Summary of PK data for EZH2 inhibitors used in KARPAS-422 and HT1376 xeograft mmouse models

Tulmimetostat increases PRC2-repressed gene expression in ARID1A mutant bladder cancer cells. (

Table with antibody source information including RRID if available

In cancer patients tulmimetostat plasma exposure levels correlate with PRC2 target gene expression changes in peripheral blood.

Summary of cell viability data for bladder cancer cells treated with tulmimetostat

Table with cell line information including source and RRID

Abstract Therapeutic inhibition of Anaplastic Lymphoma Kinase (ALK) has transformed the treatment of ALK fusion-positive non-small cell lung carcinoma (NSCLC), but ALK tyrosine kinase inhibitors (TKIs) suffer from liabilities common to all orthosteric inhibitors, such as off-target kinase inhibition and resistance alleles in and around the TKI binding site. Molecular glue degraders (MGDs) that engage the CRL4CRBN E3 ligase to promote neosubstrate degradation offer an alternative therapeutic approach. Here we describe TRI-611, a potent, brain-penetrant MGD that promotes the proximity of ALK and CRBN via a unique, non-G loop degron interface that is distal from the kinase active site. TRI-611 functions by engaging CRBN to create a neosurface that interacts with the C-lobe of the ALK kinase domain. This ALK:TRI-611:CRBN ternary complex promotes ALK polyubiquitination and CRBN-dependent degradation of ALK proteins, including ALK fusions such as EML4-ALK, and oncogenic, transmembrane ALK. Cellular degradation of EML4-ALK is rapid (occurring within 1-2 hours), robust (>90% maximum degradation), and durable (recovery half-life of more than 15 hours after compound withdrawal). Cellular proteomics profiling demonstrates the profound selectivity of TRI-611 across CRBN neosubstrates and the kinome (including key off-target kinase families such as NTRK), consistent with the unique degron interface located in a less conserved region of ALK. TRI-611 treatment leads to inhibition of downstream signaling and subsequent selective anti-proliferation of ALK-positive cell lines. TRI-611 is orally bioavailable and brain penetrant, with daily oral dosing leading to deep and durable degradation of endogenous EML4-ALK and tumor regression in both subcutaneous and intracranial xenograft models of ALK-positive NSCLC. TRI-611 represents the first example of a development stage degrader targeting an oncogenic gene fusion and has the potential to expand the arsenal of meaningful therapeutic options for ALK-positive NSCLC patients. The discovery of TRI-611 additionally broadens the reach of CRBN-modulating MGDs by exploiting a previously undescribed degron. Citation Format: Andrew R. Conery, Daniel S. La, Artyom A. Alekseyenko, David Marcoux, Aaron G. Bart, Matt L. Harlow, Patrick R. Arsenault, Nico R. Cantone, Rebecca L. Casaubon, Hari B. Kamadurai, Aravind Prasad Medikonda, Duncan E. Nunes, Tim J. Wigle, Maolin Yu, Aleksandra Zagulyaeva, Christine Zarate, Kathleen I. Seyb, Patrick Trojer, Vito J. Palombella, . TRI-611, a development stage molecular glue degrader of ALK for the treatment of ALK-positive NSCLC including central nervous system metastases [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 4618.

Abstract TRI-611 is a potent, brain-penetrant molecular glue degrader (MGD) of anaplastic lymphoma kinase (ALK) that represents the first development stage MGD of an oncogenic fusion protein. By engaging ALK via a novel degron that is distal from the orthosteric tyrosine kinase inhibitor (TKI) binding site, TRI-611 has the potential to promote the degradation of ALK proteins that are either wild-type or mutant in the kinase domain, the latter of which are an important mechanism of disease progression in patients treated with ALK TKIs. Here we show through in vitro biochemical and biophysical studies that TRI-611 is equally effective at promoting the interaction of CRBN with wild-type ALK and the ALKL1196M/G1202R compound mutant protein, as well as subsequent poly-ubiquitination. Through the use of Ba/F3 cells engineered to express 30 distinct alleles of EML4-ALK, we show that TRI-611 effectively targets all tested EML4-ALK mutant proteins in cells, including those that are refractory to ALK TKIs. The broad phenotypic activity of TRI-611 is further highlighted by potent anti-proliferation of cell line and patient-derived ALK+ NSCLC models with multiple ALK TKI resistance alleles. In mouse efficacy studies, TRI-611 leads to the regression of subcutaneous xenografts of CRISPR engineered EML4-ALK-mutant NCI-H3122 cells that do not respond to ALK TKIs. Finally, we show that TRI-611 leads to the regression of an EML4-ALK mutant primary patient-derived xenograft isolated from a patient that had progressed on prior alectinib and lorlatinib. These data demonstrate the potential of TRI-611 to address a key liability of ALK TKIs that all target the same site on ALK and support the development of TRI-611 in all ALK+ NSCLC patients, including patients with wild-type ALK kinase domain and those with acquired TKI resistance mutations. Citation Format: Andrew R. Conery, Daniel S. La, Artyom A. Alekseyenko, David Marcoux, Aaron G. Bart, Matt L. Harlow, Patrick R. Arsenault, Nico R. Cantone, Rebecca L. Casaubon, Hari B. Kamadurai, Aravind Prasad Medikonda, Duncan E. Nunes, Tim J. Wigle, Maolin Yu, Aleksandra Zagulyaeva, Christine Zarate, Lauren Highfield, Nobuyuki Kondo, Aaron N. Hata, Kenneth Ngo, Jisu Lee, Prafulla C. Gokhale, Kathleen I. Seyb, Patrick Trojer, Vito J. Palombella. TRI-611, a development stage molecular glue degrader of ALK, promotes the degradation of TKI-resistant ALK fusion proteins and leads to regression of ALK TKI-refractory tumors [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 5787.

List of 365 genes (used in main Fig. 7F) that are induced in patient’s whole blood upon treatment with tulmimetostat

Tulmimetostat treatment results in greater PRC2 target gene expression induction in phenotypically sensitive ARID1A LOF cell lines

List of gene names corresponding to 132 tulmimetostat upregulated genes in responder versus non responder cell lines

Abstract Cyclin E1 (CCNE1) is a central regulator of the G1 to S-phase transition of the cell cycle and a key dependency in multiple solid tumor types, including ovarian and endometrial cancers, and promotes resistance to CDK4/6 inhibitors in HR+/HER2- breast cancer. CCNE1 lacks a druggable pocket or enzymatic activity, which has precluded attempts to develop drugs that inhibit its function. Efforts to inhibit CCNE1 activity indirectly with CDK2 kinase inhibitors have been hindered by poor selectivity and dose-limiting toxicities that prevent full pathway suppression. Here we report the discovery of a molecular glue degrader (MGD) that co-opts the CRL4CRBN E3 ubiquitin ligase to eliminate the CCNE1 protein. Single-particle cryo-electron microscopy identifies a novel non-G-loop degron on CCNE1 that drives direct recognition of CCNE1 by the MGD:CRBN:DDB1 complex without preventing CDK2 binding to CCNE1. In cell lines analyzed, MGD treatment triggers rapid, CRBN-dependent depletion of CCNE1 with complete selectivity over the rest of the proteome. MGD-induced CCNE1 degradation exerts potent and selective anti-proliferative effects, with over 500-fold greater potency in CCNE1 amplified lines compared to non-amplified lines, a substantial improvement relative to CDK2 inhibitors (∼15-fold potency window). In HR+/HER2- breast cancer cell lines with evolved resistance to the combination of a CDK4/6 inhibitor and fulvestrant, co-treatment with the MGD restores sensitivity. Oral administration of the MGD drives robust CCNE1 reduction and antitumor activity in a CCNE1-amplified xenograft model. These data suggest that directly and selectively degrading CCNE1 could provide an effective targeted therapy for CCNE1-amplified solid tumors and overcome resistance to CDK4/6 inhibitors in HR+/HER2- breast cancer. Citation Format: Benjamin M. Vincent, David Marcoux, Claudia Caligioni, Aaron G. Bart, Matt L. Harlow, Rosaline Y. Hsu, Prashant Singh, Rachel Badger, Andrew J. Ingersoll, Thomas B. Jordan, Hari B. Kamadurai, Christine Zarate, Nico R. Cantone, Aravind Prasad Medikonda, Artyom A. Alekseyenko, Duncan E. Nunes, Taras Dauzhenka, Anushka Bhagwat, David Y. Rhee, Patrick R. Arsenault, Andrew R. Conery, Alex Constan, Louis Plamondon, Tim J. Wigle, Daniel S. La, Kathleen I. Seyb, Patrick Trojer, Vito J. Palombella, . Discovery of a selective molecular glue degrader of CCNE1 for the treatment of CCNE1-amplified solid tumors and CDK4/6i-resistant HR+/HER2- breast 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 5789.

Tulmimetostat mediated cancer cell phenotypic responses are increased in ARID1A LOF contexts.

Tyrosine kinase inhibitors (TKIs) targeting anaplastic lymphoma kinase (ALK) have transformed the treatment landscape of ALK fusion-positive non-small-cell lung carcinoma (ALK-positive NSCLC), but the limited options for patients who progress on approved TKIs highlight a continued need for an orthogonal therapeutic approach1,2. TRI-611 is a potent, brain-penetrant molecular glue degrader of ALK fusion proteins with the potential to address this need. TRI-611 promotes the proximity of the ALK kinase domain and CRL4 substrate adaptor CRBN through a unique degron interface distal from the kinase active site. The unique binding interface of TRI-611 leads to selectivity across the proteome including known CRBN neosubstrates and other kinases. TRI-611 treatment induces degradation of all forms of ALK fusion proteins, including wild-type and ALK TKI-resistant versions, leading to regression of cell line and patient-derived subcutaneous and intracranial tumour models of ALK-positive NSCLC. TRI-611 can be combined with orthosteric ALK TKIs, achieving synergistic and durable tumour regressions. TRI-611 represents to our knowledge the first clinical-stage molecular glue degrader targeting an oncogenic gene fusion and has the potential to expand the arsenal of therapeutic options for patients with ALK-positive NSCLC . TRI-611 induces the degradation of ALK fusion proteins via a previously undescribed CRBN recruitment motif, and its preclinical anti-tumour activity highlights TRI-611 as a potential new way of treating ALK-positive non-small-cell lung cancer.

Synthetic pathway and characterization of tulmimetostat, a long residence time EZH2 inhibitor.

Tulmimetostat demonstrates superior level of tumor PRC2 target gene inducon and correlation with efficacy in a bladder cancer xenograft mouse model.

Tulmimetostat improves cisplatin responsiveness in chemotherapy-resistant bladder cancer cells.

Table listing experimentally determined compound binding affinities for EZH2 and EZH1 containing PRC2 protein complexes

Additional method information to describe technical aspects of the experiments described in this manuscript