CD73 (ecto-5'-nucleotidase) drives immunosuppressive and tumor progression through both enzymatic adenosine production and nonenzymatic mechanisms, limiting the efficacy of current CD73-targeted therapies, including small-molecule enzymatic inhibitors and antibodies. Here, we reported NUCC-0227579 (C79), a first-in-class proteolysis-targeting chimera (PROTAC) that degraded CD73 via the VHL E3 ligase-dependent proteasomal and lysosomal pathways. C79 eliminated CD73 at the cell surface and intracellular compartments across multiple human cancer cell lines, abolishing nucleotidase activity and more effectively reversing adenosine-mediated immunosuppression than enzymatic inhibitors. C79 enhanced NF-κB/NFAT signaling, increased IFN-γ and TNF-α production, and promoted human CD8+ T cell activation and proliferation. In addition, C79 impaired tumor cell metabolic fitness, proliferation, migration, and adhesion through non-nucleotidase-dependent mechanisms. In humanized NSG mouse models of triple-negative breast cancer, C79 significantly suppressed tumor growth while enhancing antitumor immune responses, highlighting CD73 degradation as a mechanistically distinct strategy to overcome the limitations of existing CD73-targeted therapies.

Median Fluorescence Intensity (MFI) values for pAKTS473 flow cytometry screening assay.

The essential role of mTOR in promoting tumorigenesis of many cancers makes it an attractive therapeutic target. However, catalytic mTOR inhibitors, which block both mTORC1 and mTORC2, result in activation of negative feedback loops as a resistance mechanism. Selective mTORC2 inhibitors are expected to have the desired antitumor effects without engaging resistance mechanisms; however, to date, no such mTORC2 inhibitors have been developed. Using in silico screening and medicinal chemistry optimization, we identified several small molecules that bind to the unique mTORC2 component, stress-activated protein kinase-interacting protein 1 (SIN1). We demonstrate that this SIN1 inhibitor alters posttranslational modification, protein-protein interactions, and blocks mTORC2- and rapamycin-sensitive mTORC1-mediated signaling. The SIN1 inhibitor also inhibits wild-type RAS activation and downstream MAPK signaling, as well as cell proliferation of multiple cancer cell line types. SIN1 inhibition can enhance the efficacy of FDA-approved antineoplastic agents in vitro and may provide a novel approach for the treatment of different types of malignancies.

Docked pose of hit compound NUCC-175662 bound to SIN1

Background/Objectives: Glioblastoma (GBM) remains one of the most aggressive and treatment-resistant malignancies, driven in part by heterogeneous, therapy-resistant glioma stem cells (GSCs). Improving clinical outcomes will require innovative therapeutic approaches that target unique molecular vulnerabilities. The mitogen-activated protein kinase (MAPK) pathway drives tumor progression across multiple cancers, including GBM. MAPK-interacting kinases (MNK1/2) represent MAPK downstream effectors that phosphorylate eukaryotic translation initiation factor 4E (eIF4E), a regulator of oncogenic and anti-apoptotic mRNA translation. We previously identified pharmacological MNK inhibition as a promising therapeutic strategy for GBM, but most available MNK inhibitors lack specificity. Methods: Novel MNK inhibitor compounds were developed using medicinal chemistry optimization and evaluated through molecular docking and kinome profiling analyses. Antineoplastic activity was assessed in established GBM cell lines and patient-derived glioma stem cell models cultured as 3-D neurospheres under stem cell-permissive conditions. Effects on MNK signaling, cell viability, neurosphere growth, migration, invasion, and apoptosis were analyzed using immunoblotting, flow cytometry, viability assays, wound healing assays, and 3-D invasion assays. In addition, a compound screen was performed to identify therapeutic agents that enhance MNK-targeted therapy, followed by validation using pharmacological inhibition and siRNA-mediated knockdown approaches. Results: Our next-generation MNK inhibitor NUCC-201893 exhibited high target specificity and greater potency than the lead compound NU808, effectively suppressing eIF4E phosphorylation, GBM cell viability, neurosphere growth, migration, and invasion. Compound screening identified DNA methyltransferase (DNMT) inhibition as a potent enhancer of MNK blockade. Pharmacological DNMT inhibition enhanced the cytotoxic effects of siRNA-mediated MNK1 knockdown, while concurrent pharmacological inhibition of MNKs and DNMT resulted in greater suppression of neurosphere growth and robust induction of apoptotic responses in GSCs. Conclusions: These findings identify dual MNK and DNMT inhibition as a promising combinatorial strategy that effectively triggers antineoplastic effects in GBM cells and GSCs.

Enhancer of Zeste Homolog 2 (EZH2) is the enzymatic subunit of the Polycomb Repressive Complex 2 (PRC2). It catalyzes H3K27 methylation for epigenetic silencing of tumor suppressors and critically drives prostate cancer (PCa) progression. However, inhibitors of EZH2 catalytic function (EZH2i), such as EPZ-6438, showed limited efficacy in PCa. Here, we designed and developed a series of VHL-based proteolysis-targeting chimera (PROTAC) degraders of EZH2 using EPZ-6438 as a ligand and identified PROTAC-6272 as a lead compound. PROTAC-6272 effectively degraded EZH2 and other PRC2 subunits across diverse PCa cell lines. However, PROTAC-6272 and other similar EZH2i-based PROTACs were consistently unable to decrease androgen receptor (AR), a gene that is directly activated by solo EZH2. Mechanistically, EZH2 PROTACs failed to degrade EZH2 coactivators, such as p300, due to their inability to engage EZH2 outside of the PRC2 complex. Nevertheless, PROTAC-6272 exhibited anti-proliferative activities superior to EPZ-6438 in some PCa models, wherein it induced p21 expression and cellular senescence by disrupting a methylation-independent PRC2 function. In summary, while EZH2i-based PROTACs failed to target the PRC2-independent functions of EZH2, they confer added benefits over EPZ-6438 by abolishing a polycomb-dependent but methylation-independent function of EZH2, offering therapeutic advantages in some PCa.

KMT2A-rearranged (MLL-r) leukemias are highly aggressive hematological malignancies that require improved targeted therapies. DOT1L (histone H3K79 methyltransferase) functions as a critical oncogenic driver and represents an important therapeutic target in these high-risk leukemias. However, clinical responses to the first-in-class DOT1L inhibitor pinometostat (EPZ5676) have been modest, attributed to suboptimal pharmacokinetics and, more fundamentally, to the recognition that DOT1L possesses methyltransferase-independent functions that evade catalytic inhibition. This highlights the need for strategies that abrogate the full spectrum of DOT1L activity to effectively treat these high-risk leukemias. Proteolysis-targeting chimeras (PROTACs), which induce selective degradation of the DOT1L protein rather than inhibiting its catalytic activity, have therefore emerged as a promising approach. Notably, VHL-recruiting DOT1L PROTACs, such as DOT1L808, have demonstrated improved pharmacokinetic profiles and potent antileukemic activity in preclinical in vivo models. However, these findings remain preclinical, and significant challenges including oral bioavailability, potential toxicity, and lack of clinical validation must be addressed before clinical translation. In this review, we provide an overview of the evolving understanding of the biology of DOT1L, discuss existing MLL small molecule therapies, and evaluate current advances in therapeutically targeting DOT1L, with particular focus on the targeted degradation of DOT1L as a promising therapeutic strategy for high-risk KMT2A-r leukemia.

MYC is an important, yet challenging target in oncology as it lacks traditional "druggable" pockets. Here we show that two regions of the MYC protein, the basic-Helix-Loop-Helix (bHLH) domain, and an extended MYC Box II (eMBII) come together to form a bivalent, high-affinity small-molecule MYC inhibitor (MYCi) binding site. CRISPR-tiling mutagenesis identified mutations in the vicinity of emBII and in bHLH regions that together confer MYCi resistance. Importantly, acetylation of lysine K148 in eMBII which is essential for MYC oncogenicity in vivo increased the predicted order of this region and enhanced MYCi binding affinity. Furthermore, MYCi selectively modulated the expression of the same genes regulated by lysine-acetylated MYC in cancer cells. These studies provide a rationale for the selective targeting of acetylated, oncogenic MYC with small molecules.

gene expression correlation with NCI-60 panel drug responses

NUCC-201760 inhibits other small RAS GTPase family members in addition to RAS

DOT1L is aberrantly recruited in MLL-r leukemias and serves as a critical oncogenic driver. Substantial previous work has developed catalytic inhibitors like pinometostat, which showed limited clinical efficacy. Recent work has defined nonenzymatic functions of DOT1L in promoting leukemia progression. Because these functions cannot be blocked by enzymatic inhibitors, we developed novel DOT1L-targeting PROTACs (Proteolysis-Targeting Chimeras ) to degrade the protein and thereby block all of its functions. We describe PROTACs DOT1L705 and DOT1L808 as potent and highly selective DOT1L degraders with DC50 values of 0.33 μM and 5 nM, respectively. The effect of DOT1L705 on cell viability is highly dependent on the MLL-r status of leukemia cell lines, and it retains activity against menin inhibitor-resistant cells. In vivo studies with DOT1L808 showed its ability to achieve complete tumor regression in an orthotopic leukemia model without overt toxicity. These results establish protein degradation as a promising therapeutic strategy for MLL-rearranged leukemias.

Eukaryotic translation initiation factor 4E (eIF4E) plays an essential role in driving the translation of mRNAs that promote cell proliferation. Its activity is regulated through phosphorylation at Ser209 by MNK1/2 kinases, which are downstream effectors of mitogen-activated protein kinase signaling. Elevated eIF4E phosphorylation drives malignant progression in multiple cancers, including glioblastoma, where it promotes translation of key oncoproteins responsible for tumor growth and therapy resistance. Here, we report the development and application of a direct-to-biology (D2B) platform to rapidly optimize new MNK inhibitors for cellular inhibition. This approach combines plate-based library synthesis with an in-cell Western assay to efficiently generate structure-activity relationship (SAR) data and new compounds with potent cellular inhibition. Using this platform, we synthesized and tested >150 new analogs directly in cells, identifying inhibitors with IC50 values as low as 23 nM in LN229 cells. This work establishes a D2B approach as an effective strategy for efficient kinase inhibitor optimization.

Ovarian cancer (OC) is the 8th most common cancer in women. Most patients with high-grade serous OC present with intraperitoneal disseminated disease when diagnosed, due to mild symptoms in earlier disease stages. They respond efficiently to cisplatin or taxol-based compounds in the front-line treatment; however, resistance emerges in the majority of cases, which allows tumors to progress. There is an urgent need for improved treatment to increase patients’ survival. Tissue transglutaminase (TG2) is a protein overexpressed in many solid tumors, including ovarian; it interacts with fibronectin (FN) in the extracellular matrix and promotes OC intraperitoneal dissemination. This makes TG2 a promising target for treatment. We previously identified MT4 as a promising small molecule inhibitor (SMI) able to disrupt TG2-FN protein-protein interaction, prevent cancer cell adhesion and spheroid formation, and sensitize OC cells to chemotherapeutic drugs, such as paclitaxel (Sima LE et al., Mol Cancer Ther., 2019). We recently investigated the effect of 5 new MT4 analogues on OC cells behavior. We found that compound #3011 completely prevented spheroids formation, similar to Tgm2 gene excision, while #3002 decreased cell adhesion onto FN. We used phosphoproteomics to analyze the adaptive signaling of OC cells treated with MT4 that escape treatment. New promising molecular TG2 co-targets for OC treatment have been identified as effectors of MT4 escape pathways: sirtuins and p21-activated kinases (PAKs). Combination of signaling inhibitors and TG2-directed SMIs induce OC cell death in both 2D and 3D conditions, with potential for future therapeutic use. Our multiplex IHC data shows that in OC tumors TG2 is expressed by cancer cells, as well as by α-SMA+ CAF subset. Therefore, we next sought to test the effect of TG2 drug targeting on OC cells-fibroblasts 3D co-cultures. Our results showed that MT4 prevents heterospheroid self-assembly in vitro, which indicates potential disruption in tumor-stroma communication using TG2-directed therapies. In parallel, we developed new tools to test drug combinations, as well as radiation therapy regimens for their potential to inhibit cancer spreading. We produced a new glass microfluidic chip and adapted a miniaturized mesothelial clearance assay for live cell imaging. We obtained new SKOV3 and SKOV3-TG2KO LifeAct+ stable cell lines to allow real time imaging measurements of OC cell behavior. Subpopulations were sorted based on actin-GFP expression level and characterized by cell motility quantification. Cell lines with behavior similar with parental cells were selected for further use. We currently use these tools to investigate the potential of TG2 targeting to enhance response of cancer cells to new FLASH radiation sources (Orobeti S et al, Sci. Rep., 2024), such as very high energy electrons generated with a high-intensity laser plasma accelerator system. Monica Tudor, Cristian Munteanu, Gabriela Chiritoiu, Stefana Orobeti, Alexandra Bran, Florin Jipa, Felix Sima, Gary E. Schiltz, Daniela Matei, Livia Elena Sima. Small molecule inhibitors targeting TG2 in ovarian cancer and identification of drug escape pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3867.

The cytoplasmic human Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3 or A3) cytidine deaminases G and F (A3G and A3F) can block the spread of human immunodeficiency virus (HIV). HIV counteracts this cell-intrinsic defense through a viral protein called viral infectivity factor (Vif). Vif causes proteasomal degradation of A3G and A3F proteins (A3G/F) in HIV-producing cells to ensure infectivity of virions subsequently released from these cells. Here, we optimized a lead compound reported previously to boost cellular levels of A3G. The modified analogs designed, synthesized, and evaluated here inhibit cell-mediated post-translational degradation of A3G/F, whether Vif is present or not. This increases A3G/F incorporation into Vif-positive virions to decrease viral infectivity. The compounds and processes described here can facilitate the development of new anti-HIV therapeutics whose host-targeted effect may not be evaded by resistance-conferring mutations in HIV Vif.

Indoleamine 2,3-dioxygenase 1 (IDO1) is an immunosuppressive enzyme expressed in >90% of patient-resected glioblastoma (GBM). IDO1 expression is inversely correlated with GBM patient survival. Extracellular IDO1 contributes to IDO1-mediated immunosuppression. We developed a novel protein degrader/PROTAC (NU227326) with a 4.7 nM DC50 for human IDO1 and compared its effects on extracellular IDO1 with a potent IDO1 enzyme inhibitor (BMS-986205; IC50=1.7nM) in cultured human GBM cells and in mice with human GBM. U87 or GBM43 were treated with NU227326, BMS-986205, ±human IFNg in vitro. Native PAGE, Western blotting, ELISA, and mass spectrometry evaluated IDO1 in the whole cell lysate, microvesicles, exosomes, and the vesicle-depleted extracellular fraction (VDF). NSG mice with intracranial GBM were intraperitoneally treated with IDO1 PROTAC and evaluated for intratumoral IDO1 protein levels. BMS-986205 treatment led to multiple novel IDO1 bands in the VDF under Native PAGE conditions as compared to untreated- or NU227326 treated-GBM cells. BMS-986205 treatment dramatically increased extracellular IDO1 protein levels as compared to untreated- or NU227326 treated-GBM cells. Mass spec. identified multiple differences between BMS-986205- and NU227326-treated GBM cells. Systemic IDO1 PROTAC treatment led to a significant decrease of intra-GBM IDO1 protein in vivo. Pharmacological IDO1 enzyme inhibitor treatment increases both intracellular and extracellular IDO1 protein levels. A novel IDO1 protein degrader decreases both intracellular and extracellular IDO1 protein levels. Ongoing studies aim to understand IDO1 PROTAC effects on the anti-GBM immune response in humanized mice with intracranial human GBM PDX and with a human immune system.

Tissue transglutaminase (TG2), a multifunctional enzyme involved in protein crosslinking through transamidation, fibronectin-integrin interactions and GTP hydrolysis, is upregulated in cancer. Due to its diverse functions, TG2 has been a challenging therapeutic target. Here, we investigate the use of PROteolysis TArgeting Chimeras (PROTACs) to degrade TG2 and inhibit its tumor-promoting functions in ovarian cancer models. We describe a novel family of VHL based PROTACs using a ligand that binds to the TG2 fibronectin interacting domain and a thiol ether PEG linker. Three structurally related PROTACs-P374, P404, and P405-induced significant proteasome dependent TG2 degradation at 24 h (p < 0.05), with stable effects at 48 h. These compounds also potently inhibited cell adhesion and migration (p < 0.005), outside-in signaling, and blocked TG2 enzymatic activity (p < 0.001). An unbiased evaluation using reverse phase protein array of P374-treated cells revealed 136 differentially expressed proteins, including protein networks related to cell adhesion and involved in extracellular matrix (ECM) interactions. P374 and P405 reduced omental colonization in vivo and P374 inhibited intraperitoneal tumor dissemination and growth. Visium HD based spatial profiling of human ovarian tumors identified TG2 as a highly enriched protein at the tumors invasive edge and the interface with the ECM. Together, our findings put forward novel TG2-targeting PROTACs which effectively degrade TG2, impair its functions, and block in-vivo tumor dissemination. These results highlight the potential development of TG2 degraders towards therapeutic targeting in ovarian cancer.