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

Estrogen receptor alpha and breast cancer

Breast cancer is the most common cancer and the leading cause of cancer-related death in women.¹ There are three major classes of breast cancer: hormone receptor (HR) positive/HER2 negative, HER2 positive, and triple negative. Breast cancers are also classified by their histology. Invasive ductal carcinoma is the most prevalent, occurring in 50-70% of patients. Invasive lobular carcinoma is the next most prevalent, in 5-15% of patients. Mixed ductal/lobal carcinomas and other less common histologies make up the remaining patients.²

Estrogen receptor alpha (ERα) is a hormone receptor expressed in approximately 70% of invasive breast cancers and is a key molecular target for breast cancer.^2,3 The presence of ERα characterizes the breast cancer as likely to respond to endocrine therapy. However, around 30% of ER+ breast cancers are unresponsive to endocrine therapy³ and approximately one third of patients will develop resistance to endocrine therapy.⁴

Estrogen regulates the cell cycle

Estrogen binds to ERα inducing homo-dimerization or hertero-dimerization with ERβ and subsequent translocation into the nucleus. Once inside the nucleus, ERα binds estrogen response elements (EREs) or interacts with cofactors to bind other regions of DNA.^5,6 ERα affects the expression of a wide variety of genes many of which are associated with cell cycle regulation, chromatin regulation, cell migration, and epithelial to mesenchymal transition. ERα plays a role in multiple cell cycle regulatory events including regulation of pRb, cyclin A, and cyclin D1, and inhibition of the cyclin-dependent kinase inhibitor, p27.⁷ It is well known that cell cycle dysregulation is a hallmark of cancer because it typically leads to uncontrolled cell growth illustrating how ERα clearly has oncogenic properties. Additionally, ERα is also a key regulator of growth and differentiation in breast tissue.⁷

The variety of functions of ERα can be attributed to interactions with different functional proteins. Chromatin regulators play a significant role in ERα function and have been shown to influence endocrine-therapy effectiveness in breast cancer. To identify better treatment strategies for ERα+ breast cancer, many groups investigated ERα binding proteins, and often focused on chromatin regulators. Dot1L, a histone lysine N-methyltransferase specific to H3 lysine 79, was found to be a key component of the ERα-mediated transcriptional regulation in breast cancer. Dot1L overexpression has been linked to many other cancers including leukemia, ovarian, and prostate cancer. Initial studies in cancer cell lines, mouse models, and clinical trials demonstrated that inhibition of Dot1L led to decreased growth in endocrine-responsive and -resistant breast cancer, along with leukemia and other cancers. However, in solid tumors the high concentration necessary for Dot1L inhibitors to be effective leads to side effects and low tolerability.

Dot1L, menin, and ERα co-localize to regulate gene expression

To improve the efficacy and feasibility of Dot1L inhibitors, researchers investigated combining these inhibitors with inhibitors for additional factors. One such factor to be considered is menin. Menin binds to and activates ERα, thereby regulating gene expression through interactions with additional regulatory factors. With strong support for targeting a combination of chromatin remodelers and other members of their protein complexes as an effective strategy for cancer therapy, the authors wanted to investigate whether Dot1L and menin would have potential to be one such combination.³ 

While Dot1L and menin are known to be functional partners with ERα, being co-expressed in ERα+ tumors, and individually they affect gene transcription and epigenetic control of chromatin activity, the interplay and cooperation between Dot1L and menin in ERα+ breast cancer had yet to be investigated. ChIP-WB identified 700 co-localization chromatin binding sites for Dot1L and menin. Most of these Dot1L+menin binding sites were located near enhancer regions and only a few were located near promoters. About half of the Dot1L+menin binding sites are also ERα binding sites, and a motif search through these binding sites identified a high frequency of EREs or ERE-like sequences. Alternatively, Dot1L+menin-enriched binding sites contained four enriched motifs: cell cycle dependent element (CDE), which controls cell cycle gene transcription in G2 and M; nuclear respiratory factor-1 (NRF-1), which acts with ER to modulate mitochondrial genes; and two members of the C2H2 zing finger transcription family (ZF15 and ZF36), which are recruiters of chromatin remodelers.

Transcriptomic studies were performed in the presence of pharmacological inhibitors of Dot1L (EPZ) and menin (MI-2). A common set of transcripts (231 down-regulated and 459 up-regulated) were identified in these studies. Of these transcripts, 38 contained binding sites for both Dol1L and menin. Many of the transcripts also belong to early and late estrogen responses, including cell cycle control genes.  

Inhibition of Dot1L and menin reduce breast cancer cell growth

With the knowledge that ERα, menin, and Dot1L likely interact and play an important role in the estrogen response, the authors investigated whether targeting Dot1L and menin simultaneously could present new therapeutic strategies for both estrogen-sensitive and antiestrogen (AE)-resistant breast cancers that are more effective and specific than Dot1L inhibition alone. Dot1L inhibitor (EPZ) and menin inhibitor (MI-136) each individually reduced the growth of estrogen-sensitive, tamoxifen-resistant, and fulvestrant/ICI-resistant breast cancer cells (MCF-7). When combined, these inhibitors significantly reduced growth of all luminal-like breast cancer cell models at concentrations that were suboptimal when used individually. These same findings were observed in cells with decreased Dot1L and menin expression due to siRNA silencing. Additionally, when Dot1L and menin were inhibited, a decrease in ERα expression was observed, which likely contributes to the anti-proliferative effect.  

BAZ1B is part of the Dot1L, menin, ERα complex

Interaction proteomics were used to identify the nuclear interactome of Dot1L and menin in AE-sensitive MCF-7 cells. Initial experiments identified 731 and 676 proteins that co-purified with Dot1L and menin respectively with 561 common between the two proteins. Functional enrichment analysis on the common proteins of the interactomes identified cellular processes relating to gene transcription and chromatin structure. This interactome data was then mined for networks known to be involved in chromatin dysregulation in breast cancer, and BAZ1B (Bromodomain Adjacent To Zinc Finger Domain 1B) also known as WSTF (Williams Syndrome Transcription Factor) was identified. BAZ1B is a central component of both B-WICH and WINAC chromatin remodeling complexes. The interaction between Dot1L, menin, and BAZ1B was confirmed with co-IP experiments. Using breast cancer datasets, BAZ1B was found to be expressed at higher levels in luminal-like breast cancer as compared to normal mammary tissue and was associated with decreased survival rates in ER+ breast cancer. These data suggest that BAZ1B may be an additional therapeutic target for endocrine therapy-resistant breast cancer.

Transcriptomics was performed on BAZ1B siRNA knockdown MCF-7 cells and compared to the transcriptomes of cells in which Dot1L or menin were pharmacologically inhibited. There were 123 common top regulated transcripts between the three data sets (62 down-regulated and 61 up-regulated). Knockdown of BAZ1B inhibited receptor-mediated transactivation of the reporter gene ERE-luc by down-regulating ERα expression. Complementation studies showed direct involvement of ERα in mediating BAZ1B effects. To further support BAZ1B as a therapeutic target, it was observed that growth in all the breast cancer cell models, even those modeling endocrine therapy-resistance, was reduced. This reduced growth was enhanced when BAZ1B knockdown was combined with Dot1L and menin knockdowns or inhibition.

Identifying new treatment strategies for cancer is an ongoing mission in patient care. Combinatorial therapeutics is enabling a multifaceted approach to treatment that can be manipulated based on specific cancer type. This study provides novel combinatorial targets (Dot1L+menin and Dot1L+menin+BAZ1B) for the treatment of ERα+ breast cancers that are likely to extent as treatment options to other cancers as well.

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References

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