Estrogen regulation of non-Hodgkin lymphomas and its underlying mechanisms

Most non-Hodgkin lymphoma (NHL), including diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL), show a higher male to female incidence ratio. The reason for this is not clear, but epidemiological and experimental studies have suggested a role for estrogens in NHL acting via estrogen receptor β (ERβ). My thesis aims to demonstrate the underlying molecular mechanisms of ERβ action in MCL. Secondly, I investigated genes affected in a sex-and female age-dependent manner in DLBCL to identify estrogen-regulated genes. Furthermore, except for the role of endogenous estrogens, the effect of xenoestrogen on NHL was studied. In addition, since the tumor microenvironment (TME) also contributes to tumor cell survival and drug sensitivity, aspects of the lymphoma-TME interaction were investigated. This included stromal cell-mediated ibrutinib (the Bruton tyrosine kinase inhibitor) resistance and how to overcome this resistance in MCL.

Paper I. In order to investigate ERβ-mediated effects on MCL tumors, ibrutinib-sensitive and resistant MCL cells were grafted to mice and treated with the ERβ selective agonist diarylpropinitrile (DPN). The transcriptome and chromatin binding of ERβ were studied in Granta-519 MCL tumors in response to the DPN treatment. The results showed that DPN treatment of both ibrutinib sensitive- and resistant MCL tumors in mice resulted in impaired tumor progression. Transcriptome analysis of Granta-519 tumors showed that genes affected by DPN were enriched in several biological processes, including cell-cell adhesion, endothelial mesenchymal transition, nuclear factor-kappaB signaling, vasculogenesis, lymphocyte proliferation, and apoptosis. In addition, individual genes that play a role in MCL progressions, such as SOX11 and MALAT1, were also downregulated. Furthermore, the data indicated an interaction between the lymphoma cells and the tumor microenvironment in response to the ERβ selective agonist.

Our results demonstrate how MCL is under the control of ERβ and elucidate the molecular mechanism for the impaired tumor progression. Targeting ERβ with a selective agonist may be an additional choice for treating both ibrutinib sensitive and resistant MCL tumors.

Paper II. The aim of this paper was to investigate the sex difference and estrogen effects in DLBCL. For this, we analyzed gene expression data from an RNA sequencing cohort containing 746 DLBCL samples. Our results showed that 1293 genes were differentially expressed between females and males (adj. p-value <0.05). Few genes showed common sex-regulated expression between germinal center B-cell (GCB) and activated B-cell lymphoma (ABC) DLBCL. A gene set enrichment analysis indicated that only 4 out of 13 significant pathways were common between the subtypes (adj. p-value <0.05). The number of differentially expressed genes between pre- vs. post-menopausal females in GCB and ABC subtype were 208 and 345 respectively, with only 5 being shared. When combining the differentially expressed genes between females vs. males and pre- vs. post-menopausal females, 9 genes were identified in ABC DLBCL that might be mediated by estrogens. Two of them, NR4A2 and MUC5B, showed increased and decreased expression, respectively. Interestingly, NR4A2 has been shown to work as a tumor suppressor in lymphoma, while a reduced MUC5B expression is associated with a less malignant phenotype in several cancers. NR4A2 and MUC5B expression were confirmed to be mediated by estrogens when the ABC-DLBCL cell line U2932 was grafted to mice. Our results demonstrate sex- and female age-dependent differences in gene expression between DLBCL subtypes, likely due to estrogens, which might help to explain the sex differences in DLBCL.

Paper III. Except for endogenous estrogens or synthetic ERβ selective agonists, we showed in this paper that environmental xenoestrogens like bisphenol (BPA) and the dietary isoflavone genistein can also inhibit the growth of both human MCL tumors and murine T cell lymphomas tumors in vivo by affecting lymphoma cell proliferation and apoptosis. BPA inhibited lymphoma growth at a dose safe for human exposure. In addition, soybean products rich in isoflavones might be advantageous for lymphoma patients.

Paper IV. In this study, we focused on the mechanism of stromal cells-mediated resistance of MCL cells to the Bruton tyrosine kinase inhibitor ibrutinib. Our findings demonstrated that stromal cells protected MCL cells from apoptosis induced by ibrutinib and contributed to the proliferation recovery after removing ibrutinib. Direct cell-cell adhesion rather than secreted cytokines explained this resistance, which involved impaired ibrutinib-mediated down-regulation of PI3K/AKT signaling. Using a p110α inhibitor (BYL719) or blocking VLA4 reversed the stromal cells-mediated ibrutinib resistance. In addition, combining ibrutinib with the p110α inhibitor increased the inhibition of MCL tumor growth in vivo in xenograft experiments. Our results suggest that combining ibrutinib treatment with a p110α inhibitor, or disrupting stromal cell-MCL cell interaction, may be a promising therapy to overcome stromal cell-mediated ibrutinib resistance in MCL.

List of scientific papers

I. Dan Huang, Zhiqiang Huang, Rajitha Indukuri, Chandrashekar Bangalore Revanna, Mattias Berglund, Jiyu Guan, Konstantin Yakimchuk, Anastasios Damdimopoulos, Cecilia Williams, Sam Okret. Estrogen receptor β (ESR2) transcriptome and chromatin-binding in a mantle cell lymphoma tumor model reveal the tumor suppressing mechanisms of estrogens. [Submitted]

II. Dan Huang, Mattias Berglund, Anastasios Damdimopoulos, Sam Okret. Sex- and female age dependent differences in gene expression in diffuse large B-cell lymphoma-possible estrogen effects. [Submitted]

III. Konstantin Yakimchuk, Chandrashekar Bangalore Revanna, Dan Huang, Jose Inzunza, Sam Okret. Suppression of lymphoma growth by the xenoestrogens bisphenol A and genistein. Endocr Connect. 2018 Dec;7(12):1472-1479.
https://doi.org/10.1530/EC-18-0459

IV. Jiyu Guan, Dan Huang, Konstantin Yakimchuk, Sam Okret. p110alpha Inhibition Overcomes Stromal Cell-Mediated Ibrutinib Resistance in Mantle Cell Lymphoma. Mol Cancer Ther. 2018 May;17(5):1090-1100.
https://doi.org/10.1158/1535-7163.MCT-17-0784