Journal: bioRxiv

Article Title: TFU72 is a novel and potent DNA-PKcs inhibitor for enhancing homology-directed repair gene editing

doi: 10.64898/2026.03.10.710954

Figure Lengend Snippet: A. Specificity profile of TFU72 and AZD7648 against kinases of the PI3K and PIKK families. Data is shown as half maximal inhibitory concentration (IC 50 ) against (cellular IC 50 for DNA-PKcs, biochemical IC 50 for other kinases) different kinases. B-C. Heatmap showing the top kinases inhibited by TFU72 and AZD7648 (both at 1 µM) following testing on a human kinase panel. The data is shown in descending order sorted for kinases inhibited at 50% or above relative to a target-specific inhibitor for TFU72 ( B ) or AZD7648 ( C ). D-E. Allelic HDR editing frequencies at AAVS1 locus determined by NGS analysis at D5 post RNP and ssODN editing with different concentrations of TFU72/AZD7648 as indicated ( D ). The compounds were removed either at 24 hours post editing (1 wash) or left in the cells for 5 days (0 wash). Viability of the gene edited cells as measured by CellTiter-Glo® 2.0 Assay is shown as percentage relative to the untreated sample (gene editing without compound) ( E ) (n=2). F. Allelic distribution of HDR, INDEL and WT frequencies in K562 cells edited across different gene loci (FANCF, HEK4, HBB) with or without TFU72 treatment based on NGS analysis. U denotes untreated (editing without compound), T denotes editing with TFU72 (1 µM) (n=2).

Article Snippet: The DNA-PK Inhibitor TFU72 was obtained from Merck Healthcare (Darmstadt, DE) as a 10 mM solution in DMSO.

Techniques: Concentration Assay

Journal: bioRxiv

Article Title: TFU72 is a novel and potent DNA-PKcs inhibitor for enhancing homology-directed repair gene editing

doi: 10.64898/2026.03.10.710954

Figure Lengend Snippet: iPSC, HSPC and T cells were edited at CCR5 locus using RNP and AAV6 gene editing for knock-in of short sequence (two stop codons) with different concentrations of AZD7648 and TFU72 as indicated. HDR, NHEJ, MMEJ and WT allelic frequencies were calculated at D3 post editing using Sanger Sequencing and ICE analysis (n=3). A, C, E . Scatter plots show the allelic HDR frequency in iPSC ( A ), HSPC ( C ) and T cells ( E ). B, D, F . Bar graphs show the distribution of allelic HDR, NHEJ, MMEJ and WT frequencies in iPSC ( B ), HSPC ( D ) and T cells ( F ). GT denotes gene targeting (HDR) with RNP, AAV6 editing. A and T denote AZD7648 and TFU72, respectively.

Article Snippet: The DNA-PK Inhibitor TFU72 was obtained from Merck Healthcare (Darmstadt, DE) as a 10 mM solution in DMSO.

Techniques: Knock-In, Sequencing

Journal: bioRxiv

Article Title: TFU72 is a novel and potent DNA-PKcs inhibitor for enhancing homology-directed repair gene editing

doi: 10.64898/2026.03.10.710954

Figure Lengend Snippet: iPSC ( A ), HSPC ( B ) and T cells ( C ) were edited individually at CCR5, HBB and STING1 loci with two different gRNAs at each loci using RNP and AAV6 gene editing for the knock-in of short sequence with AZD7648 and TFU72 at the indicated concentrations. Bar graphs show allelic HDR frequencies (% targeted alleles) measured at D3 post editing using Sanger Sequencing and ICE analysis (n=3). GT denotes gene targeting (HDR) with RNP, AAV6 editing. AAV6 HDR template for CCR5 locus was designed to knock-in two stop codons at the target site. At the HBB locus, the AAV6 HDR template was designed to knock-in silent mutations and correction of the sickle cell disease mutation (E6V). For the STING1 locus, the AAV6 HDR template was designed to knock-in a point mutation (V155M) along with silent mutations at the gRNA target site.

Article Snippet: The DNA-PK Inhibitor TFU72 was obtained from Merck Healthcare (Darmstadt, DE) as a 10 mM solution in DMSO.

Techniques: Knock-In, Sequencing, Mutagenesis

Journal: bioRxiv

Article Title: TFU72 is a novel and potent DNA-PKcs inhibitor for enhancing homology-directed repair gene editing

doi: 10.64898/2026.03.10.710954

Figure Lengend Snippet: A-C . iPSC ( A ), HSPC ( B ) and T cells ( C ) were edited at CCR5, HBB and STING1 loci individually using RNP and AAV6 gene editing for the knock-in of multi-kb sequence with AZD7648 and TFU72 at the indicated concentrations and at different AAV6 doses, multiplicity of infection (MOI): 500, 1000, 2500, 5000. Bar graphs show allelic HDR frequencies (% targeted alleles) measured at D3 post editing using ddPCR analysis (n=3). GT denotes gene targeting (HDR) with RNP, AAV6 editing. AAV6 HDR templates for CCR5 and HBB loci were designed to knock-in a 2.2-kb sequence consisting of UBC promoter driven GFP followed by a bGH polyA signal sequence. For the STING1 locus, the AAV6 HDR template was designed to knock-in a 1.4-kb sequence consisting of PGK promoter driven GFP followed by a sv40 polyA signal sequence.

Article Snippet: The DNA-PK Inhibitor TFU72 was obtained from Merck Healthcare (Darmstadt, DE) as a 10 mM solution in DMSO.

Techniques: Knock-In, Sequencing, Infection

Journal: bioRxiv

Article Title: TFU72 is a novel and potent DNA-PKcs inhibitor for enhancing homology-directed repair gene editing

doi: 10.64898/2026.03.10.710954

Figure Lengend Snippet: HSPCs were edited at HBB locus using RNP, AAV6 (Hifi or WT Cas9) with AZD7648 (A, 0.5 µM) or TFU72 (T, 0.5 µM) or no treatment (U). Mock electroporated cells (M) were used as a negative control. GT denotes gene targeting (HDR) with RNP, AAV6. A . At D3 post editing, a previously characterized off-target site at Chr9 (OT1) was PCR amplified from genomic DNA and sequenced by NGS. Bar graphs show INDEL frequency at the OT1 site as determined by the CRISPResso2 tool analysis of the NGS data (n=2). WT Cas9 edited samples were assessed for the INDELs using Sanger Sequencing and ICE analysis. B . Translocation between on-target (HBB) site and OT1 off-target site was assessed by ddPCR analysis and data is shown as percentage of translocation which is the sum of 4 different translocation outcomes (n=2). C . HSPCs edited at HBB loci with Hifi Cas9 were assessed for the frequency of large deletions at the on-target site using Nanopore sequencing of a 10-kb PCR amplicon. Bar graph shows the frequency of reads with deletions of the sizes 50-1000, 1000-3000, 3000-5000 and above 5000 bp (n=3). D-E. HSPCs edited at HBB locus using RNP, AAV6 with different incubation times of AAV6 (U), AZD7648+AAV6 (A) and TFU72+AAV6 (T) were assessed for the off-target INDELs at the OT1 site as described above. D. Bar graphs show the INDEL frequency at the OT1 site at D3 post editing in samples with 12h, 24h and 72h incubation times (n=1). E. Bar graphs show the frequency of reads with deletions of the sizes 50-1000, 1000-3000, 3000-5000 and above 5000 bp as determined by Nanopore sequencing described above (n=1).

Article Snippet: The DNA-PK Inhibitor TFU72 was obtained from Merck Healthcare (Darmstadt, DE) as a 10 mM solution in DMSO.

Techniques: Negative Control, Amplification, Sequencing, Translocation Assay, Nanopore Sequencing, Incubation

Journal: Nature Communications

Article Title: DNA-PK-mediated phosphorylation of STAT6 establishes a non-canonical type 2 immunity axis to prevent macrophage senescence

doi: 10.1038/s41467-026-69996-8

Figure Lengend Snippet: A Immunoblots of pSTAT6 (pY641) and γH2AX in thioglycolate-elicited peritoneal macrophages (Thio-PM) with Etoposide (Eto) and IL-4 treatment. The right panel shows the relative intensity of pSTAT6 (pY641) ( n = 4 independent experiments). B Immunofluorescence images of staining (pSTAT6 (pY641), red; DAPI, blue) of IL-4-stimulated Thio-PMs treated with Eto. Scale bars 50 μm. Right panel shows the quantification of pSTAT6 (pY641) ( n = 3 biological replicates per group). C Immunoblots of pSTAT6 (pY641) and γH2AX in Eto-treated Thio-PMs with different inhibitors. KU55933, an inhibitor of ATM. Berzosertib, inhibitor of ATR. NU7026, inhibitor of DNA-PK. AZD7762, inhibitor of CHK1/2. The right panel shows the quantification of pSTAT6 (pY641) ( n = 3 independent experiments). D Immunoblots of pSTAT6 (pY641) and γH2AX in Eto-treated Cas9 tg/+ Thio-PMs with indicated sgRNA. The right panel shows the quantification of pSTAT6 (pY641)/tSTAT6 ( n = 3 independent experiments). E Dual luciferase reporter of STAT6 in RAW 264.7 cells treated with Eto and NU7026 ( n = 3 per group). Data are mean ± s.e.m. p-value was calculated by paired two-tailed Student’s t test ( A , D ), unpaired two-tailed Student’s t test ( B ), one-way ANOVA with Dunnett’s correction ( C , E ).

Article Snippet: Antibodies used were as follows: HSP90 (Santa Cruz, #sc-13119, 1:10,000), α-Tubulin (Sigma, #T6199, 1:10,000), STAT6 (Cell Signaling, #5397, 1:1500), pSTAT6(pY641) (Cell Signaling, #56554, 1:1000), pSTAT6(pS807) (ABclonal Tech, customized, 1:200), DNA-PK (Santa Cruz, #sc-390849, 1:500), ATM (Cell Signaling, #2873, 1:1000), ATR (Santa Cruz, #sc-515173, 1:500), CHK1 (Cell Signaling, #2360, 1:1500), CHK2 (Proteintech, #13954-1-AP, 1:1000), p16-INK4A (Abcam, #ab211542, 1:1000), γH2AX (Cell Signaling, #9718, 1:1500), BRCA1 (Thermo Fisher, #MA1-23164, 1:1000), UBE2T (Thermo Fisher, # PA576202 , 1:1000), JAK (Cell Signaling, #50996, 1:1000), STING (Cell Signaling, #13647, 1:2000), STING (pS365) (Cell Signaling, #72971, 1:1000), pTBK1 (pS172) (Cell Signaling, #5483, 1:1000), pJAK1 (pY1034/1035) (Cell Signaling, #74129, 1:1000), Ubiquitin (Cell Signaling, #3936, 1:1000), K48-specific ubiquitin (Abcam, #ab140601, 1:1000), PU.1 (Abcam, #ab227835, 1:1500), Col1a1 (Cell Signaling, #72026, 1:1000), SMAD2/3 (Cell Signaling, #8685, 1:1000), p-SMAD2 (Ser465/467)/SMAD3 (Ser423/425) (Cell Signaling, #8828, 1:1000), αSMA (Cell Signaling, #19245, 1:2000) anti-HA (Cell Signaling, #3724, 1:5000) and anti-Flag (Cell Signaling, #8146, 1:5000).

Techniques: Western Blot, Immunofluorescence, Staining, Luciferase, Two Tailed Test

Journal: Nature Communications

Article Title: DNA-PK-mediated phosphorylation of STAT6 establishes a non-canonical type 2 immunity axis to prevent macrophage senescence

doi: 10.1038/s41467-026-69996-8

Figure Lengend Snippet: A Co-IP of STAT6-Turbo and JAK1, DNA-PK, ATM, and ATR in RAW 264.7 cells with TurboID. B Proximity ligation assay (PLA) of STAT6 and DNA-PK in Thio-PMs treated with Eto. Scale bars 10 μm. C The quantification of PLA signal in ( B ) ( n = 3 biological replicates per group, with more than 100 cells). D Diagram of phosphorylated sites in mouse STAT6 after Eto treatment. E Immunoblots of pSTAT6 (pY641) and tSTAT6 in Stat6 -/- Thio-PMs expressing STAT6 mutants (WT, S807A, S807E). F Immunoblots of pSTAT6 (pY641) in STAT6-mutants-expressed Cas9 tg+ Thio-PMs with Prkdc sgRNA under IL-4 & Eto co-treatment. G Dual luciferase reporter of STAT6 in RAW 264.7 cells expressing STAT6 mutants under IL-4 treatment ( n = 3 biological replicates per group). H Immunoblots of pSTAT6 (pS807) and γH2AX in Eto-treated Cas9 tg+ Thio-PMs with Prkdc sgRNAs. I Immunoblots of pSTAT6 (pS807) and γH2AX in Eto-treated Cas9 tg/+ Thio-PMs with Ku70 or Ku80 sgRNA. J Immunoblots of pSTAT6 (pS807) and γH2AX in Eto-treated Thio-PMs pre-treated with NU7026. The right panel shows the relative intensity of pSTAT6 (pS807) ( n = 3 independent experiments). K Immunoblots of pSTAT6 (pS807) and pSTAT6 (pY641) in Thio-PMs with indicated treatment. AS1517499, an inhibitor of STAT6 (block of pY641). L Immunoblots of pSTAT6 (pS807) and tSTAT6 in Eto-treated Stat6 -/- Thio-PMs expressing STAT6 mutants (WT, Y641F). The blot results are representative of three biologically independent repeats in ( A , E , F , H , I , K and L ). Data are mean ± s.e.m. Unpaired two-tailed Welch’s t test ( C ), One-way ANOVA with Dunnett’s correction ( G ), paired two-tailed Student’s t test ( J ).

Article Snippet: Antibodies used were as follows: HSP90 (Santa Cruz, #sc-13119, 1:10,000), α-Tubulin (Sigma, #T6199, 1:10,000), STAT6 (Cell Signaling, #5397, 1:1500), pSTAT6(pY641) (Cell Signaling, #56554, 1:1000), pSTAT6(pS807) (ABclonal Tech, customized, 1:200), DNA-PK (Santa Cruz, #sc-390849, 1:500), ATM (Cell Signaling, #2873, 1:1000), ATR (Santa Cruz, #sc-515173, 1:500), CHK1 (Cell Signaling, #2360, 1:1500), CHK2 (Proteintech, #13954-1-AP, 1:1000), p16-INK4A (Abcam, #ab211542, 1:1000), γH2AX (Cell Signaling, #9718, 1:1500), BRCA1 (Thermo Fisher, #MA1-23164, 1:1000), UBE2T (Thermo Fisher, # PA576202 , 1:1000), JAK (Cell Signaling, #50996, 1:1000), STING (Cell Signaling, #13647, 1:2000), STING (pS365) (Cell Signaling, #72971, 1:1000), pTBK1 (pS172) (Cell Signaling, #5483, 1:1000), pJAK1 (pY1034/1035) (Cell Signaling, #74129, 1:1000), Ubiquitin (Cell Signaling, #3936, 1:1000), K48-specific ubiquitin (Abcam, #ab140601, 1:1000), PU.1 (Abcam, #ab227835, 1:1500), Col1a1 (Cell Signaling, #72026, 1:1000), SMAD2/3 (Cell Signaling, #8685, 1:1000), p-SMAD2 (Ser465/467)/SMAD3 (Ser423/425) (Cell Signaling, #8828, 1:1000), αSMA (Cell Signaling, #19245, 1:2000) anti-HA (Cell Signaling, #3724, 1:5000) and anti-Flag (Cell Signaling, #8146, 1:5000).

Techniques: Co-Immunoprecipitation Assay, Proximity Ligation Assay, Western Blot, Expressing, Luciferase, Blocking Assay, Two Tailed Test

Journal: Nature Communications

Article Title: DNA-PK-mediated phosphorylation of STAT6 establishes a non-canonical type 2 immunity axis to prevent macrophage senescence

doi: 10.1038/s41467-026-69996-8

Figure Lengend Snippet: A Immunoblots of pSTAT6 (pS817) and γH2AX in hMDMs under indicated treatment. B Quantification of pSTAT6 (pS817)/α-Tubulin in ( A ) ( n = 5 biological replicates per group). C mRNA expression of BRCA1 and UBE2T in hMDMs expressing hSTAT6 mutants (WT, S817A, S817E) after 16-h hIL-4 treatment ( n = 5 biological replicates per group). D DNA repair capacity of THP-1-differentiated macrophages expressing hSTAT6 mutants (WT, S817A, S817E). The right panel shows the quantification ( n = 3 independent experiments). E mRNA expression of P16-INK4A and P21-CIP1 in THP-1-differentiated macrophages expressing hSTAT6 mutants (WT, S817A, S817E) under Eto-induced senescence ( n = 3 biological replicates per group). F Phagocytosis assay of THP-1-differentiated macrophages expressing hSTAT6 mutants (WT, S817A, S817E). All groups had 30-min phagocytosis period. The right panel shows the quantification ( n = 4 biological replicates per group). G Representative image of lung sections from control and COPD donors. Scale bar 100 μm. H Quantification of P16 + cells in lungs from control and COPD donors ( n = 6 biological replicates per group). I Quantification of P16 + macrophages (CD68 + ) in lungs from control and COPD donors ( n = 6 biological replicates per group). J Immunoblots of pSTAT6 (pS817), P16-INK4A and γH2AX in lungs from control, smoker, and COPD donors ( n = 4 biological replicates per group). K Quantification of pSTAT6 (pS817)/tSTAT6 in lungs from control, smoker, and COPD donors ( n = 4 biological replicates per group). L Spearman’s correlation between pSTAT6 (pS817)/tSTAT6 and γH2AX in human lungs from control ( n = 10), smoker ( n = 4), and COPD donors ( n = 10). M Spearman’s correlation between pSTAT6 (pS817)/tSTAT6 and P16-INK4A in human lungs from control ( n = 10), smoker ( n = 4), and COPD donors ( n = 10). Data are mean ± s.e.m. p -value was calculated by paired two-tailed Student’s t test ( B , C and D ), one-way ANOVA with Dunnett’s correction ( E , F and K ), unpaired two-tailed Student’s t test ( H and I ), two-tailed Spearman’s correlation coefficient analysis ( L and M ).

Article Snippet: Antibodies used were as follows: HSP90 (Santa Cruz, #sc-13119, 1:10,000), α-Tubulin (Sigma, #T6199, 1:10,000), STAT6 (Cell Signaling, #5397, 1:1500), pSTAT6(pY641) (Cell Signaling, #56554, 1:1000), pSTAT6(pS807) (ABclonal Tech, customized, 1:200), DNA-PK (Santa Cruz, #sc-390849, 1:500), ATM (Cell Signaling, #2873, 1:1000), ATR (Santa Cruz, #sc-515173, 1:500), CHK1 (Cell Signaling, #2360, 1:1500), CHK2 (Proteintech, #13954-1-AP, 1:1000), p16-INK4A (Abcam, #ab211542, 1:1000), γH2AX (Cell Signaling, #9718, 1:1500), BRCA1 (Thermo Fisher, #MA1-23164, 1:1000), UBE2T (Thermo Fisher, # PA576202 , 1:1000), JAK (Cell Signaling, #50996, 1:1000), STING (Cell Signaling, #13647, 1:2000), STING (pS365) (Cell Signaling, #72971, 1:1000), pTBK1 (pS172) (Cell Signaling, #5483, 1:1000), pJAK1 (pY1034/1035) (Cell Signaling, #74129, 1:1000), Ubiquitin (Cell Signaling, #3936, 1:1000), K48-specific ubiquitin (Abcam, #ab140601, 1:1000), PU.1 (Abcam, #ab227835, 1:1500), Col1a1 (Cell Signaling, #72026, 1:1000), SMAD2/3 (Cell Signaling, #8685, 1:1000), p-SMAD2 (Ser465/467)/SMAD3 (Ser423/425) (Cell Signaling, #8828, 1:1000), αSMA (Cell Signaling, #19245, 1:2000) anti-HA (Cell Signaling, #3724, 1:5000) and anti-Flag (Cell Signaling, #8146, 1:5000).

Techniques: Western Blot, Expressing, Phagocytosis Assay, Control, Two Tailed Test