Characterization of TNG348: A Selective, Allosteric USP1 Inhibitor That Synergizes with PARP Inhibitors in Tumors with Homologous Recombination Deficiency

Abstract

Inhibition of the deubiquitinating enzyme USP1 can induce synthetic lethality in tumors characterized by homologous recombination deficiency (HRD) and represents a novel therapeutic strategy for the treatment of BRCA1/2-mutant cancers, potentially including patients whose tumors have primary or acquired resistance to PARP inhibitors (PARPi). In this study, we present a comprehensive characterization of TNG348, an allosteric, selective, and reversible inhibitor of USP1. TNG348 induces dose-dependent accumulation of ubiquitinated protein substrates both in vitro and in vivo. CRISPR screens show that TNG348 exerts its antitumor effect by disrupting the translesion synthesis pathway of DNA damage tolerance through RAD18-dependent ubiquitinated PCNA. Although TNG348 and PARPi share the ability to selectively kill HRD tumor cells, CRISPR screens reveal that TNG348 and PARPi do so through discrete mechanisms. Particularly, knocking out PARP1 causes resistance to PARPi but sensitizes cells to TNG348 treatment. Consistent with these findings, combination of TNG348 with PARPi leads to synergistic antitumor effects in HRD tumors, resulting in tumor growth inhibition and regression in multiple mouse xenograft tumor models. Importantly, our data on human cancer models further show that the addition of TNG348 to PARPi treatment can overcome acquired PARPi resistance in vivo. Although the clinical development of TNG348 has been discontinued because of unexpected liver toxicity in patients (NCT06065059), the present data provide preclinical and mechanistic support for the continued exploration of USP1 as a drug target for the treatment of patients with BRCA1/2-mutant or HRD cancers.

Graphical abstract

Figure 1.

TNG348 is an allosteric USP1i. A, Chemical structure of TNG348. B, Cryo-EM structure of TNG348 (green) bound to the USP1 + UAF1 + Ub-VS complex (gray, purple, and orange, respectively). C, TNG348 binding pocket in USP1; selected residues in close contact with the inhibitor are labeled. The catalytic cysteine 90 is depicted at the lower left; the dashed orange line indicates its covalent attachment to the Ub-VS. D and E, TNG348 causes dose-dependent accumulation of USP1 substrates. D, MDA-MB-436 cells were treated for 24 hours with indicated doses of TNG348, and samples were collected for Western blot analysis. E, Combined densitometric quantifications of Western blot signals of three independent experiments shown in D. F–H, Plasma exposure and induction of USP1 substrates upon TNG348 dosing correlate in vivo. Mice-bearing MDA-MB-436 tumors were exposed to TNG348 at indicated doses for 5 days. F, Plasma samples from three to four mice were taken at indicated timepoints after last dose; TNG348 levels were quantified by mass spectrometry. TNG348 concentrations were corrected with plasma protein binding from NOD-SCID mice. The dashed line represents the average viability IC₅₀ of 68.3 nmol/L. G and H, Tumors were harvested from three to four mice for each condition and processed for Western blotting against ub-PCNA and PCNA (G) and ub-FANCD2 (H). Protein levels were measured by densitometry of three to four different mice, and error bars represent the SEM.

Figure 2.

TNG348 is a selective USP1i. A, TNG348 only inhibits USP1 in vitro in a panel of 47 protein DUBs. Activity of TNG348 was tested at 10 μmol/L against the Ubiquigent DUBprofiler panel. USP12, USP46, and USP1 (in red), which all require UAF1 for activity, are labeled. B, TNG348 was assayed in vitro against the Eurofins KINOMEscan panel of 468 kinases at a single-point 10 μmol/L concentration. Only ULK3 (in red) was found to be inhibited at 89% of control binding. C, The activity of recombinant USP1 WT or V156K mutant protein was assayed in vitro. The V156K mutant shows ∼50% reduction in activity but maintains similar Km for Ub-Rhod-110 substrate to WT enzyme. RFU, Relative fluoresence units. D, The USP1 V156K mutant protein is not inhibited by TNG348 in in vitro ub-Rhod-110 assay (TNG348 lost >200-fold potency). E, The USP1 V156K mutant completely abolishes the antitumor growth effect of TNG348. Endogenous USP1 was knocked out with two USP1 gRNAs in Cas9-expressing MDA-MB-436 cells, and CRISPR-resistant WT or V156K mutant cDNA was concurrently expressed. The viability at the indicated TNG348 dose was monitored in 14-day clonogenic assays.

Figure 3.

TNG348 activity is enriched in HRD cell lines and acts through a distinct mechanism from PARPi. A and B, TNG348 activity is elevated in BRCA-mutant or HRD cells. TNG348 activity was assayed in a panel of 62 breast and ovarian cell lines by 10–21-day clonogenic assays using 10-point threefold serial dilutions of TNG348 with an 8 μmol/L top dose. Examples are shown in A. B, The average AUC from biological duplicates of technical duplicates was calculated from nonlinear regression of the dose–response and normalized so that the highest possible response is assigned a value of 1 and no response, 0. Cell lines were grouped according to the HRD status; HR proficient (HRD negative, HRD−) cells without BRCA1/2 mutations in black, and cell lines that carry a BRCA1/2 mutation or that are BRCAwt but HRD positive (HRD+, ScarHRD > 65) in red and blue, respectively. Cells line names are shown on the right from most sensitive (top) to least sensitive (bottom) to TNG348. Statistical significance was calculated with a two-tailed unpaired Student t test. ***, P < 0.001. C and D, Volcano plot showing CRISPR screening results comparing DMSO and 200 (C) or 100 nmol/L (D) TNG348-treated Cas9-expressing UWB1.289 and MDA-MB-436 cells. Dashed line: P value = 0.05. E and F, Volcano plots showing CRISPR screening comparing DMSO and 1 μmol/L (E) or 10 nmol/L (F) olaparib-treated Cas9-expressing UWB1.289 and MDA-MB-436 cells. A focused gRNA library targeting DNA damage response genes was used for C–F. Labeled in red are genes known to cause resistance to USP1i when lost or involved in the ub-PCNA pathway. In blue are genes known to confer resistance to PARPi when knocked out. G and H, RAD18 suppresses the TNG348 sensitivity of UWB1.289 (G) and MDA-MB-436 (H) cell lines. Endogenous RAD18 was knocked out in Cas9-expressing cells, and the KO efficiency was validated, along with the impact on ub-PCNA levels, by Western blotting after 24 hours DMSO or TNG348 treatment (left). The impact on viability was monitored by clonogenic assays (right). NTC, Non-target control.

Figure 4.

TNG348 synergizes with PARPi in BRCA1/2-mutant and HRD cell lines. A and B, Volcano plots of whole-genome CRISPR screens in BRCA1mut COV362 (A) and HCC1395 (B) cell lines comparing gene mutations that sensitize (left) or cause resistance (right) to TNG348. PARP1 is labeled in blue, and members of the BRCA1-A complex are labeled in gray. Dashed line: P value = 0.05. C, TNG348 synergizes with olaparib in COV362 and HCC1395 cell lines. Viability was measured by CellTiter-Glo after 7 days of growth. D and E, Combination of TNG348 with olaparib (D) or saruparib (E) was tested in a panel of 69 breast and ovarian cell lines. The average Bliss synergy score for each cell line was calculated across an entire 9 × 9 matrix, and data are presented as the average of two biological replicates. Cell lines were categorized according to the HRD status; HR-proficient (HRD−) cells without BRCA1/2 mutations (black), and cell lines that carry a BRCA1/2 mutation or that are BRCAwt but HRD+ (ScarHRD > 65) in red and blue, respectively. Statistical significance was calculated with a two-tailed unpaired Student t test. **, P value < 0.01. F, TNG348 induces PARylation. UWB1.289 cells were treated for 24 hours with indicated drugs, and samples were collected for subcellular fractionation experiments followed by Western blotting with indicated antibodies. G–J, Olaparib synergy comparison among ART558 (POLQi), RP3500 (ATRi), and TNG348 in the UWB1.289 cell line. G, Average Bliss synergy score was calculated over a 4 × 4 portion of the dose–response matrices (0.04–1.1 μmol/L for olaparib, TNG348, and ART558 and 0.45–12.3 nmol/L for RP3500). H–J, Viability assay examples of olaparib combination with TNG348 (H), RP3500 (I), and ART558 (J) plotted in G. Error bars: SEM.

Figure 5.

TNG348 drives strong in vivo antitumor activity in combination with PARPi and can overcome PARPi resistance. A–G, The growth of PDX models BR-05-0028 (A), PA1338 (B), ST4139 (C), CTG-0012 (D), HBCx-11 (E), ST1213 (F), and HBCx-14 (G) was monitored in mice during vehicle, TNG348, PARPi, or combination treatment. Average tumor volume from at least four mice from each arm is shown. Error bars represent SEM. See Supplementary Table S14 for additional information on models. H and I, TNG348 can resensitize tumors to PARPi. The model from A was subjected to prolonged treatment to olaparib until tumor regrowth was observed. A tumor regrowing from a single animal was further passaged under constant olaparib pressure to create an olaparib-resistant model. H This model is cross-resistant to several PARPi. I, Tumor growth following vehicle, TNG348, olaparib, or combination treatment in the PARPi-resistant model was monitored for 27 days. Error bars represent SEM of eight different animals for each arm. mpk, milligrams per kilogram. Statistical significance was calculated using two-way ANOVA followed by the Tukey multiple comparisons test. Only vehicle to combination comparison is shown, see Supplementary Table S13 for other comparisons. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.