Regulatory Qualification of a Cross-Disease Digital Measure: Benefits and Challenges from the Perspective of IMI Consortium IDEA-FAST.

Background: Innovative Medicines Initiative (IMI) consortium IDEA-FAST is developing novel digital measures of fatigue, sleep quality, and impact of sleep disturbances for neurodegenerative diseases and immune-mediated inflammatory diseases. In 2022, the consortium met with the European Medicines Agency (EMA) to receive advice on its plans for regulatory qualification of the measures. This viewpoint reviews the IDEA-FAST perspective on developing digital measures for multiple diseases and the advice provided by the EMA. Summary: The EMA considered a cross-disease measure an interesting and arguably feasible concept. Developers should account for the need for a strong rationale that the clinical features to be measured are similar across diseases. In addition, they may expect increased complexity of study design, challenges when managing differences within and between disease populations, and the need for validation in both heterogeneous and homogeneous populations. Key Messages: EMA highlighted the challenges teams may encounter when developing a cross-disease measure, though benefits potentially include reduced resources for the technology developer and health authority, faster access to innovation across different therapeutic fields, and feasibility of cross-disease comparisons. The insights included here can be used by project teams to guide them in the development of cross-disease digital measures intended for regulatory qualification.

Reverse Engineering of Digital Measures: Inviting Patients to the Conversation.

Background: Digital measures offer an unparalleled opportunity to create a more holistic picture of how people who are patients behave in their real-world environments, thereby establishing a better connection between patients, caregivers, and the clinical evidence used to drive drug development and disease management. Reaching this vision will require achieving a new level of co-creation between the stakeholders who design, develop, use, and make decisions using evidence from digital measures. Summary: In September 2022, the second in a series of meetings hosted by the Swiss Federal Institute of Technology in Zürich, the Foundation for the National Institutes of Health Biomarkers Consortium, and sponsored by Wellcome Trust, entitled "Reverse Engineering of Digital Measures," was held in Zurich, Switzerland, with a broad range of stakeholders sharing their experience across four case studies to examine how patient centricity is essential in shaping development and validation of digital evidence generation tools. Key Messages: In this paper, we discuss progress and the remaining barriers to widespread use of digital measures for evidence generation in clinical development and care delivery. We also present key discussion points and takeaways in order to continue discourse and provide a basis for dissemination and outreach to the wider community and other stakeholders. The work presented here shows us a blueprint for how and why the patient voice can be thoughtfully integrated into digital measure development and that continued multistakeholder engagement is critical for further progress.

The MMS22L-TONSL heterodimer directly promotes RAD51-dependent recombination upon replication stress.

Homologous recombination (HR) is a key pathway that repairs DNA double-strand breaks (DSBs) and helps to restart stalled or collapsed replication forks. How HR supports replication upon genotoxic stress is not understood. Using in vivo and in vitro approaches, we show that the MMS22L-TONSL heterodimer localizes to replication forks under unperturbed conditions and its recruitment is increased during replication stress in human cells. MMS22L-TONSL associates with replication protein A (RPA)-coated ssDNA, and the MMS22L subunit directly interacts with the strand exchange protein RAD51. MMS22L is required for proper RAD51 assembly at DNA damage sites in vivo, and HR-mediated repair of stalled forks is abrogated in cells expressing a MMS22L mutant deficient in RAD51 interaction. Similar to the recombination mediator BRCA2, recombinant MMS22L-TONSL limits the assembly of RAD51 on dsDNA, which stimulates RAD51-ssDNA nucleoprotein filament formation and RAD51-dependent strand exchange activity in vitro Thus, by specifically regulating RAD51 activity at uncoupled replication forks, MMS22L-TONSL stabilizes perturbed replication forks by promoting replication fork reversal and stimulating their HR-mediated restart in vivo.

CRL4(WDR23)-Mediated SLBP Ubiquitylation Ensures Histone Supply during DNA Replication.

To maintain genome integrity and epigenetic information, mammalian cells must carefully coordinate the supply and deposition of histones during DNA replication. Here we report that the CUL4 E3 ubiquitin ligase complex CRL4(WDR23) directly regulates the stem-loop binding protein (SLBP), which orchestrates the life cycle of histone transcripts including their stability, maturation, and translation. Lack of CRL4(WDR23) activity is characterized by depletion of histones resulting in inhibited DNA replication and a severe slowdown of growth in human cells. Detailed analysis revealed that CRL4(WDR23) is required for efficient histone mRNA 3' end processing to produce mature histone mRNAs for translation. CRL4(WDR23) binds and ubiquitylates SLBP in vitro and in vivo, and this modification activates SLBP function in histone mRNA 3' end processing without affecting its protein levels. Together, these results establish a mechanism by which CUL4 regulates DNA replication and possible additional chromatin transactions by controlling the concerted expression of core histones.

The Replisome-Coupled E3 Ubiquitin Ligase Rtt101Mms22 Counteracts Mrc1 Function to Tolerate Genotoxic Stress.

Faithful DNA replication and repair requires the activity of cullin 4-based E3 ubiquitin ligases (CRL4), but the underlying mechanisms remain poorly understood. The budding yeast Cul4 homologue, Rtt101, in complex with the linker Mms1 and the putative substrate adaptor Mms22 promotes progression of replication forks through damaged DNA. Here we characterized the interactome of Mms22 and found that the Rtt101(Mms22) ligase associates with the replisome progression complex during S-phase via the amino-terminal WD40 domain of Ctf4. Moreover, genetic screening for suppressors of the genotoxic sensitivity of rtt101Δ cells identified a cluster of replication proteins, among them a component of the fork protection complex, Mrc1. In contrast to rtt101Δ and mms22Δ cells, mrc1Δ rtt101Δ and mrc1Δ mms22Δ double mutants complete DNA replication upon replication stress by facilitating the repair/restart of stalled replication forks using a Rad52-dependent mechanism. Our results suggest that the Rtt101(Mms22) E3 ligase does not induce Mrc1 degradation, but specifically counteracts Mrc1's replicative function, possibly by modulating its interaction with the CMG (Cdc45-MCM-GINS) complex at stalled forks.

RNAi-based screening identifies the Mms22L-Nfkbil2 complex as a novel regulator of DNA replication in human cells.

Cullin 4 (Cul4)-based ubiquitin ligases emerged as critical regulators of DNA replication and repair. Over 50 Cul4-specific adaptors (DNA damage-binding 1 (Ddb1)-Cul4-associated factors; DCAFs) have been identified and are thought to assemble functionally distinct Cul4 complexes. Using a live-cell imaging-based RNAi screen, we analysed the function of DCAFs and Cul4-linked proteins, and identified specific subsets required for progression through G1 and S phase. We discovered C6orf167/Mms22-like protein (Mms22L) as a putative human orthologue of budding yeast Mms22, which, together with cullin Rtt101, regulates genome stability by promoting DNA replication through natural pause sites and damaged templates. Loss of Mms22L function in human cells results in S phase-dependent genomic instability characterised by spontaneous double-strand breaks and DNA damage checkpoint activation. Unlike yeast Mms22, human Mms22L does not stably bind to Cul4, but is degraded in a Cul4-dependent manner and upon replication stress. Mms22L physically and functionally interacts with the scaffold-like protein Nfkbil2 that co-purifies with histones, several chromatin remodelling and DNA replication/repair factors. Together, our results strongly suggest that the Mms22L-Nfkbil2 complex contributes to genome stability by regulating the chromatin state at stalled replication forks.

Proteasome-mediated protein processing by bidirectional degradation initiated from an internal site.

The proteasome is a barrel-shaped protease that conceals its active sites within its central cavity. Proteasomes usually completely degrade substrates into small peptides, but in some cases, degradation yields biologically active protein fragments. Some transcription factors are generated from precursors by this activity, but the mechanism of proteasomal protein processing remains unclear. Here we show that proteasomal processing of the yeast NFkappaB-related transcription factors Spt23 and Mga2 is initiated by an internal cleavage, followed by bidirectional proteolysis of the polypeptides. Studies with protein fusions indicate that stable proteolytic fragments are generated if the protein contains tightly folded structures that prevent the complete degradation of the protein. Furthermore, we provide evidence that the ability of the proteasome to initiate proteolysis from an internal site and to proceed via bidirectional polypeptide degradation may be relevant for the complete degradation of proteins as well.