LY3023414 is a small molecule capable of increasing transcription of NMD targets.

A) Schematic of HTS used to identify inhibitors of NMD. Mutant transcripts are represented by a smaller length in the cartoon for illustrative purposes only. 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 DMSO control). Full screen results are presented in Figure S4. C) Targeted RNA sequencing results of isogenic RPTec knockout clones treated with the 8 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, is presented only in Figure S8. D) TP53 western blot on RPE TP53 224, containing a homozygous TP53 mutation, using the four hit compounds that validated in RPTec isogenic lines. E) Western blot analysis of full-length TP53α and isoform TP53β after treatment with two NMD inhibitor lead candidates. 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) qPCR showing 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 5 µM of test compound for 16 hours.

Inhibiting NMD in cancer cells increases broad expression of truncated gene mRNA and protein.

A) Mutant transcript recovery rates for genes containing heterozygous indel mutations based on RNA-sequencing results in cell lines treated with 5µM LY3023414 for 16 hours. Strict inclusion criteria were used, such that only mutations with sufficient sequencing coverage are shown (see methods).Recovery is defined as at least 2-fold increase over DMSO treatment. B) Targeted high coverage RNA- sequencing confirms recovery of mutant transcript levels in NCI-H358 and LS180 cancer cell lines treated with LY3023414. RNF43 and DROSHA contain common heterozygous 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 hours post-treatment. Student’s t-test for target genes are all p < 0.05, while the null hypothesis holds for RNF43 (common SNP).

Novel 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 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 5µM novel inhibitor KVS0001, LY3023414 or SMG1i-11 for 24 hours. F) Western blot of phosphorylated UPF1 on three cell lines treated with KVS0001, SMG1i-11, or 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 IP injection of vehicle or 30mg/kg KVS0001 and tumors harvested 16hrs post IP treatment. All genes shown contain heterozygous out-of-frame truncating mutations except RNF43 and DROSHA which serve as controls (contain heterozygous SNP’s).

KVS0001 treatment induces targetable cell surface presentation of peptides known to be downregulated by NMD.

A) MHC class I HLA presentation of mutant specific peptide sequences from NCI-H358 and B) LS180 cells by quantitative HPLC-Mass Spectrometry. 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 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) IFN-γ levels over baseline based on ELISA in a co-culture assay with NCI-H716_A24 and NCI-H2228 cells, 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, NMD inhibitor and human CD3+ T-cells.

I n 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 5µM of NMD inhibitor KVS0001 or 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). 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. Difference is statistically significant after day 10 based on one-way 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.