Targeting neddylation sensitizes colorectal cancer to topoisomerase I inhibitors by inactivating the DCAF13-CRL4 ubiquitin ligase complex

Abstract

Colorectal cancers (CRCs) are prevalent worldwide, yet current treatments remain inadequate. Using chemical genetic screens, we identify that co-inhibition of topoisomerase I (TOP1) and NEDD8 is synergistically cytotoxic in human CRC cells. Combination of the TOP1 inhibitor irinotecan or its bioactive metabolite SN38 with the NEDD8-activating enzyme inhibitor pevonedistat exhibits synergy in CRC patient-derived organoids and xenografts. Mechanistically, we show that pevonedistat blocks the ubiquitin/proteasome-dependent repair of TOP1 DNA-protein crosslinks (TOP1-DPCs) induced by TOP1 inhibitors and that the CUL4-RBX1 complex (CRL4) is a prominent ubiquitin ligase acting on TOP1-DPCs for proteasomal degradation upon auto-NEDD8 modification during replication. We identify DCAF13, a DDB1 and Cullin Associated Factor, as the receptor of TOP1-DPCs for CRL4. Our study not only uncovers a replication-coupled ubiquitin-proteasome pathway for the repair of TOP1-DPCs but also provides molecular and translational rationale for combining TOP1 inhibitors and pevonedistat for CRC and other types of cancers.

Introduction

Colorectal cancer (CRC) remains one of the most frequent malignancies with 177,000 new cases and 58,000 deaths per year in the USA¹, with one-fifth of CRC patients presenting metastatic disease (mCRC). Currently, irinotecan in combination with fluorouracil is the first-line chemotherapy for mCRC^2,3. Novel molecular targets for combination with irinotecan are needed to improve CRC treatment.

Irinotecan is a prodrug converted within the cell to its active metabolite SN38, a potent camptothecin (CPT)-based drug targeting topoisomerase I (TOP1)^2,4. TOP1 is nuclear enzyme that relieves DNA torsional stress arising from replication, transcription, and chromatin compaction and relaxation^5,6. It dissipates DNA supercoils by cleaving one strand of the DNA double helix, allowing the broken strand to rotate (swivel) around the other strand. While cutting the DNA, TOP1 forms a transient catalytic intermediate termed TOP1 cleavage complex (TOP1cc) through a phosphotyrosyl linkage between the catalytic tyrosine residue of TOP1 and the 3’ end of the DNA. TOP1ccs are self-reversed upon resealing of the DNA break. CPT analogs trap TOP1ccs by binding at the interface between the enzyme and the DNA thereby occluding the resealing of the DNA break^7,8. The resulting irreversible TOP1ccs (which we refer to as TOP1 DNA-protein crosslinks or TOP1-DPCs), if left unrepaired, pose a serious threat to the genome as their bulky protein constituent blocks all chromatin-based processes and most importantly DNA replication⁹.

The ability of cancer cells to repair TOP1-DPCs is a key for their resistance to camptothecins^10,11. A pivotal step is the proteolysis of TOP1-DPCs, which enables tyrosyl-DNA phosphodiesterase 1 (TDP1) to hydrolyze the otherwise concealed phosphotyrosyl bond and endonucleases such as the Mre11-Rad50-Nbs1 complex to excise the DPCs by cleaving the adjacent DNA backbone^12,13,14,15. Although not fully understood, the proteolysis can be catalyzed by the ubiquitin-proteasome pathway (UPP) or by proteases including SPRTN or FAM111A, both of which are activated upon DNA replication collisions^16,17,18,19. Nonetheless, it remains largely unknown whether and how the UPP is activated against TOP1-DPC and regulated. We recently demonstrated that SUMOylation recruits the ubiquitin ligase RNF4 for the ubiquitylation and sequent proteasomal degradation of TOP1-DPCs in a replication- and transcription-independent manner²⁰. Because the SUMO pathway does not fully account for TOP1-DPC ubiquitylation and proteasomal degradation, it therefore can be conjectured that a parallel UPP partakes in TOP1-DPC removal in the context of DNA replication.

Neddylation is a post-translational modification by which the ubiquitin-like protein NEDD8 covalently targets substrate proteins through an enzymatic cascade akin to ubiquitylation^21,22. Neddylation plays a crucial role in cell viability and development and has been implicated in the repair of DNA damage such as DNA double-strand breaks^23,24,25. It is yet unknown whether neddylation plays a role in the repair of DNA-protein crosslinks.

In this work, we identify pevonedistat (PEV), a first-in-class inhibitor of neddylation recently approved for the treatment of high-risk myelodysplastic syndromes (HR-MDS), as synergistically cytotoxic with TOP1 inhibitors by high-throughput screens in human colorectal carcinoma cells. Neddylation activates the cullin 4 (CUL4)-RBX ubiquitin ligases (CRL4), which leads to K48-linked polyubiquitylation of TOP1-DPCs and their proteasomal degradation during DNA replication. We also discover that DCAF13, an understudied family member of the DDB1- and CUL4-asscoiated factors, binds the TOP1 core domain on nascent chromatin through its WD40 repeats, linking TOP1-DPCs to DDB1-CRL4 for the DPC ubiquitylation. These findings reveal a salient role of neddylation in the repair of TOP1-DPCs associated with DNA replication. They also provide proof of principle for combining pevonedistat with TOP1 inhibitors to treat CRCs and potentially other types of cancers.

Results

High-throughput screening identifies the synergistic combination of pevonedistat and TOP1 inhibitors in CRC cells

To identify effective small molecule inhibitors against CRC, we first assessed 2480 oncology-focused, approved, and investigational drugs in a library termed the NCATS Mechanism Interrogation Plate (MIPE) 5.0²⁶ in HCT116 human colorectal carcinoma cells. The library exploits redundancy by including multiple inhibitors of well-explored targets while encompassing mechanistic diversity, targeting more than 860 distinct molecular targets. We identified pevonedistat (PEV, also known as MLN-4924), as exhibiting a desired cytotoxicity profile (Fig. 1a and Supplementary Data S1). PEV is an adenosine monophosphate (AMP) mimetic, which forms a stable covalent adduct with NEDD8 in the catalytic pocket of the NEDD8-activating enzyme (NAE) heterodimer by competing with AMP and reacting with thioester-linked NEDD8 bound to the enzyme’s catalytic cysteine²⁷. This leads to inhibition of neddylation, a ubiquitin-like modification driving important biological processes including cell cycle regulation, viability and tissue development²¹. PEV is a first-in-class inhibitor currently in multiple clinical trials²⁸….