Heterobifunctional Molecules Research Articles

Abstract 1144: Orally bioavailable SMARCA2 degraders with exceptional selectivity and potency

Abstract Background: SMARCA2 (BRM) and SMARCA4 (BRG1) are two mutually exclusive DNA-dependent ATPases of the SWI/SNF complex, which function in mobilizing nucleosomes to regulate transcription, DNA replication/repair and chromosome dynamics. SMARCA4 is known to be mutated in number of cancers lacking targetable oncogenes, with SMARCA4-mutant patient population representing 10%-20% of NSCLC, 100% small cell ovarian cancer (hypercalcemic type), 28% skin cancer, 16% glioma and 14% colon cancer. Genetic studies have established the necessity of SMARCA2 for survival of tumor cells lacking SMARCA4. Although genetic silencing of SMARCA2 leads to potent anti-proliferative activity in SMARCA4-deficient cancer cell lines, pharmacological studies with a probe capable of binding to SMARCA2 and SMARCA4 bromodomains have failed to recapitulate such anti-proliferative effects. This prompted us to evaluate targeted protein degradation as an alternate approach to target SMARCA4 altered cancers. Methods and Results: A variety of hetero bi-functional molecules were synthesized by conjugating selective SMARCA2/4 bromodomain inhibitors with either VHL or CRBN E3-ligase specific ligands. Rational design approach guided by our proprietary ternary complex modeling algorithm, ALMOND (ALgorithm for MOdeling Neosubstrate Degraders) resulted in the identification of highly selective SMARCA2 degraders. The lead compound, AU-19820 showed > 10000-fold selectivity for SMARCA2 degradation versus other homologous proteins in tested cell lines. AU-19820 demonstrated potent anti-proliferative activity in SMARCA4 mutant but not in SMARCA2/4 proficient cell lines. This compound displayed favorable IV PK profile in rodents along with clean CYP profile. Additionally, the lead compound exhibited significant tumor growth inhibition in RERF-LC-A1 (SMARCA4 mutant lung cancer) xenograft model when dosed via i.v. route. Efficacious exposures were well tolerated with excellent tumor penetration. The compound also demonstrated moderate oral bioavailability in mouse. Efforts are in progress to improve this further by SAR modifications and exploiting prodrug approach. Conclusions: Potent and extremely selective SMARCA2 protein degraders were identified by conjugating SMARCA2/4 inhibitors with known VHL or CRBN ligands. SMARCA2 vs SMARCA4 selectivity handles have been very well explored with expandable SAR. Lead compound also displayed a synthetic lethality phenotype in SMARCA4 mutant cancer models while sparing SMARCA2/4 proficient ones. Further optimization of the oral bioavailability and evaluation of efficacy through oral route as well as intermittent IV dosing are planned. Citation Format: Chandrasekhar Abbineni, Leena Khare Satyam, Bilash Kuila, Ashok Ettam, Khaji Abdul Rawoof, Sreevidya MR, Sandeep Vitthal Dukare, Suraj T. Gore, Rakesh P. Nankar, Vijay Kamal Ahuja, Charamanna KB, Megha Goyal, Kiran Aithal, Samiulla DS, Subhendu Mukherjee, Thomas Antony, Sanjeev Giri, Shekar Chelur, Kavitha Nellore, Girish Daginakatte, Murali Ramachandra, Susanta Samajdar. Orally bioavailable SMARCA2 degraders with exceptional selectivity and potency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1144.

Selective targeting of non-centrosomal AURKA functions through use of a targeted protein degradation tool

Targeted protein degradation tools are becoming a new therapeutic modality, allowing small molecule ligands to be reformulated as heterobifunctional molecules (PROteolysis Targeting Chimeras, PROTACs) that recruit ubiquitin ligases to targets of interest, leading to ubiquitination and destruction of the targets. Several PROTACs against targets of clinical interest have been described, but detailed descriptions of the cell biology modulated by PROTACs are missing from the literature. Here we describe the functional characterization of a PROTAC derived from AURKA inhibitor MLN8237 (alisertib). We demonstrate efficient and specific destruction of both endogenous and overexpressed AURKA by Cereblon-directed PROTACs. At the subcellular level, we find differential targeting of AURKA on the mitotic spindle compared to centrosomes. The phenotypic consequences of PROTAC treatment are therefore distinct from those mediated by alisertib, and in mitotic cells differentially regulate centrosome- and chromatin- based microtubule spindle assembly pathways. In interphase cells PROTAC-mediated clearance of non-centrosomal AURKA modulates the cytoplasmic role played by AURKA in mitochondrial dynamics, whilst the centrosomal pool is refractory to PROTAC-mediated clearance. Our results point to differential sensitivity of subcellular pools of substrate, governed by substrate conformation or localization-dependent accessibility to PROTAC action, a phenomenon not previously described for this new class of degrader compounds.

IRAK4 degradation abrogates cytokine release and improves disease endpoints in murine models of IL-33/36- as well as Th17-driven inflammation

Abstract Interleukin-1 receptor associated kinase 4 (IRAK4) plays a critical role in TLR and IL-1R mediated inflammation through its catalytic and scaffolding functions, making it an attractive target for the treatment of immune-inflammatory diseases. Kymera has developed oral heterobifunctional molecules that selectively target IRAK4 for degradation by the ubiquitin-proteasome pathway. These degraders have broad and potent activity in-vitro against LPS + IL-1β-induced proinflammatory cytokine and chemokine production that is superior to IRAK4 kinase inhibitors. We evaluated their efficacy in comparison to kinase inhibition in-vivo in both mechanistic, and disease models of skin and CNS inflammation. IRAK4 degraders were administered to mice in models of skin inflammation induced by either IL-33 or IL-36, as well as in an EAE model of Th17-mediated CNS inflammation. In IL-33- and IL-36-induced skin inflammation, IRAK4 degradation resulted in dose-dependent decreases in ear thickness as well as local and systemic cytokines/chemokines that was superior to kinase inhibition. In MOG-induced EAE, IRAK4 degradation was superior to kinase inhibition and significantly reduced disease scores to levels comparable to a S1P receptor agonist. These data demonstrate the broad activity of IRAK4 degradation and its superiority over kinase inhibition in alleviating inflammation induced by IL-1 family cytokines as well as antigen-dependent Th17-mediated inflammation in models relevant to human disease.

Discovery of thalidomide-based PROTAC small molecules as the highly efficient SHP2 degraders.

SHP2, a non-receptor tyrosine phosphatase, plays a pivotal role in numerous oncogenic cell-signaling cascades like RAS-ERK, PI3K-AKT and JAK-STAT. On the other hand, proteolysis targeting chimera (PROTAC) has emerged as a promising strategy for the degradation of disease-related protein of interest (POI). SHP2 degradation via the PROTAC strategy will provide an alternative startegy for SHP2-mediated cancer therapy. Herein we described the design, synthesis and evaluation of a series of thalidomide-based heterobifunctional molecules and identified 11(ZB-S-29) as the highly efficient SHP2 degrader with a DC50 of 6.02nM. Further mechanism investigation illustrated that 11 came into function through targeted SHP2 protein degradation.

Small-Molecule Degraders beyond PROTACs-Challenges and Opportunities.

Targeted protein degradation (TPD) is a recent strategy, utilizing the cell's proteostasis machinery to deplete specific proteins. This represents a paradigm shift in early drug discovery, away from occupancy-driven to event-driven mechanisms.Recent efforts have focused on the development of proteolysis-targeting chimeras (PROTACs). These heterobifunctional molecules combine a target-specific binding moiety linked to an E3 ligase ligand and trigger selective ubiquitination of the target protein, marking it for proteasomal degradation. While these molecules can be highly efficacious, they generally have unfavorable physicochemical properties due to their large size.In contrast, smaller molecules that induce degradation could represent an attractive, simple option to overcoming the limitations of both traditional modulators and PROTACs. These molecules may have a range of mechanisms: recruitment of an E3 ligase (molecular glues), introduction of hydrophobic areas, or inducing local unfolding, each of which triggers degradation.We recently completed a high-throughput screen of 111,000 compounds in a cellular HiBiT assay in an effort to identify such molecules. Preliminary analysis indicates that we have been able to identify alternative small-molecule degraders. We highlight methods for triage, characterization, selectivity, and mode of action. In summary, we believe that these types of small-molecule degraders, which may possibly have more acceptable physicochemical properties than the inherently larger heterobifunctional molecules, are an exciting approach for inducing TPD, and we illustrate that a general screening approach can be successful in identifying useful start points for developing such molecules.

Evaluating Ligands for Ubiquitin Ligases Using Affinity Beads.

Proteolysis-targeting chimera (PROTAC®) protein degraders are heterobifunctional small molecules that bind a specific target protein on one end and a specific ubiquitin ligase enzyme (E3) on the other, thereby driving intracellular degradation of the target protein via the ubiquitin-proteasome system. PROTACs and other small molecule protein degraders are being developed as potential therapeutics for several diseases, with the first PROTACs having entered the clinic for cancer treatments in 2019. While humans express approximately 600 E3s, only a few have been used for protein degrader technology. A major challenge to designing degraders based on additional E3s is the development of quality ligands for other E3s. Most methods to screen for novel ligands employ purified forms of the protein of interest. Ligands discovered in this manner are typically subsequently evaluated in cultured cells. Optimal ligands efficiently cross biological membranes and interact specifically with the protein of interest, which can be assessed by a variety of cell-based methods. Functionality and specificity of ligand-protein interactions can also be evaluated using cell or tissue extracts and affinity beads based on the ligand, as described here. E3 affinity beads described herein are based on conjugation of the potential E3 ligand to biotin and commercially available streptavidin agarose with high affinity for biotin.

PROTACs to address the challenges facing small molecule inhibitors

Small molecule inhibitors of proteins represent important medicines and critical chemical tools to investigate the biology of the target proteins. Advances in various -omics technologies have fueled the pace of discovery of disease-relevant proteins. Translating these discoveries into human benefits requires us to develop specific chemicals to inhibit the proteins. However, traditional small molecule inhibitors binding to orthosteric or allosteric sites face significant challenges. These challenges include drug selectivity, therapy resistance as well as drugging undruggable proteins and multi-domain proteins. To address these challenges, PROteolysis TArgeting Chimera (PROTAC) has been proposed. PROTACs are heterobifunctional molecules containing a binding ligand for a protein of interest and E3 ligase-recruiting ligand that are connected through a chemical linker. Binding of a PROTAC to its target protein will bring a E3 ligase in close proximity to initiate polyubiquitination of the target protein ensuing its proteasome-mediated degradation. Unlike small molecule inhibitors, PROTACs achieve target protein degradation in its entirety in a catalytical fashion. In this review, we analyze recent advances in PROTAC design to discuss how PROTACs can address the challenges facing small molecule inhibitors to potentially deliver next-generation medicines and chemical tools with high selectivity and efficacy. We also offer our perspectives on the future promise and potential limitations facing PROTACs. Investigations to overcome these limitations of PROTACs will further help realize the promise of PROTACs for human benefits.

Proteolysis-targeting chimeras mediatethe degradation of bromodomain and extra-terminal domain proteins.

Bromodomain and extra-terminal domain (BET) protein family plays an important role in regulating gene transcription preferentially at super-enhancer regions and has been involved with several types of cancers as a candidate. Up to now, there are 16 pan-BET inhibitors in clinical trials, however, most of them have undesirable off-target and side-effects. The proteolysis-targeting chimerastechnology through a heterobifunctional molecule to link the target protein and E3 ubiquitin ligase, causes the target's ubiquitination and subsequent degradation. By using this technology, the heterobifunctional small-molecule BET degraders can induce BET protein degradation. In this review, we discuss the advances in the drug discovery and development of BET-targeting proteolysis-targeting chimeras.

Recent advances in epigenetic proteolysis targeting chimeras (Epi-PROTACs).

PROteolysis TArgeting Chimeras (PROTACs) are heterobifunctional molecules that trigger the poly-ubiquitination of the protein of interest (POI) inducing its degradation via the recruitment of the ubiquitin-proteasome system, thus suppressing the POI's intracellular levels and indirectly all its functions. Recently, one of the fields where the protein knockdown induced by PROTACs has demonstrated to serve as a promising biochemical tool and to provide new opportunities for drug discovery is the epigenetics (epi-PROTACs). A full inhibition of the functions of all domains of a specific epigenetic POI (e-POI), rather than just the block of its catalytic/single domain activity, is in fact a new more effective modality to hit an e-POI and, in principle, the complex it belongs to, and potentially to treat the related diseases, first cancer. In this review, we will present the most relevant progresses made, especially in the last two years, in the application of PROTACs technology to the three main classes of e-POIs: "writers", "erasers" and "readers". Emphasis will be devoted to the medicinal chemistry aspects of the epi-PROTACs design, preparation, and optimization and to the comparison with small molecule epi-drugs for both epi-targets functional annotation and potential anticancer therapy purposes.

Abstract 1754: First in class orally bioavailable BETBRD degraders

Abstract Background: Inhibition of Bromodomain and extra-terminal (BET) family proteins by small molecules is actively being pursued as a therapeutic strategy in the clinics. Targeted protein degradation is an emerging therapeutic modality that has shown initial promise in the clinic. BET protein degradation has inherent advantages over inhibition viz. expansion of indication scope and amenability to intermittent dosing schedules. While many BET degraders have been disclosed earlier, inferior pharmacokinetic properties limit their further development. Methods and Results: We have designed and synthesized various hetero bi-functional molecules by conjugating novel and selective BET BRD ligands with VHL or CRBN ligands. This exercise led to the identification of several potent and selective BRD4 protein degraders with activity in a wide range of hematological and solid tumor cell lines. We have profiled one of the lead compounds extensively in vitro to gain insights on the mechanism of action. The lead compound showed lasting effect on BET protein abundance post compound washout while leading to apoptosis. This compound has favorable IV PK profile in rodents had has clean CYP and hERG profiles. Additionally, the lead compound exhibited significant tumor growth inhibition in MV4-11 xenograft model when dosed via i.v. route. Both QD and Q48h dosing regimens were well tolerated and produced efficacy in mouse models. Further SAR in the linker portion resulted in compounds with lower iv clearance and oral bioavailability either as prodrugs or as such. The proteomics study revealed a high selectivity towards BET proteins for the lead compound. Conclusions: Potent and selective BET protein degraders were identified by conjugating novel BET BRD ligands with both VHL and CRBN ligands. Optimization of these first generation BET degraders led to improved metabolic stability, translating into low iv clearance in rodents. Further evaluation of these compounds as prodrugs resulted in good oral exposures. Lead compounds from both the series have low iv clearance and are orally bioavailable in a simple formulation. We believe these compounds serve as valuable tools to fully understand the clinical scope of BET degraders. Citation Format: Chandrasekhar Abbineni, Mahaboobi Jaleel, Subhendu Mukherjee, Sivapriya Marappan, Nirbhay Kumar Tiwari, DS Samiulla, AB Aravind, Naveen R Kumar, Indu Bansal, Raghurami B Reddy, NVM Rao Bandaru, Akhila Srinivas, Janith Mary Maben, Suraj Tgore, Avainash Kumar, Rakesh P. Nankar, Chandranath D. Naik, Thomas Antony, Kavitha Nellore, Sanjeev Giri, Girish Daginakatte, Shekar Chelur, Olli Törmäkangas, Gerd Wohlfahrt, Mari Björkman, Elina Mattila, Laura Ravanti, Anu Moilanen, Murali Ramachandra, Susanta Samajdar. First in class orally bioavailable BETBRD degraders [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1754.

Abstract LB-088: A STAT3 selective targeted protein degrader decreases the immunesuppressive tumor microenvironment and drives antitumor activity in preclinical models

Abstract Signal transducer and activator of transcription 3 (STAT3) has been implicated in multiple aspects of tumorigenesis. In addition to increasing cancer cell proliferation and survival, constitutively activated STAT3 is proposed to regulate cross-talk between tumor, stroma and immune cells to promote immune-evasion. STAT3 activity in tumors promotes the production of immune-suppressive factors that activate STAT3 in diverse immune-cell subsets. Mechanistically, genetic studies support a direct role of activated STAT3 in regulating myeloid cell differentiation to contribute to an immune-suppressed tumor microenvironment (TME) (Kortylewski et al.; Nat. Med. 2005 and Curr. Opin. Immunol. 2008) Therefore, STAT3 is a highly attractive target for immune-oncology. Here, we demonstrate that the degradation of STAT3 with a potent and selective STAT3 degrader reverses immune suppression in preclinical models. KYM-003 is a heterobifunctional molecule that hijacks the ubiquitin-proteasome system (UPS) for rapid STAT3 degradation. KYM-003 robustly degraded STAT3 in both human peripheral blood mononuclear cells (hPBMC)-derived monocytes and lymphocytes with DC50 < 100 nM. Degradation in hPBMCs was highly selective for STAT3 vs >10,000 other detected proteins (including all other STAT family members) as evaluated by deep tandem mass tag proteomics. IL-6 treatment of hPBMCs upregulates STAT3 phosphorylation, resulting in transcription of genes involved in myeloid cell-mediated immune suppression, such as IL-10 and CD163, a marker of M2 macrophage differentiation. Treatment of cells with KYM-003 at DC90 for 6 hours abrogated the IL-6 induced up-regulation of immune-suppressive gene signatures consistent with a role of STAT3 in mediating an immune suppressive environment by regulating macrophages and other myeloid cells. In several tumor cell lines, degradation of STAT3 by KYM-003 led to strong downregulation of PD-L1. Importantly, in the BioMap StroNSCLC (DiscoverRx), a co-culture system composed of NCI-H1299 lung cancer cells, hPBMCs and primary human fibroblasts that models immune-suppressed TME biology, KYM-003 treatment decreased angiogenic and immune-suppressive cytokines, including VEGF and IL-10, and promoted the pro-inflammatory anti-tumor cytokines IL-2, IFNg and TNFa. Lastly, administration of KYM-003 to mice bearing syngeneic tumors exhibited anti-tumor activity as monotherapy in the CT26 syngeneic model. We have shown that targeting STAT3 for degradation may have a role in restoring an immune-permissive environment in tumors by both rescuing the suppressed immunologic microenvironment and directly downregulating immune checkpoint signals in tumor cells. These data support STAT3 degraders as a promising new therapeutic modality as immune-oncology agents.Citations: Kortylewski et al (2005) Nat. Med, 11(12):1314-21. Kortylewski et al (2008) Curr. Opin. Immunol, 20(2):228-33 Citation Format: Alfredo Csibi, Bin Yang, Yogesh Chutake, Karen Yuan, Michele Mayo, Veronica Campbell, Alice McDonald, Scott Rusin, Kirti Sharma, Hari Kamadurai, Henry Li, Mike Sintchak, Sean Zhu, Sharon Townson, Anthony Slavin, Haojing Rong, Phillip Liu, Chris De Savi, Jared Gollob, Duncan Walker, Nan Ji, Nello Mainolfi. A STAT3 selective targeted protein degrader decreases the immunesuppressive tumor microenvironment and drives antitumor activity in preclinical models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-088.

Abstract 1949: In vivo profiling of BET degraders establishes optimal pharmacological properties that showcase distinct biological differences from BET inhibitors

Abstract Targeted protein degradation (TPD) with heterobifunctional small molecules is emerging as a promising new therapeutic modality in oncology. We sought to understand how TPD compares versus targeted protein inhibition. We used BRD4 as a surrogate target given literature showing BET degraders have a global suppressive effect on transcription compared to BET inhibitors that preferentially affect superenhancers. We selected the BET degrader CFT-743 from a library of CRBN-biased degraders based on its specificity, potency and optimum in vivo properties that enable a therapeutic window. While subtle changes in chemistry around CFT-743 had negligible impact on vitro potency, the effect on therapeutic index was significant. In vitro, CFT-743 is more potent than a BET inhibitor (AZD5153) and a CDK9 inhibitor (dinaciclib) in 100% and 97% of 38 leukemia lines tested. In mice bearing HL60 tumors, CFT-743 has similar efficacy as dinaciclib (81% regression versus 99% tumor growth inhibition (TGI)). Pre-dosing mice with the CRBN binding molecule pomalidomide significantly attenuates CFT-743 activity (TGI 40%) rendering the observed efficacy on par with BET inhibitor CPI-203 (TGI 2%) and thus confirming superior efficacy is dependent on BET degradation. In Kasumi1 and THP1 tumor models, CFT-743 efficacy is more like CPI-203 (regressions in Kasumi1 and strong TGI/moderate regressions in THP1) than dinaciclib (moderate regression and 0% TGI). In RS4;11 tumors CFT-743 promotes complete regression whereas dinaciclib and CPI-203 display modest efficacy (80% and 58% TGI). Pharmacokinetic and pharmacodynamic studies in mice bearing MV4;11 tumors demonstrate that a high initial plasma exposure of CFT-743 followed by fast compound clearance is sufficient to drive rapid loss of BRD4 in tumors (<90% of baseline by 1hr post dose) and induction of apoptosis. BRD4 expression levels are unable to recover likely due to the substantial wave of cell death, indicated by cleaved Caspase 3 and rapid decline in tumor volume. In normal tissues, CFT-743 promotes a similar rapid and near complete loss of BRD4, however; protein levels begin to recover ≤ 6hr post dose. This pharmacodynamic behavior provides a rationale as to why CFT-743 is efficacious and well tolerated. Collectively, these data highlight the in vivo features important for efficacy and tolerability with BET degraders and establish the relationships between degrader pharmacokinetics, target protein degradation and efficacy. Citation Format: Brendon Ladd, David Cocozziello, Mark E. Fitzgerald, Ryan E. Michael, Ellen F. Vieux, Linda Lee, Marta Issa, Jacob D. Feala, Roman V. Agafonov, Eun Sun Park, Christopher G. Nasveschuk, David A. Proia. In vivo profiling of BET degraders establishes optimal pharmacological properties that showcase distinct biological differences from BET inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1949.

Understanding and Improving the Membrane Permeability of VH032-Based PROTACs.

Proteolysis targeting chimeras (PROTACs) are catalytic heterobifunctional molecules that can selectively degrade a protein of interest by recruiting a ubiquitin E3 ligase to the target, leading to its ubiquitylation and degradation by the proteasome. Most degraders lie outside the chemical space associated with most membrane-permeable drugs. Although many PROTACs have been described with potent activity in cells, our understanding of the relationship between structure and permeability in these compounds remains limited. Here, we describe a label-free method for assessing the permeability of several VH032-based PROTACs and their components by combining a parallel artificial membrane permeability assay (PAMPA) and a lipophilic permeability efficiency (LPE) metric. Our results show that the combination of these two cell-free membrane permeability assays provides new insight into PROTAC structure–permeability relationships and offers a conceptual framework for predicting the physicochemical properties of PROTACs in order to better inform the design of more permeable and more effective degraders.

Abstract B50: Targeting EWS/FLI fusion oncoprotein stability/degradation in Ewing sarcoma

Abstract Fusion-transcription factors (fusion-TFs) represent a class of oncoproteins that drive tumorigenesis by activating an aberrant transcriptional program to promote the development and survival of cancer cells. Unlike fusion oncoproteins involving readily druggable proteins such as kinases, fusion-TFs are difficult to therapeutically target. Recent studies have suggested protein degradation as a novel strategy in targeting oncogenic transcription factors. However, the molecular and functional mechanisms that regulate fusion-TFs’ degradation/stability are unknown. Ewing sarcoma is the second most common bone cancer in children and is driven by EWS-ETS fusion-TFs, most commonly EWS/FLI (~85%). EWS/FLI acts as a pioneer transcription factor to activate oncogenic gene expression by chromatin remodeling that drives the tumorigenesis and survival of Ewing sarcoma tumors. Despite this, there are no clinically validated targeted therapies against EWS/FLI. Ewing sarcoma has a relatively quiet genome and lacks recurrent mutations, which hinders target identification for therapeutic intervention. Thus, we examined whether a protein degradation strategy can be exploited to target EWS/FLI by investigating the molecular and functional mechanisms by which EWS/FLI protein stability/degradation is regulated. In a genome-scale, flow cytometry-based CRISPR-Cas9 screen, we discovered that tripartite motif containing 8 (TRIM8) is a novel E3 ubiquitin for EWS/FLI. Biochemical studies showed that TRIM directly binds and ubiquitinates EWS/FLI for proteasome-dependent degradation. Moreover, we identified TRIM8 as a top enriched and selective dependency in Ewing sarcoma in a genome-scale CRISPR-Cas9 depletion screen. We determined that TRIM8 knockout increased EWS/FLI protein levels and induced apoptosis of Ewing sarcoma cells in vitro and reduced tumor growth in vivo. Consistent with TRIM8 as an E3 ligase for EWS/FLI, overexpression of TRIM8 reduced EWS/FLI protein levels and decreased growth. TRIM8 lacking the E3 ligase domain (TRIM8E3) functioned as a dominant negative, leading to increased EWS/FLI and decreased growth of Ewing sarcoma cells. To investigate whether upregulated EWS/FLI protein expression is mediating the TRIM8 knockout phenotype, we engineered Ewing sarcoma cell line by concurrently knocking out endogenous EWS/FLI using CRISPR and expressing degradable EWS/FLI (FKBP12F36V-EWS/FLI) using the dTAG system. The dTAG molecule is a heterobifunctional molecule that specifically binds to FKBP12F36V and an E3 ligase complex for targeted protein degradation. Using this degradable EWS/FLI cell model, we showed that TRIM8 suppression phenotype can be rescued by degrading the upregulated EWS/FLI protein expression to control levels. Our results demonstrate that the protein stability of fusion-TF oncoproteins is highly regulated to maintain the expression at a precise level and highlight the critical importance of oncogene dosage in cancer cell survival. Citation Format: Bo Kyung Alex Seong, Shan Lin, Katherine Donovan, Amanda Robichaud, Bjorn Stolte, Emily Wang, Neekesh Dharia, Behnam Nabet, Federica Piccioni, Nathanael Gray, Eric Fischer, Kimberly Stegmaier. Targeting EWS/FLI fusion oncoprotein stability/degradation in Ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B50.

Chemical Knockdown of MicroRNA with Small-Molecule Chimeras.

This concept article introduces the emerging area of small-molecule chimeras (SMCs) for knocking down microRNAs (miRNAs), which are endogenous gene silencers involved in diverse pathological processes. Compared with agents for genetic knockdown, small-molecules hold significant promise in this field due to their ideal pharmacokinetic and pharmacodynamic properties. The SMCs introduced here are hetero-bifunctional molecules comprising small-molecule binders (SMBs) of miRNAs and chemical functionalities that either directly cleave RNAs or recruit ribonucleases to destroy RNAs. Binding of SMBs to miRNAs brings SMCs' chemical functionalities close to the miRNA, eventually causing miRNA degradation. Compared with parent SMBs, SMCs exhibit remarkably enhanced potency and specificity in miRNA inhibition. The development and application of SMCs for miRNAs will be discussed.

588 Identification of highly potent and selective Interleukin-1 receptor associated kinase 4 (IRAK4) degraders for the treatment of hidradenitis suppurativa

Interleukin-1 receptor associated kinase 4 (IRAK4) plays a central role in myddosome signaling via kinase and scaffolding functions, making it an attractive target for the treatment of TLR- and IL-1R-driven inflammatory diseases. IL-1 family cytokines and TLRs, are central to the pathophysiology of hidradenitis suppurativa (HS), a Th1- and Th17-mediated neutrophilic, chronic inflammatory skin disease. Kymera has developed orally administered hetero-bifunctional molecules that selectively target IRAK4 for degradation and elimination by the ubiquitin proteasome pathway.

Molecular mechanisms of thalidomide and its derivatives.

Thalidomide, originally developed as a sedative drug, causes multiple defects due to severe teratogenicity, but it has been re-purposed for treating multiple myeloma, and derivatives such as lenalidomide and pomalidomide have been developed for treating blood cancers. Although the molecular mechanisms of thalidomide and its derivatives remained poorly understood until recently, we identified cereblon (CRBN), a primary direct target of thalidomide, using ferrite glycidyl methacrylate (FG) beads. CRBN is a ligand-dependent substrate receptor of the E3 ubiquitin ligase complex cullin-RING ligase 4 (CRL4CRBN). When a ligand such as thalidomide binds to CRBN, it recognizes various ‘neosubstrates’ depending on the shape of the ligand. CRL4CRBN binds many neosubstrates in the presence of various ligands. CRBN has been utilized in a novel protein knockdown technology named proteolysis targeting chimeras (PROTACs). Heterobifunctional molecules such as dBET1 are being developed to specifically degrade proteins of interest. Herein, we review recent advances in CRBN research.

PROteolysis TArgeting Chimeras (PROTACs) as emerging anticancer therapeutics.

Using PROteolysis TArgeting Chimeras (PROTACs) to degrade proteins that are important for tumorigenesis has emerged as a potential therapeutic strategy for cancer. PROTACs are heterobifunctional molecules consisting of one ligand for binding to a protein of interest (POI) and another to an E3 ubiquitin (E3) ligase, connected via a linker. PROTACs recruit the E3 ligase to the POI and cause proximity-induced ubiquitination and degradation of the POI by the ubiquitin proteasome system (UPS). PROTACs have been developed to degrade a variety of cancer targets with unprecedented efficacy against a multitude of tumor types. To date, most of the PROTACs developed have utilized ligands to recruit E3 ligases that are ubiquitously expressed in both tumor and normal tissues. These PROTACs can cause on-target toxicities if the POIs are not tumor-specific. Therefore, identifying and recruiting the E3 ligases that are enriched in tumors with minimal expression in normal tissues holds the potential to develop tumor-specific/selective PROTACs. In this review, we will discuss the potential of PROTACs to become anticancer therapeutics, chemical and bioinformatics approaches for PROTAC design, and safety concerns with a special focus on the development of tumor-specific/selective PROTACs. In addition, the identification of tumor types in terms of solid versus hematological malignancies that can be best targeted with PROTAC approach will be briefly discussed.

Small-molecule PROTACs: novel agents for cancer therapy.

Proteolysis-targeting chimera (PROTAC) is a new technology to selectively degrade target proteins via ubiquitin-proteasome system. PROTAC molecules (PROTACs) are a class of heterobifunctional molecules, which contain a ligand targeting the protein of interest, a ligand recruiting an E3 ligase and a linker connecting these two ligands. They provide several advantages over traditional inhibitors in potency, selectivity and drug resistance. Thus, many promising PROTACs have been developed in the recent two decades, especially small-molecule PROTACs. In this review, we briefly introduce the mechanism of PROTACs and focus on the progress of small-molecule PROTACs based on different E3 ligases. In addition, we also introduce the opportunities and challenges of small-molecule PROTACs for cancer therapy.

Two-Stage Strategy for Development of Proteolysis Targeting Chimeras and its Application for Estrogen Receptor Degraders.

Proteolysis targeting chimeras (PROTACs) have emerged as useful chemical probes and potential therapeutics by taking advantage of the ubiquitin-proteasome system to degrade intracellular disease-associated proteins. PROTACs are heterobifunctional molecules composed of a target protein ligand, E3 ubiquitin ligase ligand, and a linker between them. The generation of efficient PROTACs requires screening of many parameters, especially the lengths and types of the linkers. We report our proof-of-concept study using a two-stage strategy to facilitate the development of PROTACs against the estrogen receptor (ER). In stage one, a library of close to 100 PROTACs was synthesized by simply mixing a library of ERα ligands containing a hydrazide functional group at different positions with a preassembled library of E3 ligase ligands bearing different types and lengths of linkers with a terminal aldehyde group in a 1:1 ratio. Cell-based screening occurred without further purification, because the formation of the acylhydrazone linkage is highly efficient and produces water as the only byproduct. Compound A3 was the most potent ER degrader in two ER+ cell lines (DC50= ∼ 10 nM, Dmax= ≥ 95%). Stage two involved transformation to a more stable amide linker to generate a more drug-like molecule. The new compound, AM-A3, showed comparable biological activity (DC50 = 1.1 nM, Dmax = 98%) and induced potent antiproliferation (IC50= 13.2 nM, Imax= 69%) in MCF-7. This proof-of -concept study demonstrates that the two-stage strategy can significantly facilitate the development of PROTACs against ER without the tedious process of making large numbers of PROTACs one by one. It has the potential to be expanded to many other targets.