Selective USP7 inhibition elicits cancer cell killing through a p53-dependent mechanism

Ubiquitin specific peptidase 7 (USP7) is a deubiquitinating enzyme (DUB) that removes ubiquitin tags from specific protein substrates in order to alter their degradation rate and sub-cellular localization. USP7 has been proposed as a therapeutic target in several cancers because it has many reported substrates with a role in cancer progression, including FOXO4, MDM2, N-Myc, and PTEN. The multi-substrate nature of USP7, combined with the modest potency and selectivity of early generation USP7 inhibitors, has presented a challenge in defining predictors of response to USP7 and potential patient populations that would benefit most from USP7-targeted drugs. Here, we describe the structure-guided development of XL177A, which irreversibly inhibits USP7 with sub-nM potency and selectivity across the human proteome. Evaluation of the cellular effects of XL177A reveals that selective USP7 inhibition suppresses cancer cell growth predominantly through a p53-dependent mechanism: XL177A specifically upregulates p53 transcriptional targets transcriptome-wide, hotspot mutations in TP53 but not any other genes predict response to XL177A across a panel of ~500 cancer cell lines, and TP53 knockout rescues XL177A-mediated growth suppression of TP53 wild-type (WT) cells. Together, these findings suggest TP53 mutational status as a biomarker for response to USP7 inhibition. We find that Ewing sarcoma and malignant rhabdoid tumor (MRT), two pediatric cancers that are sensitive to other p53-dependent cytotoxic drugs, also display increased sensitivity to XL177A.


Supplementary Figure Captions
between free USP7 and XL041 USP7 bound, and the deuterium incorporation plots for all 81 peptic peptides that were compared between USP7 free (red) and XL041bound (blue). On the x axis is the time in minutes and on the y-axis is the relative uptake (Da). The maximum y-axis value for each plot represents the theoretical maximum amount of D that can be incorporated into a peptide. Values represent the mean of two individual measurements; error bars, s.d. B. USP7 coverage map showing all the common peptic peptides that were compared between free USP7 and XL177A USP7 bound, and the deuterium incorporation plots for all 105 peptic peptides that were compared between USP7 free (red) and XL177A bound (blue). C. Chiclet representation of the HDX MS data. The peptides from panel A and B are represented from N to C terminus (top to bottom) at each time point (left to right). The deuterium level for each bound state was subtracted from that of the protein alone and colored according to the legend shown. The horizonal dotted lines are placed to help orient the readers to notice the regions of difference between the two states. Note that in the HDX MS data set the protein sequence numbering is different and Cys34 is actually Cys223. Figure S7. Snapshot of the XL188-USP7 construct (red) and the XL177A-USP7 construct (orange) superimposed on each other after 150 ns of simulation time shows the benzyl moiety of XL177A in a similar confirmation to that of XL188.     were treated with sgRNAs targeting the indicated genes, then allowed to grow for 17 days, with luminescence readings taken at the indicated timepoints. WT:KO ratio is equivalent to the FF:Renilla ratio.       A. T L D  10  20  30  40  50  60  70  80 90 100 A  110  120  130  140  150  160  170  180 190 200 T  210  220  230  240  250  260  270  280 290 300  T L D  10  20  30  40  50  60  70  80 90 100 110  120  130  140  150  160  170  180 190 200 T  210  220  230  240  250  260  270  280 290 300

A B
A. Cell titerglo shows SB1_F_057 demonstrates no differential response based on TP53 status in Ewing sarcoma cell lines. B. Cell titerglo shows SB1_F_057 demonstrates no differential response in TC32 TP53 knock out cells. SB1_F_057 shows no differential response based on TP53 status

A B
A. Cell titerglo shows SB1_F_057 demonstrates no differential response based on TP53 status in Ewing sarcoma cell lines. B. Cell titerglo shows SB1_F_057 demonstrates no differential response in TC32 TP53 knock out cells. SB1_F_057 shows no differential response based on TP53 status A549 RKO Figure S13

Supplementary Synthetic Methods
Synthesis of XL177A, XL177B, XL112, XL041, and XL058 (see Figure S15): Step 1 (Synthesis of S1): 7-nitroquinazolin-4(3H)-one (1.55g, 8.1mmol) and tert-butyl- Step 2 (Synthesis of S2): Compound S1(2.4g, 6.0mmol) was suspended in 20mL solvent (EtOH/AcOH=1:1). 4 eq. of Fe powder was added in portions. The mixture was stirred for 1 hour at 55°C. Then the reaction was cooled down to room temperature, and filtered through a pad of Celite. The filtrate was concentrated under reduced pressure to afford the crude product, which was then purified by flash chromatography (10%MeOH in EtOAc) to afford 2.1g product S2 (93%) 1  Step 3 (Synthesis of S3): Compound S2 (2.1g, 5.6mmol) was dissolved in anhydrous 10mL dichloromethane under N2 at 0°C. 3.0 eq. of Et3N was added. Then 3-bromopropionyl chloride (1.15g, 6.7mmol) was added dropwise. The mixture was stirred at 0°C for 1 hour, then quenched with MeOH, and concentrated under reduced pressure. The solid residue was directly used for the following step without further purification. The crude product from last step was dissolved in 10mL MeOH, then 3.0eq of Et3N was added. Into the stirred mixture was added 1-methylpiperazine (0.67g, 6.7mmol) dropwise. After the addition completed, the mixture was stirred for 1 hour at 50°C. Then the reaction mixture was cooled down to room temperature and concentrated under reduced pressure, then directly subjected to HPLC purification (MeOH/H2O with 4‰ TFA) to afford 2.1g product S3 (73% in two steps) 1  Step 4 (Synthesis of S4 (R 1 =(S)-Bn, R 2 =Cl)): S3 (0.53g, 1.0mmol) was dissolved in 3mL DCM, then 5mL 4M HCl in 1,4-dioxane was added in portions. The solution was stirred for 1 hour at room temperature. Then the mixture was concentrated under reduced pressure, and left on high vacuum overnight to remove residual acid.
Then the product (0.11g, 0.25mmol) was dissolved in 3mL DMF, and basified by adding 10 eq of Et3N.