Disordered expression of the epidermal growth factor receptor (EGFR) has been associated with induction of DNA repair genes (e.g. XRCC1, ERCC1) and resistance to radiation and genotoxic drugs. However, our previous work showed that EGFR inhibition did not affect O6-methylguanine-DNA methyltransferase (MGMT)-mediated resistance. In order to block uncoupled events associated with EGFR and MGMT, we designed MR30, a single molecule termed “combi-molecule” that contains a quinazoline arm targeted to EGFR and an O6-benzylguanine (O6-BG) moiety to block MGMT. Molecular analysis of the mechanism of action of its two arms showed that: (a) it could block EGFR phosphorylation, (b) down-regulate the RAF-MAPK and the PI3K-AKT pathways, and (c) covalently modify MGMT through S-benzylation, as confirmed by MALDI analysis of a direct binding assay with isolated MGMT, (d) it induced a dose-dependent down-regulation of MGMT in lung and melanoma cells. The pleiotropic mechanism of action of MR30 culminated into strong growth inhibition (IC50: 0.018-6.02 μM), with superior activity when compared with an equimolar combination of gefitinib (a clinical EGFR inhibitor) and O6-BG (a known MGMT inhibitor). Pulse exposure experiments were required to attenuate the contribution of EGFR inhibition to the strong potency of MR30, thereby allowing to achieve the dose level required to sensitize cells to temozolomide (TMZ). Indeed, MR30 significantly sensitized EGFR-MGMT co-expressing cells to TMZ (p<0.05-0.0001). The results in toto suggest that MR30 is the first prototype of agents that may be used against tumours addicted to EGFR and to sensitize resistant tumours co-expressing EGFR and MGMT to TMZ.

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The median-effect principle proposed by Chou and Talalay is the most effective approach to parameterize interactions between several agents in combination. However, this method cannot be used to evaluate the effectiveness of equimolar drug combinations, which are comparative references for dual-targeting molecular design. Here, using data acquired through the development of “combi-molecules” blocking two kinases (e.g., EGFR-c-Src and EGFR-c-Met), we established potency indices for equimolar and dual-targeted inhibitors. If the fold difference (κ) between the IC50 of the two individual kinase inhibitors was >6, the IC50 of their equimolar combination resembled that of the more potent inhibitor. Hence, the “combi-targeting” of the two kinases was considered “imbalanced” and the combination ineffective. However, if κ ≤ 6, the IC50 of the combination fell below that of each individual drug and the combi-targeting was considered “balanced” and the combination effective. We also showed that combi-molecules should be compared with equimolar combinations only under balanced conditions and propose a new parameter Ω for validating their effectiveness. A multi-targeted drug is effective if Ω < 1, where Ω is defined as the IC50 of the drug divided by that of the corresponding equimolar combination. Our study provides a methodology to determine the in vitro potency of equimolar two-drug combinations as well as combi-/hybrid molecules inhibiting two different kinase targets.

Poly (ADP-ribose) polymerase (PARP) inhibitors are a novel therapeutic approach for the selective targeting of tumours with BRCA1/2 hereditary deficiencies. BRCA1/2 are two key proteins involved in homologous recombination (HR) repair. Although the therapeutic benefit of PARP inhibitors has now been proven in the clinic in patients with BRCA1/2 mutations, much effort has been deployed to expand the use of PARP inhibitors beyond tumours harbouring inherited deficiencies in HR- mediated DNA repair. Several combination modalities with agents that can induce HR deficiency and exploit the concept of “contextual synthetic lethality” have been reported for PARP inhibitors. Here we exploited the contextual synthetic lethality between PARP and the epidermal growth factor receptor (EGFR) to design and synthesize a novel dual EGFR targeted PARP inhibitor, termed ZSMR06. The results showed that: a) ZSMR06 is capable of inhibiting EGFR and PARP in whole cells in the nanomolar range (36-63 nM); b) it was selectively potent against BRCA2 mutant and EGFR expressing isogenic cells; c) as monotherapy, ZSMR06 was extremely potent with growth inhibitory activities superior to that of an equimolar combination of olaparib (a clinical PARP inhibitor) and gefitinib (a clinical EGFR inhibitor) in a large panel of tumour cell lines; d) ZSMR06 strongly potentiated the effect of temozolomide (TMZ) and induced significantly stronger (p&lt;0.05-0.001) growth inhibition than olaparib + TMZ combination in cells expressing O6-methylguanine-DNA methyltransferase (MGMT), an enzyme that confers a robust resistance to TMZ; e) ZSMR06 selectively potentiated TMZ on EGFR expressing cells; f) it was able to strongly sensitize bladder cancer cells to radiation (DMF50= 2.96-9.5). These results in toto showed that ZSMR06 due to its dual mechanism of action is capable of behaving as a strong potentiator of TMZ and radiation in resistant cells. Its potency in non-BRCA1/2 mutant tumour cells indicates that it may be the first prototype of hybrid molecules capable of expanding the use of PARP inhibition beyond BRCA1/2 mutations. Citation Format: Martin Rupp, Zhor Senhaji Mouhri, Ursula Stochaj, Bertrand Jean-Claude. Tandem targeting of poly (ADP-ribose) polymerase (PARP) and epidermal growth factor receptor (EGFR) as a novel strategy for enhancing radio- and chemosensitivity of refractory tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2925.

Over the past decade, PARP inhibition has been actively pursued as a novel approach for the selective therapy of tumors with BRCA1/2 mutations. The therapeutic benefits of PARP inhibitors have now been proven in the clinic against BRCA1/2 mutant ovarian cancers. This is hitherto limited to BRCA1/2 mutations, which only accounts for 5-10% of all cancers with hereditary mutations in the homologous recombination pathway. Therefore, new strategies are not only required to enhance the potency of PARP inhibitors but also to expand their use beyond BRCA mutation. While several combination modalities have been reported for PARP inhibitors, the concept of targeted PARP inhibitor has not yet been explored. Here using our novel combi-targeting approach, we report on the design of PARP inhibitors targeted to EGFR, a tyrosine kinase receptor overexpressed in several solid tumors. Recently, reports on the relationship between EGFR, PARP and BRCA have begun to emerge, one of which described a contextual synthetic lethality between EGFR and PARP (PloS one 7.10 (2012)). Here we report on the design and synthesis of novel PARP-EGFR combi-molecule based on structural modification of olaparib as a PARP inhibitor warhead and the quinazoline moiety for targeting EGFR. The results showed that: (a) it is capable of inducing a dose-dependent inhibition of PARP in isolated enzyme assay, (b) it induced a dose-dependent inhibition of EGFR in an isolated kinase assay, (c) it showed a dose-dependent inhibition of EGFR phosphorylation and downstream signaling in whole-cell assay, (d) it was selectively potent towards BRCA2 mutant and also EGFR-overexpressing cell lines, (d) it was extremely potent with activities superior to that of olaparib or gefitinib alone and their corresponding equimolar combination in three established triple negative breast cancer cell lines, (e) subcellular distribution analysis showed that it was abundantly localized in the perinuclear region. These results in toto suggest that this new combi-molecule could be developed as a single drug modality emulating the combination of PARP and EGFR inhibitors with the added benefit of being targeted to EGFR-expressing tumor cells. Citation Format: Zhor Senhaji Mouhri, Martin Rupp, Bertrand J. Jean-Claude. A novel combi-molecule engineered to target the putative synthetic lethal interactions between the epidermal growth factor receptor (EGFR) and poly(ADP-ribose)polymerase (PARP) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4035. doi:10.1158/1538-7445.AM2017-4035