David A. Rosa

A STAT inhibitor patent review: progress since 2011.

Introduction: The clinical utility of effective direct STAT inhibitors, particularly STAT3 and STAT5, for treating cancer and other diseases is well studied and known. Areas covered: This review will highlight the STAT inhibitor patent literature from 2011 to 2015 inclusive. Emphasis will be placed on inhibitors of the STAT3, STAT5a/b, and STAT1 proteins for cancer treatment. The review will, where suitably investigated, describe the mode and the site of inhibition, list indications that were evaluated, and rank the inhibitor’s relative potency among compounds in the same class. The reader will gain an understanding of the diverse set of approaches, used both in academia and industry, to target STAT proteins. Expert opinion: There is still much work to be done to directly target the STAT3 and STAT5 proteins. As yet, there is still no direct STAT3 inhibitor in the clinic. While the SH2 domain remains a popular target for therapeutic intervention, the DNA-binding domain and N-terminal region are now attracting attention as possible sites for inhibition. Multiple putative STAT3 and STAT5 inhibitors have now been patented across a broad spectrum of chemotypes, each with their own advantages and limitations.

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.

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.

CD133 + brain tumor-initiating cells are dependent on STAT3 signaling to drive medulloblastoma recurrence

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

Disarming an Electrophilic Warhead: Retaining Potency in Tyrosine Kinase Inhibitor (TKI)‐Resistant CML Lines While Circumventing Pharmacokinetic Liabilities

Pharmacologic blockade of the activation of signal transducer and activator of transcription 3 (STAT3) in tyrosine kinase inhibitor (TKI)‐resistant chronic myeloid leukemia (CML) cell lines characterized by kinase‐independent resistance was shown to re‐sensitize CML cells to TKI therapy, suggesting that STAT3 inhibitors in combination with TKIs are an effective combinatorial therapeutic for the treatment of CML. Benzoic acid‐ and hydroxamic acid‐based STAT3 inhibitors SH‐4‐054 and SH‐5‐007, developed previously in our laboratory, demonstrated promising activity against these resistant CML cell lines. However, pharmacokinetic studies in murine models (CD‐1 mice) revealed that both SH‐4‐054 and SH‐5‐007 are susceptible to glutathione conjugation at the para position of the pentafluorophenyl group via nucleophilic aromatic substitution (SNAr). To determine whether the electrophilicity of the pentafluorophenyl sulfonamide could be tempered, an in‐depth structure–activity relationship (SAR) study of the SH‐4‐054 scaffold was conducted. These studies revealed that AM‐1‐124, possessing a 2,3,5,6‐tetrafluorophenylsulfonamide group, retained STAT3 protein affinity (Ki=15 μm), as well as selectivity over STAT1 (Ki>250 μm). Moreover, in both hepatocytes and in in vivo pharmacokinetic studies (CD‐1 mice), AM‐1‐124 was found to be dramatically more stable than SH‐4‐054 (t1/2=1.42 h cf. 10 min, respectively). AM‐1‐124 is a promising STAT3‐targeting inhibitor with demonstrated bioavailability, suitable for evaluation in preclinical cancer models.

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.

The ERBB-STAT3 Axis Drives Tasmanian Devil Facial Tumor Disease

The marsupial Tasmanian devil (Sarcophilus harrisii) faces extinction due to transmissible devil facial tumor disease (DFTD). To unveil the molecular underpinnings of DFTD, we designed an approach that combines sensitivity to drugs with an integrated systems-biology characterization. Sensitivity to inhibitors of the ERBB family of receptor tyrosine kinases correlated with their overexpression, suggesting a causative link. Proteomic and DNA methylation analyses revealed tumor-specific signatures linked to oncogenic signaling hubs including evolutionary conserved STAT3. Indeed, ERBB inhibition blocked phosphorylation of STAT3 and arrested cancer cells. Pharmacological blockade of ERBB signaling prevented tumor growth in a xenograft model and resulted in recovery of MHC class I gene expression. This link between the hyperactive ERBB-STAT3 axis and MHC class I mediated tumor immunosurveillance provides mechanistic insights into horizontal transmissibility and led us to the proposition of a dual chemo-immunotherapeutic strategy to save Tasmanian devils from DFTD.

MiR-337-3p controls hepatobiliary gene expression and transcriptional dynamics during hepatic cell differentiation

Transcriptional networks control the differentiation of the hepatocyte and cholangiocyte lineages from embryonic liver progenitor cells and their subsequent maturation to the adult phenotype. However, how relative levels of hepatocyte and cholangiocyte gene expression are determined during differentiation remains poorly understood. Here, we identify microRNA (miR)‐337‐3p as a regulator of liver development. miR‐337‐3p stimulates expression of cholangiocyte genes and represses hepatocyte genes in undifferentiated progenitor cells in vitro and in embryonic mouse livers. Beyond the stage of lineage segregation, miR‐337‐3p controls the transcriptional network dynamics of developing hepatocytes and balances both cholangiocyte populations that constitute the ductal plate. miR‐337‐3p requires Notch and transforming growth factor‐β signaling and exerts a biphasic control on the hepatocyte transcription factor hepatocyte nuclear factor 4α by modulating its activation and repression. With the help of an experimentally validated mathematical model, we show that this biphasic control results from an incoherent feedforward loop between miR‐337‐3p and hepatocyte nuclear factor 4α. Conclusion: Our results identify miR‐337‐3p as a regulator of liver development and highlight how tight quantitative control of hepatic cell differentiation is exerted through specific gene regulatory network motifs. (Hepatology 2018;67:313‐327).

Abstract 1246: Development of STAT3 dual-targeting strategies for the treatment of pancreatic cancer

Pancreatic cancer remains a largely incurable disease, with patients facing the worst 5-year survival rate of any cancer. The challenge is to identify the molecular effectors that regulate the survival of pancreatic ductal adenocarcinoma (PDAC) cells, to devise molecular-targeted strategies that are effective in the metastatic setting, and overcome the protective role of the tumor-associated fibrosis and stroma. Strategies targeting multiple molecular effectors in PDAC are likely going to make a bigger impact. Thus, we are identifying molecular targets or synthetic lethal pairs that regulate critical pro-survival or pro-invasive pathways in PDAC. Constitutively activated Signal Transducer and Activator of Transcription 3 (STAT3) protein has been found to be a key regulator of pancreatic cancer and a target for molecular therapeutic intervention. To better model the tumor and its microenvironment, we utilized ex vivo 3-Dimensional (3D) cultures of patient-derived pancreatic cancer cells in the absence and presence of cancer-associated fibroblasts (CAFs). We can quantitate the inhibitory effect on both the tumor and CAFs as they are labeled with different fluorescent markers. In this co-culture model, inhibition of tumor growth is maintained following STAT3 inhibition in the presence of CAFs. We screened several STAT3 small molecule inhibitors, derived from the SH-4-54 class of STAT3 inhibitors, and found inhibition of pancreatic cancer cell proliferation in the low μM range. Our inhibitors bind the STAT3 protein potently, as shown by SPR, and demonstrate no effect in a kinome screen. In vitro studies demonstrated potent cell killing as well as inhibition of STAT3 activation in the 3D co-culture model. We have previously reported that Ref-1 (redox factor-1) regulates STAT3 activity through its redox function and blocking STAT3 through phosphorylation and redox inhibition synergizes for PDAC cell killing. In our 3D co-culture system, Ref-1 inhibitor, APX3330 decreases tumor area and intensity in a dose-dependent manner. The addition of APX3330 to STAT3 pathway inhibition via Ruxolitinib (Rux, Jak 2 inhibitor) or direct STAT3 inhibitor potentiated the killing effect in the tumor. However, the combination treatment did not appear to sensitize CAF cells, suggesting that targeting of Ref-1 and the STAT3 pathway is more specifically targeting tumor cells. Utilizing APX3330, Rux, and our lead STAT3 inhibitors, we evaluated the effects of Ref-1/STAT3 inhibition in PDAC low passage patient-derived cell lines. The activity of STAT3 and specificity of lead compounds was assessed by immunoblotting for levels of phosphorylated proteins including STAT3 (Y705) and STAT5 (Y694) in 3D culture. These studies establish the rationale for the development of STAT3 dual-targeting strategies for the treatment of pancreatic cancer and suggest that Ref-1 and STAT3 may be a synthetic lethal pair. Citation Format: Melissa L. Fishel, Michelle L. Grimard, Mark R. Kelley, David A. Rosa, Andrew Shouksmith, Gary Tin, Ji Park, Patrick T. Gunning. Development of STAT3 dual-targeting strategies for the treatment of pancreatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1246.