Search

Lanthanide-doped upconversion nanoparticles (UCNPs) possess the remarkable ability to convert multiple near-infrared (NIR) photons into higher energy ultraviolet-visible (UV-vis) photons, making them a prime candidate for several advanced applications within the realm of nanotechnology. Compared to traditional organic fluorophores and quantum dots (QDs), UCNPs possess narrower emission bands (fwhm of 10-50 nm), large anti-Stokes shifts, low toxicity, high chemical stability, and resistance to photobleaching and blinking. In addition, unlike UV-vis excitation, NIR excitation is nondestructive at lower power intensities and has high tissue penetration depths (up to 2 mm) with low autofluorescence and scattering. Together, these properties make UCNPs exceedingly favored for advanced bioanalytical and theranostic applications, where these systems have been well-explored. UCNPs are also well-suited for bioimaging, optically modulating chemistries, forensic science, and other state-of-the-art research applications. In this review, an up-to-date account of emerging applications in UCNP research, beyond bioanalytical and theranostics, are presented including optogenetics, super-resolution imaging, encoded barcodes, fingerprinting, NIR vision, UCNP-assisted photochemical manipulations, optical tweezers, 3D printing, lasing, NIR-II imaging, UCNP-molecule nanohybrids, and UCNP-based persistent luminescent nanocrystals.

This file contains a detailed description of the synthesis as well as the characterization of lead compounds PG-S3-001, PG-S3-002, and PG-S3-003. Results pertaining to the interaction of PG-S3-001 with relevant kinases is also included; Table 1. Results of Kinome Screen for Kinases involved in Regulation of STAT3.

Supplementary Figure S1. Stat5 expression correlates with the expression of anti-apoptotic genes. Supplementary Figure S2. STAT5-deficient NK cells possess aberrant transcription factors expression. Supplementary Figure S3. Loss of STAT5 alters granzyme, perforin and IFN-gamma production. Supplementary Figure S4. STAT5-deficient NK cells induce tumor promotion. Supplementary Figure S5. Loss of STAT5 increases expression of the pro-angiogenic factor VEGF-A. Supplementary Figure S6. Peripheral splenic NK cells produce VEGF-A which does not impact NK cell numbers, proliferation or maturation but enhances tumor progression in lymphoid tumor models. Supplementary Figure S7. Tumor-infiltrating NK cells and macrophages are localized around blood vessels. Supplementary Figure S8. IL-2 stimulated human NK cells mirror the murine data.

Supplementary Methods, Figure Legends

Histone deacetylase 6 (HDAC6) is an emerging clinical target for the treatment of several hematological cancers and central nervous system disorders. HDAC6 catalyzes the deacetylation of lysine residues on substrates such as tubulin, with profound implications in key cellular processes, including cellular motility and migration. This critical deacetylation activity occurs at the catalytic domain 2 (CD2) of HDAC6, and small molecule inhibitors of HDAC6 are designed to target CD2. We briefly highlight previously reported strategies for recombinant bacterial expression and purification of the HDAC6 CD2. We aim to discuss competition assays that have been used to evaluate the potency of potential HDAC6 inhibitors against CD2 via displacement of pre-bound fluorescent HDAC-probes. Moreover, we elaborate on previous protocols that have been employed in inhibitor screening and present an HDAC6-selective probe that also enables rapid and reliable high-throughput screening of new chemical entities designed to target the HDAC6 CD2.

The aberrant activity of histone deacetylases (HDACs) across a broad range of cancers and other disease indications has led to the development of small-molecule inhibitors that target one or more members of the HDAC protein family. Emerging HDAC inhibitors that show promise in drug discovery programs must be assessed across a range of in vitro assays to establish an inhibitor profile for potency and cellular selectivity towards target HDAC(s) as well as preliminary absorption, distribution, metabolism, and excretion (ADME) features. Here we provide an overview of methods to determine a subset of pivotal in vitro drug-like parameters for HDAC inhibitors (HDACi). We initially describe protocols for parallel artificial membrane permeability assays (PAMPA) to evaluate the passive permeability of small molecules across lipid membranes. Subsequently, we elaborate on cytotoxicity assays using CellTiter-Blue to determine HDACi-induced cell death in healthy/diseased cellular models. We next focus on assessing the target engagement of inhibitors with the appropriate HDAC isoforms in a cellular environment via Western blotting of acetylated HDAC substrates. Finally, we provide detailed guidelines on how to assess the metabolic stability of HDACi through whole blood stability assays. Collectively, these assays provide an overview of the permeability, selectivity, and stability of the HDAC inhibitor under development.

Schistosomiasis is a prevalent yet neglected tropical parasitic disease caused by the Schistosoma genus of blood flukes. Praziquantel is the only currently available treatment, hence drug resistance poses a major threat. Recently, histone deacetylase 8 (HDAC8) selective inhibitors have been proposed as a viable treatment for schistosomiasis. Herein, we report the phenotypic screening of a focused library of small molecules of varying HDAC isozyme-inhibition profiles, including eight HDAC8 inhibitors with10-fold selectivity in comparable functional inhibition assays and IC

Proximal multi-site phosphorylation is a critical post-translational modification in protein biology. The additive effects of multiple phosphosite clusters in close spatial proximity triggers integrative and cooperative effects on protein conformation and activity. Proximal phosphorylation has been shown to modulate signal transduction pathways and gene expression, and as a result, is implicated in a broad range of disease states through altered protein function and/or localization including enzyme overactivation or protein aggregation. The role of proximal multi-phosphorylation events is becoming increasingly recognized as mechanistically important, although breakthroughs are limited due to a lack of detection technologies. To date, there is a limited selection of facile and robust sensing tools for proximal phosphorylation. Nonetheless, there have been considerable efforts in developing optical chemosensors for the detection of proximal phosphorylation motifs on peptides and proteins in recent years. This review provides a comprehensive overview of optical chemosensors for proximal phosphorylation, with the majority of work being reported in the past two decades. Optical sensors, in the form of fluorescent and luminescent chemosensors, hybrid biosensors, and inorganic nanoparticles, are described. Emphasis is placed on the rationale behind sensor scaffolds, relevant protein motifs, and applications in protein biology., This review details optical detection methods for proximal multi-site phosphorylation, a critical post-translational modification in protein biology.

Targeted protein degradation has emerged as a transformative therapeutic modality for the treatment of human diseases. The clinical successes of approved protein degraders like lenalidomide and thalidomide in cancers and immune disorders, combined with the recent clinical debut of investigational heterobifunctional degraders, have demonstrated the potential of this pharmacological approach to expand the druggable proteome and improve patient outcomes. Molecular glue degraders are a class of protein degraders that operate by recruiting target proteins to cellular degradation machinery via noncanonical protein-protein interactions, inducing the destruction of the target protein. While heterobifunctional degraders consist of two distinct protein-binding moieties connected by a linker, molecular glue degraders contain a single pharmacophore and are thus more synthetically accessible, ligand-efficient, and often possess more drug-like physicochemical properties. In this chapter, we will explore the history of the field-from its conception to the recently accelerating discovery of novel glue degrader mechanisms-and contemplate its trajectory towards rational design with the emergence of new methods for protein quantification and high-throughput assays to screen for novel degraders.

Antibiotic resistance is a major problem for world health, triggered by the unnecessary usage of broad-spectrum antibiotics on purportedly infected patients. Current clinical standards require lengthy protocols for the detection of bacterial species in sterile physiological fluids. In this work, a class of small-molecule fluorescent chemosensors termed ProxyPhos was shown to be capable of rapid, sensitive, and facile detection of broad-spectrum bacteria. The sensors act via a turn-on fluorescent excimer mechanism, where close-proximity binding of multiple sensor units amplifies a red shift emission signal. ProxyPhos sensors were able to detect down to 10 CFUs of model strains by flow cytometry assays and showed selectivity over mammalian cells in a bacterial coculture through fluorescence microscopy. The studies reveal that the zinc(II)-chelates cyclen and cyclam are novel and effective binding units for the detection of both Gram-negative and Gram-positive bacterial strains. Mode of action studies revealed that the chemosensors detect Gram-negative and Gram-positive strains with two distinct mechanisms. Preliminary studies applying ProxyPhos sensors to sterile physiological fluids (cerebrospinal fluid) in flow cytometry assays were successful. The results suggest that ProxyPhos sensors can be developed as a rapid, inexpensive, and robust tool for the "yes-no" detection of broad-spectrum bacteria in sterile fluids.

Protein prenylation is a critical mediator in several diseases including cancer and acquired immunodeficiency syndrome (AIDS). Therapeutic intervention has focused primarily on directly targeting the prenyltransferase enzymes, FTase and GGTase I and II. To date, several drugs have advanced to clinical trials and while promising, they have yet to gain approval in a medical setting due to off-target effects and compensatory mechanisms activated by the body which results in drug resistance. While the development of dual inhibitors has mitigated undesirable side effects, potency remains sub-optimal for clinical development. An alternative approach involves antagonizing the upstream mevalonate pathway enzymes, FPPS and GGPPS, which mediate prenylation as well as cholesterol synthesis. The development of these inhibitors presents novel opportunities for dual inhibition of cancer-driven prenylation as well as cholesterol accumulation. Herein, we highlight progress towards the development of inhibitors against the prenylation machinery.

Over the past decade, covalent kinase inhibitors (CKI) have seen a resurgence in drug discovery. Covalency affords a unique set of advantages as well as challenges relative to their non-covalent counterpart. After reversible protein target recognition and binding, covalent inhibitors irreversibly modify a proximal nucleophilic residue on the protein via reaction with an electrophile. To date, the acrylamide group remains the predominantly employed electrophile in CKI development, with its incorporation in the majority of clinical candidates and FDA approved covalent therapies. Nonetheless, in recent years considerable efforts have ensued to characterize alternative electrophiles that exhibit irreversible or reversibly covalent binding mechanisms towards cysteine thiols and other amino acids. This review article provides a comprehensive overview of CKIs reported in the literature over a decade period, 2007-2018. Emphasis is placed on the rationale behind warhead choice, optimization approach, and inhibitor design. Current FDA approved CKIs are also highlighted, in addition to a detailed analysis of the common trends and themes observed within the listed data set.

We recently demonstrated that Cav1 (caveolin-1) is a negative regulator of Stat3 (signal transducer and activator of transcription-3) activity in mouse fibroblasts and human lung carcinoma SHP77 cells. We now examined whether the cellular context may affect their levels as well as the relationship between them, by assessing Cav1 and Stat3-ptyr705 amounts in different cell lines. In MDA-MB-231, A549, and HaCat cells, Cav1 levels were high and Stat3-ptyr705 levels were low, consistent with the notion of a negative effect of endogenous Cav1 on Stat3-ptyr705 levels in these lines. In addition, manipulation of Cav1 levels revealed a negative effect in MCF7 and mouse fibroblast cells, while Cav1 upregulation induced apoptosis in MCF7 cells. In contrast, however, line MRC9 had high Cav1 and high Stat3-ptyr705 levels, indicating that high Cav1 is insufficient to reduce Stat3-ptyr705 levels in this line. MCF7 and LuCi6 cells had very low Cav1 and Stat3-ptyr705 levels, indicating that the low Stat3-ptyr705 can be independent from Cav1 levels altogether. Our results reveal a further level of complexity in the relationship between Cav1 and Stat3-ptyr705 than previously thought. In addition, we demonstrate that in a feedback loop, Stat3 inhibition upregulates Cav1 in HeLa cells but not in other lines tested.

Background/aim The differentiation of the mouse breast epithelial cell line HC11 is known to require confluence as well as the addition of hydrocortisone, insulin and prolactin. Materials and methods Since confluence, which triggers the engagement of the cell-to-cell adhesion molecule E-cadherin, induces a dramatic increase in the activity of signal transducer and activator of transcription-3 (Stat3), we examined the role of Stat3 in HC11 cell differentiation. Results Stat3 inhibition abolished differentiation, indicating that Stat3 activity is critically required. However, expression of the mutationally activated form of Stat3 (Stat3C), rather than promoting, it was found to block cell differentiation, even when expressed in low levels, and in the absence of full neoplastic conversion. Conclusion The strength of the E-cadherin/Stat3 signal is key for the outcome of the differentiation process.

Hyper-activated STAT5B variants are high value oncology targets for pharmacologic intervention. STAT5BN642H, a frequently-occurring oncogenic driver mutation, promotes aggressive T-cell leukemia/lymphoma in patient carriers, although the molecular origins remain unclear. Herein, we emphasize the aggressive nature of STAT5BN642H in driving T-cell neoplasia upon hematopoietic expression in transgenic mice, revealing evidence of multiple T-cell subset organ infiltration. Notably, we demonstrate STAT5BN642H-driven transformation of γδ T-cells in in vivo syngeneic transplant models, comparable to STAT5BN642H patient γδ T-cell entities. Importantly, we present human STAT5B and STAT5BN642H crystal structures, which propose alternative mutation-mediated SH2 domain conformations. Our biophysical data suggests STAT5BN642H can adopt a hyper-activated and hyper-inactivated state with resistance to dephosphorylation. MD simulations support sustained interchain cross-domain interactions in STAT5BN642H, conferring kinetic stability to the mutant anti-parallel dimer. This study provides a molecular explanation for the STAT5BN642H activating potential, and insights into pre-clinical models for targeted intervention of hyper-activated STAT5B.

Inhibition of STAT phosphorylation is recognized as a viable therapeutic strategy for disrupting tumorigenesis. Constitutive STAT phosphorylation is found with high frequency in a number of primary tumor types, while non-cancer cells exhibit low basal activity, providing an exploitable therapeutic window. STAT activation involves phosphorylation of the SH2 domain by a number of tyrosine kinases followed by STAT dimerization and translocation to the nucleus. By blocking the cognate binding site, STAT SH2-domain inhibitors can impede kinase-mediated de novo STAT phosphorylation. Assessing for inhibitors of STAT phosphorylation has previously been conducted exclusively in cellulo using Western blot analysis. However, while providing useful in cellulo efficacy, it is not possible to conclude that inhibition is due to a direct blockade of STAT protein. Here we developed a functional assay that directly reports the blockade of phosphorylation as a result of inhibitor interaction with STAT proteins. We have optimized reaction conditions for the functional assay and validated the assay against known STAT5B ligands, including peptides and small molecule inhibitors. As part of the study, we have also identified several sites of STAT5B phosphorylation by Abl kinase. This assay will serve to delineate the functional mechanism of STAT binders in vitro and deconvolute the mechanism of phospho-STAT inhibition observed in Western blot analysis.

Histone deacetylase 6 (HDAC6) is involved in multiple regulatory processes, ranging from cellular stress to intracellular transport. Inhibition of aberrant HDAC6 activity in several cancers and neurological diseases has been shown to be efficacious in both preclinical and clinical studies. While selective HDAC6 targeting has been pursued as an alternative to pan-HDAC drugs, identifying truly selective molecular templates has not been trivial. Herein, we report a structure-activity relationship study yielding TO-317, which potently binds HDAC6 catalytic domain 2 (Ki = 0.7 nM) and inhibits the enzyme function (IC50 = 2 nM). TO-317 exhibits 158-fold selectivity for HDAC6 over other HDAC isozymes by binding the catalytic Zn2+ and, uniquely, making a never seen before direct hydrogen bond with the Zn2+ coordinating residue, His614. This novel structural motif targeting the second-sphere His614 interaction, observed in a 1.84 A resolution crystal structure with drHDAC6 from zebrafish, can provide new pharmacophores for identifying enthalpically driven, high-affinity, HDAC6-selective inhibitors.

Epigenetic targeting has emerged as an efficacious therapy for hematological cancers. The rare and incurable T-cell prolymphocytic leukemia (T-PLL) is known for its aggressive clinical course. Current epigenetic agents such as histone deacetylase (HDAC) inhibitors are increasingly used for targeted therapy. Through a structure-activity relationship (SAR) study, we developed an HDAC6 inhibitor KT-531, which exhibited higher potency in T-PLL compared to other hematological cancers. KT-531 displayed strong HDAC6 inhibitory potency and selectivity, on-target biological activity, and a safe therapeutic window in nontransformed cell lines. In primary T-PLL patient cells, where HDAC6 was found to be overexpressed, KT-531 exhibited strong biological responses, and safety in healthy donor samples. Notably, combination studies in T-PLL patient samples demonstrated KT-531 synergizes with approved cancer drugs, bendamustine, idasanutlin, and venetoclax. Our work suggests HDAC inhibition in T-PLL could afford sufficient therapeutic windows to achieve durable remission either as standalone or in combination with targeted drugs.

Simple Summary Leukemic cells show differential sensitivity to apoptosis induction by the clinically relevant drug hydroxyurea. Since resistance to hydroxyurea can pose a therapeutic problem, we searched for mechanisms that protect such cells from the toxic effects of hydroxyurea. We used proteomics followed by mass spectrometry to accomplish this task and noted a loss of the RAF1 kinase in cells that are killed by hydroxyurea. Pharmacological inhibition of RAF1 and its target BCL-XL show that these proteins suppress apoptosis induction. Furthermore, inhibition of their upstream regulators BCR-ABL1 (in chronic myeloid leukemia cells) and FLT3-ITD (in acute myeloid leukemia cells) plus hydroxyurea produced favorable results. This approach may benefit patients that are not successfully treated with tyrosine kinase inhibitors. Taken together, we provide novel insights into strategies that eliminate chronic and acute myeloid leukemia cells with combinations of clinically established and currently tested pharmaceutical agents. Abstract The ribonucleotide reductase inhibitor hydroxyurea suppresses de novo dNTP synthesis and attenuates the hyperproliferation of leukemic blasts. Mechanisms that determine whether cells undergo apoptosis in response to hydroxyurea are ill-defined. We used unbiased proteomics to uncover which pathways control the transition of the hydroxyurea-induced replication stress into an apoptotic program in chronic and acute myeloid leukemia cells. We noted a decrease in the serine/threonine kinase RAF1/c-RAF in cells that undergo apoptosis in response to clinically relevant doses of hydroxyurea. Using the RAF inhibitor LY3009120, we show that RAF activity determines the sensitivity of leukemic cells toward hydroxyurea. We further disclose that pharmacological inhibition of the RAF downstream target BCL-XL with the drug navitoclax and RNAi combine favorably with hydroxyurea against leukemic cells. BCR-ABL1 and hyperactive FLT3 are tyrosine kinases that causally contribute to the development of leukemia and induce RAF1 and BCL-XL. Accordingly, the ABL inhibitor imatinib and the FLT3 inhibitor quizartinib sensitize leukemic cells to pro-apoptotic effects of hydroxyurea. Moreover, hydroxyurea and navitoclax kill leukemic cells with mutant FLT3 that are resistant to quizartinib. These data reveal cellular susceptibility factors toward hydroxyurea and how they can be exploited to eliminate difficult-to-treat leukemic cells with clinically relevant drug combinations.

Histone deacetylases (HDACs) are an attractive therapeutic target for a variety of human diseases. Currently, all four FDA-approved HDAC-targeting drugs are nonselective, pan-HDAC inhibitors, exhibiting adverse side effects at therapeutic doses. Although selective HDAC inhibition has been proposed to mitigate toxicity, the targeted catalytic domains are highly conserved. Herein, we describe a series of rationally designed, conformationally constrained, benzanilide foldamers which selectively bind the catalytic tunnel of HDAC8. The series includes benzanilides, MMH371, MMH409, and MMH410, which exhibit potent in vitro HDAC8 activity (IC50 = 66, 23, and 66 nM, respectively) and up to 410-fold selectivity for HDAC8 over the next targeted HDAC. Experimental and computational analyses of the benzanilide structure docked with human HDAC8 enzyme showed the adoption of a low-energy L-shaped conformer that favors HDAC8 selectivity. The conformationally constrained HDAC8 inhibitors present an alternative biological probe for further determining the clinical utility and safety of pharmacological knockdown of HDAC8 in diseased cells.

Dysregulated Histone Deacetylase (HDAC) activity across multiple human pathologies have highlighted this family of epigenetic enzymes as critical druggable targets, amenable to small molecule intervention. While efficacious, current approaches using non-selective HDAC inhibitors (HDACi) have been shown to cause a range of undesirable clinical toxicities. To circumvent this, recent efforts have focused on the design of highly selective HDACi as a novel therapeutic strategy. Beyond roles in regulating transcription, the unique HDAC6 (with two catalytic domains) regulates the deacetylation of α -tubulin; promoting growth factor-controlled cell motility, cell division, and metastatic hallmarks. Recent studies have linked aberrant HDAC6 function in various hematological cancers including acute myeloid leukaemia and multiple myeloma. Herein, we report the discovery, in vitro characterization, and biological evaluation of PTG-0861 (JG-265), a novel HDAC6-selective inhibitor with strong isozyme-selectivity (∼36× ) and low nanomolar potency (IC50 = 5.92 nM) against HDAC6. This selectivity profile was rationalized via in silico docking studies and also observed in cellulo through cellular target engagement. Moreover, PTG-0861 achieved relevant potency against several blood cancer cell lines (e.g. MV4-11, MM1S), whilst showing limited cytotoxicity against non-malignant cells (e.g. NHF, HUVEC) and CD-1 mice. In examining compound stability and cellular permeability, PTG-0861 revealed a promising in vitro pharmacokinetic (PK) profile. Altogether, in this study we identified a novel and potent HDAC6-selective inhibitor (∼4× more selective than current clinical standards – citarinostat, ricolinostat), which achieves cellular target engagement, efficacy in hematological cancer cells with a promising safety profile and in vitro PK.

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction of differentiation of two mouse breast epithelial cell lines, HC11 and EpH4, and their use to study complementary stages of mammary gland development and neoplastic transformation. The HC11 mouse breast epithelial cell line originated from the mammary gland of a pregnant Balb/c mouse. It differentiates when grown to confluence attached to a plastic Petri dish surface in medium containing fetal calf serum and Hydrocortisone, Insulin and Prolactin (HIP medium). Under these conditions, HC11 cells produce the milk proteins β-casein and whey acidic protein (WAP), similar to lactating mammary epithelial cells, and form rudimentary mammary gland-like structures termed "domes". The EpH4 cell line was derived from spontaneously immortalized mouse mammary gland epithelial cells isolated from a pregnant Balb/c mouse. Unlike HC11, EpH4 cells can fully differentiate into spheroids (also called mammospheres) when cultured under three-dimensional (3D) growth conditions in HIP medium. Cells are trypsinized, suspended in a 20% matrix consisting of a mixture of extracellular matrix proteins produced by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, plated on top of a layer of concentrated matrix coating a plastic Petri dish or multiwell plate, and covered with a layer of 10% matrix-containing HIP medium. Under these conditions, EpH4 cells form hollow spheroids that exhibit apical-basal polarity, a hollow lumen, and produce β-casein and WAP. Using these techniques, our results demonstrated that the intensity of the cadherin/Rac signal is critical for the differentiation of HC11 cells. While Rac1 is necessary for differentiation and low levels of activated RacV12 increase differentiation, high RacV12 levels block differentiation while inducing neoplasia. In contrast, EpH4 cells represent an earlier stage in mammary epithelial differentiation, which is inhibited by even low levels of RacV12.

Caveolin-1 (Cav1) is an integral plasma membrane protein and a complex regulator of signal transduction. The Signal Transducer and Activator of Transcription-3 (Stat3) is activated by a number of receptor and non-receptor tyrosine kinases and is positively implicated in cancer. Despite extensive efforts, the relationship between Cav1 and Stat3 has been a matter of controversy. We previously demonstrated that engagement of E- or N-cadherin or cadherin-11 cell to cell adhesion molecules, as occurs with confluence of cultured cells, triggers a dramatic increase in the levels of tyr705 phosphorylated i.e. activated Stat3, by a mechanism requiring the cRac1 small GTPase. Since confluence was not taken into account in previous studies, we revisited the question of the relationship between Cav1 and Stat3-ptyr705 in non-transformed mouse fibroblasts and in human lung carcinoma cells, by examining their effect at different cell densities. Our results unequivocally demonstrate that Cav1 downregulates cadherin-11, by a mechanism which requires the Cav1 scaffolding domain. This cadherin-11 downregulation, in turn, leads to a reduction in cRac1 and Stat3 activity levels. Furthermore, in a feedback loop possibly through p53 upregulation, Stat3 downregulation increases Cav1 levels. Our data reveal the presence of a potent, negative regulatory loop between Cav1 and cadherin-11/Stat3, leading to Stat3 inhibition and apoptosis.

The transcription factor STAT5 is an essential downstream mediator of many tyrosine kinases (TKs), particularly in hematopoietic cancers. STAT5 is activated by FLT3-ITD, which is a constitutively active TK driving the pathogenesis of acute myeloid leukemia (AML). Since STAT5 is a critical mediator of diverse malignant properties of AML cells, direct targeting of STAT5 is of significant clinical value. Here, we describe the development and preclinical evaluation of a novel, potent STAT5 SH2 domain inhibitor, AC-4–130, which can efficiently block pathological levels of STAT5 activity in AML. AC-4–130 directly binds to STAT5 and disrupts STAT5 activation, dimerization, nuclear translocation, and STAT5-dependent gene transcription. Notably, AC-4–130 substantially impaired the proliferation and clonogenic growth of human AML cell lines and primary FLT3-ITD+ AML patient cells in vitro and in vivo. Furthermore, AC-4–130 synergistically increased the cytotoxicity of the JAK1/2 inhibitor Ruxolitinib and the p300/pCAF inhibitor Garcinol. Overall, the synergistic effects of AC-4–130 with TK inhibitors (TKIs) as well as emerging treatment strategies provide new therapeutic opportunities for leukemia and potentially other cancers.

The development of STAT protein-specific inhibitors has been the focus of a number of drug discovery programs. STAT activation occurs through phosphorylation at the STAT SH2 domain, resulting in dimerization, translocation to the nucleus, and transcription of proliferative genes. Due to the functional significance of the SH2 domain in mediating multiple components of the STAT signalling cascade, many libraries of inhibitors have been designed to target the SH2 domain. This has triggered the requirement for effective high-throughput screening platforms for analyzing binding by larger chemical libraries to STAT proteins. Herein, we present strategies for the development of a high-throughput thermal denaturation-based assay for identifying STAT inhibitors as well as high-yielding recombinant expression and purification of untagged STAT1, STAT3, and STAT5 proteins. This assay reports changes in the fluorescence of a labelled peptide bound to the STAT protein as a function of increasing temperature. STAT inhibitors which displace the labelled peptide elicit a change in the melt profile, which is quantitatively determined as a change in the area under the curve. This assay offers an alternative, but complimentary, high-throughput screening strategy for identifying new inhibitors of STAT proteins as well as characterizing further, the mode of inhibition by existing libraries of compounds.

The labeling of proteins with ubiquitin/ubiquitin-like (Ubl) proteins is crucial for several physiological processes and in the onset of various diseases. Recently, targeting ubiquitin protein labeling has shifted toward the use of allosteric mechanisms over classical activity-based approaches. Allosteric enzyme regulation offers the potential for greater selectivity and has demonstrated less susceptibility to acquired resistance often associated with active site inhibitors. Furthermore, the isoform diversity among E1 activating, E2 conjugating, E3 ligase, and deubiquitinating (DUB) enzymes offers an ideal platform for modulating activity via allostery. Herein, we have reviewed allosteric inhibitors of the ubiquitin E1-E2-E3 and DUB enzymatic cascade developed over the past decade with a focus on their mechanisms of action. We have highlighted the advantages as well as the challenges associated with designing allosteric modulators of the ubiquitin labeling machinery, and the future promise in targeting th...

STAT5B, a ubiquitious transcription factor, has been implicated in the onset and progression of several cancers. Since the inhibition of STAT activity holds significant therapeutic potential, there is a need to develop high-throughput biophysical screening platforms to rapidly identify high affinity binders of STATs. Biophysical assays would benefit from the efficient and cost-effective production of high purity, full-length STAT proteins. Herein, we have sampled a large region of protein expression and purification space that has substantially increased recombinant STAT5B protein yields from Escherichia coli. The identity of STAT5B was confirmed by Western blotting analysis, while the results of a fluorescence polarization assay indicated that the purified protein is correctly folded and functional. A thermal shift assay was employed to assess the effect of various osmolytes on the stability of the protein. The protein expression conditions identified in this study allowed for more efficient and higher recovery of soluble STAT5B protein, which will enable a broad range of biophysical studies and facilitate high-throughput STAT5B drug screening.

Src Homology 2 (SH2) domains arose within metazoan signaling pathways and are involved in protein regulation of multiple pleiotropic cascades. In signal transducer and activator of transcription (STAT) proteins, SH2 domain interactions are critical for molecular activation and nuclear accumulation of phosphorylated STAT dimers to drive transcription. Sequencing analysis of patient samples has revealed the SH2 domain as a hotspot in the mutational landscape of STAT proteins although the functional impact for the vast majority of these mutations remains poorly characterized. Despite several well resolved structures for SH2 domain-containing proteins, structural data regarding the distinctive STAT-type SH2 domain is limited. Here, we review the unique features of STAT-type SH2 domains in the context of all currently reported STAT3 and STAT5 SH2 domain clinical mutations. The genetic volatility of specific regions in the SH2 domain can result in either activating or deactivating mutations at the same site in the domain, underscoring the delicate evolutionary balance of wild type STAT structural motifs in maintaining precise levels of cellular activity. Understanding the molecular and biophysical impact of these disease-associated mutations can uncover convergent mechanisms of action for mutations localized within the STAT SH2 domain to facilitate the development of targeted therapeutic interventions.

[Image: see text] The HDAC inhibitor 4-tert-butyl-N-(4-(hydroxycarbamoyl)phenyl)benzamide (AES-350, 51) was identified as a promising preclinical candidate for the treatment of acute myeloid leukemia (AML), an aggressive malignancy with a meagre 24% 5-year survival rate. Through screening of low-molecular-weight analogues derived from the previously discovered novel HDAC inhibitor, AES-135, compound 51 demonstrated greater HDAC isoform selectivity, higher cytotoxicity in MV4-11 cells, an improved therapeutic window, and more efficient absorption through cellular and lipid membranes. Compound 51 also demonstrated improved oral bioavailability compared to SAHA in mouse models. A broad spectrum of experiments, including FACS, ELISA, and Western blotting, were performed to support our hypothesis that 51 dose-dependently triggers apoptosis in AML cells through HDAC inhibition.

Signal transducer and activator of transcription 5B (STAT5B) is constitutively activated in multiple cancers as a result of hyperactivating mutations or dysregulation of upstream effectors. Therapeutic strategies have predominantly targeted the Src homology 2 (SH2) domain to inhibit STAT phosphorylation, a prerequisite for STAT5B transcriptional activation. An alternative approach for STAT5B pharmacologic inhibition involves targeting the DNA-binding domain (DBD). However, this strategy remains relatively unexplored and is further hindered by the lack of a high-throughput in vitro engagement assay. Herein, we present the development and optimization of a STAT5B DBD fluorescence polarization (FP) assay, which facilitates rapid screening of small molecules targeting the STAT5B DBD though displacement of a fluorescently labelled oligonucleotide. The assay can generate a complete DNA-binding profile in 10 min, with signal stability up to 2 h, and minimal changes under a range of conditions including 10 % (v/v) glycerol, 15 % (v/v) DMSO, 1 mM NaCl, 0.02 % (w/v) BSA, and 1 mM EDTA. This assay is compatible with both unphosphorylated and phosphorylated STAT5B and demonstrates suitability for high-throughput screening with a Z′ factor of 0.68 ± 0.07 and a signal to noise ratio of 6.7 ± 0.84.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, incurable cancer with a 20% 1 year survival rate. While standard-of-care therapy can prolong life in a small fraction of cases, PDAC is inherently resistant to current treatments, and novel therapies are urgently required. Histone deacetylase (HDAC) inhibitors are effective in killing pancreatic cancer cells in in vitro PDAC studies, and although there are a few clinical studies investigating combination therapy including HDAC inhibitors, no HDAC drug or combination therapy with an HDAC drug has been approved for the treatment of PDAC. We developed an inhibitor of HDACs, AES-135, that exhibits nanomolar inhibitory activity against HDAC3, HDAC6, and HDAC11 in biochemical assays. In a three-dimensional coculture model, AES-135 kills low-passage patient-derived tumor spheroids selectively over surrounding cancer-associated fibroblasts and has excellent pharmacokinetic properties in vivo. In an orthotopic murine model of pancreatic cancer, AES-135 prolongs survival significantly, therefore representing a candidate for further preclinical testing.

Summary The marsupial Tasmanian devil (Sarcophilus harrisii) faces extinction due to transmissible devil facial tumor disease (DFTD). To unveil the molecular underpinnings of this transmissible cancer, we combined pharmacological screens with an integrated systems-biology characterization. Sensitivity to inhibitors of ERBB tyrosine kinases correlated with their overexpression. Proteomic and DNA methylation analyses revealed tumor-specific signatures linked to the evolutionary conserved oncogenic STAT3. ERBB inhibition blocked phosphorylation of STAT3 and arrested cancer cells. Pharmacological blockade of ERBB or STAT3 prevented tumor growth in xenograft models and restored MHC class I expression. This link between the hyperactive ERBB-STAT3 axis and major histocompatibility complex class I-mediated tumor immunosurveillance provides mechanistic insights into horizontal transmissibility and puts forward a dual chemo-immunotherapeutic strategy to save Tasmanian devils from DFTD. Video Abstract, Graphical Abstract, Highlights • Tasmanian devil proteome and DNA methylome reveal modulated tumor environment • Identification of cancer-driving hyperactive ERBB-STAT3 axis as a druggable target • ERBB-STAT3-driven suppression of MHC class I genes may facilitate immune evasion • Successful pharmacological treatments of Tasmanian devil tumors in a xenograft model, Kosack et al. identify the ERBB-STAT3 signaling axis as a central molecular driver of Tasmanian devil transmissible facial tumors. Inhibition of ERBB or STAT3 prevents tumor growth in xenograft models and restores MHC class I expression, suggesting a chemo-immunotherapeutic strategy to save Tasmanian devils.

Aggressive breast cancer subtypes utilize system xc-, a membrane antiporter, to import cystine for glutathione synthesis and maintenance of redox homeostasis, in turn releasing glutamate as a metabolic pro-nociceptive by-product. Metastatic breast cancers establish themselves at distal sites including bone, where changes in extracellular glutamate levels contribute to cancer-induced bone pain. We previously established that stearically blocking system xc- activity with sulfasalazine delays the onset of nociceptive behaviours and that xCT, the functional antiporter subunit, is positively regulated by signal transducer and activator of transcription 3 (STAT3). In the current investigation, a murine xenograft cancer-induced bone pain model was applied to examine whether pharmacological inhibition of phosphorylated STAT3 (pSTAT3) induces changes in nociception. A high glutamate-releasing, xCT/pSTAT3 over-expressing human breast cancer cell line was selected for injection into the distal epiphysis of the right femur of female nude mice. A 14-day regimen of intraperitoneal injections with either vehicle or the novel STAT3 inhibitor DR-1-55 commenced three weeks after initial intrafemoral bone injection. Nociceptive behaviours were temporally monitored by automated von Frey, dynamic weight bearing and open-field testing for the duration of the study, beginning at the baseline. Prior to sacrifice and at ethical end point, tumour-induced osteolytic lesions were radiographically assessed. Treatment with DR-1-55 significantly delayed the onset and severity of spontaneous and induced nociceptive behaviours, also decreasing human SLC7A11 ( xCT) mRNA levels in tumour-bearing limbs without altering osteolysis. In addition, two pro-inflammatory cytokines released by this cell line, interleukin 6 and interleukin 1β, were also down-regulated at the mRNA level in response to DR-1-55 treatment in vivo, with lower human interleukin 6 levels detected in the host circulation. This study demonstrates that targeting pSTAT3 may be a viable therapeutic means to manage cancer-induced bone pain, alone or in combination with stearic system xc- blockers.

Medulloblastoma (MB), the most common malignant paediatric brain tumor, is currently treated using a combination of surgery, craniospinal radiotherapy and chemotherapy. Owing to MB stem cells (MBSCs), a subset of MB patients remains untreatable despite standard therapy. CD133 is used to identify MBSCs although its functional role in tumorigenesis has yet to be determined. In this work, we showed enrichment of CD133 in Group 3 MB is associated with increased rate of metastasis and poor clinical outcome. The signal transducers and activators of transcription-3 (STAT3) pathway are selectively activated in CD133+ MBSCs and promote tumorigenesis through regulation of c-MYC, a key genetic driver of Group 3 MB. We screened compound libraries for STAT3 inhibitors and treatment with the selected STAT3 inhibitors resulted in tumor size reduction in vivo. We propose that inhibition of STAT3 signaling in MBSCs may represent a potential therapeutic strategy to treat patients with recurrent MB.

Despite dramatic improvements in treatment and survival, acute lymphoid malignancies remain the leading cause of death in children with cancer and a highly lethal malignancy in adults.[1] Acute lym...

The Signal Transducer and Activator of Transcription 3 (STAT3) oncogene is a master regulator of many human cancers, and a well-recognized target for therapeutic intervention. A well known STAT3 inhibitor, S3I-201 (NSC 74859), is hypothesized to block STAT3 function in cancer cells by binding the STAT3 SH2 domain and disrupt STAT3 protein complexation events. In this study, liquid chromatography tandem mass spectrometry analysis revealed that STAT3, in the presence of S3I-201, showed a minimum of five specific sites of modification, cysteine's 108, 259, 367, 542, and 687. Moreover, a prepared fluorescently labeled chemical probe of S3I-201 (DB-6-055) revealed that S3I-201 non-specifically and globally alkylated intracellular proteins at concentrations consistent with S3I-201's reported IC50. These data are consistent with the hypothesis that S3I-201 is a sub-optimal probe for interrogating STAT3-related cell biology.

STAT5 is a member of the signal transducer and activator of transcription family of proteins and is widely recognized as an oncogenic master regulator of hematological malignancies. To date, therapeutic approaches to attenuate aberrant activity have focused on upstream kinase targets such as JAK2, Bcr-Abl, Flt3, and Flt3-ITD. To date, there is no STAT5-selective inhibitor in clinical development. More recently, PROTAC approaches have been deployed successfully against the STAT3 isoform and demonstrated efficacy and safety in preclinical models. We herein report the identification of a potent and selective, non-PROTAC, small molecule degrader of STAT5 protein, JPX-0750. Upon binding, JPX-0750 covalently and selectively binds to a cysteine residue on STAT5 via an electrophilic warhead, which induces profound destabilization of STAT5. The unfolding/destabilization effect can be observed via Western blot, isothermal denaturation of purified protein and HDX exchange. In a range of AML and TPLL cell lines exposure to JPX-750 at nM concentrations leads to a rapid and dose-dependant degradation of both phospho-STAT5 and total STAT5 with resultant IC50's between 80-200 nM. Washout experiments determined that exposure to JPX-0750 at 1 µM for 2-4 h is sufficient to degrade STAT5, inhibit downstream targets, and induce apoptosis. Moreover, in the same washout experiments, 50% STAT5 recovery post-washout required >72 h suggesting a long pharmacodynamic effect. Against STAT3, degrading effects are observed later, 24 h post washout (6 h treatment), indicating that anti-STAT5 activity is more rapid in AML cells and in line with the inherent stability of each protein. No effect was observed on either STAT1 or pSTAT1 upon exposure to JPX-750. Importantly, compared to standard AML cell lines JPX-750 demonstrated similar low nM potency in 25/30 primary AML blasts and TPLL patient samples, including those with poor prognostic markers. JPX-750 exhibits a large therapeutic window for AML versus pooled human fibroblasts and hematopoietic stem cells (ca 100 fold). In a preclinical MV4;11 luciferase model, structurally related JPX-700, at 5 mg/kg (IP, qd) significantly reduced leukemic burden, suppressed tumour dissemination to both the lung and liver, and had no effect on body weight, organ histology or blood parameters. In summary, JPX-750 represents a new class of potent and selective small molecule degraders of STAT5 protein. Citation Format: Ji Sung Park, Gary Tin, Elvin D. de Araujo, Anna Orlova, Helena Sorger, Florian Grebien, Elizabeth Heyes, Mulu Geletu, Ruth Villalonga, Angel Sampedro, Abootaleb Sedighi, Marco Herling, Satu Mustjoki, Mohammad S. Eram, Siawash Ahmar, Richard Moriggl, Jeff A. O'Meara, John Proudfoot, Kay Noel, Dziyana Kraskouskaya, Roman Fleck, Patrick T. Gunning. A potent and selective small molecule degrader of STAT5 for the treatment of hematological malignancies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-108.