Deciphering the machinery involved in stability of the transcription factor HOXA9.

Informations projet

HOXA9 degradome

N° de convention de subvention: 702642

DOI

10.3030/702642

Projet clôturé

Date de signature de la CE 9 Mars 2016

Date de début 7 Novembre 2016

Date de fin 5 Septembre 2020

Financé au titre de

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Coût total

€ 239 860,80

Contribution de l’UE

€ 239 860,80

Coordonné par

DEUTSCHES KREBSFORSCHUNGSZENTRUM HEIDELBERG
Germany

Periodic Reporting for period 2 - HOXA9 degradome (Deciphering the machinery involved in stability of the transcription factor HOXA9.)

Période du rapport: 2018-11-07 au 2019-11-06

Résumé du contexte et des objectifs généraux du projet

The majority of small molecular drugs work as inhibitors designed for occupancy-based strategies. Drugs are designed to bind to the catalytical pocket of an enzyme and block its activity. However only a fraction of human proteins have catalytical pockets capable of this type of intervention.

Molecular glue degraders are small molecular drugs which induce interactions between proteins of interest (POI) with E3 ligases, resulting in the ubiquitination and degradation of the POI by leveraging protein – protein interactions between POI and E3 ligase.

My project seeks to identify and characterize novel molecular glue degraders. Drug-induced protein degradation holds promise for future targeted cancer treatments. This may ultimately result better outcomes for cancer patients, relieving an economic burden on the health care system.

Conclusions of the action

We developed a novel approach for molecular glue identification by correlation of the drug toxicity across multiple cancer cell lines with the expression levels of E3 ligases. This method enabled us to identify a new molecular glue – kinase inhibitor CR8. This approach can be used for newly generated datasets and hopefully will enable the discovery of additional molecular glues.

Previously described molecular glue degraders (thalidomide or indisulam analogs) induce interaction between the target and substrate receptor (DCAF). Kinase inhibitor CR8 is the first example of the published molecular glue degrader which induces the interaction between target and substrate adaptor. CR8 also modifies the function of the CDK12, transforming it from the kinase to drug-induced substrate receptor, linking DDB1 to the target for ubiquitination. Finally, cycK does not interact directly with CR8 and it is a primary substrate for degradation.

By dissecting and describing the molecular mechanism for BI-3802, which induces BCL6 polymerization, we provide structural frameworks for a rational design for a new generation of BCL6 targeting drugs. BI-3802 is a compound characterized with unique tri-modality, (1) BI-3802 inhibits BCL6 (prevents the interaction between BCL6 and co-repressor BCOR), (2) indices BCL6 polymerization (removes BCL6 from chromatin) and (3) initiates BCL6 degradation (promotes SIAH1 ubiquitination and subsequent proteasome degradation). The unique combination of the modalities determines BI-3802 excellent pharmacology.
We described a new drug modality - drug-induced polymerization. Structural characterization for the BI-3802 mechanism paves a potential path to rationally optimized new generation of BCL6 drugs as well as design the therapies to targeting other cancer dependencies by employing targeted polymerization mechanism.

In aggregate, projects which lead to the characterization of CR8 and BI-3802 as molecular glues demonstrated that modification of the compound surface-exposed moieties can induce gain-of-function glue properties, which provides rationality to modify the surface-exposed inhibitors to transform it into molecular glues. The surface of the protein is changed upon the introduction of the compounds that bind to the target. Since in every cell there are more than 20.000 different proteins with multiple surfaces, a chemical binder to the target can induce complementary to the protein surface of one of the other expressed proteins. Our work suggests there are more compounds that induce new protein interactions.

Travail effectué depuis le début du projet jusqu’à la fin de la période considérée dans le rapport et principaux résultats atteints jusqu’à présent

To characterize the scope of drug-induced degradation by novel IMiD derivatives, we employed a library of 6,572 C2H2 zinc fingers fused with green fluorescent protein (GFP) to observe protein degradation. This approach enabled us to identify zinc finger proteins degraded by CC-122 (avadomide) and CC-220 (iberdomide). CC-122 and CC-220 resulted in the degradation of distinct and unique patterns of C2H2 zinc fingers. The zinc finger ZKSC5 was exclusively degraded by CC-122, while the zinc finger IKZF2/4 was exclusively degraded by CC-220. Those results were published in Sievers QL, Petzold G, Bunker RD, Renneville A, Słabicki M, Liddicoat BJ, Abdulrahman W, Mikkelsen T, Ebert BL, Thomä NH. Defining the human C2H2 zinc finger degrome targeted by thalidomide analogs through CRBN. Science 362, 2018.

To identify novel molecular glue degraders, we correlated the viability data for 4,518 drugs with the expression levels of 499 E3 ligase components and identified that the toxicity of the kinase inhibitor CR8 correlates with the expression levels of an adaptor protein, DDB1. We showed that CR8 binds to CDK12 and induces a novel interaction with DDB1, resulting in the ubiquitination and degradation of CDK12 binding partner cycK. We demonstrated that this kinase inhibitor can gain glue activity by the chemical modification of the solvent exposed moiety. Our study suggest that other drugs can possess molecular glue activity. This work was published in Słabicki M*, Kozicka Z*, Petzold G*, Li Y, Manojkumar M, Bunker R, Donovan KA, Sievers QL, Koeppel J, Suchyta D, Sperling AS, Fink EC, Gasser JA, Wang LR, Corsello SM, Sellar RS, Jan M, Gillingham D, Scholl C, Fröhling S, Golub TR, Fischer ES, Thomä NH, Ebert BL The CDK inhibitor CR8 acts as a molecular glue degrader depleting cyclin K. Nature. 2020; *These authors contributed equally.

BCL6 is a master transcription factor, when overexpressed in certain blood cells, can lead to the development of lymphoma. Turning off BCL6 in these cells could lead to the regression of disease. Recently a compound was published, which induces BCL6 degradation by unknown mechanism. To elucidate this mechanism, we fused BCL6 to a green fluorescent protein (GFP) and observed the appearance of distinct BCL6-GFP-containing foci within minutes of degrader treatment, followed by their subsequent degradation. To understand this phenomenon, we purified recombinant BCL6 protein and observed that, upon degrader treatment, BCL6 formed long coil shaped filaments in vitro. Finally, we identified SIAH1, an E3 ubiquitin ligase which is required for this drug-induced BCL6 degradation and toxicity. A manuscript describing this work is accepted for publication Słabicki M*, Yoon H*, Koeppel J*, Nitsch L, Burman SSR, Di Genua C, Donovan KA, Sperling AS, Hunkeler M, Tsai JM, Sharma R, Guirguis A, Zou C, Chudasama P, Gasser JA, Miller PG, Scholl C, Fröhling S, Nowak RP, Fischer ES, Ebert BL. Small molecule-induced polymerization triggers degradation of BCL6. Nature.*These authors contributed equally

Progrès au-delà de l’état des connaissances et impact potentiel prévu (y compris l’impact socio-économique et les conséquences sociétales plus larges du projet jusqu’à présent)

CR8, a kinase inhibitor, binds to CDK12 and induces an interaction with adaptor protein DDB1. The gluing mechanism to DDB1 offers a unique opportunity to increase CR8 specificity as a CDK12 inhibitor. Potentially this mechanism could be employed to increase specificity for other kinase inhibitors.

In both of projects, we demonstrated that a small molecule inhibitor which has no effect on protein degradation can be used as a potent small molecule degrader due to the modification of a solvent exposed moiety. In the case of CR8, small molecule induces a new interaction with DDB1 and in the case of BCL6 it induces an interaction with an adjacent BTB domain dimer. These findings provide a rationale for the modification of the exposed drug moieties to gain novel functions, which might improve future of targeted drug therapy.

Molecular glue degrader