The KDM5/JARID1 family of Fe(II)-and ␣-ketoglutarate-dependent demethylases remove methyl groups from tri-and dimethylated lysine 4 of histone H3. Accumulating evidence from primary tumors and model systems supports a role for KDM5A (JARID1A/RBP2) and KDM5B (JARID1B/PLU1) as oncogenic drivers. The KDM5 family is unique among the Jumonji domain-containing histone demethylases in that there is an atypical insertion of a DNA-binding ARID domain and a histone-binding PHD domain into the Jumonji domain, which separates the catalytic domain into two fragments (JmjN and JmjC). Here we demonstrate that internal deletion of the ARID and PHD1 domains has a negligible effect on in vitro enzymatic kinetics of the KDM5 family of enzymes. We present a crystal structure of the linked JmjN-JmjC domain from KDM5A, which reveals that the linked domain fully reconstitutes the cofactor (metal ion and ␣-ketoglutarate) binding characteristics of other structurally characterized Jumonji domain demethylases. Docking studies with GSK-J1, a selective inhibitor of the KDM6/ KDM5 subfamilies, identify critical residues for binding of the inhibitor to the reconstituted KDM5 Jumonji domain. Further, we found that GSK-J1 inhibited the demethylase activity of KDM5C with 8.5-fold increased potency compared with that of KDM5B at 1 mM ␣-ketoglutarate. In contrast, JIB-04 (a paninhibitor of the Jumonji demethylase superfamily) had the opposite effect and was ϳ8-fold more potent against KDM5B than against KDM5C. Interestingly, the relative selectivity of JIB-04 toward KDM5B over KDM5C in vitro translates to a ϳ10 -50-fold greater growth-inhibitory activity against breast cancer cell lines. These data define the minimal requirements for enzymatic activity of the KDM5 family to be the linked JmjNJmjC domain coupled with the immediate C-terminal helical zinc-binding domain and provide structural characterization of the linked JmjN-JmjC domain for the KDM5 family, which should prove useful in the design of KDM5 demethylase inhibitors with improved potency and selectivity.

Collective invasion, the coordinated movement of cohesive packs of cells, has become recognized as a major mode of metastasis for solid tumors. These packs are phenotypically heterogeneous and include specialized cells that lead the invasive pack and others that follow behind. To better understand how these unique cell types cooperate to facilitate collective invasion, we analyzed transcriptomic sequence variation between leader and follower populations isolated from the H1299 non-small cell lung cancer cell line using an image-guided selection technique. We now identify 14 expressed mutations that are selectively enriched in leader or follower cells, suggesting a novel link between genomic and phenotypic heterogeneity within a collectively invading tumor cell population. Functional characterization of two phenotype-specific candidate mutations showed that ARP3 enhances collective invasion by promoting the leader cell phenotype and that wild-type KDM5B suppresses chain-like cooperative behavior. These results demonstrate an important role for distinct genetic variants in establishing leader and follower phenotypes and highlight the necessity of maintaining a capacity for phenotypic plasticity during collective cancer invasion.

The estrogen pathway promotes growth of breast cancer, the leading cancer diagnosis among US women. The antiestrogen therapy Tamoxifen has been the mainline therapy for the 70% of women whose tumors are estrogen receptor alpha (ERα) positive for over thirty years. Understanding how ERα is regulated, particularly in the context of Tamoxifen, is essential for the treatment of breast cancer. In previous work, we showed that SetD7 mediated methylation of ERα at lysine 302 (K302) is a key mediator of estrogen receptor alpha stability. Downregulation of SetD7 or mutation of K302 increased the rate of ERα turnover resulting in a compromised estrogen driven transcriptional response. However, the precise mechanism by which lysine 302 methylation regulates ERα stability and transcriptional activity is not yet known. In this study, we used a novel small molecule inhibitor of SetD7, (R)-PFI-2, as a chemical probe to further investigate the role of K302 methylation in the regulation of ERα. Treatment of ERα expressing MCF7 breast cancer cells with (R)-PFI-2 resulted in a dose-dependent shift in the 66kDa ERα species to a slower migrating form as detected by protein electrophoresis and immunoblotting. Molecular mass estimates of this slower migrating form are consistent with a sumoylation event. Interestingly, (R)-PFI-2-induced accumulation of the slower migrating form was only observed upon inhibition of the proteasome with MG132, suggesting that this alternately modified form of ERα represents an intermediate in the pathway to degradation. A similar time-dependent shift to the same slower migrating form was observed upon estrogen depletion of MCF7 cells stably knocked down for SetD7, but not in control cells. Furthermore, an inverse relationship was observed between endogenous levels of SetD7 and the levels of modified ER in two strains of MCF7 cell line that differ in their sensitivity to Tamoxifen. These data suggest that methylation of ERα at K302 by SetD7 may stabilize ERα by blocking another post-translational modification, possibly sumoylation, necessary for its turnover. The relationship between ER methylation, sumoylation and Tamoxifen sensitivity will be discussed. Citation Format: Elizabeth L. Zoeller, Dalia Barsyte-Lovejoy, Peter J. Brown, Dafydd R. Owen, Cheryl H. Arrowsmith, Paula M. Vertino. Regulation of estrogen receptor turnover by lysine 302 methylation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2105. doi:10.1158/1538-7445.AM2014-2105

Histone modifying enzymes are often dysregulated during carcinogenesis and are major contributors to the development of oncogenic features, including proliferation, drug resistance, and metastasis. Given these roles, histone modifiers are promising new targets for oncological therapeutics. One of the enzyme families at the center of this area of research is the lysine demethylase family KDM5, for which several inhibitors are in development. KDM5A and KDM5B are frequently overexpressed or mutated in human non-small cell lung cancer (NSCLC). In overexpression studies, KDM5B promotes invasion and migration of NSCLC cells, whereas invasion and migration of NSCLC cells were decreased following knockdown of KDM5B. Furthermore, in patients with NSCLC, KDM5B is expressed at higher levels in brain metastasis sites when compared to both normal tissues and primary tumors. These findings suggest a role for KDM5B in lung cancer and metastatic spread. However, the precise role of KDM5 family members in lung cancer invasion and metastasis is not known. Recently, we discovered that KDM5B is differentially expressed in cell subtypes within a 3D model of NSCLC collective cell invasion. In this model isolated single cells at the forefront of invasive branches (leader cells) express more 2-4 fold more KDM5B protein than the cells following (follower cells), while KDM5A and KDM5C are evenly expressed across both cell types. KDM5B mRNA expression is similar across cell types suggesting that the differential protein expression is mediated at the posttranscriptional level. Interestingly, global H3K4me3 levels are decreased in leader cells as compared to follower cells, supporting the idea that the lysine demethylases targeting this residue may be expressed at higher levels in leader cells. Collectively, these data suggest that KDM5B could be contributing to initiation of invasion at the primary site and thus, promoting metastasis. Given the rising prominence of therapeutic inhibitors of KDM5 family members, a better understanding how KDM5B contributes to cell invasion may lead to a new approach to the prevention of metastasis. Citation Format: Elizabeth L. Zoeller, Jessica Konen, Joshua Bell, Emily Summerbell, Jeanne Kowalski, Adam Marcus, Paula Vertino. Regulation of invasion by lysine demethylase 5B in non-small cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1962. doi:10.1158/1538-7445.AM2017-1962