Retraction of "Identification of Purine-Scaffold Small-Molecule Inhibitors of Stat3 Activation by QSAR Studies".

[This retracts the article DOI: 10.1021/ml100224d.].

Quality of Life and Treatment-Related Side Effects in Patients With HR+/HER2- Advanced Breast Cancer: Findings From a Multicountry Survey.

BACKGROUND: Quality of life (QOL) is a critical factor in decision-making for advanced breast cancer (ABC). There is a need to improve how QOL and treatment-related side effects (SEs) that impact it are clinically assessed. We examined healthcare professionals' (HCPs') and patients' perspectives on the importance of QOL discussions and the impact of SEs on QOL in clinical settings. PATIENTS AND METHODS: A cross-sectional online survey was conducted (7/2020-5/2021) among oncologists, nurses, and patients with HR+/HER2- ABC in 7 countries. RESULTS: The survey was completed by 502 HCPs and 467 patients. Overall, 88% of oncologists and 49% of patients recalled QOL discussions at follow-up. In the first- through fourth-line (1L, 2L, 3L, and 4L) settings, respectively, 48%, 57%, 79%, and 85% of oncologists reported QOL was very important; 73% and 45% of patients receiving 1L and 2L treatment and 40% receiving 3L+ treatment indicated QOL was important. Patients reported that insomnia, anxiety, back pain, fatigue, diarrhea, hot flashes, low sexual interest, and loss of appetite had a moderate/severe impact on QOL. Of patients experiencing certain SEs, ≥64% did not discuss them with HCPs until there was a moderate/severe impact on QOL. In patients receiving a CDK4/6 inhibitor, SEs, including insomnia, diarrhea, back pain, and fatigue, had a moderate/severe impact on QOL. CONCLUSIONS: This survey discovered disconnects between HCPs and patients with ABC on the importance of QOL discussions and the impact of SEs on QOL. These data support the use of ABC-specific QOL questionnaires that closely monitor SEs impacting QOL.

Quality of life with ribociclib versus abemaciclib as first-line treatment of HR+/HER2- advanced breast cancer: a matching-adjusted indirect comparison.

Background: A cyclin-dependent kinase 4/6 inhibitor (CDK4/6i) + endocrine therapy is recommended as first-line treatment for hormone receptor-positive/human epidermal growth factor receptor 2-negative (HR+/HER2-) advanced breast cancer (ABC). Quality of life (QoL) is an important endpoint that affects treatment decisions. Understanding the relevance of CDK4/6i treatment on QoL is gaining importance given use in earlier treatment lines for ABC and an emerging role in treating early breast cancer in which QoL may be more impactful. In the absence of head-to-head trial data, a matching-adjusted indirect comparison (MAIC) permits comparative efficacy between trials. Objective: In this analysis, patient-reported QoL for MONALEESA-2 [ribociclib + aromatase inhibitor (AI)] and MONARCH 3 (abemaciclib + AI) was compared using MAIC with a focus on individual domains. Design: An anchored MAIC of QoL comparing ribociclib + AI versus abemaciclib + AI was performed using data from the European Organization for Research and Treatment of Cancer quality of life questionnaire (QLQ)-C30 and BR-23 questionnaires. Methods: Individual patient data from MONALEESA-2 and published aggregated data from MONARCH 3 were included in this analysis. Time to sustained deterioration (TTSD) was calculated as the time from randomization to a ⩾10-point deterioration with no later improvement above this threshold. Results: Patients from the ribociclib (n = 205) and placebo (n = 149) arms of MONALEESA-2 were matched with patients from the abemaciclib (n = 328) and placebo (n = 165) arms of MONARCH 3. After weighting, baseline patient characteristics were well balanced. TTSD significantly favored ribociclib versus abemaciclib in appetite loss [hazard ratio (HR), 0.46; 95% confidence interval (CI), 0.27-0.81], diarrhea (HR, 0.42; 95% CI, 0.23-0.79), fatigue (HR, 0.63; 95% CI, 0.41-0.96), and arm symptoms (HR, 0.49; 95% CI, 0.30-0.79). TTSD did not significantly favor abemaciclib compared with ribociclib in any functional or symptom scale of the QLQ-C30 or BR-23 questionnaires. Conclusions: This MAIC indicates that ribociclib + AI is associated with better symptom-related QoL than abemaciclib + AI for postmenopausal patients with HR+/HER2- ABC treated in the first-line setting. Trial registration: NCT01958021 (MONALEESA-2) and NCT02246621 (MONARCH 3).

Chronic Inhibition of STAT3/STAT5 in Treatment-Resistant Human Breast Cancer Cell Subtypes: Convergence on the ROS/SUMO Pathway and Its Effects on xCT Expression and System xc- Activity.

Pharmacologically targeting activated 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 cancer cells. We have previously shown that blocking STAT3/5 using the small molecule inhibitor, SH-4-54, which targets the SH2 domains of both proteins, increases xCT expression, thereby increasing system xc- activity in human breast cancer cells. The current investigation demonstrates that chronic SH-4-54 administration, 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 triple-negative MDA-MB-231 cells, with the inverse occurring in estrogen-responsive T47D cells. This "ying-yang" effect is linked with a shifted balance between the phosphorylation status of STAT3 and STAT5, intracellular ROS levels, and STAT5 SUMOylation/de-SUMOylation. STAT5 emerged as a definitive negative regulator of xCT at the transcriptional level, while STAT3 activation is coupled with increased system xc- activity. We propose that careful classification of a patient's breast cancer subtype is central to effectively targeting STAT3/5 as a therapeutic means of treating breast cancer, particularly given that xCT is emerging as an important biomarker of aggressive cancers.

Efficient Routes to a Diverse Array of Amino Alcohol-Derived Chiral Fragments.

Efficient syntheses of chiral fragments derived from chiral amino alcohols are described. Several unique scaffolds were readily accessed in 1-5 synthetic steps leading to 45 chiral fragments, including oxazolidinones, morpholinones, lactams, and sultams. These fragments have molecular weights ranging from 100 to 255 Da and are soluble in water (0.085 to >15 mM).

Applying Small Molecule Signal Transducer and Activator of Transcription-3 (STAT3) Protein Inhibitors as Pancreatic Cancer Therapeutics.

Constitutively activated STAT3 protein has been found to be a key regulator of pancreatic cancer and a target for molecular therapeutic intervention. In this study, PG-S3-001, a small molecule derived from the SH-4-54 class of STAT3 inhibitors, was found to inhibit patient-derived pancreatic cancer cell proliferation in vitro and in vivo in the low micromolar range. PG-S3-001 binds the STAT3 protein potently, Kd = 324 nmol/L by surface plasmon resonance, and showed no effect in a kinome screen (>100 cancer-relevant kinases). In vitro studies demonstrated potent cell killing as well as inhibition of STAT3 activation in pancreatic cancer cells. To better model the tumor and its microenvironment, we utilized three-dimensional (3D) cultures of patient-derived pancreatic cancer cells in the absence and presence of cancer-associated fibroblasts (CAF). In this coculture model, inhibition of tumor growth is maintained following STAT3 inhibition in the presence of CAFs. Confocal microscopy was used to verify tumor cell death following treatment of 3D cocultures with PG-S3-001. The 3D model was predictive of in vivo efficacy as significant tumor growth inhibition was observed upon administration of PG-S3-001. These studies showed that the inhibition of STAT3 was able to impact the survival of tumor cells in a relevant 3D model, as well as in a xenograft model using patient-derived cells. Mol Cancer Ther; 15(5); 794-805. ©2016 AACR.

Carcinoembryonic Antigen Cell Adhesion Molecule 1 long isoform modulates malignancy of poorly differentiated colon cancer cells.

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.

Identification of Bidentate Salicylic Acid Inhibitors of PTP1B.

PTP1B is a master regulator in the insulin and leptin metabolic pathways. Hyper-activated PTP1B results in insulin resistance and is viewed as a key factor in the onset of type II diabetes and obesity. Moreover, inhibition of PTP1B expression in cancer cells dramatically inhibits cell growth in vitro and in vivo. Herein, we report the computationally guided optimization of a salicylic acid-based PTP1B inhibitor 6, identifying new and more potent bidentate PTP1B inhibitors, such as 20h, which exhibited a > 4-fold improvement in activity. In CHO-IR cells, 20f, 20h, and 20j suppressed PTP1B activity and restored insulin receptor phosphorylation levels. Notably, 20f, which displayed a 5-fold selectivity for PTP1B over the closely related PTPσ protein, showed no inhibition of PTP-LAR, PRL2 A/S, MKPX, or papain. Finally, 20i and 20j displayed nanomolar inhibition of PTPσ, representing interesting lead compounds for further investigation.

STAT3 pathway regulates lung-derived brain metastasis initiating cell capacity through miR-21 activation.

Brain metastases (BM) represent the most common tumor to affect the adult central nervous system. Despite the increasing incidence of BM, likely due to consistently improving treatment of primary cancers, BM remain severely understudied. In this study, we utilized patient-derived stem cell lines from lung-to-brain metastases to examine the regulatory role of STAT3 in brain metastasis initiating cells (BMICs). Annotation of our previously described BMIC regulatory genes with protein-protein interaction network mapping identified STAT3 as a novel protein interactor. STAT3 knockdown showed a reduction in BMIC self-renewal and migration, and decreased tumor size in vivo. Screening of BMIC lines with a library of STAT3 inhibitors identified one inhibitor to significantly reduce tumor formation. Meta-analysis identified the oncomir microRNA-21 (miR-21) as a target of STAT3 activity. Inhibition of miR-21 displayed similar reductions in BMIC self-renewal and migration as STAT3 knockdown. Knockdown of STAT3 also reduced expression of known downstream targets of miR-21. Our studies have thus identified STAT3 and miR-21 as cooperative regulators of stemness, migration and tumor initiation in lung-derived BM. Therefore, STAT3 represents a potential therapeutic target in the treatment of lung-to-brain metastases.

Signal transducer and activator of transcription 3 and 5 regulate system Xc- and redox balance in human breast cancer cells.

System Xc- is a cystine/glutamate antiporter that contributes to the maintenance of cellular redox balance. The human xCT (SLC7A11) gene encodes the functional subunit of system Xc-. Transcription factors regulating antioxidant defense mechanisms including system Xc- are of therapeutic interest, especially given that aggressive breast cancer cells exhibit increased system Xc- function. This investigation provides evidence that xCT expression is regulated by STAT3 and/or STAT5A, functionally affecting the antiporter in human breast cancer cells. Computationally analyzing two kilobase pairs of the xCT promoter/5' flanking region identified a distal gamma-activated site (GAS) motif, with truncations significantly increasing luciferase reporter activity. Similar transcriptional increases were obtained after treating cells transiently transfected with the full-length xCT promoter construct with STAT3/5 pharmacological inhibitors. Knock-down of STAT3 or STAT5A with siRNAs produced similar results. However, GAS site mutation significantly reduced xCT transcriptional activity, suggesting that STATs may interact with other transcription factors at more proximal promoter sites. STAT3 and STAT5A were bound to the xCT promoter in MDA-MB-231 cells, and binding was disrupted by pre-treatment with STAT inhibitors. Pharmacologically suppressing STAT3/5 activation significantly increased xCT mRNA and protein levels, as well as cystine uptake, glutamate release, and total levels of intracellular glutathione. Our data suggest that STAT proteins negatively regulate basal xCT expression. Blocking STAT3/5-mediated signaling induces an adaptive, compensatory mechanism to protect breast cancer cells from stress, including reactive oxygen species, by up-regulating xCT expression and the function of system Xc-. We propose that targeting system Xc- together with STAT3/5 inhibitors may heighten therapeutic anti-cancer effects.

Changes in signal transducer and activator of transcription 3 (STAT3) dynamics induced by complexation with pharmacological inhibitors of Src homology 2 (SH2) domain dimerization.

The activity of the transcription factor signal transducer and activator of transcription 3 (STAT3) is dysregulated in a number of hematological and solid malignancies. Development of pharmacological STAT3 Src homology 2 (SH2) domain interaction inhibitors holds great promise for cancer therapy, and a novel class of salicylic acid-based STAT3 dimerization inhibitors that includes orally bioavailable drug candidates has been recently developed. The compounds SF-1-066 and BP-1-102 are predicted to bind to the STAT3 SH2 domain. However, given the highly unstructured and dynamic nature of the SH2 domain, experimental confirmation of this prediction was elusive. We have interrogated the protein-ligand interaction of STAT3 with these small molecule inhibitors by means of time-resolved electrospray ionization hydrogen-deuterium exchange mass spectrometry. Analysis of site-specific evolution of deuterium uptake induced by the complexation of STAT3 with SF-1-066 or BP-1-102 under physiological conditions enabled the mapping of the in silico predicted inhibitor binding site to the STAT3 SH2 domain. The binding of both inhibitors to the SH2 domain resulted in significant local decreases in dynamics, consistent with solvent exclusion at the inhibitor binding site and increased rigidity of the inhibitor-complexed SH2 domain. Interestingly, inhibitor binding induced hot spots of allosteric perturbations outside of the SH2 domain, manifesting mainly as increased deuterium uptake, in regions of STAT3 important for DNA binding and nuclear localization.

Inhibiting aberrant signal transducer and activator of transcription protein activation with tetrapodal, small molecule Src homology 2 domain binders: promising agents against multiple myeloma.

The signal transducer and activator of transcription (STAT) proteins represent a family of cytoplasmic transcription factors that regulate a pleiotropic range of biological processes. In particular, Stat3 protein has attracted attention as it regulates the expression of genes involved in a variety of malignant processes, including proliferation, survival, migration, and drug resistance. Multiple myeloma (MM) is an incurable hematologic malignancy that often exhibits abnormally high levels of Stat3 activity. Although current treatment strategies can improve the clinical management of MM, it remains uniformly incurable with a dismal median survival time post-treatment of 3-4 years. Thus, novel targeted therapeutics are critically needed to improve MM patient outcomes. We herein report the development of a series of small molecule Stat3 inhibitors with potent anti-MM activity in vitro. These compounds showed high-affinity binding to Stat3's SH2 domain, inhibited intracellular Stat3 phosphorylation, and induced apoptosis in MM cell lines at low micromolar concentrations.

A 2,6,9-hetero-trisubstituted purine inhibitor exhibits potent biological effects against multiple myeloma cells.

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's <5µM). 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.

Potent Targeting of the STAT3 Protein in Brain Cancer Stem Cells: A Promising Route for Treating Glioblastoma.

The STAT3 gene is abnormally active in glioblastoma (GBM) and is a critically important mediator of tumor growth and therapeutic resistance in GBM. Thus, for poorly treated brain cancers such as gliomas, astrocytomas, and glioblastomas, which harbor constitutively activated STAT3, a STAT3-targeting therapeutic will be of significant importance. Herein, we report a most potent, small molecule, nonphosphorylated STAT3 inhibitor, 31 (SH-4-54) that strongly binds to STAT3 protein (K D = 300 nM). Inhibitor 31 potently kills glioblastoma brain cancer stem cells (BTSCs) and effectively suppresses STAT3 phosphorylation and its downstream transcriptional targets at low nM concentrations. Moreover, in vivo, 31 exhibited blood-brain barrier permeability, potently controlled glioma tumor growth, and inhibited pSTAT3 in vivo. This work, for the first time, demonstrates the power of STAT3 inhibitors for the treatment of BTSCs and validates the therapeutic efficacy of a STAT3 inhibitor for GBM clinical application.

Identification of Purine-Scaffold Small-Molecule Inhibitors of Stat3 Activation by QSAR Studies.

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.

Inhibiting aberrant Stat3 function with molecular therapeutics: a progress report.

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.

Binding interactions in early- and late-stage amyloid aggregates of TTR(105-115).

One of the central aims of amyloid research is to identify chemical and structural features that confer amyloidogenic propensity. In this study, we use Saturation Transfer Difference (STD) NMR spectroscopy to acquire an atom-specific map of the interactions between soluble and aggregated Transthyretin peptide (TTR(105-115)) in early- and late-stage amyloidogenesis. Atomic Force Microscopy (AFM) was used to monitor the transition of early-stage samples, containing protofilaments, to late-stage samples composed of fully-mature fibrils. Progressive aggregation was accompanied by an increase in the correlation time tau(c) of soluble TTR(105-115) as indicated by (1)H NMR line broadening, but no significant change in the (1)H chemical shifts. The STD profile of backbone amide protons is in good agreement with an earlier computational study predicting hydrogen bonding propensity for each residue in small TTR(105-115) aggregates (Paci et al., J. Mol. Biol. (2004) 555-569). The STD profiles of C(alpha) and C(beta) protons identify a central aliphatic region of the peptide, Ala108-Leu111, that plays a crucial, but different role in early- and late-stage amyloidogenesis. In general, the STD profiles of early and fully-mature samples are dissimilar, suggesting different mechanisms of self-assembly in protofilaments and mature amyloid fibrils. The early-stage mechanism appears to be more dependent on main-chain hydrogen bonding, while the late-stage mechanism involves an increased number of interactions between bulky side chains.