^{Transcriptomics and Time-Course Proteomics Reveal Pclaf As an Inducer of Resistance Against Pevonedistat in Diffuse Large B-Cell Lymphoma}

Neddylation, a post-translational modification which regulates the activity or function of proteins but does not mediate their degradation by the 26S proteasome, attaches ubiquitin-like protein NEDD8 (Neural- precursor cell- expressed developmentally downregulated 8) to the substrates via enzymatic cascades. Cullins are classical substrates and inhibiting neddylation could hamper the activation of Cullin-RING ligases (CRLs) and further impede the ubiquitination.

Recently, it has been found that neddylation overactivation is prevalent in malignancies, and hence, targeting neddylation is an attractive antitumor strategy. Pevonedistat, a first-in-class small-molecule inhibitor specific for neddylation, exhibited good activity in preclinical models, but meanwhile its resistance was reported, which might be a dominant factor limiting its single-drug efficacy. Herein, we sought to elucidate the mechanism of Pevonedistat resistance in diffuse large B-cell lymphoma (DLBCL) and propose a novel combination strategy to improve therapeutic efficacy.

Firstly, we constructed acquired Pevonedistat-resistant cell lines of DLBCL (OCI-LY8-Pevonedistat resistant, OCI-LY8-PR; OCI-LY3- Pevonedistat resistant, OCI-LY3-PR) by incremental concentration approach. The resistance to Pevonedistat was authenticated by resistance index and its specificity was substantiated by cross resistance test to its downstream proteasome inhibitors, MG132 and Bortezomib. Subsequently, we examined the phenotypic differences between the parental and resistant cells. Pevonedistat significantly inhibited neddylation modification of Cullin, accompanied with the enhanced apoptosis and cell cycle arrest at G2/M phase in parental cells, which, nevertheless, was inapparent in resistant cells.

To unravel the mechanism of acquired resistance, we conducted transcriptomics between the parental and Pevonedistat-resistant cells, which suggested genes regulating cell cycle and DNA damage repair upregulated in resistant cells. For further exploration, we employed time-course proteomics, in which parental and resistant cells were treated without or with Pevonedistat for 2, 8, 24 hours and collected for mass spectrum, respectively. The cluster analysis of proteomics suggested that the proteins overexpressed in the resistant lines in all time points were significantly enriched in biological processes such as chromosome assembly and cell cycle, which were almost consistent with transcriptomics. However, the proteins highly expressed in the parental cells were mainly enriched in the metabolism of a variety of organic substances (Figure 1). Subsequently, we mainly targeted the upregulated proteins in the resistant cells, among which, PCNA clamp-associated factor (PCLAF), a PCNA-binding partner, was screened out for its great expression difference between resistant and parental cells. We confirmed PCLAF was upregulated in the resistant cells by immunoblotting and PCLAF overexpression entitled the parental cells resistance against Pevonedistat. We further explored the mechanism of PCLAF-mediated Pevonedistat resistance in DLBCL. The ubiquitination of PCLAF was hindered in Pevonedistat-resistant cells and the accumulation of PCLAF facilitated DNA damage repair and rescued DLBCL cells from cell cycle arrest after Pevonedistat treatment. These suggested that PCLAF mediated Pevonedistat resistance in DLBCL.

Subsequently, we sought to propose a combination therapeutic regimen based on PCLAF-driven Pevonedistat resistance. Given PCLAF functions in DNA damage repair, via drug library screening, we found a DNA damage repair inhibitor, Niraparib tosylate, significantly ameliorated DLBCL cells' resistance against Pevonedistat. The efficacy of combination was validated both in vitro and in vivo.

In conclusion, this study identified that PCLAF overexpression mediated the resistance against Pevonedistat in DLBCL and proposed a potential combination strategy to improve Pevonedistat resistance.