Sam Okret

Fig S1: Ibrutinib induces apoptosis and inhibits proliferation of Mino and Rec-1 cells but the effect is impaired by stromal cells. Fig S2: Stromal cells confer ibrutinib resistance by a tumor cell non-autonomous mechanism. Fig S3: Disrupting MCL cell interaction with stromal cells by blocking the adhesion molecule VLA4 overcomes ibrutinib resistance in vitro. Fig S4: Attachment to stromal cells supports MCL cell regrowth after ibrutinib removal. Fig S5: Stromal cells do not influence ibrutinib-mediated down-regulation of NF-κB target genes but impair ibrutinib-mediated down-regulation of pAKT signaling. Fig S6: p110α inhibition overcomes stromal cell-mediated ibrutinib resistance by restoring the ability of ibrutinib to down-regulate pAKT levels. Fig S7: Effects of ibrutinib or BYL719 on mice weight and expression of NF-kB target genes in the MCL tumors. Fig S8: p110α inhibitor BYL719 or ibrutinib do not induce MS-5 stromal cell apoptosis.

Supplementary Table S1 showing QPCR primers used in the study

For most lymphomas, including diffuse large B-cell lymphoma (DLBCL), the male incidence is higher, and the prognosis is worse compared to females. The reasons are unclear; however, epidemiological and experimental data suggest that estrogens are involved. With this in mind, we analyzed gene expression data from a publicly available cohort (EGAD00001003600) of 746 DLBCL samples based on RNA sequencing. We found 1293 genes to be differentially expressed between males and females (adj. p-value < 0.05). Few autosomal genes and pathways showed common sex-regulated expression between germinal center B-cell (GCB) and activated B-cell lymphoma (ABC) DLBCL. Analysis of differentially expressed genes between pre- vs. postmenopausal females identified 208 GCB and 345 ABC genes, with only 5 being shared. When combining the differentially expressed genes between females vs. males and pre- vs. postmenopausal females, nine putative estrogen-regulated genes were identified in ABC DLBCL. Two of them, NR4A2 and MUC5B, showed induced and repressed expression, respectively. Interestingly, NR4A2 has been reported as a tumor suppressor in lymphoma. We show that ABC DLBCL females with a high NR4A2 expression showed better survival. Inversely, MUC5B expression causes a more malignant phenotype in several cancers. NR4A2 and MUC5B were confirmed to be estrogen-regulated when the ABC cell line U2932 was grafted to mice. The results demonstrate sex- and female reproductive age-dependent differences in gene expression between DLBCL subtypes, likely due to estrogens. This may contribute to the sex differences in incidence and prognosis.

Mantle cell lymphoma (MCL) is a non-Hodgkin lymphoma with one of the highest male-to-female incidence ratios. The reason for this is not clear, but epidemiological as well as experimental data have suggested a role for estrogens, particularly acting through estrogen receptor β (ESR2). To study the ESR2 effects on MCL progression, MCL cells sensitive and resistant to the Bruton tyrosine kinase inhibitor ibrutinib were grafted to mice and treated with the ESR2-selective agonist diarylpropionitrile (DPN). The results showed that the DPN treatment of mice grafted with both ibrutinib-sensitive and -resistant MCL tumors resulted in impaired tumor progression. To identify the signaling pathways involved in the impaired tumor progression following ESR2 agonist treatment, the transcriptome and ESR2 binding to target genes were investigated by genome-wide chromatin immunoprecipitation in Granta-519 MCL tumors. DPN-regulated genes were enriched in several biological processes that included cell–cell adhesion, endothelial–mesenchymal transition, nuclear factor-kappaB signaling, vasculogenesis, lymphocyte proliferation, and apoptosis. In addition, downregulation of individual genes, such as SOX11 and MALAT1, that play a role in MCL progression was also observed. Furthermore, the data suggested an interplay between the lymphoma cells and the tumor microenvironment in response to the ESR2 agonist. In conclusion, the results clarify the mechanisms by which estrogens, via ESR2, impair MCL tumor progression and provide a possible explanation for the sex-dependent difference in incidence. Furthermore, targeting ESR2 with a selective agonist may be an additional option when considering the treatment of both ibrutinib-sensitive and -resistant MCL tumors.

Acquired resistance to cancer drugs is common, also for modern targeted drugs like the Bruton tyrosine kinase (BTK) inhibitor ibrutinib, a new drug approved for the treatment of the highly aggressive and relapsing mantle cell lymphoma (MCL). The tumor microenvironment often impacts negatively on drug response. Here, we demonstrate that stromal cells protect MCL cells from ibrutinib-induced apoptosis and support MCL cell regrowth after drug removal by impairing ibrutinib-mediated downregulation of PI3K/AKT signaling. Importantly, the stromal cell–mediated ibrutinib resistance was overcome in vitro by inhibiting AKT activity using the PI3K catalytic p110α subunit–specific inhibitor BYL719. This was seen both for MCL cell lines and primary MCL cells. Furthermore, inhibition of p110α activity by BYL719 potentiated the ability of ibrutinib to inhibit MCL tumor growth in vivo in a mouse xenograft model. The stromal cell–mediated ibrutinib resistance was found to be due to a direct interaction with MCL cells and involves the integrin VLA-4, as disrupting stromal cell–MCL cell interaction using a VLA-4 blocking antibody abrogated the ibrutinib resistance. This suggests that combined treatment with ibrutinib and a p110α inhibitor, alternatively by disrupting stromal cell–MCL cell interaction, may be a promising therapeutic strategy to overcome stromal cell–mediated ibrutinib resistance in MCL. Mol Cancer Ther; 17(5); 1090–100. ©2018 AACR.

Diffuse large B-cell lymphoma (DLBCL) shows a higher incidence in males versus females. Epidemiological studies have shown that female gender is a favorable prognostic factor, which may be explained by estrogens. Here we show that when grafting human DLBCL cells to immunocompromised mice, tumor growth in males is faster. When treating mice grafted with either germinal center or activated B-cell like DLBCL cells with the selective estrogen receptor β (ERβ) agonist diarylpropionitrile, tumor growth was significantly inhibited. Furthermore, nuclear ERβ1 expression analysis in primary DLBCL’s by immunohistochemistry revealed expression in 89% of the cases. Nuclear ERβ1 expression was in a univariate and multivariate analysis, an independent prognostic factor for adverse progression-free survival in Rituximab-chemotherapy treated DLBCL (p = 0.02 and p = 0.04, respectively). These results suggest that estrogen signaling through ERβ1 is an interesting future therapeutic target for treatment of DLBCL, and that ERβ1 expression can be used as a prognostic marker.

Inhibition of estrogen biosynthesis enhances lymphoma growth in mice

It has been known for decades that lowering the circulating glucocorticoid (GC) concentration as in Addison's disease or after removing the adrenals results in thymus enlargement, largely due to thymocyte expansion. This has been attributed to the loss of the proapoptotic effects on thymocytes by adrenal GCs. Here, we test this concept and report that ACTH directly controls thymic growth post‐adrenalectomy (ADX) independent of the proapoptotic effect of GCs. This was supported by the finding that ADX caused thymus enlargement and a 1.7‐fold ( P < 0.001) increase in thymocyte number in GR ^LckCre mice resistant to GC‐induced thymocyte apoptosis, similar to the increase seen in wild‐type mice (2.2‐fold; P < 0.01). We show by immunostaining that melanocortin receptor subtype 2, which selectively binds ACTH, is partly expressed on the thymic epithelium. Furthermore, ACTH in comparison to vehicle induced a 2.0‐fold ( P < 0.01) increase in fetal thymic organ culture thymocyte numbers in vitro and enhanced 2.2‐fold ( P < 0.05) the expression of delta‐like ligand 4, a factor that supports T‐cell development. Additionally, adrenalectomized GR ^LckCre mice treated with ACTH under conditions that repressed endogenous ACTH secretion showed increased thymocyte cellularity (1.9‐fold; P < 0.01) and splenic naive T‐cell numbers (2.5‐fold; P < 0.001) compared to when treated with PBS. Altogether, our results show that ACTH directly controls thymocyte homeostasis independent of GCs. These results revise the old paradigm behind compensatory thymus growth following ADX, now demonstrating that ACTH has a central role in regulating thymocyte expansion when systemic GC concentration is low.—Talaber, G., Tuckermann, J. P., Okret, S. ACTH controls thymocyte homeostasis independent of glucocorticoids. FASEB J. 29, 2526‐2534 (2015). www.fasebj.org

Estrogen receptor β (ERβ) is expressed in immune cells and studies have suggested an antiproliferative function of ERβ. We detected ERβ expression in murine T- and human B-cell lymphoma cell lines and analyzed the effects of estradiol and selective ERβ agonists on lymphoma growth in culture and in vivo. Treating the cells with estradiol had minor effects on cell growth, whereas the selective ERβ agonists diarylpropionitrile (DPN) and KB9520 showed a strong antiproliferative effect. When grafting mice with murine T-cell lymphoma cells, male mice developed larger tumors compared with female mice, a difference that was abolished following ovariectomy, showing estrogen-dependent growth in vivo. To investigate whether lymphoma growth may be inhibited in vivo by ERβ agonist treatment, mice grafted with murine lymphoma cells were treated with DPN or KB9520. Both ERβ-selective agonists strongly inhibited lymphoma growth. The reduced tumor size seen following either DPN or KB9520 treatment was due to reduced proliferation and increased apoptosis. Our results show an ERβ ligand-dependent antiproliferative effect of lymphoma cells expressing endogenous ERβ and that lymphoma cell growth in vivo can efficiently be inhibited by ERβ agonists. This suggests that ERβ agonists may be useful in the treatment of lymphomas.