Identification of nonsense-mediated decay inhibitors that alter the tumor immune landscape

Abstract

Despite exciting developments in cancer immunotherapy, its broad application is limited by the paucity of targetable antigens on the tumor cell surface. As an intrinsic cellular pathway, nonsense-mediated decay (NMD) conceals neoantigens through the destruction of the RNA products from genes harboring truncating mutations. We developed and conducted a high-throughput screen, based on the ratiometric analysis of transcripts, to identify critical mediators of NMD in human cells. This screen implicated disruption of kinase SMG1's phosphorylation of UPF1 as a potential disruptor of NMD. This led us to design a novel SMG1 inhibitor, KVS0001, that elevates the expression of transcripts and proteins resulting from human and murine truncating mutations in vitro and murine cells in vivo. Most importantly, KVS0001 concomitantly increased the presentation of immune-targetable human leukocyte antigens (HLA) class I-associated peptides from NMD-downregulated proteins on the surface of human cancer cells. KVS0001 provides new opportunities for studying NMD and the diseases in which NMD plays a role, including cancer and inherited diseases.

Keywords: cancer biology; chromosomes; drug repurposing; gene expression; high-throughput screen; human; immunotherapy; mouse; next-generation sequencing; nonsense-mediated decay.

Figure 1.. LY3023414 is a small molecule capable of increasing transcription of nonsense-mediated decay (NMD) targets.

(A) Schematic of high-throughput screen (HTS) used to identify inhibitors of NMD. Mutant transcripts are represented by a smaller length in the cartoon for illustrative purposes only. All small molecules were tested at 10 μM. (B) Mutant RNA reads relative to wild-type reads for the top 8 hits from the HTS. The dotted line represents the minimum fraction required to be considered a hit (>5 standard deviations above dimethyl sulfoxide [DMSO] control). Full screen results are presented in Figure 1—figure supplement 4. (C) Targeted RNA-sequencing results of isogenic RPTec knockout clones treated with the eight best hits from the HTS at 10 µM. The dotted line represents a relative RNA expression level of 1, equal to that of DMSO-treated wells. Data for ceritinib, which did not validate on any line, are presented only in Figure 1—figure supplement 8. (D) TP53 western blot on RPE TP53 224, containing a homozygous TP53 mutation, using the four hit compounds that validated in RPTec isogenic lines at 10 μM. (E) Western blot analysis of full-length TP53α and isoform TP53β after treatment with two NMD inhibitor lead candidates at 10 μM. TP53β (expression known to be controlled by NMD) as well as mutant TP53 are prominently induced by LY3023414 whereas full length is not. Note that RPE TP53 223 is a heterozygous knockout clone with one near wild-type allele whereas RPTec TP53 588 contains a homozygous TP53 indel mutation. (F) Quantitative real-time PCR (qPCR) showing 10 μM LY3023414 treatment causes increased expression of the NMD controlled alternative transcript for TP53, TP53β, in parent cell lines for RPE1 and RPTec. Significance determined by Student’s t-test. Unless indicated otherwise cells were exposed to test compound for 16 hr.

Figure 1—figure supplement 1.. Next-generation sequencing results depicting genomic mutations at the CRISPR target area in STAG2 (top left) and TP53 (top right) isogenic cell line clones in the RPE1 cell line which were used in subsequent experiments.

Western blot showing STAG2 protein loss in four independent clones of which only clones 2 and 8 were used in subsequent experiments (bottom left). Note that STAG2 clone 8 has two independent biallelic indel events. The mutation in exon 15 was used for scoring relative transcript levels in the screen and for subsequent work. Immunohistochemistry (IHC) results confirming p53 protein loss in RPE TP53 221 (bottom right).

Figure 1—figure supplement 2.. Fold change in RNA expression levels from whole transcriptome RNA-sequencing data for STAG2 and TP53 knockout clones in the RPE1 cell line background.

RPE1 TP53 clone 221, RPE1 STAG2 clone 2, and RPE1 STAG2 clone 8 were used in the high-throughput screen (HTS). RPE1 TP53 clone 223 and RPE1 TP53 clone 224 are used in Figure 1—figure supplement 6. Note clone 223 has a 9-bp in-frame deletion in one allele and an out-of-frame deletion on the other allele, presumably accounting for the higher level of expression.

Figure 1—figure supplement 3.. RNA transcript level changes based on quantitative real-time PCR (qPCR) in STAG2 and TP53 knockout clones treated with the known nonsense-mediated decay (NMD) inhibitor emetine at 12 mg/ml.

Red arrows indicate the cell line containing the truncating mutation in the gene being assessed. Error bars show standard deviation of three biological replicates. All changes are statistically significant by Student’s t-test (p < 0.05).

Figure 1—figure supplement 4.. Primary screen results from high-throughput assay.

The x-axis represents all 2658 compounds screened at 10 µM, y-axis shows ratio of mutant to wild-type reads for each of the three isogenic cell lines. Higher values indicate more mutant RNA reads, representing inhibition of nonsense-mediated decay (NMD). The dotted line at 0.46 is the cutoff for a hit to be called (5 standard deviations above dimethyl sulfoxide [DMSO]-treated samples). Colored data points demarcate the eight hit compounds in the three screened cell lines.

Figure 1—figure supplement 5.. Emetine and dimethyl sulfoxide (DMSO) control sample data from the high-throughput screen (HTS) for each of the three clones used as measured by deep-targeted RNA-sequencing.

Six DMSO and two emetine (12 mg/ml) samples were included in each dosing plate for a total of 198 DMSO and 66 emetine measurements in each boxplot.

Figure 1—figure supplement 6.. Knockout status for isogenic cell lines used in this study.

(A) Immunohistochemistry (IHC) staining of TP53 in RPTec TP53 knockout clones. (B) Western blot against STAG2 on RPTec STAG2 knockout clones demonstrating successful knockout at the protein level of all 10 clones. Out-of-frame indels were confirmed in all clones by next-generation sequencing (NGS) (data not shown). The arrow indicates the expected size for full-length STAG2 protein. (C) IHC staining of TP53 protein on RPE1 TP53 knockout clones. Clone 223 contains the same truncating mutation found in clone 224 on allele 1 and has a 9-bp in-frame deletion that preserves some full-length TP53α and TP53β isoform expression on allele 2 (see Figure 1E). Note that the IHC image shown for RPE1 parent (control) in part C is the same image shown in Figure 1—figure supplement 1.

Figure 1—figure supplement 7.. Protein schematic cartoons showing indel mutation site and expected size of various TP53 knockout clones used in this study.

Note: RPE TP53 223 has an in-frame deletion in the DNA-binding domain.

Figure 1—figure supplement 8.. Fold change in mutant RNA transcription levels for STAG2 and TP53 in three knockout cell lines from the RPtec background containing out-of-frame indels targeted by nonsense-mediated decay (NMD).

Each line was treated with each of the eight hit compounds from the screen. The 10 µM dose is also shown in Figure 1C.

Figure 2.. Inhibiting nonsense-mediated decay (NMD) in cancer cells increases broad expression of truncated gene messenger RNA (mRNA) and protein.

(A) Mutant transcript recovery rates for genes containing heterozygous indel mutations based on RNA-sequencing results in cell lines treated with 5 µM LY3023414 for 16 hr. Strict inclusion criteria were used, such that only mutations with sufficient sequencing coverage are shown (see methods). Recovery is defined as at least two-fold increase over dimethyl sulfoxide (DMSO) treatment. (B) Targeted high coverage RNA-sequencing confirms recovery of mutant transcript levels in NCI-H358 and LS180 cancer cell lines treated with 5 µM LY3023414. RNF43 and DROSHA contain common heterozygous single-nucleotide polymorphisms (SNPs) and the mutant allele refers to the non-reference genome allele. Error bars indicate 95% confidence limits. (C) Western blot analyses of NCI-H358 cells showing mutant and wild-type protein levels in EXOC1 and (D) SPTAN1 with and without 5 µM LY3023414 treatment. The black arrow indicates the expected size of the mutant protein. The C-terminal SPTAN1 antibody is downstream of the out-of-frame indel mutation and is not expected to identify the mutant allele. (E) Fold change in the number of mutant RNA transcripts from deep-targeted RNA-sequencing of heterozygous mutated genes in NCI-H358 and LS180 xenografts treated by oral gavage with 60 mg/kg LY3023414 assayed 16 hr post-treatment. Student’s t-test for target genes are all p < 0.05, while the null hypothesis holds for RNF43 (common SNP).

Figure 3.. Novel nonsense-mediated decay (NMD) inhibitor KVS0001 is SMG1 specific and induces expression of NMD-targeted genes in vitro and in vivo.

(A) Fraction of mutant allele transcripts in genes with heterozygous indels previously established in this study as sensitive to NMD inhibition. Results show mutant levels after siRNA treatment targeting kinases inhibited by LY3023414. RNF43 and DROSHA are common heterozygous single-nucleotide polymorphisms (SNPs) (shaded gray) and serve as negative controls. (B) Fraction of mutant allele transcripts in genes with truncating mutations known to be sensitive to NMD inhibition after siRNA treatment with siUPF1 or non-targeting siRNA. Data from deep-targeted RNA-sequencing. (C) Structure of novel NMD inhibitor KVS0001. (D) Targeted RNA-sequencing on three genes with heterozygous, out-of-frame, indel mutations in LS180 cancer cells treated in a dose–response with KVS0001 or SMG1i-11. RNF43 serves as a control (common heterozygous SNP) and the mutant allele refers to the non-reference genome allele. (E) Western blot of EXOC1 protein in NCI-H358 cells treated with 5 µM novel inhibitor KVS0001, LY3023414, or SMG1i-11 for 24 hr. (F) Western blot of phosphorylated UPF1 on three cell lines treated with 5 µM KVS0001, SMG1i-11, or dimethyl sulfoxide (DMSO). Note that total UPF1 and p-UPF1 were run on different gels, loading controls correspond to indicated gel. (G) Fold change in the number of mutant allele transcripts measured by targeted RNA-seq in genes containing heterozygous out-of-frame indel mutations in NCI-H358 or (H) LS180 subcutaneous xenografts in bilateral flanks of nude mice. Mice were treated once with intraperitoneal (IP) injection of vehicle or 30 mg/kg KVS0001 and tumors harvested 16 hr post IP treatment. All genes shown contain heterozygous out-of-frame truncating mutations except RNF43 and DROSHA which serve as controls (contain heterozygous SNPs).

Figure 3—figure supplement 1.. RNA expression levels of kinases post siRNA targeting in NCI-H358 and LS180 cells by quantitative real-time PCR (qPCR).

Cells are treated with siRNAs targeting genes known to be inhibited by LY3023414. Error bars represent 95% confidence intervals.

Figure 3—figure supplement 2.. Slider plots showing mutant allele fraction relative to total reads measured by deep-targeted RNA-sequencing for in vitro treated LS180 (top in red) or NCI-H358 (bottom in blue) cells with 5 µM LY3023414 (labeled LYO) or a previously described SMG1 inhibitor SMG1i-11 at 1 µM.

Gene names are shown in the boxes above each slider plot, genes highlighted in gray are common single-nucleotide polymorphisms (SNPs) and serve as a negative control (not expected to change). In the case of the control SNPs, the mutant allele refers to the non-reference genome allele. TRIM21 did not show a large change in expression with either small molecule, while ANLN did not show a difference with LY3023414 but did respond to SMG1i-11. The remaining genes responded to nonsense-mediated decay (NMD) inhibition by both LY3023414 and SMG1i-11.

Figure 3—figure supplement 3.. Mutant protein expression only occurs in the presence of nonsense-mediated decay (NMD) inhibtion.

(A) Western blot showing SPTAN1 expression after treatment with dimethyl sulfoxide (DMSO), 5 µM LY3023414, or 1 µM SMG1 inhibitor SMG1i-11 (lanes 1, 2, and 3, respectively, for each antibody). The arrow indicates the expected size of the mutant NMD-targeted protein. Antibody ab75755 (Abcam) binds C-terminal to the out-of-frame indel and does not show mutant protein as expected. Antibody A301-249 (Bethyl) is polyclonal and also did not bind mutant protein. Antibody ab11755 (Abcam) is located N-terminal to the indel and does display mutant protein expression. (B) Western blot showing EXOC1 expression after treatment with DMSO, 5 µM LY3023414, or 1 µM SMG1i-11 (lanes 1, 2, and 3, respectively). The arrow indicates the expected size of the mutant (NMD-targeted) protein.

Figure 3—figure supplement 4.. Biophysical properties for novel SMG1 inhibitor KVS0001.

Figure 3—figure supplement 5.. Kinase specifity for KVS0001.

(A) Kinativ assay results for KVS0001 at 100 nM and (B) 1 µM run with biological replicates showing KVS0001 specificity against the known kinome. Results are based on the average between two unique peptides for each kinase.

Figure 3—figure supplement 6.. (A) Slider plots showing mutant allele fraction measured by deep-targeted RNA-sequencing for genes from NCI-H358 (top in red) and (B) LS180 (bottom in blue) treated in a dose–response in vitro with novel nonsense-mediated decay (NMD) inhibitor KVS0001.

DROSHA and RNF43 are common heterozygous single-nucleotide polymorphisms (SNPs) and serve as a negative control. In the case of the control SNPs, the mutant allele refers to the non-reference genome allele. Only the highest two concentrations were tested on LY3023414 which served as a positive control in this experiment. The dotted line indicates the mutant expression with dimethyl sulfoxide (DMSO) treatment and the solid line is a reference for equal expression of both the wild-type and mutant alleles.

Figure 3—figure supplement 7.. Mutant protein expression in presence of nonsense-mediated decay (NMD) inhibtion.

(A) Western blot showing expression of LMAN1 in LS180 cells treated with dimethyl sulfoxide (DMSO) (lane 1), KVS0001 at 5 µM (lane 2), or SMG1i-11 at 1 µM (lane 3). Arrows indicate expected size of wild-type and mutant LMAN1 protein. (B) Stain free loading control image for gel.

Figure 4.. KVS0001 treatment induces targetable cell surface presentation of peptides known to be downregulated by nonsense-mediated decay (NMD).

(A) MHC class I HLA presentation of mutant specific peptide sequences from NCI-H358 and (B) LS180 cells by quantitative HPLC–mass spectrometry treated with dimethyl sulfoxide (DMSO) or 5 µM KVS0001. The gene name, type of mutation (in parenthesis), and presented peptide are shown on the y-axis for each gene. Colors indicate different ions. (C) TP53 gene structure and mutant DNA sequence for NCI-H716 and NCI-H2228 cancer cell lines, both contain a homozygous splice site mutation in TP53. Capital letters represent exonic sequence; lowercase letters represent intronic sequence. DNA mutation reflected by gold bases. (D) Western blot against TP53 in the presence or absence of 5 µM NMD inhibitor in NCI-H716_A24 and NCI-H2228 cell lines. NCI-H2228 has an expected size of 46.6 kDa and NCI-H716 of 34.7 kDa. (E) Interferon (IFN-γ) levels over baseline based on enzyme-linked immunosorbent assay (ELISA) in a co-culture assay with NCI-H716_A24 and NCI-H2228 cells, 1.25 µM NMD inhibitor, human CD3+ T-cells, and bispecific antibody for TP53 and CD3. Chemotherapy (5-fluorouracil) is shown as a control. (F) Cell killing based on luciferase levels in a co-culture assay in NCI-H716 cells with and without A24 expression, treated with TP53-CD3 bispecific antibody, 1.25 µM NMD inhibitor and human CD3+ T-cells.

Figure 4—figure supplement 1.. DNA and protein sequences for the wild-type and mutant alleles of (A) EXOC1, (B) RAB14, and (C) ZDHHC16 genes.

The mutant protein sequences caused by the out-of-frame indel are highlighted in red and the boxes indicate the peptides presented on the cell surface and identified by mass spectrometry in cells treated with 5 µM of KVS0001 (see Figure 4A and B).

Figure 4—figure supplement 2.. Heavy peptide loading controls and endogenous (light) peptide presentation of genes in LS180 and NCI-H358 treated with dimethyl sulfoxide (DMSO) or 5 µM KVS0001.

Data are from quantitative HPLC–mass spectrometry. Note: y-axis scale changes between samples. Tables on the right show relative increase in peptide presentation with KVS0001.

Figure 4—figure supplement 3.. Waterfall plot of publicly available TP53 RNA expression (as shown by FPKM) for 675 cancer cell lines.

The two cell lines used in this study are highlighted in red and were in the bottom quartile of TP53 expression.

Figure 4—figure supplement 4.. Western blot of TP53 on NCI-H716 and NCI-H2228 cells treated with 5 or 7.5 µM of nonsense-mediated decay (NMD) inhibitor, 1 µM SMG1i-11, or 200 mg/ml chemotherapy, showing controls related to Figure 4D.

LYO is LY3023414 and 5-FU is 5-fluorouracil. HEK293 parent cells are shown as a control (wild-type TP53 protein).

Figure 5.. In vivo treatment of murine tumors with KVS0001 yield differential tumor growth compared with vehicle treatment.

(A) Fold change in RNA transcript levels in LLC or (B) RENCA cells treated in vitro with 5 µM of nonsense-mediated decay (NMD) inhibitor KVS0001 or dimethyl sulfoxide (DMSO). Orange bars indicate genes with homozygous indel mutations potentially targeted by NMD. Purple bars show genes with no mutations but that are known to have their normal transcription levels controlled by NMD. Green bar is a control gene that should not change with treatment. The dotted line shows relative expression of DMSO treatment (equal to 1). * indicates significantly different from untreated by Student’s T-test. (C) Treatment schedule for syngeneic tumor mouse experiments. (D) Average tumor size of LLC (left) and RENCA (right) syngeneic tumors in immune-competent mice (n = 8) treated with 30 mg/kg KVS0001 or vehicle control IP. Difference is statistically significant after day 10 based on one-way analysis of variance (ANOVA) with Dunnett’s test p < 0.001 (p < 0.05 for day 23 RENCA data point) for both tumors tested. (E) Average tumor size of LLC (left) and RENCA (right) in immunodeficient mice (n = 8) treated with 30 mg/kg KVS0001 or vehicle control. Error bars show 95% confidence intervals in all plots.

Figure 5—figure supplement 1.. Mouse weights for (A) C57BL/6N and (B) BALB/c tumor-bearing mice treated with 30 mg/kg KVS0001 or vehicle control intraperitoneal (IP) daily.

N = 8 for all arms.

Figure 5—figure supplement 2.. Mouse tumor size as measured by calipers for mammary fat pad placed syngeneic tumor models treated daily with 30 mg/kg KVS0001 or vehicle IP.

(A) Tumors with low/moderate and (B) high indel mutational loads are shown. No results presented in this figure supplement are statistically significant. Error bars show 95% confidence intervals.

Chemical structure 1.. Flowchart describing synthesis for KVS0001.