Design, synthesis, and evaluation of BCL-2 targeting PROTACs.

BCL-2, a member of the BCL-2 protein family, is an antiapoptotic factor that regulates the intrinsic pathway of apoptosis. Due to its aberrant activity, it is frequently implicated in haematopoietic cancers and represents an attractive target for the development of therapeutics that antagonize its activity. A selective BCL-2 inhibitor, venetoclax, was approved for treating chronic lymphocytic leukaemia, acute myeloid leukemia, and other hematologic malignancies, validating BCL-2 as an anticancer target. Since then, alternative therapeutic approaches to modulate the activity of BCL-2 have been explored, such as antibody-drug conjugates and proteolysis-targeting chimeras. Despite numerous research groups focusing on developing degraders of BCL-2 family member proteins, selective BCL-2 PROTACs remain elusive, as disclosed compounds only show dual BCL-xL/BCL-2 degradation. Herein, we report our efforts to develop BCL-2 degraders by incorporating two BCL-2 binding moieties into chimeric compounds that aim to hijack one of three E3 ligases: CRBN, VHL, and IAPs. Even though our project did not result in obtaining a potent and selective BCL-2 PROTAC, our research will aid in understanding the narrow chemical space of BCL-2 degraders.

Revisiting the antiangiogenic mechanisms of fluorinated thalidomide derivatives.

Introduction of fluorine into bioactive molecules has attracted much attention in drug development. For example, tetrafluorination of the phthalimide moiety of immunomodulatory drugs (IMiDs) has a strong beneficial effect on the ability to inhibit angiogenesis. The neomorphic activity of E3 ligase complexes is induced by the binding of IMiDs to cereblon. We investigated that a set of eight thalidomide analogs, comprising non- and tetrafluorinated counterparts, did not induce the degradation of neomorphic substrates (IKZF3, GSPT1, CK1α, SALL4). Hence, the antiangiogenic activity of fluorinated IMiDs was not triggered by neosubstrate degradation features. A fluorine scanning of non-traditional IMiDs of the benzamido glutarimide chemotype was performed. By measuring the endothelial cell tube formation, no angiogenesis inhibitors were identified, confirming the narrow structure-activity window of IMiD-induced antiangiogenesis.

The proteogenomic landscape of multiple myeloma reveals insights into disease biology and therapeutic opportunities.

Multiple myeloma (MM) is a plasma cell malignancy of the bone marrow. Despite therapeutic advances, MM remains incurable, and better risk stratification as well as new therapies are therefore highly needed. The proteome of MM has not been systematically assessed before and holds the potential to uncover insight into disease biology and improved prognostication in addition to genetic and transcriptomic studies. Here we provide a comprehensive multiomics analysis including deep tandem mass tag-based quantitative global (phospho)proteomics, RNA sequencing, and nanopore DNA sequencing of 138 primary patient-derived plasma cell malignancies encompassing treatment-naive MM, plasma cell leukemia and the premalignancy monoclonal gammopathy of undetermined significance, as well as healthy controls. We found that the (phospho)proteome of malignant plasma cells are highly deregulated as compared with healthy plasma cells and is both defined by chromosomal alterations as well as posttranscriptional regulation. A prognostic protein signature was identified that is associated with aggressive disease independent of established risk factors in MM. Integration with functional genetics and single-cell RNA sequencing revealed general and genetic subtype-specific deregulated proteins and pathways in plasma cell malignancies that include potential targets for (immuno)therapies. Our study demonstrates the potential of proteogenomics in cancer and provides an easily accessible resource for investigating protein regulation and new therapeutic approaches in MM.

Heterobifunctional Ligase Recruiters Enable pan-Degradation of Inhibitor of Apoptosis Proteins.

Proteolysis targeting chimeras (PROTACs) represent a new pharmacological modality to inactivate disease-causing proteins. PROTACs operate via recruiting E3 ubiquitin ligases, which enable the transfer of ubiquitin tags onto their target proteins, leading to proteasomal degradation. However, several E3 ligases are validated pharmacological targets themselves, of which inhibitor of apoptosis (IAP) proteins are considered druggable in cancer. Here, we report three series of heterobifunctional PROTACs, which consist of an IAP antagonist linked to either von Hippel-Lindau- or cereblon-recruiting ligands. Hijacking E3 ligases against each other led to potent, rapid, and preferential depletion of cellular IAPs. In addition, these compounds caused complete X-chromosome-linked IAP knockdown, which was rarely observed for monovalent and homobivalent IAP antagonists. In cellular assays, hit degrader 9 outperformed antagonists and showed potent inhibition of cancer cell viability. The hetero-PROTACs disclosed herein are valuable tools to facilitate studies of the biological roles of IAPs and will stimulate further efforts toward E3-targeting therapies.

Direct-to-biology, automated, nano-scale synthesis, and phenotypic screening-enabled E3 ligase modulator discovery.

Thalidomide and its analogs are molecular glues (MGs) that lead to targeted ubiquitination and degradation of key cancer proteins via the cereblon (CRBN) E3 ligase. Here, we develop a direct-to-biology (D2B) approach for accelerated discovery of MGs. In this platform, automated, high throughput, and nano scale synthesis of hundreds of pomalidomide-based MGs was combined with rapid phenotypic screening, enabling an unprecedented fast identification of potent CRBN-acting MGs. The small molecules were further validated by degradation profiling and anti-cancer activity. This revealed E14 as a potent MG degrader targeting IKZF1/3, GSPT1 and 2 with profound effects on a panel of cancer cells. In a more generalized view, integration of automated, nanoscale synthesis with phenotypic assays has the potential to accelerate MGs discovery.

Accessing three-branched high-affinity cereblon ligands for molecular glue and protein degrader design.

The Petasis borono-Mannich reaction was employed for an alternative entry towards three-branched cereblon ligands. Such compounds are capabable of making multiple interactions with the protein surface and possess a suitable linker exit vector. The high-affinity ligands were used to assemble prototypic new molecular glues and proteolysis targeting chimeras (PROTACs) targeting BRD4 for degradation. Our results highlight the importance of multicomponent reactions (MCRs) in drug discovery and add new insights into the rapidly growing field of protein degraders.

Leveraging Ligand Affinity and Properties: Discovery of Novel Benzamide-Type Cereblon Binders for the Design of PROTACs.

Immunomodulatory imide drugs (IMiDs) such as thalidomide, pomalidomide, and lenalidomide are the most common cereblon (CRBN) recruiters in proteolysis-targeting chimera (PROTAC) design. However, these CRBN ligands induce the degradation of IMiD neosubstrates and are inherently unstable, degrading hydrolytically under moderate conditions. In this work, we simultaneously optimized physiochemical properties, stability, on-target affinity, and off-target neosubstrate modulation features to develop novel nonphthalimide CRBN binders. These efforts led to the discovery of conformationally locked benzamide-type derivatives that replicate the interactions of the natural CRBN degron, exhibit enhanced chemical stability, and display a favorable selectivity profile in terms of neosubstrate recruitment. The utility of the most potent ligands was demonstrated by their transformation into potent degraders of BRD4 and HDAC6 that outperform previously described reference PROTACs. Together with their significantly decreased neomorphic ligase activity on IKZF1/3 and SALL4, these ligands provide opportunities for the design of highly selective and potent chemically inert proximity-inducing compounds.

SUMOylation inhibition overcomes proteasome inhibitor resistance in multiple myeloma.

Proteasome inhibition is a highly effective treatment for multiple myeloma (MM). However, virtually all patients develop proteasome inhibitor resistance, which is associated with a poor prognosis. Hyperactive small ubiquitin-like modifier (SUMO) signaling is involved in both cancer pathogenesis and cancer progression. A state of increased SUMOylation has been associated with aggressive cancer biology. We found that relapsed/refractory MM is characterized by a SUMO-high state, and high expression of the SUMO E1-activating enzyme (SAE1/UBA2) is associated with poor overall survival. Consistently, continuous treatment of MM cell lines with carfilzomib (CFZ) enhanced SUMO pathway activity. Treatment of MM cell lines with the SUMO E1-activating enzyme inhibitor subasumstat (TAK-981) showed synergy with CFZ in both CFZ-sensitive and CFZ-resistant MM cell lines, irrespective of the TP53 state. Combination therapy was effective in primary MM cells and in 2 murine MM xenograft models. Mechanistically, combination treatment with subasumstat and CFZ enhanced genotoxic and proteotoxic stress, and induced apoptosis was associated with activity of the prolyl isomerase PIN1. In summary, our findings reveal activated SUMOylation as a therapeutic target in MM and point to combined SUMO/proteasome inhibition as a novel and potent strategy for the treatment of proteasome inhibitor-resistant MM.

Proteomic profiling reveals CDK6 upregulation as a targetable resistance mechanism for lenalidomide in multiple myeloma.

The immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide are highly effective treatments for multiple myeloma. However, virtually all patients eventually relapse due to acquired drug resistance with resistance-causing genetic alterations being found only in a small subset of cases. To identify non-genetic mechanisms of drug resistance, we here perform integrated global quantitative tandem mass tag (TMT)-based proteomic and phosphoproteomic analyses and RNA sequencing in five paired pre-treatment and relapse samples from multiple myeloma patients. These analyses reveal a CDK6-governed protein resistance signature that includes myeloma high-risk factors such as TRIP13 and RRM1. Overexpression of CDK6 in multiple myeloma cell lines reduces sensitivity to IMiDs while CDK6 inhibition by palbociclib or CDK6 degradation by proteolysis targeting chimeras (PROTACs) is highly synergistic with IMiDs in vitro and in vivo. This work identifies CDK6 upregulation as a druggable target in IMiD-resistant multiple myeloma and highlights the use of proteomic studies to uncover non-genetic resistance mechanisms in cancer.

Generation of a lenalidomide-sensitive syngeneic murine in vivo multiple myeloma model by expression of CrbnI391V.

The immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide are approved drugs for the treatment of multiple myeloma. IMiDs induce cereblon (CRBN) E3 ubiquitin ligase-mediated ubiquitination and degradation of Ikaros transcription factors Ikaros (IKZF1) and Aiolos (IKZF3), which are essential for multiple myeloma. However, because of a single amino acid substitution of valine to isoleucine in mouse CRBN at position 391, mice are not susceptible to IMiD-induced degradation of neosubstrates. Here, we report that expression of human CRBN or the CrbnI391V mutant enables IMiD-induced degradation of IKZF1 and IKZF3 in murine MOPC.315.BM.Luc.eGFP and 5T33MM multiple myeloma cells. Accordingly, lenalidomide and pomalidomide decreased cell viability in a dose-dependent fashion in murine multiple myeloma cells expressing CrbnI391V in vitro. The sensitivity of murine cells expressing CrbnI391V to IMiDs highly correlated with their dependence on IKZF1. After transplantation, MOPC.315.BM.Luc.eGFP cells expressing murine CrbnI391V induced multiple myeloma in mice, and treatment with lenalidomide and pomalidomide significantly delayed tumor growth. This straightforward model provides a proof-of-concept for studying the effects of IMiDs in multiple myeloma in mice, which allows for in vivo testing of IMiDs and other CRBN E3 ligase modulators.

Systematic exploration of different E3 ubiquitin ligases: an approach towards potent and selective CDK6 degraders.

Cyclin-dependent kinase 6 (CDK6) is an important regulator of the cell cycle. Together with CDK4, it phosphorylates and inactivates retinoblastoma (Rb) protein. In tumour cells, CDK6 is frequently upregulated and CDK4/6 kinase inhibitors like palbociclib possess high activity in breast cancer and other malignancies. Besides its crucial catalytic function, kinase-independent roles of CDK6 have been described. Therefore, targeted degradation of CDK6 may be advantageous over kinase inhibition. Proteolysis targeting chimeras (PROTACs) structurally based on the cereblon (CRBN) ligand thalidomide have recently been described to degrade the targets CDK4/6. However, CRBN-based PROTACs have several limitations including the remaining activity of immunomodulatory drugs (IMiDs) on Ikaros transcription factors as well as CRBN inactivation as a resistance mechanism in cancer. Here, we systematically explored the chemical space of CDK4/6 PROTACs by addressing different E3 ligases and connecting their respective small-molecule binders via various linkers to palbociclib. The spectrum of CDK6-specific PROTACs was extended to von Hippel Lindau (VHL) and cellular inhibitor of apoptosis protein 1 (cIAP1) that are essential for most cancer cells and therefore less likely to be inactivated. Our VHL-based PROTAC series included compounds that were either specific for CDK6 or exhibited dual activity against CDK4 and CDK6. IAP-based PROTACs caused a combined degradation of CDK4/6 and IAPs resulting in synergistic effects on cancer cell growth. Our new degraders showed potent and long-lasting degrading activity in human and mouse cells and inhibited proliferation of several leukemia, myeloma and breast cancer cell lines. In conclusion, we show that VHL- and IAP-based PROTACs are an attractive approach for targeted degradation of CDK4/6 in cancer.

A MedChem toolbox for cereblon-directed PROTACs.

A modular chemistry toolbox was developed for cereblon-directed PROTACs. A variety of linkers was attached to a CRBN ligand via the 4-amino position of pomalidomide. We used linkers of different constitution to modulate physicochemical properties. We equipped one terminus of the linker with a set of functional groups, e.g. protected amines, protected carboxylic acids, alkynes, chloroalkanes, and protected alcohols, all of which are considered to be attractive for PROTAC design. We also highlight different opportunities for the expansion of the medicinal chemists' PROTAC toolbox towards heterobifunctional molecules, e.g. with biotin, fluorescent, hydrophobic and peptide tags.

The Hippo pathway effectors YAP and TAZ promote cell growth by modulating amino acid signaling to mTORC1.

YAP and TAZ are transcriptional co-activators and function as the major effectors of the Hippo tumor suppressor pathway, which controls cell growth, tissue homeostasis, and organ size. Here we show that YAP/TAZ play an essential role in amino acid-induced mTORC1 activation, particularly under nutrient-limiting conditions. Mechanistically, YAP/TAZ act via the TEAD transcription factors to induce expression of the high-affinity leucine transporter LAT1, which is a heterodimeric complex of SLC7A5 and SLC3A2. Deletion of YAP/TAZ abolishes expression of LAT1 and reduces leucine uptake. Re-expression of SLC7A5 in YAP/TAZ knockout cells restores leucine uptake and mTORC1 activation. Moreover, SLC7A5 knockout cells phenocopies YAP/TAZ knockout cells which exhibit defective mTORC1 activation in response to amino acids. We further demonstrate that YAP/TAZ act through SLC7A5 to provide cells with a competitive growth advantage. Our study provides molecular insight into the mechanism of YAP/TAZ target genes in cell growth regulation.