Goff, P. | Search bioRxiv

bioRxiv2026

Genetic or pharmacological disruption of the MSH3 Y245/K246 IDL binding pocket slows CAG repeat expansion

Recent genetic studies have shown somatic expansion of the CAG repeat is the key process driving Huntingtons disease (HD) pathogenesis. Recognition of insertion deletion loops (IDLs), lesions prone to form within the CAG repeat, by Muts{beta} (MSH3/MSH2) is thought to be the primary event in the expansion process. This starts a cascade that leads to error prone repair and incorporation of additional CAG units into the repeat. In vitro data shows MSH3 binds IDLs through a DNA binding pocket formed by MSH3 residues Y245/K246. In this study, we investigated the significance of this DNA binding motif in CAG repeat expansion using cell lines harboring long, unstable HTT CAG repeats. Genetic disruption of the MSH3 Y245/K246 motif significantly reduced DNA interaction, exhibited MMR deficiency in a frameshift mutator assay and abrogated repeat expansion in a U2OS cell line expressing mutant HTT exon 1. Pharmacological blockade of this site using a small molecule targeting the DNA binding pocket similarly reduced DNA binding and repeat expansion in a U2OS cell line. Crucially, this molecule also slowed CAG repeat expansion in medium spiny neurons derived from HD patient-iPSCs. Targeting of the MSH3 IDL binding pocket may represent a possible therapeutic strategy.

neuroscience↗

bioRxiv2021

Differential effects of high versus low linear energy transfer (LET) radiation on type-I interferon (IFNβ) and TREX1 responses

PurposeCancer cells produce innate immune signals following radiation damage, with STING pathway signaling as a critical mediator. High linear energy transfer (LET) radiations create larger numbers of DNA double-strand breaks (DSBs) per unit dose than low-LET radiations and may therefore be more immunogenic. We studied the dose response characteristics of pro-immunogenic type-I interferon, interferon-beta (IFN{beta}), and its reported suppressor signal, three-prime repair exonuclease 1 (TREX1), in vitro with low-LET x-rays and high-LET fast neutrons. MethodsMerkel cell carcinoma cells (MCC) were irradiated by graded doses of x-rays (1-24 Gy) or fast neutrons (1-8 Gy). IFN{beta} was measured as a function of dose via ELISA assay, and exonuclease TREX1 expression via immunofluorescence microscopy. The Monte Carlo damage simulation (MCDS) was used to model fast neutron relative biological effectiveness for DSB induction (RBEDSB) and compared to laboratory measurements of the RBE for IFN{beta} production (RBEIFN{beta}) and TREX1 upregulation (RBETREX1). RBEIFN{beta} models were also applied to radiation transport simulations to quantify the potential secretion of IFN{beta} in representative clinical beams. ResultsPeak IFN{beta} secretion occurred at 5.7 Gy for fast neutrons and at 14.0 Gy for x-rays, i.e., an effective RBEIFN{beta} of 2.5 {+/-} 0.2. The amplitude (peak value) of secreted IFN{beta} signal did not significantly differ between x-rays and fast neutrons (P > 0.05). TREX1 signal increased linearly with absorbed dose, with a four-fold higher upregulation per unit dose for fast neutrons relative to x-rays (RBETREX1 of 4.0 {+/-} 0.1). Monte Carlo modeling of IFN{beta} suggests Bragg peak-to-entrance ratios of IFN{beta} production of 40, 100, and 120 for proton, alpha, and carbon ion beams, respectively, a factor of 10-20-fold higher compared to their corresponding physical dose peak-to-entrance ratios. The spatial width of the Bragg peak for IFN{beta} production is also a factor of two smaller. ConclusionHigh-LET fast neutrons initiate a larger IFN{beta} response per unit absorbed dose than low-LET x-rays (i.e., RBEIFN{beta} value of 2.5). The RBE value for IFN{beta} is quite similar to data reported in the literature for DSB induction and cellular, post-irradiation micronucleation formation for neutrons and x-rays. The increased IFN{beta} release after high-LET radiation may be a contributing factor in stimulating a systemic anti-tumor, adaptive immune response (abscopal effect). However, our results indicate that TREX1 anti-inflammatory signaling in vitro for MCC cells is larger per unit dose for fast neutrons than for x-rays (RBETREX1 of 4.0). Given these competing effects, additional studies are needed to clarify whether or not high-LET radiations are therapeutically advantageous over low-LET radiation for pro-inflammatory immune signaling in other cell lines in vitro and for in vivo cancer models.

cancer biology↗