Pharmacological inhibition of HPK1 synergizes with PD-L1 blockade to provoke antitumor immunity against tumors with low antigenicity.

Immune checkpoint inhibitors have significantly transformed the landscape of cancer therapy. Nevertheless, while these inhibitors are highly effective for certain patient groups, many do not benefit due to primary or acquired resistance. Specifically, these treatments often lack sufficient therapeutic efficacy against cancers with low antigenicity. Thus, the development of an effective strategy to overcome cancers with low antigenicity is imperative for advancing next-generation cancer immunotherapy. Here, we show that small molecule inhibitor of hematopoietic progenitor kinase 1 (HPK1) combined with programmed cell death ligand 1 (PD-L1) blockade can enhance T-cell response to tumor with low antigenicity. We found that treatment of OT-1 splenocytes with HPK1 inhibitor enhanced the activation of signaling molecules downstream of T-cell receptor provoked by low-affinity-antigen stimulation. Using an in vivo OT-1 T-cell transfer model, we demonstrated that combining the HPK1 inhibitor with the anti-PD-L1 antibody significantly suppressed the growth of tumors expressing low-affinity altered peptide ligand of chicken ovalbumin, while anti-PD-L1 antibody monotherapy was ineffective. Our findings offer crucial insights into the potential for overcoming tumors with low antigenicity by combining conventional immune checkpoint inhibitors with HPK1 inhibitor.

Hepatoprotective effects of baicalein against liver ischemia-reperfusion injury and partial hepatectomy in a rat model.

BACKGROUND: Baicalein is the main active flavonoid in Scutellariae Radix and is included in shosaikoto, a Kampo formula used for treating hepatitis and jaundice. However, little is known about its hepatoprotective effects against hepatic ischemia-reperfusion injury (HIRI), a severe clinical condition directly caused by interventional procedures. We aimed to investigate the hepatoprotective effects of baicalein against HIRI and partial hepatectomy (HIRI + PH) and its potential underlying mechanisms. METHODS AND RESULTS: Male Sprague-Dawley rats received either baicalein (5 mg/kg) or saline intraperitoneally and underwent a 70% hepatectomy 15 min after hepatic ischemia. After reperfusion, liver and blood samples were collected. Survival was monitored 30 min after hepatic ischemia and hepatectomy. In interleukin 1ß (IL-1ß)-treated primary cultured rat hepatocytes, the influence of baicalein on inflammatory mediator production and the associated signaling pathway was analyzed. Baicalein suppressed apoptosis and neutrophil infiltration, which are the features of HIRI + PH treatment-induced histological injury. Baicalein also reduced the mRNA expression of the proinflammatory cytokine tumor necrosis factor-α (TNF-α). In addition, HIRI + PH treatment induced liver enzyme deviations in the serum and hypertrophy of the remnant liver, which were suppressed by baicalein. In the lethal HIRI + PH treatment group, baicalein significantly reduced mortality. In IL-1ß-treated rat hepatocytes, baicalein suppressed TNF-α and chemokine mRNA expression as well as the activation of nuclear factor-kappa B (NF-κB) and Akt. CONCLUSIONS: Baicalein treatment attenuates HIRI + PH-induced liver injury and may promote survival. This potential hepatoprotection may be partly related to suppressing inflammatory gene induction through the inhibition of NF-κB activity and Akt signaling in hepatocytes.

Linear dichroism reveals the perpendicular orientation of DNA bases in the RecA and Rad51 recombinase filaments: A possible mechanism for the strand exchange reaction.

Linear dichroism spectroscopy is used to investigate the structure of RecA family recombinase filaments (RecA and Rad51 proteins) with DNA for clarifying the molecular mechanism of DNA strand exchange promoted by these proteins and its activation. The measurements show that the recombinases promote the perpendicular base orientation of single-stranded DNA only in the presence of activators, indicating the importance of base orientation in the reaction. We summarize the results and discuss the role of DNA base orientation.

A conserved Ctp1/CtIP C-terminal peptide stimulates Mre11 endonuclease activity.

The Mre11-Rad50-Nbs1 complex (MRN) is important for repairing DNA double-strand breaks (DSBs) by homologous recombination (HR). The endonuclease activity of MRN is critical for resecting 5'-ended DNA strands at DSB ends, producing 3'-ended single-strand DNA, a prerequisite for HR. This endonuclease activity is stimulated by Ctp1, the Schizosaccharomyces pombe homolog of human CtIP. Here, with purified proteins, we show that Ctp1 phosphorylation stimulates MRN endonuclease activity by inducing the association of Ctp1 with Nbs1. The highly conserved extreme C terminus of Ctp1 is indispensable for MRN activation. Importantly, a polypeptide composed of the conserved 15 amino acids at the C terminus of Ctp1 (CT15) is sufficient to stimulate Mre11 endonuclease activity. Furthermore, the CT15 equivalent from CtIP can stimulate human MRE11 endonuclease activity, arguing for the generality of this stimulatory mechanism. Thus, we propose that Nbs1-mediated recruitment of CT15 plays a pivotal role in the activation of the Mre11 endonuclease by Ctp1/CtIP.

Human Rad51 Protein Requires Higher Concentrations of Calcium Ions for D-Loop Formation than for Oligonucleotide Strand Exchange.

Human Rad51 protein (HsRad51)-promoted DNA strand exchange, a crucial step in homologous recombination, is regulated by proteins and calcium ions. Both the activator protein Swi5/Sfr1 and Ca2+ ions stimulate different reaction steps and induce perpendicular DNA base alignment in the presynaptic complex. To investigate the role of base orientation in the strand exchange reaction, we examined the Ca2+ concentration dependence of strand exchange activities and structural changes in the presynaptic complex. Our results show that optimal D-loop formation (strand exchange with closed circular DNA) required Ca2+ concentrations greater than 5 mM, whereas 1 mM Ca2+ was sufficient for strand exchange between two oligonucleotides. Structural changes indicated by increased fluorescence intensity of poly(dεA) (a poly(dA) analog) reached a plateau at 1 mM Ca2+. Ca2+ > 2 mM was required for saturation of linear dichroism signal intensity at 260 nm, associated with rigid perpendicular DNA base orientation, suggesting a correlation with the stimulation of D-loop formation. Therefore, Ca2+ exerts two different effects. Thermal stability measurements suggest that HsRad51 binds two Ca2+ ions with KD values of 0.2 and 2.5 mM, implying that one step is stimulated by one Ca2+ bond and the other by two Ca2+ bonds. Our results indicate parallels between the Mg2+ activation of RecA and the Ca2+ activation of HsRad51.

Free-Standing Molecularly Thin Amorphous Silica Nanosheets.

Recent progress in 2D materials has initiated new fields of molecularly thin amorphous materials with mysterious properties and structures. However, designed synthesis of molecularly thin amorphous silica still remains a challenge; whether free-standing molecularly thin amorphous silica nanosheets can exist is unclear. Here, this issue is addressed by using a new chemical protocol; solid-state surfactant lamellae with ordered alkyl-chain arrangements can serve as superior templates guiding free-standing amorphous silica nanosheets. Simple sonication of the lamellar hybrids allows exfoliation into monolayer amorphous silica nanosheets with 0.9 nm thickness. In addition, the nanosheets show the distinctive feature of high colloidal stability that enables atomic layer engineering of silica nanocoatings and dielectric nanofilms. The approach may shed new light on the properties and applications of old silica.

Rrp1 translocase and ubiquitin ligase activities restrict the genome destabilising effects of Rad51 in fission yeast.

Rad51 is the key protein in homologous recombination that plays important roles during DNA replication and repair. Auxiliary factors regulate Rad51 activity to facilitate productive recombination, and prevent inappropriate, untimely or excessive events, which could lead to genome instability. Previous genetic analyses identified a function for Rrp1 (a member of the Rad5/16-like group of SWI2/SNF2 translocases) in modulating Rad51 function, shared with the Rad51 mediator Swi5-Sfr1 and the Srs2 anti-recombinase. Here, we show that Rrp1 overproduction alleviates the toxicity associated with excessive Rad51 levels in a manner dependent on Rrp1 ATPase domain. Purified Rrp1 binds to DNA and has a DNA-dependent ATPase activity. Importantly, Rrp1 directly interacts with Rad51 and removes it from double-stranded DNA, confirming that Rrp1 is a translocase capable of modulating Rad51 function. Rrp1 affects Rad51 binding at centromeres. Additionally, we demonstrate in vivo and in vitro that Rrp1 possesses E3 ubiquitin ligase activity with Rad51 as a substrate, suggesting that Rrp1 regulates Rad51 in a multi-tiered fashion.

Two auxiliary factors promote Dmc1-driven DNA strand exchange via stepwise mechanisms.

Homologous recombination (HR) is a universal mechanism operating in somatic and germ-line cells, where it contributes to the maintenance of genome stability and ensures the faithful distribution of genetic material, respectively. The ability to identify and exchange the strands of two homologous DNA molecules lies at the heart of HR and is mediated by RecA-family recombinases. Dmc1 is a meiosis-specific RecA homolog in eukaryotes, playing a predominant role in meiotic HR. However, Dmc1 cannot function without its two major auxiliary factor complexes, Swi5-Sfr1 and Hop2-Mnd1. Through biochemical reconstitutions, we demonstrate that Swi5-Sfr1 and Hop2-Mnd1 make unique contributions to stimulate Dmc1-driven strand exchange in a synergistic manner. Mechanistically, Swi5-Sfr1 promotes establishment of the Dmc1 nucleoprotein filament, whereas Hop2-Mnd1 defines a critical, rate-limiting step in initiating strand exchange. Following execution of this function, we propose that Swi5-Sfr1 then promotes strand exchange with Hop2-Mnd1. Thus, our findings elucidate distinct yet complementary roles of two auxiliary factors in Dmc1-driven strand exchange, providing mechanistic insights into some of the most critical steps in meiotic HR.

Low Young's Modulus TiNbSn Alloy Locking Plates Accelerate Osteosynthesis in Rabbit Tibiae.

A new beta TiNbSn alloy with a low Young's modulus of approximately 40 GPa has been developed to resolve the stress shielding by Young's modulus divergence. In this study, the efficacy of TiNbSn alloy locking plates on bone repair is compared to that of commercially pure titanium (CP-Ti). The TiNbSn alloy and CP-Ti, which have Young's moduli of 49.1 GPa and 107 GPa, respectively, were compared. Male Japanese white rabbits were anesthetized, and osteotomy and osteosynthesis with locking plates were performed on the right tibia. The bone repair was assessed using micro-computed tomography (CT), histomorphometry, immunohistochemistry, and mechanical testing. Micro-CT, histomorphometry, immunohistochemistry, and mechanical testing were performed four weeks after osteotomy. Six weeks after surgery, micro-CT and mechanical testing were performed. Micro-CT analysis at four weeks after surgery showed that the intramedullary fracture callus in the TiNbSn alloy group had more bone volume and numerous bridging structures compared to the CP-Ti group (CP-Ti vs. TiNbSn alloy, 34.3 ± 13.1 mm3 vs. 61.3 ± 19.6 mm3, p = 0.02; mean ± standard deviation). At four weeks post-osteotomy, the healed tibia showed significantly higher strength in the TiNbSn alloy group compared with CP-Ti (CP-Ti vs. TiNbSn alloy, 81.3 ± 31.2 N vs. 133.7 ± 46.6 N, p = 0.04). TiNbSn alloy locking plates had a more positive impact on bone formation and bone strength restoration than the CP-Ti locking plates during the early phase of bone healing.

Enterolith Treated with a Combination of Double-Balloon Endoscopy and Cola Dissolution Therapy.

A 71-year-old woman with rheumatoid arthritis who had been taking NSAIDs for many years consulted our hospital for abdominal pain. She was diagnosed with a small bowel obstruction due to an enterolith according to an abdominal CT scan that showed dilation from the enterolith in the small intestine on the oral side. It was considered that the intestinal stone was formed due to stagnation of intestinal contents and had gradually increased in size, resulting in an intestinal obstruction. We performed antegrade double-balloon endoscopy (DBE) to observe and remove the enterolith. We used forceps and a snare to fracture the enterolith. During this attempt, we found a seed in the center of the enterolith. Since the intestinal stone was very hard, cola dissolution therapy was administered from an ileus tube for 1 week. The following week, DBE was performed again, and it was found that the stone had further softened, making attempts at fracture easier. Finally, the enterolith was almost completely fractured. Intestinal stenosis, probably due to ulcers caused by NSAIDs, was found. Small bowel obstruction with an enterolith is rare. In this case, it was considered that the seed could not pass through the stenotic region of the small intestine and the intestinal contents had gradually built up around it. It has been suggested that DBE may be a therapeutic option in cases of an enterolith. Further, cola dissolution therapy has been shown to be useful in treating an enterolith, with the possible explanation that cola undergoes an acid-base reaction with the enterolith. In summary, we report, for the first time, treatment of an enterolith with a combination of DBE and cola dissolution therapy, thereby avoiding surgery and its risks.

Real-world data on NGS using the Oncomine DxTT for detecting genetic alterations in non-small-cell lung cancer: WJOG13019L.

Considering the increasing number of identified driver oncogene alterations, additional genetic tests are required to determine the treatment for advanced non-small-cell lung cancer (NSCLC). Next-generation sequencing can detect multiple driver oncogenes simultaneously, enabling the analysis of limited amounts of biopsied tissue samples. In this retrospective, multicenter study (UMIN ID000039523), we evaluated real-world clinical data using the Oncomine Dx Target Test Multi-CDx System (Oncomine DxTT) as a companion diagnostic system. Patients with NSCLC who were tested for a panel of 46 genes using the Oncomine DxTT between June 2019 and January 2020 were eligible for enrollment. Patients from 19 institutions affiliated to the West Japan Oncology Group were recruited. The primary endpoint of the study was the success rate of genetic alteration testing in four driver genes (EGFR, ALK, ROS1, and BRAF) using the Oncomine DxTT. In total, 533 patients were enrolled in the study. The success rate of genetic alteration testing for all four genes was 80.1% (95% CI 76.5%-83.4%). Surgical resection was associated with the highest success rate (88.0%), which was significantly higher than that for bronchoscopic biopsy (76.8%, P = .005). Multivariate analysis revealed a significant difference for surgical resection alone (P = .006, 95% CI 1.36-6.18, odds ratio 2.90). Although the success rate of genetic alteration testing immediately after Oncomine DxTT induction was not sufficient in this study, optimizing specimen quantity and quality may improve the use of driver gene testing in clinical settings.

High-Sensitivity Amperometric Dual Immunoassay Using Two Cascade Reactions with Signal Amplification of Redox Cycling in Nanoscale Gap.

Here, we report a high-sensitivity dual immunoassay using Lumulus amebocyte lysate (LAL) and blood coagulation cascade reactions with redox cycling in a nanoscale-gap electrode. Endotoxin and factor XIa were used as the label molecules for the immunoassay of two types of analytes to induce the LAL and coagulation cascade reactions, respectively, when each corresponding analyte existed in the sample solution. In addition to the signal amplification by the cascade reactions, we employed redox cycling in a nanoscale gap to achieve a highly sensitive assay. The nanoscale-gap electrode amplifies the amperometric signals from p-aminophenol liberated from artificial substrates in the final steps of the cascade reactions. First, the cross reaction between the LAL and coagulation cascade reactions was investigated. The results indicated that these cascade reactions did not efficiently proceed in a single solution owing to the cross reaction. Therefore, we selected to induce two cascade reactions in different solutions by bisecting the beads after the immunocomplex formation on the beads. The cross reactions of factor XIa with the LAL cascade reaction and of endotoxin with the coagulation cascade reaction were investigated. The effects of these cross reactions were revealed to be negligible by bisecting the beads before inducing the cascade reactions. Finally, a dual immunoassay for goat and human immunoglobulin G was performed, for which the limits of detection were 70 pg/mL (470 fmol/L) and 1.0 ng/mL (6.6 pmol/L), respectively. Thus, our dual immunoassay provides a sensitive platform for clinical diagnosis requiring detection of multiple analytes.

Highly Sensitive Electrochemical Immunoassay Using Signal Amplification of the Coagulation Cascade.

Here, we report a highly sensitive immunoassay for human immunoglobulin G (IgG) that uses signal amplification of the coagulation cascade. Z-Phe-Pro-Lys-p-nitroaniline (FPK-pNA) was used as a substrate for thrombin activation in the last step of the coagulation cascade. During the coagulation cascade, pNA is liberated from FPK-pNA and can be detected electrochemically. Using square wave voltammetry with a glassy carbon electrode, we demonstrated that pNA can be quantified in a solution modeling the coagulation cascade prepared by mixing FPK-pNA and pNA. Characterization of the reactivity of thrombin toward FPK-pNA revealed that thrombin efficiently reacted with FPK-pNA. Subsequent characterization of factor XIa activity of factor XIa-labeled antibody revealed that factor XIa was not inactivated during labeling. Finally, a coagulation cascade-based immunoassay for human IgG was performed using a factor XIa-labeled antibody on magnetic beads. The limit of detection for human IgG was 5.0 pg/mL (33 fM) indicating that the coagulation cascade can amplify the immunoassay sensitivity compared to immunoassay using a thrombin-labeled antibody as a condition without a coagulation cascade. Coagulation cascade-based immunoassay was also highly selective. In the near future, we will report a highly sensitive immunoassay for the simultaneous detection of multiple analytes using a coagulation cascade-based immunoassay and Limulus amebocyte lysate reaction-based immunoassay we previously reported.

ß-type TiNbSn Alloy Plates With Low Young Modulus Accelerates Osteosynthesis in Rabbit Tibiae.

BACKGROUND: Ti6Al4V alloy, which is commonly used for biomedical applications, has a Young modulus (110 GPa) that is higher than that of human cortical bone (11 to 20 GPa). Using an implant with a material with a low Young modulus that enhances load sharing by the bone even more than those made of Ti6Al4V could be beneficial for bone healing and further reduce the potential for stress shielding. A new ß-type TiNbSn alloy has a low Young modulus of approximately 40 to 49 GPa. However, whether the new titanium alloy with a lower Young modulus is advantageous in terms of fracture healing has not been assessed, and a small-animal model seems a reasonable first step in its assessment. QUESTIONS/PURPOSES: To assess the impact of a TiNbSn alloy plate with a lower Young modulus compared with a Ti6Al4V alloy plate on fracture healing, we evaluated: (1) bony bridging and callus volume, (2) new bone formation and remaining cartilage tissue, (3) osteoblast activity in the callus, and (4) mechanical strength and stiffness of the callus in bending. METHODS: Fracture plates manufactured from TiNbSn and Ti6Al4V alloys, which have Young moduli of 49 GPa and 110 GPa, respectively, were compared. The main reason for using rabbits was the high reliability of the three-point bending mechanical test of the rabbit tibia. Forty-two male Japanese white rabbits weighing 2.8 to 3.4 kg were anesthetized. A 5-cm skin incision was made on the medial side in the mid-diaphysis of the right tibia. Eight-hole plates were used, which were 42 mm long, 5 mm wide, and 1.2 mm thick. Plate fixation was performed using three proximal and three distal screws. After the plate was installed, an osteotomy was performed using a 1-mm-wide wire saw to create a standardized tibial transverse osteotomy model with a 1-mm gap. Bone healing was quantitatively assessed by two nonblinded observers using micro-CT (bony bridging and callus volume), histomorphometry (new bone formation and remaining cartilage tissue), immunohistochemistry (osteoblast activity), and mechanical testing (mechanical strength and stiffness in bending). Measurements on nondemineralized specimens were descriptive statistics due to their small number. Four weeks after osteotomy and fixation, 30 rabbits were euthanized to undergo micro-CT and subsequent mechanical testing (n = 12), histomorphometry and immunohistochemistry with demineralized specimens (n = 12), and histomorphometry with a nondemineralized specimen (n = 6). Eight weeks postoperatively, 12 rabbits were euthanized for micro-CT and subsequent mechanical testing. RESULTS: Intramedullary fracture calluses treated with TiNbSn alloy plates had larger bone volumes and more numerous bridging structures than those treated with Ti6Al4V alloy plates at 4 weeks after osteotomy (Ti6Al4V alloy versus TiNbSn alloy: 30 ± 7 mm 3 versus 52 ± 14 mm 3 , mean difference 22 [95% CI 9 to 37]; p = 0.005; ICC 0.98 [95% CI 0.95 to 0.99]). Histologic assessments demonstrated there was greater new bone formation (total callus: Ti6Al4V versus TiNbSn: 16 ± 4 mm 2 versus 24 ± 7 mm 2 , mean difference 8 [95% CI 1 to 16]; p = 0.04; ICC 0.98 [95% CI 0.93 to 0.99]; intramedullary callus: Ti6Al4V versus TiNbSn: 6 ± 4 mm 2 versus 13 ± 5 mm 2 , mean difference 7 [95% CI 1 to 13]; p = 0.02; ICC 0.98 [95% CI 0.95 to 0.99]) and a higher number of osteocalcin-positive cells (Ti6Al4V alloy versus TiNbSn alloy: 1397 ± 197 cells/mm 2 versus 2044 ± 183 cells/mm 2 , mean difference 647 [95% CI 402 to 892]; p < 0.001; ICC 0.98 [95% CI 0.95 to 0.99]) in the TiNbSn alloy group than in the Ti6Al4V alloy group. At 4 weeks after osteotomy, both bone strength and stiffness of the healed bone in the TiNbSn alloy group were higher than those in the Ti6Al4V alloy group (maximum load: Ti6Al4V alloy versus TiNbSn alloy: 83 ± 30 N versus 127 ± 26 N; mean difference 44 [95% CI 8 to 80]; p = 0.02; stiffness: Ti6Al4V alloy versus TiNbSn alloy: 92 ± 43 N/mm versus 165 ± 63 N/mm; mean difference 73 [95% CI 4 to 143]; p = 0.047). Eight weeks after osteotomy, no between-group differences were observed in the strength and stiffness of the healed bone. CONCLUSION: The results of this study indicate that TiNbSn alloy plate with a lower Young modulus resulted in improved bone formation and stiffer callus during the early phase (4 weeks after surgery) but not the later phase (8 weeks after surgery) of bone healing. CLINICAL RELEVANCE: An overly stiff plate may impair callus formation and bone healing. The TiNbSn alloy plate with a low Young modulus improves the early formation of new bone and stiff callus at the osteotomy site compared with the Ti6Al4V alloy plate in the healing process, which may promote bone repair. TiNbSn alloy may be a promising biomaterial for fracture treatment devices. Further research to address concerns about the strength of TiNbSn alloy plates, such as fatigue life and plate fracture, will be necessary for clinical applications, including mechanical tests to verify fatigue life and validation in larger animals with greater body weight.

Cytoskeletal Protein 4.1G Is Essential for the Primary Ciliogenesis and Osteoblast Differentiation in Bone Formation.

The primary cilium is a hair-like immotile organelle with specific membrane receptors, including the receptor of Hedgehog signaling, smoothened. The cilium organized in preosteoblasts promotes differentiation of the cells into osteoblasts (osteoblast differentiation) by mediating Hedgehog signaling to achieve bone formation. Notably, 4.1G is a plasma membrane-associated cytoskeletal protein that plays essential roles in various tissues, including the peripheral nervous system, testis, and retina. However, its function in the bone remains unexplored. In this study, we identified 4.1G expression in the bone. We found that, in the 4.1G-knockout mice, calcium deposits and primary cilium formation were suppressed in the trabecular bone, which is preosteoblast-rich region of the newborn tibia, indicating that 4.1G is a prerequisite for osteoblast differentiation by organizing the primary cilia in preosteoblasts. Next, we found that the primary cilium was elongated in the differentiating mouse preosteoblast cell line MC3T3-E1, whereas the knockdown of 4.1G suppressed its elongation. Moreover, 4.1G-knockdown suppressed the induction of the cilia-mediated Hedgehog signaling and subsequent osteoblast differentiation. These results demonstrate a new regulatory mechanism of 4.1G in bone formation that promotes the primary ciliogenesis in the differentiating preosteoblasts and induction of cilia-mediated osteoblast differentiation, resulting in bone formation at the newborn stage.

Renadirsen, a Novel 2'OMeRNA/ENA® Chimera Antisense Oligonucleotide, Induces Robust Exon 45 Skipping for Dystrophin In Vivo.

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease caused by out-of-frame or nonsense mutation in the dystrophin gene. It begins with a loss of ambulation between 9 and 14 years of age, followed by various other symptoms including cardiac dysfunction. Exon skipping of patients' DMD pre-mRNA induced by antisense oligonucleotides (AOs) is expected to produce shorter but partly functional dystrophin proteins, such as those possessed by patients with the less severe Becker muscular dystrophy. We are working on developing modified nucleotides, such as 2'-O,4'-C-ethylene-bridged nucleic acids (ENAs), possessing high nuclease resistance and high affinity for complementary RNA strands. Here, we demonstrate the preclinical characteristics (exon-skipping activity in vivo, stability in blood, pharmacokinetics, and tissue distribution) of renadirsen, a novel AO modified with 2'-O-methyl RNA/ENA chimera phosphorothioate designed for dystrophin exon 45 skipping and currently under clinical trials. Notably, systemic delivery of renadirsen sodium promoted dystrophin exon skipping in cardiac muscle, skeletal muscle, and diaphragm, compared with AOs with the same sequence as renadirsen but conventionally modified by PMO and 2'OMePS. These findings suggest the promise of renadirsen sodium as a therapeutic agent that improves not only skeletal muscle symptoms but also other symptoms in DMD patients, such as cardiac dysfunction.

Clinical utility of liquid biopsy for EGFR driver, T790M mutation and EGFR amplification in plasma in patients with acquired resistance to afatinib.

BACKGROUND: Cell-free DNA (cfDNA) genotyping in plasma using the cobas EGFR Mutation Test v2 (cobas) is the first liquid biopsy as a companion diagnosis to identify the EGFR T790M mutation (T790M) after the failure of treatment of EGFR-tyrosine kinase inhibitors (TKIs) (1st generation, gefitinib [G] and erlotinib [E] and 2nd generation, afatinib [A]). This study investigated the clinical utility of a liquid biopsy for patients who acquired resistance to afatinib. METHODS: We prospectively collected plasma from 51 patients who had acquired resistance to afatinib between April 2015 and November 2016 to evaluate the frequency of T790M by cobas and digital droplet PCR (UMIN000025112). Additionally, we retrospectively reviewed 38 patients who tested by cobas in plasma after G/E failure to compare for T790M detection between A and with G/E. RESULTS: The detection rate of EGFR-driver and T790M in plasma in patients treated with A (A group) as a first-line EGFR-TKI was lower than with G/E followed by A (G/E→A group), although the differences were not significant (EGFR-driver: 41% [A] vs. 67% [G/E→A], P=0.1867; and T790M: 8% [A] vs. 17% [G/E→A], P=0.5798). In first-line setting, the detection rate for EGFR-driver and T790M in plasma by cobas was lower in A group than in G/E group, although there was no significant difference (EGFR-driver: 34% [A] vs. 52% [G/E], P=0.2072; and T790M: 10% [A] vs. 27% [G/E], P=0.1161). CONCLUSION: The detection of EGFR-driver and T790M in plasma by cobas in patients treated with afatinib might be lower than with G/E in a real-world setting.

Phase II study of multidisciplinary therapy combined with pembrolizumab for patients with synchronous oligometastatic non-small cell lung cancer TRAP OLIGO study (WJOG11118L).

BACKGROUND: Synchronous oligometastatic non-small cell lung cancer (NSCLC) is generally characterised by the limited number of metastases at the time of diagnosis. Several clinical trials have shown that local ablative therapy (LAT) at all sites of the disease might be beneficial for patients with oligometastatic NSCLC. In recent years, the combination of programmed cell death 1 (PD-1) inhibitors or programmed cell death ligand 1 with cytotoxic chemotherapy has become a new standard treatment for patients with metastatic NSCLC. Furthermore, multisite LAT would inherently reduce the overall tumour burden, and this could promote T cell reinvigoration to enhance the efficacy of PD-1 inhibitors. Few studies have evaluated the efficacy of the combination of PD-1 inhibitors with LAT at all sites of disease. The aim of the present multicentre single-arm phase II study is to evaluate the efficacy of LAT at all sites of disease following standard platinum doublet chemotherapy with pembrolizumab in patients with oligometastatic NSCLC. METHODS: Thirty patients with synchronous oligometastatic NSCLC will be enrolled in the trial. All patients will receive 2-4 cycles of a systemic treatment including pembrolizumab and chemotherapy as induction therapy. Patients who will receive LAT will be determined by a multidisciplinary tumour board, including medical oncologists, radiation oncologists, and thoracic surgeons. LAT will be administered at all sites of disease within 21-56 days of the last dose of induction therapy and will be followed by maintenance therapy within 42 days of the last day of LAT. The primary endpoint is the progression-free survival (PFS) rate of 24 months from the date of initiation of LAT. The secondary endpoints are toxicity, response to induction therapy, PFS, overall survival, and the frequency of LAT. DISCUSSION: This study will provide novel data on the efficacy and safety profile of the combination of LAT and chemotherapy plus immune-checkpoint inhibitors in patients with synchronous oligometastatic NSCLC. If the primary endpoint of this study is met, extensive phase III studies further assessing this strategy will be recommended. TRIAL REGISTRATION: jRCT identifier: jRCTs041200046 (date of initial registration: 28 October 2020).

Swi5-Sfr1 stimulates Rad51 recombinase filament assembly by modulating Rad51 dissociation.

Eukaryotic Rad51 protein is essential for homologous-recombination repair of DNA double-strand breaks. Rad51 recombinases first assemble onto single-stranded DNA to form a nucleoprotein filament, required for function in homology pairing and strand exchange. This filament assembly is the first regulation step in homologous recombination. Rad51 nucleation is kinetically slow, and several accessory factors have been identified to regulate this step. Swi5-Sfr1 (S5S1) stimulates Rad51-mediated homologous recombination by stabilizing Rad51 nucleoprotein filaments, but the mechanism of stabilization is unclear. We used single-molecule tethered particle motion experiments to show that mouse S5S1 (mS5S1) efficiently stimulates mouse RAD51 (mRAD51) nucleus formation and inhibits mRAD51 dissociation from filaments. We also used single-molecule fluorescence resonance energy transfer experiments to show that mS5S1 promotes stable nucleus formation by specifically preventing mRAD51 dissociation. This leads to a reduction of nucleation size from three mRAD51 to two mRAD51 molecules in the presence of mS5S1. Compared with mRAD51, fission yeast Rad51 (SpRad51) exhibits fast nucleation but quickly dissociates from the filament. SpS5S1 specifically reduces SpRad51 disassembly to maintain a stable filament. These results clearly demonstrate the conserved function of S5S1 by primarily stabilizing Rad51 on DNA, allowing both the formation of the stable nucleus and the maintenance of filament length.

Propensity score analysis of overall survival between first- and second-generation EGFR-TKIs using real-world data.

We constructed a data set of EGFR-mutant non-small-cell lung carcinoma (NSCLC) patients, and compared the overall survival of first-generation (1G), and second-generation (2G) EGFR-tyrosine kinase inhibitors (TKIs) in clinical practice using a propensity score. We reviewed the clinical data of consecutive EGFR-mutated NSCLC patients who received EGFR-TKI therapy between January 2008 and August 2017 at 11 institutions in Japan. The primary endpoint was overall survival (OS). When comparing OS between 1G and 2G EGFR-TKIs, propensity score analyses were performed using 2 methods: matching and inverse probability of treatment weighting (IPTW). (Clinical Trial information: UMIN000030121) In total, 1400 patients from 11 institutions were enrolled in this study, and the data from the 1366 patients who received only EGFR-TKI therapy were analyzed (gefitinib [GEF], N = 732; erlotinib [ERL], N = 416; afatinib, N = 218). Median OS times (months [95%CI]) were 29.7 [27.5-33.5] in the 1G group (gefitinib, 32.0 [28.1-35.8]; erlotinib, 27.5 [23.9-31.7]), and 38.6 [32.2-NR] in the 2G group (afatinib), respectively. The trend of longer OS for afatinib against 1G EGFR-TKIs remained, even after adjusted by propensity score. (unadjusted, hazard ratio [HR] 0.676, P = .0023; adjusted by IPTW, HR 0.685 P < .0001; adjusted by matching, HR 0.725, P = .0418). Exploratory analysis showed that OS using the 2G EGFR-TKI was superior to that of the 1G EGFR-TKIs, suggesting the potential of sequential therapy of 2G EGFR-TKI followed by osimertinib. (HR 0.419, P = .0519) Real-world data analysis using 1354 data records, using propensity scoring, indicated that 2G EGFR-TKI had a trend of longer OS compared with 1G EGFR-TKIs.