KRAS G12C mutated advanced non-small cell lung cancer (NSCLC): Characteristics, treatment patterns and overall survival from a Danish nationwide observational register study.

OBJECTIVES: We aimed to characterize the advanced NSCLC population in terms of KRAS G12C prevalence, patient characteristics, and survival outcomes after the introduction of immunotherapies. MATERIALS AND METHODS: We identified adult patients diagnosed with advanced NSCLC between January 1, 2018 and June 30, 2021 using the Danish health registries. Patients were grouped by mutational status (any KRAS mutation, KRAS G12C, and KRAS/EGFR/ALK wildtype [Triple WT]). We analyzed KRAS G12C prevalence, patient and tumor characteristics, treatment history, time-to-next-treatment (TTNT), and overall survival (OS). RESULTS: We identified 7,440 patients of whom 40% (n = 2,969) were KRAS tested prior to the first line of therapy (LOT1). Among the KRAS tested, 11% (n = 328) harbored KRAS G12C. More KRAS G12C patients were women (67%), smokers (86%), had a high (≥50%) level of PD-L1 expression (54%), and more frequently received anti-PD-L1 treatment than any other group. From the date of the mutational test result, OS (7.1-7.3 months) was similar between the groups. OS from LOT1 (14.0 months) and LOT2 (10.8 months), and TTNT from LOT1 (6.9 months) and LOT2 (6.3 months) was numerically longer for the KRAS G12C mutated group compared to any other group. However, from LOT1 and LOT2, the OS and TTNT were comparable when stratifying the groups by PD-L1 expression level. Regardless of the mutational group, OS was markedly longer for patients with high PD-L1 expression. CONCLUSION: In patients diagnosed with advanced NSCLC after the implementation of anti-PD-1/L1 therapies, the survival in KRAS G12C mutated patients is comparable to patients with any KRAS mutation, Triple WT, and all NSCLC patients.

Poly(ADP-ribosyl)ation links the chromatin remodeler SMARCA5/SNF2H to RNF168-dependent DNA damage signaling.

Ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) arising in native chromatin elicit an RNF8/RNF168-dependent ubiquitylation response, which triggers the recruitment of various repair factors. Precisely how this response is regulated in the context of chromatin remains largely unexplored. Here, we show that SMARCA5/SNF2H, the catalytic subunit of ISWI chromatin remodeling complexes, is recruited to DSBs in a poly(ADP-ribose) polymerase 1 (PARP1)-dependent manner. Remarkably, PARP activity, although dispensable for the efficient spreading of γH2AX into damaged chromatin, selectively promotes spreading of SMARCA5, the E3 ubiquitin ligase RNF168, ubiquitin conjugates and the ubiquitin-binding factors RAD18 and the RAP80-BRCA1 complex throughout DSB-flanking chromatin. This suggests that PARP regulates the spatial organization of the RNF168-driven ubiquitin response to DNA damage. In support of this, we show that SMARCA5 and RNF168 interact in a DNA damage- and PARP-dependent manner. RNF168 became poly(ADP-ribosyl)ated after DNA damage, while RNF168 and poly(ADP-ribose) chains were required for SMARCA5 binding in vivo, explaining how SMARCA5 is linked to the RNF168 ubiquitin cascade. Moreover, SMARCA5 was found to regulate the ubiquitin response by promoting RNF168 accumulation at DSBs, which subsequently facilitates efficient ubiquitin conjugation and BRCA1 assembly. Underlining the importance of these findings, we show that SMARCA5 depletion renders cells sensitive to IR and results in DSB repair defects. Our study unveils a functional link between DNA damage-induced poly(ADP-ribosyl)ation, SMARCA5-mediated chromatin remodeling and RNF168-dependent signaling and repair of DSBs.

Effects of 2 weeks lower limb immobilization and two separate rehabilitation regimens on gastrocnemius muscle protein turnover signaling and normalization genes.

BACKGROUND: Limb immobilization causes a rapid loss of muscle mass and strength that requires appropriate rehabilitation to ensure restoration of normal function. Whereas the knowledge of muscle mass signaling with immobilization has increased in recent years, the molecular regulation in the rehabilitation of immobilization-induced muscle atrophy is only sparsely studied. To investigate the phosphorylation and expression of candidate key molecular muscle mass regulators after immobilization and subsequent rehabilitation we performed two separate studies. METHODS: We immobilized the lower limb for 2 weeks followed by the in-house hospital standard physiotherapy rehabilitation (Study 1). Secondly, we conducted an intervention study using the same 2 weeks immobilization protocol during which protein/carbohydrate supplementation was given. This was followed by 6 weeks of rehabilitation in the form of resistance training and continued protein/carbohydrate supplementation (Study 2). We obtained muscle biopsies from the medial gastrocnemius prior to immobilization (PRE), post-immobilization (IMMO) and post-rehabilitation (REHAB) and measured protein expression and phosphorylation of Akt, mTOR, S6k, 4E-BP1, GSK3ß, ubiquitin and MURF1 and mRNA expression of Atrogin-1, MURF1, FOXO1, 3 and 4 as well as appropriate housekeeping genes. RESULTS: In both studies, no changes in protein expression or phosphorylation for any measured protein were observed. In Study 1, FOXO3 and FOXO4 mRNA expression decreased after IMMO and REHAB compared to PRE, whereas other mRNAs remained unchanged. Interestingly, we found significant changes in expression of the putative housekeeping genes GAPDH, HADHA and S26 with immobilization in both studies. CONCLUSIONS: In neither study, the changes in muscle mass associated with immobilization and rehabilitation were accompanied by expected changes in expression of atrophy-related genes or phosphorylation along the Akt axis. Unexpectedly, we observed significant changes in several of the so-called housekeeping genes GAPDH, HADHA and S26 with immobilization in both studies, thereby questioning the usefulness of these genes for normalization of RNA data purposes in muscle immobilization studies.