair efficiency. Therefore, designing specific inhibitors targeting key enzymes in the DNA repair pathway is an effective way to induce synthetic lethality with chemotherapy or radiotherapy in cancer therapeutics. This study reviews the general pathways involved in DNA repair in cancer cells and the potential proteins that could be targeted for cancer therapeutics.

SLFN5-mediated chromatin dynamics sculpt higher-order DNA repair topology.

Repair of DNA double-strand breaks (DSBs) elicits three-dimensional (3D) chromatin topological changes. A recent finding reveals that 53BP1 assembles into a 3D chromatin topology pattern around DSBs. How this formation of a higher-order structure is configured and regulated remains enigmatic. Here, we report that SLFN5 is a critical factor for 53BP1 topological arrangement at DSBs. Using super-resolution imaging, we find that SLFN5 binds to 53BP1 chromatin domains to assemble a higher-order microdomain architecture by driving damaged chromatin dynamics at both DSBs and deprotected telomeres. Mechanistically, we propose that 53BP1 topology is shaped by two processes: (1) chromatin mobility driven by the SLFN5-LINC-microtubule axis and (2) the assembly of 53BP1 oligomers mediated by SLFN5. In mammals, SLFN5 deficiency disrupts the DSB repair topology and impairs non-homologous end joining, telomere fusions, class switch recombination, and sensitivity to poly (ADP-ribose) polymerase inhibitor. We establish a molecular mechanism that shapes higher-order chromatin topologies to safeguard genomic stability.

METTL16 antagonizes MRE11-mediated DNA end resection and confers synthetic lethality to PARP inhibition in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers. Characterization of genetic alterations will improve our understanding and therapies for this disease. Here, we report that PDAC with elevated expression of METTL16, one of the 'writers' of RNA N6-methyladenosine modification, may benefit from poly-(ADP-ribose)-polymerase inhibitor (PARPi) treatment. Mechanistically, METTL16 interacts with MRE11 through RNA and this interaction inhibits MRE11's exonuclease activity in a methyltransferase-independent manner, thereby repressing DNA end resection. Upon DNA damage, ATM phosphorylates METTL16 resulting in a conformational change and autoinhibition of its RNA binding. This dissociates the METTL16-RNA-MRE11 complex and releases inhibition of MRE11. Concordantly, PDAC cells with high METTL16 expression show increased sensitivity to PARPi, especially when combined with gemcitabine. Thus, our findings reveal a role for METTL16 in homologous recombination repair and suggest that a combination of PARPi with gemcitabine could be an effective treatment strategy for PDAC with elevated METTL16 expression.

Biomarkers for Predicting Abiraterone Treatment Outcome and Selecting Alternative Therapies in Castration-Resistant Prostate Cancer.

Approximately one-third of patients with metastatic castration-resistant prostate cancer (CRPC) exhibited primary abiraterone resistance. To identify alternative treatment for abiraterone nonresponders, we performed drug discovery analyses using the L1000 database using differentially expressed genes identified in tumor biopsies and patient-derived xenograft (PDX) tumors between abiraterone responders and nonresponders enrolled in PROMOTE trial. This approach identified 3 drugs, including topoisomerase II (TOP2) inhibitor mitoxantrone, CDK4/6 inhibitor palbociclib, and pan-CDK inhibitor PHA-793887. These drugs significantly suppressed the growth of abiraterone-resistant cell lines and PDX models. Moreover, we identified 11 genes targeted by all 3 drugs that were associated with worse outcomes in both the PROMOTE and Stand Up To Cancer cohorts. This 11-gene panel might also function as biomarkers to select the 3 alternative therapies for this subgroup of patients with CRPC, warranting further clinical investigation.

ASTE1 promotes shieldin-complex-mediated DNA repair by attenuating end resection.

The shieldin complex functions as the downstream effector of 53BP1-RIF1 to promote DNA double-strand break end-joining by restricting end resection. The SHLD2 subunit binds to single-stranded DNA ends and blocks end resection through OB-fold domains. Besides blocking end resection, it is unclear how the shieldin complex processes SHLD2-bound single-stranded DNA and promotes non-homologous end-joining. Here, we identify a downstream effector of the shieldin complex, ASTE1, as a structure-specific DNA endonuclease that specifically cleaves single-stranded DNA and 3' overhang DNA. ASTE1 localizes to DNA damage sites in a shieldin-dependent manner. Loss of ASTE1 impairs non-homologous end-joining, leads to hyper-resection and causes defective immunoglobulin class switch recombination. ASTE1 deficiency also causes resistance to poly(ADP-ribose) polymerase inhibitors in BRCA1-deficient cells owing to restoration of homologous recombination. These findings suggest that ASTE1-mediated 3' single-stranded DNA end cleavage contributes to the control of DSB repair choice by 53BP1, RIF1 and shieldin.

MET Amplification Attenuates Lung Tumor Response to Immunotherapy by Inhibiting STING.

Immune checkpoint blockade (ICB) has revolutionized cancer therapy. However, the response of patients to ICB is difficult to predict. Here, we examined 81 patients with lung cancer under ICB treatment and found that patients with MET amplification were resistant to ICB and had a poor progression-free survival. Tumors with MET amplifications had significantly decreased STING levels and antitumor T-cell infiltration. Furthermore, we performed deep single-cell RNA sequencing on more than 20,000 single immune cells and identified an immunosuppressive signature with increased subsets of XIST- and CD96-positive exhausted natural killer (NK) cells and decreased CD8+ T-cell and NK-cell populations in patients with MET amplification. Mechanistically, we found that oncogenic MET signaling induces phosphorylation of UPF1 and downregulates tumor cell STING expression via modulation of the 3'-UTR length of STING by UPF1. Decreased efficiency of ICB by MET amplification can be overcome by inhibiting MET. SIGNIFICANCE: We suggest that the combination of MET inhibitor together with ICB will overcome ICB resistance induced by MET amplification. Our report reveals much-needed information that will benefit the treatment of patients with primary MET amplification or EGFR-tyrosine kinase inhibitor resistant-related MET amplification.This article is highlighted in the In This Issue feature, p. 2659.

ZNF423 modulates the AMP-activated protein kinase pathway and metformin response in a single nucleotide polymorphisms, estrogen and selective estrogen receptor modulator dependent fashion.

OBJECTIVES: We previously discovered that the single nucleotide polymorphisms (SNP) rs9940645 in the ZNF423 gene regulate ZNF423 expression and serve as a potential biomarker for response to selective estrogen receptor modulators (SERMs). Here we explored pathways involved in ZNF423-mediated SERMs response and drugs that potentially sensitize SERMs. METHODS: RNA sequencing and label-free quantitative proteomics were performed to identify genes and pathways that are regulated by ZNF423 and the ZNF423 SNP. Both cultured cells and mouse xenograft models with different ZNF423 SNP genotypes were used to study the cellular responses to metformin. RESULTS: We identified ribosome and AMP-activated protein kinase (AMPK) signaling as potential pathways regulated by ZNF423 or ZNF423 rs9940645 SNP. Moreover, using clustered regularly interspaced short palindromic repeats/Cas9-engineered ZR75-1 breast cancer cells with different ZNF423 SNP genotypes, striking differences in cellular responses to metformin, either alone or in the combination of tamoxifen, were observed in both cell culture and the mouse xenograft model. CONCLUSIONS: We found that AMPK signaling is modulated by the ZNF423 rs9940645 SNP in estrogen and SERM-dependent fashion. The ZNF423 rs9940645 SNP affects metformin response in breast cancer and could be a potential biomarker for tailoring the metformin treatment.

Reciprocal regulation of RIG-I and XRCC4 connects DNA repair with RIG-I immune signaling.

The RNA-sensing pathway contributes to type I interferon (IFN) production induced by DNA damaging agents. However, the potential involvement of RNA sensors in DNA repair is unknown. Here, we found that retinoic acid-inducible gene I (RIG-I), a key cytosolic RNA sensor that recognizes RNA virus and initiates the MAVS-IRF3-type I IFN signaling cascade, is recruited to double-stranded breaks (DSBs) and suppresses non-homologous end joining (NHEJ). Mechanistically, RIG-I interacts with XRCC4, and the RIG-I/XRCC4 interaction impedes the formation of XRCC4/LIG4/XLF complex at DSBs. High expression of RIG-I compromises DNA repair and sensitizes cancer cells to irradiation treatment. In contrast, depletion of RIG-I renders cells resistant to irradiation in vitro and in vivo. In addition, this mechanism suggests a protective role of RIG-I in hindering retrovirus integration into the host genome by suppressing the NHEJ pathway. Reciprocally, XRCC4, while suppressed for its DNA repair function, has a critical role in RIG-I immune signaling through RIG-I interaction. XRCC4 promotes RIG-I signaling by enhancing oligomerization and ubiquitination of RIG-I, thereby suppressing RNA virus replication in host cells. In vivo, silencing XRCC4 in mouse lung promotes influenza virus replication in mice and these mice display faster body weight loss, poorer survival, and a greater degree of lung injury caused by influenza virus infection. This reciprocal regulation of RIG-I and XRCC4 reveals a new function of RIG-I in suppressing DNA repair and virus integration into the host genome, and meanwhile endues XRCC4 with a crucial role in potentiating innate immune response, thereby helping host to prevail in the battle against virus.

LRRK2 inhibition potentiates PARP inhibitor cytotoxicity through inhibiting homologous recombination-mediated DNA double strand break repair.

PARP inhibitors induce DNA lesions, the repair of which are highly dependent on homologous recombination (HR), and preferentially kill HR- deficient cancers. However, cancer cells have developed several mechanisms to transform HR and confer drug resistance to PARP inhibition. Therefore, there is a great clinical interest in exploring new therapies that induce HR deficiency (HRD), thereby sensitizing cancer cells to PARP inhibitors. Here, we found that GSK2578215A, a high-selective and effective leucine-rich repeat kinase 2 (LRRK2) inhibitor, or LRRK2 depletion suppresses HR preventing the recruitment of RAD51 to DNA damage sites through disruption of the interaction of RAD51 and BRCA2. Moreover, LRRK2 inhibition or depletion increases the susceptibility of ovarian cancer cells to Olaparib in vitro and in vivo. In clinical specimens, LRRK2 high expression is high related with advanced clinical characteristics and poor survival of ovarian cancer patients. All these findings indicate ovarian cancers expressing high levels of LRRK2 are more resistant to treatment potentially through promoting HR. Furthermore, combination treatment with an LRRK2 and PARP inhibitor may be a novel strategy to improve the effectiveness of LRRK2 expression ovarian cancers.

DOCK7 protects against replication stress by promoting RPA stability on chromatin.

RPA is a critical factor for DNA replication and replication stress response. Surprisingly, we found that chromatin RPA stability is tightly regulated. We report that the GDP/GTP exchange factor DOCK7 acts as a critical replication stress regulator to promote RPA stability on chromatin. DOCK7 is phosphorylated by ATR and then recruited by MDC1 to the chromatin and replication fork during replication stress. DOCK7-mediated Rac1/Cdc42 activation leads to the activation of PAK1, which subsequently phosphorylates RPA1 at S135 and T180 to stabilize chromatin-loaded RPA1 and ensure proper replication stress response. Moreover, DOCK7 is overexpressed in ovarian cancer and depleting DOCK7 sensitizes cancer cells to camptothecin. Taken together, our results highlight a novel role for DOCK7 in regulation of the replication stress response and highlight potential therapeutic targets to overcome chemoresistance in cancer.

USP13 regulates the replication stress response by deubiquitinating TopBP1.

The DNA replication stress-induced checkpoint activated through the TopBP1-ATR axis is important for maintaining genomic stability. However, the regulation of TopBP1 in DNA-damage responses remains unclear. In this study, we identify the deubiquitinating enzyme (DUB) USP13 as an important regulator of TopBP1. Mechanistically, USP13 binds to TopBP1 and stabilizes TopBP1 by deubiquitination. Depletion of USP13 impedes ATR activation and hypersensitizes cells to replication stress-inducing agents. Furthermore, high USP13 expression enhances the replication stress response, promotes cancer cell chemoresistance, and is correlated with poor prognosis of cancer patients. Overall, these findings suggest that USP13 is a novel deubiquitinating enzyme for TopBP1 and coordinates the replication stress response.

Mixed-lineage leukemia protein modulates the loading of let-7a onto AGO1 by recruiting RAN.

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The deubiquitinase USP36 Regulates DNA replication stress and confers therapeutic resistance through PrimPol stabilization.

PrimPol has been recently identified as a DNA damage tolerant polymerase that plays an important role in replication stress response. However, the regulatory mechanisms of PrimPol are not well defined. In this study, we identify that the deubiquitinase USP36 interferes with degradation of PrimPol to regulate the replication stress response. Mechanistically, USP36 is deubiquitinated following DNA replication stress, which in turn facilitates its upregulation and interaction with PrimPol. USP36 deubiquitinates K29-linked polyubiquitination of PrimPol and increases its protein stability. Depletion of USP36 results in replication stress-related defects and elevates cell sensitivity to DNA-damage agents, such as cisplatin and olaparib. Moreover, USP36 expression positively correlates with the level of PrimPol protein and poor prognosis in patient samples. These findings indicate that the regulation of PrimPol K29-linked ubiquitination by USP36 plays a critical role in DNA replication stress and chemotherapy response.

USP52 regulates DNA end resection and chemosensitivity through removing inhibitory ubiquitination from CtIP.

Human C-terminal binding protein (CtBP)-interacting protein (CtIP) is a central regulator to initiate DNA end resection and homologous recombination (HR). Several studies have shown that post-translational modifications control the activity or expression of CtIP. However, it remains unclear whether and how cells restrain CtIP activity in unstressed cells and activate CtIP when needed. Here, we identify that USP52 directly interacts with and deubiquitinates CtIP, thereby promoting DNA end resection and HR. Mechanistically, USP52 removes the ubiquitination of CtIP to facilitate the phosphorylation and activation of CtIP at Thr-847. In addition, USP52 is phosphorylated by ATM at Ser-1003 after DNA damage, which enhances the catalytic activity of USP52. Furthermore, depletion of USP52 sensitizes cells to PARP inhibition in a CtIP-dependent manner in vitro and in vivo. Collectively, our findings reveal the key role of USP52 and the regulatory complexity of CtIP deubiquitination in DNA repair.

The bromodomain containing protein BRD-9 orchestrates RAD51-RAD54 complex formation and regulates homologous recombination-mediated repair.

Homologous recombination (HR) is important for error-free DNA double strand break repair and maintenance of genomic stability. However, upregulated HR is also used by cancer cells to promote therapeutic resistance. Therefore, inducing HR deficiency (HRD) is a viable strategy to sensitize HR proficient cancers to DNA targeted therapies in order to overcome therapeutic resistance. A bromodomain containing protein, BRD9, was previously reported to regulate chromatin remodeling and transcription. Here, we discover that following DNA damage, the bromodomain of BRD9 binds acetylated K515 on RAD54 and facilitates RAD54's interaction with RAD51, which is essential for HR. BRD9 is overexpressed in ovarian cancer and depleting BRD9 sensitizes cancer cells to olaparib and cisplatin. In addition, inhibitor of BRD9, I-BRD9, acts synergistically with olaparib in HR-proficient cancer cells. Overall, our results elucidate a role for BRD9 in HR and identify BRD9 as a potential therapeutic target to promote synthetic lethality and overcome chemoresistance.

CHK2-FOXK axis promotes transcriptional control of autophagy programs.

Autophagy is an evolutionarily conserved catabolic process, which plays a vital role in removing misfolded proteins and clearing damaged organelles to maintain internal environment homeostasis. Here, we uncovered the checkpoint kinase 2 (CHK2)-FOXK (FOXK1 and FOXK2) axis playing an important role in DNA damage-mediated autophagy at the transcriptional regulation layer. Mechanistically, following DNA damage, CHK2 phosphorylates FOXK and creates a 14-3-3γ binding site, which, in turn, traps FOXK proteins in the cytoplasm. Because FOXK functions as the transcription suppressor of ATGs, DNA damage-mediated FOXKs' cytoplasmic trapping induces autophagy. In addition, we found that a cancer-derived FOXK mutation induces FOXK hyperphosphorylation and enhances autophagy, resulting in chemoresistance. Cotreatment with cisplatin and chloroquine overcomes the chemoresistance caused by FOXK mutation. Overall, our study highlights a mechanism whereby DNA damage triggers autophagy by increasing autophagy genes via CHK2-FOXK-mediated transcriptional control, and misregulation of this pathway contributes to chemoresistance.

ZFP161 regulates replication fork stability and maintenance of genomic stability by recruiting the ATR/ATRIP complex.

DNA replication stress-mediated activation of the ATR kinase pathway is important for maintaining genomic stability. In this study, we identified a zinc finger protein, ZFP161 that functions as a replication stress response factor in ATR activation. Mechanistically, ZFP161 acts as a scaffolding protein to facilitate the interaction between RPA and ATR/ATRIP. ZFP161 binds to RPA and ATR/ATRIP through distinct regions and stabilizes the RPA-ATR-ATRIP complex at stalled replication forks. This function of ZFP161 is important to the ATR signaling cascade and genome stability maintenance. In addition, ZFP161 knockout mice showed a defect in ATR activation and genomic instability. Furthermore, low expression of ZFP161 is associated with higher cancer risk and chromosomal instability. Overall, these findings suggest that ZFP161 coordinates ATR/Chk1 pathway activation and helps maintain genomic stability.