Identification and characterization of MAM03055A: A novel bivalent sigma-2 receptor/TMEM97 ligand with cytotoxic activity.

Sigma-2 receptor/transmembrane protein 97 (TMEM97) is upregulated in cancer cells compared to normal cells. Traditional sigma-2 receptor agonists induce apoptosis and autophagy, making them of interest in cancer therapy. Recently, we reported a novel metabolically stimulative function of the sigma-2 receptor, showing increased 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction and stimulation of glycolytic hallmarks. 6-Substituted analogs of the canonical sigma-2 receptor antagonist, 6-acetyl-3-(4-(4-(4-fluorophenyl)piperazin-1-yl)butyl)benzo[d]oxazol-2(3H)-one (SN79), produce both metabolically stimulative and cytotoxic effects. Here, we compare the activities of two related compounds: 6-amino-3-(4-(4-(4-fluorophenyl)piperazin-1-yl)butyl)benzo[d]oxazol-2(3H)-one (CM571), the 6-amino derivative of SN79, which binds with high affinity to both sigma-1 and sigma-2 receptors, and 1,3-bis(3-(4-(4-(4-fluorophenyl)piperazin-1-yl)butyl)-2-oxo-2,3-dihydrobenzo[d]oxazol-6-yl)thiourea (MAM03055A), a homo-bivalent dimer of CM571. MAM03055A resulted from the degradation of 3-(4-(4-(4-fluorophenyl)piperazin-1-yl)butyl)-6-isothiocyanatobenzo[d]oxazol-2(3H)-one (CM572), the cytotoxic 6-isothiocyanato SN79 derivative. MAM03055A exhibited high affinity and strong preference for sigma-2 receptors (sigma-1 Ki = 3371 nM; sigma-2 receptor Ki = 55.9 nM). Functionally, MAM03055A treatment potently induced cell death in SK-N-SH neuroblastoma, MDA-MB-231 breast, and both SW48 and SW480 colorectal cancer cell lines, causing proapoptotic BH3 interacting-domain death agonist (BID) cleavage in SK-N-SH cells. Conversely, CM571 induced metabolic stimulation. CM571 bound reversibly to both receptors, while MAM03055A bound pseudo-irreversibly to sigma-2 receptors and caused residual cytotoxic activity after acute exposure and removal of the compound from the media. Interestingly, MAM03055A induced a time-dependent loss of sigma-2 receptor/TMEM97 protein from cells, whereas monomer CM571 had no effect on receptor levels. These results suggest that monovalent and bivalent sigma-2 receptor ligands in this series interact differently with the receptor, thus resulting in divergent effects.

pH-Gated Succinate Secretion Regulates Muscle Remodeling in Response to Exercise.

In response to skeletal muscle contraction during exercise, paracrine factors coordinate tissue remodeling, which underlies this healthy adaptation. Here we describe a pH-sensing metabolite signal that initiates muscle remodeling upon exercise. In mice and humans, exercising skeletal muscle releases the mitochondrial metabolite succinate into the local interstitium and circulation. Selective secretion of succinate is facilitated by its transient protonation, which occurs upon muscle cell acidification. In the protonated monocarboxylic form, succinate is rendered a transport substrate for monocarboxylate transporter 1, which facilitates pH-gated release. Upon secretion, succinate signals via its cognate receptor SUCNR1 in non-myofibrillar cells in muscle tissue to control muscle-remodeling transcriptional programs. This succinate-SUCNR1 signaling is required for paracrine regulation of muscle innervation, muscle matrix remodeling, and muscle strength in response to exercise training. In sum, we define a bioenergetic sensor in muscle that utilizes intracellular pH and succinate to coordinate tissue adaptation to exercise.

HIF-independent synthetic lethality between CDK4/6 inhibition and VHL loss across species.

Inactivation of the VHL tumor suppressor gene is the signature initiating event in clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, and causes the accumulation of hypoxia-inducible factor 2α (HIF-2α). HIF-2α inhibitors are effective in some ccRCC cases, but both de novo and acquired resistance have been observed in the laboratory and in the clinic. Here, we identified synthetic lethality between decreased activity of cyclin-dependent kinases 4 and 6 (CDK4/6) and VHL inactivation in two species (human and Drosophila) and across diverse human ccRCC cell lines in culture and xenografts. Although HIF-2α transcriptionally induced the CDK4/6 partner cyclin D1, HIF-2α was not required for the increased CDK4/6 requirement of VHL-/- ccRCC cells. Accordingly, the antiproliferative effects of CDK4/6 inhibition were synergistic with HIF-2α inhibition in HIF-2α-dependent VHL-/- ccRCC cells and not antagonistic with HIF-2α inhibition in HIF-2α-independent cells. These findings support testing CDK4/6 inhibitors as treatments for ccRCC, alone and in combination with HIF-2α inhibitors.

Cyclin D-CDK4 relieves cooperative repression of proliferation and cell cycle gene expression by DREAM and RB.

The retinoblastoma protein (RB) restricts cell cycle gene expression and entry into the cell cycle. The RB-related protein p130 forms the DREAM (DP, RB-like, E2F, and MuvB) complex and contributes to repression of cell cycle-dependent genes during quiescence. Although both RB and DREAM bind and repress an overlapping set of E2F-dependent gene promoters, it remains unclear whether they cooperate to restrict cell cycle entry. To test the specific contributions of RB and DREAM, we generated RB and p130 knockout cells in primary human fibroblasts. Knockout of both p130 and RB yielded higher levels of cell cycle gene expression in G0 and G1 cells compared to cells with knockout of RB alone, indicating a role for DREAM and RB in repression of cell cycle genes. We observed that RB had a dominant role in E2F-dependent gene repression during mid to late G1 while DREAM activity was more prominent during G0 and early G1. Cyclin D-Cyclin-Dependent Kinase 4 (CDK4)-dependent phosphorylation of p130 occurred during early G1, and led to the release of p130 and MuvB from E2F4 and decreased p130 and MuvB binding to cell cycle promoters. Specific inhibition of CDK4 activity by palbociclib blocked DREAM complex disassembly during cell cycle entry. In addition, sensitivity to CDK4 inhibition was dependent on RB and an intact DREAM complex in both normal cells as well as in palbociclib-sensitive cancer cell lines. Although RB knockout cells were partially resistant to CDK4 inhibition, RB and p130 double knockout cells were significantly more resistant to palbociclib treatment. These results indicate that DREAM cooperates with RB in repressing E2F-dependent gene expression and cell cycle entry and supports a role for DREAM as a therapeutic target in cancer.

Divergent Cytotoxic and Metabolically Stimulative Functions of Sigma-2 Receptors: Structure-Activity Relationships of 6-Acetyl-3-(4-(4-(4-fluorophenyl)piperazin-1-yl)butyl)benzo[d]oxazol-2(3H)-one (SN79) Derivatives.

Sigma-2 receptors, recently identified as TMEM97, have been implicated in cancer and neurodegenerative disease. Structurally distinct sigma-2 receptor ligands induce cell death in tumor cells, linking sigma-2 receptors to apoptotic pathways. Recently, we reported that sigma-2 receptors can also stimulate glycolytic hallmarks, effects consistent with a prosurvival function and upregulation in cancer cells. Both apoptotic and metabolically stimulative effects were observed with compounds related to the canonical sigma-2 antagonist SN79. Here we investigate a series of 6-substituted SN79 analogs to assess the structural determinants governing these divergent effects. Substitutions on the benzoxazolone ring of the core SN79 structure resulted in high-affinity sigma-2 receptor ligands (K i = 0.56-17.9 nM), with replacement of the heterocyclic oxygen by N-methyl (producing N-methylbenzimidazolones) generally decreasing sigma-1 affinity and a sulfur substitution (producing benzothiazolones) imparting high affinity at both subtypes, lowering subtype selectivity. Substitution at the 6-position with COCH3, NO2, NH2, or F resulted in ligands that were not cytotoxic. Five of these ligands induced an increase in metabolic activity, as measured by increased reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) in human SK-N-SH neuroblastoma cells, further supporting a role for sigma-2 receptors in metabolism. Substitution with 6-isothiocyanate resulted in ligands that were sigma-2 selective and that irreversibly bound to the sigma-2 receptor, but not to the sigma-1 receptor. These ligands induced cell death upon both acute and continuous treatment (EC50 = 7.6-32.8 µM), suggesting that irreversible receptor binding plays a role in cytotoxicity. These ligands will be useful for further study of these divergent roles of sigma-2 receptors.

Synthetic Lethality Screens Using RNAi in Combination with CRISPR-based Knockout in Drosophila Cells.

A synthetic lethal interaction is a type of genetic interaction where the disruption of either of two genes individually has little effect but their combined disruption is lethal. Knowledge of synthetic lethal interactions can allow for elucidation of network structure and identification of candidate drug targets for human diseases such as cancer. In Drosophila, combinatorial gene disruption has been achieved previously by combining multiple RNAi reagents. Here we describe a protocol for high-throughput combinatorial gene disruption by combining CRISPR and RNAi. This approach previously resulted in the identification of highly reproducible and conserved synthetic lethal interactions (Housden et al., 2015).