Inhibition of the histone demethylase, KDM5B, directly induces re-expression of tumor suppressor protein HEXIM1 in cancer cells

Abstract

BackgroundThe tumor suppressor actions of hexamethylene bis-acetamide (HMBA)-inducible protein 1 (HEXIM1) in the breast, prostate, melanomas, and AML have been reported by our group and others. Increased HEXIM1 expression caused differentiation and inhibited proliferation and metastasis of cancer cells. Historically, HEXIM1 has been experimentally induced with the hybrid polar compound HMBA, but HMBA is a poor clinical candidate due to lack of a known target, poor pharmacological properties, and unfavorable ADMETox characteristics. Thus, HEXIM1 induction is an intriguing therapeutic approach to cancer treatment, but requires better chemical tools than HMBA.MethodsWe identified and verified KDM5B as a target of HEXIM1 inducers using a chemical proteomics approach, biotin–NeutrAvidin pull-down assays, surface plasmon resonance, and molecular docking. The regulation of HEXIM1 by KDM5B and KDM5B inhibitors was assessed using chromatin immunoprecipitation assays, RT-PCR, western blotting, and depletion of KDM5B with shRNAs. The regulation of breast cancer cell phenotype by KDM5B inhibitors was assessed using western blots, differentiation assays, proliferation assays, and a mouse model of breast cancer metastasis. The relative role of HEXIM1 in the action of KDM5B inhibitors was determined by depleting HEXIM1 using shRNAs followed by western blots, differentiation assays, and proliferation assays.ResultsWe have identified a highly druggable target, KDM5B, which is inhibited by small molecule inducers of HEXIM1. RNAi knockdown of KDM5B induced HEXIM1 expression, thus validating the specific negative regulation of tumor suppressor HEXIM1 by the H3K4me3/2 demethylase KDM5B. Known inhibitors of KDM5B were also able to induce HEXIM1 expression, inhibit cell proliferation, induce differentiation, potentiate sensitivity to cancer chemotherapy, and inhibit breast tumor metastasis.ConclusionHMBA and 4a1 induce HEXIM1 expression by inhibiting KDM5B. Upregulation of HEXIM1 expression levels plays a critical role in the inhibition of proliferation of breast cancer cells using KDM5B inhibitors. Based on the novel molecular scaffolds that we identified which more potently induced HEXIM1 expression and data in support that KDM5B is a target of these compounds, we have opened up new lead discovery and optimization directions.

Transcriptome and interactome analyses identify theTP53interacting geneRCCD1as a candidate susceptibility gene at the 15p26.1 breast and ovarian cancer risk locus

“…NR2F1 (n uclear receptor subfamily 2 group F, member 1) binds directly with ESR1 and is a member of the nuclear hormone receptor family of steroid hormone receptors, consistent with the underlying etiology and known role of steroid hormones in the risk of ovarian cancer 1,2 . Finally, KDM5B ( JARID1B ) regulates EMSY via its interaction with the micro-RNA miR-31 74 and induces re-expression of HEXIM1 , which was also enriched in in RCCD1 trans -interacting regions in ovarian cancer cells 75 .…”

Non-epigenetic induction of HEXIM1 by DNMT1 inhibitors and functional relevance

“…Our laboratory has invested considerable effort in the identification of agents that can induce HEXIM1 expression. In addition to unique HEXIM1 inducers like the compound 4a1 17 we have determined that inhibitors of the histone demethylase, KDM5B, also upregulated HEXIM1 expression 33 . 5-AzadC, as well as other agents that induce release P-TEFb, have been reported to upregulate HEXIM1 gene transcription 9 , 25 .…”

“…Western blotting analyses. Total protein was extracted from cells and subjected to western blot analyses as previously described 33 . Blots were cut into strips to minimize the amounts of antibodies required.…”

Lysine Demethylases: Promising Drug Targets in Melanoma and Other Cancers

“…Another recently identified KDM5 inhibitor is PBIT, shown to specially target and inhibit KDM5B ( Sayegh et al, 2013 ). In the context of cancer, PBIT treatment inhibited proliferation of breast cancer by derepression and upregulation of the tumor suppressor HEXIMI in vitro ( Montano et al, 2019 ). PBIT is yet to be tested in clinical trials.…”