Therapeutic targeting of histone lysine demethylase KDM4B blocks the growth of castration-resistant prostate cancer.

Epigenetics is an emerging mechanism for tumorigenesis. Treatment that targets epigenetic regulators is becoming an attractive strategy for cancer therapy. The role of epigenetic therapy in prostate cancer (PCa) remains elusive. Previously we demonstrated that upregulation of histone lysine demethylase KDM4B correlated with the appearance of castration resistant prostate cancer (CRPC) and identified a small molecular inhibitor of KDM4B, B3. In this study, we further investigated the role of KDM4B in promoting PCa progression and tested the efficacy of B3 using clinically relevant PCa models including PCa cell line LNCaP and 22Rv1 and xenografts derived from these cell lines. In loss and gain-functional studies of KDM4B in PCa cells, we found that overexpression of KDM4B in LNCaP cells enhanced its tumorigenicity whereas knockdown of KDM4B in 22Rv1 cells reduced tumor growth in castrated mice. B3 suppressed the growth of 22Rv1 xenografts and sensitized tumor to anti-androgen receptor (AR) antagonist enzalutamide inhibition. B3 also inhibited 22Rv1 tumor growth synergistically with rapamycin, leading to cell apoptosis. Comparative transcriptomic analysis performed on KDM4B knockdown and B3-treated 22Rv1 cells revealed that B3 inhibited both H3K9me3 and H3K27me3 demethylase activities. Our studies establish KDM4B as a target for CRPC and B3 as a potential therapeutic agent. B3 as monotherapy or in combination with other anti-PCa therapeutics offers proof of principle for the clinical translation of epigenetic therapy targeting KDMs for CRPC patients.

Histone lysine dimethyl-demethylase KDM3A controls pathological cardiac hypertrophy and fibrosis.

Left ventricular hypertrophy (LVH) is a major risk factor for cardiovascular morbidity and mortality. Pathological LVH engages transcriptional programs including reactivation of canonical fetal genes and those inducing fibrosis. Histone lysine demethylases (KDMs) are emerging regulators of transcriptional reprogramming in cancer, though their potential role in abnormal heart growth and fibrosis remains little understood. Here, we investigate gain and loss of function of an H3K9me2 specific demethylase, Kdm3a, and show it promotes LVH and fibrosis in response to pressure-overload. Cardiomyocyte KDM3A activates Timp1 transcription with pro-fibrotic activity. By contrast, a pan-KDM inhibitor, JIB-04, suppresses pressure overload-induced LVH and fibrosis. JIB-04 inhibits KDM3A and suppresses the transcription of fibrotic genes that overlap with genes downregulated in Kdm3a-KO mice versus WT controls. Our study provides genetic and biochemical evidence for a pro-hypertrophic function of KDM3A and proof-of principle for pharmacological targeting of KDMs as an effective strategy to counter LVH and pathological fibrosis.

Protein determinants of phage T4 lysis inhibition.

Genetic studies have established that lysis inhibition in bacteriophage T4 infections occurs when the RI antiholin inhibits the lethal hole-forming function of the T holin. The T-holin is composed of a single N-terminal transmembrane domain and a ~20 kDa periplasmic domain. It accumulates harmlessly throughout the bacteriophage infection cycle until suddenly causing permeabilization of the inner membrane, thereby initiating lysis. The RI antiholin has a SAR domain that directs its secretion to the periplasm, where it can either be inactivated and degraded or be activated as a specific inhibitor of T. Previously, it was shown that the interaction of the soluble domains of these two proteins within the periplasm was necessary for lysis inhibition. We have purified and characterized the periplasmic domains of both T and RI. Both proteins were purified in a modified host that allows disulfide bond formation in the cytoplasm, due to the functional requirement of conserved disulfide bonds. Analytical centrifugation and circular dichroism spectroscopy showed that RI was monomeric and exhibited ~80% alpha-helical content. In contrast, T exhibited a propensity to oligomerize and precipitate at high concentrations. Incubation of RI with T inhibits this aggregation and results in a complex of equimolar T and RI content. Although gel filtration analysis indicated a complex mass of 45 kDa, intermediate between the predicted 30 kDa heterodimer and 60 kDa heterotetramer, sedimentation velocity analysis indicated that the predominant species is the former. These results suggest that RI binding to T is necessary and sufficient for lysis inhibition.

Regulation of TFEB and V-ATPases by mTORC1.

Mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is an important, highly conserved, regulator of cell growth. Ancient among the signals that regulate mTORC1 are nutrients. Amino acids direct mTORC1 to the surface of the late endosome/lysosome, where mTORC1 becomes receptive to other inputs. However, the interplay between endosomes and mTORC1 is poorly understood. Here, we report the discovery of a network that links mTORC1 to a critical component of the late endosome/lysosome, the V-ATPase. In an unbiased screen, we found that mTORC1 regulated the expression of, among other lysosomal genes, the V-ATPases. mTORC1 regulates V-ATPase expression both in cells and in mice. V-ATPase regulation by mTORC1 involves a transcription factor translocated in renal cancer, TFEB. TFEB is required for the expression of a large subset of mTORC1 responsive genes. mTORC1 coordinately regulates TFEB phosphorylation and nuclear localization and in a manner dependent on both TFEB and V-ATPases, mTORC1 promotes endocytosis. These data uncover a regulatory network linking an oncogenic transcription factor that is a master regulator of lysosomal biogenesis, TFEB, to mTORC1 and endocytosis.

Interplay between pVHL and mTORC1 pathways in clear-cell renal cell carcinoma.

mTOR complex 1 (mTORC1) is implicated in cell growth control and is extensively regulated. We previously reported that in response to hypoxia, mTORC1 is inhibited by the protein regulated in development and DNA damage response 1 (REDD1). REDD1 is upregulated by hypoxia-inducible factor (HIF)-1, and forced REDD1 expression is sufficient to inhibit mTORC1. REDD1-induced mTORC1 inhibition is dependent on a protein complex formed by the tuberous sclerosis complex (TSC)1 and 2 (TSC2) proteins. In clear-cell renal cell carcinoma (ccRCC), the von Hippel-Lindau (VHL) gene is frequently inactivated leading to constitutive activation of HIF-2 and/or HIF-1, which may be expected to upregulate REDD1 and inhibit mTORC1. However, mTORC1 is frequently activated in ccRCC, and mTORC1 inhibitors are effective against this tumor type; a paradox herein examined. REDD1 was upregulated in VHL-deficient ccRCC by in silico microarray analyses, as well as by quantitative real-time PCR, Western blot, and immunohistochemistry. Vhl disruption in a mouse model was sufficient to induce Redd1. Using ccRCC-derived cell lines, we show that REDD1 upregulation in tumors is VHL dependent and that both HIF-1 and HIF-2 are, in a cell-type-dependent manner, recruited to, and essential for, REDD1 induction. Interestingly, whereas mTORC1 is responsive to REDD1 in some tumors, strategies have evolved in others, such as mutations disrupting TSC1, to subvert mTORC1 inhibition by REDD1. Sequencing analyses of 77 ccRCCs for mutations in TSC1, TSC2, and REDD1, using PTEN as a reference, implicate the TSC1 gene, and possibly REDD1, as tumor suppressors in sporadic ccRCC. Understanding how ccRCCs become refractory to REDD1-induced mTORC1 inhibition should shed light into the development of ccRCC and may aid in patient selection for molecular-targeted therapies.

Exploring a glycolytic inhibitor for the treatment of an FH-deficient type-2 papillary RCC.

BACKGROUND: A 24-year-old woman presented with a 45 cm complex cystic renal mass, which was resected. The tumor was a type-2 papillary renal cell carcinoma (pRCC-2), and several nodules remained. The patient was treated with mammalian target of rapamycin complex 1 (mTORC1) inhibitors, but after 5 months the tumor had progressed. Genetic testing of the patient revealed a novel heterozygous germline mutation in the gene encoding fumarate hydratase (FH), an enzyme of the tricarboxylic acid (TCA) cycle. As the tumor exhibited loss of heterozygosity for FH and markedly reduced FH activity, and in the absence of other established therapies, treatment with the glycolytic inhibitor 2DG (2-deoxy-D-glucose) was explored. INVESTIGATIONS: CT, histology, immunohistochemistry, genetic studies, 2-deoxy-2-(¹8F)fluoro-D-glucose (¹8FDG)-PET/CT, FH enzymatic assays, reconstitution experiments and in vitro studies of the effects of 2DG on FH-deficient tumor cells. DIAGNOSIS: pRCC-2 arising in a patient with a novel germline FH mutation and de novo hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome progressing after mTORC1 inhibitor therapy. MANAGEMENT: Surgical resection of the renal mass, treatment with mTORC1 inhibitors followed by 2DG. Unfortunately, 2DG was not effective, and the patient died several weeks later.

The N-terminal transmembrane domain of lambda S is required for holin but not antiholin function.

The lambda S gene encodes a holin, S105, and an antiholin, S107, which differs by its Met-Lys N-terminal extension. The model for the lysis-defective character of S107 stipulates that the additional N-terminal basic residue keeps S107 from assuming the topology of S105, which is N-out, C-in, with three transmembrane domains (TMDs). Here we show that the N terminus of S105 retains its fMet residue but that the N terminus of S107 is fully deformylated. This supports the model that in S105, TMD1 inserts into the membrane very rapidly but that in S107, it is retained in the cytoplasm. Further, it reveals that, compared to S105, S107 has two extra positively charged moieties, Lys2 and the free N-terminal amino group, to hinder its penetration into an energized membrane. Moreover, an allele, S105(DeltaTMD1), with TMD1 deleted, was found to be defective in lysis, insensitive to membrane depolarization, and dominant to the wild-type allele, indicating that the lysis-defective, antiholin character of S107 is due to the absence of TMD1 from the bilayer rather than to its ectopic localization at the inner face of the cytoplasmic membrane. Finally, the antiholin function of the deletion protein was compromised by the substitution of early-lysis missense mutations in either the deletion protein or parental S105 but restored when both S105(DeltaTMD1) and holin carried the substitution.

Rz/Rz1 lysis gene equivalents in phages of Gram-negative hosts.

Under usual laboratory conditions, lysis by bacteriophage lambda requires only the holin and endolysin genes, but not the Rz and Rz1 genes, of the lysis cassette. Defects in Rz or Rz1 block lysis only in the presence of high concentrations of divalent cations. The lambda Rz and Rz1 lysis genes are remarkable in that Rz1, encoding an outer membrane lipoprotein, is completely embedded in the +1 register within Rz, which itself encodes an integral inner membrane protein. While Rz and Rz1 equivalents have been identified in T7 and P2, most phages, including such well-studied classic phages as T4, P1, T1, Mu and SP6, lack annotated Rz/Rz1 equivalents. Here we report that a search strategy based primarily on gene arrangement and membrane localization signals rather than sequence similarity has revealed that Rz/Rz1 equivalents are nearly ubiquitous among phages of Gram-negative hosts, with 120 of 137 phages possessing genes that fit the search criteria. In the case of T4, a deletion of a non-overlapping gene pair pseT.2 and pseT.3 identified as Rz/Rz1 equivalents resulted in the same divalent cation-dependent lysis phenotype. Remarkably, in T1 and six other phages, Rz/Rz1 pairs were not found but a single gene encoding an outer membrane lipoprotein with a C-terminal transmembrane domain capable of integration into the inner membrane was identified. These proteins were named "spanins," since their protein products are predicted to span the periplasm providing a physical connection between the inner and outer membranes. The T1 spanin gene was shown to complement the lambda Rz-Rz1- lysis defect, indicating that spanins function as Rz/Rz1 equivalents. The widespread presence of Rz/Rz1 or their spanin equivalents in phages of Gram-negative hosts suggests a strong selective advantage and that their role in the ecology of these phages is greater than that inferred from the mild laboratory phenotype.

The T4 RI antiholin has an N-terminal signal anchor release domain that targets it for degradation by DegP.

Lysis inhibition (LIN) of T4-infected cells was one of the foundational experimental systems for modern molecular genetics. In LIN, secondary infection of T4-infected cells results in a dramatically protracted infection cycle in which intracellular phage and endolysin accumulation can continue for hours. At the molecular level, this is due to the inhibition of the holin, T, by the antiholin, RI. RI is only 97 residues and contains an N-terminal hydrophobic domain and a C-terminal hydrophilic domain; expression of the latter domain fused to a secretory signal sequence is sufficient to impose LIN, due to its specific interaction with the periplasmic domain of the T holin. Here we show that the N-terminal sequence comprises a signal anchor release (SAR) domain, which causes the secretion of RI in a membrane-tethered form and then its subsequent release into the periplasm, without proteolytic processing. Moreover, the SAR domain confers both functional lability and DegP-mediated proteolytic instability on the released form of RI, although LIN is not affected in a degP host. These results are discussed in terms of a model for the activation of RI in the establishment of the LIN state.

Periplasmic domains define holin-antiholin interactions in t4 lysis inhibition.

Bacteriophage T4 effects host lysis with a holin, T, and an endolysin, E. T and E accumulate in the membrane and cytoplasm, respectively, throughout the period of late gene expression. At an allele-specific time, T triggers to disrupt the membrane, allowing E to enter the periplasm and attack the peptidoglycan. T triggering can be blocked by secondary infections, leading to the state of lysis inhibition (LIN). LIN requires the T4 antiholin, RI, and is sensitive to the addition of energy poisons. T is unusual among holins in having a large C-terminal periplasmic domain. The rI gene encodes a polypeptide of 97 residues, of which 72 are predicted to be a periplasmic domain. Here, we show that the periplasmic domain of RI is necessary and sufficient to block T-mediated lysis. Moreover, when overexpressed, the periplasmic domain of T (T(CTD)) was found to abolish LIN in T4 infections and to convert wild-type (wt) T4 plaques from small and fuzzy edged to the classic "r" large, sharp-edged plaque morphology. Although RI could be detected in whole cells, attempts to monitor it during subcellular fractionation were unsuccessful, presumably because RI is a highly unstable protein. However, fusing green fluorescence protein (GFP) to the N terminus of RI created a more stable chimera that could be demonstrated to form complexes with wild-type T(CTD) and also with its LIN-defective T75I variant. These results suggest that the function of the unusual periplasmic domain of T is to transduce environmental information for the real-time control of lysis timing.