Supplementary Table S5 lists the wtheta and p-values for each gene in the TNG348 vs DMSO CRISPR screen in the UWB1.289 cell line from Figure 3C

Immune profiling in HT29 humanized mouse model

Supplementary Table S7 lists the wtheta and p-values for each gene in the olaparib vs DMSO CRISPR screen in the UWB1.289 cell line from Figure 3E

Homozygous deletion of the methylthioadenosine phosphorylase (MTAP) gene occurs in 10-15% of all human cancers and up to 50% of high-grade malignant gliomas, representing one of the largest opportunities for precision oncology. Loss of MTAP leads to the accumulation of 5'-methylthioadenosine (MTA), which sensitizes tumor cells to inhibition of protein arginine methyltransferase 5 (PRMT5). Herein we describe the discovery of TNG456, a potent and highly selective MTA-cooperative PRMT5 inhibitor that is brain penetrant in preclinical species and currently in Phase I/II clinical studies for the treatment of advanced or metastatic solid tumors with MTAP loss, with a focus on glioblastoma.

Supplementary Table S4 lists the area under the curve values for each cell line in the TNG348 single agent cell line panel shown in Figure 3A-B.

NanoString profiling of knockout lines

ABSTRACT PRMT5 is a type II arginine methyltransferase that forms an active complex with methylosome protein WDR77 (MEP50) to catalyze the symmetric dimethylation (SDMA) of arginine residues in proteins that regulate biological roles including apoptosis, DNA damage response and RNA processing. Some of the best characterized PRMT5 substrates are the small nuclear ribonucleoproteins SNRPB, SNRPD1 and SNRPD3, which are critical for spliceosome assembly and RNA splicing fidelity. MTAP-deleted cancers exhibit increased sensitivity to PRMT5 inhibition due to elevated levels of methylthioadenosine (MTA), a natural inhibitor of PRMT5. This vulnerability is exploited by MTA-cooperative PRMT5 inhibitors, exemplified by TNG908 and TNG462 which selectively target PRMT5 in MTAP-deleted cells while sparing MTAP-wildtype (WT) cells. Consistent with this mechanism, treatment with TNG908 in preclinical studies induces widespread splicing alterations in MTAP-deleted cancer models, with minimal effects in MTAP-WT cells. These splicing changes are consistent across diverse MTAP-deleted tumor types, including glioblastoma, pancreatic, and non-small cell lung cancer, indicating a histology-agnostic response to PRMT5 inhibition. Moreover, treatment of MTAP-WT cells with exogenous MTA mimics the splicing alterations observed with PRMT5 inhibition, as does pharmacologic inhibition of MTAP further supporting a mechanistic link between MTA accumulation, PRMT5 modulation, and aberrant splicing. Given that MTAP deletions occur in approximately 10–15% of human cancers, the identification of a robust RNA splicing signature offers a valuable pharmacodynamic biomarker for monitoring the activity of PRMT5 inhibitors. This splicing-based readout may also serve as a predictive biomarker of therapeutic response, offering greater specificity than global SDMA levels. Collectively these data suggest that a PRMT5-dependent RNA splicing signature can monitor the pharmacodynamic activity of MTA-cooperative PRMT5 inhibitors in MTAP-deleted cells.

TNG917 plasma pharmaco kinetic parameters

Supplementary Figure S1 shows additional biochemical and in vivo exposure data to support Figure 1

When tumor suppressor genes are lost through chromosomal deletion, the deletion of adjacent genes can generate therapeutic vulnerabilities. MTAP is frequently co-deleted with the chr9p21 tumor suppressor gene CDKN2A, creating a synthetic lethal dependency on protein arginine methyltransferase 5 (PRMT5). Telomeric to MTAP lies focadhesin (FOCAD), whose loss induces dependency on the HBS1-like translational GTPase (HBS1L)-protein pelota homolog (PELO) ribosome rescue complex for translational maintenance. FOCAD is deleted in ∼1 out of 3 MTAP-deleted cancers. We screened an immunomodulatory imide drug (IMiD)-focused diversity library and identified a weak hit that bound cereblon (CRBN), promoted HBS1L-CRBN-compound complex formation, and induced E3-ligase-dependent HBS1L ubiquitination and degradation. Guided by cryo-EM structures and proteome selectivity, we developed TNG961, a potent, selective HBS1L degrader that disrupts the HBS1L-PELO complex, inducing translational arrest, unfolded protein response activation, and growth inhibition in FOCAD-negative models. Oral administration of TNG961 regresses FOCAD-negative xenografts, including PRMT5 inhibitor-refractory models, establishing HBS1L degradation as a strategy to exploit FOCAD loss and supporting the clinical evaluation of TNG961 as a first-in-class precision oncology therapeutic. SIGNIFICANCE:FOCAD deletion, frequently co-occurring with MTAP/CDKN2A loss, creates a synthetic lethal dependency on the HBS1L-PELO ribosome rescue complex. TNG961, a first-in-class molecular glue degrader of HBS1L, enforces translational arrest and drives tumor regressions in FOCAD-negative models, including PRMT5 inhibitor-refractory tumors, establishing a novel precision oncology strategy for chromosome 9p21 co-deletion contexts.

Supplementary Table S12 lists the bliss synergy score values for each cell line in the TNG348 and saruparib combination cell line panel shown in Figure 4E.

Transcriptomic profiling in CT26 syngeneic mouse model

Supplementary Figure S5 shows additional data supporting Figure 5 including mouse body weight following TNG348, PARPi or combination treatment, additional in vivo experiments with lower TNG348 doses, an in vivo experiment in a BRCA1/2wt PDX model, and plasma concentration comparisons between TNG348 or PARPi alone versus their combination.

Immune profiling in HT29 humanized mouse model

Abstract Loss of tumor suppressor genes frequently results in co-deletion of neighboring genes, generating tumor-specific collateral vulnerabilities. On chromosome 9p21, deletion of CDKN2A/B commonly extends to loss of the adjacent MTAP gene creating a known dependency on PRMT5. Telomeric to MTAP lies FOCAD, which is co-deleted in 20-40% of MTAP-deleted cancers. Through unbiased combinatorial and genome-wide CRISPR screens, we discovered that loss of FOCAD, whose protein product stabilizes the SKI complex to maintain mRNA homeostasis, creates a pronounced dependency on the HBS1L/PELO ribosome rescue complex for translational integrity. To exploit this vulnerability, we developed TNG961, a first-in-class, orally bioavailable molecular glue degrader that selectively targets HBS1L. TNG961 induces formation of an HBS1L-TNG961-CRBN ternary complex, leading to potent degradation of HBS1L and secondary destabilization of its binding partner PELO. A 2.9 Å cryo-EM structure reveals the molecular glue binding mode underlying this tricomplex. A genome-wide drug anchor screen confirmed strict CRBN-dependence and SKI-complex-based sensitivity to TNG961, with proteomic profiling demonstrating exquisite selectivity for HBS1L. Functionally, TNG961 induces robust growth inhibition selectively in FOCAD-deficient models with ∼100-fold selectivity across 5 isogenic cell line pairs. This selective vulnerability is preserved in a larger parental cell line panel of > 90 lines (7-day CellTiter-Glo assay) and further supported by a multiplexed PRISM screen of > 900 models. Mechanistically, TNG961 induces translational arrest, activation of the unfolded protein response, and growth inhibition selectively in FOCAD-deficient models. In vivo, oral administration of TNG961 drives dose-dependent degradation of HBS1L and produces tumor stasis or regression across multiple FOCAD-deficient xenograft models and tumor histologies, including in the PRMT5 inhibitor-refractory setting. Because FOCAD is located on the antisense DNA strand relative to CDKN2A/B, we investigated the functional impact of partial truncations of FOCAD and translated these findings toward a prospective biomarker strategy for patient selection. IND-enabling studies are complete with a clean safety profile supporting a starting dose within the active range. These data establish targeted degradation of HBS1L as a novel therapeutic approach for FOCAD-deficient cancers and expand the landscape of actionable vulnerabilities arising from 9p21 tumor-suppressor loss. Citation Format: Douglas A. Whittington, Frank J. Bruzzese, Charlotte B. Pratt, Preksha Shahagadkar, Lauren Catherine M. Martires, Alice Tsai, John P. Maxwell, Katherine Lazarides, Matthew R. Tonini, Minjie Zhang, Samuel R. Meier, Colin Liang, Patrick McCarren, Yong Liu, John Zhang, Margaret A. Wyman, Yi Yu, Adam S. Crystal, Ed Wu, Jannik N. Andersen, Hilary Elaine Nicholson. TNG961: A selective oral molecular glue degrader of HBS1L for the treatment of FOCAD-deficient cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr ND03.

TNG917 potently induces CXCL10 secretion.

TNG917 is well tolerated in mouse studies.

Supplementary Table S3 shows information on Cryo-EM data collection, refinement and validation