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Objective Nearly 20%–29% of patients with colorectal cancer (CRC) succumb to liver or lung metastasis and there is a dire need for novel targets to improve the survival of patients with metastasis. The long isoform of the Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1-L or CC1-L) is a key regulator of immune surveillance in primary CRC, but its role in metastasis remains largely unexplored. We have examined how CC1-L expression impacts on colon cancer liver metastasis. Design Murine MC38 transfected with CC1-L were evaluated in vitro for proliferation, migration and invasion, and for in vivo experimental liver metastasis. Using shRNA silencing or pharmacological inhibition, we delineated the role in liver metastasis of Chemokine (C-C motif) Ligand 2 (CCL2) and Signal Transducer and Activator of Transcription 3 (STAT3) downstream of CC1-L. We further assessed the clinical relevance of these findings in a cohort of patients with CRC. Results MC38-CC1-L-expressing cells exhibited significantly reduced in vivo liver metastasis and displayed decreased CCL2 chemokine secretion and reduced STAT3 activity. Down-modulation of CCL2 expression and pharmacological inhibition of STAT3 activity in MC38 cells led to reduced cell invasion capacity and decreased liver metastasis. The clinical relevance of our findings is illustrated by the fact that high CC1 expression in patients with CRC combined with some inflammation-regulated and STAT3-regulated genes correlate with improved 10-year survival. Conclusions CC1-L regulates inflammation and STAT3 signalling and contributes to the maintenance of a less-invasive CRC metastatic phenotype of poorly differentiated carcinomas.

The Met receptor tyrosine kinase is activated or genetically amplified in some gastric cancers, but resistance to small-molecule inhibitors of Met often emerges in patients. We found that Met abundance correlated with a proliferation marker in patient gastric tumor sections, and gastric cancer cell lines that have MET amplifications depended on Met for proliferation and anchorage-independent growth in culture. Inhibition of Met induced temporal changes in gene expression in the cell lines, initiated by a rapid decrease in the expression of genes encoding transcription factors, followed by those encoding proteins involved in epithelial-mesenchymal transition, and finally those encoding cell cycle-related proteins. In the gastric cancer cell lines, microarray and chromatin immunoprecipitation analysis revealed considerable overlap between genes regulated in response to Met stimulation and those regulated by signal transducer and activator of transcription 3 (STAT3). The activity of STAT3, extracellular signal-regulated kinase (ERK), and the kinase Akt was decreased by Met inhibition, but only inhibitors of STAT3 were as effective as the Met inhibitor in decreasing tumor cell proliferation in culture and in xenografts, suggesting that STAT3 mediates the pro-proliferative program induced by Met. However, the phosphorylation of ERK increased after prolonged Met inhibition in culture, correlating with decreased abundance of the phosphatases DUSP4 and DUSP6, which inhibit ERK. Combined inhibition of Met and the mitogen-activated protein kinase kinase (MEK)-ERK pathway induced greater cell death in cultured gastric cancer cells than did either inhibitor alone. These findings indicate combination therapies that may counteract resistance to Met inhibitors.

The mild and efficient deblocking of aryl benzyl ethers with TFA is reported. Cleavage was fastest with ortho-electron-withdrawing groups on the phenolic ring, which we have attributed to a proton chelation effect, furnishing the deprotected phenols in excellent yields. The corresponding para-methoxybenzyl, allyl and iso-propyl ethers were also cleanly removed under these conditions. In addition, the selective aryl benzyl ether debenzylation in the presence of benzyl ester, Cbz carbamate and Boc carbamate functionalities was also observed.

This is the first of a two-part Editorial by the Guest Editors of the ChemBioChem and ChemMedChem joint Special Issue on Protein-Protein Interactions. Part 2 can be accessed via http://dx.doi.org/10.1002/cmdc.201600158; the complete issue can be viewed here: bit.ly/cbcVIppi.

Signal transducer and activator of transcription 3 (STAT3) protein is involved in regulatory functions in cell proliferation, differentiation and survival, and is linked to cancer phenotype and tumorigenesis. Towards developing new methodologies for screening STAT3 interactions, the electrochemical method based on the use of redox active protein was proposed. The electrochemical signal, due to the redox (ferrocene)-labeled STAT3 protein immobilized on a gold surface, was modulated due to protein dimerization with the unlabeled STAT3 molecule. The dramatic decrease in current density from 2.7 μA cm(-2) to 0.5 μA cm(-2) was observed following the STAT3-ferrocene-STAT3 dimerization. The electrochemical approach was further extended for screening the potential dimerization inhibitors. Previously published potent salicylic acid derivatives were the most promising candidates for inhibition of STAT3 dimerization in this assay. We expect that other SH2-containing proteins may be monitored by the proposed electrochemical method.

The conjugation of drug or molecular recognition motif to a hydrophobic fatty entity, for purpose of drug-membrane localization, has been a molecular strategy utilized for targeted inhibition of pathways involved in diseased cells. In general, membrane-anchored inhibitor structures have been composed of either a lipid or sterol group coupled via a broad range of inert linkers to either a peptide or small molecule protein recognition agent. Whilst not adhering to the molecular paradigms of modern medicinal chemistry, this approach has afforded peptidic-based therapeutics with improved cellular and in vivo efficacy, leading to more selective targeting of membrane associated protein targets and the effective immobilization of cytosolic signaling proteins through membrane anchorage. The evidence suggests that membrane-anchored peptidic inhibitors are more selective, potent, structurally rigid, and possess enhanced cell permeability profiles as compared to their non-lipidated precursors. This perspectives article will review the application of lipid or sterol conjugation to peptide inhibitors (lipo-molecules) to circumvent the poor cell permeability and metabolic labilities associated with peptidic therapeutics. In addition, the concept of protein-membrane anchorage as a novel drug modality for inhibiting cytosolic signaling protein motility in cells will be reviewed and its merits as an approach to inhibiting protein complexation, protein nuclear translocation and their potential for more effective targeting of membrane associated targets.

Src homology 2 (SH2) domains are 100 amino acid modular units, which recognize and bind to tyrosyl-phosphorylated peptide sequences on their target proteins, and thereby mediate intracellular protein–protein interactions. This review summarizes the progress towards the development of synthetic agents that disrupt the function of the SH2 domains in different proteins as well as the clinical relevance of targeting a specific SH2 domain. Since 1986, SH2 domains have been identified in over 110 human proteins, including kinases, transcription factors, and adaptor proteins. A number of these proteins are over-activated in many diseases, including cancer, and their function is highly dependent on their SH2 domain. Thus, inhibition of a protein's function through disrupting that of its SH2 domain has emerged as a promising approach towards the development of novel therapeutic modalities. Although targeting the SH2 domain is a challenging task in molecular recognition, the progress reported here demonstrates the feasibility of such an approach.

Pharmacologically targeting activated signal transducer and activator of transcription 3 (STAT3) and/or STAT5 has been an active area of cancer research. The cystine/glutamate antiporter system xc- contributes to redox balance and export of intracellularly produced glutamate in response to up-regulated glutaminolysis in aggressive breast cancer cells. We have previously shown that blocking STAT3/5 using the novel small molecule inhibitor SH-4-54, designed to target the SH2 domains of both proteins, increases expression of xCT, which encodes the active component of system xc-, thereby increasing antiporter activity in human breast cancer cells. The current investigation demonstrates that chronic treatment with SH-4-54, followed by clonal selection of treatment-resistant MDA-MB-231 and T47D breast cancer cells, elicits distinct subtype-dependent effects. xCT mRNA and protein levels, glutamate release, and cystine uptake are decreased relative to untreated passage-matched controls in the triple-negative MDA-MB-231 SH-4-54-resistant clones, with the inverse occurring in estrogen-responsive T47D cells. This “ying-yang” effect is linked with a shifted balance between phosphorylated STAT3 and STAT5, intracellular levels of reactive oxygen species (ROS), STAT5 SUMOylation/de-SUMOylation, and the expression of STAT3/5 target genes (assessed by NextGeneration RNA sequencing). STAT5 emerged as a definitive negative transcriptional regulator of xCT, while STAT3 activation was coupled with increased system xc- activity. Specifically, inhibiting constitutive STAT3 phosphorylation in MDA-MB-231 cells induces ROS, thereby affecting the SUMO pathway by favoring de-SUMOylation and STAT5 phosphorylation. Activated STAT5 is then able to serve as a repressor by binding to its recognition sequence within the xCT promoter, reducing xCT expression and thereby destabilizing an important redox balancing mechanism by limiting cystine uptake through system xc-. In contrast, in ERα-positive cells that initially respond to STAT5-mediated signaling, STAT3 becomes activated and xCT expression is up-regulated in response to chronic SH-4-54 treatment, potentially leading to a more aggressive cancer subtype. Further destabilizing the cellular redox status may therefore be critical to produce clinically meaningful outcomes linked to chronic treatment with potent STAT3/5 inhibitors like SH-4-54. We assessed this notion by treating SH-4-54-resistant MDA-MB-231 clones with specific ROS-inducing reagents, including capsazepine, bleomycin, and paclitaxel, which produced different effects on cell viability. We propose that careful classification of a patient's breast cancer subtype is central to therapeutically targeting STAT3/5 as a means of treating breast cancer, particularly given that xCT is emerging as an important biomarker of aggressive cancers. Citation Format: Linher-Melville K, Nashed MG, Ungard R, Haftchenary S, Gunning PT, Singh G. Chronic inhibition of signal transducer and activator of transcription 3/5 in treatment-resistant human breast cancer cell subtypes: Convergence on the reactive oxyten species/SUMOylation pathway and its effects on xCT expression and system xc- activity [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-03-13.

Src homology 2 (SH2) domains are 100 amino acid modular units, which recognize and bind to tyrosyl-phosphorylated peptide sequences on their target proteins, and thereby mediate intracellular protein–protein interactions. This review summarizes the progress towards the development of synthetic agents that disrupt the function of the SH2 domains in different proteins as well as the clinical relevance of targeting a specific SH2 domain. Since 1986, SH2 domains have been identified in over 110 human proteins, including kinases, transcription factors, and adaptor proteins. A number of these proteins are over-activated in many diseases, including cancer, and their function is highly dependent on their SH2 domain. Thus, inhibition of a protein's function through disrupting that of its SH2 domain has emerged as a promising approach towards the development of novel therapeutic modalities. Although targeting the SH2 domain is a challenging task in molecular recognition, the progress reported here demonstrates the feasibility of such an approach.

A focused library of hetero-trisubstituted purines was developed for improving the cell penetrating and biological efficacy of a series of anti-Stat3 protein inhibitors. From this SAR study, lead agent 22e was identified as being a promising inhibitor of MM tumour cells (IC50's5μM). Surprisingly, biophysical and biochemical characterization proved that 22e was not a Stat3 inhibitor. Initial screening against the kinome, prompted by the purine scaffold's history for targeting ATP binding pockets, suggests possible targeting of the JAK family kinases, as well for ABL1 (nonphosphorylated F317L) and AAK1.

The labeling of proteins with small ubiquitin (Ub) and ubiquitin-like (Ubl) modifiers regulates a plethora of activities within the cell, such as protein recycling, cell cycle modifications, and protein translocation. These processes are often overactive in diseased cells, leading to unregulated cell growth and disease progression. Therefore, in systems where Ub/Ubl protein labeling is dysregulated, the development of drugs to selectively and potently disrupt Ub/Ubl protein labeling offers a targeted molecular approach for sensitizing these diseased cells. This Perspective outlines the progress that has been made in the context of inhibitor development for targeting Ub/Ubl pathways.

Chronic myeloid leukemia (CML) is a stem cell disease characterized by BCR-ABL1. Most patients in chronic phase (CP) CML achieve long-lasting molecular responses when treated with BCR-ABL1 tyrosine kinase inhibitors (TKI). However, resistance against TKI occurs in a subset of patients. Several molecular mechanisms, including BCR-ABL1 mutations, contribute to TKI resistance. For imatinib-resistant patients, second- and third generation TKI, including nilotinib, dasatinib, bosutinib, and ponatinib, are available. Using these drugs, it is now possible to cover most of the known BCR-ABL1 mutations, including the multi-resistant mutation T315I. Ponatinib, a third generation TKI, induces growth-inhibitory effects in drug-resistant patients even if T315I is expressed. However, not all mutant forms of BCR-ABL1 are responsive to ponatinib. Moreover, it has been described that multiple secondary mutations in BCR-ABL1, especially T315I-involving compound mutations, confer resistance against ponatinib. Furthermore, resistance against TKI may develop independent of BCR-ABL1 mutations. Therefore, drug combinations covering a broad range of targets, are currently under investigation with the aim to overcome drug resistance in advanced CML. Bardoxolone methyl (CDDO-Me) is an oleanane triterpenoid that has been described to induce ROS generation and to suppress a number of survival-related molecules, including AKT, mTOR, and STAT3. The aim of the current project was to evaluate the anti-leukemic effects of CDDO-Me in TKI-resistant CML cells. As assessed by 3H-thymidine uptake experiments, CDDO-Me was found to inhibit growth of various CML cell lines, including K562, an imatinib resistant sub-clone of K562, KU812, and imatinib-resistant KCL22 cells (IC50: 0.1-0.5 µM). These effects were accompanied by induction of apoptosis as assessed by staining for AnnexinV and propidium iodide. Furthermore, CDDO-Me was found to block the growth of Ba/F3 cells harboring the BCR-ABL1 mutations T315I, E255K, G250E, H396P, or F359V as well as Ba/F3 cells expressing TKI-resistant compound mutations, such as T315I/E255V, T315I/F311L, T315I/F359V, or T315I/G250E (IC50: 0.1-0.25 µM). The anti-proliferative effects of CDDO-Me were also confirmed in primary CML cells isolated from 13 patients with chronic phase (CP) CML (4 TKI-resistant patients, 3 with BCR-ABL1 mutations), one in blast phase (BP), and one suffering from ponatinib-resistant Ph+ ALL harboring BCR-ABL1T315I/E255K. IC50 values were comparable between samples isolated from freshly diagnosed patients (IC50: 0.1-0.5 µM) and samples isolated from heavily pre-treated patients, (IC50: 0.1-0.5 µM) suggesting that BCR-ABL1 mutations do not influence responses to this drug. In consecutive experiments, CDDO-Me was found to produce synergistic growth-inhibitory effects when combined with second- or third-generation BCR-ABL1 TKI. The combination ´CDDO-Me+ponatinib´ was found to be effective in Ba/F3 cells expressing various BCR-ABL1 mutations, including T315I-involving compound mutations. We also found that the combination ´CDDO-Me+TKI´ leads to simultaneous dephosphorylation of STAT3 and STAT5. To clarify whether this drug action contributes to the synergistic drug-interactions observed, we performed experiments with shRNA directed against STAT3 or STAT5 and the specific STAT5-inhibitor AC-3-019. Knockdown of STAT3 was found to produce synergistic effects with TKI and with AC-3-019 in K562 and KCL22 cells, whereas STAT5-knockdown sensitized CML cells against CDDO-Me, pointing to a new effective concept of dual STAT3+STAT5 inhibition. However, CDDO-Me was also found to increase expression of heme-oxygenase-1 (HO-1), a heat-shock-protein known to trigger drug resistance and cell survival in CML cells. We therefore combined CDDO-Me with the HO-1 inhibitor SMA-ZnPP, which also resulted in synergistic growth-inhibitory effects in human CML cells and BCR-ABL1+Ba/F3 cells. Moreover, SMA-ZnPP was found to sensitize KU812 cells and Ba/F3 cells expressing BCR-ABL1T315I/F311L against the combination ´CDDO-Me+TKI´. Together, combined targeting of STAT3, STAT5, and HO-1 overcomes multiple forms of TKI resistance in highly resistant CML clones expressing BCR-ABL1T315I or T315I-containing compound mutations. Whether such drug combinations are effective in vivo in TKI-resistant patients remains to be elucidated. Disclosures Hoermann: Ariad: Honoraria; Gilead: Research Funding; Amgen: Honoraria; Novartis: Honoraria. Konopleva:Reata Pharmaceuticals: Equity Ownership; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Stemline: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Calithera: Research Funding. Deininger:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; CTI BioPharma Corp.: Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Research Funding; Gilead: Research Funding; Celgene: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees. Lion:Amgen: Honoraria; Pfizer: Honoraria; Ariad: Honoraria; Novartis: Honoraria, Research Funding; BMS: Honoraria. Valent:Ariad: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Deciphera Pharmaceuticals: Research Funding; Amgen: Honoraria.

The proteasomal pathway of protein degradation involves two discrete steps: ubiquitination and degradation. Blocking protein degradation by inhibiting the proteasome has well described biologic effects and proteasome inhibitors are approved for the treatment of multiple myeloma and mantle cell lymphoma. In contrast, the biological effects and potential therapeutic utility of inhibiting the ubiquitination cascade and the initiating enzyme UBA1 are less well understood. UBA1 is the initial enzyme in the ubiquitination cascade and initiates the transfer of ubiquitin molecules to target proteins where they are degraded by the proteasome. Here, we review the biological effects of UBA1 inhibition and discuss UBA1 inhibitors as potential anti-cancer agents. Similar to proteasome inhibition, blocking UBA1 elicits an unfolded protein response and induces cell death in malignant cells over normal cells. Chemical UBA1 inhibitors have been developed that target different regions of the enzyme and inhibit its function through different mechanisms. These molecules are useful tools to understand the biology of UBA1 and highlight the potential of inhibiting this target for the treatment of malignancy.

Src Homology 2 (SH2) domains are the paradigm of phosphotyrosine (pY) protein recognition modules and mediate numerous cancer-promoting protein-protein complexes. Effective SH2 domain mimicry with pY-binding coordination complexes offers a promising route to new and selective disruptors of pY-mediated protein-protein interactions. We herein report the synthesis and in vitro characterization of a library of coordination complex SH2 domain proteomimetics. Compounds were designed to interact with phosphopeptides via a two-point interaction, principally with pY, and to make secondary interactions with pY+2/3, thereby achieving sequence-selective discrimination. Here, we report that lead mimetics demonstrated high target phosphopeptide affinity (K(a) ∼ 10(7) M(-1)) and selectivity. In addition, biological screening in various tumor cells for anticancer effects showed a high degree of variability in cytotoxicity among receptors, which supported the proposed two-point binding mode. Several receptors potently disrupted cancer cell viability in breast cancer, prostate cancer, and acute myeloid leukemia cell lines.

Computer-aided lead optimization derives a unique, orally bioavailable inhibitor of the signal transducer and activator of transcription (Stat)3 Src homology 2 domain. BP-1-102 binds Stat3 with an affinity ( K D ) of 504 nM, blocks Stat3–phospho-tyrosine (pTyr) peptide interactions and Stat3 activation at 4–6.8 μM, and selectively inhibits growth, survival, migration, and invasion of Stat3-dependent tumor cells. BP-1-102–mediated inhibition of aberrantly active Stat3 in tumor cells suppresses the expression of c-Myc, Cyclin D1, Bcl-xL, Survivin, VEGF, and Krüppel-like factor 8, which is identified as a Stat3 target gene that promotes Stat3-mediated breast tumor cell migration and invasion. Treatment of breast cancer cells with BP-1-102 further blocks Stat3–NF-κB cross-talk, the release of granulocyte colony-stimulating factor, soluble intercellular adhesion molecule 1, macrophage migration-inhibitory factor/glycosylation-inhibiting factor, interleukin 1 receptor antagonist, and serine protease inhibitor protein 1, and the phosphorylation of focal adhesion kinase and paxillin, while enhancing E-cadherin expression. Intravenous or oral gavage delivery of BP-1-102 furnishes micromolar or microgram levels in tumor tissues and inhibits growth of human breast and lung tumor xenografts.

Molecular approaches to inhibit STAT5 signaling have been hailed as a viable targeted anticancer therapy. In particular, many drugs and drug candidates have been developed to successfully inhibit upstream effectors of STAT5 by indirectly targeting cell surface receptors and protein kinases (FLT-3, JAK2, and BCR-ABL). Indirect strategies have yielded potent agents, such as imatinib, AC2207, and EXEL823 which effectively silence STAT5 activity but which suffer from off-target effects and toxicity. This article will focus on reviewing the current literature pertaining to direct inhibitors of STAT5 protein and assess the prospects for a future STAT5-targeting therapeutic.

Multifunctional nanoparticles integrated with imaging modalities (such as magnetic resonance and optical) and therapeutic drugs are promising candidates for future cancer diagnostics and therapy. While targeted drug delivery and imaging of tumor cells have been the major focus in engineering nanoparticle probes, no extensive efforts have been made towards developing sensing probes that can confirm and monitor intra-cellular drug release events. Here, we present quantum dot (Qdot)-iron oxide (IO) based multimodal/multifunctional nanocomposite probe that is optically and magnetically imageable, targetable and capable of reporting on intra-cellular drug release events. Specifically, the probe consists of a superparamagnetic iron oxide nanoparticle core (IONP) decorated with satellite CdS:Mn/ZnS Qdots where the Qdots themselves are further functionalized with STAT3 inhibitor (an anti-cancer agent), vitamin folate (as targeting motif) and m-polyethylene glycol (m-PEG, a hydrophilic dispersing agent). The Qdot luminescence is quenched in this nanocomposite probe (“OFF” state) due to combined electron/energy transfer mediated quenching processes involving IONP, folate and STAT3 agents. Upon intracellular uptake, the probe is exposed to the cytosolic glutathione (GSH) containing environment resulting in restoration of the Qdot luminescence (“ON” state), which reports on uptake and drug release. Probe functionality was validated using fluorescence and MR measurements as well as in vitro studies using cancer cells that overexpress folate receptors.

More than 50 new inhibitors of the oncogenic Stat3 protein were identified through a structure-activity relationship (SAR) study based on the previously identified inhibitor S3I-201 (IC₅₀ =86 μM, K(i) >300 μM). A key structural feature of these inhibitors is a salicylic acid moiety, which, by acting as a phosphotyrosine mimetic, is believed to facilitate binding to the Stat3 SH2 domain. Several of the analogues exhibit higher potency than the lead compound in inhibiting Stat3 DNA binding activity, with an in vitro IC₅₀ range of 18.7-51.9 μM, and disruption of Stat3-pTyr peptide interactions with K(i) values in the 15.5-41 μM range. One agent in particular exhibited potent inhibition of Stat3 phosphorylation in both breast and multiple myeloma tumor cells, suppressed the expression of Stat3 target genes, and induced antitumor effects in tumor cells harboring activated Stat3 protein.

Aberrant activation of oncogenic signal transducer and activator of transcription 3 (STAT3) protein signaling pathways has been extensively implicated in human cancers. Given STAT3’s prominent dysregulatory role in malignant transformation and tumorigenesis, there has been a significant effort to discover STAT3-specific inhibitors as chemical probes for defining the aberrant STAT3-mediated molecular events that support the malignant phenotype. To identify novel, STAT3-selective inhibitors suitable for interrogating STAT3 signaling in tumor cells, we explored the design of hybrid molecules by conjugating a known STAT3 inhibitory peptidomimetic, ISS610 to the high-affinity STAT3-binding peptide motif derived from the ILR/gp-130. Several hybrid molecules were examined in in vitro biophysical and biochemical studies for inhibitory potency against STAT3. Lead inhibitor 14aa was shown to strongly bind to STAT3 (KD = 900 nM), disrupt STAT3:phosphopeptide complexes (Ki = 5 μM) and suppress STAT3 activity in in vitro DNA-binding activity/ electrophoretic mobility shift assay (EMSA). Moreover, lead STAT3 inhibitor 14aa induced a time-dependent inhibition of constitutive STAT3 activation in v-Src transformed mouse fibroblasts (NIH3T3/v-Src), with 80 % suppression of constitutively-active STAT3 at six hours following treatment of NIH3T3/v-Src. However, STAT3 activity recovered at 24 hours after treatment of cells, suggesting potential degradation of the compound. Results further showed a suppression of aberrant STAT3 activity in NIH3T3/v-Src by the treatment with compound 14aa-OH, which is the non-pTyr version of compound 14aa. The effect of compounds 14aa and 14aa-OH are accompanied by a moderate loss of cell viability.

Aberrant activation of signal transducer and activator of transcription (Stat) 3, a member of the STAT family of proteins, is prevalent in numerous human cancers and is now widely recognized as a critical molecular abnormality and a master regulator of tumor processes. Thus, the identification of potent and selective Stat3 inhibitors will have a high commercial potential as anticancer drugs, given the many tumors in which Stat3 is implicated.This review covers the structures and activities of direct inhibitors of Stat3 protein activity described in the patent literature since the research field's inception in 2001. The patents reviewed include peptide and peptidomimetic compounds, small molecules, oligonucleotides and platinum-based Stat3 inhibitors.Readers will gain an understanding of how Stat3 protein function has been inhibited by a wide variety of structurally diverse therapeutic compounds. Readers will learn about which classes of patented Stat3 inhibitors are most advanced toward clinical trials, and will be exposed to the proposed mechanisms of inhibition and scope of their application in treating human cancers.Numerous groups have shown that in vivo administration of inhibitors of activated Stat3 induce human tumor regression in xenograft models. Indeed, the growing number of preclinical studies in numerous cancer types, as well as the first Phase 0 clinical trial of a Stat3 inhibitor, suggest that Stat3 is a valid and exciting therapeutic target for molecular inhibitors.

Aberrantly activated signal transducer and activator of transcription 3 (Stat3) protein plays a master regulatory role in the progression and survival of human cancers through the upregulation of target protooncogenes. Numerous human cancers, including breast, ovarian, prostate, leukemia, lymphoma, multiple myeloma, and brain cancers have been shown to harbor constitutively active Stat3 protein resulting in the expression of protooncogenes. The transcriptionally active Stat3-Stat3 protein homodimer has been extensively targeted as a means to suppress the aberrant Stat3 function in human cancer. This review will outline the recent progress made toward identifying drug-like compounds capable of effectively inhibiting aberrant Stat3 signaling through the disruption of Stat3 protein-protein interactions.

Protection/activation strategy is applied to the preparation of the target compounds with high regioselectivity.

To facilitate the discovery of clinically useful Stat3 inhibitors, computational analysis of the binding to Stat3 of the existing Stat3 dimerization disruptors and quantitative structure-activity relationships (QSAR) were pursued, by which a pharmacophore model was derived for predicting optimized Stat3 dimerization inhibitors. The 2,6,9-trisubstituted-purine scaffold was functionalized in order to access the three subpockets of the Stat3 SH2 domain surface and to derive potent Stat3-binding inhibitors. Select purine scaffolds showed good affinities (K(D), 0.8-12 μM) for purified, nonphosphorylated Stat3 and inhibited Stat3 DNA-binding activity in vitro and intracellular phosphorylation at 20-60 μM. Furthermore, agents selectively suppressed viability of human prostate, breast and pancreatic cancer cells, and v-Src-transformed mouse fibroblasts that harbor aberrant Stat3 activity. Studies herein identified novel small-molecule trisubstituted purines as effective inhibitors of constitutively active Stat3 and of the viability of Stat3-dependent tumor cells, and are the first to validate the use of purine bases as templates for building novel Stat3 inhibitors.

Signal transducer and activator of transcription protein 3 (STAT3) is a latent cytosolic transcription factor that is widely recognized as being a master regulator of the cellular functions that lead to the cancer phenotype. Constitutively activated STAT3 protein activity is routinely observed in human cancers, promoting uncontrolled cell proliferation and suppressing apoptosis. Until relatively recently, inhibition of STAT3 transcriptional activity was achieved indirectly via suppression of upstream kinase activators and extracellular cytokine and (or) growth factor stimuli. However, activated STAT3 forms transcriptionally functional STAT3–STAT3 dimers, providing a valid juncture for targeted downstream molecular inhibition. STAT3's prominent role in cancer has seen a decade of innovative and novel approaches to targeting constitutively active STAT3 protein–protein complexes. This mini-review outlines the progress made towards identifying molecular agents capable of silencing aberrant STAT3 signalling through the disruption of STAT3 complexation events.

A facile, efficient and mild synthesis of 2,6,9-tri-substituted purines is presented, starting from commercially available 2-amino-6-chloropurine, which employs sequential N9 then N2 Mitsunobu reactions as key steps. Importantly, our synthetic approach to N2-functionalization of the purine nucleus obviates the harsh conditions required by the traditional nucleophilic aromatic substitution of a 2-halo group with primary amines. Benzylic, allylic, propargylic and aliphatic alcohols all coupled in very good to excellent yields in both Mitsunobu reactions. Significantly, excellent chemoselectivity and N9-regioselectivity were observed for the first coupling, and reactions were complete within 15 min at room temperature. Our novel methodology may be readily adapted to furnish N 9 -mono- or N 2 , N 9 -di-functionalized guanine analogues, and the utility of our protocol is further demonstrated by the efficient synthesis of the CDK inhibitor bohemine.

Signal transducer and activator of transcription 3 (Stat3) protein is a cytosolic transcription factor that relays signals from receptors in the plasma membrane directly to the nucleus, and is routinely hyperactivated in many human cancers and diseases.[1] Regarded as an oncogene, Stat3 is well-recognized as a master regulator of cellular events that lead to the cancer phenotype, making this protein viable target for molecular therapeutic design.[2] Stat3 inhibitors have included peptides,[3–4] peptidomimetics,[5–9] small molecules[10–14] and metal complexes.[15] Despite significant advances in Stat3 inhibition,[1] truly potent (in vivo), isoform-selective, small molecule Stat3 agents have not been readily forthcoming; this is likely due in part to the challenge of disrupting protein–protein interactions.[16]

Signal-Transducer-and-Activator-of-Transcription 3 (STAT3) protein plays an important role in the onset of cancers such as leukemia and lymphoma. In this study, we aim to test the effectiveness of a novel peptide drug designed to tether STAT3 to the phospholipid bilayer of the cell membrane and thus inhibit unwanted transcription. As a first step, STAT3 proteins were successfully labelled with tetramethylrhodamine (TMR), a fluoresc ent dye with suitable photostability for single molecule studies. The effectiveness of labelling was determined using fluorescence correlation spectroscopy in a custom built confocal microscope, from which diffusion times and hydrodynamic radii of individual proteins were determined. A newly developed fluorescein derivative label (F-NAc) has been designed to be incorporated into the structure of the peptide drug so that peptide-STAT3 interactions can be examin ed. This dye is spectrally characterized and is found to be well suited for its application to this project, as well as other single-molecule studies. The membrane localization via high-affinity cholesterol-bound small-molecule binding agents can be demonstrated by encapsulating TMR-labeled STAT3 and inhibitors within a vesicle model cell system. To this end, unilaminar lipid vesicles were examined for size and encapsulation ability. Preliminary results of the efficiency and stability of the STAT3 anchoring in lipid membranes obtained via quantitative confocal imaging and single-molecule spectroscopy using a custom-built multiparameter fluorescence mi croscope are reported here. Single-molecule spectroscopy, Fluorescei n derivative, STAT3, Fluorescence co rrelation spectroscopy (FCS), Forster resonance energy transfer (FRET)

We herein report the comprehensive characterization of the spectral and single-photon fluorescence properties of a recently synthesized fluorescein derivative and its biotinylated analog. The fluorophore displays significant increases in photostability compared to the known fluorescein label fluorescein isothiocyanate (FITC), as well as superb pH independence. This fluorescein variant has two readily accessible functional groups (aniline NH2 and phenol OH) that can be activated or blocked independently and can serve, for instance, as a fluorescent bridge between two different recognition motifs. Excellent single-photon counting fluorescence data demonstrates that it is also a particularly appropriate probe for single-molecule studies of biological interactions.

The mild and efficient deblocking of aryl benzyl ethers with TFA is reported. Cleavage was fastest with ortho-electron-withdrawing groups on the phenolic ring, which we have attributed to a proton chelation effect, furnishing the deprotected phenols in excellent yields. The corresponding para-methoxybenzyl, allyl and iso-propyl ethers were also cleanly removed under these conditions. In addition, the selective aryl benzyl ether debenzylation in the presence of benzyl ester, Cbz carbamate and Boc carbamate functionalities was also observed.

The signal transducers and activators of transcription (STATs) are a class of transcription factor proteins that regulate cell growth and survival by modulating the expression of specific target genes.[1] A total of seven different STAT isoforms, encoded in distinct genes, have been identified in mammalian cells. Stat3, a member of the STAT family, has been identified in an increasing number of tumor cell lines. Stat3 drives malignant progression through the misregulation of key proteins, including cell survival proteins such as Bcl-xL and Mcl-1, cell cycle regulators such as cyclin D1/D2 and c-myc, and inducers of angiogenesis such as vascular endothelial growth factor (VEGF).[2] In contrast to normal cells, where Stat3 activation is rapid and transient, neoplastic cells are found to display constitutive Stat3 activation that, once inhibited, correlates with suppression of both cell transformation and growth, and induction of apoptosis.[3-8] While STAT signaling is just one of many pathways compromised in oncogenesis, interruption of this pathway is sufficient to block cell transformation; this suggests that these cells have an irreversible dependence on constitutively active Stat3 for survival. Numerous reports highlight the relevance of persistent Stat3 activation in human cancers; abnormal levels of Stat3 activation have been observed in breast,[9,10] ovarian,[9] prostate,[11] haematological,[12] and head and neck cancer cell lines.[13] These investigations suggest that agents designed to disrupt Stat3 signaling hold considerable promise for the prevention and treatment of human cancers.

Stat3 is hyperactivated in many human tumors and represents a valid target for anticancer drug design. We present a novel small-molecule Stat3 inhibitor, S3I-M2001, and describe the dynamics of intracellular processing of activated Stat3 within the context of the biochemical and biological effects of the Stat3 inhibitor. S3I-M2001 is an oxazole-based peptidomimetic of the Stat3 Src homology (SH) 2 domain-binding phosphotyrosine peptide that selectively disrupts active Stat3:Stat3 dimers. Consequently, hyperactivated Stat3, which hitherto occurs as "dotlike" structures of nuclear bodies, undergoes an early aggregation into nonfunctional perinuclear aggresomes and a late-phase proteasome-mediated degradation in malignant cells treated with S3I-M2001. Thus, S3I-M2001 inhibited Stat3-dependent transcriptional regulation of tumor survival genes, such as Bcl-xL. Furthermore, Stat3-dependent malignant transformation, survival, and migration and invasion of mouse and human cancer cells harboring persistently activated Stat3 were inhibited by S3I-M2001. Finally, S3I-M2001 inhibited growth of human breast tumor xenografts. The study identifies a novel Stat3 inhibitor, S3I-M2001, with antitumor cell effects mediated in part through a biphasic loss of functional Stat3. The study represents the first on intracellular Stat3 stability and processing following inhibition by a small molecule that has significant antitumor activity.

Protein conjugation with the UFM1 small protein modifier promotes cellular survival in times of ER stress, which is a common occurrence within diseased cells. We have developed the first selective inhibitor of UBA5, the E1 activating enzyme responsible for initiating UFM1 conjugation. Based on the crystal structure of human UBA5, we identified acidic residues in close proximity to the ATP pocket that we chose to target using a zinc-coordinated polyazamacrocycle. This coordination complex was appended to adenosine through alkyl linkers of varying length in order to probe for optimal inhibition of UBA5 activity. Through this small structure-activity relationship (SAR), we identified our lead inhibitor 5C-Z that disrupts UBA5-mediated UFM1 conjugation in vitro through a non-competitive mode of action (IC50 = 4.1 μM, 95% Confidence Interval (C.I.) = 2.1-7.7 μM). Our compound demonstrates selectivity for UBA5 over other E1 enzymes in vitro, as well as over nearly 100 human kinases as evaluated using a kinome screen, despite containing adenosine within its structure. Currently, we are investigating the structural determinants that are indispensible for inhibitory activity. We are making iterative changes by varying each component of 5C-Z including the metal centre, the polyazamacrocycle, the linker, the ribose as well as the nitrogenous base. To date, we have shown that other transition metals are not as active as the zinc (II) centre, increasing the size of the macrocycle abolishes activity and reducing the linkage from the polyazamacrocycle to the linker also significantly abrogates activity. Investigating the role of each functional group on 5C-Z will help delineate the mechanism by which it selectively inhibits UBA5 activity and may shed light in progressing towards a more drug-like small molecule inhibitor. Citation Format: Stacey-Lynn Paiva, Sara da Silva, Mathew Bancerz, Mulu Geletu, Andrew M. Lewis, Jijun Chen, Yafei Cai, Julie L. Lukkarila, Honglin Li, Patrick T. Gunning. The investigation of structural determinants that mediate selective inhibition of UBA5, the UFM1 activating enzyme. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-C19.

Cells that undergo higher protein turnover, such as pancreatic secretory cells and cancerous cells, have the propensity to undergo endoplasmic reticulum (ER) stress. If left uncorrected, ER stress can result in the initiation of apoptosis. To avoid these fates, cells have developed support systems to counteract the apoptotic effects of ER stress, such as conjugation of certain stress-associated proteins with the ubiquitin-fold modifier 1 (UFM1) ubiquitin-like protein. Our research has recently focused on the discovery and in vitro validation of the first selective inhibitor of the UFM1 pathway, 5C-Z, which selectively targets the UFM1 E1 enzyme (UBA5) over other related enzymes and a panel of over 90 human kinases. This novel strategy of inhibiting UBA5 and subsequent UFMylation would make cancer cells that are highly dependent on this system more susceptible to pharmacological disturbances in ER homeostasis, which could lead to the use of milder drug dosing strategies. Our current efforts are focused on studying the effects of 5C-Z on intracellular signalling, the cellular distribution of proteins in the UFM1 pathway, and phenotypic changes within a lung cancer cell lung line (Sk-Luci6). Treatment of lung cancer cells that exhibit high levels of UBA5 protein expression results in decreased cell proliferation (EC50 = 216.9 μM, 95% C.I. = 211.5 - 222.5 μM), yet does not induce cell death in other diseased (A549) or healthy (MRC9) lung cells (up to 200 μM). The decreased cellular proliferation upon 5C-Z treatment mirrors the effect of treatment with UBA5 siRNA. Using immunocytochemistry, it appears that treatment with 5C-Z also induces changes in both cellular morphology and the distribution of UBA5-like staining within the cell. Furthermore, there is an apparent decrease in UFM1-like co-localization with its E2 conjugating enzyme (UFC1) after prolonged incubation with 5C-Z, although further intracellular analysis is required to confirm this observation. Preliminary work also indicates that these cellular effects may also be selective against the UFM1 pathway compared to related ubiquitin-like labelling pathways. We are currently evaluating the effects of 5C-Z on ER stress signalling pathways connected to the UFM1 labelling system. Citation Format: Sara R. da Silva, Mulu Geletu, Fiza Javed, Stacey-Lynn Paiva, Andrew M. Lewis, Honglin Li, Patrick T. Gunning. Cellular characterization of the selective inhibition of UBA5 by organometallic, adenosine-based inhibitors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A13.

STAT5 transcription factors gain increasing attention as essential drivers in the development of myeloproliferative and lymphoid diseases. STAT5 is often hyper-activated due to deregulated upstream kinase signaling. Additionally, recurrent gain-of-function mutations in the SH2 domain of STAT5 were described, implying its importance in oncogenic transformation independent of driver kinase mutations. To date, various tyrosine kinase inhibitors (TKI) are in the clinics or in clinical trials. However, TKI treatment is often accompanied by resistance development, cytotoxicity as a result of poor kinase selectivity, as well as cardiovascular toxicity. Hence, considering the role of STAT5 in hematopoietic cancers, it is reasonable to directly target STAT5 as a transcriptional regulator. To inhibit STAT5, we used a small inhibitory molecule (compound X) binding to the SH2 domain of the protein, subsequently resulting in the disruption of STAT5-phosphopeptide interactions. We tested the efficacy of X in STAT5 driven AML/CML cell lines and patient samples. The inhibitor efficiently suppressed transcriptional activity of STAT5, kinase-mediated phosphorylation, dimerization, nuclear translocation, DNA binding and STAT5-mediated gene expression. Importantly, we detected down regulation of c-MYC, Cyclin D1, Cyclin D2, and MCL-oncoproteins, which are important inhibitors of apoptosis and regulators of the cell cycle. Furthermore, X induced extensive apoptosis in MPN cell lines as well as robust apoptotic cell death in patient-derived cells. Our findings indicate that X is a potent and selective inhibitor of STAT5 and provides a lead structure for further chemical modifications for clinical development to improve existing therapies in MPNs.

The synthesis of a heteroditopic receptor which exhibits positive cooperativity for the binding of phosphate ion pairs under physiological conditions. Optimised complementarity between crown ether host and metal guest leads to increased binding affinity, Ka.

Three new azamacrocyclic-cyclophane hybrid receptors L(1), L(2), and L(3) have been synthesized that incorporate either 1,4,7,10-tetraazacyclododecane (cyclen) or 1,4,7-triazacyclononane (tacn) unit(s) tethered via a short amidic spacer to an electron donor and a H-bonding crown ether polycycle. The crown ether is designed to act as a host toward biologically relevant guests, whereas the macrocycle can coordinate a zinc(II) or a copper(II) ion. The pK(a) of this bound water in the zinc(II) complex of L(1) and L(2) is approximately 7.5. Isothermal calorimetry experiments carried out on [ZnL(1)(L2)(OH(2))](CF(3)SO(3))(2) and [Zn(2)L(2)(OH(2))(2)](CF(3)SO(3))(4) in buffered water (pH 7.4) at 25 degrees C show that the host strongly binds a series of phosphate derivatives. In comparison, the complex [CuL(3)(OH(2))(2)](CF(3)SO(3))(2) is a poor receptor toward phosphate substrates.

The synthesis of a modular hybrid receptor containing both an aza macrocycle and crown ether is described; a complete thermodynamic characterisation of the binding properties, in water, of the zinc(II) complex of the receptor towards phosphate is presented and the parameters are compared to those of the aza macrocycle precursor.

We have identified STAT3 as a convergence point for oncogenic signaling in tyrosine kinase inhibitor (TKI)-resistant chronic myeloid leukemia (CML) lacking BCR-ABL1 kinase domain mutations. In addition, we found that STAT3 activity contributes to disease in other myeloid disorders, including acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPNs). Utilizing TKI-resistant CML as a model system, we identified BP-5-087 as a small molecule inhibitor of STAT3 that reduces STAT3 phosphorylation and nuclear transactivation (Eiring et al. Leukemia, 2014). Binding of BP-5-087 to the STAT3 SH2 domain was initially assessed using fluorescence polarization (FP) assays and high-resolution computational docking simulations. To further validate the binding motif of BP-5-087, we conducted time-resolved electrospray ionization mass spectrometry/hydrogen-deuterium exchange experiments. Fold-change in deuterium uptake was analyzed for 68 STAT3 peptides representing 71% sequence coverage, and mapped onto the crystal structure of STAT3. This analysis precisely defined the binding epitope for BP-5-087 within the STAT3 SH2 domain. We next tested the effects of BP-5-087 in several myeloid malignancies using relevant disease models. (i) CML stem and progenitor cells from TKI-resistant patients without kinase domain mutations were treated with BP-5-087 ex vivo, using short-term liquid culture, clonogenic and LTC-IC assays. BP-5-087 treatment significantly reduced colony formation by CML stem and progenitor cells (p The in vivo activity of BP-5-087 was next evaluated in a murine model of JAK2 V617F-induced MPN. Briefly, Balb/c bone marrow was transduced with JAK2 V617F-GFP, followed by injection into lethally irradiated recipients. After disease induction, mice were treated with BP-5-087 (25 mg/kg) by once-daily oral gavage. No toxicities were observed after 40 days of treatment in BP-5-087-treated mice. While BP-5-087 did not significantly reduce the percentage of GFP+ cells, there was a 41% reduction of spleen weight in BP-5-087-treated mice compared to vehicle-treated controls (p To advance the lead optimization of our STAT3 inhibitor series, we instituted a comprehensive screening cascade. We first developed a computational model (quantitative structure-activity relationship, QSAR) to guide and prioritize selection of new inhibitor candidates for synthesis. Compounds are initially ranked using a methanethiosulfonate (MTS)-based cell viability assay in a TKI-resistant, STAT3-dependent CML cell line (AR230R). Inhibition of STAT3 is confirmed using a cell-based STAT3 reporter assay and an in vitro FP-based binding assay. Optimization of potency is balanced by the goals of reducing molecular weight (MW) and calculated LogP (cLogP) compared to BP-5-087 (MW: 694.8; cLogP: 7.3). Compounds with improvements in these categories are then subject to toxicity testing utilizing clonogenic assays with CD34+ CB cells. Non-toxic compounds are evaluated for their pharmacokinetic profile in Balb/c mice and tested for activity in primary samples from CML, AML and MPN patients. These activities have directed us to a lead compound, AM-1-124, which displays significant improvements in potency, MW, cLogP, and in vivo half-life compared to BP-5-087. AM-1-124 had minimal effects in the CB toxicity assay and induced apoptosis in primary AML patient samples at 2-fold lower concentrations than BP-5-087. With AM-1-124 as our current lead compound, we are continuing our iterative evaluation of novel STAT3 inhibitors utilizing our screening cascade. Design and testing of optimized, orally active inhibitors will enable further evaluation of STAT3 as a target in animal models of myeloid leukemia and will justify the clinical development of these compounds for patients in need of new targeted therapies. Disclosures Deininger: BMS, Novartis, Celgene, Genzyme, Gilead: Research Funding; BMA, ARIAD, Novartis, Incyte, Pfizer: Advisory Board, Advisory Board Other; BMS, ARIAD, Novartis, Incyte, Pfizer: Consultancy.

Among other cellular processes, gene expression is regulated by epigenetic histone modifications. Histone methyltransferases catalyze the transfer of the methyl group from S-adenosylmethionine to specific lysine residues on histones. Mixed lineage leukemia 1 (MLL1) is a methyltransferase that methylates lysine 4 on histone H3 (H3K4me3) and is an important regulator of the haemopoietic system. Deregulation of MLL1 is often associated with acute myeloid and lymphoid leukemias and was proposed as a novel therapeutic target. WD40 repeat protein 5 (WDR5) is a component of the multiprotein MLL1 complex that is essential for its methyltransferase activity. Thus, therapeutic intervention of the WDR5/MLL1 interaction may lead to possible novel therapeutic agents for MLL-dependent leukemias. Using a structure-based design approach our structure activity relationship studies identified selective and cell-permeable compounds that bind to WDR5 with low nanomolar affinities. In future chemical biology studies these molecules will serve as valuable molecular probes to dissect the biological role of WDR5.

The NEDD8-activating enzyme (NAE) initiates neddylation, the cascade of post-translational conjugation of NEDD8 onto target proteins. Cullin proteins are major substrates of the neddylation pathway, and are involved in diverse cellular processes. MLN4924, a selective NAE inhibitor, has displayed preclinical anti-tumor activity in vitro and in vivo, and promising clinical activity has been reported in patients with refractory hematologic malignancies. Here, we sought to understand the mechanisms of resistance to MLN4924 in leukemic cells. MLN4924 shows potent activity in K562 human leukemia cells (EC50 = 100 nM). In contrast, by exposing K562 cells to increasing concentrations of MLN4924 over 6 months, we selected a population of cells resistant to MLN4924 (R-K562MLN; EC50 > 2 µM). R-K562MLN cells proliferated similarly to parental K562 cells, and remained resistant to MLN4924 even after culturing in the absence of MLN4924 for 5 weeks. Compared to parental cells, R-K562MLN cells were similarly sensitive to a broad spectrum of chemotherapeutic drugs, including daunorubicin, a classical substrate of the multidrug efflux transporter P-glycoprotein. Thus, the mechanism of resistance to MLN4924 did not appear to be related to generalized multidrug efflux. While basal levels of NEDD8-conjugated cullin proteins were equivalent in parental and resistant cells, NEDD8-cullins were diminished in parental cells treated with 25 nM MLN4924. In contrast, no appreciable reduction in NEDD8-cullins was observed in R-K562MLN cells after treatment with up to 250 nM MLN4924. However, knockdown of NEDD8 by shRNA was cytotoxic to R-K562MLNcells, suggesting that NEDD8 conjugation remains essential for the survival of resistant cells. To further probe mechanisms of MLN4924 resistance in these cells, we sequenced the coding region of the NAE catalytic subunit UBA3 from K562 and R-K562MLN cells, and identified a mutation in codon 310 [ATT (Isoleucine, I) > AAT (Asparagine, N)] in R-K562MLN cells. Biochemical analyses using recombinant wild-type and I310N NAE complexes indicated that the I310N mutation alters biochemical properties of the NAE, increasing the enzyme’s affinity for ATP while decreasing its affinity for NEDD8. The I310N NAE complex was ∼7-fold less sensitive to MLN4924 (IC50 = 225 nM) compared to wild-type NAE (IC50 = 32 nM), providing a mechanistic basis for MLN4924 resistance in R-K562MLNcells through the UBA3 I310N mutation. To investigate whether another malignant leukemic cell line employs a similar on-target resistance mechanism, we similarly selected a population of U937 cells resistant to MLN4924 (R-U937MLN). Parental U937 cells were sensitive to MLN4924 (EC50 = 25 nM), while R-U937MLN cells were more than 40-fold more resistant (EC50 > 1 µM). Sequencing of the coding region of UBA3 in U937 and R-U937MLN cells revealed a point mutation in codon 352 [TAT (Tyrosine, Y) > CAT (Histidine, H)] only in R-U937MLN cells. As for the UBA3 I301N mutation, the Y352H mutation conferred a ∼10-fold decrease in the sensitivity of the NAE to MLN4924 (IC50= 138.5 nM and 13 nM for Y352H and wild-type, respectively) through decreased NEDD8 and increased ATP affinities. These findings suggest that the Y352H mutation, like I310N, is sufficient to provide MLN4924 resistance in leukemia cells while sustaining adequate NAE function for leukemic cell survival. We next found that R-K562MLN cells were cross-resistant to other NAE-selective inhibitors derived from Compound 1, a pan-E1 inhibitor. Nevertheless, compared to parental cells, R-K562MLN cells were not refractory to Compound 1 (EC50 = 27 nM and 81 nM, respectively). The cytotoxicity of Compound 1 towards MLN4924-resistant cells might be explained by its inhibition of other E1 enzymes. Indeed, Compound 1 diminished the abundance of ubiquitinated proteins in R-K562MLNcells similarly to its effects in K562 cells, while NEDD8-cullins in the MLN4924-resistant cells were unaffected. Overall, as MLN4924 continues to be evaluated clinically, our work provides insight into the mechanisms of MLN4924 resistance, which may facilitate the development of more effective second-generation NAE inhibitors. Disclosures: Petroski: Allostere, Inc.: Consultancy, Equity Ownership.

Despite significant advances in the treatment of Multiple Myeloma (MM), it remains incurable and thus, novel therapeutic strategies are critically needed. Prior work has demonstrated that the transcription factor STAT3 is aberrantly activated in a number of human tumors and drives hallmark oncogenic processes such as proliferation, survival, angiogenesis and drug resistance. As such, this master regulator of malignant processes has become the focus of therapeutic targeting in human tumors that harbor constitutive STAT3 activation, including MM. In the present studies, we characterize a newly developed small molecule, BP-4-018. The inhibitory mechanism of BP-4-018 is based on its ability to function as a phosphotyrosine mimetic, and thus bind the STAT3 SH2 domain to inhibit STAT3 phosphorylation (pSTAT3), STAT3 dimerization and DNA binding. Based on the 3D crystal structure of STAT3, SH2 docking simulations and fluorescence polarization assays, BP-4-018 demonstrates high affinity binding to the SH2 domain of STAT3. Using a panel of molecularly heterogeneous human myeloma cell lines (HMCLs), BP-4-018 demonstrated potent and broad anti-tumor activity in MTT assays, with IC50 values in the low μM range (1.91 μM - 6.48 μM). These anti-MM effects are mediated through the induction of the apoptosis as evidence by Annexin V+ staining, PARP cleavage and activation of caspases 3 and 9. Using patient derived bone marrow (BM) cells, BP-4-018 was shown to retain its anti-MM activity against HMCLs cultured in the presence of a BM stroma, induce apoptosis in primary MM tumor cells, and importantly, displayed little to no toxicity against non-malignant BM cells. Using cell-based assays, we show that BP-4-018 inhibits basal and cytokine-induced pSTAT3, as well as STAT3 transcriptional activity in HMCLs that stably express a STAT3-driven luciferase reporter construct. Consistent with these effects, western blot analyses of BP-4-018-treated cells reveal decreased expression of STAT3 target genes including c-Myc, Mcl-1 and Bcl-xL. Interestingly, BP-4-018 did not demonstrate preferential inhibition of cytokine induced STAT3 phosphorylation over that of STATs 1 or 5 suggesting that this compound may in fact be a pan-STAT inhibitor. BP-4-018 does not however inhibit the phosphorylation of other proteins whose regulation is SH2-dependent including AKT, ERK, SRC and LYN. In vitro combinatorial studies revealed that BP-4-018 is synergistic in combination with dexamethasone (CI=0.62), melphalan (CI=0.94), lenalidomide (CI=0.82) and is highly synergistic with bortezomib (BTZ) (CI=0.37). To examine the potential mechanisms of combinatorial synergy with BTZ, we evaluated the effects of BTZ on STAT3 signaling in HMCLs. BTZ treatment alone augmented STAT3 transcriptional activity and led to the accumulation of Mcl-1 protein, which was reversed when cells were co-treated with BP-4-018. Furthermore, HMCLs expressing a dominant negative STAT3 mutant display increased sensitivity to BTZ. Collectively these data suggest that the combinatorial synergism with BTZ is at least in part related to BP-4-018-mediated inhibition of STAT3. Finally, we used a novel STAT3-driven luciferase xenograft model of MM to show that BP-4-018 (15 mg/kg/day, PO) reduces STAT3-driven luciferase activity in vivo, significantly delays tumor growth as a single agent, and in combination with BTZ, induces marked and in some animals complete tumor regression. Neither BP-4-018 alone or in combination elicited significant toxicities as assessed by weight loss, and more extensive pharmacodynamic, pharmacokinetic and toxicology studies of BP-4-018 are currently underway. Taken together, our results highlight the promising therapeutic potential of BP-4-018 and support the continued development of targeted therapies for MM with a focus on aberrant STAT signaling. Disclosures: No relevant conflicts of interest to declare.

Mutations in the BCR-ABL1 kinase domain are a well-established mechanism of tyrosine kinase inhibitor (TKI) resistance, but fail to explain many cases of clinical TKI failure. In the remaining patients, resistance occurs via activation of alternative signaling pathways that maintain survival despite BCR-ABL1 inhibition (BCR-ABL1-independent resistance). STAT3 mediates TKI resistance in chronic myeloid leukemia (CML) cells cultured in the presence of bone marrow-derived factors (Bewry et al., 2008; Traer et al., 2012; Nair et al., 2012), and also plays a critical role in survival of CML cells with BCR-ABL1-independent resistance (Eiring et al. #31, ASH 2012). While targeting transcription factors is notoriously difficult, our combination of synthetic chemistry, in vitro reporter assays, and computational modeling has led to a low micromolar mechanism-based STAT3 inhibitor, which, in combination with TKIs, shows promise as a treatment for CML patients with BCR-ABL1-independent resistance. The original compound of the series, SF1-066 (10 µM; Fletcher et al., 2009), combines with TKIs to reduce survival of CML CD34+ cells exhibiting BCR-ABL1-independent resistance (Eiring et al. #31, ASH 2012). To improve the potency and selectivity of SF1-066, we synthesized successive STAT3 inhibitor libraries and ranked candidates by structure-activity relationship using a luciferase-based reporter screen (Kraft et al. #2445, ASH 2012). This reporter assay quantifies STAT3 transcriptional activity in TKI-resistant AR230R cells, which grow in the continuous presence of imatinib (1.0 µM), lack BCR-ABL1 kinase domain mutations, and exhibit high levels of pSTAT3Y705, thereby enabling convenient, high-throughput screening for potency and selectivity in the context of endogenous STAT3 activation. Among three sequential STAT3 inhibitor libraries, BP5-087 emerged as the new lead compound. Fluorescence polarization assays verified that BP5-087 was 5-fold more effective than SF1-066 in outcompeting an SH2 peptide probe, and computational simulations predicted better overall binding of BP5-087 (-9.6 kcal/mol) versus SF1-066 (-7.6 kcal/mol) to the STAT3 SH2 interface. In AR230R cell growth assays, BP5-087 was effective at a 5-fold lower dose compared to SF1-066, with minimal effects on TKI-sensitive parental controls. Therefore, we tested BP5-087 in the context of primary TKI resistance. BP5-087 (1 µM) in combination with imatinib (2.5 µM) reduced colony formation and increased apoptosis of CD34+ cells from CML patients with BCR-ABL1-independent resistance. These cells have no BCR-ABL1 kinase domain mutations and undergo BCR-ABL1 kinase inhibition as detected by immunoblot analyses. In contrast, BP5-087 had no effect on CD34+ cells from newly diagnosed CML patients or normal individuals. Immunofluorescence demonstrated that dual treatment of TKI-resistant CML CD34+ cells resulted in reduced levels of nuclear pSTAT3Y705, consistent with an inhibitor of STAT3 dimerization. In more primitive CML stem cells, long term culture-initiating cell (LTC-IC) assays revealed that neither inhibitor alone had any effect on colony formation of primitive LTC-IC progenitors, whereas imatinib (2.5 µM) in combination with BP5-087 (1.0 µM) reduced LTC-IC colony formation by 66%. Consistent with this observation, immunofluorescence showed high levels of pSTAT3Y705 in primitive TKI-resistant CD34+CD38- cells when cultured in the presence but not absence of TKIs. To test the feasibility of BP5-087 for in vivo use, we treated mice orally with BP5-087 (25 mg/kg/day) for 4 weeks and observed no changes in body weight, peripheral blood cellularity, or bone marrow colony forming ability. Mass spectrometry confirmed that BP5-087 is orally bioavailable. In summary, BP5-087 is a systematically-derived, direct inhibitor of STAT3 that, in combination with TKIs, reduces survival of CML cells with BCR-ABL1-independent resistance. Further rounds of structure-activity optimization may reveal an inhibitor with a clinically-relevant effective concentration. Disclosures: Deininger: Bristol Myers Squibb: Advisory Boards Other, Consultancy, Research Funding; Ariad Pharmaceuticals: Advisory Boards, Advisory Boards Other, Consultancy; Novartis: Advisory Boards, Advisory Boards Other, Consultancy, Research Funding; Celgene: Research Funding; Gilead Sciences: Research Funding.

As a master regulator of the malignant process, the oncogenic transcription factor STAT3 has become the focus of therapeutic targeting in human tumors that harbor constitutive STAT3 activity, including Multiple Myeloma (MM). In the present studies, we employ genetic and chemical tools to further validate STAT3 as a therapeutic target in MM and investigate a novel small molecule STAT3 inhibitor, BP-4-018, with promising in vitro and in vivo anti-MM activity. Using three molecularly heterogeneous human myeloma cell lines (HMCLs), we show that shRNA-mediated knockdown (KD) of STAT3 induces apoptosis, as evidenced by increased Annexin V+ staining and PARP cleavage. Consistent with this biological readout, we observed a decrease in protein expression of STAT3-regulated survival genes including c-Myc, Mcl-1, Bcl-2 and Bcl-xL in STAT3 KD cells. These findings are therapeutically relevant as treatment of HMCLs with BP-4-018 displays broad anti-myeloma activity with IC50 values in the low μM range (1.91-6.48 μM). This rationally designed STAT3 SH2 domain-binding compound induces apoptotic responses in HMCLs and primary MM cells. Mechanistic studies reveal that BP-4-018 dose-dependently inhibits constitutive STAT3 phosphorylation (pSTAT3). Moreover, in selected HMCLs the dose of BP-4-018 required to inhibit pSTAT3 correlated precisely with their respective sensitivity to BP-4-018. HMCLs expressing a STAT3-driven luciferase reporter construct confirmed inhibition of STAT3 transcriptional activity following BP-4-018 treatment, which was associated with a decrease in protein expression of STAT3 target genes. In cell-based assays, BP-4-018 did not demonstrate selectivity for inhibiting pSTAT3 over that of STATs 1 or 5. Importantly however, BP-4-018 did not inhibit phosphorylation of other SH2-dependent proteins including AKT, ERK, SRC and LYN. In vitro combinatorial studies reveal that BP-4-018 is highly synergistic with bortezomib (Btz; CI=0.37). Preliminary data suggests that this interaction may be related to Btz-induced STAT3 activation and induction of Mcl-1 accumulation as concomitant treatment with BP-4-018 inhibited Btz-induced Mcl-1 accumulation. Supporting these findings, cells expressing a dominant negative STAT3 mutant demonstrate enhanced sensitivity to Btz. Finally, we report findings from in vivo efficacy studies using a novel STAT3-driven luciferase xenograft model of MM. We show that BP-4-018 (15 mg/kg/day, PO) reduces STAT3-driven luciferase expression in vivo, significantly delays tumor growth when administered as a single agent, and in combination with Btz, promotes tumor regression without significant toxicities. Taken together, our results highlight the validity of STAT3 as a therapeutic target in MM and support continued study of BP-4-018 and its analogs for the treatment of MM. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A260. Citation Format: Danielle C. Croucher, Zhi Hua Li, Brent DG Page, Ellen Wei, Patrick T. Gunning, Suzanne Trudel. BP-4-018, a novel inhibitor of STATs demonstrates anti-myeloma activity in preclinical models: Therapeutic implications of targeting aberrant STAT3 signaling in multiple myeloma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A260.

Aberrant STAT3 signaling is prevalent in hematologic malignancies such as Multiple Myeloma (MM) and is recognized as a master regulator of tumor processes such as survival, angiogenesis, and drug resistance. Significant efforts have therefore focused on therapeutic targeting of STAT3 as an anti-cancer strategy. Based on rational structure-based design, we present pre-clinical findings for a newly developed small molecule STAT3 inhibitor, SH-4-54. SH-4-54 was designed to block the SH2 domain of STAT3 by mimicking its native phosphopeptide binding sequence and in turn prevent phosphorylation (activation) and disrupt transcriptionally active STAT3:STAT3 dimerization. SH-4-54 has been shown to have a KD for STAT3 protein of 300 nM by surface plasmon resonance and inhibits STAT3 SH2 domain-phosphopeptide interactions as assessed by a fluorescence polarization assay. The anti-MM activity of SH-4-54 was assessed using MTT assays against a panel of molecularly heterogeneous human myeloma cell lines (HMCLs), revealing potent and broad effects on cell viability with 10/15 HMCLs displaying IC50 values < 10 μM. Moreover, SH-4-54 induced apoptotic responses in HMCLs as evidenced by increased Annexin V staining. Promising results were observed for the effects of SH-4-54 on primary patient-derived myeloma cells. Using flow cytometric analysis of CD138 as a marker of myeloma cells and Annexin V staining, SH-4-54 was found to significantly reduce the percentage of CD138+/Annexin V negative, viable myeloma cells, while displaying little to no toxicity against the non-malignant (CD138−) cell fraction. Supporting the predicted molecular mechanisms of SH-4-54 activity, immunoblot analysis revealed that SH-4-54 inhibits constitutive STAT3 phosphorylation, but does not alter STAT3 protein levels. Furthermore, using HMCLs engineered to express a STAT3-driven luciferase reporter construct, SH-4-54 was found to significantly reduce STAT3 transcriptional activity, and consistently induce a reduction in proteins that encode STAT3 target genes such as c-Myc. Taken together, our results highlight the promising therapeutic potential of SH-4-54 and support the continued development of targeted therapies for MM with a focus on aberrant STAT signaling. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C246. Citation Format: Zhi Hua Li, Sina Haftchenary, Danielle Croucher, Patrick T. Gunning, Suzanne Trudel. SH-4-54, a novel small-molecule inhibitor of STAT3, demonstrates significant anti-tumor activity against multiple myeloma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C246.

Constitutive activation of Stat3, a member of signal transducer and activator of transcription (Stat) family of proteins, occurs with a high frequency in human tumors and contributes to carcinogenesis and tumor progression. Compelling evidence shows aberrant activation of Stat3 constitutes a point of convergence in oncogenic tyrosine kinase signaling, functioning as a master regulator of events crucial for tumorigenesis and malignant progression. We present a series of novel small-molecules that abrogate constitutive activation of Stat3 in human tumors, including glioma and breast cancer. The small-molecule, SH5-07, inhibits in vitro Stat3 DNA-binding activity, with IC50 value of 3.9 μM. Further, treatment with SH5-07 induces dose-dependent inhibition of constitutively-active Stat3 in human glioma U251MG and U373MG and breast cancer MDA-MB-231 cells. Moreover, treatment with low micromolar SH5-07 of U251MG, U373MG, and MDA-MB-231 cells blocks constitutive Stat3 phosphorylation, DNA-binding, and transcriptional activities by as early as 30 minutes. Furthermore, the inhibition of aberrantly-active Stat3 by SH5-07 blocks the anchorage-dependent and independent growth, survival, migration, and invasion in vitro of human glioma and breast cancer cells. Data together indicates SH5-07 is a promising small-molecule Stat3 inhibitor and a suitable drug candidate for clinical development. Citation Format: Peibin Yue, Sina Haftchenary, Patrick T. Gunning, James Turkson. A novel small-molecule inhibitor of Stat3 induces antitumor cell effects in human glioma and breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3253. doi:10.1158/1538-7445.AM2013-3253

Abstract 576 Stat3 appears to play a critical role in sustaining and generating memory B-cells and long-lived plasma cells (PCs) the normal counterparts of myeloma cells. In contrast to the transient nature of Stat3 activation in normal cells, many hematological tumors including multiple myeloma (MM) harbor constitutive Stat3 activity. Aberrant STAT signaling is recognized as a master regulator of direct and indirect tumor processes including proliferation, apoptosis, invasion, angiogenesis and cancer inflammation and thus Stat3 represents an important therapeutic target. Using structure-based design, targeting the phosphotyrosine (pY)-SH2 domain interactions that stabilizes active Stat3 dimers, phosphopeptides have been identified that block Stat3 dimerization and DNA binding activity. Peptidic agents however suffer from limited cell permeability and metabolic lability, properties that have restricted their practical application in vivo at therapeutic doses. Here we describe the dependence of myeloma tumors on Stat3 and characterize the anti-tumor activity of BP-2-047, a novel, non-phosphorylated small molecule with reduced peptidic character. We analyzed the activity of Stat3 using a Stat3 gene expression index based on Stat3 target genes in 60 human myeloma cell lines (HMCLs). Several available myeloma cell lines (XG6, XG7, JJN3 and UTMC2) demonstrated elevated STAT3 gene expression signatures (2.3 to 14.8) with confirmed constitutive Stat3 phosphorylation by Western blot analysis. RNA interference revealed that Stat3 is essential for XG6, JJN3, and UTMC2 survival. We similarly analyzed the activity of Stat3 in publicly available gene expression profile data sets derived from newly diagnosed MM patients. Approximately a third of patients demonstrated a functionally significant level of Stat3 signature, comparable to those in the Stat3-dependent cell lines. We next evaluated the anti-myeloma activity of the BP-2-047 in cell-based assays against a panel of HMCLs with high and low STAT3 activity. BP-2-047 delivered a desirable activity profile for targeted therapy. Consistent with its predicted mechanism of action, we observed dose-dependent inhibition of Tyr705Stat3 phosphorylation in XG6, XG7, JJN3 and UTMC2 cells, presumably through the blockade of Stat3 binding to the pTyr motifs of gp130 and the prevention of de novo phosphorylation by JAKs. Using a luciferase reporter assay we confirmed inhibition of Stat3 transcriptional activity in MM cells stably expressing the Stat3-dependent luciferase reporter (pLucTKS3). Inhibition of transcription was associated with decreased expression of short half-life proteins, c-Myc and Mcl-1. BP-2-047 inhibited viability of myeloma tumors with IC50 values of 3.1–7.4 microM, in cell lines with constitutive Stat3 activity. Inhibition of Stat3 phosphorylation resulted in induction of dose-dependent apoptosis as determined by PI/annexin V staining and PARP cleavage. However, cells were resistant to BP-2-047-induced apoptosis when co-cultured with bone marrow stroma cells. Identification of factors that confer survival advantage in the presence of stroma is in progress. Exposure of MM patient derived bone marrow mononuclear cells to < 15 microM BP-2-047 preferentially induced apoptosis of CD138 + MM cells. In contrast, BP-2-047 at concentrations of 30 microM failed to inhibit normal bone marrow-derived CD34 colony formation. In vitro combination studies in cell lines demonstrate synergy with dexamethasone, bortezomib and lenalidomide. Studies assessing in vivo activity of BP-2-047 as a single agent and in combination with bortezomib against Stat3 activated myeloma tumors in xenograft mice are ongoing and will be reported. These data suggest that Stat3 may represent an important therapeutic target in a subset of myeloma patients and supports innovative drug development with a focus on Stat3. Disclosures: No relevant conflicts of interest to declare.

Abstract 31 In CML, TKI resistance in the absence of BCR-ABL1 kinase mutations is mechanistically unclear. Since extrinsic signals from the bone marrow (BM) microenvironment protect CML cells from TKIs in a STAT3-dependent manner (Bewry et al. 2008; Traer et al. 2011), we hypothesized that overt resistance may occur when CML cells maintain intrinsic STAT3 activation in the absence of extrinsic signals. We also asked whether combined targeting of STAT3 and BCR-ABL1 could produce synthetic lethality to overcome TKI resistance. To model extrinsic BCR-ABL1 kinase-independent resistance, we grew CML cell lines and CMLCD34+ progenitor cells from newly diagnosed patients in the presence of conditioned medium (CM) derived from HS-5 BM stromal cells. To model intrinsic resistance, we used the imatinib-resistant K562R and AR230R cell lines. Our models of TKI resistance proliferate in 1.0–2.5 μM imatinib and exhibit pSTAT3Y705 activation despite suppression of BCR-ABL1 kinase activity. To investigate the role of pSTAT3Y705 in TKI resistance, we used shRNA-mediated knockdown (shSTAT3), a dominant-negative mutant (dnSTAT3), and pharmacologic STAT3 inhibitors developed in our laboratories to block STAT3 activity. Compared to scrambled controls in the presence of HS-5 CM, shSTAT3 reduced colony formation (33.8% reduction, p Preliminary immunofluorescence data indicates that pSTAT3Y705 levels are higher in drug-resistant CML CD34+CD38− stem cells than in CD34+CD38+ progenitor cells, suggesting a potential role for pSTAT3Y705 in TKI resistance of primitive CML stem cells. To investigate whether pharmacologic STAT3 inhibition in combination with TKIs could overcome resistance, we tested a library of compounds that interfere with STAT3 dimerization. The most selective of these inhibitors, S3I-201.1066 (SF1-066), was biochemically shown to disrupt STAT3 dimerization and transcriptional activation. Compared to cells treated with 1.0 μM imatinib alone, addition of 10 μM SF1-066 impaired the clonogenicity of K562R cells by 68.6% (p We next developed a series of 14 second-generation STAT3 inhibitors and tested them using a luciferase reporter harboring sequential STAT3 cis-inducible elements to measure endogenous STAT3 activity in AR230R cells. Two lead compounds, BP2-047 and BP3-163, reduced luciferase activity by 69.2% (p Altogether, our data identifies STAT3 as a universal target in TKI-resistant CML cells, and suggests that simultaneous pharmacologic inhibition of STAT3 and BCR-ABL1 may be a suitable therapeutic strategy for treatment of patients with TKI resistance despite inhibition of BCR-ABL1 kinase activity. Disclosures: No relevant conflicts of interest to declare.

Abstract 2445 Constitutive activation of signal transducer and activator of transcription 3 (STAT3) correlates with drug resistance and a poor prognosis in many cancers. STAT3 signaling is mediated by phosphorylation at tyrosine-705 (p STAT3Y705), dimerization, and nuclear transactivation. In chronic myeloid leukemia (CML), pSTAT3Y705 is demonstrable under two distinct resistance scenarios: (1) extrinsic resistance, in which BCR-ABL1 kinase-independent survival signals originating from the bone marrow (BM) microenvironment activate pSTAT3Y705 in a JAK2- or TYK2-dependent manner, and (2) intrinsic resistance, in which BCR-ABL1 kinase-independent signals activate pSTAT3Y705 in response to kinase inhibition. Based on these observations, we identified TKI-resistant CML as an excellent model for developing and optimizing pharmacologic STAT3 inhibitors. Using K562 and AR230 CML cells that are resistant to 1 μM imatinib (K562R and AR230R; intrinsic resistance) and primary CML CD34+ progenitor cells exposed to BM stromal-derived conditioned medium (CM; extrinsic resistance), we examined the effects of direct pharmacologic inhibition of STAT3 in TKI-resistant CML. Here, we report the design and validation of next-generation STAT3 inhibitors identified through computational modeling and screening in AR230R CML cells expressing high levels of pSTAT3Y705. We initially examined the effects of an established STAT3 inhibitor, S3I-201.1066 (SF1–066). K562R or AR230R cells were treated with 1 μM imatinib and/or 10 μM SF1–066, followed by culture in methylcellulose medium and scoring after 14–16 days. Combination treatment reduced the clonogenicity of K562R and AR230R cells to 31.4% (p0.05). Next, CD34+ cells from newly diagnosed CML patients (n=4) were cultured for 96 hours in the presence of CM and treated with 2.5 μM imatinib, 10 μM SF1–066 or both. Equal numbers of cells were then plated in colony forming assays. Imatinib combined with SF1–066 reduced colony formation to 54.8% (p Computational modeling informed the design and synthesis of a second-generation, SF1–066-based library. We evaluated compounds with shared molecular functionalities using AR230R cells expressing a luciferase reporter containing sequential STAT3 sis-inducible elements (AR230R-SIE). At 10 μM, we found two inhibitors, BP2–047 and BP3–163, that reduced luminescence by 69.2% (p To account for the reduced luminescence in AR230R-NEG cells and analyze for more potent inhibitors, we performed the luciferase assay with candidate inhibitors at 5 μM. We identified BP5–087 and BP5–088, each with increased potency and STAT3 selectivity compared to SF1–066. These compounds decreased luminescence in AR230R-SIE cells by 36.1% (p0.05). Confirmation of STAT3 binding was obtained using fluorescence polarization assays, in which the EC50 values of BP5–087 and BP5–088 measured 8.5 μM and 4.6 μM, respectively. Studies with mouse and human liver microsomes also revealed that BP5–087 and BP5–088 exhibit enhanced metabolic stability compared to SF1–066. Interestingly, treatment with BP5–087 or BP5–088 (both at 1 and 5 μM) in CML CD34+ progenitors grown in CM showed increased cytoplasmic accumulation of pSTAT3Y705 compared to controls. Thus, we identified BP5–087 and BP5–088 as two of the most potent small-molecule binders of STAT3 reported. These compounds are promising frontrunners toward new therapies for TKI-resistant CML and other diseases in which STAT3 activation drives malignant phenotypes. Disclosures: No relevant conflicts of interest to declare.