Resistance mechanisms to TP53-MDM2 inhibition identified by in vivo piggyBac transposon mutagenesis screen in an Arf ^−/− mouse model

Abstract

Inhibitors of double minute 2 protein (MDM2)-tumor protein 53 (TP53) interaction are predicted to be effective in tumors in which the TP53 gene is wild type, by preventing TP53 protein degradation. One such setting is represented by the frequent CDKN2A deletion in human cancer that, through inactivation of p14ARF, activates MDM2 protein, which in turn degrades TP53 tumor suppressor. Here we used piggyBac (PB) transposon insertional mutagenesis to anticipate resistance mechanisms occurring during treatment with the MDM2-TP53 inhibitor HDM201. Constitutive PB mutagenesis in Arf −/− mice provided a collection of spontaneous tumors with characterized insertional genetic landscapes. Tumors were allografted in large cohorts of mice to assess the pharmacologic effects of HDM201. Sixteen out of 21 allograft models were sensitive to HDM201 but ultimately relapsed under treatment. A comparison of tumors with acquired resistance to HDM201 and untreated tumors identified 87 genes that were differentially and significantly targeted by the PB transposon. Resistant tumors displayed a complex clonality pattern suggesting the emergence of several resistant subclones. Among the most frequent alterations conferring resistance, we observed somatic and insertional loss-of-function mutations in transformation-related protein 53 (Trp53) in 54% of tumors and transposonmediated gain-of-function alterations in B-cell lymphoma-extra large (Bcl-xL), Mdm4, and two TP53 family members, resulting in expression of the TP53 dominant negative truncations ΔNTrp63 and ΔNTrp73. Enhanced BCL-xL and MDM4 protein expression was confirmed in resistant tumors, as well as in HDM201-resistant patient-derived tumor xenografts. Interestingly, concomitant inhibition of MDM2 and BCL-xL demonstrated significant synergy in p53 wild-type cell lines in vitro. Collectively, our findings identify several potential mechanisms by which TP53 wild-type tumors may escape MDM2-targeted therapy.drug resistance | MDM2 inhibitor | piggyBac screen | cancer A mong the genes most commonly altered in human cancer, regardless of tumor type, are tumor protein 53 (TP53) tumor suppressor (1) and CDKN2A (INK4a/ARF) (2). The latter gene encodes two tumor suppressor proteins: p16INK4a (3), an inhibitor of cyclin D-dependent kinases that, at least in part, regulates the function of the retinoblastoma protein (RB), and p19ARF (4), a negative regulator of double minute 2 protein (MDM2) function that activates TP53, thereby inducing cell cycle arrest or apoptosis. TP53 protein levels are regulated through MDM2-mediated degradation.Toward the goal of reactivating TP53 in cells harboring inactivating upstream pathway alterations, compounds inhibiting the interaction between MDM2 and TP53, preventing TP53 degradation, have been discovered. Such agents induce TP53 reactivation in tumors in which the TP53 gene is wild type (5-8).Although preclinical studies of such MDM2 inhibitors have demonstrated significant antitumor activity, tumors commonly relapse (9, 10).Rapid emergence of resistance is...

Targeting Resistance against the MDM2 Inhibitor RG7388 in Glioblastoma Cells by the MEK Inhibitor Trametinib

“…Acquired resistance mechanisms are still not understood, especially in glioblastoma. Potential mechanisms leading to acquired MDM2 inhibitor resistance are p53 mutations (21)(22)(23)(24) as well as enhanced B-cell lymphoma-extra-large (Bcl-xl) or MDM4 protein expression, offering the opportunity to be targeted by the addition of specific inhibitors (25).…”

A therapeutic patent overview of MDM2/X-targeted therapies (2014–2018)

“…Additionally, only in a subset of p53 wt cells that respond to MDM2 antagonists, the apoptosis program is induced, allowing for the successful cancer cell elimination [6,102,103]. Moreover, the induction of cell quiescence with limited cell elimination by apoptosis leads to the selection of drug-resistant clones, as first shown for nutlin-3 [104][105][106], and then for the more recent, clinically-evaluated MDM2 antagonists, such as SAR405838 [107,108], HDM201 [109] and RG7388 [110]. For this reasons, there is now a clear trend for the design of drug combination studies that would involve MDM2 antagonists.…”

Mouse models in the era of large human tumour sequencing studies

“…Additionally, as mutagenesis remains constitutively active in these models, TIM has also been used to identify drivers of metastasis formation [97] and acquired resistance to drug treatments [98,99]. Finally, new computational approaches based on RNA-sequencing data have been developed to improve target gene prediction and offer additional insight into the effects of insertions on the expression of the affected gene [100,101].…”