Design and optimization of oestrogen receptor PROTACs based on 4-hydroxytamoxifen.

In the last four decades, treatment of oestrogen receptor positive (ER+) breast cancer (BCa), has focused on targeting the estrogenic receptor signaling pathway. This signaling function is pivotal to sustain cell proliferation. Tamoxifen, a competitive inhibitor of oestrogen, has played a major role in therapeutics. However, primary and acquired resistance to hormone blockade occurs in a large subset of these cancers, and new approaches are urgently needed. Aromatase inhibitors and receptor degraders were approved and alternatively used. Yet, resistance appears in the metastatic setting. Here we report the design and synthesis of a series of proteolysis targeting chimeras (PROTACs) that induce the degradation of estrogen receptor alpha in breast cancer MCF-7 (ER+) cells at nanomolar concentration. Using a warhead based on 4-hydroxytamoxifen, bifunctional degraders recruiting either cereblon or the Von Hippel Lindau E3 ligases were synthesized. Our efforts resulted in the discovery of TVHL-1, a potent ERα degrader (DC50: 4.5 nM) that we envisage as a useful tool for biological study and a platform for potential therapeutics

Functional interplay between ER and MAF transcription factors in breast cancer bone metastasis

[eng] Metastatic breast cancer (BCa) is the leading cause of death in BCa patients. Current therapies for BCa aim at cell proliferation and, therefore, latent disseminated cells are able to escape treatment. For this reason, there is an urgent need to understand the molecular mechanisms by which BCa cells invade distant tissues to develop new therapeutic opportunities. It was recently discovered that the v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog (MAF) transcription factor drives specifically bone colonization in estrogen receptor (ER)-positive BCa patients. MAF was shown to transcriptionally regulate genes that support metastatic functions, thereby emerging as an attractive target for the prevention of BCa metastasis to the bone. However, the nuclear localization of MAF, its lack of enzymatic activity and its intrinsically disordered structure made this transcription factor a challenging therapeutic target and brought to the forefront the potential of MAF-transcriptionally controlled proteins as well as MAF-interacting partners to become pharmacological targets for the prevention of bone metastasis. For this reason, in the present thesis, we performed a proximity-dependent biotin identification (BioID) screen to identify MAF-interacting partners that may cooperate with this transcription factor to promote bone colonization. BioID proximity labeling captured the MAF interactome in BCa cells, mainly comprised of chromatin-remodeling proteins that may influence histone modification to generate transcriptionally active or repressive chromatin structures for gene expression regulation. Moreover, ER, the main driver of ER+ BCa, emerged as a MAF interactor. MAF-positive tumors treated with bisphosphonates, a major therapeutic option for the treatment of bone metastasis, were associated with increased adverse outcomes in premenopausal patients, thereby supporting a role for estrogen (E2), the main ER ligand, in the modification of MAF-positive tumors behavior. Given this observation, we considered the MAF-ER interaction of major interest and explored the molecular mechanisms underlying the cross-talk of both transcription factors. Gene expression analyses revealed a MAF-E2 signature, which suggested that MAF modulates the E2 response either by triggering a switch of target genes or by potentiating the expression of specific E2-responsive genes. Interrogation of the chromatin landscape in BCa cells led to the observation that MAF expression was associated with increased chromatin accessibility. Moreover, assessment of ER binding to chromatin showed an increase in ER recruitment to DNA regulatory regions depending on MAF presence. Thus, our results demonstrate that MAF recruits chromatin-remodeling proteins to generate accessible chromatin regions, which result in an increase of ER recruitment to chromatin potentially to induce the expression of genes that confer metastatic properties

Rapamycin-induced miR-21 promotes mitochondrial homeostasis and adaptation in mTORC1 activated cells

mTORC1 hyperactivation drives the multi-organ hamartomatous disease tuberous sclerosis complex (TSC). Rapamycin inhibits mTORC1, inducing partial tumor responses; however, the tumors regrow following treatment cessation. We discovered that the oncogenic miRNA, miR-21, is increased in Tsc2-deficient cells and, surprisingly, further increased by rapamycin. To determine the impact of miR-21 in TSC, we inhibited miR-21 in vitro. miR-21 inhibition significantly repressed the tumorigenic potential of Tsc2-deficient cells and increased apoptosis sensitivity. Tsc2-deficient cells’ clonogenic and anchorage independent growth were reduced by ∼50% (p<0.01) and ∼75% (p<0.0001), respectively, and combined rapamycin treatment decreased soft agar growth by ∼90% (p<0.0001). miR-21 inhibition also increased sensitivity to apoptosis. Through a network biology-driven integration of RNAseq data, we discovered that miR-21 promotes mitochondrial adaptation and homeostasis in Tsc2-deficient cells. miR-21 inhibition reduced mitochondrial polarization and function in Tsc2-deficient cells, with and without co-treatment with rapamycin. Importantly, miR-21 inhibition limited Tsc2-deficient tumor growth in vivo, reducing tumor size by approximately 3-fold (p<0.0001). When combined with rapamcyin, miR-21 inhibition showed even more striking efficacy, both during treatment and after treatment cessation, with a 4-fold increase in median survival following rapamycin cessation (p=0.0008). We conclude that miR-21 promotes mTORC1-driven tumorigenesis via a mechanism that involves the mitochondria, and that miR-21 is a potential therapeutic target for TSC-associated hamartomas and other mTORC1-driven tumors, with the potential for synergistic efficacy when combined with rapalogs