Bromodomains (BD) are epigenetic readers of histone acetylation involved in chromatin remodeling and transcriptional regulation of several genes including protooncogene cellular myelocytomatosis ...(c-Myc). c-Myc is difficult to target directly by agents due to its disordered alpha helical protein structure and predominant nuclear localization. The epigenetic targeting of c-Myc by BD inhibitors is an attractive therapeutic strategy for prostate cancer (PC) associated with increased c-Myc upregulation with advancing disease. MT-1 is a bivalent BD inhibitor that is 100-fold more potent than the first-in-class BD inhibitor JQ1. MT-1 decreased cell viability and causes cell cycle arrest in G0/G1 phase in castration-sensitive and resistant PC cell lines in a dose-dependent fashion. The inhibition of c-Myc function by MT-1 was molecularly corroborated by the de-repression of Protein Kinase D1 (PrKD) and increased phosphorylation of PrKD substrate proteins: threonine 120, serine 11, and serine 216 amino acid residues in β-Catenin, snail, and cell division cycle 25c (CDC25c) proteins, respectively. The treatment of 3D cell cultures derived from three unique clinically annotated heavily pretreated patient-derived PC xenografts (PDX) mice models with increasing doses of MT-1 demonstrated the lowest IC
in tumors with c-Myc amplification and clinically resistant to Docetaxel, Cabazitaxel, Abiraterone, and Enzalutamide. An intraperitoneal injection of either MT-1 or in combination with 3jc48-3, an inhibitor of obligate heterodimerization with MYC-associated protein X (MAX), in mice implanted with orthotopic PC PDX, decreased tumor growth. This is the first pre-clinical study demonstrating potential utility of MT-1 in the treatment of PC with c-Myc dysregulation.
The cellular cytotoxicity of APY0201, a PIKfyve inhibitor, against multiple myeloma was initially identified in an unbiased
chemical library screen. The activity of APY0201 was confirmed in all 25 ...cell lines tested and in 40% of 100
patient-derived primary samples, with increased activity in primary samples harboring trisomies and lacking t(11;14). The broad anti-multiple myeloma activity of PIKfyve inhibitors was further demonstrated in confirmatory screens and showed the superior potency of APY0201 when compared to the PIKfyve inhibitors YM201636 and apilimod, with a mid-point half maximal effective concentration (EC
) at nanomolar concentrations in, respectively, 65%, 40%, and 5% of the tested cell lines. Upregulation of genes in the lysosomal pathway and increased cellular vacuolization were observed
following APY0201 treatment, although these cellular effects did not correlate well with responsiveness. We confirm that PIKfyve inhibition is associated with activation of the transcription factor EB, a master regulator of lysosomal biogenesis and autophagy. Furthermore, we established an assay measuring autophagy as a predictive marker of APY0201 sensitivity. Overall, these findings indicate promising activity of PIKfyve inhibitors secondary to disruption of autophagy in multiple myeloma and suggest a strategy to enrich for likely responders.
There is substantial evidence that in addition to nicotine, other compounds found in tobacco smoke significantly influence smoking behavior. Further, recent years have seen an explosion in the ...availability of non-combusted products that deliver nicotine, such as e-cigarettes and “home-brew” vaping devices that are essentially unregulated. There are many thousands of compounds in tobacco smoke alone, and new products are constantly introducing new compounds. Uncovering which of these compounds are active, across multiple smoking-relevant subtypes of the nicotinic acetylcholine receptor (nAChR) that influence tobacco/nicotine addiction, requires a high-throughput screening (HTS) approach. Accordingly, we developed a panel of HTS-friendly cell-based assays, all performed in the same cellular background and using the same membrane potential dye readout, to measure the function of the α3β4-, α4β2-, and α6β2-nAChR subtypes. These subtypes have each been prominently and consistently associated with human smoking behavior. We validated our assays by performing pilot screening of an expanded set of the Prestwick FDA-approved drug library. The screens displayed excellent performance parameters, and moderate hit rates (mean of 1.2% across all three assays) were achieved when identifying antagonists (chosen since effects of endogenous antagonists on consumption of nicotine/tobacco products are under-studied). Validation rates using an orthogonal assay (86Rb+ efflux) averaged 73% across the three assays. The resulting panel of assays represents a valuable new platform with which to screen and identify nAChR subtype-selective compounds. This provides a resource for identifying smoking-related compounds in both combusted and non-combusted tobacco products, with potential relevance in the search for additional smoking-cessation therapies.
Quiescin sulfhydryl oxidase 1 (QSOX1) is an enzyme overexpressed by many different tumor types. QSOX1 catalyzes the formation of disulfide bonds in proteins. Because short hairpin knockdowns (KD) of ...QSOX1 have been shown to suppress tumor growth and invasion
and
, we hypothesized that chemical compounds inhibiting QSOX1 enzymatic activity would also suppress tumor growth, invasion, and metastasis. High throughput screening using a QSOX1-based enzymatic assay revealed multiple potential QSOX1 inhibitors. One of the inhibitors, known as "SBI-183," suppresses tumor cell growth in a Matrigel-based spheroid assay and inhibits invasion in a modified Boyden chamber, but does not affect viability of nonmalignant cells. Oral administration of SBI-183 inhibits tumor growth in 2 independent human xenograft mouse models of renal cell carcinoma. We conclude that SBI-183 warrants further exploration as a useful tool for understanding QSOX1 biology and as a potential novel anticancer agent in tumors that overexpress QSOX1.
Quiescin sulfhydryl oxidase 1 (QSOX1) is a highly conserved disulfide bond-generating enzyme that is overexpressed in diverse tumor types. Its enzymatic activity promotes the growth and invasion of ...tumor cells and alters extracellular matrix composition. In a nude mouse-human tumor xenograft model, tumors containing shRNA for QSOX1 grew significantly more slowly than controls, suggesting that QSOX1 supports a proliferative phenotype in vivo. High throughput screening experiments identified ebselen as an in vitro inhibitor of QSOX1 enzymatic activity. Ebselen treatment of pancreatic and renal cancer cell lines stalled tumor growth and inhibited invasion through Matrigel in vitro. Daily oral treatment with ebselen resulted in a 58% reduction in tumor growth in mice bearing human pancreatic tumor xenografts compared to controls. Mass spectrometric analysis of ebselen-treated QSOX1 mechanistically revealed that C165 and C237 of QSOX1 covalently bound to ebselen. This report details the anti-neoplastic properties of ebselen in pancreatic and renal cancer cell lines. The results here offer a "proof-of-principle" that enzymatic inhibition of QSOX1 may have clinical relevancy.
Deregulation of the TNF-like weak inducer of apoptosis (TWEAK)-fibroblast growth factor-inducible 14 (Fn14) signaling pathway is observed in many diseases, including inflammation, autoimmune ...diseases, and cancer. Activation of Fn14 signaling by TWEAK binding triggers cell invasion and survival and therefore represents an attractive pathway for therapeutic intervention. Based on structural studies of the TWEAK-binding cysteine-rich domain of Fn14, several homology models of TWEAK were built to investigate plausible modes of TWEAK-Fn14 interaction. Two promising models, centered on different anchoring residues of TWEAK (tyrosine 176 and tryptophan 231), were prioritized using a data-driven strategy. Site-directed mutagenesis of TWEAK at Tyr176, but not Trp231, resulted in the loss of TWEAK binding to Fn14 substantiating Tyr176 as the anchoring residue. Importantly, mutation of TWEAK at Tyr176 did not disrupt TWEAK trimerization but failed to induce Fn14-mediated nuclear factor κ-light chain enhancer of activated B cell (NF-κB) signaling. The validated structural models were utilized in a virtual screen to design a targeted library of small molecules predicted to disrupt the TWEAK-Fn14 interaction. 129 small molecules were screened iteratively, with identification of molecules producing up to 37% inhibition of TWEAK-Fn14 binding. In summary, we present a data-driven in silico study revealing key structural elements of the TWEAK-Fn14 interaction, followed by experimental validation, serving as a guide for the design of small molecule inhibitors of the TWEAK-Fn14 ligand-receptor interaction. Our results validate the TWEAK-Fn14 interaction as a chemically tractable target and provide the foundation for further exploration utilizing chemical biology approaches focusing on validating this system as a therapeutic target in invasive cancers.
Background: Aberrant TNF-like weak inducer of apoptosis (TWEAK)-fibroblast growth factor-inducible 14 (Fn14) signaling is observed in inflammation, autoimmune diseases, and cancers.
Results: An integrated computational and experimental study identified small molecule inhibitors of TWEAK-Fn14 interaction.
Conclusion: The TWEAK-Fn14 interaction is tractable and can be inhibited by small molecules.
Significance: This is the first evidence of small molecules targeting TWEAK-Fn14 signaling.
Seventy-six FDA-approved oncology drugs and emerging therapeutics were evaluated in 25 multiple myeloma (MM) and 15 non-Hodgkin's lymphoma cell lines and in 113 primary MM samples. Ex vivo drug ...sensitivities were mined for associations with clinical phenotype, cytogenetic, genetic mutation, and transcriptional profiles. In primary MM samples, proteasome inhibitors, dinaciclib, selinexor, venetoclax, auranofin, and histone deacetylating agents had the broadest cytotoxicity. Of interest, newly diagnosed patient samples were globally less sensitive especially to bromodomain inhibitors, inhibitors of receptor tyrosine kinases or non-receptor kinases, and DNA synthesis inhibitors. Clustering demonstrated six broad groupings of drug sensitivity linked with genomic biomarkers and clinical outcomes. For example, our findings mimic clinical observations of increased venetoclax responsiveness in t(11;14) patients but also identify an increased sensitivity profile in untreated patients, standard genetic risk, low plasma cell S-Phase, and in the absence of Gain(1q) and t(4;14). In contrast, increased ex vivo responsiveness to selinexor was associated with biomarkers of poor prognosis and later relapse patients. This "direct to drug" screening resource, paired with functional genomics, has the potential to successfully direct appropriate individualized therapeutic approaches in MM and to enrich clinical trials for likely responders.
Multiple myeloma (MM) is a malignancy that is often driven by MYC and that is sustained by IRF4, which are upregulated by super-enhancers. IKZF1 and IKZF3 bind to super-enhancers and can be degraded ...using immunomodulatory imide drugs (IMiD). Successful IMiD responses downregulate MYC and IRF4; however, this fails in IMiD-resistant cells. MYC and IRF4 downregulation can also be achieved in IMiD-resistant tumors using inhibitors of BET and EP300 transcriptional coactivator proteins; however, in vivo these drugs have a narrow therapeutic window. By combining IMiDs with EP300 inhibition, we demonstrate greater downregulation of MYC and IRF4, synergistic killing of myeloma in vitro and in vivo, and an increased therapeutic window. Interestingly, this potent combination failed where MYC and IRF4 expression was maintained by high levels of the AP-1 factor BATF. Our results identify an effective drug combination and a previously unrecognized mechanism of IMiD resistance.
These results highlight the dependence of MM on IKZF1-bound super-enhancers, which can be effectively targeted by a potent therapeutic combination pairing IMiD-mediated degradation of IKZF1 and IKZF3 with EP300 inhibition. They also identify AP-1 factors as an unrecognized mechanism of IMiD resistance in MM. See related article by Neri, Barwick, et al., p. 56. See related commentary by Yun and Cleveland, p. 5. This article is featured in Selected Articles from This Issue, p. 4.
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