Ribosomes are abundant, large RNA–protein complexes that are the source of all protein synthesis in the cell. The production of ribosomes is an extremely energetically expensive cellular process that has long been linked to human health and disease. More recently, it has been shown that ribosome biogenesis is intimately linked to multiple cellular signalling pathways and that defects in ribosome production can lead to a wide variety of human diseases. Furthermore, changes in ribosome production in response to nutrient levels in the diet lead to metabolic re-programming of the liver. Reduced or abnormal ribosome production in response to cellular stress or mutations in genes encoding factors critical for ribosome biogenesis causes the activation of the tumour suppressor p53, which leads to re-programming of cellular transcription. The ribosomal assembly intermediate 5S RNP (ribonucleoprotein particle), containing RPL5, RPL11 and the 5S rRNA, accumulates when ribosome biogenesis is blocked. The excess 5S RNP binds to murine double minute 2 (MDM2), the main p53-suppressor in the cell, inhibiting its function and leading to p53 activation. Here, we discuss the involvement of ribosome biogenesis in the homoeostasis of p53 in the cell and in human health and disease.

Background: Antibody drug conjugate (ADC) technologies provide selective tumor killing with increased efficacy and less off-target toxicity than standard chemotherapy. Clinical validation of this approach for solid tumors has been limited, with trastuzumab emtansine, which targets HER2+ breast cancer, being the only ADC approved for treatment of solid tumors to date. Other ADCs targeting solid tumor indications have shown promise, including mirvetuximab soravtansine (MR), an ADC directed at folate receptor α (FRA). Clinical benefit has been observed in patients with high levels of FRA expression, demonstrating that FRA can be successfully targeted by this therapeutic modality. However, the anti-tumor activity for MR appears to be limited to patients whose tumors express the highest levels of FRA only. IKS01 is an ADC composed of an FRA-targeting antibody conjugated via Iksuda’s proprietary PermaLink conjugation technology, which provides fully stable conjugation thereby avoiding payload release during circulation, to the highly-potent FGX2-62 payload. FGX2-62 is a next generation pyrridinobenzodiazepine DNA mono-alkylator with pM potency in a range of tumor indications. IKS01 has been shown to have improved efficacy in both high and moderate/low FRA-expressing tumor xenograft models, compared to MR. We have extended these findings to show efficacy in an additional low FRA-expressing ovarian model, OVCAR3, and to confirm the FRA-dependence of IKS01 efficacy in the low FRA-expressing platinum-resistant OV-90 model. Methods: Conjugation of FGX2-62 to the FRA-binding antibody Isumab01 generated the ADC, IKS01, with a drug to antibody ratio (DAR) of approximately 2. FRA expression levels in cell lines used in mouse xenograft studies were determined by flow cytometry. IKS01 anti-tumor efficacy was evaluated in the low FRA-expressing ovarian adenocarcinoma OV-90 (platinum resistant) and OVCAR-3 human cell line-derived xenograft models. An FRA-targeting benchmark ADC with a format that has shown clinical efficacy was included for comparison. Target dependence was demonstrated in the OV-90 model by comparing the efficacy of IKS01 to an isotype control antibody conjugated to FGX2-62 (DAR of approximately 2). Results: IKS01 is highly effective in causing tumor regressions in the low FRA-expressing tumor models (OVCAR3 and platinum-resistant OV-90) in vivo, at doses that are well tolerated and where the benchmark ADC showed limited or no activity. The target-dependence of IKS01 efficacy in the OV-90 model was demonstrated by comparison to a non-targeting ADC which showed minimal efficacy compared to IKS01. Conclusions: The combination of a stable conjugation technology and a novel highly-potent DNA monoalkylator has led to the development of an ADC format that has shown target-dependent anti-tumor efficacy in a pre-clinical model of platinum-resistant ovarian cancer with low levels of FRA expression. These preclinical data suggest that IKS01 is a promising therapeutic candidate for the treatment of FRA-positive ovarian cancer, even when the level of surface expression of FRA is low. Citation Format: Jenny Thirlway, Adam Lodge, Andria Pelava, Daniel J Williamson, Davide Carta, Majid Al Nakeeb, Justyna Mysliwy, Paul J.M. Jackson, David E Thurston, Robert J Lutz. IKS01, a next generation antibody drug conjugate, shows target-dependent efficacy in a platinum-resistant tumor model with low levels of folate receptor α expression [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C023. doi:10.1158/1535-7163.TARG-19-C023

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Two of the four human ubiquitin-encoding genes express ubiquitin as an N-terminal fusion precursor polypeptide, with either ribosomal protein (RP) RPS27a or RPL40 at the C-terminus. RPS27a and RPL40 have been proposed to be important for the induction of the tumour suppressor p53 in response to defects in ribosome biogenesis, suggesting that they may play a role in the coordination of ribosome production, ubiquitin levels and p53 signalling. Here, we report that RPS27a is cleaved from the ubiquitin-RP precursor in a process that appears independent of ribosome biogenesis. In contrast to other RPs, the knockdown of either RPS27a or RPL40 did not stabilise the tumour suppressor p53 in U2OS cells. Knockdown of neither protein blocked p53 stabilisation following inhibition of ribosome biogenesis by actinomycin D, indicating that they are not needed for p53 signalling in these cells. However, the knockdown of both RPS27a and RPL40 in MCF7 and LNCaP cells robustly induced p53, consistent with observations made with the majority of other RPs. Importantly, RPS27a and RPL40 are needed for rRNA production in all cell lines tested. Our data suggest that the role of RPS27a and RPL40 in p53 signalling, but not their importance in ribosome biogenesis, differs between cell types.

Ribosome biogenesis is one of the biggest consumers of cellular energy. More than 20 genetic diseases (ribosomopathies) and multiple cancers arise from defects in the production of the 40S (SSU) and 60S (LSU) ribosomal subunits. Defects in the production of either the SSU or LSU result in p53 induction through the accumulation of the 5S RNP, an LSU assembly intermediate. While the mechanism is understood for the LSU, it is still unclear how SSU production defects induce p53 through the 5S RNP since the production of the two subunits is believed to be uncoupled. Here, we examined the response to SSU production defects to understand how this leads to the activation of p53 via the 5S RNP. We found that p53 activation occurs rapidly after SSU production is blocked, prior to changes in mature ribosomal RNA (rRNA) levels but correlated with early, middle and late SSU pre-rRNA processing defects. Furthermore, both nucleolar/nuclear LSU maturation, in particular late stages in 5.8S rRNA processing, and pre-LSU export were affected by SSU production defects. We have therefore uncovered a novel connection between the SSU and LSU production pathways in human cells, which explains how p53 is induced in response to SSU production defects.