An RNA Damage Response Network Mediates the Lethality of 5-FU in Clinically Relevant Tumor Types.

5-fluorouracil (5-FU) is a successful and broadly used anti-cancer therapeutic. A major mechanism of action of 5-FU is thought to be through thymidylate synthase (TYMS) inhibition resulting in dTTP depletion and activation of the DNA damage response. This suggests that 5-FU should synergize with other DNA damaging agents. However, we found that combinations of 5-FU and oxaliplatin or irinotecan failed to display any evidence of synergy in clinical trials, and resulted in sub-additive killing in a panel of colorectal cancer (CRC) cell lines. In seeking to understand this antagonism, we unexpectedly found that an RNA damage response during ribosome biogenesis dominates the drug's efficacy in tumor types for which 5-FU shows clinical benefit. 5-FU has an inherent bias for RNA incorporation, and blocking this greatly reduced drug-induced lethality, indicating that accumulation of damaged RNA is more deleterious than the lack of new RNA synthesis. Using 5-FU metabolites that specifically incorporate into either RNA or DNA revealed that CRC cell lines and patient-derived colorectal cancer organoids are inherently more sensitive to RNA damage. This difference held true in cell lines from other tissues in which 5-FU has shown clinical utility, whereas cell lines from tumor tissues that lack clinical 5-FU responsiveness typically showed greater sensitivity to the drug's DNA damage effects. Analysis of changes in the phosphoproteome and ubiquitinome shows RNA damage triggers the selective ubiquitination of multiple ribosomal proteins leading to autophagy-dependent rRNA catabolism and proteasome-dependent degradation of ubiquitinated ribosome proteins. Further, RNA damage response to 5-FU is selectively enhanced by compounds that promote ribosome biogenesis, such as KDM2A inhibitors. These results demonstrate the presence of a strong RNA damage response linked to apoptotic cell death, with clear utility of combinatorially targeting this response in cancer therapy.

DDX18 prevents R-loop-induced DNA damage and genome instability via PARP-1.

R loops occur frequently in genomes and contribute to fundamental biological processes at multiple levels. Consequently, understanding the molecular and cellular biology of R loops has become an emerging area of research. Here, it is shown that poly(ADP-ribose) polymerase-1 (PARP-1) can mediate the association of DDX18, a putative RNA helicase, with R loops thereby modulating R-loop homeostasis in endogenous R-loop-prone and DNA lesion regions. DDX18 depletion results in aberrant endogenous R-loop accumulation, which leads to DNA-replication defects. In addition, DDX18 depletion renders cells more sensitive to DNA-damaging agents and reduces RPA32 and RAD51 foci formation in response to irradiation. Notably, DDX18 depletion leads to γH2AX accumulation and genome instability, and RNase H1 overexpression rescues all the DNA-repair defects caused by DDX18 depletion. Taken together, these studies uncover a function of DDX18 in R-loop-mediated events and suggest a role for PARP-1 in mediating the binding of specific DDX-family proteins with R loops in cells.

The injury response to DNA damage in live tumor cells promotes antitumor immunity.

Although immune checkpoint blockade (ICB) has strong clinical benefit for treating some tumor types, it fails in others, indicating a need for additional modalities to enhance the ICB effect. Here, we identified one such modality by using DNA damage to create a live, injured tumor cell adjuvant. Using an optimized ex vivo coculture system, we found that treating tumor cells with specific concentrations of etoposide, mitoxantrone, or doxorubicin markedly enhanced dendritic cell­mediated T cell activation. These immune-enhancing effects of DNA damage did not correlate with immunogenic cell death markers or with the extent of apoptosis or necroptosis; instead, these effects were mediated by live injured cells with activation of the DNA-PK, ATR, NF-κB, p38 MAPK, and RIPK1 signaling pathways. In mice, intratumoral injection of ex vivo etoposide­treated tumor cells in combination with systemic ICB (by anti-PD-1 and anti-CTLA4 antibodies) increased the number of intratumoral CD103+ dendritic cells and circulating tumor-antigen­specific CD8+ T cells, decreased tumor growth, and improved survival. These effects were absent in Batf3−/− mice and in mice in which the DNA-damaging drug was injected directly into the tumor, due to DNA damage in the immune cells. The combination treatment induced complete tumor regression in a subset of mice that were then able to reject tumor rechallenge, indicating that the injured cell adjuvant treatment induced durable antitumor immunological memory. These results provide a strategy for enhancing the efficacy of immune checkpoint inhibition in tumor types that do not respond to this treatment modality by itself.

Atlas Drugged.

Vasaikar et al. report a comprehensive proteogenomic analysis of 110 colon cancer samples to identify a variety of potential signaling and metabolic targets, neoantigens, and biomarkers. This resource helps expand our understanding of the fundamental pathophysiology of this tumor type, and future mechanistic studies should help guide novel therapeutic strategies for colon cancer treatment.

PARP-1-dependent recruitment of cold-inducible RNA-binding protein promotes double-strand break repair and genome stability.

Maintenance of genome integrity is critical for both faithful propagation of genetic information and prevention of mutagenesis induced by various DNA damage events. Here we report cold-inducible RNA-binding protein (CIRBP) as a newly identified key regulator in DNA double-strand break (DSB) repair. On DNA damage, CIRBP temporarily accumulates at the damaged regions and is poly(ADP ribosyl)ated by poly(ADP ribose) polymerase-1 (PARP-1). Its dissociation from the sites of damage may depend on its phosphorylation status as mediated by phosphatidylinositol 3-kinase-related kinases. In the absence of CIRBP, cells showed reduced γH2AX, Rad51, and 53BP1 foci formation. Moreover, CIRBP-depleted cells exhibited impaired homologous recombination, impaired nonhomologous end-joining, increased micronuclei formation, and higher sensitivity to gamma irradiation, demonstrating the active involvement of CIRBP in DSB repair. Furthermore, CIRBP depleted cells exhibited defects in DNA damage-induced chromatin association of the MRN complex (Mre11, Rad50, and NBS1) and ATM kinase. CIRBP depletion also reduced phosphorylation of a variety of ATM substrate proteins and thus impaired the DNA damage response. Taken together, these results reveal a previously unrecognized role for CIRBP in DSB repair.

Shared and restricted T-cell receptor use is crucial for carbamazepine-induced Stevens-Johnson syndrome.

BACKGROUND: Stevens-Johnson syndrome (SJS) and its related disease, toxic epidermal necrolysis (TEN), are life-threatening drug hypersensitivities with robust immune responses to drugs. Despite the strong HLA predisposition to drug hypersensitivities, such as HLA-B∗1502 to carbamazepine (CBZ)-induced SJS/TEN, it remains unknown whether particular T-cell receptors (TCRs) participate in recognition of small drug/peptide-HLA complexes. OBJECTIVE: Using the strong HLA predisposition in patients with CBZ-induced SJS/TEN as a model, we aimed to study the use of TCR repertoire in patients with drug hypersensitivity. METHOD: We enrolled patients with CBZ-SJS/TEN, tolerant control subjects, and healthy subjects who had no history of CBZ exposure. We isolated PBMCs from the subjects, cultured CBZ-specific T cells, and globally investigated the expression level and third complementarity-determining region length distribution of the TCR profile. We further assessed the pathogenic role of the disease-specific clonotype using real-time PCR-based tests and functional analysis. RESULTS: On drug stimulation, CBZ-specific CD8(+) T cells were expanded in vitro and activated to release granulysin. Notably, VB-11-ISGSY was identified as the most predominant clonotype and shared among different subjects. This clonotype was present in 16 (84%) of 19 patients with SJS/TEN, absent in all 17 tolerant patients, and present at a low frequency in healthy subjects (4/29 [14%]). CBZ-specific cytotoxicity could be primed in vitro in the PBMCs of healthy subjects who are carriers of HLA-B∗1502 and VB-11-ISGSY; this cytotoxicity could be blocked by an anti-TCR-VB-11 antibody. Furthermore, a single T-cell clone expressing VA-22-FISGTY/VB-11-ISGSY showed significant cytotoxicity against HLA-B∗1502-positive antigen-presenting cells and CBZ. CONCLUSION: This study establishes the key role of the TCR in the pathogenic mechanism of SJS/TEN, explains why some HLA-B∗1502 carriers are tolerant to CBZ, and provides a biomarker profile for drug hypersensitivity.

Surgical outcome of volar plate arthroplasty of the proximal interphalangeal joint using the Mitek micro GII suture anchor.

BACKGROUND: The mechanism of injury in dorsal dislocation is usually a hyperextensive stress simultaneous with some degree of longitudinal compression. Operative treatment is indicated for those unstable and reduction is not achieved. We report the surgical outcome of volar plate arthroplasty of the proximal interphalangeal (PIP) joint using the Mitek Micro GII suture anchor. METHODS: We reviewed the medical records of 20 patients with acute or chronic dorsal dislocation or subluxation of the PIP joint who were managed using the Mitek Micro GII suture anchor over the past 5 years by the same surgeon (J.T.S.). Fourteen patients had acute injuries (<4 weeks before surgery) and six patients had chronic injuries (average interval of 4.1 week from injury to surgery; range, 1-8 weeks). The patients had persistent pain and loss of range of motion after trauma, and the reductions were still unstable. RESULTS: All patients were evaluated an average of 25 months postoperatively (range, 12-30 months). The average arc of motion of the PIP joints of the fingers was 82 degrees . There were no obvious perioperative complications, and no patient reported pain at rest or with activity. CONCLUSION: Volar plate arthroplasty using the Mitek Micro GII suture anchor is an effective treatment choice for acute or chronic PIP joint dorsal dislocation or subluxation.

Extensor-tendons reconstruction using autogenous palmaris longus tendon grafting for rheumatoid arthritis patients.

BACKGROUND: The purpose of the study is to retrospectively review the clinical outcome of our study population of middle-aged RA patients who had suffered extensor-tendon rupture. We reported the outcome of autogenous palmaris tendon grafting of multiple extensor tendons at wrist level in 14 middle-aged rheumatoid patients. METHODS: Between Feb. 2000 to Feb. 2004, thirty-six ruptured wrist level extensor tendons were reconstructed in fourteen rheumatoid patients (11 women and three men) using autogenous palmaris longus tendon as a free interposition graft. In each case, the evaluation was based on both subjective and objective criteria, including the range of MCP joint flexion after surgery, the extension lag at the metacarpophalangeal joint before and after surgery, and the ability of the patient to work. RESULTS AND DISCUSSION: The average of follow-up was 54.1 months (range, 40 to 72 months). The average range of MCP joint flexion after reconstruction was 66 degrees . The extension lag at the metacarpophalangeal joint significantly improved from a preoperative mean of 38 degrees (range, 25 degrees -60 degrees ) to a postoperative mean of 16 degrees (range, 0 degrees -30 degrees ). Subjectively all patients were satisfied with the clinical results, and achieved a return to their level of ability before tendon rupture. We found good functional results in our series of interposition grafting using palmaris longus to reconstruct extensor tendon defects in the rheumatoid patients. CONCLUSION: Reconstruction for multiple tendon ruptures is a salvage procedure that is often associated with extensor lag and impairment of overall function. Early aggressive treatment of extensor tendon reconstruction using autogenous palmaris longus tendon as a free interposition graft in the rheumatoid wrist is another viable option to achieve good clinical functional result.

Combined extraforaminal and intradiscal cement leakage following percutaneous vertebroplasty.

STUDY DESIGN: A case report and review of the literature are presented. OBJECTIVE: To describe an extremely rare case of combined extraforaminal and intradiscal cement leakage in different vertebral levels following percutaneous vertebroplasty. SUMMARY OF BACKGROUND DATA: Cement leaks in vertebroplasty are relatively common but generally not clinically significant. To our knowledge, this is the first report of extraforaminal cement leakage inducing radiculopathy combined with intradiscal cement leakage evoking acute adjacent compression fracture. METHODS: A 78-year-old woman with L2 and L5 osteoporotic compression fractures received vertebroplasty. Two weeks after surgery, the patient presented severe low back pain radiating to the right thigh, with associated weakness and numbness in the right thigh and lower leg. Roentgenographic images revealed cement leakage into the right extraforamen of L2-L3 as well as leakage into L4-L5 disc with acute adjacent compression fracture of L4. RESULTS: Surgical intervention was required to relieve discomfort. One-stage posterior approach was performed: right L2-L3 intertransverse process approach with removal of extraforaminal leaked cement and posterior instrumentation from L3-L5 and posterior fusion. The severe low back pain, leg pain, and neurologic deficit associated weakness all improved after surgery. CONCLUSION: Although considered a minimally invasive procedure, percutaneous vertebroplasty with polymethylmethacrylate is not risk free. Intractable neurologic complications can occur if it is not performed by experienced physicians under appropriate indications and cautionary safeguards.

Long-term results of transpedicle body augmenter in treating burst fractures.

BACKGROUND: Short-segment fixation alone to treat thoracolumbar burst fractures is common but it has a 20-50% incidence of implant failure and rekyphosis. A transpedicle body augmenter (TpBA) to reinforce the vertebral body via posterior approach has been reported to prevent implant failure and increase the clinical success rate in treating burst fracture. This article is to evaluate the longterm results of short-segment fixation with TpBA for treatment of thoracolumbar burst fractures. MATERIALS AND METHODS: Patients included in the study had a single-level burst fracture involving T11-L2 and no distraction or rotation element with limited neurological deficit. Patients in the control group (n = 42) were treated with short-segment posterior instrumentation alone, whereas patients in the augmented group (n = 90) were treated with a titanium spacer designed for transpedicle body reconstruction. The followup was 48-101 months. The radiographic and clinical results were evaluated and compared by Student's t test and Fisher's exact test. RESULTS: The blood loss, operation time and hospitalization were similar in both the groups. The immediate postoperative anterior vertebral restoration rate of the augmented group was similar to that of the control group (97.6% ± 2.4% vs. 96.6% ± 3.2%). The final anterior vertebral restoration was greater in the augmented group than in the control group (93.3% ± 3.4% vs. 62.5% ± 11.2%). Immediate postoperative kyphotic angles were not significantly different between the groups (3.0° ± 1.8° vs. 5.1° ± 2.3°). The final kyphotic angles were less in the augmented group than the control group (7.3° ± 3.5° vs. 20.1° ± 5.4°). The augmented group had less (P < 0.001) implant failure [0% (n=0) vs. 23.8% (n=10)] for the control group) and more patients (P < 0.001) with no pain or minimal or occasional pain (Grade P1 or P2) than the control group [90.0% (n=81) vs. 66.7% (n=28)]. All patients in the augmented group and 39 (92.8%) patients in the control group experienced neurological recovery to Frankel Grade E. Three patients in the control group had improvement to Frankel Grade D from Frankel Grade C, but later had deterioration to Frankel Grade C because of loosening and dislodgement of the implant. CONCLUSION: Posterior body reconstruction with TpBA can maintain kyphosis correction and vertebral restoration, prevent implant failure and lead to better clinical results.