Abstract Vaccinia-related kinases (VRKs) are a family of serine/threonine kinases involved in a variety of cellular processes, including cell signaling, chromatin modification, nuclear envelope dynamics, and cell cycle progression. More than 60% of glioblastomas and nearly all neuroblastomas have low expression of the VRK2 gene resulting in deficient VRK2 activity. VRK1 has been identified as a paralog synthetic lethal target in these VRK2-deficient cancers, with the potential for indication expansion into additional cancer types. Here, we show that inhibition of VRK1 leads to a concomitant loss of Barrier to Autointegration Nuclear Assembly Factor 1 (BANF1 or BAF) phosphorylation leading to aberrant nuclear envelope formation and downstream loss of cellular viability. As a target, VRK1 is both tractable and structurally-enabled, with chemical series capable of achieving >4000-fold biochemical selectivity against its paralog VRK2 and >70-fold viability selectivity in VRK2 isogenic cell line pairs. Compounds show strong correlations between biochemical, cellular target engagement, pharmacodynamic, and functional viability assays. Furthermore, in vivo tolerability studies suggest that VRK1 inhibitors are well-tolerated in immunocompromised mice. Preclinical validation studies support the development of VRK1 inhibitors for the treatment of patients with VRK2-deficient tumors such as glioblastoma and neuroblastoma. Citation Format: Kiera M. Vassallo,Kevin M. Cottrell,Katarzyna B. Handing,Mu-Sen Liu,Alvin Lu,Alice Tsai,Maria Dam Ferdinez,Patrick McCarren,Sirimas Sudsakorn,Jannik N. Andersen,Kimberly J. Briggs. VRK1 inhibition leverages paralog synthetic lethality to selectively target VRK2-deficient cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3090.

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.

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.

Perturbation of LIG1 induces proportional PAR accumulation in BRCA1-mutated cells in immunofluorescence and in-cell western assay formats. A) In-cell Western blotting of PAR on treatment day 4 in MDA-MB-436 cells engineered to express a DOX-inducible LIG1 cDNA with endogenous LIG1 knockout. B) Model validation with Western blotting. C) Quantification of relative NAD+ as deduced by total NAD (NADt)/NADH ratio normalized to +DOX condition (t-test sgLIG1 +DOX vs. -DOX p < 0.0001).

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.

CRISPRi system does not reduce viability of BRCA mutant cells using an intron-targeting control guide RNA (sgITC). Representative image (A) and quantification (B) of colony formation assay (treatment day 14) in MDA-MB-436 (BRCA1 mutant) cells engineered to express a DOX-inducible CRISPRi guide RNA targeting an intron (sgITC). Knockdown was validated by Western blot (representative image in C, quantification in D). FC, fold-change.

Abstract Vaccinia-related kinases (VRKs) are a family of serine/threonine kinases that regulate diverse cellular processes, including transcription factor activity, chromatin remodeling, nuclear envelope formation, and cell-cycle progression. Among them, VRK1 has emerged as a paralog-selective synthetic lethal target in cancers with low VRK2 expression, encompassing nearly all neuroblastomas and over 60% of glioblastomas, with potential for therapeutic expansion into additional tumor types. Here, we describe the biochemical, biophysical, and cellular assays that enabled the discovery and optimization of novel inhibitors of VRK1 activity found through variety of binding screens, activity-based screens, and rational design. High-resolution crystal structures of both VRK1 and VRK2 in complex with inhibitors revealed their binding poses, identified key interactions, and confirmed orthosteric binding. Medicinal chemistry optimization yielded chemical series capable of achieving >4000X selectivity over the VRK1 paralog VRK2 in a biochemical assay conducted with a physiologically relevant ATP concentration, and >70X selectivity for VRK2-deficient cells in cellular viability assays. Notably, strong correlations between biochemical potency, cellular target engagement, pharmacodynamic response, and functional viability supported both the specificity of the chemical matter and the robustness of the assay platform. Together, these findings establish VRK1 as a tractable and structurally enabled target and demonstrate that high paralog selectivity in cells can be achieved through orthosteric inhibition. This work provides the strong foundation necessary for structure-guided optimization of VRK1 inhibitors with potential therapeutic applications in cancers with low VRK2 expression, such as glioblastoma and neuroblastoma. Citation Format: Katarzyna B. Handing, Kevin M. Cottrell, Mu-Sen Liu, Patrick McCarren, Brett Williams, Kiera M. Vassallo, Alvin Lu, Alice Tsai, Maria Dam Ferdinez, Sirimas Sudsakorn, Brian McMillan, Jannik N. Anderson, William D. Mallender, Wenhai Zhang, John Maxwell, Kimberly J. Briggs. Hit finding and assay enablement for VRK1, a paralog synthetic lethal kinase target in VRK2-deficient cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 484.

LIG1 inactivation is lethal in BRCA1-mutant MDA-MB-436 cells, and is rescued by exogenous expression of wild-type BRCA1. A) Representative colony formation assay (treatment day 14) using DOX-inducible shRNA against LIG1, PLK1 (pan-lethal control), or a control sequence in MDA-MB-436 cells stably infected with cDNA encoding wild-type BRCA1 or a control vector. B) Model validation with Western blotting. C) Western blots assessing LIG1 protein level at assay endpoint.

Withdrawal of Doxycycline leads to LIG1 depletion that is sustained through the study. Image (A) and quantification (B) of western blotting data from tumors of immunodeficient mice grafted with MDA-MB-436 cells engineered to express a DOX-inducible CRISPR-resistant LIG1 cDNA with endogenous LIG1 knockout. Samples depicted are from endpoint (day 29 post-grouping).

Quantification of western blots for dTAG-mediated LIG1 degradation. MDA-MB-436 cells were engineered to express a CRISPR-resistant LIG1 cDNA fused to either the FKBP12mut (dTAG) degron (degradable) (A) or the FKBP12wt degron (non-degradable) (B), with knockout of endogenous LIG1. Cells were then treated for 4 days with vehicle (DMSO) or the indicated doses of the VHL-targeting PROTAC dTAGv1 and then subject to immunoblotting for LIG1, HA tag, and a loading control. FC, fold-change.

LIG1 inactivation is lethal in HRD+ BRCA1/2 wild-type HCC1806 cells. A) Representative colony formation assay (treatment day 11) using DOX-inducible shRNA against LIG1, PLK1 (pan-lethal control), or a control sequence in HCC1806 cells. B) Western blots assessing LIG1 protein level in the models from (A) (treatment day 4).

Cell lines with damaging mutations in BRCA2 are dependent on LIG1. A) Analysis of DepMap data (28) comparing LIG1 dependence of cell lines with damaging mutations in BRCA2 vs. BRCA2 wild-type cell lines. ***p=0.0004 unpaired t-test. B,C) Representative image (B) and quantification (C) of colony formation assay in DLD1 wild-type (WT) and BRCA2-/- cells transfected with siRNAs targeting the LIG1 gene or a control sequence. D,E) Representative image (D) and quantification (E) of Western blot assessing LIG1 knockdown at endpoint for the colony formation assay in (B).

TNG260 increases antigen presentation in STK11-deficient cells

Antibodies and dilutions for western blotting

In-vivo immune sensitizer screen in STK11-isogenic MC38 tumors

TNG260 treatment does not affect proportion of intratumoral Treg cells

Chromosomal deletion of tumor suppressor genes can lead to collateral loss of nearby genes, creating therapeutic opportunities for synthetic lethal targeting. One example is the frequent co-deletion of MTAP with CDKN2A/B on chromosome 9p, which creates a dependency on PRMT5 for cell survival. Located telomeric to CDKN2A and MTAP is FOCAD, a gene encoding a protein essential for stabilizing the SKI complex. The SKI complex is responsible for degrading aberrant mRNAs, thereby maintaining mRNA homeostasis. Loss of FOCAD disrupts SKI complex function, resulting in ribosome stalling on aberrant mRNA transcripts. These stalled ribosomes are dependent on the HBS1L/PELO complex for rescue and recycling, establishing a synthetic lethal interaction between HBS1L/PELO and FOCAD loss. TNG961 is a first-in-class molecular glue that targets HBS1L for degradation, representing a novel therapeutic strategy for FOCAD-deleted tumors. In vitro, TNG961 treatment leads to a dose-dependent decrease in HBS1L protein levels, which in turn destabilizes the portion of PELO involved in ribosome rescue. TNG961 displays proteome-wide selectivity for HBS1L, with no change in SALL4 protein level as determined by western blot. TNG961-induced HBS1L degradation and PELO destabilization can both be rescued by inactivation of CRBN confirming mechanism, which is further supported by cryoEM data. In engineered HiBiT knock-in cells, treatment with TNG961 induces maximal HBS1L degradation within 4 hours, without affecting GSPT1 protein levels. This selective degradation translates to potent growth inhibition in FOCAD-deleted cells, with ∼100-fold selectivity compared to FOCAD-proficient cells across 5 isogenic pairs. This selective vulnerability is preserved in a large cell line panel of over 90 models. Finally, oral administration of TNG961 induces dose-dependent degradation of HBS1L and subsequent destabilization of PELO protein in FOCAD-deleted xenograft models, resulting in tumor stasis or regression across multiple lineages. Notably, TNG961 remains effective in a pancreatic model that has become refractory to PRMT5 inhibitor treatment, supporting its potential utility in PRMT5i-resistant settings. TNG961 has been nominated as a development candidate and is currently in IND-enabling studies. Katherine Lazarides, Minjie Zhang, Frank J. Bruzzese, Douglas A. Whittington, Charlotte B. Pratt, Lauren Catherine M. Martires, John P. Maxwell, Alice Tsai, Colin Liang, Patrick McCarren, Yong Liu, John Zhang, Matthew R. Tonini, Margaret A. Wyman, Xinyuan Wu, Jannik N. Andersen, Hilary E. Nicholson. TNG961: A selective oral molecular glue degrader of HBS1L inducing tumor regression in naïve and PRMT5i-refractory FOCAD-deleted models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr B027.