Combinatorial targeting of a chromatin complex comprising Dot1L, menin and the tyrosine kinase BAZ1B reveals a new therapeutic vulnerability of endocrine therapy-resistant breast cancer

Abstract

Background Targeting vulnerabilities of cancer cells by inhibiting key regulators of cell proliferation or survival represents a promising way to overcome resistance to current therapies. In breast cancer (BC), resistance to endocrine therapy results from constitutively active or aberrant estrogen receptor alpha (ERα) signaling to the genome. Targeting components of the ERα pathway in these tumors represents, therefore, a rational way toward effective new treatments. Interaction proteomics identified several proteins associated with ERα in BC cells, including epigenetic complexes controlling gene transcription comprising the scaffold protein menin and the histone methyltransferase Dot1L. Methods We combined chromatin immunoprecipitation, transcriptome sequencing, siRNA-mediated gene knockdown (kd), pharmacological inhibition coupled to cellular and functional assays and interaction proteomics in antiestrogen (AE)-sensitive and AE-resistant human BC cell models to: map menin and Dot1L chromatin localization, search for their common and specific target genes, measure the effects of single or combinatorial knockdown or pharmacological inhibition of these proteins on cell proliferation and survival, and characterize their nuclear interactomes. Results Dot1L and menin associate in MCF-7 cells chromatin, where they co-localize in a significant fraction of sites, resulting in co-regulation of genes involved, among others, in estrogen, p53, HIF1α and death receptor signaling, regulation of cell cycle and epithelial-to-mesenchymal transition. Specific inhibitors of the two factors synergize with each other for inhibition of cell proliferation of AE (tamoxifen or fulvestrant)-sensitive and AE-resistant BC cells. Menin and Dot1L interactomes share a sizeable fraction of their nuclear partners, the majority being known BC fitness genes. Interestingly, these include B-WICH and WINAC complexes that share BAZ1B, a bromodomain protein comprising a tyrosine–protein kinase domain playing a central role in chromatin remodeling and transcriptional regulation. BAZ1B kd caused significant inhibition of ERα expression, proliferation and transcriptome changes resulting in inhibition of estrogen, myc, mTOR, PI3K and AKT signaling and metabolic pathways in AE-sensitive and AE-resistant BC cells. Conclusions Identification of a functional interplay between ERα, Dot1L, menin and BAZ1B and the significant effects of their co-inhibition on cell proliferation and survival in cell models of endocrine therapy-resistant BC reveal a new therapeutic vulnerability of these aggressive diseases.

Combinatorial targeting of menin and the histone methyltransferase DOT1L as a novel therapeutic strategy for treatment of chemotherapy-resistant ovarian cancer

“…Recently, evidences of complementary activities of menin and DOT1L inhibitors in NPM1-mutant and MLLrearranged leukaemia have been demonstrated [40], [10]. Moreover, the same result was obtained for estrogen receptor-positive breast cancer cells where synergic effect of pharmacological blockade of these two proteins on the proliferation has been shown and it was highlighted that menin represents DOT1L and ERα co-factor of in this cancer type [11]. Considering the profound effect of DOT1L inhibition on proliferation of PEO1 and PEO4 cells that has been evaluated in our previously published study where we demonstrated that treatment of OC cells with DOT1L inhibitors EPZ004777, EPZ5676 and SGC0946 induced a concentration-dependent inhibition of cell growth in PEO1 and PEO4 cells [4], we investigated whether the interaction between the two proteins occurs also in this cancer type and to estimate the effect of their simultaneous inhibition on proliferation of OC cells.…”

“…Lately, a pro-proliferative role of menin in multiple cancer types including leukaemia, hepatocellular carcinoma, breast, endometrial and prostate cancers has been demonstrated [ 13 ], [ 28 ], [ 29 ], [ 30 ], [ 31 ]. Moreover, elevated expression of gene encoding for menin protein ( MEN1 ) has been found in endometrioid and breast cancers, [ 30 ], [ 11 ], whereas in hepatocellular and prostate cancer MEN1 overexpression has been correlated to disease progression [ 28 ], [ 31 ]. In order to investigate menin role in OC development, we analysed RNA-seq data from ICGC [ 16 ] and GTEx expression profiling datasets and found out that MEN1 expression level is increased in ovarian tumours in comparison to normal tissues (Fig.…”

“…4 E, F). Moreover, such pathways as IL-8, ovarian cancer and estrogen receptor signalling pathways, previously described to be deregulated upon inhibition of DOT1L alone [ 4 ] or in combination with menin pharmacological blockade in other biological contexts [ 11 ] were also influenced. Altogether, these results indicate that combinatorial treatment of OC cells with menin and DOT1L inhibitors induces an additive antiproliferative effect on the cell growth, driven by pronounced deregulation of gene expression and signalling pathways.…”

“…Scaffold protein menin, encoded by multiple endocrine neoplasia 1 gene ( MEN1 ) and known to be involved in histone modification and epigenetic gene regulation, has been shown to physically bind DOT1L in leukaemia and breast cancer cells [ 10 ], [ 11 ]. Moreover, menin interaction with MLL is involved in development of acute leukaemias with translocations of the MLL gene [ 12 ], [ 13 ], as well as of solid tumours, including castration-resistant prostate cancer [ 14 ] and hepatocellular carcinoma [ 15 ].…”

Sini San inhibits breast cancer cell migration and angiogenesis via the HIF 1 /VEGF pathway

“…However, for women who have been diagnosed, survival has improved, but median survival is still very low. Most metastatic or recurrent breast tumors are incurable, and dramatically improving the survival rate remains the main aspiration of researchers [2]. Angiogenesis is the basic process of tumor malignant progression, and it plays an important role in early tumor metastasis [3].…”